Báo cáo Nghiên cứu khoa học Methods of phytophthora isolation from soil, root and infested plant of black pepper and other crops

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.