Interactions between root-knot nematode Meloidogyne javanica and Fusarium wilt disease, Fusarium oxysporum f.sp. Melonis in different varieties of melon

Fusarium oxysporum f.sp. melonis and root-knot nematode (Meloidogyne javanica) are destructive pathogens on cucurbits in Varamin area of Iran. The interaction between two pathogens was studied on local melon cultivars, Garmsar and Sooski. Inoculum of Meloidogyne javanica was prepared on susceptible cultivar, Rutgers using single egg mass method in greenhouse. Inoculum of Fusarium oxysporum f.sp. melonis (race 1) was prepared using Richard solution. A concentration of 2 x 10(5) micro conidia of fungus and 2000, 3000, 4000, 5000 eggs of nematode was used in 1 kg of autoclaved soil. Plants were inoculated with nematode at 2-3 leave stage then with fungus 2 weeks after nematode inoculation. The experiment was conducted in factoriel design based on CRD with 20 treatments, including varieties in 2 levels (Garmsar and Sooski), nematode in 5 levels (0, 2000, 3000, 4000, 5000 eggs) and fungus in 2 levels (presence and absence) and 3 replicates. The index that evaluated were growth index including fresh and dry weight of shoot and root, height, Fusarium wilt index and root gall index. Results of this experiment showed that all of treatments comparison to control were significantly different (p = 0.05) in growth index. Combination of fungus and nematode (5000 eggs) caused the most decrease in growth index on Garmsar and Sooski.     

Temperature effect on Fusarium oxysporum f.sp. melonis survival during horticultural waste composting

AIMS: The aim of this work was to study the effect of high temperatures generated during composting process, on the phytopathogen fungus Fusarium oxysporum f.sp. melonis. This investigation was achieved by both in vivo (semipilot-scale composting of horticultural wastes) and in vitro (lab-scale thermal treatments) assays. METHODS AND RESULTS: Vegetable residues infected with F. oxysporum f.sp. melonis were included in compost piles. Studies were conducted in several compost windrows subjected to different treatments. Results showed an effective suppression of persistence and infective capacity, as this process caused complete fungal elimination after 2-3 days of composting. In order to confirm the effect of high temperature during this process, in vitro experiments were carried out. Temperature values of 45, 55 and 65 degrees C were tested. All three treatments caused the elimination of fungal persistence. Treatment at 65 degrees C was especially effective, whereas 45 degrees C eliminated fungal persistence only after 10 days. CONCLUSIONS: The composting process is an excellent alternative for the management of plant wastes after harvesting, as this procedure is able to suppress infective capacity of several harmful phytopathogens such as F. oxysporum f.sp. melonis. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium oxysporum f.sp. melonis is a plant pathogen fungus specially important in the province of Almería (south-east Spain), where intensive greenhouse horticulture is very extended. High temperatures reached during composting of horticultural plant wastes ensure the elimination of phytopathogen microorganisms such as F. oxysporum f.sp. melonis from vegetable material, providing an adequate hygienic quality in composts obtained.     

Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95-1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95-1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.     

Differential interaction between melon cultivars and race 1.2 of Fusarium oxysporum f.sp. melonis

One hundred and ten melon accessions from different origins were evaluated for resistance to race 1.2 of Fusarium oxysporum f.sp. melonis (Fom). Artificial inoculations were performed and plants were scored for presence or absence of symptoms. The screening revealed that sources of resistance to this fungus are limited. However, a few resistant accessions were found. The accessions 'Kogane Nashi Makuwa', 'C-211' and 'C-40' from Japan provided the highest resistance to both patotypes of race 1.2 of Fom which seems to confirm that the Far East is a rich area in resistances to this race. We also detect an interesting degree of resistance in 'BG-5384', a Portuguese accession that may be classified as belonging to the botanical variety inodorus.     

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.     

Plant colonization by the vascular wilt fungus Fusarium oxysporum requires FOW1, a gene encoding a mitochondrial protein

The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. The pathogenicity mutant B60 of the melon wilt pathogen F. oxysporum f. sp. melonis was isolated previously by restriction enzyme-mediated DNA integration mutagenesis. Molecular analysis of B60 identified the affected gene, designated FOW1, which encodes a protein with strong similarity to mitochondrial carrier proteins of yeast. Although the FOW1 insertional mutant and gene-targeted mutants showed normal growth and conidiation in culture, they showed markedly reduced virulence as a result of a defect in the ability to colonize the plant tissue. Mitochondrial import of Fow1 was verified using strains expressing the Fow1-green fluorescent protein fusion proteins. The FOW1-targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also showed reduced virulence. These data strongly suggest that FOW1 encodes a mitochondrial carrier protein that is required specifically for colonization in the plant tissue by F. oxysporum.     

甜瓜根系分泌物中酚酸物质对尖孢镰孢菌的化感效应

采用HPLC法对甜瓜根系分泌物进行分离鉴定,检测到甜瓜根系分泌物中含有没食子酸、邻苯二甲酸、丁香酸、水杨酸、阿魏酸、苯甲酸和肉桂酸7种酚酸物质,通过外源添加法研究该类物质对尖孢镰孢菌的化感效应.室内试验结果表明: 阿魏酸、苯甲酸、肉桂酸在0.1、0.25 mmol·L^-1处理浓度下能够显著促进尖孢镰孢菌的孢子萌发,水杨酸则对孢子萌发具有一定的抑制作用;丁香酸、阿魏酸在菌丝培养后期表现出较强的促进作用.盆栽结果显示,在0.05、0.1和0.5 mmol·L^-1处理浓度下肉桂酸、阿魏酸、苯甲酸可显著促进甜瓜枯萎病病情.
     

Induced Resistance in Cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2–2 by Infection of the Cotyledons

Localized infection in cucumber cotyledons with Colletotrichum lagenarium induced resistance against infection after challenge inoculation with Rhizoctonia solani AG2–2 and Fusarium oxysporum f. sp. cucumerinum in the roots. The plants were unprotected in soil that was infested heavily with R. solani or in contact with the mycelium, and induced resistance was not observed. Wounding of the root also negated the effect of induced resistance to F. oxysporum .     

AM真菌和镰刀菌对西瓜根系几种酶活性的影响*

在温室盆栽条件下研究了丛枝菌根(Arbuscular Mycorrhiza, AM)真菌Glomus versiforme和西瓜枯萎镰刀菌Fusarium oxysporum f.sp. niveum对西瓜根系中过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3-葡聚糖酶和几丁质酶活性的影响。结果表明,接种AM真菌的西瓜根系中4种酶的活性均高于对照,先接种G. versiforme,后接种F. oxysporum f.sp. niveum处理的4种酶的活性均高于只接种F. oxysporum f.sp. niveum 的处理,且酶的活性峰值出现较早。表明接种G. versiforme 能预先诱导这4种酶的产生,提高其活性,从而提高西瓜对F. oxysporum f.sp. niveum侵染的抗性。接种G. versiforme的感枯萎病西瓜品种“郑杂5号”酶的增加幅度大于抗病品种“京欣1号”的接种处理,说明G. versiforme对提高感病西瓜品种酶活性的作用更大。     

Interaction between Meloidogyne incognita and Agrobacterium tumefaciens or Fusarium oxysporum f. sp. lycopersici on Tomato

Agrobacterium tumefaciens stimulated and Fusarium oxysporum f. sp. lycopersici inhibited development and reproduction of Meloidogyne incognita when applied to the opposite split root of tomato, Lycopersicon esculentum cv. Tropic, plants. The lowest rate of nematode reproduction occurred after 2,000 juveniles were applied and the fungus was present in the opposite split root. The effects of all three pathogens alone on the growth of roots and shoots of tomato plants were evident, but M. incognita had a greater effect alone than did either of the other pathogens. The length of split roots was reduced by the infection of M. incognita and A. tumefaciens or F. oxysporum f. sp. lycopersici. The number of galls induced by nematodes on roots was higher where the bacterium was applied and lower where the fungus was applied to the opposite split root.     

新疆甜瓜Fom-2基因同源序列的克隆及分析

近年来甜瓜枯萎病蔓延迅速,危害严重,分离克隆甜瓜抗病基因,利用转基因技术改良甜瓜抗病品性是一种从根本上解决甜瓜枯萎病危害的新途径。根据已知Fom-2基因序列设计特异引物,采用RT-PCR技术从新疆甜瓜抗病性品种黄旦子中扩增出580bp的cDNA片段,序列分析表明它与已发表Fom-2基因具有高度同源性基因片段,命名为X-Fom-2。以X-Fom-2为探针对新疆甜瓜黄旦子、伽什瓜、红心脆、金丽-2号进行Southem blot和Northem blot,结果表明,X-Fom-2以多拷贝形式存在。X-Fom-2在抗病品种黄旦子、金丽2号有表达且在病原菌诱导后增强,而在感病品种伽什瓜、红心脆中没有表达。     

Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 show increased susceptibility to a group of fungal and oomycete pathogens

The behaviour of Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 was investigated in response to fungal and oomycete infections. The importance of NpPDR1 in plant defence was demonstrated for two organs in which NpPDR1 is constitutively expressed: the roots and the petal epidermis. The roots of the plantlets of two lines silenced for NpPDR1 expression were clearly more sensitive than those of controls to the fungal pathogens Botrytis cinerea , Fusarium oxysporum sp., F. oxysporum f. sp. nicotianae , F. oxysporum f. sp. melonis and Rhizoctonia solani , as well as to the oomycete pathogen Phytophthora nicotianae race 0. The Ph gene-linked resistance of N. plumbaginifolia to P. nicotianae race 0 was totally ineffective in NpPDR1 -silenced lines. In addition, the petals of the NpPDR1 -silenced lines were spotted 15%–20% more rapidly by B. cinerea than were the controls. The rapid induction (after 2–4 days) of NpPDR1 expression in N. plumbaginifolia and N. tabacum mature leaves in response to pathogen presence was demonstrated for the first time with fungi and one oomycete: R. solani , F. oxysporum and P. nicotianae . With B. cinerea , such rapid expression was not observed in healthy mature leaves. NpPDR1 expression was not observed during latent infections of B. cinerea in N. plumbaginifolia and N. tabacum , but was induced when conditions facilitated B. cinerea development in leaves, such as leaf ageing or an initial root infection. This work demonstrates the increased sensitivity of NpPDR1 -silenced N. plumbaginifolia plants to all of the fungal and oomycete pathogens investigated.     

The arms race between tomato and Fusarium oxysporum

The interaction between tomato and Fusarium oxysporum f. sp. lycopersici has become a model system for the study of the molecular basis of disease resistance and susceptibility. Gene-for-gene interactions in this system have provided the basis for the development of tomato cultivars resistant to Fusarium wilt disease. Over the last 6 years, new insights into the molecular basis of these gene-for-gene interactions have been obtained. Highlights are the identification of three avirulence genes in F. oxysporum f. sp. lycopersici and the development of a molecular switch model for I-2, a nucleotide-binding and leucine-rich repeat-type resistance protein which mediates the recognition of the Avr2 protein. We summarize these findings here and present possible scenarios for the ongoing molecular arms race between tomato and F. oxysporum f. sp. lycopersici in both nature and agriculture.     

大蒜根系分泌物的化感作用

以苍山白蒜和蔡家坡紫蒜为材料,采用水培方法收集根系分泌物,研究了2个大蒜品种的根系分泌物对莴苣种子发芽和幼苗生长及对黄瓜枯萎病菌、西瓜枯萎病菌的化感效应.结果表明:2个大蒜品种的根系分泌物对莴苣种子发芽和幼苗生长均表现为低浓度(0.1、0.2 g·mL-1)促进、高浓度(0.4、0.6 g·mL-1)抑制,高浓度时蔡家坡紫蒜的抑制作用大于苍山白蒜;对黄瓜枯萎病菌和西瓜枯萎病菌的菌丝生长及孢子萌发均表现为抑制作用,随着根系分泌物浓度的提高,抑制作用增强,其中黄瓜枯萎病菌较敏感,且蔡家坡紫蒜的抑制作用大于苍山白蒜.     

Visualization of interactions between a pathogenic and a beneficial Fusarium strain during biocontrol of tomato foot and root rot

The soilborne fungus Fusarium oxysporum f. sp. radicis-lycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50-fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.     

Susceptibility of gerbera and chrysanthemum varieties (Gerbera jamesoni and Chrysanthemum morifolium) to Fusarium oxysporum f.sp. chrysanthemi

In 2002, gerbera plants (cv Kaliki) were observed exhibiting symptoms of a wilt in a soilless cultivation at Albenga area (Northern Italy). A similar wilt was also observed in the Sanremo area (Northern Italy) on cv Red Bull, Anedin and Gud finger grown in soil. The same observations were carried out in 2004 in SW Spain where gerbera plants showing wilt symptoms were observed in soilless crops. In all cases, the planting material originated from the Netherlands. Recently on the base of experimental trials F. oxysporum f. sp. chrysanthemi was recognized as the causal agent of wilts of gerbera both in Italy and in Spain. The aim of this experimental work was the evaluation of the resistance/susceptibility of available cultivars of chrysanthemum and gerbera to the Fusarium wilt. The pathogenicity of two isolates of Fusarium chrysanthemi obtained from infected gerbera (Gerbera jamesonii) and chrysanthemum (Chrysanthemum morifolium) plants was tested on several varieties both of gerbera and chrysanthemum in 2004-2006. In 2004 and 2005 respectively 54 and 30 cultivars of chrysanthemum and 57 and 55 of gerbera were tested, while in 2006 only 53 cultivars of gerbera were tested. The results showed that respectively in 2004 and 2005 67 and 33 % of chrysanthemum cultivars were highly resistant to F. chrysanthemi obtained from chrysanthemum while 57 and 53 % were highly resistant to strain isolated from gerbera. In 2004, 2005 and 2006 47, 65 and 75 % of gerbera cultivars were highly resistant to F. chrysanthemi obtained from chrysanthemum and 48, 56 and 72 % were highly resistant to the strain isolated from gerbera.     

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.     

Rhizosphere soil microorganism populations and community structures of different watermelon cultivars with differing resistance to Fusarium oxysporum f. sp. niveum

Fusarium wilt is an increasingly serious disease of watermelon that reduces crop productivity. Changes in microorganism populations and bacterial and fungal community structures in rhizosphere soil of watermelon cultivars resistant or susceptible to Fusarium oxysporum f. sp. niveum were investigated using a plate culture method and PCR-DGGE analysis. Plate culture showed that populations of culturable bacteria and actinomycetes were more abundant in the rhizosphere of the resistant watermelon cultivar than the susceptible cultivar, but the fungi population had the opposite pattern. Populations of Penicillium , Fusarium , and Aspergillus were significantly lower in the resistant cultivar than the susceptible cultivar at the fruiting and uprooting stages (p?< 0.05). Pattern matching analysis generated the dendrogram of the DGGE results indicating the relatedness of the different resistant watermelon cultivars and their corresponding rhizosphere microbial communities. Further sequencing analysis of specific bands from DGGE profiles indicated that different groups of bacteria and fungi occurred in the rhizosphere of different watermelon cultivars. Our results demonstrated that plant genotype had a significant impact on soil microbial community structure, and the differences in the rhizosphere microbial community may contribute to the differences in resistance to F. oxysporum f. sp. niveum.