瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测

通过测定黄瓜黑星病菌(Cladosporium cucumerinum)rDNA的ITS序列,比对近缘种及瓜类几种重要病原菌的ITS序列,设计出特异性引物HX-1/HX-2,经过对引物HX-1/HX-2PCR条件的优化,可以扩增出1条190bp的黄瓜黑星病菌特异性DNA条带,灵敏度达到1pg/μL。进一步将引物HX-1/HX-2和瓜类枯萎病菌、瓜类蔓枯病菌特异检测引物Fn-1/Fn-2、Mn-1/Mn-2组合,建立三重PCR体系,可一次检测出瓜类黑星病菌、瓜类枯萎病菌、瓜类蔓枯病菌3种瓜类植物重要的病原菌。建立了可以应用于田间瓜类黑星病菌PCR检测技术和瓜类主要病害三重PCR检测技术,对瓜类病害的诊断和防治具有重要的指导作用。     

不同提取条件对葡萄叶提取物抑制两种病原菌活性的影响

以黄瓜枯萎病菌和辣椒枯萎病菌作为供试菌种,采用生长速率法对葡萄叶不同溶剂提取物和不同提取方法提取物的抑菌活性进行测定。结果表明,葡萄叶不同溶剂提取物对供试菌种均表现出抗菌效果,并筛选出最佳提取溶剂是95%的乙醇,而最佳提取条件是提取温度40 ℃、提取时间和方式是超声波提取30 min,提取料液比1∶8(g/mL)。     

Induction of mutants of Fusarium oxysporum f. sp. lycopersici with altered virulence

Mutants ofFusarium oxysporum f. sp.lycopersici were obtained by UV irradiation. The mutants of race 1 and race 2 caused disease symptoms on plants with resistance genes against the corresponding wild type strains. Mutants of race 1 of the pathogen were stable, whereas mutants of race 2 lost the ability to cause disease symptoms in plants carrying the 1–2 resistance gene, after prolonged maintenenance on potato dextrose agar. Mutants of race 1 resembled race 2 in pathogenicity and they were vegetatively compatible with race 2, but no longer with race 1. These results suggest that the isolated strains with an altered virulence pattern have mutations in loci involved in avirulence.     

黄瓜枯萎病菌拮抗菌的筛选、鉴定和防效测定

为获得对黄瓜枯萎病菌Fusarium oxysporum f. sp. cucumerinum(FOC)具有拮抗作用的生防菌株,采用稀释涂布平板和平板生长对峙法从健康黄瓜根际土壤中分离、筛选对FOC具有强拮抗能力的拮抗细菌,并从形态学观察、生理生化特性、全细胞脂肪酸分析及16S rRNA和gyr B基因序列相似性对拮抗能力最强的菌株进行鉴定,同时测定拮抗菌无菌过滤发酵液对FOC菌丝生长、孢子萌发的抑制作用,并评价其发酵液对盆栽和大田黄瓜枯萎病的防治效果。研究结果显示,从健康黄瓜根际土壤中分离获得23株对FOC具有拮抗作用的细菌,其中菌株FJ17-4对FOC抑菌作用最强,鉴定为贝莱斯芽胞杆菌Bacillus velezensis。FJ17-4抑制FOC引起菌丝畸形、扭曲、膨胀、皱缩、缠绕等异常现象。10%无菌过滤液对FOC菌丝生长和孢子萌发的抑制率分别为70.96%和85.40%。50倍发酵液、菌体悬浮液和无菌过滤液盆栽防治效果分别为70.20%、58.87%和47.80%,大田防治效果分别为69.53%、58.46%和36.12%。综上,FJ17-4能有效抑制FOC,对黄瓜枯萎病具有显...     

Genetic diversity in Fusarium oxysporum f.sp. dianthi and Fusarium redolens f.sp. dianthi

Pathogenic isolates were selected representing all known vegetative compatibility groups (VCGs) and races of Fusarium oxysporum sensu lato from Dianthus spp. On basis of differences in the internal transcribed spacer region of the ribosomal DNA, six VCGs were classified as F. oxysporum f.sp. dianthi and four as F. redolens f.sp. dianthi. All VCGs of F. oxysporum f.sp. dianthi were characterized by unique restriction fragment length polymorphisms (RFLPs), unique overall esterase profiles, and unique virulence spectra, supporting a clonal lineage concept. Two VCGs of F. oxysporum f.sp. dianthi nevertheless comprised more than one race, but races within the same VCG shared the same distinct overall virulence spectrum. VCGs belonging to F. redolens f.sp. dianthi also had unique RFLPs and unique virulence spectra, but had grossly identical esterase profiles. Three new races (9, 10 and 11) are described for F. oxysporum f.sp. dianthi, and four for F. redolens f.sp. dianthi. Two races previously considered lost were recovered; race 7 was identified as a member of VCG 0021 of F. oxysporum f.sp. dianthi while race 3 was identified as a distinct VCG and race of F. redolens f.sp. dianthi. A summary of races and VCGs in F. oxysporum f.sp. dianthi and F. redolens f.sp. dianthi is presented.     

Detection and quantification of Fusarium oxysporum f. sp. cucumerinum in environmental samples using a specific quantitative PCR assay

The rapid and reliable identification and quantification of pathogens is essential for the management of economically important plant diseases. Fusarium oxysporum f. sp. cucumerinum is the soil borne fungus responsible for Fusarium vascular wilt of cucumber. In this study, we report the development of a specific and reliable real-time quantitative PCR assay and the development of an ultra-sensitive diagnostic pseudo-nested PCR assay. The capacity of the PCR assays to accurately identify and quantify Fusarium oxysporum f. sp. cucumerinum was experimentally tested by the development of standard curves from serial dilutions of copy numbers in a range of complex environmental DNA samples. The amplification efficiency, sensitivity and reproducibility of the qPCR assays were not significantly affected by the presence of any of the non-target background DNA tested. In quantitative real-time PCR, as few as 100 copies could be reliably quantified, and in simple and pseudo-nested PCR as little as 10 pg and 10 fg, respectively, could be detected. This rapid and sensitive qPCR method can be used to facilitate investigations into plant–pathogen interactions, epidemiology, and disease management practices.     

家畜沤肥浸渍液对黄瓜枯萎病的防治及作用机理探析

 家畜沤肥浸渍液对黄瓜枯萎病具有一定的防治效果,猪粪、马粪和牛粪沤肥浸渍液的防治效果分别为58.9%~92.5%、18.6%~72.1%和38.5%~72.8%。机理研究结果表明:(1)沤肥浸渍液对黄瓜枯萎病菌的小孢子萌发具有明显的抑制效果,马粪、牛粪、猪粪沤肥浸渍原液的抑制率分别达到89.4%、98.6%和90.3%,抑制率随稀释度增加而减小;(2)沤肥浸渍液中的部分微生物对黄瓜枯萎菌具有一定的拮抗作用。     

Sensitivity of Fusarium oxysporum f. sp. cubense to phosphonate

Phosphonate (0.1 mM) significantly reduced growth of Fusarium oxysporum f. sp. cubense (Foc) race 4 grown at an optimal phosphate concentration of 0.3 mM in vitro. At higher phosphate concentrations, closer to physiological conditions within the plant, the sensitivity of Foc race 4 to phosphonate was greatly reduced, with 25 mM phosphonate required to reduce growth by 50% at 1 mM phosphate. Two isolates of Fusarium oxysporum f. sp. dianthi and another race of Foc, race 1, were shown to be similar to Foc race 4 in their sensitivity to phosphonate, while another species of Fusarium, F. avenaceum , was more sensitive to phosphonate in vitro.     

Induced resistance in tomato plants against Fusarium wilt invoked by Fusarium oxysporum f.sp. dianthi

Tomato plants, susceptible toFusarium oxysporum f. sp.lycopersici, were inoculated by immersing the roots in a conidial suspension ofF. oxysporum f. sp.lycopersici race 1,F. oxysporum f. sp.dianthi race 2 or a mixture of both fungi. Plants inoculated withF. oxysporum f. sp.lycopersici showed disease symptoms after 2 weeks, whereas plants inoculated withF. oxysporum f. sp.dianthi or a mixture of both fungi remained symptomless for over 7 weeks, the duration of the experiment. In another experiment root systems of plants were split and each half was separately inoculated. One half was firstly inoculated withF. oxysporum f. sp.dianthi or treated with water, followed after a week by a second inoculation of the other half withF. oxysporum f. sp.lycopersici or by a water treatment. The disease symptoms in the half firstly inoculated withF. oxysporum f. sp.dianthi were significantly delayed, compared to plants of which that half had been treated with water. BecauseF. oxysporum f. sp.dianthi reduced disease symptoms caused byF. oxysporum f. sp.lycopersici without any direct interaction with this pathogen, it is concluded thatF. oxysporum f. sp.dianthi is able to induce resistance againstF. oxysporum f. sp.lycopersici in tomato plants.     

Selection methods for determining resistance of carnation cultivars to Fusarium oxysporum f.sp. dianthi

The level of resistance of carnation ( Dianthus caryophyllus ) cultivars to wilt caused by Fusarium oxysporum f.sp. dianthi was compared in root-dip-inoculated plants grown in pots (filled with tuff or sandy soil) in a greenhouse and plants grown in a field where the soil was artificially infested with the fungus. In the field, wilt symptoms appeared first in susceptible and subsequently in resistant cultivars; none was immune. Variations in the level of resistance were expressed either by different percentages of wilted plants (i.e. disease incidence) or by delayed disease progress as compared to a susceptible cultivar. The range of disease severity in the field, ranked on a scale from 0 to 4, was highly and significantly correlated with the percentage of diseased plants. The greenhouse test was unreliable as a predictor of the degree of resistance observed in the field. Similar wilt levels in the greenhouse and the field were found only in susceptible cultivars.     

Involvement of phenol metabolism in resistance of Dianthus caryophyllus to Fusarium oxysporum f.sp. dianthi

Three carnation cultivars were investigated for the effect of stem inoculation withFusarium oxysporum f.sp.dianthi on production of phenolic compounds and on fungistatic activity. Carnation stems were characterized by a complex mixture of cell wall-bound phenolics, several of which occurred in considerable amounts. Only very low amounts of phenolic compound were present in the vacuoles.Infection withF. oxysporum f.sp.dianthi induced the production and accumulation of a number of new compounds, both free in the cell sap and bound to the cell wall. In addition, the stem extracts acquired germination-inhibiting properties for conidia of the fungus. The accumulation of several phenolics and the fungistatic activity were roughly correlated to the degree of resistance of the three cultivars. Part of the differences in their resistance toF. oxysporum f.sp.dianthi might be due to an inhibition of the conversion of phenolic acid-type precursors into phytoalexins in the more susceptible cultivars.Samenvatting Drie anjercultivars werden onderzocht op de effecten van stengelinoculatie metFusarium oxysporum f.sp.dianthi op de produktie van fenolische verbindingen en op de fungistatische activiteit van stengelextracten. Anjerstengels bleken een complex mengsel van celwand-gebonden fenolzuren te bevatten, waarvan er verschillende in vrij grote hoeveelheden voorkwamen. Daartegenover waren de hoeveelheden fenolische verbindingen in de vacuole bij gezonde anjerstengels erg laag.Infectie metF. oxysporum f.sp.dianthi induceerde de produktie en accumulatie van een aantal nieuwe verbindingen in het celsap, alsook gebonden aan de celwand. Tevens bleek de remming van de kieming van conidiën vanF. oxysporum f.sp.dianthi onder invloed van deze stengelextracten sterk verhoogd, in vergelijking met de remming veroorzaakt door extracten van gezonde stengels. De accumulatie van een aantal fenolen en de verhoging van de fungistatische activiteit van de extracten waren in grote lijnen gecorreleerd met de uit de praktijk bekende resistentie van de drie cultivars. De verschillen in resistentie tegenF. oxysporum f.sp.dianthi zouden deels kunnen berusten op remming van de omzetting van precursors (fenolzuren) in fytoalexinen in de meer vatbare cultivars.     

一种评价甘蓝枯萎病菌转化子致病力的方法

为建立甘蓝枯萎病菌(Fusarium oxysporumf.sp.conglutinans)转化子致病力评价方法,从接种方法、甘蓝品种、苗龄、病原菌接种体浓度等几方面探索,建立了一种评价甘蓝枯萎病菌转化子致病力差异的体系。结果表明:蘸根法和伤根法均适于甘蓝枯萎病菌转化子致病力的评价,其中伤根法更优;其他适合甘蓝枯萎病菌转化子致病力评价的因素有:甘蓝品种为‘中甘21’,苗龄为三叶期,甘蓝枯萎病菌孢子悬浮液浓度为孢子含量1×106个/mL。该致病力评价方法的建立为甘蓝枯萎病菌转化子致病力衰弱或增强突变体的筛选提供了方法支持,也为下一步尖孢镰刀菌致病机理的解析奠定了基础。     

Diastereomer-selective resistance in Cladosporium cucumerinum to triazole-type fungicides

Diastereomers of triazole-type fungicides behaved differently with respect to their fungitoxicity towards wild-type strains of Cladosporium cucumerinum and strains resistant to ergosterol biosynthesis inhibitors. With increasing resistance to the fungicide ‘as a whole’, the difference in resistance to the two diastereomers of triadimenol became progressively smaller, whereas for the other triazole derivatives, this difference became progressively larger. The possible consequences of such phenomena for the practical use of diastereomeric fungicides are discussed.     

Association of induction of protease and chitinase in chickpea roots with resistance to Fusarium oxysporum f.sp. ciceri

To improve the breeding of chickpea varieties with resistance to Fusarium wilt, an attempt was made to analyse the biochemical basis of disease resistance by measuring levels of chitinase, β-1,3-glucanase, protease and proteinase inhibitor activities in dry and soaked seeds and in root and shoot tissues of wilt-resistant and wilt-susceptible cultivars. Marginal variation was observed in the levels of the candidate proteins in dry or soaked seeds. Chitinase activity was higher in roots than in shoots or cotyledons. No proteinase inhibitor activity was detected in root and shoot tissue of any of the cultivars. When the levels of these proteins were analysed in resistant (Vijay) and susceptible (JG-62) cultivars during development of wilt by growing plants in soil infested with F. oxysporum f.sp. ciceri , race 1, both cultivars showed induction of chitinase activity in the roots. However, the induced activity in JG-62 (3.82 U g⁻¹) was equivalent to the constitutive level in Vijay (3.90 U g⁻¹) and much lower than that induced in Vijay (5.18 U g⁻¹). Induction of protease activity was observed only in root extracts of Vijay when challenged by the pathogen. The root extract of Vijay showed in vitro antifungal activity in a plate assay. Simultaneous induction of proteolytic and chitinolytic activities specifically in the resistant cultivar was correlated with antifungal properties of root extracts effective in conferring resistance.     

Genetic diversity in Fusarium oxysporum f. sp. melonis*

Fusarium oxysporum f. sp. melonis is an important vascular wilt pathogen of melon. Races 1, 2 and 1–2 of this fungus have been identified in Portugal by pathogenicity tests with appropriate hosts. The aim of this research was to examine the relationships between different races of F. o. melonis of Portuguese and French origin through analysis of random amplified polymorphic DNAs (RAPDs). DNA fingerprint profiles were developed for all the accessions. Each isolate showed 5–10 DNA bands with each of the 16 primers employed. A total of 126 bands was obtained. The size of amplified DNA fragments generated with these primers ranged from 0.5 to 3.2 kb. A phenogram based on the Jaccard coefficient of similarity was computed by the unweighed pair group method using arithmetic averages (UPGMA). It was found that Portuguese race 2 is very similar to French race 1, while French race 2 is the most dissimilar being clearly separated from all other races. The genetic diversity of these isolates is also being studied for vegetative compatibility by using the nit mutant system.