University of Florida, Fort Lauderdale Research and Education Center, Fort Lauderdale, Florida 33314, USA

Background and objectives
Gaeumannomyces graminis var. graminis is a pathogen of Poaceae species, grown in tropical, subtropical and southern temperate climates, that causes a serious root rot of warm-season turfgrasses in the USA. One biological control strategy would be to obtain G. g. graminis strains that are non-pathogenic for use as post-fumigation soil treatments. Fungal morphology, physiology and pathogenicity can be altered using mutagenesis or protoplasting techniques [1, 2]. The objectives of this project were to obtain and characterize strains of G. g. graminis using these techniques.

Materials and methods
The G. g. graminis isolate selected for use was FL-39, an isolate with demonstrated pathogenicity on turfgrasses and wheat, that consistently produced fertile perithecia. Three methods were used to obtain strains: (i) protoplasts generated from mycelia (32 strains); (ii) protoplasts generated from mutagenized mycelia (127 strains); and (iii) mutagenized mycelia (11 strains). The mutagen was N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Rates ranged from 3 to 50 g/ml. These strains were characterized for differences in morphology, growth rate and pathogenicity from the original FL-39 isolate. Pathogenicity assays used wheat as the host. The in vitro assay used surface-sterilized wheat seed germinated and grown on water agar. The greenhouse assay used wheat grown in non-sterile vermiculite.

Results and conclusions
The two distinguishing characteristics used to identify G. g. graminis, lobed hyphopodia and ascospore size, were lost in 26 strains. No perithecia were produced by these strains, and lobed hyphopodia were replaced by structures with an amorphous periphery enclosing a dark, dense substrate in the centre. An additional 17 strains maintained production of normal hyphopodia but no longer produced perithecia. Remaining strains had normal hyphopodia and produced fertile perithecia. Only 11 strains had wheat disease rating values that were less than the maximum value of 5 (dead plants) in the initial pathogenicity screening using the water agar/wheat seedling assay; control treatment values were consistently rated as 1 (healthy plant). All 11 strains were obtained from regenerated protoplasts of mutagenized mycelia, either 12.5 or 25 g/ml of MNNG.

Two of these 11 strains subsequently died in storage. The remaining nine strains were subjected to further evaluations and comparisons with the parent FL-39 isolate. Six strains caused significantly less root disease than the parent isolate in the water agar/wheat seedling assay. Nine strains caused significantly less root disease than the parent isolate in the greenhouse wheat seedling assay. FL-39 had root disease rating values of 5 (1=healthy roots; 5=black, rotted roots). Only three strains had root disease rating values that were equal to or less than 2 in both pathogenicity assays. These strains were considered to be non-pathogenic and could only be detected in the root segment adjacent to the crown of the plant (0-2.5 cm), whereas all other strains and the parent isolate had colonized the entire length of the root system. This apparent lack of root colonization by the non-pathogenic strains will probably preclude their use as biological control agents.

The three non-pathogenic strains possess distinct cultural characteristics that distinguish them from the parent isolate FL-39. Two strains (FL-39-106 and -150) develop a water-soaked appearance after 5-7 days' growth on PDA. Strain FL-39-111 cultures sink into the PDA such that the surface is no longer smooth but composed of ridges. This strain also has significantly slower growth than parent isolate FL-39. All three strains still produce lobed hyphopodia, but none produces perithecia. The absence of a sexual stage may mean that the mutant will fail to cross with wild-type isolates. Prospects for biocontrol are discussed.

1. Boland GJ, Smith EA, 1991. Phytopathology 81, 766-770.
2. Yang, HA, Zhou J, Sivasithamparam K, O'Brien PA, 1994. FEMS Microbiology Letters 115, 83-86.