【作者】 郑露；

【导师】 黄俊斌；

【作者基本信息】 华中农业大学 ， 植物病理学， 2010， 博士

【摘要】 大蒜白斑病是近年来在湖北省当阳地区发现的一种毁灭性病害。自2005年以来,该病在当阳市10万亩大蒜生产区大面积发生,年均减产20%-70%,严重影响了大蒜的产量和品质。本文通过形态学和分子生物学方法将大蒜白斑病病原鉴定为茄匍柄霉Stemphylium solani。在此基础上进一步对大蒜白斑病的病原学、防治技术以及毒素作用机理进行了研究。主要研究结果如下：1.从湖北省当阳市大蒜种植基地采集具有典型症状的病叶,用常规组织分离法获得Stemphylium sp菌株31个,Alternaria sp菌株33个。从每种菌中选取代表性菌株进行致病性测定,结果表明,分别用Stemphylium sp.菌株DY-1、DY-5和DY-6的菌丝块或分生孢子悬浮液接种,叶片发病率均为100%；而用Alternaria sp.菌株DY-P-1和DY-W-1的菌丝块或分生孢子悬浮液接种大蒜叶片,均未表现任何症状。菌株DY-1、DY-5和DY-6的形态学特征相似,在PSA平板上分生孢子褐色,具1-3个横隔膜,中间隔膜颜色较深,有2-7个纵(斜)隔膜,大小29-58(45)×14-28(22)μm,长宽比为2：1；分生孢子梗顶端囊状膨大,长达169μm；经产孢表型培养,可观察到分生孢子以内壁芽生式从产孢梗的顶端囊状膨大体处产生；在自然寄主上,分生孢子大小为27-48(39)×15-30(21)μm;参照Simmons和Ellis等的分类方法,将菌株DY-1、DY-5和DY-6鉴定为茄匍柄霉S. solani.rDNA-ITS序列分析结果表明,3个供试菌株的5.8S rDNA及两侧的ITS区序列与S. solani的同源性达99%。采用neighbor-joining法构建匍柄霉不同种的系统发育树,发现菌株DY-1、DY-5和DY-6的序列与GenBank中S. solani(AF203449、AF203450)、S. nabarii(AY372679)和S. subglobuliferum(AY751454)的遗传距离最近。2.采用测定菌丝生长情况对大蒜白斑病菌生物学特性进行研究。结果表明,菌株DY-5在PSA平板上培养,5-35℃范围内均能生长,最适生长温度为20～30℃,pH值4-10均可生长,pH值为6-8时生长最佳；在Czapek液体培养基中培养,病菌对测试碳源中淀粉的利用效果最好,而对甘露醇利用最差,在测试氮源中,对谷氨酸利用最佳,对尿素利用最差；菌丝的最低致死温度为55℃、10min。另外,采用台盼蓝染色法研究了大蒜白斑病菌的侵染特性,结果表明,接种2h后孢子即可萌发；6h后可形成多根芽管(多至11根)；接种8h后芽管在气孔附近分支形成侵染菌丝,从气孔侵入到寄主细胞中；12h后侵染菌丝不断分化可从多个气孔侵入；24 h后病菌在气孔中形成大量菌丝,侵入率显著增加。3.研究了大蒜白斑病菌毒素的基本性质及专化性。试验结果表明,PSB液体培养基是大蒜白斑病菌产毒的最佳培养基；毒素滤液经甲醇或丙酮处理后,小分子物质保留了毒素的致病活性；经蛋白酶或高温处理,毒素致病活性基本不变；乙酸乙酯和正丁醇对毒素的萃取能力最强,而氯仿和苯的萃取能力相对较弱；pH值对毒素滤液的致病活性有显著影响,且随pH值升高,其致病活性逐渐降低。另外,通过测定不同大蒜品种和寄主植物对S. solani的感病性及其对毒素的敏感性,可确定大蒜白斑病菌毒素为一种非寄主专化性毒素。4.采用薄层层析(TLC)和高效液相色谱(HPLC)法对大蒜白斑病菌毒素进行分离纯化,并结合化学鉴定、紫外可见吸收光谱(UV-VIS)、红外光谱(IR)、质谱(MS)、元素分析以及核磁共振(NMR)等分析方法鉴定毒素结构。结果表明,对24种TLC展开系统进行筛选,得到其最佳展开剂为乙酸乙酯：石油醚：甲醇=4：1：0.35,可分离得到13个组分,其中组分4为主要致病组分；组分4经HPLC纯化制备,共可收集到100 mg棕红色毒素纯品,将其命名为SS-toxin。在SS-toxin的结构鉴定中,通过化学反应可初步判断该毒素是一种酚类物质,而不是酯、生物碱、多肽、蛋白质或氨基类化合物；毒素在215 nm、264nm和458nm处有紫外可见吸收峰；在红外光谱中可看出,毒素结构中有酚-OH(3400 cm-1)、-CH3(2946 cm-1、1449 cm-1、1395 cm-1)、芳香环(3092 cm-1、1595 cm-1)、-CO (1670 cm-1)和酮-C=O(1640 cm-1)存在的伸缩振动；根据ESI-MS和元素分析结果得到SS-toxin的分子式为C16H16O8(分子量336)；进一步结合毒素的1H-NMR谱,将SS-toxin初步鉴定为7-甲氧基-2-甲基-1,2,3,4-四氢-1,2,3,4,5-五羟基蒽醌(7-methoxy-2-methyl-1,2,3,4-tetrahydro-1,2,3,4,5-pentahydroxy anthraquinone)5.为明确大蒜白斑病菌毒素的致病机理,研究了毒素组分SS-toxin对寄主植物生理活性、大蒜叶片细胞超微结构及叶片质膜系统的影响。结果表明,SS-toxin对抗、感品种的浓度-剂量曲线分别为Y=0.0546X+0.2966 (R2=0.9694, P<0.05)和Y=0.0871X+3.3893 (R2=0.9774, P<0.05);毒素对大蒜根、芽生长及根尖有丝分裂均有抑制作用,其中对根、芽的抑制中浓度分别为64.9和178.5μg ml-1;大蒜叶片经毒素处理3d后,其叶绿素a、叶绿素b及总叶绿素含量均有所下降,说明毒素与病害的症状发展有密切联系；电镜观察结果表明,用毒素处理感病品种叶片2h后,细胞质膜内陷,细胞壁有轻微变形,6h后,线粒体脊数量减少,且出现空泡化,12h后,叶绿体膜肿大,片层结构出现解体,核膜部分消失,24 h后,细胞壁明显断裂,而其它结构无明显变化；用毒素处理抗病品种叶片,12h后,质膜开始出现内陷,细胞壁肿胀变形,24h后,少数线粒体空泡化,其他细胞结构无明显变化,推测质膜为SS-toxin的早期作用位点。采用两相法制备大蒜抗、感品种叶片的质膜微囊,并通过特异性酶抑制剂测得抗感品种质膜的相对纯度均在70%以上。抗、感品种质膜经不同浓度毒素处理后,其H+-ATPase水解活力变化较为明显,随处理浓度升高,酶活性逐渐降低；用最高浓度200 g m1-1毒素处理后,抗、感品种质膜H+-ATPase的相对活性分别降低至56.4%和41.4%；另外,抗、感品种质膜NADH氧化速率和Fe(CN)63-还原速率也均受抑制,以Fe(CN)63作为电子受体时,抗、感品种质膜NADH相对氧化速率分别降低至45.8%和43.1%；以NADH作为电子供体时,抗、感品种质膜Fe(CN)63相对还原速率分别下降至45.4%和18.9%。H+-ATP酶和标准氧化还原系统可能为SS-toxin在大蒜叶片细胞质膜上的作用位点。6.2006～2008年对大蒜白斑病的流行规律和防治技术进行研究,结果表明,2006-2007年病害于11月5日开始发生,至收获期病指达60.6,其中11月、12月和次年3月为发病高峰期,病指增长率分别为37%、14%和13%,而2007-2008年病害的发生推迟至11月24日,至收获期病指仅为27.5。大蒜白斑病菌越夏研究结果表明,病残体在温室(25℃,30% RH)、冰箱(4℃,90%RH)、土表、土表下10 cm和土表下20 cm条件处理165 d后,菌丝体均能存活,存活率分别为40.0%、2.9%、25.7%、17.1%和5.7%,统计病残体中分生孢子萌发率发现,处理165 d后仅温室和冰箱处理可观察到分生孢子萌发,萌发率分别为10.8%和5.7%。2006-2007年品比试验中,青秆软叶和软软叶抗病性最强,其RAUDPC分别为10.0和10.4,而红七星和温二早最为感病,RAUDPC分别为67.3和64.4。2007-2008年供试品种中,自选-2、青秆软叶和软软叶最为抗病,RAUDPC分别为2.7、3.4和3.9,而泰糯-1和山东济南最感病,RAUDPC分别为33.0和32.8。2006-2007年种子处理苗期防病试验中,种子处理剂2.5%适乐时悬浮种衣剂(0.05 g a.i.kg-1)和50%福美双可湿性粉剂(1.25 g a.i. kg-1)拌种防效最好,其RAUDPC值分别为23.0和23.7；且2007-2008年调查仍以适乐时和福美双的防效最好,其RAUDPC值分别为5.7和6.3。在PSA平板上测定9种杀菌剂对大蒜白斑病菌菌丝生长的抑制作用,以40%福星乳油的抑菌效果最好,ECso为0.4μg ml-1；30％爱苗乳油、50%多菌灵可湿性粉剂、10%世高水分散粒剂和20.67%万兴乳油抑菌效果次之,其EC50分别为1.0μg ml-1、1.4μg ml-1、2.0μg ml-1和2.7μg ml-1。2006-2007年田间药效试验中,40%福星乳油4000倍液和75%代森锰锌可湿性粉剂1000倍液、2000倍液防效最好,其RAUDPC分别为11.5、13.0和14.6；2007-2008年,40%福星乳油4000倍液、75%代森锰锌可湿性粉剂1000倍液和20.67%万兴乳油1000倍液的防效最好,RAUDPC分别为4.4、4.7和5.2,与对照药剂甲基托布津存在显著差异(P<0.05)。更多还原

【Abstract】 Leaf blight of garlic is a destructive disease in Hubei province, China. Symptoms were observed on infected leaves in Dangyang County from autumn 2005 to spring 2009, with the diseased area estimated to be over 7000 ha. Garlic yield was reduced by 20% on average with up to 70% yield losses in some fields.Leaf blight of garlic has become the main disease limiting garlic production and quality. In primary work, the causal agent of garlic leaf blight was identified as Stemphylium solani from cultural and morphological characteristics, and subsequent analysis of the internal transcribed spacer region of ribosomal DNA.And then, aetiology, epidemiology and integrated control of garlic leaf blight caused by S. solani, and pathogenic mechanism of phytotoxin produced by the pathogen were also studied in this paper. The main research results were listed as follows:1.Diseased leaves with leaf blight symptoms were collected from fields in the main garlic production areas of Dangyang County in Hubei province, China. Thirty-one Stemphylium sp.isolates and 33 Alternaria sp. isolates were obtained using usual tissue isolation method. The Stemphylium sp. isolates DY-1,DY-5 and DY-6, and Alternaria sp. isolates DY-P-1 and DY-W-1 were used for pathogenicity test. By inoculation with fungal plugs or conidial suspension, white spots were observed on inoculated leaves for isolates DY-1,DY-5 and DY-6, but no symptoms were seen on garlic plants treated with isolates DY-P-1 and DY-W-1.The isolates DY-1,DY-5 and DY-6 were transferred to PSA plates. On PSA, conidiophores were up to 169μm long. Conidia were pointed at the swollen apex of each conidiophore, brown, with one to three dark coloured transverse septa and distinctly constricted at median septa, two to seven longitudinal or oblique septa, 29-58(45)×14-28(22)μm (mean) and mean length/width ratio was 2.0.On naturally diseased leaves of garlic, the size of conidia were ranged from 27-48(39)×15-30(21)μm. Based on cultural characteristics and fungal morphology of Simmons and Ellis, the isolates were identified as S. solani. Genomic DNA was extracted from three isolates, and sequences of rDNA-ITS were obtained. Comparison with sequences in GenBank showed 99% similarity with S. solani. By neighbor-joining method, the phylogenetic tree of the ITS1-5.8S-ITS2 sequences from Stemphylium species was constructed. Stemphylium solani (AF203449、AF203450), S. nabarii (AY372679), S. subglobuliferum (AY751454) and isolates DY-1,DY-5 and DY-6 were grouped together.2.Biological characteristics of S. solani isolate DY-5 were tested by measuring mycelial growth in different conditions.For the growth of mycelia on PSA medium, the range of temperature was 5-35℃,with the optimum of 20-30℃.The favorable pH value was 4-10, with the optimum pH 6-8.Among the tested carbon and nitrogen sources in Czapek liquid medium, mannitol and urea were not good for the growth of mycelia. Starch and glutamate were the most favorable for its growth. The lethal temperature for the mycelium was 55℃for 10 minutes.The infection process of S. solani on susceptible garlic leaves was studied by light microscopy technique. The infection process was investigated by light microscopy from 2 to 120 h after inoculation (AI).Spore germination was observed within 2 h AI and up to 11 germtubes formed within 6 h AI. The pathogen invaded the garlic leaf only through stomates. Infection hyphae were observed inside stomates within 8 h AI on garlic leaves, and more stomates were infected by secondary hyphae within 12 h AI. Abundant hyphae were produced within 24 h AI and more frequent infection was observed within 24,36,48,72,96 and 120h after inoculation.3.To determine the property of the toxins, the culture filtrates were treated in various ways. No toxin could be detected in the precipitates after adding methanol or acetone. After partitioning with normal butanol or ethyl acetate, the most toxins were detected in solvent fractions.The toxic activity in culture filtrates was stable even after incubation at 121℃for up to 30 min. Culture filtrates treated with either both proteinase K or pepsin was still toxic to garlic leaves.Filtrates incubated at pH 2 to 4 for 24 hr and then re-adjusted to pH 7 showed the most toxic activity in the leaf necrosis assay, while the toxic activity decreased with increasing pH from 5 to 13.Pathogenicity of S. solani to various crops and garlic cultivars, and toxicity of its culture filtrates indicated that toxin(s) produced by S. solani were non-host-specific. 4.A non-host-specific phytotoxin from culture filtrate of S. solani was isolated by using TLC and HPLC. Preparative analytical TLC plates spotted with toxin samples were developed separately in 24 solvent systems. The most bands (total 13 fractions) from the toxin on TLC plates was obtained with ethyl acetate/sherwood oil/methanol (4:1:0.35, vol/vol/vol), and this optimal solvent system was used for preparative TLC.Only the fraction with the highest toxicity, the fourth fraction, was collected. This fourth fraction was then purified further with preparative HPLC, and the purified phytotoxin, named SS-toxin(100 mg), appeared as red-brown crystals. Based on chemical identification, the reaction was positive in ferric chloride test. The SS-toxin exhibited UV absorption maxima at 215 run,264 nm and 458 nm, and this indicated an aromatic structure for the toxin. The IR spectrum displayed characteristic absorptions for hydroxyl groups (3400 cm-1),methyl groups (2946 cm-1,1449 cm-1 and 1395 cm-1),aromatic rings (3092 cm-1 and 1595 cm-1),unchelated CO (1670 cm-1) and conjugated CO (1640 cm-1).The result of ESI-MS together with elemental analysis suggested the molecular formula of C16H16O8 (Mw:336). The structure of SS-toxin was further identified as 7-methoxy-2-methyl-1,2,3,4-tetrahydro-1,2,3,4,5-pentahydroxyanthraquinone or its derivate with 1H-NMR spectrum.5.The physiological assay, transmission electron microscopy and bioassay of standard redox system in the plasma membrane were used to study pathogenic mechanism of SS-toxin. Regression lines for susceptible and resistant cultivars are, respectively, as follows:Y=0.0871X+3.389, R2=0.9774,P<0.05,and Y=0.0546X+ 0.2966, R2=0.9694, P<0.05.The root growth of susceptible cv. Changbanpo was more susceptible to SS-toxin than the shoot growth,and the EC50 values were 64.9 and 178.5μg ml-1,respectively. The numbers of actively dividing cells root tips were also decreased. Chlorophyll content reduction by the SS-toxin detected at all tested concentrations in susceptible cv. Changbanpo, indicated that the symptoms of garlic leaf blight is related with the toxin produced by pathogen. In leaf cells of the susceptible cultivar, the earliest toxin-induced ultrastructural changes were detected 2 h after toxin treatment in the plasma membranes and cell walls, and these modifications became more severe and more frequent up to 24 h of toxin exposure. The number of mitochondrial cristae was reduced in leaves of the susceptible cultivar by 6 h after treatment with the toxin. Disordered chloroplast lamellae, swollen chloroplasts and disrupted nuclear membranes appeared after 12 h of exposure to the toxin for the susceptible cultivar. No apparent change of rough endoplasmic reticulum was observed even after 24 h. In leaf cells of the resistant cultivar, the earliest effects (plasma-membrane invagination and cell wall changes) were detected 12 h after toxin treatment. The numbers of mitochondrial cristae were reduced by 24 h. Other organelles including chloroplast, nuclear membrane and rough endoplasmic reticulum were seemingly not affected in leaf cells of the resistant cultivar during the first 24 h of exposure.The plasma membranes prepared by aqueous polymer two-phase partitioning from leaves of susceptible and resistant cultivars, showed no significant contamination by other membrane systems, as confirmed by assays tested in the presence of selective inhibitors. Total H+-ATPase activities were more than 70% inhibited by vanadate in both cultivars.The H+-ATPase activities of plasma membranes, isolated from both cultivars, were inhibited by SS-toxin in a dose-dependent manner. When the concentration of toxin was increased from 0.1up to 200 g ml-1,the relative activity of H+-ATPase in plasma membrane extracts of resistant cultivar decreased from 92.1% to 56.4% and for susceptible cultivar, from 93.4% to 41.4%. The NADH oxidation and Fe(CN)63- reduction rates of plasma membranes from both cultivars were inhibited by SS-toxin at all tested concentrations ranging from 0.1 to 200 g ml-1,and the inhibition appeared to be proportional to the concentration of toxin. Using ferricyanide as an electron receptor, the relative NADH oxidation rate in the resistant or susceptible cultivar was reduced to 45.8% or 43.1%,respectively, after treatment with SS-toxin. When NADH was used as an electron donor, the relative Fe(CN)63" reduction rate in the resistant or the susceptible cultivar was decreased to 45.4% and 18.9%, respectively. Our results suggest that, under in vitro condition, the plasma membrane ATPase and standard redox system can be both the cellular targets of SS-toxin.6.Epidemiology, cultivar resistance, and chemical controls of garlic leaf blight were investigated throughout the 2006 to 2008 growing seasons in Dangyang County, Hubei province to improve to disease control methods. In 2006/2007, the initial symptoms of small white spots were observed on 5 November, and a high disease severity was also recorded at final harvest. The months of November, December and March had the highest rates of disease increase, with 37%,14% and 13%,respectively. In 2007/2008, the occurrence of leaf blight was delayed until 24 November, and leaf blight increased very slowly with DSI reaching only a maximum of 27.5 by final harvest. For all treatments (glasshouse, cool storage, in the field on the soil surface, buried in soil at 10 cm or buried at 20 cm), S. solani was consistently recovered from diseased plant debris 165 days after treatment while viable conidia of S. solani were detected only in debris kept in the glasshouse(10.8%) or under cool storage (5.7%). The warm condition and soil surface were suitable for survival of the pathogen. There were significant differences in blight severity between garlic cultivars in the field and cultivars Qingganruanye, Ruanruanye and Zixuan-2 were among the most resistant. For, fungicidal seed treatments, fludioxonil (0.05 a.i. g kg-1) and thiram (1.25 g a.i. kg-1) had significant efficacy. Inhibition of mycelial growth of S. solani by nine fungicides was assayed on amended PSA medium. Flusilazole EC (40%) had the highest inhibitory effect with the 50% effective concentration (EC50) at 0.4μg ml-1.The next most inhibitory were 30% difenoconazole/propiconazole EC,50% carbendazim WP,10% difenoconazole WG and 20.67% flusilazole/famoxadone EC with EC50 values of 1.0,1.4,2.0,2.7μg ml-1, respectively. Fungicidal applications in the field were effective in controlling leaf blight, and 40% flusilazole EC,20.67% flusilazole/famoxadone EC or 75% mancozeb WP had the highest efficacy in reducing leaf blight severity.更多还原