Biological control of rice blast (Magnaporthe oryzae) by use of Streptomyces
Streptomyces are one of the most attractive sources of biologically active substances such as vitamins, alkaloids, plant growth factors, enzymes and enzyme inhibitors (1-3). Soil Streptomycetes are of the major contributors to the biological buffering of soils and have roles in
decomposition of organic matter conductive to crop production (4), (5). Studies even show that use of Streptomycetes enhances growth of the crop plants (6). The search for new principles in biocontrol of plant pathogens different from the classical used fungicides, is of world wide concern (7-9). Biological control of plant diseases is slow, gives few quick profits, but can be long lasting, inexpensive and harmless to life. Rice blast, caused by Magnaporthe oryzae (anamorph Pyricularia oryzae Cav.), is considered the most important disease of rice because of its world-wide distribution and the resulting severe yield loss (10). At the present research 100 isolates of Actinomycetes were isolated from agricultural soils of Kerman of Iran and screened against M. oryzae in vitro. The objective of the present study was also to isolate Streptomyces isolates having antagonistic properties with the aim that they can serve as gene donors in developing resistant transgenic plants and use as soil amendments as biofertilizer or biofungicide in biological control of the tested pathogen. From all tested isolates of Actinomycetes, 10 Streptomyces isolates showed high in vitro anti rice-blast activity.
MATERIALS AND METHODS
Culture media and preparation of pathogen: Pure culture of Magnaporthe oryzae was obtained from Mr. Padasht, Iran Rice Research Institute (IRRI), Rasht. The pathogen was maintained on potato dextrose agar (PDA, Difco-39 g PDA [L.sup.-1] of distilled [H.sub.2]O, pH 7.2). Casein glycerol agar (CGA) was prepared from basic ingredients as described by Kuster and Williams (11) and used as Actinomycetes culture.
Soil sampling and isolation of streptomycetes: Soil samples were collected from grassland, orchards and vegetable fields in different localities of Kerman provinces, Iran. Several samples randomly were selected from the mentioned localities using an open-end soil borer (20 cm in depth, 2.5 cm in diameter) as described by Lee and Hwang (12). Soil samples were taken from a depth of 10-20 cm below the soil surface. The soil of the top region (10 cm from the surface) was excluded. Samples were air-dried at room temperature for 7-10 days and then passed through a 0.8 mm mesh sieve and were preserved in polyethylene bags at room temperature before use. Samples (10 g) of air-dried soil were mixed with sterile distilled water (100 mL). The mixtures were shaken vigorously for 1 h and then allowed to settle for 1 h. Portions (1 mL) of soil suspensions (diluted [10.sup.-1), were transferred to 9 mL of sterile distilled water and subsequently diluted to [10.sup.-2], [10.sup.-3], [10.sup.-4], [10.sup.-5], [10.sup.-6]. Inocula consisted of adding aliquots of [10.sup.-3], [10.sup.-6] soil dilutions to autoclaved CGA (1, 25 m[L.sup.-1] CGA) at 50[degrees]C before pouring the plates and solidification. Three replicates were considered for each dilution. Plates were incubated at 30[degrees]C for up to 20 days. From day 7 on, Streptomyces colonies were isolated on CGA, incubated at 28[degrees]C for one week and stored refrigerated as pure cultures before use. For screening studies 100 pure Streptomyces isolates were collected.