Pathogenetic Diversity Of Colletotrichum Spp. On Apple In China

Bitter rot of apple is one of the main diseases of apple production, causing heavy fruit orleaves drop and post-harvest fruit rot in storage. It can cause enormous economic loss to bothgrowers and consumers.The aim of this study was to clear pathogen species constitution, analyze the geneticstructure of pathogens of apple bitter rot in the main apple producing areas of Liaoning,Shandong, Henan and Shaanxi Province in China. The pathogen species of Colletotrichumisolated from hosts around orchards were identified systematically and analyzed their effect asthe primary infection sources of apple bitter rot. The main results were summarized asfollows.1. A total of285samples from bitter rot and Glomerella leaf spot were collected and98isolates were obtained in the main apple producing areas of Liaoning, Shandong, Henan andShaanxi Province in China from2010to2012. These isolates were characterizedmorphologically and five gene regions (ITS, ACT, GAPDH, CHS-1and TUB2) of them weresequenced. Combining morphological, cultural and pathogenic characters, phylogeneticanalysis indicated that isolates of Colletotrichum spp. on apple were grouped into sevenspecies including one new taxon. Among them, isolates from bitter rot were grouped into sixspecies: C. alienum, C. fructicola, C. gloeosporioides, C. nymphaeae, C. siamense and onenew species, C. orientalis. And C. siamense was the predominant pathogen associated withbitter rot, occupying62.2%of the total strains. Isolates from leaf spot were grouped into twospecies, C. aenigma and C. fructicola. And C. fructicola was the predominant pathogenassociated with Glomerella leaf spot.Pathogenicity test showed that all strains of seven species were pathogenic by woundedinoculation. And six strains isolated from bitter rot were pathogenic on apple fruit bynon-wounded inoculation, showing typical symptom of bitter rot. There were differencesamong various species. The strongest isolates was strain F11PGZH02from C. alienum with100%pathogenicity rate. The strains from GLS were pathogenic on Qinguan apple butnon-pathogenic on Fuji apple. It revealed that there was significant difference in resistance ofGLS among different apple variety. 2. To discuss the population genetic diversity of pathogens associated with apple bitterrot at the molecular level, the orthogonal design was used to optimize ISSR (inter-simplesequence repeat)-PCR reaction related factors. ISSR primers were screened based onoptimized ISSR-PCR system, and applied to analyze the genetic diversity of67strains ofColletotrichum spp. causing bitter rot of apple. A total of65polymorphic bands wereamplified with10screened primers, and the polymorphic loci percentage was100%.Clustering analysis based on ISSR markers revealed that tested strains were clustered intoseven groups at0.76genetic similarity coefficient by UPGMA, including C. aenigma, C.alienum, C. fructicola, C. gloeosporioides, C. nymphaeae, C. siamense and C. orientalis,corresponding with multigene phylogenetic analysis. It indicated that ISSR molecularmarker had obvious discrimination of interspecies and intraspecies genetic variations amongColletotrichum spp. and was suitable for genetic diversity analysis of pathogens causing applebitter rot.3. The results of genetic diversity of67strains from different geographical groupsshowed that Nei’s gene diversity index (H) and Shannon’s information index (I) wererespectively greater than0.2and0.3, which indicated that the genetic diversity all over Chinawas considerably abundant. The results of population genetic structure of three geographicalgroups showed that Gst of the population from Bohai gulf area was highest, indicating that thegenetic diversity of this population was the most abundant and genetic differentiation amonggroups was bigger. The number of migrants per generation (Nm) of this group was less than1,indicating that gene flow among groups were restrained. The Gst of the other population werelower but Nm were higher, indicating that genetic differentiation were lower and geneticvariations existed within groups. There was significant gene flow within groups of Old courseof Yellow river and Northwestern loess plateau. The total Nm was0.72, indicating that therewas pathogen exchange among three geographical groups.4. From2011to2012, a total of85samples with anthracnose were collected fromRosaceae, Leguminosae and Solanaceae plants in Shaanxi Province, and41strains ofColletotrichum spp. were obtained. ITS phylogenetic analysis of21strains showed thatisolates of Colletotrichum spp. from different hosts were grouped into eight species.Multigene phylogenetic tree of five strains with similar taxa revealed that the taxa of strainsfrom pear, chili and kiwifruit were similar to Colletotrichum spp. from apple. Pathogens fromC. gloeosporioide and C. fructicala can infect not only apple but also pear, chili and kiwifruit.Clustering analysis from different hosts based on ISSR markers revealed that29strains wereclustered into eight groups at0.68genetic similarity coefficient, corresponding with multigene phylogenetic analysis. Pathogenicity test showed that10strains of six taxa werepathogenic by wounded inoculation. And strains of C. acutatum, C. salsolae and C.simmondsii were non-pathogenic on apple fruit by non-wounded inoculation, while strains ofC. fructicola, C. gloeosporioides and C. fiorinae were pathogenic on apple, showing typicalsymptom of bitter rot. It comfirmed that other hosts of pear, chili and kiwifruit around orchardwere potential primary infection source of apple bitter rot.This study is the first detailed investigation about pathogens of apple bitter rot in China.The pathogenetic diversity of Colletotrichum spp. was defined based on an international newpolyphasic approach by employing both multigene phylogenetic analyses and morphologicalcharacters. We found that ISSR molecular marker, as a supplementary means of sequenceanalysis, can differentiate both interspecies and intraspecies genetic variations amongpathogens causing apple bitter rot and is suitable for analysis of genetic diversity, geneticstructure and pathogen dissemination in Colletotrichum spp. Combining multigenephylogenetic analyses, ISSR molecular marker and pathogenicity test, pathogens ofanthracnose from different hosts around orchard were determined as potential primaryinfection source of apple bitter rot. The above outcome found the basis for effective controland plant pathology of apple bitter rot.