Anastomosis Groups Identification Of Rhizoctonia Spp.from Two Vegetables

Spinach and lettuce are two important kinds of leafy vegetables, which are grown in a lot of countries. Spinach were introduced to China1300years ago and the wide cultivating areas are fruitful. The annual output accounted for about90%of the total world production. Fan town in Taian is one of the largest base of spinach in the middle of the Shandong province.Due to years of planting,although Rhizoctonia sp.has been considered a very important pathogen.This disease usually infects plant roots and basal part of stem causing severe rot and leaves yellowing and even the whole plant of plants dead and results in significant economic losses.This study collected a large number of samples,aiming to explore the study of anastomosis group of Rhizoctonia sp. from spinach and lettuce,laying a foundation for further control the harm of the disease.The study completed anastomosis groups identification and pathogenicity test of Rhizoctonia sp.from vegetables disease,the main research results are as follows:A collection of225fungi isolates in Shandong Province from spinach were identified as Rhizoctonia sp.The spinach isolates caused hypocotyl and root rot symptoms. Hyphal anastomosis reactions and DNA sequence analyses of ribosomal internal transcribed spacers showed that all spinach isolates anastomosed with multinucleate Rhizoctonia AG4subgroup HG-Ⅰ,as well as with subgroup HG-Ⅲ and binucleate Rhizoctonia AG-A and AG-F. The group AG4-HG-Ⅲ was the dominant (68.44%of total isolates), followed by AG4-HG-Ⅰ (21.78%), while twenty isolates were AG-A and there were only two AG-F isolates. The5.8S rDNA-ITS sequence analysis showed that the isolates could be distinctly separated based on their AG types. The isolates of the same AG (or sub-AG) showed high sequence identity. In China,there are few reports about the occurrence of R.solani causing disease in spinach.A collection of54fungi isolates from lettuce were identified as Rhizoctonia sp.The lettuce isolates caused hypocotyl and root rot symptoms. Hyphal anastomosis reactions and DNA sequence analyses of ribosomal internal transcribed spacers showed that all lettuce isolates anastomosed with multinucleate Rhizoctonia AG1-IB,AG4subgroup HG-I,as well as with subgroup HG-Ⅲ and binucleate Rhizoctonia AG-A. The group AG1-IB was the dominant (42.59%of total isolates), followed by AG4-HG-I (38.89%), while six isolates were AG4-HG-Ⅲ (11.11%of total isolates) and there were only four AG-A(7.41%) isolates.Phylogenetic analysis among isolates belonging to different AGs was studied based upon5.8S rDNA-internal transcribed spacer (ITS) sequence. The results revealed that the isolates of the same AGs (or sub-AG) had a high sequence similarity (98～100%) and different AGs were significant different. So using5.8S rDNA-ITS analysis, we could easily identify the AGs of unknown isolate. Based on the phylogenetic tree which was constructed with25representative measured isolates from spinach and9isolates belonging to different fusion groups from GenBank, we found that the34isolates could be divided into3large branches. The two subgroups of AG-4(AG4-HG-Ⅰ and AG4-HG-Ⅲ) constituted the first branch; the second branch included AG-F isolates; AG-A isolates constituted the third branch. From the further analysis of the genetic relationship among the three branches, we could find that binucleate Rhizoctonia AG-F isolates have close genetic relationship to AG-4isolates, but Rhizoctonia AG-A isolates have the relatively far relationship.Based on the phylogenetic tree which was constructed with11representative measured isolates from lettuce and5isolates belonging to different fusion groups from GenBank, we found that the34isolates could be divided into3large branches. The two subgroups of AG-4(AG4-HG-Ⅰ and AG4-HG-Ⅲ) constituted the first branch; the second branch included AG1-IB isolates; AG-A isolates constituted the third branch. From the further analysis of the genetic relationship among the three branches, we could find that AG1-IB isolates have close genetic relationship to AG-4isolates, but Rhizoctonia AG-A isolates have the relatively far relationship.Used the methods of soil inoculation for pathogenicity identification,the30spinach isolates of Rhizoctonia sp. and several representative strains of AG4-HG-Ⅰ、AG4-HG-Ⅲ、 AG-F and AG-A were be test in the Seedling stage of potato. All multinucleate isolates had strong pathogenicity in general,30isolates of R. solani was divided in3groups:Strong pathogenic strains、Secondary pathogenic strains and Weak pathogenic strains by disease index. The AG4-HG-Ⅰ、AG4-HG-Ⅲ and had strong pathogenicity, while the binucleate Rhizoctonia AG-A and AG-F have the lowest pathogenicity.Used the methods of soil inoculation for pathogenicity identification,the31lettuce isolates and31lettuce isolates of Rhizoctonia sp. and several representative strains of AG4-HG-I、AG4-HG-Ⅲ、. AG1-IB and AG-A were be test in the Seedling stage of potato. The AG4-HG-Ⅰ and AG1-IB isolates had strong pathogenicity in general,31isolates of R. solani was divided in3groups:Strong pathogenic strains、Secondary pathogenic strains and Weak pathogenic strains by disease index. The AG4-HG-I and AG1-IB had strong pathogenicity, the AG4-HG-Ⅲ had secondary pathogenicity,while the binucleate Rhizoctonia AG-A have the lowest pathogenicity.With three pathogenic AG4-HG-Ⅲ strains (SR-6,SR-8,SR-101)against pathogenicity determination of pakchoi which rotates with the spinach crop according to Koch’s postulates.After inoculation, the symptoms of pakchoi plants near the ground is constricted parts browning, wilting leaves, seedlings infected lower leaves yellow, brown stem base to produce oval lesions.The results showed that the three strains infected pakchoi and caused diseases.Pakchoi is suitable to host the bacteria, which makes a difference to prevention and treatment of spinach diseases.