Variation Of RDNA Internal Transcribed Spacer Sequences In Rhizoctonia Cerealis

Wheat sharp eyespot is a soil-borne fungal disease,occurring in many different regions around the world.The pathogen is Rhizoctonia cerealis,which belongs to binucleate Rhizoctonia spp.R.cerealis is difficult to obtaine asexual or sexual spores,and there is no report to prove whether two of the nuclei are homogeneous or heterogeneous,which made it difficult to reserch on molecular genetics and pathogenic mechanism of R.cerealis.Although ITS sequences heterogeneity had been reported,the regular and reason had not been discussed in detail.In our study,we had analyzed the ITS sequences obtained from single protoplast isolates(SPIs),and investigate the heterogeneity of ITS to establish if the ITS heterogeneity is associated with the heterogeneity of nucleus,and establish if the ITS is reliable for anastomosis group or subgroup identification despite heterogeneity existence in R.cerealis.Follows are the mian conclusions:1.By using the technique of protoplast preparation and regeneration,we obtained several single protoplast isolates from WK-207,WK-137,WK-159,respectively.The mycelia were stained with 4′,6-diamidino-2-phenylindole(DAPI).The nuclei of the three test strains and their SPIs were observed and photographed using fluorescence microscope.The results showed that SPIs were similar with their original strains in aspect of colony,mycelial morphology and nuclear phase.2.One hundred and sixty-nine ITS sequences were obtained in all,include 89 ITS sequences from WK-207,50 ITS sequences from WK-137,30 ITS sequences from WK-159.ITS sequences polymorphism was analyzed.The results showed that a set of variations were found in ITS sequences from three different test strains,including GC content,ITS length and single nucleotide polymorphism(SNPs),and the variations were also found in the same strain as well as in the different ITS copies of same SPI.3.The mutation rates are different between ITS1 region and ITS2 region.In our study,the ITS2 region seemed to be more prone to base variation,but the ITS1 region was moreprone to base deletion,among R.cerealis WK-207 、 WK-137 、 WK-159.Furthermore,transitions dramatically exceed transversions in both regions.4.Eighty-nine ITS sequences of WK-207 cluster into two separated clades in phylogenetic tree,which reviewed that two different nucleus could be existed in one cell.It provides an evidence for nuclei heterogeneity by sequence analyzing in R.cerealis.5.By using DNAMAN,we analyzed the sequence similarity,which was up to 99% or more for all of three strains.In addition,by sequence phylogenetic analyzing,169 ITS sequences all belonged to AG-D Ⅰ anastomosis subgroup.The results indicated that the variation of ITS did not affect identification of anastomosis group,even at the level of subgroups.This study analyzed the ITS heterogeneity of R.cerealis in detail,which were found in different test strains,or in the same strain as well as in the different ITS copies of same SPI.And the variation was related in nuclei heterogeneity.Although the ITS heterogeneity existed,it is still accurent in identificating of anastomosis group and subgroups in R.cerealis.