2.2.69
INVESTIGATION OF THE RHIZOCTONIA SHEATH DISEASE COMPLEX IN RICE

S BANNIZA1, HC TURNER2, A JOHANSON2, DM DAVIES1, PD BRIDGE1 and MA RUTHERFORD1

lCABI Bioscience UK Centre Egham, Bakeham Lane, Egham, Surrey TW20 9TY, UK; 2NRI, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK

Background and objectives
The fungal pathogens Rhizoctonia solani, R. oryzae and R. oryzae-sativae cause sheath blight, sheath spot and aggregated sheath spot of rice, respectively, and may occur on rice concurrently. In the early stages of diseases development, when disease recognition and the implementation of appropriate management strategies are necessary, disease symptoms are extremely similar. Little is known about the interactions among the three species, and while R. solani is known to be a serious problem in rice, knowledge about the economic importance of the other two species is scarce. Identification of the species by their morphologies is possible but requires a certain degree of expertise in the genus Rhizoctonia. For this reason a range of biochemical and molecular genetic approaches were evaluated as potential diagnostic tools with the objective of selecting suitable methods to characterize the nature of the sheath disease complex.

Materials and methods
Isolates of the three species were collected during surveys in West Africa and South-east Asia and through requests to various national institutes in the regions. A representative set of isolates was selected for detailed studies. Isolates were tested for pathogenicity on rice plants under glasshouse conditions using colonized millet grains as infection units [1]. The production of species-specific zymograms of pectic enzymes was screened by polyacrylamide gel electrophoresis (PAGE) on a pectin gel [2]. A probe was derived from R. solani using primers that amplified a region of the mitochondrial DNA, and was tested for species specificity. Species-specific PCR primers were developed from the internal transcribed spacer (ITS) region rDNA and tested on pure cultures as well as infected plant material (Johanson, et al. unpublished data).

Results and conclusions
Results of all approaches enabled the differentiation of the three species of Rhizoctonia. Pathogenicity testing showed that isolates of R. solani were more virulent than isolates of the R. oryzae and R. oryzae-sativae. Lesions on plants inoculated with this species developed more rapidly and were more pronounced than with the other two species. However, lesion morphology appears not to be adequate for species diagnosis. Comparison of pectic enzyme production of the different species revealed that isolates of R. solani had characteristic zymograms with two mobile galacturonase bands, while in both other species only one weak and less mobile band of this enzyme appeared. Hybridization with the probe showed that although the probe hybridized with isolates of all 3 species, fragments of different seizes were involved in the different species. Based on sequence variations within the ITS region, species-specific PCR primers were obtained which allowed a clear identification of the three species, in pure culture as well as with infected material. Clear identification of isolates in pure culture as well as on infected plant material allowed the study of interactions of the three species in the sheath disease complex.

References
1. Banniza S, Rutherford MA, Bridge PD, Holderness M, Mordue JEM, 1996. Proceedings of the Brighton Crop Protection Conference Vol. 1, pp. 399-404.
2. Cruikshank RH, 1990. Mycological Research 94, 938-946.