Collaboration for Agriculture and Rural Development (CARD) Program 56 METHODSOFPHYTOPHTHORAISOLATIONFROMSOIL,ROOTANDINFESTEDPLANTOFBLACKPEPPERANDOTHERCROPS Project title: Management ofPhytophthora 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 ofPlant Protection Project Implementing organisations: Vietnamese Institution : National Institute ofPlant 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 isolationof 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 ofPhytophthora 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 andblack 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 cropsplant at highland of Northern and Central Vietnam. The diversity of climate also is suitable conditions for development ofPhytophthora 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 ofPlant 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 ofPhytophthora 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 ofblack pepper, quick death of durian, bud rootof pineapple, wilt death of litchi, downy mildew of tomato and potato, stem canker of citrus, black strip of rubber… However, researches ofPhytophthora fun gus were limitation because it is difficult to isolate this fungus. Results obtained from training courses helped us to conduct special subject “Method ofPhytophthoraisolationfromsoil,rootandinfested plants.” 2. Research materials andmethods 2.1. IsolationofPhytophthorafrom soil planting blackpepperandblackpepperroot 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 blackpepper 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 IsolationofPhytophthorafrom soil planting blackpepperandblackpepperroot 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 isolationofPhytophthora - Treatment 1 (one time purification). Petals were released three times (5 days interval) into cups containing Phytophthorainfested 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 IsolationofPhytophthora sp. caused blackpepper quick wilt disease 3. 1 .1 Effect of dif ferent baits on trapping ofPhytophthoraPhytophthora slowly grown on medium, therefore it was easily competed by other microorganisms. Phytophthora was difficult to directly isolate frominfested parts of plants, specially infested roots and soil. Using t rap bait to determine the samples contained Phytophthoraand 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 ofPhytophthora . Petals released into soil androot solution could be trapped from 42 to 46% samples. Table 1.Effect of different baits on trapping ofPhytophthora causing blackpepper 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 Blackpepper 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 andisolationofPhytophthora 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 andisolation 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 ofisolationof fungi from rose petal was higher than that ofother flower (34%). Collaboration for Agriculture and Rural Development (CARD) Program 59 Table 2. Effect of differently petal baits on trap andisolationofPhytophthora 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 Phytophthoraisolation a. Suppressive ability of Viben 50BTN chemical Viben 50BTN (Benomyl 95%) at 0.003 - 0.005% concentration have high effect on isolationofPhytophthora (6.9 - 11 .1%) when compared to other concentrations (Table 3). Table 3. Effect of Viben 50BTN chemical on isolationofPhytophthorafrominfested 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 ofother microorganisms and therefore enhancement ofPhytophthoraisolation percentage (38.9%). Other concentrations were not or lower effect on suppression of saprophyte microorganisms, resulting to the isolationofPhytophthora was very poor (Table 4). Table 4. Effect of Tachigaren 30L on Phytophthoraisolation 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 Phytophthoraisolation a. Rose bengan anti - bacteria Rose beng an at dosage of 10 mg/l has a high effect on isolationofPhytophthora (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 isolationof 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 isolationofPhytophthora in orchids, rubber and pineapple is effe ct on suppression of bacteria (Table 5) Table 5. Effect of RH anti - drug on Phytophthoraisolation 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 Phytophthoraisolation 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%) andother medium adding Hymexazol (50mg/l), pimaricin (10mg/l) and Rifampicin (50mg/l) have high effect on directly Phytophthoraisolationfrom infeste d roots (15.6 - 21.1%) (Table 6). Table 6. Effect of combination of chemical and anti - drug on Phytophthoraisolation 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 Phytophthoraisolation In soil androot 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 ofPhytophthora in water would be stuck pet al, develop mycelia and formed sporangium. Those sporangium produced zoospore. In order to enhance the effect ofPhytophthoraisolationfrom soil and root, purification of bait trap has been carried out many times. The result of experiment showed that effe ct ofisolation at fourth time purification was threefold compared to control (Table 7). Table 7. Effect of bait trap purification on Phytophthoraisolation 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 Collaboration for Agriculture and Rural Development (CARD) Program 61 3.2 IsolationofPhytophthorafrom soil androotof several crops The results obtained fromisolationofPhytophthora on black pepper, this fungus has been isolated in several plants (Table 8). Table 8. Effect of rose petals on Phytophthoraisolationfrom soil androotof several cropsCrops Solution Soil Root Total of soil s amples Sample isolated (%) Total ofroot 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 Phytophthoraisolationfrom soil ranged from 18.1 to 25%, meanwhile Phytophthoraisolationfrom roots rang ed from 15.0 to 26%. 3.3 IsolationofPhytophthora caused root rot and bud rot of pineapple 3.3.1 Effect of media and sample types on Phytophthoraisolation 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 ofisolation 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, Phytophthorainfested 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 Phytophthoraisolation 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 ofPhytophthora 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 plantand 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 Phytophthoraisolation 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 Phytophthoraisolation The result showed that all of disease source can be isolated Phytophthora by petal trap with percentage ofinfested sample ranged from 56% (infested base leaf) to 90% (infested soil). Table 11. Effect of disease sample source on Phytophthoraisolation Disease source Bait trap sample Infested sample Infested sample Percentage (%) Infested soil 50 45 90 Infestedroot 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 ofinfested sample ranged from 56% (infested base leaf) to 90% (infested soil). 4. Conclusions and recommendation s 4.1 Conclusions Using methodsof zoospore trap by rose petal and combination with bait trap purification could be increased effect ofPhytophthoraisolationfrom soil androotofblackpepper quick wilt disease. This method also help a gricultural technician and farmer can be quickly diagnose Phytophthora fungus caused quick wilt disease ofblack 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 ofPhytophthora fungi occurred in othercrops 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 ofPhytophthora in Southeast Asia , Australian Centre for In ternational Agricultural Research Canberra, 235 pp. 3. Drenth, A. and Sendall, B. (2004), “Isolation ofPhytophthorafrom infected Plant Tissue andsoil,and Principles of Species Identification”. In “ Diversity and Management ofPhytophthora 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. . 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. 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. 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