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.     

Genetic and pathogenic diversity within Ascochyta rabiei(Pass.) Lab. populations in Pakistan causing blight of chickpea (Cicer arietinum L.)

Pathogenic and genetic diversity in Ascochyta rabiei populations in Pakistan were evaluated. Biological pathotyping of 130 A. rabiei isolates (obtained from hierarchically collected samples) was conducted on a set of three chickpea differentials, i.e. ILC 1929 (susceptible), ILC 482 (tolerant) and ILC 3279 (resistant), under controlled conditions. Disease severity data were recorded 12 days after inoculation. Statistical analysis grouped the isolates into three pathotype classes. Four isolates belonged to pathotype I (least aggressive), 79 isolates to pathotype II (medium aggressive) and 47 isolates to pathotype-III (highly aggressive).Genetic analysis was performed using RAPDs and oligonucleotide fingerprinting, where Hinf I-digested DNA was hybridized to the³²P-endlabeled oligonucleotide probes (CAA)₅, (GAA)₅, (GA)₈, (CA)₈and (GATA)₄. Dendrograms produced by cluster analysis discriminated 46 genotypes in the A. rabiei population of Pakistan. Genetic distances and relatedness between isolates were calculated. At a genetic distance of 0.3, genotypes were divided into six distinct genotype groups A, B, C, D, E and F containing 16, 11, 2, 5, 5 and 7 isolates, respectively. Most of the genotypes were area specific or predominated in certain areas but did not belong to a distinct pathotype, while most of the aggressive isolates (pathotype III) occurred in Northern Punjab and in the North Western Frontier Province.     

Glomus intraradices, Pseudomonas alcaligenes, and Bacillus pumilus: effective agents for the control of root-rot disease complex of chickpea (Cicer arietinum L.)

The effects of Glomus intraradices, Pseudomonas alcaligenes and Bacillus pumilus on the root-rot disease complex caused by the root-knot nematode Meloidogyne incognita and the root-rot fungus Macrophomina phaseolina in chickpea was assessed by quantifying differences in the shoot dry mass, pod number, nodulation, and shoot content of chlorophyll, nitrogen, phosphorus and potassium. Inoculation of plants with G. intraradices, P. alcaligenes and B. pumilus alone and in combination significantly increased shoot dry mass, pod number, and content of chlorophyll, nitrogen, phosphorus and potassium in plants inoculated with pathogens over that in the uninoculated control plants. P. alcaligenes caused a greater increase in shoot dry mass, pod number, chlorophyll, nitrogen, phosphorus and potassium in plants with pathogens than did G. intraradices or B. pumilus. Combined application of G. intraradices, P. alcaligenes and B. pumilus to plants inoculated with pathogens caused a greater increase in shoot dry mass, pod number, nitrogen, phosphorus, and potassium than did an application of P. alcaligenes plus B. pumilus or of G. intraradices plus B. pumilus. Root colonization by G. intrardices was high when used alone, while inoculation with the pathogens reduced root colonization by G. intraradices. In the presence of P. alcaligenes and B. pumilus, root colonization by G. intraradices increased. In plants inoculated with just one antagonist, P. alcaligenes reduced galling and nematode multiplication the most, followed by G. intraradices, then B. pumilus. The greatest reduction in galling, nematode multiplication and root-rot was observed when both bacterial species and G. intraradices were applied together.     

青海东部小麦条锈菌转主寄主小檗资源调查与鉴定

 青海省是中国小麦条锈病的重要流行区。研究证实小檗是小麦条锈菌的转主寄主。然而,关于青海省分布的小檗能否作为小麦条锈菌转主寄主鲜有报道。本研究对青海东部的小檗资源进行调查,并对该地区小檗能否作为小麦条锈菌转主寄主进行了室内人工鉴定。结果表明:有10种小檗在青海东部常见且分布广泛,其中匙叶小檗、置疑小檗、松潘小檗、甘肃小檗和近似小檗,是小麦条锈菌的转主寄主小檗的新种类。本研究对进一步研究青海东部条锈菌有性生殖与毒性变异和小麦条锈病的流行奠定了基础。     

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...     

甘蓝枯萎病菌1号和2号生理小种的快速检测与鉴定

 甘蓝枯萎病菌生理小种传统鉴定方法费时费力,不能满足生产的要求,因此需要建立一种快速、可靠的分子检测技术。本研究在甘蓝枯萎病菌1号和2号生理小种基因组测序的基础上,通过比较基因组学方法筛选1、2号生理小种各自的特异基因片段并设计引物,并分别以10个甘蓝枯萎病菌1号生理小种菌株、2个2号生理小种菌株、7个尖孢镰刀菌其他专化型菌株及4个外围菌株DNA为模板进行常规PCR扩增,筛选出甘蓝枯萎病菌1号和2号生理小种特异性引物,同时引入尖孢镰刀菌通用引物W106R/W106S,建立起一步三重PCR检测甘蓝枯萎病菌1、2号生理小种的分子检测技术。结果表明,该分子检测技术实现了在一次PCR反应中快速、准确地同步检测出甘蓝枯萎病菌DNA、罹病甘蓝组织和土壤中的甘蓝枯萎病菌1号和2号生理小种,对检测甘蓝植株是否感染枯萎菌及甘蓝种植区土壤是否受到枯萎菌的污染有实用价值。     

基于URP-PCR多态性片段的苦瓜枯萎病菌特异性检测技术的建立

 通过URP(Universal Rice Primers)-PCR分析尖孢镰孢菌苦瓜专化型基因组DNA扩增片段多态性,筛选检测尖孢镰孢菌苦瓜专化型的特异性引物,并建立了基于该引物的PCR检测方法。结果表明,特异性引物为FOMM-SPF/FOMM-SPR, PCR检测体系为25 SymbolmAL,包括2SymboltB Green Taq Master Mix 12.5 SymbolmAL,10 mmol·L^-1 的上下游引物各1 SymbolmAL,模板DNA 1 SymbolmAL,灭菌去离子水补足至25 SymbolmAL;PCR程序为95℃预变性3 min,94℃变性15 s,57℃退火30 s,72℃延伸20 s,共30个循环,循环结束后72℃延伸5 min;特异性扩增片段大小294 bp,检测灵敏度为2 ng·μL^-1 DNA或50个孢子·500 mg^-1土壤。该引物及其检测方法对尖孢镰孢菌苦瓜专化型的检测特异性好、灵敏度高,可以从土壤和植物样品中快速准确地检测出苦瓜枯萎病菌,无需病原菌的分离培养和致病性检测,对苦瓜枯萎病的早期诊断和预警及有效防控具有重要的指导意义。     

The basis of host recognition in Fusarium oxysporum f. sp. lycopersici

Filtrates from shake-cultures of Fusarium oxysporum f. sp. lycopersici race 1, concentrated to 20% of the original volume, caused cell death in tomato leaf protoplasts from near-isogenic lines corresponding to the compatible cultivar/race reactions of whole plants. Maximum activity was found in late log phase cultures on Czapek-Dox supplemented with 2% casamino acids. Selective toxicity was associated only with the protein fraction of the culture filtrate. LD₅₀ values for susceptible Ace and Moneycross to F. oxysporum f. sp. lycopersici race 1 culture filtrates were 1·92 and 0·36 μg protein ml⁻¹. Corresponding values for cvs Royal Ace and MM161, each containing the I-gene conferring resistance to race 1, were >350. Culture filtrates from F. oxysporum f. sp. lycopersici race 2 gave LD₅₀ values of 2·34 and 2·08 μg protein ml⁻¹ on cvs Ace and Royal Ace, both susceptible to race 2. The LD⁵⁰ of cv. Ace to a non-pathogenic isolate of F. xysporum f. sp. lycopersici was > 350. Culture filtrates from non-host formae of F. oxysporum were 9–149-fold less toxic on cv. Ace. Protoplasts from Pisum sativum, Lactuca sativa, Zea mays, Gossypium barbadense and Solanum melongena, all non-hosts of F. oxysporum f. sp. lycopersici, were 6–175 times less sensitive to F. oxysporum f. sp. lycopersici filtrates than susceptible tomato. The putative toxins lycomarasmin and fusaric acid showed no differential toxicity to I⁺ and I⁻ tomato protoplasts. The results are discussed in the wider context of host-pathogen interaction in which specificity is considered as the recognition of susceptibility by a proteinaceous toxic metabolite of the pathogen. This hypothesis is further extended to include the specificity of F. oxysporum formae and races.     

陕西省小麦白粉菌群体毒性结构及遗传多样性

 为明确陕西省小麦白粉菌群体的毒性频率和遗传多样性,利用34个含有已知抗白粉病基因的小麦品种(系)和5对多态性ISSR分子标记,分别对2016年渭南、西安、咸阳、宝鸡、汉中和安康等6市的15个乡镇160个小麦白粉菌单孢子堆菌株进行毒性频率分析。结果显示:供试小麦白粉菌群体对Pm1、Pm2、Pm3b、Pm3c、Pm3e、Pm3f、Pm6、Pm7、Pm8、Pm19和Pm1+2+19的毒性频率在60%~100%之间,表明这些抗性基因已丧失抗性,在生产上已经丧失利用价值,对Pm4b、Pm24、Pm2+6、Pm2+Mld、Pm2+6+?、Pm4b+Mli、Pm"Era"、Pm"XBD"和Pm21的毒性频率低于20%,表明这些抗性基因抗性良好,可在生产中利用。选取其中93个单孢子堆菌株进行遗传多样性分析,结果表明白粉菌地理群体间遗传距离在0.020 4~0.103 7之间,其中宝鸡和渭南群体的遗传距离最近,汉中和咸阳群体的遗传距离最远。群体间遗传变异占总体变异的12.82%,群体内遗传变异占87.12%,表明遗传变异主要来自于群体内。Mantel Test分析表明,小麦白粉菌群体间遗传距离与地理距离相关性不大。     

尖孢镰刀菌番茄专化型中SNARE蛋白FolSso1的功能分析

 SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptor)蛋白保守存在于丝状真菌中,在膜泡转运的过程中起着关键的作用。番茄枯萎病是由尖孢镰刀菌番茄专化型(Fusarium oxysporum f. sp. lycopersici,Fol)引起的,严重威胁着番茄的生产。我们使用反向遗传学的方法来研究番茄枯萎病菌中SNARE蛋白FolSso1的功能,实验结果发现FolSSO1的基因缺失突变体菌丝生长速率降低,且产孢数量减少。另外,FolSSO1基因的缺失导致突变体相较于野生型菌株对细胞壁压力与细胞膜压力更加敏感。然后,在番茄果实和番茄植株的致病性实验中,我们发现FolSSO1的缺失并没有引起Fol致病性显著的变化。综上所述,本研究发现FolSSO1可以调控Fol营养生长,繁殖和对环境压力的响应过程,然而对Fol的致病过程并没有显著的调控作用。     

利用香蕉水培系统测定枯萎病病原菌致病力

 为建立一种评价香蕉枯萎病病原菌(Fusarium oxysporum f.sp. cubense)菌株间致病力差异的体系,在改进的香蕉水培系统基础上,对影响香蕉枯萎病菌致病力的接种菌液浓度、类型及处理方式等因素开展分析,同时与不同菌株盆栽测定结果进行比较以验证该方法的可靠性。结果表明:在香蕉水培系统下,将摇培5 d的菌液稀释10倍以上,使孢子初始浓度为1×10⁶ cfu·mL^-1,直接加入该菌液50 mL即可用于致病力评价,且不同菌株用该测定方法与盆栽测定的致病力结果基本一致。该方法的建立为香蕉枯萎病菌致病力的评价提供了快速简便的方法,也为下一步解析尖孢镰刀菌致病相关基因功能和致病机理及抗病品种的选育奠定了基础。     

尖孢镰刀菌番茄专化型中SNARE蛋白FolSso1的功能分析

 SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptor)蛋白保守存在于丝状真菌中,在膜泡转运的过程中起着关键的作用。番茄枯萎病是由尖孢镰刀菌番茄专化型(Fusarium oxysporum f. sp. lycopersici,Fol)引起的,严重威胁着番茄的生产。我们使用反向遗传学的方法来研究番茄枯萎病菌中SNARE蛋白FolSso1的功能,实验结果发现FolSSO1的基因缺失突变体菌丝生长速率降低,且产孢数量减少。另外,FolSSO1基因的缺失导致突变体相较于野生型菌株对细胞壁压力与细胞膜压力更加敏感。然后,在番茄果实和番茄植株的致病性实验中,我们发现FolSSO1的缺失并没有引起Fol致病性显著的变化。综上所述,本研究发现FolSSO1可以调控Fol营养生长,繁殖和对环境压力的响应过程,然而对Fol的致病过程并没有显著的调控作用。     

鄂西北地区小麦条锈菌主要流行小种的寄生适合度研究

 为准确预测条锈菌流行小种在鄂西北地区的变异动态,本研究采用抗性组分法测定了CYR32、CYR33对当地主栽品种的寄生适合度。结果表明:决定鄂西北小麦条锈菌最重要的寄生适合度组分依次为条锈菌的侵染能力、扩展能力和产孢能力;建立了小麦条锈菌寄生适合度属性3个主成份的数学模型;从总体结果看CYR32、CYR33均表现较高相对寄生适合度,且CYR32略高于CYR33,但相比感病对照品种和外来品种,供试小种对当地主栽品种鄂麦352和襄麦55表现出较低的寄生适合度。     

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。     

恶霉灵与溴菌腈混配对香蕉枯萎病菌的抑制效果

分别以孢子萌发法与菌丝生长法测定了噁霉灵与溴菌腈5种不同配比混配制剂对香蕉枯萎病菌的毒力.结果表明,不同配比的噁霉灵与溴菌腈的混配制剂都有不同程度的增效作用,其中以噁霉灵∶溴菌腈=2∶1的混配制剂增效作用最为显著,孢子萌发法和菌丝生长法的共毒系数分别为220和262.按噁霉灵∶溴菌腈=2∶1制成的45%噁霉灵*溴菌腈WP处理蕉园土中的孢子和菌丝,300～225 mg/L药液对孢子和菌丝的抑制率均达95%以上;处理香蕉枯萎病菌接种过10天的盆栽香蕉组培苗,20天后的防效达80%,比25%溴菌腈WP 625 mg/L和90%噁霉灵SP 300 mg/L的防治效果分别提高53%和14%.     

草莓枯萎病菌对多菌灵的抗性及其抗性菌株生物学特性

为评价草莓枯萎病菌对多菌灵的抗性风险,从山东有代表性的草莓种植区,采集、分离了4个草莓枯萎病菌菌株,分别在含多菌灵的PDA平板培养基上进行继代培养,诱导获得抗性菌株。采用菌落生长速率法测定抗性菌株ZY-D的抗性稳定性及其对其它杀菌剂的交互抗性,比较了抗性菌株ZY-D与敏感菌株ZY的生物学特性差异。结果显示:选育至45代,抗性菌株ZY-D对多菌灵的抗性达53.91倍,抗性能够在无药条件下稳定遗传,4个抗性菌株与敏感菌株ZY具有同样的致病力;抗多菌灵菌株ZY-D对甲基硫菌灵等7种药剂表现出了不同程度的交互抗性;抗性菌株ZY-D与敏感菌株ZY的适宜生长温度均为25℃,适宜pH均为9;在不同的碳、氮源条件下,ZY-D与ZY的菌落直径和产孢量存在差异。     

小麦白粉病菌对喹氧灵和苯菌酮的敏感基线及药剂的生物活性

采用离体叶段法,分别测定了从河北、河南、湖北、陕西和四川5省分离的53个小麦白粉病菌单孢菌株对苯菌酮和喹氧灵的敏感性,并分析了白粉病菌对三唑酮和苯菌酮以及喹氧灵之间的 交互抗性。结果表明:小麦白粉病菌群体对苯菌酮和喹氧灵的平均EC50值分别为(0.001 9±0.000 6) 和(0.013 1±0.002 0) mg/L,苯菌酮比喹氧灵具有更高的抑菌活性;小麦白粉病菌对三唑酮和苯菌酮与喹氧灵之间均不存在交互抗性(R2值分别为0.102 6和0.491 9);室内盆栽试验结果显示:接种前1 d和接种后1d施药,苯菌酮和喹氧灵对小麦白粉病的保护与治疗作用防效分别为92.21%、84.25%和82.43%、70.25%,表明这2种药剂不仅具有优异的保护作用,同时还具有较好的治疗作用。     

实时荧光定量PCR检测土壤西瓜枯萎病菌体系的建立

正西瓜枯萎病是由尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)引起的一种土传维管束病害,在各个时期均可发生,对西瓜产业构成严重威胁。该病具有传播途径多样、暴发快速、应急防治难等特点,因此及时准确监控土壤中的病原菌是防治西瓜枯萎病的关键。实时荧光定量PCR(quantitative real-time PCR,q PCR)技术能进行定量