Different mechanisms of Trichoderma virens‐mediated resistance in tomato against Fusarium wilt involve the jasmonic and salicylic acid pathways

In the present study, we investigated the role of Trichoderma virens (TriV_JSB100) spores or cell‐free culture filtrate in the regulation of growth and activation of the defence responses of tomato (Solanum lycopersicum) plants against Fusarium oxysporum f. sp. lycopersici by the development of a biocontrol–plant–pathogen interaction system. Two‐week‐old tomato seedlings primed with TriV_JSB100 spores cultured on barley grains (BGS) or with cell‐free culture filtrate (CF) were inoculated with Fusarium pathogen under glasshouse conditions; this resulted in significantly lower disease incidence in tomato Oogata‐Fukuju plants treated with BGS than in those treated with CF. To dissect the pathways associated with this response, jasmonic acid (JA) and salicylic acid (SA) signalling in BGS‐ and CF‐induced resistance was evaluated using JA‐ and SA‐impaired tomato lines. We observed that JA‐deficient mutant def1 plants were susceptible to Fusarium pathogen when they were treated with BGS. However, wild‐type (WT) BGS‐treated tomato plants showed a higher JA level and significantly lower disease incidence. SA‐deficient mutant NahG plants treated with CF were also found to be susceptible to Fusarium pathogen and displayed low SA levels, whereas WT CF‐treated tomato plants exhibited moderately lower disease levels and substantially higher SA levels. Expression of the JA‐responsive defensin gene PDF1 was induced in WT tomato plants treated with BGS, whereas the SA‐inducible pathogenesis‐related protein 1 acidic (PR1a) gene was up‐regulated in WT tomato plants treated with CF. These results suggest that TriV_JSB100 BGS and CF differentially induce JA and SA signalling cascades for the elicitation of Fusarium oxysporum resistance in tomato.     

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.     

Purification and characterization of an exo-polygalacturonase from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici

Abstract An exo-polygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with citrus pectin, using preparative isoelectric focusing. The enzyme, denominated PG2, had an apparent M _r of 74000 Da upon SDS-PAGE. The pI of the main PG2 isoform was 4.5, and pH and temperature optima were 5.0 and 55 °C, respectively. PG2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by anaysis of degradation products. The enzyme was N-glycosylated. The N-terminal amino acid sequence, L-A-F-N-V-P-S-K-P-P, has no identity to other known polygalacturonases.     

Partial resistance of tomato to Phytophthora infestans is not dependent upon ethylene,jasmonic acid,or salicylic acid signaling pathways

We compared tomato defense responses to Phytophthora infestans in highly compatible and partially compatible interactions. The highly compatible phenotype was achieved with a tomato-specialized isolate of P. infestans, whereas the partially compatible phenotype was achieved with a nonspecialized isolate. As expected, there was induction of the hypersensitive response (HR) earlier during the partially compatible interaction. However, contrary to our expectation, pathogenesis-related (PR) gene expression was not stimulated sooner in the partially compatible interaction. While the level of PR gene expression was quite similar in the two interactions, the LeDES gene (which encodes an enzyme necessary for the production of divinyl ethers) was expressed at a much higher level in the partially compatible interaction at 48 h after inoculation. Host reaction to the different pathogen genotypes was not altered (compared with wild type) in mutant tomatoes that were ethylene-insensitive (Never-ripe) or those with reduced ability to accumulate jasmonic acid (def-1). Similarly, host reaction was not altered in NahG transgenic tomatoes unable to accumulate salicylic acid. These combined data indicate that partial resistance in tomato to P. infestans is independent of ethylene, jasmonic acid, and salicylic acid signaling pathways.     

Species-specific primers for Fusarium redolens and a PCR-RFLP technique to distinguish among three clades of Fusarium oxysporum

The currently available morphological and molecular diagnostic techniques for Fusarium redolens and the three phylogenetic clades of Fusarium oxysporum are problematic. Aligned translation elongation factor 1 alpha (TEF-1 alpha) gene sequences from these species and their close relatives were used to design F. redolens-specific primers, and to identify restriction sites that discriminate among the three clades of F. oxysporum. The F. redolens-specific primers distinguished this species from all others included in the study. There were three TEF-1 alpha-RFLP patterns among formae speciales of F. oxysporum. These PCR-RFLP patterns corresponded with the three clades. These techniques provide simple and inexpensive diagnostic methods for the identification of F. redolens and members of the three clades of F. oxysporum.     

Co-denitrification by the denitrifying system of the fungus Fusarium oxysporum

Abstract Nitrogen compounds such as azide, salicylhydroxamic acid, and possibly ammonium ions were converted to nitrous oxide (N₂O) or dinitrogen (N₂) by Fusarium oxysporum under denitrifying conditions. Nitrogen atoms in these compounds were combined with another nitrogen atom from nitrite to form a hybrid N₂O species. The fungus exhibited much higher converting activities as compared with similar reactions catalyzed by bacterial denitrifiers. We thus propose the phenomenon be called co-denitrification, which means that such nitrogen compounds are denitrified by the system induced by nitrite (or nitrate) but are incapable by themselves of inducing the denitrifying system.     

Selection of bacteria able to control Fusarium oxysporum f. sp. radicis-lycopersici in stonewool substrate

AIMS: Tomato foot and root rot (TFRR), caused by Fusariumoxysporum f. sp. radicis-lycopersici (Forl), is an economically important disease of tomato. The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive root-colonizing bacteria from total rhizosphere soil samples. METHODS AND RESULTS: A total of 216 potentially enhanced bacterial strains were isolated from 17 rhizosphere soil samples after applying a procedure to enrich for enhanced root tip colonizers. Amplified ribosomal DNA restriction analysis, in combination with determination of phenotypic traits, was introduced to evaluate the presence of siblings. One hundred sixteen strains were discarded as siblings. Thirty-eight strains were discarded as potential pathogens based on the sequence of their 16S rDNA. Of the remaining strains, 24 performed equally well or better than the good root colonizer Pseudomonas fluorescens WCS365 in a competitive tomato root tip colonization assay. Finally, these enhanced colonizers were tested for their ability to control TFRR in stonewool, which resulted in seven new biocontrol strains. CONCLUSIONS: The new biocontrol strains, six Gram-negative and one Gram-positive bacteria, were identified as three Pseudomonas putida strains and one strain each of Delftia tsuruhatensis, Pseudomonas chlororaphis, Pseudomonas rhodesiae and Paenibacillus amylolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a fast method for the isolation of bacteria able to suppress TFRR in stonewool, an industrial plant growth substrate. The procedure minimizes the laborious screens that are a common feature in the isolation of biocontrol strains.     

Fusaric acid contributes to virulence of Fusarium oxysporum on plant and mammalian hosts

Fusaric acid (FA) is amongst the oldest identified secondary metabolites produced by Fusarium species, known for a long time to display strong phytotoxicity and moderate toxicity to animal cells; however, the cellular targets of FA and its function in fungal pathogenicity remain unknown. Here, we investigated the role of FA in Fusarium oxysporum, a soil‐borne cross‐kingdom pathogen that causes vascular wilt on more than 100 plant species and opportunistic infections in humans. Targeted deletion of fub1, encoding a predicted orthologue of the polyketide synthase involved in FA biosynthesis in F. verticillioides and F. fujikuroi, abolished the production of FA and its derivatives in F. oxysporum. We further showed that the expression of fub1 was positively controlled by the master regulator of secondary metabolism LaeA and the alkaline pH regulator PacC through the modulation of chromatin accessibility at the fub1 locus. FA exhibited strong phytotoxicity on tomato plants, which was rescued by the exogenous supply of copper, iron or zinc, suggesting a possible function of FA as a chelating agent of these metal ions. Importantly, the severity of vascular wilt symptoms on tomato plants and the mortality of immunosuppressed mice were significantly reduced in fub1Δ mutants and fully restored in the complemented strains. Collectively, these results provide new insights into the regulation and mode of action of FA, as well as on the function of this phytotoxin during the infection process of F. oxysporum.     

Hop,an active Mutator-like element in the genome of the fungus Fusarium oxysporum

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporum by its transposition into the niaD target gene. Two insertions within the final exon, in opposite orientations at the same nucleotide site, have been characterized. These elements, called Hop, are 3,299 bp long, with perfect terminal inverted repeats (TIRs) of 99 bp. The sequencing of genomic copies reveals a 9-bp target site duplication and no apparent sequence specificity at the insertion sites. The sequencing of a cDNA indicates that Hop does not contain an intron and encodes a putative transposase of 836 amino acids. The structural features (length, TIRs size, and 9-bp duplication), together with the presence of conserved domains in the transposase, strongly suggest that Hop is a Mutator-like element (MULE). Hop is thus the first active member of this family found beyond plants. The high rate of excision observed indicates that Hop is very active and thus represents a promising efficient tagging system for the isolation of fungal genes. The distribution of Hop elements within the Fusarium genus revealed that they are present in different species, suggesting that related elements could be present in other fungal genomes. In fact, Hop-related sequences have been identified in the survey of the entire genome sequence of three other ascomycetes, Magnaporthe grisea, Neurospora crassa, and Aspergillus fumigatus.     

Effect of flavonoids on the development of Fusarium oxysporum f. sp. lycopersici

Abstract

In the present study the effect of flavonoid compounds on the germination and fungal growth of the soil-borne tomato pathogen Fusarium oxysporum f. sp. lycopersici was studied. Out of 12 flavonoid compounds only myricetin and luteolin exhibited a low stimulating activity on microconidia germination of Fusarium oxysporum f. sp. lycopersici, whereas the other flavonoids tested were inactive when applied at five different concentrations. In our study the tested flavonoids affect fungal growth differently to microconidia germination. Individual flavonoid concentrations resulted in a small increase of fungal growth, but the lowest flavonoid concentrations showed an inhibiting effect on fungal growth for all flavonoids tested. There is evidence to suggest, that low flavonoid concentrations exhibit slight antimicrobial properties against Fusarium oxysporum f. sp. lycopersici.     

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     

Induced systemic resistance in tomato by non-pathogenic Fusarium species for the management of Fusarium wilt

Isolates of non-pathogenic Fusarium moniliforme (Fu3, Fu7 and Fu24), F. oxysporum (Fu2, Fu4), F. solani (Fu25) and F. merismoides (Fu1) that were found to be effective in reducing wilt incidence in tomato were tested for their potential to elicit induced systemic resistance (ISR) in tomato. Talc formulations of these isolates derived from liquid fermentation as well as cell elicitors of these cultures were tested. Changes in the phenol and total protein contents and activities of peroxidase and polyphenol oxidase were studied. Isolate Fu3 induced more phenol and total protein contents as well as activities of peroxidase and polyphenol oxidase. Elicitors of Fu2 induced more of these compounds and enzymes. Although Fu1, Fu4 and Fu24 were found to give good control against Fusarium wilt incidence in an earlier study, they were less effective in inducing these defense related compounds. Peroxidase activity was increased when plants were treated with Fu3, Fu4, Fu7, Fu24 and Fu25, whereas polyphenol oxidase activity was increased only with the isolate Fu3 and elicitor of Fu2. It is suggested that ISR was the mode of action for the isolates Fu2 and Fu3, whereas for the other isolates, the mode of action may be root colonisation, competition for nutrition and so on. The role of ISR with non-pathogenic isolates of Fusarium spp. is discussed.     

Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans

Aims: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. Methods and Results: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 μg ml^?1 suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 μg ml^?1. Conclusions: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. Significance and Impact of the Study: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.     

Purification and Characterization of an Alkaliphilic Choline Oxidase of Fusarium oxysporum

Uptake of cesium, potassium, and rubidium by Rhodococcus erythropolis CS98 and Rhodococcus sp. strain CS402 followed Michaelis-Menten saturation kinetics. The K_m’s for uptake of these monovalent cations by R. erythropolis CS98 and Rhodococcus sp. strain CS402 were 136 and 436μM for Cs⁺, 65 and 101μM for K⁺, and 102 and 113μM for Rb⁺, respectively. These values were significantly lower than those of Rhodobacter capsulatus and the Kup system in Escherichia coli. Potassium was a competitive inhibitor of cesium uptake by these strains, suggesting that cesium was accumulated by the potassium transport system. Although an uncoupler, FCCP, inhibited the cesium transport system, this system was not repressed by high concentrations of potassium in both Rhodococcus strains. However, the specificity in both Rhodococcus strains was different from the Trk system. These results suggest that the potassium transport system which can transport cesium in both Rhodococcus strains may be novel.     

水杨酸、茉莉酸和乙烯介导的防卫信号途径相互作用的研究进展

乙烯、水杨酸和茉莉酸是植物体内主要的几个防御信号途径,也是研究比较多的几个信号途径。很多试验证明不同的防御信号途径相互间存在相互作用,他们或相互抑制,或相互促进。从这三种信号途径相互间的作用,及作用的联系点进行综述。     

Molecular diversity of native Trichoderma isolates against Fusarium oxysporum f. sp. lycopersici (Sacc.). A casual agent of Fusarium wilt in tomato (Lycopersicon esculentum Mill.)

Fusarium wilt in tomato caused by Fusarium oxysporum is the one of the problematic diseases. In this study, 12 native Trichoderma isolates were isolated from different land use types in Rayalaseema region of Andhrapradesh, India and were tested for antagonistic activity against F. oxysporum using dual culture method; the maximum inhibition occurred in WT₂ (78.4%) compared to the control. Molecular characterisation using random amplified polymorphic DNA (RAPD) technique reported 91.8% polymorphism among 12 isolates of Trichoderma. Internal transcribed spacer (ITS) region of rDNA amplification with genus-specific ITS1 and ITS4 universal primers produced amplicon size from 569 bp in all the isolates. The study resulted in identification of good competitive Trichoderma isolates against F. oxysporum. A relationship was found between the polymorphism showed by the Trichoderma isolates and their hardness to F. oxysporum during antagonism. Also, exhibition of sufficient genetic polymorphism aids further exploitation in genomic fingerprinting.     

Stomatal responses to jasmonic acid, linolenic acid and abscisic acid in wild-type and ABA-deficient tomato plants

Wild-type and abscisic acid (ABA) -deficient (sitiens) tomato plants were used to analyse the effects of abscisic acid (ABA), butyric acid (BA), jasmonic acid (JA) and linolenic acid (LA) on assimilation and transpiration rates in detached leaves taking up those substances into the transpiration stream. BA did not affect assimilation and transpiration rates. ABA decreased assimilation and transpiration in both wild-type and ABA-deficient mutants. JA reduced the assimilation rate in both lines but induced a significant reduction of transpiration in the wild type only. The response to LA in both lines was slower than that to JA.