Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Effect of Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense on Induction of Defense Enzymes and Phenolics in Banana

The effect of Pseudomonas fluorescens treatment and Fusarium oxysporum f. sp. cubense inoculation on induction of phenylalanine ammonia-lyase (PAL), peroxidase (POX), chitinase, -1,3-glucanase and accumulation of phenolics in banana (Musa sp.) was studied. When banana roots were treated with P. fluorescens strain Pf10, a two-fold increase in phenolic content in leaf tissues was recorded 3 – 6 d after treatment. Challenge inoculation with F. oxysporum, the wilt pathogen, steeply increased the phenolic content in P. fluorescens-treated banana plants. Significant increase in POX activity was detected 6 – 9 d after P. fluorescens treatment. PAL, chitinase and -1,3-glucanase activities increased significantly from 3 d after P. fluorescens treatment and reached the maximum 6 d after treatment. Challenge inoculation with F. oxysporum further increased the enzyme activities. These results suggest that the enhanced activities of defense enzymes and elevated content of phenolics may contribute to bioprotection of banana plants against F. oxysporum.     

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.     

29个香蕉基因型遗传多样性的SRAP分析

采用SRAP分子标记技术对29个香蕉品种(系)的多样性进行研究,结果显示,64对SRAP引物中筛选出25个多态性较高的引物组合,共扩增出324条条带;UPGAM聚类图显示所有供试的29个香蕉品种(系)可分为2个类群且与基因型相一致;实验结果与形态、农艺性状标记分类基本一致。研究表明,SRAP技术可有效运用于香蕉基因型的遗传和育种研究。     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.     

Prevalence of Fusarium species of the Liseola section on selected Colombian animal feedstuffs and their ability to produce fumonisins

A total of 57 samples of feedstuffs commonly used for animal nutrition in Colombia (corn, soybean, sorghum, cottonseed meal, sunflower seed meal, wheat middlings and rice) were analyzed for Fusarium contamination. Fusarium fungi were identified at species level by means of conventional methods and the ability to produce fumonisins of the most prevailing species was determined. A total of 41 of the feedstuffs analyzed (71.9%) were found to contain Fusarium spp. Most contaminated substrates were corn (100%), cottonseed meal (100%), sorghum (80%), and soybean (80%). Wheat middlings and rice showed lower levels of contamination (40% and 20%, respectively), while no Fusarium spp. could be isolated from sunflower seed meal. The most prevalent species of Fusarium isolated were F. verticilliodes (70.8%), F.␣proliferatum (25.0%), and F. subglutinans (4.2%). All of them correspond to section Liseola.Production of fumonisins on corn by the isolated Fusarium was screened through liquid chromatography. Almost all strains of F. verticilliodes (97.1%) produced FB1 (5.6–25,846.4 mg/kg) and FB2 (3.4–7507.5 mg/kg). Similarly, almost all strains of F.␣proliferatum (91.7%) produced fumonisins but at lower levels than F.␣verticilliodes (FB1 from 6.9 to 3885.0 mg/kg, and FB2 from 34.3 to 373.8 mg/kg), while F. subglutinans did not produce these toxins. This is the first study in Colombia describing toxigenic Fusarium isolates from␣animal feedstuffs.     

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

Summary With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible (Red River), one tolerant (UC 105) and three resistant (Davis UC 82, Heinz, UC 90) tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo — resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.     

Isolation of Endophytic Actinomycetes From Roots and Leaves of Banana (Musa Acuminata) Plants and Their Activities Against Fusarium oxysporumf. sp. cubense

Two hundred and forty-two actinomycete strains were isolated from the interior of leaves and roots of healthy and wilting banana plants. Most of them were streptomycetes, Streptomyces griseorubiginosus-like strains were the most frequently isolated strains. Community analysis demonstrated increased actinomycete diversity in wilting leaves compared to that in healthy leaves, similar actinomycete communities were found in wilting and healthy roots. Screening of the isolates for antagonistic activity against Fusarium oxysporumf. sp. cubenserevealed that the proportion of antagonistic streptomycetes in healthy roots was higher than that in wilting roots (P < 0.01), but no difference was found between antagonistic strains isolated from healthy and wilting leaves. The potential biological control of Panama disease of banana by endophytic streptomycetes, especially Streptomyces griseorubiginosus-like strains was discussed.     

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.     

In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

韭菜对香蕉枯萎病菌生长及香蕉枯萎病发生的抑制作用

结合实验室抑菌试验和大棚人工接菌盆栽试验,研究韭菜对香蕉枯萎病菌4号生理小种(Foc4)的拮抗作用及其对香蕉枯萎病发生的防控效果.结果显示:离体条件下,韭菜粗提取液显著抑制Foc4菌丝的生长,造成菌丝变形、细胞的解体;也能显著抑制孢子的萌发并导致孢子失去活性.大棚盆栽试验中,韭菜处理的巴西香蕉苗枯萎病发病率降低70%,病情指数降低86.9%;韭菜处理的广粉1号粉蕉苗枯萎病的发病率降低76.7%,病情指数降低93.4%.研究表明,韭菜对Foc4有很高拮抗效果,而且对香蕉枯萎病有很高的防控作用.     

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.     

Evaluation of Streptomyces sp. strain g10 for suppression of Fusarium wilt and rhizosphere colonization in pot-grown banana plantlets

Streptomyces sp. strain g10 exhibited strong antagonism towards Fusarium oxysporum f.sp. cubense (Foc) races 1, 2 and 4 in plate assays by producing extracellular antifungal metabolites. Treating the planting hole and roots of 4-week-old tissue-culture-derived Novaria banana plantlets with strain g10 suspension (10⁸ cfu/ml), significantly (P<0.05) reduced wilt severity when the plantlets were inoculated with 10⁴ spores/ml Foc race 4. The final disease severity index for leaf symptom (LSI) and rhizome discoloration (RDI) was reduced about 47 and 53%, respectively, in strain g10-treated plantlets compared to untreated plantlets. Reduction in disease incidence was not significant (P<0.05) when plantlets were inoculated with a higher concentration (10⁶ spores/ml) of Foc race 4. Rhizosphere population of strain g10 showed significant (P=0.05) increase of more than 2-fold at the end of the 3rd week compared to the 2nd week after soil amendment with the antagonist. Although the level dropped, the rhizosphere population at the end of the 6th week was still nearly 2-fold higher than the level detected after 2 weeks. In contrast, the root-free population declined significantly (P=0.05), nearly 4-fold after 6 weeks when compared to the level detected after 2 weeks. Neither growth-inhibiting nor growth-stimulating effects were observed in plantlets grown in strain g10-amended soil.     

Changes of tomato rhizosphere microflora following application of the herbicide diphenamid to soil infested with Fusarium oxyspomm f.sp. lycopersici

Significant variations were detected in species composition between untreated rhizosphere and nonrhizosphere soils of tomato plants. Application of different concentrations of active ingredient of the herbicide diphenamid (5–250 ppm) to these soils caused significant alterations in species assemblages as compared with untreated soils. Also variations in species composition were denoted between treated rhizosphere and non-rhizosphere soils.Diphenamid concentrations of 10–100 ppm significantly affected microbial counts in soil and rhizosphere of tomato plants. Counts have been stimulated at diphenamid concentrations ranging from 10–50 ppm for fungi and 10–100 ppm for bacteria. At concentrations higher than the upper limits of these ranges, R/S values were not significantly affected.The results also indicated that Fusarium oxyspomm f.sp. lycopersici populations were unaffected by diphenamid at the recommended field rate (10 ppm). Above this concentration and within the conditions of the experiment, the pathogen maintained its population at detectable inocula. Population counts of Aspergillus candidus, a species reported to be able to degrade diphenamid, were high in both treated rhizosphere and non-rhizosphere soils.     

The effects of Fusarium oxysporum f.sp. lycopersici (Sacc.) Snyder & Hansen on tomato in diphenamid-treated soil

Pre-emergence soil application of the herbicide diphenamid in concentrations exceeding the normal field rate increased the resistance of tomato plants towards infection by the wilt fungus Fusarium oxysporum f.sp. lycopersici. This was detected as significant increases in the percentage emergence of seedlings although growth parameters of the raised seedlings were reduced. Treated plants exhibited no wilt symptoms, although the pathogen maintained its population at detectable levels in the rhizosphere of tomato plants. However, the growth inhibition caused by diphenamid alone was much less than that reported for the combined application of pathogen and herbicide. Growth activities of F. oxysporum f.sp. lycopersici were inhibited by high concentrations of diphenamid in vitro. It is possible that the biodegradation of this herbicide by species such as Aspergillus candidus (present in substantial counts in treated rhizospheres) was one of the causes of increased tolerence of the pathogen to the herbicide in situ.     

Development of DArT markers and assessment of diversity in Fusarium oxysporum f. sp. ciceris,wilt pathogen of chickpea (Cicer arietinum L.)

Background

Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt of chickpea is highly variable and frequent recurrence of virulent forms have affected chickpea production and exhausted valuable genetic resources. The severity and yield losses of Fusarium wilt differ from place to place owing to existence of physiological races among isolates. Diversity study of fungal population associated with a disease plays a major role in understanding and devising better disease control strategies. The advantages of using molecular markers to understand the distribution of genetic diversity in Foc populations is well understood. The recent development of Diversity Arrays Technology (DArT) offers new possibilities to study the diversity in pathogen population. In this study, we developed DArT markers for Foc population, analysed the genetic diversity existing within and among Foc isolates, compared the genotypic and phenotypic diversity and infer the race scenario of Foc in India.

Results

We report the successful development of DArT markers for Foc and their utility in genotyping of Foc collections representing five chickpea growing agro-ecological zones of India. The DArT arrays revealed a total 1,813 polymorphic markers with an average genotyping call rate of 91.16% and a scoring reproducibility of 100%. Cluster analysis, principal coordinate analysis and population structure indicated that the different isolates of Foc were partially classified based on geographical source. Diversity in Foc population was compared with the phenotypic variability and it was found that DArT markers were able to group the isolates consistent with its virulence group. A number of race-specific unique and rare alleles were also detected.

Conclusion

The present study generated significant information in terms of pathogenic and genetic diversity of Foc which could be used further for development and deployment of region-specific resistant cultivars of chickpea. The DArT markers were proved to be a powerful diagnostic tool to study the genotypic diversity in Foc. The high number of DArT markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of diversity in the Foc population present in India, as well as provided support to know the changing race scenario in Foc population.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-454) contains supplementary material, which is available to authorized users.     

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。     

Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique

Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique. The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments. Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant, indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied.