Báo cáo nghiên cứu khoa học " METHODS OF PHYTOPHTHORA ISOLATION FROM SOIL, ROOT AND INFESTED PLANT OF BLACK PEPPER AND OTHER CROPS " doc

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 Co
de:
CARD
052/04 VIE
Author(s):
Nguyen Van Tuat
1
, Pham Ngoc Dung
1
, Nguyen Thi Ly
1
, Le Thu Hien
1
1
National 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 ser
ious 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 r
ose 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 techniq
ues 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.
S

everal 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 blac
k 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 significantl
y 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 large
ly 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
crop
s 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 R
esearch 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 imp
rove 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
dise
ases 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
fun
gus
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 in
to 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
-
25
0
C 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 H
2
O), potato carrot agar
(PCA
: 20g potato, 20g carrot, 20g agar,
1000ml H
2
O) and corn meal agar (CMA: 200g
corn meal, 20g agar, 1000ml H
2
O).
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 petri
es) 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 flask
s.
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.
D
iscussions
3.1
Isolation of
Phytophthora sp.
caused
black pepper quick wilt disease
3.
1
.1
Effect of dif
ferent 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 t
rap

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 dif
ferent 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
contaminat
ive 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 hig
hest 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 beng
an 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 effe
ct 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 isolat

ion 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 infeste
d 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 + Tachi
garen 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 solu
tion, 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 pet
al, 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 effe

ct 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 bait
trap
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
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(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
s
amples
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 rang
ed 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 bee
n 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
isolatio
n
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 abilit
y 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 re
sult 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 trap
sample
Infested sample
Infested sample
Percentage (%)
Disease plant
30
30
100
Non
-
disease plant near di
sease
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 trap
sample
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 recommendation
s
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
a
gricultural 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
Phytopht
hora
.
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 Pha
n 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 In
ternational
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 Asi
a
”, Australian Centre for
International Agricultural Research
Canberra, pp. 94
–
102.
4.
Erwin, D.C. and Riberrio O.K (1996)
Phytophthora
diseases worldwide. 562
pp.