• TABLE OF CONTENTS
HIDE
 Front Cover
 Abstracts of papers presented at...
 Etiology of sorghum stalk rot complex...
 Pathogenicity of sarocladium oryzae...
 Control of helminthrosporium leaf...
 A method to accommodate non-blight...
 Screening of various plant exercises...
 Role of motility in apple blossom...
 Effect of neem oil on survival...
 neem seed derivatives for preventing...
 Transmission of rice tungro bacilliform...
 Information for contributors
 Back Cover














Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00090520/00027
 Material Information
Title: Journal of tropical plant pathology
Series Title: Journal of tropical plant pathology.
Alternate Title: Journal of Philippine phytopathology
Philippine phytopathology
Physical Description: v. : ill. (some col.) ; 26 cm.
Language: English
Creator: Philippine Phytopathological Society
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
College Laguna
Publication Date: January-June 1985
Frequency: semiannual
regular
 Subjects
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
 Notes
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00027
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 54382605
issn - 0115-0804

Table of Contents
    Front Cover
        Front Cover 1
        Front Cover 2
    Abstracts of papers presented at the twenty-second annual meeting of the Philipine phytopathological society, Mountian State Agricultural College, La Trinidad, Benguet, may 2-4 1985
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
    Etiology of sorghum stalk rot complex in Mindanao
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
    Pathogenicity of sarocladium oryzae and factors affecting sheath rot development on rice
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
    Control of helminthrosporium leaf spot of wheat (triticum aestivum) with mancozeb and thiophanate methyl
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
    A method to accommodate non-blight weather in the analysis of potato late blight epidemics
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
    Screening of various plant exercises for toxicity to meloidogyne incognita and radopholus similis
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
    Role of motility in apple blossom infection and tactic response of erwina amylovora to organic acids in plant nectar extracts
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
    Effect of neem oil on survival of the brown rice planthopper, nilaparvata lugens (stal) (homoptera: delphacidae) and on grassy stunt and ragged stunt visrus transmission
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
        Page 87
    neem seed derivatives for preventing rice tungro virus transmission by the green leafhopper, nephotettix virescens (distant)
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
    Transmission of rice tungro bacilliform and sperical viruses by nephotettix virescens distant
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
    Information for contributors
        Page 110
    Back Cover
        Page 111
        Page 112
Full Text














VOLUME 21 JANUARY and


CON'

Abstracts of Papers Presented at the Twer
Philippine Phytopathological Socih
College, La Trinidad, Benguet, May 2

Etiology of Sorghum Stalk Rot Complex
nan and Tricita H. Quimio. ..... .

Pathogenicity of Sarocladium oryzae an(
Development On Rice K.C. Chuke

Control of Helminthosporium Leaf Spol
With Mancozeb and Thiophanate IV
Lapis. ................. ...

A Method to Accommodate Non-Blight V\
Late Blight Epidemics A.B. Molina

Screening of Various Plant Extracts for Ti
and Radopholus similis Ruth S. Gu


Role of Motility in Apple Blossom Infectic
amylovora to Organic Acids in Plan
and S.M. Ries .......... .....

Effect of Neem Oil on Survival of the Rice
ilnanc It.nll Ilnmrnnn-ar. nal.nhr


IE 1985 NUMBERS 1 and 2


JTS

second Annual Meeting of the
Mountain State Agricultural
985 ................. 1-19

lindanao Naomi G. Taigo-
. . ........... ... 20-27

actors Afferting Sheath Rot
D.B. Lapis ............ 28-33

Wheat (Triticum aestivum)
ri A.M. Sinohin and D.B.
.................... 34-47

ler in the Analysis o; potatoo
Sand D.R. Mac Kenzie ..... 48-53

ty to Meloidogyne incognita
n and R. G. Davide ....... 54-64


d Tactic Response of Erwina
sctar Extracts R.G. Bayot
S.. ................. 65-79

iwn Planthopper, Nilaparvata
) and on Grassy Stunt and
Saxena and Z.R. Khan..... 80-87


im M6











Official Urgan or me rnmppme rnytopamoiogicai oaucety, me.

BOARD OF DIRECTORS 1984-85


DR. TIBURCIO T. REYES 0 President
DR. AGUSTIN B. MOLINA, JR. 0 Vice President
MISS LINA C. LAPITAN Secretary
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MR. JOSE M. BANDONG 0 Auditor
MR. VALENTINO G. BALAOING 0 Ex-Officio
DR. ROMULO G. DAVIDE Board Member
DR. DELFIN B. LAPIS 0 Board Member
MR. FAUSTO L. NUQUE 0 Board Member
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DR. ARCADIO J. QUIMIO Board Member




EDITORIAL BOARD



DR. ARCADIO J. QUIMIO Editor-in-Chief
DR. MARINA P. NATURAL 0 Associate Editor
DR. FAUSTINO T. ORILLO Associate Editor



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Makati,
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Victorias Milling Co., In(


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ABSTRACTS OF PAPERS PRES]
ANNUAL MEETING OF THE PHI
SOCIETY, Mountain State Aj
Benguet, I


Effect of Neem Oil on Survival of the
Rice Brown Planthopper, Nilaparvata
lugens Stal (Homoptera: Delphacidae)
and on Grassy Stunt and Ragged Stunt
Virus Transmission. R.C. Saxena and
Z.R. Khan. International Rice Research
Institute (IRRI) Los Baffos, Laguna.

Neem (Azadirachta indica A. Juss)
seed oil was found highly effective in
educingg the survival of brown plant-
lopper, Nilaparvata lugens Stal (Homo-
ptera: Delphacidae) and in suppressing
transmissionn of grassy stunt and ragged
stunt viral diseases of rice. Generally,
nsect survival and disease transmission
decreased with increasing neem oil con-
:entrations. After 3 days of exposure,
he insect failed to transmit the viruses
o plants sprayed with 50% neem oil,
comparedd with control plants where
rirus transmission was successful. Thus,
ieem seed oil possesses potential for
controll of brown planthopper and its
transmitted grassy stunt and ragged stunt
'iral diseases of rice.

k Search for Primary Determinant of
Irown Spot Disease of Rice. P. Vidhya-
ekaran, E.S. Borromeo and T.W. Mew.
RRI, Los Banos, Laguna.

A toxin was isolated and purified from
culture filtrate of Helminthosporium
oryzae, the rice brown spot pathogen. It
produced typical symptoms of the
disease. Host range of the pathogen
nd toxin were identical. The same toxin
would be detected in the fungus-inocu-
sted rice leaves. A highly virulent isolate
producedd more toxin than the less viru-
ent one in vitro. When the virulent
isolate lost its virulence due to repeated


FED AT THE TWENTY-SECOND
PPINE PHYTOPATHOLOGICAL
cultural College, La Trinidad,
2-4 1985


subculturing, it also lost its ability to pro-
duce the toxin. The toxin conditioned
the leaf tissues to permit colonization by
a non-toxin producing avirulent isolate. It
reduced phenolic content and peroxi-
dase and phenylalanine ammonialyase
activities in rice leaves. Phenylalanine
(0.3%) induced resistance to both the
pathogen and toxin. These results suggest
that the toxin may be the primary deter-
minant of the disease.

Efficacy and Economics of Fungicide
Usage Against Helminthosporium Leaf
Spot of Wheat (Triticum aestivum).
A.M. Sinohin and D.B. Lapis, University
of the Philippines at Los Bafios (UPLB),
College Laguna.

Manganese ethylene bisdithiocarba-
mate plus zinc (Dithane M-45) and
thiophanate methyl (Fungitox 70 WP)
were evaluated against Helminthosporium
leaf spot of wheat caused by Helmin-
thosporium sativum. The two fungicides
were sprayed at different rates and fre-
quencies. Leaf spot development was
significantly controlled by Dithane M-45
at all rates and frequencies of application.
Two to three sprayings provided the most
effective control without any phytotoxic
effect. Grain yield was significantly
higher with Dithane M-45 at 1.0 and 1.5
kg/ha in all spraying frequencies. Black
point incidence was significantly reduced
by Dithane M-45 at all rates and frequen-
cies. Fungitox 70 WP was found in-
effective against the pathogen at any of
the rates and frequencies used. Wheat
production was found profitable with
Dithane M-45. At P5.20 per kg of wheat,
the return over cash cost (ROCC) and net
-atiim (NTR nhtnined ware pQQ 51 and









Philinninp Phvtnnathnlnorv


P594.51, respectively with a return of
investment (ROI) at 8.4 percent.

Epidemiology of Wilt Disease of Coconut
in Socorro, Oriental Mindoro I. Disease
Increase with Space and Time. Nemesia
C. San Juan, O.S. Opina, Erlene C. Con-
cibido, R.G. Abad and J.G. Nuevas, Jr.
Philippine Coconut Authority (PCA)
Davao City and UPLB, College, Laguna.

Past and present occurrences as well
as severity of the wilt disease of coconut
('Socorro Wilt') in the municipality of
Socorro, Oriental Mindoro were mapped
out. The disease is concentrated in
6 adjoining 'barangays'. The disease
occurring in a "jump spread" pattern of
spatial spread is also sporadically present
in other 6 barangays. The geographical
range of the disease was observed up to
approximately 12 km radius from the
coconut farm where it was first spotted
in the early 1960s.
The relationship of disease incidence
to distance from the source was examined
by regression analysis using two models
of disease spread. Correlation between
disease incidence (Y) and distance (X)
from the inoculum source was given
as Y = 0.49x- 1.25 for Gregory's model
and Y = 0.35 e :3xLfor Kiyosawa &
Shiyomi's model. The spatial spread was
better explained by the former model.
The disease progress curves depict a
negative exponential curve. Using regres-
sion analysis, the disease progression
can he explained by both simple interest


Green Leafhopper, Nephotettix virescens
Distant. R.C. Saxena, Z.R. Khan and
N.B. Bajet, IRRI, Los Baios, Laguna.

Neem (Azadirachta indica A. Juss)
seed oil and cake showed potential for
preventing rice tungro virus transmission
by the green leafhopper, Nephotettix
virescens Distant. Neem oil and neem
cake disrupted the normal feeding
behavior of N. virescens. The insect
shifted from phloem to xylem ingestion
on rice plants treated with neem oil or
neem cake, or kept in an area permeated
with neem oil odor. Protection of rice
seedlings grown in soil incorporated with
150 and 250 kg neem cake per ha against
rice tungro virus was comparable to that
conferred by Furadan treatment. The
reduction in the leafhoppers ability to
transmit the tungro virus on neem oil
or neem cake treated plants could be
attributed to a reduction in phloem
feeding and associated aberrations in the
insect feeding behavior.

Role of Motility in Apple Blossom In-
fection and Tactic Response of Erwinia
amylovora to Organic Acids in Plant
Nectar Extracts. R.G. Bayot and S.M.
Ries, UPLB, College Laguna and Univer-
sity of Illinois, Urbana, Ill.

A nonmotile isolate (Nm22) and its
motile revertant (Mr22) of Erwinia amy-
lovora were obtained after treating cells
of the wild type (Isolate 110) with 0.3


plained by the former gencity or ootn Isoin
a rate of "Socorro wilt" the wild type when ii
as 0.00452 to 0.06029 shoots of apple seed
r using the simple interest more infection was re
model while apparent inoculated with Mr22
ranged from 0.156 to isolate. The chemot
)er year based on logistic E. amylovora to nei
plant species with v
reidtance. nr mnmepntil


Xrn1 'I1














weakly attractive while more susceptible
plants had nectar extracts that were
strongly attractive to E. amylovora.
Nectar extracts were fractionated using
anion and cation exchange resins and
the organic acid fraction was silylated
and chromatographed on a 3% SE-52
column. The organic acids attracting
E. amylovora in nectar extracts appear
to be either fumaric or malic acid or a
mixture of both.

Epidemiology of the Wilt Disease of
Coconut in Socorro, Oriental Mindoro II.
Ecological Factors Affecting Disease
Development. Nemesia C. San Juan,
O.S. Opina, Erlene C. Concibido,
R.G. Abad, and J.G. Nuevas, Jr. PCA,
Davao City and UPLB, College, Laguna.

Variance and multiple regression ana-
lyses showed certain degree of relation-
ship of disease incidence to coconut age
level, relative dominance of some weed
species, namely; Imperata cylindrica,
Mikania cordata and Paspalum conju-
gatum, incidence of the rhinoceros
beetle, Oryctes rhinoceros, soil acidity,
exchangeable bases and textural grade
of soil. The direct and indirect roles ol
these ecological factors in disease deve-
lopment, however, have to be established
yet.

Toxicity Screening of Various Plani
Extracts against Meloidogyne incognita
and Radopholus similis and Character.
ization of their Nematicidal Compo
nents. Ruth Sarra-Guzman and R.G
Davide, Isabela State University, Cabagan
Isabela and UPLB, College, Laguna.

Solvent extracts of eight plant species
were screened for toxicity against two
important nematode species, namely; M.
incognita and R. similis. Most plant
extracts showed indications of toxicity
against the nematodes in the bioassay
and inoculation tests. In some cases


Lflt..l.ll ,wlI.U. rtjZt L mt.N E l J AR U fl
Rice. A.K.M. Shahjahan, H.U. Ahmed
N.R. Sharma and S.A. Miah, IRRI
Los Baiios, Laguna.


ana rour srs le. 13xiu, 13x13, zLXIz
and 25x15 cm and four N rates ie. 0, 45
90 and 135 kg/ha were used during the
Boro season. There were interactions bet-
ween the SPs and amount of N applied on
ShB severity and yield of rice. In general,
wider SPs decreased disease incidence
and increased the yield. Higher N appli-
cation increased the yield, but it also
increased the disease incidence. Severe
disease incidence at high N level was
reduced by wider spacing without much
sacrifice in yield. The results suggest thai
it is possible to manage ShB by growing
plants with optimum spacing and nitro,
gen fertilization.

Incidence of Stalk Rot as Influenced by


LPL Ir IU1l 1 IV


_--uaia-t IVY-









Philippine Phytopathology


Weed Control Practice. Teresita C. Bay
cag and O.R. Exconde, University
Southern Mindanao (USM), Kabaca
North Cotabato and UPLB, Collef
Laguna.


The study was conducted to determii
a) the occurrence and severity of stalk r
of corn, b) the population of the path
gen in the soil, and c) know the yield kl
due to stalk rot as affected by differe
weed control practices.

The occurrence and severity of sta
rot were not significantly affected I
different weed control practices studied
However, plots subjected to off-barri
and hilling-up operations gave slight
higher percentage infection and severe
of stalk rot..
The population of the causal pathog
Erwinia carotovora var. chrysanthei
Dye in the field was not significantly i
fected by the different weed control pra
tices employed. However, its inoculu
density decreased as the plants mature
Plots with high percentage stalk r
incidence showed a correspondingly hil
percentage yield reduction. The redu
tion in yield due to stalk rot was high
from plots with off barring and hill
up operations compared to plots whi,
were left undisturbed.

Field Symptoms, Spread, and Signs of tl
Banded Leaf and Sheath Blight Disea
on Corn Hybrids in Mindanao. A.S. Ga
cia, R.G. Reloba and F.B. Castro, Bure;
of Plant Industry (BPI) Davao Cit
Field svmntom srions and snread i


Data obtained indicated that visib
symptoms were observed 25-30 days aft,
planting (mid-whorl). The lowermost le;
sheaths first show disease manifestatic
by the presence of irregular brown,
blotches with reddish brown margin
These lesions increase in size and coi
taminate the upper leaf sheaths, eventual
ly killing the blades. Infection contim
until all the leaves and ears premature]
dried.
The mycelia radiate and spread u]
ward to the blades and cause bandir
corresponding to the area invaded within
a 24-hour-period. The invaded portic
first show water soaked appearance, late
becoming dirty white and appear as ban(
with length ranging from 1.30 to 1.E
inches across the leaf sheaths and le;
blades wherever initial infection occurred
Mycelia tend to condense and fon
sclerotial bodies on the outer part of tl
dried leaf sheaths and blades infected b
the pathogen. These sclerotia are fir
white later become brown with varyir
sizes from 1.4 mm to 2.7mm.
Survey and Identification of Seedbor
Fungi and Viruses of Field Legume
Elenita M. Gutierrez, BPI, Manila.

Seven fungal genera were isolated froi
the seeds of mungo, cowpea, peanut an
soybean using the blotter test and 1
genera were isolated using the agar plal
test. Results showed that more storaj
fungi were isolated than field fungi.
Seed transmission test was done t
identify viruses of peanut (Arachis hype
-- T \ A I I


4









Abstracts of Papers


ter method (International Rules for Seed
Testing, 1966). Seeds were sown in petri
dishes containing 3 layers of moistened
filter paper as substrate and incubated
under alternating cycles of near ultra
violet (NUV) light and darkness at room
temperature. After 8 days of incubation,
the seeds were examined for the presence
of seed-borne fungi under the stereo
binocular microscope.
Results showed that the major patho-
gens of rice, Drechslera oryzae, (causal
agent of Helminthosporium leaf spot)
Trichoconia padwickii (Stackburn disease)
and Fusarium moniliforme (Bakanae
disease and foot rot) were the most pre-
valent pathogens recorded in the samples.
Pyricularia oryzae, another serious patho-
gen was present only as weak infection.
Other fungal organisms carried in many
samples were Curvularia lunata, Curvula-
ria palescens and Nigrospora oryzae.


(RTBV) and rice tungro spherical virus
(RTSV) in almost all the samples tested
especially on the symptomatic leaves.
Double infection of tungro and rice


BPI, Manila.
Seventy five (75) rice seed samples
from the provinces of Abra, Ilocos Norte,
Ilocos Sur, La Union, Pangasinan were
used in this investigation.
Four hundred (400) seeds of each
sample were tested by the standard blot-


genera were observed namely;Aspergillus,
Curvularia, Duosporium, Drechslera,
Fusarium, Paecilomyces, Penicillium, Pes-
talotia, Phaeotrichoconis, Rhizoctonia-
like, Sclerotium and Trichoderma. The
probable identity of the consistently
observed and predominant fungi from
the greenhouse were Penicillium italicum,
P. oxalicum, P. minioluteum, P. funicu-
losum and P. verruculosum. Curvularia
lunata, Rhizoctonia-like organisms and
Phaeotrichoconus sp. were most frequent-
ly isolated from the ponds.
Etiology and Control of"Bugtok" Disease


Jan. & June 1985













UI aua aU"U uDlllla. rt.a. Ujisi'i DF1,
Davao City.

Survey of "Bugtok" disease in Minda-
nao revealed that the disease is wide-
spread and destructive, reducing yield
from 26.61 to 38.53 percent and a threat
to cooking banana production in the
country.
Examination of twenty five diseased
plants indicated that symptoms were
restricted in the shrivelled ovule, pulp of
the fruit and in the vascular system at the
loculus, pedicel and serial stem.
Pure culture of the bacterium con-
sistently isolated .from infected tissues
when artificially inoculated to healthy
plants produced typically bugtok symp-
toms. The causal organism was identified
as Pseudomonas solanacearum E.F. Smith.
The pathogen gains entry at the tip of
the ovary, through the cavities from the
micropyl leading to the ovule and be-
coming systemic invade the vascular sys-
tem of the pedicel and serial stem.
Plants were susceptible to infection at
flowering period during the female phase.
The pathogen was not seedpiece borne
but infected fruits were found to be in-
fective. A seasonal fluctuation on the in-
cidence of the disease was recorded with
no incidence in some months. This could
possibly be brought about by the absence
or reduced number of insects carrying the
pathogen and sources of inoculum.
Bagging and periodic removal of dried
leaves showed significant reduction of
diseased bunches.


Etiology of Orange Leaf. G.B. Jonson,
F.C. Sta. Cruz and H. Hibino, IRRI,
Los Bafios, Laguna.

Rice plants showing orange leaf-like
symptoms were collected at 6 locations in
the Philippines and tested for transmis-
sion by vectors and presence of disease
agent.
Recilia dorsalis nymphs were given 2


source. Average 13 percent of the insects
transmitted the disease agent with incuba-
tion period ranging from 15-33 days. The
pathogen was persistent in the vector.
About 10% of the infective insects were
able to transmit the disease continuously
until its death. Retention period ranged
from 18-43 days.
The incubation period in the plant was
10-28 days. In addition to symptoms
reported for orange leaf, ragged and
twisted leaves on inoculated seedlings
were also observed. Infected seedlings
died within 3-4 weeks from symptom
appearance.
Ultrathin sections of leaf tissues were
obtained from rice plants infected with
each isolate and examined under the
electron microscope. Mycoplasma-like
organisms (MLO) were observed in sieve
tubes of all samples. The MLO were
bounded with a unit membrane and pleo-
morphic, 50-1100 nm in diameter. This
indicates that the pathogen causing
orange leaf in the Philippines is not a
virus but most likely a MLO.

Mycorrhizae in a Rice-Based Cropping
System. A.M. Rosales, Lina L. Ilag, T.W.
Mew and F.A. Elazegui, IRRI, Los Baflos,
Laguna.

Mycorrhizae are mutually beneficial
associations between plant roots and
certain fungi. A survey of field soils from
Manaoag, Pangasinan; Guimba, Nueva
Ecija; Claveria, Misamis Oriental, and
IRRI, Los Bafios, Laguna with varying
cropping histories was carried out. Results
showed that lowland ricefields contained
vesicular-arbuscular mycorrhizal fungal
spores but these were present in lower
numbers than in upland areas. The highest
number of spores was found in Claveria
and the least was noted in IRRI fields.
Species belonging to the genera Glomus,
Gigaspora and Sclerocystis were observed
in the various areas sampled. Greenhouse
experiments showed that peanut, mung-


k JujYYuv &aI *Y--U---- b


V~









Abstracts of Papers


bean, cowpea, rice and corn were taller
and greener in pots to which soil con-
taining spores of mycorrhizal fungi was
added than plants grown in uninoculated
soil. Results taken 44 days after sowing
showed that inoculated cowpea, peanut
and rice plants had significantly higher
plant heights than the uninoculated ones.
The roots of inoculated rice and peanut
had a greater weight than the uninocu-
lated plants. Mycorrhizal cowpea and
mungbean had higher yield than non-
mycorrhizal plants.

Detection of Xanthomonas campestris pv.
oryzae from Seeds of Infected Rice Plants.
M.R. Baraoidan, C.M. Vera Cruz and T.W.
Mew, IRRI, Los Bafios, Laguna.

Several methods were evaluated for
their sensitivity to detect X. campestris
pv. oryzae from rice seeds. IR36 plants
were artificially infested with bacterial
strain PX079 by injection method at
panicle exertion. At maturity, the seeds
were examined for the presence of the
bacteria by direct isolation, phage-plaque
count test, and growing-on method.
Bacteria with yellow colonies were
isolated from rice grains and seedlings of
IR36. Results on diagnostic bacteriolo-
gical, phage and pathogenicity tests indi-
cated that all yellow colonies isolated
from seeds were not X. campestris pv.
oryzae. X.C. pv. oryzae was recovered
only from infected panicle branches.


TKM6 A Source of Resistance to Rice
Tungro Spherical Virus. R.D. Daquioag,
P.Q. Cabauatan and H. Hibino, IRRI,
Los Bafios, Laguna.

Leaf samples were collected from all
plants of 26 IR varieties in the tungro
nursery at IRRI (WS, 1983; DS, 1984)
and tested for the presence of tungro-
associated viruses.
It was noted that IR20, IR26, IR30
and IR40 were infected mostly with


RTBV alone indicating their resistance
to RTSV during both seasons. Their
common parent is TKM6. This variety
and other varieties used as parents in the
breeding of IR20, IR26, IR30 and IR40
were evaluated to determine the source of
resistance of RTSV. Test seedlings were
inoculated individually in test tubes by
one viruliferous leafhopper (Nephotettix
virescens) that were allowed access to
TN1 plants infected with either RTBV
and RTSV or RTSV alone. The presence
of tungro-associated viruses in the inocu-
lated seedlings were determined by latex
test. Results showed that TKM6 (IRRI
Ace. No. 237) were not infected with
RTSV but were mostly infected with
RTBV alone. This indicates that the
resistance gene to RTSV in the four IR
varieties originated from TKM6.

The Production of Downy Mildew Re-
sistant Phil 6723 Subclones through
Tissue Culture. C.L. Morales, T.S. Jereza
and R.D. Pillado, Philippine Sugar Com-
mission (Philsucom), Negros Occidental.

Plants were regenerated from sus-
ceptible sugarcane commercial variety
Phil 6723 via tissue culture. The derived
plants together with the donor variety
were screened for downy mildew to be
able to isolate resistant clones. Some
clonal isolates were found to be resist-
ant to the disease.
The clonal isolates were comparable to
the donor variety in some agronomic
characters as well as in tonnage and sugar
yield. Significant improvements were ob-
served in some isolates in terms of num-
ber of millable stalks and brix reading.

Reaction of IR Varieties to Tungro Under
Different Disease Pressure. R.C. Cabuna-
gan, Z.M. Flores and H. Hibino, IRRI,
Los Bafios, Laguna.

Field trials to evaluate the reaction
nf rID iforfntin tn titmnrn,-ara rp nnnrt+al


Jan. & June 198.1









Philippine Phytopathology


in WS 1984 at IKK1 and luimna, Nueva
Ecija. Three week-old test seedlings were
transplanted singly and subjected to natu-


leaf samples were tested infectional
nation serology test for delayed


I resistance was indic
or retarded develop


disease maturity of nematodes in W-8(
At the In susceptible cultivars,
, r ID- . t__ .. i - .. ..


15% infections, respectively. Varieties
with high vector resistance had very low
tungro infection. Latex agglutination test
of randomly sampled leaves revealed
that IR22 was generally infected with
both rice tungro bacilliform (RTBV) and
spherical viruses (RTSV) followed by
IR42 and IR36. Generally, the other
varieties were not infected or infected by
RTSV at low level.
At the Guimba trial where high
disease pressure prevailed, high tungro
infection was obtained not only in IR22,
IR36 and IR42 but also in IR62. The
other varieties had low infection. The
presence of tungro-associated viruses
revealed the same trend as that at IRRI
trial. However, increased infection to
bpth RTBV and RTSV, and RTSV alone


inoculation. The number of eggs per
egg mass and size of egg laying females
obtained from susceptible cultivars were
significantly greater compared to those
from the resistant cultivars.

Seedling and Adult Plant Resistance of
Rice Varieties to Isolates of Bipolaris
oryzae (Breda de Haan) Shoemaker.
K.K. Baloch and J.M. Bonman, IRRI,
Los Bahios, Laguna.

Fifteen selected rice varieties were
screened for resistance to three isolates
of Bipolaris oryzae, the brown spot
pathogen. Lesion number, lesion size, and
percent diseased leaf area (% DLA) was


:se results indicate that varieties newest esoins ((.o cm-), te smau
lesions (1.6 mm2) and the lowest D:
high insect resistance can escape 6 s sse e w
on in the field resulting to low (12.2%). IR 36 was susceptible w
infection than those with low or numerous lesions (1.8/cm2) of great
ate resistance, and that RTSV size (3.6 mm2) constituting higher D
ed as an independent virus in both (60%). There was no evidence of phy,
logical races. Individual panicles
3 varieties were inoculated by injecti
at booting and by spraying at flower:
I Post-Infectional Resistance in and milk growth stages. Injection
Potato to Meloidogyne spp. R.M. booting stage caused the most gr


VoL 21









Abstracts of Papers


since grain discoloration symptoms were
equally severe in the three varieties, but
Tetep showed less grain weight loss.
Severe grain discoloration caused by
isolates of B. oryzae was associated with
increased grain sterility, decreased grain
weight, and decreased seed germination.

Analysis of Virulence of the Rice Bac-
terial Blight Pathogen. C.M. Vera Cruz
and T.W. Mew, IRRI, Los Bafos, Laguna.

A total of 490 isolates of Xanthomo-
nas campestris pv. oryzae from rice in
the Philippines from 1972-1984 were
analyzed for their virulence on bacterial
blight differential varieties. A shift in
virulence frequency over the years was
noted. In the last two years, the per-
centage of pathogen population with
virulence to the Xa4 gene for bacterial
blight resistance was 95.3% and 69.7%
for 1983 and 1984, respectively. Com-
ponents of the population with virulence
to Xa4 and other genes were also evident.
Phenotypically, the bacterial pathogen
was homogeneous and there was no cor-
relation between virulence and bacterio-
logical properties. Few features, however,
tend to partially differentiate them from
each other.

Components of Rate-Reducing Resistance
in Sorghum to Tar Spot. Elsa P. Paderes
and S.C. Dalmacio, UPLB, College,
Laguna.

A greenhouse and laboratory study
was conducted to determine the com-
ponents of rate-reducing resistance in
some tar spot-resistant sorghum geno-
types.
Seven sorghum lines showing suscep-
tible and resistant reactions were used
in this experiment. Seeds were planted
in one-meter-row plots and artificially
inoculated about a month after planting
using sections of Phyllachora-infected
leaves attached to the individual leaves


of the test plants.
Observation showed that the incu-
bation period (the time from inoculation
to the appearance of stromata) in re-
sistant lines was delayed by 21.4%,
initial sporulation period (number of days
from the first appearance of stromata
and initial production of spores) by
12.23%, and infectious period (length
of time stromata sporulate) was reduced
by 31-69% relative to a susceptible line,
Acc. 1127. Likewise, spores produced
in resistant lines were 83-93% less than
Acc. 1127. This could be attributed to
their hypersensitive reaction type which
tends also to delimit the expansion of
stromata.
The implication of these results in
the evaluation and development of
disease resistant varieties was discussed.

Cross-Inoculation Studies of Pyricularia
oryzae from Rice and Other Hosts. Alita
O. Mackill and J.M. Bonman, IRRI,
Los Bafios, Laguna.

Cross-inoculation studies were con-
ducted to investigate the existence of
Pyricularia oryzae isolates that can infect
grass weeds and rice cultivars.
Grass weeds and rice exhibiting
typical blast symptoms were collected
in the field. About 100 isolates were
collected from grass weeds and rice and
tested using injection inoculation tech-
nique. Isolates with a strong pathogenic
reaction were selected and used in further
spray-inoculation tests.
Several rice cultivars were susceptible
to isolates from Rottboellia exaltata,
Echinochloa colona, and Leersia hexan-
dra. Similarly, Brachiaria distachya, E.
colona, Leptochloa chinensis, R. exaltata,
and L. hexandra were susceptible to some
isolates originating from rice. These
weeds, as collateral hosts of the pathogen,
can provide inoculum for rice blast epi-
demics. Of the five grass weeds suscep-
tible to P..oryzae, B. distachya, and R.


Jan. & June 1985








Philippine Phytopatholog


VoL L


Institute (BRRI) since 1977. Germplasrr
and advance breeding lines from BRR1
and the International Rice Researcl
Institute (IRRI) were tested against tung
ro (Tg), bacterial blight (BB), blast (Bl)
Sheath blight (Shb), and Stem rot (SR'
in the field with artificial inoculation
Out of about five thousand material:
tested, only 34 were found to be resistant
(R) to more than one disease and 18 tc
three or more diseases. The entries show
ing R-mod. resistant (MR) reactions t(
three or more diseases are: Among thi
IR lines IR272-Mala J-15 (Tg, BB, B1
SR), and IR2053-375-1-1-5 (Tg, BB, SR)
among the BR lines BR4 (Tg, BB
SR), BR5 9Tg, BB, SR), BR9 (Tg, BB
Bl, SR), BR12 (BB, B1, SR), BR 16(BB
Bl, SR), BR51-45-5 (Tg. B1, SR), 46-5
HR65 (Tg, BB, Shb, SR), BR51- 46-5-
HR65 (Tg, BB, Shb, SR), BR51.
196-2 (BB, Bl1, Shb, SR), BR52-87-1.
HR36 (Tg, BB, SR), BR171-2B-8 (BB, B l
SR) and BR319-1 (BB, B1, SR), and
among the germplasm materials-cisadane
(BB, B1, SR), Kataribhog (Tg, BB, Shb),
and MRC (603-303 (BB, Bl, SR).
Serological Blockage of Rice Tungre
Virus Complex Transmission by Nephc
tettix virescens. H. Hibino and P.C
Cabauatan, IRRI, Los Baios, Lagunm

The infectivity of rice tungro bacilli
form (RTBV) and spherical (RTSV
viruses was neutralized by feeding viruli
ferous green leafhopper (GLH), Nepho
tettix virescens Distant through mem
brane in solutions of antiserum (IgG) ti
respective viruses. When GLH fed 1U
hours on anti-RTBV IgG diluted 25 time
with two per cent sucrose solution
prepared in 0.01 M phosphate buffet
RTBV infectivity was completely neutral
ized. All infected plants contained RTS1
alone. When GLH fed on anti-RTSV Ig(
for the same duration and concentration
RTSV infectivity was markedly reduced
Only four to five per cent of the infected
plants contained both RTBV and RTS1


exaltata are unreported previously.

Effect of Plant Age on Components of
Rice Blast Resistance. C.O. Torres and
J.M. Bonman, IRRI, Los Banios, Laguna.

The effect of plant age on components
of rice blast resistance in cultivars C22
and Denorado was studied. Plants of
different ages were obtained by staggered
planting and were inoculated with Pyri-
cularia oryzae isolate P06-6 (25,000
spores/ml). The number (LN) and size
(LS) of lesions were ascertained 10 days
after inoculation. Cumulative spore pro-
duction (CSP), latent period (L), and
infections (IP) were determined by daily
counting of spores produced from ran-
domly selected lesions. In both cultivars,
LN was maximum on plants inoculated
9 days after seeding (DAS) and decrease
dramatically with increased plant age up
to 48 DAS. LN was always less on Deno-
rado than on C22. Plant age did not
greatly affect LS in both cultivars. On
C22, plant age did not affect CSP but on
Denorado, CSP decreased with increasing
plant age. LP was similar on C22 regard-
less of plant age. However, with Deno-
rado LP was longer on older plants. IF
increased with plant age on C22 while
the reverse was observed with Denorado.
The reduced LN, lower CSP, longer LP
and shorter IP of Denorado with in-
creasing plant age perhaps contributes
to the durable resistance of this cultival
in the farmers fields.

Identification of Germplasms With Mul-
tiple Disease Resistant in IR and BR
lines in Bangladesh. S.A. Miah, A.K.M.
Shahjahan, M.A. Haque, M.A. Nahar
M.M. Hossain, M.A.T. Mia, N.R. Shar.
man, H.U. Ahmed and S.I. Akanda BRRI
Bangladesh and IRRI, Los Banios, Laguna

Searching for resistant sources tc
different diseases has been the majoi
thrust at the Bangladesh Rice Research









Abstracts of Papers


but none with RTSV alone. GLH that fed
on normal serum transmitted both viruses.
On the other hand, when RTV-carrying
GLH fed on anti-RTSV IgG with same
concentration for 16 hours and then
transferred to RTBV-infected plant for
eight hours, RTBV transmission occurred.
All infected plants contained RTBV
alone. These results showed that serolo-
gical blocking of RTBV/RTSV transmis-
sion by GLH was effective as far as
testing varieties for resistance to RTBV
ace it can not be transmitted from
plants infected with RTBV alone. Further-
more, the study showed that RTSV need
not be infective in order to help in RTBV
transmission or RTSV itself may not be
the helper component for RTBV trans-
mission.

Multiplication of Rice Ragged Stunt
Virus in the Brown Planthopper Vector
and its Distribution in Infected Plant.
Amara Parejarearn and H. Hibino. IRRI,
Los Bahos, Laguna.

Virus-free brown planthopper (BHP)
nymphs were allowed to feed on rice
ragged stunt virus (RSV)infected plants
for 4 days and held on healthy TN1
seedlings. Insects were homogenized with
0.02 phosphate buffered-saline (pH 6.5)
containing 0.05% Tween plus 2% poly-
vinyl-pyrrolidone. Extracts were tested in
ELISA and relative amount of RSV
antigen was indicated as absorbance at
405 nm.
The amount of RSV in insect body
increased gradually up to 6th day after
the termination of acquisition access
and then declined. The result indicates
that RSV acquired by the BPH retain
its antigenicity for 2 days and that
RVS multiplication start at 6 days after
the initiation of acquisition access. It
is evident that RSV is propagative in
the BPH.
In a separate experiment, roots, leaf-
sheaths and leafblades of 30-day-old


TN1 plants infected with RSV were
separately homogenized w/v in 4 volumes
phosphate buffered saline solution
(pH 7.4). In another set of samples, the
leaves and sheaths per tiller were num-
bered based on leaf position from older
to younger and were homogenized
separately. Extracts were directly tested
in ELSA to compare relative amount
of virus.
Virus antigen was detected more in
sheath than in roots and leaves. Regard-
less of tiller number, the second and third
sheath exhibited higher mean absorbance
value than other parts. RSV was also
detected in the youngest leaf and sheath
which did not show any visible symptom.
However, no RSV was detected in the
oldest leaves of some samples.

Phytotoxic Polysaccharides Produced by
Virulent and Avirulent Strains of Xantho-
monas campestris pv. oryzae. P. Vidhya-
sekaran, Menchu Alvenda and T.W. Mew,
IRRI, Los Bainos, Laguna.

Both virulent and avirulent isolates
produced extracellular polysaccharide
(EPS) in culture which induced typical
wilting symptoms on rice seedlings. There
was no apparent difference in the inten-
sity of wilt induction by EPS produced
by the different strains. But the acid
fuchsin fed through rice roots reached
the tip of the leaf faster in avirulent
strain-produced EPS treated plants than
in virulent strain-produced EPS treated
ones. Electrolyte leakage was less in all
EPS treated plants and much more
reduction was observed in virulent strain-
produced EPS treated plants. The EPS
contained glucose, mannose, glucuronic
acid, pyruvic acid and O-acetyl groups.
There was not much difference in the
sugar content but glucuronic acid,
pyruvic acid and 0-acetyl were signifi-
cantly more in the EPS produced by
virulent strains than that produced by
avirulent strains. The importance of


Jan. & June 1985









Philippine Phytopathology


these differences in pathogenesis i
discussed.

Penicillium anatolicum: A Potential I
control Agent Against Cyst Nemato
Globodera rostochiensis (Woll.). Glk
C. Molina and R.G. Davide, UP
College, Laguna.

Bioassay of crude extract from P. a
tolicum showed that the fungus produ
by-products with nematicidal actii
against G. rostochiensis. Separation
isolation of nematicidal fractions w
done by preparative thin layer chrom,
graphy. Four fractions were isolated fr
the crude extract. These were tes
against the cyst nematodes at 3 corn
trations using the immersion technic
Toxicity of the fractions was express
in percent mortality taken 24 and 48
after immersion of the test nemato
in nematicidal fractions. Fraction
showed highest toxicity with 88
mortality. Percent mortality of fract
2 did not vary significantly with thosE
Nemacur 10G and Furadan 3 G.

Concentration of Rice Tungro Spher
Virus in Tungro-Infected Rice. N.B. Bi
R.D. Daquioag, P.Q. Cabauatan and
Hibino, IRRI, Los Baiios, Laguna.

The concentration of rice tungro sl
rical virus (RTSV) in tungro-infested'
chung Native 1 (TN1) plants was de
mined by enzyme-linked immunosorb
assay (ELISA). TN1 seedlings were inc
lated with the tungro-associated vin
using Nephotettix virescens which wer
lowed an acquisition access to tungro
fected TN1 plants. At 15 days after inc
lation (DAI), the seedlings were classic
as rice tungro bacilliform virus (RTB
infected, RTSV-infected, or RTBV
RTSV.-infected using ELISA. Measu
amounts (in grams) were obtained fi
each group of plants at 15-day intern
homogenized, and appropriately dilu


in buffer. Similarly, known amou
(in O.D. units) of purifed RTSV I
were diluted two-fold in buffer, place
separately wells and tested in EIJ
simultaneously with the plant extr;
served as standard. The amount
RTSV in doubly-infected plants
always higher than in those infected w
RTSV alone with concentrations rang
from 0.02-0.65 and 0.03-0.34 O.D. un
respectively. Highest amounts appea
to be at 45-75 and 60 DAI for dout
and singly-infected plants, respective
These results suggest faster multi
cation of RTSV in plants doubly infec
with RTBV than in plants infected w
RTSV alone. The above phenomer
may partly explain the more sev
symptoms expression in RTBV + RTI
infected than in RTBV-infected plain

Yield Loss Assessment and Control
Meloidogyne incognita Chitwood in Fl
Cured Tobacco (Nicotiana tabacum
1.N. Rifien and R.G. Davide, Philipp
Tobacco Research Training Center
UPLB, College, Laguna.

Yield loss in flue-cured tobacco due
Meloidogyne incognita Chitwood and
control were assessed in Batac, Ilo
Norte from November, 1982 to M
1983. Flue-cured tobacco cultivar PF
(Balikbayan) was inoculated m
different population levels (500, 10
5000 and 10000 eggs/hill of M. incog)
to determine the loss in yield and as!
the efficacy of three namaticides
nematode control. The effectiveness
Paecilomyces lilacinus Thorn. Samson
biological control agent against M. inc
nita was also evaluated.
The three nematicides used and tl
rates of application were: oxamyl, 1.25
a.i./ha; phenamiphos, 0.85 kg a.i./ha;
carbofuran, 1.41 kg a.i./ha. The fun
was mass produced in sterilized w;
hyacinth; one liter of the fungus inc
lum was equally applied to 16 toba


1i /









Abstracts of Papers


ealngs.
There was a negative relationship
tween the population of root-knot
matode with plant height, number of
.rvested leaves, cured leaf weight and
p (all plant parts above the ground) dry
eight of tobacco. Yield loss due to
imatode densities of 500 to 10000 eggs/
11 in the soil was 0.4 to 1.5 t/ha. This
as equivalent to 12 to 14% of the yield
Plants which were not inoculated with
incognita.
Yield was increased by as much as
'7% with the use of oxamyl, and
i% with the use of phenamiphos com-
ired with the untreated control. Oxamyl
as also the most effective in reducing
e population of nematode in the soil
Sto 60 days after transplanting (DAT).
*eated plants performed better when
e nematicides were applied immediate-
after transplanting. Seedlings with
Us before transplanting and those in-
cted with nematodes after transplanting
formed similarly as long as they were
heated with nematicide.
The nematophagous fungus, P. lilaci-
is, either applied by soil drenching,
il mixing or dipping the seedlings for
ie hr in the fungal suspension increased
ant height, stem diameter and top dry
sight of tobacco at 45 DAT. The fungal
pension was 1L/16 seedlings. P. lila-
2us also decreased the nematode
population significantly by 66 to 75%
ter 45 DAT. This indicates a bright
ospect for the fungus as a biological
ntrol agent for root-knot nematodes in
bacco.

prospect of Weather Analyses in Sche-
aling Fungicide Application in the
management of Potato Late Blight. A.B.
lolina, Jr. UPLB, College, Laguna.

Microclimatic data were collected in
eld experiments carried out in La Tri-
dad and Atok, Benguet during the dry
id wet seasons of 1984. There existed


ration in tavorability for late blight
Ivelopment in the dry season while the
At season showed to be all throughout
vorable for disease development. Using
e Wallin System to determine the
vorability for late blight infection,
i4 cumulative severity values (CSV)
:re recorded during the wet season
wanting while a mean of 77 CSV was
:orded during the dry season. Atok area
oved to have more favorable conditions
r disease development than in La Trini-
d. Fungicide application schedules de-
'ed from weather analyses resulted to
auction in application frequency by as
ich as 50 percent of the calendar-base
iedule during the dry season, but not
ring the wet season.

velopment of an Integrated Control
approach for Cyst Nematode. T. Kha-
d, E.A. Versola and M. Bondad, BPI
guio City.

A four year survey of cyst nematode
cidence in the potato growing areas of
banquet and Mt. Province showed that
it of 621 farms, 31 were infested at
trying degrees. These farms were
stricted to elevations of 1,700 and
ore meters above sea level.
The potato cyst nematode was ob-
rved to complete one life cycle in appro-
mately 70 to 75 days in two different
cations. Reproduction began when the
imulative "daydegrees" reached 1,006
id 1,200 degree levels.
A significant reduction of 42% in cyst
matode population was observed when
susceptible potato variety was harvested
) days after planting compared to 90
ys.
Cyst nematode resistant variety Bero-
ia reduced cyst population by 65.67%
while susceptible variety Isola caused a
fold increase in cyst population. Carrots,
ibbage and Fennel (non-host crops)
duced cyst population at varying
:grees.


13









Philippine Phytopathology


Integrated Control of Root Knot Nema-
tode (Meloidogyne sp.) in Potato Seed-
ling Nurseries. S.A. Raymundo and
R. Salas. International Potato Center
(CIP), Lima, Peru.

One of the serious pests that could
threaten the successful adoption of the
utilization of true potato seed (TPS)
in the warm humid tropics is root knot
nematode. Although soil solarization has
been found to effectively control this
pest, it is likely that longer lasting control
can be attained if this method is in-
tegrated with others, e.g., application of
the nematode controlling fungus, Paeci-
lomyces lilacinus. The results obtained
showed that when solarization was in-
tegrated with the application of P. lila-
cinus, the degree of control was drama-
tically improved compared with the
degree of control with solarization alone
or P. lilacinus alone. After two seasons of
testing the average root galling index on
non-treated control, 30 days solarizatiorn,
P. lilacinus, and 30 days solarization +


disease even up to 1U days storage.
The data also showed that as a result
of the heart-rot control, Alliette treated
plants showed remarkable improvement
of root and top growth.Generally, higher
percentage increase in growth was ob-
tained in plants treated with the fungi-
cide and stored for 3-4 days prior to
planting.

Comparison of Soil Solarization and
Dazomet for the Control of Root Knot
Nematode (Meloidogyne sp.) Under Field
Conditions. S.A. Raymundo, J. Alcazar,
and P. Salas, CIP Lima, Peru.


With the increasing acreage planted to
potato (Solanum tuberosum) in the
warm, humid tropics, severe damage by
various pests including root knot nema-
todes are bound to be more commonly
observed. Three durations of soil solari-
zation namely 15 days, 30 days and 60
days were compared with the application


14













e D, Manzate 200, Orthocide 50 WP, T.C. Aballa and T.W. Mew, IRRI, Los
licur and Tilt 250 EC were evaluated Bafios, Laguna.
linst Helminthosporium leaf spot of
eat on UPLBW-2 under field con- Three methods of inoculation to in-
ions. All fungicides were found duce bacterial leaf streak infection were
ective in reducing the incidence of evaluated. Among the methods studied,
disease. However, the effect of spraying was the most efficient in ob-
Sfungicides varied from one rate to training higher infection. The leaf rub
their Manzate D was found as the and modified rope methods did not
'st effective at a rate above the re- increase disease intensities and were
amended in reducing leaf infection more laborious than the spraying method.
t Tilt 250 EC consistently had lower By using the spray method of inocu-
f infection at all rates tested. In re- nation, a linear relationship between
:ing grain infection, Tilt 250 EC inoculum and disease severity was noted.
all rates was also found effective com- At the inoculum level of 109 cells/ml
ed to the others. With respect to yield, infection of all eight rice varieties tested
fungicide treatments gave significantly showed a higher leaf area infection than
her yield than the control, however, that of low inoculum
y were not statistically significant with levels. Distinct varietal differences in
h other. response to the inoculum levels were
observed.
New Evaluation Scale for Sheath Blight
Rice. B.L. Candole, R.C. dela Pefia, Control of Tomato Foliar Diseases With
V. Ain and T.W. Mew, IRRI, Los Chlorothalonil, Mancozeb, Cupric Hydro-
fios. xide, and Mancozeb+ Cupric Hydroxide.
A.J. Quimio, UPLB, College, Laguna.
A new scale for evaluating rice re-
:ance to sheath blight was developed
I tested in 2 upland evaluation nur- The comparative effectiveness of Chlo-
ies. The new scale differs from the rnthalonil Mancozeh Cunric hydroxide.


eath and not on the relative position
the upper-most lesion along the sheath
-a. Varietal difference due to leaf
Fection was, therefore, an added cri-
rion in the evaluation of resistance.
other feature of the new scale is the
nsideration of a relative damage value
r each grade which was computed arbi-
irily relative to yield loss. Varietal
actions to sheath blight infection were
:arly differentiated.


effects of methods of Inoculation and
loculum Densities on Varietal Resist-
Ice to Bacterial Leaf Streak Disease.


ily-Uctober, 1984) cropping at me
P. Los Baiios Central Experiment
nation. All treatments had Lannate
or Azodrin 202 plus Agri-sticker in the
rmulations and were applied at 7 days
cle.
Cercospora leaf mold (Cercospora
ligena) and bacterial spot (Xantho-
nas campestris pv. vesicatoria) were
: destructive foliar diseases observed
the experiment. The effectiveness
the test fungicides were, therefore,
iluated against them.
Cupric hydroxide (1.4 gr. a.i/L),
ilorothalonil (1.25 gr. a.i./L), Man-
zeb (1.84 gr. a.i./L) and Mancozeb









Philippine Phytopathology


(1.4 gr. a.i./L) plus Cupric hydroxide
(1 gr. a.i./L) increased fruit yield over
the control (no fungicide) by 76, 79, 83,
and 127%, respectively. The data indi-
cated that Mancozeb plus Cupric hydro-
dixe were very effective against the
combined infections of Cercospora leaf
mold and bacterial spot considered the
most destructive foliar diseases affecting
tomato grown in the lowlands of Luzon
during the rainy season.

Control of Cucumber Downy Mildew
With Metalaxyl-Mancozeb. A.J. Quimio,
UPLB, College, Laguna.

Reduced spray applications of Metala-
xyl-Mancozeb were evaluated against cu-
cumber downy mildew during the 1984
rainy season (July-October, 1984) crop-
ping at the U.P. Los Baios Central
Experiment Station. The treatments
compared were: (1) 4 cycles Mancozeb
(1 gr. a.i./L) followed by 2 cycles Meta-
laxyl (10%) Mancozeb (48%) 0.7 gr.
a.i./L, (2) 2 cycles Mancozeb followed by
2 cycles of Metalaxyl-Mancozeb (3) 2
cycles Mancozeb followed by 4 cycles
Metalaxyl-Mancozeb, and (4) 7 cycles
Mancozeb (control treatment). All treat-
ments had Lannate L or Sevin 85S plus
Agri-sticker in the formulations.
Based on fruit weight and fruit num-
ber data, 2 applications of Metalaxyl-
Mancozeb after 2 or 4 sprays of Man-
cozeb was as effective as Mancozeb
applied 7 times at 7 days cycle. Optimal
protection of the leaves from the downy
mildew disease, however, was not ob-
served in all treatments.

Crown Sheath Rot of Rice. N.G. Fabellar
and T.W. Mew. IRRI, LosBanos, Laguna.

A rice disease characterized by yellow-
ing and subsequent drying-up of the
affected leaf blades and sheath, dark
brown to black lesions on leaf sheaths,


perethicial beaks protruding, observed
as dark spots on infected leaf sheaths,
and aborted panicle exertion was ob-
served at IRRI. Survey of some Philip-
pine provinces indicates that the disease
is endemic and its damage should be
ascertained.
The fungus was identified as Gaeu-
mannomycis graminis var. graminis, by
its symptoms produced, measurement
of ascospore, ascus and perithecia and
formation of lobate hyphopodia as
compared with its nearest related spe-
cies. Furthermore, the Kock's postulate
had been satisfied using ascospores as
inoculum.

A Method of Inoculation for Varietal
Evaluation to Bacterial Blight. R.C. Reyes
and T.W. Mew, IRRI, Los Bahos, Laguna.

An improved method of inoculation
was developed to assess rice germplasms
for resistance to bacterial blight. Inocu-
lum was prepared from 72-hour old
culture grown on potato semi-synthetic
agar medium. The bacterial cells were
suspended and adjusted to an optical
density of 0.2. Inoculation was done
by spraying the bacterial suspension to
the seedlings three days before trans-
planting at 21 days after sowing. To en-
hance infection, tap water was sprayed
to the plants one day after transplanting.
The incidence and severity was scored at
21 and 28 days after inoculation (DAI).
A total of 522 entries were evaluated by
this method, 26.0% showing a score of 1
at 21 DAI. At 28 DAI, the entries with a
disease score of 1 decreased to 8.0%.
Many entries also showed symptoms of
kresek. The number of entries showing
kresek was increased in proportion to the
entries with a higher scores.


Rice Grassy Stunt and Rice Ragged Stunt
Virus-Carriers in Brown Planthoppers
Caught by Light Trap. Z.M. Flores, H.


Vol. 21










Abstracts of Papers


[ibino and J. Perfect, IRRI, Los Bafios,
aguna.

Brown planthoppers (BPH) were
| t 1.


agged stunt-infective N. lugens varied
rom 0-7.3% and 0- 39.8%.
A steady increase in percentage in-
ective N. virescens and a relatively high
lumber of insects collected throughout
he year was obtained in 1975 when


an. & June 1985









18 Philippine Phytopathology Vol 21



study sites gave low RTV incidence, leaf- tan F, Manzate D, Manzate 200, Ortho-
hopper density, and percentage infective cide 50 WP, and Vinicur at recommended
leafhoppers. rates and Tilt 250 EC at 0.4 and 0.6 li/ha
formulated product were evaluated as
SMindprotectant and eradicant sprays against
Sorghum Stalk Rot Complex in Mindanao: Helminthosporium leaf spot.
Etiology and Development as Affected by Tilt 250 EC at 0.6 li/ha was found
Various Cultural Managements Practices more effective in both protective and
andWeather Factors. Naomi G. Tangonan eradicative tests compared with the other
and Tricita H. Quimio, USM, Kabacan, fungicides. However, a higher eradicative
North Cotabato and UPLB, College, activity against Helminthosporium leaf
Laguna. spot was observed as compared to its
protective effect. All fungicides were
Two sorghum crops, a main crop and a effective against the disease in both tests.
ratoon, were raised to determine the
pathogens causing the sorghum stalk rot Preliminary Evaluation of Fungicides for
complex and the effects of various cultu- the Control of Rhizoctonia-like Isolates
ral practices and prevailing weather condi- from Azolla spp. Rosalinda Perez Garcia
tions on its occurrence and development, and Liwanag P. Sales, UPLB, College,
Results showed that 3 fungal pathogens Laguna.
were consistently associated with the
disease. These were Colletotrichum grami- Three fungicides (Brestan, PCNB, Rov-
nicola (Ces.) Wil., Fusarium moniliforme ral) at seven concentrations each were
Sheld. and Rhizoctonia bataticola (Taub.) evaluated for their efficacies against Rhi-
Butler. Because of their synergistic rela- zoctonia-like fungus in in vitro laboratory
tionship to each other, their combined tests. Eight Rhizoctonia-like isolates from
effect resulted to an etiological phenome- various propagation ponds were inocu-
non the sorghum stalk rot complex. lated on PDA infused with 8, 5, 10, 15,
n on nlnrwino uarinin hioh nnnilatinn 90 15 and Sfl nnm nf anh finoirirlp Th<


larly, high levels ot NPK tertilizers alsi
significantly resulted to a high percentage
infection of stalk rot in both main cro]
and ratoon. The effects of weather factor
showed that high temperature slow
down infection rate but not beyond ai
optimum level; high rainfall exerts
depressive effect on infection rate while
longer sunshine duration accelerates ii


Nursery Evaluation of Fungicides Agains
Helminthosporium Leaf Spot on Wheal
Mina C. Kamatoy and D.B. Lapis, UPLE
College, Laguna.

UPLW-2 wheat variety was planted ii
nursery plots. Six fungicides namely Cai


was markedly inhibited at 5 ppm, almost
no growth at 10 ppm and higher concern
trations. Growth inhibition by Rovral was
significantly better than PCNB anc
Brestan at any concentration, but PCNI
was better than Brestan. Brestan had the
least inhibition of growth but still better
than the control.


Growth, Sporulation, and Pathogenicit
of Cercospora nicotianae Ell. & Ev. E.C
Espinosa and Lina L. Ilag, UPLB, College
Laguna.

Isolates of C nicotianae from Batac
Ilocos Norte and Los Bafios were used ii
the experiment. Isolation was easier whei









Abstracts of Papers


the disinfection period in 10% sodium
hypochlorite was prolonged from to 5
minutes and grown in carrot dextrose
agar. Better sporulation was obtained by
streaking a loopful of conidial suspension
instead of planting mycelial bits on agar
slant.
The conidia of both isolates were mor-
phologically similar. They were slender to
filiform, with truncate base and subacute
tip. The conidiophores were shorter but
wider than the conidia. The conidial and
conidiophore length were highly variable
whereas, their width were more or less
constant.
The isolates formed abundant spores
in V-8 juice agar. Maximum conidial yield
was observed 5 to 10 days after inocula-
tion. Potato dextrose agar, cornmeal agar,
carrot dextrose agar, and oatmeal agar
supported the best mycelial growth of the
isolates. Cabbage agar, tobacco agar,
onion agar and tobacco sucrose agar
which supported sporulation maintained
the least vegetative growth of the isolates.
V-8 supported fair growth and higher
conidial yield.


sources tested supported sporulation of
the two isolates but all were able to sus-
tair mycelial growth. Monosaccharides
(fructose, galactose and glucose) and
disaccharides (sucrose, lactose and mal-
tose) supported the best mycelial growth
of the two isolates. The trisaccharide,
raffinose and polysaccharide dextrin
provided better growth of isolate LB
only. All the nitrogen sources supported
vegetative growth of the two isolates.
Incubation period was 5 to 8 days
after inoculation in the three flue-cured
tobacco cultivars, regardless of inocula-
tion technique, inoculum density, and
frequency of mist application employed.
Pricked plants produced the most
number of lesions in the three flue-cured
tobacco cultivars. Spraying alone or in
combination with carborundum powder
applied as dust prior to spraying yielded
comparable number of leaf spots, indi-
cating the ineffectiveness of carborundum
as a "pre-disposing agent".
The number of leaf spots increased
with increasing inoculum density. Ino-
culum densities of 5,000 and 10,000
spores/ml produced lesion densities which


aI u4ljVllUin J1 IU lmily 1cUal wt groUwl Rul-.
nd soorulation ranked from 4 t4n + 1 arown il


:80C. Red light and total darkness stimu- applied 2 days after incubation of ino-
ited sporulation of the isolates but not culated plants in wet polyethylene bags
egetative growth. enhanced the number of leaf spots
None among the carbon and nitrogen under greenhouse condition.


Jan. & June 1985









Inc.


ETIOLOGY OF SORGHUM STA


NAOMI G. TARGONA


Respectively, Assistant Dean, Colleg
nao Kabacan, North Cotabato, and Assoc
College of Agriculture, University of th
Laguna, Philippines, 3720.
Portion of the Ph.D. dissertation of

ABR

Studies were conducted to identi
in Mindanao and to determine the path
tion to one another.
Results showed that three fungi
each other and with the stalk rot coi
(Cesati) Wilson, causing a "red rot" syrr
Sheldon and Macrophomina phaseolina
Butler,]for causing "charcoal rot" of so
to one another, their combined effect
sorghum stalk rot complex. Larger lessic
associated together and when the plants
house.



INTRODUCTION

Sorghum (Sorghum bicolor (L)
Moench) as food and feedgrain crop has
immense potentials in the country. The
crop grows very well in areas where
rainfall is erratic during the growing
season or where moisture supply is too
limited for other dry season crops to give
a decent yield. It can compare very well
with corn in its nutritive value. PCARR
(1975) cited that the livestock and feed
miller demand in Mindanao alone is
approximately 20,000 cavans of grains
per month.
Despite the fact that Mindanao is
engaged in an extensive sorghum pro-
duction, limited studies, if not entirely
wanting, has been done on diseases of
sorghum particularly sorghum stalk rot,
considered an endemic disease. This
disease is fast becoming an economically


ROT COMPLEX IN MINDANAO


ad TRICITA H. QUIMIO


Agriculture, University of Southern Minda-
Professor, Department of Plant Pathology,
hilippines at Los Bafios, (UPLB) College,

senior author submitted to UPLB.

ACT

e pathogens causing the stalk rot complex
city of each of the isolated fungus in rela-

thogens were consistently associated with
i. These were Colletotrichum graminicola
1 in sorghum stalks, Fusarium moniliforme
si) Gold.[ Rhizoctonia batalcola (Taub.)
n. Because of their synergistic relationship
Ited to an etiological phenomenon the
developed when two or three pathogens were
:inoculated in the field than in the green-




important disease especially in Mindanao
because the prevailing weather and cli-
matic conditions seem to provide an ideal
environment for its occurrence and
development.
Sorghum stalk rot in the Philippines
was first reported by Karganilla and
Elazegui (1970) to be caused by two
plant pathogens, viz., Rhizoctonia solani
Kuhn and Erwinia carotovora var. chry-
santhemi Dye. In 1978, Dalmacio noted
for the first time the occurrence of char-
coal rot of sorghum in Mindanao and
identified the causal pathogen as Macro-
phomina phaseolina (Tassi) Goid [Rhi
zoctonia bataticola (Taub.) Butler]. Pam-
plona (1980) confirmed its prevailing
occurrence to be a common phenomenon
regardless of variety, season, soil type
and time of planting. Sorghum hybrids
were notably observed as predominantly
susceptible'to the disease.








Etiology of Sorghum Stalk Rot


Enriques and Tangonan (1981) report-
ed the occurrence of a sorghum stalk rot
complex at the Southern Mindanao Agri-
cultural Research Center (SMARC) expe-
rimental area in USM, Kabacan, North
Cotabato. They noted that the disease
was caused by a complex of three plant
pathogens, viz., Fusarium moniliforme
Sheldon, Rhizoctonia solani and Erwinia
sp.
Since it appears that different causal
organisms are reported to be causing the
stalk rot complex, the objective there-
fore of the study was to confirm, iden-
tify and study the pathogenicity of each
of the isolated organisms causing stalk
rot of sorghum noting any synergistic
relationship among isolated organisms.

MATERIALS AND METHODS

Preliminary Survey and Disease Diagnosis
A survey on the occurrence, distribu-
tion and development of stalk rot on
sorgum fields at SMRAC and its vicini-
ties was made sometime in January 1983.
Field symptoms characteristic of sorghum
stalk rot were monitored and specimens
of infected tissues collected in clean
plastic bags. These were brought to the
laboratory for routine microscopic exami-
nation. Initial isolation work was done a
and the pure cultures of each isolate
maintained in potato dextrose agar (PDA)
slants. All the organisms isolated were
tagged as possible pathogens involved in
the disease complex.

Isolation and Purification

Infected stalks were washed in running
tap water to rid them of plant debris and
some soil particles. Splitting the stalks in
halves lengthwise, small bits of the infect-
ed tissues (2-3 sq mm) were surface
sterilized with 5% chlorox and rinsed in
three changes of sterile distilled water.
The sections were planted onto previous-
ly plated PDA and incubated in room


temperature. Mycelial growth radiating
from the tissues was transferred to newly
plated PDA and pure cultures were kept
in test tube slants.
At the same time, some of the infect-
ed sorghum stalks cut into convenient
sizes of 3-6 cm were allowed to sporulate
in a moisture chamber. After 3-5 days,
spore and mycelial growth were examined
under a stereoscopic dissecting micros-
cope, and using forceps, these were trans-
ferred to plated PDA and allowed to grow
in the laboratory at room temperature.
Pure cultures were also made from the
isolates.
Sonie stalks were tested for bacterial
infection by putting a cut portion in a
beaker of distilled water. A loopful of
the resulting bacterial suspension was
transferred to a test tube with 10 ml
distilled water. Serial dilutions were then
performed and a loopful was streaked on
plated nutrient agar (NA). Colonies were
selected and pure cultures maintained in
NA tube slants. To check if the bacterial
isolate was a plant pathogen, maceration
test was done on healthy sorghum stalks
cut into convenient size. The positive
result of this test was clue that the
bacterial isolate was pathogenic.

Pathogenicity Tests

Test Plants Used

IPB Hybrid sorghum seeds were sown
simultaneously in the greenhouse and
field. The seedlings were later thinned out
to maintain one to three plants per hill/
pot as the case may be. Clay pots (ca.
1 foot rim diameter) with the soil was
disinfested with 10% formaldehyde. A
nursery-like set-up was prepared in the
field with the seeds sown in rows spaced
at 75 cm and the plants distanced at 25
cm apart from each other on the furrows.
Prior to sowing, the area was thoroughly
prepared by plowing once, harrowed
twice and thereafter the furrows were


Vol. 21









Philippine Phytopathology


also disinfested with 10% formaldehyde.
The cultural requirements of the plants
were adequately provided.

Inoculum Preparation

Each isolate properly labeled and
tentatively coded pending identification,
was cultured on wooden toothpicks in
honey-peptone medium (1 g peptone, 5
ml honey, and 94 ml distilled water).
Toothpicks were packed pointed ends up,
into wide-mouth screw-capped bottles,
and were sterilized at 15 psi for 20 mi-
nutes. Two loops of its mycelial sclero-
tial-conidial/spore suspension made from
stock cultures of each of the isolates
were seeded into each 100 ml of sterilized
cooled honey-peptone medium. The me-
dium was shaken thoroughly to allow
even inoculum distribution and poured
under aseptic conditions, in a transfer
chamber, into the wide-mouthed bottles
(about 20 ml/bottle), containing the
sterile toothpicks so that the level of me-
dium in the bottle covered about one
third of the length of the toothpick. The
bottles were incubated at 350C for 7 days
at which time the toothpicks were co-
vered with mycelia and conidia/sclerotia
of the isolates and ready for use in inocu-
lation (Rao et al., 1980).

Method of Inoculation

Following the procedure of Rao and
his co-workers (1980) the sorghum plants
were inoculated 2 to 4 weeks after
flowering. A fungus-and/or bacterium-in-
fected toothpick was inserted into a hole
punched into the stalk with a thin iron
needle. Care was taken to insure that the
toothpick did not emerge through the
other side of the stem for this would
promote rapid drying of the inoculum.
All three isolates were inoculated se-
parately including all their possible com-
binations to find out the one causal
pathogen and/or group of pathogens


causing the sorghum stalk rot complex.
In all cases, the set-up was replicated 3
times with 6 plants per isolate-inoculum.
Disease development through the
characteristic symptom of sorghum stalk
rot, was carefully observed and recorded.
Percentage infection and lesion length
(in cm) were taken 15-30 days after ino-
culation. Stalks were split into halves
lengthwise to assess extent of rotting
based on the scale below (after Rose-
now and Rao, 1980). The organisms
present were re-isolated and pure cultures
were maintained primarily to compare
them with the initial or first group iso-
lates, thereby ascertaining identity.
0 less than one internode affected
1 one internode affected, but rot
does not pass through any nodal
area
2 two internodes
3 more than two
4 more than three internodes, some-
times with sclerotia
5 extensive rotting, shredding, scle-
rotia and death
The percentage infection index was
computed using the formula:
i infection + 2n2 + 3n3 + 4n4 + 5n5
% infection = x 100
5N

where:

N number of rotted stalks sampled

In + ... 5ns number of stalks showing
the scale 1, 2, 3, 4, 5, respectively.
5 represents the highest scale



RESULTS

Symptomatology

The occurrence of initial characteris-
tic symptoms of sorghum stalk rot was
observed as early as 35 days after plant-
ing. Disease ratings were taken at physio-


Jan. & June 1985









Etiology of Sorghum Stalk Rot


logical maturity of the plants. Infected
plants were observed and noted in all the
treatment combinations.
Typical field external symptoms of
sorghum stalk rot was seen as character-
ized by "red rot" phase and irregularly
discolored lesions (Fig. la), at times wa-
tersoaked in appearance, brownish to
blackish both inside and outside, hence
the description "charcoal stalk rot."
Stalks infected with stalk rot showed
a variation in appearance and color
when split open lengthwise. This was
obviously due to the different patho-
gens associated with the disease (Fig.
Ib).


Fig. 1 a & b. Characteristic external (a)
and internal (b) symptoms of sor-
ghum stalk rot complex caused by
various pathogens.


In most cases, infection takes place
up to the upper portion of the sorghum
plant, extending to the peduncles
and eventually affecting the panicle
heads and grains (Fig. 2). In greenhouse-
inoculated plants, this resulted to an
arrested or very poor development of the
panicle heads/grains.
IN


Fig. 2. Stalk rot symptom/infection ex-
tending up to the peduncle, and
grains.

The Sorghum Stalk Rot Complex

Charcoal Stalk Rot

Symptoms: The external symptoms of
this disease are premature ripening fol-
lowed by lodging of the stalk as the plant
approaches maturity, with the break usual-
ly occurring just above the crown. Black
powder-like masses of the causal fungus
commonly occur on infected stalks when
cut lengthwise. These structures, the
scleiotial bodies, may also be found on
the surface of the sorghum stalk. When
the stalk is split at the ground level, the
inside presents a shredded appearance.
The pith has disintegrated and the fibro-
vascular bundles are separated into indi-


Jan. & June 1985


...... ----









Philippine Phytopathology


VoL 21


vidual strands. The shredded interior of
the stalk appears to be dusted with the
fine charcoal. The shredded condition
may extend up in the stalk to a height of
10 to 12 inches and out into the roots at
least 12 inches. The dustlike particles
seen in the stalk are the overwintering
bodies of the fungus. The fungus enters
the feeding roots from the soil and travels
toward the crown. High temperature and
low moisture, particularly at heading
time, tended to favor charcoal rot.

Cause: Macrophomina phaseolina
(Tassi) Gold. [Rhizoctonia bataticola
(Taub.) (Butler)]. M. phaseolina is the
strain that produces both pycnidia and
sclerotia while R bataticola is the sclero-
tial bodies later turn black and are formed
in 7-10 days in culture with thick cottony
mycelia. When sclerotial bodies (40-45 to
150-250 u in diameter) are sectioned, the
distinguishing compact and pseudopa-
renchymatous tissue becomes apparent.
Elongately branched and septated hyphae
measure 9.5-10.5 u. M. phaseolina sur-
vives primarily as sclerotia that are close-
ly associated with residue. Pycnidia are
not known to commonly occur but
would afford another means of overwin-
tering. During high soil temperature,
30C and above, and low soil moisture
(80% available soil moisture and below)
the sclerotia germinate to form infection
hyphae that penetrate below-ground
plant parts. Charcoal rot generally occurs
in localized areas, therefore it is probable
that only sclerotia are disseminated by
any means that carries soil or residue.
Temperature and moisture are very spe-
cific for infection to occur, possibly the
high temperature and low soil moisture
could predispose sorghum to infection.


Fusarium Stalk Rot


Symptoms: Plants die prematurely and


lodge, breaking over at the ground line.
The heads are dull in appearance in con-
trast to the bright sheen of heads of
healthy plants. The grain is light-weight.
The pith has disintegrated and largely dis-
appeared in the area where the stalk has
broken over. In contrast to charcoal rot,
there is no dark, dusty matter on the vas-
cular bundles. Instead, the rotted area
may be simply white, or, in some case,
there is considerably red pigmentation
associated with the rot.
Although stalk rot is ordinarily ac-
companied by root rot, symptoms may
not be noticed under irrigation and ade-
quate soil fertility. The cortical tissue of
roots is first decomposed, then the vas-
cular tissue. Older roots are often totally
destroyed, leaving the plant with little
anchorage. Such plants can be easily
pulled out of the soil or toppled over by
wind. Newly formed roots have lesions of
various sizes and shapes that vary in
colors from a light tan, brick red, pink or
black. Usually the disease is most serious
during cool, wet weather following hot,
dry weather as plants near maturity.
The fungus grows from the roots and
crown into the stalk. At first, leaves turn
brown. The outside of the nodes may
have dark lesions. Inside the stalk, the
pith at first is water-soaked and even-
tually becomes disintegrated with only
the vascular bundles intact that are pink,
red or reddish purple. Later, infected
interior stalk tissue becomes a deep red
color. A white to pink mycelium may
grow on the outside of the nodes during
dump weather.

Cause: Fusarium moniliforme Shel-
don. F. moniliforme survives as chlamy-
dospore-like structure and possibly my-
celium in or on sorghum residues. The
macroconidia are 3-6 septated, sickle-
shape with pointed ends, thin walled and
elongated curved measuring 31.5-65.5 x
4-4.5 u. Microconidia are in chains, one
celled, fusiform to clavate and measure









Etiology of Sorghum Stalk Rot


7.5-10.5 x 3-3.5 u. F moniliforme is
characterized by a white cottony myce-
lial growth becoming brownish and felt-
like. Aerial hyphae grew abundant on
PDA. It sporulates within 5-7 days in
culture.

Red Rot

Symptoms: The anthracnose often pre-
cedes the stem rot phase. Initially no
symptoms show on the stem, but if it is
split, parts of the internal tissue are seen
to be water-soaked and discolored, the
actual coloration depending usually on the
genotype of the plant. These discolored
areas may be mottled, or solid, and maybe
continuous through the infected inter-
note. The nodal tissue is rarely discolored.
In susceptible varieties, the internode
tissue dries and shrinks, and surface
lesions develop and sporulate. Acervuli
can be found on both surfaces of the leaf,
on the leaf midrib, peduncle, sheats,
branches and grain.
The fungus often invades the inter-
nodes of the upper part of the stem but
the stem fall over with the break most
commonly occurring in the lower third
region.

Cause: Colletorichum graminicola
(Ces.) Wilson. The typical conidia
measuring 21.5-40.5 x 5-6.5 u are typical-
ly one-celled, hyaline and falcately cylin-
drical with blunt or rounded ends. Coni-
diophores are short, dark colored, un-
branched and aseptate. Mycelia are exten-
sively branched, elongate, and septated.
C graminicola has a characteristic whitish
mycelium turning yellowish and beco-
ming dark colored as it ages: it produces
thick and abundant aerial hyphae and
conidia. C graminicola sporulates within
5-7 days in culture.

Pathogenicity Tests

Table 1 lists the three fungal patho-


gens and their various combinations con-
sistently isolated from stalk rot-infected
sorghum from both the main crop and
ratoon sorghum plants. These fungi were
identified as Colletotrichum graminicola,
Fusarium moniliforme and Macrophomi-
na phaseolina (Rhizoctonia bataticola).
Each fungus is capable of causing sor-
ghum stalk rot by itself. Nevertheless, the
simultaneous presence of all three on
infected sorghum stalks is a very common
phenomenon observed. Of the three, C
graminicola caused significantly bigger
lesions both under greenhouse and field
conditions with a mean of 14.94 and
21.60 cm, respectively. F moniliforme
produced the smallest lesion both under
greenhouse and field with means of 13.45
and 13.54 cm, respectively. M phaseo-
lina had means of 14.88 and 17.24 cm for
greenhouse and field conditions, respec-
tively. All three pathogens caused signifi-
cantly larger lesions when compared to
each other. The combinations of C gra-
minicola / F moniliforme / M. phaseo-
lina; C graminicola / F moniliforme; C
graminicola / M. phaseolina under green-
house conditions produced significantly
larger lesions with means of 19.90, 18.28
and 16.17 cm, respectively. This suggests
that such favorable or synergistic relation-
ship caused a more severe or advanced
stalk rot infection and/or lesion develop-
ment. Of the isolates, F moniliforme
was observed to "associate" better with
the other two pathogen/isolates in
causing significantly larger lesions.
A bacterial isolate believed to be Er-
winia carotovora var. chrysanthemi
was also isolated from stalk rot-infected
sorghum. However, in this study, the
general observation was that only in
rare instances did it seem to be asso-
ciated with other fungal pathogens,
hence, may not be included as part of
the disease complex. It must be pointed
out, that bacterial stalk rot of sorghum
also did occur un SMARC sorghum fields
as a disease entity by itself in addition to


Jan. & June 1985









Philippine Phytopathology


Table 1. Lesion length (cm) of various
dated with sorghum stalk ro
under greenhouse and field c
bato, 1983-84.


ISOLATE
Greenho


C graminicola 14.84
F. monilforme 13.45
M phaseolina 14.88

C graminicola 18.28
F. moniliforme

C graminicola 16.17
M. phaseolina

F. moniliforme 19.90
M phaseolina

C graminicola -
F. moniliforme 17.10

Average 14.34


aMeans are for average of 3 replicatic
not significantly different (a = 0.05, DMRT).
the stalk rot complex caused by C grn
minicola, F moniliforme,.and M phaseo
in synergistic fashion.


DISCUSSION
Stalk rot of sorghum has become ii
creasingly important since it was firn
generally observed in SMARC in 1971
Most of the stalk rot diseases used to t
attributed to the fungus causing charco;
rot, but now it seems that several oth(
fungi may be involved. Some of their
apparently invaded the plant through
openings caused by insects or by mechi
nical injuries. Bacteria also invaded th
stalk and thus helped bring about
watersoaked, and later a rotted condition


ited pathogens and their combinations ass
mplex and inoculated on healthy sorghu
itions. USM-SMARC, Kabacan, North Col



,OCATIONa
Field AVERAGE


21.60c 18.27c
13.541 13.50e
17.24efg 16.96d

24.33b 21.31b


19.04de 17.61c


24.17b 22.03f



49.41a 33.26a

21.17b



deans within a column having a common letter a

The symptoms of stalk rot may va
with the cause and location of the initi
infection. Infections in the middle
lower part of the stalk, especially whi
they occurred through wounds near tl
base of the stalk usually were mo
destructive. External symptoms of su<
infections may at first consist of a water
soaked appearance of the stalk, with
without red or purple discoloration,
streaks on the surface of the stalk and
the veins of the sheaths and leaves. Late
one may see poorly developed kernel
premature ripening, and frequently
softening at the base of the stalk. The i
side of the stalk may show watersoak4
or discolored pith, or both, and
streaking of the vascular bundles i
fibers. The inside of the ronts of affect


zo









_. -t C0- L0_6_ Ca.-It D-*


,%,


ats likewise usually appeared water-
ked and discolored and frequently
tips of the diseased roots were dead.
At the SMARC and in nearby sorghum
wing areas of North and South Cota-
o, the occurrence of sorghum stalk
was a common observation. Although
endemic nature did not seem to cause
ring damage to sorghum production,
potential of the disease as becoming
nomically important is not a far-off
lity.
On both the main crop and ratoon sor-
un, stalk rot was noted to be caused
a complex of the three fungal patho-
s. Notwithstanding, pathogenicity
;s in greenhouse and field conditions
wed that each of the above pathogens
s capable of causing stalk rot by itself
when they infected sorghum simul-
eously in a synergistic fashion, damage
severity of infection, as measured by
on length, was more pronounced.
However, in other cases where sor-
im stalk rot was caused by a bacterial
hogen, no disease complex was appa-
t. Erwinia carotovora var. chrysan-
mi Dye, its previously reported
hogen(Karganilla and Exconde, 1972),
erally occurred separately and deve-
ed as a distinct stalk rot pathogen,
ice excluded in the disease complex.
sults of pathogenicity tests in bacteria
ected sorghum stalks revealed that it
s caused by E. carotovora var. chry-
!themi. The same pathogen was re-
rted by Bayacag (1983) to be causing
Ik rot of corn in SMARC fields.


LITERATURE CITED

YACAG, TERESITA C. 1983. Control
of stalk rot of corn through cultural
practices, SMARC Ann. Report 1982.
Univ. Southern Mindanao, Kabacan,
North Cotabato. p. 11

RIQUEZ, B.M. and NAOMI G. TA-
NGONAN. 1981. Occurrence and deve-
lopment of sorghum stalk rot as
affected by fertilizer rate and popu-
lation density. SMARC Monitor 2 (2):
12. Univ. Southern Mindanao, Kaba-
can, North Cotabato.

.RGANILLA, A.D. and F.A. ELAZE-
GUI. 1970. Local diseases of sorghum.
Philipp. Phytopathol. 6: 83.

.RGANILLA, A.D. and O.R. EXCON-
DE. 1972. Bacterial stalk rot of corn
and sorghum. Philipp. Phytopathol.
8: 4 (Abstr.)

ARR, 1975. The Philippines recom-
mends for sorghum. Philippine Coun-
cil Resources Research, Los Bafios,
Laguna. 46 p.

.O, K.N., V.S. REDDY, R.J. WIL-
LIAMS and L.R. HOUSE. 1980. The
ICRISAT charcoal rot program. In
Proc. Workshop on Sorghum Diseases,
Hyderabad, India. 468 p.

)SENOW, D.T. 1980. Stalk rot resist-
ance breeding in Texas. In ICRISAT
Proc. Intern. Workshop on Sorghum
Diseases, Hyderabad, India, 468 p.









Philippine Phytopathological Society, Inc.
1985 Phil. Phytopath. 21: 28-33


PATHOGENICITY OF SAROCLADIUM OR YZAE AND FACTORS
AFFECTING SHEATH ROT DEVELOPMENT ON RICE



K. C. CHUKE and D. B. LAPIS

Respectively, Graduate Research scholar, Department of Plant Pathology, The
International Rice Research Institute (IRRI) and Associate Professor, Department of
Plant Pathology, College of Agriculture, University of the Philippines at Los Bailos
(UPLB), College, Laguna, Philippines. 3720

Portion of the Master's thesis of the senior author submitted to UPLB.






ABSTRACT

Studies on the pathogenicity of Sarocladium oryzae revealed that the pathogen
infected rice plants at all stages of growth, but was most destructive after the booting
stage. The pathogen infected the sheath, the midrib of the leaf blade, and the grains.
Disease symptoms vary with the variety and tissue infected.
The optimum temperature and relative humidity for infection and disease develop-
ment at the heading stage was 30 C 5 C and 70%, respectively. Heavy nitrogen applica-
tion enhanced disease development. Unbalanced nutrition (N,P,K) which resulted to poor
growth also favored disease development.
Resistance to the disease differed with variety rather than with rice types.


Sheath rot of rice, caused by Saro-
cladium oryzae (Sawada) W. Gams & D.
Hawksw., was first recorded and des-
cribed as early as 1922 in Taiwan, China.
Since then, it has been reported in many
rice growing countries all over the world.
Earlier observation, the disease was con-
sidered a problem of relatively little eco-
nomic importance. Several reports of
yield losses and damage indicated, how-
ever, that the disease could be a poten-
tial threat to rice production. In Japan,
a disease incidence of 10-20% was report-
ed (Kawamura, 1940). Chen (1957)
reported yield within the range of 3 to
20%, and 10-20% losses were experienced


in India (Amin et al., 1974). Mohan and
Subramanian (1977) pointed out that, in
general, yield reductions were 6-57.5%,
depending on the susceptibility of the
rice cultivar. In 1979, the disease was
prevalent on IRRI varieties and breeding
lines at the IRRI farm (IRRI, 1980). In-
jection at the boot stage produced a
sheath rot infection of 84.4% and reduced
panicle production by 28.1% and grain
yield by 75.0% (Estrada et al, 1979).
The effect of the disease was found
to be related to rice sterility (Chien and
Huang, 1979) and chaffy grain formation
(Murty et al., 1980).









Pathogenicity of Sarocladium Oryzae


MATERIALS AND METHODS

Pathogenicity at Different Growth
Stages and Leaf Sheath Age

To observe seed transmission, natural-
ly infected seeds of TN1 and RGS-20
rice varieties were planted in sterilized
soil in beakers, which were covered with
other beakers, and sealed with masking
tape to avoid contamination. Ten days
after planting, each seedling was exam-
ined for symptoms and signs under a
stereomicroscope.
For pathogenicity tests, seedlings and
plants at tillering stage were inoculated
under laboratory and screenhouse con-
ditions using intact and excised plant
parts. The isolate S-8101 of S. oryzaq,
from IR1487-273-1-1, was used in all
inoculations.
Seeds of IR20, RGS-20, IR9224-
117-2-3, and IR442-2-58, and IR13429-
109-2 previously disinfected with 1%
sodium hypochlorite, were planted in the
petri dishes lined with moistened filter
paper and subjected to fluorescent light
at room temperature (26-28 C). When
the coleoptile of germinating seeds were
about 1 cm long, they were pricked
with needles and sprayed with spore
suspension of the fungus. The inoculated
seedlings were incubated under intermit-
tent dark and light conditions. One week
later, they were evaluated for disease
symptoms and signs.
Testing at tillering stage was done
by uniformly wounding the inner surface
of the leaf sheaths with a syringe, inocula-
ting using the spore-drop method, the
leaf sheaths at different leaf positions,
and incubating the plants in the moist
chamber. After 24 hours, they were trans-
ferred to the greenhouse benches. After 2
weeks, the infected leaf sheaths at the dif-
ferent leaf positions were counted.
To determine if natural infection
could occur at the tillering stage, 2-week-
old seedlings grown from disinfected


seeds in the plastic trays were kept in
naturally diseased conditions for 3 weeks
in the screenhouse. Symptoms were ob-
served and confirmed by incubating the
diseased tissues for 3 days in plastic
plates lined with moist filter paper and
examining tissues under the microscope
for the presence of spores and conidio-
pores. Rice plants with brown lesions
were collected from rice fields, and then
were incubated and examined under the
microscope for the presence of conidia
and conidiophores of the fungus.
At booting, the plants were inoculated
by injecting a spore suspension into the
boots. Another set of plants were injected
with distilled water and then sprayed
with a spore suspension to compare the
symptoms induced by the different
methods.

Factors Affecting Infection, Severity,
and Development of the Disease

Effect of temperature and relative
humidity. Two breeding lines and 2 va-
rieties of rice were grown in clay pots
in the greenhouse. At early booting stage,
the plants were transferred to the phyto-
tron with constant temperatures of 20,
25, 30, 35 C, and 20/15, 25/20, 30/25,
35/30, 40/35 C fluctuating day/night
temperatures at a constant relative hu-
midity of 70%. Another set of plants
at the early booting stage of growth were
subjected to 50, 70, and 90% relative
humidities at 30/28 C day/night tempera-
tures.
Effect of macronutrients (N-P-K)
on disease severity. To test the effect of
plant nutrients, rice varieties IR36 and
RGS-20 were grown in plastic trays,
each filled with 10 kg Maahas clay with
different fertilizer combinations: 160-60-
60, 120-60-60, 60-60-60, 0-60-60, 120-
0-60, 120-60-0, and 0-0-0 kg nitrogen-
phosphorus-potassium per hectare. A trial
on timing of fertilization was also con-
ducted in the screenhouse: 1) 60-60-60


Jan. & June 1985













(NPK) as basal application followed by RESULTS AND DISCUSSION
topdressing of 60 kg N/ha at booting;
and 2) 60 kg N/ha as basal application
and 60-60-60 kg NPK/ha as topdressing Pathogenicity at Different Growth Stages
at booting. of the Rice Plants and Ages of the Leaf
In all trials, inoculation was by injec- Sheath
tion of spore suspension into rice boots.
Scoring was at 30 days after inoculation. Tests on seed transmission of the
The fertilizer trial had 3 replications in disease showed that the disease was
CRD. highly transmissible through naturally
Effect of host differential reaction of infected TN1 and RGS-20 seeds. Infection
Japonica and Indica rices to the patho- percentages were 75.3% for TN1 and
gen. Resistance of the rice type to the 83.7% for RGS-20. Early symptoms were
disease was tested in the screenhouse. observed on the emerging leaves and as
Twelve varieties each of japonica and the disease advanced, the second and
indica rices were grown in concrete beds third leaf sheaths also became infected.
in the screenhouse where many diseased Under a stereomicroscope, conidia and
plants were maintained. To ensure good conidiospores in dewdrops were observed
infection, 20 plants from each variety on the seeds and stalks of the young
at booting stage were inoculated. About plants.
30 days after inoculation, scoring was At tillering stage, infection by artificial
done based on the following scale: inoculation was severe (Fig. 1). Dark
0 No symptoms on the flag leaf brown lesions formed and expanded
sheaths. from the points of inoculation. Nume-
1 Sheath has the brown or purple rous conidia and conidiospores were












Disease index (%)
awed by a spray of spore suspension Voriey:
:essfully induced the disease. The mIR1487-372-1
[al symptoms on the flag leaf sheaths RR224-117-2-3-3-2
e light brown lesions without definite m TNi
gins. As the plants became older, the 70
iptoms developed into two types:
he panicles emerged slowly or incom- 60
ely, giving typical sheath rot symp- 50
is with dark to dark brown irregular 4
)ns; 2) the panicles emerged faster,
showed light brown lesions with 3
:finite margins. In China, the latter zo -
called "purple sheath" or "purple
ith with yellow leaves" and is as-
ed to genetic phenomenon or to O 2o0 /25c /30o 350c
halosporium spp. (Zhang, 1983), Temperahue
es, thrips, and other causal agents Fig. 1. The response of rice sheath rot
en, 1981; Wang, and Li, 1981; Zhang, to different temperatures.
.3). The symptoms on rice grains Dise (%)
e dark brown spots similar to those V-- --
grains infected by HelmithosporiumIR w87-37-1
zae, the brown leaf spot disease. 4-1172-33-2
70 : TN
0o-
:tors Affecting Infection, Severity and
relopment of the Disease









Philippine Phytopathology


Disease index (%)


30-


10-
to


Variety:
.R IR 1487-372-1
N RGS-20
frNl
IR36









m\
-II '




I-- 50% ---- 1O% -I--- 90%--
Relative humidity


Fig. 3. Effect of relative humidity on the
development of rice sheath rot.


Effect of fertilization (NPK) on
disease severity.
The present study demonstrated that
the application of inappropriate com-
binations of fertilizers NPK resulted in
increased disease severity. Disease indices
were lowest at the rate of 0-60-60 and
60-60-60 kg NPK. Increased nitrogen
application resulted in considerable in-
crease in disease severity (Fig. 4). The
disease decreased to a minimum with the
reduction of the nitrogen fertilizer to a
certain level, or. with the proper combina-
tions of the other elements. Statistical
analysis showed that the disease seems to
increase with zero fertilizer application
on IR36, but not on susceptible RGS-20.
At high nitrogen with low phosphorus or
potassium, the disease slightly decreased.
Separate applications of fertilizers
reduced disease severity.
Statistical analysis indicated inter-
actions between the fertilizer and rice


N o=. o

N 2 oo =
Rates and levelof fertilizer combination
Fig. 4. Effect of N, P, K fertilizers on
disease index of rice sheath rot.
varieties. The response of the disease to
nutrient factors differed with the rice
variety tested. The increase in disease
with increase in fertilizer levels varied
significantly between varieties. In IR36
disease increase was slight at all levels
of the fertilizers; in RGS-20, infection
and disease development increased with
changes in levels of fertilizers. Present
study on fertilizer effect on the disease
confirmed previous reports that disease
increased with increasing amounts of
nitrogen fertilizers (Chen and Chien,
1964; Sung et al., 1975).

Like other fungal and bacterial disease
of rice, the sheath rot disease has been
associated with favorable nutrition of the
rice plants. In many studies on fungal
and bacterial diseases of rice, nitrogen,
phosphorus, and potassium had been
reported to affect disease development.
Soil conditions and drought stress like-
wise have been noted to affect disease
development in both irrigated and upland
rice.
Effect of host the differential react-
tion of japonica and indica rices to the
pathogen. Tests with 12 rice varieties
each of japonicas and indicas tested
under conditions of high disease pressure
showed that each rice type had a wide


Vol. 21









Pathogenicity of Sarocladium Oryzi


range of disease indices. In japonica rice, KAWAMURA, E. 1940. A note on the
the disease indices ranged from 24.1 to sheath rot of rice plant with special
88.3%, and in indica rice, the indices were reference to its causal organism,
25.7 to 87.5%. The experiment indicated Acrocylindrium oryzae Saw. Ann.
that the disease reactions varied mainly Phytopathol. Soc. Japan 10:55-59
among the varieties rather than between (in Japanese).
rice types. The finding was different from
the results obtained by Chen and Chieu IRRI (INTERNATIONAL RICE RE-
(1964), and Chien and Huang (1979). SEARCH INSTITUTE). 1980. Annual
Report for 1979. Los Bafios, Laguna,
LITERATURE CITED Philippines.

AMIN, K.S:, B.D. SHARMA, and C.R. MOHAN, R. and C.L. SUBRAMANIAN.
DAS. 1974. Occurrence in India of 1977. Influence of nitrogen and
sheath rot of rice caused by Acrocylin- sheath rot disease on sugar content
of two paddy varieties. Current
drium. Plant Dis. Reptr. 58 (4): 358- of two paddy vaetes. urrent
360. Science 46 (2):753-755.

MURTY, V.S.T., O.P. VEDA, R.G.
CHEN, C.B. 1981. Field diagnosis of MRTY, VS.T, O.P. VDA, R.G.
purple sheath symptom of rice disease. SATPUTHE, and R. KAHSYAP. 1980.
Fujian Nonye Keji 5:27-28. Sheath rot incidence and chaffy grain
percentage on some popular rices.
CHEN, M.J. 1957. Studies on sheath rot IRRN 5(5):7.
of rice plant. J. Agr. for Taiwan 6:84- SUNG, J.M., K.H. LEE and S.C. KIM.
102 (with English Summary). SUNG, J.M., K.H. LEE and S.C. KIM.
S1975. Studies on the occurrence
CHEN, C.C. and C.C. CHIEN. 1964. and control of sheath rot of rice
Some observations on the outbreak caused by Acrocylindrium oryzae
of rice sheath rot disease. J. Taiwan Sawada. The Research Reports of the
Agr. Res. 13(2): Office of Rural Dev. 17:93-98.

CHIEN, C.C. and C.H. HUANG. 1979. WANG, M.C. and S.Z. LI. 1981. The
The relation between sheath rot and observation on disease development
the sterility of rice plant. J. Agr. Res. and yield loss of rice purple sheath
China 28(1):7-16. disease. Jiensi Non-Ye 11:7-8.

ESTRADA, V.A., L.M. SANCHEZ, and ZHAN, G.C. 1983. "Purple sheath" or
PAT CRILL. 1979. Evaluation of "purple sheath with yellow leaves" of
screening methods for sheath rot rice disease. Zejiang Nonye Keziu 2.
resistance of rice. Plant Dis. Reptr.
63:908-911.


Jan. & June 1985













CONTROL OF HELMINTHOSPORIUM LEAF SPOT OF
WHEAT (TRITICUM AESTIVUM) WITH MANCOZEB
AND THIOPHANATE METHYL



A.M. SINOHIN and D.B. LAPIS



Respectively, Research Associate and Associate Professor, Department of Plant
Pathology, College of Agriculture, University of the Philippines at Los Baflos (UPLB),
College, Laguna, Philippines. 3720
Portion of the MS thesis of the senior author submitted to UPLB.



ABSTRACT

Mancozeb (Manganese ethlylene bisdithiocarbamate plus zinc; Dithane M-45)
and thiophanate methyl (Fungitox 70 WP) were evaluated against Helminthosporium
leaf spot of wheat caused by Helminthosporium sativum. The two fungicides were
sprayed at different rates and frequencies.
Leaf spot development was significantly controlled by Dithane M-45 at all rates
and frequencies of application. Two to three sprayings provided the most effective con-
trol without any phytotoxic effect.
Grain yield was significantly higher with Dithane M-45 at 1.0 and 1.5 kg fp/ha in
all soravine frequencies.













disease may result in yield loss exceeding
60%. In the Philippines, no study has yet
been conducted on the damage or loss
due to the disease although its presence
has always been observed in the wheat
experimental and pilot areas of the
country.
In temperate countries, H. sativum
caused seedling blight, root and crown
rot and head blight responsible for con-
siderable annual loss to the crop (Wood,
1962). Christensen (1925) reported about
75% of the plants in numerous wheat
fields in Minnesota succumbed to root
and basal stem rot. Mead (1933) esti-
mated the loss to be 70% at emergence
and 60% in yield when sprouted imma-
ture wheat seeds were inoculated with
the pathogen.
The pathogen also attacked the grains
causing seed discoloration known as
black point (Fig. 3) (Dastur, 1932).
Fig. 1. Diseased leaves of wheat showing
the advanced (A) and early (B)
symptoms of Helminthosporium
lo- t" __ f









Philippine Phytopathology


located (Nene et al., 1968). Infected
grains lost its quality and market value.
Like the brown rust and stem rust,
Helminthosporium disease affected the
quality of flour from infected grains
(Konechnaya and Kamelina, 1977).
Temperature and moisture appeared
to be the most important factors that
influence severity of leaf spot disease
of wheat. Infection of detached leaves
was quicker and more intense at 25 C
than at 20 C or 30 C (Nema and Joshi,
1973). They also noted that under field
condition, the development of leaf spot
symptoms in inoculated plants com-
menced in humid condition or with a
mean relative humidity (RH) of 53-64%.
On the other hand, rapid development
of leaf spot was recorded by Mishra
and Chourasia (1976) under environ-
mental condition of 26-30 C with 75%
RH. Researchers, however, agree that
under field condition, wheat plants are
most susceptible at a later stage of
growth or after the flowering stage.
Inoculation tests showed maximum in-
fection recorded on 40-60-day old
seedlings.
Helminthosporium leaf spot is severe
in the Philippines during the wheat-
growing months of December to March.
The disease had been observed almost
every year in the field trials at San
Mateo and Ilagan, Isabela; Dingras,
Ilocos Norte and in the Central Expe-
riment Station, UP at Los Bafios, College,
Laguna (B.D. Ona, wheat researcher,
Institute of Plant Breeding'). Work on
selection of varieties for Helminthospo-
rium leaf spot and foot rot resistance
is being conducted at the Department
of Plant Pathology, UP at Los Bafios.
Several workers abroad particularly
in USSR, Czechoslovakia and India
reported efficient control of Helminthos-
porium leaf spot through chemical means
(Chattophadhyay and Chakrabarti, 1961;

-YPersonal Communication


Kernkamp et al. 1970, 1974; Bidari
and Govindu, 1975; Liska and Drotarova,
1976; Ismail and Michalikova, 1977;
Lokendra, 1978, Pidoplichko and Andre-
eva. 1980; and Luz and Vieira, 1982).
No studies have yet been done on the
aspect of chemical control on H. sativum
in the country. In view of the serious
threat of this disease to the government
program on wheat research and its sub-
sequent intensive production the study
was conducted to 1) compare the efficacy
of zinc ion-manganese ethylene bisdithio-
carbamate (80% a.i.) and thiophanate
methyl (70% a.i.), 2) determine the most
effective rate and frequency of appli-
cation, 3) determine the phytotoxicity
of the chemical treatment and rates of
application, and 4) determine the eco-
nomics of chemical application.
The study was conducted at the up-
land field of the Central Experiment
Station, University of the Philippines
at Los Bafios, in College, Laguna from
December 1983 to March 1984.

MATERIALS AND METHODS

Wheat Variety Used
Wheat variety UPLW-2 (Trigo 2) was
used in the study.

Field Preparation
The field was plowed once and har-
rowed two times. After the last har-
rowing, a tractor-mounted furrower was
used to make furrows and were spaced
30 cm apart at about 5 cm deep.

Planting
All plots were properly labelled
before planting. The seeds were sown
at the rate of 9 g per 3 m row which was
approximately 125 kg/ha or a total of
90 g seeds per plot. The seeds were even-
ly distributed in the furrows and covered
uniformly with 2 cm soil.


VoL 21









of Helminthosporium Leaf S


Vacant spaces of about 1-1.2 m were
*ovided between blocks to serve as alley-
ays. There was no vacant space between
Ijacent plots within the same block. To
minimize the effect of unplanted alley
i the adjacent experimental plot, two
lard rows were planted along the
leyside of the plot. Approximately
000 m2 was used in the study.

cultural Practices
Complete fertilizer (14-14-14) and
rea (45-0-0) at the rate of 90-30-30 kg
PK/ha were applied over the furrowed
eld just before seeding.
To ward off insect interference two
*otective sprayings with an insecticide,
rodan, at the rate of 946 ml formulated
*oduct (fp)/ha were undertaken. First
id second sprayings were done at the
egetative and reproductive stage, respec-
vely.
The surrounding areas were regularly
leaned to suppress rat infestation. Con-
nuous rat control throughout the crop
eriod was provided by sustained baiting
'ith chronic toxicant, Racumin. The bait
mixture consisted of 1 part toxicant per
9 part of milled rice, the baiting material
sed. Trapping was also conducted to
:duce rodent damage.
Weeding between rows commenced at
bout 2 weeks after seedling emergence
sing a light hoe. One week after the first
feeding trowel was used for handweeding
between plants within rows. Spot weed-
ig followed up to one month after
mergence.
Overhead irrigation with perforated
ipe was employed. The experimental
field was irrigated after planting and once
very two weeks and whenever necessary
until about 2 weeks after heading.

Fungicide, Rate and Frequency of
Application
An 80% zinc manganese ethylene bis-
ithicanrhamate (Dithane M-45) and 70%


liophanate methyl (Fungitox 70 WP)
ere used in the study. Dithane is a pro-
!ctant while Fungitox is a systemic
ingicide. Including the control, four
ites were tested viz., a) recommended
ite (RR) or 1.0 kg fp/ha; b) 0.5 below
R or 0.5 kg fp/ha; c) 0.5 above RR or
.5 kg fp/ha and d) control. Three appli-
ition frequencies (Ix, 2x, 3x) of each
ingicide treatment were made. Sprayings
ere started at the flagleaf emergence
age until the milking stage. Distilled
ater was sprayed the control plots.
method and Time of Inoculation
Plots representing each treatment were
tificially inoculated by spraying spore
Ispension from a 9-day old culture ofH.
rtivum, calibrated at 40,000-50,000
pores per ml, up to the point of run-off.
ioculations were done 24 hours after
te first fungicide spraying.
liar Infection Rating
Disease evaluation was done two
eeks before harvest. Twenty-five flag-
aves taken at random per treatment
,r replication were evaluated. Rating
as done by counting the Helminthos-
)rium leaf spot lesions per flag leaf.
rain Yield
Six middle rows using 2.9 of the 3-
leter row were used in recording ob-
:rvations and gathering of yield data.
Grain yield was determined from the
harvest of the inner 2.9 m of the 6 middle
>ws. Harvesting was done five weeks
after heading. The total length of the
>ws harvested per plot with complete
and was 17.4 m (6 rows x 2.9 m/row).
orrected plot yield for missing seg-
Lents was computed using the following
)rmula:
Weight of grains
harvested x 17.4 m
rected plot yield =
Total length of
rows harvested


37









PhilioDine Phytopathology


The yield in metric tons (MT)/ha
14% moisture was calculated using
following formula:

Corr
Yield (MT/ha) = or



x
l,(


The plot area was 4.35 m2 wi
row in the plot were spaced 30
(0.3 m) apart such that:

2.9m x 1.5m = 4.35 m2

Seed Discoloration or Black Point Ral
Black point evaluation comment
after determination of yield at 1
moisture content was completed. I
colored, shrivelled seeds were selec
from 50 g samples. Percent seed
fection was determined by using the
lowing formula:

Weight of infect
seeds (g) x 10(
Percent black point = 50 g
50 g


a) Gross Return (GR) = Yiek

b) Total Production Costs (TPC

c) Return Over Cash Costs (RO,

d) Net Return (NR) = Gross F


e) Return on Investment (ROI]


I plot yield (g) 10,000 m2/ha
x
0 g/kg plot area (4.35 m2)


T 100-actual moisture (%)
x
cg/MT 86



Experimental Design
The experiment was set up in a st
split plot design at 3 x 3 m per I
replicated three times. Fungicides, r,
and frequencies of applications w
randomly assigned to the main plot, s
plot and sub-sub plot, respectively.
An appropriate analysis of varia
was used. Differences among mean val
were tested using the Duncan's Multi
Range Test (DMRT).

Cost and Benefit
Consideration for the economic side
each of the chemical treatment inclu
the use of the following relationships:



/ha) x Farm Gate Price of wheat per kg

Gross Return Production Cost (Cash)

= Gross Return ProductionCosts(Cash)

rn Total Production Costs

Net Return
x 100
Total Production Costs


1Q









Control of Helminthosporium Leaf Spot


RESULTS

Efficacy of the Fungicides
Effect on Disease Development
A significant reduction in the number
of lesions was observed with Dithane at
all rates and frequencies of application
as compared to the untreated control
(Table 1). Disease developments in treat-
ments with one or two sprayings were not


significantly different from each other at
all rates of application. An interaction
between rate and frequency was observed
when Dithane was applied three times.
Likewise, at the same frequency, the
lowest number of lesions was counted,
where 0.5 and 1.5 kg fp per ha differed
significantly. However, the level of infec-
tion did not differed significantly be-
tween 1.0 and 0.5 and so with 1.0 and
1.5 kg fp per ha.


Table 1. Helminthosporium leaf spot infection and percent control on UPLW-2 wheat as
affected by rate and frequency of application of Dithane M-45 and Fungitox

FUNGICIDE RATE LESION PERCENT
(kg fp/ha) FREQUENCY COUNTa CONTROL


Dithane M-45












Fungitox











Distilled
water


'Each figure represents a mean of 3 replications.
Means with the same letter are not significantly different at 5% level with DMRT.


23.3 bc
22.0 bc
8.9 de

22.0 bc
13.4 cd
3.7 ef

23.9 bc
14.7 cd
3.0 f

76.9 a
57.2 a
53.5 a

62.7 a
55.8 a
71.9 a

101.3 a
84.5 a
67.3 a


62.3 a


62.6
63.1
85.7

64.7
78.5
94.1

61.6
76.5
95.2

-23.5
8.1
14.0

-0.6
10.4
-15.5

-62.6
-35.6
-8.0


0


Control


Jan. & June 1985









Philippine Phytopathology


On the other hand, Fungitox seemed
to have no perceptible effect against
leaf spot development. On the other hand,
it appeared to favor disease develop-
ment as exhibited by the negative per-
cent control of the pathogen. Fungitox-
treated plants gave higher lesion counts
than the control. Lesion counts ranged
from 53 to 101 as compared to the
control with only 62.3 lesions.


Effect on Yield

Dithane at 1.0 and 1.5 kg fp/ha at all
frequencies of spraying produced grain
yield significantly better than the rest of
the treatments (Table 2). The same fun-
gicide at 0.5 kg fp/ha and all Fungitox
treatments consistently outyielded the
untreated control although no statistical
difference was noted.


Table 2. Estimated grain yield and percent yield increase of UPLW-2 wheat as affected
by rate and frequency of applications of Dithane M-45 to control Helminthospo-
rium leaf spot.

RATE COMPUTEDa YIELD
FUNGICIDE (kg fp/ha) FREQUENCY YIELD INCREASE
(MT/ha) (%)


Dithane M-45











Fungitox












Distilled
water


aEach figure represents mean of 3 replications.
Means with the same letter are not significantly different at 5% level with DMRT.


9.2
7.9
19.7

24.3
32.7
30.6

32.6
28.6
31.3

7.8
10.6
12.4

11.5
10.9
16.1

15.6
5.4
11.8


1.2 b
1.1 b
1.3b

1.4 a
1.6 a
1.5 a

1.5 a
1.5 a
1.5 a

1.1 b
1.2 b
1.2 b

1.2 b
1,2 b
1.2 b

1.2 b
1.lb
1.2 b


1.0 b


Control


Vol. 21









Control of Helminthosporium Leaf Spot


Effect on Black point

With Dithane, a marked reduction of
black point incidence at all rates and fre-
quencies of application was evident
(Table 3), although control ranged
from 37.3 to 60.2% only. Interaction
between rate and frequency was observed
at 0.5 and 1.5 fp/ha where the highest
percent control was obtained after three
spray applications. However, the control


obtained at that rate and frequency
is still insignificant when compared to the
other rates and frequencies of application.
Fungitox failed to exhibit any signi-
ficant reduction in seed discoloration.
A significantly higher incidence of seed
infection was even observed with Fungi-
tox treatments compared to the un-
treated control.


Table 3. Helminthosporium black point incidence and percent control on UPLW-2
wheat as affected by rate and frequency of applications of Dithane M-45 and
Fungitox.

RATE PERCENT PERCENT
FUNGICIDE (kg fp/ha) FREQUENCY BLACK CONTROL
POINT a


Dithane M-45












Fungitox












Distilled
water


5.2 f
5.9 ef
6.7 ef

6.3 ef
7.3 ef
6.5 ef

8.1 e
8.2 e
5.4 f

13.5 cd
16.1 b
11.2 d

14.8 bc
12.5 cd
11.5 d

19.5 a
21.8 a
12.3 d


13.1 cd


Control


60.2
54.6
48.5

51.5
43.9
49.7

43.0
37.3
58.7

-2.9
-23.4
14.4

-13.2
4.1
11.8

-49.4
-66.7
6.3


aEach figure represents mean of 3 replications.
Means with the same letter are not significantly different at 5% level with DMRT.


Jan. & June 1985









Philippine Phytopathology


Phytotoxicity of the Chemical
No phytotoxicity was observed in any
of the treatments.

Economic Analysis
Table 4 shows the estimated cost
and return per hectare. Treatments with
Dithane gave a return over cash costs


(ROCC) of P994.51at P5.20/kg. Net
return ((NR) of the same treatment was
P594.51 with a corresponding return on
investment (ROI) of 8.4%. When seeds
were sold at a commercial price of
P4.20/kg, the operator would be losing
from P480.07 and P880.07 based on
ROCC and NR, respectively. On the other
hand, much higher losses would result


Table 4. Estimated cost and return per hectare of wheat sprayed with Dithane M-45 to
control Helmenthosporium leaf spot.

AMOUNT
fIEMS
Treated Controlb


Gross Income
a. at P5.20/kg
b. at P4.20/kg

Production Cost
Cash:
Labor
Supplies and Materials

Non-cash:
Interest on capital
Total Production Cost/kg
Average Production Cost/kg

Return Over Cash Cost
a. at t5.20/kg
b. at P4.20/kg

Net Return (Profit)
a. at P5.20/kg
b. at P4.20/kg

Return on Investment %
a. at P5.20/kg
b. at i4.20/kg


P 7,667.82
6,193.24


2,260.00
4,413.31


400.00
7,073.31
4.80


994.51
-480.07


594.51
-880.07


8.40
-12.44


a Estimated yield = 1,474.58 kg/ha
bEstimated yield = 1,040.85 kg/ha


5,412.47
4,371.61


2,238.00
3,830.00


364.08
6,431.08
6.18


-655.53
-1,606.39


-1,019.61
-2,060.47


-15.85
-32.03


Vol. 21









Helminthosporium Leaf Sp


e Dithane not used in the production.

DISCUSSION
The efficacy of Dithane against Hel-
Ithosporium leaf spot confirmed the
[ings of Kernkamp et al. (1974).
;e the first fungicide application was
e prior to inoculation, Dithane pro-
!d a protective effect on the leaf
tivating most of the spores that
led on the surface. It was possible
: few spores were able to colonize
host as reflected on the few number
lesions.
Fhe observed differences in the disease
elopment with all Dithane treatments
e a function of the interaction bet-
n rate and frequency. Within rate,
ter frequency of sprayings gave the
protection effect. The sublethal
still gave lower infection but at
ler frequency of applications.
however, Evans (1971) suggested that
of the causes of fungicide tolerance
ablethal dosage. Under dosage or rate
er than the recommended seemed to
mote tolerant population develop-
it as well as resistance to the fungi-
Hence, it was suggested that the use
'ungicides should be as high as possible
h due considerations to phytotoxicity
economic costs (Dekker, 1976).
)n the other hand, recent theory
is to suggest that underdosage appli-
on of fungicide prevents high select-
pressure (Nelson, 1983). Further-
re, sublethal dosage promote com-
ition among the most virulent and less
lent strains in the pathogen popu-
ans, thus stabilizing selection.
The above discussions seemed to
gest a rate of application that would
affect the ecological balance. But
h the present tight economic situation
with the increasing cost of importing
crop, the impact of crop protection
he yield and eventually on the return
unit input should not also be over-
ked.


The failure of Fungitox to effect a
gnificant control of the. leaf spot in-
ction was contrary to the findings of
ska & Drotarova (1976) and Rusko
976). The results of the study colla-
)rated with that of Edgington et al.
971) who found that benzimidazoie
impounds like benomyl are ineffective
;ainst Phycomycetes, some Basidiomy-
tes and dark spored members of Deute-
imycetes. It is interesting to note that
ingitox have the same mode of action
the above chemical group. More so,
e imperfect stage ofH. sativum belongs
the last group of the pathogen, the
suteromycetes. Slower uptake, detoxi-
:ation or other metabolic factors may
;o explain the ineffectiveness (Erwin,
173).
Furthermore, the observed high
ease level with Fungitox seemed to
ggests that it provided a favorable
vironment for the pathogen. The
mechanics involved could be one of the
allowing: a) provide substrate for the
thogen; b) eliminate organisms com-
titive to H. sativum; and c) change
e physiology of the host making it
ore susceptible.
The relation of injury to loss is
fected by a variety of factors, both
ternal and external (Zadoks, 1979).
ie significant effect of Dithane on
eld suggests a significant role of the
ternal factor, the environment.
The magnitude of the loss also depend
i the physiological time of injury
adoks, 1979). Increased grain yield of
ieat was evident when the flag leaf was
otected from its emergence until the
iot stage (Cook, 1980). With Dithane,
Section of the flag leaves came in late to
Fect a significant reduction in photo-
nthesis that would consequently affect
ain yield. The disease was prevented
om spreading to the flagleaf after
oculation.
Leaf spot pathogen does more than
zt rednpe the c~nnnitv to nhntncvn.


43









'nmuppme rnytopathology


thesize resulting to a reduction In yield.
H. sativum together with other seedborne
pathogenslike Alternatia alternate, affect
the quality of the grain harvested in terms
of seed discoloration known as black
point (Huguelet and Kiesling, 1973).
Black point disease has probably been
present in the Philippines for quite some-
time since wheat was planted but there
was no mention yet of the disease in our
literature.
Dithane gave a significant control of
black point. On the other hand, favor-
able weather condition for seed infection
at about harvest time aggravated seed
discoloration of the Fungitox treatments
and the control. With an inoculum den-
sity of 5000-10,000 conidia per ml and
48-hr period of high humidity, seed
infection would be more favored (Hugue-
let and Kiesling, 1973).
In view of the above discussion, late
spraying of fungicide is suggested es-
pecially if atmospheric humidity be-
comes high towards the ripening stage.
Due consideration, however, should be
given to the Fertilizer and Pesticide
Authority (FPA) ruling regarding spray
interval before harvest for the safety
of the consumer. Another option would
be the use of cultivars resistant to the
disease. Some varieties appear to be more
susceptible to black point than others
(El-Helaly, 1947). An extensive test
against black point on the existing
number of varieties should be included
in the ongoing screening program.
The cost and return analysis generally
showed that wheat production with
disease control was profitable. This was
so primarily due to the higher yield
obtained compared to the control. Aside
from that, the average production cost
per kg of wheat with disease control
was lower compared to that of the
control.
The losses incurred were primarily
due to the lower yield of the crop. The
low yield failed to compensate for the


-rllB11 FIUUUILUII LuaL. IUVWC VC, yltau la
not a limiting factor in making wheat
production attractive to the farmers
(Lales, 1981). Production cost which
is largely affected by price increases can
be trimmed down by proper cultural
management.
Therefore, to grow the crop, incentives
should be provided by the government
to the Filipino farmers to venture into
wheat production. Higher farm price
is the best incentive for a farmer to
work and produce. It requires no moni-
toring by the government and there-
fore without additional bureaucratic
over head (Zobel, 1984). Price support
for some of the major inputs like ferti-
lizers and chemicals would also help trim
down high production costs.
However, if the farmer owns the land,
utilizes family labor and has enough cash
and so pays no interest, the computed
ROCC is the actual cash receivable
after harvest. Although the returns will
be higher than the cost, still this will not
be sufficient to convince the farmers to
engage in wheat production. Farmers
invest their resources only where the
rate of return from investment is suffi-
ciently high. Farmers are expecting a
rate of return of around 2:1 on their
investment in farming inputs (Herdt
and Jayasuriya, 1981). A reasonable
and stable price support is therefore need-
ed for wheat to have a competitive ad-
vantage over other cultivated upland
crops.

SUMMARY AND CONCLUSION
The efficacy of Dithane at various
rates and frequencies of application to
control disease development was shown
in the experiment conducted under Los
Bafios conditions. At all rates, two or
three applications gave superior control
of the disease. No phytotoxic effects
were observed in all treatments.
A significantly high grain yield was
obtained with Dithane at 1.0 and 1.5


/OL ZI









Jan. -A Juna 198


Control of Helminthosporium Leaf Spot


FVWL11 HI~a LV aJFL"y -iuaull*
fungicide to effect maximum control of Indian Council Agr. Res. New Delhi.
black point.
Fungitox was found ineffective against DASTUR, J.F. 1932. Foot rot and black
the pathogen at any of the rates and point disease of wheat. Central Pro-
frequencies used in the experiment. vince Agr. Livestock, India 2:275-
Economic analysis revealed the profit- 282.
ability of using Dithane under a certain
set of conditions. However, further DEKKER, J. 1976. Acquired resistance
studies related to cultural management to fungicides. Ann. Rev. Phytopathol.
specifically in the aspect of disease con- 14:405-428.
trol should be conducted to obtain the
maximum yield potential of the existing DOBRETSOV, A.N. 1962. Helminthos-
cultivar. This would consequently trim podium disease of wheat in Siberia
down high production costs. Wheat pro- and its control p. 11-14. In Y.L. Nene
duction in the Philippines would also be and V.K. Agarwal. 1978. Some seed-
economically feasible if government sup- borne diseases and their control.
port is provided. In this way wheat can Indian Council Agr. Res., New Delhi.
have a competitive advantage over other
cultivated upland crops. EDGINGTON, L.V., K.L. KHEW and
G.L. BAKDON. 1971. Fungitoxic


LIAEKA1UKtl l11I E
BIDARI, V.B. and H.C GOVINDU. 1975.
In vitro evaluation of fungicides
against three isolates of Helminthos-
porium sativum Pamm., King and
Bakke on wheat in Karnataka State.
Mysore J. Agr. Sci. 9:95-98.

CHATTOPADHYAY, S.B., and CHA-
KRABARTI. 1962. Application of
fungicides in the control of secondary
air borne infection of Helminthospo-
rium oryzae Breda de Haan. Indian
PhytopathoL 14:88-92.

CHRISTENSEN, J.J. 1925. Physiologic
specialization and mutation in Helmin-
thosporium sativum Phytopathology


pounds. Phytopathology 61:42-44.

EL-HELALY. 1947. The black point
disease of wheat. Phytopathology
37:773-780.

ERWIN, D.C. 1973. Systemic fungi-
cides. Disease control translocation
and mode of action. Ann. Rev. Phyto-
pathol. 11:389-422.

EVANS, E. 1971. Problems and progress
in the use of systemic fungicides.
Proc. British Insec. Fungi. 6th Conf.
3:758-763.

HERDT, R.W. and S.K. JAYASURIYA.
1981. The economics of insect control









Philippine PhytopatholoVy


Annual Conf. Pest Control Council of some chemicals against three phy-
of the Phil. Inc. 49-66. topathogenic fungi. Indian Botan.
Soc. 53:191-195.
HUGUELET, J.E. and R.L. KIESLING.
1973. Influence of inoculum compo- LUKHMENEV, V.P. 1976. Root rots of
sition on the black point disease of spring wheat. (Abstr.) Rev. Plant
durum wheat. Phytopathology 63: Pathol. 56-579.
1220-1225.
LUZ, W.C., and J.C. VIEIRA. 1982. Seed
ISMAIL, I.N.K., and A. MICHALIKOVA. treatment with systemic fungicides to
1977. Effect of phenolic and other control Cochliobolus sativus on barley.
compounds on the germination of Plant Dis. Rept. 66:135-136.
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in vitro. (Abstr.) Rev. Plant Pathol. MEAD, H.W. 1933. Seed injury and
56:992. infection by Helminthosporium sati-
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KERNKAMP, M.F., R. KROLL and W.C. Grain Exhibition and Conference
WOODRUFF. 1970. Wild rice disease 22:278.
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1- - --- __ 1 -. -1 n - '71l-


VOL Z.









Control of Helminthosporium Leaf Spol


Jan. & June 19f


IUlll iU VU FLl VL-U1L- VVALJI Dy or ---ol -- -. .
temic and complex action against root
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triticana and Helminthosporium sati-













A METHOD TO ACCOMMODATE
ANALYSIS OF POTAT



A.B. MOLINA, JI



Respectively, Assistant Professor, I
culture, University of the Philippines at
3720 and, Professor and Head, Departmt
Louisiana Agricultural Experiment St;
Center, 302 Life Science Building, Baton
Portion of the Ph.D. dissertation
vania State University, University Park, P.



A

A method was developed to adju:
rity values (SV) as epidemiological tim(
Severity values are arbitrary values cal
and are said to indicate the likelihood
method the cumulative SV's were used
ion analysis of disease progress curves.
days of the logistic model permitted
curves from two growing seasons of hig
mise as a method to distinguish host a
otherwise might be obliterated by chan




INTRODUCTION

The apparent infection rate (r-value)
is one measure of epidemic progression
over time when adjusted for multiple
infection (Vanderplank, 1963). It can be
calculated as the linear regression coe-
fficient of the logit transformation of
disease severity proportion regressed on
time. Since first proposed for epidemic
analyses by Vanderplank (Vanderplank,
1963), the r-value has been used widely
by plant pathologists for classifying
epidemics. It has also been used to com-
pare some elements of an epidemic such
as host resistance (Frye, 1975; Krause,


NON-B LIGHT WEATHER IN THE
.ATE BLIGHT EPIDEMICS



id D.R. Mac KENZIE



rtment of Plant Pathology, College of Agri-
Baios (UPLB), College,Laguna, Philippines.
If Plant Pathology and Crop Physiology, The
i, Louisiana State University Agricultural
ge, LA 70803.
he senior author submitted to the Pennsyl-
5802.



RACT

periods of "non-blight" weather using seve-
he analysis of potato late blight epidemics.
ed from specific environmental parameters
ate blight infection for a given day. By this
ie independent variable in the logit regress-
use of SV's rather than the conventional
-oved -resolution of some disease progress
variable weather. This procedure offers pro-
athogen characteristics from field data that
a weather patterns during a growing season.




et al, 1975; Latin, 1980; Shaner, 1973;
Vanderplank, 1968) pathogen variation
(Mackenzie, 1980) and some disease
control strategies (Mackenzie, 1981;
Merril, 1962). Some authors have defined
one form of host resistance as rate re-
ducing resistance (Nelson, 1978; Shaner
1973). Such cultivars are defined as
having lower r-values than do others.
Additionally, r-values have been used
to judge the relative parasitic fitness of
different isolates of specific pathogens
(Mackenzie, 1978).
One limitation of the apparent in-
fection rate is the great variation of this
parameter from season to season or













IromI lMocaIun to locauon. Ine r-valu
is sensitive to changes in environmental
conditions, especially weather Wag
goner, 1965; Wallin and Waggone
1950). This characteristic makes the
comparison of r-values, from epide
mics obtained from different sets o
environmental conditions, virtually im
possible.
The need to relate specific environ
mental factors and disease development
led Wallin and colleagues (Villareal
1980; Waggoner, 1965; Wallin, 1962
Wallin and Waggoner, 1950) to developt
the daily severity values (SV) which cha
racterizes the contribution of weather tc
the likelihood of infection of potato b3
Phytophthora infestans (Mont.) DeBari


presented nere are oasease assessment
recorded from plots of the potato cultival
Kennebec of four separate experiment!
carried out in the growing seasons ol
1981 and 1982 at the Pennsylvania State
University Research Farm, Rock Springs,
Centre County, PA.
The epidemic progression in the un-
sprayed plots among experiments were
compared. Considering that different
environmental conditions prevailed
among location/season experiments, these
separate experiments were considered a!
treatments for the purpose of this study
Because of the separate goals of eacl
experiment, some experiments had fewej


Jan. Ja rune 170


Memoo to Accommoaate Non-nugni wear










Philippine Phytopathology


judgements used to supplement statis-
tical evaluations of the two procedures.
The regression coefficients obtained from
the four epidemics by the two methods
of analysis were subjected to analyses
of variance to determine whether the
r-values differed among experiments.


RESULTS

Comparison of Apparent Infection Rates
Within Epidemics
Figures 1 and 2 present the logit
transformed disease progress curves for


0.6

-2.0

x
S-4.5

-7.0

-9.5


0.5

-2.0

-4.5


0 18 27 36 45 54
CUMULATIVE SEVERITY VALUES
Fig. 1. Logit transformed potato late
blight severity values plotted
against time in days (A) and
cumulative severity values (B)
and associated statistics for cul-
tivar Kennebec 1982 (Experi-
ment I).

both days and CSV. These specific
epidemics were selected to demonstrate
how the use of CSV's, as the independent
variable in the analysis of these epidemics,
improved the goodness of fit both statis-
tically and visually. Larger coefficients
of determination (R2) and smaller


standard deviations of the regression were
two statistical measures of the better fits
of apparent infection rates calculated
with CSV's.

Comparison of Apparent Infection Rates
Among Epidemics
The calculated r values for the four
sets of epidemics using days as the in-
dependent variable are given in Table 1.
Analysis of variance of these values
showed a great deal of variation of the
coefficients of regression from experiment
to experiment (F ratio = 103.5**).


0.0

-3.0
x
- -6.0
0
0
_C
-9.0

-12.0


0.0

-3.0
X

0 -6.0
0


A
Y= -9 34 1 .25(DAYS)
n2= 5s.2%






0 10 20 30 40 50
DAYS


B

S- 5. .28(CSV)
2 '.8Ol0


10 20 30 40 50 60
CUMULATIVE SEVERITY VALUES
Fig. 2. Logit transformed potato late
blight severity values plotted
against time in days (A) are
cumulative severity values (B) for
cultivar Kennebec 1982 (Experi-
ment II)


Apparent infection rates calculated on
CSV's are summarized in Table 2. Ana-
lysis of variance of these values showed
significant differences for the four epi-
demics but to a lesser extend (F ratio =
13.6**). While both methods of calcu-
lating r resulted in statistically significant


=- 1O. + .26(CSV)
R2= 9.3%


Vol. 21


2


-9.0

-12.0









an. & June 1985 Method to Accommodate Non-Blight Weather 51



able 1. Apparent infection ratesa of the cultivar Kennebec in the various late blight
epidemics in 1981 and 1982.

Replications
Kperiment I H I1 IV V Meansb


1981 .33 .34 .33 .32 .33A
1981 .25 .27 .24 .29 .27 .27B
1982 .24 .26 .25B
1982 .15 .17 .14 .17 .16C


aApparent infection rates were calculated by regressing logit X against days as the independent
riable. The unit is unit/day.

values followed by the same letters are not significantly different at P = 0.05 as determined
r the Duncan's new multiple range test.


able 2. Apparent infection rates calculated by regressinglogit X on severity values (SV),
of the cultivar Kennebec in the various late blight epidemics in 1981 and 1982.


1981 .20 .21
1981 .22 .22
1982 .27 .29
1982 .24 .26

aApparent infection rates are given as units/
bValues followed by the same letters are n
y the Duncan's new multiple range test.

differences among experiments, the dis-
ersion of the coefficients of regression
,as much less when calculated using SV
either than days.

DISCUSSION
This study demonstrate that SV's can
e used as measures of epidemiological
.me in the assessment of epidemic deve-
apment. Weather adjusted r-values acco-
rv' .t. ftCA n na.ifiP tnrt lnnrtn t+n


1 .20 .21A
) .23 .18 .21A
.28B
3 .27 .25B



fnificantly different at P = 0.05 as determined


contributee to the development of late
)light (Krause, et al, 1975;Wallin, 1962).
As all days are not equal in their contri-
,ution to disease development, the use of
,SV's in the analyses of epidemics can
:ompensate for differences in weather as
contributingg factors in disease develop-
nent.
The SV's of Wallin (1962) and Wallin
.nd Waggoner (1950) for late blight of
>otato are intentionally weather depend-
:nt. Thus. they can be used to standar-









Philippine Phytopathology


dize calculated r-values between epide-
mics. Theoretically all other things be-
ing equal, different epidemics developing
under different weather conditions would
be expected to yield identical r-values
when based on CSV's. However, the SV's
of Wallin apparently do not perfectly
account the effects of weather on the
development of epidemic. Hence, the dif-
ference in r-values. However greater dif-
ferences were noted when days were
used as the independent variable.
This method offers promise for several
applications of importance to disease
forecasting and management. More
meaningful measures of host resistance
and pathogen variation is one application.
Another would be in more precise fore-
casting of disease development and pre-
dicting disease "outbreaks". This would
result from better knowledge of the con-
tributions of cultivar differences, the
potential variability of the pathogen
population, and the effects of weather
on epidemic development.


LITERATURE CITED
FRY, W.E. 1975. Integrated effects of
polygenic resistance and protective
fungicide on the development of
potato late blight. Phytopathology
65:908-911.

HYRE, R.A. 1954. Progress in forecasting
potato late blight in Northern Maine.
Am. Potato Jour. 32: 119-125.

JAMES, W.C. 1971. An illustrated series
of assessment keys for plant diseases,
the preparation and usage. Can. Plant
Dis. Survey 51:39-65

KRAUSE, R.A., LB. MASSIE and R.A.
HYRE. 1975. Blitecast: A compute-
rized forecast of potato late blight.
Plant Dis. Reptr. 59: 95-98.

LATIN, R.X. 1980. Factors in potato


that reduce the rate of epidemic
increase of potato late- blight. Ph.D.
Thesis. The Pennsylvania State Uni-
versity, University Park, Pa.

NELSON, R.R. 1978. Genetics of hori-
zontal resistance to plant diseases.
Annual Rev. Phytopathol. 10: 359-
378.
MacKENZIE,. D.R. 1981. Scheduling
of fungicides applications for the
management of potato late blight.
Plant Disease 65: 349-399.
MacKENZIE, D.R. 1980. The prob-
lem of variable pests. Pages 183-213
In: F.G. Maxwell and P.R. Jennings,
eds., Breeding Plants to Insects. John
Wiley and Sons, New York, 693 pp.

MacKENZIE, D.R. 1978. Letter to the
editor: Estimating parasitic fitness.
Phytopathology 68: 9-13.

MERRILL, W. 1962. The oak wilt epide-
mic in Pennsylvania and West Virginia:
An analysis. Phytopathology 57:1206-
1210.

SHANER, G. 1973. Evolution of slow-
mildewing resistance of Knox wheat in
the field. Phytopathology 63: 867-
872.

VANDERPLANK, J.E. 1963. Plant
Diseases: Epidemics and Control Aca-
demic Presss, New York, 349 pp.

VANDERPLANK, J.E. 1968. Disease
Resistance in Plants. New York Aca-
demic Press. 206 pp.

VILLAREAL, R.L 1980. The slow leaf
blast infection in rice (Oryza sativa).
Ph.D. Thesis. The Pennsylvania State
University, University Park, Pa.

WAGGONER, P.E. 1965. Microclimate
and plant disease. Annual Rev. of
Phytopathol. 3: 103-126.


VoL 21










Method to Accommodate Non-Blight Weather


demics in the United Statesand Can
da. Am. Potato Jour. 39: 306-312.


S 17J .U. 1
S develop
thora


Suppl. 190: 19-23.


Jan. & June 19E


S--l -- -Pl
lots. Plant I


I









Y, Inc.


SCREENING OF VARIOUS PLA
Meloidogyne incognito



RUTH S. GUZM



Respectively, Associate Professor, (
Cabagan, Isabela and Professor, Departmi
University of the Philippines at Los Bafios
Portion of the Ph.D. dissertation oft



ABS

Solvent extracts of eight plant q
important nematode species, namely: M.
showed indications of toxicity against tl
tests. In some cases the effects of the ex
nematicides like fenamiphos and aldicarb.
The crude extracts of Anthocepi
crasdpes (water lily) and Allium cepa (o
against the nematodes. The active nemal
identified as a phenolic aldehyde in A.
ketone in the extract of A. cepa.



INTRODUCTION

Yield losses due to nematode attacks
are considerable in most agricultural and
horticultural crops in different countries.
When all crops are included, an average
annual loss of approximately 10 percent
or higher under severe conditions has
been estimated (Sasser, 1976). In view of
this, various control measures against
namatode attacks on these crops have
already been developed. For commercial
crops, such as banana, citrus and pine-
apple, nematicides are widely used to
control nematodes. On semi-annual and
annual crops, cultural control, such as
crop rotation and planting of resistant
varieties, is usually practiced to reduce


EXTRACTS FOR TOXICITY TO
ND Radopholus similis



md R.G. DAVIDE




ge of Agriculture, Isabela State University,
)f Plant Pathology, College of Agriculture,
LB), College, Laguna, Philippines. 3720
enior author submitted to UPLB.



ACT

s were screened for toxicity against two
ognita and R. simils. Most plant extracts
ematodes in the bioassay and inoculation
i on the nematodes were comparable with
chinensis (Kaatoang-bangkal), Eichonia
) gave the best results in the toxicity test
11 compounds found in the extracts were
rnsas, carboxylic acid in E. crassipes and




nematode damage.
As plant parasitic nematodes in-
creasingly become destructive to various
economic crops, development of effec-
tive control methods of the more impor-
tant genera, such as Meloidogyne, Roty-
lenchulus, Radopholus, Tylenchulus,
Protylenchus and others, becomes a
primary concern. Chemical control alone
using commercial nematicides cannot be
Solely depended upon as a number of
them have already been banned from
use due to toxicity and pollution prob-
lems. Thus, alternative control measures
against nematodes are now being investi-
gated. Plants are being seriously examined
as potential sources of organic com-
pounds with pesticidal properties (Menn,









screening or various Plant Extracts


IfY0). indeed, dilterent societies m the
world have continually employed plants
to kill or repel pests since civilization
began (Secoy and Smith, 1983). Residues
and plant extracts of a number of higher
plants exhibit nematicidal activity by
reducing population levels of some nema-
tode species.
This study involved screening of eight
local plant species that have been sus-
pected or observed to possess nematicidal
properties. It included testing of crude
(solvent) extracts of these plant species
against the nematodes Meloidogyne in-
cognita Chit. and Radopholus similis
(Cobb) Thorne out of which extracts of
four species were analyzed for their
nematicidal properties.

MATERIALS AND METHODS

Test plants. Plants used for the nema-
ticidal screening included those which
had previously shown some degree of
biological activity against nematodes. The
list of the test plants are found in Table 1.
Test nematodes. Meloidogyne incog&
nita and Radopholus similis were selected


as tioassay orgamsms to determine the
nematicidal activity of plant extracts.
The choice was based on their extensive
distribution, wide host ranges and the
great economic importance of their host
plants. *
Second-stage larvae of M. incognita
and larvae and adults of R. similis were
used in the determination of toxicity.
Mass culture of test nematodes. Popu-
lation build-up of the test nematodes was
done in the greenhouse, using tomatoes
and bananas as host plants for M. incog-
nita and R. similis, respectively. Infected
plant roots served as the source of bio-
assay organisms. For M. incognita, a
modified Baermann funnel method using
a petri dish was used to obtain a constant
and ample supply of second-stage larvae
serving as bioassay organisms (American
Phytopath Society, 1978). Similarly,
mass culture for R. similis larvae and
adults was also done in vitro using carrot
discs in sterilized water agar media
(O'Bannon, et al., 1968).
Preparation and extraction of crude
extracts. Plant samples were collected in
and around the vicinity of the UP at Los


Common Name

nsis Kaatoang-bangkal
p.
um Mangkit


Jan. & June 1985













Baiios. Bulb materials, such as the com- established a trend for toxicity of these
mon garlic and onion (var. Red Creole) extracts against the nematodes.
were procured from the Los Bafios
market. Determination of Toxicity
Leaf samples ofA. chinensis, D. gange-
ticum, A. vulgaris, E. crassipe and L. To determine toxicity of these solvent
leucocephala were collected and separate- extracts, both in vitro and in vivo methods
ly air-dried. Dried leaves were finely were tested.
ground using the Wiley mill with a 20-mm Nematode mortality tests. Emulsified
mesh screen. The ground sample was plant extracts were tested at 10,000 ppm
soaked in chloroform enough to cover it on pure populations of the two test
in wide-mouthed bottles. The chloroform nematodes in the laboratory. Each treat-
extract was a thick slurry, which was ment (ca. 300 nematodes after calibration)
then filtered with suction and exhaustive- was replicated three times. Comparative
ly washed with fresh chloroform. The checks were Nemacur (fenamiphos),
combined filtrate was concentrated in Temik (aldicarb) and Rotenone, obtained
vacuo at about 38 C using a rotary evapo- from the U.S.E.P.A. and were all tested
rator. The product was a residual thick at 200 ppm and 500 ppm. Control treat-
syrup, which was subsequently taken up ments had water alone and did not con-
in 1000 ml of 95% ethyl alcohol while tain the extract solution. Nematode mor-
being heated in a water bath (38 C) and tality of M incognita, and R. similis was
allowed to cool for at least 10 minutes. determined at 24 and 48 hours after
To precipitate the more polar compounds, treatment and computed using the


was duucu. 111i resuILIanL mixture was
again suction-filtered and concentrated
in vacuo. The watery solution was then
exhaustively extracted with successive
volumes of chloroform, dried over
sodium sulfate and concentrated in vacuo
to yield the crude extract.
Fresh bulbs of A. sativum and A. cepa
and freshly harvested mature roots of
L. leucocephala and M. oleifera were cut
into small pieces and macerated. Mace-
rated bulb tissues were soaked in chloro-
form and allowed to stand for at least 3
days with occasional agitation. Root
tissues were allowed to stand for at least
a month. The filtrate was extracted suc-
cessively with fresh chloroform or hexane
to separate the aqueous fraction. The
combined filtrate was concentrated in
vacuo at about 38 C to yield the crude
extract.
The solvent extracts being insoluble in
water had both acetone and hexane as
carrier solvents in the toxicity tests. Basic
toxicity tests using water extracts of the
plants were made earlier in the study and


%Mortality Treatment Controlx 100
100 Control
Hatchability tests for M. incognita.
Three sterilized egg masses were im-
mersed in the emulsified extract con-
tained in small petri dishes. Three re-
plications were made for each treatment.
Hatched larvae were obtained after 4
days of incubation and analyzed for
percentage reduction as follow:
SRed o Control Treatment
%Reduction = ------ x 100
Control
Soil drench experiment. Extracts dis-
solved in acetone were pipetted directly
to the rhizosphere of 3-week old tomato
plants grown in baked soil previously
inoculated with M. incognita Inoculation
was done by distributing the nematode
suspension (ca. 1000 after calibration of
nematode suspension) evenly around the
rhizosphere of the plant. Three weeks
after treatment, plants were allowed to
dry harvested and evaluated The number









Screening of Various Plant Extracts


ot galls was counted and analyzed for
percentage reduction.
Inoculation experiment. For R. similis,
nematodes (ca. 300 after calibration of
nematode suspension) incubated for
48 hours in the different extracts were
inoculated to 3-week old banana seed-
lings. The nematodes were inoculated
by pipetting into holes around the base
of the plant. Three replications were
made for each treatment.
Five weeks after inoculation, roots
were carefully washed, dried and eva-
luated. Assessment parameters used were
number of lesions in the root and num-
ber of nematodes recovered per 400 cc
and population density of nematodes
recovered per gram of root tissue.
For M. incognita, 2-week old tomato
seedlings grown in baked soil were
treated with nematodes incubated for
48 hours in the different plant extracts.
Treatments were soil-injected around the
rhizosphere of the plant. Three repli-
cations were made per treatment.
One month after inoculation, roots
were carefully harvested and evaluated
for nematode infection. Assessment para-
meters were number of galls per plant,
number of egg masses per plant and
number of nematodes recovered per root
system. Percent reduction was also
computed.
To determine the number of nema-
todes which have penetrated the root
tissue, harvested roots of assay plants
were first stained.

Isolation of Nematicidal Components
Fractionation and bioassay of the
crude extracts. Plant extracts which
showed promising results after prelimi-
nary bioassay were further separated.
Fractionation of these extracts was
carried out by preparative thin layer
chromatography. Fractions were col-
lected by manual separation of each
distinct band. Each band was separate-
ly scraped off, powdered and extracted


twice with acetone. Clean-up was done
using appropriate solvents. A. chinensis,
A. vulgaris, and E. crassipes were deve-
loped in a hexane-acetone solvent sys-
tem, while A. cepa was crystallized in
acetone from its hexane solution. The
separated fractions were then concen-
trated in vacuo and further bioassayed.
Characterization of nematicidal com-
ponents. Active components were cha-
racterized by determining their solu-
bilities in different organic solvents.
To further characterize the nematicidal
substances, the spectroscopic techniques
used were infra-red spectroscopy in order
to detect and identify functional groups
of the purified extract and ultraviolet
spectroscopy principally to determine
the presence of conjugated systems.

RESULTS AND DISCUSSION

Determination of Toxicity

Nematode mortality tests. Among the
plant species tested, A. cepa gave the
highest mortality after 48 hours in both
hexane and acetone, comparing favorably
with the treatment Nemacur at 500 ppm
and 200 ppm (Table 2) which under-
standably had the highest ranking based
on Duncan's Multiple Range Test. Rote-
none and the other plant species which
were bioassayed also exhibited a trend for
toxicity against the test nematodes. A.
chinensis also showed good results and
was even sometimes more effective than
the pure rotenone. For the R similis
mortality tests, aldicarb (Temik) was
included as a standard as this is a com-
mercial nematicide used in banana
plantations where the nematode, R.
similis abound. In Table 3, A. cepa
also showed a high mortality rate com-
parable with results obtained with aldi-
carb after 48 hours. Other treatments
also showed some promise as toxicity
was also exhibited in varying degrees
in the same 48-hour period.
The non-similarity of the results


Jan. & June 1985









rniuynipii tnly LuparLJurt~yvl.2


UULa-ntV.. LL tlt, ifaoty UI &-lt, .haiatVtfl-L
test species used suggests the specificity
of action of the plant extracts. Varia-
bility in toxic action could also be due
to differing rates of degradation. In
comparing standard plant protection
agents and naturally-occurring com-
pounds, it must not be overlooked that
practically all natural toxins are com-
pletely degraded in a short period,
leaving no residue. Even nicotine, which
is quite comparable with the very highly
toxic phosphoric acid esters, is quickly
rendered innocuous (Beye, 1980). Low
mortality values in R. similis compared
to M. incognita (Tables 2 and 3) may
also be attributed to their physical and
physiological differences. For M. incog-
nita, newly-hatched second-stage larvae
were used, which were smaller and pro-
bably more vulnerable to the treatment,
whereas in R. similis, adults were used
which were sturdier.
Hatchability tests. Extracts of all the
test plants significantly reduced the
hatchina ahilitv nf M inmnowitn Pa.e


U1iiXti,1it VAiiaw,LO. Jk3irlllllUjadl iy, L LIl
aggregate data taken for A. cepa indicated
minimum infection as shown by the low
number of nematodes recovered. This
could mean that this particular plant
extract has a high toxicity quotient for
root-knot nematodes. Meanwhile, per-
centage reduction in lesion number and
a corresponding decrease in R similis
population densities in soil and banana
root tissues are shown in Table 7.
Generally, treatment results of the
various plant extracts were highly signi-
ficant when compared to the standard
nematicide checks in terms of biological
efficacy manifested in a reduction in the
number of lesions and number of nema-
todes recovered from soil and root
tissues.

Isolation of Nematicidal Components

Fractionation and bioassay of extract
components. Crude extracts of A. chinen-
sis, A. vulgaris, E. crassipes and A. cepa
were further studied as they showed


m lable o. Uecreasea rootcnot mcidence rate fractions snowea that Fractions i
was noted following treatment with the to 8 were all toxic, albeit in varying


VOL 21















PLANT SPECIES/
TREATMENT


A. chinensis
D. gangeticum
A. vulgaris
E. crassipes
L. leucocephala (leaves)
L. leucocephala (roots)
M. oleifera
A. sativum
A. cepa
Nemacur (500 ppm)
Nemacur (200 ppm)
Rotenone (500 ppm)
Rotenone (200 ppm)

a%Mortality= Treatment control x 100
100- control
Percentage mortality taken as an average of 3 rep
Means having the same letters are not significantly


PERCENTAGE N
Acetone-Soluble

4 Hr 48Hr


).2 bc 76.8 b
4.7 d 4.5 f
3.5 bc 32.9 e
5.5 bc 37.8 de
5.6 bc 57.0 c
7.3 b 60.5 c
).9 bc 29.7 e
3.5 bc 53.3 c
).0 b 97.7 a
5.7 a 98.5 a
5.6 a 96.6 a
3.5 cd 47.6 ed
2.9 cd 36.5 de




s; data were corrected for natural death using Abt
erent at 5% level (DMRT).















PLANT SPECIES/
TREATMENT


A. chinensis
D. gangeticum
A. vulgaris
E. crassipes
L. leucocephala (leaves)
L. leucocephala (roots)
M. oleifera
A. sativum
A. cepa
Nemacur (500 ppm)
Nemacur (200 ppm)
Rotenone (500 ppm)
Rotenone (200 ppm)
Temik (500 ppm)
Temik (200 ppm)

a y Treatment- control
%Mortality = x 100
100- control
Percentage mortality taken as an average of 3 rej
Means having the same letters are not significant]


PERCENTAGE
Acetone-Soluble
34 Hr 48 Hr


16.7 cd 27.8 de
44.0 b 54.8 c
12.2 de 20.8 def
8.8 de 11.5 ef
20.9 cd 28.6 de
10.8 de 21.7 def
8.4 de 16.9 def
19.0 cd 21.3 def
26.2 c 75.7 b
17.2 cd 28.3 de
26.7 c 32.4 d
2.6 e 22.9 def
9.4 de 9.8 f
76.6 a 97.0 a
55.4 b 96.9 a




:es; data were corrected for natural death using Ab
fferent at 5% level (DMRT).









acreenmg oi various riant rxtracts


Data ai
Means


wing the same letters are not significantly different at 5% level (DMRT).
Effects of crude extracts of different plant species and nematicides applied as
_;i -i /^ ;-j- -_ ;* :__~-in i-i k__-*_ _i-_j- -r -.._- -


nematodes and root galls.
PERCENTAGE REDUCTIONa
PLANT SPECIES/
TREATMENT S No. of Nematodes No. of Galls Per
in Root Tissues Root System

A. chinensis 88.9 ab 84.1 ab
D. gangeticum 72.2 b 61.4 cd
A. vulgaris 86.1 ab 77.3 abcd
E. crassipes 86.1 ab 79.6 abc
L. leucocephala (leaves) 75.0 ab 59.1 d
M. oleifera 83.3 ab 75.0 bcd
A. sativum 77.8 ab 70.5 bed
A. cepa 77.8 ab 65.9 bed
Temik (500 ppm) 97.2 a 95.5 a
Nemacur (500 ppm) 86.1 ab 84.1 ab

a Control treatment
% Reduction = Control-treatment x 100
Control
Data are average of four replicates. Means having the same letters are not significantly different
at 5% level (DMRT).


Jan. at june Iva;,





























55.2 abc
9.4 d
100.0 a
90.8 ab
84.5 ab
54.7 abc
82.5 ab





rel (DMRT).














PERCENT)

PLANT SPECIES/ Lesion Nemati
TREATMENT Number in

A. chinensis 77.0 ab 77.
D. gangeticum 80.2 ab 62
A. vulgaris 65.5 ab 72
E. crassipes 78.9 ab 63,
L. leucocephala (leaves) 83.8 ab 3,
L. leucocephala (roots) 70.4 ab 76.
M. oleifera 85.1 ab 82,
A. sativum 87.3 ab 79.
A. cepa 80.3 ab 68.
Nemacur (500 ppm) 79.1 ab 88,
Nemacur (200 ppm) 13.8 c 4,
'Crf\l -_-N Qo n z c7


Kotenone izuu ppm) JI.J U
Temik (500 ppm) 92.2 a


a% Reduction = Control- Treatment x 100


61,









Philippine Phytopathology


degrees. In the hatchability tests, LITERATURE CITED
Fractions 1 and 3 showed the highest
percentage reduction. ABBOT, W.S. 1925. A method of com-
Characterization of nematicidal cor- putting the effectiveness of an in-
ponents. The active fractions from A. secticide. J. Econ. Entom. 18: 265-67.
chinensis, E. crassipes and A. cepa were 265-67.
further characterized. Infra-red and ultra-
violet spectroscopic data suggest the AMERICAN PHYTOPATHOLOGICAL
presence of a ketone in the A. cepa SOCIETY. 1978. Methods for evaluat-
crystal, a phenolic aldehyde in the A. ing plant fungicides, nematicides and
chinensis Fraction 2 and a carboxylic bactericides. American Phytopath So-
acid in E. crassipes Fraction 1. However, city and the Society of Nematologists
further investigations of these promising St. Paul, Minn, U.S.A. 141 pp.
nematicidal fractions need to be done,
using more refined bioassay methods in BEYE, F. 1978. Insecticides from the
order to obtain wider data base. vegetable kingdom. Plant Research
It should be noted that low toxicity and Development. 7:13-31.
data of other plant extracts used in this
study do not necessarily exclude them MENN, J.J. 1980. Contemporary fron-
from further investigation. Due to lack tiers in chemical pesticides research.
of information about the concentration J. Agri. Food Chem. 28:2-8.
of active materials in the extracts, a wide
range of doses should be assayed for O'BANNON, J.H. and A.L. TAYLOR.
results to be conclusive. 1968. Migratory endoparasitic nema-
Implications of this research show todes reared on carrot discs. Phyto-


and thus could help lessen some of the
current problems of synthetic pesticide
use, such as pest resistance, environ-
mental contamination and bio-magnifi-
cation. Synthesis of chemicals patterned
after these natural plant products could
be a progressive and ecologically sound
strategy for crop protection.


SASSER,
nf nhl


J.N. 1976. Nematode diseases


the Environment, Vol. 2, Marcel
Dekker Inc. New York, pp. 187-253.

SECOY, D.M. and A.E. SMITH. 1983.
Use of plants in control of agricultural
and domestic pests. Econ. Bot. 37:
28-57.


VoL 21









Philippine Phytol


ROLE OF MOTILITY IN APPLE BLOSSOM INFECTION AND
TACTIC RESPONSE OF ERWINIA AMYLOVORA TO
ORGANIC ACIDS IN PLANT NECTAR EXTRACTS




R.G. BAYOT and S.M. RIES


Respectively, Researcher, National Crop Protection Center, University of the Philip-
pines at Los Bafios (UPLB), College, Laguna 3720 and Associate Professor, Department
of Plant Pathology, University of Illinois, Urbana, Ill. 61801.
Portion of the Ph.D. dissertation of the senior author submitted to the University


attractive wnile mc
E. amylovora. Some
resins and the orgai
column. The organi
fumaric or malic a
varied depending up


1lliily plallt pdL11Ugc~lIu Udclid. 111 ltUI tcsa V~n IYDuax o lsui asau1lt5, -.5 .
presence of sufficient moisture, most al., 1961; Huang and Goodman, 1970).
plant pathogenic bacteria that infect The factors affecting its motility (Ray-
kahnta Orm-nnl n -int nrtfe noPnralll, ntPr mundo and Ries 1 OR1 and chemntaYxi


ings or mechanical wounds (Crosse, et. al.
1972; Mizukami and Wakimoto, 1969).
Motility and chemotaxis confer survival
advantages to certain bacterial species
(Kelman and Hruschka, 1973; Smith and
Doetsch, 1969) and may increase the
infection potential of bacterial plant


studied extensively under laboratory
conditions.
One interesting observation is that
most Rosaceous plants are susceptible to
E. amylovora infection while some are
resistant or immune (Shaw, 1934; Tho-
mas, 1931 ;Van Der Zwet and Keil, 1979).


Local Society, Inc.









Philippine Phytopathology


Resistance of plants to a pathogen is pro-
posed to be either structural or biochem-
ical in nature (Agrios, 1978). Resistance
through biochemical defense is due to the
presence or the absence of certain chemi-
cal compounds in plant cell that inhibit
the growth of certain pathogens (Day,
1974). The fact that E. amylovora is at-
tracted to apple nectar extracts (Raymun-
do and Ries, 1981) is intriguing and raises
the question whether or not susceptibility
or resistance of certain Rosaceous plants
to fire blight is related to the degree of
attractiveness of their nectar to E. amy-
lovora.
When apple nectar extracts were frac-
tionated using anion and cation exchange
resins(Raymundo and Ries, 1981), E.
amylovora was more attracted to the
organic acid fraction than to the amino
acid fraction and not to the neutral or
basic fraction. They also found that the
pathogen was not attracted to sugars but
was attracted to the organic acids malate,
fumarate, succinate and oxaloacetate, and
to the amino acid aspartate.
The objectives of the study were:
(1) to determine whether motility aids
E. amylovora in its invasion of apple
blossoms, (2) to determine the tactic
response of E. amylovora to nectar
extracts of plants with varying degrees
of resistance to the pathogen, and (3) to
find out which organic acids known
to attract E. amylovora are present in
nectar extracts of Rosaceous plants.

MATERIALS AND METHODS

Effect of Motility on Blossom Infection

Media and bacterial isolates. Modified
Emerson's medium (MEM) (Reinhardt
and Powell, 1960), modified Miller and
Schroth (MMS) medium and chemotaxis
medium (Raymundo and Ries, 1981)
were prepared and used in subsequent
tests. Semi-solid tryptone medium (STM)
rnntninAd 1 fl% trvntnne (Difcno and


0.4% agar (Armstrong, et. al., 1967). All
media were adjusted to pH 7. Chemotaxis
medium was filter-sterilized.
A rifampicin-resistant isolate of E.
amylovora (Isolate 110) was mutagenized
following a modification of the procedure
of Ordal and Adler (1974). One ml of a
24-hr-old culture grown in MEM broth
was transferred to 20 ml MEM broth, and
this culture was grown in a shaker bath
for 12 hours at 23 C. Cells were harvested
by centrifugation, at 5000 rpm in an
SS-34 (Sorvall) rotor for 5 min., and then
washed in 10 mM MgSO4 and resus-
pended in MMS to a concentration of
about 7 x 108 cells per ml (O.D. = 0.22 at
590 nm). Ethyl methanesulfonate was
added to the suspension to give a con-
centration of 0.3 M and the suspension
was mixed in a vortex mixer, incubated
for 1 hour at room temperature (23 to
25 C), centrifuged, and washed twice
with MMS. The suspension was diluted
1:1000 in MEM broth, incubated in a
shaker bath for 24 hours, diluted again in
MEM (1:50) and small drops of the
suspension placed at the center of STM
plates. All plates were incubated over-
night at room temperature. The center of
each colony was touched with a transfer
needle to obtain nonmotile cells, which
were re-inoculated into the center of
another STM plate. This process was
repeated daily four times. The final
transfer was done by streaking into MEM
agar plates and portion of a colony that
developed was suspended in a drop of
chemotaxis medium and observed under
a phase contrast microscope for motility.
Colonies with nonmotile cells were
cultured and used in subsequent tests.
Nonmotile isolates were grown in
STM for 72 hours. Colonies that pro-
duced revertants were evident by the
appearance of "flares" of bacteria spread-
ing away from the inoculation point.
Cells were taken from the edges of such
flares and were repeatedly transferred
in STM plates daily. Final transfer was


VoL 1l









Kole o0 Motilty in Apple Blossom infection


UIIU, y OLLIafeLm 111 MIfLyl piaLCJ JVIUL-
nies with highly motile cells as viewed
under a phase contrast microscope were
cultured and used in subsequent tests.

Testing for rifampicin resistance and
pathogenicity. Isolate Nm22 and Mr22
were streaked on MEM plates containing
500 ug/ml rifampicin to determine if the
rifampicin-resistance trait was retained
after mutagenesis. The pathogenicity of
isolates was tested and compared with the
wild type on green-house-grown Jonathan
apple seedlings. All isolates were grown
on MEM plates for 24 hours at 30 C, the
cells removed with sterile distilled water
and the concentration adjusted to appro-
ximately 1 x 108 cells per ml (O.D. =
0.075). Young shoots were inoculated
with a hypodermic needle.

Motility Assay
a. Measurement of colony diameter
on STM. Twenty-four-hour-old colonies
of Nm22 and Mr22 were touched sepa-
rately with the point of a transfer needle
and placed on STM plates by stab ino-
culation. Two stab inoculations were
made per plate and each isolate was re-
plicated four times. A replicate was the
average growth in diameter of both colo-
nies. Colony diameter was measured after
18 hours of incubation at room tempera-
ture.
b. Capillary assay. This assay was
performed using a modified Raymundo
and Ries (17) technique. Sterile glass
slides were used in preparing the assay
chambers and 0.25 ml of a bacterial
suspension (4 x 107 cells/ml) in motility
medium was placed in each chamber.
Assay chambers were incubated for 10,
20, 30 and 45 minutes at room tempe-
rature. The number of bacteria in a
capillary at the end of the incubation
period is a function of their rate of
movement; the more vigorously motile
the cells, the more bacteria will enter
the capillary (17). Each treatment was


replcatead our times ana me number
of bacteria per capillary was based on
duplicate plate counts.
Response of isolates to apple nectar
extract. Nectar extract was collected
from newly opened flowers (cv. Jona-
than) using a modification of the method
of Raymundo and Ries (16). A collection
flask attached to a vacuum source was
used instead of an Eppendorf pipet. This
was prepared by fitting a bent 1 ml glass
pipet into a rubber stopper. The pipet
was heated and while molten pulled to
make it a long and slender tip. Extracts
were immediately filter-sterilized, placed
in vials and stored at -20 C.
The tactic response of Mr22 and
Nm22 to apple nectar extract was com-
pared to that of the wild type (Isolate
110). The same procedure as in the
capillary motility assay was followed
except that some capillaries contained
a filter-sterilized 10-1 dilution of apple
nectar extract in chemotaxis medium,
while others contained chemotaxis
medium alone. The assay chambers were
incubated for 30 to 45 minutes at room
temperature. Each treatment was repli-
cated six times and the number of bac-
teria per capillary was based on dupli-
cate plate counts. The relative response
values were obtained by dividing the
response in capillaries containing nectar
extract by the response in the control
capillaries.

Inoculum preparation and blossom
inoculation. The Mr22 and Nm22 isolates
were grown separately in 25 ml MMS in a
shaker bath at 23 C. Cells were harvested
after 20 to 24 hours and centrifuged in an
SS-34 rotor (Sorvall) at 4000 rpm for
8 minutes. To maintain high motility, the
cells were resuspended in a solution con-
taining 10-3 M MgSO4 and 10-4 M
EDTA. Bacterial concentrations were
adjusted to 1 x 107 cells per ml during
the spring 1981 trial and to 5 x 103,
5 x 10s and 3 x 107 cells per ml during


Jan. & June 1985









Fhilippme rnytopatnoiogy


S-. .... ll ut U lII. L.L. .. IIIIvlII
concentration for detection of differences
in blossom infection between isolates.
Care was taken during suspension and
handling of inocula to minimize flagellar
breakage.
Flower clusters containing at least four
flower buds were selected and bagged
with 5 x 10 x 30 cm open-ended plastic
bags 2 to 3 days before inoculation. Ino-
culations were made by inserting the
nozzle of a hand sprayer into the open
end of the plastic bag; applying appro-
ximately 6 ml of inoculum per blossom
cluster to the point of run-off, and then
stapling the bag closed to prevent rapid
drying. The bags were removed 2 days
after inoculation. Treatments were
arranged in a randomized complete block
design with each tree considered a block
or replicate. Each treatment was repli-
cated ten times with 10 flower clusters
per replicate during the spring 1981
trial and six times during the spring
1982 trial.
Disease assessment. Disease was
assessed 7 to 10 days after inoculation,
when the infection was clearly visible
on the blossoms but had not progressed
to the base of the petiole, by taking the
total number of flowers infected and
dividing by the total number of flowers
inoculated. Disease ratings were expressed
as percentages and transformed using
arcsin.
Unless otherwise indicated, statistical
analysis were done at P = 0.05. Means
were compared using Fisher's Least
Significant Difference Test.


Response of E. amylovora to Various
Nectar Extracts
Sources of nectar extracts. Twenty-
one species of Rosaceous plants be-
longing to eleven genera and Hibiscus
rosa-sinensis, a Malvaceous plant, were
used as sources of nectar extracts. Except
for Prunus persica which was monitored


m 1iy an plants were monitored m
1982 for their flowering dates. Nectar
extracts were collected from newly opened
flowers of each species as described
above. All nectar extracts were filter-
sterilized and stored at -20 C in sterile
vials. Due to difficulty in locating the
nectaries of strawberry flowers, 250 to
300 flowers were immersed individually
into 10 ml of double distilled water for
20 seconds. The resulting flower leachate
was filtered to remove pollen and other
solid impurities and stored at -20 C.
Blossom and shoot tip inoculation.
Three to five blossom clusters per plant
were covered with 5 x 10 x 30 cm open-
ended plastic bags and inoculated with
E. amylovora (Isolate 110) at 1 x 10s
cells per ml (O.D. = 0.08) using a hand
sprayer. The open end of the plastic bag
was stapled to prevent rapid drying of
inoculum. The plastic bags were removed
after two days. Young shoot tips of
H. rosa-sinensis, Malus arnoldiana, Malus
x atrosanguinea, Malus floribunda, Malus
hupehensis, Prunus triloba and Pyrus
calleryana also were inoculated using
hypodermic needle. The blossoms and/or
shoots were monitored for 14 days for
fire blight symptoms. Plants that did not
show symptom after this time were
considered resistant or immune. Plants
were considered moderately resistant
when flower infection did not progress
to the shoots or twigs (the shoots or twigs
remained healthy after the infected
flowers dropped off). Those plants whose
flowers were blighted but the progress of
infection down the petioles was slow,
were considered moderately susceptible.
Plants were considered susceptible when
flower infection progressed rapidly to
the young shoots and the entire twig or
shoot was blighted.
Flower clusters and shoots that
exhibited symptoms were removed and
brought to the laboratory for reisolation.
Isolations were made using MEM supple-
mented with 500 ug/ml rifampicin. The


VOl. 21









Role of Motility in Apple Blossom Infection


pathogenicity of all isolates was tested on
.v. Jonathan apple seedlings grown in the
greenhouse. Seedlings were inoculated
with hypodermic needles. The same
concentrationn as in blossom inoculation
was used.
Capillary assay. Nectar extracts ob-
:ained from the different plant species
were diluted ten-fold with chemotaxis
medium and filter-sterilized. The chemo-
:axis assay described earlier was used.
Each treatment was replicated four
timess and arranged in a completely ran-
lomized design. Two controls, one con-
sisting of chemotaxis medium alone and
one of a ten-fold dilution of cv. Jonathan
lectar extracts, were included in each
riall run. Four nectar extracts were com-
pared with each control in each trial run.
This sample size could be handled most
easily without introducing too much
variation in incubation time (30 to 45
minutes) for capillaries. The response
:hen was expressed as a relative response
)y taking the ratio of the values in each
treatmentt over the blank value (no
ittractant) and as a response relative to
cv. Jonathan by taking the ratio of the
values in each treatment over the cv.
lonathan value.
The tactic response of E. amylovora
.o the nectar extract was considered
strong when the value relative to Jona-
han nectar extract was higher than
15% and considered weak when this
ralue was lower than 50%. Tactic res-
ponse with values between 50 and 85%
elative to Jonathan nectar extract was
consideredd intermediate or moderate.

Fractionation of nectar extracts.
)owex 2 format was prepared by wash-
ng the Dowex 2-x8 (50-100 mesh),
:hloride form, twice with 2 N sodium
brmate (11). Excess format was re-
noved by washing thrice with double
distilled water. Dowex 50W-x8 (50-100
nesh), hydrogen form, was also washed
wice with 2 N formic acid and washed


thrice with double distilled water. Nectar
extracts collected from flowers of plants
with varying degrees of resistance or sus-
:eptibility to E. amylovora were frac-
ionated following a modification of the
procedure of Kuksis and Prioreschi (11).
Nectar extracts were absorbed separately
an a column (10 x 1 cm) of Dowex 2
Formate and washed with 40 ml double
distilled water. Column flow was adjusted
:o 1.0 to 1.2 ml per minute. The organic
icids and/or amino acids were eluted
with 40 ml of 6 N formic acid. The for-
nic acid was removed by drying in a
otary evaporator at 45 to 50 C. The
esidue was redissolved in 5 ml double
distilled water and passed through a
:olumn (10 x 1 cm) of Dowex 50,
hydrogen form. The column was then
washed with 50 ml double distilled water.
'he eluate and the washing were com-
)ined and evaporated to dryness in a
rotary evaporator. Final drying of sam-
ples was done in a desiccator containing
nhydrous calcium sulfate and phos-
phorus pentoxide.
Preparation of trimethylsilyl (TMS)
esters. The silylation reaction was carried
)ut following the procedure of Boland
Ind Garner (4). The organic acid fraction
'rom each nectar extract source was
lissolved in 0.5 ml pyridine. Then,
).5 ml N,0-bis (trimethylsilyl) acetamide
BSA) was added and the mixture shaken
mtil a clear solution was obtained. The
:ontainer, tightly stoppered, was heated
n a water bath for 15 minutes at 70 C.
The solution was allowed to cool, con-
:entrated by drying with nitrogen gas
,ubbled through the solution and the
rolume adjusted to 0.2 ml with pyridine.
standard organic acids were silylated
individually and in combination in a
similar manner except that 1 ml each of
pyridine and BSA were used.

Gas chromatography. A Varian 2100
gas chromatograph equipped with a dual
Flame ionization detector was used. The


lan. & June 1985









Philippine Phytopathology


flow of carrier gas (helium) and hydrogen
was maintained at 40 ml per minute while
oxygen was maintained at 300 ml pei
minute. The injector, detector and co-
lumn temperatures were maintained at
220, 270 and 150 C, respectively. Chro-
matograms were recorded on a Varian
Aerograph Model A-25 strip-chart re-
corder. A pre-conditioned glass column
(1.8 m x 4 mm i.d.) packed with 3% SE-
52 coated on DMCS-treated, acid-washed,
80-100 mesh Chromosorb was used. Sam
ples were injected with a 5 ul Hamiltor
syringe. The reproduccibility of the
injection procedure was checked by
determining the standard deviation of the
peak areas of four consecutive injections
using 5 ug samples of TMS esters of
fumaric, malic and succinic acids. One
microliter of solvent pyridinee) was
injected between sample injections to
prevent ghosting (25). TMS esters of


extracts and chromatographed. Percent
recovery of each acid was determined
by dividing the peak areas with the res-
pective peak areas obtained from similar
amount of samples that were silylated
but not subjected to similar treatments
as the nectar extract. All samples were
chromatographed on the same day under
the same conditions. The procedure was
repeated using 5 and 10 mg of each
compound.

RESULTS

Effect of Motility on Blossom Infection
Isolation of nonmotile and motile
revertant mutants. Many of the isolates
tested were slightly motile as indicated
by their broader colony diameter in
STM. Isolate Nm22, however, did not


settings. Maiomnc .
:sent in relatively 16 -
S--- Mr22
F extracts of legu- 14 --o Nm22
as included as one F
. Benzoic acid was 12-
ie if this repellent 10 -
in nectar extracts 3


Vol. 21













rhese results were confirmed using the 10
:apillary assay wherein the number of o90 -
4m22 cells that entered the capillaries so- Mr22
lid not increase significantly with in- 70
:reasing incubation time whereas a sig-
lificant increase in cell accumulation 60 -
)er capillary was observed with the 50-
dr22 isolate (Fig. 1). The average num- E
,er of Nm22 and Mr22 cells that accu-
nulated per capillary after 10 minutes a o
vas approximately 30 and 540, respect- 20
vely. After 45 minutes, only 120 Nm22
ellss were recorded per capillary com- lo
,ared to 1650 cells for Mr22.
Pathogenicity tests. The Mr22 and 5sx1 5x10 3x10o
im22 isolates did not lose resistance to Inoculum Concentration (cells/ml)
ifampicin after mutagenesis. Both iso- Fig. 2. Blossom infection (%) in cultivar
ates were as pathogenic as the wild type. Jonathan (1982 trial) after inocu-
iymptoms were observed 4 days after lation with Mr22 and Nm22 at
noculation and infection progressed three inoculum concentrations.
lown the shoots at the same rate for all The percentage infection was on
three isolates. per individual flower basis, deter-
Response of isolates to apple nectar mined by taking the total number
extract. The Mr22 isolate was as chemo- of flowers infected and dividing
actic towards apple nectar extract as by the total number of flowers
he wild type. In the presence of 10-1 inoculated Each number repre-
lilution of the apple nectar extract, sents the mean of six replicates
..- .... t. lf1 l ,,- n ,.ltv,,t-rc, nr ronli-.


vild type, 19,300 and 2450 cells entered
he capillaries in the presence and absence
)f 10" dilution of nectar extract, res-
,ectively, giving a mean relative response
ralue of 7.9. The Nm22 isolate did not
espond chemotactically to the nectar
extract, with 350 and 300 cells being
recorded in capillaries without and with
iectar extract, respectively.
Blossom infection by Mr22 and Nm22
solates. During the spring 1981 trial,
blossom infection ranged from 48.0%
:o 86.3% with a mean of 71.6% in blos-
;oms inoculated with isolate Mr22 and
from 28.6% to 66.7% with a mean of
38.6% in blossoms inoculated with iso-
late Nm22 when a concentration of
1 x 107 cells per ml was used. These


In spring 1982, significantly higher
infection was recorded in blossoms ino-
.ulated with motile revertant isolate
it 5 x 105 and 3 x 107 but not 5 x 103
ellss per ml than with the nonmotile
solate (Fig. 2).

Response of E. amylovora to Various
Nectar Extracts
Significantly more E. amylovora cells
entered in the capillaries containing the
nectar extracts, including those of H.
rosa-sinensis and P. persica, which have
not been reported to be susceptible to
this pathogen, than in the control capil-
laries (chemotaxis medium alone). How-
ever, differences in the degree of response
were detected among the nectar extracts









Philippine Phytopathol


within each sampling group. Nectar <
tracts from Amelanchier arborea, Ci,
neaster lucidus, Crataegus mollis, F
garia virginiana, Malus x atrosanguin,
M. hupehensis, Malus sylvestris cv. R
Delicious, Malus x Zumi var. calocarq
Pyracantha coccinea and P. triloba h
response values comparable to or higl
than the Jonathan nectar extract. Neci
extracts of Crataegus crusgalli, Cotone,
ter multiflorus, Crataegus viridis, Ch
nomeles japonica, M. amoldiana, ,
floribunda, Malus sylvestris cv. Gold
Delicious, P. calleryana and Pyrus coi
munis gave lower response values th;
cv. Jonathan nectar extract. Three specii
namely; H. rosa-sinensis, P. persica ai
S. japonica gave a response value low
than 50% relative to cv. Jonathan. On t1
basis of relative response values, nect
extracts of C crusgalli, H. rosa-sinens,
P. communis, P. persica, S. japonica ai
cv. Golden Delicious gave values low
than 5.
Response of the different spec
to inoculation. Except for H. roc
sinensis and S. japonica, the flower
of all species inoculated with the path
gen were susceptible. The severity
infection, however, varied. In son
species such as C, japonica, M. floi
bunda, Malus x Zumi var. calocarp
P. calleryana and P. triloba, only t]
flowers were blighted while the shoo
and twigs remained healthy after tl
infected flowers dropped off. Flowe
and young fruits of C lucidus, C. mte
tiflorus and P. coccinea also were blight(
but the progress of the disease was slow
In the case of strawberry, some frui
were infected as a result of flower in,
culation.
The most susceptible species include
A. arborea, C. mollis, E. racemosa, Malt
x atrosanguinea and M. sylvestris a
Jonathan on which flower infection
progressed rapidly to the young school
and the entire twig was blighted.
No infection was observed on voun


shoots of P. triloba, H. rosa-sinensis a
P. calleryana when inoculated by neec
injection. Limited infection was observe
however, on young shoots of M. flo
bunda, while entire shoots of M. arn
diana, Malus x atrosanguinea and
hupehensis were blighted.
In all cases of infection, the pathog
was reisolated in MEM supplement
with 500 ug rifampicin per ml. T
disease also was reproduced when t
resulting isolates were needle-inject
into the young shoots of cv. Jonath
seedlings in the greenhouse.

Relationship between susceptibility
resistance of the hosts and attractivene
of nectar extracts to E. amylovora. TI
different plant species used in the stuck
can be placed artibrarily into five grou
based on their response to inoculate
and the pathogen's chemotactic respon
to their nectar extracts (Table 1). TI
first group of plants, namely: H. rosa
sinensis, P. persica and S. japonica, we
immune (nonhost) and their nect
extracts were weakly attractive to tl
pathogen. There was no infection c
H. rosa-sinensis and S. japonica wh(
both the flowers and shoots of the fc
mer and flowers of the latter were in
culated with E. amylovora. P. person
was not included in the list of speci
infected by E. amylovora (14). The p
thogen was weakly attracted to nect;
extracts of these species as indicate
by the lower relative response value
Values relative to cv. Jonathan nect,
extract also was low. The second grou
of plants which include C japonica
Malus x Zumi var. calocarpa and A
triloba were moderately resistant bi
their nectar extracts were attractive t
the pathogen while the third group c
plants such as M. floribunda and P. ca
leryana were moderately resistant an
their nectar extracts were moderate


72









Role of Motility in Apple Blossom Infection


Table 1. Relationship between susceptibility or resistance of the host and the degree of
chemotactic response of Erwinia amylovora to the nectar extracts


Source of Response to Response relative Relative
nectar extract inoculation to Jonathan response
nectar extract (%)a
Hibiscus Resistant 29.1 40
rosa-sinensia L.
Prunus persica L. Not determined 35.4 4.9
Batsch
Spiraeajaponica L. Resistant 37.8 3.9
Chaenomeles japonica Moderately 79.7 9.0
(Thunb.) Lindl. resistant
Maluz x Zumi var Moderately 121.4 7.1
calocarpa resistant
Prunus triloba Lindl. Moderately 94.5 10.1
resistant
Malus floribunda Sieb. Moderately 62.7 7.1
resistant
Pyrus calleryana Moderately
Decne cv. Bradford resistant
Cotoneaster Moderately 56.2 5.8
multiflorus Bge susceptible 66.2 7.1
Crataegus viridis cv. Moderately
Winter King susceptible 62.2 7.0
Crataegus crusgalli L. Susceptible 67.6 4.6
Exochorda racemosa Susceptible 51.6 5.3
(Lindl.) Rehd.
Pyrus communis L. Susceptible 58.2 3.9
Amelanchier arborea Susceptible 86.3 5.2
Cotoneaster lucidus Susceptible 94.5 10.6
Schlecht..
Crataegus mollis Susceptible 90.7 6.1
(Torrey & Gray)
Scheele
Fragaria virginiana Susceptible 90.9 5.5
Duch.
Malus arnoldiana Susceptible 88.4 9.2
(Rehd.) Sarg.
Malus x atrosanguinea Susceptible 104.9 6.1
Malus hupehensis Susceptible 85.8 9.2
Malus sylvestris Mill. Susceptible 9.3c
cv Jonathan


Jan. & June 1985










Philippine Phvtovatholov


Table 1. continued

Source of Response to Response relative Relative
nectar extract inoculation to Jonathan response
nectar extract (%)a


Malus sylvestris Mill. Susceptible 80.2 4.7
cv. Red Delicious
Pyracantha coccinea Susceptible 107.5 6.5
Roem. cv. Lalandi


aRatio of number of bacteria in capillaries containing a certain nectar extract over the number
of bacteria in capillaries containing the cv. Jonathan nectar extract multiplied by 100.
bRatio of number of bacteria in capillaries with nectar extract over the number of bacteria in
capillaries containing chemotaxis medium alone (control).
CThis figure was based on twelve trials with four replicates per trial and the number of bacteria
per capillary was based on duplicate plate counts. All other figures were the mean of two trialswith
four replicates per trial. All assays were run for 30 to 45 minutes at 23 to 25 C with .4 107, cells per
ml outside the capillaries.
attractive to the pathogen. The fourth 2.0, 2.6, 3.5, 4.1, 6.6, 9.1 and 12.9 mi-
group of plants (C. crusgalli, C. multi- nutes were recorded for malonic, benzoic,
florus, C. viridis, E. racemosa and P. succinic, fumaric, oxaloacetic, malic and
communis) were susceptible to moderate- tartaric acids, respectively. The sample
ly susceptible but their nectar extracts injection techniques was highly repro-
were moderately attractive to the oatho- ducible as indicated by the small stand-


ceptible and their nectar extracts were
strongly attractive to the pathogen. Most
of the species tested were in this group.
The results that agree with the hypothe-
sis, that resistant or immune .plants pro-
duce nectar that is weakly attractive and
the susceptible plants produce nectar that
is strongly attractive to E. amylovora are
those obtained from the first, third and
fifth groups.

Gas chromatography. The SE-52 co-
lumn was sensitive enough to detect small
amounts of organic acids present in nec-
tar extracts. Well-separated, sharp peaks
for benzoic, fumaric, malic, malonic
and succinic acids and short, broad peaks
for oxaloacetic and tartaric acids were
obtained when run isothermally at 150
C (Fig. 3). Based on two trials with two
observations ner trial, retention times of


range tested (2 to 10 mg samples of the
organic acids). The mean recoveries of
the organic acids after subjecting to simi-
lar treatments as nectar extracts are
shown in Table 2. Recoveries of 71.7%,
88.4% and 67.9% were obtained for fu-
maric, malic and succinic acids, respec-
tively, when 2 mg sample was used.
These are the values used as the correct-
ion factors for estimating the amounts
of fumaric and/or malic acids detected
in the nectar extracts tested.
Fumaric and malic acids were tenta-
tively identified in the nectar extract ol
M. floribunda but only the latter was
-+.-+-A ;n + -, +- ___*- _- r _C r


7A


me 1985


















3
2 4







6








2 4 6 8 10 12 14
Retention time (minutes)
Fig. 3. Chromatogram of a standard mixture of organic acids as trimethylsilyl derivatives
on 3% SE-52 column run isothermally at 150 C. Peak identification: (1) malonic
acid; (2) benzoic acid; (3) succinic acid; (4) fumaric acid; (5) oxaloacetic acid;
(6) malic acid; and (7) tartaric acid.
nathan (Fig. 4). A peak that appeared to DISCUSSION
correpsond to fumric acid was observed
in nectar extracts of H. rosa-sinensis, M. In order to test the hypothesis that
sylvestris cv. Red Delicious and P. caller- motility enhances the infection potential
yana. Trace amounts of fumaric and of bacterial pathogens such as E. amylo-
malic acids were also detected in nectar vora, it was necessary to have motile and
extract of S. japonica. Benzoic acid was nonmotile isolates that are near isogenic.
not detected in any of the nectar extracts The nonmotile isolate was as pathogenic
tested. The concentration of malic acid as either the wild type or the motile
in nectar extracts of cv. Jonathan and M. revertant when injected into Jonathan
floribunda was approximately 6.1 x 10-4 apple seedling shoots. This indicates that
and 1.2 x 10-4 M, respectively. The con- motility is not a requisite for pathogenity
centration of fumaric acid detected in in E. amylovora. The same conclusion
nectar extracts of H. rosa-sinensis, M. was reached by Panopoulos and
floribunda, M. sylvestris, cv. Red Deli- Schroth (1974) with Pseudomonas
cious and P. calleryana was approxi- phaseolicola.
mately 1.2 x 10-4 M, 1.1 x 10-4 M, 1.0 x The chemotactic response of Mr22
10-4 M and 7.0 x 10-5 M, respectively. towards Jonathan nectar extract was


lan. & June 1985


Motility in Apple Blossom lnf










Philippine Phytopatholog


able 2. Amount ot organic acia recol
nectar extract


Organic acid
2




Fumaric 71.7
Malic 88.4
Malonic 81.2
Succinic 57.9

aKnown amounts of organic acids
passed through anion and cation exchange
silylated by adding 1 ml N,0-bis (trimethyl)
70 C, the volume adjusted to 0.2 mil using py
percent recoveries were determine
jected to similar treatments as nectar extract
amount of sample) multiplied by 100. Each fi
trial:

comparable to the wild tvoe sug-esti


l (UO) when subjected to similar treatments


Amount of acid used (mg)

5 14

Percent recovered

58.0 73
70.2 58
85.4 93
69.5 82


combined, dissolved in 5 ml double distilled water
s, dried in a desiccator, dissolved in 1 ml pyridir
amidee and heated in a water bath for 15 minutes
e. Control acids were silylated only.

, dividing the peak areas of the acids that were su
the respective peak areas of the control acids (sai
was the mean of two trials with two observation I


for attracting E. amylovora to the neci


VoL


/D





















A 1









6 9 3 6 9
Retention time (minutes) Retention time (minutes)

ig. 4. Chromatogram of trimethylsilyl derivatives of organic acids isolated from nectar
extracts of (A) cv. Jonathan and (B) Malus floribunda. Four ml of Jonathan and
3 ml of M. floribunda nectar extracts were fractionated separately using anion
and cation exchange resins, dried, dissolved in 0.5 ml pyridine and silylated by
adding 0.5 ml N, 0-bis(trimethylsilyl) acetamide. The solutions were concentrated
by evaporation under nitrogen, the volume adjusted to 0.2 ml and 4 ul samples
chromatographed on 3% SE-52 column run isothermally at 150 C Peak identifica-
tion: A(1) malic acid; B(I) fumaric acid; and (2) malic acid.
Sfumaric acid was detected but the nectar extracts tested.
ithogen's chemotactic response was
igh. It is possible that other attract- Whether or not the concentration of
it organic acids (Raymundo and chemo-attractants present in nectar ex-
eis, 1980) extract in amounts that tracts of Rosaceous plants undergo the
)uld not be detected by the method same diurnal fluctuations as the sugar
sed in this study and these amounts (Ivanoff and Keit, 1941), due to an in-
lay be acting in an additive manner, verse relationship between sugar concen-
dditivity of chemotactic response tration in the nectar and atmospheric
as observed in Salmonella when two humidity (Thomas and Ark, 1934), is not
imuli aspartate and ribose were known. In this study, no specific time of
)mbined (Rubik and Koshland, day was set for blossom collection and
978). Raymundo and Ries (1980) nectar extraction. Also, due to the te-
emonstrated that all attractants of dious process involved in nectar extrac-
amylovora share a single chemore- tion, some of the collected blossoms were
eptor and therefore attractants kept in the refrigerator (in plastic bags)
iould be additive. It is also possible for as long as 3 days before nectar could
iat the concentration of aspartic be extracted.
cid, the amino acid known to
attract E. amylovora (Raymundo and LITERATURE CITED
ies, 1980), is higher in cv. Red
)elicious nectar extract than in other AGRIOS, G.N. 1978. How plants defend













themselves against pathogens. Pages blossoms. J. Agr. Res. 6
75-85 in: Plant Pathology. 2nd ed.
Acad. Press, New York. 703 pp. KELMAN, A., and HRU
1973. The role of motility
ARMSTRONG, J.B., ADLER, J., and taxis in the selective inci
DAHL, M.M. 1967. Nonchemotactic rulent bacteria in still br
mutants of Escherichia coli. J. Bacte- of Pseudomonas solanacea
riol. 93:390-398. Microbiol. 76: 177-188.

BILLING, E., BAKER, L.A.E., CROSSE, KUKSIS, A., and PRIOR
J.E., and GARRETT, C.M.E. 1961. 1967. Isolation of Krebs
Characteristics of English isolates of from tissues for gas chroi
Erwinia amylovora (Burr.) Winslow, et Anal. Biochem. 19: 468-4E
al. J. Appl. Bacteriol. 24: 195-211.
MIZUKAMI. T. and WAK


BULANU, K.L., and GARNER, G.B.
1973. Determination of organic acids
in tall fescue (Festuca arundinacea)
by gas liquid chromatography. J. Agr.
Food Chem. 21: 661-665.

rT ARK R R 10Q; n--nlo o .io Cfm


i7U7. LjplIUriulugIy allu UUIIIm
bacterial leaf blight of rice. Ann.
Phytopathol. 7: 51-72.

RDAL, G.W., and ADLER, J. 1
Isolation and complementation


... ... .. ........ . 1" r ..... J 6UU
chromatography. Crop Science 9: 341-
343. PANOPOULOS, N.J., and SCHF
M.N. 1974. Role of flagellar m
CROSSE, J.E., GOODMAN, R.N., and in the invasion of bean leav
SHAFFER, W.H., JR. 1972. Leaf Pseudomonas phaseolocila. Pn
damage as a predisposing factor in the thology 64: 1389-1398.
infection of apple shoots by Erwinia
amylovora. Phytopathology 62: 176- PARKINSON, J.S. 1977. Behavioral
182. tics in bacteria. Ann. Rev. (
11: 397-414.
DAY, P.R. 1974. Gene function in host- RAYMUNDO, A.K., and RIES,
parasite interaction. Pages 111-153 In; 1980. Chemotaxis of Erwinia
Genetics of Host-Parasite Interaction. lovora. Phytopathology 70:
WH Cram P ..-- C-A a, t C__>


HUANG, J.J., 2
1970. Morpho
of normal ro


K.N. 1981. Motility of Erwinia
acture Phytopathology 71:45-49.
amen-
ora. J. REINHARDT, J.F., and PO
10in rllltllr mprlii fn


IVANUFF, N.N., and KEIT, U.W. 1941. lovora. Phytopatholovy 50: 68!









Jan. & June 1985 Role of Motility in Apple Blossom Infection 79



tion, and inversion of response in bac- THOMAS, H.E. 1931. Plants affected by
trial sensing of chemical stimuli. fire blight. Phytopathology 21: 425-
Proc. Natl. Acad. Sci. U.S.A. 75: 435.
2820-2824.
VAN DER ZWET, T., and KEIL, H.L.
SHAW, L. 1934. Studies on resistance of VAN DER ZWET, T., and KEIL,
apple and other Rosaceous plants to 1979. Host range. Pages 29.36 in: Fire
apple and other Rosaceous plants to .
fire blight. J. Agr. Res. 49: 283-313. Blight: A Bacterial Disease of Rosa-
ceous Plants. United States Dept.
SMITH, J.L., and DOETSCH, R.N. 1969. Agric. Handbook No. 510, Washing-
Studies on negative chemotaxis and ton, D.C., U.S. A. 200 pp.
survival value of motility in Pseudo-
monas fluorescent. J. Gen. Microbiol. ZAURA, D.S., and METCOFF, J. 1969.
55: 379-391. Quantification of seven tricarboxylic
acid cycle and related acids in human
THOMAS, H.E., and ARK, P.A. 1934. urine by gas-liquid chromatography.
Nectar and rain in relation to fire Anal. Chem. 41: 1781-1787.
blight. Phytopathology 24: 682-685.









Philippine Phytopathological Society, Inc.
1985 Phil. Phytopath. 21: 80-87


EFFECT OF NEEM OIL ON SURVIVAL OF THE RICE BROWN
PLANTHOPPER, NILAPAR VATA LUGENS (Stal) (HOMOPTERA:
DELPHACIDAE) AND ON GRASSY STUNT and RAGGED STUNT
VIRUS TRANSMISSION


R. C. SAXENA and Z. R. KHAN

Respectively, Entomologist and Postdoctoral Fellow, Department of Entomology,
The International Rice Research Institute (IRRI), Los Baisos, Laguna, Philippines. 3720.


ABSTRACT

Neem (Azadirachta indica A. Juss) seed oil was found highly effective in reducing the
survival of brown planthopper, Nilaparvata lugens (Stal) (Homoptera: Delphacidae) and
in suppressing transmission of grassy stunt and ragged stunt viral diseases of rice. Ge-
nerally, insect survival and disease transmission decreased with increasing neem oil
concentrations. After 3 daysof exposure, the insect failed to transmit the viruses to plants
sprayed with 50% neem oil, compared with control plants where virus transmission was
successful. Thus, neem seed oil possesses potential for control of brown planthopper and
its transmitted grassy stunt and ragged stunt viral diseases of rice.





Insect damage to plants results from serious rice pest and vector of persistent
direct feeding or from indirect transmis- grassy stunt virus (GSV) and ragged
mission of pathogens during feeding, stunt virus (RSV) (Saxena et al., 1981,
Therefore, antifeedants, which reduce Saxena et al., 1984, Heyde et al., 1984).
or disturb feeding activities of insects The incidence of RSV was reported to
by rendering plants unattractive or have been reduced significantly in rice
unpalatable, offer a novel approach in fields sprayed periodically with 12%
pest and vector management. Centuries crude neem oil (Saxena et. al., 1981).
before synthetic insecticides became We tested the effect of different concen-
available, farmers in India protected crops trations of neem oil on the survival of
with natural repellents derived from N. lugens and on its ability to transmit
the fruits and leaves of the neem tree, GSV and RSV diseases in neem oil-
Azadirachta indica A. Juss (Saxena, sprayed rice seedlings.
1983). Mariappan and Saxena (1983,
1984) reported that neem seed-oil ad- MATERIALS AND METHODS
versely affected the survival of green
leafhopper, Nephotettix virescens The crude oil expelled from decor-
(Distant), and its transmission of semi- ticated neem seeds (1983 crop) was ob-
persistent rice tungo virus. Laboratory tained from C.M. Ketkar, Advisor to the
__A--* r n -i ;I -1 ____ -i 11-* -. - _* __ -T I . ,









1985 Effect of Neem Oil on Survival of the Rice Brown Planthopper


I uy IvdillldJPdul dIIU ua Jl
days old, hopper-sus- 5% level.
native 1 (TN1) rice
yed separately with
s at a rate of 0.5
sing a quick spray N. lugen
chemicall Co., Rock mission
ours before exposure
s sprayed with 1.6% Table
control Each treated on N. lu
in a glass test tube mission.
:d with polyvinyl cap increasing


RESULTS

vival and GS\


ows the effect
survival and
ct survival de
-m oil concern


-:ary lower than


iviva aiiu uiaaa yJ~7o 1
ere conducted se- on 1'
tunt and ragged plants
pn.V -.,r


* wv aa oiaiiivan l
5, and 50% ne
iree days after


inoculation access feeding. After 24 trol.
rs, each surviving viruliferous insect Transmission of GSV 2 by the viruli-
transferred to another freshly treated ferous N. lugens generally decreased with
ling while inoculated seedlings from increasing neem oil concentrations (Fig.
i treatment were transplanted, for 2). One-day inoculation feeding on con-
ase development, in an insect-proof trol seedlings infected 22% plants, which
henhouse, in separate plots (50 x 50 was significantly more than the percen-
for each replication, arranged in a tage of infection at all neem oil concen-
lomized complete block design. Suc- trations (Table 1). After 2 days of feed-
ive inoculation access feeding of the ing, 19% of the control seedlings were
ivors on treated plants was continued infected; significantly less infection oc-
1 4 days. GSV 2 symptoms in inocu- curred at 12% and higher neem oil con-
i seedlings were observed twice centrations. After 3 days of feeding,
r 12 days and after 3 weeks. RSV significantly less seedlings were infected
ase symptoms were observed after at 25% neem oil concentration than the
lays. control. No infection occurred on 50%
'he experiment for each disease was oil-sprayed plants.
icated 4 times, using 40 viruliferous
cts and 40 treated seedlings for each N. lugens Survival and RSV Transmission
ication of 6 treatments. A total of
0 viruliferous insects and 4784 treat- Survival of N. lugens and RSV trans-
seedlings, including those of succes- mission decreased on neem oil-treated
feelings, were used in the two ex- seedlings (Table 2, Fig. 1, 2). One day













Table 1. Survival of N. lugens and its ability to transmit grassy stunt virus strain 2 ((
neem oil. a


lI 1AaV ^ IrVc


(%) (%)


3 88 a
6 88 a
12 90a
25 82 a
50 61 b
0 94 a
(control) b

In a column, means followed by a co
aAverage of 4 replications.
bControl treatment was sprayed with 1.6% "


(%) (%) (%)


11 b 86 ab 7 a
9b 78 ab 10a
7b 69b 3b
7b 70b 2b
10b 31c 2b
22 a 93 a 19 a



in letter are not significantly different at 5% level b


ol" in water










Dil on Survival of the Rice E


T T.-u.oiA
*^ rz0.96**







Y=89.0-1.11X 40
r 0.97**
n= 6
-r *
20
2C

8C
I I I1
S6C
9= 86.2-1.06X 6
r = 0.98**
n=6 4C


2C


I I I I 0
0 3 6 12 25 50
Concentration (%)
1. Effect of different concentrations of n
viruliferous N. Lugens females after ex
days, and of ragged stunt viruliferous
(e) 3 days on neem oil-treated rice seed,

seedlings treated with 50% neem oil.
was significantly less than the sur-
percentage on the control (93%).
:t survival remained high after two
,) and three (73%) days of feeding
control seedlings. However, insect i
val in treatments involving 50%
1 oil after 2 days of feeding and 25 1
50% neem oil after 3 days of feeding
significantly reduced compared with
control. Other treatments of low
I oil concentrations did not differ
ficantly from the control.
ne-day inoculation access feeding
:ted 22% of the control seedlings;


r 0.97**
n=6
0

0 0








I I I


Y z=74.2-1.27X
n=6








6 12 25 50
Concentration(%)
oil on the survival of rice grassy stunt
ure of (a) 1 day (b) 2 days, and (c) 3
2les after exposure of (d) 2 days and
$.

ficantly less infection occurred at
or higher neem oil concentrations.
r 2 days of feeding, 13% of the
:roi seedlings were infected, which
significantly higher than on 25%
n oil-sprayed plants. No infection
irred in seedlings sprayed with 50%
n oil. After 3 days of feeding, signi-
itly less seedlings were infected at all
n oil concentrations compared to
:rol.

DISCUSSION

'he complexities of the control of


3














Table 2. Survival of N. lugens and its al


1 4
Neem oil conc. Survival
(%) (%)

3 89 a
6 88 a
12 89 a
25 75 a
50 57 b
(control) b 93 a



In a column, means followed by a comn
a Average of 4 replications.
b Control treatment was sprayed with 1.6% "


r to transmit ragged stunt virus (RSV) on TP


2 days

2 infection Survival RSV 2 ini
(%) (%) (%)

10ab 84a 9a
9 ab 76 a 7 al
5 b 79 a 7 al
6b 63a 3 b
7b 31b Oc
22 a 83 a 13 a



better are not significantly different at 5% level by

ol" in water.









Effect of Neem Oil on Survival of the Rice Brown Planthopper


Transmission (%)
25
SGSV (Ist day)
9Y 7.16+14.50( +-)
15 R2=0.95**
n=6

5-


Concentration (%)


Fig. 2. Effect of different concentrations of
in neem oil-treated rice seedlings.

N lugens and of the viral diseases of rice
plants it transmits have increased in
recent years due to the insect's resistance
to several insecticides and resurgence
with others in the fields (Heinrichs et al.,
1979; Saxena et at, 1981). Planting hop-
per-resistant rice varieties as an alternative
strategy has not been very successful
because of development and occurrence
of N. lugens biotypes in several rice grow-
ing countries (Athwal and Pathak, 1972;
Mochida, 1978; Kalode and Krishna,
1979;Pathak and Khush, 1979).
Various mineral oils are reported to re-
duce insect transmission of stylet-borne
plant viruses (Bradley et al., 1962, Van-
derveken 1968a; Bhargava and Khurana,
1969; Dubey and Nene, 1974). These
mineral oils accumulate on the anticlinal
wall of the leaves and act as interfering
agent in virus transmission (Simons and
Beasley, 1977). Although mineral oils
protect crops from insect transmission of


Transmission (%)
1 GSV (3rd day)


Concentration (%)


neem oil on GSV 2 and RSV transmission


stylet-borne viruses, they do not inhibit
the transmission of pesistent viruses
(Vanderveken, 1968b). A possible reason
is that the feeding behavior of the vector
is .not modified by oil treatment (Vander-
veken, 1973; Peters and Lebbink, 1973).
In a review of the innovative methods
of controlling insect-transmitted viral
diseases, Simons (1981) recommended
the use of oil formulations with naturally
occurring antifeedants derived from
plants. In this approach, we used neem oil
not only as an interfering agent that
reduces viral transmission but also as an
insect repellent with antifeedant proper-
ties. Saxena et al. (1981) reported that
the feeding duration of N. lugens on
neem oil-treated rice plants decreased by
0.93 min/h for every 1% increase in neem
oil concentration, while search and
avoidance of feeding sites increased
correspondingly. Recently, Heyde et al.
(1984) and Saxena et al. (1984) found


- S.O0.042X


n=6


1 t p I 1


-- A


Jan. & June 1985














that the food intake by N. lugens was
significantly reduced on rice plants
sprayed with neem oil. Thus, observed
adverse effects of neem oil on the survival
ofN. lugens and reduction in its ability to
transmit persistent GSV 2 and RSV can
be ascribed to the insect's reduced
feeding.


LITERATURE CITED

ATHWAL, D.S., and M.D. PATHAK.
1972. Genetics of resistance to rice
insects. In Rice Breeding. International
Rice Research Institute, Los Banos,
Laguna, Philippines. 739 pp.

BHARGAVA, K.S., and S.M. PAUL
KHURANA. 1969. Papaya mosaic
control by oil sprays. Phytopath, Z.
64:338-343.

BRADLEY, R.H.F., C.V. WADE, and
F.A. WOOD. 1962. Aphid transmis-
sion of potato virus Y inhibited by oils.
Virology 18:327-329.

DUBEY, G.S., and Y.L. NENE. 1974.
Aphid transmission of cowpea mosaic
virus as influeficed by oil sprays.
Indian Phytopath 27:325-330.

HEINRICHS, E.A., R.C. SAXENA, and
S. CHELLIAH. 1979. Development and
implementation of insect pest man-
agement system for rice in tropical
Asia. pp. 208-248. In Sensible Use of
Pesticides, Food and Fertilizer Tech-
nology Center, Taipei, Taiwan, Repub-
lic of China. 250 pp.

HEYDE, J.V.D., R.C. SAXENA, and H.
SCHMUTTERER. 1984. Neem oil and
neem extracts as potential insecticides
for control of hemipterous rice pests.
In Proc. 2nd Int. Neem Conf., Rauisch
Holzhausen Castle, 1983 (in press).
KALODE MR and T KRISHNA


1979. Varietal resistance of brown
planthopper in India. pp. 187-199. In
Brown Planthopper: Threat to Rice
Production in Asia. International Rice
Research Institute, Los Banos, Laguna,
Philippines. 369 pp.

MARIAPPAN, V., and R.C. SAXENA.
1983. Effect of custard-apple oil and
neefi oil on survival of Nephotettix
virescens (Homoptera: Cicadellidae)
and on rice tungro virus transmission.
J. Econ. Entomol. 76:573-576.


MARIAPPAN, V., and R.C. SAXENA.
1984. Effect of mixtures of custard-
apple and neem oil on survival of
Nephotettix virescens (Homoptera:
Cicadellidae) and on rice tungro virus
transmission. J. Econ. Entomol. 77:
Cicadellidae) and on rice tungro virus
transmission J. Econ. ntomol. 77:
519-521.

MOCHIDA, O. 1978. Brown planthopper
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Indonesia. Rep. Coop. CRIA-IRRI
Program Sukamandi, West Java, Indo-
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PATHAK, M.D., and G.S. KHUSH. 1979.
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national Rice Research Institute. pp.
285-301. In Brown Planthopper:
Threat to Rice Production in Asia.
International Rice Research Institute,
Los Banos, Laguna, Philippines. 369
pp.

PETERS, D., and G. LEBBINK. 1973.
The effect of oil on the transmission
of pea enation mosaic virus during
inoculation probes. Entomol. Exp.
Appl. 16:185-190.

SAXENA, R.X. 1983. Naturally occur-
ring pesticides and their potential. pp.
'? r 1 -l -1 -1. 1 11 -"1


OI:lM-* nhr.rkA4-1l-r









on Survival of the R


istry and World Food Supplies: The
New Frontiers, CHEMRAWN II, Per-
gamon Press, Oxford and New York.
664 pp.

SAXENA, R.C., N.J. LIQUID, and H.D.
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potential antifeedant for the control
of the rice brown planthopper, Nila-
parvata lugens pp. 171-188. In H.
Schmutterer, K.R.S. Ascher, and H.
Rembold (eds.), Natural Pesticides
from the Neem Tree (Azadirachta
indica A. Juss). Proc. 1st Int. Neem
Conf., Rottach-Egern, 1980. 297 pp.

SAXENA, R.C., P.B. EPINO, TU CHENG-
WEN, and B.C. PUMA. 1984. Neem,
chinaberry and custard-apple: antifee-
dant and insecticidal effects, of seed
oils on leafhopper and planthopper
pests of rice. In Proc. 2nd Int. Neem
Conf., Rauisch-Holzhausen Castle,
1983 (in press).

SIMONS, J.N. 1981. Innovative methods
of control for insect-transmitted plant
viral diseases. pp. 169-178. In J.J.



ACKNOW]

We thank H. Hi
for providing us w
2) and ragged stu
and Zenaida M. F
recording the numt


McKelvy, Jr., B.F. Elridge and K.
Maramorosch (eds.), Vectors of Di-
sease Agents, Interaction with Plants,
Animals and Man. Praeger Publishers,
New York. 229 pp.

SIMONS, J.N., and C.A. BEASLEY, 1977.
Visualization of oil on leaf surfaces -
a technique. J. Econ. Entomol. 70:
307-308.

VANDERVEKEN, J. 1968a. Effect of
mineral oils on aphid transmission of
beet mosaic and beet yellow viruses.
Virology 34:807-809.

VANDERVEKEN, J. 1968b. Importance
des relations vector-virus dans I'inhi-
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d'emulsions huileuses. Annls. Epiphyt.
19:141-146.

VANDERVEKEN, J. 1973. Recherche du
mechanism de I'inhibition de la
transmission aphidienne des phytovi-
rus par des substances huileuses.
Parasitica 29:1-15.



GEMENT

, virologist, IRRI,
rassy stunt (strain
virus source plants
; for assistance in
f infected plants.


N.n Z T.... IQQ<













NEEM SEED DERIVATIVES FOB
TRANSMISSION BY THE GREI
VIRESCEI

R. C. Saxena, Z.1

Respectively, Entomologist, Rese;
Research Fellow, Department of Plani
Institute (IRRI), Los Bafios, Laguna, Phil



AB!
Neem (Azadirachta indica A. Juss]
ting rice tungro virus transmission by the
tant). Neem oil and neem cake disrupt
The insect shifted from phloem to xylerr
or neem cake, or kept in an arena permea
lings grown in soil incorporated with 15(
virus was comparable to that conferred b:
hoppers ability to transmit the tungro v
could be attributed to a reduction in ph
insect feeding behavior.

The complexities of the control of
the green leafhopper, Nephotettix vires-
cens (Distant) (Homoptera: Cicadellidae),
and its transmitted rice tungro virus have
increased in recent years in the Philip-
pines. The pest's resistance to several
insecticides has led to an increase in in-
secticidal treatments which are not only
uneconomical but further aggravate the
problems of undesirable residues, envi-
ronmental pollution, and harmful effects
on nontarget organisms (Heinrichs and
Rapusas, 1983). Even repeated insecti-
cidal treatments in fields with high vector
population fail to control the tungro virus
disease, as the insect can readily transmit
the semipersistent virus before it is killed.
The alternative strategy of growing leaf-
hopper resistant varieties, though free
of adverse side effects and highly success-
ful against N. virescens until a few years
ago, is now ineffective. Also, resistance
to the vector cannot always be correlated
with resistance to the virus. For instance,
'IDR' i; mrlAra- l- ra.itnt tA AT.;--.


EVENING RICE TUNGRO VIRUS
.EAFHOPPER, NEPHOTETTIX
)ISTANT)


han, and N.B. Bajet


Fellow, Department of Entomology and
hology, The International Rice Research
!es. 3720



ACT

oil and cake showed potential for preven-
n leafhopper, Nephotettix virescens (Dis-
normal feeding behavior of N. virescens.
stion on rice plants treated with neem oil
ith neem oil odor. Protection of rice seed-
250 kg neem cake/ha against rice tungro
adan treatment. The reduction in the leaf-
n neem oil or neem -cake treated plant
feeding and associated aberrations in the


cens, but susceptible to tungro virus
(Ling, 1972; Rapusas and Heinrichs,
1982). Recently, the most popular rice
variety 'IR36', which is planted in almost
11 million ha all over the world (Anony-
mous, 1982), has succumbed to the rice
tungro virus in many locations in the
Philippines. The tungro virus-susceptibi-
lity of a variety such as 'IR36', which has
a broad genetic base that includes a wild
rice, Oryza nivara Sharma and Sastry, and
has been claimed to be resistant to several
major insect pests and diseases (Swamina-
than, 1982), is of serious concern to rice
scientists all over Asia. Biological control,
in spite of a fairly large complex of
parasites and predators (Chandra, 1980),
has not succeeded because the routine
insecticidal treatments directed at the
pest destroy its natural enemies as well.
Also, in some instances, the crop is al-
ready damaged even before the predators
and parasites arrive and multiply in a field
(R.C. Saxena, unpublished report).









vatives for Preventin


from direct feeding or from indirect
transmission of disease organisms during
feeding, antifeedants offer a novel ap-
roach in vector and disease management
(Mariappan and Saxena, 1983). Antifeed-
ants are not necessarily toxic to insects
but prevent or reduce their feeding. In a
recent review of innovative methods of
control for insect-transmitted plant viral
diseases, Simons (1981) postulated the
importance of using oil formulations with
naturally occurring antifeedants derived
from plants.
Insect antifeedant properties of the
Indian neem tree Azadirachta indica A.
Juss are well known and have been re-
viewed in depth (Ketkar, 1976, Saxena
et al., 1981a,b). Centuries before commer-
cial insecticides were available, neem
derivatives were used in Indian subconti-
nent to protect agricultural crops from
insect attack (Saxena,et al. 1984). Neem
tree is widely distributed in most of the
rice-producing countries in Asia and its
seed oil and cake can be easily obtained
at low cost by the average farmer. In
India alone, there are about 14 million
trees (Ketkar, 1976). The tree becomes
fully productive in about 10 years. In
one season, a single tree produces 30 to
50 kg of neem fruits; 30 kg of neem seeds
yield 6 kg of neem oil and 24 kg of neem
cake. The potential of such an abundant,
easily available, and inexpensive natural
product was not fully exploited for pest
management because of the advent and
acceptance of highly effective, broad-
spectrum synthetic insecticides. The grow-
ing awareness of hazards associated with
the use of synthetic insecticides has re-
cently evoked a worldwide interest in
pest control agents of plant origin.
Neem oil has a strong garlicky odor
which can be perceived from a distance.
Recently, Mariappan and Saxena (1983)
demonstrated the potential of neem oil
against N. virescens and its transmitted
tungro disease. Repeated trials at the
IRRI Experimental Farms showed that
vepn iintntrted nlntt nlnnted tn a qmn-


ceptible rice variety but surrounded by
neem oil-sprayed plots, had generally
a lower incidence of tungro than in-
dependent, untreated plots planted to
the same susceptible rice variety (R.C.
Saxena, unpublished data). Also, tungro
incidence was generally lower in rice
fields applied with a mixture of neem
cake and urea than in urea-treated con-
trol plots (Saxena et al. 1984). We,
therefore, investigated how N. virescens
feeding behavior is affected by neem oil
odor and on neem oil or neem cake-
treated rice plants. We also investigated the
effect of neem cake application to rice
plants on transmission of rice tungro
bacilliform (RTBV) and rice tungro
spherical virus (RTSV) particles.


MATERIALS AND METHODS

Electronic Recording of N. virescens
Feeding on Neem Oil-Treated Plants

Crude neem oil expelled from decor-
ticated neem seeds (1983 crop) was ob-
tained from C.M. Ketkar, advisor to the
Neem Cake Manurial Project, Poona,
India. The oil was diluted with acetone
and tested at four concentrations -
1.25, 2.5, 5, and 10%. Thirty-day-old
'Taichung Native 1' (TN1) rice plants
susceptible to leafhoppers were used in
the study. Using a quick-spray atomizer
(Pierce Chemical Co., Rockford, II.
61105), each plant was sprayed with
neem oil at a dosage of 1 ml per plant 3 h
before it was offered to the insect for
feeding. Each plant received a different
concentration of neem oil spray. Plants
treated with acetone served as the control.
To determine the insect's feeding
behavior, we used a dc variant of an
electronic device (Khan and Saxena
1984b) for recording insect feeding.
A 5-cm fine (18 um) gold wire (Tanaka
Denshi, Kogyo, K.K. Tokyo, Japan) was
attached by a small quantity of silver


Jan. & June 1985









PhilioDine Phvtooatholomv


paint (Litsilber 200, Demetron, D-6450,
Hanau, W. Germany) to dorsum of an
8-to 10-h-old female reared on virus-
free, 45-day-old TN1 rice plants. Before
attaching the wire, the insect was anes-
thetized with carbon dioxide gas. The
insect was starved but water-satiated for
2 h and then placed on an intact leaf
blade of a treated or control TN1 plant.
The gold wire was connected directly to
the negative input terminal of a transis-
torized, automatic, null-balancing dc
chart recorder having 250-mm recording
width and input resistance of 1 MR2
(Unic -der, Pantos, Nippon Denshi, Ka-
gaku, Japan). The voltage source consist-
ed of two 1.5V dc batteries connected in
a series. The positive battery terminal
was connected with the plant roots
through a moistened filter paper and an
aluminum foil (Fig. 1). The negative
battery terminal was connected directly
to the positive input terminal of the chart
recorder. The recorder pen was adjusted
to the chart baseline and insect feeding















rice leaf

aluminum foil

moist f ilter paper
beaker


Fig. 1. Schematic diagram of circuit anc
on rice plants IRRI 1984


was monitored for 180 min. A chart
speed of 1.5 cm/min at 500 m V ampli-
fier power was adequate for distinguish-
ing various waveforms and associated
voltage reversals. Each treatment, includ-
ing the control, was replicated 10 times
(i.e., 10 different individuals on 10 dif-
ferent plants).

Effect of Neem Oil Odor on the Feeding
Behavior of N. virescens

Neem oil was diluted with 1.66%
aqueous 'Teepol' solution and tested at
3, 6, 12, and 25% concentrations. Twen-
ty day old potted TN1 plants were used
in the study. A filter paper disk (9 cm
diameter) was placed on a medially perfor-
ated plastic petri dish (9 cm diameter)
around the base of each plant. One ml of
neem oil solution was applied on the filter
paper disk without contaminating the rice
plant. In the control, the filter paper disk
was applied with 1 ml of 1.66% 'Teepol' or
1 ml of paraffin oil. The plant along with










STRIP CHART

RECORDER



(+)






tipment for recording N. virescens feeding


Vnl 21









privaftivAp fnr Prevent


the pot was then covered with an inverted
clear plastic container (14 cm high, 12
cm diameter) so that neem oil odoi
permeated around the enclosed rice plant
A 5-cm fine (18 um) gold wire was at
tached by a small quantity of silver pain
to the dorsum of an 8- to 10-h-old N
virescens female reared on TN1 plants
The insect, starved for 2 hours (but watei
satiated), was then introduced into the
plastic container through a small media
hole at its top and placed on the enclosed
plant (Fig. 2). The feeding activity of the
insect was monitored for 180 min, usinl
a dc variant of the electronic monitoring
device for insect feeding (Khan anc
Saxena 1984b), at a chart speed of 1.5
cm per min. Each treatment, including
controls, was replicated 10 times, using
10 different females and 10 differen-
plants.

Effect of Neem Cake on Transmissioi
of Rice Tungro Virus Particles

Ten day old TN1 rice seedlings
grown in soil applied with neem cake al
a rate of 25, 50, 75, 150, and 250 kg/ha
were used in this study. There were tw(
control checks. One set of control plant:
did not receive any neem cake treatment
whereas other control plants were applied
with Furadan granules at a rate of 0.75 kl
ai/ha.
Tungro-infected plants containing
both RTBV and RTSV particles wern
obtained from H. Hibino, virologist
IRRI, and used as source plants. Newly
emerged N. virescens females, reared or
virus-free 45 day old TN1 plants in thi
insectary, were allowed a 3-day acquisi
tion access feeding on the source plants
Each treated or control seedling wa
placed in a glass test tube (15 x 1.5 cm
covered with polyvinyl cap and arranged
in racks by treatment in a randomized
complete block design (RCBD). Two viru
liferous adults were released into eacl


cnart recorder


gold wire


clear plastic
container


neem oil-treated
filter paper disk
dc batteries
Plastic petri dish


clay pot



Fig. 2. Schematic diagram of assembly
used during electronic recording
of N. virescens feeding on rice
plants with background odor oJ
neem oil, IRRI, 1984.
for 24 h. The inoculated seedlings frorr
each treatment were transplanted foi
disease development inside an insect-
proof cage in separate plots (50 x 50 cm]
for each replication, arranged in a RCBD
The experiment was replicated 4 times
using 80 viruliferous insects and 4C
treated seedlings for each replication. A
total of 2240 viruliferous insects anc
1120 seedlings were used in the ex.
periment. The inoculated seedling!
were harvested after 13 days and each
seedling was processed for enzyme
linked immunosorbent assay (ELISA'
for detecting RTBV or RTSV particles
Antisera to RTBV and RTSV used has a
titer of 1:1280 (T. Omuro, unpublished'
and 1:640 (P.Q. Cabauatan and H. Hibino
unpublished ), respectively, in ring inter
face tests. The immunoglobulin G (IgG;
fraction of the sera was obtained b)
ammonium sulfate precipitation and
further purified by dialysis and DEAE.
cellulose column chromatography (Clark
and Adams, 1977). Conjugation of alka-
I;- _"_-U_-_ /T-.., I7TT 0 C '___


_













mical Co., St. Louis, MO) with IgG was
done as described by Clark and Adams
(1977). The unlabelled and enzyme-
labelled IgGs were suspended in 0.05 M
carbonate buffer, pH 9.6 and 0.02 M
phosphate buffered saline, pH 7.2 con-
taining 0.05% Tween 20 (PBS-T), res-
pectively.
Plant tissues were homogenized with
1 ml PBS-T using a leaf tissue press-
grinder (Erich Pollahne, 3015 Wennigsen
am Diester, FRG). The homogenate was
directly tested in ELISA using the pro-
cedure described by Clark and Adams
[1977) with slight modifications. Briefly,
Immulon II Microtiter plates (Dynatech
Laboratories, Alexandria, VA) were sensi-
tized with 1 jg and 4 kg anti-RTSV IgG,
respectively, per ml buffer per well and
incubated at 4C for 3 h. Each well of
the plate was then filled with 200 Al of
homogenate and incubated as above for
overnight. The IgG-enzyme conjugate
vas then added and incubated at room
temperaturee for 4 h. The enzyme sub-
strate, p-nitrophenylphosphate at 1 mg/
nl 10% diethanolamine, pH 9.8, was
idded and incubated for 30 min to 1 h
it room temperature. The reaction was
stopped by adding 50 ul of 3 N NaOH
)er well and evaluated visually and the
ibsorbance subsequently determined at
t05 nm using the Microelisa Minireader
dR 590 (Dynatech). The control was
lomogenate from rice plants exposed
o the insect. Any absorbance greater
han the mean absorbance of the control
4 standard deviation of the mean of
he control was considered a positive
action (Bajet, 1983).
Thirty day old plants grown in soil
applied with neem cake at a rate of
50 kg per ha were used for electronical-
y recording the feeding behavior of N.
irescens. The treatments with neem
ake as well as the control were repli-
ated 15 times, using 15 different indivi-
uals and 15 different plants.
All pynprimpntal rlta av*r nt


insect feeding behavior on neem cake-
treated plants were analyzed using the
Duncan's (1951) multiple range test
(DMRT). Data on the feeding behavior
of N. virescens on neem cake-treated
and control plants were compared using
the t test.

RESULTS
The waveform patterns associated with
N. virescens feeding on control rice plants
were similar to those recorded by Chang
(1973) for the sugarcane leafhopper,
Perkinsiella saccharicida Kirkaldy. Wave-
form patterns for N. virescens feeding on
rice plants sprayed with neem oil solution
of different concentrations are shown in
Fig. 3. Xylem feeding was differentiated
from phloem feeding by stronger voltage
signals in the former. Phloem feeding was
significantly (P<0.05, DMRT) longer on
control plants than on neem oil-treated
plants (Table 1). Phloem feeding decreased
progressively with increased concentra-
tion of neem oil (Fig. 4). The decrease in
phloem feeding was accompanied by a
corresponding, significant (P <.05,
DMRT) increase in the frequency of
probing the salivation period, and the
xylem feeding in all treatments, except
an plants sprayed with 1.25% neeni oil.
The waveform pattern recorded for
phloem feeding was erratic on plants
sprayed with 5 and 10% neem oil, as
evidenced by repeated voltage reversals
,Fig. 3d, Pi?). In spite of increased
xylem feeding on neem oil-treated plants,
the total duration of intake from the
,ascular tissue was significantly (P<0.05,
3MRT) less than on control plants
Table 1). On plants sprayed with 10%
teem oil, the insects became restless,
)robed repeatedly, salivated profusely,
md fed mainly from xylem (Fig. 3e and
). After about 150 min of feeding
Activity, the insects became very weak
lue to increased restlessness. This was
ilso evidenced by assumption of an


Philinnina Phvgfrmnh-lan.


-- _-









Neem Seed Derivatives for Preventing Rice Tungro


Fig. 3.


Electronically recorded waveforms during N. virescens feeding on TN1 rice
plants sprayed with (a) acetone (control) and (b) 1.25% (c) 2.5% (d)5%(e,f)
10%, neem oil, IRRI, 1984.


Table 1. Means of various events in feeding bouts ofN. virescens on rice plants sprayed
with different concentrations of neem oil during 180 min. a IRRI, 1984.


Oil conc. b Probes Salivation Phloem Xylem Total
(%) (no.) (min) ingestion ingestion ingestion
(min) (min) (min)


Oil cone.
10 91 a 34.5 a 5.6 e 28.7 a 34.3 b
5 58 b 27.5 b 12.8 d 23.6 b 36.4 b
2.5 35 c 22.2 c 22.7 c 15.6 c 38.4 b
0 (acetone only) 19 d 13.9 d 45.4 a 9.7 d 55.1 a

Means wihin a column followed by the same letter are not significantly different [P< .05;
Duncan's (1951) multiple range test].
a Average of 10 replications, each replication using a new plant and a new insect.
b All the neem oil concentrations were prepared in acetone.


Jan. & June 1985









Philippine Phytopathology


1.25 25 5 10 1.25 2.5 5
Concentration (%) Concentration (%)

Fig. 4. Effects of different neem oil concentrations on
IRRI, 1984.


men tip touching the leaf surface.
Neem oil as a background odor also
disrupted N. virescens feeding. The
waveform patterns recorded during in-
sect's feeding showed that all events,
such as probing frequency, duration of
salivation, phloem feeding, xylem drink-
ing, and the total ingestion period, were
significantly (P<0.05, DMRT) different
from controls when >6% neem oil
solution was applied to filter paper disk
kept in the background of the rice plant
(Table 2, Fig. 5). Phloem feeding de-
creased progressively with increased con-
centration of neem oil. The decrease
in phloem feeding was accompanied by
a corresponding significant (P<0.05,


1252.5 5 10
Concentration (%)








5)



10


feeding activity of N. virescens,


DMRT) increase in the frequency of
probing, the salivation period, and xylem
ingestion in all treatments with >6%
neem oil. In spite of increased xylem
ingestion, the total intake by the insect
was significantly (P<0.05, DMRT) less
from plants with filter paper disks treated
with >6% neem oil than that from con-
trol plants.
Application of neem cake reduced the
seedling infection by both RTBV and
RTSV particles (Table 3). Ninety per cent
of the untreated control seedlings had
RTBV particles, but significantly
P <.05, DMRT) less infection occurred
in seedlings applied with > 75 kg neem
cake/ha or 0.75 kg ai Furadan/ha. The


VoL 21









ivatives for Preventin


Table 2. Means of various events in feeding
of different concentrations of net

Oil cone. b Probes Salivatic
(%) (no.) (min)



25 125 a 30.9;
12 116a 28.1
6 77 b 23.01
3 47 c 15.4
0 (1.66% Teepol) 43 c 14.5
0 (Paraffin oil) 41 c 13.5,


Means within a column followed by the
Duncan (1951) multiple range test]
a Average of 10 replications, each replication us
monitored for 180 min.
bAll neem oil concentrations were prepared in a,




P Probe
S Solivatlon
PI Phloem Ingestion
XI Xylem Ingestion
H 40 seconds p p P
(Rightto Left) S














nnl /l 7F/n nP nil RR OR

Fig. 5. Electronically recorded waveform
with background stimulus of (a)
"d /dl I u I 5M "Dom nil TRP IIOR,


s ofN. virescens on rice plants in presence
I as a background stimulus.a IRRI, 1984.

Phloem Xylem Total
ingestion ingestion ingestion
(min) (min) (min)


2.7 e 31.3 a 33.9 c
8.7 d 22.2 b 30.9 c
18.7 c 19.2 c 37.9 c
40.4 b 10.9 c 51.3 b
51.8 a 9.4 c 61.3 a
47.5 ab 9.1 c 56.6 ab


letter are not significantly different [P < 0.05;

new plant and a new insect;feeding activity was

is 1.66% Teepol solution.






)Sp

ssP. P PPSP P

















% 'Teepol' (b) paraffin oil (c)6%, neem oil


ann A llnn 19S









Philinnine Phvtnnnthnlnav


Table 3. Per cent infection with rice tur
spherical virus (RTSV) particles <
with neem cake or Furadan granu


Neem cake
kg/ha RTBV


150 42 c
150 42 c
75 65 b
50 85 ab
25 75 ab
0 (untreated) 90a
Furadan granules 22 c
(0.75 kg ai/ha)


In a column, means followed by a common
the Duncan's multiple range test.
a Average of 4 replications.

per cent infection of RTBV in Furadan-
treated seedlings was comparable to that
in seedling treated with 150 or 250 kg
neem cake/ha. Likewise, infection of
seedlings with RTSV alone or RTBV +
RTSV was significantly (P<0.05, DMRT)
less in treatments with 150 and 250 kg
neem cake/ha as compared to that in
untreated control seedlings. The pro-
tection against infection by virus par-
ticles was comparable to that conferred
by Furadan treatment.
The waveform patterns for N. vires-
cens feeding on rice plants grown in
soil incorporated with 250 kg neem
cake/ha (Table 4, Fig. 6) was significantly
different from those on the control
plants. Phloem feeding decreased signi-
ficantly (P<0.01, t test) with a signi-
ficant (P<0.01, t test) increase in xylem
ingestion on neem cake-treated plants.
However, there was no increase in the
frequency of probing and duration of


bacilliform virus (RTBV) and rice tungro
N1 rice seedlings grown in soil incorporated
a IRRI, 1984.


:r cent seedling infected with
RTSV (RTBV + RTSV)


30 cd 28 bcd
32 bcd 28 bcd
62 a 45 abc
48 abcd 43 abc
55 abc 50 ab
58 ab 58 a
22d 13d



er are not significantly different at 5% level by




salivation period on neem cake-treated
plants.


DISCUSSION

Nitrogenous fertilizers and pesticides
are essential but costly agrochemical
inputs for obtaining high yields in
modem rice varieties. Although applica-
tion of high rates of N fertilizer is known
to favor pest build up, a significant
reduction in fertilizer use cannot be
envisaged without compromising yield.
Pesticides are also effective in increasing
gross returns from the rice produced;
however, net returns from their use are
not as predictable, particularly during
the wet cropping season.
The average rice farmer in developing
countries of Asia is chronically short of
investible capital and is reluctant to un-
dertake the risk of costly insecticide


rr-l rr









vitivec for Preventin<


Table 4. Means of various events in feeding
control rice plants during 180 min,

Neem cake Probes Sal
(no.) (mir


250 kg/ha 19.2
control (untreated) 21.1ns

ns = not significant; ** = significant at 0.01 1
a Average of 15 replications, each replication usir

O


P PPs
PP S SP




P, I



P Probe
S -Salivation
Pi-Phloem ingestion
Xi-Xylem ingestion
H 40 seconds
(Righttoleft)
Xi





Fig. 6. Electronically recorded waveform,
TN1 plants (control) and b TN1 I
neem cake/ha. IRRI, 1985.

applications. These farmers either spend
little on insecticides or resort to under-
losing (Litsinger et. al., 1980). As the cost
of synthetic commercial insecticides pro-
gressively rise, it is feared that their use
will decline further, resulting in lower
yields. In a study of farm-level constraints


its ofN. virescens on neem cake-treated or
IRRI, 1984.

on Phloem Xylem Total
indigestion ingestion
(min) (min)

1.4 32.4
ns 42.0** 11.2**


of probability by t test.
Lew plant and a new insect.



pp S
S P
P







P PP
P
PP S "
p p Ss S S

S S Xi







ring N. virescens feeding on a untreated
s grown in soil incorporated with 250 kg


to high yields in rice in Asia, Herdt
(1979) concluded that the amount of rice
being lost to insects is substantial, but the
current pest control technology is too
costly. Identification and evaluation of
low-cost, effective, and locally available
substitutes for crop protection is. there-


Ian. & June 1985









Ihilippine PhytopatholoV 2


fore, urgently needed.
Although N. virescens is still con-
trolled effectively with insecticides in the
tropics (Heinrichs, 1979), Nephotettix
cincticeps Uhler, a closely-related pest
species in Japan, became resistant to
organophosphates (Kojima et al.. 1963;
Iwata and Hama, 1971; Kiritani, 1972)
and carbamate insecticides over a relative-
ly short span of time (Asakawa and Kaza-
no, 1976). Only a few insecticides are now
effective against N. cincticeps because it
has developed cross-resistance. Also, these
insecticides are toxic to the pest's natural
enemies, making integrated control dif-
ficult (Kiritani, 1976). Extensive use of
insecticides in the tropics may also lead
to the development of insecticide resist-
ance in N. virescens.
The likelihood of an insect pest de-
veloping resistance against plant deriva-
tives comprising an array of chemicals
causing diverse behavioral and physiolo-
gical effects is much less than when in-
secticides based on single chemical ac-
tive ingredient are used. Insect control
action by neem derivatives has been at-
tributed to a number of novel chemical
compounds (Jacobson, 1981; Kraus et.
at, 1981; Morgan, 1981).
The feeding response of an insect
after it has alighted on a plant involves
the initial feeding response, and the
continued feeding response (Beck, 1965;
Dethier, 1966). The initial feeding res-
ponse of leafhoppers is defined as probing
(application of proboscis to the plant),
salivation, and insertion of its stylets into
the food substrate. Duration of feeding
from the target tissue and food intake
defines the continued feeding response.
Neem oil and neem cake had a pro-
found effect on the feeding activity of
N. virescens. We observed that neem oil,
when sprayed on rice plants, or in an
arena with rice plants, changed the in-
sect's feeding behavior. These changes in
the feeding activity of N. virescens were
comparable to these associated with its


feeding behavior on resistant rice varie-
ties. The insect is primarily a phloem
feeder on susceptible varieties, but
switches over to xylem feeding on resist-
ant varieties (Auclair et al., 1982, Khan
and Saxena, 1984a). Increased probing
frequency also has been observed during
N. virescens feeding on resistant rice
varieties (Rezaul Karim, 1978).
Similar shift in the insect's feeding
behavior was also observed on rice plants
grown in soil incorporated with 250 kg
neem cake/ha; increase in the frequency
of probing and duration of salivation
were, however, not recorded. This effect
indicates that, even after expulsion of
oil from seeds, antifeedant principals
persisted in neem cake and were trans-
located into the rice plant from treated
soil. Gill and Lewis (1971) reported
systemic antifeedant action of three neem
products azadirachtinn [a tetracyclic tri-
terpenoid an ethanolic extract from
neem seeds, and an aqueous suspension of
ground seed kernel) against the desert
locust, Schistocerca gregaria Forskal,
which caused only slight damage to bean
plants grown in treated soil. Bean seed-
lings grown from seeds soaked for 24 h
in a solution of 0.01% azadirachtin, 0.1%
alcohol extract, or 0.1% aqueous kernel
extract were also protected against feed-
ing damage by S. gregaria adults for 1
week after germination.
A possible reason for the shift of N.
virescens feeding from phloem to xylem
on rice plants treated with neem oil or
neem cake, or kept in an arena permeated
with need oil odor, could be the harmful
effects of neem allelochemics, which may
enter the insect body during feeding or
may be perceived by the insect olfactory
system. The insect increased drinking
from xylem vessels on plants treated with
neem derivatives or kept in an arena per-
meated with neem oil odor may be due to
the insect's efforts to excrete harmful
chemicals from the body. In hemipterans,
drinking helps maintain a sufficiently


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