Diversity Of Fungi Causing Citrus Branch Diseases In China

As the most important country for citrus production and consumption in the world,China is one of the original centers of citrus with a long history of citrus cultivation and variety selection.During its growth,citrus is unavoidable to be infected by various pathogens.The branch fungal diseases are one of the important diseases that have been affecting the citrus industry in China for a long time.It could affect tree vigor at different levels or even cause the death of the whole tree.However,species and dominant species of pathogenic fungi causing citrus branch diseases remain unclear.In this study,we collected 571 samples with symptoms of shoot dieback,canker,gummosis and decay of branches and trunks mainly from Zhejiang,as well as from other 13 citrus-producing provinces in China.A total of 978 single-spore isolates were isolated.They were initially identified as families Botryosphaeriaceae and Diatrypaceae,and genera Diaporthe,Colletotrichum,Fusarium,Alternaria and Botrytis.In this study,the exact species in Botryosphaeriaceae,Diaporthe and Diatrypaceae were identified based on multigene phylogenetic analysis and morphology,and the distribution and pathogenicity of these species were evaluated.The main findings obtained are reported as follows:1.Botryosphaeriaceae are important pathogens causing citrus branch diseases,with abundant speciesA total of 111 single-spore isolates of Botryosphaeriaceae were isolated from diseased branch samples with symptoms of dieback,gummosis,canker and decay collected from Zhejiang,Chongqing,Fujian,Guangdong,Guangxi,Hunan,Jiangxi,Shaanxi and Shanghai.Phylogenetic analyses based on the combined sequences of the internal transcribed spacer region(ITS),the translation elongation factor 1-alpha gene(tef1),the beta-tubulin gene(tub2)and the DNA-directed RNA polymerase II second largest subunit(rpb2),111 isolates were identified as 18 species in seven genera.Among them,12 species have been reported,including Botryosphaeria dothidea,B.fabicerciana,Diplodia seriata,Dothiorella alpina,Do.plurivora,Lasiodiplodia citricola,L.iraniensis,L.microconidia,L.pseudotheobromae,L.theobromae,Neodeightonia subglobosa,Neofusicoccum parvum;six species were newly described,named Do.citrimurcotticola sp.nov.,L.guilinensis sp.nov.,L.huangyanensis sp.nov.,L.linhaiensis sp.nov.,L.ponkanicola sp.nov.and Sphaeropsis linhaiensis sp.nov.Among them,Botryosphaeria dothidea(28.8%)was the most abundant species,followed by L.pseudotheobromae(23.4%),which was the most widely distributed species on citrus.Pathogenicity tests indicated that all the 18 species of Botryosphaeriaceae were pathogenic to the in vitro shoots of Citrus reticulata,and most of the species were also pathogenic to the in vivo shoots of Cocktail grapefruit,but the pathogenicity was different among species.In general,the species of Lasiodiplodia were more virulent than other species in Botryosphaeriaceae.In addition,the growth test showed that Lasiodiplodia species grew fastest on the PDA medium,and could tolerate the high temperature since they could grow even at 40℃.Therefore,we concluded that Lasiodiplodia spp.were the dominant species in Botryosphaeriaceae to cause citrus branch diseases.2.Diaporthe fungal species are abundant on citrus branches,and D.citri is the most important pathogenicA total of 195 single-spore isolates of Diaporthe were isolated from diseased branch samples collected from Zhejiang,Chongqing,Hubei,Hunan,Jiangxi,Sichuan,Shanghai and Yunnan.Multigene phylogenetic analysis based on ITS,calmodulin(cal),histone H3(his3),tef1 and tub2,indicated that these isolates belonged to 20 species,including 14 known species,named D.arecae,D.caryae,D.celeris,D.citri,D.citriasiana,D.compacta,D.eres,D.fulvicolor,D.hongkongensis,D.melitensis,D.novem,D.sojae,D.unshiuensis,D.velutina;six species were firstly described in this study,named D.blunticola sp.nov.,D.curvatura sp.nov.,D.gammata sp.nov.,D.infecunditas sp.nov.,D.jishinensis sp.nov.and D.ruiliensis sp.nov.In terms of the isolated number,D.citri was the most abundant species,accounting for 68.2% of the total number of obtained isolates in this study,followed by D.eres(8.7%)and D.arecae(4.1%).Pathogenicity test showed that D.citri produced the longest lesions in the shoots of C.paradisi,and the highest incidence contrasting to other species.Taken together,we concluded that D.citri accounted for the dominant species in the genus Diaporthe that caused citrus branch diseases.3.Diatrypaceae fungi are not significant pathogens of citrus branch diseasesFifty-four single-spore isolates of Diatrypaceae were obtained from the diseased citrus branch samples collected from Zhejiang,Guangxi,Hunan,Jiangxi and Shanghai.Phylogenetic analyses based on ITS and tub2 gene showed that the 54 isolates belonged to five species in three genera,including Diatrypella japonica,Dia.pseudooregonensis,Dia.vulgaris,Paraeutypella citricola and Peroneutypa scoparia.Among them,Pe.scoparia has the widest geographical distribution and the highest obtained ratio,followed by P.citricola.The pathogenicity of these five species on detached C.paradisi branches was weak,so it was speculated that these fungi might be mild pathogens or saprophytes and had limited influence on citrus industry in China.In summary,this investigation preliminarily clarified the main pathogenic fungal groups causing citrus branch diseases in China.We found that Lasiodiplodia spp.were the dominant species in Botryosphaeriaceae,D.citri was the dominant species in Diaporthe,while species in Diatrypaceae were not important for citrus branch diseases.In addition,12 new fungal species were described in this study,11 species were firstly reported for relating to citrus worldwide,and nine species were firstly reported to citrus in China.These works partially clarified and enriched the knowledge of the diversity of pathogenic fungi related to citrus branch diseases,and laid a theoretical foundation for studies on the epidemiology and control technology of citrus branch diseases.