Front Cover
 Analyzing the monocyclic process...
 Components of resistance to cercospora...
 Inoculum sources of rice tungro...
 Effect of herbicides on the pathogenicity...
 Seedcoating with trichoderma viride...
 Bacterial crown gall: A new disease...
 Occurrence, seed transmission and...
 Stem rot of saiago (Wikstroenia...
 Phytopathological note: Impatiens...
 Abstracts of papers presented during...
 Announcing for commercial...
 Biocon II comparative effects with...
 Back Matter
 Back Cover

Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00090520/00034
 Material Information
Title: Journal of tropical plant pathology
Series Title: Journal of tropical plant pathology.
Alternate Title: Journal of Philippine phytopathology
Philippine phytopathology
Physical Description: v. : ill. (some col.) ; 26 cm.
Language: English
Creator: Philippine Phytopathological Society
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
College Laguna
Publication Date: January-June 1993
Frequency: semiannual
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00034
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 54382605
issn - 0115-0804

Table of Contents
    Front Cover
        Front Cover 1
        Front Cover 2
    Analyzing the monocyclic process in sheath blight of rice under semi-controlled conditions
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Components of resistance to cercospora canescens Ellis and Martin in mungbean [Vigna radiata (L.) Wilcheck var. radiata] and Blackgram [Vigna mungo (L.) Hepper]
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
    Inoculum sources of rice tungro viruses
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
    Effect of herbicides on the pathogenicity of root-knot nematodes (meloidogyne incognite) on soybean [gylcine max (L.) Merr.] and amaranthus spinosus
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
    Seedcoating with trichoderma viride Pers. to control sclerotium wilt in munbean [Vigna radiata (L.) Wilczek]
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
    Bacterial crown gall: A new disease of rose in the Philippines
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
    Occurrence, seed transmission and identification of colletotrichum species causing pepper anthracnose in the Philippines and varietal screening for resistance
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
    Stem rot of saiago (Wikstroenia lanceolata L.) I. Etiology and effect of envriomental factors on growth and sporulation of botryodiplodia theobromae in culture
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
    Phytopathological note: Impatiens sultanii: Host of sclerotium rolfsii and meloiodogyne incognita
        Page 101
        Page 102
    Abstracts of papers presented during the annual convention of the pest management council of the Philippines, Cebu City, May 4-7, 1993
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
        Page 109
        Page 110
        Page 111
        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
        Page 117
        Page 118
    Announcing for commercial development
        Page 119
    Biocon II comparative effects with some nematicides on biological control of plant parasitic nematodes
        Page 120
        Page 121
    Back Matter
        Page 122
    Back Cover
        Page 123
        Page 124
Full Text


iu :

:- n


IRECTORS 1993-1994

Vice-Pres, dent
R Auditor
IED Business Manager
Board Memrber
IDO Board Member
Board Me' mber
S Board Merber
A Board Mernber
Immediate Past President

flra w u. w I I na
RII-QTIn A 7n0l11 IA A


Subscriptions: Communications should be addressed to the TREASURER, P.P.S. : Department c'
Plant Pathology, UPLB, College, Laguna 4031. Philippine Phytopathology, published ser ,annually, is the
ff i'i.nlllil1 f 'k- Pkdo -i- ~ tr\ Lun C-- f -_

_ _

hm V
-I I

'hilipp. Phytopathol. 1993, Vol. 29: 1-16


R.M. LEANO, D.B, LAPIS, and S. SAVARY ;; : '

Respectively, Instructor, College of Agriculture, Nueva
Viscaya State Institute of Technology, Bayombong, Nueva Viscaya; ,,"
Professor, Department of Plant Pathology, University of the. .
Philippines, Los Barios; ORSTOM Visiting Scientist, RPantf PRhpo y!-
Division, International Rice Research Institute, Los lai0ost-Lagurna,

Keywords: Rice sheath blight, Rhizoctonia solani,
epidemiology, monocyclic

Rice trap plants, used as probes, were exposed in quadrats
that were inoculated with sheath blight (Rhizoctonia solani Kuhn) to
study the spread of the disease. The effects of leaf wetness regime,
leaf contact frequency, and strength of inoculum source were

manipulated in the quadrats by covering therm
different durations (leaf wetness), planting hil
(leaf contacts), and varying the amount and pl
the canopy (source strength). Each expel
successive batches of trap plants.

The infection efficiency increased wit
wet and dry daily cycles. Incidence and sev
increased with increased crop density. Incr
initial inoculum tend to increase disease incide
number of infection points. Most disease va
treatments involving placement of initial inoc
compared to placement of the same amount c

over time.


Sheath blight (ShB) is a fungal
disease of rice caused by Rhizoctonia
olani Kuhn (Thanatephorus cucumeris)
'rank) Donk. Under favorable
conditions, the disease causes lesions
n leaf sheaths, which coalesce, and
pread to the upper leaf sheaths and on
ie leaf blades. In the last three

Fferent spacings
t of inoculum in
involved three

decades, as modern, semidwarf
itrogen-responsive cultivars were
itroduced, the economic importance of
heath blight increased in many rice
rowing regions of the world (Teng,
990). In both lowland and upland rice
reduction areas, 25-50% yield loss
would be incurred in rice as the disease
evelops on the flag leaf (Kannaiyan
nd Prasad, 1978). Roy (1979) found

that 36% yield loss could be incurred at
tillering stage and 11.73% at booting
stage inoculation (Tsai, 1974). In the
Philippines, the range of yield losses in
farmers' field were reported as
negligible (0.4%) to 23% depending on
the variety and the nitrogen input to the
crop (Ou and Bandong, 1976).

Detailed experiments are
necessary to study and quantify
epidemiological mechanisms. Details of
the different environmental factors
associated with ShB epidemiology can
be derived from experiments under
semi-controlled conditions where the
host, the pathogen and some
environmental factors can be
manipulated. Factors possibly affecting
ShB epidemics such as leaf wetness,
crop density and leaf contact
frequency, and the strength of inoculum
source, have been selected for
screenhouse experiments. These factors
have therefore been artificially
manipulated (stimulus), in order to
measure the disease response (Zadoks,
1972). Several biotic and abiotic factors
can be manipulated in quadrats of rice
hills that represent field plots. The
disease response can be quantified by
the use of trap plants that can probe
the conduciveness of a given
environment in a quadrat.
Measurements of disease parameters
can be derived from both the trap
plants and the quadrat. Knowledge on
the dynamics of sheath blight can help
in developing a simulation model of ShB
epidemiology that can be used to
formulate methods for successful
management of sheath blight.

Most epidemiological studies on
sheath blight have been conducted in
temperate countries. In Japan, detailed
ecological studies on sheath blight have
been conducted to develop a
computerized forecasting system.

Philipp. Phytopathol. 1993, Vol. 29: 1-16

computerized forecasting system of
yield losses due to sheath blights
(BLIGHTAS). However, this cannot be
used under tropical conditions where
effects of climate on sheath blight
epidemiology might strongly differ from
that of temperate regions. Some
aspects of sheath blight epidemiology in
the tropics have already been studied
by many researchers. However,
information on the effect of leaf
wetness duration, the amount of initial
inoculum, and some cultural practices
on sheath blight spread are still lacking.
The experiments were conducted from
March to May, 1992 in the
screenhouse of the International Rice
Research Institute, Los Baios, Laguna,
Philippines, with the following
objectives: to develop an
epidemiological method for the study of
monocyclic processes in sheath blight
and to establish functional relationships
between different leaf wetness
durations and sheath blight spread,
between different crop density
treatments and sheath blight spread,
and different levels of initial inoculum
and sheath blight spread.


Experimental Environment, Land
Preparation, and Crop Establishment

Experiments (Table 1) were con-
ducted in a screenhouse enclosed with
wire mesh, with an average tempe-
rature of 31.7/28.50C, relative humidity
of 69.3/72%, and light intensity of
690/1050 watt/m2, inside and outside
of the screenhouse (Fig. 1). The experi-
mental area was plowed and harrowed
ten days before transplanting. Basal
application of 80 kg N/ha [urea (45-0-
0)] was incorporated during the final
harrowing and levelling, a day before
transolantina. A short-culmed rice

able 1. Screenhouse experiments with their corresponding treatments.


1 A non-inoculated quadrats; no water spray
and no caging
Leaf Wetness
B inoculated quadrats; no water spray and
no caging
(5 reps.)
C inoculated quadrats; with water spray and
12 hours (1 night) caging

D inoculated quadrats; with water spray and
24-hour (2 nights) caging

E inoculated quadrats; with water spray and
36-hour (3 nights) caging

F continuous wetness; treatment E plus
water spray at day time, every hour for
3 days

2 A 15 cm x 15 cm spacing (49 hills/m2)
B 15 cm x 20 cm spacing (42 hills/m2)
Crop Density C 20 cm X 20 cm spacing (36 hills/m2)
D 25 cm x 25 cm spacing (25 hills/m2)
(4 reps.)

3 A 2.5 g of inoculum on the stems

Strength of B 2.5 g of inoculum on the leaves
source C 2.5 g of inoculum on the stems and on
leaves (5 g/hill)
(5 reps.)
D 5 g of inoculum on the stems

E 5 g of inoculum on the leaves

F 5 g of inoculum on the stems and on
the leaves (10 g/hill)

G 5 g of inoculum on the stems and
2.5g on leaves

H no inoculum

2.2-- I - - - . -- -. --- .

Philipp. Phytopathol. 1993, Vol. 29: 1-16

Irrigated land

G Concrete pavement

Plastic cage

Figure 1. Structural features of the screenhouse.
A. The screenhouse
B. The quadrat

Wire screen

Philipp. Phytopathol. 1993, Vol. 29: 1-16

bed, was used in all experiments. Plants
were transplanted ten days after
sowing at six seedlings per hill, 20 x 20
cm spacing, except for spacing
treatments in experiment 2 (Table 1).
At maximum tillering stage, plants were
topdressed with 40 kg N/ha (urea).

Inoculum Preparation and Inoculation

Sheath blight inoculum was
prepared following Mew and Rosales
(1986). Isolate AG-1-LR-1 of
Rhizoctonia solani Kuhn was mass
cultured in PDA plates for 5 days. Rice-
grain-hull (RGH) was thoroughly mixed
at 1:5 ratio. The mixture was soaked in
water for 2 hours. Heat resistant
bottles, measuring 8 x 20 cm, were
filled with the RGH mixture to 80
percent, covered with aluminum foil,
and tied with rubber bands. The RGH-
filled bottles were autoclaved at
100,000 Pascal at 1210C for 2 hours.
A 5 day-old culture of R. solani in PDA
plates was inoculated to RGH mixture
at 1:4 (agar plate:RGH bottles) ratio
after cooling. The inoculated mixture
was used as source of ShB inoculum
after 10 days of incubation at room
temperature. Source hills were
inoculated with ShB at maximum
tillering stage, 40 days after
transplanting. Except for experiment 3,
stems and leaves were inoculated with
5 grams of ShB inoculum following the
insertion method of Yoshimura and
Nishizawa (1954). The inoculum was
placed directly at the base of each hill
above the water line. In all experiments,
leaves in each hill were held together by
a rubber band tied at about 15 cm
below the uppermost leaves and
removed seven days after inoculation.
To enhance infection, inoculated plants
were sprayed with water three times a
day and covered with plastic cages
every night for seven consecutive days.

Quadrat and Trap Plant

An experimental unit was
composed of a quadrat of 3 x 3 hill; 8
source hills and 1 trap plant. The latter
was a disease-free hill that had been
grown separately, and then
transplanted at the center of the
quadrat. The source hills are inoculated
plants surrounding the trap plant (Fig.
2). The quadrat was used to represent
field plots where several biotic and
abiotic factors could be manipulated.
The trap plant was used to probe the
conduciveness of a given environment
i.e., manipulated conditions prevailing in
the quadrat, for disease spread. Any
changes on the trap plant with regard
to ShB severity, number of infection
points, and incidence reflect the
conduciveness of the environment to
disease in the quadrat. Each quadrat
was sprayed with water and covered
with plastic cage for 12 hours, every
night for 3 days except for leaf wetness
treatments in experiment 1 (Table 1).
After the duration of exposure, the trap
plant was transferred into pots, sprayed
with water and covered with plastic
cage for another 3 days.


Three experiments were
conducted to test the effect of a series
of environmental factors on the spread
of sheath blight using the trap plant and
the quadrat. The treatments (Table 1) in
all experiments were aimed at
manipulating the quadrats prior to and
during the exposure of the trap plants.
Each treatment was represented by
quadrats randomly distributed in
replicates. Leaf wetness duration in
experiment 1 was manipulated by
covering the quadrats with plastic
cages. In experiment 2, contact
frequency between plant tissues (leaves


manipulation of
leaf wetness

Quadrat "
A (infected plants) o\ \!

trap plant husbandry \

- N supply
- water supply o
- variety source

inoculation .
manipulation of:
position of lesions / 0
number of lesions
0 0

To 0 0

Figure 2. Design for analyzing epidemiological processes in sheath blight und
semi-controlled conditions.

ana snearns) was manipulatea oy ne quaaraTs. une source niii was
varying the density of hills. In the first randomly chosen and five of its tillers
two experiments, 5 grams of ShB were assessed for ShB. On each of the
inoculum per hill was used. This trap plant, all the tillers and their leaves
amount was either decreased or were assessed for ShB after the 3-day
increased, and placed at different incubation in pots. All variables used in
positions on source plants in ShB assessment (Table 3) were
experiment 3. analyzed using a repeated-measures
ANOVA (Madden, 1986). Arc-sine
Experimental Design transformation was applied on data
gathered from variables 1 and 4 while
All experiments were laid out in for variable 3, log transformation was
a randomized complete block design, used to normalize the distribution of
Except for experiment 2, quadrats were values (Gomez and Gomez, 1984).
separated by a row of border hills while
alleys, 30 cm wide were provided to
separate replications. In experiment 2, RESULTS
the quadrats were assigned to im x 1m
plots with hills planted at spacing Experiment 1
similar to that of the assigfbd quadrat.
Plots and replications were separated Effect of Leaf Wetness on the Spread of
by 40-cm wide alleys. Three batches of Sheath Blight
trap plants were exposed successively
into quadrats at 3-day intervals. The effects of leaf wetness
regimes on sheath blight development
Collection and Analysis of Data on trap plants are presented in Table 4.
Significant differences were found
Four variables (Table 2) for among the treatments for all variables,
disease measurements were considered except for severity on stems (Ss).
simultaneously because one variable Disease parameters on trap plants were
may not sufficiently reflect the factors usually highest in treatment E
that contributed to the production of [intermittent wet and dry (12/12 hrs)
new lesions. A few infection points periods] followed by treatment F
could for instance result into a high (continuous wetness). Treatment E was
severity. Infectiojn efficiency was found significantly different from other
quantified as the ratio of the number of treatments for all variables, except for
infection points on the trap plant to the Incidence on tillers (Nit/Nt) and Ss.
number of infection points on the
source hills. Infection points on the Batches of trap plants were
stems and on the leaves were observed to strongly differ in sheath
considered to account for the spread of blight development, as represented by

plJIa ILOD y ICaI-LU-lc I aiiu IC a-LI- I 3ic LI i
contacts. Sheath blight severity, count
of infection points, and incidence on
tillers (Table 2) were gathered from the
source hills and from the trap plants.
The source hills were assessed for ShE
prior to exposure of the trap plants in

-ae use vug _- -usn -.
among batches was observed for all
variables, particularly infection
efficiency (IE), which is highest in batch
1 (mean = 0.61), followed by batch 2
(mean = 0.09) and batch 3 (mean =
0.7) (Table 5).

. ..... l .Y .u i -, V ., .. -I ,

Table 2. Operational definition of the va


1. Incidence The

2. Infection point A t

3. Infection The
efficiency poi

4. Severity The

Table 3. List of variables used for sheatl







Philipp. Phytopathol. 1993. Vol. 29: 1-16

bles used in ShB assessment.


itio of the total number of infected
over the total number of tillers

cal sheath blight lesion which
r may not expand and coalesce
either lesions.

itio of the total number of infection
on the trap plants over the total
3r of infection points on the source hills.

percent area covered by sheath blight
s on the host tissues.

light assessment.


icidence on tillers %

everity on stem %

everity on leaves %

otal infection points on
teams and leaves number

Infection efficiency

Phiipp. Phytopathol. 1993, Vol. 29: 1-16

Table 4. Effect of leaf wetness on S
point on stem and leaves am


A 0.10 a 0.004

B 0.14 ab 0.003

C 0.39 c 0.01C

D 0.31 ab 0.02C

E 0.38 b 0.03C

F 0.45 c 0.03C

1Based on 5 replications and 3 batches.
2A = non-inoculated quadrats; no water spray ai
B = no water spray and no caging
C = with water spray and 12-hour (1 night) cag
D = with water spray and 24-hour (2 nights) ca
E = with water spray and 36-hour (3 nights) ca
F = continuous wetness

3Nit/Nt = mean % incidence on tillers (arc-sine t
Ss = mean % severity on stems (arc-sine transit
SI = mean % severity on leaves (arc-sine transf
IPs+IPI = mean number of infection points on s
IE = mean infection efficiency ratio of the nurT
points on the trap plants to the number of infect
on the source hills.

Column means marked with common letters are
different at 5% level of LSD.

The interaction of batches wit
treatments (A x B) was not significar
for any variable except IE (F = 3.4
P <0.01). The strong A x B interaction
on IE indicates that treatments ar
ranked differently among batches (Tabi
5). Treatment D ranked second in bate
1, fourth in batch 2 and third in batc
3. Treatments E and B consistently
ranked first and fifth, respectively, in a
batches. Rank of treatment C i
batches 1 and 2 is third while treatmer
F ranked second in batches 2 and 3.

incidence on tillers, severity and intectioi
fection efficiency.1


0.08 c 0.78 c

0.08 c 0.89 c 0.04 c

0.20 bc 2.25 b 0.23 b

0.33 b 2.57 b 0.25 b

0.48 a 3.57 a 0.50 a

0.30 b 2.81 ab 0.25 b

o caging

i,^ ; ? .

s and leaves .^ -

of infection


Experiment 2

Effect of Crop Density on the Spread c
Sheath Blight

There were no significar
differences among crop densil
treatments with respect to SI, IPs
IPI, and IE (Table 6). Nit/Nt, however
significantly increased with increasing
crop density. It was higher in treatmer
A (mean = 0.53, 15 x 15 cm spacin!
than treatments C (mean = 0.17, 20

20 cm spacing) and D (mean = 0.15,
25 x 25 cm spacing). A similar effect
was observed with respect to Ss i.e.,
increase in sheath blight severity on the
stem as crop density was increased.

A strong batch effect was
observed on severity on leaves (SI),
total number of infection points on stem
and leaves (IPs + IPI), and IE as
indicated by their variance ratios
significant at P< 0.01; Values of some
disease variables decreased among the
batches of trap plants. This effect was
not noted for Nit/Nt and Ss. No
significant interaction of treatments
with batches (A x B) was noted for any
of the variables.

Experiment 3

Effect on the Strength of Inoculum
Source on the Spread of Sheath Blight

Table 7 shows the effect of the
strength of the inoculum source on
sheath blight spread. Significant
+ n-----+ -.f- ,-,+ I nf ll -n 4 -- Kli i+/M+

Philipp. Phytopathol. 1993, Vol. 29: 1-16

There was a significant batch
effect (F = 4.76, P < 0.05) on IPs +
IPI. This suggests a decrease of
infection points within the source hills
of the quadrats with the successive
batches of trap plants. The interaction
of treatments with batches (A x B) was
significant (F = 1.94, P < 0.05) on SI
i.e., ranking of treatments varied among
batches. Treatment F had highest mean
in batches 1 (0.70) and 2 (0.71), but
not in batch 3 where it ranked fourth
(mean = 0.37). In treatment G, mean
in batches 1 (0.61) and 2 (0.51) ranked
third but had the highest mean in batch
3 (0.46). Consistent ranking in all
batches was observed for treatments C
and H which ranked fifth and eight,
respectively. There was a decline of
treatment means from the first to the
third batch in most of the variables.
This decline was strong in SI (Table 8)
with mean across treatments of 0.44 in
batch 1, 0.41 in batch 2 and 0.35 in
batch 3. Strong block effect was

and IPs + IPI (F = 5.92,,

< 0.01).

Philipp. Phytopathol. 1993, Vol. 29: 1-16

Slaole u. inTeciion eTTiciency OT sneain migni wiin interaciion o0 Lreadimtens d
trap plant exposures to quadrat after inoculation.1


B 0.10 0.02 0.002 0.04 c
C 0.59 0.10 0.010 0.23 b
D 0.65 0.04 0.070 0.25 b
E 1.20 0.14 0.170 0.50 a
F 0.51 0.13 0.110 0.25 b

MEAN5 0.61 0.09 0.070

1Based on 5 replications.

2A = non-inoculated quadrats; no water spray and no caging
o ...... .. ..... .__ .. .. ;___

th water spray and 24-hour (2 nights) caging
th water spray and 36-hour (3 nights) caging

ip plant exposed to quadrat 7 days after inoculation
p plant exposed to quadrat 10 days after inoculation
p plant exposed to quadrat 13 days after inoculation

ted across batches and replications

ited across treatments and replications

6. Effect of crop density on ShB incidence on tillers, severity and
point on stem and leaves and infection efficiency.1


A 0.53 a 0.20 a 0.32 3.15
B 0.36 ab 0.19 a 0.43 3.24
C 0.17 b 0.07 b 0.41 3.13

1Based on 5 replications and 3 batches
2A = 15 cm x 15 cm spacing (49 hills/m2)
B = 15 cm x 20 cm spacing (42 hills/m2)
C = 20 cm x 20 cm spacing (36 hills/m2
D = 25 cm x 25 cm spacing (25 hills/m2)
3Nit/Nt = mean % incidence on tillers (arc-s
Ss = mean % severity on stems (arc-sine tr;
SI = mean % severity on leaves (arc-sine tr;
IPs+IPI = mean number of infection points i
IE = mean infection efficiency ratio of the
points on the trap plants to the number of in
Column means marked with common letters
different at 5% level of LSD.

ems and leaves (log-transformed)
ler of infection
on points on the source hills.
ot significantly

Table 7. Effect of strength of inoculum
and infection point on stem an


A 1.31 ab 0.32

B 0.71 c 0.16

C 1.08 abc 0.21

D 0.76 c 0.18

E 0.89 bc 0.18

F 0.95 abc 0.21

G 1.46 a 0.34

H 0.59 c 0.11

1 Based on 5 replications and 3 batches
A = 2.5 g of inoculum on the stems
B = 2.5 g of inoculum on the leaves
C = 2.5 g of inoculum each on the stems
D = 5 g of inoculum on the stems
E = 5 g of inoculum on the leaves
F = 5 g of inoculum each on the stems an
G = 5 g on the stems and 2.5 g of inocult
H = no inoculuvi

3Nit/Nt = mean % incidence on tillers (arc
Ss = mean % severity on stems (arc-sine
SI = mean % severity on leaves (arc-sine
IPs+IPI = mean number of infection point
IE = mean infection efficiency ratio of th
points on the trap plants to the number of
on the source hills.

Column means marked with common lette
different at 5% level of LSD.

Phiipp. Phytopathol. 1993, Vol. 29: 1-16

irce on ShB incidence on tillers, severity
saves and infection efficiency1


0.28 d 3.08 b 0.86

0.53 ab 4.51 a 0.74

0.40 c 4.21 a 0.51

0.24 d 3.09 b 0.75

0.48 bc 4.24 a 0.52

0.59 a 4.62 a 0.84

0.53 ab 4.87 a 0.84

0.14 d 1.87 c

on the leaves (5 g/hill)

i the leaves (10 g/hill)
)n the leaves (7.5 g/hill)

e transformed)
stems and leaves (log-transformed)
mber of infection
action points

*e not significantly

Philipp. Phytopathol. 1993, Vol. 29: 1-16

Table 8. Leaf severity with interaction o
quadrat after inoculation.1


A 0.18

B 0.65

C 0.41

D 0.29

E 0.60

F 0.70

G 0.61

H 0.06

MEAN5 0.44

1Based on 5 replications (arc-sine transfer
A = 2.5 g of inoculum on the stems
B = 2.5 g of inoculum on the leaves
C = 2.5 g of inoculum each on the stems
D = 5 g of inoculum on the stems
E = 5 g of inoculum on the leaves
F = 5 g of inoculum each on the stems an
G = 5 g on the stems and 2.5 g of inoculi
H = no inoculum

31 = trap plant exposed to quadrat 7 day.
2 = trap plant exposed to quadrat 10 day
3 = trap plant exposed to quadrat 13 day

4Calculated across batches and replication

5Calculated across treatments and replica

the water-related variables, leaf
wetness duration often has a direct
influence on pathogen activity. In some
pathosystem, leaf wetness periods are
often regarded as synonymous with
infection periods and can account for
I- .. . . = r L ....s :- I : .--

eatments and trap plant exposures to

2 3 MEAN4

).25 0.42 0.28 d

).49 0.45 0.53 ab

1.46 0.34 0.40 c

1.19 0.25 0.24 d

1.53 0.29 0.48 bc

1.71 0.37 0.59 a

1.51 0.46 0.53 ab

1.13 0.23 0.14 d

1.41 0.35

on the leaves (5 g/hill)

on the leaves (5 g/hill)

n the leaves (10 g/hill)

er in the leaves (7.5 g/hill)tion

er inoculation
ter inoculation
ter inoculation

The first experiment (Table 4),
with strong differences between leaf
wetness treatments, shows that leaf
wetness regimes play an important role
in ShB development and spread.
Comparison of treatment means
: 1:_-* L-A ^L D L .1 -1._

number of infectious lesions on the constant, favorable climatic conditions
source hills have declined during the There is high contact probability among
exposure of the second and third the plants in dense planting so the
batches of trap plants. This would disease is easily spread from plant t
mean that lesions in this experiment plant (Premalatha Dath, 1990).
were infectious only for 3 to 10 days
after inoculation. Zadoks and Schein The effect of crop density wa
(1979) called this phenomenon in the mild on severity on the leaves, tot;
epidemic process as "removal", the number of infection points, an
transition from the infectious to the infection efficiency. Although conta(
non-infectious state. Lesions that are no frequency is higher among the leave
longer infectious are removed from the than on the stems, temperature an
epidemic process. relative humidity below the canopy ar
usually more favorable for Sh
The interaction between development (Ou, 1985). This coul
treatments and batches on infection explain why the crop density variatio
efficiency suggests that treatment had a significant effect on the stem
effects differed among batches of trap (incidence and severity) and not on th
plants. Infection efficiency on trap leaves.
plants subjected to continuous wetness
(treatment F) ranked fourth in batch 1 Strong differences were foun
and second in batches 2 and 3. among the batches of trap plants o
Continuous wetness therefore appears severity on the leaves, total number (
to slow down the removal process i.e., infection points and infection efficiency,
contribute in prolonging the infectious For incidence and severity on th
period of the inoculum. stems, batch effect was not significant
This could be attributed to th
Crop and canopy densities frequency of contact between th
influence the microclimate within source hills and the trap plant where
canopies and has been shown to contact among the leaves is higher tha
increase ShB incidence and severity contact between the stems.

Philipp. Phytopathol. 1993, Vol. 29: 1-1

LUI l \

. I

ulueu uy piulunlyeu

sLU Ilb lily Ut! uue Lu LIIC IIUI L UMllll I
exposure (3 days) of the trap plants in 1978;
Li i r J---

l10 VVILI IIIllly I GJI I LO \ViV y
^^^~ "

1r-- -1---

I 11C OLfIrU b IIe Ue 1 iiy ai n y II -l- llI
infection efficiency from batch 1 to frequency among healthy

I experiment (1
ice on tillers
were significant
g treatments.
y were higher
lent A) and
lent B) spacin
20 x 20 cm (C
icing treatment

Philipp. Phytopathol. 1993, Vol. 29: 1-16

The results of the third
experiment (Table 7) indicate that the
amount of inoculum in the source
(strength) affects ShB spread. The
effect of amount of initial inoculum was
significant on incidence, severity on the
leaves, and on the total number of
infection points. Except for infection
efficiency, all variables had highest
mean values on either treatments F or G
which correspond to an inoculum
amounf of 10 g and 7.5 g per hill,
respectively. All other treatments with
low amount of initial inoculum had also
low mean values in all variables. For
severity on the leaves and the total
number of infection points, significant
differences were found when
treatments F and G were compared to
treatments A, D and H. This indicated
that increasing the amount of initial
inoculum from 2.5 to 7.5 and 10 g
induce ShB severity, particularly the
incidence on tillers, severity on leaves,
and the number of infection points. A
similar result expressed by using area
under the disease progress curve was
obtained by Sharma (1989).

Positioning of inoculum on the
leaves had a stronger effect on disease
intensity than positioning the same
amount of inoculum on the stems. The
growth habit of transplanted rice
(Vergara, 1979) allows contact
between hills. During the third
experiment, it was observed that leaf-
to-leaf and leaf-to-sheath contacts
markedly increased from early to
maximum tillering stages. The
frequency of leaf-to-leaf contacts was
higher than the frequency of leaf-to-
sheath contacts. This probably explains
the higher ShB severity on trap plants
exposed in quadrats that had been
inoculated at the leaf level.

The interaction between
batches and treatments with respect to
severity on the leaves is indicative of
changed treatment effects in each

batch. For instance, treatment F (10 g
of inoculum per hill) had the highest
mean in batches 1 and 2, bUt not in
batch 3 where it ranked fourth (Table
8). Treatment G (7.5 g of inoculum per
hill), had the third mean severity on
leaves in batches 1 and 2, but had the
highest mean in batch 3.

The quadrat and the trap plant
were used to understand the effects of
a series of environmental factors on the
dynamics of ShB. This approach
revealed that under semi-controlled
conditions, intermittent leaf wetness,
increased contact frequency, increased
amount of initial inoculum, and
positioning of inoculum at the level
favors ShB spread. The methodology
can be used to address a number of the
rice-ShB pathosystem.


FRY, W.E. 1982. Principles of plant disease
management. Academic Press Inc.,
London. 378 p.
GOMEZ, K.A. and A.A. GOMEZ. 1984. Statistical
procedures for agricultural research.
2nd ed. IRRI book. John Wiley & Sons.
Singapore. 680 p.
HASHIBA, T. and T. IJIRI. 1989. Estimation of
yield loss and computerized forecasting
system (BLIGHTAS) for rice sheath
blight disease. Tropical Agriculture
Research Series No. 22: 163-172.
KANNAIYAN, S. and N.N. PRASAD. 1978.
Adultharai Rep. 2(1): 137-138.
KANNAIYAN, S. and N.N. PRASAD. 1983. Effect
of spacing on the spread of sheath
blight disease of rice. Madras Agric. J.
70: 135-136.

MADDEN, L.V. 1986. Statistical analysis and
comparison of disease progress curves:
In: K.J. Leonard and W.E. Fry (eds.)
Plant disease epidemiology Population
dynamics and management. Vol. 1. pp-
MEW, T.W. and A.M. ROSALES. 1986.
Bacterization of rice plants for control of
sheath blight caused by Rhizoctonia
solani. Phytopathol. 76: 1260-1264.

Jr J. aIIU J.IVI. D 4,11I
due to sheath
Res. Newsl. 1(

J, S.H. 1985. I
Kew, Surrey, E

ROYLE, D.J. and D.R. BUTLER. 1986.
Epidemiological significance of liquid
water in crop canopies and its role in

ZADOKS, J.C. 1972. Methodoloav


11: pp 139-156.

RUIIll. IVIQU. I lit




**1 J*LtflIU1r

1 ralaata ana vig

i leaf spot (CLE

ungbean (Vigna radiata (L.) Wilczek most economic:
.l *i* J L ** J."

-,-. --~-----



i'"'" ~L'~ ~'~I


down" with time (Lantican, 1984)
After thorough screening of work
germplasm collections and advance
breeding lines of V. radiata, researcher:
have not been able to identify stron!
and stable resistant gene(s) against thi
pathogen (AVRDC, 1978).

Mungbean, being self-pollinated
is markedly uniform genetically. It!
improvement, particularly with the
application of modern technology, ma
have further narrowed the genetic base
of cultivars, rendering them morn
vulnerable to diseases.

Related Vigna species ar
known to possess various desirable
traits and can be exploited to broader
the genetic base of mungbean
Blackgram (Vigna mungo (L.) Hepper) i`
a closely related species that posses,
many of the attributes desired fo
mungbean improvement, particularly'
stable genes of the quantitative type fo
CLS resistance (Singh, 1981; AVRDC
1973 and 1978).

In the course of germplasry
evaluation for CLS resistance
significant variation in genotypE
responses to CLS in mungbean anc
blackgram has been observed
Mungbean and blackgram varieties wit[
varying levels of resistance anc
susceptibility to CLS have beer
identified. Identification of components
contributing to resistance to CLE
infection and elucidation of the genetics
of resistance are very important in the
development of varieties with effective
and durable type of resistance.

This study was conducted tc
characterize the CLS reaction o-
selected varieties of V. radiata and V.
mungo, and determine the components
of resistance to C. canescens.


Preparation of test plants. Fivi
V. mungo varieties (Acc. Nos. 25
1250, 22, 1130, 1228), threi
moderately resistant (IPB M82 6-8
Pagasa 5, Pagasa 7) and oni
susceptible (NCM 53) varieties of V
radiata were used to determine thi
components of resistance to C
canescens in screenhouse and in fielk

For screenhouse evaluation, thi
seeds were sown in heat-disinfestet
soil contained in 20 cm clay pot. Threi
seeds were sown in each pot. Ten poti
were planted to each variety, each po
representing one replication. The
experiment was arranged in
Completely Randomized Design (CRD).

For field evaluation of disease
development, the seeds were planted ir
2 m row plots, at a distance of 0.25 n
between hills and 1.0 m between rows
The plots were laid out in a Randomizec
Complete Block Design (RCBD) with ;
replications. Spreader rows of NCM 5.
(susceptible check), which were
severely diseased during the course o
the experiment, were planted around(
each block four weeks before the tes
entries were planted to ensure
constant supply of airborne conidi;
throughout the field experiment.

The plants in both experiment,
were maintained following the
recommended cultural practices o
growing mungbean.

Culture of the fungus anc
inoculum preparation. The fungus was
isolated from CLS-infected mungbear
leaves collected from the field. The
leaves were surface-disinfected' witf
0.5% sodium hypochlorite for 2-2

minutes and were incubated for 48-72 cumulative number of co
ours, in Petri dishes lined with of lesion until lesions

nidia per mm-

one by picking conidia arising from during which lesions sporulate; and g)
onidiophores and plating on mungbean Defoliation time when all the lesions
eed decoction agar (MSDA). Single were removed through defoliation.
pore isolates from pure cultures as
vell as subcultures were maintained on Disease development. In the
/ISDA slants and incubated for 14 days field evaluation, weekly disease ratings
t 27C. Spore suspension was were made using the disease severity
preparedd by adding sterile distilled scale (Fig. 1) adopted by the Legumes
vater to 14-day old cultures of the Pathology Group of the Institute of
ungus. Spore density was adjusted to Plant Breeding, UP at Los Banos,
10,000/ml. College, Laguna. Twenty sample plants
per variety per replication were
Inoculation procedure. Three randomly selected for observation. The
veeks after planting, the test plants same 20 plants were used for disease
vere uniformly inoculated late in the assessment throughout the experiment.
afternoon by spraying directly on the
power surface of the first trifoliate Data analysis. Square-root
eaves. In the screenhouse experiment, transformed data were used to stabilize
II test plants were covered with the variances prior to statistical
moistened polyethylene bags overnight analysis. Correlation analysis was used
nd a wet soil condition was maintained to examine the association among per-
o provide the necessary moisture for cent infection and components of resis-
ifection. In the field evaluation, regular tance to C. canescens. Discriminant and
vaterina of plants was done to provide Stenwise Discriminant (SAS. 1985)

nd relative humidity were also

Components of resistance. In
he screenhouse experiment, one leaf
ier plant or a total of three leaves per
lot (replicate) were selected for
observation. The following data were
recorded: a) Incubation period the
lumber of days from inoculation until
he appearance of the first lesions; b)
.atent period the number of days from
appearance of lesions until the onset of
populationn; c) Lesion size the
diameter of the lesion measured at 2
lays intervals starting when the lesion
vas already visible until the lesion
;oalesced; d) Lesion density the
lumber of lesions per cm per unit
ime; e) Sporulation capacity the

ind resistance reaction to the disease
lased on their percent infection and
componentss of resistance. Regression
analysis was used to identify which
resistance components can best predict
resistance to C. canescens in the field
n terms of final disease severity.


diseasee Development

The development of CLS in the
ield over the course of the season on
nungbean and blackgram varieties is
>resented in Table 1. Representative
subsets of the same data are
graphically illustrated as disease
wogress curves in Figs. 2 to 5.



< 1% 1-5% (

Figure 1. Assessment key for estimate
on mungbean leaves.

Table 1. Percent disease severity on
portions of Vigna radiata mun!

6 13 20

Acc 1130 1 0.00 0.00 0.46

Acc 1228 1 0.00 0.00 0.40

Acc 1250 1 0.00 0.00 0.28

Acc 25 1 0.00 0.00 0.53

Acc 22 1 0.00 0.00 0.40

IPB M82 6-8 1 1.83 9.45
2 9.01

NCM 53 1 1.42 10.35
2 9.06

Pagasa 5 1 1.38 10.96
2 5.79

Pagasa 7 1 1.48 10.54
2 8.96

1Acc 1130, 1228, 1250, 25 and 22 are V. mungo. IPB

2 1 denotes lower 1/3 leaf-bearing portion of the plan

e lower 1/3 and upper 2/3 leaf-bearinc

34 44 49 55 64 71

9 1.96 3.78 7.10 12.10
3.80 5.80 8.61 10.61

0 3.50 5.55 7.33 12.00
3.75 5.10 7.24 9.48

1 0.94 0.99 1.69 2.97
7.07 3.75 6.82

4 4.55 1.43 1.60 4.51 9.00 11.00
3.05 5.75 12.50 18.50 14.51

2 1.22 1.57 3.60 7.53
2.36 2.78 5.94 8.87

5 34.50 42.31 57.8.867.21 97.84 100.00

5 41.19 50.70 64.34 80.39 99.62 100.00

1 32.36 41.92 55.51 74.61 99.39 100.00

7 40.37 52.39 74.36 82.20 98.74 100.00

6 6-8, Pagasa 5, Pagasa 7 and NCM 53 are V. radiata.

Phipp. Phytopathol. 1993, Vol. 29: 17-29

Disease severity (%)

Acc 25 a)
-+- Acc 25 (b)
-- NCM 53 (a
NCM 53 (b

4 13 20 27 34 44
Days after Inoculation
a = lower canopy
b = upper canopy

Figure 2. The development of CLS
NCM 53 and Acc 25.

49 55 64 71

in the field over the course of the season on

Disease severity (%)

a = lower canopy
b = upper canopy

Days after Inoculation

Figure 3. The development of CLS in the field over the course of the season on
NCM 53 and Acc 22.

Philipp. Phytopathol. 1993, Vol. 29: 17-29

Disease severity (%)

a = lower canopy
b = upper canopy

Figure 4. The development of CLS
NCM 53 and Pag-asa 7.

Days after Inoculation

in the field over the course of the season on

Disease severity (%)

6 13 20 27 34 44 49 55 64 71

a = lower canopy
b = upper canopy

Days after Inoculation

Figure 5. The development of CLS in the field over the
NCM 53 and Pag-asa 5.

course of the season on

lilipp. Phytopathol. 1993, Vol. 29: 17-29 23

The disease rating used time was only 6 to 12 percent, much
characteristically separated the infection less than the 100 percent infection in
f the lower 1/3 from that of the upper mungbean varieties.
/3 leaf-bearing portions of the plant
uch that when the disease reached the Components of Resistance to C.
pper portion, disease severity was canescens
iken as the average percent infection
f the whole plant. Different components affected
the expression of resistance to C.
The blackgram varieties canescens (Table 2). Host resistance in
Khibited delayed onset of infection, V. mungo (Acc 22, Acc 25 and Acc
*nger time for CLS to reach the upper 1250) operates by increasing the
13 leaf bearing portion of the plant, latency and incubation time of the
id reduced total amount of disease pathogen. Considering both incubation
severity at harvest compared to the time and latent period, it takes only
susceptible (NCM 53) and moderately about 12.23 days from inoculation for
sistant (IPB M82 6-8, Pagasa 5 and the pathogen to sporulate on NCM 53
agasa 7) mungbean varieties, while it takes 19.57, 20.86 and 19.97
days, respectively, on Acc 22, Acc 25
CLS infection was observed on and Acc 1250. This is considered one
lungbean varieties 6 days after of the most effective types of
ooulation while blackgram varieties resistance as it decreases infection rate
owed a much delayed disease onset (Parlevliet, 1979). IPB M82 6-8, Pagasa
t 20 days after inoculation. At this 5, Pagasa 7, Acc 1130 and Acc 1228
point (20 days after inoculation), CLS delayed latent period by only 7, 3, 4,
as already progressed to the upper 2/3 11 and 34%, respectively. These were
af-bearing portion of mungbean not considered significantly different
varieties. This was 34 to 41 days from that of the susceptible variety,
earlier than the blackgram varieties. NCM 53.

Under highly favorable NCM 53 had the highest
conditions for disease development in number of lesions per leaf with an
ie present study, resistance possessed average of 122.33 lesions or 4.17
v Pagasa 5, Pagasa 7 and IPB M82 6- lesions per cm2. Lesion density
was apparently ineffective. These increased at the rate of 9.41 lesions per
varieties showed disease reactions day. Although no significant differences
milar to the susceptible variety, NCM were observed among the other
3, and by the end of the season, mungbean cultivars, Pagasa 7
percent infection reached as high as numerically had the highest lesion
00%. number per leaf with 115.03 lesions
prior to defoliation or with 3.15 lesions
Given the same favorable per cm2. Pagasa 5 had the fastest rate
conditions for disease development, of lesion number increase with 7.05
considerable dearee of resistance exists lesions oer day Droducinq 3.95 lesions

III vaii~il~~. Illo uc3iay~u
.. I ,I

ble 2. Disease severity and comparison among component traits of resistance
C. canescens in V. radiata and V. mungo varieties1.

Percent Incuba- Latent Lesion No. of Highest Sporu- Infectious Defo-
iety2 Infection tion Period Size Lesions/ No. of lation Period liation
Period (days) (mm2) cm2 Lesions/ Capacity (days) (days)
(days) Leaf

M82 6-8 100a 5.93b 6.73cd 6.27a 3.78ab 94.55ab 626.46bc 13.47cd 26.67

asa 5 100a 6.10b 6.27d 6.87a 3.95a 105.73a 877.63ab 15.07bc 24.30
asa 7 100a 5.93b 6.37cd 7.10a 3.15abc115.03a 1089.80a 17.03b 29.37

M 53 100a 6.13b 6.10d 6.10ab 4.17a 122.33a 1047.80ab 11.13d 21.00

22 9.23cb 9.60a 9.97ab 5.93ab 2.22c 81.93ab 773.34abc 21.90a 38.62

25 12.86b 10.13a 10.53a 4.40bc 2.53c 84.03ab 793.03abc 23.43a 41.57

1250 6.83c 9.70a 10.27ab 2.52d 2.32c 61.50b 471.79c 24.57a 41.67
1130 10.50cb 5.93b 6.80cd 2.95cd 2.73bc 96.43ab 742.00abc 25.30a 39.70

1228 9.50cb 7.27b 8.17bc 3.67cd 2.92abc 62.67b 771.67abc 24.30a 39.43

(%) 5.00** 13.81** 12.86** 19.61**21.34** 21.96** 27.21** 10.91** 6.85

letter are not significant at alpha = 0.05 by DMRT.
2Acc 1130, 1228, 1250, 25 and 22 are V. mungo. IPI

day. Acc 22 had the least number i
lesions with 2.22 lesions per cm
increasing at a rate of 3.28 lesions p,
day. Acc 25 and Acc 1130 on t1
average, had higher number of lesior
per leaf compared to NCM 5:
However, their rates of lesion formatic
were slower such that at the end of tl
infection period, Acc 25 and Acc 113
had 31% and 21% fewer lesion:
respectively, relative to the susceptib
variety, NCM 53.

Pagasa 7 sustained the large:
lesions with an average of 7.10 mrr
with lesion expansion rate of 0.39 mrr
per day. This is not significant
different from the rates in othi
mungbean varieties. Among blackgrai
accessions, there was a considerab
reduction in lesion expansion

2 6-8, Pagasa 5, Pagasa 7 and NCM 53 are V. radiata.

particularly in Acc 1250 which include
the appearance of only small lesions c
the leaves as well as slowing of lesic
growth over time.

Blackgram accessions had low,
lesion density and lower lesion diamet,
compared to mungbean varieties. The,
parameters indicate a restricted growl
of the organism in the host tissues.
reduction in disease several
consequently is observed since tt
successful development of sporulatir
lesion after initial infection decrease
with a decreasing rate of lesion growtl
The higher number of lesions and fast
rate of lesion expansion on mungbee
varieties are indications of the inabilil
of the host to inhibit establishment an
further growth of the pathogen in tr
tissues. The defense mechanism


esen in ine moaeraxely resistant
rieties is insufficient to restrict the
velopment of the pathogen.

Sporulation proceeded at a
ster rate in mungbean causing higher
id of infections. Infectious period
tended up to 17 days from initial
orulation in Pagasa 7. Lesions on
ackgram accessions sporulated 55%
cc 1250) and 24% (Acc 25) less
an NCM 53. Sporulation rates were
inificantly lower by 61-77% and 48-
1% compared to NCM 53 and Pagasa
respectively. The reduction in
orulation by the pathogen in
ickgram accessions can be attributed
effect of host resistance in limiting
sion size and consequently reducing
a total lesion surface available for
orulation (Parlevliet, 1979). Infectious
riod of blackgram varieties extended
to 10-14 days more than NCM 53
d 8-10 days more than the other
ungbean varieties. It was observed
at as long as the leaves were intact
d have not senesced, the lesions
intinued to sporulate. However,
orulation proceeded at a much lower
te which somehow slowed down the
ogress of an epidemic because of the
duction in the total number of
fectious units produced.

Defoliation of the susceptible
iriety NCM 53 occurred 21 days after
oculation while the leaves of other
ungbean varieties abscissed 24-29
lys after inoculation. Defoliation in
ackgram varieties ranged from 38-41
iys, which were delayed by 81-95%
lative to NCM 53. The fast lesion
owth and tremendous sporulation in
e susceptible variety could have
iysiologically hastened abscission.

relationship of Resistance Components

Incub.ation period and latent


related (r = 0.985) (Table 5).
ferences in incubation period and
ent period are often assumed to
lect differences in growth rate of the
thogen in the host (Parlevliet, 1979).
high lesion density, there was a
ortening of the incubation period (r =
).769) and the increase in total sporu-
ion (r = 0.788). Incubation period
d latent period were also strongly and
sitively correlated with defoliation
riod (r = 0.711; r = 0.802) due to
section by C. canescens. At higher
ion counts per unit leaf area, a cycle
disease development maybe
mpleted over a significantly shorter
riod. Since lesion density was
gatively correlated (r = 0.811) with
foliation, decreasing lesion density
r unit leaf area would correspondingly
lay leaf loss over time.

As with latent period, lesion
e is assumed to reflect growth rate
the pathogen in the host and there-
e, its spore production. Resistant cul-
ars having significantly smaller
.ions due to restricted growth and
ionization of C. canescens in the host
sues would therefore have lower
ore yield compared to their suscep-
le counterpart. A significant positive
rrelation between lesion size and
ore production (r = 0.661) was

The infectious period showed a
gative correlation with lesion size (r
0.811) and lesion density (r = -
765). At high infection load, the
lives were apparently exhausted
oner, resulting in hastened defoliation
d early termination of spore pro-
ction. Delayed defoliation resulting
)m low infection load significantly


Except for sporulation capa
nthar tnmnnnAnt- warp -innific;



I Mile (3. 1-orreiaxo

ri coemTIciei

1. % Infection 1.600** -0.715*

its Tor c


n -70r

period 1.000** 0.985

3. Latent period 1.000

4. Lesion size

5. Lesion density

6. Sporulation

7. Infectious

8. Defoliation

The correlation between any two compc

ns = not significant
= significant at alpha = 0.05
** = significant at alpha = 0.01

Table 4. Regression of percent in

ponents of resistance to C. canescens.

4 5 6 7 8

0.830** 0.890** 0.577ns -0.943** -0.955

-0.425ns -0.769* -0.469ns 0.613ns 0.711

-0.518ns -0.833**-0.563ns 0.705* 0.802

1.000** 0.617ns 0.661* -0.811* -0.787

1.000** 0.788** -0.765* -0.811

1.000** -0.510ns -0.575

1.000** 0.980


its of resistance is based on 30 observations.

tion of lesion density and defoliation.



discriminate between resistance and susceptible reactions to CLS.


Ilation O.bZa'" U.4/Z

in size 0.481** 0.519

on Density U.454" U.4ot^"

rulation Capacity 0.392** 0.608**

Wilk's Lambda the likelihood ratio statistic for testing the hypothesis that the mear
lasses on the selected variables are equal in the population (SAS, 1985).

Average Squared Canonical Correlation close to one if all groups are well separ
f all or most directions in the discriminant space show good separation for at least 2 gr

- significant at alpha = 0.0001

to percent infection. The with lesion density (LD) and defolia
in the total amount of time (D) contributing 7% and 8
observed among the variability in Y, respectively, after
varieties could be explained effect of other variables have b
mbined effects of the various eliminated.
ts of resistance. Resistance
lescens tends to be linearly According to Parlevliet (19
with a longer latent period, lesion density affects the reproduce
ion density and decreased rate of the pathogen resulting
i. Parlevliet (1979) noted that reduced disease severity. He fur
nponents directly affect the noted that the number of success
- -A+, _4f +t,. .l+-,rnn infnf-+ti-rn in t+rme nf cnrn1iln

riod and lesion size do not affect the
productive rate of the pathogen
ectly. They tend, however, to be
wrongly correlated with observed latent
riod and spore production,
spectively. Regression analysis
owed that lesion density and
foliation time accounted for 90.30%
the total variability observed in
ease severity caused by
- - ----- -<-r.i-l TA,- --A-1 V

Ionization. Defoliation time is
gatively correlated with lesion density
= -0.811). The ability of the plant to
lay defoliation is certainly one of the
measures of resistance in mungbean. In
ast mungbean varieties, the leaves
main functional instead of senescent
ter the maturity of the first pods. The
olonged reproductive behavior of
ungbean (60% of the total lifespan)
.rrmit th+h nrnAllrtinn nf carnnrl nnrl



contribute 6 to 38% of the total y
(Garcia, 1979).

An efficient field screer
which hopefully integrates
cumulative effects of all facl
contributing to resistance is t
warranted. In peanut, Johnson et
(1986) suggested that assessments
percent diseased leaflet or perc
defoliation taken at the right time we
be easier, faster and would provide
much relevant information about f
performance of genotypes being tes
for infection caused by early leaf sF
Such method can be adopted
screening mungbean/blackgram lines
CLS resistance.

This study clearly showed 1
evaluating the resistance compone
of host-pathogen interactions is
important step toward the selection
parents to be used in the develop
of more durable and stable resist
varieties or hybrids. Based on the c
gathered, linear functions were derin
which would discriminate varin
resistant reaction from that of
susceptible reaction to CLS. The lin
discriminant function for CLS resista
is: Y = -51.818 + 0.297 (IP) + 3.,
(LP) + 1.755 (LS) + 1.901 (LD)
0.271 (SC) + 12.636 (D) 0.694 (T
where IP is incubation period; LP
latent period; LS is lesion size; LC
lesion density; SC is sporulal
capacity; D is defoliation time and "
is infectious period. The lin
discriminant function for suceptib
reaction to CLS is: Y = -39.718
0.692 (IP) + 3.164 (LP) + 3.008(
+ 5.225 (LD) + 0.366 (SC)
110.032 (D) 0.978 (TLS). These
the sets of quantitative variables 1
best differentiate the varie
evaluated. Stepwise Discrimin
analysis (Table 5) showed I
defoliation time, lesion size and dens

d and sporulation capacity were
variables that best reveal
differences among these varieties.
e Determining lesion size
S degree of sporulation, however,
I difficult tasks under field condition
S Since the host resistance reaction v
f sufficiently explained by the paramet
t lesion density and defoliation tir
I these two components can be relia
S used in CLS assessment. Lesion den,
I and defoliation time are frequer
1 correlated with other resistal
components. Thus, selection
r resistance response based on these t
r parameters would reflect the effects
several mechanisms. This resistance
presumably more durable and effect
t (Parlevliet, 1979).

I From the data gathered, it v
f observed that the degree of associate
t of some components varied. Thi
t variations probably arise from gem
a differences in these components (\
S der Plank, 1963; Berger, 19'
I Parlevliet and Van Ommeren, 197
a This opens the possibility
r accumulating resistance genes
quantitative resistance. This appro,
i to plant disease control stresses
acceptable level of disease which i
) have little or no effect on yield i
s quality.
I Useful genes for resistance
S CLS appear to be present in blackgri
r Interspecific hybridization can be
r desirable long-term program to trans
CLS resistance into mungbean i
) bring the two species into a comrr
gene pool.


Progress Report for 1972. AVR
Shanhua, Taiwan. p. 19.

Progress Report for 1975. AVRDC,
Shanhua, Taiwan.

Progress Report for 1977. AVRDC,
Shanhua, Taiwan. p. 90.

ERGER, R.D. 1977. Application of
epidemiological principles to achieve
plant disease control. Annu. Rev.
Phytopathol. 15: 165-183.

IARCIA, C.T. 1979. Physiological and agronomic
response of mungbean (Vigna radiata
Wilczek cv. "Pagasa") to nitrogen and
phosphorus fertilization and inoculation
under varying rates of irrigation. M.S.
Thesis, Univ. Philipp. Los Bahos,
College, Laguna, Philippines.

1986. Relationship between
components of resistance and disease
progress of early leaf spot on Virginia-
type peanut. Phytopathology 76: 495-

ANTICAN, R.M. 1984. Advances in mungbean
breeding in the Philippines and
prospects for further improvements.
D.L. Umali Professorial Chair Lecture,
October 2, 1984. Dept. of Agron., Univ.
of the Philipp., Los Banos, College,


4ANGABAN, F.L. and M.P. NATURAL. 1988.
Variation in Cercospora, causal
organism of mungbean leaf spot in the
Philippines. Proc. of the 20th Annual
Convention of the Pest Control Council
of the Philippines.

4EW, I-PIN C., T.C. WANG and T.W. MEW.
1975. Inoculum production and
evaluation of mungbean varieties for
resistance to Cercospora canescens.
Plant Dis. Rep. 59: 397-401.

Partial resistance of barley to leaf rust,
Puccinia hordei. II. Relationship between
field trials, microplot tests and latent
period. Euphytica 24: 293-303.

'ARLEVLIET, J.E. 1979. Components of
resistance that reduce the rate of
epidemic development. Annu. Rev.
Phytopathol. 17: 203-222.

JUEBRAL, F.C. 1974. Some diseases of legumes
in the Philippines and their control.
Handout. IRRI/MCTB. p. 1128.

Personal Computers, version 6 ed. Cary,
NC: SAS Inst. INC. 378 pp.

;INGH, D. 1981. Breeding for resistance to
diseases in greengram and blackgram.
Theor. Appl. Genet. 59: 1-10.

/AN DEER PLANK, J.E. 1963. Plant disease:
Epidemic and control. Academic Press,
New York and London. 349 pp.




Assistant Scientist,
Pathologists, Division of Plant
Research Institute, Los Banos, L

Keywords: Rice tungi

Naturally infected stubble
virus inoculum sources for leafl
insect mortality varied with r
infected singly or dually with ric
rice tungro spherical virus (RT!
harvest. The presence of RT
indicates that some wild ricE
Greenhouse inoculation of 5 wile
RTBV and RTSV and RTBV aloi
nigropictus, N. malayanus, and
infection mostly by RTSV on O.
minute and 0. eich,
P. coarctata were not infected r
used. Virus recovery from RTS
virescens resulted to very low t
Native 1 seedlings. High absorb;
weed species D. setigera and L.
tungro viruses but no virus part
microscopy. Back inoculation fro
infection. N. nigropictus failed
preferred hosts E. glabrescens ar
also failed to do the same to its i


Tungro is the most important
viral disease of rice in South and
Southeast Asia and it is caused by rice
tungro bacilliform virus (RTBV) and rice
tungro spherical virus (RTSV) (Hibino et
al., 1979). The viruses are transmitted
by leafhoppers Nepthotettix cincticeps
(Uhler) (Hibino, 1963); N. malayanus
Ishihara et Kawase (IRRI, 1973);



*search Assistant, and Plant
athology, The International Rice
juna, Philippines.

viruses, epidemiology, inoculum

of different rice cultivars served as
pper vectors. Virus recovery and
cultivars. Volunteer rice plants
ungro bacilliform virus (RTBV) and
) harbor leafhopper vectors after
Sin field-collected 0. rufipogon
species are infected in nature.
ce species using RTSV alone; both
as virus sources and Nephotettix
virescens as vectors resulted to
rachyantha and RTBV alone on O.
reri. 0. granu/ata and
ardless of vector and virus source
infected 0. brachyantha using N.
ismission on susceptible Taichung
ces in ELISA were obtained in the
exandra after inoculation with the
e was trapped by immunoelectron
D. setigera to rice did not result to
:o transmit tungro viruses to its
E. colona. Likewise, N. malayanus
:ural host L. hexandra.

N. nigropictus (Stal) (Rivera and Ling,
1968); N. parvus Ishihara et Kawase
(Rivera et al., 1972); N. virescens
(Distant) (Rivera and Ou, 1965; Ling,
1966); and Recilia dorsalis
(Motschulsky) (Rivera et al., 1969). N.
cincticeps is distributed only in the
temperate and subtropical regions of
Asia while the other Nephotettix
species are distributed in the tropics
(Inoue, 1986). Transmission of RTBV

Philipp. Phytopathol. 1993, Vol. 29: 30-41

by leafhoppers is dependent on RTSV
but RTSV can be transmitted alone.
N. virescens transmits both viruses
more efficiently than the other vector
species (Ling, 1972; Hibino, 1983).

Laboratory tests and field
surveys showed that N. virescens is
restricted to rice, N. malayanus to
Leersia hexandra and N. parvus to
Isachne globosa. N. nigropictus is
polyphagous, occurring on L. hexandra,
Echinochloa colona, E. crus-gal/i,
Hymenachne pseudointerrupta and rice
(Inoue, 1986). Because of the wide
host range of the vectors, it is
suspected that plants other than rice
may play an important role in the
tungro epidemiology.

Under intensified rice cropping
systems with continuous or staggered
plantings, diseased stubbles, weeds and
standing rice crops act as sources of
inoculum for the carryover of tungro
viruses (Mukhopadhyay et al., 1986).
Volunteer plants and wild rice species
may also play an important role in
disease survival between cropping

In this study, rice stubbles,
volunteer rice, wild rice species, and
weeds reported as hosts of the leaf-
hopper vectors were studied in the
greenhouse and in the field to examine
whether they are alternative hosts of
the tungro viruses or they serve as po-
tential sources of virus inoculum. Preli-
minary results have been published (Flo-
res et al., 1989; Tiongco et al., 1992).



Tungro vectors, N. virescens,
N. nigropictus, and N. malayanus were
used in the transmission tests.
N. virescens was reared continuously

for several generations on Taichung
Native I (TN1) plants in the greenhouse.
N. nigropictus and N. malayanus were
collected from the field and reared on
E. glabrescens and L. hexandra,

Acquisition and Inoculation

Adult insects were given acqui-
sition feeding periods of 1-4 days on
TN1 plants infected with both RTBV
and RTSV or RTSV alone. The insects
were then introduced into the potted
test plants in Mylar cages or test tubes
for inoculation feeding periods of 1-7


All inoculated plants were in-
dexed by latex test (Omura et al.,
1984) or enzyme-linked immunosorbent
assay (ELISA) (Bajet et al., 1985) 3-4
weeks after inoculation. Plant extracts
with high absorbance in the ELISA were
further observed under the electron
microscope (Milne, 1984).

Virus Recovery Test

Back inoculations to rice were
conducted using virus-free insects
which were obtained by introducing
newly emerged adults to young TN1
seedlings in pots grown in insect-free
cages. The seedlings were changed
twice a day for 2 days. The virus-free
insects were introduced into the test
plants for 3-4 days virus acquisition
feeding. The insects were then given a
24-hr inoculation feeding on TN1 seed-
lings in test tubes. Three weeks later,
the presence of tungro viruses on ino-
culated TN1 seedlings was determined

Rice Stubble

Greenhouse study. Stubbles of
cultivars IR22, IR26, IR36 and IR54

taken from the field and transferred to
individual pots in the greenhouse. The
stubbles with new growths were
indexed by ELISA to determine virus
infection. Stubbles of IR22 and IR36
were used as sources of both RTBV
and RTSV, IR26 as source of RTBV
alone and IR54 as source of RTSV
alone. N. virescens was used to acquire
both viruses from the infected stubbles
for 4 days and thereafter it was used to
inoculate 7 day-old TN1 seedlings in
test tubes for 1 day. The inoculated
seedlings were planted in pots and
indexed by the latex test after 3 weeks.

Field study. Eighty stubbles of
IR64 and 40 stubbles of TN1 naturally
infected with both RTBV and RTSV as
confirmed by ELISA were individually
caged in the field and used as virus
sources 2 weeks after harvest. Twenty
male N. virescens were introduced into
each stubble. The mortality rate of the
insects was determined the following
day by counting the number of
remaining insects. The infectivity of
insects was tested on TN1 seedlings in
test tubes with a 24-hr inoculation
feeding. In another test, N. virescens
and N. nigropictus were collected by a
sweep net from 1 to 4 week-old
stubbles in the field and their infectivity
were tested. Inoculated seedlings were
assayed by latex test 3 weeks later.

Volunteer Rice

Volunteer rice plants from
spilled and germinated seeds after
threshing were taken from various
threshing sites in the 1990 dry season
crop in Magarao and Pili, Camarines
Sur; Barotac Nuevo, Iloilo; and Isulan,
Sultan Kudarat. The sites were spread
over an area of 2-5 ha in tungro-
endemic fields. Number of tungro
vectors in 3 to 4 week-old volunteer
rice plants was determined by 10
sweeps of a 33-cm diameter insect net.

Tungro virus infection was determined
by taking 10-20 batches of 10
seedlings from each site and subjecting
them separately to ELISA.

Wild Rice

Wild rice plants having
phenotypic characteristics similar to
those of grasses and which may be
growing unnoticed in the field were
tested as possible alternative hosts of
the tungro viruses. The wild rice plants
were obtained from the Division of
Plant Breeding, Genetics, and
Biotechnology, IRRI, and kept in insect-
proof cages. The plants were assayed
for presence of the tungro viruses using
ELISA. Virus-free plants were then
propagated and maintained in the
greenhouse for the following inoculation

Single inoculation of RTBV and
RTSV. N. virescens were given 3-day
acquisition feeding on TN1 plants
infected with both RTBV and RTSV. Six
to 13 plants of 0. brachyantha A.
Chev. et Roehr., 0. eichingeri A. Peter,
0. granulata Nees et Arn. ex Watt.,
0. minute J.S. Presl. ex. C.B. Presl.,
and Porteresia coarctata (Roxb.)
Tateoka were inoculated at 5
insects/plant for 5 days. Ten TN1
seedlings were inoculated using one
insect/seedling for 1 day as a control.
Inoculated wild rice and TN1 plants
were assayed for tungro viruses one
month after inoculation. Two to four
uninoculated plants of each test species
were used as control for ELISA test.
Those considered with positive
infection by ELISA were further
examined by IEM.

Two-fold inoculation. Wild rice
plants were inoculated with RTSV alone
and subsequently with both RTBV and
RTSV 14 days after the first inoculation
to determine their resistance to RTSV
using other Nephotettix species.

Philipp. Phytopathol. 1993, Vol. 29: 30-41 3

N. nigropictus and N. malayanus fed for (L.) Nees, Panicum repens L., an
3 days on RTSV-infected TN1 plants Paspalum distichum L. The plants wer
were used in the first inoculation. The indexed for the presence of tungr
same plants were inoculated 2 weeks viruses using ELISA. Virus-free plant
later by N. virescens previously fed for were propagated as test plants fc
3 days on RTBV and RTSV-infected greenhouse inoculation.
plants. Two N. nigropictus and 3 each
of N. malayanus and N. virescens were Four to 15 plants of each wee
used per seedling in the 5-day species were inoculated by N. virescen
inoculation period. Four to six plants of previously fed for 3 days on TN1 plant
each species including TN1 plants infected with both RTBV and RTS\
serving as control were inoculated. Ten insects were introduced into
Before the second inoculation, test Mylar cage with the test plant for a 5 t
plants were assayed 14 days after 7-day inoculation feeding. Ten TN
inoculation for RTSV infection using seedlings serving as control were give
ELISA. Plants were again assayed 28 1-day inoculation feeding using
days after the second inoculation, insect/seedling. Inoculated plants wer
assayed for rice tungro viruses usin
Virus recovery from wild rice. ELISA and IEM.
Test plants infected with any of the
viruses after the two-fold inoculation In another test, a total of 4
test were used as virus sources. Adults E. glabrescens and 17 E. colona plan
of virus-free N. virescens were given a grown from seeds were inoculated 2
4-day acquisition feeding on infected days after germination by N. nigropictL
test plants. The test was repeated using and N. virescens previously fed c
the same plants as virus sources. RTSV-infected plants for 3 days. Eac
plant was inoculated by 5 viruliferoi
Infection in natural population, insects for 5 days. The plants wei
Four vegetative samples of 0. officinalis assayed using ELISA one month afti
Wall ex Watt collected from an area 25 inoculation.
km east and 20 km west of Zamboanga
City, Mindanao, were indexed by ELISA Inoculation into L. hexandr,
to determine tungro infection in the Different inoculation tests were done c
natural habitat of the wild rice species. L. hexandra, a natural host of /
Likewise, 20 vegetative samples of 0. malayanus and N. nigropictus (InoL
rufipogon Griff. taken from the inland and Ruay-aree, 1977; Inoue, 1986).
province of Bukidnon, Mindanao, were
also assayed. No rice plant was planted Different acquisition feedir
within few kilometers from the periods. Adults of N. nigropictus which
collection sites, were allowed 1, 2, and 4 da,
acquisition feeding on RTBV and RTS'
Weeds infected TN1 plants were used
inoculate 10-12 test plants
. . . B : ...d. I-I--- "I'r 4- 4 1 -t

- o r% r i- L ------- RA..--- __ U __lr

III~ Iviivvviiiy ~~~viru ui

'hilipp. Phytopathol. 1993, Vol. 29: 30-41

ifected plants. Tungro virus infection and RTSV sources, 25% of the insects

mined using EL

SA one month transmitted RTBV and RTSV, 28%

L. hexandra was also doubly transmitted RTSV alone. Eight percent
noculated following the methods used of insects fed on RTSV-infected IR54
n wild rice. Six plants were inoculated stubbles transmitted RTSV. No
n each treatment using N. malayanus transmission occurred when RTBV-
md N. nigropictus as vectors, infected IR26 stubbles were used as
virus sources.
Continuous exposure to virus
infection. Seven L. hexandra plants Field study. Infectivity of N.
vere inoculated by 3 N. virescens confined on IR64 and TN1
nalayanus/plant in Mylar cages for 5 stubbles was 51% and 63%,
lays. Plants were then transferred into respectively. Leafhopper mortality was
in insect cage with 2 TN1 plants higher on IR64 stubbles (76%) than on
infected with both RTBV and RTSV. TN1 stubbles (51%).
adultss of N. malayanus were
introduced into the cage and allowed to Leafhopper density in 1 week-
nultiply. Test plants inside the cage and 4 week-old TN1 stubbles was
vere disturbed 2-3 times a week to higher compared with IR64 stubbles.
lisperse the insects. L. hexandra plants Field leafhopper populations on TN1
vere assayed using ELISA 3 weeks stubbles have low levels of infectivity
ifter the initial inoculation in the Mylar while those collected from IR64
:age and 2, 3 and 6 weeks after the stubbles were not infective (Table 1).
introduction of the insects into the
:age. Infectivity of 10 adult insects was Volunteer Rice
ilso assayed 7 weeks after the insects
vere released into the cage. Higher population density of
leafhopper vectors was found in Sultan
Virus recovery from weeds. Kudarat compared with that in
sack inoculation tests were conducted Camarines Sur and Iloilo. Higher level of

-he insects were given 3 days
acquisition feeding on D. setigera and 1
lay inoculation feeding on TN1
;eedlings (2 insects/seedling).


lice Stubble

Greenhouse study. Of the N.
'irescens fed on IR22 stubbles infected
vith both RTBV and RTSV, 60%
transmitted RTBV and RTSV, 17%
transmitted RTBV alone and 14%
transmitted RTSV alone to TN1
*eedlings. Using IR36 stubbles as RTBV

loilo and Sultan Kudarat. No infection
vas obtained in samples from
mariness Sur (Table 2).

Vild Rice

Single inoculation of RTBV and
ITSV. Using N. virescens as vector, 2
>ut of 7 0. brachyantha, 3 out of 8 0.
'ichingeri, and 4 out of 12 0. minute
Ilants were infected with RTBV alone
Table 3). These infected plants, except
or some 0. brachyantha, showed slight
allowingg and stunting. Further
examinationn of the plant extracts by
EM revealed the presence of RTBV

'hilipp. Phytopathol. 1993, Vol. 29: 30-41

Fable 1. Infectivity of N. virescens and N
week-old IR64 and Taichung Nati

(NO.) (F
1-week ol

IR64 4 1

TN1 31 3

4-week ol

IR649 45

TN1 14 4

'Number of N. virescens and N. nigropictus collected

-One insect inoculated per one TN1 seedling in test

3B+S = infected with both RTBV and RTSV; B =
RTSV alone, indexed by ELISA.

Table 2. Tungro vector density and pt
volunteer rice plants in three I


Camarines Sur 5 4.4

loilo 12 2.5

Sultan Kudarat 16 19.0

1Number of leafhopper/10 sweeps of insect net.

2Batches of 10 seedlings were assayed separately b

gropictus collected Trom i weeK-ana 4
1 (TN1) stubbles.

)2 _- -........- -

0 0 0

0 7 0


0 0

1 3 0

10 sweeps of an insect net.

for one day.

fected with RTBV alone; and S = infected with

mntage RTBV and RTSV infection in
vinces in the Philippines, dry season,

TESTED2 ----

77 0 0 0

239 3.7 0.4 175

317 4.4 1.9 21.3


36 Philipp. Phytopathol. 1993, Vol. 29: 30-4

Table 3. Absorbance at 405 nm in ELISA and observation by IEM of wild rice an(
weed species inoculated with RTBV and RTSV using N. virescens a:

PLANT SPECIES PLANTS SCORE ------- -------------

Porteresia coarctata 8 0.00 (0) 0.00 (0) X X
Oryza brachyantha 5 -0.00 (0) 0.00 (0) X X
0. brachyantha 2 + 0.09-0.10 (0) 0.00 (0) +
O. eichingeri 5 0.00-0.02 (0) 0.00 (0)
0. eichingeri 3 + 0.16-0.17 (0) 0.00 (0) +
0. granulata 6 0.00-0.02 (0) 0.00 (0)
0. minute 8 0.00-0.06 (0) 0.00-0.01 (0)
0. minute 4 + 0.13-0.35 (0) 0.00-0.03 (0) +
0. sativa
cv. TN1 (CK) 10 + 0.42-0.69 (0) 0.48-0.79 (0) + +
Digitaria setigera 10 0.00-0.06 (0-.02) 0.02-0.15 (.03-.05) X X
D. setigera 4 -0.08-0.14 (0-.02) 0.18-0.44 (.03-.05)
Leersia hexandra 11 0.00-0.04 (0) 0.00 (0)
L. hexandra 3 0.14-0.53 (0.00) (0) 07-0.34 (0)
Panicum repens 10 0.01-0.11 (.01-.06) 0.00 (0) X X
Paspalum distichum 15 0.00-0.04 (0) 0.00-0.08 (.0-.02) X X
glabrescens 4 -0.00 (0) 0.00-0.01 (0) X X
Eriochloa procera 5 -0.00 (0) 0.00-0.04 (.01-.03) X X
Leptochloa chinensis 5 -0.00 (0) 0.00 (0) X X

1+, symptoms observed; no symptom observed.

2Range of absorbance of extracts from inoculated and uninoculated plants (in parenthesis).

+, virus particles observed; -, no virus particles observed and X, no observation.

Two-fold inoculation. In the first 0. granulata and L. hexandra after the
inoculation, RTSV-viruliferous N. first and second inoculations (Table 4)
malayanus infected 60% of O. Similar results were obtained where
brachyantha. All test plants were N. nigropictus was used in the firs-
infected with RTSV after the second inoculation (Table 5).
inoculation of both RTBV and RTSV by
N. virescens. No RTSV infection was Virus recovery from wild rice
recorded in 0. eichingeri and 0. minute RTSV was transmitted by N. virescens
after the first inoculation. However, from 0. brachyantha to 1 out of 54
33% of both species became infected TN1 seedlings tested. However,
with RTBV alone after the second successful recovery of RTSV by 2E

4). un Ii1 plants, /ou/o was infected second 1
with RTSV after the first inoculation virus soi

.lldl UbIIy Lin Sdme plants ad

After the second inoculation. No tungro virus fromI RTBV-infectecI
after the second inoculation. No tungro virus from RTBV-infectec

Philipp. Phytopathol. 1993, Vol. 29: 30-41

respectively. This indicate that the
plants were not infected with RTSV.

Infection in natural population.
One isolate of 0. rufipogon from
Bukidnon was found to be infected by
RTBV alone when assayed by ELISA.
No discernible tungro symptom was
observed in the infected isolate. The
assay of 24 tillers of the infected O.
rufipogon isolate revealed 92%
infection. No infection occurred in the
other isolates. This is the first report of
tungro infection in wild rice taken from
its natural habitat in the Philippines.


Inoculation to different species.
Among the 7 weed species tested,
some plants of D. setigera and L.
hexandra recorded high absorbances in
the ELISA tests. However, further IEM
examinations of these plants revealed
no virus particles (Table 3). All TN1
plants were infected by both RTBV and

No infection was observed in 42
E. glabrescens and 17 E. colona plants
grown from seeds and inoculated by
RTSV-viruliferous N. nigropictus and N.

Inoculation to L. hexandra

Different acquisition feeding
periods. No infection was obtained in
L. hexandra after inoculation by
N. nigropictus and N. malayanus
following acquisition feeding on RTBV
and RTSV-infected TN1 plants.

There was also no infection in
L. hexandra inoculated by RTSV-
viruliferous N. nigropictus, followed by
- -- --- A :L--. 0-rDW -4 1

Continuous exposure to virus
infection. L. hexandra plants inoculated
in Mylar cage and later exposed
continuously to RTBV and RTSV-
infected TN1 plants with N. malayanus
in a cage for 7 weeks did not become
infected by either of the tungro viruses.
Infectivity tests of the adult N.
malayanus 7 weeks after the initial
exposure to the virus sources proved
negative, indicating failure of the
insects to acquire the viruses from TN1

Virus recovery from weed. Back
inoculation test showed that none of
the 12 TN1 seedlings became infected
after having been inoculated for 1 day
by N. virescens (2 insects/seedling)
previously fed for 3 days on D.


Knowldge on how a pathogen
survives in the field is critical in the
formulation of a sound disease manage-
ment strategy. In these experiments,
the vectors acquired tungro viruses
from stubbles of some rice cultivars but
virus recovery and insect mortality
varied with cultivars. This shows that
infected stubbles can act as reservoir
for tungro viruses. A low level of
infectivity was observed in leafhoppers
collected from the stubbles, which may
be due to the mature condition of the
virus sources (i.e. stubbles). Certain
factors may reduce the carryover of
the disease in stubbles. These are:
a) the availability of water which affects
regrowth of the stubbles, b) destruction
of the inoculum sources and vector
habitat by plowing and puddling, c) the
retention of the virus in the stubbles
Arn\l~ c~n ,ni,+k 1Tr-FAnr

38 Philipp. Phytopathol. 1993, Vol. 29: 30-41

Table 4. Percentage of plants infected by tungro viruses after the first inoculation of
RTSV by N. malayanus and second inoculation two weeks later of both
RTBV and RTSV by N. virescens.


Porteresia coarctata 6 0 0 0 0

Oryza brachyantha 5 60 0 0 100

0. eichingeri 6 0 0 33 0

O. granulata 3 0 0 0 0

0. minute 6 0 0 33 0

Taichung Native 1 4 75 100 0 0

Leersia hexandra 6 0 0 0 0

1Inoculated with 3 N. malayanus/plant after 3 days acquisition feeding on RTSV source plants. ELISA was done 14
days after inoculation.

2Same plants were inoculated with 3 N. virescens/plant after 3 days acquisition feeding on RTBV and RTSV-source
plants. ELISA was done 28 days after inoculation.

Table 5. Percentage plants infected by the tungro viruses after the first inoculation
by RTSV-viruliferous N. nigropictus and second inoculation two weeks later
by RTBV and RTSV-viruliferous N. virescens.


Porteresia coarctata 6 0 0 0 0

Oryza brachyantha 4 75 0 0 100

0. eichingeri 6 0 0 33 0

0. granulata 4 0 0 0 0

0. minute 7 0 0 43 0

Taichung Native 1 4 25 100 0 0

Leersia hexandra 6 0 0 0 0

1Inoculated with 2 N. nigropictus/plant after 3 days acquisition feeding on
days after inoculation.

RTSV source plants. ELISA was done 14

2Same plants were inoculated with 3 N. virescens/plant after 3 days acquisition feeding on RTBV-and RTSV-source
plants. ELISA was done 28 days after inoculation.

Philipp. Phytopathol. 1993, Vol. 29: 30-41

fecundity and survival rates of vectors
in the stubble might be low.

The importance of volunteer rice
in the tungro disease cycle has been
given little attention in the past. Our
study shows that volunteer rice plants
were infected with tungro viruses and
they also harbored leafhopper vectors.
Infected volunteer rice may serve as a
source of tungro virus and as a tempo-
rary refuge and source of food for the
vectors in harvested fields. However,
their contribution to tungro epidemics,
has to be quantified.

Several workers have investiga-
ted the possibility of wild rice plants
and weeds as alternative hosts of
tungro viruses (Wathanakul, 1964;
Rivera et al., 1969; Inoue and Ruay-
Aree, 1977; Rao and Anjaneyulu,
1978; Tarafder and Mukhopadhyay,
1979; Anjaneyulu et al., 1982; 1988;
Parejarearn et al., 1990; Khan et al.,
1991). In this study, 0. brachyantha,
0. eichingeri, and 0. minute were
artificially infected with either RTBV or
RTSV. Leafhopper recovery tests of the
tungro viruses from infected wild rice
plants showed that they are poor sour-
ces of inoculum. Anjanyulu et al.
(1982) reported virus recovery from O.
brachyantha and 0. eichingeri only at
certain times after inoculation, while
tungro viruses were recovered from
other wild rice species at all test
periods after inoculation. This indicates
that not all wild rice can serve as ideal
virus source. The occurrence of RTBV
infection in 0. rufipogon growing in its
natural habitat shows that this species
can be infected by the tungro viruses in
nature. 0. rufipogon is a perennial plant
and once infected may serve as a
source of virus over a long period of

Weeds as alternative hosts of
tungro viruses and as sources of virus
inoculum were not convincingly estab-
lished in this study. N. nigropictus failed
to transmit the tungro viruses to
E. glabrescens and E. colona and
N. malayanus to L. hexandra. These are
natural hosts of both vectors (Inoue and
Ruay-Aree, 1977; Inoue, 1986). Conti-
nuous exposure of L. hexandra to
tungro-infected rice plants in a cage
with N. malayanus did not result to rice
tungro infection. In addition, infectivity
tests of insects confined in the cage
with both L. hexandra and tungro-infec-
ted plants proved negative. These indi-
cate that L. hexandra may not be a host
of the tungro viruses or that the viruses
were not transmitted due to the insects'
feeding preference. N. malayanus pre-
fers to feed on L. hexandra than on rice
(Kim et al., 1986). Back inoculation by
N. virescens to rice using D. setigera
plants as virus sources resulted in
negative transmission. Results of the
above experiments are not consistent
with the results of earlier investigations
which reported positive detection on
some weeds including D. setigera and
L. hexandra by ELISA (Anjaneyulu et
al., 1988; Parejarearn et al., 1990;
Khan et al., 1991). In this study, virus
particles were observed by IEM in
samples of wild rice and TN1 plants but
not from samples of D. setigera and L.
hexandra. However, the latter samples
have high absorbances in ELISA
suggesting non-specific reaction or
there is a very low concentration of the
tungro viruses in the samples. A more
sensitive diagnostic method should be
used to investigate virus infection in
weeds. Recent progress on nucleotide
sequence analysis of RTBV (Kano et al.,
1992) may help in the use of poly-
merase chain reaction (PCR) for detect-
ing small amounts of RTBV in rice

plants and insect vectors. PCR techno-
logy may resolve the conflicting results
on weeds.

In the present study, rice plants
(stubbles, volunteer rice), and to some
extent, wild rice species served as
hosts of tungro viruses in the field.
Their contribution to the spread of
tungro disease in asynchronously
planted rice may be very important.


K. MOODY. 1988. Host plants of rice
tungro (RTV)-associateed viruses. Int.
Rice Res. Newsl. 13(4):30-31.

S.K. SINGH. 1982. Experimental host
range of rice tungro virus and its
vectors. Plant Dis. 66: 54-56.

1985. Enzyme-linked immunosorbent
assay to diagnose rice tungro. J. Plant
Prot. Trop. 2(2): 125-129.

Recovery of rice tungro virus (RTV) from
rice stubbles. Int. Rice Res. Newsl.
14(3): 35-36.

HIBINO, H. 1983. Transmission of two rice
tungro-associated viruses and rice
waika virus from doubly or singly
infected source plants by leafhopper
vectors. Plant Dis. 67: 774-777.

HIBINO, H., N. SALEH, and M. ROECHAN. 1979.
Transmission of two kinds of rice
tungro-associateed viruses by insect
vecitrs. Phytopathology 69: 1266-

INOUE, H. 1986. Biosystematic study on the
genus Nephotettix occurring in Asia.
Bull. Kyushu Natl. Agric. xp. Stn. 24(2):

Phlipp. Phytopathol. 1993, Vol. 29: 30-41

INOUE, H. and S. RUAY-AREE. 1977. Bionomics
of green leafhopper and epidemics of
yellow orange leaf virus disease in
Thailand. Trop. Agric. Res. Seer. 10:

(IRRI). 1973. Annual Reeport for 1972.
P.O. Box 933, Manila, Philippines. 246

1992. Nucleotide sequence of capsid
protein gene of rice tungro bacilliform
virus. Arch. Virol. 124: 157-163.

R.D. DAQUIOAG. 1991. Rice and weed
hosts of rice tungro-associated viruses
and leafhopper vectors. Plant Dis. 75:

1986. Levels of resistance of rice
cultivars and the weed Leersia hexandra
L. to Nephotettix malayanus Ishihara et
Kawase and N. virescens (Distant). Crop
Prot. 5(6): 400-405.

LING, K.C. 1966. Nonpersistence of the tungro
virus of rice in its leafhopper vector,
Nephotettix impicticeps. Phytopathology
56: 1252-1256.

LING, K.C. 1972. Rice virus diseases.
International Rice Research Institute,
P.O. Box 933, Manila, Philippines. 142

MILNE, R.G. 1984. Electron microscopy for the
identification of plant viruses in vitro
preparations. Methods Virol. 7: 87-120.

Epidemiology and control of rice tungro
virus disease in West Bengal. Pages
142-153 in Rice hoppers, hopperborne
viruses and their Integrated
Management. S. Mukhopadhyay and
M.R. Ghosh, eds. Bidhan Chandra,
Krishi Viswavidyalaya, West Bengal,

Y. SAITO. 1984. Detection of rice
viruses in plants and individual insect
vectors by latex flocculation test. Plant
Dis. 68: 374-378.

1990. Hosts of rice tungro-associated
viruses (RTVs) in Thailand. Int. Rice
Res. Newsl. 15(6): 21-22.

RAO, G.M. and A. ANJANEYULU. 1978. Host
range of rice tungro virus. Plant Dis.
Rep. 62: 955-957.

and K.C. LING. 1972. New vectors of
rice tungro and yellow dwarf. Philipp.
Phytopathol. 8:10.

RIVERA, C.T. and K.C. LING. 1968. Transmission
of rice tungro virus by a new vector,
Nephotettix apicalis. Philipp.
Phytopathol. 4: 16.

Suscept range of rice tungro virus.
lhilipp. Phytopathol. 6: 16-17.

RIVERA, C.T., K.u. LING, S.H. OU, and V.M.
AGUIERO. 1969. Transmission of two
strains of rice tungro virus by Recllha
dorsalis. Philipp. Phytopathol. 5: 17.

RIVERA, C.T. -and S.H. OU. 1965. Leafhopper
transmission of "tungro" disease of rice.
Plant Dis. Rep. 49: 127-131.

Potential of weeds to spread rice tungro
in West Bengal, India. Int. Rice. Res.
Newsl. 4(1): 11-12.

H. KOGANEZAWA. 1992. Tungro
viruses in volunteer rice plants. Int. Rice
Res. Newsl. 17(4): 20.

WATHANAKUL, L. 1964. A study on the host
range of tungro and orange leaf viruses
of rice. M.S. Thesis, University of the
Philippines, College of Agriculture, Los
Banos, Laguna, Philippines. 35 pp.

(Meloidogyne incognita
max (L.) Merr.] AND /


Portion of the B.S. Thesis

Respectively, former n
Department of Plant Protection, V
Baybay, Leyte.

Keywords: Root-knot I
Amaranthus spinosus

Glyphosate and pendimel
ppm reduced gall and egg mass ii
the roots of soybeans and A. spi
potent than pendimethalin against
in A. spinosus. The herbage and
considerably decreased at herbici
ppm. Glyphosate at the same c
heavier productive soybean pods
days after application, very slight
and pendimethalin to soybean an


vveeus ana
nematodes are among
>f soybean that ca
important broadleaf v
vith soybeans ar
pinosus L., A. hybrid
-onyzoides L., Mimi

plant parasiic
the serious pests
iuse low yield.
/eeds associated
re Amaranthus
'us L., Ageratum

'ngulata L. (Holm et al., 19//). In the
ropics, the growth of A. spinosus can
;ause more than 50% yield loss in
oybean (Hinson and Hartwig, 1982).

Root-knot nematode
Meloidogyne incognita (Kofoid and
Vhite) Chitwood] is also a serious pest
if soybean [Glycine max (L.) Merr.].

aranthus spinosus L.


he senior author.

*r student and Professors,
'as State College of Agriculture,

iatode, herbicides, soybean,

n herbicides at 400 and 600
es, and nematode population in
us. Glyphosate appeared more
e nematode in soybean but not
It weights of A. spinosus were
concentrationss of 400 and 600
entrations produced more and
an the other treatments. At 7
no phytotoxicity of glyphosate
. spinosus was observed at all

Veeds can serve as primary or alternate
losts of this pest. Broadleaf weeds are
consideredd primary hosts of nematodes
Davide et al., 1974). Valdez (1968)
claimedd that A. spinosus is a good
alternate host of root-knot nematode.

The use of herbicides can help
armers obtain the optimum yield of
;rops. Glyphosate (N-phosphonomethyl
Ilycine) and pendimethalin [N-(11-
ienzenamine] are herbicides which are
effectivee in controlling noxious weeds
associated with soybean and other
eguminous crops (Thomson, 1983).
3lyphosate is a relatively non-selective
road spectrum post-emergence
erbicide which is applied to the


iliage. It is translocated throughout the 400, 600 ppm) of each gl
arial and underground plant parts, pendimethalin. The 2 x
endimethalin is a pre-emergence experiment arranged in

yphosate and
5 factorial

r best results. It is not translocated in was used in both experiments.
ants following root or shoot
sorptionn. Egg masses of M. incognita
were separated from the galled roots of
Multiple pest control is ideal and infested tomato plants and used as
'sirable but little work has been done inocula for infesting soybean and A.
i this subject. Such approach is spinosus. Twenty egg masses were
tremely useful to farmers for placed in clean vials with water and
introlling pests attacking their crops. poured into three holes bored around
eeds and plant parasitic nematodes the base of 20 day-old test plants.
which cause serious damage to Seven days after inoculation, the two
ybean may be controlled using such herbicides at the above mentioned
iproach. levels were incorporated into the soil.
The untreated plants served as the
Studies on the effects of control.
.rbicides on nematodes and weeds
sociated with soybean are very At 90-100 days after
lited. Information about the nature of inoculation (mature stage of test
!rbicide effects on nematode plants), soybean and A. spinosus were
ithogenicity has not been fully carefully removed from the pots and
iderstood. Furthermore, growth of separated from the soil to expose the
eeds and soybean and change in root- root system. A one gram root sample
lot nematode population as influenced was obtained from each test plant, cut
, glyphosate and pendimethalin are into pieces, wrapped with cotton gauze,
>t well documented. Results of this tied separately and stained by dipping
udy may provide effective weed and into boiling acid fuchsin lactophenol for
matode control, thus, an additional 3 minutes.
-_1 I--~-- -:-^-~-

This study is aimed to
determine the effects of glyphosate and
endimethalin on the growth and yield
f soybean and A. spinosus infested
with root-knot nematode and on the
oot-knot nematode population, root
all and egg mass production in these
wo plant species.


Two sets of experiments using
oybean (var. CES 434) and A.
pinosus as test plants were

I-J jJt|FU I tI V I I *-~I*Il IIU III I
/stem of the test species were
observed. The data gathered on
oybean and A. spinosus were herbage
id root weight, plant height at
arvest, number of productive and non-
roductive soybean pods per plant,
average number of soybean seeds per
od, yield of productive soybean pods
er plant and herbicide injury rating.


effect on Soybean

-4~f --- - ;-4; -/n nri'e

herbicides significantly influenced root pendimethalin were n

nematode population (Table 1). At low which obtained the highest valid
concentrations (50-100), glyphosate Glyphosate at similar concentrations i
and pendimethalin exerted similar parently decreased root weight. TI
effects compared with the untreated observation agrees with the results
control, i.e. moderate to severe galling, Griffin and Anderson (1978) that rc
high egg mass indices and nematode weight of nematode infested tome
population. At high dosages, however, plants decreased when trifluralin (a,a
parameter values considerably trifluoro-2,6-dinitro-N,N-dipropyl-p-
decreased. This implies that both toluidine) was applied at 0.56 kg/
herbicides at high concentrations are due to suppressed nematode develc
detrimental to nematode growth and its ment in the roots.
root gall and egg mass production. This
observation concur with the report of Regardless of concentrate
King et al., (1977) that herbicides EPTC used, glyphosate and pendimetha
(S-ethyl dipropylthiocarbamate) and ver- significantly reduced root weight
nolate (S-propyl dipropylthiocarbamate) soybean but not plant height a
applied at 4-20 mg/kg reduced gall den- herbage weight (Table 2). Glyphose
sity. Likewise, Osman and Viglierchio was more effective than pendimetha
(1981) revealed that dip treatment of in inhibiting gall production by t
tomato roots with oryzalin (3,5-dinitro- nematode and consequently in reduci
N4,N4-dipropylsulfanilamide) at concen- root weight of the plants.
tration of 10-800 ug/ml significantly
reduced nematode development as Yield components. The numk
determined by less galls and larvae and grain yield of productive po
present in the roots. The activity of showed significant differences amo
oryzalin is attributed to herbicide treatment combinations (Table :
incorporation into nematode DNA which Plants treated with 600 ppm glyphosi
arrests differentiation of cells and produced the highest number
interfere with e..-nrvnnonepqi< (Fvqnn nrnriitivp nnrl fnllinwAor h\ thn

Concentrations of bol
herbicides significantly reduced gall an
egg mass indices and nematoc
population in soybean roots (Table 1
However, glyphosate appeared mor
potent to the nematode tha

Some agronomic characteristic.
Plant height and herbage weigI
of soybean were not significantly
affected by M. incognita inoculation
and herbicide treatments. Howeve
all treatments generally cause
considerable reduction in root weigI
Tkhl ? 91 Rrnnt nrainhtc nf riln1

significantly more productive pods th;
the untreated control. The least numb
of productive pods was produced I
the nematode-infested plants treat(
with 100 ppm pendimethalin which
were not significantly different from tl
untreated plants. The number of no
productive pods and average number
seeds per pod did not significantly diff
among treatment combinations.

The highest yield of production
pods was obtained in plants appliR
with 400 ppm glyphosate (Table 3
Like the number of productive pods, tl
I tnm\ ne*f 1.Ir~-l- 'C ^- I %jJ nke rt nr J+ 'I ('tA

Philipp. Phytopathol. 1993, Vol. 29: 42-53

Table 1. Root gall and egg mass indices and nematode population in soybean roots
infested with M. incognita and treated with different concentrations of
glyphosate and pendimethalin.1





Control (0)







11n a column, means followed by a common letter are not significantly different at 5% level by DMRT.

2Rating scale used: 1 = no gall or egg mass (none);
than 100 galls or egg masses (severe).

the untreated control. An inverse
relationship between yield performance
of the treatments and gall production by
the nematodes was observed. Galling of
roots can cause disruption of xylem and
phloem vessels which can reduce trans-
location of food materials and water,
and finally result in low yield (Loveys
and Birds, 1973; Meon et al., 1978).
The preceding observation conforms
with the report of Krauss and Noel
(1982) that alachlor (2-chloro-2',6'-
diethyl-N-methoxymethyl acetanilide) at
0.125 g/ml suppressed nematode

2 = trace; 3 = slight; 4 = moderate; 5 = more

parasitism in
increasing yield.

soybean thereby

The high yield obtained from
plants treated with 400 or 600 ppm
glyphosate implies that these
treatments reduced or suppressed
nematode development. Conversely,
treatments with pendimethalin and
glyphosate at low concentrations were
less effective in inhibiting nematode
activity which in turn led to low yield.
Yield increase may also depend on the
nematicides used in controlling

4.6 a

4.4 a
4.0 a
2.6 b
2.2 bc
1.6 c

4.8 a
4.6 a
4.0 a
2.8 b
1.0 c

4.4 a

4.0 a
4.0 a
2.2 b
1.8 c
0.8 c

4.4 a
4.0 a
3.8 a
3.0 b
1.0 c

125.0 a

114.2 a
101.2 a
50.6 bc
32.4 cd
21.6 d


120.6 a
115.4 a
70.0 b
52.2 b
17.0 d


Philipp. Phytopathol. 1993, Vol. 29: 42-53

Table 2. Plant height, herbage and root weight of soybean
M. incognita and treated with different concentrations of

infested with
glyphosate and


Control (0) 50.1 9.9 24.4 a


50 51.4 11.0 16.3 b
100 47.6 12.2 15.2 b
200 48.6 12.5 11.5 bc
400 38.9 9.2 8.2 c
600 36.2 7.2 6.6 c

Ave. 45.5 10.5 11.6


50 45.8 9.2 24.3 a
100 47.8 7.8 23.2 a
200 45.7 10.4 15.7 b
400 42.2 7.6 6.7 c
600 40.0 8.8 5.7 c

Ave. 44.2 8.8 15.1

lIn a column, means followed by a common letter are not significantly different at 5% level DMRT.

nematodes and probably the nematode
species. Herbicides such as vernolate,
metribuzin [4-amino-6-tert-butyl-3-
(methylthio)-as-triazine-5(4H)-one], tri-
fluralin, or metribuzin plus trifluralin
gave effective control of Heterodera
glycines and produced higher yield of
soybeans (Krauss and Noel, 1982).

Concentration of glyphosate sig-
nificantly increased the number and
yield of productive pods (Table 3).
Pendimethalin slightly increased these
yield parameters but the values were
not significantly different from the un-
treated plants. The number of non-

productive pods and average number of
seeds per pod were similar for both

Herbicide injury rating. Very
slight to no injury was observed in all
treatments at 7 days after herbicide
application (Table 4). However, at 27
days after herbicide application, very
slight discoloration of leaves at 400 and
600 ppm glyphosate and 600 ppm
pendimethalin was noted. Lower
concentrations (50, 100 and 200 ppm)
of either herbicide gave statistically
similar effects compared with the
control. Robles and Fabro (1985)


able 3. Yield components of soybean infested with M. incognita and treated with
different concentrations of glyphosate and pendimethalin.1


Control (0) 0.3 40.4 e 13.4 c 2.4


50 0.3 61.8 bc 29.7 ab 2.4
100 0.3 -4 .de 14.9 c 2.4
200 0.3 5 c 22.3 b 2.4
400 0.4 68.4 b 34.6 a 2.3
600 0.2 89.4 a 32.8 a 2.4

Ave. 0.3 63.4 26.8 2.4


50 0.4 45.0 de 18.7 c 2.4
100 0.3 35.8 e 11.7 c 2.3
200 0.3 442 e 16.2 c 2.5
400 0.4 51.6cd 2.6 b 2.5
600 0.3 49.8 d 20.1 bc 2.4

Ave. 0.3 45.3 17.9 2.4

l._ 1.._ ._ ._ _-. .- -.-.-_1 L.. -- _ -_ -+ - -, -, + ...mf~ i tk riffar- ^- t at r 0/_ lI. [ M API T

reported that pendimethalin at 1.0
.g/ha and cloproxydim [(E,E)-2-1-(3-
;loro-2-propenyl) oxy-iminobutyl-5-2-
ehtylthio) propyl-3-hydroxy-2-
;yclohexen-1-one] at 0.25-0.75 kg/ha
ire not phytotoxic to soybean and
>eanut. Pendimethalin is generally not
oxic to plants because it is photo-
lecomposed within 7 days after herbi-
:ide application (Parochetti and Dee,
1978). Moreover, Helling (1976) noted
hat dinitroaniline compounds are degra-
led by dealkylation and reduction reac-
:ions leading to less or no phytotoxicity
:o plants. Carter and Camper (1975)
ilso observed that trifluralin, did not

dversely affect Pseudomonas species
lue to biological degradation of the her-
oicide. On the other hand, the result of
lesa-Garcia et al., (1984) about the
olerance of broad bean (Vicia faba L.)
o glyphosate is confirmed by the
observationn mentioned earlier. Less or
o1 phytotoxicity of glyphosate could be
explainedd by the degradation of such
herbicide into inactive metabolites and
ts high degree of soil binding (Sprankle
?t al., 1975). Furthermore, Anderson
1983) claimed that glyphosate is
strongly adsorbed by soil colloids and
ias little or no phytotoxicity following
;oil application.

Table 4. Herbicide injury rating at 7 da,
with M. incognita and treated
and pendimethalin.


Control (0)







1In a column, means followed by a common let
rating scale used: 1 = no injury; 9 = completely

Effect on A. spinosus L.

Gall and egg mass indices and
nematode population in roots
Glyphosate and pendimethalin at 51
ppm resulted in production of mor
galls and egg masses in the roots which
were statistically similar to that of th
untreated control (Table 5). High
levels of the herbicide significant
decreased the root gall and egg mas
indices. The greatest reduction of thes
parameters was noted at the highest
----- --- /'- ^ ---1 -X -:".--

and pendimethalin
could suppress th
n 1ll1e nrl nrnriirt

t high

ition of roc
nin mnQQac

ith different concentrations of glyphosatE


1.0 b

1.0 b
1.0 b
1.4 a
1.2 ab


1.0 b
1.0 b
1.0 b
1.0 b
1.4 a


are not significantly different at 5% level by DMRT

This observation conforms with the
results of Krauss and Noel (1982) tha
trifluralin and metribuzin at 100 uy/m
were effective in reducing nematode
fecundity. They concluded tha
reduction in number of eggs and larval
was due to interference of CO2 fixation
and inhibition of lateral root emergence,

High nematode density in thi
roots was observed at 50, 100 and 20(
ppm of both herbicides (Table 5). Plant:
treated with these concentration:
showed no significant difference fron
the control. However, concentration:
of 400 and 600 ppm of the tw<
herbicides substantially reduce
----+ A- -- I-+__ +k, -

Philipp. Phytopathol. 1993, Vol. 29: 42-53

Lower nematode recovery in the plant
roots at higher concentrations (400 and
600 ppm) may be due to mechanical
exclusion by the mature root system or
to greater synthesis of repellent or toxic
substances by the plant as reported by
Castillo et al., (1972) in peanut.

The results indicate that the
number of root galls and egg masses as
well as the nematode population in the
roots could be reduced by the
application of glyphosate and pendime-
thalin at concentrations of 400 and 600
ppm. These agree with the findings of
Osman and Viglierchio (1981) that dip
treatments of tomato roots with
oryzalin at concentrations of 200-800
ug/ml significantly reduced nematode
development as evidenced by less galls
and larvae in the roots.

Unlike in soybean, the effect of

A. spinosus was significantly heavier
than the other treatments (Table 6).
Reduction in root weight was noted as
the level of each herbicide was
increased. However, it appears that
glyphosate is more inhibitory to root
growth than pendimethalin at lower
concentrations but less inhibitory at
higher dosages.

The heavier root weights of
plants treated with low concentrations
(50, 100 and 200 ppm) of the
herbicides may be attributed to higher
nematode population and consequently
more galls produced on these plants
(Table 5). These galls apparently had
contributed to the weight of the roots.
The same explanation holds true for the
lower root weight of plants at higher
concentrations. This observation agrees
with the results obtained by Griffin and
. .... -

Philipp. Phytopathol. 1993, Vol, 29: 42-53

Table 5. Root gall and egg mass indices and nematode population in roots of A.
spinosus infested with M. incognita and treated with different
concentrations of glyphosate and pendimethalin.1





Control (0)







3.8 a

3.8 a
3.0 bc
3.2 c
2.4 cd
2.2 d

4.0 a
3.6 ab
3.0 bc
2.2 d
1.4 e

4.2 a

4.0 a
3.4 ab
3.0 b
1.4 cd
1.2 d

4.0 a
3.6 a
2.8 b
2.0 b
1.2 d

108.6 a

94.4 a
76.2 a
83.4 a
19.4 b
19.2 b


87.4 a
84.4 a
79.8 a
21.8 b
10.8 b


11n a column, means followed by a common letter are not significantly different at 5% level by DMRT.
2Rating scale used: 0 = no gall or egg mass (none); 5 = more than 100 galls or egg masses (severe).

Dee (1978) noted that pendimethalin is
also degraded and photodecomposed
rapidly on the first 7 days after

Overall results indicate that
glyphosate must be applied at high
dosages (400 and 600 ppm) in order to
control nematode growth and
development as well as minimize its
pathogenicity to soybean and
A. spinosus. Application of herbicide is
a potential tool in the formulation of
nematode management strategies.

Extensive studies should be
done to adequately evaluate the
mechanism of action of both herbicides
in reducing nematode pathogenicity and
to identify the compatible components
that could be integrated for pest
management in soybean. Herbicides
which can effectively control both plant
parasitic nematodes and weed species
should be identified. Furthermore, field
experiments must be conducted to
accurately assess the effects of the
herbicides on nematode pathogenicity
and plant growth.

Philipp. Phytopathol. 1993, Vol. 29: 42-53

Table 6. Plant height, herbage and root weight of A. spinosus infested with M.
incognita and treated with different concentrations of glyphosate and


Control (0)














78.5 a

72.8 ab
57.9 bc
48.7 c
48.0 c
43.7 c


84.2 a

64.1 bc
56.1 cd
49.3 cde
36.5 efg
31.9 efg


79.5 a

71.7 ab
57.6 bc
51.3 c
50.9 c


90.8 a

77.8 ab
44.7 def
28.0 fg
19.0 g


1In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Philipp. Phytopathol. 1993, Vol. 29: 42-53

Table 7. Herbicide injury rating on A. spinosus infested with M. incognita and
treated with different concentrations of glyphosate and pendimethalin.



Control (0)






Ave. 1.1

'In a column, means followed by a common letter are not significantly different at 5% level, DMRT; rating
scale used: 1 = no injury; 9 = completely destroyed.


ANDERSON, W.P. 1983. Principles of Weed
Science. 2nd ed. West Publishing Co.,
Minnesota, U.S.A. 295 pp.

CARTER, G.E. and N.D. CAMPER. 1975. Soil
enrichment studies with trifluralin. Weed
Sci. 23: 71074.

MORRISON. 1972. The nature of peanut
resistance to Meloidogyne hapla. Phil.
Phytopath. 7: 15-27.

1974. Host-index of plant parasitic
nematodes in the Philippines. College,
Laguna. 32 pp.

EVANS, I.M. and P.R. GROSS. 1978. 5-
Bromodeoyuridine does not affect
development of the sea urchin, Arbacia
punctulata. Exptl. Cell Res. 114: 85-93.

GRIFFIN, G.D. and J.L. ANDERSON. 1978.
Response of trifluralin on pathogenicity
of Meloidogyne hapla on tomato and
alfalfa. Pit. Dis. Reptr. 62: 32-33.

HELLING, C.S. 1976. Dinitroaniline herbicides in
the soil. J. Environ. Qual. 5: 1-15.

HINSON, K. and E.E. HARTWIG. 1982. Soybean
production in the tropics. FAO Pit.
Protection Paper. Rome, Italy. 113 pp.

J.P. HERBERGER. 1977. The World's
Worst Weeds. Univ. Press. Hawaii,
Honolulu. pp. 55- 101.

INGRAM. 1977. Effect of two
thiocarbamate herbicides on seventy of
disease caused by Meloidogyne
arenana. J. Nematol. 9: 274-275

1.0 b

1.0 b
1.0 b
1.0 b
1.0 b
1.2 ab

1.0 b
1.0 b
1.0 b
1.0 b
1.4 a

Philipp. Phytopathol. 1993, Vol. 29: 42-53

KRAUSS, RM. and G.R. NOEL. 1982. Effect of
preemergence herbicides on Heterodera
glycines population dynamics and yield
of soybean. J. Nematol. 14: 452

LOVEYS, B.R. and A.F. BIRDS. 1973. The
influence of nematodes on
photosynthesis in tomato plants.
Physiol. Pit. Path. 13: 525-529.

MALEFYT, T. and W.B. DUKE. 1984.
Pendimethalin phytotoxicity to powell
amaranth (Amaranthus powellii L.).
Weed Sci. 32: 520-529.

1978. Change in free proline following
infection of plants with either
Meloidogyne javanica and
Agrobacterium tumefaciens. Physiol.
Pit. Path. 12: 251-256.

TORRES. 1984. Phytotoxicity and yield
response of broad bean (Vicia faba L.)
to glyphosate. Weed Sci. 32: 445-450.

Herbicide effects on nematode diseases.
J. Nematol. 12: 544-545.

PAROCHETT, J.V. and G.W. DEE. 1978.
Photodecomposition of eleven
dinitroaniline herbicides. Weed Sci. 26:

ROBLES, R.P. and L.E. FABRO. 1985.
Postemergence control of Rottboellia
exaltata by cloproxydim in soybean
(Glycine max) and peanut (Arachis
hypogaea). Proc. 16th Pest Contr.
Counc. Phil. Annual Conf., BSU, La
Trinidad, Benguet. 16 pp.

1973. Toward molecular mechanisms of
developmental processes. Annual Rev.
Biochem. 42: 601-646.

1975. Rapid inactivation of glyphosate
in the soil. Weed Sci. 23: 224-228.

THOMSON, W.T. 1983. Agricultural chemicals -
Herbicides. Thomson Publications,
Fresno, CA., U.S.A. pp. 49-222.

VALDEZ, R.B. 1968. Survey, identification and
host parasite relationship of root-knot
nematode occurring in some parts of the
Philippines. Phil. Agric. 51: 802-824.



Respectively, former Un
Professor of the Department o
College of Agriculture, ViSCA, Be

Keywords: Trichoderma

Conidial suspensions o
seedcoated in mungbean to asso
Sclerotium rolfsii Sacc. In the
107/ml or greater of T. viride c
S.' rolfsii infection. Use of 10
significantly decreased incider
treatments and a relative prol
respectively; 109 conidia/ml prov
than benomyl application (34.4
T. viride with S. rolfsii and E
inoculation gave a relative pro
respectively. T. viride treatment
mungbean seedlings in terms of
T. viride did not affect the germi
seedcoating with T. viride conid
mungbean from infection by
significantly higher than the
fungicides benomyl (12.21%) an
grain yield was increased by (
seedcoating over the yield obtained


Sclerotium rolfsii Sacc. which
causes wilt disease is one of the most
common pathogens attacking
mungbean. This disease can reduce the
yield of mungbean by attacking the
plant near the soil line causing wilting

Philipp. Phytopathol. 1993, Vol. 29: 54-66



graduate Student and Associate
lant Protection, Visayas State
ay, Leyte 6521-A, Philippines

le; biological control; Sclerotium

rrichoderma viride Pers. were
their biocontrol efficacy against
experiments, seedcoating with
dia protected mungbean against
and 109 conidia/ml resulted in
of Sclerotium wilt among
:ion of 51.13% and 54.48%,
1 seed protection that was better
)). Simultaneous application of
ication 5 days after S. rolfsii
tion of 66.00% and 67.81%,
also enhanced the growth of
an height increments (45-68%).
on of coated seeds. In the field,
suspension of 109/ml protected
rolfsii by 37.69% which was
el of protection provided by
naneb (14.94%). Consequently,
Skg/ha with T. viride conidial
rom the untreated control.

(Opina, 1978). Single infection can
cause severe plant damage because this
pathogen extensively possesses
ectotrophic mycelial growth where a
single thallus can cover a considerable
surface area. Relatively low population
densities of the pathogen can produce
severe diseases because it spreads
through or over the soil (Baker and

S. rolfsii has been common in
mungbean grown after wetland rice
(Elazegui and Mew, 1983).

Chemical control is usually
suggested against S. rolfsii. For
instance, Cabunagan and Soledad
(1977) reported that certain fungicides
like captain (Orthocide), chloroneb
(Tersan SP), Bravo (chlorothalonil),
maneb (Manzate), and Thiophanate
methyl (Fungitox) inhibited growth of S.
rolfsii at 1500 and 2000 ppm
concentrations. Benomyl has also been
found effective against S. rolfsii of rice
(Ou, 1985). However, the continuous
use of pesticides can result in the
development of pesticide resistance,
destroy beneficial fungi or lead to
environmental pollution. Due to the
adverse effects of pesticide, it is
important to explore the use of
biological control in crop protection.

One of the most promising
groups of microorganisms for the
biological control of soilborne plant
pathogens is the Trichoderma species.
Trichoderma spp. are found to be highly
antagonistic to S. rolfsii and Rhizoctonia
solani Kuhn in vitro and in greenhouse
tests. For instance, T. harzianum
decreased incidence of R. solani
infection in cotton (Elad et al., 1980),
radish (Lifshits et al., 1985), pea
(Lifshits and Windham, 1986) and rice
(Rosales, 1985); T. harzianum reduced
incidence of stem rot in peanut
(Backman and Rodriguez-Kabana,
1975); T. aureoviride and T. glaucum
reduced damping-off due to S. rolfsii
infection in cowpea (de la Pefia et al.,
1986) and R. solani infection in corn
(Varela, 1988); and T. glaucum
significantly controlled foot rot caused
by S. rolfsii in wheat (Neypes et al.,
1988). Of the several species tested,
culture filtrates of T. viride and T.
harzianum inhibited from 16 to 84% the
conidial germination of vegetable
ana+hannnc nllrh n A ltmrnri; hrnccfa


solani, Fusarium oxysporum f. sp.
:opersici and Cercospora canescens
Icantara, 1987).

However, in most field
)assays of Trichoderma spp.,
)control of targeted soilborne fungal
thogens were achieved but required a
*mendous amount of inoculum source
the biocontrol agent. For instance,
rnandez (1988) applied T. aureoviride
th its rice bran culture medium at
10 kg/ha to obtain adequate control of
rolfsii in peanut. Results of the work
Elad et al., (1980) suggested 1,000
/ha of wheat bran cultures of
harzianum to control R. solani and
rolfsii in beans, 600 kg/ha to control
e same pathogen in cotton, and 750
/ha for tomato damping-off control.
jeed, application of such bulky
sterial can be a hindrance for farmers
adopt the biocontrol technology.
ius, the efficacy of T. viride by seed
?atment using pot and field plot
periments was evaluated for the
ntrol of Sclerotium wilt in mungbean.

This study was conducted to
termine the most effective conidial
ncentration of T. viride seed
?atment for the control of S. rolfsii; to
aluate the most appropriate time of
viride application against S. rolfsii
section; and to compare the degree of
ntrol to be provided by Trichoderma
%atment with that of standard
ngicidal treatment.


elation, Maintenance and Mass
oduction of Sclerotium rolfsii

Sclerotial bodies of S. rolfsii
ere collected from a mungbean field
Fested with the pathogen. Sclerotial
dies were surface-sterilized by
:ping in 0.1% sodium hypochlorite
amlrlh fnr 1R5 carnndc Thoa wara

rinsed in three changes of sterile wat
and blotted dry in a sterile absorbe
paper. The sclerotial bodies we
planted to a petri dish containir
solidified potato dextrose agar (PD,
and incubated in the laboratory. Whe
growth was observed, a portion of tl
mycelium was cut and asceptical
transferred to PDA slants where th<
were maintained.

Mass production of S. rolf,
used in the experiment was done in ri<
grain-rice hull (1:3 v/v) medium. Tw
thirds of each 0.5-liter glass jar w<
filled with rice grain-rice hull mediu
plus 150-ml water and was cover
with an aluminum foil for sterilization
the pressure cooker at 20 psi for 2
minutes. The medium in each jar wi
seeded with agar block of 2 week-o
cultures of S. rolfsii and the pathogf
was allowed to grow for two weel
prior to inoculation. This mediu
containing S. rolfsii served as tt
source of sclerotial bodies and mycel
for inoculation or soil infestation.

Bioassay of Different Isolates
Trichoderma Species Against S. rolfsii

Different Trichoderma specify
were used in this study, namely:
aureoviride Rifai, T. viride Pers.,
harzianum Rifai, Trichoderma sp. fro
corn and Trichoderma sp. from peanu
Except for T. harzianum, all isolate
were collected from different fields
Leyte. They were tested in tf
laboratory to determine the mo:
promising Trichoderma species i
isolates in controlling S. rolfsii.

Agar discs were obtained froi
2 week-old plated cultures of tl
different Trichoderma species an
S. rolfsii using a sterilized 5-mi
diameter cork borer. Agar discs <
Trichoderma cultures were pair

priviuubiy piaUeu rU/. raireu ag
discs were positioned 3 cm apart 4
plated PDA. The treatments we
replicated 3 times in a complete
randomized design. One week later, tl
degree of antagonism of Trichodern
against S. rolfsii was measured usil
the rating scale developed by Bell et a
(1982) as follows:

1 Trichoderma complete
overgrew the pathogen and covered tl
entire medium;

2 Trichoderma overgrew
least 2/3 of the medium surface;

3 Trichoderma and S. rolf,
each colonized approximately 1/2 of tl
medium surface (more than 1/3 ar
less than 2/3);

4 S. rolfsii colonized at lea
2/3 of the medium surface ar
appeared to withstand encroachment I
Trichoderma; and

5 S. rolfsii complete
overgrew Trichoderma and occupied ti
entire medium surface.

Since the preliminary te
showed that the Trichoderma isolatE
can cause lysis of mycelia of S. rolfs
the method of measuring lysis zor
was devised. Plates containing 20 r
potato dextrose agar were seeded wi
agar blocks of S. rolfsii. After 3 days ,
incubation under ambient roo
conditions, a 5-mm agar culture disc i
each of the Trichoderma test isolatE
was placed at the center. Lysis
mycelia of S. rolfsii was manifested t
outward gradual clearing from tl
center and 3 weeks after, the lysis zor
was measured. Degraded hyphae i
S. rolfsii was confirmed under ligl
microscope. Each test isolate wi
replicated 5 times in a complete

)ecies or isolate against S. rolfsii was fo
sed in this study.

T. viride. The treatments were as
linw "

io rice grain-rice null mealum
maintenance, Mass Production and only
reparation of Trichoderma Inoculum T1 S. rolfsii (S.r.) in rice grain-
rice hull medium
T. viride, the species which was T2 105 conidia/ml + S.r. in
*und effective against S. rolfsii in the rice grain-rice hull medium
eliminary bioassay test, was T3 106 conidia/ml + S.r. in
maintained on PDA slants by serial rice grain-rice hull medium
ansfer at one month interval to fresh T4 107 conidia/ml + S.r. in
)A slants. Two week-old plated rice grain-rice hull medium
iltures served as conidial source for T5 108 conidia/ml + S.r. in
oculation. rice grain-rice hull medium
T6 109 conidia/ml + S.r. in
Different conidial concentrations rice grain-rice hull medium
T. viride were determined using a T7 Benomyl (1.5 g a.i./l)
?macytometer. Ten ml of sterile water
as poured on each plate and the Ten seeds were sown per cup.
mnidia were scraped gently with a wire The treatments were arranged in a
op. Concentration of conidia on stock completely randomized design
ispension was first determined and replicated 10 times. This set-up was
ter diluted to obtain the desired repeated once and placed in the field
concentrations Inncnin with thp with rnilv tamnarnatir rnnninn frnm

surfactant for every 10 ml of the mm based on the data gat
-rslh1rn QiKnan^,n +k- \/;CZfA DAfACA %A1-+[k

-- --

In niRinTQT Warp WTATorTn ranillariv I no

ce wilting w;

PnrT F

:ing and
S -- --

termination of the Most Effective seed germination of 93.0% was
viride Conidial Concentration in determined to correct the data on pre-
introlling S. rolfsii emergence rotting and post-emergence
Two week-old rice grain-rice
II cultures of S. rolfsii were placed on Determination of the Time of
tray in the screenhouse and the Application of T. viride for Most
medium with S. rolfsii were broken into Effective Control of S. rolfsii Infection
ices. Fifty ml of the inoculum was
xed thoroughly with 350 ml sterile The most effective
il and the mixture was placed each in concentration, 109 conidia/ml, of
5-liter plastic cups ready for planting. T. viride found in the earlier part of the
study was used. Two week-old rice
Ten grams of mungbean seeds grain-rice hull cultures containing
ir. Mg50-10A) found to be the most mycelia and sclerotial bodies of S. rolfsii



sterile soil. -itty ml ot tne inoculum per
350 ml of sterile soil was placed in

were nanaweeaea one monrn ar
. .., .

planting, protect plants' from aphid (Apt,
craccivora) and pod borer (Marui
Ten grams of mungbean seeds testulalis) infestation. The plants we
(var Mg50-10A) were soaked in 10 ml not fertilized.
of 109 conidia/ml suspensions of T.
viride for 30 minutes. Ten seeds were Soil Infestation with S. rolfsii
sown per cup.
Plots were artificially infesti
The treatments were the with 450 ml of 2 wk-old rice grain-ri,
following: sterile water-treated seeds hull (1:3 v/v) cultures of S. rolfsii. TI
planted to S. rolfsii in rice grain-rice hull inoculum plus culture medium we
culture medium (untreated control); T. broadcasted evenly along furrows ai
viride-treated seeds planted with S. covered with thin soil before sowii
rolfsii + rice grain-rice hull culture the mungbean seeds. Plots with
medium at the same time; T. viride- artificial infestation with S. rolfsii we
treated seeds planted 5 days ahead of provided to check on the amount
S. rolfsii inoculation; and T. viride- natural inoculum present in the area.
treated seeds planted 5 days after S.
rolfsii inoculation. Seed Treatments

The treatments were replicated Seed treatments were done I
10 times in completely randomized soaking seeds for 30 minutes with: (
design. The set-up was also placed in 109 conidia of T. viride/ml; (2) 1.5
the field. The plants were watered a.i. of benomyl/liter; (3) 1.8 g a.i.
daily. The number of pre-emergence maneb/liter; and (4) sterile tap water
rotting and post-emergence wilting was untreated control. Maneb was include
gathered 25 days after planting. because it was proven effective again
S. rolfsii (Cabunagan and Soleda
1977) and is commonly available in tl
FIELD EXPERIMENT market. Conidia from 2 wk-old pota
dextrose agar cultures of T. viride w
Field plot experiment was used as seed treatment materials. Fi
conducted to further evaluate the drops of Tween 80 were added per 1(
efficacy of T. viride to control S. rolfsii ml of T. viride conidial suspension.
using the most effective concentration ratio of 2:1 (g seeds: ml of T. viri,
of 109 conidia/ml. conidial suspension) was used sin
this was enough to fully moisten t
Field Plot Preparation and Culture of seeds. Each treatment was replicated
Test Plants times and arranged in a randomiz
complete block design (RCBD).
Plots measuring 1.5 x 2.5 m

corrected based on the mean 86% seed 108 and 109 conidia per ml gave the
armination on plots without the lowest total disease incidence of 43.9%
sthogen. Harvested grains were sun- and 40.97%, respectively. Seedcoating
tried for 3 consecutive days and grain with Trichoderma at 107 conidia/ml
'eights obtained served as the basis (63.01%) was comparable with
ir computing yield per hectare. benomyl treated seeds (59.03%).
andomly selected 100 seed samples Higher proportion of pre-emergence
om the first and second primings were rotting than of post-emergence wilting
/eighed for each treatment. Data were was observed among the treatments.
ialyzed using Randomized Complete Generally, T. viride coated seeds
lock Design with three replications, provided higher protection from pre-
reatment means were compared emergence rotting than from post-
illowing the Duncan's Multiple Range emergence wilting (Table 3). The
est (DMRT). degree of protection provided by
T. viride increased with increasing
conidial concentrations of seedcoating
RESULTS AND DISCUSSION suspensions (108 and 109/ml), giving
the highest protection (30.25% and
Based on the degree of 37.18%) relative to untreated control.
Itagonism using the Bell et al., (1982)
der all of thp ininate. were fnirnd RpdrInatinn with 1n9

)ility to lyse S. ro/fs
-ichoderma sp. from F
viriidp wAer fnlind mn

ii mycelia, protection provided by benomyl seed
eanut and treatment.
st effective

respective antagonists and/or protection provided by T. viride,
lycoparasites can be a better indicator conidial suspensions of 10s and 109/ml
f effectiveness for the biocontrol of were most effective with control
ingal pathogens. T. viride was used efficacy of 51.13% and 54.48%,
because it was already speciated respectively. Benomyl s#d treatment
compared to that of Trichoderma taken had control efficacy of 34.41% vwich
*om peanut. was comparable only with 107
conidia/ml (29.99%). Conidial
concentrations of 105/ml (1.43%) and
POT EXPERIMENTS 106/ml (0.24%) provided little
protection that was not significantly
Since S. rolfsii causes pr, different from untreated control. Dela
mergence rotting and post-emergence Pefia et al., (1986) obtained similar
tilting in mungbean, these two results using T. glaucum. When they
arameters were used in comparing treated the mungbean seeds with 105
S---r A _n +k- +rmI n+-+-I r --nk-A--- IrnI 1 r A nk +t, -Inn

Philipp. Phytopathol. 1993, Vol. 29: 54-66

Table 1. Degree of antagonism exhibited by the different Trichoderma isolates
against S. rolfsii in vitro.1


T. harzianum 3.00 a 2.18 c
T. aureoviride 2.33 ab 3.07 c
Trichoderma (corn) 2.33 ab 7.12 b
Trichoderma (peanut) 2.00 b 17.16 a
T. viride 2.00 b 18.71 a

CV (%) 15.47 10.18

1Means with the same letter are not significantly different at 5% level by DMRT.
2Rating based on the method described by Bell et al. (1982) with 1 as most antagonistic and 5 as least
Average of 5 replicates taken after 3 weeks of incubation.

Table 2. Mean percentage of rotten mungbean seeds and diseased plants due to
S. rolfsii 25 days after planting as influenced by seed treatment with
different Trichoderma viride conidial concentrations.1





Rice grains-rice hull
S. rolfsii alone
105 conidia/ml + S.
106 conidia/ml + S.
107 conidia/ml + S.
108 conidia/ml + S.
109 conidia/ml + S.

0.00 d
80.00 a

56.67 ab

77.74 a

30.00 c

26.02 c

27.96 c
49.03 b

0.00 e

0.00 c
10.00 b

32.04 a

12.04 b

33.01 a

17.96 b

13.01 b
10.00 b

90.00 a

88.71 a

89.78 a

63.011 b

43.98 cd

40.97 d
59.03 bc

CV (%) 25.03 30.84 12.12

1Values averaged across two trials were corrected based on 93% seed germination; data for ANOVA and DMRT were
square-root transformed; in a column, means with the same letter are not significantly different at 5% level by DMRT.

liner simultaneously witn, b days
before or 5 days after S. rolfsii
ioculation had 13.98%, 14.95% and
2.26% incidence, respectively, which
tere significantly lower than the
1.08% incidence in the untreated
control (Table 4). Based on post-
mergence wilting, all treatments did
ot vary significantly from the untreated
control except for T. viride seed
eatment applied 5 days before
. rolfsii inoculation. Since the S. rolfsii
oculation was done after planting, the
athogen inoculum placed on the
Jrface resulted to more propagules
batting in contact directly with the
ssue of the seedlings above ground. T.
ride propagules around the seedcoat
would not protect seedlings from being
fected above the soil line, hence, the
*eater post-emergence wilt incidence
ian in the other treatments. Control
'ficiency of T. viride applied either
multaneously with or 5 days after S.
Ifsii inoculation was 66.00% and
7.81%, respectively (Table 5). Forty
iur and 46 percent of the overall
ficacy was due to protection from
*e-emergence rotting due to T. viride
application simultaneously with and 5
ays after S. rolfsii inoculation. Growth
: mungbean seedlings expressed as
ean height was enhanced by T. viride
gardless of time of application (Table
i. Seeding height was increased by
3% and 61% when T. viride was
>plied simultaneously with and 5 days
ter S. rolfsii inoculation, respectively.
iis is consistent with the findings of
aker et a., (1984). When they added
harzianum mixed with propagative
edium at a rate higher than 105 CFU/g
>il, there was a significant increase in
eight, height, and flower production
chrysanthemum and petunia
>mpared with non-treated control.
milarly, Keifeld and Chet (as cited by
let, 1987) found an earlier
*rmination and increase in plant


dish and bean seedlings upon adding
.harzianum. The mechanism behind
lis phenomenon is not well understood
ut Chet, (1987) speculated that
richoderma may affect the plant by
xcreting a regulating hormone which
nay in turn increase the growth rate or
ie efficiency of the nutrient uptake.

Mungbean seeds coated with
ie different conidial concentrations of
. viride without S. rolfsii showed
higher percentage of healthy seedlings
Table 7). This was comparable with
ie untreated control (rice grain-rice hull
medium), an indication that T. viride did
ot adversely affect seed germination
id seedling growth. However,
treated seeds planted in S. rolfsii-
fested soil resulted in significantly less
umber of healthy seedlings compared
'ith T. viride-treated seeds.


Microplots used to test field
'ficacy of T. viride have an average
background disease incidence of 0.54%
)mpared with 80.73% disease
cidence in plots artificially infested
ith S. rolfsii. All seed treatments
gnificantly reduced disease incidence
je to infection by S. rolfsii compared
ith the untreated control (Table 8).
ie total disease incidence in plots
)wn to seeds coated with T. viride
)nidial suspension was 50.30% which
as lower than those treated with
enomyl (70.87%) and maneb
;8.67%). Of the total damping-off
cidence, pre-emergence seed rotting
:counted for 45.00-67.67%
otection confirming the result
)tained in the earlier pot experiment.

Seeds coated with T. viride
nidia gave 37.69% protection which
as significantly higher compared with

laDle .. nelauve proULCULIUn piUVlueu
against infection by S. rolfsii.1


S. rolfsii alone 0.00 d

105 conidia/ml 0.91 d

106 conida/ml 0.21 d

107 conidia/ml 14.28 c

108 conidia/ml 30.25 at

109 conidia/ml 37.18 a

Benomyl 28.58 b

CV (%) 25.03

1Values were corrected based on 93% seed germination;
a column, means with the same letter are not significantly

2Total control efficacy = [(%Total Diseased plants in Sr
Total Diseased plants in Sr alone] x (100).

Table 4. The effect of time of applii
infection on mungbean taken :


S. rolfsii alone 51.08 a

Simultaneous T.v.
and S.r. 13.98 b

T.v. 5 days before
S.r. 14.95 b

T.v. 5 days after
S.r. 12.26 b

C.V. (%) 29.54

1Values were corrected based on 93% seed germination;
a column, means with the same letter are not significantly
2T.v. stands for Trichoderma viride and S.r. for Sclerotiur

rnlmpp. rnylopamol. i.uj, vol. Lz: q.-oo

seed treatment of Trichoderma viride


0.00 c 0.00 d

0.52 c 1.43 d

0.03 c 0.24 d

15.71 a 29.99 c

20.88 a 51.13 ab

17.30 a 54.48 a

5.83 b 34.41 bc

30.84 12.12

a for ANOVA and DMRT were square-root transformed, in
ferent at 5% level by DMRT.

e % Total Diseased plants in a Treatment) divided by %

ion of Trichoderma viride on S. rolfsii
days after planting.1


7.74 b 58.82 a

6.02 b 20.00 b

35.05 a 50.00 a

6.67 b 18.93 b

31.46 18.80

a for ANOVA and DMRT were square-root transformed; in
ferent at 5% level by DMRT.


Philipp. Phytopathol. 1993, Vol. 29: 54-66

days a

Table 6. Mean height of mungbean
Trichoderma viride and Scleroi


S. rolfsii alone

Simultaneous T.v.
and S.r.

T.v. 5 days ahead
of S.r.

T.v. 5 days after

CV (%)

1Values were corrected based on 93% seed germination
Means with the same letter are not significantly different

2T.v. stands for Trichoderma viride and S.r. for Sclerotiun

ants as affected by varying time of
7 rolfsii inoculations taken 2 weeks after


8.62 c

14.47 a 68.00

12.49 b 45.00

13.92 a 61.00


ta for ANOVA and DMRT were square-root transformed.
Y. level by DMRT.


Table 7. Mean percentage of healthy
Trichoderma viride at differ
with Sclerotium rolfsii taken "

Philipp. Phytopathol. 1993, Vol. 29: 54-66

ungbean plants after seedcoating with
conidial concentrations and inoculating
seks after planting.1

Table 9. Protection provided by seed t


T. viride + Sr 33.7,
Benomyl + Sr 11.6E
Maneb + Sr 14.3E
Control (Sr alone) 0.0(

In a column, means with the same letter are not

Table 10. Yield of mungbean as affec
conidia/ml) and with fungicid,


T. viride + Sr 1560.33 b
Benomyl + Sr 1257.67 c
Maneb + Sr 1367.33 c
Control (Sr alone) 925.33 d
CV (%) 17.88

'In a column, means with the same letter are not

Although seedcoating with T. viridE
conidial suspension was found mosi
effective, the biocontrol efficacy
obtained in the field test was indeec
lower than the one obtained in the pol
experiment using heat-disintested sandy
loam soil. This suggests that biotic-
related factors or mechanisms which
could suppress the biocontrol activities
of the introduced T. viride in the fielc
probably exist. Understanding such

iment against iniec(ion Dy o. rous5l.

Yo) WILTING (%) (%)

3.97 a 37.69 a
0.55 b 12.21 b
0.58 b 14.94 b
0.00 b 0.00 c

ificantly different at 5% level of DMRT.

by seed treatment with T. viride (10E
i control wilt caused by S. rolfsii (Sr).1.

Sr ALONE ----------
1st Priming 2nd Primir

+635.00 5.27 a 5.03 a
+332.34 5.27 a 4.93 a
+442.00 5.03 a 4.83 a
-- 5.27 a 4.73 a
4.69 5.47

ificantly different at 5% level by DMRT.

could help in finding ways to enhancE
the efficacy of the biocontrol agent.

Mungbean grain yields wer(
increased by all seed treatments
compared with the untreated control
However, T. viride conidial seedcoatinc
gave the highest yield increase at 63E
kg/ha (Table 10). This was higher thar
the yield increase obtained frorr
benomyl seed treatment (332.34 kg/ha
-1-y m 1nh A I'3 3fn L-/kh\ CrZ-

liliDD. PhvtoDathol. 1993. Vol. 29: 54-66

Trichoderma harzianum, a biocontrol
ngicides did not affect the 100-seed agent effective against Sclerotium rolfsii
eight of mungbean yield. and Rhizoctonia solani. Phytopathology
70: 119-121.
The results clearly show that ELAZEGUI, F.A. and T.W. MEW. 1983. Survival
eating seeds with conidia of T. viride of Rhizoctonia solani, Sclerotium rolfsii
a potential non-chemical means of and Fusarium sp. in dryland and wetland
fields. (Abstract) Philipp. Phytopathol.
fective plant disease control. It does 19: 1:
it only control plant pathogens such
S. rolfsii but also enhances the FERNANDEZ, S.J. 1988. Efficacy of Trichoderma
aureoviride as biocontrol agent for
owth of mungbean plants -- an Sclerotium rolfsii Sacc. causing stem rot
Iditional advantage over some (Arachis hypogea L.). M.S. Thesis,
!sticides which frequently cause Visayas State College of Agriculture,
. Baybay, Leyte. 72 p.
lytotoxicity and mortality to nontarget
*neficial organisms. LIFSHITS, R., S. LIFSHITS and R. BAKER. 1985.
Decrease incidence of Rhizoctonia solani
pre-emergence damping-off by use of
LITERATURE CITED integrated chemical and biological
controls. Phytopathology 69: 431-434.
.CANTARA, T.P. 1987. Antagonistic activities
of Trichoderma species against LIFSHITS, R. and T. WINDHAM. 1986.
vegetable fungal pathogens in vitro. Mechanisms of biocontrol pre-
Undergraduate Thesis, UP Los Bahos, emergence damping-off of pea by seed
College, Laguna. 45p. treatment with Trichoderma spp.
Phytopathology 76: 7.
,CKMAN, P.A. and RODRIGUEZ-KABANA. Phytopathology 76: 7.
1975. A system for the growth and NEYPES, M.V., LAPIS, D.B. and TELAN, I.F.
delivery of biological control agents to 1988. Trichoderma glaucum Abbott for
the soil. Phytopathology 65: 819-821. biological control of foot rot of wheat
caused by Sclerotium rolfsii Sacc.
KER, K. and L. COOK. 1976. Biological control Philipp. Agric. 71: 157-163.
of plant pathogens. W.H. Freeman and
Co., San Francisco, USA. pp. 135-169. OPINA, O.S. 1978. Consequences of intensive
and sequential cropping on Sclerotium
,KER, R., Y. ELAD and I. CHET. 1984. The rolfsii and other pathogens associated
controlled experiment in the scientific with legumes and sorghum. Ph.D.
method with special emphasis on Thesis, UPCA, College, Laguna. 149 p.
biological control. Phytopathology 74:
1019-1021. OU, S.H. 1985. Rice Diseases. Cambian News

LL, D.K., H.D. WELLS and C.R. MARKMAN. Ltd. Great Britain. Second Ed. 380 p.

1982. In vitro antagonisms of PE
Trichoderma species against six fungal
plant pathogens. Phytopathology 72:

,BUNAGAN, R.C. and B.S. SOLEDAD. 1977.
Growth and sclerotial formation of
Sclerotium rolfsii Sacc. as influenced by RC
different fungicides at varying
concentrations. (Abstract) Philipp.
Phytopathol. 13: 12-13.

IET, I. 1987. Trichoderma application, mode of
action and potential as a biocontrol
agent of soilborne plant pathogenic Vp
fungi: In Innovative Approaches to Plant
Disease Control. I. Chet, Ed. Wiley
Series in Ecological and Applied
Microbiology. John Wiley and Sons, Inc.
p. 137-160.

MEW. 1986. Trichoderma aureoviride
and T. glaucum as biocontrol agents
against damping-off of crops planted
after rice. (Abstract) Philipp.
Phytopathol. 27: 4.

)SALES, A.M. 1985. Rice straw decomposition
by Trichoderma species and its effect
on inoculum of sheath blight pathogen,
Rhizoctonia solani. Kuhu. M.S. Thesis.
University of the Philippines at Los
Banos, College, Laguna. 112p.

,RELA, R.N. 1988. Evaluation of Trichoderma
aureoviride for the control of sheath
blight of corn caused by Rhizoctonia
solani. B.S. Thesis, Visayas State
College of Agriculture, Baybay, Leyte.
30 p.

Philipp. Phytopathol. 1993, Vol. 29: 67-71



This study was support
Protection Center, College of
Philippines at Los Bafos (UPLB), C

Assistant Professor, N;
University of the Philippines at Lo!

Keywords: Crown gall, Ag

A disease resembling bac
stems of roses in one of the gard
cells were present in gall tissues
using potato dextrose agar (PDA
colonies developed within 48-7
Gram-negative, non-spore former
induced the development of tyl
inoculated onto wounded tomato
first report of the occurrence
Agrobacterium tumefaciens in the


Crown gall is a plant tumor
usually found at the base or crown of
many dicotyledonous plants but can
also develop on stems, branches or
twigs of such plants. The bacterial
etiology of crown gall was discovered
by Smith and Townsend in 1907. The
bacterium, Agrobacterium tumefaciens
(Smith and Townsend) Conn, is the
causal agent of the disease only in the
sense that it transmits very efficiently a
tumor-inducing plasmid (TiP) to
susceptible plant cells and it is this TiP
that is responsible for the



in part by the National Crop
agriculture University of the
age, Laguna, Philippines.

nal Crop Protection Center,
aios, College, Laguna 4031.

acterium tumefaciens, rosa sp.

al crown gall was observed on
in Los Bafios, Laguna. Bacterial
I were isolated into pure culture
White, circular, domed, mucoid
hours. Cells were rod-shaped,
and aerobic. Bacterial isolates
II crown gall symptoms when
ems (Cv. VC-11-1). This is the
rose crown gall caused by

infecting 643 plant species belonging to
331 genera distributed among 93
widely separated families but the crown
gall disease can be induced only in
freshly wounded tissues (Riker and
Keitt, 1926). Although the pathogen in
general has a wide host range,
individual strains have restricted host
ranges (Anderson and Moore, 1S79).
Some perennial Rosaceae, e.g. apple,
apricot, peach and rose, are among the
most susceptible to crown gall infection
(Haas et al., 1991).

Roses for commercial flower
production are susceptible to crown gall
Aicanqg anr4 dirant lncpae ram ctifffrarl

case in Israel where entire crops are
destroyed in the nursery if more than
1% of the plants have galls.

In 1988, a disease resembling
bacterial crown gall was first observed
on stems of roses planted in one of the
gardens in Los Banos, Laguna. When
the author visited the garden in the
middle of 1991, about 10% of the
plants had galls. Since bacterial crown
gall caused by A. tumefaciens is not
known to be present in the Philippines,
it was deemed necessary to determine
the cause of the disease in order to
formulate disease management strategy
and prevent the spread of the disease
to other economic crops in the country.


Collection and microscopic
examination of galls. Rose stems with
galls at different stages of development
that were still creamy white were
collected and brought to the laboratory.
Galls were washed thoroughly with tap
water and blotted dry. Thin sections of
gall tissues were mounted on slides
with distilled water and observed
microscopically for bacterial ooze. This
was further verified by Gram staining.

Isolation of bacterial pathogen.
Galls were washed with tap water,
blotted dry, surface disinfected in 10%
chlorox solution for 5 minutes and
rinsed twice in sterile water. The outer
gall tissues were removed using sterile
scalpel and the inner gall tissues were
diced and placed in about 2 ml sterile
water. These were then cut into small
pieces, allowed to stand for 30 minutes
to enable bacterial cells to move out
from the tissues and the resulting

Philipp. Phytopathol. 1993, Vol. 29: 67-71

individual colonies were streaked on
PDA slants.

Pathogenicity testing of
isolates. Two tomato seedlings (Cv.
VC-11-1) were planted in each pot
containing disinfested soil. Stems of 5
week-old tomato seedlings were
wounded using sterilized scalpel. Five
ml sterile water were added into each
tube containing 2 day-old bacterial
isolates and a loopful of the cell
suspension was deposited into each
wound. Control plants were wounded
and a drop of sterile water was
deposited into the wound but .were not
inoculated. Pots were enclosed with
polyethylene bags for 24 hours. Plants
were monitored for the development of
crown gall symptoms.

Reisolation of the pathogen.
Bacterial pathogen was reisolated from
gall formed on tomato stems following
the procedure described for bacterial
isolation from rose gall tissues.

Precautionary measures were
observed in the handling and disposal
of materials used in the experiment.
Test plants and gall tissues were
burned after each use. Cultures of the
pathogen, soil and other materials used
in the experiment were sterilized in a
pressure cooker before disposing them.


At the time of the survey in
1991, about 10% of the rose plants
had galls ranging from 1-4 cm in
diameter (Fig. 1). In some cases as
many as 5 galls were seen on one
stem. Majority of the galls developed on
or near the cut portion of the stem


Fig. 1. Crown gall tumors on rose Fig. 2. Colony growth characteristics
stems found in one garden in of bacteria' isolated from rose
Los Banos, Laguna. galls (after 3 days).

stems rub each other during inclement

Microscopic examination of gall
tissues. Microscopic examination of gall
tissues prior to isolation revealed the
presence of bacterial cells in the form of
bacterial ooze. Determining bacterial
presence in gall tissue is a requisite for
successful isolation of the pathogen
because some galls may no longer
contain bacterial cells. This is due to
the fact that oncogenic strains of the
bacterium contain large plasmids of
about 150-230 kilo bases (Zaenen et
al., 1974; Van Larebeke, et al., 1974),
part of which (T-DNA) is transferred to
the plant cell during oncogenic
transformation and maintained there
indefinitely (Chilton et al., 1977). Upon
integration of the T-DNA into the plant

the bacterial pathogen. The pathogen
was isolated successfully on PDA and
D-1 agar. White, circular, domed,
mucoid colonies developed within 48 to
72 hours on PDA (Fig. 2). The
bacterium did not grow on crystal violet
pectate medium (CVP). Colonies were
not yellow on YDC and did not produce
fluorescent pigment on King's medium
B. Bacterial cells were Gram-negative,
rod-shaped, non-spore-forming and
aerobic. These characteristics conform
with that of the genus Agrobacterium
(Moore et al., 1988).

Pathogenicity tests and
reisolation of the pathogen. Initial
symptoms of crown gall were already
visible on inoculated wounds of tomato
stems (Cv. VC-11-1) after 14 days.
Galls were already well developed 21

-----rr- -----r-------- I----


monins Irg. i), vvounaea DUT terestics
uninoculated tomato stems did not had be

similar o mne rose gall isolates



Fig. 3. Crown gall tumors formed on tor
months after inoculation with bai

S(Cv. VC-11-1) stems 21 days (a) and 3
ia isolated from rose galls (b).


.Nqw. ft-A.-M d.

Philipp. Phytopathol. 1993, Vol. 29: 67-71

These tests had satisfied Koch's DE CLEENE, M. and J.DE LEY. 1976. The host
range of crown gall. Bot. Rev. 42: 389-
postulates and proved that the crown 466.
gall of rose found in the Philippines is
causedd by A. tumefaciens. This is the FARKAS, E. and J.H. HAAS. 1985. Biological
control of crown gall in rose nursery
first report of the occurrence of rose stock. Phytoparasitica 13: 121-127.
crown gall in the country. The disease
was probably introduced into this HAAS, J.H., AIDA ZVEIBIL, D. ZUTRA, EDNA
country through imported rose cuttings 1991. The presence of crown gall of
or planting materials latently infected grape incited by Agrobacterium
with the crown gall bacterium. tumefaciens Biovar 3 in Israel.
Phytoparasitica 19: 311-318.
Whether this pathogen can HAMILTON, R.H. and M.Z. FALL. 1971. The loss
persist or survive in its new of tumor-initiating ability in
environment remains to be seen Agrobacterium tumefaciens by
incubation at high temperature.
because there are strains of A. Experientia 27: 229-230.
tumefaciens that could loss their TIP
and are rendered non-tumorigenic when MOORE, L.W., C.I. KADO and H. BOUZAR. 1988.
II. Gram-negative bacteria. A.
cultured at high temperature (Hamilton Agrobacterium. In: Laboratory Guide for
and Fall, 1971). Identification of Plant Pathogenic
Bacteria. 2nd Edition. N.W. Schaad
(ed.). APS Press, The American
CONTROL RECOMMENDATIONS Phytopathological Society, St. Paul,
Minnesota, pp. 16-36.
The possible establishment and RIKER, A.J. and G.W. KEITT. 1926. Studies on
spread of the crown gall bacterium to crown gall and wound overgrowth of
other plants and to other parts of the apple nursery stock. Phytopathology 16:
country can be minimized, if not totally 765-808.
prevented, by proper monitoring of SMITH, E.F. and C.O. TOWNSEND. 1907. A plant
planting materials and established tumor of bacterial origin. Science 25:
plants in the nursery or garden for 671-673.
crown gall symptoms. All infected VAN LAREBEKE N., G. ENGLER, M. HOLSTERS,
plants must be removed and burned. S. VAN DEN ELSACKER, I. ZAENEN,
Cutting or running tools must be dis- R.A. SCHILPEROORT and J. SCHELL.
Cutting or running tools must be dis- 1974. Large plasmid in Agrobacterium
infested in formaldehyde or 70% tumefaciens essential for crown gall-
alcohol solution after cutting each stem inducing activity. Nature 252: 169-170.
LITERATURE CITED Supercoiled circular DNA in crown gall-
inducing Agrobacterium strains. J. Mol.
ANDERSON, A.R. and L.W. MOORE. 1979. Host Biol. 86: 109-127.
specificity in the genus Agrobacterium.
Phytopathology 69: 320-323. ACKNOWLEDGEMENT
BRAUN, A.C. 1978. Plant tumors. Biochimica et
i nhsr Ac t, C1A, 1r- -1Q1 .. ..



U V jrV II, II iI L-r/ II I -l IIII Ii/I E--loll


University Researcher, Institute of
Plant Breeding, UPLB, College, Laguna.

Keywords: Pepper anthracnose, Colletotrichum capsici,
C. gloeosporioides, seed transmission, varietal screening

Anthracnose infecting fruits of hot and sweet peppers was
found in 13 out of 19 provinces surveyed in the Philippines. High
disease incidence was observed in Batangas and Cavite and very low
in provinces where pepper is grown during the dry season usually
after rice. C. capsici and C. gloeosporioides were found associated
with infected pepper fruits. Morphological and cultural
characteristics of both pathogens were described. C. capsici
occurred predominantly in infected pepper fruits compared with C.
gloeosporioides. Mixed infection usually occurred in some fruit
samples. Seedborne nature of anthracnose pathogens has been
demonstrated. The proportion of infected seeds was directly related
with the severity of fruit infection by C. capsici. Out of 71 pepper
entries screened for resistance to C. capsici and C. gloeosporioides
under laboratory conditions, only accessions A-148 and C 01172
gave the lowest disease incidence and severity by both pathogen 10
days after inoculation. Of.70 pepper accessions grown under natural;
field conditions only 3 accessions were found free of C. capsici


epper in the Philippines. Malabanan Isolation and Identification of
1926) and Subang (1956) identified Colletotrichum spp.
ie fungus as C. nigrum.. Ocfemia
1931) reported C. phomoides as the Diseased pepper fruits were
pathogen causing pepper anthracnose brought to the laboratory for isolation

. pnomon
species ii

n the us
uch contr
adopted b
economic r
ultivars is
control alt,
icidence i
late and
species E
id viruler


disease Sur

'ere surve
verity of

'i as the
in the

ease in
ily based
lot been
due to
?arch for
introl is
idy was
ine the
f pepper
s, 2) to
local and
rials for


ng areas
nnce and
? on fruit

e were i
3 incubate
isture cha
itions wer
in sterile
s plated c
-s, germin;
ad-up unc
Using a fi
re maintain
in an in
are tested
ing them ir
ipe fruits
:ivar. Repr
ausal orgi
3n detached
okra, snare
host rang
were ident
by Sutton
:onidial m<
:ics and pa


? fruits o
a were c
were grol
,75 and
'he seeds
icallv extr;

ice of
hrs in
is was
;ses of
water. A
ar and
e. Pure
i green
e also
ra and
he key
which is

er cv.
m the
ling to
t area
i fruit

,tv nf nonnor XA- riatiarmi

as aeiermmnea.
Incidence of Anthracnose
:reening for Resistance
Anthracnose infecting fruits of
Pathogenicity test of isolates. sweet and hot pepper was found in 13
I isolate of C. capsici and out of 19 provinces surveyed (Table 1).
gloeosporioides were inoculated into The disease incidence can be as high as
*tached fruits of susceptible pepper 80% and practically absent in some
. Matikas. Severity of infection provinces. High incidence was observed
as determined after 10 days of in Batangas, Cavite, La Union, Nueva
cubation. Virulent isolates were Viscaya and Laguna provinces, The
entified for subsequent varietal disease was not recorded in the
:reening tests. provinces of Pangasinan, Ilocos Norte,
Ilocos Sur, Benguet, Davao del Norte
Greenhouse screening. Local, and South Cotabato in spite of the large
produced and improved breeding lines pepper plantings. The main reason for
hot and sweet pepper were grown in the absence or low incidence of the
e screenhouse. Healthy ripe fruits disease is believed to be due to
ere harvested and brought to difference in the production systems.
e laboratory for inoculation. The test Farmers in Batangas, Cavite and Laguna
uits were washed in running water, usually grow pepper throughout the
ot-dried and 10 fruits were sprayed year especially during the rainy season.
sing a de Vilbiss atomizer No. 15, with In provinces where the disease was not
)nidial suspension of virulent isolate recorded, farmers grow pepper only
andardized at 1 x 106 spores/ml. during dry season particularly after rice
ie inoculated fruits were placed side crop is harvested, hence, the condition
/ side on a piece of welded wire is too dry for disease development.
ipport that was put inside a 5 x 17 x
4 cm plastic box containing 50 ml Identification of Colletotrichum spp.
:erile water. After the box lid was
aced tightly, the set up was incubated Based on morphological and
: room temperature for 10 days. The cultural characteristics of single spore
cidence and severity of infection were cultures taken from sporulating lesions
*corded 7-10 days after inoculation, of diseased pepper fruits, C. capsici and
C. gloeosporioides are the causal
Field screening. Ripe pepper pathogens of pepper anthracnose in the
uits from UPLB experimental station Philippines. The conidia of C. capsici

laboratory. A total OT IUU Truits were
andomly taken from each accession
ind the incidence and severity of
mnthracnose were determined. The
species of the causal fungus was
identified through microscopic
examination of the spores following the
cey developed by Sutton (1980).

acuie apex io.v vu.U x o.v -+.J u
while those of C. gloeosporioides were
cylindrical, ellipsoidal with obtuse
apices tapering slightly at the base,
sometimes constricted in the center,
8.0 17.0 x 2.7 4.5 u. On PDA, the
colonies of C. capsici were initially
white to buff pink or white to greenish

rr ----r-----~'

Philipp. Phytopathol. 1993, Vol. 29: 72-83

Table 1. Incidence of pepper anthracnose on fruits collected from 19
provinces in the Philippines and occurrence of Colletotrichum


Tranca, Bay Sweet 30 100 0
Tranca, Bay Hot 25 45.3 62.2
Lamot, Calauan Sweet 2 100 0
Looc, Calamba Hot 5 0 100
UPLB, College Sweet 10 0 100

Pinagulingan-bata, Hot &
Lipa City Sweet 80 81 25
Janopol, Tanauan Hot 5 0 100

Tuklong, Kawit Sweet 30 0 100
Buenavista, Gen.
Trias Hot 10 75 100

Dila-dila, Sta.
Rita Hot 10 40 0
Sta. Barbara,
Bacolor Hot 0 0 0
San Juan, Sta.
Rita Hot 0 0 0

San Jacinto,
San Manuel Hot 2 100 0

Bacag, Villasis Hot 0 0 0
Lipay, Villasis Hot 0 0 0

Cabalo, San Antonio Sweet 0 0 0
San Gabriel, San
Antonio Sweet 5 100 0
Seminoglan, San
Narciso Sweet 10 100 0
Beddeng, San Hot &
Narciso Sweet 0 0 0
Linsungan, San
Marcelino Sweet 0 0 0

Philipp. Phytopathol. 1993, Vol. 29: 72-83

Table 1. Continued...


La Union
Kaukalan, Taloy Sweet 25 40 100

Nueva Ecija
Campus, Talavera Hot 2 100 0
Burnay, Talavera Hot 0 0 0
Pambuan, Gapan Hot 0 0 0
Poblacion, San Jose Hot 0 0 0
Abar, San Jose Hot 25 100 0

Tumpik, Kapangan Sweet 0 0 0
Aso, Kapangan Sweet 0 0 0

Ilocos Sur
Cabaroan, Sta. Hot &
Catalina Sweet 0 0 0
Barbara, Magsingal Sweet 0 0 0

locos Norte
Baay, Batac Hot & Sweet 0 0 0
Garasgas, Batac Hot 0 0 0
DMMSU, Batac Hot 0 0 0

ISU, Echague Hot & Sweet 0 0 0

Aurora Hot 5 100 0
Santiago Hot 1 100 0
Roxas Hot 15 100 0

Nueva Vizcaya
NVSIT, Bayombong Hot & Sweet 0 0 0
Barat, Bayombong Hot 0 0 0
Busilak, Bayombong Hot 40 100 0
Bilanse, Dupax
del Norte Hot 15 100 0
Indiana, Bambang Hot 10 100 0
Salinas, Bambang Sweet 25 100 0

Camarines Norte
Vinzons Hot 1 0 100
Labo Hot 1 0 100
Daet Hot 0 0 0

Philipp. Phytopathol. 1993, Vol. 29: 72-83

Table 1. Continued...


Polangu Hot & Sweet 20
Oas Hot
Legazpi Hot
Daraga Hot

Camarines Sur
Iriga Hot
Pili Hot

Davao del Norte
Tamayong Hot

South Cotabato
San Jose, Gen. Santos Hot
San Isidro, Gen.
Santos Hot
Balauan Hot

1Cc, Colletotrichum capsici; Cg, Co

gray. Abundant setae were observed in
cultures of C. capsici while no setae
were noted on C. gloeosporioides. Pale
to salmon-orange spore masses were
frequently observed in C. gloeospo-
-ioides. These results conformed with
the findings of Quimio (1977) that
C. capsici is one of the causal organism
of pepper anthracnose in the
Philippines. Mah (1985) and Kadu et
al., (1978) likewise identified C. capsici
as the causal fungus of pepper
anthracnose in Malaysia and India, res-
pectively. Hadden and Black (1988)
similarly reported that
C. gloeosporioides and C. capsici were
the major incitants of pepper
anthracnose in Louisiana, USA.

The disease symptom caused
:y C. capsici is usually associated with

(%) CC CG

100 0
15 100 0
10 100 0
10 100 0

10 100 0
0 0 0

0 0 0

0 0 0

0 0 0
0 0 0

otrichum gloeosporioides

the presence of black specks which are
arranged in concentric rings on
the surface of the sunken lesions giving
a target-board appearance. C.
gloeosporioides produces salmon-
colored gelatinous mass on the surface
of the lesion.

Host range test showed that
C. gloeosporioides could infect
detached fruits of tomato, guava,
banana and snapbeans but not eggplant
while C. capsici infects fruits of tomato,
guava, eggplant, okra and snapbeans
but not banana.

Occurrence of the
Colletotrichum spp. Colletotrichum
capsici occurred predominantly in
infected pepper fruits compared with
C. gloeosporioides (Table 1). High

Table 2. Percent seedborne infection of
cv. Matikas using blotter meth<


0 (Healthy)






1Average of 4 replications; means having the sa
by DMRT.

frequency of occurrence of C. capsici
was observed in Laguna, Isabela, Nueva
Viscaya and Albay while
C. gloeosporioides predominated in
Batangas, Cavite and Laguna. In some
instances both organisms occurred in
the same pepper fruit.

Seedborne Test

Results showed that
anthracnose pathogens are seedborne.
Severity of fruit infection by C. capsici
is directly related with the proportion of
infected seeds (Table 2). Hot pepper
fruits with 10, 30, 50, 75 and 100%
disease severity resulted to 10.2, 59.0,
59.0, 80.5, and 100% seed infection,
respectively. The results conform with
the findings of Grover and Bansal
(1968). They reported that pepper
seeds obtained from diseased fruits
carry the nathoaen both in and on the

Philipp. Phytopathol. 1993, Vol. 29: 72-83

capsici in naturally infected hot pepper


0 0.0 e

0 10.2 d

6 59.0 c

0 59.0 c

1 80.5 b

2 100.0 a

letter are not significantly different at 5% level

contaminated seeds could be a very
efficient way of introducing the disease
in areas where it is not known to occur.

Screening for Resistance

Pathogenicity of isolates.
Eighteen C. capsici and 1
C. gloeosporioides single spore isolates
were tested for their ability to cause
infection on pepper fruits. Results
showed that 5 of the C. capsici isolates
gave severe infection on both green and
red pepper fruit while 3 of the
C. gloeosporioides isolates gave 100%,
and 80-90% disease severity on the
red and green fruits (Table 3).
The results showed that there are
variations in virulence between the
two species of Colletotrichum and
among isolates of same species. This
suggests the presence of races or
strains within the species. These results

Table 3. Disease severity of 18 isc
C. gloeosporioides on green a


C. capsici

PP 11-1 Zambales
11-2 Zambales
12-2 Lipa, Batangas
12-1 Lipa, Batangas
6-5 Sta. Rita, Pamp
6-4 Sta. Rita, Pamp;
12-5 Lipa, Batangas
7-1 Kawit, Cavite
6-1 Sta. Rita, Pamp.
7-3 Kawit, Cavite
6-3 Sta. Rita, Pamp.
6-2 Sta. Rita, Pamp;
12-4 Lipa, Batangas
7-2 Kawit, Cavite
5 Los Baios, Lagi
1 Bay, Laguna
4 Bay, Laguna
2 Bay, Laguna

C. gloeosporioides

PP 13-2 Lipa, Batangas
13-1 Lipa, Batangas
14-1 Gen. Trias, Cavi
14-3 Gen. Trias, Cavi
8-3 Labo, Camarine,
8-1 Labo, Camarine,
14-2 Gen. Trias, Cavi
3 Los Banos, Lagi
9-1 Vinzons, Cam. r
9-2 Vinzons, Cam. r
10 Guimaras, Iloilo

Average of 10 fruits per isolate.

red-ripe truits ot hot pepper cv. Matikas.


100 100
100 100
100 100
100 90
a 100
a 100 90
100 70
100 100
a 100 88.9
100 100
a 100 90
a 90 80
80 10
80 80
70 44
50 10
30 40
10 40

100 40
100 80
100 80
100 70
)rte 100 10
orte 80 90
80 40
80 30
:e 60 30
:e 60 50
40 40

Philipp. Phytopathol. 1993, Vol. 29: 72-8

Hp 87-311-5 B 55 10 100 100
15-2-17 56 45 100 100

C 01826 90 80 88 65

Table 4 Continued...


Hp 87-298-2 91
Red Santaka B 94
Hp 87-297-B 91
15-3-36 94
Iriga, Albay A 94
A-121 9
A-103 9:
Pp 67 9:
A-122 9:
A-160 9'
Iriga, Albay B 9!
Hp 87-284-3 91
A-128 9'
15-2-20 9
Sta. Catalina 9
A-119 9
C4 10'
15-2-21 10
13-2-11-1 10
Hp 87-291 10
15-3-25 10
A-153 10
15-2-26 10
13-2-11-4 10
Hp 87-281-9 10
15-3-20 10
Hp 87-311-5 10
Iberica, Labo 10
Hp 87-291-1 10

a Average of 10-20 fruits per entry; NT =


70 100 100
75 100 100
80 100 100
80.5 100 100
60.5 100 100
87 100 100
93 100 100
81 100 100
100 100 96
95 100 100
45 100 100
80 100 100
13 100 100
50 100 100
NT 100 100
93 100 100
90 100 100
74 100 100
100 100 100
93 100 100
58 100 100
83 100 100
80 100 100
85 100 100
95 100 100
80 100 100
100 100 100
100 100 100
80 100 100


Philipp. Phytopathol. 1993, Vol. 29: 72-83

Table 5. Field screening of 70 pepper accessions for resistance to C. capsici.1

(%) (%)

2182 0 2192 55
222 0 2201 57.1
40 0 Matikas 58
2195 10 Pp 52 58.7
2204 10 9 60
2187 11.8 2191 62.5
2106 12 36 63
2091 15.2 65 65
2104 15.2 55 66
2093 162092 66.7
2220 17.6 57 67
6 17 58 68
29 17.2 21 70
18 18 30 70
2105 19 12 71
4 20 33 72.2
14 25 59 73.3
2186 25.5 8 73.7
2213 36.4 39 74
39 33.3 34 77
13 41 38 78
20 43.2 11 78
15 44 10 79
7 44 35 80
5 50 49 80
24 50 42 82
2 50 47 83.2
2181 50 60 83.4
Sinagtala 51 28 84
27 52.2 53 84.1
56 53 48 90
2 53 50 96.1
1 53.2 17 100
44 53.4 16 100
3 55.1 52 100

'Average of 100 fruits per entry.

Philpp. Phytopathol. 1993, Vol. 29: 72-83

Laboratory screening. Seventy
one pepper entries consisting of
12 from AVRDC, 1 from Thailand,
2 from Malaysia and 56 from
Philippines were screened for resistance
to virulent isolates of capsici and
C. gloeosporioides in the laboratory
using the plastic box moist
chamber and spray spore suspension
technique. The results showed that only
accessions A-148 and 01172 gave the
lowest disease incidence and severity
for both pathogens (Table 4). All other
entries were found susceptible to
both pathogens. The results suggest
that there is very limited sources of
resistance against anthracnose of
pepper. Similarly, Kadu et al., (1978)
reported that there is wide variation in
resistance of pepper varieties to
C. capsici and they mentioned difficulty
in finding resistant variety.

Field evaluation. Three
accessions namely Acc. 2182, 222
and 40 were free of pepper anthracnose
(Table 5). Disease incidence
ranged from 10 to 100% on the other
entries. Microscopic examination of
field infected pepper fruits showed
constant association of C. capsici while
C. gloeosporioides was practically
absent. The results conform with
that of the laboratory evaluation
suggesting a narrow sources of
resistance against anthracnose.


GROVER, K. K. and BANSAL, R. D. 1968. Occur-
rence and overwintering of
Colletotrichum piperatun on Capsicun
frutescens in India. Indian
Phytopathology 23: 664-668.
HADDEN, J.F. and BLACK, L.L. 1988. Anthrac-
nose of pepper caused by
Colletotrichum spp. PhD Thesis.
Louisiana State University, Baton Rouge.
KADU, I. K., MORE, B.B. and UTIKAR. P.G. 1978.
Field reaction of chilli germplasm to
anthracnose. Indian Phytopath. 31:378-
MAH, S. Y. 1985. Anthracnose fruit rot
(Colletotrichum capsici) of chilli
(Capsicum annuum): causal pathogen,
symptom expression and infection
studies. Teknol. Sayur-sayuran Jil. 1:
MALABANAN, D. B. 1926. Anthracnose of
pepper. Philipp. Agr. 14: 491-501.
OCFEMIA, G. 0. 1931. Notes on some economic
plant diseases in the Philippine Islands
II. Philipp. Agr. 19: 581-589.
QUIMIO, T. H. 1977. Species of Colletotrichum in
the Philippines. Nova Hedwigia 28: 543-
SANTOS, L. G. and V. G. FUNTILA. 1966. The
control of anthracnose of sweet pepper.
Philipp. J. Plant Ind. 31:61-69
SUBANG, N. M. 1957. Ripe rot of pepper. UPCA
Monthly Bull. 22:8.
SUTTON, B. C. 1980. The Coelomycetes.
Commonwealth Mycological Institute
G. B. p. 523-537.
VERMA, M. L. 1973. Comparative studies ont
virulence of isolates of four species of
Colletotrichum parasitic on chillies.
Indian Phytopathology 26: 28-31.

Philipp. Phytopathol. 1993, Vol. 29: 84-100



Portion of the Ph.D Dissertation of the senior author
submitted to the Graduate School of the University of the
Philippines at Los Banos.

Respectively, University Researcher, Institute of Plant
Breeding and Professor, Department of Plant Pathology,
University of the Philippines at Los Baios, College, Laguna 4031.

Keywords: salago, stem rot, Botryodiplodia theobromae,
factors affecting growth and reproduction

Stem rot of salago was characterized by wilting and drying
of leaves followed by browning, drying of stem and defoliation.
Numerous stroma containing pycnidia were formed on the stem
surface as the disease progressed. Wound was essential for stem
rot infection and development. Mycelia were found more effective
inoculum than pycnidiospores.

Pycnidia and pycnidiospores in culture, inoculated, and
naturally infected salago stems were characterized. The isolated
fungus was identified as Botryodiplodia theobromae Pat. Mycelial
growth, pycnidia and pycnidiospore yields were affected by agar
media, light, pH and temperature. Yeast extract agar, continuous
light, UV light, 25C and slightly acidic to slightly alkaline conditions
enhanced sporulation of B. theobromae. The fungus germinated best
at 25 to 30C, at 36 to 98% relative humidity and produced the
longest germ tube at 20C and 98% RH.

This study reports the first
in the Philippines.


Salago (Wikstroemia lanceolata
L.) of the Family Thymelaceae is a slow
growing shrub that thrives abundantly
in the mountainsides and hillsides,
along roadbanks, under coconut
plantations and along rock crevices

occurrence of stem rot of salago

throughout the country. It grows on
any kind of soil and can withstand
adverse conditions such as drought,
typhoons and continuous rain. The
tough bark of the crop is the source of
high and superior quality fibers. These
fibers are excellent raw material for the
manufacture of high grade paper for

Philipp. Phytopathol. 1993, Vol. 29: 84-100

bank notes, paper money, stencil, linen,
air mail paper, security and onion skin
papers, art paper, fishing nets and lines,
clotheslines, sacks, bond paper for legal
documents, certificates, cords, strainer,
mosquito nets, bags, hats, wallets,
sachets, fans and ropes (Yao, 1985).
Wikstroemia ovata has medicinal values
(ERDB, 1985). The fresh barks are
sources of relief for broncial cattarh.

The Philippines is one of the
major exporters of salago fiber in Asian
countries. In 1981, about 7,714 bales
of salago fibers were exported to
Taiwan, Japan and Korea valued at US
$499,405. A 20% export decline the
following year was noted due to the
non-replanting of harvested area,
farmers' lack of knowledge on the
commercial value of the crop and the
difficulty of propagating salago. In
1987, however, the country again
exported 5,793 bales to Taiwan and
Japan valued at US $0.42 million
(Family School Hillyland Development
Project/UPLB, 1988).

Although there are 20 species
of salago, only four are common in the
Philippines. These are W. lanceolata
Merr., W. indica (L.) C. E. Mey,
W. meyeniana Wart. and W. ovata C. E.
Mey. Within each species, numerous
varieties are reportedly known to occur.

Previous observation showed
that salago was relatively free from
insect pests and diseases. But when the
crop has been propagated on a large
scale for commercial production and for
reforestration,, it has succumbed to
diseases caused by microorganisms
especially when the plants are grown in
nurseries. The most common and
destructive disease is stem rot. Its
occurrence was first observed in 1987
on salago seedlings grown in nurseries
_ A ... I x_ ALn L _

brought to the Institute of Plant
Breeding, UP at Los Bafos, almost all
plants succumbed to stem rot. This
disease threatens the commercial
growing and production of salago as an
export fiber crop, hence this study.

The study was conducted to: 1)
isolate and test the pathogencity of the
causal organism, 2) describe the
symptoms of stem 'pot in naturally and
artificially inoculated salago plants, 3)
identify the causal pathogen and 4)
determine the effect of environmental
factors on growth and sporulation of
the fungus in culture.


Isolation and Pathogenicity Test

The causal organism was
isolated from the diseased stem tissues
using the tissue planting technique.
Surface-sterilized stem sections were
planted in potato dextrose agar (PDA)
plates. The plates were incubated at
170 foot-candles at 30C until the
growth of the fungus became visible.
The culture was purified by subsequent
transfers of mycelia to PDA slants and
pure cultures were maintained for use
in subsequent studies.

Two methods of inoculation
involving wounding and non-wounding
of the stem were tried in the
pathogenicity test. For the wounding
method, the epidermis of the stem was
scraped with flamed and sterilized
scalpel while the stem were kept intact
for the non-wounding method. Mycelial
agar disc and pycnidiospores were used
as inocula. Mycelial agar discs (7 mm
diameter) taken from the periphery of
48 hr-old PDA plate culture were placed
on wounded and non-wounded portions

Philipp. Phytopathol. 1993, Vol. 29: 84-10

were observed daily
t of symptoms. Early
ptoms of the disease
Re-isolations of the

inoculated plants were done. diameter was measured 24 hr at

Philipp. Phytopathol. 1993, Vol. 29: 84-100

from the UV source. After exposure to
UV light, plates were exposed to 40
watt fluorescent tube at 170
footcandles and 30 C for 14 days.

Effect of temperature. The
mycelial agar disc was placed on
the plated YEA (pH 6.5) and the
seeded plates were incubated at
continuous darkness at 15, 20, 25, 30,
35, and 40 C.

Conidial Germination and Germ Tube

Effect of temperature. Pycnidia
from 14 day-old culture were
suspended in sterile distilled water
contained in test tube. The pycnidia
were gently squeezed with stirring rod
and then thoroughly shaken in a Vortex
mixer. The spore suspension was
pipetted in water agar plate. The plate
was rotated to evenly disperse the
spores and then incubated at 5, 10, 15,
20, 25, 30, 35 and 40 C. The number
of germinated and non-germinated
spores was taken after 4 hrs of
incubation. Length of germ tube of 25
spores was measured using Filar

Effect of relative humidity.
About 5 ml of the pycnidiospore
suspension were pipetted on water agar
plate. The opened plates were then
enclosed in plastic containers
containing solutions with the following
relative humidity (%) levels: 36 (54.0%
H2SO4), 49 (44.0% H2SO4), 70
(26.5% CaCI2), 85 (17.1% CaCI2), 98
(water). One hundred spores were
counted and the number of germinated
spores recorded. The germ tube length

(um) of 25 spores were measured at 2,
3, and 4 hrs of incubation.


Symptomatology'and Signs

Under field condition, symptoms
of stem rot consisted of wilting of
leaves, browning, reddening and drying
of the stem and later wilting of the
plant as the disease progressed (Fig.
la-c). The leaves above the point of
infection first turned yellow, later
brown and then fall off. The dried
leaves showed necrotic spots (Fig. 1d).
Complete defoliation of plant occurred
15 to 16 days after the appearance of
wilting. Browning of the stem extended
above the point of initial infection.
Sometimes the lower portion of the
infected stem or branch recovered and
continued to grow. The brown bark of
infected stem turned black and the
fibers rotted and disintegrated while the
woody part of the stem became
discolored (Fig. 2b-c). The disease can
affect any part of the plant at any stage
of plant growth. The symptoms
produced on inoculated and naturally
infected plants were similar. Wilting and
defoliation of the plant were observed
24 to 48 hrs after mycelial inoculation.

Numerous black stroma
containing erumpent pycnidia were
visually seen on the surface of infected
stem (Fig. 2a and 2d). Thee pycnidia
continued to develop until the whole
plant was covered with these
structures. The tissues beneath the
epidermis showed the impressions of
pycnidial structures which contained


Fig. 1. Symptoms of salago stem
complete defoliation of infect

Fig. 2. Symptoms and signs of s
erumpent pycnidia on sui
disintegration of fibers, c)

a) initial wilting, b) drying of leaves,
plant, d) necrotic spots on dried leaves.

go stem rot a) black stroma containil
e of infected stem, b) browning al
*coloration of woody portion of stem,

merous pycnidiospores. Similar (Wang and Pinckard, 1973). Adeniji
igal structures were seen on (1970a) and Krupinsky (1983)
ificially inoculated plants. mentioned that B. theobromae is a
wound parasite. Wounding is necessary
Plation and Pathogenicity Test for infection and disease establishment.

The fungus constantly The high incidence of stem rot
sociated with the disease was easily could also be due to moisture stress.
lated into pure culture using tissue Less moisture in the bark favored
hinting technique on PDA. The mycelia growth of B. theobromae (Riffle, 1978).
the fungus were evident 2 to 3 days Furthermore, Bier (1964) and
er tissue planting. Schoeneweiss (1975) noted that
factors contributing to moisture stress
Artificial inoculation revealed in a host increase susceptibility to
it the mycelia of the fungus readily certain canker pathogens.
ected salago test plants. High
rcentage of infection (98.3%) was The death of the stem above
trained by wounding the stem. the point of inoculation confirms the
>culation of intact stems resulted in finding of Krupinsky (1982) on Siberian
3% infection (Table 1). Wilting elm. The occurrence of the symptoms
curred 24 hrs after inoculation and 24 hrs after inoculation suggested that
mplete browning of the stem and the isolate used for inoculation was
foliation of the plant occurred after 2 virulent and aggressive.
3 days.
Morphology of the Fungus
No infection was observed on
)unded and non-wounded stems Hyphae and chlamydospores in
ing pycnidiospores. Similar observa- culture. Hyphae were branched,
n was reported by Olunloyo and septate,4nultinucleate and hyaline when
uruoso (1975) on cashew and Lewis young but colored when old. Hyphal
378) on sycamore inoculated with cells ranged from 5.0 u 7.4 x 21.8 u -
nidia of B. theobromae. However, the 49.5 u, with a mean of 6.2 u x 39.1 u.
ter found that the fungus colonized Chlamy-dospores were produced in old
e host but no canker was produced. culture grown in YEA slant. They were
dark, thick-walled and round to oval in
Wounding of tissues was found shape.
cessary for infection and successful
development of rot in cotton bolls by Pycnidia and conidia in culture
Vlodia gossypina (Beguico, 1979), rot and in host. Pycnidia in agar culture
sweet potato tubers by D. tubericola were globose, black and occurred singly
arsud, 1979) and canker in Siberian or in groups within a stroma (Fig. 3a).
n by B. hypodermia (Krupinsky, Stroma was simple, with single or no
83). The low incidence of infection opening. Spores were released from the
non-wounded stem could be pycnidia in dry matrix or cirrhi. The
:ributed to the thickness of the white cirrhi released one-celled, hyaline
idermal cells that might hinder pycnidiospores. In one month-old
netration of invading hyphae. This cultures, the cirrhi were black and were
aracter had hpbn imnlicated in made-un of two-celled. dark-colored

Table 1. Effect of wounding on stem rot infection of salago using mycelia.a,

Inoculation Method Infection (%)1

Wounding 98.3 a

Non-wounding 8.3 b

Control 0 c

1Mean of 3 replications, 20 plants per replicate; means having different letter are significantly different a
5% level by LSD.

Table 2. Mycelial growth, pycnidial and pycnidiospore production of B. theobromat
at different culture media incubated at 30 C and 170 footcandles.1

Colony Number (after 14 days)
Agar Medium Diameter -------------
after 24 hrs Pycnidia Pycnidiospore/20
(mm) pycnidia (x103)

V-8 juice 75.2 a 133.2 cd 256.5 d

Yeast extract 63.5 b 222.8 a 2619.0 a

Potato dextrose 59.7 c 123.5 de 482.5 c

Malt extract 54.0 d 141.0 cd 92.0 e

Czapek's dox 49.8 e 167.5 b 1346.0 b

Glycerine 44.7 f 99.2 e 146.0 de

Sweet potato
sucrose 44.3 f 152.5 bc 193.2 d

Water 34.8 g 32.2 f 94.0 e

Corn meal 30.0 h 29.2 f 188.0 d


Philipp. Phytopathol. 1993, Vol. 29: 84-100

Pycnidia from naturally and
artificially infected stem tissues were
uniloculate or multiloculate in black
stroma (Fig. 4). Some uniloculate
pycnidia have ostiole with short straight
and rounded neck. The diameter of the
pycnidia from agar plate, inoculated
stem and naturally infected stem ranged
from 600 to 1369 u, 115 to 580 u, and
118 to 1112 u, with mean of 888.0 u
418.3 u and 335.3 u, respectively. The
pycnidia from naturally infected stem
were the biggest and significantly larger
than those from agar plate and
inoculated stem.

Pycnidiospores were elliptical,
obovate to ovate. Immature
pycnidiospores were hyaline and
one-celled. Mature pycnidiospores were
dark-colored and two-celled with
longitudinal striations.

The spore length .from agar
plate ranged from 13:7 to 41.8 u, 7.9
to 22.5 u from inoculated stem while
11.9 to 22.7 u from naturally infected
stem with mean of 21.8, 18.8, 18.5 u,
respectively. The spore width from agar
plate ranged from 5.8 to 13.5 u, 7.0 to
15.8 u in inoculated stem, while 7.6 to
12.1 u in naturally infected stem with
mean of 11.1, 12.3, 9.8 u,
respectively. The mean spore length
from agar plate was significantly longer
than those from the other two sources.
Mean spore width from naturally
infected stem was significantly
narrower than those from agar plate
and inoculated stem.

The shape bf the pycnidia and
pycnidiospores of, he fung from agar
culture, inoculates stem ani naturally
infected stem were similar. However,
differences in size may be attributed to
the differences in the amount, kind and
availability of nutrients from these three
sources. The bigger pycnidia produced

in YEA indicated that this substrate
provided the nutrients needed by the
fungus. The larger pycnidia produced
on naturally infected stem also
indicated that the fungus obtained more
nutrients from old stems (about 1.5 yr-
old) than from the inoculated stems (6

Identity of the Fungus

The pycnidiospores of
Botryodiplodia sp. isolated from
infected salago stem and from agar
culture were elliptical, hyaline, one-
celled when immature; brown, two-
celled with longitudinal striations when
mature. These characteristics were
similar to B. theobromae isolated from
cassava (Lozano and Booth, 1976),
banana (Meredith, 1961) and apple
(Goos et al., 1961). The size of the
pycnidiospores which ranged from 15-
25 u x 6-13 u, fell within the range of
17-20 u x 10-13 u, 21.5-31,5 u x
10.5-17,25 u as reported by Wardlaw
(1932) and Rosario (1954). The
pycnidia from salago were similar to
that of B. theobromae from banana and
cacao, with emphasis on formation of
either uniloculate or multi-loculate
pycnidium. Both isolates were black,
round and sunken and simple in culture
(Filer, 1969; Nowell, 1926; Wardlaw,
1932; Goos et al., 1961; Lozano and
Booth, 1976). Isolates from salago and
B. theobromae from apple, banana,
dugwood and cacao produced
chlamydospores and red pigment in
culture incubated at 37C (Goos et al.,
1961; Satour et al., 1969a; Meredith,
1961; Mullen, 1987; Voorhees, 1942).
Both isolates have an optimum
temperature for mycelial growth in
culture of 25-30C (Wardlaw, 1932;
Adeniji, 1970a). Based on these
information, the fungus isolated from
salago was identified as Botryodiplodia
theobromae Pat.

Fig. 3. Cross section of stromata of Bo


rnlupp. rnyropainol. I a, vol. Li: o- iuu

odiplodia theobromae from agar culture.


Philipp. Phytopathol. 1993, Vol. 29: 84-100

ct of Environmental Factors on continuous light and alternate 1E
wth and Sporulation darkness and 9 hrs light (A
gave the biggest colony. The sm;
Effect of culture media. The colony was produced in alternate
lest colony diameter was observed hrs light and 9 hrs darkness (ALD1
V-8 JA (75.2 mm) and the lowest continuous darkness. The hic
CMA (30.0 mm) (Table 2). pycnidial and pycnidiospore counts
nidiospore production was signifi- occurred in YEA exposed
tly higher on yeast extract agar than continuous light as compared
- ---* -, J:-^1: LJ:..L _- -1I--:_ 1:--4 J:J.:- M Tl-- A:-.

degree ot sporulation occurred on V-8
JA, SPSA and CMA while very few
spores were produced on MEA and

Bigger colony was produced on
V-8 JA but yielded fewer pycnidia and
pycnidiospores. CMA produced the
smallest colony and lowest number of
pycnidia. However, it produced more
pycnidiospores than MEA and GA
which yielded bigger colony and higher
number of pycnidia. The color of the
media changed from yellow to gray and
black after 14 days of incubation. Very
abundant, aerial mycelial growth was
associated with YEA, PDA, and
Czapeck's dox agar but sparse and
appressed on sweet potato sucrose,
water and corn meal agar. Pycnidia
were dispersed but tend to be
concentrated on the edges and top of
the cover plate on YEA, PDA, CDA and

Change in the color of the
media could be due to the change in the
pH during growth of the fungus.
Differences on the distribution of
pycnidia and their location in culture
was greatly influenced by the
composition of carbon and nitrogen
sources (Wardlaw, 1932; Stevens,
1933; Satour et al., 1969a and 1969b).

Effect of light. There were
significant differences in colony
diameter, pycnidial and pycnidiospore

et al., (1962) and Halos (1970) but
not with Bequico (1979). This could be
due to the duration of exposure to
either light or darkness. No pycnidia
were formed by exposing the culture
for 14 days in continuous darkness.
Similar observation was reported by
Wardlaw (1932) on B. theobromae
isolated from banana. While the
pycnidial count was higher on ALD1
and ALD2, it gave lower spore yield.
Formation and distribution of pycnidia
were affected by light (Satour et al.,

Exposure to ultraviolet (UV)
light improved pycnidial production and
sporulation of the fungus (Table 4).
Increasing the length of exposure to UV
light increased the number of pycnidia
and pycnidiospores. The highest
number of pycnidia (419.8) was
observed on cultures exposed for 11
days to UV light. Pycnidial counts from
cultures exposed for 11 ands14 days to
UV light were significantly higher
compared with other treatments. Three
days exposure to UV light gave the
lowest pycnidial count. Cultures
exposed to UV light for 9, 11 and 14
days yielded high pycnidiospore count
while 3 hrs exposure gave the lowest

Means in column with same letter

are not significantly different at 5% level using DMRT.

Phiipp. Phytopathol. 1993, Vol. 29: 84-

Effect of pH. The fungus At 40 C, the isolate produced pin
juced bigger colonies and more peach red pigment on YEA at the I
nidia in medium with pH ranging of inoculum. The isolate prok
n 5.0 to 7.0 (Table 5). Mycelial produced certain chemical that diffl

more alKaline. Myceiiai grown was
optimum at pH 5.0 to pH 6.5 while
pycnidial and pycnidiospore production
was optimum at pH 5 to 7 and pH 5.5
to 8, respectively. The results suggest
that the fungus can tolerate wide pH
range without adverse effect on growth
and reproduction.

Effect of temperature. B.
theobromae did not grow at 15C and
40C (Table 6). Cultures exposed to 30
C gave the highest colony diameter
(39.8 mm) followed by those exposed
to 25C (34.2 mm) and 35C (30.3 mm).
The fungus produced pycnidia and
pycnidiospores only at 25C. These data
suggest that the fungus will produce
pycnidia upon exposure to continuous
darkness if the incubation temperature
is 25 C. This result is consistent with
the findings of other investigators.
Adeniji (1970b) observed that B.
theobromae produced maximum growth
at 25C on PDA-Difco but failed to grow
at 35 C. The failure of B. theobromae to
produce pycnidia at 20, 30 and 35C
was indicative of the interaction of light
and temperature. Moreover, the data
suggested that light is essential for
pycnidial and pycnidiospore production
of B. theobromae.

The color of the medium
changed from pale orange yellow to
tilleul buff at 20 C, to light grayish olive
at 25 C and to pale gull gray at 30 C
and 35 C. At 20 C, the middle portion
of the culture darkened and at 25, 30
and 35 C the mycelia became grayish

(1925) found isolates of B. theobromae
producing red pigment on culture
exposed at 37 C.

Conidial Germination and Germ Tube

Effect of temperature. The
pycnidiospores of B. theobromae
started to germinate 2 hrs after
incubation at 25 to 35C (Table 7). The
highest mean percentage germination
was noted at 30C (67.2%) followed
at 25C (62.8%). Within 3-4 hrs,
germination occurred at 20 and 40C.
Regardless of the temperature, the
highest mean percentage germination
was observed after 4 hrs of incubation
(62.2%). No germination was noted at
1 hr of incubation.

One to two germ tubes may be
produced per spore. Generally,
immature spores produce single germ
tube while mature spores produced two
germ tubes, one from each cell.

Regardless of the period of
incubation, the longest and shortest
germ tube was produced at 30 C (20.5
u) and 40 C (6.3 u), respectively (Table
8). Germ tube elongation did not vary
significantly at 20, 25 and 35 C. The
length of germ tube increases about 12
times from 2 to 3 hr of incubation
compared with a 2-fold increase from 3
to 4 hr of incubation. This indicates
that the initial germ tube elongation is
very rapid.

Pp. rnytopaJuIl. IWWU, VOl. Le; o0- EUu

Fable 5. Mycelial growth, pycnidia and I
grown in yeast extract agar al


5.0 42.2 a

5.5 43.5 a

6.0 44.2 a

6.5 42.2 a

7.0 36.8 b

7.5 33.8 bc

8.0 32.5 c

8.5 26.2 d

9.0 24.5 d

Means in column with common letters are not signil

Fable 6. Mycelial growth, pycnidial and
grown in yeast extract again

(C) (MM)

15 0 e

20 15.7d

25 34.2 b

30 39.8 a

35 30.3 c

40 0 e

Means in column with common letters are ntq sign

nidiospore production of B. theobromae
3 C, 170 footcandles and different pH

Pycnidia Pycnidiospore/20
pycnidia (x103)

72.5 abc 78.0 c

77.5 abc 169.5 a

81.5 ab 108.0 abc

70.5 abc 152.0 a

110.5 a 121.0 abc

56.2 bc 140.0 ab

55.5 bc 108.0 abc

49.2 c 88.0 bc

23.0 d 53.0 d

itly different at 5% level using DMRT.

nidiospore production of B. theobromae
t continuous darkness and different

Pycnidia Pycnidiospore/20
pycnidia (x103)

0 b O b

0 b Ob

56.2 a 2.0 a

Ob b

Ob b

0 b b

ntly different at 5% level using DMRT.

Philipp. Phytopathol. 1993, Vol. 29: 84-100

Table 7. Percentage germination of L
and temperature levels1

(C) 1

15 0

20 0

25 0

30 0

35 0

40 0
Mean 0 d 22.9 c

'Means with common letter are not significantly c

Table 8. Length of germ tube (u) of I
incubation periods and tempe

(C) 1

20 0

25 2.8

30 6.7

35 1.4

40 0
Mean 1.8 a

'Means with common letters are not signifantly di

heobromae at different incubation period:

2 3 4 MEAN

0 0 0 Oe

0 67.3 95.0 40.6 c

52.7 98.0 100.0 62.7 a

74.0 96.0 99.0 67.2 a

10.7 90.0 96.0 49.2 b

0 54.7 83.0 34.4 d
51.Ob 62.2 a

*ent at 5% level using DMRT.

nidiospore of B. theobromae at differen-
ures levels

2 3 4 MEAN

10.3 38.9 12.6 b

14.3 31.3 12.1 b

26.4 48.8 20.5 a

13.2 35.1 12.4 b

11.1 14.5 6.4 c
12.5 b 28.1 c

mnt at 5% level using DMRT.

Fable 9. Percentage germination of
incubation times and relative hu

(C) 1

36 0 7'

49 0 7'

70 0 8.

85 0 8!

98 0 8!
Mean Oc 8

Means with common letters are not significantly difi

Fable 10. Length of germ tube (u) of py<
incubation times and relative hu

(%) 2

36 5.2

49 4.7

70 4.8

85 5.3

98 4.6
Mean 4.9 a

Means with common letters are not significantly difi

Philipp. Phytopathol. 1993, Vol. 29: 84-100

ryodiplodia theobromae at different

3 4 MEAN

97.7 94.6 67.4 a

90.7 92.7 64.7 a

90.0 92.0 66.7 a

92.3 94.0 67.9 a

93.3 93.0 68.0 a
b 92.8 a 93.2 a

it at 5% level using DMRT.

liospores of B. theobromae at different


23.1 14.1 a

21.4 13.2 a

23.9 14.4 a

22.7 14.0 a

24.3 14.4 a
23.1 b

it at 5% level using DMRT.

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