The biology and ecology of Nomuraea rileyi (Farlow) Samson

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Title:
The biology and ecology of Nomuraea rileyi (Farlow) Samson
Physical Description:
x, 83 leaves : ill. ; 28 cm.
Language:
English
Creator:
Kish, Leslie Paul, 1944-
Publication Date:

Subjects

Subjects / Keywords:
Nomuraea rileyi   ( lcsh )
Fungi imperfecti   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1975.
Bibliography:
Includes bibliographical references (leaves 77-82).
Statement of Responsibility:
by Leslie Paul Kish.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000317742
notis - ABU4569
oclc - 08871560
System ID:
AA00003528:00001

Full Text












THE BICLOGY AND ECOLOGY OF Nomuraea riL-:- .i (FA!LCW SGAMSON!


By

Leslie Paul Kish











A IESEH' r .TION SL5-MITTED TO THE GDUA'IE COUNCIL Or


DEGE O DOCTCR OF PFHIL3 OSPi{





.;i'~LT :LTY OF FLORII-
1975




















I wish tc express my gratitude tc my supervisory committee and the

faculties of thi Eotany and Enri:' 'r,-,i Dezpartmer~rs for their counsel and

encouragement during this research project. The technical assistance of

the staff of the Pathology laboratory of -t-te Department of Entomology is

gratefully acknowledged.























TABLE CF A.


ACKNOWTLE.L ,E" ;- .S....................


LIST OF T L". ............ ...... .....


LIST OF FICRES!.................. ..


ABSTRACT . . .......


,T. ICTION ................... .....


:iatorlcal Perspective .........


Ecological Perspective.........


:f ~ ~A.1, .!',_; l,' ;AI,?; lA- . ..


a.sic Bioloogy of the Pathogen..


Cultural Characverist-ic..


Gen.eral..............


Effect of pH.........


Effect of Light and D


Effect of Temperature


Decreased C.::.- ,>7. ns


Conidial Qermination.


)


.............



.............


.............







.............


.............
.............


1. 11 .. .......



-............


.............



ark...........


............


ar..........


.............


ion.-,S.a.......


Conidial Production and Di


Coridial rrappiong .. .


Ccnidial Froduction per Cadaver.......................


Morpholcgy, and Development of Pathogen in Host ............


Influence of Integumen .... ........................


Cntogeny of Pathcgen. ... ..............................




iii


. . . ii


.. .............. vii


....................v ii
.................... v ii


lx



































. . . 6



. .... .. .. . .
..... .... ..... ...


. .










Sexual Reprfducion. .............................. -..

Fungal Ecology. ......... ......... .................. ..... 10

The Environment and Dispersal, ability, Infection and
Sporulation................................................ 10

Airborne Conidial Density ............................. 10

The F'r~ 'is Overwinter ...................... ........... 10

liability ...................................... 10

The Adult Moth and Dissemination .................... 11

Infection from Leafborne Conidia...................... 11

Infection and Humidity................................ 12

Infection and Temperature ............................ 12

Infection, Trrmp-rature and 'letness..................... 13

Conidiophorous Formation and Sccrulati)on.............. 13

Sporulation and Humidity.............................. 14

Irrigation............................................ 14

Infection and Fungicidal Spray....................... 15

Meteorological Monitori '. Systems.......................... 16

Sampling for Populatior and Tnfection Rates................ 17

Host Plant Considerations................................. 17

Soybean Variety ...................................... 17

Cultural Practices ................. .................. 18

OBSERVATIONS ...................................................... 19

Basic Biology of the Pathogen................................. 19

Cultural Characteristics................................... 19

General .............................................. -.

Effect of p ............ .. ........................... 19

Effect of Light and Darl .............................. 1

Effect of Tremperature................................. 19

iv










Decreased OxySen Tension ............................. 20

Conidial Germnin tion ................................. 2

Conidial Production end Dispersal......................... 20

Airborne Conidial Incidence........................... 20

Conidial Production per Cadaver...................... 21

:'r '.lo'y and Development of Pathoge n in ost............. 23

Influence of Host Integument.......................... 23

Mode of Infection.................................... 23

Ontogeny of Pathogen ................................. 23

Sexual Reproduction...................................... 24

Fungal Ecology ................................................. 24

Life Cycle Relationships................................. 24

Dynamics of an Epizootic .................................. 25

The Environnient and ii3perrsal, Viability, Infection
and Sporulation ............................... ............ 26

Airborne Conidial Density............................ 26

The Fungus Overwinter................................ 28

Viability................................ ............ 28

The Adult Moth and Dissemination..................... 28

Infection from Lea~borne Conidia..................... 28

Infection and Humidity ............................... 28

Infection and Temperature............................. 30

Infection, T2mperature and 3'...e..r: r ................... 30

Conidiophorous Formation and Sporulation............. 30

Sporulation and Humidity............................. 32

Irrigation........................................... 32

Infection and Fungicidal Spray....................... 35

DISCUSSION............................................................ 37

V









BI-LT (&9GA1PITY .........................

B ''17 APHiL L .... ... ... .. ... ... ... .. ... ... ... ..
















LIST OF TABLES


Page

Table 1. Conidial production of Nomuraea rileyi
on Pseudoplusia includens........................... 22

Table 2. Infection of Pseudoplusia includes by
Nomuraea rileyi and subsequent sporulation.......... 29

Table 3. Infection of soybean looper larvae by conidia
stored for three months under various conditions
of temperature and wetness.......................... 31

Table 4. Conidiophorous formation and conidiogenesis
under natural field conditions...................... 33

Table 5. Sporulation of Nomuraea rileyi on cadavers
of VBC pinned to soybean plants in the field........ 34

Table 6. Infection of larvae on soybean treated with
Benolate compared with infection in an
untreated plot ...................................... 36

Table 7. Meteorological conditions compared with
significant periods of infection and sporulation.... 45
















LIST OF FIGURES


Figure 1.



Figure 2.


Figures 3-6.

Figures 7-8.

Figure 9.


Figure 10.


Figure 11.


Figures 12-13.


Figure 14.


Figure 15.


Figure 16.


A chronology of airborne conidial
densities, air movement, foliage
wetting and precipitation......................

Conidial production relative to
insect surface area............................

Ontogeny of pathogen in host...................

Conidiophorous and conidial development........

Selected life cycle relationships
in a soybean agroecosystem....................

VBC population and infection rates
at the Quincy station, 1973....................

VBC population and infection rates
at the Quincy station, 1974 ...................

Adult moth showing conidia
attached to legs...............................

Air movement at the Quincy
station, 1973..................................

Foliage wetting at the Quincy
station, 1973..................................

Precipitation at the Quincy
station, 1973.................................


viii








A btr-act of Dissertati' on iPre-et'd to h Graduate C,:l
of th University of Florida i: ,e.r.. r.uslL ent of the e, C irem
for the DeFg--e f Doctor oL' philosophy


THE BIOLOGY AND ECOLOGY OF Nomuraea rileyi (FARLOW) SAMSON

By

Leslie Paul 7-:t.

December 1975

Chai.r-an: Dr_. Leland Shanor
oajYr Department: Botany

Bas~i biological and ecological aspects of the entorcgenous patho-

gen Iomiuraea rileyi (Fungi Lmperfecti) are investigated.

Cult;rai isolates from cadavers of the velvetbean cazerpiller grew

tnd sporula t-Ud on Sabouraud Mlaltose Agar in the pH range 5-9 in both

ig.Lht and dark regimers and under reduced oxygen tension. Opttrj-.

growtn d and s:orjlation occurs between 1.5 and 25C 5.

Conidia ge-niinated within 3 hours when pieces of host integu iren.t

were placed with them in a drop of water.

The time from exposure to the pathogen to death of the host average.

6 days. Infection occurs t rough t'he inteum,-- nt ~d also tb ingestion o

.onidia through feeding. ,-.e fuwn-.' ramifies throughout the host bcdy

via the hemiocoe. Conidiogenesis occurs in the 4? hoLrs foll2cirng death

at relative humidities above ,'.-. The number of conidia produced was

directly related to host size at death.

Field expericenr.s demonstrate iri'ect i:, -spor'- tlor, and dissem-

ination to be highly sensitive to meteorlogical condi bions and host pop-

LarTionc dernitieo. Field data indic'tde t~L.t sor,'- ideas concerning opti-

mn!i conditions for an epizootic, such as generally wet, relr conditions,

must be qualified before being applied s a getreral rule. Some










conditions may even be highly detrimental to the progression of various

phases of the disease. Aspects of natural epizootics of the pathogen

observed and monitored during 1973-75 are presented and discussed as to

their significance in the development of an integrated pest management

program for soybean.
















Historical Fersonective

The activity of entomogenous fungi has been noted mn-rl Tincs over

the years. !.-.-e fungi occur ~-ith such frequency and irfe-t -uch a vide

range of hcats that accounts of their activity are widespread throughout

the literaaturc.

.-. first type of insect infection definitely dentified ea to ic,-

microbiall nasure 'was? that of a fungus, demonstrated by Bassi (1835) on

a silwc-rm -adaver. Other accounts can be foud in the literature

describing what are, obviously fungal infections of insects such as t rise

described by Gray (1958) and the vegetable wasps and plant worfs of

Cooke (150o). Since Bassi, entomogenous f-ngi and their activities have

been described tine and again by many investigators. For instance, the

genus kno-rn today as ntomophthora -was first described as mniusa on the

common hcusefly by Cohn (1855). Other entonop-hthoraceous infecicns

were described on hosts by Fresenius (1856. 1858), Bruner (1833), Arthur

(1838), Thaxter (1888), Skaife (1925), Sawyer (1931), Ullyet- and

Schncrken (1940), and others over the last 35 years. Additional aspects

of ento!:opaethogenic i'fngi have been published since their original de-

criLoticn. More detailed historical accounts can be found in Steinhaus

(1956", 1.1ler-Kg!l.-r (1965), Madelin (1966), and Ferron (1975). Exten-

sive host. c-aT-alogues have been published by Sawda- (1919), Petch (1930,

1933), Chsrles (1941), Petch and Bisby (1950), Leatherdale (1958). 'us-

tafSson, (1965), and MlacLeod and "~il:,er- -ger ( "73). MYcolo'ical










investigations include t he vc.':.i..s .i.: of S.ccarc (1884 ::'i,*

10.--.ter 18538-193), Fawcett (1907-1948), Spe,.^ini (19S-1917', ?P;-.

(1907-1950), and Mains (19?9-1958).

Since Metchnk(off infected the larvae of Aniscplia austr,,.-a Tbst.

with spares of MetErrhi ziu anisopiae (Metch. ) Scr. in 1.879, .andreds,

possibly itoucsnds, of attempts have been made ':o control insect pests

utilizing fu 4i. imong these ire accounts f iahiiu. (r l-

stschik 18.86, 1 -8, Rorer 1910, 191.3, Frieder-:ihs 1913, Groenew-ege 1916,

Ru.gers 1916, Stevensorn 1918, ergula 1.31, SMr ions 1939), EntoLcphthora

(Howarnd 19C2, Speare and Colley 1912, Speare 1922, lustan 1924),

Aschersonir: ('. -.tson 1915), Isaria (Quayle anld Ti'or 1915'), and Beauveria

(Lugger 188~, Snow 1890, 1895, 1896-97, Lefebvre 1931, Soirrett et al.

1937, Beall et al. 1939, and Jaynes -and Marucci 1947). Txcellent reviews

are found in Steinhaus (1949) and r,.-- anrd Hussey (1972).

Probably the best summation of efffort. inn biological control was

that of the *e:fiZ.nent insect patho:lAgist E.A. Steinhsaus (1949, p. 666):

r.questionably, tho greatest stumbling block to the
widespread use of microargariisms ir. the artificial
control of insects is man's colossal ignorance of the
subject and his inability to control the various en-
vironmental factors that play thj dominant role in the
ecology of the diseases cc-icerr.e. It s euld be
repeatedly emphasized That th. itcr ..ol control. of
certain insects is being on.ino lyv ma Snined in
nature without 'he .Lei or tre i.lierference of msn.
In other cases, natural ep lcctics of disease break
o0.Ut from time to time :,J. :,=c.at;ntall, rluce pop-
ulations of insects. 'erfrore, Aheabher :te economic
entomologist wishes to recoLnize it or not, microbial
control of insects is effe;tvne, does ta.]e place in
-nature, and is of great .ve-'-all i-.port';ar. e.

That Steinhaus should address this evaluation of insect biological con-

trol to economic entomologists was -perceptiv. in ;two respects: (I) prob-

ably more than anyone in his ti-me. ':. r: u s r-: .>-:?:d the necessity









cf an interdi s lary approach o bil:?Jl control; (2) the ?C.ui:r.:ics

of food prodi.ction f-or a continuously raisin world population dic .ae

that biological control be developed to Its fullest potential, partic-

ularly for basic food crops as well as others of increasing importance.

A crop presently increasing in isprtnce is soybean, which consti-

tutes a basic source of protein for man both directly as food and indi-

rectly through i-s use as fodder for domestic animals. cTh uniqueness

of this plent i.es in its ability riTh the aid of bacteria, to fix atmos-

pheric nitrogen in the process of amino acid and protein sin :esis.

An increasi T dependence on this crop has rendered agriculture sen-

sitive to csybean pests. In Florida, where soybean production increased

to almost 30C,O ^ acres ii 1973, the velvetbesn caterpillar, A.tiarsi-

germmtalis Hiibner, is the most significant pest, having the capaSili y

to defoliate a crop in a ma.ttr of days.

Fortir.ately, this pe-st has its own enemies. One of the most de-

structive is the en.comogencus fru-is Nomuraea rileyi (Farlow) Samson

which under certain condi tio;ns in Florida can decimate the caterpillar

population causing mortality rates approaching 100%.

The f'n.s kv. lrnown today as Nomuraea rileyi was first described as

Nomuraea prasina by Maublanc (1903). Sawada (1919) transferred Nomuraea

prasina to the genus Spicaria. A long series of misinterpretations con-

cerning the concept of the genus Spicaria, originating with Harz (3.871)

and given _valic L.y by Saccardo (1886), culminated in the transfer of

Botryis rileyi to Spicaria rileyi (Charles 1936). After 1936, Spicaria

existed as an ra-biguous taxon, based on at leat two generic concepts

until Kish et al. (1974) transferred Spicaria rileyi to Nomuraea and

placed Spicaria prasina in synoncmiy.









Ecolo{! cal Pvr[:- i I -;

Under natural field conditions in .florid, phytophagous pest pop-

ulations begin a rapid increase within a few weeks of floweri.n of the

host plaits. As the larval population grows, defoliation increases. A

month after flowering, the popu nation of Antircarsi ('EC) may reach

population levels of 20 larvae per row lout, composed of large lroers

of third, fourth, and fifth instar larvae. It. is during this peak that

def.oliatiion damage exceeds the economic threschld.

The fungus also makes its appearance seen after the so;L't~ flowers,

but infections increase slowly in relation to the increase in pest pop-

ulation. From several weeks to a month after its appearance in the field,

the rate of infection explodes, engulfing the entire VBC population.

Unfortunately, with rare e-xception, the increasing inf el`t n curve

intersects the decreasing population curve approximately 2 weeks after

insect population levels have reached the damage threshold. From a bio-

logical control standpoint, it is this lag time between the initiation

of economic damage and the build-up of the fungus to epizootic propor-

tions that constitutes the difference between effective and ["e ff l-.tive

control.

The purpose of this research was to determi.re the complete fungal

life cycle of N. rileyi from both the morphological and ontogenetic stand-

point and to clarify ecological relationships and the extent and sig-

nificance of their variability.















-., -:-: AND MA',,T17 S
: --- f- ,,- --
Basic B-..... ;.. P t ."

Cultural characteristics

General--Living cultures of N. rileyi were inoculated on disposable

petri plates containing Sabouraud Maltose Agar with 1% yeast extract

(SBY) and on slants containing the same medium. The fungus was routinely

maintained in culture on this medium for most laboratory experimentation.

Effect of pH--The pH of SBY agar was adjusted vrith KOH an.d/or lac-

tic acid and buffered at pH 4,5,6,7,8, and 9, utilizing a 10% phosphate

buffer. Media buffered at individual pH values were poured into plates

and inoculated '-ith conidia of N. rileyi. Three replications were made

at each pH ralue. Cultures were held under ambient conditions.

Effect of Light an-. Dark--Culture plates containing SBY medium were

inoculated with conidia of N. rileyi. Five plates were inoculated and

grown in constant light: five plates were covered with aluminum foil and

grown in constant darkness: five plates were grown under a mixed light

regimen of 12 hours of light and 12 hours of darkness. All cultures

were held at 250C.

Effect of Temperature--Plates of SBY were inoculated with conidia

of N. rileyi and five replicates placed under each of the follc ~lii con-

stant temperatures: 50C, 100C, 150C, 30C, and 34C. Inoculated plates

were grov.w in a 12 hour light regimen. Addition. l plates were covered

with aluminum foil and held in constant darkness at '00 and 340C. Light

grown cultures were observed periodically for several weeks. Cultures









gro'n in darkness were 2Lserved only after terminaion fl the ex:..r;r.nt

(3 weeks).

Decreased Oxygen Tension--Tne effect of decreased oxygen tension on

growth and spor-ulation was investigated. Sabouraud vMIaltose Agar with

yeast extract was poured into Erlenmeyer flasks and inoculated with

conidia of N. rileyi. Half of the flasks were covered with aluminum

foil; all flasks were placed under vacuum. Cultures were kept under

vacuum for 2 months.

Conidij' il '-::r,'I.a.:i'i --Ei.j'.t Riddell mounts (Riddell 1950) were

placed in moist chambers. Each contained one agar block, approximately

1 cm' of the following media: water agar (WA), water agar with neopep-

tone (WAN), water agar with maltose (W,'I), water agar with yeast extract

(;.i.'), water agar with r:.-:,-tone and maltose (WANMl), and Sabouraud Mal-

tose Agar with yeast (SBY). Each was inoculated with conidia of Nomuraea

and Flaced on a laboratory counter under standard laboratory ccndciitions

of light and. Temperature. MounZts were examined hourly for conidial ger-

mination during the first 6 hours and daily thereafter for 9 days.

Conidia were placed on two additional SBY Riddell mounts in a moist

chamber, covered with aluminum foil, and placed overnight in a cabinet.

The following day mounts were examined for conidial germination.

Conidial Production and Dispersal

Conidial Trapping--In order to determine the relative density of air-

borne conidia over the soybean field on a seasonal, daily, and hourly

basis, conidial trapping mechanisms were placed in the field during the

third week of July 1974, approximately 6 weeks after the soybean were

planted. Traps were placed in both a dry plot and an irrigated plot, and

at a point midway between plots. Conidial density was sampled contin-









uously hourlyl) t rough-:'t the growing season until :-e first week in

October. Conidia ware -rapped on microscope slides v;hich -ere covered

(along the entire length of one side) with a strip of clear dual adhesive

tape. Each slide contained conidia sampled over 24 hours recorded in

24 bands. Slides were collected at 48 hour intervals.

Exposed slides were transported to the laborat.:- in. dustproof

cases. Hourly conidial counts (or estimation where large numbers of

conidia were encountered) were made and graphed. In order to minimize

errors due to incomplete banding (equipment malfunction or improper ori-

entation of the edge of the slide to the intake aperture), each hourly

count was averaged with the preceding hour's count and with the two sub-

sequent hours. Hourly data were utilized in raw form (unaveraged) for

correlation of substantial hourly increases and/or decreases of conidia

with meteorological data.

Conidial Production per Cadaver--In order to determine the number

of conidia produced on a cadaver, larvae of various sizes of Pseudoplusia

includes (Walker) were dusted with conidia and reared on media until

death occurred. Upon death, cadavers were measured in length and diameter

at a point one segment anterior to the first abdominal proleg. From

these linear measurements, surface area of the cadaver was tabulated.

The cadavers were then placed in a moist chamber for 3 to 5 days

during which conidiophorous and conidial formation occurred. The cadavers

were removed from the moist chambers and individually placed in a known

volume of water containing one drop of ordinary dishwashing liquid as a

wetting agent. After repeated agitation, a sample of the water-conidia

suspension was placed on a Bright-Line hemacytometer and the conidia

counted. From the volume containing a known number of conidia on the









hem~ac- .m. neer, ;he l.OTi co-iOi'k iln s.-s]"r.slon was determined. r-lc

derived in this rmanner, the nzribber of coni,'a on -he cadaver -as divided

by insect surface area in order to ,determine .c'nidial production per

unit surface area. Conidial prcduac2ion on various sized larvae as well

as production versus tine over a period of 3 to 5 days were determined.

All conidial data were subjected to standard regression analysis (Stele

and Torrie 1960) to determine the equation for ccnidial production per in-

sect. For complete details concerning methods, see fish and Allen (1976).
" -p... .. .. ,- .. -, r:., : _rcn .

Effect of Insect Integument--In order to determine the effect, if

any, of the presence of integument on conidial germination, molt integu-

ments were collected from various size larvae of P. includes and stored

in sterile vials in a refrigerator.

Conidia were placed in a drop of distilled water to which a small

piece of inzegumrent was added. This mixture was then covered with a

cover slip and placed in a moist chamber. The same procedure was used

for testing small, medium, and large larval integuments. The control

contained conidia in distilled water without the incorporation of pieces

of insect integument. Slides were observed at 3C-minute intervals for

the first 4 hours aid again after 24 and 48 hours.

Ontogeny of Pathogen--To determine the chronological and ontogenetic

sequence of infection, larvae were fixed at varying lengths of time after

dusting with conidia. Cadavers were dehydrated to butanol and imbedded

in paraffin. After microtoming, the sections were stained in either

congL red or lactophenol cotton blue. The sections were examined for

germinated and ungerminated conidia on the integument, penetration -nyphae,

and localization and form of the fungus inside the insect's body.










To determine the mode of infection, you-g larvae were isolated in a

weight boaT, and briefly and lightly touched with a camel hair ruLsh con-

taining conidia. The larvae '.ere placed in a closed ice cream >'onainer

which was kept moist with wet paper toweling in Th e ottom and maintained

without food overnight. The following day the larvae were removed from

the container, washed. vigorously with running tap water, and placed in

a new cup containing fresh rearing media. A second group of larvae were

placed on media containing conidia. After feeder: overnight larvae were

removed from the containers, washed vigorously with rurr:i. tap water,

and placed on media without conidia. Control larvae were not dusted.

All larvae were reared for 10 days and infection recorded within the

three groups. Three repetitions were made using a total of 60 larvae.

In order to obtain further evidence of mode of infection, 10 larvae

were fed one microdrop of an aqueous conidial suspension, administered

directly into the larva's mouth with a finely drawn glass pipette. A

second group of 10 larvae were fed one microdrop of water without conidia.

Ten additional larvae were inoculated with a microdrop of the conidial

suspension placed on the back of each larva. Each of the three groups

were placed in individual containers on a standard pinto bean diet and

observed for 10 days.

Portions of young larvae were also periodically squash-mounted after

dusting with conidia. Such mounts were generally limited to hemocoelomic

fluids and small portions of the body stained with lactophenol cotton

blue and examined under high power of a compound microscope.

Sexual Reproduction

Cultures of N. rileyi isolated from lepidopterous larvae from Brazil,

Israel, Florida, South Carolina, Illinois, 'isz-zsippi. Arkansas, Louisi-










ana, Iisscuri, and a stock culture frcm the Centraal-bureau voor Schirn-

meicultures (Baarn, The Netherlands) .re inoculated on a plate of SBY

in n attempt to induce sexual reproduc'tionr. One trial was held at stan-

dard laboratory conditions; a second trial was placed in an incubator at

90%' relative humidity, 320C night temperature and 380C dayBSti~ tempera-

ture (12 hours). Cultures v;ere observed for 3 months.

Fungal EccTL :."

Tne Envi.:_ojr.ent S and Dispereal, Yiability, Infection, and Sporulation

Airborne Conidial Density--Conidial density over the field was mon-

itored. continuously throughout the 1974 soybean growing season. Sea-

sonal, daily, and hourly density data were tabulated and correlated with

daily and hourly meteorological data. For correlative purposes, hourly

air movement was tabulated on a A--hour average. Fol"ia.; 'ettinL.g, drying,

and precipitation were plotted hourly as raw data. All data were plot-

ted on equivalent chronological scales and analyzed for correlation.

Ccrrelative ancmalies were examined from raw data charts.

.-." _.-.2. -, o- 'i...r--In order to determine a potential _of living

insects or cadavers in the soil as substrates for fungal overwintering,

approximately 48 cubic feet of soil in a soybean field were examined.

Lepidoptercus L ests in the field had urier.gcr.e an epizootic of N. rileyi

the previous year. The soil was selectively screened for the presence

of lepidopterous larvae, cadavers, and plant detritus. All stubble and

living and dead insects were examined in the laboratory for presence of

the pathcgen.

Viability--An attempt was made to determine the viability of coni-

dia exposed to the environment ir the soil through winter. Soil samples

were taken on 1 April 1974 from a field planted in soybean in 1973. The









VBC population in this field had undergone an epizootic of N. rileyi

lasting from August until mid October of 1073. The soil was screened,

placed in water suspension, and selectively centrifuged to retrieve coni-

dia. A drop of distilled water r containing conidia and a. piece of in-

sect integument was placed on a microscope slide, held in a moist cham-

ber, and observed at 30-minute intervals for 4 hours.

Adult Moth. and Conidial Dissemination--An attempt was made to deter-

mine the role, if any, of the adult moth in dissemination of the fungus

under natural conditions.

Potted soybean plants were raised in greenhouses during the winter

of 1974-75, Adult moths of both P. includes and A. gemmatalis were seg-

regated by sex, dusted i.th conidia of N. rileyl, and simultaneously re-

leased in the same ratio. Twenty-five mated females of each species were

released in one greenhouse and 25 males with 25 unmated females of each

species were released in a second greenhouse. One hundred mated female

moths of each species were released without dusting to serve as a control.

in a third greenhouse. Plants were inspected daily for populations of

healthy and diseased larvae.

Moths collected in the field during an epizootic in 1975 were trans-

ported to the laboratory in individual vials and fixed. Various body

parts were examined for conidia of N. rileyi.

Infection from Leafborne Conidia--A sample of laboratory-reared lar-

vae were fed leaves collected randomly from a field that had undergone an

epizootic of N. rileyi in 1974 to determine if initial infection could

result from conidia borne on leaves. Leaves were collected twice weekly

in the field, transported to the laboratory, and placed in rearing cham-

bers to serve as the only diet for larvae initially fed on media. Leaf









feeding began the second we5k1 of July 1975, a few days before any infec-

tion was no ted in the field. Larvae were observed for fungal infection

for 10 consecutive days.

Infection and Humidity--The effect of humidity on i.-fectin 7-as

investigated with the aid of an environmental chamber providing control

of temperature, photoperiod, and relative humidity.

Fifteen cups of rearing medium, each containing 3 larvae, o.re

dusted with conidia harvested directly from cadavers. Five test groups,

each containing 45 larvae, lvere dusted daily at 0500, 0800, 1300. 1530

and 2000 hours. After dusting, each cup of larvae was placed in .".ns

environmental chamber under a photoperiod corresp...i'' closely t day

length during July in Florida, a daytime temperature of 32C, and a

nighttime temperature of 2100, also tp:.' .-l for July. IHumidity was varied

for each trial, consisting of 225 larvae dusted at the times previc-'u1.

cited. Trials were run at 90%, 70%, and 50% relative humidity. An

undusted control group of 40 larvae was also placed in the chamber under

corresponding variations of humidity.

Total death counts were recorded for a period of 10 days after

dusting. The number of days from dusting to death of the first larva was

also recorded.

Infection and Tenmerature--I1'-. effect of below average temperatures

on infection was investigated utilizing the environmental chamber in the

laboratory. The photoperiod was held constant on a diurnal sequence of

16 hours of light. Relative humidity vas held above 90%.

Twenty cups of rearing medium, each containing 3 larvae, aere dusid

with conidia obtained directly from a cadaver. The cups were capped and

placed in the environmental chamber at 10C. A control group of 46 lar-










vae was set up in the saae maner, d c-d ~i-l cri..a, but retained

at 250C. Death due to the fungus \ws recorded or. a daily basis for both

groups for 10 days.

After 10 days, the dusted larvae subjected to the lower temperatures

were removed from the environmental chamber and maintained under ambient

conditions. For 10 additional days, death due to the fungus was recorded

for the same groups of larvae.

Infection. Temperature and Wetness--Conidia were harvested from sev-

eral cadavers in April 1974. The conidia were divided into four equal

lots and stored in small vials. One vial was placed in a refrigerator

with temperatures not exceeding 50C. Another was placed in a freezer

with temperatures not warmer than -160C. Approximately 2 ml of distilled

water were added to a third vial which was stored under ambient conditions.

The fourth vial, containing dry conidia at ambient conditions served as a

control.

On 10 July 1974, conidia were removed from storage and applied (as

dust or suspension in the case of the vial with water) to 3-day-old

larvae placed in cups of rearing medium, each containing three larvae.

Larvae were held on the medium for 10 days at which time total death due

to fungal infection was recorded.

Conidiophorous Formation and Sporulation--Approximately 5 row-feet

of soybean in a field undergoing an epizootic were enclosed by a cheese-

cloth tent. Over a period of 3 days, VBC larvae present on the plants

were tagged upon dying. The time of death was recorded. Cadavers were

observed each morning and evening thereafter through conidiophorous forma-

tion and conidiogenesis or until they were lost to physical or biotic fac-

tors. Times of conidiophorous (white stage) and conidial (green stage)










formation were recorded on the tags. '.,e.e data "wer correlated with

environmental conditions.

From 5 September 1973 through 20 October 1973, fourth and fifth

instar VBC larvae, freshly killed by Nomuraea, were pinned to leaves

in the field. Half of the larvae were pinned as close as possible to

the top of the canopy while the other half were pinned well in the middle

of the leafy section. Total cadavers undergoing sporulation as well as

comparisons between cadavers in the top and middle of the canopy were

made after cadavers were placed in the field. Sporulation was correlated

with weather data.

Sporulaticn and Humidity--In order to determine the effect of rela-

tive humidity on sporulation, dead larvae were held under the regimens

of relative humidity at which they were infected. Cadavers were incu-

bated at 90'5, 70%, and 50% relative humidity for 3 to 4 days after death.

During this time, the appearance of conidia imparted a green color to the

cadaver. Sporulation was considered to be optimum when conidia were pro-

duced in such large numbers that they settled around the cadaver giving

the appearance of a ring of green dust. Production of fewer conidia was

termed partial sporulation.

Irrigation--The effect of irrigation on conidial dispersal was in-

vestigated. Various sized larvae were dusted with conidia and held on

rearing medium until death. Upon death, insect surface area was computed

in the manner previously described for determination of conidial produc-

tion per cadaver. The cadavers were placed in a small, numbered weight

boat and maintained in a moist chamber for 5 days to insure maximum coni-

dial development. The number of conidia per cadaver was computed utilizing

a standard regression equation.










Cadavers, which in all cases adhered to the weight boat durii- con-

idiophorous and conidial development, were transported to the field in

covered petri plates to avoid conidial loss to the air during transport.

Once in the field, the weight boats were removed and carefully taped to

rigid stems of soybean plants. Cadavers were placed in both an irri-

gated and nonirrigated (check) plot.

Pieces of filter paper of known surface area were placed directly

below one cadaver in both the check and irrigated plots to determine if

conidia were washed to the ground. Both cadavers were the same size.

The irrigation system was activated for 2 hours and total precipi-

tation -was recorded. The cadavers in both plots were removed from the

plants, placed in individual vials, and capped. The filter papers be-

neath the cadavers were also placed in vials.

Each cadaver w'as subjected to the procedure outlined above for de-

termination of the number of conidia present after an exposure to the

elements in the field for a 2 hour period. Total conidia on each cada-

ver was tabulated and those cadavers exposed to irrigation were compared

with those of the check plot. The number of conidia collected on the

filter paper of the check and irrigated plots was determined.

Infection and Fungicidal Spray--To determine the effect of the

application of phytopathogenic fungicides on the course of an epizootic,

a one-third acre plot of soybean was sprayed (with Benolate applied at

a rate of 1 pound per acre) at the first sign of flowering (13 August

1975) and again 3 weeks later. A control plot adjacent to the Benolate

plot was untreated. Population counts and infection rates, extrapolated

from a -sairl of larvae held for 5 days in rearing cups, were computed

bi-weekly for both plots. Sampling was accomplished by the shake cloth









method (Barnes and Jones 1970).

Meteorological Monitoring Systemsir

Field experiments during 1973 and 1974 r;ere carried out at the

Agricultural Research and Education Center in C'uinc, Florida. Macroen-

vironmental monitoring facilities of the National Oceanographic Atmos-

pheric Administration Environmental Study Services Center in cooperation

with the University of Fiorida's Institute of Food and Agricultural

Sciences provided daily data on the following parameters:

(1) Solar radiation

(2) Pan evaporation

(3) Soil temperature

(4) Air temperature

(5) Eainfall and intensity

(6) Dew duration

(7) 's'ii; speed and direction

(8) Atmospheric pollutants

Microclimatological equipment and facilities of the Department of

Fruit Crops, University of Florida, in these field experiments included

the following:

(1) Sensitive wind speed and direction sensors having a stall

speed of less than one mile per hour

(2) Atkins lithium chloride sensors for dew point determination

(3) Dew duration sensors

(4) Air and soil temperature thermocouples

(5) Esterline Angus recorders for dew duration sensors

(6) Esterline Angus recorders for wind seed and direction

(7) Leeds and Northrop 24-point thermoccuplle -ze.eperature










(d) Honeywell recorder for dew point sensor temperatures

Environmental sampling was conducted continuously or at hourly intervaLs.

In 19`75, the environmental monitoring system was moved to a soybean

field at the University of Florida's horticultural farm approximately

10 miles nort-hwerst of :ainesville, Florida, where insect po-r:la:ion and

pathogen research were continued.

Data for the 3 :'er were reduced and analyzed. o correlate memcor-

logical data with infection and spor-lation inputs, various avera;gir.g

systems were employed to make the date reflect- tre_,ds during ti'e periods

considered sigi.ificar.t for analysis. Significant periods were based on

infection and sporulation data,

Sampling for Fopulatio a-nd Infection Rates

P- ul'sicn srempling during the soybean gro'r"irg seascr was =acom-

plished by t.'e a:ke cloth method. In 1974 and 1975, 1'' roC..feeT prer

acre were sampled in a pattern determine to be igni.fic.it by computer

analy:,i.: (Perscnal communication 1974, W.W. Menke, Department of Indus-

trial Management, Clemscin University, Clemson, South Carolina). Sam-

pling was conducted bi-v:eekly and pest populations computed on a row-

foot basis.

A sample of larvae, collected in the field by the shake cloth method,

was retained for laboratory observation in order to determine tne percen-

tage of fungal infection in the host population. The larvae were retained

for 5 days to i_':sure that infection occurred naturally in the field and

not as a consequence of collection methods or laboratory contiamiriation.

Death due to Noruraea was recorded daily.

Host, Planr, Consic.erations

Soybean Variety--All experimental plots were pLanted in Bragg soybean






18


during 1-73, 197L, and 1975.

Cultural Practice--Soybean were planted each year in 36 inch rows

at a rate of 12 seeds per row-foot. Cultivation was employed in Quincy

To control weeds in 1973 and 1974 and was generally completed twice be-

fore flowering. Soybean plots at the horticultural farm and experiment

station grounds on the University of Florida campus were treated with

Lasso herbicide at a rate of 25 pounds per acre.

















Basic Biology of the Pathogen

Cultural Characteristics

General--!-omuraea rileyi grew and sporulated slowly on SBY medium.

Under standard laboratory conditions the cultures first formed yeast-

like pustules, flesh tone in color, giving rise, in several days, to

short, densely packed, white conidiophores. The mycelial stage did not

spread over the culture medium and long-branching L,:.;i? were not evi-

dent. Cultures sporulated slowly, begirnnin at the center of the colony

and gradually spreading to the periphery until, after several weeks, the

entire f-unal iat bore a dense layer of green conidia. Cultures groin

under alternating light and dark photoperiods showed no evidence of di-

urnal perioicity for conidiogenesis.

Effect of ph--Groath and sporulation occurred at all pH ranges

tested, except at pH 4. Spcrulation was heavy at pH 6, 7, and 8, but '7as

slower and light on media of pH 5 and 9. Cultures growing on media of

pH 9 underwent a conspicuous and extended yeast-like phase.

Effect of Light and LDark--All cultures grew and sporulated.

Effect of Temperature--Nomuraea grew and sporulated slowly between

50C and 15C. Cultures grown at 300C and 340C remained yeast-like and

turned red. After several days, structures resembling sterile ascocarps

developed, but a week later cultures appeared to be disintegrating and

growth was arrested. Conidiophores and conidia developed within a few

days following removal of cultures from elevated temperatures.









Cultures grown in light and darkness and held under elevated temp-

eratures reach ted similarly, with the exception that the fungus grown in

the darkness lacked the red pigmentation.

Decreased Oxygen Tension--Cultures grown in full light and in dark-

ness grew and sporulated under decreased oxygen tension.

Conidial Germrination--Conidial germination took place in 4 hours at

a rate of 50% on WAMY at room temperature. Of the other media, SBY,

WA-MI, and WANT promoted a germination rate between 10 and 25% after 4

hours following inoculation. Occasional germination took place on W1i

and WAN in 4 hours. Conidia did not germinate on WA and WAY. Conidia

on SBY and VANY did not form typical germ tubes, but enlarged and became

irregularly shaped, thin walled, and yeast-like in appearance. The

growth of germ tubes on WAM and WAN was arrested after one day and no

further growth took place. Conidia germinated overnight in darkness at

room temperature.

Germ tubes were always singular and polar.

Conidial Productlon and Dispersal

Airborne Conidial Incidence--Conidia of N. rileyi first appeared in

the soybean field on 26 August 1974 in extremely small numbers and only

during several hours of the afternoon. The first larvae to die of

Nomuraea infecticn were collected on the same day. Conidial density re-

mained low (less than 100 during any given hour) until 5 September when

several hourly conidial counts surpassed 1000 per hour. By 16 September,

hourly conidial counts over 16,000 were recorded, increasing to over

20,0C00 y 21 Sep-emnber and to. over 70,000 for a one hour period of 27

September. Conidial density over the field gradually diminished to a

daily high of just over 1,000 for several hours on 3 October when the









traps were removed from the field.

Because of equipment malfunctions, only conidial density data from

the spore trap in the dry plot were deemed efficientlyl y complete for

correlative purposes. Conidial counts by day and by hour throughout the

period between 26 August and 3 October are presented graphically in

Figure 1.

Conidial Production per Cadaver--Data indicate that a definite rela-

tionship exists between cadaver size and conidial production.

Insect size for 14 trials ranged from 14 x 1 mm to 32 x 3 nm,

approaching the lower and upper limits for insect size at time of death.

Because of their age and the development time of the pathogen, small lar-

vae had to be infected soon after emergence from the egg. Larger larvae

became infected 7 to 10 days prior to prepupal development.

The curve (Fig. 2) representing the average data for the counting

trials was estimated by a standard regression analysis and falls within

a 99% confidence limit for conidial production values per insect surface

area. The equation fitting the curve is Y = -0.07544 + 0.00586(X) +

0.0000259(X2), ;here Y equals conidia x 109 and X equals insect surface

area in mn-. The variation in conidial production throughou- the sample

size range was determined at + 5 x 107 conidia at the P = 0.05 level.

This level allows a maximum 20% counting error for cadaver sizes at the

lower extreme of the curve, diminishing to 1.2% at the top of the curve.

Conidial production per unit surface area decreased with decreasing

cadaver size (Table 1). Because volume increases as a cube function of

linear measurements, and surface area increases only as a square func-

tion, larger insects provided a greater volume of nutrients per unit sur-

face area.









TABLE 1. Conidial production of Nomuraea rileyi on Pseuidoplusia includes.


Trial Reps Insect Size Surface Area Total conidia Conidia/rm2
(mm ( 10) x 106)


32 x

32 x

29 x

25 x

20 x

19 x

22 x

20 x

19 x

17 x

16 x

15 x

14 x

14 x


3

3

3

3

2.5

2.5

2



1

1

1

1

1

1
1


301

301

273

235

157

149

138

125

59

53

50

47

43

43


4.05

3.93

3.55

2.80

1.53

1.54

1.00

0.99

0.34

0.22

0.29

0.34

0.27

0.24


13.44

13.03

13.02

11.93

9.71

10.33

7.26

7.89

5.81

4.11

5.74

7.33

6.22

5.59


* Falling outside confidence limits.


---~-











influence of Host Inte1r' t--Conidia er-snated on all slides with

integument present. Some germ tubes were discernable after 2.5 hours,

however, most germination took place between 3 and 3.5 hours.

Conidia were observed to germinate along a distance gradient. Those

conidia nearest a piece of integument germinated first; conidia more dis-

tant from the in;t',-gme:.t germinated in sequence relative to their prox-

imity to the int. r ieit.

Mode of Infection--Within 7 days after application of conidia to the

integument of the larvae. 87.5% of the larvae had died of .nmuraea infec-

tion. Mortality was 100% after 10 days. Those larvae which fed for 24

hours on media dusted with conidia suffered no mortality during the first

7 days and 4( after 10 days. All controls survived without disease inci-

dence during the same time period.

Larvae which were force-fed a conidial suspension of Nomuraea suffered,

30% mortality in 8 days, the same infection rate of those larvae on which

conidia had been applied directly to the cuticle. All control larvae

developed to maturity.

Ontogeny of the Pathogen--Conidia in contact with the insect integu-

ment germinated in less than 8 hours (Fig. 3). Conidia and germ tubes

became appressed to the integument in most cases but infecting germ tubes

which penetrated the integument from some distance, were observed 15 hours

after dusting. In many cases, the integument surrounding the point of

penetration became blackened and ulcerous (Fig. 4). Once within the epi-

cuticle, the furg-s lysed large cavities in the exocuticle and endocuticle

and showed no marked inclination to penetrate and grow perpendicularly to

the surface of the epicuticle. The fungus maintained filamentous growth









while penetrati:.- the integu-enT. After penetrating the hemocoel, the

mycelium fragmented and multiplied in a yeast-like fashion (Fig. 5).

After 4 days the host became sluggish and moribund. Squash mounts

demonstrated the spread of the fungus throughout the hemocoel, partic-

ularly in the posterior segments, and the host tissues near points of

penetration. The fungus did not appear to have an affinity for the gut

region, and did not invade tissues of the head until after death.

A few hours before death of the host, the fungal cells elongated and

became fusiform (Fig. 6).

Just prior to death, the insect usually climbed'to the upppermost

leaves of the host plant and a sticky secretion issued from the anus,

firmly cementing the insect to the plant. In many cases, the insect

died with the anterior portion of its body in an elevated position.

Under conditions of relative humidity above 70%, conidiogenesis

commenced 24 to 36 hours after the cadaver turned white with the matura-

tion of conidiophores (Fig. 7). Forty-eight hours after conidial for-

mation, the cadaver appeared green because of the large numbers of

conidia produced (Fig. 8).

Sexual Reproduction

All isolates grew and formed conidia. However, mycelia of the vari-

ous isolates remained separate.

No sexual fruiting bodies were observed nor could any be induced to

develop.

Fungal Ecology

LZie Cycle Relationships

Life cycle relationships within the soybean agroecosystem are depic-

ted for the VBC in Figure 9. Relationships of other soybean pests were









similar in most respects but differed slightly in time of appearance.

The life cycle of the soybean, Glycine max, (L.) Merr., is basic to

all other cycles in the system. The growing season in Florida extends

from planting, usually in early June, to early October when the plants

become senescent. From this time until the following growing season,

the soybean exist only in seed form. For most of the year, the dead

plants figure in a significant detritis cycle w.thithin the system.

As depicted on Figure 9, the VBC made its appearance in early July,

though some insects were occasionally seen earlier. The adult moths ovi-

posited beneath the leaves from mid-July tl]rch September. Between Sep-

tember and the following July, the insect was absent from the soybean

system but ;was occasionally found on alternate leguminous plants not

killed by freeze.

The appearance of Nomuraea in the system was tertiary to that of the

plants and lepidopterous larvae. Seemingly dependent on host density,

the f':!.u;.s made its appearance on a relatively few larvae in early Aug-

ust. By the third week in August it had become firmly established and

had infected nearly 100% of the VBC. Through abundant infection, sporu-

lation, and dissemination, the fungus perpetuated itself through what may

be depicted as a short cycle, which lasted until mid-October when the

beans were harvested. Survival mechanism of the fungus through the winter

was not determined.

Dynamics of an Epizootic

Early in August 1973, interactions between principal organisms became

firmly established. Moths appeared and deposited their eggs beneath the

leaves. The fungus was only occasionally found in isolated parts of the

field, while the first generation larval population density was less than









one per row-foot. Infection rates amcng larval population of 3-5 per

row-foot (Fig. 10) rose steadily to 22% and 24%, respectively, in two

plots by 24 August and approached ICCI by the end of the month. The VBC

population reached a peak of approximately six larvae per row-foot in

the two plots on 22 August and fell precipitously to less than one per

row-foot on 19 September. During this time, infection rates in the field

were 'l'". After 30 September infection rates dropped sharply approach-

ing the 60% range by 10 October when sampling was discontinued. Larval

population increased slightly in one plot after 30 September but continued

to decrease in the other plot.

The rapid increase and lateral spread of the disease to epizootic

proportions emanated from "hot spots" of infected cadavers in the field

and was dependent on -,:.essfZul progression of the fungal life cycle

through three major stages of development, each sensitive to biotic and

physical factors within the system.

The Environment and Dispersal, Viability, Infection, and Sporulation

Ai:b:.: "r.id.'al r c:.t-'+.---As presented in Figures 1 and 11, conidial

density over the field was directly related to (1) the infection rate in

the host population (2) the daily hours of foliage wetting and drying

(3) precipitation and (4) air movement.

Velvetbean caterpillar larvae were first observed on 16 August. Lar-

val density rose to approximately one larva per row-foot on 26 August,

the first day infection was noted among field collected larvae and when

conidia first appeared in the spore traps. A total of five conidia were

recovered over a period of 13 hours. On 1 September, the larval popula-

tion reached four larvae per row-foot with an infection rate of less than

6%. No conidia were recovered on that day. The larval population









peaked at more than six larvae per row-fcct on 10 SepIeicber while the

infection rate climbed to 39%. The spcr trap i.:c b-etween the two

plots collected over 55,C00 conidia in a 23-hour period. On 19 Septem-

ber, infection rate reached 98% of the larvae in the field and the coni-

dial count was well over 100,000 for a 24-hour period extending into 20

September

Daily airborne conidial densities were also directly related to the

foliage wtting .. Conidial densities demonstrated a periodicity of rel-

atively low counts at night when the foliage was wet and higher counts

during the day when, with the exception of rain, the foliage was dry.

Precipitation data (Fig. 1), when correlated with conidial densities,

indicate that, almost without exception, rain is followed by lower air-

borne conidial densities (7, 9, 10, 12, and 16 September). The notable

exception to this observation was the effect of a trace to several hund-

redths of an inch of rain which seemed to be followed by increased con.id-

ial counts (26, 27, and 28 September).

Air movement, like foliage wetting, demonstrated a periodicity dur-

ing the observations (Fig. 1). Air movement was consistently higher

during the daylight hours than at night. High conidial counts correlated

with periods of high air movement in combination with dry foliage.

Peak hours of conidial density usually occurred during periods of gusty

winds associated with thunderstorms or passing weather fronts; there

were a total of eight such fronts during September 1974 at the Quincy

station. Of particular note is the early afternoon of 27 September

(1300 hours) during the passage of a warm front accompanied by high winds.

Conidial counts were estimated at over 150,000 between 1200 and 1400

hours and contributed to the hirhest daily total recorded in 1974.









_i_ _-_r C__er"-ner--io trace of cadavers could be found in the

selectively screened soil. Four living larvae in a prepupal state were

recovered and tentatively identified as VBC. The insects held in moist

chambers in the laboratory ,ppatea in one week.

Viability--Conidia obtained from soil samples did not germinate

after 4 hours.

Adult Moth and Dissemination--Female moths released into the green-

houses oviposited on the plants. Two weeks after releasing the moths,

l-arvae cf b th the VBC and soybean loopers were observed, however, no

infection cf the larvae by Nomuraea occurred.

Conidia were observed on the legs of moths collected in the field

during an epic-ctic of Nomuraea in September 1975. The conidia were

adherent to the tarsal claws and scattered among the leg scales (Figs.

12-13 ).

Infection from Leafborne Conidia--Larvae fed on field collected soy-

bean leaves did not beccre infected by Nomuraea until 26 August even

though laborat cry reared lots of field collected larvae occasionally

became infcted beginning the week of 14 July. At the time of the first

infection on the leaf fed larvae the infection in the field, as computed

from laborat-ory based estimates of field collected larvae, was 1%.

Infection and Humidity--Infection data for the humidity trials are

given in Table ". The average infection rate at 90% relative humidity

was 64%, and at '70 relative humidity was 68%. No infection occurred at

50',. tAltho/ugh some variation in infection rates among groups dusted at

various times .di occur, no diurnal pattern was established.

At 90% arnd 70% relative humidity, the first larvae died 129 hours

after dusting (5.4 days).

No deaths due to Nomuraea were recorded in the controls at any








TABLE 2. I1nfetiCon f Pseudoplusia includes by 'i-nu.rs. r:.e-'i and subsequent
sporulation.


Humidity setting Dusting Time Percent Infection Percent Sporulation


90+ 2000 70 83 (t)

0230 67 75 (t)

0330 67 75 (t)

0800 49 100 (t)

70 2000 80 19 (t) 36 (p)

0100 55 0 (t) 64 (p)

0330 64 0 (t) 69 (p)

0800 80 0 (t) 64 (p)

1700 64 0 (t) 76 (p)

50* 2000 0 0

0100 0 0

0330 0 0

0800 0 0

1700 0 0


* three replications with identical results
(t) total
(p) partial









',i- dt, level.


Infection and Temperature--No infection of larvae dus ted and main-

tained at 10CC was noted after 10 days. A control group, held under am-

bient temperatures, z;fVfered 767 mortality during ihe same time period.

When the test group of larvae ,was removed from the influence of the

lower temperature, t3" died of Ncmuraea infection within the following 10

days. The control group suffered an additional mortality on the elev-

enth day after which h all control larvae pupated without additional mor-

tality.

Infection, Temperature, and Wetness--Results of reinfectivity studies

utilizing ccnidia stored over 3 months under wet and dry conditions and

at various temperature regimens are given in Table 3. Conidia stored

under cold, dry -onditions maintained the ability to reinfect the host

after more -than 3 months, while those stored under ambient temperatures

were considerably less effective in causing infection. Conidia stored in

water were not viable after the 3-month period.

Conidiophorous Formation and Sporulation--A total of eight VBC larvae

died between 9 September and 11 September. Three of the eight cadavers

were lost to predation or unknown causes. All larvae except one died

during the night and conidiophores were conspicuous by 0900 hours the

next morning. ornidiophorous formation took place in 15 hours in all

cases in which the larvae died at night. The hours of foliage wetting

are given in Figure 1 which shows that during the period 11-14 Septem-

ber at least 8 hours of foliage wetting occurred each night with accom-

panying high huIridity that favored conidial formation. No apparent rea-

son is known why sporulation took place on some cadavers on 13 September

while others, which had died at the same time, underwent sporulation










TABLE 3. Infection of Soybean Ico.,er larvae
by conidia stored for three months
under various conditions of temp-
erature and wetness.


Conidia Stored Infection after 10 days



-16C 64%

500 62%

250C (dry) 7%

250C (wet) 0%









a day later. A chrcnol .-. of fungal development is given in Table 4.

Of the 227 cadavers placed in the field during Sect-,en~:r and October

of 1973, 61% turned green with at least partial conidial production.

Sporulation was significantly higher daring September in both the upper

and lower canopy. The sporulation rate relative to time, position in

the canopy, and total precipitation during the observation period is

given in Table 5. With the exception of those placed in the field on

26 September, more cadavers underwent snorulation in the -:.ddie of the

canopy than at the top, if sporulation occurred at all. Sporulation was

complete on 37% of the cadavers; partial sporulation occurred on 24% of

the cadayv.-r- at the top of the canopy and 46% of the cadavers in the

middle.

Data demonstrate that precipitation occurred on 10 different days

during the period 5-30 September while the period 1-20 October had only

one day of precipitation.

Sporulation and Humidit ---- :ults of sporulation experiments at var-

ious humidities are given in Table 2. No conidial formation took place

below 70% relative humidity. Optimum conidial production occurred at,

90% relative humidity or above.

Irrigation-Irrigat--ri ion resulted in a total of 0.72 inches of precip-

itation in the 2-hour period.

Cadavers exposed to irrigation lost an a.'cra'e of 91% of the conidia

calculated to be present. During the same time period, those cadavers

not exposed to irrigation lost 49% of the conidia. Size of the cadavers

ranged from 13.6 x 1.2 mm to 30 x 3 mm.

After irrigation, the filter paper below the cadaver in the irrigated

plot contained 2,115 conidia per mm2. In contrast, the paper below the









TABLE 4. Conidiophorous formation .ana ccnidiagenecis
under natural field conditions.


Cadaver Date/Time of Death White Green



1 night 9/9-10 9/10 9/13

2 night 9/9-10 9/10 9/13

3* night 9/10-11 9/11

A* night 9/10-11 9/11

5 night 9/10-11 9/11 9/14

6 night 9/10-11 9/11 9/13

7 night 9/10-11 9/11 9/14

8* morning 9/11


* lost due to predation

















-p

*0
O,









*H
I)

F-

























0
0
rFi





05


















H
o0






-'


0







C-
u






r?
O
0








0,
(^

d
*0











-a

*r\
S-1


0 0 0 0 0 0 o .C C 0 0 0 0 0 0
0 n 0 tO a' L> 0'7 0 0 L-N- C- C\2 0 0 0 0
















0 0 0 0 o0 0 0
1-4 il rE

















0 -P 0 4-' 3} 0 + 0 0 0 -P 0 -P 0 t -
P0 P 0 0 -P 0 0 0 4 0 -0- 00









cadaver in the dry plot contained 272 conidia per r= or one-seventh of

the total found on the ;e:r in the irrigated plot.

Infection and Fungicidal Spray--P"or .o a treat ment of Benolate on

13 August 1975, six larvae infected with Nomuraea had been found. The

infection rate on 11 August, based on laboratory rearirg records of field

collected larvae, was 55 in the test plot and slightly over 1% in the

check plot. The infection rate on 14 August was 0Q for the Benolate

plots and 9% in the check plots. On 19 August, the Benolate plot in-

fection rate had risen to 2' and the check plot infection rate had climbed

to 20%. From 25 August until 3 September, no deaths due to the fungus

occurred in laboratory reared lots of larvae collected from the Benolate

plot, while infection rates in the check plot were 42% on 25 August, 63%

on 29 August, and 55% on 1 September.

Population and infection data for the two plots are given in Table 6.









TABLE 6. Infection of larvae on s:.:1-,. Treated with Beno-
late compared with infection n in an treated plot.



Sample Date Plot Larvae/Row Ft Infection Rate


7/16/75



7/23/75



7/30/75



8/6/75



8/11/75



8/14/75



8/19/75



8/21/75



8/25/75



8/29/75



9/1/75


0.36

0.35

0.56

0.54

1.05

1.10

1.63

2.35

4.99

3.96

3.16

2.62

1.56

1.82

1.04

1.94

1.86

1.63

1.46

1.12

2.00

1.14


0%

3%

0%

0%

0%

0%

0%

2%

5%

1%

0%

9%

2%

20%

4%

31%

0%

42%

0%

63%

0%

56%


* treated with Benolate















DISCUSSION

The consequences of overpopulation in relation to food production

have become increasingly apparent in recent years. Production has in-

creased, particularly in the industrialized nations, largely because of

man's ability to overcome limiting, environmental pressures. This

success has neither been without substantial cost and effort (i.e.

today's eergy consuming technology), nor can it be rated as unqualified

in view of widespread food shortages. A realistic evaluation cf food

production for a world population increasing to infinity faces stark

recognition of -he inevitable. This earth, with limited sourcesce, will

support only a limited population. As the population limit is approached,

man's alternatives are to soften the impact in underdeveloped countries

through massive food sharing programs, which can only delay a final,

difficult transition to a steady state population.

Methods used to increase food production fall within a few categor-

ies: (1) improved cultural practices; (2) improved genetic hybrids;

(3) massive application of fertilizer; and (4) chemical control of pests

and pathogens. All of these methods have finite limits, or stated

differently, are governed by the economic laws of diminishing returns.

Not only is it disheartening to recognize the limitations of our capabil-

ities in controlling our environment, but it is also disillusioning to

realize that some of our efforts are creating ecological problems far

worse in long term consequences than the short term problems they are

designed to alleviate. The polluting effects of agricultural chemicals,










particularly DDT dichlorodiphenyltrichloroethanee) and other chlorinated

hydrocarbon pesticides, have resulted in more stringent control or, in

some cases, banning of some of our most effective control agents. In-

creased food production as a consequence of chemical control must be

foregone in ever-increasing measures for man's long term survival.

Obviously, some program which is effective, economical, and ecologically

sound must be developed to effect control where agrochemicals are banned

or found undesirable.

The potential of biological control of insects has been actively

investigated since the latter part of the 19th century, but has met with

only limited success. An integrated approach to pest control, incor-

porating reproducible, predictive control capabilities is yet to be

formulated.

Although there have been attempts in the past to predict plant

disease (Cook 1949, van der Plank 1963, Waggoner and Horsfall 1969),

there is no precedent for such an approach for insect pests of food crops.

Many instances of biological control of insects by artificial dissem-

ination of fungal spores or infected cadavers appear in the literature.

The reader is referred to accounts in Steinhaus (1949) and Burges and

Hussey (1971). More extensive work, utilizing a combination of fungi

and weak insecticides, has been reported by Russian investigators (apud

Bucher, 1964).

It becomes apparent, as one reviews the literature, that inputs from

the ecosystem as a unity, incorporated into an integrated pest management

program, are necessary to make biological control a consistently reliable

control measure.

Natural control by N. rileyi is ineffective from a biological control









standpoint for 2 reasons: (1) nan's !acK of control over natural condi-

tions which may or may not be favorable for d-eieloment of an epizootic

and (2) a matter of timing which allo,-s for severe defoliation during

the 10 days to 2 week delay between the peak of the pest population and

the occurrence of the fungus in epizootic proportions.

The potential of N. rileyi as a biological control agent in a sys-

tematic program has been virtually unexplcred. Although various aspects

of its basic biology have been reported by Getzin (1961), the mode of

penetration and ontogeny of Nomuraea have not been investigated previously.

The mode of infection and development of Nomuraea do not seem to

be far different from Metarrhizium as reported by McCauley et al. (1968)

or Beauveria reported by Fargues and Vey (1974). Nomuraea appears to

differ frcm Metarrhizium in that infection via the gut can occur even

though it does not seem to be the primary route.

Although germinating conidia were not tested for enzymatic activity,

production of such enzymes has been reported for several species of Ento-

mophthora Gabriel (1968) and for Metarrhizium McCauley et al. (1968).

It is probable that when infection by Nomuraea is studied from a physio-

logical standpoint, enzymes will be found that aid in the infection

process. Further studies on an ultrastructure level such as those

accomplished on Metarrhizium by Zacharuk (1970) are needed for Ncmuraea.

Information concerning pathogenicity and field observations of

epizootics has been reported by Watson (1915), Hinds and Osterberger

(1931), Charles (1938), Gudauskas and Canerday (1966), Behnke and Paschke

(1966), Burleigh (1972), Allen et al. (1971), and Ignoffo et al. (1975).

In its basic biology, Nomuraea does not distir'uish itself from the

majority of entomogenous fungi in particular, or from 7 -;:i I.perfecti









in general.

As with so many entomopathogens, however, Nomuraea exhibits those

peculiarities of growth common to organisms of a pathogenic nature.

These include slow growth and sporulation, slow germination of conidia,

and a requirement for a medium rich in protein and carbohydrates. Some

of the more categorical features include a preference for lepidopterous

larvae as hosts and the common occurrence of epizootics in populations

of larvae which feed on legume crop plants.

Although no sexual reproduction was observed or could be induced,

the formation of reddish, sterile ascocarp-like structures at elevated

temperatures suggestss that this fungus, under proper conditions yet to

be elucidated, may undergo sexual reproduction and ascocarp formation

similar to that found in the Hypocreales. In this order, which contains

several entomopathogens, formation of a phialosporic asexual state is

common,

A single cadaver of P. includes, 32mm in length and 3mm in diameter,

produces in excess of four billion conidia under optimum conditions.

Such an abundant production of propagules suggests that this is a

mechanism evolved to compensate for the apparent frailty of conidia

under natural environmental conditions.

A number of factors regulate the pathogen in the field: (1) the

lack of longevity of conidia which have a half-life of 2 days (Personal

communication 1975, C.M. Ignoffo); (2) the apparent lack of an over-

wintering spore or large host population during the winter; (3) the

dilution factor of conidia disseminated by wind; and (4) the critical

phases of catch and infection which are dependent on a combination of

biotic and physical parameters. The above factors operate to limit the









viability of sL conidia produced. The irect and indirect effects of

long-range dispersal present special problems in determining the manner

in which the fungus infects larval r'.iltions in areas cultivated for

the first time. The" dispersal of fungi by moths has been reported by

Mioreno et al. (1972). Conidia of N. rileyi have been observed on the

legs of moths collected in the field during an epizootic. Since VBC

moths are known to migrate on a seasonal basis, there is no logical rea-

son to doubt that they may carry their own control measures as they

travel fro one host plant population to another. Evidence exists that

the VBC in particular may be present south of Orlando, Florida, all year-

around, and migrations of these pests from low density overwintering pop-

ulations may initiate or at least contribute to local populations during

the growing season north of Orlando (Personal communication 1974, G.L.

Greene, Agricultural Research and Education Center, Quincy, Florida).

In more northern, continental climates, diapausing larvae may serve as

overwintering hosts or the pathogen may pass the winter in a resistant

asexual or sexual spore stage.

The daily fluctuation of conidial densities over the field correlate

well with meteorological conditions, host population density, and infec-

tion rates. Conidia did not appear in spore traps in 1974 or 1975 until

the host population had reached one larva per row-foot and the first

deaths due to Nomuraea infection had been observed among field sampled,

laboratory reared larvae. Seasonally, airborne conidial density increases

with increasing infection rates. On a daily basis, high hourly densities

correlate with dry foliage and high air movement. As indicated by field

experiments; utilizing irrigation and supported to a great extent by pre-

cipitation and conidial count data, the fi.'dr 'cphcbic conidia are readily









washed to the ground as a result of rain, heavy dew, or irriga.Ton.

Potential for contact with the host and subsequent infection by conidia

is diminished. Clerk (1968) reported that aqueous extracts of soil

actually inhibited the germination of conidia of Beauveria bassiana and

.t 3l'.'-hiziuam anisopliae.

The viability of conidia in the soil over winter is further put in

doubt by the results of reinfectivity studies. Only completely dry

conidia or those stored at reduced temperatures maintained the ability to

infect the host. Those conidia stored at ambient temperatures were con-

siderably less active than those stored at low temperatures.

The effect of the fungicide treatment on the infection rate within

the host population was dramatic. Two weeks after the first application

of Denolate, Nomuraea had become firmly established in the check plot yet

was rarely encountered in the treated plot immediately adjacent. Two

striking consequences of the fungicide treatments were evident: (1) Al-

though the larval population at the test plot initially fell, a month

later after two applications of Benolate it had increased. (2) There

was no natural control by Nomuraea in the test plot.

One can immediately see the negative economic effects of the fungi-

cide treatment. First, the cost of the fungicide application and second,

after a month, the insect population was higher and no natural control

was established. In addition, the fungicide treatment, routinely conduc-

ted by farmers regardless of the incidence of infection, was unnecessary

as no phytopathogens became established in any of the plots during 3

vear- of field ;ork.

The effects of pesticides have received increasing attention by

several investigators and indirect effects have been reported as harmful









to various aspects 'f the agroecosystem (Fargues 1973; Person.al '-o.uni-

caticn 1975, C.i. Ignoffo). Other investigators, however, are experimen-

ting with the utilization of pesticides in conjunction with biological

control measures (Telenga 1964) and some, particularly Russian workers,

claim significant progress (apud Bucher 1964). It is clear from experi-

mental evidence that further research must be conducted on the effects

and beneficial utilization of agrochemicals.

Meteorological data of 1973 were reduced and observations and para-

meter values analyzed for correlations. Only those dynamics of the bio-

tic and physical environment indicating correlative relationships are

discussed below.

To correlate meteorological data with infection and sporulation in-

puts, an averi;;!. system was devised to reflect trends during time peri-

ods considered significant for analysis of infection and sporulation data.

Daily air movement (Fig. 14) and foliage wetting (Fig. 15) were plotted

as a mean for 7 consecutive days, the approximate time period from infec-

tion of the host to its death. The purpose was to examine each day as

part of a 7-day period, rendering a daily parameter guide of the infection-

to-death period. Such an approach was necessary because collections

were made at approximately 7-day intervals and determination of the spe-

cific date of infection was impossible. Precipitation was also plotted

as a mean for 7-day periods (Fig. 16) for reasons similar to those

applicable to air movement and foliage wetting. Thus, any one point on

these graphs indicates the mean air movement, mean hours of foliage wet-

ting, or total precipitation for the 7-day period beginning on any given

day.

Significant periods were based on infection and sporulation data.









..-.ty-; verb percent of the insects collect & or 31 A;1'-t (extrapolated

fron 1C fourl iLstar larvae) died within the 31 August 6 September

period. This means that an insect that died on 31 August was infected on

25 August; those dying on 1 September were infected on 26 August, etc.

Due to this high mortality rate, the period 25 August thrcigh 31 August

was significant in that all conditions were favorable for infection. By

the saLc reasoning, the period from 28 September through 4 October was

less tha) o-timal for infection because larvae collected on 4 October

suffered only 833 mortality in the following 7 days.

After insect death, but before the mycelium protruded from the in-

tegimentL the insects were pinned to leaves in the field and observed for

development of conidia. Significant periods for sporulation were de+er-

mined in a similar manner to those deemed significant for infection.

Meteorological conditions during significant periods are presented

in Table 7. It can be readily seen that only when environmental param-

eters are observed collectively can significant correlations be made.

A combination of high air movement, little precipitation, and approx-

imately 10 hours of foliage wetting favored infection during the period

from 25 August through 31 August. It is apparent that dissemination of

conidia is an extremely important preliminary step to the actual spread

of disease. Conidia were dispersed well only under relatively dry,

windy conditions. Not only did such conditions not exist during the

period from 2 September to 4 October, but any conidia present were prob-

ably mechanically beaten or washed from foliage by heavy rains.

It also is apparent that optimum conditions for infection are not

necessarily op-imnum for sporulation. With the exception of high air

movement during the period from 31 August to 6 September, (which was











TABLE 7. Meteorological conditions ccMpared with significant periods of
infection and sporulation.


Air Movement
(mli)


Total Precipitation
(In)


IFEcTI::

Period:

Period:



Period:

Period:

SPORULATION

Period:

Period:


8/25-8/31

9/28-10/4



8/31-9/6

10/4-10/10



9/13-9/19

9/19-9/25


45/day (78 daily high)

23/day (44 daily high)



72/day

48/day



41/day

41/day


0.40

2.00



1.50

0.00



0.30

0.00


_


_ __ I I


_ __ I _I_










TABLE 7 extended


Mean Foliage Wetting
(hr )


10.5

19.0


Day Precipitation
(in)


5 dry + 0.10 + 0.30

(1.4) 3 dry + 1.12 + 0.50 + 0.20 + 0.26




3 dry + 0.30 + 0.25 + 0.50 + 0.60

all dry




4 dry + 0.04 + 0.08 + 0.22


all dry


_ __ __


_ ~ ______ ___


__ __ __ __









probably insignificant to sporulation under the wet conditions that exis-

ted), conidiogenesis was favored by daily precipitation and long hours of

foliage wetting.

Field data indicate that some ideas concerning optimum conditions

for an epizootic, such as generally wet, rainy conditions, must be quali-

fied before being applied as a general rule. Some may even be highly

detrimental to the progression of various phases of the disease. Work

is continuing to identify both optimum and poor conditions for various

phases of an epizootic.

Since the reduction of the majority of 1974 data, the following basic

hypotheses and assumptions relative to the VBC, Nomuraea, and their inter-

relationships in the agroecosystem have been formulated. Most are -ased

on experimental evidence or observations; a few lack such support bu1 are

being actively investigated.

A. The fungus, which is insect host-dependent in all cases, survives the

winter in three possible ways, depending on latitude.

1. From central to south Florida and on coastal margins of the

Gulf Coastal Plain where freezing temperatures do not kill

all suitable host plants of the insects, the fungus maintains

a low population density.

2. In northern inland latitudes, the fungus may overwinter by a

resistant, sexual, or asexual stage.

3. Although somewhat doubtful, conidia may survive in the soil

for extended periods.

E. Conidiai dispersal occurs in several ways.

1. Conidia become attached to the bodies of migrating moths (re-

sulting in intermediate to long range dispersal).









2. Conidia are dispersed locally by wind.

C. Lateral spread of the disease emanates from "hot spots" in the field.

It is dependent, to some unkr.ovn extent, on host-insect density.

D. Meteorological conditions simultaneously promote and retard various

phases of fungal development and the epizootic.

1. Dry, windy conditions promote conidial dispersal

a. Increased conidial densities result from dry, windy condi-

tions but are also dependent on host population density

and infection rates.

2. Dry, windy conditions retard germination and infection as

such, but promote infection if followed by humid conditions,

provided no excess of free water exists.

3. Fungal ontogeny within the host body, up to and including

death of the host, is independent of weather conditions.

4. Conidiophores will form independently of fluctuations in

external humidity as long as the cadaver does not undergo

dessication rapidly after death of the host.

5. Under normal conditions, development of conidiophores and/or

conidia can be arrested for several days to a week or longer,

then resume when conditions again favor their development.

6. The minimum relative humidity at which conidial production

takes place is 70%. As relative humidity increases, conidial

production increases.

7. Rain and foliage wetting promote conidiophorous formation

and conidiogenesis.

8. Conidia on cadavers will be washed to the ground by an exten-

ded light rain, a brief heavy rain, or long hours of heavy









foliage vettrin by dew.

9. A. alternation of wet and dry conditions is necessary for

spread of infection.

10. An excess of free water during the height of an epizootic

will have little net effect on the course of the epizootic.

11. An excess of free water in the early stages of an epizootic

(infection rate approximately less than 10%) may retard the

spread of infection if it follows conidial formation but pre-

cedes conidial dispersal.

12. The alternation of short periods of foliage wetting and

high humidity with longer periods of dry, windy conditions

favor the increase and spread of infection.

E. Insect susceptibility to infection may show seasonal and/or diurnal

periodicity.

Environmental data as well as laboratory and field data concerning

the host and pathogen are being pooled for development of a pathogen sim-

ulator model. The model, when completed, will enable investigators to

predict with reasonable accuracy and consistency the development of the

soybean uLg.-.: .-tem under a given set of conditions.

Natural control of a pest population within a well-defined system

is not cormon. The consistency of occurrence and high level of population

control make No~muraea a candidate to become the first fungus incorporated

into a successful biological control program.

Results of this study demonstrate the neccessity for recognizing

the pathogen in relation to the physical and biotic environment within

which it plays a role. It is clear that an intricate and dynamic balance

exists between :.'.raea and the biotic community and that the full range









and variety of stimuli-response relationships must be s m-died both sep-

arately and collectively before they can be manipulated for our benefit.





















*H ,



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Plate I

Fig. 3. Germ tube development after 8 hours on the host intejumenrt.
x 1750.

Fig. 4. Cross section of host showing blackened, ulcercus in--~jrer.t
in the area of hyphal penetration, x 650.

Fig. 5. Cross section of host showing yeast-like fungal cells in the
hemocoel. x 300.

Fig. 6. Fusiform fungal cells as seen in squash mounts of the host near
death. x 650.














F-


f:r


I~5
(U -,


Ag4y \-















Plate II

Fig. 7. Cadaver 24 hours after death showing conidiophores which look
white in mass. Black areas are sclerotized tissue surrounding
the original points of infection. x 4.

Fig. 8. Cadaver showing dense covering of conidia. x 1.3.


















. i...















Fig. 9. Selected life cycle relationships in a soybean agroecosystem.












































10-15 September


itiiii- Fungus--long cycle

E.'-A' Soybean--growing season

r'.ii_ Soybean--detritis cycle

:: ?'Fungus--short cycle

I ZZ ;VBC cycle




































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Plate III

Fig. 12. -arsai claw of Anticarsia gemmtalis adult showing attached
and associated conidia c'). x 650.

Fig. 13. Leg scales of Anticarsia gemmatalis adult showing attached
conidia. x 650.




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BIOGRAPHICAL :'Z 7I

Leslie Paul Kish was born November 28, 1944, in Johnstown, Penn-

sylvania, The fifthh of six sons and daughters of Joseph and Evelyn

(Tomasel) Kis. He was graduated from Blacklick Township High School,

Belsanio, Penrsylvania, in 1962; following graduation he entered the U.S.

Air Force and served until honorably discharged in 1966.

After graduation from Okaloosa-Walton Jr. College, Valpariso, Flor-

ida, in 1968, he entered the University of Florida where he recei-.ed

the Bachelor of Science degree in 1970 and the Master of Science degree

in 1971. He is the author of several publications desainU- with the

cytology, mc .'i l:i.g, ecology, and taxonomy of coprophilous and entomo-

genous fungi.

He is married to the former Clara Dianne Pitts of Milligan, Florida,

and they are ,he parents of one daughter, Heather Kathleen.















I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Leland San-or, Chairman
Professor of Botany



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





's W. Krnoroungri
associate Professor if Botany



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.





Henry C./ ldrich
Associate Professor of Botany










I certify that I have read this study and that in my opinion it
conforms to accepTable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philcsophy.




Dan ft. Griffin I
AssDciate Professor of~ ts-ny



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presen-taion and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




George E. Allen -
Professor of Entomology



This dissertation was submitted to th-e raduate Faculty of the tollee7
of Agriculture and to the Graduate Ccun4cil, and was accented as partial
fulfillment of the requirements for the degree of D'c.tor of Philosophy.

December, 1975


Dean, Collee of tAgriUi.ture


Dean, Graduate School































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