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.     

Ultrastructural and cytochemical characterization of elicitor-induced structural responses in tomato root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici

Commercial chitosan and laminarin, as well as -glucans, isolated from either Phytophthora megasperma f.sp. glycinea or Saccharomyces cerevisiae, were applied to decapitated tomato (Lycopersicon esculentum Mill.) plants and evaluated for their potential to induce defense mechanisms in root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A significant decrease in disease incidence was monitored in elicitor-treated plants as compared to water-treated plants. No difference was detected in the capacity of the elicitors under study to confer enhanced protection against pathogen attack. Ultrastructural investigations of the infected root tissues from watertreated (control) plants showed a rapid colonization of all tissues including the vascular stele. Fungal ingress was lways associated with marked host cell disorganization and cell wall alteration. In root tissues from elicitortreated plants, restriction of fungal growth to the epidermis and the outer cortex, decrease in pathogen viability, and formation of numerous wall appositions at sites of attempted penetration were the main features of the hostpathogen interaction. The wall appositions were found to vary greatly in their appearance from multi-textured to multi-layered structures, from elongated deposits to hemispherical protuberances. Application of various goldcomplexed probes to root tissue sections revealed that callose, pectin and phenolic-like compounds (likely lignin) were the main components of the newly-formed barriers. By contrast, cellulose appeared confined to outer or intermediate layers resembling the host cell wall in terms of structure and architecture. In the absence of fungal challenge, the cytologically visible consequences of elicitation were restricted to a discrete deposition of electron-opaque substances in the vacuoles of some cells, and wall appositions were not detected. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm ignal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by an exogenous elicitor.Abbreviations AGL Aplysia gonad lectin - FORL Fusariumoxysporum f.sp. radicis-lycopersici The authors wish to thank Sylvain Noël for excellent technical assistance and Drs. J.P. Geiger and Michel Nicole (ORSTOM, Montpellier, France) for providing the purified laccase. This work was supported by a grant from the FCAR-CQVB (Fonds Québécois pour la Formation de Chercheurs et l'Aide à la Recherche and Centre Québécois de Valorisation de la Biomasse) and by a contract from the Company Tourbières Premier Ltée, Rivière-du-Loup, Québec.     

Vermicompost can suppress Fusarium oxysporum f. sp. lycopersici via generation of beneficial bacteria in a long-term tomato monoculture soil

Plant and Soil - Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) has severely decreased global tomato production. Organic amendments are widely applied to suppress Fol all over...     

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.     

Integrated and biological control of gladiolus corm rot and wilt caused by Fusarium oxysporum f.sp. gladioli

An isolate of Trichoderma virens Miller, Giddens & Foster, carboxin and a combination of both were evaluated for the control of gladiolus corm rot and wilt caused by Fusarium oxysporum f.sp. gladioli in glasshouse and field experiments. All treatments significantly reduced disease incidence in both glasshouse and field conditions. T. virens gave control at least as good as carboxin in all experiments. Control was significantly improved in two field experiments by combining the biological and chemical treatments.     

Chitinase-gold complex used to localize chitin ultrastructurally in tomato root cells infected by Fusarium oxysporum f. sp. radicis-lycopersici, compared with a chitin specific gold-conjugated lectin

Summary A cytochemical technique for the ultrastructural localization of chitin in tomato root cells infected byFusarium oxysporum f. sp.radicis-lycopersici is reported. Chitinase was complexed to colloidal gold and thin sections were incubated with the enzyme-gold complex. This technique yielded a more uniform distribution of gold particles over the fungus wall, compared to that obtained with the lectin-gold technique. Both techniques revealed no labelling of the fungus cytoplasm, except for organelles resembling Woronin bodies. No significant labelling of either healthy or infected root cells was seen except for the secondary walls of vessels and, occasionally, that of adjoining parenchyma cells. The importance of this technique in studying the development of the pathogen within host cells is discussed.     

Effects of the tomato pathogen Fusarium oxysporum f. sp. radicis-lycopersici and of the biocontrol bacterium Pseudomonas fluorescens WCS365 on the composition of organic acids and sugars in tomato root exudate

The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bacterial biocontrol strain Pseudomonas fluorescens WCS365, and of both microbes, on the amounts and composition of root exudate components of tomato plants grown in a gnotobiotic stonewool substrate system were studied. Conditions were selected under which introduction of F. oxysporum f. sp. radicis-lycopersici caused severe foot and root rot, whereas inoculation of the seed with P. fluorescens WCS365 decreased the percentage of diseased plants from 96 to 7%. This is a much better disease control level than was observed in potting soil. Analysis of root exudate revealed that the presence of F. oxysporum f. sp. radicis-lycopersici did not alter the total amount of organic acids, but that the amount of citric acid decreased and that of succinic acid increased compared with the nontreated control. In contrast, in the presence of the P. fluorescens biocontrol strain WCS365, the total amount of organic acid increased, mainly due to a strong increase of the amount of citric acid, whereas the amount of succinic acid decreased dramatically. Under biocontrol conditions, when both microbes are present, the content of succinic acid decreased and the level of citric acid was similar to that in the nontreated control. The amount of sugar was approximately half that of the control sample when either one of the microbes was present alone or when both were present. Analysis of the interactions between the two microbes grown together in sterile tomato root exudate showed that WCS365 inhibited multiplication of F. oxysporum f. sp. radicis-lycopersici, whereas the fungus did not affect the number of CFU of the bacterium.     

Effect of root exudates of mycorrhizal tomato plants on microconidia germination of Fusarium oxysporum f. sp. lycopersici

The effect of root exudates from mycorrhizal and non-mycorrhizal tomato plants on microconidia germination of the tomato pathogen Fusarium oxysporum f. sp. lycopersici was tested. Microconidia germination was enhanced in the presence of root exudates from mycorrhizal tomato plants. Tomato plants were colonised by the arbuscular mycorrhizal fungus Glomus fasciculatum, indicating that alterations of the exudation pattern depended on the degree of root AM colonisation. Testing the exudates from plants with a high and a low P level revealed that the alterations of the root exudates from mycorrhizal plants, resulting in a changed effect on microconidia germination, are not due to an improved P status of mycorrhizal plants.     

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

Summary With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible (Red River), one tolerant (UC 105) and three resistant (Davis UC 82, Heinz, UC 90) tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo — resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.     

Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.     

Time-course study of the accumulation of hydroxyproline-rich glycoproteins in root cells of susceptible and resistant tomato plants infected byFusarium oxysporum f. sp.radicis-lycopersici

The accumulation of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants is thought to be involved in the defense response to pathogens. An antiserum raised against deglycosylated HRGPs from melon was used for studying the subcellular localization of these glycoproteins in susceptible and resistant tomato (Lycopersicon esculentum Mill.) root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A time-course of HRGP accumulation revealed that these glycoproteins increased earlier and to a higher extent in resistant than in susceptible cultivars. In the compatible interaction, increase in HRGPs was largely correlated with pathogen invasion and appeared to occur as a result of wall damage. In the incompatible interaction, HRGPs accumulated in the walls of uninvaded cells, thus indicating a possible role in the protection against fungal penetration. The occurrence of substantial amounts of HRGPs in papillae, known to be physical barriers formed in response to infection, and in intercellular spaces provides additional support to the concept that such glycoproteins play an important role in disease resistance.     

Genetic and virulence variation in Fusarium oxysporum f. sp. cepae causing root and basal rot of common onion in Iran

Root and basal rot of common onion (Allium cepae L.) caused by Fusarium oxysporum f. sp. cepae is one of the most important diseases causing tremendous losses in onion‐growing areas worldwide. In this study, random amplified polymorphic DNA (RAPD), intersimple sequence repeats (ISSR) and virulence studies were conducted to analyse 26 F. oxysporum f. sp. cepae isolates obtained from the main onion‐growing regions of Iran, including Fars, Azerbaijan and Isfahan states. Cluster analysis using UPGMA method for both RAPD and ISSR markers revealed no clear grouping of the isolates obtained from different geographical regions, and the isolates were observed to derive probably from the same clonal lineage. Pathogenicity test indicated that all F. oxysporum f. sp. cepae isolates were pathogenic on onion; however, virulence variability was observed among the isolates. The grouping based on virulence variability was not correlated with the results of RAPD and ISSR analyses.     

A formulation of neem cake seeded with Bacillus sp. provides control over tomato Fusarium crown and root rot

Fusarium crown and root rot (FCRR) caused by the fungus Fusarium oxysporum f. sp. radicis-lycopersici is a damaging soil-borne disease of tomato. A plant growth rhizobacterium, Bacillus sp. strain HN09 isolated from neem tree rhizosphere soil, was shown to inhibit the growth, germination and development of normal morphology of the FCRR pathogen. A substantial level of disease control was achieved in greenhouse trials by soil supplementation with a preparation of neem cake seeded with HN09. Dry sterilisation of neem cake before fermentation gained comparable disease control effect as those in the unsterilised treatment, whereas moist sterilisation treatment decreased the effect significantly. This bioformulation also led to significantly raised activities in tomato genes encoding pathogenesis-related proteins, hence providing an effective alternative for the control of FCRR, reducing the need for chemical fungicide and fertilisers that impact the environment.     

Antagonistic bacteria of composted agro-industrial residues exhibit antibiosis against soil-borne fungal plant pathogens and protection of tomato plants from Fusarium oxysporum f.sp. radicis-lycopersici

Rhizospheric and root-associated/endophytic (RAE) bacteria were isolated from tomato plants grown in three suppressive compost-based plant growth media derived from the olive mill, winery and Agaricus bisporus production agro-industries. Forty-four (35 rhizospheric and 9 RAE) out of 329 bacterial strains showed in vitro antagonistic activity against at least one of the soil-borne fungal pathogens, Fusarium oxysporum f.sp. radicis-lycopersici (FORL), F. oxysporum f.sp. raphani, Phytophthora cinnamomi, P. nicotianae and Rhizoctonia solani. The high percentage of total isolates showing antagonistic properties (13%) and their common chitinase and β-glucanase activities indicate that the cell wall constituents of yeasts and macrofungi that proliferate in these compost media may have become a substrate that favours the establishment of antagonistic bacteria to soil-borne fungal pathogens. The selected bacterial strains were further evaluated for their suppressiveness to tomato crown and root rot disease caused by FORL. A total of six rhizospheric isolates, related to known members of the genera Bacillus, Lysinibacillus, Enterobacter and Serratia and one RAE associated with Alcaligenes faecalis subsp. were selected, showing statistically significant decrease of plant disease incidence. Inhibitory effects of extracellular products of the most effective rhizospheric biocontrol agent, Enterobacter sp. AR1.22, but not of the RAE Alcaligenes sp. AE1.16 were observed on the growth pattern of FORL. Furthermore, application of cell-free culture extracts, produced by Enterobacter sp. AR1.22, to tomato roots led to plant protection against FORL, indicating a mode of biological control action through antibiosis.     

Combined application of Pseudomonas fluorescens strain LRB3W1 with a low dosage of benomyl for control of cabbage yellows caused by Fusarium oxysporum f. sp. conglutinans

An antagonistic bacterium, Pseudomonas fluorescens strain LRB3W1, inhibited the fungal growth of Fusarium oxysporum f. sp. conglutinans in vitro and suppressed cabbage yellows caused by F. oxysporum f. sp. conglutinans. Under glasshouse conditions, the bacterium survived at ca. 10⁶-10⁷ CFU g^-1 in the cabbage rhizosphere for 4 weeks after the initial application. The chemical fungicide, benomyl, did not suppress the disease severity at low concentration (1 or 10 µg mL^-1). However, the disease severity was decreased by the combined application of a low dosage of benomyl with strain LRB3W1. Combined application of a low dosage of benomyl with strain LRB3W1 was more effective than treatment with the bacterium alone. The survival of strain LRB3W1 was not influenced by the presence of benomyl. This combined use of the biocontrol agent, strain LRB3W1, and a fungicide, benomyl, should be an attractive approach for suppressing cabbage yellows in sustainable agriculture because of the reduced chemical dosages needed for disease management.     

An isozyme marker for resistance to race 3 of Fusarium oxysporum f. sp. lycopersici in tomato

Summary The levels of erucic acid and other fatty acids in seeds of microspore-derived spontaneous diploid plants from crosses between low and high erucic acid parents were examined. The analysis confirmed that erucic acid is simply inherited and is determined by two genes that act in an additive manner. The effects of the genes for erucic acid on the levels of the other fatty acids was also determined and many significant correlations were found. In particular, erucic acid levels were negatively correlated with oleic acid and linoleic acid levels. The study also illustrates several advantages of using haploidy to analyze the inheritance of agronomically important traits. In particular, the number of phenotypic classes is smaller in androgenic populations and differences between classes are greater than in an F₂ population.     

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.     

Use of β-Glucuronidase Activity to Quantify the Growth of Fusarium oxysporum f. sp. radicis-lycopersici during Infection of Tomato

The β‐glucuronidase (gus) reporter gene was integrated into the phytopathogenic fungus Fusarium oxysporum f. sp. radicis‐lycopersici (FORL) in a co‐transformation experiment using the hygromycin B resistance (hph) gene as selective marker, which resulted in the generation of 10 mitotically stable transformants. One transformant, F30, was selected based on the results of prior detailed characterization of the 10 transformants for growth rate, conidia production and pathogenicity in comparison with the wild‐type strain. A strong positive correlation was found between GUS activity and accumulated biomass of in vitro‐grown fungus and therefore GUS activity was used to study fungal growth quantitatively in two tomato lines. Although a parallel increase in lesion development and GUS activity was noted for both tomato lines, a correlation between the GUS activity and disease progression was not always possible. Interestingly, the levels of GUS activity obtained for the more resistant line were higher than those obtained for the susceptible line, indicating that disease progression in tomato caused by FORL may not be related only to the amount of fungal biomass within the root tissue.     

Chitosan Treatment: An Emerging Strategy for Enhancing Resistance of Greenhouse Tomato Plants to Infection by Fusarium oxysporum f.sp. radicis-lycopersici

The potential of chitosan, a non-toxic and biodegradable polymer of beta -1,4-glucosamine, for controlling fusarium crown and root rot of greenhouse-grown tomato caused by Fusarium oxysporum f.sp. radicis-lycopersici (FORL) was investigated. The amendment of plant growth substratum with chitosan at concentrations of 12.5 or 37.5 mg l-1 significantly reduced plant mortality, root rot symptoms and yield loss attributed to FORL. Maximum disease control was achieved with chitosan at 37.5 mg l-1, when plant mortality was reduced by more than 90% and fruit yield was comparable with that of non-infected plants. In the absence of FORL, chitosan did not adversely affect plant growth and fruit yield. Cytological observations on root samples from FORL-inoculated plants revealed that the beneficial effect of chitosan in reducing disease was associated with increased plant resistance to fungal colonization. In chitosan-treated plants, fungal growth was restricted to the epidermis and the cortex. Invading hyphae showed marked cellular disorganization, characterized by increased vacuolation and even complete loss of the protoplast. The main host reactions included the formation of structural barriers at sites of attempted fungal penetration, the deposition of an opaque material (probably enriched with phenolics according to its electron density) in intercellular spaces and the occlusion of xylem vessels with tyloses, polymorphic bubbles and osmiophilic substances. Although chitosan may also have antifungal properties, the ultrastructural observations provide evidence that chitosan sensitizes tomato plants to respond more rapidly and efficiently to FORL attack. Chitosan has the potential to become a useful agent for controlling greenhouse diseases caused by soil-borne pathogens.