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Bioformulations and Nano Product from Chaetomium Cupreum CC3003 to Control Leaf Spot of Rice Var. Sen Pidoa in Cambodia

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Bioformulations and Nano Product from Chaetomium Cupreum CC3003 to Control Leaf Spot of Rice Var. Sen Pidoa in Cambodia International Journal of Plant Biology 2016; volume 7:6413 Bioformulations and nano Pidoa.1 It has been reported that C. lunata caused leaf spot for the first time in India, and Correspondence: Huyly Tann, Department of product from Chaetomium that symptom showed brown leaf spot and Plant Production Technology, Faculty of cupreum CC3003 to control finally blight. Moreover, it has been demon- Agricultural Technology, King Mongkut’s Institute leaf spot of rice var. Sen Pidoa strated that C. lunata caused many symptoms of Technology Ladkrabang Bangkok, in rice e.g. grain discoloration,2 leaf spot,3 Chalongkrung Road, Ladkrabang, Bangkok in Cambodia black kernel and seedling blight.4 Sheath rot of 10520, Thailand. rice was reported for the first time in Tamil Tel.: +855.698.98655/+855.128.98655. E-mail: [email protected] Huyly Tann, Kasem Soytong Nadu, India.5 Curvularia lunata causing leaf Department of Plant Production spots on Sorghum bicolor was also reported for Key words: Brown leaf spot; Chaetomium sp.; 6 Technology, Faculty of Agricultural the first time in Pakistan. Biological control of Rice. Technology, King Mongkut’s Institute of plant diseases has widely contributed to the Technology Ladkrabang Bangkok, reduction of the use of toxic chemical fungi- Acknowledgements: this is a part of PhD research Thailand cides by farmers that pollute the environment and corresponding author would like to express and Harmon-target organisms. Chaetomium his sincere thanks to all advisory committee for sp., belonging to the Ascomycota, has been their encouragement of this research. reported as a biocontrol agent against several 7,8 Contributions: the authors contributed equally. Abstract plant pathogens. Chaetomium globosum and C. cupreum have been successfully applied to Conflict of interest: the authors declare no poten- control rice blast caused by Pyricularia oryzae.9 tial conflict of interest. Curvularia lunata was isolated from leaf The objective was to evaluate Chaetomium spot of rice var. Sen Pidoa and tested for path- cupreum CC3003 as a biocontrol agent to con- ogenicity. Chaetomium cupreum CC3003 Received for publication: 16 January 2016. trol leaf spot of rice var. Sen Pidoa caused by Accepted for publication: 25 January 2016. expressed antifungal activity against C. lunata Curvularia lunata. in dual culture test. Hexane-crude extract, This work is licensed under a Creative Commons EtOAc-crude extract and methanol-crude onlyAttribution-NonCommercial 4.0 International extract from C. cupreum inhibited sporulation License (CC BY-NC 4.0). of C. lunata with ED50 of 6.41, 0.83 and 7.81 Materials and Methods g/mL, respectively. Pot experiment revealed ©Copyright H.Tann and K. Soytong., 2016 that plant heights in treated with a spore sus- Licensee PAGEPress srl, Italy μ use International Journal of Plant Biology 2016; 7:6413 pension of C. cupreum, bioformulation of C. Isolation of pathogen and patho- doi:10.4081/pb.2016.6413 cupreum, nano product from C. cupreum and genicity test tebuconazole were not significantly different The pathogen was isolated from leaf spots of when compared to the inoculated control. rice var. Sen Pidoa in Cambodia by using the lowing previous studied methods.11,12 The Disease reduction compared to the inoculated tissue transplanting technique which followed antagonistic fungus and pathogen were cul- control from treatment with a spore suspen- the method of Tann et al.,10 and morphological- tured on PDA at room temperature (30-32ºC) sion of Chaetomium, bioformulation of C. ly identified. A pure culture of the puta- for 7 days. A 0.5 cm diameter sterilized cork cupreum, nano product from C. cupreum and tivepathogen was tested for pathogenicity. A borer was used to remove an agar plug at the tebuconazole ranged between 41.66% to Completely Randomized Design (CRD) was periphery of thepathogen and antagonistic 58.33%. Field experiment indicated that chem- performed with 4 replications. The rice var. fungus colonies. The agar plug of the pathogen ical method was decreased leaf spots infection Sen Pidoa was used for the pathogenicity test. was transferred to one side of a PDA plate and by 60%, followed by organic method (40%) and commercialThe pathogen inoculums of C. lunata was cul- an agar plug of an antagonistic fungus to the GAP methods (40%) , respectively. The chemi- tured on potato dextrose agar (PDA) and incu- opposite side. PDA plate with a single plug of cal and GAP methods were significantly higher bated for 10 days at room temperature approx- an antagonistic fungus or thepathogen served in grain weight than the organic method when imately (30-33ºC). The inoculums was adjust- as the controls. All plates were incubated at compared to the non-treated control. This is ed to 1×106 spores/mL before spray-inoculat- room temperature and abnormal spores and the first report using C. cupreum toNon control leaf ing 20-day-old rice seedlings. Brown leaf spot normal spores of pathogen in each treatment spot of rice var. Sen Pidoa caused by C. lunata symptoms were monitored and evaluated were recorded under a binocular compound in Cambodia. using a disease index as follows: 1=no symp- microscope. Data included colony diameter toms 0%, 2=small blighted spots 1-25%, (cm) and the number of pathogen spores 3=dead cells in the area of blighted spots (1-2 which were counted using by haemacytometer. mm) which turned brown 26-50%, 4=expand- Percentage inhibition of colony growth and- Introduction ed oval-shaped lesions (1-2 cm) and cell death spore production of C. lunata were computed in the center of lesion 51-75% and 5=diseased according to the following formula: % inhibi- Rice (Oryza sativa L) is one of the major area over 76 %. tion (colony diameter or spore production of food crops in Asia where it is the daily diet pathogen in control plate − colony diameter or more than in other regions of the world. The Dual culture antagonistic test spore production of pathogen in the dual cul- major problems causing reductions in the Chaetomium cupreum CC3003 wasobtained ture plate) / colony diameter or spore produc- quality and quantity of rice include pathogens from Assoc. Prof. Dr. Kasem Soytong. This tion of pathogen in control plate × 100. Colony and insect pests. Observation and preliminary promising antagonist was tested for inhibition diameter and spore production were statisti- disease diagnosis found that a leaf spot of rice of C. lunata causing brown leaf spot of rice cally computed using analysis of variance. caused by Curvularia lunata has become one var. Sen Pidoa. The experiment was done Treatment means were compared using of the most serious diseases of this crop in using the dual culture antagonistic test which Duncan’s Multiple Range Test (DMRT) at Cambodia especially in the rice var. Sen was arranged in a CRD with 4 replications, fol- P=0.05 and 0.01. [International Journal of Plant Biology 2016; 7:6413] [page 59] Article In vitro antifungal metabolites from var. Sen Pidoa were soaked in sterile water for (0.75 kg/plot) and buprofezin 25%WP (30 g/20 Chaetomium cupreum CC3003 24 hours in moisten paper until germination, L) together with tebuconazole (20 cc/20 L) every 20 days until harvest. Disease index of against Curvularia lunata then planted into pots (3 seedlings per pot). The 15-day-old rice seedlings were inoculated leaf spot was recorded as in the pot experi- Fungal growth and crude extracts by wounding leaves and applying a 1×106 ment. Other data collected were plant height C.cupreum CC3003 was cultured in potato dex- spore/mL; three wounded leaves/seedlings (cm), number of tillers, panicle number/plant, trose broth (PDB) and incubated at room tem- were done. Each treatment was applied as panicle length (cm) and panicle weight (g), perature (28-30°C) for 4 weeks. Fungal bio- mentioned above at every 15 days until har- grain weight/plot (kg) and dried hay weight masses were removed from the liquid by vest. (kg). Data were computed by analysis of cheesecloth filtration and dried over night at ANOVA and treatment means were compared 28-32°C. The extraction was performed by the Preparation of nano-particles from using DMRT at P=0.05 and P=0.01. method described by Kanokmedhakul et al.13 The air-dried fungal biomass was ground and Chaetomium cupreum CC3003 extracted with hexane (1:1 vol) and incubated Nano-particles from C. cupreum CC3003 by shaking for 24 hat room temperature. The were obtained from Joselito Dar and Kasem Results Soytong (KMITL, Bangkok, Thailand) who solvent was separately taken out of the marc by Isolation of pathogen pathogenicity filtration through filter paper (Whatman firstly investigated these new nano-particles No.4). The marc from hexane extraction was which were developed and characterized nano test extracted with ethyl acetate (EtOAc) and fol- materials loaded with active compounds from Curvularia lunata was isolated from leaf lowed with methanol (MeOH) using the same Chaetomium sp. Crude extracts from C. spot of rice in var. Sen Pidoa in this study and procedure as hexane. The solvents were sepa- cupreum CC3003 were used in this study. The demonstrated to be pathogenic on this host. rately evaporated to yield crude hexane, EtOAc extracts were incorporated into poly acetic acid and MeOH extracts. and electro spin at 25-30 kv. The product from Dual culture antagonistic test In vitro antifungal metabolites from C. cupreum CC3003 was pale orange in color. Chaetomium cupreum CC3003 significantly Chaetomium cupreum CC3003 against Scanning electron microscope images revealed inhibited C. lunata causing leaf spot of rice in Curvularia lunata was done by using the poi- that the nano material from C. cupreum meas- the dualonly culture test; spore production of son agar method.14 The experiment was done ured 171 nanometers.
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