Study On Pathogen And Its Pathogenicity Of Alfalfa Anthracnose

Anthracnose is a kind of destructive,widespread fungus disease that causes economic losses of many plants,and this disease is also the one of factors that limits production of alfalfa.But there are few studies on alfalfa anthracnose in China at present,and it is hardly known the information about the pathogen species,pathogenicity,distribution area,disease severity and quality losses.Studies are needed in order to obtain the information above,and to find out the control strategy.In this present study,the cultivated alfalfa in northwest(Gansu,Ningxia and Xinjiang provinces),southwest(Yunnan and Sichuan provinces),and north(Inner Mongolia and Heilongjiang provinces)of China were used as research material.Disease investigations,pathogens isolation,morphological and molecular identification,pathogenicity tests,and the conventional nutrition tests of alfalfa anthracnose were carried out through field trial,laboratory trial,and greenhouse trial from 2014 to 2018,and obtain the main results as follows.1.There were 16 surveyed areas in 7 provinces in total,alfalfa anthracnose caused by five Colletotrichum spp.was found in 8 surveyed areas in 6 provinces.Alfalfa anthracnose caused by C.trifolii occurred in Gansu(Jiuquan),Ningxia(Yinchuan),and Xinjiang(changji).Alfalfa anthracnose caused by C.truncatum occurred in Inner Mongolia(Chifeng)and(Shaerqin).Alfalfa anthracnose caused by C.destructivum occurred in Sichuan(Xindu).Alfalfa anthracnose caused by C.americae-borealis occurred in Yunnan(Xiaoshao),Gansu(Minle)and Inner Mongolia(Shaerqin).Alfalfa anthracnose caused by C.incanum occurred in Inner Mongolia(Chifeng).Among these Colletotrichum species,it is the first report that alfalfa anthracnose caused by C.incanum in the world,and alfalfa was the new host out of soybean.It is also the first report that alfalfa anthracnose caused by C.americae-borealis in China.2.According to the colony characteristics,morphological characteristics andpolygenic phylogeny,it is determined that five Colletotrichum spp.have three different shape conidia and belong to four species complex in Colletotrichum,namely C.americae-borealis and C.destructivum belong to destructivum complex with straight conidia.C.truncatum and C.incanum respectively belong to truncatum complex and spaethianum complex with curved conidia.C.trifolii belong to orbiculare complex with cylinder conidia.In addition,the related sequences of reported pathogen JL01 of alfalfa anthracnose in Jilin were re-analysed and studied and confirmed that strain JL01 was C.americae-borealis rather than C.lini,therefore it has not reported that alfalfa anthracnose caused by C.lini in China.To test the effect of temperature and pH on fungal growth,representative strains of each Colletotrichum species were used and it was found that temperature range from 25 to 30 °C and pH range from 5 to 7.The morphology study also comfirmed that the conidium production from setal apices was only fould in C.trifolii and C.truncatum,and conidia from conidiogenous cells was common in other species.3.Through field investigation,it was found that alfalfa anthracnose caused symptoms and damage mainly on stems,sometimes on leaves,and also on root crown when the disease was serious.The symptoms of anthracnose caused by different pathogens from each region was basically the same without significant differences.Among the 6 provinces,8 anthracnose areas,the alfalfa anthracnose occured in cultivated in Gansu(Jinta),Ningxia(Yinchuan),and Inner Mongolia were the most serious,with 39.5% to 61.7% incidence and 32.7% to 45.4% disease severity.While the alfalfa anthracnose occured in Inner Mongolia(Shaerqin),Sichuan(Xindu),Gansu(Minle),Yunnan(Xiaoshao)the effects were relatively mild with less than 30%disease seveirty.The conventional nutrition,18 kinds of amino acid between,and dry weight between anthracnose-diseased and healthy plants from serious areas were examined,and the analysis results indicated that the dry weight and the crude protein of healthy plants were significant higher than infected plants(P<0.05),and the loss of dry weight were 17.05% to 22.35%,and the loss of crude protein were 18.2% to30.03%.The ADF and the NDF of healthy plants were significant lower than for infected plants(P<0.05),and there is no significant difference in crude fat between healthy and infected plants(P>0.05).In general,the total amino acid and essential amino acid content of anthracnose-infected plants were significantly lower than that of healthy plants(P < 0.05),and the loss of these two measures were 24.85% to30.26% and 21.37% to 30.86%,respectively.In addition,the redundancy analysis ofthe occurrence of anthracnose and dry weight,conventional nutrients and amino acids showed that the dry weight,crude protein,crude ash,lignin,total amino acid and essential amino acid were negatively correlated with the occurrence of anthracnose,while ADF and NDF were positively correlated with the occurrence of anthracnose.4.For the pathogenicity tests confirmed that five Colletotrichum spp.from alfalfa were the pathogens that caused anthracnose on different growth stage of alfalfa including seeds,4-days-old seedlings and 4-weeks-old plants.Moreover,it had great influence on seeds germination and seedlings growth,therefore these pathogens were the possible reason for the alfalfa death in fields from sowing to germination.On the whole,the pathogenicity of six Colletotrichum spp.strains to alfalfa were C.trifolii(JQLYZ21)> C.truncatum(CFLYZ34)> C.destructivum(XDLYZ45)> C.americae-borealis(SEQ2LYZ14)> C.incanum(CFL2YZ41)> C.americae-borealis(MLLYZ28).5.Through disease investigation,pathogens isolation and identification,and pathogenicity,it is determined that alfalfa stem blight caused by Nothophoma sp.in Xichang,Sichuan was the new diseases,unknown anywhere else in the world.Verticillium wilt of alfalfa caused by Verticillium alfalfae in Gansu,and root rot of alfalfa caused by Cylindrocladium sp.in Chifeng were new records from China.While Verticillium wilt of alfalfa is also a quarantined disease in China.