Front Cover
 Front Matter
 Abstracts of papers presented at...
 Gray leafspot of tomato and pepper...
 Detection and partial characterization...
 The influence of rust control on...
 Effects of meloidogyne incognita...
 Growth and sporulation of pseudocercospora...
 Effect of four nematicides on nematode...
 Properties of a virus causing mosaic...
 Note: Powdery mildew of tomato...
 Detection of several Philippine...
 Back Cover

Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00090520/00023
 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 1980
Frequency: semiannual
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
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: VID00023
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
    Front Matter
        Front Matter 1
        Front Matter 2
    Abstracts of papers presented at the seventeenth annual meeting of the Philippine phytopathological society inc., Cebu City, 23-26 April, 1980
        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
    Gray leafspot of tomato and pepper caused by stemphylium iycopersici (Ejoji) yamamoto
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
    Detection and partial characterization of a downy mildew spore germination inhibitor in corn leaf extracts
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
    The influence of rust control on the yield of a soybean
        Page 38
        Page 39
        Page 40
        Page 41
    Effects of meloidogyne incognita and rotylenchus reniforms on grapes
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
    Growth and sporulation of pseudocercospora psophocarpi (Yen) deighton at different media, temperature and pH
        Page 49
        Page 50
        Page 51
        Page 52
    Effect of four nematicides on nematode control and yiel of cabbage
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    Properties of a virus causing mosaic in elephantopus mollis BHK
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
    Note: Powdery mildew of tomato caused by erysiphe cichoracearum DC. ex. Mecat
        Page 67
        Page 68
        Page 69
        Page 70
    Detection of several Philippine plant viruses by the counterimmunoelectrophoretic assay
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
    Back Cover
        Page 97
        Page 98
Full Text

Ill I n l

-i" A '

= mmH m mm m n N m m I n m


w 1OwY W


racts of Paoer

isnot~ of

Detection and Partial Characterization of a Downy Mildew Spore Germi-
nation Inhibitor in Corn Leaf Extracts H.G. Buenaflor and E.M.T.
Mendoza ..... ................................... 27-37

The Influence of Rust Control on the Yield of Soybean A. L. Piamonte and
F. C. Quebral ...................................... 38-41

Effects of Meloidogyne incognita and Rotylenchus reniformis on Grapes -
R. Sarra-Guzman and R. G. Davide .................... 42-48

Growth and Sporulation of Pseudocercospora psophocarpi (Yen) Deighton
at Different Media, Temperature and pH A. R. Pua and
L. L. lag ......................................... 49-52

Effect of Four Nematicides on Nematode Control and Yield of Cabbage -
R. G. Davide and R.A. Zorilla ........................... 53-58

Properties of a Virus Causing Mosaic in Elephantopus mollis BHK L. T.
Talens and A. D. Talens ................... ............. 59-66

NOTE: Powdery mildew of Tomato Caused by Erysiphe cichoracearum
DC. ex. Mecat T.H. Quimio & M.M. Nadayao ............... 67-70

Detection of Several Philippine Plant Viruses by the Counterimmunoelectro-
phoretic Assay L. T. Talens and A. D. Talens ............... 71-95




President -
Vice-President -
Secretary -
Treasurer -
Auditor -
Ex-Officio -
Board Members -


American Cyanamid Company, Princeto

Bayer Philippines, Inc., 622 Shaw Blvd.,

Canlubang Sugar Estate, Canlubang, Lagl

Hijo Plantation, Inc., Tagum, Davao del

Schering AG/Berlin, West Germany (Agr
855 Pasay Road, Makati, M(

DRS 1980-81



lew Jersey, USA.

idaluyong, Metro Manila


emical Division) 5th Floor, Evekal Bldg.,

Victorias Milling Co., Inc. Victoria, Negros Occidental



Official Organ of the Philippine Phytopathological Society, Inc.

TRICITA H. QUIMIO, Editor-in-Chief, Department of Plant Pathology
UPLB, College, Laguna
S. C. DALMACIO, Associate Editor, UPLB, College, Laguna
M. B. CASTILLO, Associate Editor, UPLB, College, Laguna
C. A. BANIQUED, Business Manager, BPI, Manila

/ -,

SOCIETY, CEBU CITY, 23-26 April,

Detection of several Philippine plant Optimum temperature for growth
viruses by the counter-immunoelectro- was found to be 10-30C, no growth
phoretic assay. L. T. Talens and A. C. occurring at 5 C and beyond 35 C.
Dolores-Talens, IPB. It was found to grow fast in the dark
and also under intermittent light-dark
Counter-immunoelectrophoretic assay combinations but was induced to pro-
(CIEPA) was successfully used to detect duce more spores utilizing UV light.
six Philippine plant viruses infecting Spore germination was set at 10 and
leguminous plants cowpeaa and mung- 35 C, with infection in rice plants going
bean), solanaceous plants (pepper, tobac- as high as 93-94% at 30-35 C.
co and tomato) and orchids. It was RH at 98.7 to 100% at 20-35 C fa-
demonstrated to be a specific, sensitive, vored conidial germination. The same
reliable, rapid and simple technique for was, however, inhibited at all tempera-
the routine diagnosis of these viruses, tures tried with RH set below 98.7%.
Likewise, CIEPA was useful in the diag- Moreover, conidia failed to germinate at
nosis of virus diseases with single or any RH at temperatures of 5-15 C and
mixed infections in a 30-minute single- 40 C.
step operation. Fusarium moniliforme survived on
The technique was capable of identi- seeds of IR42 and IR38 at 20 C seven
fying three morphological types of plant months after storage. It also survived at
viruses, namely: the isometric particles 20-25 and 30 C on seeds of IR 5929-13-3.
of cowpea mosaic virus, mungbean No survival occurred on seeds of IR
mottle virus, and southern bean mosaic 9846-215-3 and IR 9703-41-3 at tempe-
virus; the rigid rod-shaped particles of ratures of 20-35 C.
the orchid and yellow strains of tobacco The minimum inoculation level re-
mosaic virus; and the semi-flexuous rod- quired to cause "bakanae" infection
shaped particles of Cymbidium mosaic was 1/2:10 (v/v), infection increasing
virus, at 1/2:10 to 3:10 but declining at 4:1C
and 5:10. It was noted to survive foi
Studies on the etiology, epidemiology 155 days in soil in a number considerably
and control of the bakanae disease of reduced to cause infection in seedlings.
rice in the Philippines. F. L. Nuque, The following were found resistant
T. Vergel de Dios, L. M. Sanchez, J. M. to at least 33 "bakanae" isolates: IR20
Bandong and J. P. Crill, IRRI. IR26, IR32, IR38, IR44 and IR45
IR32 and IR44 were the most resistant
Bakanae, an important rice disease to all isolates.
in the Philippines, caused by Gibberella Studies on pathogenic races of F. mo
fujikuroi (Fusarium monififorme), was niliforme yielded six race groups of whicl
studied. isolate F2 was the most virulent.

Philippine Phytopathology

Benomyl 50% WP at 5-10 g/kg seed,
applied either in dry or slurry form re-
duced bakanae incidence. Benomyl and
Homai at 20 and 40 g/kg seed applied
as seed treatment effectively controlled
bakanae. Soaking of seedlings in Beno-
myl suspension of 1.0 g/liter for 4 hours
also controlled the disease. Soaking
naturally-infested IR40 seeds in Benomyl
suspension at 1, 2 and 4 g/liter suspen-
sion for 18 and 48 hours considerably
reduced disease incidence.

Evaluation of some sugarcane cultural
practices on parasitic nematode popula-
tion and sugarcane yield. F. T. Gar-
gantiel and F. C. Barredo, VICMICO.

Some of the cultural practices used
in this experiment were found to reduce
parasitic nematode population to within
tolerable levels. Burning of trash, inter-
cropping with mungbean, and trash far-
ming affected nematode population.
However, it was only with the inter-
action of trash burning and intercrop-
ping that significant increase in number
of millable stalks, tonnage and sugar
yield were attained.
Seven genera of plant parasitic nema-
tode were observed. Pratylenchus was
the most prevalent followed by Helico-
tylenchus, Hoplolaimus, Xiphinema,
Tylenchorhynchus, Ciconemoides and

Variation in chromosome number and its
implications to virulence and pathogeni-
city of the rice blast fungus. K.V.S.R.
Kameswar Row, J. P. Crill and F. L.
Nuque, IRRI.

The inheritance of blast resistance
was studied using four blast isolates
(750778, T72, T63 and 2017 belonging
to three race groups) and five rice varie-

ties (Tetep, Carreon, IR36, Sensho and
KTH-17). Isolate T-63 exhibited a consis-
tent, resistant or susceptible reaction on
the varieties and was considered an un-
stable isolate.
The process of reproduction in the rice
blast fungus Pyricularia oryzae was found
to be by atypical mitosis. A variation
in the number of chromosome from 2-12
was observed in hyphal, conidiophore,
and conidium cells. The most frequently
observed were those with three and six
chromosomes. Changes in chromosome
number probably result in duplication or
deletion of genes for pathogenicity or
The segregating populations of rice
evaluated for resistance with an unstable
isolate could not be fitted into any
Mendelian ratio as could be done with a
stable isolate. Such variable behavior
among the isolates was explained based
on the usual cytological processes ob-
served inP. oryzae.

Properties of a virus causing mosaic in
Elephantophus mollis Bhk. L. T.
Talens and A. C. Dolores-Talens, IPB.

The viral agent causing mosaic in
Elephantophus mollis was investigated.
E. mollis mosaic virus was found to be
mechanically transmissible to Musa tex-
tilis, Capsicum annuum, several species
of Nicotiana, Tetragonia expansa and
three species of Chenopodium. Cheno-
podum amaranticolor, C. murale and
C. quinoa were local lesion host plants.
The symptoms incited by E. mollis
mosaic virus infection in N. ghttinosa
were diagnostic.
The properties of the virus in crude
sap were found as follows: dilution
end-point at 10'2; thermal death point
at 50-55C; and longevity in vitro was 3
hours. Electron microscopy of negatively-

Vol. 16

Abstracts of Papea

stained specimen preparations showed
isometric particles 30 nm in diameter.
The successful transmission of
E. mollis mosaic virus into abaca provides
the first direct experimental evidence of
an unstable, isometric virus causing
mosaic in this important export fiber
plant of the Philippines. This virus might
prove to be the answer to the long-
standing puzzle regarding the viral etio-
logy of the abaca mosaic disease in the

Notes on powdery mildews of weeds
hitherto unreported in the Philippines. -
A. S. Garcia.

The occurrence of powdery mildew.
on 11 weed species and 16 other cultiva
ted crops were observed and reported
for the first time in Mindanao, Philip
The powdery mildew infecting Casik
tora Linn., also infected mungbean
producing the typical symptoms of the
Examinations of the infected plan
parts seem to indicate that there are tw(
groups of powdery mildews occurring
in the weed species collected and culti
vated crops observed based on the plan
parts affected and the manner of tho
cutting of the spores.

Occurrence and identity of tobacco mo
saic virus and Cymbidium Mosaic Vimn
infections in orchids. N. Chansilpa ani
L. T. Talens, IPB.

The occurrence and identity of to
bacco mosaic virus (TMV-O) and Cymbi
dium mosaic virus (CYMV) infections ii
Philippine orchids was investigated. Inde
xing of virus infections by serological tes
of 117 orchids belonging to 16 genera in

dicated that 36.7% of the samples tested
were infected singly or mixedly with
Virus infections were not detected in
Aeridis, Arundina, Ascocenda, Bulbophy-
llum, Epidendmm, Renanthera, Spatho-
glottis, Trichoglottis and Vanda. Of 43
orchids which were found to be infec-
ted with viruses, 6.9%was due to TMV-O
infections alone, 58.2% was due to
CYMV, and 34.9% was due to mixed
TMV-O and CYMV infections.
Cattleya and its related genera accoun-
ted for 41.9% of those infected, followed
by Dendrobium (20.9%), Oncidium
(13.9%), Aranda (11.6%), Cymbidium
(4.7%), Arachis (2.3%), Phalaenopsis
(2.3%) and Vandopsis (2.3%). Many of
the infected orchids were of commercial
value and were noted to be imported
from foreign countries.

Unreported virus disease of bamboo
(Dendrocalamus latflorus (Munro) Mc
Clre) in the Philippines and proof of it
transmission. L. V. Magnaye and F. R
Celebrar, BPI.

A viral disease was recorded for the
first time in the Philippines on intro
duced "Machiku" bamboo originating
from Taiwan. Some of the observed cha
racteristic symptoms induced on diseased
bamboo plants are: mosaic-like patter
on leaves, necrotic streaks on culms, vas
cular discoloration, hardening anc
apparent dehydration of shoots resulting
to its poor quality and eventual shorten
ing of internodes.
The virus is mechanically transmitted
to bamboo (D. latiflorus) as well as tc
corn (Zea mays L.) and aguingay (Rott
boelia exaltata Linn.)
Results of electron microscopic exa
mination based on the leaf-dip prepare
tions by Dr. Dante A. Benigno of thi

Jan-June 1981

I'hilipp Phytopathoogy

Department of Plant Pathology, Univer-
sity of the Philippines at Los Baflos re-
vealed the association of flexuous rod
particles with mosaic.

Properties of a vims causing mosaic in
cucurbits. L. T. Talens, IPB.

A virus isolated from field-grown
cucurbits at the University of the Philip-
pines at Los Baflos experiment station
incited severe mosaic, dark green blisters,
and pronounced leaf distortion and
deformation in cucumber, muskmelon,
squash on inoculated leaves of Cheno-
podium amaranticolor. No symptoms
developed nor was virus recovered from
inoculated Nicotiana glutinosa, N. ta-
bacum cvs Samsun and White Burley,
Petunia hybrida, and Vigna unquiculata
cvs Red and All Season.
The physical properties of the virus
were as follows: infectivity was retained
after dilution to 10'2 but not to 10"3;
infectivity was not abolished after heating
for 10 min at 55 C, but at 60 C; and
infectivity was retained after storage for
6-9 days at 25 C.
Electron microscopy of negatively-
stained specimen preparations revealed
flexuous rod-shaped particles 700-750
nm in length. Based on these properties,
the virus isolate was identified as water-
melon mosaic virus-2. Positive diagnosis
by serology was not made due to lack
of reference antiserum.

Further studies on the properties of a
nmoio.cauing vim in wild passion
vine (Pasflora feetida L.). L. T.
Talens and LolitaP. Martinez.

The viral etiology of a disease in wild
passion vine (Passiflom foetida L.) exhi-
biting mosaic, yellow spotting, leaf dis-
tortion and formation, and reduction

in leaf size and growth was investigated.
The properties of the virus in crude sap
were found to be as follows: thermal
inactivation point, 65-70 C; dilution
end-point, 104; and longevity in vitro,
1 day at 25 C.
The virus was mechanically transmis-
sible to P. edulis, Nicotiana benthamania,
and Chenopodium amaranticolor, and
C. quinoa, the two Chenopodium species
being local lesion host. Plant species
belonging to the families Aizoceae,
Compositae, Graminae, and Leguminosae
were not susceptible to the virus nor
was virus recovered from symptomless
Electron microscopy of negatively-
stained specimen preparations revealed
rigid rod-shaped particles. Serological
tests showed no reaction when antigen
preparations were tested against several
reference TMV-antisera.

Survey and identification of diseases
affecting coffee in Mindano. A. S.

From 1976 to 1979 a series of field
trips to the different provinces in Minda-
nao was carried out to survey and identi-
fy diseases affecting the growth of
Robusta coffee planted in commercial
scale. Actual recording and diagnosis
were done from the eight selected Robus-
ta coffee plantations. These are located
in the provinces of Davao Oriental,
Agusan del Norte, Misamis Oriental,
Bukidnon and Davao City.
There are six diseases of Robusta
Coffee with economic importance. These
are the anthracnose, pink disease, algal
spot, thread blight, rust and eye spot.
The symptoms, distribution and
extent of infection are presented and

VoL 16

Abstracts of Papem

Occurrence of mungbean scab in the
Philippines. L. L. lag and V. M. Marfil,

A mungbean disease identical to the
scab disease reported in 1976 in Indo-
nesia was observed to occur in Bukid-
non, Isabela, Pampanga and the Central
Experiment Station of U.P. at Los
Baflos, College, Laguna.
The symptoms include stunting, leaf
curling, and a shothole effect on the
leaves. Leaf lesions are brown to reddish
brown with the centers of old lesions
turning gray or grayish white. Stem
lesions are round to elliptical, somewhat
raised, with reddish brown margins and
grayish white centers. Lesions in imma-
ture pods start as sunken elliptical or
irregular dark brown areas with gray
centers. As the pod matures the lesions
become raised and appear grayish.
The pathogen is Sphaceloma sp.
Maximum sporulation was observed in
mungbean leaf extract-oatmeal agar
(pH 8) after 8 days of incubation at 20 C.
All the 14 mungbean varieties that
were screened for resistance to scab
were found susceptible.

Economic hosts of Pseudomonas sola-
naceaum EFS isolates from abaca. -
A. R. Rillo.

Eleven economic plants belonging to
five families were artificially and natu-
rally inoculated with P. solanacearum
isolates from abaca.
Potato, castor bean, eggplant, tobac-
co, tomato, diploid banana, abaca and
heliconia were infected upon artificial
inoculation with the organism. Under
natural conditions, however, only castor
bean, tomato, and eggplant were infected.
The isolates failed to infect abaca
under natural conditions, suggesting the

possible role of biotic or abiotic agents
in disease occurrence.
The results of the study showed that
the organism exists as a variable species
with various pathogenic potential in

Sweet potato tuber rot disease. M. K.
Palomar, A. D. Solis and H. S. Bandala,

Tuber rot or Java black rot, caused
by Diplodia theobromae, is the most
prevalent storage disease of sweet potato.
Affected tubers become dry, hard and
coal-black in color. Isolations made
from stored sweet potato revealed a high
incidence (71%) of D. theobromae
alone and in combination with other
fungi. The occurrence of Asperglus,
Rhizopus and Fusarium in tubers was
D. theobromae was artificially ino-
culated to tubers from 169 varieties/
accessions of sweet potato. Results
showed that 78 were resistant, 13 were
moderately resistant and 78 were suscept-
ible to tuber rot. BNAS 51 was used as
susceptible check throughout the expe-

Bacterial foot rot of rice. T. W. Mew
and M. B. Rosario, IRRI.

Present study indicated that the di-
sease is caused by a bacterium very
similar to Erwinia chrysanthemi causing
bacterial corn stalk rot. Isolates from
both rice and corn incited typical stalk
rot on both crops by methods of cross-
inoculation. They were also similar in
general bacteriological characteristics to
E. chrysanthemi. Four colony types
were distinguished on PSA medium.
A distinct blue pigment was produced
by the corn isolates on slides of corn

Jan-June 1980

ina -, VrLn mtlMna

stalks at 20 C but not above. The blue were separately inoculated by spraying
pigmentation may be a specific charac- to nine new Japanese differential varieties
teristic of the corn isolates. Other tests, with specific genes for resistance. Record-
however, including colony morphology ing of reactions was made 1 week after
on crystal violet pectate medium, growth inoculation using a 0 to 4 scale, with 0
at 36 C, alpha-methyl glucoside and as immune and 4 as susceptible. Forty
phosphatase reactions all indicated that one races were identified, based on the
the rice isolates from the Philippines were reaction of the differentials. Race 102
similar to those isolated from rice in was the most prevalent with 12 isolates
Japan and Korea, and those from followed by race 100 and race 103 with
E. chrysanthemi of corn stalk rot. 8 and 7 isolates, respectively. Five races
of P. oryzae races 003, 103, 107,
137 and 303, were identical to those
Race identification of Cereospor oryzae. reported in Japan.
- B. A. Estrada, L. M. Sanchez, F. L.
Nuque and J. P. Crill, IRRI.

Four hundred thirty-seven varieties Interaction between rice (Oryza satim)
Four hundred thirty-seven varieties
and IRRI breeding lines artificially ino- and cowpea (Vig unquiculta) and
Xenthomonas oryzae. K. Tsuchiya,
culated with C oryzae in the field were K.
T. W. Mew and S. Wakimoto, IRRI.
evaluated during the 1978 dry season.
Following natural field infection, disease
reactions were evaluated, then 49 varie-
ties and breeding lines were selected for Lesons of bacte b on
further studies in the greenhouses. From leaves initiated by virulent strains were
the reactions of these different varieties reduced by mixing or pre-infection with
inoculated with various isolates obtained an avirulent or weakly virulent mutant
from IRRI, Boilo and Pangasinan, eight strain. The inhibitory effect was not ob-
varieties were selected to comprise a served on rice leaves infected with a
tentative set of narrow brown leaf spot virulent prior to an avirulent strain. No
differential varieties. These are IR8, hypersensitive reaction (HR) was in-
Zenith, IR20, Delitus, IR26, Southern duced on rice leaves of a resistant variety
Red Rice, IR9129-159-3-2-3-3 and MI infected with either avirulent or weakly
273. virulent strains of X. oryzae, or in an
Preliminary results indicated the pre- incompatible rice-bacterium combination.
sence of six race groups composed of HR on cowpea was induced by virulent
16 races with CA and CC as the most strains exemplified by different virulence
predominant races. groups and virulent induced mutants.
Only living cells of X. oryzae at 2-5
x 106 cells/ml induced by avirulent
Pathogenic races of Pyrkicuari oryzae strains on cowpea was either delayed or
identified by Japanese differential with not induced at all. None of the heat-
specific R-genes. S. P. Ebron, F. L. killed bacteria, cell-free supernatant, or
Nuque and J. P.Crill, IRRI. polysaccharide fraction prepared from
bacterial cultures of virulent strains
~f\ rhl*jt tA iml4Jta. a F P jMn 4iAirA A lt

VAI itf

Ahma eft nf PMaTm

which are infected with tungro. Results had titers of 1:1024 for TMV-U and
of 65 randomized sampling on the 1:4096 to 1:8192 for CMYV. Compa-

Isification in 10% chloroform (v/v)
low speed (5,000 rpm for 15 mi-
*s) centrifugation, precipitation with

atically infected leaves as source of cow-
pea mosaic virus inoculum. With two
flexuous rod-shaped viruses whose identi-
tv was not established. Chenonodium


v- ..--. non

biological purification of each virus iso- between 8, 16 and 24 hours IAT. Posi-
late was attained by single lesion isolation tive transmission was obtained at 10 and
in appropriate local lesion host. 15 min. AAT; however, the acquisition
access time which showed serial trans-
mission was at 30 minutes.

Studies on the capacity of N. lugens
to transmit rice ragged stunt disease on
rice plants. 1. Effect of different acqui- Search for other insect vectors of rice
sition and inoculation access times on ragged stunt disease. V. M. Aguiero,
percentage infective insect. R. D. R. D. Daquioag and K. C. Ling, IRRI.
Daquioag and V. M. Aguiero, IRRI.

Possible insect vectors of ragged
Virus-free second instar nymphs were stunt disease of rice, such as Nepho-
used in this study. Rice ragged stunt- tettix nigropictus, N. virescens, Recilia

as virus source, and 5- to 7-day old TN1 tested by using daily serial transmiss

ADtractU 01 rapeOs

Meteorological conditions as they in-
fluence the natural occurrence and dis-
tribution of legume diseases. N. S. Franje
and E. E. Danlag, CMU.

The effect of meteorological condi-
tions on the occurrence and distribution
of legume diseases (viz. anthracnose,
Cercospora leaf spot, powdery mildew,
rust and Rhizoctonia) is very apparent.
Although the optimum relative humi-
dity needed for maximum disease pro-
duction was not attained, it was evident
that the average relative humidity, air
temperature, evaporation, rainfall and
others taken prior to disease rating con-
tributed to the degree of infection on
different legumes.
The concentration of airborne coni-
dia, on the other hand, had been ob-
served to influence the degree of infec-
tion as shown by the number of spores
trapped taken eight days before the
rating schedule.

Ecology of the rice sheath blight patho-
gens: parasitic survival. T. W. Mew,
N. G. Fabellar and F. A. Elazegui, IRRI.

weVus. raimugoemiy siL snuwcu uaLI uLr
weed isolates were highly virulent on
rice. It is obvious that these weeds are
not only hosts of the fungus but also
serve as inoculum source to infect rice.

Ecology of the rice sheath blight patho-
gen: saprophytic survival. T. W. Mew,
A. M. Rosales and F. A. Elazegui, IRRI.

The saprophytic survival of Thanete-
phorus cucumeris was further studied by
burying healthy rice straw pieces (2-3 cm
apart from interode) in unsterilized soil
of dryland origin. The soil was mixed
with different amounts of inoculum and
incubated for one month under con-
dition of submergence, with 100%
moisture and condition with 50% moist-
ure saturation. A lower percentage of
rice straw was colonized by the fungus
under submergence than in 50% satura-
tion. When infected rice straw pieces
were buried in soil of dryland origin,
recovery of the fungus was lower in
unsterilized than in sterilized soil. In
unsterilized soil, at 50% moisture satu-
ration, a higher percentage of the straw
was colonized not only by the fungal

culum was broadcast into tne neia, ootn "" '. -- '"" "--"" "" "-""-. U""~*
rice and weeds became infected. Many pathogen and finally by the sheath blight
weed species were found as susceptible pathogen alone.
as rice. A survey of IRRI farm indicated
that many weeds in the rice fields of both
wetland and dryland, in the graminae Screening for bacterial blight resistance
family were naturally infected with in the Bicol region. B. A. Zaragosa,
Thanatephorus cucumeris (Frank) Donk. R. C. Reyes and T. W. Mew, IRRI.
The symptoms on the weed hosts were
identical to those on rice. In fallow fields During the wet season 1979, two sets
under wetland conditions, sclerotial of screening tests for resistance to bacte-
bodies and sometimes the basidial stage rial blight were made at the Bicol Rice
of the fungus could be observed on the and Corn Experiment Station at Pili,

Ja--June lYP

PhlUnninA Phvtnnathn lon

Camarines Sur using inoculum from emas group varieties were susceptible to
laboratory cultures and that from natu- types I to IV, but only Zenith and
rally infected leaves. One set, consisting Nigeria 5 were resistant to types III and
of 1217 IRRI materials from the General I, respectively. Ten varieties of Wase
Screening Nursery and the International Aikoku group, two of them, Shin 2 and
Rice Observational Nursery was inocu- Lead Rice were susceptible to all the
lated with pure cultures of Xanthomonas pathotypes. Six of them, Nagomasari,
oryzae from the Plant Pathology Depart- Ortiglia, Chukei 314, Kuntlan, Chugoku
ment of the International Rice Research 45 and 70X46, were resistant to all
Institute. Results showed 27.5% suscept- types. TKM 6 was resistant to type I
ible; 14.6% moderately susceptible; but susceptible to the other three, while
17.6% moderately resistant and 40.4% Nakashin 120 was resistant to types I,
resistant. In the other test using 126 II and III but susceptible to IV. Three
entries from Cooperative Irrigated low- varieties, Jamica, Zenith G713 and
land rice performance tests of the Phi- Himekei 16 belonging to Java group
lippine Seedboard, artificial inoculation showed resistant reactions to the four
was done using suspension from sup- types. Amareriyo of this varietal group
nosedlv diseased cut leaves from blight was susceptible to all the types, though

Vnl. 16

Jma-Juma 1980 Absbascof PpS

carrying no gene for resistance in Japan, on the basis of this reaction, that strain
to all the four pathotypes in the Philip. of virulence group II might be heter
pines. On the other hand, Wase Aikoku genous. Subsequently, a test designed
3 having the Xa3 and Java carrying three confirm the resistance or susceptibility
genes: Xal, Xa3, and Xa-kg for resistance Cas 209 to Group II strains demonsti
in Japan were resistant to the four Phi- ted that the group could be fourth
lippine pathotypes. classified into two groups based
IRRI differentials, however, showed their virulence to Cas 209 in addition
different reactions to Japanese patho- their reaction on other varieties of tl
types. IR8, carrying no functional gene differentials. Among 16 of Group
for resistance in the Philippines, was strains evaluated, 5 of them were virule
resistant to pathotypes II, III and V in to Cas 209 and 11 were avirulent. C
Japan. IR20, conditioned by a domi- 209, therefore, serves as a distinct varie
nant gene Xa4 for resistance was re- to differentiate Group II strains. Wh
sistant to pathotypes I and V, but sus- it is susceptible to Groups I and I
ceptible to pathotypes II, III and IV. strains, it is completely resistant to sor
IR1545-339 and DV85 conveying their and susceptible to others in Group .

The results indicate that rice diff
rentials developed in Japan and at IRI
are individually very distinct in the
reactions to the pathotypes in Japan as
the Philippines. Likewise, the five path
types in Japan and four in the Phili
pines were individually distinct in vir
lence to the combined set of rice diff

CAS 209 A new differential rice varie
for virulence of Xanthomonas oryzae
T. W. Mew, C. M. Vera Cruz and R.
Reyes, IRRI.

The effort to identify rice variety
suitable for differentiation of the vir
lence of X. oryzae was continued sin
1976. Cas 209 (IRRI Ace. No. 1579
from Senegal was one of them. Cas 2(
was susceptible to strains of virulen
Groups I and III, but gave variat
reaction to Group II strains. It was st
ceptible to PX079 but resistant to PX8(
both Group H isolates. It was suspect

Reaction of four msgarcane varieties t
the root knot nematode Meloidogym
incognita Chitwood. P. E. Patayo
and N. S. Franje, CMU.

Four sugarcane varieties viz, Alunai
Phil. 56-226, Phil. 61-11 and Phil. 60-1
exhibited different reactions to tt
root-knot nematode, Meloidogyne incoj
In all the varieties, nematode inocul:
tion did not affect plant height. Tl
height of inoculated sugarcane w:
about the same as those of the none ini
culated control. Only Phil. 60-19 show
a highly significant decrease in root as
top weights at 1120 and 2016 inoculu
The nematode population increase
significantly in all inoculated varieties
The percentage increase in 1120 inoci
lum level was relatively higher than
2016 inoculum level. At 1120 inoculu
level, Phil. 61-11 increased by 50.6
followed by Alunan 50.3%, Phil. 60-1
45.0% and Phil. 56.22 40.6%. Sugarcal

iinnan Phvtnoathnonav

emed to be a favorable host for Meloi-
igyne incognita
The response of the roots of the four
garcane varieties to the penetration of
Le root knot nematode was significant.
Among the four varieties inoculated,
lil. 60-19 had the highest number of
ematodes recovered per gram of roots.
lil. 56-226 ranks second, Alunan ranks
lird and Phil. 61-11 had the lowest,
ith a mean of 7.67. In the assessment
' the parameters considered in this
udy, Phil. 60-19 appears to be a sus-
,ptible variety to the root knot nema-

study of some components of hori-
atal resistance to rice blast disease.
R. L. Villareal, W. R. Coffman, D. R.
acKenzie and R. R. Nelson, IRRI.
The study of the behavior of the rice
irieties Tetep, IR442-2-58, Gogowierie,
ourado Precose, IRAT 13 and 1021
loculated with Pyricularia oryzae iso-
tes T-9, T-27 and 78-116-2 under con-
olled environment in the phytotron
characterized the major components of
te pathogenesis of the rice blast which
tributess to the horizontal resistance
IR) in rice. These components include
orulation capacity (SC), latent period
P), disease efficiency (DE), and lesion
ze (LS).
The results showed that IRAT 13,
ogowierie, Tetep, 1021 and Dourado
recose possess different levels of HR.
ligh levels of HR were clearly demons-
.-A :- TYD AT 121 nr.A r-Anr;a T+

58, the susceptible check. Varietal
fferences in the various components
'HR were also observed.
action of Japanese field resistant
e varieties to eight races of Pyriculari
yzae. S. P. Ebron, F. L. Nuque and
P. Crill, IRRI.
Fifty-one rice varieties with field resis-
nce and specific R-genes from Japan
ere inoculated with races number 002,
10, 102, 103, 104, 106 and 107 consist-
g of 20 isolates each. The varieties dif-
red from resistant to susceptible in their
action to the different race-isolates.
irieties Sorachi, Todoro Wase, Tatsumi-
ochi, Matsumae, Ishikari, etc. were
sistant to all races with susceptibility
dex of 1.00. Varieties Ishioka 3 and
ao mochi were the most susceptible
th both susceptibility index of 3.90.
within specific race number 102, isolates
28, T-54, T-55, and T-61 induced
fferent reactions on Suzuhara-mochi.
ikuto Norin 24, Ishioka 3, Sensho and
ikoton suggesting that these isolates,
though classified in the same race
oups differ in their virulence. Within a
oup of varieties with specific R-genes
i-a, Pi-i and Pi-a, Pi-i), reactions to
fferent race isolates varied from R, M,
Id S indicating that other unknown R-
nes are involved which cannot be de-
cted by races in Japan.

,reening of rice varieties and lines for
ast resistance. C. Q. Torres, F. L.
uque and J. P. Crill, IRRI.

1. 16

Abstracts for Papers

of resistance to blast were IR533-13-1-1;
IR1416-128-5-8; IR4547-6-2-5; IR3273-
289-2-1473; IR4563-52-1-3-6; IR5929-
12-3; IR559-4-1-1; Carreon, and Tadu-
Likewise, 86 varieties with known
field resistance and R-genes in Japan
were tested twice at IRRI blast nur-
sery. Many of the field resistant varie-
ties varied in reaction from moderately
resistant to very susceptible. Some
varieties within the group exhibited
type 3-4 reactions, others, 5-6 reactions
and the rest 7-9 reactions. Within a
group of varieties having specific R-genes
(Pi-a, Pi-k, Pi-m) differences in reaction
varied from R, M, or S, indicating that
other R-gene (s) are involved which were
not detected by races in Japan and sug-
gesting that the genotypes are diverse
among varieties within each group.

Component technology evaluation for
some diseases of dryland crops grown
before and after rice. F. A. Elazegui
and T.W. Mew, IRRI.
Control measures for common plant
diseases of dryland crops established
before and after rice at the IRRI Crop-
ping Systems Outreach Sites were eva-
luated based on small farmers' ability
for their adoption. Chemical control
using reduced rates of fungicides suffi-
cient to lower the inoculum or disease
pressure was initiated since low rates
entail less cost-input.
Seed treatment with Ridomil 25 WP
at the rate of 0.125 g/kg seed was
observed to control downy mildew of
corn planted before rice in Pangasinan.
The disease incidence was lower in
Macapuno variety and in early planting.
The control on sweet corn was reflected
statistically on yield gained. For grain
legumes, greenhouse evaluation of fungi-
cides applied as seed treatment against

damping-off indicated that Dithane M-45,
a commercially available fungicide was
significantly active on Pythium and
Rhizoctonia damping-off. Vitavax 200
and 300, both experimental fungicides
significantly controlled Sclerotium
damping-off. For powdery mildew of
mungbean, spraying with Benlate at the
rate of 0.15 g/liter of water controlled
the disease significantly. Insignificant
differences in yield between the treated
and untreated plots could be due to late
occurrence of the disease.

Daconil 2787 W-75 and Topsin Methyl
70 WP against three foliage diseases of
giant cavendish bananas. M. O. San
Juan, TRRC.
A field experiment was conducted for
1 year (1978) in the banana plantation
of Davao Fruits Corporation in La Fili-
pina, Tagum, Davao del Norte, to test the
efficacy of Daconil and Topsin against
sigatoka, black leaf streak and freckle or
black spot caused by Mycosphaerella
musicola Leach, M. ffiensis Deighton and
Phyllostietina musarum Cooke, respec-
Statistical analysis on disease develop-
ment, functional leaves at fruit emer-
gence and at harvest, pycnidia counts,
and fruit production showed no differen-
ces among the treatments. The foliage
diseases were effectively controlled by
Daconil and Daconil-Topsin just as those
sprayed with Dithane-Benlate-Oil-Triton-
Water mixture (control).
Comparative cost analysis based on the
actual cost of chemicals and aerial spra-
ying-charges for the duration of the
experiment showed a total of P2,936.31
per hectare per year of the Dithane-Ben-
late-Oil-Triton-water mixture (control),
while the Daconil treatment had a saving
of P268.30 per hectare per year and
P1,138.30 per hectare per year for the

Ja-June 1980

Philippine Phytopathology

Daconil-Topsin treatment.
The fungicides used in this experi-
ment particularly Daconil and Daconil-
Topsin can also be used profitably in
small areas either at low or high vo-
lume sprays with power sprayers. Aside
from being economical, these chemicals
are easy to mix unlike spray mixture
wherein spray oil is used.

Chemical control of Cercospora leaf
mold in tomato. P. M. Halos and G. C.
Molina, UPLB.

Field test of chemicals showed that
Cercospora leaf mold of tomato can be
effectively controlled by several fungi-
cides. These are Delsene MX, Benlate,
Daconil and Zincofol. Comparatively,
Delsene MX is the best among them since
it consistently controlled the disease even
half the recommended rate.

Chemical control of bottom rot on
cabbage. P. M. Halos and G. C. Molina,
Six fungicides namely, Daconil, Man-
zate 200, Zincofol, Delsene MX, Benlate
and Curzate M were tested for their effi-
cacy against bottom rot of cabbage
caused by Rhizoctonia solani. The three
rates tested, namely, recommended rate,
half the recommended rate and twice the
recommended rate, provided significant
control of the disease. Of the six fungi-
cides tested, Manzate 200 at twice the
recommended rate (30.4 g/20 li water)
gave the best control. Delsene MX and
Zincofol also showed promising control
of the disease.

Chemical control of Sclerotium seedling
blight on rice. S. D. Merca, M. C. Rush,
J. M. Bandong and J. P. Crill, IRRI.

A method was devised for screening

seed-protectant fungicides for activity
against S. rolfsii The fungicides were
initially tested at rates of 500 and 1000
g formulated chemical/100 kg seeds for
the liquid formulations. Effective fun-
gicides were Vitavax 74 WP, Vitavax
200 (vitavax-thiram, 37.5-37.5 WP), Vita-
vax 300 (vitavax-captan, 37.5-37.5 WP),
Uniroyal H719, Rovral 50 WP, and
Elanco EL-228 9.46 EC. These com-
pounds increased stands 10-fold or more
over than that produced by untreated
seeds. Buckman TCMTB 30 EC and
Boots 40542 40 EC were moderately
effective, increasing stands 4-fold over the
control. Benlate 50 WP and Terra-Coat
L-205 23.2 EC were not effective. The
Vitavax products were used in dosage
experiments and all three formulations
gave effective control at rates as low as
10 g formulated per 100 kg seeds. EL-228
was phytotoxic at the rate tested (300
& 600 ml 100 kg seeds) with plants
stunted and the leaves shortened and

Chemical control of neck blast under
upland conditions. C. Q. Torres, J. M.
Bandong, F. L. Nuque and J. P. Crill,
The effectiveness of eight fungicides
namely: EDPD Thalide, Benomyl, Man-
cozeb, CGA 49104, M 7007, PB20,
M6953, Cusul and RH 2161 for con-
trolling neck blast under upland con-
ditions was evaluated in two experi-
ments. The rates of the materials used
per hectare were 1,1,2,2,1 kilogram,
6 and 1 liters, respectively. The fungi-
cides plus sticker (Triton AE (3.2 1/ha)
were sprayed twice at early flowering
and one week later. The percentage of
neck blast was used as the criterion for
evaluating the efficacy of the chemicals.
All the fungicides in the first experi-
ment gave effective control of neck blast

Vol. 16

Jan-Jne 1980 Abstracts of Papers 15

under heavily infective conditions in the the 20 g/kg rate giving about 91% and
upland with significant differences among 70% control of leaf blast, respectively.
treatments. EDPD ranked first followed Hoe 25986 (75% WP) and Homai or
by Mancozeb in the control of neck NF-68 (80% WP) each applied at the
blast. In the second experiment, fungi- rate of 40 and 20 g formulation per kg
cides effectively controlled neck blast seeds also exhibited systemic action and
and significant differences among treat- moderate degree of leaf blast control.
ments were noted. However, their effec-
tiveness was different from the first expe- Seed treatment with systemic fungi-
riment and Benomyl was the most effec- cides for sheath blight control in rice.
tive followed by EDPD in controlling L. M. Sanchez, J. M. Bandong, B. A.
neck blast. Estrada and J. P. Crill, IRRI.

Seed treatment with systemic fungi-
cides for leaf blast control in rice. Seedsofthe sheath blight-susceptible
C. Q. Torres, J. M. Bandong, F. L. Nuque variety, R137 were treated wth fungi-
and J. P. Crill, IRRI. cides either as dry powders or slurrys.
The pesticides and seeds were placed in
Fungicide samples, especially coded a glass jar about twice the volume of the
compounds in the early developmental seeds to be treated, the jar was capped,
stages, were used as seed treatments to shaken by hand to disperse the chemical,
determine their efficacy for systemic and placed onto a jar mill for 10 minutes
control of blast caused by Pyricularia or until the seeds were thoroughly coated
oryzae, at seedling stage. Seeds of the with the fungicide. The treated seeds
blast-susceptible line IR442-2-58 were were planted in heavily seeded rows in
treated with the fungicides as dry plastic trays. Phytotoxic effects were
powders or by slurry treatment and were noted from germination until two weeks
grown in rows either in plastic trays or after seeding. The test plants were inocu-
directly in the blast nursery beds. Test lated by spreading a rice-grain culture of
seedlings were exposed to heavy ino- the sheath blight fungus between rows of
culum in the nursery and covered with the seedlings 2 weeks after seeding. To
plastic sheets for at least 3 consecutive insure rapid sheath blight infection, the
nights 2 weeks after seeding. Covering inoculated seedlings were kept moist
the plants prolonged the dew period and and covered with polyethylene plastic
increased the infection by P. oryzae. sheets for at least 3 consecutive nights.
Plants were observed for phytotoxic Biweekly disease readings were made,
effects for 1 to 2 weeks after seeding. by counting the total number of healthy
The number of blast lesions per seedling and infected leafsheaths and leafblades
determined 3 weeks after seeding and at per row, until 8 weeks after seeding.
weekly intervals until 8 weeks after Benomyl applied as Benlate 50 WP,
seeding. at the rate of 40 g formulation per kg
A new coded compound, Hoe 00550 seed, gave the most effective sheath blight
75 wp (Hoecst Co.) was effective at rates control (about 70%) among the chemi-
of 40 and 20 g formulation per kg seeds. cals tested. Benlate 50 wp at 20 g and
The fungicide gave effective leaf blast Cercobin M 70 wp at 40 g formulation
control for as long as 8 weeks. The 40 per kg seeds gave an intermediate level
g/kg seed rate was more effective than of sheath blight control.

Philipp. Phytopathol 16: 16-26
Received for publication: 2 June 1980


Nenita L. Opina and Tricita H. Quimio

Research Associate, Institute of Plant Breeding, and Associate Professor, Dept. of
Plant Pathology, University of the Philippines at Los Baflos, College, Laguna, respec-

Portion of the M.S. thesis of the senior author.


An extensive survey of gray leafspot of solanaceous crops particularly
tomato, pepper and eggplant was made on different vegetable growing areas
of the Philippines but the disease was found to occur only in Los Bafios,
Laguna. Five isolates were obtained from diseased tomato and four from
pepper. All isolates caused symptoms similar to gray leafspot incited by
species of Stemphylium.
Despite significant differences in morphological details, all isolates
were identified as S. lycopersici (Enjoji) Yamamoto. Conidial dimensions
of all isolates fall within the limits of S. lycopersici and S. solani Weber
but not of S. botryosum Wallr. The isolates were further distinguished
from S. solani and S. botryosum by having verrucose conidial cell wall with
1-4 constrictions on the cross-septa and conidiophore swellings ranging
from 1-8.
Cross inoculation experiments showed that all isolates were pathoge-
nic to all the tomato and pepper varieties tested. Reaction test showed that
the isolates varied in their virulence to several varieties of tomato.
The nine isolates so far obtained are identified as S. lycopersici
(Enjoji) Yamamoto. The significant variation among isolates could be
partially attributed to the existence of physiologic race. However, races
could not be identified with certain degree of confidence in this study.

A leafspot of tomato caused by a
species of Stemphylium was first ob-
served in January, 1979 at the tomato
germplasm collections and variety trial
plots of the UPLB Central Experiment
Station. The disease was causing prema-
ture defoliation with alarming rapidity
in some tomato successions. Researchers
at the Dept. of Plant Pathology had
collected the disease (CALP No. 7951-52)
and tentatively identified the isolated
causal organism as Stemphylium lycoper-

sici (NRCP-DPP-CC No. 182), (Quimio,
Solanaceous crops, particularly tomato
and pepper, are very important vegetable
crops in the Philippines. The Institute of
Plant Breeding is conducting a massive
breeding work to develop varieties of
these crops that are high yielding and
resistant against common diseases. Defi-
nitely, the presence of this apparently
new unreported disease on tomato
in the Philippines will complicate breed-

Gray leafipot by Stemphyihum lycopersici

ing works of these crops. Proper identi-
fication of the causal agent as well as its
distribution and pathogenicity is, there-
fore, of paramount importance.
This study was conducted in the
Dept. of Plant Pathology, UPLB, with
the following specific objectives:
1) to survey the occurrence of this
disease on tomato and pepper in the
Philippines 2) to identify the species
of Stemphylium infecting these plants,
3) to determine its pathogenicity on the
various isolates of tomato and pepper.


Survey and Collection of Diseased Plants
Gray leafspot of tomato and pepper
was surveyed in various localities of the
Philippines where these crops are widely
grown such as Ilocos Norte, Laguna,
Ilocos Sur, Mountain Province, Panga-
sinan, Batangas, Cavite, Bukidnon and
Misamis Oriental. Diseased specimens
were brought to the laboratory for exa-
mination and isolation of the causal

Isolation of the Causal Agent
Causal agent of the gray leafspot of
tomato and pepper was isolated using
tissue culture method. Isolated cultures
were kept in PDA agar tubes, allowed to
sporulate and used for subsequent mor-
phological and pathogenecity tests.

Morphological Studies
Cultures of the nine isolates of Stem-
phylium were grown in PDA medium
for observations and measurements of the
conidia and conidiophores. Other conidial
and conidiophore morphology such as
shape, color, septation, sculpture of co-
nidial epispore, shape of conidial apex
and nodulation of conidiophore were
noted for each isolate.

Spore suspension of each isolate stan-
dardized into 3 x 104 spores per ml were
inoculated into foliage of 4-week old
tomato plants using de Vilbiss atomizer.
Plants were kept in moist chamber for
2-3 days.
Disease reaction was taken 7 days
after inoculation using the following
rating index:
0 no visible symptom
1 spots up to 0.5 mm in diame-
ter, 10% lower plant leaves
2 spots small, up to 1.0 mm in
diameter, 25% of lower plant
leaves affected
3 spots moderate in size, up to
1.5 mm in diameter, 50%
of plant leaves affected
4 spots coalescing more than
75% of plant leaves affected,
lower leaves brown and defo-
Ratings 0-2 were considered resistant,
2.1-3 moderately resistant, 3.1-4 mode-
rately susceptible and 4.1-5 susceptible.

Identification of Species
Species identification was based on
gross morphology, cultural characteris-
tics, cross-inoculation and differential
pathogenicity as reported by various
authors (Weber, 1930) and on a key
made by Ellis (1971).


Survey and Collection of Diseased Plants
Survey from eight provinces in the
Philippines failed to show the occurrence
of gray leafspot disease on tomato and
pepper. The disease was observed and
collected only from various cultivars
of tomato and pepper at various plots
within the campus of the University of

Jan-June 1980

Phiippine Phytopathdolyo

the Philippines at Los Bafios (Table 1).
It is possible that the disease was intro-
duced through the various germplasm
collections of the Institute of Plant
Breeding at the UPLB.

The symptoms usually appear on
leaves, rarely on petioles and never on
fruits. The size of the spots varies from
0.5 to 10 mm in diameter. The spots
are circular to irregular in outline and

first appear as minute sunken, brownish
to black specks or sometimes watery
gray in color on the upper and lower
sides of the leaves and occasionally
surrounded by chlorotic halo. As the
lesions enlarge, they may coalesce to
form a larger spot. The central dead
area of the spots turns grayish brown
while the margin often turns dark
brown or black. The center of the lesions
may crack leaving a shothole appearance
(Fig. 1).

Fig. 1. Typical symptoms of Stemphylium sp. on A) tomato, B) pepper.

Five isolates from tomato and 4 from
pepper were maintained and considered
for further study (Table I). The isolates
were kept in PDA agar tubes under mine-
ral oil and transferred to agar plates

whenever necessary.

Morphological features of the 9 iso-
lates grown in PDA medium are presented
in table 2 and figure 2. The muriform co-

Vol. 16

Table I. Stemphylium isolates from tomato and pepper collected at separate locations within the UPLB campus


ST 6-1 Tomato Re 2 Nov., 1979 RRC, CES (old site~, UPLB
ST 14 Tomato R9 Dec., 1979 CES (new site), UPLB
ST 30-1 Tomato Marikit Feb., 1980 Agronomy Plots, UPLB
ST 31-2 Tomato 79-115 Feb., 1980 Agronomy Greenhouse, UPLB
ST 32-2 Tomato VC 11-1 Feb., 1980 Demonstration Plot, UPLB
SP 5-1 Pepper Pp 6 Dec., 1979 CES (new site), UPLB
SP 8 Pepper Gp 2 Dec., 1979 RRC, CES (old site), UPLB
SP9-1 Pepper Keystone Florida Dec., 1979 Agronomy Plots, UPLB
SP 15 Pepper HpP-40 March, 1980 RRC, CES (old site), UPLB

VnlA 1

nidia varied from ovate, ellipsoidal, ob- bearing conidia. The conidiophores conti-
clavate to subspherical. They are buffy nued to grow through the terminal swel-
brown when young and olive brown when ling (percurrent type) resulting to a
old with rounded bases often with pro- series of nodular swellings, spaced at
minent basal scar. The conidial cell wall irregular intervals along their length
were all verrucose. Major constrictions (Fig. 3B). Conidial size varied significant-
varied from 1-4 while apices were found ly among isolates (table 3) the length
to be pointed, mostly round in culture. ranging from 31.6 46.7 p and the
The conidiophores were unbranched width 17.7 27.0 p. The conidiophore
to loosely branched, straight to flexuous size ranged from 135.0 258.2 x 4.5 -
with vesicular swell on tips (Fig. 3A) 6.3 p.


Il:uira DIr~rs~ril~mr

Table 2. Morphological features of the nine isolates of Stemphylium sp.

Cell Cross-septa Apex

ST 6-1 Verrucose 1-2 constrictions rounded and po

ST 14 -ditto- 1-2 constrictions mostly pointed
ST 30-1 -ditto- 1-3 constrictions mostly pointed
ST 31-2 -ditto- 1-3 constrictions rounded and po
ST 32-2 -ditto- 1-3 constrictions mostly pointed
SP 5-1 -ditto- 1-3 constrictions mostly pointed
SP 8 -ditto- 14 constrictions mostly pointed
SP 9-1 -ditto- 1-3 constrictions mostly rounded
SP 15 -ditto- mostly 2 constrictions mostly rounded

Philippine Phytopathology

Fig. 3. A) lose-up of conidiophore tip shoi
B) Percurrent conidiophore type.


On the basis of morphological fea-
tures, as well as pathogenecity tests,
the nine isolates of Stemphylium studied
appear to be identical despite slight
variation in size and virulence to tomato
:ultivars. The literature indicates that
:onidial and conidiophore dimensions of
Stemphylium vary within the species.
For instance, S. lycopersici have been
reported to have 21-60 x 12-24 1p and
50-84 x 16-23 p conidial dimensions
[Yamamoto, 1960; Ellis, 1971) while
:onidia of S. floridanum are 36 x 12 p1
40.5 x 13.4 pL and 48 x 16 A, (Hannon
and Weber, 1955; Bashi, 1974; and
Graham and Zeiders, 1960). S. solani
have 48.08 x 22.4 p, 37 x 18 pi, 42.8
x 15.8 j and 48 x 21 p conidial dimen-
sions (Weber, 1930; Bashi, 1974; Hannon
and Weber, 1955, and Braverman, 1968)
while S. botryosum have 30 x 16.5,
30 x 20 j, 34 x 21 pand 32 x 21 1 coni-
dial dimensions (Rotem& Wahl, 1966;
Graham and Zeiders, 1960 and Braver-
man, 1968).

, swelling typical of Stemphylium.

Species of Stemphylium have long
been known to attack a variety of hosts.
S. floridanum is pathogenic on a num-
ber of tomato varieties, pepper, egg-
plant, wild soda apple and possibly
gladiolus but failed to infect petunia,
tobacco, china aster and white potato
(Hannon and Weber, 1955). Braverman
(1968) claimed that S. botryosum incites
mild infection on pepper and tomato
while Neegaard (1945) reported tomato
to be susceptible to S. botryosum.
However, studies made by Graham
and Zeiders (1960) showed tomato
plants were not attacked. They reported
that S. botryosum is pathogenic to alfal-
fa, clover, crown vetch, blue lupine and
fescue. On the other hand, S. solani
is very pathogenic to tomato and mode-
rately on pepper (Braverman, 1968, and
Graham and Zeiders, 1960).
Based on reports of Hannon and
Weber (1955); Rotem and Bashi (1977);
Yamamoto (1960) and Ellis (1971),
the nine isolates of Stemphylium sp.
could be classified into one species. Des-
pite differences among isolates, general

OL 10

Jan-June 1981

r qleafot by Stemphylcm lycop

resemblance was noted on the different
studies made.
On the basis of gross morphology,
the isolates closely resemble S. tycoper-
sici, which is synonymous to S. florida-
nun according to Yamamoto (1960),
than any other species of Stemphylium.
Conidial dimensions of the isolates fall
within the limits of S. lycopersici and
S. botryosum but not of S. solani where
the dimension is relatively smaller. Based
on the key to the identification of spe-
cies made by Ellis (1971), the ratio of
conidial length and width of S. lycoper-
sici is 3:1 or more, whereas in S. solani
ratio is not more than 2:1.
The species identification of the
isolates is further narrowed down on the
basis of their conidial and conidiophore
features. The most striking differences
among species Stemphylium are the coni-
dial cell wall architecture, constriction
of cross-septa, nodular swellings of co-
nidiophore and the presence of ascige-
rous stage. Based on these criteria, all
isolates generally possessed verrucose

conidial cell wall with 1-4 constriction
on the cross septa. The nodular swelling
of the conidiophore range from 1-8 anw
ascigerous stage is unknown to all iso
lates. All these criteria conformed witl
morphological features of S. lycopersic
described by various authors. S. solan
possesses round to pointed conidi,
with smooth to reticulate cell wal
and with one deep constrictions o0
the median septum whereas S. botryo
sum have echinulate always round ape)
and mostly one constriction on tlu
conidia. On the other hand, S. lycoper
sici have verrucose conidia with 1-M
constrictions, usually 2-3. Nodular swel.
lings of the conidiophore are present ir
both S. lycopersici and S. solani. The
former is characterized by 1-7 swellings
while the latter is characterized by noi
visible nodular swellings in mature co
nidiophores. Nodular swelling is absent
in S. botryosum. Ascigerous stage of
S lycopersici and S. solani is unknown,
while known to appear in some cultures
of S. botryosum.

Philippine Phytopathology

Table 3. Average conidia and conidiophore sizes of the nine isolates of Stemphylium sp.


ISOLATE Lengtha Width-! Length!a Width-/
(u) (u) (u) (u)

ST 6-1 31.6 g 18.9 d 258.2 a 4.9 cd
ST 14 37.6 c 19.3 cd 192.3 cd 4.9 cd
ST 30-1 34.8 e 17.7 e 162.5 e 4.8 de
ST 31-2 33.5 f 18.9 d 241.4 b 4.9 cd
ST 32-2 37.7 c 19.0 d 186.0 d 5.0 c
SP 5-1 41.4 b 18.2 e 175.5 d 4.6 ef
SP 8 46.7 a 27.0 a 175.0 d 5.3 b
SP 9-1 34.0 ef 21.9 b 135.0 f 6.3 a
SP15 35.7 d 20.0 c 206.5 c 5.2 bc

aAverage measurements having common letter comprise a group that are not significantly
different at 5%level.

Table 4. Pathogenecity of nie isolates of Stemphylium sp. on pepper and tomato

2029 RV 4 California 74-61-15-1

ST 14 ++A- ++ ++ +++
ST30-1 +++ ++ ++ +++
ST 31-2 +++ +++ ++ +++
ST 32-2 +++ + ++ +++
ST6-1 +++ ++ ++ +
SP5-1 +++ ++ +++ +++
SP15 +++ ++ +++ +++
SP8 + +++ +
SP9-1 + +++ +
J no visible symptom.
+ Tiny spots, most often do not enlarge.
++ Spots more than 1 mm diameter, often enlarge to bigger lesion.
+++ Spots occasionally coalesce to form larger lesion.

Vol 16

Table 5. Virulence test of 9 isolates of Stemphylium sp. to different tomato varieties and lines

R4-2043 R10-3034 VC9-1-2-9 RV 4 RV 6 RV 7 RV 9 C32d Florida 2432 Florida Dale

ST 14 0 0 0 3 0 0 0 0.22 1 1
ST 30-1 0 1.75 1 4.25 0 0.75 1 1 1.25 0
ST 31-2 0 0 0.22 4.0 1 1 1.25 0 0 0
ST 32-2 0 0 0 0.22 0 0 0 0 0 0.22
ST 6-1 0 0.22 0 2.75 0 0 0 0 0 0
SP 5-1 0 0 0 3.5 0 0 0.5 0 0 0
SP 15 0 0.75 0 2.75 0 0 0 0 0 0.22
SP8 0 0 0 0 0 0 0 0 0 0
SP9-1 0 0 0 0 0 0 0 0 0 0

a 0 no visible symptom
1 spots up to 0.5 mm; 10% lower plant leaves affected.
2 spots small, up to 1.0 mm in diameter; 25%lower plant leaves affected
3 spots up to 1.5 mm in diameter; 50%plant leaves affected.
4 spots occasionally coalescing, about 2 mm in diameter; 75% of plant leaves affected; lower leaves yellow.
5 spots coalescing; more than 75% of the plant leaves affected; lower leaves brown and defoliated.
0-2 = Resistant; 2.1-3 = Moderately Resistant; 3.1-4 = Moderately Susceptible; 4.1-5 = Susceptible.


Philippine Phytopatholo

utum jionaranum sp. nov. rnytopatnology. 4.:11-10.

NEERGAARD, P. 1945. Danish species of Altemaria and Stemphylium. E. Munka;
Copenhagen, Denmark. 560 p.

ROTEM, J., Y. COHEN and I. WAHL. 1966. A new tomato foliage disease in Is
caused by Stemphylium bortyosum. Can. J. Pl. Sci. 46:265-270.

ROTEM, J. and E. BASHI. 1977. A reivew of the present status of the Stemphyli
complex in tomato foliage. Phytoparasitica. 5:45-48.

QUIMIO, T. H. 1979. Illustrated genera of Philippine Plant Pathogenic Fungi. NRCP ]
ject IE-92. Annual Report, June.

WEBER, G. F. 1930. Gray leafspot of tomato caused by Stemphylium solani sp. r
Phytopathology. 20:513-518.

YAMAMOTO, W. 1960. Synonymous species of Altemaria and Stemphylium in Jai
Trans. Mycol. Soc. Japan. 2:92-93.

Philipp. Phytopathol. 16: 27-37
Received for publication: 10 Sept. 1980


Hilda G. Buenaflor and Evelyn Mae T. Mendoza

Former Research Associate and Research Biochemist, respectively, Biochemistry
Laboratory, Institute of Plant Breeding, College of Agriculture, University of the
Philippines at Los Bafios, College, Laguna. HGB is now with the Entomology Dept.,
International Rice Research Institute, Los Bafios, Laguna. Please address correspon-
dence to EMTM.
This study was supported by grants from the Philippine Government to the
Institute of Plant Breeding, UPLB.


Conditions for sampling, incubation procedures and a bioaasay for
the spore germination inhibitor of Sclerospora philippinensis were estab-
lished. Dilutions (1:50) of aqueous crude extracts of 3-day old leaves of
resistant lines pH 101 and pH 102 restricted spore germination from 5 to
47%. Higher spore germination levels, 48 to 70%, were obtained with
extracts from the susceptible varieties popcorn and Philippine supersweet.
Water control gave about 83% germination. The majority of the germina-
ted spores incubated with resistant line extracts had short stubby germ
tubes with knobs at the tip, in contrast to the long and slender germ tubes
of spores incubated in extracts of susceptible varieties or water. Compara-
ble values were obtained using crude extracts of 4-day old pH 101 uninfec-
ted leaves and leaves extracted 24 hours after infection. Spores incubated
with extracts of 5-day old seedlings infected for 48 hours had slightly higher
levels of germination.
The spore germination inhibitor was also detected in leaf diffusates
from infected and uninfected leaves. Decreasing or increasing the pH of
crude extract with dilute acid or base resulted in an increase in spore ger-
mination although germination decreased at higher pH's.
The inhibitor was found to be heat stable and dialyzable, with mole-
cular weight of about 1,400 daltons but could be bound to larger mole-
cules like proteins.
The possible role of this inhibitor in corn resistance to S. philippinen-
sis is discussed.

The downy mildew disease of maize Philippines resulting in yield losses of
(Zea mays (L.) caused by Sclerspora 40 to 60% and as high as 80 to 100%
philippinensis Weston is considered as the under severe conditions (Weston, 1920;
most destructive corn disease in the Barredo and Exconde, 1973). Penetra-

Philippine Phytopathology

tion of corn leaves by the fungus occurs
about 2 hours after inoculation and is
strictly via the stomata, by either the
germ tube or hypha followed by inter-
cellular invasion of mesophyll cells
(Dalmacio and Exconde, 1969). Effec-
tive infection occurs under moist, dark
and low temperature conditions for at
least 4 hours after inoculation
(Barredo and Exconde, 1973). Corn
plants have been found to increase in
resistance to the disease with age.
The thrust in the control of this
disease has been in breeding more re-
sistant corn varieties. Work in this area
for the last few years has led to the
development of several downy mildew
resistant varieties, namely, Phil DMR 1,
Phil DMR 2, MIT Var 2, Phil DMR
Composite 1 and Phil DMR Composite 2.
This study was undertaken to inves-
tigate the mechanism 6f resistance of
corn plant to downy mildew.


Preparation of Test Plants, Crude Leaf
Extracts and Mature Spore Suspensions

Test Plants. Fifty seeds per variety
of downy mildew resistant inbred lines
(pH 101 and pH 102) and susceptible
varieties (popcorn and Philippine super-
sweet corn) were planted in seedboxes
measuring 15 in x 20 in x 4 in. Four
seedboxes per variety were used in each
Crude Leaf Extract. Leaves of desired
maturity were harvested, washed gently
with distilled water and excess moisture
blotted with filter paper. Leaves were
cut into small pieces and homogenized

in a chilled Waring blendor (1 g leaves:
5 ml distilled water) for three 15 second
periods at number 5 speed. The homo-
genate was centrifuged for 10 minutes
at 12,000 x g in a Sorvall RC5 Centri-
fuge at 4 C. The clear supernate was
collected and used immediately or stored
at 0 C. Leaf extracts had a pH of 5.5-5.7.
Mature Spore Suspension. The con-
ditions used were based on previous
studies of spore maturation and infection
by S. philippinensis (Barredo and Excon-
de, 1973; Dogma, 1975). Infected corn
leaves were obtained from the field or
from previously inoculated plants in pots
late in the afternoon. These were placed
in a container filled to about one-fourth
with water and exposed to a fluorescent
lamp (180 ft. c.) overnight. The following
day, the leaves were cleaned gently to
remove old downy mildew growth, put
in a moist black plastic bag, and
incubated at 23 C. Spores were collected
and dispersed in a small amount of dis-
tilled water after 8 to 9 hours of incuba-
tion. For inoculation purposes a spore
density of 65,000 per ml was used.

Bioassay for Spore Germination Inhibitor

Five pl of a suspension containing 100-
200 spores was incubated in 20 pi of
diluted crude extract or water (control).
The slides were placed in a well-sealed
moist plastic container. Germination was
allowed to take place overnight in an
incubator at 23 C. The percent germina-
tion of spores was determined using a
light microscope. About 100-200 spores
were counted for each determination
done in four replicates. Spores with germ
tubes equal or longer than the spore
length were considered germinated.

Vol. 16

Spore Gernination Inhabitor

Properties of Spore Germination Inhibitor

Extracts of pH 101 resistant inbred
line and popcorn leaves were used in
studying the properties of the spore
germination inhibitor.
Heat Stability. Crude leaf extracts
(1:5 dilution) of the resistant and sus-
ceptible plants were placed in a boiling
water bath for 15 minutes. Bioassays
were performed after treatment.
Dialyzability. Crude leaf extracts (1:5
dilution) were dialyzed for 24 hours
at 4 C. The retentate, undialyzed leaf
extracts and watercontrol were bio-
Molecular Size. Concentrated lyophi-
lized crude leaf extract of the resistant
pH 101 inbred line was subjected to gel
filtration using Sephadex G-50 column
(2 x 35 cm), pressure head of about
20 cm H20, at 4 C, with 0.02 M NaC1
as eluant. Trypsin, lysozyme, bacitracin
and bromophenol blue were used as
molecular weight markers.
Effect of pH. The influence of pH
on spore germination was determined
by incubating mature spores in crude
leaf extracts whose pH had been adjusted
with 0.01 Hcl or NaOH solutions prior
to bioassay.

Leaf Diffusate Collection Method

Leaves of 3-day old seedlings were
placed on a solution containing 5% suc-
rose and 20 ppm kinetin. Several drops
of water or spore suspension (65,000
spores/ml) were placed on each with a
small pipette. The leaves were then
placed in a sealed moist chamber and
incubated at 230C in the dark for about
15 hours. After incubation, these were
exposed to a light of 104 ft. c. density

for 30-45 minutes at room temperature.
Several drops were withdrawn from the
leaf surface and centrifuged to remove
the spores. The supernate was collected
and bioassayed for spore germination

Protein Determination

Protein concentration in crude ex-
tracts was measured according to the
method of Lowry et al. (1951). Crystal-
lized and lyophilized bovine serum
albumin (Sigma Chemical Co.) was used
as standard.


Crude extracts of popcorn leaves
with a concentration greater than 1:50
inhibited spore germination to a large
degree (Table 1). Hence, at high concen-
trations any spore germination inhibi-
tor activity of crude extract of the
resistant line could be masked.
Using 3-day old seedlings, spore
germination was significantly inhibited
by the preparations from resistant inbred
line (Table 2) by an average of 30% more
than extracts from susceptible varieties.
The majority of germinated spores
incubated in extracts of resistant plants
had short, stubby germ tubes with
knoblike formation at the tip. In contrast,
spores incubated in extracts of suscep-
tible plants and water control had long
and slender tubes. Occasionally, abnor-
mal germ tube growth similar to that
observed in resistant plant extracts
was seen in spores germinated in sus-
ceptible plant extract preparations.
One-month old plants were also
harvested and crude extracts prepared
from their leaves. These extracts had

Jan-June 1980

ilippine Phytopatholosy


gure 1. Fractionation of lyophilized 3-day old pH 101 leaves on Sephadex G-50.
1:50 dilution of crude extract was applied on a Sephadex G-50 column (2 x 32
cm) and eluted with 0.02 M Na at an operating pressure of 20 cm H20 at 4 C.

spore germmauon


opcom 1:5

lean spore germination of two replicates.

nilar effect on percent spore germina-
on as that of water control (Table 2).
terestingly, 1 month old plant was
ore resistant to the downy mildew
Ithnaen than the 3-day old eedlino.


To determine if more of the inhibi-
r could be produced upon infection,
lay old seedlings were inoculated.
,tracts from infected and uninfected
mts, 24 hours after infection, had


oe Germination Inhabitor

) significant difference in their capa- To test whether the inoculum used
ty to allow spore germination (Table 3). was sufficient to infect the plants, ano-
Ktension of incubation time to 48 hours their set of test plants were infected simul-
lowed that infected leaves allowed even taneously with the above. Chlorotic
*eater spore germination than infected specks were produced after one day
aves, indicating less spore germination at the site of infection on inoculated
hibitor activity, test plants and these developed to streaks
Comparable protein levels in crude after 5 days. Fifteen days after inocula-
rtracts within each treatment were tion, 83, 5, 70 and 6% infection were
obtained, hence, any difference in results observed in popcorn, pH 101; Philippine
mnot be attributed to variation in supersweet and pH 102 test plants,
degree of extraction. respectively.

able 2. Effect of crude extracts of corn leaves on Sclerospora philippinensis spore

MI Extract (0%)


32 Philippine Phytopthology V

Table 3. Effect of rude extracts of uninfected and infected corn leaves on
Sclerospom phtiippinensi spore grrminationa


A. Uninfected vs. Infected
4-day old leaves

56 g

bated with mature spores as described in

)nly values within each treatment followed
tistical analysis for each treatment was done

Effect of pH on Spore Germination

When & phlippinensis spores we
incubated in crude extract of pH 1(
leaves titrated to various pH's wil
0.01 N HCI or NaOH, percent gem
nation increased at pH's lower and high
than pH 5.7 of the untreated extrac
Germination was highest at pH 6.
73.0%, and decreased to 28% at p
11.0 (Table 5).

Some Properties of the Spore
Germination Inhibitor

Size. The small (< 10,000) molecul
weight of the spore germination inhit
tor was indicated by the loss of activil

B. Uninfected vs. Infected
5-day old leaves

pH 101 uninfected
pH 101 infected

a 1:50 dilutions of crude extracts were i
bMean values from at least four replicat,
by different letters differ significantly.
using the completely randomized design

Leaf Diffusate Experiments

Leaf diffusates from both uninfect
and infected resistant and susceptit
detached leaf samples showed an increa
in ability to inhibit spore germination
incubation time was increased (Table 9
Furthermore, there was a greater amou
of spore germination inhibitor in infect
leaves versus uninfected leaves, both res
tant and susceptible. The difference V;
more marked after 42 and 66 hours
incubation. However, diffusates fro
both resistant and susceptible test leai
showed similar effects on spore germir
tion. Uninfected popcorn sample aft
42 hours even had a higher spore germir
tion inhibitor activity than the resista

Spore Germination Inhabitor

Table 4. Percentage of germination of Sclerospora philppinensis spores in diffusates
obtained from detached leaves of susceptible and resistant coma


18 54.5 50.9 51.8 46.0
(6.5)b (4.0) (6.9) (4.2)

42 34.4 8.9 53.0 12.3
(3.9) (3.8) (5.7) (3.9)

66 25.1 16.3 27.8 13.6
(3.8) (4.1) (4.0) (3.8)

a Spore germination average of two replicates
bValues in parentheses are pH of diffusates.

Table 5. Effect of pH on spore germination inhibitor of pH 101 crude extract


4.6 41.2
5.5 48.5
5.7b 5.2
6.6 73.7
7.5 66.6
8.5 38.3
9.6 46.2
11.0 28.0
Water control pH 5.6 82.1

a pH of crude extract was adjusted by titrating with either 0.01 N HCI or NaOH.
bpH of ph 101 crude extract.

Jan-June 1980

lifppine Phytopathology

F crude extracts after dialysis (Table 6). of a water-soluble spore germination
determination of the molecular weight inhibitor in young seedlings of both
f the inhibitor was done by passing resistant lines (pH 101 and pH 102)
H 101 extract through a Sephadex and susceptible varieties (popcorn and
-50 column. Two areas of inhibitor supersweet), although a significantly
Activity corresponding to average mole- greater activity is consistently obtained
ilar weights of 24,000 and less than in the resistant plant. Spore germination
,400 were obtained (Figure 1). These .values for the crude extract of pH 101,
actions produced abnormal growths on e.g., could vary from 5.2 to 47%. How-
owny mildew spores similar to those ever, when compared to control and pop-
bserved with crude extracts. corn extract in the same set of experi-
Heat stability. Heating the crude ex- ment, the inhibitor was always higher
act increased the spore germination in the more resistant plant extract. Inhi-
hibitor activity of popcorn crude ex- bition of spore germination by some
act suggesting activation of performed antifungal substances has been previously
Inhibitor (Table 7). This is similar to that reported to account partly for the resis-
bserved with DIMBOA (2,4-dihydroxy- tance of some sugar beet varieties to
-methoxy-l,4-benzoxazin-3-one), a re- downy mildew caused, by Peronaspora
stance factor in corn against European farinosa f. sp. betae (Russell, 1972)
mr borer which is hydrolyzed from its and bean leaves (Vicia faba L.) to
active glucoside form by a glucosidase Botrytis cinerea (Purkayastha and
id also by heat (Klun et al., 1967). Deverall, 1965).
A, n.,;raafiao+inn de~n r~0~s01A *1,~n *h

idicating the preinfectional or non-
hytoalexin character of this factor.
furthermore, no increase in spore germi-
ition inhibitor activity was observed
ren after 2 days of infection. Presu-
ably the amount of preinfectional spore
wrminatinn inhihitnr ie miffeifant tn nrar-

This study represents the first serious
attempt to elucidate the biochemical
mechanism of corn resistance to downy
iildew. Our results show the occurrence

oL 16

xre Germination Inhabitor

'able 6. Dialyzability of spore germination inhibitor"


Before Dialysisb

Control 81
Popcorn 61
ph 101 24

After Dialysis

Control 81
Popcorn 83
ph 101 80

1:5 crude extract was dialyzed against water overnight at 4 C. The retentate's volume
was measured, diluted to 1:50 with water and incubated with mature spores as indicated
in Methods.
'The protein content of popcorn and ph 101 before dialysis was 18.5 and 19.8 mg/ml,

Control 84 84

Popcorn 87 22

ph 101 10 27

a 1:50 crude extracts were heated in a boiling water both for 15 minutes. After cooling
down to room temperature, the samples were incubated with mature spores as
described in Methods. Popcorn and ph 101 extracts had 27.5 and 30.0 mg protein/ml.
bMean values of at least four replicates.

kn-June 1980

JO rmuppme rnyrpamotogy Vol.

In these samples, there was an increase The spore germination inhibitor wa
in the inhibitor activity with longer not detected in extracts of one mont
incubatiqn periods, a phenomenon which old plants, both resistant and susceptible
occurs for both phytoalexins and pre- This suggests a different mode of resil
infectional resistance factors. Notably tance mechanism for older or mature
and rather expectedly, maximal inhibi- corn plants. Notably corn exhibits various
tion was observed in a shorter incubation degrees of resistance to European cor
time with infected leaves than with borer at different stages of maturity
uninfected ones. However, there was no (Guthrie, et al., 1960). DIMBOA
difference in inhibitor activity between known to account for the first broo
the pH 101 and popcorn samples unlike resistance of corn at the whorl stage a
that obtained when crude extracts were though its chemical basis of resistance fc
used. Perhaps, this is due to the artificial the pollen-shedding stage of the plant i
environment of the detached leaf in this unknown (Klun and Robinson, 1969'

Jan-June 1980 Spore Germination Ihhabitor 37

KLUN, J. A., C. L. TIPTON and T. A. BRINDLEY. 1967. 2,4-dihydroxy-7-methoxy-1,
4-benxazin-3-one (DIMBOA), an active agent in the resistance of maize to the
European corn borer. J. Econ. Entomol. 10:1529-1533.

KLUN, J. A. and J. F. ROBINSON. 1969. Concentration of two 1,4-benzoxazinones in
dent corn at various stages of development of the plant and its relation to resis-
tance of the host plant to the European corn borer. J. Econ. Entomol. 62:214-220.

LIM, S. M., J. D. PAXTON and A. L. HOOKER. 1968. Phytoalexin production in corn
resistant to Helminthosporium turcicum. Phytopathology. 58:720-721.

LOWRY, O. H., N. J. ROSEBROUGH, A. L. TARR and R. L. RANDALL. 1951. Protein
measurement with the Folin Phenol reagent. J. Biol. Chem. 193:265-275).

MACE, M. E. and J. A. VEECH. 1973. Inhibition of Helminthosporium turcicum spore
germination by leaf diffusates from northern leaf blight-susceptible and resistant
corn. Phytopathology. 63:1393-1394.

PURKAYASTHA, R. P. and B. J. DEVERALL. 1965. The detection of antifungal subs-
tances before and after infection of beans (Vicia faba L.) of Botrytis spp. Ann.
Appl. Biol. 56:269-277.

RAMILO, C. A. and W. G. PADOLINA. 1976. Comparison of phenolics in downy mildew
resistant and susceptible varieties of corn. Kimika 1, 33-49.

RAYMUNDO, A. D. and O. R. EXCONDE. 1972. Some factors affecting reaction of six
corn varieties and synthetics to downy mildew. UPCA Rice and Corn Project
Annual Report, 1971-1972.

RUSSEL, G. E. 1972. Components of resistance to diseases in sugar beet. In F. G. H.
Lupton, G. Jenkins, and R. Johnson (eds.). The Way Ahead in Plant Breeding,
Proc. 6th Congress of EUCARPIA (European Association for Research in Plant
Breeding). pp. 99-107.

WESTON, W. H. 1920. Philippine downy mildew of maize. J. Agr. Res. 19:97-122.

PhFippi ePhytopatho. 16: 38-41
Received for publication: 12 June 1980


A. L. Piamonte and F. C. Quebral

Respectively, Senior Research Assistant and Professor, Department of Plant
Pathology, University of the Philippines at Los Baflos, College, Laguna.


Paired plot spraying experiment using the combination of Dithane
M-45 and Plantvax 75W was conducted at the UPLB Central Experiment
Station to determine the influence of rust on the yield of soybean.
Observations on the incidence of rust infection showed that TK No. 5
was infected earlier than Clark 63, indicating that the former is more sus-
ceptible to rust infection than the latter.
Based on the International Working Group on Soybean Rust rating
system, the mean disease rating of the protected plots of both varieties
was 123 for bottom third, while 311 or no pustules nor lesions on the
upper third leaves. The unprotected plots had an average rating of 142 and
333 for bottom third and upper third leaves, respectively.
The protected plots gave a significantly higher yield compared to
the unprotected. TK No. 5 yielded 503 kg while Clark 63 had 748 kg over
the unprotected. A significant increase of 47% for TK No. 5 and 30%
for Clark 63 was obtained in the protected over the unprotected. Yield
reduction was due to lower number of pods/plant, number of seeds/pod
and seed weight.

Soybean rust caused by Phakopsora
pachyrhizi Syd. is one of the most se-
rious problems of soybean production
throughout the country. Its occurrence
cannot be neglected knowing that it can
reduce the yield from 30% to as much as
80% (Ballon and Legaspi, 1968). Under
field conditions favoring infection and
disease development, soybean rust can
cause complete defoliation of the crop
before its normal maturity with con-
sequent reduction in yield (Singh, 1977).
Numerous chemicals have been tested
for controlling soybean rust. Batoon and
Rubia (1966) stated that Shell Copper,
Copper Lonacol and Fungicides 658
gave significant control of the disease.
Likewise, Torres (1976) reported that

for both wet and dry seasons Dithane
M-45 reduced rust infection and caused
significant increase in yield by 64%
and 24% for TK No. 5 and Clark 63,
respectively. Yang (1977) in Taiwan,
obtained a 50% yield reduction when
soybean plants were not protected with
Dithane M-45. He further stated that
this fungicide provided better control
of rust as reflected by higher yields,
while Singh and Thapliyal (1977) in
India, reported that Dithane M45 or
Dithane Z 78 could partially control
the disease.

The present study was conducted to
determine the influence of rust control
on the yield of soybean.

t Control on Soybean


rhis study was conducted at the
tral Experiment Station, U.P. at
Bafios during the dry season of
'6. The experimental area was appro-
lately 165 square meters. The plants
e planted on November 12, 1976,
;ed 60 cm apart between rows and
Lnged in Randomized Complete Block
ign with a 4-row 6-meter plot per
Ltment, replicated four times. Each
location consisted of two treatments
designated as protected (P) and
protected (U). Two varieties were used
his study, namely TK No. 5 and Clark
The plants with a seeding rate of
44 kg/ha were thinned to approxi-
tely 300,000 plants per hectare.
*adan 3G was used for the control
insects at the rate of 1 bag per hectare,
ile Azodrin 168, Thiodan and Mala-
in were used in succession every 7
's up to maturity, and pre-emergence
bicide, Treflan E.C. was used to
itrol the weeds. Ammonium sulfate
i applied once at planting time.
One month after planting the expe-
[ental plants were inoculated with
ire suspension of P. pachyrhizi and 1
r after, the first fungicide spray was
lied. Subsequent sprays were made
ry 7 days thereafter up to January
77. After the first application, 15
nts per treatment were randomly
ected on a two center row which
re then properly tagged.
Two fungicides were used in this
dy. They were Dithane M-45 and
ntvax 75W, applied in combina-
n at the rate of 2 lbs and 0.2 lbs per
0 gal of water, respectively. This
igicidal combination was applied at
ekly intervals for 2 months for a
:al of five applications.
The plants were rated for disease

erity 2 days after the last spray appli-
ion using the three-digit scheme
ing, 1977b). One month later, the
nts were harvested and yield recorded
I projected into hectare basis.


At the time of the first spray, the
nts were at the flowering stage. One
-k after, bacterial pustules were ob-
ied on all treatments, especially on
No. 5 variety. However, the bacterial
asion was overrun by rust pustules in
week's time which resulted to the
action of the unprotected plots of
th varieties. Rust pustules were evident
soybean leaves 2 weeks after inocu-
on. On the unprotected plots of TK
. 5, rust pustules progressed rapidly
ich resulted in the defoliation of the
lets. However, the protected plot
gained green until maturity.
After the third spray application,
rk 63 was not as badly affected as
No. 5. The former exhibited some
pree of tolerance against the disease.
Two weeks after the third applica-
n, TK No. 5 was heavily affected
rust. The leaves of the unprotected
nts turned yellow and some were
eady defoliated. On the other hand,
Ferences between the protected and
protected plants of Clark 63 were
rious. Those protected were still
en, while the unprotected turned
low due to P. pachyrhizi
One week after the last spraying
'th fungicide application) the plants
the protected plots of TK No. 5 were
lerally still green, although some
ves began to turn yellow. Generally,
. No. 5 defoliated earlier than the
rk 63, indicating that this variety
C No. 5) was very susceptible to
t infection.


0Phihopine Phvtovatholouv

The mean disease rating of protect
ted plots of TK No. 5 was 133 for th
bottom third leaves while 311 for th
upper third leaves measured from th
ground level (Table 1). On the othe
hand, the unprotected plots had ai
average rating of 142 and 333 for bot
tom third and upper third leaves, res
pectively. The upper third leaves of th4
protected plots showed no rust pustules
The results indicate the effectiveness
of Dithane M-45-Plantvax 75W combi
nation in suppressing the development o
the soybean rust fungus. It was observe(
that soybean rust infection began on th4
lower leaves and the failure of the pro
tectant fungicides to spray the lowel
leaf surfaces caused the infection on the

Table 1. Comparative disease ratings of t
(Dry Season, 1976)a

Variety Treatment I

Clark 63 Protected


TK No. 5 Protected


a Average of 15 plants per treatment
b IWcRR =rtin avatom V(Yna 1977 hl

protected plots.
The protected plots of both variety
gave significantly higher yield compare
to the unprotected plots (Table 2). 0
TK No. 5, there was an increase of 1.9
on the number of pods developed pc
plant, while 5.98 grams on the numb(
of seeds/ plant. Moreover, on the 100
seed weight, there was still an increase
of 69.50 grams over the unprotected
Similar results were also observed o
Clark 63 in that the protected plots gay
higher yield than the unprotected plot:
The average projected yield/ha of bot
varieties was very promising. TK No.
had an increase of 503.66 kg, while
Clark 63 had 748.08 kg over the unprc

soybean varieties in paired plot experiment

it Location Disease rating

Upper 311
Middle 211
Bottom 123

Upper 333
Middle 233
Bottom 143

Upper 311
Middle 222
Bottom 133

Upper 333
Middle 233
Bottom 143


Rust Control on Soybean

Table 2. Influence of rust control on the yield of soybean (Dry season, 1976)

Variety and No. of No. of Weight of Weight of 1000 Average
Treatment pods/ seeds/ seeds/ seeds/ projected
plant" plant plant (g) treatmentb yield (kg)/ha

TKNo. 5

Protected 14.48* 24.71m 3.42* 151.50** 1072.45**
Unprotected 12.49 18.73 1.46 82.00 568.79
Increase (%) 13.00 24.00 57.00 45.80 46.96

Clark 63
Protected 28.54** 50.77** 7.09** 136.25" 1910.52**
Unprotected 22.44 36.66 3.46 125.50 1162.44
Increase (%) 21.00 27.00 51.00 7.00 37.15

a Average of 4 replications, 15 plants/treatment
bAverage of 4 replications
*Significantly different at .05 level of probability
**Significant at .01 level of probability
nsNot significant


BALLON, F. B. and B. M. LEGASPI. 1969. Reaction of promising soybean hybrids and
standard varieties to rust. Philipp. Phytopathol. 4:2 (Abstr.).

BATOON, C. R. and LUZ RUBIA. 1966. Chemical control of soybean rust. Philipp.
Phytopathol. 3:1 (Abstr.).

SINGH, B. B. and P. N. THAPLIYAL. 1977. Breeding for resistance to soybean rust in
India. Proc. Asia-Oceania Soybean Rust Workshop/Seminar. Feb. 28-March 4,
1977. Manila, Philippines.

TORRES, C. Q. and F. C. QUEBRAL. 1976. Comparative effects of five fungicides
against soybean rust. Tropical Grain Legume Bulletin No. 6. Ibadan, Nigeria.

YANG, C. Y. 1977a. Soybean Rust in Eastern Hemisphere. Proc. Asia-Oceania Soybean
Rust Workshop/Seminar. Feb. 28-March 4, 1977. Manila, Philippines.

YANG, C. Y. 1977b. The IWGSR rust rating system. Soybean Rust Newsletter 1(1).4-6.

Jan-June 1980

Philippine Phytopathol 16: 42-48
Received for publication: 2 July 1980


Ruth M. Sarra-Guzman and R. G. Davide

Respectively, Assistant Professor, College of Agriculture, Isabela State University,
and Associate Professor, Department of Plant Pathology, College of Agriculture, Uni-
versity of the Philippines at Los Bafios, College, Laguna.

Portion of the M.S. Thesis of the senior author.


Inoculation of three varieties of grapes, namely: Black Ribier, Red
Cardinal and Brazilian IAC 971-1, with Meloidogyne incognita at 0, 10, 20
and 30 egg mass per plant and Rotylenchulus reniformis at 0, 5,000, 10,000
and 15,000 larvae per plant resulted in significant differences in root and
top growths and degree of root infection between the inoculated plants and
the non-inoculated controls.
Histopathological examination of diseased root tissues showed
M. incognita individuals in different feeding orientations and induced the
formation of synctia. Likewise, R. reniformis was observed feeding at the
pericycle and also induced the formation of similar but smaller syncytia.

Grapes, which used to be an exotic
and temperate fruit crop, are now success-
fully grown in some parts of the Philip-
pines. It is becoming a promising agri-
cultural venture (PCARR, 1975a) and
because of its high profitable returns
(Katigbak and Gonzales, 1973), a num-
ber of growers have now gone into com-
mercial production, particularly in the
provinces of Cebu, Batangas, Davao,
Cotabato and Zamboanga. In more
successful vineyards, where improved
knowledge of grape culture has been
applied, harvests have been relatively
high (Matienzo, 1973).
As more hectarage are developed for
grape-growing, pests and diseases specific
to the crop could become a serious prob-
lem. In fact, at present, such diseases as
downy mildew, anthracnose, root knot
and other diseases caused by nematode
infections are seriously affecting some

vineyards. These diseases are considered
as the major limiting factors in the grape
industry (PCARR, 1975b).
This study was conducted to evaluate
the effects of Meloidogyne incognita
Chitwood and Rotylenchulus reniformis
Linford & Oliveira on three locally-
grown grape varieties, namely: Red Car-
dinal, Black Ribier and Brazilian hybrid
IAC 971-1 and to investigate the extent
of root infection.


Species identification
Root-knot and reniform nematode-
infected grape root samples from Cebu
were washed with tap water and cut
1 mm long, and stained for 3 minutes
in boiling acid-fuchsin lactophenol.

to and R. reniformis on Gra

ined roots were placed in vials with
ar lactophenol for at least 24 hrs
ore dissecting the nematodes under a
reoscopic microscope for identifi-
ion. Species identification of Meloido-
le was done by examining several
-ineal patterns of adult females and
npared with those described by
orne (1962). For Rotylenchulus, spe-
s was identified using taxonomic
ys and species descriptions (Linford
l3iveira, 1940: Thorne, 1962).

culation test
M. incognita and R. reniformis were
ted for their pathogenic potentials
grapes. Three-month old rooted
things of three grape varieties, namely:
ick Ribier, Red Cardinal and Brazilian
C 971-1, were used as test plants.
e cuttings were grown singly in heat-
rilized soil in 20 cm diam clay pots.
oculation of test plants was done on
week old cuttings. Prior to inocula-
n, egg masses of M. incognita were
rface-sterilized in 0.1% streptomycin
Ifate. For M. incognita the inoculum
*els were 0, 10 and 20, 30 egg masses
aiform size) per plant, whereas for
reniformis, inoculum levels of 0,
)00, 10,000 and 15,000 larvae were
ed per plant. Each level was replicated
e times for each grape variety. All
)culated plants were randomly arranged
d kept in the greenhouse for 3 months.
After 3 months, the effect of nema-
de infection on the growth of the
mts was determined by taking fresh
eights of roots and tops. The degree
root galling caused by M. incognita
is determined by using the following
11 index ratings: 1 = no gall (0%);
= trace galling (1-25%); 3 = slight
ling (25-50%); 4 = moderate galling
0-75%); 5 = severe galling (75-100%).

Histological studies were done on
matode-infected roots. For M incog-
a, samples were taken from galled
)ts (Fig. 1) whereas for R. reniformis,
:tions were taken from parasitized areas
arest the root tip. Diseased sections
re cut crosswide about 10 mm, dehy-
ited in a graded series of tertiary butyl
ohol concentrations and infiltrated in
raffm. Longitudinal and transverse
:tions were cut 12 u thick using a rota-
microtome. Cut sections were
united on glass slides and stained with
ranin-fast green using the method
scribed by Sass (1964). Cellular effects
nematode feeding on the root tissues
:re examined microscopically.
ecies identification
Based on the examination of several
rineal patterns and comparisons of the
ecimens with Thorne's descriptions of
- species, the root-knot nematode spe-
s used in the study was identified as
incognita and the reniform nematode
s Rotylenchulus reniformis Linford
oculation test
R. reniformis inoculated on Black
bier variety at different levels signi-
:antly affected plant growth (Table 1).
lere was a significant reduction in top
d root weights at- 10,000 and 15,000
rels as compared with the control.
apparently the 5,000 level was still low
cause any detectable damage on the
Likewise, the varieties Black Ribier,
azilian and Red Cardinal also showed
me significant differences in their
3wth responses to M. incognita at dif-
rent inoculum levels (Table 2). The 20
d 30 eggmass level gave significant
auction in top and root growth in the


jr.uupFuMu K. t.PraMijnuy


Fig. 1. A portion of Red Cardinal gn
root system showing galls cau
by M. incognita.

Table 1. Effect of Rotylenchulus reniJ
different levels of inoculuma

Inoculum level TOP WEIG
per plant
(no. of larvae) Mean

0 1.34 a

5,000 1.35ab

10,000 1.11 c

15,000 1.23 c

aData are means of five replicates
log (x + 1). Means with different letter
at 5%level with Duncan's multiple rang

j vaneues. However, tne gau maex i
Brazilian was much lower than R
Cardinal and Black Ribier.

Histopathology and symptomatology
M. incognita infection of roots caus
gall formation. Histopathology of t
galls showed the nematode's orientati
in feeding, manner of penetration a
the damage rendered to the host tissu
Infected tissues were usually observe
in young lateral roots near the root r
(Fig. 2). Cross section of infected roi
showed that the pericycle appeared
be the primary site of feeding and soui
of nutrition for both nematodes. Forn
tion of syncytia is a characteristic res
of infection by both nematode speci
However, syncytia of R. reniformis (F
3) were found to be relatively small
than those induced by the root-knot 1
matode, M. incognita (Fig. 4). The wit
and length of the multinucleate cA
of R. reniformis ranged from 1.43-1.
u and .62-2.64 u, respectively, with
average width of 1.05 u and an aver,

is on top and root weights of Black Ribier a

Decrease Decrease
(%) Mean (%)

1.39 a

1.25 ab 10.0

16.0 1.05 c 24.0

8.0 1.21 b 13.0

transformed value based on common
column indicate significant difference


Iwa au A. renuurrmas on %J'

g. 2. Nematode feeding on portions of Red
ofM. incognita (arrow). B. adult female


. 3. A longitudinal section of a grape
root showing R. eniformisinduced
syncytia (X0000).

L -

rdinal grape roots. A. an adult female
1. reniformis (arrow).

4. A longitudinal section of a grape
root showing M. incognita-induced
syncytia near the vascular tissues
(arrow) (X10000).


Table 2. The effect of Meloid

Inoculum level per
(No. of egg masses)


ans with differ

7.62 c 24.74 c 4.8 37.0 a

rent letters in column are significantly different at 5% level, DMR

bridge (1975) have noted that this is a characteristic result of infection b
a common response of host plants to the nematode.


BIRCHFIELD, W. 1972. Differences in host-cell response to the reniform nematode
Phytopathology. 62:747 (Abstr.).

BIRD, A. F. 1974. Plant response to root-knot nematode. Ann. Rev. of Phytopatho

KATIGBAK, J. U. and C. L. GONZALES. 1973. Economics of growing grapes. An. Husl
and Agr. J. 8:24-25.

LIFORD, M. B. and J. M. OLIVEIRA. 1940. Rotylenchulus reniformis, nov. gen. n. sp
a namatndpt nnracitf nfn rnAtnf alminthA1 nr WU/ah PDrr'r T/C A.

JLf r .-Ajr-tn ru D P-4r4-J.M; A-

hsn--hno 1

infection by endoparasitic sessile nen
todes. Moreover, Bird (1974) report
that this process involves a chain
events starting with nuclear and nucleol
enlargement followed by cell wall brea
down, synchronous mitosis and incorp
ration of adjacent cells. Similar obsen
tions have also been reported by Ras]
et at (1973) in California, where thr
species of the root-knot nematoi
have been found to infect grape roc
These syncytia of grapes, as infecti4
of the host tissues advances, may seo
affect the vascular bundles and th
may result in a partial or complex
blocking of the flow of water and nt
rients from the soil.
Likewise, R. reniformis was all
observed to affect top and root weight i
the inoculated plants. The nemato(
infection of young root tips in grap
is consistent with the observation i
Birchfield (1972) who reported th
the nematodes showed no specificil
to the region of maximum invasion bi

length of 1.52 u. The nematodes we
mostly observed in the inner corti
The tissues responded to M. incogn
infection by changing from normal cA
to highly specialized syncytia, or gi0
cells which are sometimes referred to
multinucleate transfer cells. In the ci
of grape roots, dimensions of syncy
formed in infected tissues ranged fr(
.47-1.99 u in width and 0.57-3.33 u
length and 1.70 u average diameter. T
nematode was usually observed in t
cortical and cambium layers.


The results of the inoculation te
indicate that M. incognita and R re
formis can suppress the vegetative grow
of grape plants. The root-knot nemato
was observed to cause trace to modern
galling in all the varieties tested. Histoi
gical studies of the infected galled ro(
revealed that syncytia or giant cells a
formed around the head of the feedi
_ _-3_ "_^ T .- j-_- -I I

Philippine Phytopathology

MAI, W., H. H. LYON and T. H. KRUK. 1960. Pictorial key to genera of plant-parasite
nematodes. Ithaca. N. Y., Cornell Univ. p. 55.

PCARR. 1975 a. The Philippine Recommends for Grapes. p. 70.

PCARR. 1975b. Proceedings of Grape Researchers meeting. Dec. 12-13, 1975. Cebu City.

RASKI, D. J., W. B. HART and A. N. KASIMATIS. 1973. Nematodes and their control
in vineyards. Calif. Agr. Expt. Sta. Ext/Serv. Circular 533. p. 3.

REBOIS, R. V., P. A. MADEEN and B. J. ELDRIDGE. 1975. Some ultra-structural
changes induced in resistant and susceptible soybean roots following infection by
R. renifonnis J. Nematol. 7:133-138.

THORNE, G. 1961. Principles of Nematology. New York. McGraw-Hill. 553 p.

SASS, J. E. 1964. Botanical microtechnique (3rd ed.). Ames. The Iowa State Univ.
Press. 228 p.

Vol. 16

Phiipp. Phytopathol 16: 49-52
Received for publication: 10 July 1980


Araceli R. Pua and Lina L. lag

Research Associate, Institute of Plant Breeding and Associate Professor, Depart-
ment of Plant Pathology, University of the Philippines at Los Baflos, College, Laguna.


Pseudocercospora psophocarpi (Yen) Deighton formed the highest number
of conidia in winged bean leaf-extract-agar medium after 21 days.
Abundant sporulation was noted at 25 and 30 C with maximum spore
production at 30 C. No growth was observed at 35 C. Conidial production was
observed from pH 5.5 to 8.0 although sporulation at various pH levels was tem-

Welles (1925) observed a species of
Cercospora on winged bean in the Philip-
pines and noted that the pathogen caused
serious damage on this host. Four decades
later Borlaza and Roldan (1964) reported
a Cercospora leafspot which they attri-
buted to a new species, C. aranetae.
Deighton in 1976 considered C aranetae
similar to C psophocarpi which Yen in
1967 found in Singapore and thereby
renamed the causal fungus, Pseudocer-
cospora psophocarpi (Yen) Deighton
(Price, 1978).
The disease has been observed to
affect only the leaves. The initial symp-
tom is the appearance of tiny yellow-
ish areas. Spots are usually angular which
turn black on the undersurface of the
leaf when the fungus sporulates. Spots
on the upper surface appear smooth,
pale brown, becoming darker brown with
The present study is based on an
isolate of P. psophocarpi obtained from
the Central Experiment Station, Univer-
sity of the Philippines at Los Bafos,

Laguna. The morphology of the patho-
gen and the symptoms caused are similar
to those previously reported [Borlaza
and Roldan (1964), Deighton (1976)].


Conidia from the lower surface of
infected leaves were scraped off with
a sterile scalpel and aseptically placed
in a test tube containing sterile distilled
water. Two loopfuls of the suspension
were streaked on V-8 juice agar slant.
Cultures were incubated at room tempe-
rature (23-300C).

A pathogenicity test was carried out
to ascertain that the isolate was the
cause of winged bean leaf spot. The ino-
culum used was a conidial suspension
from 10-day old cultures grown in V-8
juice agar. Both lower and upper leaf
surfaces of 6-month old winged bean
plants were sprayed with the inoculum.

Phfllmine Phvtooatholoav

vTnt i1

Media* 6 days 9 days

V-8 16.5 67.5
MEA 0.0 5.0
CZA 0.0 6.5
GlyA 28.5 76.0
Onion A 3.5 27.0
WLEOA 41.5 196.0
CTHA 36.0 287.0
PDA 0.0 0.0
WLEOA +D 9.5 65.5
WGPOA 0.0 7.0

Onion A

12 days 15 days 18 days

65.5 46.5 36.5
6.0 21.0 48.5
2.5 0.5 99.5
83.0 53.5 59.5
40.0 42.5 43.0
182.0 275.0 428.5
275.0 235.5 242.5
2.5 1.5 0.5
55.0 111.5 135.5
29.5 125.5 344.5

V-8 Juice agar
Malt extract agar
Czapek's agar
Glycerine agar
Onion agar
Winged bean leaf extract oatmeal agar
Carrot tuber homogenized agar
Potato dextrose agar
Winged bean leaf oatmeal agar + dextrose
Winged bean pod extract oatmeal agar

Philippine Phytopathology

Table 2. Sporulation of Pseudocercospora psophocarpi as affected by temperature and
pH (spores/ml)

15 C 20 C 25 C 30 C 35 C

5.5 0 2,000 22,500 57,500 no growth
6.0 0 1,500 18,500 159,000 no growth
6.5 500 500 10,500 122,000 no growth
7.0 500 500 29,500 55,500 no growth
7 0 3 500 27 000 38000 no growth

I Jlt J.. J I V I- IatII. J ..UtJt LU .It LI UJ. J"lltUJt5 AU "Ji ala. jl. D UI5 I r UUIIUA LLI.1 %U.,
Minneapolis, Minn.

WELLES, C. C. 1925. Taxonomic studies of the genus Cercospora in the Philippine
T-l----.J- 1 -- ---- T n....la "1'1. r ( 't L 1 0

Vol. 16

rnuipp rnytopa

R. G. Davide and R. A. Zorilla

actively Associate Professor and Research Assistant, Departm
University of the Philippines at Los Bafios, College, Laguna.

irted by the National Crop Protection Center Research Fund.



Cabbage is widely grown in Nueva creased cabbage yield by 50.8% when
Ecija, Ilocos, Benguet and Mountain applied at the rate of 7.5 kg a.i. per
Province in Luzon and in some parts of hectare.
the Visayas and Mindanao. The 1972 This study was conducted to deter-
report of the Bureau of Agricultural Eco- mine the effectiveness of four nema-
, I f - ----.. 1... T'-_'1- t f l .... 1A fNf

are piantea to caooage m me rnuppmes
with yields varying from 1,511 to 8,388
kilograms per hectare. Among the factors
responsible for the wide variation in
yield are pests and diseases. It has been
demonstrated locally and abroad that
nematodes such as Pratylenchus pene-
trans, Trichodonms christie, and Meloi-
dogyne incognita could cause significant
yield reductions in cabbage (Christie
& Perry, 1951; Acedo & Rhoade, 1968;
dela Rosa & Davide, 1969; Davide &
Quebral, 1970). In a nematicide test
conducted in Atok, Benguet Davide
& Comedis (1972) showed that Temik
10G controlled Pratylenchus sp. and
Trichodorus sp. and significantly in-

ruiauain 3u anu vyuaeL luu, aganuiL
Meloidogyne incognita and other nema-
todes associated with lowland cabbage
and also to evaluate their effects on yield.


The experiments were conducted in an
area with sandy loam soil near the green-
house of the Department of Plant Patho-
logy, College of Agriculture, U.P. at Los
Baflos. The area was divided into 1 x 4
meter sublots, each lined with concrete
cement blocks. A 30-cm space was pro-
vided between plots as a passageway.
Using a randomized complete block
design, the following nematicides were


Phlippine Phytopathology

tested: Temik 10G (10% 2-methyl-2
(methylthio) propionaldehyde O-(methyl
carbamoyl) oxime), Vydate 10G (10%
S-methyl 1-(dimethyl carbamoyl)-N-
[(methyl carbamoyl)] Thioforminidate),
Furadan 3G (3% 2,3-dihydro 2,2-dime-
thyl-7-benzofuranyl methyl carbamate),
and Mocap 10G (10% O-ethyl S, S,
dipropyl phosphorodithioate). The rate
used was 40 g per 4 sq m plot for Temik
10G, Vydate 10G and Mocap 10G while
Furadan 3G was used at 60 g per plot.
Each chemical was evenly spread and
incorporated into the top 8 cm soil a
day before planting the test plants. For
pre-treatment determination of the soil
nematode population, a total of 400 cc
soil samples were collected from three
sites per plot and after treatment, sub-
sequent sampling was done at monthly
intervals until harvest. The area was pre-
viously planted to susceptible tomato
for 3 months to insure adequate build-
up of nematode population.
A day after treatment, each plot was
planted with 7-week old cabbage seed-
lings (vars. KY for the first trial and KK
for the 2nd and 3rd trials) grown in small
plastic bags where each plant was ino-
culated with two egg masses of M. incog-
nita before planting to insure uniform
infection of the plants. Each treatment
including the control was replicated
three times and each replicate plot had
two rows of cabbage planted at 30 cm
apart. Each row contained eight plants
giving a total of 16 plants per plot.
Throughout the growing period, the
plants were regularly fertilized with
ammonium sulfate and sprayed with
insecticide Sevin or Thiodan and fungi-
cide Benlate to prevent insect attack and
fungal infection. All soil samples collec-
ted were processed for nematodes in the
nematology laboratory using the sieving-
Baermann funnel combination. To deter-

mine the degree of root-knot nematode
infection after harvest, each plant was
carefully uprooted and root gall index
was taken as follows: 1 for no gall; 2 for
trace (1-25%), 3 for slight (25-50%),
4 for moderate (50-75%) and 5 for severe
(75-100%). The effect of the treatment
on yield was determined by weighing
individual cabbage head at harvest and
the data were later summarized and sta-
tistically analyzed for significance test.
The study consisted of three trials from
July 1978 to March, 1980.


Effect of the treatment on root-knot
nematode infection
The mean gall index ratings of the
plants in three trials clearly indicate that
the nematicidal treatment had consider-
ably reduced the root-knot nematode
infection (Table 1). Generally, the mean
gall index ratings were much lower in
the first trial than in the succeeding
trials. This may be due to the fact
that the experiment which was conduc-
ted during the rainy months (July to
October) when too much rainwater in
the soil could have affected the nema-
tode population. However, in the second
and third trials when the experiments
were conducted towards the dry season
(November-March), most plants in the
non-treated control showed moderate
to severe infection. In this case, Temik
10G-treated plants had the lowest gall
index (1.6) and more plants showed no
galls; Vydate 10G-and MocaplOG-treated
had trace to slight galling index, while
majority of the plants treated with Fura-
dan 3G had slight galling.

Effects on other parasitic nematodes
Evidently, all the nematicide treat-
ments greatly reduced the populations of

Vol 16

Four Nematicides on Cabbage

Mocap 10G 1.8 2.7 2.1
Furadan 3G 1.8 2.9 2.7
Control 2.4 4.6 4.6

a KY variety was used in the first trial and KK for the second and third trials.
bThe gall index ratings are as follows: 1 for no galls; 2 for trace (1-25%); 3 slight
(25-50%); 4 for moderate (50-75%) and 5 for severe (75-100%). Data for each trial
are means of 3 replications.

Jan-June 198(

BPhBlinhia tPhwtm~mthdlA

Temik 10G 12.7 a 31.2 10.4 a 87.5 17.5 a 61.9
Vydate lOG 11.7 b 21.1 9.5 ab 72.8 14.4 b 32.6
Mocap 10G 9.9 c 2.9 8.7 bc 56.8 13.8 bc 27.8
Furadan3G f1.6 b 20.0 8.3 c 49.8 13.5 bc 24.7
Control 9.6 c 5.5 d 10.8 d

aData of each trial are mean of three plot replicates. Means followed by the same letters
in a ,nllimn arp nnt ainnificantlv lifF Aff ant at / la.al .,+k TLDT

LrAl I&

Four Nematicides on Cabbage

Table 2. Mean nematode counts before and after soil treatments with

nematicides on cabbage variety KK a

Nematodes per 400 cc soil
Treatment and Before After Treatment
Nematode Genus Treatment Ist month 2nd month 3rd month

Temik lOG

Meloidogyne b

Vydate lOG


Mocap lOG


Furadan 3G








Meloidogyne 1,220.0
Helicotylenchus 48.6
Rotylenchubus 79.3
Trichodons 9-3
Pratylenchus 13.6
Hoplolaimus 2.6
Total 1,373.4

183 11.6 9.6
6.6 7.6 8.3
7.6 7.6 8.3
0.6 0 03
0.6 23 1.6
2.0 0.6 0.6

35.7 29.7 28.7

303 25.6 16.3
12.0 10.6 12.0
13.0 10.0 12.6
13 0 1.3
0 23 23
1.0 2.0 1.6
57.6 50.5 46.1

74.5 77.0
113 11.6
14.6 18.0
0 0
1.0 1.6
.3 0.6
101.8 108.8

106.3 97.3
14.6 153
11.0 13.0
1.6 1.6
1.6 .6
.6 1.0
135.7 128.8

1,379.0 1,548.6
733 71.6
84.6 973
15.0 14.6
15.3 15.6
2.3 3.0
1,569.5 1,750.7




aData from third trial.
bSecond stage larvae.

Jan-June 1980

Philippine Phytopatholol


ACEDO, J. R. and R. A. RHODE. 1
gica 14:1 (Abstr.).

CHRISTIE, J. R. and G. V. PERRY. 1
of the genus Trichodorus. Scien<

with vegetable crops in Benguet.

DAVIDE, R. G. and A. COMEDIS. 1,
Atok, Benguet. Phil. Agric. 55:2

parasitic nematodes on vegetable

DI SANZO, C. P. 1973. Nematode resl

RHOADES. H. L. 1977. Comparison


Lesion nematode injury to cabbage. Nemal

. A root disease of plants caused by a nemal

970. Some plant parasitic nematodes associ;
ric. at Los Bafios. 9:12-14.

. Chemical control of nematodes on cabbaE

1969. Pathogenicity test of four genera of p
ips. Phil. Phytopathol. 5:29-38.

se to carbofuran. J. Nematol. 5:22-27.

granular and transparent water application
oiling the sting nematodes Belonolaimus lo
Inn ,i-ri Qnr Plnrrli. "'.410NI_ N.



L. T. Talens and Angelita (

Virus Biocontrol Laboratory, Institute c
)pines at Los Bafios, College, Laguna, Philipp


The viral agent causing mosaic in E,
gated. E. mollis mosaic virus was found t
to abaca (Musa textiles), Capsicum annuu
Tetragonia expansa, and three species o
amaranticolor, C. murale and C. quinoa w
symptoms incited by E. mollis mosaic viri
The properties of the virus in cru
dilution end-point at 10-2; thermal death
in vitro was at 3 hours. Electron microscot
preparations showed isometric particles 30 1
The successful transmission of E.
provides the first direct experimental evi
virus causing mosaic in this important expi
This virus might prove to be the answer tc
ing the viral etiology of the abaca mosaic di

Le occurrence of a mosaic disease the
n uncultivated broad-leaf weed nu
hantophus mollis BHK.) commonly of
I growing in abaca and coconut ob
nations in Southern Luzon was first ful
ved by del Rosario in 1963. Al- fe,
gh the viral etiology of the disease are
established then, a limited study evi
ie biological properties of the virus mi
reported only recently (Protacio, Kc
). The disease received little atten- cal
despite claim by Protacio (1977) vil
a viral agent was transmitted to
I and coconut causing mosaic and
ig-cadang types of symptoms, res- pr
vely. ap
-cause E. mollis grows wild in wide sy



'lant Breeding, University of the

hantophus mollis was investi-
ie mechanically transmissible
several species of Nicotiana,
7henopodium. Chenopodium
local lesion host plants. The
infection in N. glutinosa were

sap were found as follows:
nt at 50-55C; and longevity
if negatively-stained specimen
in diameter.
His mosaic virus into abaca
Ice of an unstable, isometric
fiber plant of the Philippines.
e long-standing puzzle regard-
ie in the Philippines.

icol region where abaca and coco-
re two major plantation crops and
a desirability to confirm Protacio's
ration in 1977, we investigated
r the biological and morphological
'es of E. mollis mosaic virus. We
iterested in acquiring experimental
ice that other viruses) can cause
c in abaca (Musa textilis Nee).
(1968) and Smith (1972) indi-
that two morphological types of
s incited mosaic in abaca.

is report describes the physical
rties of E. mollis mosaic virus, its
transmissibility, host range and
tomatology, and particle morpho-

Particle morphology was deterrn
using partially purified specimen pre]
Virus indicator plants tions. Specimens were deposited <
Seeds of test plants belonging to collodion-coatqd 400-mesh elec
Aizoceae (Tetragonia expansa), Chenopo- micrscope copper grids and were sta
diaceae (Chenopodium amaranticolor, with 2% neutral phosphotungstate I
C. murale, C quonia), Amaranthacea to examination in a JEOL 100-U


were collected

abaca plants. Aphid trans
were brought to the Cotton i
Institute of Plant collected fi
y of the Philippines the Depar
were kept in potted Aphids wei
imigation with Dow- to 10-15 m
was maintained in infected E.
n Tetragonia expansa. aphids wen
issues showing symp- E. mollis a
actions were excised inoculation
heat-sterilized mortar killed by sl
A phosphate buffer, drin accnrd

r in Elenhantnnhus molli.q

1 vein-banding (Fig. la). Arti- local lesions.
xculated E. mollis plants main- showed system
he greenhouse showed a yellow begins from t
lb). At an advanced stage of ding outward
action, plants became stunted secondary vei
Emerging leaves were reduced a severe nec
id nale areen or chlorotic in premature de

la. Close-up of an infected leaf Fig lb. Artificially-inoculated E. mollis
showing mosaic symptoms and plant with mosaic and yellow
roon vpin-hnndinr snottinei


Phllionine Phvtonatholci

rig. Z. LucuI Aus5tnis IrLlcu oUy c. m,
mosaic Virus infection on inoc
ted leaf of Chenopodium ama
ticolor. Some lesions were
rounded with a necrotic r

Abaca, Musa textilis (Fig. 9), seedl
developed mild mosaic symptoms wl
was sometimes followed with the app
ance of light green to yellow strn
running perpendicular to the mii
of the leaf (Fig. 9).
The following plant species were
infected with E mollis mosaic vi
Gomphrena globosa, Cucumis sati
C melo, C pepo, Phaseolus hma
P mungo, Vigna radiata, V. unguicul
V. umbellata and Zea mays. No atten
were made to check these symptom
plants for E. mollis mosaic virus
back-inoculation into local lesion hc

Physical properties
The properties of the virus in cr
sap of infected E. mollis were as folio
infectivity was abolished after heating
50-55 C; infectivity was lost after c
tion to 10-3 but not to 10'2; and in
tivity was not detected after 3 h(
of storan at 25 CV

- *. ta wslUIF|ItVI IIf, 1Jc/!ullJ s{mLC-U
E. mollis mosaic virus infeci
on inoculated leaf of Chenopodi

Aphid transmission
In several experiments on non-pel
tent transmission of E. mollis mo!
virus using Aphis gossypii as the veci
short feeding probes by the vector
peared sufficient to transmit the vi
inciting mosaic symptoms in E. mc
seedlings. When infected plants w
back-indexed on C amaranticolor, k
lesions typical of the virus infecti
were noted.

Particle morphology
Electron microscopy of specimen I
parations negatively-stained with
neutral phosphotungstate revealed 1
and empty isometric particles 30 nm
diameter (Fig. 10). The majority of
virus particles were noted to be emp
The low proportion of full to eml
particles might be related to the 1
infectivity of partially purified vi

:i ~4


:in Elephantophus mollis



gg. 4-8. Symptoms produced by E. mollis m
(4), Nicotiana glutinosa (5), N. rustic

ic virus infections in Capsicum annuum
N. rustic (7), and Tetragonia expansa


Philippine Phytopathok

Fig. 9. Symptoms produced by E. I
mosaic virus infection in a
Musa textilis. Note pale-gree
yellow streaks running parallel
the veins from the midrib tt
margin of the leaf


is Fig. 10. Electron micrograph of E.
2, mosaic virus. Note the preset
o empty (e) and full (f) virus
h cles. The majority of partic
re empty.

able cadang-cadang type of sym]
in onn/' llnt

runy transrttea me virai agent ino virus possesses biological and ph:
abaca and coconut causing mosaic and properties and morphological fea
cadang-cadang types of symptoms, res- comparable to those of the virus
pectively. So far as is known, no attempts cribed previously (Celino, 1940; 1
were ever made to confirm the results Ocfemia, 1953). Celino (1940, 1
of Protacio's seemingly insignificant and Ocfemia (1953), however, did
observations in 1977. We have not visualize by electron microscopy
pursued the transmission of E. mollis morphology of the viral agent. Tc
mosaic virus into coconut for several knowledge, Protacio (1977) did
reasons among which the viroid etiology examine his virus isolate in the elei
of cadang-cadang (Randles, 1975) ap- microscope and, therefore, this is
peared established. This does not pre- first report of the transmission ol
clude the possibility, however, that isometric particles of E. mollis m
E. mollis mosaic virus as Protacio (1977) virus into abaca. Furthermore,
claims might in fact cause indistinguish- properties of the virus in crude


ic in Elephantophus mollis

nely: (1) the virus is unstable, The isometric particles of E. mollis
the virus occur in low concentra- mosaic virus and the flexuous rod-shaped
a in leaf extracts, and (3) the virus particles of abaca mosaic virus (Eloja
Sa low thermal stability, are almost and Tisley, 1963) might shed some light
ntical to those of cucumber mosaic into Ocfemia's (1953) notations that
I tomato aspermy viruses (Smith, abaca mosaic virus possessed features
72). Several attempts to identify similar to bean yellow mosaic virus and
mollis mosaic virus by agar gtl solu- tomato or chrysanthemum aspermy virus.
diffusion (Ouchterlony, 1962) and Interestingly, bean yellow mosaic virus
interimmunoelectrophoretic (Talens, and tomato or chrysanthemum aspermy
79) assays using reference antiserum viruses possessed flexuous rod-shaped
inst cucumber mosaic virus have and spherical particles (Smith, 1972),
ed thus far. respectively. In our limited transmission
The literature on the mosaic disease experiments using 4 strains of sugarcane
abaca records several weed species mosaic virus, the development of mosaic
ich can be infected with a virus from symptoms was not observed suggesting
Id-infected abaca. Likewise, viruses that the virus described by Eloja and
'bored by these weed species have Tisley (1963) might be an unusual strain
mn transmitted into abaca. These weeds of sugarcane mosaic virus.
1w wild in abaca plantations through- Finally, the control of mosaic in abaca
t the Philippines. Unfortunately, the will depend on a thorough knowledge of
al etiology of the diseases of weeds its precise viral etiology. The finding of
I abaca had not been systemically the isometric particles of E. mollis
estimated except perhaps for a strain mosaic virus as causing certain mosaic
sugarcane mosaic virus which had types in this important export fiber plant
en described to cause abaca mosaic of the Philippines could be the significant
ease (Eloja and Tisley 1963). Thus, evidence needed to re-examine the abaca
Mosaic disease of abaca, as Kent mosaic virus disease complex. We are
)54) had described it, appears to now attempting to study the stabilization
'olve a complex of several viruses or of the virus and to prepare specific anti-
us strains. serum for serodiagnostic purposes.


)S, L., D. J. HAGEDORN, and L. QUARTZ. 1960. Suggested procedures for inter-
national identification of legume viruses. T. P1. Ziekten. 66:328-343.

,LINO, M. S. 1940. Experimental transmission of the mosaic of abaca or Manila hemp
(Musa textilis Nee.) Philipp. Agric. 29:379-413.

ILINO, M. S. 1956. Mechanical transmission of the abaca mosaic virus. Philipp. Agric.


Philinnine Phvtoonathoh

ELOJA, A. L. and T. W. TINSLEY. 1963. Abaca mosaic virus and its relations
sugarcane mosaic. Ann. Appl. Biol. 51:253-258.

KENT, G. C. 1954. Abaca mosaic. Philipp. Agric. 37:555-557. i7.

OCFEMIA, G. 0. 1953. Note on the occurrence of the mosaic disease in the I
Experiment Station in Banao, Guinobatan, Albay. Philipp. Agric. 37:84-87.

OUCHTERLONY, 0. 1962. Diffusion-in-gel methods for immunological analysis
Progr. Allergy. 6:30-154.

PROTACIO, D, B. 1977. Evidence of cadang-cadang disease as coming from weed
phantophus mollis. Philipp. Phytopathol. 13:1 (Abstr.).

RANDLES, J. W. 1975. Association of two ribonucleic acid species with cadang-c,
disease of coconut palm. Phytopathology. 65:163-167.

SMITH, K. M. 1972. A textbook of plant virus diseases. 3rd ed. Acad. Press. 68

TALENS, L. T. 1979. Rapid detection of tobacco mosaic virus infections in on
Philipp. Phytopathol. In press.


pp Phytopathol


Tricita H. Quimio and Matea M. Nadayao

Associate Professor and former Graduate Student, Department of Plant Patholo-
f, University of the Philippines at Los Bafios, College, Laguna, respectively.

Powdery mildew is a very common veloped basipetally from the meriste-
sease of tomato in the Philippines. The matic region of the conidiophore, re-
sease is usually observed during the maining abstricted and passively attached
y summer months when considerable to the conidiophore until dislodged by
mage in the form of wilting during air currents or by disturbance of the
vere infection results. The disease was leaves.
st reported in the Philippines by Based on a key utilizing conidial stages
sinking (1918), who identified the of the organism (Yarwood, 1978), the
usal organism as Erysiphe sp. (Oidium above descriptions fit both Erysiphe
). Although no detailed study has ever cichoraceaum and Sphaeotheca fulige-
en made on the disease, subsequent na, except that no cylindrical fibrosis
ports recorded the causal organism as bodies characteristic of the latter were
eveilula taurica (= Oidiopsis taurica observed on the tomato isolates. Like-
ev.] Salm. (Orillo et al., 1959, PCARR, wise, conidia of E. cichoraceaum germi-
>75, and Quebral, 1971). nate by a single germ tube with incons-
From an investigation by the authors picuous appresoria, while germ tubes of
1978-79, it is here reported that a S. fuligena are forked (Sitterly, 1978).
tusal organism of powdery mildew of The host range study (Table 2) showed
imato in the Philippines is Erysiphe that the powdery mildew organism from
choraceamm DC ex Mecat, the same tomato infects okra (Hibiscus esculentus
rganism attacking a majority of cul- L.), white potato (Solanum tuberosum
vated crops, such as the family Sola- L.), papaya (Carica papaya L.) and
iceae (Dixon, 1978), Compositae tobacco (Nicotiana tabacum L.). The
)ixon, 1978) and the family Cucurbita- above four host species are reported
eae (Sitterly, 1978). The conidial for E. cichoracearum (USDA Host
age, Oidium, is, however, the only Index, 1960). No infection, however,
arm observed. Based on critical morpho- occurred on eggplant, mungbean, cow-
igical studies of the asexual stage of a pea, mulberry, squash and watermelon.
sw collections (Table 1), the following The latter two crops are also reported
descriptions were obtained (Fig. 1.): hosts of E. cichoracearum but it is
3nidiophore, 88.38 x 9.02 y, un- possible that the tomato powdery mil-
ranched, composed of 3-4 cylindrical dew strain used was not infective on
ells, hyaline, arisingOut of superficial these hosts and/or more repeated inocula-
yphae; conidia 38.13 11 x 15.67 p1 tions on several host varieties should have
arrel-shaped, chain-forming, hyaline, de- been done.

Table 1. Collection of powdery mildews on different varieties of tomato from dif:
places in the Philippines


UPLB, Laguna Marikit 6-13-79
VC-11-1 7-6-79
VC48-1 8-23-79
Cebu T 8-27-79

Ilagan, Isabela Pearl Ha

Dingras, Ilocos Norte C-11D

Tnril favan Pitv A T71'




Acc. 292 11-21-7!
Acc. 320 11-21-75
Acc. 303 11-21-75

,^ Leveillula taurica (= Oidiopsis j
Swas definitely not the ones collect
Studied, since the characteristic n
S' logy of Oidiopsis conidiophoree
South of the stomata and non-chi
"' -- ming) was never observed from
collections made.

m !
S~iS^ ik/^.I

rig. j. Lonraopnore ana conmaa of me

70 Philppine Phytopathology VoL 16

QUEBRAL, F. C. 1971. Some common diseases of tomato. Agriculture at Los Bafios.

REINKING, 0. A. 1918. Philippine economic plant diseases. Philippine J. Sci.

SITTERLY, W. R. 1978. Powdery Mildews of Cucurbits. In Spencer, D. M. (ed.). The
Powdery Mildews. pp. 359-377. Acad. Press, New York.

USDA. 1960. Index of Plant Diseases in the United States. USDA Plant Dis. Survey.
UTnAhnk^L 1112 n

YARDWOOD, C. E. 1978. History and Taxonomy of Powdery Mildews. In
M. (ed.). The Powdery Mildews. pp. 1-32. Acad. Press, New York.

licaton: 5 Nov. 1980


L. T. Talens and Angelita C. Dolores-Talens

iocontrol Laboratory, Institute of Plant Breeding, University of
s Baflos, College, Laguna.
hors are grateful to Mr. Nopchai Chansilpha for his assistance.
d the best paper in Plant Pathology in 1980 by Philippine Phytc
at the Annual National Conferences of the Pest Control Counm
,id at Hotel Mercedes, Cebu City, 23-26 April 1980. The paper
Ling form.

Counterimmunoelectrophoretic assay (CIEPA) was successfully used
to detect six Philippine plant viruses infecting legumes cowpeaa and mung-
bean), solanaceous plants (pepper, tobacco and tomato), and orchids. It
was demonstrated to be a specific, sensitive, reliable, rapid and simple tech-
nique for the routine diagnosis of these viruses. Likewise, CIEPA was useful
in the diagnosis of virus diseases with single or mixed infections in a single-
step, 30-minute operation.
The technique was capable of identifying three morphological types
of plant viruses, namely: the isometric particles of cowpea mosaic virus,
mungbean mottle virus, and southern bean mosaic virus; the rigid rod-
shaped particles of the orchid and yellow strains of tobacco mosaic virus;
and the semi-flexuous rod-shaped particles of Cymbidium mosaic virus.
Results of CIEPA compared well with, if not better than, those of
microprecipitin and agar gel double diffusion tests. With the exception of
the yellow strain of tobacco mosaic virus and southern bean mosaic virus
in which infectivity assay was consistently found to be somewhat more
--nnitive CIIT.PA semed anual or better than infectivity asav for tnhac-

Philippine Phytopathology

The remarkable characteristic of
serological reaction is the specific inter-
action of antigenic determinants that are
resident in the virus structural proteins
and its homologous antiserum. For
instance, antibodies react only with the
antigen which was used for immuniza-
tion or with closely-related antigens.
Serological procedures such as
microprecipitin test under paraffin oil
(van Slogteren, 1955), agar gel double
diffusion test (Ouchterlony, 1962),
radial-immuno-diffusion test (Shepard,
1974; Purcifull and Batchelor, 1977),
bentonite-flocculation test (Bozicevich
et al., 1960), latex-flocculation test
(Singer and Plotz, 1956, Bercks and
Querfurth, 1969; 1971), enzyme-linked
immunosorbent assay (ELISA) test
(Voller et al., 1977), serologically spe-
cific electron microscopy (Ball, 1974;
Brlansky and Derrick, 1974; Derrick,
1973; Derrick and Brlansky, 1976), and
others (Ball, 1974) have been developed
and used specifically in the control and/
or prevention of virus diseases through
indexing procedures (Hollings, 1965),
meristem culture and/or thermotherapy
(Kunkel, 1935; 1936; Morel, 1948;
Bawden, 1964; Hollings, 1965), virus
diagnosis (Wetter, 1965), and surveillance
of viruliferous insect vectors (Gera et aL,
1978; Clarke and Converse, 1980).
Of the various serological tech-
niques, immunoelectrophoresis in agar
gels has not been a popular tool in plant
virus diagnosis. Its application, however,
in the clinical detection of microbial
infections in human beings is commonly
known. Wetter (1965) surmised that
immunoelectrophoresis has little value
except perhaps in the assessment of virus
purity or in the determination of the
electrophoretic homogeneity or hetero-
geneity of purified virus suspensions.
Recently, Talens et aL (1979)

in attempting to adopt a fast and routine
serodiagnostic procedure for mass testing
of imported orchids for TMV-O infec-
tion demonstrated that counterimmuno-
electrophoretic assay (CIEPA) was a
highly practical and valuable tool. Its
application in plant virus disease diag-
nosis has not been fully explored else-
The study described here attempts
to establish the optimum experimental
conditions in CIEPA test, to determine
its sensitivity and specificity in detec-
ting plant viruses of three different
morphological types, to compare its
sensitivity and specificity in detecting
plant viruses of three different morpho-
logical types, to compare its sensitivity
with two commonly-used serological
techniques (microprecipitin and agar gel
double diffusion tests) and infectivity
assay, and to demonstrate its applica-
tions in virus survey, virus identification
and mass-indexing.


Virus source
Several virus isolates originally ob-
tained from field-infected legumes (cow-
pea and mungbean), solanaceous (pepper,
tobacco and tomato) crop plants, and
orchids were used. They were cowpea
mosaic virus, CPMV-Sb (Talens, 1978),
mungbean mottle virus, MMV (Talens,
1978), cowpea strain of southern bean
mosaic virus, SBMV-cs (Talens, 1979),
orchid strain (Rillo, 1978) and yellow
strain (Talens, unpublished observation)
of tobacco mosaic virus, TMV-O and
TMV-ys, respectively, and Cymbidium
mosaic virus (Rillo, 1978).

Virus propagation and assay host plants
The virus isolates were propagated
in their appropriate host plants as

VoL 16

follows: which was use in the propagation o
(1) MMV on Vigna radiata (L.) TMV-O, every leaf was inoculated
Wilzcek cv CES-ID 21 14-21 days after with virus inoculum known to incit,
inoculation of 10-14-day old seedlings, the production of at least 200 loca
(2) CPMV-Sb on Vigna unguicu- lesions.
lata (L.) Walp. cv Red 14-21 days after
inoculation of 10-14-day old seedlings. Inoculation technique
(3) SBMV-cs on Vigna unguicu- Virus inoculum consisted of it
lata (L.) Walp. cv Blackeye 21-28 days fected leaf tissue homogenates whicl
after inoculation of 10-14-day old seed- were routinely diluted (1:10, v/v) ii
lings. 0.25 M phosphate buffer, pH 7.5. I
(4) TMV-ys on Nicotiana some instances, 1% mercaptoethanol o
tabacum cv White Burley and/or Lyco- 0.11% thioglycollic acid was added t<
persicon esculentum Mill. cv Marikit minimize virus inactivation. An abra
21-28 days after inoculation of 2-3- sive aid (Celite) was then added to
month old plants. final concentration of 1% (w/v). Celit
(5) TMV-O on Gomphrena glo- aided virus transmission by providing
bosa L. 10-14 days after inoculation of the wound for virus attachment ani
plants at the 4-6-leaf stage of develop- penetration.
ment. Inoculation was performed b;
(6) CYMV on Cymbidium or- rubbing two to three fully-expandei
chids and Tetragonia expansa L. 28-35 leaves of test plants with a cotton swal
days after inoculation of 12-24-month soaked in the virus-Celite suspension
old and 2-4-month old plants, respec- Inoculated leaves were rinsed thorough
tively. ly with tap water to minimize damage
Infectivity assays were performed due to rubbing and chemical toxicity
using the following assay host plants: Infected leaf tissues were hai
MMV on Cyamopsis tetragonoloba L., vested when symptoms of virus infec
CPMV-Sb on Chenopodium quinoa L., tions were evident. These symptom
SBMV-cs on Vigna unguiculata (L.) consisted of mottling, mosaic, veir
Walp. cv Clay. TMV-ys on Nicotiana banding, chlorotic to light green ring
glutinosa L., TMV-O on Gomphrena and spots stunted growth, reduction
globosa L., CYMV on Cassia occidentalis in leaf size which was sometimes ac
L. companies by distortion and defoi
Propagation and assay host plants mation and, as in the case of TMV-C
were grown in potted soil sterilized local lesions were produced only o;
by fumigation with methyl bromide inoculated leaves of Gomphrena.
(Dowfume). Test plants were inoculated Harvested leaf tissues were kep
at their most susceptible stages which inside air-tight plastic bags and wer
were usually 10-14 days after seeding stored frozen at -20 C until needed
for the legumes cowpeaa and mung- Frozen leaf tissues were usually prc
bean), 2-4 months for the non-legumes cessed after 1-2-day storage at -20 (
(Gomphrena, Cassia, Lycopersicon,
Nicotiana, Tetragonia, and Chenopo- Virus purification
dium), and 12-24 months for Cymbi- The virus isolates were purifie
dium orchids. In the case of Gomphrena according to the following schedules

74 Philippine Phytopathology Vol. 16

(1) MMV was extrated in 0.25 M phos- at 4 C. Low speed centrifugation was at
phate buffer, pH 7.5 containing 1% 5,000 rpm for 15-20 minutes in a Spinco
mercaptoethanol, clarified by emulsifi- SW 27 type rotor; high speed centrifu-
cation with 10% chloroform and low gation was at 27,000-30,000 rpm in a
speed centrifugation, and purified by Spinco Fixed Angle Type 30 rotor for
two cycles of differential high speed 2-3 hours. A differential cycle of centri-
centrifugation. Purified virus was re- fugation consisted of one high and low
suspended in 0.025 phosphate buffer, speed centrifugations. The centrifugation
pH 7.5 and finally clarified by low time was usually longer for the isometric
speed centrifugation. (2) CPMV-Sb was (MMV, CPMV-Sb, and SBMV-cs) viruses;
extracted in 0.1 M phosphate buffer, shorter, for the rigid rod-shaped (TMV-O
pH 7.0 containing 1% mercaptoetha- and TMV-ys) and semi-flexuous rod-
nol, clarified by treatment with 8.5% shaped (CYMV) viruses.
butanol (v/v) and low speed centri- Purified virus suspensions were
fugation, and purified by two cycles of stored at 4 C after addition of 0.025%
differential high speed centrifugation. NaN3 as a preservative. In other instan-
Purified virus was resuspended in 0.01 M ces, purified virus solutions were diluted
phosphate buffer, pH 7.0 and finally in buffered 50% glycerine and stored at
clarified by low speed centrifugation. -20 C. The addition of glycerine pre-
(3) SBMV-cs was extracted, clarified, vented freezing minimizing virus dena-
and purified in the same fashion as turation due to alternate freezing and
MMV. (4) TMV-O and TMV-ys were thawing.
extracted in 0.1 M phosphate buffer,
pH 7.5, clarified by emulsification with Serology
10% chloroform and low speed centri- Antisera against these virus iso-
fugation, concentrated by precipitation lates were prepared by immunization
with 6% polyethylene glycol (PEG) of rabbits with purified virus suspensions
6000 and 4% NaCI and low speed cen- containing 1-5 mg/ml protein. Six week-
trifugation, and purified by two cycles ly intravenous injections were adminis-
of differential high speed centrifugation. tered through the marginal ear veins. This
Purified virus was resuspended in 0.01 was commonly followed by a rest period
M phosphate buffer, pH 7.5 and finally ranging from 2-6 weeks. Finally, a boos-
clarified by low speed centrifugation. ter shot was given and blood samples
(5) CYMV was extracted in 0.5 M borate, were collected 7-10 days after the last
pH 8.2 containing 1% mercaptoethanol, injection. Periodic bleedings were also
clarified by emulsification with 10% carried out to follow the progress of anti-
chloroform and low speed centrifugation, body production. Bleeding was made
concentrated by precipitation with 6% by making a horizontal slit along the
PEG 6000 and 4% NaCI and low speed marginal ear veins with a sharp razor
centrifugation, and purified by two cycles blade.
of differential high speed centrifugation. Blood was allowed to clot by
Purified virus was resuspended in 0.05 standing for 2 hours at room tempe-
M borate, pH 8.2 and finally clarified rature or by storage at 4 C for 12-16
by low speed centrifugation. hours. Antisera were carefully aspirated
All centrifugations were performed with a Pasteur pipet after centrifugin
in a Spinco Model L5-65 ultracentrifuge clotted blood at 10,000 rpm for 15-20

lutes. Antisera were kept frozen at 48238). The bottom of all wells were
SC in 50% glycerine, sealed with a small drop of melted agar
Antiserum titer was determined before use.
no thea m;,nraeniitfn faet (van Dsantant wnrea Inarld into the

moistened cotton swabs were placed
inside the covered plastic Petri dishes.
This was found to be suitable, less cum-
bersome, and economical. Results of
titrations were read 2 hours after incu-
bation at room temperature and again 18-
24 hours after incubation at 4 C.

Ouchterony agar gel double diffusion
and counterimmunoelectrophoresis
The medium used in all serological
tests by agar gel double diffusion and
CIEPA tests contained 1.0% Noble agar
dissolved in 0.05 M borate buffer, pH
8.5, 0.025% NaN,, and 0.85% NaC1.
Agar was melted by heating suspension
in boiling water bath for 45-60 minutes.
In the Ouchterlony agar gel dou-
ble diffusion test, 12.0 ml of melted
agar was pipetted into 15 x 100 mm
scratch-free glass Petri dish. Wells (4-6
mm in diameter and spaced 4-6 mm edge
to edge) were cut with a cork borer or
with a Grafar Auto-Gel Punch (Grafar
Corp., Detroit, MI 48238) in a standard
pattern of six wells around a center well.
The bottom of all wells were sealed with
a small drop of melted agar prior to
deposition of appropriate antiserum and
antigen samples.
In CIEPA test, 10-12 ml of melted
agar was casted into each set of three
glass slides 25 x 75 mm in size. The slides
were placed in an immune frame holder
tightly fitted onto the immune leveling
table set of the LKB Immunoelectro-
phoresis Model 3946. Wells (4 mm in
diameter) were cut with a Grafar Multiple
Gel Punch (Grafar Corp., Detroit, MI

the wells. In instances where additional
quantities of test materials were to be
tested, the size of the antigen wells
were made larger to accommodate 500-
microliter quantities. The size of the
antiserum wells were always kept cons-
Electrophoresis was run at a cons-
tant current of 10 mA per 25 x 75 mm
agar slide. The direct current passing
through the agar bed might be increased
to 15 mA to shorten electrophoretic
runs which were normally scheduled for
30 minutes. In both cases, some heating
took place and this might be unsuitable
for heat-unstable viruses. Power was
supplied with a BioRad power supply
or with a Buchler voltage and current
regulated power supply Model No.
Recording of results. Thirty
minutes after each CIEPA run or 2448
hours after agar gel double diffusion test
was found sufficient time for the forma-
tion of precipitation zones. Agar gel
double diffusion plates and CIEPA agar
slides were photographed using a dark-
field illuminator. The illuminator con-
sisted of a rectangular box fitted with a
circular 40-watt fluorescent lamp and an
elevated flat black central area. High
contrast Kodak film 5069 was used for
photographic recording purposes.

Rectivity and specificty of prepared
All antiserum preparations were
found free of detentahle antihndiei tn

Vol. 16

lilippine Phytopathology

uninfected antigens (crude tissue homo-
genates from homologous and hete-
rologous source plants) or to extracts
which were concentrated approximate-
ly ten-fold by high speed centrifugation
at 100,000 g for 3 hours and 4C.
Antiserum samples used at a di-
lution of 1:80 to 1:120 reacted visibly
against homologous or closely-related
but not to heterologous purified virus
suspensions (Fig. 1). In further CIEPA
test, the precipitin lines were quite dis-
tinct when antigen concentrations ranged
from 1 to 500 nanogram protein per ml.
The precipitin lines (Fig. 1, MMV and
SBMV) tended to appear more diffused
and thick at high antigen concentrations.
In the case of CPMV (Fig. 1), the preci-
pitin zone was also diffused for two
reasons: firstly, the antigen concentra-
tion deposited in the antigen well was
high and, secondly, CPMV consisted of
two electrophoretic forms.
At high antiserum concentration,
the position of the precipitin lines was
closer to the antigen well. Likewise, the
lines were generally diffused. Hence,
adjustment of antigen and antiserum
concentrations for use in CIEPA could
be an important step in the precise
detection and identification of mixed
virus infections.
To further confirm the specificity
of the prepared antisera, cross-absorp-
tion tests were conducted. This was
performed by mixing diluted antisera
with an excess of homologous and
heterologous antigens or vice-versa. After
overnight incubation at 4 C, the residual
antigen or antibody titer was determined
in CIEPA test. It is clear from figure 2
that virus-specific antigens were removed
only with homologous but not with
heterologous antisera.
Antiserum prepared against TMV-O
(Fig. 1) reacted against both TMV strains

whereas anti-TMV-ys reacted only against
homologous antigen preparation. In cross-
absorption tests (Fig. 2), TMV-O antigen
was completely absorbed by TMV-O
and TMV-ys antisera whereas TMV-ys
antigen was completely absorbed by
homologous antisera and only partially
by TMV-O antiserum. These observations
demonstrated the close but not identical
relationships between the two strains
of TMV.

Experimental conditions of CIEPA
To establish the optimum condi-
tions required for CIEPA test, it was
essential to determine the zones of equi-
valences for the different virus-antiserum
systems. Preliminary experiments
revealed that purified virus at 100-250
nanogram protein per ml and prepared
antisera diluted at 1:80 to 1:120 pro-
duced sharp precipitin lines.
Of several buffer (0.05 M phos-
phate, pH 6.0 to 8.0; 0.05 M tris- HCI,
pH 8.0 to 9.0; 0.05 M borate, pH 8.0
to 9.0; and 0.05 M acetate, pH 6.0)
solutions tested tris or borate buffers
were suitable in all CIEPA tests. With
these buffer systems, the precipitin lines
usually became prominent in 30 minutes
from the start of CIEPA run using 10mA
direct current per 25 x 75 mm agar
slide. The electrophoretic run could be
shortened by increasing the constant
current to 15 mA. However, this opera-
tion posed the danger of thermal dena-
turation particularly in the case of
unstable viruses. In this particular study,
however, this was not observed to impede
in the appearance of precipitin zones.

Comparative sensitivity of CIEPA, micro-
precipitin and agar gel double diffusion
The small isometric particles of
CPMV-Sb, SBMV-cs, MMV readily dif-

Detection of Plant Virus by CIEPA








45 2
3 ~ J


Fig. 1. Photographs of CIEPA illustrating immunoreactivities and immunospecificities
of prepared antisera. Numbered wells contained specific antisera against the
following viruses: 1 TMV-O; 2 = TMV-ys; 3 = CPMV-Sb; 4 = MMV; 5 SBMV-
cs; and 6 = CYMV. Unlabeled wells contained virus indicated on left-hand side of

)t e..rf1

Jan-June 1980



Philippine Phytopathology




Fig. 2. Photographs of cross-absorption tests of prepared antisera against homologous
and heterologous purified virus preparation. Residual virus antigen was deter-
mined by CIEPA test using antisera against virus indicated on left-hand side of
photographs. 1 = TMV-ys; 2 = TMV-O; 3 = CPMV-Sb; 4 = MMV; 5 = SBMV-cs;
and 6 = CYMV.

Vol. 16


r` "

3 d~ (5
P ~L

Piiluppiac Thytopathbol yo

Fig. 3. Photographs of CIEPA test demonstrating its sensitivity. Numbered wells con-
tained virus concentration expressed in nanogram per ml. Unlabeled wells
contained antisera specific against virus indicated on left-hand side of photo-

Vol. 16

Detection of Plant Virus by CIEPA

Correlation between CIEPA and infectivi-
ty assay local lesion host plants
In preliminary comparative sensiti-
vity assessment of CIEPA and infectivity
assay in which purified TMV-O suspen-
sion was used, the results suggested that
CIEPA performed as well as infectivity
assay. For instance, CIEPA detected 1
nanogram protein of purified TMV-O,
and the same concentration of TMV-O
produced two local lesions per leaf of
inoculated Gomphrena globosa plant.
Because it was thought that the other
viruses were not as stable as TMV, com-
parison of the sensitivity of CIEPA with
that of infectivity assay was conducted
only utilizing freshly-prepared leaf tissue
homogenates which were then serially
diluted in appropriate buffer solution.
The virus content of diluted virus pre-
parations were then determined by both
CIEPA and infectivity assay.
As shown in table 2, results of
CIEPA and infectivity assay were com-
parable in most cases except perhaps
as in the case of the common strain of
TMV and SBMV-cs. With these two
viruses which are shown to be present
in high concentrations in infected leaf
tissues and which has a high specific
infectivity index (number of local lesions
per milligram virus protein), the infecti-
vity assay was several-fold more sensi-
tive than CIEPA. However, with CPMV-
Sb, CIEPA was more sensitive than
infectivity assay. The higher sensitivity
offered by CIEPA in the detection of
CPMV-Sb might be attributed to the
fact that this particular virus codes for
the synthesis of three different sedimen-
ting, virus specific proteins (top, middle,
and bottom components), of which only
the bottom component was infectious.
Consequently, the specific infectivity
index was generally lower than that of
TMV or SBMV-cs and, therefore, it

would account for the higher sensitivity

Detection of mixed virus infections by
To demonstrate the applications
of CIEPA in the diagnosis of mixed
virus infections, mixtures of different
viruses were prepared and analyzed by
CIEPA. As shown in figure 4, mixture of
TMV-O and CYMV separated as two
discrete precipitin lines. The TMV-O
precipitin band (Fig. 4-3) was thick
due to high virus protein concentration
applied into the antigen depot. The
CYMV protein (Fig. 4-2) migrated
faster resulting in the separation of the
two viruses (Fig. 4-1). Further CIEPA
tests (Fig. 5) dearly demonstrate that
certain mixtures of viruses can be detec-
ted simultaneously in a single-step opera-
tion which normally lasted for only 30
minutes. Figures 5-5 to 5-10 contained
mixture of two different viruses; Figures
5-11 to 5-13 had three different viruses;
and Figure 5-14 contained four different
viruses, all of which could infect legumi-
nous plants. It is evident that as many
as three different viruses (Fig. 5-12) could
be diagnosed by CIEPA. Figure 5-14
showed the remarkable reproducibility of
CIEPA's ability to separate virus mix-
tures. This same figure also shows that
the fourth virus in the mixture (Fig. 5-14)
co-migrated with thick faster moving
Results of CIEPA tests undoubted-
ly illustrate its capacity to detect viruses
of different morphological types in a
mixture and in a short period of time.
This is in comparison with other serolo-
gical tests which usually take about 2
hours for the microprecipitin test (van
Slogteren, 1955) and several days for the
ELISA test (Voller et aL, 1977). Fur-
thermore, with these serological tests

Jan-June 1980

82 Philippine Phytopathology VoL 16

Table 2. Comparative sensitivity of CIEPA and infectivity assay for the detection of several Philip-
pine plant viruses.

Reciprocal of end point titer
Virus CIEPA Infectivity assay

Mungbean mottle virus 640 1280
Cowpea mosaic virus 320 160
Southern bean mosaic virus 160 640
Tobacco mosaic virus yellow strain 320 5120
Tobacco mosaic virus orchid strain 160 160
Cymbidium mosaic virus 80 160

Fig. 4. Photograph of CIEPA test of TMV-O and CYMV in single and mixed virus
suspensions. Unlabeled wells contained mixed TMV-O and CYMV antiserum.
Numbered wells contained (1) mixture of TMV-O and CYMV, (2) CYMV alone,
and (3) TMV-O alone.

virus mixture cannot be detected simul- in figures 5-1 to figures 5-4 deposited in
taneously in a single-step procedure. separate wells can be employed. In all
In situations where CIEPA tests cases, CIEPA is certainly a rapid, repro-
using polyvalent antisera (Figs. 5-5, 5-7, ducible, sensitive, and specific serodiag-
5-9, 5-10, 5-11, 5-13, and 5-14) cannot nostic technique with wide applications
distinguish the viruses as single entities, in applied plant virus research in the
monovalent antisera such as those shown Philippines or elsewhere.

ion of Plant Vinys by CIEPA

S5. Photographs of CIEPA tests of individ
viruses (5-10), mixtures of three vin
(14). Unlabeled wells contained mi)
viruses. Labeled wells contained (1) T
cs, (5) TMV-CPMV, (6) TMV-SBM\

Id indexing of virus infections by
To illustrate the practical utiliza-
n of CIEPA as a routine serological
hnique in virus disease diagnosis, small
ces of leaf tissues were excised from
urally-infected orchid, eggplant, cu-
*bit, pepper, tobacco, and tomato,
nts. Except for some forty orchids
ich were kindly provided by Chan-
lor E. Q. Javier and Dr. G. G. Divina-
cia of the University of the Philip-
ies at Los Bafios, all source plants for
us indexing were selected at random
I were collected at the experimental
its of the University. In eggplants and
;urbits, we were specifically attemp-

virus suspension (1-4), mixtures of two
(11-13), and mixture of four viruses
e of antisera specific against all four
*ys; (2) CPMV-Sb, (3) MMV, (4)SBMV-
7) TMV-MMV, (8) CPMV-SBMV, (9)
14) TMV-CPMV-MMV-SBMV, antigen

g to detect a strain of TMV only. In
case of solanaceous crop plants, we
e looking for viruses other than
V. For instance, we would like to
)w if field-grown tomatoes are infec-
with a virus similar to that reported
Talens (1979). In one particular virus
ease of wild passion vine (Passiflora
rida L.) which was reported pre-
isly by del Rosario et al. (1966),
used CIEPA to determine if the rigid
-shaped particles found in leaf dip
cimen preparations were serologically
ted to TMV. In this case, no preci-
n line formed between Passiflora
saic virus and antiserum preparations
inst the common, yellow, and legume


Philippine Phytopath

strains or IMV.
Results of CIEPA tests of ap]
rently virus-infected orchids which 1
longed to Chancellor E. Q. Jav
(Fig. 6) and Dr. G. G. Divinagra
(Fig. 7-8) showed that many of th4
imported orchids were infected w
TMV-O, CYMV or both. Twenty six
40 different orchids tested were fou
to be infected singly or mixedly w
TMV-O and CYMV. In most instance
virus infections can be detected in
parts of the afflicted plant including t
roots (Fig. 6 and Fig. 8) and flow
which showed color breaking.
In two Cattleya cv Queen Siri
orchids which was bought by Chancel
E. Q. Javier (Fig. 6-1) and Dr. G.
Divinagracia (Fig. 7-4), mixed vi
infections with TMV-O and CYb
was detected. This observation suggest
that Queen Sirikit orchids original
from the same infected mother stc
that had been used for mass-propagati
through meristem culture. It does r
exclude the likelihood that these
chids became infected as they matui
in commercial nurseries. For instan
two Sir Edward McDonald (Fig. (
and Fig. 6-4) orchids were infected w
CYMV and TMV-O-CYMV, respective
The absence of a precipitin line agaii
TMV-O antiserum in figure 6-3,howev
might be due to low virus concentratic
In any case, the high incidence of vii
infections in imported orchids confirm
a previous report (Rillo, 1978) and
affirmed the view (Divinagracia, person
.communication) that these viruses mi
be controlled through indexing pro
dures and eradication of diseased plan
The detection of virus infections
roots or flowers confirms Rillo's (19'
observation and suggests possible mc
of virus spread in nurseries through rc
contact and use of contaminated cutti

tools in marketing of cut orchid flow
In serious cases of severe root infection
excessive root cell damage inflicted
the invading virus could account for p
mature death of highly susceptil
Field-grown tomato plants wi
found to be infected with TMV, 1
identity of the strains could not
established by CIEPA due to lack of
ference antisera against numerous stra
of TMV. One hundred tomato plant sa
pies for CIEPA test were infected v
TMV, 48 of which are shown in figure
Flowers and seeds of infected tom;
plants were also found to contain 1
virus. In pepper, 13 of 56 samples si
jected to CIEPA test were infected w
TMV. Leaf tissues of eggplants a
cucurbits did not contain detectal
quantities of TMV antigen. The thi
ness of the precipitin lines shown
figure 9 indicates the amount of virus
leaf tissue extracts. Results of this pa
cular survey of TMV infections in th
agricultural crops were obtained in app
ximately 2-4 hours which would norma
take 4-10 days through infectivity ass
This does not take into account 1
length of time and the tedious proc
involved in the preparation of app
private assay host plants.
In one tomato plant, a virus isol
was obtained which did not react w
antiserum prepared against TMV-ys
CIEPA test. This particular isolate incil
the development of mosaic symptoms
Datura metel (Fig. 10), Lycopersic
esculentum (Fig. 11), Nicotiana bent
mania (Fig. 12). N. glutinosa (Fig. 1
N. tabacum cv Samsun (Fig. 14) and,
Tetragonia expansa (Fig. 15) virus inf
tion was manifested as chlorotic sp
later becoming necrotic. Based on th
properties, this virus isolate appeal
similar to that reported previously


1of Pant Virm by CIEPA



6. Photographs of four Cattleya orchi<
CYMV) indexing by CIEPA test of le
upper unlabeled wells contained an
contained anti-CYMV. Labeled wells
orchids L6 and R6 contained extra

1-4) sampled for virus (TMV-O and
L) and root (R) tissue samples. Black
MV-O; black lower unlabeled wells
stained tissue extracts from sampled
from apparently uninfected Cattleya




Fig. 7. Photographs of CIEPA tests of
plants belonging to Dr. G. G.
at Los Baios. Unlabeled well
CYMV. Wells labeled C conta
ration. Numbered wells contain
Cattleya, (3) Uninfected Dendi
cattleya, (6) Grammatophyllui
drobium (10) Aeridis, (11) An
Well labeled O contained CYMh

f tissue extracts prepared from several orch
inagracia of the University of the Philippir
contained mixed antisera against TMV-O ai
I mixed TMV- O and CYMV antigen prel
extracts from (1) Vandopsis, (2) Uninfect
um (4) Cattleya cv Queen Sirikit, (5) Laeli
(7) Dendrobium (8) Dendrobium, (9) De
a, and (12) Archnis.
which was tested against anti-CYMV(Randle!


....--- iffl ... .. ...J -


The CIEPA was also employed to
etect the presence of TMV antigen
i single seeds of infected tomatoes
Fig. 16a to Fig. 16c). In efforts to
establish a TMV-decontamination pro-
edure of infected tomato seeds, several
chemicals and detergents were tested.
'his was done by soaking 100% virus-
ontaminated tomato seed lots for 2
ours at room temperature, and wash-
rg thoroughly treated seeds with water
rior to CIEPA test. Results of this par-
cular study are shown in figures 16-1 to
6-5. Figures 16d to figure 16.f con-
ained extracts from 10 untreated infec-
ed seeds. Figure 16C contained TMV
It is evident that batches of 10
infected tomato seeds (Figure 16d-16f)


TMV-As rb j

,YMV-As -41

g. 8. Photographs of CIEPA tests of ten
collection. Leaf and root tissue sa
TMV-O (AT) and anti-CYMV( A).
as unlabeled wells contained speci
contained TMV-O (T) and CYMV ((
logous and heterologous antisera.

;ontamen IMV. wnen comparaoe oat-
.hes of tomato seeds were treated with
chemicalss and detergents (Table 3), only
pyrollidine (5%) and 6N HC1 were
shown to be effective in degrading the
rirus (Fig. 16-1 and Fig. 16-2). Seed
treatment with 1N HCI (Fig. 16-3), 1
SDS (Fig. 164), and 50% Teepol(Fig.
16-5) did not appear to degrade the
rirus. In seeds treated with SDS and
Feepol, two precipitin lines were noted.
Fhis was due to the presence of aggre-
gated virus particles.
While treatment with 5% pyrol-
idine and 6N HCI successfully degraded
:he virus, such treatments were found
:o reduce seed germination (Table 3)
drastically. It might be necessary to
decrease the treatment time with these
wo chemicals or else test other che-


hid plants from Dr. G. G. Divinagracia's
les were tested separately against anti-
mbered wells contained virus sap where-
antiserum preparations. Lettered wells
antigens which were tested against homo-

I ....... I -, -~lr

mnuppme rnytopau

ture TMV in infected tomato seed
In the case of 1% SDS, 50% Teep(
and IN HC1, the treatment time cou
be extended to completely denatu
TMV. We are currently testing the
possibilities in attempts to find an effe
tive virus decontamination procedu
without affecting the germination
tomato seeds. Furthermore, we wou
like to show evidence that these deconi
mination procedures result in the lo
of infectivity of TMV.


The successful production of sp
cific antisera against several Philippim
plant viruses provided the convenie
model systems for investigating tl
applicability of CIEPA in mass-indexi
of orchids, in virus disease survey, in tl
determination of virus infection
tomato seeds and in the assessment
virus decontamination procedures
TMV-infected tomato seeds and,
general, in the rapid diagnosis of disea
either with single or mixed virus infe
The development, demonstratic
and adoption of a simple, sensiti
reliable reproducible, specific and rap
laboratory technique in virus detectii
and identification has an important cc
tribution in applied plant virus research
One of the main justifications in t
genesis of such a technique is the ecor
mic- advantage to be gained from eai
diagnosis of virus infections in import
agricultural crops and in the intercept
of potentially-dangerous viruses as int
national exchanges of germplasms I
come a common occurrence among im
tutions and plant scientists. In the Phil
pines, the availability of a routine sei
diagnostic tool occupies an imports

place when one considers current gover
ment's thrusts for increased productic
in food and energy sources in the 198C
and production constraints of which tl
frequent occurrences of viruses infectii
major cultivated crop plants are the mo
important. The frequent incidence ar
widespread occurrence of viruses som
times in mixed infections coupled wil
the lack of information regarding the
effects on crop productivity and qualil
and their possible widespread dessimin
tion through the use of contaminate
seedstocks have accounted for the lo
productivity of agricultural crops
farmer's field. With a simple and rap
virus detection and identification tec
nique, seed-borne viruses can be inte
cepted, timely application with ch
micals to ward off viruliferous insect
and surveillance of insect vectors
virus carriers could save a particul
crop from partial or complete failure
Our comparison of the CIEP
test with two serological technique
(microprecipitin and agar gel doub
diffusion) and infectivity assay for tl
detection of several isometric, rigid ro
shaped, and semi-flexuous rod-shape
viruses suggests that CIEPA can be us(
with confidence. The sensitivity
CIEPA definitely exceeded that of tl
microprecipitin and agar gel diffusic
tests. Furthermore, CIEPA test tak
only 30 minutes while the other two ser
logical tests require several hours befo
any indication of a positive reaction ci
be visualized. When CIEPA and micr
precipitin tests are compared with ag
gel diffusion test, the amount of anti
rum required for the former is larger th:
the latter test. For instance, in agar F
diffusion test using a six-well gel patte
six separate samples can be tested again
a small volume of reference antiserui
In the case of CIEPA and microprecipit


Detection of Plant Virus by CIEPA

CL~ (j )Y-)
LO ~d

4. -AA.N&, A& Al

Fig. 9. Photographs of CIEPA tests of leaf extracts from 48 tomato plants which were
indexed for TMV infections. Numbered wells contained virus sap from field-
infected tomato plants. Unlabeled wells contained antiserum against the yellow
strain of TMV. Density and thickness of precipitin lines indicate virus concentra-
tion in crude sap. Note 100% infection as evidenced by the presence ofprecipi-
tin lines in all 48 samples.

Jan-June 1980





Fig. 10-15. Symptoms incited by a viru
The occurrence of the virus
and then transmission exp
symptoms of virus infections
(11), Nicotiana benthamania
(14), and Tetragonia expansa



late obtained from field-infected tomato:
ate was detected firstly through CIEPA tes
ients. The tomato virus isolate produce
Datura metel (10), Lycopersicon esculentun
), N. glutinosa (13), N. tabacum cv Samsul

chemicals and detergents on seed gem

No. of seed
Treatment sed

5% Pyrollidine 98
1N HCL 82
6N HCI 92
1% Sodium dodecyl
sulfate 85
50%Teepol 72
Untreated seeds 104

tests, six separate virus extracts must be
tested against six small volumes of refe-
rence antiserum. However, with CIEPA,
mixtures of viruses can be detected using
a single antigen sample tested against a
polyvalent antiserum preparation. With
rod-shaped viruses, CIEPA performed
better than agar gel diffusion test not
only in terms of sensitivity but also in
separating virus mixtures by electrically
forcing them to move through the small
pores of the 1% agar gel. Lack of anti-
serum against viruses belonging to the
Polyvirus group with particles ranging
from 700-800 nm hindered our efforts to
extend CIEPA's application to this lar-
gest and important group of viruses.
Comparison of CIEPA with infec-
tivity assay showed varying degrees of
sensitivity depending upon specific infec-
tivity of the virus. For instance, TMV-O
and CPMV-Sb can be detected with consi-
derable degree of certainty by CIEPA and
results of CIEPA agreed well with those
of infectivity assay. In the case of TMV-
ys and SBMV-cs, infectivity assay was
found to be more sensitive than CIEPA
presumably for two reasons, one of
which is their high specific infectivity


No. of seeds Percent Seed
germinating Germination

42 42.9
66 80.0
15 16.3

74 87.1
61 84.7
83 79.8

index and the second being their occur-
rence in high concentration in infected
leaf extracts. The problem with infec-
tivity assay, however, is that it is slow and
requires much greenhouse space, and in
a virus testing program where many
plants are to be tested, CIEPA might
prove to be more useful and economical
in the long run. The employment of
CIEPA in virus indexing eliminates the
tedious process of maintaining and caring
of assay host plants. With several modi-
fications in the extraction procedure
and in the size of flat agar gel bed, bet-
ween 800-1000 individual samples can
be analyzed in a day.
The results of field indexing of
orchids by CIEPA confirmed our pre-
vious observation (Talens et al., 1979)
regarding its practical value in large-
scale testing of orchids for TMV-O
infections, one of two most common
viruses infecting orchids throughout the
world. Experimental evidence was also
presented to demonstrate that mixed
infections with TMV-O and CYMV, the
other notorious virus infecting orchids,
can be detected in a single-step 30-
minute operation using mixed TMV-O

fhilppmine PhytopathoogV



Fig. 16. Photographs of CIEPA tests to
single seed (a-c) and to show
degradation of TMV (1-5) in
extracts from ten infected tom;
contained extracts from infected
lidine, (2) 6N HC1, (3) IN HC1
trained reference TMV antigen.

and CYMV antisera. Through CIEPA
testing, we confirmed the prevalence
of these two viruses in many imported
orchids (Rillo, 1978) and that these
viruses can be detected in all parts of the
d-iseased orchid plant. Consequently, we
suggest observance of strict phytosanitar5
practices in orchid nurseries and making
certain that plants do not crowd or touch
each other including the roots.
With the use of CIEPA, we demons-
trated that mixtures of two or more viru-
ses belonging to three morphological
types (isometric, rigid-rod, and semi-
flexuous) can be separated. Separation of
virus mixtures by CIEPA is feasible so
long as sufficient differences in electro-

5 C

lonstrate the detection of TMV infection ii
effect of chemicals and detergents on th
cted tomato seeds. Fig. 16d-16f contained
seeds. Antigens used in wells numbered 1-:
imato seeds treated as follows: (1) 5% pyrol
) 1% SDS and (5) 50% Teepol. Well C con

phoretic mobility exist among the viruse
in a given mixture.
The CIEPA test is useful and rapic
in obtaining information regarding the
occurrence of TMV infections in field
grown tomatoes, pepper, and tobacco
The detection of TMV in infected tomato
seeds and testing of TMV-decontamina
tion through chemical or detergent treat
ment further illustrate the power and
versatility of the technique. Other appli.
cations of CIEPA pertain to the acquisi.
tion of information regarding virus con.
centration and antiserum titer. When used
with intragel cross-absorption test, CIEPA
will aid in serovar definitions of Philip-
pine plant viruses particularly TMV and

VoL 14

1 of Plant Virus by Ca

its strains and in establishing serological
relationships of Philippine plant viruses
with those reported elsewhere. The tech-
nique has tremendous potential applica-
tion in plant quarantine work where rapid
ind simple procedure is sought.
A key question which remains to
be answered pertains to the application
)f CIEPA in the diagnosis of diseases
causedd by numerous flexuous rod-shaped
viruses (Gibbs et al., 1970, 1971). Unfor-
unately, we have not pursued this
projectt due to lack of specific antisera.
n this regard, several approaches can
be pursued. For instance, the pore cha-
acteristics of the agar gel bed can be
enlarged by mixing agar with other suit-
ible bed materials such as agarose or
>olyacrylamide gel. Another strategy
which has successfully worked out in
theirr virus systems consisted of degrading
he virus into active protein subunits
Purcifull and Batchelor, 1977; Shepard,
1974) and employing these subunits
directly in serological tests or, alter-
iatively, antiserum is produced against
:he degraded virus protein prior to its
ise in serological test.


3ALL, E. M. 1974. Serological tests for the:
Committee, Amer. Phytopathol. Soc.,

BAWDEN, F. C. 1964. Plant viruses and vir
York. 361 pp.

IERCKS, R. and G. QUERFURTH. 1969.
den Latextest zum serologischen Nach
Zeitschrift. 65:243-256.

detection of distant serological relatic

We have not compared the sensi-
ivity of CIEPA with ELISA test (Voller
it al., 1977) simply for the reason that
he chemicals needed for the latter have
iot arrived. It would not be surprising
f ELISA test turns out to be more sen-
itive. However, if rapidity and simpli-
:ity are sought in a serodiagnostic tech-
lique, CIEPA will definitely outclass
iLISA test.
The key in virus disease control lies
n early and rapid detection, provision
md maintenance of virus-free mother
tock and virus-tested seeds, and surveil-
ance of insect vectors. Counterimmuno-
.lectrophoretic technique has proved
highly successful in the detection and
identification, and separation of virus
fixtures infecting several cultivated
:rops. The demonstration of its useful-
tess in several aspects of plant virus
research is realized here and not else-
vhere. Further explorations of its use in
irus diseases of abaca, citrus, potato,
ice, and other major agricultural crops
nust await until antisera against these
viruses have been produced.


itification of plant viruses. Plant Virology
Paul, Minnesota. 31 pp.

a diseases. 4th ed. Ronald Press Co., New

eitere methodische Untersuchungen uber
s pflanzenpathogener Viren. Phytopathol.

TH. 1971. The use of latex test for the
hips between plant viruses. J. Gen. Virol.

an-June 1980

94 Philippine Phytopathology VoL

BERCKS, R., R. KOENIG, and G. QUERFURTH. 1972. Plant virus serology. In Ka,
C. I. and H. O. Agrawal (Eds.). Principles and Techniques in Plant Virology,
466-490. van Nostrand Reinhold Co., N. Y.

BOZICEVICH, J., H. A. SCOTT, and M. M. VINCENT. 1963. The bentonite flocculat
test for detection of plant viruses and titration of antibody. Proc. Soc. Exp. B
Med. 114:794-798.

BRLANSKY, R. H. and K. S. DERRICK. 1974. Serologically specific electron mici
scopy: detection and identification of tomato spotted wilt virus and icosahedi
plant viruses in crude extracts. Proc. Amer. Phytopathol. Soc. 1:21 (Abstr

CLERKE, R. G. and R. H. CONVERSE. 1980. Enzyme-linked immunosorbent assay
detect potato leaf roll virus in potato tubers and viruliferous aphids. Plant
Disease. 64:43-45.

DERRICK, K. S. 1973. Quantitative assay for plant viruses using serological specific el
tron microscopy. Virol. 56:652-653.

DERRICK, K. S. and R. H. BRLANSKY. 1976. Assay for viruses and mycoplasma usi
serologically specific electron microscopy. Phytopathol. 66: 815-820.

GERA, A. A., G. LOBENSTEIN, and B. RACCAH. 1978. Detection of cucumber moss
virus in viruliferous aphids by enzyme-linked in immunosorbent assay. Virolog

GIBBS, A. J., B. D. HARRISON and A. F. MURANT. 1970-1971. CMI/AAB. Descriptil
of Plant Viruses.

HOLLINGS, M. 1965. Disease control through virus-free stock. Ann. Rev. Phytopathe

KUNKEL, L. O. 1935. Heat treatment for the cure of fellows and rosette of neac

KUNKEL, L. O. 1936. Heat treatment
of peach. Phytopathol. 26:809-83(

MOREL, G. M. 1948. Recherches sur I
tissue vegetaux. Ann. Epiphyties (1

OUCHTERLONY, O. 1962. Diffusion-
Progr. Allergy. 6:30-154.

dium dodecyl sulfate (SDS)-treatei
Agric. Exp. Stn. Tech. Bull. No. 78

the cure of yellows and other virus disease

ilture associee de parasites obligatoires et d
). 14:123-124.

el methods for immunological analysis. I]

OR. 1977. Immuno-diffusion tests with sc
ant viruses and plant viral inclusions. Florid
9 pp.

luuiecWon 1O rian VI vus oy LurYA

L KUSAKIU, M. b., U. A. 15tnNILiNU, ana j
in the Philippines. 1. Passiflora foetida L.

,LO, E. P. 1978. Isolation and identificati
Laeliocattleya "Randy" and chlorotic li
in the Philippines. M. S. Thesis. Unpublisg

EPARD, J. F. 1974. Gel diffusion method
viruses X, S, and M. Montana State Agric

GER, J. M. and C. M. PLOTZ. 1956. Th
serological diagnosis of rheumatoid arthri

ENS, L. T. 1978. Mungbean viruses in the
mottle in mungbean. Philipp. Phytopathc

LENS, L. T. 1978. Cowpea viruses in the I
virus in cowpea, Vigna unguiculata (L.) A

LENS, L. T. 1979. Tomato viruses in the I
tomato, Lycopersicon esculentum Mill. P

LENS, L. T. 1979. Cowpea viruses in the I
of a cowpea strain of southern bean

LENS, L. T., N. CHANSILPA, and A. C.
tion of tobacco mosaic virus in orchids. F

N SLOGTEREN, D. H. M. 1955. Serologic
paraffin oil. Proc. of the 2nd Conferenc
gen. pp. 51-54.

LLER, A., D. E. BIDWELL, and A. BAR1
sorbent assay. Flowline Press. Gurnsey, I

TTER, C. 1965. Serology in virus-disease i

. LIS.UJ. 1y virus alseases oI weeas
lipp. Agric. 48:95-112.

of the virus causing mosaic disease on
streak on Oncidium "Golden Shower"

)r the serological detection of potato
p. Stn. Bull. 662. 72 pp.

tex-fixation test. I. Application to the
Amer. J. Med. 21:888-892.

lilippines. I. Identity of a virus causing

ppines. I. Identity of a mosaic-causing
Philipp. J. Crop Sci. In Press.

ppines.I. Properties of a virus infecting
)p. Phytopathol. In Press.

ppines. III. Isolation and identification
saic virus. Philippine Phytopathol. In

ILORES-TALENS. 1979. Rapid detec-
pp. Phytopathol. In Press.

nicro-reactions with plant viruses under
i Potato Virus Diseases. Lisse-Wagenin-

TT. 1977. The enzyme-linked immuno-
.43 pp.

nosis. Ann. Rev. Phytopathol. 3:19-42.

-June IBOU

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Manuscripts must be reports of original research, except meritorious reviews and
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accept or reject the manuscript is final.

The manuscript should be typed on one side of 8 x 11 inch paper, double spaced

The author's name should follow the title. Author's position, institutional address,
and acknowledgements should follow the author's name.

Papers other than Notes maybe organized conveniently under: Abstract, Introduc-
tion, Materials and Methods, Results, Discussion, (or Results and Discussion) and
Literature Cited.

In the text, citations should be by name-and-year system, e.g. Ou and Nuque
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