西瓜砧木种传尖孢镰刀菌粗毒素研究

采用乙酸乙酯和乙醚萃取法提取2株西瓜砧木种传镰刀菌粗毒素中的镰刀菌酸,并用高效液相色谱(HPLC)进行了定量分析;测定了镰刀菌酸粗毒素对西瓜砧木幼苗的致萎蔫作用.结果表明,2株种传尖孢镰刀菌粗毒素中都可检测到镰刀菌酸,质量浓度分别为69.9μg/mL和842.3 μg/mL.用2株种传尖孢镰刀菌粗毒素处理西瓜砧木幼苗根部能引起幼苗枯萎,且西瓜砧木幼苗的萎蔫程度随着粗毒素中镰刀菌酸浓度的升高和处理时间的延长而加重.关于西瓜砧木种传尖孢镰刀菌粗毒素中镰刀菌酸含量的检测及其引起幼苗萎蔫为国内首次报道.     

镰刀菌对大蒜根系分泌物的敏感性与其致病力相关分析

试验采用菌丝生长速率法测定了大蒜根系分泌物对3种供试植物病原镰刀菌的抑菌活性, 并进一步分析了18株从腐烂蒜瓣上分离的尖孢镰刀菌和12株从小麦赤霉病样分离的禾谷镰刀菌对大蒜根系分泌物的敏感性及致病力之间的关系。研究结果表明, 大蒜根系分泌物对供试镰刀菌均具有抑制活性, 但从腐烂蒜瓣上分离的尖孢镰刀菌对根系分泌物的敏感性低于其他菌株。致病力分析结果表明, 供试的18株尖孢镰刀菌均能使蒜瓣发病, 但致病力与其对根系分泌物的敏感性无明显相关性; 供试的禾谷镰刀菌中对根系分泌物不敏感的4株菌株能侵染蒜瓣, 但敏感性高的菌株不能侵染蒜瓣, 且根系分泌物对禾谷镰刀菌的抑制率与禾谷镰刀菌致病力之间呈显著的负相关。这表明大蒜根系分泌抑菌物质是根系抵御镰刀菌侵染的重要机制, 但一些菌株能对根系分泌物产生抗性, 从而侵染大蒜。综上所述, 大蒜根系分泌物对镰刀菌具有抑制活性, 可以利用大蒜和其他作物间作或轮作控制镰刀菌枯萎病的发生和蔓延, 但长期利用大蒜轮作或间作控制土传病害可能面临镰刀菌对大蒜根系分泌物产生抗性, 导致防效降低的风险。     

烟草镰刀菌根腐病病菌致病粗毒素的研究

本试验测定了经尖孢镰刀菌（Fusarium oxysporum）和茄病镰刀菌（F.solani）粗毒素浸泡后烟草幼苗抗病相关酶系的活性及细胞膜透性的变化,以研究毒素对烟草的致病作用。结果表明,引起烟草根腐病的尖孢镰刀菌和茄病镰刀菌的毒素属于非寄主专化性毒素（NHST）。烟草幼苗经毒素处理12～60 h,其过氧化物酶（peroxidase, POD）和多酚氧化酶（polyphenol oxidase, PPO）的活性都迅速升高,随后下降,幼苗出现不同程度的萎蔫。50%的毒素液处理幼苗根部54 h后,根部细胞膜的损伤率比对照均增加了80%,叶片的细胞膜损伤率比对照增加了90%。另外毒素处理能抑制烟草种子胚根的生长,使幼苗萎蔫,与病原菌直接接种表现相同,说明毒素是病原菌侵染烟草导致发病的一个重要因子。     

大白菜枯萎病病原镰刀菌种类的初步研究

 2014~2016年,由于栽培管理及重茬等原因,大白菜枯萎病在我国大面积发生,造成了巨大的经济损失。为了明确引起大白菜枯萎病的病原菌,本课题组从山东、内蒙古、河北、甘肃等大白菜主产区采集了具有典型枯萎病症状的病样,并对样品中的病原菌进行了分离和鉴定。形态学鉴定结果表明:分离物分别具有尖孢镰刀菌 (Fusarium oxysporum)、茄病镰刀菌 (F. solani) 和木贼镰刀菌 (F. equiseti)的形态学特征。柯赫氏法则验证结果表明:3种病原菌均能使大白菜发病,且发病症状与田间症状一致。此外,基于病原菌的rDNA-ITS和mt SSU序列的测序比对,3种病原菌与尖孢镰刀菌、茄病镰刀菌和木贼镰刀菌的同源性分别达99%~100%,这与形态学鉴定结果相一致。尖孢镰刀菌引起白菜枯萎病为国内首次报道,而茄病镰刀菌和木贼镰刀菌引起白菜枯萎病为国内外首次报道。     

丛枝菌根真菌对接种尖孢镰刀菌后黄瓜 根系次生代谢物的影响

采用灌根法对4周龄的黄瓜幼苗接种尖孢镰刀菌Fusarium oxysporum f.sp.cucumerinum,研究了不同取样时期AM真菌Glomus versiforme对植株根系黄酮和总酚含量及叶片中POD和PPO酶活性的影响.结果表明:菌根化植株可通过生物量的补偿和体内次生代谢物含量的改变来提高抗病性.即接种F.oxysporum f. sp. cucumerinum的第9天,菌根化植株根系和地上部生物量分别比对照高出50%和31.7%,此时,对照发病率达15%,而菌根化植株生长始终正常;在病原菌的侵入初期和后期,菌根化植株根系中黄酮和总酚含量高于对照;在植株发病期,菌根化植株仍可通过根系中含量相对较低的黄酮和总酚来抵御病原菌的侵害;与对照相比,抗病性较强的菌根化植株叶片中POD酶活性变化较为缓和,且PPO酶活性一直较高.     

河北廊坊大豆枯萎病病原镰刀菌的分子鉴定

 为明确河北廊坊中国农科院植保所试验基地大豆孢囊线虫病田内大豆枯萎病病原镰刀菌的种类,对362份罹病枯萎大豆植株进行病原真菌分离,得到335株真菌;使用镰刀菌通用引物鉴定出镰刀菌(Fusarium spp.) 279株,占分离菌株83.3%;镰刀菌特异性引物、测序等分子生物学技术结合形态学特征进一步鉴定镰刀菌种类,鉴定出尖孢镰刀菌(F. oxysporum)189株,占分离菌株56.4%;茄病镰刀菌(F. solani)67株占20.0%、禾谷镰刀菌(F. graminearum)16株占4.8%、木贼镰刀菌(F. equiseti)3株、层出镰刀菌(F. proliferaum)2株、燕麦镰刀菌(F. avenaceum)和厚孢镰刀菌(F. chlamydosporum)各1株;致病性测试结果表明数量最多的尖孢镰刀菌(F. oxysporum)中约92.8%菌株具有不同程度的致病力;这些结果表明该试验基地大豆枯萎病的优势病原菌为尖孢镰刀菌(F. oxysporum);研究结果可为大豆枯萎病的防治提供科学依据,并为大豆孢囊线虫与尖孢镰刀菌复合侵染大豆的研究奠定基础。     

非无菌操作下分离尖孢镰刀菌的培养基

 尖孢镰刀菌(F.oxysporum)是一类非常重要的土传病原真菌,寄主范围广,可引起100多种植物的萎蔫型病害。     

西瓜枯萎病菌镰刀菌酸对西瓜苗作用机制的初步探讨

 镰刀菌酸是由镰刀菌属中那些能引起多种植物萎蔫的病原菌所产生的一种非特异性毒素,从发现至今已有60多年了,但关于它在病程中作用问题的研究还不多,而且意见也不一致。     

尖孢镰刀菌侵染对马铃薯光合效率和叶绿素荧光参数影响

为研究枯萎病对马铃薯光合特性的影响,在幼苗期接种尖孢镰刀菌Fusarium oxysporum后,统计抗病品种陇薯10号和感病品种新大坪的病情指数,并测定叶绿素含量、光合及荧光参数。结果表明,尖孢镰刀菌侵染30 d后马铃薯表现出枯萎病症状,叶绿素含量显著降低,其中叶绿素a含量降幅最大,抗病品种陇薯10号比对照降低9.64%,感病品种新大坪比对照降低14.24%。尖孢镰刀菌侵染后马铃薯光合效率显著降低,侵染30 d后,抗病品种陇薯10号净光合速率比对照降低39.56%,感病品种新大坪比对照降低47.13%。病株的光响应曲线参数光补偿点、暗呼吸速率和表观量子效率都显著提高;而光饱和点和最大净光合速率都显著低于对照,表明尖孢镰刀菌侵染缩小了马铃薯对光能的利用有效范围。病株CO2响应曲线参数CO2饱和点、最大净光合速率和羧化效率分别显著低于对照;病株CO2补偿点和光呼吸速率反而升高,说明碳同化过程受到尖孢镰刀菌的限制。暗适应下的初始荧光、最小荧光、最大荧光、PSII最大光化学效率、光适应下PSII最大光化学效率及实际光化学效率、光化学猝灭系数、非光化学猝灭系数和光合电子传递速率均显著低于对照,...     

基于RPA恒温扩增技术对大豆尖孢镰刀菌的快速检测

尖孢镰刀菌侵染大豆引起枯萎病,导致大豆严重减产。为实现对大豆上尖孢镰刀菌的快速检测,基于重组酶聚合酶扩增（RPA）技术开展了试验研究,根据大豆尖孢镰刀菌的特异基因设计了正反引物各3个,并通过两两组合筛选出最佳引物对,实现了在恒温（39℃）条件下,反应15～20 min,即可检测90 fg/μL以上浓度的尖孢镰刀菌,且与大豆疫霉菌、大豆灰斑菌等均无交叉反应。本研究建立的大豆尖孢镰刀菌检测法,相较于传统PCR法和实时定量PCR法具有快速、便捷、即时观察的特点,为该病原的快速检测提供了新的技术手段。     

常熟地区蚕豆枯萎病病原菌鉴定及其致病力初探

2000、2001年在江苏省常熟地区采集了有典型枯萎症状的蚕豆标样各50个,获94个镰刀菌单孢菌株。经鉴定分别属于尖孢镰孢(Fusarium axysporum)、燕麦镰孢(F.avenaceum)、串珠镰孢(F.moniliforme )、木贼镰孢(F.equiseti)、三线镰孢(F.tricinctum)、禾谷镰孢(F.graminearum)和茄镰孢(F.solani),其中尖孢镰孢、燕麦镰孢、串珠镰孢、木贼镰孢为该地区蚕豆镰刀菌枯萎病的主要病原菌。测定了48个镰刀菌菌株对蚕豆的致病力,尖孢镰孢、木贼镰孢、串珠镰孢和燕麦镰孢对蚕豆的致病力都较强。用蚕豆枯萎病菌和棉花枯萎病菌交叉接种棉花和蚕豆,结果表明两者存在着较强的交互侵染能力。     

尖孢镰刀菌的遗传多态性

 尖孢镰刀菌是重要的植物维管束病原真菌。近年来,有关该菌的遗传多态性研究报道很多,本文着重综述了利用营养体亲和群、DNA多态性技术研究尖孢镰刀菌专化型、小种及其相互关系等方面的进展,同时介绍了对棉花枯萎病菌、瓜类枯萎病菌及尖孢镰刀菌小种起源等的研究概况。     

Use of a Nitrate-Nonutilizing Mutant and Selective Media to Examine Population Dynamics of Fusarium oxysporum f. sp. spinaciae in Soil

ABSTRACT Determining the population density of the spinach wilt pathogen Fusarium oxysporum f. sp. spinaciae in soil with conventional Fusarium-selective media is quite difficult because nonpathogenic strains of F. oxysporum also grow on those media and are indistinguishable from the pathogen. Therefore, a nitrate-nonutilizing (nit) mutant of the pathogen and corresponding selective media were tested in an experimental approach to determine the population density of the pathogen. Colony forming units of the pathogen were countable after soil-dilution plating onto nit mutant-selective media MMCPA, CMP, and CGMBP. Colony forming units of wild-type Fusarium spp. were countable using a wildtype Fusarium-selective medium, GMBP. By combining nit mutant- and wild-type-selective media, the population densities of pathogenic and nonpathogenic F. oxysporum in the same soil could be measured selectively. This method was useful in studying population dynamics of the pathogen after different soil treatments. Soil disinfested with hot water or chloropicrin was amended with the nit mutant pathogen, and subsequent changes in population densities of the pathogen were compared with those in nontreated field soil. The pathogen rapidly proliferated in disinfested soil and wilt developed faster than in nontreated soil. When a nonpathogenic isolate of F. oxysporum was added at high density to sterilized soil prior to the pathogen, growth of the pathogen was greatly suppressed. Nonpathogenic F. oxysporum could not, however, reduce the density of preexisting pathogen.     

Disease Development Following Infection of Tomato and Basil Foliage by Airborne Conidia of the Soilborne Pathogens Fusarium oxysporum f. sp. radicis-lycopersici and F. oxysporum f. sp. basilici

ABSTRACT Fusarium oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato, and F. oxysporum f. sp. basilici, the causal agent of Fusarium wilt in basil, are soilborne pathogens capable of producing conspicuous masses of macroconidia along the stem. The role of the airborne propagules in the epidemics of the disease in tomato plants was studied. In the field, airborne propagules of F. oxysporum f. sp. radicis-lycopersici were trapped with a selective medium and their prevalence was determined. Plants grown in both covered and uncovered pots, detached from the field soil, and exposed to natural aerial inoculum developed typical symptoms (82 to 87% diseased plants). The distribution of inoculum in the growth medium in the pots also indicated the occurrence of foliage infection. In greenhouse, foliage and root inoculations were carried out with both tomato and basil and their respective pathogens. Temperature and duration of high relative humidity affected rate of colonization of tomato, but not of basil, by the respective pathogens. Disease incidence in foliage-inoculated plants reached 75 to 100%. In these plants, downward movement of the pathogens from the foliage to the crown and roots was observed. Wounding enhanced pathogen invasion and establishment in the foliage-inoculated plants. The sporulation of the two pathogens on stems, aerial dissemination, and foliage infection raise the need for foliage protection in addition to soil disinfestation, in the framework of an integrated disease management program.     

In planta and soil quantification of Fusarium oxysporum f. sp. ciceris and evaluation of Fusarium wilt resistance in chickpea with a newly developed quantitative polymerase chain reaction assay

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.     

Sporulation of Fusarium oxysporum f. sp. lycopersici on Stem Surfaces of Tomato Plants and Aerial Dissemination of Inoculum

ABSTRACT Plants exhibiting symptoms of wilt and xylem discoloration typical of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici were observed in greenhouses of cherry tomatoes at various sites in Israel. However, the lower stems of some of these plants were covered with a pink layer of macroconidia of F. oxysporum. This sign resembles the sporulating layer on stems of tomato plants infected with F. oxysporum f. sp. radicis-lycopersici, which causes the crown and root rot disease. Monoconidial isolates of F. oxysporum from diseased plants were assigned to vegetative compatibility group 0030 of F. oxysporum f. sp. lycopersici and identified as belonging to race 1 of F. oxysporum f. sp. lycopersici. The possibility of coinfection with F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was excluded by testing several macroconidia from each plant. Airborne propagules of F. oxysporum f. sp. lycopersici were trapped on selective medium in greenhouses in which plants with a sporulating layer had been growing. Sporulation on stems was reproduced by inoculating tomato plants with races 1 and 2 of F. oxysporum f. sp. lycopersici. This phenomenon has not been reported previously with F. oxysporum f. sp. lycopersici and might be connected to specific environmental conditions, e.g., high humidity. The sporulation of F. oxysporum f. sp. lycopersici on plant stems and the resultant aerial dissemination of macroconidia may have serious epidemiological consequences. Sanitation of the greenhouse structure, as part of a holistic disease management approach, is necessary to ensure effective disease control.     

Zinc Improves Biocontrol of Fusarium Crown and Root Rot of Tomato by Pseudomonas fluorescens and Represses the Production of Pathogen Metabolites Inhibitory to Bacterial Antibiotic Biosynthesis

ABSTRACT Crown and root rot of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici is an increasing problem in Europe, Israel, Japan, and North America. The biocontrol agent Pseudomonas fluorescens strain CHA0 provides only moderate control of this disease. A one-time amendment of zinc EDTA at 33 mug of Zn(2+)/ml to hydroponic nutrient solution in soilless rockwool culture did not reduce disease when used alone, but did reduce disease by 25% in the presence of CHA0. In in vitro studies with the pathogen, zinc at concentrations as low as 10 mug/ml abolished production of the phytotoxin fusaric acid, a Fusarium pathogenicity factor, and increased production of microconidia over 100-fold, but reduced total biomass. Copper EDTA at 33 mug of Cu(2+)/ml had a similar effect as zinc on the pathogen in vitro; it reduced disease when used alone, and increased the biocontrol activity of CHA0 in soilless culture. Ammonium-molybdate neither improved the biocontrol activity of CHA0 nor affected production of fusaric acid or microconidia. Strain CHA0 did not degrade fusaric acid. Fusaric acid at concentrations as low as 0.12 mug/ml repressed production by CHA0 of the antibiotic 2,4-diacetylphloroglucinol, a key factor in the biocontrol activity of this strain. Production of pyoluteorin by CHA0 was also reduced, but production of hydrogen cyanide and protease was not affected, suggesting that fusaric acid affects biosynthesis at a regulatory level downstream of gacA and apdA genes. Fusaric acid did not affect the recovery of preformed antibiotics nor did it affect bacterial growth even at concentrations as high as 200 mug/ml. When microbial meta-bolite production was measured in the rockwool bioassay, zinc amendments reduced fusaric acid production and enhanced 2,4-diacetylphloro-glucinol production. We suggest that zinc, which did not alleviate the repression of antibiotic biosynthesis by fusaric acid, improved biocontrol activity by reducing fusaric acid production by the pathogen, which resulted in increased antibiotic production by the biocontrol agent. This demonstrates that pathogens can have a direct negative impact on the mechanism(s) of biocontrol agents.     

Interactions Between Meloidogyne artiellia, the Cereal and Legume Root-Knot Nematode, and Fusarium oxysporum f. sp. ciceris Race 5 in Chickpea

ABSTRACT In the Mediterranean Basin, Fusarium oxysporum f. sp. ciceris and the root-knot nematode Meloidogyne artiellia coinfect chickpea. The influence of root infection (after inoculation with 20 nematode eggs and second-stage juveniles per gram of soil) by two M. artiellia populations, from Italy and Syria, on the reaction of chickpea lines and cultivars with partial resistance to Fusarium wilt (CA 252.10.1.OM, CA 255.2.5.0, CPS 1, and PV 61) and with complete resistance to F. oxysporum f. sp. ciceris race 5 (CA 334.20.4, CA 336.14.3.0, ICC 14216 K, and UC 27) was investigated under controlled conditions. In genotypes with partial resistance, infection by M. artiellia significantly increased the severity of Fusarium wilt, irrespective of the fungal inoculum density (3,000 or 30,000 chlamydospores per gram of soil), except in cultivar CPS 1 at the lower fungal inoculum density. In genotypes with complete resistance to Fusarium wilt, infection by M. artiellia overcame the resistance to F. oxysporum f. sp. ciceris race 5 in CA 334.20.4 and CA 336.14.3.0 but not in ICC 14216 K, irrespective of the fungal inoculum density, and overcame the resistance in UC 27 only at the higher inoculum density. Infection by the nematode significantly increased the number of propagules of F. oxysporum f. sp. ciceris race 5 in root tissues of genotypes with complete resistance to Fusarium wilt, compared with roots that were not inoculated with the nematode, irrespective of the fungal inoculum density, except in ICC 14216 K, in which this effect occurred only at the higher inoculum density. Reproduction of an M. artiellia population from Syria in the absence of F. oxysporum f. sp. ciceris race 5 was significantly higher than that of a population from Italy in all tested chick-pea genotypes except ICC 14216 K. However, there was no significant difference between the reproduction rates of the two nematode populations in plants infected with F. oxysporum f. sp. ciceris race 5, irrespective of the fungal inoculum density and the reaction of the genotypes to the fungus.     

Induced Resistance by Penicillium oxalicum Against Fusarium oxysporum f. sp. lycopersici: Histological Studies of Infected and Induced Tomato Stems

ABSTRACT Tomato (Lycopersicon esculentum) plants of 'Lorena' were induced with a conidial suspension (10(7) conidia per ml) of Penicillium oxalicum before inoculation with Fusarium oxysporum f. sp. lycopersici, the wilt pathogen. Histological changes occurred in plants under both growth chamber and glasshouse culture conditions and there was a reduction of disease severity. In noninduced plants, the pathogen produced almost a complete loss of cambium (75 to 100% reduction), an increase in the number of bundles, and a decrease in the number of xylem vessels (20% reduction), in which the diameter also was reduced by 20 to 30% in hypocotyls and epicotyls. The percentage of vessels colonized by F. oxysporum f. sp. lycopersici was positively correlated to the area under the disease progress curve (AUDPC). However, plants induced with P. oxalicum showed less disease, did not lose the cambium, had a lower number of bundles, and had less vascular colonization by F. oxysporum f. sp. lycopersici (35 to 99%). These effects also were observed in 'Precodor', which is susceptible to races 1 and 2 of F. oxysporum f. sp. lycopersici, and partially in 'Ramón', which is resistant to both races. Renewed or prolonged cambial activity that led to the formation of additional secondary xylem could be one of the reasons for disease reduction in P. oxalicum-induced tomato plants.