Front Cover
 Front Matter
 Abstracts of papers presented at...
 Fruit rot of pepper caused by fusarium...
 Various heat treatments for aflatoxin...
 Pathologic reactions of cotton...
 Comparison of the effects of different...
 Cowpea little leaf disease in the...
 Pre-harvest antrancnose in...
 Pre-harvest antrancnose in...
 Pythiums from Philippine soils
 Phytopathological note: effect...
 Back Matter
 Back Cover

Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00090520/00019
 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 1977
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: VID00019
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
        Front Matter 3
        Front Matter 4
    Abstracts of papers presented at the fourteenth annual meeting of the Philippine phytopathological society inc., Bacolod City, 18-20 May, 1977
        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
    Fruit rot of pepper caused by fusarium spp.
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
    Various heat treatments for aflatoxin decontamination in mungbean (vigna radiata
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
    Pathologic reactions of cotton grown in soil infested with rotylenchulus reniformis
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
    Comparison of the effects of different meloidogyne incognita inocula on baguio bean (phaseolus vulgaris L.)
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
    Cowpea little leaf disease in the Philippines: Possible viral etiology as detected by immunodiffusion technique
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
    Pre-harvest antrancnose in mango
        Page 50
        Page 51
        Page 52
    Pre-harvest antrancnose in mango
        Page 53
    Pythiums from Philippine soils
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
    Phytopathological note: effect of KCI fertilization on coconut leaf spot diseases and yield
        Page 74
        Page 75
        Page 76
        Page 77
    Back Matter
        Page 78
        Page 79
        Page 80
    Back Cover
        Page 81
        Page 82
Full Text



arbide (Philippines), Inc., P. 0. Box 56, Commercial Center Post Offit
rtro Manila



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

Subscriptions: Communications should be addressed to the Treasurer, P. P. S. c/o Department
of Plant Pathology, UPLB, College, Laguna 3720. Philippine Phytopathology, published semi-annually
(January and June), is the official organ of the Philippine Phytopathological Society, Inc. It is sent
free to members in good standing and to Sustaining Associates. For others, it is P25 per year or
P12.50 per copy (domestic) and $25 per year or $12.50 per copy elsewhere, postage free and payable
in advance. Membership in the Philippine Phytopathological Society, Inc.: Information regarding
membership will be supplied by the Secretary upon request. Page Charge: The editorial board reserves
the right to charge some authors a preset amount for each published page commensurate upon the
payment capabilities of their research projects or supporting institutions. Advertisements: Rates may
be secured from the Business Manager. No endorsement of any statement of claims made in adver-
tisements is assumed by this Journal or by the Philippine Phytopathological Society, Inc.

Transmission of Rice Grassy Stunt essential to have some way of specifying
ease by Three Biotypes ofNilaparvata the level of field infection when a
;ens. K. C Ling and V. M. Aguiero, variety is tested. For this particular pur-
R/. pose, the term "tungro pressure" is
The vector of rice grassy stunt disease, proposed here. It tentatively refers to
>wn planthopper (Nilaparvata lugens), the average tungro reaction of other rice
Three current biotypes. These biotypes varieties or lines, based on the interna-
Fer in ability to attack rice varieties tional scale for resistance to rice tungro,
;sessing different resistant genes to the that are being grown adjacent to a test


i tested on Taichung Native 1. The (IR5, IR8, IR:
ilts showed no significant differences and IR1561-2u
ong the three biotypes in percentage the tungro press
active transmitters, duration of the cular variety,

[R 28, IR30, IR34,
tested increased as
increased. For a parti-
relationship between

sage, length of retention period, and approximately the same whether the
nber of infected seedlings per insect. tungro pressure was applied in one or
e test insects of the three biotypes many rows. However, the rate of increase
e similar in life span, nymphal stadia, in field reaction of varieties to the
It number, and molt interval, increase in tungro pressure was not
constant. Among IR varieties tested,
Field Reaction of IR Varieties to IR34 is the most resistant to tungro
rgro Disease. K. C Ling, E. R. according to results obtained in 1975
ngco and R. D. Daquioag, IRRI. and 1976.
The reaction of a rice variety to tungro

been standardize
e described in t
1 System for Rib

oj Laaang-Laaang uvsease
from Weed Elephantopus
I Protacio, BPI.

ous mollis mosaic virus

inivivj inciie two istimct aise
variptu tn thZ +iunarn A .....

2 Philippine Phytopathology Vol

cadang-cadang symptoms on coconut. be not affected by the factors suct
The prominent veins strain (EMMV2) numbers of insects released, var
produces typical mosaic symptoms but times of releasing insects, intervals (
distinct from the Davao form of abaca than 300 minutes) between ins
mosaic. When introduced into young release, numbers of rice hills in the
rn1nnt candlinoa it alqn indue.s mottline and healthy versus tungro-dise;

. 13

into chlorotic streaks with age. Mixture of insects varied among rice varieties tested,
Bokingan and E. mollis strains produce and among rice plants of different sizes

abaca 10-50 on b. moms ana z/-ou on Kace Maiaay m me rnmppmes: u
coconut. Distribution and possible Cause. -
H. T Begonia, BPI.
Dispersal of Nephotettix virescens. -
K. C Ling and E. R. Tiongco, IRRI. The name given to the new rice disease
in the Philippines was infectious gall
The spread of rice tungro disease in a disease. The disease was first observed in
field is determined by the dispersal, December, 1976 in North Cotabato,
movement, and migration of tungro- Mindanao. Extensive surveys from
viruliferous rice green leafhoppers. Results January to May, 1977 by the Bureau of
from studies on the dispersal of Nepho- Plant Industry revealed that the disease
tettix virescens in cages indicate that the also occurs in Davao del Norte, Davao
dispersal as a function of distance in del Sur, Sultan Kudarat, South Cotabato,
terms of the number of rice hills (x) inter- Zamboanga del Sur, Zamboanga City,
evening between the point of release and Misamis Occidental, Ozamis City, Agusan
the rice hill where the insects are found, del Norte, Agusan del Sur, Butuan City,
can be expressed by Y = a (1 + X)-b, Surigao City, and Bukidnon, in the
where Y = percentage of total number Mindanao Island. Only rice farms in
of insects per hill. The dispersal as function Laguna and Nueva Ecija in Luzon have
of distance and duration can be expressed been found to have the disease. Several
by: rice cultivars were infected with varying
-0.17d degrees of severity namely: IR38, IR36,
Y= 69.8e-0.23d(1 + X)-3.6e IR30, IR26, IR1561, IR28, IR29, IR20,
IR32, C4-636, and BPI-76. Of the 3 latest
where e = 2.718; d = duration, ranging v s i t f
h .r...n 1 ani 11 A.V" and Y = distance varieties introduced to farmers (IR32,

s[ItIDUuUII U ulu 1IIac ta U1n 1 1 -
an area. empty grains; branching at the ui
nodes; twisting of some leaves; prese
dispersal of the insects seemed to of elongated galls on the nether sur

Populations of Plant Parasitic Nema- """'""" ."... .... j.
odes Associated with Cropping Patterns todes Associated with Different 1
nder Different Rice-Growing Environ- Forms and Cropping Patterns in Iloil
tents in Manaoag, Pangasinan. M. B. M B Castillo, M. B. Arceo and J
astillo, M. B. Arceo and J. A. Litsinger, Litsinger, UPLB-IRRI.
'PLB-IRRI. A survey was conducted to deten
the populations of plant parasitic ne
todes in different land forms and c
Plant parasitic nematode populations ping patterns of rainfed upland and
rere determined in cropping patterns land areas in Oton, Tigbauan, and L
under different rice-growing environ- Iloilo. Soil texture ranges from cla
ients in Manaoag, Pangasinan. Field clay loam and pH from 6 to 7.2. (
gumes and vegetables were the standing cropping patterns of three-year dura
rops at sampling time. Nematodes were and with legumes or other non-gran
extracted from soil and root samples. ceous upland crops as standing crops
feloidogyne spp. and Rotylenchulus spp. considered. Nematodes were extra
redominated over the other plant para- from replicated composite samples (30
tic nematodes. Differences in nematode soil and 1 g roots) by the sieving-Baerni
populations among environments were funnel technique; those in the roots
associated with duration of flooding, stained with acid fuchsin-lactophenol.
he highest populations were detected in loidogyne spp. and Rotylenchulus
on-flooded upland fields. Isolated predominated over the other p
addies in drainage ways among these parasitic nematodes. From the mosi
pland fields yielded surprisingly low the least nematode-infested, the I
populations. In these areas, one crop of forms were as follows: unbunded I
addy rice undergoes two to three levee; unbunded summit; bunded suni
months of flooding. This was apparently and bunded river levee; plain, plateau
efficient to effectively control the nema- slope; and drainage way. The effect
)des, although soil erosion from sur- land form was evidently related \
fundingg slopes would be a source of duration of flooding. Lowland
infestation each rainy season. In one cropping was associated with redu
iwland area, farmers periodically flood nematode populations. Apparently,
leir fields to alleviate the iron and zinc crop of lowland rice was sufficient
deficiencies in the soil. Although popula- effectively control the nematodes in
ons were reduced, relatively higher more flooded lowland areas. In the no
)pulations were observed in this area. flooded areas, high populations resu
he normal rainfed paddies, where rice is from continuous cropping of upl
iltivated under three months of flood- crops. Dissemination of nematodes fi

4 Philippine Phytopathology Vol. 13

the more elevated to the less elevated in controlling nematodes for four months
land form could have accounted for the after treatments.
higher populations in the unbunded river BHC and Bux 3G were effective only
levee than in the unbunded summit. in the first two months, however, during
So S N o t the first month these chemicals gave
Effect of Some Nematicides on the .
t of m e Atcihigher nematode control with 90.1% and
Control of Nematodes Attacking Sugar-
cane and on Seedpiece Germination and 9
Yield. T. T Reyes and E. D. Beguico, Treatments with Nemagon 10G gave
UPLB. the highest seedpiece germination with
85.9%. This was followed by Furadan 3G
Laboratory and field tests were con- with 83.9%. The control had 68.8% ger-
ducted to determine the effect of mination.
Bunema, Bux 3G, Furadan 3G, BHC,
Nemagon G, uHostathin 5G, C d The yield in tons of cane was highest
Nemagon 10G, Hostathion 5G, and .
em on oto ad in the Nemagon lOG-treated plots with
Temik 10 on the control of nematodes
an average of 3.75 tons/400m- or 94
attacking sugarcane and on seedpiece tons/a. s as fooe raan
tons/ha. This was followed by Furadan
germination and yield. The soil screen 3G with 3.66 tons/400m2 or 91.5 tons/ha
technique was used to test the effective- and Teik G with 3.51 ton/4 or
and Temik 10G with 3.51 tons/400m2 or
ness under laboratory condition of the
87.7 tons/ha. The average yield in the
chemicals against the nematodes co- control plots was 2.76 tons/400 or 69
control plots was 2.76 tons/400m2 or 69
only encountered in the field, such as
Criconemoides, Helicotylenchus, Hoplo- tons/ha.
laimus, Xiphinema, Hemicriconemoides, Based on the ratio of piculs sugar per
Tylenchorhynchus, Pratylenchus, Tricho- ton of cane, Furadan 3G had the highest
dorus, Meloidogyne incognita and M (1.99) followed by Temik 10G and the
javanica. In the field test, the chemicals control (1.91). The lowest was obtained
were applied in the furrows at planting with Nemagon 10G the control (1.91).
time. The germination percentage was The lowest was obtained with Nemagon
assessed two months after planting and 10G treatment (1.82). However, the
the yield, 11 months after. Soil sampling differences were insignificant. The dif-
was done monthly for four months after ferences in percent sucrose of the juice
planting. The laboratory test consisted were also non-significant. The percent
of quadruplicate pots while the field test sucrose of juice obtained range from
consisted of four replicate plots each 88.02 to 91.20%.
measuring 16 x 24 m arranged in ran- Furadan 3G gave the highest yield
domized complete block design, increase in sugar over the control with
Results of the laboratory test showed 27.6% or a net income of P3,415.11 per
that Nemagon 10G was the most effec- hectare. Nemagon 10G and Temik 10G
tive with a mean control of 95.4%. This increased sugar yields by 23.0% (a gain
was followed by Hostathion 3G and of P2,094.05) and 21.3% (a profit of
Furadan 3G with 76.9% and 69.4%, P2,164.77). BHC gave 20.6%(P2.008.803
respectively. Bunema was the least profit) sugar yield increase over the
effective with 47.2% control. The nema- control.
tode genera also reacted differently to the Pathogenic Strains of Xanthomonas
nematicides used. oryzae in the Philippines. T. W. Mew
In the field test, Temik 10G, Nemagon and C. M. Vera Cruz. IRRI
10G and Furadan 3G remained effective The virulence patterns of X. oryzae

Jan. & June 1977 Abstract of Papers 5

isolates collected in the Philippines from are present in an area before the parti-
1963 to 1976 were evaluated. Four host cular host genotype is widely planted.
varieties were used: IR8 which has not PX071 is one of the isolates that was
shown genes for bacterial blight resis- found in Palawan in 1974; some isolates
tance; IR20, which has the dominant of the Isabela strain were collected in
gene Xa4; IR1545-339, which carries the Davao in 1963.
recessive gene xa5; and DV85, one gene
of which is recessive and similar to xa5, Characteristics of Pseudomonas sola-
and another one dominant but different nacearum Strain that Infect Ginger
from xa4. (Zingiber officinale) in the Philippines. -
Ee Yu Kam and A. J. Quimio, UPLB.
The results show clearly that pathoge-
nic strains or races exist in the population Eight ginger isolates of Pseudomonas
of X. oryzae in the Philippines. Most of solanacearum from five provinces in the
the 83 isolates belong to one group that Philippines were studied for their i) host
we call the common strain, exemplified ranges, ii) ability to utilize disaccharides
by isolate PX061. Host genotypes carrying of hexose alcohols, iii) colony mor-
either one of the two major genes (Xa4 or phology on tetrazolium chloride agar,
xa5) are resistant to that strain. The nutrient agar and potato dextrose agar
Isabela strain is exemplified by the isolate plus 1% peptone, iv) ability to produce
PX079 collected in Davao; the host geno- the melanin pigment in a tryosine-
type carrying the recessive gene xa5 is containing agar medium, v) ability to
resistant. The third group of isolates, cause hypersensitive reaction in tobacco
which makes up a small portion of the leaves, and vi) susceptibility to five
present collection and is typified by bacteriophage isolates.
the PX079 collected in Davao; the host All the isolates oxidized the hexose
genotype carrying the recessive gene alcohols only and therefore belonged to
xa5 is resistant. The third group of iso- biochemical type 4 produced hyper-
lates, which makes up a small portion of sensitive reaction in tobacco leaves, were
the present collection and is typified by virulent to tobacco, eggplant, sweet
PX071 from the Palawan area, over-, potato, peanut, lima bean,
pepper, potato, peanut, lima bean,
comes the resistance of host genotypes cowpea, diploid and triploid banana, and
that carry both the dominant and reces- Heliconia, under natural conditions. With
sive genes. It appears that PX071 is more artificial inoculation, all the isolates
compatible with genotypes having xa5 tested were avirulent to tobacco, tomato,
than those with xa4. It always produces eggplant, sweet pepper, peanut, lima
more lesions on IR1545-339 than on bean, cowpea, castor bean and diploid
IR20. DV85 is resistant to all isolates. A banana. All caused severe wilt of ginger in
variety like IR8, with no apparent genes both artificial and natural inocula-
for resistance to bacterial blight, is tions. They showed variability in melanin
susceptible to the three groups of iso- production and differences in sus-
lates. All isolates under study produced ceptibility to five phage isolates. Three
lesions on IR8, but isolates such as isolates were weakly virulent to '20-29'
PXO10, which have weak virulence, tomato, four isolates were virulent to
produced considerably fewer lesions than 'Yellow plum' tomato and three isolates
others. showed varying degrees of virulence
The results also confirmed that isolates to castor bean under natural conditions.
compatible with a certain host genotype With artificial inoculation, all isolates

showed varying degrees of virulence to the seedlings in all methods ot ap-
potato, two isolates were weakly virulent plication. The bacterial antagonist, on the
to eggplant, five isolates were weakly other hand, had no apparent inhibitory
virulent to abaca and triploid banana, all effect on growth and consistently re-
were weakly virulent toHeliconia and the duced the number of wilted seedlings in
two isolates tested were weakly virulent all methods of application except the
to 'Giant Cavendish' banana, seed-soaking technique. The average re-
Attempts to differentiate them from ductions of wilted seedlings were from
isolates of other host plants based on 62%to 31%, from 72% to 46%, and
colony characteristics and phage sus- from 63% to 45% by root-dipping, seed-
ceptibiity were not successful. Cross ling-watering, and soil mixing methods,
ceptibility were not successful. Cross
inoculation studies showed that all respectively. The reductions were con-
isolates from other host plants were not sistently greater in lightly infested soil
isolates from other host plants were not
than in heavily infested soil.
able to cause typical wilt of ginger. than in heavily infested soil.
Based on these, it is proposed that the The bacterial antagonist was identified
Based on these, it is proposed that the a a s of B b o
P. solanacearum which infects ginger in as a species of Bacillus based on its
P. solanacearum which infects ginger in morphological and cultural characteristics.
the Philippines be classified as a new race, and
race 4 according to existing race classifi- I grew ra ly an ormed s n
cation of the pathogen with the ginger hours or less on agar media, had no
a a a aiioa iffr ia hot harmful effect on tomato seedlings and
plant as an additional differential host. effectively reduced tomato wilt, in
naturally or artificially P. solanacea-
Antagonistic Microorganisms from To- rum-infested soils. Consequently it has
mato Rhizosphere for Biological Control the potential for use as a biological
of Pseudomonas solanacearum. S. P. control agent for P. solanacearum.
Tong-LingandA. J. Quimio, UPLB.
Inoculation of Soybean Seedlings with
Xanthomonas phaseoli var. sojense by
Cotyledon-Pricking Technique. J. M.
A total of 106 fungi, bacteria, and Casyao and A. J. Quimio, UPLB.
actinomycete were isolated from rhizo-
sphere soils of tomato plants grown in
different substrates. About 12% of them One-week old soybean seedlings were
showed antagonism to Pseudomonas inoculated by pricking the cotyledons
solanacearum on agar media. Of these, with a needle inoculator dipped into a
three fungi and one bacterial antagonists suspension of the bacterial pustule
were selected and tested for their effec- pathogen, Xanthomonas phaseoli var.
tiveness in controlling tomato wilt sojense. In susceptible plants, bacterial
in soil artificially or naturally infested ooze may be noted at the inoculation
with P. solanacearum. The spores and sites three days after inoculation. This
mycelial fragments or cell suspensions of was followed by yellowing, and finally
the antagonists were applied to the premature falling-off of the cotyledons
seedlings by seed-soaking, root-dipping, within the next four days. In resistant
seedlingwatering, and soil-mixing me- varieties, these reactions were much less
thods. pronounced. With the inoculation tech-
The three fungal antagonists re- nique, it was possible to distinguish
tarded the normal growth and failed to between susceptible and resistant varietal
reduce incidence of bacterial wilt among reactions to bacterial pustule within seven

Jan. & June 1977 Abstract of Papers 7

days after inoculation or fourteen days other hand, no spore was released during
after seed germination. The critical nights when there was no dew.
inoculum concentration of the isolate Under controlled environmental con-
used for differentiating varietal reactions editions in the phytotron, the infection of
was equivalent to a cell suspension rice leaves by P. oryzae started with a
showing 0.30 O.D. at 425 nm wavelength. minimum period of 6 hours leaf wetness.
Air-spora at the UPLB-CA Experiment The number of lesions increased up to a
Station (Wet Season, 1975). F. C. maximum of 22 hours continuous leaf
QuebralandA. L. Piamonte, UPLB. wetness in the dew chamber. A positive
linear correlation existed between period
The diurnal periodicity of the air- of leaf wetness (X) and the probable
borne fungal spores was studied using number of lesions on the leaves (Y). Of
silicon-coated slides exposed to an the two varieties tested, KTH-17 had
automatic volumetric spore trap. Air- more lesions than Peta and the following
borne fungus spores were studied for two equations were developed to estimate
kinds and numbers in 24-hour daily the number of lesions for the two varie-
exposures from June, 1975 to November, ties, respectively:
1975 (wet season). (1) Y= -22.59+7.76X- 0.87X2
Curvularia and Cercospora had day + 0.0520X3 0.000938X4
time peaks usually from 9:00 a.m. to (2) Y=-5.17 + 1.87 X 0.25X2
2:00 pjn. and 8:00 a.m. to 12:00 noon, + 0.0165X3 0.00029X4
respectively. Fusarium spores showed a When infected seedlings (after 24
diurnal periodicity with low daytime hours of incubation) were kept in dew
numbers increasing to a maximum at chamber for different lengths of time
night usually at 10:00 p.m. Cladospo- during P. oryzae colonization of the rice
rium had twin peaks, namely at 9:00 p.m. leaves, the daily 2-hour period of leaf
and at 5:00 a.m. wetness for 4-5 days resulted in the
Powdery mildew and rust spores maximum number of lesions. The lesion
were insignificant during the wet season, size increased with increments in the
No definite diurnal pattern was period of leaf wetness. Also, the time of
observed on the two unidentified fungus lesion appearance was shorter when the
spores. period of leaf wetness was prolonged.

Effect of Leaf Wetness (Dew Period) Virulence Pattern and Aggressiveness
on Spore Release, Infercion and Colo- of Isolates of Sclerospora philippinensis
nization of Rice by Piricularia oryzae Weston on Maize in Bukidnon. A. R.
Car. M. I. El Rafei and S H. Ou. Josue and O. R. Exconde, UPLB.

In a two-week dry season expe- The virulence, aggressiveness, and spo-
riment under upland conditions at relation of 15 isolates of Sclerospora
IRRI blast nursery, it was found that the philippinensis Weston collected from
spores released from infected rice leaves corn growing areas in Bukidnon were
always started after the formation of the studied under controlled environmental
dew on the leaves. There was a great in- conditions.
crease in number of spores released Infective conidia obtained from single
as the dew period increased. On the spore cultures of 15 isolates were inocu-

with 14 different types of virulence pension from 4-day old cultures. Inc
patterns. The most virulent was isolate 14 ted plants were placed in a dew cha
with a mean percentage infection of at 25 C for different periods of time

isolate a, witn j.oo percent. inc meI
systemic infection among isolates ar
among test materials differed significantly:
Variability in aggressiveness of 1
isolates was observed using TX 60
Hooker B, and PF 87-4-3. Isolate 3 w;
the most aggressive resistant inbred T
601, causing systemic infection in only
days after inoculation. Majority of tl
isolates caused systemic infection 7 da:
after inoculation regardless of the inbrei
Isolate 14 had the highest spore coui
with 15,761 spores/ 25 sq mm whi
isolate 3 had the least with 13,898 spore
25 sq mm. Spore counts among isolat
did not differ significantly.

The Clipping Technique of Screenin
for Leaf Scald Resistance in Rice. S.
Ou, F. L. Nuque and S. P. Ebron, IRRI.

The leaf scald disease of rice (Rhy
chosporium oryzae) has recently gain(
increasing importance in many cou
tries. An attempt was made to develc
methods for screening varieties resistai
to the disease.
In increasing for resistance by artil
cial inoculation, the production of larl
quantities of spores is required. Ti
organism, when isolated from disease
tissues to pure cultures, consists large
of mycelium with small sporulatih
sectors. However, by transferring i
streaking the pinkish spore sectors (
potato dextrose agar, large quantities i
spores were produced at temperature
between 20 to 25 C.

I ucw pcIiou. -a uit ucw Jpcluu w
Increased, the number of lesions al
increased, with the maximum lesii
number produced after 24 hours d(

Comparing the two methods of ir
culation, by spraying, the third a
fourth fully opened leaves were infect
but the first and the second leaves we
not. In contrast, all the leaves inoculat
by the clipping method were infect
regardless of leaf position. Therefore, t
S clipping method of inoculation was mc
efficient and this could be used in lai
scale screening for resistance under fil

More than 400 varieties grown in t
blast nursery were inoculated using t
clipping technique. Preliminary resu
indicated that varieties differed in re,
tion to leaf scald. The lesion length
inoculated varieties ranged from 0.2
2.4 cm two weeks after intermediate
susceptible to leaf scald disease.

S Quantitative Resistance ofRice to A
Disease and its Effect on Disease De
S opment. Sang-Won Ahn and S. H.
Rice varieties react to the blast fungi
Pyricularia oryzae Cav., not only qui
tatively, i.e., resistant or susceptible
S specific races based on lesion type, b
S also quantitatively, i.e., number
F Qualitative resistance of variety m
S studied by inoculating speci
monoconidial isolates (races). Quanti
tive resistance of the rice plant to bli

Abstract of Papers

international and Philippine race differen-
al varieties and several breeding lines and
varieties to natural infection in the blast
irsery and by artificial inoculation with
onoconidial isolates.
High positive correlation was observed
between quantitative resistance and the
iectrum of qualitative resistance to races
)th under natural infection and in ar-
ficial inoculation. Varieties resistant to
ore races of the fungus have higher
vels of quantitative resistance. Resis-
nce of a variety to a specific race or
rulence gene is a component of total
lantitative resistance. Therefore, quanti-
tive resistance is an expression of the
teraction between the spectrum of
salitative resistance of a variety to the
ngal population consisting of various
ces. Quantitative resistance appears
nilar to horizontal or general resistance J
field reaction but differs from the
tter when riceplants are artificially
oculated with specific races of the i
The higher level of quantitative resis-
ace in some breeding lines observed in
[tural infection was confirmed by ino-
lating pathogenic isolates which re-
Ited in smaller number of lesions. The
rel of quantitative resistance was
gher in certain progenies than in either s
rent. r
Higher level of quantitative resistance i
iuced the infection efficiency and
:ondary infection rate of the fungus. It C
;o reduced the rate of increase in c
population ofP. oryzae and consequently, s
laily decrease in disease incidence. t
Isolation, Sporulation and Inoculation
r Cercospora oryzae. B. A. Estrada t
d S. H. Ou, IRRI. 1
The isolation of C. oryzae (Sphaeru- v
a oryzae) from diseased leaves and t

obtaining sufficient number of spores
or a longer period on culture medium
>ose some problems in studying the
disease. This led to the modification of
he usual tissue plating and spore-drop
methods and the adoption of a new
method known as the "Node Technique"
or isolation and the prune juice-stem
rode medium for sporulation. The
improved technique and the modified
medium proved very efficient, thus pa-
ing the way for greenhouse inoculation
nd field screening studies of the disease.
After inoculating a spore-suspension
rom a 12-day old culture on the 30-day
Id IR2307-64-2-2 seedlings in the green-
ouse, we found that symptoms began to
ppear 21 days later. The number of
esions formed reached the maximum at
bout 30 days after inoculation. Using
'ur different stages of IR1487-372-1-1
lants (39, 60, 81 and 102 days after
ceding), we found that C. oryzae can
ifect rice plant at all stages of growth.
he incubation period in all stages of
ce growth was approximately three
eeks. The long incubation period
id the slow development of the lesions
lay account for the late appearance
fthe disease in the fields.
In a field trial during the 1976 wet
;ason, two susceptible, three inter-
lediate and five resistant varieties prev-
iusly observed under natural conditions
ere inoculated late in the afternoon
ring the late booting stage. Lesion
)unts on flag leaves of the 10 lines
lowed marked differences in reaction to
ie disease. Natural infection showed a
milar trend in reaction of the different
res. Thus, the present results confirmed
.evious findings on the reaction of
lese lines under natural conditions.
ie results of field inoculation, likewise
iggested the possibility of screening
rieties and promising lines for resis-
nce under field conditions.



Phytotoxicity of Filtrates from
Cultures of Stagonospora sacchari Lo and
Ling. M. Guevarra, M. Rosario and
P. M Halos, UPLB.

Filtrates from cultures of Stagonos-
pora sacchari in Richard's medium
exhibited toxic effects when bioassayed
for activity. The toxic agent has the
features of a pathotoxin in that it appears
to play an important causal role in
the development of leaf scorch disease.
Scorching symptoms induced by the
toxin duplicated those of sugarcane
plants infected by the pathogen. The
toxic metabolite affected the susceptible
test variety (Phil 6111) but not the
resistant variety (Phil 58260) in the leaf
cutting, shoot and root assays. The
toxicity of the culture filtrates was
intimately correlated with pathogenicity
of the causal organism.
The toxic agent exhibited a low order
of biological activity in the root and
shoot assays. Roots of susceptible sugar-
cane were minimally inhibited by 1:1,00C
dilution of the culture filtrates. The toxin
appears to be non-selective for root
and shoot growths in cor, and sorghum
were also inhibited at the same concern.
tration. The toxic material is stable at
boiling temperature.

Field Mold of Rice Grains. S. H. On
T. Vergel De Dios, IRRI.

Rice grains in the fields priorto harvest
are contaminated with many fung
(molds). In severe cases, the panicle
appear grayish instead of the natural
golden 'brown color. Attempts wen
made to determine the fungi involved
and the frequency of their occurrence
both outside and inside endospermm) the
grains and degree of damage to the endo
More than 100 rice varieties and breed
in linc m ornwn during the dry and we

seasons in replicates tor yielo ooserva-
tions at IRRI were sampled. Fifty to 100
grains of each variety/line were tested for
the presence of the molds. To detect the
fungi of the whole grain, the grains were
washed in sterile water and plated on
PDA. For detecting fungi in the endo-
sperms, the glumes were removed, the
endosperms were surface-disinfested with
calcium hypochlorite and then plated on

All whole grains were contaminated
with two to four kinds of fungi. More
than 10 different molds were found, but
the most common ones were Alternaria
(Trichoconis) sp. (80%) Curvularia spp.
(88%), AFsarium spp. (80%) and Nigros-
pora sp. was low (6%).

About 67% of the endosperms were
infected during the dry season and 75% in
the wet season. The predominant fungus,
Alternaria sp. had 58% occurrence durinE
the dry and 47% in wet season. Fusarium
sp. was more frequent in wet (20%) than
in dry (2%) season. Other fungi varied
from 1 to 6% in occurrence.

These fungi caused damage varying
from small discolored spots to complete
rotting of the endosperms. The damage
from these fungi may increase during
storage. Whether the Alternaria sp.
produce mycotoxins remains to be inves

Comparative Ability of Various Agri-
cultural Commodities to Support Afla-
toxin Formation. L L. lag, UPLB.

Various cereals, fruits, vegetables
nuts, spices and pasture grasses wen
tested for their ability to support
aflatoxin formation.

The highest amounts of aflatoxin Bi
were observed in dried coffee bean:
(36,764 ppb), rice grains (15,000 ppb)


Abstract of PaDers

cashew nuts (10,417 ppb), sweet potato
tubers (6,000 ppb), peanut kernels (750
ppb), corn grains (483 ppb), cassava
tubers (357 ppb), and coconut meat (202
ppb). Small amounts of aflatoxins B2
and G1 were formed in some substrates.
The other agricultural commodities
that were tested supported the formation
of aflatoxin in varying amounts, often
below 100 ppb.
No aflatoxins were formed in the fol-
lowing commodities in spite of the luxu-
riant growth of Aspergillus flavus Link
ex Fries: fruits of bitter gourd and bottle
gourd, snap bean, string bean, cabbage
head, ginger rhizome, pineapple fruit,
mustard leaves, sugar beet, leaves of Para
grass, Buffel grass, Guinea grass and other
pasture grasses. A. flavus did not grow in
inoculated garlic cloves, consequently, no
aflatoxin was formed in this substrate.

Postharvest Control of Papaya Fruit
Rots by Hot Thiabendazole Suspension. -
A. J. Quimio andM. P Batalla, UPLB.

Tests were made to determine the effi-
cacy of hot thiabendazole suspensions
in controlling post-harvest papaya fruit
rots. In vitro studies showed that the
common papaya fruit rot pathogens in
the Philippines, namely, Colletotrichum
gloeosporioides Penz., Fusarium solani
(Mart) Sacc., Botryodiplodia theobromae
Pat., Phytophthora parasitica Dastur,
and Rhizopus stolonifer (Ebr. ex Fr.)
Blind were more sensitive to and easily
eradicated by 10 min exposure to hot (50
C) suspension of thiabendazole than
either hot water (50 C) treatment or
conventional thiabendazole dip treatment
at room temperature alone.
Treatment of naturally infected fruits
of the "Cavite" papaya cultivar with
hot (50 C) water containing 100, 250,
and 500 ppm thiabendazole for 10 min.
gave highly significant reduction of fruit

rots when compared with the conventio-
nal chemical dip treatment at room
temperature. The degree of ripening at
room temperature and eating quality of
the hot thiabendazole-treated fruits were
not affected. The data indicated that
lower concentrations of thiabendazole,
when used as 50 C suspensions at 10 min
dipping time, can effectively control
postharvest fruit decay.

Postharvest Control of Papaya Fruit
Rots by Hot Water Treatment. A. J.
Quimio and M. P. Batalla. UPLB.

Studies were made to determine the
efficacy of hot water treatment in con-
trolling postharvest diseases of papaya.
Experiments on the thermal sensitivity
of fresh fruits of "Cavite" and "Solo"
papaya cultivars showed that they may be
soaked into 53 C water bath for 20 min
without affecting their physical appear-
ance and eating quality. This hot water
treatment schedule was found to be near in
vitro thermal death points of the common
fruit rot pathogens attacking papaya in
the Philippines which were: 10 min for
mycelia and 53 C, 20 min for spores of
Colletrotrichum gloeosporioides Penz;
53 C, 20 min for mycelia and spores of
Fusarium solani (Mart.) Sacc.; 53 C, 10
min for mycelia and spores of Botryo-
diplodia theobromae, Pat.; 53 C, 10 min
for mycelia of Phytophthora parasitica
Dastur; and 53 C, 20 min for spores and
mycelia of Rhizopus stolonifer (Ebr. ex
Fr.) Lind.
Hot water treatment at 53 C for 20
min of naturally infected "Cavite"
papaya fruits showed that it could signi-
ficantly reduce the postharvest fruit
decay up to 100% This treatment,
however, was not effective against decay
due to Aspergillus and Botryodiplodia.

The Influence of Fruit Maturity
in Banana on Rots in Transit and

)ine Phytopatholog

Storage. -- J. M. Dangan and A. A
Pordesimo, UPLB.
The degree of maturity has some in
fluence on the degree of rotting ii
Latundan fruits. Mature fruits with ;
ripening index of 6, have high disease
index during the first week of storage ii
corrugated cartons and perforated
plastic bags. No rotting was observed ii
either immature or fairly mature fruit!
However, during the second week o
observation, high disease index was als
recorded on fairly mature fruits but onl
moderate rotting was discernible o
immature fruits. On the other hand, fruit
maturity in Bungulan has no appreciable
influence on the degree of rotting. A
fruits, irrespective of degree on maturity)
showed high disease index at a low riper
ing index of 2 during the first week c

High ripening index was observe
during the first week of storage in corrt
gated cartons and perforated plastic
bags for mature Latundan fruits. Hig
ripening index in Bungulan occurred
during the third week. All fruits stored i
non-perforated plastic bags remained gree
up to the termination of the experiment
Four genera of fungi associated wit
banana fruit rot were isolated fror
rotting crown, stalk, and peel tissues c
Latundan and Bungulan bananas. Th
most prevalent were Colletotrichum musa
(Berk. & Curt.) v. Arx, Botryodiplodi
theobromae Pat., Thielaviopsis musarut
Riedl, and Fusarium spp.
Leaf Blight and Gray Spot of Coconi
and Their Chemical Control. M. I
San Juan and J. B. Rebigan. Twin Rive
Research Center.
The leaf blight ot coconut caused 1
Helminthosporium halodes Deschsl. an
the gray spot caused by Pestalotia palm
rum Cke. are the most common ar
important diseases on coconut seedling

Close spacing between plants makes the
conditions conducive to rapid disease
development especially on very suscepti
ble varieties. The Yellow Dwarf (Yellov
King) variety was used in the experiment
Spread of the diseases in the nurser)
was checked through the application o
effective fungicides. Among the fungi
cides tested and found to be effective
were Daconil 2787 W75, Kocidi
101, Dithane M-45, Captex 50 WP
Delsan. Grapefun, Vitigran Blue, anm
Brassicol 50 in their order of effective
ness. These fungicides did not differ sig
nificantly at 5%level.
The systemic fungicides, namely Ben
late 50WP (Benomyl) and Topsin Methy
(Thiophanate-methyl) were ineffective
against the leaf blight and gray spo

Growth and Sclerotial Formation
Sclerotium rolfsii Sacc. as Influenced b
Different Fungicides at Varying Concei
trations. R. C. Cabunagan and B. ,
Soledad, Central Philippine Universit

A study on the effectivity of different
fungicides against Sclerotium rolfsii,
fungus causing seedling blight of rice we
carried out in the laboratory and in th
The bioassay showed that incorpor;
tion of fungicidal solutions to the plate
PDA inhibited mycelial growth and sch
rotial formation and that the extent c
inhibition varied among concentration
and fungicides tested. Higher degrees c
inhibition were exerted by Ceresa
followed in descending order by Hinosai
Maneb, Cosan and Fungitox at bot
1,5000 and 2,000 ppm concentration
Fungicides Milcurb, Cupravit and Vitigra
I Blue showed lesser degrees of inhibitio
of both mycelial growth and scleroti
S formation but were significant (P
S 0.05) different from the control.


Abstract of Fapers

ine eiiecluviy or me live iungiciaes in
the bio-assay was not confirmed under
field condition. Except for Ceresan, the
effects of Maneb, Cosa, Hinosan and
Milcurb before and after sowing did not
significantly (P = 0.05) differ from
control both in terms of disease severity
and growth rates of IR1561 dapog rice
Because of the apparent effectivity of
the 200 ppm Ceresan for the control of
rice seedling blight caused by Sclerotium
rolfsii both under laboratory and field
condition its use is strongly recom-
Biological Control of Mungbean Stem
Rot Caused by Fusarium solani f sp.
phaseoli. I. G. Catedral and P. M.
Halos, UPLB.
An experiment was conducted'to veri-
fy the antagonistic activity of Tricho-
derma viride against Fusarium solani f. sp.
phaseoli Likewise, the host parasite-
antagonist relationships as modified by
the substrates were also studied.

Trichoderma viride successfully con-
trolled the stem rot of mungbean caused
by Fusarium solani f. sp. phaseoli probab-
ly through antibiosis. The degree of con-
trol afforded was comparable to that of
soil sterilization with methyl bromide.
Incorporation of the carbohydrate rich
sorghum residues also checked the
disease even without the addition of the

antagonist. On the other hand, the use of
nitrogen-rich mungbean residues en-
hanced the disease even in the presence of
the antagonist. Thus, the stem rot disease
of mungbean could be successfully
controlled by the antagonist, Trichoder-
ma viride or by cultural manipulation.
Growth and Sclerotial Formation of
Sclerotium rolfsii Sacc. as influenced by
Varying Concentrations of Different Pre-
Emergence Herbicides. R. F Sebidos
and B. S. Soledad, Central Philippine
A bioassay on the inhibitory effect of
Sclerotium rolfsii Sacc., a soil borne
fungus and the causal organism of seed-
ling blight in rice was carried out in the
laboratory. The experimental treatments
consisted of 1000, 2000, and 3000 ppm
of the three different pre-emergence her-
bicides namely: Gesatop, Diuron and
TOK plus a check fungicide; Ceresan.
Among pre-emergence herbicides
tested, Diuron at all concentrations tes-
ted, was the most effective in controlling
mycelial growth and sclerotial formation
of Sclerotium rolfsii. Because of the
apparent effectivity of Diuron, we strong-
ly recommend its use against Sclerotium
rolfsii and other related soil-borne plant
pathogens from the standpoint of toxi-
city, economy, and broad spectrum of


ieceivea ror pu

r17Xll i XAJ V.L A L, a XJ


Former student and Associate Pro
logy, College of Agriculture, U.P. at Los
Portion of an undergraduate thesi
ment of the B.S. Agriculture degree.


Fruit rot of pepper (Capsicu
occurs widely in the field, after
market. The lesions were initially
of brown. Under moist condition:
the affected surface, and in the ai
soft water mass. In dry condition
became shrivelled.
The organism isolated from
tified as Fusarium oxysporum Scl
Wr. according to the taxonomic s
to be pathogenic to all the varieties
F. oxysporum and F. solan
Both species grew at different pf
ponses may explain their ubiqui
lence of Fusarium rot in Philippine

Pepper (Capsicum annuum L.) is oft
subject to fruit rots. A common fruit i
is caused by Fusarium species which c
cause serious damage not only in t
field but also in storage.
The rotting is usually incited by t
fungus during the various phases of t
marketing process through injuries in
dental to harvesting, packaging, transpo
ing and marketing of the produce.
Many species of this fungus are co
mon in soils suitable for growing vej
table crops. Consequently, most fr
crops such as pepper are contaminat
by Fusarium when harvested eventhoul
they may not yet be infected. Soi
pepper fruits that are found near or
the surface on the soil often become
fected by Fusarium since this fungus c

%. Av..," su.J lJ. a l,. vi. j rj IL J .


fessor, respectively. Department of Plant Pi
Banos, College, Laguna.
; presented by the senior author in partial ft


i annuum L.) caused by species of Fusarium
harvest in transit, in storage, and in the
pale in color, then turned to various shades
, whitish or pinkish mycelia developed on
vanced stage, the entire fruit turned into a
is, the affected pepper lost moisture and

typicallyy diseased pepper fruits were iden-
ect and Fusarium solani (Mart.) Appel and
stem of Snyder and Hansen. Both proved
of pepper tested.
grew and sporulated from 20 C to 30 C.
levels ranging from pH 5 to 8. These res-
aus distribution in nature and the preva-
fruits and vegetables.

in live for a number of years in the soil a
At it spreads rapidly by means of soil pa
in cles conveyed from one place to anoti
Le by rains, floods, tools, or other agents.

This study was conducted at the
Le apartment of Plant Pathology, College
he Agriculture, University of the Philippi
:i- at Los Bafios, from April, 1975 to Ji
t- 1976 to identify the species of Fusari
responsible for the rotting of pepper p
a- based on the morphological and cult
e- characteristics of the causal fungus,
it to ascertain the effects of different t4
d peratures, pH, and various media on
h growth and sporulation of the fungus.
in Isolation to pure cultures

1, Ir II

Fruit Rot of Pepper

The experimental organisms were
isolated from diseased pepper fruits by
the tissue planting method.
Study of morphological characteristics
The morphological characteristics of
each fungus, grown in PDA and kept
under ordinary laboratory room condi-
tions (average temperature, 27.5 C),
were determined.
Conidia were stained with Nigrosin,
examined and measured. Spore measure-
ments were made with a filar micrometer
under high power (400 x magnification).

Chlamydospore production was stimu-
lated by placing a small piece of the fun-
gus together with some of the PDA
medium on which the fungus was growing
in 10 ml distilled water and allowing this
to stand for about 7 to 10 days (Alexan-
der, 1966).

For photomicroscopy, stains such as
lactophenol cotton blue, methylene blue,
and phenol red were used as mounting
media. Observations were made fairly
rapidly as the macronidia tend to swell
due to liquid imbibition.
Growth and sporulation in different

The experimental organisms were sepa-
rately grown in water agar, potato dex-
trose agar, Czapek's agar, Mycophil
agar, and V-8 juice agar (Tuite, 1969). A
6-mm agar disc cut with a cork borer
from the radiating mycelia of a 3 to
5-day old culture placed at the center of
the petri plate was used to start each co-
lony. Three plates were provided for
each medium and colony diameters were
measured in millimeters everyday for one
week. Color changes in the mycelium and
in the medium were determined based on
R. Ridgway's (1916) "Color Standard
and Color Nomenclature". All cultures
were kept at ordinary laboratory room

temperature (average of 27.5C).
To determine the degree of sporula-
tion, a spore suspension was made by
adding 10 ml or 20 ml of sterile water
to each petri plate. The surface of the
fungal growth was scraped gently with a
wireloop to disperse the spores. From the
suspension, an aliquot was placed in a
haemacytometer and the number of
macroconidia and microconidia were
counted separately.
Growth and sporulation at different

The growth of each fungus in relation
to temperature was studied by growing it
in V-8 juice agar medium and incubating
the inoculated petri dishes at different
temperatures: 0 C, 10 C, 20 C, 30 C, and
room temperature (average 27.5 C).
Three cultures of each isolate were kept
at each temperature and the radial spread
of the colony in each petri dish was
measured after 7 days. The number of
conidial production was determined with
a haemacytometer.
Growth and sporulation at different pH
The fungi were separately grown in
PDA adjusted to initial pH levels of 5.0,
5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. The cultures
were incubated under ordinary labora-
tory conditions for 7 days. The daily
linear growth and degree of sporula-
tion were determined.

Pathogenicity tests
To demonstrate the pathogenicity of
the organisms isolated from decaying
pepper fruits, inoculation experiments
were conducted with hot and sweet
varieties of pepper. The following lines
were tested: line isolated from a cross
between Batangas Native and World
Beater, line isolated from a cross bet-
ween Batangas Native and California
Wonder, Hungarian Wax, and some

Jan. & June 1977

Philippine Phytopa

strains or -rasites were usea. Inese were
surface-disinfected in 20% Chlorox for 1
minute, rinsed twice with tap water, and
air-dried. The test materials were divided
into four sets, one set containing at least
two pieces. They were inoculated with a
sterilized dropper using the following
treatments: 1) not inoculated, not
pricked, 2) not inoculated, pricked,
3) inoculated, not pricked and 4) inocula-
ted, pricked. These were incubated in
moist perforated plastic bags and kept
at room temperature inside incubation
chambers. The controls were placed in
separate chambers. Observations for the
appearance of symptoms were made daily
for two weeks.

Fig. 1. Growth of F. sola8
and d exhibited a
whereas b and c hac
and pionnotal types

KUiUL 1 ANlu ULiaLU iU31N

Isolation to pure cultures
From the initial isolations from na-
turally diseased tissues, two types of cul-
tures resulted which exhibited the dual
phenomenon described by Hansen (1938).
One was characterized by an abundance
of aerial mycelia (figs. la and ld). The
conidia were ordinarily produced in spo-
rodochia. Occasionally, cultures were
found with abundant aerial mycelia but
few macronidia, termed as the mycelial
type. Other cultures, called the pion-
natal type, had little or no aerial my-

in various agar media; a
nycelial type of growth
Combination of mycelial
>f growth.

Vol. 13

Fruit Rot of P

celium but had numerous macrocon.
dia. As cultures of the latter type agec
they usually produced a slimy appearance
A mass transfer of culture resulted int
a mixture of mycelial and pionnota
types (figs. lb / Ic). This was probably
due to a reassortment of nuclei perpetua
ted from the outset (Goddard, 1939).

Identification of Species
The isolates in this study were iden
tified as F oxysporum Schlect, and F
solani (Mart.) Appel and Wr., based or
the taxonomic system of Snyder anc

Fusarium oxysporum. Microconidii
numerous, dominantly 0 septate. Somi
ellipsoidal whereas other were ovoid
fusoid in shape. The microconidial stage
was often produced.
Macroconidia were fine and elongate(
with thin walls, finely pointed at thei
extremities or gradually attenuated to
ward both ends. Septation frequencie:
varied from 3 to 6; 3 septate 52%, 4 -
septate (33.5%), 5 septate (12.5%)
and 6 septate (2%).
Sclerotial bodies were found after 2I
hours of incubation in ordinary laborato
ry temperature. They were bluish-blaci
in color, globose or in cauliflower-like
Chlamydospores were found singly oi
in pairs. They were either conidial oi
Fusarium solani. Microconidia were
also abundant but bigger in size than the
microconidia of F oxysporum. The)
were mostly one-celled.
Macronidia were slightly curved, stock)
and with thick walls, slightly constricted
at apex or not ending by a fine point
They were somewhat pedicillate. The 2 -
septate macroconidia were more common
about 89% and the 3 septate conidia

were about 11%.
Scleorotial bodies were not observed.
Chlamydospores were found singly or
in pairs. They were either mycelial or
Spore measurements. Measurements
of 200 macronidia from 20-day old PDA
cultures showed that the length of macro-
conidia of F. oxysporum ranged from
20.5 to 83.0 u and the width from 1.5 to
2.49 u..The majority ranged from 27.5
to 34.0 u in length and 2.5 to 3.49 u in
width. The average size was 44.05 u x
3.415 u.
The length of macroconidia ofF. solani
ranged from 20.5 to 48 u and the width
from 2.5 to 7.49 u. The majority ranged
from 27.5 to 34.0 u in length and 3.5
to 4.49 u in width. The average size of
the 200 macroconidia of F solani was
32.92 x 4.585 u.
The macroconidia of F. oxysporum
were longer than those of F solani but
the latter's macronidia were wider than
those of the former.

Cultural Characteristics

Growth and sporulation in different
media. Tables 1 and 2 show differences in
the growth rate of the two fungi in various
substrates. In general, the widest colony
diameters were formed in PDA and V-8
juice agar followed by Czapek's agar and
then by Mycophil agar. Water agar was
not a good growth medium for both
isolates. Mycelial growth in water agar
was sparse, finely scattered, thin and
barely visible even though linear exten-
sion as measured in water agar was similar
to that in Mycophil agar.
Aerial mycelia were present and typi-
cally well developed in all cultures with
the exception of those growing in water

Jan. & June 197

X?1 -10

Phillnnine. Phutnnathnlnur~

Table 1. Colony diameter (in mm) formed bJ


1 2

PDA 15.8 32.0
Water agar 6.0 8.0
Czapek's 9.0 20.0
Mycophil 9.0 22.0
V-8 juice 15.16 31.6

'Average of 3 plates/medium/day.

Table 2. Colony diameter (in mm) formed bj

1 2
PDA 8.5 20.5 26.
Water agar 6.0 19.0 31,
Czapek's 7.5 19.5 25,
Mycophil 9.0 22.0 34
V-8 juice 8.5 23.6 33

'Average of 3 plates/medium/day.

F. solani exhibited excellent growth in
PDA. This medium turned pale pinkish
buff. On the other hand, F. oxysporum
exhibited excellent growth in V-8 juice
agar wherein pale vinaceous pink mycelia
covered the entire petri plate with a loose
cottony growth 3 to 5 mm in height.
This medium became shell pink. Color
changes were observed in the mycelia and
the various agar media with the excep-
tion of water agar in which the mycelium
and the medium remained colorless.
It was observed that the medium
which gave the most abundant mycelial

Fusarium oxysporum in various media1


4 5 6 7

5.0 59.3 82.5 87.5 90
;7.5 53.3 62.5 67.5 70
2.6 45.0 63.3 77.3 85
.3.3 46.3 56.5 66.0 70
7.0 64.5 79.0 88.3 90

Fusarium solani in various media'

)ays of incubation

1 4 5 6 7
5 43.6 59.5 67.5 83.0
3 46.3 55.6 61.0 71.6
5 38.0 51.5 59.3 78.0
) 45.6 57.0 68.0 70.5
) 47.0 59.0 70.0 80.0

growth also gave the greatest number of
spore counts (table 3), with the exception
of mycophil agar in which more spores
were formed than in Czapek's agar when
in fact, Czapek's agar was a better me-
dium than mycophil for mycelial growth.
Both isolates yielded more microconidia
than macroconidia.
Growth and sporulation at different
temperatures. Mycelial growth was
maximum at room temperature (average
of 27.5 C) for F oxysporum, 30 C for
F. solani (fig. 2). It appears that the op-
timum temperature for the mycelial

Fruit Rot of P

Table 3. Macroconidia and microconidia (>
and F. solani in various media

F. oxysporum
Macro Micro
conidia conidia

PDA 13.50 280.50

Water agar 0 1.0

Czapek's 0.72 61.25

Mycophil 0.77 111.0

V-8 juice 2.25 554.0




6 0




Fig. 2. Linear growth (mn
F. solani incubated

106 per ml) formed by Fusarium oxysporun

F. solani
Total Macro Micro Total
conidia conidia

294.00 11.56 13.50 25.06

1.0 0.01 0.25 0.26

61.97 0.09 1.0 1.09

111.77 0.02 1.5 1.52

556.25 0.42 9.25 10.67



[URE (C)

) of F. oxysporum and
at different temperatures.

J.- R, .I,,, 1 QT7

lilippine Phytopathology

growth of both fungi lies somewhere
around 27-30 C. At 0 C the growth of
both fungi were completely inhibited.
However, at 10 C, F oxysporum was able
to grow at a slow rate whereas F solani
remained dormant.
Most fungi are unable to grow at 35-40
C. Mycelia are easily killed by high tem-
peratures and many fungi die slowly
when held in culture at temperatures
just above the maximum for growth
(Cochrane, 1958). Flsarium is extremely
sensitive to heat and cold. It causes very
slight infection at 7.7 C and 36 C but
severe infections between 19 to 32.19
with optimum at 27.75 C (Chupp, 1960).
Temperature plays a very important role
in the growth, sporulation and pathogene-
city of fungi.
Table 4 shows a marked increased in
sporulation from 20 to 30 C. Sporulation
was greatest at 30 C. Both organisms were
not able to sporulate at 10 C. It was observ-
ed that F. oxysporum sporulated more
than F solani at all the temperatures
From the results obtained for linear
growth and sporulation, it was noted

Table 4. Number of macroconidia and mic
porum and F. solani at various ten

F. oxysporu
Macro Micro
conidia conidia

LO C 0. 0
20 C 0.75 250.5
10 C 10.70 613.5

room tem-
Ave. of
17.5 C) 13.50 280.5

that linear growth was in general directly
related to sporulation, that is, the greater
the colony diameter, the more spores
were formed by the organisms.
There were some differences in the
linear growth rate of the two fungi
from pH 5.0 to pH 8.0 (tables 5 & 6).
Both species had a wide pH range in
terms of mycelial growth. This may be
due to their capacity to adapt and grow
at different pH concentrations.
F oxysporum sporulated best at pH
5.0 and least at pH 8.0 (table 7). This
indicates that acidic conditions were
preferred for sporulation by this fungus.
F solani sporulated best at pH 6.0 and
least at pH 5.0. The two species varied in
their optimum pH requirement as pH 5.0
was favorable for spore production for F
oxysporum but was unfavorable for F
solani. It was noted that the pH range
for sporulation was narrower than that
for vegetative growth, specially with
respect to macroconidial formation of F

Microconidia were at all times formed
in greater abundance than macroconidia.
No macroconidia were formed by F.

conidia (x 106) formed by Fusarium oxys-

I F. solani

total Macro Micro Total
conidia conidia

0 0 0 0
51.25 4.38 16.0 20.38
24.2 18.29 80.25 98.54

)4.0 11.56 13.50 25.06

Vol. 13

Jan. & June 1977

laote 3. cotony aameter (mnn) of Fusari

1 2

5.0 8 15.0 3
5.5 8 16.0 31
6.0 8 13.6 31
6.5 9 16.0 41
7.0 13 20.3 4;
7.5 8 17.3 4
8.0 8 18.0 4:

'Average of 3 cultures.

Table 6. Colony diameter (mm) of Fusariu

1 2 3

5.0 10 13.0 35.3
5.5 8 23.3 36.3
5.0 8 13.6 36.0
5.5 8 14.6 36.0
7.0 9 12.0 37.0
7.5 9 13.0 37.0
3.0 9 13.0 34.0

' Average of 3 cultures.

m oxysporum grown at a:rjerent intma pH


3 4 5 6 7

6 48.6 62.3 62.3 75.0
0 54.3 68.6 79.0 86.6
3 47.6 64.6 70.3 79.0
0 41.6 66.0 69.6 76.3
6 64.0 78.0 83.6 89.3
3 63.3 79.0 82.6 91.5
0 55.0 72.3 84.6 89.3

i solani grown at different initial pH levels


4 5 6 7

47.0 61.3 73.0 86.6
47.0 63.0 75.3 87.5
49.5 64.0 72.3 87.0
59.5 65.6 78.3 89.5
50.0 66.0 75.3 91.5
49.0 65.8 77.3 88.0
48.0 64.5 78.6 88.3

Fruit Rot of

22 Philippine Phytopathology Vol. 13

Table 7. Number of spores (x 106) formed by Fusarium oxysporum and F. solani at
various pH

F. oxysporum F. solani

pH Macro Micro Total Macro Micro Total
conidia conidia conidia conidia

5.0 4.52 685.0 689.52 0 0.50 0.50
5.5 3.0 317.5 320.5 0 3.25 3.25
3.0 11.0 155.5 166.5 11.90 44.5 56.4
3.5 5.9 187.5 193.4 11.7 32.5 44.2
7.0 4.0 292.5 296.5 6.83 37.75 44.58
7.5 2.2 141.0 143.2 3.1 12.5 15.6
3.0 1.0 39.5 40.5 9.5 26.25 35.75

Fusarium rot of pepper appeared first gent odor was occasionally produced.
as small watersoaked spots which were The tissues became soft and within few
either circular or irregular in outline. days the whole fruit collapsed.
The lesions were generally brown and In every inoculation experiment re-
somewhat sunken due to the rapid drying isolation of the fungus from infected
and shrinking of the tissues. Under very fruits was done. In all cases, the morpholo-
moist conditions, the lesions commonly gical characteristics of the reisolated
became covered with whitish or pinkish -,..,, ...... :,A;,,., ..,:.1, i,..... -r +1.

Lu a waitcy, uctayou, uoUWIIIII-sl-UIa
fruit due to the coalescence of several
lesions. However, in relatively dry incuba-
tion chambers, although lesions may
occur after three days of inoculation,
infection was slow and diseased tissues
remained fairly firm. The symptom was a
sunken, wrinkled dry rot on the inocu-
lated portion.
Fusarium decay on sweet varieties of
pepper was hastened by the association
of some forms of bacteria which appeared
a few days after infection. A fruity pun-


tion of chlamydospore formation by
natholoov 56:353-354.

Stoessl, Unwin, and Ward (1973)
revealed that some species of Fusarium,
including F oxysporum and F. solani,
could degrade capsidiol, the antifungal
sesquiterpene induced in pepper fruits to
the less toxic capsenone.

Species of Fusarium are often con-
sidered "wound" or as weak pathogens.
However, their ability to grow rapidly
under a wide range of environmental con-
ditions more than make up for their
supposed "weakness".


GOLD, and W.C. SNYDER. 1966. Induc-
tsarium solani in sterile soil extracts. Phyto-

unrult Itot 01

LUtjr;nKAlrit V.W. 1i9a. rnysiology or
524 pp.
GODDARD, M. 1939. "Studies on varial
sarium graminearum Schwabe)". R
taxonomy and pathogenicity of Fuw
Phytopathology 39:359-376.
HANSEN, H.N. 1938. The dual phenomel
RIDGWAY, R. 1916. Color standard an<
43 pp.
SNYDER, W.C. and H.N. HANSEN. 194
Bot. 27: 64-67.
SNYDER, W.C. and H.N. HANSEN. 1941.
to section Martiella. Ibid. 28:783-74
reference to Discolor and other secti
species and their perfect stages. Phyt
from plants. Fungal oxidation of ca]
Fusarium species according to the ta
University Park, Pennsylvania State 1
TUITE, J. 1969. Plant Pathological Meth
WOLLENWEBER, H.W. 1913. Studies on
BAILEY, 1925. Fundamentals for

ingi. New York. John Wiley and bons, In

>n in Giberella saubinettii (Mont.) Sacc. (Fi
r. of J.W. Oswald. 1949. Cultural variation
-ium species associated with cereal root rot

n in imperfect fungi. Mycologia 30: 442-45!
color nomenclature. Washington, D.C. Illu

The species concept in Fusarium. Amer. .

ie species concept in Fusarium with refereni

15. The species concept in Fusarium wi
is. Ibid. 32:657-666.
65. Current status of taxonomy in Fusariu,
pathology 55:833-837.
ARD. 1973. Post-infectional fungus inhibitic
idiol in pepper fruit. Phytopathology 63:22

58. A pictorial guide to the identification <
mnomic system of Snyder and Hansen.
diversity Press. Illus. 51 pp.
is, fungi and bacteria. Burgess Publ. Co. 23

he Fusarium problem. Phytopathology 3:2

axonomic studies of Fusarium. J. Agr. Re

Jan. & June 1977



Researcher, National Crop Protection
department of Plant Pathology, College c
aguna 3720.
The major portion of this paper was I
ients for the degree of Master of Science ir
t Los Banos, College, Laguna.


Various heat treatments such as
autoclaving and boiling, and oven-dry
effects on reducing or eliminating th
mungbean seeds.



enter, and Associate Professor, respectively,
Agriculture, U.P. at Los Banos, College,

.sented as partial fulfillment of the require-
lant Pathology, College of Agriculture U.P.


Ailing, autoclaving, combination of
eating were tested to determine their

and Mitchell, 1946). In ly disposed of in the local
tries where meat is in markets as dry seeds.
id, therefore, expensive, conditions of harvesting.



Lusln, i1iungucan may oe nvaaea heavily
y toxicogenic strains of the common
told Asperigullus flavus and this results
1 the elaboration of a highly toxic class
f mycotoxins commonly referred to as
flatoxins (Wogan, 1965). Aflatoxins are
group of highly poisonous, carcino-
enic compounds and potent hepato-
oxins which cause tumorous changes in
ie liver. The chemical and biological
effects of the four principal members of
is group of mycotoxins, aflatoxins
!1, B2, Gi and G2, have been reviewed
y Wogan (1966).

Although initial reports indicated that
flatoxin is heatstable, later studies de-
lonstrated that aflatoxin can be par-
ally degraded at elevated temperatures.
ischbach and Campbell (1965) reported
iat temperatures of 300 C or above are
required to decompose aflatoxin from
agricultural products.

Newberne et al. (1966) reported that
Jtoclaving contaminated peanut meal
destroyed or eliminated part of aflatoxin
:tivity with no measured diminution in
ie nutrient value as feed. In the same
ear, Coomes et al. also observed that
utoclaving (15 psi, 121 C) moistened
eanut meal samples containing 7000
g/kg of aflatoxin for four hours reduced
oxin level to 350 ug/kg.

Mann et al. (1967) reported that on
ilseed meals, definite reductions of
latoxin were obtained at 100 C and in-
easing moisture content. The greatest
Auction in aflatoxins Bi plus B2 was
:hieved by heating the oilseeds aiming
)% moisture for 120 min. at 100 C.
bout 34% reduction in aflatoxin (111 to
3 ppb) was obtained when contaminated
eanut was heated in a similar manner.

Lee et al. (1969) reported that the
rerage reduction in the aflatoxin content
f peanuts ranged from 45 to 83%,

iepenumg on me roastng conoitons ana
he level of toxin in the raw kernels.
Fhe degree of reduction in aflatoxin
:ontent in both oil and dry-roasted
peanuts was greater at the highest aflato-
:in contamination levels.
Although much has been published on
he decontamination of aflatoxin in many
agricultural commodities, nothing has
een reported on mungbean. Since it is a
ich source of protein for man and ani-
nals, it is therefore, important that we
investigate the effect of various heat
treatments in mungbean seeds contain-
ng aflatoxin for the results of this ex-
eeriment can serve as part of the solution
o improve the quality of locally availa-
>le foods and feeds.
This study was conducted at the De-
iartment of Plant Pathology from April
976 to September 1977. Thin-layer
hromatography which was used in de-
ecting aflatoxin was done at the Afla-
oxin Laboratory of the Department of
:hemistry, College of Sciences and Hu-
nanities, College, Laguna.


The procedure for the extraction of
flatoxin by Pons and Goldblatt (1965)
tas followed. However, the presence of
interfering pigments created a problem
luring the subsequent thin layer chroma-
ography analysis so that clean-up proce-
lures by Eppley et al. (1968) were
employed. For qualitative and quantita-
ive analysis of aflatoxin, TLC (thin-
syer chromatography) was employed.
The sample extract and aflatoxin
standard were separately spotted in a
lass plate coated with silica gel or Adsor-
osil-1. The bottom of the plate was
lipped in a developing solvent for 10-15
nin. to allow the solvent to move
through the spots of the sample and the
standard. The various compounds inthe.

~~'~'Y"U'UV'VL ~1U

DJULO wLu 3tx paLaL..u uwl --, -.. I. .n a 6 m Unn5 WE'- t IL. U S'L J'J*AZE6,
relative adsorbabilities, as they moved each flask was inoculated with 30 ml
with the solvent through the silica gel. spore suspension of the A. flavus isolate
After this development process the plate and incubated for 7 days at ambient
was dried and examined under a long- room temperature (29-33 C). The flasks
wave (365nm) ultraviolet lamp viewer, were rated for amount of fungal growth
The presence of aflatoxin and the and sporulation to correlate aflatoxin
amounts present were determined by com- content with growth. Fifty-gram quanti-
paring with the standard the color inten- ties of the inoculated mungbean with
sity distance travelled by the fluorescent moisture contents ranging from 44-59%
spots that appeared under UV light, and known to contain certain levels of
aflatoxin were used. Three autoclaving
Computation of the amount of aflatoxin temperatures were tested: 100 C (0 psi),
in the sample extract 121 C(15 psi) and 126 C (20 psi) for 0.5,
Sfrm u 1.0, 2.0 and 3.0 hours. Unheated sam-
The formula used by Pens and Gold-
blatt (1965) was employed: aples served as controls. After the heat
treatment, the samples were reassayed for
aflatoxin content.
Amount of aflatoxin (ppb) =
(Vs) (Cs) (S. D.) x 1000 Decontamination of Aflatoxin by Boiling
(X) (W) (0.7)* Only and by Autoclaving plus Boiling

*(Valid only for 175/250 aliquot of sam- A portion of the inoculated mungbean
pie extract) from the sample for autoclaving study
where: was used. Fifty grams of inoculated
(Vs) ul of aflatoxin standard for mungbean were suspended in a known
which color intensity of standard spot volume of boiling water (100 C) for vary-
matches intensity of the color spot of the ing periods (0.5, 1.0, 2.0, 3.0 hr). The
sample volume of water used was increased pro-
) cn o a i portional to length of the boiling periods
(Cs) conc n of f n such that the final volume after the heat-
the standard. in ua/ml 0.5 ue/mlBI and .

extract is diluted for TLC analysis, in ul
(X) volume of sample extract spot
ted, in ul
(W) sample weight in grams

Decontamination of Aflatoxin by Auto
Triplicate 300-gm samples of CES-5
mungbean seeds were separately placed ii
3000-ml Erlenmeyer flasks. In each flask
300 ml of distilled water were added
allowed to stand for 2 hr with frequent

Five 50-gram samples of inoculated
mungbean were autoclaved at 121 C (1I
psi) for 15 min. The autoclaved mung
bean was suspended in a known volum(
of boiling water for different boiling
periods (0.5, 1.0, 2.0, 3.0 hr). Thin
layer chromatography and fluorescence
under ultraviolet light were used fo:
aflatoxin determination.

Aflatoxin Decontamination by Oven
Dry Heat Treatment
The possible destruction of aflatoxir

.J a;'J LlY' Ltn. L..l.SLn 111 tI I..tJ.,I I aco aIuItaVIln.5il bLIIIhni.UI 1i 4jl a Liu U jIUU
also tested. For each 250-gram CES 55 of heat treatment increased, a correspond-
mungbean seed variety, 50 ml of dis- ing decrease in aflatoxin content was ob-
tilled water were added. Each sample was served until no more aflatoxin was de-
placed in a 3000-ml Erlenmeyer flask, tected after 3 hrs of heating. These
allowed to stand for 2 hr with frequent results confirm the observations of
mixing and then separately inoculated Coomes et al. (1966) who demonstrated
with 50 ml spore suspension of A, flavus that sterilization by autoclaving at 15
isolate. The flasks were incubated for 7 lb/in2 (120 C) of wet toxic groundnut
days at ambient room tempetarure. After meals resulted in a progressive reduction
7 days, twenty-two 50-gm samples were of aflatoxin with time. They also repor-
dishes. Five different oven temperatures ted that autoclaving of pure aflatoxin Bi
(150, 180, 200, 220, 250 C) and four at 15 lb/in2 (120 C) for 4 hr gave metha-
different heating periods (0.5, 1.0, 2.0, nol-soluble, nonfluorescent reaction pro-
3.0 hr) were employed. Untreated sam- ducts in which no traces of starting ma-
ples (control) were also included and trials could be detected by thin-layer

I a part of aflatoxi
ninution in the nuti

ation of

rilized ii
study h
a 44-599
Its on t
toxin de
e unheat
29 ppb e
inged a

effect of

Aflatoxin (Bl) content (ppl
Temperature and Pressure
100 C (0 Dsi) 121 C (15 psi)

43 29

VIQall .1 . .- ik. Iyo In -t,. u 1 --
no differences in appearance from that of
the control but, the aflatoxin content
(57 ppb) of the treated sample was re-
duced by 55.60% when compared to that
of the control. When the samples were
further subjected to boiling temperature
for different periods, the mungbean be-
came very soft and the color was markedly
different from that of the control.

Table 2 presents the data obtained in
this experiment. It is evident that the
treatments described have substantially
reduced or eliminated the aflatoxins from
the inoculated mungbean as measured by
TLC assay.
In general, as the boiling and auto-
claving plus boiling processes were pro-
longed, progressive reductions in the
aflatoxin content were observed. This is
similar to the finding of Mann et al.
(1967) who reported that oilseed meals
heated at 100 C showed marked reduc-
tions in aflatoxin levels with increasing
periods of heating and increasing mois-

xins B, plus B2 practically achieved was
about 44 ppb obtained by heating the
oilseed meals for 120 min. at 100 C with
moisture content of 20% About 34% re-
duction in aflatoxin (111 to 73 ppb) was
obtained when contaminated peanut was
heated in similar manner.
It is apparent that moisture enhances
aflatoxin destruction by heat because
boiling (100 C) for 1.0, 2.0, 3.0 hr reduced
the aflatoxin content of contaminated
mungbean seeds to levels considered
acceptable where oven-dry heat treatment
at higher temperatures did not reduce the
toxin below the tolerance level of 30 ppb
set by FAO and WHO. It is likely that the
first change is the hydrolytic opening of
the lactone ring of aflatoxin Bi (Figure 1)
to form a substituted O-coumaric acid
(Figure lb) followed by decarboxylation
of the acid (Figure Ic). Some investigators
who performed experiments on aqueous
hydrolysis of the toxin gives support to
this (Coomes et al. 1966; Newberne et al.,

Aflatoxin (Bl) content (ppb)
Length of heat
treatment (hr) Boiling only' Autoclaving+boiling2

0.5 36 14
1.0 21 11
2.0 13 0
3.0 0 0
Unheated (control) 129

1 Boiled at 100 C for 0.5, 1.0, 2.0, 3.0 hr.
2The samples were first autoclaved at 121 C (15 lb/in2) for 15 min and then

rr"'~- "~ 1V11111--r~~lJ

- -- -~-

I T'bnnnpmnto~ ,,incn

o 0


0' ^ -' ^ Heat
0 0' ^ OCH3 4

(a) HO

0 (
o (b)



Fig. 1. Aqueous hydrolysis of aflatoxin E
by decarboxylation (c).

:a) forming O-coumaric acid (b) followed

Tr-- A- IN.._ I n 17


Effect of Oven-Dry Heat Treatmen
't-^ A*n-_:_ 10rj- :_ n-A..,

culated Mungbean and were not fit to be eaten. Similarly,
The A. flavus isolate grew more luxu- Lee et al. (1968) reported that although
riantly in raw than in autoclaved mung- almost complete reduction in aflatoxin
bean when incubated at ambient room content (92 to 98% ) was observed in
temperature (29-33 C) for 7 days. How- peanuts roasted for 5 min at 400 F, the
ever, the aflatoxin formed was less (107 peanuts were burnt and would not be
ppb) compared to the amount produced considered acceptable.
in the sterilized mungbean (129 ppb). The practical application of most of
This shows that growth is not necessarily these treatments need further investigation
related to the amount of toxin forma- since some treatments changed the ap-
tion. pearance and possibly other properties of
There was reduction in the aflatoxin the mungbean. Studies in texture changes

similar results were observe in samples
heated at 150 C for 2.0 and 3.0 hr.
Completed reduction in aflatoxin content
was observed in seeds heated at 200 C for
3.0 hr and at 220 C and 250 C for all
time periods tested (Table 3, Appendix

Fable 3. Effect of oven-dry heat treatment

Length of heat ----
treatment (hr) 150 181

0.5 79
1.0 70
2.0 7
3.0 7
Jnheated (control) 107

nutritional value of the product is not
impaired by the decontamination pro-
cess and that the aflatoxin decontamina-
ted mungbean contains toxic by-products.

on the aflatoxin (B1) content in inoculate,

flatoxin (Bl) content (ppb)

C 200 C 220 C 250(

) 7 0 0
L 4 0 0
1 3 0 0
7 0 0 0

of proteins with their nutritive value. Nutrition Abstr. and Rev. 16: 249-278.
OOMES, T.G., D.C. GROWTHER, A.J. FEUELL and B.J. FRANCES. 1966. Experi-
mental detoxification of groundnut meals containing aflatoxin. Nature 209:406-407.

ni__ n .

11 __


lan. & June 1977 Aflatoxin Decontamination in Mungbean 31

?PLEY, R.M., L. STOLOFF and A.D. CAMPBELL. 1968. Collaborative study of"A
versatile procedure for assay of aflatoxins in peanut products", including preparatory
separation and confirmation of identity. J. Ass. Off. Agric. Chem. 51:67-73.
:SCHBACH, H. and A.D. CAMPBELL. 1965. Note on detoxification of the aflatoxin.
J. Assoc. Offi. Agr. Chemists 48:28.
tion and chemical composition of munghean. Philipp. J. Sci. 93:47-56.
3E, L.S., A.F. CUCULLU, A.O. FRANZ, JR. and W.A. PONS, JR. 1969. Destruction
of aflatoxin in peanuts during dry and oil roasting. J. Agr. Food Chem. 17:45-453.
ANN, G.E. L.B. CODIFER, JR. and F.G. DOLLEAR. 1967. Effect of heat on aflatoxin
in oilseed meals. J. Agr. Food Chem. 15: 1090-1092.
EWBERNE, P.M., R. RUSSO and G.N. WOGAN. 1966. Pathol. Vet. 3: 331-340. In:
Aflatoxin-A summary of recent works by F. G. Peers. Trop. Sci. 9:186-203.
)NS, W.A., JR. and L.A. GOLDBLATT. 1965. The determination of aflatoxin in cotton-
seed products. J. Am Oil Chemists' Soc. 42: 461-475.
OGAN, G.N. 1965. Mycotoxins in foodstuffs. M.I.T. Press. Cambridge, Mass. p. 291.
OGAN, G.N. 1966. Chemical nature and biological effects of the aflatoxins. Bacteriol.
Rev. 30:460-470.

Philipp. PhytopathoL 13:32:37 (1977)
Received for publication: 4 April, 1978



Associate Professor and Research Assistant, respectively, Department of Plat
Pathology, College of Agriculture, U.P at Los Banos, College, Laguna.
This study was supported by UPLBCA-PCARR Project No. 170-3 and UPLB-IP
project on cotton.


Cotton plants grown singly in 23-cm diam clay pots containing 4.4
liters of soil infested with varying numbers of active and egg stages of Roty-
lenchulus reniformis exhibited pathologic reactions five months after planting.
In soil initially infested with approximately 200, 300 and 500 nematodes,
root weight was reduced by 30.8%, 35.1% and 48.0%, respectively. At both
300 and 500 infestation levels, reduction in number of bolls produced was
Plants grown singly in small plastic cups containing 250 cc soil initially
infested with approximately 76 active and egg stages of the nematode showed
reductions in root weight, top weight and height of 47.6%, 32.3% and 27.6%,
respectively, two months after planting. Their roots were necrotic and their
leaves distinctly chlorotic. Nematode population increased by 1.8 times during
the two-month period. When a different, but similarly treated, set of plants
were transplanted from the cups to the 13-cm diam clay pots and grown for
another four months, root weight, height and number of bolls produced were
reduced by 33.3% 49.3% and 28.8% respectively. During the six-month
cropping period, nematode population increased by 21.8 time&

Pests and diseases are among the maior to 1977 in the cotton-growing areas o

the United States where active nematolh
gical investigations have been conducted
the disease called root knot, caused b
the root-knot nematode, Meloidogyr
spp., ranked second only to wilt in impo
tance (Presley, 1954). The genera Rot
lenchulus, Pratylenchus, Belonolaimu
and Hoplolaimus have also been imp]
cated with damage on cotton (Presle,
1954; Krusberg and Sasser, 1956; Bird
field, 1962).
A survey that we conducted in 197

most widely-distributed, having been o
served in all areas surveyed, and hil
population densities were frequently a
sociated with stunting of plants. It
believed to be the most important nem
tode pest of cotton in the country
The genus Meloidogyne was limited
distribution and the negative results o
tained from inoculation experiment
(Begonia and Davide, 1968;Valdez, 196
Toledo and Davide, 1969; Castillo, 197
indicated resistance of cotton to tl

Reactions of Cotton With R. reniformis

isolates of the six locally existing root-
knot nematode species tested. Studies
are underway to determine the parasitic
capabilities of the other genera of nema-
todes found associated with cotton roots.

The present investigation was de-
signed to confirm the susceptibility of
cotton to the reniform nematode, Roty-
lenchulus reniformis, and to determine the
feasibility of using nematode-infested soil
as inoculum in determining the relative
pathologic reactions of the crop to the


Reactions of cotton grown in potted
soil infested with varying numbers of
R. reniformis

Field population of R. reniformis
collected from, soils around mung 'bean
roots at the IRRI Experiment Station,
College, Laguna was hand picked and
increased on mung bean for several
generations in pots. Nematode-infested
soil containing pieces of roots from this
nematode culture was thoroughly mixed
in a cement microplot. Twenty 300-cc
samples were randomly obtained from
the mixture and separately processed
for nematodes using the sieving-Baer-
mann funnel technique. To account for
the egg population, bioassay of each
sample was also made following the
procedure reported earlier. 'Castillo, et al., i
1977). The bioassay consisted of collect-
ing the sieved soil and root fragments
caught in the coarse sieve in a 5-cm diam
clay pot and growing three mung bean
seedlings in it for 14 days. The soil and
whole root systems in each pot were then
re-processed for nematodes using the
sieving-Baermann funnel and root staining
techniques. During the bioassay period,
the nematode-infested soil was placed in

36-liter capacity plastic containers and
kept moist at all times inside a green-

The average number of R. reniformis
recovered per 300-cc sample was 34,
40% of which was recovered through
bioassay. Based on this, five different
ratios of nematode-infested and baked
soil mixture, namely 0:1, 1:4, 1:1.5, 1.5:
1, and 1:0, were placed in 23-cm diam
clay pots (4.4-liter capacity) to obtain
the following nematode population den-
sities per pot: (0 check), 100, 200, 300,
and 500. Each population density was
replicated 10 times.

Three cotton (Deltapine 16) seeds
were planted per pot. As soon as one
seedling emerged, the other two seeds
in the pot were immediately dug out.
The pots were then randomly arranged
on a cement floor outside the green-
house. Fertilization with ammonium
sulfate was done by side dressing at one
and three months after planting. The
plants were allowed to grow for five
months, after which data on root
weight, top weight and number of bolls
produced were collected. Number of
bolls was considered instead of weight
because some bolls were still green at the
termination of the experiment.

Reactions of cotton initially grown in
small plastic cups containing nematode-
free soil and soil infested with R. reni-

The need for an inoculation tech-
nique for mass screening of cotton for
resistance to R. reniformis necessitated
the investigation on the use of small
amounts of nematode-infested soil as
source of nematode inoculum in de-
termining the relative reactions of cotton

Jan. & June 1977

Philippine Phytopathology

to infections by the nematode.
The same R. reniformis population
used in the preceding experiment was
used in this study. However, the nema-
todes were of a different generation and
was increased on mung bean and pigeon
pea in cement microplots. Sixty 250 cc
soil samples containing pieces of roots
were collected from the mixture of
nematode-infested soil. Of these samples,
20 were randomly obtained and processed
for nematodes, using the sieving-Baermann
funnel and bioassay techniques earlier
described to determine the average
number of nematodes present in each
sample. The bioassay period was 21
The remaining 40 samples were
each placed in plastic cups (250-cc
capacity) and each cup planted to one
cotton (Deltapine 16) seed. To serve
as checks, 40 cups were filled with baked
soil and also planted to cotton. The cups
were arranged into two groups on a
cement floor outside the greenhouse.
Each group consisted of 20 cups with
cotton seeds planted in baked soil and
20 cups with cotton seeds planted in
nematode-infested soil.

Two months after planting, data on
root weight, top weight and plant height
were collected from the plants in one
group of cups. The soil and whole root
system in each cup were processed for
nematodes using the sieving-Baermann
funnel and staining techniques and the
nematodes present quantified.

The plants in the remaining group of
cups were transplanted singly, with the
soil intact, in 13-cm diam clay pots to
which baked soil was added. Fertilization
with ammonium sulfate was done by side
dressing after transplanting and at one
month thereafter. At about one month
after transplanting, however, severe leaf

infestation by insects (scale insects,
thrips and mealy bugs), aggravated by
Ascochyta blight, set in despite foliar
spraying with recommended pesticides.
This resulted in poor plant growth. Boll
production was delayed and so the crop-
ping period was extended to six months.
Data on root weight, plant height, number
of bolls produced, and nematode recover-
ies from soil and roots in each pot were
then collected. No data on top weight
were gathered because plants were alrea-
dy at the stage of senescense and most
leaves had fallen to the ground at the
end of the sixth month. As in the pre-
ceding experiment, number of bolls
was considered instead of weight because
some bolls were still green at the termina-
tion of the experiment.


Reactions of cotton grown in potted
soil infested with varying numbers of
R. reniformis

No apparent difference in root nec-
rosis between cotton plants grown in
nematode-infested soil were observed.
Compared to the check, significant
reductions in root weight of 30.8% 35.1%
and 48.0% resulted in plants grown in
soil infested with approximately 200, 300
and 500 R. reniformis, respectively
(Table 1). Top weight of plants did not
significantly vary among plants grown in
soils with different infestation levels. At
both 300 and 500 infestation levels,
significant reductions of 39.3% in the
number of bolls produced were ob-

The result of this experiment indi-
cates the susceptibility of cotton to
R. reniformis. It appears that a density
of at least 300 active and egg stages of the
nematode per 4.4 liters of soil (equivalent

Vol. 13

Table 1. Reactions of cotton grown in
numbers of Rotylenchulus ren

Approximate number
of nematodes present Root weig
in the soil (g)

0 (check) 10.3b
100 8.7b
200 7.la
300 6.6a
500 5.3a

aData are means of 10 replicates; di
5% level with DMRT; numbers in parent.

to 27.3 per 400 cc soil) was the critical
level of the nematode in pots and lower
densities were still within the tolerance
limit. The possible influence on nema-
tode density of the 14-day storage of
the nematode-infested soil in plastic con-
tainers, while representative samples were
being bioassayed, cannot be ruled out.
This influence, however, is believed to be
insignificant since the soil was kept
moist at all times.
The present finding also suggests the
feasibility of using nematode infested
soil in determining the relative nematode
density-yield loss relationship on cotton.
Reactions of cotton initially grown in
small plastic cups containing nematode-
free soil and soil infested with R. reni-
The average initial nematode density
in 250 cc nematode-infested soil contained
in small plastic cups was 76. Of this num-
ber, 50 were recovered through the sieving-
Rnarmnnn finnAl tPrhninmP nnrl *?

9tted 4.4 liters of soil infested with varyir
)rmis for five months

Top weight Number c
(g) bolls

70.4a 14.5b
75.3a 15.9b
75.0a 15.1b

74.0a 8.8a
61.9a 8.8a

erent letters indicate significant difference :
es are percentage reductions compared to tf

through bioassay. This nematode densit)
increased by 1.8 times (134) after two
months and by 21.8 times (1,654) aftei
the plants were transplanted in 13-crr
diam. clay pots and allowed to grow foi
another four months. The poor plan
growth towards the end of the cropping
period is believed to have adversely in
fluenced the nematode density at the
termination of the experiment.
Compared to cotton plants grown ir
nematode-free soil, pathologic reaction!
were exhibited by those grown in nema
tode-infested soil (Table 2). Reduction!
in root weight, top weight and plan
height of 47.6% 32.3% and 29.7%
respectively, were noted two month!
after planting. A more important obser
ovation was that all plants grown in nema
tode-infested soil showed root necrosis
their leaves distinctly chlorotic and the
reduction in plant height was very appa
rent. Thus, the usefulness of the inocula
tion technique employed in this experi
- I- 4r__ :._ 4,'

T a T 4

D AAj-lmn iP r .0 4-. 17:4.u D -,r:*_

dle 2. Recoveries of Rotylenchulus reniforr
plant reactions at two different period

Number of nematodes
recovered 2 and 6
months afer

-owing medium 2 months 6 month;

soil (check) 0 0
1 infested
w/ about 76
R. reniformis 134 1,654

aData are means of 20 replicates; initii
lerimental units.
bCounts from both soil and whole roc

CNumbers in parenthesis are percentage
*and **, significantly different from t

s from cotton initially grown in 250 cc nm


2 months after planting

Root weight Top weight Height
(g) (g) (cm)

2.1 41.8 20.2

1.1** 28.3** 14.2**
(47.6) (32.3) (29.7)

nematode counts and data collected from tl

system in each cup (after 2 months) and in i

reductions compared to the check.
e check at 5% and 1% levels, respectively.

Reactions of Cotton With R reniformis

R. reniformis in cotton was indicated.
Preliminary findings from a cooperative
screening for multiple adversity resistance
in segregating populations of cotton being


BEGONIA, D.T. and R.G. DAVIDE. 1968.
to three isolates of Meloidogyne incog,
3IRCHFIELD, W. 1962. Host-parasite relat
ium hirsutum. Phytopathology 52:86
3ASTILLO, M.B. 1976. Relative suscepti
crops to inoculation with Meloidogyne
of determining populations of Rotyl
through bioassay. Philipp. Agr. 61:64-(
nematode in cotton roots. Phytopatho
'RESLEY, J. T. 1954. Cotton diseases a
Bull. No. 1745. 21p.
tOLEDO, R. and R. G. DAVIDE. 1969.
crops to infection by Meloidogyne jav
TALDEZ, R.B. 1968. Survey, identification
nematodes occurring in some parts of t

conducted at the Institute of Plant Breed-
ing (IPB) concurred with this observa-


ithologic reactions of field and fiber crops
a. Philipp. Phytopathol. 4:52-73.
is of Rotylenchulus reniformis on Gossyp-
ity, resistance and tolerance of selected-
)p. in pots. Philipp. Phytopathol. 12:17-23.
LITSINGER. 1977. Increased efficiency
chulus reniformis and Meloidogyne acrita

. Host-parasite relationships of the lance
gy 46:505-510.
methods of control. U.S.D.A. Farmer's

actionss of different vegetable and field
ca. Philipp. Phytopathol. 5:45-51.
nd host-parasite relationships of root-knot
SPhilippines. Philipp. Agr. 51:802-824.

Jan. & June 1977



B. K. LIM an

Graduate student and Associate Pro
logy, College of Agriculture, U.P at Los BR


Five kinds of Meloidogyne inc
mately 1000 eggs and/or larvae, wer
(Phaseolus vulgaris L. ) plants grown I
Plants inoculated with newly-1
Inoculations with isolated egg mas:
and isolated eggs resulted in moder
tode-infested soil exhibited very sE
nematode-infested soil caused sign:
(55.2% ), tops (43.9%) and seed yi<
experiment (55 days after inoculatic
resulted in plants inoculated with r
in plants inoculated with isolated e
pieces and isolated eggs almost dout
hv K 1 timrn in nlants arown in nema



ssor, respectively, Department of Plant Path
as College, Laguna.


nita inocula, each containing approxi-
;ested for their effects on Baguio bean
gly in 18-cm diam clay pots.
ched larvae had the least root galling.
, egg masses attached to root pieces
Stalling, while plants grown in nema-
re galling. Among the inocula, only
,ant reductions in weights of roots
s (52.5% ). At the termination of the
i, no increase in number of nematodes
vly-hatched larvae, while the numbers
masses, egg masses attached to root
d. The number of nematodes increased
de-infested soil.

tions involving root-knot nematodes, In I.Uo Yf autI were satisfactory mocula
various techniques of infecting plant and superior to larvae, fragmented egg
roots have been used. Sasser (1951) masses or eggs collected by blender and
used small gelatin capsules, each contain- centrifugation techniques.
ing a single egg mass and little moist sterile
soil. Godfrey (1931), Dropkin (1957), The present study was designed to
Bird (1959) and Dropkin and Boone compare the effects of M incognita egg
(1966) introduced larval suspensions onto masses, isolated eggs, larvae, and nema-
the root tips. Barrons (1938) and Bailey tode-infested soil on Baguio bean, a
(1941) grew their test plants in a mixture nematode-susceptible crop. The result of
of sterile soil and cut pieces of roots this investigation will reveal which of
containing nematode galls. Isolated egg these inocula is best to use in studies on
masses or egg masses still attached to root the relative effects of different root-knot
pieces and thoroughly mixed with baked nematode densities on susceptible crops.
soil also served as inocula in many inocu-
lation experiments. Hussey and Barker
(1973) compared five different methods MATERIALS AND METHODS
of preparing and introducing inocula of
Meloidogyne spp. and found that eggs Source of nematode inocula and test plant

n two ted of collecting the sieved !
b) egg root fragments that collected in
heated mesh screen in 5-cm diam clay
n the which a small amount of baked
I eggs added. One mung bean (MG-5C
Itrices was then planted in each pol
nutes, weeks after planting, the soil in i
repto- was again processed for nematc
nd (e) the whole root system stained 1
mine the nrrinc P nf nAomna+tn

the 60-
pots to

Jan. & June 1977

m. incognita Inocula on Baguio Bean

in, Phaseo-

S -6 v -r1-ur L-., vai. -Ullt; llCUt ,
as test plant. Seeds were germin
seedbox containing baked soil.

Preparation of nematode ino
inoculation procedure

-- I

i test

d in tl

was fill

adv were the fi

mLIL JLJ- 11
bioassay coi

e introduced
Baguio bean
/ in 18-cm
ked soil. The

sses, ai

tct of

total ot approximately ,UZY eggs were
ta o a t eg todes were retained, the rest were dis
present in the three egg masses introduced.
In the case of egg and larvae inocula, sus-
pensions containing approximately 1,000 Each nematode inoculum was intro

ippme rnytopatnoiogy

greenhouse with a temperature range of
26 to 35C. They were protected from
foliar pests by spraying at 10-day inter-
vals with recommended pesticides.

Collection of data
Fifty-five days after inoculation, data
on root galling and weights of roots,
tops and dry seed yields were collected.
Root galling was rated on a 1 to 5
severity scale. The nematode population
densities in soil and roots of each pot
were also determined following extrac-
tion through the sieving-Baermann funnel
and root staining techniques and bio-

Table 1. Reactions of potted Baguio bean I
approximately 1,000 Meloidogyne

Inoculum ratingY Root

Check (Nema-

assay procedure discussed earlier.


Reactions of Baguio bean plants to
inoculation with approximately 1,000
M. incognita varied with the kind of
inoculum used (Table 1). Plants inocu-
lated with newly-hatched larvae had the
lowest root gall rating. Inoculation with
isolated egg masses, egg masses attached
to root pieces and isolated eggs resulted
in moderate galling, while plants grown in
nematode-infested soil exhibited very
severe galling. Among the nematode
inocula, only the nematode infested soil

plants to different inocula, each containing
Sincognita, and nematode recoveryx

Plant weights (g) Number of
times the
Seed nematodes
Top yield increased

Isolated egg
masses 3.0b 21.1b 58.5b 14.5b 1.8b
Egg masses
attached to
root pieces 3.0b 20.6b 57.9b 14.6b 1.8b
Isolated eggs 3.2b 20.0b 59.2b 14.8b 1.9b
larvae 1.7a 23.2b 62.1b 15.2b 1.0a
soil 5.0c 10.3a 34.6a 7.5a 5.1c
(55.2) (43.9) (52.5)

XData are means of five replications; different letters indicate significant
differences at 5% with DMRT; numbers enclosed in parentheses are percentage reduc-
tions compared to the check.

YBased on relative percentages of galled portions of root systems determined as
follows: 1, 0% (none); 2, 1-25%(light); 3, 26-50%(moderate); 4, 51-75% (severe); and
5, over 75% (very severe).


dan. & June 19TI M. incognita Inocula on Baguio Bean 41

caused significant reductions in weights than those grown in nematode-infested
of roots (55.2%), tops (43.9%) and seed soil, where the nematodes were in the
yields (52.5%) of inoculated plants. Based relatively undisturbed state. While it was
on nematode recovery 55 days after true that plants in nematode-infested
inoculation or at the termination of the soil were attacked immediately after
experiment, no increase in number of transplanting, in contrast with the
nematodes was noted in plants inoculated others which were given four days to get
with newly-hatched larvae, while the established before the nematodes were
numbers in plants inoculated with isolated introduced close to their roots, the fact
egg masses, egg masses attached to root that the nematodes in nematode-infested
pieces and isolated eggs almost doubled. soil still had to find the roots, perhaps
The highest increase in number of nema- more or less evened up the situation.
todes (5.1x) was observed in plants grown
in nematode-infested soil. Under the conditions of the experi-
ment, it appears that among those tested,
Undoubtedly, the differences in plant nematode-infested soil is the best inocu-
reactions to the different inocula and in lum to use in relating density of M.
nematode recoveries were partly related incognita and perhaps of other plant
-..;t namtnd-. -llnznAr|l oftar ;nrilnn ..,...

42 Philippine Phytopathology Vol. 13

GODFREY, G.H. 1931. Some techniques used in the study of the root-knot nematode,
Heterodera radicicola. Phytopathology 21:323-329.
HUSSEY, R.S. and K.R. BARKER. 1973. A comparison of methods of collecting inocula
of Meloidogyne spp., including a new technique. Plant Dis. Reptr. 57:1025-1028.
SASSER, J.N. 1951. The use of gelatine capsules for making single egg mass inoculations
with the root-knot nematode (Meloidogyne spp.) Phytopathology 41: 564 (Abstr.).

Philion. Phvtonath


L.T. T

Research Virologist, Institute of Plan
Los Banos, College, Laguna, Philippines.
Grateful appreciation is expressed to 1
This investigation was supported by
Government to the Institute of Plant Bre


Based on double immunodiffus
extracts of cowpea showing little leaf
specific antigens which reacted strong]
gose mosaic virus (BRMV), bean pod r
virus (BGMV), broad bean wilt virus-
southern bean mosaic virus (SBMV-cs
precipitin lines which fused together st
particles measuring 25 and 30 nm in
microscope. The presence of viral anti
possibility that at least two spherical-s
in cowpea.

The first report of a disturbance in
cowpea, Vigna unguiculata (L.) Walp.,
called "little leaf' disease was made by
Benigno and Paje (1975).The incidence
of the disease was observed in experi-
mental plots of the University of the
Philippines at Los Banos. Later, the
disease was found to be widespread
wherever cowpea was being cultivated in
the country.
Several viruses have been isolated
from cowpea, namely: cowpea mosaic
virus, CPMV (Bock, 1973; Lovisolo
and Conti, 1966), cowpea chlorotic
mottle, CCMV (Kuhn, 1964), cowpea
mild mottle, CMMV (Brunt and Kenten,
1973), cowpea strain of southern bean
mosaic virus, SBMV-cs (Fulton and
Scott, 1977; Shepherd and Fulton,
1962), cowpea strain of bean yellow



Breeding, University of the Philippines al

Is. Adelaida Lacuata for technical assistance
. research grant provided by the Philippine
eding, University of the Philippines at Lo,


on test in agar plates, clarified leaf
syndrome was found to contain virus-
y with antisera made against bean ru-
ottle virus (BPMV), blackgram mottle
'ype I (BBWV), and cowpea strain of
. BRMV, BPMV, and BGMV showed
ggesting serological identity. Isometric
diameters were noted in the electron
lens and virus-like particles raised the
iaped viruses caused little leaf disease

mosaic virus, BYMV-cs (Anderson,
1959), cowpea strain of cucumber
mosaic virus, CMV-cs (Anderson, 1955),
a virus isolated from Arkansas (She-
pherd, 1963; Shepherd, 1964) and
cowpea yellow mosaic virus isolated in
Nigeria (Wells and Deba, 1961) which
appeared to be serologically related to
bean pod mottle virus (BPMV), and
cowpea strain of tobacco mosaic virus,
TMV-cs (Lister and Tresh, 1955).
These viruses caused different types of
mosaic and mottle disease. However,
none of these viruses has been described
to cause little leaf disease in cowpea.
With limited information of the viral
etiology of the little leaf disease, the
present investigation was undertaken to
determine by serodiagnosis, the virus
(es) which are present in detectable

9 (1977)

Philippine Phytopathology

amounts in leaf extracts of infected
cowpea plants. Hopefully, this would
yield clues that can be used in isolation
strategies, and in attempting to re-
produce the little leaf disease under
greenhouse condition.

Viruses and antisera
The source of virus antigens came
from field-grown cowpea plants infec-
ted with the little leaf disease. Infected
leaf tissues were triturated in a heat-
sterilized mortar and pestle. The homo-
genate was squeezed through cheesecloth
and clarified by centrifugation at
10,000 g for 10 minutes. The clear,
brown supernatant fluid served as
antigen source.
The reference antisera were kindly
supplied by Dr. J. P. Fulton (BRMV,
cs, CMV-cs), Dr. J. K.I Uyemoto (BBWV-
Type I) and Dr. R. J. Shepherd (SBMV-
cs). Except for the latter antiserum
against SBMV-cs, all arrived in excellent
condition. Anti-SBMV-cs contained
granular precipitate and was cloudy.
Serological tests were conducted in
1.0% Ionagar (Oxoid No. 2) gel plates
using an eight-member, 6-cm antiserum
well pattern surrounding a central 8-cm
antigen depot. To test two different
batches of antigen preparations and to
save valuable antisera, a second set of
eight-members 8-cm antigen wells were
made surrounding the antiserum wells.
The distance between the antigens and
antisera was 3 mm.
The ionagar was dissolved in 0.01M
tris-HC1, pH 7.2 containing 0.85% NaC1,
and 0.2% sodium azide.
The immunodiffusion plates were
incubated at 30 C for 24-48 hr. For
longer period of time, they were kept
at 4C.

Electron Microscopy
A drop of the brown virus solution
was placed on a carbon-reinforced,
collodion-coated copper grid. After
about two minutes the excess fluid was
removed by touching the grid-edge with
a piece of filter paper. The specimen
was stained with neutral 2% phospho-
tungstate and viewed in a JEOL electron
microscope at a magnification of about


In the experimental plots of the
University of the Philippines at Los
Banos, virus-diseased cowpea plants
exhibiting various types and combina-
tions of symptoms such as green mosaic,
yellow mosaic, rugose mosaic, vein-
banding, puckering of foliage, necrosis,
and stunted plant growth were often-
times visualized. The little leaf disease
(Fig. 1) seemed peculiar in that affected
plants can be spotted with ease parti-
cularly if they became infected with
the viruses shortly after emergence from
the soil.

Afflicted plants showed severe stun-
ted growth, deformed, wrinkled and
puckered leaves, and excessively-reduced
size of the foliage which tended to roll.
The shortened intemodes and profused
axillary growth gave rise to the bunchy
appearance at the growing points of the
plant. Severely-infected plants did not
produce pods.

Like Benigno and Paje (1975),
efforts to transmit the viruses by me-
chanical inoculation techniques failed.
Insect transmission using Aphis cracci-
vora Koch. proved ineffective. Growing-
on tests using virus indicator plants have
not been useful in yielding much-
needed information about the virus

Vol. 1;



I:~~:rr ~I)lrrf~nnflrnlr\rt~r

7 A 3

Fig. 2. Agar gel diffusion pattern of clarified sap of cowpea

n. & June 1977 Cowpea Leaf Disease 47

le 1. Reaction of several reference antisera against leaf extracts of cowpea infected
with little leaf disease

Antisera Antigen source

4V-Fulton +++ +++
MV-Fulton +++ +++
MV-Fulton +++ +++
NV-Type I-Uyemoto +++ +++
dV-cs-Fulton + ++
dV-Arkansas-Fulton -

+Relative strength of precipitation reaction
-No visible precipitation reaction

al-shaped virus-like particles in
ected with little leaf leaf disease.
tively stained with 2% neutral

particle sizes which measured 25

F ig. 3. Electron micrograph of sphere
clarified sap of cowpea plant ih
Sample preparations were net
Note the presence of twc
nm (a) and 30 nm (b).

linnine Phvtonatholouv

syndrome. The fusion of the precipitii
lines and absence of spur formation witl
BPMV, BRMV, and BGMV appear t(
indicate antigenic similarity. This observa
tion is in disagreement with that o
Fulton and Scott (1977) on the serolo
gical unrelatedness of BGMV with othe
members of the beetle-transmitted viru
(Fulton and Scott, 1977; Pathak, 1974)
Further reciprocal serological tests ar
needed to determine if the BGMV detec
ted in cowpea little leaf disease possess
serological properties similar to member
of the comovirus group.

The detection of BBWV-Type
antigen is somewhat surprising because th
virus has not been described to attack]
cowpea under field and greenhouse cor
Electron microscopic examinations o
clarified extracts provide visual evidence
that virus-like, isometric particles which]
measured 25 and 30 nm in diameter wer
present. Rigid rod-shaped particles similar
to those reported previously by Benign
and Paje (1975) were not observed. Suc]
discrepancy is not clear. In this study, th
virus preparations used for the immune
diffusion and electron microscopi
studies were the same. No further treal
ment of the antigen source other than th
brief clarification step by low spee


ANDERSON, C. W. 1955. Vigna and Crc
on a strain of cucumber mosaic
Rptr. 39:346-348.
ANDERSON, C.W. 1959. Vigna and Croi
mission tests with aphids and beetle
BENIGNO, D.A. and L.L. PAJE. 1975. 1
cowpea. Agr. at Los Banos 14:3.
BOCK, K. R. 1973. East African strain i
Biol. 74:75-83.
BRUNT, A. A. and R. H. KENTEN. 1i
virus infecting cowpea in Ghana. An

centrifugation was performed.
The reason for the failure to transm
the little leaf disease could be attribute
to the inability of the viruses in the inm
culum obtained from little leaf-infecte
cowpea plants to initiate infection whe
simultaneously introduced into an unil
fected and susceptible host plant. Abortii
infection might be assumed to resu
from competition for virus attachmer
and penetration sites, inadequate vin
inoculum, virus inactivation, or specif
vector inolvment.
Efforts are currently being undertake
to isolate the viruses as single entities
Preliminary results indicate that BgMV
indeed present in the little leaf extrac
Attempts will be undertaken to rein,
culate the different virus isolates i
various combinations and inoculatic
strategies in order to reproduce the litt
leaf disease in cowpea.
The little leaf disease of cowpea is <
great economic importance. Yield lo
could be as high as 100% if cultivate
plants succumb to these viruses in the
seedling stage. Furthermore, the litt
leaf disease has been observed to I
present in mungbean and beans. Ti
identification of the viruses in cowpi
little leaf disease is a step forward
successful methods of control.


talaria viruses in Florida. I. Preliminary repi
irus obtained from- cowpea plants. Plant E

ilaria viruses in Florida. V. Comparative tra
Phytopathology 49: 117-118.
little leaf: a new and destructive virus disease

f cowpea aphidborne mosaic virus. Ann Ap

73. Cowpea mild mottle: a newly recognize
k. Appl. Biol. 74:67-74.


Cowpea Leaf

JALE, W. T. 1949. Observations on a vir
Biol. 36:327-333.
FULTON, J.P. and H.A. SCOTT. 1977. Be
transmission by beetles. Fitopatologia
CUHN, C. W. 1963. Field occurrence and
bean mosaic virus. Phytopathology 53
(UHN, C.W. 1964. Purification, serology,
pathology 54:853-857.
LISTER, R. M. and J.M. THRESH. 1955..
by a strain of tobacco mosaic virus. N;
LOVISOLO, 0. and M. CONTI. 1966. Id
mosaic virus. Neth. J. Plant Path. 72:1
)UCHTERLONY, 0. 1962. Diffusion-in-
Progr. Allergy 6:30-154.
'ATHAK, H.C. 1974. Seed-borne plant vin
testing. Copenhagen. 155 pp.

disease of cowpea in Trinidad. Ann. Appl.

rugose mosaic and related viruses and their
rasileira 2:9-16.
propertiess of the cowpea strain of southern
d properties of a new cowpea virus. Phyto-

mosaic disease of leguminous plants caused
ire 175:1047-1048.
itification of an aphid-transmitted cowpea
methods for immunological analysis. I.

s-identification and diagnosis in seed health

SHEPHERD, R. J. 1964. Properties of a mosaic virus of cowpea and its relationship to
the bean pod mottle virus. Phytopathology 64:466-473.
SHEPHERD, R.J. and R. W. FULTON. 1962. Identity of a seed-borne virus of cowpea.
Phytopathology 52:489-493.
WELLS, D. G. and R. DEBA, 1961. Sources of resistance to the cowpea yellow mosaic
virus. Plant Dis. Reptr. 45:878 831.


RAPAiv*rd fnr nub



Product Development Specialist,
Products Division.
Paper presented to the Philippine Ph
of the Pest Control Council of the Philif
International Convention Center, Metro M


Fungicide application one we
days before harvest gave good pre-
trichum gloeosporioides) in mango
developed per inflorescence with 1
2.64 g/liter of Daconil 75WP, Dith.
harvest gave good anthracnose co

With the opening of the export market
for mango, its growing and production
has been given support by the goverr
ment. New plantings on a plantatio
type scale have been going on in differed
parts of the country as an anticipation
of growing export demand. With thi
move from both public and private
sector, an efficient and cost-effectiv



nion Carbide Philippines Inc., Agricultui

topathological Society 9th Annual Conferen
ines held on May 3-6, 1978 at the Philippil


after bud break at fruit setting and 10
irvest control of anthracnose (Colleto-
During the rainy months, more fruits
ngicide treatment. Application rate at
*e M45 and Manzate 80, 10 days before
rol, eliminating laborious post-harvest

and Smoot and Segall (1963) reported
good anthracnose control with hot wate
bath treatment at temperature of 13C
1320F (54.5-560C) for 5 minutes. Prc
longed exposure at this temperature
range for 10 minutes shows moderate
scald-like heat treatment injury as re
ported by Spalding and Reeder (1972)
Good anthracnose control with Tri

rroolem or mango growers is low yiela g/li. was also effective. These findings
of marketable fruits due to minimal fruit were also reported by Bondad (1974).
setting and fruit development, as a con-
sequence of fungal attack. This problem Pordesimo et a1.(1972) and Pordesimo
becomes more pronounced during wet and Barredo (1976) evaluated fungicides
becomes more pronounced during wet
months which highly favor anthracnose applied as pre-harvest treatment against
infection on leaves, flowers, newly formed postharvest disease on mango. Their
and mature fruits. Heavy infection results suggested protecting fruits starting
eventually lead to excessive fruit drop- from the field and immediately after
ping, thus, reducing the number of harvest for exportable quality mango.
harvestable fruits. Despite these studies, however, it is a
rarity for most contractors and growers
Successful postharvest anthracnose to use fungicides because emphasis has
control using chemicals and hot water always been on insect control. Likewise,
treatment has been reported by several postharvest treatment is laborious and
workers. Pennock and Maldonado (1962), limited to special post-treatment rooms

Lpril, 1978

Jan. & June 1977 Pre-harvest Anthracnose Control 51

and laboratories. Therefore, this study Anthracnose ratings were taken a week
conducted in Davao, Laguna and Nueva after storage using the following index:
Ecija aimed to generate use pattern tech-
nology for pre-harvest anthracnose con- 1 = 0-5 spots on the fruit surface
trol in mango under field conditions. 2 = 6-10 spots on the fruit surface
3 = 11-15 nnots on the fruit surface

52 Philippine Phytopathology Vol. 13

Table 1. Anthracnose rating on mango fruits treated with Daconil 75 WP and Cupravit,
(Davao City, Menzi Farms) (1976)

Disease Ratinga
Treatment Rate Mean % Control
(g/li) I III

Daconil 75WP 2.64 1.4 1.6 1.0 1.33a 65.00
Cupravit 5.28 2.6 2.8 2.2 2.53b 33.42
Sevin 85S 5.28 4.0 3.5 3.9 3.8bc -

aFigure entry average of 10 fruits/replicate.

Table 2. Anthracnose ratings and average number of fruits that developed inflorescence
on various fungicide/insecticide treatment. (Sta. Rosa, Laguna) (1976)

Treatment Rate Disaease Fruit/ % Control Yieldb
(g/li) Rating Panicle (Basket)

Daconil 75WP 2.64 1.05 3.6a 67.0 19.0a
Dithane M45 2.64 1.05 3.2a 67.0 18.5a
Manzate 80 2.64 1.2 3.4a 62.0 18.5a

aFigure entry mean of 4 replication.
bBasket containing 90 fruits on the averaE

Table 3. Anthracnose rating and fruit deve
cide-insecticide combination. (M,

Treatments Rate Spray
(g/li.) Interval

Daconil 75WP 2.64 weekly
Daconil 75WP 2.64 bi-weekly
Manzate 80 2.64 weekly
Manzate 80 2.64 bi-weekly
Sevin 85S 5.28 weekly

aSevin 85S tank mix as standard insecticic
bFigure entry mean of 4 replication.

'ment of mango treated with various fungi-
TC, Munoz, Nueva Ecija) (1976)

sease Fruit/ % Control Yield
citing Panicles (Basket

1.0 3.9 51.21 67a
..05 3.4 48.28 66a
1.2 3.2 41.46 62al
..6 2.8 21.00 59b
1.05 1.4 33c


P.-.ha..c..t A nthran

gave significantly low incidence of an-
thracnose compared to Sevin 85S alone.
This result agrees with that of Pordesimo
and Barredo (1976) and Pordesimo, et al.
(1972), that postharvest diseases can be
minimized with proper pre-harvest fun-
gicide application. It is also evident that
flowers left unprotected by fungicide,


IONDAD, N. D. 1974. A note on the coi
myl and thiabendazole. Philipp. J. Sc
4cMILLIAN, R. T., Jr. 1973a. Enhancem
bining copper with NU Film. Proc. F
IcMILLIAN, R. T., Jr. 1973b. Control c
with systemic and non-systemic fung
reduce anthracnose decay. J. Agr. Ur
harvest dip of mango fruits against
Pest Control Council Conf., 16-18
8 (Abstr.)
sprays on mango fruit setting and poi
MOOT, J. J. and R. H. SEGALL. 1963.
anthracnose. Plant Dis. Reptr. 47: 73
fected by f&fngicides and heat treatmi

especially during rainy months, set few
fruits, illustrating the beneficial effect
)f additional input on fungicide. Like-
vise, the carry-over effect of fungicide
applicationn 7-10 days before harvest on
*ostharvest diseases showed that labo-
ious postharvest treatment can be


I of postharvest disease of fruits with ben(
of anthracnose control on mango by con
Itate Hort. Soc. 85:268-270.
ithracnose and powdery mildew of mang
as. Trop. Agric. 50: 245-248.
i. Hot-water treatment of mango fruits I
Puerto Rico 46; 272-283.
. M. HALOS. 1972. Pre-harvest and pos
Ithracnose and fruit rots. Proc. 34d Nat
il 1972. Teachers' Camp, Baguio City. ]

1976. Effect of fungicide and insecticid
rvest rots. Philipp. Phytopathol. 12:51-59,
it water as a postharvest control of mang
'2. Postharvest disorders of mangoes as a
Plant Dis. Reptr. 56: 751-753.

e" o

Received for public

problems. parasitism in. higher plants, mainly,
ies of Pythium were originally of and stems. These cause tissue rot
,_ .. . damnino-nff. Frz77i (195Q6 wnrl

,auc ium UL UlaV;ac i l1iiisjy tiaue, ul~s
identification became important 1
pathologists. Sideris (1932) published e:
tensively his taxonomic studies of P.
thium spp. isolated in Hawaii. His pr
posed classification system generally w;
not accepted by other mycologists b
cause his species separation was vagi
and indefinite, and later workers four
that he had described the same speci
under different names.

Middleton (1943) later compiled i
extensive monograph of the genus P
thium, with complete mycological trea
ment, host records and illustrations. H
key to the species reflects the importai

is well-illustrated and in general follov
Middleton's concept of the genus. Hendr
and Campbell (1970) actually examine
10,000 isolates of Pythium from 2,1C
soil samples from various parts of tl
U.S. and concluded that certain speci,
form species complex with character
distinct only at the. extremes but which
merge at a medium point.

While keys to the species of Pythiu
are not lacking, it is of extreme impc
tance that we have also a data on tl
tropical species of the organism as the
occur in the Philippines. With the dev
lopment of specialized technique f

rytniums rrom rnmm

igation was prompted by sonme other watermolds lik
study of the genus Pythium Dictyuchus, etc. In some cases
nes. The start, therefore, of Pythium was isolated without t
project supported by the "contaminants". With experi
arch Council of the Philip. can determine by the naked
us to survey, name, and colony is pure Pythium: most
Terent species of Pythium Pythium colonies appear m
ippine soils. This informa- slimy than other watermolds. N
I to contribute toward a examination, however, were al'
anrlino af thp enpeioc and ted to exactly differentiate th

lant pathogen. This report gives the
description or morphology of the isolated
ythium species and presents a simple
ey for ease of their identification.


collection of Soil Samples and Isolation
A collection of soil samples from some
provinces in the Philippines was under-
aken in 1976-77(Plate I).The soil samples

ately in running water to remove cling-
ig soil debris and transferred to fresh,
terile distilled water. Hyphal filaments
tere then cut with a sharp blade or scal-
el, blot on sterile tissue or filter paper
hen planted on plated water agar. Cases
f mixed cultures did not present much
problems because Pythium hyphae grew
iuch faster than the other watermolds. A
iece of the agar with the advancing
yphal tips was then cut and transferred
3 new agar plates for further examination
nd identification.


ine Phytopathology


a i.
(.- O"
3 ~ \

ft~ t


de a refrigerator if possible until portion of the fungal growth, F
dy for identification to species. To a dish of sterile water, and addi
other preserve and minimize drying up, halved sorghum seeds.
-ilized mineral oil was used and shown Identification to Species
be generally successful in preservation
cultures. The method consisted of The presence of sexual as



rymniums rrorm rnui

Auiuu. iss ,aoo Vl Ul flit-us.y nLwUrY Ve
n inducing sexual reproduction, cultures
vere grown in V-8, commeal and oatmeal


solution and Identification
A total of 500 soil samples were col-
ected at random at representative locali-
ies in the Philippines. Nineteen species
tere identified from a total of around
00 isolates. No attempt was made on the
uantitative distribution of these species
either was there an attempt to name
ew species. The works of Middleton
1943) and Waterhouse (1968) were
sed in the identification of the nine-
-en species. Described below are the
isolated species, all of which, except for
Sdebaryanum, are described for the first
me in the Philippines. A key to isolated
ecies of Pythium is presented after
iese descriptions. All the cultures are
eposited at the Fungal Culture Collection
FCUP-103) of the Dept. of Plant Patho-
>gy, UP at Los Bafios, Philippines.


Pythium acantophoron Sideris Myco-
aisa. 24-36 1932

Ivote: inls species is characterized by
he presence of irregular and dendroid
ateral hyphae and by the complete
absence of sporangia. It is reported only
rom Hawaii by Sideris (1932).

.. Pythium allantocladon Sideris, My-
cologia 24:27-28. 1932

Hyphae very fine, branched, 2.75-8.2
Im in diam, produce allantoid struc-
ures measuring 7-8 urn in diam which
occasionallyy produce sexual organs;
porangia spherical to subspherical, ter-
ninal and intercalary, irregular in size,
2-24 um diam, sometimes arising from
dlantoid hyphae; oogonia subspherical,
mooth and thick-walled, 14.8-20 um in
liam, sometimes in clusters; antheridia
nonoclinous and diclinous, antheridial
ell clavate, 1 per oogonium, often apan-
Irous; oospore spherical, aplerotic, single,
mooth and thin-walled, sometimes borne
t the tip of an allantoid body, 11.2-14
m in diam.
On soil, collected 1977, Benguet,
sabela, Laguna, Negros, Quezon, Tarlac,
nd Zamboanga.
Note: The chief distinguishing charac-
eristic of this species is the production
f allantoid structures which either
terminate vegetatively or occasionally
produce sexual organs.

1.6-4.8 um in diam, hyphal laterals pre- 1915.
sent, slightly dendroid, terminal and
intercalary; sporangia not observed; zoo- Hyphae branched, 2.3-7.5 um in diam;
spore not observed; oogonia spherical, sporangia inflated, filamentous, digitate
terminal and intercalary, measuring and lobulate element of varying length
30-46.7 um in diam, with conical pointed and diameter; zoospore reniform, la-
spines measuring 5.8-8.7 um in diam; terally biciliate, measuring 6.7-10.5 um in
antheridia typically monoclinous, rarely diam upon encystment; oogonia spherical,
liclinous, 1 per oogonium; oospore smooth and thick-walled, terminal and
splerotic, spherical, single,smooth-walled, intercalary, measuring 15.9-26.8 um in
measuring 2643.5 um in diam. diam; antheridia monoclinous, sometimes
On soil, collected 1977, Agusan. diclinous, intercalary and terminal, 1-2


58 Philippine Phytopathology Vol. 13

per oogonium, antheridial cell barrel or Note: The shape of the sporangium
dome-shaped, cylindrical or broadly cla- and the position of vesicle separate P.
vate; oospore aplerotic, single, smooth carolinianum from P. ultimum, the for-
and moderately thick-walled, measuring mer having spherical sporangia and stalked
15-17 um in diam. vesicle; the latter, ellipsoidal sporangia
and sessile vesicle.
On soil, collected 1977, Benguet,
Bohol, Cagayan, Camarines Norte, Ilocos 6. Pythium debaryanum Hesse, Inaug.
Note, Laguna, Leyte, Negros, Pangasinan, Dissert. Halle, 1874.
Rizal and Tarlac. Hyphae branched, septated, 2.10-4.3
P. aphanidermatum is one of the most ur in diam, irregular swollen interca-
common species of Pythium isolated lary bodies present; sporangia spherical
from Philippine soils, to irregular in shape, terminal and inter-
4. Pythm b i S, M calary 16.5-22 urn in diam; zoospores
4. Pythum butler Subramaniam, Mem.- reniform, laterally biciliate, measuring
p1919. 6.8-9.7 um upon encystment; oogonia
spherical, smooth, terminal and inter-
Hyphae fine, septated in old cultures, calary measuring 13.8-26 um in diam;
measuring 1.754.32 um in diam, irre- antheridia monoclinous and diclinous,
gular hyphal swellings present; sporangia 1-4 per oogonium, antheridial cell cla-
inflated, filamentous, terminal and inter- vate which adheres to the oogonium
calary; zoospores reniform, laterally bici- along their entire length and arising from
late, measuring 5.4-7.2 um in diam upon a long stalk from a neighboring branch;
encystment; oogonia spherical, inter- oospore spherical to subspherical, aple-
-nI- cmn-th anrl thin.wallpr mpn- -1-* --Al .: 4- -.n1rl.A

Jan. & June 1977 Pythiums From Philippine Soils 59

nous, usually single, rarely two per um in diam, hyphal swellings present;
oogonium; oospore aplerotic, smooth, sporangia spherical, subspherical, pyriform
contains several reserve globules, mea- to irregular in shape, papillary protru-
suring 9-17.8 um in diam. berances of variable sizes present; zoo-
On soil, collected 1977, Agusan, spores not observed; oogonia smooth,
Cagayan, Cebu, Ilocos Norte, Isabela, irregular in shape and size, hyphal swel-
Laguna. lings present at oogonial stalk, 15-18.5
um in diam; antheridia 1-2, antheridial
Note: P. deliense is closely allied to cell clavate, typically monoclinous or
P. a cell clavate, typically monoclinous or
P. aphamdermatum. Oogonia and
P. aphanideatum. ogonia and diclinous; oospore occasionally aplerotic,
oospores are very similar in size and shape sooh and tic alle, becoin irre
but P. deliense shows the characteristic smooth and thick-walled, becoming irre-
but P. deliense shows the characteristic gularly waed when mature, with one
Sf e t gularly warted when mature, with one
curving of the oogonia towards the
S large central globule, measuring 13-15.9
straight-stalked antheridium. Beside it is
um in diam.
also in this species that we encounter
clusters of oogonia. On soil, collected 1977, Camarines
8. Pythium inflatum Matthews, Studies Sur.
on the genus Pythium. 1931.
r -^ _.- nri ... ...- .. -r~ .. . 'r_-

rporangia composed of inflated fila-
nentous and dactyloid elements; zoo-
spores reniform, laterally biciliate, formed
n a vesicle, borne on an evacuation tube,
measuring 6.4-7.8 um in diam upon
:ncystment; oogonia spherical to sub-
spherical, smooth and thin-walled, inter-
:alary measuring 14.7-18 um in diam;
mntheridia usually diclinous, sometimes
nonoclinous, 2-4 per oogonium, antheri-
lial cells crook-necked; oospore plerotic,
single, 12.8-16 um in diam, smooth and
On soil, collected 1977, Negros.
Note: P. periilum possessing also
liclinous antheridia differs from P.
nflatum in having the antheridial cells
rising from a single antheridial branch.
Vhile in P. arrhenomanes the antheri-
lial branches are frequently divided and
he cells are many up to 25 per oogonium,
n P. inflatum, the antheridial branches
.re simple and the cells are 102 per oo-
1. Pythium irregulare Buisman, Med.
Phytopath. Lab. 11:1-51. 1927

U1 VVuguiu1 VA UIC IuIInlci vady 111 Wilupe.
Dogonial protruberances are present al-
though more or less very irregular. Some
reports on this species show definite
spines of various sizes and shapes on the
oogonial wall.

The sporangia vary much in shape
from spherical to subspherical, elliptical,
ovoid, pyriform or truncate.

O0. Pythium monospermum Pringsheim,
Jb. Wiss. Bot. 1:288-290, 298-299,

Hyphae branched, septated in old
:ultures, 1.76-5.7 um in diam, often with
numerous bud-like lateral outgrowths,
:atenulate hyphal swellings present; spo-
rangia filamentous, terminal and inter-
:alary, branched or unbranched, delimi-
:ed by a septum; zoospore reniform,
aterally biciliate, measuring 6.2-15 um
ipon encystment;oogonia spherical, inter-
:alary and terminal, smooth and thin-
valled, 14-23 um in diam; antheridia
nonoclinous and diclinous, 1-3 per oogo-
lium, antheridial cell club-shaped arising
rom lone ooeonial stalk: oosvore single.

liDDine PhvtoDathologv

walled 10.5-19 um in diam.
On soil, collected 1977, Albay, Ba-
tangas, Baguio City, Bohol, Ilocos Sur,
Laguna, Mandaue City and Pangasinan.
Note: Often mistaken for P. gracile,
P. monospermum differs in having ple-
rotic oospores and antheridia both mono-
clinous and diclinous. It differs from
P. aphanidermatum not only in the type
of oospore but also in having not inflated

11. Pythium myriotylum Dreschler, Jour.
Wash. Acad. 20:398-418. 1930.

Hyphae branched, septated in old
cultures, measuring 2.3-7.8 um in diam,
hyphal swellings present; sporangia infla-
ted, filamentous lobulate or digitate lateral
elements, terminal or intercalary; zoo-
spores reniform, laterally biciliate, 7.6-13.5
um in diam upon encystment; oogonia
spherical, smooth and thin-walled, ter-
minal or intercalary, measuring 13.5-30.8
um in diam; antheridia usually diclinous,
sometimes monoclinous 3-6 per oogo-
nium, antheridial cell clavate, crook-neck-
ed, arched; oospore aplerotic, single,

On soil, collected 1977, Bontoc.
Note: P. myriotylum differs from P.
aphanidermatum in having more dicli
nous antheridia than monoclinous anc
the antheridial cells crook-necked anc

12. Pythium oedochilum Drechsler, Jour.
Wash. Acad. Sci. 20:398-418. 1930.

Hyphae branched, measuring 2.1-7.1
um in diam, catenulate hyphal swelling
present; sporangia ovoid to obovoid
irregular in shape, subspherical, typically
acrogenous, sometimeslateral, intercalary
proliferous, papillate; zoospore reniform
laterally biciliate, measuring 10.5-16.'
um in diam upon encystment; oogoni

subspherical, typically terminal, inter
calary, measuring 17.5-34 um in diam
antheridia diclinous, sometimes mono
clinous, 1-3 per oogonium, antheridia
cells curved, elongated, cylindrical ant
wavy; oospore aplerotic, single, smooth
and thick-walled measuring 14.5-30 um is

On soil, collected 1977, Benguet
Bohol, Bukidnon, Camarines Sur, Laguna
La Union, Pampanga, Pangasinan, Quezor
City, & Zamboanga.

Note: Elongate, cylindrical, curve<
and wavy antheridium is typical ani
characteristic of P. oedochilum.

13. Pythium polytylum Dreschler, Jour
Wash. Acad. Sci. 20:415416. 1930

Hyphae branched, 1.75-6.22 um ii
diam, catenulate hyphal swellings present
appresoria abundant at hyphal apex
sporangia spherical to subspherical
measuring 22.7-30.5 um in diam, with
papilla, acrogenous, intercalary, prolific
rous; zoospore reniform, laterally bic:
late, measuring 23-32 um in diam oc
gonia spherical to subspherical, smoot
and thin-walled, acrogenous, measurin
23-32 um in diam; antheridia most
diclinous, sometimes monoclinous, 1-
per oogonium, antheridial cell curves
elongate cylindrical, wavy in profile
antheridial stalk rarely exhibiting hel
coid involvements; oospore apleroti<
single, measuring 23.5-27.5 um in diary
smooth and moderately thick-wallec
containing several reserve globules.

On soil, collected 1977, Albay, Bengue
Cagayan Iloilo and Isabela.
14. Pythium proliferum de Bary, Jahi
Wiss. Bot. 2: 169-192. 1860.
Hyphae branched with hyphal swe
lings; sporangia spherical to ovoid, tern
inal, measuring 28.5-42 um in diam, ore


Pythiums From PhilipI

liferous; zoospore reniform, laterally bici- Hyphae branched, septated, mea-
liate, measuring 6.5-10 um in diam upon during 1.72-4.24 um in diam; sporangia
encystment; oogonia spherical, smooth or spherical to subspherical, pyriform and
with a short apical papilla, terminal or spindle shaped, 10-22.5 um in diam;
intercalary; antheridia monoclinous, dic- zoospores not observed; oogonia spherical
linous, diclinous and hypogynous, 1-4 per to subspherical, 10.5-19.3 um in diam,
oogonium, sessile or stalked, antheridial spines conical with obtuse apices or more
cell clavate; oospore spherical, aplerotic, commonly digitate 4.74-9.0 um long,
thick-walled, measuring 17.5-26 um in intercalary and terminal; antheridia typi-
diam. cally monoclinous though sometimes dic-
On soil, collected 1977, Zamboanga. linous, terminal, 1-2 per oogonium, ori-
ginating close to the oogonium 9.4-20 um
Note: This species readily produces long and 2.4-4.85 um wide; oospore
proliferous papillate sporangium. It differs plerotic, single, smooth-walled 9.0-19.8
from other nrnlifernui nrnranocial n.ariae

others contain several.
Note: Pythium spinosum is distin-
15. Pythium rostratum Butler, Mem. guished from other echinulate oogonial
Dept. Agr. India Ret. 1:1-60. 1907. forms with plerotic oospores by the char-
Hyphae very fine, irregularly branched, acteristic long digitate, obtuse oogonial
measuring 1.0-6.2 um in diam; sporan- protruberances.
gia terminal, intercalary, spherical to
subspherical, smooth and thin-walled, 17. Pythium tardicrescens Vanterpool,
measuring 9.25-23.2 um in diam; zoo- Ann. Appl. Biol. 25:528-543.
spores reniform, laterally biciliate; oogonia
intercalary, terminal, spherical to sub- Hyphae iguarly branched, septated
Hyphae irregularly branched, septated
spherical, smooth and thin-walled, mea- when mature, measuring 1.92-4.15 um in
during 8.6-29.8 um in diam; antheridia diam, hyphal swellings present; spo-
monoclinous, single, delimited and within rangia inflated, filamentous, toruloid or
the oogonial stalk, antheridial cell small, moderately lobed; zoospores not ob-
clavate, 2.3-7.6 um wide and 2.69-12.3
clavate, 2.3-7.6 u wide and 2.69-12.3 served; oogonia spherical, terminal and
um long; oospore plerotic, smooth-
Swalled intercalary, measuring 14-26 um in diam;
antheridia usually monoclinous, some-
On soil, collected 1977, Mandaue times diclinous 1-3 per cogonium, anthe-
City,Misamis Oriental. ridial cell clavate and crook-necked, not
quite inflated; oospore aplerotic, single,
Note: The most distinctive charac- smooth with a moderately thick wall,
teristic of Pythium rostratum is the an- measuring 17.8-8.24 um in diam.
theridium, typically monoclinous, and On soil, coected 1977, Laguna.
the antheridial cell extremely short,
reduced to lateral swelling immediately Note: P. tardicrescens may be dis-
adjacent to the oogonium. tinguished from P. aphanidermatum by
16. Pythium spinosum Sawada, Jour. its antheridial cells which are typically
Nat. Hist. Soc. Formosa. 16:199-212. terminal, not greatly inflated and varying
1926. in number from one to 6.


Philippine Phytopathology

18. Pythium ultimum Trow, Ann. Bot.

Hyphae long, slender, branched, septa-
ted 1.35-5.25 um in diam; sporangia sphe-
rical and terminal, 9.25-23. 2 um in diam,
intercalary; oogonia smooth, terminal,
spherical, rarely intercalary, 14.5-18.2um;
antheridia 1-2 per oogonium; oospore
aplerotic, single, spherical 13.5-19.2 um
in diam with a smooth and thick wall.
On soil, collected 1977, Cavite and
Note: Like Pythium debaryanum, P.
ultimum is also a parasite of a large varie-
ty of hosts and is of wide occurrence. It
is distinguished from P. debaryanum in
having antheridia originating immediately
adjacent to oogonium and are swollen.
19. Pythium undulatum Petersen, Bot.

Tidssk. 29:345440. 1909; Ann
Mycol. 8:494-560. 1910.

Hyphae fine, measuring 1.2-2.75 um in
diam; sporangia acrogenous, ellipsoidal,
sometimes with a small apical papilla,
meausring 95-122 um long and 54.2 um
wide, proliferous, the subsequent sporan-
gia usually formed within the primary
one ; zoospores reniform, laterally bici-
liate, 12.2-16.8 um in diam, formed in a
sessile apical vesicle; sexual reproduction
not observed.
On soil, collected 1977, Benguet,
Camarines Norte, Laguna and Pangasi-

Note: Pythium undulatum differs
from P. carolinianum in the shape of its
sporangium: ellipsoidal for P. undulatum
and spherical for P. carolinianum.


Fig. 1. Pythium acanthophoron. (A) Hyphal laterals,
(B-C) Intercalary oogonia, (D) Oogonium with
diclinous antheridium, (E) Oogonia with
monoclinous antheridia.

Vol. 13

Pythiums From Philippine Soils

Fig. 2. Pythium allantocladon. (A)
Hyphae with allantoid bodies
producing spherical sporangia,
(B) oospore at the tip of an
allantoid body, (C) Oogonium
with monoclinous antheri-
dium, (D) Clusters of oogo-
nia with monoclinous anthe-
ridia, (E) Oogonia with dic-
linous antheridia








Fig. 3. Pythium aphanidermatum. (A)
Terminal filamentous sporan-
gium, (C-D) Oogonia with
diclinous antheridia, (E-F)
Oogonia with monoclinous
antheridia, (G-H) Intercalary


Jan. & June 1977

ilippine rnytopatnology


ra a

sporangia, (F-H) Oogonia v
E knob-shaped antheridia cell

F 6 H


Jan. & June 1977 Pythiums From Philippine Soils 65

A B c
C p

Pythium debaryanum. (A-D)
Hyphal swellings, (E) Termi-
mal and intercalary sporangia,
(F) Oogonium with diclinous
antheridia, (G) Oogonium with E
monoclinous and diclinous an-
theridia (H) Intercalary oogo-
lum. F




Fig. 7. Pythium deliense. (A-B
ferent types of sporanj
(C-D) Oogonia with m
nous antheridia, (E)
nium with diclinous a
dium, (F) Clusters of c
with monoclinous anti

Philippine Phytopatholuog~

A 1

Fig. 8. Pythium inflatum (A! Hyphal
swellings, (B) Intercaia. 'ilarnen-
tous sporangium, (C) Oogonium
with monoclinous anthei-riia. (D)
Oogonium with both mtnoclirlous
and diclinous antheridi .Ej 00-
iniu with monoliia<



Fig 9. Py thium monospermum. (A) Inter-
ca!ayv and terminal sporangia, (B)
Catt-nulate hyphal swellings and
bud-like lateral out-growths, (C-E)
Ac:ogenous oogonia with both
monoclinous and diclinousanthe-

B /f-


0 E

VC1. c '


Pyrhlu:r.'- From Philippi w S .;-i



/ I
ii /



i !~


Fig.10. Pythium myrtotylhum (A, De-
veloping sporangia, (B) Hyphal
swellings, (C) Louulate sporan-
gia, (D-E) Oogonia with mono-
ciAnoui and detlinous antherk-
dia. (F) Oogonmum with dich-
nous, lobate and crook-necked
ant:hsedia iG) Oogocniarn ith:
dielinous antheridia.


?-~ ,,

i ;r



Fi3 II P, ;':,: oedoi' rn (A-B)
Main-.1^ oogonia vi;!; dic!i;'ous
rnthri 'ia, (C) Onltgr'irrm with
mcndlinouF .-r diclinous
antheridia, (D) Appressoria,
(E) Ovoid and subspherical



c Ir


Philippine Phytopathology



Fig. 13. Pythium proliferum. (A-B) Pro-
liferating sporangia, (C-D) Sphe-
rical and ovoid sporangia, (E)
Hypogynous antheridium, (F)
Oogonium with monoclinous
and diclinous antheridia.


Fig.12.Pythium polytylum. (A) Hy-
phal swellings, (B) Spherical
and subspherical sporangia, (C'
Sporangium showing prolife-
ration, (D) Oogonium with
monoclinous antheridia, (E)
Oogonium with monoclinous
and diclinous antheridia, (F)
Oogonium with diclinous an-



Vol. 13



Pythiums From Philippine Soils

Fig.14. Pythium rostratum. (A-C) Dif-
ferent types of sporangia, (D)
Oogonium with monoclinous
and diclinous antheridia, (E-G)
Terminal and intercalary oo-
gonia with monoclinous anthe-


B c



Fig.15. Pythium spinosum. (A) Inter-
calary oogonium, (B) Oogoni-
um with diclinous antheridium,
(C) Oogonium with monocli-
nous antheridium, (D-E) Inter-
calary sporangia, (F) Pyriform

Jan. & June 1977





Fig.17. Pythium ultimum. (A) Oogon-
ium with amphigynous antheri-
dium, (B-C) Oogonia with
monoclinous and paragynous
antheridia, (D) Aplerotic oo-
gonium with monoclinous and
diclinous antheridia, (E-G) In-
tercalary and terminal

B Fig.16. Pythium tardicrescens.
phal swellings. (B)
sporangium, (C-D)
with monoclinous and d
antheridia, (E) Oogonit
monoclinous antheridiu

A B c



Tr. i.... i. i

Pythiums From Philippi:






Fig.19. Pythium irregulare. (A)
swellings, (B-C) Differen
of sporangia, (D) Monocli
theridium, (E) Diclinous
dium, (F) Mature roug]
oospores in irregularly


Philippine Phytopathology


Sporangia present
Sprangia spherical
Sexual reproduction present
Allantoid bodies produced on hy
Allantoid bodies not produced
Oogonium smooth
Oospore plerotic, antheridi
monoclinous or hypogynoi
Oospore aplerotic
Sporangium proliferous
Oospore containing s
Oospore containing s
Sporangium sphere
Sporangium ovoid
Sporangium not prolifei
Antheridium sessile,
adjacent to oogon
Antheridium stalked
immediately adjac
Oogonium echinulate
Oospore plerotic, oogonial
digitate and long.....
Oospore aplerotic, oogonia
mammiform ........
Sexual reproduction absent
Sporangia prolate ellipsoidal, vesicle
Sporangia spherical, vesicle stalked.
Sporangia filamentous
Sporangia undifferentiated from veget
Sporangia densely inflated, toruloid
Oospore plerotic ...........
Oospore aplerotic
Irregular hyphal swellings and
antheridia common ... .
Irregular hyphal swellings and
not common
Antheridium monoclinous
Oogonial branched curv
Oogonial branched not
Antheridial cell typio
1 to 2 per oogoni
Antheridial cell typii
1 to 6 per oogoni
Antheridium diclinous, oci
Sporangia not known, oogonia spiny...


a ........... .. P. allantocladc

n usually
.............. P. rostratum

gle reserve globule. .... P. proliferum
veral reserve globule
al................ P. polytylum
.......... ... P oedochilum
iginating immediately
m .......... ... P. ultimum
lot originating
it to oogonium....... P. debaryanum

................. P. spinosum
protruberances not
................. P. irregulare

essile ............. P. undulatum
................. P. carolinianum

ive hyphae, not inflate. P. monospermum
r lobed
................. P. inflatum

................. P. butler
ypogynous antheridia

occasionally diclinous
1 toward antheridia .... P. deliense
irved toward antheridia
fly intercalary,
n ................ P. aphanidermata
lly terminal,
n ................ P. tardicrescens
.................. P.myriotylum
................. P. acantophoron

Vol. 1?

'ythiums From Philippine Soils


'REZZI, M.J. 1956. Especies of Pythium 1
Argentina. Rev. Invest. Agr. T.X. No. 2
iENDRIX, F. F. Jr. and W. A. CAMPBEI
Pythium species in soils in the container
iENDRIX, F. F. Jr. and W. A. CAMPBELL. :
of Phytopath. 11:77-98.
dIDDLETON, J.T. 1943. The taxonomy, hi
genus Pythium. Memoirs of the Torrey
3IDERIS, C. P. 1932. Taxonomic studies in
gia 24:14-61.
[EODORO, N. G. 1935. Enumeration of I
WATERHOUSE, G.M. 1968. The Genus Py
gical Paper. 110. 50 p.


topathogenas identificados en la Republica
3uenos Aires. 241.
L. 1970. Distribution of Phytopthora and
al United States. Can. J. Botany 48:377-84.
)73. Pythium as plant pathogens. Ann. Rev.

it range, and geographic distribution of the
botanical Club. 20:1-171.
he family Pythiaceae. II. Pythium. Mycolo-

tilippine fungi. Bureau of Printing. Manila.
hium Pringsheim. Commonwealth Mycolo-

ian. D& une ir.t



R. G. ABAD a

Research Supervisors, Crop Protect
Agricultural Research Branch, Philippine (

Leaf diseases, like other disturbances,
reduce the efficiency of foliage in a crop's
photosynthetic function. In effect, pre-
cocity and yield are affected. In coconut,
the most common leaf diseases are leaf
spots notably those caused by the fungus
Pestalozzia palmarum Cke .
Despite the widespread occurence of
coconut leaf spot diseases, they are consi-
dered of minor importance due perhaps
to their being non-lethal and may be
because their presence in relation to
growth and yield has not been clearly
demonstrated. The large numbers of
poorly managed coconut landholdings in
the country considerably affected by
these leaf disorders are evident. Such
situations cannot however, be ignored
if good returns are expected of coconut
In the control of fungal diseases, the
use of fungicides usually come to one's
mind first. In dealing with leaf spot
diseases of coconut, application of fun-
gicides may not be feasible, particularly
on tall mature palms. Fertilizer studies
at the Davao Research Center in Bago-
Oshiro, Davao City, however, presented
some circumstantial evidences on the
relationship of leaf spots and the yield
performance of palms.

Leaf Spot Density In Relation To Earliness
of Bearing

T! w'\ d'is-losed hb Ab;d i't al (197")



an and Agro-Soils Divisions, ie-:pecti\ely.
)conut Authority, Bago-Oshiro, Davao City

from a six-year experiment of 'ie Davao
Research Center, that palms heavily in-
fected with leaf spot flowered relatively
late than less affected ones. Ir this par-
ticular experimental area, 37 per cent of
the less leafspot infected palms were
already bearing after four years fiom trans-
planting while the highly infected ones
had only 15 per cent. On the sixth year,
93 per cent of the former were already
bearing compared to only 54 per cent in
the latter.
Through regular fertilization with
potassium chloride (KCI), occurrence of
leaf spot diseases was minimized (Table
1 and Fig. 1) which likewise have re-
sulted to the promotion of precocity.
Application of nitrogen and phosphorus
fertilizers favored disease development.

Production From Heavily and Less Leaf
Spot-Affected Palms

In another two and a half-years ex-
periment of the PCA-ARB involving full-
bearing Laguna palms, the preventive
effect of KCI on the occurrence of leaf
spots was likewise observed. Based on a
one-year record of leaf spot ccunL con-
ducted regularly from ten mic-leaflet
samples of the 14th leaf, an average
of 193 spots per leaflet was nored from
KCI-untreated trees while only i spots
per leaflet was observed on the aggregate
KCI-fertilizcd palms (Table 2). however,
no significant differences wer noted
:.:iong treatments, KC(..

F ij c~al >CNote, iCJI FerAiizati.

able 1. Leaf :pot count per leaflet (6-year oi

(NPK Combination)2



1Results based on 10 middle leaflet san
he youngest fully expanded frond). Means
!antly different at 5% level (Duncan's Multipkl
'alues. Lifted from Abad et al (1975).

2Per tree annually from field-planting:
;uperphosphate, and Ko, K1 and K2 = 0, 15(
Ifter two years and three folds thereafter up 1

Lower yield was, as expected, noted
rom palms severely affected by leaf
pots with an average of 142.8 nuts/tree
)r 22.78 kg copra/tree. The highest yield
was obtained with 1.66 kg KCI/tree.
It is not, however, asserted that leaf
spots are directly responsible for poor
yield. Rather, it is emphasized that their
presence in significant degree should
serve as an indicator for poor crop
nutrient status or less yield, since fungi
musing leaf spots particularly Pestalozzia
Palmarum are generally more common
>n unthrifty palms because they are
physiologically weak to resist fungal


The widespread presence of foliage
disorders on field-planted coconut should,
as earlier stated, be regarded as signs of
poor nutrition. Proper fertilization is
therefore needed to improve the growth

I palms)'

Leaf Spot
Count (No.)


ples from leaf rank 9 (9th leaf starting from
followed by the same letter are not signifi-
Range Test). Analysis based on transformed

N1 = 400 g ammonium sulfate, Pi = 300 g
and 300 g KCI, respectively. Rates doubled
o the 6th year.

disease and increasing the yield. This
operation is easy and less expensive.
Application at the rate of 3.33 kg KCI/
tree/year gave a net income ofP2,542/ha
within 2 years while the 1.66 kg KCI/
tree/year gave yield :P2,379 net
profit, which is about P800 more per
hectare annually 'over the palms re-
ceiving no KCI (Magat, 1975)*.

Existing coconut farms or plantations
may be fertilized with 1.5 to 2.0 kilos
ammonium sulfate (21% nitrogen) in
combination with 1.6 to 2.5 kilos KCI
(60% K2 0 and 44% CI) per tree per year.
These rates hold true especially for in-
land-upland coconuts, even in the absence
of leaf analysis. Weed control and good
drainage are a must.

For palms below six years of age under
similar conditions, PCA-ARB recommends

"Copra price used: P1.30/kilo (1974);
I56 rlms/lh ect a r

r -.c

ilippine rhytopathology

Portions of leaflets from untreated and KCI treated ti
Note the numerous spots of different sizes on leaflets f
trees without KCI.

Vol. 13

Phvtnnatholnieal Notp: K

average teaj spor reaarng p

CI/tree/ Nut/tree
ar ) (no.)

0 142.8
83 161.8


^^ ^^



2 Blank

166 14.6b
178 10.4b
198* 9.0b
130** 10.7b


ava.sb. vIMasiw *V.IItwu C U W7 e aCsi jac.l a ~ny V s lv iJas- b ... ay unaIlJculr a, s /v 1SCv

i is bro
n radius
I- zation
2 Fol
rl ha rw

b) 3 to 5 year old palms sami
lizer combination but at doubled
The recommended fertilizer place

1975. Ir
ogen, ph
)p Sci. E


ver thi
of the

tter crc

Republic of the Philippines
Department of Public Works, Transportation and Communications

(Required by Act 2580)

The undersigned, TRICITA H.
PATHOLOGY published semi-annually
duly sworn in accordance with law, hereb:
management, circulation, etc., which is r'
wealth Act No. 201.


Business Manager: CEFERINO A. BAN:
Office of Publication: DEPT. of PLANT I

In case of publication other than <
lated of the last issue dated JANUARY &

1. Sent to paid subscribers .....
2. Sent to others than paid subscrit
T o ta l ...........

Baiios, the affiant exhibiting his Residen
Laguna on February 14, 1978

Doc. No. 475
Page No. 95
Book No. XXV
Series of 1978

NOTE: This form is exempt from the pa:

English, College, Laguna, after having be
submits the following statement of owners
uired by Act 2580, as amended by Commc


UPLB-CA, College, Lagu
1UED BPI, San Andres, Manila
CITYT, INC. UPLB-CA, College, Lagu
G HOUSE 373 Quezon Ave., Q.C.

ily, total number of copies printed and cir(
UNE, 1974

. . ... . .. 120
*s .. .......... ... 380
............ 500

*fore me this 12th day of May, 1978 at 1
Certificate No. 80913523 issued at Los BaL

Notary Public
Until December 31, 1978
P.T.R. No. 0049101
Los Baios, Laguna

nent of documentary stamp tax.

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