Molecular differentiation and virulence profiling of Fusarium oxysporum f. sp. lycopersici infecting tomato using ITS-RFLP markers

Under climate chamber conditions, suppression of tomato wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL) was obtained by spraying a suspension of Phytophthora cryptogea (Pc) zoospores on the green parts of two tomato cultivars, Danish Export (susceptible) and Elin F1 (moderately resistant). Direct competitive or antagonistic interaction between Pc and FOL in the soil or on the root surfaces was ruled out, but not interaction within the stems of plants. After Pc application followed by FOL inoculation, the two cultivars showed no wilt disease symptoms during the 50 days following FOL inoculation, whereas the FOL‐control plants were destroyed by wilt within about 40 days. Over a period of 7 weeks, both Pc and FOL were detected inside the stems of plants, for all treatments. Pc and FOL were also found inside the petioles of plants, except for one treatment, Pc application followed by challenge inoculation with FOL. Systemic induced resistance was assumed to be responsible for the observed elimination of disease incidence. It was concluded that, under climate chamber conditions, Pc application to the green parts of tomato plants is a viable alternative for control of disease incidence caused by FOL.

BackgroundThis study addresses the critical issue of Fusarium wilt in tomatoes, caused by Fusarium oxysporum f. sp. lycopersici (FOL), a severe fungal pathogen responsible for global yield losses. Conventional control measures, such as resistant crop varieties and chemical fungicides, have limitations due to environmental concerns and the risk of pathogen resistance. As a sustainable alternative, this study aims to explore the biocontrol potential of the bacterial strain Sh-17, focusing on its lipopeptides (LPs) to effectively suppress FOL.ResultsThis study demonstrated the antifungal capability of the Sh-17 strain, obtained from a tomato field, against FOL. Through 16S rDNA gene sequence analysis and phenotypic evaluation, Sh-17 was identified as Bacillus subtilis Sh-17. During the disease control assay using in vitro petri dishes, Sh-17 showed promising plant growth-promoting and disease-control capabilities in seedlings when tomato seeds were inoculated with both Sh-17 and FOL. Subsequently, the lipopeptide extract derived from Sh-17 showed strong antifungal properties in a dose-dependent manner, with complete inhibition of FOL at a concentration of 3500 µg mL−1. Furthermore, it was observed that LPs decreased the amount of ergosterol, which affects the stability and general structure of the plasma membrane. The genomic DNA of Sh-17 was subjected to PCR screening, which revealed the presence of genes responsible for the biosynthesis of antifungal LPs. Furthermore, LC–MS analysis identified distinct LPs, such as surfactins, fengycin, iturins, bacilysin, and bacillomycin derivatives in the crude LPs extract of Sh-17. Moreover, microscopic analyses (fluorescent/TEM) demonstrated morphological abnormalities and even death of the hyphae and spores of the phytopathogen upon its interaction with LPs.ConclusionsB. subtilis Sh-17 exhibits strong antifungal properties against FOL and supports seedlings health by protecting them from pathogen infestation. The LPs produced by Sh-17 inhibit FOL growth in a dose-dependent manner by disrupting the pathogen’s cellular structures and proved to be an effective biocontrol agent against Fusarium wilt in tomatoes.Graphical abstract

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is a devastating soilborne disease in tomatoes. Magnesium oxide nanoparticles (MgO NPs) induce strong immunity against Fusarium wilt in tomatoes. However, the mechanisms underlying this immunity remain poorly understood. Comparative transcriptome analysis and microscopy of tomato roots were performed to determine the mechanism of MgO NP-induced immunity against FOL. Eight transcriptomes were prepared from tomato roots treated under eight different conditions. Differentially expressed genes were compared among the transcriptomes. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that in tomato roots pretreated with MgO NPs, Rcr3 encoding apoplastic protease and RbohD encoding NADPH oxidase were upregulated when challenge-inoculated with FOL. The gene encoding glycine-rich protein 4 (SlGRP4) was chosen for further analysis. SlGRP4 was rapidly transcribed in roots pretreated with MgO NPs and inoculated with FOL. Immunomicroscopy analysis showed that SlGRP4 accumulated in the cell walls of epidermal and vascular vessel cells of roots pretreated with MgO NPs, but upon FOL inoculation, SlGRP4 further accumulated in the cell walls of cortical tissues within 48 h. The results provide new insights into the probable mechanisms of MgO NP-induced tomato immunity against Fusarium wilt.

Studying the mechanisms through which endophytic fungi confer protection to host plants against parasites will contribute toward elucidating the endophytic fungi-plant-pathogen relationship. In this study, we evaluated the effects of endophytic Beauveria bassiana on the antioxidant activity, oxidative stress, and growth of tomatoes infected with the fusarium wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL). Tomato seedlings were inoculated with B. bassiana conidia and then contaminated with FOL experimentally. Four treatments (Control [T1], FOL only [T2], B. bassiana only [T3], and B. bassiana and FOL [T4]) were assessed. The plants from the B. bassiana and FOL treatment (T4) were significantly taller (DF = 3, 56; p < 0.001) and produced more leaves and aerial part biomass than those treated with only FOL (T2). Remarkably, plants in the two treatments with FOL (T2 and T4) had the lowest antioxidant activities; meanwhile, plants from the FOL treatment (T2) had the lowest ROS (superoxide and hydroxyl radicals) contents. Broadly, strong positive correlations between ROS and all the plant growth parameters were recorded in this study. While the current results revealed that the endophytic entomopathogen B. bassiana enhanced antioxidant capacity in plants, it did not improve the antioxidant capacity of F. oxysporum-infected plants. It is possible that the pathogenic FOL employed a hiding strategy to evade the host immune response and the antagonistic actions of endophytic B. bassiana. In conclusion, B. bassiana inoculum enhanced the growth of tomatoes infected with FOL, induced higher oxidative stress in both F. oxysporum-infected and -uninfected tomatoes, and improved antioxidant activities in plants inoculated with B. bassiana only.

ABSTRACTSimultaneous infestation with root-knot nematodes (RKN) and Fusarium oxysporum f. sp. lycopersici (FOL) leads to formation of a disease complex that increases crop losses than effect of either RKN or FOL. In this study a management programme involving plant resistance, biological control agents, and neem was carried out to manage RKN and fusarium wilt disease complex. The biological control agents were Purpureocillium lilacinum (PL) and Trichoderma harzianum (TH) while the RKN was Meloidogyne javanica. In vitro dual culture plates were set up to test the interaction of biological control agents and FOL. Greenhouse experiments were conducted using two tomato cultivars Rambo F1 and Prostar F1. The treatments were; PL, TH, PL–TH, neem, PL neem, TH neem, and PL–TH neem. Each treatment was replicated four times and the treatments set up in a randomised complete block design in the greenhouse. Inhibition of FOL mycelial growth by TH and PL was 51.9%, and 44% respectively by the ninth day in vitro culture plates. In the cultivar, Prostar F1, the treatments PL–TH, PL, and TH in the presence or absence of neem had a FOL disease severity score significantly lower than the untreated control. Host resistance sufficed to prevent infection of Rambo F1 with FOL. The treatments PL–TH, PL and TH reduced FOL propagules and M. javanica juveniles in the roots and performed even better when combined with neem in both tomato cultivars. Therefore, a host that is resistant combined with biological control agents and organic amendments can be used in the management of RKN and FOL in tomato production.

Tomato is a popular vegetable widely grown in the tropics, which is mainly attacked by fusarium wilt incited by Fusarium oxysporum f. sp. lycopersici (FOL). In the present scenario, an ecofriendly alternative strategy such as use of fungi from rhizosphere is being explored to combat the phytopathogen invasion. This study was carried out to evaluate the efficacy of Trichoderma asperellum MSST to promote the growth and yield parameters of tomato S-22, a susceptible variety. This study was also undertaken to manage fusarium wilt disease under in vitro and in vivo conditions. Significant increase in vegetative parameters like root length, shoot length, plant weight and chlorophyll content 60 days after sowing (DAS) was observed. There was reduction in the incidence of fusarium wilt in tomato up to 85%. Increase in the level of total phenol, peroxidase, polyphenoloxidase and phenylalanine ammonium lyase activity at 10th day of pathogen inoculation showed enhancement of plant defence mechanism by T. asperellum MSST against FOL. Overall study revealed that isolate MSST was proven to be potential biocontrol agent showing induced resistance against FOL.

Fusarium wilt is one of the most important diseases that affects various plants and is caused by the fungus Fusarium oxysporum. Fusarium wilts in tomato plants are caused by Fusarium oxysporum f. sp. lycopersici (FOL), whether in fields or greenhouses. The tomato plant is considered one of the most important plants. This disease, however, affects its agricultural production. The current research aimed to diagnose Fusarium species that were isolated from infected tomato plants from different regions in Iraq by using molecular and morphological methods and to study the evolutionary relationship between the studied species, as twenty Fusarium isolates were obtained from infected tomato plants. Genomic DNA was amplified, using primers ITS1 and ITS4 to amplify ITS region, as there were differences between the results of the morphological diagnosis, which were based on the study of morphological features, and the molecular diagnosis which was based on the ITS region. Depending on the morphological features, F7 and F8 isolates were diagnosed as F. oxysporum, while the molecular diagnosis of the ITS regions showed that they were F.incarnatum and F.proliferatum respectively. For the rest of the isolates, the morphological and molecular diagnoses were consistent with Fusarium oxysporum. All isolates were recorded in the Gen Bank, and were given their accession numbers. An evolutionary tree was also made for the studied isolates to know the degree of genetic convergence and divergence between them. The current study concluded that the tomato plants in the field and greenhouse from different regions in Iraq were infected with different Fusarium species, with Fusarium oxysporum f. sp. lycopersici, being the most common species, was widely spreading among the identified isolates. Conventional methods based on morphological features take time and may result in incorrect identification of closely related species. Henceforth, Fusarium species may be correctly identified by using molecular methods based on ITS sequences.

ABSTRACTNine non-pathogenic bacterial isolates, recovered from Datura metel organs and able to colonise the internal stem tissues of tomato cultivar Rio Grande, were screened for their ability to suppress tomato Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL), and to enhance plant growth. S33 and S85 isolates tested were found to be the most effective in decreasing Fusarium wilt severity by 94–95% compared to FOL-inoculated and untreated control. A significant enhancement of growth parameters was recorded on tomato plants inoculated or not with FOL. Both isolates were characterised and identified using 16S rDNA sequencing genes as Stenotrophomonas sp. str. S33 (KR818084) and Pseudomonas sp. str. S85 (KR818087). Screened in vitro for their antifungal activity towards FOL, these isolates led to 38.7% and 22.5% decrease in pathogen radial growth and to the formation of an inhibition zone of 12.75 and 8.37 mm respectively. Stenotrophomonas sp. str. S33 and Pseudomonas sp. str. S85 were found to be chitinase-, protease- and pectinase-producing strains but unable to produce hydrogen cyanide. Production of indole-3-acetic acid-like compounds, phosphate solubilising ability and pectinase activity were investigated for elucidating their plant growth-promoting traits and their endophytic colonisation ability.

Evaluation of the Resistance and Differential Induction of Chitinases in Tomato in Response to Inoculation with Fusarium oxysporum f. sp.lycopersici Fusarium oxysporum f. sp. lycopersici (FOL) causes Fusarium wilt in tomato, a disease that significantly affects its production. This project investigated the response of different tomato genotypes to Fusarium wilt, aiming at selecting sources of resistance, as well as identifying the differential chitinase secretion during infection between resistant and susceptible cultivars. Results show that the BHRS cultivar proved to be resistant to the disease and there was increased chitinolytic activity in the roots of this genotype six days after inoculation (dai).

Elicitation of plant defense enzymes against Fusarium oxysporum f. sp. lycopersici in tomato plant using a novel rhizobacteria Providencia rettgeri MSS2

RETRACTED: Biocontrol of Fusarium wilt in tomato by Trichoderma spp

The use of novel isolates of Trichoderma with efficient antagonistic capacity against Fusarium oxysporum f. sp. lycopersici (FOL) is a promising alternative strategy to pesticides for tomato wilt management. We evaluated the antagonistic activity of 30 isolates of T. asperellum against 4 different isolates of FOL. The production of extracellular cell wall degrading enzymes of the antagonistic isolates was also measured. The random amplified polymorphic DNA (RAPD) method was applied to assess the genetic variability among the T. asperellum isolates. All of the T. asperellum isolates significantly reduced the mycelial growth of FOL isolates but the amount of growth reduction varied significantly as well. There was a correlation between the antagonistic capacity of T. asperellum isolates towards FOL and their lytic enzyme production. Isolates showing high levels of chitinase and β-1,3-glucanase activities strongly inhibited the growth of FOL isolates. RAPD analysis showed a high level of genetic variation among T. asperellum isolates. The UPGMA dendrogram revealed that T. asperellum isolates could not be grouped by their anta- gonistic behavior or lytic enzymes production. Six isolates of T. asperellum were highly antagonistic towards FOL and potentially could be used in commercial agriculture to control tomato wilt. Our results are consistent with the conclusion that understanding the genetic variation within Trichoderma isolates and their biochemical capabilities are required for the selection of effective indigenous fungal strains for the use as biocontrol agents.

The spaceflight environment imparts unique selective pressures on the plants and microbes of plant growth chambers on the International Space Station (ISS), which generally manifests through genetic signatures associated with a heightened response to stress. Terrestrially, a baseline understanding of the gene fitness response for any plant growth-promoting microbe when in a tripartite relationship with host and pathogen is currently unknown and is important to characterize before closed-environment spaceflight implementation. To that end, this study evaluated the behavior of an ISS plant habitat isolate of Burkholderia contaminans as tomato seeds transitioned to seedlings and assessed gene fitness during challenge with Fusarium oxysporum f. sp. lycopersici (FOL), the causal agent of Fusarium wilt. Using a seed film delivery method vetted for spaceflight, B. contaminans was applied to Solanum lycopersicum cv. Red Robin seeds. Green fluorescent protein (GFP)-tagged B. contaminans was primarily found to localize at the shoot-root junction and was detected on shoots. Upon FOL challenge, B. contaminans population levels remained stable, and despite harboring antifungal and plant growth-promoting capacity, these properties were not conferred in response to FOL in the tissue culture environment. To probe mechanisms underlying the bacterial-fungal interaction between B. contaminans and FOL in the tomato root zone, a genome-wide transposon mutant library was developed for the B. contaminans isolate. Transposon sequencing (Tn-Seq) analysis revealed that the type II secretion system (T2SS) was critical for root zone establishment, whereas a Nudix hydrolase was specifically important for responding to FOL infection and provided further confirmation that antifungal and siderophore-producing gene clusters were not.IMPORTANCEThis study is the first to evaluate the genetic fitness of a Burkholderia contaminans International Space Station (ISS) isolate in the plant root zone in association with the obligate pathogen Fusarium oxysporum f. sp. lycopersici (FOL). This isolate of B. contaminans establishes in the tomato root zone, does not confer plant growth promotion in tissue culture, but is persistent in the tomato root zone when challenged with FOL through stress-adaptation mechanisms rather than direct antifungal antagonism. The response of B. contaminans in the host root zone when in the presence of the pathogen suggests the microbe is primed to counter stress, which may further confer an advantage in the spaceflight environment.

Five primer/probe sets to identify the tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL), and its three races selectively were designed based on the rDNA-intergenic spacer and avirulence genes. Real-time PCR using genomic DNA from mycelia and soil DNA with the primer/probe sets allowed the successful identification of FOL and its races.

BackgroundFusarium wilt biocontrol using endophytic microorganisms may represent a potentially attractive and environmentally safe alternative since endophytes could better limit disease incidence and severity through inhibition of the systemic fungus progress.Main body of the abstractTwenty-three endophytic bacterial isolates, naturally associated with Solanum sodomaeum and Solanum bonariense, were evaluated for their ability to control Fusarium wilt of tomato induced by Fusarium oxysporum f. sp. lycopersici (FOL) and to promote plant growth. Selected endophytic isolates were screened in vivo, using the root dipping and the culture substrate drenching methods. The most bioactive isolates were subjected to morphological and biochemical characterization and subsequent identification through 16S rDNA sequencing genes. Seven isolates (Stenotrophomonas maltophilia S23, S24, S26 and S28; Bacillus sp. SV81; Azotobacter chroococcum S11; and Serratia marcescens S14) were found to be the most efficient in reducing disease severity by 82–96% over control. Treatments with these isolates led to a significant enhancement in growth parameters, estimated at 45.5–61 and 24.2–70.5% than the control, in tomato plants infected or not with FOL, respectively. Diffusible and volatile metabolites released from bacterial cultures had significantly limited FOL radial growth. All isolates were positive for indole-3-acetic acid (IAA) production. S. marcescens S14, S. maltophilia S28, and Bacillus sp. SV81 exhibited a positive phosphate solubilization activity. Production of chitinase, protease, pectinase, and hydrogen cyanide were also investigated.Short conclusionThis study clearly demonstrated that endophytic bacteria recovered from these 2 Solanum species can be explored as promising biocontrol agents active against FOL and are able to enhance tomato growth.