| Apple core rot is a main fruit disease.It causes preharvest fruit drop,fruit core rot and fruit decay during storage,which seriously affected apple fruit quality,reduced apple production and brought fruit growers,apple sellers,and consumers enormous economic losses.In China,Trichothecium roseum is a dominating agent of apple core rot.Apple core rot occurred commonly in open core cultivar.Red Fuji is belonging to an intermediate cultivar.In early fruit growth stage,apple core was closed,the sinus became open as fruit expansion which made carpel cavities open.Current gaps in comprehensive understanding of infection periods and the infection parts of pathogens impede progress toward effective management for diseases in apple production.Our study choosed dominating agent Trichothecium roseum in China as target pathogen,and studied infection characteristic and pathogenic mechanism in Fuji cultivar,to clear the pathogenic mechanism of T.roseum,and provide theoretical bases for the control of apple core rot.The findings in our study are as following:Apple core rot occurs in fruit carpel cavities,special structure of fruit creates difficulties for studying infection process.Our study initially constructed PEG-mediated genetic transformation system of T.roseum and green fluorescent tagged strains: Young hyphae were acquired by cultivating conidia of T.roseum in yeast extract peptone dextrose liquid broth.The hyphae were digested by a mixed liquor of driselase and lyase to obtain protoplasts of T.roseum.By using PEG mediated protoplast transformation,an exogenous plasmid carrying a zeocin screening marker and a green fluorescent gene was inserted into genome of T.roseum and resulted resistant transformants.The green fluorescent protein(GFP)tagged TR45 strain was obtained from screening plates.Comparing with wild type strain,hyphal growth,sporulation quantity and pathogenicitiy of TR45 strain did not change obviously.The fluorescent of TR45 strain was still stable after 10 generations of subculturing in the medium without selective markers.We observed infectious hyphae with obvious fluorescent intensity in infected apple fruit tissues.These results provided useful tools for further studying gene functions and infection characteristics of T.roseum.By using GFP tagged strain TR45,infection and extension characteristics in different flower tissues,clayx tubes,intervening tissues and carpel cavities by T.roseum were studied.The results showed as follows.T.roseum can infect petals,anthers,filaments,and separated styles of apple.Numerous conidia produced on infected flower debris.In two weeks after full bloom,flower debris with many hyphae and conidia of T.roseum were wrapped in the calyx tube with sepal closure.In three weeks after full bloom,T.roseum entered a united style via a unified stylar top fissure,and extended in stylar fissures inside of the unified stylar towards fruit intervening tissues,and further extended extending fissures between style base and carpel cavities.At 60 days after full bloom(dafb),hyphae extended at inter tissues of carpel cavities but still did not enter carpel cavities.At 100 dafb,the sinus,extending fissure and carpel cavities were connected with the expansion of sinus,and hyphae entered carpel cavities.In carpel cavities,hyphae ramified on carpel walls,and infected flesh tissues via cracks on carpel cavities,and caused core rot.In addition,from 60 dafb to harvest time,most conidia of T.roseum did not germinate in calyx tubes.We inferred that T.roseum may not infect or enter styles after 2 months.These results indicated that the unified stylar top fissure is the main infection court of T.roseum entering apple core.The pathogen has to pass through several passages including stylar fissures,extending fissures,and cracks on carpel cavities and the sinus opening to finish flesh infection.Besides flower infection,T.roseum also could infect the calyx basin of bagged apples and the propagules in the calyx basin were potential pathogenic source of apple core rot.Infection characteristics of T.roseum infection which resulted black spots on the fruit were studies.We found that the percent of conidial germination of T.roseum was only 2% by treating in sterile distilled water for 12 h,the percent of conidial germination reached 99% by treating in apple juice solutions.The percent of germination on lenticels and microcracks was significantly higher than surrounding apple pericarp.The result showed that the ingredient of apple juice could significantly promoted conidial germination.Percents of conidial germination reached 100% for 18 h under 95% relative humidy conidition.The result showed infection of T.roseum requires relative low humidity.On the apple fruit,T.roseum did not directly penetrate fruit pericarp,hyphae infected fruit via stomata,lenticels,and microcracks,These results showed the microenvironmental humidity provided by apple bags,especially plastic bags with poor air permeability,can easily satisfy the humidity requirement of T.roseum and finish its infection cycles.This could partly explain the reason that apple fruit easily occurred black spots after bagging.Apple black spot mainly occurred on calyx basin.The disease affected appearance quality of fruit,and propagules from black spots may be also potential pathogenic source of apple core rot.When T.roseum infected apple pericarps and flesh tissues,hyphae only colonized in intercellular space between hypodermal cells and parenchymal cells,which indicated that T.roseum have an intercellular extension property,could not penetrate host cell walls;By further observation,in the stylar fissure extension,hyphae did not penetrate endoepidermal cells of stylar fissues.In extending fissure extension of intervening tissues,hyphae did not penetrate inner cell walls of extending fissures.In carpel cavities,hyphae only infected flesh cells via cracks on the carpel cavity wall.These results supported that T.roseum could not penetrate host cell walls.This may be the main reason that T.roseum did not directly cause fruit rot in their pathway,only hyphae entered carpel cavities and caused core rot.The functions and pathogenic roles of trichothecin produced by T.roseum were studied.By deletion of trichodiene synthase encoding gene(Tri5)of T.roseum,and observation of the effects of exogenous trichothecin on pathogenicity of T.roseum.Our results showed that ΔTrTri5 mutant without producing trichothecin lost full pathogenicity on fruit,when ΔTrTri5 mutant restored trichothecin production ability by reverting Tri5 gene,its pathogenicity was also restored.Exogenous adding trichothecin restored pathogenicity of ΔTrTri5 mutant;The fruit pericarp did not show obvious symptom with small amout trichothecin(10 μg/mL)application.Inoculations with ΔTrTri5 mutant and small amout trichothecin promoted pathogenicity of ΔTrTri5 mutant.This result indicated that trichothecin play an important role in T.roseum pathogensis.Application of the concentration of 100 μg/mL and 500 μg/mL of trichothecin did not cause symptomatic changes of fruit tissues.By transmission electron microscopy,plastid membrane of flesh cells broke,and plastid degraded,cell membrane and vacuole membrane accumulated high density electrons.These results indicated that trichothecin had phytotoxic functions,and was the key pathogenic factor in T.roseum pathogensis.Our study found that stylar top fissure,a natural opening in the top of unified style,is the kep part of core infection of apple fruit by T.roseum.After entering the opening,pathogen spreads along stylar fissure,extending fissure of style,sinus,and lastly infects fresh via cracks on carpel cavities resulting core rot.Besides,we specified that trichothecin is a phytotoxic function,and is a key pathogenic factor of T.roseum infection for apple core rot.This study has theoretical and practical significance for designing targeted new fungicides,interaction mechanisms between T.roseum and apple host and making targeted control strategies of apple core rot... |
