With fungal diseases posing a major threat to agricultural production, the application of fungicides to control related diseases is often considered necessary to ensure the world\'s food supply. The search for new bioactive agents has long been a priority in crop protection due to the continuous development of resistance against currently used types of active compounds. Heterocyclic compounds are an inseparable part of the core structures of numerous lead compounds, these rings constitute pharmacophores of a significant number of fungicides developed over the past decade by agrochemists. Among heterocycles, nitrogen-based compounds play an essential role. To date, diazole (imidazole and pyrazole) and diazine (pyrimidine, pyridazine, and pyrazine) derivatives make up an important series of synthetic fungicides. In recent years, many reports have been published on the design, synthesis, and study of the fungicidal activity of these scaffolds, but there was a lack of a comprehensive classified review on nitrogen-containing scaffolds. Regarding this issue, here we have reviewed the published articles on the fungicidal activity of the diazole and diazine families. In current review, we have classified the molecules synthesized so far based on the size of the ring.

Clonostachys (Bionectriaceae, Hypocreales) species are common soil-borne fungi, endophytes, epiphytes, and saprotrophs. Sexual morphs of Clonostachys spp. were placed in the genus Bionectria, which was further segregated into the six subgenera Astromata, Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella. However, with the end of dual nomenclature, Clonostachys became the single depository for sexual and asexual morph-typified species. Species of Clonostachys are typically characterised by penicillate, sporodochial, and, in many cases, dimorphic conidiophores (primary and secondary conidiophores). Primary conidiophores are mononematous, either verticillium-like or narrowly penicillate. The secondary conidiophores generally form imbricate conidial chains that can collapse to slimy masses, particularly on sporodochia. In the present study, we investigated the species diversity within a collection of 420 strains of Clonostachys from the culture collection of, and personal collections at, the Westerdijk Fungal Biodiversity Institute in Utrecht, the Netherlands. Strains were analysed based on their morphological characters and molecular phylogeny. The latter used DNA sequence data of the nuclear ribosomal internal transcribed spacer regions and intervening 5.8S nrDNA (ITS) and partial 28S large subunit (LSU) nrDNA and partial protein encoding genes including the RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) and β-tubulin (TUB2). Based on these results, the subgenera Astromata, Bionectria, Myronectria and Zebrinella are supported within Clonostachys. Furthermore, the genus Sesquicillium is resurrected to accommodate the former subgenera Epiphloea and Uniparietina. The close relationship of Clonostachys and Sesquicillium is strongly supported as both are inferred phylogenetically as sister-genera. New taxa include 24 new species and 10 new combinations. Recognition of Sesquicillium distinguishes species typically forming a reduced perithecial stroma superficially on plant tissue from species in Clonostachys often forming well-developed, through bark erumpent stromata. The patterns of observed perithecial wall anatomies, perithecial wall and stroma interfaces, and asexual morph diversifications described in a previously compiled monograph are used for interpreting ancestral state reconstructions. It is inferred that the common ancestor of Clonostachys and Sesquicillium may have formed perithecia superficially on leaves, possessed a perithecial wall consisting of a single region, and formed intercalary phialides in penicilli of conidiophores. Character interpretation may also allow hypothesising that diversification of morphs occurred then in the two genera independently and that the frequently stroma-linked Clonostachys morphs evolved together with the occupation of woody host niches and mycoparasitism. Taxonomic novelties: New species: Clonostachys aurantiaca L. Zhao & Crous, Clonostachys australiana L. Zhao & Crous, Clonostachys bambusae L. Zhao & Crous, Clonostachys buxicola L. Zhao & Crous, Clonostachys cylindrica L. Zhao & Crous, Clonostachys ellipsoidea L. Zhao & Crous, Clonostachys flava L. Zhao, Crous & Schroers, Clonostachys fujianensis L. Zhao & Crous, Clonostachys fusca L. Zhao, Crous & Schroers, Clonostachys garysamuelsii L. Zhao & Crous, Clonostachys hongkongensis L. Zhao & Crous, Clonostachys longiphialidica L. Zhao, Crous & Schroers, Clonostachys obovatispora, L. Zhao & Crous, Clonostachys palmae L. Zhao, Crous & Schroers, Clonostachys parasporodochialis L. Zhao & Crous, Clonostachys penicillata L. Zhao, Crous & Schroers, Clonostachys reniformis L. Zhao & Crous, Clonostachys vacuolata L. Zhao, Crous & Schroers, Clonostachys venezuelae L. Zhao, Crous & Schroers, Mycocitrus synnematus L. Zhao & Crous, Nectriopsis didymii L. Zhao & Crous, Sesquicillium intermediophialidicum L. Zhao & Crous, Sesquicillium neerlandicum L. Zhao & Crous, Sesquicillium symmetricum L. Zhao & Crous. New combinations: Mycocitrus coccicola (J.A. Stev.) L. Zhao & Crous, Mycocitrus coxeniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium essexcoheniae (Y.P. Tan et al.) L. Zhao & Crous, Sesquicillium lasiacidis (Samuels) L. Zhao, Crous & Schroers, Sesquicillium phyllophilum (Schroers) L. Zhao, Crous & Schroers, Sesquicillium rossmaniae (Schroers) L. Zhao, Crous & Schroers, Sesquicillium saulense (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium sesquicillii (Samuels) L. Zhao, Crous & Schroers, Sesquicillium spinulosisporum (Lechat & J. Fourn.) L. Zhao & Crous, Sesquicillium tornatum (Höhn.) Schroers. New synonyms: Clonostachys aranearum W.H. Chen et al., Clonostachys chuyangsinensis H. Yu & Y. Wang, Clonostachys eriocamporesiana R.H. Perera & K.D. Hyde, Clonostachys granuligera (Starbäck) Forin & Vizzini, Clonostachys indica Prasher & R. Chauhan, Clonostachys spinulosa R.H. Perera et al., Clonostachys squamuligera (Sacc.) Forin & Vizzini, Clonostachys wenpingii (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang. Epitypes (basionyms): Fusidium buxi J.C. Schmidt ex Link, Verticillium candelabrum Bonord. Citation: Zhao L, Groenewald JZ, Hernández-Restrepo M, Schroers H-J, Crous PW (2023). Revising Clonostachys and allied genera in Bionectriaceae. Studies in Mycology 105: 205-266. doi: 10.3114/sim.2023.105.03.

Cotton Verticillium wilt is mainly caused by the fungus Verticillium dahliae, which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of V. dahliae from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for V. dahliae. Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of V. dahliae, which was validated using several closely related Verticillium spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/μL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method\'s user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/μL genomic DNA of the V. dahliae, and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in V. dahliae and will contribute to the development of field-deployable diagnostic productions.

The soybean production area is expanding in Uzbekistan. Soybeans were planted on an area of 10 thsd ha and the harvest amounted to 30 thsd metric tons in 2023 (IPAD, https://ipad.fas.usda.gov/countrysummary). Macrophomina phaseolina (Mp) is a soil- and seed-borne fungal pathogen causing economically important diseases of legume crops (Pennerman et al. 2024). Drought stress and a warm climate are favorable to this pathogen (Irulappan et al. 2022). Under these conditions, its microsclerotia survive for a longer period and become more virulent (Chamorro et al. 2015). In August 2022, typical symptoms of charcoal rot were observed in about 25% of \"Orzu\" soybean cultivar affecting 6 ha located on the experimental base \"Durmon\" of our institute. Diseased plants displayed the following charcoal rot symptoms: leaves turn yellow, then wilt, die, and remain attached to the plant; the lower portion of the stem and tap root have a light gray or ashy black discoloration; tiny black specks on the lower stem and root; after splitting the stem, it has the appearance of fine charcoal powder. In order to determine the causal agent of these symptoms, a total of 17 diseased plants were collected from focal lesions in soybean plantings. From each plant, twelve sections of stem and root tissue were selected, cut into small 5-mm pieces, and surface sterilized with 1% sodium hypochlorite for four minutes, then rinsed three times with sterile distilled water. The disinfected tissues were dried on sterile filter paper for 5 min and placed on PDA Petri plates, which were incubated in an incubation chamber for 3 days (16 h light (26oC) and 8 h dark (18oC)). Fungi were subsequently subcultured on PDA and incubated for 7 days to obtain pure cultures. Six monohyphal colonies were purified. The colonies showed dense growth, with a gray initial mycelium becoming darker with aging. After 8 days on PDA, black-colored microsclerotia with spherical to oblong shapes were observed. On average, they measured 60 µm in width and 130 µm in length (n = 30). From six isolated monohyphal colonies, one has been chosen for molecular-genetic identification. Molecular-genetic analysis was conducted by amplification and sequencing of the ITS region with the ITS1 and ITS4 primers (White et al. 1990). The resulting sequence was deposited in the NCBI database under accession number OQ073450. After BLAST analysis (Altschul et al. 1990) it was 100% identical with the reference sequences of Mp (accession MT039671, MT039663 and MH496040) isolated in sugar beet, maize and sunflower, respectively, from Serbia. In order to verify the pathogenicity, soybean seedlings (cv. Orzu) were dipped into spore suspension (1 × 107 spores/ml) of sequenced strain R-17 for 1 minute and transferred to a 15 cm diameter plastic pot with 350 g of sterilized soil mix. After 25 days, the inoculated plants showed classic charcoal rot symptoms, while the control plants remained healthy. The pathogen was successfully reisolated from the infected seedlings onto PDA, fulfilling Koch\'s postulate. The identity of the re-isolated strain was confirmed by morphological features and sequencing of the ITS region. It should be noted that in Uzbekistan, Mp has not been documented in any plants. Therefore, according to our knowledge, this is the first report of this fungus affecting soybean plants in Uzbekistan. Since molecular-genetic analysis of the R-17 strain showed clustering with strains from Serbia, we speculate that there may have been a recent introduction of Mp from Serbia into Uzbekistan. This assumption is additionally confirmed by the fact that Serbia is the largest seed exporter in Uzbekistan. The increase in charcoal rot disease poses a major challenge to soybean production in Uzbekistan. Understanding the genetic diversity of Mp can be utilized to manage this disease, improve soybean yield, and help soybean breeding programs in Uzbekistan.

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Synthetic chemical pesticides are primarily used to manage plant pests and diseases, but their widespread and unregulated use has resulted in major health and environmental hazards. Using biocontrol microbes and their bioactive compounds is a safe and sustainable approach in plant protection. In this study, a furoic acid (FA) compound having strong antibacterial activity against soil-borne phytopathogenic bacterium Ralstonia solanacearum [causal agent of bacterial wilt (BW) disease] was isolated from Aspergillus niger and identified as 5-(hydroxymethyl)-2-furoic acid compound through spectroscopic analyses (liquid chromatography-mass spectrometry (MS), electron ionization MS, and NMR). The SEM study of bacterial cells indicated the severe morphological destructions by the FA compound. The FA was further evaluated to check its potential in enhancing host resistance and managing tomato BW disease in a greenhouse experiment and field tests. The results showed that FA significantly enhanced the expression of resistance-related genes (PAL, LOX, PR1, and PR2) in tomato and caused a significant reduction (11.2 log10 colony-forming units/g) of the R. solanacearum population in soil, resulting in the reduction of bacterial wilt disease severity on tomato plants and increase in plant length (58 ± 2.7 cm), plant biomass (28 ± 1.7 g), and root length (13 ± 1.2 cm). The findings of this study suggested that the fungus-derived FA compound can be a potential natural compound of biological source for the soil-borne BW disease in tomato.

Necrotrophic phytopathogens pose a serious challenge to the productivity of several crops causing seedling damage, pre- and post-emergence damping-off and root rot thus reducing plant growth and yield. They are known to gain nutrition by secreting a diverse array of hydrolytic enzymes and thereby causing extensive host plant tissue maceration. Amongst the diverse hydrolases, proteases play a pivotal role in the necrotrophic mode of nutrition and thereby in determining pathogenic virulence. Host plants often counteract the necrotrophic proteolysis events by proteins (peptides), particularly through protease inhibitors (PIs). PIs play an important role in host innate immunity function by functioning as anti-metabolic proteins inhibiting the activity of phytopathogenic secretory proteases. Their abundance in plant storage organs explains their anti-nutritional interaction which stalls pathogenic invasion. PIs, therefore, constitute potential candidates that can be deployed as effective antimicrobials in agriculture, particularly against necrotrophic soil-borne pathogens. The present review traces the progress made in the identification of PIs from plants, and their inhibitory potential against necrotrophic phytopathogens and explores prospects of utilizing these molecules as effective anti-necrotrophic formulations for disease management.

Rusty root rot is a severe disease in ginseng (Panax ginseng C. A. Mey) production caused by Ilyonectria robusta. The severity of the disease may be related to the residual ginsenosides in soil. In order to elucidate the response mechanism between Rg1 treatment and the occurrence of ginseng rust, we performed growth, reproduction and transcriptome analysis on treated Rg1. The results showed that Rg1 significantly promoted the mycelial growth and sporulation compared with the control, and aggravated the disease symptoms of Panax ginseng. A total of 6708 transcripts out of 213 131 annotated genes identified from global transcriptomic analysis were differentially expressed in Ilyonectria robusta grown during the Rg1 treatment. These genes were found to be related to the carbon-nitrogen metabolism, transport and assimilation. Many of these genes were also associated with pathogenicity based on the Phi-base database. Several transcription factors were related to specific biological processes, such as nitrogen utilization. The current results revealed that Rg1 played a major role in the development of rusty root rot by promoting fungal cell growth and affected the expression of genes required for pathogenesis. Rg1 could aggravate the invasion of Ilyonectria robusta on ginseng root, which preliminarily revealed the reason for the aggravation of rusty root rot in ginseng soil-borne.

Avocado root rot caused by the oomycete Phytophthora cinnamomi is a severe disease that affects avocado production in Mexico and worldwide. The use of biological control agents such as Trichoderma species isolated from places where the disease is always present, represents an efficient alternative to reduce losses. Thus, the objective of this research was to evaluate the biocontrol ability of 10 endophytic Trichoderma spp. strains against P. cinnamomi tested both in vitro and in the greenhouse. The endophytic Trichoderma spp. were recovered from Persea americana and Cinnamomum verum roots, isolated and purified on potato-dextrose-agar medium. Ten strains were identified by phylogenetic reconstruction of the internal transcribed spacer region of rDNA sequences as T. asperellum (T-AS1, T-AS2, T-AS6, and T-AS7), T. harzianum (T-H3, T-H4, and T-H5), T. hamatum (T-A12), T. koningiopsis (T-K8 and T-K11), and P. cinnamomi (CPO-PCU). In vitro dual-culture assay, the percentage of inhibition of radial growth (PIRG) between Trichoderma spp. and P. cinnamomi strains was measured according to the Bell\'s scale. PIRG results indicated that T-AS2 reached the highest value of 78.32%, and T-H5 reached the lowest value of 38.66%. In the greenhouse, the infection was evaluated according to the percentage of disease incidence. Plants with the lowest incidence of dead by avocado root rot were those whose seedlings were inoculated with T-AS2 and T-AS7, resulting in only 5% death by root rot caused by P. cinnamomi. The disease incidence of seedlings with wilt symptoms and death decreased more than 50% in the presence of Trichoderma spp. Relying on the results, we conclude that T. asperellum and T. harzianum contribute to the biocontrol of soil-borne pathogenic oomycete P. cinnamomi.

The soil-borne, plant-pathogenic Ralstonia solanacearum strain OE1-1 produces and secretes methyl 3-hydroxymyristate (3-OH MAME) as a quorum sensing (QS) signal, which contributes to its virulence. A global virulence regulator, PhcA, functioning through the QS system, positively regulates the expression of ralA, which encodes furanone synthase, to produce aryl-furanone secondary metabolites, ralfuranones. A ralfuranone-deficient mutant (ΔralA) is weakly virulent when directly inoculated into tomato xylem vessels. To investigate the functions of ralfuranones, we analysed R. solanacearum transcriptome data generated by RNA sequencing technology. ΔralA expressed phcB, which is associated with 3-OH MAME production, and phcA at levels similar to those in strain OE1-1. In addition, ΔralA exhibited down-regulated expression of more than 90% of the QS positively regulated genes, and up-regulated expression of more than 75% of the QS negatively regulated genes. These results suggest that ralfuranones affect the QS feedback loop. Ralfuranone supplementation restored the ability of ΔralA cells to aggregate. In addition, ralfuranones A and B restored the swimming motility of ΔralA to wild-type levels. However, the application of exogenous ralfuranones did not affect the production of the major exopolysaccharide, EPS I, in ΔralA. Quantitative real-time polymerase chain reaction assays revealed that the deletion of ralA results in the down-regulated expression of vsrAD and vsrBC, which encode a sensor kinase and a response regulator, respectively, in the two-component regulatory systems that influence EPS I production. The application of ralfuranone B restored the expression of these two genes. Overall, our findings indicate that integrated signalling via ralfuranones influences the QS and virulence of R. solanacearum.