Mechanism And Resistance Induced On Marssonina Apple Blotch

Marssonina apple blotch, caused by Diplocarpon mali Y. Harada&Sawanura （anamorph:Marssonina coronariae （Ellis&J. J. Davis） J. J. Davis）, is one of the most severe diseases ofapple （Malus×domestica）. This disease leads to premature defoliation in the main regions ofapple production, thereby weakening tree vigor and affecting the size, color, quality, andquantity of the fruit. However, the knowledge of mechanism on marssonina apple blotch ispoorly documented that has become an impediment to effectively control the disease in theorchard. In this study, we previously evaluated the resistance of28cultivars and39Malusspecies or biotypes after inoculating leaves with conidial suspensions of D. mali in vitro andvivo. Using M. sieversii and M. prunifolia cv. Donghongguo, we investigated the infectionprocess of D. mali on apple leaves, and monitored the levels of hydrogen peroxide, activitiesof antioxidant and pathogenesis-related enzymes, expression of marker genes for salicylicacid （SA）, jasmonic acid （JA）, and ethylene （ET） in pathways for defense regulation, and theconcentrations of18types of phenolic comounds during incompatible and compatibleinteractions. We also examined whether abiotic stress, plant hormones, and melatonin couldimprove resistance to Marssonina apple blotch. Our goal was to offer useful information fordeveloping and optimizing the disease management. The main results are as follows:1. Using artificial inoculations, the resistance of28cultivars and39Malus species orbiotypes to Marssonina apple blotch were evaluated. Of the28apple cultivars tested here, five（‘Pinova’,‘Honeycrisp’,‘Pink Lady’,‘Qinguan’, and ‘Redchief Delicious’） proved to beresistance genotypes and a low incidence of infection. The39species and biotypes showedsignificant differences in their degree of resistance to D. mali. Eight accessions wereclassified as resistant resources, including M. toringoides （Rehd.） Hughes cv.Wushanbianyehaitang, M. sieversii Ledeb. cv. Xinjiangyepingguo, M. sieboldii （Reg.） Rehd.cv. Sanyehaitang （xinannongda）, M. yunnanensis Schneid. cv. Dianchihaitang, M. spectabilisBorkh. cv. Haitanghua, M. sieversii （Ledeb.） Roemer cv. Yepingguo9, M. sieversii （Ledeb.）Roemer cv. Zhongshuhongguozi, and M. sieboldii （Reg.） Rehd. cv. Tengchongsanyehaitang.Those that proved most resistant are potential resources in programs for resitance breedingand disease management.2. The germination and growth of D. mali on leaves of resistance and susceptible Malusplants was disclosed by fluorecence and electromicroscopy.‘Naganofji No.2’ and M. prunifolia cv. Donghongguo, which were susceptible to Marssonina apple blotch, with highergermination of condia on their leaves surface. The pathogen penetrated the cuticle either bygerm tube or by formation of appressoria. Compared with the susceptible plants, the resistantplants ‘Qinguan’ and M. sieversii significantly suppress condia germinated. On their leavessurface, largely condia were wizened. Seven days post inoculation （dpi）, abundant hyphaewas observed in leaves tissue of ‘Naganofji No.2’, but fewer in ‘Qinguan’. These resultsdemonstrate that the disease resistance to Marssonina apple blotch of host plants responsed asinvading-resistance and conidia germinating inhibition.3. A ROS burst occurred in the infected plants of disease-resistance M. sieversii but notin those of the susceptible M. prunifolia. Compared with the uninfected M. sieversii control,infected plants of that species had a significant H₂O₂burst at2dpi, and then maintained thathigh level throughout the plant-pathogen interaction. Infected plants of M. prunifolia hadsignificantly lower H₂O₂concentrations at4,6, and10dpi when compared with the controlfor that species. This meant that ROS burst has a key role in the resistance response by M.sieversii. We observed a large increase in SOD activity and suppression of APX activity in M.sieversii plants at the early stage of infection, suggesting that this species produced andaccumulated much more H₂O₂. However, in M. prunifolia, the significant rise in APX andCAT activities during that early stage may have explained why those plants had no ROS burst.4. Compared with the uninfected M. sieversii plants, chitinase activity in the infectedplants increased rapidly after6dpi. For M. prunifolia, activity did not differ significantlybetween infected and uninfected plants, except at Day20, meaning that chitinase respondedearlier in M. sieversii. Relative expression followed a similar trend, with up-regulation ofchitinase transcripts occurring8d earlier in M. sieversii. Furthermore, β-1,3-glucanaseactivities and transcription levels were higher in M. sieversii than in M. prunifolia under eitherinoculated or uninoculated conditions. This demonstrated that chitinase and β-1,3-glucanasewere more effective in limiting pathogen growth and invasion in M. sieversii.5. Using M. sieversii and M. prunifolia cv. Donghongguo, we monitored theconcentrations of18types of phenolic compounds, transcripts for key enzymes in thephenylpropanoid pathway, and activities by phenylalanine ammonia lyase （PAL）, polyphenoloxidases （PPO）, and peroxidases （POD） when plants interacted with D. mali. We found gallicacid, epicatechin, and ρ-coumaric acid showed sensitivity to inoculation with D. mali. Levelsof gallic acid followed opposite trends in M. sieversii and M. prunifolia after inoculation.Compared with the uninfected M. sieversii control, infected plants of that species had asignificantly high concentration of gallic acid at4dpi, which was then maintained throughoutthe remainder of that interaction period. Infected plants of M. prunifolia had significantly lower gallic acid concentrations after4dpi than did the control for that species. In bothspecies, infection with D. mali enhanced their concentrations of epicatechin and reduced theirlevels of ρ-coumaric acid. PAL activity in M. sieversii plants was dramatically elevated after10dpi, peaking to3.14-fold higher than the control at20dpi. For M. prunifolia, PAL activitygradually increased before being decreased in infected plants.6. Up-regulation of the SA and JA pathway, along with suppression of the ET pathway,proved to be essential to resistance by M. sieversii plants. PR1and PR5were strongly inducedby D. mali only in M. sieversii, indicating that effective manipulation of the SA pathway wasessential for conferring resistance by that species. The transcription of COI1and PLD wasinduced in the incompatible interaction of M. sieversii-D. mali but not in the susceptible M.prunifolia plants, meaning that this JA pathway was also essential to the resistance by M.sieversii. However, we also found that JA-signaling acted antagonistically on SA-dependentdefenses such that, during the later stages of infection, expression of the JA markers COI1andPLD was increased while that of PR1and PR5in the SA pathway was decreased. ERF3wasinduced by D. mali in the susceptible genotype M. prunifolia but not in the resistant M.sieversii. We verified this theory by pre-treating those plants with exogenous ACC, and foundthat such an application made them even more susceptible to D. mali. By contrast, exogenousSA and JA improved the resistance of M. prunifolia plants, which was consistent with ourconclusion that the up-regulation of the SA and JA pathway, along with suppression of the ETpathway enable plants of M. sieversii to be resistant to D. mali.7. Pre-treatment with exogenous melatonin improved resistance to D. mali by susceptiblegenotype M. prunifolia. Pre-treatment enabled plants to maintain intracellular H₂O₂concentrations at steady-state levels and enhance the activities of plant defense-relatedenzymes, possibly improving disease resistance. Because melatonin is safe and beneficial toanimals and humans, exogenous pre-treatment might represent a promising cultivationstrategy to protect plants against this pathogen infection.