Pectic activities from Fusarium oxysporum f. sp. melonis: Purification and characterization of an exopolygalacturonase

Abstract: Fusarium oxysporum f. sp. melonis produced an extracellular enzymic mixture with high pectic activities, (at least an exopolygalacturonase, an endopolygalacturonase and two lyases) in a medium with glucose and pectin as carbon sources. An exopolygalacturonase from this crude enzyme preparation was purified 23.8 times by Sephadex G-200 and ion-exchange HPLC. It had a K _m of 6 mM, a M _r of 58 000, a p I of 6.4, optimum pH of 5 and was stable in the 3.5–6.5 pH range. This enzyme preferentially hydrolysed polygalacturonic acid, showing only 5% activity on pectin, and did not exhibit the activity of an endoenzyme.     

Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis

Abstract Fusarium oxysporum f. sp. melonis produces extracellular endo-1,4-β-xylanase and β-xylosidase when grown in shaken culture at 26°C in a mineral salts medium containing oat spelt xylan and glucose as carbon sources. Endo-1,4-β-xylanase was purified 251 times from 5-day-old culture filtrates, by Sephacryl S-200, ion exchange and gel filtration HPLC. The purified sample yielded a single band in SDS polyacrylamide gels with a molecular mass of 80 kDa on electrophoretic mobility and 83 kDa by gel filtration behavior. High activity of the endo-1,4-β-xylanase against xylan was observed between 5 and 8 pH, and between 40 and 60°C, the optimum pH and temperature being 5.0 and 50°C, respectively. Kinetic properties of the enzyme are similar to those of other fungal xylanases, showing high affinity towards oat spelt xylan with a K _m of 1 mM expressed as xylose equivalent.     

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。     

Identification of resistance against Fusarium oxysporum f.sp. lini in linseed germplasm

Screening of germplasm/varieties was made to find out the sources of resistance against F. oxysporum f. sp. lini. Screening was conducted on 78 available germplasm/varieties during 2003–2004 and 2004–2005 in rabi season of linseed under natural conditions. Out of total 78 entries, 27 cultures were found to be resistant to disease as the disease incidence in these cultivars were between 0 and 10%. Twenty-three cultivars fell in moderately resistant category with 10.1–25% wilt incidence. Nine genotypes were found moderately susceptible sho'wing 25.1–50% disease incidence, 14 genotypes were found susceptible showing 50.1–75% and 6 genotypes were found highly susceptible to disease (above 75%).     

A rapid serological test for detecting Fusarium oxysporum f.sp. narcissi in Narcissus

Polyclonal antiserum was elicited against a strain of Fusarium oxysporum f.sp. narcissi (GCRI80/26) and a specific and sensitive enzyme-linked immunosorbent assay developed. Antiserum raised to cell wall fractions gave better recognition than that to cytoplasmic fractions. Recognition was equally good in artificially and naturally infected bulbs. Little cross-reactivity in bulb tissue was shown by three other bulb-rotting fungi. Nine isolates of F. oxysporum f.sp. narcissi from a wide geographic area gave similar results in an indirect ELISA of mycelial extracts, although some cross-reactivity was observed with two other Fusarium spp. Four Fusarium spp. and four other fungi showed little cross-reactivity. Ten days after inoculation the pathogen was readily detected in the base plate area of three Narcissus cultivars and points remote from the inoculation site in the most susceptible cultivar. A direct correlation was observed between positive results in the enzyme-linked immunosorbent assay and recovery of the pathogen on selective medium.     

Biological control of Fusarium oxysporum f. sp. radicis-cucumerinum by some Trichoderma harzianum isolates

The purpose of this study was to investigate the effects of isolates T₂₂, T₉ and T₆ of Trichoderma harzianum on isolate F₄₂ of Fusarium oxysporum f. sp. radicis-cucumerinum. The effect of T. harzianum isolates on controlling disease was examined under greenhouse conditions. Results showed that these three isolates, respectively, had the high effect on inhibition of pathogen growth. In examining the severity of disease in the greenhouse, it was found that isolate T₂₂ had the greatest effect on controlling the pathogen. The results of volatile compounds showed that these isolates, respectively, were effective in reducing mycelial growth of isolate F₄₂. The highest peroxidase activity was observed on the fourth day in isolate T₆ and the highest phenylalanine ammonia lyase enzyme activity was observed on the fifth day in isolate T₂₂. Based on the results, isolate of T₂₂ showed the greatest effect on the induction of resistance against F₄₂ and may be a successful agent for biological control of root and stem rot of cucumber.     

棉枯萎病菌异核体及其不同表型分离子在基因转录水平上的差异

采用银染mRNA差异显示技术,研究了棉花枯萎病菌异核体菌株(Ag149)及其两个不同表型分离子(Ag149-Ⅰ和Ag149-Ⅲ)之间差异表达的基因,观察到在300～700bp之间出现了19个差异条带,经反向RNA印迹,证实其中2条差异带为阳性：编号为C6的差异条带在Ag149和Ag149-Ⅰ菌株中呈高表达,而编号为G5的差异条带在菌株Ag149和Ag149-Ⅲ中呈高表达。这两条差异片段经测序后在GenBank中分别进行同源比较,发现由C6片段编码的氨基酸序列与多种生物(包括动物、植物和微生物)NADH脱氢酶的第6个亚基有不同程度的同源性(30％～70％);而编码G5片段的氨基酸序列与龟裂链霉菌(Streptomyces rimosus)的OtrB(tetracycline efflux protein)蛋白有35％的同源性。首次证明在异核体菌株及其不同表型分离子之间存在基因转录水平差异,为深入研究丝状真菌异核体形成的分子遗传机制提供了线索。     

Effect of flavonoids on the development of Fusarium oxysporum f. sp. lycopersici

Abstract

In the present study the effect of flavonoid compounds on the germination and fungal growth of the soil-borne tomato pathogen Fusarium oxysporum f. sp. lycopersici was studied. Out of 12 flavonoid compounds only myricetin and luteolin exhibited a low stimulating activity on microconidia germination of Fusarium oxysporum f. sp. lycopersici, whereas the other flavonoids tested were inactive when applied at five different concentrations. In our study the tested flavonoids affect fungal growth differently to microconidia germination. Individual flavonoid concentrations resulted in a small increase of fungal growth, but the lowest flavonoid concentrations showed an inhibiting effect on fungal growth for all flavonoids tested. There is evidence to suggest, that low flavonoid concentrations exhibit slight antimicrobial properties against Fusarium oxysporum f. sp. lycopersici.     

利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核

西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。     

Fungus gnats vector Fusarium oxysporum f.sp. radicislycopersici1

Fungus gnat adults transported Fusarium oxysporum f.sp. radicis-lycopersici from Petri dish culture and infected host plants to the roots and hypocotyls of healthy tomato and bean plants. The source of the fungus did not affect the ability of fungus gnats to transport the fungus to healthy hosts. The presence of fungus gnat larvae in media in which young tomato plants were grown did not increase the incidence of plant infection by the pathogen. Fungus gnat adults appear to aid in the dissemination of F. oxysporum f.sp. radicis-lycopersici.     

Population dynamics of Fusarium oxysporum f. sp. raphani in soils of different fungistatic capacity

Abstract Population dynamics of Fusarium oxysporum f. sp. raphani PEG-4 with a resistant marker to antibiotic hygromycin B was investigated in soils of different fungistatic capacity. Germination of PEG-4 was significantly higher in a soil amended with chemical fertilizer (CF-soil) than in a soil amended with farmyard manure (FYM-soil). Thus it was concluded that CF-soil was weakly fungistatic, and FYM-soil was strongly so. The fates of PEG-4 spores in the soils were similar when PEG-4 was introduced at initial densities of 1 10¹ to 10⁷ g⁻¹. However, alteration of environmental conditions, which might induce the germination of PEG-4 spores, resulted in the soils having different effects on the fate of PEG-4. Survival of PEG-4 in CF-soil increased compared with that in control conditions, but that in FYM-soil decreased.     

Control of chickpea wilt (Fusarium oxysporum f.sp. ciceris) using Trichoderma spp. in Ethiopia

Thirty-two Trichoderma isolates were collected from soils grown with chickpea in central highlands of Ethiopia. The eight isolates were identified by CAB-International as Trichoderma harzianum, T. koningii and T. pseudokoningii. In in vitro tests, all Trichoderma isolates showed significant (P < 0.05) differences in their colony growth and in inhibiting the colony growth of Fusarium oxysporum f.sp. ciceris, race 3. In potted experiment, four Trichoderma isolates were tested as seed treatment on three chickpea cultivars (JG-62 susceptible, Shasho moderately susceptible and JG-74 resistant) against F. oxysporum f.sp. ciceris, race 3. The result showed that T. harzianum and unidentified Trichoderma isolate T23 significantly reduced wilt severity and delayed disease onset. The degree of wilt severity and delay of disease onset varied with chickpea cultivars. Our study revealed that biological control agents such as Trichoderma can be a useful component of integrated chickpea Fusarium wilt management.     

Anatomical and biochemical aspects of interaction between roots of chickpea and Fusarium oxysporum f. sp. ciceris race 2

Roots of the susceptible “JG-62” and resistant “WR-315” chickpeas (Cicer arietinum L.) were inoculated with a conidial suspension of Fusarium oxysporum f. sp. ciceris. Anatomical and biochemical studies were carried out in a time-course manner to elucidate the infection process and plant defence reactions. Scanning electron microscope images revealed fungal colonisation in the root hair region. Early occurrence of fungal biofilms associated with the infected “JG-62” root epidermis was also visualised. After 96 h of inoculation, a gradual accumulation of polysaccharide positive deposits was observed in the xylem vessels of the infected “JG-62” roots. Fungal mycelium was observed in the vessel lumen of infected “JG-62” after 22 days of inoculation. Due to fungal invasion during this period, some of the vessels also appeared collapsed in “JG-62”, whereas vessels in “WR-315” remained intact. The host plant defence responses specifically linked to the susceptible interactions were the induction of ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase in roots and shoots.     

Selection of bacteria able to control Fusarium oxysporum f. sp. radicis-lycopersici in stonewool substrate

AIMS: Tomato foot and root rot (TFRR), caused by Fusariumoxysporum f. sp. radicis-lycopersici (Forl), is an economically important disease of tomato. The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive root-colonizing bacteria from total rhizosphere soil samples. METHODS AND RESULTS: A total of 216 potentially enhanced bacterial strains were isolated from 17 rhizosphere soil samples after applying a procedure to enrich for enhanced root tip colonizers. Amplified ribosomal DNA restriction analysis, in combination with determination of phenotypic traits, was introduced to evaluate the presence of siblings. One hundred sixteen strains were discarded as siblings. Thirty-eight strains were discarded as potential pathogens based on the sequence of their 16S rDNA. Of the remaining strains, 24 performed equally well or better than the good root colonizer Pseudomonas fluorescens WCS365 in a competitive tomato root tip colonization assay. Finally, these enhanced colonizers were tested for their ability to control TFRR in stonewool, which resulted in seven new biocontrol strains. CONCLUSIONS: The new biocontrol strains, six Gram-negative and one Gram-positive bacteria, were identified as three Pseudomonas putida strains and one strain each of Delftia tsuruhatensis, Pseudomonas chlororaphis, Pseudomonas rhodesiae and Paenibacillus amylolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a fast method for the isolation of bacteria able to suppress TFRR in stonewool, an industrial plant growth substrate. The procedure minimizes the laborious screens that are a common feature in the isolation of biocontrol strains.     

芽孢杆菌B1、B2对豌豆尖镰孢菌抗菌机理的研究

同、异步培养结果表明：芽孢杆菌B1、B2对豌豆尖孢镰孢菌(Fusarium oxysporum Schl f．sp．pisi)有很强的抑菌作用。经B1、B2无菌液处理后的病原菌由灰白色变为白色,气生菌丝增多且纠结成团。抗菌显微特征是：导致病菌孢子和菌丝体膨大、畸形、原生质凝聚、孢子不萌发或萌发异常、菌丝生长点产生大量泡囊、生长受阻,后期菌丝体断裂、泡囊破裂、原生质外泄。B1、B2无菌液中蛋白含量分别为1795．53μg/mL和1345．93μg／mL,各含一种抗菌蛋白,其分子量分别为103．5kD(B1)和127．6kD(B2)。     

Variability in Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea

The variability in cultural characteristics and the virulence among three isolates of Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea was studied under laboratory conditions. The three isolates (Foc-1, Foc-2 and Foc-3) did not show any significant difference in their mycelial dry weight production at any temperature regimes, pH level or the growth media tested. The radial growth on PDA also did not differ significantly in the three isolates. However, some quantitative differences were noted in colony characters and septations in macroconidia of these isolates. The isolate Foc-1 exhibited dull white, thin and flat hairy growth, spreading out like thread, Foc-2 showed a white fluffy colony with irregular aerial margin, while Foc-3 exhibited a pinkish white, slightly fluffy colony with regular margin. Conidia also differed with regard to septation. Three to six septa were present in Foc-2, while there were 2–3 in isolates Foc-1 and Foc-2. These isolates differed significantly with regard to their virulence on test varieties. Isolate Foc-1 was more virulent that Foc-2 or Foc-3 and produced abundant spores.     

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。     

Screening of fungal antagonists against yellows of cabbage caused by Fusarium oxysporum f. sp. conglutinans

 Screening of fungal antagonists against yellows of cabbage caused by Fusarium oxysporum f. sp. conglutinans was carried out. We obtained 78 seed-borne fungal isolates from 20 kinds of vegetable roots. Fifty-five soilborne fungal isolates were obtained from the surface-sterilized roots of seven vegetables. Twelve isolates were from field soil using a baiting method. By in vitro and in vivo screening, two seedborne species of Penicillium (S-34) and P. citrinum (S-59), and four soilborne Epicoccum nigrum (TC-33), Fusarium solani (SS-6, CM02), and F. oxysporum f. sp. lactucae (F-9501) suppressed yellows of cabbage effectively. Reductions in disease incidence ranged from 28% to 63%. Received: August 8, 2001 / Accepted: August 28, 2002 Present address:Resource Development Division, Biological Resource Center, National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0812, Japan Tel. +81-438-52-2384; Fax +81-438-52-2329 e-mail: paku-jyuyon@meti.go.jp Acknowledgments We are grateful to Dr. T. Aoki (National Institute of Agrobiological Sciences, MAFF) for identifying Fusarium species, and Dr. Kyung-min Kim (Kyungbuk University, Korea) for advising with the statistical analysis. Correspondence to:J.-Y. Park