Fusarium Oxysporum F. Sp. Niveum Research Articles

Management of Fusarium wilt of watermelon (Citrullus lanatus) using Bacillus subtilus and Bacillus cereus

Fusarium wilt of watermelon [Citrullus lanatus (Thunb.) Matsumura & Nakai] is caused by Fusarium oxysporum f. sp. niveum (FON), is the most severe soil-borne disease under the prevailing conditions of Punjab. Present study was carried out during 2018–19 and 2019–20 at the pot house and research farm of Punjab Agricultural University, Ludhiana, Punjab to evaluate the antagonistic activity of 15 isolates of Bacillus spp. against FON. These isolates were screened by using dual culture assay and among them, Bacillus subtilus (B1) and Bacillus cereus (B3) showed maximum inhibition percentage i.e. 57.00 and 58.22, respectively. Similarly, these isolates (B1 and B3) showed maximum activity of chitinase (2.31 and 2.16 unit/ml) and glucanase (1.72 and 1.79 unit/ml). Further, Talc-based bio- formulations of B1 and B3 isolates were evaluated in pot house and research farm of Punjab Agricultural University, Ludhiana, Punjab. Amongst all the treatments, Bacillus subtilis (B1) and Bacillus cereus (B3) as seed + soil @15 g treatment showed maximum inhibition of FON. Therefore, based on our findings, Bacillus subtilis (B1) and Bacillus cereus (B3) used as seed + soil treatment was able to effectively manage the fusarium wilt in watermelon under pothouse and field conditions.

Tacrolimus analogue produced by Bacillus amyloliquefaciens HSSN09 suppresses watermelon Fusarium wilt by antagonizing FON

Watermelon Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (FON) is one of the most devastating diseases in watermelon production. Biological control approaches are efficient and environmentally-friendly, thus have aroused broad interest. However, only a few biocontrol factors/molecules have been characterized due to varied genomes and diversified secreted secondary metabolites of biocontrol microorganisms. We report the screen and identification of a Bacillus amyloliquefaciens strain HSSN09 with high antagonism to FON and watermelon growth promotion capacity. B. amyloliquefaciens HSSN09 and its secondary metabolites showed inhibition on FON mycelial growth, spore formation and spore germination. Metabolomes of the highly antagonistic strain B. amyloliquefaciens HSSN09, medium antagonistic strain B. amyloliquefaciens FGW53, weakly antagonistic strain B. subtilis JC03 and non-antagonistic strain B. cereus FGW109 were compared with LC-MS/MS. By antagonistic assay, tacrolimus was screened out among the 531 molecules that appeared in the sequencing results of HSSN09 metabolome but not in that of the non-antagonistic strain without significant inhibition on the growth of FON. Among the four tested Bacillus strains, tacrolimus analogue was only observed in the supernatant of HSSN09 by HPLC and MALDI-TOF-MS. We further confirmed the ability of tacrolimus to suppress FON and promote watermelon seedling growth.

Studies on Fusarium wilt of Watermelon by using Antifungal Agents: An Invitro in Sights

Watermelon (Citrullus lanatus) is highly prone to Fusarium wilt, caused by a deadly soil-borne pathogen called Fusarium oxysporum f. sp. niveum (FON). Changes in production practices in crops, reduction in usage of fumigation with methyl bromide, crop rotation activities, and increasing human population, led to a resurgence of more virulent strains of FON. When resistant varieties are unavailable, the disease must be managed through a combination of integrated disease control practices. We conducted in vitro screening of botanicals where Allium cepa exhibited maximum inhibition of 80.32 and 82.62 percent at 5 and 10 percent concentrations, respectively. In vitro evaluation of bioagents revealed the maximum percent inhibition by T. harzianum (83.03%) followed by B. subtilis, A. flavus, P. fluorescence, T. viride, and A. niger. Fungicides by Poisoned Food Technique showed that a combination of carbendazim +mancozeb was effective in mycelial inhibition of 99.74 and 99.81 percent at 500 and 1000 ppm concentration respectively. The knowledge obtained from these in vitro findings can be applied in the field at a large scale for reducing and managing watermelon wilt disease.

A Biocontrol Strain of Serratia plymuthica MM Promotes Growth and Controls Fusarium Wilt in Watermelon

Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (FON), is a predominant and devastating soil-borne disease that results in significant yield losses in watermelon cultivation. In this study, a strain MM isolated from the herbage rhizosphere soil, exhibited an inhibition rate of 65.46% against FON, leading to mycelial collapse, atrophy, and deformation. In pot experiments, strain MM effectively controlled Fusarium wilt of watermelon, showing a control efficacy of 74.07%. Through morphological observation and 16S rDNA gene sequencing, strain MM was identified as Serratia plymuthica. Additionally, S. plymuthica MM demonstrated antagonistic activity against eight plant pathogens, indicating that MM had broad-spectrum antifungal activity. The strain also exhibited the ability to synthesize siderophores and indole acetic acid (IAA), both of which are growth-promoting compounds. Moreover, strain MM secreted various extracellular enzymes, including protease, chitinase, β-glucanase, and cellulase. This ability allowed S. plymuthica MM to readily colonize watermelon roots and promote seedling growth. Inoculation with S. plymuthica MM increased the activity of PAL, POD, PPO, and CAT enzymes associated with watermelon defense. Furthermore, qRT-PCR analysis revealed up-regulation of LOX, POD, PAL, ClPR3, and C4H genes, which are related to plant disease resistance. The results indicated that S. plymuthica MM enhances watermelon plants’ resistance to FON by activating the JA, SA, and shikimic acid phenylpropanoid–lignin synthesis pathways. Gas chromatography–mass spectrometry (GC-MS) analysis of S. plymuthica MM culture supernatant identified piperazinedione, pyrrolo[1,2-a]pyrazine-1,4-dione, and octadecenamide as the main antimicrobial substances. Overall, S. plymuthica MM shows promise as a biocontrol agent against Fusarium wilt of watermelon, suggesting its potential for the development of a new biocontrol agent.

Control effect of root exudates from mycorrhizal watermelon seedlings on Fusarium wilt and the bacterial community in continuously cropped soil.

Watermelon (Citrullus lanatus) is susceptible to wilt disease caused by Fusarium oxysporum f. sp niveum (FON). AMF colonization alleviates watermelon wilt and regulates the composition of root exudates, but the effects of mycorrhizal watermelon root exudates on watermelon Fusarium wilt is not well understood. Root exudates of watermelon inoculated with AMF (Funeliformis mosseae or Glomus versiformme) were collected in this study. Then the root exudates of control plants and mycorrhizal plants were used to irrigate watermelon in continuous cropping soil, respectively. Meanwhile, the watermelon growth, antioxidant enzyme activity, rhizosphere soil enzyme activities and bacterial community composition, as well as the control effect on FON were analyzed. The results indicated that mycorrhizal watermelon root exudates promoted the growth of watermelon seedlings and increased soil enzyme activities, actinomyces, and the quantity of bacteria in rhizosphere soil. The proportion of Proteobacteria and Bacteroides was decreased, and the proportion of Actinobacteria, Firmicutes, and Chloroflexi in rhizosphere soil was increased when the seedlings were watered with high concentrations of mycorrhizal root exudates. The dominant bacterial genera in rhizosphere soil were Kaistobacter, Rhodanobacter, Thermomonas, Devosia, and Bacillus. The root exudates of mycorrhizal watermelon could reduce the disease index of Fusarium wilt by 6.7-30%, and five ml/L of watermelon root exudates inoculated with F. mosseae had the strongest inhibitory effect on watermelon Fusarium wilt. Our results suggest mycorrhizal watermelon root exudates changed the composition of bacteria and soil enzyme activities in rhizosphere soil, which increase the resistance of watermelon to Fusarium wilt and promoted the growth of plants in continuous cropping soil.

Draft Genomes of Five Fusarium oxysporum f. sp. niveum Strains Isolated from Infected Watermelon from Texas with Temporal and Spatial Differences

Fusarium oxysporum f. sp. niveum (FON) is a soilborne fungal pathogen of watermelon that has significantly impacted production worldwide. In the United States, watermelon growers can experience 30 to 100% yield loss from this soil pathogen. In this study, we present five draft genome sequences of FON-race 2 isolates collected from four different counties within Texas, spanning more than 23 years. The estimated genome sizes of the de novo assembled genomes were W2 = 49 Mb, COM = 48 Mb, HAR = 49 Mb, STORM = 49 Mb, and PRU = 48 Mb. These sizes were on par with the previously sequenced FON races 0 and 2 from Texas, which ranged from 49 to 54 Mb. Gene prediction program AUGUSTUS predicted 13,554 genes in W2, 13,658 genes in COM, 13,678 genes in HAR, 13,821 genes in STORM, and 13,347 genes in PRU. The information from our five genome drafts contributes to the southeast genomic dataset of FON for comparative genomic analysis. Furthermore, the sequences are beneficial in the understanding of changes within the FON genome over time. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Thymol Induces Cell Death of Fusarium oxysporum f. sp. niveum via Triggering Superoxide Radical Accumulation and Oxidative Injury In Vitro

Fusarium oxysporum f. sp. niveum (FON) causes watermelon wilt that is one of the major disease-causing yield losses of watermelon. Sustainable development of agriculture requires controlling watermelon wilt disease with good environmental performance. One important approach is to identify environmental-friendly compounds with inhibitory activity against FON. Thymol is a plant-derived compound that is safe for ecology. Little is known about the application of thymol in agriculture. In this study, we studied the inhibitory activity of thymol against FON by using morphological, physiological, and histochemical approaches. Thymol significantly inhibited colony diameter of FON in a dose-dependent manner, with EC50 at 21 µg/mL. Thymol at 10, 21, and 35 µg/mL decreased the fresh weight of FON mycelia by 29.0%, 50.6%, and 69.5%, respectively. Microscopic observation revealed irregular damage and loss of shape of mycelia upon thymol exposure. Thymol induced the accumulation of superoxide radical in mycelial cells and accompanied increased activity of antioxidative enzymes (SOD, superoxide dismutase; CAT, catalase). Thymol induced membrane permeability was indicated by lipid peroxidation and electrolyte leakage (increased by 29–58%) in mycelial cells. These results suggested that thymol induced oxidative damage in mycelia, which may be one of the possible reasons for thymol-induced mycelial cell death observed with fluorescent detection. Thymol decreased the production of conidia and inhibited the germination of conidia. Thymol induced superoxide radical accumulation, lipid peroxidation, and cell death in conidia as well. All of these results revealed the inhibitory activity of thymol against FON, which may have resulted from the superoxide radical-induced oxidative injury in both conidia and mycelia of FON.

Micronutrients Affect Expression of Induced Resistance Genes in Hydroponically Grown Watermelon against Fusarium oxysporum f. sp. niveum and Meloidogyne incognita

The soil-borne pathogens, particularly Fusarium oxysporum f. sp. niveum (FON) and southern root-knot nematode (RKN, Meloidogyne incognita) are the major threats to watermelon production in the southeastern United States. The role of soil micronutrients on induced resistance (IR) to plant diseases is well-documented in soil-based media. However, soil-based media do not allow us to determine the contribution of individual micronutrients in the induction of IR. In this manuscript, we utilized hydroponics-medium to assess the effect of controlled application of micronutrients, including iron (Fe), manganese (Mn), and zinc (Zn) on the expression of important IR genes (PR1, PR5, and NPR1 from salicylic acid (SA) pathway, and VSP, PDF, and LOX genes from jasmonic acid (JA) pathway) in watermelon seedlings upon inoculation with either FON or RKN or both. A subset of micronutrient-treated plants was inoculated (on the eighth day of micronutrient application) with FON and RKN (single or mixed inoculation). The expression of the IR genes in treated and control samples was evaluated using qRT-PCR. Although, significant phenotypic differences were not observed with respect to the severity of wilt symptoms or RKN galling with any of the micronutrient treatments within the 30-day experimental period, differences in the induction of IR genes were considerably noticeable. However, the level of gene expression varied with sampling period, type and concentration of micronutrients applied, and pathogen inoculation. In the absence of pathogens, micronutrient applications on the seventh day, in general, downregulated the expression of the majority of the IR genes. However, pathogen inoculation preferentially either up- or down-regulated the expression levels of the IR genes at three days post-inoculation depending on the type and concentration of micronutrients. The results demonstrated here indicate that micronutrients in watermelon may potentially make watermelon plants susceptible to infection by FON and RKN. However, upon infection the IR genes are significantly up-regulated that they may potentially aid the prevention of further infection via SA- and JA-pathways. This is the first demonstration of the impact of micronutrients affecting IR in watermelon against FON and RKN infection.

Salicylic acid remodeling of the rhizosphere microbiome induces watermelon root resistance against Fusarium oxysporum f. sp. niveum infection.

Fusarium wilt disease poses a severe threat to watermelon cultivation by affecting the yield and quality of the fruit. We had previously found that the rhizosphere microbiome has a significant impact on the ability of watermelon plants to resist Fusarium wilt development and that salicylic acid (SA) is closely related to this phenomenon. Therefore, in this study, the role of SA as a mediator between plants and microbes in activating resistance against Fusarium oxysporum f. sp. niveum (FON) infection was explored through physiological, biochemical, and metagenomic sequencing experiments. We demonstrated that exogenous SA treatment could specifically increase some beneficial rhizosphere species that can confer resistance against FON inoculation, such as Rhodanobacter, Sphingomonas, and Micromonospora. Functional annotation analysis indicated that SA application significantly increased the relative abundance of glycoside hydrolase and polysaccharide lyase genes in the microbiome, which may play an essential role in increasing plant lipids. Moreover, network interaction analysis suggested that the highly expressed AAC6_IIC gene may be manipulated through SA signal transduction pathways. In conclusion, these results provide a novel strategy for controlling Fusarium wilt in watermelons from the perspective of environmental ecology, that is, by manipulating the rhizosphere microbiome through SA to control Fusarium wilt.

The Improved Biocontrol Agent, F1-35, Protects Watermelon against Fusarium Wilt by Triggering Jasmonic Acid and Ethylene Pathways.

Watermelon Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (FON), is one of the most important diseases, and has become a major limiting factor to watermelon production worldwide. Previous research has found that the improved biocontrol agent, F1-35, had a high control efficiency to watermelon Fusarium wilt. In this study, the control efficiency of F1-35 to watermelon Fusarium wilt was firstly tested, and the control efficiency was 61.7%. Then, we investigated the mode of action of F1-35 in controlling watermelon Fusarium wilt. Using a pairing assay, we found that F1-35 did not inhibit the normal growth of FON. To know more about the interaction between F1-35 and watermelon root, the protein expressions of roots after 12, 24, and 48 h post-inoculation were examined. A total of 1109 differentially expressed proteins were obtained. KEGG analysis found that the most differentially expressed proteins occurred in alpha-linolenic acid metabolism, cysteine and methionine metabolism, plant–pathogen interaction, and the MAPK signaling pathway to the plant. A further analysis of differentially expressed proteins showed that F1-35 triggered the jasmonic acid and ethylene pathways in watermelon. To validate our results, the qRT-PCR was used to analyze the gene expression levels of PAL, LOX1, and CTR1. The gene expression results showed that those genes, which were positive correlated with the JA pathway, were up-expressed, including PAL and LOX1, and the negative associated gene, CTR1, was down-expressed. In conclusion, the improved biocontrol agent, F1-35, improves the resistance of watermelons to FON by triggering the JA and ET pathways.

Transcriptomic and metabonomic insights into the biocontrol mechanism of Trichoderma asperellum M45a against watermelon Fusarium wilt.

Watermelon (Citrullus lanatus) is one of the most popular fruit crops. However, Fusarium wilt (FW) is a serious soil-borne disease caused by Fusarium oxysporum f. sp. niveum (FON) that severely limits the development of the watermelon industry. Trichoderma spp. is an important plant anti-pathogen biocontrol agent. The results of our previous study indicated that Trichoderma asperellum M45a (T. asperellum M45a) could control FW by enhancing the relative abundance of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of watermelon. However, there are few studies on its mechanism in the pathogen resistance of watermelon. Therefore, transcriptome sequencing of T. asperellum M45a-treated watermelon roots combined with metabolome sequencing of the rhizosphere soil was performed with greenhouse pot experiments. The results demonstrated that T. asperellum M45a could stably colonize roots and significantly increase the resistance-related enzymatic activities (e.g., lignin, cinnamic acid, peroxidase and peroxidase) of watermelon. Moreover, the expression of defense-related genes such as MYB and PAL in watermelon roots significantly improved with the inoculation of T. asperellum M45a. In addition, KEGG pathway analysis showed that a large number of differentially expressed genes were significantly enriched in phenylpropane metabolic pathways, which may be related to lignin and cinnamic acid synthesis, thus further inducing the immune response to resist FON. Furthermore, metabolic analysis indicated that four differential metabolic pathways were enriched in M45a-treated soil, including six upregulated compounds and one down-regulated compound. Among them, galactinol and urea were significantly positively correlated with Trichoderma. Hence, this study provides insight into the biocontrol mechanism of T. asperellum M45a to resist soil-borne diseases, which can guide its industrial application.

Variation of Soil Microbial Community and Sterilization to Fusarium oxysporum f. sp. niveum Play Roles in Slightly Acidic Electrolyzed Water-Alleviated Watermelon Continuous Cropping Obstacle.

It is critical to exploit technologies for alleviating watermelon continuous cropping obstacle which frequently occurs and results in the limiting production and economic losses of watermelon. This study aimed to explore the effects of slightly acidic electrolyzed water (SAEW) on watermelon continuous cropping obstacles. The results showed that SAEW significantly improved the growth of watermelon seedlings cultivated in continuous cropping soil and caused a mass of changes to the diversity of the soil microbial community. Compared with Con, SAEW decreased the diversity index of bacteria by 2%, 0.48%, and 3.16%, while it increased the diversity index of fungus by 5.68%, 10.78%, and 7.54% in Shannon, Chao1, and ACE index, respectively. Besides, the enrichment level of Fusarium oxysporum f. sp. niveum (FON) was remarkably downregulated by 50.2% at 14 days of SAEW treatment, which could decrease the incidence of Fusarium wilt disease. The wet and dry weights of FON mycelia in the fluid medium were depressed more than 93%, and the number of FON colonies in continuous cropping soil was reduced by 83.56% with SAEW treatment. Additionally, a strong correlation between watermelon, FON, and SAEW was presented by correlation analysis. Furthermore, the content of endogenous reactive oxygen species (ROS) was over quadruply increased by SAEW, which may contribute to the sterilizing effect of SAEW on FON. Taken together, our findings demonstrated that exogenous SAEW could alter the soil microbial diversity and decrease the accumulation of FON, which improved the growth of watermelon seedlings and finally alleviated continuous cropping obstacles of watermelon.

Changes of microbiome in response to sugars in a wilt pathogen-infested soil

Sugars are frequently and abundantly found in root exudates, but influence of specific sugars on the fate of soil-borne pathogens, microbiome structure, and particularly microbial interactions are not well understood. A 42-day of microcosm incubation was conducted with two soils: a natural watermelon Fusarium wilt pathogen (i.e., Fusarium oxysporum f. sp. niveum (FON))-infested soil (Low-FON soil) and the soil further receiving the wilt pathogen inocula (High-FON soil). Both soils were supplemented with four simple sugars before incubation. The results show that, in both soils, FON was enriched by all sugars although co-living with tremendously diverse microbes; and bacterial richness, evenness, and diversity were decreased and bacterial community structure was changed by all sugars. Bacterial richness and evenness were negatively correlated with FON quantity in both Low-FON and High-FON soils, indicating that FON may tend to live in soil with low alpha-diversity. In both Low-FON and High-FON soils, the sugar-spiked networks had more links, higher density, larger modules, and shorter harmonic geodesic distance, suggesting greater potentials for microbial interaction and niche-sharing. The positive links between some of the keystone taxa and FON indicates that these keystone taxa may have promoted FON. This may be one of reasons why FON could proliferate vigorously after sugar supplementation.

Evaluation of Pic-clor 60 [choloropicrin pre-mixed with 1,3 dicholoropropene] and soil-applied fungicides for the Fusarium wilt management in watermelon

Watermelon (Citrullus lanatus var. lanatus) yield losses due to Fusarium wilt can be considerable and have been increasing in the United States, mainly due to the emergence of the new races of Fusarium oxysporum f. sp. niveum (FON). One of the potential management strategies to reduce Fusarium wilt is the use of fumigants and soil-applied fungicides. Field experiments were conducted in 2018 and 2020 to evaluate fumigant Pic-clor 60 (choloropicrin pre-mixed with 1,3 dicholoropropene) with post-plant soil-applied fungicides (prothioconazole and pydiflumetofen) for their effectiveness against Fusarium wilt. The field was fumigated 21 days before transplanting, and three applications of either fungicide were made at 14-day intervals via a drip irrigation system starting a day after transplanting. In 2018, based on the disease ratings at harvest maturity, all treatments significantly reduced final disease incidence compared to the non-treated control, and the highest reduction was observed when the fumigant was applied at 336.26 kg/ha along with either fungicides. The application of fumigant, together with either fungicides, had significantly higher reduction in disease incidence compared to the stand-alone application (fumigant or fungicides). Also, Pic-clor 60 at 336.26 kg/ha provided a higher reduction in disease incidence than Pic-clor 60 at 280.21 kg/ha. In 2020, results showed a similar trend to 2018, where Pic-clor 60 at 336.26 kg/ha in combination with either prothioconazole or pydiflumetofen had significantly lower final disease incidence. The reduction in disease incidence in Pic-clor 60 at 336.26 kg/ha was higher compared to a lower application rate of 280.21 kg/ha, either when applied alone or in combination with either fungicides. The results presented here are the first to demonstrate that the fumigant Pic-clor 60 along with soil-applied fungicides, pydiflumetofen or prothioconazole may provide an effective management option for Fusarium wilt of watermelon.

First Report of Fusarium wilt disease on Watermelon Caused by Fusarium oxysporum f. sp. niveum (FON) in Malaysia.

Fusarium wilt disease incited by Fusarium oxysporum f. sp. niveum (FON) is the utmost devastating soil-inhabiting fungal pathogen limiting watermelon (Citrullus lanatus) production in Malaysia and globally. The field disease survey of fusarium wilt was carried out during December 2019 and November 2020, in three major production areas (3 farmer fields per location) in Peninsular Malaysia namely, Mersing, Serdang and Kuantan and disease incidence of 30 and 45%, was recorded for each year, respectively. Infected watermelon plants showed symptoms such as vascular discoloration, brown necrotic lesions to the soil line or the crown, one-sided wilt of a plant, or a runner or the whole plant. Infected root and stem tissues, 1-2 cm pieces were surface sterilized with 0.6% NaOCl for 1 minute followed by double washing with sterile water. The disinfected tissues were air-dried and transferred onto semi-selective Komada's medium (Komada 1975) and incubated for 5 days. The fungal colonies produced were placed on potato dextrose agar (PDA) to attain a pure culture and incubated at 25±2℃ for 15 days. The pure fungal colony was flat, round and light purple in color. Macroconidia were straight to slightly curved, 18.56-42.22 µm in length, 2.69-4.08 µm width, predominantly 3 septate and formed in sporodochia. Microconidia measured 6.16-10.86 µm in length and 2.49-3.83 µm in width, kidney-shaped, aseptate and were formed on short monophialides in false-heads. Chlamydospores were single or in pairs with smooth or rough walls, found both terminally or intercalary. To confirm their pathogenicity, two-week-old watermelon seedlings (cv. NEW BEAUTY) were dipped into spore suspension (1 ˟ 106 spores/ml) of representative isolates of JO20 (Mersing), UPM4 (Serdang) and KU41 (Kuantan) for 30 second and then moved into 10 cm diameter plastic pots containing 300 g sterilized soil mix. Disease symptoms were assessed weekly for one month. Control seedlings were immersed in sterile distilled water before transplanting. The inoculated seedlings showed typical Fusarium wilt symptoms like yellowing, stunted growth, and wilting, which is similar to the farmer field infected plants. However, the seedlings inoculated by sterile distilled water remained asymptomatic. The pathogen was successfully re-isolated from the infected seedlings onto Komada's medium, fulfilling the Koch's postulate. For the PCR amplification, primers EF-1 and EF-2 were used to amplify the tef1-α region. A Blastn analysis of the tef1-α sequences of the isolates JO20 (accession nos. MW315902), UPM4 (MW839560) and KU41 (MW839562) showed 100% similarity; with e-value of zero, to the reference sequences of F. oxysporum isolate FJAT-31690 (MN507110) and F. oxysporum f. sp. niveum isolate FON2 790-2 (MN057702). In Fusarium MLST database, isolates JO20, UPM4 and KU41 revealed 100% identity with the reference isolate of NRRL 22518 (accession no. FJ985265). Though isolate FJ985265 belongs to the f. sp. melonis, earlier findings had revealed Fusarium oxysporum f. sp. are naturally polyphyletic and making clusters with diverse groups of the Fusarium oxysporum species complex (O'Donnell et al. 2015). The isolates JO20, UPM4 and KU41 were identified as F. oxysporum f. sp. niveum based on the aligned sequences of tef1-α and molecular phylogenetic exploration by the maximum likelihood method. To the best of our knowledge, this is the first report of F. oxysporum f. sp. niveum as a causative pathogen of Fusarium wilt disease of watermelon in Malaysia. Malaysia enables to export watermelon all-year-round in different countries like Singapore, Hong-Kong, The United Arab Emirates (UAE), and Netherlands. The outburst of this destructive soil-borne fungal pathogen could cause hindrance to watermelon cultivation in Malaysia. Thus, growers need to choice multiple management tactics such as resistant varieties, cultural practices (soil amendments and solarization), grafting, cover crops and fungicide application to control this new pathogen.

Molecular Characterization of Laboratory Mutants of Fusarium oxysporum f. sp. niveum Resistant to Prothioconazole, a Demethylation Inhibitor (DMI) Fungicide.

Fusarium oxysporum f. sp. niveum (FON) is the causal agent of Fusarium wilt in watermelon, an international growth-limiting pathogen of watermelon cultivation. A single demethylation inhibitor (DMI) fungicide, prothioconazole, is registered to control this pathogen, so the risk of resistance arising in the field is high. To determine and predict the mechanism by which FON could develop resistance to prothioconazole, FON isolates were mutagenized using UV irradiation and subsequent fungicide exposure to create artificially resistant mutants. Isolates were then put into three groups based on the EC50 values: sensitive, intermediately resistant, and highly resistant. The mean EC50 values were 4.98 µg/mL for the sensitive, 31.77 µg/mL for the intermediately resistant, and 108.33 µg/mL for the highly resistant isolates. Isolates were then sequenced and analyzed for differences in both the coding and promoter regions. Two mutations were found that conferred amino acid changes in the target gene, CYP51A, in both intermediately and highly resistant mutants. An expression analysis for the gene CYP51A also showed a significant increase in the expression of the highly resistant mutants compared to the sensitive controls. In this study, we were able to identify two potential mechanisms of resistance to the DMI fungicide prothioconazole in FON isolates: gene overexpression and multiple point mutations. This research should expedite growers’ and researchers’ ability to detect and manage fungicide-resistant phytopathogens.

The Transcriptomic Profile of Watermelon Is Affected by Zinc in the Presence of Fusarium oxysporum f. sp. niveum and Meloidogyne incognita.

Zinc (Zn) accumulation and deficiency affect plant response to pests and diseases differently in varying pathosystems. The concentrations of Zn in plants aid in priming defense signaling pathways and help in enhanced structural defenses against plant pathogens. Studies are lacking on how concentrations of Zn in watermelon plants influence defense against two important soil-borne pathogens: Fusarium oxysporum f. sp. niveum (FON) and southern root-knot nematode (RKN, Meloidogyne incognita). In this study a comparative transcriptomics evaluation of watermelon plants in response to high (1.2 ppm) and low (0.2 ppm) levels of Zn were determined. Differential transcript-level responses differed in watermelon plants when infected with FON or RKN or both under high- and low-Zn treatment regimes in a controlled hydroponics system. Higher numbers of differentially expressed genes (DEGs) were observed in high-Zn-treated than in low-Zn-treated non-inoculated plants, in plants inoculated with FON alone and in plants inoculated with RKN alone. However, in the co-inoculated system, low-Zn treatment had higher DEGs as compared to high-Zn treatment. In addition, most DEGs were significantly enriched in hormone signal transduction and MAPK signaling pathway, suggesting an induction of systemic resistance with high-Zn concentrations. Taken together, this study substantially expands transcriptome data resources and suggests a molecular potential framework for watermelon-Zn interaction in FON and RKN.

Effects of Exogenously applied palmitic acid on systemic resistance against Fusarium oxysporum f. sp. niveum in watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai)

e studied the effects of exogenously applied palmitic acid (PA) on reactive oxygen species (ROS) production, defense-related enzyme activity and malondialdehyde (MDA) content of roots and analyzed the response of defense-related genes in Fusarium oxysporum f.sp.niveum (FON) inoculated watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai). Our results showed that as compared to control, PA reduced the morbidity of Fusarium wilt by 33.3 % and disease severity by 53.2 %, increased the level of ROS of watermelon roots at early infection stage and the activity of defense related enzymes, and decreased the MDA content of watermelon roots. Moreover, PA up-regulated the expression of disease defense-related genes. These results suggested that PA promoted the accumulation of ROS and up-regulated the transcript level of defense-related genes and eventually induced the systemic resistance in watermelon against FON.

Inactivated pathogenic mycelia as a biocontrol agent against Fusarium wilt and its effects on continuously cropped watermelon

ABSTRACT Fusarium wilt is a widespread disease causing serious production losses in watermelon (Citrullus lanatus L.). Accordingly, control agents to manage this disease should be developed to ensure the sustainability of watermelon cultivation. In this study, inactivated Fusarium oxysporum f. sp. niveum (FON) mycelia were used as a decoy inoculation agent, and the effects of this treatment on Fusarium wilt and its possible underlying mechanisms were investigated. Inoculation with inactivated FON mycelia significantly decreased rhizosphere FON number and Fusarium wilt disease severity, and this might be closely related to the observed improved abundances and community structures of native beneficial rhizosphere microorganisms. In addition, inactivated FON mycelia inoculation significantly enhanced soil enzyme activity levels and plant systemic resistance, which promoted resistance to Fusarium wilt and thus watermelon growth. These results demonstrated that inactivated FON mycelia could be used as a novel biocontrol agent to alleviate Fusarium wilt and increase yields in watermelon.

Microbial assembly and association network in watermelon rhizosphere after soil fumigation for Fusarium wilt control

Soil fumigation is an effective method to control soil-borne diseases like Fusarium wilt, however the processes and mechanisms driving microbial community reestablishment and pathogen suppression in the rhizosphere after fumigation remain poorly understood. In this study, we examined the dynamics of the rhizosphere microbiome and microbial network associations across different watermelon development stages and plant statuses (i.e. healthy and dead) after soil fumigation and organic fertilizer (OF) application in plastic shelters with Fusarium oxysporum f. sp. niveum (FON) heavily infected. Our results showed that fumigation treatments significantly reduced Fusarium wilt disease incidence and pathogen abundance, meanwhile, decreased soil microbial metabolic activity, fungal biomass and diversity. Bacterial community recovered from fumigation suppression in a short period, while fungal suppression was longer lasting, resulting in decreased fungi to actinomycetes (F/A) and fungi to bacteria (F/B) ratios in PLFA profiles. We further found some bacterial families, such as Actinospicaceae within Actinobacteria, Haliangiaceae, Rhizobiaceae and uncultured Rhodospirillales within Proteobacteria, Sporolactobacillaceae and Limnochordaceae within Firmicutes were greatly enriched after fumigation and might potentially contribute to pathogen suppression. The fumigation treatments significantly reduced microbial network complexity and the percentage of fungal nodes in comparison to un-fumigated control treatment. In contrast, a more complex microbial network was observed in the rhizosphere soil of healthy plants than that in the soil surrounding dead plant roots within fumigation treatments. Furthermore, healthy plant rhizosphere significantly enriched potential beneficial and nitrogen cycle-related bacterial phyla like Gemmatimonadetes, Verrucomicrobia, and Nitrospirae. More interestingly, Fusarium were markedly enriched in the rhizosphere soil of healthy plants and mainly represented by non-FON Fusarium like F. verticillioides and F. solani, implying a potential niche competition between FON and nonpathogenic Fusarium species in the rhizosphere of healthy watermelon. Taken together, our results provide vital information on the reconstruction of microbial communities and potential interactions between plant and its beneficial consortium after fumigation, which is instructive to develop more systematic strategies through targeting both beneficial and pathogen-similar taxa to improve disease control and soil suppression.