香蕉枯萎病菌myosin-1基因的功能分析及外施dsRNA介导的抗性评估

 香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense,Foc)引起的香蕉毁灭性土传病害,其中 4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。SMART在线软件分析myosin-1基因具有肌球蛋白马达蛋白(myosin motor domain, MMD),肌动蛋白尾结构TH1(myosin tail)和 Src家族同源结构域SH3(src homology domain 3),与禾谷镰刀菌中氰烯菌酯靶标基因myosin-5具高度的蛋白同源性,相似性高达83%。利用Split-marker基因重组技术获得Foc4的myosin-1基因敲除突变体,Δmyosin-1突变株丧失了对氰烯菌酯的敏感性,菌丝生长缓慢,产孢量减少且孢子畸形,对香蕉致病力严重下降,证实myosin-1是氰烯菌酯在Foc4中的作用靶标基因。外施靶向myosin-1体外转录的dsRNA,能抑制菌丝的生长,降低菌丝活性;菌丝膨胀扭曲分枝增多,出现典型的球状结构,与氰烯菌酯处理后的表型一致。在盆栽活体人工接种实验中,体外施用dsRNA可以明显抑制枯萎病外部症状的发展,推迟发病时间,赋予寄主抗性,结果说明体外施用dsRNA可以作为新型杀菌剂防治香蕉枯萎病。综上,myosin-1基因作为氰烯菌酯在Foc4中的靶标基因具有高度的序列保守,在调控菌丝生长发育,产孢以及致病力等方面发挥重要作用,而外施dsRNA具有防治香蕉枯萎菌的巨大潜力。     

钾肥与生物有机肥配施防治香蕉枯萎病效果初探

通过盆栽试验研究了生物有机肥与氯化钾和硫酸钾配施防治香蕉枯萎病的效果。试验结果表明,两种肥料配施促进了香蕉苗生长,降低了香蕉枯萎病病情指数,提高了防病效果,生物有机肥与KCl和K2SO4配施防病效果比单施生物有机肥分别高60%和90%。利用T-RFLP分析土壤细菌DNA多样性,生物有机肥与钾肥配施提高了细菌三个遗传多样性指数,增加了土壤中芽胞杆菌种类。FAME分析也发现生物有机肥以及与钾肥配施促进革兰氏阳性细菌和放线菌繁殖,抑制革兰氏阴性细菌和真菌生长。生物有机肥与钾肥配施,优势互补,改善了土壤微生物群落结构,有利于提高防病效果。     

Fusarium wilt of banana: biology,epidemiology and management

Fusarium oxysporum is a soil borne hyphomycete that causes vascular wilts in several crop plants. A variety of remedial measures such as the use of fungicides, soil amendments and biological antagonists have proved insufficient in controlling F. oxysporum. Ever since it was first reported in banana crop, the only effective control strategy known is planting of resistant cultivars. However, presumably due to the high mutation rates and rapid co-evolution with its host, Fusarium wilt has surmounted host defense barriers and has already begun infecting even the resistant Cavendish varieties that dominate export markets worldwide. Transgenic banana plants showing enhanced resistance to Fusarium wilt have been developed in recent past, but they remain largely confined to the laboratory. The importance of banana as source of food and income in developing countries world over and the need to develop Fusarium wilt tolerant cultivars by novel biotechnological approaches is detailed herein. In this communication, we review the biology and management of Fusarium wilt in banana with the aim of providing the baseline of information to encourage much needed research on integrated management of this destructive banana crop disease problem.     

Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the rhizosphere of healthy banana plants

One of the most serious diseases of banana is fusarium wilt, caused by Fusarium oxysporum f.sp. cubense ( Foc ). The objectives of this study were to isolate and identify nonpathogenic F. oxysporum strains from soils suppressive to banana wilt, and to determine the diversity of these isolates. More than 100 Fusarium strains were isolated from the rhizosphere of banana plants and identified to species level. Pathogenicity testing was carried out to confirm that these isolates were nonpathogens of banana. A PCR-based RFLP analysis of the intergenic spacer region of the ribosomal RNA operon was used to characterize the nonpathogens. The isolates were also compared with isolates of Foc from South Africa and the known biological control isolate of F. oxysporum , Fo47. The species-specific primers FOF1 and FOR1, in addition to morphological features, were used to confirm the identity of F. oxysporum isolates included in the PCR-RFLP analysis. Twelve different genotypes could be distinguished, identified by a six-letter code allocated to each isolate following digestion with the restriction enzymes Hae III, Hha I, Hin fI, Msp I, Rsa I and Scrf I. Eleven of these included nonpathogenic F. oxysporum isolates, and these groups could all be distinguished from the genotype that included Foc . Fo47 was included in one of the genotype groups consisting of nonpathogenic F. oxysporum isolates from South Africa.     

Sequence variation in the putative effector gene SIX8 facilitates molecular differentiation of Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc), causal agent of fusarium wilt of banana, is among the most destructive pathogens of banana and plantain. The development of a molecular diagnostic capable of reliably distinguishing between the various races of the pathogen is of key importance to disease management. However, attempts to distinguish isolates using the standard molecular loci typically used for fungal phylogenetics have been complicated by a poor correlation between phylogeny and pathogenicity. Among the available alternative loci are several putative effector genes, known as SIX genes, which have been successfully used to differentiate the three races of F. oxysporum f. sp. lycopersici. In this study, an international collection of Foc isolates was screened for the presence of the putative effector SIX8. Using a PCR and sequencing approach, variation in Foc‐SIX8 was identified which allowed race 4 to be differentiated from race 1 and 2 isolates, and tropical and subtropical race 4 isolates to be distinguished from one another.     

Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Fusarium oxysporum f. sp. cubense race 4

Fusarium wilt (Panama disease), caused by the fungus Fusarium oxysporum f. sp. cubense race 4 (Foc race 4), is one of the most destructive diseases affecting banana (Musa). Early and accurate detection of Foc race 4 is essential to protect the banana industry. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the detection of Foc race 4 based on a SCAR marker sequence. The detection limit for this assay was 10 fg per 25 μl reaction in pure culture and DNA amplification was completed within 60 min. The assays detected 69 different isolates of Foc race 4 from geographically distinct counties in China, and no cross-reaction was observed with other fungal pathogens. When 26 infected and eight healthy looking but infested banana samples naturally from different fields were examined, the detection rate of LAMP was 100 %. The LAMP assay developed in this study was simple, fast, sensitive, and specific, and can be used in the field to detect Foc race 4 in infected banana plant tissue in resource-poor settings.     

Comparative analysis of pathogenic and nonpathogenic Fusarium oxysporum populations associated with banana on a farm in Minas Gerais,Brazil

Fusarium wilt is one of the most devastating diseases on banana. The causal agent, Fusarium oxysporum f. sp. cubense (Foc) is genetically diverse and its origin and virulence are poorly understood. In this study, pathogenic Foc isolates and nonpathogenic F. oxysporum isolates from Minas Gerais in Brazil were compared using EF‐1α and IGS sequences. This allowed the examination of the origin and evolutionary potential of Foc in a country outside the region of origin of the banana plant. Two different sequence types were found among Foc isolates. One appeared to be of local origin because it was identical to the sequence type of the largest group of nonpathogenic isolates. To explore if the ‘local’ Foc isolates had acquired pathogenicity either independently through coevolution with the host, or through horizontal gene transfer (HGT) of pathogenicity genes from other, probably introduced, Foc isolates, the presence and sequence of putative SIX effector genes were analysed. Homologues of SIX1, SIX3 and SIX8 were found. SIX1 sequences were identical and exclusively found in all pathogenic isolates, while variable ratios of sequences of multicopy gene SIX8 were found among nonpathogenic and different pathogenic isolates. This observation supports the HGT hypothesis. Horizontal transfer of genes between isolates of F. oxysporum has important implications for the development of reliable diagnostic tools and effective control measures. Full genome sequencing is required to confirm HGT and to further unravel the virulence mechanisms of forma specialis cubense.     

Evaluating the potential of soil management to reduce the effect of Fusarium oxysporum f. sp. cubense in banana (Musa AAA)

European Journal of Plant Pathology - Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt in banana (Musa AAA). Foc Race 1 devastated the subgroup Gros Michel during the first half of the...     

大蕉枯萎病病原菌鉴定及TEF-1α序列分析

 近年来,大蕉枯萎病在广东省东莞市发生严重,为了有效控制病害发生蔓延,生产上急需明确大蕉枯萎病的病原。本研究收集了我国华南地区的12株大蕉枯萎病病原菌及19株包括1号及4号生理小种的单孢菌株,以来源于澳大利亚的1号、2号、3号和亚热带4号生理小种以及4株非病原尖孢镰孢菌作对照,通过病原菌形态鉴定、致病性测定、4号小种(Foc 4)及热带4号小种(TR4)的分子特异检测、以及基于翻译延伸因子(TEF-1α)序列的系统发育分析,对大蕉枯萎病病原菌进行鉴定。同时,对我国华南地区不同来源的香蕉枯萎病病原菌的遗传发育关系及致病性分化情况进行了研究。结果表明:(1)引起大蕉枯萎病的病原菌主要是1号生理小种或者是与1号生理小种亲缘关系较近的一个新的系统发育谱系,该谱系可能为 1 号生理小种变异演化而来;(2)大蕉枯萎病病原菌对大蕉和粉蕉都有较强的致病力,但不能侵染香蕉;我国的1号小种存在一定的分化,其中有一个类群只能感染粉蕉,另一个类群既能感染粉蕉也能感染大蕉;(3)大蕉与粉蕉枯萎病的病原菌在致病性及遗传发育关系上都存在一定的交叉和分化。     

The potential of nonpathogenic Fusarium oxysporum and other biological control organisms for suppressing fusarium wilt of banana

The aim of this study was to evaluate the ability of nonpathogenic F. oxysporum and Trichoderma isolates from suppressive soils in South Africa to suppress fusarium wilt of banana in the glasshouse. Several biological control agents and commercial biological control products were included in the study. The isolates were first screened in vitro on potato dextrose agar. In glasshouse evaluations, the fungal and bacterial isolates were established on banana roots before they were replanted in pathogen-infested soil, while the commercial biocontrol agents were applied as directed by the supplier. Banana plantlets were evaluated for disease development after 7 weeks. In vitro tests showed none of the nonpathogenic isolates suppressed Fusarium oxysporum f.sp. cubense ( Foc ), while slight suppression was observed with the two Trichoderma isolates. Results of the glasshouse evaluations revealed that two of the nonpathogenic F. oxysporum isolates, CAV 255 and CAV 241, reduced fusarium wilt incidence by 87·4 and 75·0%, respectively. The known biological control agent Fo47 did not suppress Foc significantly. Pseudomonas fluorescens strain WCS 417, known for its ability to suppress other fusarium wilt diseases (WCS 417), reduced disease incidence by 87·4%. These isolates should be further evaluated for potential application in the field, independently and in combination.     

A molecular diagnosis method using real-time PCR for quantification and detection of Fusarium oxysporum f. sp. cubense race 4

The Fusarium genus causes devastating plant diseases worldwide, in which Fusarium oxysporum is the most serious crop pathogen. Disease monitoring is the basis of integrated pest management of any disease. The lack of rapid, accurate, and reliable device to detect and identify plant pathogens is one of the main limitations in integrated disease management. This study describes an efficient and quantifiable diagnosis method for the specific detection of F. oxysporum f. sp. cubense (Foc) race 4 in field-infected banana. With the optimized PCR parameters using the SCAR (sequence characterized amplified region) primers FocSc-1/FocSc-2 and a real-time PCR strategy, the developed method showed high reproducibility and was very sensitive to detect extremely low quantities of Foc genomic DNA (gDNA). We also found that Foc gDNA in severely symptomatic banana pseudostems and leaves were 6946-fold and 26.69-fold more than in those of mild-symptomatic banana, respectively.     

香蕉枯萎病拮抗菌贝莱斯芽胞杆菌的筛选鉴定及其生物学特性

香蕉枯萎病是目前香蕉产业面临的毁灭性病害,目前尚无特别有效的防治药剂.为发掘香蕉枯萎病的生防菌资源,本研究通过初筛和复筛获得两株对香蕉枯萎病菌具有较强拮抗活性的菌株Blz67和Blz02.依据形态学观察及生理生化测试结果,并结合16S rRNA和gyrA基因序列分析,将两株拮抗菌鉴定为贝莱斯芽胞杆菌Bacillus v...     

Mitigation of vanillic acid-promoted faba bean Fusarium wilt by faba bean–wheat intercropping

Long-term continuous monocropping of faba beans increases the incidence of faba bean wilt, while faba bean–wheat intercropping can effectively control it. This study aimed to understand the underlying mechanism of faba bean–wheat intercropping for the control of Fusarium oxysporum and vanillic acid (VA)-promoted occurrence of faba bean wilt. The occurrence of faba bean wilt was investigated among the monocropped and intercropped plants of faba beans in a field experiment. The contents and types of phenolic acids were examined in the rhizosphere soil. Monocropped and intercropped faba beans were examined under the dual stress of F. oxysporum and different concentrations of VA (0, 50, 100, 200 mg/L) to understand the alleviating mechanism of faba bean–wheat intercropping. Exogenous addition of high concentrations of VA significantly inhibited the growth and reproduction of F. oxysporum, but under the dual stress of F. oxysporum and different concentrations of VA, it significantly inhibited the defence enzymes of faba bean roots, stems, and leaves, and rhizosphere soil enzymes. Interestingly, faba bean–wheat intercropping alleviated VA stress and thereby the incidence and disease index of faba bean Fusarium wilt by improving plant resistance and soil enzyme activity. The dual stress of F. oxysporum and VA promotes the occurrence of Fusarium wilt by damaging the defence system of the faba bean root system and rhizosphere soil environment. However, faba bean–wheat intercropping effectively alleviates the autotoxicity of VA by improving the physiological and biochemical resistance of faba beans and soil enzyme activities, and thus controls the occurrence of Fusarium wilt.     

香蕉枯萎病菌1号小种分泌蛋白与效应子的预测与分析

 由尖孢镰刀菌古巴专化型1号小种(Fusarium oxysporum f. sp. cubense race 1,Foc1)引起的香蕉枯萎病是香蕉生产上的毁灭性病害之一。分泌蛋白作为一类重要致病因子,在Foc1与香蕉互作过程中起着重要作用。本文利用SignalP、WoLF PSORT、TargetP、TMHMM和big-PI Predictor等生物信息学软件,对Foc1全基因组编码的15 438条蛋白质氨基酸序列进行了分泌蛋白及效应子的预测分析。结果表明,Foc1全基因组编码蛋白中有988个经典分泌蛋白,占编码蛋白总数的6.40%。蛋白特征分析表明,其氨基酸长度主要集中在101~500个氨基酸(占总数的71.26%),信号肽长度集中在17~20个氨基酸(占61.94%),信号肽切割位点以SPaseⅠ型为主(占92.91%)。碳水化合物酶类(CAZymes)分析表明,有281个分泌蛋白属于CAZymes,其中以糖苷水解酶家族最多。对细胞壁降解酶类分析表明,有27个分泌蛋白属于纤维素降解酶类,73个属于果胶降解酶类,42个属于木聚糖降解酶类。以氨基酸长度≤400和半胱氨酸残基数≥4为筛选条件,发现Foc1经典分泌蛋白中有378个候选效应子。qRT-PCR分析表明,7个候选效应子均在香蕉组织提取物诱导后获得上调表达,从实验角度证实了其为真正的效应子。     

A molecular diagnostic for tropical race 4 of the banana fusarium wilt pathogen

This study analysed genomic variation of the translation elongation factor 1α (TEF‐1α) and the intergenic spacer region (IGS) of the nuclear ribosomal operon of Fusarium oxysporum f. sp. cubense (Foc) isolates, from different banana production areas, representing strains within the known races, comprising 20 vegetative compatibility groups (VCG). Based on two single nucleotide polymorphisms present in the IGS region, a PCR‐based diagnostic tool was developed to specifically detect isolates from VCG 01213, also called tropical race 4 (TR4), which is currently a major concern in global banana production. Validation involved TR4 isolates, as well as Foc isolates from 19 other VCGs, other fungal plant pathogens and DNA samples from infected tissues of the Cavendish banana cultivar Grand Naine (AAA). Subsequently, a multiplex PCR was developed for fungal or plant samples that also discriminated Musa acuminata and M. balbisiana genotypes. It was concluded that this diagnostic procedure is currently the best option for the rapid and reliable detection and monitoring of TR4 to support eradication and quarantine strategies.     

Functional analysis of the G-protein α subunits FGA1 and FGA3 in the banana pathogen Fusarium oxysporum f. sp. cubense

The asexual fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of fusarium wilt in bananas (Musa spp.). This fungus poses a threat to banana production throughout the world. Here, two Foc genes, fga1 and fga3, were functionally characterized. These genes encode proteins homologous to the G-protein α subunits GPA1 from Saccharomyces cerevisiae and MAGC from Magnaporthe grisea, respectively. The deletion of fga1 leads to a phenotypic defect in colony morphology and reductions in vegetative growth, conidiation and pathogenicity against the banana plant (Musa spp. cv. Brazil), which was not observed for the Δfga3 deletion mutant. Intriguingly, both Δfga1 and Δfga3 deletion mutants showed declines in intracellular cyclic AMP levels and increases in heat resistance, suggesting that FGA1 regulates growth, development, pathogenicity, and heat resistance, whereas FGA3 modulates heat resistance, potentially through the cAMP-dependent protein kinase A pathway. These findings offer insights into the roles of the G-protein α subunits in the development and pathogenicity of the fungus Foc.     

香蕉枯萎病菌TR4原生质体转化和基因敲除体系构建

正香蕉枯萎病是一种典型的维管束和土传真菌病害,也是最具毁灭性的植物病害之一~([1])。香蕉一旦感病,很难结果,而且目前尚无有效的防治措施~([1])。因此,研究植物病原菌侵染进程和致病机制,可以拓宽思路以寻求植物病害防治新途径。香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f.sp.cubense,Foc)侵染所致~([1])。对生产影响最大的Foc是侵染包括大蜜哈、     

Fusarium oxysporum f. sp. canariensis: evidence for horizontal gene transfer of putative pathogenicity genes

Fusarium wilt is a serious disease of the date palm Phoenix canariensis, caused by Fusarium oxysporum f. sp. canariensis (Foc). A previous study that characterized and compared the genetic diversity of the Australian Foc population with international strains suggested that the Australian population may have had an independent evolutionary origin. The current study compared the species phylogeny of the Australian and international populations and determined that Foc is not monophyletic, separating into three supported lineages across the two phylogenetic species of the Fusarium oxysporum species complex. This confirms an independent evolutionary origin for Foc in Australia. However, phylogenetic analysis of the putative pathogenicity genes Secreted In Xylem (SIX) did not reveal any separation of the Australian and international Foc strains. Furthermore, there was very low SIX sequence diversity within Foc. Horizontal gene transfer is argued to be the most parsimonious explanation for the incongruence between the species and SIX gene phylogenies.     

Biological control agents (BCAs) of verticillium wilt: influence of application rates and delivery method on plant protection,triggering of host defence mechanisms and rhizosphere populations of BCAs

Verticillium dahliae causes severe yield reductions in a variety of important annual crops worldwide. Control of verticillium wilt has relied on soil fumigation; however, the use of the main soil fumigant, methyl bromide, has been banned in the European Union since 2010, creating a demand for novel crop protectants. As such, the use of biocontrol agents (BCAs) is an appealing management strategy. Prerequisites for the development of a successful BCA are an understanding of the modes of action of the antagonist, its ecological fitness and an efficient and economically feasible delivery system. Therefore, two BCAs (Paenibacillus alvei K165 or the nonpathogenic Fusarium oxysporum F2) and two release strategies (seed coating or amendment of the transplant soil plug) were assessed against verticillium wilt of aubergine (eggplant). Mixing the transplant soil plug with K165 or F2, at a rate of 10 and 20% (v/v), respectively, reduced verticillium wilt symptom development. Furthermore, a positive correlation was revealed between the release strategy and the BCA rhizosphere population. Correlation analysis also showed that disease severity was negatively correlated to the rhizosphere size of the BCA population. In addition, qPCR analysis showed that both BCAs induced the expression of the pathogenesis‐related (PR) proteins PR1 and PR4 in the stem of aubergines before and after inoculation with V. dahliae in a manner that suggests a link with the rhizosphere size of the BCA population.     

Nonspecific toxins as components of a host‐specific culture filtrate from Fusarium oxysporum f. sp. cubense race 1

Bananas and plantains (Musa spp.) are among the most important crops in the world providing staple food for hundreds of millions of people. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc). Despite the fact that there is very little known on the role of microbial metabolites in the molecular mechanism of Foc infections, it has been proposed that the toxins fusaric acid and beauvericin produced by Foc play an important role during pathogenesis. The aim of this contribution was to study the toxic components of culture filtrates (CF) of Foc and to isolate the extracellular microbial metabolites involved in the plant response. An in vitro bioassay was used to evaluate the production of phytotoxic metabolites as well as the specificity of culture from a strain of Foc belonging to VCG 01210 (race 1). A host‐specific CF was obtained and the phytotoxic compounds characterized as fusaric acid, beauvericin and fumonisin B1. Despite the presence of these nonspecific toxins, a water‐soluble extract from the CF induced protection to the main phytotoxic fraction, measured by lesion area. This hydrophilic fraction induced a fast and strong response of just jasmonic acid (JA)‐dependent defence genes rather than salicylic acid (SA)‐ and ethylene (ET)‐response genes in resistant cultivars. Extracellular proteins isolated from CF of Foc provide an important source for further investigations on the molecular basis of the interaction between Foc and banana.