JSM TSCHEN

Department of Botany, National Chung Hsing University, Taichung 40227, Taiwan, ROC

Background and objectives
Both nonpathogenic and hypoviruient Rhizoctonia isolates have been shown to protect various plants from damping-off and other diseases caused by virulent strains of Rhizoctonia [1]. The occurrence of plasmids in R. solani is well established[2], but their role in pathogenicity or virulence is unknown. Chitinases and related enzymes induced in plant tissues may have lytic activity against invading fungi but enhanced chitinase activity in host plants inoculated with suppressive strains of R. ;solani has not been reported. The objectives of this study were to determine the effects of plasmids in R. ;solani isolates on their virulence and their ability to suppress disease caused by highly virulent isolates, and to measure chitinase activity in radish seedlings after inoculation with suppressive isolates of R. ;solani.

Results and conclusions
Electrophoresis of fungal total DNA showed that 11 out of the 13 R. ;solani isolates carried plasmids, but that isolates CHU344 and CHU345 did not. Plasmid-carrying isolates grew faster than the two non-plasmid-carrying strains when they were grown on PDA and on glycine and threonine media (pH values 5.0 and 10.0). Virulence of the 13 R. ;solani isolates ranged from 0.04 to 1.0, based on percentage of seedlings damped-off by each.

Twelve strains of R. ;sofa were used to preinoculate radish seedlings. Eight of the isolates, namely, CHU341, CHU344, CHU345, CHU346, CHU347, CHU348, CHU350 and CHU353 suppressed damping-off caused by isolate CHU352, with suppressive index values of 0.60, 0.88, 0.48, 0.17, 0.56, 0.25, 0.69 and 0.23, respectively.

The size of chromosomal DNA appears to be greater than 23.1 ;kbp. The plasmid is approximately 2.6 ;kbp and the dimer of plasmid is approximately 5.2 ;kbp. The ultrastructure of the plasmid DNA from R. ;solani CHU341 (pCHU341), observed by transmission electron microscopy, showed that the plasmid is linear and both terminals have hairpin structures. In replication, plasmid DNA begins to make a replicate origin near the terminus of the plasmid and this origin enlarges to become a replication bubble. The Y-form replicate fork is extended by the replication bubble.

Total proteins in the radish seedlings increased by about one-tenth, whereas the specific activity of chitinase increased tenfold in crude extracts, when the seedlings were inoculated with suppressive strains CHU341, CHU344 and CHU345. We have found that the plasmids were usually in virulent strains. Plasmids may be involved in the growth of R. ;solani and protection against environmental stress; they may cause increased infectivity of R. ;solani. We demonstrated that most isolates of R. ;solani AG4, independent of their virulence or plasmid content, can be used as biocontrol agents to protect seedlings from infection by virulent R. ;solani by preinoculation. However, the use of the virulent isolates for biocontrol in the agriculture practice is not recommended. In the present study, we demonstrated for the first time the chitinase activity in radish seedlings induced by preinoculation with suppressive isolates of R. ;solani. In this case, the suppressive isolates of R. ;solani may serve as chitinase inducers which result in protection of seedlings from infection by virulent isolates.

References
1. Sneh B, Ichielevich-Auster M, Plaut Z, 1989. Canadian Journal of Botany 67, 2135-2141.
2. Jabaji-Hare SH, Burger G, Forget L, Lang BF, 1994. Current Genetics 25, 423-431.