Front Cover
 Table of Contents
 Front Matter
 Stenocarpella disease complex in...
 Stenocarpella disease complex in...
 Phytophthora nicotianae var. parasitica...
 Evaluation of multilines for bacterial...
 Cultural and morphological characterization...
 Suppression of two levels of meloidogyne...
 Abstracts of papers presented during...
 Back Matter
 Back Cover

Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00090520/00047
 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-December 2004
Frequency: semiannual
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00047
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
    Table of Contents
        Table of Contents
    Front Matter
        Front Matter
    Stenocarpella disease complex in corn. I. Disease progress as affected by site on inoculation and inoculum concentration
        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
    Stenocarpella disease complex in corn. II. Losses as affected by site of inoculation and inoculum concentration
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
    Phytophthora nicotianae var. parasitica Water as the casual agent of leaf blight and crown rot of cordyline terminalis Kunth
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
    Evaluation of multilines for bacterial leaf blight control in rice
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
    Cultural and morphological characterization of cercospora canescens Ellis and Martin, the cause of leafspot of mungbean (Vigna radiata (L.) Wilczek
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    Suppression of two levels of meloidogyne incognita infection in tomato roots by two formulations of vesicular-arbuscular mycorrhizal (VAM) inoculant
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
    Abstracts of papers presented during the 35th anniverary and annual scientific meeting of the pest management council of the Philippines Inc., held at Amigo Terrace Hotel, Iloilo City on March 16-19, 2004
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
    Back Matter
        Page 84
    Back Cover
        Page 85
        Page 86
Full Text


Formerly Philippine Pt

January Decemb<

Published by
The Philippine Phytopathologic
c/o Department of Plant f
UP Los Banos, College,
4031 Philippines



"R 1&2

Society, Inc.


S;": | Published by the Phil



,sident R. A. Zorilla
e President G.A. Peralta
:retary P.G. Gonzales
,asurer F.M. dela Cueva
jitor O.S. Opina
siness Manager R.V. Abgona
LO L.A. Lando
ard Members C.M. Vera Cruz
M.P. Natural
T.O. Dizon
MaA.G. Maghuyop
-Officio E.B. Gergon


Associate Editors

Circulation Manager


AgChem Manufa(
Bayer Cr(
Trans World 1
Syngenta Ph
DuPont Crc

Cover photo

Dine Phytopathological Society, Inc.



President P.G. Gonzale
Vice President F.M. dela Cu
Secretary T.U. Dalisay
Treasurer L.M. Dolores
Auditor O.S. Opina
Business Manager R.G. Bayot
PRO MaA.G. Magi
Board Members T.O. Dizon
C.J.R. Cuma
C.M. Vera Cr
M.B. Palacp<
Ex-Officio R.A. Zorilla


eodora.O. Dizon
Iizaldo G. Bayot
;hristian Joseph R. Cumagun
eodora O. Dizon


ring Corporation
I., Inc.
ding Co., Inc.

JME 40 NUMBER 1 & 2


:arpella disease comr
-cted by site of inoculh
wvra and AD Ravmiu

lex in corn. I. Disease
nation and inoculum co

;arpella disease complex in corn. II. Losse
,d by site of inoculation and inoculum conce
vera and AD Raymundo

>hthora nicotianae var. parasitica Water as 1
of leaf blight and crown rot of Cordyline ter
jay, GG Divinagracia, ON Bayot and TO Dil

nation of multilines for bacterial leaf blight cor
\bien, MC Abalos, MP Femando

al and morphological characterization of Ce
cens Ellis and Martin, the cause of leafspot
i radiata (L.) Wilczek
rrater and FM dela Cueva

session of two levels of Meloidogyne incogni
ato roots by two formulations of vesicular-al
rhizal (VAM) inoculant
wurinaria, JI Orajay and MB Brown

acts of Papers presented during the 35th Ant
annual Scientific Meeting of the Pest ManagE
Philippines, Inc. held at Amigo Terrace Hot
S16-19, 2004



;e progress

s as

the causal

zon 26-34

ntrol in rice

of mungbean


ita infection


niversary and
ement Council
el, Iloilo City on



.a a a..nl I- *a Asl.a 6a6A ,

ar infection. Ear rot severity reached 12.67% and 1

INTRODUCTION 24 to 300C with a minimum temperatu
^% gA-.. --asa. 1L .. .

1996 to October 1996 (Season B).
Presently, one of the diseases
affecting the crop that has received worldwide Description of the Experimental Area
attention is a complex caused by
Stenocarpella macrospora (Earle) Sutton The soil in the experimental area was
(formerly Diplodia macrospora Earle) (Latterell analyzed prior to planting. The soil properties
and Rossi, 1983). The disease was and analytical procedures used are presented
previously considered of minor economic in Table 1 (PCARRD, 1980).
importance but recently became alarming as it
affects not only the leaves but also the stalks Experimental Design and Treatments
and the ears (Turner and Bell, 1978).
The experiment was laid out in
In the Philippines, D. macrospora was factorial design arranged in a randomized
recorded in 1931 first as causing leaf spot complete block design with three replications.
disease (Stevens and Celino, 1931) and stalk Seven sites of inoculation, leaf, stalk, ear, leaf
and ear rot diseases in 1987, the latter as and stalk, leaf and ear, stalk and ear and leaf,
high as 29% in some farms in South Cotabato stalk and ear served as factor A. The five
(Dalmacio and Lozano, 1987). inoculum levels (Uninoculated, 1 x 104, 2 x
104, 3 x 104 and 4 x 104 conidia/ml) were
The nature of relationship between assigned as factor B. Each sub-plot had an
the leaf blight phase and the other phases of area of 3.0 x 5.0 m2. A one-meter pathway
the disease is not fully understood although was provided between main plots and 0.5 m
stalk rot and ear rot infections appear to be pathway between sub-plots. Plots were
dependent upon leaf infection (Dalmacio and thinned to a density of 53,333 plants per
Lozano, 1987). hectare. All data were collected on the two
center rows of the four-row plot.
Limited information can be found
regarding the development of the disease, Cultural Management Practices
particularly on the influence of weather factors
and on the method of evaluating disease Land Preparation
severity and amount of inoculum to create
maximum disease pressure on the hpst. The experimental area was
These are key information in screening for thoroughly plowed and harrowed twice until a
host resistance. well-pulverized soil was attained. Furrows at
a distance of 0.75 m apart were prepared.
This study, therefore, was done to IPB 929 seeds were sown within furrows at a
evaluate the effect of the amount of inoculum distance at 0.25 m per hill. Thinning was
and plant part inoculated on the development done to one plant per hill one week after
of the disease, emergence.

MATERIALS AND METHODS Fertilizer Application

Disease Development in Relation to Site of The fertilizer rate 60-60-0 was applied
Inoculation and Amount of Inoculum based on the soil analysis done by the Soil
Concentration and Plant Tissue Laboratory (SPTL) at the
Department of Soil Science, Central
Time and Place of Study Mindanao University.

The field experiments were conducted Split application o0 fertilizer was done.
at the Agricultural Experiment Station, Central One half of N and all of P were applied basally
Mindanao University, Musuan, Bukidnon. The during planting and the remaining N was side
first season experiment was conducted from dressed at about 25 days after sowing.

I _ _ __ _

Stenocarpella disease complex

Cultural Operation

Hand weeding was done at 15, 30
and 45 days after emergence. Periodic
spraying of Karate (Deltamethrine) insecticide
was done to prevent corn borer damage.

Furadan 3 G (Carbofuran) was
incorporated in the soil at 10kg/ha before
planting to protect the seeds from red ants
and other insects.

Mass Production and Preparation
of Inoculum

Mass production of inoculum was
done using plated oatmeal agar incubated for
at least 30 days to allow abundant production
of pycnidia. Spore suspensions were
prepared either by pricking the harvested
pycnidia using 2 needles or by macerating
cultures using a Warring blender and filtering
through cheesecloth. The resulting
suspension was adjusted to the desired
concentration for inoculation.

Concentration of Fungal Inoculum

Four spore concentrations were
prepared. These were 1 x 104 conidia/ml, 2 x
10' conidia/ml, 3 x 104 conidia/ml and 4 x 104
conidia/ml. Using a haemacytometer, the
highest spore concentration of 4 x 104
conidia/ml was determined and served as
stock concentration. The other concentrations
were prepared from this original suspension
by ratio and proportion.

Inoculation Methods and Inoculation Sites

Plants were leaf-inoculated at 35,
stalk-inoculated at 55 and ear-inoculated at 70
days after emergence (2 weeks after silk
emergence). With a disposable syringe, 1 ml
of the different spore concentrations was
dropped at various inoculation sites. The
whori method of inoculation was used in
inoculating leaves of 35 day-old corn plants.
The inoculation on the stalk was done by
injecting the spore concentration above the
nail-punctured fourth node below the
developing ear of the 55 day-old plants. The
inoculated parts were wrapped with parafilm
to prevent immediate drying of inoculum. The

ear inoculation was done by injecting the
spore concentration at the outt end, 2 weeks
after silk emergence. Al inoculations were
made during the late afternoon.

Data Gathering

Leaf Blight Rating. Disease
development was initially assessed 14 days
after inoculation and at 14-day intervals
thereafter until harvest. Ten plants within
each subplot were tagged randomly and used
for disease assessment. Time of assessment
was recorded as number of days after
inoculation (DAI). Rating was done by
measuring the lesion size and counting the
number of lesions starting from the first
symptom appearance on the whorl and at 14
day-intervals. The data obtained were
converted to percentage diseased leaf area
per plant by measuring the total leaf area of
10 plants after anthesis. The position of each
sampled leaf was determined by numbering
the leaves from bottom to the top of the corn
plants. The values were averaged to
determine the percentage disease for each

lesion size x number of lesions

% disease =

average leaf area

Apparent infection rate was calculated
using Vander Plank's equation (Vander Plank,

1 X 2 X
r = [------] (loge --- oge ----]
t2-tl 1 -x2 I-x

where: r = rate of infection
tl = initial time of disease
t2 = final time of disease
log, = natural logarithm
xl = percent disease at ti
x2 = percent disease at t2

Stalk Rot Rating. The severity of
stalk rot infection was assessed at 14-day
intervals after inoculation using five border
plants in each sub-plot. The stalks were split-
opened longitudinally through the inoculated

area and the extent of rotting was assesse

The extent of rotting was determine
based on the scale used by Rosenow ar
Rao (1980) where: 0 = less than or
internode affected, 1 = one internode affect
but does not pass through any nodal area, 2
2 intemodes, 3 = more than two intemodes,
= more than three intemodes and 5
extensive rotting, shredding and death.

The percentage infection index wi
computed using the equation:

% stalk Ono + n + 2n2 +3n + 4n4+5n
rotting= .x
where: N n'number of rotted stalks, -"
Ono + ........5ns = stalks
showing the scale of 0,
1, 2, 3, 4, 5, respectively
5 represents the highest scale

Ear Rot Rating. Disease severity
individual 10 sampled ears was determine<
based on the scale used by Klapproth ar
Hawk (1991) with modification; where: 0 = r
visible infection, 1 = 1 to 25% infect
kernels, 2 = 26 to 50% infected kernels, 3
51 to 75% discolored and infected kernels ar
ears fully covered with mycelium, 4 = 76%
100% discolored and infected kernels ai
ears fully covered with mycelium and 5 = i
kernels and cob completely rotted.

The percentage infection index w;
computed using the equation:

%ear Ono+ inl +2n + 3n + 4n4 + 5n
rotting= -x'
where: N = number of infected kernels
On, + ........5ns = ears
showing the scale of 0,
1, 2, 3, 4, 5, respectively
5 = represents the highest scale

Data Analyses

Data obtained were analyzed usir

experimental data analysis. Fisher's Least
Significant Difference (FLSD) was used in
comparing the treatment means.


Effect of Inoculum Concentration of
Stenocarpella macrospora and Site of
Inoculation on the Progression of the
Leaf Blight, Stalk Rot and Ear Rot
Complex in Corn

Leaf Blight Severity

Leaf blight symptoms were observed
as early as 3 days after whorl inoculation
during both cropping seasons. The lesion
appeared as pin size and irregular white spots
around the inoculation area above the whorl.

The disease increased both in
Seasons A and B (Fig. 1 and 2). The disease
progression was fast on plants that were
whorl-inoculated (leaf and its combination
sites) with high amount of conidial suspension
(4 x 104) compared to the control. Plots that
were not inoculated on the leaf (stalk and
stalk-and-ear) showed natural infection that
was significantly lower than the infection in the
inoculated plots.

During Season A, the disease
increased steadily over time. The apparent
increase of tissue blighted was observed from
28 to 56 days after inoculation regardless of
the site of inoculation and the amount of
inoculum. The disease was evidently high on
plots that were whorl-inoculated with various
amounts of inoculum. The apparent infection
rates-(r) were estimated at 0.04003, 0.0434,
1 0.0587, 0.0366, 0.03962, 0.04867 and
0.03502 per day in leaf, stalk, ear, leaf-and-
stalk, leaf-and-ear, stalk-and-ear and leaf-
stalk-and -ear inoculation sites, respectively.

During Season B, the disease
progress curve increased gradually from 14 to
42 days after inoculation and then abruptly
increased at 56 until 70 DAI.
Control plots among the different sites
of inoculation showed natural leaf blight

estimated as 0.0462, 0.0543, 0.0691, 0.0489,
0.0520, 0.0647 and 0.0467 per day at leaf,
stalk, ear, leaf-and-stalk, leaf-and-ear, stalk-
and-ear and leaf-stalk-and-ear inoculation
sites, respectively.

Stalk Rot Severity

The severity of stalk- rot varied in
seasons (Fig. 3 & 4). During Season A, the
percentage stalk rotting (14 DAI) was
significantly higher (40%) in plots stalk-
inoculated with various amounts of inoculum
(1 x 104, 2 x 104, 3 x 104 and 4 x 104
conidia/ml) compared to the control (0%).
Stalk rot was not observed on plants that were
not inoculated on the stalk (leaf and leaf-and-
ear combination). There was not much
change in stalk rotting at 21 days after
inoculation. Significant increase was noted at
28 DAI with concentrations 3 x 104 (58.33 %)
and 4 x 104 conidialml (60.33%) compared
with the control (0%). Leaf-and-stalk
inoculation combination had 58.33% at the
same amount of inoculum. The stalk-and-ear
and leaf-stalk-and-ear have 65.00, 66.67 and
65.00 and 68.33%, respectively.

A similar trend was observed during
the season B, although the range was fr-m 0
(control) to 60.00 % with the highest inoculum
level (4 x 104 conidia/ml).

A significant difference on stalk rot
severity was observed among the various
inoculum concentrations compared with the
control. The percentage stalk rotting
increased with an increased inoculum

Percentage Ear Rot Infection

The site of inoculation and inoculum
concentration influenced the degree of ear rot
infection in both cropping seasons (Fig. 5).
The percentage ear rot infection was higher
during Season A compared to that of Season

In aH instances, ear inoculated plants
l i_ ,m, ,lii,*-, u-;6k trsiui- o rldffta rA4

af-and-ear, stalk-and-ear and leaf-stalk-
d-ear) exhibited a significantly higher ear
infection with an increasing amount of
culum compared with the control.

During Season A, plants inoculated in
rs and in combination with various plant
rts exhibited the highest percentage ear rot
action with an increasing amount of
culum ranging from 96.33 to 100%
spared to the control (4.33 to 12.67%). The
ferent inoculum level, however, did not
fer significantly on their effect on
rcentage ear rot infection.

Plants inoculated on the leaf with
ious amount of inoculum showed
parable ear rot infection with the control
its ranging from 10.33% to 12.67%. Stalk
culated plots exhibited similar level of
rcentage ear rot infection (10 to 15%) but
ry significantly with the control (2%). Similar
nd of percentage ear rot infection was
ted on leaf-and-stalk site of inoculation.

During Season B, percentage ear rot
action was significantly higher in plots
iculated with varying inoculum
ncentrations compared to the control.
ants inoculated in the ear alone exhibited
t highest percentage ear rot infection (97%)
h the highest amount of inoculum (4 x 10
nidialml) compared with the control (10%).
tween the different levels of inoculum,
ever, no significant difference was
served among each other. Ear rot infection
:reased with increasing inoculum


Effect of Inoculum Concentration of
Stenocarpella macrospora and
its of Inoculation on the Progression of
the Leaf Blight, Stalk Rot and
Ear Rot Complex in Corn

Leaf Blight Severity. S. macrospora
n be extremely destructive as it can cause
ee phases, namely leaf blight* stalk rot and

__ _ _


time during the two seasons, a characteristic
of a polycyclic disease. The increase
however, was generally low despite the
varying amounts of inoculum available for the
onset of the disease. Nevertheless, it was
evident that an increase in the amount o
inoculum induces a corresponding increase ir
disease progress. It follows that the higher the
initial inoculum the faster the development ol
the disease with respect to time (Ward et al

All plants inoculated on the leaf site
and in combination with other plant parts
exhibited a leaf blight severity higher thai
those inoculated on the stalk and ear alone
during both seasons.

The low level of disease observed
may be attributed to the resistance of the hosi
to the leaf blight phase and to unfavorable
weather factors. This was evident as more
lesions developed only on the inoculated
leaves but only few on upper leaves. Hosl
kind and weather factors greatly affect the
progress of the disease evident as more
lesions developed only on the inoculated
leaves but only few on upper leaves. As
mentioned by Vander Plank (1963), the
development of the disease is affected by foui
important factors, namely; infection,
sporulation, latent period and loss ot
infectious tissues. The susceptibility oi
resistance of the host to the pathogen affects
each of these processes. In addition, the
lower levels of disease and low rate ol
progress may indicate an inhibition ot
pathogen development or poor hosi
colonization in resistant cultivars as
expressed by the development of few lesions
despite the availability of inoculum (Berger el
al, 1977).

Under environmental conditions
favorable for disease development, small
differences in the number of lesions, lateni
period and sporulation contributed a
significant effect in disease severity and rate
of disease progress (Ringer and Grybauskas,
1995). In addition, the development of
secondary infections that were influenced by
weather conditions determined the severity of

~uIIUU upaillu mE l 1o;1.

Stalk Rot. Results indicated the
absence of stalk rot development in plants
inoculated on the leaves. Stalk rotting
developed only on parts inoculated with
varying inoculum levels on the stalk and in
combination with other plant parts in both
seasons. The stalk rotting, however, was
slightly higher during season A than during
season B. In addition, the percentage of stalk
rot increased with an increase in the amount
of inoculum (1 x104, 2 x 104, 3 x 10 and 4
x104 conidia/ml).

The absence of stalk rot in plants
inoculated on the leaves agrees with the
findings of Olantinwo et al. (1999). However,
they emphasized that the zero incidence of
stalk rot does not indicate a non-existence of
stalk rot but it could indicate unfavorable
conditions for stalk rot development at the
time of disease assessment In addition, cor
is most susceptible when inoculated just
before the time of pollen production and
thereafter, depending upon the variety

Ear Rot Severity. Site of inoculation
and inoculum level significantly influenced the
degree of ear rot severity. In all instances,
plants inoculated on the ear and in
combination with other plant parts showed
significantly more severe ear rot infection
compared with those inoculated on the
leaves, stalk and its combinations in both
cropping seasons. In addition, ear severity
increases with an increase in the amount of
inoculum level (1 x104, 2 x 104, 3 x 10' and 4
x104 conidia/ml).

The results indicated that cor ears
are susceptible to the ear rot pathogen, S.
macrospora at the time of inoculation. The
period just prior to and during silking is critical
for ear development (Prine, 1971) and less
disease was obtained when inoculation was
made at later dates (Ullstrup, 1970). This
period is the beginning during which rapid dry
matter accumulation in the kemels continues
at essentially a constant daily rate until all the
kernels are fully dented (Hanway, 1963).

.aMnn r~ naia dieAin

Likewise, the severity of Diplodia ear rot was
directly proportional to the concentration of
spores in the inoculum (Ullstrup, 1970).

Although, ear rot infection was low on
leaf- and stalk-inoculated plants alone and in
combination, this indicated that ear rot
infection occurs when the leaves and stalks
are infected with the pathogen. This is also
an indication that conidia from the leaves may
be deposited near the base of the earshoot,
germinate and penetrate the outer husks
causing ear infection (Dalmacio and Lozano,
1987). The deposition of conidia may be due

maydis) compared as pathogens of
com. Plant Dis. 67: 725-729.

Epidemiology of Stenocarpella
macrospora (Earle) Sutton on Maize
in the Mid-altitude Zone of Nigeria. J.
Phytopathol. 147: 347-352.

PCARRD. 1980. Standard Methods of
Analysis for Soil, Plant Tissue, Water
and Fertilizer. Philippine Council for
Agriculture and Resources Research
and Development (PCARRD), Farm

Alovera and Raymundo
VANDER PLANK JE. 1963. Plant Diseases:
Epidemics and Control. Academic
Press, New York. 349pp. ACKNOWLEDGMENT

WARD JMJ, EL STROMBERG, DC This study was supported by the
NOWELL and FW NUTTER JR. Central Mindanao University (CMU)

. ulay it
il imp
action. P

ie chemi


e K (pprr

IpJU n% ul00a0 vl viuvelUpllaIll
ice in maize
)is. 83:884-895.

operties of the soil and the methods u



Ljidl aI J.



H meter
No. 2 Method
otometer Met

- 40



14 2< 42 5i
DayI alter Inoculation (D)AI)
lxat'i(r 04003)




14 ,2 42 56 70
Days after Inoculation (I)AI)
Stalk (r --.0434)

1! 2" 2 56 70
DA illr l"o ulllion (IDAl)
I enl';f kld lr 0 30 6 )

Legend: r --Apparent Infection Rate
CO-- Conto
C2 2 x 104 conidia/ml

14 4 5- 70
)u lr Inocu ( A)\9 )
I iant tI ar (di .03 12)

C1 1 10 lconidia/ml
C4 4 x 10 conidia/ml

I I' 42 5( 70
) i)ls i .l oculalion ( I ) \ 1
S il. a o r 0.1 ,"

C3 3 x 10 conidia/ml

Figure 1 Disease progress curves of corn leaf blight caused by Stenocarpella macrospora
(Ear e) Sutton at various sites of inoculation from November 1995 to March 1996
(SeasOn A),

1 2S 42 56 70
uY after Inoculaliion (DAI
Fiar (r- .0587)


-40 ClI
30 e'C2
F 30 C3
20 m NEt C4
10 -

14 28 42 56 70
Days after Inoculation (DAI)
Leaf(r =.0462)

,2 40
5 30
: 10

14 28 42 56 '0
Days after Inoculation (DAI)
Eai (r =0691)

m 30

14 28 42 56 70
Days after Inoculation (DAD)
Leaf and Stalk (r =.0489)

I 40

14 28 42 56 70
Days after Inoculation (DAI)
Leaf. Stalk and Ear (r -.0467)

m 30
14 28 42 56 70
Days after Inoculation (DA
IStalk (r= (0543)

50 -
m 30
14 28 42 56 0
Days after Inoculation (IDA
Stalk and Ear (r =.0647.

2 20
14 28 42 56 70
Days after Inoculation (DAI)
Leaf and Ear (r =.0520)

Legend: r Apparent Infection Rate
CO Control
C3 3 x 104 conidia/ml

C1 -1 x 104 conidia/ml
C4 4 x 104 conidia/ml

C2 2 x 104 conidia/ml

Figure 2. Disease progress curves of corn leaf blight caused by Stenocarpella macrospora
(Earle) Sutton at various sites of inoculation from June to October 1996 (Season B)

080 01- D 80 Q Leaf
7 28 DAI 70 14 DAI iStalk
6 60 0 Ear
6(1 Leaf & Stalk
50 UW Leaf & Ear
4(1 40 Q Stalk & Ear
3(0 30 i Leaf, Stalk & Ear

J 1 4j
10 10

CO Cl C2 C3 C4 CO Cl. C2 C3 C4

Inoculum Concentration Inoculum Concentration

80 "
7021 DAI
50- '
1 40(


CO Cl C2 C3 C4

Inoculum Concentration

Where: CO -Control C1 1 x 104 conidia/ml C2 -2 x 104 conidia/m!
C3 3 X 104 conidia/ml C4 4 X 104 conidia/mi
DAI days after inoculation

Figure 3. Stalk rot severity of corn due to Stenocarpella macrospora (Earle) Sutton as influenced by
site of inoculation and inoculum concentration from November 1995 to March 1996
(Season A,.

Site of inoculation

C(0 ( 2 (i3 C4

() C'I (2 ('3 C4
Inoculum i on(centraiion

21 DA \I

lnoculumnl ("c.tciC.t liO6lI

(CO Cl ('2 C3 C4
Inoculum Concentration

Where: CO Control C1 1 x 104conidia/ml
C3 3 x 104 conidia/ml C4 4 x 104 conidia/ml
DAI days after inoculation

C2 2 x 104 conidia/ml

Figure 4. Stalk rot severity of cor due to Stenocarpella macrospora (Earle) Suiton as influenced
by site of inoculation and inoculum concentration from June to October 1996 (Season B).

Site of inoculation

~.~ i ,, I
,S() ||1 I B 1 o ill

0 1 I .. 11
W r C -Cnrl1 1 x10 coidami C 2 x 104 n. id am

('O (il (2 C(3 C4
Inoculum Concentration

Season A

(i Cl C2 (C3 '4
Inoculuin Concentratioii
Season l1

Where: CO Control C1 1 x 104 conidia/ml C2 2 x 104 conidia/ml
CO 3 x 104 conidia/ml C4 4 x 104 conidia/ml



Portion of the Ph. D. dissertation of the senior author submitted to the Graduate School,
University of the Philippines Los Baflos (UPLB), College, Laguna.

Respectively, former graduate student, Department of Plant Pathology, UPLB, College,
Laguna now Professor, Department of Plant Pathology, CMU, Maramag, Bukidnon, and
Professor, Department of Plant Pathology, UPLB, College, Laguna

Yield and yield components were significantly influenced by site of
inoculation and level of inoculum. Yield varied in both seasons. Yield
ranged from zero to 9.02 tons/ha during season A and from zero to 7.6
tons/ha during season B. Zero yield (poor quality of kernels due to thick
mvcelial growth of the funausa was obtained amann nlntA inneti.latd in tha

alone or in combination with other plant parts had 100% loss regardless of
the amount of inoculum.

Using disease severity during different and crop growth stages as
predictors, the following response-surface models of yield loss (Y) were
developed for Seasons A and B, with R2 values of 0.852 and 0.83,

1) Y = 10.0 + 0.104 X(Ls) + 0.040Xssa4) + 0.021XsR2a) + 0.929(ER),
2) Y = 18.487 + 0.182X(sR28) +0.92 XER)

where LB56 is leaf blight severity at 56 days after inoculation (DAI) or (91
days after emergence (DAE), SR14 is stalk rot severity at 14 DAI or (69
DAE), SR28 is stalk rot at 28 DAI or (83 DAE), and ER is ear rot severity at
harvest (ER)

Keywords: corn, Stenocarpella macrospora, ear rot, stalk rot

INTRODUCTION corn production as it can cause ear rotting
aside from blighting of leaves and recently
Although reports on Stenocarpella, stalk rot (Dalmacio and Ldzano, 1987). Ear
previously known as Diplodia, are limited rot incidence has been reported to reach as

Alovera and Raymundo

Yield loss data due Diplodia ear rot The experiment was laid out in
disease are scanty. In Zambia, where D. factorial design arranged in a randomized
macrospora Earle is the most common complete block design with three replications.
pathogen of corn, losses of up to 75% were Seven sites of inoculation, leaf, stalk, ear, leaf
recorded especially in pscx unwfertilized crops and stalk, leaf and ear, stalk and ear and leaf,
(Logan 1n74. Similarly, Chambers (1988) stalk and ear served as factor A. The five
~urded a yield loss (grain wt/plant) of as inoculum levels (Un-inoculated, 1 x 104, 2 x
high as 97% from inoculation of D. 104, 3 x 104 and 4 x 104 conidia/ml) were
macrospora made 10 days after mid-silk. assigned as factor B. Each sub-plot had an
area of 3.0 x 5.0 m2. A one-meter pathway
Llano and Schieber (1980) reported was provided between main plots and 0.5 m
appreciable losses in yield, stored grain and pathway between sub-plots. Plots were
poultry feed on infected grain while Marasas thinned to a density of 53,333 plants per
and van der Westhuizen (1979) indicated 1 hectare. All data were collected on the two
5 % kernel infection, center rows of the four-row plot

In the Philippines, Franje (2000) Mass Production and Preparation
recorded different yield losses of two varieties, of Inoculum.
CMU Var 12 and GSI 40 M, and stages of
growth most critical to inoculation. CMU Var Mass production of inoculum was
12 had 17.73% yield loss when inoculation done using plated oatmeal agar incubated for
was done during the whorl stage and at at least 30 days to allow abundant production
flowering stage while GSI 40M had 11.83% of pycnidia. Spore suspensions were
and 7.94% during 25% silking and blister prepared either by pricking the harvested
stages of growth, respectively. pycnidia using two needles or by macerating
cultures using a Warring blender and filtering
Due to the fact that the disease is a through cheesecloth. The resulting
complex involving three phases, differences in suspension was adjusted to the desired
losses can be expected especially when these concentration for inoculation.
phases occur at different times and under
conditions of varying amounts of inocula. So Concentration of Fungal Inoculum
far, the consequences of possible interactions
amonq these phases are not known. This Four spore concentrations were

therefore, wi
termine the
elation and li
Id compone
as model as
talk rot and 4


The experin
Alovera and
study on
irpella dises
i utilized in c

hired. These
onidia/ml, 3
ia/ml. Usi
st spore i
ia/ml was
prepared ft
to and propc

elation Mett

Plants i
avs after en

nd 4 x
neter, 1
4 x

tion Sitb

ad at
s after !

r ...... .. I --- .' ....... 7 """ J"-' -' - ~~--- - -- -_- '

Stenocarpella disease complex

stalk was done by injecting the spore
concentration above the nail-punctured fourth
node below the developing ear of the 55 day-
old plants. The inoculated parts were
wrapped with parafilm to prevent immediate
drying of inoculum. The ear inoculation was
done by injecting the spore concentration at
the butt end, 2 weeks after silk emergence.
All inoculations were made during the late

Corn ears were manually harvested
and shelled at their maturity date. Yield and
yield components were obtained from the 1.5-
m x 5.0-m square area two inner rows at each
sub-plot. The weight of shelled grains and
percentage moisture content were determined
using the Steinlite Moisture Tester. Twenty
randomly selected ear samples were used in
measuring yield components. Yields were
converted to tons per hectare at 14% moisture
content as follows:

Grain yield (ton/ha) was computed
based on the equation:

axb 100-MC GW
Adjusted Plot Yield = x -- x -
c 86 EW

Adjusted Plot yield
Adjusted Yield/ha = --- x 1 ha
Harvestable Area

where: a = Fresh weight of ears per plot
b = Stand count per plot
c = Perfect Stand
MC = Moisture Content
GW = Grain weight of 20 ear
samples (shelled)
EW = Ear weight of 20 samples

The values for percentage loss were
calculated from the yield of inoculated and
control plots within the subplots using the

YL = x 100

where: YL = yield loss
YH = yield of healthy plants
YD = yield of diseased plants

Yield loss assessment was based on
the poor quality of ears as indicated by the
shriveling and discoloration of kernels and the
presence of thick mycelial growth of the
fungus in cobs and ears. As a result, ears
were discarded and gain weight was not

Yield, yield parameters and yield loss
data were subjected to analysis of variance
(ANOVA). The regression method of SPSS
Ver 8.0 Windows software was used to
develop a model showing the relationship
between disease seventy rating of various
plant parts, taken in Part I of the study, with
percent yield toss. The disease severity rating
of various plant parts affected served as the
independent variable and percentage yield
loss as the dependent variable.


Grain Yield (tonslha) as Affected by Site of
Inoculation and Inoculum Concentration

Grain yield per hectare as affected by
Stenocarpella disease complex varied in both
seasons (Table 1).

The yield during season A ranged
from 0 to 9.02 tons/ha. The zero yield (poor
quality due to thick mycelial growth of fungus
in the kernels and cob) was obtained in the
plots inoculated in the ear and in combination
with various sites of inoculation regardless of
the amount of inoculum (1 x 10', 2 x 104, 3 x
104 and 4 x 104 conidia/ml) applied as
compared with the un-inoculated (control).
These ears were covered with heavy mycelial
growth extending into the grains and cobs. At
harvest, ears were badly-infected, moldy and
unfit for feed. These were discarded.

Alovera and Raymundo

Among the inoculated plants that
produced quality grain yield, the leaf-
inoculated had the highest yield. The yield,
though, decreased with the application of an
increasing amount of inoculum as compared
with the control. Yields obtained on the stalk
and leaf-and-stalk inoculated combinations
were comparable among the different levels of
inoculum. It is apparent that grain yield
decreased as the inoculum concentration

Among the different sites of
inoculation, the leaf site showed the highest
yield of 9.02 tons/ha. This was significantly
followed by stalk (6.60 tons/ha), leaf-and-
stalk (5.24 tons/ha), leaf-and-ear (5.23
tons/ha), stalk-and-ear (4.63 tons/ha), ear
(4.31 tons/ha) and leaf-stalk-and-ear (4.29

A similar trend of yield was obtained
during Season B though it ranged from 0 to
7.60 tons/ha. Plants inoculated in the ear and
in combination with various plant parts
significantly did not give any good quality yield
in various amounts of inoculum (1 x 104, 2 x
104, 3 x 104 and 4 x 104 conidia/ml) applied
compared with the control.

Ear Diameter

The different inoculation sites and
inoculum concentrations showed a highly
significant effect on the average ear diameter
(cm) during Season A (Table 1). Plots
inoculated with various amounts of inoculum
concentration irrespective of the sites showed
smaller ear diameter compared with the un-
inoculated (control). The reduced size of ear
diameter is an indication of the injurious effect
of the pathogen.

Among the inoculated plant parts, the
leaf, stalk and in combination site (leaf-and-
stalk) applied with the lowest inoculum level
(1 x 104 conidia/ml) showed wider ear
diameter ranging from 4.00 to 4.27cm
compared with those ear-inoculated and all its
combination sites ( 3.52 to 3.70 cm).

The ear diameter was unusually
smaller on all ear-inoculated treatments
regardless of the amount of inoculum applied.
This reduced ear diameter was due to the
activity of the pathogen causing severe ear
rotting. The ear husks were pasted together
making it difficult to remove the husks.
Between the varying levels of inoculum,
however, no significant difference on ear
diameter was observed. It was apparent,
though, that ear diameter generally decreased
with an increased level of inoculum

During Season B, however, neither of
the different factors showed significant effect.
Nevertheless, plots inoculated with various
amounts of inoculum concentration
irrespective of the sites showed small ear
diameter compared with the uninoculated
control. Likewise, ear diameter was small with
an increase level of inoculum. Ears of ear-
inoculated plants and in combination with
other plant parts generally had small ear
diameter compared with those inoculated on
the leaf, stalk and its combination site.

Ear Length

The ear length of corn in both
cropping seasons was not significantly
influenced by sites of inoculation and
inoculum concentrations or its interaction.

Apparent differences on ear length,
however, were observed between the two
cropping seasons (Table 1). Corn ears were
shorter during Season A, which ranges from
12.61 to 14.99cm compared with those.ears
during Season B ranging from 13.91 to 15.25

Weight of 1000 Kernels

The weight of 1,000 seeds during
Seasons A and B ranged from 0 (poor kernel
quality) to 274.02 g and 0 (poor kernel
quality) to 260.50g, respectively (Table 1).

The ear-inoculated plant and in
combination with other plant parts of
inoculation significantly did not give any

warpella disease complex

quality grain weight regardless of the amount showed an increase in the amount of loss with
of inoculum concentrations (1 x 104, 2 x 104, an increase in the amount of inoculum (1 x
3 x 104 and 4 x 104 conidia/ml) applied 104, 2 x 104, 3 x 104 ano 4 x 104 conidia/ml)
compared with.the control (uninoculated) in ranging from 8.86 to 48.31%, 11.09 to 52.83%
both seasons. These ears rendered seed and 22.52 to 53.51%, respectively.
weight that were not of good quality since
these are covered with thick mycelial growth Similar trend of crop loss was
and pycnidia, hence, considered as discarded observed during Season B. The leaf-and-ear,
seeds. In contrast, the mean weight of seeds ear, stalk-and-ear and leaf-stalk-and-ear
on leaf, stalk, and leaf-and-stalk inoculated inoculated plants showed a corresponding
plants in both seasons showed varied grain yield reduction of 100% among the different
weight. The un-inoculated (control) plot on inoculum concentrations aoplied (1 x 104, 2 x
leaf inoculated site during the dry season had 10', 3 x 104 and 4 x 104 conidia/ml). Leaf-
higher grain weight (262.10g) compared with inoculated pants showed an increase yield
those inoculated with the varying amounts of loss with an increase !n the amount of
inoculum (lx104 conidia/ml) (261.52 g). inoculum ranging from 5.10 to 25.683%. The
During Season B, the leaf uninoculated plot stalk-inoculated had loss ranging from 38.38
had low grain weight (249.25g) compared with to 51.547% while the leaf-and-stalk
the three inoculum levels (1x104, 2 x104, and combination had loss ranging from 47.81 to
3 x104 conidia/ml) with 260.50, 254.49 and 61.033%. Among the different levels of
249.36g, respectively. On stalk inoculated inoculum concentration, however, no
plots, likewise, uninoculated (control) plot had significant differences were observed.
low grain weight (245.50g) compared with
those applied with increasing amounts of Relationship of Crop Loss and Leaf Blight,
inoculum, 248.64 (1 x 104 conidia/ml), 258.67 Stalk Rot and Ear Rot at Different Sites of
(2 x 104 conidia/ml), 253.78 (3 x 104 Inoculation and Inoculum Concentration
conidia/ml) and 258.04 g (4 x 104 conidia/ml).
The difference, however, were not statistically The different sites of inoculation and
significant. amount of inoculum greatly influenced the
disease severity complex exhibited by plant
Percent Yield Loss as Affected by Site and the degree of yield reduction incurred.
of Inoculation and Inoculum
Concentration of S. macrospora During Season A, leaf blight severity
was higher in leaf-inoculated plants ranging
The different sites of inoculation and from 16.38 (control) to 38.58% (4 x 10
amounts of inoculum increased the degree conidia/ml) and ear rot infection of 10.33
of yield losses during both Seasons A and B (control) and 12.67 % ( 4 x 104 conidia/ml)
(Table 2). In both cropping seasons, higher which incurred a corresponding percentage
losses were obtained on ear-inoculated reduction of 8.86 to 48.31 %/. Stalk rot was
plants and in combination with other plant not observed at this site.
parts regardless of the amount of inoculum
(1 x104, 2 x 104, 3 x 104 and 4 x 104 On stalk-inoculated plants, the leaf
conidia/ml) compared to the control. blight, stalk rot and ear rot complex occurred
which incurred an increase in loss with an
Losses as much as 100% were increase in the amount of noculum, 11.093
recorded in ear-inoculated plants and in (1 x104 ) to 52.83 % (4 x104 ). Ear-inoculated
combination with other plant parts. These plants and in combination with other plant
ears were covered with thick mycelial parts produced grain yield that were heavily
growth, hence of poor quality and were infected with mycelial growth in all levels of
considered as discarded, inoculum, hence incurred a 100% reduction.

During Season A, plants inoculated in Durina Season B, leaf-inoculated

Alovera and Raymundo

Leaf blight infection ranged from 8.96% rot (SR28) and ear rot (ER).
(control) to 18.33 % (4 x 10 conidia/ml) while
ear rot ranged from 5.33% (control) to 18.33% As a result, the following response-
(4 x 104 conidia/ml). Stalk rot was not surface models for estimating percent losses
observed at this site. The combined effect of due to the diseases for Seasons A and B,
leaf blight and ear rot incurred an increase in respectively, were obtained:
loss with an increase level of inoculum Y = 10.0 + 0.104 X(LB6) +
ranging from 5.51 to 25.68% (Table 2). 0.040X(sR14) + 0.021X(sR28) + 0.929X(ER)
and Y = 18.487 + 0.182 X(SR28) + 0.92
The stalk-inoculated plants showed X(ER)
complex symptoms of leaf blight, stalk rot and
ear rot severity. The level of leaf blighting These models gave corresponding
among the various inoculum concentrations, significant R2 values of 0.85 and 0.83. In
however, was very low and was comparable addition, the F values in the analysis of
with the control. The stalk rot ranged from 0 variance and t-values in the t-statistics for
(control) to 60.0% (4 x 104 conidia/ml) while each parameter estimate were highly
the ear rotting raged from 9.667 to 17.667%. significant.
This disease complex reduced the yield from
38.38 (1 x 104 conidia/ml) to 50.35% (4 x 104 Evidence showed that grain yield was
conidia/ml). significantly affected by sites of inoculation
and amount of inoculum level. There was no
Similar trend was observed on the quality grain yield obtained in all ear-
combination of leaf-stalk-and-ear inoculation inoculated plants regardless of the amount of
except that leaf blighting was higher with inoculum applied in both seasons. This only
31.183% at the highest inoculum level (4 x indicated that inoculum level significantly
104 conidia/ml) and a corresponding yield reduced the yield per ha for both Seasons A
reduction of 61.03%. and B. The ear husk tissues turned brown
prematurely with mycelial growth extending
Stalk rotting was not observed in ear- into the grains and cobs while the leaves of
inoculated plants and in combination with leaf the plants were still green.
at the time of assessment. The leaf blight in
all levels of inoculum was low and in all Prine (1971) indicated that the period
instances similar with the control. The just prior to and during silking is critical to ear
combination of ear-leaf inoculation, however, development. Hanway (1963) also indicated
showed higher level of leaf blighting in all that starch began to accumulate in the
inoculum levels compared with the control. endosperm at this time. This is the beginning
All these sites of inoculation had higher ear rot of the period of which rapid dry matter
infection in all levels of inoculum were ears accumulation in the kernels continues at
were moldy and caused yield loss as much as essentially constant daily rate until all the
100%. kernels are fully dented. At this stage, the
rate of dry matter accumulation by the plant
Model for Yield Loss. The search for has begun to decline. Hence, this stage of
the best independent variables for estimating growth during which ear inoculation was made
percentage loss (PL) was achieved using the greatly affected corn gain yield.
regression analysis of the SPSS Ver 8
Windows software. During Season A, the Stalk-inoculated plants also gave
best set of independent variables was the lower grain yield comparable with those that
combination of leaf blight severity at 56 days were leaf-inoculated. However, the
(LBs6) after inoculation (DAI) or (91 days after combination of leaf-and-stalk inoculation
emergence (DAE)), stalk rot at 14 DAI (SR14) showed significant lower yield compared with
or (69 DAE), stalk rot at 28 DAI (SR28) or (83 leaf-inoculated plants but comparable with
DAE) and ear rot at harvest (ER). During the stalk-inoculated plants. At these sites of

Fni..fUIpIrI UIwo w %AI UIII IA

il.U;uIdUUo yraFil yeluu UI plnidi lgllludnimluy
decreased as the amount of inoculum
increased suggesting that the different
amounts of inoculum can cause significant
differences in yield. This also indicated that a
combinationn of leaf and stalk infection cause
Greater effect on yield. Fajemisin and Hooker
1974) showed that stalk rot susceptibility was
increased as a consequence of leaf blight
infection. This reduction in yield is due to
decreased in functional leaf area due to
prematuree drying of the leaves and leaf
sheaths. The stalk acts as transitory carbon
storage organ, the carbon being used to build
loral pieces surrounding the kernels and the
arbon filling kernels originate from
)hotoassimilates accumulated after anthesis
Cliquet et al, 1990). These photoassimilates
stored in the stalks and in the leaves before
anthesis could be remobilized for ear
Development (Setter and Meller, 1984). Prioul
?t al. (1990) showed that less than 10% of the
;arbon assimilated in the kernels comes from
mobilization from different organs. This was
supported by the study of Daynard et al.
1969) showing reduction in stalk dry weight
beginningg 2 to 3 weeks after mid-silking which
corresponded to a period of high ear dry
natter accumulation. Hence, the presence of
iecrotic lesions in single intemodes may
nterrupt the physiological processes and may
cause severe reduction in yield. Moreover,
nuch of the water and nutrient taken up by
roots does not reach the above ground part of
tie plant resulting in barren stalks, smaller
ears or premature drooping of ears and plants
appear to mature early (Christensen and
Nilcoxon, 1966).

Michaelson (1957) demonstrated that
stalk rot reduced the yield of shelled corn by 2
to 22% depending on the pathogen, the
hybrid, the plant parts inoculated, the number
of infections per plant, the time of inoculation
and the season. He also emphasized that
losses were greater when the hybrids were
inoculated 9 to 10 weeks before harvest,
rather than 5 to 6 weeks before harvest.

The different sites of inoculation and
inoculum concentrations showed significant
effect on the ear diameter and weight of 1000

liameter compared to the inoculated plots.

Among the sites of inoculation, the
eaf, stalk and the combination of leaf-and-
stalk inoculated plants showed bigger ear
diameter with low inoculum level compared
with those inoculated on the ears and in
combinationn with other plant parts.

The ear diameter was smaller on all
har-inoculated treatments regardless of the
amount of inoculum concentration compared
vith those of the control plots. This reduced
iar diameter is due to the activity of the
)athogen causing severe ear rotting. Ear
tusks were pasted together with mass of
nycelial growth and pycnidia extending into
he cob.

On the other hand, leaf, stalk and
eaf-stalk-inoculated treatments had varied
eight of 1000 grains compared to the
control During Season A, the weight of grains
vas low on stalk-inoculated compared with
he leaf-inoculated plants. The reduction in
eight of grains was probably brought about
>y the stress during or after silking (Claasen
and Shaw 1970) resulting to the Inhibition of
transport of the photoassimilates into the ears
hat were necessary for grain-filling period.

The higher grain weight on leaf-
noculated plants alone could be due to the
power percentage infection observed. This
indicated further that the active photosynthetic
area was not greatly affected by the blight
)hase of the disease.

The amount of inoculum significantly
increased the percentage crop reduction
respective of the site of inoculation in both
seasons. Plants inoculated in the ear and in
combination with other plant parts showed
100% reduction regardless of the amount of
noculum. This result indicates that ears were
nore vulnerable to pathogen attack than other
plantt parts. This is supported by high severity
Af the ear rot infection accompanied by the
education in ear size and diameter. Heavily
infected ears were hard and husks were
pasted together with heavy white mycelial
__ -& La- L. .I- .-..A. J rl.. __. .4.. .__:_

Alovera and Raymunao

were moldy and practically of no value.
Bayles (1977) stated that the events prior to
or immediately following anthesis affected
grain yield This is supported by reports of
Jones and Clifford (1978) that the severity of
ear rot infection depends on the time at which
infection occur, the prevailing environmental
conditions and on the susceptibility of the

The presence of lesions on the stalk
has a great influence on yield reduction.
Lesions in single intemode may have
interrupted the physiological processes in
plants. Daynard et al. (1969) reported a
decline in stalk dry weight beginning at the 2
to 3 weeks after mid silking which
corresponded to the a period of high ear dry
matter accumulation. The reduction in dry
weight appeared to be due to a movement of
soluble carbohydrates into the ear. The
activity of the pathogen in the stalks may
cause blockage in the transport of assimilates
needed in filling the ears thereby limits the
weight of the ears. In addition, the nutrient
taken up by the roots does not reach the
above ground parts of the plant resulting in
barren stalks, smaller ears or premature
dropping of the ears. This reduction in the
weight of kernels had contributed to grain
yield losses (Perkins and Pedersen, 1987).

Yield losses from 2 to 22% has been
recorded by Michaelson (1957) which depend
upon the hybrid, the plant part inoculated and
the time of inoculation and the season. A
97% yield loss (grain wtlplant) was recorded
from an inoculation of D. macrospora 10 days
after midsilk (Chambers, 1988) while Logan
(1974) reported losses as much as 75%
especially in poor unfertilized crops.

Model for Crop Loss

S. macrospora is a pathogen that
causes three phases of disease, leaf blight,
stalk rot and ear rot. Considering that these
phases occur at different parts of the plant
and at various crop growth stages, the
surface-response model was deemed
appropriate for us.

Leaf blight severity at 56 days (LBss)
after inoculation (DAI) or (91 days after

emergence (DAE), stalk rot at 14 DAI (SR14)
or (69 DAE), stalk rot at 28 DAI (SR28) or (83
DAE) and ear rot at harvest (ER) proved to be
strong descriptors for percentage loss and
can be considered to be vital in the grain-
filling activity. The inclusion of all these
variables in the model was considered
important which might represent every
independent aspects of variation in the
depended variable. In addition, it significantly
increase the R2 value of the models.

The criteria used to select the best
fitting models included the coefficient of
determination (R2), which indicates the
proportion of the total variation explained by
the model (P>0.01), F-statistic, which test the
significance of the regression model (P>0.01)
and T-statistics of each partial regression
coefficient (P>0.01), which test the
contribution of each partial regression
coefficient to the model.

Among the different predictors, ear rot
severity proved to have the greatest effect on
percentage loss in both seasons.

During Season A, leaf blight severity
at 56 days (LB5s) after inoculation (DAI), stalk
rot at 14 DAI (SR14) and stalk rot at 28 DAI
(SR28) corresponded to 91, 69 and 83days
after emergence (DAE), respectively. These
periods corresponded to the highest
increased in leaf blight severity and stalk rot at
2 to 3 weeks after silking. According to
Hanway (1962), the rate of dry, matter
accumulation and its redistribution into the
various parts of corn plant is essentially a
linear function of time. Early season growth
was essentially to all leaves and leaf sheaths
and the late season into the grain. However,.
less potential yield of grain will be attained if
the net assimilation rate is decreased by any
factors such as moisture deficiency late in the
season or to the leaf area prematurely
reduced by some factors that include


Stenocarpella disease complex in
corn. I. Disease progression as
affected by site of inoculation and

Stenocarpella disease complex

inoculum concentration. J. Trop. Pit.
Pathol. 40:1-13.

BAYLES RA. 1977. Poorly filled grains in the
cereal crop. I. The assessment of
poor grain filling. J. Nat. Inst. Agric.
Bot. 14: 232-240.

CHAMBERS KR. 1988. Effects of time of
inoculation on Diplodia stalk and ear
rot of maize in South Africa. Maize
Abstr. 5 (5): 388.

1966. Stalk Rot of Corn. Monograph
3. The American Phytopathological
Society. 59 pp.

CLAASEN MM and RH SHAW. 1970. Water
deficit effects on corn. Vegetative
components. Agron. J. 62:649-652

MARIOTTI. 1990. C & N mobilization
from stalk and leaves during kernel
filling by 3C & 1SN tracing in Zea
mays L. Plant Physiol. 94: 1547-1553.

Notes on leaf blight, ear rot and stalk
rot of corn caused by Diplodia
macrospora Earie in the Philippines.
Philipp. Phytopathol. 23: 22 -23.

1969. Contribution of stalk soluble
carbohydrates to grain yield in corn
(Zea mays L.) Crop Sci. 9: 831-834.

Predisposition to Diplodia stalk rot in
com affected by three
Helminthosporium leaf blights.
Phytopathology 64: 1496 -1499.

FRANJE NJE. 2000. Yield loss assessment
due to Diplodia macrospora Earle
inoculated at different growth stages
of CMU Var 12 and GSI 40M.
Unpublished Undergraduate Thesis.
Central Mindanao University,
Musuan, Bukidnon. 79pp.

HANWAY JJ. 1962. Corn growth and
composition in relation to soil fertility:
Growth of different plant parts and
relationships of both leaf weight and
grain yield. Agron. J. 54:144-148

HANWAY JJ. 1963. Growth stages of corn
(Zea mays L.). Agron. J. 556 -491.

Cereal Diseases, Their Pathology and
Control. BASF United Kingdom
Limited. Agrochemical Division.

- LLANO A and E SCHIEBER. 1980. Diplodia
macrospora of corn in Nicaragua.
Plant Dis. 64: 797.

LOGAN J. 1974. Plant Pathology in Zambia.
PANS 20: 169- 176.

WESTHUIZEN. 1979. Diplodia
macrospora: The cause of a leaf
blight and cob rot of maize (Zea
mays) in South Africa.
Phytophylactica. 11: 61 64.

MICHAELSON MF. 1957. Factors affecting
development of stalk rot of corn
caused by Diplodia zeae.
Phytopathology 47: 499-503.

Disease development and yield
losses associated with northern leaf
blight on corn. Plant Dis. 71: 940-943.

PRINE GM. 1971. A critical period for ear
development in maize. Crop Sci. 11:

DUQUE. 1990. Relationships
between carbohydrate metabolism in
eat and adjacent leaf during grain
filling in maize genotypes. Plant
Physiol. Biochem 28: 485-493.

Reserve carbohydrates in maize stem
(14C ) glucose and (14C) sucrose

Alovera ana Kaymunao

uptake characteristics. Plant Physic
75: 617-622.

diseases caused by Diplodia. Philipi
Agric. 20: 370 374.

Stenocarpella disease complex

Table 1. Yield and yield parameters of corn as influenced by site of inoculation and inoculum
concentration from November 1995 to March 1996 (Season A) and June to October
1996 (Season B)

Ear Diameter Ear Length Weight of 1,000 Adjusted.
(cm) (cm) Seeds (g) Yield (tonslha)
TREATMENT Season Season Season Season Season Season Season Season

1 x104
2x 104
3 x 104
4x 104
1 x104
2x 104
3 x 104

1 x 104
4 x 104
Leaf & Stalk
1 x104
2 x104
Leaf & Ear
1 x104
2x 104
4 x 104
Stalk & Ear
1 x 104
2 x 104
3x 104
4x 1' 4
Leaf, Stalk & Ear
1 x104
4 x 104















13.36 15.10 262.17 249.25 9.02
13.14 14.86 261.52 260.50 7.85
13.70 14.66 254.76 254.49 7.88
13.78 14.28 254.16 249.36 6.97
13.68 14.65 252.99 248.93 5.21

14.00 15.01 274.02 245.50 6.55
13.37 14.73 251.38 248.64 6.02
13.16 14.63 264.47 258.67 4.75
13.13 14.62 251.65 253.78 4.71
13.63 14.79 236.05 258.04 3.98

13.45 14.82 246.69 253.30 4.31
14.02 15.13 0.00 0.00 0.00
13.76 15.25 0.00 0.00 0.00
13.23 14.90 0.00 0.00 0.00
13.25 15.05 0.00 0.00 0.00

13.57 14.87 270.96 251.27 5.24
12.61 14.03 248.64 251.76 3.42
13.21 14.89 249.39 251.09 3.62
13.05 15.12 230.47 252.95 3.24
12.87 14.31 238.96 250.29 2.91

13.57 15.00 257.50 258.54 5.23
14.21 13.91 0.00 0.00 0.00
13.44 14.57 0.00 0.00 0.00
14.22 15.07 0.00 0.00 0.00
13.57 14.12 0.00 0.00 0.00

13.23 14.94 250.52 248.57 4.63
13.34 14.38 0.00 0.00 0.00
13.21 14.02 0.00 0.00 0.00
13.22 13.91 0.00 0.00 0.00
13.22 14.65 0.00 0.00 0.00

13.99 14.19 253.30 251.70 4.29
13.76 14.09 0.00 0.00 0.00
14.99 14.66 0.00 0.00 0.00
13.55 15.24 0.00 0.00 0.00
13.76 14.37 0.00 0.00 0.00
NS NS 14.71 9.576 1.78









--- ---

Stenocarpella disease complex

Table 2. Yield losses in corn as influenced by site of inoculation and amount of inoculum

concentration from November
October 1996 (Season B)

1995 to March 1996 (Season A) and from June to

Site of Inoculation Inoculum Season A Season B
Concentration (November to March) (June to October)







FLSD .05





1 X 104
2X 104
3X 104
4X 104

1 X 104
2X 104
3X 104
4X 104

1 X 104
2X 104
3X 104
4X 104

1 X 104
3X 104
4X 104

1 X 104
2X 104
3X 104
4X 104

1 X104
2X 104
3X 104
4X 104

1 X104
2X 104
3 X 104
4X 10







Portion of the undergraduate thesis of tt

'Assistant Professor, Adjunct Profe
respectively, Department of Plant Pathology, 2
Breeding, College of Agriculture, University of tl

This study was conducted ti
blight and crown rot of Cordyline te
foliage ornamental crop. Infected h1
'Pink Top' and 'Hawaiian Compacta'
Breeding and Dept. of Horticulture
Lucban, Quezon, respectively were
isolated using the modified tissue pi
structures was induced by mating
Cultural and morphological chara
particular incubation temperatures, i
measurements of the sexual and
Following the key to identification
isolates were identified as Phytoph
(syn. P. parasitica Dast.) Furthermo
demonstrated. To our knowledge, 1
about this species infecting ti plants

Key words: Cordyline terminalis, crown rot, leaf


The ornamental industry has an excellent
potential to become one of the Philippine's top
dollar earners considering its wide diversity of
exotic flora as well as the tropical
environment, conducive for year-round
propagation. Cordyline terminalis, commonly
known as ti plant of Hawaiian good-luck-plant,
is just one of those very promising ornamental
crops. Grown mainly for their attractive
foliage, this comprises a large number of
indigenous and newly introduced varieties and
hybrid cultivars, coming in various forms,
shapes, colors and sizes. They are utilized in
bouquets, flower arrangements, home



senior author.

r, former University Research Associate,
search Associate Professor, Institute of Plant
3hilippines Los Baflos, College, Laguna

identify the causal organism of leaf
inalis or ti plant, a potential export
'es of C. terminalis 'Lemon Lime',
m nurseries of the Institute of Plant
UPLB and from a farmer's field in
collected and the causal organism
ting technique. Formation of sexual
; isolates in V-8 juice agar blocks.
ristics like growth responses to
ting reactions and morphology and
;exual structures were examined.
species of Phytophthora, the three
Pra nicotianae var. parasitica Water
their heterothallic nature was also
s is the first report in the country

1ht, Phytophthora nicotianae var parasitica

gardens, parks and golf courses as accents,
borders or hedge plants.

In 1980, a leaf disease was observed
from the cultivar Hawaiian Compacta grown in
Laguna (Divinagracia, 1980). Further survey
revealed that it was also widespread in other
provinces such as Quezon, Cavite, Batangas,
Iloilo, Cebu and Negros Occidental
(Divinagracia and Bayot, 1998). Initially, the
disease appears as greenish, irregular and
water-soaked lesions on both young and
mature leaves near the soil surface. Later,
infected portions become dry and papery,
shredded and eventually fall off (Fig. la).

ajay et a.

rolonged wetness often leads to appearance
f whitish mycelium on the leaf surface
:ig.lb). In most cases, the crown becomes
Mtten leading to plant death, or if not, causing
ie development of side branches which
Iters the overall shape of the plant. Any of
ie damages mentioned due to the disease
reduces the aesthetic qualities of ti plant and
,nders them unattractive to buyers.
icidences may reach up to 80% during
periods of frequent rainfall and with
jsceptible varieties (Divinagracia and Bayot,

Initial isolation of the causal organism
om specimens obtained at the Institute of
hllt Breeding, UPLB consistently yielded
hytophthora sp. based on cultural
characteristics and morphology of the asexual
ructures. Pathogenicity was also confirmed.
lentification of the organism up to species
vel however, was not fully accomplished
cause the isolate failed to produce sexual
ructures by itself in culture (Divinagracia and
ayot, 1998). This led to the idea that it was a
eterothallic species, and therefore, another
late with an opposite mating type is

The genus Phytophthora consists of
wveral species known as pathogens of a
ide array of plants. In the Philippines, past
udies on ornamental diseases focused
ainly on the characterizations of the
rmptoms and survey of their occurrence.
tailed studies on the identification of causal
ganism are limited. This study was done to
entify the species of Phytophthora causing
af blight and crown rot of C. terminalis in the
lilippines. Knowing its exact identity and
iture are very important in formulating
fective management strategies against the


elation of the Causal Organism

Diseased leaf samples of three different
Itivars of C. terminalis namely 'Lemon
ne', 'Pink Top' and "Hawaiian Compacta'
,re collected from the nurseries of the
stitute of Plant Breeding and Dept. of
)rticulture in UP Los Baftos, and from a

irmers neia in Lucoan, uuezon. omaii
actions of the leaves were cut from the
advancing margins of the lesions, surfaced
:erilized with 0.05% NaOCI solution, rinsed
nd then soaked in sterile distilled water for
Iree days. Soaking was done to induce the
iycelial growth of the organism
'hytophthora sp. being water "mold"). The
andard tissue plating technique was
modified to facilitate isolation. Plated water
gar was cut perpendicularly to create four
actions. The agar from each section was
gently lifted and the rinsed tissues with
lycelial growth were placed under. Surface
iycelial growth was then picked and
ansferred onto potato dextrose (PDA) slants
ir culture and storage.

lentification of the Causal Organism

The subsequent procedures were done-
s required by the key to the species
entification of Phytopthora by Stamps et al.
990). Growth of the isolates on V-8 juice
gar (Miller, 1955) at temperatures 27.5, 30.0,
2.0 and 37.00C was observed and measured
daily. Asexual structures (sporangia,
Ilamydospores and hyphae) of the isolates
stainedd from 14-day old cultures on the
ime medium were characterized and
measured. Sexual structures on the other
md were produced by mating the isolates on
jar blocks mounted on sterile microscope
des supported by plastic straws in the Petri
sh lines with moistened filter paper. The
orphology of antheridia, oogonia and
)spores that formed were also characterized
id measured. For each structure, 50
imples were randomly selected and
measured using a filar micrometer.


The causal organism was readily isolated
ing the modified tissue planting technique.
ie mycellum grew though and spread on the
iar surface and left the contaminants, which
ire mostly bacteria beneath.

characterization of the Isolates

Growth at different incubation
mperatures. The three isolates grew in all.
nperatures tested. Growth at 370C,

temperatures. The average growth rate at this
temperature was 5.13, 4.71 and 5.25 mm/day
for the Lucban, IPB and Horticulture isolates,
respectively (Table 1).

Morphology and morphometrics of
asexual and sexual structures. Table 2
summarizes the measurements of asexual
and sexual structures of the three isolates of
Phytophthora sp.

Hyphae. Coenocytic and hyaline. Width
ranged from 5.41-8.71um for Lucban, 5.87-
8.53 um for IPB and 6.06-8.08 um for
Horticulture isolates.

Sporangia. Markedly papillated. Shaped
varied from nearly spherical to ovoid and
obpyriform or pear-shape. Most of them were
terminally located, though intercalary ones
also exist. Length and breath measurements:
43.52 and 32.56 um for the Lucban, 43.50
and 34.00 for IPB and 44.61 and 34.10 um for
the Horticulture isolates. Length to breadth
ratio: 1.34, 1.28 and 1.32 for the three isolates
in the same order, respectively (Fig. 2a-f).
Sporangiophores were developed in a simple

Chlamydospores. Terminal and intercalary;
spherical and large; abundantly produced on
culture medium after about 14 days of
incubation. Diameters: 27.77, 28.58 and 30.39
urn for the Lucban, IPB and Horticulture
isolates, respectively. Wall thickness
exceeded 2.00 um for all the isolates (Fig. 2g-

Sexual structures were not formed in
same strain mating. Likewise, no sexual
reproduction was observed in the pairing of
IPB and Horticulture isolates. Instead, such
were only formed when the Lucban isolate
was paired with either the IPB or the
Horticulture isolates. During sexual
reproduction, the oogonium grew through the
antheridium and matured above it, later
forming the oospore inside (Fig. 3-a). This is
known as the amphigynous type of oospore
development, and seen as the oogonial stalk
encircled by the antheridium.

stalks all centric. Diameter 25.66 um for the
Lucban-IPB mating and 26.31 um for the
Lucban-Horticulture mating.

Antheridia. Hyaline, and mostly roundish to
cylindrical. Diameter 9.53 um for the Lucban-
IPB mating and 9.46 um for the Lucban-
Horticulture mating.

Oospores. Globose with smooth walls (Fig.
3b-c). Considered plerotic based on the
definition of Hawksworth et al. (1995) since
approximately 80-90% of the total oogonia
volume was occupied by the oospore (Fig. 1k-
n). Diameter: 21.91 um for the Lucban-IPB
mating and 22.57 um for the Lucban-
Horticulture mating.

Identification to Species Level

Stamps et al (1990) divided the genus
Phytophthora into seven taxonomic groups
according to major morphological differences.
The three isolates were all qualified under the
second group for having markedly papillate
sporangia, heterothallic sexual compatibility
and amphigynous type of oospores
development. There are 16 species (including
varieties) listed in this group. With no other
key features namely the ability to grow at
temperature higher than 35 C and sporangia
having broad/roundish base, the identification
was further narrowed down to into P.
nicotianae var nicotianae (P.n.n) and P.
nicotianae var. parasitica (P.n.p)

Table 3 presents the summary of
comparison of the isolates with the two-
closely related varieties of P. nicotianae. Note
that the characters of the isolates such as
range of shape of sporangia and antheridia
and hyphal width were more similar to P.n.p
than in P.n.n. In addition, characters
describing P.n.n like the early formation of
chlamydospores and hyphal swellings and the
eccentric oogonial stalks were not observed in
all of the isolates. The only features found not
consistent with the description of P.n.n was
the plerotic oospores. This, however, was
given no weight at all because of some
inconsistencies among the description,
definition and figures presented in the
available mycological references (Stamps et

r ~~~~v~~~~~~VU ----

UI, I uYvV, I la lO VV LI II tU al, I r,
Waterhouse, 1956 and 1963).

The three isolates therefore were all
identified as P. nicotianae Brenda de Haan
var. parasitica Water. (syn. P. parasitica
Dast.). The organism belongs to Family
Pythiaceae, Order Peronosporales and Class
Oomycetes, of the Phylum Oomycota under
Kingdom Stramenopila (Alexopoulos et al,
1996). The taxonomy and nomenclature of
this species was and is still chaotic taking its
root from the mistakes in the original
description (Ho and Jong, 1989). The original
description of Breda de Haan was based in
harmonious elements: the sporangia
illustrated belong to P. nicotianae but the
oogonia resemble another organism, which
was possibly Pythium. That explains the
inconsistencies on taxonomic references
about the plerotic oospores as noted above.
P. parasitica was described afterward as a
separate species due to several "distinct"
morphological features (not noticing the
mistakes in the original description). Later on,
the variety nicotianae and parasitica were
introduced and attached to both species to
separate isolates infecting tobacco and those
that are plurivorous. This made the
nomenclature even more confusing. Different
approaches and methods were made in order
to establish the relationship of those isolates:
morphological (Ho and Jong, 1989),
biochemical (Oudemans and Coffey, 1991),
numerical (Hall, 1993), serological (Cristinizio
et al, 1983) and RFLP analysis of
mitochondrial and chromosomal DNA. Their
studies lead to a single conclusion that the
species called P. nicotianae and P. parasitica
are the same and the separation of P.
nicotianae into two varieties cannot be
justified. Under the International Code of
Botanical Nomenclature, the name P.
nicotianae being the first name given has
priority over P. parasitica and must be used in
preference although many are still familiar
with P. parasitica. (CABI, 2001). It was is not
the aim of this paper to resolve the taxonomic
chaos within the genus Phytopthora or the
species of concern, therefore, we decided to
retain the name P. nicotianae var parasitica
following the name used in the key (Stamps et
al, 1990).

,UCGVCaI U IcdIou3i Ul VI Iil Iial
plants worldwide were attributed to this
species. For C. terminalis growing in Hawaii,
Trujillo et al (1975) reported this species
causing leaf spot on the cultivar Green L'ai. In
the University of Florida's Pest Control Guides
for C. terminalis, P. parasitica was likewise
listed as the one causing the same disease in
ti plant, as well as in schefflera, spathiphyllum,
dieffenbachia and philodendron. Its existence
in the Philippines was reported long before
but are mostly associated with food crops like
eggplant, pepper, pineapple, papaya,
watermelon, cantaloupe, coconut and citrus
(Tangonan, 1999). Dizon et al. (1997)
reported this as one of the pathogens causing
the stem rot complex of periwinkle. Its
pathogenicity to other commercially important
ornamental crops is just another aspect that
warrants a thorough investigation. Knowledge
on this aspect will have a bearing on what
crops and ornamentals can be grown side by
side or alternately with ti plants. This will be a
very important concern for the growers.

The identified pathogen is known and
was shown to be a heterothallic species. This
means that it requires a compatible strain or
an opposite mating type of the same species
to produce a sexual structures or oospores
(intraspecific mating). The IPB and
Horticulture isolates probably posses the
same mating type (the respective places of
isolation being in one locality) that is why no
mating between them occurred. On the other
hand, the Lucban isolate provided the
opposite mating type that resulted to
formation of oospores when paired with either
of the two isolates (Lucban, Quezon, the
place of isolation for the latter is
geographically far from UP Los Baios).
Oospores are very important in the life cycle
of this organism because they provide a
mechanism of surviving adverse condition and
are sure sources of inocula when favorable
environment is restored (Agrios, 1997). But
more importantly, mating to produce oospores
provides a potential source of genetic
variation (Ko, 1998). Literature also points out
the occurrence of sexual reproduction
between morphologically and physiologically
distinct heterothallic species of Phytophthora
interspecificc hybridization) under certain
conditions (Savane et al 1968: Kn 19801

-1-i-Y -1 -'

I Ii ^ lv l l ;Iafr I1 I.ijtJLI i

matings can provide a mechanism of creatir
more genetic variants, which may result
change in pathogenicity and host rang
Precautions should be carefully observed ,
as not to introduce a sexually compatib
strain that may be harbored by plants/so
grown from other places. Such risks can 1
minimized by choosing healthy plantir
materials and by excluding the soil if possib
when transporting planting material
Application of right fungicides to starting pla
materials may also be necessary to eradica
whatever inocula are being harbored.


AGRIOS GN. 1997. Plant Pathology. 4th e
Academic Press. California.

BLACKWELL. 1996. Introducto
Mycology. 4th ed., John Wiley ar
Sons, Inc. New York 868 p.

NOVIELLO. 1983. Differentiation
some Phvtoohthora snecies 1

Facult. de Scier
Napoli, Portici 17:

oresis. Annale del
icie del'Universit

CD-ROM. CABI Publishin(
Wallingford. UK.

DIVINAGRACIA GG. 1980. Diseases (
important flowering omamenta
plants. PCARRD Annual Report 49 p

Disease management in selected
exportable foliage plants. UPLE
PCARRD and DOST Annual Repo
48 p.

Symptomatology and pathogen
associated with the stem rot disease
complex in periwinkle. Phi
Phytopathol. 33:68-71.

* rr lwrr v lls I I ,l5 I* I I 11-.1 | *d V I I I
and DN PEGLER. 199
AINSWORTH and Bisby's Dictiona
of Fungi. 8th ed. Wallingford. CA
International, New York 616 p.

HO HH and SC JONG. 1989. Phy^ophtho,
nicotianae (P. parasitica). Mycotaxc

KO WH. 1980. Sexuality, evolution and orig
of Phytophthora. Plant Prot. Bu
(Taiwan) 22:141-151.

KO WH. 1998. Sexual reproduction
pythiaceous fungi. Bot. Bull. Aca
Sinica 39:81-86.

MILLER PM. 1955. V-8 juice agar as
general purpose medium for fungi ar
bacteria. Phytopathology 45:461-46;

revised systematic
papillate Phytophtho
nn irtn7uma annlu

s of twelh
a cnarioc hacc

ME GALLEGLY. 1968. Homothallisrr
heterothallism and interspecifi
hybridization in the genu
Phytophthora, Phytopatholog

Revised tabular key to the species c
Phytophthora. 2d ed. Mycol. Par
162. Commonw. Mycol. Inst., Kev
Surrey, England 28 p.

TANGONAN NG. 1999. Host Index of Plar
Diseases in the Philippines. 3r ec
PhilRice, Mufioz, Nueva Ecijc
Philippines. 408 p.

SWINDALE. 1975. Phytophthora le.
spot of ti plant. Plant Dis. Repti

WATERHOUSE GM. 1956. The gent
Phytophthora. Misc. Publ. 1:
Commonw. Mycol. Inst., Kew. Surre
England. 120 p.

WATERHOUSE GM. 1963. Key to tU
species of Phytopthora de Bar
Mycol. Pap. 92. Commonw. Mycc
Inst., Kew, Surrey, England. 22. P.



This study was supported by DOS

-~;- ?i



flrziga af tal

Figure 2. Asexual (a-i) structures of the three isolates of Phytophthora sp. obtained from
14-day old cultures on V-8 juice agar: intercalary (a-b) and terminal papillate
sporangium (c-f) with shape either obpyriform (c), ovoid (d-e) or nearly spherical
(f); intercalary (g) and terminal (h-i) chlamydospores. (Figures not presented
in scale to fit the page).

Table 1. Growth of three isolates of Phytophthora sp. on V-8 juice agar at different incubation

Isolate Temperature Radial growth (mm)" Ave growth
(C) Day 1 Day 2 Day 3 Day 4 (mm/day)c
.ucban 27.50 11.25 9.92 7.83 8.63 9.41
30.00 12.33 9.34 8.50 7.46 9.41
32.00 12.33 10.25 8.42 7.63 9.66
35.00 12.20 10.55 8.60 7.40 9.69
37.00 9.44 6.81 2.31 1.95 5.13
PB 27.50 9.67 9.16 7.17 6.00 8.00
30.00 10.67 8.66 8.50 7.17 8.75
32.00 10.75 8.83 7.59 8.08 8.81
.r nn 11 RA 7 A9 7 97 -a 1, r7 R QA

Mean of six measurements (growth diameter of each of the three replicates measured twice
perpendicular to each other)
Computed using this formula: Radial Growth aayn = diameter nd, diameter dgn-,
Mean of four computed radial growth rates.

'able 2. Measurements (um) of asexual and sexual structures of three isolates of Phytophthora

Structures Measurements (um)a
Lucban IPB Horticulture
k. Asexual

34 Phytophthora nicotia

Table 3. Comparison of the two varieties of Phytophthora nicotianaea and the three isolate
under study.

spherical +
obpyriform +
obturbinate +
45 um long
45 to 75 um long +
early +
- wall 2-3 um or more
Hyphae swellings
on agar +
in water +
uniform, < 5 um +
- irregular, tough, < 9 um
Sexual structures
spherical +
ellipsoid or avoid
<30 um +
30 to 40 um +
stalk often eccentric +

a Only important characters that would highlit
nicotianae were presented.
b Taken from the Revised Tabular Key to the ;
Legend: + = character expected/observed
= character not observed

n.p Lucban IPB Horticulture

+ + + +

+ + + +
4- + -+ +

+ + + +
+ + + +

+ + + +

+ __+ + +
n.p Lucban x IPB Lucban x Horticulture

+ + +
+ + +

+ + +

the differences between the two varieties of P

:ies of Phytophthora (Stamps et a/, 1990)

Journal of Tropical Plant Pathology 40:35-47



Plant Breeding and Biotechnology Division, Philippine Rice Research Institute Maligaya,
3119 Science City of Muiioz, Nueva Ecija. Current Address: Texas A & M University,
Agricultural Research and Extension Center, 1509 Aggie Drive, Beaumont, TX 77713, USA

Several genes for bacterial leaf blight (BLB) are now available and
being pyramided in several genetic backgrounds like IR64 and in the
process of the development, lines with single gene and gene combination
can be generated. These lines are typical isolines that can be tapped in the
multilines. Through Asian Rice Biotechnology Network (ARBN), elite lines
with different genes were developed and exchange of lines was initiated.
Two linas from indonesia, Bio 1 and Bio 2, containing Xa-5 and Xa-7,
respectively i4R 54 baxckqrounds and 3 lines also with IR 64 background
from PhilRice but "orsri-n6?ng Xa 6 and Xa-21 were evaluated together with
mixtures of these fines for ttren'e sreaso:rs.

AR3:2-19-3-4 had the hSaihet average yield of 3.60 tiha across wet
season of 2Cu1 ani 2092, Joihowed by AR32-19-3-3 wh. 3.55 "/ha, both
significantly higher th~in t. two checks, PSB Rc.2 an:d Ri4. Due to the
absence of diseae- pi-s'::-, rHowev.;r, dry sea-son dat. showed
comparative ys 'u"s' '? An -F y'2 5: severely

affected hmf o::3 ~r n-i :- -) ::-

ni~rn tu rm i n it. ?:!::j

+ -'5F' a d
*r,;i w 'e e r d -

K ywvord>: Daa1t *'i i t. ,I ,, l ,

on heter_ n ew s. ':;. ; .. ....

line system beca '; i;.i;- r ...
variation or non- icrr' :'. i .
flourished (Hartn.r,. i .. .; :, ;..,. ;'
the planting of rewer genotypes. be-am.-:e .. ;
trend in industrial agriculture This. however,
enhanced vulnerability to abiotic and Dioe:c
stresses favoring further resistance
breakdown. Cultivation of mixtures within arn;

eweeOnr se:Re^s r, -':iowni ^*ci: ss early as
S' sease losses
S,69) but it was
,.r d- d "; : L. mited : studies
have bnen ccnduc : ntoned i,1 the fvst
:;:::? {rowing :i Fre, 39) but several
studies followed {'.' ", ':195) and mch
mor.: in recent ye: (Garrett and Mu.ndt,
1999). The general theme, however, was that
the crops should have heterogeneity for
disease resistance that could be achieved in
several ways like in multilines, variety mixture
or line mixture.

Tabien, Abalos and Fernando

Multilines concept as opposed to
purelines (Browing and Frey, 1969) proposed
50 years ago, was designed for small grains,
wheat, oat and barley to control continental
rust fungi and powdery mildew. It was
generally a mixture of varieties and defined as
reconstitutable composite of phenotypically
similar but genotypically dissimilar lines
(Jensen, 1965). Multilines usually yields like
or better than the mean of their components,
and can provide fairly stable and reliable high
levels of production (Wotfe, 1985). This
stability could be due to disease control or
compensation between host genotypes for
damage caused by abiotic, stresses or
genotype by environment interactions. Use of
multilines has been in practice in thousand
bectares of wheat, oat or barley in Russia,
Germany and Canada. The most successful
US peanut varieties 'Florigiant' and
'Florerunner' and winter wheat variety
'Stephens' are all composed of several lines.
In rice, the first successful large scale use of
variety mixture was for blast control as
reported by Zhu et al. (2000). Planting of blast
susceptible glutinous and resistant inbreds or
hybrids reduced blast severity in glutinous by
an average of 94 percent.

Multilines can be line mixture, variety
mixture or mixture of elite lines of nearly
identical host like isolines or near-isogenic
lines. The first two types have been popular in
small grain crops but not isolines. Isolines, the
line products from 4-6 backcrossing that differ
in few traits like disease resistance genes are
useful components like varieties in multiline
development. Backcrossing to improved
popular rice varieties like IR64, PSB Rc14 and
BPD Ri-10 has been done and elite lines have
been generated (Tabien et al, 2003) and other
teams of the Asian Rice Biotechnology
Network (ARBN) are currently developing
isolines using DNA marker-aided selection
(Vera Cruz C., personal communication). At
present, two lines in IR64 genetic background
having single bacterial blight resistance (BLB)
genes from Indonesia have been released as
new varieties in 2002 and ready for
collaborative evaluation (Bustamam, M.
personal communication). Since these
isolines were developed independently and
are not yet evaluated in other ecosystem,
these can be potential components of a

multiline or a new variety. Moreover, these
contain different genes with differential
reaction to various BLB races.

Like in rice blast control, use of
resistant varieties has been the method of
control for BLB. More than two dozen genes
for BLB have been identified and being
transferred to popular varieties (Kameswara
Rao and Jena Lakshminarasu, 2002).
However, due to homogeneity even on the
gene being used in the breeding programs,
resistance breakdown has been reported.
Varieties with gene Xa-4 were devastated by
Isabela strain in the 60s. To prolong the use
of these genes, gene pyramiding is currently
on-going using marker-aided selection
(Tabien et al. 2002). The elite lines from
marker-aided selection (MAS) are nearly
isogenic lines since these were generally
developed through backcrossing. Each line
has specific resistance gene or gene
combination transferred using DNA marker.

The PhilRice and Indonesian elite
lines having single or pyramided BLB genes
can be components of a multiline, thus their
combination can be evaluated. The objectives
of this study, therefore, were to evaluate yield
potential of the elite lines derived form MAS
and to evaluate the combination of these
multilines and susceptible varieties like IR64
in controlling BLB.


The experiment was conducted at
PhilRice-Maligaya, Science City of Muioz for
two wet seasons (WS) 2001 and 2002, and
one dry season (DS), 2002. Two lines from
Indonesia, Biol and Bio 2 in IR64 background
containing Xa-5 and Xa-7, respectively
courtesy of Dr. M. Bustamam of Research
Institute for Food Crops Biotechnology
(RIFCB) of Indonesia and three lines also in
IR64 background developed by PhilRice were
evaluated together with mixtures of these
lines or with IR64. Two popular varieties IR64
and PSB Rc28, were used as yield and
susceptible checks, respectively. The
treatments laid-out in Randomized Complete
Block Design (RCBD) with three replications
were as follows:

2. Bio-2 (IR64 with Xa-7 from Indonesia)
3. AR32-19-3-4 (IR64 with Xa-5 and Xa-21
from Philippines)
4. AR32-19-3-3 (IR64 with xa-5 and Xa-21
from Philippines)
5. AR32-19-3-2 (IR64 with xa-5 from
6. Combination of Bio-1 and Bio-2 (1:1 ratio,
xa-5 and Xa-7 combination)
7. Combination of AR32-19-3-3 lines and
IR64 (1:1 ratio, Rx S)
8. Combination of AR32-19-3-4 lines and
IR64 (1:1 ratio, Rx S)
9. AR32-19-3-3 and AR32-19-3-2 (1:1 ratio
10. IR64 (Susceptible check)
11. PSB Rc 28 (Susceptible and yield check)

Seedlings grown in seedbed for 21 days
were transplanted in 3 x 4 m plots using 20 x
20 cm distance of planting. Replanting of
missing hills was done a week after
transplanting. Fertilizers were applied at the
rate of 90-30-30 kg/ha N, P205 and K20
during DS and 60-30-30 kg/ha N, P205 and
K20 during WS. Nitrogen application was
split (at transplanting and at panicle initiation)
while all P and K were applied at
transplanting. No insecticide was applied
during the growing period but weeding was
done when necessary. Other cultural
management practices were done as need
arises. Yield and other agronomic data, grain
quality test and reaction to different pests
were gathered following the national
cooperative testing protocol (NCT
Guidelines, 1996). Data gathered were
analyzed using analysis of variance and the
means were compared using Duncan's
Multiple Range Test (DMRT) of the software
IRRI Stat.


Grain Yield

Table 1 shows the yields of purelines
and multilines for three seasons. For two WS
seasons, purelines AR 32-19-3-3 and AR32-
19-3-4 were the two high yielding entries and
closely followed by AR32-19-3-2. Among the
multilines, 1:1 mixture of AR32-19-3-3 and
AR-19-10Q--9 nd rnmhinntinn of AR32-19-3-3

were comparable to the yields of the pure
stand isolines. The pureline Bio 1 performed
better than Bio 2 but both isolines had lower
yields than two IR64 derived lines from
PhilRice. All pure isolines and some multilines
were high yielder have higher yields than the
check varieties.

During WS, on yearly basis, the same
trend can be observed although very low yield
was recorded in 2002 due to prevalence of
pest and diseases, particularly blast. The
lowest mean yield among purelines was
generally obtained in AR32-19-3-2 but when
combined with AR32-19-3-3, it had better
yield than most of the purelines and multilines.
This line as pureline, although numerically
higher than the two checks during WS, was
statistically comparable with IR64. At high
pests and disease pressures, most of the
lines were comparable with the checks except
the two lines, AR32-19-3-3 and AR32-19-3-4
that are currently at the national testing.

During the DS, in the absence of high
pest and disease pressure, the highest yield
of 9.29 t/ha was obtained from the original
IR64. It was closely followed by Bio 2, AR32-
19-3-3 and its sister line AR32-19-3-4. In the
WS, AR32-19-3-2 had the lowest yield among
purelines. The mixture and purelines tended
to cluster and these were found statistically
similar with IR64.

Seasonal variation is clearly
manifested in the data and was supported by
the analysis of variance. The highest yield
was obtained during the DS and nearly just
half during WS. Across three seasons data
showed that the sister lines, AR32-19-3-4 and
AR32-19-3-3 that are near varietal release
and both having Xa-21 and xa-5 genes for
various races of BLB (Tabien et al 2003) have
consistent high yields. These are followed by
Bio 1 with xa-5 for BLB. Among the multilines,
AR32-19-3-3 either in combination with
original IR64 or its sister line, AR32-19-3-2
showed the best potential and comparable
with Bio 1.

Reaction to BLB and Other Diseases


Tabien, Abalos and Fernando

Table 2 shows the reaction to BLB
and sheath blight of elite pureline and line
mixtures or multilines during WS. Except for
AR32-19-3-2, all purelines had resistant
reaction to BLB due to the genes transferred
to these -lines using DNA markers. The
original but susceptible IR64 when combined
with any of the three AR32 lines had lower
infection, thus were rated moderately
resistant. Although the initial rate of infection
was not gathered, it was noted that the lines
had slow disease progress and the lesions
were smaller and fewer. Consistent reaction
was noted for all treatments in two WS trials
except AR32-19-3-2 and AR32-19-3-3 that
changed from MR to MS. Reactions to
stemborer (SB) were similar for all treatments
either pure or multiline (data not shown) but
not for sheath blight (ShB). Only Bio 1, all
three AR32 lines, PSB Rc28 and the
combination of original IR64 and AR32-19-3-3
had intermediate reaction to ShB. All the rest
were found susceptible. Figure 1 shows the
field stand and BLB reaction of pure stand
elite lines and IR64.

Agronomic Traits

Some agronomic data taken in
purelines and multilines are presented in
Table 3. Longest maturity was obtained from
lines developed in Indonesia while the rest
were very close to the original IR64. The
multilines, however, had generally shorter
maturity although some are near the average
of the components. The height of the mother
tillers was highest in Bio 2 and its
combinations with Bio 1 but the shortest were
from the purelines AR32-19-3-3 and AR32-19-
3-4. In most cases, the multilines had height
nearly equal to the original IR64. Highest tiller
production and highest number of panicle
were noted in Bio 1 but the highest
percentage productive tillers were from Bio 2.
Most multilines had fewer panicles and tiller
count, thus had the lowest percentage
productive tillers.

Grain Quality

Seeds harvested during the 2002 DS
were'sampled and analyzed in the Analytical
Service Laboratory at the Rice Chemistry
Division of PhilRice to evaluate the grain

quality of isolines and the combination of
isolines. In general, the grain quality
parameters were comparable to the original
pure stand IR64 (Table 4) although the
highest percentage brown rice and head rice
recovery was obtained in Bio 1 and the
highest total milled rice was from AR32-19-3-
4. The components of all multilines were
comparable to one another, thus the grain
quality gathered was fairly consistent and
comparable with the grain qualities of IR64,
the most popular rice in the country. Figure 2
shows the grains of mixture and pure IR64.


Yield, Genetic Diversity and Resistance

Monoculture is very common at the
species level nearly 100 years ago but this
expanded in different levels, reducing the
number of species of varieties within species
and even genetic differences- within the
varieties. Rice was not an exemption. Few
varieties dominated certain areas, country and
even the world. Typical example is Banay-
banay having nearly all areas planted with
IR64 and so with our rice fields in the country,
in general. Breakdown of resistance is likely
and disease control seems not feasible at all
in severe cases like in tungro. There is crop
strength through diversity (Wolfe, 2000) and
the use multilines or cultivar mixture is an
option to solve some of these problems.

Reports have indicated that
increasing genetic diversity in disease
endemic areas can enhance disease control.
It was shown in China that a combination of
resistant and susceptible rice varieties can
reduce blast severity in the area (Zhu et al,
2000). Although in smaller scale, our three
seasons study on the use multilines for control
of bacterial leaf blight showed promising
results. All mixtures had yield advantage of
19-42% over the original BLB susceptible
IR64 during WS where BLB is most prevalent.
Although the yields of pure stand isolines
were 10-61% higher than pure IR64, this may
not be durable or stable in the long run. The
isolines with single gene or pyramided genes
that are effective to several if not all races of
BLB (Tabien et al, 2003) can be like the Xa-4


)minated IR varieties that had its breakdown

The stability of multilines can be due
the presence of both vertical (VR) and
)rizontal resistance (HR) (Browing and Frey,
)69). Multilines may have several VR genes
at reduce initial infection (Xo) and have HR
at reduce rate of infection (r). It was noted
at Bio 1 and AR32-19-3-2 were both
estimated to have xa-5 gene but their reaction
BLB races present in the site of the study
offered and these could be due to the
esence of HR genes. Moreover, these were
veloped independently and were exposed
different pressure, thus may have different
ce non-specific resistance genes for other
ces or pathogens as found in wheat (Garrett
id Mundt, 1999). It was reported also that if
R was incorporated to near-isogenic lines
;e what was done in the five isolines
,aluated, and if the recurrent parent IR64
is pure line HR which is likely, Xo and r may
h reduced further (Leonard, 1969).

The susceptible varieties mixed with
line can withstand the pressure of the
sease as observed in three multilines
evaluated. This could be due to lower
section rate and slower disease
'velopment mentioned above. Resistant
ies act as barrier to the spread of the
athogens, thereby decreasing the number of
fective dispersal (Browing and Frey, 1969).
is like brush fire that its spread is dependent
i the available green grasses in the area. All
ixtures evaluated were found having
moderate resistance to BLB, markedly less
isceptible than pure stand of IR64 and the
ghly susceptible PSB Rc28. Since the
imponents differed in susceptibility, the
ixture restricts the spread of the disease
lative to the mean of their components. The
suits of the initial infections may have
lique effect in restraining the spread of the
Ithogen population (Wolfe, 1985). It can be
>ted that two of the multiline evaluated were
e simplest mixture as defined by Leonard
969), a combination of highly resistant
,R32 lines) and susceptible line (IR64). As
edicted in the model, there was a reduction
disease due to 50% reduction in the
portion of susceptible tissues per unit area
the multilines tested. This Darticular

>mbination of lines is of economic interest
nce the susceptible host genotype IR64 has
iperior agronomic traits that merit protection
rough deployment in combination with a
sistant genotype like AR32 lines listed
)ove. This will prolong the use of IR64 and
rather enhance its popularity.

The yield levels of the four multilines
iried. Two had yield higher than the mean of
e components while the other two had yield
wer than the mean yield of the components
ata not shown). It was reported that in most
Ises, the yield of the components was equal
higher than the mean component and
rely lower than the components (Wolfe,
)85) but the magnitude varies with the
nature of the components. Performance of a
ie in pure stand is not indicative of its
performance in the mixture. AR32-19-3-4 had
e highest pure stand yield but it had lower
eld when combined with IR64. Conversely,
R32-19-3-2 having the lowest pure stand
eld performed better in combination with its
ster line AR32-19-3-3. It can be noted that
e three sister lines may have high genotypic
milarities but all behave differently in
ultilines or mixtures. AR32-19-3-3 seems to
Sa good combiner but not Bio 1, Bio 2 and
R32-19-3-4. Reports have shown that very
w cultivars posses good ecological
imbining ability, those when in mixture
creases yield (Allard and Adams, 1969,
lott and Mundt, 1990). It was common only
i lines that eco-evolved for many
generations like sister lines but not like Bio 1
id Bio 2 that were selected in different
>pulations. In most cases, genotypes had
od combining ability if they are good
impetitors (yield higher when bordered by
other genotype) and good neighbors
Iduce yield increase on different bordering
,notype) (Finckh and Mundt, 1992).

Data on agronomic traits under no
ease indicate competition among
imponents. It was noted above that

ultilines had always lower tiller and panicle
lunts and majority of these was not
oductive. This condition will also affect
sease response since an increase or
crease in competition for resources can

reported that severity of disease in mix
was affected not only by selection
epidemiological effects but also on
genotype of the companion.

Genetic backgrounds are likely
influence host diversity effect on dis
(Garrett and Mundt, 1999) unless
components of the mixture are isolines
these were estimated to account for 25
the total host-diversity effect (Wolfe a
1981). Considering, however, that the iso
evaluated in this study were designed to
the IR64 genetic background, the varia
observed can be attributed to other geno
components that make a single indiv
unique. Just comparing the performance
the three sister lines support this compete
These lines maybe phenotypically like I
but absolute similarity is less I
considering gene segregation and
selection made in their develop
Specifically, AR32-19-3-2 had xa-5 gen,
BLB while AR32-19-3-4 and AR32-19-3-2
pyramided lines having xa-5 and Xa-21 g
effective to nine races of BLB. The former
rated moderately susceptible (MS) while
pyramided lines were found resistant (1
the field. Combination of MS and R lines
shown beneficial since their mixture
nearly comparable yield with the pyrarr
lines. In wheat, the mixture of R and MS
resulted to suppression of fungal disea.
MS as the season progresses (Garrett
Mundt, 1999). The same scenario was
observed in our plots for two wet sea,
thus it was rated better in resistance thar
susceptible checks.

Isolines in Multiline Development

The evaluated isolines are som
the available lines with great potential
multiline development. However, ,
backcrossing is very common in bree
programs, more valuable and important
are possibly unexplored. Backcrossing i
effective breeding method in gene substiti
or transfer to increase usefulness
successful varieties like IR64.

There are two ways to use isolini
the development of multilines, the 'clean i
and 'dirty nrnn' annrnanh fMarcAhall 1Q77\

es The combination of Bio 1 and Bio2 is a ty
id dirty crop approach wherein the compor
ie carry different single resistance gene
none of the lines are resistant to all ki
races while a combination of AR32-1!
to and AR32-19-3-4 that was not evaluate
We this study is an example of clean
ie approach where the component lines v
id be resistant to all prevalent races of
of pathogen. The simplest mixture as mentil
al, by Leonard (1969) is the combination
es and S. These are the mixtures of IR64 w
'P lines and R with MS evaluated in this s
is Our results showed that for two sea,
)ic simple mixture had higher yield than dirty
al approach but the latter had better
of resistance. Since both mixtures contain
n. or plants that can be regularly attacked b
4, pathogen, these can stabilize the
ly structure of the pathogen population
te would have an effect similar to polygenic
it. specific or horizontal resistance (Marshall
or Pryor, 1978). This strategy may extend
re useful life of strong resistance genes
as including those that have broken in the
as (Van der Plank, 1963), thus the genes liki
ie 5 and Xa-21 currently popular BLB gen(
in rice breeder can be useful in longer dun
as and the defeated genes like Xa-4 can sti
id useful in developing simple multilines for
,d control.
in As mentioned, there are se,
id isolines that can be tapped in the mull
so development. These lines, however, sh
s, be evaluated thoroughly since not all lines
ie be combined effectively. Combining at
prevalent races, genes available, mati
grain qualities are some factors to consid
the mixture development.

of Use of Multilines: Yield and Disease cc
e The use of multiline is simple
ig inexpensive strategy for disease manager
Is that can be added or integrated with (
in strategies. This can further improve efficii
on and reduce chemical dependence. More(
of multilines provide stable and reli
production compared to the pure stand w
was partly due to beneficial effect
in compensation (Wolfe, 1985). Report fui


Evaluation of multilines

pyramiding of resistance genes due to stability
of disease control.

Generally, the mechanism involved in
using multiline as strategy in stable disease
control can be attributed to: (1) decrease the
spatial density of the susceptible plants. In 1:1
ratio used in this study, the number of
susceptible plants per unit area was reduced
by 50% which further lessen the amount of
bacterial cells per unit area; (2) barrier effect
provided by the resistant plants that fill
between susceptible plants. The resistant line
in the mixture acted as deterrent in the spread
of the pathogen to susceptible plants; (3)
resistance induction by non-pathogenic
spores. Due to induced resistance, the
pathogenic spores that land on the same
area may have limited reproductive capacity
or may not infect at all. It was reported that
the combined effects of these three
mechanisms is low as observed in our limited
data but the multiplicative effect over several
generations of the pathogen will be larger that
can limit disease spread.

The big advantage of multilines as
disease management option is often
associated with reduction in yield losses. But
even though these lines may not best pureline
vari" '. Mixing offers an opportunity to
com- -e wider range of important traits that
ma. not be available or achievable to be
prc- nt in a single crop genotype. Multilines
c also reduce risk of yield losses due to
Sbiotic and abiotic stresses, reduce yield
-:ability caused by cultivar and environment
Fraction and take advantage of good
Dining ability or yield compensation in
ure grown in variable environment
,,rett and Mundt, 1999).

if Multilines: Other Issues

Maintenance of quality produce is one
Stie drawbacks of using multilines.
r,,we er, our results showed that this could
easily be dealt with by critical selection of
( -mponents. The isolines from popular
v'rietics like IR64 are potential candidates in
lultilines development. Since these are
early iike original the IR64 as shown in
agron mic and grain quality traits, the grains
will b(- uniform so with maturity and plant

height, the traits important in grain processing
and harvesting, respectively. Mixture should
be compatible in agronomic and quality
characteristics to minimize harvesting and
marketing difficulties associated with mixtures.

Rice farmers tend to use the same
seed harvested in their field for several
seasons. However, drift may occur in
multilines favoring some components of the
mixture. To maintain the effectiveness of the
multilines, these have to be reconstituted, or
the mixture changed after 2-3 cycles at most.
This will also maintain the original harvest
quality observed in the multilines.

Generation of lines from backcrossing
generally limits the breeders to improve or use
the older varieties of proven performance.
However, as shown above, several types can
be generated in the process. All lines had its
unique performance and these can be
evaluated to determine the best combination.


The isolines generated in the
backcrossing program both in conventional or
DNA marker aided selection aimed to transfer
or combine the genes are potential
components of multilines for BLB control.
Initially, the combination of AR32-19-3-2 and
AR32-19-3-4 is the most promising.
Combination of resistant line with a typical
susceptible variety like IR64 is another option
to prolong the usefulness of popular varieties,
those with proven good performance. The
results herein presented indicated the
potential of multilines in BLB control but
additional or detailed studies have to be
conducted to make use of the full potential of
this strategy in BLB control.


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Population studies in predominantly
self-pollinating species. Xlh.
Intergenotypic competition and
population structure in barley and
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Multiline cultivars as a means of

Tabien, Abalos and Fernando

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CC MUNDT. 1999.
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HARLAN JR. 1972. Genetics disaster. J.
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breeding for durable resistance to
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MARSHALL, D.R. 1977. The advantages and
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Multiline varieties and disease control.
I. The "dirty crop" approach with each
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GUIDELINES. 1996. Philippine Rice
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and EBR TADLY. 2002. Preliminary
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RAPUSAS, JP Rilon and LS
Sebastian. 2003. DNA marker-aided
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JENKINS. 1981. The use of cultivar
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Jenkyn JF and Plumbs RT (eds).
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WOLFE MS. 1985. The current status and
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Genetic diversity and disease control
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Evaluation of multilines


The authors would like to thank
ARBN-IRRI for financial support, Dr.
Bustamam for sharing Bio 1 and Bio 2, the

Analytical Service Laboratory of PhilRice for
grain quality test and staff of Plant Breeding
and Biotechnology division for technical
support during the conduct of this study.

Table 1. Grain yield (t/ha) of elite lines in pure stand and multilines (PhilRice-Maligaya 2001-2002)

Lines Yield (t/ha)
2001 2002 Average 2002DS Average
WS1 WSla ofWS *Across
Bio 1 (IR64 with xa-5 from Indonesia) 3.95cd 2.31 bcd 3.13 8.81 5.02
Bio 2 (IR64 with Xa-7 from 3.87cd 1.78abc 2.83 9.17 4.94
AR32-19-3-4 (IR64 with xa-5 and Xa- 4.33de 2.67d 3.50 9.11 5.37
21 from Philippines)
AR32-19-3-3 (IR64 with xa-5 and Xa- 4.65e 2.48cd 3.56 9.16 5.44
21 from Philippines)
AR32-19-3-2 (IR64 with xa-5 from 3.45bc 1.36a 2.41 7.64 4.15
Combination of Bio 1 and Bio 2 (1:1 3.98cd 1.59ab 2.78 8.62 4.73
Combination of AR32-19-3-3 and 4.49de 1.77abc 3.13 8.94 5.07
IR64 (1:1 ratio)
Combination of AR32-19-3-4 and 3.53bc 1.84abc 2.69 8.97 4.78
IR64 (1:1 ratio)
AR32-19-3-3 and AR 32-19-3-2 4.29de 2.06bcd 3.18 8.85 5.07
IR64 (Susceptible check) 3.07ab 1.30a 2.18 9.29 4.55
PSB Rc28 (Susceptible and yield 2.50a 1.55ab 2.02 8.96 4.33
SMeans followed by the same letter are not significantly different at 5% level by DMRT
From three replicates, # with no pest pressure. @ with high pressure of bacterial leaf blight
(BLB), sheath blight (ShB), blast and stem borer (SB).
Evaluation of multilines

44 Evaluation of multilines

Table 2. Field reaction to bacterial leaf blight (BLB) and sheath blight of elite lines in pure stand
and multilines (PhilRice-Maligaya, 2001-2002).

Lines/Multilines _Field Reaction
2001 WS 2002 WS
BLB Sheath Blight BLB Sheath Blight
Bio 1 (IR64 with xa-5 from Indonesia) R I R I
Bio 2 (IR64 with Xa-7 from Indonesia) R S R, S
AR32-19-3-4 (IR64 with xa-5 and Xa-21 R I R I
from Philippines)
AR32-19-3-3 (IR64 with xa-5 and Xa-21 R R
from Philippines)
AR32-19-3-2 (IR64 with xa-5 from MS I MS
Combination of Bio 1 and Bio 2 (1:1 ratio) S..R S R S
Combination of AR32-19-3-3 and IR64 MR I MR I
(1:1 ratio)
Combination of AR32-19-3-4 and IR64 MR S MR S
(1:1 ratio) __
AR32-19-3-3 and AR 32-19-3-2 (R/MS) MR S MS S
SIR64 (Susceptible check) S S SS S
PSB Rc28 (Susceptible and yield check)I S S I___ __
R- resistant, MR moderately resistant, MS -moderately susceptible, I-intermediate, S-

Table 3. Agronomic data of elite lines in purestand and multilines (PhilRice-Maligaya 2002 DS).

Lines/Multilines Agronomic Parameters
Maturity Plant No. of No. of %
SHeight Panicles Tillers Productiv
SI (cm)j __ __e Tillers
SBio 1 (IR64 with xa-5 from Indonesia) _120 95 26 30 67.00
Bio 2 (IR64 with Xa-7 from 120 101 21 23 91.30
AR32-19-3-4 (IR64 with xa-5 and Xa- 116 88 20 25 80.00
21 from Philippines) _
AR32-19-3-3 (IR64 with xa-5 and Xa- 112 I 88 15 19 78.95
21 from Philippines) i
AR32-19-3-2 (IR64 with xa-5 from 122 91 21 24 87.50
Philippines) _ _-0 ...
Combination of Bio 1 and Bio 2 (1:1 120 1 20 23 86.96
Combination of AR32-19-3-3 and i 20G + '9 24 79.17
IR64(1:1 ratio) i
Combination of AR32-19-3-4 and 113 957 1 22 77.27
iR64 (1:1 ratio) 73
AR32-19-3-3 and AR 32 19-3-2 113 i 17 23 73.91
(R/MS) 1 _
IR64 (Susceptible check) 113 95 20 23 86.96
PSB Rc28 (Susceptible and yield 113 '91 17 .1 80.95
check) _____, _

Evaluation of multilines

Table 4. Physical properties of grains from purelines and multilines, 2002 DS


Bio 1 (IR64 with xa-5 from
Bio 2 (IR64 with Xa-7 from

Brown Rice (%)

Total Milled Rice (%)

Average Class




Head Rice (%)



-+ Cl a




~- -4- 4 --------




AR32-19-3-4 (IR64 with xa-5 and 78.87 F 68.57 G1 47.83 G2
Xa-21 from Philippines)
AR32-19-3-3 (IR64 with xa-5 and 78.75 F 68.86 G1 49.68 G1
Xa-21 from Philippines)
AR32-19-3-2 (IR64 with xa-5 76.43 F 66.02 G1 48.87 G1
from Philippines)
Combination of Bio 1 and Bio 2 78.18 F 66.34 G1 39.30 G3
(1:1 ratio)
Combination of AR32-19-3-3 and 78.46 F 66.72 G1 48.43 G1
IR64 (1:1 ratio)
Combination of AR32-19-3-4 and 73.30 P 64.16 G2 49.78 G1
IR64 (1:1 ratio) __
AR32-19-3-3 and AR 32-19-3-2 78.64 F 68.96 G1 50.27 G1
IR64 (Susceptible check) 77.98 F 68.29 G1 48.38 G1

PSB Rc28 (Susceptible and yield
Check) ______
F-fair, P premium, G1 -Grade 1,



G2 Grade 2, G3 Grade 3


F "

i ioR

Figure 1. Field reaction of IR64 and p


stand of elite line during wet season trials.


Figure 2. Full and dehulled grains of IR64 E
leaf blight (BLB) resistant lines.

mixture of IR64 and elite bacterial




Graduate Research Assistant1 and U
College of Agriculture, University of the Pt
(2Corresponding author e-mail address: fmdcu

The variability of Cercosp
mungbean leafspot was evaluate
characterization. A total of 29 isolat
growing regions in Luzon.

Seven groups were gene
mungbean seed decoction agar (MS
9.75 mm, conidial lengths from
ranged from 3.00 8.68 pm. Dunca
18, 19 and 15 groupings based oi
conidial width, respectively. I
geographical origin.

Pathogenicity tests were c
ability of the fungal isolates. All
mungbean susceptible variety.
incubation period, lesion diameter
isolates. Virulence parameters wei
and do not reflect the locality where

Cultural and morphological
the isolates and not always dire
aggressiveness of the fungus.

Keywords: Cercospora leafspot, Cercospora c


Cercospora leafspot (CLS) is a serious
ease of mungbean in the Philippines. It is
vere during the rainy season which can cause
al crop failure on susceptible varieties but can
so occur during the dry months. The leaf spots
e circular to irregularly shaped with tan or gray
- -A_ AJ s..: L .-. f- A- -ii



ersity Researcher2, Institute of Plant Breeding,
pines at Los Banos, College, Laguna 4031.

i canescens, a fungus that causes
using cultural and morphological
were collected from major mungbean

:ed based on color observed on
k). Colony diameter ranged from 3.82
).00-137.33 pm while conidial width
Multiple range Test (DMRT) revealed
:olony diameter, conidial length and
>upings were not dependent on

ducted to determine the pathogenic
;olates were pathogenic to NCM 53
significant differences in terms of
I lesion density were exhibited by the
negatively correlated with each other
plates were collected.

laracters were highly variable among
f correlated with the virulence and

escens, mungbean

part of the entire leaf turns brown, dries ul
even defoliates. If infection is not arre!
defoliation can occur before harvesting
Philippines Recommends for Mungbean, 1991

The disease is one major constrain
mungbean production and is endemic ir
mungbean-growing areas. Yield reduction dt
CLS in the Philippines Was reported to be 23'(

Ferrater and dela Cueva

when leaf defoliation reached 75% (Quebral,

CLS is caused by the fungus Cercospora
canescens Ellis and Martin. The conidia of C.
canescens in culture and in host tissues are
acicular with truncate base and narrow tip;
hyaline, straight or curved; multiseptate, usually
straight or slightly curved; light brown; measuring
183 x 6.0 pm on the average.

Foliar diseases caused by fungal
pathogens can be controlled by fungicides.
However, excessive use of chemicals poses
problems to health and the environment. The use,
of resistant varieties coupled with sound practices
is recommended because it is safe, economical
and environment-friendly. It was observed that
some varieties that were resistant to a certain
strain of a pathogen in one location become
susceptible when deployed to other areas.
Mangaban and Natural (1988) reported that there
are morphological and pathogenic variations
within the different isolates of C. canescens but
the extent of variability has not been fully

This study was conducted to evaluate the
cultural and morphological variability of the fungal
isolates and to compare pathogenic
characteristics of the isolates on susceptible


Collection of Cercospora Leafspot-
Infected Leaves

Mungbean leaves with typical spot
symptoms were collected from different
mungbean growing areas in Luzon (Figure 1). The
diseased samples were placed in plastic bags and
brought to the laboratory. The isolates were
labeled properly according to the place of origin.

Preparation of Mungbean Seed Decoction
Agar (MSDA) Medium

One hundred (100) grams of mungbean
seeds were boiled in distilled water until cooked.
The decoction was measured and enough water
was added to bring the volume to 1000 ml.

The solution was added with 20 g agar-
agar and cooked in the microwave to dissolve the
agar completely. It was dispensed in test tubes at
10 ml each, covered with cotton plugs and
sterilized for 20 min at 15 psi.

The autoclaved tubes were placed in
slanting boards until medium congealed. The
solidified medium was stored in the refrigerator
until use.

Culture of the Fungus

To isolate the fungus, CLS-infected
mungbean leaves were surface-sterilized with 1%
sodium hypochlorite for 1 minute, rinsed 3x with
sterilized distilled water and blotted dry with
sterilized filter paper. These were incubated for
48-72 hours in Petri dishes lined with moistened
filter paper to allow conidial production. Lesions
were then examined for the presence of conidia
under a dissecting microscope (30x). Isolation of
the causal pathogen was done by picking
conidium arising from conidiophore (single spore
isolation technique) using a moistened wire loop
and streaked on MSDA slant

Single spore isolates from pure cultures
as well as subcultures were maintained on MSDA
slants overlaid with mineral oil after incubation for
7 days at 240C under continuous light (30 Watts).

Preparation of Spore Suspension

Seven-day old cultures of the pathogen
were used as inocula. Spore suspension of the
test organism was prepared aseptically. The slant
cultures were flooded with sterile distilled water
and the surface scraped gently with a sterile wire
loop. The fungal suspension was strained
through double-layered sterile cotton gauze into
sterile 250 ml beakers. The spore density was
adjusted to 50,000/mi (Lapis, 1991).

Cultural and Morphological Analysis
of Pathogen Variation

The CLS susceptible mungbean seed
variety NCM 53 was sown in 20 cm clay pot with
heat-disinfected soil in the screen house. The
seeds were allowed to germinate until the first
trifoliate leaves appeared. Five test plants per pot
were grown with 4 pots per isolate. The test

Cultural and morphological 50

plants were uniformly inoculated with spore seeded at the center of a petri dish containing
suspension (standardized at 50,000 spores/ml) of MSDA. The plates were maintained at 280C for 7
each isolate late in the afternoon. Tween 80 was days afterwhich, colony diameter was determined.
added at 1% v/v of inoculum suspension to
enhance attachment of the spores to the leaves. RESULTS AND DISCUSSION
Using a hand sprayer, the spore suspension was
directly sprayed on the lower surface of the first Collection and Purification
trifoliate leaves. After inoculation all test plants of Fungal Isolates
were covered with moistened polyethylene bags.
A wet soil condition was maintained to provide the A total of 29 isolates of Cercospora
necessary moisture for the establishment of the canescens were obtained for this study (Table 1).
organism (Telan, 1991). Four isolates were collected from Pangasinan,
two from La Union, sixteen from Isabela and
Control pots were prepared for each seven from Cagayan.
isolate by spraying pure distilled water to the test
plants. All control pots were grouped separately Mungbean is being planted as post rice
away from the inoculated plants. crop in Central Luzon, post garlic and onion in
Ilocos Region while in Cagayan Valley mungbean
Data on incubation period (number of is intercropped with corn and leguminous plants
days from inoculation to symptom appearance), like cowpea and sitao.
lesion diameter and lesion size were gathered.
Morphological and Cultural Characterization
Lesion diameter was determined by fitting of the Fungal Pathotype
a foot ruler on the lesion. Initial measurement
was done when the lesion was already visible and Colonies were slightly raised and round in
was continued until it attained maximum size or shape in MSDA (Figure 2). The 29 isolates were
until defoliation (Mangaban and Natural, 1988). grouped into 7 on the basis of the color observed
on MSDA (Table 2). Majority of the isolates
Lesion number or lesion density was (34.48%) belong to group I with cement gray
determined by counting all the lesions in each color. Other isolates belong to the following color
plant using a hand counter and taking the average groupings: Group II (charcoal gray 17.24%),
among the three test plants for each isolate, group III (stone gray 17.24%), group IV (asphalt
10.34%), group 5 (bronze green 10.34%),
The design followed in these tests was for group 6 (yew green 6.9%) and group 7 (laurel
a nested experiment in a Completely Randomized green 3.45%). Groups I and II comprised of
Block Design (RCBD). isolates from Cagayan Valley and'Central Luzon.
Group III consisted of a single isolate from Central
Characterization of Different Isolates Based on Luzon and 4 isolates from Cagayan Valley.
Cultural and Morphological Characteristics Groups V and VI consisted of isolates from
Cagayan Valley and Ilocos Region.
Spore suspensions of the test isolates
were inoculated aseptically in Petri dishes All isolates produced intense red
containing MSDA in 3 replications and were pigmentation on MSDA medium. This may be
incubated at 240C under continuous light (30 attributed to cercosporin, a unique toxin produced
watts). by Cercospora species infecting plants (Jenns
and Daub, 1995).
The cultures were closely observed for 7-
15 days for their growth characteristics such There were significant differences
colony color, size and shape as well as spore observed in colony diameter of the isolates (Table
size. Color was based on Maerz and Paul's 3). Colony size ranged from 3.82-9.75 mm in
(1950) Dictionary of Color. diameter. Among the isolates plated onMSDA,
Mc16-CV produced the biggest colony size
A 3 mm mycelial disc from 7 day-old (9.75mm). This was not significantly different with
culture of each isolate on MSDA was aseptically MC21-CV ).bOmm) and was followed by isolate

Mc22-CV (8.90mm). These 3 isolates were all
collected from Cagayan Valley. The smallest All isolates were pathogenic to NCM 53
colony size on the other hand, was noted in Mc24- and caused typical symptoms. The first visible
CV with colony diameter of 3.82mm. However, symptom was the appearance of reddish brown
this was not significantly different from that of spots manifested on the upper surface of the
Mc39-CV, Mc27-CL, Mc29-CL and Mc11-CV with leaves. The individual lesions were water-soaked,
colony sizes of 3.95, 3.92, 3.925 and 3.90 mm, nearly circular with irregularly shaped margins and
respectively. The group with the smallest colony with gray or white centers.
diameter consisted of isolates collected from
Cagayan Valley and Central Luzon. It can be Different degrees of aggressiveness were
inferred from these data that colony diameter did noted among isolates (Table 6). Symptoms
not group organisms according to the place of started to appear 2-6 days after inoculation
origin, depending on the isolate. Isolates Mc16-CV and
Mc4-CV were the most aggressive since they
Significant differences were also produced earliest lesions on susceptible
observed in conidial length (Table 4) and conidial mungbean line. It took only about 2 days for
width (Table 5) of the isolates studied. Among the these isolates to exhibit symptoms. The rest of
isolates, Mc37-CV had the longest conidial length them showed infection 3, 4, 5 and 6 days later.
but this was not significantly different from Mc41- Mc16-CV and Mc4-CV also gave the highest
CV and Mc27-CL having lengths of 136.00 Pm lesion counts with an average of 59.78 and 52.33
and 131.99 pm, respectively. The isolates with lesions per plant, respectively (Table 7). Although
the shortest conidia were Mc21-CV and Mc24-CV these two isolates yielded a correlation coefficient
which are not significantly different with each of r = 1.0, the rest of the isolates yielded a
other having conidial length of 41.70 pm and correlation coefficients of r = -0.82201. Thus,
39.00 gPm, respectively. The isolate with the incubation period and lesion density, on the
highest width was Mc48-CV with 8.68 Pm but this overall observation were negatively correlated.
was not significantly different with Mc37-CV and T w a c
Mc41-CV with conidial width measurements of There was also considerable variationin
lesion diameter among the isolates studied (Table
8.67 Pjm and 8.53 Pim, respectively. The isolates 8). Mc21-CV gave the biggest lesion size of 4.97
with the least conidial width were Mc7-CV and mm while Mc24-CV had the smaest esion
Mc39-CV with similar width of 3.00 pm. Isolates diameter. Although Mc21-CV had the widest
with the widest and narrowest width were all lesion diameter, it did not yield the highest number
collected from Cagayan Valley. Figure 5 shows of lesions per plant (only 37.33 lesions). It was
the different conidial length and width observed on noted then that lesion diameter is not correlated
representative isolates. with lesion density.

Duncan Multiple Range Test revealed 17, Computation of correlation coefficients
17 and 16 groupings based on colony diameter, revealed that lesion density and lesion diameter
conidial length and width, respectively. It is a were weakly correlated at r = 0.226115. Lesion
striking observation that Cagayan Valley (CV) diameter and incubation period were negatively
isolates were present in all groupings and shown correlated (r = -0.15738). Although this result
to pair with isolates of other regions (CL and IR) in needs to be further verified with repeated
all morphological characters studied, experiments, this initial finding has confirmed
previous analyses (Mangaban and Natural, 1988;
Pathogenic Variability Studies Lapis, 1991) that these pathogenicity characters
were loosely correlated and vary considerably
To assess the relationship between depending on the isolate.
phylogeny and virulence, all isolates were
inoculated on mungbean susceptible variety NCM To assess if grouping patterns existed
53 and were maintained in the screenhouse. among isolates when evaluated based on
Data gathered were incubation period, lesion virulence data, DMRT was computed and
density and lesion diameter, analyzed. It was shown that separate groupings
existed among virulence characters and that

Cultural and morphological

groupings were mixtures of isolates from different

From the results of morphological and
pathogenicity studies, it can be deduced that
cultural characters are not directly related to the
virulence and aggressiveness of C. canescens.
Groupings were different in each parameter being

When morphological and pathogenicity
characters were aligned with each other, a varying
pattern was exhibited by the isolates. However, a
few isolates exhibited a remarkable pattern. Mc4-
CV which has the shortest conidia gave higher
lesion number and bigger lesions. This isolate
also demonstrated greater aggressiveness
because it infected mungbean test plants as early
as 2 days after inoculation. This showed that
conidial length is not a significant factor in the
pathogenicity of the isolate.

This was further exhibited by Mc21-CV
which also got the shortest conidia similar to Mc4-
CV but gave bigger colonies on MSDA and larger
lesions on mungbean test plants.

On the other hand, Mc16-CV which was
of medium length conidia gave the largest
colonies and highest number of lesion counts. It
also obtained considerably wider lesions on NCM
test plants and shortest incubation period.

It can be suggested that colony diameter
may be related with the aggressiveness and
pathogenicity of certain isolates but not the length
and width of its conidia.

Cercospora canescens comprised a
number of isolates that differ culturally as well as
pathogenically and this great variability made the
task of classifying the isolates into groups difficult.
Moreover, variability also existed among isolates
from a particular locality as
exhibited by the isolates used in this study. Initial
studies of Mangaban and Natural (1988) using
eight isolates produced similar results. Several
studies (Adams, 1988; Sneh, et al., 1991) also
found complex variation patterns in appearance in
culture, physiological and morphological
characteristics, growth, pathogenicity, host range
and virulence observed among and within
different fungal strains. These methods also do
not offer reliable information on genetic variation

or taxonomic relationships.


morphology and virulence are phenotypic
characters and do not necessarily reveal the
underlying genetic homologies among the isolates
(Alexopoulous and Mims, 1979). Virulence genes
which control pathogen specificity towards a
particular host or host variety are under intense
selection pressure and comprise only a very small
part of the fungal genome (Flor, 1971).

Problems associated with studying
different levels of genetic diversity are better
addressed by the use of molecular techniques.
By the application of newer techniques based on
the polymerase chain reaction, it is now possible
to screen large numbers of field isolates and
reveal more accurate and specific relationships.


ADAMS GC. 1988. Thanatephlorus cucumis
(Rhizoctonia solani) a species complex of
wide host range. In: Advances in Plant
Pathology (eds. D.S. Ingram and D.H.
Williams), vol.6, Genetics of Plant
Pathogenic Fungi (eds. G.S. Sinda).
Academic Press, London, U.K. pp.

Introductory Mycology. John Wiley and
Sons, New York.

FLOR HH. 1971. Current status of the gene-for
gene hypothesis. Annual Review of
Phytopathology 9: 275-296.

JENNS AE and ME DAUB. 1995.
Characterization of mutants of
Cercospora nicotianae sensitive to the
toxin cercosporin. Phytopathology 85:

LAPIS CB. 1991. Components and
inheritance of quantitative resistance
to Cercospora canescens Ellis and
Martin in mungbean (Vigna radiata (L.)
Wilczek var. radiata) and Blackgram
(Vigna mungo (L.) Hepper). M.S.
Thesis. UPLB, College, Laguna,

Variation in Cercospora, causal


Ferrater and dela Cueva 53

organism of mungbean leaf spot in the TELAN IF. 1991. Survey, bioassay and
Philippines. Paper presented in the greenhouse evaluation of medicinal
19th Annual Convention of the Pest plants for fungal diseases of
Control Council of the Philippines held mungbean. Ph.D. Thesis. UPLB,
at Cebu City on May 3-7, 1988.. College, Laguna.

legumes in the Philippines and their MUNGBEAN. 1991. The Philippines
control. Handout. IRRI/MTCP. Recommends Series No. 25-A. 117p.
Philippines: PCARRD.
Anastomosis groups of multinucleate ACKNOWLEDGMENT
Rhizoctonia species. In: Identification
of Rhizoctonia species, pp. 67-73. APS This study is supported by the
Press. St. Paul, MN, USA. Department of Agriculture-Bureau of Agricultural
Research (DA-BAR)

ltuitl uai E8 Hi

N8: L 86Afe BAl 81
: R8Ra :FGRI : IR RR MeF8R
8 ^R8R:Ui :R H IlRGR IR
S88; '8 R~ : FIAgR 8
j R8R 1" N 9 1 IRR 8
-881 1 NOR' R o8r
8 ER 8' U
p:Rj a RA

tural and morphological 55

Die 2. Groupings of 29 Cercospora canescens isolates on mungbean seed decoction agar (MSDA)
based on colony color


)-CV Central Luzc
1-CV Cagayan Va
r-CV Central Luzc
3-CV Cagayan Va






Bronze green

Mc13-CV Cagayan Valley ((
Mc14-CV Ilocos Region (IR:

Laurel green Mc4-CV Ce


Charcoal gray

Charcoal gray

56 Cultural and morphological

Table 3. Colony diameter of the different Cercospora canescens isolates

Mc16-CV 9.75a Mc25- CV 5.58kij
Mc21- CV 9.50a Mc18- CV 5.22jjl
Mc22- CV 8.90b Mc37- CV 5.20kjl
Mc20- CV 3.02c Mc41- CV 5.20kjl
Mcl3- CV 7.50dc Mc47- CV 5.00ki
Mc14- CV 7.38de Mc15- CV 4.98kl
Mcl- CV 7.28de Mc28-CL 4.62m
Mc4- CV 7.12de Mc33-IR 4.12nm
Mc23- CV 6.80fe Mc7- CV 4.05nm
Mc48- CV 6.45fg Mc39- CV 3.95n
Mc26-CL 6.30fgh Mc27-CL 3.92n
Mc2- CV 5.88igh Mc29-CL 3.92n
Mc19- CV 5.85igh Mcl1-CV 3.90n
Mc31-IR 5.70ijh Mc24- CV 3.82n
Mc12- CV 5.68ijh
*Average of 10 colonies per isolate; means followed by a common letter are not significantly different at
5% level with DMRT.

Table 4. Conidial length (pm) of the different Cercospora canescens isolates rown on mungbean
seed decoction agar


Mc37-CV 137.33a Mcl 1- CV 67.80ihgf
Mc41- CV 136.00a Mc25- CV 67.50ihgf
Mc27-CL 131.99a Mc23- CV 62.67ihgj
Mc48- CV 124.00ab Mc13- CV 62.10ihgj
Mc33-IR 115.99bc Mc1- CV 59.40ihkj
Mc26-CL 115.50bc Mc47- CV 57.00ilkj
Mc29-CL 114.668bc Mc15- CV 49.80mlkj
Mc28-CL 110.10c Mc22- CV 47.70mlk
Mc12- CV 91.30d Mc14- CV 46.80mlk
Mc31- IR 78.67edf Mc39- CV 45.00ml
Mc7- CV 78.60edf Mc18- CV 44.70ml
Mcl6- CV 78.00edf Mc20- CV 44.40ml
Mc19- CV 77.334edf Mc21- CV 41.70m
Mc2- CV 75.00egf Mc4- CV 39.00m
Mc24- CV 71.40ehgf
*Average of 10 colonies per isolate; means followed by a common letter are not significantly different at
5% level with DMRT.

able 5. Conidial width (pm) of the different Cercospora canescens isolates grown on mungbean
seed decoction agar


Mc48-CV 8.68a Mc 3- CV 4.80jigh
Mc37- CV 8.67a Mc21- CV 4.50kjigh
Mc41- CV 8.53a Mc25- CV 4.50kjigh
Mc27-CL 7.87ba Mc18- CV 4.50kjigh
Mc31-IR 7.07bcd Mc20- CV 4.20kjilh
Mc33-IR 6.94bcd Mc12- CV 4.20kjilh
Mc19- CV 6.67becd Mc28-CL 4.20kjilh
Mc23- CV 6.67becd Mc29-CL 4.20kjilh
Mc4- CV 6.60becd Mc26-CL 3.90kjil
Mc22- CV 6.60becd Mc14- CV 3.90kjil
Mc2- CV 6.30fecd Mc24- CV 3.30kl
Mc16- CV 5.70fegd Mc47- CV 3.30kl
Mc15- CV 5.40fegh Mc7- CV 3.001
Mcl- CV 5.10figh Mc39- CV 3.001
Mcl CV 4.80jigh
Average of 10 conidia per isolate; means followed by a common letter are not significantly
different at 5% level with DMRT.

able 6. The different isolates of Cercospora canescens with their corresponding incubation period

PERIOD (days) (days)
Mcl6-CV 2 Mc19- CV 5
Mc4- CV 2 Mc39- CV 5
Mc13- CV 3 Mc47- CV 5
Mc15- CV 4 Mc37- CV 5
Mc12- CV 3 Mc23- CV 5
Mc2- CV 3 Mc29-CL 4
Mcl 1-CV 4 Mcl- CV 4
Mc25- CV 4 Mc41- CV 4
Mc14- CV 3 Mc27-CL 6
Mc21- CV 4 Mc33-IR 6
Mc20- CV 4 Mc26-CL 5
Mc28-CL 4 Mc18- CV 5
Mc48- CV 5 Mc7- CV 5
Mc22- CV 5 Mc31-IR 6
Mc24- CV 5

- -1 I* I I V. Iae

iie i. Lesion Uenbsity UiIIUiere i IsJIaia ui ru

Mc16-CV 59.78 a
Mc4- CV 52.33 ba
Mc13- CV 49.33 bc
Mc15-CV 48.22 bcd
Mc12- CV 46.33 becd
Mc2- CV 45.78 becd
Mcli- CV 42.00 ecd
Mc25- CV 39.00 ed
Mc14- CV 37.89 e
Mc21- CV 37.33 e
Mc20- CV 25.33 gf
Mc28- CL 21.89 gfh
Mc48- CV 21.78 gfh
Mc22- CV 21.44 gfh
Mc24- CV 20.44 gifh
averagee of 3 plants; means followed by a common
Yo level with DMRT.

ile 8. Mean lesion diameter (mm) of different Cen
susceptible variety.


Mc21-CV 4.97a
Mc26-CL 4.70ba
Mc27-CL 4.60bac
Mc28-CL 4.50bdac
Mc4- CV 4.47ebdac
Mc15- CV 4.40ebdacf
Mc29-CL 4.37ebdgcf
Mc11-CV 4.10ebdighcf
Mc20- CV 4.07ejdighcf
Mc19- CV 4.01ejkdighcf
Mc22- CV 3.93ejkdighlf
Mcl- CV 3.87ejkighlf
Mc47- CV 3.87ejkighlf
Mc16- CV 3.87ejkighlf
Mc23- CV 3.84jkighlf
Average of 30 lesions per isolate; means followed
% level with DMRT.

PUifJa UatIWUiaUt il III 1t4IVI uo IIIunlyIcdIIl ouucLpuuL

Mc19- CV 19.78 jgifh
Mc39- CV 19.22 jgifh
Mc47- CV 18.56 jgifh
Mc37- CV 17.89 jgih
Mc23- CV 17.00 jgih
Mc29-CL 16.89 jgih
Mcl-CV 15.00 ikh
Mc41-CV 13.00 jlikh
Mc27-CL 11.33 jlik
Mc33-IR 11.00 jlik
Mc26-CL 10.33 jlk
Mc18- CV 6.22 Ik
Mc7- CV 5.78 Ik
Mc31-IR 5.11 I

Letter are not significantly different at

cospora canescens isolates in NCM 53 mungbear

Mc14- CV 3.83jkighlf
Mc7- CV 3.77jkighl
Mc13- CV 3.77jkighl
Mc48- CV 3.73jkihl
Mc39- CV 3.67jkihl
Mc2- CV 3.60jkimhl
Mc25- CV 3.50jkiml
Mc33-IR 3.47jkml
Mc12- CV 3.47jkml
Mc31-IR 3.40nkml
Mc37- CV 3.40nkml
Mc41- CV 3.37nml
Mc18- CV 3.03nm
Mc24- CV 2.83n

by a common letter are not significantly different a

Journal of Tropical Plant Pz



Portion of the undergraduate thesis o

'Former undergraduate thesis studel
Plant Pathology, 2Associate Research Profe!
Microbiology (BIOTECH), University of
3Corresponding author. E-mail: jiorajay@ufl.(

The effect of pre-coloni
arbuscular mycorrhizal (VAM)
Meloidogyne incognita inoculated
greenhouse experiment. The ton
MycoVam or mycorrhizal root inc
the control. One month after,
inoculated with 1000 and 5000 eg!
the experiment, plant growth an
nematode.count and mycorrhizal 4

Results showed that ino(
mycorrhizal inoculant did not sigr
On the other hand, MycoVAM a
galling by about 73 and 62%, resp
at both low and high level of nem;
soil and root system were also
treatment by as much as 94 an(
was found more effective in suppi
at high nematode inoculum dens
treatment also resulted to 86% cc
was significantly higher than root

Keywords: VAM fungi, Meloidogyne incognih


The tomato (Lycopersicc
esculentum Mill.) plant is a major vegetab
crop that has achieved tremendous popular
over the last century. It is grown in outdo,
fields, greenhouses and net houses


%JAY1 3 and MB BROWN2

e first author.

Ind Asst. Professor, respectively, Department c
, National Institute of Biotechnology and Applie
SPhilippines Los Bafios, College, LagunE

ion of tomato roots by vesicular-
igi on the eventual infection of
low and high levels was studied in a
Plants were pre-treated with either
ant, with untreated plants serving as
*y were either left uninoculated or
>f M. incognita per plant. At the end of
field parameters were assessed and
>nization determined.

ttion with either of the two types of
:antly increase plant growth and yield.
mycorrhizal root inoculant reduced
:ively, indicating suppressed infection
Je inoculation. Nematode count in the
gnificantly decreased by mycorrhizal
)%, respectively. MycoVam treatment
;ing M. incognita infection particularly
than mycorrhizal root inoculant. This
lization of tomato root system, which

>ot knot, tomato

the top ranking vegetables in the wor
particularly in 'terms of production ar
importance (Alam et al, 1994). In year 200
the Philippines produced 148,100 metric tor
of tomatoes amounting to PhP 1.18 B (Ph
Statistical Yearbook, 2001). The fruit, which

.rinaria, Orajay and Brow

be processed in a variety of ways (Villareal

Plant parasitic nematodes, primarily)
Meloidogyne incognita constitute one of the
major problems in tomato production
worldwide. This sedentary endoparasitic
species is capable of rapid multiplication no!
only in tomato but also in a wide range ol
crops causing an estimated loss of 77 billion
dollars annually. Characteristic symptom ol
infection appears on the root system. Infected
roots swell at the point of invasion anc
develop into the typical root galls that are al
least twice as large as the diameter of a
healthy root. Several galls may develop as
subsequent new infections take place, giving
the root a rough and clubbed appearance
Usually, infected roots remain shorter, show
various stages of necrosis and develop rotting
as a result of secondary infection by othel
pathogens. Impairment of the root systerr
results to poor uptake of water and nutrients,
making the plant very prone to midday wilting
(Agrios, 1997).

The development and use ol
alternative methods to chemical control gets
more attention these days among researchers
and growers alike, with increasing public
awareness about the latter's perceived
potential hazards to human health and
environment. Management strategies
continually explored include various cultural
practices, biological control and even genetic
engineering of the plant to make it resistant to
nematodes. One of the most promising
approaches is the use of vesicular-arbusculai
mycorrhizal (VAM) fungi. These fungi form a
symbiotic association with plants. The fungi
gain carbohydrates from plant roots but in
return, they enhance plant uptake of inorganic
nutrients particularly phosphorus (Harrier and
Watson, 2004). VAM fungi are members ol
the Zygomycete Order Glomales belongING
to any of the genera Gigaspora,
Scutellospora, Glomus, Sclerocystis,
Acaulospora and Entrophospora (Morton and
Benny, 1990).

These fungi, although generally
accepted as growth enhancer, are not really

established as biocon agents of plan
pathogens. Reports in literature an
inconsistent when it comes to the effect o
VAM fungi on plants' responses to invadin(
pathogens like plant parasitic nematodes. Fo:
and Spasoff (1972) found that the VAIK
fungus Gigaspora gigantea increased th4
susceptibility of tobacco to the nematode. 1i
another study, Atilano et al (1976) observe(
that Glomus etunicatum or Gigaspor
margarita even caused an eight-fold increase
in the number of nematode eggs per gram o
root and pods of peanut compared with th(
nonmycorrhized plants. On the contrary, othe
researchers reported that pre-inoculation o
plants with VAM fungi markedly hampe
nematode penetration of roots (MacGuidwin
1985). Mycorrhizal treatment also reduced thE
number of nematodes that develops int(
adults as well as root gall severity and tota
nematode density in different crop!
(Baltruschat et al, 1973; Hussey an(
Roncadori, 1982; Jaizme-Vega et al, 1997). Ih
addition, VAM fungi enhanced plant vigor
masking the effect of initial nematode infection
(Roncadori and Hussey, 1977; Umesh et al
1988; and Siddiqi and Mahmood, 1995). Ir
the Philippines, researchers of BIOTECH it
UPLB are conducting efficacy trials of VAN
fungi against soil borne pathogens of different
crops, and are also developing them intc
commercial products for farmers.

Since VAM fungi's direct association
is with the host root, and the nematode
suppression is basically and highly dependent
on this established symbiotic association, it i.
important to know if the increased nematod(
inoculum density would affect the persistence
of fungi-root association and the degree o
suppression it can manifest when challenge(
by higher level of nematode inoculation. Thi,
study therefore was done specifically t(
determine the efficacy of the two types o
VAM inoculant developed at BIOTECH ir
suppressing root knot of tomato; compare the
level of suppression of VAM-treated plants a
two levels of M. incognita inoculation; and t(
determine the effect of increased level o
nematode inoculation on the persistence o
pre-established mycorrhizal network in tomato

on were assessed
killing of the whole
ng the number
im of roots (J2 tI
:id fuchsin, and
jes (newly hatch
0 cc of soil as
I- /+*.*J

per plant. Each 10% KOH, ;
fas replicated eight fuchsin in I;
pletely Randomized one-step ste

sferred to clay pots pre
of sterilized soil-sand tote
plantss were watered line

One month after VAP
nlantc wurn innrlllfw i frt ith no

inoculation, statistics
iftria anne narranntq

aluckdimrtiupf wrhilap Fv

experiment. The suspension was inoculation turned out to be significant, the
standardized to deliver 1000 eoas/ ml and mean of their combined effect was subjected

j Ia1.
i gree
f, UP
s: M
lots c
Sof t

I nere was no SignlnIGan IFILraciluIUn ue lua u WeyIILL WIIIMUL IItcoVitII y IIILUcaiUlly
detected between -the type of mycorrhizal actual root production. Weight alone seemed
ioculant and levels of nematode inoculation to be not a reliable indicator of the effect of
or all the growth and yield parameters RKN inoculation on root growth. Root length,
assessed. Tables la and b present all growth which can be digitally scanned and measured
ind yield parameters assessed as influenced (Pan and Bolton, 1991; Kaspar and Ewing,
y type of mycorrhizal inoculant and levels of 1997), can be used as an additional or an
nematode inoculation, respectively, alternative parameter.

Results of this experiment showed Effect of Mycorrhizal Treatment on the
hat mycorrhizal inoculation did not Establishment and Population Build-up of
significantly affect growth and yield of tomato M. incognita on Tomato 'Yellow Plum'
Yellow Plum' as plants treated either with
IlycoVam or root inoculant had means Unlike the growth and yield
comparable with those of control plants. parameters, the observed galling severity and
Similar were the findings of Elsen et al (2002) nematode densities were results of interaction
n banana where the arbuscular mycorrhizal between the form of mycorrhizal inoculant and
ungi they used had no significant effect on nematode inoculation level. Hence, data were
he shoot weight, shoot diameter, plant height analyzed and means presented per treatment
Ind root weight of test plants. They attributed combination in Table 2. Overall, percent
his to low mycorrhizal colonization of the galling and number of nematodes in soil and
oots as plants were harvested quite early. In root system correlated with the level of
contrast Halos and Zorilla (1979) and Orolfo nematode inoculation, which is somewhat
1982) found that mycorrhiza-treated tomato expected in experiments involving an
plantss were taller and had higher yield than increasing level of nematode inoculation
he untreated plants. This apparently is the (Mulyadi, 1987; Khalequzzaman, 2003).
visual effect of mycorrhizal treatment as a Regardless of VAM inoculant, plants
esult of enhanced crop nutrition (Harrier and inoculated with 5,000 eggs had more severe
Vatson, 2004). In this present study, the root galling, higher number of migratory
observation could be due to the use of pots stages (juveniles and males combined) in the
hat were not large enough to allow full soil, and higher number of sedentary stages in
expansionn of the root system. Shoot growth the root system than those inoculated with
and yield are directly related to root 1,000 eggs. In uninoculated plants, neither
reduction, the latter being responsible for galls nor nematodes were detected in both
nutrient and water uptake. Plant development soil and root system, affirming the validity of
can be influenced by container size and in the treatments done. These data clearly show
general, as size decreases, plant leaf area, that pre-inoculation of tomato roots by VAM
ind shoot and root biomass decrease fungi did not prevent infection of roots by
Cantliffe, 1993). Hence, the expected growth hatched juveniles of M. incognita. VAM fungi
enhancement effect of VAM on treated plants are not pathogens of nematode (Harrier and
wouldd have been masked by the limiting effect Watson, 2004) and certainly, do not attack
)f small pot size. juveniles entering root tissues or those
sedentary stages that are already in. It can
Nematode inoculation, either at 1,000 therefore be assumed that the numbers of
)r 5,000 levels likewise did not affect hatched juveniles that actually penetrated the
significantly all of the assessed parameters roots of VAM-treated and untreated plants are
except root weight. In the latter, inoculated not different at all as reported by Suresh et al
omato roots weighed significantly higher (1985) on tomato and Smith et al (1986a) on
:orr pared with the uninoculated ones. This is cotton, which explains this correlation
luite different from what was expected since between inoculum density and galling severity
nematode infection and feeding normally and nematode density with o; without VAM
retard root development (Agrios, 1997). Gall fungi.
ormation obviously results in the enlargement
of orimarv roots. whic.. 7ould have added to

Suppression of two Ik

This study also showed the ability
VAM fungi to significantly reduce galling a
nematode counts in roots and soil of plar
under the same level of inoculum. F
instance, at 1,000 eggs inoculum lev
MycoVAM and root inoculant treated plar
had 6 and 9% root galling respective
compared with 22% in untreated plan
These correspond to 73 and 59% reduction
the two VAM inoculants, respectively. At t
higher level of nematode inoculum, cont
plants had 63% galling while treated plar
only had 18 and 24 %, an equivalE
reduction of 71 and 62% by MycoVAM a
root inoculant, respectively. Interestingly, t
galling severity in the VAM-treated plar
inoculated with 5,000 eggs was comparable
that of control plants inoculated with 1,0
eggs. This reduced galling w
complemented by significant decrease in t
number of nematodes detected in root system
and soil medium of VAM-treated plan
Furthermore, it was shown that the relati
efficacy of the two VAM fungi inoculants
suppressing M. incognita was not affected
increasing nematode inoculum level frc
1,000 to 5,000 based on the perce
reduction. Although gall severity a
nematode numbers in root system and s
increased with the inoculum level, the perce
reduction in those parameters turned out to
even consistently higher at 5,000 than
1,000 levels, suggesting a higher degree
suppression. There seems to be
maximization of the benefits of the symbio
relationship in situ leading to mc

Since VAM fungi did not prevent rc
infection by M. incognita, the significant
lower number of nematodes found
mycorrhized roots could be due to a differE
mechanism. The most likely one is t
alteration in the host root in response to t
fungi that made them poorer hosts compare
with non-mycorrhized plants. Francl (19
and Borowicz (2001) qualified it a! an induc
resistance. For example, Morandi (19t
noted greater phytoalexin production
mycorrhized than in non-mycorrhizd soybe
roots. Such compounds have been pr3viou;
associated with an incompatible rott-kn,

colonization was increased levels of lignil
and phenols, which were associated wi
reduced reproduction of M. javanica (
tomato (Singh et al., 1990). On the special
effects of mycorrhizal colonization to tl
nematodes, Smith et al (1986b) reported th
the rate of development of second-sta(
juveniles (J2) of M. incognita to egg-layir
females was delayed when the nematode w.
added 28 days after planting to soil load(
with G. intraradices but not when it w;
inoculated at planting time. In another stud
MacGuidwin et al (1985) showed that the tin
required for the inoculated J2 of M. hapla
mature in mycorrhized onion roots was abo
1,000 degree hours longer than in no
mycorrhized roots. In this present study, tt
plants were exposed to nematodes for tv
months, duration which under tropic
conditions could allow two generations
nematode (one cycle takes about four weeks
In the case of mycorrhized tomato roots, tt
lengthening of the maturation period of tr
nematode probably caused lesser nematode
that reached reproductive maturity. Let
number of mature nematodes would definite
reduce the population size of the secor
generation. In addition, the lowered e,
production of the M. incognita feeding in tl
mycorrhized roots as observed by Caling
al (1989) could also have contributed to ti
overall reduction in the number of nematode

Between the two forms of mycorrhiz
inoculants, MycoVAM treatment suppress<
better the nematode build-up than ro
inoculant. This was particularly observed
higher level of nematode inoculum whe
MycoVAM-treated plants had only 31 and 1;
nematodes per 100 cc of soil and per gram
roots respectively, or 94 and 70% reducti(
on those parameters relative to the untreat(
plants. Treatment with root inoculant result(
only to 91 and 54% reduction, again on tho:
parameters respectively at 5,000 inoculu
level. It cannot be exactly rstt-,,int
however, if this higher reduction by MycoVA
treatment was really due to the formulatic
per se or to amount of VAM fungi propagulI
contained by the product applied at tl
recommended rate (5 g for MycoVAM and 0
g for root inoculant). Propagules introduced

Laurinaria, Orajay and Brown

quantified in this study, but it is possible that
MycoVAM delivered more propagules,
resulting to faster colonization of root system
as the data on the next section shows.

Effect of High and Low Levels of
Subsequent M. incognita Inoculation on
Mycorrhizal Colonization of Tomato Roots

The extent of mycorrhizal
colonization of roots was determined using
the grid line intersect method. Analysis
showed no significant interaction between the
form of mycorrhizal inoculant and level of
nematode inoculation. This simply suggests
that forms of mycorrhizal inoculant and levels
of nematode inoculation independently
influence further mycorrhizal colonization of
the roots.

Table 3a shows that the form of
inoculant could influence the extent of
mycorrhizal colonization of roots. Plants
treated with MycoVAM showed higher
percentage of the roots colonized than those
treated with root inoculant. This significant
difference in root colonization may account for
the observed higher reduction in nematode
number inside the roots of tomato exposed to
higher inoculum level as discussed earlier.
On the other hand, the level of subsequent
nematode inoculation appeared not a
significant factor in further root colonization by
VAM fungi (Table 3b). Neither the 1,000 nor
the 5,000 level of inoculation resulted to
mycorrhizal root colonization significantly
different from that of the nematode-free

In literature, contrasting reports can
be found with regard to the effect of
nematodes on the development of VAM fungi
in the roots, but these could be attributed to
complex and specific interactions among host
plant, species of nematode and VAM fungi,
and experimental conditions. Nematodes
could impede mycorrhizal establishment in the
roots as suggested by the increased VAM
infection following treatment of soil with
nematicides (Bird et al, 1974; Germani et al,
1980; Menge, 1982). In another study, neither
sporulation nor root infection by G. margarita
was affected by the presence of M. incognita
in soybean roots (Carling et al, 1989). In our

study, take note that the tomato plants, which
were still seedlings at that time, were exposed
to VAM inoculant four weeks before the
nematodes. Francl (1993) stated that in a
nematode-mycorrhizal fungi interaction, the
first organism to arrive at and colonize the
habitable sites within the root system would
have the advantage. In that case, it was the
VAM fungi that got this perceived advantage
in such a way that subsequent nematode
inoculation, whether low or high, did not affect
anymore the already established fungal
structures. It cannot be concluded however, if
this will still hold true once challenged by an
even higher level of nematode inoculum. This
would be an interesting subject to study.

Our present study affirmed the
beneficial effect of mycorrhizal treatment as
far as the reduction of galling and number of
root-knot nematode are concerned. Between
the two forms of mycorrhizal inoculants being
produced by BIOTECH, MycoVAM was
shown to be more effective in reducing root-
knot nematodes especially at higher inoculum
density than the root inoculant, maximizing
the benefits of the symbiotic relationship. This
was correlated with the higher percentage of
root system colonized in plants treated with
MycoVAM, which was not at all affected by
either levels of nematode inoculation. It is
important to stress however, that VAM-
treatment must be made before plants get
exposed to nematodes. For example,
seedbeds can be treated with mycorrhizal
inoculant to allow its establishment that will
eventually confer certain level of resistance to
invading root-knot nematode.


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Table la. Effect of type of mycorrhizal inoculant on growth and yield of tomato 'Yellow Plum'
Under greenhouse conditions

Type of Shoot height Shoot dry Total fruit weight Root fresh
inoculant (cm) weight (g) (g) weight (g)

MycoVam 112.67 16.93 19.98 14.88

Root inoculant 114.88 18.29 17.06 14.95

Control 113.67 18.22 19.22 13.77

Table lb. Effect of levels of Meloidogyne incognita inoculation on growth and yield
of tomato 'Yellow Plum' under greenhouse conditions

Inoculum level Shoot height Shoot dry Total fruit weight Root fresh weight
(eggs/pot) (cm) weight (g) (g) (g)

0 117.00 16.92 18.34 11.40 a

1,000 111.06 18.53 19.48 15.39 b

5,000 113.15 18.00 18.44 16.81 b
Means followed by the same letter are not significantly different according to Tukey's HSD
at 5% level.

treated with two forms of mycorrhizal inoculant and levels of nematode inoculation

rpe of RKN % % Juveniles/ % Nematodes/ %
culant inoculation Galling2 Reduction4 100 cc Reduction4 g roots3 Reduc
level(eggs/pot) soil3

oVAM 0 Oa 0a 0 a

1,000 6b 73 13 b 88 71 b 6

5,000 18 c 71 31 cd 94 125 c 7(

t 0Oa Oa Oa

1,000 9b 59 21 bc 79 86 b 5:

5,000 24 c 62 47 d 91 189 d 5

itrol 0 Oa 0a 0 a

1,000 22 c 98 e 183 cd

5,000 63 d 540 f 413 e
sans of 8 replicates. Means in a column followed by the same letters are not significantly
ferent according to Tukey's HSD test (P < 0.05).
means of original data were presented but they were arcsine transformed first prior to analysis.
,presents all sedentary stages. Means of original data were presented but they were log10
S1) transformed first prior to analysis.
percent reduction at the same RKN inoculation level. Computed using the formula:
% reduction = control -VAM treated x 100

CI mnnrec

Table 3a. Mycorrhizal colonization of tomato
mycorrhizal root inoculant.



Mycorrhizal root inoculant

1 Means of original data were presented but
analysis. Means followed by the same let
by Tukey's HSD.

Table 3b. Mycorrhizal colonization of tomatc
Meloidogyne incognita

Level of inoculum (eggs per pot)

0 (uninoculated)

1,000 (low)

5,000 (high)
1 Means of original data were presented I
analysis. Means were not significantly diffe

roots 11 weeks after treatment by MycoVAM ar

% Mycorrhizal Root Colonization 1

86.09 a

72.70 b

0.00 c
sy were arcsine transformed prior to statistical
are not significantly different at 5 % level

>ots 7 weeks following three levels of inoculation

% Mycorrhizal Root Colonization '



: they were arcsine transformed prior to statistic
it at 5% level by Tukey's HSD.



Optimization of Fungicide Application foi
the Control of Blossom Blight of Mango
(Mangifera indica L.) Caused by
Colletotrichum gloeosporioides (Penz).
M.R.A.Nabua (Department of Plant Pathology,
College of Agriculture, UP Los Bahos,
College, Laguna) Best Undergraduate
Thesis Award

Experiments were performed in three mange
production systems, namely, BelCris Farm,
Digos City, ECJ Farms, Hagonoy, Davao del
Sur and SODACO Farm, Malungon,
Sarangani to determine the protectant and
systemic fungicides with outstanding
performance against blossom blight anc
establish the best frequency and timing ol
spray application. The experimental
treatments were evaluated on the basis o0
blossom blight severity and yield components
Experimental results indicated strong
evidence to conclude that there exist a group
of fungicides, which can be used effectively
against blossom blight under low anc
extremely high disease pressures. Under low
disease pressure, all the test fungicides were
effective in suppressing blossom blight,
however, only the systemic fungicides hac
provided satisfactory floral protection undei
high disease pressure. Azoxystrobin anc
Difenoconazole performed outstandingly
among the systemic fungicides. Results alsc
showed enough reason to believe that propel
timing of spray application can significantly
suppress blossom blight even at a reduce
spray frequency. Under low disease pressure
one spray application of Mancozeb oi
Difenoconazole between 21 to 28 days aftei
flower induction (DAFI) can adequately
suppress blossom blight to an acceptable
level. Under high disease pressure, one tc
two spray applications of Azoxystrobin within
10 to 28 DAFI provided sufficient flora
nrotectionl from blossom blight. One spray
-- Z -119 --- -- A -

ITY ON MARCH 16-19, 2004

increased if it is applied at the early stage of
floral development (10 DAFI). The observed
protective effects afforded by protectant and
systemic fungicides were consistently
translated into better fruit set, significant
increased in fruit retention and ultimately
higher fruit yield.

Molecular and Cellular Responses of
Plants to Diseases: Nicotiana species as
Model Hosts. Paul H. Goodwin (Associate
Professor, Department of Environmental
Biology, University of Guelph, Ontario, NIG
2WI Canada)

A number of model plant-microbe systems
have been developed to examine the
molecular basis of plant disease-resistance
and susceptibility. Nicotiana species make
good model hosts as they are easy to
genetically manipulate and are widely studied
at a molecular level in plant-virus interactions.
Nicotiana benthamaiana has been used in
the development of virus-induced gene
silencing (VIGS), where a plant gene is
silenced through RNA interference by
infecting a plant with a recombinant virus
containing a portion of the plant gene. We are
studying the response of plants infected by
the hemibiotrophic fungi, Colletotrichum
destructivum and C. orbiculare. These
pathogens of Nicotiana species were
transformed with the green fluorescent protein
gene to make them easier to monitor in the
host. Genes selected for analysis were
chosen based on their frequency of
appearance in expressed sequence tag
libraries developed from several different
Colletotrichum-infected plants. The
expression of selected N. benthamaiana and
N. tabacum genes were examined following
infection by Colletotrichum, and some of
these were silenced by VIGS. Examples to be
discussed are glutathione S-transferase
genes and genes related to ethylene.

Leaf Scald, A New Disease of Sugarcane in
the Philippines. M.P. de Ocampo, F.M. dela
f .... n r 1 -Ar M a :- -

(Institute of Plant Breeding (IPB), College ol
Agriculture, UPLB, College, Laguna anc
PHILSURIN Research Station, VMC
Compound, Victorias City)

A selective medium (Modified Wilbrink's
Medium) was used in the isolation ol
Xanthomonas albilineans (Ashby) Dowson,
which causes leaf scald disease oi
sugarcane. MWM medium supported higl
plating efficiencies of the pathogen. The
growth rate, morphology and pigmentation ol
colonies on the medium were used to identify
the pathogen. Likewise, cultural, biochemica
and physiological characterization were done
to characterize the pathogen. Pathogenecity
tests were performed using manua
decapitation of cane tops above the growing
point, usually between the 3m and 4th dewlap
followed by application of infected juice oi
pure culture of the bacterium Polymerase
chain reaction using specific primers, PGBL 1
and PGBL 2, designed on the basis of a
multiple sequence alignment amonc
sequences of Xanthomonas albilineans was
employed. PCR amplification of infected juice
and pure culture of the bacterium yielded a
288pbp DNA product.

The selective isolation and nucleic
acid base method (polymerase chain reaction
confirmed the presence of leaf scald cause
by Xanthomonas albilineans, in some
commercial sugarcane fields in the

Biological Control of Head Rot Disease
Caused by Rhizoctonia solani Kuhn. 0
Cabbage (Brassica oleracea var. capitata
Using Microbial Antagonists. B.D. Acaba
Jr. (Leyte State University)

Tne study was conducted in vitro, pot anc
field experiments to: 1) collect, isolate an<
screen potential microbial antagonists against
Rhizoctonia soiani in vitro; 2) determine the
most promising and effective antagonis
against R. solani in pot experiments and 3
evaluate the most effective antagonist a.
biocontrol agent against head rot disease o
cabbage under field condition.
rTh. A;rifn*rnt ntnnnircte vhihitea

megaterium, Trichoderma viride anc
Trichoderma sp. (onion isolate) significantly
reduced radial growth, caused disease
suppression and were highly antagonistic to
R. solani. Trichoderma sp. (onion isolate)
applied at the rate of 1.33 tons/ha and 1.99
tons/ha. obtained low disease incidence and
higher disease reduction of 87% and 90%,
respectively compared with the fungicide
(Mancozeb) at 62% reduction only. Yield
obtained using the two rates were 0.56 kg and
0.59 kg marketable head per pot and were
higher compared to the fungicide (Mancozeb).
Highest net income or gross margin was
obtained in Trichoderma sp. (onion isolate)
treated plants at 75 g and 50 g with Php 3.44
and Php 2.87 per head compared to fungicide
(Mancozeb) with Php 0.45.

Head rot incidence was significantly
reduced when Trichoderma sp. (onion isolate'
was applied two or three times at 10 WAP
Three applications of the antagonist had the
lowest head rot ranging from 3% to 4% bu'
not significantly different to the fungicide
(Mancozeb). Yield of marketable heads dic
not differ with each other. Incidence of heac
rot disease in the field was high whict
resulted to less marketable heads and lowel
yield. The antagonists failed to suppress
completely the head rot disease in the fielc
which resulted to negative net return or gross

Control of Cassava Bacterial Blighl
Caused by Xanthomonas campestris pv
manihotis Using Bacterial Antagonists
M.K. Palomar and M.B. Posas (Leyte State

The effects of different concentrations of three
promising bacterial antagonists applied a,
protectant were evaluated for the control o
bacterial blight of cassava caused b,
Xanthomonas campestris pv. manihotis. It
potted experiment, the protective effect of th(
three promising bacterial antagonists namely
Pseudomonas fluorescens, Pseudomona,
sp. and Bacillus sp. increased with the
increase in level of inoculum concent -:tio;
regardless of the method of inoculation sed
Field test of the most effective concenr atiot
(1.3 x 108 cfu/ml) showed no interaction
between the method of inoculation and

Ahctrar4t r


promising bacterial antagonist us
Xanthomonas campestris pv. n
highly significant reduction of pei
infection. percent disease i

;ed to control
ranihotis. A
cent disease

inoculum density of L. theobromae, the
greater the percent infected roots, thus the
lower the percent disease control. Regardless
rf inrwnlnlm rAnncitine rf TriFhnrwArm~ nn 17rt

observed on plants treated with any of the
three promising bacterial antagonists.

VarifIanIlnn Trial fnr fhe (Pntnrnl Af Tine

percent infected roots and percent was conducted to evaluate the effic
control in both 50 and 100 g inoculum Virtuoso 10 AS (Bacillus subtilis QS
L. theobromae. The higher the Strain) for the control of manoo anthract

n blight. Virtuoso 10 AS (Bacillus incidence. Plant tissues were prepared as in
s QST 713 Strain) also exhibited laboratory experiment. Soil mixture was
nt performance in reducing incubated in sealed plastic bags for 15 days,
:nose infection on harvested fruits. On aerated for 1-2 days. Placed in seedling bags
ior hand with Flcinnp mannifarh thp (.FiO n p ah) and nianted with tomato

as. monnoreo weemy.

tion of Crucifer Wastes as The results of the laboratory
:..-jk-k Bjk DerajXl BWl PAnCMni nvnrriramntoe chnwjr4 +hpat Qracn n wxtafr


potential to suppress Ks population ranging Anthracnose (Colletotrich
from 6 to 15 fold three weeks after gloeosporioides Penz.). G.A. Peralta, I
incorporation compared with untreated soil Saavedra and R.C. Suiza (NCPC-CA,UF
when the soil mixture was wrapped in nylon and Syngenta Phil.)
cloth. When the soil mixture was incubated in
sealed containers the broccoli roots and The study was conducted from Decem
shoots and radish leaves reduced Rs 1999 to May 2001. The first experimental
population in the soil by 72 fold three weeks up was conducted in a mango orchard
after incorporation compared with the control. Brgy. Palangue III, Naic, Cavite fr
December 1999 to May 2000 while
In greenhouse test no wilting was second experiment was conducted in
observed in soil treated with cabbage leaves, mango orchard at Brgy. Calumpang Lej
Broccoli roots or leaves, cauliflower roots or Indang, Cavite from December 2000 to N,
leaves, mustard roots or leaves and radish 2001. The experiment was set-up in RC
leaves while the untreated soil had a mean with 4 replications. The test chemicals Y
wilt incidence of 42 %. applied five times after flower induction: at
22, 38, 55 and 101 days after flower induct
In Majayjay field trial (mid elevation) (DAFI). The study was conducted to evalu;
with sandy loam soil, there was reduction of the efficacy of Acibenzolar-S-Methyl
bacterial wilt incidence in plots treated with Induction of Resistance in Mango (Mangffi
broccoli (cv. Nomad) roots (12.8 %), broccoli indica L.) to Anthracnose (Colletotrich,
(cv. Nomad) leaves (16.7 %), broccoli (cv. gloeosporioides Penz.)
Patriot) roots (15.6 %), broccoli (cv. Patriot)
leaves (11.1 %), cauliflower (Milkyway) roots Acibenzolar-S-Methyl showed
(17.7 %), and cauliflower (whiteflash) roots + phytotoxic effects on mango leaves, flow
leaves (19.4 %) compared with untreated and fruits during the trials. In both tri
control plots (47.2 %) eight weeks after mango trees sprayed with Acibenzolar
transplanting. In Liliw field trial (mid-elevation) Methyl have lower percent disease sever
with sandy loam soil, plots treated with compared to the untreated control. T
broccoli (Nomad), Radish (Speedy) and performance of Acibenzolar-S-Methyl
Mustard (Chinese) had wilt incidence of 5.2 higher rates was comparable to I
%, 8.4 %, and 11.5 %, respectively, compared standards, benomyl and difenoconazole.
with 28.1 % in untreated plots. lower rate. the performance was comparal
to the standard, mancozeb. Percent coni
At the NCPC bacterial wilt nursery obtained from the highest rate of Acibenzol
(low elevation) with clay loam soil, plots S-Methyl treated trees was more than 80%.
treated with broccoli (Nomad), cabbage medium rates the product performance w
(Scorpio), and radish (Speedy) wastes had comparable to the standards. At lower rate
wilt incidence of 4.5 %, 10.2 %, and 14.2 %, the efficacy of Acibenzolar-S-Methyl was i
respectively, compared with 31.8 % in as effective as the standards, benomyl a
untreated control plots 3 weeks after Difenoconazole but performed better th
transplanting In another trial, significant mancozeb. Percent anthracnose infection

, -

differences in wilt incident
were observed only in
plastic sheets after
biofumigants. Untreated
incidennr of RR 4 % wh


an tt

Abstracts of papers

Fruit set at 45 and fruit retention count
at 60 and 75 DAFI showed that Acibenzolar-
S-Methyl treated trees have a significantly
higher fruit set and retention count than the
untreated control and were comparable to the
standards, benomyl and Difenoconazole at
higher rates. At lower rate the product
performance was comparable to the standard,
mancozeb. Highest fruit retention count at 75
DAFI was obtained from Acibenzolar-S-Methyl
treated trees at 5.0 ml. a.i./100 li.
Acibenzolar-S-Methyl treated trees produced
significantly more marketable fruits than the
untreated control. At 2.50 and 5.0 ml a.i./100
li., the product's performance was comparable
to the standards, benomyl and
Difenoconazole and significantly excellent
than the standard, mancozeb. At lower rate
the product performance was comparable to
the standard fungicide, mancozeb.

Mimizing Sugarcane Disease Introduction
Through Strict Implementation of
Quarantine Protocols. F. M. De la Cueva, M.
B. Palacpac, M. P. de Ocampo, C. R. Untal,
L. Gelia and R. T. Luzaran (IPB-CA, UPLB,
Post-Entry Quarantine Station, Los Bafos,
Laguna and PHILSURIN)

The threat of introduction of new diseases or
new strains of pathogens is always a part of
sugarcane germplasm exchange. As part of
sugarcane varietal improvement project, post-
entry quarantine procedures for sugarcane
were developed and implemented. Foreign
varieties were observed for 24 months inside
the greenhouse where routine disease
detection and diagnosis were being done.
Sensitive and specific diagnostic assays for
pathogens were developed/adapted and
applied. Open field quarantine was also
established in an isolated area where religious
monitoring of introduced varieties were
performed. Materials that exhibited typical
symptoms and signs of diseases such as
mosaic, yellow leaf, grassy stunt and leaf
scald were rogue out and burned. Certified
disease-free materials were hot water-treated
and multiplied for multi-location evaluation.

Expression of Resistance of 'Pisang Jari
Buaya' .and 'Yangambi Km 5' to
Radophilus similis Thorne (1949) Under In
vitro Conditions. J. I. Orajay, A. Elsen and

D. E. Waele (Dept. of Plant Pathology-CA,
UPLB and Katholicke Univ. Leuven, Belgium)

An experiment was performed to monitor the
expression of resistance of 'PJB' and
'Yangambi km 5' in time. 'Grand Naine' was
included as the susceptible reference.
Plantlets grown in MS rooting medium were
inoculated with manually-picked gravid
females of Radopholus similis population from
Uganda. They were incubated at 280C with
16-hour photoperiod. Roots were harvested
2,4,8,12 and 16 weeks after inoculation. At
each period of analysis, juvenile male and
female nematodes were counted separately
from roots and medium. Also, root sections
were sampled and processed for histological
analysis to monitor expression of resistance
mechanisms as proposed in literature.
Significant interactions were found between
factors time and cultivar for most of the
parameters. At week 8, differences in the final
population R. similis among the three cultivars
were observed. 'PJB' had a significantly lower
total nematode counts than 'Grand Naine',
while 'Yangambi' was neither different from
any of the two. The results show that
resistance in 'PJB' and 'Yangambi km 5' is
expressed as slowing down of nematode
population build-up rather than complete
inhibition of multiplication. Toward week 16,
total nematode counts of both cultivars were
already comparable with that of 'Grand

Staining of root sections of both
inoculated and uninoculated plantlets with
toluidine blue revealed the presence of
lignified/suberized cell walls in the central
cylinder and endodermis of 'PJB'. This was
similarly observed though at a lesser extent,
in 'Yangambi km 5'. Its detection in
uninoculated plantlets confirmed the
constitutive nature of this mechanism of
resistance. Such thickenings were not
observed in 'Grand Naine'. Occurrence of
phenolic cells in the stele and cortex were
observed in all of the three cultivars, but the
expected massive accumulation of such cells
in 'Yangambi km 5' as an induced response
was not detected. The fact that there was a
slowing down of nematode reproduction in the
two cultivars suggests that other mechanisms

Abstracts of papers

of resistance are operating which needs to be
probed further.

In Vitro Screening for Resistance to
Radopholus similis Thome (1949) in
Selected Philippine Musa Cultivars. J. I.
Orajay, A. Elsen and D. E. Waele (Dept. of
Plant Pathology-CA, UPLB nd Katholicke
Univ. Leuven, Belgium)

Four Philippine Musa cultivars namely
'Serorita', 'Pamoti-On', 'Matavia' and Pisang
Lemak Manis' were screened under in vitro
conditions for resistance to Radopholus similis
population from Uganda. The cultivar 'Grand
Naine' was used as the susceptible check.
Plantlets were grown in glass jars containing
rooting medium for four weeks. They were
inoculated with 25 manually picked gravid
females of R. similis from monoxenic cultures
in alfalfa callus. Ten replicates were prepared
for each cultivar. They were incubated at 280C
with 16-hour photoperiod. After eight weeks,
nematodes were extracted both from the root
system and medium by maceration-sieving
method. Volume of suspension was adjusted
to 50 ml and the number of juveniles, males,
females, separately determined based on 3 x
2 ml sub samples. Final population of R.
similis was significantly lower in cultivars
'Sefiorita' and'Pamoti-On' than in 'Grand
Naine', while 'Matavia' and Pisang Lemak
Manis' had nematode counts not significantly
different from the susceptible check. This
present in vitro screening study confirmed the
resistance of the two cultivars which was
previously detected under greenhouse
experiments, and further established the
suitability of 'Grand Naine' as a susceptible
reference cultivar for Musa screening studies.

Screening for Resistance of Improved
Banana Hybrids Against Sigatoka. L. E.
Herradura, M. A. Alforque and A. B. Molina

Sigatoka leaf spot disease is one of the most
important constraints of banana production as
it significantly reduces fruit yield and quality,
and the high cost associated to its control.
Since chemical control is expensive beyond
the means of small scale growers, and its
negative impact to the environment and
human health, the Musa germp'ism

improvement program of INIBAP through its
network of Musa breeding programs
worldwide focused on to the development of
disease-resistant varieties. Several improved
Musa varieties/hybrids were made available
for field evaluation against Sigatoka leaf spot
disease in different environmental conditions
worldwide. The BPI-DNCRDC received
several accessions and together with two
popular local cultivars evaluated them against
Sigatoka leaf spot disease under local

The different varieties were planted in
the field in a replicated Randomized Complete
Block Design (RCBD). Disease rating was
done using INIBAP Technical Guidelines for
evaluation of germplasm for resistance to
Mycosphaerella leaf spot diseases. Disease
rating parameters, in terms of Youngest Leaf
Spotted (YLS), Disease Development Time
(DDT) and Disease Severity (DS) showed that
some varieties suffered less infection than
others did. The most susceptible was the local
cultivar Lakatan while Cardaba and the
resistant checks showed high resistance to
the disease. Several FHIA varieties exhibited
high degree of resistance to Sigatoka leaf
spot. Results indicate the potential of some of
the improved varieties for managing Sigatoka
leaf spot disease.

Sustainable Fresh Market Tomato
Production Utilizing Sod-Based Strip
Tillage and Resistant Cultivars. J. R. Rich,
S. M. Olson, and M. T. Momol (University of

Fresh market tomatoes (Lycopersicon
esculentum) were grown in established
bahiagrass (Paspalum notatum) utilizing strip-
tillage techniques in eight field trials over four
years in northern Florida U.S.A. Cultivated
strips were made into bahiagrass sod and
tomatoes transplanted into the strips. Main
plots consisted of tomato cultivars with
variable levels of resistance to soilbome
fungal diseases and nematodes, and
presence or absence of soil applied
fumigation. Tomatoes were drip-irrigated and
trellised but only limited polyethylene mulch
was applied. Production factors explored were
1) sod management; 2) strip-tillage widths; 3)
fertilizer rates; 4) soil-borne disease incidence

ir field evaluation was
small Diot trials An ir

i o IIIY"-od.la WUityl, OILGIIII, irIiUL;tlIUDJ U" cIULUcIII atll
almn n.ndr irtai aennrhibr ar'il clirl nnt uarv eiinnifirnntflr

*ll *l V6 w EI ~f, C el 01 0 IVJ I I1 V IUPVJI
petition to the tomatoes. Tilled strips in The larval development, cocoon
1.8 m-wide-rows were subsequently production and silk yield and quality were
eased to 75-cm-wide. Nitrogen rates seriously affected when silkworm was fed with
:al of northern Florida tomato production MRR-infected leaves. Silkworm larvae fed
) kg/ha) was found adequate for good with MRR-infected leaves had high larval
ato yields. Soil-borne disease incidence mortality and longer larval duration, was
low and comparable to conventionally efficient feeder and produced lighter cocoons
ited tomatoes fumigated with standard with silk filaments that frequently break during
hyl bromide treatment. Root-knot reeling. The number of cocoon per liter,
latodes were found in areas of the percent reelable cocoon, cocoon shell
iagrass that previously contained percentage, denier, renditta and reelability
idleaf weeds, but this was addressed were not affected by MRR.
g resistant tomato cultivars. In the large-
e farmer trial, yields were lower in strip-till Disease management interventions
atoes but profit was higher due to fewer such as host resistance and cultural practices
its compared to the conventional planting. such as sanitary pruning and branch thinning
strip tillage tomato production is radically were evaluated under normal mulberry
rent from the currently used high input production systems. Field screening of 19
em, and it offers potential as a mulberry cultivars against MRR yielded one
ainable, profitable and lower input resistant cultivar (Alfonso), five moderately
native. resistant (S13, C6, M. Local, Sa6, and SRDC2)
and 13 susceptible cultivars.
d Loss Assessment of Silk Production
Development of Disease Management Component analysis of resistance
Itegies Against Mulberry (Morus alba showed that the resistant reaction conferred
i.) Red Rust Caused by Aecidlum morl by resistant and moderately resistant cultivars
clay. A.T. Gonzales and 0. S. Opina was attributed to its ability to limit or suppress
IMMSU, Sericulture Research and sporulation capacity, prolong latent period and
elopment Institute and UPLB, Department shorten the infectious period of the fungus.
lant Pathology)
Sanitation pruning, the cutting back of
study was conducted to determine the branches and total removal of remaining
ct of mulberry red rust (MRR) on the yield leaves significantly delayed the development
quality of mulberry leaves (cv. Batac), of MRR, but such delay was not translated
al growth and development of silkworms, into an increase in mulberry yield. The

Abstracts of papers

Mungbean (Vigna radiate (L) Wilczek), J.B.
Ferrater and F.M. dela Cueva (Plant
Pathology Laboratory, Institute of Plant
Breeding (IPB), College of Agriculture, UPLB,
College, Laguna)

The extent of variation among the different
isolates of Cecospora canescens, the causal
fungus of mungbean leafspot was evaluated
using cultural characterization, pathogenecity
tests and rep-PCR (repetitive sequence-
based polymerase chain reaction). A total of
32 isolates were collected from mungbean
growing regions of Luzon and Mindanao and
isolated using single spore isolation
technique. This study was conducted to
determine and characterize the extent of
variation and to delineate the phylogenetic
relationships among the isolates.

Seven groups were generated based
on color observed on mungbean seed
decoction agar. Colony diameter ranged from
3.82-9.75 mm, conidial lengths from 39.0 -
137.33 pm while conidial width ranged from
3.0 8.68pm. Duncan's Multiple Range Test
(DMRT) revealed 18, 19 and 15 groupings
based on colony diameter, conidial length and
conidial width respectively. Groupings were
not dependent on geographical origin.

All isolates were pathogenic to
mungbean susceptible variety, NCM 53.
Significant differences in incubation period,
lesion diameter, and lesion density were
observed. Virulence parameters were
negatively correlated with each other and do
not reflect the origin of the isolates. Cultural
and morphological characters were not
always directly correlated to virulence and
aggressiveness of the fungus.

Genetic diversity was assessed using
rep-PCR fingerprinting. Three sets of primers
(BOX, ERIC and REP) corresponding to
conserved repetitive elements were used to
generate genomic fingerprints.

Cluster analysis of composite re-PCR
data revealed 28 haplotypes among 29
isolates that were grouped into 7 clusters at
the 50% similarity level. Groupings of the
isolates tend to correlate with their geographic

Nei's diversity index of the composite
data obtained using rep-PCR primers was
high at 0.70 which implies high level of
genetic diversity This considerable variation
could lead to the emergence of new resistant
strains of Cecospora canescens.

Genetic Characterization of Near-lsogenic
Lines for Blast Resistance with Indica-
type Line, IR49830-7-1-2-2 Genetic
Background. L. A. Ebron, Y. Fukuta, T. Imbe,
H. Kato, H. Tsunematsu, M. J T. Yanoria, R.
Ohsawa and M. Yokoo (IRRI, Department of
Plant Pathology)

We have developed eight kinds of near-
isogenic lines (NILS) for blast resistance with
the genetic background of indica-type line
IR49830-7-1-2-2 by using recurrent backcross
breeding. Eight different kinds of single blast
resistance genes-Pik, Piz, Piz-5, Pita-2, Pi3(t),
Pi5(t), and Pi9(t) were introduced in each
NIL using selected Philippine isolates of the
blast fungus Pyricularia grisea Sacc. The
genotypes of the recurrent parent, IR49830-
7-1-2-2, and each NIL were confirmed by
differential system and allelism test. This was
carried out by inoculating F2 plant populations
derived from the crosses with monogenic lines
and NILs with CO 39 genetic background as
differentials with avirulent and virulent blast
isolates. Four genes Pia, Pib, Pik-s, and
Pita were found in IR49830-7-1-2-2, and the
target gene was introduced in each NIL.
These NILs were designated as IRBLk-Ku/RL,
and IRBL9-W/RL.

Biological Control of Soil-borne Pathogens
in Onion Using Trichoderma spp. D. K. M
Donayre, R. M. Gapasin and S. A. Miller
(PhilRice, Crop Protection Division;, LSU,
Department of Pest Management and
OARDC, OSU, Department of Plant

The efficacy of Trichoderma viride and
Trichoderma sp. (T5-onion isolate) against
soil-borne pathogens (Sclerotium cepivorum,
Fusarium oxysporum f.sp. cepae and Phoma
terrestris infecting onion cv. Rushmore was
determined in two field studies during the
2003 dry season, from January to April 2003

the PhilRice Central Experiment Station in
I Bongabon, Nueva Ecija. P
Incidence of S. cepivorum and F. s4
sporum f.sp. cepae was observed and ol
arded at 30 and 60 DAT. Higher incidence al
both diseases were higher in plots ol
:ulated with the organisms but no w
agonists added into the soil. Incidence was e
rally lower when the antagonists, T. ta
le and T5 isolate, and fungicide (Maneb) cl
e added to the soil inoculated with the fr
logens. It was generally lower at 60 DAT. v,
i at 30 DAT except in the treatment with
ixysporum f.sp. cepae and fungicide where
Jence was similar at both plant ages. P
:hoderma population in plots with the L;
agonists was slightly higher at 30 DAT pi
1 at 60 DAT. is
Yield was highest from the D
noculated plots but significantly differed F1
r from those inoculated with organisms te
ie, S. cepivorum + the two antagonists, mr
pathogens with Maneb. The effects of in
i antagonists and the fungicide on the in
j of onion were stronger on S. cepivowm o
i on F. oxysporum f.sp. cepae as shown pi
the higher yield obtained with the latter rn
anism. r
The two antagonists reduced root s4
action caused by P. terrestris. But results 1I
wed that the fungicide used had higher al
action in root infection than the gi
igonists. T viride population was higher at
DAT than at 30 DAT except with the
itment with T5 isolate alone. Disease tti
erity did not vary much among the di
tments with the pathogens and the bi
agonists as well as with the fungicide. si
d however, did not differ much among the th
tments. w
liminary Risk Assessment of Pesticide p1
Selected Water Pathways of Laguna G c!
Pera'a L M. Varca. C M. Bajet, L E. di
tro, M -. Navarro, and B. B. Quintana to
;PC, A.-PLB) o01
r sii identification, available information, to
ticic usage and other relevant data st
jirec for the risk, assessment were in

views and surveys and the use of map.
minary risk assessment using Pesticide
ict Ranking Index or PIRI follows, for the
cted cases with best available data. PIRI's
active is to determine which among an
( of pesticides has the greatest potential
contaminating the environment and the
!r pathways, and deserves a closer
Nation for ecolotoxicological studies. It
s into account the selected pesticides
nical properties, application rates and
jency, climatic seasons and soil

The project sites are in the towns of
sanjan, Lumban, Liliw, and Calauan,
nme. The land use area is basically
ted to agricultural crops. The topography
generally from level to gently sloping to
ilating. Amount of rainfall from January to
ember 2001 was around 1951.70 mm.
the same period, the minimum
merature is about 23.50C while the
imum is around 31.8 OC. Pesticide usage
ie selected sites comprised largely of
ticides mostly pyrethroids,
nophosphates and organochlorines. In
ly rice the frequency of spray application
ed from 1-2 per cropping season (4
ths). In vegetables the number of
cation ranged from 2-10 per cropping
on (4-5 months). And on pineapples, 15-
imes per cropping season that last for
it a year is the normal practice of most

PIRI's results on paddy rice showed
the selected pesticides applied during the
season cropping, have less impact on
ground and surface water, than the wet
.on planting. Mobility impact is greater
toxicity hazards furthermore, the surface
r is more affected than ground water. 2,4
*rbicide gave the highest relative pollution
ntial, followed by cypermethrin, lambda
lothrin and then deltamethrin in
ending order. On low land vegetables.
ity impact is greater than mobility impact
surface water. But on high land
,tables, mobility impact is greater than
ity and the most affected catchment is the
ice water. Fungicides top the mobility

mobility, and surface water is more affec
than ground water.

Utilization of Virus-Free Planting Mated
in Increasing the Productivity
Sweetpotato (Ipomoea batatas L.)
Central Luzon. L. M. Dolores and J.
Ferrater (IPB-CA, UPLB)

Sweetpotato is an important cash crop
Central Luzon one of the biggest supplier
sweet potato in the Philippines. In addition
its being traditional food use, the crop
emerging as an industrial crop for va
added foods (i.e. noodles), feeds and stai
products. Like most any other cro
sweetpotato is very susceptible to virus
The virus may occur in single or in mi>
infections in plants causing significant yi
losses and low quality of sweetpotato. Yi
losses of more than 50 % have report
been due to the use of virus infected plant
materials (Jayasinghe and Laranang, 1999).

By using pathogen-tested tiss
culture seed pieces Laranang and Nava
(2000) have shown a substantial increase
storage root yield and weight of vines as
result of the reduced and delayed incidence
camote virus complex.

Nonetheless, the potential bene
that can be derived from using sweetpot
clean planting materials (CPM) has not i
been fully established since it is still in its ee
stage. Moreover,CPMs are not exempt
from being infected with viruses having
normal reinfection rating under high disea
pressure Laranang and Navarro (2000)

Hard evidence that will showcase t
advantages and benefits of using Cle
Planting Material (CPM) Technology
sweetpotato root production has not been fi.
realized by majority of farmers, hence tl
study was conducted.

Partial Characterization and Molecui
Cloning of Sweetpotato Feathery Mot
Virus (SPFIIV). L. M. Dolores G. N. Yebr
and M. G. Colle (IPB-CA, UPLB)

v l v lg U. I It''-.a 113 II IVJLUC VIUo \ r |1
has been roentified as one of the m
important constraints in sweetpot
production. The virus has been noted in lal
sweetpotato ields in Central Luz
Spreading over most of the sweetpot
areas, it has led to substantial yield loss
and the loss of an important variety cal

Several SPFMV isolates have be
characterized based on differential reactic
to diagnostic hosts, Ipomoea setosa z
ipomoea nil. The virus was purified fn
mechanically inoculated i. nil using Cessii
chloride (Cscl) step gradient centrifugat
giving a faint opalescent band near the botti
of the centrifuge tube. The virus yield rang
from 9-30 mg/kg with A260nm/A280nm re
of around 1.2. The purified virus A
infectious and exhibited flexuous rod partic
typical of a poty virus under an elect

Further characterization of 1
SPFMV isolates by reverse transcript
polymerase chain reaction (RT PCR) of I
purified virus resulted to the amplification
the coat protein gene of SPFMV using 2 si
of primers designed to amplify the partial a
full length coat protein gene of SPFMV. T
expected PCR product sizes of 400bp and '
kb for partial and full length CP, respective
were obtained and successfully cloned usi
the TOPO TA cloning kit of INVITROGEN.

Such results on the characterizati
and cloning of SPFMV would be very useful
illuminating its position within the potyvir
group. Moreover, information on the molecu
aspects of the virus would help facilitate t
development of rapid and sensiti
techniques for virus detection a
identification that are important in monitor
virus infection in the field.

National Repository, Multiplication ai
Dissemination Centers (NRMDCs): A
Instrument to Enhance the Distribution
improved Pest and Disease Resista
Musa varieties in Asia and the Pacit
!.Van den Bergh, M.A.G Maghuyop, V.N. Ri
dnd A.B. Molina Jr. (INIBAP)

ult crops in tne world, but their production is
variously threatened by many pest and
disease problems, among which black and
allow sigatoka, fusarium wilt and nematodes.
he members of the Banana Asia and the
pacific Network (BAPNET) of the Intemational
network for the Improvement of Banana and
lantain (INIBAP have identified pests and
diseases as the main constraint to Musa
reduction in Asia and have appealed to
IIBAP to mobilize resources to address such

Different institutions began banana
breeding programs to overcome these
diseases, and a number of high yielding, pest-
nd disease-resistant varieties were
developed. Although the major Musa breeding
programs are located outside Asia, many of
le new hybrids being produced by these
programs maybe of interest for production in

The International Musa Testing
program (IMTP) was established in 1989 as a
orld-wide collaborative effort coordinated by
JIBAP to evaluate, in multi-locational trials
round the world, such elite Musa varieties
produced by breeding programs as well as
promising germplasm accessions from the
JIBAP collection, in order to obtain
formation on their resistance/tolerance to
lack and yellow sigatoka, fusarium wilt and
ematodes. The aim is to identify banana and
lantain hybrids resistant to these pests and
diseases, which would meet local
squirements and with which small-scale
irmers could replace existing susceptible
The availability of the improved
materials for wide distribution is limited by the
apability of the INIBAP Transit Centre (ITC)
respond to the many requests for materials
worldwide. To complement the IMTP, National
eposiory, Multiplication and Dissemination
enter (NRMDC) were identified in all
APNET countries to provide access to the
ew, improved hybrids and superior varieties
developed by breeding programs all over the
oorld, multiply them locally, and make them
available to Asia and Pacific countries for
national yield performance evaluation by
esearchers/farmers and eventual adoption by

le region Trom oursiae ana Detween
countries in the region, is carried out
according to the FAO/PGRI Guidelines for the
;afe Movement of Musa Germplasm.

Twenty-one accessions were already
aimed over to Bangladesh, China, India,
idonesia, Papua New Guinea, the
'hilippines, Sri Lanka, the South Pacific
commissionn and Vietnam. While a Letter of
Agreement (LOA) was signed with Cambodia,
lalaysia, Thailand and the Taiwan Banana
research Institute.

diseases of Sampaguita. V. R. Daquioag, M.
1. Navasero, M. V. Navasero, R. Boncodin
nd D. Campilan (Dept. of Plant Pathology,

,ix common diseases of sampaguita,
asminum sambac, are documented. These
re anthracnose caused by Colletotrichum
loeosporioedes; Fusarium rot caused by
:usarium sp.; Phytophthora rot caused by
'hytophthora sp. yellow ringspot mosaic
caused by a virus; sooty mold caused by
'apnodium sp. and algal spot caused by
'ephaleuros virescens. Disease symptoms as
iey occur in the field are described. Some
observations of growers are discussed.

cloningg of the DNA 3 of the Bunchy Top
lanavirus in Abaca. D. E. V. Villamor, L. C.
ialvez, P. M. Barcial and N. B. Bajet (Dept. of
'lant Pathology-CA, UPLB)

banana bunchy top nanavirus (BBTV) causes
ne of the most serious diseases of banana.
i very similar disease, called abaca bunchy
op, also affects abaca and the causal virus or
iruses have been assumed but not proven to
e similar, if not the same viruses. The BBTV
enome has six components with the open
leading frame (ORF) of DNA 3 coding for the
oat protein gene. Two outwardly extending
irimers for the DNA 3 of BBTV (Wanichakom
!t al.2000. Arch. Virol 145:593-602) were
ised in a polymerase chain reaction (PCR) to
simplify the putative DNA 3 of the bunchy top
lanavirus in abaca (AbaBTV). A PCR product
if about 1000 base-pairs (bp) was amplified
rom the total nucleic acid extracts of bunchy
op infected banana and abaca, but none from


banana and abaca tissues. This PCR produ(
was cloned into a topoisomerase activate
vector and three representative clones coming
from each of the BBTV-infected banana an
AbaBTV-infected abaca were selected<
Analysis of the selected clones by colon
PCR confirmed the presence of a 1000 b
insert. The result of this study provide
evidence on the presence of the virus genom
DNA 3 component and, in general, th
similarity of the two viruses infecting the tw
crops at the molecular level. This is also th
first report on the cloning of one particule
DNA component of the bunchy top virus i
abaca. Nucleotide sequence analysis of thes
clones is further pursued to determine th
degree of similarity between the two DNA

Reactions of Different Banana Cultivare t
Nematode Infection in Quezon Province. F
A. Zorilla, T. O. Dizon, D.C. Pantastico, J.
Orajay and F.S. De la Cruz (NCPC, IPB
Dep't. of Plant Pathology CA. UPLB)

Survey of population, prevalence an
distribution of parasitic nematodes associate
with different banana cultivars wer
conducted in nineteen (19) barangays (
seven (7) towns in Quezon Province. Roc
damage assessment expressed a
percentage dead roots, percentage roc
necrosis and nematode counts per 10 gram
roots from each sample were obtained. Fiv
nematode genera were found associated wit
banana cultivars. These are Radopholu
similis. which were found abundant in som
cultivars, followed by Pratylenchut
Helicotylenchus, Rotylenchulus reniformit
Meloidogyne incognita was also foun
prevalent in other cultivars. Root damage
assessment data showed that a range of 0 t
45% dead roots and percentage necrosis c
2 5-62.0 % were noted.

Banana RDE Collaborative Project ii
Luzon: Distribution and Evaluation c
Improved Musa and Popular Loca
Varietlts to Rehabilitate Local Banan
ndtustry. M. A. G. Maghuyop, E A. Anit,
Van drn B ergh, V. N. Roa, J. E Eusebio an
.A Molina Jr. (INIBAP-AP, and PCARRD)

programmes within INIBAP network hav
made significant accomplishments resulting t
a number of new, high yielding and disease
resistant varieties of banana and plantair
These varieties include both dessert an
cooking types that may have good potentii
for smallholder production in the Philippines
The project aims to make available th
improved varieties to small-scale farmer
through the collaborative projects wit
PCARRD and several state universities i
Luzon. These varieties were transferred fror
INIBAP international genebank to th
Philippines through an appropriate Materii
Transfer Agreement (MTA). A total of 32,19
tissue-cultured planting materials consisting c
five improved hybrids and two local
preferred cultivars were distributed to fiv
state universities and colleges in Luzon where
on-campus demonstration trials were set u
aside from on-farm verification trials i
farmers' fields in four agro-ecological zone!
The introduced disease-resistant varieties ar
being evaluated as alternative c
complimentary cultivars to rehabilitate th
local banana industry, which is beset with
number of disease problems. Appropriate
production system for optimum yield is als
being developed. Other activities include
training of project staff and farmer cooperator
and field visits.

Effect of Nitrogen on Bulb Rot Incidence 1
Onion During Storage. S. E. Santiago, D. 1
Eligio, R. T. Alberto and S. A. Miller (PhilRice
Crop Protection Division; CLSU, Departmer
of Pest Management; and OARDC, Ohi
State University)

The influence of varying levels of nitroge
application on the incidence of bulb rc
disease in onion under storage was evaluate
using 'Red Creole' and 'Yellow Grane)
varieties. The study was conducted i
farmer's field in Talavera, Nueva Ecija fror
December 2002 to March 2003 for the pre
harvest phase and in the cold storage i
Cabanatuan City (post-harvest phase) fror
April September 2003. Yield of 'Yelloi
Granex' with no N application (control) wa
significantly higher (22.2 tons/ha) than yield
from 50 N, 100 N and 200 N treatments
Highest yield in 'Red Creole' was recorded in


e treatment with 50 N (17.22 tons/ha). For
e post-harvest phase, onion bulbs were
aced in net bags with net capacity of 20
I/bag and stored at room temperature (270C)
id cold storage room (0C). 'Yellow Granex'
ared at 270C room temperature lasted for
ur weeks (100% rottina) while in 'Red

! weeks in storage. E
om was terminated on
jh percentage of sour
ivus. A. niler Burkhol

ulbs stored in cold
the 20t week with
d bulbs. Asperqillus

ganisms of bulb rot under storage. A. flavus
as the predominant bulb rot microorganism
'Red Creole' and A. niger in 'Yellow Granex'
room temperature while Fusarium sp. was
evalent in 'Yellow Granex' at OPC.

tegrated Management of Anthracnose
:olletotrIchum gloeosporioldes (Penzig)
inzig & Sac.), A Disease of Increasing
iportance in Onion. R. T. Alberto, M. V.
jca and S. A. Miller (CLSU, Department of
ast Management, PhilRice, Crop Protection

Division and OARDC, OSU, Department of
Plant Pathology)

A field study was conducted at PhilRice
Central Experiment Station in Maligaya,
Science City of Munoz, Nueva Ecija to
develop integrated management strategies
against anthracnose of onion. Different
combinations of disease management and
cultural practices showed that wider spacing
(18 x 20 cm) of onion seedlings with low
nitrogen (60 kg/ha) application and Mancozeb
application at 7 days interval significantly
reduced the incidence, severity and AUDPC
of anthracnose of onion. However, the yield
did not differ significantly with the other
treatments except with the treatment with no
Mancozeb, standard spacing and low

smbership In the PPS is prerequisite to publishing in the Journal of Tropical Plant Pathology, C
least one author must be a members of the Society. The editorial Board, however, may rels
s rule for contributions of exceptional merit. It may also invite distinguished scientists t
tribute articles of interest tot eh Society.

manuscript, must be reports of original research, excerpt of meritorious reviews, and should n(
Ive been published elsewhere. The decision of the Editorial Board to accept or rejei
manuscript is final.

ie manuscript should be typed on one side of 81/2x11 inch paper, double spaced throughout.

ipers other than Notes may be organized conveniently under: Title, Author(s), Authors; position
id address, Abstract, Key words, introduction, Materials and Methods, Results, Discussion ((
msults and Discussion) and Literature Cited, Acknowledgement.

the test, citations should be by name-and year system, e.g. Molina (1996), Ou and Nuqu
980), Ou et al. (1990) if more than two authors.

erature citation should be in alphabetical order. Do not cite unpublished work; it should appei
footnote. Biological Abstracts' 1968 List of Serials with Title Abbreviations must be consulted i
Ibreviating the names of journals. Examples of abbreviations: Phil. Phytopathol., J. Mol. Biol
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