Synthetic community derived from grafted watermelon rhizosphere provides protection for ungrafted watermelon against Fusarium oxysporum via microbial synergistic effects.

BACKGROUND: Plant microbiota contributes to plant growth and health, including enhancing plant resistance to various diseases. Despite remarkable progress in understanding diseases resistance in plants, the precise role of rhizosphere microbiota in enhancing watermelon resistance against soil-borne diseases remains unclear. Here, we constructed a synthetic community (SynCom) of 16 core bacterial strains obtained from the rhizosphere of grafted watermelon plants. We further simplified SynCom and investigated the role of bacteria with synergistic interactions in promoting plant growth through a simple synthetic community. RESULTS: Our results demonstrated that the SynCom significantly enhanced the growth and disease resistance of ungrafted watermelon grown in non-sterile soil. Furthermore, analysis of the amplicon and metagenome data revealed the pivotal role of Pseudomonas in enhancing plant health, as evidenced by a significant increase in the relative abundance and biofilm-forming pathways of Pseudomonas post-SynCom inoculation. Based on in vitro co-culture experiments and bacterial metabolomic analysis, we selected Pseudomonas along with seven other members of the SynCom that exhibited synergistic effects with Pseudomonas. It enabled us to further refine the initially constructed SynCom into a simplified SynCom comprising the eight selected bacterial species. Notably, the plant-promoting effects of simplified SynCom were similar to those of the initial SynCom. Furthermore, the simplified SynCom protected plants through synergistic effects of bacteria. CONCLUSIONS: Our findings suggest that the SynCom proliferate in the rhizosphere and mitigate soil-borne diseases through microbial synergistic interactions, highlighting the potential of synergistic effects between microorganisms in enhancing plant health. This study provides a novel insight into using the functional SynCom as a promising solution for sustainable agriculture. Video Abstract.

How different of the rhizospheric and endophytic microbial compositions in watermelons with different fruit shapes.

Fruit shape is an important character of watermelon. And the compositions of rhizospheric and endophytic microorganisms of watermelon with different fruit shape also remains unclear. To elucidate the biological mechanism of watermelon fruit shape formations, the rhizospheric and endophytic microbial community compositions between oval (OW) and circular watermelons (CW) were analyzed. The results showed that except of the rhizospheric bacterial richness (P < 0.05), the rhizospheric and endophytic microbial (bacterial and fungal) diversity were not statistically significant between OW and CW (P > 0.05). However, the endophytic microbial (bacterial and fungal) compositions were significantly different. Firstly, Bacillus, Rhodanobacter, Cupriavidus, Luteimonas, and Devosia were the unique soil dominant bacterial genera in rhizospheres of circular watermelon (CW); In contrast, Nocardioides, Ensifer, and Saccharomonospora were the special soil dominant bacterial genera in rhizospheres of oval watermelons (OW); Meanwhile, Cephalotrichum, Neocosmospora, Phialosimplex, and Papulaspora were the unique soil dominant fungal genera in rhizospheres of circular watermelon (CW); By contrast, Acremonium, Cladosporium, Cryptococcus_f__Tremellaceae, Sodiomyces, Microascus, Conocybe, Sporidiobolus, and Acremonium were the unique soil dominant fungal genera in rhizospheres of oval watermelons (OW). Additionally, Lechevalieria, Pseudorhodoferax, Pseudomonas, Massilia, Flavobacterium, Aeromicrobium, Stenotrophomonas, Pseudonocardia, Novosphingobium, Melittangium, and Herpetosiphon were the unique dominant endophytic bacterial genera in stems of CW; In contrast, Falsirhodobacter, Kocuria, and Kineosporia were the special dominant endophytic genera in stems of OW; Moreover, Lectera and Fusarium were the unique dominant endophytic fungal genera in stems of CW; By contrast, Cercospora only was the special dominant endophytic fungal genus in stems of OW. All above results suggested that watermelons with different fruit shapes exactly recruited various microorganisms in rhizospheres and stems. Meanwhile, the enrichments of the different rhizosphric and endophytic microorganisms could be speculated in relating to watermelon fruit shapes formation.

Antagonistic effect of Bacillus and Pseudomonas combinations against Fusarium oxysporum and their effect on disease resistance and growth promotion in watermelon.

AIMS: We aimed to develop an effective bacterial combination that can combat Fusarium oxysporum infection in watermelon using in vitro and pot experiments. METHODS AND RESULTS: In total, 53 strains of Bacillus and 4 strains of Pseudomonas were screened. Pseudomonas strains P3 and P4 and Bacillus strains XY-2-3, XY-13, and GJ-1-15 exhibited good antagonistic effects against F. oxysporum. P3 and P4 were identified as Pseudomonas chlororaphis and Pseudomonas fluorescens, respectively. XY-2-3 and GJ-1-15 were identified as B. velezensis, and XY-13 was identified as Bacillus amyloliquefaciens. The three Bacillus strains were antifungal, promoted the growth of watermelon seedlings and had genes to synthesize antagonistic metabolites such as bacilysin, surfactin, yndj, fengycin, iturin, and bacillomycin D. Combinations of Bacillus and Pseudomonas strains, namely, XY-2-3 + P4, GJ-1-15 + P4, XY-13 + P3, and XY-13 + P4, exhibited a good compatibility. These four combinations exhibited antagonistic effects against 11 pathogenic fungi, including various strains of F. oxysporum, Fusarium solani, and Rhizoctonia. Inoculation of these bacterial combinations significantly reduced the incidence of Fusarium wilt in watermelon, promoted plant growth, and improved soil nutrient availability. XY-13 + P4 was the most effective combination against Fusarium wilt in watermelon with the inhibition rate of 78.17%. The number of leaves; aboveground fresh and dry weights; chlorophyll, soil total nitrogen, and soil available phosphorus content increased by 26.8%, 72.12%, 60.47%, 16.97%, 20.16%, and 16.50%, respectively, after XY-13 + P4 inoculation compared with the uninoculated control. Moreover, total root length, root surface area, and root volume of watermelon seedlings were the highest after XY-13 + P3 inoculation, exhibiting increases by 265.83%, 316.79%, and 390.99%, respectively, compared with the uninoculated control. CONCLUSIONS: XY-13 + P4 was the best bacterial combination for controlling Fusarium wilt in watermelon, promoting the growth of watermelon seedlings, and improving soil nutrient availability.

Potential of melatonin and Trichoderma harzianum inoculation in ameliorating salt toxicity in watermelon: Insights into antioxidant system, leaf ultrastructure, and gene regulation.

Melatonin (MT) is an extensively studied biomolecule with dual functions, serving as an antioxidant and a signaling molecule. Trichoderma Harzianum (TH) is widely recognized for its effectiveness as a biocontrol agent against many plant pathogens. However, the interplay between seed priming and MT (150 µm) in response to NaCl (100 mM) and its interaction with TH have rarely been investigated. This study aimed to evaluate the potential of MT and TH, alone and in combination, to mitigate salt stress (SS) in watermelon plants. The findings of this study revealed a significant decline in the morphological, physiological, and biochemical indices of watermelon seedlings exposed to SS. However, MT and TH treatments reduced the negative impact of salt stress. The combined application of MT and TH exerted a remarkable positive effect by increasing the growth, photosynthetic and gas exchange parameters, chlorophyll fluorescence indices, and ion balance (decreasing Na+ and enhancing K+). MT and TH effectively alleviated oxidative injury by inhibiting hydrogen peroxide formation in saline and non-saline environments, as established by reduced lipid peroxidation and electrolyte leakage. Moreover, oxidative injury induced by SS on the cells was significantly mitigated by regulation of the antioxidant system, AsA-GSH-related enzymes, the glyoxalase system, augmentation of osmolytes, and activation of several genes involved in the defense system. Additionally, the reduction in oxidative damage was examined by chloroplast integrity via transmission electron microscopy (TEM). Overall, the results of this study provide a promising contribution of MT and TH in safeguarding the watermelon crop from oxidative damage induced by salt stress.

Streptomyces-triggered coordination between rhizosphere microbiomes and plant transcriptome enables watermelon Fusarium wilt resistance.

The use of microbial inoculant is a promising strategy to improve plant health, but their efficiency often faces challenges due to difficulties in successful microbial colonization in soil environments. To this end, the application of biostimulation products derived from microbes is expected to resolve these barriers via direct interactions with plants or soil pathogens. However, their effectiveness and mechanisms for promoting plant growth and disease resistance remain elusive. In this study, we showed that root irrigation with the extracts of Streptomyces ahygroscopicus strain 769 (S769) solid fermentation products significantly reduced watermelon Fusarium wilt disease incidence by 30% and increased the plant biomass by 150% at a fruiting stage in a continuous cropping field. S769 treatment led to substantial changes in both bacterial and fungal community compositions, and induced a highly interconnected microbial association network in the rhizosphere. The root transcriptome analysis further suggested that S769 treatment significantly improved the expression of the MAPK signalling pathway, plant hormone signal transduction and plant-pathogen interactions, particular those genes related to PR-1 and ethylene, as well as genes associated with auxin production and reception. Together, our study provides mechanistic and empirical evidences for the biostimulation products benefiting plant health through coordinating plant and rhizosphere microbiome interaction.

A CRISPR/LbCas12a-based method for detection of bacterial fruit blotch pathogens in watermelon.

Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila, Pseudomonas syringae, and Xanthomonas campestris. The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/µL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli, and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli, is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli.

The SUMOylation pathway regulates the pathogenicity of Fusarium oxysporum f. sp. niveum in watermelon through stabilizing the pH regulator FonPalC via SUMOylation.

SUMOylation is a key post-translational modification, where small ubiquitin-related modifier (SUMO) proteins regulate crucial biological processes, including pathogenesis, in phytopathogenic fungi. Here, we investigated the function and mechanism of the SUMOylation pathway in the pathogenicity of Fusarium oxysporum f. sp. niveum (Fon), the fungal pathogen that causes watermelon Fusarium wilt. Disruption of key SUMOylation pathway genes, FonSMT3, FonAOS1, FonUBC9, and FonMMS21, significantly reduced pathogenicity, impaired penetration ability, and attenuated invasive growth capacity of Fon. Transcription and proteomic analyses identified a diverse set of SUMOylation-regulated differentially expressed genes and putative FonSMT3-targeted proteins, which are predicted to be involved in infection, DNA damage repair, programmed cell death, reproduction, growth, and development. Among 155 putative FonSMT3-targeted proteins, FonPalC, a Pal/Rim-pH signaling regulator, was confirmed to be SUMOylated. The FonPalC protein accumulation was significantly decreased in SUMOylation-deficient mutant ∆Fonsmt3. Deletion of FonPalC resulted in impaired mycelial growth, decreased pathogenicity, enhanced osmosensitivity, and increased intracellular vacuolation in Fon. Importantly, mutations in conserved SUMOylation sites of FonPalC failed to restore the defects in ∆Fonpalc mutant, indicating the critical function of the SUMOylation in FonPalC stability and Fon pathogenicity. Identifying key SUMOylation-regulated pathogenicity-related proteins provides novel insights into the molecular mechanisms underlying Fon pathogenesis regulated by SUMOylation.

Fine mapping of ClLOX, a QTL for powdery mildew resistance in watermelon (Citrullus lanatus L.).

KEY MESSAGE: ClLOX, is located on chromosome 2 and encodes a lipoxygenase gene, which induced watermelon powdery mildew resistance by inhibiting pathogen spread. Powdery mildew is one of the most severe fungal diseases reducing yield and quality of watermelon (Citrullus lanatus L.) and other cucurbit crops. Genes responsible for powdery mildew resistance in watermelon are highly valuable. In this study, we first identified the QTL pm-lox for powdery mildew resistance in watermelon, located within a 0.93 Mb interval of chromosome 2, via XP-GWAS method using two F2 populations. The F2:3 families from one of the F2 populations were then used for fine-mapping the pm-lox locus into a 9,883 bp physical region between 29,581,906 and 29,591,789, containing only two annotated genes. Of these, only ClG42_02g0161300 showed a significant differential expression between the resistant and susceptible lines after powdery mildew inoculation based on RNA sequencing (RNA-seq) and qRT-PCR analysis, and is designated ClLOX. Derived Cleaved Amplified Polymorphic Sequence (dCAPs) markers were developed and validated. In addition, our tests showed that the resistance was anti-spread rather than anti-infection of the pathogen. This study identified a new resistance gene (ClLOX), provided insights into the mechanism of powdery mildew resistance, and developed a molecular marker for watermelon breeding.

Sensitivity of Meloidogyne incognita, Fusarium oxysporum f. sp. niveum, and Stagonosporopsis citrulli to Succinate Dehydrogenase Inhibitors Used for Control of Watermelon Diseases.

Watermelon is affected by diseases such as Fusarium wilt, gummy stem blight, and root-knot nematode (RKN). Succinate dehydrogenase inhibitors (SDHIs) with potential fungicide and nematicide activity provide the opportunity to control multiple diseases with one compound. In this study, we aimed to determine the sensitivity of Meloidogyne incognita race 4 (MI4), Fusarium oxysporum f. sp. niveum (FON), and Stagonosporopsis citrulli (SCIT) to existing SDHIs: benzovindiflupyr, fluopyram, cyclobutrifluram, and pydiflumetofen. All SDHIs had fungicidal activity against 19 SCIT isolates in mycelial growth assays, but isolates were most sensitive to pydiflumetofen (median EC50 = 0.41 µg/ml). Most of the 50 FON isolates tested were sensitive to cyclobutrifluram for mycelial growth (median EC50 = 4.04 µg/ml) and conidial germination (median EC50 = 0.2 µg/ml) assays but were not sensitive to fluopyram. MI4 was most sensitive to cyclobutrifluram for egg hatch (mean EC50 = 0.0019 µg/ml) and J2 motility (mean EC50 = 1.16 µg/ml) assays but was not sensitive to pydiflumetofen. Significant positive correlations between the sensitivity of SCIT (mycelial growth) and FON (mycelial growth and conidial germination) for cyclobutrifluram and benzovindiflupyr (SCIT r = 0.88; FON r = 0.7; P < 0.0001) and cyclobutrifluram and pydiflumetofen (SCIT r = 0.83; FON r = 0.67 and 0.77; P < 0.0001) indicate a potential for cross-resistance between these SDHIs for these fungal pathogens. Overall, results suggest that cyclobutrifluram may be used for managing RKN, whereas it should be used judiciously for Fusarium wilt of watermelon and gummy stem blight due to the existence of insensitive isolates to the fungicide.

Degradation of α-Subunits, Doa1 and Doa4, are Critical for Growth, Development, Programmed Cell Death Events, Stress Responses, and Pathogenicity in the Watermelon Fusarium Wilt Fungus Fusarium oxysporum f. sp. niveum.

The ubiquitin-proteasome system (UPS) regulates protein quality or control and plays essential roles in several biological and biochemical processes in fungi. Here, we present the characterization of two UPS components, FonDoa1 and FonDoa4, in watermelon Fusarium wilt fungus, Fusarium oxysporum f. sp. niveum (Fon), and their biological functions. FonDoa1 localizes in both the nucleus and cytoplasm, while FonDoa4 is predominantly present in the cytoplasm. Both genes show higher expression in germinating macroconidia at 12 h. Deletion of FonDoa1 or FonDoa4 affects vegetative growth, conidiation, conidial germination/morphology, apoptosis, and responses to environmental stressors. FonDoa1, but not FonDoa4, positively regulates autophagy. The targeted disruption mutants exhibit significantly attenuated pathogenicity on watermelon due to defects in the infection process and invasive fungal growth. Further results indicate that the WD40, PFU, and PUL domains are essential for the function of FonDoa1 in Fon pathogenicity and environmental stress responses. These findings demonstrate the previously uncharacterized biological functions of FonDoa1 and FonDoa4 in phytopathogenic fungi, providing potential targets for developing strategies to control watermelon Fusarium wilt.

Differential plant cell responses to Acidovorax citrulli T3SS and T6SS reveal an effective strategy for controlling plant-associated pathogens.

Acidovorax citrulli is a gram-negative plant pathogen that employs the type Ⅲ secretion system (T3SS) to infect cucurbit crops and cause bacterial fruit blotch. This bacterium also possesses an active type Ⅵ secretion system (T6SS) with strong antibacterial and antifungal activities. However, how plant cells respond to these two secretion systems and whether there is any cross talk between T3SS and T6SS during infection remain unknown. Here, we employ transcriptomic analysis to compare cellular responses to the T3SS and the T6SS during in planta infection and report distinctive effects on multiple pathways. The T3SS-mediated differentially expressed genes were enriched in the pathways of phenylpropanoid biosynthesis, plant-pathogen interaction, MAPK signaling pathway, and glutathione metabolism, while the T6SS uniquely affected genes were related to photosynthesis. The T6SS does not contribute to the in planta virulence of A. citrulli but is critical for the survival of the bacterium when mixed with watermelon phyllosphere bacteria. In addition, T3SS-mediated virulence is independent of the T6SS, and the inactivation of the T3SS does not affect the T6SS-mediated competition against a diverse set of bacterial pathogens that commonly contaminate edible plants or directly infect plants. A T6SS-active T3SS-null mutant (Acav) could inhibit the growth of Xanthomonas oryzae pv. oryzae significantly both in vitro and in vivo and also reduce symptoms of rice bacterial blight. In conclusion, our data demonstrate the T6SS in A. citrulli is nonpathogenic to the plant host and can be harnessed as a pathogen killer against plant-associated bacteria. IMPORTANCE Chemical pesticides are widely used to protect crops from various pathogens. Still, their extensive use has led to severe consequences, including drug resistance and environmental contamination. Here, we show that an engineered T6SS-active, but avirulent mutant of Acidovorax citrulli has strong inhibition capabilities against several pathogenic bacteria, demonstrating an effective strategy that is an alternative to chemical pesticides for sustainable agricultural practices.

FonTup1 functions in growth, conidiogenesis and pathogenicity of Fusarium oxysporum f. sp. niveum through modulating the expression of the tricarboxylic acid cycle genes.

The Tup1-Cyc8 complex is a highly conserved transcriptional corepressor that regulates intricate genetic network associated with various biological processes in fungi. Here, we report the role and mechanism of FonTup1 in regulating physiological processes and pathogenicity in watermelon Fusarium wilt fungus, Fusarium oxysporum f. sp. niveum (Fon). FonTup1 deletion impairs mycelial growth, asexual reproduction, and macroconidia morphology, but not macroconidial germination in Fon. The ΔFontup1 mutant exhibits altered tolerance to cell wall perturbing agent (congo red) and osmotic stressors (sorbitol or NaCl), but unchanged sensitivity to paraquat. The deletion of FonTup1 significantly decreases the pathogenicity of Fon toward watermelon plants through attenuating the ability to colonize and grow within the host. Transcriptome analysis revealed that FonTup1 regulates primary metabolic pathways, including the tricarboxylic acid (TCA) cycle, via altering the expression of corresponding genes. Downregulation of three malate dehydrogenase genes, FonMDH1-3, occurs in ΔFontup1, and disruption of FonMDH2 causes significant abnormalities in mycelial growth, conidiation, and virulence of Fon. These findings demonstrate that FonTup1, as a global transcriptional corepressor, plays crucial roles in different biological processes and pathogenicity of Fon through regulating various primary metabolic processes, including the TCA cycle. This study highlights the importance and molecular mechanism of the Tup1-Cyc8 complex in multiple basic biological processes and pathogenicity of phytopathogenic fungi.

Fusarium oxysporum f. sp. niveum Pumilio 1 Regulates Virulence on Watermelon through Interacting with the ARP2/3 Complex and Binding to an A-Rich Motif in the 3' UTR of Diverse Transcripts.

Fusarium oxysporum f. sp. niveum (Fon), a soilborne phytopathogenic fungus, causes watermelon Fusarium wilt, resulting in serious yield losses worldwide. However, the underlying molecular mechanism of Fon virulence is largely unknown. The present study investigated the biological functions of six FonPUFs, encoding RNA binding Pumilio proteins, and especially explored the molecular mechanism of FonPUF1 in Fon virulence. A series of phenotypic analyses indicated that FonPUFs have distinct but diverse functions in vegetative growth, asexual reproduction, macroconidia morphology, spore germination, cell wall, or abiotic stress response of Fon. Notably, the deletion of FonPUF1 attenuates Fon virulence by impairing the invasive growth and colonization ability inside the watermelon plants. FonPUF1 possesses RNA binding activity, and its biochemical activity and virulence function depend on the RNA recognition motif or Pumilio domains. FonPUF1 associates with the actin-related protein 2/3 (ARP2/3) complex by interacting with FonARC18, which is also required for Fon virulence and plays an important role in regulating mitochondrial functions, such as ATP generation and reactive oxygen species production. Transcriptomic profiling of ΔFonPUF1 identified a set of putative FonPUF1-dependent virulence-related genes in Fon, possessing a novel A-rich binding motif in the 3' untranslated region (UTR), indicating that FonPUF1 participates in additional mechanisms critical for Fon virulence. These findings highlight the functions and molecular mechanism of FonPUFs in Fon virulence. IMPORTANCE Fusarium oxysporum is a devastating plant-pathogenic fungus that causes vascular wilt disease in many economically important crops, including watermelon, worldwide. F. oxysporum f. sp. nievum (Fon) causes serious yield loss in watermelon production. However, the molecular mechanism of Fusarium wilt development by Fon remains largely unknown. Here, we demonstrate that six putative Pumilio proteins-encoding genes (FonPUFs) differentially operate diverse basic biological processes, including stress response, and that FonPUF1 is required for Fon virulence. Notably, FonPUF1 possesses RNA binding activity and associates with the actin-related protein 2/3 complex to control mitochondrial functions. Furthermore, FonPUF1 coordinates the expression of a set of putative virulence-related genes in Fon by binding to a novel A-rich motif present in the 3' UTR of a diverse set of target mRNAs. Our study disentangles the previously unexplored molecular mechanism involved in regulating Fon virulence, providing a possibility for the development of novel strategies for disease management.

Bacillus altitudinis-Stabilized Multifarious Copper Nanoparticles Prevent Bacterial Fruit Blotch in Watermelon (Citrullus lanatus L.): Direct Pathogen Inhibition, In Planta Particles Accumulation, and Host Stomatal Immunity Modulation.

The nano-enabled crop protecting agents have been emerging as a cost-effective, eco-friendly, and sustainable alternative to conventional chemical pesticides. Here, the antibacterial activity and disease-suppressive potential of biogenic copper nanoparticles (bio-CuNPs) against bacterial fruit blotch (BFB), caused by Acidovorax citrulli (Ac), in watermelon (Citrullus lanatus L.) is discussed. CuNPs are extracellularly biosynthesized using a locally isolated bacterial strain Bacillus altitudinis WM-2/2, and have spherical shapes of 29.11-78.56 nm. Various metabolites, such as alcoholic compounds, carboxylic acids, alkenes, aromatic amines, and halo compounds, stabilize bio-CuNPs. Foliar application of bio-CuNPs increases the Cu accumulation in shoots/roots (66%/27%), and promotes the growth performance of watermelon plants by improving fresh/dry weight (36%/39%), through triggering various imperative physiological and biochemical processes. Importantly, bio-CuNPs at 100 µg mL-1 significantly suppress watermelon BFB through balancing reactive oxygen species system, improving photosynthesis capacity, and modulating stomatal immunity. Bio-CuNPs show obvious antibacterial activity against Ac by inducing oxidative stress, biofilm inhibition, and cellular integrity disruption. These findings demonstrate that bio-CuNPs can suppress watermelon BFB through direct antibacterial activity and induction of active immune response in watermelon plants, and highlight the value of this approach as a powerful tool to increase agricultural production and alleviate food insecurity.

Palmitoyl Transferase FonPAT2-Catalyzed Palmitoylation of the FonAP-2 Complex Is Essential for Growth, Development, Stress Response, and Virulence in Fusarium oxysporum f. sp. niveum.

Protein palmitoylation, one of posttranslational modifications, is catalyzed by a group of palmitoyl transferases (PATs) and plays critical roles in the regulation of protein functions. However, little is known about the function and mechanism of PATs in plant pathogenic fungi. The present study reports the function and molecular mechanism of FonPATs in Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt. The Fon genome contains six FonPAT genes with distinct functions in vegetative growth, conidiation and conidial morphology, and stress response. FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for Fon virulence on watermelon mainly through regulating in planta fungal growth within host plants. Comparative proteomics analysis identified a set of proteins that were palmitoylated by FonPAT2, and some of them are previously reported pathogenicity-related proteins in fungi. The FonAP-2 complex core subunits FonAP-2α, FonAP-2ß, and FonAP-2µ were palmitoylated by FonPAT2 in vivo. FonPAT2-catalyzed palmitoylation contributed to the stability and interaction ability of the core subunits to ensure the formation of the FonAP-2 complex, which is essential for vegetative growth, asexual reproduction, cell wall integrity, and virulence in Fon. These findings demonstrate that FonPAT1, FonPAT2, and FonPAT4 play important roles in Fon virulence and that FonPAT2-catalyzed palmitoylation of the FonAP-2 complex is critical to Fon virulence, providing novel insights into the importance of protein palmitoylation in the virulence of plant fungal pathogens. IMPORTANCE Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, is one of the most serious threats for the sustainable development of the watermelon industry worldwide. However, little is known about the underlying molecular mechanism of pathogenicity in Fon. Here, we found that the palmitoyl transferase (FonPAT) genes play distinct and diverse roles in basic biological processes and stress response and demonstrated that FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for virulence in Fon. We also found that FonPAT2 palmitoylates the core subunits of the FonAP-2 complex to maintain the stability and the formation of the FonAP-2 complex, which is essential for basic biological processes, stress response, and virulence in Fon. Our study provides new insights into the understanding of the molecular mechanism involved in Fon virulence and will be helpful in the development of novel strategies for disease management.

Bio-Functionalized Manganese Nanoparticles Suppress Fusarium Wilt in Watermelon (Citrullus lanatus L.) by Infection Disruption, Host Defense Response Potentiation, and Soil Microbial Community Modulation.

The use of nanofabricated materials is being explored for the potential in crop disease management. Chemically synthesized micronutrient nanoparticles (NPs) have been shown to reduce crop diseases; however, the potential of biogenic manganese NPs (bio-MnNPs) in disease control is unknown. Here, the potential and mechanism of bio-MnNPs in suppression of watermelon Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (Fon) are reported. Bio-MnNPs are synthesized by cell-free cultural filtrate of a waterrmelon rhizosphere bacterial strain Bacillus megaterium NOM14, and are found spherical in shape with a size range of 27.0-65.7 nm. Application of bio-MnNPs at 100 µg mL-1 increases Mn content in watermelon roots/shoots and improves growth performance through enhancing multiple physiological processes, including antioxidative capacity. Bio-MnNPs at 100 µg mL-1 suppress Fusarium wilt through inhibiting colonization and invasive growth of Fon in watermelon roots/stems, and inhibit Fon vegetative growth, conidiation, conidial morphology, and cellular integrity. Bio-MnNPs potentiate watermelon systemic acquired resistance by triggering the salicylic acid signaling upon Fon infection, and reshape the soil microbial community by improving fungal diversity. These findings demonstrate that bio-MnNPs suppress watermelon Fusarium wilt by multiple ex planta and in planta mechanisms, and offer a promising nano-enabled strategy for the sustainable management of crop diseases.

QTL associated with resistance to Stagonosporopsis citrulli in Citrullus amarus.

Gummy stem blight (GSB) is a fungal disease affecting cucurbit crops, including watermelon (Citrullus lanatus), leading to significant yield losses. The disease is caused by three Stagonosporopsis species, of which Stagonosporopsis citrulli is the most common in the southeastern United States. Currently no gummy stem blight-resistant watermelon cultivars are available to growers. In this study, QTL-seq in an interspecific population developed from Sugar Baby × PI 189225 (Citrullus amarus) identified QTL on chromosomes 2, 5, 9 and 11. A novel QTL on chromosome 5 (Qgsb5.2) associated with resistance to S. citrulli (PVE = 13.3%) was confirmed by genetic mapping. KASP marker assays were developed for selection of Qgsb5.2 to allow breeders to track the allele contributing resistance to GSB, reducing the need for laborious phenotyping. Pyramiding different GSB resistance QTL could be a useful strategy to develop GSB resistant watermelon cultivars.

Bama Pig Manure Organic Fertilizer Regulates the Watermelon Rhizosphere Bacterial Community to Inhibit the Occurrence of Fusarium Wilt Under Continuous Cropping Conditions.

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum is an important manifestation of continuous cropping barrier, which causes the quality and yield of watermelon to decline. In early stage of this study, the organic fertilizer fermented by Bama pig manure applied in soil was proved to significantly inhibit the occurrence of disease by improving the structure of soil microbial community. However, the mechanism was not clear. The high-throughput sequencing technology, combined with network and PICRUSt2 function analysis was used to investigate it. MiSeq sequencing showed that the bacterial community of organic fertilizer treated soil was composed of 34 phyla and 768 genera, the number of genera was higher than that of sterile water treated soil. Fertilization significantly increased the diversity and changed the composition of bacterial community based on alpha, beta diversity, and ANOSIM/Adonis analysis. LEfSe species difference and network analysis showed that fertilization improved the relative abundance of bacteria with biological control or plant growth promotion characteristics in soil, such as Sphingomonas, Halobacillus, Nocardioides, and enhanced the interaction between rhizosphere bacteria, made the network structure more complex. PICRUSt2 also revealed fertilization promoted the bacterial function, such as metabolism and genetic information processing. These results showed that the pig manure organic fertilizer might reduce the occurrence of Fusarium wilt by regulating bacterial community, interaction, and functional metabolism in watermelon rhizosphere soil.

Acidovorax citrulli Effector AopV Suppresses Plant Immunity and Interacts with Aromatic Dehydratase ADT6 in Watermelon.

Bacterial fruit blotch (BFB) is a disease of cucurbit plants caused by Acidovorax citrulli. Although A. citrulli has great destructive potential, the molecular mechanisms of pathogenicity of A. citrulli are not clear, particularly with regard to its type III secreted effectors. In this study, we characterized the type III secreted effector protein, AopV, from A. citrulli strain Aac5. We show that AopV significantly inhibits reactive oxygen species and the expression of PTI marker genes, and helps the growth of Pseudomonas syringae D36E in Nicotiana benthamiana. In addition, we found that the aromatic dehydratase ADT6 from watermelon was a target of AopV. AopV interacts with ADT6 in vivo and in vitro. Subcellular localization indicated ADT6 and AopV were co-located at the cell membrane. Together, our results reveal that AopV suppresses plant immunity and targets ADT6 in the cell membrane. These findings provide an new characterization of the molecular interaction of A. citrulli effector protein AopV with host cells.

Essential Acidovorax citrulli Virulence Gene hrpE Activates Host Immune Response against Pathogen.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli. The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli. Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli, thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.