嫁接西瓜对西瓜枯萎病菌侵染的防御机制研究

为了揭示嫁接提高西瓜抗枯萎病的机制,该研究以嫁接西瓜为材料,采用扫描电镜观察了枯萎病菌侵染下寄主的组织结构变化,荧光定量分析了相关防卫基因的表达,比较了嫁接西瓜对枯萎病菌侵染的抗感反应。结果显示:(1)枯萎病菌侵染后,与自根西瓜相比,嫁接西瓜的根部木质部导管通过快速形成膜状物、侵填体及细胞壁增厚阻塞菌丝入侵;自根西瓜防御反应较嫁接西瓜晚,严重侵染时薄壁细胞降解,导管组织脱落导致维管系统空洞,从而使植株呈现萎蔫症状,该现象在嫁接西瓜中没有发现。(2)枯萎病菌侵染后,嫁接西瓜比自根西瓜具有较高的防卫基因表达水平,其中:嫁接西瓜中,CHI、APX和PPO基因的表达随枯萎病菌侵染时间的延长而升高,而PAL呈现先升高后降低的表达趋势,但仍高于本底表达;自根西瓜中,仅PPO基因在枯萎病菌侵染后表达上调,而其他基因的表达则是先升高后降低,与嫁接西瓜中的PAL基因表达一致。研究表明,嫁接植株一方面通过快速的组织结构响应,另一方面从转录水平提高了相关防卫基因的表达,最终使植株具有抗病性;推测防御基因在嫁接植株与枯萎病菌互作中的强烈诱导响应可能是嫁接植株抗病的分子机制之一。     

Biological Control ofMonilinia laxaandFusarium oxysporumf. sp.lycopersiciby a Lytic Enzyme-ProducingPenicillium purpurogenum

Biological control experiments were conducted with the lytic enzyme-producing fungusPenicillium purpurogenumagainst the plant pathogensMonilinia laxaandFusarium oxysporumf. sp.lycopersici.Applications ofP. purpurogenumto peach shoots previously inoculated withM. laxareduced lesion length and extent of pathogen colonization of shoots by 90 and 80% (P ≤ 0.05), respectively, comparable to the level of disease control obtained with the fungicide captan. Disease severity in tomato plants inoculated withF. oxysporumf. sp.lycopersiciwas decreased by 30% (P ≤ 0.05) with the biological treatment. The fungusP. purpurogenumproduced β-1,3-glucanase and chitinase activities in liquid culture that were inducible by cell walls and live mycelium ofM. laxabut not ofF. oxysporumf. sp.lycopersici.Crude filtrates or crude enzyme preparations ofP. purpurogenumcultures with lytic enzyme activities produced lysis of hyphae and spores ofM. laxaandF. oxysporumf. sp.lycopersici.These lytic effects were strong inM. laxaand ended in complete dissolution of mycelium. The induction of lytic enzymes byM. laxaand the effects of lytic enzymes on mycelia of the pathogens in relation to the different degrees of biological control obtained are discussed.     

The application of high-throughput AFLP's in assessing genetic diversity in Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc) is responsible for fusarium wilt of bananas. The pathogen consists of several variants that are divided into three races and 21 vegetative compatibility groups (VCGs). Several DNA-based techniques have previously been used to analyse the worldwide population of Foc, sometimes yielding results that were not always consistent. In this study, the high-resolution genotyping method of AFLP is introduced as a potentially effective molecular tool to investigate diversity in Foc at a genome-wide level. The population selected for this study included Foc isolates representing different VCGs and races, isolates of F. oxysporum f. sp. dianthi, a putatively non-pathogenic biological control strain F. oxysporum (Fo47), and F. circinatum. High-throughput AFLP analysis was attained using five different infrared dye-labelled primer combinations using a two-dye model 4200s LI-COR automated DNA analyser. An average of approx. 100 polymorphic loci were scored for each primer pair using the SAGA^MX automated AFLP analysis software. Data generated from five primer pair combinations were combined and subjected to distance analysis, which included the use of neighbour-joining and a bootstrap of 1000 replicates. A tree inferred from AFLP distance analysis revealed the polyphyletic nature of the Foc isolates, and seven genotypic groups could be identified. The results indicate that AFLP is a powerful tool to perform detailed analysis of genetic diversity in the banana pathogen Foc.     

香蕉枯萎病田间分布型及病原菌在植株上的分布

为探讨香蕉枯萎病大田病株及其体内尖孢镰刀菌(Fusarium oxyporum f.sp.cubense)的分布情况,首先对大田病株的发病情况进行调查,通过分布频次检验、聚集指标测定、Taylor幂法则、Iwao m*-m模型等对田间病株的空间分布型进行研究,在此基础上,检验聚集均数λ,分析其聚集原因。同时,在香蕉植株不同部位取样,检测病原菌在植株体内的分布情况。结果表明:香蕉枯萎病大田病株的理论分布符合聚集类型,各项聚集度指标均满足C1、I0、m*/m1、CA0、K0。大田病株的空间图式也趋于聚集分布,聚集程度随着种群密度升高而升高,病株间互相吸引,以病株群为单元在蕉地分布均匀,其相对聚集度随种群密度变化的速率为(11.0962+0.1752)m,密度越高,相对聚集度随密度变化速率越大。这种聚集分布是环境作用导致。建立最适理论抽样数模型后,根据一定置信水平下的允许误差值可估测相应发病情况时所配套的最适理论抽样数,且随着病情加重,配套抽样数随之减少。在进行序贯抽样时,假如累计病情等级高于判据上限即可视为防治蕉地,若累计病情等级低于判据下限可视为安全蕉地,如果累计病情等级在判据上限和下限之间,需增加抽样量,但可以理论抽样模型中的最大抽样量终止抽样。最适宜的抽样方法为棋盘式取样法和单、双对角线取样法。此外,枯萎病菌在香蕉植株体内的分布因样地发病程度和植株部位不同而有显著差异,植株球茎的平均含菌量显著高于其它部位。     

大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验

【目的】采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌,探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性,为发掘和应用防病、抗逆优良菌株提供理论基础。【方法】采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选,显微观察法研究筛选菌株引起病原菌菌丝变化,通过菌株培养特征、理化特性和16S r DNA序列同源性分析确定菌株系统发育地位,比色法测定DD174耐盐碱性,盆栽试验验证防病效果。【结果】经复筛和代谢液试验有5株拮抗性较强菌株,被作用病原菌菌丝畸形、细胞壁消失、自溶,菌丝基部加粗、分支增多,呈树根状;菌丝被菌苔包埋而溶解、断裂,菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2T(GQ292772)和B.thuringiensis ATCC 10792T(AF290545),菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958T(X95923)。DD174能耐受6%盐浓度,p H 10生长良好,具有一定耐盐碱能力。DD174处理组防治效果达76.32%,其他防效均在62%以上,可作为棉花枯萎病的生防菌株资源。【结论】大豆根瘤内存在棉花枯萎病内生拮抗细菌,其中有些菌株具有一定耐盐碱能力,对棉花枯萎病病原菌及病害有一定抑菌和防病作用。     

棉花抗枯萎病相关转录因子基因的克隆与表达

以枯萎病菌诱导棉花基因表达谱中获得的差异表达bZIP作为探针,采用电子克隆结合RT-PCR方法从棉花抗枯萎病品种‘中棉所12’中克隆了1个TGA转录因子基因,命名为GhTGA2.2。序列分析表明,该基因的cDNA全长1 356bp,编码451个氨基酸,预测分子量为50.04kD,等电点为5.85,含有保守的bZIP结构域。系统进化树分析表明,GhTGA2.2属于bZIP亚家族的TGA转录因子,与拟南芥AtTGA2、烟草NtTGA2.2亲缘关系最近。qRT-PCR分析表明,经枯萎病菌诱导后,GhTGA2.2基因在抗病品种中呈上调表达,随处理后时间的推移,其相对表达量呈先升高后降低的趋势,并于处理后24h表达量达到最大;水杨酸诱导后1h,GhTGA2.2基因相对表达量迅速增加;茉莉酸和乙烯诱导后GhTGA2.2基因的相对表达量明显降低,呈下调表达。研究推测,GhTGA2.2基因可能通过水杨酸信号传导途径参与对枯萎病菌的防御反应。     

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3 kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease. Received: 1 June 1998 / Accepted: 25 December 1998     

尖孢镰刀菌亚麻专化型Folprp4基因参与调控菌丝生长和分生孢子发生

目的: 通过对尖孢镰刀菌中Folprp4基因的鉴定,揭示其在尖孢镰刀菌中的功能及致病相关性。方法: 基于同源重组原理,根据测定出的Folprp4基因序列,应用Split-Marker重组技术构建含有潮霉素抗性基因(hph)的基因缺失盒。将基因缺失盒经PEG介导转化到野生型原生质体中,在含有潮霉素B的TCC培养基上筛选转化子,通过PCR正负筛查获得Folprp4基因缺失突变株(ΔFolprp4)。构建含有Folprp4基因的载体pZDH1,并将其转化到敲除突变体中进行互补测验。结果: 与野生型(hm)和异位插入突变体(ecFolprp4)相比,敲除突变体菌丝生长受到严重阻碍,当野生型和异位插入突变体长满整个平板时,敲除突变体菌落呈小点状。敲除突变体的另一个显著变化是ΔFolprp4的分生孢子产量显著下降。侵染实验表明,ΔFolprp4对亚麻幼苗的毒力显著降低。互补实验表明,该互补载体的回复子(Folprp4-C)在菌落形态、生长速率、分生孢子产量和毒力方面均恢复到了野生型菌株。结论: Folprp4基因与尖孢镰刀菌的菌丝生长、分生孢子发生和致病性有关。     

尖孢镰孢菌果胶裂解酶基因家族鉴定及侵染表达模式分析

[背景]番茄枯萎病是番茄生产中常见的土传真菌病害.[目的]为鉴定番茄枯萎病基因组果胶裂解酶基因家族,明确该基因家族在侵染过程表达模式.[方法]采用生物信息学方法鉴定了番茄枯萎病尖孢镰孢菌(Fusarium oxysporum f sp.Lycopersici)基因组内PEL基因家族,并分析了基因结构、染色体定位及三级结...     

拮抗棉花枯萎病菌的地黄内生细菌筛选、鉴定和促生潜能

[目的]为了筛选棉花枯萎病菌拮抗性菌株资源,从药用植物地黄根块中分离内生细菌,分析优良菌株的促植物生长特性和耐盐碱特性,发掘优良菌株资源,为研发棉花枯萎病生防菌剂提供参考价值.[方法]采用平板对峙法对分离内生细菌进行棉花枯萎病菌拮抗性试验,荧光显微镜观察法研究病原菌菌丝的变化、分光光度计法测定吲哚乙酸(IAA)含量和1...     

棉花抗枯萎病相关ERF-B3亚组转录因子的克隆与表达

以拟南芥ERF-B3亚组转录因子的AP2/ERF结构域为探针,利用NCBI中的棉花(Gossypium hirsutum)EST数据库,通过电子克隆结合RT-PCR方法,从枯萎病菌诱导后的高抗枯萎病品种‘中棉所12’克隆到1个与抗枯萎病相关的ERF-B3亚组转录因子基因GhB301。序列分析结果显示,GhB301基因cDNA全长593 bp,开放阅读框384 bp,编码127个氨基酸,含有一个保守的AP2/ERF结构域。实时荧光定量PCR检测该基因的表达显示,枯萎病菌诱导后,GhB301基因在棉花根中、抗病品种中优势表达;在乙烯、水杨酸、干旱、低温及高盐的诱导下表达量均发生变化。研究结果表明,GhB301基因可能参与了棉花对病原菌、激素及非生物胁迫的应答反应。     

Evidence for conspecificity of Piper methysticum forst. f. and Piper wichmannii C. DC

Morphological, chemical, cytological and genetic evidence demonstrating the absence of taxonomic distinction between Piper methysticum and Piper wichmannii are reviewed. Piper methysticum is not a separate species, but rather a group of sterile cultivars selected from somatic mutants of P. wichmannii. As P. methysticum was described first (1786), it has priority and P. wichmannii (1910) is superfluous. A new subspecific classification is suggested that makes a distinction between the sterile cultivars (P. methysticum var. methysticum) and the wild populations (P. methysticum var. Wichmannii).     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

Inheritance and genetic mapping of resistance to Alternaria alternata f. sp. lycopersici in Lycopersicon pennellii

The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.     

Colonization of root tissues and protection against Fusarium wilt of rye (Secale cereale) by nonpathogenic rhizosphere strains of Fusarium culmorum

Colonization of rye (Secale cereale) tissues by nonpathogenic rhizosphere Fusarium culmorum isolates DEMFc2 and DEMFc5 and a pathogenic strain DEMFc37, and their effect on plant fresh weight were studied in pot experiments. Both rhizosphere isolates colonized the epidermis and the cortex but were not found in vessels, while the pathogen colonized all three layers of root cells. The numbers of pathogen CFU isolated from plant tissues were much higher than those of the rhizosphere isolates in spite of the same number of macroconidia used as inoculum (1 × 10⁵ g⁻¹ of soil). Inoculation of seedlings with DEMFc2 resulted in a 20% increase, with DEMFc5 in more than a 20% reduction, and with DEMFc37 in a 38% reduction of shoot fresh weight of 14-day-old plants. Pre-colonization of plants with (either of) the rhizosphere isolates and subsequent inoculation with the pathogen resulted in plant weights the same as those observed in plants inoculated with the rhizosphere strain alone. The disease severity index for shoots of plants pre-colonized with DEMFc2 was reduced from class 4 (86% diseased plants) observed for plants inoculated with the pathogen alone to class 2 (average of 8% diseased plants) when pre-treated with the rhizosphere strain. The CFU number of the pathogen isolated from the interior of roots of plants pre-colonized with the rhizosphere isolates was as low as 10% of the number isolated from plants inoculated with the pathogen alone. A study of in vitro interactions between the rhizosphere isolates and the pathogen suggests that changes in plant colonization by the pathogen and its effect on fresh weight of plants pre-colonized with the rhizosphere isolates were not connected with inhibition of its growth by a direct action of the rhizosphere isolates. The results suggest that strain DEMFc2 can be considered as a potential biocontrol agent.     

Biocontrol of Collar Rot Disease of Betelvine ( Piper betle L.) Caused by Sclerotium rolfsii by Using Rhizosphere-Competent Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N

Collar rot disease of betelvine (Piper betle L.) caused by Sclerotium rolfsii is difficult to control by conventional means by use of chemicals; therefore, use of biocontrol agents is desirable. In the present study, 186 bacterial strains of different morphological types were screened for their biocontrol activity against S. rolfsii under in vitro conditions. Two strains, Pseudomonas fluorescens NBRI-N6 and P. fluorescens NBRI-N, were selected for further studies because of their ability to inhibit the mycelial growth of the pathogen significantly. Spontaneous rifampicin-resistant (Rif^r) derivatives of P. fluorescens NBRI-N6 and P. fluorescens NBRI-N showing growth rate and membrane protein composition comparable to the wild type were selected to facilitate their monitoring in the rhizosphere. Field trials demonstrated that strain P. fluorescens NBRI-N6 was better than P. fluorescens NBRI-N in increasing the yield of betelvine significantly, whereas a consortium of the two strains controlled the disease more than either of the strains. The screening method should prove useful in identifying rhizosphere bacteria with the greatest potential for controlling diseases caused by phytopathogenic fungi. RID= ID= <E5>Correspondence to:</E5> C.S. Nautiyal; <E5>email:</E5> nautiyalnbri&commat;yahoo.com Received: 5 August 2002 / Accepted: 7 October 2002     

Seedling vaccination by stem injecting a conidial suspension of F2, a non-pathogenic Fusarium oxysporum strain, suppresses Verticillium wilt of eggplant

Several bacterial and fungal strains have been evaluated as biocontrol agents (BCAs) against Verticillium dahliae. In these studies, the BCAs were applied as a root drenching inoculum; however, this application method may have an adverse effect on the native, beneficial for the plants, microbial community. In the present study, it was evaluated whether endophytic application by stem injecting a conidial suspension of the non pathogenic Fusarium oxysporum F2 strain, isolated from a V. dahliae suppressive compost amendment, could reduce significantly Verticillium wilt symptom development in eggplants. It was revealed that stem injection of F2 seven days before transplanting the seedlings to soil infested by V. dahliae microsclerotia resulted in reduced disease severity compared to the control treatment. To visualise F2 ramification into the plant vascular system eggplant stems were injected with an EGFP transformed F2 strain. It was shown that F2 colonises effectively the plant vascular tissues over a long period of time as it was assessed by F2 DNA levels. In parallel, qPCR analysis showed that the application of F2 reduced significantly the amount of V. dahliae DNA in the stem tissues compared to the control treatment.     

贝莱斯芽孢杆菌SH-1471发酵条件优化及其番茄枯萎病的防治效果

【目的】贝莱斯芽孢杆菌(Bacillus velezensis) SH-1471是一株兼具防控作物土传病害、促进土壤养分转化以及促进作物生长等功能的菌株(保藏编号：CCTCC No. M 2022923,专利号：ZL 2022 1 1479280.X)。挖掘其潜在的生物活性并探究其最适发酵条件,是推进该菌株产业化和商业化开发的有效手段之一。【方法】结合形态学、分子生物学、16SrRNA基因和gyrB基因对菌株SH-1471进行分类地位鉴定;利用PCR技术,对菌株抗生素合成基因检测;使用平板对峙试验和发酵液抑菌试验测定菌株抑菌广谱性;并测定其体外产酶、解磷、解钾、固氮及产铁载体能力;以菌株发酵液OD600值和抑菌率为指标,通过设计单因素试验和响应面优化试验,探究菌株的最佳发酵配方和最佳发酵条件;采用室内盆栽试验测定优化前后菌株发酵液对番茄植株的促生效果及其对番茄枯萎病的防治效果。【结果】经鉴定,SH-1471为贝莱斯芽孢杆菌(Bacillus velezensis),具有srfA、fenB、ituA、ituD和bymA等抗生素合成基因,对番茄枯萎病菌(Fusarium oxysporum...