Identification Of Pathogens Causing Main Leaf Spot Diseases On Loquat And Analysis Of Fungal Diversity

Loquat(Eriobotrya japonica Lindl.)is a subtropical evergreen fruit tree native to China.It is favored by consumers for its tasty and nutritious fruits.Widespread loquat leaf spot disease(LLSD)occurrence has limited the development of loquat industry with increasing cultivation areas.Additionally,it is difficult to distinguish the exact LLSD according to the symptoms in fields,because there are many different kinds of LLSD but the symptoms on leaves are similar.Besides,more than one pathogen exit in one kind of LLSD.It has been reported that grey spot disease,ring spot disease,anthracnose,tip leaf spot disease and round spot disease are main LLSDs in China.In this study,we aim to precisely identify pathogens and analyze the fungal diversity of the main LLSDs throughout the year and different regions using combined traditional methods of isolation and identification(harboring colony and spore morphological observation,rDNA-ITS sequence analysis and pathogenicity test)and Miseq high-throughput sequencing.This study will lay the foundation for the control of LLSD.Following were the main results of the research.1.To isolate the causal pathogens,diseased leaves were collected from Chongqing at different growth stages(spring,summer and autumn).The pathogens were isolated using traditional methods of isolation and identification.It indicated that more than one pathogen caused LLSD based on identification and isolation frequency.The pathogens of grey spot disease might be Colletotrichum(40.00%),Alternaria(20.00%),Phoma(10.00%)and Pestalotiopsis(6.67%);Leptosphaeria microscopica(6.67%)might commonly cause ring spot disease,which is first reported to cause ring spot disease on loquat in China;The pathogens causing anthracnose on leaves might be Alternaria(19.23%),Phoma(17.95%),Pestalotiopsis(15.38%)and Colletotrichum(14.10%);The pathogens resulting in tip leaf spot disease might be Alternaria(23.91%),Colletotrichum(21.74%)and Phoma(16.30%);Colletotrichum(40.98%)and Alternaria(18.03%)might commonly cause round spot disease on loquat.Moreover,the isolation frequency of one LLSD was different at different growth stages.2.To analyze the fungal diversity in Chongqing at different growth stages,totally 365 isolates were obtained by traditional methods of isolation and identification.All the isolates belonged to Ascomycota(98.08%)and Basidiomycota(1.92%).There were 23 genera and Colletotrichum(24.66%),Alternaria(20.00%),Phoma(13.70%),Pestalotiopsis(11.23%),Epicoccum nigrum(5.21%)and Fusarium(5.21%)were high-frequency(isolation frequency > 5%).The isolates with highest isolation frequency which were Phoma(23.16%)?Alternaria(26.47%)and Colletotrichum(41.79%)were different in spring,summer and autumn.It showed that the kind of predominant pathogen changed by the timeframe in Chongqing.3.The causal pathogens of 5 main LLSDs were isolated and identified by traditional methods in different regions(Chongqing,Sichuan and Fujian Province)of China.In total,242 isolates belonging to 16 genera were obtained and Colletotrichum(53.11%),Pestalotiopsis(12.81%),Fusarium(10.74%),Phoma(7.44%)and Alternaria(5.79%)were high-frequency isolates.The isolation frequency of pathogens showed difference,but the kinds of pathogens were similar from different regions,especially the predominant genera.Colletotrichum ranked the first in all of the three regions,and the isolation frequencies were 48.75%,60.53% and 51.16%,respectively.It indicated that the fungal diversity of LLSD in different regions was similar.4.Diseased leaves of 5 main LLSDs at different growth stages were collected in Chongqing to study the fungal diversity using Miseq platform.The main results were(1)260 genera were sequenced which mainly belonged to Ascomycota(78.33%)and Basidiomycota(19.39%),24 genera of which such as Pestalotiopsis,Colletotrichum and Alternaria were predominant genera(relative abundance >1%).The relative abundance of 24 predominant genera accounted for 88.38% of all the sequenced genera;(2)The fungal composition of LLSDs at different growth stages was different.147 genera were obtained from spring shoots,13 genera of which were predominant.162 genera were obtained from summer shoots,16 genera of which were predominant.157 genera were obtained from autumn shoots,16 genera of which were predominant.There were 85 shared genera at three growth stages,8 genera of which were predominant.The relative abundance of 8 predominant genera constituded 53.67%,57.31% and 78.9%,respectively;(3)Principal Co-ordinates Analysis(PCoA)showed that the fungal diversity and relative abundance of the same LLSD at different growth stages were different,which was basically consistent with traditional isolation results.5.Diseased leaves of 5 main LLSDs in different regions were collected in autumn to compare the fungal diversity using Miseq platform.The main results were(1)223 genera were sequenced,27 genera of which were predominant genera,accounting for 85.20% of the total sequences;(2)The fungal composition of LLSDs in different regions was different.156 genera were obtained from Chongqing Province and there were 16 predominant genera.135 genera were obtained from Sichuan Province and there were 14 predominant genera.133 genera were obtained from Fujian Province and there were 15 predominant genera.The three regions shared 75 genera and only Pestalotiopsis,Ascochyta,Colletotrichum and g__k__Fungi(not annotated)were predominant.(3)There exited significant difference between the samples in Fujian and the other two regions(P<0.05).The composition and abundance of dominant genera were significantly different,among which g__f__Glomerellaceae,Chaetospermum,Cladosporium and g__o__Pleosporales showed the most significant difference.In summary,the occurrence of LLSD was not caused by a single pathogen,but triggered by mixed pathogens.The Miseq high-throughput sequencing technology was applied to the analysis of fungal composition and diversity for the first time in this research.Both of the Miseq sequencing results and traditional isolation and identification results showed there were differences in the composition and relative content of the same LLSD at different growth stages and in different regions.Moreover,more detailed and comprehensive information of fungal community could be obtained by Miseq sequencing.It is suggested that Miseq high-throughput sequencing technology can be employed to analyze the fungal diversity of plant diseases,and combined with traditional isolation and identification methods,the diseases caused by fungi may be predicted and diagnosed timely,which could guarantee the effective prevention and control of diseases.