蔬菜土传病原菌三重PCR检测体系的建立与应用

【目的】 针对生产中危害严重的3种蔬菜土传病原菌瓜果腐霉（Pythium aphanidermatum）、尖镰孢（Fusarium oxysporum）、大丽轮枝菌（Verticillium dahliae）,建立三重PCR（triplex PCR）检测体系,为蔬菜土传真菌病害的早期诊断和鉴定提供技术与方法。【方法】 筛选3种病原菌特异性引物组合,通过设定不同的PCR退火温度、引物浓度、循环次数以及延伸时间,探索影响三重PCR扩增的因素,建立蔬菜土传病原菌三重PCR检测体系,并对体系灵敏度进行检测。为了验证建立体系的稳定性,选择2×Taq Master PCR Mix（北京博迈德生物技术有限公司）和TaKaRa Taq酶（大连宝生物工程有限公司）2个不同公司的试剂,分别利用C1000 Touch ^TM型（赛默飞世尔科技有限公司）与Aeris ^TM型（新加坡艺思高科技有限公司）热循环仪进行三重PCR反应,比较检测结果的可重复性。对田间采集的35份病害样本和149份土壤样本,分别进行三重PCR和病原菌分离检测,以确定所建立得三重PCR检测体系的适用性。 【结果】 三重PCR反应体系中引物对AsAPH2B/AsPyF、FOF1/FOR1、VActF/VActR可分别扩增出瓜果腐霉、尖镰孢、大丽轮枝菌长度为163、328和530 bp的特异性目的片段。反应体系（25 μL）：0.12 μmol·L ^-1 AsAPH2B/AsPyF,0.16 μmol·L ^-1 FOF1/FOR1,0.24 μmol·L ^-1 VActF/VActR,2×Taq Master PCR Mix 12.5 μL,退火温度为60.8℃,35个循环。该体系对病原菌纯培养物的检测灵敏度为10 ^-1ng·μL ^-1,对土壤中大丽轮枝菌、尖镰孢和瓜果腐霉的检测灵敏度分别为10 ⁵、10 ⁶个孢子/g土壤和10 ^-2mg菌丝/g土壤。分别采用2×Taq Master PCR Mix和TaKaRa Taq酶,利用C1000 Touch ^TM型和Aeris ^TM型热循环仪进行三重PCR,扩增结果一致,说明三重PCR体系检测稳定性好。对田间采集的病害和土壤样本进行检测,25份病害样本和71份土壤样本中检测出带菌,三重PCR检测与病原菌分离培养结果一致。 【结论】 本研究建立的瓜果腐霉、尖镰孢、大丽轮枝菌三重PCR检测体系具有灵敏度高、稳定性和重复性好的特点,能够快速、准确地检测田间病株及其根围土壤中的瓜果腐霉、尖镰孢、大丽轮枝菌,为蔬菜土传病害的早期预防和流行监测提供了有效的技术手段。     

单味与复方中草药提取物抑制镰刀菌的比较研究

本试验以禾谷镰刀菌(Fusarium graminearum)和串珠镰刀菌(Fusarium verticillioides)为供试菌株,从26种中草药水提物中筛选出6种对两种镰刀菌有抑制效果的药物,通过不同的组方配伍,利用生长速率法检验不同组方对镰刀菌的抑制效果。试验显示:对串珠镰刀菌抑菌效果最强的为肉豆蔻-五味子两药配伍组,抑菌率为70.55%(P <0.05),比单药和三药配伍最优组分别高11%、17%。对禾谷镰刀菌抑菌作用最强的是肉豆蔻-诃子-石榴皮三药配伍组,抑菌率为77.91%(P <0.05),比两药配伍最优组高58%,较单药最优组提高90%;结果表明中草药提取物组方后可显著提高抑菌活性。     

The Effect of Inoculation Treatment and Long-term Application of Moisture on Fusarium Head Blight Symptoms and Deoxynivalenol Contamination in Wheat Grains

Fusarium head blight (FHB) is an important disease of wheat, which can result in the contamination of grains with mycotoxins such as deoxynivalenol (DON). Artificial inoculation of flowering ears with conidial suspensions is widely used to study FHB diseases. Our goal was to compare four inoculation treatments in which a conidial suspension was sprayed on flowering ears and to study the effect of the application of moisture during kernel setting and filling with a mist-irrigation system. Ten wheat genotypes were inoculated with a DON-producing Fusarium culmorum strain. Inoculation treatments varied in time of application of the inoculum (morning or evening) and in the method of controlling humidity during inoculation (bagging or mist irrigation). A wet season was simulated with a mist-irrigation system, keeping the crop canopy wet for at least 26 days after flowering. The severity of FHB symptoms (area under disease progress curve (AUDPC)), yield loss and DON contamination in the grains were determined. AUDPC data obtained with the different inoculation treatments were highly correlated (r=0.85–0.95). Mist irrigation after inoculation resulted in a higher mean disease severity, but in a overall lower toxin contamination as compared to the non-irrigated treatments. Genotypic differences in DON accumulation were present: for one wheat line toxin contamination significantly increased when irrigated, while two genotypes accumulated significantly less toxin. The closest relationships (r=0.73–0.89) between the visual symptoms and the DON content were obtained under moderate mean infection pressure. This relation between visual symptoms and the DON content deteriorated at higher infection levels.     

Fusarium confers protection against several mycelial pathogens of pepper plants

Inoculation of nonhost pepper ( Capsicum annuum ) plants with the tomato wilt pathogen, Fusarium oxysporum f.sp. lycopersici (FOL), caused no symptoms and the fungus was not recovered from any part of the plant. FOL, however, partially protected pepper plants from subsequent infection with Phytophthora capsici , Verticillium dahliae or Botrytis cinerea by significantly reducing the percentage of diseased plants and the appearance and intensity of symptoms. FOL did not inhibit the mycelial growth of these pathogens in vitro . The protection induced by FOL against Botrytis was inhibited by 1-methylcyclopropene (MCP), an inhibitor of ethylene perception, suggesting the involvement of this hormone in the signalling of FOL-induced resistance. The activities of β-1,3-glucanase and peroxidase 48 h after FOL induction were similar to those in control plants. Chitinase activity, however, was higher in the stems of plants inoculated with FOL. A study of the levels of phenolic compounds revealed that cell-wall-bound phenolics were more abundant in plants treated with FOL, especially in stems, while soluble phenolic contents did not differ.     

苯并噻二唑甲酸三氟乙酯诱导仙客来抗枯萎病的研究

为了明确新型化合物苯并噻二唑甲酸三氟乙酯对土传病害的诱导抗病作用,本文研究了该化合物诱导仙客来对枯萎病的抗病效果。通过叶面喷施苯并噻二唑甲酸三氟乙酯可诱导仙客来对枯萎病的抗病效果,在仙客来移栽缓苗后7d叶面喷施该化合物,不同浓度对枯萎病的抗病效果不同,其中100mg.L-1的处理,在诱导后25、30和35d的抗病效果分别为82.90%、58.59%和64.17%;在仙客来成株枯萎病发病初期用该化合物诱导,抗病效果仍可达63.91%,显著高于噁霉灵处理（47.63%）。该研究将为寻求通过叶面喷雾施药防治园艺作物土传病害的新途径起到参考作用。     

草莓枯萎病菌抗多菌灵突变体ZY-R的生理生化特性研究

为明确抗多菌灵草莓枯萎病菌(ZY-R)菌株的生理生化特性,分析其可能的抗性机制,以课题组获得的抗多菌灵达53.91倍的ZY-R菌株及其敏感菌株(ZY)为研究材料,比较了ZY-R与ZY在苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活力及相对渗率等方面的差异。结果表明:以清水和不同浓度多菌灵处理两菌株1.5~24 h后,ZY-R体内的POD和PAL活力始终高于ZY的;敏、抗菌株间POD活力的差异远大于PAL。在处理时间内,PAL活力先上升后下降,6 h达到最高值;而POD活力一直处于上升状态,24 h达到最高值,且敏、抗菌株之间的差异达到最大,25 mg/L条件下ZY-R的POD活力为ZY的2.26倍;此外,与ZY相比,ZY-R可以渗出更多的电解质。     

晋南地区黄瓜、黑籽南瓜死秧的病原菌鉴定及品种抗性研究

经分离、培养对不同菌种培养性状的观察,确定了侵染黄瓜、黑籽南瓜造成死秧的镰刀菌主要为尖镰孢菌黄瓜专化型、尖镰孢菌西瓜专化型、串珠镰刀菌和腐皮镰孢菌4种。经致病性测定,4种镰刀菌均能侵染黄瓜,引起发病造成死秧,可分为强致病类型和中强致病类型。经抗病性鉴定,黑籽南瓜种子只有南瓜4号为耐病品种;黄瓜种子也只有津优31号为耐病品种。     

应用尖孢镰刀菌毒素滤液鉴定大豆品种抗病性初探

引起大豆根腐病的尖孢镰刀菌粗毒素不仅能使大豆根部产生类似病菌侵染引起的症状,而且能抑制大豆幼根的生长和对大豆幼苗具有致萎作用;用毒素滤液处理大豆胚根和幼根,测定大豆不同品种抗病性,用盆栽土壤作对照,试验结果基本一致。利用毒素滤液测定不同品种对大豆根腐病的抗病性,可应用于大量的初筛工作。     

抗香蕉枯萎病菌拮抗菌的鉴定及其定殖

通过逐步提高药物浓度方法,获得了抗利福平400μg/ml、对香蕉枯萎病菌拮抗活性稳定的T2WF和W10标记菌株。初步确定两菌株为芽孢杆菌属细菌。采用灌根接种法,在香蕉苗根际土和香蕉苗体内定殖结果表明,拮抗菌液接种浓度为5×104cfu/ml,两菌株均可从香蕉根际土和香蕉体内分离到,并在香蕉体内定殖和传导。在香蕉根际土、根部、球茎和假茎中,T2WF标记菌的菌量高峰分别出现在接种后5、5、11和3d,分别为707、437、273、117cfu/mg;而W10标记菌的菌量高峰分别出现在接种后5、3、5和5d,分别为784、260、253、110cfu/mg。两菌株在香蕉根际土和香蕉体内1~15d的消长动态均有一个共同的“由升到降”趋势。从数量和数量变动幅度上看,香蕉根部的菌量明显高于茎部的菌量。     

Physiologic races ofFusarium oxysporum f.sp.melonis in the southeastern anatolia region of turkey and varietal reactions to races of the pathogen

Thirty-four isolates ofFusarium oxysporum f.sp.melonis (F.o.m.) obtained from 205 fields in melon-producing areas in the southeastern Anatolia Region of Turkey were identified on the basis of colony morphology and pathogenicity by the root dip method. In this region the mean prevalence of wilt disease was 88.1% and the mean incidence of disease was 47.5%. Physiologic races 0, 1, 2, and 1,2 of the pathogen were determined by their reactions on differential melon cultivars ‘Charentais T,’ ‘Isoblon’, ‘Isovac’ and ‘Margot’ in the greenhouse. Race 1,2, representating 58.8% (20/34) of all isolates, was widely distributed. Of the other pathogenic isolates, eight were identified as race 0, five as race 1, and one as race 2. This is the first report of physiologic races ofF.o.m. in Turkey. Of 44 melon cultivars tested in the greenhouse for resistance toF.o.m. races, 36 were found to be moderately resistant to race 0, 17 were susceptible to race 1,2, 34.1% were highly resistant to race 1, and 52.2% had moderate resistance to race 2. http://www.phytoparasitica.org posting July 16, 2002.