• TABLE OF CONTENTS
HIDE
 Front Cover
 Rice sheath blight spread: A case...
 Trichoderma species for the biocontrol...
 Bugtok disease of cooking bananas:...
 Control of Rotylenchulus reniformis...
 Identification of citrus trist...
 Phytopathological note: Occurrence...
 Phytopathological note: Concentrating...
 Back Cover














Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00090520/00035
 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 1994
Frequency: semiannual
regular
 Subjects
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
 Notes
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00035
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
    Rice sheath blight spread: A case study on focal expansion in plant disease epidemiology
        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
    Trichoderma species for the biocontrol of sweet pepper stem rot (Sclerotium rolsfii Sacc)
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
    Bugtok disease of cooking bananas: I. Etiology and diagnostic symptoms
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
    Control of Rotylenchulus reniformis on Cotton (Gossipium hirsutum L.) through Organic manuring mulching and intercropping
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
    Identification of citrus tristeza
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
    Phytopathological note: Occurrence of rice dwarf disease in the Philippines
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    Phytopathological note: Concentrating genes for Philippine downy resistance and desirable agronomic traits in corn
        Page 59
        Page 60
        Page 61
        Page 62
    Back Cover
        Page 63
        Page 64
Full Text









Official Publication of the Philippine Phytopathologiccal Society, Inc.


)IRECTORS- iIV. I I I 994-
)IRECTORS 1994-1995


President RUSTICO A. ZORILLA
Vice-President NESTOR G. FABELLAR
Secretary LINA C. LAPITAN
Treasurer NENITA L. OPINA
Auditor CEFERINO A. BANIQUED
Business Manager LINA C. LAPITAN
Board Member AVELINO D. RAYMUNDO
Board Member ANGELITA D. TALENS
Board Member RIZALDO G. BAYOT
Board Member ARTURO MANZANILLA
Board Member ELENITA G. SISON
Immediate Past President TEODORA O. DIZON

PHILIPPINE PHYTOPATHOLOGY
EDITORIAL BOARD 1994-1995


OSCAR S. OPINA
RIZALDO G. BAYOT
TEODORA 0. DIZON



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'" ,' l


ditor-in-Chief
associate Editor
associate Editor
















3., INC.

ssed to the TREASURER, P.F.S c'o Department o
.ine Phytopathology, published semi-annually, is the
Society, Inc. It is sent free- to members in gooc
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IILII I IlL IIII IVI






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


RICE SHEATH BLIGHT SPRE/
EXPANSION IN PLANT


NANCY P. CASTILLA, F.A.

Research Assistant, Senior Assistan
IRRI-ORSTOM Shuttle Research Project o
Entomology and Plant Pathology Division,

Key words: Epidemiology, factors a
efficiency, Rhizoctonia solani, rice sheath


ABS

The effects of nitrogen si
leaf wetness regime and plar
the spread of rice sheath bliC
were studied under field and !

Field experiments were cc
and dry seasons with three nit
kg N/ha) as main plots and fil
subplots in a split-plot design
nitrogen supply to the crop I
focal spread of the disease. T
(lower vs. upper layer of the i
longer than the effect of inocu
in the leaf wetness and in the
are the driving factors in the i

The inoculum efficiency i
nitrogen content of the host
contact frequency, was studied
accumulation of wet and d
content and higher plant cc
efficiency.

The apparent contradictii
nitrogen content of the host
the positive effect of nitroge
spread illustrates the comply
components of a pathosysten

INTRODUCTION

Sheath blight has become one of
the most important diseases in several
rice-growing regions during the last two
decades (Teng et al., 1990). The disease
**_ j =--'-- *.l _- --_


A UASk I UUY UN I-ULAL
EASE EPIDEMIOLOGY


4ZEGUI, and S. SAVARY

dentist and Visiting Scientist, respectively;
haracterization of Rice Pest Constraints;
ernational Rice Research Institute

:ting spread, focus expansion, inoculum
ht


kCT

y, inoculum source structure,
>-plant contact frequency on
causedd by Rhizoctonia solani
controlled conditions.

cted during the 1992-93 rainy
an input levels (0, 80 and 120
ioculum source structures as
I eight replications. Increasing
strong effect on the rate of
effect of inoculum positioning
opy) on the focus area lasted
amount (5 vs 15 g). Changes
itact frequency between hills
ad of sheath blight.

heath blight, as influenced by
Int, leaf wetness regime and
monocyclic experiments. The
>eriods, decreasing nitrogen
icts increased the inoculum


between the negative effect of
it on inoculum efficiency and
supply to the crop on disease
nteractions among individual


19851. Mycelia (Mew et al., 1980;
Premalatha Dath, 1985) and sclerotia
from the soil (Gangopadhay and
Chakrabarti, 1980; Belmarretal., 1987)
cause primary infection in rice. The
disease spreads by means of mycelia
growing from an infected tissue to


IL5 I TdIy UL dl., I ZIUJ






2


The increasing importance of sheath mainplots: no fertilizer, 80 kg N/ha, anc
blight has been attributed to the changes 120 kg N/ha. Nitrogen was applied,ir
in cultural practices associated with the the form of urea. The subplots were
introduction of high-yielding varieties, represented by different structures of the
The application of high levels of nitrogen inoculum sources: 5 g at the upper layei
fertilizer to increase yield (Kannaiyan and of the plant canopy, 5 g at the lower layer
Prasad, 1979; Lee and Rush, 1983; 15 g at upper layer, 15 g at the lowei
Rajan, 1986; Marchetti and Bollich, layer, and a non-inoculated control. Ar
1991), close plant spacing (Kannaiyan individual plot consisted of 15 x 15 hills,
and Prasad, 1983), and the high-tillering with the central source hill representinC
capacity of high-yielding varieties the inoculum source (Fig. 1). The
(Hashiba and Ijiri, 1989) favor sheath inoculum consisted of a 1/5 rice grain
blight development. Continuous hull mixture colonized by a lowland rice
cropping, characterizing intensive rice isolate of the fungus belonging tc
production, favors the accumulation of anastomosis group 1. Inoculations at the
initial inoculum over several cycles,and lower layer were done by inserting the
subsequent sheath blight development. inoculum at the base of the source plan
and at the upper layer by inserting the
Sheath blight causes 5.0 to 50% inoculum at the center of the upper leaves
reduction in grain yield in the Philippines of the plant canopy and holding the
(Ou et al., 1973). Approximately 85% inoculum together by a rubber band.
of the country's total rice area is planted Inoculations were made during the
to high-yielding varieties (IRRI, 1991), maximum tillering stage (50 days aftei
suggesting that sheath blight is likely to transplanting). Both experiments hac
remain a potential threat to rice produc- eight replications.
tion in this country.

A better understanding of the rice- Data Collection
sheath blight pathosystem is useful to


,11t ,LII UlIyilL ~ IR t
many interacting f
affecting one of the
acteristics of the pi
temporal behavior.
here address the ef
factors, both in tei
ponents and spat
ment.


lIll
S plain
wet
S is
con
disc
sou
areas
and


ty on the
contend
quency. A
site of
ased pla
bout a p


ivycoiogical Zociety, 1 YbJ). lo measu
MATERIALS AND METHODS focus area, the maximum distance
Lesions from the source plant along tl
1. ANALYZING THE FOCAL SPREAD rows (length) and across rows (widt
OF SHEATH BLIGHT IN THE FIELD (Fig. 1) were measured at week
: -.. I. .-I R A _ ._ __..._ . .. ---_ I


cxperimeniai design ana Layout

Two experiments were conducted
at IRRI during the rainy season (Aug.-
Nov. 1992) and dry season (Feb.- March
1993). IR72, a semi-dwarf, high-yielding
variety, was planted with a 20- x 20-cm
spacing. Both experiments were laid out
in a split-plot design (Fig. 1), with the


uLIII a LIIIUUyiiUUL LIle uLIUpp yII sWaidoUI
starting from 12 days after the source
establishment. Assuming that the focus
expands with an elliptical shape, the area
of focus (a) was computed as: a = (l.w)/
4, where I and w are the focus length
and width in cm, respectively.

The initial sheath blight severity of






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


Main Plot: Nitrogen Supply
NO: no nitrogen input
N1: 40 kg N/ha basal application
40 kg N/ha at maximum tillering
N2: 40 kg N/ha basal application
Rep 8 Rep 7 40 kg N/ha at maximum tillering
40 kg N/ha at booting


Subplot: Inoculum Structure
C: uninoculated
L5: 5g inoculum at the upper layer
S5: 5g inoculum at the lower layer
Rep 6 Rep 5 L15: 15g inoculum at the upper layer
S15: 15g inoculum at the lower layer








Rep 4 Rep 3
across
C 00000oooo000ooo
0000000000000000
000000000000000ooooooooooooooo
000000000000000
20.4m S5 00000000000000
20.4m $5 ooooooooooooooo
Soooooooooooooo Source plant
S along oooo000ooo 00oooo
L15 ooooooooooooooo
000000000000000
000 00000 000 0000
5-- 000000000000000
000000000000000
3.2m NO N2 0 0oooo oooooooo120cm
3.2m NO N1 N2 ooooooooooooool
Rep Rep2 20cm


Fig. 1. Experimental design and layout for analyzing the focal spread of sheath blight
in the field. An experimental unit consists of a plot with 15 x 15 hills.






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


assessing the percentage of leaf and stem
irea covered by sheath blight lesions on
a sample of five tillers. The first assess-
nent was done 12 days after source
establishment.

Leaf wetness in the plant canopy

>f the plant canopy to hold moisture. :
Assessments were done in the upper,
middle, and lower layers of the canopy. .
.eaf wetness assessments were taken ten .
:imes on ten hills in each nitrogen
treatment The amount of leaf wetness
Nas assessed at 0600 hours following
a four-point assessment scale (Fig. 2),
here 0 = dry, 1 = few, scattered big
Iroplets, 2 = thin film of small droplets
ind 3 = dense film of fine droplets. :
.: .*:


Contact frequency between plants
was assessed by counting the number
of leaf-to-leaf contacts and leaf-to-sheath
contacts of a reference hill with its
neighboring hills.

Hills were sampled during the stem
elongation, booting, and ripening stage Fi
for determination of nitrogen content by
the Kjeldahl method (Varley, 1966). One
hill in each nitrogen level was sampled
per replicate.

Data Analyses
Variation in focus area was
analyzed using two-way ANOVA (Gomez
and Gomez, 1984), with nitrogen input
levels as main plot and inoculum source
structure as subplot treatment. Inoculum
source structure was further partitioned
into the amount (5 g vs. 15 g) and the
location (upper vs. lower layer of the
canopy). To analyze focus expansion over
time, cross-sectional analyses (Zadoks,
1972) of the focus area were performed
for each of the six observations.

The effect of nitrogen supply on the
mean leaf wetness rating of ten hills was
analyzed using one-way ANOVA. The
area under the progress curve (AUPC) of
nitrogen content in the leaves and the
AUPC of the number of leaf-to-leaf and
leaf-to-sheath contacts were computed
using trapezoidal integration and analyzed


0 1 2 3

g. 2. Assessment scale for rating
leaf wetness in the plant
canopy, where: 0 = dry, 1 =
with a few scattered big
droplets, 2 = with a thin film
of small droplets, and 3 =
with a dense film off fine
droplets.



Multiple regression analyses of the
combined data from the rainy and dry
season experiments were performed
using SAS (SAS, 1985; 1991). The mean
rate of focus expansion (R) was norma-
lized by natural logarithm transformation.
It was computed using the formula:
n-1
(ai+ a / (ti t.)
i=1
R=
n-1
where n = tne number of observations
(6 for each experiment), a = the focus
area in cm2 at sample time i, and t = the
number of days after inoculation at
sample time i.






Philipp. Phytopathol. 1994, Vol. .EJ1): 1-13


Table 1. List of explanatory variables used in the multiple regression analyses.


VARIABLE DESCRIPTION UNIT


N Area under the progress curve of the
nitrogen content in the leaves %.day

W Overall wetness of the canopy throughout
the cropping season (product of mean
leaf wetness rating and mean daily rainfall
during the cropping season)

C Area under the progress curve of the number
of leaf-to-leaf and leaf-to-sheath contacts %.day

S Initial sheath blight severity on the sheath of
the source plant %

L Initial sheath blight severity on the leaves
of the source plant %


The log-transformed mean rate of epidemiological attributes of the quadrat
focus expansion (F) was used as the were manipulated according to th(
variable to be explained and the variables, variable being investigated. To probe the
as listed in Table 1, as regressors. conduciveness of the quadrats to sheath
Multicollinearity between explanatory blight spread, healthy trap plants wen
variables was determined by analyzing transferred to the center of the quadrat
the Pearson correlation coefficients, After three days, the trap plants weri
variance inflation factors (VIF), and removed from the center of the quadra
eigenvalues generated by SAS (SAS and potted. After a three-day incubation
1985; 1991). period, the number of infection point:
was counted. An infection point i:
II. ANALYZING THE EPIDEMIOLOGI- defined as a typical sheath blight lesioi


CONDITIONS

Experimental Design and Layout

Experiments were conducted und
semi-controlled conditions followii
Leaio et al. (1993) to determine t
relationship between inoculum efficient
(IE) and leaf wetness regime, conta
frequency, and nitrogen content. IR72,
semi-dwarf, high-yielding variety, w;
planted in the screenhouse. Unle;
otherwise specified, all experiments h.
a 20- x 20- cm spacing and a fertiliz
rate of 120 kg N/ha. Quadrats of 3 x
hills were inoculated at the leaf level ar
base of the tillers with 5 g of rice grai
hull mixture colonized by the fungus. TI


The experiments were conducted
using a randomized complete block
design with three successive batches
of trap plants. A quadrat represented
the basic experimental unit.

Relationship between Leaf Wetness
Regime and Inoculum Efficiency in Sheat#
Blight

The experiment was conducted
during the 1993 dry season (Feb. to Apri
1993) with the following leaf wetness
regimes as treatments: permanently dr
(TO), wet for one night (T1), wet for tw(
consecutive nights (T2), wet for threi









wet (PW). Leaf wetness was achieved
during night time by spraying the
quadrats with water and covering them
with plastic cages at about 1700 hours.
Permanent wetness was achieved by
continuously spraying the quadrats with
water in the day time and covering the
cages at night time. The experiment had
five replications.

Relationship between Nitrogen Supply to
the Crop and Inoculum Efficiency in
Sheath Blight

The experiment was conducted in
June to July 1992 with eight replications
and three nitrogen input levels as
treatments. To manipulate the nitrogen
supply to the crop, trap plants were
grown in plots with three nitrogen input
levels: no nitrogen (NO), 60 kg N/ha (N1),
and 90 kg N/ha (N2). At maximum
tillering stage, the trap plants were
transferred to the center of the quadrats
fertilized at the rate of 120 kg N/ha.

Relationship between Contact Frequency
and Inoculum Efficiency in Sheath Blight

The experiment was conducted in
Sept. to Oct. 1992 with five replications.
To vary the contact frequency, hills were
transplanted at 14 days after sowing in
a delimited area at 18 x 18 cm spacing.
During maximum tillering stage, plants
were transplanted in the main
experimental area according to the
following plant spacings: 15 x 15 cm,
15 x 20 cm, 20 x 20 cm, and 25 x 25
cm. The number of leaf-to-leaf and leaf-
to-sheath contacts in each plant spacing
treatment was counted following the
methodology adopted for the field
experiment.

Data Collection and Analyses

During each probing period, one
quadrat hill was randomly chosen and
five of its tillers were sampled for leaf
area and stem area (cm2) measurements.
The number of infection points on all the
leaves and stem of each tiller was


Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


The variable considered in all the
experiments was inoculum efficiency (IE),
computed as: IE = LD / LDq, where LD
is the lesion density on the leaves and
stems of the trap plant and LDq is the
average lesion density on the quadrat.
Inoculum efficiency was calculated during
each probing period. It measures the
spread of the disease from the infected
hills in the quadrat to the healthy trap
plant, and corrects for the differences in
the area of the source and trap plants
across the different treatments and
batches. Inoculum efficiency represents
the progeny/parent ratio, in terms of
lesions present on the quadrat and on
the trap plants.

Inoculum efficiency was analyzed
using repeated-measures ANOVA
(Madden, 1986), where batch is
considered as the subplot and the
experimental treatments as main plots.


RESULTS

I. ANALYZING THE FOCAL SPREAD
OF SHEATH BLIGHT IN THE FIELD

Figures 3 and 4 show the progress
of the focus area (cm2) throughout the
crop growth period during the rainy
and dry seasons, respectively. Focus
area was generally wider and increased
faster during the rainy season than in the
dry season. The spread was generally
faster in plots with high nitrogen supply,
and in plots where the source plants were
inoculated with high inoculum levels or
inoculated at the upper layer. Focus area
expanded exponentially during the rainy
season and in plots with high nitrogen
supply. A decrease in the focus area
(focus exhaustion) occurred during the
dry season and in plots receiving little or
no nitrogen supply.

The cross-sectional ANOVA showed
that nitrogen sunnlv to thp rrnn affartrl






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


cropping season. This indicates tha
natural infections and interplo
interference were negligible during bott
experiments. The effect of inoculur
positioning on the focus area lasted
longer than the effect of the amount o
inoculum. Inoculum positioning affected
the focus area until 33 days afte
inoculation during the rainy season,an(
until 56 days after inoculation during th
dry season. The effect of the amount o
inoculum on focus area lasted only unti
19 days after inoculation during the rain)
season and was erratic in the dry seasor
(Table 3).
The A IJPC of the numher of leaf-to


There were significant differences i
IE among the different plant spacing!
with the IE significantly higher in close
plant spacings than in wide plan
spacings (Fig. 6a). Inoculum efficiency
in interrupted leaf wetness treatment
(T3) was significantly higher than i
continuous dryness or in continuou
wetness treatments (Fig. 6b). Inoculur
efficiency declined with an increase i
nitrogen supply (Fig. 6c). Inoculur
efficiency at 60 and 90 kg N/ha (wit
1.80 and 2.17% nitrogen conten
respectively) was significantly lower tha
no N supply (with 1.70% nitroge
content), with a twofold difference







'hilipp. I'hltop(athul. 1994 ,ol. 30l)). 1-


focus area (cm2)
3000
NO

2000-


1000


0 i
10 20 30 40 50 60



15000
N1

10000-


5000-



10 20 30 40 50 60



30000
N2




20000 /





10000



S1L5
S-* S15

o ----* S5
0
10 20 30 40 50 60
number of days after source establishment



Fig. 3. Focus expansion during the rainy season, L15 = 15 g inoculum at the upper
layer of the source plant L5 = 5 g inoculum at the upper layer; S15 = 15g
:C.1 ."- 1-1- r [ ,- : .1. ." *1^ l^






t'hilipp. Phytopathol. YY94, Vol. U(l.): 1-15


focus area (cm2)
500
SNO
250-

0--
10 20 30
1500
N1


1000




500



0 -i
10 20 30
3000

N2






2000








1000









10 20 30
number of days afte




Fig. 4. Focus expansion during the dry si
N2 = 120kgN/ha. L15 = 15 g ir
L5 = 5 g inoculum at the uppe
S5 = 5 g inoculum at the base.


40 50 60















40 50 60



























S15
40 50 60 S5













40 5o 60 ----- S5
)urce establishment



on. NO = no N supply; N1 = 80 kg N/ha,
ulum at the upper layer of the source plant
yer; S15 = 15 g inoculum at the base;














Observation

Source of Variation df 1 =12 2

MS


Replication 7 0.27 0.09

Nitrogen Level 2 4.25** 3.68

Error (a) 14 0.19 0.10

Inoculation Method (I) 4 185.13** 242.34

A2: Rest 3 1.46"* 1.14

Positioning (P) 1 ".C "* 2.80

Amount (A) 1 0.97 0.62

PxA 1 0.39 0.00

Control vs. A2 1 736.15'* 965.96

Nxl 8 0.49** 0.37

Error (b) 84 0.13 0.09

Total 119

'Based on natural log-transtormed data; = P< 0

Table 3. Cross-sectional ANOVA for focu

Observation

Source of Variation df 1 =12 2

MS

Replication 7 0.44 0

Nitrogen Level 2 6.81 11

Error (a) 14 0.06 0.

Inoculation Method (I) 4 143.87" 161

A2: Rest 3 1.73** 1

Positioning (P) 1 4.73 3

Amount (A) 1 0.44* 0

Pxk 1 0.001 0.

Control vs. A2 1 570.30*' 643.

Nxl 8 0.48"* 0

Error (b) 81 0.08 0

Total 119

'Based on natural log-transformed data; =p < 0.(


rniiupp. rlnyupaunoI. 17Y., vul. Juvl;. i-10



rea during the rainy season'.


nber and days after source establishment

3 3 = 27 4 = 33 5 = 47 6 = 56

MS MS MS MS


1.01* 1.22** 2.21 5.89

5.38"* 11.00"* 20.44** 56.06**

0.28 0.12 0.98 2.47

261.24** 282.39** 323.14*" 340.40 *

0.33* 0.26 0.60 0.55

0.70* 0.52* 0.60 0.33

0.14 0.20 1.20 1.30

0.16 0.07 0.01 0.03

1043.98"* 1128.76** 1290.76** 1359.95**

0.37** 0.73** 1.45** 3.57**

0.11 0.13 0.49 0.85



** = P< 0.01.

rea during the dry season1.


nber and days after source establishment

3 3 = 27 4 = 33 5 = 47 6 = 56

MS MS MS MS

0.36 0.21 0.33 0.29

8.90** 12.92"* 20.80 14.91**

0.16 0.15 0.72 0.96

165.23** 176.98** 180.37"* 177.80'*

0.98* 1.10** 2.62** 2.52

S 2.75"* 1.71** 3.37* 4.97**

0.17 1.59** 4.28** 1.91

0.001 0.01 0.20 0.69

658.00** 704.61"* 713.62** 703.63"

S 0.69** 0.83** 1.43** 1.36"

0.08 0.12 0.33 0.51






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15





AUPC of the number of leaf-to-leaf (AUPCLLC) and leaf-
to-sheath (AUPCLSC) contacts
rIn-


AUPCLLC, DS


S.. .........-.
, -


r .................................** **"
0...........---- _--h---------B
------------


I
AUPCLSC, RS


I
AUPCLSC, DS


0 I I I I
0 40 80 12C

Leaf wetness rating
2.50-i


----- AUPCLLC, Rainy
Season ( RS)


....."...0........


AUPCLSC, RS


--..0--- AUPCLLC, Dry
Season (DS)
----A---- AUPCLSC, DS


----- Rainy Season
........ ........ Dry Season


N supply (kg N/ha)


Fig. 5. Effect of N supply on the contact frequency among hills (a) and the mean leaf
wetness rating of the plant canopy (b).


I
AUPCLLC, RS


2.25-


2.00-


1.75-


1.50-


1.25-


~---


.-"






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


15x15 15x20 20x20 25x25
Plant spacing (cm)
0.20
b

0.15 TO = permanently dry

T1 = wet for one night
0.10- LSDo.05
T2 = wet for two consecutive nights

0.05- T3 = wet for three consecutive nights

PW = permanently wet
0.00
TO T1 T2 T3 PW
Leaf wetness regime
0.20
C

0.15-


0.10-


0.05- LSD 0.05


0.00- i
0 60 90
Nitrogen supply (kg /ha)
Fig. 6. Relationship between plant spacing (a), leaf wetness regime (b), nitrogen supply
(c) and the inoculum efficiency in sheath blight.






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


Smne contact oT germ tuoe content may have a neg
surface. Jones (1986) effect on inoculum efficie
most fungal pathogens be compensated by the
ods to penetrate its host. contact frequency amonc


he results indicate that CARISSE, 0. and A. C.
t indirectly affects the rate 1992. Influence
vision via the capacity of wet periods, rel
)y to retain moisture and and temperature
ency, as shown by its carrots by Cerce
, with these two variables Phytopathology
effect on R2 of its removal






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15


GANGOPADHYAY, S. and N. K. LEANO, R. M., N. P. CASTILLA, D. B
CHAKRABARTI. 1982. Sheath LAPIS and S. SAVARY. 1993.
bliaht of rice. Rev. Plant Pathol. A simple methodoloav for


GOMEZ, K. A. and A. A. GOMEZ. 1984.
Statistical Procedures for
Agricultural Research. 2nd ed.
John Wiley and Sons, New York.
680 p.

HASIBA, T. and T. IJIRI. 1989.
Estimation of yield loss and
computerized forecasting system
(BLIGHTAS) for rice sheath blight
disease. Trop. Agric. Res. Ser.
22:163-173.

IRRI. 1991. World Rice Statistics 1990.
International Rice Research
Institute. Los BaFios, Philippines.
320 p.

JONES, A. L. 1986. Role of wet periods
in predicting foliar diseases. In:
Plant Disease Epidemiology.
Population Dynamics and
Management. Vol. 1. K. J.
Leonard and W. E. Fry (eds.). Vol.
1. pp. 87-100.

KANNAIYAN S. and N. N. PRASAD.
1979. Influence of nitrogen
fertilization on the development
of sheath blight. IRRN 4(3):14.

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.

KOZAKA, T. 1961. Ecological studies
on sheath blight of rice plant
caused by Pellicularia sasakii
(Shirai) S. Ito, and its chemical
control. Chugoku Agric. Res.
20:1-133.

LEANO, R. M. 1993. Ecological factors
associated with sheath blight
epidemiology and yield loss in
rice (Oryza sativa L.). M.S.
Thesis, University of the Philip-
pines, College, Laguna. 82 p.


(ShB) epidemiologic processes
under semicontrolled conditions.
IRRN 18 (3):42-43.

LEE, F. N. and M. C. RUSH. 1983. Rice
sheath blight: A major rice
disease. Plant Dis. 67:829-832.

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. 55-86.

MARCHETTI, M. A. and C. N. BOLLICH.
1991. Quantification of the
relationship between sheath
blight severity and yield loss in
rice. Plant Dis. 75:773-775.

MEW, T. W., N. G. FABELLAR, and F. A.
ELAZEGUI. 1980. Ecology of
the rice sheath blight pathogen:
saprophytic survival. IRRN 5
(4):16.

Ou, S. H. 1985. Rice Diseases, 2nd ed.
Commonwealth Mycological
Institute, Surrey, England. 379 p.

Ou, S. H., J. M. BANDONG and F. L.
NUQUE. 1973. International
Rice Research Conference. IRRI,
Los Baios, Philippines, April 23-
27, 1973.

PREMALATHA DATH, A. 1985. A better
criterion in rating the reaction of
rice cultivars against sheath
blight. Indian Phytopathol.
38:678-682.

RAJAN, C. P. D. 1986. Influence of
sheath blight (ShB) on agronomic
traits at different N levels. IRRN
11(1 ):23.






Philipp. Phytopathol. 1994, Vol. 30(1): 1-15









TRICHODERMA SPECIES FOR THI
STEM ROT (SCLERC


R. A. PAP


Associate Professor, Department of
Agriculture (ViSCA), Baybay, Leyte.

Key words: Biological control, Sclerotiu
species


ABS


Ten fungal isolates were t


....





i ~-r-~~~~-~- -~ -1 ----- -\-I---


of Trichoderma are reported antagonistic (i) First is the conventional dual culture
to S. rolfsii (Papavizas, 1985). Many method described by Bell et al. (1982).
successful control in the greenhouse Agar discs were obtained from 2-week
were reported (Chet, 1987) but very few old plated cultures of the different isolates
have developed simple, practical and and S. rolfsii using a sterile 5-mm cork
economical application technologies borer. Agar discs were paired individually
(Lapis, 1992). The large amount of with that of S. rolfsii on a previously
biocontrol agent applied in the field to plated PDA. Paired agar discs were
get adequate control remains the positioned 3 cm apart. One week later,
foremost obstacle to its adoption by the degree of antagonism against S rolfsii
farmers (Adams, 1990). This study was was measured using the 5-point scale
conducted to identify the most described by Bell et al., (1982) as follows:
antagonistic Trichoderma isolate; to find
the best substrate for conidial production, Scale 1 Antagonist overgrew
and to assess its biocontrol efficacy completely the patho-
using seed treatment and seedling dip gen and covered
methods of application, the entire medium;
Scale 2 Antagonist overgrew
at least 2/3 of the
MATERIALS AND METHODS medium surface;
Scale 3 Antagonist and S.
Isolation and Mass Production of S. rolfsii rolfsii each colonized
about 1/2 of the
Sclerotial bodies of S. rolfsii were medium surface;
collected from infected sweet pepper, Scale 4 S. rolfsii colonized at
surface-sterilized by dipping in 0.1% least 2/3 of the
sodium hypochlorite for 5 minutes, medium surface and
blotted dry, and aseptically planted to a appeared to with-
petri dish containing acidified potato stand encroachment
dextrose agar (PDA). One to two weeks by the antagonist;
after, agar block was obtained at the edge and
of mycelial colony, and transferred to PDA Scale 5 S. rolfsii completely
slants for pure culture maintenance. overgrew the antago-
nist and occupied
Mass inoculum production of the entire medium
S. rolfsii was done in rice grain-rice hull surface.
(1:3V/v) culture medium. Two-thirds of
each of 500-ml glass jars was filled with (ii) The second method of bioassay
the medium, added 125 ml water, and was developed to measure the relative
covered with an aluminum foil for capacity of isolates to lyse mycelia of S.
sterilization at 20 psi for 30 minutes. The rolfsii. Plates containing 25 ml PDA were
sterilized medium was seeded with agar seeded near the center with agar discs
block from 2-week old cultures of S. of S. rolfsii. Three days later, 5-mm agar
rolfsii. Two-week old rice grain-rice hull culture disc of each Trichoderma or
cultures of the pathogen were used as Gliocladium isolate was placed at the
the source of sclerotial bodies and center. Lysis zone as a result of
mycelia for all soil infestation in the mycoparasitism was measured 12 days
succeeding experiments. after seeding the candidate biocontrol
agent. Zones of lysed mycelia of the
Bioassay of Antagonism pathogen were regressed with i;me to
estimate the rate of lysis due to each
Nine local isolates of Trichoderma sp. antagonist. Each test isolate was
and one for Gliocladium sp. were replicated five times in a completely
bioassayed against S. rolfsii in the randomized design.
laboratory (Table 1). The bioassay of











pepper stem rot pathogen, Scd



ISOLATE ORIGIN


Trichoderman sp. (P-1) Matalom, Leyte
Trichoderma sp. (C-1) Punta, Baybay, Leyte
Trichoderma sp. (C-2) Malabang, Lanao del Sur
T. harzianum (Th-1) DPP, ViSCA, Baybay, Leyte
T. harzianum (Th-2) DPP ViSCA Baybay, Leyte
T. viride Matalom, Leyte
T. glaucum UP Los Banos, Laguna
T. aureoviride UP Los Banos, Laguna
T. pseudokoningii UP Los Baios, Laguna
Gliocladium sp. UP Los Baios, Laguna

Based on the five-point rating scale described by Bel
2 Average of 5 replicates taken 12 days after seeding tl
rolfsii.
SBased on regression analysis of zones of lysis against
old plated cultures of S. rolfsii).

The most antagonistic isolate
against S. rolfsii was used in the
succeeding experiments.

Substrates for Conidial Production

Trichoderma sp. (P-1) anc
Gliocladium sp. were found mosi
antagonistic to S. rolfsii. Trichoderma
isolate (P-1) was used because its
growth characteristics and conidia
longevity have recently been established
(Paningbatan et al., 1992).

A total of 14 agricultural
by-products and weeds were tested as
substrates for conidial production,
namely: corn grits (white corn),
corn bran, rice grains, rice bran, rice hulls,
rice straw, sweet potato tuber
(cv Red Wonder), sweet potato leaves,
taro tuber (cv Kalpao), taro leaves, ipil-
ipil leaves, Micania cordata leaves,
Gliricidia sepium leaves and sawdust.
Potato dextrose agar was used as
standard culture substrate. Leaves, rice
straw and tubers were chopped finely and
were used while still fresh. All the
substrates were sundried for three
consecutive days and dry weights
were used as basis for conidial


- -----rrV_ __W -- - --- r --


r degree of antagonism against the sweet
ium rolfsii.


YG LYSIS ZONE2 LYSIS RATE3 DAYS TO REACH
(mm) (mm/day) 10 mm LYSIS ZONE


O a 35.33 a 3.88 2.6
3 ab 30.67 b 3.42 2.9
0 ab 8.67 c 0.40 25.0
0 ab 3.00 d 0.52 19.2
O ab 0.00 d 0.00
O a 10.00 c 1.16 6.3
O a 31.00 b 3.16 3.2
0 ab 8.67 c 0.38 26.3
0 c 0.00 d 0.00
0 a 35.00 a 3.86 2.6

(1982) with 1 as the most antagonistic and 5 the least.
ididate antagonists on 1 week old plated PDA cultures of S.

(4, 7, 19, 12 days after seeding Trichoderma sp. on 3-day


production. Ten grams of each substrate
was put in 250-ml glass jars and 20-25
ml water was added. Glass jars were
covered with aluminum foil and sterilized
at 20 psi for 30 minutes. One ml of spore
suspension taken from 2-week old PDA
cultures of Trichoderma sp. was put
aseptically to each substrate-filled glass
jars. Temperatures varied from 20.5 C
to 35.4 C during the incubation period.
Conidial yield was counted using a
hemacytometer after two weeks of
incubation. Treatments were replicated
five times and arranged in a completely
randomized design.
SCREENHOUSE EXPERIMENTS

Biocontrol of the Disease in the Seedbed

Preparation of seedbeds. Fourteen-
cm clay pots were used as seedbed with
baked sandy loam soil. The soil was
infested using rice grain-ricehull 2-week
old cultures of S. rolfsii. The inoculum
source was mixed thoroughly with the
soil in a 1/7 ratio (v/v). Seedbed with
uninfested soil served as control.

Seed treatments. Five different
conidial concentrations (105, 101, 107,
108, 109 conidia/ml) were used as seed






rnuispp. rnytopainol. 1Y4, vol. .Ai>l):Io-L3


treatment preparations. One drop of
Tween 80 per 10 ml of suspension was
added as surfactant. Fifty seeds of
sweet pepper (All Season EW Select
variety) were soaked in 5 ml of each
suspension for 15 minutes and sown in
a seedbed. Maneb at 1.5 g a..i. per liter
served as fungicidal check. Untreated
control was also provided. The
temperature and relative humidity ranged
from 20.7-32.8 C and 66-98%, res-
pectively. Healthy seedlings were
counted one month after sowing.
Disease incidence was recorded one
month after sowing by counting rotten
seeds and wilted seedlings. Treatments
were replicated five times in a completely
randomized design. Seed viability was
taken to correct the percent disease
incidence.

Biocontrol of the Disease After
Transplanting

Preparation of planting materials.
Sweet pepper seeds were sown on
wooden seedbox (30 cm x 50 cm x 15
cm) with baked clay loam soil. The
seedbed was watered daily when
necessary. The seedlings were
transplanted one month after sowing.
The soil vas infested with S. rolfsii
following the same procedure described
in the previous experiment.

Seedling dip with conidial
suspension of Trichoderma. One hundred
seedlings (1-month old) were dipped in
30 ml conidial suspension for five
minutes using the same concentrations
used in the seed treatment experiment.
Ten seedlings were transplanted per pot.
Wilted seedlings were counted daily for
two weeks (disease severity after two
weeks remained the same), and the level
of disease severity was computed based
on the cumulative number of rotten
seedlings. Maneb at 1.5 g a.i. per liter
was used as fungicidal check. Treatment
with Trichoderma (10' per ml) alone was
included to determine any adverse effect
on the sweet pepper seedlings.


HELD PLOT EXPERIMENT

The efficacy of Trichoderma sp. was
further evaluated in the field using the
seedling dip method and the most
effective concentration of 109 conidia/
ml.

Plot Preparation and Culture of Test
Plants

Sandy loam field plots
(1.0 x 2.5 m) were cultivated, pulverized,
furrowed at 0.50 m apart, and
transplanted with seedlings at 40 cm
apart. Test plants were watered
whenever necessary, weeded three
weeks after transplanting and sprayed
with malathion after one month at
recommended rate to control aphid
infestations. Each seedling was basally
applied with 3 grams of complete fertilizer
(12-12-12).

Soil Infestation with S. rolfsii

Plots were artificially infested with
400 ml of 2-week old cultures of S rolfsii.
The inoculum source was broadcasted
evenly along furrows and covered with
thin soil before transplanting. Only sterile
culture medium was added to uninfested
plots to determine the background
infection.

Seedling Dip Treatments

One-month old seedlings after
sowing were gently uprooted and then
treated with the following:
(1) 109 conidia iml of Trichoderma
(dip) + S. rolfsii

(2) 109 conidia/ml of Trichoderma
(dip) alone

(3) Maneb at 1.5 g a.i./liter (dip) +
S. rolfsii

(4) Maneb at 1.5 g a.i./liter (drench)
+ S. rolfsii

(5) S. rolfsii alone

(6) Untreated control










A ratio of 100 seedlings/30 ml of
suspension was enough to moisten the
base of the stem and roots of the
seedlings for a 5-minute dip. Maneb at
1.5 g a.i./liter was used as seedling dip
and drench fungicide. Drenching with
the fungicide has been recommended
(Sherf and MacNab, 1986) and there-
fore served as standard check. Each
sweet pepper transplant received 20
ml of maneb as drench. Three months
after transplanting, diseased plants
were counted. Cumulative number of
diseased plants and weight of
harvested marketable fruits were used
to compare treatments. The treatments
were replicated three times, and
arranged in a randomized complete
block design. Weather data were
obtained from the ViSCA PAG-ASA
Weather Station.

RESULTS AND DISCUSSION

Most Antagonistic Isolates

Eight out of nine Trichoderma
isolates were antagonistic against
S. rolfsii based on the bioassay of Bell et
al. (1982). However, based on the lytic
ability, only Trichoderma sp. (P-1) and
Gliocladium sp. were regarded as most
antagonistic. Isolates of T harzianum
(Th-2) and T pseudokoningii did not lyse
the mycelia of S. rolfsii; sclerotial bodies
were formed. The method of measuring
the extent of lysed mycelia quantifies the
level of antagonism better than the dual
culture bioassay method. This confirms
earlier finding involving four isolates of
three different species of Trichoderma
tested against S. rolfsii isolated from
mungbean (Paningbatan et al., 1992).

Trichoderma sp. (P-1) and
Gliocladium sp. can lyse mycelia of the
pathogen at the rate of 3.88 and 3.86
mm/day, respectively (Table 1). Isolate
P-1 lysed mycelia of S rolfsiithree times
faster than T viride, the species found
to be most antagonistic to S. rolfsii of
mungbean (Paningbatan et al., 1992). It
wou'd take an average of 2.6 days to
lyse a 10-mm zone while T viride would
take 6.3 d6ys. Trichoderma sp. (P-1) was
used in subsequent experiments.


Philipp. Phytopathol. 1994, Vol. 30(1):16-25


Table 2. Conidial yields of Trichoderma
sp. grown for two weeks on the
different substrates based on
dry weights.


SUBSTRATE


Corn grits (wnite corn)
Corn bran (white corn)
Rice grains
Rice bran
Rice hull
Rice straw
Sweet potato tuber
Sweet potato leaves
Taro tuber
Taro leaves
Ipil-ipil leaves
Micania cordata leaves
Gliricidia sepium leaves
Sawdust
Potato dextrose agar2


CONIDIA (x 109)/g1


0.95 c
0.40 f
0.63 e
0.24 g
0.01 j
0.02 i
0.80 d
0.00 j
4.00 b
0.01 j
0.20 h
0.01 j
0.03 j
0.00 j
20.39 a


SAverage of 2 trials with 5 replications. Means
with the same letter are not significantly different
at P = 0.05 by DMRT
2 Based on 8.667 x 109 conidia per 25-mi plated
dextrose agar (PDA) prepared from 17 g of
commercial dehydrated PDA (Difco Laboratories,
Michigan, USA) per liter of water.


Substrate for Conidial Production of
Trichoderma sp.

Solid media for experimental
production of Trichoderma inoculum
include bark pellets, sawdust, wheat
bran, barley grains, and pellet
formulations (Papavizas, 1985). In the
Philippines, rice bran is a recommended
substrate because of its availability
(Castro, 1990; Fernandez, 1988). Lately,
rice bran is mixed with cassava starch
(1:3 w/w) as a binding agent to pelletize
the Trichoderma inoculum (Cumagun,
1992).

Taro tuber, corn grits, sweet potato
tuber, rice grains, and corn bran were
found to be better substrates for conidial






Philipp. Phytopathol. 1994, Vol. 30(1):16-25


Table 3. Comparative conidia (x 109)/ peso ratios of the different dried substrates
based on their existing market prices.


CONIDIA/ COST/KG (f~ CONIDIA/
SUBSTRATE GRAM PESO
(x 109) Material Processing 4 Total RATIO

Rice bran 0.244 5.00 0 5.00 48.8
Rice grain 0.633 5.00 0 5.00 127.0
Corn bran 0.400 5.00 0 5.00 80.0
Corn girts 0.956 9.00 0 9.00 106.2
Sweet potato tuber 0.800 23.32' 10.01 33.34 24.0
Taro tuber 4.005 18.502 7.87 26.37 151.9
Potato dextrose agar 20.392 4894.183 0 4894.18 4.2

' Based on 0.300 dry/fresh weight ratio at P7.00/kg of fresh tuber.
2 Based on 0.378 dry/fresh weight ratio at P7.00/kg fresh tuber.
3 Present cost P2,200/1b
4 Processing cost was computed from 0.267 manhour/kg fresh weight at P11.36 manhour labor on
P90.90/manday.


Table 4. Effects of conidial concentration of Trichoderma sp. applied as seed
treatment on stem rot incidence of sweet pepper one month after sowing.1


% DISEASE BIOCONTROL2
TREATMENT INCIDENCE% EFFICACY (%)


105 conidia/ml +
106 conidia/ml +
107 conidia/ml +
108 conidia/ml +
109 conidia/ml +
Maneb + '
Trichoderma alone
Sr alone
Untreated check


15.75 b
16.67 b
14.29 bc
12.38 c
4.76 d
15.75 b
2.33 e
24.76 a
1.43 e


36.39
32.67
42.29
50.00
80.78
(36.39)3


' Average of two trials inside the screenhouse with 5 replications. In a column, means with the same
Letter are not significantly different at P = 0.05 by DMRT. Means were corrected based on 84%
viability of seeds.
2 Computed by the formula, BE = (DSa-DT)/DSa* 100; where: BE= biological efficacy, DSa = disease
incidence of Sr alone and DT = disease incidence of treatment.
3 Efficacy of the fungicide check.


production of Trichoderma sp. than rice
bran (Table 2). Among these substrates,
taro tuber appeared the best for conidial
production (4 x 109/g), 16.4 times better
than rice bran (2.4 x 108/g). Conidial
yield of Trichoderma sp. on PDA remained
highest at 2 x 1010/g. Based on the
conidia/peso ratio, however, taro tuber,
rice grains, corn grits, corn bran, rice bran


and sweet potato tuber are cheaper than
using PDA (Table 3). For each peso
invested, taro tuber (1.5 x 1011 conidia/
peso) supported 36 times the conidia
produced on PDA and three times the
conidia produced on rice bran. Shifting
to taro tubers would reduce our
dependents on the imported PDA as a
substrate for conidial production.










Table 5. Effect of conidial concentration
stem rot incidence of sweet p
screenhouse.'


DI1
TREATMENT INCIDI

105 conidia/ml + Sr 8
106 conidia/ml + Sr
107 conidia/ml + Sr
108 conidia/ml + Sr ,
109 conidia/ml + Sr
Maneb + Sr
Sr alone
Trichoderma alone
109 conidia/ml
1 Average of two trials with 5 replications. Means %
significantly different at P = 0.05 by DMRT.
2 Computed by the formula, BE = (DSa-DT) DSa 1
efficacy, DSa = disease incidence of Sr alone and
treatment.
3 Efficacy of the fungicide check.

Seed Treatment with Trichoderma sp. in
Seedbed

Seed treatment with 109 conidia/ml
of Trichoderma sp. reduced disease
severity from 24.76% to 4.76% or a
biocontrol efficacy of 80.78% (Table 4).
The efficiency was 55% better than the
protection by maneb. Seed treatments
with 107 and 108 conidia/ml gave
comparable protection with that of
maneb. Disease severity in seedbeds
treated with Trichoderma sp. alone did
not vary significant compared with
untreated control indicating that it is
not deleterious to the seeds and
seedlings. Similarly, T. viride was
found to have no adverse affect on
treated mungbean seeds (Paningbatan
et al., 1992). Seed treatment with
Trichoderma sp. can mean freedom
from disinfesting seedbed soil.


Seedling Dip to Protect Sweet
Pepper After Transplanting

Seedling dip using conidial
suspension of Trichoderma sp. was
evaluated as a practical method of
annlvinn the antannnikt ppdilinns


Philipp. Phytopathol. 1994, Vol. 30(1):16-25


ichoderma sp. applied as seedling dip on
,r two weeks after transplanting in the



;E BIOCONTROL2
E (%) EFFICACY (%)

10.64
19.15
21.28
46.81
76.50
(23.40)3




ie same letter are not

there: ".' = biological
= disease incidence of



dipped in 109 conidia/ml for 5 minutes
before transplanting into infested soil
with 94% disease potential gave the
lowest (22%) disease incidence (Table
5). Biocontrol efficacy was 76.50%,
3.3 times better than the maneb
(23.40%) (Table 5). Trichoderma
conidial suspension alone did not harm
the sweet pepper seedlings when used
as seedling dip.
Field Evaluation of Seedling Dip

Field plots used in this experiment
have no background incidence of stem
rot while artificially infested plots have
72.23% disease incidence (Table 6).
The seedling dip method using 109
conidia/ml of Trichoderma sp. provided
73.04% biocontrol efficacy relative to
the untreated (Sr alone) plots. The
results confirmed the effectiveness of
the seedling dip method in pot
experiment. Maneb formulation when
used as seedling dip could not give
significant protection from stem rot
disease in the field but drenching every
transplanted seedling had comparable
effect compared to seedling dip with
Trichoderma sp.
The average fruit yield in
unprotected lots (Sr alonpl with 77 93 0/






?hilipp. Phytopathol. 1994, Vol. 30(1):16-25


Fable 6. Incidence of sweet pepper stem rot in the field due to infection by Sclerotium
rolfsii (Sr) after 45 days as affected by dipping in 109/ml conidial suspension
of Trichoderma sp. prior to transplanting.

DISEASE BIOCONTROL FRUIT YIELD
TREATMENT INCIDENCE % EFFICACY (%) (t/ha)

rrichoderma (Dip) + Sr 19.47 b 73.04 7.51 b
Mlaneb (Dip) + Sr 66.57 a (7.84)1 2.39 c
Maneb (Drench) + Sr 27.57 b (61.83) 6.48 b
Sr alone 72.23 a 1.89 c
Trichoderma alone 0 c 9 95 a
Jntreated Control 0 c 9.64 a

Efficacy of the fungicide check.


Fable 7. Manhour and bioprotectant formulation requirement of the different methods
of controlling stem rot compared to fungicide application.


METHOD MANHOURS/HA LITER/HA
Seed Treatment (foor Seedbed)1
Trichoderma sp. 0.75 0.25
Maneb 0.75 0.25

Seedling Dip (Before Transplanting)2
Trichoderma sp. 10.74 15.00
Maneb 10.74 15.00

)rench, Recommended for'Maneb3 140.97 1000.00

About 120-gram seeds are needed per hectare with 50,000 plants.
SLiquid suspension number of seedlings ratio of (30 ml:100 seedlings).
SAt the rate of 20 ml per transplant.


disease incidence was 1.89 tons/ha For instance, Strashnow et al. (1985)
compared with the 7.51 and 6.48 tons/ reported that a formulation of






Philippi Phi Ipathol. 1994, Vol. 30(1):16-2'


Seed treatment and seedling dip BACKMAN, P.A. and RODRIGUEZ-
with conidial preparations of Trichoderma KABANA. 1975. A system tor
sp. are practical and easy-to-follow the growth and delivery of
methods for biocontrol of sweet pepper biological control agents to the
stem rot in the seedbed and in the field. soil. PhLyopathology 65: 819-821.
Little amount of conidial suspension is
needed to treat seeds and seedlings per BELL, D.K., H.D. WELLS and C.R.
hectare (Table 7). It is also less time MARKMAN. 1982. In vitro
consuming, requiring only 7.6% of the antagonism of Trichoderma species
time spent in drenching maneb, the against six fungal plant
recommended fungicide to control stem pathogens. Phytopathology 72:
rot pathogen. 379-382.


ichoderma spp.,
environmental pol
rays used to c,
afspot of peanut
development of strz


Trichodei


piuauuion oUT ungic


(Panir


rmers.


TERATURE CITED


)AMS, P.A. 1990. The
mycoparasites fo


ATIENZA, M. 1927. Sclerotium disease
of tomato and Deooer. Philion.


of Trih
biologi


,STRO, I


blight.
Lagun;

IET, I. 19
to plal
and S(

4D, Y., I. (
Trichc
biocon
Sclerc
Rhizoc
70: 11

RNANDEZ
Tricho
biocon
rolfsii
peanu
M.S.
Leyte.

"ALES, L.i
fungic
-I-


a means
against she
loratory stL
3aios, Lagu

Iss product
and evaluate
tion rates i
ol of she
J.P. Los Bai


/e approach
control. Wi
p. 137-1(

.ATAN. 191
rzianum,
ective agai
Ifsii a
hytopatholc


. Efficacy
eoviride
)r Scleroti,
3 stem rot
ypogaea I
CA, Bayb


Side effect
secticides


0-,..l-.-.. I -..a- Al A -


A ,


,, .. o _









4APAVIZAS, G.C. 1985. Trichoderma
and Gliocladium: biology, ecology
and potential for biocontrol. Ann.
Rev. Phytopathol. 23: 23-54.


HERF, A.F. and A.A. MACNAB. 1986.
Vegetable diseases and their
control. John Wiley and Sons, Inc.,
N.Y. 728 p.

iTRASHNOW, Y., Y. ELAD, A. SWAP and
I. CHET. 1985. Integrated control
of Rhizoctonia solani by methyl
bromide and Trichoderma
harzianum. Plant Pathol. 34: 146-
151.


'hilipp. Phytopathol. 1994, Vol. 30(1):16-25


.APIS, D.B. 1992. The prospect of
mycoparasites for the biological
control of plant pathogens in the
Philippines. UPLB Foundation
Professorial Lecture, 7 Dec. 1992,
UP Los Baios, College, Laguna.

'ANINGBATAN, R.A., M.G. MARAMARA
and A.C. VILLAR. 1992.
Seedcoating with Trichoderma
viride on biocontrol of Sclerotium
wilt in mungbean. Paper presented
during the Regional R & D Review
and Planning Workshop on June
2-4, 1992. UEP, Catarman,
Northern Samar.






Philipp. Phytopathol. 1994, Vol. 30(1):26-34


BUGTOK DISEASE OF COOKING BANANAS: I. ETIOLOGY AND
DIAGNOSTIC SYMPTOMS

CONCEPCION E. SOGUILON, LYDIA V. MAGNAYE and MARINA P. NATURAL


A portion of the MS thesis of the senior author submitted to the University of the
Philippines at Los Baios (UPLB), College, Laguna.

Supported jointly by IDRC-PCARRD-UPLB-BPI Banana (Philippines) Project.

Respectively, Senior Agriculturist, Supervising Agriculturist; Davao National Crop
Research and Development Center, Bago Oshiro, Davao City; and Associate Professor,
Department of Plant Pathology, UPLB, College, Laguna.

Key words: Banana, bugtok disease, etiology, Pseudomonas solanacearum,
symptomatology

ABSTRACTS

Bugtok disease of cooking bananas was conclusively found
to be caused by Pseudomonas solanacearum E.F. Smith. Results
of cultural, morphological, biochemical and pathogenicity tests
revealed striking similarities between the bugtok bacterial isolates
and P solanacearum from other hosts. Bacterial cells were
Gram negative, rod-shaped and measured about 0.5 to 1 um by
1.5 to 4 um. Colonies on TZCA were irregular, convex and fluidal
with or without pink centers. Artificially inoculated tomato
seedlings and banana plantlets wilted. Bugtok symptoms on
fruits were reproduced in artificially inoculated inflorescence.

Two distinct symptom types were observed from field
infected fruits; namely, the reddish brown and the black
discoloration of the fruit pulp. In either case, the fruit pulp was
hard especially in areas that were discolored. Discoloration
was intense in the core of the fruit. Vascular tissues in the
pedicels, fruits and stems, and leaf sheaths were also discolored
becoming less intense in parts that are far from the fruits.

If the male inflorescence (heart) was detached from the
bunch, a bugtok infected plants can not be differentiated from a
healthy plant. With the male bud still attached, bugtok infected
plants can be recognized because the bracts fail to dehisce,
giving it a loose and dry appearance. Healthy male inflorescence
appear compact and succulent as the older bracts dehisce
successively at maturity.

INTRODUCTION augment the income of farmers and to
supplement their nutritional requirements.
Banana is considered as one of the The common dessert varieties are Lakatan,
most important fruit crops in the Seiorita, and Latundan and the cooking
Philippines. It ranks number one in terms varieties are Saba and Cardaba. The
of export revenues and area planted corporate farms that cater to the strict
(Valmayor, 1990). Bananas are grown all requirements of the export market grow
over the country as a backyard crop to only a single variety, usually Giant









rendish, in large tracts of land. These
stations are found in the four provinces
Mindanao, namely, Davao del Norte,
iao del Sur, South Cotabato, and
amiss Oriental.

Sint early 1950s, backyard growers
n Mindanao have been bothered by a
ease locally known as bugtok. Roperos
)65) first reported this disorder and
Digested that it was caused by a
:terium. In 1969, Zehr and Davide
scribed a similar disease which was
led tibagnol by the local folks in
naguete City, Negros Oriental. They
o isolated and pathogenicity-tested a
:terium. Plant pathologists from the
iana industry have noted that bugtok
nptoms are similar to the symptoms
banana fruits infected with
oolanacearum which causes the moko
ease or bacterial wilt of bananas.

Bugtok has now become a
lespread disease of cooking Banana
ba and Cardaba varieties) in Mindanao.
as caused the virtual abandonment of
all-and medium-sized farms planted to
se varieties. There is, therefore, a felt
id to study the disease in more detail
>rder to formulate measures to manage
disease.

This paper aims to i) provide
dences that bugtok is caused by
solanacearum, and ii) describe
gnostic symptoms of the disease.


MATERIALS AND METHODS

nptom Description

In order to identify bugtok infected
nts without dissection, plants from
turally infected fields at Calinan,
iugao, Tagakpan and Biao, Matina,
vao were closely observed. The external
)earance of plants were compared with
ilthy plants.

Banana plants suspected to be
acted with bugtok were dissected. If
its showed intense discoloration, the
ter parts like peduncle, fruit stem,
_1 ^ _ _ ^ J __ _ _


27


e dissected. The extent of browning
iese parts were noted.

ation of the Pathogen

Method I. Five mm2 sections from
it parts like the fruit pulp, peel, fruit
ich and fruit stem showing
:oloration were immersed in sterile
tilled water. A loopful of the
pension was streaked dn previously
ed tetrazolium chloride agar (TZCA).
es were incubated and observed for
trial growth.

Method II. Cut ends of the fruit
ch were washed with detergent and
water, and disinfected with 50%
nmercial bleach (5% sodium
ochlorite). The exposed surface was
-off using a flame-sterilized knife, dried
wrapped with moistened polyethylene
to induce bacterial exudates. Fruit
uncles were incubated for 3 to 14
's or until bacterial exudates were
n from the cut ends. A loopful of
exudate was suspended in five ml
rile distilled water for 10 to 20
iutes, thoroughly mixed and a
pful was streaked on the surface of
previouslyy plated and dried TZCA.
,se were incubated for three to five
's at room temperature. Single
onies were purified and maintained
slants. Stock cultures were
intained in sterile distilled water.
hogenicity Tests

Tomato seedlings Three week old
iato seedlings (cv. VC 11) were
culated by stem injection or root
going. Approximately 0.1 ml of 108
any forming units (CFU) per ml was
cted into the base of each test plant,
;eedlings were uprooted and dipped in
trial suspension for 30 seconds, then
isplanted. Control plants were injected
h or their roots dipped in sterile distilled
ter.

Banana plantlets Tissue-cultured
iana cv. Cardaba and field-collected
kers of banana cv. Cavendish were
cted with 10 ml bacterial suspension
Itaining about 108 CFU per ml into the






28


Control plants were injected with 10 rr
sterile distilled water.

Banana inflorescence Three newly
emerged inflorescence of banana c\
Abuhon were first punctured with a sharia
force. Then 10 ml of bacterial suspension
were injected to the punctured inflores
cence. The inoculated inflorescence wa
bagged with cheesecloth to exclud
insects. Inflorescence injected with steril
distilled water served as control.

Characterization and Identification
of the Pathogen

Morphological characters of th
isolated bacteria were observed usin'
stained and unstained preparations
Gram-stained cells were measured
Cultural characteristics of the isolate,
bacteria were noted after growing oi
TZCA. Physiological characteristics wer
also observed following the test
recommended by Hildebrand et al. (1 988
for non-fluorescent pseudomonads
Characteristics of the isolated bacteria
were compared with published character
of R solanacearum.

After it was established that th
bugtok bacterium was P solanacearum
biovar classification according to Haywan
li nr n A\ -l I- -


intense in fruit pedicels and fruit bunct
becoming less intense in parts far fror
the fruits.

External diagnostic symptoms c
bugtok were observed only in plant
where the male buds 'heart) were lel
undetached f.on; the bunch. The outer
bracts covering the male bud were loos<
dry and undehisced (Fig. 2). In health
male buds, the old bracts normally fal
off, thus buds appear compact an
succulent until harvest. Table
confirms the observation that plant
bearing dry, loose and undehisced bract
in the male inflorescence are infected. Th
length of the male axis from the fru
bunch to the male bud had no influence
in the incidence of bugtok. Fifty there
plants from four locations in Dava
showing symptoms in the mal
inflorescence were positive for bugtol
Such symptom can, thus, be used t
diagnose bugtok infected plants.

In the Philippines, farmers usually
harvest the male inflorescence after th
fruits have set and used as vegetable<
When this hannonp ;tanrinn hiintn






29


Fig. 1. Typical symptom of bugtok in unripe fruits
showing intense discoloration of the pulp
especially at the core (B) compared with healthy
fruits (A).














,








Fig. 2. External diagnostic symptoms of bugtok can
be seen only in plants with undetached male
inflorescence (heart): In diseased plants, the
old bracts do not dehisce (A) giving a dry,
loose appearance; healthy bunch (B) shows a
clean, long male axis bearing the male
inflorescence.






Philipp. Phytopathol. 1994, Vol. 30(1):26-34


inoculated to Cavendish suckers caused
wilting six days after stem injection.
Isolation Control plants did not wilt.


Initial attempts to isolate the
pathogen by standard isolation techniques
were unsuccessful. Parts of infected
fruits, peduncle or fruit stem immersed in
water did not show bacterial ooze.
Streaking a loopful of suspension in plated
TZCA failed to recover bacteria.

Bacterial pure cultures were obtained
only when infected male axis and
detached male inflorescences were
incubated for 3 to 14 days or until
exudates from the cut ends were visible
(Fig. 3a). Later, it was found that bacterial
isolation was also possible by directly
streaking on agar medium the milky
substance oozing from detached bracts
of the male inflorescence (Fig. 3b).

Colonies that developed on TZCA
after 48 to 72 hours incubation at
about 28 to 30 C were one to four mm
in diameter, irregular, convex and
fluidal with or without pink formazan
(Fig. 4) typical of P. solanacearum.

Pathogenicity Test

Typical bugtok symptoms were
reproduced in fruits after inoculating the
newly emerged inflorescence with bacteria
(Fig. 1). Fruits were discolored and
vascular browning was evident in fruit
stems and peduncles. The bracts covering
the male inflorescence did not dehisce but
were loose and dry.

All the 18 isolates inoculated to
tomato seedlings either by stem injection
or by root dip resulted in wilting.
Incubation period for stem injected tomato
seedlings was as early as one day which
continued to progress until the seedlings
died. Tomato plants inoculated by root
dipping wilted after 2 to 3 days (Table 2).

Six selected bugtok isolates
inoculated by injection to Cardaba
plantlets also resulted in wilting.
Incubation period was longer since wilting
was observed from 6 to 12 days after
inoculation. Two selected isolates


Bacteria were reisolated from
artificially inoculated plants. The colony
characteristics were similar to the original
isolates.

Pathogen Characterization
The 18 bugtok isolates were
aerobic Gram-negative rods, about
0.5 pm to 1.0 um by 1.5 to 4.0 pm. All
isolates were catalase positive,
produced hydrogen sulfide from
cysteine and able to elicit a
hypersensitive response in tobacco cv.
White Burley. All isolates failed to
liquefy gelatin after 14 days, did not
produce levan from sucrose and did not
form melanin pigment in 0.1% L-
tyrosine medium (Table 3).
Results of these tests indicate that
the bacterium belongs to the genus
Pseudomonas. Pathogenicity tests in
tomato and banana plantlets caused the
plants to wilt, thus we conclude that the
bacterium is P solanacearum, Race 2. All
isolates failed to utilize the disaccharides
lactose, maltose, and cellobiose, and the
sugar alcohols e.g. mannitol, sorbitol and
dulcitol, thus considered biovar 1 based
on Hayward's classification.

To confirm the identify, cultures were
provided to various laboratories for
molecular analysis [Seal et al., 1993;
Alvarez et al., 1993; Eden-Green, 1993;
Ilagan and Raymundo, 1993 (personal
communication)]. They have all confirmed
the similarity of the bugtok bacterium to
P solanacearum.


DISCUSSION

Bugtok disease shows similarities in
fruit symptoms to the blood disease of
bananas in Java, Indonesia (Eden-Green
and Satratmadja, 1990) and moko
disease. Bugtok affected plants, however,
appear normal externally and do not show
leaf discoloration nor wilting. Bugtok is
very common in farmers' backyard where
cultivars Saba and Cardaba are planted.
Moko is emerging as a problem in






Philipp. Phytopathol. 1994, Vol. 30(1):26-34


Fig. 3. Bacterial exudates from infected male axis (a) incubated for
two weeks and from freshly opened, infected male inflorescence
(b).


Fig. 4. Colonies of bacteria growing on tetrazolium chloride agar 72
hours after streaking on plates.






32 Philipp. Phytopathol. 1994, Vol. 30(1):26-34


Table 2. Incubation period (days) of the different bacterial isolates from bugtok-infected
cooking bananas inoculated on tomato and banana.


TOMATO CARDABA FIELD-GROWN
ISOLATE SEEDLINGS PLANTLETS CAVENDISH
NO. ---------------------------------------- --------------------- ---------------------
Root dip Injection Injection Injection


6-17

12-27
_1/

7-14

6-10


11-14


6-23


6-27


7-19


" Symbol = no data









Bible 3. Results of the diagnostic tests
conducted on the bugtok
isolates compared to the moko
isolates.

Diagnostic Bugtok Moko
Test Isolates Isolates

'am staining
elanin formation slight
,S production + +
italase reaction + +
van formation
elatin liquefaction + +
ist for 02 requirement aerobic aerobic
arch hydrolysis
veen 80 hydrolysis + +
oxidation of hexose alcohols

lactose
maltose
cellobiose
Kidation cf dissacharides
mannitol
sorbitol
dulcitol
ithogenicity test

tomato seedlings + +

tissue-culture derived
Cardaba plantlets + +
Cardaba shoots
hypersensitive reaction
on tobacco +


commercial plantations where the cultivar
iant Cavendish is grown in monoculture.
iant Cavendish is grown in monoculture.
here is a possibility that the bugtok
:rains became highly specialized in
cooking bananas. A still unknown vector
probably disseminates the bacterium via
ie inflorescence, thus the bugtok
/mptoms. Saba and Cardaba are very
ill cultirvars. Perhaps, colonization of
ie lower parts of the plant takes a long
me, thus, wilting does not occur and
ie maiden suckers or followers remain
isease-free if planted in isolation.
whether the moko isolates in the
hilippines originated from the bugtok
olates is a matter of speculation.
ugtok is an old disease of cooking
fAnrtn rE r-n r+trIA ,


nerica. With these findings, ideas on
e origin of moko in the Philippines will
obably change. Further studies are
iing conducted to determine the
lationship between bugtok and moko
rains.


TERATURE CITED

_VAREZ, A.M., J. BERESTECKY, J.I.
STILES, S.A. FERREIRA and A.A.
BENEDITC. 1993. Serological and
molecular approaches to
identification of Pseudomonas
solanacearum strains from Heliconia.
pp. 62-69, In: Hartman, G.L. and
A.C. Hayward, (eds.). Bacterial
wilt. ACIAR Proceedings No. 45,
381 p.
JDDENHAGEN, I.W. 1986. Bacterial
wilt revisited. In: Persley, G.J.
(ed.). Bacterial wilt disease in Asia
and the South Pacific, ACIAR.
Proceedings No. 13: pp. 126-139.

)EN-GREEN, S.J., and H.
SASTRAATMADJA. 1990. Blood
disease of banana present in Java.
FAO Plant Protection Bulletin 30:49-
50.

AYWARD, A.C. 1964. Characteristics
of Pseudomonas solanacearum. J.
Appl. Bacteriol. 27:265-277.

ILDEBRAND, D.C., M.N. SCHROTCH
and D.C. SANDS. 1988.
Pseudomonas. pp. 60-80 In:
Schaad, N.W. (ed.). Laboratory
Guide for Identification of Plant
Pathogenic Bacteria. APS Press, St.
Paul Minnesota, 164 p.

3PEROS, N.I. 1965. Note on the occur-
rence of a new disease on cooking
banana in the Philippines. Coffee
and Cacao Journal 8:135-136.

EAL, S., L. JACKSON and M. DANIELS.
1993. Development of molecular
diagnostic techniques for detection
Pseudomonas solanacearum and
identification of subgroups within
the species. pp. 97-105. In:










(eds.). Bacterial Wilt. ACIAR
Proceeding No. 45, 381 p.

VALMAYOR, R.V. 1990. Bananas and
Plantains in the Philippines. In:
Valmayor, V., (ed.). Banana anc
plantain R & D in Asia and the
Pacific: Proceedings of a Regiona
Consultation on Banana anc
Plantation R & D Networking
Manila and Davao, 20-24
November 1989. Montpelliel
France: INIBAP. 189 p.
ZEHR, and R.G. DAVIDE. 1969. An
investigation of the cause of the
"Tapurok" disease of cooking
bananas in Negros Oriental.
Philip. Phytopathol. 5:1-5.






Philipp. Phytopathol. 1994, Vol. 30(1):35-43


CONTROL OF ROTYLENCHU,
(GOSSYPIUM HIRSUTUI
MANURING, MULCHIN


L.Q. MARASIGAN, A. DEL

Portions of the BS and MS theses of

Respectively, former BS and MS st
Pathology, University of the Philippines a

Key words: cotton, intercropping, m;


ABS

On potted cotton, the efi
of Leucaena leucocephala, G
and Asystacia gangetica and
rates of 6.3 and 12.6 g/pot
investigated. All the treatr
population. E. amaura at high
phenamiphos applied at the rn
high rates of G. sepium, low r,
of rice straw exerted co
phenamiphos. Least reduction
low rate of mulching with rice
Leucaena leaves.

In the field experiment
manuring at the high rate (5.C
was demonstrated. Manuring
and sawdust and low (2.5 ton
and intercropping with con
population in cotton. Phenamil
provided the best nematode
cotton.

Although the treatment
nematodes, they failed to incre
intercrop may be a better co
while organic amendments and
deficiency due to the actions o


INTRODUCTION

A survey conducted in the cotton-
growing areas of the Philippines
hnwvarl tho nrv/alant neenrintinn ^-f


i RENIFORMIS ON COTTON
.) THROUGH ORGANIC
\ND INTERCROPPING


RUZ and M.B. CASTILLO

first two authors.

ts and Professor, Department of Plant
Baiios, UPLB, College, Laguna.

ng, mulching, R. reniformis.


CT

tity of manuring with leaves
dia sepium, Eulalia amaura
:hing with rice straw at the
.reniformis population was
s reduced the nematode
ite was more effective than
If 5 kg a.i./ha. The low and
if A. gangetica and high rate
-able effects to that of
population was provided by
aw and soil incorporation of


ie nematicidal effects of
s/ha) with G. sepium leaves
ie high rate with cow dung
) and high rates of coir dust
so reduced R. reniformis
at the rate of 7.5 kg a.i./ha
itrol and yield increase in


educed the population of
the yield of cotton. Corn, as
titor for nutrients and light
ching may result to nitrogen
:omposers.


the reniform nematode, Rotylenchulus
reniformis with cotton (Castillo, 1977).
-igh populations of the nematode were
frequently associated with plant






36 Philipp. Phytopathol. 1994, Vol. 30(1):35-43


be the most important pest of cotton in This study was designed to
the country. Significant growth and confirm the suppressive effect of
yield reductions were observed in a amending soil with plant leaves and
subsequent pot experiments using cow dung, mulching with rice straw
infested soil as source of nematode and intercropping with corn on
inoculum (Castillo and Sevilla, 1977). populations of R. reniformis on potted
and field cotton. It was also aimed to
Amending soil with plant leaves compare the effectiveness of the above
for nematode control was first treatments with that of the nematicide-
investigated by Linford et al. (1938). insecticide phenamiphos.
They showed that addition of
pineapple leaves to the soil controlled
the root-knot nematodes on cowpea. MATERIALS AND METHODS
Later, Duddington (1957) reported that
infection of oats by cereal root Pot experiment
nematodes was reduced when
chopped cabbage leaves were Pooled nematode-infested soil
incorporated into infested soil prior to collected from the cotton-growing
planting. Singh et al. (1967) and Singh areas of Pangasinan, Nueva Ecija,
and Sitaramaiah (1971) observed Ilocos Sur and Ilocos Norte were mixed
reduction in Meloidogyne javanica with baked soil in three 1 x 1 m
population on okra and tomato after cement microplots and grown to
sawdust application, cotton (Gossypium hirsutum L.) for one
cropping period. The generic
Van der Laan (1956) observed a composition and mean density of the
slow rate of the golden cyst nematode nematodes were determined through
development when farmyard manure the routine extraction procedure
was added to soil. Calinga (1979) (combination of sieving, using the 45
indicated the potentials of soil pm pore sieve as the terminal sieve
incorporation of animal dungs, namely, and Baermann funnel techniques) and
cow dung, horse dung, carabao Hung bioassay described by Castillo et al.
and pig aung for R. reniformis co.. rol. (1977). Based on nematode recoveries
Castillo (1985) cited chicken dung as from ten 300 cm3 randomly obtained
an efficient soil amendment for soil samples, the mean populations of
nematode control and in increasing nematodes were 581.5, 33.0, 18.5,
crop yield. This was subsequently and 9.5 for R. reniformis,
confirmed by Duhaylongsod and Helicotylenchus, Hoplolaimus, and
Castillo (1989), Duque (1988) and Rotylenchus, respectively. The
Karmacharya and Castillo (1987) in Rotylenchulus species was identified
field and pot experiments, as R. reniformis, following the key
provided by Dasgupat et al. (1968).
Watson (1949) reported that after
application by mulching of almost any Leaves, including succulent stems
vegetable matter that will decay, the of ipil-ipil [(Leucaena leucocephala
growth of such a sensitive plant as (Lam.) de Wit], kakawate (Glericidia
okra in soil heavily infested with root sepium Sted.), Sta. Anastacia
knot was improved. [Asystacia gangetica (L.) T. Anders]
and Manila grass [Eulalia amaura
Castillo et al. (1976) reported the (Buese) Ohwi] were chopped to 3 cm
suppressive effect of corn on in length. Rice (Oryza sativa L.) straw










nrnnnir rntnrilk PycA(nt rice


weeV IIIIAtU UpcdadItIly vvilLm IL IIOLruu- oone. rresence o0 ne1imidUUt III Li l
infested soil in 13-cm diameter pots. roots was determined by staining in
Two rates, namely, 6.3 g and 12.6 g, acid-fuchsin lactophenol and clearing in
were applied. Rice straw, applied at plain lactophenol following the
the same rates, was used as mulch, procedure by McBeth et al. (1941).
rather than as soil amendment because Identification and quantification of
it is normally used as mulch. nematode were done with the use of
Phenamiphos (Nemacur 10G), a stereoscopic and dissecting
nematicide-insecticide, was applied at microscopes.
the rate of 8.2 g/pot, approximately
equivalent to 5 kg a.i./ha. To serve as Field experiment
one of the checks, a pot containing
only nematode-infested soil was A field experiment to determine
provided. The other check was a pot the effect of amending soil with fresh
containing baked soil from the same leaves of kakawate and ipil-ipil, cow
batch of soil used in the other dung, sawdust, coir dust, mulching
treatments. Each treatment was with rice straw and intercropping with
replicated five times, corn on populations of R. reniformis in
cotton was conducted during the dry
The soil in each pot was kept season (November to May) at the
moist and covered with plastic for 2 Mariano Marcos State University
weeks. Subsequently, five seeds of (MMSU), Batac, Ilocos Norte.
cotton cv. Deltapine 16 were planted
in each pot. The seedlings were The experimental area was
thinned to one plant per pot 2 weeks approximately 34 m x 39 m, previously
after seeding. The delay in seeding grown to eggplant. Preliminary
was done to avoid phytotoxicity of the nematode determination in the area
organic materials on the germinating yielded tive nematode genera, namely,
seedlings. The pots were arranged in a Rotylen .bulus, Helicotylenchus,
. . . .... o_ _~_- ... _-_ _L --


greenhouse witn a temperature range
of 27 to 32 C. Control of insects and
diseases were provided by alternate
foliar spraying with endosulfan
(Thiodan) and mancozeb (Dithane M-
45), whenever necessary. Basal
application of complete (14-14-14)
inorganic fertilizer and at monthly
intervals thereafter, at the rate of 90
kg N, 90 kg K20 and 90 kg P205/ha
for the duration of the experiment was
done. The plants were allowed to grow
for 5 months.

Data on R. reniformis recoveries
per 250 cm3 soil and 1 g roots from
each pot were collected at the


Inese gienteaf, nuiyrl Ilr
were identified as i
S. I ...


LvuC / u JiLa IL. O. I lvW .

Land preparation was done by a
tractor. Four 10 m x 32 m blocks
separated by a 1.0 m pathway were
prepared, each with 16 4 x 4 m plots,
0.2 m apart. Mean initial density of R.
reniformis per 300 cm3 soil,
determined from 10 randomly collected
samples from each block was 92,
based on recovery using the routinary
sieving and Baermann funnel and
l ? . .I =" - -


fu1






Philinn Phvtnnathnl 100d VMl An(i\l.A1_41


randomly assigned to the 16 plots of feeder roots were randomly collected
each block. Because there were only from each plot. These subsamples
15 treatments, one plot in the corner were pooled and a composite 300 cm3
of each block was excluded in the soil sample and 1 g root sample were
randomization. All the organic obtained. Nematodes from the soil and
materials (when used as manure) and roots were extracted in the same
phenamiphos were evenly applied in manner as in the pot experiment,
15-cm wide furrows then covered with except that the sieved soil and root
soil. Plant leaves were chopped to fragments caught in the sieves were
about 4 cm length before application, assayed on mungbean to account for
Rice straw was also chopped and the egg population. Identification and
spread evenly on the planting sites. quantification were done in the same
The organic materials were applied at manner as in the pot experiment.
two rates: 2.5 and 5 tons/ha. Yields in the three center rows of each
Phenamiphos (Nemacur 10G) was plot were considered.
applied at 7.5 kg a.i./ha. A nontreated-
check plot was also provided. Cotton RESULTS
cv. Deltapine 16 was seeded at the
rate of five seeds/hill in the five Pot experiment
furrows. Distances between furrows
and hills were 75 cm and 25 cm, The results of the pot experiment
respectively. In the treatment with to determine the effect of soil
each corn row as an intercrop, each amendments, rice straw mulch and
row of cotton was between two rows phenamiphos on nematode recoveries
of corn (cv. DMR 2). Three corn seeds from cotton five months after planting
were planted per hill, with 50-cm are shown in Table 1. Only
distance between hills. Thinning of R. reniformis was observed in high
cotton and corn seedlings to two and numbers (93.5%). Other nematode
one per hill, respectively, was done genera were not considered because
one month after planting. they accounted for only 6.5% of the
nematode populations.
Fertilization with complete (14-14-
14) inorganic fertilizer at the rate of All treatments reduced the R.
15.4 kg N, 15.4 kg P205 and 15.4 reniformis population compared to the
K20/ha was done one month after nematode-infested control. Except
planting. This was followed a month Sta. Anastacia and kakawate leaves,


rate of 41.41 kg N/ha. Off-barring






. ..... pp. 1A REJUtIufLul. i77t, VUIU.


Table 1. Ettect ot soil amendment witn plant leaves ana muicning wiLn rnue bUav
on populations of Rotylenchulus reniformis on potted cotton five months
after planting.'


APPLICATION RATE NEMATODE DENSITY
TREATMENT (g/pot) (No./200 cm3 soil and 1 g root)



Ipil-ipil leaves
6.3 396.4 g


1Data are rr
significantly differ
(V'-x ) values.


: replicates.
/el with DMRT.
,0.


110.
150.


132.
88.


111.
96.


176.
117.



111.


748.


nilar letter
>ased on






Philipp. Phytopathol. 1994, Vol. 30(1):35-43


Table 2. Effect of amending soil with plant leaves, cow dung, sawdust and coir
dust, mulching with rice straw and intercropping with corn on populations
of Rotylenchulus reniformis on field cotton and on yield five months after
planting.



NEMATODE DENSITY COTTON
TREATMENT APPLICATION RATE (No./200 cm3 soil YIELD
(tons/ha) and 1 g root) (tons/ha)


Inil-inil leaves


Kakawate leaves



Cow dung


Sawdust


Coir dust


Rice straw mulch


2.5
'5.0


Intercropping with corn
(var. DMR 2)

Phenamiphos
(7.5 kg a.i./ha)

Nematode-infested soil
(check)


not significantly


509.2 ab
231.7 ab


376 ab
136 a


455.7 ab
184.7 a


435.7 ab
132.2 a


183.2 a
145.0 a


360.0 ab
212.2 ab


56.5 a


10.5 a


764 b


1.339 bc
1.048 ab


1.375 bc
1.088 ab


1.054 ab
1.062 ab


.979 ab
1.298 bc


1.135 a-c
1.220 bc


1.301 bc
1.237 bc


.568 a


1.717 c


1.223 ab


'Data are means of four replicates. Means followed by the same letters are
different at 5% level with DMRT.










il amendment with ipil-ipil leaves.

eld experiment

The result of the field experiment
determine the effect of soil
nendments and rice straw mulch at
e rate of 2.5 and 5.0 tons/ha and
tercropping with corn and
lenamiphos at the rate of 7.5 kg
i./ha on nematode recoveries from
itton five months after planting is
lown in Table 2. Data on


nsidered since populations of o
matode genera were very
tailingg about 5%).

Intercropping with corn


her
ow


wdust (5.0 tons/ha), coir dust (2.5
id 5.0 tons/ha) and kakawate leaves
.0 tons/ha) reduced R. reniformis
)pulations on cotton (Table 2). These
eatments were equally effective and
imparable with the effect of
ienamiphos at the rate of 7.5 kg
i./ha., low and high rates of rice
raw mulch and ipil-ipil leaves, low
te (2.5 tons/ha) of cow dung,
iwdust and kakawate leaves were not
anificantly different with nematode-
fested soil.

Despite the nematode controls
ovided by the treatments, only
ienamiphos increased the yield of
>tton.

DISCUSSION


The pot experiment showed the
oppressive effects of manuring or
rending soil with leaves of ipil-ipil,
ikawate, Manila grass and Sta.
nastacia and mulching with rice
raw on populations of R. reniformis
i cotton. The effectivity of amending


ate (9.1 tons/ha) was substantiated
y the results of the field experiment.
: was shown that amending soil at the
igh rate (5.0 tons/ha) with cow dung,
awdust and low and high rates of coir
ust and intercropping with corn can
control the R. reniformis populations.
Ihese observations concurred with
hose of other workers (Linford et al.,
938; Duddington, 1957; Singh et al.
967; Singh and Sitaramaiah, 1971;
fan der Laan, 1956; Calinga, 1979;
Vatson, 1949; Castillo et al., 1976).

The efficiency of soil amendments
o control R. reniformis population
suggestedd that any organic materials
Idded to the soil will control
nematodes. Generally, the high rate of
application was more effective than the
ow rate in the field experiment. Sayre
1971) attributed the reduction in
nematode populations due to soil
amendment to the direct toxicity of the
products of decomposition, an increase
n the number of natural enemies and
:he alteration of plant-nematode
relationships through the effect on soil
fertility. Other plausible explanations
:or the nematicidal effect of organic
materials include ammonification
Walker, 1969) and heat generation
During decomposition (Sussman, 1982;
3uhaylongsod and Castillo, 1989).

Despite the nematode control
provided by the treatment, only
phenamiphos application increased the
seedcotton yield. Intercropping of corn
with cotton did not increase the -yield
since the nematode-suppressive effect
of the treatment was perhaps offset by
the reduced plant growth due to
competitionn for nutrients and light by
the taller corn.

According to Follet .et al. (1981),
nitrogen deficiency usually occurs in
soils amended with organic materials









soil with the organic materials in the
field experiment was probably due to
the nitrogen deficiency resulting from
competition for nitrogen between the
plants and microorganisms. Chicken
dung was effective in reducing
nematode populations perhaps because
of its richness in nitrogen (Calinga,
1979; Duhaylongsod and Castillo,
1989). Reyes and Davide (1975) also
noted highest percentages of
occurrence of nematophagous fungi in
chicken dung, compared to other
dungs.

LITERATURE CITED

CALINGA, R.H. 1979. Field and
laboratory studies on the
effectiveness of dungs for the
control of plant parasitic
nematodes on cowpea. M.S.
thesis, Department of Plant
Pathology, UPLB, College, Laguna.

CASTILLO, M.B. 1977. Survey,
biology and control of nematodes
attacking cotton. Annual Report
No. 2, PCARR Project No. 170-4,
UPLB-CA, College, Laguna, 18 pp.

CASTILLO, M.B. 1985. Some studies
on the use of organic soil
amendments for nematode
control. Philipp. Agric. 68:76-93.

CASTILLO, M.B., N.B. BAJET and R.R.
HARWOOD. 1976. Nematodes in
cropping patterns. I. Populations
of Rotylenchulus reniformis on
successively monocultured crops.
Philipp. Agric. 288-294.

CASTILLO, M.B., M.S. ALEJAR and
J.A. LITSINGER. 1977.
Increased efficiency of
determining populations of
Rotylenchulus reniformis and
Meloidogyne incognita through
bioassay. Philipp. Agric. 61: 64-
69.


Philipp. Phytopathol. 1994, Vol. 30(1):35-43


CASTILLO, M.B. and N.C. SEVILLA.
1977. Pathologic reactions of
cotton in soil infested with
Rotylenchulus reniformis. Philipp.
Phytopathol. 13:32-37.

DASGUPTA, D.R., D.J. RASKI and
S.A. SHER. 1968. A revision of
the genus Rotylenchulus Linford
and Oliveira, 1940 (Nematoda:
Tylenchidae). Helminthol. Soc.
Wash. Proc. 35:169-191.

DUDDINGTON, C.L. 1957. The friendly
fungi. London. Faber and Faber
18 p.

DUHAYLONGSOD, R.D. and M.B.
CASTILLO. 1989. Plant parasitic
nematode populations and tomato,
Lycopersicon esculentum Mill.
reactions in soil amended, with
fresh and composted organic
materials. MSU J. 1: 34-44.

DUQUE, R.C. 1988. Comparative
effectiveness of chicken dung,
Bioact, urea and phenamiphos in
controlling nematodes and
increasing yield of potted tomato
(Lycopersicon esculentum Mill.).
M.S. Thesis, Department of Plant
Pathology, UPLB, College, Laguna.

FOLLET, R.H., L.S. MURPHY and R.L.
DONAHUE, 1981. Fertilizers and
soil amendments. Prentice Hall,
Inc., Englewood Cliffs, New
Jersey, 557 p.

KARMACHARYA, B.B. and M.B.
CASTILLO. 1987. Plant
nematode populations and tomato
(Lycopersicon esculentum Mill.)
yield in field plots treated with
chicken dung, sawdust and urea.
Philipp. Agric. 69:289-305.

LINFORD, M.B., F. YAP and J.N.
OLIVEIRA. 1938. Reduction of
soil populations of the root-knot









nematode during decomposition of
organic matter. Soil Sci. 45: 127-
140.

MCBETH, C.W., A.L. TAYLOR and
A.L. SMITH. 1941. Note on
staining nematodes in root tissue.
Helminthol. Soc. Wash. Proc.
8:26.

REYES, T.T. and R.G. DAVIDE. 1975.
Fungi for biological control of
plant parasitic nematodes. UPCA
Tech. Bull. No. 31-51.

SAYRE, R.M. 1971. Biotic influence in
soil environment, pp. 235-256
In: B.M. Zuckerman, W.F. Mai
and R.A. Rohde. Plant parasitic
nematodes: I. Morphology,
anatomy, taxonomy and ecology.
Acad. Press, New York and
London.

SINGH, R.S., B.S. SINGH and S.P.S.


1971. Control of root knot
through organic and inorganic
amendments of soil: Effects of
sawdust and inorganic nitrogen.
Indian J. Nematol. 1: 80-84.

SUSSMAN, V. 1982. Easy
composting. Rodale Press,
Emmaus, PA. 135 p.

VAN DER LAAN, P. 1956. The
influence of organic manurina on










STRAINS IN PUMMELO


LORNA E. HERRADURA, N.B. BAJET and LYDIA V. MAGNAYE

A portion of MS thesis of the senior author submitted to the Graduate School,
University of the Philippines at Los Baios, College, Laguna.

Respectively, Plant Pathologist, Davao National Crop Research and Development
Center (DNCRDC), Bureau of Plant Industry (BPI), Bago Oshiro, Davao City,
Assistant Professor, Department of Plant Pathology, UPLB, College, Laguna anc
Supervising Agriculturist, DNCRDC, BPI, Bago Oshiro, Davao City.

Key words: Citrus, Closterovirus, cross protection, ELISA, strain
identification, tristeza.


ABSTRACT

Isolates of citrus tristeza virus (CTV) collected from
various citrus plantations in Davao and Batangas induced wide
range of symptoms and severity on different citrus cultivars.
These isolates were free of citrus greening and exocortis
pathogens when subjected to biological indexing. Enzyme-
linked immunosorbent assay (ELISA) tests confirmed the
isolates as CTV. On key lime (Citrus aurantifolia) as indicator
host, the isolates were classified into mild and severe strains.
Mild strain exhibited mild vein clearing while severe strain


not induce any symptom or
orange (C. aurantium) that sE
the mild and severe strai
antibodies of CTV dwarf strai

Pummelo plants cv Ma(
strain (pummelo 169) did no
months after challenge ino
pummelo P-03 and pummel
inoculated with the severe s
with the mild strain showed
additional evidence for the
country. The study also der
can be a viable alternative cor


INTRODUCTION

Among the virus and virus-like
diseases, tristeza caused by citrus
tristeza virus (CTV), remains as one of


ie leaves and stems of sour
Sas indicator host. Extracts of
reacted to the monoclonal
:TV-D).

anes pre-inoculated with mild
low any severe symptom five
nation by the severe strains,
%T. Similar pummelo cultivars
ns but without pre-inoculation
iere symptoms. This provided
sence of CTV strains in the
istrated that cross protection
il for CTV in pummelo.


the most serious disease problems in
many citrus-growing areas around the
world (Bar-Joseph et al., 1979;
Hamilton, 1985). It has significantly
affected citrus production throughout


rniuplp. rny~topatnoi. JYY4, V01.rrl~~~J






Philipp. Phytopathol. 1994, Vol. 30(1):44-5


the world by lowering the quantity and
quality of yield and by killing more than
50 million trees (Bar-Joseph et al.,
1981). The disease is widely
distributed in South Africa, South
America, India, Australia and
Southeast Asia. In the Philippines, the
disease was first reported in 1958 as
affecting calamondin (Citrus
madurensis Lour.) in the Bicol Region
(Bigornia and Calica, 1961). Previous
studies also showed that CTV infected
other citrus species and is widely
distributed in the country (Benigno,
1976; Cortez and Celino, 1968; Del
Rosario and Alaban, 1965). Results of
survey conducted in Region XI
(Mindanao) showed that the disease is
common in all citrus plantations and
severely affecting pummelo trees
(Magnaye and Herradura, 1990).

Several control methods have
been tried to minimize tristeza but
cross protection appeared to be the
most promising method. Cross
protection has been based on the
discovery'tff mild strains of CTV that
prevent symptom expression of the
severe strains in citrus plants. The
results of virus-induced protection of
citrus against CTV demonstrated the
commercial significance to the citrus
industry in Brazil (Costa and Muller,
1980; Grant and Costa, 1951). Cross
protection is presently utilized in
controlling tristeza in America,
Australia, India, Israel, Japan and
South Africa (Hamilton, 1985).
However, despite the success obtained
in other countries, cross-protection has
never been adopted in the Philippines
to control CTV. This paper presents
the isolation of strains commonly
associated with the different citrus
types, identification of strains using
biological and serological techniques
and identification of mild strains with
cross-protection potential.


MATERIALS AND METHODS

CTV Characterization

Citrus samples were collected
from the different citrus growing areas
of Luzon and Mindanao. Symptoms
observed on the leaves and stems ol
the different cultivars were recorded
Budsticks of the different citru,
species were collected and biologically
indexed for citrus greening anc
exocortis pathogens by graft
inoculation (Childs, 1968) on szinkorr
and etrog citron, respectively. For each
citrus sample, three test plants anc
one control were used. Plants testec
negative for greening and exocortis
pathogens were subjected to ELISA tc
confirm the presence of CTV using
polyclonal antibody from Florida.

The plants were subjected to
biological indexing and serology for
further characterization of the isolates.
For biological indexing, two indicator
plants, sour orange and key lime were
used in the assay to determine the
presence of seedling yellows and stem
pitting strains, respectively (Salibe and
Giacometti, 1984).

Serological Characterization of CTV by
ELISA

The indirect ELISA (Clark et al.,
1986), double antibody sandwich
(DAS-ELISA) and indirect-double
antibody sandwich (I-DAS-ELISA)
ELISA techniques (Tsai et al., 1990)
were used to identify the presence of
strains among the isolates.

The procedure described by Clark
et al. (1986) was followed with some
modifications for the detection of CTV
by indirect ELISA. Briefly, antigen was
coated to the plate at 100 ul/well,
incubated overnight, and washed with
PBS-T 3-5 times. Blocking solution
containing 0.2% Carnation non-fat milk










was added at 150 ul/well, incubated
for 1 hr at room temperature, and
washed as above.

For the DAS-ELISA (Su, personal
communication, 1993), a CTV
monoclonal antibody (CTV-McAb
3E10) was diluted 500 times with
coating buffer, and used for coating
the ELISA plate by adding 100 ul to
each well. The plate was incubated
and washed following the I-DAS-ELISA
procedure described above. Antigen
preparation was the same as that of I-
DAS-ELISA procedure. Extract was
added to each well at 100 ul, and
incubated at 37 C for 2 to 3 hr or at 4
C overnight. Unbound antigen was
washed as above. Alkaline
phosphatase-labelled antibody (anti-
CTV McAb 3E10) solution in PBS-T
containing 0.2% non-fat Carnation milk
was added at 100 ul/well and
incubated for 2 hr at 37 C. Unbound
antibody conjugate was washed as
above. Fresh substrate solution was
added at each well at 100 ul/well,


were calculated and analyzed
statistically using Chebychev's
inequality (Sutula et al., 1986). The
nhknrknntn -nhl niA c r...lm lnt. a m cn-


Philipp. Phytopathol. 1994, Vol. 30(1):44-53


A = M+4 SD, where A = absorbance
value, M = mean absorbance of
healthy control, SD = standard
deviation of absorbance of healthy
control plants and 4 = constant factor
for 95% probability level. Absorbance
reading higher than this value was
considered positive.

Cross Protection Tests

Mild CTV isolates identified
through biological indexing and ELISA
were used in cross protection tests.
Pummelo cv Magallanes was budded
onto calamandarin rootstock infected
with the mild strain serving as the
protective strain. After one year, it was
challenge-inoculated with severe aphid-
transmitted CTV isolate. Ten plants
were used for each trial with two
plants without challenge inoculation to
serve as control. Five months after
challenge inoculation, they were
evaluated to determine the effect of
the severe isolate on the leaves and
stems of test plants. Bark was stripped
10 cm from the bud union to observe
for stem pitting. Another batch of
challenge-inoculated plants was done
but the severe isolate was inoculated
two months after inoculation of
protective strain. Pummelo 169 (mild
isolate) was used as the protective
strain since it caused mild vein clearing
and no stem pitting on key lime. The
challenge strains were two aphid-
transmitted isolates from pummelo
(pummelo AT and P-03) which were
considered severe because they
developed stem pitting on the test
plants and on key lime. The
development of stem pitting and
number of pits per square centimeter
were used as indicator of variation
among CTV isolates (Van Vuuren et
al., 1993).






Philipp. Phytopathol. 1994, Vol. 30(1):44-5


RESULTS AND DISCUSSION

Symptomatology of Different Isolates

Different strains collected frorr
Luzon and Mindanao showed variec
symptoms depending on the citrus
species. On mandarin cultivars, CTs
usually induced vein corking in ladu,
vein clearing and stem pitting in king
mandarin, and leaf cupping, veir
corking and stem pitting in pummelc
(Table 1).

Biological Assay

Biological assay was used tc
determine the variations of th(
isolates. Isolates were classified as
mild and severe on the basis of their
reaction to key lime (Wallace et al.,
1956). The isolates can be broadly
grouped into two strains (Table 2);
those exhibiting mild vein clearing anc
without stem pitting on key lime as
exemplified by the pummelo 169
isolate, and those causing severe anc
pronounced vein clearing as well as
pitting on stem and branches of key
lime e.g. pummelo P-03 and pummelc


density of stem pitting may be
doubled. Isolate pummelo P-03 did nol
..~+nnnno+n mc sn,;+h ra


bark bt induced an average of 2:
pits/cm on matured stems.

The isolates did not show observ
able symptoms on sour orange twi
months after inoculation. This indicate
the absence of seedling yellows.

Serological Technique

Extracts of the mild and sever
isolates reacted to the there
monoclonal antibodies obtained fron
Dr. H. J. Su of National Taiwai
University. Nine isolates (Pummelo AT
Florida GF, Ladu and six isolates front
calamansi) showed positive reaction ti
the three monoclonal antibodies
namely, 3E10, 10E3, and 4G12 (Tabl
3). The positive reactions indicate tha
the isolates belong to the CTV-D strai
as described by Su (1981). The result:
of serological tests are consistent witl
the result of biological indexing on ke'
lime. The severe isolates showed
symptoms such as vein corking in ladi
and pummelo and severe vein clearin!
and stem pitting in calamansi (Table 1)
The results showed that there i
variability among CTV population in th
country and that the presence c
severe and mild strains wa
demonstrated. Greater fractions a
isolate reacted to the antibodie
against the Taiwan strains compared
with the Florida isolates indicating tha
the CTV in the Philippines may b
more closely related to those a
Taiwan strains. Likewise, the sever
CTV-D strains was also detected i
most of the samples tested.

Cross Protection

Challenge-inoculation of the mil
isolate, pummelo 169, by the sever
isolate, pummelo P-03, did not produce
any visible symptoms on both th
leaves and stems of test plants (Tabl
4). Leaf development was normal wit










from Luzon and Mindanao.

SPECIES/CULTIVAR CODE

Pummelo A


Pummelo Pummelo (H

Pummelo P-C

Pummelo (1 6

Pummelo (Tdp

Calamansi Calamansi (-

Calamansi Calamansi 1


Calamansi Calamansi 2


Calamansi Calamansi 3


Calamansi Calamansi 4


Calamansi Calamansi 5


s on citrus species and cultivars obtainec


ORIGINAL SYMPTOM

severe leaf cupping; stem
pitting; reduction in size

,UPLB) symptomless

leaf cupping; stem pitting

leaf cupping

symptomless

i, UPLB) moderate vein clearing

atangas) severe vein clearing;
stem pitting

itangas) severe vein clearing;
stem pitting

itangas) severe vein clearing;
stem pitting

itangas) severe vein clearing;
stem pitting

it+n 0\ C. at ra t -n -nlar=nn









lime.


ISOLATE STRAIN TYPE KEY LIME



Pummelo AT severe vein clearing and stem pitting

Pummelo TdPm mild vein clearing

Pummelo P-03 severe vein clearing and stem pitting

Pummelo 169 mild vein clearing

Calamansi Dvo 1 severe moderate vein clearing and
stem pitting

Calamansi Dvo 2 severe moderate vein clearing and
stem pitting

Perante orange severe moderate to severe vein
clearing

Valencia mild vein clearing

King mandarin (BKM) severe moderate vein clearing and
mild stem pitting

Ponkan (TdPn) mild none


Plants inoculated with the severe strain
showed leaf cupping symptom. Pitting
was not observed on the partially
opened stem which is also a
characteristic symptom of severe CTV
strains. Challenge-inoculation by the
severe strain gave similar results on
the test plants challenge-inoculated
after one year. When evaluated 4 to 5
months after challenge-inoculation,
mild isolates of pummelo and
grapefruit did not show any pitting
symptom on the opened stem. The
mild isolate was capable of protecting
the plant from the effects of the severe
strain of CTV when introduced two
months before challenge-inoculation as


shown by the absence of the severe
symptom.

The mild pummelo isolates where
challenge-inoculated by the severe
pummelo isolates prevented severe
strains to develop severe symptoms
Results from the study showed thai
cross protection can be a viable control
alternative of CTV on pummelo in the
country especially in Mindanao where
pummelo is a major fruit crop. The
study also provided additional evidencE
of the existence of CTV strains in he
country.










monoclonal antibodies and indir







SAMPLES 3E'



Pummelo (AT) 2.

Florida GF 0.

Ladu (nursery) 1.

Calamansi 1 1.

Calamansi 2 1.


rent citrus tristeza virus samples using
-double antibody sandwich ELISA.



ABSORBANCE1

Monoclonal Antibody

10E3 4G12



+ 0.97 + 0.35 +

+ 0.16 + 0.11 +

+ 0.61 + 0.28 +

+ 0.43 + 0.97 +

+ 0.50 + 0.90 +


Calamansi 4 1.71 + 0.52 + 0.6;

Calamansi 5 1.63 + 0.88 + 0.41

Taiwan GF 0.01 0.01 0.0;

Pummelo nurseryv) 0.01 0.01 0.OC


1.60 + 0.41 + 0.10 +

0.01



negative reaction.


Healthy

















PUMMEl

TEST NO. Leaves




1 Normal

2 Normal

3 Normal

4 Normal

5 Normal

6 Normal

7 Normal

8 Normal

9 Normal

10 Normal

11 (mild isolate
only) Normal

12 (challenge
strain
only) Cupping



Symbol + = with stem pitting; = no


Phlllpp. Fhytopathol. 1994, Vol. 30(1):44-3J


IIIIIICIIU IIIIU ,L U VVILII IIIlIU IOUICLV %I U1iI1lm l*
severe strains (P-03 and AT) five months




LO P-03 PUMMELO AT
-------------..---------- --
Stem' Leaves Stem'




Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal



Normal +




+ Cupping +



stem pitting










LITERATURE CITED

BAR-JOSEPH, M., S.M. GARNSEY and
D. GONZALVES. 1979. The
closterovirus, a distinct group
elongated plant viruses. Adv.
Virus Res. 25:93-168.

BAR-JOSEPH, M., C.N. ROISTACHER,
S.M. GARNSEY and D.J. GUMPF.
1981. A review on tristeza, an on-
going threat to citriculture. Proc.
Int. Soc. Citrict. 1:419-423.


BENIGNO, D.A. 1976. Plant
diseases in the Philippines.
Diseases of Tropical Crops.
Symp. Trop. Agr. Res.,
1976.


virus
Virus
Proc.
Sept.


BIGORNIA, A.E. and C.A. CALICA.
1961. Tristeza in the Philippines.
pp. 101-105. In: Proc. 2nd Conf.
of 10CV. W.C. Price (ed.) Univ. of
Florida Press, Gainesville, F.L.

CHILDS, J.F.L. (ed.). 1968. Indexing
procedures for 15 virus diseases
of citrus trees. USDA Handbook
No. 333. 96 p.

CLARK, M.F., R.M. LISTER and M.
BAR-JOSEPH. 1986. ELISA
Techniques. pp. 743-760. In:
Methods in Enzymology, Vol. 118.
A Weissbach and H. Weissbach
(ed.), Univ. of Florida Press,
Gainesville, F.L.

CORTEZ, R.E. and C.S. CELINO. 1968.
Distribution of tristeza virus in the
Philippine citrus. Proc. of the 4th
Conf. of IOCV. J.F.L. GCilds (ed.),
Univ. Florida Press, Gainesville,
FL.

COSTA, A.S. and G.W. MULLER.
1980. Tristeza control by cross
protection. Plant Dis. 64:541-583.


Philipp. Phytopathol. 1994, Vol. 30(1):44-53


DEL ROSARIO, M.S. and C. ALABAN.
1965. Virus diseases of citrus in
the Philippines. Proc. 3rd Conf. of
IOCV W.C. Price (ed.) Univ. of
Florida Press, Gainesville, FL.

GRANT, T.J. and A.S. COSTA. 1951.
A mild strain of the tristeza virus
of citrus. Phytopathology 41:114-
122.

HAMILTON, R.I. 1985. Using plant
viruses for disease control. Hort.
Science 20:848-852.

HERRADURA, L.E. 1993. Isolation and
partial characterization of citrus
Tristeza Closterovirus in the
Philippines. MS Thesis, UPLB, 87
P.

MAGNAYE, L.V. and L.E.
HERRADURA. 1990. Survey,
selection and indexing of citrus
candidate parent trees. Bureau of
Plant Industry, Manila.
(Unpublished).

SALIBE, A.A. and D.C. GIACOMETTI.
1984. Evidences that tristeza and
stem pitting are different viruses
or components of the same
complex. pages 76-80. In: Proc.
9th Conf. of IOCV S.M. Garnsey,
L.W. Timmer, and J.A. Dodds
(ed.). Univ. California Riverside,
C.A.

SU, H.J. 1981. A tristeza virus strain
causing dwarf of pomelo and
grapefruit. Proc. Int. Soc.
Citriculture. Vol. 1:423-426.

SUTULA, C.L., J.M. GILLETT, S.M.
MORRISSEY and D.C. RANSDELL.
1986. Interpreting ELISA data and
establishing the positive-negative
threshold. PI. Dis. 70:722-726.






Philipp. Phytopathol. 1994, Vol. 30(1):44-53


TSAI, M.C., H.J. SU, and S.M.
GARNSEY. 1990. Citrus tristeza
virus (CTV) strains and identifica-
tion by monoclonal antibodies,
with emphasis on Southeast Asia
isolates. In: Proc. 4th International
Asia Pacific Conference on Citrus
Rehab. B. Aubert, J. Montyaporn
and D. Buangsuwon, (eds.). Chiang
Mai, Thailand.

VAN VUUREN, S.P., R.P. COLLINS
AND J.V. DA GRACA. 1993.
Evaluation of citrus tristeza virus
isolates for cross protection of
grapefruit in South.Africa. PI. Dis.
77:24-28.


WALLACE, J.M., P.C.J. OBERHOLZER
and J.D.J. HOFMEYER. 1956.
Distribution of viruses of trristeza
and other diseases of citrus in
propagative material. PI. Dis.
Reptr. 40:3-10.


ACKNOWLEDGEMENT

This study was supported by the
Philippine Council for Agriculture
Natural Resources Research and
Development (PCARRD) Scholarship to
L. E. Herradura and in part by a
research grant to Narceo B. Bajet by
the Department of Science and
recchnology (DOST), Grant #
8946721, and by the Bureau of Plant
Industry, Department of Agriculture.










PHYTOPATHOLOGICAL NOTE: I
DISEASE IN TI


P.Q. CABAUATAN, R.C. CABUNAG/
KOGAI

Respectively, Assistant Scientists, F
Entomology and Plant Pathology Divisic
Los Baios, Laguna, Philippines.

Key words: Electron microscopy, lea
serology, symptomatology

ABSI

Symptoms of rice dwarf <
Midsayap, North Cotabato. In
exhibited fine chlorotic streak;
Electron microscopic observati
plants revealed the presence
particles about 65 nm in c
symptomatic leaves gave str
antiserum in rapid immunof
double antibody sandwich
assay (DAS-ELISA). Positil
obtained with Nephotettix a
virescens and Recilia dorsalis
the vector with an incubation
was also transmitted via the
N. nigropictus.
INTRODUCTION

Rice dwarf disease is the oldest
tnnurn wirni rdicacao in tho w/nrlrl


Philipp. Phytopathol. 1994, Vol. 30(1):54-58


OCCURRENCE OF RICE DWARF
E PHILIPPINES


SFILOMENA C. STA. CRUZ and H.
ZAWA

search Assistant, and Plant Virologist,
, International Rice Research Institute,


hopper transmission, rice dwarf disease,


ACT

;ease were observed in rice in
cted plants were stunted and
or specks on the leaf blades.
I of clarified sap from infected
of rice dwarf virus (RDV)
meter. Leaf extracts from
ig positive reaction to RDV
er paper assay (RIPA) and
izyme-linked immunosorbent
transmission of RDV was
7ropictus but not with N.
The virus was persistent in
period of about 15 days. RDV
igs of viruliferous females of

(Nasu, 1963), N. virescens (Xie et al.,
1981) and Recilia dorsalis (Fukushi,
1937). The virus is persistent in the
vector with an incubation period that
ranged from 4 to 58 days in N. cincticeps
(lida et al., 1972) and 9 to 42 days in R.
Dorsalis (Shinkai, 1962). The virus is









1V I I IIIpJIJ I ICGO U III ULII UII l l III GI II *JI II IUSl O1 a l CO*l I
country. However, in August 1992 we
collected tungro-infected rice from Second instar nymphs
hilRice substation in Midsayap, North N. nigropictus, N. virescens
otabato, to study tungro variability in the R. dorsalis were given four days ac
hilippines. When the plants were to RDV-infected TN1 plants, transfh
(amined closely at IRRI, one plant (cv to healthy TN1 seedlings for
tri Merah) from this collection showed days and then individually confined
usual virus-like symptoms (e.g. chlorotic a 6-day old TN1 seedling in a test 1
:reaks similar to rice dwarf disease) in Insects were transferred dailh
edition to tungro symptoms. These healthy TN1 seedlings until 1
symptoms have been observed in the died. Inoculated seedlings v
ame station since 1990. Further surveys transplanted in pots and kept in screi
id leaf sample collections in September cages until symptoms develop
992 revealed that dwarf-like symptoms Percentage infective insects, v
,ere prevalent in the station and in the incubation period in the insect
surrounding rice fields. A study was transmission pattern were determine
ierefore conducted to determine the each insect species. A total of 400 in,
entity of the disease. A preliminary of each species were tested
port has been published (Cabauatan et transmission.
., 1993).
To test for transovarial pas,
of the virus, 2nd to 3rd instar nympi
MATERIALS AND METHODS N. nigropictus were allowed aci
to a diseased plant for four days
irus Source Propagation then reared on healthy TN 1 seedlings
id Symptomatology they became adult. All females 1
selected and confined on hea
The original virus isolate came TN1 seedlings to lay eggs for three c
om PhilRice substation in Midsayap, Nymphs were tested individually
orth Cotabato. The plant was stunted RDV transmission immediately ;
nd exhibited dark green foliage with fine emergence from the eggs. Percent
hlorotic specks or streaks on the leaf infective nymphs and virus incubi
lades. When leaf extracts from this plant were noted.
rere tested in DAS-ELISA, it was
)und to be infected with rice tungro Electron Miscoscopy
acilliform virus (RTBV) and rice dwarf
irus (RDV). Since RTBV alone is non- Five grams of infected leaves
ansinissible (Cabauatan and Hibino, homogenized in 15 ml of 0.
985), rice dwarf virus was propagated phosphate buffer, pH 7.2, filt
1 rice cv. Taichung Native 1 (TN1) using through cheesecloth and centrifuge
iis plant as virus source. Second instar 5,000 rpm for 10 minutes. Ca
ymphs of N. nigropictus were allowed tetrachloride was added to
access to this virus isolate for four days, supernatant (20% final concentration]
ansferred to healthy TN1 seedlings for mixture was homogenized for one m
2 days virus incubation and then and centrifuged for 10 minute
onfined with 6 to 7-day old TN1 5,000 rpm. The supernatant
eedlings in a mylar cage for 24 hours, again centrifuged at 40,000 rpm fc
ioculated seedlings were transplanted in minutes. The pellet was suspei
ots and kept in insect-proof cages until in 0.5 ml buffer and was used
symptoms developed. Inoculated plants electron microscopy. A drop of






56 Philipp. Phytopathol. 1994, Vol. 30(1):54-58


Serology chlorotic specks or interveinal chlorotic
streaks. Infected plants developed dark
All leaf samples with dwarf-like green foliage. Stunting symptom was mild
symptoms collected from Midsayap were on TN1. Unlike RDV in Japan, the RDV
individually homogenized in phosphate isolate from Midsayap did not induce
buffer (0.1 M, pH 7.2) and were directly excessive tillering. Infected plants have
tested in DAS-ELISA using the method of slightly reduced tillering. Plants infected
Hibino et al. (1988). Healthy leaves were at seedling stage survived until maturity
processed similarly and were used as and produced panicles with filled but
control. Rapid immunofilter paper assay discolored grains. Plants grown from
was also applied to detect RDV antigen seeds of infected plants did not show
in leaf extracts as described by Tsuda et symptoms of RDV.
al. (1992). The antisera used in the
experiment was given by H. Hibino, Leafhopper Transmission
National Agriculture Research Center,
Japan. Of the three leafhopper species
tested, only N. nigropictus transmitted
RDV (Table 1). About 10 percent of the
RESULTS AND DISCUSSION insects tested were infective. The virus
was persistent in the vector with an
Symptomatology incubation period that ranged from 8 to
23 days (ave. 15 days). The transmission
The first symptom of RDV infection pattern was consecutive. RDV was also
appeared as minute white specks or congenitally transmitted to the offspring
streaks along the veins of emerging leaves of females (transovarial passage) that had
about 9 to 10 days after inoculation of 6- previous access to RDV-infected plants.
day old TN1 seedlings. Streaking About 10 percent of the nymphs that
symptoms became more conspicuous 3 hatched from eggs of females that had
to 4 weeks after inoculation (Fig. 1). Later, access to infected plants were infective.
almost all emerging leaves exhibited either Eight percent of them transmitted RDV
immediately after emergence from the
eggs.



Table 1. Transmission of rice dwarf virus
by three species of leafhoppers.


INSECTS INSECT INCUBATION
SPECIES TESTED TRANSMITTED PERIOD IN
(NO.) (NO.) (DAYS)


N. nigropictus 400 40 8-23

N. virescens 400 0

R. dorsalis 400 0

Transovarial passage
N. nigropictus 579 60 0-2
Fig. 1. Rice leaves infected with rice
dwarf virus showing chlorotic 1/ Insects hatched from eggs of all adults that have
spekcs or streaks (right) and previous access to RDV-infected plants.
healthy leaf (extreme left).





Philipp. Phytopathol. 1994, Vol. 30(1):54-58 57


The transmission characteristics of Serology
the Philippine RDV isolate agree fairly well
with those previously reported (Fukushi, Leaf extracts of all symptomatic
1969). However, in our tests, the leaves collected from Midsayap reacted
Philippine isolate was not transmitted by positively to RDV antiserum in both RIPA
N. virescens and R. dorsalis which were and DAS-ELISA. Strong positive reactions
reported as vectors of RDV in other were observed even at 1000x dilution of
countries (Xie et al., 1981; lida et al., the infected sap in both tests. None of
1972). The difference may be due to the the healthy controls reacted to RDV
difference in insect colony used or antiserum.
difference in strain of RDV. So far, N.
nigropictus is the only identified vector The above results clearly indicated
of RDV in the Philippines. This leafhopper that the dwarf-like disease in Midsayap
species has graminaceous weeds as the was indeed caused by RDV. To our
main host and rice is only an alternative knowledge, this is the first report of RDV
host. In our survey in Midsayap we found occurrence in the tropics. Although
a few plants of Echinocloa sp. infected dwarf has appeared in early literatures
with RDV as indicated by serological tests. about virus diseases in the Philippines
This indicates that this weed species may (Reyes, 1957), it was later shown to be
also serve as a reservoir of RDV. Several tungro based on symptomatology and
species of weeds were also reported to virus-vector relations (Ling, 1972). To
be infected with RDV (lida, 1969). One date, RDV is found only in North Cotabato,
way to control RDV and its vector Mindanao, based on our limited survey
therefore is to eliminate their weed hosts. conducted in 1993.

Electron Microscopy
LITERATURE CITED
Electron microscopic observation of
clarified infected sap revealed the CABAUATAN, P.Q., H. KOGANEZAWA,
presence of RDV particles about 65 nm R.C. CABUNAGAN and F.C. STA.
in diameter (Fig. 2). The size and shape CRUZ. 1993. Rice dwarf (RD), a new
of the particles were similar to those virus disease in the Philippines. Int.
previously reported (lida et al, 1972). Rice Res. Newsl. 18:50.
CABAUATAN, P.Q. and H. HIBINO. 1985.
Transmission of rice tungro bacilliform
and spherical viruses by Nephottetix
virescens Distant. Philipp.
Phytopathol. 21: 103-109.

FUKUSHI, T. 1934. Studies on the dwarf
disease of the rice plant. Imp. Acad.
Proc.-(Japan) 13:328--331.

FUKUSHI, T. 1937. An insect vector of
the dwarf disease of the rice plant.
Imp. Acad. Proc. (Japan) 13: 328-
331.

IUKUSHI, T. 1969. Relations between
propagative rice viruses and their
vectors. p. 279-301. In: K.
Maramoroch (ed.) Viruses, vectors
Fig. 2. Electron micrograph of rice dwarf and vegetation. Interscience
virus particles stained with uranyl Publishers, New York.
acetate.










, E. SHIKATA, I. KIMURA and PARK, R.K., Y.T. JUNG, Y.D. JIN, D.K.
MOTO. 1960. Electron and D.H. LEE. 1982. Studies o
opic studies on the rice dwarf current status of rice virus disc
Janan Acad. Proc. 36:352- in Yeongnam area (In Korean),


R.D. DAUUIUA(, K.U.
AN and D. DAHAL. 1988.
e to rice tungro spherical
ce. PI. Dis. 72:843-647.

SHINKAI and I. KIMURA.
ce dwarf virus. CMI/AAE
n of plant viruses. No. 102.

., M.H. HEU, D.N.
)HAR and R.B. PRADHAN.
ymptom resembling those
varf disease in KathmandL


MtfYtb, U.ivi. Io/. nice
in the Philippines. F/
Bull. 6:17-19.

REYES, G.M., B.M. LEGA
MORALES. 1959. Pr
on the dwarf or stunt
of rice in the Philippi
J. Agric. 24:27-43.

SHINKAI, A. 1962. Stu
transmission of rice vir
(In Japanese, English s


I I A .Q.

- ^l^ -,^t TCI ifA C N


s, Laguna, Philippines.


and K. TOIV
detection ani
for plant vin


LIdlIl IIII VIIUS UI~DOU Dc UI I Li; plaln
in Japan (In Japanese; English XIE, L.H., Q.Y. LIN and J.R. GUO. 1981.
summary). Kyushu Agr. Exp. Sta. A new insect vector of rice dwarf
Bull. 8:153-349. virus. Int. Rice Res. Newsl. 6: 14.






hilipp. Phytopathol. 1994, Vol. 30(1):59-6


PHYTOPATHOLOGICAL NOTE: CONCENTRATING GENES FOR
PHILIPPINE DOWNY MILDEW RESISTANCE AND
DESIRABLE AGRONOMIC TRAITS IN CORN


A. D. RAYMUNDO and B. J. CALILUNG, JR.

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

Respectively, Associate Professor, Department of Plant Pathology, and University
Research Associate, IPB, UPLB, College, Laguna, Philippines.

Key words: Downy mildew, recurrent selection, resistance


ABSTRACT

Following a simple phenotypic recurrent s e I e c t i on
technique, two corn populations which are highly resistant to
Peronosclerospora philippinensis (Weston) Shaw causing
Philippine downy mildew, were developed. These populations,
designated as CPRP1 and CPRP3, were from base populations
earlier formed out of the resistant plants ot the F2 generation of
commercially-available hybrids. After several cycles of selection
under high inoculum pressure, a significant increase in the level
of resistance was observed in both populations when compared
to the reaction of the original F2 population. As each population
is highly diverse genetically, visual selection for agronomic
characters such as plant height, ear height, general vigor, ear
size, and tassel size, was likewise done. Selection for resistance
to other pathogens that attack the crop at subsequent stages
of growth is possible in these populations.

INTRODUCTION the growing season due to the usual
practice of staggered planting which
In the Philippines, downy mildew ensures a continuous source of inoculum.
causedd by Peronosclerospora P philippinensis also is able to tide over
,hilippinensis (Weston) Shaw persists as adverse conditions by infecting and
:he most important disease in corn. surviving in other host plants. Modern
althoughh metalaxyl fungicidal seed corn cultivars with DMR genes and
treatmentt (Exconde and Molina, 1978) hybrids that are normally treated with
and the incorporation of downy mildew metalaxyl are planted in less than 10% of
resistance (DMR) genes in modern the total area utilized for corn. The F,
:ultivars (Aday, 1974) have been shown generation of these hybrids, whose
:o be extremely effective, sporadic pedigrees trace back to susceptible foreign
epidemics are common occurrences and parentage, has been harvested and planted
n many cases the yield losses inflicted by farmers. Epidemics have been
lave been significant, observed to be severe in these plantings
since these F2s are no longer protected
The persistence of downy mildew has by metalaxyl. As resistant cultivars appear
been predicted (Raymundo et al., 1993). to provide the best and most sustainable
Vlany factors contribute to this condition disease management strategy it is
ind these include the availability of the imperative that new germplasm pools be
:orn cron for a orolonoed period during developed to serve as sources of











resistance genes.

This paper reports on th
development of corn populations with hig
degree of resistance to P philippinensi
and good agronomic characteristics.


MATERIALS AND METHODS

The population improvement
methodology, modified from phenotypi
recurrent selection, used in developing tw
source populations with downy milde\
resistance and good agronomi
characteristics is presented in Figure 1.


RESULTS AND DISCUSSION

Two corn populations, designate
CPRP (Corn Pathology Resistan



RESISTANT F2 PLANTS FROM 17
COMMERCIAL HYBRIDS AND --- S
OPEN-POLLINATED VARIETIES BULK- S
SIBBED





PLA


VISUAL SELECTIONFOR VIGOR, PLANT
HEIGHT, EAR HEIGHT, AND OTHER RES
AGRONOMIC CHARACTERS DONE RA\
BEFORE POLLINATION E




I


RE


EVALUATION O
DOWNY MI
RANDOMIZED



Fig. 1. Population improvemei
corn populations with
~nfrrrnr.,~rif k. ~hr .n+nr.,.


Philipp. Phytopathol. 1994, Vol. 30(1):59-62


Population) 1 and CPRP 3, were formed
out of the resistant plants of the F2
generation of commercially-available
hybrids by bulk-sibbing. After several
cycles of recurrent selection under high
inoculum pressure, a significant increase
in the level of resistance was observed in
both populations when compared to the
reaction of the original F2 population (Fig.
2 and Table 1). Three cycles of selection
in CPRP1 population reduced the level of
downy mildew by 87% while two cycles
in CPRP3 appeared enough to bring down
infection by 86%. Visual selection for
desirable agronomic characteristics
resulted in the reduction of plant height
and ear height, increase in ear size and
tassel size, and improvement in general
plant vigor and standability.

The few cycles of selection needed
to improve the level of resistance to




'E POPULATION
CYCLE O
SUSCEPTIBLE SWEET CORN
4, SPREADER ROWS AS SOURCE
OF INOCULUM

ISCEPTIBLE
TS DISCARDED



TANT PLANTS
OM-MATED BY
LK-SIBBING
| EARS HARVESTED, KERNELS AT BOTH ENE
OF EAR DISCARDED, ONLY EAR ROT-FREE
SAMPLES SELECTED FOR NEXT PLANTING(

CYCLE 1



AT PROCESS


CYC{ES OF SELECTION IN
)EW NURSERY USING
DMPLETE BLOCK DESIGN



methodology adopted in developing
downy mildew resistance and good
'Q










30





15 +CPRPC2

10

5
0 W---Y x "
5 10 15 21 27 32 37
Days Alter Emergence
ig. 2. Disease progress of Philippine
downy mildew on different cycles
of recurrent selection in corn
populations of CPRP, and CPRP3.


able 1. Percent infection of Philippine
downy mildew on different
cycles of recurrent selection in
corn populations CPRP,, and
CPRP3 at one month after plant
emergence.


POPULATION PERCENT
INFECTION

Cycle 0 33.46 a
CPRP3C1 5.24 b
CPRP1C1 4.91 b
CPRP1C3 4.51 b
CPRP3C2 4.41 b
CPRP1C2 2.04 b

lean followed by the same letter are not significantly
fferent at 5%.
philippinensis appears to indicate a high
egree of heritability of the character.
Inly a few genes with additive effects
ave been estimated to govern downy
mildew resistance, a quantitatively-
iherited character (Kaneko and Aday,
980). Borges (1987) reported that
general combining ability effect is more
nportant than specific combining ability
i a diallel cross of six inbred lines and
hat maternal and reciprocal effects
contributed to variation indicating the role


61


f cytoplasm factors. Simple selection
procedures, such as mass selection and
henotypic recurrent selection, have been
proposed for use with characters governed
y a few genes with strong additive
effects (Randle et al., 1984; Raymundo,
991; Gardner, 1978).

Simultaneous selection for agronomic
iaracters of importance resulted in
gnificant improvement. As Miles et al.
980) asserted, mass selection schemes
an be effective in producing gains in
distancee to leaf blight, stalk rot, and yield
atential in corn populations. These
:hemes provide the possibility for
electing for resistance to other pathogens
iat attack the corn crop at subsequent
:ages of growth. Evaluation for reaction
Such diseases as Rhizoctonia sheath
ight, bacterial stalk rot, and other leaf
seases are easily accomplished with the
appropriate artificial inoculation
*chniques Raymundo and Pascual, 1989;
taymundo et al., 1993).

A corn population with improved
eld potential and disease resistance
wouldd provide a source of lines combining
>r both characters (Miles et al., 1980).
uch a population is needed in current
hilippine breeding programs that tend to
Ignore the importance of host plant
distancee (Raymundo et al., 1993). The
itrogression of foreign germplasm,
roven to be susceptible to many
digenous pathogens, has exacerbated
ie disease situation which is already
ivored by the ideal tropical environment.
focus on multiple disease and insect
sst resistance is an imperative that
iould have long been recognized.

Baldos (1994) underscored the
nportance of an adequate level of
distancee to pests and diseases for the
ill exploitation of heterosis in crosses
between tropical and temperate
ermplasm in hybrid corn breeding. He
suggested more collaborative work
between public and private corn breeders
i population improvement programs to
overcome this limitation. The present
aper proposes that the inclusion and
active participation of plant pathologists
nd entomologists in corn breeding






62


programs and that higher priority mu
be given to multiple disease and pe
resistance.These are some of the mo
specific things that should be consider
seriously in order to attain a reliable ar
sustainable approach in minimizing tt
downy mildew and other pest problem:


LITERATURE CITED

ADAY, B. A. 1974. The Philippir
program on breeding for resistance
to downy mildew of maize. Pro
Symp. on Downy Mildew of Maiz
Trop. Agr. Res. Ser. No. 8, pp. 20
219.

BALDOS, D. P. 1994. The hybrid coi
technology: An assessment of
progress. Philipp. Agric. 77: 77-81

BORGES, 0. L. 1987. Diallel analysis
maize resistance to sorghum dowr
mildew. Crop Sci. 27: 178-180.

EXCONDE, O. R. and A. B. MOLINA, JI
1978. Note: Ridomil (Ciba-Geigy),
seed dressing fungicide for tt
control of Philippine corn dowr
mildew. Philipp. J. Crop Sci. 3:6C
64.
GARDNER, C. 0. 1978. Populatic
improvement in maize. In: Mai;
Breeding and Genetics, D. B. Walde
ed., pp. 207-228. Wiley, New Yor


KANEKO, K. and B. A. ADAY. 198C
Inheritance of resistance to Philippin
downy mildew of maize
Peronosclerospora philippinensis
Crop Sci. 20: 590-594.

MILES, J.W., J.W. DUDLEY, D. G. WHITE
and R.J. LAMBERT. 1980. Improvin
corn population for grain yield an
resistance to leaf blight and stalk ro
Crop Sci. 20: 247-251.

RANDLE, W. M., D. W. DAVIS, and J. \
GROTH. 1984. Improvement an
genetic control of partial resistance
in sweet corn to corn leaf rust.
Am. Soc. Hort. Sci.: 109: 777-781

RAYMUNDO, A. D. 1991. Geneti
variability for resistance to Puccini
polysora Underw. in corn. Philipl
Phytopathol. 27: 36-40.

RAYMUNDO, A.D. and C.B. PASCUAL
1989. Screening and evaluatio
techniques for resistance to down
mildew in corn and sorghum. Proc
Symposium-Workshop on Evaluatio
of Host Plant Resistance t
Pathogens and Insect Pests in Cor
and Sorghum. Univ. So. Mindanat
Kabacan, No. Cotabato, July 20-21
1989.

RAYMUNDO, A.D., B.J. CALILUNG, JR
A. T. AQUINO, and D.P. BALDOS
1993. Evaluation of multiple disease
resistance of the F2 generation c
corn hybrids. Philipp. Agric. 76:13
19.











a Philippine 'hytopatnology or at least one autnor mu
ty. The Editorial Board, however, may relax this rule i
of exceptional merit. It may also invite distinguished
es of interest to the Society.

scripts must be reports of original research, except n
Id have not been published elsewhere. The decision of
at or reject the manuscript is final.

manuscript should be typed on one side of 8' x 11 inc
ighout.


Biol., Plant Dis. Reptr., J. Agr. Res., Amer. J. Bot.

lowledgements should be placed at the end of the article i.e. a


-s should be numbered consecutively, and each typed on a separ
have descriptive headings and should be understandable withoi
ext. Lower case superscript letters are to be used for footnotes I
rining tables should follow Literature Cited and should be nun


res should add clearly to an understanding of the paper. The si
s of figures (graphs, line, drawings, and photographs) should
lal page. Combine illustrations in composite cuts when possible
unit to correspond with the text figure reference, using cons
,rals. Label each illustration in pencil on the reverse side with the
author's name. Legends for figures should be typed together on a
1 page following the tables.

atest journal of Philippine Phytopathology for more details on
rs to oe submitted to the journal.


re number
irate num-





I




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