• TABLE OF CONTENTS
HIDE
 Front Cover
 Front Matter
 Plant parasitic nematodes associated...
 Effects on tylenchulus semipenetrans...
 Survey of higher plants for fungicidal...
 Latent infection in mango caused...
 Heritability of differences in...
 Cowpea viruses in the Philippines:...
 Inhibition of pathogens of fields...
 Identity of a strain of cowpea...
 Temperature relations of philippine...
 Identity of the bacterium associated...
 A review: Predicting the outbreak...
 Note: Occurrence of a witches broom...
 Back Matter
 Back Cover














Group Title: Journal of Tropical Plant Pathology
Title: Journal of tropical plant pathology
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00090520/00021
 Material Information
Title: Journal of tropical plant pathology
Series Title: Journal of tropical plant pathology.
Alternate Title: Journal of Philippine phytopathology
Philippine phytopathology
Physical Description: v. : ill. (some col.) ; 26 cm.
Language: English
Creator: Philippine Phytopathological Society
Publisher: Philippine Phytopathological Society
Place of Publication: Philippines
College Laguna
Publication Date: January 1979
Frequency: semiannual
regular
 Subjects
Subject: Plant diseases -- Periodicals -- Philippines   ( lcsh )
Plants, Protection of -- Periodicals -- Philippines   ( lcsh )
Genre: periodical   ( marcgt )
 Notes
Dates or Sequential Designation: v. 1, no. 1 (January 1965)-
General Note: Title from cover.
General Note: "Official publication of the Tropical Plant Pathology."
 Record Information
Bibliographic ID: UF00090520
Volume ID: VID00021
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 54382605
issn - 0115-0804

Table of Contents
    Front Cover
        Front Cover 1
        Front Cover 2
    Front Matter
        Front Matter 1
        Front Matter 2
    Plant parasitic nematodes associated with grapes in cebu and batangas
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
    Effects on tylenchulus semipenetrans cobb and other pathogens on citrus in the Philippines
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
    Survey of higher plants for fungicidal properties against pyricularia oryzae Cav
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
    Latent infection in mango caused by colletotrichum gloeosporioides
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
    Heritability of differences in virulence bewteen races 2 and 3 of cochliobolus carbonum
        Page 47
        Page 48
        Page 49
        Page 50
    Cowpea viruses in the Philippines: II. Isolation and identification of cowpea strain of cucumber mosiac virus
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
    Inhibition of pathogens of fields legumes by Mimosine
        Page 58
        Page 59
        Page 60
        Page 61
    Identity of a strain of cowpea mosaic virus in winged bean (psophocarpus tetragonolobus L.)
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
    Temperature relations of philippine solanaceous isolates of pseudomonas solanacearum
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
    Identity of the bacterium associated with bacterial brown spot of phalaenopsis orchids
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
    A review: Predicting the outbreak of bacterial blight of rice by the bacteriophage method
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
    Note: Occurrence of a witches broom disease of mungbean in the Philippines
        Page 86
        Page 87
    Back Matter
        Page 88
    Back Cover
        Page 89
        Page 90
Full Text
SMhytopathologyAA
hytopathology











THE PHILIPPINE PHYTOPATHOLOGICAL SOCIETY, INC.
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Subscriptions: Communications should be addressed to the Treasurer, P. P. S. c/o Department
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PLANT PARASITIC NEMATODES ASSOCIATED
WITH GRAPES IN CEBU AND BATANGAS

R. G. DAVIDE, RUTH M. SARRA arid LUCIANA R. MARANAN

Associate Professor, former Graduate Assistant and former Research Assistant,
departmentt of Plant Pathology, College of Agriculture, University of the Philippines
t Los Banos, College, Laguna.
This study was supported by NFAC Project FAR-016-73.
The authors wish to express their sincere thanks and appreciation to the BPI Per-
onnel of Region VII in Cebu, particularly to Director E. Gianzon, Director C. Lucero,
dr. R. Diang, Mr. Francisco Mirasol, and Mr. A. Paradela for their assistance in our survey
a Cebu. The assistance of Mr. F. T. Gargantiel in the identification and counting of
nematodes in some soil samples is gratefully acknowledged by the authors.

ABSTRACT











rent areas but with relatively lower
Soil samples were collected from population densities were Pratylenchus,
various grape areas in Cebu and Meloidogyne and Xiphinema. Ocassion-
Batangas. The soil sample size was about ally, Scutellonema, Hoplolaimus, Paraty-
600-800 cc per sampling site and the lenchus Criconemoides, and Tylencho-
number of samples collected per area rhynchus were also observed in some
depended largely on the size of the samples.
vineyard. At least 10-20 samples were
obtained from 1-2 hectares of grapes. Results of the nematode analysis of
These samples were taken 30 cm from the soil samples from Batangas are presented
base of the plant at 15-30 cm deep. All in table 2. Eleven genera of plant para-
soil samples were placed in plastic bags sitic nematodes were detected in the soil
and brought to the nematology laborato- samples collected from the different
ry, Department of Plant Pathology, areas. Their occurrence and population
UPLB-CA, for nematode analysis. density also varied considerably in the
different localities. The varieties of
The soil samples were processed using grapes grown in these areas were more
the sieving-funnel method. Sub-samples or less similar with those in Cebu.
of 400 cc per sampling site were used Evidently, except for the genera Hemi-
for the nematode analysis. After 48 cycliophora and 7Tyenchulus, the
ir in the funnels, the nematodes were same genera of nematodes found
drawn out into the vials and stored in in Cebu were observed in Batangas.
4 C refrigerator. Later, the nematodes Likewise, R. reniformis was the most
were placed in a calibrated counting dish commonly observed and usually present


nd counted.

RESULTS

Results of the nematode survey
1 Cebu are summarized in Table 1.
en genera of plant parasitic nema-
)des were found associated with grapes
varieties Red Cardinal, White Malaga,
lack Ribier, etc.) in the different areas
Irveyed. Their population density and
distribution varied considerably in the
different places. In general, Rotylenchu-
,s and Helicotylenchus were more
revalent than the other nematode gene-
a. The highest population density
If R. reniformis was obtained in soil
samples from Tungkop, Minglanilla, Cebu
rith an average of 803.9 individuals per
00 cc soil. Other genera that were more


)il) was obtained in Tanauan. Its lowest
ensity of 12.8 nematodes per 400 cc
Ail was obtained in the soil samples from
rea II in BalayanJ.elicotylenchus,Hoplo-
imus, Hemicycliophora, Meloidogyne,
id Tylenchorhynchus were also found
iore frequently than the other genera
at their population densities were rela-
rely lower, compared with Rotylen-
tulus, M. incognita and R. reniformis,
different stages of development, were
)served in feeding positions in roots
ig. 1). In addition, root galls of varying
ces were found in M. incognita -
fected roots (Fig. 2).

DISCUSSION

This study shows that nematodes
re associated with grapes in Cebu and









Table 1. Occurrence and distribution of plant parasitic nematodes associated with grapes in Cebu


Mean nematode count/400 cc soil
Mean nematode count/400 cc soil


Locality


Helico- Rotylen- Praty- Xiphi- Meloi- Scutel- Hoplo- Pamty- Crico- Tylencho-
tylenchus chulus lenchus nema dogyne lonema laimus lenchus nemoides rhynchus


A. Northern Cebu


Liloan 394.8 2429.0
Magay, Compostela 384.20 488.7
Jogan, Consolacion 253.15 157.4
Lamac, Consolacion 1.7 0
BPI Mandaue Expt. Sta. 26.3 848.2
Casili, Consolacion 321.0 1692.0
Banilad, Mandaue 18.0 741.2
Tipolo, Mandaue 1.2 2.5


4.8 5.0
0 15.0
0.2 0
0 0.7
0.1 12.5
0 0
0.2 452.0
3.0 0


0 0 0
2.6 0 0
0 0 0
0 0 1.0
0.9 0 0
0 53.3 0
0 4.2 0
0 0 0


B. Southern Cebu
Tungkop, Minglanilla
Villadolic, Carcar


803.9 4081.3 0.9
32.4 713.9 29.2


2.4 0
0 0


















Table 1. (continued)


Abogon, Sibonga
Lagtang, Taliay
Linao, Talisay
Sibonga
Poblacion, Argao
Malinglingon, Argao
Bagakay, Sibonga
Oeana, Carcar
Cogon, Carcar
Cogon, Nag
Cogon, Pardo


2.5 1087.5 3.3
0 387.0 0.3
6.7 597.0 0
274.1 170.3 5.7
305.5 344.5 5.5
10.9 19.6 48.6
221.5 50.2 5.2
106.0 221.0 6.5
276.0 25.5 20.7
87.5 26.5 5.2
3.7 147.0 46.7


0 3.4 0
0 0 0
0 0 0
4.2 6.0 0
0 0 0
0 0 0
00 0 0
0.2 0 0
0 0 0
7.0 0 0
0.3 0 0








Table 2. Distribution and population density of plant parasitic nematodes associated with grapes in Batangas.


Mean nematode count/400 cc soil

Location Rotylen- Helicoty- Hoplo- Hemicy- Xiphi- Tylencho- Tylen- Meloi- Praty- Crico- Paraty-
chulus lench u laimus cliophora nema rhynchus chulus dogyne lenchus nemoides lenchus
Nasugbu


3.6 6.1 0 1.3 0
0.1 1.3 0 17.2 1.7
4.0 0.2 0 20.2 3.8


49.0 0.2
0.8 0.16

28.3 2.4

2.0 0


0.2 0
0.2 0


0.5 0


13.3 14.4
2.6 1.3


5.4 21.4
0.2 0


3.6 0 0 0



7.0 0 0 0


0 1 4.3 0 0 0


3.3 3.8 0.9 0.2


0.4 0.3


0.5 0


0 0.2 0 0 0 0 0 0


Areal
Area II
Area HI

Balayan
Area I
Area II

Malvar

Tanauan


292.8
134.7
788.6



55.7
12.8

490.7

2049.3





CA






















CARDINAL


1 and White Malaga root systems showing galls due








Philippine Phytopathology


Batangas. The nematode genera reported
abroad as causing damage on grapes,
namely Meloidogyne, Pratylenchus, and
Xiphinema were also detected in many
grape farms. In some cases, trace to
slight root gall formation was already
observed in such varieties as Red Car-
dinal and White Malaga (Fig. 2). How-
ever, there has been no attempt to evaluate
the extent of damage due to these nema-
todes on grapes, since in most of the areas
surveyed, the plants were either just
a few months old or 2-3 years old. Evi-
dently, at this age, populations of these
nematodes were still relatively so low to
cause serious damage.


though it has not been reported on
grapes abroad, it is possible that under
our conditions, this nematode could
become of economic importance.

The presence of almost identical
genera of nematodes in Cebu and Ba-
tangas may be partly explained by the
fact that most planting materials mainly
grown in plastic containers with unsteri-
lized soil came from Cebu. On the other
hand, the presence of T. semipenetrans
in some samples from Batangas could
be due to the fact that a few years ago,
these areas were planted to citrus which
was wiped out later by a disease complex
partly caused by T. semivenetrans and


8














Philipp. Phytopathol. 15: 9-22
Received for publication: 3 September, 1974



EFFECTS OF TYLENCHULUS SEMIPENETRANS COBB
AND OTHER PATHOGENS ON CITRUS
IN THE PHILIPPINES

ID 'nATTi ANT' A r- nTT A RORA











MATERIALS AND METHODS ments; 1) T. semipenetrans plus C. sal-
monicolor, the causal fungus of pink
Inoculation experiments disease, 2) T. semipenetrans plus Fusa-
The following experiments were rium sp. isolated from citrus root rot,
conducted to determine whether T. 3) T. semipenetrans alone, and 4) non-
semipenetrans and the other genera of inoculated controls.
plant parasitic nematodes can cause
serious damage to citrus plants. Seed- T. semipenetrans of approximately
lings of different citrus varieties such 5,000 individuals were inoculated to the
as Ladu, Szinkom, Calamansi, and Po- plants 3 days ahead of the fungus pa-
melo were grown in 15 cm diam clay thogens. This was done by placing the
pots filled with sterilized soil. When they nematode suspension into two small
were about 6 months old, they were holes about 5-cm deep near the base
inoculated with known population levels of the plant. A 10-day old culture of
of T. semipenetrans. Other genera of C salmonicolor and Fusarium sp. was
nematodes such as Xiphinema, Helico- used. Suspensions of the mycelia and
tylenchus, Scutellonema, Paratylenchus, spores of these fungi were separately
Pratylenchus, Diptherophora, Tylencho- prepared and used as inocula. These
rhynchus, and Criconemoides were also were inoculated into the plants by pla-
inoculated separately on either of the cing 10 cc of the suspension into two
ahnv m ntinnirtn .itnic variptio. Pa. holes. 4 cm deen. located onnnsite


reduction of linear top growth and popu.
lation counts of nematodes in the soi
and roots of citrus. T. semipenetran,
was extracted from 1 gram root sample
by the Waring blendor and sieving me
thods.

On the other hand, the nematode
from the soil samples (400 cc) wer
extracted by using the combined sievin
and the Baermann funnel methods. Th
nematodes were drawn off the funnel
into small vials within 78 hours. Late
they were placed in small plastic dishes
and examined for nematodes under
stereoscopic microscope.

Interactions of T. semipenetrans wit
other fungus pathogens of citrus
Seedlings of Calamansi were growl
in 15-cm diam. clay pots filled witl
sterilized soil and when they were abou
6 months old they were placed into fou


were kept in the greenhouse bench oi
top of inverted pots and spaced abou
30 cm apart to avoid cross contamina
tion of the different treatments. Thre,
months after inoculation, the rate o
reproduction of T. semipenetrans wa
determined by examining the nematode
in the soil and roots of the test plants.


In another experiment, the effect o
Phytophthora sp., which was isolate<
from the rotted portion of a citrus root
on the rate of reproduction of T. semi
penetrans was also determined usini
6-month old Szinkom seedlings as tes
plants. The same procedure was used a
in the other experiments above, excep
that the suspension of Phytophthon
sp. was introduced into the soil through
two 15 cm glass tubings, placed opposite
each other, 5 cm deep into the soil nea
the base of the plant. Five replicate









Effects of T Semipenetrans Cobb


and roots of the test plants were deter-
mined 4 1/2 months after inoculation.

Interaction of T. semipenetrans and the
citrus greening pathogen
The greening disease is believed to
be the cause of citrus decline in Batangas
and other provinces. This disease has
long been recognized by Martinez
and Wallace (1967a, 1967b) as caused
by a virus. Recently, however, Martinez
et al. (1970) reported that the cause of
the greening disease was evidently not a
virus but a mycoplasma-like organism.
In our survey last year, we observed that
in most cases, the population density of
T. semipenetrans was relatively higher
in trees showing severe symptoms of
greening. Whether this can be demons-
trated experimentally is not known,
hence, this study was made.
Twelve-month old Ladu seedlings,
grown in 20-cm diam clay pots filled
with sterilized soil, were budded with
Szinkom identified by A.L. Martinez of
the Bureau of Plant Industry Lipa Expe-
riment Station to be infected with the
greening pathogen. Three months later,
these budded plants were inoculated


with approximately 15,000 individuals
of T. semipenetrans. Of these budded
plants, six showed traces of greening and
nine showed moderate to severe greening
in 3 1/2 months after inoculation. At
this period, 200 cc soil samples were
taken from each pot and the nema-
todes were extracted by using the com-
bined sieving and Baermann funnel
methods.

RESULTS

Inoculation experiments
Table 1 represents the results of the
first inoculation experiment using dif-
ferent isolates of T. semipenetrans
from different provinces. It is evident
from the results that 12 months after
inoculation growth of the Ladu seedlings
was greafly affected by the nematode
inoculation. Based on the percentage
reduction in plant height, the different
isolates showed some degree of variations.
For instance, the isolate from Nueva
Viscaya caused a reduction of top growth
by 53.4% whereas the isolate from
Batangas (Lipa) caused 42.0% reduction
of top growth on Ladu seedlings.


Table 1. Effect of Tylenchulus semipenetrans isolates on the
seedlings


linear height of Ladu


Nematode Plant Decrease
count after height in height (cm)
12 months (cm)

Nueva Vizcaya 86,400 56.6 a 53.4
Nueva Ecija 46,625 62.6 a 48.5
Cagayan 94,125 65.0 ab 46.5
Batangas 44,500 70.5 a 42.0
Control 0 121.6 c -

aThe initial inoculum level was about 16,000 nematode individuals per plant.
bMeans with letters in common do not differ significantly (P = 0.05).


January, 1979









Philippine Phytopathology


Likewise, on Calamansi seedlings, T.
semipenetrans from Bicol and Batangas
seriously affected the height of the
plants. As shown in Table 2 at the ino-
culation of 50,000 nematodes per plant,
the isolate from Lipa, Batangas caused
a reduction of top weight by 41.4% 7.5
months after inoculation, while the


isolates from Camarines Norte and
Camarines Sur caused a reduction of
22.9% and 16.1%, respectively. Exam-
ination of soil and roots of the inocula-
ted plants revealed heavy infestation of
the soil and severe infection of the roots
(Fig. 1), resulting in a considerable
reduction in height (Fig. 2). The graph


Table 2. Effect of Tylenchulus semipenetrans on the height of Calamansi seedlings 7.5
months after inoculation with 50,000 nematodes per plant


Source of Plant Per cent
nematode heighta reduction
isolate (cm) in height

Lipa, Batangas 43.08 41.04 a
Camarines Norte 56.36 22.86 b
Camarines Sur 61.27 16.14 b
Control 73.07

aMean of five replications.

bThe same letters indicate non-significant difference at 5% level.


Fig. 1. Portion of Calamansi root heavily attacked by Tylenchulus semipenetrans Cobb.
(See arrows).


Vol. 15


V ii








Effects of T Semipenetrans Cobb


MONTHS AFTER INOCULATION
(T. sUMpNETrANS)


Fig. 3. Height response of Calamansi seedlings to T. semipenetrans at an inoculum level of 50,000
nematodes per plant


0 12000
g 30000
80 Mn CONTROL

TO

60



S40

to,

20

10


0 2

MONTHS AFTER INOCULATION
(T SEMIPENETRANS)
Fig. 4. Height response of Calamansi seedlings to
different isolates of T. semipenetrans at 50,000
nematodes per plant


January, 1979
























TLEMCHlUWS SMIPNETRWS CONTROL


Philippine Phytopathology


Vol. 15


.- 4.1.-
.1"Wh -


I *


- T. SEMIPENETRANS


CONTROL


Fig. 2. Effect of T. semipenetrans Cobb (Lipa isolate) on the top and
roots of Calamansi seedlings, 7.5 months after inoculation with
50,000 nematodes per plant.


inoculation as shown in Fig. 4, there
was a gradual reduction in growth of
Calamansi seedlings inoculated with T.
semipenetrans isolates from Camarines
Sur and Lipa, Batangas at the 50,000
level. Again, there was an indication
that isolates from the different pro-


in Fig. 3, likewise, shows the relative effect
of T. semipenetrans at the 50,000 inocu-
lum level per plant on the growth of Cala-
mansi seedlings. At 11 months after inocu-
lation, more than 50% of the linear top
growth of the plants was reduced by nema-
tode inoculation. As shown in Fig. 4 there








Effects of T Semipenetrans Cobb


vinces have varying pathogenic capabili-
ties on Calamansi seedlings. For instance,
the isolate from Lipa, Batangas was more
virulent, based on the reduction of height
of the plants, than those from Camarines
Norte and Camarines Sur. This could be
an indication of the existence of physio-
logic races of T. semipenetrans.
Effect of low population levels of '7
semipenetrans on different citrus varieties.
The graph in Fig. 5 indicates that
per plant inoculum levels of 12,000


whereas at 30,000 level there was appa-
rent reduction of linear top growth of
the plants 7 months after inoculation.
A similar trend was observed in other va-
rities as shown in Table 3. In Pomelo
and Ladu seedlings inoculated with
10,000 T. semipenetrans per plant,
there was an increase of 22.3% and
8,8% in height, respectively, These dif-
ferences, however, were not statistically
significant. On the other hand, Szinkom
seedlings showed a reduction in height by


0 2 7


MONTHS AFTER INOCULATION
(T SEMIPENETRANS)

Fig. 5. Growth response of Calamansi seedlings at
10 12,000 and 30,000 inoculation levels of
T. semipenetrans per plant.


and 30,000 of T. semipenetrans have
varying effects on the top growth of
Calamansi seedlings. There is indication
of stimulation effect on top growth
of the plants at 12,000 inoculum level,


12.0% as compared with the non-ino-
culated plants. These findings seem to
idicate that the different varieties had
varying degrees of susceptibility or
tolerance to the nematode infection.


January, 1979








Philippine Phytopathology


Table 3. Effect of Tylenchulus semipenetrans at 10,000 inoculum level per plant on the
growth of Pomelo, Szinkom, and Ladu seedlings 7 months after inoculation1


Citrus variety Root wt. (g) Top wt. (g) Plant ht. (cm)

Ladu
a. Inoculated 21.60 13.33 62.8
b. Non-inoculated 26.50 10.00 57.3
Szinkom
a. Inoculated 17.50 17.2 70.90
b. Non-inoculated 24.25 22.0 80.55
Pomelo
a. Inoculated 15.7 18.7 50.2
b. Non-inoculated 13.00 14.90 41.1

1Data are the mean of six replicate plants. Statistical analysis of the data showed
no significant difference between the inoculated and non-inoculated plants (P = 0.05).


Effect of T. semipenerrans isolate from
Aparri, Cagayan on the growth of Ladu
seedlings
In this study the inoculum level used
was 50,000 nematodes per plant and data
were gathered 5 months after inocula-
tion. The results showed a reduction of
28.7% in root weight and 173% in
height (Table 4). Again, this illustrates


that at high inoculum level the nema-
todes could cause serious damage on
citrus plants.
Effect of different citrus varieties on the
rate of reproduction of T. semipenetrans

It was observed in the survey study
that field population of T. semipenetrans
varied with the different citrus varieties


Table 4. Effect of Tylenchulus semipenetrans isolate from Aparri, Cagayan on the
growth of Ladu seedlings 5 months after inoculation with 50,000 nematodes
per plant


Treatment Root wt. Per cent Plant Percentage
(g) reduction height reduction
(cm)

Inoculated Plants 10.16* 28.7 34.16* 17.32
Non-inoculated Plants 14.25 41.32 -


Data are
aMeans of six replications.
*Significant at 5% level


Vol. 15












varying degrees of susceptibility or tole- citrus. For instance, in Pomelo seedlings,
rance to the nematode infection. It was citrus. For instance, in Pomelo seedlings
not known whether these effects could the nematode population had increased
be demonstrated experimentally, so it was over its initial inoculum level of 1.000
decided, therefore, that this study be per plant by 151.0% in 2 1/2 months as
conducted. The results of this study compared to 19.2% population increase
are summarized in Table 5. The data in Szinkom, 67.9% in Ladu, and 75.7% in
represent the total nematode counts in Calamansi.
both soil and roots of the test plants
2 1/2 months after inoculation. The ratq Inoculation with other nematode genera
of reproduction of the nematodes varies In Table 6, the data show that

Table 5. Rate of reproduction of Tylenchulus semipenetrans on different varieties of
citrus 2% months after inoculation with 1,000 nematodes per plant



Nematode count Percentage
Citru variety Soil Root Total population
increase

Szinkom 701.80 490.60 1,192.40 19.24
Ladu 255.40 1,424.00 1,679.40 67.94
Calamansi 369.50 1,388.00 1,757.50 75.75
Pomelo 1,643.00 867.33 2,510.33 151.03

SMeans of five replications.
Table 6. Results of inoculation of different nematode genera isolated from various citrus
plantations in Oriental Mindoroa


Nematode genera No. of nematode No. of nematode
Inoculated recovered
per plant 4 months
after


~"""Ul)


~-~L"Y VI 1 YI~C~UO W












Hoplolaimus, Pratylenchus, Xiphinema, of these genera were still found in the
Diptherophora, and Helicotylenchus did soil samples 3 months after inoculation.
not reproduce in citrus root. On the other
hand, the population of T. semipenetrans
had increased more than three times its ori- Effect of the interaction of T. semi-
ginal inoculum level from 2,506 to 6,128 penetrans and some fungus pathogens
after 4 months. The same trend of results of citrus
was observed from the genera collected Under field conditions, citrus trees
from Davao (Table 7) and Cebu (Table8) are not only vulnerable to attack by
except that in some cases few individuals nematodes but also to other pathogens

Table 7. Results of inoculation with different nematode genera obtained from a citrus
plantation in Mandug, Davao Citya


No. of nematodes No. of nematodes
Nematode genera inoculated recovered


Criconemoides 10
Hemicycliophora 60
Pratylenchus 30
Tylenchorhynchus 80
Helicotylenchus 100
Paratylenchus 50
Tylenchulus semipenetmns 10,000



aSzinkom seedlings were used as test I

Table 8. Results of inoculation with differ
plantation in Colawin, Cebu.


No. of I
Nematode genera inoci
per

Paratylenchus 48:
Scutellonema 211
Diphtherophora 31
Pratylenchus 6:
Xiphinema 56
Criconemoides 21
Tylenchulus semipenetrans 3,00(


aSzinkom seedlings were used as test I


0
6
4
4
32
0
17,388



ints.

it nematode genera obtained from a citrus



natodes No. of nematodes
rted recovered
mt 3 months after inoculation

6
9
0
0
4
3
4,275


Lnts.









Effect of T Semipenetrans Cobb


bThe percentage population increase was based on the counts of the T. semi-


January, 1979








Philippine Phytopathology


the presence of Fusarium sp., the increase
was only 45.7%. Likewise, there was a
remarkable increase (155.3%) in the po-
pulation density of the nematodes in
the presence of Phytophthora sp. (Table
10). However, the development of a pink
disease, foot rot or root rot disease in
the inoculated plants was not observed.
This may be due to the greenhouse
conditions which were probably not
favorable for the development of the
disease. The data only nearly indicate
the favorable effects of these fungus
pathogens on the reproduction or multi-
plication of T. semipenetrans.

Influence of greening pathogen of citrus
on the rate of reproduction of T. semi-
penetrans
It has been observed in the survey
that in most cases, the population density
of T. semipenetrans was relatively higher
in plants with severe greening than in
plants which were healthy-looking or
with only traces of greening. This study
was, therefore, conducted to determine
whether the greening pathogen has any
effect on the reproduction of the ne-

Table 11. The recovery of Tylenchulus
the greening pathogena



Plant condition


Moderate to revere greening

Healthy-looking to trace greening

aData were taken 3% months after
replicate plants.
bThe inoculum level was 15,000 nen
CBased on the population counts in
**Significant at 1% leveL


matoaes. lne results are presented in
Table 11. Based on these data, there is
evidence suggesting a favorable influence
of the greening pathogen on the rate
of reproduction of the nematodes. In
plants with moderate to severe greening,
the population density of T semipe-
netrans had been remarkably increased
by 335.8% over those in the healthy
looking or with only traces of greening.

DISCUSSION
The observation in the present study
that it took longer period to detect any
growth differences in the nematode
inoculated plants was in agreement
with the report of Van Gundy and Tsao
(1963). They found that it would take
6 months to obtain sufficient growth of
citrus seedlings to show significant
differences in growth and nematode
population increase.
The results of the inoculation expe-
riment using isolates of T. semipenetrans
from Camarines Norte, Camarines Sur,
and Lipa, Batangas indicate the possible
existence of biotypes or physiological
races of the nematodes in that they exhi-

emipenetrans in Ladu seedlings infected wil



Nemno~e Percentage
count population
(200 cc soils) increasee

80.00** 335.8

18.33 -

inoculation and these are the means of si

todes per plant.
ie healthy to trace greening plant conditions


/ol. 15











bited varying degrees of virulence on
the test plants. A more thorough study
to define these races will have to be
conducted in the future. The existence of
biotypes of T. semipenetrans in Califor-
nia has been reported by Baines et al.
(1967, 1969). These investigators showed
two distinct biotypes of T. semipene-
trans, based on the ability of the isolates


nematodes was greatly favored by the
presence of these pathogens. Observations
by some workers abroad also indicated
that greater reduction in growth of
citrus plants could be attributed to the
combinedd infections of Fusarium solani,
F. oxysporum and T. semipenetrans
[O'Bannon et al., 1967: O'Baninon,
1966). On the other hand, Feldmesser,


different varieties of citrus could also had a much lower percentage of Fusa-
influence the rate of reproduction of rium infection whereas those feeder roots
T. semipenetrans. For instance, the severely infected with the nematodes
nematode reproduced at a much faster had the highest incidence of F. solani
rate in Pomelo as shown by their high and F oxysporum. Likewise, Van Gundy
population density, than in Calamansi. and Tsao (1963) showed that F. solani
However, Calamansi gave a higher popu- infection on citrus seedlings became
nation density of the nematodes than severe in the presence of T. semipene-
Szinkom. These findings provided a trans. The present study, however, has
possible explanation why in our survey not determined the effect of the nema-
last year, the population density of tode infection on the incidence of Fusa-
T. semipenetrans significantly varied rium or Phytophthora on the inoculated
among the different citrus varieties, plants.
These results would also mean that our There was also evidence which suggests
citrus varieties have varying degrees of a possible interaction between the green-
susceptibity to nematode infection. ing pathogen, a mycoplasma-like
The inoculation experiment with i ng p athogen, a myoplasma-like
other genera such as Hoplolaimus, Pra- organism (Martinez et al, 1970, and
tylenchus, Xiphinenm, Diphtherophora, T. semi-penetrans. It was shown in the
tylenchus, ty phinema, Diphtherophora, inoculation experiments that the nema-
Helicotylenchus, Paratylenchus, and Cri- todes had a higher rate of reproduction in
conemoides showed neither evidence of severely infected wi the green-
root damage nor any increase in nema- pateee inft with the gree
tode population of Szinkom seedlings. ing pathogen than in plants with traces
On the contrary, there was a considerable of greening. This may indicate that the
decrease in the nematode population physiological changes in the roots
beyond the inoculum level which is an brought about by the infection of the
indication that Szinkom seedlings were greening pathogen had provided a favor-
unsuitable hosts for these nematodes. able biochemical environment for the
In the interaction studies between nematodes to reproduce.


~r _d 'F OI-____21-^ ~_L1












I TTED A TT


two biotypes of citrus nematode on citrus and on some other hosts. J. Nematc
1: 150-159.
BAINES, R.C., T. MIYAKAWA, and R.H. SMALL. 1967. Biotypes of the citrus nem;
tode (Tylenchulus semipenetrans) and their effects on resistant rootstock. Nem
tologica 14: 147. (Abstr.)
BAINES, R.C. 1950. Citrus-root nematode investigations. Helminthol. Abstr. 19: 87.
COBB, N.A. 1941. Citrus-root nematode. J. Agr.Res. 2: 217-230.
DAVIDE, R.G. and A.G. DELA ROSA. 1971. The association of plant parasitic nem,
todes with the citrus decline in the PhiliDDines. PhiliDDine Aer, 55: 26-66.


semipenetrans into rough lemon rc
roots. Phytopathology 52: 9. (Abs
MARTINEZ, A.L. and J.M. WALLACI
citrus in the Philippines. Philipp4 PI
MARTINEZ, A.L. and J.M. WALLACE,
Philippines and transmission of th
Dis. Reptr. 51: 692-695.

MARTINEZ, A.L., D.M. MORA and A.
of citrus greening disease in the
biotics. Plant Dis. Reptr. 54: 1007
O'BANNON, J.H., C.R. LEATHERS
Tylenchulus semipenetrans and
Phytopathology 57: 414-417.
O'BANNON, J.H., C.R. LEATHERS ai
the interacting between citrus nem
spp. to infection to rough lemon ((
TRINIDAD, A.G. and R.G. DAVIDE.
citrus decline in Batangas. Agr. Los BI
VAN GUNDY, S.D. and P.H. TASO.
nematode Tylenchulus semipenetrn
VAN GUNDY, S.D. and P.H. TASO.
Fusarium solani as influence by ti
pathology 53: 488-489.


ts and in soil and its relation to Fusarium in
.)
1967a. Studies of the leaf mottle-yellows
it Industry 32: 153-164.
L967b. Citrus leaf-mottle-yellows disease in
casual virus by a psyllid Diaphorina citri. PI


. ARMEDILLA. 1970. Supression of sympt,
iilippines by treatment with tetracycline a
009.
id H.W. REYNOLDS. 1967. Interactions
isarium spp. on rough lemon (Citrus lim

I H.W. REYNOLDS. 1966. The relationship
ode (Tylenchulus semipenetrans) and Fusar
trus limon) Dis. Abstr. 27 B-20.
369. Plant parasitic nematodes associated %
os 9: 12-14.
963. Infesting citrus seedlings with the cil
s. Phytopathology 53: 228-229.
963. Growth reduction of citrus dseelings
citrus nematode and other soil factors. Phi









A


Received for publication: 15 May, 1978

SURVEY OF HIGHER PLANT,
AGAINST PYRICL

D. B. LAPIS and

Associate Professor, Department of I
sity of the Philippines at Los Banos, Collh
Dumaguete City, respectively.
Portion of Master's thesis of the junii

ABS

Ninety three plants were scree
cularia oryzae, the causal organism
inhibitory to the growth of the test f
Of the 19 plant extracts, six
at different dilutions of the superna
the effect of time. In most cases, i
solution; only Impatiens balsamina
The precipitate was more active tha
tion of Pseudocalymna alliaceum w
cantly more active than its precipit
extracts decreased with time.
I. balsamina, 1, alliaceum, Allil
their assayed in vivo to test the eff
peutant or both, and the effect of ti
of extracts.
I. balsamina was found active
alliaceum, A. sativum and T. ereci
Spray intervals of 8 days was found
of I. balsamina and 4 days for theral
A. sativum and T. erecta.

The acceptance of fungicides as part-
ners to herbicides and insecticides as an
agricultural input have been recognized
for several decades. But, the extensive
use of the former has been hindered by
several factors, foremost, is the cost-
benefit factor. In the present decade, the
cost of these chemicals has tremendously
increased and is continuously increasing
to become prohibitive to small farmers.
Aside from these factors and due to
intensive cropping, crop protection pro-
grams and demand for the availability of
the recommended fungicides are
somewhat affected. In some instances,
they are not available.


FOR FUNGICIDAL PROPERTIES
ARIA OR YZAE CAV

;DNA DUMANCAS

mt Pathology, College of Agriculture, Unive
e, Laguna and Instructor, Siliman Universit

author.

RACT

ad for fungicidal property against Pyri-
f rice blast. Of these, 19 were found
igus.
ere further assayed to test the activity
nt and precipitate of the extracts, and
e extracts were active at 1:10 aqueous
vas found active up 1:1000 dilution.
the supernatant liquid with the excep-
arein its supernatant liquid was signifi-
e. Generally, the activity of the plant

i sativum and Tagetes erecta were fur-
t of the extracts as protectant, thera-
e interval of spraying on the effectivity

Sa therapeutant and protectant while P.
were found to be therapeutant only.
Necessary to obtain the protective effect
utic effect of I. balsamina, P. alliaceum,


been found in wide application are the
inorganic fungicides. These fungicides
which are generally used by crop
producers often leave toxic residues to
the soil, water, atmosphere and most
especially on plants and plant products
eaten by animals and men. This is pol-
lution and positively endangers the lives
of mankind.
The cost, availability, environmental
pollutions, and hazard to human and ani-
mal health posed by the aforementioned
group of fungicides, are good bases for
investigation. Hence this study is focused
on searching for naturally-occurring anti-
fungal substances from higher plants that
h-- ot-h+ir4r na;noi+ D j-*-a I thrniloh m











vitro and in vivo tests

MATERIALS AND METHODS

Selection and maintenance of plant
materials
The selection of the plants was based
on reports and scientific findings with
special reference to local species. Most of
the plants chosen for the screening
purpose are maintained at the College of
Sciences and Humanities Hortorium, UP
at Los Bafios.

Laboratory screening ofplant materials
Ninety three (93) plants were screened
for its activity against P. oryzae (Table 1).
Isolation of the test fungus. The test
fungus was isolated using the tissue
Culture method as described by Riker
and Riker (1936). After 1 to 2 days of
incubation, the fungus was transferred to
potato dextrose agar (PDA) slants and
maintained at 28 C with continuous light.
This served as the pure culture and source
of inoculum used in the assay.

Preparation of inoculum and spore
suspension. A 14-day old culture of
P. oryzae was used as the source of
inoculum. Calibrated spore' suspension of
the test organism was prepared asepti-
cally. Slant cultures were flooded with
sterile distilled water and surfaces were
scraped gently with a sterilized transfer
needle. The suspension was decanted
through a double layer of sterilized gauze
into a sterilized 250-mi erlenmeyer
flask. Spore concentration was determined
using a hemacytometer, following the
procedure described by Sharvelle (1961).
Spore concentration was standardized at
5000 to 6000 spores/ml.

Preparation of spore-seeded agar plate.
Using a sterilized pipette, 2 ml of the cali-
brated spore suspension was seeded into a
sterile petri plate. Ten ml of PDA was


poured into the seeded plate. The plate was
rotated to facilitate even distribution
of organism within the agar. Then the
agar was allowed to solidify.


Preparation of crude extracts. Plant
materials were collected the day before
or on the day of the assaying. Plant
materials collected the day before were
placed individually in plastic bags and
were stored in the refrigerator. In all
cases, only the leaves were utilized
unless otherwise indicated.

Twenty grams of the plant material
was used in preparing the crude extract.
The weighed plant material was washed
with 10% chlorox, rinsed three times
with sterile distilled water, then homo-
genized with 40 ml sterile water in a
Waring Blendor. The homogenous
mixture was filtered through a double
layer of sterilized gauze.using a sterilized
250-ml capacity erlenmeyer flask as a
receiver. The filtrate served as the crude
concentrated extract.


Assay of effectivity of the extracts.
The effectivity of the extracts of each
plant materials was assayed using the fol-
lowing procedure: Sterile chromato-
graphic paper discs with a diameter of 6
mm was impregnated with the plant ex-
tract by dipping. Four impregnated discs
were transferred equidistantly to the
spore-seeded agar plate previously pre-
pared under aseptic conditions.
The plates were properly labelled
and incubated under room temperature.
Zone of inhibition was measured after 2,
4 and 8 days of incubation.
A series of bio-assay discs impregnated
with sterile distilled water served as
controls.
All treatments were replicated three
times.


i4


------rr----- --J ---r---------~~










* Plants for Fungicidal Prope


ble 1. List of plant species screened for fui

Family Scienti


anthaceae Asystasis gar
Barleria crist
Graptophylh

liaceae Allium cepa
A. sativum L

naranthaceae Amaranthus
Aerva lanata

lacardiaceae Mangifera in

nonaceae Anona squan

iocyanaceae Catharanthui
Nerium indic
Plumiera acu
Tabernaemor

aceae Amorphopha

Isaminaceae Impatiens ba

sellaceae Baella rubm ]


Icidal property'

: Name Common
and/or Local

tica (L.) T. Anders asistasia
SL. violeta
* pictum (L.) Griff. barabas or ati

onion (bulb)
garlic (cloves)

inosus L. uray
.) Juss. apug-apugan

a L. mango

;a L. atis

,seus (L.) Don. sirsirika
i Mill. adelpha
nata Ait. kalachuchi
ina pandacaqui Poir.. pandakaki,

is campanulatus Roxb. pungapong

mina L. kamantigue

Alugbati


noniaceae Crescentia cujete L. calabasa
Pseudocalymna aliaceum Lam.) Sandwitz garlic vine
Tecoma stands (L.) HBK yellow alder

nbacaceae Ceiba pentandra (L.) Gaerta. Kapok

raginaceae Symphytum officinale L. comfrey

asalpinaceae Cassia alata L. acapulco
Piliostigma malabaricum Roxb.) Benth alibangbang

icacaceae Carica papaya L. papaya

nmelinaceae Zebrina pendula Schmizl. wandering dew

npositae Ageratum conyzoides L. bulak-manok
Artemisia vulgaris L. damong-maria
Bulmea balsamifem D.C. Prodr. sambong
Tagets erecta L. marigold

ivolvulaceae Wedelia trilobata (L.) Hitchc. wedelia
Ipomoea aquatic Forsk. kangkong
I. batatas (L.) Lamk. camote


25












Crassulaceae Bryophyllum pinnatum Kurz. kataka-taka

Cruciferae Brassica integrifolia O.E. Schulz. mustard
B. oleracea var capitata L. cabbage (he
Raphanus sativus L. radish

Cucurbitaceae Gymnopetalum chinense (Lour. Merr.) tamleng
Momordica chamntia L. ampalaya

Elaeocarpaceae Muntingia calabura L. datiles

Erythroxylaceae Erythozylym coca Lam. cocaine

Eurphorbiaceae Antidesma pentandrum Blanco Merr. binayoyong
Euphorbia hirta L. kolindrina
E. pulcherrima Wild. poinsetia
Jatropha gossypifolia L. tua-tua or d
J. podagrica Hook gout plant
Manihot esculenta Grantz kamoteng-k
Mallotus philippinensis (Lam.) Muell. Arg. banato
Pedilanthus tithymaloides var. variegatus lady's slippi
(L.) Poit.

Graminae Cymbopogon citratus (D.C. ex Nees.) Stapf. tanglad
(Poaceae)

Labiatae Coleus blumei Benth. mayana
(Lamiaceae) Mentha arvensis L. herba bueni
Pogostemon cablin (Blco.) Benth. ablin

Lythraceae Lagertroemia speciosa (L.) Pers. banaba

Malvaceae Hibiscus esculentus L. okra
H. rosa-sinensis L gumamela

Mimosaceae Samanea saman (Jacq.) Merr. akasia

Moringaceae Moringa oleifem L. malunggay

Musaceae Musa x paradisiaca L. latundan (ri
Musa x paradisiaca L. latundan (u

Myrtaceae Psidium guajava L. guava

Nyctaginaceae Mirabilisjalpa L. alas-cuatro

Oleaceae Jasminimum multiflorum L. sampaguitai

Oxalidaceae Averrhoa bilimbi L. kamias

Pandanaceae Pandamus odoratissimus L. f. pandan

Papilionaceae Derris elliptica (Roxb.) Benth. tubli
Dolichos tablab L. batao


^jU


L -









Higher Plants for Fungicidal Properties


Table 1. (continued)


Piperaceae


Plumbaginaceae


Piper-lalot D.C.

P. auriculata Lain


Portulaceae Portulaca oleracea L.
Talinum traingulare (Jacq.) Gaertn.


lalot


plumbagin-bughaw


kulasiman
talinuin


Rubiceae



Sapotaceae

Solamaceae



Sterculiaceae


Tiliaceae


Verbenaceae


Zingiberaceae


Gardenia jasminoides Ellis.
Ixora chinesis Lam.
L coccinea U L.

Citrus madurensis Lour.

Cestrum nocturnum L.
Datura metel L.
Lycopersicum esculentum Mil.


Corchorus olitorius L.


Leucosyke capitellata (Poir.) Wedd.

Callicarpa candicans (Burm.) Hochr.
Clerodendrum intermedium Chain.
C. quadriculare (Blco.) Merr.
C. siphonathus R. Br.
Lantana camera L.
Premna odorata Blco.
Stachytarpheta jamaicensis (L.) VahL
Vitex negundo L.


Zingiber offinicale Roxc.


rosal (flower)
santan-tsina
santan-pula

calamansi

dama de noche
talong-punai
tomato


saluyot

alagasi


tigau
kasongpangil
bagawak
music bush
wild lantana
alagao
candi-dcandihan
lagundi

ginger (rhizomes)


Dilution assay

Crude extracts found promising in the
above test were assayed for their effecti-
vity in different concentrations. 1:10,
1:100 and 1:1000 dilutions were prepared
from the concentrated crude extract and
assayed separately following the same
procedure as in the screening of the plant
materials. Discs impregnated with sterile
distilled water served as control.
All treatments were done in three trials
of three replicates each.

Further assay of promising extracts

A further assay of the promising ex-


tracts was conducted simultaneously with
the assay of the different dilutions. The
crude sap was centrifuged for 10 to 15
minutes at 1400 to 1500 rpm. The super-
natant was separated from the preci-
pitate by decantation. The precipi-
tate was diluted with 2 to 5 ml of sterile
distilled water. The amount of water
added was determined by the amount of
the precipitate collected to make a 1:1
concentration.
The supernatant and the diluted
precipitate were assayed separately for ef-
fectivity following the same procedure as
in the above.

In-vive bioassay in the greenhouse


January, 1979











Test Plants. IR-8 variety as test plant RESULTS
was grown in nursery plot measuring
1 meter x 20 meters. The nursery plot Screening of plant materials
was subdivided into 1 meter x 1 meter
subplots spaced 1 meter apart. The sub- Nineteen plants were found active
plots have seven rows with each row against P. oryzae in the preliminary assay
being planted to 5 grams of seeds, of 93 plant materials (Table 2).
The number of plots represents the
number of plant extracts that were Dilution and longevity assay of the plant
chosen from the in-vitro assay. extracts
The treatments were completely ran-
domized with three replicates. Three trials Six from the 19 plant materials found
were conducted, inhibitory to P. oryzae were further as-
Spraying of the crude extract. A sayed for their activity at different dilu-
modified Hudson Lektric sprayer attached tions. The size of the inhibition zone and
to a pressure/vacuum pump was used in availability of plant materials were the
spraying. The pressure was maintained at factors considered in selecting the plants
5 psi. Each treatment was sprayed with for further assay.
10 ml of the concentrated crude extract. The result of the dilution assay of the
Therapeutic effect. The crude extract different plant materials to evaluate their
was sprayed 1, 2, 4 and 8 days after in- inhibitory property to the growth of P.
oculation. Plots sprayed with distilled oryzae is shown in table 3. The concentra-
water served as control, ted crude extracts of all the plant
Protective effect. The plants were materials were significantly more active
sprayed with the extracts 1, 2, 4 and 8 than their diluted forms. I. balsa-
days before inoculation. This assayed the mina was active up to 1:1000 dilu-
protective potential as well as the longevity tion T. erecta was active at 1:100 dilution
of the extracts in preventing infection, but showed the same activity as 1:10 dilu-
Spraying was done when the seedlings tion. All of the plant materials except
were 2 weeks old for protective and the- E. pulcherrima were active against the
rapeutic effects. growth of P. oryzae at 1:10 dilution.
Inoculation. Infected rice leaves were In all cases, the plant extracts decreased
finely chopped and served as the inoculum its activity in suppressing the growth of
source. The inoculum was broadcasted in the test organism with an increase in
the space between the first and the dilution.
second row and between the sixth and
the seventh row of the rice seedlings in In me concentrated form, there were
the plot. significant differences among treatments,
Gathering of data. Data were collected- with the exception of S. oficinal
at the necrotic stage of the disease. In the with the exception of S. off-
















other.
The result of the longevity assay of
the plant extracts against the growth
of P. oryzae is shown in table 3. The In the case of P. alliaceum, valuo
growth activity of I. balsamina on the within the different dilutions did n<
2nd day and on the 4th day were not significantly change with time excel
significantly different, but showed a sig. for 1:10 dilutions wherein the zone (
nificant decrease on the 8th day P. inhibition measurements decreased sign
alliaceum, A. sativum and T. erecta ficantly with time. On the 2nd day, tl
showed a significant decrease in its ac- zone of inhibition as affected by tl
tivity with time. The activities of S. concentrated extract did not diffi
officinale and E. pulcherrima did not significantly with 1:10 dilution. On tl
significantly change with time. 4th and 8th days, the activity of tl
concentrated extract and 1:10 dilutio
When the measurement was done was significantly different from eac
on the 2nd day, I. balsamina and P. other.
alliaceum did not significantly differ,
as well as A. sativum and T. erecta. The activity of the concentrate
On the 4th day, A. sativum and T. erecta extract of A. sativum cloves decrease








30 Philippine Phytopathology Vol. 15

Table 2. Plant species showing activity against Pyricularia oryzae based on the zone of
inhibition


Plant Diameter of Plant Diameter or
species inhibition species inhibition
zone (mm) zone (mm)

A. sativum 45.00 T. erecta 25.30
L balsamina 30.00 S. officinale 25.00
A. capitellata 29.18 S. jamaicensis 24.33
B. pinnatum 27.00 C. siphonanthus 24.00
K pulcherrima 27.00 A. pentandrum 23.70
D. eleptica 26.33 J. gossypifolia 23.33
P. alliaceum 26.25 A. balsamifera 21.58
V. negundo 26.25 B. integrifolia 16.6.6
E. coca 25.42 T. pandacaqui 15.58
L batatas 25.33

Average of three replicates, each replicate with four discs or an average of 12 discs.

Table 3. Average zone of inhibition (mm) of Pyricularia oryzae showing the fungicidal
property of the plant species at different dilutions, measured at different days



Plant D Dilution
materials A
ecies Cone. 1:10 1:100 1:1000 Control
species Y

I. balsamina 2 30.01 22.66 20.07 17.31 0.00b
4 27.89 20.80 18.33 16.68 0.00
8 26.81 19.84 15.61 14.26 0.00

P. alliaceum 2 45.00 45.00 0.00 0.00 0.00
4 45.00 30.52 0.00 0.00 0.00
8 45.00 22.33 0.00 0.00 0.00

A. sativum 2 42.36 23.97 0.00 0.00 0.00
4 38.86 16.03 0.00 0.00 0.00
8 30.31 14.44 0.00 0.00 0.00

T. erecta 2 26.53 21.41 19.72 0.00 0.00
4 21.97 18.49 16.501 0.00 0.00
8 17.40 13.12 10.44 0.00 0.00

S. officinale 2 24.30 14.12 0.00 0.00 0 00
4 25.08 16.39 0.00 0.00 0.00
8 26.47 16.36 0.00 0.00 0.00

E, pulcherrima 2 24.14 0.00 0.00 0.00 0.00
4 26.18 0.00 0.00 0.00 0.00
8 26.86 0.00 0.00 0.00 0.00

aAverage of three trials with three replicates per trial.
bFigures not connected by lines are significantly different (0.05) by Duncan's
Multiple Range Test.
















E. pulcherrima was active against hand, exhibited an inactive supernatant
P. oryzae only in concentrated extract, against P. oryzae
the activity of which did not change
significantly with time.
The activity of the supernatant
Assay of supernatant liquid and precipi- and the precipitate of I. balsamina, T.
tate of the plant extracts erecta and S officinale against P. oryzae
was the same. However, the precipitate
A further assay of the promising of E. pulcherrima was as active as the
extracts was conducted simultaneously precipitate of I. balsamina. Both the su-
with the assay of the different dilutions. pematant and the precipitate of
T ._--.-* ..


L;ulllpitlu


assayed separately to further evaluate natant a
the property of the toxic compound. sativum
larger zon
The effect of the assay of the super- ceum aga


the precipitate of A.
racts gave significantly
Sinhibition next to P. allia-
the growth of P. oryzae.


plant extract on the growth of P. oryzae In Vivo bioassay in the greenhouse
is shown in table 4. Generally, there was
no difference between the two compo- The fungicidal substances from A.
nents of the plant extracts in their sativum, P. alliaceum, I. balsamina,

Table 4. Average zone of inhibition (mm) of Pyricularia oryzae treated with the supe
natant liquid and the precipitate of the plant extracts



Plant Supernatant Treatments Control
anriup linuid Precinitate


39.12a 0.00
32.47b 0.00a
23.74cd 0.00a
19.09d 0.00a


P. alliaceum
A. sativum


Y


S. -,-d - c C









N


Id R


Test.









January, 1979 Higher Plants for Fungicidal Properties 33

alliaceum extracts were deposits of dark are 2-methoxy-l-napthoquinone from I.
green materials which adhered to the balsamina, allyl isothiocynate from B.
leaf surfaces for 3 days. The spray resi- integrifoila (Pryor et aL 1940) and allicin
dues of T. erecta and P. alliaceum were from A. sativum (Cavallito et at 1945).
still clearly visible on the foliage even
after 3 days.
The significant decrease of the zone
DISCUSSION of inhibition with time as affected by
T. erecta, P. alliaceum and A. sativum
The result of the screening of plant corroborates with previous findings
materials for fungicidal property is (Lapis and' Dumancas, 1978). However,
an indication that the occurrence of subs- there was an increase in the activity of
tances having active agents is not un- S. officinale and E. pulcherrima on the
common among higher plants. Of the 19 4th and 8th days after seeding suggestive
plant materials that showed active prin- of slow diffusion of the active compo-
ciples against P. oryzae, six species have nent into the medium effecting slower
been reported to possess antibacterial action against the test organism. This is
and/or antifungal properties. These were further supported by the inactivity of
B. pinnatum, T. erecta, I. batatas, B. the supernatant of the extract.
integrifolia, I. balsamina (Walker, et. al The inactivity of the supernatant
1937; Pryor et. al. 1940) and A. sativum suggests a low solubility in water of the
(Ark and Thompson, 1959; Tansey and active component of the leaf, thus the
Appleton, 1975). So far, only few com- very slow diffusion into the medium.
pounds have been isolated from the
plants under study. The active compounds As in the results with H. oryzae


Table 6. Average number of blast lesion on rice plant treated with the crude extracts
after inoculation'

Days after Inoculation
Treatments 1 2 4 8 Average

I. balsamina 1.95 1.27 2.41 19.30 6.23
P. alliaceum 2.17 2.66 4.39 13.11 5.732
A. sativum 5.07 4.54 7.33 18.84 8.95
T. erecta 3.08 5.08 6.97 16.98 8.03
Control 6.78 6.09 9.18 29.36 12.85

Average 3.81 3.93 6.06 19.64


'Average of 3 trials with 4 replicates per trial and 10 plants per replicate.
2 Figures not connected by lines are significantly different at 0.05 level by Duncan's
Multiple Range Test.









Philippine Phytopathology


(loc. cit) the activity of the active com-
ponent is likely to be influenced by the
methods employed. From this, the in-
activity in the diluted form of the plant
extracts to control growth of P. oryzae
is perhaps due to the inability of water to
dissolve the active compound in the plant
materials.

A spray interval of 1 or 2 days before
inoculation of P. oryzae gave the same
effect as in the previous study with H.
oryzae (loc cit.) Similarly, the effect of
the spray interval 4 and 8 days before ino-
culation were not significantly different
but differed significantly with those of 1
2 days, thus suggesting that spraying the
plants with the crude extracts, 1 and 8
days or at weekly interval, is enough to
give a protective effect with balsamina.

The effects of spraying the crude
extracts after inoculation at 1- and 2-day
intervals did not differ significantly, but
significantly differed with 4 and 8 days,
thus again suggesting that a closer
interval of 4 days is necessary to get the


expective therapeutic effect of I. balsa-
mina, P. alliaceum, A. -sativum and
T. erecta.


The presence of active principles in
higher plants is influenced by several
factors as discussed in the previous
paper (loc. cit.). From the present results,
it wih be noted that some plant materials
showed a larger zone of inhibition during
the screening assay than those of the
results in the dilution and further assay
of the supernatant and the precipitate,
corroborating the factors influencing the
presence of active components in the
plant.


Further studies in the extraction,
synthesis, mode of action, longevity and
factors influencing the presence of the
active compound, dosage and economics,
is suggested to be able to recommend
the possibility of successful large-scale
production for crop protection against
diseases in plants.


LITERATURE CITED

ARK, P.A. and J.P. THOMPSON. 1959. Control of certain diseases of plants with anti-
biotics from garlic. (Allium sativum L.). Plant. Dis. Reptr. 43:276-282.
CALVALLITO, C.J., J.H. BAILEY and J.S. BUCK. 1945. The antibacterial principle of
Allium sativum. III. Its precursor and "Essential oil of Garlic." J. Amer. Chem.
Soc.67:1032.
LAPIS, D.B. and E.E. Dumancas. 1978. Fungicides activity of crude plant extracts against
Helminth osporium oryzae. Philipp. Phytopathol. 14:23-37.
LUGOD, G.C. and J.V. PANCHO. ca. 1966. Medicinal plants in the College of Agriculture
Arboretum and vicinity. College, Laguna: UPCA Press, 47 p.
PRYOR, D.E., J.C. WALKER and M.A. STAHMAN. 1940. Toxicity of allyl isothiocyanate
vapor to certain fungi. Am. J. Bot. 27:30-38.
RIKER, A.J. and R.S. RIKER. 1936. Introduction to Research on plant Diseases. St.
Louis, Chicago: 117 p.
SHARVELLE' E.C. 1961. The nature and uses of modern fungicides. Minnesota: Burgess
Publishing Co., 308 pp.
TANSEY, M.R. and J.A. APPLETON. 1975. Inhibition of Fungal Growth by Garlic
Extract. Mycologia 67:409-413.
WALKER, J.C., S. MORRELL and H.H. FOSTER. 1937. An antifungal substance in the
corn plants and its effect on the growth of two stalk rotting fungi. Nature 183:341.


Vol. 15
















hilipp. Phytopathol. 15:35-46
received for publication: 28 September, 1978

LATENT INFECTION IN MANGO CAUSED BY
COLLETOTRICHUM GLOEOSPORIOIDES

VICTORIA R. DAQUIOAG and TRICITA H. QUIMIO

Research Assistant and Assistant Professor, respectively, Department of Plant
ethology, College of Agriculture, U.P. at Los Banos, College, Laguna.
Portion of the thesis of the senior author presented for graduation for the degree of
aster of Science in Agriculture, U.P. at Los Banos.

ABSTRACT

The presence of latent fungal infection and its histopathology were
determined on naturally-infected and artificially-inoculated mango fruits.
Artificially wounded mango fruits still attached to the tree formed
calluses whether or not spores of C. gloeosporioides were introduced. Seem-
mingly these calluses served as a barrier which inhibited hyphal ramification
and extensive colonization. No typical lesions were observed on unwounded
attached fruits until they were fully ripened. Appressoria and h phae were
the forms in which the fungus survived the period of latency.
Unwounded mature green fruits inoculated while still attached to the
tree produced anthracnose lesions 3 days after picking and incubating at room
conditions. This clearly indicates detachment as one of the pre-requisites for
symptom development.
Parallel experiments in the laboratory on detached fruits showed that
typical lesions developed on wounded immature fruits 4 days after inocula-
tion; wounded mature green fruits, 48 hr, and wounded ripe fruits, 24 hr.
On unwounded detached fruits, typical lesions were also observed on im-
mature ones at about 13 days; on mature green fruits, more than 8 days and
on ripe ones at 4 days after inoculation.








Philippine Phytopathology


ceding penetration. Infection hyphae can
also be produced from appressoria. These
appressoria remain viable on the surface
of the suscept for sometime, but infec-
tion will not yet take place. As soon as
the infection hypha penetrates the
suscept's cuticle and the outer epidermal
cell wall, this could be the start of patho-
genic relationship. However, time may
pass between penetration and the start
of such relationship. This stage is referred
to as latent or incipient infection. In this
condition, actual infection has taken
place, though macroscopically not yet
discernible, but further development of
the infection hypha is delayed.
Most of the works on latent infection
has been done with tropical fruits and
apples. In banana, papaw (or papaya),
mango, citrus, avocado, cacao and tomato
fruits, Colletotrichum spp. produce latent
infections. Wardlaw and Leonard (1936)
demonstrated #he presence of C. gloeos-
porioides in tle skin of green mango and
papaw. C. gloeosporioides was isolated
from the skin of mango, avocado and
citrus fruits. (Baker, 1938); and from the
skin of apparently sound grape fruit in
storage, cacao and tomato (Baker, 1938;
and Baker and Wardlaw, 1937).
Apparently, detailed information re-
garding the mechanism of latent infection
in mango by C. gloeosporioides is in-
adequate. This investigation is necessary
in order to establish the precise nature of
latent infection, mode and time of entry
and spread of the fungus. This would also
provide a sound biological basis for adapt-
ing and programming suitable control
measures to minimize postharvest wast-
age, and to a considerable extent, help
ensure the successful export of the fruit.


MATERIALS AND METHODS

Culture of C. gloeosporioides was ob-


trained from mango fruit lesions using
tissue culture technique and grown on
yeast extract agar. Spore masses were
washed-off from 7- to 14-day old agar
cultures with sterile distilled water. The
concentrated spore suspension was in-
troduced on wounded or unwounded
attached and detached fruits.


Inoculations on attached fruits

A mango (cv. Carabao) tree growing in
the orchard of the Horticulture Depart-
ment, UP at Los Bafios was used as source
of test fruits in the experiment. Two
weeks after fruit set, 24 fruits were chosen
and marked. They were surface-sterilized
by wiping with wad of cotton wet with
1:1000 HgC12 and were rinsed with tap
water.
Eight fruits still attached to the tree
were wound-inoculated. Wound inocula-
tion was performed by making super-
ficial punctures in small areas on the
mango fruits and subsequently introduc-
ing the inoculum of C. gloeosporioides on
the wound. The punctures were made by
an improvised inoculator composed of
several pins stuck to a rubber stopper oc-
cupying approximately a 7-mm diam
(Quimio and Quimio, 1974). Wound
spots were made^near the base, middle
and above the tip of one side of the
fruits. The inoculum suspension was
dropped on each wounded area. Another
set of eight attached fruits were inocula-
ted by allowing spore suspension to flow
freely from the base of one side of the
fruit without wounding. The line of flow
was marked with pentel pen in order to
trace the path of the inoculum. The re-
maining eight fruits were also wounded
but drops of sterile distilled water were
placed on them to serve as checks. Fruits
were enclosed in moist plastic bags with
the aid of stapler and masking tape to
minimize contamination and at the same


Vol. 15








Latent Infection in Mango


time serve as incubation chamber.
Symptom development on the fruits
was observed daily. Every observable
pathologic reaction of the fruits was noted.
Treated fruits were sampled or picked 1,
2, and 3 weeks after inoculation for histo-
logical study of latent infection. Histolo-
gical study of latent infection was done
by employing the Paraffin technique.
Tissues were fixed in FAA, dehydrated in
a tertiary butyl alcohol series, and infil-
trated and embedded in paraffin. Sections,
15 u thin, were cut with the use of a rotary
miprotime, stained and mounted in
Canada balsam. Safranin-fast green com-
bination of stain was used.
One-half the number of the inoculated
fruits were left intact on the tree in order
to determine whether or not ripeness is a
prerequisite for infection. Inoculated
fruits picked during their mature stages
were observed in the laboratory^for pos-
sible infection. This was done to find out
how detachment can influence disease
development.


Inoculation on detached fruits
Parallel inoculations were done on
mango fruits of the same age and variety
using the same inoculum and inoculation
techniques as in the experiment on at-
tached fruits. Inoculated fruits were laid
on plastic trays and incubated in plastic
bags supplied with abundant moisture.

RESULTS

On attached wounded fruits
Callus formation on inoculated and
control fruits were observed 2 days after
inoculation. These corky structures were
somewhat raised, and perhaps served as
barrier in the tissues for penetration and
extensive colonization of the pathogen
(Fig. 1). Sections from wounded fruits
harvested 6 days after inoculation re-
vealed that the fungal hyphae were pre-
sent within the phellem cells. Arrested
hyphal growth were observed in the
mesocarp wherein these corky structures










"


Fig. 1. Callus formation on inoculated attached mango fruit. The
somewhat raised corky structures could be clearly seen 2 days
after inoculation (x 1/2).


January, 1979











were formed. The depth of fungal pene- observed on intact mango fruits during
tration was limited by the presence of the 3-week observation. However, the re-
the host barrier in the form of a ligni- maining wounded attached fruits showed
fled layer of cells encompassing the typical anthracnose lesions 3-4 months
wounded area (Fig. 2). The cells appeared after inoculation. At this time, the fruits
as reddish brown spots and were associ- were fully ripened.
ated with hyphal growth. These observa-
tions were likewise noted on sections cut On attached unwounded fruits
from the wounded fruits harvested at the The fruits remained apparently
2nd and 3rd week intervals. Neither signs healthy 3-1/2 months after inoculation.
nor symptoms of C. gloeosporioides were However, anatomical examination of ino-




































Fig. 2. Transverse section of the mesocarp showing the formation of corky or ligni-
fied cells enclosing the artificial wound. Note that the hyphal growth and
depth of penetration is somewhat limited around this area. This photo-
micrograph was taken 1 week after inoculation (X79).











































Fig. 4. Cross section of the exocarp of attached un- Fig. 5
wounded immature fruit showing the more or
less circular appressorium and the lower
appressorial wall buckled inward (X1250).








Pktpnt Tnfpetinn in Manarn


uits, 48 hr (Fig. 6); and ripe fruits, 24
r (Fig. 7).


The typical symptoms of anthracnose
n immature fruits were noticeable at
)out 13 days after inoculation; mature
reen fruits, more than 8 days (about 200 4


















Fig. 6. Symptom development of an
mango (cv. Carabao) fruits froi
that typical lesions appeared 48















24

Fig. 7. Symptom development of anth
Carabao) fruits from 24 to 96 1
lesions formed 24 hr after inocul


r) (Fig. 8); and ripe ones, 4 days
'ig. 9).
In a water film on the surface of the
*een fruit spores of C. gloeosporioides
:rminated within 12 hr after inoculation
id 20% germination was observed.
erm tubes seemed to be longer than















72 96

hracnose on mature green wounded
24 to 96 hr after inoculation. Note
r after inoculation (X 1/4).















72 96

acnose on ripe wounded mango (cv.
After inoculation. Note the typical
tion (X 1/4).


AI





rrI.MF4,


11~5,rU


L~~ ~ \:ir~' 8'UL TJ


.6'


1(6


(\U


m


- ~ w .J -- Im II


B


ki9Art


I


















- gamzea ana auscolorea. I ne lesions Turnea
cuticle by mechanical means for further black and covered with orane-colored
.. ... ..black and covered with oranite-colored


c - -- ----- -- - -
soria. At 8 days, se
served on the ripen
time, hypha of the 1
penetrated the epi
cell. This epidermal
vity and penetrated
the cell. Conidia we
one-celled, sometin
upon termination.
fection had advar
t;C/ena ramlelinr vi;Qhl


ecks were ob- Spores
ts. During this pension of
ted spores also exocarp si
tissue of the fruits. Ge
resumed acti- about 12 i
llulose wall of 96 hr, slenw
ine, elongated, which coni
coming septate ing on the
9th day, in- tissue (Fig.
roughout the was obser
inns Ilesionsn cuticle. TI


tTT...1. -.. ., 4- U :-,.-- --A .1nnmt-*A .-A-rni1 -1.lle Th-ii npn--ri.


I sus-
n the
ripe
>egan
18 to
uced
mify-
' the
tgress
ermal
liallv





















turned black occupying a that the cork layers may inhibit further
er, the hyphae developed invasion of the fungus into the starchy
)ut in all directions. Acer- mesocarp. When the invading fungus
was observed 7 days secretes hydrolytic enzymes to break
n (Fig. 11). down the cell walls, the damaged cells







SS .








Latent Infection in Mango 45

attached fruits inoculated and ripe mango fruits did not result in the
nature green and mature production of lignified periderm. On
id to show any indication papaya fruit, Stanghellini and Aragaki
te fungus, however, had (1966) attributed this to the higher deg-
ria on fruits that were de- ree of susceptibility of detached than the
and 3 weeks after inocu- attached fruits. They also opined that cer-
iserving the same pheno- tain metabolism normally supplied by the
aoo fruit Binyamini and leaves and present only in low concentra-
(1972) theorized that tions in fruits are not available or/are in-
contained inhibitors of hibited once the fruit has been detached
lent. Barell and Bamell or reaches a certain stage of maturity.
hiakravarty (1957) also This is believed to be the first report on
>e banana fruit contained susceptibility being associated with the
:h as tannins, which in- inability of fruits to respond without sup-
lopment of G. musarium. pression by chemical or physical inhibi-
work of Simmonds, Ver- tors, to normal wound-healing processes.
ined that the green peel
it contained substances Appressoria may not be a requisite
richum sp. for penetration. This was shown by the
production of germ tube from spores
it study, both wounded which directly penetrated the suscept's
I attached mango fruits tissues. However, in some instances,
icnose lesions when fully penetration of the suscept's tissues was
s picked or detached du- affected through infection peg produced
ire green stages showed fromappressoria.
when incubated in the
days. Hence, there is no In adopting postharvest control meas-
s to be present so that in- ure, a thorough knowledge of the mode
& tb.. e .t. in









Philippine Phytopathology


LITERAL

BAKER, R.E.D. 1938. Studies in the path
of latent infections in developing frui
BAKER, R.E.D. and C.W. Wardlaw. 1937.
1. On the types of infection encou
Bot. (n.s.) 1:59-65.
BARNELL, H.R. and E. BARNELL. 1945
of tannins within the banana and the
ripening. Ann. Bot. (n.s.) 9:77-99.
BINYAMINI, N. and M. SCHIFFMANN-:
fruit due to Colletotrichum gloeospor
CHAKRAVARTY, T. 1957. Anthracnose
Massee), with reference to latent ii
40:337-345.
HALOS, P.M. 1970. Sporulation of Diplod
infected mango fruits. (Unpublishec
QUIMIO, A.J. and T.H. QUIMIO. 1974. Po
nose by hot water treatment. Philipp.
STANGHELLINI, M.E. and ARAGAKI,
callose deposition to anthracnose


JRE CITED

genicity of tropical fungi. 2. The occurreno
i. Ann. Bot. (n.s.) 2:0,9-31.
studies in the pathogenicity of tropical fungi
.tered in the storage of certain fruits. Ann

Studies in tropical fruits 16. The distribution
changess in their condition and amount during

ADEL. 1972. Lacent infection in avocado
)ides. Phytophatology 62:592-594.
of banana (Gloeosporium musarum Cke, e
sectionn in storage. Trans. Brit. Mycol. Soc

i natalensis Pole-Evans and histopathology o
M.S. Thesis. U.P. College, Laguna. 64 p
;harvest control of Philippine mango anthrac
Agr. 58:138-146.
1966. Relation of periderm formation am
evidence in papaya fruit. Phytopatholog


1:621-654.


Vol. 15














HERITABILITY OF DIFFERENCES IN VIRULENCE BETWEEN
RACES 2 AND 3 OF COCHLIOBOLUS CARBONUM

S. C. DALMACIO, D. R. MACKENZIE, and R. R. NELSON

Former Graduate Assistant, Associate Professor and Evan Pugh Professor, respec
tively, Department of Plant Pathology, The Pennsylvania State University, University
Park 16820. Present address of senior author: Department of Plant Pathology, Univer
sity of the Philippines at Los Banos, College, Laguna, Philippines.
Contribution No. 1045, Department of Plant Pathology, the Pennsylvania Agri
cultural Experiment Station.
ABSTRACT

Two isolates of Cochliobolus carbonum inciting fleck (Race 2) and
linear lesion (Race 3) reactions on the corn inbred Pa33 were crossed to study
the heritability of the differences. Analysis of 40 ascospore progenies indi-
cated that the differences in lesion length between Race 2 and Race i is a
highly heritable factor (nearly 80%) controlled by as many as five genes.
Moreover, further analysis indicated that significant epistatic effects were
present, precluding narow sense heritability estimates without some exten-
sive backcrossing.
Isolates of Cochliobolus carbonum paper reports quantitative heritability dif-
Nelson (Helminthosporium carbonum ferences in lesion length between selected
Ullstrup) incite three pathogenicity types isolates of Race 2 and Race 3 of C.
on selected maize genotypes. All of the carbonum.
three reactions have been designated as
races with blight type lessons as Race 1 MATERIALS AND METHODS
and flecks as Race 2 on specific inbreds
(e.g., inbred Pr) Nelson & Ullstrup, Two isolates of C. carbonum, one


with this pathogen have shown that the
ability to incite a blight reaction (vs.
flecks) is controlled by a single gene
(Nelson and Ullstrup, 1963). Traditional
genetic studies into the mode of inheri-
tance of Race 3 reactions have been
inconclusive (Dalmacio, 1976). The ob-
servations that isolates inciting linear
lesions varied greatly in their ability to
incite longer or shorter lesions and that
some ascospore progenies of Race 3 X
Race 3 parentage gave only fleck type
lesions led to the hypothesis that isolates
inciting fleck to linear reactions differ


heritability of these racial Mtterences.
Crossing and ascospore isolation pro-
cedures as described elsewhere (Dalma-
cio, 1976 and Nelson, 1959) were
followed.
Spore suspensions of 7-day old cul-
tures were prepared and sprayed into the
leaves of 20- to 30 day- old plants of the
maize inbred Pa 33 using a small hand
sprayer at a constant pressure of 700
g/cm2. Inoculated plants were incubated
for 48 hr in a chamber with an inter-
mittent mist system that operated for
10 of every 30 minutes. Subsequently,









rnllppine rnytopatnology


times, each time representing a replicate.
Seven days after inoculation, 20 random-
ly selected lesions were measured for
lesion length on the third or fourth leaf
for each of the 40 progeny isolates.
Analysis of variance, an estimate of
'broad' sense heritability, estimation of
the number of effective factors (genes),
and tests for epistatic effects were made
according to the methods described by
Burnett (1975) and MacKenzie (1971).

RESULTS AND DISCUSSION

Table 1 presents a two-way analysis
of variance values for lesion length. The
broad sense heritability (h2) is de-
fined as h2 = v2g/v2p where v2g is the
genotypic variance and v2p is the pheno
typic variance given as the sum of the
environmental variance (v2e) and the
genotypic variance (v2g). The narrow
sense heritability estimate is given as the
ratio of the additive genotypic variance
(v2a) to the phenotypic variance (v2p)
where v2g = v2a -- v2I. In haploid
organisms such as C carbonum, it is

Table 1. Analysis of variance for lesion
ascospore progenes derived from
num

Source of Degree of Sums of I
Variation Freedom Squares I

Between
Progeny 39 115.37

Within
Progeny 40 13.45

Total 79 128.80

v2g is the environmental variance;
replications and h2 the broad sense herital
**Denotes a significant difference at tl


effects and hence, no v2 D. The effects of
epistasis (v21) can be estimated as the
distortion of the normal distribution
from the expected by the additive
genetic variance (v2 a).
Differences between Races 2 and 3
in C. carbonum measured as lesion
length proved to be a highly heritable
trait. The broad sense estimate of heri-
tability (h2) was 79.6%, indicating a con-
siderable measure of genetic association.

Estimates of the number of genes
controlling the differences between Race
2 and Race 3 in C. carbonum were cal-
culated by two methods. The first was
by parental differences and the second
by progeny extreme differences as given
by the formula:

(P 1 P2)2
k=
4v2g

andk= (Tf(L) -f(S)2

4 v2g


!ngth on maize inbred Pa33 incited by 4.
,ace 2 x Race 3 cross of Cochliobolus carbc


?an Components F-test h2
uares of Variancea


96 v2e+ r(v2g) 8.81** 79.6%


336 vke



Sis the genotypic variance, r the number a
lity estimate.
0.01 level for an F-test.


vol. 10











Heritability of Differences


respectively, where k is the number of
virulence genes, PF and P2 the lesion
length means of the parents and fl (L)
and f1 (S) the means of the progeny
isolates inciting the longest and shortest
lesion, respectively, and v2 g the genoty-
pic variance. Both methods of estima-
tion assume that all of the genes contri-
buting to the trait are concentrated in
one parent and all of the alternative
alleles in the other parent. It is also
assumed that the genes are unlinked.
Failure of these assumptions will cause


ZIO
W '


C3
w8




Cl)6
u,)


-J4
0


an underestimate of k (the number of
genes). The estimate of number of genes
(k) were 3.5 and 5.0 by the two me-
thods, respectively.


The frequency distribution for lesion
length of the 40 progenies of the Race
2 X Race 3 cross is presented in figure
1. Inspection of the distribution showed
an abundance of progeny isolates inciting
intermediate lesion length. None exceed-
ed those incited by the Race 3 parent









P2







_ t


I 2 3 4 5 6


LESION LENGTH (mm)


Fig. 1. Frequency distribution of lesion lengths among 40 ascospore progenies derived
from a cross of Cochliobolus carbonum Race 2 x Race 3 and tested on maize
inbred Pa33. Symbols P1 and P2 are for mean parental types.


........ ......i:iiii
........... ....ii::ii~
...........if~ifi3
..........:::l l :''l :'i'iiii


:.;.:,:.:,:,;. ,;.:.?;,;.:,;,: :.:,:.;,:.;.;.:.;,:.:,;.;,:.:,:,: ,:.;.:.;.:.:.;,:.2:.:.;.;,;.;,:.:.: ;.:,:,:,:*:*:,:,:,:,:,:.:,:,:.:,:,


January, 1979


. .. ... ... ...
.... ... .... ...
............



......... . .~
....... ...

.... ... ....


..........i~l~
........ ........
.... .............~i:~i~i:~
......... ..:j:::::i::


.................
:- :X:X"". '.x~i
..................iiiii j
..................l~iiiii
........ ........
........ .....ii~ii


?f^^^


.............iii
......
XX~2~~i
. ...... .......



X.:
X X..-:::;;

.................~
... ... ... ...
:X .`''..' I..........
...........iii~ii
X.j:::::.::::::
.........i~~i~iii


.............~
.... ... ... ...
X.,~iiiii I~~
.. .. .

.... .........i
X~~i~ii
..........ii
..............~
................


....... ... .P,~i~~::
............iiji iii
.................~iii
X: X:iiiiiiiiiiii
X.:ii:iiiiiijiiii
Wiiiiiii llii ::


.. ............. .l::i
...........li
..........ii~i
................t~
..................:::i::?
........... .. ...::~:::
...........:::;:--:
.......... .......
...... ........i~
................liii~i
...............iiii~ii
.................i'iiili
X : : '. : -X.Ni.:::~
..................iii~i
X:i~iiii~ii j~i









Philinnina PhwtnnathnlnV


while 25% of the progeny incited lesic
within the range of those incited by I
Race 2 parent.

The skewed progeny distribution i
virulence (Fig. 1) suggested nonalle
interaction (or epitasis). This effect a
estimated by the formula:


I -T-
2

where I is the epistatic effect, PI a
P2 are the parental means for lesi'
length and fl is the progeny met
Significant epistatic effect is prese
when I, the difference between the I
rental means and the progeny mean,
positive and negative, within the lim
of error. If zero, all the genetic variant
is additive. I was estimated to be 1.:
+ 0.47, indicating the presence of ep
tasis.

A narrow sense heritability estims
for these data could not be made for t
above virulence study since it was i1


s epistatic effects. As discussed by Burn
D (1975) -the most convenient meth
of partitioning these variances is
backcrossing.
r
: We present these findings as 4 i
i monstration of analytical methods tl
can be applied to help explain pathol
variation. Our efforts to sort out 1
genetic factors by traditional Mendeli
genetics were fruitless. By the applicati
of these quantitative statistical tei
niques we have been able to provide
assessment of the genetic system cc
I trolling the differences of Race 2 a
I Race 3 of C. carbonum.

t We view these differences betwe
S C. carbonum Race 2 and Race 3 w
i strong feelings of apprehension. C
I evidence suggests that expression
relatively few genes (3-5) to Race
S has significantly heightened the viruler
of this once innocuous pathogen
maize. We suggest that the potent
exists for the expression of a few mc
genes. The consequence of this coi
be the development of new variants
this pathogen which would possess t


BURNETT, J. H. 1975. Mycogenetics;An introduction to the


DALMACIO, S. C. 1976. Genetic studies of pathogenicity type, virulence and sexu
reproduction in Cochliobolus carbonum. Ph. D. Thesis, The Pennsylvania Sta
University, University Park, Pennsylvania. 78 p.
MACKENZIE, D. R. 1971. Qunatitative inheritance of fungicide tolerance in a nature
population of Cochliobolus carbonum. Phytopathology 61:471-475.
NELSON, R. R. 1959. Cochliobolus carbonum, the perfect stage of Helminthosporiu
carbonum. Phytopathology 49:807-810.
NELSON, R. R., and A. J. ULLSTRUP. 1961. The genetics of pathogenicity in Cochli
bolus carbonum. Phytopathology 51:1-2.
NELSON, R. R., M. H. BLANCO, S. C. DALMACIO, and B. SHAIN MOORE. 197
A new race of Helminthosporium carbonum on corn. Plant Dis. Reptr. 57:822-821


Rfn















COWPEA VIRUSES I
H. ISOLATION AND IDEN'
STRAIN OF CUCUM

L. T. I


fHE PHILIPPINES:
ICATION OF A COWPEA
R MOSAIC VIRUS.

.ENS


he Philippines at Los Banos, College, Laguna.
This investigation was supported by research funds provided by the Philippine
government to the Institute of Plant Breeding, University of the Philippines at Los
lanos, College, Laguna. Philippines.

ABSTRACT

A cowpea virus isolate which was found to be transmitted by mechani-
cal inoculation and aphids (Aphids craccivora Koch. and Aphis gossypii
Glover) was investigated.
The thermal instability, positive aphid transmission, morphological
feature, and serological property, and ability of the virus incite mosaic
symptoms identify the virus as a strain of cucumber mosaic virus.
The cowpea strain of cucumber mosaic virus possessed the following
in vitro properties: dilution end-point at 10-4, thermal inactivation point at
70 C; and longevity in vitro for 3-5 days at 25 C. The virus consisted of
isometric particles 28-30 nm in diameter. It reacted in agar gel serological
reaction against reference cucumber mosaic virus antiserum preparations.


Lb L, 1ubUJ
cultivate(
eing system:


'Talens, L. T. 1978. Cowpea viruses in the Philippines: I. Identity ofia mosai,
I:~n :-rt ., :* -^,,./* T7;-n ,. >.. ..i_+^/T \ u r~i T^ K * m:I:-*-I 1 117- o-:




























uwmsg-uii i-i uaum5 Electron
species particularly leaf diD. c


-.. -. --- neura pnospnorungsiate. A JIUL-IUU
test. type electron microscope was used fi
Local lesions which developed in C. observation of virus morphology.
amaranticolor and/or C. quinoa were
excised and repeatedly passed in these Serological test in Ouchterlony(196:
same hosts to obtain the virus isolate. agar gel diffusion plate employing ant
Leaves containing numerous lesions were serum against several legume virus
removed and'ground in a sterilized mortar known to infect cowpea was carried ou
and pestle with 0.05 M phosphate buffer, These antisera were kindly provided b
pH 7.5. The phosphate-buffered homo- Dr. S. Tolin of Virginia Polytechn:
genate containing 1% Celite was rubbed Institute, Blacksburg, Virginia, Dr. J. I
on primary leaves of 10-14 day old Uyemoto of Kansas State Universitl
red cowpea seedlings in which virus Manhattan, Kansas, Dr. R. J. Shepher
induced the appearance of veinal chlorosis of the University of California, Davi
and mosaic symptoms 6-14 days after California, and Drs. H. A. Scott and JP
mechanical inoculation. C amaranticolor Fulton of the University of Arkansa
and/or C. quinoa served as assay host Fayetteville, Arkansas.
plants in tests of the stability properties Agar gel consisting of 1.0% ionaga
of the virus and occasionally in checking dissolved in 0.05 M tris-HCI, pH 7.5 an
the biological purity of the virus isolate.
.025% sodium azide was used. An eight
well pattern surrounding a central depc
s identification was prepared. The peripheral well
The identity of the virus isolate was were charged with reference antisera; th
based on studies ofitsin vitro properties, central well, with antigen being tested
morphological feature, and serological Virus-specific antigen preparations consi:
property. ted of crude cowpea leaf extracts which
was previously clarified by low spee
The properties of the virus in crude centrifugation at 5,000 g for 10 min.
sap were determined according to Bos


Ca,


DU:1::. D----,-- nlrm





!a Viruses in the Philippines


Sa


Rir ,


iptoms induced by the cowpe;
ind in C. quinoa (2).


- that they might be of value
gnosis. In Vigna unguiculata


itoms similar
i nlants were


primary leaves which was later accom- virus-specific was obtained in
Sd by veinal chlorosis and eventual inoculation tests onto C. amaranti


listing of clarified crude sap and partially
purified virus suspensions contained iso-
netric particles 28-30 nm in diameter
'Fig. 5). No other particle types of virus-
ike morphology were seen.
Crude virus sap from infected leaves
ised as source of virus antigen reacted


ninosae, and Solanaceae (Talens, manus-
:ript in preparation).
Studies on the properties of the virus in
,rude sap extracted from infected cowpea
leaf tissues revealed that: (1) infectivity
was retained after dilution to 10-3 but









Philippine Phytopathology


Fig. 3. Symptoms induced by the cowpea virus isolate on uninoculated
trifoliate leaf of cowpea, (Vigna unguiculata (L.) Walp. cv. Red)


Fig. 4. Agar gel double diffusion reaction of cowpea leaf extracts (A and
B) which were infected with the cowpea virus isolate using Aphis
craccivora (A) and A. gossypii (B).


Vol. 15








Cowpea Viruses in the Philippines.


021v agar gel diffi


le to Uuchterlony agar gel .- k-i .- ". "Yr
nd specific reference anti- can transmit the virus in
ble. fashion and the seemi
range exhibited by the
s of particle morphology, manuscript in preparati
ire, and aphid transmission that this virus might be
nt manner,one of the virus portance not only in co


report appeared to be the tirst descrip
tion of CMV in cowpea in the Philippines
Unlike other strains of CMV which have


pea (Benigno and Quebral. 1977; Talens,
in press" ) CMV-cs may aggravate what
might have been a tolerable level of


January, 19


A-









Philippine Phytopathology


Fig. 6. Agar gel double d
between the cowpei
reference antiserun
well was charged wi
ral wells with (a) co
ber mosaic virus-'
virus-Inouye, (d) i
(e) been pod mott]
virus-antiserum prel


sion serological reaction
us isolate (AG) and several
-eparations (a-h). Central
larified virus sap; periphe-
*a mosaic virus (b) cucum-
i, (c) cucumber mosaic
hern bean mosaic virus,
irus, (f) blackgram mottle
tions.


virus infection in cowpea.
Further study is clearly needed to nation-wide cultivation. Likewise, investi-
determine the occurrence of CMV-cs gations on its nature and properties
under natural condition and its mode of should be done in order to establish its
spread particularly in cowpea cultivars relationship with other isometric, non-
which have been recommended for persistent, aphid-borne legume viruses.


LITERATURE CITED

BENIGNO, D. A. and L. L. PAJE. 1975. Little leaf: a new destructive disease of cowpea
Agr. Los Banos 14:3.
BENIGNO, D. A. and F. C. QUEBRAL. 1974. The virus diseases of vegetables and legume!
in the Philippines. Proc. Conf. on Plant Prot. Trop. and Sub-Trop. Areas, BPI
Manila, p 570-582.


, 11


Vol. 1b









January, 1979


Cowpea Viruses in the Philippines. II


BOS, L., D. J. HAGEDORN, and L. QU
national identification of legume vinr
OUCHTERLONY. 0. 1962. Diffusion-ir
Progr. Allergy 6:20-54.
TALENS, L. T. 1977. Cowpea little leaf <
as detected by immunodiffusion tect


'Z. 1960. Suggested procedures for inter-
T. P1. Ziekten 66:328-343.
methods for immunological analysis. II.

se in the Philippines: possible viral etiology
ie. Philipp Phytopathol. 13:43-49.









Philipp. Phytopathol. 15:58-61
Received for publication: 26 March, 1979



INHIBITION OF PATHOGENS OF FIELD LEGUMES BY MIMOSINE

M.D. EBUENGA, LINA L. ILAG and
EVELYN MAE T. MENDOZA

Respectively, Research Assistant, National Crop Protection Center; Senior Plant
Pathologist and Senior Biochemist, Institute of Plant Breeding, College of Agriculture
University of the Philippines at Los Banos, College, Laguna.
This study was supported by the Institute of Plant Breeding, University of the
Philippines at Los Banos.
ABSTRACT

The effects of three mimosine concentrations (0.1%, 0.2%, and 0.8%) in-
corporated in various culture media on the growth of nine pathogens of
peanut, mungbean and soybean were studied.
Mimosine inhibited mycelial growth of Colletotrichum lindemuthianum
(Sace. and Magn.) Bri. and Cav., Sclerotium rolfsii Sacc., Cercospora canes-
cens Ell. and Mart., Diplodia natalensis P. Evans., and an Altemaria sp. patho-
genic on soybean. The compound also inhibited conidial germination of
Cercospora personata (Berk and Court.) Ell. and Everh. and uredospore ger-
ination of Phakopsora pachyrhizi Syd. and Puccinia arachidis Speg. The
formation of sclerotial bodies of S. rolfsii was completely inhibited in all
treatments with mimosine.
Some reduction in the multiplication of cells of Xanthomonas phaseoli
(E.F. Smith) var. sojense (Hedges) Starr. and Burk. was observed in the pre-
sence of mimosine.


Mimosine [beta (N)[ 3 hydroxy-4
pyridone] alpha-amino propionic acid] is
a non-protein amino acid present in the
seed, leaf and other parts of Leucaena
leucocephala (Lam.) de Wit and in
Mimosa pudica L.
Mimosine causes depilatory and other
toxic effects on animals particularly the
ruminants, rats, mice, pigs and poultry
(Thompson et al., 1969; Hegarty et al.,
1964; Brewbaker and Hylin, 1964;
Grounce et al., 1962; Dewreede and
Wayman, 1970; Labadan et al., 1969). It
was also found to be toxic to certain
plants such as mungbean seedlings
(Thompson et al., 1969) and Vicia faba
(Pritchard and Court, 1968).

Little is known on the effect of mi-


mosine on microorganisms particularly
plant pathogens. Suda (1960) noted that
the compound exerted an antagonistic
action against indole acetic acid and cer-
tain amino acids during the growth of the
bacterium Escherichia coli.
MATERIALS AND METHODS

The effects of mimosine were tested
on the following pathogens: Colletotri-
chum lindemuthianum (Sacc. & Magn.)
Bri. & Cav., Sclerotium rolfsii Sacc.,
Cercospora canescens Ell. & Mart., and
Diplodia natalensis P. Evans, all from
mungbean; Puccinia arachidis Speg. and
Cercospora personata (Berk & Court)
Ell & Everh., both from peanut; Phakop-
sora pachyrhizi Syd., Alternaria sp. and
Xanthomonas phaseoli (E.F. Smith) var.








Pathogens of Field Legumes


sojense (Hedges) Starr. & Burk. from
soybean.

The media used in the tests were
infused with 0.0%, 0.1%. 0.2% or 0.3% mi-
mosine which was previously dissolved
in 0.1 N HCL. The pH of all the media
was adjusted to 6.5 after the addition of
mimosine. The effect of mimosine on the
linear extension of fungal mycelia was
carried out in potato dextrose agar (PDA)
with the desired mimosine concentrations.
The experiments on the effect of mimo-
sine on uredospore germination of
P. pachyrhizi and P. arachidis were
conducted in water agar with various
concentrations of the toxin. Observa-
tions on conidial germination of C
personata as affected by mimosine was
made in tap water. The bacterium, X.
phaseoli var. sojense was cultured in
nutrient broth with the desired levels
of mimosine.


The effect of the compound on my-
celial growth was determined by
measuring the daily increase in colony
diameter of the cultures in PDA. The
percentages germination of conidia and


uredospores were determined by counting
the number of germinated spores per
microscopic field relative to the total
number of spores ii the field. The effect
of the various mimosine concentrations
on X. phaseoli var. sojense was studied
by turbidimetric measurements with a
Spectronic 20 and by counting the
colonies formed in nutrient agar infused
with various concentrations of mimo-
sine.

All tests were conducted at least twice
in triplicates per treatment.

RESULTS AND DISCUSSION

All the mimosine concentrations ad-
versely affected mycelial extension and
spore germination of the fungal patho-
gens tested (Tables 1 and 2). The higher
mimosine concentrations were generally
more toxic. However, the lowest mimo-
sine concentration (0.1%) was sufficient
to substantially reduce the growth of
practically all the pathogens tested.
The degree of inhibition caused by
mimosine varied with the test micro-
organism and the concentration of the


Table 1. Average colony diameter (mm) of five fungal pathogens in PDA with various
concentrations of mimosine *


Mimosine concentration (%)
Pathogen
0.0 0.1 0.2 0.3

C. Undemuthtanum 40.5 33.3 31.8 29.0
C. cenescens 18.0 16.6 14.0 11.4
Alternar.a sp. 48.0 3.0 3.0 3.0
. rolflU 39.8 25.3 3.0 3.0
D. nataleuns 47.5 22.3 23.0 22.7


*The data shown for C. Jndemuthfanum, Alternari sp., and C. acneecens were taken after
incubation for 5 days whereas those for S. rolfVs and D. natalensr were made after 2 days of incuba-
tion due to the rapid mycelial expansion of the latter two fungi which entirely covered the surface of
the control plates after the 2nd day of incubation.


January, 1979








'hilippine Phytopathology


able 2. Percentage spore germination of thr
mimosine concentrations



ithogen 0.0


. pachyrhizi 47.0


legume pathogens as affected by various



imosine concentration (%)
0.1 0.2 0.3

19.5 6.9 3.0


P. arachidis 38.6 9.8 5.5 6.b
C. personata 67.9 61.1 60.3 12.5





toxin. Alternaria sp. was the most sensi- centration (0.1%) caused 27.5 and
tive to mimosine wherein 0.1% mimosine 28.8% decline in germination, in P.
caused more than 90% reduction in pachyrhizi and P. amchidis, respectively
mycelial extension (Table 1). Another (Table 2). C. personata was less sensitive
highly sensitive pathogen was S. rolfsii to 0.1 and 0.2%,, but 0.2%, mimosine
wherein 0.1 and 0O2% mimosine caused caused 55.4% reduction in germination.
about 38 and 93% reduction in my- percentage germination.
celial growth, respectively. Moreover, the
formation of sclerotial bodies by the Mimosine also inhibited X. phaseoli
fungus was completely inhibited in all var. sojense as shown by turbidimetric
the concentrations tested. The linear measurements and the plate count
expansion of D. natalensis was also inhi- method. The average numbers of bacte-
bited by the toxin during the first 2 rial colonics/cm2 in nutrient agar with
days of incubation (Table 1) but appre- 0.1%, 0.2% and 0.3% mimosine were 32,
ciable aerial mycelial growth was ob- 32.5 and 60, respectively, compared
served in the mimosine-treated media to 75 in the control plates with no mi-
during the succeeding days. C. lin- mosine.
demuthianum and, to a lesser extent,
C. canescens were also inhibited. We have observed that unlike other
leguminous plants, the stems, leaves,
A separate experiment showed that an pods and seeds of ipil-ipil or L. leucoce-
isolate of Rhizoctonia.solani from soy- phala are relatively free of diseases of
bean was inhibited by 0.02% mimosine any kind. It is a great temptation to at-
and did not grow at all in PDA with tribute this apparent resistance to the
0.2% toxin. mimosine which we found toxic in
varying degrees to all the legume patho-
Uredospore germination of the rust gens tested.We are further tempted to
fungi, P. pachyrhizi and P. arachidis suggest that a practical use of mimosine
Uwa pytrpmplV tun1tl7 f th YP nron;A +nA n nhrieipn ;.n r4 no nn n+-1 m-,r h


LIubfli. 1n1C ILUWCNL rMnnosinv con- onrt


Vol. 15









nT- 1 - --- 0 Ja T


LITER/


BREWBAKER, J.L., and J.W. HYLIN.
Leucaena species and related mimo
CROUNSE, R.C., and D.D. MAXWELL
hair by mimosine. Nature 194: 694
DEWREEDE. S.. and O. WAYMAN. 19


PURE CITED


1964. Variations in mimosine content am<
eae. Crop Sci. 5: 348-349.
nd H. BLANK. 1962. Inhibition of growth
i95.
n wffpat nf mimncinf on th~ r1t fptill R


HEGARTY, M.P., P.C. SHINCKEL and R.D. COURT. 1964. Reaction of sheep to the
consumption of Leucaena glauca Benth and to its toxic principle mimosine. Aust.
J. Agr. Res. 15: 153-167.

LABADAN, M.M., T.A. ABILAY, A.S. ALEJAR, and V.S. PUGTILAN. 1969, The effect
of feeding high levels of ipil-ipil (Leucaena leucocephala) leaf meal on comb and
testis growth of single comb white leghorn cockerels. Philipp. Agr. 53: 402-410.
PRITCHARD, A.J., and R.D. COURT. 1968. The cytological effects of mimosine.
Cytologia 33: 73-81.
THOMPSON, J.F., C.J. MORRIS and I.K. SMITH. 1969. New natural amino acids. Ann.
Rev. Biochem. 38: 137-153.


3-n-,onr 1Q7C












Phlipp. Phytopathol. 15: 6268
Received for publication: 26 March, 1979

IDENTITY OF A STRAIN I
WINGED BEAN (PSOPHOCI

L. T. TALENS and


Virologist and Research Associat
Institute of Plant Breeding, University of
This investigation was supported
Government to the Institute of Plant I
Banos, College, Laguna.

AB

An abnormal condition in wi
L.) which consisted of prominent
tapered ends, green bands, and ser
inoculations of phosphate-buffered
plants resulted in the isolation a
mosaic virus. Identification was baa
an Arkansas strain of cowpea mos
of virus particles consisting of isom
diagnostic reaction of Chenopodi
consisted of local reaction followed


Winged bean (Psophocarpus tetragono


OF COWPEA MOSAIC VIRUS IN
RPHUIS TETRAGONOLOBUS L.)

. C. DOLORES-TALENS


,respectively, Virus Biocontrol Laboratoi
he Philippines at Los Banos, College, Laguna.
ly research grant provided by the Philippi
reading, University of the Philippines at L


TRACT

ged bean (Psophocarphus tetragonolobus
thickening of the veins and leaves with
ted laminae was investigated. Mechanical
homogenate onto selected virus indicator
Id identification of a strain of cowpea
id on positive serological reaction against
c virus antiserum, morphological feature
trickc particles 25-28 nm in diameter, and
m amaranticolor and C. quinoa which
>y systemic invasion.

and a filamentous virus causing necrotic
mosaic. which is not seed-borne. The onlI


substitute source of plant proteins for the "'""' """""""" 5--. -,- ,-
humid tropics. It is presently ranked third sible occurrence of viruses in winged bean
among the priority commodity crops as in the Philippines was based on a com-
established'by the Philippine Council for munication to the Director of the Insti-
Agriculture Research and Resource tute of Plant Breeding from plant scien-
(CA ). While appearing better tists of the Rockeffeler Foundation in
(PCARR). While appearing better in
amino acid composition than most. Jakarta, Indonesia in June 1978. The
legumes, winged bean lacks nutritional communication indicated that winged
bean seed introductions UG 1, 19, and
levels of cystine and methionine (Kap- bean seed introductions UG 19, and
siotis, 1968; Cey et al., 197. 100 might harbor viruses. The identity of
siotis, 1968; Cerny et al., 1971). t
these viruses was not known.
Like many other cultivated crop plants, O o w b
Observations of winged bean plantings
winged bean does not appear to escape
S bat the Institute of Plant Breeding for the
infection by fungi, nematodes, and viru- at e P t r
she's. Fauquet et al. (1978) described two past 2 years suggested the presence of
viral diseases of winged bean in the Ivory both types of diseases, based on field
Coast. Their investigations revealed two symptoms. Unfortunately, the possible
particle-types: an isometric virus causing viral il of these maladies were not
.:... .. .... ... 64, ... A +.a +, pursued further.









Strain of Cowpea Mosaic Virus


the probable viral agents) involved in an
abnormal condition in winged bean being
grown in potted soil for seed multiplica-
tion. The abnormalities consisted of signi-
ficant reduction in plant growth and
architecture, severe distortion of the
leaves, prominent thickening of the veins
and leaves, rugged green edges, and veinal
yellowing (Fig. 1). When clarified leaf
tissue homogenate was used as antigen
source in Ouchterlony agar gel serological
test, a positive precipitin reaction.was
observed against a reference anti-cowpea
mosaic virus-Arkansas strain. Cowpea
mosaic virus was recently identified in
cowpea in the Philippines (Talens, in
press").
This report describes the isolation and
identification of cowpea mosaic virus in
winged bean in the Philippines. Identifica-
tion was based on morphological feature,


serological property, and diagnostic res-
ponse of selected virus indicator plants


MATERIALS AND METHODS

Virus source, test plants, and inoculation
Potted winged bean apparently infec-
ted with a virus disease was used. Leaf
tissues (Fig. 1) were excised and homo-
genized in a sterile mortar and pestle
containing 0.25 M phosphate buffer, pH
7.5 and 1% Celite. Seedlings of Cheno-
podium amaranticolor Coste et Reyn.,
C. quinoa Willd., Vigna unguiculata
(L.) Walp. cv. Red, and V. sinensis
(L.) Hassk. ssp. sesquipedalis (L.) v.
Eselt were used as virus indicators. Virus
inoculum consisting of phosphate-buf-
fered (0.025 M, pH 7.5) homogenate of
infected leaf tissues with 1% Celite was


Fig. 1. Symptoms on infected winged bean leaves from which a strain of cowpea
mosaic virus was isolated.
'Talens, L. T. 1978. Cowpea viruses in the Philippines: I. Identity of a mosaic-
causing virus in cowpea, Vigna unguiculata (L.) Walp. In press at Philipp. J. Crop Sci.


January, 1979









VU1. I


rubbed on the foliage of test plants.
Excess inoculum and abrasive was washed
with running tap water.

Virus identification
The procedure for virus identification
was based on studies of particle morpho-
logy by electron microscopy, agar gel
serological reaction using reference anti-
serum preparations against several legume
viruses, and response of selected host
plants to virus infection.
Electron microscopy of concentrated
virus preparation negatively stained with
2% neutral phosphotungstate was
done. A JEOL-100 U type electron
microscope was used.
Ouchterlony agar gel immunodiffu-
sion test (Ouchterlony, 1962) in
1.0% ionagar dissolved in 0.01 M tris-HC 1
buffer, pH 7.5 containing 0.85% NaCI
and 0.025% NaN3 was utilized. A six-well
pattern containing antisera to several























Fig. 2. Symptoms induced in Chenopo-
dium amaranticolor upon inocula-
tion with phosphate-buffered leaf
homogenate from infected winged
bean leaves.


legume viruses surrounding a center,
antigen depot was prepared.

RESULTS AND DISCUSSION

Mechanical inoculations of C. amaran
ticolor (Fig. 2) and C. quinoa (Fig. 3-4
resulted in the appearance of chlorotic
local lesions 6-8 days post-infection
Virus infection appeared to spread system.
mically in C. quinoa (Fig. 4) resulting ir
the appearance of bright yellow color.
tion on the uninoculated leaves (Fig. 4).
V. sinensis ssp. sesquipedalis (Fig. 5) and
V. unguiculata (Fig. 6) had no lesions on
inoculated leaves but developed severe
mosaic and leaf distortion. Infected
plants were severely stunted.
When clarified infected leaf extracts
serving as virus antigen was placed in again
gel diffusion plates, a positive precipitin
reaction was observed only with anti-cow-
pea mosaic virus (Fig. 7). No other visible























Fig. 3. Symptoms induced in Chenopo-
dium quinoa upon inoculation
with phosphate-buffered leaf
homogenate from infected winged
bean leaves.


i ~'Y~t'F 1 IJ YVYUYVV6~












--F



















Pig. 4. Systemic symptoms consisting of bright yellow coloration spreading from the
basal end of Chenopodium quinoa leaves.






























5

Fig. 5. Symptoms induced by cowpea mosaic virus infection
on Vigna sinensis ssp. sesquipedalis.


.... j l . .







Philippine PhytopatholoV


Fig.


Agar gel double diffusion serolo-
gical reaction between the winged
bean virus isolate (AG) and refe-
rence antisera (a-f). Central well
was charged with clarified virus
antigen; peripheral wells with (a)
cucumber mosaic virus, (b) cow-
pea mosaic virus-Arkansas strain,
(c) southern bean mosaic virus-
cowpea strain, (d) bean pod
mottle virus, (e) broad bean wilt
virnl and If\ hann aitrna mneaid


i




Fig.


ns induced by cowpea mosaic virus infec-
Vigna unguiculata cv. Red.


Vonl









Strain of Cowpea Mosaic Virus


reaction was seen when antisera against
southern bean mosaic, broad bean wilt,
bean pod mottle, blackgram mottle,
mungbean mottle, bean rugose mosaic,
quail pea mosaic, cucumber mosaic, to-
bacco mosaic, bean yellow stipple, and
tobacco ringspot viruses were employed.
Serological test.for other viruses infect-
ing legumes was not performed due to
lack of antiserum samples.

Electron microscopy of negatively-
stained specimen preparations revealed
isometric particles 25-28 nm in diameter
(Fig. 8). Two types of particle morpho-
logy were observed: full and empty
virions.


tempts have been made to describe the
virus disease problems eversince. The
occurrence and identity of cowpea
mosaic virus in winged bean can not be
overlooked. With the current thrusts in
agricultural intensification and diversifica-
tion programs, of which winged bean has
been tagged as the third priority crop by
PCARR, winged bean production program
must take note of the effect of cowpea
mosaic virus infection as well as other
viruses which have been reported to attack
winged bean. While the use of resistant
cultivars is widely-considered to
be the best solution to combat these
virusess, it suffers a limitation in that
no investigations have ever been con-


Fig. 8. Electron micrograph of the winged bean isolate of cowpea
mosaic virus negatively stained with 2% neutral phospho-
tungstate.


Investigations on the disease problems
of winged bean in the Philippines have
been reported in the early 1900 (Baker,
1916; Reinking, 1918). No serious at-


ducted to find out the viruses) infecting
winged bean in the Philippines. Clearly,
efforts should be made to identify the
viral etiology of certain winged bean
diseases.


January, 1979









no


LITERAL

BAKER, C.F. 1916. Additional notes on
Forester 5: 73-78.
CERNY, K., M. KORDYLAS, F. POSPID
Nutritive value of the winged beal
Nutr. 26: 293-299.
FAUQUET, C. D. LAMY and J. C. TH(
on winged bean(Psophocarphus tet
tional Working Group on Legume Vir
KAPSIOTIS, G. D. 1968. Chemical analysis
REINKING, O. A. 1918. Philippine econoi


RE CITED

hilippine plant diseases. Philipp. Agr. and

L, O. SVABENSKY, and B. ZAJIC. 1976
(Psophocarphus palustris Desv.). British J.

FVENEL. 1978. Two viral diseases isolated
gonolobus) in Ivory Coast. Abstr. (Interns
es. Zurich, Switzerland.
n winged bean. FAO: Rome.
ic plant diseases. Philipp. J. Sci. 13:165-216.


~ilir~i~n Pk~~stl.~l~m











Received for publication: 10 May, 19791

TEMPERATURE RELATIONS
ISOLATES OF PSEUDO

A. J. QUIMIC
Assistant Professor, Department of P1
sity of the Philippines at Los Banos Coll
Agriculture Research Center, Japan (TARC)
Supported by the TARC UPLB Pro
SABS1

Wide variations in the in-vitro o
Paeudomonas solnacearum isolates,
plant, pepper, white potato, tobacco
optimum ranging from 81.0 to 86.5 (
maximum 87.0 to 45.0 C. Majority
temperature optima ranging from 88.
for growth of isolates from localities
sea level, were not different from the
elevations below 100 ft above s lea
host and temperature requirements foi


Pseudomoms soladaerum E. F.
Smith, the cause of bacterial wilt of sola-
naceous crops and other plants and con-
sidered as one if not the moat impor-
tant bacterial pathogen in the world, is
noted for its pathogenic variability (Kel-
man, 1953; Buddenhagen and Kelman,
19 A \^ 1- -1-- '&__ -


)F PHILIPPINE SOLANACEOUS
IONAS SOLANACEARUM

and H. TABEI
int Pathology, College of Agriculture, Univer-
ege, Laguna and Plant Pathologist, Tropical

gram.
RACT

ardlu tempemture for growth of 61
aD belonging to Race 1, from egg-
Sand tomato, were observed; with the
, the minimum 8.5 to 15.5 C, and the
(68%) of the isolates, however, had
Sto 84.5 C. The cardinal temperatures
of high elevations, e.g. 4,999 ft above
e of isolates coming hom phees with
rel. There was no correlation between
growth.


isolates of P. solanaceaum and provides
additional evidence for its variable intra-
specific characters.

MATERIALS AND METHODS


ilt tolerance (Granada and Sequeira, provinces of the Philippines, from toma-
975; Hayward, 1976), pigment produc- to, eggplant, pepper, tobacco and white
on (Thurston, 1963; El-Helaly et a. potato, from the National Culture Collec-
969; French and Sequeira, 1970); tion of P. solnaceaum of the Depart-
rious culture and physiological proper- ment of Plant Pathology, University of
es (Buddenhagen and Kelman, 1964; the Philippines at Los Baflos were used in
ehr, 1970; Hayward, 1964, 1975; the study.
aneroni and Doudoroff, 1971; Harris, Culture medium
972; Hayward, 1976) and temperature Kelman's (1954) differential medium
lations(Kelman, 1953; Harrison, 1961; (10 g peptone, 10g dextrose, Ig casein
ayward 1964; Quinion et al. 1964; hydrolysate, 10 mg 2, 3, 5, trephenyl-
ayward, 1976). tetrazolium chloride (0.5% solution),
This paper reports on the tempe. 18 g agar, 1000 ml) distilled water
Iture relations of Philippine solanaceous was used for isolations and cleaning of









Philinninp Phutnnthanln-r


stock cultures; fresh inocula was pre-
pared using the same medium without
the tetrazolium salt.
Determination of cardinal temperatures
for growth
The tests were carried out in Kelman's
liquid medium without the tetrazolium
salt; inoculum consisted of 24-hr liquid
culture in the same medium.
The minimum, optimum and maxi-
mum temperatures for growth of the
different isolates were determined using
the Temperature Gradient Incubator
Model TN-3 (Toyo Kagaku Sangyo Co.,
Ltd.); this equipment uses special L-
shaped culture test tubes in a continuous
and constant shaking culture system with
30 different gradient temperatures. The
incubator was set at temperature gra-

Table 1. Cardinal temperatures forgrowth
isolates

Isolate
Origin (Code) Host

Cagayan p2 Pepper
TInhal. T.RQQ rp',,P t


dients of 3.5 to 45 C for the experiments.
Optimum temperatures for growth
were recorded with a Spectrophotometer
(Spectronic 20, Bausch & Lomb) at
425 nm, 5-6 hr after inoculation). Mini-
mum and maximum growth temperatures,
on the other hand, were checked 2 days
after inoculation. The temperature in
each test tube was monitored 4 times a
day.

RESULTS AND DISCUSSION

The optimum growth temperature of
the solanaceous isolates ranged from 31.0
to 36.5 C; the minimum temperature
3.5 to 15.5 C; and the maximum 37.0
to 45.0 C (Table 1).



f some Philippine Pseudomonas solanacearur



Optimum Minimum Maximur
nQ n 1 in o rK


LE60 Tomato 34.0 12.5 39.5
SM6 Eggplant 35.0 12.0 41.0
Nueva Vizcaya E10 Eggplant 33.0 12.5 39.5
T44 Tomato 34.0 8.5 41.0
Benguet ST3 Potato 31.5 3.5 39.5
ST7 Potato 33.0 15.0 38.5
II5C Potato 33.5 13.5 39.5
LE50 Tomato 34.0 6.5 41.0
LE46 Tomato 33.5 10.0 39.5
La Union NT15 Tobacco 33.0 12.0 39.5
NT8 Tobacco 34.0 10.5 43.0
N15 Tobacco 35.0 12.0 38.5
Nueva Ecija LE61 Tomato 31.0 10.5 41.0
LE43 Tomato 33.0 8.0 41.0
NT13 Tobacco 33.0 8.5 39.5
LE42 Tomato 34.0 10.0 41.0


70









,nas Rnlonnarrn,,c lcl.t~.


ile 1. (continued)


gasinan TSC Tomato
TBAY Tomato
E24 Eggplant
rpanga P4 Pepper
E23 Eggplant
TPAM Tomato
acan LE35 Tomato
ezon CF4 Sweet pepper
runa SM2 Eggplant
WP5 Potato
LE100 Tomato
T15RRC Tomato
LE11 Tomato
LE63 Tomato
NT10 Tobacco
E3 Eggplant
CF3 Sweet pepp
T1RRC Tomato
ST8 Potato
LE64 Tomato
ay CF6 Sweet pepper
lo LE62 Tomato
T51 Tomato
E14 Eggplant
rte LE59 Tomato
gros Occidental WP2 Potato


31.0 13.0 39.0
35.0 8.5 39.5
35.0 8.0 40.5
34.0 12.5 43.0
35.0 15.0 40.0
36.5 12.5 45.0
33.0 12.0 39.5
35.0 8.5 41.0
33.0 8.0 39.5
33.0 12.0 41.0
33.0 8.5 41.0
33.5 10.5 41.5
34.0 13.0 39.0
34.0 12.5 41.0
34.0 8.5 41.0
34.0 10.5 39.5
34.0 10.5 38.0
35.0 8.0 41.0
35.0 11.0 41.0
36.0 6.5 45.0
34.0 10.5 43.0
34.0 11.0 41.0
34.0 3.5 39.0
34.0 9.5 41.0
33.0 15.5 38.5
33.0 10.0 38.5


LE13 Tomato 34.0 13.5 39.0
T21 Tomato 34.0 5.0 45.0
T56 Tomato 34.5 8.5 39.5
amis Oriental T3-1 Tomato 31.5 4.5 36.5
T2-1 Tomato 34.0 6.5 41.0
T2-2 Tomato 36.5 4.5 45.0
ao del Norte LE17 Tomato 33.0 10.0 38.0
LE19 Tomato 35.0 11.5 40.0
LE18 Tomato 35.5 9.0 45.0
th Cotabato T47 Tomato 34.0 10.0 41.0
p3 Pepper 34.0 11.0 43.0





For the purpose of classification, the however, had optimum temperatures of
plates may be grouped based on opti- 33.0 to 34.5 C (Table 2). There was no
im temperature for growth as follows: correlation between the optimum tem-
31.0-31.5 C, b) 33.0-33.5 C, c) 34.0- perature range and minimum or maxi-
.5 C, d) 35.0 to 35.5 C, and 3) 36.0- mum temperatures for growth. However,
.5 C. Majority of the isolates (68%), isolates with the highest optimum tem-









Philippine Phytopathology


Table 2. Classification of Pseudomonas solanacearum isolates based on optimum tem-
perature for growth


Isolates


LE61, ST3, TSC, 31
E10, LE17, LE33, LE35, LE43,
LE59, LE100, NT13, NT15, P1,
P2, SM2, ST7, T15RRC, WP2,
WP5, II5c
CF3, SF6, E3, E14, LE11,
LE13, LE30, LE38, LE42,
LE44a, LE50, LE60, LE62,
LE63, NT8, NT10, P3, P4, T21,
T44, T47, T51, 2-1, 56
F4,E23,E24, LE18,LE19,
LE46, N15, SM6, ST8, T1RRC,
TBAY,
LE64, TPAM, 2-2


No. of isolates


4( 6.06%)



17 (25.76%)




28 (42.42%)


13 (19.70%)
4( 6.06%)


Total 66


perature (36.0 -36.5 C) had also the
highest maximum temperature (45.0 C)
for growth.
The data also showed that with the
exception of the tomato isolates the
range of temperature optima of isolates
from the same host plant was quite
narrow; potato isolates, optimum growth
temperature ranged from 33.0 to 33.5 C;
pepper isolates, 33.0 to 34.0 C; tobacco
isolates, 33.0 to 34.0 C; eggplant isolates,


33.0 to 35.0 C and tomato
to 35.5 C (Table 3).


isolates, 31.0


The cardinal temperatures for growth
of isolates from high elevations such as
those from La Trinidad, Benguet (eleva-
tion 4,999 ft), Claveria Misamis Oriental
(elevation 3,020 ft) and Philipps, Bukid-
non (2,215 ft) were not different from
those of isolates coming from elevations
below 100 ft above sea level such as those


Table 3. Relationships between optimum temperature for growth of Pseudomonas
solanacearum isolates from various host plants


Isolate Optimum
Host plant (Code) Origin temp.


Nueva Vizcaya
Laguna
Laguna
Iloilo
Pampanga
Pangasinan
Isabela


Temperature
(C)

31.0-31.5
33.0-33.5



34.0-34.5




35.0-35.5


36.0-36.5


Eggplant


E10
SM2
E3
E14
E23
E24
SM6


33.0
33.0
34.0
34.0
35.0
35.0
35.0


Vol. 15
















P3 South Uotabato 64.u
P4 Pampanga 34.0
CF3 Laguna 34.0
CF4 Quezon 35.0
Potato ST3 Benguet 31.5
ST7 Benquet 33.0
WP2 Negros Occidental 33.0
WP5 Laguna 33.0
II C Benguet 33.0
ST8 Laguna 35.0
Tobacco NT13 Nueva Ecija 33.0
NT15 La Union 33.0
NT10 Laguna 34.0
NT8 La Union 34.0
N15 La Union 35.0
Tomato LE61 Nueva Ecija 31.0
TSC Pangasinan 31.0
31 Misamis Oriental 31.5
LE17 Davao 33.0
LE33 Isabela 33.0
LE35 Bulacan 33.0
LE43 Nueva Ecija 33.0
LE59 Leyte 33.0
LE100 Laguna 33.0
T15RRC Laguna 33.5
LE11 Laguna 34.0
LE13 Bukidnon 34.0
LE30 Isabela 34.0
LE42 Nueva Ecijia 34.0
LE44a Nueva Ecija 34.0
LE50 Benguet 34.0
LE60 Isabela 34.0
LE62 Iloilo 34.0
LE63 Laguna 34.0
QA A


2-1
LE38
56
LE19
T1RRC
TBay
LE18
LE46
LE62
TPAM
2-2


Misamis Oriental 34.0
Nueva Ecija 34.5
Bukidnon 34.5
Davao 35.0
Laguna 35.0
Pangasinan 35.0
Davao 35.5
Benguet 35.5
Laguna 36.0
Pampanga 36.5
Misamis Oriental 36.5










Philippine Phytopathology


isolates from Isabela, Pampanga, Panga-
sinan and Laguna; there are no correla-
tions, therefore, between in-vitro tempe-
rature growth requirements and the
isolate's origin based on elevations
(table 4). The data also suggest that P.
solanacearum found at high elevations
probably came from the lowlands.
The temperature optima observed for
the Philippine solanaceous isolates were
quite variable. However, the dominant
populations would have optimum growth


temperatures from\ 33.0 to 34.5 C. Inde-
pendent observations by different workers
abroad indicate the \same variability of
optimum growth temperatures of Pseu-
domonas solanacearum (Quiflon, et al.
1964; Kelman, 1953). Isolates conform-
ing to race 3 in the system of Buddenha-
gen et al. (1962) which have markedly
lower temperature optimum of 27-28 C
(Harrison, 1961, Hayward, 1964, Thurs-
ton, 1963) do not occur in the Philip-
pines as yet.


Table 4. Cardinal temperatures for growth of Pseudomonas solanacearum isolated
from different elevations


Isolate
Elevation Province (Code) Host plant


Temperature

Opt. Min. Max.


Benguet ST3
ST7
II5C
LE50
LE46
Claveria 31
Misamis 2-1
Oriental 2-2
Bukidnon P1
LE13
T21
56
Isabela LE33
LE30
LE60
SM6
Pampanga P4
E23
TPAM
Pangasinan TSC
TBay
E24
Laguna LE11
LE63
LE64


4,999 ft




3,020 ft


2,215 ft



Below 100 ft


Potato
Potato
Potato
Tomato
Tomato
Tomato
Tomato
Tomato
Pepper
Tomato
Tomato
Tomato
Tomato
Tomato
Tomato
Eggplant
Pepper
Eggplant
Tomato
Tomato
Tomato
Eggplant
Tomato
Tomato
Tomato


31.5
33.0
33.5
34.0
35.5
31.5
34.0
36.5
33.0
34.0
34.0
34.5
33.0
34.0
34.0
35.0
34.0
35.0
36.5
31.0
35.0
35.0
34.0
34.0
36.0


39.5
38.5
39.5
41.0
39.5
36.5
41.0
45.0
39.0
39.0
45.0
39.5
38.5
39.5
39.5
41.0
43.0
40.0
45.0
39.0
39.5
40.5
39.0
41.0
45.0


Vol. 15












LITERATE

UDDENHAGEN, I. W. and A. KELMAN. I
bacterial wilt caused by Pseudomon
2: 203-230.
L-HELALY, A. F., M. K. ABO-EL-DAHAE
nesis in cultures of Pseudomonas solana
RENCH, E. R. and L. SEQUEIRA. 1970.
Central and South America: a comparal
rRANADA, G. A. and L. SEQUEIRA. 19'
Pseudomonas solanacearum from tobac
[ARRIS, D. C. 1972. Intra-specific variati
292. In H. P. Maas Geesteranus (ed.) I
Wageningen, The Netherlands. 365 p.
[ARRISON, D. E. 1961. Bacterial wilt of pc
studies on the causal organism, Psei
Australian J. Agr. Res. 12: 854-871.
[AYWARD, A. C. 1964. Characteristics of P
7: 265-277.
[AYWARD, A. C. 1975. Biotypes of Pseud
Plant Pathol. Soc. Newsl. 4:9-11.
[AYWARD, A. C. 1976. Systematics and rel
L. Sequeira and A. Kelman (eds). P
Workshop on the Ecology and Contre
solanacearum. Raleigh, North Carolina.
:ELMAN, A. 1953. The bacterial wilt caum
ture review and bibliography. N. C. Agi
ALLERONI, N. J. and M. DOUDOROF
DNA homologies of Pseudomonas sc
696.
IUINON, V. L., M. ARAGAKI and M. ISH
tionships of 3 strains ofPseudomonm
54: 1096-1099.
'HURSTON, D. H. 1963. Bacterial wilt oi
381-390.
;EHR, E. I. 1970. Cultural, physiological ai
ippine Pseudomonas solanacearum. Phi


E CITED

54. Biological and physiological aspects of
solanacearum. Ann. Rev. Phytopathol.

nd M. A. EL-OORANI. 1969. Chromoge-
arum. J. Phytopathol. UAR. 1: 1-11.
rains of Pseudomonas solanacewum from
a study. Phytopathology 60: 506-512.
Characteristics of Colombian isolates of
. Phytopathology 65: 1004-1009.
I in Pseudomonas solanacearum. p. 289-
oc. 3rd Int. Conf. Plant Pathogenic Bact.,

Itoes, I. Field symptoms of the disease and
omonas solanacearum variety asiaticum.

domonas solanacearum. J. Appl. Bacteriol.

onas solanacearum in Australia. Australian

ionships of Pseudomonas solanacearum. In
c. First Intern. Planning Conference and
of Bacterial wilt caused by Pseudomonas
56 p.
I by Pseudomonas solanacearum. A litera-
!xp. Sta. Tech. Bull. 99, 194 p.
1971. Phenotypic characterization and
nracearum. J. Gen. Microbiol. 107: 690-

S1964. Pathogenicity and serological rela-
solanacearum in Hawaii. Phytopathology

potatoess in Colombia. Am. Potato J. 40:

biochemical properties of isolates of Phil-
p. Phytopathol. 6: 29-43.


,-;-- Q-1 -------- T.-]-+-


-1













IDENTITY OF THE BA
BACTERIAL BROWN SP(


A. J. QUIM
Assistant Professor, Department o
sity of the Philippines at Los Banos I
Agriculture Research Center, Japan (TAI

Supported by the TARC UPLB i
ABSI
The morphological, cultural, p
of the causal organism of bacterial
disease hitherto unreported in the I
organism identified as Pseudomonas c


In the early rainy season of 1975, th
authors observed a bacterial brown spc
disease affecting the Phalaenopsis orchid
of the Floriculture Division of the De
apartment of Horticulture, Universit
of the Philippines at Los Baflos, Colleg4
Laguna. A survey for the disease in th


TERIUM ASSOCIATED WITH
'OF PHALAENOPSIS ORCHIDS


) and H. TABEI
Plant Pathology, College of Agriculture, Uni
Illege, Laguna and Plant Pathologist, Trop
:).

ogram.
ACT
dsiological and biochemical character
brown spot of Phalaenopsis orchids, a
ilippines, were studied and the causal
tleyae (Pavarino) Savulescu.


plate streaking technique using.nutrien
agar (NA) from diseased Phalaenopsi
leaves collected from the campus of th
University of the Philippines at Los Bafoi
College, Laguna. The isolates were puri
fled by repeated streaking and isolating.
single colony isolates on NA.


MATERIALS AND METHODS
The following tests and studio
'he causal hactArillnum iunre Afl>a h, rnmlain4A.


I I_ _





Brown Spot of Phalaenopsis


I-


I-


Lte I. Bacterial brown spot of Phalaenopsis: 1) early symptom, 2) advanced sympto
and 3) causal bacterium.


77








7 8


rnmppme rnytopatnolog


'eCn morpnology and gram stain rea
tions Cell shape, size and arrangement
were studied in hanging drop preparation
and methylene blue stained smears froi


Nitrate reduction Reduction i
nitrates was tested in both Difco nutriel
broth and nutrient agar plus 0.1% sodiu:
nitrate. Sulfanilic acid and a-naphthyl


wa CLCU usnU:g Ue eaud bromthymol
gnated filter paper method. tone and bromthymol
were also tested for hydro- used (Difco Manual,


recwoync acanvuy uraaDury s me
thod of testing for pectolytic enzyme:
was used (Bradbury, 1970).
Indole production Indole produce
tion from tryptone broth was tested usinj
vanilla as a test reagent according to th(
method of Roessler and McClung (1943).
Production of pigment Clara',
medium (Clara, 1934) and Medium B ol
King, Ward, and Raney (King, et al
1954) were used in this test.


sterilized with the basal medium; th4
basal medium has a 1% concentration o
the carbon compound.
Cardinal temperatures for growth -
The optimum, minimum, and maximun
temperatures for growth were determine<
using the Temperatures Gradient Incuba
tor Model TN-3 (Toyo Kagaku Sangyc
Co., Ltd.). The optimum temperature
was recorded 5-6 hr after inoculation
with a Beckman 20 Spectrophotometer








Bacterial Brown Spot of Phalaenopsis Orchids


The minimum and maximum temperatures
for growth were determined 2 days after
inoculation.

RESULTS AND DISCUSSION

Initial symptoms of the disease deve-
loped within 48 hr after inoculation of
Phalaenopsis leaves. Typical symptoms of
the disease were well developed in 5 to 7
days from inoculation.

Results of studies on morphological,
cultural, physiological, and biochemical
characters of the bacterium are summa-
rized below:
Rods: 0.5. x 1.0-2.0 microns, with
rounded ends, occurring singly or occas-
sionally in pairs, motile by means of a
single polar flagellum (Plate 1:3).
Gram negative
Poly-B-hydroxybutyrate, positive,
Aerobic
Nutrient agar colonies: Grayish white,
circular, convex, entire, smooth, butyrous.
Nutrient agar slant: Growth grayish
white, filiform, non-flourescent, odorless,
brittle to viscid, medium unchanged.
Nutrient broth: Growth odorless,
moderate clouding, viscid sediment, thin
pellicle.
Fermi's solution: Good growth
Chromogenesis: No pigment in Clara's
medium or on Medium B of King, Ward
and Raney.
Gelatin not liquefied
Fresh milk and Litmus milk unchanged
Nitrates reduced to nitrites.
Indole production, negative.
Hydrogen sulfide production, negative.
Pectolytic activity, positive.
Starch hydrolyzed.


Glucose metabolized fermentatively in
Hugh and Leifson's medium.
Levan formation, negative.
Oxidase. reaction, positive.
Acid but no gas from fructose, galac-
tose, glucose, glycerol, mannitol, sorbitol
and xylose.
No acid or gas from arabinose, dulcitol,
esculin, inositol, inulin, lactose, maltose,
mannose, raffinose, rhamnose, salicin, so-
luble starch, sucrose and trehalose.
Optimum temperature 29.5 C, mini-
mum temperature 7.0 C,, maximum
temperature 39.5 C.

A bacterial brown spot disease of
orchids had been reported in California
and Italy (Ark and Thomas, 1946; Ark,
1950; Ark and Starr, 1951), causing oc-
casional losses in nurseries. The symptoms
of this disease which is caused by Pseudo-
monas cattleyae (Pavarino) Savulescu,
resemble those of the disease reported
here.
The characters of the bacterium
under study belong to the genus Pseudo-
monas (Doudoroff and Pallerone, 1975)
and are similar to those of P. cattleyae
(Synonyms: Bacterium cattleyae Pava-
rino; Phytomonas cattleyae (Pavarino)
Ark and Thomas). As described in the 7th
Edition of Bergey's Manual of Deter-
minative Bacteriology (1957), P. cat-
tleyae has characters which overlap
with those of P. dysoxyli Hutchinson,
P. heianthi (Kawamura) Savulescu and
P. melopthora Allen and Ricker. How-
ever, they differ from each other by their
host specificity; Pseudomonas dyxosyli
attacks a tree belonging to the family
Meliaceae, while P. helianthi and P.
melopthora infect sunflower and apple,
respectively. In the 8th edition of the
manual (Bergey's Manual of Determi-
native Bacteriology, 1974), P. desoxyli


January, 1979









Philippine Phytopathology


and P. helianthi are treated as "may be
synonyms, biotypes, pathotypes, or
varieties" of P. syringae van Hall but it
also "may even deserve independent
specific rank". Similarly, in this manual,
P. cattleyae and P. melopthora are in-
cluded among those "nomen-species which
has been incompletely described but
which appear to conform to the generic
definition" of Pseudomonas. In the


absence of a comprehensive comparative
study between these apparently rela-
ted "species", the authors conclude
that the bacterium causing brown spot
of Phalaenopsis orchids under study
is Pseudomonas cattleyae (Pavarino) Savu-
lescu. Stock cultures of this bacterium
are deposited at the Bacterial Disease
Laboratory of the Department of Plant
Pathology, UPLB.


LITERATURE CITED

ANONYMOUS. 1953. Difco Manual (9th edition) Difco Laboratories, Detroit, Michigan.
350 p.
ARK, P. A. 1950. Destructive diseases of orchids in California. Orchid Digest 14:174-178.
ARK, P. A. and H. E. THOMAS. 1946. Bacterial leaf spot and bud rot of orchids caused
by Phytomonas cattleyae. Phytopathology 30:695-698.
ARK, P. A. and M. P. STARR. 1951. Bacterial diseases of orchids. Plant Dis. Reptr.
35:42-43.
BRADBURY, J. F. 1970. Isolation and preliminary study of bacteria from plants. Rev.
Plant Path. 49:213-218.
BREED, R. S., E.G.D. MURRAY, and N. R. SMITH. (Eds.) 1957. Bergey's manual of
determinative bacteriology, Baltimore: William and Wilkins Co.
BUCHANAN, R. E. and N. E. GIBBONS. (Eds.) 1974. Bergey's manual of determinative
bacteriology. Baltimore: William and Wilkins Co. 1268 p.
BURDON, K. J. 1946. Fatty material in bacterial and fungi revealed by staining dried
fixed slide preparations. J. Bact. 52:665-677.
CLARA, F. M. 1934. Comparative study of the green flourescent bacterial pathogens.
New York (Cornell) Agric. Expt. Sta. Mem. 159. 34 p.
HUGH, R. and E. LEIFSON. 1953. The taxonomic significance of fermentative versus
oxidative metabolism of carbohydrates by various Gram negative bacteria. J.
Bact. 66:24-26.
KELMAN, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum
to colony appearance on a tetrazolium medium. Phytopathology 44:693-695.
KING, E. O., W. K. WARD, and D. E. RANEY. 1954. Two simple media for the demons-
tration of pyocyanic and fluorescin. J. Bact. 66:24-26.
ROESSLER, W. G. and L. S. McCLUNG. 1943. Suggested method for use of vanilla as a
test reagent for indole and skatole production by bacteria. J. Bact. 45-413 (Abst.)
SOCIETY OF AMERICAN BACTERIOLOGISTS. 1957. Manual of Microbiological
Methods. New York: McGraw Hill Book Co. 315 p.


Vol. 15














OUTBREAKK OF E


r -r- ypuvnOllu uaL
litions. Precipitation during the early The subje
vth stage of the rice plant, submer- forecasting h
:e of seedlings in the nursery, flood- and Mizukar


light
gami
and
Vaki-









Philippine Phytopathology


increase in phage population during the
incubation period. Tagami et al. (1964)
used this method to detect the seasonal
changes of X. oryzae on rice and in irri-
gation water during the rice-growing
season. The method was, however, too
complicated for practical application in
--A-,-iv-n rliaAa n..-- +-.aa1r T I.tr Taiami


cal siulles, ume viruicnce u ulu pnagw,
isolated in tropical Asia was similar t(
that of either the OPi group or the OP2
group (Goto, 1965; Singh et al. 1970
Kim and Wakimoto, Wakimoto and Ue
matsu, unpublished data). However, the
host range varied and differed from thai
of the Japanese strains (Wakimoto


ge specific to X. oryzae in the paddy p of the ecfic location (Bangk
I or irrigation water increases as the Thailand) has changed.
.A.a in-0. e r art n ltIhrAalr nf


common and widely distributed, and were transplanted from nurseries in w
ing to the OP, group, which differs no phages were detected in the surro
n OPN mornhologicallv and serolori- __ .-. ..... _..


82



















)st o0 A. oryzae. In some nurseries, (plaque forming unit)/ml based on plaque
)hage was detected early and in counts (Yoshimura. 1963).
nce, outbreak of the disease in
Idy field at the later growth stage In Kyushu, Japan, the phage popula-
)e predicted (Tagami and Mizu- tion increased rapidly in July with the
,962; Tagami, 1962), unless wea- number exceeding 103 pfu/ml and
editions became drastically differ- reached the peak in August at 10s pfu/ml.
n the normal patterns. In fields where no phage could be detec-
ted until the end of July, onset of the
pan, over 95% of the nurseries are disease was delayed until mid-October.
ent prepared in indoor nursery
so the method may not be appli- In the tropics, phage could some-
times be detected in fields during the
off-season (dry season) and the disease
ri Lanka, the phage population would occur in the main season. In
series was highly correlated with Thailand, if the phage number was less
:rity of kresek, the wilting stage than 103 pfu/ml in August (off-season),
erial blight which occurs during the disease may occur but will not be
: few weeks after the seedlings are severe. However, if the number exceeds
nted from the nurseries (Shige- 103 pfu/ml at maximum tillering stage
id Tabei, 1969; Watanabe, 1975). in October, a disease outbreak may be
predicted in the following season (Tabei









Philippine Phytopathology


rice bacterial blight outbreak in tropical
Asia were carried out mostly in experi-
ment stations or in areas with good irri-
gation systems, like the Central Plains of
Thailand (Emachit and Mew, personal
communication). Hence, the bacterio-
phage method of predicting the outbreak
of bacterial blight may be limited to irri-
gated fields. Because accuracy of the
method was influenced significantly by
bacteriophage population determination
in irrigation canals or channels, to apply
the method under rainfed conditions
requires further study. Similarly, pre-
dicting disease outbreak would be en-
hanced if it could be coupled with yield
forecast. In either area of information
concerning the rice bacterial blight in
tropical Asia, our knowledge is still very
meager. The forecasting of disease out-
breaks or epidemics would be a contri-
bution to the prediction of crop yields.
Yield forecasts are useful if remedies can
be applied to prevent the disease epide-
mics (Waggoner, 1960). Our knowledge
of disease appraisal, however, is still too


limited to be able to quantify yield losses
caused by bacterial blight in tropical
Asia. In addition, chemical control of
the rice bacterial blight is not exercised
in the tropics because it is neither feasible
nor available to the farmers. Even though
prediction might be possible, little or
nothing can be done to prevent its occur-
ence.

CONCLUSIONS

The forecasting system of bacterial
blight is not practiced in tropical Asia.
Little work has been done under expe-
rimental conditions using the bacterio-
phage method. Rice cultivation in most
of tropical Asia is rainfed, and this kind
of rice culture limits the use of the
method. As irrigated rice in the tropics
gradually increases and organized rice
farming systems become important in
rice production in the tropics, scientists
may find the method useful after re-
assessing the procedures under specific
agroenvironmental conditions.


LITERATURE CITED

BARKER, R. and R. W. HERDT. 1978. Rainfed lowland rice as a research priority An
economist's view. Paper presented for the International Rice Research Conference
on April 17-21, 1978. IRRI, Los Banos, Philippines.
GOTO, M. 1965. Bacteriophages of Xanthomonas oryzae, the pathogen of bacterial leaf
blight of rice, collected in the Philippines. Bull. Fac. Agr. Shizuoka Univ. 15:31-38.
GOTO, M. 1969. Ecology of phage bacteria interaction in Xanthomonas oryzae (Uyeda
et Ishiyama) Dowson. Bull. Fac. Agr. Shizuoka Univ. 19:3167.
ISAKA, M. and S. NOSAKI. 1961. Strains and lysotypes of Xanthomonas oryzae col-
lected from Fukui Prefectures (in Japanese with English summary). Proc. Assoc.
Plant Protect., Hokuriku 9:30-32.
ITO, H. and S. TAKAHASHI. 1964. On the properties of Xanthomonas oryzae bacterio-
phages distributed in Shyonai area. Yamagata Prefecture (in Japanese). Prefecture
(in Japanese). Proc. Assoc. Plant. Protect., Kita Nihon 15:40-41.
KOBAYASHI, K. 1960. A study on the type of bacteriphage of leaf blight bacterium of
rice plant (Xanthomonas oryxae) in Kumamoto Prefecture (in Japanese). Proc.
Assoc. Plant Protect., Kyushu 10:98-99.
MIZUKAMI, T. and S. WAKIMOTO. 1969. Epidemiology and control of bacterial leaf
blight of rice. Ann. Rev. Phytopathol. 7:51-72.


Vol. 15










nianr flithrea I< nf R-f-tai


SHIGEMURA, C. and H. TABEI. 1969. N
Ceylon. Int' Rice Comm. Newsl. 18::
SING, K. G., UEMATSU, T. and S. WAKID
and phage from Malaysia. Ann. Phyt
SRIVASTAVA, D. N. 1972. Bacterial bligh
TABEI, H. andS. EAMCHIT. 1974. Strains
in Thailand. JARQ 8:122-123.
TAGAMI, Y., KUHARA, S., KURITA, T.,
population of Xanthomonas oryzae ]
of bacterial blight (in Japanese). P
TAGAMI, Y., KUHARA, S., KURITA, T.,
Epidemiological studies on the bact
(Uyeda et Ishiyama) Dowson. II. (in
Agr. Exp. Stn., 10:23-50.
TAGAMI, Y. and T. MIZUKAMI. 1962. H
blight of rice caused by Xanthomor
Japanese with English summary). Sp
insect pests. Plant Protect. Div. Min. j
TAGAMI, Y. 1962. Ecological studies on
of rice (in Japanese). Ph.D. thesis, Ky
WAGGONER, P. E. 1960. Forecasting ep
Pathology. 3:291-313. Academic Pres
WATViMn Vt 10-A -lArm.:~.l .._ -U.,


isures to control "kresek" disease of rice i
-22.
)TO. 1970. Studies on Xanthomonas oryzao
,athol. Soc. Japan 36: 56-64.
if rice. Indian Phytopath. 25:1-16.
Xanthomonas oryzae and its bacteriophage

ad SEKIYA, N. 1958. Relation between th4
age in paddy field water and the occurrena
!. Assoc. Plant Protect., Kyushu 4:63-64
UJII, H., SEKIYA, N., and T. SATO. 1964
al leaf blight of rice Xanthomonas oryzae
>anese with English summary). Bull. Kyushu

;orical review of the researches on bacteria
oryzae (Uyeda et Ishiyama) Dowson (ir
ial report for forecasting plant diseases ani
r. and Forest., Japan No. 10, pp. 1-112.
e occurrence of bacterial leaf blight disease
hu Univ., Japan. pp. 1-171.
emics. In Horsfall and Dimond (ed.). Plan
New York.
*. Tr .*


ation of the presence of Xar
se with English summary).

Af strains of Xanthomonas


iHIMURA, S. and Y. TAGAMI. 1967. Forecasting and control of bacterial leaf bli
of rice in Japan. In Rice dise s and their control by growing resistant varieties
their mianures. Tronieal Aar.r a. Sar No 1 29.-2R


- .... ... I .-












NOTE: OCCURRENCE OF )
MUNGBEAN ID

D. R. A

Associate Professor and Chairman,
Agriculture, University of the Philippines a







During a survey of the virus diseases


tu1.
recognized by t
earance. The le
cupped or twist
gether close to


Attempts to look for the specific
vector of this disease are in progress.


PITCHES' BROOM DISEASE OF
:HE PHILIPPINES

IENIGNO

departmentt of Plant Pathology, College o:
los Banos, College Laguna.


































Fig. 1. Mungbean or green gram (Vigna
radiata), dimunutive, and bunch-
ed. susDected to be caused
by a mycoplasna-like organism,
(MLO).















ERF

Vol. 14(Nos. 1 & 2)

page 64. Second and third sentences <
third paragraph.


TA










Should read:
However, data on fiber yields from the
treated plots, which could have served as
the ultimate basis for effective nematode
control were not taken. Unlike in abaca,
nematode control in bananas are reflected
by fruit production with due considera-
tion on the chemical residue resulting
from the nematicide application.


ERF

Vol. 14 (Nos. & 2)

page 64. Second and third sentences
third paragraph.



















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Department of Public Works, Transportation and Communications
BUREAU OF POSTS
Manila

SWORN STATEMENT
(Required by Act 2580)


The undersigned, TRICITA H. QUIMIO, editor of PHILIPPINE PHYTO-
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duly sworn in accordance with law, hereby submits the following statement of ownership,
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Editor-in-Chief

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