Identification And Diversity Study Of The Pathogenetic Fungi On The Diseased Walnut Branches From Yunnan Province

Walnut is an important woody oil-bearing plant,which is abundant of economic and ecological significance in China.As the biggest walnut production province,the planting areas and yield as well as production value of walnut in Yunnan rank the top in China.However,blind expansion of the walnut planting areas and factors such as extensive management and abnormal climate make frequent walnut diseases and pests which greatly endanger the prosperity and development of walnut industry in Yunnan Province.Based on comprehensive field investigations and diseased sample collections in the main production areas including 18 counties from ten Prefectures in Yunnan Province,the study was carried out through traditional identification method covering indoor freehand slicing,tissue isolation together with micro-examination.In the meantime,rDNA-ITS sequencing was supplemented to identify parts of the separated strains difficult to identify through morphology.On the basis of identified pathogens on the diseased walnut branches,Shannon-Wiener Index and Simpson’s Index were used for diversity analysis on the pathogens so as to more comprehensively recognize the current epidemic situations of the walnut branch diseases and pathogen species in the main walnut production regions in Yunnan province.The results would supply basis for control and prevention of walnut branch diseases in Yunnan.Meanwhile,the study on the pathogenic fungi of walnut branch diseases would be enriched in order to set foundations for deepening walnut disease research.The results showed as follows:1.Based on the previous investigation,233 diseased walnut branch samples were collected from 18 Counties of ten Prefectures,the main walnut productive areas in Yunnan Province,among which we collected the most samples in Chuxiong and Qujing Prefectures,66 and 64,respectively,the least samples in Wenshan Prefecture,only one sample.Through freehand slicing of screened 225 diseased branches,232 identified specimens were obtained.2.Morphological identification of the 232 specimens with 27 genera containing31 species Fungi belonging to Ascocomycota(nine genera containing ten species),Basidiomycota(five genera containing five species)and Mitosporic Fungi(13 genera containing 16 species)and two species belonging to two genera of parasitic plants were identified.Among them,16 species subordinated to 11 genera as new pathogenic fungi were first discovered endangering walnut branches.They are Eutypella stellulata,Nectria cinnabarina,Tubercularia vulgris,Ganoderma lobatum,Trametes hirsuta,Didymosporium liquidambaris,Helminthosporium multiseptatum,H.pseudomicrosorum,H.velutinum,Fusicoccum sp.,Libertella sp.,Elsinoe sp.,Diatrypella sp.1,Diatrypella sp.2,Aplosporella sp.and a new species Pestalosphaeria juglandis.3.The identification by slicing diseased branches showed that four genera(Phomopsis,Dothiorella,Fusicoccum and Helminthosporium)were with widest distribution which distributed in more than eight counties,and with highest frequency of occurrences,12.93%,12.07%,13.36%,and 12.5%,respectively.Botryosphaeria and Libertella with frequency of 7.76% followed,among which the former genus distributed wider in six counties while the latter was narrowly distributed in two counties.4.After tissue isolation,145 strains were obtained by screened 200 diseased walnut branches except samples caused by Basidiomycota.Through morphological identification,the fungi were under 14 genera containing 14 species and six unknown species.The identification results by slicing and isolation were coincided with Phomopsis,Phoma,Dothiorella,Fusicoccum,Libertella and Eutypella.5.The isolation results illustrated that Phomopsis juglandina with the highest frequency of isolation was 39.31%,widely distributing in nine counties.The results through slicing and isolation were showing that Phomopsis was the predominant genus causing walnut branch disease in Yunnan Province.6.The diversity analysis by Shannon-Wiener Index and Simpson’s diversity Index manifested that the pathogenic diversity of different areas obviously differed.The pathogen species and quantities of Qujing Prefecture were at the top with the highest richness,followed by Zhaotong,Baoshan,Lincang,Dali,but the data of Kunming,Lijiang and Nujiang were less than the above-mentioned Prefectures.7.Five series of branch diseases in the study were twig blight,canker,bark rot,wood rot and parasitic disease,among which twig blight was the most wildly distributed.The diversity analysis of Shannon-Wiener and Simpson index revealed that twig blight was the most serious walnut branch disease in Yunnan Province.