Isolation And Identification Of Endophytes Of Mulberry And Pathogen Of Alfalfa In Shaanxi Province

Mulberry Leaf is not only the feed of silkworm,but also is a new kind of forage resource because of its high crude protein content,various mineral nutrients,rich vitamins,good palatability and high digestibility.Plant endophytes have various ecological functions,including promoting plant growth and improving plant resistance.Alfalfa is one of the most widely cultivated and important perennial leguminous grasses in the world,which has the characteristics of high crude protein content and high yield.With the increasing of cultivated area,disease has become an important factor restricting the yield and quality of alfalfa.It is an important foundation for industrial development and healthy breeding to define the types and harms of these two kinds of feed plant pathogenic bacteria and endophytic fungi,which has important theoretical and practical value.This study collected alfalfa disease samples and healthy mulberry samples from four counties(Yangling District,Bin County,Dingbian County and Yuyang District)of Shaanxi Province form 2015 to 2017.The pathogenic isolates were obtained by tissue isolation methods and were identified based on morphological characteristics and gene sequencing analysis.The pathogenicity of isolates were carried out by using WA plate and sprinkling the root.The main contents and conclusions of this study are as follows:1.The isolation and purification of endophytes from the healthy branches and leaves of Hongguo 2 from Yangling District were collected by tissue block separation method.Identification of isolates by using morphological characteristics and gene sequencing analysis,totally 10 isolates were obtained.That belonged to 7 gengra,namely,Colletotrichum sp.,Phoma sp.,Phomopsis sp.,Peyronellaea sp.,Fusarium sp.,Mucor sp.,and Xylaria sp.The results showed that the endophytes of Hongguo 2 in Yangling District were distributed in 7 genera,and the separation rate was higher in the genus of Phoma sp,Colletotrichum sp and Fusarium sp.2.The results of the investigation on the diseases of alfalfa growing region in Shaanxi Province from 2015 to 2017 show that: The brown spot and leaf spot were the main alfalfa diseases in Bin County,among which the 2015 incidence was the highest,43.89% and 54.09% respectively.Investigation of alfalfa diseases in Dingbian County in 2015-2016,the spot blight was the most serious,the September 2016 incidence of up to 55.29%;The brown spot was the most serious disease in Yuyang District in 2015,incidence is up to 55.5%.Alfalfa rust,root rot,black stalk,powdery mildew and the harm of the disease have occurred,but the frequency is lower than the spot and leaf spot.The results showed that alfalfa disease occurred in Shaanxi province,which was characterized by many disease types,long time,brown spot and leaf spot as the main disease,while the other diseases had less damage.3.The results showed that four species were identified from the sample of alfalfa root rot,including Fusarium avenaceum,Fusarium solani,Fusarium incarnatum and Fusarium oxysporum.Fusarium avenaceum wad isolated from all of 4 locations in Shaanxi Province,Fusarium solani was isolated from 3 locations in Binxian County,Dingbian County and Yuyang District,Fusarium oxysporum and Fusarium oxysporum were isolated in Binxian and Yangling Districts,respectly.The results showed that: all strains were pathogenic.The pathogenicity of Fusarium solani DB2 strain from Dingbian was significantly higher than that of Fusarium avenaceum DB1 strain.There was no significant difference in the disease index between the isolates the other strains.The pathogenicity test of live inoculation also validated that pathogens BX1,BX2,BX3 and YL2 had pathogenicity to alfalfa plants.At the same time,the four pathogenic Fusarium strains of alfalfa root rot were also first reported in Shaanxi Province.The results showed that the dominant pathogenic species of alfalfa root rot in Shaanxi Province was Fusarium avenaceum,followed by Fusarium solani.4.In 2017,we found a new disease--alfalfa leaf blight in Yangling Districts.Isolation,purification,pathogenicity determination and biological characteristics of pathogenic bacteria after collecting disease samples,and we obtained a pathogenic strains,Rhizoctonia solani.The result of biological chatacteristics showed that the optimum pH for tis growth was 7.Dark conditions conducive to the growth of the pathogen.Soluble starch,not fructose was the optimal carbon source for mycelia growth of this pathogen.The optimal nitrogen source was potassium nitrate rather than ammonium nitrate.The results showed that the pathogen that causes alfalfa leaf blight was Rhizoctonia solani.