First Record of Fusarium Wilt of Tobacco in Greece Imported as Seedborne Inoculum

Leaf yellowing and brown discoloration was observed in tobacco plants cv. Burley TN97 in tobacco fields of central Greece in 2002. Fusarium oxysporum f. sp. nicotianae was isolated from symptomatic plants and Koch's postulates were fulfilled. The pathogenicity of the isolated fungus was examined on five tobacco cultivars (Burley TN97, BurleyB21, VirginiaBE9, Virginia Niki and Anatolika KE26/2). The pathogen was present in tobacco seed batches imported in 2000 and 2001, which indicates that the infected seed is most probably the primary source of the disease in Greece. As Fusarium oxysporum f. sp. vasinfectum can also cause vascular wilt in tobacco, the hypothesis that the isolated F. oxysporum strain belongs to f. sp. vasinfectum was excluded by a pathogenicity test to cotton cv. Acala SJ‐2. This is the first report of F. oxysporum f. sp. nicotianae in Greece and the second in the European Union, although the seedborne nature of the pathogen has not been previously reported in Europe.     

Effect of Simulated Soil Solarization and Organic Amendments on Fusarium Wilt of Rocket and Basil Under Controlled Conditions

Pot trials were carried out under controlled conditions to evaluate the effectiveness against Fusarium wilt of rocket (Fusarium oxysporum f.sp. conglutinans) and basil (F. oxysporum f.sp. basilici) of soil amendments based on a patented formulation of Brassica carinata defatted seed meal and compost, combined or not with a simulation of soil solarization. The soil solarization treatment was carried out in a growth chamber by heating the soil for 7 and 14 days at optimal (55–52°C for 6 h, 50–48°C for 8 h and 47–45°C for 10 h/day) and sub‐optimal (50–48°C for 6 h, 45–43°C for 8 h and 40–38°C for 10 h/day) temperatures similar to those observed in summer in solarized soil in greenhouses in Northern Italy. Two subsequent cycles of plant cultivation were carried out in the same soil. Even at sub‐optimal temperature regimes, 7 days of thermal treatment provided very valuable results in terms of disease control on both rocket and basil. In general, the thermal treatment was more effective against F. oxysporum f.sp. basilici than against F. oxysporum f.sp. conglutinans. Control of Fusarium wilt of rocket is improved with 14 days of thermal treatment. The combination of organic amendments with a short period of soil solarization (7 or 14 days), although not providing any improvement to the level of disease management, did significantly increase biomass and positively affected yield.     

Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil

We developed two species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Fusarium spp. and Mycosphaerella spp., two pairs of species-specific primers, Fn-1/Fn-2 and Mn-1/Mn-2, were synthesized. After screening 24 isolates of F. oxysporum f. sp. niveum, 22 isolates of M. melonis, and 72 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Fn-1/Fn-2 primers amplified only a single PCR band of approximately 320 bp from F. oxysporum f. sp.niveum, and the Mn-1/Mn-2 primers yielded a PCR product of approximately 420 bp from M. melonis. The detection sensitivity with primers Fn-1/Fn-2 and Mn-1/Mn-2 was 1fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with either Fn-1/Fn-2 and or Mn-1/Mn-2, two nested PCR procedures were developed, and the detection sensitivity increased 1000-fold to 1ag. The detection sensitivity for the soil pathogens was 100-microconidia/g soil. A duplex PCR method, combining primers Fn-1/Fn-2 and Mn-1/Mn-2, was used to detect F. oxysporum f. sp. niveum and M. melonis in plant tissues infected by the pathogens. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogens directly in soil samples. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Common Bean Reaction to Fusarium oxysporum f. sp. Phaseoli, the Cause of Severe Vascular Wilt in Central Africa

During the September‐December season of 1990, severe symptoms of Fusarium wilt were for the first time observed on a popular climbing bean (Phaseolus vulgaris L.) cultivar. G 2333. introduced within the previous 5 years. Seventy‐three bean genotypes were screened for resistance lo the disease, using artificial inoculation. The effect of inoculation density on the reaction of four selected genotypes was also investigated. Of the 29 climbing bean genotypes evaluated, 19 were resistant, including 11 of the 15 pre‐release or released cultivars. Of the 44 bush bean cultivars evaluated, 28 were resistant, five were intermediate and 11 were susceptible. All susceptible cultivars showed vascular discoloration. In both susceptible and resistant genotypes, the fungus spread almost equally from the entry points in inoculated roots to the base of the plants, but colonization and vertical spread within the vascular system were markedly less in resistant than in susceptible cultivars. At 20 and 30 cm above soil level, the fungus was only recovered from susceptible cultivars. Increasing inoculum density from 10² to 10⁷ conidia/ml did not affect the resistance of cultivars RWR 950 and G 685 but. in the susceptible cultivars G 2333 and MLB‐48‐89 A. it resulted in early appearance, high incidence and severity of the disease.     

Biological Control of Fusarium Wilt in Tomato Caused by Fusarium oxysporum f. sp. lycopersici by AMF Glomus intraradices and some Rhizobacteria

In the present study, the effects of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Schenck & Smith and four rhizobacteria (RB; 58/1 and D/2: Pseudomonas fluorescens biovar II; 17: P. putida; 21: Enterobacter cloacae), which are the important members of the rhizosphere microflora and biological control agents against plant diseases, were examined in the pathosystem of Fusarium oxysporum f. sp. lycopersici [(Sacc) Syd. et Hans] (FOL) and tomato with respect to morphological parameters (fresh and dry root weight) and phosphorous (P) concentration in the roots. Treatments with single and dual inoculation with G. intraradices and RB strains reduced disease severity by 8.6–58.6%. Individual bacteria inoculations were more effective than both the single AMF and dual (G. intraradices + RB) inoculations. In addition, the RB and G. intraradices enhanced dry root weight effectively. Significant increases in root weights were recorded particularly in the triple inoculations compared with single or dual inoculations. Compared with the non‐treated controls all biological control agents increased P‐content of treated roots of plants. Colonization with RB increased especially in triple (FOL + G. intraradices + RB) inoculations whereas colonization of G. intraradices was significantly decreased in treatment of FOL + G. intraradices compared with triple inoculations. The results suggest that suitable combinations of these biocontrol agents may ameliorate plant growth and health.     

Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporum

A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum.     

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.     

Biocontrol of Fusarium Wilt and Growth Promotion of Tomato Plants Using Endophytic Bacteria Isolated from Solanum elaeagnifolium Stems

Seven culturable bacterial isolates, obtained from the internal stem tissues of Solanum elaeagnifolium and successfully colonizing the internal stem tissues of tomato cv. Rio Grande, were screened for their in vivo antifungal activity against Fusarium oxysporum f.sp. lycopersici (FOL) and their growth‐promoting potential on tomato plants. SV101 and SV104 isolates, assessed on pathogen‐challenged tomato plants led to a significant decrease (77–83%) in Fusarium wilt severity and vascular browning extent (76%), as compared to the inoculated and untreated control. Isolates enhanced growth parameters on pathogen‐challenged and unchallenged tomato plants. SV104 and SV101 isolates were most effective in suppressing disease and enhancing plant growth. These two isolates were identified as Bacillus sp. str. SV101 ( KU043040 ) and B. tequilensis str. SV104 ( KU976970 ). They displayed antifungal activity against FOL; pathogen growth was inhibited by 64% and an inhibition zone (11.50 and 19.75 mm) against FOL could be formed using whole cell suspensions. SV101 and SV104 extracellular metabolites also inhibited FOL growth by 20 and 55%, respectively, as compared to control. B. tequilensis str. SV104 was shown to produce protease, chitinase, pectinase, IAA and siderophores. Bacillus sp. str. SV101 displayed pectinase activity and was found to be an IAA‐producing and phosphate‐solubilizing agent. To our knowledge, this is the first study reporting on S. elaeagnifolium use as a potential source of potent biocontrol and plant growth‐promoting agents.     

Effects of Commercial and Indigenous Microorganisms on Fusarium Wilt Development in Chickpea

The purpose of this research was to determine whetherBacillus subtilis,nonpathogenicFusarium oxysporum,and/orTrichoderma harzianum,applied alone or in combination to chickpea (Cicer arietinumL.) cultivars ‘ICCV 4’ and ‘PV 61’ differing in their levels of resistance to Fusarium wilt, could effectively suppress disease caused by the highly virulent race 5 ofFusarium oxysporumf. sp.ciceris.Seeds of both cultivars were sown in soil amended with the three microbial antagonists, alone or in combination, and 7 days later seedlings were transplanted into soil infested with the pathogen. All three antagonistic microorganisms effectively colonized the roots of both chickpea cultivars, whether alone or in combination, and significantly suppressed Fusarium wilt development. In comparison with the control, the incubation period for the disease was delayed on average about 3 days and the final disease severity index and standardized area under the disease progress curve were reduced significantly between 14 and 33% and 16 and 42%, respectively, by all three microbial antagonists. Final disease incidence only was reduced byB. subtilis(18–25%) or nonpathogenicF. oxysporum(18%). The extent of disease suppression was higher and more consistent in ‘PV 61’ than in ‘ICCV 4’ whether colonized byB. subtilis,nonpathogenicF. oxysporum,orT. harzianum.The combination ofB. subtilis+T. harzianumwas effective in suppressing Fusarium wilt development but it did not differ significantly from treatments with either of these antagonists alone. In contrast, the combination ofB. subtilis+ nonpathogenicF. oxysporumtreatment was not effective but either antagonist alone significantly reduced disease development.     

Detection and Quantification of Fusarium oxysporum f. sp. niveum Race 1 in Plants and Soil by Real-time PCR

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/μl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.     

Occurrence and correlations of nematodes,Fusarium oxysporum and edaphic factors on banana plantations

Problems caused by nematodes and Fusarium wilt (Panama disease) on banana plantations are responsible for yield losses and are limiting to its cultivation. In the state of Goias, there is little information about the nematode occurence on this crop, and its relation with the incidence of Fusarium oxysporum f.sp. cubense (Foc). This research had the purpose to identify the occurrence of plant‐parasitic nematodes on banana plantations and to verify its correlation with the Fusarium wilt and with the soil attributes (pH, texture, nutrients). Twelve banana orchards in the state of Goias were sampled in the municipalities of Anapolis, Caiaponia, Goiatuba, Itaguaru, Itumbiara (two areas), Jatai, Morrinhos, Ouro Verde, Palestina, Taquaral and Uruana. All sampled areas, except Morrinhos, revealed contamination with Foc, and all areas had different genera of nematodes either in the soil or in the roots samples. Meloidogyne sp., Helicotylenchus sp. and Rotylenchus sp. were the main genera of plant‐parasitic nematodes found in the samples, with Meloidogyne sp. and Rotylenchus sp. being the most dominant and abundant genera. The presence of Pratylenchus sp. increases the population levels of F. oxysporum. Helicotylenchus sp. is highly correlated with high concentrations of Mn. High population density of Meloidogyne sp. was found in irrigated areas with low concentrations of P, Ca, Mg and soil pH.     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

Biocontrol Activity of Aspergillus terreus ANU-301 against Two Distinct Plant Diseases,Tomato Fusarium Wilt and Potato Soft Rot

To screen antagonistic fungi against plant pathogens, dual culture assay (DCA) and culture filtrate assay (CFA) were performed with unknown soil-born fungi. Among the different fungi isolated and screened from the soil, fungal isolate ANU-301 successfully inhibited growth of different plant pathogenic fungi, Colletotrichum acutatum, Alternaria alternata, and Fusarium oxysporum, in DCA and CFA. Morphological characteristics and rDNA internal transcribed spacer sequence analysis identified ANU-301 as Aspergillus terreus. Inoculation of tomato plants with Fusarium oxysporum f. sp. lycopersici (FOL) induced severe wilting symptom; however, co-inoculation with ANU-301 significantly enhanced resistance of tomato plants against FOL. In addition, culture filtrate (CF) of ANU-301 not only showed bacterial growth inhibition activity against Dickeya chrysanthemi (Dc), but also demonstrated protective effect in potato tuber against soft rot disease. Gas chromatography-tandem mass spectrometry analysis of CF of ANU-301 identified 2,4-bis(1-methyl-1-phenylethyl)-phenol (MPP) as the most abundant compound. MPP inhibited growth of Dc, but not of FOL, in a dose-dependent manner, and protected potato tuber from the soft rot disease induced by Dc. In conclusion, Aspergillus terreus ANU-301 could be used and further tested as a potential biological control agent.     

Genetics of resistance to Fusarium oxysporum f.sp. cucumerinum races 1 and 2 in cucumber line Wisconsin-2757

The mode of inheritance of resistance to Fusarium oxysporum f.sp. cucumerinum races 1 and 2 in Wisconsin-2757 (WI-2757), a gynoecious cucumber (Cucumis sativus L.), was determined by analysing segregation of F₁, F₂ and BC₁ populations of crosses with susceptible cultivar Straight-8. Resistance to either race 1 or race 2 in WI-2757 was conferred by a single dominant gene. In allelism tests, resistance to either race in WI-2757 was determined by the gene Fcu-1, which also confers resistance in line SMR-18.     

Race Profiling and Molecular Diversity Analysis of Fusarium oxysporum f.sp. ciceris Causing Wilt in Chickpea

Seventy isolates of Fusarium oxysporum f.sp. ciceris (Foc) causing chickpea wilt representing 13 states and four crop cultivation zones of India were analysed for their virulence and genetic diversity. The isolates of the pathogen showed high variability in causing wilt incidence on a new set of differential cultivars of chickpea, namely C104, JG74, CPS1, BG212, WR315, KWR108, GPF2, DCP92‐3, Chaffa and JG62. New differential cultivars for each race were identified, and based on differential responses, the isolates were characterized into eight races of the pathogen. The same set of isolates was used for molecular characterization with four different molecular markers, namely random amplified polymorphic DNA, universal rice primers, simple sequence repeats and intersimple sequence repeats. All the four sets of markers gave 100% polymorphism. Unweighted paired group method with arithmetic average analysis grouped the isolates into eight categories at genetic similarities ranging from 37 to 40%. The molecular groups partially corresponded to the states of origin/chickpea‐growing region of the isolates as well as races of the pathogen characterized in this study. The majority of southern, northern and central Indian populations representing specific races of the pathogen were grouped separately into distinct clusters along with some other isolates, indicating the existence of variability in population predominated by a single race of the pathogen. The present race profiling for the Indian population of the pathogen and its distribution pattern is entirely new. The knowledge generated in this study could be utilized in resistance breeding programme. The existence of more than one race, predominated by a single one, in a chickpea cultivation zone as supported by the present molecular findings is also a new information.     

Vibrational, 1H-NMR spectroscopic,and thermal characterization of gladiolus root exudates in relation to Fusarium oxysporum f. sp. gladioli resistance

Fourier transform Raman (FT Raman) and IR (FTIR) and (1)H-NMR spectroscopies coupled with differential scanning calorimetry (DSC) were applied to the characterization of root exudates from two cultivars of gladiolus (Spic Span and White Prosperity) with different degrees of resistance and susceptibility to Fusarium oxysporum gladioli, the main pathogen of gladiolus. This work was aimed at correlating the composition of root exudates with the varietal resistance to the pathogen. Spectroscopic analysis showed that White Prosperity root exudate differs from Spic Span root exudate by a higher relative amount of the aromatic-phenolic and sugarlike components and a lower relative amount of carbonylic and aliphatic compounds. DSC analysis confirmed the spectroscopic results and showed that White Prosperity root exudate is characterized by an aromatic component that is present in a higher amount than in the Spic Span root exudate. The results are discussed in relation to the spore germination tests showing that White Prosperity, which is characterized by a remarkable resistance toward F. oxysporum gladioli, exudes substances having a negative influence on microconidial germination of the pathogen; root exudates from Spic Span, one of the most susceptible cultivars to F. oxysporum gladioli, proved to have no effect. White Prosperity's ability to inhibit conidial germination of F. oxysporum gladioli can be mainly related to the presence of a higher relative amount of aromatic-phenolic compounds.     

Induced resistance to Botrytis cinerea in Capsicum annuum by a Fusarium crude elicitor fraction,free of proteins

Fusarium oxysporum f. sp. lycopersici (FOL) induces resistance in pepper against the airborne pathogen Botrytis cinerea and the soil‐borne pathogen Verticillium dahliae. However, its practical use is limited due to its pathogenicity to other crops. In this study we tested several fractions of a heat‐sterilised crude FOL‐elicitor preparation to protect pepper against B. cinerea and V. dahliae. Only the protein‐free insoluble fraction of the preparation reduced B. cinerea infection. However, none of the fractions reduce V. dahliae symptoms. The insoluble protein‐free fraction induced expression of defence genes in the plant, namely a chitinase (CACHI2), a peroxidase (CAPO1), a sesquiterpene cyclase (CASC1) and a basic PR1 (CABPR1). Even though the CASC1 gene was not induced directly after treatment with the insoluble fraction in the leaves, it was induced after B. cinerea inoculation, showing a priming effect. The insoluble protein‐free FOL‐elicitor protected pepper against the airborne pathogen through a mechanism that involves induced responses in the plant, but different to the living FOL.     

香蕉枯萎病菌粗毒素对地衣芽胞杆菌生长和培养液上清蛋白组成的影响

本文研究了香蕉枯萎病菌4号生理小种湛江菌株(Foc 4-zj)产生的粗毒素对地衣芽胞杆菌R21菌株生长及其培养液中蛋白组成变化的影响。实验结果表明, Foc 4-zj的粗毒素能够抑制R21菌株的生长, 缩短其生长周期; 减少培养液上清蛋白含量以及改变蛋白质的种类; 低剂量的粗毒素有利于拮抗蛋白的积累, 而高剂量的粗毒素则相反。