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Mechanisms of Resistance to Southern Chinch Bug, Blissus insularis Barber (Hemiptera

Permanent Link: http://ufdc.ufl.edu/UFE0021706/00001

Material Information

Title: Mechanisms of Resistance to Southern Chinch Bug, Blissus insularis Barber (Hemiptera Blissidae), in St. Augustinegrass
Physical Description: 1 online resource (134 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antibiosis, antixenosis, blissus, catalase, epg, feeding, lignin, lipoxygenase, peroxidase, ppo, salivary, scb, sclerenchyma, stenotaphrum
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, is the most commonly grown lawn grass in Florida. The southern chinch bug (SCB), Blissus insularis Barber (Hemiptera: Blissidae), is the most serious insect pest of St. Augustinegrass. Host plant resistance has long been one of most successful management tools to control the SCB. Resistance to SCB has been identified in the St. Augustinegrass lines ?Floratam?, ?FX-10? and ?NUF-76?. Floratam was once resistant but is now susceptible. This research investigated the mechanisms of SCB resistance in FX-10 and NUF-76. Choice and no-choice tests and ovipositional and developmental studies indetified strong antixenosis in FX-10 and NUF-76 and antibiosis in NUF- 76. FX-10 and NUF-76 had significantly higher peroxidase activity and NUF-76 had significantly higher polyphenol oxidase activity 5 and 8 d after infestation compared to uninfested controls. FX-10 had significantly higher lipoxygenase activity 3, 5 and 8 d after infestation compared to uninfested controls. Native gels stained for peroxidase indicated that some isozymes in FX-10 and NUF-76 were induced 5 and 8 d after infestation. Electrical penetration graph studies showed that the SCB made more frequent probes, spent longer time on pathway related activities on FX-10 and NUF-76, compared to the susceptible Floratam and Palmetto. Significantly higher number of salivary sheaths was observed in the outermost leaf sheath in FX-10 and NUF-76. The total lignin content of the axillary shoot and the thickness of the epidermal layer in SCB resistant and susceptible St. Augustinegrasses did not differ significantly. TEM studies on the leaf sheath showed scelerenchyma cells with thicker cell walls in NUF-76 and FX-10 than in Floratam and Palmetto suggesting that the sclerenchyma cells around the vascular bundle in FX-10 and NUF-76 could serve as an impediment to the SCB stylet penetration. From this research, I conclude that the strong antixenosis found in FX-10 and NUF-76 and antibiosis in NUF-76 could be due to a combination of induction of oxidative enzymes, thicker sclerenchyma cells around the vascular bundle and possible presence of feeding deterrents or toxins in phloem sap or sieve tube blocking mechanisms.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cherry, Ronald H.
Local: Co-adviser: McAuslane, Heather J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021706:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021706/00001

Material Information

Title: Mechanisms of Resistance to Southern Chinch Bug, Blissus insularis Barber (Hemiptera Blissidae), in St. Augustinegrass
Physical Description: 1 online resource (134 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: antibiosis, antixenosis, blissus, catalase, epg, feeding, lignin, lipoxygenase, peroxidase, ppo, salivary, scb, sclerenchyma, stenotaphrum
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, is the most commonly grown lawn grass in Florida. The southern chinch bug (SCB), Blissus insularis Barber (Hemiptera: Blissidae), is the most serious insect pest of St. Augustinegrass. Host plant resistance has long been one of most successful management tools to control the SCB. Resistance to SCB has been identified in the St. Augustinegrass lines ?Floratam?, ?FX-10? and ?NUF-76?. Floratam was once resistant but is now susceptible. This research investigated the mechanisms of SCB resistance in FX-10 and NUF-76. Choice and no-choice tests and ovipositional and developmental studies indetified strong antixenosis in FX-10 and NUF-76 and antibiosis in NUF- 76. FX-10 and NUF-76 had significantly higher peroxidase activity and NUF-76 had significantly higher polyphenol oxidase activity 5 and 8 d after infestation compared to uninfested controls. FX-10 had significantly higher lipoxygenase activity 3, 5 and 8 d after infestation compared to uninfested controls. Native gels stained for peroxidase indicated that some isozymes in FX-10 and NUF-76 were induced 5 and 8 d after infestation. Electrical penetration graph studies showed that the SCB made more frequent probes, spent longer time on pathway related activities on FX-10 and NUF-76, compared to the susceptible Floratam and Palmetto. Significantly higher number of salivary sheaths was observed in the outermost leaf sheath in FX-10 and NUF-76. The total lignin content of the axillary shoot and the thickness of the epidermal layer in SCB resistant and susceptible St. Augustinegrasses did not differ significantly. TEM studies on the leaf sheath showed scelerenchyma cells with thicker cell walls in NUF-76 and FX-10 than in Floratam and Palmetto suggesting that the sclerenchyma cells around the vascular bundle in FX-10 and NUF-76 could serve as an impediment to the SCB stylet penetration. From this research, I conclude that the strong antixenosis found in FX-10 and NUF-76 and antibiosis in NUF-76 could be due to a combination of induction of oxidative enzymes, thicker sclerenchyma cells around the vascular bundle and possible presence of feeding deterrents or toxins in phloem sap or sieve tube blocking mechanisms.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cherry, Ronald H.
Local: Co-adviser: McAuslane, Heather J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021706:00001


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14b3632966857b9c459f7403d41ede9a633aac25







MECHANISMS OF RESISTANCE TO SOUTHERN CHINCH BUG, Blissus insularis
BARBER (HEMIPTERA: BLISSIDAE), IN ST. AUGUSTINEGRASS




















BY

MURUGESAN RANGASAMY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008



































2008 Murugesan Rangasamy
























To my beloved mother, Thirumathi K. Kaliammal, and father, Thiru C. Rangasamy Gounder









ACKNOWLEDGMENTS

I pay my sublime obeisance to my esteemed parents, wife Priya, and daughter Sudhikssha

whose love, sacrifice and moral support helped me to put this tireless venture to a fruitful result.

To express the feelings in my heart, I put on record my deepest gratitude and profoundest

indebtedness to my advisors, Dr. Ron Cherry and Dr. Heather McAuslane, for their guidance and

making my dissertation worthy of presentation. Their constant attention, patient helping, sense

of dedication and search for perfection are underlying factors which will be part of my memory

forever. I consider myself fortunate to be their disciple. I am obliged to the members of my

advisory committee, Dr. Bala Rathinasabapathi, Dr. Eric Schemlz, Dr. Russell Nagatta, and Dr.

Elaine Backus for their support, guidance and valuable suggestions which led to a successful

completion of my research and compilation of this dissertation.

I give special thanks to Ms. Debbie Boyd Mr. Alwin Wilson and Ms. Shan Wu, our

biological scientists for their technical assistance. I sincerely thank Dr. Marty Marshall for use

of his spectrophotometer, Ms. Karen Kelly and Dr. Sharron Mathews for helping with electron

microscope, Dr. Louis Jack for his help with the EPG workshop. I duly acknowledge my lab

mates, Karla Addesso, Amit Sethi, and Jennifer Mayor, for their critical comments and

suggestions on my research and presentations. No appropriate word could be traced in the

lexicon to avouch my friends Bhuvana, Chandramohan, James, Karthik, Ravi, Sabari, Subbu,

Sureshbabu and others who helped me both concentrate and relax during my PhD program. All

may not be mentioned but none is forgotten.









TABLE OF CONTENTS
page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST O F TA BLE S ............................................................................................. .............

LIST OF FIGURES ................................. .. ..... ..... ................. .9

A B S T R A C T ............ ................... ............................................................ 1 1

CHAPTER

1 L IT E R A TU R E R E V IE W ............................................................................... .................. 13

T axonom y of the C hinch B ugs .............................................................................. ........ 13
H ost Plants and Feeding Preferences......................................................................... ... ... 15
Feeding H abit and N ature of D am age ........................................................................ ...... 16
O viposition and D evelopm ent .............. ................................................................ .............. 17
Seasonal Wing Polymorphism and Movement..... .............. ..................18
M anagem ent of Southern Chinch Bugs......... ......................................... ..... ............... 19
C cultural M methods .............................................................................................. .......... 20
B biological C control A gents ......... .................................... ....................... ............... 20
Chem ical C control M ethods.................. .......... ......... ... ................................. 21
St. Augustinegrass and Host Plant Resistance against SN.,,itl in Chinch Bugs .............23
Breeding Resistant Varieties against Chinch Bugs, Continuing Missions...................26
Refining the Screening Techniques ................................................... ............... ... 29
Other Species of Chinch Bugs and Host Plant Resistance............................................29
Characterization of Resistance against Chinch Bugs in Buffalograss.............................30
Chinch Bug Resistance in Sorghum ..................................................................... 31
Investigating the Mechanisms of Southern Chinch Bug Resistance in St.
A ugustinegrasses...................................................... ................... .......... ...... 31

2 CATEGORIES OF RESISTANCE IN ST. AUGUSTINEGRASS LINES TO
SOUTHERN CHINCH BUG (HEMIPTERA: BLISSIDAE).........................................33

In tro du ctio n ................... .......................................................... ................ 3 3
M materials and M methods ................................... ... .. .......... ....... ...... 35
P plants and Insects .........................................................................35
C h oice T ests ............................................................................. 3 6
N o-C choice T ests ................................................................... 37
O vipositional Studies..................................................... 38
Developmental Studies ............. ... ............. ............................... ..........38
Statistical A analysis ........................ ................ .. .. ... ......... .... .. .....39
R e su lts ......... ....... .. ............. .. ........................................................... 3 9
C choice T ests. ..................................................................39
N o-C choice T ests ................................................................... 40









O vipostional Stu dies........... ...... ............................................ .............. ......... ........ 4 1
Developmental Studies ........ ..... .............. ............ ...... ........ ... ...............41
D discussion ..................................................................................42

3 OXIDATIVE RESPONSES OF ST. AUGUSTINEGRASSES CHALLENGED BY
SOUTHERN CHINCH BUG, Blissus Insularis BARBER (HEMIPTERA:
B L IS S ID A E ) ..................................................................................................................... 4 7

In tro du ctio n ................... ...................4...................7..........
M materials and M ethods ............... ........................................ .............. ...... 48
P lants, Insects, Sam ple C ollection....................................................................... .......48
S am p le P rep aration ......... ...... .................................................................. ....... ............... 4 9
E stim action of T total P protein .............................................................................................50
Isozyme Profile Studies .................... ........ ........ ...............51
Statistical A n aly sis ................................................................52
R results ........... ......... .................................... 53
T total P protein C content ...............................................................53
Peroxidase Activities ................................. ............................ ...........53
Polyphenol Oxidase A activities ......................................................... 53
Lipoxygenase A activities ........................ ....................... .... ....... .......... 54
Catalase A activities ................................................... ............. ............ 54
Isozym e Profile Studies ...................................................................... ........54
D iscu ssio n ................................................... ...... .......................... .............................5 5

4 QUANTITATIVE ANALYSIS OF FEEDING BEHAVIOR OF SOUTHERN CHINCH
BUG, Blissus insularis BARBER (HEMIPTERA: BLISSIDAE), ON RESISTANT
AND SUSCEPTIBLE ST. AUGUSTINEGRASSES.................. ......................... 68

In tro d u ctio n ................... ...................6...................8..........
M materials and M methods ...................................... .. ......... ......... .....70
P la n ts .............. .... ...............................................................7 0
In sects ..........................................................................................7 0
Wiring and Acclimating of the Insects.......................... ......... .................70
EPG Equipment and Data Acquisition...... ........................................71
W aveform Categories ............................................................ ...... ....72
Definition of Non-Sequential Parameters .................................................... ...............73
Statistical A n aly sis ................................................................73
R e su lts ................... ...................7...................4..........
C o h o rt L ev e l ........................................................................................................7 4
E v e n t L ev e l ........................................................................................................7 5
P ro b e L ev e l ........................................................................................................7 5
In se ct L ev e l ..............................................................................7 6
D iscu ssio n ................... ...................7...................7..........







6










5 ROLE OF LEAF SHEATH LIGNIFICATION AND ANATOMY IN RESISTANCE
AGAINST SOUTHERN CHINCH BUG, Blissus insularis BARBER (HEMIPTERA:
BLISSIDAE) IN ST. AUGUSTINEGRASS LINES................................... ...............87

Introduction ................. ...................................... ............................87
Materials and Methods ....................... ................... ............. ....89
Staining for Salivary Sheaths ................................................ .............................. 90
Lignin Quantification ....................... .............................. .. ................ 91
Staining for L ignin ................................................................9 1
Transm mission Electron M icroscopy ....................................................... ...............92
Experim ental Design and Statistical Analysis...................................... ............... 92
R esults............ ......... ........................ ... ................................ ................. 93
D discussion ........... ..................................... ............................ ................ 94

6 S U M M A R Y .............................. ............ ............................. ................ 10 8

L IST O F R E F E R E N C E S ............................ ...........................................................................115

B IO G R A PH IC A L SK ETCH .............. ................................................... ........... ........... 134









LIST OF TABLES


Table page

2-1 Distribution (mean proportion SEM) of adult southern chinch bugs among five St.
Augustinegrass lines in a choice situation at varying time intervals after release *..........44

2-2 Mean number ( SEM) of excretory spots deposited per living chinch bug on St.
Augustinegrass lines in a no-choice situation at varying time intervals after release .......45

2-3 Mean number ( SEM) of eggs produced per female alive on St. Augustinegrass
lines at w eekly intervals after release ........................................... ......................... 46

4-1 Durations and frequencies of probing parameters of EPG data recorded on four
southern chinch bug resistant and susceptible St. Augustinegrass lines..........................82

4-2 Total number of major waveform events (TNWE) on each of four southern chinch
bug-resistant or -susceptible St. Augustinegrass varieties (n = 22). .................................82

4-3 Mean waveform durations per event (WDE) for major waveforms recorded for
southern chinch bug feeding on resistant and susceptible St. Augustinegrasses ..............83

4-4 Mean duration of major waveforms per probe (WDP) on each of four southern
chinch bug-resistant or -susceptible St. Augustinegrasses. ............................................. 83

4-5 Mean number of major waveform events per insect (NWEI) on each of four southern
chinch bug-resistant or -susceptible St. Augustinegrass varieties. ................................83









LIST OF FIGURES


Figure page

3-1 Total protein content in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of
southern chinch bug feeding. ..... ........................... .......................................... 60

3-2 Levels of peroxidase (POX) specific activity levels in St. Augustinegrass lines 1, 3,
5, and 8 d after initiation of southern chinch bug feeding. ..............................................61

3-3 Levels of polyphenoloxidase (PPO) specific activity in St. Augustinegrass lines 1, 3,
5, and 8 d after initation of southern chinch bug feeding. ............................................62

3-4 Levels oflipoxygenase (LOX) specific activity in St. Augustinegrass lines 1, 3, 5,
and 8 d after initiation of southern chinch bug feeding. ..................................................63

3-5 Levels of catalase (CAT) specific activity in St. Augustinegrass lines 1, 3, 5, and 8 d
after initiation of southern chinch bug feeding. ....................................... ............... 64

3-6 Native gels stained for POX activity in St. Augustinegrass lines 1, 3, 5, and 8 d after
initiation of southern chinch bug feeding. .............................................. ............... 65

3-7 Native gels stained for PPO activity in St. Augustinegrass lines 1, 3, 5, and 8 d after
challenge by southern chinch bug ......................................................................... ...66

3-8 Native gels stained for LOX activity in St. Augustinegrass lines 1, 3, 5, and 8 d after
challenge by southern chinch bug ......................................................................... ...67

4-1 EPG waveforms produced by southern chinch bug feeding on St. Augustinegrass,
recorded using an AC/DC EPG monitor............................ .. ....... .....................84

4-2 Total percentage of time spent on total wave form duration (TWD) by southern
chinch bugs on resistant and susceptible St. Augustinegrasses in different waveform
categories ................................................................................. 85

4-3 Waveform duration per insect (WDI) for major waveforms on resistant and
susceptible St. A ugustinegrasses. ........................................................... .....................86

5-1 St. Augustinegrass stolon A. with the axillary shoot (as); B. Parts of axillary shoot,......98

5-2 Transverse section of outermost St. Augustinegrass leaf sheath showing southern
chinch bug salivary sheath stained with McBride's stain (400X). ...................................99

5-3 Number of southern chinch bug salivary sheaths found on the outermost leaf sheath
of axillary shoots ............................................................... ... .... ........ 100









5-4 Number of southern chinch bug branched salivary sheaths on the outermost leaf
sheath of axillary shoots from southern chinch bug-resistant and -susceptible St.
A ugustinegrasses ...................................... ..................... ...... ................ 101

5-5 Total lignin content of axillary shoot in southern chinch bug-resistant and -
susceptible St. A ugustinegrasses. ............................................ ............................ 102

5-6 Transverse sections of outermost leaf sheath vascular bundles stained for lignin in
southern chinch bug-resistant and -susceptible St. Augustinegrasses (400X)...............03

5-7 Transverse sections of the epidermal layer of the outermost leaf sheath in southern
chinch bug-resistant and -susceptible St. Augustinegrasses (400X) .............................104

5-8 Thickness of epidermal layer of outermost leaf sheath in southern chinch bug-
resistant and -susceptible St. Augustinegrasses .............................................................105

5-9 Transmission electron micrographs (7000X) of the sclerenchyma cells of the
outermost leaf sheath of St. Augustinegrasses resistant and susceptible to southern
ch in ch b u g ...................................... ................................................... 10 6

5-10 Percentage of cell wall area relative to total cell area of sclerenchyma of the
outermost leaf sheath of southern chinch bug-resistant and -susceptible St.
A ugustinegrasses........................................................................... ...... ....... 107









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MECHANISMS OF RESISTANCE TO SOUTHERN CHINCH BUG, Blissus insularis
BARBER (HEMIPTERA: BLISSIDAE) IN ST. AUGUSTINEGRASS

By

Murugesan Rangasamy

May 2008

Chair: Ronald H. Cherry
Cochair: Heather J. McAuslane
Major: Entomology and Nematology

St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, is the most commonly

grown lawn grass in Florida. The southern chinch bug (SCB), Blissus insularis Barber

(Hemiptera: Blissidae), is the most serious insect pest of St. Augustinegrass. Host plant

resistance has long been one of most successful management tools to control the SCB.

Resistance to SCB has been identified in the St. Augustinegrass lines 'Floratam', 'FX-10' and

'NUF-76'. Floratam was once resistant but is now susceptible. This research investigated the

mechanisms of SCB resistance in FX-10 and NUF-76. Choice and no-choice tests and

ovipositional and developmental studies indetified strong antixenosis in FX-10 and NUF-76 and

antibiosis in NUF- 76. FX-10 and NUF-76 had significantly higher peroxidase activity and

NUF-76 had significantly higher polyphenol oxidase activity 5 and 8 d after infestation

compared to uninfested controls. FX-10 had significantly higher lipoxygenase activity 3, 5 and 8

d after infestation compared to uninfested controls. Native gels stained for peroxidase indicated

that some isozymes in FX-10 and NUF-76 were induced 5 and 8 d after infestation. Electrical

penetration graph studies showed that the SCB made more frequent probes, spent longer time on

pathway related activities on FX-10 and NUF-76, compared to the susceptible Floratam and









Palmetto. Significantly higher number of salivary sheaths was observed in the outermost leaf

sheath in FX-10 and NUF-76. The total lignin content of the axillary shoot and the thickness of

the epidermal layer in SCB resistant and susceptible St. Augustinegrasses did not differ

significantly. TEM studies on the leaf sheath showed scelerenchyma cells with thicker cell walls

in NUF-76 and FX-10 than in Floratam and Palmetto suggesting that the sclerenchyma cells

around the vascular bundle in FX-10 and NUF-76 could serve as an impediment to the SCB

stylet penetration. From this research, I conclude that the strong antixenosis found in FX-10 and

NUF-76 and antibiosis in NUF-76 could be due to a combination of induction of oxidative

enzymes, thicker sclerenchyma cells around the vascular bundle and possible presence of feeding

deterrents or toxins in phloem sap or sieve tube blocking mechanisms.









CHAPTER 1
LITERATURE REVIEW

Chinch bugs are in the family Blissidae and are specialized to feed on plant sap. In the

United States, there are four economically important species and subspecies, i.e. Blissus

insularis Barber, B. leucopterous leucopterous (Say), B. leucopterous hirtus Montandon and B.

occiduus Barber feeding on grasses and related crop plants such as corn, sorghum and wheat. In

Florida, the southern chinch bug, B. insularis, is a serious insect pest of St. Augustinegrass,

Stenotaphrum secundatum (Walt.) Kuntze. The taxonomy of the pest chinch bugs has been

difficult and will be reviewed before concentrating on B. insularis, the subject of this

dissertation.

Taxonomy of the Chinch Bugs

Blissus leucopterous was first described in the United States by Say in 1831 from a single

specimen collected from the coast of Virginia. Although chinch bugs were collected from New

York and Massachusetts, no economic damage was noticed (Fitch 1856). The first report of

damage in the northeastern United States surfaced when Linter (1883) noticed chinch bug

damage in a meadow of timothy grass at Hammond, New York. The name B. leucopterous was

commonly applied to all chinch bugs collected in the United States until the early 1890s when

Montandon (1893) described Blissus hirtus based on a single specimen collected from Hazelton,

Pennsylvania. Blissus hirtus was certainly the form referred to as B. leucopterous by Fitch and

Linter.

Although Montandon (1893) gave species status to hirtus, Barber (1918) did not consider

hirtus as a separate species. Based on geographical location, body form and wing

polymorphism, Barber (1918) classified Blissus leucopterous into three varieties, i.e. 1. Blissus

leucopterous var. hirtus Mont., which he stated was common to the highlands of United States









and Canada, characterized by the presence of macropterous and brachypterous forms. 2. Blissus

leucopterous var. arenarius Barber, found in Sandy Hook, New Jersey on a species of sand

grass, characterized by being narrower and longer with only macropterous forms and, 3. Blissus

leucopterous var. insularis Barber commonly found in Punta Gorda, Florida, characterized by a

shorter and narrower body form than the typical B. leucopterous. The three varieties mentioned

by Barber were subsequently called subspecies, races or synonymies by many authors, which led

to confusion among researchers and with Barber himself.

Based on biological, morphological and cytological evidence Leonard (1966) considered

B. leucopterous, insularis and arenarius as distinct species but retained hirtus at the subspecies

level. He described arenarius and maritimus as subspecies of B. arenarius, and hirtus and

leucopterous as subspecies of B. leucopterous.

Barber (1918) also described B. occiduus from specimens collected from Ft. Collins,

Colorado, and Germino, New Mexico. The reported distribution of B. occiduus includes

California, Colorado, Montana, and New Mexico in the United States and Alberta, British

Colombia, Manitoba and Saskatchewan in Canada (Bird and Mitchner 1950, Slater 1964).

Blissus occiduus is a relatively smaller chinch bug having conspicuous wing polymorphism and

is an important insect pest of buffalograss, Buchloe dactyloides (Nuttall) Engelmann (Baxendale

et al. 1999).

Blissus insularis Barber is referred to here by its Entomological Society of America

approved common name, southern chinch bug (Bosik 1997). The ESA approved common names

of the other economically important species of chinch bugs include hairy chinch bug, chinch bug

and western chinch bug for B. leucopterous hirtus Montandon, B. leucopterous leucopterous

(Say), and Blissus occiduus Barber, respectively (Bosik 1997).









Host Plants and Feeding Preferences

Newell and Berger (1922) recorded the chinch bug for the first time in Florida on St.

Augustinegrass. They noticed the insect at the base of the grass surrounding brown areas of

lawns and determined it to be B. leucopterous, the same species that attacks cereals. Beyer

(1924) considered B. leucopterous to be a serious pest in Florida because it was feeding on St.

Augustinegrass, the grass grown generally as lawngrass over the entire state. However, Howard

(1888) and Webster (1907) did not consider St. Augustinegrass as a host among the grasses

damaged by the chinch bug.

Wilson (1929) reported that chinch bug caused extensive damage to St. Augustinegrass in

the southeastern United States. He observed that no other cultivated plants were infested where

B. leucopterous seriously damaged St. Augustinegrass. Hamilton (1935) reported the chinch bug

B. leucopterous as a pest of lawns and golf courses in the eastern United States.

Waldon (1936) found B. leucopterous hirtus damaging lawns in Connecticut. Maxwell

and MacLeod (1936) also reported that the same species attacked 19 different varieties of

lawngrass. Kuitert and Nutter (1952) described for the first time the chinch bugs in St.

Augustinegrass as B. leucopterous insularis and Kerr (1966), in a short note, mentioned that

insularis and leucopterous differed morphologically. Kerr (1966) also believed that the chinch

bug damaging lawns in Florida could be elevated to species status because B. insularis had

distinct morphological features and it was not found on corn and other cereals. Kelsheimer and

Kerr (1957) found B. leucopterous insularis severely damaging torpedo grass (Panicum repens

L.), occurring occasionally on centipede grass [Eremochloa ophiuroides (Munto)], and rarely on

Bermuda grass [Cynodon dactylon (L.) Pers.]. Blissus leucopterous insularis was considered to

be the second most expensive plant- feeding arthropod in Florida (Kerr 1966). In Florida, St.









Augustinegrass was the only grass to suffer severe damage by chinch bugs although the bugs

feed also on zoysia grass (Zoysia spp.), and bahia grass (Paspalum notatum Fluegg). However

most of the damage on these other grass species occurred when they were grown near St.

Augustinegrass with large bug populations (Kerr 1966).

Cherry and Nagata (1997) studied the ovipositional preference of southern chinch bug with

four cultivars of St. Augustinegrass ('Bitter Blue', Floratam, FX-10 and 'Seville') and four

other lawn grasses (P. notatum, C. dactylon, E. ophiuroides and Z. japonica). They concluded

that southern chinch bug resided in and oviposited on St. Augustinegrass cultivars more than on

other lawngrasses. Three cultivars of St. Augustinegrass, Bitter Blue, Seville and Floratam,

favored high adult survival rates and the lowest survival rates were recorded on P. notatumm, C.

dactylon, and E. ophiuroides.

Feeding Habit and Nature of Damage

Beyer (1924) found that chinch bugs pushed themselves into the laterals of the close-fitting

leaves of St. Augustinegrass and feeding caused a reddish stain on the damaged plant parts.

Affected plants had reduced growth and became dwarf. Watson (1925) found that infested

lawngrass turned brown in patches and then died out completely. He also reported that the plants

around the brown patch area exhibited yellowing symptoms and were infested with chinch bugs

sucking plant sap. Wilson (1929) designated the injury as diebackk" because of the progressive

nature of the damage. The gregarious feeding habit of the bugs caused the plant to wither. The

bugs abandoned the withered plants and moved into neighboring healthy plants. Lawns on dry

sandy soils were found more vulnerable to insect attack probably because of reduced vigor of the

plants and rapid multiplication of bugs. Kerr (1966) found the bugs aggregated in scattered

patches rather than distributed evenly. Before moving into a new area, the bugs killed the plants









in the infested area completely. Short et al. (1997) reported that the injury first occurs in water

stressed areas or in areas of full sunlight and infested lawns display circular discolored patches.

Cherry (2001) reported that southern chinch bugs were extremely aggregated in yellow damaged

St. Augustinegrass within areas of light and moderate infestations whereas only a few southern

chinch bugs were present in the grass 5 m or more away from an infestation.

Oviposition and Development

Beyer (1924) reported that the female chinch bugs laid their eggs near the roots of the

grass. The eggs were pushed into protected places, often into crevices at grass nodes or almost

out of sight between leaf blades where they come together at their base. Adults continued to

oviposit for many consecutive weeks (Wilson 1929, Kerr 1966).

Fecundity was estimated by Beyer (1924) to vary from 105 to 250 eggs or to average 250

eggs per female by Kerr (1966). Eggs are small and oval-shaped with a blunt end from which

four small projections extend (Stacy 2001). Eggs are first pale white in color but turn amber and

eventually red before hatching. The egg period varies widely with estimates of 9 to 13 days

(Wilson 1929), 7 to 11 days (Kuitert and Nutter 1952), 2 weeks (Eden and Self 1960), 11 to 33

days (Komblas 1962), and 112 weeks in summer but a month or more in winter (Kerr 1966). The

first nymphal instar has yellow body color which changes to red with a faint band across the

abdomen. The later instars are black with a narrow white band (Stacy 2001).

Nymphs take an average of 28 to 30 days (Wilson 1929, Kuitert and Nutter 1952, Eden

and Self 1960) to reach maturity. Wilson (1929) reported a pre-oviposition period of 5 to 13

days with the average of 8 days while Eden and Self (1960) reported 7 to 10 days.

Males survive for an average of 42 days and the females for 70 days. The generation time

has been estimated variously as 45 to 56 (Wilson 1929, Kuitert and Nutter 1952, Eden and Self









1960). Beyer (1924) described six nymphal stages and two generations with an overlapping

third generation during the summer in Florida while Kerr (1966) reported only five nymphal

instars. Wilson (1929) found mixed populations of adults and all stages of nymphs in spring

because of the extended period of oviposition. The adults mated in spring after a hibernation

period and started laying eggs (Eden and Self 1960).

Seasonal Wing Polymorphism and Movement

Polymorphism in chinch bug wings was reported by Van Duzee (1886). He noticed that

the shortwinged brachypterouss) form ordinarily predominated except under hot and dry summer

conditions, when the longwinged (macropterous) form predominated. This led him to believe

that the chinch was a tropical longwinged species. Webster (1907) reported that short- and

longwinged forms were found intermixed along seacoasts and northern sections of the United

States. He insisted that this condition must have been brought about by disuse of wings for a

period of time but not by temperature of the habitat. Wilson (1929) reported that both

macropterous and brachypterous southern chinch bugs occur in Florida and the relative number

of these two forms varied with season. Komblas (1962) concluded that population density could

be a reason for wing polymorphism because nymphs grown under crowded conditions were

more likely to develop into macropterous adults than were uncrowded nymphs. Numbers of

macropterous adults of southern chinch bug in south Florida were higher in summer and fall,

when population densities were also higher than they were in winter and spring seasons (Cherry

2001). However these data did not explain the exact cause for macroptery. Cherry (2001)

believed that various factors like heritability, population density and host plant condition might

determine wing polymorphism based on the reports that macroptery in the oriental chinch bug,

Cavelerius saccharivorus Okajima was density dependant (Denno and Peterson 2000) and was









strongly influenced by seasonal factors like day length and temperature (Fujisaki 2000). Leonard

(1966) found migration to be an influencing factor in wing polymorphism. Reinert and Kerr

(1973) noted that although both macropterous and brachypterous forms are found in the southern

chinch bug, the latter was predominant. Cherry and Wilson (2003) reported that the

macropterous adults have reduced fecundity and egg laying rates compared to brachypterous

females. Kerr (1966) reported that the major difference between B. leucopterous in northern

states and B. insularis in Florida was the absence of large spring flights of B. insularis. Although

some of the longwinged forms took flight, nothing equaled the mass flight of B. leucopterous

from overwintering quarters. Kerr (1966) noted that B. insularis moved from one lawn to

another mainly by walking and streams of bugs could be seen moving out from heavily infested

areas. The bugs were able to cover several hundred feet in a half-hour period of time.

Management of Southern Chinch Bugs

Southern chinch bugs can easily be managed if populations are detected early in a season's

population growth. Infestation can be detected easily by parting the grass near yellowed areas

and examining the base of the turf and the soil surface for chinch bugs. Heavily infested areas

would have large number of bugs migrating across sidewalks and driveways. Flotation and

vacuum techniques can also be used to identify areas of infestation (Congdon and Buss 2002).

There are preventive and curative methods available to control southern chinch bug. Preventive

methods such as cultural methods will avoid occurrence of insect problems and curative methods

will control the insects once their infestation is identified.









Cultural Methods

Cultural control is manipulation of crop production and management techniques to reduce

or avoid pest damage to plants. Cultural control manipulations are based on habitat management

and require a thorough understanding of the ecosystem in which the pest thrives.

Many insect pests of lawns live in thatch. Thatch is the layer of dead plant material found

between the green tops of the grass plant and soil. Proper mowing helps to prevent the thatch

from building up. St. Augustinegrass lawns should be mowed to a height of 3 to 4 inches

(Congdon and Buss 2002). The lawns should be mowed weekly during the growing season and

not more than 35 to 45 percent of the leaf blade should be removed at the time of mowing.

When the thatch is thicker than 1 cm, vertical mowing of the lawn could help. Good aeration

combined with top-dressing could increase microbial decay of dead grasses thus reducing the

thickness of thatch (Congdon and Buss 2002).

Southern chinch bugs prefer dry hot environments thus prolonged drought or water stress

can encourage problems. Water stress and dry weather increase the chances of survival for

nymphs and adult chinch bugs by reducing the occurrence of insect diseases. On the other hand,

excessive watering reduces microbial activity thus increasing the thickness of the thatch.

Regular inspection for dry or water stressed areas during summer is necessary (Congdon and

Buss 2002). Irrigating the lawn to a depth of 6 inches at a time is advised because frequent

watering induces shallow rooting and in turn higher chinch bug damage (Merchant and Crocker

1998).

Biological Control Agents

Reinert (1978b) reported on natural enemies of the southern chinch bug in Florida. His list

included a scelionid egg parasitoid, Eumicrosoma benefica Gahan, the only parasitoid found,









lygaeid predators (big-eyed bugs) Geocoris uliginosus (Say) and G. bullatus Say, anabid, Pagasa

pallipes Stal., anthocorids Xylocoris vicarius (Reuter) and Lasiochilus pallidulus Reuter, a

dermapteran, Labidura riparia Pallas, and a reduvid, Sinea spp. He has also found ant

predators, primarily Solenopsis geminata (F.), a spider, Lycosa sp., and a fungus, Beauveria

bassiana (Balsomo) Vuillemin. Reinert (1978b) concluded that the main biological agents

capable of keeping the chinch bug population under control were E. benefica, G. uliginosus, X

vicarius, and L. riparia. Big-eyed bugs and anthocorids are similar to chinch bugs in both size

and shape. Eumicrosoma benefica is capable of parasitizing up to 50 percent of the chinch bug

population (Hellman and Mathias 2002). The effectiveness of the predators mainly depends on

the number and dosage of insecticidal sprays. Although various species of ants were reported to

be predators of chinch bugs (Wilson 1929, Kerr 1966, Reinert 1978b), the red imported fire ant

(Solenopsis invicta Buren) was most often associated with southern chinch bug infestations in

Florida (Cherry 2001). Beneficial nematodes were unable to give consistent results when tested

against chinch bugs (Merchant and Crocker 1998).

Chemical Control Methods

Although fertility and water management, thatch control and use of resistant varieties give

considerable reduction in incidence of chinch bugs, heavy damage accompanied by high insect

populations necessitates the use of chemical control. It is essential to locate the problematic

areas. If 20 to 25 chinch bugs per square foot area are detected, insecticide application is

recommended (Congdon and Buss 2002). Spot treatment is enough in case of minimal

infestation but treating the whole area is necessary if the infestation is heavy (Congdon and Buss

2002). It is also important to visit the treated areas to see whether the bugs are controlled or not.

Repeated sprays may be required because the eggs are not killed by most of the available









insecticides (Nagata and Cherry 2003). Pesticides available for professional use include

acephate, chlorpyriphos, diazinon, carbaryl, bifenthrin, cyfluthrin, cypermethrin, deltamethrin,

lambda-cyhalothrin, permethrin and imidacloprid.

Repeated use of insecticides increases possible development of insecticide resistance

(Reinert and Portier 1983). The first report of chinch bug resistance to chlordane came in 1953

when Wolfenbarger (1953) in Miami found that chlordane failed to control the population of

chinch bugs. Afterwards reports continued to surface on the resistance of chinch bugs against

DDT in Sarasota (Kerr and Robinson 1958), and in Miami (Kerr 1958), and against parathion in

Fort Lauderdale (Kerr 1960) as well as other organophosphates like chlorpyriphos and diazinon

(Reinert and Niemczyk 1976). Reinert (1982) reviewed the resistance to other chlorinated

hydrocarbons and organophosphates. In 1983, Reinert and Portier investigated the distribution

and level of southern chinch bug resistance against organophosphates and carbamates. They

found that the level of resistance against chlorpyriphos was 3.4-fold to 3.19 x 108-fold along a 70

km coastal area of Broward and Palm Beach counties in Florida. A 9.25-fold level of resistance

to propoxur, a carbamate insecticide, was also demonstrated.

Cherry and Nagata (2005) reported for the first time that the southern chinch bug had

developed resistance to bifenthrin, a commonly used synthetic pyrethroid to control chinch bugs

in Florida. In recent years, increased use of insecticides further aggravated the problem. Cherry

and Nagata (2007) collected southern chinch bugs from 10 different urban areas in Central and

South Florida and tested their resistance to different classes of insecticides. They found varying

level of southern chinch bug resistance to deltamethrin, imidacloprid, and lambda-cyhalothrin

and, for the first time also confirmed the resistance to bifenthrin. Cherry and Nagata (2007) also

noted that the level of insecticide resistance varies depending on the location. Most populations









in Florida are still susceptible to these insecticides, however, the situation is alarming and further

underlines the importance of improving natural host plant resistance.

St. Augustinegrass and Host Plant Resistance against Southern Chinch Bugs

St. Augustinegrass is grown extensively in southern coastal states and California (Crocker

et al. 1982). The world's first record of planting of St. Augustinegrass was on 11th November

1880, as turf bordering an avenue at A.M Reed's Mulberry Grove plantation, at Yukon, Orange

Park, Florida (Works Progress Administration 1939).

After the Second World War, the market for St. Augustinegrass grass increased

dramatically and during the late 1960s varieties like 'Floratine', 'Florida Common' and

'Roselawn' were commercially available for lawns. McGregor (1976) estimated that the area

under turfgrass in Florida was more than 3, 68,700 ha, of which St. Augustinegrass accounted for

more than 46 percent of the home lawns. Carter and Duble (1976) estimated that 56 percent of

the lawns in Texas were in St. Augustinegrass but 96 percent in the Gulf coastal areas. The area

in St. Augustinegrass sod production was 3,100 hectares in 1974 (Florida Department of

Agriculture and Consumer Services 1976) and 13,400 hectares in 1991 (Hodges et al. 1994). St.

Augustinegrass is estimated to cover more than half a million hectares in Florida (Hodges et al.

1994) and accounts for 85% of the sod industry in Florida with a value of $262 million annually

(Haydu 2005).

The leaves of St. Augustinegrass have round-tipped leaf blades of 5 to 14 mm width that

are distichously arranged (Busey 2003). St. Augustinegrass is the most versatile of the warm

season grasses and is able to survive well in acid or alkaline soils, soils with low organic matter,

and soils with poor or good drainage. It is salt tolerant and has few disease and nematode









problems requiring little maintenance. It is also able to perform well in full sunlight and

moderate shade (Crocker 1993).

St. Augustinegrass expanded rapidly into warm coastal regions as a lawngrass mainly

because of human migration. However a new mosaic virus called St. Augustine Mosaic Disease

(SAD), which was first reported in 1966, in Texas, challenged the popularity of St.

Augustinegrass. The disease was caused by the St. Augustine decline strain of Panicum Mosaic

Virus (SAD-PMV) (Lee and Toler 1973, Niblett et al. 1977) and the virus spread throughout the

St. Augustinegrass growing regions of Texas (Toler and Walla 1976) and Louisiana (Holcomb et

al. 1972). Horn et al. (1973) estimated a $100 million loss due to the disease and in the same

year, almost 90 percent of the St. Augustinegrass in Corpus Christi, Texas was affected.

St. Augustinegrass researchers from Florida and Texas realized that the virus could not be

controlled therapeutically because of the ability of the virus to spread through vegetative

propagation and mechanically through lawn mowers. Although the virus was not found in

Florida, imminent spread from the neighboring states was anticipated. Accessions of St.

Augustinegrass were screened for virus resistance under greenhouse conditions and the Floratam

variety was found resistant to the virus in 1968 (Horn et al. 1973). Floratam displayed a high

level of resistance under greenhouse conditions and for 24 consecutive months under field

conditions as well.

Reinert and Kerr (1973) reported that southern chinch bugs were able to damage seriously

or kill whole lawns and were considered to be the most serious insect pest of St. Augustinegrass.

Kerr (1966) rated the southern chinch bug as one of the most economically important arthropod

in Florida second only to citrus mite, in terms of the cost of control measure (Strobel 1971).

Because of the destructive nature of this pest, experiments were started to find resistant varieties









of St. Augustinegrass. In 1972, Reinert reported that nine accessions of St. Augustinegrass had

seasonal mean populations of 1.6 or fewer bugs per 0.1 m2. In 1974, Reinert and Dudeck

identified three St. Augustinegrass accessions resistant to southern chinch bug. They screened

11 promising accessions along with the available commercial cultivars using confined tests and

reported that accessions FA-108, Floratam and FA-118 were highly resistant. The resistance was

characterized as antibiosis because adults and nymphs suffered 50 percent mortality and

surviving adults had reduced fecundity. One of these three accessions, Floratam was released

jointly by the University of Florida and Texas A&M University (Horn et al. 1973). The

resistance to SAD virus and chinch bugs, superior color, ability to cover the ground fast and

perform well under the conditions of Florida and Texas made Floratam more preferred than all

other commercial varieties.

Because of the coarse-textured leaves of Floratam, the researchers continued their search

for fine-textured St. Augustinegrass lines (Reinert 1978a) and also for lines having combined

resistance to SAD and southern chinch bug (Bruton et al. 1979, Reinert et al. 1980, Crocker et al.

1982). Despite a decade of research, the only line advanced and released was Floralawn (FA-

108) which is fast-growing with fine-textured leaves and combined resistance to SAD and

southern chinch bug (Dudeck et al. 1986).

Breakdown of Floratam Resistance

After its release in 1973, Floratam quickly expanded in commercial sod production and

accounted for 77 percent of St. Augustinegrass sod production in Florida (Busey 1986). Many

investigations since the germplasm release confirmed its resistance to the southern chinch bug

(Reinert et al. 1980, Crocker et al. 1982, Reinert et al. 1986) and because of its vegetative

reproduction and poor seed production, genetic breakdown of the resistance was considered to be

unlikely to occur (Busey 1979). Nevertheless, by 1987, Busey and Center discovered a









widespread population of chinch bugs that had overcome the resistance of Floratam and

Floralawn St. Augustinegrasses. Because of the association of polyploidy and host resistance in

St. Augustinegrass and the ability of the chinch bugs to overcome the host plant resistance, the

population that overcame Floratam resistance was dubbed the polyploid damaging population

(PDP). Busey and Center (1987) tested a local population of standard southern chinch bug and

the PDP population with these resistant varieties and found that the PDP adults had higher

longevity and oviposition rate than the standard adults when confined on Floratam and Floralawn

varieties. They concluded that the phenomenon observed was due to variability in the insect and

not the grass. Busey and Coy (1988) conducted a survey among sod producers and county

agents in Florida and found that Floratam-killing southern chinch bugs were widespread. The

county agents considered the southern chinch bug as the most serious insect pest of St.

Augustinegrass while the sod producers thought that caterpillars were more serious than chinch

bugs. Busey and Coy (1988) concluded that chinch bug continued to be the most limiting factor

in St. Augustinegrass-established areas, because the southern chinch bug was damaging in most

counties. Nagata and Cherry (2003) collected southern chinch bug populations from nine cities

or their suburbs in Florida and found all populations capable of overcoming the resistance in

Floratam.

Breeding Resistant Varieties against Chinch Bugs, Continuing Missions

Crocker et al. (1989) realized the necessity of continuing the effort to develop more

resistant varieties of St. Augustinegrass in Texas after seeing the reports of the ability of the PDP

of southern chinch bug to attack Floratam in Florida (Busey and Center 1987). Crocker et al.

(1989) tested varieties and accessions for resistance against Texas strains of southern chinch bug

and SAD-PMV. The results showed that Floratam still was strongly antibiotic to southern









chinch bug. Thus it was concluded that the breakdown of Floratam antibiosis in Florida was

absent or not widespread in Texas. They concluded that the populations of southern chinch bugs

from Texas and Florida might not respond uniformly to future varieties.

The susceptibility of Floratam was a major concern to the turfgrass industry in Florida at

the time it was reported (Busey and Center 1987) but there was no information on how the PDP

adapted to Floratam antibiosis. Busey (1990a) found that host adaptability in PDP was heritable

and retained in subsequent generations irrespective of the previous host. The populations from

various regions differing in ability to attack hosts were one potentially interbreeding population

and capable of exchanging a particular genetic trait. The other important discovery was that

oviposition rates of PDP on Floratam were much greater than previously reported (Busey and

Center 1987) suggesting an improvement in host adaptation when the colony was maintained on

the resistant host. Vegetative propagation (genetically homogenous in subsequent generations)

in St. Augustinegrass offered a steady selection pressure for southern chinch bug adaptation.

Also the long distance transport of sod increased the gene exchange between the PDP and

standard populations thus host adaptability was expected to occur (Busey 1990a).

Busey (1990b) continued his quest for resistant varieties against the PDP of southern

chinch bug and reported that PDP chinch bugs grown on FX-2, FX-10, and FX-33 exhibited

shorter adult longevity and fewer cumulative lifetime eggs than insects grown on Floratam. Both

laboratory and field PDP exhibited high mortality on FX-33. In addition to confined tests, Busey

(1990b) also used the production of excrement as an indicator of feeding activity. As a result of

this research, a southern chinch bug resistant and coarse-textured breeding line, FX-10, the only

recognizable St. Augustinegrass cultivar with pubescence was registered (Busey 1993). This

variety performed well under seasonal drought, was notably resistant to PDP and standard









populations of southern chinch bug, and had moderate resistance to Pyricularia leaf spot.

Because of the course texture and other negative characteristics such as slow ground coverage,

susceptibility to weeds, FX-10 was never extensively grown in Florida (Nagata and Cherry

2003). The other problem with FX-10 was difficulty in transferring its resistance to southern

chinch bug into other desirable lines because of non-viable seeds. All commercially available St.

Augustinegrass cultivars with resistance to southern chinch bugs like Floratam, Floralawn and

FX-10 are polyploids and have coarse appearance. Although diploid (2n=18) lines from St.

Augustinegrass produce viable seeds and have fine-textured leaves they have never been

accepted widely because of their poor resistance against chinch bugs (Nagata and Cherry 2003).

Nagata and Cherry (2003) made a breakthrough in their quest for the southern chinch bug

resistance when they reported that NUF-76, a diploid St. Augustinegrass selection, and NUF-216

both had resistance against southern chinch bug. Of the fourteen lines inspected for resistance,

the mortality of the bugs was significantly higher on NUF-216 and NUF-76 lines than on any

other line. Although the mortality on NUF-216 and NUF-76 was not significantly different from

FX-10, the mortality on these three lines was higher than that on any other line. Interestingly,

high variation in mortality between different locations was observed on some St. Augustinegrass

lines and this variation was attributed to the irregular response of the chinch bugs within Florida

to both host plant resistance as reported by Busey and Center (1987) and insecticide resistance

reported by Reinert and Portier (1983). After finding a diploid St. Augustinegrass resistant to

southern chinch bug, Nagata and Cherry (2003) expect that the transfer of the desirable

characters with chinch bug resistance into other lines will be easier in the near future.









Refining the Screening Techniques

Chinch bug resistance in varieties like Floratam and Floralawn was termed antibiosis

because these cultivars produced higher mortality in confined tests. However, the exact

mechanism of the resistance could not be explained by the confined tests. Busey and Center

(1987) complained that the mortality data on the susceptible 'Florida Common' were

inconsistent when the experiments were conducted for different periods of time on the same host.

The crowding of chinch bugs released on sprigs of experimental grass and the variation among

the field and laboratory populations of the chinch were the reasonable causes discussed. Busey

(1990b) used visual rating of the excreta as a criterion for screening the germplasm. However,

realizing that mere visual examination of excreta could not be an accurate selection criterion,

Busey and Zaenker (1992) postulated they could weigh the excreta. Because the excreta adhered

to surfaces like sidewalls of the glass culture tubes, antistatic film and aluminum foil (Busey

1990b), it was determined that the collection and weighing of the excreta was possible. The

antistatic film was rolled inside and toward the top of each culture tube and the tubes were

inclined. They observed that more than 99 percent of the excreta was deposited on the film and

not on the glass or the grass. The films were removed and dried at 240C for several days before

weighing. They reported that the excreta weight was highly correlated with oviposition rate and

crowding of up to 20 bugs per sprig did not influence excreta production.

Other Species of Chinch Bugs and Host Plant Resistance

Host plant resistance to control chinch bugs has been explored in other crops such as

sorghum and in other turfgrasses such as buffalograss. Buffalograss is a perennial warm season

grass species native to the American Great Plains (Wenger 1943, Beard 1973). It is a fine

textured low growing turfgrass, which spreads vegetatively by stolons (Nuland et al. 1981,









Falkenberg-Borland and Butler 1982, Riordan et al. 1996). In recent years, buffalograss earned

popularity as an alternative turfgrass because it is drought tolerant and has few pest and disease

problems (Heng-Moss et al. 2002). One of the reported insect pests of buffalograss is B.

occiduus whose hosts include barley, Hordeum sp., corn, Zea mays L., oats, Avena sativa L.,

sugarcane, Saccharum officinarum L., wheat, Triticum aestivum L., bromegrass, Bromus spp.,

and various native grasses including buffalograss (Ferris 1920, Parker 1920, Farstad et al. 1951).

Currently very few control measures are available for chinch bugs and other insect pests in

buffalograss (Baxendale et al. 1999). Extensive differences exist among buffalograss selections

in their resistance to mealy-bugs, Tridiscus sporboli and Trionymus sp., suggesting the

possibility of also breeding chinch bug resistance in buffalograss (Johnson-Cicalese et al. 1998).

Based on greenhouse and field evaluations of eleven lines, NE91-118, 'Tatanka', 'Bonnie Brae'

and 'Cody' had high to moderate resistance to B. occiduus whereas NE84-45-3 and 378 were

highly susceptible (Heng-Moss et al. 2002).

Characterization of Resistance against Chinch Bugs in Buffalograss

The basis of resistance in the four chinch bug resistant buffalograsses was categorized

(Heng-Moss et al. 2002). Among the cultivars investigated, they found NE91-118, 'Cody' and

'Tatanka' exhibiting moderate-to-high levels of tolerance and 'Bonnie Brae' exhibiting

moderate-to-low levels of tolerance to B. occiduus based on insect damaging ratings and

functional plant loss indices. They suggested studying parameters such as height of regrowth,

dry weight and yield clippings, root length, and tillerings that all represent plant aesthetics, an

important aspect in assessing the tolerance of turfgrass to chinch bugs. The antibiosis tests

showed no differences among the resistant and susceptible cultivars of buffalograss thus ruling

out the possibility of any influence on the growth and development of B. occiduus by the









resistant cultivars. This is inconsistent with earlier discoveries reporting that resistance against

southern chinch bug was antibiosis (Reinert and Dudeck 1974, Reinert et al. 1980). However,

cultivars like NE91-118 and 609 were not preferred by chinch bugs in choice tests suggesting the

presence of antixenosis. These results supported the earlier discovery of antixenosis resistance in

NE-609 to the mealybugs T. sporoboli and Trionymus sp. SEM studies showed differences in

trichomes densities among resistant and susceptible lines. Leaf blades of the resistant lines like

NE91-118, NE-120, 378 and 609 had few trichomes and were characterized as glabrous

(Johnson-Cicalese et al. 1998).

Chinch Bug Resistance in Sorghum

Chinch bugs that damage sorghum and small grains were recorded as B. leucopterous

leucopterous (Webster 1909). The concept of host plant resistance as an important tool for

managing chinch bugs in sorghum plants became evident when Dahms (1948) reported the

"milos" sorghum were very susceptible to chinch bugs, the "feteritas" were susceptible and the

"kafirs" and sorgoss" were resistant. During the early 1980s resistance to chinch bugs in

sorghum lines and hybrids under field conditions was reported. However, Meehan and Wilde

(1989) realized the importance of screening the sorghum lines under greenhouse or growth

chamber conditions during the periods of low, natural field infestations. Under greenhouse

conditions the commercial sorghum hybrids and lines were subjected to nymphs and adults at the

seedling stage. Plant mortality for hybrids or lines was inconsistent between choice and no-

choice tests. However, the days from infestation to death of plants in studies done in growth

chambers were in agreement with the results from earlier field tests.

Investigating the Mechanisms of Southern Chinch Bug Resistance in St. Augustinegrasses

Thus far, studies on the mechanisms of resistance in St. Augustinegrass to southern chinch

bug have been confined to those done on Floratam. Reinert and Dudeck (1974) and Reinert









(1978) reported that Floratam and 10 other St. Augustinegrass accessions exhibited antibiosis to

southern chinch bug based on adult and nymphal mortalities. Reinert et al. (1980) and Crocker

et al. (1989) used the term 'antibiosis' to describe the resistance to southern chinch bug in St.

Augustinegrass lines. Unfortunately, none of the above-mentioned reports explained the exact

cause of the nymphal and adult mortality. Busey and Zaenker (1992) quantified the feeding

response of southern chinch bug on St. Augustinegrass as weight of excreta produced and

frequency of confined bugs on the grass as opposed to off the grass. They found significant

differences among hosts in excreta weight within 24 h after releasing the insects and a strong

correlation between excreta weight and oviposition rate. Although they proposed a new

resistance bioassay technique they did not determine the exact resistance mechanism operating in

St. Augustinegrass against southern chinch bug.

The goal of this study is to determine more precisely the mechanisms of southern chinch

bug in both polyploid ('FX-10') and diploid (NUF-76) St. Augustinegrass lines. This goal will

be achieved by the following objectives:

* Objective 1. To determine the modality of resistance to southern chinch bug in St.
Augustinegrass.

* Objective 2. To compare oxidative responses of southern chinch bug resistant and
susceptible St.Augustinegrass lines to B. insularis infestation.

* Objective 3. To study precise feeding behavior of southern chinch bugs on susceptible and
resistant St. Augustinegrass lines using electrical penetration graph technique (EPG)

* Objective 4. To determine the role of lignification and leaf sheath anatomy in southern
chinch bug resistance in St. Augustinegrass.









CHAPTER 2
CATEGORIES OF RESISTANCE IN ST. AUGUSTINEGRASS LINES TO SOUTHERN
CHINCH BUG (HEMIPTERA: BLISSIDAE)

Introduction

St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze is the most widely used

lawn grass in tropical and sub-tropical climatic regions (Sauer 1972). It is the primary turfgrass

in Florida and is grown on approximately 70% of all lawns (Busey 2003). The southern chinch

bug, Blissus insularis Barber is the most serious insect pest of St. Augustinegrass (Crocker

1993). Southern chinch bugs suck the sap from the grass until it withers and dies, turning from a

healthy green color to brown-yellow. Infestation typically occurs first in water stressed areas

and along edges of the lawn or areas in which grass is growing in full sunlight (Short et al.

1995). The annual cost of southern chinch bug management and losses in Florida alone was

estimated at $5 million in 1983 (Hamer 1985) and is undoubtedly much more today. The

economic importance of this insect has increased as it has developed resistance against several

insecticides (Reinert and Portier 1983, Cherry and Nagata 2005).

'Floratam', a polyploid variety of St. Augustinegrass with resistance to southern chinch

bug, has been available in Florida and Gulf Coast areas since its release in 1973 (Horn et al.

1973). Reinert and Dudeck (1974) classified the resistance to southern chinch bug found in

'Floratam' as antibiosis because insects on 'Floratam' exhibited more than 50 percent adult and

nymphal mortalities and surviving insects had reduced fecundity compared to insects reared on

susceptible varieties of St. Augustinegrass. However, Busey and Center (1987) reported

instances of southern chinch bugs overcoming 'Floratam' resistance in Florida, beginning in

1985. They named this resistance-breaking population of the southern chinch bug, the

polyploid-damaging population. Nagata and Cherry (2003) collected southern chinch bug

populations from nine cities or their suburbs in Florida and determined the distribution of the









polyploid-damaging population to be widespread. This problem seems to be confined to Florida

however because, in 1989, Crocker et al. (1989) reported that there was no evidence of the

polyploid-damaging population of southern chinch bug in Texas.

The lessened resistance in 'Floratam' prompted plant breeders to continue their search for

resistance to the southern chinch bug. 'FX-10', a polyploid St. Augustinegrass, was registered in

1993 (Busey 1993) with resistance against the southern chinch bug, but because of its very

coarse texture, toughness and poor ground coverage, it was never extensively grown (Nagata and

Cherry 2003). Nagata and Cherry (2003) reported no significant difference in southern chinch

bug mortality among 'FX-10', NUF-216 and NUF-76. The latter two lines are products of the

University of Florida's turf-breeding program. Mortality on all three lines was significantly

higher than that on 'Floratam' and 'Floratine'. This research was significant because Nagata and

Cherry (2003) identified, for the first time, a diploid St. Augustinegrass line, NUF-76, with

resistance to the southern chinch bug; all previous resistant lines were polyploids. Diploid lines

produce viable seeds allowing recombination of characters and selection of new lines. Diploid

lines also have narrow leaf blades, which gives them a much finer and more aesthetically

pleasing texture. As of yet, it is not known by which mechanism resistance to southern chinch

bug operates in these resistant lines.

Understanding the mechanisms of resistance in St. Augustinegrass to southern chinch bug

is crucial. This knowledge may allow turf breeders to design appropriate screening assays,

which would expedite their development of insect resistant varieties. The objective of this

research was to characterize the mechanisms of southern chinch bug resistance in the polyploid

('FX-10') and diploid (NUF-76) St. Augustinegrass lines. To meet this objective we conducted

choice and no-choice tests to examine adult chinch bug preference for and survival on resistant









and susceptible St. Augustinegrass lines. We also quantified reproduction of adult chinch bugs

and studied development and survival of immature chinch bugs on susceptible and resistance

lines.

Materials and Methods

Plants and Insects

St. Augustinegrass lines selected for the experiments included NUF-76 (resistant diploid),

'FX-10' (resistant polyploid), 'Floratam' (once resistant polyploid), 'Bitter Blue' (susceptible

polyploid) and 'Palmetto' (susceptible diploid). Plant material was supplied by R. Nagata

(Everglades Research and Education Center, Belle Glade, FL). Rooted sprigs from single node

cuttings were grown in plastic pots (12.5 cm diameter x 12.5 cm depth) containing a potting

medium of 1:1 (v/v) sand and Fafard mix #2 (Conrad Fafard, Agawam, MA). Test plants were

6 to 8 weeks old. Plants were fertilized with Osmocote Plus 15-9-12 (Scotts-Sierra Horticultural

Products Company, Marysville, OH) at the rate of 5g/kg of growing medium. Stolons

approximately 30 to 40 cm in length with a minimum of three nodes were used for testing.

Nagata and Cherry (2003) reported that most populations of southern chinch bugs in

Florida had overcome 'Floratam' resistance. Thus for all experiments, southern chinch bugs

were collected from Royal Palm Beach, Palm Beach Co., FL. Preliminary trials were conducted

to determine their ability to develop on 'Floratam'. The insects were collected by vacuuming St.

Augustinegrass lawns as described by Cherry (2001) and then sorting through debris in the

laboratory for fifth instars and adults (depending on the needs of the experiment). After

collection, insects were provided with fresh 'Floratam' stolons as needed and maintained at 180C

in 20-liter plastic buckets.









Choice Tests

Choice tests were conducted to study the behavior of southern chinch bugs on resistant and

susceptible lines of St. Augustinegrass. Arenas were constructed from circular polypropylene

food storage containers measuring 33.6 (top diameter) x 33.0 (bottom diameter) x 10.6 cm

(height) (Rubbermaid 3907, Wooster, OH) that were modified in the following manner. Five

holes of 6-mm diameter, equally spaced around the perimeter of the arena and 3 cm below the

open edge were drilled. A channel was then cut from the open edge of the container to the hole

drilled in the side. The majority of the lid surface area was removed, leaving approximately 3

cm around the sealing edge. Anti-virus insect screen (hole size of 0.0105 x 0.0322 inches,

Green-Tek, Edgeton, WI) covered the opening in the lid to facilitate aeration.

A moist paper towel was placed on the bottom of the arena to prevent desiccation of

insects. Plant stolons, still attached to potted plants, were passed through the channels to rest in

the holes on the side of the arena. The portion of the stolons in contact with the plastic arena was

wrapped with parafilm strips (American National Can, Greenwich, CT) so that the plant would

not be damaged. After positioning the stolons, the channels were sealed with clear cellophane

tape to prevent southern chinch bugs from escaping. Plants were watered as needed. NUF-76,

'FX-10', 'Floratam', 'Bitter Blue' and 'Palmetto' were evaluated in this choice test.

Twenty chinch bug adults (10 males and 10 females) of undetermined age were released at

the center of the arena and the lid was replaced. The location of the insects was recorded as 'on'

one of the five lines or 'off all plants at 24 h, 48 h, 72 h and 120 h after release. The

experimental design was a completely randomized block design. The experiment was conducted

in a laboratory room that was maintained at 30 +10C and 14:10 L:D h photoperiod and was

replicated 15 times.









No-Choice Tests

No-choice tests were conducted to investigate the possibility of antibiosis in resistant St.

Augustinegrass lines. One stolon of each line (NUF-76, 'FX-10', 'Floratam', 'Bitter Blue' and

'Palmetto'), still attached to the plant, was introduced into separate rectangular arenas. The

arena was made of a food storage container (Rectangle 1.8 qt, Rubbermaid #7A23, Wooster,

OH) with dimensions of 27 cm x 15 cm x 5.7 cm. One hole was drilled on the 15-cm side and a

channel was cut as described in the choice-test section so that a stolon could be passed unharmed

to the interior of the box. The majority of the lid surface area was removed leaving

approximately 3 cm around the sealing edge. Anti-virus insect screen was glued over the

opening to facilitate aeration. The insects were starved for 12 h before the start of the

experiment

A moist paper towel was placed on the bottom of the arena to prevent desiccation of

insects. A sheet of heavy-duty aluminum foil (Reynolds Kitchens, Richmond, VA) was cut to

the size of the arena and placed on the moist paper towel. Twenty chinch bug adults (10 males

and 10 females) were released inside the arena. The exact locations of feeding of the adults such

as leaf sheaths, intemodes, or nodal regions, were observed 24 h, 48 h, 72 h and 120 h after

release. The aluminum foil sheet was replaced after each observation and the number of

excretory spots was recorded with the naked eye. Use of aluminum foil sheets to collect the

southern chinch bug excreta was originally reported by Busey and Zaenker (1992). The

experimental design was a randomized complete block design with 15 replicates. The

experiment was conducted in a laboratory room that was maintained at 30 +10C and 14:10 L:D h

photoperiod. Plants were watered as needed.









Ovipositional Studies

Studies were conducted to investigate the effect of St. Augustinegrass lines on fecundity of

southern chinch bugs. Rectangular arenas used in no-choice tests were used for this experiment

and St. Augustinegrass lines used included 'FX-10', NUF-76 and 'Floratam'. In order to obtain

adults of similar age, fifth instar nymphs were selected from the field collection and then

provided with fresh 'Floratam' runners. Insects that molted to the adult stage within a 5-d period

were selected for the experiment. Five pairs of adults were released into the arena. A week after

release, surviving chinch bugs were transferred gently using a mouth aspirator from one arena to

a new arena with an undamaged stolon from the same individual plant. Adult mortality was

recorded and the stolons were dissected to count the number of eggs. This was continued for a

total of 4 weeks. This experiment was conducted at the Envirotron, IFAS, University of Florida

that was maintained at 30 1IC. Plants were watered as needed. The experimental design was a

randomized complete block design with 13 replications.

Developmental Studies

This experiment was conducted to study the influence of St. Augustinegrass lines on

growth and development of southern chinch bug nymphs. The experimental set up was as

explained in the no-choice test except the arena size was 28 x 16 x 11 cm (1 x w x h)

(Rubbermaid #3901, Wooster, OH). Five pairs of adults (0-5 d old) were released into the arena.

After 32 d boxes were opened and stolons were dissected to count the number of nymphs and

adult chinch bugs surviving. This experiment was conducted at the Envirotron, IFAS, University

of Florida that was maintained at 30 +fC. Plants were watered as needed. The experimental

design was completely randomized block design with 11 replications.









Statistical Analysis

The distribution of chinch bugs in choice tests among the five lines taken over a period of

5 d was analyzed as a repeated measures design using mixed model analyses (PROC MIXED,

SAS Institute 1999) after data were transformed by arcsine \x. In no-choice tests, the numbers

of excretory spots counted over a period of 5 d were analyzed as a repeated measures design

using mixed model analyses (PROC MIXED). In ovipositional studies, the number of eggs laid

per female was transformed by I (x + 0.375) to correct for nonhomogenity of variances and data

were analyzed using a repeated measures analysis with a mixed model (PROC MIXED). For the

developmental studies, the number of nymphs produced and adults surviving after 32 d was

analyzed using PROC GLM (SAS Institute 1999). When significant F-tests were obtained,

means were compared using Tukey's test with a significance level of P < 0.05.

Results

Choice Tests.

More chinch bugs were observed on the three susceptible lines than the two resistant lines

at three out of four observation times (Table 1). Southern chinch bug adults showed significant

preference for susceptible lines at 24 h, 72 h and 120 h after release (F = 10.43; df = 4, 70; P <

0.0001). Forty-eight hours after release, the proportion of the chinch bugs on plants found on

'FX-10' was not significantly lower than the proportion counted on susceptible plants. NUF-76

had a significantly lower proportion of chinch bugs on it than on all susceptible lines except

'Bitter Blue'. The preferences of chinch bugs for different lines did not differ among time

periods (F = 0.43; df= 3, 207; P = 0.7300). Between 69 and 72 % of the released adults were

found either feeding or resting on a stolon; the remaining adults were either off the plant or dead.

The proportions of chinch bugs not found on the plants were 0.30, 0.27. 0.28, and 0.31 at 24 h,









48 h, 72 h, and 120 h after release, respectively. A significant interaction was detected between

St. Augustinegrass lines and time after release (F = 2.1; df = 12, 207; P = 0.0157). This

significant interaction indicates that, in general most lines had a similar proportion infestation

over time. However 'Palmetto' was relatively more infested early in the experiment whereas

'Floratam' was relatively more infested later in the experiment. Mortality of adults in the arenas

was not analyzed statistically but ranged between 5 and 10% (one or two insects out of 20).

Adult chinch bugs were observed feeding on all plants, including the resistant 'FX-10' and NUF-

76, and fecal spots were noticed underneath all plants.

No-Choice Tests

On most of the observation periods, 40 to 45 % of the adult chinch bugs were found inside

the leaf sheaths, a few were observed on nodal and intemodal regions, and the remaining 55 to

60% of the insects were off the plant. The number of excretory spots counted differed

significantly among St. Augustinegrass line (F = 27.16; df = 4, 56; P < 0.0001) (Table 2). The

number of excretory spots found under 'FX-10' was consistently significantly less than under the

susceptible lines at all the time periods. At 48 h and 72 h, the mean number of excretory spots

produced on NUF-76 was approximately half the number found on the susceptible lines,

however the differences were not statistically significant. At 120 h the mean number of excretory

spots produced on NUF-76 was significantly lower than found on the susceptible lines. There

were significant differences among time periods (F= 6.19; df = 3, 135; P < 0.0006). In general

production of excretory spots was greatest at 24 h, most likely because insects were starved for

12 h before the start of the experiment, and declined afterwards. There was no significant

interaction of time with St. Augustinegrass line (F = 1.23; df = 12, 135; P = 0.27).









Ovipostional Studies

Southern chinch bug adults confined on 'Floratam' produced 11 and five times more eggs

than those on 'FX-10' and NUF-76, respectively (F = 57.6; df = 2, 36; P < 0.0001) (Table 3).

There were significant effects due to time and the interaction between St. Augustinegrass lines

and time (time: F = 9.79; df = 3, 108; interactions: F = 5.24; df = 6, 108; P < 0.0001). This

interaction was due to the higher mean number of eggs laid during the second and third weeks of

the experiment compared to the first and fourth weeks on 'Floratam' and 'FX-10' but higher

oviposition on NUF-76 on the first and third weeks than the second and fourth weeks. During

the third and fourth weeks of the experiment, fewer females were alive on 'FX-10' and NUF-76

than on "Floratam' (F = 32.77; df= 2, 108; P < 0.0001) (Table 3). However, during the first

week, numbers of female alive on different lines did not differ significantly whereas during the

second week fewer females were alive on 'FX-10' than on NUF-76 and 'Floratam'. The eggs

were often found inside the folded emerging leaf or partially opened leaf sheaths and

occasionally on adventious roots.

Developmental Studies

The five pairs of adults released on 'FX-10', NUF-76 and 'Floratam' produced an average

of 9.5 3.6 (mean SEM), 20.3 6.7, and 180.5 20.1 nymphs, respectively, after 32 d.

Nymphal production on 'FX-10' and NUF-76 was significantly less than that on 'Floratam' (F=

284.7; df= 2, 53; P < 0.0001). The number of adults surviving on 'FX-10' (0.8 0.3) and NUF-

76 (1.9 0.6) was significantly less than that on 'Floratam' (8.0 0.4) (F = 71.90; df = 2, 20; P

< 0.0001). Most of the nymphs were first and second instars; a very few third instar nymphs

were found on 'Floratam'. Thus we assume that the adults observed after 32d were the

originally released adults rather than offspring.









Discussion

Choice tests, no-choice tests, ovipositional and developmental studies were helpful to

determine the presence or absence of antixenosis and antibiosis in NUF-76 and 'FX-10', chinch

bug-resistant St. Augustinegrass lines. In choice tests, southern chinch bugs significantly

preferred the susceptible lines to both 'FX-10' and NUF-76, suggesting the presence of

antixenosis in the resistant lines. The finding of significantly fewer excretory spots under 'FX-

10' in no-choice tests confirms the presence of antixenosis. However, the mean number of

excretory spots found under NUF-76 was not consistently different from the susceptible lines.

At 24 h, NUF-76 was not significantly different from any of the susceptible lines however at 48

h and 72 h, the mean number of spots found under NUF-76 was significantly different from

'Palmetto', a susceptible diploid, and at 120 h the mean number of spots were significantly

different from all the susceptible lines. Our data indicate a trend that feeding of southern chinch

bug on NUF-76 is reduced over time. While reduced feeding (antixenosis) on 'FX-10' and

NUF-76 could impact chinch bug survival it is unlikely to have been the sole reason for the low

survivorship of chinch bugs on these lines documented previously (Nagata and Cherry 2004).

Adults released on 'FX-10' and NUF-76 laid many fewer eggs than on 'Floratam'. The

reduction in fecundity could be due to reduced feeding on 'FX-10'and NUF-76 shown in the no-

choice tests but was so extreme that it is more likely that reduced fecundity is due to a nutritional

inadequacy for egg development or some toxin that influenced reproduction (i.e., antibiosis).

Studies on the mechanisms of resistance in St. Augustinegrass to southern chinch bug have

been confined to those done on 'Floratam'. Reinert and Dudeck (1974) and Reinert (1978)

reported that 'Floratam' and 10 other St. Augustinegrass accessions exhibited antibiosis to

southern chinch bug. Based on adult and nymphal mortalities, the mechanism of resistance in









'Floratam' was categorized as antibiosis. Reinert et al. (1980) and Crocker et al. (1989) used the

term 'antibiosis' to describe the resistance to southern chinch bug in St. Augustinegrass lines.

Unfortunately, none of the above-mentioned reports explained the exact cause of the nymphal

and adult mortality.

Resistance to other pest chinch bugs has been studied. Heng-Moss et al. (2003)

investigated the mechanisms of resistance to western chinch bug (Blissus occiduus Barber) in

buffalograss (Buchloe dactyloides (Nuttall) Engelmann). They found an array of modalities of

resistance among germplasm tested (antixenosis, antibiosis and tolerance) but reported no

morphological or biochemical differences to account for these different susceptibilities.

Our studies confirm that both 'FX-10' and NUF-76 have a high level of southern chinch

bug resistance. However, because of the course texture and other negative characteristics such as

slow ground coverage, proneness to weeds, 'FX-10' was never extensively grown in Florida

(Nagata and Cherry 2003). Another problem with 'FX-10' was difficulty in transferring its

resistance to southern chinch bug into other desirable lines because of non-viable seeds. Being a

diploid and therefore much easier to cross, NUF-76 is a very important source of chinch bug

resistance for turfgrass breeding programs. NUF-76 allows very little population growth and

should need many fewer applications of insecticides. Studies are underway to understand how

'FX-10' and NUF-76 are morphologically or biochemically different from 'Floratam' and other

chinch bug-susceptible lines and how the polyploid damaging population of southern chinch bug

overcame resistance in 'Floratam'.









Table 2-1. Distribution (mean proportion SEM) of adult southern chinch bugs among five St.
Augustinegrass lines in a choice situation at varying time intervals after release *
Proportion ofB. insularis found on St. Augustinegrass lines*
Lines Time after chinch bug release
24 h 48 h 72 h 120 h
'FX-10' 0.15 + 0.03b 0.18 + 0.03ab 0.14 + 0.02b 0.10 + 0.02b
NUF-76 0.09 0.03b 0.10 0.03b 0.08 0.02b 0.11 + 0.04b
'Floratam' 0.23 0.03a 0.25 0.03a 0.29 0.03a 0.29 0.05a
'Bitter Blue' 0.21 0.04a 0.21 0.04ab 0.23 0.04a 0.23 0.04a
'Palmetto' 0.33 0.02a 0.27 0.02a 0.25 0.02a 0.27 0.02a
* Proportions calculated as no. of chinch bugs on one St. Augustinegrass line divided by the
number of chinch bugs counted on all lines. Those chinch bugs off the plants at a particular
observation time were not included in the totals. Means within the same column with a letter in
common are not significantly different (a = 0.05) by Tukey's test.









Table 2-2. Mean number ( SEM) of excretory spots deposited per living chinch bug on St.
Augustinegrass lines in a no-choice situation at varying time intervals after release
Number of excretory spots per living insect*
Lines Time after chinch bug release
24 h 48 h 72 h 120 h
'FX-10' 1.7 + 0.3b 1.5 + 0.3c 1.3 + 0.3c 1.7 + 0.4b
NUF-76 3.6 + 0.5a 2.0 0.2bc 2.6 0.3bc 2.6 + 0.5b
'Floratam' 5.2 + 0.5a 3.9 + 0.4a 3.5 + 0.3ab 5.1 + 0.5a
'Bitter Blue' 3.9 + 0.5a 3.6 + 0.3ab 4.0 0.5ab 4.7 + 0.5a
'Palmetto' 4.0 0.4a 3.9 + 0.3a 4.4 + 0.3a 5.4 + 0.6a
* Number of excretory spots produced during a certain time period was calculated as the number
of spots divided by number of adults alive that time period. Means in a column with the same
letter in common are not significantly different (a = 0.05) by Tukey's test.










Table 2-3. Mean number ( SEM) of eggs produced per female alive on St. Augustinegrass lines
at weekly intervals after release
Weeks after chinch bug release
Lines Number of eggs per live female
1 2 3 4
'FX-10' 1.4 + 0.6c 2.2 + l.b 2.5 1.7b 0.7 + 0.5b
NUF-76 6.5 + 1.4b 2.5 0.8b 5.4 + 3.4b 0.8 + 0.6b
'Floratam' 13.9 + 1.6a 25.3 + 2.4a 28.3 6.3a 9.2 + 2.6a
Number of live females
'FX-10' 4.15 + 0.25a 1.30 + 0.5b 0.69 0.4b 0.46 0.3b
NUF-76 4.07 + 0.32a 3.15 + 0.4a 1.46 + 0.5b 0.30 0.2b
'Floratam' 4.61 + 0.1a 4.15 + 0.2a 4.0 0.2a 3.58 + 0.4a
* Means in a column with the same letter in common are not significantly different (a = 0.05) by
Tukey's test.









CHAPTER 3
OXIDATIVE RESPONSES OF ST. AUGUSTINEGRASSES CHALLENGED BY
SOUTHERN CHINCH BUG, BLISSUS INSULARIS BARBER (HEMIPTERA: BLISSIDAE)

Introduction

St. Augustinegrass, Stenotaphrum secundatum (Walt.), is the most widely used turfgrass in

tropical and subtropical climatic regions (Sauer 1972). It is a commonly grown residential

turfgrass species in Florida and is estimated to cover more than one million acres. St.

Augustinegrass accounts for 85% of total sod production in Florida and is worth more than $262

million annually. The southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae), is

the most serious insect pest of St. Augustinegrass (Crocker 1993). The annual cost of southern

chinch bug management and losses in Florida alone was estimated at $5 million in 1983 (Hamer

1985) and is undoubtedly much more today. The economic importance of this insect is further

increased by its development of resistance to several key insecticides (Reinert and Portier 1983;

Cherry and Nagata 2005, Cherry and Nagata 2007).

Historically, host plant resistance has been one of the most successful pest management

tools for this insect. Floratam, a polyploid variety of St. Augustinegrass with resistance to

southern chinch bug, has been available in Florida and Gulf Coast areas since its release in 1973

(Horn et al. 1973). However, in 1985 a population of chinch bug was reported to have overcome

Floratam's resistance in Florida (Busey and Center 1987). Nagata and Cherry (2003) collected

southern chinch bug populations from nine widely-dispersed cities or their suburbs in Florida

and found all populations capable of overcoming the resistance in Floratam.

As potential alternatives to Floratam, two southern chinch bug resistant St. Augustine lines

were developed: a polyploid line, 'FX-10' (Busey 1993) and a diploid line, NUF-76 (Nagata and

Cherry 2003). However, the mechanisms of resistance in these lines are unknown. Deciphering

these mechanisms will greatly facilitate our efforts to breed cultivars with durable chinch bug









resistance in St. Augustinegrass. Although the mechanisms of plant resistance against phloem-

feeding aphids have been studied extensively (Thompson and Goggin 2006), aphids feed on their

host plants by probing intracellularly, while southern chinch bugs probe intercellularly, likely

resulting in more and different kinds of cellular damage and plant response.

In our recent investigations, we showed that both 'FX-10' and NUF-76 deterred feeding of

southern chinch bugs (antixenosis) (Rangasamy et al. 2006). NUF-76 also had adverse effects

on the biology of the insect (antibiosis) (Rangasamy et al. 2006), suggesting a metabolic basis

for insect resistance. One of the most prominent plant responses to insect herbivore attack is the

induction of oxidative enzymes such as polyphenol oxidase (PPO), peroxidase (POX),

lipoxygenase (LOX) and catalase (CAT) (Green and Ryan 1972; Hildebrand et al. 1986; Felton

1989; Felton et al. 1994a b; Stout et al. 1999; Constabel et al. 2000; Chaman et al. 2001; Ni et al.

2001; Heng-Moss et al. 2004). These enzymes, because of their potential roles in the synthesis

of defense compounds and/or in oxidative stress tolerance, have often been implicated in plant

resistance to insect herbivores.

The objective of this investigation was to compare the enzymatic responses of resistant and

susceptible lines of diploid and polyploid St. Augustinegrass to feeding by a population of

southern chinch bugs that has overcome the resistance of Floratam. Our study focused on the

induction of oxidative enzymes POX, PPO, CAT and LOX in four cultivars of St.

Augustinegrass following the introduction of southern chinch bugs.

Materials and Methods

Plants, Insects, Sample Collection

St. Augustinegrass lines selected for comparison included NUF-76 (resistant diploid), FX-

10 (resistant polyploid), Floratam (susceptible polyploid), and 'Palmetto' (susceptible diploid).

Both polyploid and diploid lines were included to determine whether the ploidy level has any









role in resistance to southern chinch bugs. Rooted sprigs from single node cuttings were grown

in plastic pots (12.5 cm diam. x 12.5 cm depth) containing a 1:1 (v/v) mixture of sand and Fafard

mix #2 (Conrad Fafard, Agawam, MA). Plants were fertilized with Osmocote Plus 15-9-12

(Scotts-Sierra Horticultural Products Company, Marysville, OH) at the rate of 5 g kg-1 of

growing medium. Stolons used in experiments were approximately 30 to 40 cm in length, with a

minimum of three nodes, located on plants that were 6 to 8 weeks old.

Southern chinch bugs were collected from Royal Palm Beach (Palm Beach County, FL)

for all experiments. The insects were collected by vacuuming southern chinch bug-infested St.

Augustinegrass lawns as described by Cherry (2001). In the laboratory, fifth instars and adults

were separated from the debris and insects were provided with fresh Floratam runners and

maintained at 180C in 20-1 plastic buckets. Twenty adult chinch bugs (1:1 sex ratio) were

confined in organdy sleeve cages on the youngest three nodes of one runner of a St.

Augustinegrass plant. The chinch bugs were removed from the runner before collecting the

sample for protein analysis. A 2-g amount of sample including leaf sheaths and blades, excised

from each runner, was collected 1, 3, 5, and 8 d after infestation and frozen immediately in liquid

nitrogen. The samples were stored at -800C until analyses. Five replicate plants were infested

for each line at each time period along with an equal number of control plants whose runners

were confined in cages without chinch bugs.

Sample Preparation

Frozen St. Augustinegrass samples were ground into powder using a mortar chilled with

liquid nitrogen. Soluble proteins were extracted using 5 ml 20 mM HEPES (4-(2-

hydroxymethyl)-l-piperazineethanesulfonic acid) buffer (pH 7.2) containing protease inhibitors

[0.3 ml/g plant tissue, a mixture of 1 piM bestatin, 0.2 mM 4-(2-aminoethyl) benzenesulfonyl

fluoride, 1 pM pepstatin A, 1 pM E-64, 10 [M leupeptin and 1 mM 1, 10-phenanthroline









(Sigma, St. Louis, MO) and 1% (wt/v) polyvinylpyrrolidone]. The homogenate was filtered

through a 4X muslin cloth and centrifuged at 10,000 x g for 10 min at 40C. The supernatant was

collected, desalted using a PD-10 desalting column (Sephadex G-25M, Amersham Biosciences

Inc., Piscataway, NJ), and stored at -800C until protein and enzyme analyses.

Estimation of Total Protein

Total protein content and enzyme activities were determined using a Beckman Model DU

640 Ultraviolet-Visible spectrophotometer. The total protein content was determined by

following the modified method of Lowry et al. (1951) using the Folin-Ciocalteau phenol reagent

(Pierce Chemical, Rockford, IL) and bovine serum albumin as a standard.

Enzyme Activities

The soluble POX specific activity was measured with guaiacol (Sigma, St. Louis, MO) as a

substrate. Increase in absorbance at 470 nm was monitored for 2 min using a protocol modified

from Hildebrand et al. (1986) and Hori et al. (1997). The reaction was started by adding 10Cl of

30% (v/v) H202 to a quartz cuvette containing 100 pl 250 mM HEPES-KOH, pH 7.0, 5 pl

protein extract, 300 pl 18 mM guaiacol, and 585 il distilled water. The POX specific activity

(liM mg protein- min-) was calculated using the molar absorptivity of guaiacol at 470 nm (F =

26.6 M-1 cm-1).

Catalase-specific activity was determined following a modified protocol from Hildebrand

et al. (1986). The reaction was started by adding 200 pl protein extract to a quartz cuvette

containing 100 pl 250 mM HEPES-KOH, pH 8.0, 100 pl 75 mM H202 (Sigma, St. Louis, MO)

and 600 [l distilled water. The reaction was monitored at 240 nm for 2 min and the CAT

specific activity was determined using the molar absorptivity of H202 at 240 nm (s = 43.6 M-1

cm-1) and expressed as moles mg protein- min'.









PPO activity was measured using a protocol modified from Hori et al. (1997). The

reaction was started by adding 25 pl of protein extract to a cuvette containing 100 pl 250 mM

HEPES-KOH, pH 6.0, 500 pl 1.6% catechol (Sigma, St. Louis, MO) and 375 pl distilled water.

PPO activity was monitored at 470 nm for 2 min and calculated as the change in A470 mg protein

1 min-1

LOX activity was determined using a protocol modified from Skorzynska-Polit and Krupa

(2002). Linoleic acid (Fluka, St. Gallen, Switzerland) substrate was prepared by following a

protocol described in Koch et al. (1992). A 10 mM stock solution of sodium linoleate was

prepared by mixing 14 pl linoleic acid, 14 pl Tween-20 and 2 ml oxygen-free distilled water by

drawing back and forth with a Pasteur pipette which produced a milky emulsion. The emulsion

was cleared on addition of 0.11 ml 0.5 M NaOH. The volume was then made up to 5 ml with

oxygen-free distilled water and dispensed to 2-ml screw-capped vials which were flushed with

N2 and stored at -200C. The reaction was started by adding 10 pl 10 mM linoleic acid to a quartz

cuvette containing 50 pl protein extract, 100 pl 250 mM HEPES-KOH (pH 7.0) and 860 pl

distilled water. The change in absorbance at 234 nm was recorded for 5 min. The LOX activity

was expressed as the change in A234 mg protein- min-.

Isozyme Profile Studies

Isozyme expression of POX, PPO and LOX was studied using native gel electrophoresis

(Mini-Protean III, Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of protein (30

Gg/well) were loaded into 10-well 4.5 % (wt/v) and 7.5% (wt/v) polyacrylamide discontinuous

gels for POX and PPO and 5.0% (wt/v) and 12.5% (wt/v) gels for LOX. Tris-glycine (pH 8.3)

continuous buffer system was used to run the gel for 120 min at 30 mA at 40C. POX and PPO

isozymes were stained using a protocol modified from Vallejos (1983) and LOX isozymes were

stained according to a protocol by Heinisch et al. (1996).









Gels to be stained for POX activity were incubated in 50 mM sodium acetate buffer (pH

5.0) at 25 2C on a platform shaker set at 50 rpm. After 10 min of incubation, 20 il 30% H202

and 10 mg 4-chloronaphthol (dissolved in 0.5 ml methanol) were added to the buffer.

Peroxidase activity was visualized as dark blue bands after approximately 20 min. Gels to be

stained for PPO activity were soaked in 20 mM HEPES (pH 7.2) buffer containing 5 mM DL-3-

(3,4-dihydroxyphenylalanine) (DOPA, Sigma, St. Louis, MO) for 30 min at 25 2C on a

platform shaker set at 50 rpm. PPO activity was visualized as dark brown bands on a clear

background after 30 min.

Gels to be stained for LOX activity were briefly rinsed in distilled water and incubated

for 30 min in linoleic acid substrate solution at 40C on a platform shaker set at 50 rpm. The gel

was rinsed again in distilled water, and then 20 ml of the staining solution was added. It was

incubated on a shaking platform set at 50 rpm at 25 2C for 10 min. LOX activity was

observed as pink bands. The substrate solution was prepared by mixing 50 pl linoleic acid and

50 [l 95% ethanol using a magnetic stirrer in a 2-ml screw cap vial at 40C by exclusion of light.

The solution was transferred into a 50-ml flask and the volume was made up to 50 ml with 0.2 M

sodium borate buffer (pH 9.0). The staining solution was prepared by mixing 0.5g N, N-

dimethyl-p-phenylenediamine, 4.5 ml methanol and 0.5 ml acetic acid (1:1, acid : water) and the

volume was made up to 50 ml with distilled water.

Statistical Analysis

The study was set up using a completely randomized design with five replications. The

means of enzyme activities in polyploid lines (FX-10 and Floratam) and diploid lines (NUF-76

and Palmetto) were analyzed separately using PROC GLM (SAS 1999) and Tukey's test was

used for mean separations. In the two-way ANOVA model, time after treatment and grass lines

were considered fixed effects, while replicate plants were random.









Results


Total Protein Content

There were no significant differences in total extractable protein content between chinch

bug-infested and control plants for any of the St. Augustinegrass lines at any time periods after

infestation (P> 0.05, n=5) (Figure 3-1).

Peroxidase Activities

FX-10 and NUF-76 plants infested with southern chinch bug had nearly two-fold higher

POX-specific activities 5 and 8 d after infestation compared to their respective controls (F =

52.68, df = 3, 64, P < 0.0001, n = 5) (Figure 3-2). Two-way ANOVA found no significant

interaction between St. Augustinegrass lines and time (F = 1.8, df = 9, 64, P < 0.07). POX

activities in infested FX-10 plants on 5 and 8 d were significantly higher than levels in the

susceptible polyploid, Floratam, whether infested or uninfested. The southern chinch bug-

susceptible Floratam and Palmetto did not have any significant difference in POX specific

activity between infested and control plants at any of the time periods studied (F = 1.69, df= 3,

64, P = 0.08) (Figure 3-2).

Polyphenol Oxidase Activities

The southern chinch bug-infested NUF-76 plants had significantly higher PPO activity 5

and 8 d after infestation compared to their respective control plants (F = 21.39, df = 3, 64, P <

0.05, n = 5) (Figure 3-3). There were no significant differences in PPO specific activity between

the southern chinch bug-infested and control plants of FX-10, Floratam (F = 1.98, df = 3, 64, P =

0.1255) and Palmetto (F = 1.61, df = 3, 64, P = 0.10) at any of the time periods after infestation.

With the exception of NUF-76 plants 5 and 8 d after infestation, there were no significant

differences in PPO activities among infested and control NUF-76 and Palmetto plants.









Lipoxygenase Activities

FX-10 plants infested with southern chinch bug had significantly higher LOX specific

activity 3, 5 and 8 d after infestation compared to their respective control plants (F = 312.96; df =

3, 64; P < 0.0001, n = 5) (Figure 3-4). There was a significant interaction between St.

Augustinegrass line and time (F = 25.46, df = 9, 64; P < 0.0001). LOX specific activity in

southern chinch bug-infested FX-10 plants peaked 3 d after infestation when it was 2-fold higher

than 1 d after infestation, and then declined 5 and 8 d after infestation. The LOX activities in

southern chinch bug-infested and control Floratam plants were not significantly different.

However chinch bug-infested FX-10 plants had significantly higher LOX activity than

uninfested FX-10 controls, Floratam controls and FX-10 and Floratam infested plants on 1 and 3

d (Figure 3-4). Two-way ANOVA found no difference in LOX activities between FX-10

controls, Floratam control and infested plants at any of the time periods studied. There were no

significant differences in LOX specific activities between southern chinch bug-infested and

control NUF-76 and Palmetto plants (F =1.69, df = 3, 64, P = 0.18).

Catalase Activities

There were no significant differences observed in CAT specific activity between southern

chinch bug-infested and control plants at any time period after infestation in any of the St.

Augustinegrass lines studied (Figure 3-5).

Isozyme Profile Studies

Native gels stained for POX-specific activity showed five isozymes in FX-10; two of them

were induced 5 and 8 d after infestation compared to their respective controls (Figure 3-6).

There were three POX isozymes in NUF-76 with no apparent difference between infested and

control plants. There were three POX isozymes in Palmetto and five in Floratam with no

difference between infested and control plants at any of the times after infestation (Figure 3-6).









Native gels stained for PPO identified two isozymes in NUF-76, Palmetto and Floratam

and three in FX-10 with no apparent difference between southern chinch bug-infested and

controls plants at any of the time periods studied (Figure 3-7). There were two LOX isozymes

seen in NUF-76, four in Palmetto, three in FX-10, and one in Floratam (Figure 3-8). There were

no apparent differences in LOX isozyme intensity or number between southern chinch bug-

infested and controls plants.

Discussion

There was no significant difference in extractable total protein content of southern chinch

bug-infested and control St. Augustinegrass plants at any time after infestation, although

southern chinch bug-infested FX-10 plants showed an overall increase in protein content over

time. Our results agree with those of Heng-Moss et al. (2004) who found no increase in total

protein content after infestation by the western chinch bug, Blissus occiduus, of resistant

Buffalograss varieties 'Cody' and 'Tatanka'. However Ni et al. (2001) reported a significant

increase in total protein content in wheat varieties infested with the Russian wheat aphid,

Diuraphis noxia (Mordvilko), and the corn leaf aphid, Rhopalosiphum padi (L.), in contrast to an

earlier report of a two-fold decrease in total protein 2 d after infestation by D. noxia (Van der

Westhuizen and Pretorius, 1995).

We found both chinch bug-resistant lines, FX-10 and NUF-76 had a nearly two-fold

increase in POX specific activity levels 5 and 8 d after infestation compared to uninfested control

plants. Our results agreed with many previous reports that POX specific activity levels increase

after herbivore infestation (Hildebrand et al. 1986; Felton et al. 1994a, b; Van der Westhuizen et

al. 1998; Ni et al. 2001; Chaman et al. 2001; Heng-Moss et al. 2004; Khattab, 2007). Lupin

varieties resistant to green peach aphid, Myzuspersicae (Sulzer), had significantly different









levels of POX and PPO activity, protease inhibitors and soluble phenolics. However, the levels

of PPO and POX were not increased after the aphid infestation (Cardoza et al. 2005)

Herbivore damage is known to induce production of H202 in plants (Dowd and Lagrimini,

1997). The level of H202 in plants is regulated by oxidative enzymes such as POX and CAT,

and it has been demonstrated in vitro that POX activity is dependent on the supply of H202

(Duffey and Felton, 1991). An increase in POX activity in insect-resistant plants may detoxify

the peroxides, thus reducing plant tissue damage compared to susceptible plants (Hildebrand et

al. 1986). Peroxidases are involved in plant cell wall-building processes, by mediating the

oxidation of hydroxycinnamyl alcohols into free radical intermediates, cross-linking of

polysaccharides and extension monomers, lignification and suberization (Chittoor et al. 1999).

Brisson et al. (1994) suggested that the oxidative cross-linking of the plant cell wall could

enhance its ability to slow pathogen ingress. Such a reinforcement of the cell wall in response to

insect feeding may serve as a mechanical barrier and prevent the insect's stylets from reaching

their feeding sites, phloem sieve elements in the case of southern chinch bug. An enhancement

of the POX isozyme activity in maize (Zea mays L.) increased resistance to herbivores and

pathogens (Dowd and Lagrimini, 1997). Increased POX activities in FX-10 and NUF-76 in our

study suggest that these two lines may have increased cell wall lignification.

Plant catalases scavenge H202 that occurs in mitochondria and peroxisomes and reduce

them into H20 and 02. Zhu-Salzman (2004) found that the CAT genes were down-regulated in

resistant sorghum plants infested with greenbug and speculated that the reduced CAT activity

could help the plant to maintain high H202 levels thus causing damage to the insect midgut. Park

et al. (2006) also found that CAT genes were down regulated in greenbug resistant sorghum

lines. However, Heng-Moss et al. (2004) reported a loss in CAT activity in response to feeding









by B. occiduus on susceptible buffalograss varieties. Our study found no association between

CAT-specific activity levels and southern chinch bug resistance in St. Augustinegrasses, and

supports previous reports using other plant-insect combinations indicating no relationship

between CAT activity and resistance (Faccioloi, 1979; Matkovics et al. 1981; Hildebrand et al.

1986).

The association of PPO activity with host plant resistance to insects has been reported in

many plants including tomato, potato, coffee and poplar (Duffey and Felton, 1991; Constabel et

al. 1996; Wang and Constabel, 2004; Chaman et al. 2001; Thipyapong et al. 2006). In our

studies, only NUF-76 had significantly higher levels of PPO specific activity as a result of

southern chinch bug infestation, whereas FX-10 showed no such induction. Heng-Moss et al.

(2004) found no association between PPO activity in buffalograsses and their resistance to B.

occiduus.

Our studies showed that FX-10 had a two-fold higher LOX-specific activity 3 d after

southern chinch bug infestation, and a reduced but still significantly higher activity 5 and 8 d

after infestation compared to the control plants (Figure 3-4). Increased activity of LOX has been

reported in soybean in response to two-spotted spider mite infestation (Hildebrand et al. 1986)

and attack by larvae of Helicoverpa zea (Boddie) (Felton et al. 1994b). The increased levels of

LOX activity caused oxidative damage in midgut epithelial cells ofH. zea larvae and reduced

their growth (Felton et al. 1994b). Bi et al. (1997) showed significant increase in LOX in H. zea

damaged cotton squares along with altered phenolic contents. Gene expression studies showed

that LOX transcripts were strongly elicited in tomato plants in response to attack by the aphids,

Macrosiphium euphorbiae (Thomas) and M persicae (Fidantsef et al. 1999) and were locally

upregulated in Nicotiana attenuata after Macrosiphium nicotianae Blackman infestation









(Voelckel et al. 2004). LOX-deficient N. attenuata plants, when planted into native habitats,

became more vulnerable to adapted herbivores such as Manduca sexta (L.) and attracted new

opportunistic herbivores, such as Empoasca spp. (Kessler et al. 2004).

Lipoxygenases are ubiquitous enzymes that catalyze the hydroperoxidation of

polyunsaturated fatty acids having cis, cis-pentadiene moieties (Hildebrand et al. 1986). Linoleic

acid and linolenic acid are the major LOX substrates in plants (Hildebrand et al. 1986). Lipid

peroxidation by LOX results in formation of fatty acid hydroperoxides which may enzymatically

and/or chemically be degraded to highly reactive aldehydes, y-ketols, epoxides (Gardner, 1991),

hydroxyl radicals, singlet oxygen, superoxide ion and peroxyl, acyl and carbon-centered radicals

(Kanofsky and Axelrod, 1986). Gardner (1979) mentioned that the interactions between lipid

hydroperoxides and proteins results in protein-protein cross linking, and amino acid damage.

These chemical changes in proteins may affect the assimilation of amino acids of insects. These

lipid peroxidation end products could be repellent to insect feeding and operate via antixenosis or

be toxic and function via antibiosis (Felton et al. 1994b). Both modalities of resistance have

been reported previously in St. Augustinegrass against the southern chinch bug (Rangasamy et al

2006). Shukle and Murdock (1983) demonstrated that LOX could directly act as a defense in

soybean against insect attack. Lipoxygenases are also involved in inducible herbivore resistance;

the LOX oxidation of linolenic acid is the first step in jasmonic acid signaling pathway, which

has been implicated in coordination of direct plant defense such as production of oxidative

enzymes and protease inhibitors (Staswick and Lehman, 1999, Mao et al. 2007) and indirect

plant defense such as production of volatiles (Dicke et al. 1999; Thaler et al. 1999). Rayapuram

and Baldwin (2006) investigated the performance ofM. sexta larvae on N. attenuata plants

silenced in LOX3 expression and jasmonic acid signaling and found that LOX3-mediated









defense in wild plants reduced larval growth, consumption and frass production compared to the

mutant. Mao et al. (2007) transformed maize plants with the wheat oxalate oxidase gene and

reported that the up-regulated LOX transcripts and 14-fold higher free phenolics in transgenic

plants were positively associated with their resistance to the European corn borer (Ostrinia

nubilalis H.) compared to the control plants.

Based on the results from our research, we associate the increased levels of POX and LOX

in FX-10 and POX and PPO in NUF-76 with southern chinch bug resistance in these lines. The

increased POX and LOX activities could be a cause of antixenosis in FX-10 and NUF-76 and

antibiosis in NUF-76 found in our previous studies (Rangasamy et al. 2006, Chapter 2).

Increased POX activity seems to be due to the induction of specific isozymes. Together, our

results support the hypothesis that oxidative enzymes play a vital role in herbivore resistance in

plants. Our data suggest that identification of genes encoding inducible PPO, POX, and LOX

enzymes could be the first step in understanding southern chinch bug resistance in St.

Augustinegrass and will be valuable markers for breeding southern chinch bug resistant lines.

The presence of different kinds of southern chinch bug resistance in FX-10 and NUF-76 is in

agreement with our previous studies (Rangasamy et al. 2006, Chapter 2). Our research supports

the hypothesis that chinch bug resistance in St. Augustinegrass has a metabolic basis and

highlights the possible involvement oflignification and oxidative enzymes in secondary product

synthesis in chinch bug resistance in St. Augustinegrass.













--*-- Infested
Control



-i





--*-- Infested
Control


1 3 5


--*-- Infested
- Control


11 -

10

9

8

7


--*-- Infested
- Control


8 1 3 5
Time after infestation (d)


Figure 3-1. Total protein content in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of
southern chinch bug feeding. A. FX-10, SCB resistant polyploid. B. Floratam, SCB
susceptible polyploid. C. NUF-76, SCB resistant diploid. D. Palmetto, SCB
susceptible diploid. The values shown are mean + SE of five replicates.












1.6 1.6
1.6- Infested -- Infested
1.4 Control -- Control
1.2 1.2
S1.0 ----- 1.0
S0.8 0.8
0.6 0.6
E 0.4 0.4
0.2 0.2
S0.0 0.0
1.6 -- Infested 1.6
1.4 Control 1.4 -'- Infested
S1. 12 Control
1.2 1.2
1.0 ---1.0
S0.8 0.8
0.6 0.6 ---
0 0.4 0.4
0.2 0.2
0.0 0.0
1 3 5 8 1 3 5 8
Time after infestation (d)


Figure 3-2. Levels ofperoxidase (POX) specific activity levels in St. Augustinegrass lines 1, 3,
5, and 8 d after initiation of southern chinch bug feeding. The values shown are mean
+ SE of five replicates. Means marked with differ significantly between control and
infested treatments (P < 0.05, Tukey's test).

















--- Infe.ted Inested
S-Control Control
.4 4

.% :-- i^ 2 : 3 i



E 1 1
0

5 5
-, --- Infested -- Infested
S4 Control Control
----------4
o 3 3




a 1
Q)



0 0
1 3 5 8 1 3 5 8
Time after infestation (d)


Figure 3-3. Levels ofpolyphenoloxidase (PPO) specific activity in St. Augustinegrass lines 1, 3,
5, and 8 d after initation of southern chinch bug feeding. The values shown are mean
+ SE of five replicates. Means marked with differ significantly between control and
infested treatments (P < 0.05, Tukey's test).


- Infested


r r.~r~~r~l













16
----'- Infested
S- Control 12
S1 2



J.O
/ -------.. 1.0
/ 0,8

0.4
0.2
0 0
1.6
--'-- Infested 1 4
-t- Control
12
1.0
08
0.6


02
1.0


1 3 5


8 1
Time after infestation (d)


--*-- Infested
Control











--A-- Infested
Control


3 5


Figure 3-4. Levels of lipoxygenase (LOX) specific activity in St. Augustinegrass lines 1, 3, 5,
and 8 d after initiation of southern chinch bug feeding. The values shown are mean +
SE of five replicates. Means marked with differ significantly between control and
infested treatments (P < 0.05, Tukey's test).









/.U 7.0
---- Infested --- Infested
Control Control
6.5 6.5


-- --- -
F 6.0 6.0


5.5 5.5

0.0 0.0
c ------------ 0.0 -----------
<
7.0 7.0
6. -0-- Infested ---- Infested
S-0- Control -o- Control
6.5 6.5


6.0 6 __---- 6.0


5.5 5.5


0.0 0.0
1 3 5 8 1 3 5 8

Time after infestation (d)
Figure 3-5. Levels of catalase (CAT) specific activity in St. Augustinegrass lines 1, 3, 5, and 8 d
after initiation of southern chinch bug feeding. The values shown are mean SE of
five replicates. Means marked with differ significantly between control and infested
treatments (P < 0.05, Tukey's test).











Scl
T C


8d
T C


Id
T C


3d
T C


5d 8 ld
T C T C


4-
,4...


1-.


Floratam


4...-
4-...


NUF-76 Palmnetto


Figure 3-6. Native gels stained for POX activity in St. Augustinegrass lines 1, 3, 5, and 8 d after
initiation of southern chinch bug feeding. T and C refer to the infested (i.e., treated)
and control samples, respectively. Arrows indicate the number of POX isoforms.


I cl
T C


3 c
T C


FX-10









Id 3 d
T C T


5d
C T C


Sd Id
T C T C


3d
TC


5d 8d
T C T


Figure 3-7. Native gels stained for PPO activity in St. Augustinegrass lines 1, 3, 5, and 8 d after
challenge by southern chinch bug. T and C refer to the infested (i.e., treated) and
control samples, respectively. Arrows indicate the number of PPO isoforms.


I4-










1 cl 3cl cl 8 cl
T CT C T CT C


1 cl 3cl cl 8 cl
T CT CT C T


4-


C


I"


Figure 3-8. Native gels stained for LOX activity in St. Augustinegrass lines 1, 3, 5, and 8 d after
challenge by southern chinch bug. T and C refer to the infested and control samples,
respectively. Arrows indicate the number of LOX isoforms.


I -71: i l Ll









CHAPTER 4
QUANTITATIVE ANALYSIS OF FEEDING BEHAVIOR OF SOUTHERN CHINCH BUG,
BLISSUS INSULARIS BARBER (HEMIPTERA: BLISSIDAE), ON RESISTANT AND
SUSCEPTIBLE ST. AUGUSTINEGRASSES

Introduction

St. Augustinegrass is the most widely planted turfgrass in Florida and other Gulf coast

states. St. Augustinegrass is estimated to cover more than one and a half million acres in Florida

(Hodges et al. 1994) and accounts for 85% of the sod industry in Florida with a value of $262

million annually (Haydu et al. 2005). The southern chinch bug, Blissus insularis Barber, is the

most serious insect pest of St. Augustinegrass (Crocker 1993). The annual cost of southern

chinch bug management and losses in Florida alone was estimated at $5 million in 1983 (Hamer

1985) and is undoubtedly much more today. The economic importance of this insect is further

augmented by its development of resistance to several key insecticides (Reinert and Portier 1983,

Cherry and Nagata 2005, 2007).

Historically, host plant resistance has been one of the most successful pest management

methods for this insect. 'Floratam', a polyploid variety of St. Augustinegrass with resistance to

southern chinch bug, has been available in Florida and Gulf Coast areas since its release in 1973

(Horn et al. 1973). However, by 1985 a population of chinch bug had overcome Floratam's

resistance in Florida (Busey and Center 1987). Although significant progress has been made in

identifying new sources of southern chinch bug resistance in St. Augustinegrass lines, such as the

polyploid FX-10 (Busey 1993) and the diploid NUF-76 (Nagata and Cherry 2003), the

mechanisms of resistance in these lines are unknown. Rangasamy et al. (2006) investigated

categories of resistance to southern chinch bug in FX-10 and NUF-76 and reported high levels of

antixenosis in both lines and possible antibiosis in NUF-76 (Chapter 2). Higher mortality of

southern chinch bugs has been reported on resistant FX-10 and NUF-76 than on susceptible









Floratam, Palmetto and Bitter Blue (Nagata and Cherry 2003); however, the cause of mortality is

still unknown. The mortality may be caused by toxic secondary metabolites present in phloem

sap, lack of adequate nutrition after sustained feeding or starvation due to reduced feeding on

resistant varieties. Gaining a better understanding of the feeding behavior of southern chinch

bugs on these resistant and susceptible St. Augustinegrasses is essential to elucidate further the

mechanisms of resistance in FX-10 and NUF-76.

Studying the feeding behavior of insects with piercing and sucking mouth parts is more

difficult compared with insects with chewing mouthparts and requires specialized techniques.

McLean and Kinsey (1964) developed a unique technique, the electronic feeding monitor which

was later referred to as the electrical penetration graph (EPG) (Tjallingii 1985) to study the

feeding behavior of hemipterans. The EPG monitor has gone through many modifications and

improvements (Brown and Holbrook 1976, Kawabe and McLean 1980, Kimsey and McLean

1987, Backus and Bennett 1992, Backus and Bennett, unpublished) and today is the most

rigorous technique for studying feeding behavior and plant interactions of hemipterans. The

basic principles of EPG have been well described by Walker (2000). In brief, the EPG monitor

consists of a voltage source and an amplifier which are connected to each other. Both the insect

and plant are connected to the EPG monitor thus becoming a part of the circuit. The insect is

connected to the amplifier using a very thin gold wire and silver conducting paint and the plant is

connected to the voltage source in the EPG monitor using a conductive probe (an uninsulated

stiff copper wire). Whenever the insect attempts to probe or inserts its stylet into the plant, the

circuit becomes complete and the current flows from the voltage source through the plant, the

insect, the amplifier (via its input resistor) and then back to the voltage source (Walker 2000).

The plant and the insect act as the biological component of the circuit and resist current flow.









The changes in resistance in the insect-plant interface, as well as biopotentials generated, are

measured as electrical signals and converted into waveforms which are unique to the insect's

activities inside the plant.

In the research reported here, we used the EPG technique to investigate the stylet probing

of southern chinch bugs on St. Augustinegrass. The St. Augustinegrasses selected for our studies

included FX-10 (resistant polyploid), NUF-76 (resistant diploid), Floratam (susceptible

polyploid), and 'Palmetto' (susceptible diploid).

Materials and Methods

Plants

Plants were grown as described in Rangasamy et al. (2006) (Chapter 2). Stolons used in

experiments were approximately 30 to 40 cm in length with a minimum of three nodes and were

cut from plants that were 6 to 8 weeks old.

Insects

Southern chinch bugs were collected from Royal Palm Beach (Palm Beach County, FL) by

vacuuming infested St. Augustinegrass lawns as described by Cherry (2001a). Adults and

nymphs were collected by suction into a modified WeedEater Barracuda blower/vacuum

(Poulan/Weedeater, Shreveport, LA). In the laboratory, fifth instars were separated from the

debris and provided with fresh Bitter Blue runners and maintained at 260C in 20-1 plastic buckets

with a moist paper towel at the bottom to avoid desiccation of insects. Only newly emerged 3-5

d old adult females were used for the experiment.

Wiring and Acclimating of the Insects

Insects were removed from the colony and starved for 3 h before wiring. A 12.7-[m-

diameter (0.0005 in.) gold wire (Sigmund Cohn Corp., Mt. Vernon, NY) was glued to the









pronotum of the insect using silver conductive paint (Ladd Industries, Burlington, VT). The

insect was held immobile during application of the wire tether by placing its abdomen in a

Pasteur pipette tip under low vacuum suction pressure. After wiring, the insects were allowed to

rest on a moist paper towel and acclimate to the wire for a 1-h period before recording.

EPG Equipment and Data Acquisition

A four-channel AC-DC electrical penetration graph (EPG) monitor (Backus and Bennett,

unpublished data) was used for recording the feeding behavior of southern chinch bug on St.

Augustinegrass. Plants, insects and the head-stage amplifiers were all placed in a Faraday cage,

constructed of 1.27-cm mesh galvanized hardware metal cloth (LG Sourcing Inc., N.

Wilkesboro, NC), to avoid any external electrical noise. An input impedance level of 107 Q was

used for all four channels and all the test plants were supplied with a 10 mV, 1000 Hz AC

substrate voltage. The pots containing the plants were placed on wooden blocks to electrically

isolate them from the Faraday cage. A selected runner from each St. Augustinegrass plant was

laid down along a Plexiglas stage measuring 20 cm length x 5 cm width and fixed in place using

strips of Parafilm (Pechiney Plastic Packaging, Menasha, WI). The Plexiglas stage was held

horizontally by an alligator clip connected to a "helping hand" holder (van Sickle Electronics, St.

Louis, MO). The use of the horizontal stage allowed insects that had left the plant the

opportunity to return by walking to the plant (unless they fell off the stage and hung suspended

from their wire tether). The changes in electrical resistance as well as biopotentials during stylet

probing were amplified, rectified and digitized at a rate of 100 samples per second using a DI-

720 analog-to-digital board as described by Almedia and Backus (2004) and recorded using a

Dell Pentium notebook with WinDaq Pro software (Dataq Instruments, Akron, OH). All

recording started at 1600 h (in an isolated room at 26 + 2C with photoperiod of L18:D6) and









continued for 24 h simultaneously on four St. Augustinegrass lines; probing was artificially

terminated for insects with mouthparts still inserted in the plant at 24 h. There were 35

recordings per treatment; each consisting of all four St. Augustinegrasses arranged randomly

inside the Faraday cage and connected individually to each of the four channels in the monitor.

However, recordings where one or more of the insects within the replicate were not active or

dead were discarded, leaving 22 replicates that were analyzed statistically. Waveform

Characterization and Measurements

The waveforms produced by southern chinch bug feeding on St. Augustinegrass were

categorized based on stereotypical patterns (Backus et al., ms. in prep.). In brief, there are four

waveform families (Z, G, H, and I), and six waveform types (Z1, Z2, G1, G2, II and 12) within

those families (Figurel). For the purpose of comparing the waveforms produced on southern

chinch bug resistant and susceptible St. Augustinegrasses, only the major waveform categories of

Zl, Z2, P (pathway, G1 + G2 + H) and I (ingestion, II + 12) were analyzed in this research. The

duration measurements for each waveform were made with WinDaq /Pro +Waveform Browser

software at a compression of 10 (2 sec/div.) using 2 sec as the threshold duration for a particular

waveform event. The files were copied into a Microsoft Excel file developed for EPG analysis

by van Giessen and Jackson (1998) and modified for B. insularis.

Waveform Categories

The following waveform categories used in the analysis are depicted in Figure 4-1.

Z1: Insects off the plant,

Z2: Non-probing baseline,

Gl: variable-frequency and high-amplitude waveforms; always occur at the beginning of a
probe after Z2 and followed by G2, presumably associated with stylet pathway activities,

G2: hump-like, high-amplitude putative pathway waveform,









* H: low-amplitude putative pathway waveform, always preceded by G2,

* I: Putative ingestion, very uniform, repetitive (7-8 Hz) waveform with peaks and waves
(Figure 4-1).

E. A. Backus (personal communication) suggests that II and 12 may be associated with

either active and passive ingestion or salivation and ingestion, respectively. No biological

correlation for either stylet activities or location has been established to differentiate between II

and 12 waveform types at this time; however it is likely that at least part of this ingestion is from

phloem.

Definition of Non-Sequential Parameters

Non-sequential parameters used in this study were defined by Backus et al. (2007). A

probe is defined as all behaviors occurring from the beginning of stylet penetration probing into

plant tissue until stylet withdrawal. A waveform event is defined as a continuous, uninterrupted

occurrence of one waveform type (Almeida and Backus 2004, Backus et al. 2007). An

accumulator parameter for an insect is designated "by insect" and a parameter averaged across

the N insects assayed is termed "per insect".

Statistical Analysis

Statistics of waveforms from the experiment were analyzed using statistical analysis

software (SAS 1999) and the procedure described by Almeida and Backus (2004) and Backus et

al. (2007). The descriptive statistics for feeding behaviors were compiled at the heuristic levels

of waveform event, probe, insect and cohort as described in Backus et al. (2007). A cohort-level

waveform analysis will show the total behavioral repertoire of the southern chinch bug

population tested. Non-sequential parameters for EPG data were the same as those named and

described in Backus et al. (2007). In brief, the basic parameters of EPG data at the event level

are the number of waveform events per probe (NWEP) and waveform duration per event (WDE).









The values in these parameters are combined to achieve parameters at the probe level, such as

the number of probes per insect (NPI), and probing duration per probe (PDP). Probe level values

were combined for parameters at the insect level, such as probing duration per insect (PDI),

waveform duration per insect (WDI), and number of waveform events per insect (NWEI). When

more than one waveform event occurs during a probe, the probe-level data provide sequence

information, transition information or both (Backus et al. 2007). The insect-level data provide

behavioral information on individual insects as well as of an average insect in the cohort.

Restricted maximum likelihood estimation (REML-ANOVA) (PROC MIXED, SAS 1999)

was used to find out whether the frequencies or durations of each waveform type or overall

probing were significantly different on the four St. Augustinegrass lines, per event, per probe,

per insect or per cohort (Backus et al. 2007). When required, means were then separated using

the LSMEANS statement (SAS 1999). Frequency and duration data were square root- and log-

transformed, respectively, before ANOVA to improve homogeneity of variances. Differences

were considered significant at a = 0.05.

Results

Cohort Level

Southern chinch bugs spent only 28% and 46% of their total 24-h plant access time in

stylet probing (total probing duration, TPD) on FX-10 and NUF-76, respectively (combination of

P and I, Table 4-1; Figure 4-2). However, chinch bugs confined on Floratam and Palmetto spent

56% and 66%, respectively, of their access times in probing. More probing events (TNPE) were

recorded from chinch bugs confined on FX-10 and NUF-76 than on Floratam and Palmetto

(Table 4-1). Overall, ingestion duration was highest on Palmetto, followed by Floratam, then

NUF-76 and lowest on FX-10 (Figure 4-3). It is also valuable to note that only a third of stylet









penetrations in which pathway waveform P began successfully transitioned to the ingestion

phase (I1) on FX-10 and NUF-76, whereas nearly half of the probes on Floratam and Palmetto

resulted in ingestion waveforms (Table 4-2). To statistically analyze the data, cohort level data

were subdivided to event level and rebuilt through probe and insect levels.

Event Level

Probing duration per event (PDE) is the averaged duration of all events, regardless of

waveform type. In general, the longest events were made on FX-10, intermediate on Floratam

and Palmetto, and shortest events were made on NUF-76 (F = 46.22; df = 3, 2500; P < 0.0001)

(Table 4-1). A waveform event is defined as the smallest, continuous duration of a particular

waveform type. The duration of each Z1 waveform (i.e., time southern chinch bugs were off the

plant) was the longest on FX-10, intermediate on Palmetto and NUF-76 and shortest on Floratam

(F = 3.38; df = 3, 111; P = 0.02) (Table 4-3). The duration of Z2 waveform (non-probing,

walking or resting on the plant) per event was not significantly different among FX-10, Floratam

and Palmetto; however, the duration of Z2 was shorter on NUF-76 compared to the others (F =

45.62; df = 3, 7640; P = 0.0001). The longest duration of the pathway waveform type (P)

occurred on FX-10 whereas the shortest pathway waveform event occurred on Floratam;

intermediate durations were recorded on NUF-76 and Palmetto, which were not significantly

different from one another (F = 29.51; df = 3, 11000; P = 0.0001). The durations of ingestion

waveform event (II, 12 waveforms) on Floratam and Palmetto were four and two times longer

than on FX-10 and NUF-76, respectively (Ii: F= 96.44; df = 3, 2772; P = 0.0001, 12: F= 29.45;

df = 3, 672; P = 0.0001) (Table 4-3).

Probe Level

Southern chinch bugs feeding on resistant FX-10 and NUF-76 produced significantly more

probes per insect (NPI) than bugs feeding on susceptible Floratam and Palmetto (F = 17.64; df=









3, 172; P = 0.0001) (Table 4-1). In addition, the mean duration of probes (PDP) on FX-10 and

NUF-76 were only one-third to one-half as long as those on Floratam and Palmetto; the duration

of probes on the latter two were not significantly different (F = 46.22; df = 3, 25000; P = 0.0001)

(Table 4-1). Considering waveforms on a per probe basis, the insects spent the longest time in

pathway activities (P waveforms) on FX-10 and the shortest time on Floratam however, no

difference was observed between NUF-76 and Palmetto (F = 7.56; df= 3, 697; P = 0.0001)

(Table 4-4). The mean durations of ingestion waveforms (both II, 12) during a probe on FX-10

were only one third that of the duration on Floratam. For II, duration per probe on FX-10 was

not significantly different from that on NUF-76 but both FX-10 and NUF-76 were significantly

different from the intermediate duration on Floratam and the longest probe duration on Palmetto

(F = 18.77; df= 3, 450; P= 0.0001). The longest durations of 12 on Floratam and Palmetto were

not significantly different, but both were greater than the intermediate duration of 12 on NUF-76

and the shortest duration on FX-10 (F = 15.03; df= 3, 257; P = 0.0001) (Table 4-4).

Insect Level

There were no significant differences in the number of times that insects walked off the

plant (Z1), on a per insect basis, among the four St. Augustinegrass lines (F = 0.71; df = 3, 45; P

= 0.51) (Table 4-5). However, the duration of the times off the plant was longer for FX-10,

NUF-76 and Palmetto than for Floratam (Figure 4-3). There was no significant difference

observed among FX-10, NUF-76 and Palmetto (Z1, F= 8.92; df = 3, 45; P = 0.0001). The

numbers of nonprobing events on the plant (Z2, F= 8.62; df = 3, 84; P = 0.0001) were higher on

FX-10 and NUF-76 than on Floratam and Palmetto (Table 4-5). The duration of nonprobing

waveforms (Z2) was longest on FX-10, significantly different from the intermediate durations

observed on Floratam and NUF-76 and the shortest duration on Palmetto (Z2, F= 8.92; df = 3,









84; P = 0.0001) (Figure 4-3). Chinch bugs confined on FX-10 and NUF-76 produced

significantly more pathway waveform events per insect (F = 5.75; df = 3, 84; P = 0.0013) (Table

4-5) that were of longer duration (F = 8.23; df = 3, 84; P < 0.0001) (Figure 4-3) than those

produced by insects confined on Floratam and Palmetto. Fewest pathway waveform events per

insect were produced on Palmetto whereas an intermediate number of pathway waveform events

per insect was observed on Floratam (Table 4-5). Although the number of ingestion (11 and 12)

waveform events per insect did not differ among the four St. Augustinegrass varieties (F = 0.60;

df = 3, 84; P = 0.6168) (Table 4-5), the mean durations of ingestion waveform I1 were three and

two times longer on Floratam and Palmetto than on FX-10 and NUF-76, respectively (F = 11.63;

df = 3, 84; P < 0.0001) (Table 4-3). The mean durations of ingestion waveform 12 were four and

two times longer on Floratam and Palmetto than on FX-10 and NUF-76, respectively (F = 9.82;

df= 3, 84; P < 0.0001) (Figure 4-3).

Discussion

For the first time, the EPG technique has been used for studying the feeding behavior of

southern chinch bug on St. Augustinegrasses. EPG results revealed that the southern chinch

bugs were off the plant (Z1 waveforms) on FX-10 and NUF-76 for a longer time than on

Floratam and Palmetto. These results agree with our previous studies which showed that fewer

southern chinch bugs were found on resistant FX-10 and NUF-76 than on Floratam, Palmetto

and Bitter Blue in a choice experiment, suggesting the presence of strong antixenosis (Chapter 2;

Rangasamy et al. 2006). When the insects were on the resistant FX-10 and NUF-76 plants, they

spent more time in non-probing activities, such as resting or walking, than in ingestion than

when on susceptible Floratam and Palmetto. The short durations of sustained ingestion on FX-

10 and NUF-76 and long duration of sustained ingestion on Floratam and Palmetto also









corroborate our previous results (Rangasamy et al. 2006; Chapter 2), which showed that

significantly fewer excretory spots were produced by chinch bugs fed on FX-10 and NUF-76

than on the susceptible varieties.

Successful insect colonization on a host plant depends on host plant acceptability. For

phloem-feeding, piercing and sucking insects such as aphids, host acceptability depends partly

on the ability of the insect's stylets to reach the phloem and partly on the quantitative and

qualitative properties of phloem sap (Klingauf 1987). Tarn and Adams (1982) and Dreyer and

Campbell (1987) discussed that aphid stylet penetration on resistant plants is often characterized

by more frequent, but shorter probes, and longer waveforms associated with pathway activities.

We found that southern chinch bugs confined on resistant FX-10 and NUF-76 probed more

frequently and that their pathway waveforms were longer than on the susceptible Floratam and

Palmetto. The durations of ingestion, on per event, per insect and per probe levels, were all

significantly reduced in chinch bugs feeding on the resistant St. Augustinegrass varieties

compared to the susceptible varieties. Thus, the present study clearly demonstrates that southern

chinch bugs spent longer on FX-10 and NUF-76 in activities that produced the pathway

waveforms (P) and less time in activities that resulted in ingestion waveforms (II, 12). These

results indicate the presence of a physical barrier around the vascular bundle or chemical

deterrents from the phloem of the resistant cultivars. Similar to our results, the aphids, Myzus

persicae (Sulz.) and Nasonovia ribisnigri (Mosley), produced many probes, longer waveforms

associated with extracellular stylet pathway activities and shorter waveforms associated with

phloem ingestion on resistant lettuce plants, Lactuca sativa L., suggesting that aphid resistance

might involve both mesophyll and phloem factors (Montllor and Tjallingii 1989).









Our results suggest that southern chinch bug can perceive significant differences among

the four St. Augustinegrasses before their stylets reach the phloem sieve elements; thus, the

resistance factors in FX-10 and NUF-76 may reside in tissue encountered by the chinch bug

stylets prior to reaching the vascular bundle. Dreyer and Campbell (1984) and Campbell and

Dreyer (1985) found that a chemical modification of pectin in the middle lamella of some

sorghum varieties, Sorghum bicolor L. (Moench) made it difficult for the intercellular stylet

penetration by certain biotypes of greenbug, Schizaphis graminum (Rondani). It is possible that

a similar resistant mechanism occurs in the resistant St. Augustinegrasses. Transmission electron

microscopic studies on St. Augustinegrass leaf sheath anatomy revealed that the sclerenchyma

cells in NUF-76 and FX-10 have thicker cell walls (Chapter 5) which may serve as a physical

barrier and prevent or make it difficult for southern chinch bug stylets to reach the phloem sieve

elements.

Resistance factors residing in sieve elements are often indicated by short durations of

ingestion waveforms (Campbell et al. 1982, Ryan et al. 1987, van Helden and Tjallingii 1993,

Cole 1994, Caillaud et al. 1995, Paul et al. 1996, Klingler et al. 1998, Sauge et al. 1998,

Kaloshian et al. 2000, Tjallingii 2006). Jiang and Walker (2007) found that the silverleaf

whitefly, Bemisia argentifolii Bellows and Perring, terminated its ingestion waveforms more

often and spent reduced amount of time in ingestion on whitefly-resistant alfalfa clones than on

susceptible clones, and suggested that this ingestion behavior could be due to the chemical

constituency of the phloem sap, such as the presence of toxins or repellents or lack of stimulants.

The shorter duration of ingestion waveforms could also be caused by a physical blockage of

phloem sap by callose deposition causing a long term plugging (Eschrich 1975) or by phloem-

specific proteins as a temporary defense measure or by other sieve tube sealing mechanisms (van









Bel 2003). As evidence for the blockage of sap flow, Jiang and Walker (2007) reported that on

one of the whitefly-resistant alfalfa clones, phloem sap ingested per unit time (correlated with

honeydew production) was reduced and a longer lag time was observed between the last drop of

honeydew produced and the termination of ingestion waveform. They speculated that the

whitefly might have tried to continue the ingestion even after a possible phloem blockage and the

last observed drop of honeydew, but eventually gave up because of difficulty in ingestion.

In our research, the phloem sap in FX-10 and NUF-76 did not stimulate sustained feeding,

as shown by reduced duration of ingestion waveforms compared to those on Palmetto and

Floratam. The shorter duration of ingestion waveforms could be due to the presence of

resistance factors in phloem sap or blockage of sieve elements and possibly could be an

important component of southern chinch bug resistance in FX-10 and NUF-76. Although we

have no direct evidence that the resistant St. Augustinegrasses have phloem-sealing mechanisms

or feeding deterrents in phloem sap, stylectomy technique has been used to explore these

possibilities in aphids and it could also be tried in southern chinch bugs. Caillaud et al. (1995)

found that the resistance factors for the aphid, Sitobion avenae (Fabr.), were present in the sieve

elements of some lines of Triticum monococcum L. and inhibited phloem sap ingestion by the

aphid. Caillaud and Niemeyer (1996) investigated the reasons for the rejection of some lines of

T. monococcum by S. avenae. They found no significant difference in the concentration of

hydroxamic acids, a family of aphid resistance factors in cereals, between the favorable T

monococcum line Tm47 and the resistant lines, Tm46 and Tm44, suggesting that the rejection of

aphid resistant lines was not due to these chemical factors examined. They collected phloem sap

from young seedlings of aphid resistant and susceptible T. monococcum and found that the

success in stylectomy, the proportion of stylets resulting in longer exudations (>1 min), the









average duration of exudation and the final volume of phloem sap exuded, were significantly

higher in the aphid-susceptible genotypes than the resistant ones. This suggests that phloem-

sealing caused the aphid to reject feeding in the aphid-resistant T. monococcum lines.

Our previous investigations showed that southern chinch bugs did not prefer resistant lines

and their oviposition and development were affected on them (Rangasamy et al. 2006, Chapter

2). The current study, which employed EPG technique to elucidate the feeding behavior of

chinch bugs, showed that the southern chinch bugs made more probes, took longer time to reach

the phloem sieve elements and spent less time on ingestion in resistant lines, suggesting that

these resistant lines may have some physical or biochemical factors) impeding the stylet's

uptake from the phloem. While not previously reported on homopterans, chemical analysis of

St. Augustinegrass phloem sap, collected using the stylectomy technique, could be used to

investigate the presence of feeding deterrents or role of sieve tube sealing mechanisms in

southern chinch bug resistance in FX-10 and NUF-76. Considering the economic importance of

St. Augustinegrass and the continuing race to find chinch bug resistant varieties, our current

research provides exact details on feeding behavior of southern chinch bugs and will eventually

further elucidate the mechanisms of resistance.












Table 4-1. Durations and frequencies of probing parameters of EPG data recorded on four southern chinch bug resistant and
susceptible St. Augustinegrass lines. Values within rows followed by the same letter are not significantly different at a
0.05 separated by LSD means test. *Mean SE.


Variables


Total probing duration
in min (TPD)
Total no. of probing
events (TNPE)
Probing duration per
event (PDE) (min)*
No. of probes per insect
(NPI)*
Probing duration per
o probe (PDP) (min)*
Probing duration per
insect (PDI) (min)*


FX-10


8922

2291

9.94 + 0.59a

101.37 + 8.08a

4.01 + 0.23c

1034.45 + 44.32


St. Augustinegrasses
Floratam NUF-76


17819

1545

8.98 + 0.61b

68.96 + 6.22b

11.75 1.39a

630.19+ 58.35


14909

2571

6.53 + 0.49c

114.37 + 13.20a

5.93 + 0.71b

762.33 + 71.18


Palmetto


21082

1352

7.85 + 0.57b

59.69 + 6.83b

16.06 + 2.15a

482.03 + 66.18


Table 4-2. Total number of major waveform events (TNWE) on each of four southern chinch bug-resistant or -susceptible St.
Augustinegrass varieties (n = 22).


Major waveforms


insect off the plant
non-probing, insect walking
pathway
ingestion


FX-10
33
2258
2253
744
134


No. of waveform events (TNWE)
Floratam NUF-76
21 36
1524 2535
1534 2532
630 806
170 185


P values


<0.0001

<0.0001

<0.0001

0.7833


Palmetto
25
1327
1314
596
187










Table 4-3. Mean waveform durations per event (WDE) for major waveforms recorded for southern chinch bug feeding on resistant
and susceptible St. Augustinegrasses. Values within the rows with the same letter are not significantly different at a = 0.05
separated by LSD means test.


Major waveforms

insect off the plant
non-probing, insect walking
pathway
ingestion


Waveform duration per event (WDE) (min) (mean SE)
FX-10 Floratam NUF-76 Palmetto
60.41 + 18.37a 18.62 + 9.95c 39.43 17.75b 44.14 + 12.12a
9.20 + 0.52a 8.85 0.6a 6.06 + 0.42b 7.16 + 0.52a
1.50 + 0.30a 1.25 + 0.04c 1.29 + 0.05b 1.28 + 0.05b
4.28 0.31c 17.72+ 1.40a 10.24 0.80b 21.99 1.92a
6.69 0.58b 20.34+ 1.93a 10.29 + 1.37b 27.46 4.18a


Table 4-4. Mean duration of major waveforms per probe (WDP) on each of four southern chinch bug-resistant or -susceptible St.
Augustinegrasses. Values within rows followed by the same letter are not significantly different at a = 0.05 separated by
LSD means test


Major waveforms


pathway
ingestion


FX-10
1.51 + 0.06a
5.19 + 0.49c
7.46 + 0.73c


Waveform duration per probe (WDP) (min) (mean SE)
Floratam NUF-76
a 1.17 0.04c 1.33 0.06b 1
15.18 1.59b 7.32 0.88c 2(
19.18 + 2.33a 12.45 + 1.77b 21


Palmetto
.44 + 0.09b
).45 + 2.84a
.66 3.16a


Table 4-5. Mean number of major waveform events per insect (NWEI) on each of four southern chinch bug-resistant or -susceptible
St. Augustinegrass varieties. Values within rows followed by the same letter are not significantly different at a = 0.05
separated by LSD means test.


Major waveforms

insect off the plant
non-probing, insect walking
pathway
ingestion


No. of waveform events per insect (NWEI) (mean SE)
FX-10 Floratam NUF-76 Palmetto
2.20 + 0.46 2.10 + 0.51 3.00 + 0.60 2.09 + 0.40
102.64 + 8.10a 69.28 6.21b 115.23 13.29a 60.32 6.84b
140.41 + 12.64a 108.05 9.30b 154.36 17.05a 89.63 9.07c
33.82 + 5.13 28.64 + 2.52 36.64 + 4.07 27.10 + 3.21
6.10 0.83 7.73 + 1.15 8.41 + 1.80 8.50+ 1.37


P values

0.5111
<0.0001
<0.0013
0.4018
0.6168


P values

0.0200
<0.0001
<0.0001
<0.0001
<0.0001


P values

<0.0001
<0.0001
<0.0001
















I 4: I s1 / .,:ld 044 .I^ ^^^^^









.... . .. ; .. .

SCBPalnettolD 7 Subv 10 xv AC SCBPalettolO 7 Subv 10 v AC SCBPae1ettotl0 7 Subv 10

Figure 4-1. EPG waveforms produced by southern chinch bug feeding on St. Augustinegrass,
recorded using an AC/DC EPG monitor. Major waveform categories: Z2 baseline,
Gl and G2 high-amplitude pathway waveforms, H low-amplitude pathway
waveform, Il and I2 ingestion waveforms.











I Z1 FXA-l Zi Floratam
13% 6% 1%

1546% / 43%



1521
S6% 10%
Z1 NUF-76 Z1 Palmetto
5% 4%

3 ,.Z2
Z2 1 ._, .30%

49% 58%\

14% 1 8%


Figure 4-2. Total percentage of time spent on total wave form duration (TWD) by southern
chinch bugs on resistant and susceptible St. Augustinegrasses in different waveform
categories. Z Insects off the plant, Z2- insects on the plant, resting, walking; P -
combined pathway waveforms (G1+G2+H), I combined ingestion waveforms
(11+12).












1200
a
m w FX-O1
S1000 a Floratam
SNUF-76
4-
S800-( b r Palmetto
800 b
*U a
600 b


400
a ~a

I 200 a a b b


0
Zl Z2 P II 12

Waveforn categonie:.

Figure 4-3. Waveform duration per insect (WDI) for major waveforms on resistant and
susceptible St. Augustinegrasses. Z1 insects off the plant, Z2 insects on the plant,
resting, walking; P combined pathway waveforms (G1+G2+H), I combined
ingestion waveforms (11+12). Values (mean SE, n = 22) with the same letter in a
waveform category are not significantly different at a = 0.05 separated by LSD means
test.









CHAPTER 5
ROLE OF LEAF SHEATH LIGNIFICATION AND ANATOMY IN RESISTANCE AGAINST
SOUTHERN CHINCH BUG, BLISSUS INSULARIS BARBER (HEMIPTERA: BLISSIDAE) IN
ST. AUGUSTINEGRASS LINES

Introduction

St. Augustinegrass, Stenotaphrum secundatum, is the most widely used turfgrass in

tropical and subtropical climatic regions (Sauer 1972). It is a commonly grown residential

turfgrass species in the southern United States, covering more than one million acres and

accounting for 85% of the sod industry in Florida alone, with a value estimated at $262 million

(Haydu et al. 2005). The southern chinch bug is the most serious insect pest of St.

Augustinegrass (Crocker 1993). The annual cost of southern chinch bug management and losses

in Florida alone was estimated at $5 million in 1983 (Hamer 1985). Management of this insect

has further been challenged by its development of resistance to several key insecticides (Reinert

and Portier 1983, Cherry and Nagata 2005, Cherry and Nagata 2007).

Historically, host plant resistance has been one of the most successful management tools

for this insect. 'Floratam', a polyploid variety of St. Augustinegrass with resistance to southern

chinch bug, has been available in Florida and other Gulf Coast states since its release in 1973

(Horn et al. 1973). Within a decade after its release, in 1985, a population of chinch bug was

reported to have overcome Floratam's resistance in Florida (Busey and Center 1987).

Significant progress has been made in identifying new sources of southern chinch bug resistance

in St. Augustinegrass lines. However the registered polyploid FX-10 with a high level of

resistance to southern chinch bug (Busey 1993), is coarse and tough and never became popular

among growers (Nagata and Cherry 2003). The discovery of southern chinch bug resistance in

diploid NUF-76 was considered important because of its fine texture and slow growth (Nagata

and Cherry 2003). However, the mechanisms of resistance in these lines are unknown.









Rangasamy et al. (2006) investigated categories of resistance to southern chinch bug in FX-10

and NUF-76 and found high levels of antixenosis in both lines and possible antibiosis in NUF-76

(Chapter 2). Electrical penetration graph (EPG) studies revealed that southern chinch bugs took

longer to reach the phloem sieve elements presumably and spent less time on ingestion on FX-10

and NUF-76 compared to their susceptible controls, Floratam and Palmetto, respectively

(Chapter 4). Relatively more stylet probings per insect on FX-10 and NUF-76 than on Floratam

and Palmetto also suggested the presence of stylet penetration impediments around the vascular

bundle in resistant varieties. In addition, the short duration of ingestion on FX-10 and NUF-76

suggested the presence of feeding deterrents within phloem sap and/or sieve tube plugging

mechanisms.

Lignin content of plant tissues has often been correlated with resistance to insects and plant

pathogens. Blum (1968) identified lignin as a major resistance factor in sorghum against shoot

fly, Atherigona variasoccata. Shoot fly resistant sorghum plants had highly lignified and

thickened cells around the vascular bundles in the young leaves of the central whorl preventing

penetration by larvae. Toughness of plant tissues has often been considered as a major factor in

their unsuitability to insect feeding (Feeny 1970, Coley 1983). Leaf beetles (Plagiodera

versicolora (Laicharting)) feeding on mature Salix sp. leaves succumbed to increased mandible

wear compared to those feeding on young leaves which resulted in reduced leaf consumption and

beetle fecundity (Raupp 1985). Bark beetles, Dendroctonus micans Hugel feeding on spruce

trees with higher bark lignin content had reduced larval weight, survival and development rate

(Wainhouse 1998). Solanum integrifolium plants transformed with horseradish peroxidase gene

had 30% higher lignin content compared to wild-type plants and a high level of resistance to corn

earworm (Heliothis armigera (Hibner), Lepidoptera: Noctuidae) (Tsuduki et al. 2006). Our









previous investigation into the activities of oxidative enzymes such as peroxidase, lipoxygenase,

catalase and polyphenol oxidase in response to the feeding of southern chinch bug on resistant

and susceptible St Augustinegrass lines revealed that the chinch bug-resistant FX-10 had

increased peroxidase activity 5 and 8 d after southern chinch bug infestation (Chapter 3).

Because plant peroxidases play diverse roles in plants including lignin biosynthesis in the cell

wall (Lagrimini et al. 1987) and oxidation of toxic compounds (Gasper et al. 1982) and because

lignification of cells could be an impediment to insect pests (Coors 1987, Buendgen et al. 1990,

Wainhouse et al. 1990, Wainhouse et al. 1998, Tsuduki et al. 2006), we hypothesized that

increased lignification could be a possible component of southern chinch bug resistance in these

St. Augustinegrass lines.

The objectives of this research were to investigate the possibility that increased

lignification and differences in leaf sheath anatomy could account for resistance in diploid NUF-

76 and polyploid FX-10 St. Augustinegrasses. Specifically, we compared stylet probing

frequencies, leaf sheath lignin content and vascular bundle anatomy among two southern chinch

bug-resistant and two susceptible St. Augustinegrass varieties.

Materials and Methods

Plants

St. Augustinegrass varieties used in the following experiments included FX-10 (resistant

polyploid), Floratam (susceptible polyploid), NUF-76 (resistant diploid) and Palmetto

(susceptible diploid). The plants were grown as described by Rangasamy et al. (2006). Runners

used for the stylet probing study and lignin staining were 30 to 40 cm in length with a minimum

of four nodes; runners used for the lignin quantification had a minimum of 15 nodes.









Insects

Southern chinch bugs were collected from Royal Palm Beach (Palm Beach County, FL).

The insects were collected by vacuuming St. Augustinegrass lawns as described by Cherry

(2001a) and then sorting through debris in the laboratory for fifth instars and adults. After

collection, insects were provided with fresh Floratam stolons and maintained at 180C in 20-1

plastic buckets with a moistened paper towel at the bottom to increase humidity within the

bucket.

Staining for Salivary Sheaths

Visual observations suggested that chinch bugs were residing in and feeding on the axillary

shoot. Hence, we focused our studies on the axillary shoot which consisted of an outermost leaf

sheath and several layers of inner leaf sheaths with leaf blades and meristematic tissues (Figure

5-1). Ten chinch bug adults (5 males and 5 females) were confined for 48 h in organdy sleeve

cages to the third youngest node of one runner of each St. Augustinegrass plant. The chinch

bugs were removed from the plant before collecting the sample for staining for salivary sheaths.

Southern chinch bug-infested axillary shoots were excised from the plants and placed in

McBride's staining solution. The staining solution was prepared using 0.2% (wt/v) acid fuchsin

in a 1:1 mixture of 95% (v/v) ethanol and glacial acetic acid (Sigma-Aldrich, St. Louis, MO)

according to the methods of Backus et al. (1988). The plant tissues were stained for 24 h at 24 +

2C, then placed in the clearing solution and autoclaved for 15 min at 1200C and 15 psi. The

clearing solution was prepared using 99% (v/v) glycerol, 85% (v/v) lactic acid and distilled water

(1:1:1, v/v/v, Sigma-Aldrich, St. Louis, MO) (Backus et al. 19988). The stained salivary sheaths

in each leaf sheath within an axillary shoot were counted under a dissection microscope at 40X

(Figure 5-2)









Lignin Quantification

Total lignin content of uninfested axillary shoots was quantified using the Klason method

(Kirk and Obst 1988). Fresh axillary shoot were air dried and ground using a Wiley mill

(Thomas Scientific, Swedesboro, NJ) to pass through a 20-mesh screen. A 200-mg sample was

weighed into a small beaker and 1 ml of 72% (v:v) H2SO4 per 100 mg sample was added. The

sample-acid mixture was digested by placing in a water bath at 300C for 1 h and was stirred

frequently to get a complete solution. After exactly 1 h, the solution was diluted using 28 ml of

distilled water for each 1 ml of acid and transferred to an Erlenmeyer flask. The diluted solution

was then hydrolyzed in an autoclave at 1200C for 1 h and filtered through a 50-ml Pyrex Gooch

crucible with fritted disc of 4 5.5 [im porosity (Sigma-Aldrich, St. Louis, MO). The filtrate

was saved for estimating the acid-soluble lignin. The Klason lignin residue was washed with hot

water to remove the acid. The Klason lignin was then dried to constant weight at 1050C and

expressed as a percent dry weight. The acid-soluble lignin present in the filtrate was determined

spectrophotometrically at 205 nm using an absorptivity value of 11OL/g-cm (extinction

coefficient) to calculate the total lignin content (Ehrman 1996).

Staining for Lignin

Fresh sections of 10-15 [tm of the outermost leaf sheath of an axillary shoot were prepared

using a cryostat (MICROM HM 505E, Microm Loborgerate GMBH, Strasse, Germany) and

stained with 2% (wt/v) phloroglucinol (Sigma-Aldrich, St. Louis, MO) solution in ethanol/water

(95/5 v/v) for 2 min followed by treatment with 50% (v/v) HC1 for 1 min for the Weisner

reaction (Rastogi and Dwivedi 2006). The stained sections were photographed at 400X under an

epifluorescence light microscope microscope (Olympus BH2-RFCA, Olympus America, Inc.,

Lake Success, NY, USA).









Transmission Electron Microscopy

The axillary shoot from the fourth youngest node of each of the four St. Augustinegrasses

was collected and cut into small segments of 1-2 mm in length and fixed in Trump's EM fixative

(1% wt/v glutaraldehyde 4% wt/v paraformaldehyde in phosphate buffer saline (PBS), pH 7.3),

incubated in 2% (wt/v) osmium tetroxide and then dehydrated in a graded ethanol-acetone series

(25%, 50%, 75%, 95%, 100% (v/v) ethanol, 100% (v/v) acetone). After dehydration, the tissues

were embedded with Spurr's epoxy resin (Spurr 1969) at concentrations of 30%, 50%, 75%,

100% and placed in an oven at 600C for 48 h. Ultra-thin sections of 1 [m were made using an

ultramicrotome (EM-UC 6, Leico Microsystems, Bannockburn, IL) equipped with a glass knife

(Type 7801B, LKB Knife Maker, Rockville, MD). The ultra-thin sections were post-stained

with 2% (wt/v) uranyl acetate and viewed in a Hitachi H7000 electron microscope (Hitachi,

Tokyo, Japan) operated at an accelerating voltage of 75 kV. To measure the thickness of the

epidermal layer of the leaf sheath, photomicrographs of leaf sheath cross-sections were taken

using a light microscope at 400X and the epidermal layer thickness was measured using Adobe

Photoshop CS2 (Adobe Systems Inc., San Jose, CA). The proportion of the sclerenchyma cell

wall area relative to the total sclerenchyma cell area was calculated from the TEM micrographs

(3500X) of the leaf sheath vascular bundle using the SPOT image analysis program (Version 3.5,

Diagnostic Instruments, Inc. Sterling Heights, MI). Twenty five sclerenchyma cells were

measured for each of the three replicate plants in each of the four St. Augustinegrass lines.

Experimental Design and Statistical Analysis

The experiment to investigate the salivary sheath distribution was a completely

randomized design with four treatments (FX-10, Floratam, NUF-76, and Palmetto) and 20

replications. The samples for lignin staining and electron microscopic studies were collected









from three replicate plants of each of the four St. Augustinegrasses. Parameters such as number

of salivary sheaths in each leaf sheath layer relative to the outermost leaf sheath, total lignin

content, leaf sheath epidermal layer thickness and the proportion of sclerenchyma cell wall area

relative to total cell area were analyzed using PROC GLM (SAS 1999). When appropriate,

means were separated using Tukey's test at a significance level of a = 0.05.

Results

Most of the 20 axillary shoots examined from each of the four St. Augustinegrass lines

contained five leaf sheaths, arranged in concentric whorls. Significantly more salivary sheaths

were counted on the outermost leaf sheath layer in FX-10 and NUF-76 than on Floratam and

Palmetto (F = 16.78; df = 3, 76; P < 0.0001) (Figure 5-3) and these salivary sheaths were

branched more often in FX-10, Floratam and NUF-76 compared to Palmetto (F = 2.58; df= 3,

39; P < 0.0080) (Figure 5-4). A significantly smaller proportion of salivary sheaths reached the

innermost layer of FX-10 and NUF-76 axillary shoots compared to Floratam and Palmetto (F=

63.16; df = 3, 76; P < 0.0001) (Figure 5-3).

There was no significant difference in total axillary shoot lignin content between FX-10

and Floratam or between NUF-76 and Palmetto, although polyploid lines had significantly more

lignin than the diploid lines (F = 23.22; df = 3, 15; P = 0.0002) (Figure 5-5). Staining for lignin

did not show any distinct difference between southern chinch bug-resistant and -susceptible St.

Augustinegrass lines (Figure 5-6). In addition, there was no significant difference in the

thickness of the leaf sheath epidermal layer among the St. Augustinegrass lines studied (F=

1.17; df = 3, 6; P = 0.28) (Figs. 5-7, 5-8). However, TEM studies on the leaf sheath vascular

bundle indicated significantly thickened sclerenchyma cell walls in NUF-76 and FX-10

compared to Floratam and Palmetto (Figure 5-9). Quantification of cell wall thickness in









sclerenchyma cells confirmed these observations (Figure 5-10). The cell walls accounted for a

much larger proportion of total sclerenchyma area in the resistant St. Augustinegrasses than in

the susceptible grasses (F = 4.65; df = 3, 6; P = 0.0181) (Figs. 5-9, 5-10).

Discussion

Chinch bugs, among other hemipterans, lay down a salivary sheath produced by gelling

saliva during the intracellular passage of their stylets through plant tissue. Painter (1928) noted

the presence of a 'stylet-sheath' during feeding of the common chinch bug (B. leucopterous

leucopterous), a pest of sorghum, Sorghum bicolor (L.) Moench. The production of salivary

sheaths has often been used as a quantitative measure of homopteran feeding (Bowling 1979,

Marion-Poll et al. 1987, Bing et al. 1991). The research reported herein investigated the possible

presence of impediments to stylet penetration and access to phloem in southern chinch bug-

resistant St. Augustinegrass lines.

In this research, I found a significantly higher number of salivary sheaths observed on the

outermost leaf sheath layers in FX-10 and NUF-76 than on susceptible Floratam and Palmetto,

corroborating our EPG studies which indicated that chinch bugs probed significantly more often

on FX-10 and NUF-76 than on the susceptible varieties (Chapter 4). Oxidative enzymes such as

peroxidase, lipoxygenase and polyphenol oxidase had increased activities in FX-10 and NUF-76

plants infested by southern chinch bugs compared to infested Palmetto and Floratam plants

(Chapter 3); the induction of these enzymes could be due to the mechanical damage resulting

from the higher number of probings on the leaf sheath. EPG studies (Chapter 4) showed that

southern chinch bugs spent more time on pathway waveforms in FX-10 and NUF-76 which is

corroborated by the presence of more branched salivary sheaths on FX-10 and NUF-76. The

large proportion of salivary sheaths reaching the innermost leaf sheath on Floratam and Palmetto









demonstrates a strong preference by the southern chinch bugs for the intercalary meristematic

tissues, which are continuing to differentiate and are covered by the mature outermost leaf

sheaths. In other insect systems, Ni and Quisenberry (1997) found no significant difference in

the number of salivary sheaths of Russian wheat aphid Diuraphis noxia (Mordvilko) between

resistant ('Halt') and susceptible ('Arapahoe') varieties of wheat, Triticum aestivum L.

However, more salivary sheaths were initiated through leaf stomata on the aphid-resistant 'Halt'

than on the susceptible 'Arapahoe', suggesting the possible presence of antixenotic epidermal

characters such as leaf toughness and epicuticular waxes on 'Halt' leaves. Bing et al (1991)

similarly observed that 57% of the time the stylets of the bird cherry-oat aphid, Rhopalosiphum

maidis (L.) entered through stomata in maize, Zea mays L, plants in the late whorl stage however

only 8% of the stylets penetrated through stomata in the seedling stage. This suggested that the

higher rate of stomatal entry of stylets was due to easier penetration. Anderson et al. (2006)

found that Blissus occiduus Barber probed more frequently on buffalograss, a preferred host,

than on less-preferred sorghum and St. Augustinegrass.

We found that a smaller proportion of the initiated salivary sheaths reached the innermost

layer of the axillary shoot in resistant FX-10 and NUF-76 than they did in the susceptible

varieties. Based on the results from the EPG studies (Chapter 4) and stylet probing distribution,

these probes were probably terminated during pathway phase prior to ingestion (presumably

from phloem). We postulated that lignified tissue may be impeding stylet progress towards or

access to the tissue in which the southern chinch bug prefers to feed. Painter (1928) noted that

the phloem tissue was the target of the stylets of the common chinch bug. Because of a strong

preference by the common chinch bug to feed within the leaf sheath (Painter 1928) and our own

observations of feeding by southern chinch bug (Chapter 2), we quantified the total lignin









content of the leaf sheath and stained the leaf sheaths to look for quantitative and qualitative

differences, if any.

In St. Augustinegrass, the vascular bundle is surrounded by the highly lignified

sclerenchyma cells (Figure 5-3). Sclerenchyma cells have thickened secondary cell walls which

contain cellulose, hemicelluloses and lignin. In general, the lignin content of sclerenchyma cell

wall ranges from 18 35% (wt/wt) (Mauseth 1988) and the presence of lignin provides a stable

protection to the vascular bundle. Our TEM studies found a larger proportion of leaf sheath

sclerenchyma cell wall area in FX-10 and NUF-76 relative to the total cellular area consistent

with our hypothesis that the sclerenchyma cells may serve as a physical barrier to stylet

penetration.

Our previous studies suggested that chinch bug resistance mechanisms of the polyploid

line FX-10 may be different in certain aspects than that found in NUF-76 since NUF-76 showed

antibiosis but both lines had antixenosis (Rangasamy et al. 2006). It is interesting to note here

that both resistant lines had thicker sclerenchyma cell walls compared to the susceptible lines,

suggesting that NUF-76 may have additional features for antibiosis. A greater proportion of

sclerenchyma cell wall area was observed on NUF-76, a diploid line with total lignin content less

than the polyploids FX-10 and Floratam. It may be that deposition of lignin in a few

sclerenchyma cells with thick cell walls rather than total lignin is important as a barrier to the

southern chinch bug stylets.

In summary, our results showed that stylet penetration to innermost leaf sheaths occurred

more frequently in susceptible lines than in resistant lines, suggesting an impediment to stylet

penetration. However, this impediment is not due to differences in their epidermal layer or total

lignin content. Based on qualitative and quantitative analyses of TEM of vascular bundles, the









resistant lines FX-10 and NUF-76 had a greater degree of lignification in sclerenchyma cells

around the vascular bundles. We propose that thicker sclerenchyma cells around the vascular

bundles is one of perhaps several features of the resistance in St. Augustinegrass lines to

southern chinch bug and may be useful as a marker in breeding for southern chinch bug

resistance.























Figure 5-1. St. Augustinegrass stolon A. with the axillary shoot (as); B. Parts of axillary shoot, a
outermost leaf sheath with leaf blade, b prophyll, c second leaf sheath layer with
leaf blade, d third leaf sheath layer with leaf blade, e axillary meristem.























Ij



Figure 5-2. Transverse section of outermost St. Augustinegrass leaf sheath showing southern
chinch bug salivary sheath stained with McBride's stain (400X). ep epidermal layer,
pc parenchyma cells, ss salivary sheath, sc sclerenchyma cells, ph phloem, xy
xylem.













25 1b
C-%

30 16 --0-






FX-10 Floratam NUF-76 Palmetto FX-10 Hlonrtain NUF-76 Palmetto
St Augustinegrass lines St. Augustinegrass lines
Figure 5-3. Number of southern chinch bug salivary sheaths found on the outermost leaf sheath
of axillary shoots (A) and the proportion of salivary sheaths reaching the innermost
sheath (B) of southern chinch bug-resistant and -susceptible St. Augustinegrasses.
The values shown are mean SE. Means followed by the same letter are not
significantly different at a = 0.05 level separated by Tukey's test (n = 20).














61 a
a
4? a







4-
01


SFX-10 Floratam NUF-76 Palietto

St. Augusmtinegrass lines
Figure 5-4. Number of southern chinch bug branched salivary sheaths on the outermost leaf
sheath of axillary shoots from southern chinch bug-resistant and -susceptible St.
Augustinegrasses. The values shown are mean + SE. Means followed by the same
letter are not significantly different at a = 0.05 level separated by Tukey's test (n =
14).












-- a
5a

b b


^ I 1 '








FX-10 Floratam NUF-76 Palmetto
St. Augustinegrass lines
Figure 5-5. Total lignin content of axillary shoot in southern chinch bug-resistant and -
susceptible St. Augustinegrasses. The values shown are mean SE of six replicates.
Means followed by the same letter are not significantly different at a = 0.05 level
separated by Tukey's test (n = 6).











A B C D

.... ., .. -, Xph

1, SC

Sbc




III! .1




















southern chinch bug-resistant and -susceptible St. Augustinegrasses (40X) (n = 3).
A. FX-10, B. Floratam, C. NUF-76, D. Palmetto. ph phloem, xy xylem cells, sc -
sclerenchyma cells, bc bundle sheath cells.






























I f' rN Jl. I 11
Figure 5-7. Transverse sections of the epidermal layer of the outermost leaf sheath in southern
chinch bug-resistant and -susceptible St. Augustinegrasses (400X) (n = 3). A. FX-10,
B. Floratam, C. NUF-76, D. Palmetto. ep epidermal layer, pc parenchyma cells.










S160
140
I 120
S100
m 80
j 60
40
I 20
20


FX-10 Flointam NUTF-li Palmetto


St. Augustinegrass lines
Figure 5-8. Thickness of epidermal layer of outermost leaf sheath in southern chinch bug-
resistant and -susceptible St. Augustinegrasses. Means (+ SE) followed by the same
letter are not significantly different at a = 0.05 level separated by Tukey's test (n = 3).











thick, highly lignified
secondary cell wall
of scleronchyma cell


L ~. .240T M .. L ZA:V
Figure 5-9. Transmission electron micrographs (7000X) of the sclerenchyma cells of the
outermost leaf sheath of St. Augustinegrasses resistant and susceptible to southern
chinch bug (n = 3). A. FX-10, B. Floratam, C. NUF-76, D. Palmetto.












100
171
-4
o o a
S80 b


60












St. Agustin6egr... lines
S40 ).


40




FX-10 Floratani NUF-76 Palnetto

St. Augustinegras8 lines
Figure 5-10. Percentage of cell wall area relative to total cell area of sclerenchyma of the
outermost leaf sheath of southern chinch bug-resistant and -susceptible St.
Augustinegrasses. Means ( SE) followed by the same letter are not significantly
different at a = 0.05 level separated by Tukey's test (n = 3).









CHAPTER 6
SUMMARY

St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, is the most commonly

grown lawn grass in Florida and other Gulf coast states.. It is estimated to cover more than one

and a half million acres in Florida and accounts for 85% of the sod industry in Florida, worth

$262 million annually. The southern chinch bug, Blissus insularis Barber (Hemiptera:

Blissidae), is the most serious insect pest of St. Augustinegrass. The nymphs and adult chinch

bugs feed on the inside of the leaf sheath; the infested plants turn yellow and eventually die.

Repeated use of insecticides led to development of insecticide resistance in southern chinch bug

to chlorinated hydrocarbons, organophosphates, carbamates, synthetic pyrethroids and

insecticides with novel modes of action, such as imidacloprid and lambda-cyhalothrin. Host

plant resistance has long been one of the successful management tools for control of the southern

chinch bug.

'Floratam', a polyploid St. Augustinegrass variety with initially high level of resistance to

southern chinch bug was released in 1974. However, 12 years after its release, a population of

southern chinch bug from Florida overcame Floratam's resistance. FX-10, another southern

chinch bug resistant polyploid variety was registered in 1993 with a high level of resistance to

southern chinch bug. However, it never became popular among growers because of its coarse

texture, slow ground coverage and susceptibility to weeds. In 2003, NUF-76, a diploid St.

Augustinegrass line was reported to have a high level of southern chinch bug resistance. Its

discovery was considered as a breakthrough in breeding for southern chinch bug resistance

because of its diploid genome, making the transfer of southern chinch bug resistance easier.

Understanding the mechanisms of southern chinch bug resistance in resistant St. Augustinegrass

is essential for maintaining host plant resistance and the development of future cultivars with









better resistance. The main objective of this research was to investigate mechanisms of southern

chinch bug resistance in FX-10 and NUF-76.

Choice and no-choice tests and ovipositional and developmental studies were conducted to

determine the categories of resistance in FX-10 and NUF-76 to the southern chinch bug. When

adult chinch bugs had a choice among attached stolons of three susceptible lines (Floratam, and

'Bitter Blue', a susceptible polyploid, and 'Palmetto', a susceptible diploid) and the two resistant

lines, chinch bugs were found significantly more often on the susceptible lines over a 5-d

exposure period. This result indicated the presence of antixenosis factors in the resistant lines

FX-10 and NUF-76. In a no-choice study, chinch bugs produced fewer excretory spots

(suggesting less ingestion) on FX-10 and NUF-76 than on the susceptible lines. The no-choice

study confirmed a high level of antixenosis in FX-10, a moderate level of antixenosis in NUF-76,

and possible antibiosis in NUF-76. Ovipositional and developmental studies conducted using

Floratam, FX-10 and NUF-76 demonstrated that the adults released on Floratam produced more

eggs and offspring than adults confined on FX-10 and NUF-76.

Higher mortality of southern chinch bugs has been reported on resistant FX-10 and NUF-

76 than on susceptible Floratam, Palmetto and Bitter Blue (Nagata and Cherry 2003); however,

the cause of mortality is still unknown. Understanding the mechanisms of chinch bug resistance

in these two cultivars is essential for future turfgrass breeding programs. Plant defenses against

herbivores are usually characterized as constitutive or induced. Constitutive defense is always

present in plants whereas induced defense occurs only in response to herbivore attack which

further reduces or deters the herbivory. Induced plant defense could be direct when they have a

direct negative effect on the herbivore or indirect when the natural enemies of the herbivores are

attracted. Increased activities of oxidative enzymes such as polyphenol oxidase, peroxidase, and









lipoxygenase are often correlated with induced plant defense to insects. FX-10 and NUF-76 had

significantly higher peroxidase activity 5 and 8 d after infestation and NUF-76 had significantly

higher polyphenol oxidase activity 5 and 8 d after infestation compared to control plants. FX-10

had significantly higher lipoxygenase activity 3, 5 and 8 d after infestation compared to control

plants. Catalase activities did not differ significantly between infested and control plants in any

of the lines tested. Native gels stained for peroxidase indicated that some isozymes in FX-10 and

NUF-76 were induced 5 and 8 d after infestation. Isozyme profiles of polyphenol oxidase and

lipoxygenase did not differ between control and infested FX-10, NUF-76 and Floratam. The

increased levels of peroxidase and lipoxygenase in FX-10 and peroxidase and polyphenol

oxidase in NUF-76 are associated with southern chinch bug resistance in these lines.

Southern chinch bugs are intracellular feeders. Induction of these oxidative enzymes could

involve a general plant response to mechanical damage caused by the chinch bug stylets and also

include hypersensitive response of cells around the site of stylet penetration and reactions

specific salivary proteins. Increased oxidative enzyme activities could cause a combination of

induced plant defense mechanisms such as enhanced plant toughness through lignification, poor

nutritional quality and toxicity. The increased toughness could potentially be caused by

increased peroxidase activities. Peroxidases and polyphenol oxidases are known to produce

protein binding quinones thus reducing the nutritional quality of the plants. Subsequent to the

generation of activated products due to the action of these oxidative enzymes, reactions may

occur that could potentially result in cross-linking or polymerization of materials toughening the

plant tissues, and nutrient complexes which are relatively unavailable and in cell death. The

reduced oviposition, development and survival of southern chinch bugs on FX-10 and NUF-76

could be due the poor nutritional quality caused by induced activity of these oxidative enzymes.









Lines of experimental evidence shows that plant responses to attack by hemipterans are similar

to that of plant pathogens (Kaloshian and Walling 2005). Fungal-rust resistant wheat plants

showed induction of lignin biosynthetic pathway in cells exhibiting hypersensitive reactions in

response to pathogen infection thus preventing the further hyphal development (Menden et al.

2007). St. Augustinegrasses resistant to the southern chinch bugs may also respond in the same

way and prevent further stylet penetration.

Understanding the feeding behavior of southern chinch bugs on the resistant FX-10 and

NUF-76 is important to elucidate the mechanisms of resistance. For the first time, the electrical

penetration graph (EPG) technique was used to quantify southern chinch bug feeding behavior

on resistant and susceptible St. Augustinegrass lines. Southern chinch bugs made more frequent

probes, produced longer-duration waveform events for pathway-related behaviors (searching for

an ingestion site) and spent less time in ingestion-related waveforms on FX-10 and NUF-76,

compared to the susceptible Floratam and Palmetto. Relatively more stylet probes per insect on

FX-10 and NUF-76 than on Floratam and Palmetto suggest the presence of stylet penetration

impediments around the vascular bundle in resistant varieties. In addition, the short duration of

presumed phloem sap ingestion on FX-10 and NUF-76 suggests the possible presence of

resistance factors in phloem sap or blockage of sieve elements. A significantly higher number of

salivary sheaths were observed in the outermost leaf sheath in FX-10 and NUF-76, which further

supports the possible presence of a physical barrier or hypersensitive reactions. Also, the

significantly greater number of branched salivary sheaths in FX-10 and NUF-76 corroborates our

EPG studies that the southern chinch bugs spent more time on pathway related activities (during

which the salivary sheath is constructed) on these two resistant lines.









Plant peroxidases play diverse roles in plants such as lignin biosynthesis, scavenging

hydrogen peroxides, and oxidation of reduced toxic compounds and 3-indoleacetic acid (IAA).

EPG studies showed that southern chinch bugs probe more frequently on FX-10 and NUF-76 but

their stylets took a longer time to reach the phloem sieve elements. Based on the above results, I

hypothesized that lignin content and/or leaf sheath anatomy may play a role in southern chinch

bug resistance in FX-10 and NUF-76. However, there was no significant difference between

FX-10 and Floratam or between NUF-76 and Palmetto in total lignin in the axillary shoot.

Therefore, total lignin content does not appear to influence southern chinch bug resistance;

however, subtle differences in lignin structure may be important. In general, lignins are made of

polymerized phenolic monolignols such as p-coumaryl-, conferyl- and sinapyl-alcohols which

are generated through the phenyl propanoid pathway (Barber and Mitchell 1997). The lignins in

graminaceous plants are characterized byp-hydroxy-phenyl, guaicyl, and syringyl units (Iiyama

et al. 1990). In rust-resistant wheat plants, total lignin content was increased in response to

pathogen infection and the increase was exclusively due to increase in syringyl units thus

changing the syringyl to guaiacyl ratio from 0.55 to 1.43(Menden et al. 2007). Previous studies

showed that the increase in syringyl-rich lignin or pure syringyl lignin content occurs in plants

exhibiting hypersensitive-resistance responses to plant pathogens (Mitchell et al. 1999). The

total lignin content of the axillary shoot in southern chinch bug resistant and susceptible St.

Augustinegrasses did not differ significantly. However, a change in the ratio of these lignin

monomers could have occurred in cells around the stylet pathway, thus preventing further stylet

penetration and leading to an increased number of stylet probes on southern chinch bug resistant

plants, as shown by both the EPG and salivary sheath distribution studies. There was no

significant difference in the thickness of the epidermal layer among any of the St.









Augustinegrasses studied. Transmission electron microscopic studies on axillary shoot anatomy

revealed that the cell walls of the highly lignified sclerenchyma cells in the leaf sheath

contributed a greater percentage of total cell area in NUF-76 and FX-10 than in Floratam and

Palmetto, suggesting that the sclerenchyma cells around the vascular bundle in FX-10 and NUF-

76 could serve as an impediment to the southern chinch bug stylet and prevent them from

reaching the phloem sieve elements. Thus, these results show for the first time that at least part

of southern chinch bug resistance mechanism is a physical impediment to stylet penetration into

the vascular bundles.

The presence of thicker sclerenchyma cells in FX-10 and NUF-76 appears to be the

constitutive plant defense mechanism against the southern chinch bug and the reason why the

southern chinch bugs probe more and change direction more frequently, producing branched

salivary sheaths, on these resistant lines. More probing could result in more cellular damage thus

leading to a release of plant defense signaling molecules such as salicylic and jasmonic acid.

The induction of oxidative enzymes in FX-10 and NUF-76 could be an induced plant defense

mechanism and could cause an adverse effect on southern chinch bug survival and development

on these resistant lines either by reduced phloem sap ingestion or reduced food absorption.

Apart from the excessive cellular damage, more probes also causes deposition of more salivary

proteins into the damaged plant tissues. The role of salivary proteins in southern chinch bug-St.

Augustinegrass interactions is not known. However, considering the diverse role of insect

salivary proteins in plants, it is possible that an excessive deposition of salivary proteins on the

resistant cultivars may activate the induced plant defense mechanism in FX-10 and NUF-76. In

susceptible plants, these salivary proteins could act as suppressors of plant defense.









This research suggests that the resistant mechanisms formerly found in Floratam were

different from any of the mechanisms described above in FX-10 and NUF-76. When Floratam

was resistant, the cellular damage and the presence of salivary proteins may have been detected

by plant receptors and resulted in the activation of induced plant defense. However, over time,

the southern chinch bugs could have evolved to make a change in the nature or content of

salivary protein which is not recognized by the receptors in the plant leading to susceptibility.

The susceptibility in Palmetto, a diploid, and Bitter Blue, a polyploid, may be due the absence of

receptors to perceive the southern chinch bug attack. It is possible that the resistant mechanisms

present in each St. Augustinegrass are unique because of the genetic dissimilarities between the

lines.

From this research, I conclude that FX-10 and NUF-76 have strong antixenosis to southern

chinch bug, while NUF-76 has antibiosis. The southern chinch bug resistance in FX-10 and

NUF-76 could be due to a combination of induction of oxidative enzymes, thicker sclerenchyma

cells around the vascular bundle, and possible presence of feeding deterrents or toxins in phloem

sap or sieve tube blocking mechanisms. Studies outlined here will serve as a solid foundation to

further test: (a) phloem sap properties for potential antifeedants or altered nutritional properties,

(b) plant metabolic responses including insect-induced polyphenoloxidase, peroxidase and

lipoxygenase, and (c) sieve tube blocking mechanisms in FX-10 and NUF-76. The results have

important implications for breeding St. Augustinegrass lines with improved resistance to

southern chinch bugs.









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BIOGRAPHICAL SKETCH

Murugesan Rangasamy was born in Erugalur, a village 20 miles south of Salem, India in

1975 and is the only child of his parents. He graduated from Mallasamudram higher secondary

school in 1992 and earned his Bachelor's degree in Agriculture in 1999 from the Tamil Nadu

Agricultural University, Coimbatore India. After graduating with the bachelor degree in

agriculture, Murugesan worked as a Marketing Development Trainee with EID Parry (India) Ltd.

Farm Input Division for eight months and involved in promoting an environmentally friendly

insecticide, Neemazal, among cotton growers. He was the recipient of a prestigious Junior

Research Fellowship awarded by Indian Council of Agricultural Research for doing graduate

studies in Entomology at the Punjab Agricultural University, Ludhiana, India, where he earned

his master's in 2001. Upon graduation with Master's, he worked as a Senior Research Fellow on

the World Bank sponsored National Agricultural Technology at the Indian Agricultural Research

Institute, New Delhi, India. His research project aimed to develop insect and disease resistant

cole crops such as cauliflower and cabbage. His undaunted zealous to work on insects,

especially on host plant resistance, landed him at the University of Florida with an opportunity to

do PhD in Entomology. Murugesan investigated mechanisms of southern chinch bug resistance

in St. Augustinegrasses. After earning his PhD in 2007, he is planning to continue his career in

research on insect plant interactions. Murugesan has always been a meritorious student

throughout his carrier and recipient of many scholarships and fellowships for his studies which

otherwise would have been extremely difficult.

Murugesan has been married to Shunmuga Priya Jeevaratchagan, an Accounting

Coordinator at the University of Florida Financial Services, for 212 years and the couple are

blessed with a baby girl, Sudhikssha Murugesan, who will be turning one year on February 20,

2008.





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1 MECHANISMS OF RESISTANCE TO SOUTHERN CHINCH BUG, Blissus insularis BARBER (HEMIPTERA: BLISSIDAE), IN ST. AUGUSTINEGRASS BY MURUGESAN RANGASAMY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Murugesan Rangasamy

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3 To my beloved mother, Thirumathi K. Kaliammal, and father, Thiru C. Rangasamy Gounder

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4 ACKNOWLEDGMENTS I pay m y sublime obeisance to my esteemed pa rents, wife Priya, and daughter Sudhikssha whose love, sacrifice and moral supp ort helped me to put this tireles s venture to a fruitful result. To express the feelings in my heart, I put on record my deepest gratitude and profoundest indebtedness to my advisors, Dr. Ron Cherry an d Dr. Heather McAuslane, for their guidance and making my dissertation worthy of presentation. Their constant at tention, patient helping, sense of dedication and search for perfection are underl ying factors which will be part of my memory forever. I consider myself fortunate to be thei r disciple. I am obliged to the members of my advisory committee, Dr. Bala Rathinasabapathi, Dr. Eric Schemlz, Dr. Russell Nagatta, and Dr. Elaine Backus for their support, guidance and va luable suggestions which led to a successful completion of my research and co mpilation of this dissertation. I give special thanks to Ms. Debbie B oyd Mr. Alwin Wilson and Ms. Shan Wu, our biological scientists for their t echnical assistance. I sincerely thank Dr. Marty Marshall for use of his spectrophotometer, Ms. Karen Kelly and Dr Sharron Mathews for helping with electron microscope, Dr. Louis Jack for his help with the EPG workshop. I duly acknowledge my lab mates, Karla Addesso, Amit Sethi, and Jenni fer Mayor, for their critical comments and suggestions on my research and presentations. No appropriate word could be traced in the lexicon to avouch my friends Bhuvana, Cha ndramohan, James, Karthik, Ravi, Sabari, Subbu, Sureshbabu and others who helped me both conc entrate and relax during my PhD program. All may not be mentioned but none is forgotten.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAP TER 1 LITERATURE REVIEW.......................................................................................................13 Taxonomy of the Chinch Bugs............................................................................................... 13 Host Plants and Feeding Preferences...................................................................................... 15 Feeding Habit and Nature of Damage....................................................................................16 Oviposition and Development................................................................................................ 17 Seasonal Wing Polymorphism and Movement....................................................................... 18 Management of Southern Chinch Bugs..................................................................................19 Cultural Methods.............................................................................................................20 Biological Control Agents...............................................................................................20 Chemical Control Methods..............................................................................................21 St. Augustinegrass and Host Plant Resi stance against South ern Chinch Bugs ..............23 Breeding Resistant Varie ties against Chinch Bugs Continuing Missions ......................26 Refining the Screening Techniques................................................................................. 29 Other Species of Chinch Bugs and Host Plant Resistance.............................................. 29 Characterization of Resistance against Chinch Bugs in Buffalograss ............................. 30 Chinch Bug Resistance in Sorghum ................................................................................31 Investigating the Mechanis m s of Southern Chinch Bug Resistance in St. Augustinegrasses.................................................................................................................31 2 CATEGORIES OF RESISTANCE IN ST. AUGUSTINEGRASS LINES TO SOUTHERN CHINCH BUG (HEM IPTERA: BLISSIDAE)................................................33 Introduction................................................................................................................... ..........33 Materials and Methods...........................................................................................................35 Plants and Insects............................................................................................................ 35 Choice Tests....................................................................................................................36 No-Choice Tests.............................................................................................................. 37 Ovipositional Studies.......................................................................................................38 Developmental Studies.................................................................................................... 38 Statistical Analysis.......................................................................................................... 39 Results.....................................................................................................................................39 Choice Tests....................................................................................................................39 No-Choice Tests.............................................................................................................. 40

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6 Ovipostional Studies........................................................................................................ 41 Developmental Studies.................................................................................................... 41 Discussion...............................................................................................................................42 3 OXIDATIVE RESPONSES OF ST. AUGUSTI NEGRASSES CHALLENGED BY SOUTHERN CHINCH BUG, Blissus Insularis BARBER (HEMIPTERA: BLISSIDAE)...........................................................................................................................47 Introduction................................................................................................................... ..........47 Materials and Methods...........................................................................................................48 Plants, Insects, Sample Collection................................................................................... 48 Sample Preparation..........................................................................................................49 Estimation of Total Protein............................................................................................. 50 Isozyme Profile Studies...................................................................................................51 Statistical Analysis.......................................................................................................... 52 Results.....................................................................................................................................53 Total Protein Content......................................................................................................53 Peroxidase Activities....................................................................................................... 53 Polyphenol Oxidase Activities........................................................................................ 53 Lipoxygenase Activities..................................................................................................54 Catalase Activities...........................................................................................................54 Isozyme Profile Studies...................................................................................................54 Discussion...............................................................................................................................55 4 QUANTITATIVE ANALYSIS OF FEEDING BEHAVIOR OF S OUTHERN CHINCH BUG, Blissus insularis BARBER (HEMIPTERA: BLISSIDAE), ON RESISTANT AND SUSCEPTIBLE ST. AUGUSTINEGRASSES.............................................................68 Introduction................................................................................................................... ..........68 Materials and Methods...........................................................................................................70 Plants......................................................................................................................... ......70 Insects..............................................................................................................................70 Wiring and Acclimating of the Insects............................................................................ 70 EPG Equipment and Data Acquisition............................................................................ 71 Waveform Categories...................................................................................................... 72 Definition of Non-Sequential Parameters....................................................................... 73 Statistical Analysis.......................................................................................................... 73 Results.....................................................................................................................................74 Cohort Level....................................................................................................................74 Event Level.................................................................................................................... ..75 Probe Level......................................................................................................................75 Insect Level................................................................................................................... ..76 Discussion...............................................................................................................................77

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7 5 ROLE OF LEAF SHEATH LIGNIFICATI ON AND ANAT OMY IN RESISTANCE AGAINST SOUTHERN CHINCH BUG, Blissus insularis BARBER (HEMIPTERA: BLISSIDAE) IN ST. AU GUSTINEGRASS LINES.............................................................. 87 Introduction................................................................................................................... ..........87 Materials and Methods...........................................................................................................89 Staining for Salivary Sheaths.......................................................................................... 90 Lignin Quantification......................................................................................................91 Staining for Lignin.......................................................................................................... 91 Transmission Electron Microscopy................................................................................. 92 Experimental Design and Statistical Analysis .................................................................92 Results.....................................................................................................................................93 Discussion...............................................................................................................................94 6 SUMMARY..........................................................................................................................108 LIST OF REFERENCES.............................................................................................................115 BIOGRAPHICAL SKETCH.......................................................................................................134

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8 LIST OF TABLES Table page 2-1 Distribution (mean proportion SEM) of adult southern chinch bugs am ong five St. Augustinegrass lines in a choice situation at varying time interval s after release *.......... 44 2-2 Mean number ( SEM) of excretory spots deposit ed per living chinch bug on St. Augustinegrass lines in a no-choice situation at varying tim e intervals after release....... 45 2-3 Mean number ( SEM) of eggs produced per fem ale alive on St. Augustinegrass lines at weekly intervals after release................................................................................ 46 4-1 Durations and frequencies of probing pa ram eters of EPG data recorded on four southern chinch bug resistant and susceptible St. Augustinegrass lines............................ 82 4-2 Total number of major waveform events (TNWE) on each of four southern chinch bug-resistant or -susceptible St. Au gustinegrass varieties (n = 22)................................... 82 4-3 Mean waveform durations per event (WDE) for major waveforms recorded for southern chinch bug feeding on resistan t and susceptible St. Augustinegrasses. .............. 83 4-4 Mean duration of major waveforms per probe (WDP) on ea ch of four southern chinch bug-resistant or -susceptible St. Augustinegrasses................................................ 83 4-5 Mean number of major waveform events per ins ect (NWEI) on each of four southern chinch bug-resistant or -susceptible St. Augustinegrass varieties..................................... 83

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9 LIST OF FIGURES Figure page 3-1 Total protein content in St. Augustinegra ss lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. .............................................................................................60 3-2 Levels of peroxidase (POX) specific activ ity levels in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. ................................................ 61 3-3 Levels of polyphenoloxidase (PPO) specific activity in S t. Augustinegrass lines 1, 3, 5, and 8 d after initation of southern chinch bug feeding.................................................. 62 3-4 Levels of lipoxygenase (LOX) specific ac tivity in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. ....................................................63 3-5 Levels of catalase (CAT) specific activity in St. Augus tinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. .................................................................64 3-6 Native gels stained for POX activity in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. .........................................................................65 3-7 Native gels stained for PPO activity in St. Augustinegrass lines 1, 3, 5, and 8 d after challenge by southern chinch bug. .....................................................................................66 3-8 Native gels stained for LOX activity in St Augustinegrass lines 1, 3, 5, and 8 d after challenge by southern chinch bug. .....................................................................................67 4-1 EPG waveforms produced by southern ch inch bug feeding on St. Augustinegrass, recorded using an AC/DC EPG monitor. ........................................................................... 84 4-2 Total percentage of tim e spent on to tal wave form duration (TWD) by southern chinch bugs on resistant and susceptible St Augustinegrasses in different waveform categories..................................................................................................................... ......85 4-3 Waveform duration per insect (WDI) for m ajor waveforms on resistant and susceptible St. Augustinegrasses....................................................................................... 86 5-1 St. Augustinegrass stolon A. with the axilla ry shoot (as); B. Part s of axillary shoot, ....... 98 5-2 Transverse section of outermost St. A ugustinegrass leaf sheath showing southern chinch bug salivary sheath staine d with McBrides stain (400X). .................................... 99 5-3 Number of southern chinch bug salivary sheaths found on the out erm ost leaf sheath of axillary shoots..............................................................................................................100

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10 5-4 Number of southern chinch bug branched salivary sheaths on the outerm ost leaf sheath of axillary shoots from southern chinch bug-resistant and -susceptible St. Augustinegrasses..............................................................................................................101 5-5 Total lignin content of axillary shoot in southern chinch bug-resistant and susceptible St. Augustinegrasses. .................................................................................... 102 5-6 Transverse sections of outermost leaf sheath vascular bundles stained for lignin in southern chinch bug-resistant and -s usceptible St. Augustinegrasses (400X) ................. 103 5-7 Transverse sections of the epidermal layer of the outerm ost leaf sheath in southern chinch bug-resistant and -susceptib le St. Augustinegrasses (400X)............................... 104 5-8 Thickness of epidermal layer of outermo st leaf sh eath in s outhern chinch bugresistant and -susceptible St. Augustinegrasses...............................................................105 5-9 Transmission electron micrographs (7000X ) of the sclerenchym a cells of the outermost leaf sheath of St. Augustinegrasse s resistant and suscep tible to southern chinch bug........................................................................................................................106 5-10 Percentage of cell wall area relative to total cell area of sclerenchym a of the outermost leaf sheath of southern ch inch bug-resistant and -susceptible St. Augustinegrasses..............................................................................................................107

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MECHANISMS OF RESISTANCE TO SOUTHERN CHINCH BUG, Blissus insularis BARBER (HEMIPTERA: BLISSIDA E) IN ST. AUGUSTINEGRASS By Murugesan Rangasamy May 2008 Chair: Ronald H. Cherry Cochair: Heather J. McAuslane Major: Entomology and Nematology St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, is the most commonly grown lawn grass in Florida. The southern chinch bug (SCB), Blissus insularis Barber (Hemiptera: Blissidae), is the most serious in sect pest of St. Augustinegrass. Host plant resistance has long been one of most successf ul management tools to control the SCB. Resistance to SCB has been identified in the St. Augustinegrass lines Floratam, FX-10 and NUF-76. Floratam was once resistant but is no w susceptible. This research investigated the mechanisms of SCB resistance in FX-10 and NUF-76. Choice and no-choice tests and ovipositional and developmental studies indetified strong antixe nosis in FX-10 and NUF-76 and antibiosis in NUF76. FX-10 and NUF-76 had significantly higher pero xidase activity and NUF-76 had significantly higher polyphenol oxida se activity 5 and 8 d after infestation compared to uninfested controls. FX-10 had si gnificantly higher lipoxygenase activity 3, 5 and 8 d after infestation compared to uninfested contro ls. Native gels stained for peroxidase indicated that some isozymes in FX-10 and NUF-76 were induced 5 and 8 d after infestation. Electrical penetration graph studies showed that the SCB made more frequent probes, spent longer time on pathway related activities on FX-10 and NUF-76, compared to the susceptible Floratam and

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12 Palmetto. Significantly higher number of salivar y sheaths was observed in the outermost leaf sheath in FX-10 and NUF-76. The total lignin content of the axil lary shoot and the thickness of the epidermal layer in SCB resistant and su sceptible St. Augustinegrasses did not differ significantly. TEM studies on the leaf sheath showed scelerenchyma cells with thicker cell walls in NUF-76 and FX-10 than in Floratam and Pa lmetto suggesting that the sclerenchyma cells around the vascular bundle in FX-10 and NUF-76 could serve as an impediment to the SCB stylet penetration. From this research, I conclu de that the strong antixenosis found in FX-10 and NUF-76 and antibiosis in NUF-76 could be due to a combination of induction of oxidative enzymes, thicker sclerenchyma cells around the vasc ular bundle and possible presence of feeding deterrents or toxins in phloem sap or sieve tube blocking mechanisms.

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13 CHAPTER 1 LITERATURE REVIEW Chinch bugs are in the fam ily Blissidae and ar e specialized to feed on plant sap. In the United States, there are four economically important species and subspecies, i.e. Blissus insularis Barber, B. leucopterous leucopterous (Say), B. leucopterous hirtus Montandon and B. occiduus Barber feeding on grasses and related crop plants such as corn, sorghum and wheat. In Florida, the southern chinch bug, B. insularis is a serious insect pe st of St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze. The taxonomy of the pest chinch bugs has been difficult and will be reviewed before concentrating on B. insularis, the subject of this dissertation. Taxonomy of the Chinch Bugs Blissus leucopterous was first described in the U nited States by Say in 1831 from a single specimen collected from the coast of Virginia. Although chinch bugs were collected from New York and Massachusetts, no economic damage wa s noticed (Fitch 1856). The first report of damage in the northeastern United States su rfaced when Linter ( 1883) noticed chinch bug damage in a meadow of timothy gra ss at Hammond, New York. The name B. leucopterous was commonly applied to all chinch bugs collected in the United Stat es until the early 1890s when Montandon (1893) described Blissus hirtus based on a single specimen collected from Hazelton, Pennsylvania. Blissus hirtus was certainly the form referred to as B leucopterous by Fitch and Linter. Although Montandon (1893) gave species status to hirtus Barber (1918) did not consider hirtus as a separate species. Based on geographical location, body form and wing polymorphism, Barber (1918) classified Blissus leucopterous into three varieties, i.e. 1. Blissus leucopterous var. hirtus Mont. which he stated was common to the highlands of United States

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14 and Canada, characterized by the presence of macropterous and brachypterous forms. 2. Blissus leucopterous var. arenarius Barber, found in Sandy Hook, New Jersey on a species of sand grass, characterized by being narrower and lo nger with only macropterous forms and, 3. Blissus leucopterous var insularis Barber commonly found in Punta Go rda, Florida, characterized by a shorter and narrower body form than the typical B. leucopterous. The three varieties mentioned by Barber were subsequently called subspecies, races or synonymies by many authors, which led to confusion among researchers a nd with Barber himself. Based on biological, morphological and cytological evidence Leonard (1966) considered B. leucopterous, insularis and arenarius as distinct species but retained hirtus at the subspecies level. He described arenarius and maritimus as subspecies of B. arenarius, and hirtus and leucopterous as subspecies of B. leucopterous. Barber (1918) also described B. occiduus from specimens collected from Ft. Collins, Colorado, and Germino, New Mexico. The reported distribution of B. occiduus includes California, Colorado, Montana, and New Mexico in the United States and Alberta, British Colombia, Manitoba and Saskatchewan in Cana da (Bird and Mitchner 1950, Slater 1964). Blissus occiduus is a relatively smaller chinch bug having conspicuous wi ng polymorphism and is an important insect pest of buffalograss, Buchloe dactyloides (Nuttall) Engelm ann (Baxendale et al. 1999). Blissus insularis Barber is referred to here by it s Entomological Society of America approved common name, southern chinch bug (B osik 1997). The ESA approved common names of the other economically important species of chinch bugs include hairy chinch bug, chinch bug and western chinch bug for B. leucopterous hirtus Montandon, B. leucopterous leucopterous (Say), and Blissus occiduus Barber, respectively (Bosik 1997).

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15 Host Plants and Feeding Preferences Newell and Berger (192 2) recorded the chin ch bug for the first time in Florida on St. Augustinegrass. They noticed the insect at th e base of the grass surrounding brown areas of lawns and determined it to be B. leucopterous, the same species that attacks cereals. Beyer (1924) considered B. leucopterous to be a serious pest in Florida because it was feeding on St. Augustinegrass, the grass grown generally as lawngr ass over the entire state. However, Howard (1888) and Webster (1907) did not consider St. Augustinegra ss as a host among the grasses damaged by the chinch bug. Wilson (1929) reported that chinch bug caused extensive damage to St. Augustinegrass in the southeastern United States. He observed that no other cultivated plants were infested where B leucopterous seriously damaged St. Augustinegrass. Hamilton (1935) reported the chinch bug B leucopterous as a pest of lawns and golf courses in the eastern United States. Waldon (1936) found B leucopterous hirtus damaging lawns in Connecticut. Maxwell and MacLeod (1936) also reported that the same species attacked 19 di fferent varieties of lawngrass. Kuitert and Nutter (1952) described for the firs t time the chinch bugs in St. Augustinegrass as B leucopterous insularis and Kerr (1966), in a short note, mentioned that insularis and leucopterous differed morphologically. Kerr (1966) also believed that the chinch bug damaging lawns in Florida could be elevated to species status because B. insularis had distinct morphological features and it was not f ound on corn and other cereals. Kelsheimer and Kerr (1957) found B leucopterous insularis severely damaging torpedo grass ( Panicum repens L.), occurring occasionally on centipede grass [ Eremochloa ophiuroides (Munto)], and rarely on Bermuda grass [ Cynodon dactylon (L.) Pers.]. Blissus leucopterous insularis was considered to be the second most expensive pl antfeeding arthropod in Florida (Kerr 1966). In Florida, St.

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16 Augustinegrass was the only grass to suffer seve re damage by chinch bugs although the bugs feed also on zoysia grass ( Zoysia spp.), and bahia grass ( Paspalum notatum Fluegg). However most of the damage on these other grass specie s occurred when they were grown near St. Augustinegrass with large bug populations (Kerr 1966). Cherry and Nagata (1997) studi ed the ovipositional preference of southern chinch bug with four cultivars of St. Augustinegrass (Bitter Blue Floratam, FX-10 and Seville) and four other lawn grasses (P. notatum, C. dactylon, E. ophiuroides and Z. japonica) They concluded that southern chinch bug resided in and ovipos ited on St. Augustinegrass cultivars more than on other lawngrasses. Three cult ivars of St. Augustinegrass, Bitt er Blue, Seville and Floratam, favored high adult survival rates and the lowest survival rates were recorded on P. .notatum, C. dactylon, and E. ophiuroides Feeding Habit and Nature of Damage Beyer (1924 ) found that chinch bugs pushed themse lves into the laterals of the close-fitting leaves of St. Augustinegrass and feeding caused a reddish stain on the damaged plant parts. Affected plants had reduced growth and becam e dwarf. Watson (1925) found that infested lawngrass turned brown in patches and then died out completely. He also reported that the plants around the brown patch area exhibited yellowing sy mptoms and were infested with chinch bugs sucking plant sap. Wilson (1929) designated the in jury as dieback because of the progressive nature of the damage. The gregarious feeding ha bit of the bugs caused th e plant to wither. The bugs abandoned the withered plan ts and moved into neighboring healthy plants. Lawns on dry sandy soils were found more vulnerable to insect at tack probably because of reduced vigor of the plants and rapid multiplication of bugs. Kerr (1966) found the bugs aggregated in scattered patches rather than distributed ev enly. Before moving into a new area, the bugs killed the plants

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17 in the infested area completely. Short et al. (199 7) reported that the injury first occurs in water stressed areas or in areas of full sunlight and infested lawns display circular discolored patches. Cherry (2001) reported that southern chinch bugs were extremely aggregated in yellow damaged St. Augustinegrass within areas of light and mode rate infestations whereas only a few southern chinch bugs were present in the grass 5 m or more away fr om an infestation. Oviposition and Development Beyer (1924) reported that the fe m ale chinch bugs laid their eggs near the roots of the grass. The eggs were pushed into protected places, often into crevices at grass nodes or almost out of sight between leaf blades where they come together at th eir base. Adults continued to oviposit for many consecutive weeks (Wilson 1929, Kerr 1966). Fecundity was estimated by Beyer (1924) to vary from 105 to 250 eggs or to average 250 eggs per female by Kerr (1966). Eggs are small and oval-shaped with a blunt end from which four small projections extend (St acy 2001). Eggs are first pale wh ite in color but turn amber and eventually red before hatching. The egg period va ries widely with estimates of 9 to 13 days (Wilson 1929), 7 to 11 days (Kuitert and Nutter 1952), 2 weeks (Eden and Self 1960), 11 to 33 days (Komblas 1962), and 1 weeks in summer but a month or more in winter (Kerr 1966). The first nymphal instar has yellow body color which changes to red with a faint band across the abdomen. The later instars are black with a narrow white band (Stacy 2001). Nymphs take an average of 28 to 30 days (Wilson 1929, Kuitert and Nutter 1952, Eden and Self 1960) to reach maturity. Wilson (1929 ) reported a pre-oviposit ion period of 5 to 13 days with the average of 8 days while Eden and Self (1960) repor ted 7 to 10 days. Males survive for an average of 42 days and the females for 70 days. The generation time has been estimated variously as 45 to 56 (W ilson 1929, Kuitert and Nutter 1952, Eden and Self

PAGE 18

18 1960). Beyer (1924) described six nymphal stages and two generations with an overlapping third generation during the summer in Florida while Kerr (1966) reported only five nymphal instars. Wilson (1929) found mixed populations of adults and all stages of nymphs in spring because of the extended period of oviposition. Th e adults mated in spring after a hibernation period and started laying eggs (Eden and Self 1960). Seasonal Wing Polymorphism and Movement Polym orphism in chinch bug wings was repor ted by Van Duzee (1886). He noticed that the shortwinged (brachypterous) form ordinarily predominated except under hot and dry summer conditions, when the longwinged (macropterous) form predominated. This led him to believe that the chinch was a tropical longwinged speci es. Webster (1907) reported that shortand longwinged forms were found intermixed along s eacoasts and northern sections of the United States. He insisted that this condition must have been brought about by disuse of wings for a period of time but not by temperature of the habitat. Wilson (1929) reported that both macropterous and brachypterous southern chinch bugs occur in Florida and the relative number of these two forms varied with season. Kombla s (1962) concluded that population density could be a reason for wing polymorphism because nymphs grown under crowded conditions were more likely to develop into macropterous adu lts than were uncrowded nymphs. Numbers of macropterous adults of southern chinch bug in south Florida were highe r in summer and fall, when population densities were also higher than they were in wi nter and spring seasons (Cherry 2001). However these data did not explain th e exact cause for macroptery. Cherry (2001) believed that various factors like heritability, population density and hos t plant condition might determine wing polymorphism based on the reports that macroptery in the oriental chinch bug, Cavelerius saccharivorus Okajima was density dependant (Denno and Peterson 2000) and was

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19 strongly influenced by seasonal factors like day length and temperat ure (Fujisaki 2000). Leonard (1966) found migration to be an influencing f actor in wing polymorphism. Reinert and Kerr (1973) noted that although both macropterous and brachypterous forms are found in the southern chinch bug, the latter was predominant. Ch erry and Wilson (2003) reported that the macropterous adults have reduced fecundity and egg laying rates compared to brachypterous females. Kerr (1966) reported th at the major difference between B. leucopterous in northern states and B. insularis in Florida was the absence of large spring flights of B. insularis. Although some of the longwinged forms took flight nothing equaled th e mass flight of B. leucopterous from overwintering quarters. Kerr (1966) noted that B. insularis moved from one lawn to another mainly by walking and streams of bugs c ould be seen moving out from heavily infested areas. The bugs were able to cover severa l hundred feet in a half-hour period of time. Management of Southern Chinch Bugs Southern chinch bugs can easily be m anaged if populations are detected early in a seasons population growth. Infestation can be detected easily by parting the gras s near yellowed areas and examining the base of the turf and the soil surface for chinch bugs. Heavily infested areas would have large number of bugs migrating acro ss sidewalks and driveways. Flotation and vacuum techniques can also be used to iden tify areas of infestation (Congdon and Buss 2002). There are preventive and curative methods availabl e to control southern chinch bug. Preventive methods such as cultural methods will avoid occu rrence of insect problems and curative methods will control the insects once their infestation is identified.

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20 Cultural Methods Cultural control is m anipulation of crop produc tion and management techniques to reduce or avoid pest damage to plants. Cultural cont rol manipulations are base d on habitat management and require a thorough understand ing of the ecosystem in which the pest thrives. Many insect pests of lawns live in thatch. That ch is the layer of dead plant material found between the green tops of the grass plant and so il. Proper mowing helps to prevent the thatch from building up. St. Augustinegrass lawns shoul d be mowed to a height of 3 to 4 inches (Congdon and Buss 2002). The lawns should be mo wed weekly during the growing season and not more than 35 to 45 percent of the leaf bl ade should be removed at the time of mowing. When the thatch is thicker than 1 cm, vertical mowing of the lawn could help. Good aeration combined with top-dressing could increase microbi al decay of dead grasses thus reducing the thickness of thatch (Congdon and Buss 2002). Southern chinch bugs prefer dry hot environm ents thus prolonged drought or water stress can encourage problems. Water stress and dry w eather increase the chances of survival for nymphs and adult chinch bugs by reducing the occurrence of insect diseases. On the other hand, excessive watering reduces microbial activity th us increasing the thickness of the thatch. Regular inspection for dry or water stressed areas during summer is necessary (Congdon and Buss 2002). Irrigating the lawn to a depth of 6 inches at a time is advised because frequent watering induces shallow rooting and in turn hi gher chinch bug damage (Merchant and Crocker 1998). Biological Control Agents Reinert (1978b) reported on natura l enem ies of the southern chinch bug in Florida. His list included a scelionid egg parasitoid, Eumicrosoma benefica Gahan, the only parasitoid found,

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21 lygaeid predators (big-eyed bugs) Geocoris uliginosus (Say) and G. bullatus Say, anabid, Pagasa pallipes Stal., anthocorids Xylocoris vicarius (Reuter) and Lasiochilus pallidulus Reuter, a dermapteran, Labidura riparia Pallas, and a reduvid, Sinea spp He has also found ant predators, primarily Solenopsis geminata (F.), a spider, Lycosa sp., and a fungus, Beauveria bassiana (Balsomo) Vuillemin. Reinert (1978b) concluded that the main biological agents capable of keeping the chinch bug population under control were E. benefica G. uliginosus X. vicarius, and L. riparia. Big-eyed bugs and anthocorids are si milar to chinch bugs in both size and shape. Eumicrosoma benefica is capable of parasitizing up to 50 percent of the chinch bug population (Hellman and Mathias 2002). The eff ectiveness of the predators mainly depends on the number and dosage of insecticidal sprays. A lthough various species of ants were reported to be predators of chinch bugs (Wilson 1929, Kerr 1966, Reinert 1978b), the red imported fire ant ( Solenopsis invicta Buren) was most often associated with southern chinch bug infestations in Florida (Cherry 2001). Beneficial nematodes were unable to give consistent results when tested against chinch bugs (Merch ant and Crocker 1998). Chemical Control Methods Although fertility and water m anagement, thatch control and use of resi stant varieties give considerable reduction in incide nce of chinch bugs, heavy damage accompanied by high insect populations necessitates the use of chemical control. It is essential to locate the problematic areas. If 20 to 25 chinch bugs pe r square foot area are detected, insecticide application is recommended (Congdon and Buss 2002). Spot tr eatment is enough in case of minimal infestation but treating the whole area is necessa ry if the infestation is heavy (Congdon and Buss 2002). It is also important to vi sit the treated areas to see whethe r the bugs are controlled or not. Repeated sprays may be required because the eggs are not killed by most of the available

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22 insecticides (Nagata and Cherry 2003). Pesticides available for professional use include acephate, chlorpyriphos, diazinon, ca rbaryl, bifenthrin, cyfluthri n, cypermethrin, deltamethrin, lambda-cyhalothrin, permethrin and imidacloprid. Repeated use of insecticides increases possi ble development of insecticide resistance (Reinert and Portier 1983). The first report of chinch bug resistance to chlordane came in 1953 when Wolfenbarger (1953) in Miami found that chlordane faile d to control the population of chinch bugs. Afterwards reports continued to surface on the resistance of chinch bugs against DDT in Sarasota (Kerr and Robinson 1958), and in Miami (Kerr 1958), and against parathion in Fort Lauderdale (Kerr 1960) as well as ot her organophosphates like chlorpyriphos and diazinon (Reinert and Niemczyk 1976). Reinert (1982) reviewed the resistance to other chlorinated hydrocarbons and organophosphates. In 1983, Reiner t and Portier investig ated the distribution and level of southern chinch bug resistance against organophos phates and carbamates. They found that the level of re sistance against chlorpyriphos was 3.4-fold to 3.19 108-fold along a 70 km coastal area of Broward and Palm Beach counties in Florida. A 9.25-fold level of resistance to propoxur, a carbamate insectic ide, was also demonstrated. Cherry and Nagata (2005) reported for the first time that the southern chinch bug had developed resistance to bifenthri n, a commonly used synthetic pyret hroid to control chinch bugs in Florida. In recent years, increased use of inse cticides further aggravated the problem. Cherry and Nagata (2007) collected southern chinch bugs from 10 different urban areas in Central and South Florida and tested their re sistance to different classes of insecticides. They found varying level of southern chinch bug resistance to deltamethrin, imid acloprid, and lambda-cyhalothrin and, for the first time also confirmed the resistance to bifenthrin. Cherry and Nagata (2007) also noted that the level of insectic ide resistance varies depending on the location. Most populations

PAGE 23

23 in Florida are still susceptible to these insectic ides, however, the situati on is alarming and further underlines the importance of improvi ng natural host plant resistance. St. Augustinegrass and Host Plant Re sistance against Southern Chinch Bugs St. Augustinegrass is grown extens ively in southern coastal states and California (Crocker et al 1982). The worlds first record of planting of St. Augustinegrass was on 11th November 1880, as turf bordering an avenue at A.M Reeds Mulberry Grove plantation, at Yukon, Orange Park, Florida (Works Progress Administration 1939). After the Second World War, the market for St. Augustinegrass grass increased dramatically and during the late 1960s variet ies like Floratine, F lorida Common and Roselawn were commercially available for lawn s. McGregor (1976) estimated that the area under turfgrass in Florida was more than 3, 68,700 ha, of which St. Augustinegrass accounted for more than 46 percent of the home lawns. Cart er and Duble (1976) estima ted that 56 percent of the lawns in Texas were in St. Augustinegrass but 96 percent in the Gulf coastal areas. The area in St. Augustinegrass sod production was 3,100 hectares in 1974 (Florida Department of Agriculture and Consumer Services 1976) and 13,400 hectares in 1991 (Hodges et al. 1994). St. Augustinegrass is estimated to cover more than ha lf a million hectares in Florida (Hodges et al. 1994) and accounts for 85% of the sod industry in Florida with a value of $262 million annually (Haydu 2005). The leaves of St. Augustinegrass have round-tipp ed leaf blades of 5 to 14 mm width that are distichously arranged (Busey 2003). St. Augus tinegrass is the most versatile of the warm season grasses and is able to survive well in acid or alkaline soils, soils with low organic matter, and soils with poor or good draina ge. It is salt tolerant and has few disease and nematode

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24 problems requiring little maintenanc e. It is also able to pe rform well in full sunlight and moderate shade (Crocker 1993). St. Augustinegrass expanded rapidly into warm coastal regions as a lawngrass mainly because of human migration. However a new mosa ic virus called St. Augustine Mosaic Disease (SAD), which was first reported in 1966, in Texas, challenged the popularity of St. Augustinegrass. The disease was caused by the St Augustine decline strain of Panicum Mosaic Virus (SAD-PMV) (Lee and Toler 1973, Niblett et al. 1977) and the viru s spread throughout the St. Augustinegrass growing regions of Texas (Tol er and Walla 1976) and Louisiana (Holcomb et al 1972). Horn et al (1973) estimated a $100 million loss due to the disease and in the same year, almost 90 percent of the St. Augustinegrass in Corpus Christi, Texas was affected. St. Augustinegrass researchers from Florida and Texas realized that the virus could not be controlled therapeutically because of the abilit y of the virus to spread through vegetative propagation and mechanically through lawn mo wers. Although the virus was not found in Florida, imminent spread from the neighbori ng states was anticipate d. Accessions of St. Augustinegrass were screened for virus resistan ce under greenhouse conditions and the Floratam variety was found resistant to the virus in 1968 (Horn et al 1973). Floratam displayed a high level of resistance under greenhouse conditions and for 24 consecutive months under field conditions as well. Reinert and Kerr (1973) reported th at southern chinch bugs were able to damage seriously or kill whole lawns and were cons idered to be the most serious in sect pest of St. Augustinegrass. Kerr (1966) rated the southern chinch bug as one of the most economically important arthropod in Florida second only to citrus mite, in terms of the cost of control measure (Strobel 1971). Because of the destructive nature of this pest, ex periments were started to find resistant varieties

PAGE 25

25 of St. Augustinegrass. In 1972, Reinert reported that nine accessions of St. Augustinegrass had seasonal mean populations of 1.6 or fewer bugs per 0.1 m2. In 1974, Reinert and Dudeck identified three St. Augustinegra ss accessions resistant to southe rn chinch bug. They screened 11 promising accessions along with the available co mmercial cultivars using confined tests and reported that accessions FA-108, Floratam and FA118 were highly resistant. The resistance was characterized as antibiosis because adults and nymphs suffered 50 percent mortality and surviving adults had reduced fec undity. One of these three accessions, Floratam was released jointly by the University of Florida and Te xas A&M University (Horn et al. 1973). The resistance to SAD virus and chinch bugs, superi or color, ability to cover the ground fast and perform well under the conditions of Florida and Texas made Florat am more prefer red than all other commercial varieties. Because of the coarse-textured leaves of Flor atam, the researchers continued their search for fine-textured St. Augustinegrass lines (Reine rt 1978a) and also for lines having combined resistance to SAD and southern chinch bug (Bruto n et al. 1979, Reinert et al. 1980, Crocker et al. 1982). Despite a decade of research, the only lin e advanced and released was Floralawn (FA108) which is fast-growing with fine-textured leaves and combined resistance to SAD and southern chinch bug (Dudeck et al. 1986). Breakdown of Floratam Resistance After its release in 1973, Floratam quickly expanded in commercial sod production and accounted for 77 percent of St. Augustinegrass sod production in Florida (Busey 1986). Many investigations since the germpl asm release confirmed its resist ance to the southern chinch bug (Reinert et al 1980, Crocker et al 1982, Reinert et al 1986) and because of its vegetative reproduction and poor seed production, genetic breakdow n of the resistance was considered to be unlikely to occur (Busey 1979). Neverthele ss, by 1987, Busey and Center discovered a

PAGE 26

26 widespread population of chinch bugs that ha d overcome the resistance of Floratam and Floralawn St. Augustinegrasses. Because of the association of polyploidy and host resistance in St. Augustinegrass and the ability of the chinch bugs to overcome the host plant resistance, the population that overcame Floratam resistan ce was dubbed the polyploid damaging population (PDP). Busey and Center (1987) tested a local population of standard southern chinch bug and the PDP population with these resistant varietie s and found that the PDP adults had higher longevity and oviposition rate than the standard adults when conf ined on Floratam and Floralawn varieties. They concluded that the phenomenon obs erved was due to variability in the insect and not the grass. Busey and Coy (1988) conducte d a survey among sod producers and county agents in Florida and found that Floratam-killin g southern chinch bugs were widespread. The county agents considered the southern chinch bug as the most serious insect pest of St. Augustinegrass while the sod producer s thought that caterpillars were more serious than chinch bugs. Busey and Coy (1988) concluded that chinch bug continued to be the most limiting factor in St. Augustinegrass-established areas, because the southern chinch bug was damaging in most counties. Nagata and Cherry (2003) collected southern chinch bug populations from nine cities or their suburbs in Florida and found all populati ons capable of overcoming the resistance in Floratam. Breeding Resistant Varieties against Chinch Bugs, Continuing Missions Crocker et al. (1989) realized the necessity of continuing the effort to develop m ore resistant varieties of St. Augustinegrass in Texas af ter seeing the reports of the ability of the PDP of southern chinch bug to attack Floratam in Fl orida (Busey and Center 1987). Crocker et al. (1989) tested varieties and accessions for resistan ce against Texas strains of southern chinch bug and SAD-PMV. The results showed that Flor atam still was strongly antibiotic to southern

PAGE 27

27 chinch bug. Thus it was concluded that the br eakdown of Floratam antib iosis in Florida was absent or not widespread in Texas. They conclu ded that the populations of southern chinch bugs from Texas and Florida might not respond uniformly to future varieties. The susceptibility of Floratam was a major con cern to the turfgrass industry in Florida at the time it was reported (Busey and Center 1987 ) but there was no information on how the PDP adapted to Floratam antibiosis. Busey (1990a) f ound that host adaptability in PDP was heritable and retained in subsequent generations irrespecti ve of the previous host. The populations from various regions differing in ability to attack hos ts were one potentially interbreeding population and capable of exchanging a particular genetic trait. The other important discovery was that oviposition rates of PDP on Floratam were much greater than previously reported (Busey and Center 1987) suggesting an improvement in host adaptation when the colony was maintained on the resistant host. Vegetative propagation (gen etically homogenous in subsequent generations) in St. Augustinegrass offered a steady selection pressure for southern chinch bug adaptation. Also the long distance transport of sod incr eased the gene exchange between the PDP and standard populations thus host adaptability was expected to occur (Busey 1990a). Busey (1990b) continued his quest for resistan t varieties against the PDP of southern chinch bug and reported that PDP chinch bugs grown on FX-2, FX-10, and FX-33 exhibited shorter adult longevity and fewer cumulative lifetime eggs than in sects grown on Floratam. Both laboratory and field PDP exhibited high mortality on FX-33. In addi tion to confined tests, Busey (1990b) also used the production of excrement as an indicator of f eeding activity. As a result of this research, a southern chinch bug resistant and coarse-textured breeding line, FX-10, the only recognizable St. Augustinegrass cultivar with pubescence was registered (Busey 1993). This variety performed well under seasonal drought, was notably resistant to PDP and standard

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28 populations of southern chinch bug, and had moderate resistance to Pyricularia leaf spot. Because of the course texture and other negativ e characteristics such as slow ground coverage, susceptibility to weeds, FX-10 was never extensively grown in Florida (Nagata and Cherry 2003). The other problem with FX-10 was difficulty in transferring its re sistance to southern chinch bug into other desirable lines because of n on-viable seeds. All commercially available St. Augustinegrass cultivars with resistance to southe rn chinch bugs like Floratam, Floralawn and FX-10 are polyploids and have coarse appearance. Although diploid (2n=18) lines from St. Augustinegrass produce viable seeds and have fi ne-textured leaves they have never been accepted widely because of their poor resistance against chinch bugs (Nagata and Cherry 2003). Nagata and Cherry (2003) made a breakthrough in their quest for the southern chinch bug resistance when they reported that NUF-76, a diploid St. Augustinegrass selection, and NUF-216 both had resistance against southern chinch bug. Of the fourteen lines inspected for resistance, the mortality of the bugs was significantly higher on NUF-216 and NUF-76 lines than on any other line. Although the mortality on NUF-216 a nd NUF-76 was not significantly different from FX-10, the mortality on these thr ee lines was higher than that on any other line. Interestingly, high variation in mortality between different lo cations was observed on so me St. Augustinegrass lines and this variation was attri buted to the irregular response of the chinch bugs within Florida to both host plant resistance as reported by Busey and Center (1987) and insecticide resistance reported by Reinert and Portier ( 1983). After finding a diploid St Augustinegrass resistant to southern chinch bug, Nagata and Cherry (2003) expect that the transf er of the desirable characters with chinch bug resistance into ot her lines will be easie r in the near future.

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29 Refining the Screening Techniques Chinch bug resistance in varieties like Fl oratam and Floralawn was termed antibiosis because these cultivars produced higher mortality in confined tests. However, the exact mechanism of the resistance could not be explai ned by the confined tests. Busey and Center (1987) complained that the mortality data on the susceptible Florida Common were inconsistent when the experiment s were conducted for different periods of time on the same host. The crowding of chinch bugs released on sprigs of experimental grass and the variation among the field and laboratory populations of the chinch were the reasonable caus es discussed. Busey (1990b) used visual rating of the excreta as a cr iterion for screening the germplasm. However, realizing that mere visual examination of excr eta could not be an accura te selection criterion, Busey and Zaenker (1992) postulated they could we igh the excreta. Because the excreta adhered to surfaces like sidewalls of th e glass culture tubes, antistatic film and aluminum foil (Busey 1990b), it was determined that the collection and weighing of the excreta was possible. The antistatic film was rolled inside and toward the top of each culture tube and the tubes were inclined. They observed that more than 99 per cent of the excreta was deposited on the film and not on the glass or the grass. The films were re moved and dried at 24C for several days before weighing. They reported that the excreta weight was highly correlated w ith oviposition rate and crowding of up to 20 bugs per sprig di d not influence excreta production. Other Species of Chinch Bugs and Host Plant Resistance Host plant resistance to contro l chinch bugs has been explor ed in other crops such as sorghum and in other turfgrasses such as buffalogr ass. Buffalograss is a perennial warm season grass species native to the American Great Pl ains (Wenger 1943, Beard 1973). It is a fine textured low growing turfgrass, which spread s vegetatively by st olons (Nuland et al 1981,

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30 Falkenberg-Borland and Butler 1982, Riordan et al 1996). In recent years, buffalograss earned popularity as an alternative turfgrass because it is drought tolerant and has few pest and disease problems (Heng-Moss et al. 2002). One of the reported insect pests of buffalograss is B. occiduus whose hosts include barley, Hordeum sp ., corn, Zea mays L., oats, Avena sativa L., sugarcane, Saccharum officinarum L., wheat, Triticum aestivum L., bromegrass, Bromus spp., and various native grasses includi ng buffalograss (Ferris 1920, Parker 1920, Farstad et al. 1951). Currently very few control measures are availabl e for chinch bugs and other insect pests in buffalograss (Baxendale et al. 1999). Extensive differences ex ist among buffalograss selections in their resistance to mealy-bugs, Tridiscus sporboli and Trionymus sp., suggesting the possibility of also breeding chin ch bug resistance in buffalogra ss (Johnson-Cicalese et al. 1998). Based on greenhouse and field evaluations of el even lines, NE91-118, Tatanka, Bonnie Brae and Cody had high to m oderate resistance to B. occiduus whereas NE84-45-3 and 378 were highly susceptible (Heng-Moss et al. 2002). Characterization of Resistance again st Chinch Bugs in Buffalograss The basis of resistance in the four chinch bug resistant buffalograsses was categorized (Heng-Moss et al. 2002). Am ong the cultivars investigate d, they found NE91-118, Cody and Tatanka exhibiting m oderate-to-high levels of tolerance and B onnie Brae exhibiting moderate-to-low levels of tolerance to B. occiduus based on insect damaging ratings and functional plant loss indices. They suggested stu dying parameters such as height of regrowth, dry weight and yield clippings, root length, and tillerings that all represent plant aesthetics, an important aspect in assessing th e tolerance of turfgrass to ch inch bugs. The antibiosis tests showed no differences among the resistant and su sceptible cultivars of buffalograss thus ruling out the possibility of any influence on the growth and development of B. occiduus by the

PAGE 31

31 resistant cultivars. This is inconsistent with earlier discoveries reporti ng that resistance against southern chinch bug was antibiosis (Reinert and Dudeck 1974, Reinert et al. 1980). However, cultivars like NE91-118 and 609 were not preferred by chinch bugs in choice tests suggesting the presence of antixenosis. These results supported the earlier discovery of an tixenosis resistance in NE-609 to the mealybugs T. sporoboli and Trionymus sp. SEM studies showed differences in trichomes densities among resistant and susceptible li nes. Leaf blades of the resistant lines like NE91-118, NE-120, 378 and 609 had few trichomes and were characterized as glabrous (Johnson-Cicalese et al. 1998). Chinch Bug Resistance in Sorghum Chinch bugs that damage sorghum and small grains were recorded as B. leucopterous leucopterous (Webster 1909). The concept of host plant resistance as an important tool for managing chinch bugs in sorghum plants became evident when Dahms (1948) reported the milos sorghum were very suscep tible to chinch bugs, the feter itas were susceptible and the kafirs and sorgos were resistant. Duri ng the early 1980s resistance to chinch bugs in sorghum lines and hybrids under field conditions was reported. However, Meehan and Wilde (1989) realized the importance of screeni ng the sorghum lines under greenhouse or growth chamber conditions during the periods of low, natural field infestations. Under greenhouse conditions the commercial sorghum hybrids and lines were subjected to nym phs and adults at the seedling stage. Plant mortality for hybrids or lines was inconsistent between choice and nochoice tests. However, the days from infestatio n to death of plants in studies done in growth chambers were in agreement with the results from earlier field tests. Investigating the Mechanisms of Southern Chin ch Bug Resistance in St. Augustinegrasses Thus far, studies on the m echanisms of resist ance in St. Augustinegrass to southern chinch bug have been confined to those done on Florat am. Reinert and Dudeck (1974) and Reinert

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32 (1978) reported that Floratam and 10 other St. Augustinegrass accessions exhibited antibiosis to southern chinch bug based on adult and nymphal mort alities. Reinert et al. (1980) and Crocker et al. (1989) used the term antib iosis to describe the resistance to southern chinch bug in St. Augustinegrass lines. Unfortunately, none of th e above-mentioned reports explained the exact cause of the nymphal and adult mortality. Bu sey and Zaenker (1992) quantified the feeding response of southern chinch bug on St. Augustin egrass as weight of excreta produced and frequency of confined bugs on the grass as oppos ed to off the grass. They found significant differences among hosts in excreta weight within 24 h after releasing th e insects and a strong correlation between excreta weight and ovipos ition rate. Although they proposed a new resistance bioassay technique they did not determ ine the exact resistance mechanism operating in St. Augustinegrass against southern chinch bug. The goal of this study is to determine more precisely the mechanisms of southern chinch bug in both polyploid (FX-10) and diploid (NUF-76) St. Augustinegra ss lines. This goal will be achieved by the following objectives: Objective 1. To determine the modality of resist ance to southern chinch bug in St. Augustinegrass. Objective 2. To compare oxidative responses of southern chinch bug resistant and susceptible St.Augustinegrass lines to B. insularis infestation. Objective 3. To study precise feeding behavior of s outhern chinch bugs on susceptible and resistant St. Augustinegrass lin es using electrical penetr ation graph technique (EPG) Objective 4. To determine the role of lignificatio n and leaf sheath anatomy in southern chinch bug resistance in St. Augustinegrass.

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33 CHAPTER 2 CATEGORIES OF RESISTANCE IN ST. AUGUSTINEGRASS LINES TO SOUTHERN CHINCH BUG (HEMIPTERA: BLISSIDAE) Introduction St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze is the m ost widely used lawn grass in tropical and sub-trop ical climatic regions (Sauer 1972) It is the primary turfgrass in Florida and is grown on approximately 70% of all lawns (Busey 2003). The southern chinch bug, Blissus insularis Barber is the most serious insect pest of St. Augustinegrass (Crocker 1993). Southern chinch bugs suck the sap from th e grass until it withers a nd dies, turning from a healthy green color to brown-yello w. Infestation typically occurs first in water stressed areas and along edges of the lawn or areas in which gr ass is growing in full sunlight (Short et al. 1995). The annual cost of southern chinch bug management and losses in Florida alone was estimated at $5 million in 1983 (Hamer 1985) and is undoubtedly much more today. The economic importance of this insect has increased as it has developed resistance against several insecticides (Reinert and Portie r 1983, Cherry and Nagata 2005). Floratam, a polyploid variety of St. Augustin egrass with resistance to southern chinch bug, has been available in Florida and Gulf Co ast areas since its release in 1973 (Horn et al 1973). Reinert and Dudeck (1974) classified th e resistance to southern chinch bug found in Floratam as antibiosis because insects on Floratam exhibited more than 50 percent adult and nymphal mortalities and survivi ng insects had reduced fecundity compared to insects reared on susceptible varieties of St. Augustinegrass. However, Buse y and Center (1987) reported instances of southern chinch bugs overcoming F loratam resistance in Florida, beginning in 1985. They named this resistance-breaking population of the southern chinch bug, the polyploid-damaging population. Nagata and Cherry (2003) co llected southern chinch bug populations from nine cities or th eir suburbs in Florida and dete rmined the dist ribution of the

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34 polyploid-damaging population to be widespread. This problem seems to be confined to Florida however because, in 1989, Crocker et al. (1989) reported that there was no evidence of the polyploid-damaging population of southern chinch bug in Texas. The lessened resistance in Floratam prompted plant breeders to continue their search for resistance to the southern chinch bug. FX-10, a polyploid St. Augustinegra ss, was registered in 1993 (Busey 1993) with resistance against the so uthern chinch bug, but because of its very coarse texture, toughness and poor ground coverage, it was never extensively grown (Nagata and Cherry 2003). Nagata and Cherry (2003) reported no significant differenc e in southern chinch bug mortality among FX-10, NUF-216 and NUF-76. The latter two lines are products of the University of Floridas turf-breeding program. Mortality on all three lines was significantly higher than that on Floratam and Floratine. Th is research was significant because Nagata and Cherry (2003) identified, for the first time, a diploid St. Augustineg rass line, NUF-76, with resistance to the southern chinch bug; all previo us resistant lines were polyploids. Diploid lines produce viable seeds allowing recombination of ch aracters and selection of new lines. Diploid lines also have narrow leaf blades, which give s them a much finer and more aesthetically pleasing texture. As of yet, it is not known by which mechanism resistance to southern chinch bug operates in these resistant lines. Understanding the mechanisms of resistance in St. Augustinegrass to southern chinch bug is crucial. This knowledge may allow turf breeders to design appropriate screening assays, which would expedite their development of insect resistant varieties. The objective of this research was to characterize the mechanisms of southern chinch bug resi stance in the polyploid (FX-10) and diploid (NUF-76) St Augustinegrass lines. To m eet this objective we conducted choice and no-choice tests to examine adult chin ch bug preference for and survival on resistant

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35 and susceptible St. Augustinegrass lines. We al so quantified reproduction of adult chinch bugs and studied development and surv ival of immature chinch bugs on suscep tible and resistance lines. Materials and Methods Plants and Insects St. Augustinegrass lines selected for the expe riments included NUF-76 (resistant diploid), FX-10 (resistant polyploid), F lo ratam (once resistant polyploid), Bitter Blue (susceptible polyploid) and Palmetto (susceptible diploid) Plant material was supplied by R. Nagata (Everglades Research and Educati on Center, Belle Glade, FL). R ooted sprigs from single node cuttings were grown in plastic pots (12.5 cm diameter .5 cm depth) containing a potting medium of 1:1 (v/v) sand and Fafard mix #2 (C onrad Fafard, Agawam, MA). Test plants were 6 to 8 weeks old. Plants were fertilized with Os mocote Plus 15-9-12 (Scotts-Sierra Horticultural Products Company, Marysville, OH) at the ra te of 5g/kg of growing medium. Stolons approximately 30 to 40 cm in length with a mi nimum of three nodes were used for testing. Nagata and Cherry (2003) reported that mo st populations of southern chinch bugs in Florida had overcome Floratam resistance. Th us for all experiments, southern chinch bugs were collected from Royal Palm Beach, Palm Beach Co., FL. Preliminary trials were conducted to determine their ability to develop on Florata m. The insects were collected by vacuuming St. Augustinegrass lawns as described by Cherry (2001) and then sorting through debris in the laboratory for fifth instars and adults (depe nding on the needs of the experiment). After collection, insects were provided w ith fresh Floratam stolons as needed and maintained at 18C in 20-liter plastic buckets.

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36 Choice Tests Choice tests were conducted to study the behavior of southern chinch bugs on resistant and susceptible lines of St. Augustinegrass. Arenas were constructed from circular polypropylene food storage containers meas uring 33.6 (top diameter) 33.0 (bottom diameter) .6 cm (height) (Rubbermaid 3907, Wooster, OH) that we re modified in the following manner. Five holes of 6-mm diameter, equally spaced around th e perimeter of the arena and 3 cm below the open edge were drilled. A channel was then cut from the open edge of the container to the hole drilled in the side. The majority of the lid surface area was removed, leaving approximately 3 cm around the sealing edge. Anti-virus inse ct screen (hole size of 0.0105 0.0322 inches, Green-Tek, Edgeton, WI) covered the opening in the lid to facilitate aeration. A moist paper towel was placed on the bottom of the arena to prevent desiccation of insects. Plant stolons, still attached to potted pl ants, were passed through the channels to rest in the holes on the side of the arena. The portion of the stolons in contact with the plastic arena was wrapped with parafilm strips (American Nationa l Can, Greenwich, CT) so that the plant would not be damaged. After positioning the stolons, the channels were sealed with clear cellophane tape to prevent southern chinch bugs from escap ing. Plants were watered as needed. NUF-76, FX-10, Floratam, Bitter Blue and Palme tto were evaluated in this choice test. Twenty chinch bug adults (10 males and 10 females) of undetermined age were released at the center of the arena and the lid was replaced. The location of the insect s was recorded as on one of the five lines or off all plants at 24 h, 48 h, 72 h and 120 h after release. The experimental design was a completely randomized block design. The experiment was conducted in a laboratory room that was maintained at 30 1C and 14:10 L:D h photoperiod and was replicated 15 times.

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37 No-Choice Tests No-choice tests were conducted to investigate the possi bility of antibiosis in resistant St. Augustinegrass lin es. One stolon of each line (NUF-76, FX-10, Floratam, Bitter Blue and Palmetto), still attached to the plant, was in troduced into separate rectangular arenas. The arena was made of a food storage containe r (Rectangle 1.8 qt, Rubbe rmaid #7A23, Wooster, OH) with dimensions of 27 cm 15 cm 5.7 cm. One hole was drilled on the 15-cm side and a channel was cut as described in the choice-test section so that a stolon could be passed unharmed to the interior of the box. The majority of the lid surface area was removed leaving approximately 3 cm around the sealing edge. Anti-virus insect screen was glued over the opening to facilitate aeration. The insects were starved for 12 h before the start of the experiment A moist paper towel was placed on the bottom of the arena to prevent desiccation of insects. A sheet of heavy-duty aluminum fo il (Reynolds Kitchens, Richmond, VA) was cut to the size of the arena and placed on the moist pape r towel. Twenty chinch bug adults (10 males and 10 females) were released inside the arena. The exact locations of feeding of the adults such as leaf sheaths, internodes or nodal regions, were observed 24 h, 48 h, 72 h and 120 h after release. The aluminum foil sheet was repla ced after each observation and the number of excretory spots was recorded with the naked eye. Use of aluminum foil sheets to collect the southern chinch bug excreta was originally reported by Busey and Zaenker (1992). The experimental design was a randomized complete block design with 15 replicates. The experiment was conducted in a laborator y room that was maintained at 30 1C and 14:10 L:D h photoperiod. Plants were watered as needed.

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38 Ovipositional Studies Studies were conducted to investigate the eff ect of St. Augus tinegrass lines on fecundity of southern chinch bugs. Rectangular arenas used in no-choice tests were used for this experiment and St. Augustinegrass lines used included FX-10, NUF-76 and Flora tam. In order to obtain adults of similar age, fifth instar nymphs we re selected from the field collection and then provided with fresh Floratam runners. Insects th at molted to the adult stage within a 5-d period were selected for the experiment. Five pairs of a dults were released into the arena. A week after release, surviving chinch bugs were transferred gently using a mouth aspirator from one arena to a new arena with an undamaged stolon from the same individual plant. Adult mortality was recorded and the stolons were dissected to coun t the number of eggs. This was continued for a total of 4 weeks. This experiment was conducte d at the Envirotron, IFAS, University of Florida that was maintained at 30 1C. Plants were watered as ne eded. The experimental design was a randomized complete block design with 13 replications. Developmental Studies This experim ent was conducted to study th e influence of St. Augustinegrass lines on growth and development of southern chinch bug nymphs. The experimental set up was as explained in the no-choice test except the arena size was 28 16 11 cm (l w h) (Rubbermaid #3901, Wooster, OH). Five pairs of adults (0-5 d old) were released into the arena. After 32 d boxes were opened and stolons were di ssected to count the number of nymphs and adult chinch bugs surviving. Th is experiment was conducted at the Envirotron, IFAS, University of Florida that was maintained at 30 1C. Plants were watered as needed. The experimental design was completely randomized bl ock design with 11 replications.

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39 Statistical Analysis The distribution of chinch bugs in choice test s among the five lines taken over a period of 5 d was analyzed as a repeated m easures de sign using mixed model analyses (PROC MIXED, SAS Institute 1999) after data were transformed by arcsine x. In no-choice tests, the numbers of excretory spots counted over a period of 5 d were analyzed as a repeated measures design using mixed model analyses (PROC MIXED). In ovipositional studies, th e number of eggs laid per female was transformed by (x + 0.375) to correct for nonhomogenity of variances and data were analyzed using a repeated measures analysis with a mixed model (PROC MIXED). For the developmental studies, the number of nymphs pr oduced and adults surviving after 32 d was analyzed using PROC GLM (SAS Institute 1999 ). When significant F-tests were obtained, means were compared using Tukeys te st with a significance level of P < 0.05. Results Choice Tests. More chinch bugs were observed on the three su sceptible lines than th e tw o resistant lines at three out of four observation times (Table 1). Southern chinch bug ad ults showed significant preference for susceptible lines at 24 h, 72 h and 120 h after release (F = 10.43; df = 4, 70; P < 0.0001). Forty-eight hours after release, the pr oportion of the chinch bugs on plants found on FX-10 was not significantly lower than the proportion counted on sus ceptible plants. NUF-76 had a significantly lower proportion of chinch bugs on it than on all susceptible lines except Bitter Blue. The preferences of chinch bugs for different lines did not differ among time periods ( F = 0.43; df = 3, 207; P = 0.7300). Between 69 and 72 % of the released adults were found either feeding or resting on a stolon; the remaining adults were either off the plant or dead. The proportions of chinch bugs not found on the plants were 0.30, 0.27. 0.28, and 0.31 at 24 h,

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40 48 h, 72 h, and 120 h after release, respectively. A significant inte raction was detected between St. Augustinegrass lines a nd time after release ( F = 2.1; df = 12, 207; P = 0.0157). This significant interaction in dicates that, in general most lines had a similar proportion infestation over time. However Palmetto was relatively more infested early in the experiment whereas Floratam was relatively more infested later in th e experiment. Mortality of adults in the arenas was not analyzed statistically but ranged between 5 and 10% (one or two insects out of 20). Adult chinch bugs were observed feeding on all pl ants, including the resi stant FX-10 and NUF76, and fecal spots were noticed underneath all plants. No-Choice Tests On m ost of the observation periods, 40 to 45 % of the adult chinch bugs were found inside the leaf sheaths, a few were observed on nodal and internodal regions, and the remaining 55 to 60% of the insects were off the plant. Th e number of excretory spots counted differed significantly among St. Augustinegrass line (F = 27.16; df = 4, 56; P < 0.0001) (Table 2). The number of excretory spots found under FX-10 was consistently si gnificantly less than under the susceptible lines at all the time periods. At 48 h and 72 h, the mean numb er of excretory spots produced on NUF-76 was approximately half the number found on th e susceptible lines, however the differences were not statistically sign ificant. At 120 h the mean number of excretory spots produced on NUF-76 was significantly lower th an found on the suscep tible lines. There were significant differences among time periods ( F = 6.19; df = 3, 135; P < 0.0006). In general production of excretory spots was greatest at 24 h, most likely b ecause insects were starved for 12 h before the start of the experiment, and de clined afterwards. There was no significant interaction of time with St. Augustinegrass line ( F = 1.23; df = 12, 135; P = 0.27).

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41 Ovipostional Studies Southern chinch bug adults confined on Flora tam produced 11 and five times more eggs than those on FX-10 and NUF-76, respectively ( F = 57.6; df = 2, 36; P < 0.0001) (Table 3). There were significant effects due to time and the interaction between St. Augustinegrass lines and time (time: F = 9.79; df = 3, 108; interactions: F = 5.24; df = 6, 108; P < 0.0001). This interaction was due to the higher mean number of eggs laid during the s econd and third weeks of the experiment compared to the first and fourth weeks on Floratam and FX-10 but higher oviposition on NUF-76 on the first and third weeks than the second and fourth weeks. During the third and fourth weeks of the experiment, fewer females were alive on FX-10 and NUF-76 than on Floratam ( F = 32.77; df = 2, 108; P < 0.0001) (Table 3). However, during the first week, numbers of female alive on different lines did not differ significan tly whereas during the second week fewer females were alive on FX-10 than on NUF-76 and Floratam. The eggs were often found inside the folded emerging leaf or partially ope ned leaf sheaths and occasionally on adventious roots. Developmental Studies The five pairs of adults released on FX-10 NUF-76 and Floratam produced an average of 9.5 3.6 (mean SEM), 20.3 6.7, and 180.5 20.1 nymphs, respectively, after 32 d. Nymphal production on FX-10 and NUF-76 was significantly less th an that on Floratam ( F = 284.7; df = 2, 53; P < 0.0001). The number of a dults surviving on FX-10 (0.8 0.3) and NUF76 (1.9 0.6) was significantly less th an that on Floratam (8.0 0.4) ( F = 71.90; df = 2, 20; P < 0.0001). Most of the nymphs we re first and second instars; a very few third instar nymphs were found on Floratam. Thus we assume that the adults observed after 32d were the originally released adults rather than offspring.

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42 Discussion Choice tests, no-choice tests, ovipositional and developm ental studies were helpful to determine the presence or absence of antixenosis and antibiosis in NUF-76 and FX-10, chinch bug-resistant St. Augustinegrass lines. In choice tests, southern chin ch bugs significantly preferred the susceptible lines to both FX-10 and NUF-76, suggesting the presence of antixenosis in the resistant lines. The finding of significantly fewer ex cretory spots under FX10 in no-choice tests confirms the presence of antixenosis. However, the mean number of excretory spots found under NUF-76 was not consisten tly different from the susceptible lines. At 24 h, NUF-76 was not significantly different from any of the susceptible lines however at 48 h and 72 h, the mean number of spots found unde r NUF-76 was significantly different from Palmetto, a susceptible diploid, and at 120 h the mean number of spots were significantly different from all the susceptible lines. Our data indicate a trend th at feeding of southern chinch bug on NUF-76 is reduced over time. While re duced feeding (antixenosis) on FX-10 and NUF-76 could impact chinch bug survival it is unlik ely to have been the sole reason for the low survivorship of chinch bugs on these lines do cumented previously (Nagata and Cherry 2004). Adults released on FX-10 and NUF-76 laid many fewer eggs than on Floratam. The reduction in fecundity could be due to reduced feeding on FX-10and NUF-76 shown in the nochoice tests but was so extreme that it is more lik ely that reduced fecundity is due to a nutritional inadequacy for egg development or some toxin that influenced reproduction (i.e., antibiosis). Studies on the mechanisms of resistance in St Augustinegrass to southern chinch bug have been confined to those done on Floratam. Reinert and Dudeck ( 1974) and Reinert (1978) reported that Floratam and 10 other St. Augus tinegrass accessions ex hibited antibiosis to southern chinch bug. Based on adult and nymphal mortalities, the mechanism of resistance in

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43 Floratam was categorized as antibiosis. Reinert et al. (1980) and Crocker et al. (1989) used the term antibiosis to describe the resistance to southern chinch bug in St Augustinegrass lines. Unfortunately, none of the above-mentioned report s explained the exact cause of the nymphal and adult mortality. Resistance to other pest chinch bugs ha s been studied. Heng-Moss et al. (2003) investigated the mechanisms of re sistance to western chinch bug ( Blissus occiduus Barber) in buffalograss ( Buchloe dactyloides (Nuttall) Engelmann). They f ound an array of modalities of resistance among germplasm tested (antixenos is, antibiosis and tole rance) but reported no morphological or biochemical di fferences to account for these different susceptibilities. Our studies confirm that both FX-10 and NUF -76 have a high level of southern chinch bug resistance. However, because of the course te xture and other negative characteristics such as slow ground coverage, proneness to weeds, FX10 was never extensively grown in Florida (Nagata and Cherry 2003). Another problem with FX-10 was difficulty in transferring its resistance to southern chinch bug into other desi rable lines because of non-viable seeds. Being a diploid and therefore much easie r to cross, NUF-76 is a very important source of chinch bug resistance for turfgrass breeding programs. NU F-76 allows very little population growth and should need many fewer applications of insect icides. Studies are unde rway to understand how FX-10 and NUF-76 are morphologica lly or biochemically different from Floratam and other chinch bug-susceptible lines and how the polypl oid damaging population of southern chinch bug overcame resistance in Floratam.

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44 Table 2-1. Distribu tion (mean proportion SEM) of adult southern chinch bugs among five St. Augustinegrass lines in a choice situation at varying time interv als after release Proportion of B. insularis found on St. Augustinegrass lines* Time after chinch bug release Lines 24 h 48 h 72 h 120 h FX-10 0.15 0.03b 0.18 0.03ab 0.14 0.02b 0.10 0.02b NUF-76 0.09 0.03b 0.10 0.03b 0.08 0.02b 0.11 0.04b Floratam 0.23 0.03a 0.25 0.03a 0.29 0.03a 0.29 0.05a Bitter Blue 0.21 0.04a 0.21 0.04ab 0.23 0.04a 0.23 0.04a Palmetto 0.33 0.02a 0.27 0.02a 0.25 0.02a 0.27 0.02a Proportions calculated as no. of chinch bugs on one St. Augustinegrass line divided by the number of chinch bugs counted on all lines. Those chinch bugs off the plants at a particular observation time were not included in the totals. M eans within the same column with a letter in common are not significantly different ( = 0.05) by Tukeys test.

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45 Table 2-2. Mean number ( SEM) of excretory spots deposit ed per living chinch bug on St. Augustinegrass lines in a no-choice situation at varying time intervals after release Number of excretory sp ots per living insect* Time after chinch bug release Lines 24 h 48 h 72 h 120 h FX-10 1.7 0.3b 1.5 0.3c 1.3 0.3c 1.7 0.4b NUF-76 3.6 0.5a 2.0 0.2bc 2.6 0.3bc 2.6 0.5b Floratam 5.2 0.5a 3.9 0.4a 3.5 0.3ab 5.1 0.5a Bitter Blue 3.9 0.5a 3.6 0.3ab 4.0 0.5ab 4.7 0.5a Palmetto 4.0 0.4a 3.9 0.3a 4.4 0.3a 5.4 0.6a Number of excretory spots pr oduced during a certain time peri od was calculated as the number of spots divided by number of adu lts alive that time period. Mean s in a column with the same letter in common are not significantly different ( = 0.05) by Tukeys test.

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46 Table 2-3. Mean number ( SEM) of eggs produced per female alive on St. Augustinegrass lines at weekly interv als after release Weeks after chinch bug release Number of eggs per live female Lines 1 2 3 4 FX-10 1.4 0.6c 2.2 1.1b 2.5 1.7b 0.7 0.5b NUF-76 6.5 1.4b 2.5 0.8b 5.4 3.4b 0.8 0.6b Floratam 13.9 1.6a 25.3 2.4a 28.3 6.3a 9.2 2.6a Number of live females FX-10 4.15 0.25a 1.30 0.5b 0.69 0.4b 0.46 0.3b NUF-76 4.07 0.32a 3.15 0.4a 1.46 0.5b 0.30 0.2b Floratam 4.61 0.1a 4.15 0.2a 4.0 0.2a 3.58 0.4a Means in a column with the same letter in common are not significantly different ( = 0.05) by Tukeys test.

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47 CHAPTER 3 OXIDATIVE RESPONSES OF ST. AUGUSTI NEGRASSES CHALLENGED BY SOUTHERN CHINCH BUG, BLISSUS INSULARIS BARBER (HEMIPTERA: BLISSIDAE) Introduction St. Augustinegrass, Stenotaphrum secundatum ( Walt.), is the most widely used turfgrass in tropical and subtropical climatic regions (Sauer 1972). It is a comm only grown residential turfgrass species in Florida and is estimated to cover more than one million acres. St. Augustinegrass accounts for 85% of total sod pro duction in Florida and is worth more than $262 million annually. The southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae), is the most serious insect pest of St. Augustinegrass (Crocker 1993). The annual cost of southern chinch bug management and losses in Florida alone was estimated at $5 million in 1983 (Hamer 1985) and is undoubtedly much more today. The ec onomic importance of this insect is further increased by its development of resistance to se veral key insecticides (R einert and Portier 1983; Cherry and Nagata 2005, Cherry and Nagata 2007). Historically, host plant resistance has been one of the most successful pest management tools for this insect. Floratam, a polyploid vari ety of St. Augustinegrass with resistance to southern chinch bug, has been available in Florid a and Gulf Coast areas since its release in 1973 (Horn et al. 1973). However, in 1985 a population of chinch bug was reported to have overcome Floratams resistance in Florida (Busey and Ce nter 1987). Nagata and Cherry (2003) collected southern chinch bug populations from nine widely-d ispersed cities or th eir suburbs in Florida and found all populations capable of overcom ing the resistance in Floratam. As potential alternatives to Floratam, two southern chinch bug resistant St. Augustine lines were developed: a polyploid line, FX-10 (Bus ey 1993) and a diploid line, NUF-76 (Nagata and Cherry 2003). However, the mechanisms of resistance in these lines are unknown. Deciphering these mechanisms will greatly facilitate our e fforts to breed cultivars with durable chinch bug

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48 resistance in St. Augustinegrass. Although the mechanisms of plant resistance against phloemfeeding aphids have been studied extensively (Thompson and Goggin 2006), aphids feed on their host plants by probing intr acellularly, while southern chinch bugs probe interc ellularly, likely resulting in more and diffe rent kinds of cellular da mage and plant response. In our recent investigations, we showed that both FX-10 and NUF76 deterred feeding of southern chinch bugs (antixenos is) (Rangasamy et al. 2006). NUF-76 also had adverse effects on the biology of the insect (antibiosis) (Rangasamy et al. 20 06), suggesting a metabolic basis for insect resistance. One of the most prominent plant responses to insect herbivore attack is the induction of oxidative enzymes such as pol yphenol oxidase (PPO), peroxidase (POX), lipoxygenase (LOX) and catalase (CAT) (Green a nd Ryan 1972; Hildebrand et al. 1986; Felton 1989; Felton et al. 1994a b; Stout et al. 1999; Constabel et al. 2000; Ch aman et al. 2001; Ni et al. 2001; Heng-Moss et al. 2004). These enzymes, becau se of their potential roles in the synthesis of defense compounds and/or in oxidative stress to lerance, have often been implicated in plant resistance to insect herbivores. The objective of this investigation was to comp are the enzymatic responses of resistant and susceptible lines of diploid and polyploid St. Augustinegra ss to feeding by a population of southern chinch bugs that has overcome the resistance of Floratam. Our study focused on the induction of oxidative enzymes POX, PPO, CAT and LOX in four cultivars of St. Augustinegrass following the introdu ction of southern chinch bugs. Materials and Methods Plants, Insects, Sample Collection St. Augustinegrass lines selected for com par ison included NUF-76 (resistant diploid), FX10 (resistant polyploid), Fl oratam (susceptible polyploid), and Palmetto (susceptible diploid). Both polyploid and diploid lines were included to determine whether the ploidy level has any

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49 role in resistance to southern ch inch bugs. Rooted sprigs from single node cuttings were grown in plastic pots (12.5 cm diam. 12.5 cm depth) c ontaining a 1:1 (v/v) mixture of sand and Fafard mix #2 (Conrad Fafard, Agawam, MA ). Plants were fertilized with Osmocote Plus 15-9-12 (Scotts-Sierra Horticultural Products Company, Marysville OH) at the rate of 5 g kg-1 of growing medium. Stolons used in experiments we re approximately 30 to 40 cm in length, with a minimum of three nodes, located on plants that were 6 to 8 weeks old. Southern chinch bugs were collected from Royal Palm Beach (Palm Beach County, FL) for all experiments. The insects were collected by vacuuming southern chinch bug-infested St. Augustinegrass lawns as described by Cherry (2001) In the laboratory, fifth instars and adults were separated from the debris and insects we re provided with fresh Floratam runners and maintained at 18C in 20-l plastic buckets. Twenty adult chinch bugs (1:1 sex ratio) were confined in organdy sleeve cages on the younge st three nodes of one runner of a St. Augustinegrass plant. The chinch bugs were removed from the runner before collecting the sample for protein analysis. A 2-g amount of sample including leaf sheaths and blades, excised from each runner, was collected 1, 3, 5, and 8 d afte r infestation and frozen immediately in liquid nitrogen. The samples were stored at -80C until analys es. Five replicate plants were infested for each line at each time period along with an equal number of control plants whose runners were confined in cages without chinch bugs. Sample Preparation Frozen St. Augustinegrass sam ples were ground into powder using a mortar chilled with liquid nitrogen. Soluble pr oteins were extracted usi ng 5 ml 20 mM HEPES (4-(2hydroxymethyl)-1-piperazineethanesulfonic acid) bu ffer (pH 7.2) containing protease inhibitors [0.3 ml/g plant tissue, a mixture of 1 M be statin, 0.2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 1 M pepstatin A, 1 M E-64, 10 M leupeptin and 1 mM 1, 10-phenanthroline

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50 (Sigma, St. Louis, MO) and 1% (wt/v) polyviny lpyrrolidone]. The homogenate was filtered through a 4X muslin cloth and cen trifuged at 10,000 g for 10 min at 4C. The supernatant was collected, desalted using a PD-10 desalting column (Sephadex G-25M, Amersham Biosciences Inc., Piscataway, NJ), and stored at 80C until protein and enzyme analyses. Estimation of Total Protein Total pro tein content and enzyme activities were determined using a Beckman Model DU 640 Ultraviolet-Visible spectropho tometer. The total protein content was determined by following the modified method of Lowry et al. (1951) using the Folin-Ciocalteau phenol reagent (Pierce Chemical, Rockford, IL) and bovi ne serum albumin as a standard. Enzyme Activities The soluble POX specific activity was measured with guaiacol (Sigma, St. Louis, MO) as a substrate. Increase in absorbance at 470 nm was monitored for 2 min using a protocol modified from Hildebrand et al. (1986) and Hori et al. (1997). The reacti on was started by adding 10l of 30% (v/v) H2O2 to a quartz cuvette containing 100 l 250 mM HEPES-KOH, pH 7.0, 5 l protein extract, 300 l 18 mM guaiacol, and 585 l distilled water. The POX specific activity (M mg protein-1 min-1) was calculated using the molar abso rptivity of guaiacol at 470 nm ( = 26.6 M-1 cm-1). Catalase-specific activity was determined follo wing a modified protoc ol from Hildebrand et al. (1986). The reaction wa s started by adding 200 l protei n extract to a quartz cuvette containing 100 l 250 mM HEPES-KOH, pH 8.0, 100 l 75 mM H2O2 (Sigma, St. Louis, MO) and 600 l distilled water. The reaction wa s monitored at 240 nm for 2 min and the CAT specific activity was determined us ing the molar absorptivity of H2O2 at 240 nm ( = 43.6 M-1 cm-1) and expressed as moles mg protein-1 min-1.

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51 PPO activity was measured using a protocol modified from Hori et al. (1997). The reaction was started by adding 25 l of protein extract to a cuvette containing 100 l 250 mM HEPES-KOH, pH 6.0, 500 l 1.6% cat echol (Sigma, St. Louis, MO) and 375 l distilled water. PPO activity was monitored at 470 nm for 2 min and calculated as the change in A470 mg protein1 min-1. LOX activity was determined using a protocol modified from Skorz ynska-Polit and Krupa (2002). Linoleic acid (Fluka, St. Gallen, Switzerland) substrate was prepared by following a protocol described in Koch et al. (1992). A 10 mM stock so lution of sodium linoleate was prepared by mixing 14 l linoleic acid, 14 l Tween-20 and 2 ml oxygen-free distilled water by drawing back and forth with a Pasteur pipette which produced a milky emulsion. The emulsion was cleared on addition of 0.11 ml 0.5 M NaOH. The volume was then made up to 5 ml with oxygen-free distilled water and disp ensed to 2-ml screw-capped vials which were flushed with N2 and stored at -20C. The reaction was starte d by adding 10 l 10 mM linoleic acid to a quartz cuvette containing 50 l prot ein extract, 100 l 250 mM HEPES-KOH (pH 7.0) and 860 l distilled water. The change in absorbance at 234 nm was recorded for 5 min. The LOX activity was expressed as the change in A234 mg protein-1 min-1. Isozyme Profile Studies Isozym e expression of POX, PPO and LOX wa s studied using nativ e gel electrophoresis (Mini-Protean III, Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of protein (30 g/well) were loaded into 10-well 4.5 % (wt/v) and 7.5% (wt/ v) polyacrylamide discontinuous gels for POX and PPO and 5.0% (wt/v) and 12.5% (w t/v) gels for LOX. Tris-glycine (pH 8.3) continuous buffer system was used to run the ge l for 120 min at 30 mA at 4C. POX and PPO isozymes were stained using a protocol modifi ed from Vallejos (1983) and LOX isozymes were stained according to a protocol by Heinisch et al. (1996).

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52 Gels to be stained for POX activity were in cubated in 50 mM sodium acetate buffer (pH 5.0) at 25 2C on a platform shaker set at 50 rpm. After 10 min of incubation, 20 l 30% H2O2 and 10 mg 4-chloronaphthol (dissolved in 0.5 ml methanol) were added to the buffer. Peroxidase activity was visualized as dark blue bands after appr oximately 20 min. Gels to be stained for PPO activity were soaked in 20 mM HEPES (pH 7.2) buffe r containing 5 mM DL(3,4-dihydroxyphenylalanine) (DOPA, Sigma, St Louis, MO) for 30 min at 25 2C on a platform shaker set at 50 rpm. PPO activity was visualized as dark brown bands on a clear background after 30 min. Gels to be stained for LOX activity were brie fly rinsed in distille d water and incubated for 30 min in linoleic acid substrat e solution at 4C on a platform sh aker set at 50 rpm. The gel was rinsed again in distilled water, and then 20 ml of the staining solution was added. It was incubated on a shaking platform set at 50 rpm at 25 2C for 10 min. LOX activity was observed as pink bands. The subs trate solution was prepared by mixing 50 l linoleic acid and 50 l 95% ethanol using a magnetic stirrer in a 2-ml screw cap vial at 4C by exclusion of light. The solution was transferred in to a 50-ml flask and the volume was made up to 50 ml with 0.2 M sodium borate buffer (pH 9.0). The stai ning solution was prepared by mixing 0.5g N N dimethyl-p-phenylenediamine, 4.5 ml methanol and 0.5 ml acetic acid (1:1, acid : water) and the volume was made up to 50 ml with distilled water. Statistical Analysis The study was set up using a com pletely randomized design with five replications. The means of enzyme activities in polyploid lines (FX-10 and Floratam) and diploid lines (NUF-76 and Palmetto) were analyzed separately usi ng PROC GLM (SAS 1999) and Tukeys test was used for mean separations. In the two-way AN OVA model, time after treatment and grass lines were considered fixed effects, while replicate plants were random.

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53 Results Total Protein Content There were no significant differe nces in total ex tractable protein content between chinch bug-infested and control plants for any of the St. Augustinegrass lines at any time periods after infestation ( P> 0.05, n=5) (Figure 3-1). Peroxidase Activities FX-10 and NUF-76 plants infested with southe rn chinch bug had nearly two-fold higher POX-specif ic activities 5 and 8 d after infestati on compared to their respective controls (F = 52.68, df = 3, 64, P < 0.0001, n = 5) (Figure 3-2). Two-way ANOVA found no significant interaction between St. Augustinegrass lines and time (F = 1.8, df = 9, 64, P < 0.07). POX activities in infested FX-10 plan ts on 5 and 8 d were significan tly higher than levels in the susceptible polyploid, Floratam, whether infested or uninfeste d. The southern chinch bugsusceptible Floratam and Palmetto did not have any significant diffe rence in POX specific activity between infested and cont rol plants at any of the time pe riods studied (F = 1.69, df = 3, 64, P = 0.08) (Figure 3-2). Polyphenol Oxidase Activities The southern chinch bug-infested N UF-76 pl ants had significantly higher PPO activity 5 and 8 d after infestation compared to their respective control plants (F = 21.39, df = 3, 64, P < 0.05, n = 5) (Figure 3-3). There were no significant differences in PPO specific activity between the southern chinch bug-infested and control plants of FX-10, Florat am (F = 1.98, df = 3, 64, P = 0.1255) and Palmetto (F = 1.61, df = 3, 64, P = 0.10) at any of the time periods after infestation. With the exception of NUF-76 plants 5 and 8 d after infestation, ther e were no significant differences in PPO activities among infested and control NUF-76 and Palmetto plants.

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54 Lipoxygenase Activities FX-10 plants infested with southern chin ch bug had significantly higher LOX specific activity 3, 5 and 8 d after infestat ion com pared to their respective control plan ts (F = 312.96; df = 3, 64; P < 0.0001, n = 5) (Figure 3-4). There was a significant interaction between St. Augustinegrass line and time (F = 25.46, df = 9, 64; P < 0.0001). LOX specific activity in southern chinch bug-infested FX-10 plants peaked 3 d after infestation when it was 2-fold higher than 1 d after infestation, and then declined 5 and 8 d after in festation. The LOX activities in southern chinch bug-infested and control Floratam plants were not significantly different. However chinch bug-infested FX-10 plants had significantly highe r LOX activity than uninfested FX-10 controls, Floratam controls and FX-10 and Floratam infested plants on 1 and 3 d (Figure 3-4). Two-way ANOVA found no difference in LOX activities between FX-10 controls, Floratam control and in fested plants at any of the ti me periods studied. There were no significant differences in LOX specific activitie s between southern chinch bug-infested and control NUF-76 and Palmetto plants (F =1.69, df = 3, 64, P = 0.18). Catalase Activities There were no significant diffe rences observed in CAT specific activity between southern chinch bug-infested and control plants at any tim e period after infestation in any of the St. Augustinegrass lines studied (Figure 3-5). Isozyme Profile Studies Native ge ls stained for POX-specific activity show ed five isozymes in FX-10; two of them were induced 5 and 8 d after infestation compared to their respective controls (Figure 3-6). There were three POX isozymes in NUF-76 with no apparent difference between infested and control plants. There were three POX isozymes in Palmetto and five in Floratam with no difference between infested and control plants at any of the times after infestation (Figure 3-6).

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55 Native gels stained for PPO id entified two isozymes in NUF-76, Palmetto and Floratam and three in FX-10 with no a pparent difference between southern chinch bug-infested and controls plants at any of the time periods stud ied (Figure 3-7). There were two LOX isozymes seen in NUF-76, four in Palmetto, three in FX-10, and one in Floratam (Figure 3-8). There were no apparent differences in LOX isozyme intensity or number between southern chinch buginfested and controls plants. Discussion There was n o significant difference in extractabl e total protein content of southern chinch bug-infested and control St. A ugustinegrass plants at any tim e after infestation, although southern chinch bug-infested FX-10 plants show ed an overall increase in protein content over time. Our results agree with those of Heng-Moss et al. (2004) who found no increase in total protein content after infestat ion by the western chinch bug, Blissus occiduus, of resistant Buffalograss varieties Cody and Tatanka. Ho wever Ni et al. (2001) reported a significant increase in total protein content in wheat va rieties infested with the Russian wheat aphid, Diuraphis noxia (Mordvilko), and th e corn leaf aphid, Rhopalosiphum padi (L.), in contrast to an earlier report of a two-fold decrease in total protei n 2 d after infestation by D. noxia (Van der Westhuizen and Pretorius, 1995). We found both chinch bug-resistant lines, FX-10 and NUF-76 had a nearly two-fold increase in POX specific activity levels 5 and 8 d after infestation compared to uninfested control plants. Our results agreed with many previous reports that POX specific activity levels increase after herbivore infestat ion (Hildebrand et al. 1986; Felton et al. 1994a, b; Van der Westhuizen et al. 1998; Ni et al. 2001; Chaman et al. 2001; Heng-Moss et al. 2004; Khattab, 2007). Lupin varieties resistant to green peach aphid, Myzus persicae (Sulzer), had significantly different

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56 levels of POX and PPO activity, pr otease inhibitors and soluble phe nolics. However, the levels of PPO and POX were not increased afte r the aphid infestation (Cardoza et al. 2005) Herbivore damage is known to induce production of H2O2 in plants (Dowd and Lagrimini, 1997). The level of H2O2 in plants is regulated by oxidati ve enzymes such as POX and CAT, and it has been demonstrated in vitro that POX activity is de pendent on the supply of H2O2 (Duffey and Felton, 1991). An increase in POX activ ity in insect-resistant plants may detoxify the peroxides, thus reducing plan t tissue damage compared to su sceptible plants (Hildebrand et al. 1986). Peroxidases are involved in plant cell wall-building proce sses, by mediating the oxidation of hydroxycinnamyl alcohols into free radical intermediates, cross-linking of polysaccharides and extensin monomers, lignifica tion and suberization (Chittoor et al. 1999). Brisson et al. (1994) suggested that the oxidative cross-linking of the plant cell wall could enhance its ability to slow pathoge n ingress. Such a reinforcement of the cell wall in response to insect feeding may serve as a mechanical barrie r and prevent the insects stylets from reaching their feeding sites, phloem sieve elements in the case of southern chinch bug. An enhancement of the POX isozyme activity in maize ( Zea mays L.) increased resistan ce to herbivores and pathogens (Dowd and Lagrimini, 1997). Increased POX activities in FX-10 and NUF-76 in our study suggest that these two lines may have increased cell wall lignification. Plant catalases scavenge H2O2 that occurs in mitochondria and peroxisomes and reduce them into H2O and O2. Zhu-Salzman (2004) found that the CAT genes were down-regulated in resistant sorghum plants infested with gree nbug and speculated that the reduced CAT activity could help the plant to maintain high H2O2 levels thus causing damage to the insect midgut. Park et al. (2006) also found that CA T genes were down regulated in greenbug resistant sorghum lines. However, Heng-Moss et al. (2004) reported a loss in CAT activity in response to feeding

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57 by B. occiduus on susceptible buffalograss varieties. Our study found no association between CAT-specific activity levels and southern chin ch bug resistance in St Augustinegrasses, and supports previous reports using other plant-in sect combinations indicating no relationship between CAT activity and resistance (Faccioloi, 1 979; Matkovics et al. 1981; Hildebrand et al. 1986). The association of PPO activity with host plant resistance to in sects has been reported in many plants including tomato, potato, coffee a nd poplar (Duffey and Felton, 1991; Constabel et al. 1996; Wang and Constabel, 2004; Chaman et al. 2001; Thipyapong et al. 2006). In our studies, only NUF-76 had significan tly higher levels of PPO speci fic activity as a result of southern chinch bug infestation, whereas FX-10 showed no such induction. Heng-Moss et al. (2004) found no association between PPO activity in buffalograsses and their resistance to B. occiduus. Our studies showed that FX-10 had a twofold higher LOX-specifi c activity 3 d after southern chinch bug infestation, and a reduced but still significantly higher activity 5 and 8 d after infestation compared to the control plants (Figure 3-4). Increased activity of LOX has been reported in soybean in response to two-spotted spider mite infestation (Hildebrand et al. 1986) and attack by larvae of Helicoverpa zea (Boddie) (Felton et al. 1994b). The increased levels of LOX activity caused oxidative damage in midgut epithelial cells of H. zea larvae and reduced their growth (Felton et al. 1994b). Bi et al. (1997) showed si gnificant increase in LOX in H. zea damaged cotton squares along with altered phenolic contents. Ge ne expression studies showed that LOX transcripts were strongly elicited in tomato plants in response to attack by the aphids, Macrosiphium euphorbiae (Thomas) and M. persicae (Fidantsef et al. 1999) and were locally upregulated in Nicotiana attenuata after Macrosiphium nicotianae Blackman infestation

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58 (Voelckel et al. 2004). LOX-deficient N. attenuata plants, when planted into native habitats, became more vulnerable to adapted herbivores such as Manduca sexta (L.) and attracted new opportunistic herbivores, such as Empoasca spp. (Kessler et al. 2004). Lipoxygenases are ubiquitous enzymes that catalyze the hydroperoxidation of polyunsaturated fatty acids having cis, cis-pentadiene moieties (Hildebra nd et al. 1986). Linoleic acid and linolenic acid are the majo r LOX substrates in plants (H ildebrand et al. 1986). Lipid peroxidation by LOX results in formation of fa tty acid hydroperoxides which may enzymatically and/or chemically be degraded to highly reactive aldehydes, -ketols, epoxides (Gardner, 1991), hydroxyl radicals, singlet oxygen, s uperoxide ion and peroxyl, acy l and carbon-centered radicals (Kanofsky and Axelrod, 1986). Ga rdner (1979) mentioned that th e interactions between lipid hydroperoxides and proteins results in protein-protein cross linking, and amino acid damage. These chemical changes in proteins may affect th e assimilation of amino acids of insects. These lipid peroxidation end products could be repellent to insect feeding and operate via antixenosis or be toxic and function via antibio sis (Felton et al. 1994b). Both modalities of resistance have been reported previously in St Augustinegrass against the southern chinch bug (Rangasamy et al 2006). Shukle and Murdock (1983) demonstrated th at LOX could directly act as a defense in soybean against insect attack. Lipoxygenases are also involved in inducible herbivore resistance; the LOX oxidation of linolenic aci d is the first step in jasmonic acid signaling pathway, which has been implicated in coordi nation of direct plant defense such as production of oxidative enzymes and protease inhibitors (Staswick and Lehman, 1999, Ma o et al. 2007) and indirect plant defense such as production of volatiles (Dicke et al. 1999; Thaler et al. 1999). Rayapuram and Baldwin (2006) investig ated the performance of M. sexta larvae on N. attenuata plants silenced in LOX3 expression and jasmonic acid signaling and found that LOX3-mediated

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59 defense in wild plants reduced larval growth, consumption and fr ass production compared to the mutant. Mao et al. (2007) transformed maize plants with the wheat oxalate oxidase gene and reported that the up-regulated LOX transcripts an d 14-fold higher free phenolics in transgenic plants were positively associated with thei r resistance to the European corn borer ( Ostrinia nubilalis H.) compared to the control plants. Based on the results from our research, we a ssociate the increased levels of POX and LOX in FX-10 and POX and PPO in NUF-76 with sout hern chinch bug resistance in these lines. The increased POX and LOX activities could be a ca use of antixenosis in FX-10 and NUF-76 and antibiosis in NUF-76 found in our previous st udies (Rangasamy et al. 2006, Chapter 2). Increased POX activity seems to be due to the induction of specific isozymes. Together, our results support the hypothesis that oxidative enzymes play a vital role in he rbivore resistance in plants. Our data suggest that identification of genes encoding inducib le PPO, POX, and LOX enzymes could be the first step in understa nding southern chinch bug resistance in St. Augustinegrass and will be valuable markers for breeding southern chinch bug resistant lines. The presence of different kinds of southern chinch bug resistance in FX-10 and NUF-76 is in agreement with our previous studies (Rangasamy et al. 2006, Chapter 2). Our research supports the hypothesis that chinch bug resistance in St Augustinegrass has a metabolic basis and highlights the possible involvement of lignification and oxidative enzymes in secondary product synthesis in chinch bug resistance in St. Augustinegrass.

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60 1358 0 7 8 9 10 11 Infested Control 0 7 8 9 10 11 Infested Control Time after infestation (d) 1358 0 7 8 9 10 11 Infested Control Total protein (mg g -1 fresh weight) 0 7 8 9 10 11 Infested Control Figure 3-1. Total protein content in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. A. FX-10, SCB resistant polyploid. B. Floratam, SCB susceptible polyploid. C. NUF-76, SCB re sistant diploid. D. Palmetto, SCB susceptible diploid. The values shown are mean SE of five replicates.

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61 Figure 3-2. Levels of peroxidase (POX) specific activity levels in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. The values shown are mean SE of five replicates. M eans marked with differ significantly betwee n control and infested treatments ( P < 0.05, Tukeys test). 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Infested Control 1358 POX specific activity (moles mg protein -1 min -1 ) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Infested Control Time after infestation (d) 1358 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Infested Control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Infested Control

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62 0 1 2 3 4 5 Infested Control Time after infestation (d) 1358 PPO Specific activity (A 470 mg protein -1 min -1 ) 0 1 2 3 4 5 Infested Control 0 1 2 3 4 5 Infested Control 1358 0 1 2 3 4 5 Infested Control Figure 3-3. Levels of polyphenolox idase (PPO) specific activity in St. Augustinegrass lines 1, 3, 5, and 8 d after initation of southern chinch bug feeding. The values shown are mean SE of five replicates. Mean s marked with differ significantly betwee n control and infested treatments ( P < 0.05, Tukeys test).

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63 Figure 3-4. Levels of lipoxygenase (LOX) specific activ ity in St. Augustinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. The values shown are mean SE of five replicates. Means marked w ith differ significantly between control and infested treatments ( P < 0.05, Tukeys test).

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64 Figure 3-5. Levels of catalase (CAT) sp ecific activity in St. Augustin egrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. The valu es shown are mean SE of five replicates. Means marked with diffe r significantly between control and infested treatments (P < 0.05, Tukeys test). Time after infestation (d) 1358 0.0 5.5 6.0 6.5 7.0 Infested Control Catalase specific activity (A 240 mg protein -1 min -1 ) 0.0 5.5 6.0 6.5 7.0 Infested Control 0.0 5.5 6.0 6.5 7.0 Infested Control 1358 0.0 5.5 6.0 6.5 7.0 Infested Control

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65 Figure 3-6. Native gels stained for POX activity in St Augustinegrass lines 1, 3, 5, and 8 d after initiation of southern chinch bug feeding. T and C refer to the infe sted (i.e., treated) and control samples, respec tively. Arrows indicate th e number of POX isoforms.

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66 Figure 3-7. Native gels stained for PPO activity in St Augustinegrass lines 1, 3, 5, and 8 d after challenge by southern chinch bug. T and C refer to the infested (i.e., treated) and control samples, respectively. Arrows indicate the number of PPO isoforms.

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67 Figure 3-8 Native gels stained for LOX activity in St Augustinegrass lines 1, 3, 5, and 8 d after challenge by southern chinch bug. T and C refer to the infested and control samples, respectively. Arrows indicate the number of LOX isoforms.

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68 CHAPTER 4 QUANTITATIVE ANALYSIS OF FEEDING BEHA VIOR OF S OUTHERN CHINCH BUG, BLISSUS INSULARIS BARBER (HEMIPTERA: BLISSI DAE), ON RESISTANT AND SUSCEPTIBLE ST. AUGUSTINEGRASSES Introduction St. Augustinegrass is the m ost widely plante d turfgrass in Florida and other Gulf coast states. St. Augustinegrass is estimated to cover more than one and a half million acres in Florida (Hodges et al. 1994) and accounts for 85% of the sod industry in Florida with a value of $262 million annually (Haydu et al. 2005). The southern chinch bug, Blissus insularis Barber, is the most serious insect pest of St Augustinegrass (Crocker 1993). The annual cost of southern chinch bug management and losses in Florida alone was estimated at $5 million in 1983 (Hamer 1985) and is undoubtedly much more today. The ec onomic importance of this insect is further augmented by its development of resistance to se veral key insecticides (Reinert and Portier 1983, Cherry and Nagata 2005, 2007). Historically, host plant resistance has been one of the most successful pest management methods for this insect. Floratam, a polyploid va riety of St. Augustinegrass with resistance to southern chinch bug, has been available in Florid a and Gulf Coast areas since its release in 1973 (Horn et al. 1973). However, by 1985 a popula tion of chinch bug had overcome Floratams resistance in Florida (Busey and Center 1987). Although significant progr ess has been made in identifying new sources of southern chinch bug re sistance in St. Augustineg rass lines, such as the polyploid FX-10 (Busey 1993) and the dipl oid NUF-76 (Nagata and Cherry 2003), the mechanisms of resistance in these lines are unknown. Rangasamy et al. (2006) investigated categories of resistance to southern chinch bug in FX-10 and NUF-76 and reported high levels of antixenosis in both lines and possible antibiosis in NUF-76 (Chapter 2). Higher mortality of southern chinch bugs has been reported on re sistant FX-10 and NUF-76 than on susceptible

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69 Floratam, Palmetto and Bitter Blue (Nagata and Ch erry 2003); however, the cause of mortality is still unknown. The mortality may be caused by t oxic secondary metabolites present in phloem sap, lack of adequate nutrition after sustained feeding or star vation due to reduced feeding on resistant varieties. Gaining a be tter understanding of the feeding behavior of southern chinch bugs on these resistant and suscep tible St. Augustinegrasses is esse ntial to elucid ate further the mechanisms of resistance in FX-10 and NUF-76. Studying the feeding behavior of insects with piercing and sucking mouth parts is more difficult compared with insects with chewing m outhparts and requires speci alized techniques. McLean and Kinsey (1964) developed a unique t echnique, the electronic feeding monitor which was later referred to as the elec trical penetration graph (EPG) (Tjallingii 1985) to study the feeding behavior of hemipterans. The EPG monitor has gone through many modifications and improvements (Brown and Holbrook 1976, Kawabe and McLean 1980, Kimsey and McLean 1987, Backus and Bennett 1992, Backus and Benne tt, unpublished) and today is the most rigorous technique for studying feeding behavior and plant interactions of hemipterans. The basic principles of EPG have been well describe d by Walker (2000). In brief, the EPG monitor consists of a voltage source and an amplifier whic h are connected to each other. Both the insect and plant are connected to the EP G monitor thus becoming a part of the circuit. The insect is connected to the amplifier using a very thin gold wire and silver conducting paint and the plant is connected to the voltage sour ce in the EPG monitor using a conductive probe (an uninsulated stiff copper wire). Whenever the in sect attempts to probe or insert s its stylet into the plant, the circuit becomes complete and th e current flows from the voltage source through the plant, the insect, the amplifier (via its i nput resistor) and then back to the voltage source (Walker 2000). The plant and the insect act as the biological component of the ci rcuit and resist current flow.

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70 The changes in resistance in th e insect-plant interface, as well as biopotentials generated, are measured as electrical signals and converted in to waveforms which are unique to the insects activities inside the plant. In the research reported here, we used the EP G technique to investig ate the stylet probing of southern chinch bugs on St. Augustinegrass. The St. Augustinegrasses se lected for our studies included FX-10 (resistant polyploid), NUF-76 (resistant diploid), Floratam (susceptible polyploid), and Palmetto (susceptible diploid). Materials and Methods Plants Plants were grown as described in R angasamy et al. (2006) (Chapter 2). Stolons used in experiments were approximately 30 to 40 cm in length with a minimum of three nodes and were cut from plants that were 6 to 8 weeks old Insects Southern ch inch bugs were collected from Royal Palm Beach (Palm Beach County, FL) by vacuuming infested St. Augustinegrass lawns as described by Cherry (2001a). Adults and nymphs were collected by suc tion into a modified WeedEater Barracuda blower/vacuum (Poulan/Weedeater, Shreveport, LA). In the labo ratory, fifth instars we re separated from the debris and provided with fresh Bitter Blue runners and maintained at 26C in 20-l plastic buckets with a moist paper towel at the bottom to avoid de siccation of insects. Only newly emerged 3-5 d old adult females were used for the experiment. Wiring and Acclimating of the Insects Insects were rem oved from the colony and starved for 3 h before wiring. A 12.7-mdiameter (0.0005 in.) gold wire (Sigmund Cohn Corp., Mt. Vernon, NY ) was glued to the

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71 pronotum of the insect using silver conductive paint (Ladd Industries, Burlington, VT). The insect was held immobile during application of the wire tether by placing its abdomen in a Pasteur pipette tip under low vacuum suction pres sure. After wiring, the insects were allowed to rest on a moist paper towel and acclimate to the wire for a 1-h period before recording. EPG Equipment and Data Acquisition A four-channel AC-DC electri cal penetration graph (EPG) monitor (Backus and Bennett, unpublished data) was used for recording the feed ing behavior of southern chinch bug on St. Augustinegrass. Plants, insects and the head-stage am plifiers were all placed in a Faraday cage, constructed of 1.27-cm mesh galvanized ha rdware metal cloth (LG Sourcing Inc., N. Wilkesboro, NC), to avoid any external electrical noise. An input impedance level of 107 was used for all four channels and all the test plants were supplied with a 10 mV, 1000 Hz AC substrate voltage. The pots containing the plants were placed on wooden blocks to electrically isolate them from the Faraday cage. A selected runner from each St. Augustinegrass plant was laid down along a Plexiglas stage measuring 20 cm length 5 cm width and fixed in place using strips of Parafilm (Pechiney Plastic Packaging, Menasha, WI). The Plexiglas stage was held horizontally by an alligator clip connected to a helping hand holde r (van Sickle Electronics, St. Louis, MO). The use of the horizontal stage allowed insects that had left the plant the opportunity to return by walking to the plant ( unless they fell off the stage and hung suspended from their wire tether). The changes in electric al resistance as well as biopotentials during stylet probing were amplified, rectified and digitized at a rate of 100 samples per second using a DI720 analog-to-digital board as described by Alme dia and Backus (2004) and recorded using a Dell Pentium notebook with WinDaq Pro software (Dataq Instruments, Akron, OH). All recording started at 1600 h (in an isolated room at 26 2C with photoperiod of L18:D6) and

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72 continued for 24 h simultaneously on four St. Augustinegrass lines; pr obing was artificially terminated for insects with mouthparts still in serted in the plant at 24 h. There were 35 recordings per treatment; each consisting of a ll four St. Augustinegrasses arranged randomly inside the Faraday cage and connected individually to each of the four cha nnels in the monitor. However, recordings where one or more of the insects within the replicate were not active or dead were discarded, leaving 22 replicates th at were analyzed statistically. Waveform Characterization and Measurements The waveforms produced by southern chinch bug feeding on St. Augustinegrass were categorized based on stereotypical patterns (Backus et al., ms. in prep.). In brief, there are four waveform families (Z, G, H, and I), and six waveform types (Z1, Z2, G1, G2, I1 and I2) within those families (Figure1). For the purpose of comparing the waveforms produced on southern chinch bug resistant and suscepti ble St. Augustinegrasses, only the major waveform categories of Z1, Z2, P (pathway, G1 + G2 + H) and I (ingestion, I1 + I2) were analyzed in this research. The duration measurements for each waveform were made with WinDaq /P ro +Waveform Browser software at a compression of 10 (2 sec/div.) using 2 sec as the threshold du ration for a particular waveform event. The files were copied into a Microsoft Excel file developed for EPG analysis by van Giessen and Jackson (1998) and modified for B. insularis Waveform Categories The following waveform categories used in th e analysis are depicted in Figure 4-1. Z1: Insects off the plant, Z2: Non-probing baseline, G1: variable-frequency and high-amplitude waveforms; always occur at the beginning of a probe after Z2 and followed by G2, presumably associated with stylet pathway activities, G2: hump-like, high-amplitude putative pathway waveform,

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73 H: low-amplitude putative pathway waveform, always preceded by G2, I: Putative ingestion, very uni form, repetitive (7-8 Hz) waveform with peaks and waves (Figure 4-1). E. A. Backus (personal communication) suggest s that I1 and I2 may be associated with either active and passive ingestion or salivation and ingestion, respectively. No biological correlation for either stylet activit ies or location has been establis hed to differentiate between I1 and I2 waveform types at this time; however it is lik ely that at least part of this ingestion is from phloem. Definition of Non-Sequential Parameters Non-sequential param eters used in this study were defined by Backus et al. (2007). A probe is defined as all behaviors occurring from the beginning of stylet penetration probing into plant tissue until stylet withdrawal. A waveform event is defined as a continuous, uninterrupted occurrence of one waveform type (Almeida and Backus 2004, Backus et al. 2007). An accumulator parameter for an insect is designated by insect and a parameter averaged across the N insects assayed is termed per insect. Statistical Analysis Statistics of wavefor ms from the experiment were analyzed using statistical analysis software (SAS 1999) and the procedure describe d by Almeida and Backus (2004) and Backus et al. (2007). The descriptive statis tics for feeding behaviors were compiled at the heuristic levels of waveform event, probe, insect and cohort as described in Backus et al. (2007). A cohort-level waveform analysis will show the total behavior al repertoire of the southern chinch bug population tested. Non-sequential parameters for EPG data were the same as those named and described in Backus et al. (2007). In brief, the basic parameters of EPG data at the event level are the number of waveform events per probe (N WEP) and waveform duration per event (WDE).

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74 The values in these parameters are combined to achieve parameters at the probe level, such as the number of probes per insect (NPI), and probing duration per probe (PDP). Probe level values were combined for parameters at the insect leve l, such as probing duration per insect (PDI), waveform duration per insect (WDI), and number of waveform events per insect (NWEI). When more than one waveform event occurs during a probe, the probe-level data provide sequence information, transition information or both (Backus et al. 2007). The insect-level data provide behavioral information on individu al insects as well as of an av erage insect in the cohort. Restricted maximum likelihood estimati on (REML-ANOVA) (PROC MIXED, SAS 1999) was used to find out whether the frequencies or durations of each waveform type or overall probing were significantly differe nt on the four St. Augustinegrass lines, per event, per probe, per insect or per cohort (Backus et al. 2007). When required, means were then separated using the LSMEANS statement (SAS 1999). Frequency a nd duration data were square rootand logtransformed, respectively, before ANOVA to impr ove homogeneity of variances. Differences were considered significant at = 0.05. Results Cohort Level Southern ch inch bugs spent only 28% and 46% of their total 24-h plant access time in stylet probing (total probing duration, TPD) on FX-10 and NUF-76, respectively (combination of P and I, Table 4-1; Figure 4-2). However, chinch bugs confined on Floratam and Palmetto spent 56% and 66%, respectively, of their access times in probing. More probing events (TNPE) were recorded from chinch bugs confined on FX-10 and NUF-76 than on Floratam and Palmetto (Table 4-1). Overall, ingestion duration was highest on Palmetto, followed by Floratam, then NUF-76 and lowest on FX-10 (Figure 43). It is also valuable to note that only a third of stylet

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75 penetrations in which pathway waveform P be gan successfully transitioned to the ingestion phase (I1) on FX-10 and NUF-76, whereas nearly half of the probes on Floratam and Palmetto resulted in ingestion waveforms (Table 4-2). To statistically analyze the data, cohort level data were subdivided to event level and rebui lt through probe and insect levels. Event Level Probing duration per event (PDE) is the averaged duration of all events, regardless of waveform type. In general, the longest events were made on FX-10, intermediate on Floratam and Palmetto, and shortest events were made on NUF-76 ( F = 46.22; df = 3, 2500; P < 0.0001) (Table 4-1). A waveform event is defined as the smallest, continuous duration of a particular waveform type. The duration of each Z1 waveform (i.e., time southern chinch bugs were off the plant) was the longest on FX-10, intermediate on Palmetto and NUF-76 and shortest on Floratam ( F = 3.38; df = 3, 111; P = 0.02) (Table 4-3). The durati on of Z2 waveform (non-probing, walking or resting on the plant) per event was not significantly different among FX-10, Floratam and Palmetto; however, the duration of Z2 was shorter on NUF-76 compared to the others ( F = 45.62; df = 3, 7640; P = 0.0001). The longest duration of the pathway waveform type (P) occurred on FX-10 whereas the shortest pa thway waveform event occurred on Floratam; intermediate durations were recorded on NUF-76 and Palmett o, which were not significantly different from one another (F = 29.51; df = 3, 11000; P = 0.0001). The durations of ingestion waveform event (I1, I2 waveforms) on Floratam and Palmetto were four and two times longer than on FX-10 and NUF-76, respectively (I1: F = 96.44; df = 3, 2772; P = 0.0001, I2: F = 29.45; df = 3, 672; P = 0.0001) (Table 4-3). Probe Level Southern chinch bugs feeding on resistant FX -10 and NUF-76 produced significantly more probes per insect (NPI) than bugs feeding on susceptible Floratam and Palmetto ( F = 17.64; df =

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76 3, 172; P = 0.0001) (Table 4-1). In a ddition, the mean duration of probes (PDP) on FX-10 and NUF-76 were only one-third to one-half as long as those on Floratam and Palmetto; the duration of probes on the latter two were not significantly different ( F = 46.22; df = 3, 25000; P = 0.0001) (Table 4-1). Considering waveforms on a per probe basis, the insects spent the longest time in pathway activities (P waveforms) on FX-10 and the shortest time on Floratam however, no difference was observed between NUF-76 and Palmetto ( F = 7.56; df = 3, 697; P = 0.0001) (Table 4-4). The mean durations of ingestion waveforms (both I1, I2) during a probe on FX-10 were only one third that of the duration on Floratam. For I1, duration per probe on FX-10 was not significantly different from that on NUF-76 but both FX-10 and NUF-76 were significantly different from the intermediate duration on Florat am and the longest probe duration on Palmetto ( F = 18.77; df = 3, 450; P = 0.0001). The longest durations of I2 on Floratam and Palmetto were not significantly different, but both were greater than the intermediate duration of I2 on NUF-76 and the shortest duration on FX-10 ( F = 15.03; df = 3, 257; P = 0.0001) (Table 4-4). Insect Level There were no significant differe nces in the num ber of times that insects walked off the plant (Z1), on a per insect basis, am ong the four St. Augustinegrass lines ( F = 0.71; df = 3, 45; P = 0.51) (Table 4-5). However, the duration of the times off the plant was longer for FX-10, NUF-76 and Palmetto than for Floratam (Fi gure 4-3). There was no significant difference observed among FX-10, NUF-76 and Palmetto (Z1, F = 8.92; df = 3, 45; P = 0.0001). The numbers of nonprobing even ts on the plant (Z2, F = 8.62; df = 3, 84; P = 0.0001) were higher on FX-10 and NUF-76 than on Floratam and Palme tto (Table 4-5). The duration of nonprobing waveforms (Z2) was longest on FX-10, significantly different from the intermediate durations observed on Floratam and NUF-76 and th e shortest duration on Palmetto (Z2, F = 8.92; df = 3,

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77 84; P = 0.0001) (Figure 4-3). Chinch bugs confined on FX-10 and NUF-76 produced significantly more pathway waveform events per insect ( F = 5.75; df = 3, 84; P = 0.0013) (Table 4-5) that were of longer duration ( F = 8.23; df = 3, 84; P < 0.0001) (Figure 4-3) than those produced by insects confined on Floratam and Pa lmetto. Fewest pathway waveform events per insect were produced on Palmetto whereas an in termediate number of pathway waveform events per insect was observed on Floratam (Table 4-5) Although the number of ingestion (I1 and I2) waveform events per insect did not differ among the four St. Augustinegrass varieties ( F = 0.60; df = 3, 84; P = 0.6168) (Table 4-5), the mean durations of ingestion waveform I1 were three and two times longer on Floratam and Palmetto than on FX-10 and NUF-76, respectively ( F = 11.63; df = 3, 84; P < 0.0001) (Table 4-3). The mean durations of ingestion waveform I2 were four and two times longer on Floratam and Palmetto than on FX-10 and NUF-76, respectively ( F = 9.82; df = 3, 84; P < 0.0001) (Figure 4-3). Discussion For the first tim e, the EPG technique has been used for studying the feeding behavior of southern chinch bug on St. Augustinegrasses. EPG results revealed that the southern chinch bugs were off the plant (Z1 waveforms) on FX-10 and NUF-76 for a longer time than on Floratam and Palmetto. These results agree with our previous studies which showed that fewer southern chinch bugs were found on resistant FX -10 and NUF-76 than on Floratam, Palmetto and Bitter Blue in a choice experiment, suggesting the presence of strong antixenosis (Chapter 2; Rangasamy et al. 2006). When the insects were on the resistant FX-10 and NUF-76 plants, they spent more time in non-probing activ ities, such as resting or wa lking, than in ingestion than when on susceptible Floratam and Palmetto. Th e short durations of su stained ingestion on FX10 and NUF-76 and long duration of sustained ingestion on Floratam and Palmetto also

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78 corroborate our previous result s (Rangasamy et al. 2006; Chapter 2), which showed that significantly fewer excretory spots were pr oduced by chinch bugs fed on FX-10 and NUF-76 than on the susceptible varieties. Successful insect colonizati on on a host plant depends on hos t plant acceptability. For phloem-feeding, piercing and suckin g insects such as aphids, host acceptability depends partly on the ability of the insects stylets to reach the phloem and partly on the quantitative and qualitative properties of phloem sap (Klingauf 1987). Tarn and Adams (1982) and Dreyer and Campbell (1987) discussed that aphi d stylet penetration on resistant plants is often characterized by more frequent, but shorter probe s, and longer waveforms associat ed with pathway activities. We found that southern chinch bugs confined on resistant FX-10 and NUF-76 probed more frequently and that their pathway waveforms we re longer than on the susceptible Floratam and Palmetto. The durations of ingestion, on per ev ent, per insect and per probe levels, were all significantly reduced in chinch bugs feeding on the resistant St. A ugustinegrass varieties compared to the susceptible variet ies. Thus, the present study clea rly demonstrates that southern chinch bugs spent longer on FX-10 and NUF76 in activities that produced the pathway waveforms (P) and less time in activities that re sulted in ingestion waveforms (I1, I2). These results indicate the presence of a physical ba rrier around the vascular bundle or chemical deterrents from the phloem of the resistant cultivars. Similar to our results, the aphids, Myzus persicae (Sulz.) and Nasonovia ribisnigri (Mosley), produced many probes, longer waveforms associated with extracellular stylet pathway activities and shorter waveforms associated with phloem ingestion on resistant lettuce plants, Lactuca sativa L., suggesting that aphid resistance might involve both mesophyll and phloem factors (Montllor and Tjallingii 1989).

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79 Our results suggest that sout hern chinch bug can perceive significant differences among the four St. Augustinegrasses before their stylet s reach the phloem sieve elements; thus, the resistance factors in FX-10 and NUF-76 may reside in tissu e encountered by the chinch bug stylets prior to reaching the vascular bundle. Dreyer and Campbell (1984) and Campbell and Dreyer (1985) found that a chemical modificatio n of pectin in the middle lamella of some sorghum varieties, Sorghum bicolor L. (Moench) made it difficult for the intercellular stylet penetration by certain biotypes of greenbug, Schizaphis graminum (Rondani). It is possible that a similar resistant mechanism occu rs in the resistant St. Augustin egrasses. Transmission electron microscopic studies on St. Augustinegrass leaf sh eath anatomy revealed that the sclerenchyma cells in NUF-76 and FX-10 have thicker cell walls (Chapter 5) which may serve as a physical barrier and prevent or make it difficult for southern chinch bug stylets to reach the phloem sieve elements. Resistance factors residing in sieve elements are often indicated by short durations of ingestion waveforms (Campbell et al. 1982, Ryan et al. 1987, van Helden and Tjallingii 1993, Cole 1994, Caillaud et al. 1995, Paul et al. 1996, Klingler et al. 1998, Sauge et al. 1998, Kaloshian et al. 2000, Tjallingii 2006). Jiang an d Walker (2007) found that the silverleaf whitefly, Bemisia argentifolii Bellows and Perring, terminated its ingestion waveforms more often and spent reduced amount of time in inge stion on whitefly-resistan t alfalfa clones than on susceptible clones, and suggested that this ingestion behavior could be due to the chemical constituency of the phloem sap, such as the presence of toxins or repellents or lack of stimulants. The shorter duration of ingestion waveforms coul d also be caused by a physical blockage of phloem sap by callose deposition causing a long term plugging (Eschrich 1975) or by phloemspecific proteins as a temporary defense measure or by other sieve tube sealing mechanisms (van

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80 Bel 2003). As evidence for the blockage of sap flow, Jiang and Walker (2007) reported that on one of the whitefly-resistant alfalfa clones, phl oem sap ingested per unit time (correlated with honeydew production) was reduced and a longer lag time was observed between the last drop of honeydew produced and the termination of ingestion waveform. They speculated that the whitefly might have tried to continue the inges tion even after a possible phloem blockage and the last observed drop of honeydew, but eventually gave up because of difficulty in ingestion. In our research, the phloem sap in FX-10 and NUF-76 did not stimulate sustained feeding, as shown by reduced duration of ingestion wa veforms compared to those on Palmetto and Floratam. The shorter duration of ingestion waveforms could be due to the presence of resistance factors in phloem sa p or blockage of sieve elemen ts and possibly could be an important component of southern chinch bug resistance in FX-10 and NUF-76. Although we have no direct evidence that the resistant St. Augustinegrasses have phloe m-sealing mechanisms or feeding deterrents in phloe m sap, stylectomy technique has been used to explore these possibilities in aphids and it coul d also be tried in southern ch inch bugs. Caillaud et al. (1995) found that the resistance factors for the aphid, Sitobion avenae (Fabr.), were present in the sieve elements of some lines of Triticum monococcum L. and inhibited phloem sap ingestion by the aphid. Caillaud and Niemeyer (1996) investigated the reasons for th e rejection of some lines of T. monococcum by S. avenae. They found no significant difference in the concentration of hydroxamic acids, a family of aphid resistance factors in cereals, between the favorable T. monococcum line Tm47 and the resistant lines, Tm46 a nd Tm44, suggesting that the rejection of aphid resistant lines was not due to these chemical factors examin ed. They collected phloem sap from young seedlings of aphid resistant and susceptible T. monococcum and found that the success in stylectomy, the proportion of stylet s resulting in longer e xudations (>1 min), the

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81 average duration of exudation and the final vol ume of phloem sap exuded, were significantly higher in the aphid-susceptible genotypes than th e resistant ones. This suggests that phloemsealing caused the aphid to reject feeding in the aphid-resistant T. monococcum lines. Our previous investigations showed that southern chinch bugs did not prefer resistant lines and their oviposition and development were affect ed on them (Rangasamy et al. 2006, Chapter 2). The current study, which employed EPG technique to elucidate the feeding behavior of chinch bugs, showed that the southern chinch bu gs made more probes, took longer time to reach the phloem sieve elements and spent less time on ingestion in resistant lines, suggesting that these resistant lines may have some physical or biochemical factor(s) impeding the stylets uptake from the phloem. While not previously reported on homopterans, chemical analysis of St. Augustinegrass phloem sap, co llected using the stylectomy t echnique, could be used to investigate the presence of feeding deterrents or role of sieve tube sealing mechanisms in southern chinch bug resistance in FX-10 and NUF -76. Considering the economic importance of St. Augustinegrass and the continuing race to find chinch bug resistant varieties, our current research provides exact details on feeding behavior of southern chinch bugs and will eventually further elucidate the mechanisms of resistance.

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82 Table 4-1. Durations and frequencies of pr obing parameters of EPG data recorded on four southern chin ch bug resistant and susceptible St. Augustinegrass lines. Values within rows followed by the same le tter are not significantly different at = 0.05 separated by LSD means test. *Mean SE. St. Augustinegrasses Variables FX-10 Floratam NUF-76 Palmetto P values Total probing duration in min (TPD) 8922 17819 14909 21082 Total no. of probing events (TNPE) 2291 1545 2571 1352 Probing duration per event (PDE) (min)* 9.94 0.59a 8.98 0.61b 6.53 0.49c 7.85 0.57b <0.0001 No. of probes per insect (NPI)* 101.37 8.08a 68.96 6.22b 114.37 13.20a 59.69 6.83b <0.0001 Probing duration per probe (PDP) (min)* 4.01 0.23c 11.75 1.39a 5.93 0.71b 16.06 2.15a <0.0001 Probing duration per insect (PDI) (min)* 1034.45 44.32 630.19 58.35 762.33 71.18 482.03 66.18 0.7833 Table 4-2. Total number of major waveform events (TNWE) on each of four southern chinch bugresistant or -susceptible St. Augustinegrass varieties (n = 22). No. of waveform events (TNWE) Major waveforms FX-10 Floratam NUF-76 Palmetto Z1 insect off the plant 33 21 36 25 Z2 non-probing, insect walking 2258 1524 2535 1327 P pathway 2253 1534 2532 1314 I1 744 630 806 596 I2 ingestion 134 170 185 187

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83 Table 4-3. Mean waveform durations per ev ent (WDE) for major waveforms recorded for southern chinch bug feeding on resistant and susceptible St. Augustinegrasses. Valu es within the rows with the same le tter are not significantly different at = 0.05 separated by LSD means test. Waveform duration per event (WDE) (min) (mean SE) P values Major waveforms FX-10 Floratam NUF-76 Palmetto Z1 insect off the plant 60.41 18.37a 18.62 9.95c 39.43 17.75b 44.14 12.12a 0.0200 Z2 non-probing, insect walking 9.20 0.52a 8.85 0.6a 6.06 0.42b 7.16 0.52a <0.0001 P pathway 1.50 0.30a 1.25 0.04c 1.29 0.05b 1.28 0.05b <0.0001 I1 4.28 0.31c 17.72 1.40a 10.24 0.80b 21.99 1.92a <0.0001 I2 ingestion 6.69 0.58b 20.34 1.93a 10.29 1.37b 27.46 4.18a <0.0001 Table 4-4. Mean duration of major waveforms per probe (WDP) on each of four southern chinch bug-resistant or -susceptible St. Augustinegrasses. Values within rows followed by th e same letter are not significantly different at = 0.05 separated by LSD means test Waveform duration per probe (WDP) (min) (mean SE) P values Major waveforms FX-10 Floratam NUF-76 Palmetto P pathway 1.51 0.06a 1.17 0.04c 1.33 0.06b 1.44 0.09b <0.0001 I1 5.19 0.49c 15.18 1.59b 7.32 0.88c 20.45 2.84a <0.0001 I2 ingestion 7.46 0.73c 19.18 2.33a 12.45 1.77b 21.66 3.16a <0.0001 Table 4-5. Mean number of major waveform events per insect (NWEI) on each of four southern chinch bug-resistant or -susceptibl e St. Augustinegrass varieties. Values within rows followed by the same letter are not significantly different at = 0.05 separated by LSD means test. No. of waveform events per insect (NWEI) (mean SE) P values Major waveforms FX-10 Floratam NUF-76 Palmetto Z1 insect off the plant 2.20 0.46 2.10 0.51 3.00 0.60 2.09 0.40 0.5111 Z2 non-probing, insect walking 102.64 8.10a 69.28 6.21b 115.23 13.29a 60.32 6.84b <0.0001 P pathway 140.41 12.64a 108.05 9.30b 154.36 17.05a 89.63 9.07c <0.0013 I1 33.82 5.13 28.64 2.52 36.64 4.07 27.10 3.21 0.4018 I2 ingestion 6.10 0.83 7.73 1.15 8.41 1.80 8.50 1.37 0.6168

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84 Figure 4-1. EPG waveforms produ ced by southern chinch bug feeding on St. Augustinegrass, recorded using an AC/DC EPG monitor. Major waveform categories: Z2 baseline, G1 and G2 high-amplitude pathway waveforms, H low-amplitude pathway waveform, I1 and I2 ingestion waveforms.

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85 Figure 4-2. Total percentage of time spent on total wave form durat ion (TWD) by southern chinch bugs on resistant and susceptible St Augustinegrasses in different waveform categories. Z1 Insects off the plant, Z2 insects on the plant, resting, walking; P combined pathway waveforms (G1+G2+H), I combined ingestion waveforms (I1+I2).

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86 Figure 4-3. Waveform duration per insect (W DI) for major waveforms on resistant and susceptible St. Augustinegrasses. Z1 insect s off the plant, Z2 insects on the plant, resting, walking; P combined pathwa y waveforms (G1+G2+H), I combined ingestion waveforms (I1+I2). Values (mean SE, n = 22) with the same letter in a waveform category are not significantly different at = 0.05 separated by LSD means test.

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87 CHAPTER 5 ROLE OF LEAF SHEATH LIGNIFICATION AND ANAT OMY IN RESISTANCE AGAINST SOUTHERN CHINCH BUG, BLISSUS INSULARIS BARBER (HEMIPTERA: BLISSIDAE) IN ST. AUGUSTINEGRASS LINES Introduction St. Augustinegrass, Stenotaphrum secundatum, is the most widely used turfgrass in tropical and subtropical climatic regions (Sauer 1972). It is a comm only grown residential turfgrass species in the southern United States, covering more than one million acres and accounting for 85% of the sod industry in Florid a alone, with a value estimated at $262 million (Haydu et al. 2005). The southern chinch bug is the most serious insect pest of St. Augustinegrass (Crocker 1993). The annual cost of southern chinch bug management and losses in Florida alone was estimated at $5 million in 1983 (Hamer 1985). Management of this insect has further been challenged by its development of resistance to several key insecticides (Reinert and Portier 1983, Cherry and Nagata 2005, Cherry and Nagata 2007). Historically, host plant resistance has been one of the most successful management tools for this insect. Floratam, a polyploid variety of St. Augustinegrass with resistance to southern chinch bug, has been available in Florida and ot her Gulf Coast states since its release in 1973 (Horn et al. 1973). Within a decade after its release, in 1985, a population of chinch bug was reported to have overcome Floratams resistance in Florida (Busey and Center 1987). Significant progress has been made in identifying new sources of southern chinch bug resistance in St. Augustinegrass lines. However the regi stered polyploid FX-10 with a high level of resistance to southern chinch bug (Busey 1993) is coarse and tough and never became popular among growers (Nagata and Cherry 2003). The disc overy of southern chinch bug resistance in diploid NUF-76 was considered important because of its fine texture a nd slow growth (Nagata and Cherry 2003). However, the mechanisms of resistance in th ese lines are unknown.

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88 Rangasamy et al. (2006) investigated categories of resistance to southern chinch bug in FX-10 and NUF-76 and found high levels of antixenosis in both lines and possible antibiosis in NUF-76 (Chapter 2). Electrical penetra tion graph (EPG) studies revealed that southern chinch bugs took longer to reach the phloem sieve elements presum ably and spent less time on ingestion on FX-10 and NUF-76 compared to their susceptible controls, Floratam and Palmetto, respectively (Chapter 4). Relatively more stylet probings per in sect on FX-10 and NUF-76 than on Floratam and Palmetto also suggested the presence of st ylet penetration impediments around the vascular bundle in resistant varieties. In addition, the short duration of ingestion on FX-10 and NUF-76 suggested the presence of feeding deterrents within phloem sa p and/or sieve tube plugging mechanisms. Lignin content of plant tissues has often been correlated with resistance to insects and plant pathogens. Blum (1968) identifie d lignin as a major resistance f actor in sorghum against shoot fly, Atherigona variasoccata. Shoot fly resistant sorghum plan ts had highly lignified and thickened cells around the vascular bundles in the young leaves of the central whorl preventing penetration by larvae. Toughness of plant tissues has ofte n been considered as a major factor in their unsuitability to insect feeding (Feeny 1970, Coley 1983). Leaf beetles (Plagiodera versicolora (Laicharting)) feeding on mature Sa lix sp. leaves succumbed to increased mandible wear compared to those feeding on young leaves wh ich resulted in reduced leaf consumption and beetle fecundity (Raupp 1985). Bark beetles, Dendroctonus micans Hugel feeding on spruce trees with higher bark lignin cont ent had reduced larval weight, survival and development rate (Wainhouse 1998). Solanum integrifolium plants tr ansformed with horseradish peroxidase gene had 30% higher lignin content compared to wild-type plants and a high level of resistance to corn earworm (Heliothis armigera (Hbner), Lepidoptera: Noctuidae) (Tsuduk i et al. 2006). Our

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89 previous investigation into the activities of oxidative enzymes su ch as peroxidase, lipoxygenase, catalase and polyphenol oxidase in response to the feeding of s outhern chinch bug on resistant and susceptible St Augustinegra ss lines revealed that the chinch bug-resistant FX-10 had increased peroxidase activity 5 and 8 d after southern chinch bug infestation (Chapter 3). Because plant peroxidases play diverse roles in plants including lignin biosynthesis in the cell wall (Lagrimini et al. 1987) and oxidation of toxic compounds (Gasper et al. 1982) and because lignification of cells could be an impediment to insect pests (Coors 1987, Buendgen et al. 1990, Wainhouse et al. 1990, Wainhouse et al. 1998, Tsuduki et al. 2006), we hypothesized that increased lignification could be a possible component of southern chinch bug resistance in these St. Augustinegrass lines. The objectives of this research were to investigate the possibility that increased lignification and differences in leaf sheath anatomy could account for resistance in diploid NUF76 and polyploid FX-10 St. Augustinegrasses. Specifically, we comp ared stylet probing frequencies, leaf sheath lignin content and vascular bundle anatom y among two southern chinch bug-resistant and two susceptibl e St. Augustinegrass varieties. Materials and Methods Plants St. Augustinegrass varieties used in the following experim ent s included FX-10 (resistant polyploid), Floratam (suscepti ble polyploid), NUF-76 (resist ant diploid) and Palmetto (susceptible diploid). The plants were grown as described by Rangasamy et al. (2006). Runners used for the stylet probing st udy and lignin staining were 30 to 40 cm in length with a minimum of four nodes; runners used for the ligni n quantification had a minimum of 15 nodes.

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90 Insects Southern chinch bugs were collected from Royal Palm Beach (Palm Beach County, FL). The insects were collected by vacuuming St. Augustinegrass lawns as described by Cherry (2001a) and then sorting through debris in the la boratory for fifth instars and adults. After collection, insects were provided with fresh Floratam stolons a nd maintained at 18C in 20-l plastic buckets with a moistened paper towel at the bottom to increase humidity within the bucket. Staining for Salivary Sheaths Visual observations suggested that chinch bugs were residing in and feeding on the axillary shoot. Hence, we focused our studies on the axilla ry shoot w hich consisted of an outermost leaf sheath and several layers of inner leaf sheaths wi th leaf blades and meristematic tissues (Figure 5-1). Ten chinch bug adults (5 males and 5 females) were confined for 48 h in organdy sleeve cages to the third youngest node of one runner of each St. Augustinegrass plant. The chinch bugs were removed from the plant before collecting the sample for staining for salivary sheaths. Southern chinch bug-infested axillary shoots were excised from the plants and placed in McBrides staining solution. The staining solutio n was prepared using 0.2% (wt/v) acid fuchsin in a 1:1 mixture of 95% (v/v) ethanol and glac ial acetic acid (Sigma-A ldrich, St. Louis, MO) according to the methods of Backus et al. (1988). The plant tissues were stained for 24 h at 24 2C, then placed in the clearing solution and autoclaved for 15 min at 120C and 15 psi. The clearing solution was prepared usin g 99% (v/v) glycerol, 85% (v/v) lactic acid and distilled water (1:1:1, v/v/v, Sigma-Aldrich, St. Louis, MO) (Bac kus et al. 19988). The stained salivary sheaths in each leaf sheath within an axillary shoot we re counted under a dissection microscope at 40X (Figure 5-2)

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91 Lignin Quantification Total lignin content of uninfested axillary shoots was quantified using the Klason m ethod (Kirk and Obst 1988). Fresh axillary shoot were air dried and ground using a Wiley mill (Thomas Scientific, Swedesboro, NJ) to pass th rough a 20-mesh screen. A 200-mg sample was weighed into a small beaker and 1 ml of 72% (v:v) H2SO4 per 100 mg sample was added. The sample-acid mixture was digested by placing in a water bath at 30C fo r 1 h and was stirred frequently to get a complete solution. After ex actly 1 h, the solution was diluted using 28 ml of distilled water for each 1 ml of acid and transferre d to an Erlenmeyer flask. The diluted solution was then hydrolyzed in an autoclave at 120C for 1 h and filtered through a 50-ml Pyrex Gooch crucible with fritted disc of 4 5.5 m porosity (Sigma-Aldrich, St. Louis, MO). The filtrate was saved for estimating the acid-soluble lignin. The Klason lignin residue was washed with hot water to remove the acid. The Klason lignin was then dried to constant weight at 105C and expressed as a percent dry weight. The acid-solu ble lignin present in the filtrate was determined spectrophotometrically at 205 nm using an absorptivity va lue of 110L/g-cm (extinction coefficient) to calculate the to tal lignin content (Ehrman 1996). Staining for Lignin Fresh sections of 10-15 m of the outermost leaf sheath of an axillary shoot were prepared using a cryostat (MICROM HM 505E, Microm Loborgerate GMBH, Strasse, Germany) and stained with 2% (wt/v) phloroglucinol (Sigma-Ald rich, St. Louis, MO) solution in ethanol/water (95/5 v/v) for 2 min followed by treatment w ith 50% (v/v) HCl for 1 min for the Weisner reaction (Rastogi and Dwivedi 2006). The staine d sections were photographed at 400X under an epifluorescence light microscope microscope (Olympus BH2-RFCA, Olympus America, Inc., Lake Success, NY, USA).

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92 Transmission Electron Microscopy The axillary shoot from the fourth youngest node of each of the four St. Augustinegrasses was collected and cut into small segments of 12 mm in length and fixed in Trumps EM fixative (1% wt/v glutaraldehyde 4% wt/v paraformalde hyde in phosphate buffer saline (PBS), pH 7.3), incubated in 2% (wt/v) osmium tetroxide and then dehydrated in a graded ethanol-acetone series (25%, 50%, 75%, 95%, 100% (v/v) et hanol, 100% (v/v) acetone). Af ter dehydration, the tissues were embedded with Spurrs epoxy resin (Spurr 1969) at concentrations of 30%, 50%, 75%, 100% and placed in an oven at 60C for 48 h. Ultr a-thin sections of 1 m were made using an ultramicrotome (EM-UC 6, Leico Microsystems Bannockburn, IL) equipped with a glass knife (Type 7801B, LKB Knife Maker, Rockville, MD). The ultra-thin sections were post-stained with 2% (wt/v) uranyl acetate and viewed in a Hitachi H7000 electron microscope (Hitachi, Tokyo, Japan) operated at an accel erating voltage of 75 kV. To measure the thickness of the epidermal layer of the leaf sheat h, photomicrographs of leaf sh eath cross-sections were taken using a light microscope at 400X and the epid ermal layer thickness was measured using Adobe Photoshop CS2 (Adobe Systems Inc., San Jose, CA ). The proportion of the sclerenchyma cell wall area relative to the total sclerenchyma cell area was calculated from the TEM micrographs (3500X) of the leaf sheath vascular bundle usi ng the SPOT image analysis program (Version 3.5, Diagnostic Instruments, Inc. Sterling Heights, MI). Twenty five sclerenchyma cells were measured for each of the three re plicate plants in each of the four St. Augustinegrass lines. Experimental Design and Statistical Analysis The experim ent to investigate the salivary sheath distribution was a completely randomized design with four treatments (F X-10, Floratam, NUF-76, and Palmetto) and 20 replications. The samples for lignin staining a nd electron microscopic studies were collected

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93 from three replicate plants of each of the four St. Augustinegrasses. Parameters such as number of salivary sheaths in each leaf sheath layer relative to the outermost leaf sheath, total lignin content, leaf sheath epidermal layer thickness and the proportion of sclerenchyma cell wall area relative to total cell area were analyzed using PROC GLM (SAS 1999). When appropriate, means were separated using Tukeys test at a significance level of = 0.05. Results Most of the 20 axillary shoot s exam ined from each of the four St. Augustinegrass lines contained five leaf sheaths, arranged in concen tric whorls. Significant ly more salivary sheaths were counted on the outermost leaf sheath layer in FX-10 and NUF-76 than on Floratam and Palmetto (F = 16.78; df = 3, 76; P < 0.0001) (Figure 5-3) and th ese salivary sheaths were branched more often in FX-10, Floratam and NUF-76 compared to Palmetto ( F = 2.58; df = 3, 39; P < 0.0080) (Figure 5-4). A sign ificantly smaller proportion of salivary sheaths reached the innermost layer of FX-10 and NUF-76 axillary shoots compared to Floratam and Palmetto ( F = 63.16; df = 3, 76; P < 0.0001) (Figure 5-3). There was no significant difference in total ax illary shoot lignin content between FX-10 and Floratam or between NUF-76 and Palmetto, although polyploid lines had significantly more lignin than the diploid lines ( F = 23.22; df = 3, 15; P = 0.0002) (Figure 5-5). Staining for lignin did not show any distinct difference between sout hern chinch bug-resistant and -susceptible St. Augustinegrass lines (Figure 5-6). In additi on, there was no significant difference in the thickness of the leaf sheath epidermal layer among the St. Augustinegrass lines studied ( F = 1.17; df = 3, 6; P = 0.28) (Figs. 5-7, 5-8). However, TEM studies on the leaf sheath vascular bundle indicated significantly thickened scle renchyma cell walls in NUF-76 and FX-10 compared to Floratam and Palmetto (Figure 59). Quantification of cell wall thickness in

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94 sclerenchyma cells confirmed these observations (F igure 5-10). The cell walls accounted for a much larger proportion of total sclerenchyma area in the resistant St. A ugustinegrasses than in the susceptible grasses ( F = 4.65; df = 3, 6; P = 0.0181) (Figs. 5-9, 5-10). Discussion Chinch bugs, am ong other hemipterans, lay down a salivary sheath produced by gelling saliva during the intracellular pass age of their stylets through plant tissue. Painter (1928) noted the presence of a stylet-sheath duri ng feeding of the common chinch bug ( B. leucopterous leucopterous), a pest of sorghum, Sorghum bicolor (L.) Moench. The production of salivary sheaths has often been used as a quantita tive measure of homopteran feeding (Bowling 1979, Marion-Poll et al. 1987, Bing et al. 1991). The research reported he rein investigated the possible presence of impediments to stylet penetrati on and access to phloem in southern chinch bugresistant St. Augustinegrass lines. In this research, I found a significantly higher number of salivary sheaths observed on the outermost leaf sheath layers in FX-10 and NUF76 than on susceptible Floratam and Palmetto, corroborating our EPG studies which indicated that chinch bugs probed si gnificantly more often on FX-10 and NUF-76 than on the susceptible variet ies (Chapter 4). Oxidative enzymes such as peroxidase, lipoxygenase and polyph enol oxidase had increased act ivities in FX-10 and NUF-76 plants infested by southern chinch bugs compar ed to infested Palmetto and Floratam plants (Chapter 3); the induction of these enzymes coul d be due to the mechanical damage resulting from the higher number of probings on the leaf sh eath. EPG studies (Chapt er 4) showed that southern chinch bugs spent more time on path way waveforms in FX-10 and NUF-76 which is corroborated by the presence of more branched salivary sheaths on FX-10 and NUF-76. The large proportion of salivary shea ths reaching the innermost leaf sheath on Floratam and Palmetto

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95 demonstrates a strong preference by the southern chinch bugs for the intercalary meristematic tissues, which are continuing to differentiate and are covered by the mature outermost leaf sheaths. In other insect syst ems, Ni and Quisenberry (1997) found no significant difference in the number of salivary sheaths of Russian wheat aphid Diuraphis noxia (Mordvilko) between resistant (Halt) and susceptible (Arapahoe) varieties of wheat, Triticum aestivum L. However, more salivary sheaths were initiated through leaf stomata on the aphid-resistant Halt than on the susceptible Arapahoe, suggesting the possible presence of antixenotic epidermal characters such as leaf toughness and epicuticul ar waxes on Halt leav es. Bing et al (1991) similarly observed that 57% of the time the stylets of the bird cherry-oat aphid, Rhopalosiphum maidis (L.) entered through stomata in maize, Zea mays L, plants in the late whorl stage however only 8% of the stylets penetrated through stomata in the seedling st age. This suggested that the higher rate of stomatal entry of stylets was due to easier penetration. Anderson et al. (2006) found that Blissus occiduus Barber probed more frequently on buffalograss, a preferred host, than on less-preferred sorghum and St. Augustinegrass. We found that a smaller proportion of the init iated salivary sheaths reached the innermost layer of the axillary shoot in resistant FX10 and NUF-76 than they did in the susceptible varieties. Based on the results from the EPG st udies (Chapter 4) and st ylet probing distribution, these probes were probably terminated during pathway phase prior to ingestion (presumably from phloem). We postulated that lignified tissu e may be impeding stylet progress towards or access to the tissue in which the southern chinch bug prefers to feed. Painter (1928) noted that the phloem tissue was the target of the stylets of the common chinch bug Because of a strong preference by the common chinch bug to feed w ithin the leaf sheath (P ainter 1928) and our own observations of feeding by southern chinch bug (Chapter 2), we quan tified the to tal lignin

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96 content of the leaf sheath and stained the leaf sheaths to look for quantitative and qualitative differences, if any. In St. Augustinegrass, the vascular b undle is surrounded by the highly lignified sclerenchyma cells (Figure 5-3). Sclerenchyma ce lls have thickened secondary cell walls which contain cellulose, hemicelluloses and lignin. In general, the li gnin content of sclerenchyma cell wall ranges from 18 35% (wt/wt) (Mauseth 1988) and the presence of lignin provides a stable protection to the vascular bundle. Our TEM st udies found a larger propo rtion of leaf sheath sclerenchyma cell wall area in FX-10 and NUF-76 re lative to the total cellular area consistent with our hypothesis that the sclerenchyma cells may serve as a physical barrier to stylet penetration. Our previous studies suggested that chinch bug resistance mechanisms of the polyploid line FX-10 may be different in certain aspects than that found in NUF-76 since NUF-76 showed antibiosis but both lines ha d antixenosis (Rangasamy et al. 2006). It is interesting to note here that both resistant lines had thicker sclerenchyma cell walls compared to the susceptible lines, suggesting that NUF-76 may have additional feat ures for antibiosis. A greater proportion of sclerenchyma cell wall area was observed on NUF-76, a diploid line with to tal lignin content less than the polyploids FX-10 and Floratam. It may be that deposition of lignin in a few sclerenchyma cells with thick cell walls rather than total lignin is important as a barrier to the southern chinch bug stylets. In summary, our results showed that stylet pe netration to innermost leaf sheaths occurred more frequently in susceptible lines than in re sistant lines, suggesting an impediment to stylet penetration. However, this impediment is not due to differences in their epidermal layer or total lignin content. Based on qualita tive and quantitative analyses of TEM of vascular bundles, the

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97 resistant lines FX-10 and NUF-76 had a greater degree of lignification in sclerenchyma cells around the vascular bundles. We propose that thicker sclerenchyma cells around the vascular bundles is one of perhaps several features of the resistance in St. Augustinegrass lines to southern chinch bug and may be useful as a marker in breeding for southern chinch bug resistance.

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98 Figure 5-1. St. Augustinegrass stolon A. with the ax illary shoot (as); B. Pa rts of axillary shoot, a outermost leaf sheath with leaf blade, b prophyll, c second l eaf sheath layer with leaf blade, d third leaf sheath layer with leaf blade, e axillary meristem.

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99 Figure 5-2. Transverse section of outermost St. Augustinegrass leaf sheath showing southern chinch bug salivary sheath stained with McBr ides stain (400X). ep epidermal layer, pc parenchyma cells, ss salivary sheath, sc sclerenchyma cells, ph phloem, xy xylem.

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100 Figure 5-3. Number of southern chinch bug salivary sheaths f ound on the outermost leaf sheath of axillary shoots (A) and the proportion of salivary sheaths reaching the innermost sheath (B) of southern chinch bug-resist ant and -susceptible St. Augustinegrasses. The values shown are mean SE. M eans followed by the same letter are not significantly different at = 0.05 level separated by Tukeys test (n = 20).

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101 Figure 5-4. Number of southern chinch bug branched salivary sheaths on the outermost leaf sheath of axillary shoots from southern chinch bug-resistant and -susceptible St. Augustinegrasses. The values shown are mean SE. Means followed by the same letter are not signifi cantly different at = 0.05 level separated by Tukeys test (n = 14).

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102 Figure 5-5. Total lignin content of axillary s hoot in southern chinch bug-resistant and susceptible St. Augustinegrasses. The values shown are mean SE of six replicates. Means followed by the same letter ar e not significantly different at = 0.05 level separated by Tukeys test (n = 6).

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103 Figure 5-6. Transverse sections of outermost leaf sheath vascul ar bundles stained for lignin in southern chinch bug-resistant and -suscepti ble St. Augustinegrasses (400X) (n = 3). A. FX-10, B. Floratam, C. NUF-76, D. Palm etto. ph phloem, xy xylem cells, sc sclerenchyma cells, bc bundle sheath cells.

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104 Figure 5-7. Transverse sections of the epidermal la yer of the outermost leaf sheath in southern chinch bug-resistant and -susceptible St. A ugustinegrasses (400X) (n = 3). A. FX-10, B. Floratam, C. NUF-76, D. Palmetto. ep epidermal layer, pc parenchyma cells.

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105 Figure 5-8. Thickness of epidermal layer of outermost leaf sheath in southern chinch bugresistant and -susceptible St Augustinegrasses. Means ( SE) followed by the same letter are not signifi cantly different at = 0.05 level separated by Tukeys test (n = 3).

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106 Figure 5-9. Transmission electron micrographs (7000X) of the sclerenchyma cells of the outermost leaf sheath of St. Augustinegrasse s resistant and susceptible to southern chinch bug (n = 3). A. FX-10, B. Floratam, C. NUF-76, D. Palmetto.

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107 Figure 5-10. Percentage of ce ll wall area relative to total cell area of sclerenchyma of the outermost leaf sheath of southern ch inch bug-resistant and -susceptible St. Augustinegrasses. Means ( SE) followed by the same letter are not significantly different at = 0.05 level separated by Tukeys test (n = 3).

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108 CHAPTER 6 SUMMARY St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, is the m ost commonly grown lawn grass in Florida and other Gulf coast st ates.. It is estimated to cover more than one and a half million acres in Fl orida and accounts for 85% of the sod industry in Florida, worth $262 million annually. The southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae), is the most serious insect pest of St. Augustinegrass. The nymphs and adult chinch bugs feed on the inside of the leaf sheath; the infested plants tu rn yellow and eventually die. Repeated use of insecticides led to development of insecticide re sistance in southern chinch bug to chlorinated hydrocarbons, organophosphates, carbamates, synthetic pyrethroids and insecticides with novel modes of action, such as imidacloprid and lambda-cyhalothrin. Host plant resistance has long been one of the successful management to ols for control of the southern chinch bug. Floratam, a polyploid St. Augustin egrass variety with initially high level of resistance to southern chinch bug was released in 1974. Howeve r, 12 years after its re lease, a population of southern chinch bug from Florida overcame Flor atams resistance. FX-10, another southern chinch bug resistant polyploid variety was regist ered in 1993 with a high level of resistance to southern chinch bug. However, it never became popular among growers because of its coarse texture, slow ground coverage and susceptibility to weeds. In 2003, NUF-76, a diploid St. Augustinegrass line was reported to have a high level of southern chinch bug resistance. Its discovery was considered as a breakthrough in breeding for southern chinch bug resistance because of its diploid genome, ma king the transfer of southern chinch bug resistance easier. Understanding the mechanisms of southern chin ch bug resistance in resistant St. Augustinegrass is essential for maintaining host plant resistance and the development of future cultivars with

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109 better resistance. The main objective of this rese arch was to investigate mechanisms of southern chinch bug resistance in FX-10 and NUF-76. Choice and no-choice tests and ovipositional and developmental studies were conducted to determine the categories of resist ance in FX-10 and NUF-76 to th e southern chinch bug. When adult chinch bugs had a choice am ong attached stolons of three su sceptible lines (Floratam, and Bitter Blue, a susceptible polyploid, and Palmetto a susceptible diploid) and the two resistant lines, chinch bugs were found significantly more often on the susceptible lines over a 5-d exposure period. This result indica ted the presence of antixenosis factors in the resistant lines FX-10 and NUF-76. In a no-choice study, chin ch bugs produced fewer excretory spots (suggesting less ingestion) on FX-10 and NUF-76 than on the susceptible lines. The no-choice study confirmed a high level of antixenosis in FX -10, a moderate level of antixenosis in NUF-76, and possible antibiosis in NUF76. Ovipositional and developmental studies conducted using Floratam, FX-10 and NUF-76 demonstrated that th e adults released on Floratam produced more eggs and offspring than adults confined on FX-10 and NUF-76. Higher mortality of southern chinch bugs ha s been reported on resistant FX-10 and NUF76 than on susceptible Floratam, Palmetto and Bitter Blue (Nagata and Cherry 2003); however, the cause of mortality is still unknown. Understa nding the mechanisms of chinch bug resistance in these two cultivars is essentia l for future turfgrass breeding pr ograms. Plant defenses against herbivores are usually characterized as constituti ve or induced. Constitutive defense is always present in plants whereas induced defense occu rs only in response to herbivore attack which further reduces or deters the herb ivory. Induced plant defense coul d be direct when they have a direct negative effect on the herbivore or indirect when the natural enemies of the herbivores are attracted. Increased activities of oxidative enzymes such as pol yphenol oxidase, peroxidase, and

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110 lipoxygenase are often correlated with induced plant defense to insects. FX-10 and NUF-76 had significantly higher peroxidase activity 5 and 8 d after infestati on and NUF-76 had significantly higher polyphenol oxidase activity 5 and 8 d after infestation compared to control plants. FX-10 had significantly higher lipoxygenase activity 3, 5 and 8 d after infe station compared to control plants. Catalase activities did not differ significantly between infe sted and control plants in any of the lines tested. Native gels stained for peroxidase indicated that some isozymes in FX-10 and NUF-76 were induced 5 and 8 d after infestati on. Isozyme profiles of polyphenol oxidase and lipoxygenase did not differ between control and infested FX10, NUF-76 and Floratam. The increased levels of peroxidase and lipoxygena se in FX-10 and per oxidase and polyphenol oxidase in NUF-76 are associated with southern chinch bug resistance in these lines. Southern chinch bugs are intracellular feeders. Induction of these oxidative enzymes could involve a general plant response to mechanical damage caused by the chinch bug stylets and also include hypersensitive response of cells around th e site of stylet penetration and reactions specific salivary proteins. Incr eased oxidative enzyme activities could cause a combination of induced plant defense mechanisms such as enhanced plant toughness through lignification, poor nutritional quality and toxicity. The increas ed toughness could potentially be caused by increased peroxidase activities. Peroxida ses and polyphenol oxidase s are known to produce protein binding quinones thus redu cing the nutritional quali ty of the plants. Subsequent to the generation of activated products due to the ac tion of these oxidative enzymes, reactions may occur that could potentia lly result in cross-linking or polym erization of mate rials toughening the plant tissues, and nutrien t complexes which are relatively una vailable and in cell death. The reduced oviposition, development and survival of southern chinch bugs on FX-10 and NUF-76 could be due the poor nutritional quality caused by induced activity of these oxidative enzymes.

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111 Lines of experimental evidence shows that plant responses to attack by hemipterans are similar to that of plant pathogens (Kaloshian and Wa lling 2005). Fungal-rust resistant wheat plants showed induction of lignin biosynt hetic pathway in cells exhibiti ng hypersensitive reactions in response to pathogen infection thus preventing the further hyphal develo pment (Menden et al. 2007). St. Augustinegrasses resistant to the southe rn chinch bugs may also respond in the same way and prevent further stylet penetration. Understanding the feeding behavior of southe rn chinch bugs on the resistant FX-10 and NUF-76 is important to elucidate th e mechanisms of resistance. Fo r the first time, the electrical penetration graph (EPG) technique was used to quantify southern chinch bug feeding behavior on resistant and susceptible St. A ugustinegrass lines. Southern chin ch bugs made more frequent probes, produced longer-duration waveform events for pathway-related behaviors (searching for an ingestion site) and spent less time in i ngestion-related waveforms on FX-10 and NUF-76, compared to the susceptible Floratam and Palme tto. Relatively more stylet probes per insect on FX-10 and NUF-76 than on Floratam and Palmetto suggest the presence of stylet penetration impediments around the vascular bundle in resistant varieties. In addition, the short duration of presumed phloem sap ingestion on FX-10 a nd NUF-76 suggests the possible presence of resistance factors in phloem sap or blockage of sieve elements. A significantly higher number of salivary sheaths were observed in the outermost leaf sheath in FX-10 and NUF-76, which further supports the possible presence of a physical ba rrier or hypersensitive reactions. Also, the significantly greater number of br anched salivary sheaths in FX -10 and NUF-76 corroborates our EPG studies that the southern chinch bugs spen t more time on pathway related activities (during which the salivary sheath is construc ted) on these two resistant lines.

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112 Plant peroxidases play diverse roles in plan ts such as lignin bi osynthesis, scavenging hydrogen peroxides, and oxidation of reduced toxic compounds a nd 3-indoleacetic acid (IAA). EPG studies showed that southern chinch bugs probe more frequently on FX-10 and NUF-76 but their stylets took a longer time to reach the phloem sieve elements. Based on the above results, I hypothesized that lignin content and/or leaf sheath anatomy may play a role in southern chinch bug resistance in FX-10 and NUF-76. However, there was no significant difference between FX-10 and Floratam or between NUF-76 and Palme tto in total lignin in the axillary shoot. Therefore, total lignin content does not appear to influence s outhern chinch bug resistance; however, subtle differences in ligni n structure may be important. In general, lignins are made of polymerized phenolic monolignols such as p-coumaryl-, conferyla nd sinapyl-alcohols which are generated through the phenyl propanoid pathwa y (Barber and Mitchell 1997). The lignins in graminaceous plants are characterized by p-hydroxy-phenyl, guaicyl, and syringyl units (Iiyama et al. 1990). In rust-resistant wheat plants, total lignin content was increased in response to pathogen infection and the increase was exclusively due to increase in syringyl units thus changing the syringyl to guaiacyl ratio from 0.55 to 1.43(Menden et al. 2007). Previous studies showed that the increase in syri ngyl-rich lignin or pure syringyl lignin content occurs in plants exhibiting hypersensitive-resistan ce responses to plant pathogens (Mitchell et al. 1999). The total lignin content of the axillary shoot in s outhern chinch bug resistant and susceptible St. Augustinegrasses did not differ significantly. Howe ver, a change in the ratio of these lignin monomers could have occurred in cells around the stylet pathway, thus preventing further stylet penetration and leading to an in creased number of stylet probes on southern chinch bug resistant plants, as shown by both the EPG and salivary sheath distribution stud ies. There was no significant difference in the thickness of the epidermal layer among any of the St.

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113 Augustinegrasses studied. Transmi ssion electron microscopic studie s on axillary shoot anatomy revealed that the cell walls of the highly lignified sclerenchyma cells in the leaf sheath contributed a greater percentage of total cell area in NUF-76 and FX-10 than in Floratam and Palmetto, suggesting that the sclerenchyma cells around the vascular bundle in FX-10 and NUF76 could serve as an impediment to the southe rn chinch bug stylet and prevent them from reaching the phloem sieve elements. Thus, these results show for the first time that at least part of southern chinch bug resistance mechanism is a physical impediment to stylet penetration into the vascular bundles. The presence of thicker sclerenchyma cells in FX-10 and NUF-76 appears to be the constitutive plant defense mechanism against th e southern chinch bug and the reason why the southern chinch bugs probe more and change direction more frequently, producing branched salivary sheaths, on these resistant lines. More pr obing could result in more cellular damage thus leading to a release of plant de fense signaling molecules such as salicylic and jasmonic acid. The induction of oxidative enzymes in FX-10 a nd NUF-76 could be an induced plant defense mechanism and could cause an adverse effect on southern chinch bug survival and development on these resistant lines either by reduced phloe m sap ingestion or reduced food absorption. Apart from the excessive cellular damage, more probes also causes depos ition of more salivary proteins into the damaged plant ti ssues. The role of salivary prot eins in southern chinch bug-St. Augustinegrass interactions is not known. Howeve r, considering the dive rse role of insect salivary proteins in plants, it is possible that an excessive deposition of salivary proteins on the resistant cultivars may activate the induced plan t defense mechanism in FX-10 and NUF-76. In susceptible plants, these salivary proteins could act as suppre ssors of plant defense.

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114 This research suggests that the resistant m echanisms formerly found in Floratam were different from any of the mechanisms describe d above in FX-10 and NUF-76. When Floratam was resistant, the cellular damage and the presen ce of salivary proteins may have been detected by plant receptors and resulted in the activation of induced plant defense. However, over time, the southern chinch bugs could have evolved to make a change in the nature or content of salivary protein which is not rec ognized by the receptors in the plant leading to susceptibility. The susceptibility in Palmetto, a diploid, and Bitte r Blue, a polyploid, may be due the absence of receptors to perceive the southern chinch bug attac k. It is possible that the resistant mechanisms present in each St. Augustinegrass are unique beca use of the genetic dissimilarities between the lines. From this research, I conclude that FX-10 and NUF-76 have strong antix enosis to southern chinch bug, while NUF-76 has antibiosis. The southern chinch bug resistance in FX-10 and NUF-76 could be due to a combination of induction of oxidative enzymes, thicker sclerenchyma cells around the vascular bundle, a nd possible presence of feeding de terrents or toxins in phloem sap or sieve tube blocking mechanisms. Studies outlined here will serve as a solid foundation to further test: (a) phloem sap properties for potential antifeedants or altered nutritional properties, (b) plant metabolic responses including insect-induced polyp henoloxidase, peroxidase and lipoxygenase, and (c) sieve tube blocking mechanisms in FX-10 and NUF-76. The results have important implications for breeding St. Augustinegrass lines with improved resistance to southern chinch bugs.

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115 LIST OF REFERENCES Almeida, R. P. P., and E. A. Backus. 2004. Stylet penetration behaviors of Graphocephala atropunctata (Signoret) (Hem iptera, Cicadellidae): EPG waveform characterization and quantification. Annal. Entomol. Soc. Am. 97: 838-851. Anderson, W. G., T. M. Heng-moss, F. P. Baxendale, L. M. Baird, G. Sarath, and L. Higley. 2006. Chinch bug (Hemiptera: Blissidae) mouthpart morphology, probing frequencies, and locations on resistant and susceptible ge rmplasm. J. Econ. Entomol. 99: 212-221. Annan, I. B., G. A. Schaefers, W. M. Tingey and W. F. Tjallingii,. 1997. Stylet activity of cowpea aphid (Homoptera: Aphididae) on leaf extracts of resistant and susceptible cowpea cultivars. J. Insect Behav. 10: 603-618. Backus, E. A. 1994. History, development and applications of the AC electronic insect feeding monitor, pp. 1-51. In M. E. Ellsbury, D. E. Ullman a nd E. A. Backus (eds.), History, development and application of the AC elect ronic insect feeding monitor. Thomas Say Publications in Entomology, Entomological Society of America, Lanham, MD. Backus, E. A., and W. H. Bennett. 1992. New AC electronic insect feeding monitor for nestructure analysis of waveforms. Ann. Entomol. Soc. Am. 85: 437-444. Backus, E. A., W. B. Hunter, and C. N. Arne. 1988. Technique for staining leafhopper (Homoptera: Cicadellidae) salivary sheath and eg gs within unsectioned plant tissue. J. Econ. Entomol. 81: 1819-1823. Backus, E. A., J. Habibi, F. Yan, and M. Ellersieck. 2005. Stylet penetration by adult Homalodisca coagulata on grape: electrical penetration graph waveform characterization, tissue correlation, and possible im plications for transmission of Xylella fastidiosa Annal. Entomol. Soc. Am. 98: 787-813. Backus, E. A., A. R. Cline, M. R. Ellerseick, and M. S. Serrano. 2007. Lygus hesperus (Hemiptera: Miridae) feeding on cotton: new methods and parameters for analysis of nonsequential electrical penetration graph da ta. Annal. Entomol. Soc. Am. 100: 296-310. Barber, H. G. 1918. A new species of Leptoglossus : A new Blissus and varieties. Brooklyn Ent. Soc. Bull 13: 35-39. Barber, M. S., and H. J. Mitchell. 1997. Regulation of phenylpropa noid metabolism in relation to lignin biosynthesis in plants, pp. 243-293. In K. J. Jeon (ed.), Inte rnational review of cytology a survey of cell biology. Academic Press Inc., San Diego. Baxendale, F. P., T. M. Heng-Moss, and T. P. Riordan. 1999. Blisus occiduus Barber (Hemiptera: Lygaeidae): a new chinch bug pest of buffalograss. J. Econ. Entomol. 92: 1172-1176. Beard, J. B. 1973. Turfgrass: science and culture. Pr entice Hall, Englewood Cliffs, NJ.

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117 Busey, P. 1986. Morphological identification of St. A ugustinegrass cultivars. Crop Sci. 26: 2832. Busey, P., and B. J. Center. 1987. Southern chinch bug (Hemipte ra: Heteroptera: Lygaeidae) overcomes resistance in St. Augustineg rass. J. Econ. Entomol. 80: 608-611. Busey, P., and B. L. Coy. 1988. Vulnerability of St. Augustineg rass to the southern chinch bug. Proc. Fla. State Hort. Soc. 101: 132-135. Busey, P. 1990a. Inheritance of host adaptati on in the southern chinch bug Heteroptera:Lygaeidae). Ann. Entomol. Soc. Am. 83: 563-567. Busey, P. 1990b. Polyploid Stenotaphrum germplasm: resistance to the polyploid damaging population southern chinch bug. Crop Sci 30: 588-93. Busey, P., and E. I. Zaenker. 1992. Resistance bioassay fr om southern chinch bug (Heteroptera: Lygaeidae) J. Econ. Entomol. 85: 2032-2038. Busey, P. 1993. Registration of FX-10 St. A ugustinegrass. Crop Sci. 33: 214-215. Busey, P. 2003. St. Augustinegrass, Stenotaphrum secundatum ( Walt.) Kuntze, pp. 309 330. In M. D. Casler and R. R. Duncan (eds.), Bi ology, breeding, and geneti cs of turfgrasses. John Wiley and Sons, Inc., Hoboken, NJ. Caillaud, C., J. Pierre, B. Chaubet, and J. Di Pietro. 1995. Analysis of wheat resistance to the cereal aphid, Sitobion avenae, using electrical penetrati on graphs and flow charts combined with correspondence analysis. Entomol. Exp. Appl. 75: 9-18. Caillaud, C. M., and H. M. Niemeyer. 1996. Possible involvement of the phloem sealing system in the acceptance of a plant as host by an aphid. Cell. Mol. Life Sci. 52: 927-931. Campbell, B. C., and D. L. Dreyer. 1985. Host-plant resistance of sorghum: differential hydrolysis of sorghum pectic substan ces by polysaccharases of greenbug biotypes ( Schizaphis graminum Homoptera: Aphididae) Arch. Insect Biochem. Physiol. 2: 203215. Campbell, B. C., D. L. McLean, M. G. Kin sey, K. C. Jones, and D. L. Dreyer. 1982. Probing behavior of the greenbug (Schizaphis-graminum biotype-c) on resistant and susceptible varieties of sorghum. En tomol. Exp. Appl. 31: 140-146. Cardoza, Y. J., J. Reidy-Crofts, and O. R. Edwards. 2005. Differential interand intraspecific defense induction in Lupinus by Myzus persicae feeding. Entomol. Exp. Appl. 117: 155-163. Carter, R. P., and R. L. Duble. 1976. Variety evaluations in St. Augustinegrass for resistance to the southern lawn chinch bug, pp. 39. Tex. Agric. Exp. Stn. Prog. Rep. PR-3374C.

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134 BIOGRAPHICAL SKETCH Murugesan Rangasam y was born in Erugalur, a village 20 miles south of Salem, India in 1975 and is the only child of his parents. He graduated from Mallasamudram higher secondary school in 1992 and earned his Bachelors degr ee in Agriculture in 1999 from the Tamil Nadu Agricultural University, Coimba tore India. After graduating with the bachelor degree in agriculture, Murugesan worked as a Marketing De velopment Trainee with EID Parry (India) Ltd. Farm Input Division for eight m onths and involved in promoting an environmentally friendly insecticide, Neemazal, among cotton growers. He was the recipient of a prestigious Junior Research Fellowship awarded by Indian Council of Agricultural Resear ch for doing graduate studies in Entomology at the Punj ab Agricultural University, L udhiana, India, where he earned his masters in 2001. Upon graduation with Maste rs, he worked as a Senior Research Fellow on the World Bank sponsored National Agricultural T echnology at the Indian Agricultural Research Institute, New Delhi, India. His research proj ect aimed to develop ins ect and disease resistant cole crops such as cauliflower and cabbage. His undaunted zealous to work on insects, especially on host plant resistance, landed him at th e University of Florida with an opportunity to do PhD in Entomology. Murugesan investigated mechanisms of southern chinch bug resistance in St. Augustinegrasses. After earning his PhD in 2007, he is planning to c ontinue his career in research on insect plant interactions. Mur ugesan has always been a meritorious student throughout his carrier and recipient of many scholarships and fellowships for his studies which otherwise would have been extremely difficult. Murugesan has been married to Shunmuga Priya Jeevaratchagan, an Accounting Coordinator at the University of Florida Financial Services, for 2 years and the couple are blessed with a baby girl, Sudhikssha Murugesan, who will be turning one year on February 20, 2008.