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 ProgramMETHODS OF PHYTOPHTHORA ISOLATION FROM SOIL, ROOT AND INFESTED PLANT OF BLACK PEPPER AND OTHER CROPS Project title: Manage

Collaboration for Agriculture and Rural Development (CARD) Program

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 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 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

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

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

Total of baits

Bait trapped (%)

Total of baits Bait trapped

(%)

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%)

Table 2 Effect of differently petal baits on trap and isolation of Phytophthora

Total of baits Bait trapped

(%)

Total of baits Bait isolated

(%)

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

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

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)

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

Nguyen Van Tuat, Pham Ngoc Dung, Nguyen Thi Ly, Le Thu Hien

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 (%)

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

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 bait

trap

Bait trap isolated

Effect increase (times)

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

Total of soil samples

Sample isolated (%)

Total of root samples

Sample isolated (%)

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

samples

Percentage (%)

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

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 (%)

Non-disease plant near disease

plant

Non-disease plant far away from

disease plant

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 (%)

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),

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