Advances in submerged liquid fermentation and formulation of entomopathogenic fungi.

Entomopathogenic fungi (EPF) can be defined as beneficial multifunctional eukaryotic microorganisms that display pivotal ecological services in pest management, with some species possessing the special ability to establish mutualistic relationships with plants. Mass production of these fungi is critical to support affordable widespread commercialization and worldwide field application. Among the mass production methods explored mainly by industry, submerged liquid fermentation is a robust and versatile technology that allows the formation of different types of propagules designated for various applications in pest control. Many hypocrealean EPF are easily culturable on artificial substrates by producing single-celled structures (hyphal bodies, blastospores, and submerged conidia) or multicellular structures (mycelium and microsclerotia). Less frequently, some EPF may form environmentally resistant chlamydospores, but these structures have almost always been overlooked. A continued research pipeline encompassing screening fungal strains, media optimization, and proper formulation techniques aligned with the understanding of molecular cues involved in the formation and storage stability of these propagules is imperative to unlock the full potential and to fine-tune the development of robust and effective biocontrol agents against arthropod pests and vectors of diseases. Finally, we envision a bright future for the submerged liquid fermentation technology to supplement or replace the traditional solid substrate fermentation method for the mass production of many important EPF. KEY POINTS: • Submerged liquid fermentation (SLF) allows precise control of nutritional and environmental factors • SLF provides a scalable, robust, and cost-effective platform for mycopesticide production • Enhancing formulation, shelf life, and field efficacy of submerged propagules remain crucial • Understanding the molecular mechanisms behind submerged propagule formation is key to advancing SLF technology.

Transcription factors containing both C2H2 and homeobox domains play different roles in Verticillium dahliae.

Verticillium dahliae causes Verticillium wilt in more than 200 plant species worldwide. As a soilborne fungus, it forms melanized microsclerotia and colonizes the xylem of host plants. Our previous study revealed a subfamily of C2H2-homeobox transcription factors in V. dahliae, but their biological roles remain unknown. In this study, we systematically characterized the functions of seven C2H2-homeobox transcription factors in V. dahliae. Deletion of VdChtf3 and VdChtf6 significantly decreased the production of melanized microsclerotia, and knockout of VdChtf1 and VdChtf4 enhanced virulence. Loss of VdChtf2 and VdChtf6 increased conidium production, whereas loss of VdChtf5 and VdChtf7 did not affect growth, conidiation, microsclerotial formation, or virulence. Further research showed that VdChtf3 activated the expression of genes encoding pectic enzymes to participate in microsclerotial formation. In addition, VdChtf4 reduced the expression of VdSOD1 to disturb the scavenging of superoxide radicals but induced the expression of genes related to cell wall synthesis to maintain cell wall integrity. These findings highlight the diverse roles of different members of the C2H2-homeobox gene family in V. dahliae. IMPORTANCE: Verticillium dahliae is a soilborne fungus that causes plant wilt and can infect a variety of economic crops and woody trees. The molecular basis of microsclerotial formation and infection by this fungus remains to be further studied. In this study, we analyzed the functions of seven C2H2-homobox transcription factors. Notably, VdChtf3 and VdChtf4 exhibited the most severe defects, affecting phenotypes associated with critical developmental stages in the V. dahliae disease cycle. Our results indicate that VdChtf3 is a potential specific regulator of microsclerotial formation, modulating the expression of pectinase-encoding genes. This finding could contribute to a better understanding of microsclerotial development in V. dahliae. Moreover, VdChtf4 was associated with cell wall integrity, reactive oxygen species (ROS) stress resistance, and increased virulence. These discoveries shed light on the biological significance of C2H2-homeobox transcription factors in V. dahliae's adaptation to the environment and infection of host plants.

Microsclerotia from Metarhizium robertsii: Production, ultrastructural analysis, robustness, and insecticidal activity.

Microsclerotia (MS) are considered one of the most promising propagules for use as active ingredients in biopesticides due to their tolerance to abiotic factors and ability to produce infective conidia for the control of pests. Therefore, the objective of this research was to establish the conditions required to induce the formation of microsclerotia in Metarhizium robertsii Mt004 and to study its development process, tolerance to abiotic factors and insecticidal activity of MS-derived conidia. M. robertsii started to form hyphal aggregates after 2 days and looked more compact after 8 days. MS were mature and pigmented after 20 days. The final yield was 2.0 × 103 MS/mL and MS size varied between 356.9 and 1348.4 µm. Ultrastructure analysis revealed that mature MS contained only a few live cells embedded in an extracellular matrix. Mature MS were more tolerance to UV-B radiation, heat and storage trials than conidia from Solid State Fermentation. MS-derived conidia were as virulent as conidia against Diatraea saccharalis larvae. These results showed that MS are promising propagules for the development of more persistent and efficient biopesticides for harsh environmental conditions. Our findings provide a baseline for production and a better understanding of microsclerotia development in M. robertsii strains.

Ca2+ affects the hyphal differentiation to sclerotia formation of Athelia rolfsii.

RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+-chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2-ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.

The role of VdSti1 in Verticillium dahliae: insights into pathogenicity and stress responses.

Sti1/Hop, a stress-induced co-chaperone protein, serves as a crucial link between Hsp70 and Hsp90 during cellular stress responses. Despite its importance in stress defense mechanisms, the biological role of Sti1 in Verticillium dahliae, a destructive fungal pathogen, remains largely unexplored. This study focused on identifying and characterizing Sti1 homologues in V. dahliae by comparing them to those found in Saccharomyces cerevisiae. The results indicated that the VdSti1-deficient mutant displayed increased sensitivity to drugs targeting the ergosterol synthesis pathway, leading to a notable inhibition of ergosterol biosynthesis. Moreover, the mutant exhibited reduced production of microsclerotia and melanin, accompanied by decreased expression of microsclerotia and melanin-related genes VDH1, Vayg1, and VaflM. Additionally, the mutant's conidia showed more severe damage under heat shock conditions and displayed growth defects under various stressors such as temperature, SDS, and CR stress, as well as increased sensitivity to H2O2, while osmotic stress did not impact its growth. Importantly, the VdSti1-deficient mutant demonstrated significantly diminished pathogenicity compared to the wild-type strain. This study sheds light on the functional conservation and divergence of Sti1 homologues in fungal biology and underscores the critical role of VdSti1 in microsclerotia development, stress response, and pathogenicity of V. dahliae.

First report of Macrophomina phaseolina causing сharcoal rot in soybean (Glycine max (L.) Merr.) plants in Uzbekistan.

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.

Verticillium dahliae VdPBP1 Transcription Factor Is Required for Hyphal Growth, Virulence, and Microsclerotia Formation.

Verticillium dahliae, a fungal pathogen that affects more than 200 plant species, including tomatoes, requires specific proteins for its early steps in plant infection. One such crucial protein, VdPBP1, exhibits high expression in the presence of tomato roots. Its 313-amino acid C-terminal section restores adhesion in nonadhesive Saccharomyces cerevisiae strains. To uncover its role, we employed a combination of bioinformatics, genetics, and morphological analyses. Our findings underscore the importance of VdPBP1 in fungal growth and pathogenesis. Bioinformatic analysis revealed that the VdPBP1 gene consists of four exons and three introns, encoding a 952-codon reading frame. The protein features a 9aaTAD domain, LsmAD, and PAB1 DNA-binding sites, as well as potential nuclear localization and transmembrane helix signals. Notably, the deletion of a 1.1 kb fragment at the gene's third end impedes microsclerotia formation and reduces pathogenicity. Mutants exhibit reduced growth and slower aerial mycelial development compared to the wild type. The VdPBP1 deletion strain does not induce disease symptoms in tomato plants. Furthermore, VdPBP1 deletion correlates with downregulated microsclerotia formation-related genes, and promoter analysis reveals regulatory elements, including sites for Rfx1, Mig1, and Ste12 proteins. Understanding the regulation and target genes of VdPBP1 holds promise for managing Verticillium wilt disease and related fungal pathogens.

Kss1 of Verticillium dahliae regulates virulence, microsclerotia formation, and nitrogen metabolism.

Verticillium dahliae causes destructive vascular wilt diseases on more than 200 plant species, including economically important crops and ornamental trees worldwide. The melanized microsclerotia (MS) enable V. dahliae to survive for years in soil, thus the fungus is especially difficult to control once it has become established. Previously, we found that the mitogen activated protein kinase VdSte11 (MAPKKK) plays key roles in MS formation, penetration, and virulence in V. dahliae. In this study, two MAPK homologs of the yeast Ste7p and Kss1p were identified and characterized in V. dahliae. Deletion of VdSte7 or VdKss1 reuslted in severe defects in melaninized MS formation and virulence. Furthermore, phosphorylation assays demonstrated that VdSte11 and VdSte7 can phosphorylate VdKss1 in V. dahliae. Proteomic analysis revealed a significant change in sterol biosynthesis with a fold change of ≥ 1.2 after the deletion of VdKss1. In addition, phosphoproteomic analysis showed that VdKss1 was involved in the regulation of nitrogen metabolism. Finally, we identified VdRlm1 as a potentially downstream target of VdKss1, which is involved in regulating ammonium nitrogen utilization. This study sheds light on the network of regulatory proteins in V. dahliae that affect MS formation and nitrogen metabolism.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Multifunctional regulation of NADPH oxidase in growth, microsclerotia formation and virulence in Metarhizium rileyi.

OBJECTIVES: Microsclerotia (MS), anti-stress structures produced by many filamentous fungi, have been proven to be a great substitute for conidia in the production of insecticides within entomogenous fungi. NADPH oxidase (Nox) is a highly conserved ROS-response protein family that is widespread in eukaryotes and plays distinct roles in environmental fitness among various filamentous fungi. However, it is not clear whether the formation of MS and pathogenicity in entomogenous fungi is regulated by the Nox inside. In this study, we reported the presence of NADPH oxidase homologs in a great potential biocontrol fungus, Metarhizium rileyi, and further showed multiple biological functions. RESULTS: Three Nox homologous genes in M. rileyi showed high expression throughout the entire process of MS formation. Targeted deletion of MrNoxA, MrNoxB and MrNoxR all led to a decrease in MS yield and impaired morphology. Moreover, the anti-adversity assay showed that they are indispensable for growth, osmotic pressure and oxidative stress regulation in Metarhizium rileyi. Most importantly, △MrNoxR and △MrNoxA but not △MrNoxB showed a dramatic reduction in virulence via inoculation. The normality of appressoria might be unaffected in mutants since there are no striking differences in virulence compared with WT by topical injections. CONCLUSION: Our results revealed that NADPH oxidase plays important roles in growth regulation, MS formation and pathogenicity in M. rileyi, perhaps in the ROS response and hyphal polarity.

First Report of Root Rot Caused by Macrophomina phaseolina in Peanut Shandong Province, China.

Peanut (Arachis hypogaea L.) is a widely grown oilseed crop of great agricultural importance worldwide. In July 2022, disease symptoms were observed on peanut roots in Laixi (36º85' N, 120º54' E), Shandong Province, China. About 25% of the plants showed various symptoms, including stem and root rot and blackening, microsclerotia on the stem, yellowing and wilting of leaves, and even death. Twenty diseased plants were collected to confirm the pathogen. Symptomatic roots were cut into small pieces, disinfested with 75% ethanol for 1 min and 0.5% NaClO for 2 min, rinsed three times with sterile water, dried on sterile filter paper, and then spread on potato dextrose agar (PDA) supplemented with 100 µg/mL chloramphenicol and incubated at 25°C in the dark. At the beginning of growth, the fungus formed sparse, white mycelia, which white, then darkened with age and microsclerotia were formed in the medium after 5 days. The mycelium aggregated into black, round to oblong or irregularly shaped microsclerotia 84 to 163 µm long and 54 to 125 µm wide (n=40). These morphological characteristics were consistent with the description of Macrophomina phaseolina (Holliday and Punithalingam, 1970). Molecular identification was performed by sequencing the internal transcribed spacer (ITS) region with ITS1 and ITS4 and translation elongation factor 1-alpha (TEF) with EF1-728F/EF1-986R (Glass and Donaldson 1995) of a representative isolate SXY183. ITS (OR056369) and TEF (OR098356) of SXY183 showed 100% and 97.74% similarity with M. phaseolina (KF951622, KF951997), respectively. Phylogenetic analysis was performed using Neighbor-Joining (NJ) analysis based on the gene sequences of ITS and TEF. The fungus was identified as M. phaseolina based on molecular analysis and morphological characteristics. The pathogenicity of a representative isolate (SXY183) was tested on peanuts under greenhouse conditions. Two-week-old peanut (Huayu No. 9115) seedlings were inoculated with a mycelial plug (8 mm diameter) at the root base of each plant and cultured in a greenhouse (30°C during the day and 25°C at night, a 12-h photoperiod, and 80% RH). Ten plants were inoculated with a plug of non-colonized PDA as a control. Brown lesions were observed on the stem and root of all inoculated seedlings 7 days after inoculation, but not on the control plants. The experiment was repeated three times. M. phaseolina was re-isolated from the symptomatic root and confirmed based on morphological characteristics and DNA sequence analysis of ITS and TEF. M. phaseolina is a soil-borne fungus that is distributed worldwide and has a broad host range. Disease agent has previously been reported on several host plants such as adzuki bean, faba bean, watermelon, Plukenetia volubilis, Atractylodes lancea and Curcuma longa in China (Cai et al., 2020; Sun et al. 2016; Sun et al., 2019; Sun et al., 2020; Wang et al., 2020; Wu et al., 2022). However, this is the first report in which M. phaseolina was found to cause peanut root rot in Shandong Province, China. Our report will provide important information for studying the epidemiology and management of this disease.

The Frq-Frh Complex Light-Dependently Delays Sfl1-Induced Microsclerotia Formation in Verticillium dahliae.

The vascular plant pathogenic fungus Verticillium dahliae has to adapt to environmental changes outside and inside its host. V. dahliae harbors homologs of Neurospora crassa clock genes. The molecular functions and interactions of Frequency (Frq) and Frq-interacting RNA helicase (Frh) in controlling conidia or microsclerotia development were investigated in V. dahliae JR2. Fungal mutant strains carrying clock gene deletions, an FRH point mutation, or GFP gene fusions were analyzed on transcript, protein, and phenotypic levels as well as in pathogenicity assays on tomato plants. Our results support that the Frq-Frh complex is formed and that it promotes conidiation, but also that it suppresses and therefore delays V. dahliae microsclerotia formation in response to light. We investigated a possible link between the negative element Frq and positive regulator Suppressor of flocculation 1 (Sfl1) in microsclerotia formation to elucidate the regulatory molecular mechanism. Both Frq and Sfl1 are mainly present during the onset of microsclerotia formation with decreasing protein levels during further development. Induction of microsclerotia formation requires Sfl1 and can be delayed at early time points in the light through the Frq-Frh complex. Gaining further molecular knowledge on V. dahliae development will improve control of fungal growth and Verticillium wilt disease.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Stevia rebaudiana in Arizona, USA.

Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ, USA (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Five symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile water, and plated onto potato dextrose agar (PDA). All the five isolates displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width × 114 µm length (n=30). For molecular identification, genomic DNA was extracted from mycelia and microsclerotia of a representative isolate (Yuma) using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), translation elongation factor-1α (TEF-1α), calmodulin (CAL), and ß-tubulin (ß-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O'Donnell and Cigelink 1997), respectively. A BLAST search of sequences revealed 98.7 to 100% identity to Macrophomina phaseolina sequences (MK757624, KT261797, MK447823, MK447918). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina (Holliday and Punithaligam 1970). Sequences were submitted in the GenBank under accession numbers OP599770 (ITS), OP690156 (TEF-1α), OP612814 (CAL), and OP690157 (ß-TUB). Pathogenicity assay was performed on 9-week-old stevia plants (var. SW2267), grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth at 28°C. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty healthy plants were inoculated by soil drenching 50 ml of the inoculum per pot. Soil drenching using sterile distilled water was done on 5 non-inoculated control plants. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on all 20 inoculated plants, whereas all the 5 control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequences of ITS, TEF-1α, CAL and ß-TUB regions. Although M. phaseolina has been reported earlier on stevia in NC, USA (Koehler and Shew 2018), this is a first report from AZ, USA. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in AZ, USA in coming years.

VdTps2 Modulates Plant Colonization and Symptom Development in Verticillium dahliae.

The trehalose biosynthesis pathway is a potential target for antifungal drugs development. Trehalose phosphate synthase (TPS) and phosphatase are widely conserved components of trehalose biosynthesis in fungi. However, the role of trehalose biosynthesis in the vascular plant-pathogenic fungus Verticillium dahliae remains unclear. Here, we investigated the functions of the TPS complex, including VdTps1, VdTps2, and VdTps3 in V. dahliae. Unlike VdTps2, deletion of VdTps1 or VdTps3 did not alter any phenotypes compared with the wild-type strain. In contrast, the ΔVdTps2 strain showed severely depressed radial growth due to the abnormal swelling of the hyphal tips. Further, deletion of VdTps2 increased microsclerotia formation, melanin biosynthesis, and resistance to cell-wall perturbation and high-temperature stress. Virulence assays and quantification of fungal biomass revealed that deletion of VdTps2 delayed disease symptom development, as evident by the reduced virulence and decreased biomass of the ΔVdTps2 strain in plant stem tissue following inoculation. Additionally, increases in penetration peg formation observed in the ΔVdTps2 strain in the presence of H2O2 suggested that VdTps2 suppresses initial colonization. Our results also revealed the role of VdTps2 as a regulator of autophagy. Together, these results indicate that VdTps2 contributes to plant colonization and disease development. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Verticillium Wilt Caused by Verticillium dahliae in Grafted Tomato in Taiwan.

Grafted tomato (Solanum lycopersicum L.) is widely used to manage soil-borne diseases (Lee et.al 2010). In Taiwan, grafting on eggplant (S. melongena L.) rootstock have been extensively used to reduce bacterial wilt in tomato production. In July 2019, wilting plants were found at a cherry tomato farm (~ 0.5 ha) located at Miaoli County. About 10% tomatoes of cv. 'Mint Shine' grafted onto the eggplant rootstock displayed wilt symptoms. Numerous leaflets with chlorosis, inter-vein yellowing, V-shaped necrotic lesions and withered leaves were observed on the affected plants. Some plants eventually wilted and died. A cut at the grafting site revealed the vascular discolorations on both scion (tomato) and rootstock (eggplant). A fungus with a compact whitish colony was consistently isolated from the symptomatic vascular tissue by using acidified potato dextrose agar (PDA) plates. Two isolates, Ve2 from eggplant and Ve4 from tomato, grown on PDA plates were characterized. Both Ve2 and Ve4 grow slowly (ca. 2.6 mm/day at 28 oC) and shared almost identical cultural and morphological characteristics. They first showed whitish mycelium and cream color in reverse within 1 week. Later, numerous microsclerotia developed evenly over the colony and the reverse color turned dark black. Microscopic observations revealed hyaline hyphae with black, elongated, irregularly spherical microsclerotia measuring 31.3 to 71.5 × 16.8 to 49.0 µm (average 50.4 × 28.5 µm) on a 3-week-old PDA culture. Abundant hyaline, single-celled, ellipsoida conidia measuring 2.7 to 4.7 × 0.9 to 3.2 µm (average 3.7 × 1.9 µm) and verticillate conidiophores were observed. The fungus was identified as Verticilium dahliae based on the consistent morphological characteristics (Hawksworth et. al 1970). To confirm the identity, the internal transcribed spacer regions of ribosomal DNA, amplified by PCR with universal primers ITS4/ITS5 (White et.al 1990), were sequenced. Both strains shared the same sequences (GenBank MZ734460; MZ736637), and BLASTn searching was 100% identical to many records of V. dahliae including an ex-epitype CBS130341. Pathogenicity was tested on 3-week-old seedlings of tomato cv. 'Bonny best' and eggplant cv. 'Longship' by a root dip method (Bhat & Subbarao 2007). Eighteen plants arranged into three replications were inoculated for each host-isolate combination, and incubated in the greenhouse at 25±3℃. The pathogenicity test was repeated two times, with the result that both isolates were pathogenic to tomato and eggplant. Both isolates induced wilt symptoms in all inoculated plants within 14 days post-inoculation (DPI). Severe leaf drop, wilting and vascular discoloration in all inoculated eggplant whereas slight yellowing and mildly stunt growth in tomato were observed at 21 DPI. Koch's postulates were fulfilled by re-isolating the same fungus from both infected tomato and eggplant. All uninoculated plants remained health and no V. dahliae was isolated from them. To our knowledge, this is the first report of V. dahliae and Verticilium wilt of grafted tomato caused by this pathogen in Taiwan. This pathogen affects over 400 plant species and has resulted in significant economic losses in many regions of world (Subbarao 2020). It is important to investigate the distribution and extent of damage caused by this emerging pathogen on Solanaceous or other crops.

VdGAL4 Modulates Microsclerotium Formation, Conidial Morphology, and Germination To Promote Virulence in Verticillium dahliae.

Verticillium dahliae Kleb is a typical soilborne pathogen that can cause vascular wilt disease on more than 400 plants. Functional analysis of genes related to the growth and virulence is crucial to revealing the molecular mechanism of the pathogenicity of V. dahliae. Glycosidase hydrolases can hydrolyze the glycosidic bond, and some can cause host plant immune response to V. dahliae. Here, we reported a functional validation of VdGAL4 as an α-galactosidase that belongs to glycoside hydrolase family 27. VdGAL4 could cause plant cell death, and its signal peptide plays an important role in cellular immune response. VdGAL4-triggered cell death depends on BAK1 and SOBIR1 in Nicotiana benthamiana. In V. dahliae, the function of VdGAL4 in mycelial growth, conidia, microsclerotium, and pathogenicity was studied by constructing VdGAL4 deletion and complementation mutants. Results showed that the deletion of VdGAL4 reduced the conidial yield and conidial germination rate of V. dahliae and changed the microscopic morphology of conidia; the mycelia were arranged more disorderly and were unable to produce microsclerotium. The VdGAL4 deletion mutants exhibited reduced utilization of different carbon sources, such as raffinose and sucrose. The VdGAL4 deletion mutants were also more sensitive to abiotic stress agents of SDS, sorbitol, low-temperature stress of 16°C, and high-temperature stress of 45°C. In addition, the VdGAL4 deletion mutants lost the ability to penetrate cellophane and its mycelium were disorderly arranged. Remarkably, VdGAL4 deletion mutants exhibited reduced pathogenicity of V. dahliae. These results showed that VdGAL4 played a critical role in the pathogenicity of V. dahliae by regulating mycelial growth, conidial morphology, and the formation of microsclerotium. IMPORTANCE This study showed that α-galactosidase VdGAL4 of V. dahliae could activate plant immune response and plays an important role in conidial morphology and yield, formation of microsclerotia, and mycelial penetration. VdGAL4 deletion mutants significantly reduced the pathogenicity of V. dahliae. These findings deepened the understanding of pathogenic virulence factors and how the mechanism of pathogenic fungi infected the host, which may help to seek new strategies for effective control of plant diseases caused by pathogenic fungi.

The Phosphatase VdPtc3 Regulates Virulence in Verticillium dahliae by Interacting with VdAtg1.

Type 2C protein phosphatases regulate various biological processes in eukaryotes. However, their functions in Verticillium dahliae have not been characterized. In this study, homologs VdPtc1, VdPtc3, VdPtc5, VdPtc6, and VdPtc7 were identified in V. dahliae on the basis of homologous comparison with those in Saccharomyces cerevisiae. VdPtc2 and VdPtc4 are missing in the genome of the V. dahliae XJ592 strain. VdPtc3 is the homolog of Ptc2, Ptc3, and Ptc4 proteins in S. cerevisiae, implying that VdPtc3 may play versatile functions in V. dahliae. VdPtc3 promoted conidium development, melanin, and microsclerotium formation in V. dahliae. The ΔVdPtc3 strains showed increased sensitivity to NaCl and sorbitol and augmented the phosphorylation of p38 mitogen-activated protein kinase homolog Hog1 induced by osmotic stress. Besides, the ΔVdPtc3 strains also showed milder Verticillium wilt symptom on cotton. Furthermore, VdPtc3 interacts with VdAtg1, which modulates melanin and microsclerotium formation, as well as pathogenicity.

Antifungal effects of volatile organic compounds produced by Trichoderma koningiopsis T2 against Verticillium dahliae.

Volatile organic compounds (VOCs) produced by microorganisms are considered promising environmental-safety fumigants for controlling soil-borne diseases. Verticillium dahliae, a notorious fungal pathogen, causes economically important wilt diseases in agriculture and forestry industries. Here, we determined the antifungal activity of VOCs produced by Trichoderma koningiopsis T2. The VOCs from T. koningiopsis T2 were trapped by solid-phase microextraction (SPME) and tentatively identified through gas chromatography-mass spectrometry (GC/MS). The microsclerotia formation, cell wall-degrading enzymes and melanin synthesis of V. dahliae exposed to the VOC mixtures and selected single standards were examined. The results showed that the VOCs produced by strain T2 significantly inhibited the growth of V. dahliae mycelium and reduced the severity of Verticillium wilt in tobacco and cotton. Six individual compounds were identified in the volatilome of T. koningiopsis T2, and the dominant compounds were 3-octanone, 3-methyl-1-butanol, butanoic acid ethyl ester and 2-hexyl-furan. The VOCs of strain T2 exert a significant inhibitory effect on microsclerotia formation and decreased the activities of pectin lyase and endo-ß-1,4-glucanase in V. dahliae. VOCs also downregulated the VdT3HR, VdT4HR, and VdSCD genes related to melanin synthesis by 29. 41-, 10. 49-, and 3.11-fold, respectively. Therefore, T. koningiopsis T2 has potential as a promising biofumigant for the biocontrol of Verticillium wilt disease.

Effects of Volatile Organic Compounds Produced by Pseudomonas aurantiaca ST-TJ4 against Verticillium dahliae.

Verticillium dahliae is one of the most destructive fungal pathogens, causing substantial economic losses in agriculture and forestry. The use of plant growth-promoting rhizobacteria (PGPR) is an effective and environmentally friendly strategy for controlling diseases caused by V. dahliae. In this study, 90 mm in diameter Petri plates were used to test the effect of volatile organic compounds (VOCs) produced by different concentrations of Pseudomonasaurantiaca ST-TJ4 cells suspension on V. dahliae mycelia radial growth and biomass. The mycelial morphology was observed by using scanning electron microscopy. The conidia germination and microsclerotia formation of V. dahliae were evaluated. The VOCs with antifungal activity were collected by headspace solid-phase microextraction (SPME), and their components were analyzed by gas chromatography-mass spectrometry (GC-MS). The VOCs produced by strain ST-TJ4 significantly inhibited the growth of mycelium of V. dahliae. The morphology of the hyphae was rough and wrinkled when exposed to VOCs. The VOCs of strain ST-TJ4 have a significant inhibitory effect on V. dahliae conidia germination and microsclerotia formation. At the same time, the VOCs also reduce the expression of genes related to melanin synthesis in V. dahliae. In particular, the expression of the hydrophobin gene (VDAG-02273) was down-regulated the most, about 67-fold. The VOCs effectively alleviate the severity of cotton root disease. In the volatile profile of strain ST-TJ4, 2-undecanone and 1-nonanol assayed in the range 10-200 µL per plate revealed a significant inhibitory effect on V. dahliae mycelial radial growth. These compounds may be useful to devise new control strategies for control of Verticillium wilt disease caused by V. dahliae.

Production of Purpureocillium lilacinum and Pochonia chlamydosporia by Submerged Liquid Fermentation and Bioactivity against Tetranychus urticae and Heterodera glycines through Seed Inoculation.

Pochoniachlamydosporia and Purpureocilliumlilacinum are fungal bioagents used for the sustainable management of plant parasitic nematodes. However, their production through submerged liquid fermentation and their use in seed treatment have been underexplored. Therefore, our goal was to assess the effect of different liquid media on the growth of 40 isolates of P. lilacinum and two of P. chlamydosporia. The most promising isolates tested were assessed for plant growth promotion and the control of the two-spotted spider mite (Tetranychus urticae) and the soybean cyst nematode (Heterodera glycines). Most isolates produced > 108 blastospores mL−1 and some isolates produced more than 104 microsclerotia mL−1. Microsclerotia of selected isolates were used to inoculate common bean (Phaseolus vulgaris L.) seeds in greenhouse trials. All fungal isolates reduced the T. urticae fecundity in inoculated plants through seed treatment, while P. chlamydosporia ESALQ5406 and P. lilacinum ESALQ2593 decreased cyst nematode population. Purpureocillium lilacinum was more frequently detected in soil, whereas P. chlamydosporia colonized all plant parts. Pochonia chlamydosporia ESALQ5406 improved the root development of bean plants. These findings demonstrate the possibility of producing submerged propagules of P. chlamydosporia and P. lilacinum by liquid culture, and greenhouse trials support the applicability of fungal microsclerotia in seed treatment to control P. vulgaris pests.