应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

以3株黄瓜尖镰孢菌(Fusarium oxysporum f.sp.cucumarinum)、23株镰孢菌属(Fusariumspp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料,采用化学裂解法提取总DNA,进行PCR-RFLP和巢式PCR检测,试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种,而巢式PCR对黄瓜尖镰孢菌具有特异性.运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测,结果表明,两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌,PCR-RFLP在感病品种接种后3d即可检测到病原菌,而巢式PCR在接种后5d才能检测到病原菌.     

Purification and characterization of an exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici

Abstract An exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici , a fungus that produces root rot, was purified by gel filtration and ion exchange chromatography. It had a M _r 68 K, a pH optimum of 5.6 and an optimum temperature of 60°C. This polygalacturonase was inhibited by calcium ions and had a K _m of 0.64 mM using sodium polypectate as substrate. The exo mode of action of this enzyme was revealed by thin-layer chromatography of hydrolysed substrate.     

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。     

Effet de L'acide Caféoylshikimique des Racines du Palmier Dattier sur L'activité et la Production des Enzymes Hydrolytiques de Fusarium oxysporum f. sp. albedinis

Effect of caffeoylshikimic acid of date palm roots on activity and production of Fusarium oxysporum f. sp. albedinis cell wall‐degrading enzymes The caffeoylshikimic acid (CSA), a major phenolic compound of date palm roots, represents one of the resistance factors of the host to Fusarium oxysporum f. sp. albedinis. The CSA was tested at various concentrations (0,25 to 3 µ mol/ml) on the activity and the production of F. oxysporum f. sp. albediniscell wall‐degrading enzymes (CWDE): proteases, cellulases, pectinemethyl‐esterases (PME), polygalacturonases (PG) and polygalacturonate trans‐eliminases (PGTE). The results obtained show that CSA had very little effect on the activity of the various enzymes although it greatly reduced their production. The mycelial growth was also affected by CSA, but this does not explain why only the production of CWDE was noticeably reduced. In order to explain this differential effect of CSA on the activity and production of CWDE, in one group of experiments the effects of the products of hydrolysis of CSA (caffeic acid and shikimic acid) was tested and in another, the effect of the products of CSA (quinones) obtained by tyrosinase oxidation was investigated. The results obtained show that the shikimic acid did not have a significant effect on the activity of the CWDE but presented a weak inhibition of their production.The caffeic acid showed a larger inhibition of the activity of the various CWDE that was more than that of CSA and its inhibiting effect appeared to be more important during their production. The oxidation of CSA by tyrosinase was accompanied by a greater inhibition of the activity of the various CWDE. This inhibition was appreciable in comparison with that observed due to the effect of non‐oxidized CSA on CWDE production. In the same way, oxidation of caffeic acid provoked a greater inhibiting effect on the activity of CWDE than unoxidized caffeic acid. These results suggests that CSA generates products of hydrolysis (in particular caffeic acid) and products of oxidation (quinones) which inhibit the activity of the proteolytic, cellulolytic and pectinolytic enzymes produced by F. oxysporum f. sp. albedinis in the culture medium.     

Characterization of emerging populations of Fusarium oxysporum f. sp. conglutinans causing cabbage wilt in China

Fusarium oxysporum f. sp. conglutinans (FOC) causes Fusarium wilt, a disease of cabbage that has brought about significant economic loss throughout northern China since it was first detected in 2001. To characterize the Chinese FOC isolates, we compared the cultural characteristics, pathogenicity and races between the Chinese isolates and the type strains (race 1: 52,557 and race 2: 58,385). The Chinese FGL‐03‐6 isolate had cultural characteristics similar to those of strain 52,557, including colony growth rate, colony and spore characteristics and responses to temperature changes, while the strain 58,385 grew faster, produced more pigment and spores and was more adaptable to temperature fluctuations. The lethal temperature for all strains was 60°C, and the optimal temperatures for pathogen growth on potato dextrose agar and pathogenicity on plants were 25°C and 25 to 30°C, respectively. Tests for race and pathogenicity indicated that different cabbage cultivars had similar resistance reactions to FGL‐03‐6 and 52,557. However, the pathogenicity of FGL‐03‐6 was similar to that of 58,385, which infected quickly and caused more severe disease symptoms. This study further provides information regarding characterizing different strains of F. oxysporum f. sp. conglutinans.     

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%).     

Characterization of Isolates of Fusarium oxysporum Schlecht. f. sp. vasinfectum (Atk.) Snyd. & Hans., Races 1-6, by Cellular Fatty Acid Analysis

Fatty acid analysis, a common method for the identification of bacteria, was modified and applied to characterize isolates of Fusarium oxysporum f. sp. vasinfectum. After evaluating the fatty acid profiles by means of cluster analysis and three-dimensional plotting of the main fatty acids, the isolates were classified into five groups. Identical patterns were obtained for isolates of races 3 and 5 and for isolates of races 2 and 6. Isolates with so far unknown race determinations were arranged into their corresponding fatty acid groups.     

Gerbera jamesonii, Osteospermum sp. and Argyranthemum frutescens: New Hosts of Fusarium oxysporum f. sp. chrysanthemi

The pathogenicity of five isolates of Fusarium oxysporum obtained from infected gerbera (Gerbera jamesonii), chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.) plants was tested on some varieties of the following Compositae hosts: C. morifolium, G. jamesonii, Argyranthemum frutescens (Paris daisy) and Osteospermum sp. and compared with the host range and pathogenicity of an isolate of F. oxysporum f. sp. chrysanthemi obtained from the ATCC collection. The results indicated that isolates of F. oxysporum from G. jamesonii as well as those from A. frutescens and Osteospermum sp. belong to the forma specialischrysanthemi. The isolate from gerbera was virulent on all tested varieties of gerbera, C. morifolium, A. frutescens and Osteospermumsp. Similar results were obtained testing the isolates obtained from A. frutescens and Osteospermumsp. The strain from C. morifolium infected cultivar of gerbera, A. frutescens and Osteospermum sp. The pathogenicity of isolate of F. oxysporum f. sp. chrysanthemi obtained from the ATCC showed a different cultivar range particularly in the case of chrysanthemum and gerbera.     

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。     

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.     

茄子自毒物质对辣椒种子萌发及枯萎菌的化感效应

运用模拟的方式,采用生物测定和室内培养的方法,研究了两种茄子自毒物质香草醛和肉桂酸各浓度对辣椒种子萌发和幼苗生长的化感效应,及其对辣椒枯萎菌菌丝生长的影响.结果表明:这两种自毒物质对辣椒种子萌发和幼苗生长具有低浓度促进,高浓度抑制的化感效应.香草醛和肉桂酸对辣椒和茄子种子的化感效应存在较大差异,辣椒种子受这两种自毒物质的抑制强度明显弱于茄子种子.香草醛和肉桂酸对辣椒枯萎菌菌丝生长表现为各浓度(0.1, 0.5, 1, 4mmol/L)下均具有抑制作用,作用强度随着浓度增加而增强,肉桂酸低浓度时对菌丝生长的抑制作用即达到显著水平.     

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.     

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。     

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.     

Genetic Variability of Fusarium oxysporum f.sp. ciceris Population Affecting Chickpea in the Sudan

Fusarium wilt caused by Fusarium oxysporum f.sp. ciceris (Foc) is the most important soilborne disease of chickpea in the Sudan and many other countries. A total of 76 Foc isolates from six different chickpea‐growing states in the Sudan have been collected in this study to investigate the genetic diversity of Sudanese Foc isolates. Additional 14 Foc isolates from Syria and Lebanon were included in this study. All isolates were characterized using four random amplified polymorphic DNA (RAPD), three simple sequence repeats (SSR), five sequence‐characterized amplified region (SCAR) primers and three specific Foc genome primers. Based on the similarity coefficient, the results indicated two major clusters included seven subclusters. The isolates from the Sudan were grouped as identified as races 0, 2 and unknown races. The isolates from Syria and Lebanon were grouped together as they identified as races 1B/C and 6, respectively. This study identified a new race Foc (race 0) in the Sudan. The results of this study will be useful for breeders to design effective resistance breeding program in chickpea in the Sudan.     

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.     

SDSC-TAB和高盐沉淀法提取香蕉枯萎病菌基因组DNA的比较

以香蕉枯萎病菌菌株为试验材料,采用SDS- CTAB法和高盐沉淀法提纯香蕉枯萎病菌基因组DNA。结果表明:高盐沉淀法是适合于香蕉枯萎病菌基因组DNA提取的方法。该方法提取的DNAOD2 60 2 80值显示产物纯度较高;经琼脂糖凝胶电泳得到一条带型较宽且清晰的DNA谱带,DNA浓度较高,基本无DNA碎带;不用RNase处理,已无RNA的干扰,无需任何纯化处理即可用于PCR扩增和RAPD分析。同时对DNA提取过程中的细节问题进行了探讨与分析。     

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.