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Survey and Evaluation of Native Fungal Pathogens for Biocontrol Of J. Trop. Agric. and Fd. Sc. 37(1)(2009): 119-128 Survey and evaluation of native fungal pathogens for biocontrol of barnyard grass (Echinochloa crus-galli complex) [Bancian dan penilaian kulat patogen untuk kawalan biologi rumput sambau (Echinochloa crus-galli complex)] S. Tosiah*, J. Kadir**, M. Sariah**, A.S. Juraimi**, N.P. Lo* and S. Soetikno*** Key words: Echinochloa, barnyard grass, Exserohilum monoceras, biocontrol agent Abstract Echinochloa crus-galli is reported to be among the three most serious weeds of rice in many countries in Asia. In Malaysia, yield loss by E. crus-galli was about 41%. Selective chemical herbicides are primary means to control the grass. However, the extended use of the herbicides may develop negative implication to the environment and public health. Several fungal pathogens have been reported to attack barnyard grass (E. crus-galli complex) in various parts of the world. This study was to isolate, identify and evaluate the native fungi from diseased barnyard grass in paddy field, for potential as bioherbicide. From surveys carried out in 2003 to 2004 throughout the Peninsular Malaysia, several fungi species were identified to be associated with the diseases. A total of 82 isolates from 12 fungus genera have been isolated. Among the fungi were E. monoceras, E. longirostratum and Curvularia lunata. The fungus, E. monoceras, was consistently found associated with the disease, virulent, stable and has the ability to produce spores profusely in culture. These characteristics suggest that it can be used as biological control agent for the grass. Introduction Echinochloa crus-galli complex (barnyard Echinochloa spp. is found widespread in grass) locally known as `rumput sambau' warm temperate and subtropical regions of consists of E. crus-galli var crus-galli and the world, extending into the tropics. Hill et E. cruss-galli var formosensis. The E. crus- al. (1990), stated that Echinochloa is more galli, E. orizycola and E. colona are competitive in direct-seeded rice than in annuals species, whereas the E. stagnina transplanted rice. and E. picta are perennial species. There are five important species The E. crus-galli is reported to be of Echinochloa in the rice granary in among the three most serious weeds of rice Peninsular Malaysia namely E. crus- in many countries in Asia, and is a major galli complex, E. oryzicola, E. colona, weed in a wide range of crops throughout E. stagnina and E. picta (Azmi 1998). the tropical and subtropical regions (Holm *Strategic Resources Research Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia **Plant Protection Department, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Malaysia ***CAB International, MARDI Headquarters, Serdang P.O. Box 12301, 50774 Kuala Lumpur, Malaysia Authors' full names: Tosiah Sadi, Jugah Kadir, Sariah Meon, Abdul Shukor Juraimi, Lo Nyok Piang and Soetikno Sastroutomo E-mail: [email protected] ©Malaysian Agricultural Research and Development Institute 2009 119 Biocontrol of barnyard grass et al. 1977). In Malaysia, the yield loss by M.B. Ellis, Daclylaria dimorhospora Echinochloa crus-galli is estimated about Veenbaas-Rijks., and Exserohilum oryzae 41% (Azmi 1992). Sivan have also been isolated from E. crus- Cultural and mechanical methods galli in the Philippines (Zhang et al. 1996). have been introduced to control the grass. In Korea, Colletotrichum graminocola However, selective chemical herbicides are showed strong pathogenicity in wide widely used. Although chemical herbicides range of growth stages of E. crus-galli var are the major contributory factor to highly praticola and E. crus-galli var caudata productive modern intensive agriculture, too (Yang et al. 2000). Exserohilum monoceras heavy a reliance upon them have created was also reported as potential bioherbicide certain problems. and not pathogenic on planted rice varieties These problems have arisen due to in China (Huang et al. 2001) and in Vietnam use, misuse, increasing cost, production (Khanh 2002). Kadir et al. (2003) reported contaminants, transport accidents, storage that E. longirostarum has good control on and disposal of toxic wastes. Its leads to Rottboellia cochichinensis (itch grass) and contamination of chemical herbicides on E. crus-galli in Malaysia. surface and groundwater, spray drift and The objectives of this study were to long-term impact of herbicide residues isolate, identify and evaluate native fungi in agriculture products. Other increasing from diseased Echinochloa in Malaysia rice issue caused by the continuous use of field for potential biocontrol of barnyard chemical herbicide is the development of grass. herbicide-resistant grass. Biological control was identified as one of the alternatives to Materials and methods reduce the problems and also suitable for Collection and isolation sustainable agricultural programme. Diseased leaves of Echinochloa spp. were Research in biocontrol of E. crus-galli collected from various locations of five has been intensified in Asia in the early rice granary areas throughout Peninsular 1990s. A stemborer, Emmalocera belonging Malaysia (Perlis, Kedah, Seberang Perai, to Lepidoptera; Pyralidae, discovered in Selangor and Kelantan) in 2003 and 2004. rice field in Kedah feeds only on E. crus- Various disease symptoms observed in the galli complex, E. oryzicola, E. satgnina fields were collected and placed in plastic and E. picta (Goto 1992). Mohamed and containers. Diseases collected from different Hamed (1986) reported that Sogatodes Echinochloa species were placed separately pusanus, Homoptera; Delphacidae found in and documented. Tanjung Karang, Selangor has preference to Fresh diseased parts or lesions were feed on Echinochloa compared to rice plant. cut near the edge about 1 cm2 in size Several fungal pathogens have and surface-sterilized with 10% sodium been suggested as biocontrol agents for hypochlorite solution, shaken vigorously and Echinochloa. They are Cochliobolus lunatus rinsed thrice with sterile distilled water to (Tsukamoto et al. 1997) and Exserohilum remove any debris and traces of disinfectant. monoceras [Septosphaeria monoceras] Four leaf pieces were placed onto moisten (Zhang et al. 1996; Zhang and Watson filter paper in Petri dishes and incubated at 1997). 28 ± 2 °C. All emergent fungi were isolated Besides Exserohilum monoceras daily until no new fungi were observed. (Drechslera) Leonard & Suggs, Bipolaris Isolates were initially identified to sacchari (E.J. Butler) Shoemaker, the genus level based on their conidial Curvularia geniculata, Tracy & Earle morphology (Ellis 1971, 1976; Sivanesan Boedijn, C. lunata (Wakker) Beodijn 1987). Stock cultures were maintained var aeria (Batista, Lima & Vasconcelos) in sterile tap water in bijoue bottle and 120 S. Tosiah, J. Kadir, M. Sariah, A.S. Juraimi, N.P. Lo and S. Soetikno kept at 17 ± 2 °C. Working cultures were Screening and pathogenicity testing maintained in V8 juice agar. Detached leaves methodFor the first Fungi with distinctive characteristics screening, healthy and fully developed of saprophytes were excluded from further leaves of E. crus-galli and rice plant consideration after their initial isolation. MR 219 collected from glass house were Other isolates were tested for pathogenicity cut into 3 cm long pieces, put in separated to barnyard by fulfilling Koch's postulates. container, surface-sterilized with 10% The screening was carried out in two sodium hypochlorite mixed with 2-3 drops stages: (i) detached leaves method, and wetting agent, rinsed and placed onto moist (ii) inoculation on seedling. filter paper in a Petri dish each with three pieces of E. crus-galli or rice plant leaves. Plant production Agar plug 6 mm diameter containing Seeds of E. crus-galli were collected during mycelium or spore and mycelium were the disease symptoms scouting. The seeds cut from 7-day-old cultures on V8 juice were multiplied in glass house for further agar and put on the leaf surface inversely. use. Seeds were kept in refrigerator at 4 °C. Before putting the agar plug, the leaf pieces For the rice plant, the new variety MR 219 were pricked with needles at spots where was used throughout the studies. The seeds the plug would be placed. The Petri dishes were obtained from MARDI Seberang Perai were then incubated at room temperature, gene bank. 28 ± 2 °C. The scoring was taken by visual Echinochloa crus-galli seeds were observation based on lesion development. soaked for 4 days until germinated before Further evaluation of the fungal pathogen transplanted to pots while paddy seeds was only emphasised on isolates that gave were soaked overnight before use. Five only positive reaction to E. crus-galli. pre-germinated seeds (coleoptile and radicle visible) were planted in 10 cm diameter Whole plant methodEchinochloa crus- plastic pots filled with water-saturated clay galli and rice plant at four-leaf stage were soil collected from rice field of MARDI inoculated with 20 ml spore suspension at Tanjung Karang. Seeded pots were placed about 2.5 x 106 spore/ml concentration. in green house. Green house condition was Prior to inoculation, calibration was done to 35/24 ± 5 °C day/night temperature, and a check the optimum amounts of suspension 12 hour photoperiods. that would wet the plant evenly. This was done using tap water. The initial volume of Inoculum preparation the water was measured using measuring All isolates were grown on V8 juice agar cylinder. After spraying the plants, the and incubated at 28 ± 2 °C light/dark volume of the left over was measured again. photoperiod. For the first screening, 7-day- This was carried out a few times to ensure old cultures were used. The cultures were consistency. The average of the difference cut at the actively growing mycelium using of the initial volume and the left over was 6 mm diameter cork borer. For the second considered as the optimum volume for screening, 14-day-old cultures were used to spraying the plants. ensure sporulation. To get enough spores, The spore suspensions were prepared 5-10 petri dishes were prepared for each as simple emulsion by adding 1% v/v corn isolate.
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