Genetic Diversity And Evolution Of Rhizoctonia Solani Associated With Root Rot Of Alfalfa

Alfalfa(Medicago sativa)is the most widely planted forage crop in China and worldwide.Root rot is one of the main limiting factors in alfalfa production,which seriously affects the yield and quality of forage,and reduces its feeding value and economic value.Rhizoctonia solani is one of the major pathogens associated with root rot in alfalfa.Diseases cuased by this pathogen are difficult to control as it can survive in soil and plant residues for many years and has abundant intraspecific genetic variation.At present,there are few studies on the genetic diversity and evolution of R.solani associated with alfalfa root rot.This study firstly isolated and identified the fungal pathogens from plant samples collected in northwest China;the pathogenicity of R.solani was then evaluated by soil inoculation method,followed by analysis on the genetic diversity and phylogenics of R.solani.This study aimed to reveal the pathogenicity and evolutionary of R.solani and to provide theoretical basis for the effectively control of root rot in alfalfa.The main results are as follows:1.A total of 162 R.solani strains were isolated from root tissue,which collected from 21 alfalfa planting areas in five northwestern provinces(Gansu,Shaanxi,Ningxia,Xinjiang and Qinghai),accounting for 4.9% of the total strains numbers.The isolation frequency of R.solani isolated from Ningxia was the highest(9.0%),followed by Xinjiang(6.6%),Qinghai and Gansu(both <3.0%),and no R.solani was isolated in Shaanxi.61 representative strains of R.solani were cultured on PDA medium.After 3 days,they were divided into three groups: slow growth(38%),medium growth(54%)and rapid growth(8%).After 14 days,according to whether or not the sclerotia was formed.19 strains that formed sclerotia were divided into three types(dispersive,peripheral and central)according to the distribution of the sclerotia,42 strains without the sclerotia were divided into four types(G1-4)according to colony morphology and color.2.The pathogenicity of 61 representative strains of R.solani to Longdong alfalfa seedlings were evaluated by the method of soil inoculation and were scored with disease severity index of shoot and root,shoot height and root length,aboveground and underground biomass.The results showed that there were significant differences in the pathogenicity of different strains(P<0.05).The disease index of aboveground and root were 6.3-73.8 and 1.3-67.5 respectively.The relative reduction values of plant height and aboveground biomass were 3.8%-82.9% and 15.7%-96.4%respectively.The relative reduction values of root length and underground biomass were 78.1% and 95.7% respectively.XJR1 was the most virulent and NXR24 was the least virulent.The pathogenicity of the strains in different provinces was also different.8 strains with strong pathogenicity were 3 strains in Ningxia and 5 strains in Xinjiang;31 strains with medium pathogenicity were 14 strains in Ningxia,13 strains in Xinjiang and 4 strains in Qinghai;22 strains with weak pathogenicity were 13 strains in Ningxia and 9 strains in Xinjiang.3.The genetic evolutionary relationship of 62 R.solani strains in Ningxia,Xinjiang and Qinghai were studied by phylogenetic analysis of ITS gene sequence.The results showed that 62 strains were distributed in 5 sub-branches under2 major genetic branches.Then add the ITS gene sequence of other plant R.solani reference strains,another genetic evolutionary relationship were studied again.The result showed that 5 branches were identified as AG2 and AG4,AG5,AG-A and AG-K anastomosis group.Among them,AG-K has the highest frequency(41.9%),followed by AG-A,AG4 and AG5,and AG2 has the lowest frequency(1.6%).There was a certain correlation between the strains of different AG groups and their geographical origin,pathogenicity and morphological classification(the strains belonging to AG-A were all from Ningxia;the strains of AG5 only contain Qinghai and medium virulence strains in geographical origin and pathogenicity;the strains of AG4 were all from medium and fast-growing strains,and all produced the sclerotinia in the peripheral and dispersed form).There was no significant difference among strains on different hosts of the same AG group.