【作者】 赵华；

【导师】 黄丽丽；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2012， 博士

【摘要】 由Diplocarpon mali Y. Harada&K. Sawamura（无性世代为Marssonina coronariae（Ellis&J. J. Davis） J. J. Davis）引起的苹果褐斑病是导致我国苹果树早期落叶的主要病害，近年来在我国苹果产区中度至严重发生，流行年份重病园7⁹月间的落叶率高达80%¹00%，严重时导致苹果秋后二次开花，不但降低了苹果的产量和品质，而且极大地削弱了树势。然而由于该病菌分离培养困难，对其生物学特征、致病机理及其所致病害的发生规律等基础信息了解甚少，新药剂筛选和品种抗病性评价难以实施，致使生产上对该病害的防治一直处于盲目被动状态，无法有效控制病害发生流行。因此，本研究利用多种研究技术和方法，拟首先揭示病菌的病原学特征以及病菌与寄主的互作关系，进而开展病害的化学防治研究，从而为生产上科学、有效地控制病害提供科学依据。本论文取得了以下主要研究结果：1.通过不同分离方法和培养基获得了苹果褐斑病菌的纯培养物近300株。本研究分别采用组织块分离法、分生孢子团分离法和单孢子分离三种方法在马铃薯葡萄糖琼脂（PDA）、10%V8培养基、苹果叶片葡萄糖琼脂培养基（ALDA）和苹果叶片煎汁葡萄糖琼脂培养基（ALEDA）上皆分离得到了苹果褐斑病菌，但组织块分离法和分生孢子团分离法成功率只有10%左右，而单孢分离法污染少，成功率高达90%以上，明显优于其他两种方法。2.明确了苹果褐斑病菌的培养特征，发现其在不同培养基上25°C黑暗培养1个月后菌落大小、形态和繁殖体产生情况有明显差异。在PDA培养基上菌落直径约为7mm，黑褐色蚯蚓粪状，无气生菌丝和基内菌丝，未观察到子实体；在10%V8培养基上形成的菌落大小与PDA培养基上相当，黑褐色、边缘呈放射状，气生菌丝白色、稀疏，基内菌丝深褐色，有子实体；在ALDA培养基上菌落直径约7mm，黄褐色，气生菌丝金黄色、茂密，基内菌丝深褐色，有子实体；病菌在ALEDA培养基上菌落直径约2mm，黄褐色至黑褐色，气生菌丝少许，无基内菌丝，子实体生于菌落表面。3.通过液体振荡培养后测量菌丝干重的方法，明确了苹果褐斑病菌的生物学特征。以未加碳源和氮源的Czapek培养液为空白对照比较了6种碳源、3种氮源对病菌生长的影响，发现葡萄糖等对病菌的菌丝生长有显著的促进作用，蛋白胨是最佳氮源。比较供试的13种不同营养液发现病菌在马铃薯胡萝卜葡萄糖培养液（PCDB）、马铃薯胡萝卜蔗糖培养液（PCSB）和胡萝卜葡萄糖培养液（CDB）中的菌丝生长量和产孢量最大。以PCDB（或PCDA）为培养基质研究了温度和酸碱度等环境条件对病菌生长和繁殖的影响，结果表明病菌在低于等于5°C和高于等于30°C条件下无法生长，25°C是该病菌菌丝生长和分生孢子产生的最适温度；病菌在pH值小于等于3和大于等于9的环境条件下无法生长，pH5⁸的环境下生长良好且有利于分生孢子产生。4.建立了苹果褐斑病菌室内毒力评价方法，并利用该方法对来源于陕西省10个区县的40株病菌的毒力进行了评价，初步明确了陕西省苹果褐斑病菌群体的毒力结构。通过离体叶片定点定量悬滴接种分生孢子的方法，从4个苹果属砧木（M26、新疆野苹果、花叶海棠和山定子）和3个栽培品种（富士、嘎啦和秦冠）中筛选出发病早、发病率高、病斑较大、产生分生孢子盘数目较多的新疆野苹果作为病菌毒力评价的指示材料，以接种后10d的病斑直径作为评价指标对病菌的毒力强弱进行评价。结果显示，供试菌株的毒力差异显著，按病斑直径划分为弱毒力（0<D<2mm）、中等毒力（2mm≤D≤6mm）和强毒力（D＞6mm）三个毒力类群，其中中等毒力的菌株为优势类群，在群体中所占比例达到72.5%。但菌株的毒力差异与地域来源没有明显的相关性。5.利用荧光和电子显微镜技术首次系统揭示了褐斑病菌在苹果叶片上的侵染致病过程。发现褐斑病菌似乎通过一层胶状物质将分生孢子附着在叶片表面。接种后6h分生孢子即可在叶片上、下表皮萌发入侵，12²4h为侵入高峰。病菌既可以通过芽管直接穿透叶片角质层，也可通过在芽管顶端分化形成附着胞侵入。侵入后病菌在寄主角质层下和细胞间隙扩展，并在寄主表皮和叶肉细胞内形成像专性寄生菌一样的吸器结构，据此推测此时病菌为活体营养。至接种后5d发现有胞内菌丝存在，说明病菌很可能已经进入死体营养阶段。与此同时，观察到大量角质层下菌丝平行排列形成角质层下菌丝束（SHS），从侵染点呈辐射状向外扩展。菌丝束可不断分支、扩展并可向下生长入侵寄主组织，再次形成胞间菌丝和吸器，成为该病菌快速扩展和繁殖的主要手段。接种后7d左右叶片上出现分生孢子盘。6.通过组织细胞学技术观察了寄主细胞受侵染后产生的抗病反应特征。结果显示，侵染前期侵染点周围的表皮细胞壁荧光反应明显，有胼胝质沉积，且幼嫩叶片比成熟叶片表现得更为敏感和强烈。但病菌进入叶肉组织后，寄主细胞表现出了不同程度的病理反应，如质壁分离、叶绿体肿胀解体、细胞核消解、原生质体紊乱以及细胞死亡等细胞病理学特征，并与症状出现相吻合。7.本研究首次对不同类型杀菌剂的毒力进行了室内测定，发现有机硫杀菌剂丙森锌对病菌分生孢子萌发和分生孢子盘产生有明显的抑制作用，对菌丝生长的抑制作用相对较差，其EC₅₀分别为1.07μg/ml，1.70μg/ml和6.76μg/ml，说明其具有很好的保护作用。而三唑类杀菌剂戊唑醇和苯醚甲环唑对菌丝生长和分生孢子盘产生的抑制作用明显，戊唑醇的EC₅₀分别为0.06μg/ml和0.055μg/ml，苯醚甲环唑的EC₅₀分别为0.009μg/ml和0.024μg/ml，说明其具有很好的治疗作用。两种药剂对分生孢子萌发的抑制效果较差， EC₅₀分别为128.825μg/ml和331.131μg/ml。8.本研究还通过田间防治试验明确了陕西关中地区苹果褐斑病药剂防治的关键时期。2005-2006年不同喷药时间的田间防治试验结果表明，4月下旬至6月上旬（花后至幼果期）喷施2³次保护性杀菌剂如丙森锌、代森锰锌即可有效控制当年病害的发生和流行，防治效果达到90%以上，说明此时是保护性杀菌剂喷施的关键时期，也可能是田间病菌初侵染的高峰期。而病害发生初期（陕西关中地区6月中旬至7月中旬）是内吸性杀菌剂防治的关键时期，此时喷施1²次内吸性杀菌剂如三唑类杀菌剂戊唑醇和苯醚甲环唑，或QoI类杀菌剂嘧菌酯等，即可有效减轻当年病害的危害，病害防治效果皆可达到80%以上。更多还原

【Abstract】 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..更多还原