• TABLE OF CONTENTS
HIDE
 Front Cover
 Effects of multiple pests on crop...
 Seaweed (Eucheuma sp.) as a substitute...
 Variants of rice grassy stunt virus...
 Variants of rice grassy stunt virus...
 Effects of temperature and humidity...
 Genetic variability of resistance...
 Occurrence and development of sheath...
 Abstracts of papers and posters...














Title: Journal of Tropical Plant Pathology
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Permanent Link: http://ufdc.ufl.edu/UF00090520/00004
 Material Information
Title: Journal of Tropical Plant Pathology
Series Title: Journal of Tropical Plant Pathology
Physical Description: Serial
Language: English
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
Publication Date: 1991
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00004
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 - 1624346
electronic_oclc - 54382605
issn - 0115-0804

Table of Contents
    Front Cover
        Front Cover 1
        Front Cover 2
    Effects of multiple pests on crop growth and yield
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
    Seaweed (Eucheuma sp.) as a substitute for agar in culture media for selected fungal pathogens
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
    Variants of rice grassy stunt virus in the Philippines
        Page 20
    Variants of rice grassy stunt virus in the hilippines
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
    Effects of temperature and humidity on germination and infection of colletotrichum gloeosporioides (Penz.) Sacc. on 'Carabao' mango (Magifera indica L.)
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
    Genetic variability of resistance to puccinia polysora underw. in corn
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
    Occurrence and development of sheath blotch of rice caused by pyrenochaeta oryzae shirai ex miyake in the Philippines
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
    Abstracts of papers and posters presented during 1991 pest mnagement council of the Philippinrd annual convention; BPI. Manila
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
Full Text










PHILIPPINE PHYTOPATHOLOGY
Official Organ of the Philippine Phytopathological Society, Inc.


PHILIPPINE PHYTOPATHOLOGICAL SOCIETY, INC.
OFFICERS 1991-1992


NARCEO B. BAJET
LINA C. LAPITAN
TEODORA O. DIZON
ANGELITA C.D. TALENS
CEFERINO A. BANIQUED
OSCAR S. OPINA
JOSE M. BANDONG
AVELINO D. RAYMUNDO
NERIUS I. ROPEROS
LUISITO B. ROS
PAUL S. TENG
CASIANA M. VERA CRUZ


President
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PHILIPPINE PHYTOPATHOLOGY
EDITORIAL BOARD 1991-1992


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PAUL S. TENG


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Phytopathological Society, Ino.







EFFECTS OF MULTIPLE PESTS ON CROP GROWTH AND YIELD


Hans Pinnschmidt


Plant Pathology Division, International Rice Research Institute, Los Bafios, Laguna, Philippines.

Plenary paper presented at the Pest Management Council of the Philippines, May 8, 1991,
Bureau of Plant Industry, Malate, Metro Manila.


ABSTRACT

Damage effects of multiple pest populations need to be quantified in order to
obtain realistic yield loss predictions, loss profiles, damage thresholds, and other
means for improving decision making in IPM. Despite a considerable amount of
research done on the topic, further multifactorial experiments that use detailed
assessment methods are needed to study the specific effects of multiple pest
infestations on the performance of crops. The conventional approach of predicting
multiple pest effects for pest management purposes uses empirically obtained pest-
loss equations. These quantify the overall-effects of given pest infestation levels on
yield and are data set specific and therefore not applicable across cropping conditions
and pest situations. The rather recent dynamic pest-crop modeling approach in
contrast allows a more realistic, physiologically based mimicking of multiple pest
effects, and is transportable across cropping situations, since it is strictly mechanistic.
The relative importance of various pests, different damage mechanisms, as well as
intra and interspecific pest interactions to the resulting total yield loss can thus be
studied in detail. The practical implications of the pest-crop modeling approach
consist of predicting, creating, and comparing multiple pest-loss scenario and are
discussed with regard to decision aids for developing strategies and tactics in IPM.


I. Justification for studying multiple pest
effects

Multiple pest infestations are a situation
rather commonly encountered in farmers fields
(Johnson, 1990). For example, pests and diseases
of rice frequently occur in one or the other
combination within the same field. If substantial
crop damage and yield loss can be caused by
either of these pests, this will be even more so if
they occur" in .combination. Despite of this,
decision aids and recommendations for pest
management are usually designed for single pest
situations. This paper aims to demonstrate the
need and possible means of filling this gap in
order to improve decision making in pest
management under multiple pest conditions.

II. Economic considerations and implications

The ultimate goal of studying multiple pest
effects is the optimization of profit for the
producer at an acceptable level of risk, at
acceptable prices for the consumer, and at
minimized harmful impact on the environment


and human health. Therefore, control tactics and
strategies have to be optimized in an
economically, socially, and environmentally
acceptable way. The management
recommendations and decision tools to be used
must be based on a thorough, quantitative
knowledge of crop response to multiple pest
situations.
Multiple pest infestations, in comparison to
single pest infestations, can significantly alter
economic decision criteria. One criterion
commonly used to indicate whether pests should
be controlled or not is the economic threshold
(ET). The ET indicates the pest population or
damage level that can economically be tolerated
and does not require control measures. As
demonstrated by Palis et al. (1990), the
economic threshold for damage caused by one
pest strongly depends on the damage caused by
other pests. In the simplest case, the economic
threshold for a pest declines linearly with
increasing infestation level of another pest,
forming a so-called "iso-loss line". Multiple pest
situations can also affect the benefit obtained
from control measures. For two pests, the







functional relationship between pest infestation
levels and benefit from control measures can be
visualized in form of a benefit plane that shows
increasing benefit with increasing infestation
levels of either pest (Blackshaw, 1986). Other
economic considerations that arise from multiple
pest situations include the question whether two
or more pests can be controlled with the same
control measure and pesticide and how the
importance of key- and/or secondary pests
changes in the presence of other pests. Also, the
economic benefit of a control measure will
depend on how well each pest can be controlled.
Economic considerations mingle here with
biological and technical considerations that are
determined by the specific features and
interactions of the multiple pest-host system
under study and the available control options.

III. The need to quantify multiple pest effects

In order to obtain realistic yield loss
predictions, loss profiles, and other means for
improving decision making in IPM, the damage
effects of multiple pests need to be quantified.
The conventional approach of quantifying the
effect of multiple pest infestations on yield is by
means of pest-loss, equations or empirical
damage functions. For two pests, a simple
equation of this type would be:

y = blx1 + b2x2 (1);

where y = total yield loss, x1 and x2 =
infestation levels of two pests at a critical crop
stage, and bl and b2 = the respective damage
coefficients for x1 and x2. Equation (1) thus
represents a so-called "single point" or "critical
point model", because it relates yield loss to
infestation levels at a single critical time. It
implies that multiple pest damage effects are
simply additive, following the assumption:

y = (P1 +P2+...Pn)Y (2);
where y = total yield loss due to multiple
pest effects, Y = attainable yield, and P1, P2 ...
Pn (E [0,1]) = relative effect of each pest on
yield.
Multiple pest effects are additive only if
pests do not interact with regard to crop
response. Then, the relationship between the
sum of all pest effects and yield is linear (Fig. 1).
A representative review of literature by Johnson
(1990) however revealed that multiple pest
effects on yield were most often found to be less-
than-additive, due to antagonism among the
pests. greater-than-additive effects, due to
synergism among the pests, and additive effects
occurred less frequently.


50 100
Sum of multiple pest damage effects (%)


Figure 1: Yield as related to damage effects of multiple
pests for different types of pest-pest interaction.


The following equation accounts for
damage effects of multiple pests on yield if the
pest populations interact:.

y = Y-Y[(1-P1)(1-P2)...(1-Pn)]b (3);

where y = total yield loss due to multiple
pest effects, Y = attainable yield, P1, P2 ... Pn
(E [0,1]) = potential relative effect of each pest
on yield, and b = coefficient of interaction. With
b approaching zero, y approaches zero too, due
to antagonistic interactions among the pests. The
more synergistic interactions occur, the higher b
becomes, 'y thus approaching Y. Since b will
rarely be known, let's assume b = 1. Equation
(3) then equals the one proposed by Padwick
(1956) that is able to account for a slight
antagonism among multiple pests. The
advantage of equation (3) compared to equation
(2) consists in leading to yield loss predictions
that are always <. Y, even for high pest
infestation levels.
Multiple pest interactions have a direct
impact on the yield loss caused by each single
pest, and therefore on the total yield loss as well.
Also, economic decision criteria for control
options change. For example, antagonism among
pests can significantly lower the total yield loss
and the yield loss caused by each single pest,
compared to a situation where pest populations
do not interact. Thus, the yield loss profile
changes. As a consequence, the economic
threshold of a single pest can significantly be
raised in the presence of antagonistic pests,
while it can drastically be lowered in the
presence of synergistic pests (Fig. 2). Both,







synergism and antagonism, caneither be direct or
indirect. Indirect effects are usually mediated
through the host, while direct effects are not
(Table 1).


No Interaction


T YL.

T YL bI


Antoaoonsm


Due to




Pest A

Pest B


Pestc C
TYLa= total yield oss if pests do no interact (=65%)
T YLb= totol yield loss for antagonilic interaction among psts (= 48%)


Figure 2: Crop loss profile for three hypothetical pests
(A,B,C), assuming either no interaction or
antagonism among the pests.

An often observed example for direct
antagonism is competition and inhibition among
pests, leading to less-than-additive effects on
yield loss (e.g. Johnson et al., 1986, 1987; Karban
et al., 1987; da Luz and Bergstrom, 1987).
Indirect antagonism can be observed when one
pest induces host resistance against another pest
(e.g. Karban et al., 1987; da Luz and Bergstrom,
1987). Indirect synergism occurs when damage
caused by one pest lowers the tolerance of the
host against another pest or facilitates the entry
of another pest into the host (e.g. Rowe et al.,
1985; Lee et al., 1986; Soriano et al., 1986).
Symbiosis or commensalism might serve as an
example for direct synergism. Simulation studies
of Rabbinge and Vereyken (1980) indicated that
yield loss due to shading by saprophytes
colonizing on honeydew produced by aphids
might be much higher than the yield loss caused
by the feeding activities of the aphids alone.
The following empirical damage function
accounts for non-additive effects of two pests on
yield:

y = blx1 + b2x2 + b1,2x1x2 (4).

This function is identical with equation (1),
except for the interaction term b 12X1x2 that
accounts for interactions.between x1 and x2, with
b1,2 being the respective damage coefficient.
Empirical pest-loss equations are usually
obtained via multiple regression analysis with
yield or yield loss as the dependent variable.
Parameters characterizing the intensity of pest
infestation serve as the independent variables.
Examples for multiple pest-loss equations for


rice are provided by Khosla (1977), Teng et al.
(1990b), and Palis et al. (1990). Additional
details on the construction of pest-loss equations
in general are given by Madden (1983), Teng
(1985, 1987), Waggoner and Berger (1987), and
Walker (1987). However, empirical damage
functions only quantify the overall-effects that
pests or diseases have, without considering the
specific damage mechanisms. While useful for
purposes such as yield loss surveys, they are
usually very data set specific and can not be
applied across cropping conditions and pest
situations. For example, their applicability tends
to be very dependent on the pattern of pest or
damage development (Litsinger, 1991), which is
variable in many pest-crop systems.

IV. The need to understand multiple pest
effects

Ultimately, the damage mechanisms of
multiple pests have to be understood if
"transportable" quantitative knowledge is to be
obtained. The chronological pattern of
occurrence and severity level of pests the time
profile (Heong, 1990) certainly affects the yield
components. For example, pests or damage
occurring during the vegetative phase of
development of rice, such as leaf blast,
caterpillars, and dead hearts, might primarily
reduce the no. panicles per area and the no. of
grains per panicle. During the generative phase,
panicle blast, white heads, or rats might mainly
affect the grain weight or the no. panicles per
area. To understand the nature of multiple pest
damage, it is important to know: where on the
plant does damage occur, what kind of damage
does occur, how does the damage affect the
functioning of the plant, and how does damage
caused by one pest interfere with damage caused
by another pest? For example, a leaf spot disease
might just cover and shade the green leaf area
and thus reduce the amount of light captured,
while in virus-infected leaves, the photosynthetic

rate might be reduced. Both types of damage will
decrease the productivity of the crop. With both
diseases occurring on the same leaf, the relative
importance of the leaf spot disease might be
overridden by the effect of the virosis. Other
diseases might block the translocation of water
and/or assimilates which in turn will certainly
alter the importance of other pests that cause
other types of damage.

Such considerations inevitably lead to the
conclusion that damage mechanisms of pests and
their effects and interactions have to be studied
at the level of crop physiological processes and
variables, rather than at the crop level (Table 2).
Empirical pest-loss equations are usually
generated at the crop level and rarely at the level







of physiological determinants of crop growth,
eventhough there are some examples for this
approach (e.g. Waggoner and Berger, 1987).
Quantitative knowledge about pest- or multiple
pest effects at the level of basic crop
physiological processes and variables is however
limited. A quantitative understanding of multiple
pest damage effects wbuld require to identify the
crop physiological processes or variables affected
by the respective pests and to quantify the effects
of given pest levels on these. This would yield
functional relationships describing the effects of
given pest levels on crop variables and
physiological processes, such as leaf area,
photosynthesis, translocation, etc.. Figure 3
shows such a functional relationship for the
effect of a single pest on a hypothetical crop
physiological process. Quantitative studies of this
kind have been conducted for a number of single
pests (e.g. Rabbinge and Coster, 1984; Rabbinge
et al., 1985; Van der Werf, 1988; Heong, 1990;
Heong and Fabellar, 1988; Kenmore, 1980;
Bastiaans, 1991). Hardly, two or more pests were
studied in combination. This however would be
necessary to understand effects of pest
interactions at the physiological level.

Table 2: The mechanistic approach of tracing the
damage effects of multiple pests on
yield along the level of biological
organization.


multiple pest
level of organization: damage effects on:

crop yield
physiological radiation use efficiency
determinants radiation interception
of crop growth partitioning
rate of biomass loss
duration of productivity
crop state variables, biomass
production and flow senescence
of assimilates, growth of plant organs
crop growth processes respiration
translocation
sink strength
photosynthesis
transpiration
resource uptake
resource availability
etc.


V. Simulation of multiple pest effects

Quantitative knowledge about pest damage
mechanisms as mentioned above can be used to
simulate the damage effects of multiple pests by
means of crop simulation models. In such
models, crop physiological processes and
variables are linked in a way that crop
development, growth, and yield build-up can be
simulated. Pest damage effects can be coupled to


any of these processes or variables and thus
affect them. This mechanistic approach thus
allows to "mimick" pest effects in great detail.
Therefore, it might be more applicable for a
wider range of conditions then the use of
empirical damage functions. Various versions of
this pest-crop coupling approach were explored
by Gutierrez et al. (1975, 1976, 1977, 1983),
Boote et al. (1983), Adams (1987), Adams et al.
(1984, 1987), Roermund et al. (1986), Johnson et
al. (1987a), Johnson and Teng (1989), Benigno
et al. (1988), and others. The approach was
discussed by Teng et al. (1990, 1990a) for the
IBSNAT Ceres crop model for rice which is
currently used at IRRI to develop coupling
routines for simulating the damage effects of
various major rice pests (Pinnschmidt et al.,
1990). The specific damage effects that these
pests supposedly have are listed in Table 3.
1.0-
-\ -. under-proportional
a)
\ \
o73
':E 0.5- \
-6
-- \ proportional

N
over-proportional .
0.0 -- -,-- ,-' "-- ,
0.0 0.5 1.0
Relative pest or disease severity
Figure 3: A hypothetical crop physiological process as
affected by the proportionality of damage effects
that result from- the severity level of a
hypothetical pest or disease.

Table 3: Six selected rice pests and some of
their presumable effects on the crop.

disease or pest: effect gn crop:


leaf blast


reduction of light interception
reduction of photosynthetic rate


acceleration of leaf senescence
panicle blast reduction of grain filling
loss of panicles
sheath blight reduction of light interception
blockage of translocation
acceleration of leaf senescence


leaf folder

stem borers


reduction of leaf area
reduction of light interception
disruption of translocation
killing of growing points


plant hoppers consumption of assimilates
acceleration of leaf.senescence
enhancement of transpiration









Hypothetical progress curves of each of the
pests listed in Tab. 3 were read into the crop
model. The model simulated how processes and
variables such as leaf area, photosynthetic rate,
grain filling, leaf senescence, etc. were affected
by the infestation level and the assumed damage
effects of each pest. Parameter values, such as
feeding and damage fates, were mostly based on
a "good guess". The results showed that adding
pest species to the crop simulation led to a
steady decline of the biomass production of the
crop (Fig. 4). The consequence of any pest
scenario, such as different pest combinations,
pest levels, and patterns of pest progress, for the
performance of the crop can thus be simulated
and compared for any cropping scenario, such as
different weather patterns, varieties, cultural
practices, etc.. The results of such simulation
studies can then be used for developing decision
aids for pest management. But prior to this,
more quantitative data are needed on the actual
damage mechanisms of each pest and on pest
interactions. The approach is currently being
calibrated and validated with field data sets.
disease/pest-free
1200
& leaf blast
1000 & panicle blast

E 800
& sheath blight
S600 & leaf folder
S& stem borers
.0 & plant hoppers

200

0 20 40 60 80 100 120 140 160 180 200
days after seeding
Figure 4: Simulated biomass production under multiple
pest situations.

VI. Experimentation needed for studying the
damage effects of multiple pests

Whether conducted in the laboratory,
greenhouse, or field, multiple pest studies have
to be multifactorial. Fig. 5 shows a three-
factorial strip-split-plot design for studying the
damage effects of three different pests. Each
strip represents different pest or damage levels.
This design is especially suitable for field trials,
because creating and maintaining different pest
levels is easier than in a completely randomized
design. The range and amount of pest levels to
be obtained, the level of detail in data gathering,
and the variables to be measured depend on the
type of experiment and the objectives of the
study.


A2


C1 C2 C3

Al




A3


B1 B2 B3

Figure 5: Example of one replicate of a three-factorial
strip-split-plot design for studying the damage
effects of multiple pests. A, B, and C are
different pest species; 1, 2 and 3 are different
pests intensity levels.



"Micro-level" trials aiming at an
quantitative understanding of the nature of
multiple pest damage effects at the physiological
level can be conducted in the laboratory,
greenhouse, or field. They require a fairly wide
range of pest levels and pest combinations. The
data collection has to be rather detailed and
centers on measuring pest damage activity rates
and crop response rates, if possible daily, in
order to obtain parameters and coefficients for
pest effect simulation.
"Macro-level" trials represent classical yield
loss experiments to be conducted in the field.
They focus on the "over-all" effects of multiple
pests on crop development and yield. Thus, they
either aim at obtaining and testing empirical
damage functions or validating pest effect
simulations, and here, a rather wide range of
pest levels and pest combinations would be
required. Or they aim at testing control practices
or identifying key features of multiple pest
scenarios with regard to yield loss, and then, a
rather limited range of pest levels and pest
combinations would be sufficient. The data
gathering essentially aims at obtaining progress
curves of pest infestation or damage levels and
of crop development. Final yield and yield
components should be measured in both kinds of
trial.
Both types of experiments go hand-in-hand,
because results of micro-level trials have no
practical applicability until they were validated in
macro-level trials. On the other hand, niacro-
level trials will hardly ever yield a thorough
understanding of what pests are really doing to






the crop, unless they are supported by micro-
level trials.
Creating and maintaining different pest
levels will be crucial in such experiments.
Examples of how to obtain and maintain wide
arrays of disease or pest infestation levels are
provided by Johnson et al. (1986, 1987), Nutter
(1990), and Litsinger (1991). Furthermore, exact
measurements of pest population or damage
levels will be crucial for obtaining high quality
results. At IRRI, where we work with combined
leaf blast and leaffolder infestations, we use an
assessment key for counting leaf blast lesions of
different sizes (Fig. 6). Leaf blast can thus quite
accurately be assessed. A similar key is being
used to estimate the absolute amount of
leaffolder feeding damage (Fig. 7). From both
assessment keys, standard diagrams were also
obtained as a guide for estimating the percent
damage caused by either pest (Fig. 8 and 9).


VII. Conclusions
Multiple pest infestations impose a
challenging problem on crop protection, because
they are common for most crops and have
significant effects on crop development, yield,
and yield loss structure and thus a significant
economic iinpact. In order to optimize multiple
pest management, decision tools have to be
developed, based on yield loss predictions,
damage thresholds, and other means. This
requires that the combined effects of multiple
pest populations on yield be quantified. To
ensure the applicability of these decision tools
across cropping conditions and multiple pest
situations, more quantitative knowledge is
needed on specific damage mechanisms of pests
and on multiple pest interactions with regard to
crop response at the physiological level. This
knowledge can be used for simulating the effects
of multiple pest infestations on the performance
of crops and has to be tested against data
obtained in conventional yield loss trials in the
field. Yield loss trials can further be useful for
identifying over-all effects of multiple pests on
yield and for testing multiple pest management
practices.


Assessment Key for Leaf Blast Lesions

( cm2)


class 5


class 3


class 1


I IIlI


0.45


0.21


0.09


0.04


0.02


I cm


Figure 6: Assessment key for leaf blast lesions (cm2).


class 2


s, 4 1


class 4





















































Figure 7: Assessment key for leaffolder feeding damage (cm2).


















0.1%


1%
1%


B

9I
B


5%


A


'I



'1



10


I


6



I
30


I



Ir


I
It





Standard Diagram For Assessing


Leaf Folder


5


A
1!v


Damage


is


4


10


30


LP'.1


50 %


Figure 9: Standard diagram for assessing leaffolder damage.


1%


II
I


-






IIX. Acknowledgement

The author wishes to express his thanks to
the IBSNAT project, University of Hawaii,
CTAHR, Department of Agronomy and Soils
Science, 2500 Dole street, Krauss Hall 22, USA,
HI 96822, for the financial support during the
preparation of this paper.

IX. References

ADAMS, S. S., 1987: Simulation of sugarbeet-
powdery mildew interrelations. PhD thesis.
Univ. Calif., Davis. 476 pp.
ADAMS, S. S., ROUSE, D. I. and NICOT, P.
C., 1984: Simulation of potato early dying
disease epidemiology using coupled models
of host and pathogen development.
Phytopathology 74:1268 (Abstract).
ADAMS, S. S., ROUSE, D. I. and BOWDEN,
R. L., 1987: Performance of alternative
versions of POTWIL: A computer model
that simulates the seasonal growth of
Verticillium-infected potato. Am. Potato J.
64:429 (Abstract).
BASTIAANS, L., 1991: The ratio between
virtual and visual lesion size as a measure
to describe reduction in leaf photosynthesis
of rice due to leaf blast. Phytopathology 81:
(in press).
BENIGNO, E. A., SHEPHERD, B. M.,
RUBIA, E. G., ARIDA, G. S., VRIES, F.
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SEAWEED (Eucheuma sp.) AS A SUBSTITUTE FOR AGAR
IN CULTURE MEDIA FOR SELECTED FUNGAL
PATHOGENS

Luz B. Montesclaros and Jesusito L. Lim

Respectively, Instructor, Don Severino Agricultural College, Indang, Cavite and
Assistant Professor, Department of Plant Protection, Visayas State College of
Agriculture, Baybay, Leyte, Philippines.

Portion of BS Thesis conducted by the senior author in ViSCA, Baybay, Leyte,
Philippines.

Given the Best Undergraduate Thesis Award by the Philippine Phytopathological
Society during the 19th Annual Convention of the Pest Control Council of the
Philippines, Inc. held at Sacred Heart Center, Cebu City on May 4-7, 1988.


ABSTRACT


Seaweed or "gozo" carrageenan was found to be an excellent substitute for
commercial agar in the preparation of PDA and Onion Agar. Sclerotium rolfsii Sacc.,
Helminthosporium maydis Nish. Miy., Cercospora henningsii Allescher, Sphaceloma
batatas Saw. and Pestalotiopsis sp. exhibited best mycelial growth at 30, 40 and 45, 25
and 30, and 20 g/l "gozo" carrageenan, respectively. For good sporulation/sclerotia
formation, 30 g carrageenan liter was superior for S. rolfsii and 20 g/1 for H. naydis;
for S. batatas, 20 g/l and Pestalotiopsis spp., 40 g/l. C. henningsii did not sporulate on
both PDA and the medium with "gozo" carrageenan substitute.


INTRODUCTION

Microorganisms, like all other forms of
life, require water and. various nutrients for
growth, reproduction and survival. In
culturing microbes, these requirements are
adequately supplied in a suitable artificial
culture medium such as nutrient agar (NA),
potato dextrose agar (PDA) and tryptone
glucose yeast extract agar (TGYA)
A great variety of culture media had
been carefully concocted for particular
microbial groups. However, a certain
principle governs the composition of the
various types of culture media i.e., each
should contain a balanced mixture of the
different nutrients furnished in amounts
proportional to the biosynthetic requirements
of the microorganisms (Stainer, 1978).
Culture media are either solid or liquid
with the solid culture media containing agar
as the solidifying agent. Agar is not a
component of liquid culture media.


Agar is chiefly extracted from Gelidium
and Gracilaria species and occasionally from
other red algae such as Pterocladia,
Ceramiium, Suhlia, Ahnfeltia and Acan-
thopeltis (Duckworth et al., 1970). It was first
used in China in the 17th century and was
immediately introduced to Japan where it has
been produced extensively (Trainor, 1978).
In light of the present economic
situation in the Philippines, scientists and
researches are trying to find ways of reducing
research cost. Thus, to-,nullify the need to
import the expensive powdered agar, they are
looking into the potential of other species of
algae that are abundant in the Philippine cost
as source of agar as agar substitute.
Studies in the Philippines on the
utilization of local seaweeds which contain
agar are very limited. Vannajan and Trono
(1977) and Rodulfo et al. (1985) mentioned
that Gracilaria vemicosa locally known as
"gulamang dagat" was a good source of
"gulaman" or local agar. This "gulaman"
proved to be a good substitute for BBL,







Difco, Oxoid and Funflag agars (Fernandez
et al., 1977). Ilag et al. (1982) also proposed
a growth medium in which "gulaman" was
used instead of the imported powdered agar.
Similarly, studies on the use of local agar for
microbial cultures were reported by Rodulfo
et al. (1985) and Santiago et al. (1985). They
found that the growth of the different species
of bacteria, yeasts and fungi tested on the
local agar medium was comparable to that of
Difco agar medium.
Several workers tried to investigate the
use of carrageenan, a substance which brings
about a good gel, in microbiological media.
According to Santos (1980) carrageenan was
first isolated by Schimidt in 1844 from
Chondrus crispus. This particular material
was tried in some microbiological media by
Walker and Day in 1943 and Watson and
Aspirion in 1976.
In the Philippines, the red seaweed
Eucheuma, which is locally known as "gozo"
and farmed on a commercial scale, is the
most important industrial gum carrageenan
(Epifanio et al., 1981). That media prepared
from the Kappa carrageenan extract of
Eucheuma striatum (Schmitz) could
adequately support the growth of bacteria
was reported by Epifanio et al. in 1981. This
study was, thus, conducted to determine the
suitability of "gozo" carrageenan as a
substitute for commercially prepared agar in
culture media used for growing some of the
common fungal pathogens in the locality,
namely, Sphaceloma batatas Saw., Cercospora
henningsii Allesher, Pestalotiopsis spp.,
Sclerotium rolfsii Sacc. and Helmin-'
thosporium maydis Nish. ex Miy, the causal
organisms of sweet potato stem and foliage
scab, cassava leaf spot, gabi marginal leaf
spot, seedling damping off, and corn leaf
spot, respectively.


MATERIALS AND METHODS

Extraction of Carrageenan from Seaweed

One hundred grams of Eucheuma
previously dried until yellowish black was
placed in a 10 liter-container to which 5 liter
of water with 2 g of potassium hydroxide was
added. The mixture was cooked in a boiling
water bath for 3 hr.
The extract paste was strained through a
cheesecloth, allowed to solidify and cut into
15.2 x 2.5 cm strips. The strips were placed
in a freezer for 8-16 hr and then placed under


the sun until carrageenan, a dried porous
structure, was obtained.

Suitability of Seaweed Carrageenan

PDA with different amounts of the
seaweed carrageenan substitute (20, 25, 30,
35, 40 and 45 g) was prepared following the
standard procedure.
Five pathogens, namely, H. maydis, C.
henningsii, S. batatas, Pestalotiopsis spp., and
S. rolfsii were used to test the suitability of
seaweed carrageenan as a substitute for
commercial agar in the preparation of PDA.
A 6-mm diameter plug of the cultured
isolates, except S. batatas, was obtained and
planted at the center of the petri dish with
culture medium. For S. batatas, a loopful of
the spore suspension containing about 1.3 x
107 spores/ml was streaked on the plate.
Mycelial growth of all the isolates was
measured daily for 14 d or until the plates
were filled. Slide mounts of the cultures
were prepared daily to observe for spore
formation. Upon initial formation of spores,
a plug of the isolates, ca. 6 mm in diameter
was randomly obtained and macerated in 5
ml of 0.02% Tween 80 solution for spore
count determination using a haema-
cytometer. The spore suspension in the vial
was left for 24 hr at room temperature.
Using a microscope, one hundred spores
were counted and percent spore germination
was determined. Five replications were
provided for each concentration per
organism. All plates were incubated at room
temperature. Results were compared to
those obtained using standard PDA.

Statistical Analysis

Analysis of variance for completely
randomized design (CRD) was used to
compare differences among treatments.

RESULTS AND DISCUSSION

Growth of Different Fungi on Media with
Varying Concentrations of Seaweed
Carrageenan

Sclerotium rolfsii

S. rolfsii, when grown on PDA (control)
and the medium with different seaweed
carrageenan concentrations, exhibited sparse
radial growth on the first day. On PDA,
white aerial and straight mycelia covered








the plate in 3 d of incubation while creeping
mycelia covered the media with seaweed
carrageenan only after 4-6 d of incubation.
The rate and abundance of mycelial
growth of S. rolfsii varied with the different
concentrations of carrageenan. The
differences observed were significant in the
mycelial growth in the media with varying
concentration of seaweed carrageenan and
the control.


Helmninthosporium maydis

H. maydis on PDA and on the medium
with carrageenan developed compact cottony
and greenish black mycelia. However, it
formed bigger colonies in all concentrations
of seaweed carrageenan than in PDA after 5
of incubation (Table 2). Significant
differences among treatments were observed
starting on the second day of incubation with
40 and 45 g of carrageenan supporting the
best growth.


Table 1. Daily average mycelial expansion of Sclerotium rolfsii as influenced by different
concentrations of gozo carrageenan.

Wt. of Seaweed Colony Diameter (mm)V1
Treatment Carrageenan
No. (g) Day 1 Day 2 Day 3

1 30 11.70 a 37.00 ab 58.50 ed
2 25 13.80 a 34.00 b 56.70 ed
3 30 15.10 a 36.80 ab 62.00 ab
4 35 13.80 a 35.30 b 59.80 be
5 40 14.30 a 33.50 b 58.00 ed
6 45 14.60 a 34.80 b 54.20 d
Control3- 0 14.30 a 40.40 a 66.40 a

1/ Substituted for agar in the preparation of the growth media following the standard procedure for PDA.
2/ Average of 4 replicates; means with the same letters in each day of observation are to significantly different
at 1% with DMRT.
/ Standard potato dextrose agar.



Table 2. Daily average mycelial expansion of Helminthosporium maydis as influenced by
different concentrations of gozo carrageenan.


Treatment WL of Seawe Colony Diameter (mm) -
No. Carrageenam
(g) Day Day2 Day3 Day4 DayS Day6 Day7 Day8 Day9 Day1O Dayll Dayl2 Dayl3 Dayl4
1 20 8.40a 10.20cd 13.40d 16.20d 19.00d 21.20d 24.40ad 27.30bc 29.60cd 30.80ocd 32.60d 36.12b 37.50,b 3~60d
2 25 8.20a 12.800 13.20d 13.20d 20.20d 22.60d 25.60cd 28.80bc 29.10d 31.00 o 36.90abd 35.70bc 36.70,f 38.90f
3 30 8.80e 15.00d 19.60d 22.60abd 25.20ad 27.00abd 28.20d 29.901b 31.340d 35.90abd 38.50ab 44.76a 47.42be 49.600b
4 35 0.40a 17.40be 21.00b 26.0ab 27.60b 32.00ab 32.20b 33.50bc 35.00bc 36.30abC 38.10abd 40a.60ab 41.320d 44.16
5 40 8.80a 21.60 24.00a 29.20a 31.80e 35.40a 36.40ab 45.70 39.30ab 42.82ab 44.50ab 50.00a 51.30ab 54.40ab
6 45 0.60A 18.80ab 21.20ab 25.20ab 28.80ab 31.80ab 37.00 39.30ab 43.10a 47.10a 47.30" 51.10a 52.54a 55.30a
control. 0 8.200 12.20d 14.400d 16.20d 17.80d 19.80d 2060d 21.000 21.70d 22.30F 22.90 24.32c 24.58f 26.408

1' Substituted for ar in the preparation of the growth media following the standard procedure for PDA.
2/ Average of 5 eplicates; means with the same letters in each day of observation are not significantly different at 1 % with DMRT.
/ Standard potato dextroes agar.







Pestalotiopsis spp.

Mycelial growth of Pestalotiopsis spp. on
PDA and on the medium with 30, 35, 40 and
45 g/l carrageenan/liter was straight and
scanty. At early stages of growth, the mycelia
were glossy, trans-parent, yellowish and flat
on the medium surface. In the later stages,
white cottony growth was formed around the
seeded inoculum disc. An even thin cottony
growth on the surface of the previous growth
was formed when the fungus was about to
sporulate. On the medium with 20 and 25 g/l
carrageenan, the mycelia were straight,
creeping and transparent. Pestalotiopsis spp.
exhibited faster mycelial expansion in most
treatments than on PDA (Table 3). The
highest concentration of sea-weed
carrageenan (45 g/l) supported the lowest
mycelial growth which was not significantly
different from that on PDA.

Cercospora henningsii

Onion agar was used for growing C.
henningsii, which in accordance with the
previous findings of Martinez (1979), did not
sporulate on PDA. There was no
considerable difference ob-served in the
appearance of mycelia on the different
concentrations of carrageenan and onion
agar. The mycelia ranged from sparse to
abundant and white to dirty white or grayish.
Table 4 shows that the variations among
treatments were highly significant during the
second and third days of incubation. Fastest
growth was observed at carrageenan con-
centrations of 25 and 30 g/1. Slowest growth
was ob-tained at the lowest concentration (20
g/l) which did not vary significantly with the
control.

Sphaceloma batatas

Different concentrations of carrageenan
caused variations in the growth of S. batatas.
At lower concentrations of carrageenan (20
to 30 g/l), metallic black to metallic brown,
flat colonies grew. At higher concentrations
(35 to 40 g/l), the fungus formed flat, straw-
colored colonies during the first week of
incubation and turned brownish with
greenish tint at the side on subsequent days.
On PDA, the colony was brown to reddish
and were bigger. The growth of S. batatas
was determined by considering its
abundance, scarcity or absence on the
medium. Spores of the fungus germinated
after three days of incubation on PDA and
four days on the medium with carrageenan.
Initially, the fungus performed better on


PDA than on the medium with carrageenan
with the lowest cartageenan concen-tration
supporting the best growth (Table 5). From
the 9th to the 14th day however, PDA and
medium with carrageenan concentrations of
20 g/l exhibited the same amount of growth.
Treatment 6 which had the highest
carrageenan concentration supported the
least number of colonies with treatments 2, 3,
4 and 5 exhibiting no considerable difference
in terms of the amount of colony growth.
The above results show that the rate of
growth of the organisms varied at different
carrageenan concentrations. These
differences can be attributed to the
characteristics of the fungi themselves and to
the nutrient and moisture contents of the
medium with varied proportions of
carrageenan.

Spore/Sclerotium Count and Germination
of Fungi in Medium With Varying
Concentrations of Seaweed or "gozo"
Carrageenan

Sclerotium rolfsii

S. rolfsii formed selerotial bodies on all
concentrations of seaweed carrageenan
tested (Table 7) with the degree of sclerotial
formation varying significantly with the
concentration. The highest number of
sclerotia was observed in Treatment 3 (30
g/l) which was significantly lower compared
to that in the control.
In general, media that support extensive
growth yield the highest number of sclerotia
(Punja, 1985). Thus, the observation that
PDA which supported the greatest mycelial
growth also yielded the highest number of
sclerotia followed by Treatment 3. It was
noted, however, that sclerotial bodies formed
on the medium with carrageenan were
appreciably bigger than those on PDA. This
can be attributed to the relatively higher
protein content of carrageenan than that of
agar-agar (Table 6).
It was also observed that sclerotial body
formation started 6 days after inoculation in
Treatments 5 and 6 (40 and 45 g/l,
respectively) while only after 7 to 8 days on
PDA and in Treatments 2 and 3. At the
lowest concentration, on the other hand,
vegetative growth was prolonged with
sclerotia formation starting only on the 12th
day of incubation. The early production of
sclerotia in Treatments 5 and 6 was due to
the limited amount of available nutrients in
the media. Moreover, since water was low in
relation to the solute (especially the
carrageenan) in the media, the nutrients










Table 3. Daily average mycelial expansion of Pestalotiopsis spp. as influenced by different
concentrations of gozo carrageenan.


Treatment Wt. of Seaweed Colony Diameter (mm)Z
No. Carrageenanl/
(g) Day 1 Day 2 Day 3 Day 4 Day 5


1 20 17.00de 26.40be 47.70ab 58.30ab 72.50ab
2 25 18.80a 24.80cd 45.20bc 57.10bc 71.90cd
3 30 17.80cd 24.60de 42.70de 55.40cd 70.80de
4 35 18.60ab 27.00ab 45.10cd 58.30ab 72.30bc
5 40 18.40bc 27.20a 46.20a 60.40a 76.20bc
6 45 14.40de 19.20f 35.40f 46.80e 61.70f
Control3/ 0 13.60e 20.40ef 39.00ef 50.80de 63.00ef

1/ Substituted for agar in the preparation of the growth media following the standard procedure for PDA.
2/ Average of 5 replicates; means with the same letters in each day of observation are not significantly different at 1% with
DMRT.
/ Standard potato dextrose agar.






Table 4. Daily average mycelial expansion of Cercospora henningsii as influenced by different
concentrations of gozo carrageenan.

Treatment Wt. of Seaweed Colony Diameter (mm)2/
No. Carrageenan1'
(g) Day I Day 2 Day 3
1 20 22.00b 46.40d 60.00ef
2 25 28.00a 58.60a 74.40a
3 30 24.80ab 56.00ab 73.80ab
4 35 23.60b 50.0cd 65.00de
5 40 21.60b 51.60bc 68.00bc
6 45 22.00b 51.00bc 67.20cd
Control/ 0 22.80b 46.20d 56.20

1/ Substituted for agar in the preparation of the growth media following the standard procedure for PDA.
2 Average of 5 replicates; means with the same letters in each day of observation are not significantly different at 1%
with DMRT.
SStandard onion agar.










Table 5. Growth of Sphaceloma batatas on PDA and the medium -with different
concentrations of gozo carrageenan.U


Treatment Wt. of Seaweed
No. Carrageenan Day 3 Day 6 Day 9 Day 12 Day 14
(g)

1 20 +++ ++++++ +++++++ ++++++++

2 25 ++ +++++ ++++++ +++++++
3 30 ++ +++++ ++++++ ++++++

4 35 ++ +++++ ++++++ ++++++
5 40 ++ ++++ +++++ ++++++

6 45 ++ ++ ++ +++

Control3/ 0 + +++ ++++++ +++++++ ++++++++


/ Data taken daily of 14 days, average of 5 replicates; = absence of growth; + = amount of growth.
2/ Substituted for agar-agar in the preparation of growth media following the standard procedure for preparing PDA.

/ Standard potato dextrose agar.




Table 6. Nutrient contents of seaweed carrageenan and agar-agar (%).


Sample Nitrogen Protein Sugar Starch


Seaweed carrageenan 0.321 2.01 0.68 66.78

Agar-agar 0.1405 0.878 0.92 90.72







Table 7. Spore/sclerotium count and % spore germination on PDA and on medium with varying
concentrations of gozo carrageenan./-

Wt. of S. rolfsii H maydis Pestalotopsis spp. S. batatas C henningii
Treatment Seaweed
No. Carrageenan2' Sclerotial % .Cerfi- Spores % Gernm- Spores % Germi- Spores % Germi- Spores % Germi-
(g) bodies nation. (X10") nation (X10 ) nation (X1Z) nation (X10 nation

1 20 7.8f 100 25883 13. 2583b 21.40'
2 25 34.2' 100 222.2 15.6bc 222.2c 18.40a
3 30 86.8P 100 167.6d 18.9b 38.8e 167.6d 19.60a
4 35 76.8 100 1333' 14.4 1833 1333e 19.20a
5 40 67.8c 10 833- 14.C 475.0a 833- 19.20"
6 45 47.0d 100 355g 13.6 275.0 35'5 19.20a


Control 0 121.0' 100 302.0a .22.0a 341.6" 302.0a 23.80'


1/ Average of five replicates, means with the same letters ir eacn column are not significantly different at 1% with DMRTI
2J Subsituted for agar-agar in the preparation of the growth media following the standard procedure for PDA.
3/ Potato dextrose agar.







became less available to the organism. This
is in consonance with the observation of
Christias and Lockwood (1973) that the time
of production of sclerotia was closely related
to the time of depletion of carbohydrate and
other nutrients in the media. All sclerotia
from treatments with carrageenan
germinated upon transfer to PDA.

Helminthosporium maydis

The highest number of spores of H.
maydis was obtained from treatments which
supported lower mycelial growth (Table 2).
Treatment 1 (20 g/l) and the control yielded
the highest number of spores while
Treatment 6 (45 g/l) gave the lowest spore
number (Table 7).

Pestalotiopsis spp.

Pestalotiopsis spp. did not sporulate in
Treatments 1 and 2 but yielded the highest
number of spores in Treatment 5 which was
significantly higher than that in the control
(Table 7). Spores from all treatments with
carrageenan did not germinate after 24 hr of
incubation.

Sphaceloma batatas

S. batatas yielded spores in direct
proportion to the amount of carrageenan
added to.the medium (Table 7). The control
gave the highest spore count followed by
Treatment 1 while the lowest spore count
was observed at the highest carrageenan con-
centration (Table 7). No significant
difference in percentage spore germination
of the fungus was ob-served among the
different treatments and the con-trol.

Cercospora henningsii

The fungus failed to sporulate in all
treat-ments. Very few conidia were observed
in one of the control plates. No spores were
observed for the sporulation stage might not
have been reached yet since observation was
concluded after 14 days.. Paningbatan and
Ilag (1981) obtained considerable sporulation
of Cercospora arachidicola and C. per-
sonatum on onion agar beyond 14d of
incubation. Lastly, the temperature at which
the cultures were incubated might not have
been favorable for the or-ganisms to
sporulate.


LITERATURE CITED


BISCHOOF, T.W. and M.O. GURRAWAY.
1985. The effect of xylose on the growth
and sporu-lation of an isolate of
Bipolaris maydis race T and its relation
to pH and ammonium levels.
Phytopathology 75:138-141.

BOOTH, C. 1971. Fungal culture media. p.
49-94. In Methods in Microbiology.
Vol. 4 C. Booth, ed. Acad. Press, New
York.

CHRISTIAS, C. and J.C. LOCKWOOD.
1973. Conservation of mycelial
constituents in four sclerotium-forming
fungi in nutrient deprived conditions.
Phytopathology 63:602-605.

DUCKWORTH, M., K.C. HONG and W.
YAPHE. 1970. The agar
polysaccharides of Gracilaria species.
Carbohydrates Res. 18:1-9.

EPIFANIO, E.C., R.L. VERY, F.
UYENCO, GJ.B. CAJIPE and E.
LACERNA. 1981. Carrageenan from
Eucheuma striatum (Schmitz) in
bacteriological media. Appl. Environ.
Microbiol. 41:155-158.

FERNANDEZ, W.L. M.S. ALEJAR and
J.C. GONZALES. 1977. Coconut water
broth and agar as microbiological
culture media. (Abstr.). Kalikasan,
Philipp. J. Biol. 6:78-79.

ILAG, L.L., A.R. PUA and VA. MARFIL.
1982. Sweet potato sucrose agar an
inexpensive culture medium for fungal
growth. Philipp. Phytopathol. 18:78-88.

MARTINEZ, MA. 1979. Susceptibility of
cassava at different stages of growth of
Cercospora leaf spot disease.
Undergraduate Thesis, Visayas State
College of Agriculture (ViSCA),
Baybay, Leyte.

MERCADO, V.M. and A.N. PORDESIMO.
1968. The effect of chicken manure on
the incidence of Sclerotium disease of
vegetables. Philipp. Phytopathol. 4:75-
87.







PANINGBATAN, RA. and L.L. ILAG. WATSON, N. and D. ASPIRION. 1976.
1981. Sporulation in vitro of Substitute for agar in solid media for
Cercosporidium personatum (Berk. and common usages in microbiology. Appl.
Curt.) Deighton and Cercospora Environ. Microbiol. 31:509-513. (As
archidicola Hori from peanut. Philipp. cited by Epifanio et al., 1981. Appl.
Phytopathol. 17:21-29. Environ. Microbiol. 41:155-158).

PUNJA, Z.K. 1985. The biology, ecology and
control of Sclerotium rolfsii. Annu.
Rev. Phytopathol. 23-97-127.

RODULFO, B.R., A.M. VUELTRAN, A.C.
MONROYO, B.B. DON JUAN, F.E.
ANZALDO, F.T. CASIL, C.B.
SAMSON, F.L. MONROYO and C.M.
SANTIAGO JR. 1985. Agar-agar
extraction and purification process.
NIST J. 2:6-8.

SANTIAGO, C.M., B.M. MERCADO, B.
BLANCO-DE GUZMAN and M.B.
BIGOL. 1985. Microbiological
application of agar-agar extracted from
local seaweeds. NIST J. 2(4):9-11.

SANTOS, GA. 1980. Quality of carrageenan
and agar. p. 123-129. In Pacific
Seaweeds Aquaculture. Proceeding of a
Symposium on Useful Algae. E.A.
Abbott, M.S. Poster and L.F. Eklund
(eds.). University of Californiaa,
California.

STAINER, R.Y. 1978. What is
microbiology? p. 1-32. In Essays in
Microbiology. John Wiley and Sons
Ltd., New York, NY.

TRAINOR, F.R. 1978. Introductory
Phycology. John Wiley and Sons Inc.,
New York, NY.

TRIONE, E.J. 1974. Sporulation and
germination of Phytophthora laterales.
Phytopathology 64:1531-1533.

VANNAJAN, S. and G.C. TRONO, JR.
1977. The marine benthic algae of
Manila Bay. I. Intro-duction,
Cyanophyta and Chlorophyta. Kali-
kasan, Philipp. J. Biol. 6:33-36.

WALKER, A.W. and A.A. DAY. 1943.
Extracts from Irish moss as a substitute
for agar in bac-teriological culture
media. Food Res. 8:435-443. (As cited
by Epifanio et al., 1981. Appl. Environ.
Microbiol. 41:155-158).









VARIANTS OF RICE GRASSY STUNT VIRUS IN THE
PHILIPPINES

Gilda J. Miranda, H. Koganezawa, and N.B. Bajet


Research Assistant, Plant Virologist, International Rice Research Institute, P.O. Box
933 Manila, Philippines and Assistant Professor, University of the Philipines at Los Baios
(UPLB), 4031 College, Laguna, respectively.
Portion of the thesis of the senior author submitted to the Graduate School, UPLB, as
partial fulfillment for the degree of Master of Science.

ABSTRACT

Four distinct and stable variants or isolates of rice grassy stunt virus
(RGSV) were obtained by sequential inoculation. They were designated as M3
(very mild), M2 (moderately mild), SC (moderately severe), and S2 (severe).
These isolates differ in their symptomatology, reaction to Oryza nivara L. and
other 0. nivara-derived varieties, and transmission characteristics by
Nilaparvata lugens (Stal).


INTRODUCTION

Grassy stunt disease of rice (Oryza
sativa L) is commonly found in various rice-
growing regions in South, Southeast, and
East Asia (Ou, 1985; Hibino et al., 1985;
Hibino, 1986) causing severe damage to the
rice crop. The disease has gained importance
because its pathogen, rice grassy stunt virus
(RGSV), is persistently transmitted by one of
the most important rice pests,brown
planthopper (BPH) Nilaparvata htgens (Stal)
(Hirao et al., 1985). In the Philippines, a
major outbreak of the disease was reported
in Laguna in 1973-74 (IRRI, 1973). The
isolate type of the virus was characterized by
symptoms that included severe stunting,
excessive tillering, and an erect growth habit.
This was designated as RGSV1. In 1979,
another type of the virus was observed in the
Philippines and was designated as RGSV2. It
induced tungro disease-like symptoms
(Cabauatan et al., 1985; Hibino, 1985).
We observed that RGSV source plants
in the Virology greenhouse of the
International Rice Research Institute (IRRI),
Los Baltos, Laguna, Philippines, showed
various types of symptoms which were
different from those caused by RGSV1 and
RGSV2. These included very severe, very
mild or almost symptomless on cultivar
Taichung Native 1 (TN1). The vector
transmissibility of RGSV was low compared
to previous reports.


Understanding the biology of these
different RGSV isolates is important
particularly in screening for resistance and
generally in formulating possible control
management measures. Therefore, this study
was conducted to obtain stable and distinct
isolates by sequential inoculation and
characterize them based on a)
symptomatology, b) reaction to 0. nivara
and other 0. nivara-derived varieties, and c)
transmission characteristics by N. lugens.

MATERIALS AND METHODS

Source of Infected Plants
RGSV-infected plants maintained in the
Virology greenhouse which showed distinct
symptoms were obtained and rice plants
infected with RGSV from the field were also
collected. All samples were tested for
RGSV, rice tungro viruses, and ragged stunt
virus by latex flocculation test (LFT, see
below). Those disease sources that reacted
positively only to the anti-RGSV serum were
selected as RGSV sources.

Isolation of Variants
Virus-free second instar N. lugens were
confined on each of RGSV sources for a 4-d
acquisition access period (AAP). The
viruliferous insects were confined for another
6 d on 7 to 10-d-old TN1 seedlings and then
individually given sequential inoculation
access period (IAP) of 1 d on 7-to 10-d-old








TN1 seedlings until the insect died.
Inoculated seedlings were transplanted into
pots and grown in insect-free cages for
symptom development. Plants showing
symptoms similar to the original virus source
and those which reacted positively to the
anti-RGSV serum by LFT (See below) were
transplanted separately in pots. These were
used as virus sources for the second transfer.
The transfer and selection of RGSV isolates
continued serially until all infected plants
showed symptoms similar to those of the
original source plants.

Symptomatology
Aside from TN1, diagnostic species for
RGSV such as Shansan-sa-san and Reiho
(Hibino, 1986) were also inoculated with the
different RGSV isolates. Seven-to ten-d-old
seedlings were mass-inoculated and confined
separately in screened cages in the
greenhouse to avoid contamination.
Symptoms which developed on each cultivar
infected with the different RGSV isolates
were recorded. Infected plants were indexed
3-4 wk from inoculation by LFT (see below).


Varietal Reaction
The reaction of the four isolates to rice
varieties 0. nivara, IR42, IR54, and TN1 was
studied. The mass inoculation for testing
resistance to RGSV was carried out as
described by Ling et al. (1970). Three to four
wk after inoculation, the second youngest leaf
of each plant was sampled and indexed by
LFT (see below). The rating scale
categorized 0-30% infection as resistant, 31-
60% as intermediate, and 61-100% as
susceptible. The experiments were laid out
in a two-factor randomized complete block
(RCB) design with three replicates.

Transmission Tests
Serial daily transmission using BPH
determined virus-vector relationships of each
RGSV isolate. The relationships determined
were incubation period, percentage of active
transmitters, and retention period. First to
second instars of N. lugens nymphs were
given a 4-d AAP on a source plant from each
isolate and then individually given a 1-d
sequential IAP on 7-to 10-d-old TN1
seedlings until the planthopper died.


Figure 1. Symptoms of the different RGSV variants collected from the greenhouse and field
in about 2-month-old TN1 plants. Plants 2,4,6 and 7 designated as S2, SC, M2,
and M3, respectively, were the isolates with distinct and stable symptoms. Plant
1,3,'and 5 failed to show stable symptoms. H is health TN1 plants.







Seedlings exposed to the planthoppers were
then transplanted into pots and grown in
insect-free cages for observation of symptom
expression. The data for incubation period in
the plant and in insects were further analyzed
using probit analysis (Finney, 1971).

Latex Flocculation Test (LFT)
The LFT as described by Omura et al.
(1984) was used to assay leaf samples for
RGSV. A portion of the second youngest
leaf approximately 10 cm in length of an
inoculated plant was collected. Each sample
was homogenized separately in 1 ml 0.05 M
Tris-HC1, pH 7.2, using a combined leaf and
bud press (Erich Pollahne, Wennigsen,
Federal Republic of Germany). Sap was
mixed with sensitized latex suspension and
the mixture was shaken for 15-30 min. The
presence of RGSV antigen was determined
by the appearance of latex particle clumps
when viewed under a light microscope at
100X magnification. Sensitized latex
suspensions were also mixed with healthy
plant extracts as controls.

RESULTS
Isolation of RGSV Variants
The RGSV sources were classified
into 7 groups based on symptomatology (Fig.
1). From these, only four isolates with
distinct and stable symptoms were obtained
after a total of 5-6 series of transfers by BPH.
These were designated as very mild (M3),
moderately mild (M2), moderately severe
(SC), and severe (S2). M3, M2, and S2 were
selected from the original six greenhouse
isolates while the SC was obtained from a
field in South Cotabato.

Symptomatology
Symptoms caused by the four RGSV
isolates on 3-4-wk-old TN1 plants differed
from each other. The M3 isolate was the
mildest and induced symptoms that were
almost not discernable as plants appeared
similar to healthy ones. The M2 isolate
induced slight stunting and less tillers with an
erect growth habit. Affected leaves became
narrow and slightly yellow. TN1 plants
infected with SC isolate showed profuse
tillering. Plant height was slightly shorter
than those with M2. The leaves were narrow,
had rusty necrotic leaf spots, and showed
pale green to yellowing discoloration. The S2
isolate induced severe stunting, less profuse
tillers, and a spreading growth habit. The
leaves were narrow, yellow and had rusty
necrotic spots.


On TN1, symptoms of M3 usually
appeared from 19-38 d (ED50 = 26 d), M2
from 18-32 d (ED50 = 22 d), SC from 7-20 d
(ED50 = 14 d), and S2 from 8-19 d (ED50 =
11 d). Shansan-sa-san and Reiho plants
infected with the different isolates developed
additional symptoms which were distinct
from those of TN1. The four RGSV isolates
caused striping in the leaves on Shansan-sa-
san and premature death was often observed
on Reiho infected with SC and S2 isolates.

Varietal reaction
0. nivara and varieties IR42 and
IR54 which possess 0. nivara genes were all
resistant to the M3 and S2 isolates. These
rice species and varieties, however, were
susceptible to M2 and had intermediate
reaction to the SC isolate. TN1, which does
not possess any 0. nivara genes, also showed
resistance to the M3 isolate and intermediate
resistance to the S2 isolate but was
susceptible to both the M2 and SC isolate.
All varieties reacted to M3 and M2 isolates in
the same way as TN1 (Table 1).

Transmission Tests
M2 isolate (12.1%) obtained the
highest percent active transmission followed
by the SC and S2 isolates (6.6, 5.2%,
respectively). M3 isolate (1.3%) obtained the
lowest percent active transmission (Table 2).
All RGSV isolates were persistently
transmitted by the BPH but some insects
transmitted only once. The incubation period
of the RGSV isolates in the insect was from 5
to 26 d for both M3 and M2 with
corresponding ED50 of 11 and 8 d,
respectively. The SC and S2 isolates had
incubation periods of 5-18 and 5-15 d,
respectively, and both isolates showed the
same ED50 of 7 d. The insects remained
infective even afer molting. The transmission
patterns of all isolates were more or less
intermittent.


DISCUSSION

Four stable and distinct isolates namely
M3, M2, SC, and S2 were obtained from
among the seven varying RGSV symptoms
observed in the greenhouse and field.
So far two strains of RGSV (RGSV1 and
RGSV2) had been reported in the
Philippines. Both strains caused almost
identical symptoms when the plants were
inoculated at the seedling stage. However,
when inoculated at a later stage of growth,
RGSV2 induced tungro-like symptoms.







Table 1. Cultivar reaction to the four RGSV isolates mass-inoculated using Nilaparvata lugens and
indexed by the latex flocculation test 3-4 weeks from inoculation.


CULTIVAR RGSV isolates1

M32 M2 Sc S2

TN1 9.6 (R)ay 69.2 (S)abx 70.9 (S)ax 32.5 (I)ax

IR42 3.7 (R)az 75.7 (S)ax 51.0 (I)ay 27.9 (R)aby

IR54 15.4 (R)ay 66.1 (S)abx 48.0 (I)ax 22.5 (R)aby

Oryza
nivara 8.8 (R)ay 75.9 (S)abx 55.0 (I)ax 6.8 (R)by

1 Percent plant infection. Letters enclosed in parenthesis are the following, R = resistant; I = intermediate reaction; and S
= susceptible. Means having common letters in columns (abc) and rows (xyz) are not significantly different at the 1% level,
Respectively by DMRT.
2 M3 = very mild; M2 = moderately mild; SC = moderately severe; S2 = severe isolates.



Table 2. Transmission of the four RGSV isolates to 7-to 10-day-old TN1 seedlings by
Nilaparvata lugens.


s Proportion of2 Incubation period (ED50)3
Isolate infected plants

In BPH In plant

M3 4/311 b 5-26 a 19-38 a
M2 41/339 a 5-26 ab 19-32 b
SC 20/304 a 5-18 b 7-20 c
S2 16/309 ab 5-15 b 8-19 d

Means having a common letter are not significantly different at the 1% level by DMRT.
1 Isolates: M3 = very mild; M2 = moderately mild; SC = moderately severe; S2 = severe.
2 Number of infected plants/total number of plants inoculated using test tube inoculation. Inoculated TN1
plants were indexed using latex test 3-4 weeks from inoculation.
3 Effective incubation time calculated by quant, probit analysis, at which 50% of the total viruliferous BPH
tested transmitted the virus and 50% of the total inoculated TN1 plants showed symptoms.


Since the two strains were closely related
serologically (Cabauatan, et al., 1985),
another way to differentiate them was in their
ability to infect 0. nivara and varieties with
0. nivara genes. 0. nivara was resistant to
RGSV1 but susceptible to RGSV2 while TN1
was susceptible to both RGSV1 and RGSV2
(Cabauatan et al., 1985; Hibino et al., 1985).
Results obtained in this study showed that O.
nivara was susceptible to the M2 isolate, and
intermediately susceptible to the SC isolate
(Table 1). The M2 and SC isolates resemble


RGSV2 in this respect. However, our results
were not sufficient to prove or to disprove
that these isolates are or belong to RGSV2.
Inoculation of these isolates to aged rice
plants to examine for the appearance of
tungro-like symptoms is yet to be done. The
four isolates also differ from the original
RGSV1. TN1 and 0. nivara showed similar
reaction to each of the four isolates.
Chen and Chiu (1982) reported three
types of symptoms of RGSV, namely GSW,
GSB, and GSY in Taiwan. Among the







isolates obtained in our study, SC resembled
GSB as both induced excessive tillering in
TN1 and Shansan-sa-san. S2 resembled
GSW for the severe stunting and narrowed
leaves of TN1. Likewise, Iwasaki et al (1985)
reported the presence of mild, ordinary, and
severe strains in Japan. Our M2 and M3
isolates resemble their mild strain in
symptomatology on cultivar Reiho. Their
severe strain is also similar to our S2 isolate.
However, tests for direct comparisons to
establish relationships between the Taiwan,
Japan, and our isolates have not yet been
carried out due to plant quarantine
regulations.
The four isolates were transmitted by
the BPH in a persistent manner similar to
results of previous studies conducted in the
Philippines (Hibino et al., 1985). Likewise,
transmission efficiencies of the three BPH
biotypes and the greenhouse-reared colony
did not significantly differ from each other
(Jonson, 1990). Transmission efficiencies,
however, varied for each virus isolate. The
highest percent active transmission was
obtained with the M2 isolate, followed by SC,
S2, and M3 isolates. Percent active
transmission of M2, SC, and S2 corresponded
quite closely to the reported ranges of
percent active transmission obtained for
RGSV2 which was 5-20% (Hibino et al.,
1985). M3 on the other hand, did not fall
within the reported ranges for either RGSV1
or RGSV2 which were 26-31 and 5-20%,
respectively (Cabauatan et al., 1985; Hibino
et al., 1985).
The effective incubation period in
the plant also varied with each isolate. The
incubation period of the mild isolates
appeared to be longer than the severe
isolates. The incubation periods of M3 and
M2 appeared beyond the reported ranges of
RGSV1 and RGSV2 while both SC and S2
isolates appeared more or less in the same
ranges as those of RGSV1 and RGSV2
(Cabauatan et al., 1985; Hibino et al., 1985).
Similarly, results show that the incubation
periods in the insect of the mild isolates were
relatively longer than those of the severe
isolates though both mild and severe isolates
fell within the reported ranges of RGSV1,
RGSV2, and even the Taiwan isolates
(Cabauatan et al., 1985; Hibino et al., 1985;
Chen and Chiu, 1982).
The scope of the present study was
limited to an examination of certain
biological aspects which may not be
considered as definitive criteria for the
differentiation and identification of strains.
Hence, these variants are designated as


isolates instead of strains. Further studies on
cross protection, serology, and structural
criteria which are more sensitive and
discriminating should also be undertaken to
establish if they are considered definitively as
strains of RGSV.



Acknowledgment

The authors appreciate the
suggestions made by Drs. H. Hibino, T.W.
Mew, and V.J. Calilung during the course of
the experiment.



LITERATURE CITED


CABAUATAN, P.O., H. HIBINO, D.B.
LAPIS, T. OMURA, and T.
TSUCHIZAKI. 1985. Rice grassy
stunt virus 2: a new strain of rice
grassy stunt virus in the Philippines.
IRRI Res. Pap. Ser. No. 106. 8 pp.

CHEN, C.C. and R.J. CHIU. 1982. Three
symptomatologic types of rice virus
diseases related to grassy stunt in
Taiwan. Plant Dis. 66:15-18.

FINNEY, D.J. 1971. Probit analysis. 3rd ed.
Cambridge Univ. Press, Great
Britain. 333 pp.

HIBINO, H., P.Q. CABAUATAN, T.
OMURA, and T. TSUCHIZAKI.
1985. Rice grassy stunt virus strain
causing tungro-like symptoms in the
Philippines. Plant Dis. 69:538-541.

HIBINO, H. 1986. Rice grassy stunt virus.
AAB descriptions of plant viruses,
No. 320. Commonw. Mycol. Inst.
Surrey, U.K.

HIRAO, J., S. OYA, and H. INOUE. 1985.
Transmission of rice grassy stunt
virus (RGSV) by the brown
planthopper, Nilaparvata lhgens Stal
(Hemiptera:Delphacidae). Bull.
Kyushu Natl. Agric. Exp. Stn.
24:307-337.

INT. RICE RES. INST. (IRRI). 1973.
Annual Report for 1973. Los Banos,
Philippines. 402 pp.







IWASAKI, M., M. NAKANO, and A.
SHINKAI. 1985. Strain of rice
grassy stunt virus with different
symptoms. Ann. Phytopath. Soc.
Japan. 51:351.

JONSON, G.B. 1990. Variants of rice grassy
stunt virus in the Philippines.
Unpublished M.S. Thesis, University
of the Philippines at Los Banos,
Philippines. 56 p.

LING, K.C., V.M. AGUIERO, and S.H.
LEE. 1970. A mass screening
method for testing resistance to
grassy stunt disease of rice. Plant
Dis. Reptr. 54:565-569.

OMURA, T., H. HIBINO, T. USUGI, H.
INOUE, T. MORINAKA, S.
TSURAMACHI, CA. ONG, M.
PUTTA, T. TSUCHIZAKI, and Y.
SAITO. 1984. Detection of rice
viruses in plants and individual insect
vectors by latex flocculation test.
Plant Dis. 68:374-378.

OU, S.H. 1985. Rice diseases, pp. 45-48.
Common. Mycol. Inst. Surrey, U.K.










EFFECT OF TEMPERATURE AND HUMIDITY ON
GERMINATION AND INFECTION OF Colletotrichum
gloeosporioides (PENZ.) SACC. ON 'CARABAO' MANGO
(Mangifera indica L.)

A. BI Estrada and L. L. lag

Formerly, University Research Associate, Postharvest Horticulture Training and Research
Center and Professor, Department of Plant Pathology, University of the Philippines at Los
Banos, College, Laguna, respectively.

Portion of the Master's thesis of the senior author, U. P. at Los Banos.


ABSTRACT

Experiments were conducted to evaluate the effect of temperature (T)
and relative humidity (RH) on germination and growth of the anthracnose
fungus Colletotrichum gloeosporioides (Penz.) Sacc. on mango (Mangifera
indica L.). Results of these experiments together with actual field weather data
were used to evaluate the worthiness of the scheduled spray program employed
in a mango orchard in Dasmarinas, Cavite.
Germination and growth of C. gloeosporioides were highly sensitive to T
and RH. Germ tubes were formed and mycelia grew optimally at 25 to 300C.
Spore germination and fungal growth increased as the RH was increased from
90 to 100%. Germination on glass slides was inhibited at RH of 90% and
below even after 36 h at 300C. Germination percentage was higher and more
profuse growth were observed on leaves and fruit peels than on glass slides.
There was no relationship between the stage of fruit development and spore
germination, mycelial growth, and appressorial formation.
Appressorial formation increased with increasing T and RH. No
appressoria were formed on glass slides. Increased appressorial formation
resulted in increased severity of anthracnose infection in mango leaves. Lesions
appeared earlier and were more severe under 97.5 and 100% RH at 25 and
30 C.
Evaluation of the local standard spray program revealed that majority of
the scheduled fungicide sprays were made during low anthracnose-risk periods.


INTRODUCTION

Mango (Mangifera indica L.) is one of the
most important tropical fruits grown
commercially in the Philippines (Mendoza
and Wills, 1984) particularly the cultivar
'carabao' which is estimated to comprise
about 90% of the existing mango trees in the
country (Guianzon, 1977). Export volumes of
this commodity in 1987 amounted to
13,431,707 gross kilograms FOB valued at US
$ 12,492,571 (National Census and Statistics
Office, personal communication). This
amount, could have been larger if not for
some production and postharvest constraints,
one of which is the mango anthracnose
caused by Colletotrichum gloeosporioides


(Penz.) Sacc.. Anthracnose is the most
important field and postharvest disease of
mango (Cook, 1975; Palo, 1932). The malady
results in reduced production, decreased
quality of fruits and increased cost of input.
Although numerous researches in
mango anthracnose have been done locally
and elsewhere, adequate information on the
epidemiology of the disease is still lacking.
Mango growers follow a scheduled spraying
program intended to minimize the effect of
the disease but which may actually result in
erroneous applications with fungicide being
applied when not needed, or fungicide not
applied when actually required. Basic
information on factors affecting the
anthracnose fungus may help considerably in









efficiently and economically controlling the
disease. This study was undertaken to
evaluate the effect of temperature and
humidity on the biology of the anthracnose
fungus. Findings from these experiments
were used to assess the worthiness of the
scheduled spray program in Dasmarinas,
Cavite.

MATERIALS AND METHODS

Isolation of the Fungus

C. gloeosporioides was isolated from infected
fruits from Cavite and Batangas by tissue
planting on potato dextrose agar (PDA).
Pure cultures of the Cavite and Batangas
isolates, designated as Il and 12, respectively,
were maintained and used m subsequent
studies.

Determination of Optimum Humidity and
Temperature for Conidial Germination and
Growth on Glass Slides

A. Effect of Humidity

Humidity experiments were conducted
using bottles of approximately three-liter
capacity. Various concentrations of glycerol
in water as adapted from Doberski (1981)
were followed to adjust the humidity within
the bottles.The percentages of pure glycerol
that were used for maintaining different
relative humidity (RH) levels were as follows:
51% RH = 79.3%, 74% RH = 59.8%, 86%
RH = 41.0%, 90% RH = 31.4%, 95% RH =
18.8%, 97.5% RH = 10.0%, and 100% RH
= 0% glycerol. Each bottle contained about
600 ml of the respective water-glycerol
solutions.

Spores from 7 and 14 d old cultures of
C. gloeosporioides were brushed off from the
culture plates and streaked on clean and dry
glass slides using a watercolor brush. The
concentration of spores was not adjusted
using a haemacytometer as wetting of spores
may interfere with the aim of determining the
effect of humidity alone. The slides were
placed on screens inside the bottles. Counts
on the number of germinated and non-
germinated spores were done randomly from
100 spores in each isolate at each humidity
level after 18 and 36 h. A spore was
considered to have germinated when the
germ tube formed has exceeded half the
length of the spore.


B. Effect of Temperature

A similar set-up as in the humidity trials
was made in the temperature (T)
experiments but each bottle contained only
600 ml water to maintain high humidity
within the chamber. The bottles were stored
at 15, 20, 25, 30, 35, and 400 C. Spores of
each isolate were brushed on glass slides and
placed on the screens inside the bottles.
Percentage germination and germ tube
growth were recorded after 18 and 36 h.

Germination and Growth at Various
Combinations of Temperature and Humidity

The combined effect of T and RH on
the growth of C. gloeosporioides on leaves
and fruit peels was investigated. Different
humidity solutions namely 90, 95, 97.5 and
100% were prepared using glycerol in bottles.
The bottles were then stored at 20, 25, and
300C.

A. Spores on Leaves

Spores from 7 d old cultures of the
fungus were brushed on the upper surface of
leaf samples (about 1 month old). The end of
leaf petioles were inserted into water picks
before the samples were placed on the
screens inside the bottles. After 18 and 36 h,
leaves were taken out of the bottles and the
brushed areas were examined by a stripping
technique as described by Fitzell et al. (1984)
using a nail varnish-acetone solution (50%
v/v). Infection of leaves at the different T-
RH levels were also evaluated. Leaf samples
brushed with spores were incubated inside
the bottles and severity of lesions was
recorded after 5 d of incubation.

B. Spores on Fruit Peels

Peel samples measuring 3-4 cm long
and 1 cm wide were taken from fruits at
different stages of development e.g. 57, 76, 97
and 119 d after flower induction. The peels
were brushed with spores from 12 and then
kept inside the bottles. After 18 and 36 h, the
brushed areas of the peel were examined
using the stripping technique (Fitzell et al,
1984) with a 3:2 (v/v) ratio of nail
varnish:acetone.







Application of Laboratory Results in Pre-
and Postharvest Anthracnose Control

Records of T, RH, and duration of leaf
wetness (as measured by a thermohy-
grograph and a Lufft sensor attached to the
the thermohygrograph) from December,
1988 to February, 1989 were obtained from a
mango orchard in Dasmarinas, Cavite.
Records of rainfall occurrence were also
taken. From these data, potential high
anthracnose-risk periods during flowering
and fruit development were identified and
the worthiness of the scheduled fungicide
spraying program being followed by the
growers was determined based on the results
from the humidity and temperature studies
done in the laboratory.

RESULTS AND DISCUSSION

Determination of Qptimum Humidity and
Temperature for TCnidial Germination and
Growth on Glass Slides

A. Effect of Humidity

Germination of conidia from both
isolates on glass slides was inhibited at RH of
90% and below, even after 36 h of incubation
(Fig. 1A to D). However, some spores at
90% RH were able to absorb moisture and
swell especially after 36 h. As the RH was
increased to 95, 97.5 and 100%, a
corresponding rise in the amount of
germinated spores was observed and growth
became more profuse. A considerable
number of spores incubated at 95 and 97.5%
RH germinated even in the absence of visible
free water on the surface of the glass slides.
Sporulation occurred within 18 h at 100%
RH and after 36 h at 97.5%. No appressorial
formation was recorded in all RH levels even
after 36 h.

The age of the culture from which the
spores were taken significantly affected the
percentage of spores that germinated. A
lower percentage of conidia from 14 d old
cultures germinated compared to those from
7 d especially for isolate I1. Lenne (1978) had
similar findings using spores of C. musae. She
observed the greatest proportion of
germinated spores from 10 to 12 d old
cultures compared to those from 12 to 16 d.
This occurrence could be partly due to the
accumulation of higher levels of germination
inhibitors in older cultures. Spores of C.


gloeosporioides are known to produce a
water-soluble self-inhibitor that inhibits
germination of other spores (Lax et al, 1985).
Since more spores were likely to have been
formed in the older cultures, more of the
inhibitor may have also been produced which
may have subsequently altered spore
germination.

B. Effect of Temperature

Conidial germination of C.
gloeosporioides was highest and germ tube
growth was most rapid at 300C for both
isolates (Fig. 1E to H). When the T was
reduced to 25 and 200C or increased to 350,
there was a concomittant drop in the number
of germinated spores and the germ tubes
became less widely spread. At 150,
germination was almost nil after 18 h
incubation but it increased to about 3 and
10% for 11 and 12, respectively after 36 h with
germ tubes generally shorter than the length
of the spore. A total inhibition of
germination was noted at 400C for both
isolates even after 36 h although some spores
were swollen especially after 36 h.
Sporulation occurred only at 25 and 300C
after 18 h and at 200C after 36 h. No
appressorial formation was noted at the
different T regimes even after 36 h of
incubation. Conditions favoring germ tube
growth could have continuously occurred on
glass slides thus inhibiting the formation of
appressoria. Parberry (1981) advanced the
idea that appressorium is stimulated when
germ tube growth is inhibited by factors such
as antagonisitc microorganisms, dessication,
and inhibitory chemical. All these factors
may have been absent on glass slides.

As in the experiment on RH, the
percentage germination was statistically
higher in spores from 7 d than from 14 d old
cultures. Again, the possible presence of
higher levels of inhibitors in 14 d old cultures
may have contributed to the occurrence of
lower proportions of germinated spores from
these cultures (Lax et al, 1985).







































Relative Humidity (%) Temperature (oC)


Fig. 1. Germination of conidia from 7 and 14 d old cultures of 2 isolates
(11 and 12) of C. gloeosporioides on glass slides after 18 and 36 h
icubation under various humidities at 300C (A to D) and in a
moist chamber at different temperatures (E to H).


Germination and Growth at Various
Combinations of Temperature and Humidity

A. Leaves

Germination and Growth

Total inhibition of germination on
mango leaves was noted at 200C within the
18 h period at humidities 95% and below
(Fig. 2A toD). However, after 36 h
incubation, restricted germination was
observed only at 90% RH with about 23 and
33% conidia germinating at 95% RH for 11
and 12, respectively.

Germination of conidia at 250C and
300C was similar especially for 12 wherein no


significant differences were noted in all RH
levels except at 95% RH after 18 h. For I,
however, considerable differences in
germination at both observation periods were
recorded in RH of 95% and above. An
almost complete inhibition of germination
was noted at 90% RH within 18 h but it
reached an average of 3 and 9% for both
isolates at 250 and 300, respectively, after 36
h. As in the experiments on glass slides, germ
tube and mycelial growth on leaves became
more profuse with increasing T and RH and
with time.
Appresorial Formation

In contrast to the total inhibition of
appressorial formation observed on glass
slides, a few appressoria were formed on leaf







































90 95 97.5 100 90 95 97.5 100 90 95 97.5 100 90 95 97.5 100
Relative Humidity (%)

Fig. 2. Conidial germination (A to D) and appressorial formation (E to F)
of two isolates (11 and 12) of C. gloeosporioides on mango leaves
after 18 and 36 h of incubation under various temperature-
humidity regimes.


surfaces within 18 h incubation (Fig. 2E to
H). It occurred generally at 97.5 and 100%
RH and initiation was somewhat favored at
250C for both isolates. After 36 h incu-
bation, appressorial formation increased
markedly and reached up to approximately
60% at 300C and 100% RH for both isolates.
A decrease in temperature and humidity
effected a concomittant decrease in the
amount of appressoria formed. Total
inhibition was noted at 95% RH at 200C and
at 90% RH at 250C and 300C.

A possible reason for the development
of abundant appressoria on leaves compared
to none formed on slides is the presence on


leaves of factors inhibiting germ tube
elongation e.g. antagonistic microorganisms
or inhibitory chemicals which then promoted
appressorial formation (Parberry, 1981).
Moreover, the phenomenon of thigmo-
tropism or contact stimulus may have also
contributed to this occurrence. The germ
tube of the fungus may have recognized host
(e.g. leaf) from non-host (e.g. slide) surfaces
hence the profuse formation of appressoria
in the former. Lenne (1978) also observed
lower proportions of germinated spores
producing appressoria on glass than on
cellophane which Parberry (1981) regarded
as a closer approximation to host surface
texture than glass.







Infection of leaves

Data on the severity of anthracnose on
leaves brushed with spores and incubated for
5 d in the various T-RH regimes are in
agreement with the results obtained on the
amount of appressoria formed. An increase
in appressorial formation resulted in an
increased severity of anthracnose lesions on
the leaves for both isolates (Fig. 3). At 200C,
the anthracnose symptom appeared only as
minute specks on the surface of the leaves
and was relatively abundant at 97.5 and 100%
RH. Leaves at 95% RH had a few specks
whereas those at 90% RH were free from
infection. Symptoms at 250 and 300C varied
from minute specks to large dark brown to
black round spots which often coalesced
forming irregular lesions. The largest and
most number of lesions appeared at 100%
RH, followed by 97.5% and 95% RH. A few
minute specks were present at the two T
regimes even at the low RH of 90%.

B. Peel Surfaces

Germination and Growth

Germination of conidia brushed on the
peel surfaces of mango fruits was much
higher than that on leaves (Fig 4 A to F).
Considerable germination occurred even in
90% RH at 20 C. After 36 h of incubation,
germination in 90% RH at 300C already
reached between 55 and 75%. The higher
percentage germination on peels compared
to leaves may be due to the slightly higher
humidity in the microenvironment on the
surface of the former because of the escape
of moisture from the cut tissues of the peel.
The inducing effect of some substances
secreted by the fruit tissues to the surface of
the peel on spore germination cannot also be
discounted.
The stage of fruit development did not
affect the percentage germination of spores
as there were no differences noted in all
temperatures after 36 h. This apparently
indicates that the physical and chemical
properties of the surface of mango fruits at
different stages of development do not vary
considerably to exert a marked effect on
germination. It is not known however,
whether the stage of fruit development will
have the same effect during the penetration
phase of infection.

Germ tube and mycelial growth on peel
surfaces was generally more abundant than
on leaves. Among fruits at different stages of


development, only slight differences in the
extent of growth was observed. In general,
short germ tubes were produced in all
temperature regimes at 90% RH. As the RH
and T were increased, the growth became
more profuse. After 36 h, germ tubes were
still short at 90% RH in all temperatures but
the number of spores forming germ tubes
increased considerably. Very profuse growth
and sporulation were noted at 100% RH at
200C and at 97.5 and 100% at 250C and
300C.

Appressorial Formation

Although germination was higher and
growth was more widely spread on peel
surfaces, a much lower rate of appressorial
formation was observed compared to leaf
surfaces. This could be due to some
inhibiting substances present on the peel of
green fruits. Appressorial formation was, in
general, absent after 18 h in all humidities
and temperatures for both isolates.
Appressoria were observed after 36 h of
incubation and only at 97.5 and 100% RH
(Fig. 4G to H). The highest estimated
percentage formation of appressoria (above
40%) was in 100% RH at 300C. As in
germination, no distinct trend or pattern was
noted between appressorial formation and
the stage of fruit development.

Application of Laboratory Results in Pre
and Postharvest Anthracnose Control

When the results of the laboratory
experiments on spore germination, germ tube
growth and appressorial formation were
evaluated and related to actual field data, it
was found that some if not the majority, of
the mango growers' scheduled fungicidal
sprays were made during low anthracnose-
risk periods. The six sprays in the standard
spray regime which were based on
recommendations by experts i.e. five sprays
from flower induction to fruit set and one
application 30 d after fruit set (Fig. 5A) (R.
Bugante, personal communication; Bondad,
1989), were made when RH and T were
generally low and the duration of leaf
wetness was practically short. As an example,
in the third scheduled spray, the mean T and
RH were around 24-25 C and 70-78% (Fig.
5A), respectively, for a period of 6 d before
fungicide application. No RH above 90% was
likewise recorded at this period (Fig. 5A).
Moreover, no period of leaf wetness was
noted a day before spraying (Fig. 5B).
Another instance was in the fourth spray











100%


20C











25C











300C


Fig. 3. Anthracnose lesions on leaves brushed with spores of C.
gloeosporioides and incubated for 5 d under various temperature-
humidity regimes.


90%


95%


97.5%
























S-50
LSD LSD LSD5
40 .Q

30 C

2

Co



0
LSD 1.| L SDM



S40







90 95 97.5 100 90 95 97 5 100 90 95 97.5 100

Relative Humidity (%)

Fig. 4. Conidial germination (A to F) and appressorial formation (G to I)
of isolate 2 of C gloeosporioides on peel of mango fruit at different
stages of development after 18 and 36 h incubation under various
temperature-humidity regimes.











C

24-




12--





B
24-




12--




HIGH ANTHRAGNOSE RISK PERIODS A

40--- -\I\U J- .A \

30 RAINFALLS II -
-- --- -"-- -- --"-'- -" - - -
2 -- ------ .- --LV- -V l -
20- -

10 SCHEDULED
SPRAYS
FLOWERING FRUIT DEVELOPMENT
1 10 20 1 10 20 1 10 20


DEC

1988


JAN


FEB


1989


Fig. 5. The average temperature and relative humidity (A), duration of
leaf wetness (B) and duration of humidity at 90% and above
(C) during a 3-month period from flower induction to fruit
harvest. Vertical lines joined together at the base by a horizontal
line refer to continuous occurrences.


Oc


0
-j


80


E
60-

40


20







when the average T and RH were 200C and
66-75% respectively, for a period of 3 d
before the date of spraying. Also during this
period, no leaf wetness and RH above 90%
were recorded.

If proper timing of fungicide spraying
was followed, the 3rd and 4th applications
and even the second one could have been
skipped and spraying could have been made
during fruit development when several
periods of high anthracnose infection may
have likely occurred. One such period was
January 31 to February 1. Although the
recorded mean RH was 88 and 83% on these
dates, respectively, (Fig. 5A), a 22 h duration
of RH at 90% and above was noted during
this period (Fig. 5C). Likewise, rainfall
occurred and the Lufft sensor recorded a 26
h duration of leaf wetness (Fig. 5B).
Although the T (230C) was not optimum for
spore germination and growth, this may still
be conducive for disease development based
on laboratory results. Fungicide application
should have been made either on February 1
or 2. Other periods when risk of anthracnose
infections were high are presented on Fig.
5A.

The significance of timing fungicide
sprays may be put in question because the
present scheduled spray regime and various
postharvest treatments appears to reduce
postharvest occurrences of mango
anthracnose. However, considering that so
much is lost before harvest due to
inflorescence and fruit drops which could be
attributed to inadequate anthracnose control
during production, the proper timing of
fungicide application is necessary for
improved management of the disease. Better-
timed applications will not only increase
production by reducing preharvest losses, but
also reduce disease after harvest, and
eliminate the cost of unnecessary fungicide
sprays.


LITERATURE CITED

BONDAD, N. D. 1989. The Mango. Rex
Printing Company, Quezon City. 402
pp.
COOK, AA. 1975. Disease of-Tropical and
Sub-Tropical Fruits and Nuts. 1st ed.
Hafner Press, New York. 231 pp.

DOBERSKI, J.W. 1981. Comparative
laboratory studies on three fungal
pathogens of the Elm bark beetle
Scolytus scolytus: Effect of temperature
and humidity on infection by Becuveria
bassiana, Metarhizium anisopliae and
Paecilomyces farionosus J. Invert.
Pathol. 36:195-200.


FITZELL, R.D., C.M. PEAK and
DARNELL. 1984. A model
estimating infection levels
anthracnose disease of mango.
Appl. Biol. 104:451-458.


R.E.
for
of
Ann.


GUIANZON, E.P. 1977. Present status and
future prospects of the mango industry.
Proc. 8th Nat. Annu. Conf. Pest Control
Council Philipp., 18-20 May 1977,
Bacolod City.

LAX, A.R., G.E. TEMPLETON and W.L.
MEYER. 1985. Isolation, purification,
and biological activity of a self-inhibitor
from conidia of Colletotrichum
gloeosporioides. Phytopathology 75:386-
390.

LENNE, J.M. 1978. Studies on the biology
and taxonomy of Colletotrichum species.
PhD Thesis, University of Melbourne.

MENDOZA, D.B. and R.B.H. WILLS (ed).
1984. Mango Fruit Development,
Postharvest Physiology and Marketing
in ASEAN. ASEAN Food Handling
Bureau. 111 pp.

PALO, MA. 1932. Anthracnose and
important pest of the mango in the
Philippines with respect to blossom
spraying experiment. Philipp. J. Sci.
48:209-235.

PARBERRY, D.G. 1981. Biology of
anthracnose on leaf surfaces. In:
Microbial Ecology of the Phylloplane. J.
P. Blakeman (ed.). Academic Press,
London. 502 pp.











GENETIC VARIABILITY FOR RESISTANCE TO
Pucciniapolysora UNDERW. IN CORN

A. D. Raymundo

Supported in part by the Institute of Plant Breeding (IPB), University of the Philippines at
Los Banos'(UPLB), College, Laguna, Philippines.

Assistant Professor, Department of Plant Pathology, UPLB, College, Laguna, Philippines.

Keywords: Genetic variance, host resistance, Pucciniapolysora


ABSTRACT

The Design I mating system was utilized to estimate genetic parameters
for resistance to Puccinia polysora in corn utilizing IPB Var 1, an open-
pollinated variety.
Additive genetic variance is substantially larger than non-additive
variance. A heritability value of 63% was obtained. Simple selection
procedures may be used for improvement of levels of resistance to P. polysora.


Leaf rust, caused by Puccinia polysora
Underw. is of major importance in corn
growing areas of the tropics where high
temperature and high relative humidity occur
(Oro and Exconde, 1974). It usually appears
at silking time and losses can be very
substantial when susceptible cultivars are
grown. It has become endemic in the
Philippines since it was first reported (Reyes,
1956) but higher incidence and severity have
been observed in recent years with the
increasing use of hybrids and improved
varieties (IPB, 1982, 1989).
The genetics of host-pathogen
interaction in corn rust is an area that has not
received attention proportionate to its
relative importance in disease control.
Physiologic races of P. polysora and
inheritance of a gene for resistance have
been reported in the United States (Robert,
1962; Ullstrup, 1965) while resistance to
some races has been found in Africa (Ryland
and Storey, 1955; Storey and Ryland, 1957).
At least two genes of the Rpp series have
been reported to be in linkage (Storey and
Ryland, 1959). Moreover, Rpp of the series
was found linked to a gene for resistance to
P. sorghi, the causal pathogen of common
corn rust (Ullstrup, 1965). A single dominant
gene has been identified in the inbred line,
B1138T (Futtrell et al, 1975). Horizontal or
general resistance appears to be common


and has been utilized in rust control
(Robinson, 1973).
Evaluation of germplasm materials in
the Philippines has shown that resistance
exists in lines and in diverse populations (
Raymundo and Exconde, 1973; IPB, 1982). In
a number of cases, a slow-rusting type
characterized by a reduced infection rate has
been indicated (IPB, 1989). Breeding for
resistance has been attempted (Aquilizan et
al., 1961) but has not been sustained.
This study was undertaken to estimate
the variability for rust resistance in a corn
population and to use this estimate to
determine the best strategy to breed for
enhanced level of this trait.

MATERIALS AND METHODS

The Design I mating system (Comstock
and Robinson, 1952) was used to generate
half-sib and full-sib families from IPB Var. 1,
an open-pollinated variety. Fifty-two males
were each mated to separate sets of 4
females to produce 208 progenies. These
progenies were divided into 13 sets of 4 male
groups each and each such set of 16
progenies was assigned to a block and
replicated twice within the block. A different
randomization of the 16 progenies was made
within each of the two replications. Each plot
consisted of one 5-meter row. Rows were








spaced .75 meter and seeds were planted at
.25 m between hills at 2 seeds per hill.
Point-source plants within the
experimental plot were inoculated with P.
polysora by rubbing leaves with uredospore
dust, collected previously from infected
plants, at 3 weeks after seedling emergence
to serve as foci of infection. Infection rating,
based on percentage of leaf area affected,
was taken on a row basis, each with 20 plants,
at 3 weeks after silking time.
Data were transformed using arcsin
before conducting analysis of variance.


RESULTS AND DISCUSSION

The weather during the flowering to
maturity stages of the corn crop the time
when rust usually is most severe (Oro and
Exconde, 1974), was favorable for disease
development. Disease severity levels
observed at the time of rating were adequate
enough to detect distinct variability among
families. Percentages of infection as low as
9.9 and as high as 45.0 were recorded in some
families. The observation confirmed earlier
reports (IPB, 1989) of the moderate level of
resistance of IPB Var 1.
Although rust pustules were seen in the
whole corn canopy, the lower leaves were
observed to be infected more heavily than the
upper leaves in families that showed higher
degree of resistance. Observation until
harvest time indicated a tendency toward a
sustained low level of infection in some
families. This type of reaction which can be
described as slow-rusting has been reported
(IPB, 1989). Closer examination of pustule
types in families revealed no distinct
differences as might be expected in
hypersensitive types or physiologic resistance
(Hooker, 1967).
The analysis of variance showed
significant differences among sources of
variation (Table 1). The additive genetic
variance, 52A was substantially larger than
the non-additive genetic variance, 6 Dn which
in this study was highly negligible. Additive
variance appears to control the expression of
slow-rusting type of resistance in IPB Var 1.
In studies of the inheritance of resistance to
P. sorghi in corn, Kim and Brewbaker (1977)
found that additive gene action was the main
source of genetic variation and the resistance
levels of the parent lines were linearly related
to their crosses.


Likewise, Randle et al. (1984) reported
that in sweet corn, generation mean analyses
revealed important additive dominant and
sometimes epistatic gene action. Other host-
pathogen systems in corn such as that
involving Cercospora zeae-maydis (Thompson
et al., 1987), quantitative resistance analysis
has shown that additive genetic variance is
the major source of genetic variation.
The narrow-sense heritability value, h2,
of 63.0 is quite high (Table 2). Estimates of
heritability of resistance in many host-
pathogen systems have usually been of this
magnitude. For instance, Randle et al.
(1984) obtained estimates that ranged from
65.4 to 89.7% in quantitative partial
resistance in sweet corn to leaf rust caused by
P. sorghi. The kinds of genetic variation and
their magnitude should be known before
making decisions regarding the breeding
strategy to improve populations. Design I, a
nested mating design, has been used
extensively to estimate genetic variances for
many characters in corn (Gardner, 1976).
Estimates of additive genetic variance were
substantially larger than the comparable
estimates of dominance variance (Sprague,
1966). The magnitude of additive variance
obtained in the present study of the
quantitative resistance to P. polysora and the
heritability value estimated indicate that an
enhanced level of resistance can be achieved
by methods such as mass selection or
phenotypic recurrent selection. Conceivably,
a significant gain can be realized after a few
cycles of selection with efficient artificial
inoculation techniques. Randle et al. (1984)
has suggested that if working with additive
effects is desired in the case of P. sorghi, S1
family selection, discarding those progenies
which show a high degree of inbreeding
depression, could be used. If dominance as
well as additive variance is important then
some form of reciprocal recurrent selection
could be followed to maximize gain.
Miles et al. (1980) have extended the
utility of genetic components of variance in
improving corn populations for more than
one character. A two-stage mass selection
scheme was suggested for improving
resistance to leaf blight and stalk rot with one
cycle per year. A.similar approach can be
done with resistance to P. polysora and to a
seedling disease such as downy mildew
caused by Peronosclerospora philippinensis
(Weston) Shaw which has been known to
involve additive gene effects (Kaneko and
Aday, 1980).











TABLE 1. Analysis of variance for leaf rust resistance in corn cultivar, IPB
Var 1, in a Design I experiment.


Degrees of
Source of variation Freedom Mean Squares

Sets (S) 12 253.492

Replication in sets (R/S) 13 36.341

Males in sets (M/S) 39 144.752

Females in males in sets (F/M/S) 156 20.876

Error 195 15.859


Significant at .01 level




TABLE 2. Estimates of genetic parameters of leaf rust resistance in corn
cultivar, IPB Var 1.


Parametera Estimate



6 2m 15.49

62f 2.51

S2A 61.94

62D -51.90

h2 (narrow-sense) 0.63

a 6 2mis the variance of male effects, 6 2js the variance of female effects, 6 2As additive
genetic variance, 6 2Dis dominance variance, and h 2is heritability value.









LITERATURE CITED


AQUILIZAN, F. A., I. S. SANTOS, and C.
C. JESENA. 1961. Breeding for
resistance to corn rust. In 2nd Annual
Report, Rice and Corn Program, 1959-
60, Univ. of the Phil., Coll. of Agric.,
College, Laguna, Philippines, p. 23.

COMSTOCK, R. E. and H. F. ROBINSON.
1952. Estimation of average dominance
of genes. In J. W. Gwen (ed.).
Heterosis, Iowa State Coll. Univ.
Press, Ames, Iowa, pp. 494-516.

FUTTRELL, M. C., A. L. HOOKER, and G.
E. SCOTT. 1975. Resistance in maize to
corn rust controlled by a single
dominant gene. Crop Sci. 15: 597-599.

GARDNER, C. O. 1976. Quantitative
genetic studies and population
improvement in maize and sorghum. In
E. Pollack, O. Kempthorne and T. B.
Bailey, Jr. (eds.), pp. 475-489 Proc.
Intern. Conf. on Quant. Genetics, Iowa
State Univ., Aug. 16-21, 1976

GEIGER, H. H. and M. HEUN. 1989.
Genetics of quantitative resistance to
fungal diseases. Ann. Rev. Phytopathol.
27: 317-341.

HOOKER, A. L. 1967. The genetics and
expression of resistance in plants to
rusts of the genus Puccinia. Ann. Rev.
Phytopathol. 5: 163-182.

IPB. 1982. Annual Report. Institute of
Plant Breeding, University of the
Philippines at Los Banos, College,
Laguna, Philippines.

IPB. 1989. Annual Report. Institute of
Plant Breeding, University of the
Philippines at Los Banos, College,
Laguna, Philippines.

KANEKO, K. and B. A. ADAY. 1980.
Inheritance of resistance to Philippine
downy mildew of maize,
Peronosclerospora philippinensis. Crop
Sci. 20: 590-594.

KIM, S. K. and J. L. BREWBAKER. 1977.
Inheritance of general resistance in


maize to Puccinia sorghi Schw. Crop Sci.
17: 456-461.

MILES, J. M., J. W. DUDLEY, D. G.
WHITE and R. J. LAMBERT. 1980.
Improving corn population for grain
yield and resistance to leaf blight and
stalk rot. Crop Sd. 20: 247-251.

ORO, R. S. and O. R. EXCONDE. 1974.
Penetration and infection of corn by
Puccinia polysora Underw. Phil. Agric.
53: 50-60.

RANDLE, W. M., D. W. DAVIS and J. V.
GROTH. 1984. Improvement and
genetic control of partial resistance in
sweet corn to corn leaf rust. J. Am. Soc.
Hort. Sci. 109: 777-787.

RAYMUNDO, A. D. and O. R.
EXCONDE. 1973. Evaluation of
resistance to Puccinia polysora Underw.
of some monogenic lines of corn. In
14th Annual Report, Rice and Corn
Program, 1971-72, Univ. of the Phil.,
Coll. of Agric., College, Laguna,
Philippines.

REYES, G. M. 1956. A note on the
occurence of a species of corn rust new
to the Philippines. Araneta J. Agric. 3:
68-71.

ROBINSON, R. A. 1973. Horizontal
resistance. Rev. Plant Pathol. 52: 483-
501.

ROBERT, ALICE, L. 1962. Host ranges
and races of the corn rusts.
Phytopathology 52: 1010-1012.

RYLAND, AUDRIE K. and H. H.
STOREY. 1955. Physiological races of
Puccinia polysora Underw. Nature 176:
655.

SPRAGUE, G. F. 1966. Quantitative genetics
in plant improvement. pp. 315-351. In K
J. Frey (ed.), Plant Breeding. Iowa State
Univ. Press, Ames, Iowa.

STOREY, H. H. and RYLAND, AUDRIE
K. 1957. Resistance in maize to the
tropical American rust fungus, Puccinia
polysora Underw. I. Genes Rppl and
Rpp2. Heredity 11: 61-65.




40


STOREY, H. H. and RYLAND, AUDRIE
K. 1959. Resistance in maize to the
tropical American rust fungus, Puccinia
polysora Underw. II. Linkage of genes
Rppl and Rpp2. Heredity 13: 61-65.

THOMPSON, D. L., R. R. BERQUIST, G.
A. PAYNE, D. T. BOWMAN and M.
M. GOODMAN. 1987. Inheritance of
resistance to gray leaf spot in maize.
Crop Sci. 27: 243-246.

ULLSTRUP, A. J. 1965. Inheritance and
linkage of a gene determining resistance
in maize to an American race of
Pudcinia polysora. Phytopathology 55:
425-428.









OCCURENCE AND DEVELOPMENT OF SHEATH BLOTCH OF
RICE CAUSE BY Pyrenochaeta oryzae SHIRAI EX MIYAKE IN
THE PHILIPPINES

A.K.M. Shanjahan and T.W. Mew

International Rice Research Institute, PO Box 933, Manila, Philippines. Present Address of
senior author: Bangladesh /Rice Research Institute, Joydebpur, Bangladesh


ABSTRACT

Sheath blotch (ShB1) of rice caused by Pyrenochaeta oryzae Shirai ex
Miyake is prevalent in the Philippines. The disease produces typical oval
brownish, blotch-like lesions near the middle of the outer leaf sheaths. The
lesion enlarges as the plant matures, covers the whole sheath, and kills the leaf.
Pycnidia with protruding beaks are embedded in the sheath tissue. The disease
was present in 25% of the fields surveyed with incidence that ranged from 1 to
5% and severity from 1 to 5. All the commercially grown IR varieties were
infected but five IR cultivars varied in the rate of ShB1 development.


INTRODUCTION

The rice plant is a host of more than 70
pathogens (Ou, 1985). Some of the
pathogens are aggressive while others are
weak and considered minor pathogens. All
these diseases do not prevail in all countries
and in the same country in all seasons
(Shahjahan, et al. 1987). Modern cultivation
practices have brought about changes in the
status of some of the diseases including
occurrence of diseases that were absent or
not reported in the past.
Sheath blotch (ShB1), caused by
Pyrenochaeta oryzae Shirai ex Miyake is a
minor disease. Although it has been
reported in Japan, Burma, china, India,
Malaysia, Sierra Leone, Philippines,
Thailand, and Bangladesh (Miyake, 1910
Mori et al 1964; Ou, 1985; Shahjahan, et al
1983; Ahuja and Bhan, 1987), no information
is available on its distribution and reaction of
varieties. In a survey of the incidence of
sheath blight-like diseases in the Philippines,
ShB1 was found occurring at a high
frequency in some areas. The infected
plants were senescing earlier than the healthy
plants. The present study reports of its
occurrence and distribution in the
Philippines, and its development in different
IR-varieties.


MATERIALS AND METHODS
A. Disease Survey
A rapid survey was conducted to study
the occurrence and distribution of the disease
in six regions (Bicol, southern Tagalog,
Southern Mindanao, and Western Visayas)
of the Philippines and IRRI farm. In each
region different locations and in each
location different fields were examined.
ShB1 incidence was noted as number of
infected and healthy hills and tillers, and
severity (0 to 9 scale) of hills/spot x 10 spots
selected at random in each field. The growth
stage of the crop and the name of the
cultivars grown were also noted. Disease
samples were collected from each field
isolation of the casual pathogen.

B. Development of sheath blotch in
different IR-varieties.

The pathogen was isolated from
diseased samples collected during the survey
trip from different locations. Two isolates
were purified by hyphal tip culture and grown
in PDA for two weeks. They grew pycnidia,
pycnidia, fruiting bodies which were
embedded in the medium with the peak
protruding outside. They were identified as







P. oryzae (Ou, 1985; and Shabjahan, et al.
1983). Their pathogenicity was tested on IR
58 grown in the greenhouse.
The development of the ShB1 in five IR
varieties (IR26, IR36, IR58, IR60, and IR62)
was studied on potted plants in the
greenhouse. Seedling of the five varieties
were transplanted in 30 cm diameter earthen
pots, 3 hills/pot with five pots/variety. They
were grown with standard fertilizer (N in the
form of (NH4)2 at 90 kg/ha, P in the form of
P205 at 30 kg/ha, and K at 30 kg/ha)
applications to boot stage. Two purified P.
oryzae isolates were grown in sterilized rice
grains in 125 ml conical flasks for 10 d. The
infected grain culture was used as inoculum
for this study. A wound was made near the
middle of the outer leaf sheath (2nd from
top) with a needle and a grain culture
(inoculum was attached on the leaf sheath
with scotch tape. Varieties were placed as
the main plot and isolates as subplot, i.e.,
both the isolates were inoculated to three
tillers in each of the two hills of a pot. The
tillers in the 3rd hill served as controls, i.e.,
wounded but not inoculated. The pots were
placed on a greenhouse bench covered with
polyethylene sheets from 1700 hr to 0700 hr
to maintain high humidity. The length and
width of the sheath were measured 10 and 20
days after inoculation. The lesion and sheath
area were calculated by multiplying the mean
length and mean width of the lesion and
sheath, respectively, to determine the
proportion of diseased area. The rate of
ShB1 development was calculated using Van
Der Plank's equation (1963).



RESULTS AND DISCUSSION

ShB1, caused by P. oryzae, infects on
rice in the Philippines. It is visible usually
during the boot to heading stage of the crop.
The disease produces oval brownish, blotch-
like lesions near the middle of the outer leaf
sheath of the rice tillers (Fig. 1). The lesion
enlarges as the plant matures, covering the
whole sheath, and killing the leaf. Pycnidia
of P. oryzae upon closer examination can be
seen embedded in the diseased tissue (Fig.
2). The leaves of infected tillers eventually
senesce and dry.
The survey revealed that the disease is
prevalent in the IRRI farm and farmer's
fields in Southern Tagalog, Bicol, Western
and Eastern Visayas, and Southern
Mindanao, the main rice growing regions of
the Philippines. On the average, 25% of the


-P




Fig. 1. Typical symptom of sheath blotch of
rice caused by Pyrenochaeta oryzae.
The blotch develops on the outer leaf
sheath, oval shaped with dark brown
border.


Fig. 2. The blotch as it looks after covering
the whole sheath. Many black
pycnidia with protruding beak can
be seen in the sheath tissues.







fields or plots examined (72 out of 270) had
rice plants showing ShB1 symptoms. The
incidence varied between plots in each
location because of differences in cultivars
and growth stage of the crop at the time of
the survey. However, the incidence in some
of the fields was 15% (based on % tillers
infected) and the severity was between 1-5
(on a scale of 0-9). All the commercially
grown IR-varieties were found infected with
the disease in varying degrees in some
location or the other (Table 1.).

On artificial inoculation, the two isolates
of P. oryzae were pathogenic on IR58. The
five IR cultivars showed differences in sheath
blotch development during the first ten days
after inoculation. By 20 days this difference
was no longer apparent (Table 2). However,
the cultivars differed significantly in the rate
of sheath blotch development. IR58 showed
the highest (0.204) rate followed by IR36,
IR26, IR60, and IR62 (Table 3.) Also no


difference in virulence between the two
isolates (Fig. 3) and no interactions between
the isolates and the varieties were evident
from this study.

Ou (1985) mentioned the occurrence of
ShB1 in the Philippines, but no other
information is available on this disease. The
symptom is not easily noticeable because it
attacks the outer leaf sheaths usually during
the later growth stages and fades away with
the senescence of the outer leaves
(Shahjahan, et. al.. 1983). If the attack is
early or the cultivar is susceptible and the
environment favorable, this could cause early
senescence, affecting grain filling. The
pathogen has also been found to be
associated with rice grain discoloration (Mia,
et al. 1979). The disease has been reported
from India (Ahuja and Bhan, 1987).
However, further studies are necessary to
understand the nature of the disease.


Table 1. Occurence of sheath blotch on rice cultivars grown in different regions of the Philippines,
1985.

Loca- Fields/ Fields Stage Inci- Severity4
Regions1 tions Varieties grown plots or plots w/ of th dence3 0-9
examined infected crop % scale
(no.) tillers (%) (range) (range)

Bicol 17 IR56, IR58, IR62 75 25.3 Hd-Mat 1-10 1-4
IR-line & a local
variety
S. Tagalog 14 IRvar. & lines 60 16.7 F1-Mat 1-6 1-5
including IR36,
IR58, IR62
Mindanao 16 IR60, IR64 65 30.8 F1-Mat 1-5 1-3
(S. Cotabato) 7-tonner&IR line
Visayas 22 IR22, IR36, IR42 70 32.9 Hd-Mat 1-15 1-3

Total 69 270 26.7 Hd-Mat 1-15 1-5

/ Areas surveyed in the four regions include Bicol:Albay, Camarines Sur, and Camarines Norte; Southern Tagalog:
Laguna including IRRI farm; Southern Mindanao:South Cotabato; and Eastern Visayas:Iloilo and Leyte, respectively.
/ Hd = Heading, Mat = matured, Fl = Flowering.
3/ Incidence as % of tillers infected/field.
4/ Severity measured on a 0 to 9 scale with 0 = no blotching of sheath, 9 = 100% of the infected sheath area
blotched.









Table 2. Development of sheath blotch by two isolates of Pyrenochaeta oryzae in five IR-varieties.


Mean 1/ area2l of sheath tissue blotched, mm 2

Varieties 10 days 20 days

P.O.-1 3/ P.O.-2 3/ Mean P.O. 1 P.O. 2 Mean

IR 26 116.6 ABa 152.4 ABa 134.5 BC 746.4Aa 588.0 Aa 667.2 A

IR 36 89.2 Ba 93.4 Ba 91.3 C 451.OBa 531.0 Aa 491.0 A
IR 58 140.0 Ba 123.9 Bc 123.9 BC 715.OABa 588.8 Aa 651.9 A
IR 140.0 ABa 175.2 A a 157.6 AB 476.6ABa 483.0 Aa 479.8 A
IR 62 166.8 A a 202.8 A a 184.5 A 616.OABa 752.0 Aa 684.0 A


1/ Means of 5 replications. Means having a common letter are not significantly different by DMRT at alpha = 5%.
Capital letters compare varieties within an isolate and small letter compare isolates wihtin a variety.
2/ Area of sheath tissue affected (blotches) was calculated by multiplying the length and width of the lesion produced on
the inoculated n-2 (2nd from top) sheath of each tiller.
3/ Indicates two isolates of P. oryzae.




Table 3. Rate of sheath blotch development due to inoculation with two isolates of Pyrenochaeta
oryzae in five IR-varieties in the greenhouse.



Mean rate (r)1/ of sheath blotch development


Varieties P.O. -12/ P.O. -2 2/ Mean

IR 26 0.209 a 0.152 b 0.181 b

IR 36 0.182 a 0.195 a 0.188 ab

IR 58 0.202 a 0.205 a 0.204 a

IR 60 0.144 b 0.120 c 0.132 c

IR 62 0.143 b 0.155 B 0.149 c

Mean 0.176 0.165


1/ Mean of 5 replications, r was calculated using Van Der Plank's equation (1963). Means followed by a common letter (in
column) are not significantly different at 5% level by DMRT.
2/ Indicates isolates of P. oryzae






































Fig. 3. Sheath blotch developed by two isolates of Pyrenochaeta oryzae on IR26 in the
greenhouse.


LITERATURE CITED

AHUJA, S.C. AND U. BHAN. 1987. Sheath
blotch of rice. Int. Rice. Res.
Newslet. 12(4):31-32.

MIA, MA.T., A.K.M. SHAHJAHAN AND
SA. MIAH. 1979. Microorganisms
associated with spotted and
discoloured rice grains in
Bangladesh. Int. Rice. Res. Newslet.
4(5):8.

MIYAKE, I. 1910. Studien Uber die Pilze
der Resiplflanze in Japan. J. Coll.
Agr. Imper. Univer. Tokyo 2:237-
276.

MORI, K., T. MAKING and T. OSAWA.
1964. A leaf sheath disease of the


plant caused by Pyrenochaeta sp.
Bull. Shizuaka Prefecture Agr. Exp.
Sta. 9:25-31 (Eng. Summ).

OU, S.H. 1985. Rice diseases. Commonw.
Mycol. Inst. New England. 380 p.

SHAHJAHAN, A.K.M., H.U. AHMED. and
SA. MIAH. 1983. Occurrence of
sheath blotch of rice in Bangladesh.
Int. Rice Res. Newslet. 8(2):12.

SHAHJAHAN, A.K.M., T. DUVE and J.M.
BONMAN. 1987. Climate and rice
diseases. 125-138, In Weather and
Rice, IRRI Philippines.

VAN DER PLANK, J.E. 1963. Plant
Diseases: epidemics and control.
Acad. Press, N.Y. 349 p.








ABSTRACTS OF PAPERS AND POSTERS PRESENTED DURING
1991 PEST MANAGEMENT COUNCIL OF THE PHILIPPINES
ANNUAL CONVENTION
08-10 May 1991, Bureau of Plant Industry, Manila


TAXONOMY OF THE FUNGUS CAUSING
GALL RUST DISEASE OF Albizia falcataria
(L.) BACK. Nonitq S. Franje and
Hermogenes C. Alovera. Central Mindanao
University, Musuan, Bukidnon.

The diagnostic symptoms produced by
the rust disease in Albizia falcataria (L.) Back
includes large convoluted brown galls on
branches, stems and seed potds, smaller
blister-like galls on leaves and petioles,
dieback and defoliation which often results to
death of trees, and the characteristics
distortion of developing shoots, upward
cupping and twisting of the leaves.
The causal fungus is identified as
Uromycladium, a rust genus first reported in
New Zealand. Histopathological studies
showed three distinct stages of the fungus life
cycle in Albizia, namely: Pycnial (0), Uredial
(II) and Telial (III). Taxonomic characters of
the genus in every stage resembles that of
Uremycladium notabili. However, the sizes of
the structures produced by the fungus in
Albizia differes that of U. notabili as
previously described in Acacia by
Cunningham (1931). In addition, the
teliospsores do not germinate and the
infecting unit observed was the uredospores.
(See figures attached).
The different features mentioned
suggest that a new species of Uromycladium
is infectingAlbizia falcataria in Mindanao.

EFFECTS OF MULTIPLE PESTS ON
CROP GROWTH AND YIELD. Hans
Pinnschmidt, Division of PLant Pathology,
International Rice Research Institute, Los
Banos, Laguna.

Damage effects of multiple pest
populations need to be quantified in order to
obtain realistic yield loss predictions, loss
profiles, damage thresholds, and other means
for improving decision making in IPM.
Despite a considerable amount of research
done on the topic, further multifactorial
experiments that use detailed assessment
methods are needed to study the specific
effects of multiple pest infestations on the
performance of crops. The conventional
approach of predicting multiple pest effects


for pest management purposes uses
impirically obtained pest-loss equations that
are data-set specific and quantify the overall-
effects of given pest infestation levels on
yield. Conventional loss profiles usually do
not consider whether the combined effects of
multiple pest infestations are additive or
interactive. The rather recent dynamic pest-
crop modeling approach in contrast allows a
more realistic, physiologically based
mimicking of multiple pest effects, and is
transportable across cropping situations,
since it is strictly mechanistic. The relative
importance of various pests, different
damage mechanisms, as well as intra- and
interspecific pest interactions to the resulting
total yield loss can thus be studied in detail.
The practical implications of the pest-crop
modeling approach consist of predicting,
creating, and comparing any kind of multiple
pest-loss scenario and are discussed with
regard to decision aids for developing
strategies and tactics in IPM.

BLASTSIM.2 A MODEL FOR TROPICAL
LEAF BLAST-RICE PATHOSYSTEM. S.B.
Calvero, Jr. and P.S. Teng. Division of Plant
Pathology, International Rice Research
Institute, Los Banos, Laguna.

An attempt to understand the tropical
leaf blast-rice pathosystem via the
BLASTSIM.2 is presented. The model uses
3-dimensional matrices or response curves
aside from regression equations to explain
biological processes in the monocycle.
Simulations was further improved with the
incorporation of DEWFOR, a dew formation
model based on several micro- and macro-
climatic factors; and the receptivity factor, a
parameter to measure differences in varieties
in terms of their reaction to blast. Written in
Fortran, the model has a user-friendly
interface and runs in IPM PC compatible
microcomputers.








RELATIONSHIPS AMONG SEVERITY OF
LATE LEAFSPOT, CANOPY REF-
LECTANCE, HEALTHY LEAF AREA
DURATION AND POD YIELD IN TWO
PEANUT CULTI-VARS. V.M. Aquino and
F.M. Shokes. Institute of Plant Breeding,
UPLB.

Field studies were conducted in
Marianna, Florida in 1988 and 1989 to study
the relationships among severity of late
leafspot (caused by Cercosporidium perso-
natum), canopy reflectance, healthy leaf area
duration (HAD) and pod yield in two peanut
cultivars.
Spray treatments of chlorothalonil were
applied to establish different levels of
disease. Leaf area index (LAI) was measured
eight times during the season and disease
progression and canopy reflectance were
monitored. Percent canopy reflectance at 800
nm was measured using a hand-held
multispectral radiometer. Healthy leaf area
duration and green leaf area were calculated
from LAI and the total amount of disease.
Pod yield for Florunner, a cultivar
susceptible to late leafspot, decreased as the
duration of healthy leaf area decreased. The
percent canopy reflectance at 800 nm
decreased as the disease severity and
defoliation increased. Linear relationships
were obtained between green leaf area and
percent canopy reflectance for both cultivars.
Using a model developed earlier for
Florunner, pod yield of Florunner was
adequately predicted from HAD for all
treatments. However, the yield for Southern
Runner, a partially resistant cultivar, was
overestimated by the model for Florunner.
Results support the concept that HAD can
be used to predict yield, and to determine
yield loss due to late leafspot disease.

EVALUATION OF MULTIPLE DISEASE
RESISTANCE OF THE F2 GENERATION
OF CORN HYBRIDS. A.D. Raymundo, B.J.
Calilung, Jr. and A.T. Aquino. Dept. of Plant
Pathology and Institute of Plant Breeding,
U.P. at Los Banos.

F2 populations of commercial corn
hybrids were evaluated for reaction to
Philippine downy mildew, bacterial stalk rot,
Rhizoctonia banded leaf and sheath blight,
and Helminthosporium leaf spot.
The F2 hybrids generally, were highly
susceptible to downy mildew under high


inoculum pressure. Only four entries,
F3228F2, SMC-317F2, P3208F2, and Y8Cy-
65F2, showed infection below 30 percent.
Against bacterial stalk rot, only SMC
308F1 showed a moderately resistant
reaction. All other entries were either
susceptible or highly susceptible.
No significant differences in
Rhizoctonia blight and Helminthosporium
leaf spot infections were observed among
entries.
As many farmers currently are using F2
seeds from F1 parents whose parentage
usually is of foreign origin, there appears to
be a high risk of sustaining considerable
losses in yield due to downy mildew and
bacterial stalk rot.

EVALUATION OF Trichoderma SPP. FOR
THE CONTROL OF DAMPING-OFF OF
TOBACCO. P.N. Dipon and M. Salinas.
University of Eastern Philippines and
University of the Philippines at Los Banos.

The potential of seven species of
Trichoderma for controlling damping-off was
studied in the laboratory and greenhouse.
Growth of surface mycelium of Fusarium
oxysporum f. sp. nicotianae, Sclerotium rolfsii,
and Pythium aphanidermatum were severely
inhibited by T. hamatumn, T. harzianum, and
T. aureoviride. These antagonists overgrew
the pathogens and covered the entire portion
of the culture plate ten days after incubation.
T. hamatum and Trichodenna sp. (Batac
isolate #1) were antagonistic overgreww at
least twothirds of the medium surface)
against F. o. nicotianae and S. rolfsii, but not
against P. aphanidennatum.
Greenhouse evaluation using T.
harzianum and soil naturally infested with the
disease showed that tobacco ;damping-off
could be satisfactorily controlled up to a
period of 30 days after sowing (DAS).
Infection in treated soil was 35% at 15 DAS
(pre-emergence damping-off) and 58% in
transplantable seedlings (30 DAS). Without
the antagonist, infection of 58% at 15 DAS
and 75% at 30 DAS were obtained. The high
incidence of damping-off during the post-
emergence phase of the disease may indicate
that the antagonist alone could not contain
the disease. Very few seedlings survived at 70
DAS, 40 to 73 seedlings in treated soil and
25 in the control.







FACTORS AFFECTING THE DEVELOP-
MENT OF RHIZOCTONIA BLIGHT AND
SCLEROTIUM ROT OF AZOLLA. Marina
P. Natural and Rosella L. Mendoza. Dept. of
Plant Pathology, UPLB.
Factors affecting the development of
two major diseases of Azolla were studied in
an attempt to standardize techniques in
screening for disease resistance. The rice-rice
hull or rice-sawdust mixture were found
superior over other media tested for
inoculum production.
Five grams inoculum incubated for 1-2
weeks and spread over fronds grown in 200
cm trays increased the rate of disease
development. However, 5 g was considered
too severe in screening for resistance since all
lines became susceptible to both diseases.
Shading (35-50% sunlight), extreme soil pH
(pH 3.5 and pH 9) and optimum phosphorus
content (20 ppm) favored disease
development.

NEW FUNGAL PATHOGENS OF AZOLLA.
Marina P. Natural and Rosella L. Mendoza.
Dept. of Plant Pathology, UPLB.

Azolla samples showing rot and blight
symptoms were collected from the UPLB
Azolla Pathology greenhouse, farmers fields
and propagations ponds in Laguna, La Union
and Nueva Viscaya. Fungi belonging to 10
different genera were isolated and tested for
pathogenicity. Symptoms were reproduced
after inoculation but only Acremonium sp.,
Aspergillus sp., Curvularia sp., Botryodiplodia
and two still unidentified fungi were
reisolated from the inoculated Azolla. We
failed to reisolate Altemaria sp.
Phaeotricochonis sp., Phoma sp. and
Fusarium sp. Among these fungal pathogens,
we consider the unidentified fungus #2 (U2)
as the most destructive especially in our
greenhouse Azolla cultures. All cultures were
morphologically characterized.

ALTERNATE HOSTS OF Helminthosporium
sativum. C.B. Pascual and A.D. Raymundo.
Institute of Plant Breeding and Department
of Plant Pathology, UPLB.

Field observations showed the
association of infected weeds with the
incidence and severity of Helminthosporium
leaf spot in wheat production areas. Spores of
Helminthosporium sp. were isolated from the
collected infected weeds from fields. Cross


inoculation tests showed that Dactylodenium
aegyptium and Rottboellia cochinchinensis are
alternate hosts of H. sativum.

RESISTANCE TO DMI FUNGICIDES IN
Rhynchosporium secalis. Dela Pena, R.C.,
Hollomon, D.W., and SJ. Kendall. Division
of Plant Pathology, IRRI, Los Banos,
Laguna.

Two field trials were conducted to
determine the effects of resistance on the
performance of DMI steroll C-14
demethylation inhibitor) fungicides against
Rhynchosporium secalis. Tebuconazole
applied either alone, or in mixture with
carbendazim, significantly reduced leaf blotch
severity and increased yield of barley.
Reduced sensitivity of R. secalis to DMI
fungicides declined after fungicide
treatments. A bimodal distribution of
triadimenol sensitivity in R. secalis was
detected in contrast to basically unimodal
distribution of sensitivity to propiconazole,
tebuconazole and prochloraz. Cross-
resistance was observed between triadimenol,
propiconazole and tebuconazole but not with
prochloraz. The level of resistance to
triadimenol was higher than for the other
three DMIs.

STATUS OF CITRUS BUD-TRANSMIS-
SIBLE DISEASES IN MINDANAO. L.V.
Magnaye and L.E. Herradura. Davao
National Crop Research & Development
Center, Bureau of Plant Industry, Bago
Oshiro, Davao City.

Determining the status of the diseases
in the different citrus orchards will lead us to
the potential parent sources of clean
materials as well as to other strategies in
solving one of the existing problems which is
the presence of bud-transmissible diseases.
Results of survey and biological
indexing in the citrus growing areas of
Mindanao revealed that out of 94 trees
surveyed, no single tree is free from any
known bud transmissible disease with two or
more diseases co-existing in a single tree.
Using the conventional biological indexing,
resultant budlings which were found free of
leaf mottling and exocortis but with mild vein
clearing on Key lime were established inside
screen houses as nursery mother trees to
serve as scion sources. For continuous
indexing of NMT's and direct indexing of
potential citrus sources, side-shoot grafting









was developed in the station. This is a
modified biological indexing technique which
can give results in 2 to 6 months with a
limited number of indicator plants to be
maintained.

ISOLATION, IDENTIFICATION AND PA-
THOGENICITY TESTS OF SEED-BORNE
FUNGI ASSOCIATED WITH COWPEA
SEEDS. Edita A. Bautista and Oscar S.
Opina. Institute of PLant Breeding and
Dept. of Plant Pathology, UPLB.

Thirteen fungal species were isolated
and identified from 10 cowpea lines/varieties
using the blotter method. These were:
Aspergillus niger, Aspergillus flavus,
Aspergillus sp., 2 Fusarium sp., Curvularia sp.,
Penicillium sp., Macrophomina sp.,
Chaetomium sp., Colletotrichum sp.,
Rhizoctonia solani, Rhizopus, and
Botryodiplodia sp.
Association of the above identified
seed-borne fungi on cowpea seeds adversely
affected seed germination and seedling
emergence. Seed-borne fungi adversely
affecting seed germination appeared to
reduce root and shoot length elongation and
were shown to induce lesions on the root and
stem which progressed rapidly causing the
eventual death of affected seedlings.

ETIOLOGY OF A NEW LEAF DISEASE
OF PEANUT. Edita A. Bautista and Marina
P. Natural. Institute of Plant Breeding and
Department of Plant Pathology, UPLB.

The causal fungus, Pestalotiopsis
arachidis Satya, which was isolated from
naturally infected peanut leaves showed
similar dark brown lesions with numerous
acervuli uniformly distributed on the diseased
portion of the artificially inoculated peanut
leaves. The fungus produced both aerial and
submerged mycelia abundantly when grown
on the different media. The size of the
conidia ranges from 19.7 27.5 x 5.2 6.4 um.
They are ellipsoid or fusoid, usually 5 celled
tapering at both ends and bulging in the
region of the 3 colored cell. The fruiting
bodies appear as minute black dots and are
globose or oval.
The isolate showed different growth
characteristic and sporulation when grown on
different media. Onion agar best supported
the sporulation of the fungus and Potato
Dextrose Agar produced more abundant
mycelial growth.


SURVEY AND IDENTIFICATION OF
PEPPER ANTHRACNOSE AND VARIETAL
SCREENING FOR RESISTANCE. N.L.
Opina and R.B. Valdez. Institute of Plant
Breeding and Department of Plant
Pathology, UPLB.

Surveys for pepper anthracnose in the
field and in market places were conducted in
17 provinces of Luzon. The disease incidence
ranged from 0-80% with the highest observed
in Batangas, Cavite and Laguna and lowest in
provinces where pepper is grown after rice
during the dry season.
Pepper anthracnose is caused by
Colletotrichum capsici and C. gloeosporoides.
Mixed infections of both species were
observed in the Southern Tagalog areas but
C. gloeosporoides appears to Ipredominate. In
some provinces single infections occurred
with up to 100% infection.
Pathogenicity tests of single spore
isolates of 19 C capsici and 11 C.
gloeosporoides on green and red ripe fruits of
a susceptible hot pepper cultivar using the
plastic box moist chamber technique showed
that 5 ot the C capsici isolates gave 100%
infection on both fruit types while 3 of the C.
gloeosporoides isolates had 100% infection on
the red fruits and 80-90% in the green fruits.
Out of 48 pepper entries screened for
resistance to C capsici and C gloeosporoides,
only Accessions 01172 had the lowest number
of fruits and fruit area infected by both
isolates after 10 days from inoculation.

ETIOLOGY OF THE YELLOWING AND
WILT DISEASES OF BLACK PEPPER
(Piper nigrum L.). B.A. Zaragoza, L.L. Ilag
and M.B. Castillo. Bicol Experiment Station,
Pili, Camarines Sur and UPLB, College,
Laguna.

The cause of the yellowing and wilt
diseases attacking black pepper in the Bicol
Region was investigated and determined as
Meloidogyne incognita Chitwood. Initial
symptom was slight to general yellowing of
leaves. Wilting occurred two to three months
after heavy, continuous rains followed by
sunny, warm and dry weather. The nature of
the symptoms was similar to those caused by
a wide range of biotic and abiotic factors.
A survey for the presence of
Meloidogyne incognita in black pepper plants
from 4 provinces, 12 towns and 14 barangays
in the Bicol Region showed 64.3% of their







root samples had galls. Personal assessment
of loss in stand was 10 to 65%. Suspect fungal
pathogens were ruled out particularly
Phytophthora spp. despite a rapid isolation
technique using pimaricin-vancomycin-
pentachloronitrobenzene-hymexazol (PVPH)
selective medium.
Identification of the nematode species
was by the characteristic perineal patterns of
the adult females. Different developmental
groups of the nematode were recovered at
weekly periods after inoculation. One
complete life cycle occurred between 21 to 28
days after larval inoculation.
The histopathology of the black pepper
nematode revealed the formation of giant
cells, distorting the cortical cells, disrupting
and blocking the vascular bundles. Multiple
infection in the roots produced more galls
and giant cells, thus more damage to the
plant.

DETECTION OF FEATHERY MOTTLE
VIRUS IN SWEET POTATO. A.P. Nazarea,
L.M. Dolores, MJ.C.C. Malabanan, L.D.
Valencia, and N.B. Bajet. Institute of Plant
Breeding and Plant Pathology Dept., U.P. at
Los Banos.

Few plants in the 1990 sweet potato trial
by the Institute of Plant Breeding, UPLB
showed symptoms like mild mosaic/mottle,
downward curling/leaf distortion, and vein
clearing. Leaves of the regenerated cuttings
obtained from those plants maintained in the
greenhouse showed the same symptoms.
Fertilization induced more pronounced
symptoms and were similar to those
expressed by the non fertilized plants. Results
of transmission tests showed that leaf extracts
did not induce disease when rubbed onto
Ipomea batatas L., Chenopodium
amaranticolor Coste and Reyn., Datura metel
L., D. stramonium L., Lycopersicon
esculentum Mill., Nicotiana glutinosa L., N.
rustica L., N. tabacum L., Ocinum basilicum
L., Gomphrena globosa L., and Petunia x
hybrida Vilm. Myzus persicae Sulz. but not
Aphis gossypii Glover was able to transmit a
virus to I. batatas with the cuttings as source
plants. In indirect enzyme-linked
immunosorbent assay, extracts of the cuttings
reacted to anti-feathery mottle virus
(SPFMV) serum but not to antiserum to
sweet potato latent virus. Our results indicate
that SPFMV or a virus serologically related
to SPFMV is assocaited with, if not the cause


of the observed disease syndrome on the
sweet potatoes. Further studies on the
isolate and its distribution in the Philippines
are in progress.

YIELD LOSS DUE TO TARO FEATHERY
MOSAIC VIRUS INFECTION. R.M.
Gapasin, C.E. Sajise and M.A. Buyser.
VISCA, Baybay, Leyte.

The effect of taro feathery mosaic virus
(TFMV) infection on the growth and yield of
taro (var. kalpao) was determined using
mechanically-inocuclated and naturally
infected taro seed pieces.
TFMV-inoculated plants at earlier stage
of growth, generally showed higher disease
incidence and yield loss than at later stage. In
addition, plants infected with TFMV
produced more suckers than the uninfected
ones. Mechanical inoculation of TFMV
employed in both pot and field experiments
showed a reduction in yield of taro from 2.80
to 58.04% and 20.24 to 32.14%, respectively.
However, taro plants naturally infected
by TFMV in the field did not show reduction
in yield. Moreover, there were no significant
differences noted in the number of suckers
produced between the infected and healthy
plants.

VIRUS CONCENTRATION IN VARIETIES
RESISTANT/TOLERANT TO RICE TUNG-
RO VIRUSES. R.C. Cabunagan, Z.M. Flores,
E.C. Coloquio and H. Koganezawa, Inter-
national Rice Research Institute, Los Banos,
Laguna.

Relative amounts of RTBV and RTSV
in infected plants of Balimau Putih (Acc.
17204) tolerant; Utri Merah (Acc. 16680) -
resistant to RTSV and tolerant; TKM 6 (Acc.
237) resistant to RTSV, and Gam Pai 30-
12-15 (Acc. 831) resistant to vector, were
examined by ELISA in order to clarify the
relationship between the virus concentration
in plants and resistance/tolerance.
RTBV concentration in Balimau Putih
and Utri Merah was consistently low
compared to TKM 6 and Gam Pai 30-12-15
in which the concentration was lower than in
susceptible check TN1.
Except for Utri Merah rice tungro
viruses were detected in roots, leaf
sheath/culm and leaf blade of TN1, TKM 6,
Balimau Putih and Gam Pai 30-12-15, with
virus concentration highest in leaf blade,







followed by leaf sheath/culm and roots. In
Utri Merah, low concentration of RTBV was
detected only in leaf blades.
Utri Merah and Balimau Putih do not
show visible symptoms, even if infected.
RTBV causes tungro symptoms, low
concentration of RTBV throughout the
growth stage of these varieties can be the
basis of tolerance, although the mechanism
remains a mystery.

SENSITIVITY TO STREPTOMYCIN AND
STARCH HYDROLYSIS OF Xanthomonas
campestris pv. dieffenbachiae ISOLATES.
Lolita D. Valencia and Marina P. Natural,
Institute of Plant Breeding, UPLB.

Anthurium plants showing blight and
systemic symptoms were collected from
anthurium greenhouses around the Los
Banos area from November 1990 February
1991. Xanthomonas campestris pv.
dieffenbachiae were isolated using modified
TZC medium.
In vitro test results showed that 3 out of
57 isolates were resistant to 200 ppm
streptomycin sulfate. These isolates were
collected from greenhouses that have used
Streptomycin-based bactericides to control
bacterial blight. Forty five of the 57 isolates
were able to hydrolyze starch. Variability on
colony form and size were also observed
among isolates.

FIELD EVALUATION ON THE
DIFFERENT CONTROL METHODS
AGAINST EGGPLANT SHOOT AND
FRUIT BORER ON THE GROWTH AND
YIELD OF EGGPLANT. R. Bustamante,
P.B. Luzaran and L.T. Gruber, Bureau of
Plant Industry, Manila.

An experiment to determine an effective
control method against eggplant shoot and
fruit borer, Leucinodes orbonalis Guence
was conducted at the Philippine National
Agricultural School in Guyong, Sta. Maria,
Bulalcan in January to 1990 using parasitoid
wasp. Trichogramma, green Muscardine
fungus, Metarhizium anisopliae, Botanical
extract, (Neem + Lemon grass + Galangal),
Mechanical cutting of infested parts, farmers
practice use of commercial insecticides and
check of control following the randomize
complete block experimental design.
Plot size was 3 meters wide and 4
meters long with a spacing of 50 cm. between


plants and 100 cm. between rows. Each
treatment consisted of 28 plants per plot.
Trichogramma was released one week after
planting at 100 cards (200,000 wasps) per
hectare followed by weekly intervals. Weekly
cutting of infested shoots. Weekly spraying of
botanical extract (Neem + galangal + lemon
grass) at a proportion of 1:1:/600. Weekly
spraying of chemical following the farmers
practice of 1 liter/ha, was undertaken.
Metarhizium was sprayed around the plants
on Metarhizium plots.
Results showed that Trichogramma
treated plot gave the highest yield of 4,474
marketable fruits, followed by Thioden and
Decis-treated plot with 3,030 marketable
fruits. Botanical-treated plot gave 2,494
fruits, Metarhizium-treated plot 2,282 fruits
and Mechanical plot gave the lowest yield of
1,670 fruits. However, there were no
significant differences observed among all the
treatments used in the study (yield,
marketable fruits, damaged fruits, infested
shoots, exis holes). This could be attributed
to drought and inadequate supply of water
during the period when the experiment was
conducted.

GENETIC TRANSFORMATION OF
Xantho-monas oryzae pv. oryzicola BLS 335
BY ELECTROPORATION. M.I.A. Tudor,
A.K. Raymundo, M. Baraoidan and R.J.
Nelson, Institute of Biological Sciences,
UPLB and Division of Plant Pathology,
IRRI, College, Laguna.

Through the process of transformation,
a cell can take up naked DNA and
incorporate it in its genome to acquire an
altered genotype. This process can occur
naturally or can be induced by chemical
treatment. In Xanthomonas oryzae pv.
oryzicola, efforts to induce transformation
failed. Elecrtoporation was therefore,
attempted. Electroporation is a method that
can induce transient permeability of cell
membrane via electric field-induced for
formation. In. X.o. oryzicola BLS 335
electroporation with pBluescript (pBS)
plasmid DNA yielded transformants with
maxi .um electroporation frequencies of 6.90
x 1Q transformants per ug DNA and 8.25 x
10 transformants per recipient sell. Using a
cell concentration of 10 cell/ml,
transformation frequency was observed to be
directly proportional to DNA concentration
up to 800 ng/160 ul and with electric field
strength up to 2.5 kv. Higher levels were not








tried. Transformants were confirmed by
plasmid examination. No transformants were
obtained using the cosmid vector pLAFR5 as
donor. This was attributed to the larger size
of this plasmid, which made DNA uptake
more difficult for the organism. Improving
the methods and optimizing the conditions
could result in electroporation of larger
plasmids and in higher transformation
efficiency.

SURVEY AND DETECTION OF PEPPER
VIRUSES AND SCREENING FOR
RESISTANCE. L.M. Dolores and R.B.
Valdez, Institute of Plant Breeding, UPLB.

A survey for pepper viruses in 17
provinces of Luzon using DAS and DAC
ELISA showed the following in decreasing
order of occurence viz, ToMV/TMV, CMV,
TEV, CVMV, PVY and PeMV.
The virus isolates detected by ELISA in
the survey were characterized using
differential hosts and electron microscopy.
Two tobamoviruses that induced yellow and
green mosaic on pepper were identified as
TMV and ToMV, respectively. TMV caused
systemicmosaic on Nicotiana sylvestris while
ToMV induced necrotic local lesions on the

same host. Both virus isolates showed short
rigid rod particles under the electron
microscope. Another important virus found
was CMV which caused systemic mosaic
symptoms on Nicotiana glutinosa and
Cucumis sativus.
Out of 49 hot pepper entries screened
for resistance to TMV and ToMV 9 were
resistant to both viruses while 19 were
promising against ToMV only.

SCREENING OF MICROORGANISMS AS
POTENTIAL. BIOLOGICAL CONTROL
AGENTS FOR MANGO ANTHRACNOSE.
Glafera Janet F. Barroga and Lina L. Ilag.
University of the Philippines at Los Banos.

Microorganisms were isolated from the
surface of fruits and leaves of mango
(Mangifera indica Linn.). They were later
screened for their in vitro inhibitory effect on
the growth, sporulation and spore
germination of Colletotrichum gloeosporioides
Penz. Of the 166 bacteria and 14 fungi
isolated, 98 bacterial isolates and 3 fungal
isolates (E-12, P-10, G-14) inhibited the
mycelial growth of Colletotrichum
gloeosporioides. Clear inhibition zones were


formed between the pathogen and the
antagonistic bacteria. Faint inhibition zones
were formed between the pathogen and the
three fungal isolates. The antagonistic
bacteria reduced sporulation and spore
germination of C. gloeosporioides. Of the
three fungal isolates, only G-14 reduced the
spore concentration, while P-10 alone
reduced the spore germination of the
pathogen. Isolate E-12 was identified as
Pestalozzia sp., P-10 as Fusarium sp., while
G-14 is described as a non-sporulating fungus
with septated hyaline mycelia. The
antagonistic bacteria were characterized
according to their Gram stain reaction,
motility, and growth characteristics.

EPIDEMIOLOGY OF PEANUT STRIPE
VIRUS DISEASE IN PEANUT (Arachis
hypogaea L.). Editha M. dela Cruz, M. P.
Natural, Department of Plant Pathology,
University of the Philippines at Los Banos,
College, Laguna.

The disease progress of peanut stripe
was studied over two 1,040 sq m fields
planted with BPI-Pn9 peanut cultivar. Each
field was provided with an artificially
inoculated primary focus of 15 plant-hills at
center of approximately 6,400 plant-hills. The
disease progress pattern indicated a
continuous fashion of increase during the wet
and dry season. The disease progress curve
generated by PStV was an S-shaped curve
which is typical of a polycyclic or a compound
interest disease. According to Vanderplank's
equation of average apparent infection rate
for compound interest disease, higher rate of
infection occurred dining the dry season (r =
0.36 per unit week ) than during the wet
season (r = 0.27 per unit week ). Likewise,
higher levels of final disease incidence was
observed during the dry season (6.58 to 8.30
percent) than during the wet season (2.28 to
4.50 percent). The higher level of aphid
dispersal during the dry season possibly
contributed to higher disease incidence and
rate of infection.
The spread of PStV in space showed a
focal pattern indicating a localized spread of
the disease from plant-to-pant by non-
persistent aphid sectors. Incidence of PstV
decreased with increasing distance of annuli
from primary focus. The disease gradient of
PStV was ssteeper during the wet season (-
1.65) than in the dry season (-1.29). An
account of the estimated multiple linear
relationship between disease incidence and








environmental factors, indicated that
temperature was the most important factors
in the spread of PStV in both seasons.
Sunshine duration and aphid count were
significantly correlated to disease incidence
only in the dry season while evaporation and
rain were not significantly correlated in both
seasons. Aphis craccivora was identified as
the principal vector of PStV in the area.
The yield and yield components of
peanut were not significantly correlated to
time of PStV infection. Since the experiment
was greatly affected by adverse weather
conditions in both seasons, it is possible that
the growth and development of the plants
were more responsive to factors than the
disease. Hence, the study merits further
investigation.

GENETICS OF RESISTANCE TO
Peronosclerospora philippinensis (WESTON)
SHAW IN CORN. A.D. Raymundo and A.
Velilla, University of the Philippines at Los
Banos, College, Laguna.

The genetics of resistance to
Peronosclerospora philippinensis (Weston)
Shaw, incitant of downy mildew in corn, was
studied in dialled crosses and in full-sub
families. Likewise, several parameters of
quantitative resistance were ascertained.
A quantitative pattern of inheritance of
resistance to P. philippinensis of the additive
type was observe in the families and
generations studied. Onset of systemic
symptom appearance, rate of infection, and
area under disease progress curve were key
parameters in the expression of quantitative
resistance.

PRESENCE OF RICE TUNGRO
BACILLIFORM VIRUS (RTBV) IN XYLEM
CELLS OF TUNGRO-INFECTED RICE.
Sta. Cruz, F.C. and H. Koganezawa, IRRI,
Los Banos, Laguna.

Earlier studies have revealed that rice
tungro bacilliform virus (RTBV) and rice
tungro spherical virus (RTSV) are restricted
to phloem tissue of infected plants and it is
suggested that only phloem feeding by green
leafhopper (GLH) caused disease
transmission. On GLH-resistant cultivars,
however, it was observed that GLH feed
mainly from the xylem and transmitted
mostly RTBV. To determine the location of
RTBV. in host cells, rice seedlings of a
tungro-susceptible cultivar, TN1, and GLH-


resistant cultivar, ASD 8, wer inoculated with
rice tungro viruses using adult GLH. The leaf
blades of infected plants were collected at 30
DAI for electron microscope study. RTBV
were found in xylem parenchyma cells as well
as in phloem cells of tungro-infected TN1
and ASD 8, while RTSV was observed only
in phloem cells and around the boundary
between xylem and phloem tissues. These
results suggest that GLH can transmit RTBV
directly to xylem cells, where the virus
multiplies and causes infection. This further
supports the observation that on GLH-
resistant cultivars, GLH feeds mainly from
the xylem and transmitted predominantly
RTBV.

A POLYCYCLIC TEST FOR ASSESSING
QUANTITATIVE RESISTANCE TO RICE
BLAST. T.V. de Dios-Mew, E.L.M. Guico,
and J.M. Bonman, International Rice
Research Institute, Los Banos, Laguna.

Quantitative resistance to rice blast is
usually evaluated in greenhouse inoculations
in one disease cycle (monocylce). However,
this method requires test plants to be of the
same leaf age at inoculation and thus only a
few cultivars can be tested in one experiment.
A polycyclic test was developed so that many
lines can be tested simultaneously. Test
plants were grown in trays (150 plants/tray)
and inoculated in isolation cages 2-weeks
after sowing using dry culture segments of a
single isolate of Pyricularia grisea (= P.
oryzae). Disease was scored 2-3 weeks after
inoculation by visually estimating the
percentage disease leaf area of at least 15
randomly choose leaves per tray. Results
from the polycyclic tests were comparable to
those of the more tedious monocyclic test,
but differences between cultivars were
accentuated. The polycyclic test is a useful
alternative method for assessing quantitative
resistance to blast.

POPULATION MANAGEMENT OF RICE
ROOT NEMATODES, Hirschmanniella spp.,
with Sesbania rostrata. I.R.S. Soriano, D.M.
Matias and J.C. Prot. IRRI, Los Banos,
Laguna.

Two field experiments were conducted
to assess the effect of planting and utilization
of Sesbania rostrata as green manure on the
population of Hirschmanniella oryzae and H.
mucronata in a sequential cropping of S.
rostrata and rice. The growing of S. rostrita








regardless of incorporation as green manure
resulted to a highly significant correlation of
yield and nematode population densities. The
increase in yield of rice crop following the
planting and/or incorporation of the
leguminous crop could be due to fertilizer
effect and control of the rice root nematodes
or factors linked to nematode population
density. However, it will be more economical
to replace S. rostrata with a cash crop that
could augment the farmer's income.

PLANT PARASITIC NEMATODES ASSO-
CIATED WITH UPLAND RICE IN THE
PHILIPPINES. L.M. Villanueva, J.C. Prot
and D.M. Matias. IRRI, Los Banos, Laguna.

In order to determine the major
nematode genera associated with upland rice,
1,900 soil and root samples were collected
from eight provinces in the Philippines.
Samples were taken at preseeding and
maximum tillering stage from Batangas,
Camarines Norte, Camarines Sur, Leyte,
Misamis Oriental, Samar, Sorsogon, and
Zamboanga del Sur. Eleven species or
genera were identified: Criconemella,
Helicotylenchus, Hemicriconemoides,
Hemicycliophora, Hoplo-laimus, Meloidogyne,
Pratylenchus zeae, Roty-lenchulus renifonris,
Rotylenchus, Tylen-chorhynchus, and
Xiphinema. Criconemella, P. zeae,
Helicotylenchus, and Xiphinema were the
most frequently occurring nematode species
and genera. However, P. zeae was the most
predominant, it was recovered in high
population densities from all the fields
surveyed. This indicates that P. zeae could be
considered a potential threat to upland rice
production in the country.

DETECTION AND PURIFICATION OF
NON-CAPSID PROTEIN ON RICE
GRASSY STUNT VIRUS INFECTED RICE
LEAVES. Miranda, G.. and H.
Koganezawa, Division of Plant Pathology,
IRRI, Los Banos, Laguna.

Rice grassy stunt virus (RGSV) is
classified as a tenuivirus. This group of
viruses has a common characteristic of
producing non-capsis protein (NCP), but
evidence for NCP by RGSV is lacking.
Hence, this study was conducted to examine
the production of NCP by RGSV.
NCP of RGSV was easily detected in
infected rice plants but not in healthy ones by
using SDS-PAGE following Laemmli's


method. NCP was -purified through the
process of crystallization and solubilization
by differential pH.
Purified NCP formed needle shaped
crystals at pH 5.0 6.0. The needle-like
crystals measured ranged from .1.8 9.6 um
length and 0.04 0.4 um width. NCP is
composed of a single protein with a
molecular weight (MW) of 24 kd, while the
coat protein has a MW of 37 kd. The purified
NCP had a typical protein absorption
spectrum with maximum absorbance at 278, a
minimum absorbance at 252 and 0.6
absorbance ratio (A260/280)- The yield of
NCP was about 20 mg/100 g tissue. RGSV-
NCP is very similar to that produced by other
tenuiviruses.

THE MICROFLORA OF RICE. M.T.R.
Cerez, B. Cottyn and T.W. Mew. The
International Rice Research Institute, Los
Banos, Laguna.

The total microflora associated with rice
has been evaluated. Bacteria were isolated
from rice samples coming from 17 provinces
of the Philippines. Results showed that out of
the total 4684 isolates, 185 or 4% are plant
pathogenic which were mostly derived from
sheath (72%), followed by seed (20%) and
least from seedlings (8%). Initial phenotypic
(physiological and biochemical) tests on
these spathogens showed that 78% were
fluorescent pseudomonads which could be
Ps. marginalis, Ps. syringae pv. syringae or Ps.
filscovaginae. Included among the non-
fluorescent pathogens were isolates similar to
Ps. glumae, Ps. avenae, Ps. plantarii and
Erwinia herbicola. Further study is being
conducted as to the real identities of these
bacteria and their possible effects on the rice
plant.




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