The Etiology And Pathogenic Mechanism Of Peanut Sheath Blight

Peanut is an important oil crop and economic crop in China.It has a wide planting area and is distributed in most provinces in China.As main peanut-producing area,Liaoning Province is an important export base in the Northeast.The peanut industry has become an important way for farmers to increase production and income.Peanut area is increasing year by year.Large-scale planting and intensive production provide favorable conditions for the occurrence of pests and diseases,leading to the rise of diseases and insect pests.Peanut sheath blight was found in Liaoning Province in recent years as a new disease which has caused huge amount loss.This study investigated etiology of pathogen,infection process,main pathogenic mechanism of peanut sheath blight and obtained cDNA full-length sequence of PG gene.Results are as follows:1.Peanut sheath blight field investigation and etiology were determined.In July 2013,we first found the new disease peanut sheath blight in Xiawangjia Village,Xiawangjia Town,Liaoyang City,Liaoning Province.The symptoms are water-soaked lesions,necrosis of leaves,and web-like mycelia on plants,followed by the development of sclerotia on tissue surfaces.The disease in peanut occurs severely between mid-July and mid-August,and high humidity and high temperature are conducive to disease occurrence.The disease occurred in Liaoning and Shandong Province and the disease incidence is as high as 100%in severe cases.The pathogen was identified as Rhizoctonia solani through morphology and molecular biology,and identification by Koch's rule after isolation and purification.The pathogen of peanut,corn and rice sheath blight are all pathogenic on the three hosts respectively,but the pathogenicity is different.For hypha growth,best temperature ranged from 25³0°C and best pH was 7⁸.The optimum medium was Czapek and Oat medium.Starch and peptone were most suitable carbon and nitrogen source.Light promoted growth of hypha.For sclerotium formation,best temperature was 25³0°C and best pH was 7.The optimum medium is corn flour medium and PDA.Glucose and starch were most suitable carbon source while sodium nitrite was most suitable source.Light has no significant effect on sclerotia formation2.The infection process of R.solani on the host was studied for the first time.Through macroscopic observation,cytochemistry,microscopic and ultramicroscopic techniques,it was found that the host leaf infected by R.solani could form infection structures including appressorium and infection cushions.The pathogen infected host fast,and the lesion appeared at 48 h post inoculation?HPI?.Lesions were formed under the infection cushions.The development of the pathogen and symptoms on the peanut leaf include:hyphae from inoculum grew onto a leaf surface?6 h?.After hyphae grew in contact with the leaf surface and spread,great amount of appressorium were produced?12 h?.Great amount of white to fawn infect cushions arose?24 h?.Viscous material were produced around infection cushions,and cell membrane system in leaf surface appeared injury or death slightly;infection cushions turned into light brown and the bottom of cusion appeared to shrink;cells under infection cushion were dead massively;hyphae invade into the leaf,grew and expanded extracellularly and intracellularly with cell deformation,starch grains disappearance,plasmid separation,chloroplasts disintegration,fat droplets increase,and mitochondria disappearance?36 h?.Infection cushions became much more shrinking and color was deepened;obvious lesion appeared under infeciton cushion and the tissue became decayed.;cells in the lesion area were dead massively?48 h?.Infection cushions became more color-deepened and more shrinking.Lesions under infection cushion expanded and combined;cells in the decayed tissue area were dead massively[60 h or?and?after 60 h].3.The pathogenic mechanism of peanut sheath blight was identified for the first time.R.solani produced several cell wall degrading enzymes in vitro?in liquid culture?and in vivo?in peanut plants?.PG,PMG,Cx and?-glucosidase activities were detected in infected tissues including stalk and leaves of Baisha and Silihong peanut cultivars.PG and PMG activities were more significant.Extracts of healthy tissue showed little or no such activities.In shaken liquid cultures of R.solani in medium containing pectin or pectin plus carboxymethyl cellulose?CMC?as the carbon source?s?,PG and PMG were notably active.Significant Cx activity was detected in cultures with CMC or pectin plus CMC as the carbon source?s?.It was determined that substrate was main inducer.However,only a very low level of?-glucosidase activity was observed in cultures with any of the tested carbon sources.An increase of pH was recorded in decayed peanut tissues and liquid culture filtrates;the filtrate pH and fungal growth positively correlated.PG,PMG and Cx activity were found to correlate extremely significantly with pH and fungal growth in pectin plus CMC shaken cultures.PG,PMG and?-glucosidase in shaken cultures in medium containing pectin,PG,PMG and Cx activity in CMC medium showed significantly positive correlation with pH and fungal growth.It was preliminary determined that fungal growth and?or?pH were important infulence factors on theses enzymes.The crude enzymes extracted from liquid culture of R.solani induced decay of healthy peanut leaves.A single active PG isozyme with isoelectric point around 9.2 was detected in culture filtrates and in infected peanut tissues by the method of isoelectric focusing electrophoresis.4.The PG gene fragment from the agent of peanut sheath blight and transcription expression level of the agent R.solani inoculated on peanut leaves were investigated.The full-length cDNA sequence and CDS sequence of PG gene of the pathogen were obtained by using the RACE technique.The sequence lengths of cDNA and CDS were 1255 bp and 1095bp,respectively,including an ORF composed of 364 amino acids.The molecular weight of PG was predicted to be 37511.96 D,and the theoretical isoelectric point was 8.93.Total number of positively charged residues was 28;total number of negatively charge residues was22;the molecular formula was C₁₆₃₄H₂₅₉₀N₄₆₀O₅₃₀S₁₁,the instability index was computed to be 20.93,the aliphatic index was 74.51,and grand average of hydropathicity was-0.132.Signal peptide prediction analysis suggested that one signal peptide site existed,and the cleavage site was between amino acids 29-30.Subcellular localization was extracellular.Meanwhile phosphorylation sites analysis suggested 25 Ser sites,21 Thr sites,4 Tyr sites.After R.solani inoculated on peanut leaf,PG showed up-regulated expression at 12 h and the highest expression was at 60 h after inoculation.