Studies On Genetic Diversity,Infection Process,Fungicide Resistance And Early Detection Of Venturia Carpophila In China

China is a big country for the peach industry,with the highest cultivation area and yield in the world.However,peach scab has widely occurred in various peach-producing areas in China,resulting in the decline of peach quality and yield,posing a serious threat to the peach industry.This study collected peach scab samples from different varieties in 14 provinces in China.Pathogens were isolated,identified based on morphological observation and molecular identification.At the same time,the genetic diversity and population structure of Venturia carpophila,the causal agent of peach,mume and apricot scabs were analyzed.Besides,the infection process of V.carpophila on peach leaves was cytologically investigated.Meanwhile,a detection system was established to efficiently detect the fungicide sensitivity in V.carpophila based on the microplate method,and the sensitivities of V.carpophila to 5 fungicides were investigated.Finally,based on the ITS sequence of V.carpophila,a LAMP method was developed for rapid detection of peach scab.The obtained information may provide theoretical basis and technical support for the diagnosis and management of peach scab,lay a foundation for systematic research on mechanisms of pathogenicity in V.carpophila,and provide new ideas for breeding resistant varieties for peach scab.The main research results obtained are as follows:1)Identification and genetic diversity analysis for pathogens causing peach scab in China.From 2017 to 2019,peach scab samples were collected from 14 provinces across the country;Apple scab samples were collected from Xinjiang and Anhui;Pear scab samples were collected from Hebei;Crabapple scab samples were collected from Heilongjiang;Apricot scab samples and mume scab samples were collected from the orchard of Huazhong Agricultural University.More than 950 single-spore isolates were obtained by the single spore isolation method.Among them,126 isolates were selected as representatives for morphological and multi-locus phylogenetic analysis(ITS+LSU).Results showed that the representative isolates were identified as 4different species of Venturia genus,namely Venturia carpophila,Venturia inaequalis,Venturia nashicola and Venturia asperata.Among them,peach scab(V.carpophila),mume scab(V.carpophila),apricot scab(V.carpophila)and crabapple scab(V.asperata)were firstly reported in China.The field symptoms of peach scabs were investigated,and it was found that symptoms of peach scab in different varieties,different growth stages and different regions were different.At the same time,the symptom of peach scab was very similar with peach bacterial spot and peach black spot,especially in the early infection stage.The Koch's postulate was fulfilled to verify the 4 new record species reported for the first time in China.V.carpophila isolates isolated from peach,apricot and mume showed significant differences in growth rate,colony morphology,and sporulation.Altogether,186 isolates of V.carpophila isolated from different hosts,different geographic regions were selected for genetic diversity analysis.Results showed that the V.carpophila populations showed host specificity at species level but no specificity was observed at variety or tissue level,also populations were not correlated with geographic distributions.2)Study on the infection process of V.carpophila on peach leaves.Using light microscopy,laser-scanning confocal microscopy,scanning electron microscopy and transmission electron microscopy,we systematically investigated the infection process of V.carpophila on peach leaves for the first time.Results showed that V.carpophila mainly infected the abaxial side of peach leaves.The entire infection process can be divided into three main stages,namely the penetration stage characterized by the formation of appressoria and penetration pegs;the expansion stage characterized by the formation of sub-cuticular hyphae and stromata;and symptom appearance stage characterized by the formation of conidiophores and conidia.In brief,V.carpophila germinated at the tip of conidia and produced short germ tubes on the leaf surfaces at 2 days post-inoculation(dpi).At 3 dpi,swollen tips of germ tubes differentiated into appressoria.At 5 dpi,penetration pegs broke through the cuticle layer,the thin pegs differentiated into thick sub-cuticular hyphae in the pectin layer of the cell walls.At 10 dpi,the sub-cuticular hyphae grew horizontally and extensively colonized in the pectin layer.The primary hyphae ramified into secondary hyphae and proliferated along with the incubation.At 15 dpi,the sub-cuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells.At 30 dpi,conidiophores developed from the sub-cuticular stromata.Finally abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi.3)Detection of sensitivity of V.carpophila to five fungicides.Based on the optimization of the microplate method,the fungicide sensitivity detection system was established in V.carpophila.In brief,the MEB was selected as the best medium,the initial spore suspension concentration was used at 10⁶/m L and incubation time was 96h.Next,the sensitivity of 135 single-spore V.carpophila isolates to commonly used fungicides carbendazim,iprodione,propiconazole,azoxystrobin and boscalid were determined using the established microplate method.Results showed that the mean EC₅₀ values of tested isolates to iprodione,propiconazole,azoxystrobin and boscalid were 16.287?g/m L,0.165?g/m L,0.570?g/m L and 0.136?g/m L,respectively.The EC₅₀ values of V.carpophila isolates to above four fungicides displayed unimodal frequency distributions,indicating no resistance occurred to these fungicides.On the other hand,bimodal frequency distribution was observed for carbendazim,indicating that the V.carpophila developed resistance to carbendazim.Actually,the resistance was widely detected from all over the 14 provinces.Molecular analysis showed that the point mutation E198K of the TUB2 gene determined the high resistance,while the E198G conferred the moderate resistance.Both moderate and high resistances were stable,and the resistant isolates did not show significant fitness penalties.On the contrary,some resistant isolates showed even better competitiveness under certain stresses,indicating the high risk of spreading of resistant populations in practice.4)Development of a loop-mediated isothermal amplification(LAMP)method for the rapid detection of V.carpophila on peach.A set of LAMP primers was designed based on the internal transcribed spacer(r DNA-ITS)sequence to detect V.carpophila.Compared to the conventional PCR method,the LAMP method not only exhibited higher sensitivity and specificity in the detection of V.carpophila but also required simpler equipment and less operational time.The minimum detectable concentration of the V.carpophila genomic DNA with LAMP was 56.6 fg/?l,which was 100 times lower than the conventional PCR method.When eight fungal species including V.carpophila(23 isolates from 14 provinces)and one bacterial species were applied for LAMP detection,the developed method could specifically detect V.carpophila.Moreover,crude DNAs of peach fruit samples were applied for the LAMP detection,the diseased samples could also be correctly detected.