The CARD project coded 052/04/VIE on capacity building for Phytophthora detection and
management in horticultural crops in Vietnam has been conducted in 2005-2006 with the
participation of PPRI, SOFRI, Thua Thien-Hue CRDF and some local technicians and farmers. The
aim of this project was to improve new technology for Phytophthora fungi detection and management
as one serious plant pathogen that causing diseases on many horticultural crops.
The result of this study has helped scientist to rapidly and precisely detect Phytophthora pathogen that
causing diseases on black pepper, rubber trees, citrus tree crops, etc. Using rose flower petal as trap
for isolation of zoospores and purification of diseased samples has been successfully conducted in
laboratory. The culture media RT* including RH+Tachigaren 30L could give the success about 45.3%
in fungus isolation. Other techniques namely method of disease sampling, storage, treatment,
detection and identification of Phytophthora fungi have been successfully conducted by Vietnamese
technicians. This method can be used for early phytophthora diagnosis based on initial symptoms.
Several scientists at PPRI, SOFRI, Thua Thien –Hue CRDF have gained their knowledge in plant
pathology and after the project termination they have succeeded in getting the government funded
research projects. One of those is the study on quick death of black pepper, quick death of durian
trees, tip rot of pineapple, yellow wilt of litchi, downy mildew of tomato, stem canker of citrus and
black strip of rubber. These research findings such as disease diagnosis and management on the IPM
basis have significantly contributed to horticultural development in Vietnam.
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Collaboration for Agriculture and Rural Development (CARD) Program
56
METHODS OF PHYTOPHTHORA ISOLATION FROM SOIL, ROOT
AND INFESTED PLANT OF BLACK PEPPER AND OTHER CROPS
Project title: Management of Phytophthora Diseases in Vietnamese Horticulture
Project Code: CARD 052/04 VIE
Author(s): Nguyen Van Tuat1, Pham Ngoc Dung1, Nguyen Thi Ly1, Le Thu Hien1
1National Institute of Plant Protection
Project Implementing organisations:
Vietnamese Institution: National Institute of Plant Protection
Vietnamese Project Team Leader: Dr Nguyen Van Tuat
Australian Organization: The University of Sydney
Australia Team Leader: Professor David Guest
SUMMARY
The CARD project coded 052/04/VIE on capacity building for Phytophthora detection and
management in horticultural crops in Vietnam has been conducted in 2005-2006 with the
participation of PPRI, SOFRI, Thua Thien-Hue CRDF and some local technicians and farmers. The
aim of this project was to improve new technology for Phytophthora fungi detection and management
as one serious plant pathogen that causing diseases on many horticultural crops.
The result of this study has helped scientist to rapidly and precisely detect Phytophthora pathogen that
causing diseases on black pepper, rubber trees, citrus tree crops, etc. Using rose flower petal as trap
for isolation of zoospores and purification of diseased samples has been successfully conducted in
laboratory. The culture media RT* including RH+Tachigaren 30L could give the success about 45.3%
in fungus isolation. Other techniques namely method of disease sampling, storage, treatment,
detection and identification of Phytophthora fungi have been successfully conducted by Vietnamese
technicians. This method can be used for early phytophthora diagnosis based on initial symptoms.
Several scientists at PPRI, SOFRI, Thua Thien –Hue CRDF have gained their knowledge in plant
pathology and after the project termination they have succeeded in getting the government funded
research projects. One of those is the study on quick death of black pepper, quick death of durian
trees, tip rot of pineapple, yellow wilt of litchi, downy mildew of tomato, stem canker of citrus and
black strip of rubber. These research findings such as disease diagnosis and management on the IPM
basis have significantly contributed to horticultural development in Vietnam.
Key words: Phytophthora fungi, black pepper, horticulture crops, detection, identification
1. Introduction
The diversity of climate and geography
allowed planting many crops. The tropical
crops largely plant at Northern and Southern,
meanwhile temperate crops plant at highland
of Northern and Central Vietnam. The
diversity of climate also is suitable conditions
for development of Phytophthora spp. Some
Phytophthora species strongly destroyed many
crops in Vietnam and seriously caused loss of
yielding and economy.
The CARD project number 052/04/VIE had
been implemented from 2005-2006 with
collaboration between Australian experts and
Vietnam staffs of Plant Protection Research
Institute, Southern Fruit Research Institute, and
Thua Thien Hue Center of Fruits Research and
Development. Project has been organized
training courses and workshops for participants
from institutions, universities, extension
CARD 050/04 VIE – Market improvement for Vietnamese fruits
57
department and locations. The objective of
project to improve capacity for research and
management of Phytophthora diseases in
Vietnam. The results of project were
successful. Through knowledge obtained from
training courses, researchers have been
proposed and carried out projects of studies on
Phytophthora diseases such as quick and low
death of black pepper, quick death of durian,
bud root of pineapple, wilt death of litchi,
downy mildew of tomato and potato, stem
canker of citrus, black strip of rubber…
However, researches of Phytophthora fungus
were limitation because it is difficult to isolate
this fungus. Results obtained from training
courses helped us to conduct special subject
“Method of Phytophthora isolation from soil,
root and infested plants.”
2. Research materials and methods
2.1. Isolation of Phytophthora from soil
planting black pepper and black
pepper root by petal and leaf traps
Soil from rhizosphere at 15 cm to deep and
early disease infested rootlet samples were
collected. Soil and rootlet (one third in
volume) were placed into cups contained
distilled water. Cups were lightly stirred by
glass stick and kept at least for 2 hours to soil
and rootlets deposited. Freshly color petals
(0.5 x 0.5 cm) or black pepper leaves were
released into cups containing samples and
incubator at 20-250C for 1, 2 and 3 days. The
discolor petals were observed under
microscope to find out Phytophthora zoospore.
Discolor petals contained conidia were purified
by culturing in carrot agar (CA: 200g carrot,
20g agar, 1000ml H2O), potato carrot agar
(PCA: 20g potato, 20g carrot, 20g agar,
1000ml H2O) and corn meal agar (CMA: 200g
corn meal, 20g agar, 1000ml H2O).
2.2 Isolation of Phytophthora from soil
planting black pepper and black
pepper root by cocoa, apple, fresh
papaya fruits
Fruits were chiselled (2 cm in
diameter) and holes were smeared by moisture
soil collected from 5 cm to deep including
roots. Those fruits were covered by nylon and
placed in room temperature. Those fruits were
daily observed and margin of brown parts
occurred in fruit were cut and cultured in
media.
2.3 Using chemical and antibiotic
substrates to suppress saprophyte
microorganisms existed in samples
Nguyen Van Tuat, Pham Ngoc Dung, Nguyen Thi Ly, Le Thu Hien
58
Rose Bengal and Rifampicin with
different concentrations were used.
Experiments were conducted with three
replication (three petries) and samples trapped
were recorded.
2.4 Effect of trap purification on
isolation of Phytophthora
- Treatment 1 (one time purification).
Petals were released three times (5 days
interval) into cups containing
Phytophthora infested soil solution. The
old petals were removed and last petals
were isolated in CA medium.
- Treatment 2 (second time purification).
3 ml of solution in treatment 1 was
moved to flasks containing 200ml
sterilized water. Petals were released
three times (7 days interval) into flasks.
The old petals were removed and last
petals were isolated in CA medium.
- Treatment 3 (third time purification). 3
ml of solution in treatment 2 was moved
to flasks containing 200ml sterilized
water and petals were isolated as method
of treatment 2.
- Treatment 4 (fourth time purification). 3
ml of solution in treatment 3 was moved
to flasks containing 200ml sterilized
water and petals were isolated as method
of treatment 2.
- Treatment 5 (fifth time purification). 3
ml of solution in treatment 4 was moved
to flasks containing 200ml sterilized
water and petals were isolated as method
of treatment 2.
- Treatment 6. Control (non purification).
3. Discussions
3.1 Isolation of Phytophthora sp. caused
black pepper quick wilt disease
3.1.1 Effect of different baits on trapping of
Phytophthora
Phytophthora slowly grown on medium,
therefore it was easily competed by other
microorganisms. Phytophthora was difficult to
directly isolate from infested parts of plants,
specially infested roots and soil. Using trap
bait to determine the samples contained
Phytophthora and those trap bait will be
isolated in media (Table 1).
The result of Table 1 showed that using of
petals and fruits has a high effect on trapping
of Phytophthora. Petals released into soil and
root solution could be trapped from 42 to 46%
samples.
Table 1.Effect of different baits on trapping of Phytophthora causing black pepper quick wilt disease.
Baits
Disease collection
Soil Root
Total of
baits
Bait trapped
(%)
Total of baits Bait trapped
(%)
Rose petal 50 46.0 50 42,0
Black pepper leave 50 44.0 50 36.0
Fresh papaya fruit 50 14.0 50 16.0
Fresh cocoa fruit 50 6.0 50 8.0
CA medium 50 4.0 50 0.0
3.1.2 Effect of different petal baits on trap
and isolation of Phytophthora
Using of different color of flower as baits to
trap and isolate Phytophthora conidia (Table
2). All of petals could be attracted
Phytophthora but trap and isolation level of
petals were very different. Red rose flower
with thick petal was lightly rotten and
contaminative and could be lightly prevented
microorganisms living in water. Hence, the
ability of isolation of fungi from rose petal was
higher than that of other flower (34%).
Collaboration for Agriculture and Rural Development (CARD) Program
59
Table 2. Effect of differently petal baits on trap and isolation of Phytophthora
Bait Trap ability Isolated ability
Total of baits Bait trapped
(%)
Total of baits Bait isolated
(%)
Lagerstroemia flower 50 32 50 4
Rose mallow flower 50 39 50 6
Rose flower (red color) 50 46 50 17
Rose flower (light red color) 50 42 50 12
Loofah flower 50 34 50 3
3.1.3 Suppressive ability of chemical for
saprophyte fungi in Phytophthora
isolation
a. Suppressive ability of Viben 50BTN
chemical
Viben 50BTN (Benomyl 95%) at 0.003-
0.005% concentration have high effect on
isolation of Phytophthora (6.9-11.1%) when
compared to other concentrations (Table 3).
Table 3. Effect of Viben 50BTN chemical on
isolation of Phytophthora from infested roots
b. Suppressive ability of Tachigaren
30L (Hymexazol 30%) chemical
Tachigaren 30L chemical (a.i.
Hymexazol 30%) at concentration 0.05%
has highest effect on limitation of other
microorganisms and therefore
enhancement of Phytophthora isolation
percentage (38.9%). Other concentrations
were not or lower effect on suppression of
saprophyte microorganisms, resulting to
the isolation of Phytophthora was very
poor (Table 4).
Table 4. Effect of Tachigaren 30L on Phytophthora isolation
Treatment Total of bait traps Bait isolated (%)
Tachigaren 30L (0,1%) 100 0,0 a
Tachigaren 30L (0,075%) 100 18,0 c
Tachigaren 30L (0,05%) 100 38,9 d
Tachigaren 30L (0,025%) 100 20,2 c
Tachigaren 30L (0,01%) 100 9,1 b
V8 medium (non chemical) 100 0,0 a
CV (%) 13,2
3.1.4 Suppressive ability of anti-bacteria
chemicals for bacterial contamination
in Phytophthora isolation
a. Rose bengan anti-bacteria
Rose bengan at dosage of 10 mg/l has a high
effect on isolation of Phytophthora (16.9%),
whereas other dosages have lower effects.
Repeated experiments showed that using Rose
bengan has unstable effect on isolation,
therefore, it don’t encourage using for isolation
of Phytophthora.
b. RH anti-drug (a.i. Rifampicin 150mg)
Treatment
Root sample
isolated (%)
Viben 50BTN (0,02 %) 0.0 a
Viben 50BTN (0,01 %) 0.0 a
Viben 50BTN (0,005 %) 11.1 d
Viben 50BTN (0,003 %) 6.9 c
Viben 50BTN (0,001 %) 3.9 b
V8 medium (non chemica l) 0.0 a
CV (%) 28.2
Nguyen Van Tuat, Pham Ngoc Dung, Nguyen Thi Ly, Le Thu Hien
60
Using Rifampicin tablet (150mg) at 50mg/l has
high effect and stable on isolation (36.6%).
Application of Rifampicin at 50mg/l for
isolation of Phytophthora in orchids, rubber
and pineapple is effect on suppression of
bacteria (Table 5)
Table 5. Effect of RH anti-drug on Phytophthora isolation
Treatment Total of bait trap Bait isolated (%)
RH (10 mg/l) 100 7.0 b
RH (30 mg/l) 100 19.1 d
RH (50 mg/l) 100 36.6 e
RH (70 mg/l) 100 11.9 c
RH (100 mg/l) 100 0.0 a
V8 medium (non chemical) 100 0.0 a
CV (%) 13.3
3.1.5 Effect of combination of chemical and
anti-drug on Phytophthora isolation
Medium adding Rifampicin (50mg/l) and
Tachigaren 30L (0.05%) has a high effect on
Phytophthora isolation from rose petals.
Medium adding Viben 50BTN (0.005%),
Rifampicin (50mg/l) and Tachigaren 30L
(0.05%) and other medium adding Hymexazol
(50mg/l), pimaricin (10mg/l) and Rifampicin
(50mg/l) have high effect on directly
Phytophthora isolation from infested roots
(15.6-21.1%) (Table 6).
Table 6. Effect of combination of chemical and anti-drug on Phytophthora isolation
Treatment Bait trap isolated(%)
Root isolated (%)
Tachigaren 30L + Viben 50BTN + Rose
bengal + RH 0.0 a 0.0 a
Viben 50BTN + RH + Tachigaren 30L 22.7 b 15.6 c
RH + Tachigaren 30L 45.3 d 5.6 b
Hymexazol + Pimaricin + Rifampicin 37.3 c 21.1 d
V8 medium (non chemical) 0.0 a 0.0 a
CV (%) 12.7 25.0
3.1.6 Effect of bait trap purification on
Phytophthora isolation
In soil and root solution, there is not
only Phytophthora fungus, but also parasitic
and saprophytic microorganisms. Elimination
of those microorganisms was based on
purification of zoospores and spores in water.
Zoospores and spores of Phytophthora in
water would be stuck petal, develop mycelia
and formed sporangium. Those sporangium
produced zoospore. In order to enhance the
effect of Phytophthora isolation from soil and
root, purification of bait trap has been carried
out many times. The result of experiment
showed that effect of isolation at fourth time
purification was threefold compared to control
(Table 7).
Table 7. Effect of bait trap purification on Phytophthora isolation
Treatment Total of baittrap
Bait trap
isolated
Effect increase
(times)
First purification 100 25 1,47
Second purification 100 32 1,88
Third purification 100 44 2,58
Fourth purification 100 58 3,41
Fifth purification 100 51 3,00
Control (non purification) 100 17 1,00
Collaboration for Agriculture and Rural Development (CARD) Program
61
3.2 Isolation of Phytophthora from soil and
root of several crops
The results obtained from isolation of
Phytophthora on black pepper, this fungus has
been isolated in several plants (Table 8).
Table 8. Effect of rose petals on Phytophthora isolation from soil and root of several crops
Crops
Solution
Soil Root
Total of soil
samples
Sample
isolated (%)
Total of root
samples
Sample isolated
(%)
Durian 27 22.2 27 14.8
Aquilaria 20 25.0 20 15.0
Cafe 33 18.1 33 24.2
The Phytophthora isolation from soil ranged
from 18.1 to 25%, meanwhile Phytophthora
isolation from roots ranged from 15.0 to 26%.
3.3 Isolation of Phytophthora caused root
rot and bud rot of pineapple
3.3.1 Effect of media and sample types on
Phytophthora isolation
The result of experiment showed that
Phytophthora including P. nicotianae and P.
cinamoni have been simultaneously occurred
in three media PSM, CA and WA. However,
the effect of isolation depended on sample
types. Phytophthora was highly recorded in
fresh sample with infested samples in PSM,
CA and WA media were 86%, 54% and 14%
respectively. Meanwhile in old sample,
Phytophthora infested samples in PSM and CA
media only were 14%, 2% respectively and
specially could not appear in WA medium.
Table 9. Effect of media and sample types on Phytophthora isolation
Media Sample type
Number of
sample
isolation
Infested samples
Number of
samples
Percentage (%)
PSM
Fresh 50 43 86
Old 50 8 16
CA
Fresh 50 27 54
Old 50 0 0
WA
Fresh 50 7 14
Old 50 0 0
SPA
Fresh 50 0 0
Old 50 0 0
3.3.2 Effect of soil samples collected from
different places on ability of
Phytophthora trap
Phytophthora fungus exists and transmits in
soil by release zoospore which can be swim in
water. They infect and cause plant disease
when available condition. Zoospore trap
method has been used to know fungus in bud
rot field. The result of experiment indicated
that P. nicotianae and P. cinamoni have been
presented in disease field. Infested sample
collected from non-disease plant near disease
plant and non-disease plant far away from
disease plant were 76.67% and 36.67%
respectively (Table 10).
Nguyen Van Tuat, Pham Ngoc Dung, Nguyen Thi Ly, Le Thu Hien
62
Table 10. Effect of soil samples collected from different places on Phytophthora isolation
Place collected bait trap sample Bait trapsample
Infested sample
Infested sample Percentage (%)
Disease plant 30 30 100
Non-disease plant near disease
plant
30 23 76.67
Non-disease plant far away from
disease plant
30 11 36.67
3.3.3 Effect of disease sample source on
Phytophthora isolation
The result showed that all of disease source
can be isolated Phytophthora by petal trap with
percentage of infested sample ranged from
56% (infested base leaf) to 90% (infested soil).
Table 11. Effect of disease sample source on Phytophthora isolation
Disease source Bait trapsample
Infested sample
Infested sample Percentage (%)
Infested soil 50 45 90
Infested root 50 37 74
Infested base leaf 50 28 56
The result showed that all of disease source
can be isolated Phytophthora by petal trap with
percentage of infested sample ranged from
56% (infested base leaf) to 90% (infested soil).
4. Conclusions and recommendations
4.1 Conclusions
Using methods of zoospore trap by rose petal
and combination with bait trap purification
could be increased effect of Phytophthora
isolation from soil and root of black pepper
quick wilt disease. This method also help
agricultural technician and farmer can be
quickly diagnose Phytophthora fungus caused
quick wilt disease of black pepper. Media
adding RH and Tachigaren 30L has high effect
on Phytophthora isolation. Freshly infested
sample should be use to isolate Phytophthora.
4.2 Recommendations
This successful research result can be used as
basic methodology for sampling, detection and
identification of Phytophthora fungi occurred
in other crops in Vietnam.
REFERENCE
1. Burgess, L.W., Knight, T.E., Tesoriero, L.
and Phan Thuy Hien (2008), Diagnostic
manual for plant diseases in Viet Nam,
Australian Centre for International
Agricultural Research, 210 pp.
2. Drenth, A. and Guest, D.I. (2004),
Diversity and Management of
Phytophthora in Southeast Asia,
Australian Centre for International
Agricultural Research Canberra, 235 pp.
3. Drenth, A. and Sendall, B. (2004),
“Isolation of Phytophthora from infected
Plant Tissue and soil, and Principles of
Species Identification”. In “Diversity and
Management of Phytophthora in
Southeast Asia”, Australian Centre for
International Agricultural Research
Canberra, pp. 94 – 102.
4. Erwin, D.C. and Riberrio O.K (1996)
Phytophthora diseases worldwide. 562
pp.