Etiology, Histology And Cytology Study Of Marssonina Leaf Blotch Of Apple And Its Chemical Control

Diplocarpon mali [Y. Harada&K. Sawamura (anamorph Marssonina coronariae (Ellis&J. J. Davis) J. J. Davis] the causal agent of Marssonina leaf blotch of apple primarilyinfects apple leaves and lead to severe abscission of leaves during the growing season,resulting in reduction of quantity and quality of apples. However, the knowledge ofpathogenesis and epidemiological aspects about this destructive pathogen is poorlydocumented that has become an impediment to effectively control the disease in the orchard.In this study, we investigated the biological characteristics and infection process of D. mali onapple leaves, and evaluated the effect of chemical control in the field, which will offer usefulinformation for developing and optimizing the disease management. The main results are asfollows:1. The D. mali was isolated by using single germinated conidium, diseased leaf segmentand picking conidia mass from acervuli on the media of potato dextrose agar (PDA),10%V8, apple leaves dextrose agar (ALDA) and apple leaves extraction dextrose agar,respectively. About300isolates were obtained in total. The results showed that eachisolation method was applicable to obtain the pure culture of the fungus. However, singlespore isolation was much easier to handle, and presented less contamination thanisolation using diseased leaf segment and conidia mass from acervuli. The success rate ofsingle spore isolation was more than90%that was much higher than that of other twomethods (10%success rate).2. The in vitro growth of D. mali, such as color and diameter of colonies, and ability ofreproduction, differed on PDA,10%V8, ALDA and ALEDA media. The one-month-oldculture of D. mali on PDA was brown to black with about7mm in diameter, grewupwards like wormcast; the distinctive fruit bodies and aerial hyphae were not observedon colonies. On the medium of10%V8, the color and size of colonies were similar withon PDA. However, there were a few of white aerial hyphae and acervuli on the radialedge of the colony, and dark brown vegetative hyphae were observed in the medium aswell. On the medium of ALDA, the fungus formed flat colonies with the similar size ason PDA. The fluffy aerial hyphae on the surface were golden yellow, and the vegetative hyphae in the medium were dark brown. The fruit bodies were produced in the center ofthe colony. While, on ALEDA, about2mm colonies with brown to black color formedafter30d cultivation. It was irregular, and grew upwards as well. A few of brown aerialhyphae and acervuli were observed on the surface of colonies.3. The effects of media and environmental conditions on mycelial growth and conidialproduction of D. mali were conducted by using liquid shake culture, which revealed morebiological information about this fungus, and will help to implement high throughputscreening of fungicides, and facilitate mycological and pathological research on thefungus and Marssonina leaf blotch of apple caused by D. mali. The results showed thatpotato and carrot dextrose broth (PCDB), potato and carrot sucrose broth (PCSB) andcarrot dextrose broth (CDB) were most favourable for rapid mycelial growth and conidialproduction. All carbon sources tested (dextrose, fructose, galactose, sucrose, mmaltoseand lactose) and peptone favoured for mycelial growth, but none of carbon and nitrogensources tested stimulated conidial production significantly. The fungus did not grow at5and30°C after14d of incubation, and did not grow at pH3and9. The optimumtemperature for mycelial growth and conidial production was25C. Active mycelialgrowth and sporulation occurred at pH5–8.4. A method to estimate the strain virulence of D. mali was established by inoculatingconidial suspension on detached apple leaves, which allow us to reveal the virulencecomposition of D. mali population in Shaanxi province. Xinjiang crabapple was selectedfrom four rootstocks of Malus sp.(M26, Xinjiang crabapple, Huaye crabapple andShandingzi) and three apple cultivars (Fuji, Gala and Qinguan) as plant material forvirulence evaluation, because of its high susceptibility over other tested plant materials.The virulence differentiated among40D. mali strains collected from Shaanxi province,but this differentiation unrelated to their geographical origins. We divided the virulenceof D. mali into three levels that was weak, moderate and strong based on the lesiondiameter (D)<2mm,2mm≤D≤6mm, and D>6mm, respectively. Strains withmoderate virulence took a large proportion (72.5%) of total.5. The infection process of D. mali on apple leaves was disclosed by fluorescence andelectron microscopy. The results showed that conidia attached to leaf surface apparentlyby mucilage, and germinated on both sides of leaves6h post-inoculation (hpi). Itpenetrated the cuticle by infection peg formed either in germ tube or appressoria in6hpi.Then the typical haustorial like parasites were observed in host epidermal and mesophyllcells accompanied by extension of subcuticular and intercellular hyphae. Five dayspost-inoculation (dpi), the intracellular hyphae were observed. At the same time, the subcuticular hyphal strands (SHS) were produced as a special way for fast expanding andreproduction. About7dpi, acervuli formed on inoculated leaves. These histological andcytological informations, included including first report on haustoria and SHS production,could help us to understand more about the pathogen-host interaction mechanisms and todevelop novel resistant cultivars. This is also very important for developing andoptimizing the disease management strategies.6. The responses of host cells upon invasion of D. mali was described. On the early stage ofthe infection process, fluorescent depositions (callose) were observed on the epidermalcell wall surrounding the penetration site, which was thought to act as a physical barrierto impede microbial penetration. This phenomenon was more obvious on tender leaves.With the development of disease, the pathological changes of the host cells wereassociated with the expansion of D. mali, including plasmolysis, swelling anddisintegration of host organelles such as nuclei and chloroplasts, disaggregation of nuclei,disorganization of the protoplasm. Finally host cells were died and collapsed, and thenecrosis lesion appeared on apple leaves.7. Two triazole fungicides, tebuconazole and difenoconazole, and one dithiocarbamatefungicide propineb were investigated for their fungitoxicity on D. mail in differentdevelopmental stage in vitro, which will facilitate to reveal their mode of fuction and todesign appropriate use recommendations. The triazole fungicides showed a stronginhibition effect on mycelial growth and acervuli formation, but less efficiency onconidial germination. EC₅₀values of tebuconazole on mycelium dry weight, acervuliformation and conidial germination were0.060,0.055and128.825μg/ml, respectively,indicating their good therapeutic action. And EC₅₀values of difenoconazole were0.009,0.024and331.131μg/ml, respectively. However, dithiocarbamate fungicide propinebexhibited stronger inhibition on conidial germination and acervuli formation than onmycelial growth with EC₅₀values of each assay were1.07,1.70and6.76μg/ml,respectively, which indicated that this class of chemicals was function as protectiveagents.8. The critical period was explicit for spraying fungicides to control Marssonina leaf blotchof apple in Guanzhong region of Shaanxi province. The field trial during2005to2006showed that protectant fungicides such as propineb and mancozeb were recommended tospray two to three times from late April to early June in a growing season (the periodfrom post-anthesis to young fruit stage), which presented more than90%control efficacy,and indicated that this period probably is the primary infection stage of the fungus inGuanzhong region. The initial stage of disease prevalence was crucial for systemic fungicides. In this period, it is recommended to alternatively use fungicides, such astebuconazole, difenoconazole and azoxystrobin et al..