Fusarium Oxysporum F. Sp. Cucumerinum Research Articles

Co-Culture of White Rot Fungi Pleurotus ostreatus P5 and Bacillus amyloliquefaciens B2: A Strategy to Enhance Lipopeptide Production and Suppress of Fusarium Wilt of Cucumber.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), poses a serious threat to cucumber productivity. Compared to traditional chemical pesticides, biological control strategies have attracted more attention recently owing to their effectiveness against pathogens and their environmental safety. This study investigated the effect of white rot fungi Pleurotus ostreatus P5 on the production of cyclic lipopeptides (CLPs) of Bacillus amyloliquefaciens B2 and the potential co-culture filtrate of strains B2 and P5 to control cucumber Fusarium wilt. A PCR amplification of CLP genes revealed that B. amyloliquefaciens B2 had two antibiotic biosynthesis genes, namely, ituA and srf, which are involved in iturin A and surfactin synthesis. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that CLPs derived from strain B2 contained two families, iturin A (C14, C15) and surfactin (C12-C17). The co-culture exhibited an enhanced accumulation of iturin A and surfactin compared to the monoculture of strain B2. Furthermore, the gene expressions of ituA and srf were both significantly upregulated when co-cultured with the fungus compared to monocultures. In an in vitro experiment, the co-culture filtrate and monoculture filtrate of B. amyloliquefaciens B2 inhibited mycelial growth by 48.2% and 33.2%, respectively. In a greenhouse experiment, the co-culture filtrate was superior to the monoculture filtrate in controlling cucumber Fusarium wilt disease and in the promotion of plant growth. Co-culture filtrate treatment significantly enhanced the microbial metabolic activity and decreased the abundance of FOC in the rhizosphere soil. These results show that the co-culture of P. ostreatus P5 and B. amyloliquefaciens B2 has great potential in cucumber Fusarium wilt disease prevention by enhancing the production of bacterial CLPs.

Metabolomic and Transcriptome Analysis of the Inhibitory Effects of Bacillus subtilis Strain Z-14 against Fusarium oxysporum Causing Vascular Wilt Diseases in Cucumber

Controlling cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC) with Bacillus strains is a hot research topic. However, the molecular mechanism of Bacillus underlying the biocontrol of cucumber wilt is rarely reported. In this study, B. subtilis strain Z-14 showed significant antagonistic activity against FOC, and the control effect reached 88.46% via pot experiment. Microscopic observations showed that strain Z-14 induced the expansion and breakage of FOC hyphae. The cell wall thickness was uneven, and the organelle structure was degraded. The combined analysis of metabolome and transcriptome showed that strain Z-14 inhibited the FOC infection by inhibiting the synthesis of cell wall and cell membrane, energy metabolism, and amino acid synthesis of FOC mycelium, inhibiting the clearance of reactive oxygen species (ROS) and the secretion of cell wall-degrading enzymes (CWDEs), thereby affecting mitogen-activated protein kinase (MAPK) signal transduction and inhibiting the transport function.

Dissection of Paenibacillus polymyxa NSY50-Induced Defense in Cucumber Roots against Fusarium oxysporum f. sp. cucumerinum by Target Metabolite Profiling.

Simple SummaryPlant growth-promoting rhizobacteria (PGPR) have significant potential to enhance the tolerance of biotic and abiotic stresses and the productivity of crops. However, the mechanism of PGPR in improving plant resistance to pathogens is unclear. Recently, the newly isolated Paenibacillus polymyxa strain NSY50 was shown to considerably suppress the Fusarium wilt of cucumber plants. This study was carried out to explore the underlying mechanism of NSY50 in improving plant resistance to pathogen invasion via target metabolite profiling, and the results indicated that strain NSY50 was able to alleviate Fusarium wilt stress by activating GSH metabolism and improving redox balance. Our research findings enable a deeper understanding of P. polymyxa NSY50-induced enhanced defense against F. oxysporum in cucumber.To gain insights into the roles of beneficial PGPR in controlling soil-borne disease, we adopted a metabolomics approach to investigate the beneficial impacts of P. polymyxa NSY50 on cucumber seedling roots under the pathogen of Fusarium oxysporum f. sp. cucumerinum (FOC). We found that NSY50 pretreatment (NSY50 + FOC) obviously reduced the production of reactive oxygen species (ROS). Untargeted metabolomic analysis revealed that 106 metabolites responded to NSY50 and/or FOC inoculation. Under FOC stress, the contents of root osmotic adjustment substances, such as proline and betaine were significantly increased, and dehydroascorbic acid and oxidized glutathione (GSH) considerably accumulated. Furthermore, the contents of free amino acids such as tryptophan, phenylalanine, and glutamic acid were also significantly accumulated under FOC stress. Similarly, FOC stress adversely affected glycolysis and the tricarboxylic acid cycles and transferred to the pentose phosphate pathway. Conversely, NSY50 + FOC better promoted the accumulation of α-ketoglutaric acid, ribulose-5-phosphate, and 7-phosphosodiheptanone compared to FOC alone. Furthermore, NSY50 + FOC activated GSH metabolism and increased GSH synthesis and metabolism-related enzyme activity and their encoding gene expressions, which may have improved redox homoeostasis, energy flow, and defense ability. Our results provide a novel perspective to understanding the function of P. polymyxa NSY50, accelerating the application of this beneficial PGPR in sustainable agricultural practices.

Termite Nest Associated Bacillus siamensis YC-9 Mediated Biocontrol of Fusarium oxysporum f. sp. cucumerinum.

The antagonistic potential of bacteria obtained from the nest of Odontotermes formosanus was assessed against Fusarium oxysporum f. sp. cucumerinum (FOC). Of 30, seven termite nest-associated bacteria strains had biocontrol potential. Among them, the strain YC-9 showed the strongest antifungal activity toward FOC. Phylogenetic analysis of the 16S rRNA amplified product of YC-9 revealed its identification as Bacillus siamensis. The in vivo antifungal activity experiment showed that the application of YC-9 at 108 cfu/ml significantly reduced the cucumber wilt incidence with a control efficacy of 73.2%. Furthermore, plant growth parameters such as fresh weight, dry weight, plant height, and root height were significantly improved by 42.6, 53.0, 20.8, and 19.3%, respectively. We found that inoculation with B. siamensis YC-9 significantly increased the activity of defensive enzymes such as peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) in diseased cucumber roots, thereby raising the resistance. PCR using gene-specific primers revealed that B. siamensis YC-9 contains biosynthetic genes for known antibiotics, including bacillomycin, iturin, and surfactin. Chemical analysis of the cultivation of B. siamensis YC-9 resulted in the isolation of five metabolites, including hexadecanoic acid (1), cyclo-(L-phenylalanylglycine) (2), cyclo-(L-trans-Hyp-L-Leu) (3), C15-surfactin (4), and macrolactin A (5), the structures of which were identified by the analysis of NMR spectroscopic data and MS. Among them, the compound 4 showed significant antifungal activity against conidial germination of FOC with an IC50 value of 5.1 μg/ml, which was comparable to that of the positive control, cycloheximide (IC50 value of 2.6 μg/ml). Based on these findings, this study suggests that termite-nest associated B. siamensis YC-9 could be a potential biological control agent for integrated control of soil-borne diseases like cucumber Fusarium wilt.

Proteomic insights of chitosan mediated inhibition of Fusarium oxysporum f. sp. cucumerinum

Fusarium oxysporum f. sp. cucumerinum (FOC) infects cucumber plants, causing significant yield losses. Chitosan is a natural biodegradable compound that has antifungal properties. To understand the inhibitory mechanism of chitosan against FOC, a comprehensive proteomic study was carried out for the identification of chitosan responsive proteins (CRPs) from the mycelia of chitosan-treated FOC. Two-dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS analysis led to the identification of 62 differentially abundant CRPs. Functional classification of these CRPs revealed that most proteins were involved in metabolism and defense. Gene Ontology analysis revealed that the majority of the proteins were assigned in proteolysis and hydrolase activity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that among the biologically active pathways in chitosan-treated FOC mycelia, ‘carbohydrate metabolism’ was enriched for most of the proteins. This study gives a snapshot of the molecular basis of fungal inhibition by chitosan resulting in disease resistance in cucumber plants after inoculation with chitosan-treated FOC by explaining how chitosan restricted disease severity (i.e., down-regulating the plant cell wall degrading enzymes, FOC self-attack, hindering FOC structural and functional protein biosynthesis and DNA biosynthesis and affecting FOC transporter proteins). This study contributes to putting more weight on using the bioactive natural compound chitosan as an antifungal material instead of applying chemical fungicides in agriculture. SignificanceChitosan has been used as one of the safe and effective alternatives to fungicides in controlling cucumber vascular wilt disease caused by Fusarium oxysporum f. sp. cucumerinum (FOC) that is responsible for severe production losses. Chitosan application showed a significant decrease in wilt disease severity compared to chitosan untreated FOC and showed an efficiency of 91.7% in reducing pathogenicity. A comprehensive proteomic investigation of chitosan-responsive proteins (CRPs) from the mycelia of chitosan-treated FOC was carried out in order to better understand the inhibitory mechanism of chitosan against FOC which led us to identify 62 differentially expressed CRPs. Our proteomic study revealed CRPs in FOC involved in a variety of functions, including disease inhibition in cucumber. This study depicts what happens inside the fungus following treatment with chitosan and how chitosan played the role of the maestro in influencing the synthesis of proteins responsible for the virulence of FOC and their respective pathways, rendering FOC unable to infect the cucumber plant and lose its pathogenic potential to cause wilt disease. The efficiency of chitosan in inhibiting certain proteins or specific pathways of FOC gives a golden opportunity in controlling vascular wilt, so we highly recommend applying chitosan in disease management under greenhouse conditions or in the open field

Enhanced Biocontrol of Cucumber Fusarium Wilt by Combined Application of New Antagonistic Bacteria Bacillus amyloliquefaciens B2 and Phenolic Acid-Degrading Fungus Pleurotus ostreatus P5.

Continuous monoculture of cucumber (Cucumis sativus L.) typically leads to the frequent incidence of Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC). As potent allelochemicals, phenolic acids are believed to be associated with soilborne diseases. This study aimed to investigate the effect of single or co-inoculation of antagonistic bacteria Bacillus amyloliquefaciens B2 and phenolic acid-degrading fungus Pleurotus ostreatus P5 on the suppression of cucumber Fusarium wilt. The strain B2 was identified as B. amyloliquefaciens based on biochemical, physiological, and 16S rDNA and gyrB gene sequence analyses. Strain B2 showed indole-3-acetic acid (IAA) and siderophore production and phosphate solubilization in in vitro assays. Scanning electron microscope (SEM) imaging showed the ability of strain B2 to adhere to the root surface of cucumber. P. ostreatus P5 could effectively degrade mixed phenolic acids as its sole source of carbon and energy for growth in liquid medium. In a pot experiment, four treatments were established as follows: (1) CK, uninoculated control; (2) B2, inoculation of strain B2; (3) P5, inoculation of strain P5; and (4) B2 + P5, co-inoculation of strain B2 and strain P5. At the end of the 60-day pot experiment, the B2, P5, and B2 + P5 treatments significantly reduced disease incidence by 48.1, 22.2, and 63.0%, respectively, compared to the CK treatment (p < 0.05). All three inoculation treatments significantly increased the growth of cucumber seedlings and suppressed the FOC population compared to the control (p < 0.05). High-performance liquid chromatography (HPLC) analysis showed that total phenolic acids were decreased by 18.9, 35.9, and 63.2% in the B2, P5, and B2 + P5 treatments, respectively. The results from this study suggest that combined application of B. amyloliquefaciens B2 and P. ostreatus P5 could be a promising strategy for suppressing Fusarium wilt and improving plant growth of cucumber seedlings under continuous cropping conditions.

Comparative proteomics analyses of mycelial, conidial, and Secreted Proteins of high-pathogenic and weak-pathogenic Fusarium oxysporum f. sp. cucumerinum isolates

Fusarium oxysporum f. sp. cucumerinum (FOC) is one of the important fungi that infect cucumber plants, causing significant yield losses. Plant pathogenic fungi differ in their disease severity and aggressiveness degree with diverse infection modes governed by specialized proteins involved in signaling pathways leading to pathogenicity, the area which is less explored. To understand the pathogenicity and virulence variation of FOC isolates, a comprehensive proteomic study was done to compare the polypeptide patterns of mycelial, conidial, and secreted proteins of the highly pathogenic (hp-FOC) and weakly pathogenic (wp-FOC) isolates. Two-dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS analysis led to the identification of shared and unique proteins involved in metabolism and pathogenicity in both fungal isolates. This study may outline the biological and molecular basis of the different fungal structures to understand the mechanism of their action inside the host tissues. The determination of proteins that express the ability of the pathogen to induce pathogenesis may aid in detecting specialized inhibitors that suppress pathogenic protein activities to control the disease, this in turn may contribute to reduce the excessive use of fungicides, which further enhance the productivity, crop quality, and environmental safety.

Succinoglycan Riclin reshaped the soil microbiota by accumulating plant probiotic species to improve the soil suppressiveness on Fusarium wilt of cucumber seedlings

Biocontrol of soil-borne pathogens by recruiting soil microbiota brings forth benefits to soil quality and plant production while lowers environmental impact. Succinoglycan possesses various biological activities, but its role in soil amendment is still elusive. The succinoglycan Riclin was investigated in this study as a polysaccharide-type biocontrol agent for improving the soil suppressiveness on a typical fungal pathogen Fusarium oxysporum f. sp. cucumerinum (FOC). Results demonstrated that addition of Riclin increased the soil microbial carbon and lowered the species richness of soil fungal communities. After addition of 2.5 mg/g Riclin for 90 days, the relative abundance of Actinobacteria and Firmicutes were increased by 76.6% and 193.4%, compared with the control. Meanwhile, Proteobacteria and Ascomycota were decreased by 25.9% and 30.4%. The relative abundance of beneficial genera, namely Nocardioides, Kribbella, Streptomyces, Gaiella, Marmoricola, Bacillus, and Rhizobium, became 1.13, 5.17, 0.87, 0.45, 3.57, 4.53, and 6.30 folds higher than the control, respectively. Antagonism towards soil-borne pathogens was probably enhanced as both hydrolase activity and biosynthesis of bioactive secondary compounds were improved. Importantly, Riclin-treated soil significantly reduced the incidence of Fusarium wilt of cucumber seedlings by suppression of FOC. In conclusion, addition of Riclin was conducive to the improvement of soil suppressiveness.

Screening and identification of antagonistic bacteria from vermicompost against Fusarium oxysporum f. sp. cucumerinum

ABSTRACT Fusarium oxysporum f. sp. cucumerinum (FOC) is one of the most destructive pathogens in many cucumber-growing countries around the globe. There is still lack of high-resistant cucumber cultivars against this pathogen, and the use of fungicides is threatening the environmental conditions. However, biocontrol is an environment-friendly technique in plant protection. In this regard, the present work was aimed to screen antagonistic bacterial strains from vermicompost to against FOC in cucumber. Using the dilution-plate method, 374 bacterial strains were isolated from fresh vermicompost. A total of 28 bacterial strains showed antagonistic activity against FOC, among which strain D2 exhibited the best inhibitory effect on the growth of FOC, with the diameter of the growth inhibition zone of 28.28 mm. Then strain D2 was classified and identified based on the morphology and colony characteristics, physiological and biochemical characteristics, and 16S ribosomal DNA (rDNA) sequence homology. For molecular identification, a 1,462-bp fragment of 16S rDNA of strain D2 was obtained. Based on its morphological, physiological, and biochemical characteristics, strain D2 was identified as Burkholderia cenocepacia.

Allelopathic effects of acetone extract from celery rotten roots and rhizosphere soil on cucumber pathogen Fusarium oxysporum f. sp. cucumerinum

We studied the inhibitory effects of allelochemicals extracted from celery (Apium graveolens L.) rotten roots and rhizosphere soil on Fusarium oxysporum f. sp. cucumerinum (FOC). The allelochemicals were extracted and separated by column chromatography from the acetone extracts of celery rotten roots and rhizosphere soil. After extraction each fraction was mixed with potato dextrose agar (PDA) culture medium and then inoculated with FOC. The best fraction was screened by measuring the colony diameter and its effects on FOC was determined by evaluating the activities of antioxidant enzymes. The allelochemicals of best fractions were detected using GC-MS. The best fractions obtained after second run in column chromatography were labeled as RRA32, RRA38, RRA101, RRA102 and RRSA55, RRSA56, RRSA105, RRSA106 and they had allelopathic potential of 29.68 %, 31.97 %, 40.38 %, 41.55 % and 29.51 %, 29.47 %, 29.30 % and 32.85 % respectively. The antioxidants enzymes (peroxidase, catalase and superoxide dismutase) activities were significantly lower in treated FOC than control. Using the 8-best fractions, the GC-MS analysis yielded, total 47 compounds viz., 7 organic acids, 17 esters, 1 phenol, 1 alcohol, 1 aldehyde, 5 nitrogen-containing compounds and 2 carbides.

Fusarium oxysporum Isolates Collected from the same Geographical Zone Exhibited Variations in Disease Severity and Diversity in Morphological and Molecular Characters

Thirty Fusarium oxysporum f. sp. cucumerinum (FOC) isolates were collected from Kafr El-Sheikh governorate, Egypt to study their pathogenic variability and genetic diversity. All isolates caused the typical symptoms of wilting with different severity degrees and reduced cucumber growth parameters and crop productivity under greenhouse conditions. We have classified the isolates to different groups based on pathogenicity, morphological characters, disease quantum degrees and genetic variation. The polymorphisms obtained from RAPD and protein profiles revealed a high degree of genetic diversity and their cluster analysis was matched with the results obtained from the pathogenicity. Previously it was believed that the different collection regions of the same pathogen isolates were the cause for their pathological and morphological differences, and there was no genetic variation can be occurring between isolates as long as they were collected from the same place. In our results, the genetic diversity of FOC isolates proves that the genetic variation of fungus isolates can occur in the same geographical location.

Dragonfly-Associated Trichoderma harzianum QTYC77 Is Not Only a Potential Biological Control Agent of Fusarium oxysporum f. sp. cucumerinum But Also a Source of New Antibacterial Agents.

A strain isolated from the gut of Pantala flavescens was characterized as Trichoderma harzianum QTYC77. The strain was assessed as a potential biocontrol agent against Fusarium oxysporum f. sp. cucumerinum (FOC). Mycoparasitism and competing abilities of T. harzianum QTYC77 lead to inhibition of the mycelial growth of FOC, with the inhibition rate of 70.99%, in dual culture assays. Activities of chitinase and β-1,3-glucanase, responsible for fungal cell-wall degradation, were gradually increased and their activities were the maximum on the fifth day of fermentation with 23.20 and 1.84 U/mL, respectively. T. harzianum QTYC77 was discovered to have potent biocontrol potential with the control efficiency of 67.43% against the FOC in vivo pot experiment. Furthermore, two novel compounds azaphilone D (1) and E (2) along with three known metabolites 3-hydroxymethyl-6, 8-dimethoxycoumarin (3), harzianone (4), and pachybasin (5) were isolated and identified from T. harzianum QTYC77. Unfortunately, these metabolites did not show antifungal activities against FOC. However, both metabolites 1 and 3 displayed moderate activity against Staphylococcus aureus with disc diameters of zone of inhibition (ZOI) of 7.3 and 7.2 mm, respectively, compared with that of referenced gentamycin (ZOI = 14.5 mm). In addition, metabolite 1 possessed a moderate antibacterial activity against Bacillus subtilis with a ZOI value of 7.0 mm compared with that of positive gentamycin (ZOI = 15.2 mm). The present results suggested that T. harzianum QTYC77 was not only a potential biofungicide against FOC but also the source of new antibacterial agents.

Identification of SCAR markers linked to the Foc gene governing resistance to Fusarium oxysporum f. sp. cucumerinum in cucumber cv. SMR-18

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC) is an important soil-borne disease of cucumber in several parts of the world. In this study, we employed amplified fragment length polymorphic (AFLP) markers along with bulked segregant analysis (BSA), in order to identify structural markers linked to the Foc gene that governs resistance to FOC races 1, 2 and 3 in the resistant cv. SMR-18. From the four AFLP markers originally identified at the BSA level, only one (E12M50134 + T) maintained a linkage in coupling phase against the F2 mapping population. AFLP-mediated sequencing was employed in order to ensure the identification and sequencing of the truly linked AFLP fragment. This AFLP marker mapped at a distance of 6.0 cM from the Foc resistance locus and was successfully converted into two sequence-characterized amplified region (SCAR) co-dominant markers. SCAR marker SCE12M50B produced a single fragment linked to Foc and behaved as a dominant marker, while SCE12M50A amplified one strong fragment linked to Foc in parallel with a second faint fragment. The SCE12M50B fragment physically maps on chromosome 2 and lies 1.64 Mb (7,0 cM) away from the microsatellite marker SSR03084, which is closely linked to Ccu locus that controls resistance to scab, caused by Cladosporium cucumerinum in cv. SMR-18.

Biocidal effects of volatile organic compounds produced by the myxobacterium Corrallococcus sp. EGB against fungal phytopathogens

Myxobacteria have excellent biocontrol activity against various phytopathogens due to their rich spectrum of secondary metabolites and active predatory characteristics. In this study, the mycelial growth of Fusarium oxysporum f. sp. cucumerinum (FOC) was found to be significantly inhibited by volatile compounds (VOCs) produced by Corallococcus sp. EGB. A total of 32 compounds were identified among the VOCs produced by strain EGB, of which isooctanol exhibited the highest antifungal activity, with dosages of 3.75 and 4.0 μL/plate being sufficient to suppress FOC and Penicillum digitatum, respectively. Isooctanol was found to damage the cell wall and cell membranes of FOC and P. digitatum. Apoptosis-like cell death of FOC and P. digitatum induced by isooctanol was observed subsequently due to the accumulation of reactive oxygen species (ROS). The transcription level of genes related to cell wall integrity (CWI) pathway and redox reactions were significantly upregulated by 15- to 40-fold, indicating the stress caused by isooctanol. Postharvest storage experiments showed that the disease severity of post-harvest oranges infected with P. digitatum could be significantly reduced by isooctanol at 114.2 μL/L.

A predatory myxobacterium controls cucumber Fusarium wilt by regulating the soil microbial community

BackgroundMyxobacteria are micropredators in the soil ecosystem with the capacity to move and feed cooperatively. Some myxobacterial strains have been used to control soil-borne fungal phytopathogens. However, interactions among myxobacteria, plant pathogens, and the soil microbiome are largely unexplored. In this study, we aimed to investigate the behaviors of the myxobacterium Corallococcus sp. strain EGB in the soil and its effect on the soil microbiome after inoculation for controlling cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC).ResultsA greenhouse and a 2-year field experiment demonstrated that the solid-state fermented strain EGB significantly reduced the cucumber Fusarium wilt by 79.6% (greenhouse), 66.0% (2015, field), and 53.9% (2016, field). Strain EGB adapted to the soil environment well and decreased the abundance of soil-borne FOC efficiently. Spatiotemporal analysis of the soil microbial community showed that strain EGB migrated towards the roots and root exudates of the cucumber plants via chemotaxis. Cooccurrence network analysis of the soil microbiome indicated a decreased modularity and community number but an increased connection number per node after the application of strain EGB. Several predatory bacteria, such as Lysobacter, Microvirga, and Cupriavidus, appearing as hubs or indicators, showed intensive connections with other bacteria.ConclusionThe predatory myxobacterium Corallococcus sp. strain EGB controlled cucumber Fusarium wilt by migrating to the plant root and regulating the soil microbial community. This strain has the potential to be developed as a novel biological control agent of soil-borne Fusarium wilt.6RWdjdPf3LfQd4sE1BE91JVideo abstract.

Mixed culture fermentation between Rhizopus nigricans and Trichoderma pseudokoningii to control cucumber Fusarium wilt

Fusarium wilt, caused by Fusarium oxysporum, is a devastating soilborne fungal disease. Compared to traditional chemical pesticides, biological control has been attracting increasing attention recently, owing to its eco-friendly features. This study investigated the potential use of mixed culture fermentation of Rhizopus nigricans and Trichoderma pseudokoningii (MCF) to control Fusarium oxysporum f.sp.cucumerinum (FOC). At first, the tablet confrontation experiment using bio-control fungi and F. oxysporum showed that the suppression rate of R. nigricans and T. pseudokoningii were more than 60% higher than that of individual fermentations. Furthermore, the inhibitory effect of MCF on the pathogen was higher than that of single fermentation broth, both in vitro and in greenhouse experiment. At the molecular level, MCF had better control on the number of pathogens in the plant tissue and soil, as seen by the real-time PCR assay. In the pot experiment, increased activities of the defense enzymes involved in PAL, SOD, and POD indicated that mixed broth treatment could increase the plant-induced resistance. In the field experiment, after two-year treatment by the mixed broth, the relative control effect on cucumber Fusarium wilt was 76.5%. Through this study, we demonstrated that co-fermentation of R. nigricans and T. pseudokoningii has synergistic effect on the control of F. oxysporum, which may promote more reasonable and effective utilization of biocontrol agents in future.

Plant Primary Metabolism Regulated by Nitrogen Contributes to Plant-Pathogen Interactions.

Nitrogen contributes to plant defense responses by the regulation of plant primary metabolism during plant-pathogen interactions. Based on biochemical, physiological, bioinformatic and transcriptome approaches, we investigated how different nitrogen forms (ammonium vs. nitrate) regulate the physiological response of cucumber (Cucumis sativus) to Fusarium oxysporum f. sp. cucumerinum (FOC) infection. The metabolic profile revealed that nitrate-grown plants accumulated more organic acids, while ammonium-grown plants accumulated more amino acids; FOC infection significantly increased levels of both amino acids and organic acids in the roots of ammonium-grown plants. Transcriptome analysis showed that genes related to carbon metabolism were mostly up-regulated in plants grown with nitrate, whereas in ammonium-grown plants the up-regulated genes were mostly those that were related to primary nitrogen metabolism. Root FOC colonization and disease incidence were positively correlated with levels of root amino acids and negatively correlated with levels of root organic acids. In conclusion, organic acid metabolism and expression of related genes increased under nitrate, whereas ammonium increased the level of amino acids and expression of related genes; these altered levels of organic acids and amino acids resulted in different tolerances to FOC infection depending on the nitrogen forms supplied.

Wilted cucumber plants infected by Fusarium oxysporum f. sp. cucumerinum do not suffer from water shortage.

Fusarium wilt is primarily a soil-borne disease and results in yield loss and quality decline in cucumber (Cucumis sativus). The main symptom of fusarium wilt is the wilting of entire plant, which could be caused by a fungal toxin(s) or blockage of water transport. To investigate whether this wilt arises from water shortage, the physiological responses of hydroponically grown cucumber plants subjected to water stress using polyethylene glycol (PEG, 6000) were compared with those of plants infected with Fusarium oxysporum f. sp. cucumerinum (FOC). Parameters reflecting plant water status were measured 8d after the start of treatment. Leaf gas exchange parameters and temperature were measured with a LI-COR portable open photosynthesis system and by thermal imaging. Chlorophyll fluorescence and chloroplast structures were assessed by imaging pulse amplitude-modulated fluorometry and transmission electron microscopy, respectively. Cucumber water balance was altered after FOC infection, with decreased water absorption and hydraulic conductivity. However, the responses of cucumber leaves to FOC and PEG differed in leaf regions. Under water stress, measures of lipid peroxidation (malondialdehyde) and chlorophyll fluorescence indicated that the leaf edge was more seriously injured, with a higher leaf temperature and disrupted leaf water status compared with the centre. Here, abscisic acid (ABA) and proline were negatively correlated with water potential. In contrast, under FOC infection, membrane damage and a higher temperature were observed in the leaf centre while ABA and proline did not vary with water potential. Cytologically, FOC-infected cucumber leaves exhibited circular chloroplasts and swelled starch grains in the leaf centre, in which they again differed from PEG-stressed cucumber leaves. This study illustrates the non-causal relationship between fusarium wilt and water transport blockage. Although leaf wilt occurred in both water stress and FOC infection, the physiological responses were different, especially in leaf spatial distribution.

Isolation of a potential biocontrol agent Paenibacillus polymyxa NSY50 from vinegar waste compost and its induction of host defense responses against Fusarium wilt of cucumber

Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC) is one of the major destructive soil-borne diseases infecting cucumber. In this study, we screened 60 target strains isolated from vinegar waste compost, from which 10 antagonistic strains were identified to have the disease suppression capacity of bio-control agents. The 16S rDNA gene demonstrated that the biocontrol agents were Paenibacillus polymyxa (P. polymyxa), Bacillus amyloliquefaciens (B. amyloliquefaciens) and Bacillus licheniformis (B. licheniformis). Based on the results of antagonistic activity experiments and pot experiment, an interesting strain of P. polymyxa (named NSY50) was selected for further research. Morphological, physiological and biochemical characteristics indicated that this strain was positive for protease and cellulase and produced indole acetic acid (22.21±1.27μg mL−1) and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). NSY50 can significantly up-regulate the expression level of defense related genes PR1 and PR5 in cucumber roots at the early stages upon challenge with FOC. However, the gene expression levels of a set of defense-related genes, such as the plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family (e.g., Csa001236, Csa09775, Csa018159), 26kDa phloem protein (Csa001568, Csa003306), glutathione-S-transferase (Csa017734) and phenylalanine ammonia-lyase (Csa002864) were suppressed by pretreatment with NSY50 compared with the single challenge with FOC after nine days of inoculation. Of particular interest was the reduced expression of these genes at disease progression stages, which may be required for F. oxysporum dependent necrotrophic disease development.

Nitrate Increased Cucumber Tolerance to Fusarium Wilt by Regulating Fungal Toxin Production and Distribution.

Cucumber Fusarium wilt, induced by Fusarium oxysporum f. sp. cucumerinum (FOC), causes severe losses in cucumber yield and quality. Nitrogen (N), as the most important mineral nutrient for plants, plays a critical role in plant–pathogen interactions. Hydroponic assays were conducted to investigate the effects of different N forms (NH4+ vs. NO3‒) and supply levels (low, 1 mM; high, 5 mM) on cucumber Fusarium wilt. The NO3‒-fed cucumber plants were more tolerant to Fusarium wilt compared with NH4+-fed plants, and accompanied by lower leaf temperature after FOC infection. The disease index decreased as the NO3‒ supply increased but increased with the NH4+ level supplied. Although the FOC grew better under high NO3− in vitro, FOC colonization and fusaric acid (FA) production decreased in cucumber plants under high NO3− supply, associated with lower leaf membrane injury. There was a positive correlation between the FA content and the FOC number or relative membrane injury. After the exogenous application of FA, less FA accumulated in the leaves under NO3− feeding, accompanied with a lower leaf membrane injury. In conclusion, higher NO3− supply protected cucumber plants against Fusarium wilt by suppressing FOC colonization and FA production in plants, and increasing the plant tolerance to FA.