The Etiology And Pathogenesis Of Athelia Bombacina Causing Postharvest Fruit Rot On Pear

Pear(Pyrus spp.)is one of the main fruit trees in the world.Hebei is the main pear producing area as well as storage and distribution center in China.Huangguan Pear is one of the main cultivated and storage varieties.Besides domestic sales,it was also exported to Europe,America and Southeast Asia.Since 2016,a new disease had been found sporadically in Huangguan Pear during storage in Suning,Shenzhou and Xinji of Hebei Province.In 2018,the incidence of this unknown disease was as high as 10%,some even reached 20%,which caused serious economic losses to pear storage enterprises.As a new disease,its pathogen,biological characteristics and pathogenic mechanism had not yet been reported,which made it impossible to propose new disease prevention and control technical regulations for production.Therefore,Athelia bombacina fruit rot was took as the research object.Investigation of disease occurrence,identification of pathogenic bacteria,pathogenic biology,genome sequencing and comparative genomics analysis of pathogenic bacteria,interaction mechanism between pathogens and hosts,resistance evaluation of germplasm resources to pathogenic bacteria and determination of toxicity of indoor agents were carried out.The main results are as follows: 1.The first study confirmed that the postharvest A.bombacina fruit rot of pear was a new disease worldwide,and its pathogen was A.bombacina.The main symptoms of the A.bombacina fruit rot were light brown round spots on the fruit surface at the early stage of the disease,which could be distributed in many spots.Later,the lesion gradually enlarged,showing round or oval regular lesions with depressions.After cutting the peel of the diseased part,it could be seen that the flesh of the diseased part forms clear cavity.The diseased fruit flesh was not easy to separate from the surrounding healthy flesh.In the later stage,many lesions merge into one.The whole fruit surface was covered with white hyphae,and strong mushroom fragrance can be smelled.Sometimes it could be seen that a large number of seed layers were distributed in the fruit of the diseased part,which eventually leaded to collapse and shrinkage of the fruits.Under high humidity conditions,the lesion could rapidly been expanded and caused the whole fruits to rot.Through isolation,purification,morphological observation,pathogenicity measurement and phylogenetic analysis of ITS sequences of pathogenic figus,it was concluded that the pathogenic causing diseases of Huangguan pear during storage period were A.bombacina,belonging to Atheliaceae,Atheliales,Agaricomycetidae,Agaricomycetes,Basidiomycota.This was the first report that this pathogen could cause fruit harvesting diseases in the world.A.bombacina could infect not only pears,but also apples,strawberries,cherries,nectarines,peaches,apricots and jujubes,however,it didn’t infect blueberries,grapes,oranges and kiwifruit etc.,The infection ability of hawthorns,oranges and pomelos was weak,and the infection of oranges and pomelos was limited to the peel,not penetrate into the pulp tissue.A.bombacina had different infection ability to different pear and apple varieties.2.The biological characteristics of A.bombacina were systematically studied.The optimum medium for mycelial growth of A.bombacina was PDA,the optimum temperature growth was 25 C,the optimum p H was 7,the optimum carbon source was glucose,the optimum nitrogen source was glutamic acid,and dark treatment was beneficial to mycelial growth.The lethal temperature of mycelium was 54 C for 10 min.The optimal germination temperature of the spores was 20 ℃,the optimum p H was 6,and the best carbon source was glucose.The effect of light on spore germination was not obvious.The lethal temperature of the spores was 52 ℃ for 10 min.By studying the spore production under different conditions,the best culture conditions for the spore production of the strain were as follows: medium formula: 15 g of oat,15 g of agar,10 g of glucose,2 g of glycine,30.5 g of Ca CO3,2 g of Na CL,1000 m L of water adjusted the p H to 6.A.bombacina was inoculated into the above-mentioned culture medium,keeping the environmental humidity above 95%.After 3-5 days of dark culture,the culture temperature was adjusted to 5 ℃.After 2-3 days of culture,the culture temperature was adjusted to 20-25 ℃ again.After 3-5 days of light culture,a large number of basidiospores with identical morphology and maturity could be produced.Effective induction of basidiospores laid the foundation for later pathogenesis study.3.The whole genome sequencing of A.bombacina was performed for the first time,and a high-quality fine genome map of fungi was obtained,and comparative genome analysis was carried out.By observing the protoplast formation of four different enzymatic hydrolysates after different enzymatic hydrolysis time,the preparation conditions of A.bombacina protoplast were optimized.The high-efficiency enzymatic hydrolysate was 1.5% lysozyme and the enzymatic hydrolysis time was 2.0 h.Mononuclear strains were obtained.Compared with the original strains,the growth rate of mononucleated strains on PDA was significantly reduced,but the results of pathogenicity test showed that the strain had the same activity.The heterozygosity rate of A.bombacina ABD-3 was about 0.00% and the GC content of genome was 47.96%.It belonged to simple genome and met the requirements of sample evaluation for subsequent genome fine mapping.Pre-evaluation showed that the genome size of A.bombacina ABD-3 was about 29 Mb.Fifteen Scaffolds and 15 Contigs were obtained.The total length was 27 661 196 bp,the GC content was 48.91%,the length of N50 was 2 943 476 bp,the length of N90 was 1 878 669 bp,and the longest fragment was 3 843 316 bp.The whole genome sequencing of A.bombacina was carried out,and a high-quality fine map of the fungal genome was obtained.The gene sequence of A.bombacina ABD-3 strain were compared with the KOG database,and 4879 genes were successfully annotated.The proteins of A.bombacina ABD-3 strain were mainly related to general function prediction,post-translational modification,protein transfer,chaperone and signal transduction mechanism.By compared with the database,A.bombacina ABD-3 strain was successfully annotated with 2 807 proteins.Among them,the most of the molecular functional proteins are catalytic activity,the most of the cell components are cell membranes,and the most of the biological processes are metabolic processes and cellular processes.The number of proteins successfully annotated by KEGG database was 316,and they participated in 108 metabolic pathways.Among the 8974 proteins successfully annotated by NR,Moniliophthora roreri had the highest correlation.A total of 501 proteins were successfully annotated by the CAZyme database.The GHs protein family has the highest proportion of genes,which was 45.91%.Among them,four gene families,GH16,GH18,GH5 and GH43,had a higher proportion of GHs.4.The interaction mechanism between A.bombacina and fruits was preliminarily clarified.During the determination of soluble sugar and oxalic acid in the fermentation broth of pathogenic bacteria,it was found that with the extension of culture time,soluble sugar content first increased and then decreased,while oxalic acid content first increased and then decreased slightly.The laccase activity of pathogenic bacteria in liquid medium of enzyme production first increased and then decreased,the neutral xylanase continuously increased in the process of culture,and the polygalacturonase first increased and then basically stabilized.Compared with the control group,the content of malondialdehyde(MDA)in fruits after inoculation with A.bombacina continuously increased.Catalase(CAT)activity at different stages of the disease was significantly higher at the inoculated site than at the uninoculated site.At the early and middle stage,the activity of POD at the inoculated site was lower than that at the uninoculated site,and at the later stage,the activity of POD at the inoculated site was higher than that at the uninoculated site.Through the analysis of WGCNA co-expression network,it was found that the change trend of samples could be well reflected in the four modules of MEbrown,MEdarkturquoise,MEblack and MEdarkred.Finally,according to the correlation analysis between modules,MEbrown and the negatively correlated MEblack module were selected as the differential gene screening module.According to the data of the transcriptome differentially expressed genes,20 differentially expressed genes were screened in the MEbrown and MEblack modules,among which 5 genes were down-regulated and 15 genes were up-regulated,which were mainly related to the biosynthesis of secondary metabolites,amino acid metabolism,phenylpropanoid biosynthesis,and the pathogen-host interaction related pathways.5.Established the resistance evaluation technology system of different pear and apple germplasms against A.bombacina,and screened out the effective agents to A.bombacina.The pear and apple germplasms could be classified into five categories: high resistance(HR),resistance(R),moderate resistance(MR),susceptibility(S)and high susceptibility(HS)by cluster analysis and AD methods.Pear and apple germplasm were ranked as the best clustering points with euclidean distances of 14.0 and 10.0 respectively.Compared with AD method,clustering analysis could classify pear and apple germplasms more scientifically.Generally speaking,pear resistant disease was less germplasm,pear germplasms with high resistance accounted for only 2.5%,while apple germplasms with high resistance accounted for more than 20%.The resistance of pear and apple germplasms had no significant relationship with maturity and species.A.bombacina hyphae were sensitive to triazole fungicides such as tebuconazole,myclobutanil,difenoconazole,flusilazole and triazolone,but not to benzimidazole fungicides such as carbendazim and thiophanate,broadspectrum fungicides such as chlorothalonil and clotrimazone.Finally,according to the virulence regression equation,the effective agents were tebuconazole,myclobutanil,fludioxonil and tirafuramide,which had the best inhibitory effect on A.bombacina hyphae.EC50 were 0.027,0.048,0.054 and 0.095 mg L-1,respectively.