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.     

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.     

Biocontrol of Fusarium oxysporum f.sp. cubense tropical race 4 by formulated cells and cell-free extracts of Streptomyces griseus in sterile soil environment

The biocontrol activities of cells and cell-free extracts of Streptomyces griseus was tested against Fusarium oxysporum f.sp. cubense tropical race 4 (FOC race 4) in a sterile soil environment. They were first formulated in sodium alginate, kaolin clay and in alginate–kaolin combination, prior to introducing into sterile soil inoculated with 6 log₁₀ cfu FOC race 4 g^?1 soil. Results revealed that bioformulated cells of S. griseus, irrespective of the materials used, were generally more effective in inhibiting growth of FOC race 4 when compared to non-formulated cells of S. griseus. Kaolin was the most suitable inert material as formulation of S. griseus with kaolin effectively suppressed FOC race 4, with only 5.40 log₁₀ cfu g^?1 of FOC race 4 recovered after 20 days. Kaolin formulations also allowed good cell recovery post-formulation. Alginate was less desirable as poorer control was demonstrated, with 6.12 and 6.16 log₁₀ cfu g^?1 of FOC race 4 recovered from soils treated with alginate only and alginate–kaolin formulated S. griseus, respectively. Bioformulations did not benefit cell-free extracts at all. Our study suggests formulation of cells of S. griseus is more beneficial than cell-free extracts and kaolin is the preferred material for formulation.     

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.     

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.     

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.     

Isolation and characterization of an antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII

An antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII has been isolated and partially characterized. The antibiotic is produced throughout culture growth, with maximum amounts accumulating in the broth when the culture is in the early stationary phase of growth. The activity declines within about 30 h after the culture enters stationary phase. Purification techniques included chromatography on Amberlite XAD-2, DEAE Sephadex and SP Sephadex in addition to C18 HPLC with an average yield of 75%. This antibiotic only inhibits pathogenic strains of S. scabies that cause scab disease on potato and other tuberous vegetables and does not affect S. griseus, S. venezuelae, Actinomyces bovis, Nocardia asteroides, Clostridium perfringens, Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, Micrococcus luteus, Serratia marcescens and Escherichia coli. The antibiotic has a molecular weight of 500 or less, and is stable for weeks at acidic pH but is very labile at alkaline pH conditions. Received 18 February 1997/ Accepted in revised form 11 August 1997     

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.     

Changes in the redox status of chickpea roots in response to infection by Fusarium oxysporum f. sp. ciceris: apoplastic antioxidant enzyme activities and expression of oxidative stress-related genes

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas ( Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15–17 days after inoculation) and (iii) when all plants had developed disease symptoms (20–22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier ( i.e. GR and SOD) or to higher levels ( i.e. DAO) in the incompatible interaction. Expression of apx , cat , sod , lipoxygenase ( lox ) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier ( lox ) or higher ( actin ) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.     

Inheritance and genetic linkage of fusarium wilt {Fusarium oxysporum f.sp. cucumerinum race 1) and scab {Cladosporium cucumerinum) resistance genes in cucumber (Cucumis sativus)

The inheritance of resistance of the cucumber cv. SMR 18 to the race 1 of Fusarium oxysporum f.sp. cucumerinum, the linkage relationship between resistance to race 1 of F. oxysporum f.sp. cucumerinum, resistance to Cladosporium cucumerinum and fruit spine colour, and the reactions of several cucumber cultivars to inoculations with race 1 of F. oxysporum f.sp. cucumerinum and C. cucumerinum were examined. The inbred line Straight 8 (P,), which has white fruit spines and is susceptible to both fusarium wilt and scab was crossed with the inbred line SMR 18 (P₂), which has black fruit spines and resistance to both diseases. When F, F₂, F₃, BC₁P₁ BC₁P₂ and BC₁P₁ selfed progenies were inoculated at the cotyledon stage with a suspension of spores of race 1 of F. oxysporum f.sp. cucumerinum, the ratios of resistant to susceptible plants indicated that resistance was conferred by a single dominant gene, designated Fcu-1. When 171 BC^! plants were selfed and from each resulting F₂ family different groups of 15–25 seedlings each were tested for resistance to either disease, segregation data indicated that the Fcu-1 locus and the Ccu locus for C. cucumerinum resistance were completely linked. No evidence for linkage was found between the Fcu-1 (Ccu) locus and the B locus for fruit spine colour. Among the 59 cultivars tested at the seedling stage, 15 were susceptible, while the remainder were highly resistant to inoculations with both pathogens.