Purification and characterization of an exo-polygalacturonase from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici

Abstract An exo-polygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with citrus pectin, using preparative isoelectric focusing. The enzyme, denominated PG2, had an apparent M _r of 74000 Da upon SDS-PAGE. The pI of the main PG2 isoform was 4.5, and pH and temperature optima were 5.0 and 55 °C, respectively. PG2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by anaysis of degradation products. The enzyme was N-glycosylated. The N-terminal amino acid sequence, L-A-F-N-V-P-S-K-P-P, has no identity to other known polygalacturonases.     

Races and virulence of Fusarium oxysporum f.sp. cubense on local banana cultivars in Kenya

Virulence of 31 Kenyan isolates of Fusarium oxysporum obtained from bananas showing symptoms of Panama disease was tested against the differential banana cvs Bluggoe, Gros Michel, Dwarf Cavendish, and two other local cvs Muraru and Wang'ae. Seventeen isolates were assigned to either race 1 or race 2 of F. oxysporum f.sp. cubense (FOC). Race 4 was not apparent in this sample of 31 isolates from Kenya as none were pathogenic to cv. Cavendish, and no wilted Cavendish have been observed in field surveys in Kenya. Races could not be assigned to 12 isolates as they were virulent on more than one differential cultivar, and two were apparently not pathogenic. All isolates assigned to races 1 and 2 belonged to the VCG bridging complex 0124/5/8/20, but some other isolates belonging to this VCG complex could not be assigned to race. All five isolates assigned to VCG 01212 could not be assigned to known races. Considerable variability thus exists within FOC isolates within this region. Local cultivars of banana showed differential resistance to the pathogen. The interaction of cultivars and isolates on the level of disease was significant. Overall, cv. Wang'ae was the most susceptible to most of the isolates tested, regardless of their race, and could therefore be used as a reference cultivar in pathogenicity tests of isolates of FOC in the East African region. Of the cultivars tested that are widely grown on smallholder farms in Kenya, Muraru was the least susceptible.     

香蕉枯萎病菌4号生理小种致病相关基因foABC1的分离

通过对香蕉枯萎病菌4号小种致病突变体B1233的进一步研究,分离了被突变的致病相关基因foABC1,同源性分析及保守结构预测该基因编码一类ABC转运蛋白,其功能可能同稻瘟病菌的ABC转运蛋白一样,负责真菌毒素的泵出,或是像其他真菌的ABC转运蛋白,在病原菌侵染寄主植物时能忍耐植物因防卫反应所释放的植保素或抗毒素类物质。     

pH值对香蕉枯萎病菌4号生理小种生长的影响

【背景】香蕉枯萎病菌4号生理小种(镰刀菌)是香蕉产业的致命威胁。已有研究表明土壤pH值越高,香蕉枯萎病发病率越低,但是现有pH值对镰刀菌影响的研究大都是用强酸强碱调节pH值,pH值没有缓冲体系保护,而且尚未检测试验终点时介质的pH值。此外,关于pH值对香蕉枯萎病菌4号生理小种(Foc4)影响的研究尚不系统,难以用于指导生产实践。【目的】为系统地了解土壤酸碱度对Foc4生长的影响。【方法】在pH 3.0-11.0之间设定9个pH值梯度,模拟酸性到碱性土壤pH值条件,于室内培养条件下系统研究pH值对Foc4生长、产孢、孢子萌发的影响及其生长过程对环境pH值的影响。【结果】弱酸性至中性环境(pH 5.0-7.0)最适宜于香蕉枯萎病菌的生长、产孢和孢子萌发。弱碱性处理(pH8.0和pH9.0)孢子平均萌发率较弱酸性环境处理(pH5.0和pH6.0)下降了73.1%。与pH 6.0酸性处理相比,pH 8.0和pH 9.0处理的产孢量分别下降了52.3%和68.1%。【结论】香蕉枯萎病菌Foc4生长和萌发过程会产酸,但是在缓冲体系液体培养基中,除了pH 9.0和pH10.0处理终点培养液pH值分别下降了0.34和0.27个单位外,其它处理起始和终点的pH值无差异。说明在缓冲体系液体培养基中的研究结果可以反映环境pH值对Foc4生长和萌发的影响。在作物可以生长的pH值范围内(pH5.0-9.0),碱性和微碱性条件(pH8.0-9.0)能明显抑制Foc4生长、产孢和孢子萌发。     

Isolation and characterization of endophytic streptomycete antagonists of Fusarium wilt pathogen from surface-sterilized banana roots

A total of 131 endophytic actinomycete strains were successfully isolated from surface-sterilized banana roots. These isolates belonged to Streptomyces (n=99), Streptoverticillium (n=28), and Streptosporangium (n=2) spp. The remaining 2 isolates were not identified. About 18.3% of the isolates inhibited the growth of pathogenic Fusarium oxysporum f. sp. cubense on banana tissue extract medium. The most frequently isolated Streptomyces sp. strain S96 was similar to Streptomyces griseorubiginosus. About 37.5% of the S. griseorubiginosus strains were antagonistic to F. oxysporum f. sp. cubense. The antagonism of strain S96 was lost when FeCl(3) was introduced into the inhibition zone. In vivo biocontrol assays showed that the disease severity index (DSI) was significantly (P=0.05) reduced and mean fresh weight increased (P=0.001) in plantlets treated with strain S96 compared to those grown in the absence of the biocontrol strain. These findings indicate the potential of developing siderophore-producing Streptomyces endophytes for the biological control of fusarium wilt disease of banana.     

Investigation of the diversity of effector genes in the banana pathogen,Fusarium oxysporum f. sp. cubense,reveals evidence of horizontal gene transfer

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana‐infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole‐genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1–SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF‐1α/RPB1/RPB2 (translation elongation factor‐1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.     

Spatial distribution and association of banana (Musa spp.) Fusarium wilt (Fusarium oxysporum f.sp. cubense) epidemics with biophysical factors in southwestern Ethiopia

Abstract

Field surveys were conducted in 2014 and 2015 to determine the distribution and association of wilt incidence with biophysical factors. Spatio-temporal analysis of wilt epidemics in the farms revealed a wide distribution of the disease. Cropping year, district, cultivar, crop density, planting date, weeding, sucker and bud management, source of moisture and planting materials, presence/absence of wilt in neighboring farms, disease management practices, farmers awareness towards wilt diseases, planting date and cropping system were significantly associated with disease incidence in a multiple-variable model. A wilt incidence of ≤30% had high probability of association to good weed, sucker and male bud management practices, high level of farmers awareness towards wilt, low population density, disease-free suckers, rainfed production, absence of wilt in neighboring farms and good disease management schemes. Findings of this study showed Fusarium wilt is a major constraint and need for on-farm trainings to concerned bodies about wilt management schemes.     

香蕉枯萎菌基因组DNA提取方法的研究

以香蕉枯萎菌菌株为试验材料,在SDS～CTAB法和高盐沉淀法等基础上加以改进,对两种提纯香蕉枯萎菌基因组DNA的方法进行了比较研究。结果表明：高盐沉淀法是适合于香蕉枯萎菌基因组DNA提取的方法。该方法提取的DNA OD260／OD280的比值为1．841,DNA产量为0．81mgDNA／g菌丝体。基因组DNA经琼脂糖凝胶电泳得到一条带型较宽且清晰的DNA谱带,基本无DNA碎带;将提取的DNA直接用于PCR扩增,得到带多而且清晰、整齐、基本无拖尾的RAPD图谱。     

Evaluation of a liquid formulation of Pseudomonas fluorescens against Fusarium oxysporum f. sp. cubense and Helicotylenchus multicinctus in banana plantation

Plant growth promoting rhizobacteria (PGPR) are eco-friendly alternatives to chemical fungicides to manage plant diseases. We evaluated the efficacy of a Pseudomonas fluorescens formulation against Fusarium oxysporum f. sp. cubense and Helicotylenchus multicinctus at multiple banana plantations. Three field trials were conducted to assess the wilt incidence and the populations of nematode and bacteria in the soil treated with a liquid formulation of P. fluorescens at 2.0, 3.0 and 4.0 l ha^?1 using drip irrigation system at 60, 120, 180 and 240 days after planting. The results showed that the treatment at 4.0 l ha^?1 reduced the wilt incidence by 60 %. It also reduced the overall population of H. multicinctus by 41.3–89.0 % in the treated fields. The presence of P. fluorescens in the treated soil was 5.6 × 10⁶ cfu g^?1 of soil at the time of harvest. The treatment of biocontrol agent P. fluorescens also resulted in an overall yield increase in banana production by 36.6–46.5 % compared to the control.     

Production of monoclonal antibodies to Fusarium oxysporum f.sp. cubense race 4

The production of monoclonal antibodies (mab) to Fusarium oxysporum f.sp. cubense (Foc ) race 4 is described. Heat-killed conidia of this fungus were toxic to female Balb/c mice, but this toxic reaction was not found with fractionated hyphal walls. A simple and reproducible enzyme immunoassay using a standard 9 cm polystyrene Petri dish as a solid phase was devised for screening culture supernatant fluids. Sixteen stable hybridoma clones secreting mabs of the IgM class were isolated by fusing splenic lymphocytes from immunized female Balb/c mice with P3-NSl-Ag4-l mouse myeloma cells. Monoclonal antibodies produced by eight of the 16 hybridoma clones were selected and the specificity of the mabs was determined by an indirect immunofluorescence test. Of the eight mabs, only one displayed an exceptionally high degree of specificity to the thick-walled chlamydospores of Foc race 4. This specific reactivity allowed differentiation of Foc race 4 from other races.     

Biochemical markers as a useful tool for the early identification of Fusarium oxysporum f.sp. cubense,race 1 resistance banana clones

Abstract

Panama disease of banana (Musa spp) caused by the fungus Fusarium oxysporum f. sp. Cubense (FOC), is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Chemical control is not economically effective and is also hazardous to the environment and human health. Breeding for disease resistance is an alternative strategy, which leads to the development of resistance clones. Field evaluation is the most reliable method of screening for disease resistance, but it is demanding in terms of cost, manpower and space requirements. Another approach of screening hybrids at the sucker's stage (planting material) through biochemical markers has been found to be effective in early identification of resistant hybrids. The resistance mechanisms involving the role of phenol, PAL, oxidative enzymes like peroxidase (PO), polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase and PR-proteins like chitinase, β-1-3 glucanase were studied and they showed relatively higher activity in resistant hybrids than susceptible hybrids. Isozyme analysis of peroxidase (PO) and polyphenol oxidase (PPO) was also carried out in cultivars and hybrids, which revealed the induction of specific isoforms in the resistant hybrids upon challenge inoculation. This could be a useful tool for early identification of F. oxysporum f. sp. cubense resistance banana clones.     

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。     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

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...     

Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups

Fusarium oxysporum f. sp. cubense, the causal agent of fusarium wilt of banana (Musa spp.), is one of the most destructive strains of the vascular wilt fungus F. oxysporum. Genetic relatedness among and within vegetative compatibility groups (VCGs) of F. oxysporum f. sp. cubense was studied by sequencing two nuclear and two mitochondrial DNA regions in a collection of 70 F. oxysporum isolates that include representatives of 20 VCGs of F. oxysporum f. sp. cubense, other formae speciales, and nonpathogens. To determine the ability of F. oxysporum f. sp. cubense to sexually recombine, crosses were made between isolates of opposite mating types. Phylogenetic analysis separated the F. oxysporum isolates into two clades and eight lineages. Phylogenetic relationships between F. oxysporum f. sp. cubense and other formae speciales of F. oxysporum and the relationships among VCGs and races of F. oxysporum f. sp. cubense clearly showed that F. oxysporum f. sp. cubense''s ability to cause disease on banana has emerged multiple times, independently, and that the ability to cause disease to a specific banana cultivar is also a polyphyletic trait. These analyses further suggest that both coevolution with the host and horizontal gene transfer may have played important roles in the evolutionary history of the pathogen. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system should sexual reproduction occur. Although, no sexual structures were observed, some lineages of F. oxysporum f. sp. cubense harbored MAT-1 and MAT-2 isolates, suggesting a potential that these lineages have a sexual origin that might be more recent than initially anticipated.Fusarium oxysporum Schlechtendahl emend. Snyder and Hansen is a cosmopolitan species (9) comprised of both pathogenic and nonpathogenic isolates (20). The pathogenic isolates of F. oxysporum cause fusarium wilt of several agricultural crops, and are accordingly subdivided into formae speciales (3, 26, 55). One of the economically more important and destructive formae speciales is the causal agent of fusarium wilt (Panama disease) of banana (Musa spp.), F. oxysporum f. sp. cubense (E. F. Smith) Snyder et Hansen. This disease has been reported in all banana production regions of the world, except those bordering the Mediterranean, Melanesia, Somalia, and some islands in the South Pacific (66, 77).A range of approaches are typically employed for the characterization of F. oxysporum f. sp. cubense isolates. Based on virulence to specific banana cultivars (66, 67), the pathogen may be classified into one of three races (i.e., races 1, 2, and 4), although this designation may be contingent on environmental conditions. For instance, genetically identical isolates of F. oxysporum f. sp. cubense are classified as race 4 isolates in the subtropics and as race 1 isolates in the tropics because they cause disease to Cavendish bananas under subtropical conditions only (67, 86). Based on vegetative compatibility, F. oxysporum f. sp. cubense isolates have been separated into 24 so-called vegetative compatibility groups (VCGs) (5, 29, 47, 68). Finally, various DNA-based tools have been used to separate F. oxysporum f. sp. cubense into a number of clonal lineages that more or less correspond to their grouping based on VCGs (6, 22, 38, 59).The evolutionary history of F. oxysporum f. sp. cubense is complex. Based on the results of phylogenetic studies (4-7, 22, 38, 57, 59). F. oxysporum f. sp. cubense represent multiple unrelated lineages, some of which are more closely related to other formae speciales of F. oxysporum than to other F. oxysporum f. sp. cubense lineages (3, 57, 59). This has lead to speculations that new pathogenic forms of F. oxysporum may be derived from other pathogenic and nonpathogenic members of this species (21). Factors such as coevolution with the plant host and the spread of virulence determinants via processes such as parasexuality, heterokaryosis, and sexual recombination also have been implicated in the evolution of this pathogen (11, 36, 37, 39, 64, 65, 69). Although parasexuality and heterokaryosis are known to occur in F. oxysporum (11, 39), sexual fruiting structures have never been observed in the species and only indirect evidence for sexual recombination has been detected (82). Indeed, the organization of the F. oxysporum f. sp. cubense mating type locus (MAT) is similar to those found in the closely related Gibberella fujikuroi (Sawada) Ito in Ito et K. Kimura complex and other heterothallic ascomycetes (2, 90).Development of appropriate disease management strategies and the selection of F. oxysporum f. sp. cubense-resistant banana cultivars may benefit from a better understanding of the diversity and evolutionary history of the pathogen. Although most previous DNA-based studies provided knowledge regarding the diversity of F. oxysporum f. sp. cubense, the genetic relatedness among the lineages identified in these studies remains uncertain (22). It is also not clear how the different races and VCGs of F. oxysporum f. sp. cubense are related to one another and to other isolates of F. oxysporum. Therefore, the main objective of this study was to resolve the relationships among the F. oxysporum f. sp. cubense VCGs and determine their relationships with other formae speciales and nonpathogenic members of F. oxysporum by using a multigene phylogenetic approach (8, 32, 52, 53, 62, 75, 91). To facilitate the rapid differentiation of the various F. oxysporum f. sp. cubense lineages, we also aimed to develop a diagnostic PCR-restriction fragment length polymorphism (RFLP) procedure. To evaluate the potential of F. oxysporum f. sp. cubense to reproduce sexually, sexual crosses among isolates of opposite mating types were attempted after PCR-based detection of the MAT-1 and MAT-2 idiomorphs (34).     

Purification and characterization of a novel exopolygalacturonase from Fusarium oxysporum f.sp. lycopersici

A novel exopolygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with polygalacturonic acid, using two steps of purification: preparative isoelectric focusing and cationic exchange chromatography. The enzyme designated PG3 had an apparent M_r of 63 000±3000 Da upon SDS-PAGE and a pI of 7.0. PG3 was active within a broad range of pH from 3.5 to 9. The temperature optimum was 55°C. PG3 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The enzyme was N-glycosylated. The production of PG3 was constitutive at low levels, and synthesis was increased following induction by PGA and partially repressed by glucose.     

农杆菌介导的香蕉枯萎病菌4号生理小种转化体系的优化

香蕉枯萎病是世界范围内香蕉种植区最为严重的病害之一,严重威胁和影响着香蕉产业的发展.本文针对香蕉枯萎病病原菌的4号生理小种,建立了农杆菌介导的转化体系,确定了影响转化效率主要因子的优化体系是:农杆菌在IM培养基诱导前农杆菌OD_(600)为0.15、农杆菌经IM液体培养基诱导的时间为7 h、乙酰丁香酮(AS)浓度为150 μmol/L、Focr4孢子浓度为1×10~6个/mL、共培养时间为48 h、培养温度为25℃、诱导培养基pH值为5.5.在此条件下,转化效率能达到700～800个转化子/10~6个香蕉枯萎病菌孢子.PCR验证表明外源的T-DNA已经成功随机地整合到该病原菌基因组中.目前,应用该转化体系已获得2 300多个转化子,为后续克隆相关致病基因打下了良好基础.