| Pleurotus ostreatus is one of the widely cultivated edible mushrooms in the world.Bacterial brown blotch disease caused by Pseudomonas tolaasii is common in P.ostreatus production,which severely affects yield and quality and causes significant economic losses.Based on evaluation the resistance of different P.ostreatus varieties to bacterial brown blotch disease,this study selected two varieties with different resistance levels to investigate the mechanisms of P.ostreatus response to P.tolaasii infection and the reasons of resistance differences between different varieties.The main results are as follows:1.Thirty-two P.ostreatus varieties were inoculated with P.tolaasii,and disease indices were recorded 24 hours after inoculation.It was found that Huaping 36,Ping8129,Tekang650,and Pingzaoqiu 615 showed the best resistance to the disease,while Heimei 89,Xia Hui No.1,and Huaping 801 performed the worst in terms of resistance.2.Two varieties with the greatest resistance difference(Huaping 801 and Huaping 36)were selected,and their fruiting bodies were inoculated with P.tolaasii.Symptoms were observed at 12 hours,24 hours,and 72 hours after inoculation.It was found that the susceptible variety,Huaping 801,showed slight yellowing on the cap after 12 hours of inoculation,brown spots over the entire cap after 24 hours,severe wilting and watersoaking appearance after 72 hours,while the cap turned completely yellow.In contrast,there were no symptoms observed in Huaping 36 at 12 and 24 hours after inoculation,and only a few yellow spots appeared on the cap after 72 hours.The ultrastructure of healthy and diseased fruiting body samples at each time point was observed using scanning electron microscopy and transmission electron microscopy.The two varieties exhibited significant differences in hyphal and cell morphology.The susceptible variety had a large number of bacteria distributed on the surface,with most hypha being engulfed by bacteria,resulting in breakage and pores.In contrast,the resistant variety had very few bacteria distributed on it.Cells of the susceptible variety underwent severe deformation,producing a large number of vesicles,with the contents of the cells leaking out,and mitochondrial deformations occurring;however,the resistant variety did not exhibit any significant cell changes or only mild changes.3.Enzyme activity assays were conducted on diseased and healthy samples at different time points,revealing changes in the activities of tyrosinase,SOD(superoxide dismutase),and intracellular malondialdehyde after inoculation with P.tolaasii.The tyrosinase activity and intracellular malondialdehyde content of resistant variety were significantly lower than those of sensitive one,while the SOD activity was significantly higher,indicating that the tyrosinase of sensitive variety was activated,the degree of cellular damage was greater,and the ability to clear reactive oxygen species was weaker.4.Transcriptome sequencing was performed on both varieties at each time point,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed.The results showed that for the susceptible Huaping 801 variety,differentially expressed genes at 12 hours after inoculation were mainly enriched in biological processes such as oxidative phosphorylation,mitochondrial transmembrane transport,reactive oxygen species,and austindiol metabolism;those at 24 hours were mainly enriched in processes related to glycine,serine,and threonine metabolism,carboxylic acid metabolism,and oxidoreductase activity;those at 72 hours were mainly enriched in processes related to monooxygenase activity,glycine,serine,and threonine metabolism.In contrast,for the resistant Huaping 36 variety,differentially expressed genes at 12 hours after inoculation were mainly enriched in biological processes such as aromatic alcohol dehydrogenase activity,penicillin and cephalosporin biosynthesis;those at 24 hours were not significantly enriched in any biological processes;and those at 72 hours were mainly enriched in pathways related to toluene degradation,formate metabolism,etc.Time series analysis showed that different expression patterns of genes in Huaping 801 clustered into eight modules,while those in Huaping 36 clustered into six modules.5.Combining co-expression network analysis results and gene function annotation,a model of P.ostreatus response to P.tolaasii infection was constructed as follows: when P.ostreatus cells sense the signal of pathogen invasion,the signal is transduced to the cytoplasm through the signaling pathway.Detoxifying enzyme genes,such as glutathione S-transferase,phenylalanine,and histidine deaminase,and MFS transporter protein genes are activated.Cytochrome P450 also participates in the conversion or excretion of toxic substances outside the cell.Peroxidase genes are activated to resist oxidative damage.At the same time,DNA repair occurs in the nucleus to withstand pathogen damage,cytochrome c participates in apoptosis to maintain internal environmental stability.The membrane structure is damaged,leading to the release of phenolic substances,an increase in fatty acid content,increased membrane fluidity,activation of tyrosinase genes,catalyzing the production of melanin,causing blotch symptoms.6.Based on comparative transcriptome information,the reasons of resistance differences among P.ostreatus varieties were analyzed.Genes encoding fungal toxicity and lysozyme were significantly upregulated in resistant variety,preventing bacteria from colonizing and proliferating,while they were not upregulated in susceptible variety.The degree of cell damage was smaller in resistant variety,while the susceptibility variety had a higher degree of membrane damage with severe leakage of cell contents,mitochondrial damage,and abnormal cell growth.Tyrosinase genes were not significantly upregulated in resistant variety,and tyrosinase activity was low,which could not oxidize phenolic substances and cause blotch symptoms.SOD activity in resistant variety remained at a high level after bacterial inoculation,effectively clearing oxidative damage caused by reactive oxygen species,while the SOD activity of susceptible variety was very low in the later stages.7.Thirty candidate genes were selected from transcriptome sequencing results,which showed significant differential expression between resistant and susceptible varieties and were associated with disease resistance in other species,including genes encoding ethanol dehydrogenase,lysozyme,and serine/threonine kinase.The expression levels of these candidate genes in diseased P.ostreatus fruiting bodies of various varieties were determined,and their correlations with disease severity index were analyzed.It was found that the relative expression levels of alcohol dehydrogenase gene(gene＿6990),sphingomyelin phosphodiesterase gene(gene＿2176),and serine/threonine protein kinase gene(gene＿5188)were significantly positively correlated with disease severity index,while those of bacterial alpha-L-rhamnosidase 6 hairpin glycosidase domain(gene＿4782),lysozyme gene(gene＿6008),and alcohol dehydrogenase gene(gene＿5811)were significantly negatively correlated.Coding sequences of candidate genes with correlation were cloned in Huaping 36 and Huaping 801 varieties,and it was found that there were numerous single nucleotide polymorphism and insertions/deletions loci between the two varieties,for serine/threonine protein kinase gene(gene＿5188)and phospholipid phosphatase gene(gene＿2176),which can be used for screening molecular markers for rapid identification of P.ostreatus varieties’ resistance in future studies.This study conducted disease resistance identification on a P.ostreatus population,screened out P.ostreatus varieties resistant to brown blotch disease,and explored some disease-resistant related genes.The mechanisms of P.ostreatus response to P.tolaasii infection and the reasons of resistance differences between P.ostreatus varieties were studied,which would contribute to further research on the occurrence mechanism of bacterial brown blotch disease in edible mushrooms and provide a reference for efficient prevention and control of bacterial brown blotch disease in edible mushrooms and resistance breeding of P.ostreatus. |
PDF Full Text Request