番茄枯萎病菌和颈腐根腐病菌KASP-SNP检测技术的建立

为快速、准确地对番茄枯萎病菌Fusarium oxysporum f. sp. lycopersici(FOL)和番茄颈腐根腐病菌F. oxysporum f. sp. radicis-lycopersici(FORL)进行检测,基于尖孢镰刀菌F. oxysporum多聚半乳糖醛酸外切酶基因pgx4的单核苷酸多态性（single nucleotide polymorphism,SNP）位点,设计FORL、FOL生理小种1(FOL-R1)、2(FOL-R2)和3(FOL-R3)的竞争性等位基因特异性PCR-SNP(kompetitive allele specific PCR-SNP,KASP-SNP)引物,建立番茄颈腐根腐病菌和番茄枯萎病菌KASP-SNP检测技术,并通过与常规PCR比对及ITS与pgx4序列分析对该检测技术的可靠性进行验证。结果显示,在FORL、FOL-R1、FOL-R2和FOL-R3中存在35个变异SNP位点,设计出18对KASP-SNP引物,筛选出FORL＿KASP、FOLrace1＿KASP、FOLrace2＿KASP和FOLrace3＿KASP共4对分型清晰的...     

尖镰孢菌EST-SSR遗传多样性分析及通用性评价

为了解38株尖镰孢菌的遗传多样性并开发可在近缘镰孢菌种中通用的尖镰孢菌EST-SSR标记,利用设计和筛选的18对多态性EST-SSR引物对38株尖镰孢菌和5种近缘镰孢菌进行SSRPCR扩增,经6%非变性聚丙烯酰胺凝胶分离扩增产物,并用NTSYS软件分析供试尖镰孢菌的PCR扩增结果。结果表明,18对EST-SSR标记引物在38株尖镰孢菌中检测到75条多态性条带,多态性比率达92.6%,平均每对引物可扩增4.2条;各菌株间的遗传相似系数介于0.565~0.946之间,平均为0.721;来源于同科寄主植物群体的菌株间的平均遗传相似系数以葫芦科最大,锦葵科最小,依次为葫芦科兰科豆科亚麻科茄科锦葵科。在相似系数为0.756时,供试38株菌有35株按照不同科寄主植物聚为不同的类群,说明尖镰孢菌SSR类群的划分与其寄主来源具有一定的相关性。18对EST-SSR引物在近缘镰孢菌种中均能有效扩增的引物数及通用性比率为10对和55.6%,均显示多态性的引物有2对,占供试引物总数的11.1%。表明尖镰孢菌ESTSSR区域遗传多样性丰富,基于尖镰孢菌EST序列开发镰孢菌通用SSR标记是可行的。     

番茄枯萎病菌和青枯病菌拮抗细菌的评价

为筛选出对番茄枯萎病和青枯病有较好防效的生防菌,采用平板对峙法,以番茄枯萎病菌Fusarium oxysporum和番茄青枯病菌Ralstonia solanacearum为靶标菌,从江苏沭阳、宿迁、溧水及内蒙古海拉尔分离到的2 062株细菌菌株中筛选拮抗菌株,并采用平板对峙法、拮抗菌液灌根法、分子生物学方法进行拮抗物质检测、盆栽试验及种属鉴定。结果表明:从2 062株细菌中共筛选到21株对番茄枯萎病和青枯病具有很强拮抗作用的菌株,均能分泌蛋白酶,具有解磷作用;不能分泌几丁质酶和纤维素酶,仅4株细菌能分泌嗜铁素。拮抗细菌SY290对番茄枯萎病和番茄青枯病防效最高,分别达到74.2%和75.0%,SQ728和LS536次之,但防效均大于60%。结合各菌株形态特征、16S r DNA与gyr-B序列分析结果,菌株SY177、SY290和SQ728鉴定为解淀粉芽胞杆菌Bacillus amyloliquefaciens,菌株LS536为枯草芽胞杆菌B.subtilis。     

尖孢镰刀菌磷脂脂肪酸的遗传多样性分析

为明确尖孢镰刀菌Fusarium oxysporum不同专化型菌株的遗传多样性,利用MIDI微生物脂肪酸鉴定系统分析了来源于5个专化型的27株菌株的磷脂脂肪酸种类和含量,并进行了磷脂脂肪酸聚类分析。结果表明,供试尖孢镰刀菌中共检测到9种脂肪酸,在兰氏距离为25.98时分为3个类群,即主要脂肪酸、微量脂肪酸和偶现脂肪酸,其中主要脂肪酸有16:0、18:0、18:1cis9(ω9)和18:2cis9,12/18:0a,含量共占总磷脂脂肪酸的96.41%;微量脂肪酸有14:0、15:0、16:1cis9(ω7)和20:0,偶现脂肪酸仅有17:1(ω11)。不同专化型菌株的主要脂肪酸仅18:2cis9,12/18:0a存在显著差异,微量脂肪酸除15:0外其余3种均有显著差异。聚类分析结果表明,以供试的全部27株菌株聚类时,其磷脂脂肪酸与寄主专化型间无明显相关性,以寄主来源为同科(葫芦科或茄科)的菌株聚类时,其磷脂脂肪酸与寄主专化型间存在着一定的对应关系。     

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,...     

Defense strategies of pea embryo axes with different levels of sucrose to Fusarium oxysporum and Ascochyta pisi

The aim of this study was to compare the defense responses of embryo axes of Pisum sativum L. cv. Kwestor with different sucrose levels to pathogenic fungi, i.e. systemic acting Fusarium oxysporum f. sp. pisi and locally acting Ascochyta pisi. Embryo axes were cultured on Heller medium for 96 h. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). After inoculation of the pea embryo axes with pathogenic fungi a generally higher concentration of free radicals was detected by electron paramagnetic resonance (EPR), in comparison to non-inoculated embryo axes. The inoculation with F. oxysporum caused stronger generation of free radicals in −Si than in +Si embryo axes. A different response was observed after inoculation with A. pisi; starting from 48 h, the concentration of free radicals in +Si axes was found to be 1.5 times higher than in −Si embryo axes. The values of spectroscopic splitting coefficients for these radicals suggest that they are semiquinone radicals. The EPR method also revealed Mn²⁺ ion accumulation after 24 h of culture. Over time, high levels of these ions were recorded in +Si embryo axes inoculated with F. oxysporum, while in +Si embryo axes inoculated with A. pisi they decreased. Up to 48 h after inoculation with the pathogenic fungi, Mn²⁺ ion levels were higher in +Si embryo axes than in +Sn axes. The activity of superoxide dismutase (SOD, EC 1.15.1.1) increased in +Si embryo axes up to 72 h after inoculation with pathogenic fungi; however, it was generally lower than in +Sn axes. Catalase activity (CAT, EC 1.11.1.6) increased up to 72 h after inoculation with F. oxysporum and the values were higher than in the non-inoculated tissue. Especially high activity of this enzyme was noted in −Si embryo axes after inoculation with either F. oxysporum or A. pisi. Peroxidase activity (POX, EC 1.11.1.7) towards pyrogallol in embryo axes increased during culture; however, it was lower or similar to that in non-inoculated embryo axes. SOD, CAT and POX zymograms showed that the synthesis of new isoforms was induced after inoculation with pathogenic fungi. Peroxidase isozymes detected by the reaction with diaminobenzidine in native PAGE were intensely stained in +Si embryo axes after inoculation with pathogenic fungi. Respiratory activity of the inoculated tissues was considerably higher than in non-inoculated tissues. The respiration rate was generally much higher in +Si than in −Si embryo axes. Growth of −Si embryo axes was more significantly retarded as a consequence of inoculation than that of +Si embryo axes.These results indicate that, depending on the manner of influence of a pathogenic fungus, both similar and differing defensive strategies may be initiated and a raised sugar levels in pea tissues limit the development of F. oxysporum and A. pisi.     

Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

The antifungal glycoalkaloid -tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades -tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (>98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, -tomatine-inducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by two-dimensional gel electrophoresis. A protein with a molecular mass of 64kDa accumulated in the -tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading -tomatine to lycotetraose and tomatidine.     

Cultural characteristics, pathogenicity and genetic diversity of Fusarium oxysporum isolates from tobacco fields in Spain

Several formae speciales of Fusarium oxysporum are capable to produce disease in tobacco plants. Different authors have classified those isolates as a forma specialis or a race within on the basis of the severity of disease and host specificity. Fusarium wilt of tobacco plant in Extremadura (central Spain) tobacco fields have been recorded in the last years and F. oxysporum was isolated from symptomatic plants. The aim of our study was to characterize these F. oxysporum populations. For this purpose, the in vitro spore production and growth and the virulence (severity of disease) have been tested. Although all isolates behaved as pathogen, the virulence of isolates was different. The differences in growth could not be correlated with other characteristics but the two isolates with scarce spore production have also behaved as the weakest pathogen. We have analyzed intergenic spacer (IGS) region polymorphism of ribosomal DNA and random amplified polymorphic DNA (RAPD) markers to assess the genetic diversity within F. oxysporum isolates. These molecular analyses showed two major groups with different physiological capabilities that could reflect two different lineages. One group was characterized by medium–high sporulation, high virulence and the same IGS-RFLP pattern. The other group was more heterogeneous featuring low–medium sporulation and variable virulence and growth. This first experimental approach to pathogen population could be a good starting point for further studies including non-pathogenic isolates and a larger number of pathogen that could clarify if there are two or more genetic lineages.     

西瓜与枯萎病菌非亲和互作相关基因的分离及表达分析

 从整体水平阐明西瓜对西瓜枯萎病菌1号生理小种的抗病分子机制和抗病相关基因的表达特征,以高抗枯萎病菌1号生理小种的西瓜品种“卡红(Calhoun Gray)”为试材,接种西瓜枯萎病菌和蒸馏水的根尖组织作为测验方(Tester)和驱动方(Driver),构建西瓜枯萎病菌胁迫的SSH-cDNA正向文库。利用反向 Northern 斑点杂交技术对文库中克隆进行杂交筛选。随机测序300个阳性克隆,序列比对分析,利用RT-PCR技术分析抗病相关基因的表达特性。259条EST成功测序,167条与已知基因具有较高的同源性,占全部ESTs的65.5%,其中与抗病和防卫相关的有64条23种,占24.7%;卡红对枯萎病菌1号生理小种的抗性相关基因主要涉及抗病信号传导、抗病防卫、转录因子、次生代谢合成和细胞保护等方面;Aquaporin和Peroxidase基因在接种后表达量均增加。Calhoun Gray对枯萎病菌侵染作出的反应是全方位多方面的,抗病相关基因主要集中在系统获得性抗性反应中,获得了一些Calhoun Gray与野生西瓜PI296341在与枯萎病生理小种1互作中差异表达的基因,为深入研究西瓜与枯萎病菌互作的分子机制以及关键基因的功能分析奠定基础。     

中国华北地区瓜类尖孢镰刀菌对咪鲜胺的敏感性及抗药突变株生物学性状研究

为明确中国华北地区瓜类尖孢镰刀菌Fusarium oxysporum Schl.对咪鲜胺的抗药性现状及抗药突变株的生物学性状,采用菌丝生长速率法,分别测定了采自北京、山东、河北等地未使用过咪鲜胺的112株瓜类尖孢镰刀菌对咪鲜胺的敏感性,并通过药剂驯化的方法获得尖孢镰刀菌抗咪鲜胺突变株。结果表明:咪鲜胺对112株瓜类尖孢镰刀菌的平均EC50值为（0.030 1±0.030 4）μg/mL,菌株频率呈连续单峰曲线分布,未发现敏感性明显下降的亚群体,因此,可将该EC50值作为瓜类尖孢镰刀菌对咪鲜胺的敏感基线。药剂驯化共获得7株抗咪鲜胺突变株,其抗性倍数介于6.2~26.8之间;突变株在菌丝生长速率、菌丝干重和致病力等方面均明显低于亲本菌株,差异显著;仅突变株HG13052701-R1的抗药性可以稳定遗传,其他6株抗咪鲜胺突变株的抗药性均不能稳定遗传。室内交互抗性测定结果表明,咪鲜胺仅与戊唑醇之间有交互抗性,与多菌灵和齅霉灵之间均无交互抗性关系。研究表明,瓜类尖孢镰刀菌在药剂选择压下可以形成抗咪鲜胺群体,具有低等抗药性风险。     

番茄枯萎病菌转录因子FolMsn2的生物学功能

为探究转录因子FolMsn2在番茄枯萎病菌Fusarium oxysporum f. sp. lycopersici生长发育及致病过程中的作用,以番茄枯萎病菌野生型菌株4287为材料,利用基因敲除方法获得FolMSN2基因缺失突变体△Folmsn2及回补体菌株△Folmsn2-C,通过测定菌株的生长速率、孢子产量及致病力等表型初步分析番茄枯萎病菌中FolMSN2的生物学功能。结果显示,与野生型菌株4287相比,FolMSN2基因敲除突变体△Folmsn2生长速率显著减慢,菌株产孢量显著下降,仅为野生型菌株4287产孢量的6.7%;外界环境压力胁迫试验结果显示,FolMSN2基因缺失突变体△Folmsn2对渗透压、盐胁迫的敏感性显著提高,而对细胞壁、氧化压力胁迫变得更加耐受。此外,FolMSN2基因缺失突变体△Folmsn2对番茄苗及果实的致病力显著下降,进一步分析发现FolMSN2基因缺失影响其对玻璃纸的穿透能力;亚细胞定位结果表明,FolMsn2蛋白定位于细胞核内。表明FolMsn2参与调控番茄枯萎病菌的营养生长、无性繁殖以及对不同环境胁迫的应答过程,其可能通过影响菌丝对寄主的穿透能...     

Isolation of differentially expressed genes during interactions between tomato cells and a protective or a non-protective strain of Fusarium oxysporum

Pathogenic isolates of Fusarium oxysporum applied on a non-host plant species, as soil-borne non-pathogenic isolates, are able to protect this plant against pathogenic strains inducing wilts. Several modes of action contribute to the biocontrol activity of these protective strains; however the genetic basis of the biocontrol mechanisms is far from being understood. The aim of this study was to identify genes involved in biocontrol activity of F. oxysporum using an original model made of Fom24, a strain protective on tomato and its mutant rev157 which has lost its protective capacity. A Rapid Subtractive Hybridization (RaSH) approach was chosen to identify genes up-regulated in the protective or in the non-protective interaction when germinated conidia of either Fom24 or rev157 are confronted to tomato cell cultures. A total of 86 up-regulated sequences were generated, 42 and 44 from the protective and the non-protective interaction respectively. Homology searches led to identification of both plant and fungal genes that were grouped according to their putative functions. Among plant genes, those involved in plant response to stresses were the most abundant. Expression profiles of genes homolog to a basic endochitinase, a ferredoxine-NADP(H) reductase (FNR), an ATP synthase and the RPM1-interacting protein 4 (RIN4) were confirmed by Northern blotting. A large proportion of fungal sequences were encoding genes of unknown function; among other, those involved in response to oxidative stress and a gene putatively encoding an enolase are the most promising to further study their potential role in the protective interaction between F. oxysporum and tomato.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

大蓟总黄酮对尖孢镰刀菌甜瓜专化型生长、生理的影响及其田间防治效果

为探究大蓟总黄酮对尖孢镰刀菌甜瓜专化型Fusarium oxysporum f. sp. melonis的抑制效果,采用微波超声法提取大蓟种子中的总黄酮,并测定其对其尖孢镰刀菌甜瓜专化型生长、生理指标的影响及其田间防治效果。结果显示,大蓟总黄酮浓度为10 mg/mL时,对尖孢镰刀菌甜瓜专化型的菌丝生长抑制率和孢子萌发抑制率达到100.0%,菌丝畸变、断裂;尖孢镰刀菌甜瓜专化型OD650 nm和pH最小,分别为0.3和5.5;其电导率是对照组1.2倍。不同浓度大蓟总黄酮处理下尖孢镰刀菌甜瓜专化型呼吸强度、超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性均呈现先上升后下降的趋势,羧甲基纤维素酶和β-葡萄糖苷酶活性降低,浓度为10 mg/mL大蓟总黄酮处理尖孢镰刀菌甜瓜专化型60 h后,呼吸强度、SOD和CAT活性均降至最低,分别为7.8 mgCO2·cm-2·h-1、22.9 U/mL和20.8 U/mL。田间防治结果表明,浓度为10 mg/mL大蓟总黄酮处理下,甜瓜枯萎株数最少,防治效果最佳,为93.5%。表明大蓟总黄酮能够有效地抑制尖孢镰刀菌甜瓜专化型生长,具有进一步发展为绿色新型植物源尖孢镰刀菌甜瓜专化型防治剂的能力。     

内生真菌HND5菌株抗香蕉枯萎病菌相关非核糖体多肽合成酶基因簇的鉴定

为明确帚枝霉属内生真菌Sarocladium brachiariae HND5菌株具体的抑菌活性物质,在全基因组测序数据的基础上,利用生物信息学对抑菌活性物质合成基因簇进行定位,通过聚类分析和系统发育树分析对基因簇合成产物进行鉴定,并通过基因缺失突变构建及代谢组分析确定具体的抑菌活性物质。结果显示,HND5菌株共含有7个非核糖体多肽合成酶(non-ribosomal peptide synthetase,NRPS)基因簇,聚类分析结果表明基因簇29合成产物为噬铁素,基因簇30合成产物为类环孢霉素类抗菌多肽;系统发育树结合生物信息学结果显示基因簇30合成产物与已知环孢霉素结构差异大,为一个新型非核糖体多肽;将基因簇30 NRPS基因缺失突变后,HND5菌株丧失抑菌活性;同野生型菌株相比,基因簇30 NRPS基因缺失突变体缺失一个分子量为887.54 Da的多肽类物质。表明HND5菌株的抑菌活性物质为一个分子量为887.54 Da的新型类环孢霉素非核糖体多肽,基因簇30负责该多肽的生物合成。     

Elucidation of defence responses and signalling pathways induced in Arabidopsis thaliana following challenge with Phytophthora cinnamomi

Arabidopsis thaliana (Arabidopsis) Col-0 was inoculated with Phytophthora cinnamomi to assess the interaction and defence responses involved. Pathogen ingress and asexual reproduction occurred on root tissue but not leaf tissue. The colonisation of root tissue did not cause disease symptoms or plant death, indicating that Arabidopsis Col-0 was tolerant of the infection. The induction of several plant defence responses including the expression of defence-related genes were found, with differences displayed between inoculated root and leaf tissue. Arabidopsis defence-related gene mutant/over-expressing lines were also inoculated with P. cinnamomi but none of the lines tested exhibited a marked increase in susceptibility to the pathogen.     

烟粉虱MED隐种蜕皮激素受体基因cDNA克隆、转录与组织表达

蜕皮激素受体(ecdysone receptor,Ec R)是调控昆虫蜕皮、变态和生殖等过程中基因表达的重要调控因子。为研究Ec R在烟粉虱Bemisia tabaci生长发育中的作用,利用RT-PCR和RACE等技术扩增了烟粉虱MED隐种Ec R基因的c DNA全长序列,并利用实时荧光定量PCR技术检测其在烟粉虱不同发育时期相对表达量的变化。Bt Ec R c DNA序列全长2 844 bp,含有一个1 518 bp的开放阅读框,共编码505个氨基酸,预测蛋白分子量为56 k D,等电点为6.43,含有特殊结构功能域:DNA结合域(DBD_Ec R)和配体结合域(LBD_Ec R),该序列编码的蛋白质序列与其它已报道的昆虫Ec R蛋白序列具有很高的相似性,命名为Bt Ec R(Gen Bank登录号:KR534774)。Bt Ec R在MED隐种若虫、雌成虫各发育时期均有表达,若虫期在伪蛹前期达到最高值,成虫期呈现先升高后降低的趋势,且在羽化后第7天达到最高值,约为羽化第1天的23倍。研究结果为揭示Bt Ec R在烟粉虱整个生长发育过程中的作用提供了重要依据。