Volatilome of the maize phytopathogenic fungus Fusarium verticillioides: potential applications in diagnosis and biocontrol.

BACKGROUND: Fusarium verticillioides is a maize fungal phytopathogen and a producer of volatile organic compounds (VOCs) and fumonisin B1 (FB1). Our aim was to study the volatilome, conidial production, ergosterol and FB1 biosynthesis in maize cultures over a 30-day incubation period (5, 10, 15, 20, 25, 30 days post inoculation [DPI]). The effect of pure VOCs on the same parameters was then evaluated to study their potential role as biocontrol agents. RESULTS: In total, 91 VOCs were detected, with volatile profiles being more similar between 5 and 10 DPI compared with 15, 20, 25 and 30 DPI. Ergosterol content increased steadily with incubation time, and three growth stages were identified: a lag phase (0 to 15 DPI), an exponential phase (15 to 20 DPI) and a stationary phase (20 to 30 DPI). The maximum concentration of FB1 was detected at 25 (0.030 µg FB1/µg ergosterol) and 30 DPI (0.037 µg FB1/µg ergosterol), whereas conidial production showed a maximum value at 15 DPI (4.3 ± 0.2 × 105 conidia/µg ergosterol). Regarding pure VOCs, minimal inhibitory concentration values ranged from 0.3 mm for 4-hexen-3-one to 7.4 mm for 2-undecanone. Pure VOCs reduced radial growth, conidial production and ergosterol and FB1 biosynthesis. CONCLUSIONS: The marked resemblance between VOC profiles at 5 and 10 DPI suggests that they could act as early indicators of fungal contamination, particularly 4-ethylguaiacol, 4-ethyl-2-methoxyanisole, heptanol and heptyl acetate. On the other hand, their role as inhibitors of fungal growth and FB1 biosynthesis prove their great potential as safer alternatives to control phytopathogenic fungi. © 2024 Society of Chemical Industry.

Screening and identification of Paenibacillus polymyxa GRY-11 and its biological control potential against apple replant disease.

Apple replant disease (ARD) is a significant factor restricting the healthy development of the apple industry. Biological control is an important and sustainable method for mitigating ARD. In this study, a strain of Paenibacillus polymyxa GRY-11 was isolated and screened from the rhizosphere soil of healthy apple trees in old apple orchards in Shandong Province, China, and the effects of strain GRY-11 on soil microbial community and ARD were studied. The result showed that P. polymyxa GRY-11 could effectively inhibit the growth of the main pathogenic fungi that caused ARD, and the inhibition rates of the strain against Fusarium moniliforme, Fusarium proliferatum, Fusarium solani, and Fusarium oxysporum were 80.00%, 71.60%, 75.00%, and 70.00%, respectively. In addition, the fermentation supernatant played an active role in suppressing the growth of pathogenic fungi. The results of the pot experiment showed that the bacterial fertilizer of the GRY-11 promoted the growth of Malus hupehensis seedlings, improved the activity of protective enzymes in plant roots, enhanced the soil enzyme content, and optimized the soil microbial environment. In general, the GRY-11 can be used as an effective microbial preparation to alleviate ARD. Our study offers novel perspectives for the prevention of ARD.

First Report of Fusarium concentricum Causing Leaf Blight, Shoot Blight, Flower and Fruit Rot on Kadsura coccinea in China.

Kadsura coccinea (Lem.) A. C. Smith is a traditional medicinal plant grown in south China. Lignans and terpenoids extracted from the root, fruit and stem were found to have anti-proliferative, anti-HIV, anti-hepatitis, anti-oxidant, neuroprotective, and other pharmacological attributes for treating rheumatoid arthritis and gastroenteric et al. disorders (Yang et al., 2020). However, little is known about the biotic disorders of this evergreen climbing shrub. In surveys carried out in a 15- hectare -orchard located 23°59'55''N, 113°55'13''E, all K. coccinea plants were observed exhibiting an array of symptoms including leaf blight, shoot blight, flower rot and fruit rot from May to July of 2023, with disease incidences of 17% , 18%, 16% and 28%, respectively. A greyish -brown blight appeared predominantly on the leaf margins or tips. Light to dark brown lesions on the shoots, flower calyxes and fruits were slightly sunken, irregularly shaped, and watery, usually with white aerial mycelium. For pathogen isolation, infected tissues were cut into fragments of about 5 mm in diameter, surface sterilized with 75% ethanol for 15 s and 1% NaClO for 2 min, and then rinsed three times with sterilized distilled water. Filter paper-dried tissues were placed onto potato dextrose agar (PDA) and incubated at 28°C for 5 days. Seventy-one morphologically similar colonies were produced from 100 tissue fragments, with other tissues lacking colonies or with different colonies. Genomic DNA of 10 randomly selected isolates were extracted from 5-day-old pure cultures. Molecular characterization of the 10 subcultured strains was analyzed by sequencing three regions i.e. translation elongation factor 1-alpha (TEF-1), beta-tublin (TUB), and RNA polymerase Ⅱ second largest subunit (RPB2), amplified using primers EF1/EF2 (O'Donnell et al., 1998), T1/T22 (O'Donnell and Cigelnik 1997), and RPB2-5f2/RPB2-7cr (Liu et al. 1999). Maximum likelihood and Bayesian Inference analysis based on the dataset of the combined three sequences by MEGA 11.0 showed that nine strains were consistently identified as F. concentricum. The three sequences (OR632200, OR632199, and OR754282) of a representative strain (SGXF1) shared 99.42%, 99.62%, and 99.80% identity with those of the type strain CBS450. 97. The colonies were white to pale buff with serrated edges and sparse aerial mycelium that initially formed a loose, white cottony texture of 5-10 mm in height. After 7 d's culture at 26°C in an incubator with a photic door, dense and abundant fluffy reddish-white aerial mycelium covered the entire PDA medium of 9 cm-diameter, with alternating pale orange and reddish-grey concentric rings at center with diffusible pigments. Catenate microconidia were obovoid to fusoid-shaped, mostly 0-septate, with a flattened base, (10.98 ± 0.83) x (3.41 ± 0.15) µm (n=20), and were produced on both mono-and polyphialides, whereas macroconidia were curved and long, with a slightly beaked apical cell and a basal cell, mainly 3- to 5-septate, and (33.80 ± 0.81) x (4.31 ± 0.12) µm (n=20) in size. These morphological characteristics further indicates it to be F. concentritum (Nirenberg and O'Donnell, 1998; Xiao et al., 2023). To determine pathogenicity, three 1-y-old seedlings were wound inoculated by conidial suspension (1 × 106 conidia/mL) on different tissues and incubated at 25°C in a greenhouse with 10 replications. At 2 d after inoculation (DAI), the treated flowers gradually turned from fresh red and green to black at calyxes, with the black area then gradually expanding. At 6 DAI, part of the flowers turned black, accompanied by white mycelium and yellowish powdery spots. Similarly, leaves, stems, and fruits turned from green to dark brown at infection sites within 7 DAI. Black to brown, uneven necrotic areas, and reddish spots were observed under a dissecting microscope, with white mycelium attached at 6 DAI. The symptoms observed in lab were similar to those observed in the field, whereas three control plants remained asymptomatic. Fungal colonies identical to initial isolations were recovered from artificially infected tissues and confirmed to be F. concentricum again, thereby completing Koch's postulates. This report is the first to document F. concentricum causing disease on K. coccinea. Appropriate measures will be developed based on this study to protect this economically important plant in the field.

Antimicrobial mechanisms and antifungal activity of compounds generated by banana rhizosphere Pseudomonas aeruginosa Gxun-2 against fusarium oxysporum f. sp. cubense.

Introduction: Fusarium wilt of banana, also recognized as Panama disease, is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (FOC TR4). In recent years, strategies utilizing biocontrol agents, comprising antifungal microorganisms and their associated bioactive compounds from various environments, have been implemented to control this destructive disease. Our previous study showed that Pseudomonas aeruginosa Gxun-2 had significant antifungal effects against FOC TR4. However, there has been little scientific investigation of the antibacterial or antifungal activity. The aim of this study was to isolate, identify and evaluate the inhibition strength of active compounds in P. aeruginosa Gxun-2, so as to explain the mechanism of the strain inhibition on FOC TR4 from the perspective of compounds. Methods: The main antibacterial compounds of strain Gxun-2 were isolated, purified and identified using by fermentation extraction, silica gel column chromatography, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) techniques. The effect of the compounds on the mycelial growth, morphology and spore germination of strain FOC TR4 was observed by 96-well plate method and AGAR diffusion method. Results: Among the metabolites produced by the strain, four antifungal compounds which were identified phenazine (C12H8N2), phenazine-1-carboxylic acid (PCA) (C13H8N2O2), 2-acetamidophenol (C8H9NO2) and aeruginaldehyde (C10H7NO2S) were identified through HPLC and NMR. Of these compounds, phenazine and PCA exhibited the most pronounced inhibitory effects on the spore germination and mycelial growth of FOC TR4. Phenazine demonstrated potent antifungal activity against FOC TR4 with a minimum inhibitory concentration (MIC) of 6.25 mg/L. The half-maximal effective concentration (EC50) was calculated to be 26.24 mg/L using the toxicity regression equation. PCA exhibited antifungal activity against FOC TR4 with an MIC of 25 mg/L and an EC50 of 89.63 mg/L. Furthermore, phenazine and PCA triggered substantial morphological transformations in the mycelia of FOC TR4, encompassing folding, bending, fracturing, and diminished spore formation. Discussion: These findings indicate that strain Gxun-2 plays a crucial role in controlling FOC TR4 pathogenesis, predominantly through producing the antifungal compounds phenazine and PCA, and possesses potential as a cost-efficient and sustainable biocontrol agent against Fusarium wilt of banana in forthcoming times.

High-density genetic map construction and QTL mapping to identify genes for blight defense- and yield-related traits in sesame (Sesamum indicum L.).

Sesame (Sesamum indicum L.) is an important oilseed crop widely cultivated in subtropical and tropical areas. Low genetic yield potential and susceptibility to disease contribute to low productivity in sesame. However, the genetic basis of sesame yield- and disease-related traits remains unclear. Here, we represent the construction of a high-density bin map of sesame using whole genome sequencing of an F2 population derived from 'Yizhi' and 'Mingdeng Zhima'. A total of 2766 Bins were categorized into 13 linkage groups. Thirteen significant QTLs were identified, including ten QTLs related to yield, two QTLs related to Sesame Fusarium wilt (SFW) disease, and one QTL related to seed color. Among these QTLs, we found that SFW-QTL1.1 and SFW-QTL1.2 were major QTLs related to Fusarium wilt disease, explaining more than 20% of the phenotypic variation with LOD > 6. SCC-QTL1.1 was related to seed coat color, explaining 52% of the phenotypic variation with LOD equal to 25.3. This suggests that seed color traits were controlled by a major QTL. Candidate genes related to Fusarium wilt disease and seed color in the QTLs were annotated. We discovered a significant enrichment of genes associated with resistance to late blight. These genes could be spectral disease resistance genes and may have a role in the regulation of Fusarium wilt disease resistance. Our study will benefit the implementation of marker-assisted selection (MAS) for the genetic improvement of disease resistance and yield-related traits in sesame.

Insights on mining the pangenome of Sphingobacterium thalpophilum NMS02 S296 from the resistant banana cultivar Pisang lilin confirms the antifungal action against Fusarium oxysporum f. sp. cubense.

Introduction: Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), poses a significant global threat to banana cultivation. Conventional methods of disease management are increasingly challenged, thus making it necessary to explore alternative strategies. Bacterial endophytes, particularly from resistant genotypes, are gaining attention as potential biocontrol agents. Sphingobacterium thalpophilum, isolated from the resistant banana cultivar Pisang lilin (JALHSB010000001-JALHSB010000029), presents an intriguing prospect for combating Fusarium wilt. However, its underlying biocontrol mechanisms remain poorly understood. This study aimed to elucidate the antifungal efficacy of S. thalpophilum NMS02 S296 against Foc and explore its biocontrol mechanisms at the genomic level. Methods: Whole genome sequencing of S. thalpophilum NMS02 S296 was conducted using next-generation sequencing technologies and bioinformatics analyses were performed to identify genes associated with antifungal properties. In vitro assays were used to assess the inhibitory effects of the bacterial isolate on the mycelial growth of Foc. To explore the biomolecules responsible for the observed antagonistic activity, metabolites diffused into the agar at the zone of inhibition between Foc S16 and S. thalpophilum NMS02 S296 were extracted and identified. Results: Whole genome sequencing revealed an array of genes encoding antifungal enzymes and secondary metabolites in S. thalpophilum NMS02 S296. In vitro experiments demonstrated significant inhibition of Foc mycelial growth by the bacterial endophyte. Comparative genomic analysis highlighted unique genomic features in S. thalpophilum linked to its biocontrol potential, setting it apart from other bacterial species. Discussion: The study underscores the remarkable antifungal efficacy of S. thalpophilum NMS02 S296 against Fusarium wilt. The genetic basis for its biocontrol potential was elucidated through whole genome sequencing, shedding light on the mechanisms behind its antifungal activity. This study advanced our understanding of bacterial endophytes as biocontrol agents and offers a promising avenue for plant growth promotion towards sustainable strategies to mitigate Fusarium wilt in banana cultivation.

An effector protein of Fusarium graminearum targets chloroplasts and suppresses cyclic photosynthetic electron flow.

Chloroplasts are important photosynthetic organelles that regulate plant immunity, growth, and development. However, the role of fungal secretory proteins in linking the photosystem to the plant immune system remains largely unknown. Our systematic characterization of 17 chloroplast-targeting secreted proteins of Fusarium graminearum indicated that Fg03600 is an important virulence factor. Fg03600 translocation into plant cells and accumulation in chloroplasts depended on its chloroplast transit peptide. Fg03600 interacted with the wheat (Triticum aestivum L.) proton gradient regulation 5-like protein 1 (TaPGRL1), a part of the cyclic photosynthetic electron transport chain, and promoted TaPGRL1 homo-dimerization. Interestingly, TaPGRL1 also interacted with ferredoxin (TaFd), a chloroplast ferredoxin protein that transfers cyclic electrons to TaPGRL1. TaFd competed with Fg03600 for binding to the same region of TaPGRL1. Fg03600 expression in plants decreased cyclic electron flow (CEF) but increased the production of chloroplast-derived reactive oxygen species (ROS). Stably silenced TaPGRL1 impaired resistance to Fusarium head blight (FHB) and disrupted CEF. Overall, Fg03600 acts as a chloroplast-targeting effector to suppress plant CEF and increase photosynthesis-derived ROS for FHB development at the necrotrophic stage by promoting homo-dimeric TaPGRL1 or competing with TaFd for TaPGRL1 binding.

Maize stigmas react differently to self- and cross-pollination and fungal invasion.

During sexual reproduction in flowering plants, tip-growing pollen tubes travel from the stigma inside the maternal tissues of the pistil towards ovules. In maize (Zea mays L.), the stigma is highly elongated, forming thread-like strands known as silks. Only compatible pollen tubes successfully penetrate and grow through the transmitting tract of the silk to reach the ovules. Like pollen, fungal spores germinate at the surface of silks and generate tube-like structures (hyphae) penetrating silk tissue. To elucidate commonalities and differences between silk responses to these distinctive invading cells, we compared growth behavior of the various invaders as well as the silk transcriptome after self-pollination, cross-pollination and infection using two different fungi. We report that self-pollination triggers mainly senescence genes, whereas incompatible pollen from Tripsacum dactyloides leads to downregulation of rehydration, microtubule, and cell wall-related genes, explaining the slower pollen tube growth and arrest. Invasion by the ascomycete Fusarium graminearum triggers numerous defense responses including the activation of monolignol biosynthesis and NAC as well as WRKY transcription factor genes, whereas responses to the basidiomycete Ustilago maydis are generally much weaker. We present evidence that incompatible pollination and fungal infection trigger transcriptional reprograming of maize silks cell wall. Pathogen invasion also activates the phytoalexin biosynthesis pathway.

Bacterial seed endophytes promote barley growth and inhibits Fusarium graminearum in vitro.

OBJECTIVES: Seeds host microbes that function in plant growth and phytopathogen resistance. The aim of the work was to investigate total bacterial community in malting barley seeds and whether their bacterial seed endophytes have dual functional roles in plant growth-promotion and inhibition of Fusarium graminearum, the causative agent of Fusarium head blight (FHB) in barley. We used culture dependent and culture independent methods. RESULTS: Phylogenetic classification of seed endophytic bacteria based on sequencing data identified B. subtilis, B. licheniformis and B. pumilis as predominant subgroups. Location driven divergence in bacterial endophytic communities was evident based on a clear separation of the samples from Crookston and other location samples. The bio-primed seeds using one hundred and seventy bacterial isolates showed that 3.5% (6/170) of the bacterial isolates conferred greater than 10% increase in both root length (RL) and shoot length (SL), while 19.4% (33/170) and 26.5% (45/170) showed RL and SL specific growth effects, respectively, relative to controls. Among the six bacterial isolates that increased RL and SL, five (#29, #63, #109, #124 and #126) also significantly inhibit the growth of F. graminearum based on in vitro assays. This study identified novel seed bacterial endophytes that could be further exploited for promoting growth during seedling establishment and as biocontrol for combating the devastating scab disease.

Harnessing RNA interference for the control of Fusarium species: A critical review.

Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium-induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi-based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi-based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation.

Green and efficient extraction of an anticaner agent N-methylsansalvamide using ultrasound-assisted deep eutectic solvents from mycelia of strain Frusarium sp. R1.

N-methylsansalvamide (SA), one of cyclic pentadepsipeptides produced by several Fusarium strains, is a promising therapeutic agent for the treatment of cancer disease. In order to make sufficient amount of SA for drug development, a green and efficient extraction process of SA from the mycelia of strain Fusarium sp. R1 using deep eutectic solvent-assisted ultrasound extraction (DES-UAE) was firstly achieved in this work. Solvent screening results indicated that choline chloride-acetic acid (ChCl-Aa) was shown to be the best DES for SA extraction. Through single-factor trials, Plackett-Burman design (PBD) and BoxBehnken design (BBD) experiments, the optimal conditions for DES-UAE with the highest SA yield of 58.2 ± 1.1 mg/g were obtained as follows: ChCl-Aa ratio of 1:2.0 (M/M), water content of 16.4 %, liquid-solid ratio of 37:1 (mL/g), ultrasonic power of 175 W for 47.4 min at 46.3 °C. Compared to conventional extraction approaches, DES-UAE exhibited better SA yield since it caused more serious damage to the surface of mycelia powder on basis of scanning electron microscopy (SEM) analysis. Furthermore, molecular interaction studies suggested that SA has a variety of interactions with ChCl-Aa, including hydrogen and electrovalent bonds as well as van der Waals forces. Finally, the recovery rate of SA reached up to 99.5 % when the ratio of distilled water and DES extracts was 15:1 (V/V). These findings provide the way for large-scale production of SA.

ATP-binding cassette transporter TaABCG2 contributes to Fusarium head blight resistance by mediating salicylic acid transport in wheat.

ATP-binding cassette (ABC) transporters hydrolyse ATP to transport various substrates. Previous studies have shown that ABC transporters are responsible for transporting plant hormones and heavy metals, thus contributing to plant immunity. Herein, we identified a wheat G-type ABC transporter, TaABCG2-5B, that responds to salicylic acid (SA) treatment and is induced by Fusarium graminearum, the primary pathogen causing Fusarium head blight (FHB). The loss-of-function mutation of TaABCG2-5B (ΔTaabcg2-5B) reduced SA accumulation and increased susceptibility to F. graminearum. Conversely, overexpression of TaABCG2-5B (OE-TaABCG2-5B) exerted the opposite effect. Quantification of intracellular SA in ΔTaabcg2-5B and OE-TaABCG2-5B protoplasts revealed that TaABCG2-5B acts as an importer, facilitating the transport of SA into the cytoplasm. This role was further confirmed by Cd2+ absorption experiments in wheat roots, indicating that TaABCG2-5B also participates in Cd2+ transport. Thus, TaABCG2-5B acts as an importer and is crucial for transporting multiple substrates. Notably, the homologous gene TaABCG2-5A also facilitated Cd2+ uptake in wheat roots but did not significantly influence SA accumulation or FHB resistance. Therefore, TaABCG2 could be a valuable target for enhancing wheat tolerance to Cd2+ and improving FHB resistance.

Low molecular weight acids differentially impact Fusarium verticillioides transcription.

Fusarium verticillioides is both an endophyte and pathogen of maize. During growth on maize, the fungus often synthesizes the mycotoxins fumonisins, which have been linked to a variety of diseases, including cancer in some animals. How F. verticillioides responds to other fungi, such as Fusarium proliferatum, Aspergillus flavus, Aspergillus niger, and Penicillium oxalicum, that coinfect maize, has potential to impact mycotoxin synthesis and disease. We hypothesize that low molecular weight acids produced by these fungi play a role in communication between the fungi in planta/nature. To address this hypothesis, we exposed 48-h maize kernel cultures of F. verticillioides to oxalic acid, citric acid, fusaric acid, or kojic acid and then compared transcriptomes after 30 min and 6 h. Transcription of some genes were affected by multiple chemicals and others were affected by only one chemical. The most significant positive response was observed after exposure to fusaric acid which resulted in >2-fold upregulation of 225 genes, including genes involved in fusaric acid synthesis. Exposure of cultures to the other three chemicals increased expression of only 3-15 genes. The predicted function and frequent co-localization of three sets of genes support a role in protecting the fungus from the chemical or a role in catabolism. These unique transcriptional responses support our hypothesis that these chemicals can act as signaling molecules. Studies with gene deletion mutants will further indicate if the initial transcriptional response to the chemicals benefit F. verticillioides.

Susceptibility of Aphis craccivora (Hemiptera: Aphididae) to three entomopathogenic Fusarium species.

The black aphid (Aphis craccivora) is an insect pest that can cause significant losses to different agricultural crops. Entomopathogenic fungi can be good options for controlling this insect. Fusarium species have shown promising results in the biological control of several agricultural pests, mainly of the order Hemiptera. This study investigated the susceptibility of A. craccivora to 27 Fusarium isolates, distributed among F. sulawesiense (4), F. pernambucanum (6) and F. caatingaense (17). The viability of the conidia of all isolates was assessed by measuring their germination rate. Pathogenicity tests were conducted at 107 conidia/mL, and the best-performing isolate was further tested at different concentrations (104 to 108 conidia/mL). Data were analyzed using ANOVA, Tukey's test at 5 %, and R for calculating lethal times (LT50,90) and lethal concentrations (LC50,90). All isolates had viable conidia with germination rates between 92.67 % and 100 %. Mortality rates ranged from 17.22 % to 90.23 %. F. pernambucanum URM 7559 had the shortest lethal times (LT50 of 2.24 days and LT90 of 4.42 days), followed by F. sulawesiense URM 7555 (LT50 of 2.35 days and LT90 of 4.77 days) and F. caatingaense with LT50 of 3.93 days for URM 6784 and LT90 of 8.27 days for URM 6807. The three Fusarium species tested, especially F. pernambucanum, showed promise in the biological control of A. craccivora. Although the results are promising, additional studies are needed to evaluate the safety, field efficacy and environmental impacts of Fusarium use, focusing on the interaction with the agricultural ecosystem and the risks to non-target organisms.

Identification and molecular detection of the pathogen of Phalaenopsis leaf yellowing through genome analysis.

Moth orchids (Phalaenopsis spp.) are globally popular ornamental flowers. However, effective management strategies for Phalaenopsis leaf yellowing remain elusive, making the disease a challenging obstacle affecting moth orchids at various growth stages. This disease manifests as collar rot, leaf yellowing, leaf abscission, and eventually, plant death. The lack of effective management strategies is likely attributed to a limited understanding of the disease pathogenesis and pathogen dissemination pathways. Fusarium phalaenopsidis sp. nov. was established in this study to stabilize the classification status of Phalaenopsis leaf yellowing pathogens using molecular and morphological features. The genome of the holotype strain was sequenced and assembled, revealing its genome structures. Analyses of virulence-related elements, including transposon elements, secondary metabolite biosynthetic gene clusters, effectors, and secreted carbohydrate-active enzymes, shed light on the potential roles of three fast core chromosomes in virulence. Two species-specific primers were designed based on unique gene sequences of two virulence-related proteins through comparative genomics and BLAST screening. The specificity of these primers was validated using isolates of F. phalaenopsidis, non-target species in the Fusarium solani species complex, other Fusarium species complexes, and saprophytic fungi. These results are intended to accelerate the identification of the pathogens, facilitate the study of disease pathogenesis, and pave the way for elucidating pathogen dissemination pathways. Ultimately, they aim to contribute to the formulation of effective control strategies against Phalaenopsis leaf yellowing.

Gene markers generating polygenic resistance in melon-Fusarium wilt-FOM1.2 interaction pathosystem.

Developing melon genotypes with resistance to Fusarium oxysporum f. sp. Melonis-(FOM) race1.2 is a major goal in any breeding program. In this study, we identified the role of 11 gene markers that contribute to polygenic resistance during the FOM1.2-melon interaction. qRT-PCR analysis elucidated upregulation of candidate marker genes AMT, DXPR, Fom-2, GLUC, GalS, GRF3, MLO, PRK, RuBlsCo, TLP and WRKY in resistant 'Shante-F1' and 'Khatouni', and susceptible 'Shante-T' and 'Shahabadi' at 7, 14 and 21 days post-inoculation (dpi). We also studied changes in defence-related enzyme activity: chitinase (CHI), ß-1,3-glucanase (GLU) and peroxidase (POX) in melon roots. AMT, GLUC and DXPR transcripts were upregulatied in leaf and root tissues of the resistant 'Shante-F1' and 'Shahabadi'. Transcript levels for GalS and GRF3 increased 6.77- and 6.83-fold in roots of 'Shante-F1' at 7 dpi, whereas in PRK, TLP and WRKY theye increased by 7.84-, 5.15- and 12.26-fold at 14 dpi, respectively. However, transcript levels increased by 5.18-fold for Fom-2 and 8.46-fold for MLO at 21 dpi. Also, RBC transcript level peaked at 14 dpi with 4.9-fold increase in leaves of resistant genotypes, whereas AMT increased 2.94-fold at 21 dpi, and GLUC and DXPR increased 7.11- and 2.91-fold at 14 dpi in 'Shante-F', respectively. Defence-related-enzyme activity was also upregulated three-fold in resistant varieties. The dynamic shifts in the melon transcriptome induced by FOM1.2 emphasize that resistance mechanisms are predominantly regulated through signalling pathways involving CHI, GLU, and POX defence response. Surprisingly, the AMT gene, basically resistant to downy mildew, Pseudoperonospora cubensis; GLUC, MLO and PRK resistant to powdery mildew (Sphaerotheca fusca); TLP and WRKY resistant to Phytophthora blight (Phytophthora capsici); and GRF3 and RBC resistant to root knot nematodes (Meloidogyne spp.) were upregulated in resistant genotypes, indicating a dual role of these genes in resistance to more than one disease at a time.

A virulent milRNA inhibits host immunity by silencing a host receptor-like kinase MaLYK3 and facilitates infection by Fusarium oxysporum f. sp. cubense.

MicroRNA-like RNAs (milRNAs) play a significant role in the infection process by plant-pathogenic fungi. However, the specific functions and regulatory mechanisms of fungal milRNAs remain insufficiently elucidated. This study investigated the function of Foc-milR138, an infection-induced milRNA secreted by Fusarium oxysporum f. sp. cubense (Foc), which is the causal agent of Fusarium wilt of banana. Initially, through precursor gene knockout and phenotypic assessments, we confirmed that Foc-milR138 acts as a virulent milRNA prominently upregulated during the early stages of Foc infection. Subsequent bioinformatic analyses and transient expression assays in Nicotiana benthamiana leaves identified a host receptor-like kinase gene, MaLYK3, as the direct target of Foc-milR138. Functional investigations of MaLYK3 revealed its pivotal role in triggering immune responses of N. benthamiana by upregulating a suite of resistance genes, bolstering reactive oxygen species (ROS) accumulation and callose deposition, thereby fortifying disease resistance. This response was markedly subdued upon co-expression with Foc-milR138. Expression pattern analysis further verified the specific suppression of MaLYK3 by Foc-milR138 during the early root infection by Foc. In conclusion, Foc secretes a virulent milRNA (Foc-milR138) to enter the host banana cells and inhibit the expression of the plant surface receptor-like kinase MaLYK3, subverting the disease resistance activated by MaLYK3, and ultimately facilitating pathogen invasion. These findings shed light on the roles of fungal milRNAs and their targets in resistance and pathogenicity, offering promising avenues for the development of disease-resistant banana cultivars.

A Novel Secreted Protein of Fusarium oxysporum Promotes Infection by Inhibiting PR-5 Protein in Plant.

Fusarium oxysporum, an important soilborne fungal pathogen that causes serious Fusarium wilt disease, secretes diverse effectors during the infection. In this study, we identified a novel secreted cysteine-rich protein, FolSCP1, which contains unknown protein functional domain. Here, we characterized FolSCP1 as a secreted virulence factor that promotes the pathogen infection of host plants by inhibiting diverse plant defence responses. FolSCP1 interacted with the pathogenesis-related 5 (PR-5) protein SlPR5, a positive regulator of tomato plant immunity against multiple tomato pathogens, and effectively attenuated the antifungal activity of the tomato PR-5 protein. FoSCP1, a homologue of FolSCP1, was secreted by a F. oxysporum isolate from infected tobacco and targeted the tobacco PR-5 protein NtPR5 to suppress plant defence for further infection. In summary, our study revealed a fungal virulence strategy in which F. oxysporum secrete effectors that interfere with plant immunity by binding to the PR-5 protein of the host plant and inhibiting its biological activity, thereby promoting fungal infection.

Construction of a comprehensive meteorological risk index for wheat Fusarium head blight prediction based on more than a half-century of monitoring data.

Fusarium head blight (FHB) represents a critical threat to wheat production globally, not only reducing yields but also contaminating crops with harmful mycotoxins. This study aimed to elucidate new spatiotemporal patterns of FHB incidence and to develop a comprehensive meteorological risk index to enhance scientific prevention and control of the disease. Through the analysis of annual and decadal variations from 1965 to 2023, the study assessed FHB trends across four agricultural regions (I, II, III, and IV) in Jiangsu Province, located in the middle and lower reaches of the Yangtze River-a hotspot for FHB in China. Key findings include: Since 1965, Regions I and III consistently exhibited higher FHB incidence rates compared to Regions II and IV. Post-2000, there was a notable increase in years with high incidence rates, with Region III overtaking Region I as the region with the highest incidence. Since 2010, occurrences of FHB reaching the most severe grade (Grade 5) have surpassed those in previous decades across all regions. The study also revealed a stronger correlation between meteorological factors (cumulative precipitation, number of days with rainfall ≥ 0.1 mm, total rainy days with ≥ 2 and ≥3 consecutive days of rain, total rainy days with both average daily air temperature ≥ 15 °C and daily rainfall ≥ 0.1 mm, days with average daily relative humidity ≥ 85%, cumulative sunshine hours, and cumulative cloudy days) and the FHB incidence rates during the heading-flowering-grain filling period in Regions I, II, and III, compared to the heading-flowering period alone. This indicates that optimal temperature and high humidity during the grain filling stage significantly contribute to the final FHB incidence rates. Despite the less apparent correlation between temperature changes and disease rates, the significant warming trend observed since 2000 has likely fostered conditions conducive to the proliferation of FHB. The comprehensive meteorological risk index, constructed to incorporate key meteorological factors during the heading-flowering-filling period, showed a strong correlation with actual disease incidences. The index demonstrated fitting accuracy rates of 84.7% for Region I, 72.9% for Region II, 83.1% for Region III, and 90.9% for Region IV, underscoring its effectiveness in predicting FHB occurrences. This tool offers both convenience and practicality, providing valuable insights for strategically managing FHB risks based on local weather conditions.

Exogenous Application of dsRNA-Inducing Silencing of the Fusarium oxysporum Tup1 Gene and Reducing Its Virulence.

Fusarium oxysporum is a widespread soil-borne fungal pathogen that can infect various plants, causing wilt and root rot diseases. The root rot disease of Atractylodes macrocephala caused by F. oxysporum is among the most serious diseases associated with continuous cropping, significantly hindering its sustainable development. In this study, we aimed to investigate the effect of exogenous application of double-stranded RNA (dsRNA) on silencing the F. oxysporum Tup1 gene to reduce its virulence and to evaluate its potential application in controlling root rot disease in A. macrocephala. The Tup1 gene was amplified from the F. oxysporum genome, and different lengths of Tup1-dsRNA were designed and synthesized. The uptake of dsRNA by the fungus was verified using Tup1-dsRNA labeled with fluorescein, and in vitro dsRNA treatment experiments were conducted to assess its impact on the growth and virulence of F. oxysporum. Additionally, Tup1-dsRNA was applied to the roots of A. macrocephala to evaluate its effectiveness in controlling root rot disease. The experimental results showed that F. oxysporum could effectively uptake exogenously applied Tup1-dsRNA, significantly reducing Tup1 gene expression. All lengths of Tup1-dsRNA inhibited fungal growth and caused morphological changes in the fungal hyphae. Further plant experiments and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis indicated that Tup1-dsRNA treatment significantly reduced the incidence of root rot disease in A. macrocephala, which was supported by the reduction in peroxidase (POD) and catalase (CAT) enzyme activities, malondialdehyde (MDA) content, and proline (Pro) levels in treated root tissues. This study demonstrated that exogenous dsRNA could reduce the virulence of F. oxysporum by silencing the Tup1 gene and effectively mitigate the root rot disease it causes in A. macrocephala. The successful application of Tup1-dsRNA provided strong evidence for the potential of RNA interference (RNAi) technology in plant disease control. Future research could further optimize the design and application of dsRNA to enhance its practical value in agriculture.