• TABLE OF CONTENTS
HIDE
 Front Cover
 Front Matter
 Abstracts of papers presented at...
 The morphology of different Philippine...
 Carbon, nitrogen and vitamin nutrition...
 Translocation and movement of the...
 Effect of gamma radiation on the...
 Variability of Philippine isolates...
 Aspergillus flavus infection of...
 Inoculation of pregerminated corn...
 Development and hitropathological...
 Studies on growth, sporulation...
 Survey and pathogenicity tests...
 Evaluation of the different methods...
 Phytopathological note: Inducing...
 Back Cover














Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00090520/00016
 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 1973
Frequency: semiannual
regular
 Subjects
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
 Notes
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00016
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 54382605
issn - 0115-0804

Table of Contents
    Front Cover
        Front Cover 1
        Front Cover 2
    Front Matter
        Front Matter 1
        Front Matter 2
    Abstracts of papers presented at the tenth annual meeting of the Philippines phytopathological society Legaspi City, 16-18 May 1973
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
    The morphology of different Philippine isolates of colletotrichum falactum went
        Page 7
        Page 8
        Page 9
        Page 10
    Carbon, nitrogen and vitamin nutrition colletotrichum falacatum went
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
    Translocation and movement of the greening pathogen in citrus
        Page 16
        Page 17
        Page 18
    Effect of gamma radiation on the growth of storage rot-causing microorganism in banana under different incubation temperatures. I. Var. Lacatan
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
    Variability of Philippine isolates of colletotrichum gloeosporioides from fruits
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
    Aspergillus flavus infection of pre-harvest corn, drying corn, and stored corn in the Philippines
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
    Inoculation of pregerminated corn seeds, conidial production during the day and the use of tween 80 in inoculating Philippine corn downy mildew
        Page 42
        Page 43
        Page 44
        Page 45
    Development and hitropathological effects on meloidigyne incognita on celery
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
    Studies on growth, sporulation and spore-germination of colletotrichum gloeosporioides penz
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    Survey and pathogenicity tests of plant parasitic nematodes associated with sugarcane in negros occidental
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
    Evaluation of the different methods and rates of application of temik 10G for the control of nematodes on abaca
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
    Phytopathological note: Inducing the perfect stage of rhizoctonia solani kuhn
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
    Back Cover
        Page 81
        Page 82
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M. T. T. REYES, UPLB, College, Laguna

r: . G.ADAVIDE, UPLB, College, Laguna

NMmbers: C. T. RrVERA, IRRI, College, Laguna
H. E. KAUFFMAN, IRRi, College, Laguna

.

lilNGwo ASSOCIATES

:an Cynamid:Company, Princeton, New Jersey, U.S.A.

PIllippines, Inc. 622 Shaw Blvd., Mandaluyong, Rizal

SSugar Estate, Canlubang, Calamba, Laguna
r."'- .













PHILI

PHYTOPA!
Official Organ of the Philippine



EDITORIAL

I. J. DOGMA, JR., Editor-in-(
UPLB, Colle!

P. M. HALOS, Associate Edit

A. J. QUIMIO, Associate Edil
BUSINESS MA

D. A. BENIGNO, Business Mi

















Subscriptions. Communications should be
'nt of Plant Pathology, UPCA, College, L;
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r others, it is P25 per year orP12.50 perc
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ytopathological Society: Information rec
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i endorsement of any statement of claim,
urnal or by the Philippine Phytopathologic


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HOLOGY
ytopathological Society, Inc.



BOARD

.f, Department of Botany
Laguna

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4GEMENT

3er, UPLB, College, Laguna

















ressed to the Treasurer, P.P.S. c/o Depart-
na 3720. Philippine Phytopathology pub-
Fficial organ of the Philippine Phytopatho-
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Republic of the Philippines
Department of Public Works and Communications
BUREAU OF POSTS
Manila


SWORN STATEMENT
(Required by Act 2580)

The undersigned, PONCIANO M. HALOS, Editor of PHILIPPINE PHYTOPATHOLOGY,
jblished Semi-annually, in English, at UPCA, College, Laguna, after having been duly
vorn in accordance with law, hereby submits the following statement of ownership,
management, circulation, etc. which is required by Act 2580, as amended by Common-
ealth Act No. 201.

NAME ADDRESS

ditor PONCIANO M. HALOS UPCA, College, Laguna
business Manager DANTE A. BENIGNO UPCA, College, Laguna
owner PHIL. PHYTOPATHOLOGICAL
SOCIETY UPCA, College, Laguna
-inter AGRIX PRINTING PRESS, INC. S.U. UPLB, College, Laguna
office of Publication UPCA, Dept. of Plant Pathology, College, Laguna

In case of publication other than daily, total number of copies printed and circulated
the last issue dated January & June 1972
1. Sent to paid subscribers 120
2. Sent to others than paid subscribers 480
Total 600

PONCIANO M. HALOS
Editor-in-Chief

SUBSCRIBED AND SWORNto before me this 6th day of August, 1975 at LOS BANOS,
AGUNA, the affiant exhibiting his Residence Certificate No. 3296744, issued at
os Bafios, Laguna on May 27, 1975

Atty. ROMAN M. ALONTE
(Officer administering oath)

NOTE: This form is exempt from the payment of documentary tax.





DISTRACTS OF PAPERS PRESENTED AT THE TENTH ANNUAL MEETING
OF THE PHILIPPINE PHYTOPATHOLOGICAL SOCIETY,
LEGASPI CITY, 16-18 May 1973



Application of deep-freezing me- severe mottling symptoms. These cultivars
Sin detecting seed-borne fusaria and tolerated the presence of the virus and had
r fungi. B.A. Advincula, P. Neer- normal flowering and seed setting.
I and S.B. Mathur (UPCA; Danish SMV has a very limited host range.
tute of Seed Pathology for Develop- Only corn and sugar cane were found
Countries) susceptible to this virus. No symptoms
The standard blotter method used were observed on the following dicots:
generall seed health testing was com- Chenopodium amaranticolor, C quinoa,
d with deep-freezing method, a modi- Datura stramonium, Cucumis sativus,
:ion of the former, in counting in- Gomphrena globosa, Phaseolus vulgaris,
ions of Fusaria and other fungi in P. mungo, P. atropurpureous, Vigna si-
s of tropical and temperate origin. nensis, Glycine max, Cassia occidentalis,
fungi studied were Fusarium avena- Centrosema pubescens and Dolichos axi-
n, F culmorum, F dimerum, F mo- llaris.
orme, F nivale, F. poae, Drechslera Although SMV is readily sap trans-
tae, D. oryzae, D. sorokiniana, Nigros- missile, it is unstable in vitro. Its thermal
r oryzae, Pyricularia oryzae and Tri- inactivation point is 60 C, dilution end
conis padwickii. In general, deep-free- point is 1: 10,000 and its longevity in
method gave comparable results. vitro is only up to 17 days. Infectivity of
nting and identification were easier the virus was lost upon drying the infect-
to suppression of saprophytic flora ed leaves. One cycle of freeze-thawing
absence of roots and sprouts. also drastically reduced its infectivity.
Deep-freezing followed by incuba- In the field the corn aphid, Rhopa-
at 20 C proved much superior for losiphum maidis Fitch. is responsible for
citing infections of F nivale. Higher its dissemination. This aphid can acquire
fences of F avenaceum, F culmorum and transmit the virus within 20 minutes
F moniliforme were obtained at feeding, indicating that SMV is a non-
: irrespective of the method. Nigros- persistent virus.
oryzae was adversely affected by Serological test by ring precipitin
Jeep-freezing method. showed that the sorghum mosaic virus
antigen reacted with the antiserum of
Sorghum Mosaic: The first virus maize dwarf mosaic virus, a strain of
ise of sorghum in the Philippines. sugar cane mosaic virus.
SBenigno and A.D. Karganilla (UPLB,
ege, Laguna) An efficient method for field ino-
A virus causing mosaic and red culation of bacterial leaf blight on rice. -
A virus sing syoms on a and red Y.A. Cho, S.D. Merca and H.E. Kauffman
ping symptoms on a number of varie- (International Rice Research Institute,
and cultivars of sorghum was isolated
a Cosor-2, a variety of sorghum plant- Baos, Laguna)
last year at the Central Experiment A method for field inoculation of
ion of the U.P. College of Agriculture, bacterial leaf blight (RLRA has been modi-


LU OUlrIIUJII. vL LIL 00 .UILIVMtO t.OLtLU
the greenhouse, 5 showed moderate
stance to the virus with faint mottling
symptoms, 13 were highly susceptible
wing red striping and drying of leaves
eventual death of the plants, 15
tivars did not exhibit any red striping
drying of leaves but instead exhibited


neter plastic hose is placed in the
:ulum in a bottle and the other end is
mned to the handle so the tip is just
ve the blade. The inoculum is then
oned to the blade and the dripping
he suspension is controlled automatic-
by stretching (opening) the hose and





bending (closing) when the trimmers are
used.
Approximately 3,000 plants can be
inoculated per hour in the field with
500 ml of inoculum. An additional ad-
vantage over the use of regular scissors
is the more uniform distribution of ino-
culum from plant to plant throughout
the inoculation period.

Some biological studies of nema-
todes associated with the citrus decline
in the Philippines. R.G. Davide (UPLB,
College, Laguna)

Through a series of inoculation
experiments, it has been established that
the citrus nematode, Tylenchulus semi-
penetrans Cobb, is capable of causing


aclerospora ptlmppmensis, tie pri-
mary causal fungus of maize downy
mildew in the Philippines, is an obligate
parasite and to date has not yet been
brought into axenic culture. Because
other related species of Sclerospora can
attack maize, viz., S. sacchari and S. spon-
tanea, an artificially-produced, homoge-
nous population of S. philippinensis is
an absolute necessity in definitive basic
studies of this pathogen. Such a popula-
tion was obtained on corn seedlings
(3-4-leaf stage) using single conidia as
inoculum. The newly formed spores on
naturally diseased corn leaves were allowed
to lodge onto 20/o water agar in petri
dishes. Small blocks of agar, each with a
conidium starting to germinate, were cut
out under a dissecting binocular, and


significantly reduced by more than 50 plants were placed in plastic bags and
per cent. There was evidence that the dispensed for 6 days in an ISCO growth
degree of damage on the plants was depen- chamber adjusted to night conditions of
dent upon the population density of the 23.5 C and 970/o RH, thereafter to day
nematodes. An inoculum level of approxi- conditions of 30 C and 750/o RH. Suc-
ximately 50,000 nematodes per plant was cess was low, 130 on the average.
more destructive in shorter period of time Systemic infections became noticeable
than that of 10,000 level. 9-15 days after inoculation. Essentially
The different isolates of T. semi- the same results were obtained when
penetrans showed varying degrees of vi- single conidiophores bearing conidia were
rulence when inoculated to Ladu and used as inoculum. Here, however, the
Calamansi seedlings. This indicated a pos- chances for success were increased by as
sible existence of biotypes of the nema- many times as there are conidia per
todes in this country. conidiophore (15-20 on the average).
Results of the interaction experi- A single corn seedling infected system-
ments revealed that the rate of reproduc- ically is sufficient. The population of the
tion of T. semipenetrans was greatly in- pathogen was increased easily by inocu-
creased in the presence of other citrus lasting other seedlings by the mass-spore
pathogens such as Corticum salmonicolor, technique, but with the inoculum coming
Fusarium sp., Phytophthora sp., and the in it
greening pathogen, a mycoplasma-like or- from the infected w
ganism. a single spore.
On the other hand, results of the Survey for corn infestation by As-
inoculation experiments with the other pergillusflavus in the Philippines. L.
genera of nematodes such as Pratylenchus, Dag (UPLB, College, Laguna)
Helicotylenchus, Paratylenchus, Hemicy-
cliophora, Tylenchorhynchus, Cricone- The presence of Aspergillus fvus
moides and Diphtherophora did not show in corn prior to harvest, while drying
after harvest, and in storage was surveyed.
any sign of reproduction nor damage on
any sign of reproduction nor d ge on The fungus was rarely present in mature
roots of the inoculated Szinkom seedlings. corn in field although 80/0 infection
corn in field although 8/o infection
was observed in two instances. Newly
harvested corn that was being sun-dried
Monospore culture of Sclerospora and stored corn consistently harbored the
on corn. IJ. Dogma Jr., O.R. Exconde fungus; 1000/o infestation was commonly
and R.G. Bayot (UPLB, College, Laguna) observed.





inoculation experiments witn pure me otner tnree varieties. Ine ncreas(
Itures of A. flavus and A. parasiticus in population in X. oryzae on the lea:
)wed that under certain conditions surface generally correlates with the in
Id corn may be infected to as much as crease in disease symptoms.
/o with the aflatoxin-forming fungi.
The use of leaf extract for inocula
Growth of Sorghum vulgare L 3998 ting rice varieties against bacterial leal
d associated with microflora upon treat- blight. S.D. Merca, M.P. Natural and
ent with gamma rays. J.Z. de Jesus H.E. Kauffman (International Rice Re
search Institute, Los Bafios, Laguna)
Seeds of Sorghum vulgare L. 3998 N l ps of
:re irradiated with gamma rays from Natral p of n
ibalt-60 at different dose levels. The nas oryzae in leaf extract from infected
1,,.,. r, ~,, ~,,,,~,,,


failed to grow. Germination of the seeds
in both the 30 and 60 Krads treatment
was good while at 120 and 240 Krads
growth was strongly inhibited. Microbial
growth was also inhibited. Repetition of
irradiation treatment using dosages lower
than 30 Krads was done. The dosages were
15, 20, 25 and 30 Krads. When planted
in pots significant differences in sorghum
panicle size and grain numbers were
obtained compared with the control. The
best results were observed between 15 and
20 Krads. Data gathered showed that as
the dose levels increased from 15 Krads
up, microbial growth was controlled and
the germination of the sorghum seeds
was delayed.

Epidemiological studies of bacterial
leaf blight on resistant and susceptible
rice varieties. SD. Merca and H.E. Kauff-
man (International Rice Research Insti-
tute, Los Bafios, Laguna)

Rice varieties IR20 (R), IR8 (MS),
IR1330 (HS) and TN 1 (HS) were grown
under upland conditions and inoculated
with streptomycin resistant (PXO 22S)
Xanthomonas oryzae strain at the center
of the plots. An overhead sprinkler irri-
gation system was used to simulate rain-
fall. Data for bacterial spread on the leaf
surfaces of the plants were collected twice
a week by making leaf washings from 13
different locations within each plot. Sam-
ples of leaf washings were spotted on
streptomycin-Wakimoto agar plates (1000
ppm streptomycin sulfate + 100 ppm
nystatin). Populations of other microor-
ganisms were also observed.
Reading on visual blighting of rice
leaves were taken at weekly intervals.
Preliminary results indicate the disease
spreads much faster in IR1330 than in


are cut into small pieces and soaked in
water to allow the bacterium to ooze out.
Populations of more than 109 viable
bacterial cells per ml can easily be ob-
tained if equal volumes of water and leaf
pieces are soaked for 15 minutes. These
bacterial populations give high infection
frequency and a good disease score. If the
leaves are soaked more than four hours
the population of saprophytic bacteria
increase and inhibit the survival of
X. oryzae.
The use of the bacteria from leaf
extract eliminates the potential problem
of virulence loss when pure cultures are
used. It also evaluates the naturally oc-
curing strains of X. oryzae existing in
various areas of the country. This allows
breeding material to be screened in small
experimental stations where methods for
culturing the bacterium are not available.

Effect of temperature on the sur-
vival of Xanthomonas oryzae in seeds.
- M.P. Natural and H.E. Kauffman (Inter-
national Rice Research Institute, Los
Bafos, Laguna)

The effect of various temperature
on long term survival of Xanthomonas
oryzae in rice seeds was determined. Seeds
infected with a streptomycin-resistant
isolate were kept at 0-4 C, 20 C and 35 C
and assayed at one month intervals.
High populations of viable X. ory-
zae were maintained when seeds were
kept at 04 C. About 4.8 x 10' bacteria
per hull and 3.4 x 104 bacteria per brown
rice were recovered after 120 days of
storage. Although the population of viable
X. oryzae gradually declined, at 20 and
35 C, about 9.6 x 10 and 1.3 x 10
bacteria per hull were still recovered at
the two respective temperatures after 120





lays of storage. The results suggest that
C. oryzae can survive quite long in the
eeds even at high temperatures. Thus,
dissemination of the bacterium by seeds
nay occur although tests evaluating trans-
nission of the bacterium to growing
'lants have all been negative.

Eradication of Xanthomonas oryzae
Ishiyama and Uyeda) Down son present
r rice seeds. M.P. Natural and H.E.
:auffman (International Rice Research
institute, Los Baftos, Laguna)

Tests were conducted on eradica-
ing Xanthomonas oryzae in seeds by
ot water treatment and by soaking them
i two concentrations of the experimental
chemical TF-130 in combination with hot
rater treatment. The use of streptomycin-
esistant isolates made possible the quan-
itive determination of bacterial popula-
ions in the seeds following the various
treatments.
Results indicated that a hot water
-eatment alone maintained at 56-58 C
)r 15 minutes did not effectively eradi-
ste X. oryzae. Approximately 103 cells
er seed remained viable both in the hul
n]r krnun, At. fr. i I-mln+: rI


destructive during early flowering. Some
f the chemicals like Benlate, Hoe 17411,
opsin M, BASF 3050 and Hinosan at the
ste of 1, 1, 1, 1 kg and 1 liter, respect-
rely, found effective against rice blast
Iso showed effectiveness against sheath
light. Since both diseases could be con-
rolled simultaneously, the use of these
chemicals for control may become
practical.

DISEASE EPIDEM: A program for
computer to simulate disease epidemic. -
).S. Opina and F.C. Quebral (UPLB,
os Banos, Laguna)

Disease Epidem, a program written
i IBM 1620 Fortran II language was
developed to simulate downy mildew
epidemic at any set of weather conditions.
t employs the temperature, relative humi-
ity, wind speed, sunniness, and wetness
or each 3 hours of each day. Each 3
ours, it modulated the course of the
allowing fungal stage according to the
different effect of the weather factors
pon them: formation of conidiophores
ad spores, departure of spores on wind
r rain, finding a host, germination of the
)ores. Denetration of the host. incubation


om the hulls but not from the brown ""m'. t"",t-t,'"" a'"" ,, 1r,aL u I1""
:e. When the concentration of TF-130 a simulator.
is increased to 1000 ppm, no viable Formation of lesion in sheath blight
oryzae was recovered from either the of rice. S.H. Ou and Sonia Ebron
lls or brown rice. The results indicate (International Rice Research Institute,
at the bacterium can be eradicated Los Baflos, Laguna)
om the seed by a combination of hot
iter and chemical treatments. The lesions of sheath blight on
infected plants are separated from each
Chemical control of sheath blight other, sometimes close together, while at
rice. F.L. uque, S.H. Ou, J.M. other times, are at some distance from
ndong and T. Ebron Jr. (International one another. The mycelium of the fungus,
ce Research Institute, Los, Baflos, however, covers the entire area. We found
guna) two types of mycelium, the straight-run-
c . . ning mycelium and the short, much





:d from were resist


oserveu m pactnes m test tuoe or pern 1llr ircaula lluwVu mLIM vVly lcw
ish walls. This explains why the lesions isolates of race P150 were recovered from
f sheath blight are separated. A single the mixed culture (cage inoculated with
esion is the result of multiple infection P150 and P38). Superficially, it seemed
y many lobed cells. that population P150 diminished, a situa-
tion similar to that of stabilizing selection.
Age of rice plant in relation to However, there were also very few isolates
susceptibility to sheath blight. S.H. Ou, of race P150 recovered from the seedlings
.L. Nuque and J.M. Bandong (Interna- inoculated with P150 alone. Single co-
ional Rice Research Institute, Los Bafnos, nidial isolation tests showed that in both
aguna) cases the original races have changed to

In testing sheath blight resistance many new races. The apparent reduction
t seedling stage in the sheath blight of population of P150 is due, in fact, to
the change of races rather than stabilizing
nurseries, we found distinct resistant and
selection.
susceptible reactions among rice varieties.
however, when the same group of varieties Perfect state of Rhizoctonia solani





very slow to very fast. The type of myce- Diseased samples of rice leaves from
hum varies from straight, in strand to Diseased samples of rice leaves from
floccose. The color varies from whistrand to 13 provinces in the Philippines were
floccose. The color varies from white, collected. Single colony isolations were
buff to deep brown. Some cultures were coected Single colony isolations were
observed to produce the lobed mycelium made and 100 isolates were inoculated to
observed to produce the lobed mycelium
freely while in others scarcely or none. IR20 (R), IR790 (MS), IR944-125 (MS),
The sclerotial formation also differs great- and JC 70 (HS). Most isolates showed an
ly. Some produced sclerotia within 4 to 5 average virulence score of between 4 to 6.
days, others produced no sclerotia after 4 One isolate, however, has an average score
weeks. Sclerotia vary in sizes, numbers, of 2.5 and 4 isolates averaged 3.5. Three
shapes, and in locations inside the petri isolates averaged from 6.5 to 8 in disease
dish. Some cultures produced one or reaction.
two large sclerotia, others have many
medium-sized sclerotia and still in others T
as effused masses. The sclerotia may be Thus some variation in overall viru-
as effused masses. The sclerotia may be lence was evident among the isolates
formed near the center of the petri dish ence was evdent the soltes
or near the edges. These suggest that tested. The relationship, however, appears
cultural morphology is not a good basis to be of a horizontal nature between the
for identifying species of Rhizoctonia. host and pathogen. This concept was
further supported when more than 100
Virulence of Philippine isolates of varieties were tested against naturally
Xanthomonas oryzae. Tin Win and occurring strains of X oryzae in five
H.E. Kauffman (International Rice Re- locations in the Philippines. The varieties
search Institute, Los Baflos, Laguna) gave similar disease scores in all locations.






THE MORPHOLOGY OF DIFFER]
COLLETOTRICHUM F;

HAMIZ UDDIN AHMED AND

Senior Scientific Officer, Agriculture Divi
Bangladesh, and Associate Professor, Departmel
University of the Philippines, College, Laguna, Ph
Part of a dissertation by the senior auth
School of the College of Agriculture, University
in partial fulfillment of the requirements for th
1969.

ABSTR/

The morphological characteristics of thirty
collected from the various cane-growing areas
variations in the sizes of spores and setae among
hyaline, septate, branched and measures 3.0 to 6.0,
iu for all isolates. The setae measure 70.0 to 183
middle. The average length of setae for all isolate'
4.45 + 0.25 u. The spores are hyaline, one-cellec
and 3.8 to 7.841 in width with the average of 27.4


Colletotrichumfalcatum Went (Phy-
ilospora tucumanensis Speg.) is the causal
rganism of red rot of sugar cane. The
ingus is known to have two main types,
amely the light and dark types. The
torphological characteristics of types are
tore or less the same, but they are
distinguished on the bases of color of
lycelium, degree of sporulation, and
rulence (Abbott, 1938; Chona, 1954;
hona and Hingorani, 1951; Chona and
rivastava, 1960; Rafay, 1950). The light
rpe is more virulent than the dark type.
bbott (1933) cultured hundreds of iso-
Ites of C falcatum and reported that the
plates differed in their pathogenicity.
lhmed and Shahjahan (1972) isolated
several isolates from the different cane-
rowing areas of Bangladesh and reported
iat there was not much variation in the
morphology of different isolates. Ahmed
1973) studied many isolates collected
rom various parts of the Philippines and
sported that the isolates differed in their
irulence. The following is a study of the
lorphological characteristics of these
hilippine isolates.

MATERIALS AND METHODS

Diseased specimens were obtained
rom various sugar cane-growing areas of
he Philippines (Table 1). The fungus
ias isolated by the tissue planting and
ingle spore methods and 13 isolates were


T PHILIPPINE ISOLATES OF
,CATUM WENT

IL G. DIVINAGRACIA

n, Atomic Energy Centre, Ramna, Dacca-2,
of Plant Pathology, College of Agriculture,
)pines respectively.
presented to the Faculty of the Graduate
the Philippines, College, Laguna, Philippines
degree of Doctor of Philosophy September,


r

1 isolates of Colletotrichum falcatum Went,
the Philippines were studied. There were
well as within the isolates. The mycelium is
in diameter with the average of 4.25 j- 0.15
u in length and 3.0 to 6.0/u in width at the
113.95 9.85,u while the average width is
alcate and measure 16.0 to 41.0.u in length
5.7,u by 5.4 0.8,u.


Jar medium (50 g Quaker oats, 15 g agar
nd 1 1 distilled water) under room con-
itions. Only the imperfect form of the
ingus was studied, since no ascocarps
'ere observed. Hyphae, spores and setae
'ere measured using a filar micrometer.
olor determination was made according
Sthe color standard of Ridgway (1912).

RESULTS AND DISCUSSION

Mycelium. The mycelium was hya-
ne, septate and branched. Its diameter
mged from 3.0 to 6.0tu with an average
f 4.24 4 0.15 u for the thirteen isolates
Table 2).
Spores. The spores were hyaline,
ne-celled and falcate. They measure
5.0 to 41.0,u in length and 3.8 to 7.8/u
tirteen isolates was 27.4 + 5.7 tu while
ie average width was 5.4 -+ 0.8/u. On
ie average, spores of isolate No. 10
ere longest and those of isolate No. 13,
ie shortest.
Setae. The setae were black in
Alor and broader at the base. The length
iried from 70.0 to 183.0/u with some
iriations within the isolates. The average
ngth for all isolates was 113.95 9.85 u.
he diameter of the middle portion of
ie setae ranged from 3.0 to 6.0iu with
n average of 4.45 4- 0.25 /u.
The present findings on the mor-
hological characteristics of different iso-
Ites of C. falcatum are in accordance
*,:l*..__1 etU... -1,a. I Akht 1 0.1B






Table 1. Isolates of C. falcatum collected
Philippines


ISOLATE NO.

1
2
3
4
5
6
7
8
9
10
11
12
13







Lewton-Brain 1908; Edgerton, 195(
Martin et. al. 1961) who worked on C
falcatum and described the general cha
racteristics of the fungus. According tb
them the mycelium is hyaline, septate anm
branched; the conidia are hyaline, one
celled and falcate. Abbott (1938) an(
Martin et. al. (1961) stated that the co
nidia measure 16 to 48 a by 4 to 8 a
averaging 25 by 6,u, while conidia measure
14 +. AA- - 1_- L- .-..... L I-- -._ -


from various sugar cane-growing areas of th



PLACE OF COLLECTION

antral Experiment Station, UPCA, Los Baflo
ept. of Plant Pathology, UPCA, Los Bafios
anlubang Sugar Estate, Calamba, Laguna
jzon Experiment Station, Pampanga
da. Luisita, Tarlac
asugbu, Batangas
da. Ibaca, Pres. Roxas, Capiz
edellin, Cebu
rmoc, Leyte
umaguete, Negros Oriental
icolod, Negros Occidental
ictorias, Negros Occidental
indoro National Agricultural School, Orienta
Mindoro






of one type were narrower being 30.2 j
3.179 by 5.4 + 0.465 u while the conidi.
of other type measured 20.21 + 2.12 b3
7.17 + 0.57 ua. However, he (Lo, 1948
was not able to classify the fungus int(
types, as the morphological characteristic
described alone were not considered a
criteria for the classification of the fungus
into different types. The fungus was
grouped into different types on the base,


the setae measure 100 to 200 u. Ahmed There was a
and Shahiahan (1972) worked with siv virulence nf the ienl


marked difference in


isolates and reported that the length of and all the thirteen isolates were grouped
spores varied from 12.0 to 50.0 lu with into two categories such as the isolate
the average of 23.0- 3.0/u. According to No. 1 as highly virulent, and the rest of
them, the setae measure 100 to 200 u the isolates as weakly virulent (Ahmed,
in length. Karet. a. (1964) worked with 1973). It is evident from Table 2 that
three isolates and showed that the ave- there were variations in the ranges as well
rage length and width of the isolates were as in average length of spores and setae
29.7 by 5.1 /u, 28.5 by 5.4 u and 23.2 among different isolates; however, such
by 5.0/u. variations were not correlated with viru-
lence. The highly virulent isolate has thi
In the present study there was same size as a weakly virulent isolate
also quite a variation in the sizes of This finding differs from those of Chona
spores and setae of the different isolates, and Hingorani (1951) who worked with
But this cannot be used as basis for three isolates having different virulence
classifying the fungus. Lo (1948) in and reported that the average spore length
Taiwan, made a similar observation and for highly, intermediate and weakly viru.
reported two types of conidia and setae lent isolates were 27.1 /u, 26.6 iu and
of the fungus. According to him conidia 22 1 ai rp.netivpwi,





'nts in microns of mycelia, spores and setae of different isolates of


I (111.3)


Rn n-i iR n





and A.K.M. SHAHJAHAI
isolates of Colletotrichum falcatui

CHONA, B.L. 1954. Studies on the d
cane varieties to red rot. Indian J.

and M.K. HINGO
isolates of Colletotrichum falcatu,

and D.N. SRIVA
falcatum Went, the causal organism
13:58-65.

EDGERTON, C.W. 1958. Sugar cane an<
Baton Rouge. 301 p.

KAR, K., O.S. RANA AND S.C. GUPF
cumanensis Arx and Mull. Indian

LEWTON-BRAIN, L. 1908. Red rot o
Exp. Sta. Bull. 8: 1-44.

LO, T.T. 1948. Two types of conidia a
Cane Res. 1: 23-24.

MARTIN, J.P., E.V. ABBOTT AND C
world. Vol. 1 Elsevier Publ. Co., ,

RAFAY, S.A. 1950. Another strain of

RIDGWAY, R. 1912. Color standards


1972. Morphology and sporulation of differ
lent. Nucleus 9: 115-116.

ses of sugar cane. Relative resistance of su
r. Sci. 24: 301-315.

NI. 1951. Comparative studies of certair
Vent. Indian Phytopathol. 3:301-315.

AVA. 1960. Variations in Colletotrichun
of red rot of sugar cane. Indian Phytopathol


Diseases. 2nd Ed. Louisiana State Univ. Pre


1964. A new virulent strain of Gloremella
. Cane J. 9:27-31.

igar cane stem. Hawaiian Sug. Planters' Ass


setae of Colletotrichum falcatum Went. J. S


HUGHES. 1961. Sugar cane diseases of
terdam. 542 p.

ualospora tucumanensis. Curr. Sci. 10:25-26

i color nomenclature. Washington D.C. 43






kRBON, NITROGEN AND VITAMIN NUTRITION OF COLLETOTRICHUM
-. yr 1 1.rlI~r Tr~v "." l'~


ry important in understand- water
y and ultimately in helping were
*r_ .. ^ i -_hv qiv


a nnP liftr arrhnhvdratt.!





autoclave without pressure for ten minutes In l) +) mannose, no linear growth was
and then added to the sterilized basal observed but slight growth occurred in
medium. Where solid medium was used, liquid medium.
15 g agar was added to the liquid medium The fungus sporulated readily on
containing the above ingredients. sucrose, soluble starch, glucose, D-man-
All solid and liquid media were nitol, D-galactose, lactose, maltose, D-sor-
adjusted to pH 6.0 with either NaOH bitol, raffinose, levulose and L-arabinose
or HC1. For studies on growth, one loop- (Table 1). Sucrose was the best for spo-
ful of spore suspension with 80 to 100 rulation among the carbon sources. The
spores per microscopic field was placed organism sporulated moderately on inulin,
at the center of the petri plates on solid and poorly on L(--) rhamnose, D(--)
medium, and 0.1 ml of spore suspension mannose and dulcitol.
was used for liquid medium. All liquid The present findings on carbon
medium experiments were performed as utilization are similar with that of Ra.
still-cultures in 125 ml Erlenmeyer flasks makrishnan (1941). In the present test
containing 40 ml of the medium. The D(-) mannose did not support growth
seeded solid or liquid medium was kept while glucose was favourable for growth.
under room conditions (29 + 2 C) for the Since glucose and mannose have the same
period of investigation, empirical formula, the difference in result
Growth measurement was based on may be due to the configuration of sugars.
colony diameter from solid medium and This was also pointed out by Mathuretal.
dry weight from liquid medium. For get- (1950) when they observed differences in
ting dry weight, the mycelial mat from sporulation among hexoses.
each flask was filtered using Whatman's Nitrogen utilization from inorganic
filter paper No. 42 and then they were sources. The fungus grew best on potas.
dried to a constant weight at 80 C, cooled sium nitrate followed by sodium nitrate
in a desiccator, and weighed. and calcium nitrate (Table 2). Although
Sporulation was determined on the linear growth was poor on ammonium
solid medium. Twenty ml of medium carbonate, ammonium phosphate, am-
were poured in each petri plate and after monium sulfate and urea, it was mode.
it solidified, it was flooded with 1.5 ml rate in the liquid medium. Growth was
spore suspension of above concentration. very poor on ammonium molybdate and
The inoculum was distributed uniformly potassium nitrite.
and the plates were then kept under room The results on inorganic nitrogenous
conditions for ten days. A uniform area compounds agree with that of Rama-
from petri plate was taken randomly, and krishnan (1941). It is evident that growth
the agar medium together with the myce- of the fungus was significantly better on
lial mat and spores were placed in a test nitrate nitrogen than on ammonium nit-
tube containing a known volume of dis- rogen. Lilly and Barnett (1951) and
tilled water. The tubes were shaken to Cochrane (1958) mentioned that nitrates
obtain a homogenous snore suspension were excellent sources of nitrogen for






Table 1. Growth and sporulation of C. falcatum on different carbon sources

Colony diameter Dry weight (mg) No. of spores
Carbon source (mm) 7 days after 10 days after per cm210
seeding seedingb days after seed-
ing (lx 105)c

L-Arabinose 58.0 63.0 11.2
L (-/-) Rhamnose 66.8 18.5 3.8
Glucose 79.2 261.5 24.2
Levulose 79.4 176.75 14.3
D (+) Mannose I d 30.0 5.8
D-galactose 55.2 170.0 18.7
Sucrose 82.6 273.0 32.8
Lactose 57.2 83.5 18.1
Maltose 81.6 121.0 17.2
Raffinose 72.6 205.0 16.2
Soluble starch 73.0 195.75 30.1
lnii; "M7 n l0 n on


L--OUIUlUI I VU.0
D-mannitol 69.8

a. Average of five replications.
b. Average of four replications.
c. Average of 24 counting on six rep
d. Very little growth at the beginning






Table 2. Growth and sporulation of C.
nitrogen

Colony diameter D
Nitrogen source (mm) 7 days after 11
seedinga s<


Potassium nitrite 1 d
Potassium nitrate 76.8
Sodium nitrate 79.6
Calcium nitrate 57.4
Ammonium carbonate 52.8
Ammonium molybdate 37.6
Ammonium phosphate 62.8
Ammonium sulfate 45.2
Urea 60.6

a. Average of five replications.
b. Average of four replications.
c. Average of 24 counting on six replicatic
d. Very little growth at the beginning.


I I V. I 1 1V u.,
96.0 19.4



nations.







alcatum on different sources of inorganic


weight (mg) No. of spores
lays after per cm 10
lingb days after seed-
ing (1 x 105)c

d 0.5
J16.25 13.1
78.75 11.7
70.25 8.9
.25.25 7.8
78.75 1.0
33.5 15.2
30.5 3.0
.38.0 1.7



S.





Table 3. Growth and sporulation of C. falcatum on different amino acids

Colony diameter Dry weight (mg) No. of spores per
Amino acid (mm) 6 days after 10 days after cm210 days after
seedseedina seeding seeding (1 x 105)c

Glycine 60.2 169.25 7.8
DL-Alanine 58.0 170.0 7.3
L-Leucine 60.4 141.75 16.7
DL-serine 57.0 123.5 12.9
DL-Threonine 45.4 73.0 17.9
L-Cysteine 83.6 51.0 6.2
DL-Methionine 50.0 93.75 3.6
L-Cystine 73.0 135.0 11.5
L-Asparagine 69.8 133.5 5.3
DL-Aspartic acid 64.8 115.0 3.3
L-Glutamic acid 66.0 125.0 3.9
L-Arginine 60.6 150.0 4.8
L-Histidine 60.6 83.5 9.6
DL-Phenylalanine 47.8 123.0 11.5
DL-Tyrosine 71.2 92.75 14.1
L-Tryptophane 49.2 115.0 8.2

a. Average of five replications.
b. Average of four replications.
c. Average of 24 counting on six replications.



Table 4. Growth and sporulation of C. falcatum on different vitamins

Colony diameter Dry weight (mg) No. of spores per
Vitamin (mm) 7 days after 10 days after cm210 days after
seeding seeding seeding (1 x 105)c

Biotin 87.8 205.0 11.6
Thiamine d 76.2 176.75 9.0
Biotin & Thiamine 85.2 215.0 17.0
Riboflavin e 111.25 3.1
Ascorbic acid 24.8 137.0 1.8
Folic acid 1 e 117.0 2.3
Nicotinic acid 85.2 142.0 1.6

a. Average of five replications.
b. Average of four replications
c. Average of 24 counting on six replications.
d. Added as monohydrochloride.
e. Little growth at the beginning.


DL-tyrosine, DL-serine, DL-phenylalanine
and L-cystine. The fungus sporulated mo-
derately on L-histidine, L-tryptophane,
glycine, DL-alanine, L-cysteine and L-
asparagine, and poorly on L-arginine, DL-
methionine, DL-aspartic acid and L-glu-
tamic acid.
No other comprehensive study on


the effect of amino acids on growth and
sporulation of C. falcatum is available.
Ramakrishnan (1941) worked only with
asparagine and found that it was also a
good source of nitrogen for the growth
of this fungus. However, the findings of
the present study agree in general with
other investigators working with other





fungi (Cochrane, 1958; Leonian and Lilly, with riboflavin, ascorbic acid, folic acid
1940; Pelletier and Keitt, 1954). and nicotinic acid.
Vitamin requirements. Linear Fungi require minute amounts of
growth was almost nil with riboflavin specific organic compounds or vitamins
and folic acid, but in the corresponding for growth (Cochrane, 1958; Fries, 1965;
liquid medium the organism grew appre- Lilly and Barnett, 1951). Some fungi may
ciably (Table 4). Good linear growth but synthsize them; others are dependent on
moderate dry weight was obtained with exogenous supply. In the present study,
nicotinic acid, whereas very small colony the growth and sporulation were highly
diameter with moderate dry weight was stimulated when biotin and thiamine were
observed with ascorbic acid. The fungus added to the medium. No report is availa-
grew best in the treatments with biotin, ble in this respect for C falcatum. How.
and both biotin and thiamine. It also grew ever, many other fungi showed similar
well on the medium with thiamine alone, requirements for these two vitamins. Pyri-
The isolate sporulated best in the cularia oryzae needed thiamine and bio-
treatment where biotin and thiamine were tin for growth and sporulation (Ramos,
added together. It also sporulated well on 1961), while Phycomyces blakesleeanus
media with biotin and thiamine alone. The required thiamine for growth (Lilly and
fungus produced few spores on media Barnett, 1951).


BOURNE, B.A. 1953. Studies on sugar ca
Soc. Sug. Cane Technol. 8: 915-924.

COCHRANE, V.W. 1958. Physiology of fui

FRIES, N. 1965. The chemical environm
organic growth factors, p. 491-523.1
fungi, Vol. 1. Academic Press, New )

HUGHES, C.G. 1953. Red rot disease of
8: 924-936.

LEONIAN, L.H. and V.G. LILLY. 1940.
influencing the growth of some thia

LILLY, V.G. and H.L. BARNETT. 1951.1
Inc., New York. 464. p.

MARTIN, J.P., E.V. ABBOTT and C.G
world. Vol. 1. Elsevier Publ. Co., Ani

MATHUR, R.S., H.L. BARNETT and V.(
lindemuthianum in culture. Phytopa

PELLETIER, R.L. and G.W. KEITT. 195
acids as sources of nitrogen. Amer. J

RAMAKRISHNAN, T.S. 1941. Studies in
on C. falcatum Went. Proc. Indian A

RAMOS, J.S. 1961. Influence of biotin


red rot in the Flonda Everglaaes. rroc. Int.


John Wiley & Sons, Inc., New York. 524 p.

for fungal growth. 3. Vitamins and other
r.C. Ainsworth and A.S. Sussman (eds.) The
c.

gar cane. Proc. Int. Soc. Sug. Cane Technol


dies on the nutrition of fungi. IV. Factors
te requiring fungi. Amer. J. Bot. 27: 18-26.

biology of the fungi. McGraw-Hill Book Co.,


UGHES 1961. Sugar cane diseases of the
rdam. 542 p.

,ILLY. 1950. Sporulation of Colletotrichum
logy 40: 104-114.

Venturia inaequalis (Cke). Winnt. VI. Amino
)t. 41: 362-371.

e genus Colletotrichum. Physiological studied:
.Sci. 14: 395411.

Ad thiamine on growth and sporulation ol
1: 29. (Abstract.)






TRANSLOCATION AND NV
PATHOGI

A.L. h

Senior Plant Pathologist, B

AI

The greening pathogen failed to movie
of the stem of seedlings of Ladu, Ponkan
orange, suggesting that it is translocated in tli
buds into the seedling stocks of Ladu and Po

Translocation and movement of vi
ruses affecting citrus have not been widely
investigated so far. Nour-Eldin and El-
Banna (1965) reported that psorosis virus
can enter the xylem of sweet orange
plants and move downward through th<
ringed area of the stem and that upward
movement takes place much less, if at all.
Price (1968) demonstrated that psorosis
virus, including tristeza virus, failed tc
move upward or downward through the
girdled portion of the stem of citrus
seedlings which led him to conclude thai
both viruses are translocated in the phloem
and not the xylem. Other studies (Price,
1968; Price and Knorr, 1960) have de
monstrated that psorosis and tristeza virus-
es, after entering the plants, move rapid
ly in both directions. Salibe (1965) report-
ed that exocortis virus required 20 day;
to pass from the infected bud into nursery


VEMENT OF THE GREENING
I IN CITRUS

,RTINEZ
au of Plant Industry, Lipa City.

FRACT

pward or downward through the girdled portion
d Szinkom mandarin and Madam Vinous sweet
phloem. The pathogen passed from the inoculum
an in a period of 7 days.

portion of the stems of seedlings in anc
their group.
To determine the movement of th
greening pathogen from infected buds
seedlings of Ladu and Ponkan were inc
culated from the same sources of greenin
pathogen used in the above experiment
Each seedling received 2 inoculum bud
placed at the same height along the sterr
On the 5th day and every day onwar,
until the 16th day, 3 inoculated seedling
of each variety were cut back immediately'
below the inoculum buds. All of the tes
plants were observed for the appearance
of greening symptoms.

RESULTS AND DISCUSSION

Within 10 months following inocu
lation, all girdled plants exhibited lea
abnormalities characteristic of nutrition


UUiAtcu 4at it DurIau 01 nanL inuusiry, test plants maexea positive tor the green
Lipa City. ing pathogen. The pathogen was no1
detected in the non-inoculated portion
MATERIALS AND METHODS of any of the test plants.
Seedlings of Ladu, Ponkan and Szin- No infections occurred when t
kom mandarin and Madam Vinous sweet stem portions bearing the inoculated bud,
orange were used to test the transloca- were removed on the fifth and sixth da3
tion of the pathogen. A ring of bark 3 after the buds were placed (Table 1)
cm wide was removed from the stem of Two of the six plants were infected where
each test seedling. The seedlings were then the buds were left until the seventh day
inoculated from small trees of Szinkom There was 100 per cent infection where
on Calamandarin rootstock that were the inoculum was left in the plants 1
maintained as sources of greening patho- days or longer. In a second experiment
gen. The source trees had been previously inoc m buds were left on plants fo
infected with psylla-transmitted greening five, six andsevn days. There was nc
pathogen. Three inoculum buds were infection in the five and six day group!
inserted below the girdled portion of the but 5 of the 10 plants in the seven da)
stems of one group of seedlings, and group became infected.
thrpP hlldf Wurp nult ahnva th~ mwrllal1 Tlh. n---; -. r.: F;1,.A





by an instantaneous cut through citrus
tissue with contaminated knife or razor
blade, it would be expected that a virus-
bearing bud would not have to be in
contact with healthy tissue even one day
to cause infection.

Unless virus concentration plays
some role in the rate of movement from
inoculum buds into the supporting citrus
seedling, comparative tests with different
citrus viruses under the same conditions
might show that all of them that are
mechanically transmissible will infect at
the same rate. It seems likely that the
determining factor for infection is the
production of cellular union between
the inoculum buds and the supporting
seedling. The rate at which this unior
occurs is no doubt influenced by th<
growth condition of both the inoculurm
buds and the seedlings in which they are


move upward or downward through the
girdled portion of the stems of Ladu
Ponkan and Szinkom mandarin and Ma
dam Vinous sweet orange. Apparently
the pathogen is translocated in the phloen
in the different seedling varieties. Prici
(1968) found that neither psorosis virus
nor tristeza virus moves through thi
girdled portion of the stems of Key limit
and Pineapple sweet orange. According
to him, both psorosis and tristeza viruse:
are translocated mainly through the
phloem, probably through the sieve tubes
The greening pathogen was fount
experimentally to move from the inocu
lum buds to seedlings of Ladu and Ponkal
in 7 days. Price (1968) reported tha
tristeza virus passed from the inoculation
bud to the seedling stock within 8 to 1Y
days and psorosis virus moved into th,
plants within 9 to 17 days. He stated tha
the variation in movement of both viruse


rOOTSTOCK. In views, nuwcvcr, UI LItMe IIu- IIICLIuIll LUuoo [plaCit. I was nUt ucLi-
ings of Garnsey and Jones (1967) and mined whether vascular connections are
others that this virus is easily transmitted established in that period of time.




Table 1. Infection of seedlings from which inoculum buds were removed daily after
five days

Treated Number of days inoculum buds remained in test plants

seedlings 1

5 6 7 8 9 10 11 12 13 14 15 16

Plants Infected

Ladu 0 0 1 1 0 2 2 3 3 3 3 3
Ponkan 0 0 1 0 2 1 2 3 3 3 3 3

1 Three seedlings of each variety in each treatment





LITERATURE CITED

GARNSEY, S.M. and J.W. JONES. 1967. Transmission of exocortis virus with contami
nated budding tools. Plant Dis. Reptr. 51: 410-413.

NOUR-ELDIN, F. and M.T. EL-BANNA 1965. Distribution and movement of psorosi,
and tristeza viruses in citrus plants. p. 272-275. In W.C. Price (ed.), Proc. 3rd Conf
Intern. Organization Citrus Virol. Univ. Florida Press. Gainesville.

PRICE, W.C. 1968. Translocation of tristeza and psorosis viruses. 52-58. In J. F. L.
Childs (ed.), Proc. 4th Conf. Intern. Organization Citrus Virol. Univ. Florida Press






















































of bananas. Chemical means of krads when grown in brain infusion
ng these microbial spoilage was but 9.5 krads when inoculated and
helpful but often produced unde- diated in potato tubers.
esidues on the fruits. In the Philippines, hardly any
any researches have shown that are available on the radio-sensitivit,
i1 cnnilnop roiurl h, rtirrldrd h\, ... .- . '--^ t-^.


peacnes couli De aeiayea ior iu aays under ditterent incubation temperatures


--4 ---







































































uAuLIun agu, iuc AextraLc again, snarun- mcity alter / months of storage. Further
dextrose agar, unpeeled banana decoction study and identification of the remaining
. ... . 1 i r r* .-.- ^ o .


vere immpi


- --------- -- -- ~ ~ a rvrr.Jo.. fl










































irresppIIUlu LU LJi padL1lUgGI i ulat~iu r" ~"'"b" .. -it'"'""' '"-~ "
d described by Wardlaw (1935). The 2 days when exposed to 50 krads with


ter (mm) of fungal isolates incubated for 6


Botryodiplodia GloI
theobromae mI

90.00 7
90.00 5
Agar 89.17 7
86.20 7
^r t\, '1


Sar


/U.U3
78.87


nana Peel Decoction Agar 59.70 72.00





Average colony diameter of Botryodiplodia theobromae after exposure to di
rent doses of gamma radiation and incubation at different temperatures.

30-20C
A C DE
80- B F
G

60-


40-


20-


0-
5 10 15
NUMBER OF DAYS


AA#


60-


40-


20-


0-
5
P


F










) 15 20
dBER OF DAYS





Gloeosporium musarum. The lethal
doses of the fungus were 400, 250 and
200 krads when incubated at 30 + 2 C,
22 + 2 C, 22 2 C, and 12 + 2 C res-
pectively. The colony-forming potential
was significantly reduced when exposed
to 100 krads and incubated at 12 + 2 C
and 22 _- 2 C, while when incubated at
30 2 C, reduction of colony-forming
potential was attained only at a dose of at
least 300 krads (Fig. 2 ). Rate of growth
was significantly suppressed for 5 days
when exposed to 50 krads and incubated
it 30 + 2 C. However, when the pathogen
was exposed to 50 krads and incubated at
12 + 2 C and 22 + 2 C, rate of growth
was suppressed for 14 and 24 days, res-
pectively.
Effect of irradiation on Erwinia
sp. Different shapes and slopes of the
survival curve of the organisms were
sk....." "'".,s... "" '".:.. ..


were identified to be Gloeosporium musa-
rum and Botryodiplodia theobromae while
the pathogenic bacterium was Erwinia sp.
Gloeosporium musarum grew well on
potato dextrose agar while Botryodiplo-
dia theobromae showed good growth on
Czapek's Agar. The lethal doses of Gloeos-
porium musarum, and Botryodiplodia
theobromae when incubated at 30 + 2 C
were 400 krads and 500 krads respectively
The lethal doses decreased as the incuba-
tion temperatures decreased. Suppression
of growth rate increased up to 37 days
when the microorganisms were incubated
at 12 2 C using the same radiation
dosages. A similar case was observed on
Gloeosporium musarum where its growth
rate was significantly depressed for 5
days when exposed to 50 krads and incu-
bated at 30 2 C while when incubated
at 12 2 C, growth suppression signifi-
cantly increased to 24 days. In the case


md dextrose salt agar exhibited an expo- it was grown. in ts experiment te u10
value of Erwinia sp. varied from 5.5 to
nential survival curve with increasing dose value of Erwinia sp. varied from 5.5 to
7.55 krads.
of radiation (Fig. 3). IH-wever, a sig- From the over-all results obtained,
moidal curve was exhibited when grown Frm te resus
on trypticase soy agar. The D10 value inhibition of growth of the pathogen is
(dose required for a 10 fold reduction of possible even when a dose as low as 50
viable cells of the organism) was 7.75 krads and incubation temperature of
krads when grown on trypticase soy agar 30 2 C were used. Ths growth ihibi-
and 7.25 and 5.5 krads when grown in tion could probably be increased provided
nutrient agar and dextrose salt agar the incubation temperature is decreased.
respectively. The inhibition of growth of the pathogen
in the banana substrate may be possible
at low temperature, 12 + 2 C, assuming
DISCUSSION that the organism might exhibit higher
resistance in the banana fruit. Therefore,
Two pathogenic fungi and one bac- further studies must be done on the
trial species were isolated and identified Lacatan itself to determine the radiation
to cause rotting on Lacatan. These fungi and low temperature tolerance of the fruit.





Figure 2 Average colony diameter of Gloeosporium musarum after exposure to
different doses of gamma radiation and incubation at different temperatures.


30 4- 20C


10
NUMBER OF DAYS


5 10 15 20 25
NUMBER OF DAYS


5 10 15 20
NUMBER OF DAYS


Control

50 Krad

100 Krad

150 Krad

200 Krad

250 Krad

300 Krad

350 Krad

400 Krad

450 Krad

No growth


A

B

C

D

E

F

G

H
N.G*

N.G


22 4- 20C





Figure 3. Survival curve of Erwinia sp. when grown on various nutrient med
incubated at 30 C.


100
TRYPTICASE SOY AGAR-_-
NUTRIENT AGAR___-- ...[]
DEXTROSE SALT AGAR.__- -




10





LA I UXJlVRk 1 I I IJC


AGATI, J.A. 1922. Banana stem and fruit rot. Philippine Agriculturist 10:411-422.

ALEXOPOLOUS, C. 1962. Introductory Mycology. 2nd ed. John Wiley and Sons Inc.
New York. 613 p.

BARNETT, H.L. 1960. Illustrated genera of imperfect fungi. 2nd ed. Burgess Publ. Co.
Minneapolis. 225 p.

BEREHA, L., and G.B. RAMSEY. 1959. Effect of gamma radiation on brown rot and
Rhizopus rot of peaches and causal organism. Phytopathology 49:354-356.

M.A. SMITH, and W.P. WRIGHT. 1959. Factors influencing
the use of gamma radiation to control decay of lemons and oranges. Phytopatho-
logy 49:91-96.

__, M.A. SMITH, and W.P. WRIGHT. 1960. Gamma radiation dose response
of some decay pathogen. Phytopathology 50:474-476.

DDI EV DC Er \ I' ~ TDDAV ..,A A DADVED ULITC'rPUIr2.TL 1(AO ....


LxKttIN, Li.K. ana K.U. iUUs. 1 Yi. Fungi
Phytopathology 53:271-275.

HIRAI, T. 1939. Diseases of banana in train
Japan VIII cited in Rev. Appl. Mycol.

MEREDITH, D.S. 1960. Studies on the
causing storage rots in Jamaican banai

3ARAVACOS, G.D., L.P. HATZIPETROU,
of gamma radiation of some fruit
Quart. 3(1-2):A6-A9.

SLABYS, B.M., and A.M. DOLLAR. 196&
and Erwinia carotovora to Cobalt 60
A22-A24-

WARDLAW, C.W. 1935. Diseases of Banan;


)UnIdiCU WI1i uluWII IUL UI UUAXU uianalJu.


)ort from Formosa. Ann. Phytopath. Soc.
3:4041.

loeosporium nusarum. Cke. and Massee
i. Ann. Appl. Bio. 48:279-528.

id E. GEORGIADOU. 1962. Lethal doses
oilage microorganisms. Food Irradiation


Radiation survival of Erwinia atroseptica
mma rays. Food Irradiation Quart. 5(1-2)


Macmillan London. 615 p.





VARIABILITY OF PHILIPPINE I
GLOEOSPORIOIDi

LEOVICA M. ELA ani

Former Undergraduate Student and i
culture, U.P.L.B., College, Laguna.
Project supported by Natural Science I

ABS

Six isolates of C. gloeosporioides wei
physiological variability.
The isolates varied in the degree of t
most virulent; M-51 moderately virulent, and
media the isolates showed variable charact
growth, size of spores and density of sporulal
was shown by their differential reaction to ter
It was concluded and proven that C.g,
of a number of strains.


Colletotrichum gloeosporioides
Penz., the causal organism of a
plant disease called anthracnose, is one
of the most widely distributed plant
pathogenic fungi. Several host plants have
been recorded for the organism but in
most cases it attacks tropical fruits like
mango, papaya, citrus, and avocado. Be-
sides attacking the fruits, the fungus also
infects the blossoms, young leaves and
tender tips, twigs or branches, causing
blossom blights, leaf spots, withertip and
die-back of the twigs and branches.
Burger (1921) and Garcia (1949)
observed that C. gloeosporioides. is a
polymorphic species made up of a num-
ber of strains. This was confirmed by
Von Arx (1957). The strains have dis-
tinct cultural characteristics when grown
on artificial media. Ocfemia and Agati
(1926) also noted certain minor variations
in morphological characters and produc-
tion of conidia of C. gloeosporioides
The same variations were reported by
Shear and Wood (1913) on Glomerella
cingulata (Stonem.) Von Schrenk and
Spaulding, the perfect stage of C. gloeos-
porioides. Moreover, species of an organ-
ism often manifest variations in their
reaction to chemicals, light and temper-
ature and on pathogenicity on closely
related host plants (Melhus and Kent,
1939). Knowledge of the extent of
variability is imperative to sound
taxonomy.
This study was conducted in the
laboratory of the Department of Plant


LATES OF COLLETOTRICHUM
FROM FRUITS

FRICITA H. QUIMIO

stant Professor respectively, College of Agri-

arch Center.

LCT

tudied for their cultural, morphological and

virulence, C-1l, P-12, P-16 and M-55 being
slightly virulent. Grown on five different agar
tics, notably color and amount of mycelial
. Further evidence of variability of the isolates
rature and light.
sporioidesis a polymorphic organism made up



Pathology, UPLB, from March to Sep-
tember, 1973 to study the extent of
variability of the different isolates of
C. gloeosporioides from mango, citrus and
papaya.

MATERIALS AND METHODS

Isolation. Colletotrichum gloeospo-
rioides was isolated from naturally infec-
ted mango, citrus and papaya fruits using
tissue planting technique. Table 1 shows
the sources of the isolates used in the
study. The different isolates obtained were
reinoculated on their respective fruit host
for test of virulence. They were maintain-
ed in potato dextrose agar (PDA) for
subsequent studies of their variability.
Preparation of inoculum and ino-
culation procedures. Single spores were
used to inoculate plates for the study of
variation induced by medium. One loop-
ful of a conidial suspension from a 4-day-
old culture of each of the different iso-
lates was streaked in a thinly plated water
medium. The plates were incubated for
12 hr at ordinary room temperature
(27-30 C). With the use of the low power
objective of the microscope, the location
of each single germinating conidium was
marked at the bottom of the petri plates
with a pentel dot. A slightly flattened
needle was used to pick up and transfer,
aseptically, single germinating conidium
in small bit of agar to sterile petri plates
containing the test media.
For the study of variations induced





lmuute Juurxes UJ lsuur is uj L. gloeosponolaes useu in tri prertsaeilm y uaru triuI
virulence


ISOLATE HOST VIRULENCEa LOCALITY

C-5 Citrus X Bulacan
C-11 Citrus XXX Batangas
P-12 Papaya XXX Laguna
P-16 Papaya XXX Pangasinan
M-51 Mango XX Laguna
M-55 Mango XXX Zambales


aDisease Index (3 days after inoculation):



X ------------- Slightly virulent
XX ---------- -- Moderately virulent
XXX ------------- Severely virulent







by temperature and light, mycelial plugs Variations induced by temperature.
(6-mm diameter) obtained from the outer Each of the 6 isolates were inoculated
margin of 3-4 day-old cultures with a on YEA and incubated at 10, 15, 20, 25,
sterile cork borer were used as inoculum. 30, and 35 C without light for 6 days.
Photographs of the culture plates and
Variations induced by medium. sporulation were likewise taken as des-
The different isolates of C. ilneosnorinides .. __


act Agar (YEA


pared according to Toro (1922) while the
rest were followed from Tuite (1969).
Twenty ml of each of the sterile media
were plated, congealed, and inoculated as
previously described. The inoculated plates
were incubated for 6 days in General Elec-
tric Precision Model Incubator adjusted
to 30 C without light. Photographs of the
culture plates and photomicrographs of
the conidia were taken after the incubation
period. To determine sporulation, a 6-mm
diameter mycelial plug cut with a cork
borer was placed in a test tube containing
2 ml 1: 1000 Hgl solution and shaken
-.-* _.. ...i 1 -_ .. .. --*i i * ."


ness (CLU), alternating light and darkness
(ALD), and ordinary room conditions
(RC). Light was provided with 14-watt
cool white fluorescent bulbs. Cultures
subjected to continuous darkness were
wrapped with aluminum foil. Temperature
of incubation was 25 C except that of
room conditions. Results were recorded
as in temperature studies.

RESULTS

VIRULENCE TESTS

Pathogenicity or virulence test





ES varnc


variations inuce Dny medium. I le oI Lne uliereiit iaU1 dias LU u1nicint L ln-
6 isolates showed variable characteristics perature levels. All the isolates showed
(color and amount of aerial mycelium, best growth at 30 C. At 10 C, isolates
formation of spore masses, and colony C-5, P-12, P-16 and M-55 did not show
diameter) on both natural and synthetic any growth whereas isolates C-11 and M-
media tested (Table II). The color of the 51 showed slight growth. Isolates C-11,
colonies on the surface of the medium P-12 and M-55 showed better growth in
ranged from dirty white to salmon orange terms of colony diameter at 35 C than
in YEA; white to salmon orange in SDA, at 15 C while C-5 and M-51 had reverse
CZA, and V-8; and white to dark slate reaction. The same colony diameter of
gray in CM. Colony borders ranged from P-16 resulted from 15 C and 35 C expo-
whitish to pinkish. Nosetae were produced sure, although the latter temperature
on all media. duced much denser growth. All of the
isolates showed best growth at 30 C
The spore sizes were affected by except M-51 which grew better at 25 C
the media and exhibited variability, than at 30 C.
Average sizes of conidia were 11.86 x Striking differences can also be seen
4.11 1u; 13.64 x 4.13 u; 11.47 x 4.09 u; on the amount of sporulation of cultures
12.80 x 3.88 /u and 13.46 x 3.71 lu on exposed to different temperatures. Figure
CZA, CM, SDA, V-8, and YEA, respect- 4 shows the different reactions to sporu-
ively. The shape of the snores remained lation exhibited by the isolates to the






P-16 Very abundant, pale olive gray to 64
pallide mouse gray with whitish
border mycelial growth

M-51 Very abundant, dirty white to salmon 57
orange mycelium radiating from the
plug

M-55 Fairly abundant, olive gray with 65
whitish border mycelial growth


SDA C-5 Abundant, dull gray with whitish 64
border mycelial growth

C-ll Fairly abundant, light salmon orange 72
with whitish border mycelial growth

P-12 Fairly abundant, salmon orange with 57
pinkish border mycelial growth;
zonated

P-16 Very abundant, white fluffy 67
mycelial growth

M-51 Abundant, dirty white mycelial 52
growth

M-55 Abundant, gray mycelial growth 72


CZA C-5 Abundant, pale purplish gray mycelial 15
growth

C-ll Fairly abundant, gray with whitish 66
border mycelial growth

P-12 Abundant, salmon orange with 63
pinkish border mycelial growth

P-16 Very abundant, gray mycelial growth 67

M-51 Very abundant, fluffy white 48
mycelial growth

M-55 Very abundant, gray mycelial growth 67
CM C-5 Fairly abundant, slate gray 72
mycelial growth; zonated
C-11 Very abundant, dark slate gray 78
mycelial growth
P-12 Fairly abundant, salmon orange 65
mycelial growth
P-16 Very abundant, gray to fluffy 73
mycelial growth
P-51 Very abundant, gray to white 66
mycelial growth
M-55 Abundant, light slate gray with 65
whitish border mycelial growth






V-8 C-5 Sparse, slate gray with whitish 62
border mycelial growth

C-11 Sparse, dirty white mycelial growth 63

P-12 Sparse salmon orange mycelial 55
growth

P-16 Sparse, white mycelial growth 66

M-51 Very abundant, white mycelium 48
radiating from the plug

M-55 Fairly abundant, white mycelium 64
radiating from the plug



Colors were described according to Ridgway's color standard and color nomenclature
(1921).

After 6 days incubation at 30 C without light



ability at 20 C, 25 C and 30 C. A more there are differences in color and rate of
ble range of spore sizes was observed at mycelial growth. These may be attributed
C and 35 C. to differences in nutrients derived from
Variation induced by light condi- the different culture media used. This
ns. Variations in growth characteristics result is in harmony with the findings of
.1_ _. 3 J l^_- -I* - -.. I 1_, - _..1_ i' . ...- _- _T lr'1 \ '. i -_ . .. ... i I ... t


LU SIII1II5 p pUll, WIUllt U t 4O11U ll Ul oall ,
and dirty white to orange pink under
RC, CL, CD, and ALD, respectively. In
general, rate of growth was not affected
by the different light conditions. Sporu-
lation occurred in all cases although it was
enhanced by exposure to light and alter-
nating light and darkness with the except-
ion of one isolate which gave best sporula-
tion at CD (Fig. 6). Isolates C-5, C-11,
P-12 and M-55 sporulated best at CL
while P-16 gave best sporulation at RC.
Spore sizes were not affected by the
different light conditions.

DISCUSSIONS

A critical analysis of the C gloeos-
porioides isolates from different hosts has
shown variability not only in their patho-
genicity but also in their cultural and
norphological features such as color and
unount of mycelial growth, spore sizes
md shapes, formation of spore masses
md spore density when grown on different
nedia.
It is evident from Table II that


LrailsiernIg cultures iroun un type Uo
medium to another.
It is also apparent from the same
table that no two isolates from the same
host or isolates from different hosts have
a very close resemblance to each other
in terms of colony characteristics. How-
ever, variability in colony characteristics
alone as mentioned by Johnson (1952)
in his studies on Septoria avenue is not a
convincing proof of variability since there
is no range limit of variation set for the
organism. This may hold true with
C. gloeosporioides. Moreover, microorgan-
isms are liable to undergo changes through
several subculturings. These changes, ac-
cording to Burger (1921), Garcia (1949)
and Von Arx (1957) are plain proofs of
mutation thus, making C gloeosporioides
a polymorphic species.
The shape and size of conidium form
important taxonomic characters. The
spore sizes of C gloeosporioides vary to
some extent and as shown in the present
study, are influenced by the substrate
and environment. Yet its significance in
specific differentiation cannot be ignored.





Spore shapes are more or less constant
among the isolates.
Aside from cultural and morpho
logical variations exhibited by the differ
ent isolates of C. gloeosporioides, then
exist also some physiological difference
between them as shown in the temper
ature and light studies. These results agree
in general, with previous reports on th
physiological differences of isolates o
Fungi Imperfecti (Voorhees, 1942). Stri
king variability is shown in their sporu
lation as affected by the culture media
temperature and light. This is not enough
justification however, of variability unles
other variables are taken into consider
ation. Especially significant is the absence
of setae, a definite feature of Colletotn
chum species, on all the media tested
Ramakrishnan (1946) stated that thi
failure of such structure to form in cul
ture is a proof that their growth is some





LITERA


BRIERLEY, W.B. 1929. Variation in
2:1629-1634.

BURGER, O.F. 1921. Variations in Coll
737.

CHRISTENSEN, C.M., E.C. STAKMAN
phytopathogenic fungi. Ann. Rev. I

GARCIA, L.A. 1949. Anthracnose of the
Rico 33:2743.

JOHNSON, T. 1952. Cultural variability in

MACLEOD, D.M. 1954. Natural and cult
N.Y. Acad. Sci. Ann. 60:58-70

MELHUS, T.E. and G.C. KENT. 1939. I
N.Y. 1st ed. 493 pp.

OCFEMIA, G.O. and J.A. AGATI. 1921
and upo in the Philippine Islands. P

RAMAKRISHNAN, T.S. 1946. Studies i
Sci. 25:15-26.

RIDGWAY, R. 1912. Color standard and c
D.C. 43 p. 53 pl.


times suppressed oy me environment ant
the medium and, consequently, is not o
much taxonomic value.

Evidence presented herein indicate!
the range and the kind of variation exist
ing in C gloeosporioides. Variation is
fairly common in phytopathogenic fun
gi (Christensen et a.. 1947) as well ai
in bacteria (Brierley, 1929). Knowledg(
of the extent of variation is particularly)
important in the taxonomy of the organ
ism because the value of any taxonomi
system depends to a considerable exten
upon the validity of criteria chosen t(
separate groups. Useful systems mus
take into account the generic variation
in the organism concerned, if distinction
without difference are to be avoided
A knowledge of such variation as state
by Christensen et al. (1947) is basic to
sound taxonomy.





IRE CITED


ngi and Bacteria. Proc. Int. Cong. Pit. Sc


trichum gloeosporioides. J. Agr. Res. 20:72:


.nd J.J. CHRISTENSEN. 1947. Variation
robiol. 1:61-84.

nonaceae in Puerto Rico. J. Agr. Univ. Puerl


ptoriaavenae Frank. Can. J. Bot. 30:318-331

I variation in Entomogenous Fungi Imperfeci


ments of Plant Pathology. McMillan and Cc


The cause of anthracnose of avocado, mane
ippine Agriculturist 14:199-216.

he genus Colletotrichum. Proc. Indian Aca


or nomenclature. A. Hoen and Co., Washingtc














totrichum Cda
















































































































100 S C C




YEA SA CZA CM V-8

Culture Media
























































ig. 3. Mycelial growth of C. gloeosporio
temperature levels for 6 days withoi
000



400
aP
M51




I 200 r

100

I Io Isc
0 10C 15C
.. . .

a, 1 /lrnlr.~n.n'nnnlln ,rA C


Sisolates on YEA exposed at the differed
ght.













25C 30C 35C
eflaubo ion

eosDorioides isolates on different temerm


r


cr













































Fig. 5. Mycelial growth of C. gloeosp
conditions.










400



200
1W


aides,isolates on YEA subjected to different








P12
nmmimm Pl6
M51
[2222255 S


Fig. 6. Graph showing sporulation of 6 C. gloeosporioides isolates on different
conditions.






'ERGILLUS FLAVUS INFECTION OF PRE-HARVEST CORN, DRYING CORN
AND STORED CORN IN THE PHILIPPINES

LINA L. ILAG

Assistant Professor, Department of Plant Pathology, College of Agriculture, University
of the Philippines at Los Baihos, College, Laguna
This study was supported by U.P. FAR 024-73 and Corn 3.3 projects.

ABSTRACT

A survey for the presence of the Aspergillus flavus group in mature pre-harvest corn
;rains, in drying corn after harvest, and in stored corn showed that this group was present
poradically and in relatively low percentages of infection in field corn in six of the seven
>rovinces surveyed. Samples from Cebu corn fields were highly infected. Newly harvested corn
:hat was being sun-dried, and corn in storage had high percentages of infection.
The degree of infection was high in corn samples that were dried and/or stored as shelled
(ernels, low in corn on the cob without husks, and minimal in corn on the cob with intact
iusks. Infection was directly related to the amount of A. flavus inoculum in the air which was
least in corn fields, higher in the drying area, and highest in warehouses.
Inoculation experiments with pure cultures of A. flavus and Link and Fries A. parasiticus
Speare showed that these aflatoxin-forming fungi may infect corn in the field specially if the
ears are injured prior to inoculation.



Members of the Aspergillus flavus This study was conducted to deter-
>up, specifically A. flavus Link ex Fries mine with more clarity field infection






ken in 5.250/o NaCOl for 3 min, rinsed, uninoculated control ears were infect
and blotted dry in sterile neswpaper The inoculum here may have come fr(
circles and plates in malt salt agar (MSA) air-borne conidia. There appeared to
containing 7.50/o NaCI. The plaets were no distinct differences in the severity
observed 4-5 days later for the growth infection during the dry and wet cr
of A. flavus or A. parasiticus. seasons.
Some pricked ears were infecl
Natural infection of corn with A. not only with A. flavus and/or A. par
flavus group A 2-year (1971-1973) ticus, but also with Fusarium spec
survey was made to determine the extent Kernels that harbored the Fusarium wi
of natural infection of corn with the usually free of the aflatoxin-forming fur
aflatoxin-forming fungi. It covered select- Table 2 shows the percentage na
ed provinces in Southern and Central ral infection of pre-harvest corn by
Luzon, the Bicol region, Visayas and A. flavus group in seven provinces.
Mindanao. The survey included field corn, lowest (1.660/o) infection was in Bula<
drying corn, and stored corn. In this paper and the highest (51.500/o) in Cebu.


5I-lit t -ldll ,lVII I..ai L- .1 l .l.l.Illi
corn is corn collected while being sun
dried and harvested not more than 7 day:
previous to time of collection; store
corn is corn that had been dried anc
stored for various period of time.
Sampling for field corn was b:
random picking of 5-10 mature ears fron
each field. Approximately 500 grams pe
sample were obtained in the case of shelled
corn that were being sun-dried as well a
shelled corn in storage. For drying an
stored corn that were still on the cob
5-10 ears per sample were taken at ran
dom. The samples were tested for th
presence of the A. flavus group by takir
at random 100 kernels from each sample
These were treated with 5.250/o NaOCI
rinsed, blotted dry, and plated in MSA as
above. The plates were observed 4-5 day
later for fungal growth.

At the time of sample collection
plates with MSA were exposed for
min in the field, drying or storage are
as the case may be. This was done t(
determine the presence and relative abun
dance of A. flavus in the air around thi
area of sampling. All plates were observed
4-5 days later for the presence of A
flavus group.

RESULTS AND DISCUSSION
Standing mature corn ears inocula
ted in the field with A. flavus and A
parasiticus got infected two weeks afte
inoculation (Table I). Injured or prickei
ears were more susceptible to invasion
by the fungi although intact or unpricke
ears were also infected to a lesser degree
A small percentage of the kernels fron


ly glUWii 111 uuiaalii anlu 1t1i it/p i.
usually harvested green and consumer
as boiled corn on the cob. Cebu has lonj
been a corn-growing province where the
crop is planted year after year and harvest
ed when fully mature for milling as the
staple food of the people.
Generally, pre-harvest corn was in
fected with A. flavus group to a lesser
degree compared to drying and store
corn (Table 3). Pre-harvest corn had
high percentage (86.70/o) of sample!
that were free of aflatoxin-forming fun
gi. Most of the drying corn and store
corn samples were highly infected respect
ively with 58.12 and 63.3 average per cenl
kernels yielding A. flavus Only 2.20/o ol
the drying corn samples were not infected
These observations indicate that infect-
ion sets in mainly while corn is being
dried after harvest. Nevertheless, even ii
pre-harvest infection is much less thar
post-harvest infection the need to control
the former is essential in view of the
highly toxic nature of the toxin elaborate
by A. flavus. Nearly 250/o of the stored
corn sampled were free ofA. flavus at the
time of sampling. It is possible for these
kernels to have once harbored A. flavu.
and the fungus subsequently killed b)
lack of moisture, chemicals or othe:
causes.
Aerial sampling for A. flavus reveal
ed that the fungus was rarely present ii
the field air but was common in drying
and storage areas. Plates exposed in corr
fields had an average of 1.5 colonies,
plate. Plates exposed in drying and sto
rage areas had an average of 9.5 and 102.5
colonies/plate, respectively. Thus th<
spore load of the air was directly related








IK IA IMaNI ury season crop yvet season crop

Inoculated with A. flavus
Pricked 35.0 47.0
Not Pricked 9.0 12.0
Inoculated with A. parasiticus
Pricked 31.0 27.0
Not Pricked 11.0 16.0
Control
Pricked 4.0 3.0
Not Pricked 1.0 1.0


Table I. Infection of pre-harvest corn inoculated


with A. tlavus and A. parasiticus


PROVINCE NO. OF KERNEL!


Batangas 900
Bulacan 300
Camarines Sur 200
Cebu 300
Davao 300
Laguna 1000
South Cotabato 600





Table 3. Natural infection of

NO. OF PERCENT S
SAMPLES WITHO1
A. FLA P

ield corn 39 86.70
trying corn 12 2.20
tored corn 27 23.10

1100 kernels from each sample were tested 1


PERCENT KERNELS
ESTED YIELDING
A. FLA VUS

4.06
1.66
4.00
51.50
15.66
6.20
5.17





n with A. flavus group

IPLES AVERAGE PERCENT
KERNELS WITH
> A. FLA VUS 1

5.25
58.12
63.30

A. flavus group





to degree of A. flavus infection which
was least in the field, higher in the drying
areas and highest in warehouses (Table 3).
These findings are similar to those of
Tuite and Christensen (1957) who noted
that the aerial inoculum of A. flavus and
other storage fungi was uncommon in the
fields (wheat), moderately abundant in
country elevators where freshly harvested
grains were being handled, and much
more abundant in terminal elevator. The
degree of infection in corn that was being
sun-dried under various conditions was
examined (Table 4). Corn being dried
in areas adjacent to warehouses were more
highly infected than corn dried in the
open near private homes. This was true
for shelled corn as well as corn being
dried on the cob. As expected, the A.
flavus spore load of the air in warehouses
and adjacent areas was much higher than
that in drying areas near private houses


(Table 4).
Corn dried as shelled grains yielded
a higher percentage of A. flavus compared
to corn dried on the cob (Table 4).
Practically all shelled kernels from ware-
houses were infected with the fungus.
The corn husk appears to afford
protection from infection. Unhusked corn
dried along the sides of houses yielded
the least percentage (5.5) of infected
grains (Table 4). Similarly, corn with in-
tact husks stored along the sides or under
private houses contained less grains with
A. flavus (4.330/o infection) than shelled
corn stored in warehouses (81.750/o in-
fection). The spore load in the air may
have been a contributory factor here.
It was further observed that corn ears
from the field that were infested with
weevils or damaged by rats usually had
kernels that harbored A. flavus.


Table 4. Per cent A. flavusgroup infection in corn being sun-dried under various conditions

COMMODITY NO. OF GRAINS PERCENT INFECTION NO. OF
TESTED A. FLA VUS
Range Average COLONIES
PLATE a

Shelled corn, drying in
the open, near pri-
vate homes 700 0- 100 82.0 7.0
Shelled corn, drying in
areas adjacent ot
warehouse 300 98 -100 99.0 21.0
Corn on cob, without
husks, drying near
private houses 300 0- 29 19.5 3.0
Corn on cob, without
husks, drying adja-
cent to warehouse 300 0- 100 50.0 15.0
Corn on cob, with husks
intact, drying along
sides of private homes 400 0 9 5.5 1.25

a Plates of malt salt agar were exposed for 5 minutes in the area of sample collection.
Each figure represents the average number of colonies of A. flavus that appeared in
6 or more plates 4-5 days after exposure.


LITERATURE CITED

CHRISTENSEN, C.M. and H.H. KAUFMANN. 1969. Grain storage, the role of fungi in
quality loss. University of Minnesota Press, Minneapolis. 153 p.





CI.rNK, U.L. ana IN.U. UAVIS. iYO. Anlatoxin rormation oy Aspergtuus jIavus. In
L.A. Goldblatt, ed. Aflatoxin, scientific background, control and implications.
Academic Press, N.Y.

kNDIALAN, I.M. and L.L. ILAG. 1973. Aflatoxin production of Aspergillus flavus
Link isolates from rough rice, corn, soybean, peanut and copra. Philippine Agricul-
turist 57:254-263.

APER, K.B. and D.I. FENNELL. 1965. The genus Aspergillus. The Williams and
Wilkins Co., Baltimore. 686 p.

ANTAMARIA, P.A., A.C. PIZARRO and C.R. JACKSON. 1972. Aflatoxin contamina-
tion on raw agricultural crops and their by-products in the Philippines. Phil. Phy-
topathology 8:12-20.

JITE, J. 1961. Fungi isolated from unstored corn seed in Indiana in 1956-58. Pit. Dis.
Reptr. 45:212-215.

and C.M. CHRISTENSEN. 1957. Grain storage studies, XXIII. Time of
invasion of wheat by various species of Aspergillus responsible for deterioration
of stored grain and source of inoculum for these fungi. Phytopathology 47:265-268.





INOCULATION OF PREGERMINATED CORN SEEDS, CONIDIAL PRODUCTION
DURING THE DAY AND THE USE OF TWEEN 80 IN INOCULATING PHILIP-
PINE CORN DOWNY MILDEW

F.C. BARRED and O.R. EXCONDE

Portion of a thesis submitted by the senior author in partial fulfillment of the require-
ments for graduation with the degree of Master of Science from the College of Agriculture
U.P. Los Bafios.
Instructor and Associate Professor respectively, Department of Plant Pathology, UPCA.
The authors wish to thank the help of Dr. Otto E. Schultz, Associate Professor, Cornell
University for his valuable help and suggestions in the early part of this study.

ABSTRACT
Germinated seeds of PH-801 were readily infected when inoculated before olantine than


to a moist condition inside the growth chamber
of 23.3 and 970/o, respectively. High percent
were inoculated on a susceptible corn variety
also gave high percentage of infection.



Conventional time of inoculation of
corn downy mildew pathogen is general-
ly done early in the morning. Recent find-
ings, however, tend to indicate that this
can be modified in such a way that it can
be done during the day rather than in the
unholy hours in the morning. Although
earlier attempts to induce conidial pro-
duction of S. philippinensis have failed,
Weston (1920), Exconde et al, (1967)
obtained conidial production during the
day by wetting infected plants conti-
nuously inside a dark chamber at a temper-
ature of 19-24 C. In Taiwan, the same
findings on S. sacchari were reported by
Matsumoto and Yang (1964). These work-
ers demonstrated that sporulation occur-
red during the day provided that infected
leaves were kept in a moist chamber. They
postulated that the nocturnal habit of
the fungus is intimately associated with
relative humidity at night but not with
darkness. Similarly, Sun (1970) showed
that corn plants infected also with S.
sacchari was successfully induced to pro-
duce conidia in darkness. He observed
that 9 to 11 hours at high humidity were
required for the full development of
conidiophores and conidia of the fungus
at 24 + 1 C.
On the other hand, Ullstrup (1970)
suggested that the testing of various
adjuvants such as detergent or nutritive
substances may improve the effectiveness
of artificialiv annlied innoniilm He added


iith a night temperature and relative humidity
;e infection was obtained when these conidia
Conidia suspended in 500 ppm. of Tween 80




that a search for materials and methods
best adapted to production of inoculum
would be helpful.

This study was conducted to deve-
lop a more convenient method of inocu-
lating the Philippine corn downy mildew.

MATERIALS AND METHODS

Inoculation at different stages of
germination. Corn seeds of PH-801 were
pre-germinated for 72, 48, 24 and 12
hours. Before planting, forty pre-germina-
ted seeds from each group were sprayed
with 4 ml inoculum suspension contain-
ing about 50,000 conidia per ml. After
4 hours, each treatment was planted in
two-20 x 4 x 5 inches metal seedflats
with 20 pre-germinated seeds per seedflat.
These were incubated in the growth
chambers with a temperature relative
humidity of 30 C/750/o and 23.3 C/970/o
for the day and night conditions, respect-
ively. Percentage infection based on sys-
temic symptoms were compared after
12 days.
Induced sporulation during the day
and inoculation in the evening. Two-
downy-mildew infected leaves of PH-801,
approximately of the same age were
collected from the source area at 6, 8, 10
11 in the morning and at 12:00 high noon.
The leaves were cut into 5 cm long from
the lower, middle, and top portion of the





infected area. They were washed and
placed in Petri dishes lined with moist
tissue paper and then exposed to a prog-
rammed night temperature-relative humi-
dity of 23.3 C/970/o. Hourly examina-
tions for conidial production were made
starting at 4:00 p.m. to 10:00 p.m. Once
the conidia had been observed at the
mature stage, sample of 5 cm2 of the
infected leaf with conidia from each treat-
ment were fixed in 2 ml Farmer's solution
for conidial counting using a haemacyto-
meter. The earliest time of collection
which showed abundant conidial product-
ion was used as the basis for the gathering
of inoculum material the next morning.
Tween 80 as a wetting agent for
inoculum suspension. To counteract the
waxy surfaces of corn seedling leaves
which causes less rentention of inoculum
spray without rubbing them gently by
hand, a 500 ppm solution of Tween 80
in distilled water was used as the suspen-
ding medium of the conidia. This con-
centration was found to be the lowest
concentration of Tween 80 that could
affect wetting of corn leaves without
causing any effect on the germination of
conidia. The resulting suspension was
inoculated on forty-3-day old PH 801
corn seedlings. Check plants inoculated
without Tween 80 was provided as con-
trol. Both groups of inoculated plants
were covered with plastic bags and kept
under laboratory conditions. After 4
hours, covers were removed and the
plants were incubated under field condi-
tions for 12 days.

RESULTS

Inoculation at different stages of
germination. Sclerospora philippinensis
failed to cause infection on non-gemina-
ted seeds. On the other hand, 950/o in-
fection was obtained on seeds pre-germi-
nated for 48 and 72 hours with the form-
er having an average appearance of system-
ic symptoms of 7.8 days while the latter
for 7.1 days (Table I). The results indicate
that inoculation can be modified by ino-
culating germinated corn seeds before
planting them, thus saving time in waiting
for at least 3 days after emergence as
previously recommended by the authors
(Barredo and Exconde, 1972). One pro-
blem however, is that corn seeds do not
germinate at the same time hence selection


becomes necessary before inoculation.
Induced conidial production during
the day and inoculation at eight in the
evening. Conidial production and length
of incubation were influenced by day
light exposure prior to sporulation (Ta-
ble 2). Results of inoculation from coni-
dia produced during the day under arti-
ficial night condition in the growth cham-
ber showed 900/o systemic infection after
12 days.
Failure to induce conidial product-
ion during the day seems to be due to
the inability of most workers to consider
the length of day light exposure of the
source plants before inducing sporulation.
Weston (1923) tried but failed to obtain
conidia of S. philippinensis during the
day although he provided a layer of moist-
ure on the surface of infected plants.
Exconde et al., (1967) reported that coni-
dial production occurred during the day
by wetting infected plants continuously
inside a dark chamber at a temperature
of 19 to 24 C. The amount of conidia,
however, was not comparable to what was
produced under night condition. In the
case of S. sacchari, Matsumoto and Yang
(1964) demonstrated that sporulation oc-
curred during the day provided that
infected leaves were kept in a moist cham-
ber. They postulated that the nocturnal
habit of the fungus is associated with
relative humidity at night. In this study
when the resultant conidia was inoculated
on PH 801 corn seedlings, disease inci-
dence was 90.30/o which is comparable
with those conidia produced during the
night and inoculated early in the morning.
The finding implies that inoculation can
be programmed earlier in the evening
rather than at dawn.

Tween 80 as wetting agent. Coni-
dial inoculation with and without Tween
80 at 500 ppm showed 900/o and 400/o
infection, respectively. The high degree
of infection among the plants inoculated
with inoculum suspended in Tween 80
could be due to the high retention of
inoculum sprayed on the leaves.
The use of adjuvants to improve
the effectiveness of artificial inoculation
was suggested by Ullstrup (1970). In
the present study, Tween 80 at 500 ppm
when used as the suspending medium of
the conidia was effective in wetting the
leaves of corn seedlings.






Table 1. Appearance of systemic symptoms and percent infection on inoculated pre-
germinated seeds for 12, 24, 48 and 72 hours.

PRE-GERMINATION NO. OF PLANTS PERCENT APPEARANCE a
TIME USED INFECTION OF SYMPTOMS
(DAYS)

12 Hours 40 0
24 Hours 40 0
48 Hours 40 95 7.8
72 Hours 40 95 7.1

SAverage incubation period :-(NS )


Where: Ni : Number of plants infected.
Sa : Symptom appearance (days).
Nit Total number of plants infected.




Table 2. Induced daytime formation of conidia at a temperature of 23.3 C sampled at
different times of the day

TIME OF SAMPLING CONIDIA PRODUCED LENGTH OF
PER CM2 INCUBATION

6 A.M. 0
8 A.M. 5,555 12 hrs.
10 A.M. 27,777 11 hrs.
11 A.M. 31,111 10.5 hrs.
12 A.M. 30,000 10 hrs.





LITERATURE CITED

BARREDO, F.C. and O.R. EXCONDE. 1972. Incidence of Philippine corn downy mildew
as affected by inoculum, suscept and environment. Philippine Agriculturist 57:
232-244.

EXCONDE, O., EDNA EDRALIN and B.A. ADVINCULA. 1967. Some factors affect
ing formation and germination of spores of Sclerospora philippinensis. Proc. Fourth
Inter-Asian Corn Improvement Workshop. Lyallpur, West Pakistan.

MATSUMOTO, T. and S.M. YANG. 1964. Downy mildew of sugarcane in Taiwan. IV
Further studies on the relation of environment to sporulation. Taiwan Sugar Expt
Sta. Rept. 23: 55-62.

ULLSTRUP, A.J. 1970. Opportunities for international cooperative research on down,
mildew of maize and sorghum. Indian Phytopathol. 23:386-388.

SUN, M.H. 1970. Sugarcane downy mildew of maize. Indian Phytopathol. 23:262-26

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

1923. Production and dispersal of conidia in Philippine Sclerospora o
maize. Jour. Agr. Res. 23:239-278.





DEVELOPMENT AND HISTOPATHOL(
INCOGNITA


R.A. PEREZ and


Former Undergraduate Student and A
Plant Pathology, U.P. College of Agriculture, Cc
Undergraduate thesis of the senior autho
The authors wish to thank I.M. Samianc
acknowledgment is also due Dr. R.G. Davidi
cycle chart of Meloidogyne.

ABSTI

The development and histopathological
incognita on celery var. Utah 57-70H were inves
In an incubation room maintained at 21
larvae in seedling roots occurred nine days afi
3rd and 4th moults occurred successively on the
the larval cuticles. This indicated that the thr.
Egg masses on the posterior ends of mature fe
the life cycle was complete on the 37th.
In a growth chamber kept at 23 C, lar
seedling transplanted in petri dishes containing
introduction of 70 disinfected larvae per root
region of elongation. Inward migration appea
After penetration, the nematodes were mostly
lying parallel to the long axis of the root. Galls
their formation occurred within one day after pe
at least from hypertrophy of cortical tissues. G
were usually grouped in the vascular cylinder, bi
and disrupted the vascular tissues causing the dis



The pathogenicity and host range
)f Meloidogyne incognita attacking cele-
-y in La Trinidad, Benguet were already
determined (Castillo and Bulag, in press).
Studies on post-infection development
md the histopathology of infection are
acking.
Christie (1946) presented the follo-
wing general developmental pattern of
root-knot nematodes. The first moult
occurs inside the egg before the larva has
attained its maximum length. The first
;tage is, therefore, spent within the egg.
Further larval growth occurs after the
First moult. Upon eclosion, the second
stage larva migrates to and invades the
root, usually in the region of elongation
immediately behind the root cap. Follow-
ng initial penetration through the epider-
nis, the larva forces its way into and
nigrates through the tissue until it be-
:omes sedentary, usually near the stele.
When the larva assumes its final position,
t feeds only on cells within the reach
)f its stylet. It is in this position that the
!nd, 3rd and 4th moults occur, after
vhich the adult stage is reached. Follow-


[CAL EFFECTS OF MELOIDOGYNE
N CELERY


.B. CASTILLO


tant Professor, respectively, Department of
ge, Laguna.

id V.M. Ramos for preparing Fig. 1. Grateful
'r permission to reproduce partly his life


,T

cts of the root-knot nematode Meloidogyne
.ted.
23 C, the 2nd moult of the most advanced
inoculation with newly-hatched larvae. The
th day, during which adult females also shed
parasitic moults occurred within two days.
les were first observed on the 23rd day and

penetration of roots of two-true leaf stage
50/o agar occurred nine days following the
item. Penetration took place usually in the
to be both intercellular and intracellular.
nfined to the vascular cylinder, frequently
;re observed on the 10th day, suggesting that
ration. These galls appeared to have resulted
t cells were observed on the 11th day. They
ortical ones were also present. Thye blocked
anization of the latter.



ng the 4th moult. males egress from
he larval cuticles as motile vermiform
nematodes. The adult females remain
edentary and increase greatly in
ength and width and gradually become
>ear-shaped. Upon maturation, a gelati-
ious matrix or sac is extruded at the
posterior end of the body Maggenti and
Ulen (1960) later found this to be
through the anus Oviposition begins
nd eggs are expelled through the vulva
nd accumulate within the matrix. The
gg mass usually ruptures the cortex and
he epidermis and it can be seen usually
is whitish to yellowish brown mass on
he surface of the root.
The time involved in the develop-
nent of root-knot nematodes varies, de-
)ending on numerous factors. Knowledge
if the developmental periods, particularly
completionn of life cycle, is useful in the
formulation of control measures such as
he use of trap crops and post-planting
application of nematicides.
Investigations on the reactions of
he host tissues especially during the
arly stages of nematode infection are











st plants ana mo- me most auvanceu larvae ouserveu uuruing


WCu1 {lllxUniCU 6 U IIULO 111 LI 1 rICCIJIUUaC D w V" L" u b ..--. >. Iluru- I ua i-U
with a temperature range of 26 to 32 C. ed cuticle (s) attached to each larva was
TheM. incognita isolate used was obtained determined under an oil immersion ob-
from called celery roots collected from jective of a microscope.


nrco, La innlaaca, ise
were started from a sin
which was maintained
tomato (Lycopersicot
'2029', rainy season v
house. Roots contain
washed, soaked in w
larvae that hatched \
the use either of a 3
Baermann funnel appa
Nematode devel
ry seedlings at two-ti
transplanted singly int
pots containing heat


nguet. i e mocula
gle egg mass culture
1 and increased on
1 esculentum Mill.)
variety, in the green-
ing egg masses were
ater for 24 hr and
vere collected with
25-mesh sieve or a
iratus.
opment. Forty cele-
rue leaf stage were
) four-inch diameter
-sterilized soil and


IIIJtIaUtuImmiuy ut IIw
SSeventy second stal
I been disinfected witl
lycin sulfate were pipe
onto the root tips of ea
cry seedlings grown si
r in petri dishes. Aftc
petri dishes were plao
wth chamber with a tern
20 to 23 C. At daily inti
- 1_ _


tu I aIllu t
These roo
rated with


rvae that
3/o strep-
aseptical-
28 intact
n 0.50/o
)culation,
a lighted
ure range
, the root
ngs were
the 14th
hydrated,
sectioned


days after transplanting, an aliquot sus- staine


- --- ---- -














3rd STAGE LARVA
(10th DAY)


4th STAGE LARVA
(10th DAY)


2nd STAGE LARVA
(st to 8th DAY)


ADULTS e ,
JUST
AFTER
4th
MOULT



SHEDDING
(12th DAY) ADULTS AFTER
LARVAL CUTICLES ,

EGG-LAYING0 S '
23rd DAY

i ,,,, 1

(10th DAY)
MATURE L +















Fig. 1. Life cycle of M. incognita on celery at 21 to 23 C, based on the development
of the most advanced individuals. Developmental groups (partly after Chris-
tie, 1946):

A-a-c; B-d-h; M:i; C:j; D:k; andE-1.





















































^ -g -- ,.






Fig. 2. Longitudinal sections of celei
Sections of roots 9 and 10 d,
nematode penetration points i
root 10 days after inoculation
larvae; D, Section of a root 1i
in the vicinity of nematode feo
inoculation showing nematode
tissues.


roots infected with M. incognita. A-B,
s after inoculation, respectively, showing
the region of elongation; C, Section of a
towing orientations of feeding nematode
lays after inoculation showing a large gall
'ng site; E, Section of a root 14 days after
nlduced giant cells blocking the vascular





Table 1. Earliest periods of recovery of the different nematode developmental groups

DEVELOPMENTAL GROUPS DAYS AFTER INOCULATION

A 2
B 8
C or D 10
M 12
E 23
Aa 37

a Second generation.



Table 2. Nematode recovery and development at selected periodsa


DAYS AFTER DEVELOPMENTAL GROUPS
INOCULATION A B C or D M E TOTAL

1 to 5 5.8 0 0 0 0 5.8
6 to 10 36.0 1.4 0.2 0 0 37.6
11 to 15 41.4 8.0 6.8 0.2 0 56.4
16 to 20 31.6 14.2 15.0 0.2 0 61.0
21 to 25 22.6 19.0 18.4 0 1.6 62.6
26 to 30 18.8 17.4 30.0 0.2 8.0 74.4
31 to 35 15.6 14.0 26.4 0 10.0 66.2
36 to 40 70.4 9.2 27.2 0 16.4 123.2

a Values are means of 5 plants during the 5-day period indicated.



nematodes were most abundant in the 10th day (Fig. If). The resulting 4th
21-25 day period, after which their num- stage larva lost its stylet, the front part
ber decreased continuously through the of which remaining attached to the moult-
termination of experiment. Group C or D ed cuticle, the hind part disappearing
nematodes increased through the 26-30 (Fig. If). The cuticle retained a character-
day period and then decreased. Very istic spike-like tail although the enclosed
few group M nematodes were recovered. larva was bluntly rounded posteriorly.
The number of group E nematodes was Some nematodes had already completed
highest in the 36-40 day period. The the four moults also on the 10th day.
highest number of first generation nema- The resulting adults had their stylet
todes recovered during the 26-30 day reformed and their digestive and repro-
period was 74.4. This constituted only ductive organs were well-developed (Fig.
about 3/o of the total number of nema- 1 g-h). Adult females with shed larval


b) Observations of
tode specimens: All larvae
vermiform until the 5th
inoculation (Fig. la). Al
heean to inereanse in wi,





or by conspicuous openings extending
to the cortical region (Fig. 2 A, B). In-
ward migration appeared to be both
intercellular and intracellular. The nema-
todes were confined mostly to the vas-
cular cylinder, frequently lying parallel
to the long axis of the root (Fig. 2 B, C).
Occasionally, there were larvae with their
heads embedded in the vascular cylinder,
their bodies extending through the cortex
towards the root surface.
Nematode infections were accom-
panied by gall and giant cell formations
(Fig. 2 E). Galling, first observed on the
10th day, appeared to be brought about
by hypertrophy of cortical tissues. Three
to seven giant cells formed around the
head of each feeding nematode beginning
on the llth until the 14th day. These
giant cells occurred in the groups, usually
separated only by their much thickened
cell walls. They were present both in the
cortical and vascular regions, more com-
monly in the latter location. When in the
vascular region, the giant cells appeared
to block, disrupt and disorganize the
vascular tissues. As many as 10 nuclei
were observed per giant cell until the
14th day. These stained more deeply
than those of the surrounding normal
cells and were almost always aggregated
at the center of each giant cell. Formation
of lateral roots near nematode feeding
sites was occasionally observed.


DISCUSSION

The post-infection development of
M. incognita on celery at 21 to 23 C
followed the general pattern for root-
knot nematodes. The intervals between
the three parasitic moults (2nd, 3rd, and
4th) of individual larvae were not deter-
mined, but in the most advanced larvae
were completed within two days. The
2nd moult was first observed on the 9th
day following inoculation; the 3rd and
the 4th occurring successively on the


10th. The onset of the parasitic moults
observed was two days earlier than on
tomato at 29.4 C (Triantaphyllou and
Hirschmann, 1959) while their comple-
tion was one to three days faster than
the general observations on root-knot
nematodes on other crops (Bird, 1959;
Castillo, Russell and Morrison, 1973;
Triantaphyllou, 1959). Egg mass product-
ion and completion of M. incognita life
cycle occurred later than on other crops
at different temperatures. For instance,
egg masses were observed two and three
days later than on tomato at 20 C
(Davide, 1965) and at 29.4 C (Triantaphy-
lou and Hirschmann, 1959), respectively,
and eight days later than on ampalaya at
25.5 to 29 C (Davide, 1967). However,
reinfection by second generation larvae,
which was observed on the 37th day,
was 20 days earlier than on tomato
(Tarjan, 1952). These variations may be
attributed to the difference in the experi-
mental conditions and suitability of the
host plants.
The migration of the parasite into
the root tissues of celery seedlings grown
in 0.50/o agar at 20 to 23 C appeared to
be both intercellular and intracellular.
Gall formation was found to be due at
least partly to the hypertrophy of cortical
tissues. Since nematodes first appeared
inside the roots on the 9th day following
inoculation and gall and giant cells were
observed on the 10th and llth day,
respectively, it seemed that, under the
conditions of the experiment, galls form-
ed within one day after penetration. The
fiant cells formed within two days.
These periods somewhat concur with the
observations of Christie (1946) and others
on other crops infected with root-knot
nematodes. The presence of nematode-
induced giant cells which blocked and
disrupted the vascular tissue was apparent-
ly responsible for the disease in celery as
is the case in other root-knot-affected
plants.


LITERATURE CITED


BIRD, A.F. 1959. Development of the root-knot nematodes Meloidogyne javanica
(Treub) and Meloidogyne hapla Chitwood in the tomato. Nematologica 4:31-42.

CASTILLO, M. B., C.C. RUSSELL and L.S. MORRISON. 1973. Development of Meloi-
dogyne hapla in peanut. Phytopathology 63:583-585.






range of a root-knot nematode att
Philippine Agriculturist (in press).
tourist (in press).

CHRISTIE, J.R. 1946. Host-parasite relatior
marioni. II. Some effects of the host


', l Allr l Z JZ *. I llu ,l tlry allu lIv.iJL
acking celery in La Trinidad, Benguet.



ships of the root-knot nematode, Heteroder


DAVIDE, R.G. 1965. Influence of environment on development and sex differentiation
of root-knot nematodes. Nematologica 13:102-110.

1967. Pathogenicity and development of root-knot nematodes on ampala-
ya. Philippine Agriculturist 50:795-803.

MAGGENTI, A.C. and M.W. ALLEN. 1960. The origin of the gelatinous matrix in
Meloidogyne. Helminthol. Soc. Wash. Proc. 27:4-10.

SASS, J.E. 1964. Botanical microtechnique 3rd ed. The Iowa State University Press,
Ames. 288 p.

TARJAN, A.C. 1952. Life histories of the root-knot nematodes. Phytopathology 42:20
(Abstr.).

TRIANTAPHYLLOU, A.C. and H. HIRSCHMANN. 1959. Post-infection development
of Meloidogyne incognita Chitwood, 1949 (Nematode: Heteroderidae). Ann. Inst.
Phytopathol. Benaki, N. S. 3:1-11.






































































tpmnprntiire. the .r rnigm resumed its





Table 1. Effects of temperature on growth and sporulation of C.gloeosporioides (M61)
from mango a


TEMPERATURE COLONY DIAMETER MYCELIUM SPORULATION
OC (mm) (spores/.01 ml)
M55 M61 M55 M61

10 5 (original 5 None 0 0
size)
15 15 20 scanty, flat 8 11
20 42 45 abundant 10 48
25 55 60 fairly abundant, 15 63
30 65 78 flat
abundant 23 72
35 10 13 fairly abundant 19 68
aerial

a Recorded after 7 days of incubation on PDA. Average of 3 plates.


Effect of light. Isolate M61 in yeast Effect of different culture media.
extract agar plates was incubated at 25 C The different agar media used in this
under continuous light, continuous dark- test are the following: 1) Czapek's
-- -J r. -, ji c\riu a1. '^\ r'i _... .--_l1 -.





Table 2. Effect of light on sporulation of C. gloeosporioides

TREATMENT b COLONY DIAMETER SPORULATION
(mm) (spores/,01 ml)

Continuous light 85 1110
Continuous darkness 70 760
Alternating light and darkness
(24 hrs) 35 1260
Room condition 40 780

(30 / 2 C)


a Recorded after 7 days of incubation.
All at 25 C except room condition treatment.


Table 3. Effect of pH on mycelial growth, sporulation, and spore-germination of C.
gloeosporioides a


pH MYCELIAL DIAMETER SPORULATION SPORE GERMINATION
(mm) (spores/.Ol ml) (/o)


3 /U

6 90

7 80

8 75

9 70



a Average of 5 plates.


Table 4. Sporulation of C.gloeosporioides

CULTURE MEDIA SPOR]
M55

Czapek 122.5
Saboraud 5.0
Corn meal agar 12.5
Potato dextrose agar 12.5
Oatmeal agar 12.5
Yeast extract 520.0


a Incubation time was 5 days at 30 C (coni


120 25

135 25

58 15

50 10






different culture media a

'.01 ML
M61

110.0
30.0
52.5
65.0
65.0
770.0


ious darkness) and 3 days at room condition





Table 5. Germination of C. gloeosporioide
mango fruit juice

SUBSTRATES PERCENT
at

Plain distilled water
Malt extract
Water agar
Ripe skin juice
Unripe skin juice
Ripe flesh juice
Unripe flesh juice

a Average of 5 counting.


Observation on germination. Ger-
mination was characterized by the pro-
duction of a hyaline germ tube at either
end of the cell. Conidium about to germ-
inate shows the thinning of the portion
of the cell wall from which the germ
tube arises. At germination, the spore
appeared empty because its contents had
removed to the elongating germ tube or
prohypha.
Germination was very poor and
irregular in pure water at 24 hr. In malt
extract, spores started to germinate in
4 hr while in water, germination started
18-24 hr later. In-vivo studies show that
spores in water suspension started to
germinate on the surface of ripe and
unripe mango fruits in 5 hr.
Figure 3 shows germinated spores
of C. gloeosporioides, some exhibiting
thick-walled appresoria.


DISCUSSION
Among the general conditions
known to influence growth and sporula-
tion of fungi the most important are
temperature, light, pH, and the composi-
tion of the media. These external condi-
tions largely control the internal environ-
ment of the cells of a fungus and bring
them to a condition which allows growth
and reproduction. While these views ad-
mit a general application to all fungi,
the specific application rests upon the
detailed study of each fungus.
The present study showed that
C. gloeosporioides grows and sporulates
under a wide range of temperature from
15 C to 35 C, the optimum being


(M61) at different solutions and expressed


GE GERMINATION a
r 24 hr

10
95
)0
95
70
35
70





25-30 C. No growth occurred at 10 C
even up to three weeks of incubation.
These information would explain the
wide distribution of organism in tropical
and semi-tropical countries (Sattar and
Mallik, 1939). Low temperatures also
prolong the storage life of mango, its host
fruit (Ram Ayyar and Joshi, 1929; Chee-
ma and Gandhi, 1926; Pantastico et al
1970) without being over grown by the
anthracnose organisms that had supposed-
ly infected the fruits while on the tree.
Light is not necessary for growth,
sporulation, and spore-germination of
C gloeosporioides but it does help for
more luxuriant spore formation. Impli-
cation that mango fruits stored in the
dark can slow down sporulation Diplodia
natalensis, causal organism of Stem-end
rot of mangos (Halos, 1970) would not
hold true with C. gloeosporoides.
The ability of C gloeosporioides.
to germinate and grow slowly at pH 3
and 4 is an evidence for the reported
ability of C gloeosporioides to cause
latent infection on green mango fruits
(Simmons, 1941). The spores can tolerate
the pH of green fruit skin, germinate, and
thereby cause penetration and infection
but mycelial growth and development
is inhibited until the fruit ripens. The
ability of the spores to germinate in ex-
pressed juice of skin and flesh of green
mango fruits further supported this.

Better germination in malt extract
than in pure water, as was found in
the present study, confirmed the report
of Baker et al(1940). Both results showed
that C gloeosporioides germinates slowly
in pure water but the rate of germination









Effect of temperature on the mycelial growth of C. gloeosporioides on PDA.


Fig. 1. Effect of tem


Fig. 2. Effect of differe
on growth of 2 iso
porioides. SdA Sa
Czapek's, YEA
CMA corn meal,
PDA, potato dext


90
80
i \

I 70
M61
60 55

50


10 15 20 25 30 350C

Temperature (oC)

perature on spore-germination of C. gloeosporioides on water agar.
































nt culture media Fig. 3. Germinating spores of C. gloeos-
lates of C. gloeos- porioides showing thick-walled ap-
ibouraud's, CzA presoria.
- yeast extract,
OMA oat meal,
rose agar.





s increased when nutrient is added. The
matter study also showed that when 20/o
nalt extract was added to the infection
Irop on a ripe fruit surface, heavy infect-
on occurred after 18 hr while without
he addition of malt extract, infection


LITERATUI


BAKER, R.E.D, S.H. CROWD, and R.K.
caused by Colletotrichumgloeosporioi

IHEEMA, G.S. and S.R. GANDHI. 1926. R
and other tropical fruits. Agr. J. India.

1ALOS, P. 1970. Sporulation by Diplodia
infected mango fruits. M.S. thesis. U.P


as delayed for a further 24 hr. It is
possible that the time taken to establish
ifection after inoculation with spores in
ater can partially be attributed to the
me taken for the exosmosis of the fruits.



CITED


:KEE. 1940. A review of latent infections
and allied fungi. Trop. Agric. 27:128-132.

aeration as a means of preserving mangoes
403405.

alensis Pole-Evans and histopathology of
llege of Aeric. 64 D.


RAM AYYAR, C.S. and N.V. JOSHI. 1929. Preservation of mangoes by cold storage.
Agric. J. India. 24:124-126.

SATTAR, A. and S. MALLIK. 1939. Studies on anthracnose of mango caused byglome-
rella cingulata Stoneman (S. and V.S.). (Colletotrichumgloeosporioides Penz.) in
the Punjab. Indian J. Agric. Sci. 9:511-521.

SIMMONS, J.H. 1941. Latent infection in tropical fruits discussed in relation to the part
played by species of Gloeosporium and Colletotrichum. Proc. Roy. Soc. Qd.
52:92-120.

TUITE, J. 1969. Plant pathological methods: fungi and bacteria. Burgess Publishing Co.
Minn. 239 p.






SURVEY AND PATHOGENICITY TESTS OF PLANT PARASITIC NEMATODES
ASSOCIATED WITH SUGARCANE IN NEGROS OCCIDENTAL


Research
Plant Pathology, 1
A portion


nt and Associate Professor and Chairmal
college of Agriculture, College, Laguna.
undergraduate thesis of the senior author.


actively, Department of


Results of the survey showed that the following 13 genera of plant para
were associated with sugarcane in Negros Occidental: Pratylenchus, Helicotylenc,
hynchus, Xiphinema, Rotylenchulus, Meloidogyne, Hoplolaimus, Paratylenchi
Criconemoides, Scutellonema, Trichodorus and Hemicycliophora. Some of tl
Pratylenchus zeae, Xiphinema americanum, Tylenchorhynchus martini, Helicot
tera, Rotylenchulus reniformis, Meloidogyne incognita and Hoplolaimus galeatus
Pratylenchus was the most abundant and widely distributed genus whi
54.80/o of the total nematode population and occurred in 98.80/o of the soil sar
whereas Paratylenchus was the least distributed in the area as it occurred only i
total soil samples.
Pathogenicity test revealed that M. incognita, T. martini, P. zeae and H.
capable of infecting the sugarcane var. Phil. 62120 with a corresponding increase
by approximately 77, 53, 27 and 24 percent, respectively. Infected plants s]
growth. Xiphinema americanum and Rotylenchulus reniformis showed only sli
the test plants with no detectable increase in population three months after inoci



Sugarcane, Saccharum officinarum rily to determine the oct
L., is a major export crop in the Philip- bution and pathogenic
pines. The FAO report (1970) indicated plant parasitic nematodes
that the country has approximately sugarcane in the provir
320,000 hectares of sugarcane with an Occidental.
annual production of 16,000,000 metric


action ot the crop could hardly be


and diseases that seriously affect the crop. vey was made in Decei
Nematodes are one of these pests that January 1971 when the s
perhaps substantially affect the product- were at mid-harvesting
ion of sugarcane in this country. stage. Soil samples were i
There are reports abroad which localities in Negros Occ
indicate that nematodes may affect sugar- were taken at random 3
cane production. For instance, in Louis- near the rhizosphere and I
iana, U.S.A., Birchfiled (1953) observed bags. Then they were 1


ra were
)ulation
stunted


Lted wi
Negr


The st
1970
ie plan


__


Pr */nfl -l>l**P i-niIIC rl Cn. A +"n 4F mwn.. 4 Ulndr D-lV l-r -,. DTD1fA Fr4- mn+rln


.... p





cedures and with the aid ot taxonomic Pratylenchus, Xiphinema, Helicotylen-
keys by Mai et al(1964), Thorne (1961), chus, Tylenchorhynchus, Meloidogyne,
Sher (1966), and Tarjan (1964). Longidorus, Hoplolaimus, Paratylenchus,
After the identification, the nema- Trichodorus, Rotylenchulus, Cricone-
tode population density was determined moides, Hemicycliophora and Scutellone-
in each sample by counting the individual ma. Some of the species identified were
nematodes belonging to each genus. P. zeae Graham, T. martini Fielding,
Pathogenicity test. The pathogenic H. dihystera (Cobb) Sher, X. americanum
capabilities of the different species of Cobb, R. reniformis Linford and Olivaira,
nematodes were determined by conduc- M. incognita (Kofoid and White) Chit-
ting the following pathogenicity test. wood and H. galeatus Steiner.
Sugarcane seed pieces (var. Phil. 62120) Evidently there was considerable
were propagated in 8-inch clay pots filled variation in the distribution and popula-
with baked soil. Before planting, the tion density of the nematodes in the
seed pieces or stalks of about 6 inches different areas of Negros Occidental.
long were surface-sterilized by dipping in For instance, in Victorias Pratylenchus,
1:500 solution of mercuric bichloride for Tylenchorhynchus, Helicotylenchus, Hop-
three minutes. lolaimus, Xiphinema and Hemicycliopho-
Two weeks later, the plants were ra were the only genera detected. Of these,
inoculated with approximately 1000 indi- Pratylenchus showed a population level of
viduals each of P. zeae, T. martini, 1342 individuals as compared to 207
M. incognita and R. reniformis. Due to a Tylenchorhynchus, 78 Helicotylenchus,
limited amount of H. dihystera and 58 Hoplolaimus, 42 Xiphinema and 11
X. americanum the test plants were Hemicycliophora detected per 400 cc of
inoculated with only 500 individuals of soil samples. The highest population level
each species. Five replicate plants were of Pratylenchus (10,199 individuals) was
used for each nematode treatment. The observed in 10 samples from Binalbagan,
same number of plants served as controls. the lowest (78 individuals) in 5 samples
All inoculated plants and the controls from Villadolid. The population level of
were kept in the greenhouse on top of Xiphinema and Tylenchorhynchus was
hollow blocks to prevent cross highest in samples from Himamaylan and
contamination. Binalbagan, respectively. In La Granja,
The height of the plant and the La Carlota and Hinigaran, Rotylenchulus
weight of the top portion and root por- reniformis was the most dominant with a
tion were taken 90 days after inoculation, population count of 3,389, 1,419 and
To determine the population build-up 1,001 individuals, respectively.
of the nematodes, the soil from each The population of Meloidogyne was
pot was processed as for samples taken highest in Himamaylan with 1,276 indi-
from the field. Root samples of one gram viduals in 10 samples. In two sampling
were also obtained from plants inoculated sites in Kabankalan, Scutellonema was
withM. incognita andR. reniformis. These detected although in low population level.
were cut into small pieces, fixed in FAA The samples collected in Bago had the
for at least 48 hrs., then washed in tap highest population density of Hoplolai-
water and stained for 3 minutes in boiling mus. Likewise, Longidorus had the high-
--] .- .e.. ._ a Pt nnnillatinin drpnitv in em rnlae -lp+_t.








82.70/o, but it had a population dens
of only 7.450/o. The third is Helicotyl
chus with 750/o distribution; the fou:
is Tylenchorhynchus (74.400/o), the fi
is Longidorus (48.210/o), and the si
is Hoplolaimus (44.640/o).
In terms of population densi
Rotylenchulus ranked second to Pra
lenchus. The other genera occurred
less than 200/o of the total samples a
most of them constituted less than 1'
of the total nematode population.
Pathogenicity. The data on
mean number of nematodes recover
from the soil and root samples 90 d&
after inoculation are shown in Table
Of the six nematodes species tested
pathogenicity on the sugarcane var. P
62120, only M. incognita, T. marti
P. zeae and H. dihystera had an incre
in population density. There was a sli
decrease in the population of R. reni)


y to a number of environmental fact
such as the soil texture and pH, var
S of the crop, age of the plants at the 1
of the survey and others. These fac
L have been demonstrated to influence
distribution and population density
nematodes associated with citrus t
in the Philippines (Davide and de la R
S 1971).
That the nematodes associated i
S sugarcane in Negros Occidental are c
ble of causing considerable damage
S sugarcane had been demonstrated in
I pathogenicty test. Although the ex
S riment was somewhat limited in sco
. there was evidence that Pratylenchus z
r Tylenchorhynchus martini, Meloidog
incognita, Xiphinema americanum, R,
lenchulus reniformis and Helicotylent
e dihystera did infect the plants cau
t retardation in root and top growth. I
S severe stunting was observed in pl


in soil infested with the aforementioned is a favorable host ot these nematodes
nematode species are summarized in (Apt and Koike, 1962; Birchfield and
Table 4. Stunting of the plants inoculated Martin, 1965).
with T. martini and P. zeae was more There was evidence that the differ-
severe compared to plants infected with ent species have varying rates of repro-
H. dihystera, M. incognita, R. reniformis duction on sugarcane (Phil. 62120 var.).
and X. americanum (Fig. 1). There were However, this did not mean that the
faster rate of reproduction and higher
also indications that top and root weights faster rate of reproduction and higher
of inoculated plants were affected by the population density of the nematodes, led
nematodes to greater damage on sugarcane. The
From individual roots examined, results showed that M. incognita with a
From individual roots examined,
lesions and necrosis were commonly population increase of770/o three months
lesions and necrosis were commonly
observed on plants infected with T. mar- after inoculation had power pathogenic
tini, P. zeae and R. reniformis. Individual capabilities than Pratylenchus zeae and
roots of plants inoculated with H. dihys- Tyenchorhynchus martini which have a
tera showed an abundance of feeder roots corresponding increase in population of
and dying of the root tips. Selected indi- 27 and 53 per cent, respectively. This
vidual roots from plants infected with indicates that the plants have more tole-
X. americanum exhibited slight traces of rance to M incognita infection than to
necrosis. Galls of various sizes were ob- P. zeae or T. martini. On the other hand,
served in roots of plants inoculated with R. reniformis and X. americanum showed
M. incognita. However, most of the galls no apparent increase in population level
were small and occurred mostly in the but noticeable damage on the root sys-
feeder roots. teams was observed. The movement and
survival ofX. americanum can be affected
hv sqil with low nir-fillpd norec and high


1
The results of this study show that s
the occurrence, distribution and popula- N
tion density of plant parasitic nematodes c
associated with sugarcane in Negros Occi- I
dental vary considerably with the differ- i
ent localities. This variation may be due s



























atylenchus 4 1,726 431.50 2.38 2.67


totall number of individuals of each genus

otal number of individuals of all the genera

MLE 3. Mean population counts of nematode
(var. Phil. 62120)a



MATODE Initial
Count


ncognita 1,068.6
wrtini 1,018.4
eae 1,032.0


X 100


90 days after inoculation on sugarcane




Final Per cent
Count Increase


4,567.6b 77
2,162.8 53
1,405.4 27
1- 1 t-


dean of 5 plant replicates.

loth soil and root counts.

3oth soil and root counts.
























S32.50


i of 5 plant replicates.


stern England. Nematologica 14:197-201.

, C.R. 1961. Notes on nematodes infesting sugarcane in tt
igan mill districts. Phil. Sug. Inst. Quart. 7:16-17.

J., J.P. MARTIN, C.A. WISMER and H. KOIKE. 1969. Ner
rietal yield decline of sugarcane in Hawaii. Plant Disease 1


-I














*1
[V.i


(


watode infection on top i


'"-
: ::t
~I

B
.;-I


owing the relative e


W-!


effect of ne




MADAMBA, C.P. 1966. Plant parasitic nematodes with special emphasis on those attack-
ing sugarcane. Phil. Sug. Inst. Quart. 12:129-133.

MAI, W.F., H.H. LYON and T.H. KRUK. 1964. Pictorial key to the genera of plant para-
tic nematodes. Department of Plant Pathology, Cornell University, Ithaca, New
York.

PONCHILLIA. P.E. and D.C. NORTON. 1970. Effect of certain soil properties on the
survival and migration of Xiphinema americanum. Jour. Parasitology 4:269 (Abstr.).

RECUENCO, J.P. 1970. Parasitic nematodes associated with sugarcane. Phil. Sug. Inst.
Quart. 16:10-15.

SHER, S.A. 1966. Revision of the Hoplolaiminae (Nematoda) VI. Helicotylenchus
Steiner, 1945. Nematologica 12:1-56.

SIDDIQUI, M.R. and K.F. BROWN. 1964. Helicotylenchus retusus n. sp. (Nematoda:
Hoplolaiminae) found around sugarcane roots in Negros Occidental, Philippines,.
Proc. Helm. Soc. Wash. 31:209-211.

TARJAN, A.C. 1964. A compendium of the genus Tylenchorhynchus (Tylenchidae:
Nematoda). Proceedings of the Helmintological Society 31:270-280.

THORNE, G. 1961. Principles of nematology. McGraw-Hill. Book. Co., Inc. 553p.

WARD, C.H. 1960. Dagger nematodes associated with forage crops in New York. Phyto-
pathology 50:658 (Abstr.).




EVALUATION OF THE DIFFERENT METHODS AND RATES OF APPLICATION
OF TEMIK 10G FOR THE CONTROL OF NEMATODES ON ABACA

R.G. DAVIDE and A.L. ELOJA

Associate Professor and Chairman, Department of Plant Pathology, UPCA and formerly
Supervising Plant Pathologist, Bureau of Plant Industry, respectively.

ABSTRACT
Temik 10G has been shown to control effectively such genera and species of plant
parasitic nematodes as Rotylenchulus reniformis, Helicotylenchus sp., Pratylenchus sp.,
Meloidogyne incognita, Xiphinema sp., and Hoplolaimus sp. In the experiment at planting time,
counts made two months after treatment revealed that there was an approximately 61-96 per
cent control of the nematodes. 13-94 per cent in 6 months, and 12 to 50 per cent in 9 months
depending upon the methods and rates of application. The split method of application gave a
longer control of the nematodes than the straight method of application.
Similarly, in the one-year old abaca plantation experiment, the efficacy of Temik 10G
was dependent upon the methods and rates of application. The poke hole method was less
effective than the circular band method. This would indicate that the rate of diffusion of the
chemical in the soil is somewhat slow and hence, it must spread over a larger area to get a
better coverage and control of the nematodes.
In the two abaca experiments, the rate of 30 lbs/acre gave results which were comparable
with those of the 60 lbs/acre rate as far as the nematode control is concerned. However, at a
higher rate (60 lbs/acre) and using the poke hole method of application, Temik 10G increased
sucker production by 14.9 per cent to 29.8 per cent in the experiment at planting time and
and 6 to 23 per cent in the one-year old plantation experiment.
As a result of the nematode control by Temik 10G treatment, abaca plants showed
improvement of growth. There was approximately 1 to 8 per cent increase in growth two
months after treatment, 12-44 per cent in 6 months and 10 to 17 per cent in 9 months. Better
growth response in the circular band method of application was observed.




Temik 10G is widely used abroad This study was primarily conducted
for the control of various species of to evaluate the effectiveness of Temik
insects and nematodes. However, in the 10G when used in different method and
Philippines this chemical has not yet been rates of application to control nematodes
fully evaluated particularly its effective- attacking abaca.
ness and economic value as a nematocide. MATERIALS AND METHODS
Abaca is one of the export crops of
the Philippines. In fact for several years Treatment at planting time. The
before and after the second world war experiment
t objective of the first set of experiment
it was a leading export crop. During then the effect of straight
last few years, however, the abaca indus- and spit et t e of applying Temik str
try suffered a set back in the world st eth of applying Temik 10
at the rates of 30 and 60 Ibs/acre on
market due to the advent of synthetic at the rates of 30 and 60 lbs/acre on
fibers. It was just recently that the young abaca plants. The straight treat-
fibers. It was just recently that the s
government campaigned for the revival of ment was made by applying the whole
government campaigned for the revival of dosage of the chemical during the time
the industry due to an increasing demand of planting. For the rate of 30 lbs/acre
for Manila hemp abroad and in local 25 grams were applied in each hole about
markets. Besides, the discovery of abaca 12 inches deep and covered with a small
fiber as an excellent pulping material in amount of soil before the planting mate-
paper manufacture has encouraged pri- rial was placed, while for the 60 bs/acre,
vate enterprises to establish pulping plants. 50 grams were placed in each hole prior
50 grams were placed in each hole prior
Due to the increasing market de- to planting the abaca. The straight treat-
mands for abaca fibers abroad, the coun- ment consisted of 8 hills for each two
try is now developing its abaca industry replicates.
for maximum production. However, some
of the factors that may limit its product- In the split application method,
ion are pests and diseases and nematode only 1/2 of the total amount of each rate
is one of the pests that can attack abaca. was placed into the hole at planting time

67





and the remainder was applied two motnhs
later into the four poke holes, 6-12 inches
deep, and about one foot from the base
of the plant. This treatment consisted of
7 hills for each two replications. Soil
samples were collected before Temik
10G was applied and subsequent sampling
were made 2, 6 and 9 months after
application. Plant parasitic nematodes
were extracted from the samples, identi-
fied and counted in the nematology
laboratory of the Department of Plant
Pathology, UPCA.
Treatment in one-year old plant-
ation. To determine the reaction of older
plants to Temik 10G applied at different
methods and rates, the following experi-
ment was conducted on one-year old
abaca plantation. The rates used were
also 30 to 60 lbs/acre but the method
of application was different from the
first experiment in that the chemical was
applied either in poke hole or circular
bands. In the poke hole method, four
holes, 1-2 inches in diameter and 6-12
inches deep, were made about one foot
from the base of the plant at equal
distances from each other.
In the circular band method, the
chemical was applied into a circular canal
about 34 inches wide and 6-12 inches
deep at radius of one foot from the
base of the plant. After placing the
chemical, the holes and the circular canals
were covered with soil.
For both methods of application,
four replications were made for each rate
used and in each replication 6-plant hills
were assigned. As in the first experiment,
soil samples were collected before treat-
ment and subsequent sampling were done
2, 6 and 9 months later.

RESULTS AND DISCUSSION

Treatment at planting time. The
data obtained from this experiment are
summarized in Table 1. Counts of nema-
todes before the treatment of Temik 10G
were referred to as initial counts. The
computation on the nematode control
was based upon these initial counts.
The genera and species of plant
parasitic nematodes associated with the
abaca plants were Rotylenchulus renifor-
mis Linford and Oliviera, Meloidogyne
incognita Chitwood, Pratylenchus sp.,
Helicotylenchus sp. ana occasionally Ra-


dopholus similis Cobb,Xiphinema sp., and
Hoplolaimus sp., were observed.
As shown in the data the nematode
counts made 2, 6 and 9 months after
treatment indicate that the effectiveness
of Temik 10G in controlling the nema-
tode varies considerably with the methods
and rates used. In the straight application
method maximum control of the nema-
tode (91.60/o) was observed 2 months
after treatment, whereas in the split
application method maximum control was
obtained in 2 months at 60 lbs/acre and
in 6 months at 30 lbs/acre.
The split application, however, gave
a longer control of the nematode than
the straight application. This is shown
by the fact that with 30 lbs/acre, at 9
months after treatment, the split applica-
tion method still provided 49.90/o con-
trol of the nematode, whereas in the
straight application only 11.80/o of the
nematodes were controlled. The same
trend of results were obtained at 60 lbs/
acre at 9 months after treatment whereas
the straight application gave only 14.60/o
control as compared with the 370/o
control from split application.
As far as the rate of application
is concerned, the results indicated that
its effectiveness also varies with the me-
thod of application.
For instance, based on the counts
made after 2 and 6 months the 30 lbs/
acre rate gave a higher percentage control
than the 60 lbs/acre rate in the straight
application method, whereas in the split
application method, the 60 lbs/acre rate
gave a higher percentage of control.
However, based on the 9-month
counts the rate of 30 lbs/acre with split
application afforded a better control
(49.90/o) than 60 lbs/acre (37.00/0).
On the other hand, both rates gave almost
the same level of control in the straight
application method. In general, it seems
that the effectiveness of 30 lbs/acre would
not differ much from that of 60 lbs/acre
in controlling the nematodes.
Treatment in one-year old plant-
ation. The results of the experiment are
presented in Tables 2 and 3. Again, the
most predominant genera and species of
plant parasitic nematodes observed were
Rotylenchulus reniformis, Helicotylen-
chus sp., Meloidogyne incognita Chit-
wood and Pratylenchus sp. Occasionally
Xiphinema sp., Radopholus similis Cobb






Table 1. Effect of different rates and methods of application of Temik JOG on the
population density of plant parasitic nematodes on abaca plants when treated at
planting time

NEMATODE COUNTS/300 cc. SOILSb
APPLICATION NEMATODE Months after Treatment
Rate and Method GENERAa Initial 2 Per 6 Per 9 Per
Cent Cent Cent
Control c Control Control


Rotylenchulus
Helicotylenchus
Meloidogyne
(larvae)
Pratylenchus

TOTAL

Rotylenchulus
Helicotylenchus
Meloidogyne
Pratylenchus


260.0
30.0
160.0


40.0 2.0


281.5
12.5
37.5

21.5


185.5
17.2
154.7

75.7


490.0 41.0 91.0 353.0 28.0 432.1 11.8


185.0
51.0
21.0
12.0


269.0 105.5 61.2 28.0 89.2 126.6 49.9


Rotylenchulus
Helicotylenchus
Meloidogyne
Pratylenchus

TOTAL

Rotylenchulus
Helicotylenchus
Meloidogyne
Pratylenchus

TOTAL

Rotylenchulus
Helicotylenchus
Meloidogyne
Pratylenchus

TOTAL


126.5
80.0
9.0
20.0


105.5
12.5
75.0
12.5


121.2
3.0
73.0
3.8


235.5 47.0 80.1 206.0 12.6 201.0 14.6


170.0
40.0
45.0
20.0

275.0

70.0
135.5
10.0
61.0

277.0


118.0
7.5
40.0
7.8


11.5 96.0 17.0 93.8 173.3 37.0


188.0
91.0
13.0
11.0

303.5


141.5
95.0
20.5
13.5


822.0
24.0
363.5
27.0


270.5 1,236.5


A. 30 lbs/acre
Straight


TOTAL


B. 60 lbs/acre
Straight


Control


a Occasionally Radopholus similis, Xiphinema, and Hoplolaimus were found
b Data represent the mean of two composite soil samples from two replications with
7-8 plants each
c Per cent control was computed based on the initial counts








The data indicated that the popul
tion of the different genera of pla
parasitic nematodes were considerably r
duced by Temik 10G treatment. The r
matode population decreased continuot
ly until 6 months after treatment. Ho\
ever, 9 months later, the nematode pop
tions were already comparable with tl
pre-treatment levels but compared wil
the population increase in the check, it
evident that Temik 10G has remarkab
suppressed the nematode build up. I
shown in the data the circular bai
method gave a better control than tl
poke hole method of application. Nem
tode counts made 2 and 6 months aft
treatment showed that the poke ho
method at 30 lbs/acre provided 30.7
52.5 per cent control compared with tl
47.8 to 79.6 per cent control afforded t
the circular band method. However,
60 lbs/acre the poke hole method ga
55.4 per cent and 38.5 per cent contr
after 2 and 6 months, respectively.
On the other hand, nematode count
made 9 months after treatment reveal
that with the exception of the circul
band method at 60 lbs/acre rate, t
chemical was already getting less effect
ive in controlling the nematode. T1
population levels of the nematode hi
considerably increased in some treatment
For instance, in the circular band meth(
at 30 lbs/acre and in the poke he
method at 60 lbs/acre, the counts we
already higher than the pre-treatme
counts. However, this increase in t]
nematode population was still much low
than the population levels in the corr,
ponding controls. Evidently, based on tl
nematode counts of the controls, Tern
10G treatment had still held back tl
population level by 31 to 54 per ce
depending upon the method of application
The circular band method still gave
better control than the poke hole method
Like the first experiment, the 60 Ibs/ac:
rate did not show any better control th,
the 30 lbs/acre.
Effect of different rates and metho
of Temik 1 OG application on plant growth
To determine whether the reduction
the nematode population from the s<
has resulted in the increase in grow
of the plants, heights of the mother plan
in the one-year old plantation experime


time because the plants were later attack
ed by mosaic and bunchy-top diseases.
There is evidence from the dat;
gathered that Temik 10G treatments ha
resulted in the increase of top growth o
abaca plants. This increase in plant growth
was manifested 6 months after treatment
with Temik 10G. Measurements made
months later indicate very small increase
in plant growth which range from 0.91
8.84 per cent. In 6 months, the maximun
growth increase was 44.4 per cent. How
ever, this growth increase was reduce<
as the nematode population tended tb
increase 9 to 12 months later.
In general, the circular band me
thod which was more effective in reducing]
the nematode population gave a high
percentage of plant growth increase thai
the poke hole method. Data showed tha
the circular band method still gave 16.6'
per cent growth increase 12 months afte
treatment at 30 lbs/acre and 9.55 per cen
growth increase at 60 lbs/acre, where
in the poke hole method no plant growth
increase was observed 12 months later
Effect of Temik 10G on the sucked
production of abaca plants. To deter
mine whether Temik 10G treatment ha
any effect on sucker production, count
were made on the number of sucker
developed 6 months after treatment. Th,
data are summarized in Tables 5 and 6
There is evidence that a higher rate (6(
lbs/acre) Temik 10G can reduce sucke
production. In the treatment at planting
time, there was an increase of 29.8 pe
cent sucker production in the straight
application at 60 lbs/acre and 17.0 pe
cent increase in the split application m(
thod 6 months after treatment. At th
lower rate (30 lbs/acre) there was ni
increase in sucker production. On thi
other hand, in the split application o
30 lbs/acre an increase of 14.9 per cen
sucker production was observed.
Basically, the same trend of result
were obtained in the one-year old plan
ation experiments. The data shown ir
Table 6 indicate the effect of Temik 10C
on sucker production 12 months afte
treatment. These data showed that sucke
production was more or less influence
by the rates and methods of application
of the chemical (Fig. 1). It was also a
i n IL . _. I 1 -





TABLE 2. Effect of different rates and methods of application of Temik 10G on the
nematode population on abaca when treated one year after planting


APPLICATION
Rate and Method


NEMATODE
GENERA


Initial


NEMATODE COUNTS/300 cc SOILS a
Months after treatment
2 6 9


A. 30 lbs/acre
Poke Hole


Rotylenchus
Helicotylenhus
Meloidogyne
(larvae)
Pratylenchus


346.8 240.3 164.6 365.5


Circular Band


Rotylenchus
Helicotylenchus
Meloidogyne
Pratylenchus


222.0 116.9


45.6 230.5


B. 60 lbs/acre
Poke Hole


Rotylenchus
Helicotylenchus
Meloidogyne
Pratylenchus


TOTAL


Circular Band


C. Control


Rotylenchus
Helicotylenchus
Meloidogyne
Pratylenchus

TOTAL


Rotylenchus
Helicotylenchus
Meloidogyne
Pratylenchus


248.6 110.8 152.8 331.0


259.3
57.8
90.0
0


169.5
20.3
1.0
0


129.3
18.5
26.5
10.0


244.3
23.3
72.5
10.0


407.1 190.8 184.3 350.1


164.3
164.8
17.0
0


628.8
178.0
20.3
8.0


164.0
14.8
17.3
10.5


359.5
42.5
84.0
15.0


324.4 835.1 206.6 501.0


243.3
75.5

23.0
5.0


182.2
53.0

2.0
3.0


162.2
21.0

8.2
9.2


TOTAL


263.0
31.0

54.0
17.5


118.0
100.0
4.0
0.0


82.3
24.3
7.3
3.0


TOTAL


32.6
6.6
5.3
1.3


161.0
15.0
46.5
.0


102.8
42.8
103.0
0


120.0
13.2
14.8
4.4


214.8
15.7
88.8
11.7


TOTAL


a Data represent the mean of 4 replications with six hills each containing 4-8 plants/hill.
Hoplolaimus and Xiphinema were also found occasionally.


--







ion was obtained, whereas at 30 lbs/i
only 9.56 per cent increase was
served. On the other hand, there was
increase in sucker production in the ci
lar band method at 30 Ibs/acre.
In general, this study has gi
valuable information with regards to
effect of Temik 10G on the control
nematodes affecting abaca in the Ph
pines. It is evident in the data obtain
that the effectiveness of the chem
depends mainly on the rate and met]
of application. However, it was not kni
in this study whether, as a result of
treatment, fiber yield could also be
creased. Since the experiment was c
ducted on young plants (one-year i




Table 3. Summary data on the effec.
application on the nemat,
after planting



APPLICATION
Rate and Method Initial 2


A. 30 lbs/acre

Poke Hole 346.8 240

Circular Band 222.0 116

B. 60 lbs/acre

Poke Hole 248.6 110

Circular Band 407.1 190.

C. Control 324.4 835.


study on its effect on fiber yield
abaca be conducted.


ACKNOWLEDGEMENT

The permission of the Stati
Superintendent at the BPI Davao Expe
ment Station and the BPI Regional Dire
or for use of the experimental site
greatly appreciated by the authors. App
ciation is also expressed to Uni
Carbide Phil, Inc. for the assistance giv
in the experiment.
The help of Mr. Ruben Bicomoj
Jr. in typing the manuscript is grateful
acknowledged.




'the different rates and methods of Temik 1
population on abaca plants treated one ye



NEMATODE COUNTS/300 cc SOILS
Months After Treatment
Per cent 6 Per cent 9 Per c
Control Control Cont



30.7 164.7 52.5 365.5

47.8 45.6 79.6 230.5



55.4 152.8 38.4 331.0

50.3 184.3 54.7 350.0 11

S 206.6 501.0







old plantation


APPLICATION REPLICAT]

tate and Method I II

L. 30 lbs/acre

Poke Hole

2 months 86.20 90.21 7
6 months 125.00 139.33 10:
12 months 172.00 173.80 12,

Circular Band

2 months 90.90 81.94 9
6 months 139.67 190.33 15
12 months 177.00 160.00 19

1. 60 lbs/acre

Poke Hole

2 months 83.60 96.11 7
6 months 132.67 112.00 12
12 months 164.50 129.60 14

Circular Band

2 months 105.53 83.77 8

6 months 147.01 179.00 11
12 months 173.20 206.60 14


MEAN PER CENT
GROWTH
IV INCREASE





77.04 83.27 0.98
97.00 117.65
120.00 147.50



91.00 88.86 7.77
148.50 182.88 44.41
202.00 184.50 16.69





75.33 82.12
191.67 141.46 11.70
189.50 156.90



84.62 89.74 8.84

125.80 141.85 12.08
164.70 173.20 9.55





TABLE 5. Effect of TEMIK O1G on sucker production of abaca plant treated at planting
time


REPLICATION2 PER CENT
RATE METHOD MEAN SUCKER
(Ibs/acre) I II INCREASE


A. 30 1. Straight 22 18 20.0
2. Split 28 26 27.0 24.9

B. 60 1. Straight 37 24 30.5 29.8
2. Split 28 27 27.5 17.0

C. Control 21 26 23.5


1 Data were taken 6 months after treatment.

2 Each replication consisted of 7 hills for the straight application and 8 hills for the split
application.


TABLE 6. Effect of TEMIK 10G on the sucker production of one-year old abaca planta-
tion 12 months after treatment


RATE METHOD REPLICATION MEAN PER CENT
SUCKER
I II III IV INCREASE


A. 30 lbs/acre

Poke Hole 13.2 8.8 14.4 13.0 12.6 9.56
Circular Band 10.2 7.6 11.7 9.6 9.8

B. 60 lbs/acre

Poke Hole 17.8 16.8 11.3 11.0 14.2 23.47
Circular Band 18.3 10.4 6.2 14.0 12.2 6.08

C. Control 11.6 13.2 12.0 9.3 11.5






























ir a.


Figure 1. Effect of TEMIK O1G trea,
taken 9 months after treatn


it on the growth of one-year old abaca plan









AJ.

Assistant Professor, Department of Pla
The author wishes to thank Dr. R.T.
tory, University Park, Pennsylvania, U.S.A. fc


This paper presents an attempt to
explore further the use of synthetic,
semi-synthetic, and natural media for
inducing the perfect stage ofRhizoctonia
solani.
The following media were used
with eight isolates of R. solani (Table 1):
(1) sterile tap water; (2) modified Sabo-
raud's solution-glucose, 5 g; peptone, 2 g;
(3) modified Saboraud's solution-glucose,
5 g; KH2PO4, 2.5 g; MgSO4, 7H20, 2.5 g;
sucrose, 5 g; FeCl3, trace; (4) soil extract-
soil extract obtained from autoclaved
1 kg fairly air-dried soil 1 liter distilled
water after allowing the mixture to stand
overnight; dextrose, 1 g; yeast extract,
0.1 g; KH2PO4, 1 g; (5) potato dextrose
broth-decoction of boiled 50 g potato in
1 liter distilled water; dextose, 5 g;
(6) rye decoction; decoction of autoclaved




TABLE 1. Iso



ISOLATES
Host


C-43 Pinus resinos

C-65 Picea glauca

C-189 Brassica nive,

C-239 (?)

C-244 Plaseolus sp.

C-259 Phaseolus sp.

NC-245 Medicago fal

Va-29


MIO

thology, UPLB-CA, College, Laguna.
wood, Professor at the USDA Pasture Labora-
viding cultures of R. solani


12.4 g rye grains in 1 liter distilled water;
(7) oat decoction decoction of auto-
claved 50 g unhusked oat grains in 1 liter
distilled water; (8) fresh potato tuber,
turnip tuber, and cabbage lead discs two
to three mm. thick slices of 1 inch
diameter tuber plugs from surface dis-
infected tubers and 1 inch. diameter leaf
discs from the inner leaves of a cabbage
head. Formulations of all liquid media
were on per liter distilled water basis.
The pH was adjusted to 7.2 before sterili-
zation at 121 C for 15 min. The media
were plated at 20 ml/plate and seeded at
the center with 1-cm diameter agar-myce-
lium plugs from advancing margins of
10-day old cultures on PMYDA (extract
from boiled 200 g potato; malt extract,
1 g; yeast extract, 0.5 g; dextrose, 20 g;
agar, 17 g; distilled water to make 1 liter).




of R. solani used



ORIGIN OF CULTURES
Place


Quebec, Canada

Quebec, Canada

(?) ATCC-13248

Quebec, Canada

Costa Rica

Costa Rica

North Carolina, USA

Louisiana, USA








placed at the center of Petri plates contain
ing 20 ml sterile distilled water.
These replicates were used for eac
medium. The cultures were incubate
at 24-26 C. The plate cultures were ex;
mined directly under 100X magnificatic
to ascertain basidial stage formation an
sporulation. The experiment was repeated
once.
Perfect stage formation was ol
served only on oat decoction, Richard
medium, soil extract, and tap water. (
the 8 isolates used, only 4, name]
C-43, C-65, C-189, and C-239 formed tl
sexual stage (Table 2). All these isolate
formed hymenia on oat decoction an
Richard's medium; C43 did not fru
on soil extract and tap water, while onl
C-239 fruited on tap water. Very abui



TABLE 2. Sexual stage formation
Rhizoctonia solani on a

MEDIA
C-43


Sex Growth Sex
Form Char b Fon
(day)a (day

Tap water -
x

Modified
Richard's 17 +17
Solution xxx


extract. Hymenial formation on the rest
of the media was sparse. The morpholo-
gical characteristics of the basidial appa-
ratus of the fruiting isolates were typical
of the genus Thanatephorus described
by Talbot (1970).

The hymeniaproduced on Richard's
medium were sparse, virtually covered by
thick vegetative growth, and undistingui-
shable from lumps of lysed aerial hyphae
unless examined under the microscope.
On soil extract, the hymenia were minute,
flat, white, glistening, and somewhat
powdery on top of thin mycelium ad-
pressed to the medium (Fig. 1). Except
for a thicker mycelial growth, the hy-
menial characteristics were observed on
oat decoction.



growth characteristic of four isolates of
rent media

ISOLATES

C-189 C-239


;rowth Sex Growth Sex Groi
har. Form Char. Form Char
(day) (day)

-- 10
x x x


+17 + 17 +
LXx xxx xxx
























RFgure 1. Seventeen-day old cultures Rhizoctonia solani Kuhn (C-239) on A) potato
dextrose broth showing abundant sclerotia and thick mycelial growth, and
B) soil extract showing basidial stage formation; note the abundant whitish
and glistening small clusters of hymenia on thin mycelium.


The mycelial growth was very thin induced on solid substrates by artificially
to none on tap water; Saboraud's solu- inoculating host plants, covering rich
tion, and cabbage leaf; thin on soil non-synthetic agar cultures with unsteri-
extract; relatively thick on rye decoction, lized soils, and by transferring cultures
oat decoction, fresh potato and turnip from rich agar media to soil extract agar,
tubers; and very thick on Richard's solu- damp filter paper, or sometimes to water
tion and potato dextrose broth. All test agar (Sherwood, 1970). The present stu-
isolates produced abundant sclerotia on dy showed that a solid substrate is not
potato dextrose broth and fresh potato necessary for its sexual stage formation.
discs, generally very few to none on In addition, it could be induced on
fresh cabbage leaf, Richard's solution, synthetic media such as Richard's me-
soil extract, oat and rye decoction, and dium. The study also provides additional
none on Saboraud's solution and tap evidence for the presence of self-fertile
water. and self-sterile strains of R. solani shown
Earlier studies indicated that sexual by previous investigators (Flentji, et al.,
stage formation of R. solani could be 1970; Talbot, 1970).







Trac



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