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ANASTOMOSIS GROUPS OF RL/
J. J. TAGUBASE1 and
'Former undergraduate thesis student, Univel
lege, Laguna, presently, Science Research SI
ilRice), Mufioz, Nueva Ecija; and 2Professor, D(
The cross-infection potential an
anastomosis groups of Rhizoctonia s
relation to disease development in sel
by cultural methods.
Growth rates of mycelia, in terms
analysis, in all anastomosis groups v
showing the highest and least valuo
mycelial seeding. Only AG1-1A, AG1-1
sclerotial bodies. Sclerotial bodies wei
four days after mycelial seeding while t
ZOCTONIA SOLANI KHUN
L. D. RAYMUNDO2
ty of the Philippines Los Bafios (UPLB),
;ialist, Philippine Rice Research Institute
artment of Plant Pathology, UPLB, College,
cultural variation of the ten
ani Kuhn were determined in
ted crops and possible control
the slope in a linear regression
re high with AG3 and AG-BI
Respectively one week after
,AG3, AG7, and AG-BI formed
observed in the first four AGs
It in AG-BI was seen one month
ved between the formation of
aforementioned AGs except for
)athogenic to all test crops. All
2 Cultural variation and cross-infection in Rhizoctonla solani
Rhizoctonia solani Kuhn is a widely
distributed plant pathogen. It is a common soil
borne pathogen that has a variety of hosts,
forms sclerotia in the soil and persists for a
long period even without a host, either as thick
-walled brown hyphae or sclerotia in plant
debris (Hyakumachi et al., 1983). Together with
other Rhizoctonia species, R solani Kuhn is
widespread in the world and in both cultivated
and non-cultivated soils (Ogoshi, 1987). This
wide adaptability of the pathogen results from
its many strains that differ in host range,
virulence on a given host, type of attack,
temperature relation, survival in soil, response
to CO2, and other characteristics.
Basidiospores of Thanatephorus cucumeris
(Frank) Donk, the sexual stage ofthis pathogen,
are considered to have low genetic variability.
The complexity within this species accounts
for many failures in the past to develop resistant
varieties or crop rotation systems. Several
attempts have been made to group isolates of
this pathogen taxonomically. However,
variation in morphology, pathogenicity, and
physiology causes much confusion in the study
of taxonomy and nomenclature of R solani
At present, anastomosis of mycelia is the
criterion most widely accepted and used to
group isolates ofthis fungus (Anderson, 1982;
Suresh and Mall, 1982). Isolates of different
anastomosis groups are genetically distinct
since isolates belonging to different AGs do
not anastomose. Anastomosis groups are
somewhat similar morphologically but isolated
genetically. There are 12 anastomosis groups
(AG-1 to AG-11 and AG-BI) based on hyphal
anastomosis, cultural morphology, pathoge-
nicity and anastomosis behavior, cultural
appearance and DNA-based sequence
homology (Carling and Kuninaga, 1990; Carling
et al., 1994; Snehet al., 1991). AG-1 and AG-
2 are further divided into subgroups: AG-1 A,
AG-1 1B, AG-1 1C and AG-2-1 and AG-2-2.
There is host specificity in relation to
anastomosis groups as shown by many studies.
The distribution of AG is highly dependent on
the crop cultivated in a particular area.
Moreover, a specific AG can occupy one field
(Ogoshi and Ui, 1983). However, previous
studies contend that although AGs are isolated
genetically, isolates from one AG can also be
pathogenic to crops they are not known to
attack. Cross-pathogenicity of different AGs
gives us a better understanding of the impact
of the cropping pattern on the ecology of R
solani. If one known rotational crop is attacked
by different AGs then using this crop as part
of a rotation management scheme may render
the strategy ineffective.
In the Philippines, R. solani Kuhn is a
destructive pathogen of corn, rice, sorghum,
soybean, mungbean, and other important crops
(Pascual and Raymundo, 1988). Infected corn,
rice, and sorghum exhibit banded leaf and
sheath blight. Raymundo et al.(1994)
determined the cross infectivity of isolates of
R. solani from different host species (corn,
sorghum, wheat, cabbage, potato) that are
planted in rotation with rice or weed species in
rice fields (Rottboellia exaltata and Cyperus
rotundus), on rice cultivar, IR 72. The isolate
from cabbage showed a very low degree of
virulence on rice while no symptom of sheath
blight was caused by the potato isolate. It is
not clear as yet whether anastomosis groups
of Rhizoctonia solani from different crop
species are able to cross infect satisfactorily
as to cause epidemics. This information is
absolutely crucial in decision-making regarding
sheath blight management by crop sequence
strategies. This is important as cropping
systems are intensified to raise the level of
This study was undertaken: a) to
characterize ten AGs of R solani based on
cultural characteristics, b) to test the cross-
infection capability of these ten AGs on computed by simple linear regression using
different crops; and c) to relate the results to the equation:
the development of epidemics and to where: Y = diameter ofmycelia
management of sheath blight by cultural b = rate of increase of diameter of
methods mycelial growth
x = days after mycelial seeding
MATERIALS AND METHODS B. Cross-infection by Ten Anastomosis
Groups on Different Crop Species.
A. Cultural Variation Among 10
Anastomosis Groups of Rhizoctonia 1. Corn (Zea mays)
solani Mycelial blocks from each of the ten
anastomosis group were inoculated on
Pure cultures of isolates belonging to ten detached leaves of corn cultivar, UPCA Var.
different anastomosis groups ofR. solani from 2, in petri dishes, with filter paper immersed in
a collection at the Department of Plant solution with kinetin. The size of lesion
Pathology, University of the Philippines Los produced in these detached leaves was
Bafios, College, Laguna, were revived in potato measured daily for one week. Using the
dextrose agar (PDA). These anastomosis recorded lesion length, area-under-the-lesion-
groups ofR solani, from various host species, progression curve (AULPC) was computed
were used: 1-1A, 1-1B, 2-1, 3, 5, 6, 7, 8, 10, using the formula adapted from Campbell and
and BI. Madden (1990).
The experiment was laid out using AULPC = + (t _(+ t)
Randomized Complete Block Design (RCBD) i = 1
with three replications per anastomosis group.
Each replication consisted of four Petri dishes, where n = of assessment periods
A separate incubator was used for each Y = lesion length
replication of the set up. The cultures were t = time duration between
incubated for three weeks to allow for full 2 assessments
growth of mycelia and maximum sclerotial
production. They were observed for mycelial 2. Rice ( Oryza sativa)
characteristics such as colony color, One-month old seedlings of rice cultivar
--- _P n T 3 aru w-C _n;l__lr YD'l %xiiwk mwAlinl Mk/Gc rvf
i. Tagubase and A.D. Raymundo
rolume Jso ~ & ) .une-iuemoer cuuu
4 Cultural variation and cross-infection in Rhizoctonla solani
3. Mungbean (Vigna raatata)
Virulence test was carried out in
sterilized rice hull rice bran mixture and
sterilized course sand. About 5 grams of
inoculum from each isolate were incorporated
at the sterilized rice hull rice bran mixture
and incubated for three weeks. After this
period, the inoculated rice hull rice bran
mixture was placed in trays. Mungbean
variety NMC-53 was grown at 1.5 cm layer
of sterilized soil at 15 24 oC on the trays and
watered to field capacity every 4 days.
Percent seed germination was also noted,
based on emerged plants. The severity of
hypocotyl infection was rated using the
1 slightly diseased, lesion <3mm
2 moderately diseased, lesion 3-6mm
3- severely diseased or dead, lesion >6mm
The experiment followed a Complete
Randomized Design (CRD) with three
replications per anastomosis group and 100
mungbean seeds per replication.
4. Sorghum (Sorghum bicolor)
Sorghum seeds were planted on
the same infested soil used for the mungbean
seedling pathogenicity test. Plant emergence
and survival, based on number of living plants),
were recorded at seven and 14 days after
planting. At 14 days, plants were uprooted,
washed and rated for severity of stem infection
using the following scale:
1= no symptoms
2 = lesion (s), < 3mm
3 = lesion (s), 3 to 6mm
4 = lesion(s), > 6mm
5 = plant dead
The experiment was carried out in CRD
with three replications per anastomosis group
and 50 sorghum seeds per replication.
5. Cabbage (Brassier oleracea var.
Methods of planting, inoculation and
evaluation were the same as those followed
for the sorghum seedling pathogenicity test.
The experiment followed a CRD with three
replications and 50 seeds per replication.
Data obtained on the pathogenicity tests
except on corn, were subjected to analysis of
variance and separation of means by FLSD
(0.5) using the IRRISTAT software. Analysis
of disease severity for each crop was done
RESULTS AND DISCUSSION
I. Cultural Variation in Ten Anastomosis
Groups of R. solani.
Cultures of the ten anastomosis groups of
R. solani varied in terms of cultural
characteristics, namely color of growing
mycelium, discoloration of medium, growth
rate, color, size, and type of sclerotial
production (Tables 1 and 2). These variations
are in agreement with previous studies on the
anastomosis groups of R solani.
Pale to dark brown was the general color
of the rapidly growing mycelium in each AG.
Some colonies remained pale brown as in the
case of cultures of AG-1 1A, 2,3,8, and 10.
Cultures ofAG-6 and 7 eventually became dark
to very dark brown. Pale brown may appear
to be dull yellow as in the case of AG-5.
Parmeter et al. (1970) disclosed that any
mycelia remaining permanently white or
showing any pigmentation other than other
various shades and hues of brown are not likely
R solani. Shades of brown are the reliable
diagnostic color for mycelia and mature
sclerotia in all isolates when grown in pure
Tagubase and A.D. Raymundo Volume 36 (1 & 2) June-December 2000 5
Iture (Butler and Bracker, 1970). Sclerotia sorghum and mungbean seedlings than with
rmed by the AGs in this study were generally other AGs. AG-BI was the only sclerotia-
shade of brown and varied greatly in size, forming group that exhibited low pathogenicity
-1 1A isolal
rom corn produced p,
i-io nhrial oI l m t io ,al
'2)\ alcn t+ tp
tknro cc"' a nnstw
-Ut lVVI l, O l wa1 -J-ll L jlJ-- Fltl-lI .t A l /'-U' < a.. J LL.L. U l.cIL LIaI LL 1a s tJ y"iJLnv ,
with some brownish exudation few days after correlation between the size of sclerotia and
sclerotial initiation. Above results confirmed pathogenicity. Isolates with large sclerotia were
the findings of Hashiba and Mogi (1975) who significantly more virulent than those with
studied developmental changes in sclerotia of smaller sclerotia or without sclerotia.
the rice sheath blight fungus, R. solani AG-1. Shahjahan et al (1987), however, reported that
There were variations observed on the sizes no significant correlation exist between
and number of sclerotia formed even on the sclerotial production and virulence.
replicate cultures of each AG. This confirmed
with Fajardo'results (1984) on isolates of R AG-3 had the highest rate in mycelial
solani causing rice sheath blight. Not all AGs growth while AG-BI had the least rate. When
formed sclerotial bodies. Only AG-1 1A, AG- correlation was attempted between mycelial
1 1B, and AG 3 produced light to dark brown growth rates of each AG's with their
sclerotial bodies. GrishamandAnderson(1988) pathogenicity to tested crops, no clear
found out that AG-2 and AG-5 were sclerotia association between these two parameters
forming groups. emerged. AG-3 was only pathogenic to
mungbean, but nonpathogenic to corn and rice.
Naiki and Ui (1978) concluded that Likewise, no clear association was evident
nutritional conditions might influence inoculum between the numbers of sclerotial bodies
potential as sclerotia formed on soil are smaller produced among sclerotia-forming AGs with
than those produced in pure culture. Moreover, their pathogenicity on the different test crops.
Maier and Staffeldt (1960) reported an isolate AG-7 was highly pathogenic on sorghum,
that produced sclerotia on carrot agar but not mungbean and cabbage but non-pathogenic to
on PDA. Sclerotia were small and infrequent corn and rice. On the other hand, AG-BI was
on nutritionally poor media but generally large non-pathogenic on both rice and corn, highly
-4 -rm--n+ r4^1 -wrfl, fxrnIv 1 0 C n.tbnapnlnr tor rhlhllr rnrntl tea f+nr-alkn-m
6 Cultural variation and cross-infection in Rhizoctonia s
pathogenic, others weakly pathogenic, and the rotations with susceptible crops, suggest
rest intermediate on specific crops. Many incidence and severity of diseases cause
studies have shown host specificity in relation R solani will continue to increase (Win
to anastomosis groups (i.e. AG-1 in and Nabben, 1988; Jones and Belmar, 11
Leguminoseae and Gramineae, AG-2 on and Kim et al. 1992).
Brassiceae and Crucifereae and AG-3 on
Solanaceae). In contrast, Bolkan et al. (1985) The results of this study show that cr4
contends that hyphal AG in R. solani, which infection among AGs on crop specie
represents genetic isolation, is not host-specific, rotation with rice occurs. All of the ten,
although some tendencies are evident. The significantly reduced stands of sorgl
result of this study is in agreement with this seedlings and cabbage seedlings. AG-1
report. For example, AG-1 1A and AG-1 IB AG-1 1B, AG-3 and AG-7 significantly cai
had been characterized as pathogens reduction in post-emergence survival
belonging to Leguminaseae and Graminaea. mungbean. AG-1 1A, identified as the a
Moreover, AG-1 1A elicits sheath blight pathogen of sheath blight on rice (Belmh
of rice and leaf blight on corn. On the other al. 1987; Kim 1996; Kim et al 1988) was
hand, AG-3 prefers Solanaceae (Keiger et al to infect all the test crops. The results fin
1997; Bains and Bisht, 1995). However, the suggest that corn; sorghum, cabbage
results of this study show that AG-3 can also mungbean that showed high susceptibility
be highly pathogenic to mungbean and AG-1 1A cannot be utilized as rotation c
sorghum. These crops belong to Leguminaseae with rice. Each of these crop species is a
and Graminaeae, respectively. The to this particular AG. In rice-based and cA
observation that AG- related host specificity based cropping systems, crops that
as determined in terms of symptom develop- susceptible to AGs of R. solani other 1
ment, was relative rather than absolute, can. -AG-1 1A can be planted as rotational a
be explained by considering host plant since based on this study, these AGs
specificity and pathogenic capacity as two nonpathogenic to corn and rice. Such kinc
superimposed phenomena (Keiger etal., 1997). crop sequence strategies appear to be
most logical if the objective is to suppress
Crop sequence is one of the cultural growth ofR. solani, decrease inoculum den
management strategies adopted by most -.and consequently reduce disease severity
farmers to reduce crop losses due to plant
diseases. There have been reported studies
that demonstrate the occurrence of more than UIERATLRECfED
one AG on a particular rotational crop. Nelson
et al. (1997) reported that AG-4, AG-5, and ANDERSON NA. 1982. The genetics
AG-2-2 that are virulent on soybean were also pathology of Rhizoctonia solani. Ann.
highly virulent on rotational crops such as Phytopathol 20:329-347.
sugarbeet, dry bean, mustard and potato. BAINSPS, BISHT VS. 1995. Anastomosis
Moreover, Ruppel (1985) reported the identity and virulence of Rhizoctonia so
susceptibility of rotation crops such as small isolates collected fompotato plants in Alb
grains and corn to a root rot isolate ofR. solani Canada. PlantDis. 79:241-242
from sugarbeet. The expanding area
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mration and cross-infection in Rhizoctonia soi
J.J. Tagubase and A.D. Raymundo Volume 36 (1 & 2) June-December 2000 9
able 1. Cultural variation in ten anastomosis groups ofRhizo,
Anatomosis Days to No. of Color of Diameti
Group Sclerotial mature mature of
Production sclerotia' sclerotia scleroti
AGI-IA 4 DAMS4 20c Dark brown 1-2 mm
AGI-1B 5 DAMS 12d Hazel brown .5-3 mm
AG-3 4 DAMS 23b Brown 1.5-
AG-BI 30 DAMS 5e Light brown .5-3 mm
AG-7 5 DAMS >100a Light brown Pinhead
Meams of tee replication with four sample per replication. Means followed by
SDispesed means several to many scattered sclerolia
Aggregate means side by side to fused sclerotia
Days after myceial seeding
dia solani Kuhn on PDA medium.
Type of Site of Pigmentation of Discoloration
Production Production vegetative of
Dispersed2 Both middle Brownish Brownish
& edge white white
Aggregate3 Middle Light brown to Brown
Solitary & Underside Light brown Light brown
aggregating cover of the
Aggregate Middle Brownish No
Dispersed Center Brownish gray Dark brown
Pale yellowish Yellowish
Dark brown Dark brown
Light brown Light brown
Light brown Brown
Brownish Hazel brown
ame letter are not significantly different at LSD=2.303 (a=.0S)
10 Cultural variation and cross-infection in Rhizoctonia solani
ble 2. Myc lal variation of anastomosis groups ofRhizoctonia solani Kuhn on PDA medium
Diameter of mycelial growth(cm)'
Days after mycelial seeding
2 3 4 5 6
I t +
Average of three replications wiu tour samples each AG per replication, in 9 cm diameter petri dish.
U 9 cm, the whole plate was covered with mvcelia.
Rate of increase
of diameter of
Table 3. Pathogenicity of anastomosis groups ofRhizoctonia. solani on different crop species.
MUNGBEAN SORGHUM CABBAGE CORN RICE
Symptom Disease Symptom Disease Symptom Disease Symptom Disease Symptoms Disease
severity severity severity severity Severity
AGI-IA Damping off +++ Damping +++ Leaf rot +++ Leafblight +++ Sheath +++
AG-IB Damping off +++ Damping +++ Leaf rot +++ -
AG2 Surface + Damping ++ Leaf rot +++ -
AG3 Damping off +++ Damping ++ Leaf rot ++ -
AG5 Surface + Damping ++ Leaf rot +++ -
AG6 Surface + Damping ++ Leaf rot +++ -
AG7 Damping off +++ Damping +++ Leaf rot +++ -
AG8 Surface + Damping ++ Leaf rot +++ -
AGIO Surface + Damping ++ Leaf rot +++ -
AGB 1 Surface + Damping ++ Leaf rot +++ -
+++ highly pathogenic(51-100 % diseased)
+ moderately pathogenic(25-50 % diseased)
+ weakly pathogenic(1-25 % diseased)
- nonpathogenic, no symptoms
12 Cultural variation and cross-infection in Rhizoctonia solani
Table 4. Relative pathogenicity often anastomosis groups ofRhizoctonia solani on mungbean
% Post Points of injury listed in order
Anastomosis % emergence Injury of importance
group Emergence survival' Rating2_
AGI-IA 72.7 c 32. lbc 3 Hypocotyl, leaves
AGI-IB 87.3 ba 24.6bc 3 Leaves, hypocotyl
AG-2 93.3 a 92.6a 1 Scabbing on surface
AG-3 80.0 cb 16.4c 3 Hypocotyl
AG-5 96.0 a 100.Oa 0 no injury
AG-6 86.3 bd 100.Oa 1 scabbing on surface
AG-7 86.0 bd 36.3b 3 leaves, hypocotyl
AG-8 91.7 ad 100.Oa 0 no injury
AG-10 90.3 a 100.Oa 0 no injury
AG-B1 86.3 bd 100.0a 2 scabbing on surface
Control 99.0 a 100.Oa 0 no injury
LSD 9.4 17.3
'percent emergence and percent post emergence survival (mean number of plants per trays, 100 seeds planted) determined at 4 and
14 days following planting, respectively. Means followed by the same letter are not significantly different at (X = .05.
2 0 = healthy; 1 = slightly diseased, lesion(s) < 3 mm; 2 moderately diseased, lesion(s) 3 6 mm;
3 = severely diseased or dead, lesion(s) > 6 mm
Table 5. Relative pathogenicity often anastomosis groups ofRhizoctonia.solani Kuhn on sorghum seedlings.
% % Post-emergence
Anastomosis Emergence survival Disease severity
AG1-IA 76.0 b 38.4 c 5
AGI-IB 86.0 ab 38.7 c 5
AG-2 85.3 ab 57.3 be 4
AG-3 90.0 ab 59 be 2
AG-5 85.33ab 66.1 b 3
AG-6 85.3 ab 53.1 be 4
AG-7 77.3b 49.0 be 5
AG-8 78.0 b 57.7 be 3
AG-10 84.0ab 57.3 be 2
AG-B1 78.0b 73.8 b 4
Control 98.0 a 100.0 a 1
LSD 15.29 22.89
percent emergence and percent post emergence survival (mean number of plants per trays, 50 seeds planted) determined at 4 and 14
days following planting, respectively. Means followed by the same letters are not significantly different at (X = .05.
b Disease severity determined 14 days after planting and based on a 1 to 5 scale where 1 = no symptoms; 2 = lesion (s), < 3 mm; 3 =
lesionss, 3 to 6 mm; 4 = lesion(s), > 6 mm; 5 = plant dead.
J.J Tagubase and A.D. Raymundo
Volume 36 (1 & 2) June-December 2000 13
14 Cultural variation and cross-infection in Rhizoctonia solani
Table 6. Relative pathogenicity of ten anastomosis groups of Rhizoctonia solani on cabbage
/%Post emergence survival
AGI-IA 25.0 d 60.0c 4
AGI-IB 41.2c 68.3 bc 4
AG2 11.8 e 56.7c 5
AG3 51.5 b 55.0 c 4
AG5 37.7c 80.0ab 4
AG6 16.4de 76.7ab 5
AG7 43.6bc 76.7ab 4
AG8 16.7de 70.0bc 5
AGIO 10.4e 80.0ab 5
AGB1 18.9de 78.3ab 5
Control 100.0a 91.7a 1
LSD 8.68 15.32
SOemergence and %/opost-emergence survival (mean no. of plants per trays, 20 seeds planted) determined at 4 and 14 days
following planting, respectively. Means followed by the same letters are not significantly different at (X = .05.
b Disease severity determined at 14 days after planting and based on a 1 to 5 scale where 1 = no symptoms 2 = lesion(s) < 3mm.,
3 = lesion(s), 3 to 6 mm.; 4 = lesion(s) 76 mm. 5 = plant dead.
HOST SUITABILITY OF COMMON FIELD CROP AND WEED SPECIES TO
THE RICE ROOT-KNOT NEMATODE, MELOIDOGYNE GRAMINICOLA
E. B. GERGON1
Supported by the Integrated Pest Management Collaborative Research Support Program
(IPM CRSP) with funds from the USAID Global Bureau Grant No. LAG-G-00-93-00530-00.
'Senior Science Research Specialist, Philippine Rice Research Institute, Mufioz,
Host suitability of 39 crop species, which include 153 cultivars and
85 weed species, to Meloidogyne graminicola were tested in greenhouse
trials. Based on nematode populations in the roots and gall index rating,
Glycine max, Pisum sativum, Bejuncea czernjaew, Psophocarpus
tetragonolobus, Allium cepa, B. rapa ssp. chinensis, B. rapa ssp.
pekinensis, 5 cultivars of Lattuca sativa, 2 cultivars of Brassica oleracea,
Raphanus sativus '60 days', 1 cultivar of P vulgaris, 4 cultivars of
Lycoperv'con lycopersicum, and Triticum sativum were the good hosts of
the pathogen. All cultivars of families Cucurbitaceae, Solanaceae except
tomato, and Leguminoceae except those mentioned above were poor
hosts or nonhosts of M. graminicola and are therefore potential rotation
crops in rice-vegetable farming systems in fields infested by this
nematode. Some cultivars belonging to same species showed varying
degrees of resistance to nematode infection.
Among the weeds, all members of Cyperaceae, Ageratum conyzoides,
Eclipta prostrata, Commelina benghalensis, C. diffusa, Merremia gemella,
Mollugo pentaphylla, Ludwigia adscendens, L perennis, Aeschynomene
indica, Sesbania sesban, Macroptilium lathyroides, Cynodon dactylon,
Echinochloa colona, E. crus-gali, E. glabrescens, Ischaemum rugosum,
Leersia hexandra, Leptochloa chinensis, Paspalidium flavidum, Lindernia
hirta, Eichhornia crassipes, and Oryza sativa (Red Rice) were good hosts
of M. graminicola and should be managed properly in rice-vegetable
Kev words: Meloidogyne graminicola, root-knot,
INTRODUCTION Prot et al., 1994). The nematode has been
found in many onion farms in San Jose City
The rice root-knot nematode, Meloidogyne and Bongabon, Nueva Ecija and has been
graminicola Golden and Birchfield, 1965, has shown to reduce the quality of onions for export
been identified as a potentially serious pathogen (Gergon et al., 2001).
carried over from rice to onion (IPM CRSP,
1995). It is an important pest of rice in Asia The nematode, being soil-borne, is difficult
(Bridge et al., 1990) causing as much as 20% to control especially in areas devoted to a
to 75% yield reductions (Tandinganetal., 1996; continuous rice-onion cropping pattern.
16 Host sustainability of common field crops and weeds
Intensive cropping schemes using host crops
provide the pathogen a constant food supply
for its growth and reproduction. Aside from
rice and onion, M graminicola can survive in
several vegetables and weeds (Birchfield,
1965; Bridge et al., 1990; Gapasin and
Barsalote, 1977; Yik and Birchfield, 1979) in
the absence of the main crops.
Control of soil-borne nematodes has been
focused on cultural management practices,
which aim to reduce the build-up ofnematode
population levels in the soil. One of these
practices is crop rotation using unsuitable or
nonhost crops. The golden nematode,
Globodera rostochiensis (Roth and Thorne,
1984) and root-knotnematode (Rao et al., 1970)
infestations in the fields have been effectively
controlled through this method. In line with
this concept of disease control, this study was
conducted to: (1) determine the host status and
susceptibility to nematode damage of different
field crops and identify candidate rotation crops
for rice-based fanning system and (2) test the
infectivity of M graminicola in weed species
associated with rice-vegetable cropping
MATERIALS AND METHODS
Evaluation of host status of different
field crop and weed species was conducted
separately under greenhouse conditions.
Seeds of 39 crop species including 153 cultivars
were directly sown in 3.5 kg sterile sandy
loam soil contained in 20-cm-diameter clay
pots. One week after sowing, seedlings were
thinned to one plant per pot and inoculated
with 1,000 M. graminicola second-stage
Eighty-five weed species associated with
different rice ecosystems were collected
from different fields in Los Bafios, Laguna and
San Jose City, Nueva Ecija. Weed seedlings
were transplanted in 15-cm diameter clay pots
containing 1-kg sterile sandy loam soil and
inoculated with 500 J2s five days after
transplanting. The weeds were identified upon
appearance of inflorescence with the aid of
different references (Moody et al., 1984;
Pancho and Obien, 1995).
The inocula used m both experiments were
collected from a greenhouse culture of rice
cultivar UPL Ri-5 extracted after 24-hr
incubation period in a misting chamber. J2s
were introduced into the soil over the exposed
portion of the roots. UPL Ri-5 was sown
among test entries to serve as susceptible
check. Each species was replicated five
times with one pot per replication. The potted
plants were arranged in a Randomized
Complete Block Design and watered daily
until saturation point. The plants were fertilized
with ammonium sulfate at 80 kg Nha-'
Trellises were provided for creeping plants.
The experiment on weeds was terminated
45 days after inoculation while the different
crop species were harvested 60 days after
inoculation. During harvesting, the plants were
carefully uprooted and tops were cut off. The
roots were gently washed under running water
to remove the soil and rated for galling on a 1
to 5 scale (0 to 100% infection), as follows: 1
for no root galls, 2 for trace galling (1-10%), 3
for slight galling (11 to 30%), 4 for moderate
galling (31-50%), and 5 for severe galling (51-
100%). The roots' were then cut into 5-mm
long pieces and mixed. From this mixture, a
3-g subsample was taken and placed in a mist
chamber for 5 days to extract the nematodes.
J2s of M graminicola were collected and
counted under a steromicroscope.
Based on nematode density in the roots
and gall index rating, the host status of different
crop and weed species was defined. Crop and
weed species having numbers not significantly
different from UPL Ri-5 or whose estimated
E.B. Gergon Volume 36 (1 & 2) June-December 2000 17
nematode population inthe roots at harvest was
>90% ofthe initial inoculum and/or having gall
index greater than 3 up to 5 were considered
good hosts. Entries with estimated population
counts of >10% but <90% of initial inoculum
and/or gall indices between >2 and 3 were
considered moderate hosts. Plants having 21
J2 but 10% of initial inoculum and/or gall index
rating between >1 and <2 were considered
poor hosts. Plants that did not allow any
nematode in their roots and did not produce
any gall were considered nonhost. Means were
separated using Fisher's LSD.
Host Reaction of Different Crop
Species to M. graminicola
M. graminicola increased more than 10
times not only in the roots of UPL Ri-5 but
also in the roots of Psophocarpus
tetragonolobus 'Batangas Medium' (Fig. 1),
B. rapa L. ssp. chinensis 'B. Behi', Glycine
max, Pisum sativum 'Chinese Dark Green',
and six cultivars of Triticum sativum. All these
crop species and cultivars supported high J2s
in the roots and produced heavy root galling.
Their galled tissues contained mature females,
egg masses, males and hatched second-stage
Members of the families Cruciferaceae,
Graminaceae, Leguminoceae, and Solanaceae
displayed a wide range of reaction to M.
graminicola (Table 1). Cultivars ofBejuncea
(L.) czernjaw, B. rapa L. ssp. pekinensis,
and B. rapa L. ssp. chinensis of Cruciferaceae
were good hosts ofM. graminicola with high
population densities in their roots and gall
indices greater than 3. Raphanus sativus '60
days' and Apium graverolens 'Tall Utah' of
the same family were poor hosts, with gall
ratings of less than 2 and with less than 10%
of the initial J2s in their roots. Rice and all
cultivars of wheat of Graminaceae also
supported substantial increases in the nematode
Corn cultivars were either poor host or
nonhost of the pathogen. Onions of Alliaceae
were also preferred host of M. graminicola
Differences among cultivars were also
evident. R sativus '60 days' was a poor host
but cultivar 'Rond Ecarlate Race Loto' was a
good host. Phaseolus vulgaris 'White' was
a poor host and two of its cultivars; 'BSU#1'
and 'Bush Blue Lake' were both good hosts
of M graminicola. Similarly, cabbage
'Copenhagen Market + D64' was a good host
while 'Cabuko' and 'Irodori' were poor hosts.
All member of Cucurbitaceae, consisting
of 5 crop species and 29 cultivars, and
Solanaceae with 9 species and 26 cultivars,
were all poor hosts or nonhosts of
M. graminicola. Among the solanaceous
crops, only tomato showed variable reaction.
Three cultivars of tomato supported increased
nematode population and had gall indices
greater than 3 while the rest were moderate
Reaction of Different Weed Species
to M. graminicola.
Weed species differed in their host sta.,
toM. graminicola (Table 2). Out of 85 weed
species evaluated, 27 were good hosts, 10 were
moderate, and 48 were either poor or nonhosts
of the nematode. Among the good hosts of
M. graminicola were Echinochloa colona
and Commelina diffusa (Fig. 2), which
exhibited heavy galling. Large terminal and
root axil galls developed in C. diffusa while
abundant but smaller galls were produced in
E. colona. The other weed species that
supported nematode reproduction in their roots
were all members of Cyperaceae, Ageratum
Volume 36 (1 & 2) June-December 2000 17
18 Host sustainability of common field crops and weeds
conyzoides, Eclipta prostrata, C.
benghalensis, Merremia gemella, Mollugo
pentaphylla, Ludwigia adscendens, L.
perennis, Aeschynomene indica, Sesbania
sesban, Macroptilium lathyroides, Cynodon
dactylon, E. glabrescens, E. crus-gali.
Ischaemum rugosum, Leersia hexandra,
Leptochloa chinensis, Paspalidium
flavidum, Eichhornia crassipes, Lindernia
hirta, and Oryza sativa (Red Rice) (Table 2).
With reference to susceptible check of the
same species, Red Rice, a common
contaminant in rice seed lots, was equally
supportive of M. graminicola increase. E.
crassipes supported significantly greater
number of J2s in the roots than rice UPLRi-5.
Most of the broadleaf weeds of the
families Capparaceae, Commelinaceae,
Convolvulaceae, Euphorbiaceae, Fabaceae,
Oxalidaceae. Portulacaceae, Sphenocleaceae,
and Verbenaceae were poor or nonhosts while
the majority of the sedges and grasses were
moderate or good hosts of the nematode.
Variable reaction within families also existed
in weeds. Echinochloa ,colona and E.
glabrescens were both good hosts to M
graminicola while Digitaria ciliaris and D.
setigera of the same family were poor hosts.
Among 20 members of Poaceae
(=Gramineae), 8 species supported
reproduction of M. graminicola.
Methods of determining host reaction to
root-knot nematode infections differ (Ferris et
al., 1994,Yikand Birchfield, 1979). Roy(1977)
used good, moderate, poor, andnon-hosts based
on percent galling, type and size of galls, and
reproduction of the nematode. Growth
parameters have also been considered in
disease assessment although interpretation of
results may not be accurate (Bhat and
Subbarao 1999). Our greenhouse trials
produced four groupings of host reaction to
M. graminicola which were similar to that of
Roy (1977), although we did not consider the
type and size of galls. Good hosts of M
graminicola allow the nematodes to penetrate,
feed, reproduce, and complete their life cycles
(Rohde, 1965; Sasser and Kirby, 1979).
Consequently, greater amount of galls and
higher number of J2s are extracted from their
Reports on host status of some crops to
M. graminicola may also differ. For instance,
the report of Roy (1977) that corn is a moderate
and good host of M. graminicola disagrees
with our findings and the reports of Birchfield
(1965). Similarly, Roy (1977) listed S.
melongena as a good host while we classified
this crop species as a poor or nonhost of M.
graminicola. Differential reactions could be
attributed to variation in cultivars or in the
nematode population (Roy, 1977). Southards
and Priest (1973) also reported that good host
plants of Meloidogyne species in one part of
the world are not necessarily good hosts to all
populations of that species.
The degree of root-knot infection varied
among susceptible crops. Some crop species
supported higher nemnatode densities than
others, which is indicated by a greater amount
of J2s extracted from their roots. Onions, for
instance, are susceptible to the nematode but
population densities in their roots were lower
than in rice, mustard, or wheat. M.
graminicola reproduced on onions, as shown
by eggs, galls, and mature females with
embryonated eggs in their root systems, and
caused reduction in bulb weight and diameter
(Gergon et al., 2001). However, onions have
few and delicate roots which probably
accounted for the lesser number of nematodes
that could be extracted from them. This
variation could also lead to a different
classification of host reaction of a certain crop
to a pathogen.
Differences in host reaction to M.
graminicola were evident not only among crop
species but also among cultivars of the same
species. Differences in susceptibility among
cultivars ofPanicum maximum have also been
detected in South African populations of M
incognita (Bridge et al., 1990). This finding
indicates that a careful selection of crop
species should be made before a reliable
recommendation is given to farmers especially
if the cultivar is intended for planting in rice
root-knot-infested fields. The host status of a
certain crop being considered for rotation
should be screened against the pathogen before
inclusion in a crop rotation scheme.
Potential rotation crops in rice-vegetable
cropping systems were identified in this
experiment for the management of M.
graminicola. Cucumis sativus, Vigna
sinensis, and Capsicum frutescens have also
been reported resistant to M graminicola
(Golden and Birchfield, 1968; Roy 1977).
When planted after rice or onion, these crops
can reduce the nematode level in the soil, either
by inhibiting nematode development and
reproduction in their roots, by producing
substances toxic to the nematodes. by
stimulating a hypersensitive reaction, or by lack
of attraction (Giebel, 1974). Pepper, peanut,
mungbean, and cucumber are already used in
rotation in many rice-onion farms in San Jose
City and Bongabon, Nueva Ecija but they are
usually planted after onion. The interval
between rice and onion cropping is shorter
than the duration of these crops and so onions
are not usually benefited by the effect of crop
Many predominant weeds in rice-
vegetable system (IPM CRSP, 1995) are good
alternate hosts of M graminicola including
Echinochloa colona, Digitaria setigera,
Ischaemum rugosum, and Cyperus iria.
Cyperus rotundus, F miliacea, and E.
Volume 36 (1 & 2) June-December 2000 19
colonum have also been reported as
susceptible (Jepson, 1987; Yik and Birchfield,
1979). Most weeds in Poaceae (=Gramineae)
were good hosts of this nematode, which was
similar to the findings of Rao et al., (1970).
As its species name implies, M graminicola
infects primarily plants belonging to the
Gramineae, which also includes rice and wheat.
A. indica and S. sesban, which are used as
green manure crops also, support M.
graminicola. Some of these green manure
crops have caused the spread of the pathogen
in many rice fields (Prot, 1994; Gergon and
Prot, 1995). This shows that the presence of
susceptible weeds plays a big role on the
persistence ofM. graminicola in the field. If
these weeds are present in the area, the effect
of crop rotation or fallow period may be
compromised. High initial nematode population
levels at the start of the cropping caused by
presence of weeds will definitely affect the
growth and yield of the succeeding crop
especially if a susceptible cultivar or variety is
Several crop species serve as good hosts
M. graminicola. There are others, however,
where the nematode can not increase in
number. These are considered poor hosts and
can be used in rotation with susceptible crops
in rice-vegetable cropping systems even in
nematode-infested fields. They can reduce
the initial population levels ofM graminicola
in the soil allowing the next crop to establish
itself and avoid the severe damage that may
be brought about by early infection of this
nematode. However, recommendations of
specific crop cultivars requires further
screening against local populations of M
graminicola since differences in response to
the nematode exist among cultivars. Good
weed management is also very important since
20 Host sustainability of common field crops and weed:
the pathogen can multiply in susceptible weeds
that maybe present in the field.
BHAT RG, SUBBARAO KV. 1999. Host range
specificity in Verticillium dahliae.
BIRCHFIELD W. 1965. Host parasite relations
and host range studies of a new Meloidogyne
species in Southern USA. Phytopathology
BRIDGE J, LUC M, PLOWRIGHT R. 1990.
Nematode parasites office, pp. 69-108. InLuc
M., SikoraR A., Bridge J. (Eds.). Plant parasitic
nematodes in subtropical and tropical
agriculture. Wallingford, U.K: Commonwealth
Agricultural Bureau (CAB) International
Institute of Parasitology
FERRIS H, CARLSON HL, VIGLIERCHIO DR,
CE,JURMAA,KIRBYDW. 1993. Hoststatus
of selected crops to Meloiodgyne chitwoodi.
J. Nematol (SuppL) 25:849-857.
GERGON EB, PROTJC. 1995. Effects of water
regime on the reproduction of rice root knot
nematode, Meloidogyne graminicola, in
Sesbania spp. andAeschynomene spp. Phillip.
J. Crop Sci 20:56.
GERGON EB, MILLER SA, DAVIDE RG. 2001.
Occurrence and pathogenecity of rice root-
knot nematode (Meloidogyne graminicola)
and varietal reaction of onion (Alllium cepa).
Phillip. Agric. Sci. 84:43-50.
GIEBELJ. 1974. Biochemical mechanismsofplant
resistance to nematode: A review. J. Nematol.
GOLDEN AM, BIRCHFIELD W. 1968. Rice root-
knot nematode (Meloidogyne graminicola) as
a new pest of rice. Plant Dis. Reptr. 52:423.
IPM CRSP. 1995. Annual Report of the integrated
pest management collaborative research
supportprogram. Virginia Polytechnic Institute
and State University. U.S.A 398pp.
JEPSON SB. 1987. Identification of root-knol
nematodes (Meloidogyne species). Farnham
Royal, England Wallingford, U.K.: Common-
wealth Agricultural Bureau (CAB),
International Institute of Parasitolog. 265pp.
JR. EC. 1984. Major Weeds ofthe Philippines.
University of the Philippines Los Bafios,
College, Laguna, Philippines. 328pp.
PANCHO JV, OBIEN SR. 1995. Manual of
Ricefield Weeds in the Philippines. Philippine
Rice Research Institute, Mufioz, NuevaEcija.
PROTJC. 1994. The combination ofnematodes,
Sesbania rostrata, and rice: the two sides of
the coin. Int Rice Res. Newsl. 19:30-31.
PROTJC,VILANUEVAM, GERGONEB. 1994.
The potential of increased nitrogen supply to
mitigate growth andyield reduction of upland
rice cultivar UPL Ri-5 caused byMeloidogyne
graminicola. Fund. Appl. Nematol. 17:445-
RAO YS, ISRAELP, BISWAS H. 1970. Weedand
rotation crop plants as hosts for rice root-knot
nematode, Meloidogyne graminicola (Golden
and Birchfield). Oryza7: 137-142.
ROHDE RA. 1965. The nature of resistance in
plants to nematodes. Phytopathology 55:
ROTH R, VON TORNE E. 1984. Development of
potato cyst nematode populations, Globodera
rostochiensis (Wollenweber, 1923) Behrens,
1975 in plots of a long term crop rotation
experiment on Lovisol (Tieflehmfahlerde).
Arch. Phytopathol Pflanzenschulz 20:127-133.
ROY A. 1977. Host suitability of some crops to
Meloidogyne graminicola. Indian
e 3o (i a z) June-UecemDer zuuu Zi
SASSERJN, KIRBY ME 1979. Crop cultivars
resistant to root-knot nematodes,
Meloidogyne species with information on seed
sources. International Meloidogyne project.
Raleigh, North Carolina.
SOUTHHARDS CJ,PRIEST ME 1973. Variation
in pathogenicity of seventeen isolates of
Meloidogyne incognita. J. Nematol. 5: 63-67.
TANDINGANIC,JCPROT, RG. DAVIDE. 1996.
Influence of water management on tolerance
of rice cultivars for Meloidogyne graminicola.
Fund. Appl. NematoL 19: 189-192.
YIKCP, BIRCHFIELDW. 1979. Hoststudies and
reaction of rice cultivars to Meloidogyne
graminicola. Phytopathology 69:497-499.
22 nosi susiainaDiily or common nela crops anu weeus
Table 1. Reaction ofdifferentfield crop species
Family/Botanical Name Ct
Allium cepa L.(Onion) Red Creole
Allium sativum L (Garlic) Batangas
Apium graverolens L. (Celery) Tall Utah
Bejuncea (L.) czernjaew BPI
B. rapa L. ssp. chinensis (L.) B. Behi
Handt (Pechay) Green Boy
B. rapa L. ssp. pekinensis (Lour.) Corazon
Handt (Chinese cabbage) Early Sprin
Brassica oleracea var. Africa Cros
capitata L. (Cabbage) Cabuko
Market + D
Raphanus sativus L. (Radish) 60 days
Lactuca sativa L. (Lettuce) Batavia de
J2s Gall Index Host
/3-gm root Status4
176 n-n 3.2 g-j G
x 2,006 k-n 5.0 a G
242 m-n 2.8 -1 M
239 m-n 2.8 '-1 M
526 m-n 2.8 '-' M
17 m-n 1.8 n-q P
38 m-n 2.2 1-o M
5,411 f-n 5.0 a G
9,660 -k 5.0 a G
2,385 k-n 5.0 a G
10,400 -i 4.6 G
489 k- 3.4 f- G
7,482 f-n 5.0 a G
988 m- 3.8 d- G
7,720 f-" 5.0 a G
3,073 i-n 5.0 a G
3,627 h-n 5.0 a G
849 -n 4.2 G
489 m-n 3.0 h-k M
46 m-n 1.6 o-r
359 m-n 3.4 f-i G
280 m-n 2.8 i-I M
152 m-n 2.6 j-m M
64 "n 1.8 n-q P
44 m-n 2.0 "m- M
1,969 k-n 3.4 f-i G
10 "m- 1.4 -r P
2,508 "- 3.2 g" G
38 m-" 1.8 P
1,125 m- 4.2 b G
958 m-n 4.6 a G
6,001 f-n 5.0 a G
838 4.4 a-d G
99 m-n 2.2 '1 M
4,960 g-n 5.0 G
ie 36 (1 & 2) June-December 2000 23
Table 1. cont...
Cucumis sativus L. (Cucumber) Batangas W
: 0 n 1.2 qr P
Acc. 51001 25,891 b-c 5.0
r(If'7-. 19 R1I e-f C n
24 Host sustainability of common field crops and weeds
Table 1. cont...
Zea mays L. (Corn)
Arachis hypogaea L (Peanut)
Cajanus cajan (Pigeon pea)
Glycine max Mer. (Soybeans)
Phaseolus lunatus L. (Lima
P. mungo L. (Mungbean)
P. sesquipedalis L.
(Dry beans/ Pole sitao)
P. vulgaris L. (Snap beans)
Pisum sativum L. (Garden Pea)
Vigna unguiculata Endl.
Yellow CN 2
IPB Pn 82 70
IPB Pn 82 71
IPB Pn 82 82
IPB Pn 83 117
UPL Pn 2
#1 (Dark green)
Bush Blue Lake
CES 18 6
CES 26 1
16,600 d- 5.0
E.B. Gergon Volume 36 (1 & 2) June-December 2000 25
Table 1. cont..
V unquiculata ssp. unquiculata x
V. unguiculata ssp. sesquipedalis
(Bush sitao/Bush bean)
Abelmochus esculentus L. (Okra)
Capsicum frutescens L.
Citrullus vulgaris Schrod
Corchorus capsularia L.
Cucumis melo L. (Melon)
Daucus carota L. (Carrot)
Dolichos lablab L. (Batao)
Ipomoea aquatica Foesk
#6 (Dark green)
6 "-" 1.0
1 n 1.0
26 n"" 1.2
85 m-n 2.8
470 m-n 1.2
6 r-n 1.0
Volume 36 (1 & 2) June-December 2000 25
26 Host sustainability of common field crops and weeds
Table 1. cont..
Lycopersicon lycopersicum Mill. Apollo White 1,579 -" 4.6 G
(Tomato) Big 1,222 3.6 f" G
Bonanza 25 "" 1.8 -"' P
Improved Pope 386 1.4 P' M
KS Apollo 355 1.6 M
Maligaya 100 "n- 1.4 p-r M
Marikit 947 "mn 4.2 -e G
Obtention Inra 141 m-n 2.4 k-n M
Rocky 1,263 4.0 b G
Tabeth 103 64 -n 2.8 i-1 M
Solanum melongena L. Casino 901 0 n 1.0 r N
(Eggplant) Dumaguete 2 1.0 r P
Jackpot 0 n 1.0 r N
Long Eggplant 1 1.0 r p
Long Violet 1 1.0 r P
Thai Type 0 1.0 r N
LSD (0.05) 1,511.6 0.75
'Data are means of 5 replications. Means followed by a common letter are not significantly different by LSD test at
2 ? = unidentified entries.
3Gall Index rating on a 1-5 scale (0 to 100% infection): 1 for no root galls, 2 for trace galling (1-10%), 3 for slight
galling (11 to 30%), 4 for moderate galling (31-50%), and 5 for severe galling (51-100%). ailleproductive index
was based on Pf/Pi where Pf= final population ofJ2s extracted per plant; Pi = initial population (1000).
4G=Good host (J2s 290% of the initial inoculum (Pi) and/or gall index (GI) >3); M= Moderate host (J2s >10%
<90% Pi and/or GI >2 <3); P = Poor host (J2s>l but <10% Pi and/or GI 1l <2); N = nonhost (0 J2s, GI <1).
E.B. Gergon Volume 36 (1 & 2) June-December 2000 27
Table 2. Reaction of different weed species to Meloidogyne graminicola.
Weed species J2/3-g root1 Gall Index2 Host
Hygrophila phlomoides (Wall.) Ness 7 1.4 e" P
Trianthemaportulacastrum L. 56 J1.4 e-g M
Achyranthes aspera L. 32 1.8 d-f P
Alternanthera sessilis (L.) DC. 50 J 1.8 d-f M
Amaranthus spinosus L. 8 J 1.4 -f P
Celosia argentea L. 15 J 1.4 P
Gomphrena celosioides Mart. 12 1.4 P
Pistia stratiotes L. 0 1.0 g N
Ageratum conyzoides L. 621 Ij 5.0 a G
Bidenspilosa L. 9 J 1.4 g P
Blumea lacera (Burm. f.) DC. 5 1.8 d-f P
Chromolaena odorata (L.) R. M. King & 1 1.0 g P
Ecliptaprostrata (L.) L. 468 '- 1.8 df G
Sonchus oleraceus (L.) 1 i 1.0 g P
Synedrella nodiflora (L.) Gaertn 9 1.8 d-f P
Vernonia cinerea (L.) Less. 67 2.0 cd M
Cleome rutidosperma DC. 11 1.0 g P
Commelina benghalensis L. 524 J 5.0 a G
Commelina diffusa Burn. f. 658 J 5.0 G
Murdannia nudiflora (L.) Brenan 151 2.0 "d M
Ipomoea aquatica Forsk. 5 1.4 P
Ipomoea triloba L. 11 1.4 eg P
Merremia gemella (Burm. f.) Hallier f. 684 '' 2.6 c G
Cyperus difformis L. 1658 J 5.0 a G
Cyperus iria L. 1039 h-j 5.0 a G
Cyperus rotundus L. 1174 g" 5.0 a G
Fimbristylis miliacea (L.) Vahl 1226 g 5.0 a G
Scirpus maritimus L. 3495 e- 5.0 a G
Volume 36 (1 & 2) June-December 2000 27
28 Host sustainability of common field crops and weeds
Table 2. cont....
Euphorbia heterophylla L. 6 J 1.8 df p
Euphorbia hirta L. 6 J 1.2 P P
Euphorbia hypericifolia L. 12 J 2.0 Pe p
Phyllanthus amarus Schum. & Thonn. 4 1.2 fg P
Phyllanthus debilis Klein ex Willd. 18 1.4 e-g p
Oryza sativa L. Red Rice' 18900 b 5.0 a G
Oryza sativa L. "UPLRi-5' (Check) 19267 b 5.0 a G
Hyptis rhomboidea Mart. & Gal. 3 1.0 g P
Arachis hypogea L. (wild peanut) 96 1.8 d-f M
Ammannia baccifera L. 45 2.0 c P
Sida rhombifolia L. 2 1.8 d-
Mimosa invisa Mart 5 1.2 g P
Mimosa pudica L. 4 J 1.2 P P
Mollugo pentaphylla L. 2,647 f-h 2.4 d G
Boerhavia erecta L. 63 2.0 C" M
Ludwigia adscendens (L.) Hara 4,735 d- 5.0 a G
Ludwigia octovalvis (Jacq.) Raven 6 j 1.2 9 P
Ludwigiaperennis L. 5,550 d 5.0 a G
Oxalis corniculata L. 0 1.0 g N
Alysicarpus vaginalis (L.) DC. 30 1.4 "g P
Aeschynomene indica L. 3,958 df ".0 a G
Calopogonium muconoides Desv. 345 2.6 c M
Centrosemapubescens Benth. 0 1.0 g N
Crotolariajuncea L. 0 J 1.0 g N
Desmodium triflorum (L.) DC. 0 1.0 g N
Macroptilium lathyroides (L.) Urb. 2,940 h 2.4 cd G
Sesbania sesban (L.) Merr. 1,547 f1 5.0 a G
Cenchrus echinatus L. 3 1.4 "-g p
Chloris barbata Sw. 2 1.2 f P
Cynodon dactylon (L.) Pers. 356 5.0 a G
Dactyloctenium aegyptium (L.) Beauv. 1 J 1.4 e P
Digitaria ciliaris (Retz.)KoeL 3 1.4 P
Volume 36 (1 & 2) June-December 2000 29
Table 2. cont....
Digitaria setigera Roth ex R. & S 17 J 1.8 d-f P
Echinochloa colona (L.) Link 3,029 --g 5.0 a G
Echinochloa crus-galli (L.) Beauv. ssp. 703 5.0 a G
hispidula (Retz.) Honda
Echinochloa glabrescens 3,379 -f 5.0 a G
Munro ex Hook.f.
Eleusine indica (L.) Gaertn. 2 J 1.2 'f P
Imperata cylindrica (L.) Beauv. 27 i 1.8 d-f p
Ischaemum rugosum Salisb. 1,656 5.0 a G
Leersia hexandra Sw. 4,656 d- 5.0 a G
Leptochloa chinensis (L.) Nees 1,039 h 5.0 a G
Panicum repens L. 4 j 1.2 'g P
Paspalidiumflavidum (Retz.) A. Camus 326 i- 5.0 a G
Paspalum conjugatum Berg. 0 o 1.0 g N
Paspalum distichum L. 2 J 1.0 g P
Pennisetum purpureum Schumach. 0 o 1.0 g N
Rottboellia cochinchinensis (Lour.)W.D. 3 1.4 e-g P
Eichhornia crassipes (Mart.) Solms 34,452 a 5.0 a G
Monochoria vaginalis (Burm. f.) Presl 213 J 2.6 c M
Portulaca oleracea L. 109 h 2.4 cd M
Borreria ocymoides (Burn. F.) DC. 22 J 1.4 `'g P
Lindernia hirta (Cham. & Schlecht.) 4,600 d-e 5.0 a G
Sphenoclea zeylanica Gaertn. 18 J 1.4 e-g P
Spilanthespaniculata Wall. Ex DC. 89 J 2.0 ad M
Melochia concatenata L. 6 J 1.4 e-g P
Corchorus olitorius L. 0 j 1.2 fg P
Lantana camera L. 0 o 1.0 g N
Stachytarphetajamaicensis (L.) Vahl. 0 o 1.0 g N
LSD (0.05) 2,741 0.73
'Data are means of 5 replications. Means followed by a common letter are not significantly different by
LSD test at p=0.05.
2Gall Index rating on a 1-5 scale (0 to 100% infection): 1 for no root galls, 2 for trace galling (1-10%),
3 for slight galling (11 to 30%), 4 for moderate galling (31-50%), and 5 for severe galling (51-100%).
'G=Good host (J2s 290% of the initial inoculum (Pi) and/or gall index (GI) >3); M= Moderate host
(J2s -10% <90% Pi and/or GI >2 <3); P = Poor host (J2s2l but <10% Pi and/or GI >1 <2); N =
nonhost (0 J2s, GI <1).
Fig. 1. Meloidogyne graminicola galls on rice 'UPL Ri-5"(A) and winged bean (B)
ability of conwnon field crops and weei
.u. wsws.f VwUIUIlIC l ~u I Nw s .,UIIC-&C-IIuIM buUV y i
ig. 2. Meloidogyne graminicola galls on the root system of Echinochloa
colona (A) and Commelina
EFFECT OF ENVIRONMENTAL FACTORS ON THE GROWTH AND
SPORULATION OF Chalaraparadoxa (De Seynes) Sacc. IN CULTURE
N. SAN JUAN-BACHILLER1 and L. L. ILAG2
Imrnnn nr Tnhf r II nmIcarTrinn nr TnI cpnirr apI rnnr hnnmlr1whm 1 trn thPI rr1anldtP vrhrnn
.r .. ~..
il nrl 1-
INTRODUCTION MATERIALS AND METHODS
Stem bleeding disease of coconut in the Mycelial Growth and Sporulation
'hilippines was first reported by Lee in 1922.
t used to be a minor disease but it is now Effect of Various Culture Media. The
becoming a serious problem especially with following media were evaluated: potato
coconut hybrids. The effects of the disease dextrose agar (PDA), Czapek's solution agar
nay range from general weakened conditions (CSA), malt extract agar (MEA), nutrient agar
ifthe palhs to eventual loss in yield. In serious (NA), sucrose nitrate agar (SNA), coconut
ases, stem bleeding can lead to death ofpalms water agar (CWA), and V-8 juice agar (V-
Abad, 1983; Gapasin, 1983). 8JA). All media were sterilized by autoclaving
for 15 minutes at 1220C. A 7-mm diameter
In Indonesia, the disease had no effect on mycelial agar disc from 48-hr-old plate cultures
he number offemale flowers and nut harvested was placed at the center of the plated agar.
during a one year observation period Seeded plates were inverted and incubated at
Brahamona, 1986). 300C at a light intensity of 170-ft candles. The
colony diameter was measured after 24, 36
There are several causes of stem and 48 hr of incubation. Ten plates were used
bleeding. These include lightning injury, for each agar medium. Five plates were used
weevil attack, heavy manuring, heavy rains or to determine the mycelial growth diameter
floods, exposure to fire, and fluctuating water while the remaining five were utilized to
ible (Goonewardene, 1955; Del Rosario, 1968, determine the amount of sporulation of the
escrloea nerem is cause by clnaiara
aradoxa (De Seynes) Sacc., formerly called
'hielaviopsis paradoxa (De Seynes) von
Some environmental factors affecting
tem bleeding development are high humidity,
H (Piggot, 1964; Woodroof, 1979), heavy
ins and unfavorable soil conditions (Child,
973). Matthew and Remanandran (1980)
.ported that electrical conductivity and pH
re not directly related to the incidence of the
This study was conducted to determine the
effects of different culture media, light,
nitrogen sources, pH, temperature, and relative
humidity on the morphological and cultural
characteristics of C. paradoxa.
ay-old culture m 10 ml of sterile water. The
isc was macerated with a glass rod to get a
omogenous spore suspension. The spore
suspension was shaken every time an aliquot
tas pippeted out of the test tube. A drop of
le suspension was placed in a hemacytometer
nd the number of spores counted under a
Effect of Light. Petri dishes plated with
0 ml PDA (pH 6.0) were seeded withmycelial
gar discs (DRC isolate) at the center and
icubated under various light regimes:
antinuous light (CL), using 40 watt fluorescent
ibe at a distance of 30 cm; continuous
arkness (CD), where agar plates were
covered with used carbon paper; 12 hr-light
ad 12-hr darkness (ALD1); 16-hr light and 8
r-darkness (ALD2), and 8 hr-light and 16 hr
34 tal factors on the growth of Chalara paradox
darkness (ALD3). Three days after incubation,
diametric growth was measured. Sporulation
was measured after seven days of incubation
using a hemacytometer.
Effect of Nitrogen Sources. Ammonium
sulfate and peptone were used as nitrogen
sources both in liquid and solid media. The
liquid basal medium was prepared using the
following ingredients: glucose (10 g), potassium
phosphate (1 g), magnesium sulfate (0.5 g),
ferrous sulfate (0.2 mg), manganese chloride
(0.2 mg), biotin (0.05 mg), thiamine (0.1 mg)
and distilled water (1000 ml). Ammonium
sulfate (0.015 g) and peptone (0.02 g) were
added separately to one liter of the basal
medium. The solid agar medium was prepared
by adding 2.5% agar (Difco) to the liquid
medium. The mycelial disc (5 mm in diam.)
from the 7-day-old C. paradoxa (DRC isolate)
was transferred to a 125 ml Erlenmeyer flask
containing 75 ml liquid medium. The medium
without a nitrogen source served as control.
All the treatments were incubated in a
shaker at ordinary room temperature (28-30oC)
from 12:00 noon to 6:00 p.m. for 5, 8 and 15
days. At the end of each incubation period
the liquid medium was separated from the
mycelial mat using an ordinary strainer. The
mycelial mat was collected in aluminum foil
and dried in an incubator at 65oC until constant
dry weight was obtained. Each treatment was
replicated three times and arranged in
Completely Randomized Design.
Mycelial discs (5 mm in diam.) from the
7-day-old C. paradoxa culture were
transferred to test tubes with agar medium
prepared from the liquid medium. Fifteen days
after incubation, the degree of sporulation was
determined using a hemacytometer.
Effect ofpH. After sterilization, the PDA
medium was adjusted to the following pH: 5.5,
6.0, 6.5, 7.0, 7.5, and 8.0 by adding either sterile
IN HCI or IN NaOH and shaken thoroughly
before plating. The plates were seeded with
C. paradoxa (DRC isolate) using a 5 mm
diameter mycelial disc. The mycelial growth
diameter was measured after two days of
incubation. Sporulation was measured after
Effect of Temperature. The conidial
germination and germ tube elongation of 10
isolates (AADC, ARC, Bato, Calinan,
Camiguin, CII, DRC, La Filipina, Medina and
ZRC) of C. paradoxa were compared.
Bottles of approximately 3.5 L capacity were
used as incubation chambers. Each bottle
contained one liter of water to maintain high
humidity within the chamber. Screen
receptacles were placed inside the bottles about
one inch above the water level to hold the glass
Dry spores obtained from one-week old
cultures were placed on clean dry glass slides.
The slides were placed on the screen in the
bottles. The bottles were closed and stored at
20, 25, 30, 33 and 35C. After 24 hours of
incubation, the slides were removed from the
bottles, the germinating spores stained
immediately with lactophenol cotton blue and
the numbers of germinating spores counted by
selecting 100 spores at random. Observations
were made for both immature and mature
spores. The spores, which were hyaline and
thin-walled, were considered immature conidia,
whereas spores with thick walls, dark brown
in color and detached from the conidiophore
were considered mature spores.
Effect of Relative Humidity. Bottles of
approximately 3.5 L capacity were used as
incubation chambers. Various amounts of
glycerol were mixed with water to get the
desired relative humidity (RH) in the chamber
(Doberski, 1981). One liter of 31.4%, 18.8%,
ital factors on the growth of Chalara paradox
Volume 36 June-December 2000 35
10.0% and 0% glycerol-water solution was
placed inthe bottles to get a RH of 90%, 95%,
97.5% and 100% respectively. A screen was
placed inside the bottle to hold the glass slides.
It was about an inch above the solution. Dry
spores obtained from one-week-old cultures
(DRC isolate) were placed on clean dry glass
slides. The slides were placed on the screen
inside the bottles. The bottles were closed and
incubated at 300C.
After 24 and 48 hr of incubation the slides
were removed from the bottles. The spores
were stained immediately in lactophenol cotton
blue and observed microscopically. The
number of germinating spores was counted by
selecting at random 100 spores.
Combined Effects of Temperature and
The spores of the DRC isolate were
prepared as described above and exposed to
various RH and temperature regimes. Each
set of humidity chambers with 90, 95, 97.5 and
100% RH were incubated at 20, 25, 30, 33
and 350C for 24 and 48 hours. Spore
germination was determined microscopically
after staining with lactophenol cotton blue.
Experimental Design and Data Analysis
Experiments were conducted in
Completely Randomized Design (CRD) with
four replications. Data were analyzed
statistically and treatment means were
compared using Duncan's Multiple Range Test
RESULTS AND DISCUSSION
Mycelial Growth and Sporulation
Effect of Various Culture Media. All
the ten isolates produced abundant mycelia on
PDA and scant to almost no growth on NA
and CSA. The isolates had varied reactions
on the other media. The mycelial growth
characteristics of the Albay Research Center
(ARC) isolate is shown in Table 1. The ARC
isolate grew best on PDA and CWA, with a
mycelial growth diameter of 86.7 mm and 85.3
mm, respectively. Better growth was also
observed on VJA (83.9 mm), MEA (83.5 mm)
and on NSA (80.2 mm). Least growth was
observed on NA (31.4 mm) and CSA (30.2
mm). The color of the colonies was at first
white, becoming black after five days of
incubation (Fig. 1). Al-Hassan (1983) reported
that PDA was also the best medium for the
mycelial growth of C. paradoxa isolated from
fruit rot of date palm.
The isolates varied in their sporulation
capacity, as shown by the large discrepancies
in the amount of spores they produced on SNA
(Table 2). The AADC, Calinan, CII, Medina
and DRC isolates produced about twice as
many spores than the other five isolates.
Among the media tested, SNA appeared to be
the best medium for sporulation followed by
PDA and MEA. The isolates did not sporulate
on CSA because very scant mycelia were
produced on this medium.
Colony size at 48 hr and the number of
spores at seven days incubation were not
directly related. For instance, the widest colony
was produced on PDA, but the spore count
was much less compared to SNA. Differences
in the spore count in culture may be influenced
by the composition of carbon and nitrogen
sources (Satour, et al., 1969a and 1969b).
Effect of Light. Cultures grown under
continuous light had the widest colony diameter
(66.2 mm) (Table 3). The smallest colony
diameter (57.5 mm) was produced in
continuous darkness. The highest phialospore
and aleuriospore count also occurred on PDA
exposed to continuous light as compared with
N. S. Bachiller and L.L. llag
36 Effect of environmental factors on the growth of Chalara paradox
other light conditions. These findings agree
with the observation of Halos (1970) and
Smooth (1962). There were significant
differences in colony diameter and conidial
counts in the five light conditions tested. The
color of the medium changed from whitish to
blackish under continuous light, with masses
of spores formed. The highest number of
spores was observed in continuous light (95.5
x 104) while the lowest spore count was in
continuous darkness (51.3 x 104).
Effect of Nitrogen Source. Ammonium
sulfate was a better source of nitrogen for the
growth of C. paradoxa (compared with
peptone), as shown by the significantly higher
mycelial dry weights produced after five and
eight days of incubation (Table 4). After 15
days of incubation, no difference in mycelial
dry weight was observed between the
ammonium sulfate- and peptone-amended
medium. The slower rate of growth in the
ammonium sulfate-amended medium at the
later stages of incubation (15 days) may be
attributed to the effect of pH. According to
Garraway and Evans (1984), there is a
tendency towards a decrease in final pH of
the medium in the case of ammoniacal nitrogen
source due to the utilization of ammonium
The slower rate of growth in the peptone
amended medium is an indication that the C.
paradox isolate has difficulty in breaking
down peptone into a more readily assimilable
form of nitrogen (NO3- or NH4). The results
also suggest that the C. paradoxa isolate used
in the study prefers a more readily assimilable
source of nitrogen like ammonium sulfate,
especially in the early stages of growth.
However, variations in the pattern ofutilization
of nitrogen sources may be present even
among different isolates of the same species.
Some types of nitrogen compounds may be
used more effectively than others by particular
fungal species (Garraway and Evans, 1984).
Theoretically, no fungal growth is expected
in the medium with zero nitrogen. However,
in this study, growth was observed although at
a slower rate as compared with ammonium
sulfate- and peptone-amended medium. This
could be attributed to other sources ofnitrogen.
One possible source of nitrogen could be the
PDA disc used in the inoculation of the basal
synthetic medium. The vitamins as components
ofthe medium were another source of nitrogen.
Significantly, more spores were produced
after five and eight days of incubation in the
medium with ammonium sulfate than in the
medium with peptone (Table 5). After 15 days
of incubation, no significant difference in
sporulation of the fungus was observed
between the ammonium sulfate- and peptone-
amended medium. Very few spores were
produced in the medium where nitrogen was
Effect of pH. Mycelial growth of C.
paradoxa at all pH levels was generally very
abundant and aerial (CMI Description No. 143,
Morgan-Jones, 1966). However, a bigger
colony diameter was produced in the media
with pH ranging from 4.0 to 7.0 compared with
pH 8.0 (Table 6).
There was no difference in the number of
phialospores produced from pH 4.0 to 7.0.
However, more aleuriospores were produced
atpH 4.0 than in other pH levels. Both mycelial
growth and sporulation decreased only at pH
Since C. paradoxa can grow luxuriantly
and sporulate abundantly in a wide range of
pH, its potential for survival could be higher.
Conidial Germination and Growth on
Effect of Temperature. After 24
hours of incubation, only 2% of the spores
S. Bachiller and LL. Ilag Volume 36 June-December 2000 37
Combined Effects of Temperature and
The best temperature/RH combination for
pore germination was the 25C/100% RH
egime, where 90% and 97% of the spores
lad germinated after 24 hours and 48 hours,
respectively (Table 7). High spore germination
vas also observed at the 25C/97.5% RH
egime where 65% and 95% of the spores had
;erminated after 24 hours and 48 hours,
respectively. At 30C and 100% RH, 57%
nd 85% germination was observed after 24
ours and 48 hours, respectively. No spore
ermination was observed at 90% RH in all
temperature regimes. At 35C, 50%-65%
pore germination was observed after 48 hours
i the presence of 95%-100% RH. Only 10
nd 40% of the spores germinated after 24
nd 48 hr., respectively, at 20oC/100% RH
The different isolates of C. paradoxa,
obtained from different places in the Philippines,
ad varied reactions to different RH/
:mperature regimes as shown by their spore
termination pattern (Fig. 4). For instance,
ie two isolates AADC and ZRC did not
terminate in all temperature regimes when
icubated at 90% RH, five isolates (Calinan,
;amiguin, CII, DRC and Medina) germinated
nly at 30C with 90% RH, while the La
ilipina isolates germinated at 30, 33 and 35C
rith 90% RH.
When the relative humidity was
creased to 95%, 25C was the optimum
:mperature for seven isolates (AADC,
:alinan, Camiguin, CII, DRC, La Filipina,
ledina) and 30C was the optimum
temperature for three isolates (ARC, Bato,
RC). Only four isolates (AADC, ARC, Bato,
>RC) germinated at 20oC/95% RH regimes
mging from about 7 to 15%.
germinated at 20oC, 10% at 25C, 14% at 30C
and 7% at 33C (Fig. 2). After 48 hours of
incubation, the percentage of spore germination
ose to about 10% at 20C, 50% at 25oC, 45%
t 30oC and 20% at 33C. This shows that the
optimum temperature for spore germination of
. paradoxa (DRC isolate) is between 25C
md 30C. Only the mature spores germinated.
'he phialospores and the immature
leuriospores did not germinate even after 48
Lours of incubation m all temperature regimes.
The results obtained conform to the report
If Morgan-Jones (1966) that most spores of
athogenic fungi germinated and grew well at
5-32C. Likewise, Milanes& Herrera (1986)
reported that Thielaviopsis paradoxa isolated
rom sugar cane had optimum growth at 28C
under laboratory conditions. However, Al-
lassan (1983) reported that T. paradox
isolated from fruit rot of date palm was unable
o grow at temperatures higher than 30C.
Effect of Relative Humidity. Spore
ermination of C. paradoxa was positively
orrelated with relative humidity (Fig. 3). The
ptimum RH for spore germination was
between 97.5% and 100%. Spore germination
after 24 hours of incubation at 100% RH was
7%. This increased to about 48% germination
after 48 hours of incubation. At 97.5% RH,
pore germination was about 12% and 45%
after 24 and 48 hours, respectively. Only 1%
f the spores germinated after 24 hours of
Icubation at 90% RH. This increased to only
% after 48 hours of incubation.
Mortuza (1990) obtained similar results for
asiodiplodia theobromae and by Estrada
nd Ilag (1991) for Colletotrichum
loeosporioides where spore germination
creased as the RH was increased from 90%
38 Effect of environmental factors on the growth of Chalara paradoxa
Eight isolates had a higher percent spore
germination at the 97.5% RH/250C and 100%
RH/25C regimes. An almost similar trend
was observed at the 97.5% RH/30C and
100% RH/300C regimes (Fig. 4). Only two
isolates (ARC and ZRC) had higher percent
spore germination at the 97.5% RH/30C and
100% RH/30C regimes.
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Philippines. Coconut Today 1: 119-152.
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Thielaviopsis paradoxa and its role in date
fruitrot in Iraq. The Date Palm J. 2: 37-54.
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bleeding on the flashing and fruit of coconut
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temperature and humidity on germination and
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temperature on growth and pathogenicity of
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temperature and humidity on the germination,
growth and infection of Lasiodiplodia
theobromae (PAT.) Griff & Maubl. Cause of
stem-end rot in mango (Mangifera indica L.)
fruits. Unpublished MS Thesis, Univ. Philipp.
Los Bafios, College, Laguna, 83 p.
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SATOURMM, RK WEBSTER, WB HEWITT.
1969. A Studies on Diplodia and Diplodia-
like fungi I. Effect of carbon source on certain
taxonomic characters. Hilgardia39:601-629.
N. S. Bachiller and LL hag Volume 36 June-December 2000 39
SATOURMM, RKWEBSTER, WB HEWITT.
1969b. Studies on Diplodia and Diplodia-like
fungi II. Effect of nitrogen sources on growth,
sporulation and certain taxonomic characters.
SMOOTJJ. 1962. Effect of light on growth and
sporulation of stem-end rot fungi.
Fig 1. Mycelial growth of C
after five days ofincu
Phytopathology 52:28 (Abstr.).
WOODROOFJG. 1979. Coconuts: Production,
processing, product. AVI Publishing Co.,
Westport, Connecticut. 2 ed. 30 pp.
rlaraparadoxa (ARC isolate)
tion on different agar media
N. S. Bachiller and -LL [lag
Volurne 36 June~ecernber 2000 39
40 Effect of environmental factors on the growth of Chalara paradox
20 25 30
TE M PE RAT ,
Mi 24 hrs M 48 hl
Fig 2. Effect of temperature on spore germination of Chalara
paradoxa (DRC isolate) at 24 and 48 hr of incubation
100 97 5
RELAT'VJE -.M'DFTv ?l
Fig 4. Effect of relative humidity on spore germination of
Chalaraparadoxa (DRC isolate) at 24 and 48 hr of incubation
104 0 h
AADC Bato Camiguin DRC Medr,,A
ARC Catnan CII La Fipina ZR,
| 2oc f3 l ;'ooC 30 oC
iN 33 oC 3 :5 oc C
Ln. .. ...
Fig. 4. Effect of relative humidity and temperature on spore germination often T paradoxa isolates.
1.i 1- 1,I
h ~Pi.. LL
42 Effect of environmental factors on the growth of Chalara paradoxa
able 1. Colony diameter and mycelial growth che
different culture media.'
Agar Medium Diameter (nun)2
Potato Dextrose 86.7a
Nutrient Agar 31.4d
Malt Extract 83.5b
Sucrose Nitrate 80.2c
Czapek's Solution 30.2d
Coconut Water 85.3a
V-8 Juice Agar 83.9b
Incubated at 30C with light intensity of 170 ft-candles
2 Mean of eight plates. Taken after five days of incubation
3Taken after five days ofincubation.
Means in column with common letter are not significantly differ
"able 2. Sporulation of 10 isolates of C. parade
Fungal Isolate CSA NA
AADC 0 1.0
ARC 0 1.2
Bato 0 1.0
Calinan 0 1.8
Camiguin 0 1.4
CII 0 2.0
DRC 0 1.8
ute.u.~ajLc uj lnruturu puruuuovu (i-L laUle; UOn
Very abundant, aerial
Very sparse, appressed
Very sparse, appressed
Very abundant, slightly aerial at edges
Abundant, slightly aerial
at 5% level
on different culture media after seven days of
ut (x 104) on different media*
MEA SNA PDA
2.3 13.9 3.5
2.9 5.6 2.9
2.3 2.6 1.4
4.8 13.3 4.0
Table 3. Mycelial growth and sporulation of C. paradoxa grown in potato dextrose agar (PDA)
under different light conditions'
Cultural Colony Number of Conidia (x 104)3
Light Conditions Growth Diameter Phialospores Aleuriospores Total
Continuous Very abundant
Light (CL) aerial 66.2" 39.7a 55.8 95.5"
Continuous Darkness Abundant aerial
(CD) 57.50 15.50 35.8b 51.3c
Alternate Light and
(12 hr light, 12 hr Very abundant
darkness, ALD1) aerial 62.3b 34.5"b 38.2b 72.7b
(16 hr light, 8 hr Abundant aerial
darkness, ALD2) 61.5b 26.0b 26.7c 52.70
(8 hr light, 16 hr Abundant aerial
darkness, ALD3) 61.0b 28.5b 25.0c 53.50
SIncubated at 300C with a light intensity of 170 ft-candles
2 Mean of six replicates. Taken 24 hrs after plating
3 Mean of six replicates. Taken 7 days after plating
Means in column with common letter are not significantly different at 5% level
Table 4. Effect of nitrogen sources and incubation period on mycelial weight of C. paradoxa'.
Mycelial Weight (grams) at various incubation time dayss)
Nitrogen Source 5 8 15
Zero Nitrogen 0.0590 0.120" 0.206b
Ammonium Sulfate 0.208a 0.337a 0.349a
Volume 36 June-December 2000 43
4. S. Bachiller and L.L. Ilav
44 Effect of environmental factors on the growth of Chalara paradoxa
'able 5. Effect of nitrogen sources on the sporulation of C. paradoxa after seven days of incubation.
Spore Count (x 104) at various incubation time (days)1
Nitrogen Source 5 8 15 Mean
Means in column wi
growth and sporn
Volume 36 June-December 2000 4
Femperature/RH 24 hr 48 hr
% RH 10.0 40.0
i% RH 16.7 28.3
/ RH 0.0 13.3
o RH 0.0 0.0
% RH 90.0 96.7
i% RH 65.0 95.0
6 RH 0.0 71.7
E RH 0.0 0.0
% RH 56.7 85.0
i% RH 46.7 86.7
'RH 10.0 45.0
DETECTION OF PAPAYA RINGSPOT VIRUS (PRSV) USING
MEMBRANE IMMUNOBINDING ASSAY (MIBA)
S. R. RUSWANDI1 and N. B. BAJET2
Portion of the undergraduate thesis of the senior author submitted to the University of the
Philippines Los Bafios, College, Laguna, Philippines
'Undergraduate thesis student and 2Professor, Department of Plant Pathology, University
of the Philippines Los Bafios, College, Laguna, Philippines
Membrane immunobinding assay (MIBA) was used and compared
with indirect enzyme-linked immunosorbent assay (ELISA) in detecting
papaya ringspot virus (PRSV) in infected Carica papaya L. MIBA was
effective in detecting PRSV antigen and utilized lesser amount of
antiserum than ELISA. The highest dilution of PRSV antigen detected
was 1:10000 for both MIBA and ELISA. Detection of PRSV in field
samples gave almost the same result for both procedures. Results
suggest that MIBA can be used as an alternative diagnostic technique
for the detection of PRSV.
Keywords: Papaya ringspot, membrane immunobinding assay, detection
INTRODICTION measure that has been sought after an
Papaya (Carica papaya L.) is one of the
most important crops in the Philippines. It is a
common table food and is a good source ol
vitamin C. It also contains enzymes that break
down proteins in aqueous solution (Lin et al.,
1988). It is also commercially important. In
fact, Solo, a papaya variety, is the primary
cultivar exportedto Japan and Hongkong while
Cavite Special is the major cultivar for fresh
fruit in the local market (Pabuayon et al.,
Papaya production has been threatened
with the outbreak of papaya ringspot, a viral
disease that was first reported in the Philippines
in 1984 (Opina, 1986). In an effort to control
the disease, special quarantine measures had
been issued by the Department of Agriculture
(Rrpmnia cnrlVi llp ri 100 1 Annthpr m-ntrnl
However, its development may take sometime
because there is no ringspot resistance
gene within C. papaya. Putative resistance
gene or genes have been found in wilt
relatives of papaya but the introgression o
such genes into papaya has also been ver
difficult as these wild relatives and papaya ar
hardly compatible. Offspring or embryos o
putative hybrids of these wide crosses ar
hardly viable, even when rescued and nurture
in tissue culture media (Villegas, 2002)
Transgenic papaya that expresses the coa
protein of PRSV has also been developed an
could be an added option for the control ofthi
dreaded disease (Fitch et al., 1994).
Diagnosis and early detection ar
important in preventing the spread of infected
S.R. Ruswandi and N.B Volume 36 (1 & 2) June-December 2000 4
is the enzyme-linked immunosorbent assay or
ELISA. However, ELISA also has its
drawbacks. It was shown to have high
nonspecific reactions in uninfected tissue
extracts, which may arise from nonserological
interaction among the components of the assay
(Zagula et al., 1990). Also, the standardized
procedure for the detection of PRSV Philippine
isolates recommends an overnight incubation
period for the antigen (Eusebio et al., 1994).
The whole process usually consumes more
than ten hours and seven days before results
could be obtained. Methods to reduce the time
of processing a large number of samples while
retaining the sensitivity of the test are desirable.
In this study, another serological method called
the membrane immunobinding assay or MIBA
was employed to detect PRSV.
MATERIALS AND METHODS
Papaya leaf samples used in the
experimental were obtained in Los Bafios,
Laguna. Healthy controls were papaya
seedlings grown under greenhouse conditions.
Membrane Immunobinding Assay
The MIBA protocol sent by Professor
Roger Hull of the John Innes Institute, UK for
rice tungro was followed with some
modifications. About 0.2 g of each leaf sample
was macerated with a mortar and pestle with
the addition of phosphate-buffer saline, pH 7.4
(PBS) (0.01 M sodium/potassium phosphate
buffer, pH 7.4 containing 2 nM potassium
chloride and 0.14 M NaCl at 1:5 ratio
(wt./vol/). The leaf extracts were dispensed
into microcentrifuge tubes and centrifuged for
5 min at 14,000 rpm (Eppendorf5415C). The
supernatant was collected and transferred
into fresh microcentrifuge tubes and the pellets
were discarded. About 5 tl of each sample
was blotted into a nitrocellulose membrane
(BioRad) and the blots were air-dried. The
membrane was soaked in PBS and placed in a
tray before incubating for 2 hr at room
temperature in 20 ml antibody solution
containing PBS (pH 7.2), 5% skim (nonfat)
milk (Carnation), 0.1% Tween 20 (polyethylene
sorbitan monolaureate) and antiserum of PRSV
at 1:10,000. The incubation was done with
slow shaking in an orbital shaker (Bellco) at
After incubation, the membrane was
washed with 20 ml was solution (PBS, 0.1%
Tween 20 and 5% skim milk) three times at 10
min per wash with slow shaking (50 rpm) after
which it was re-incubated in 20 ml antibody
conjugate solution (1 ul goat anti-rabbit antibody
conjugated with alkaline phosphatase or
GARAP, Sigma Chemicals Co., St. Louis,
Missouri, USA; cat# A2168 in 1000 ul ofwash
solution) at room temperature for 2 hr with
slow shaking at 50 rpm. The membrane was
again washed as above but this time with 20
ml PBS containing 0.1% Tween 20 followed
by a final wash for 3 min with 20 ml 100 mM
Tris-HCl (pH 9.5).
The presence of bound antibody was
visualized by the addition ofthe substrate. The
substrate consisted of 0.05 mg/ml nitroblue
tetrazolium (NBT) dissolved in Iml 4 mM
magnesium chloride and 0.05 mg
bromochloroindoylphosphatase (BCIP) in 1 ml
100% dimethylformamaide (DMF). Incubation
was done while shaking at 50 rpm until the
blots have completely developed. Reactions
were terminated by washing the membrane
with distilled water and drying it in-between
filter papers (Whatman No. 3).
The protocol for ELISA was that of Dr.
Y. Amemiya, Chiba University, Japan and
Eusebio (1992) with some modifications. The
S.R. Ruswandi and N.B
Volume 36 (1 & 2) June-December 2000 C1
48 uuMLiuE. %. papaya 141mWP.W v...-
sap extraction procedure was me same as m
About 100 pl of the extracted sap was
dispensed in the wells of an ELISA plate
(Immunolon II, Dynatech Laboratories,
Alexandria, Virginia, USA) and then incubated
overnight at 40C after which the wells were
washed with PBS-T three times at 10 min per
wash. After washing, 1% skim milk in PBS-T
was dispensed at 200 ul/well and the plate was
incubated for 2 hr at room temperature and
then was washed with PBS-T as above.
After addition of the antiserum to PRSV
at 1:1000 dilution in PBS-T at 100 ul/well, the
plate was incubated for 2 hr at room
temperature then washed as above. GARAP
diluted at 1:1000 dilution in PBS-T was then
added at 100 pl/well and was again incubated
for 2 hr then washed as above. The enzyme
substrate, p-nitrophenyl phosphate (pNPP) was
dissolved in 10% diethanolamine, pH 9.8 at 1
mg/ml, dispensed into each well of the ELISA
plate and incubated for 1.5 hr followed by the
addition of 50 pl/well of3M sodium hydroxide
to terminate the reaction. pNPP was the
substrate used for alkaline phosphatase
enzyme conjugate because when it is cleaved
by the enzyme, the product turns yellow, the
intensity of which is evaluated with a
spectrophotometer or an ELISA reader at 405
Determination of the Effect of Different
Antibody Dilutions on MIBA and
One set of duplicate blots and one seL
duplicate wells of each sample were
prepared as in MIBA and ELISA, respectively.
However, different dilutions of the antibody
were used. For MIBA, the antibody was
... . .. ... I - -
UIlUltU LU AllllVC 41L ilU lUIUIVVWlU ULLULou...
1:1250,1:2500,1:5000 and 1:10000.
Determination of the Effect of Different
Antigen Dilution on MIBA and ELISA
One set of duplicate blots ana one set of
duplicate wells were also prepared as in MIBA
and ELISA, respectively. This time, the dilution
of the antigen was varied. About 0.1 g of each
leaf samples was homogenized in 0.9 ml Of
PBS to arrive at 1:10 dilution. From this
dilution, subsequent ten-fold dilutions were
madeto arrive atthe following: 1:100, 1:1000,
1:10000 and 1:100000. These dilutions were
used in both MIBA and ELISA.
Detection of PRSV in Field Samples
Using MIBA and ELISA
'Ten symptomless and ten asymptomatic
samples were collected from Batong Malake,
Los Bafios, Laguna. Papaya seedlings from
the greenhouse were used as healthy controls.
One set of duplicate blots for MIBA and one
set of duplicate wells for ELISA were
RESULTS AND DISCUSSION
The procedure for MIBA followed was
originally prepared for the detection of rice
tungro bacilliform ad rice tungro spherical
viruses. This diagnostic technique can also be
used for the detection of PRSV as shown in
the results of this study.
The sensitivities of MIBA and indirect
ELISA were compared based on the ability to
detect PRSV at varying dilutions of the
antiserum and antigen and the ability to detect
field-infected papaya samples. Indirect ELISA
of the papaya samples at varying antiserum
Volume 36 (1 & 2) June-December 2000
S.K. Kuswandl and N.
detect the virus in sample B which show
typical symptoms of PRSV. This could be d
to the presence of another virus causing ve
similar symptoms as PRSV but is serological
unrelated to it. The inability of indirect ELIK
to detect the antigen at higher dilutions oft
antiserum implies that only dilutions lower
equal to 1:1250 should be used to rea
positively in ELISA and that dilutions high
than 1:1250 will yield negative reactions.
MIBA, on the other hand, showed positi
results up to 1:10000 dilution of the antiseru
at the same antigen dilution implying that t
dlihtinn end-nnint nf their nnticpnim rn-ld
ELISA, all samples were negative at 1:1
dilution where the antigen is theoretically hig
This may be due to the fact that many plan
contain certain compounds, which can inhit
immunological or enzymatic reactions. Th
can be remedied by further diluting the samp
(Clark, 1981). In MIBA, most of he interferii
components may have been precipitated by tl
brief centrifugation prior to blotting. Sample
showed positive results in MIBA up to a dilutic
of 10-2 antigen dilutions and in ELISA at
dilution of 1:10000.
For the detection of PRSV in field sample
1 out of 10 symptomatic samples yield(
negative result. Sample B was evaluate
." "l. d A rl' I I A I
50 Detection of papaya ringspot virus
for evaluating a diagnostic technique are
reliability, sensitivity, cost, and labor. These
are important considerations especially in
countries like Philippines. MIBA, as shown in
the experiment, met all these criteria and thus
can serve as an alternative test for the
detection of PRSV.
BERGONIAHT, EVILLEGAS.1991. Eradication
and rehabilitation program in papaya ringspot
affected areas. In: First National Symposium/
Workshop on Ringspot and Other Diseases of
Papayain the Philippines. February 7-8, 1991,
Bureau of Plant Industry, San Andres, Malate,
CLARCKMT. 1981. Immunsorbentassaysinplant
pathology. Annm Rev. Phytopathol 19:83-106.
the detection of papaya ringspot virus in
Luzon, Philippines. Pilipp. Agric. 77:383-391.
D, SLIGHTOM JL,SANFORD JC. 1992.
Virus resistant papaya plants derived from
tissues bombarded with the coat protein gene
of papaya ringspot virus. Bio/Technology 10:
HSUHT,RHLAWSON. 1991. Directtissueblotting
for detection of tomato spotted wilt virus in
Impatiens. PlantDis. 75:292-295.
LINCC, HJSU, DNWANG. 1989. The control of
papayavirus inTaiwan. RO.C. Tech. Bulletin
OPINA OS. 1986. Studies on a new virus disease
of papaya in the Philippines. In: Plant Virus
Diseases of Horticulture Crops in the Tropics
and Sub-Tropics. FFTCBookSeriesNo. 33.
III MANALO. 1988. Supply and price
relationships for selected fauits and vegetables
in the Philippines. PCARRD/ASSP Funded
Proj. Rpt Dept Agr. Econ., Coll. Econ. Manag,
Univ. Philipp. Los Bafios, College, Laguna,
POWELL CA. 1987. Detection of three plant
viruses by dot-immunobinding assay.
RMLISTER. 1990. EvalnationofthreeELISA
methods as alternatives to ISEM for detection
ofthe wheat spindle streakyellow mosaicvirus.
S.R. Ruswandi and N.I
Volume 36 (1 & 21 June-December 2000
Table 1. Comparison of the reactions of test Si
Sample Description 1:250
A Healthy ND' -
B Infected ND + b
C Symptomless ND -
D Sympyomless ND -
E Symptomatic ND + b
F Symptomatic ND + 1'
G Symptomatic ND + 1b
pies in ELISA and MIBA using varying dilutions oj
650 1:1250 1:5000 1:10000
M E M E M E M
+ + + +
+ + + +
+ + + + +
+ + + + +
Table 3. Comparison of the reactions offield-infected papaya samples in EISA and MIBA
Sample Description ELISA MIBA
C Symptomatic + +
D Symptomatic + +
E Symptomatic + +
F Symptomatic + +
G Symptomatic + +
H Symptomatic + +
I Symptomatic + +
J Symptomatic + +
K Symptomatic + +
L Symptomless +
U Symptomless +
i. Ruswandi and N.B. Bajet Volume 36 (1 & 2) June-December 2000 53
g varying antiserum dilutions. Row A,
d papaya sample; Rows C and D, symp-
,mptomatic samples; Column 1, 1:250
amn 3, 1:1250 dilution; Column 4, 1:2500
umn 6, 1:10000 dilution.
A at varying antigen dilutions. Row A,
:d papaya sample; Row C, symptomless
rmples; Column 1, 1:10 dilution; Column
on, Column4, 1:0000 dilution; Column 5,
Fig 1. MIBAresults ofdifferent samples u
healthy papaya samples; Row B, info
tomless samples; Rows E, F and G,
dilution; Column 2, 1:650 dilution; Coc
dilution, Coluhmm 5, 1:5000 dilution; C
Fig 2. Reactions of different samples in M
healthy papaya samples; Row B, infec
sample; Rows D,E and F, symptomatic
2, 1:100 dilution; Column 3, 1:1000 dill
54 Detection of papaya rinaspot vi
Fig. 3. Reactions of the field sample
sample; Columns B-K, symj
sing MIBA. Column A, healthy papaya
amatic papaya samples; Columns L-U,
Detection of Dagpava rinaspot vi
At)'TF- 1RvP1UnhTTCTinN nF A4FLOMhOGYNE GRAMINICOLA ON
EB. GERGON an
Senior Science Research Specialist, Philipp
;ija; and 2Nematologist, Institut Francais de Rec
)operation (ORSTOM), France
The reproduction of Meloidogyne
the green manure crops-Sesbania, A
studied in simulated flooded and wel
tode reproduction was suppressed in
in well-drained soil. Aeschynomene nil,
A. afraspera 14054, 14143; A. pratensi
and 13058; A. aspera 13020; A. elaphi
S. rostrata 24062, S. cannabina 21074
22026, and Neptunia oleracea support
sities in their roots. Their use under
the risk of yield loss caused by M. g
crop. Only A. indica from the Philip
evenia and S. cannabina 21035 maint:
their roots in at least two experiments,
manure crops in rainfed ricefields.
Key words: Aeschynomene sp., Sesbania sp
ze root-knot nematode.
Sesbania spp. and Aeschynomene spp.
-e green manure crops that are possible
iurces ofbiolovicallv fixed nitrogen to sustain
ILLJ LIJ 1 ill V 0l
: Rice Research Institute, Mufioz, Nueva
cher Scintifique pour le Developpement en
raminicola on 42 accessions of
chynomene and Neptunia-was
rained soil conditions. Nema-
ntinuously flooded soil but not
ca 14040; A denticulata 13003;
13006; A. indica 13016, 13019,
cylon 12148; A. scambra 13015,
9. tedraptera 21164, S. speciosa
high nematode population den-
linfed conditions could increase
ninicola in the subsequent rice
ies, A. scambra, A. sensitive, A.
ed low nematode populations in
id are therefore, potential green
green manure, Meloidogyne graminicola,
Another benefit of green manure crops is
,ir potential use in nematode control. S.
strata (Germani et al., 1983), A. afraspera
rot et al., 1992), and Sphenoclea zeylanica
14.1rl- At 1 1 1 111I hnirp h--n fnlmrd t
rice production systems where green manure saturation point. All entries were replicated
crops are utilized, it is important to identify six times and arranged in a split plot design.
which species are resistant to the nematode. The three experiments were done sequentially
In this study, the reproduction of M and each experiment included cultivar UPL
graminicola in 22 accessions of Ri-5 as the control check. New entries and
Aeschynomene spp., one accession of selected species of green manure crops from
Neptunia sp., and 19 accessions of Sesbania the previous setup were included in subsequent
spp. was evaluated under simulated flooded runs.
and well-drained soil conditions.
Sixty days after inoculation, roots were
collected from each pot. The roots were
MATERIALS AND METHODS washed thoroughly and processed for
nematode extraction. Three sub samples of 3
Three experiments following same g each were placed in a misting chamber for 5
procedures were conducted in the greenhouse days to collect juveniles and males of M
in PVC pots sealed at one end and measuring graminicola. The number of nematodes per
groups: group 1 pots, intended to simulate ANOVA and treaty
I ILU L U VLUJl JJt I.JVl OjU V flW J-U. ,* TvYLzAJSJ. UI.JLtjJ (4^11 1 j / ( .J IlO 1115 1 1OU.wA tiUVl lIS J. I Lit
5,000 cm3 of autoclaved sterilized sandy loam recovery of nematode juveniles higher than the
soil (9% clay, 20% silt, and 71% sand), while initial inoculum was used as indicator of
pots in group 2 were filled with about 4,500 nematode reproduction.
cm3 of similar soil.
Seeds of Aeschynomene spp., Sesbania RESULTS AND DISCUSSION
spp. and Neptunia sp. from different countries
were scarified by soaking in concentrated The number ofM graminicola recovered
I2SO, for 30 min followed by rinsing and from roots of plants grown in well-drained soil
soaking in tap water overnight before sowing, was significantly higher than that obtained
Two days after sowing, 2,000 second stage- under flooded conditions in all experiments
juveniles (J2) of M. graminicola were (Table 1). Simulated rainfed conditions favored
introduced into two 5-cm holes made near the reproduction of the nematode as indicated by
. u IUgUII dllU r.v. l lUl VUIUIlIC I u \1 I af M JUI IC-lUd iI I U VV U1
nematode recovery from their roots indicates Among Aeschynomene spp., 8 species or
that the pathogen survived in flooded soils. 11 accessions were found comparable with
In these experiments, flooding was done UPL Ri-5 in their reaction to M. graminicola
3 days after inoculation, which allowed the in at least one experiment in well-drained soil.
nematodes to invade the roots before flooding, These were A. nilotica 14040; A. denticulata
and then the soil was never drained. Results 13003; A. afraspera 14054, 14143; A.
confirmed earlier reports that M. graminicola pratensis 13006;A. indica 13016, 13019, and
can survive in waterlogged soil as eggs in 13058; A. aspera 13020; A. elaphroxylon
eggmasses or as juveniles (Bridge and Page, 12148; and A. scambra 13015. The number
1982). Egg masses can remain viable for 14 of nematodes extracted from their roots was
months or more in waterlogged soil (Roy, 1982) not significantly different from that obtained
reported that. The low number of nematodes from UPLRi-5, which indicates that they are
recovered from the roots after continuous good hosts of M. graminicola.
flooding also indicates that continuous Aeschynomene spp. that had significantly
flooding for 57 days reduced, but did not lower nematode counts than UPLRi-5 and
eliminate, M. graminicola. The nematodes susceptible Aeschynomene spp. were A
were likely immmobilized in one site and were .indica 13071,A. scambra 13015,A. sensitive
prevented from migrating to other parts of the 12002,A. evenia 13010,A. denticulate 13003,
root system that affected further nematode A. uniflora 13158, A. ciliata 13078, A.
reproduction. Flooding has been reported to schimperi 12156, and the unidentified species
limit the ability of M graminicola to invade of Aeschynomene from Australia. A. indica
rice roots (Manser, 1968). In addition, 13071,A. scambra 13015,A. sensitive 12002,
Sesbania spp. and Aeschynomene spp. are andA. evenia 13010 maintained low nematode
stem-nodulating green manure crops well- population in their roots in two repetitions of
adapted to waterlogged soil. Under submerged the experiment.
conditions, they develop parenchymatous cells
in their roots which make their root tissues Sesbania species that did not differ
spongy (Ladha et al., 1992), a characteristic significantly from 'UPL Ri-5' in their nematode
change in root texture which may have density in the roots under simulated upland
restricted the development and the active conditions included S. rostrata 24062, S.
movement of the nematode juveniles inside cannabina 21074; S. tedraptera 21164, and
their roots. S. speciosa 22026. These species can be
considered susceptible and good hosts of M.
In experiments 1 and 2, where graminicola. S. cannabina 21035, 21037,
Aeschynomene spp. and Sesbania spp. were 21076, and 21132; S. virgata 22060; S.
equal in number, analysis showed no significant spirits 21099; S. varadero 21098; S.
difference between the two genera. Certain punctata 24043; S. emerus 22100; and S.
species of both genera, however, highly sesban 22093 harbored significantly lower
favored nematode development in well-drained nematode populations than the susceptible
soil and served as good hosts of the nematode Sesbania spp.
58 Reproduction of Meloidogyne graminicola
1990). In these environments, fields often
experience only intermittent flooding during
the cropping season allowing M. graminicola
populations to increase in high densities in the
soil. Growing of S. rostrata, S. aculeata (syn.
S. cannabina 21044), A. afraspera, and A.
nilotica, which were considered the most
promising green manure crops in lowland fields
(Ladha et al., 1992) and at the same time good
hosts of M graminicola can, therefore,
enhance the increase of the pathogen to
damaging levels in the subsequent rice crop.
Reaction to M. graminicola should
therefore be considered in selecting green
manure crops in rainfed rice cropping.
S. rostrata, which is the most popular
among these crops, is confirmed to be very
susceptible to the nematode. Continuous use
of S. rostrata can increase the population
density ofM. graminicola in nonflooded soil.
Incorporation of S. rostrata-infected roots
with inorganic fertilizer can also spread the
pathogen to clean fields. Because of the risks
involved with the use of species susceptible to
M. graminicola, all necessary precautions
must be observed when using them in
intermittently flooded fields. The maximum
benefits derived from green manuring may
not be sufficient to compensate for the loss in
yield due to the nematode in the following rice
The use of resistant green manure crops
is the most practical approach if green
manuring is to be considered in rainfed cropping
systems. Several Sesbania spp. and
Aeschynomene spp., which have been found
in this study to exhibit some degree of
resistance to the nematode in both flooded
and nonflooded conditions could be potential
green manure crops in rainfed and irrigated
ricefields infested with M. graminicola.
BECKERM, DIEKMANN KH, LADHAJK, DE
DATTASK, OTIOW JC. 1991. Effect ofNPK
on growth and nitrogen fixation of Sesbania
rostrata as green manure for lowland rice
(OryzasativaL.). Plant and Soil 132:149-158.
BECKERM, LADHA JK, OTTOW JC. 1990.
Growth and N, fixation of two stem-nodulating
legumes and their effect as green manure in
lowlandrice. SoilBiol Biochem. 22:1109-1119.
BRIDGE J, PAGE S. 1982. The rice root-knot
nematode, Meloidogyne graminicola, on deep
water rice (Oryza sativa subsp. indica). Revue
GERBERKG,SMARTJRC,ESSERRP. 1987. A
comprehensive catalogue of plant parasitic
nematodes associated with aquatic and
wetland plants. Institute of Food and
Agricultural Sciences, University of Florida,
Gainesville, Florida, USA. Agric Expt Sta Tech
GERMANIG,REVERSARG,LUCM. 1983. Effect
of Sesbania rostrata on Hirschmanniella
oryzae inflooded rice. J. Nematol. 15:269-271.
LADHAJK,PAREEKRP,BECKERM. 1992. Stem
nodulating legume-Rhizobium symbiosis and
its agronomic use in lowland rice. Adv. Soil
MANSERPD. 1968. Meloidogyne graminicola, a
cause of root-knot of rice. FAO Plant Prot.
MOHANDAS C, RAO YS, SAHU SC. 1981.
Cultural control of rice root nematodes
(Hirschmanniella spp.) with Sphenoclea
zeylanica. Proc. Indian Acad. Sci. (Animal
PROT JC. 1994. The combination of nematodes,
Sesbania rostrata, and rice: the two sides of
thecoin. Int. Rice Res. Newsl. 19:30-31.
ion ana J.u. rroxi vulull c a w I i ;.... -- I
T JC, SORIANO I, MATIAS D, SAVARY S. RAO Y S, ISRAEL P. 1972. Influence of soil tyl
.992. Use of green manure crops in control of on the activity of the rice root knot nematod
firschmaniella mucronata and H. oryzae in Meloidogyne graminicola Golden an
rrigatedrice. J. NematoL 24: 127-132. Birchfield. IndianJ. Agric. Sci. 42:744-747.
mitigate growth andyield reductions of upland
rice cultivar UPLRi-5 causedby Meloidogyne
graminicola. Fund. AppL Nematol. 17:445-454.
RAOYS, ISRAELP. 1971. Studies on nematodes
of rice and rice soils. Influence of soil chemical
properties on the activity of Meloidogyne
graminicola, the rice root knot nematode.
ROY AK. 1982. Survival of Meloidogyne
graminicola eggs under different moisture
conditions in vitro. Nematol. Mediter. 10:221-
NATANABE I, ROGER PA, LADHA JK, VAN
HOVE C. 1992. Biofertilizer germplasm
collections at IRRI. The International Rice
Research Institute, P. O. Box 933, Manila.
Table 1. Average number of second-stage Meloidogyne graminicola juveniles per gram offresh root ofAeschynomene spp and Sesbania spp. grown in flooded and
Acces- M. graminicola /g root.
sion Species (Country of Origin) First experiment Second experiment Third experiment
No. Flooded Well-drained Flooded Well-drained Flooded Well-drained
12005 .4eschynomene sp. Benth (Australia) 17 b 1,345 e 740 a 7,925 ab
A. afraspera J. Leonard(Senegal)
A. afraspera J. Leonard (Gambia)
A. afraspera Leonard (Guinea)
A. aspera Linn. (Sri Lanka)
A. ciliata (Colombia)
A. ciliata (Senegal)
A. denticulata Rudd (Brazil)
.4. denticulata Rudd (Venezuela)
A. elaphroxylon Taub (Burundi)
A. evenia W. F. Wright (Columbia)
A. indica Linn. (China)
A. indicy Linn. (India)
A. indica Hochst ex A, Rich (Madagascar)
A. indica Linn. (Philippines)
A. nilotica Taub. (Mali)
A. pratensis Donn. Smith (Brazil)
A. pratensis Donn. Smith (Senegal)
A. scabra G. Don (Zimbabwe)
A. schimperi Hochst ex A. Rich (Senegal)
A. sensitiva Swarzt (Brazil)
A. uniflora E. Mey. (Zaire)
Sesbania sp.(El Salvador)
S. cannabina Linn. & Merrill (China)
S. cannabina (Retz.) Poir (India)
S. cannabina (Retz) Poir (China)
S. cannabina (Retz.) Poir (China)
21037 S. cannabina Linn. & Merrill (China)
16 b 11,042 ab 8 c
1 b 270 e 69 b
9 b 362 e 1 c
2 b 34 e 41 c
!8 b 59 e
18 b 4,678 c-e -
16 b 14,250 a -
.1 b 63 e
1 b 528 e
Table 1. Cont.
S. cannabina (Retz.) Poir (Japan)
S. emerus (Cuba)
S. javanica Miquel (Thailand)
S. punctata Brem. & Oberm. (Madagascar)
S. punctata Brem. & Oberm. (Madagascar)
S. rostrata Brem. & Oberm.(Senegal)
S. sesban Linn. & Merrill (Philippines)
S. speciosa Taub.(Indonesia)
S. speciosa Taub. (Nigeria)
S. spirits (Cuba)
S. tedraptera Hochst. ex Baker (Kenya)
S. varadero (Cuba)
S. virgata (Brazil)
Neptunia oleracea (China)
Rice cultivar 'UPLRi-5'
16 c 3,797 c-g
740 a 7,925 ab
AVERAGE 90 B 1,828 A 83 B 4,017 A 99 B 2,463 A
'Means in a column followed by a different lower case letter are significantly different by LSD at p = 0.05.
Means in a row within an experiment followed by a different upper case letter are significantly different by
LSD at p = 0.05. indicates entry was not included in the test.
EVALUATION OF PLANT EXTRACTS FOR POST-HARVEST
MANAGEMENT OF BACTERIAL SOFT ROT OF CABBAGE
V. R. MAMARIL1 and F.C. ABARQUEZ2
Supported by the Bureau of Plant Industry, San Andres, Malate, Manila
1Supervising Agriculturist and 2Agriculturist II, Crop Research Division, bureau of Plant
Industry, San Andres, Malate, Manila
This study aimed to evaluate the preventive and control efficiency
of plant extracts against bacterial soft rot of cabbage, a post-harvest
Bio-assay experiments against Pectobacterium carotovorum subs.
carotovorum, the causal bacterium of soft rot of cabbage, showed that
alum, streptomycin, and extracts of katakataka, chives, eucalyptus, duhat
sweet potato, onion, and garlic inhibited the growth of the organism.
These effective treatments were tested similarly for their
antibacterial properties against the same organism. The te:;t used
cabbage core and carrot slices as the growing media of the bacteria.
Streptomycin, katakataka, chives, eucalyptus, duhat, and garlic showed
complete preventive and control properties on both cabbage core and
carrot slices. Sweet potato and alum treated slices showed slight
Only the promising treatments were further evaluated on whole
cabbages and under ambient storage condition. This experimental phase
revealed that streptomycin, chives, garlic, katakataka, duhat and
eucalyptus were able to prevent bacterial soft rot infection up to five
days. The preventive capability of alum lasted only for three days.
Similarly, streptomycin, garlic, duhat, and eucalyptus were able to control
bacterial soft-rot infection. Chives and katakataka treated cabbage heads
showed soft-rot infection after four days and three days, respectively.
Keywords: Bacterial soft rot, Pectobacterium carotovorum subsp. carotovorum,
post harvest disease
R. Mamaril and F.C. Abarquez Volume 36 June-December 2000 63
Bacterial soft rot, caused bythe bacterium
Pectobacterium carotovorum subsp.
carotovorum, which was previously called
Erwinia carotovora subsp. carotovora is one
if the most destructive post-harvest diseases
if vegetables, particularly cabbage
Daengsubha, 1974). The bacterium attacks
he storage organs of almost all stored vegetable
crops. The disease is characterized by watery,
imelly, soft decay of storage tissues. The
)acterium is common in soils and normally
nvades plants through injuries from harvesting,
freezing. Insects make plants susceptible to
attack. Soft rot infection, if not checked during
torage, results to heavy losses, which may
-each more than fifty percent.
The main characteristic ofP carotovorum
;ubsp. carotovorum is its ability to produce
arge quantities of pectic enzymes
Daengsubha, 1974). The Erwinias differ in
nly a few biochemical properties. However,
hese properties were distinctive enough so that
nost strains can be identified into species. In
he same report of Daengsubha (1974) on an
extensivee examination of rooted vegetables
collected from 19 towns and 20 cities across
he country, he identified five species of soft
ot bacteria. The predominant-isolate was that
f E. carotovora subsp. carotovora.
One of the key areas to consider in
preventing and controlling soft rot disease is
he use non-harmful chemicals and
)otanicals that may possess antibacterial
properties. Extensive studies on the pesticidal
properties of various plant extracts have
ieen done. For instance, Singh et al. (1993)
toted the presence of antibacterial activity
n higher plants. Rao et al. (1989) have
observed that the in vitro potency ofthe volatile
3il of Limnophilla gratissima was similar to
;hat of streptomycin With the aim of
developing a disease control strategy that
is effective, environment friendly, and
cost efficient, this study was done to screen
plant extracts that are inhibitory to the
bacterial soft-rot pathogen and to identify the
)est treatment for post-harvest control of
cabbage of soft-rot.
MATERIALS AND METHODS
Source of Inoculum. A pure culture of
carotovorum subs. carotovorum was
obtainedd from the Department of Plant
'athology, University of the Philippines Los
3afios, College, Laguna. The pure culture
vas grown in Potato Dextrose Peptone
%gar (PDPA) slants for 24 hours and the
)acterial suspension was inoculated
iseptically on cabbage core slices to test its
)athogenicity. These inoculated slices were
)laced on sterile Petri dishes lined with
noist and sterile filter paper. After 24 hours
)f incubation, a loopful of infected section
vas suspended in sterile distilled water. The
suspension was shaken in a vortex mixer.
rhen a loopful of the suspension was
treaked on plated PDPA. The plates were
ncubated for 48 hours. Single, circular,
md milky colonies were transferred to PDPA
plants. These pure cultures served as the
source of inoculum for the succeeding
Bio-assay of Plant Extracts for
3actericidal Activity. Thirty seven plants
mnown to have bactericidal properties
Quisumbing, 1978) were evaluated for
possible inhibitory effects against
Scarotovorum subs. carotovorum. These
vere as follows:
64 Post-harvest prevention and control of bacteria soft rot of cabbage
6. Sweet potato
10. Damong Maria
14. Four fingers
34. Siling labuyo
35. Tubang bakod
Premna odorata L.
Muntigia calabura L.
Cassia alata L.
Ficus elastica N.
Opuntia ficus indica
Ipomea batatas L.
Manihot esculenta Crantz
Allium schoerropassum L.
Anacardium occidentale L.
Artimisia vulgaris L.
Taraxacum officinale L.
Syzigium cumini L.
Eucalyptus globulus L.
Schefflera odorata M.
Allium sativum L.
Psidium guajava L.
Mentha arvensis L.
Portulaca oleracea L.
Bryophyllum pinnatum L.
Tagetes erecta L.
Coleus scutellarioides L.
Coleus amboinicus Lour.
Allium cepa L.
Carica papaya L.
Aloe Barnadensis M.
Blumea balsamifera L.
Capsicum frutescens L.
Jatropha curcas L.
Amaranthus spinosus L.
The plant extracts were prepared by
cutting the plant parts to be used into small
pieces, surface disinfected in 20% chlorox
solution for ten minutes and rinsed three times
in sterile distilled water. For every 50 g of
plant sample, 25 ml of 95% ethyl alcohol was
added and osterized in a blender. The
inoculated tissues were then filtered and
assayed against the test pathogen. This was
done using the paper disc method as follows:
Plates with PDPA medium were seeded with
the test pathogen and allowed to dry. Filter
paper discs were immersed into the plant
extract and placed equidistantly on the agar
V.R. Mamaril and F.C. Abarquez Volume 36 June-December 2000 65
plates previously seeded with P carotovorum
subs. carotovorum. Four discs were placed
in each plate, with four plates per treatment.
The plates were incubated at room temperature
and the zone of inhibition was measured after
24 hours. The data were analyzed statistically
using one-way ANOVA and treatment means
were compared using DMRT at 0.05 level.
Preliminary Assay for Preventive and
Eradicative Properties of Selected Plant
Extracts. The seven plant extracts that
showed inhibitory effects against the bacterial
soft rot pathogen were further tested for their
preventive and eradicative properties. These
were compared with streptomycin sulfate and
alum. Ethyl alcohol and sterile distilled water
served as control. The 20% alum was prepared
by dissolving 20 grams of alum powder in 100
ml of sterile distilled water. The streptomycin
solution was prepared by dissolving one gram
of material in 100 ml sterile water. The final
treatments were as follows:
T, Katakataka T7 Garlic
T2 Chives T8 Alum
T3 Eucalyptus T9 Streptomycin
T4 Duhat T,1 Ethyl alcohol
T, Sweet potato T1, Distilled water
In the preventive treatment, cabbage
cores and carrot slices were first immersed
in the plant extracts for 30 minutes, allowed to
air dry in sterile filter paper and inoculated
with 0.5 ml of bacterial suspension using a
syringe. The cabbage core and carrot slices
were then incubated in a moist chamber for
48 hours and evaluated for soft rot severity
using the following scale:
In the eradicative treatment, the cabbage
cores and carrot slices were first dipped into
the bacterial suspension for twenty minutes and
dried on sterile filter paper. After 16 hours.
they were immersed in the various treatments
for 30 minutes, dried and incubated in a moist
chamber. Soft rot severity was evaluated
following the same rating scale described
Post-Harvest Experiment. Newly
harvested cabbages (variety Scorpio) were
purchased from a farm in Nueva Ecija. They
were inspected and made sure that samples
were free of bacterial softrot symptoms. The
cabbages were transported to Manila. Extra
care was taken to prevent transport damage
that may predispose the products to softrot
infection. After 24 hours, the cabbages were
trimmed of the outer or wrapper leaves and
used in succeeding tests.
a. Preventive Properties of Plant
Extracts. Alum and streptomycin solutions
and the plant extracts were prepared as
described previously. The butt ends (bottom)
of the cabbage heads were surface sterilized
in 20% chlorox solution for ten minutes and
rinsed with sterile distilled water. Then the
cabbage bottoms were pricked (twenty pricks)
and dipped in the various treatments for twenty
minutes. The treated cabbages were then dried
and individually packed in perforated plastic
bags. After 24 hours, the same cabbage butts
were dipped in bacterial suspension for twenty
minutes, dried, and packed in the plastic bags.
Degree of infection on the surface of the butt
ends was recorded daily for five days. After
the fifth day, the cabbages were cut longitudally
Rate of Infection
80-100% percent Infection
V.R. Mamaril and F.C. Abarquez
Volume 36 June-December 2000 65
to check mternal alsease damage anu uegre
of infection was assessed following the ratin
scale described previously.
b. Eradicative or Curative Propertie
of Plant Extracts. The same surface
sterilization technique was followed on th
cabbage heads. The butt ends were price
(20 times) then dipped in bacterial suspension
for twenty minutes, dried, and packe
individually in perforated plastic bags. Aft(
12 hours, the end butts were dipped in tl
treatments for twenty minutes, dried, an
packed individually in the plastic bags. Intensity
of infection was recorded daily for five day:
On the fifth day, the cabbages were ct
longitudally to check internal infection. For a
post-harvest treatments, five cabbage
represent one replicate. The data wei
analyzed statistically using one-way ANOV
and treatment means were differentiated using
Duncan's Multiple Range Test at 0.05 alpf
Bioassay of Antibacterial Properties ol
Out of thirty-seven plants evaluated, onl
seven were found to have extracts that ai
inhibitory to P carotovorum sub
carotovorum. This was shown by tl
inhibition zone produced around the filter pap,
disc after 24 hours of incubation (Table 1
Chive was the most effective with inhibitic
zone of 10.34 mm. It was even more effectil
than 1% streptomycin with inhibition zone i
only 8.8 mm. The second most effective w;
garlic extract (6.8 mm), followed by eucalypti
(5.0 mm) and onion (4.0 mm). Katakatal
(3.6 mm) and duhat (3.4 mm) extracts we
as effective as 20% alum solution (3.6 mir
The least effective was that of sweet pota
Assay lU rJevenllLlvc aiu iUmiu auraLv;
Properties of Selected Plant Extracts
Since identical results were obtained i
cabbage cores and carrot slices, only cabbaj
cores were used in subsequent observation
Visual observations after 48 hours incubatic
showed that no bacterial softrot infectic
developed on cabbage cores and carrot slice
treated with chives, duhat, eucalyptus, garl
and katakataka extracts applied both ,
preventive and curative treatments (Table 2
These were comparable with 1% streptomyci
Slight bacterial softrot infection developed (
cabbage cores and carrot slices treated wi
onion extracts. Alum and sweet potato extra
treated samples had moderate soft rot infection
Untreated samples (sterile water only) h,
severe soft rot infection. Onion and swe
potato extracts were excluded in subseque
PreventiveTtreatment. Only extrac
of katakataka and garlic were comparable
1% streptomycin in preventing softrot infectic
up to 120 hours of storage (very effect
rating). Chives, duhat and eucalyptus extra
had identical effectiveness in preventing sc
rot infection (Table 3), with ratings of ve
effective up to 72 hours and effective up
120 hours based on the rating scale describe
previously. Alum (20%) was effective on
up to 72 hours. Even ethyl alcohol was qui
effective up to 48 hours.
Eradicative or Curative Treatmer
When the plant extracts and other compound
were applied 16 hours after inoculation with
carotovorum subs. carotovorum, only thr
(duhat, eucalyptus and garlic) out of five pla
extracts tested were found very effective
controlling cabbage softrot up to 120 hours
storage (Table 4). Chive extract was effect
only up to 96 hours while katakataka w
MmaIIrld adnu r.L,. mUdaiquIL E.un.. u uuIrU- nwuLm. -v- -
tective only up to 4 nlours. Alum (ZUV/o)
as effective up to 72 hours. The streptomycin
%) treatment, which served as basis for
amparison, was consistently effective up to
10 hours of storage. Cabbage heads treated
ith ethyl alcohol and the untreated control
terile water only) had already an average
)ft rot infection of 40-60% after 48 hour
ised on the rating scale described previously.
This study shows that there are few plants
ith extracts that have great potential in
jntrolling bacterial soft rot caused by
carotovorum subsp. carotovorum.
Katakataka extract appears to be most
fective when applied as a protectant and
ras able to suppress soft rot infection up to
ve days, similar to the effect of 1%
reptomycin. However, it was not effective
s a curative treatment because its effect
ras only good for 48 hours. Chive extract,
rhich was the most promising during the
litial bioassay with growth inhibition zone
greater than that of 1% streptomycin, did not
ve up to expectation as a post harvest
urative treatment. Duhat, eucalyptus, and
arlic extracts were effective both as
protective and curative treatments.
The bactericidal properties of these plant
extracts may be attributed to the various
chemicals produced by the plants. For example,
uisumbing (1978) cited the findings of
Vesmer that Eucalyptus globulus yield from
.Ito 1.96% d-a-pinene, camphene, fenchene,
Idehydes valericc, butyric, capronic),
Icohols (ethyl, amyl, isoamyl), acetic acid,
ymol, sesquiterpene, eudesmol, and
pinacarveol. Eucalyptus oil is extensively used
n medicine and is highly esteemed as a
valuable remedy of certain illnesses. He also
mentioned that the active principle in garlic is
e volatue on wmcn ranges ioum u.uvv-
009%. In addition, the bulbs contain allicin,
lisin, allyl disulphide, allyl propyldisulphide,
ulin, etc. Chive is an excellent source of
acium, phosphorous and iron. Katakataka
aves are used as an astringent, antiseptic
id counter irritant against poisonous insect
tes. The effectiveness of alum solution in
)ntrolling cabbage soft rot has been attributed
Sthe effects of AP1 or low pH (4.2) on the
icterial pathogen (Henderson and Easton,
The three plant extracts (duhat, eucalyptus
id garlic) appears to be more effective than
lum solution (15%) recommended by
orromeo and Ilag (1984) as a post harvest
)ntrol of cabbage soft rot. However, one of
te drawbacks in the use of the crude leaf
tracts of duhat and eucalyptus is the -reenish
iscoloration or residue left on the treated
cabbage. This could be overcome by improving
ie extraction procedure to remove the green
igments prior to application or by simply
imming off the discolored portion. The use
f garlic extract needs further study in the light
f fluctuating market price that sometimes the
Dst of treatment is prohibitive and ofthe strong
arlic odor emitted after treatment.
The effectiveness of 1% streptomycin
against bacterial soft rot is expected because
:is a strong broad spectrum antibiotic. It was
nly used inthis study as basis for comparison.
t's used on crops like cabbage which is
sometimes eaten raw, to control bacterial plant
athogens, is discouraged to minimize the risk
f resistance development in bacterial
athogens infecting animals and humans.
These plant extracts with protective and
radicative properties need to be studied
further, subjecting them to more rigorous tests,
nd should include cost-benefit analysis so that
farmers, traders and consumers will be assured
68 id control of bacteria soft rot of cabba
of a technology that is environment friendly, s,
and economical to use in reducing post harv
losses due to bacterial soft rot.
BORROMEO ES. 1983. Characterization andpc
harvest control of Erwinia carotovora sub
carotovora (Jones) infecting cabbage (Brass,
oleracea var. capitata L.) Unpublished Ph.
Dissertation, University of the Philippines I
Bafios, College, Laguna
DAENGSUBHAW. 1974. Vegetable soft-i
bacteria in the Philippines. Unpublished Ph.
Dissertation, University of the Philippines I
Bafios, College, Laguna
DEANS SG, WATERMAN PG. 1993. Biologil
activity of volatile oil. Volatile Oil Crol
Longman Scientific and Technical Book.
JOHNSON D. 1930. The relation of cabba
maggot and other insects to the spread a
development of soft-rot of crucife:
Phytopathology 20: 857-872
HENDERSON D, EASTONRG. 1980. Stingose:
new and effective treatment for bites a
stings. Med. Jour. Aust. 67: 146-150 (Abst
KIKOMOTO T. 1980. Ecological aspects ofthe sc
rot bacteria. Rep. Inst. Agr. Res. Taha
University, Japan, 31: 19-41.
est MENELEY JC, STANGHELLINIME. 197:
Occurrence and significance of soft-rottir
bacteria in healthy vegetable;
Phytopathology 62: 1325-1327
PEROMBELON MCM. 1972. Aquantitatii
method for assessing virulence of Erwini
carotovoravar. carotovora and E. carotovoi
var. atroseptica and susceptibility to rottir
D. of potato tuber tissues, pp.299-303. In H
Maas Geestermus (Ed.) Proc. Int Conf. Plai
S Pathol Bacteriol, 3'd Wageningen, Ti
Netherlands: Cent Agr. Pub. Doc. PUDO(
D. QUISUMBING E. 1978. Medicinal plants oftl
Philippines. Quezon City, Philippines: JM
al SINGH, PJ, BEGUM JU, CHOWDHURY I
)s. ANWARMN. 1993. Antibacterial activil
of some higher plants in Chittagon
gUniversity campus. Chittagong Universit
nd science, Bangladesh.
TAMIMI KM, BANFIELD WM. 1965
Transmission of bacterial soft-rot bacteria
A fruit flies. Phytopathology 59: 403 (Abstr.)
S TSUYAMA H. 1978. Seasonal variation in td
population of soft-rot bacteria (Erwini
carotovora var. carotovora) on the lee
t-. surface of tobacco, pp.787-793. In Proc. In
Conf. Plant Pathol. 4th, Angers 1978 ed., Sti
Pathol. Veg. Phytobacetrial, 97
Pathol. Veg. Phytobacetrial, INRA. 978pp.
id control of bacteria soft rot of cabba
V.R. Mamaril and F.C. Abarquez Volume 36 June-December 2000 69
Table 1. Plant extracts with inhibitory effects aga
'Treatment means followed by the same letters are not signit
Table 2. Degree of infection on cabbage core slic
Streptomycin No Bacterial Infection
Katakataka No Bacterial Infection
Chives No Bacterial Infection
Eucalyptus No Bacterial Infection
Duhat No Bacterial Infection
Sweet Potato Moderate Bacterial Infe
Onion Very Slight Bacterial Ir
Garlic No Bacterial Infection
Alum Moderate Bacterial Infe
Distilled Water Full Bacterial Contamiu
t P. carotovorum subsp. carotovorum.
Average Inhibition Zone '(m)
itly different from each other using DMRT at 0.05 level of
48 hours after treatment.
No Bacterial Infection
No Bacterial Infection
No Bacterial Infection
No Bacterial Infection
No Bacterial Infection
on Moderate Bacterial Infection
;tion Slight bacterial infection
No Bacterial Infection
on Moderate Bacterial Infection
ion Full Bacterial Contamination
7A Pfsy-narvest Oh-eveIRIU 4 aim GuuIuUI U1 UaU.LCIIa *JUk -I ... Id
Table 3. Soft rot rating of cabbage heads aj
extracts as preventive treatment.
Streptomycin 1.0 b
Katakataka 1.0 b
Distilled Water 3.0"
Ethyl Alcohol 1.3 b
CV (%) 15.27
'In a column, means followed by the same letters are not si
Table 4. Soft rot rating of cabbage heads ai
extracts as curative treatment.
Chives 1.0 b
Eucalyptus 1.0 b
Distilled Water 3.0"
Ethyl Alcohol 1.3"
CV (%) 15.27
'In a column, means followed by the same letters are not si
different time intervals after application of plant
Mean Degree of Infection*
72 Hours 96 Hours 120 Hours
1.0 1.0 1.0
1.01 2.0b 2.0
1.0d 1.0 1.3
1.0d 1.0 1.0"
1.0d 2.0b 2.0 d
1.0d 2.0b 2.0d
4.7a 5.0 5.0"
1.7 4.3" 5.0
2.3b 5.0a 5.0a
20.3 18.57 21.4
[icantly different from each other using DMRT at 0.05 level ol
different time intervals after application of plan
Mean Degree of Infection*
72 Hours 96 Hours 120 Hours
1.0 d 1.0 d 1.0
1.0d 2.0bc 3.0b
1.0 d 1.3 cd 1.7 b
3.0b 5.0 5.0
1.0 d 1.3 1.7 b
1.0 d 1.3 1.7 b
4.7 5.0a 5.0
1.7" 2.7b 4.0a
3.7b 5.0a 5.0a
20.3 18.57 21.4
icantly different from each other using DMRT at 0.05 level o
A CTID A -rrT f1V DA PD1IC WD 'rTi ' TT DDTDTML Dr VTUIDArTCY1T r('t"_ A I
0."%-/XJPIL 1 JX All LJimll "1. .. V KJ.UI1." A
ANNUAL SCIENTIFIC CONVENTION
COUNCIL OF THE PHILIPPINES,
Atection of papaya ringspot virus (PRSV) f
Southern Mindaonao. LE Herradura, ('
J. Magnaye and NB Bajet. Bureau of Plant k
Justry, Davao City and Department of Plant S
thology, UPLB, College, Laguna S
The occurrence of papaya ringspot
tyvirus is recorded for the first time in two d
micipalities in South Cotabato, namely; ri
ilomolok and Tupi. Symptoms observed on d
- leaves collected from a solo papaya ti
mntation at Silway 8, Polomolok, South p
atabato last Oct. 1999 were mild mottle on
ung leaves and leaf distortion. Oily streaks
,re also observed on some of the petioles fi
>m the sample trees infected. Symptoms p
agressed three months thereafter wherein o
y streaks on the petioles were very severe
d water soaked spots were observed on the
mts. On fruit, ringspot symptoms can be a
served on green fruits. Reduction and 3
lorosis of leaves were also observed. The F
mptoms observed on the leaves and fruits P
: typical of the characteristic symptoms of 1
ELISA tests conducted separately at BPI- s
iCRDC and at the Department of Plant
thology, UP Los Bafios on papaya leaf
mples gave positive results using PRSV-LB a
tisera from UP Los Bafios. The virus was fi
t detected on the papaya leaf samples from p
ilomolok when a parallel test was conducted s
BPI using PRSV atisera from Japan ii
tainted from Dr. Maoka, Okinawa, Japan, but 1
ve positive result on the positive controls 2
lich were PRSV-infected leaves from Los Bi
11 .31-" AAINVI VJIKiAKY AfNI)
OF THE PEST MANAGEMENT
IAGUIO CITY, MAY 3-6,2000
resistance studies on sweet pepper
'apsicum anuum L.) against the root-
lot nematode (Meloidogyne incognita A.).
Ruelo. Biology Department, University of
n Carlos, Cebu City.
Resistance is an ideal way to control
eases, if satisfactory levels of durable
distancee can be incorporated into culturally
sirable crop plants. Resistant varieties save
ie, effort, and money otherwise spent fighting
Three investigations are herein reported
)m the Sweet and Hot Pepper Resistance
oject which started in 1994 and is still going-
on this day.
The investigation delved into the primary
d confirmatory screening for resistance of
sweet pepper varieties of M incognita.
ur were found immune (PBC163, PBC579,
3C 631, PBC 692); four highly resistant (PBC
4, PBC 431, PBC 575, PBC 578); two
ghtly resistant (PBC 435, COO 505); all the
st were either moderately resistant,
sceptible or highly susceptible.
Two other experiments were conducted
the same time to evaluate the effects of the
[lowing on the growth and yield of sweet
pper varieties, immune PBC 631 and highly
sceptible California Wonder, which were
)culated with the test root-knot nematode:
Neem (Azadirachta indica) seed powder,
Nematode-tapping fungus (Paecilomuces
acinus), 3) Insecticidal bacteria (Bacillus
iringiensis), 4) Vermicompost produced by
I t 1 1 f .,7 /on nl _
Hostathion, b) lemiK (to compare wr
The immune characteristics of the varie
PBC 631 were adequate to ward off the roc
knot nematode. Any addition of biological at
chemical treatments to control the nematod
would be superfluous.
Although highly significant results we
observed in almost all of the parameters
growth and yield used for evaluation, 1
consistent pattern emerged with the resista
sweet pepper variety (PBC 631) as host plain
For instance, shoot growth was great
increased by Neem (324 g) and P lilacus (3:
g) but decreased by Hostathion (200.3 g); re
growth was greatly improved by vermicompc
(212.2 g) but decreased by B. thuringien
(123 g) andthe control (128 g). Best bioma
development was seen among vermicompo:
treated plants (98 g) but negatively affect
by the control plants (57 g), B. thuringien.
(58 g) and Hustathion (59 g) treatments.
Yield as seen through the fruit weig
parameter was positively increased by Tenr
(961 g) but negatively affected by conti
No significant differences amor
treatments compared with the control we
seen in two parameters to assess yield name]
growth in height and fruit number. Numerica
though P lilacanus has the tendency
increase plant height and Temik, the fn
number, but these were not statistical
With California Wonder, a high
susceptible sweet pepper variety as a hc
plant, a consistent pattern was observe
Hostathion greatly decreased gall format
(143), nematode population in roots (176), a
egg mass (84). Similarly egg mass was kc
untreated control plants had the uhghest coun
(1320, 1668 and 523), respectively.
Plant growth and development we
greatly improved by vermicompost but great
decreased in the control and by one or the oth
treatments: plant growth in height (79.4 ci
control = 57.2 g); shoot growth (285 g, contr
= 175g) root growth (165 g, reduced by Tem
= 124 g and control 127.2 g); biomass (92.4
control = 37g).
Yield was best seen with Neem in terry
of fruit weight by Neem (997 g) ai
vermicompost (990.4 g, decreased by t]
control 2555 g).
Perhaps this is the first report <
vermicompost being able to improve pla
growth and development of the high
susceptible sweet pepper variety Californ
Wonder and a resistant variety PBC 631,
terms of shoot and root growth. Neem is t
treatment used to improve positive yields
California Wonder. To effect good control
IA' incognita on a highly susceptible ho
Hostathion is the best to use. However wi
PBC 631, its resistant traits would be adequa
Rhizoctonia disease of salago and i
control. NG Tangonan, VM Escopala
USM, Kabacan, Cotabato
Rhizoctonia disease of salago caused 1
R. solani Khunn is reported for the first tin
The pathogen infected all stages of growth
salago. It causes damping-off of seedling, le
and twig blights. In seedlings, symptoms a
characterized by girdling, discoloration oft
vascular tissue, rotting of the base, and toppli
over. In mature plants, symptoms a
characterized by blighting and drying up
leaves. Affected twigs dry-up, leaves fall-(
and dieback. Whitish mycelia and abunda
sclerotia were observed on the infected twi
iracteristic symptoms 5-7 days after
culation. Nine fungicides were found
active in suppressing the growth ofR. solani J
vitro. Five were found effective in vivo
en applied as protectant and eradicant.
:ewise, six fungal biocontrol agents showed
agonistic effects against R solani in vitro. I
preening corn for resistance to Philippine
-n downy mildew. NG Tangonan, FD
ambot. USM, Kabacan, Cotabato.
Promising inbred lines, open-pollinated
rieties (OPVs), and corn hybrids from
MMYT in Mexico and from the germplasm 1
election of USM were screened in the field
identify, select and develop corn materials
h resistance to the Philippine downy mildew
M) disease. Out of the 2,236 entries/lines
eened, 56 were USM materials and 2,180
refromCIMMYT. It was noted that 1,802
ries/lines or 85.12% showed resistance to
4. Two hundred-thirty-two or 10.9%
awed intermediate reaction and 141 or
i6% exhibited susceptible reaction. The
ing scale used is the standard adopted by
ilippine downy mildew researchers.
mntrol of Phytophthora disease and
iizoctonia leaf blight of durian. NG
ngonan, CO Gonzales. USM, Kabacan,
An in vitro bioassay test of several
ngicides and augmentative biological control
ngi against Phytophtora palmivora and
lizoctonia solani causing diseases of durian
is conducted. Four Trichoderma species
d Rhizopus sp. were found antagonistic to
Stwo fungal pathogens. Effective treatments
:re further tested in the field. Fosetyl-Al
liette), metalaxy (Ridomil 25 WP), copper
droxide (funguran OH and Kocide DF), and
mcozeb (Dithane M45 and Manzate 200)
owed effectiveness both as protectant and
lophanate-metyl (topsm) ana triaaimeron
ayleton) showed effective control against
solani in both protective and therapeutic
ntrol. T. harzianum showed higher
:agonistic ability over P palmivora in durian
ten applied as protectant and theraputant.
i the other hand, all four species of
ichoderma were found effective as
otectant but only T harzianum and other
ecies showed further effectiveness when
plied as therapeutant against R. solani.
iltural control including proper spacing,
ning, thinning, water management, fertilizer
plication, crop rotation and intercropping, and
:chanical control by weeding (ring or slash)
;re also studied in relation to durian disease
cidence of Ralstonia solanacearum in
itato tubers collected in Bukidnon. MP
natural and EM Jover. UPLB, College,
By streak plating on media, R.
lanacearum was detected in 93 out of 234
bers. The isolates were confirmed by
persensitivity test in tobacco and for some
plates by PCR using primers 759/760.
venty, 40, 60 and 80% incidence were
served in 3, 27, 10 and 2 tuber samples,
spectively. The rest (52%) had less than
)% infection. Result of this study confirmed
ports that potato seeds in Bukidnon are
ghly contaminated with the bacterial
ganism. This could be the primary reason
hy incidence of bacterial wilt of potato in the
gion is very high despite the fact that potato
as introduced in some areas less than 5 years
go. The practice of farmers to keep their
vn seed pieces for the next crop could have
read the disease to new areas of production.
After planting 120 tubers in sterile soil,
any tubers rotted. Some plants were still
:ry small despite the fact that they were
r 1 i 1 ,L___~__~~L___L n --IIY;.IYI
planted almost at the same time. Some plants
have wilted from tubers that were highly
contaminated with the bacterium.
Characterization of the bacterium causing
soft rot of Phalaenopsis leaves. JL Carilo
and MP Natural. UPLB, College, Laguna
The causal bacterium was isolated from
Phalaenopsis leaves showing soft rot. The rot
is internal because the epidermal cell layer is
left contact unless pressure is applied. Once
broken, a dirty white liquid oozes out. Koch's
postulate was carried out and the purified
bacterium was used for characterization.
Morphological, cultural and physiological
characteristics revealed that the causal
bacterium is Pectobacterium carotovorum
sbsp. carotovorum (Jones 1901) Hauben et
al., 1999, formerly Erwinia carotovora
(Jonesl901) Bergey et al., 1923.
Field evaluation of Frowncide 50 SC
against major diseases of potato and
onion. RM Labadan, SP Milagrosa, and
Potato: There was a slight difference in
the result of two studies. Results at BSU
experimental farm revealed no significant
differences among the fungicidal treatments
as shown by the statistically similar PLB
infection rating. Curzate M and Daconil 75
WP-sprayed plants have numerically lower
disease rating than froncide sprayed plants.
Furthermore, plants sprayed with Curzate and
Daconil yielded more tubers than Frowncide
sprayed potatoes but was statistically similar.
Trials in Pico revealed that potato late blight
was effectively controlled by Frowncide
flowable compared with Curzate M and
Daconil 75WP. The disease rating was
significantly lowered by Frowncide, thus yield
was significantly higher than plants sprayed
Meteorological conditions during the
conduct of the studies favored plant growth as
well as the development and spread of the
diseases. No phytotoxic effect of Frowncide
op potato plant was observed.
Onion: Two field experiments for onion
were conducted at the experimental station of
the Research, Extension and Training Center,
CLSU and in farmers' fields in Barangay
Caanawan, San Jose City from November 1994
to April 1995. The objective of the study was
to determine and establish the efficacy of
Frowncide 50 SC against major onion diseases.
Three rates of Frowncide 50 SC were used
namely: 150 g ai/ha, 200g ai/ha and 250 g ai/
ha. Daconil 75 WP, at 1500g ai/ha, served as
the standard fungicide and control.
The percent leaf area infected by purple
blotch and bulb rot diseases was significantly
affected by the fungicides evaluated in both
locations. Frowncide 50 SC at 250 g ai/ha was
significantly effective in reducing purple blotch
infections. The efficacy of lower rates of
Frowncide 50 SC 150 g ai/ha and 200 g ai/ha
was comparable to the standard fungicide.
Similar results were obtained in bulb yield,
wherein plots sprayed with Frowncide 50 SC
at 250 g ai/ha produced plants which gave the
highest yield compared to other treatments.
Factors affecting the occurrence of rice
tungro disease epidemic in the
Philippines: Implications on disease
management. RC Cabunagan, N Castilla
and O Azzam. IRRI, Los Banos, Laguna.
A survey was conducted in farmers' field
inthe Philippines from 1995 to 1997. The sites
covered were Isabela, Nueva Ecija, North
Cotabato, Bohol and the Bicol Region. Tungro
disease incidence was assessed visually based
collected and indexed serologically by ELISA
For the presence of the rice tungro bacilliform
(RTBV) and rice tungro spherical (RTSV)
viruses. The relationship between production
environment variables (ecosystem, synchrony
f planting, and variety) and disease variables
[visual incidence, double RTBV and RTSV,
single RTBV, and single RTSV) was examined.
This study showed the interaction between
variety and svnchronv of Dlantine and
incidence is not only associated with high
double RTBV and RTSV infection, but also
vith high single RTSV infection. Without
serology, the importance of RTSV-infected
plants, which did not exhibit symptoms but
contributed to high overall tungro incidence,
would have been overlooked.
The results show that if the bacterial in rice-onion cropping system.
crown gall in the roses is not managed Gergon, R Gapasin, 0 Opina, J Halbr,
properly, the bacterial pathogen may spread PhilRice, Nueva Ecija, ViSCA, Baybay, I
to a nearby orchard or nursery where UPLB, College, Laguna, Pennsylvania
susceptible trees are grown. University & Ohio State University, US,
Confirmatory test of the effectiveness of Rice Hull Burning (RHB) is a tradit
various orchidaceous mycorrhizal fungi cultural practice of many onion growers i
(OMF) on in vitro-cultured orchids. MB Jose City, Nueva Ecija, Philippines main]
Brown. BIOTECH, UPLB, College, Laguna weed control and yield increase. The rest
carbonized rice hull is incorporated into th
Several Ascomycetous and Basidio- during land preparation before transplant
mycetous fungi were screened for their onion. Studies to evaluate the effect of '
effectiveness in enhancing the growth of in on rice root-knot nematode, Meloido
vitro-cultured orchids. The fungi were in the graminicola, and onion yields were condi
form of finely chopped fruiting bodies, mycelia in farmer's fields naturally infested b,
and sclerotial bodies. pathogen. Fifteen cm-thick rice hull
sufficient to reduce nematodes population
In pot experiments, mycelial fragments and the soil. The effect of heat from burning
sclerotial bodies were placed near the roots of hull on the nematodes reached up to 3(
orchid seedlings while finely chopped fruiting deep. The effect of deep plowing on nerm
bodies were incorporated with the potting population was insignificant in comparison
medium consisting of charcoal and chopped standard plowing. Increasing thickness of]
fern hips. Inoculation was done during gave a significant contribution to increa.
repotting. onion yield and production of bulbs for ex
Plots that received 30 cm-thick rice hull
Five out of 10 genera with finely-chopped 27% more large bulbs than 15 cm-thick
fruiting bodies and nine out of 37, five out of and 40% more than no RHB. Thirty m
35 isolates in mycelial form were found thick rice hull gave a yield advantage of
effective as growth enhancer of Dendrobium. over RHB and 11% cm-thick hull.
The genera of inoculants in the form of finely
chopped fruiting bodies that were found Efficacy of using hypoviru
effective were identified as Trametes, Rhizoctonia sp. to control banded leaf
Lenzites, Ganoderma, Coriolus, and Fomes. sheath blight in corn. CB Pascual,
Two of the mycelial inoculants were sclerotial Raymundo and M Hyakumachi. IPB
forms and these were identified to be Department of Plant Pathology, UP
Rhizoctonia and Sclerotium. The seven College, Laguna; Gifu University, Gifu, J,
effective isolates in mycelial form were still
unidentified due to their inability to form s Hypovirulent binucleate Rhizoctonia
PYulll cnnrip ^Y or- C -r- DL.'7 -__ .. A - --_1 1 1.
Abstract Volume 36 June-December 2000 77
susceptibility to the disease and delayed onset while all the C. papaya plants did. Finally, a
of BLSB. plate trapped antigen-ELISA confirmed that
the virus was not present in the 34 hybrids and
Corn plants inoculated with virulent RS- C. cauliflora plants but present in C. papaya
35 isolate near Rhv7-inoculated site had smaller plants. Taken together, these three tests
lesion size than leafsheath inoculated further strongly suggested that all the C. papaya
from Rhv7. There was a reduced mycelial x C. cauliflora hybrid plants are resistant to
growth in the presence of Rhv. PRSV and that this resistance was inherited
from C. cauliflora.
Breeding for resistance to papaya ringspot
virus (PRSV). PM Magdalita, RA Drew Control of damping-off and growth
and SW Adkins. IPB, UPLB, College, enhancement of pechay ( Brassica
Laguna, Griffifth University, University of pekinensis) seedlings by Trichoderma. VC
Queensland, Australia. Cuevas, AM Sinohin and JI Orajay. UPLB,
Interspecific hybridization between Carica
papaya and the resistant wild species, Carica Three species of Trichoderma were
cauliflora Jacq. was conducted to create tested for their efficacy in controlling damping-
hybrids containing resistance to PRSV. Two off of pechay. These were T. parceramosum,
thousand one hundred hybrid embryos (4.8%) T pseudokoningii and T. harzianum (UV
from 43,736 seeds were isolated 90 to 120 days irridiated). Test sites were established in Rizal
after pollination. The abortive hybrid embryos and UPLB, Laguna. Preparation of biocon
were germinated for five days only onto a agents followed the procedure developed by
medium containing 0.5-strength De Fossard Cumagun and Lapis (1993).
nutrients, plus BAP (0.25 uM), NAA (0.25
uM), GA, (10 uM), sucrose (58 uM) and agar No significant differences were detected
(8 g L-1). Using this embryo culture protocol, among the three species of Trichoderma for
resistance to PRSV in C. papaya x C. both germination and seedling survival. All of
quercifolia and in C. pubescens x C. them, however, were significantly higher than
parviflora was also developed. One thousand the control.
nine hundred eighty-one C. papaya x C.
cauliflora hybrid embryos were germinated Trichoderma-treated plots resulted to
and 485 hybrid plants were produced 73.45% and 80% control of pre-emergence
successfully. The number of main leaf veins damping-offand seedling survival, respectively.
and the leaf margin were found intermediate The control plots yielded only 14% survival.
between C. papaya and C. cauliflora, hence
these were used as morphological markers of Growth enhancement trials showed that
hybridity. Further analysis ofthe putative hybrid T. parceramosum significantly increased
plants by RAPD was undertaken to confirm height and dry weight of seedlings by 65.73%
hybridity. A pooled analysis of the RAPD and 100%, respectively. T harzianum-treated
bands consistently produced by five primers seedlings followed, increasing height and
indicated that all 120 putative hybrids analyzed weight by 53% and 84%, respectively. T
were genetic hybrids of C. papaya x C. pseudokoningii also increased growth and
cauliflora. Manual inoculation and field test was significant compared to the control but at
indicated that 34 hybrids and C. cauliflora lesser extent compared to the other two
plants did not become infected by the virus, species.
Cross infection and cultural variation in rice-based system. On the other hand, crops
Rhizoctonia solani Kuhn anastomosis harboring AGs other than AG -1A can be used
groups and implications to rice sheath in these cropping systems, as these AGs are
blight development and control. J. nonpathogenic to both rice and corn. In other
Tagubase and AD Raymundo. UPLB, cropping systems not involving either rice or
College, Laguna. corn, it appeared that a sequential scheme such
as that involving mungbean and cabbage is
The cross infection potential and cultural feasible.
variation of ten anastomosis groups of
Rhizoctonia solani Kuhn were determined in Genetic and aggressiveness variability of
relation to disease development in selected Ralstonia solanacearum affecting tomato
crops and possible implications to sheath in the Philippines. NL Opina, RG
blight development and control by cultural Maghirang, CA Relevante and AK
methods. Growth rate ofmycelia, in terms of Raymundo. IPB and IBS, UPLB, College,
the slope in a linear regression analysis, in all Laguna.
anastomosis groups were high with AG3 and
AG-BI showing the highest and least values, Atotal of 83 tomato isolates obtained from
respectively one week after mycelial seeding. different provinces were isolated and classified
Only AG1-1A, AGI-1B, AG3, AG7, and AG- into biovars. Of these, 70 isolates (84%) were
BI formed sclerotial bodies. Sclerotial bodies biovar 3 while 13 were biovar 4. The DNA of
were observed in the first AGs four days after each isolate was extracted and the DNA
mycelial seeding while those in AG-BI were ranges from 50-2000 mg/pl. The amplification
seen one month after seeding. A correlation of the genomic DNA using RAPD, ERIC,
was observed between the formation of BOX and REP primers showed a characteristic
sclerotia and the pathogenicity of the set of PCR products containing few to many
aforementioned AGs except for AG-BI which fragments of different molecular sizes and
turned out to be weakly pathogenic to all tested staining intensity between and within the
crops. All non-sclerotial forming AGs were biovar isolates. Among the primers tested
weakly to moderately pathogenic to mungbean ,Operon RAPD primers OPD 11, 13 and 18
and sorghum but were highly pathogenic to gave better genetic diversity of R.
cabbage. Only AGI-IA was able to cause solanacearum compared to ERIC, REP, and
leaf and sheath blight on corn and rice. BOX primers. RAPD primers amplified 42
bands positions. UPGMA analysis of 83
It is clear that different anastomosis groups isolates resolved 71 halophytes with 11 clusters
ofR. solani have different pathogenic potential using SAS cluster analysis. Of the 11 clusters,
to various crops species. In many cases, some 5 clusters composed of isolates from
of the AGs were not able to incite disease in Mindanao, 3 consisted of isolates from Luzon
many crops that are attacked by other AGs. and 3 clusters consisted of a mixture of
This is paramount to planning strategies of isolates from Luzon and Mindanao. The
managing sheath blight utilizing crop sequence grouping of isolates is corrected to the origin
or rotation. For instance, based on the study, of isolates. The results showed that R.
in corn-based cropping system, mungbean, solancearum is highly diverse within and
sorghum, and cabbage as rotational crops between biovars and origin. The findings on
would present a risk as the AG, in this case this research are useful in designing breeding
AG -1A, attacking corn is highly pathogenic programs against bacterial wilt tomato.
to these crops. This situation is applicable to a
Volume 36 June-December 2000 79
test of 47 isolates using classified into biovars and of these, 62 (65%)
the other hand,
wilting of 42, 2
1 virulent isolate
most of the le!
o biovar4. This
ng which isolai
of resistance in
e most resistant
which were re.
78% of to some
3, and suscepti
useful and Pai
led for Luzarai
ns of R. solana
ill strains, 4 wei
rately resistant t
Ee rest had re
ins to various str
of sugarcane i
dela Cueva, RI
* Natural. IPE
Bacolod and Del
IPLB, College, I
11 anrd 7 mill dl
Santiago and i
'..), uV.) VLk "' TO
145 varieties, boti
ip of each sample
hilRice, Nueva Ecija and Oho state Ofthese, Phil 8013, Phil 8583, Phil 7464, Phil
university, USA. 8477 and V86-550 consistently yielded positive
reaction in the test indicating the presence of
The effect of host plant resistance and Clavibacer xyli subs. xyli, the causal organism
iltural practice on the management of of ratoon stunting disease.
acterial wilt disease of egg plant was
raluated on the basis of disease incidence at Results showed that Phil 8013 was the
te end of the season and area under disease most widely cultivated variety in Negros and
progress curve (AUDPC). The variety of Panay and had the highest incidence of the
plantt significantly affected the incidence disease. Phil 8583 and Phil 7464 were also
id AUDPC of bacterial wilt. Jackpot cultivar present in almost all mill districts and these
id the lowest incidence and AUDPC of were both positive to RSD. The newly
icterial wilt compared to DPL and Abar. Soil released high yielding variety V86-550 is now
at has been cultivated and not mulched with also widely grown in Negros but observed to
ce straw gave the lowest AUDPC while soil be infected with the disease in all mil districts.
at has been cultivated and mulched generated This finding revealed that there is widespread
ie lowest bacterial incidence. occurrence of RSD in Negros and Panay and
that the disease is present in almost all varieties
To search for additional sources of regardless of location. This indicated that
distancee to bacterial wilt, isolation and strategies to properly manage the disease
assification of the causal bacterium were should be developed in such a way that yield
ade and the collected isolates were used for will not be severely affected. Planting materials
:reening for resistance. A total of 96 R. should be cleaned before multiplication and
planting materials from Negros and Panay to of simple technologicalinterventions, viz. ea
other sugarcane areas where the disease is debudding and sanitation. Implementation
not yet known to occur should be properly the IPM program in Bugtok devastated S,
monitored to prevent further spread of the plantations has resulted in a dramatic decl
disease. in the incidence of disease and a 10 to 15-f
increase in bunch yield. Cost and return anal3
Site-specific IPM for mango: The showed that the IPM program can result in
Mindanao experience. OS Opina, JR increase in farm income from P11,155
Medina and LRA de Jesus. UPLB, P20,565/ha.
Detection of ratoon stunting disease
An IPM program for mango was sugarcane. NL Opina, LM Dolores, (
developed and implemented in Menzi Farmers Molina, MP Natural and CA Relevan
Cooperative (MEFO), Mati, Davao Oriental Institute of Plant Breeding, UPLB a
to address the major pest and disease problems Department of Plant Pathology, UPI
responsible for low fruit yield and quality of College, Laguna.
mango in the Mindanao region. The IPM
program featured an integrated approach The sensitivity of polymerase ch;
utilizing two pest management strategies, reaction (PCR) to detect Clavibacter a
namely, reduction of initial inoculum or pest subs. xyli (Cxx), the causal bacterium of
infestation and reduction of rate of infection ratoon stunting disease (RSD) of sugarcane
or pest reproduction. The two strategies were fivrovascular fluid (FVF) was compa.
achieved through the integration of the with dot blot Immunoassay (DBIA) e
following control methods and tactics: cultural evaporative binding enzyme-lini
practices (e.g. pruning and sanitation), fruit immunosorbent assay (EB ELISA). PI
bagging, pest monitoring and pesticide proved to be the most sensitive assay wh
management. The implementation of the IPM could detect Cxx at 1 x 10-7dilutions from F
programfortwo trials (1997-1998) consistently samples and Cxx isolates. DBIA and
increased fruit yield by 26.9% over the usual ELISA, on the other hand, could detect Cxm
MEFO production management practices; 1 x 10-3, respectively using Cxx isolates 26
increased acceptable fruit by 10.75%; reduced
fruit rejection by 8.35% and reduced crop All sugarcane samples obtained from VI
protection inputs (pesticides) by 24.52%. Bacolod, Camarines Sur, and IPB, UPLB, I
Bafios were positive to Cxx using PCR wl
Enhancing Saba banana production 10 of the 24 FVF samples from La Carl
through Bugtok management. OS Opina, were negative to Cxx. Not all of the samp
AB Molina Jr., GC Molina, RD Daquioag showed consistent reactions to DBIA E
and DC Blanco. UPLB, College, Laguna. EB ELISA. Crude samples which exhibi
positive/negative reactions in earlier te
An IPM program for Bugtok disease of become positive to all tests after the samp
Saba banana was formulated and implemented were centrifuged. These inconsistent reacti<
in Camiguin Province to address the Bugtok obtained from DBIA and EB ELISA v
problem responsible for low yield and confirmed using PCR.
abandonment of Saba plantations in Visayas
and Mindanao. The IPM program featured Efficiency, sensitivity, and reliability of e
fr-# Vnhimp 3B JuneP-fecemnbr 2000 R1
:re extracted and prepared for assay. PCR
Is the most sensitive assay as compared to
- ELISA tests.
transformation of papaya to develop
SV resistance by microprojectile
)mbardment. PM Magdalita, LL
illanueva, LD Valencia, O
hatchawankampanih, VN Villegas. IPB,
PLB, College, Laguna and Kasetsart
Zygotic embryos (15,875) of a solo
ipaya, harvested 90-120 days after
,llination, were explanted on a somatic
abryo induction medium. Average
:rcentage somatic embryogenesis after 4
months was 40%. A batch of 3,650
ibryogenic lumps had been squashed for
ansformation. Transient expression
dies using microprojectile bombard-
ent showed the gus expression on the
)mbardment somatic embryo was optimum
ing a pressure of 1200 KPA at a distance
S12.5 cm. For stable transformation,
matic embryos were bombarded with the
mne construct containing coat protein
'P) gene of Los Bafios, Philippine isolate.
election and generation of putative
isformants is underway.
ew diseases and pests of selected
ornamental plants in the Philippines. TO
izon. IPB, UPLB, College, Lagmna
Eight ornamental plants grown in
irseries were found to be infected with new
seases caused by fungal pathogens. A
rtorious plant parasitic plant was infecting
'ussaenda flava. The symptomatology
id the pathogen were described. Origin of
e diseases and alternate hosts were also
ariability of Collectotrichum sp. isolated
Twelve isolates of Colletotrichum sp.,
use of antracnose in Aglaonema, were
Elected from various areas of Luzon, Visayas
Id Mindanao. Variability among isolates
ised on colony growth and sporulation
pacity in vitro and reaction to selected
Itivars of Aglaonema was determined.
On potato dextrose agar, all the isolates
offered with regards to colony size, rate of
ycelial growth and sporulation. Isolates 1,
, 24, 77, 81 and 82 had similar colony size,
iereas only isolate 15 had the highest spore
lunt. Isolate 20 grew faster that the other
Effect of the 12 isolates to Aglaonema cv
silver Queen" differed. Isolates 81 and 24
used the biggest lesion on detached and intact
waves, respectively. When all the isolates
ere inoculated to 12 cultivars ofAglaonema,
>ne exhibited resistant reaction. Isolates 37
duced susceptible reactions in all cultivars
bile 3 isolates caused similar reaction in all
altivars. Only 2 cultivars were found
sceptible to all isolates of Colletrotrichum.
11 other isolates and cultivars showed varying
ungal hyperparasites: An effective
)mponent of an integrated disease
management strategy for chrysanthemum
white rust. LM Villanueva and MD
oteng. BSU, La Trinidad, Benguet.
The effectiveness of two hyperparasites
'chrysanthemum white rust, Verticillium sp.
id Pecilomyces sp. was evaluated under
-eenhouse conditions at Benguet State
university, La Trinidad, Benguet.
Application of Verticillium sp. at
oculation time and two days ahead of the
development and markedly reduced
number of rust pustules in chrysanthemi
When combined with sub-lethal dose
Triforine, the hyperparasites gave a con
comparable with sequential spraying
Triforine, Mancozeb and Elemental Sul
However, Verticillium sp. alone i
significantly more effective than sin
application of Paecilomyces sp.
In the tolerant cultivar Fiji, the higl
return on investment (ROI) was obtained fi
plants sprayed with Verticillium sp. + Trifoi
followed by sequential spraying of Trifor
Mancozeb and Elemental Sulfur. Spraying
Verticillium alone gave ROI comparable
sequential spraying of the different fungicic
To determine the best time of spraying N
the two hyperparasites, another greenho
experiment was carried out. Two spray
of Verticillium after one application of
combination ofTriforine + elemental Sulfur,
the most effective followed closely by
Veticillium and Paecilomyces sp. ai
Triforine + elemental Sulfur. Outstand
control of the disease in plants treated v
above hyperparasites resulted in the quality
cutflower yield. As expected, the lov
marketable cutflowers were obtained fi
Characterization of Philippine isolates
Erwinia chrysanthemi causing bactel
stalk rot of corn. AA Gumarang and
Pascual. IPB, UPLB, College, Laguna.
Out of 112 isolates of bacteria collect
from different corn-growing areas in
Philippines, 102 were confirmed to be Erwi
chrysanthemi pv zeae based on morpholi
and standard determinative tests for Erwin
Variation was detected among isolates
sucrose, D-glucose, H2S production and gel;
ie Detection of leaf curl geminivirus
n. tomato using non-radioactive DNA prc
of LM Dolores. IPB, College, Laguna.
of A total of 22 tomato, one pepper and
Ir. squash exhibiting various types of leaf curl
is mosaic symptoms collected from tom
le growing areas of Luzon were tested for
presence of germinivirus using the Philip1
tomato leaf curl germinivirus (TLCGV) D
st probe. Of these, 19 out of 22 tomato leaf
m infected samples gave positive hybrids with
ie DNA probe. The pepper sample on the oi
e, hand, did not hybridize with the pro
of Reactions varied from strong to weak sigi
to as visualized on the nitrocellulose member
s. of the NBT x-phosphate detection metal
Squash samples with severe leaf c
th symptoms also developed positive hybrids N
se the DNA probe, which suggested the prese
Ys ofgerminivirus in squash.
Is Results from this experiment indicated
le potential importance of leaf curl disease
er the efficiency of using non-radioactive D
ig probe in detecting germinivirus in tomato.
of Endophytic fungi in rice leaves.
st Gonzales and TU Dalisay. MMMSU-SD
m Bacotan, La Union and UPLB, Colle
of Endophytic fungi associated in rice lea
al were initially studied to compare the spei
B composition and their abundance in six I
Ax Forty-three fungi of different taxa w
he isolated. From these, 11 genera w
ia identified. These were Arthrimium s
,y Aspergillus sp., Cladosporium s
2. Dactylaria sp., Penicillium s
in Microsporium sp., Nodulisporium s
in Trichoderma sp., Pestalotia sp.,
\.\/Mni/-^.o LLn'mi, ai Ii 4Xi ixC -na7 nnarpr rpnnrTe An MnRl nvnamir r nT nllnr.nv-Tn
LIl, IVLLSI L UI."LV -, -L. 4.vu V l..uv.j vI-
abundance, among the isolated endophytes.
Mak Fae Deng rice accession had the highest
percentage of isolates at 26.5% due probably
to a greater chance of trapping fungal
propagules with its tall stands and wide leaves.
Development of blast resistant near-
isogenic lines of rice for multiline
breeding. LAEbron, IRRI; T Imbe NARC.
Tsukuba, Japan; H Kato, TNAES, Akita.
Japan; H Tsunematsu, IRRI; Fukuta, IRRI.
DL Adorada, IRRI; and GS Khush, IRRI.
Los Banos, Laguna
Blast resistant near-isogenic lines (NILs]
were developed at IRRI for multiline breeding
To date, 11 major resistance genes have beer
transferred into two genetic backgrounds, IR24
ad IR49830-7-1-2-2, following the convention
backcross breeding method. Ten resistance
genes, PI-1, Oi-k, Pi-1, Pi-3, Pi5 (t), Pi 7(t)
Pi-9(t), Pita2, Pi-z and Pi-zs are common ix
both backgrounds. Pi-zi and Pi-ta wer
incorporated into IR49830-7-1-2-2 and IR24
respectively. Most of the NILs are presently
at BC F, or BC6F4 generations.
IR24 is a cultivar for irrigated ecosysten
while Ir49839-7-1-2-2 is an elite line witl
submergence tolerance for rainfed and lowland
ecosystem. The submerge tolerance in NIL;
ofIr49830-7-1-2-2 has been confirmed. These
NILs will be used to confirm their application
as multilines in tropical areas as a first field
Epidemiology and forecasting of abac;
bunchy-top. AD Raymundo and N1V
Basio. UPLB, College, Laguna.
Bunchy-top disease has often beei
managed unsuccessfully due to inadequate
understanding of its dynamics. Rate of spread
and increase have not been quantified. Thi
infection focus at the center and on a compute
model of the disease.
A 9-month observation period showed tha
bunchy-top development is slow. The earl
part of the period was characterized b.
infection of a few hills near the source o
inoculum. At this time, aphid colonies were
observed on many of the plants. In the ensuring
months, infection increased rapidly. The lay
time it takes for the disease to take off appear
to be a function of incubation time.
Initial runs with a preliminary model i
STELLA software showed big gaps ii
information pertaining to critical aspects ofth
life cycle of bunchy-top. A new model with
bunchy-top disease component and
Pentalonia nigronervosa component will b
Geo-epidemiology and integrated
management of bunchy-top and mosaic o
abaca in the Bicol Region. AD Raymundc
NB Bajet, AC Sumalde, B Cipriano, RI
Borromeo, EC Aril, LA Triman, F d
Castro, CR Rafallo, and R Tagala. UPLE
College, Laguna, and Fiber Industry Develop
ment Authority, Region 5, Legaspi City.
This papers reports about attempts a
managing bunchy-top and mosaic of abaca b
eradication and replanting utilizing the principk
of epidemiology. The premise is based on th
theory that for eradication to succeed, th
source of inoculum or disease "hot spots" mu!
first be located and then eradicate
Subsequently, replanting must be done only i
areas where the source of inoculum has bee
Data from surveys done for more than
year were inputted into a GIS software t
generate maps showing distribution and disease
The provinces of Sorsogon and
Catanduanes appeared to have been hit harder
than other areas in Bicol Region although in
less affected provinces, specific concentrations
of disease were still seen. Bunchy-top is not
only spreading fast, it intensifies at a high rate.
In some areas in Sorsogon, it has increased
more than twice within a year.
SA A.l MfrV lA At3 V tli %hJLJU l fl.ltJfl UV.Jl%, *lL 1J1L
Bicol Region. It appears that a continuing
effort over the long-term is needed if bunchy-
top and mosaic are to be contained. A systems
approach, which considered all aspects of the
abaca-virus-vector relationship including social
and political factors, will be discussed.
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