Identification And Molecular Detection Of Pathogens Causing Anthracnose On Loquat In Chongqing

Loquat(Eriobotrya japonica Lindl.)is a subtropical evergreen fruit tree originating from China.It is favored by consumers for its tasty,nutritious fruits and the positive effects on the lung,stomach spleen.However,the occurrence of anthracnose endangered the loquat production in the past few years.Anthracnose of loquat mainly infected leaves and fruits.Brown spots symptoms were observed on the leaves and fruits after infection.Syptoms on diseased fruits would rapidly expand and appear as slightly sunken,water-soaked and soft lesions.Additionally,anthracnose could also cause fruit decay after harvest.The above symtoms resulted in economic loss to growers and consumers.In addition,it was reported that the nature of latent infection were observed on loquat anthracnose and it was too late to control the disease after visual sypmtoms appeared in fields.Therefore,it is necessary to develop a rapid,responsible method to detect anthracnose of loquat.The objective of this study was to identify the pathogens causing loquat anthracnose.The diseased leaves and fruits collected in Beibei district and Hechuan district in Chongqing were placed on potato dextrose agar plate.The purified isolates were chosen for rDNA-ITS sequence and pathogenicity test.The other objective of this study was to develop a rapid,responsible detection system based on nested-PCR to provide theoretical foundation for early dection and effective control of loquat anthracnose of loquat.The main results were as follows:1.During Feb.2016 to Oct.2016,the incidence of loquat a nthracnose was investigated in the Southwest University Loquat Germplasm.It showed that the initial period of loquat anthracnose in Chongqing was early April and the disease index was3.3%.The disease outbroke during late April to early June and the disease index increased from 10.6%to 31%.During late June to late July,the loquat a nthracnose entered peak period and the disease index stab ilized around 41%.Moreover,the temperature was at 25-30℃and the relative humidity was 60%from anthracnose outbreak period to peak period.It indicated that high temperature and high humidity contributed to the spread of loquat anthracnose,especially the young leaves of summer shoots were sensitive to loquat anthracnose.2.During 2015 to 2017,218 samples with disease were collected from Southwest University Loquat Gerplasm,Southwest University 105 plantation base and plantation base of Weituo in Hechuan district.In total 406 strains were isolated from all the samples and of which 131 isolates belonged to Colletotrichum according to the colony morphology,phylogenetic analysis based on rDNA-ITS sequence.To identify the 131Colletotrichum isolates at species level,the ITS,CAL,GAPDH,CHS-1 and TUB2region of them were amplified and sequenced.With the morphological observation results,131 Colletotrichum isolates from loquat anthracnose were grouped into three species which were C.gloeosporioides(42 isolates),C.acutatum(61 isolates)and C.karstii(28 isolates).3.The pathogenicity tests were conducted on the cultivars named?Jinhua No.1?and?Huabai No.1?using spore suspension.Pathogenicity test results showed that 131Colletotrichum isolates appeared typical symptoms both on the leaves and fruits after wound inoculation and the re-isolated strains caused similar syptom as that in the fields.However,on leaves,the pathogenicity of C.gloeosporioides was highest and C.karstii was weakest.On fruits,the pathogenicity of C.acutatum was highest while C.karstii was weakest.4.Two specific nested inner primer pairs,Cg1F/R screened from 20 pairs and Ca1F/R screened from 17 pairs,were designed based on the different ribosome DNA sequence between C.gloeosporioides and C.acutatum.The nested PCR which set the universal primer pair ITS1/ITS4 as outer primer and Cg1F/R as inner primer could amplify 392 bp fragment only from C.gloeosporioides.No amplification product was observed from other tested fungi.The nested PCR which set the universal primer pair ITS1/ITS4 as outer primer and Ca1F/R as inner primer could amplify 490 bp fragment only from C.acutatum.No amplification product was observed from other tested fungi.5.In the nested PCR using Cg1F/R primer pair,the sensitivity of detection was 1pg/μL which was 10~3 times as that in the conventional PCR(1 ng/μL).Meanwhile,the sensitivity of detection in the conventional PCR using Ca1F/R was 10 pg/μL and which in the nested PCR was 10 ag/μL.The sensitivity of Ca1F/R in the nested PCR was 10~6 times as that in the conventional PCR.6.60 loquat samples with typical anthracnose symptoms and 27 samples without symptoms were collected.Of all the samples with symptoms,Colletotrichum were identified from 39 samples in total using traditional isolation and identification methods.C.gloeosporioides and C.acutatum were simultaneously isolated from 6samples,C.gloeosporioides and C.karstii were simultaneously isolated from 2samples.C.gloeosporioides,C.acutatum and C.karstii were individually isolated from 17,12 amd 2 samples,respectively.Of all the samples with symptoms,Colletotrichum were detected from 44 samples in total using nested-PCR developed in this study.C.gloeosporioides and C.acutatum were simultaneously detected from 9samples.C.gloeosporioides was individually detected from 19 samples which was consistent with traditional identification method.Moreover,C.acutatum was individually detected from 16 samples.It showed that the nested-PCR molecular detection system developed in this study was more effective than traditional method.Of all the samples without symptoms,C.gloeosporioides were identified from 2samples in total using traditional method.However,Colletotrichum were detected from9 samples using nested-PCR method.C.gloeosporioides and C.acutatum were detected from 6 and 3 samples,respectively.It showed that the nested-PCR method could detect C.gloeosporioides and C.acutatum traces on the leaves of loquat.In conclusion,it was confirmed that the causal agent of loquat a nthracnose were C.gloeosporioides,C.acutatum and C.karstii using traditional isolation and identification methods.The main causal agents were C.gloeosporioides and C.acutatu.Furthermore,the procedure of nested-PCR which set ITS1/ITS4 as outer primer,Cg1F/R and Ca1F/R as inner primer was a rapid,effective,specific and sensitive method to detect C.gloeosporioides and C.acutatum,respectively.This study provided technical support for rapid detection of pathogens and early diagnosis of loquat anthracnose.