Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3?kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease.     

Genetic Diversity of Fusarium oxysporum Populations Isolated from Tomato Plants in Tunisia

Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. radicis‐lycopersici is a new devastative disease of tomato greenhouse crops in Tunisia. Nothing is known neither about the population of this pathogen in this region, nor about the population of F. oxysporum f. sp. lycopersici the causal agent of Fusarium wilt of tomato. In order to examine the genetic relatedness among the F. oxysporum isolates by intergenic spacer restriction fragment length polymorphism (IGS‐RFLP) analysis and to elucidate the origin of the formae specialesradicis‐lycopersici in Tunisia by looking for genetic similarity of Tunisians isolates with isolates from a foreign source, the genetic diversity among F. oxysporum f. sp. radicis‐lycopersici and F. oxysporum f. sp. lycopersici populations was investigated. A total of 62 isolates of F. oxysporum, obtained from symptomless tomato plants, were characterized using IGS typing and pathogenicity tests on tomato plants. All Fusarium isolates were highly pathogenic on tomato. Fusarium oxysporum f. sp. radicis‐lycopersici isolates were separated into five IGS types. From the 53 F. oxysporum f. sp. radicis‐lycopersici isolates, 34 isolates have the same IGS types (IGS type 25), and the remaining 19 isolates were distributed into four IGS types. However, the only nine isolates of F. oxysporum f. sp. lycopersici have six different IGS types. This difference of diversity between the two formae speciales suggests that F. oxysporum f. sp. radicis‐lycopersici isolates have a foreign origin and may have been accidentally introduced into Tunisia.     

Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

Background  

Fusarium oxysporum f. sp. lycopersici is the causal agent of vascular wilt disease in tomato. In order to gain more insight into the molecular processes in F. oxysporum necessary for pathogenesis and to uncover the genes involved, we used Agrobacterium-mediated insertional mutagenesis to generate 10,290 transformants and screened the transformants for loss or reduction of pathogenicity.     

Further Investigation of the Specificity of Interactions Between Wild Lactuca spp. and Bremia lactucae Isolates from Lactuca serriola

Callus cultures derived from isogenic lines of the tomato cultivars Moneymaker and Craigella, resistant or susceptible to F. oxysporum f. sp. lycopersici, were inoculated with Fusarium oxysporum f. sp. lycopersici race 1. Fungal growth was restricted on callus derived from resistant plants, after inoculation with a conidial suspension, whereas callus derived from susceptible plants was totally overgrown by the fungus within 7 days. The concentration of the phytoalexin rishitin was significantly higher in the callus culture derived from a resistant tomato line compared with the callus culture from a susceptible line, 2 and 3 days after inoculation with mycelium. The results of the experiments were compared with experiments with whole plants. Rishitin production as well as growth of the fungus was comparable with responses in plant-fungus interaction. Therefore callus culture may be useful in studying the interaction between tomato plants and race 1 of F. oxysporum f. sp. lycopersici.     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

The intercropping partner affects arbuscular mycorrhizal fungi and Fusarium oxysporum f. sp. lycopersici interactions in tomato

Arbuscular mycorrhizal fungi (AMF) and their bioprotective aspects are of great interest in the context of sustainable agriculture. Combining the benefits of AMF with the utilisation of plant species diversity shows great promise for the management of plant diseases in environmentally compatible agriculture. In the present study, AMF were tested against Fusarium oxysporum f. sp. lycopersici with tomato intercropped with either leek, cucumber, basil, fennel or tomato itself. Arbuscular mycorrhizal (AM) root colonisation of tomato was clearly affected by its intercropping partners. Tomato intercropped with leek showed even a 20 % higher AM colonisation rate than tomato intercropped with tomato. Positive effects of AMF expressed as an increase of tomato biomass compared to the untreated control treatment could be observed in root as well as in shoot weights. A compensation of negative effects of F. oxysporum f. sp. lycopersici on tomato biomass by AMF was observed in the tomato/leek combination. The intercropping partners leek, cucumber, basil and tomato had no effect on F. oxysporum f. sp. lycopersici disease incidence or disease severity indicating no allelopathic suppression; however, tomato co-cultivated with tomato clearly showed a negative effect on one plant/pot with regard to biomass and disease severity of F. oxysporum f. sp. lycopersici. Nonetheless, bioprotective effects of AMF resulting in the decrease of F. oxysporum f. sp. lycopersici disease severity were evident in treatments with AMF and F. oxysporum f. sp. lycopersici co-inoculation. However, these bioprotective effects depended on the intercropping partner since these effects were only observed in the tomato/leek and tomato/basil combination and for the better developed plant of tomato/tomato. In conclusion, the effects of the intercropping partner on AMF colonisation of tomato are of great interest for crop plant communities and for the influences on each other. The outcome of the bioprotective effects of AMF resulting in the decrease on F. oxysporum f. sp. lycopersici disease severity and/or compensation of plant biomass does not depend on the degree of AM colonisation but more on the intercropping partner.     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Induction of defense-related proteins in tomato roots treated with Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici

Pseudomonas fluorescens isolate Pf1 was found to protect tomato plants from wilt disease caused by Fusarium oxysporum f. sp. lycopersici. Induction of defense proteins and chemicals by P. fluorescens isolate Pf1 against challenge inoculation with F. oxysporum f. sp. lycopersici in tomato was studied. Phenolics were found to accumulate in bacterized tomato root tissues challenged with F. oxysporum f. sp. lycopersici at one day after pathogen challenge. The accumulation of phenolics reached maximum at the 5^th day after pathogen challenge. In pathogen-inoculated plants, the accumulation started at the 2^nd day and drastically decreased 4 days after the pathogen inoculation. Activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) increased in bacterized tomato root tissues challenged with the pathogen at one day after pathogen challenge and activities of PAL and PO reached maximum at the 4^th day while activity of PPO reached maximum at the 5^th day after challenge inoculation. Isoform analysis revealed that a unique PPO1 isoform was induced and PO1 and PPO2 isoforms were expressed at higher levels in bacterized tomato root tissues challenge inoculated with the pathogen. Similarly, -1,3 glucanase, chitinase and thaumatin-like proteins (TLP) were induced to accumulate at higher levels at 3-5 days of challenge inoculation in bacterized plants. Western blot analysis showed that chitinase isoform Chi2 with a molecular weight of 46 kDa was newly induced due to P. fluorescens isolate Pf1 treatment challenged with the pathogen. TLP isoform with molecular weight of 33 kDa was induced not only in P. fluorescens isolate Pf1-treated root tissues challenged with the pathogen but also in roots treated with P. fluorescens isolate Pf1 alone and roots inoculated with the pathogen. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restriction of invasion of F. oxysporum f. sp. lycopersici in tomato roots.     

Host-specific Fusarium oxysporum Causes Wilt of Jojoba

Jojoba [Simmondsia chinensis (Link) Schneider] plantations in Israel originated from vegetative propagation, planted during 1991–92, have shown symptoms of wilting and subsequent death. Verticillium dahliae was only rarely isolated from these plants and artificial inoculation showed only mild disease symptoms. Fusarium oxysporum caused severe chlorosis, desiccation, defoliation and wilt in leaves of jojoba plants, resulting in plant death. Recovery of the fungus from artificially inoculated stem cuttings and seedlings showed for the first time that F. oxysporum was the primary pathogen. Inoculated cuttings exhibited wilt within 3 weeks, while in seedlings wilt occurred 10–24 weeks after inoculation. Seedlings and cuttings of jojoba which were inoculated with other Fusarium isolates originating from different crops (F. oxysporum f. sp. vasinfectum from cotton, F. oxysporum f. sp. dianthi from carnation, F. oxysporum f. sp. lycopersici from tomato and F. oxysporum f. sp. basilicum from basil) did not develop symptoms. Moreover, cotton, tomato, melon and cucumber seedlings inoculated with several virulent F. oxysporum isolates from jojoba did not show any symptoms of wilt or defoliation. These results indicate a high degree of specificity of the Fusarium isolates from jojoba; therefore, it is suggested that this isolate be defined as F. oxysporum f. sp. simmondsia.     

Population structure and linkage disequilibrium in a large collection of Fusarium oxysporum strains analysed through iPBS markers

In the current study, 160 pathogenic strains of Fusarium oxysporum collected from tomato, eggplant and pepper were studied. Eighteen inter‐primer binding site (iPBS)‐retrotransposon primers were used, and these primers generated 205 scorable polymorphic bands. The number of polymorphic bands per primer varied between 9 and 19, with a mean of 11 bands per primer. The highest polymorphism information content (PIC) value was determined as 0.27, and the lowest was 0.05. The unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram including a heat map revealed that the 160 pathogenic strains of F. oxysporum were divided into two main clusters. The first cluster mainly included F. oxysporum f. sp. capsici (FOC) and F. oxysporum f. sp. melongenae (FOMG) isolates. The second cluster mainly comprised F. oxysporum f. sp. lycopersici (FOL) and F. oxysporum f. sp. radicis lycopersici (FORL) isolates. The highest percentage of loci in significant linkage disequilibrium (LD) was detected for FOL, whereas the lowest level of LD was found for FOC, and 95.2%, 99.4%, 99.1% and 97.4% of the relative kinship estimates were less than 0.4 for FOL, FOMG, FORL and FOC, respectively. LD differences were detected among formae speciales, and LD was higher in FOL as compare to FOC species. The findings of this study confirm that iPBS‐retrotransposon markers are highly polymorphic at the intraspecific level in Fusarium spp.     

Microconidia germination of the tomato pathogen Fusarium oxysporum in the presence of root exudates

Abstract

In this study we assessed microconidia germination of the tomato pathogens F. oxysporum f. sp. lycopersici (Fol) and F. oxysporum f. sp. radicis-lycopersici (Forl) in the presence of root exudates. Tomato root exudates stimulated microconidia germination and the level of stimulation was affected by plant age. Treatment of root exudates with insoluble polyvinylpolypyrrolidone, which binds phenolic compounds, indicated that tomato root exudates contain phenolic compounds inhibitory to F. oxysporum microconidia germination. Our study indicates that tomato root exudates similarly stimulate microconidia germination of both Fol and Forl. However, individual F. oxysporum strains differ in the degree of germination response to the root exudates. Furthermore, root exudates from non-host plants also contain compounds that stimulate microconidia germination of Fol. In general, the effects of root exudates from non-host plants did not differ considerably from those of tomato. The ability of phenolic compounds to inhibit germination of Fol seems not to be plant-specific.     

Genetic and physical analysis of a YAC contig spanning the fungal disease resistance locus Asc of tomato (Lycopersicon esculentum)

The Alternaria stem canker disease of tomato is caused by the necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). The fungus produces AAL toxins that kill the plant tissue. Resistance to the fungus segregates as a single locus, called Asc, and has been genetically mapped on chromosome 3 of tomato. We describe here the establishment of a 1383-kb YAC contig covering the Asc locus and a series of plants selected for recombination events around the Asc locus. It was shown that the YAC contig corresponds to a genetic distance of at least 11.2 cM. Thus, the recombination rate in the Asc region is six times higher (123 kb/cM) than the average for the tomato genome. Furthermore, the Asc locus could be localised to a 91-kb fragment, thus paving the way for the cloning and identification of the Asc gene(s) by complementation. Received: 31 July 1998 / Accepted: 6 October 1998     

The effects of Fusarium oxysporum f.sp. lycopersici (Sacc.) Snyder & Hansen on tomato in diphenamid-treated soil

Pre-emergence soil application of the herbicide diphenamid in concentrations exceeding the normal field rate increased the resistance of tomato plants towards infection by the wilt fungus Fusarium oxysporum f.sp. lycopersici. This was detected as significant increases in the percentage emergence of seedlings although growth parameters of the raised seedlings were reduced. Treated plants exhibited no wilt symptoms, although the pathogen maintained its population at detectable levels in the rhizosphere of tomato plants. However, the growth inhibition caused by diphenamid alone was much less than that reported for the combined application of pathogen and herbicide. Growth activities of F. oxysporum f.sp. lycopersici were inhibited by high concentrations of diphenamid in vitro. It is possible that the biodegradation of this herbicide by species such as Aspergillus candidus (present in substantial counts in treated rhizospheres) was one of the causes of increased tolerence of the pathogen to the herbicide in situ.     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. III. PR-protein gene expression and ethylene evolution in tomato cell lines transgenic for phytohormone-related bacterial genes

 Previous work carried out in our laboratory has shown that, in tomato, the alteration of endogenous phytohormone equilibria through the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis can modify the active defense response to Fusarium oxysporum f. sp. lycopersici. The susceptible cv ‘Red River’ acquires a stable competence for active defense, particularly when the phytohormone equilibrium is altered in favour of cytokinins. Here, we analyse the expression of genes involved in the defense response against pathogens, i.e. pathogenesis-related (PR)-protein genes, in the susceptible ‘Red River’ and resistant ‘Davis’ cultivars transgenic for the aforementioned genes. Fungal cell-wall components, glutathione, salicylic acid and the ethylene-forming ethephon are used as “probes” for the induction of defense processes, including ethylene production. The data obtained show that the extracellular PR-proteins (acidic chitinase and PR-1 protein) that were inducible in the control tissue of the resistant ‘Davis’ cultivar and not expressed in the susceptible ‘Red River’ cultivar became constitutive in the transgenic tissues of both. On the other hand, expression of the intracellular PR-proteins (basic chitinase and β-1,3-glucanase) was found to be constitutive in all cases, both in the control and in the transgenic cell lines of the resistant and the susceptible tomato cultivars. Ethylene production was higher in ‘Davis’ than in ‘Red River’, and significantly increased in the transgenic cell lines, particularly when cytokinin synthesis was altered. Received: 25 February 1998 / Accepted: 7 April 1998     

Biological control of Fusarium wilt of tomato with <Emphasis Type="Italic">Fusarium equiseti</Emphasis> GF191 in both rock wool and soil systems

The plant growth-promoting fungus (PGPF) Fusarium equiseti GF191 was tested for its ability to control Fusarium wilt of tomato (FWT) caused by Fusarium oxysporum f. sp. lycopersici (FOL) in both a hydroponic rock wool and soil system. F. equiseti effectively controlled FWT, with protective effects based on disease severity of 66.7–88.6% in four experiments. The numbers of colony-forming units of FOL per gram fresh weight of stems were significantly reduced (P < 0.05) in plants treated with F. equiseti. Stem extracts from F. equiseti-treated and pathogen-challenged plants significantly inhibited the germination and germ-tube length of FOL microconidia and the production of FOL budding-cells. Tomatine content in tomato stems treated with F. equiseti was significantly increased compared with the non-treated control.     

Kinetic of the production of polygalacturonase and pectin lyase by two closely relatedFormae speciales ofFusarium oxysporum

The kinetic of thein vitro production of polygalacturonase and pectin lyase of two closely related fungi,Fusarium oxysporum f.sp.lycopersici andF. oxysporum f.sp.radicis-lycopersici, was examined under various culture conditions such as the source of carbon, the pH, and the age of cultures. Over a 5-day period, the production of these enzymes by various isolates of the sameforma specialis (f. sp.) ofF. oxysporum was not significantly different (P ≥ 0.05). However, the amount of the enzymes produced differed markedly between both f. sp. The different carbon sources added to the culture media, such as citrus pectin, apple pectin, tomato cell wall fragments, andd-galacturonic acid, proved to be higher pectinase inducible substrates than sucrose and glucose. For both fungi, polygalacturonase and pectin lyase activities were optimal at pH 5.0 and 8.0, respectively. Furthermore, pectin lyase production had a partial Ca²⁺ requirement in contrast to polygalacturonase production which was limited by Ca²⁺. In most experiments performed, the production of polygalacturonase appeared superior withF. oxysporum f.sp.radicislycopersici than withF. oxysporum f.sp.lycopersici. On the other hand, pectin lyase production ofF. oxysporum f.sp.lycopersici was approximately 10-fold greater than that byF. oxysporum f.sp.radicis-lycopersici in media supplemented withd-galacturonic acid.