De novo synthesis of ferrichrome by Fusarium oxysporum f. sp. cubense TR4 in response to iron starvation

Manipulation of iron bioavailability in the banana rhizosphere may suppress Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc). However, iron starvation induced by application of synthetic iron chelators does not effectively suppress Fusarium wilt. It is unclear whether Foc can subvert iron chelators and thereby evade iron starvation through the synthesis of iron-scavenging secondary metabolites, called siderophores. In vitro studies were conducted using iron-deficient growth medium and medium supplemented with a synthetic iron chelator, 2,2′-dipyridyl, to mimic iron starvation in Foc Tropical Race 4 (Foc TR4). Concentration of extracellular siderophores increased three-fold (p < 0.05) in the absence of iron. Liquid chromatography-mass spectrometry analysis detected the hydroxamate siderophore, ferrichrome, only in the mycelia of iron-starved cultures. Moreover, iron-starved cultures exhibited a reduction in total cellular protein concentration. In contrast, out of the 20 proteinogenic amino acids, only arginine increased (p < 0.05) under iron starvation. Our findings suggest that iron starvation does not cause a remodelling of amino acid metabolism in Foc TR4, except for arginine, which is required for biosynthesis of ornithine, the precursor for siderophore biosynthesis. Collectively, our findings suggest that biosynthesis of siderophores, particularly ferrichrome, could be a counteractive mechanism for Foc TR4 to evade iron starvation.     

Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10³ spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.     

The effector SIX8 is required for virulence of Fusarium oxysporum f.sp. cubense tropical race 4 to Cavendish banana

Plant pathogens employ effectors as molecular weapons to manipulate host immunity and facilitate colonization. Fusarium oxysporum f. sp. cubense is the agent of wilt disease in banana plantlets and four races of the pathogen have been identified based on the cultivar specificity. A total of 9 SIX genes have been detected in the genome of Foc TR4 and 6 genes detected in Foc1. Among these SIX genes, SIX2 and SIX8 are only detected in Foc TR4, not identified in Foc1. Expression profiles analysis revealed that SIX genes of Foc TR4 are highly induced after inoculation to Cavendish banana plantlets. Virulence analysis of the SIX2 and SIX8 knock-out mutants showed that SIX8 is required for the virulence of Foc TR4 while SIX2 has no obvious functions. Over expression of SIX8-FLAG proteins in the SIX8 knock-out mutant partly restored the virulence. Western blot analysis suggested that SIX8 could be secreted into the extracellular space and a signal peptide resided the N-terminal polypeptide sequence. This study provides some clues for further research on mechanism of SIX8 in regulating virulence of Foc TR4.     

香蕉枯萎病菌Fow1基因的克隆及序列分析

为了解Fow1基因在尖镰刀菌古巴专化型侵染香蕉过程中的作用,及其与尖镰刀菌古巴专化型生理小种1号和生理小种4号之间的致病力差异的关系,采用PCR和RT-PCR方法扩增了2个生理小种的Fow1基因,并对扩增产物进行了克隆测序及相似序列搜索和比对,还对基因编码的蛋白进行了结构预测和功能分析。研究结果表明2个生理小种Fow1基因开放阅读框均为957bp,编码318个氨基酸,基因序列和氨基酸序列差异小,而且两个生理小种Fow1基因所编码的蛋白均具有酵母线粒体载体蛋白典型的结构特征,推测Fow1基因可能为香蕉枯萎病菌在香蕉组织中定殖所必需。从Fow1基因序列及其编码蛋白的氨基酸序列看,2个生理小种致病力的差异与Fow1基因并无明显对应关系,这为进一步研究Fow1基因功能奠定了基础。     

Genetic Diversity of Fusarium oxysporum f.sp. cubense Isolates (Foc) of India by Inter Simple Sequence Repeats (ISSR) Analysis

To find out the genetic diversity of Indian Foc isolates of banana, a total of 107 isolates of Fusarium which includes 98 Foc isolates obtained from different banana growing regions of India and seven Foc isolates belong to all known VCGs obtained from Australia and two non-pathogenic Fusarium oxysporum (npFo) isolates were subjected to ISSR analysis. In the initial screening of ISSR primers, out of 34, 10 primers which generated more polymorphic bands were selected for further analysis. The Phylogenetic analysis carried out based on the fingerprints obtained through ISSR analysis indicated the presence of wide genetic diversity among the Foc isolates of India and also its polyphyletic nature. Totally, seven different clusters were obtained and these clusters differentiated the Foc isolates of India based on the races/VCGs. Besides, the cluster analysis clearly distinguished the freshly emerged Foc strain obtained from cv. Grand Naine (Cavendish-AAA) and Poovan (Mysore-AAB) from the other Foc isolates. The non-pathogenic F. oxysporum isolates which have been included for comparison purpose also clustered separately. All these above said findings indicates for the first time the discriminatory power of ISSR to clearly distinguish and separate the Foc isolates according to its race/VCGs and also its virulence. This study would be useful not only to design and develop effective management strategies but also useful for quarantine purposes.     

Differential Colonization Patterns of Bananas (Musa spp.) by Physiological Race 1 and Race 4 Isolates of Fusarium oxysporum f.sp. cubense

Fusarium oxysporum f.sp. cubense (Foc) is the causative agent of Fusarium wilt of bananas (Musa spp.). To clarify the colonization patterns of Foc in bananas, two green fluorescent protein‐tagged isolates, NT320 (race 1) and B2‐gfp (race 4), were used to follow infection of the banana varieties Pisang Awak and Brazil. Penetration and colonization of both isolates in roots of these two banana varieties were observed within 6 days, but sporulation in xylem vessels was not observed until day 30 postinoculation. Interestingly, B2‐gfp penetrated into xylem vessels of Pisang Awak banana roots more quickly than NT320, implying that the race 4 isolate is more virulent than the race 1 isolate. This result was further confirmed by comparing the disease severity of plants inoculated with NT320 with that of plants inoculated with B2‐gfp. Quantitative real‐time PCR revealed that some pathogenicity‐associated genes, including Fga1, Fhk1, Fow2 and Ste12, were upregulated by B2‐gfp during exposure to Brazil bananas, while they were either downregulated by NT320 or not significantly changed. These data might partly explain why the race 4 isolate was more virulent than the race 1 isolate.     

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.     

Occurrence and correlations of nematodes,Fusarium oxysporum and edaphic factors on banana plantations

Problems caused by nematodes and Fusarium wilt (Panama disease) on banana plantations are responsible for yield losses and are limiting to its cultivation. In the state of Goias, there is little information about the nematode occurence on this crop, and its relation with the incidence of Fusarium oxysporum f.sp. cubense (Foc). This research had the purpose to identify the occurrence of plant‐parasitic nematodes on banana plantations and to verify its correlation with the Fusarium wilt and with the soil attributes (pH, texture, nutrients). Twelve banana orchards in the state of Goias were sampled in the municipalities of Anapolis, Caiaponia, Goiatuba, Itaguaru, Itumbiara (two areas), Jatai, Morrinhos, Ouro Verde, Palestina, Taquaral and Uruana. All sampled areas, except Morrinhos, revealed contamination with Foc, and all areas had different genera of nematodes either in the soil or in the roots samples. Meloidogyne sp., Helicotylenchus sp. and Rotylenchus sp. were the main genera of plant‐parasitic nematodes found in the samples, with Meloidogyne sp. and Rotylenchus sp. being the most dominant and abundant genera. The presence of Pratylenchus sp. increases the population levels of F. oxysporum. Helicotylenchus sp. is highly correlated with high concentrations of Mn. High population density of Meloidogyne sp. was found in irrigated areas with low concentrations of P, Ca, Mg and soil pH.     

Preference of Cosmopolites sordidus for fusarium wilt-diseased banana plants

The objective of this study was to understand the role of Cosmopolites sordidus in the dispersal of Fusarium oxysporum f. sp. cubense race 1 (Foc) and more specifically to determine whether C. sordidus is preferentially attracted to Foc-contaminated banana plant material vs. Foc-free material. The attraction of C. sordidus to healthy and Foc-contaminated Gros Michel banana bulbs and pseudostems, as well as to pure cultures of Foc, was compared using four-choice olfactometers. Males and females were studied separately. After storage in a dark room, weevils were placed in the central chamber of olfactometers. After 15 min, the orientation of each of the weevil was determined according to the food source located in each of the four peripheral chambers of the olfactometer. Overall, 560 olfactometer assays were carried out. Weevils were found to prefer Foc-infected plant tissues. Weevil preference was not significantly affected by weevil sex. The attraction of C. sordidus to Foc-contaminated plant material suggests that this insect could be important for the dissemination of Foc, especially in early stages of epidemics, when Foc-infected plants are scarce.     

生物肥与甲壳素和恶霉灵配施对香蕉枯萎病的防治效果

通过盆栽试验研究了生物肥与甲壳素和恶霉灵配施防治香蕉枯萎病效果,试验结果表明,生物肥与恶霉灵配施(H+F)处理香蕉枯萎病病情指数最高,生物肥与甲壳素配施(C+F)处理病情指数最低。单独生物肥处理防病效果为32.8%,生物肥与甲壳素配施处理为42.5%,而生物肥与恶霉灵配施加重了香蕉枯萎病病情。Biolog Eco微平板研究发现,AWCD(平均每孔颜色变化率)和Shannon等4个多样性指数变化趋势与防病效果相反:防病效果好的处理,土壤细菌功能多样性指数反而低,经检测发现病原真菌(Fusarium oxysporum f. sp. cubense)可利用Biolog Eco微平板上碳源底物并发生颜色变化,干扰测定结果。T-RFLP分析土壤细菌DNA多样性,对照(灭菌生物肥)土壤中TRFs末端限制性片段最少,生物肥与甲壳素配施处理最多。与网上数据库比较,生物肥与甲壳素配施增加了土壤中芽胞杆菌种类,与恶霉灵配施降低了芽胞杆菌种类。分析发现,T-RFLP和Biolog的主成份分析载荷图具有较高一致性。因此,生物肥与生物农药甲壳素配施,从生态角度控制土传病害,优势互补,提高了土壤细菌多样性,改善了土壤细菌群落结构,有利于提高防病效果。     

氨基酸影响尖孢镰孢菌古巴专化型厚垣孢子的形成

香蕉枯萎病是由尖孢镰孢菌古巴专化型Fusarium oxysporum f. sp. cubense（Foc）侵染引起的一种土传真菌病害,已严重威胁香蕉产业的健康发展。该病菌产生的厚垣孢子可在土壤中存活多年,是香蕉枯萎病的初侵染源。本研究通过氨基酸添加试验,证明添加甘氨酸可抑制厚垣孢子的形成;通过对该病菌厚垣孢子形成前期、初期、中期和后期的转录组分析,发现氨基酸合成通路中有93个基因的表达水平在厚垣孢子形成过程中发生了显著变化;In silico 分析表明其中10个基因参与调控真菌的氨基酸合成,11个基因参与调控真菌种的生长发育和产孢,19个基因参与调控真菌种的致病性和毒素产生。由此推测,氨基酸合成通路不仅与尖孢镰孢菌古巴专化型厚垣孢子的形成相关,其有可能参与调控该病菌的致病性。     

Development of an in vitro hydroponic system for studying the interaction between banana plantlet and Fusarium oxysporum f. sp. cubense

Plant Cell, Tissue and Organ Culture (PCTOC) - Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Several experimental...     

7个香蕉品种对枯萎病的抗性评价及3种评价方法的对比

[目的] 评价香蕉自主选育品种对枯萎病病原菌尖孢镰刀菌古巴专化型（Fusarium oxysporum f. sp. cubense）热带4号小种（tropical race 4, Foc TR4）的抗性。[方法] 以浸根法和2种灌注法将Foc TR4接种于不同抗性的香蕉品种上,分析接种后香蕉的发病情况,比较筛选这3种评价方法,并评价7个香蕉品种的苗期和田间抗性水平。[结果] 采用浓度为2×10⁵ 个·mL^-1的PDB培养基Foc TR4孢子悬浮液灌注法进行香蕉苗期抗性评价更为高效可行;综合苗期和田间抗性评价结果,7个香蕉品种的抗性由高到低为：南天黄>红研3号>红研5号>红研2号>滇蕉1号>巴西蕉>红研1号。[结论] 以南天黄作为高抗品种对照,以巴西蕉作为高感品种对照条件下,红研3号和红研5号抗性为中等抗性偏强,红研2号达到中抗水平,滇蕉1号为感病,红研1号为高度感病。