Mycelial Growth Research Articles

Isolation, antioxidant and antifungal activity of furanocoumarins from the leaves of Heracleum vicinum Boiss.

In this work, coumarin compounds were extracted by ultrasonic-microwave synergistic extraction from the leaves of Heracleum vicinum Boiss.. The five furanocoumarins—pimpinellin, isopimpinellin, bergapten, xanthotoxin, and imperatorin—that were isolated from the leaves of Heracleum vicinum Boiss. were quantified using HPLC and GC, and their quantities in the aboveground sections was determined. The antioxidant activities of five furanocoumarins were assessed with ABTS radicals and DPPH radicals, with the best scavenging effects of xanthotoxin and bergapten, with half maximal inhibitory concentration (IC50) of xanthotoxin for ABTS radicals and DPPH radicals of 1.85 mg/mL and 3.95 mg/mL, respectively, and bergapten for ABTS radicals and DPPH radicals IC50 of 1.68 mg/mL and 4.61 mg/mL, respectively. The antifungal activities of five furanocoumarins against trichophyton rubrum were evaluated. The minimum inhibitory concentration (MIC) of xanthotoxin (200 μg/mL) and imperatorin (12.5 μg/mL) showed the highest inhibitory effects against trichophyton rubrum. The inhibitory mechanism of xanthotoxin was discussed. When trichophyton rubrum was treated with 200 μg/mL of xanthotoxin, the spore germination rate was 34.92%, the nucleic acid leakage was 52.22%, the inhibition of radial growth of mycelium could reach 87.87%, and the content of ergosterol was reduced by 37.37%. It indicates that xanthotoxin was found to be able to damage mycelium shape, prevent spore germination and mycelium growth, kill mycelium cells and release intracellular materials, and obstruct the production of ergosterol. These results indicate that furanocoumarin is a potent active component that has an important role in Heracleum vicinum Boiss. leaves.

Study on Enzyme Activity and Metabolomics during Culture of Liquid Spawn of Floccularia luteovirens.

To comprehensively investigate the physiological characteristics and metabolic processes of the mycelium of Floccularia luteovirens (F. luteovirens), a wild edible fungus unique to the plateau region, we conducted an in-depth analysis of the mycelium enzyme activity and metabolites during different culture periods. The activity of seven enzymes all followed a trend of initially increasing and then decreasing. The intra- and extracellular activity peaks of three hydrolases-amylase, protease, and cellulase-all occurred on the 20th day, except for the extracellular amylase, which peaked on the 15th day. In contrast, the peak activity of laccase occurred on the 10th day. Moreover, three types of oxidoreductases in the mycelium (catalase (CAT), superoxide dismutase (SOD), and 2,3,5-triphenyltetrazolium chloride (TTC)-dehydrogenase (TTC-DH)) also exhibited significant changes in activity. CAT and SOD activity reached their maximum on the 20th day, whereas TTC-DH showed high activity on both the 10th and 20th days. Through a comprehensive assessment of the evolving trends of these physiological parameters, we determined that the optimal cultivation cycle for F. luteovirens liquid spawn is 20 days. An untargeted metabolomic analysis revealed that 3569 metabolites were detected in the F. luteovirens mycelium, including a variety of secondary metabolites and functional components, with terpenoids being particularly abundant, accounting for 148 types. By comparing three different culture stages (10 days, 20 days, and 30 days), 299, 291, and 381 metabolites, respectively, showed different accumulation patterns in the comparison groups of 10d vs. 20d, 20d vs. 30d, and 10d vs. 30d. These differential metabolites were primarily concentrated in carboxylic acids and their derivatives, fatty acyl groups, organic oxygen compounds, and lipid compounds. In addition, there were several amino acids whose abundance continued to grow during culturing. The metabolism of amino acids greatly affects mycelium growth and development. This research delineates the interplay between mycelium growth and metabolism, offering empirical support for a cultivation strategy for liquid F. luteovirens, and an exploration of its metabolites for potential applications.

First Report of Basal Stem Rot of Achnatherum inebrians Caused by Fusarium pseudograminearum in China.

Drunken horse grass (Achnatherum inebrians) is a perennial bunchgrass that is widely distributed in arid and semi-arid grasslands in northwest China (Zhang et al., 2021). In July 2023, Basal stem rot was found in artificially grown drunken horse grass plots in Yuzhong County (35.85° N, 104.12° E), Gansu Province, China, with an average incidence of 5.2%. Diseased plants showed crown and basal stem rot with chocolate brown discoloration at the base of the stem and slight constriction of some basal stems. Five field's foci were surveyed and at least 6 basal stems per focus were collected. Infected basal stems were surface-sterilized (75% ethanol for 30 s and 1% NaClO for 90 s), rinsed three times with sterilized water, placed on potato dextrose agar (PDA), and incubated at 22°C in the dark for 3 days. Isolates were purified by single spore cultures (Leslie and Summerell, 2006). The average mycelial growth rate was 4.8 to 7.5 mm/day at 25°C on PDA, and the colonies produced aerial mycelium varying from rose to yellow white, and rose to burgundy pigment diffused into the agar. Macroconidia of the isolates were produced on carnation leaf agar (CLA) incubated under black light and observed to be abundant, but no microconidia were found. Macroconidia were relatively slender, curved to almost straight, commonly 3-6 septate, averaging 30.1 × 3.8 μm (n=50). The morphological characteristics of this fungus fully fit the description of F. pseudograminearum (Aoki and O'Donnell, 1999). To obtain the phylogenetic support, DNA of three representative isolates YZ-Y-1, YZ-Y-2 and YZ-Y-3 was extracted by using an HP Fungal DNA Kit (D3195), and a portion of the RNA polymerase II second largest subunit (RPB2) gene and elongation factor 1 alpha (EF1-α) gene were amplified using primers RPB2-5f2 and RPB2-7cr (O'Donnell et al. 2010) and EF1 and EF2 (O'Donnell et al. 1998), respectively. Results of sequences were deposited in GenBank (accession nos. PP179044 to PP179049). A nucleotide BLAST search revealed RPB2 and EF1-α sequences to be 99.8 and 100% similar to the corresponding sequences of the ex-type strain NRRL 28062 F. pseudograminearum accessions numbers MW233433 and MW233090, respectively. For pathogenicity tests, 15 μl of conidia suspension (1×106 conidia/ml) was inoculated into the stem bases of 10 healthy drunken horse grass seedlings (around 3 weeks old) using a sterile syringe, then wrapped with moistened sterile gauze, while the other 10 drunken horse grass seedlings were injected with sterile water as a control. All seedlings were placed in a greenhouse with a plastic cover at 15-22°C and 90-100% relative humidity. All inoculated drunken horse grass seedlings showed symptoms similar to those of natural infection with stem basal rot, whereas uninoculated drunken horse grass seedlings remained healthy after 15 days. Fungi re-isolated from the basal stems of inoculated plants were confirmed phenotypically and molecularly as F. pseudograminearum. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of drunken horse grass in China. The disease has become a potential threat to the growth of drunken horse grass in China.

Direct antifungal activity of methyl salicylate on soft rot caused by Rhizopus stolonifer and its membrane targeting mechanism

Methyl salicylate (MeSA) plays a significant role in various physiological metabolic processes of plants, however, reports on the antifungal activity were rare. This study aimed to investigate the direct antifungal activity of MeSA against Rhizopus stolonifer and to explore its antifungal mechanism at the membrane targeting level. Our study demonstrated that MeSA had antifungal activity. MeSA inhibited the mycelial growth and spore germination of Rhizopus stolonifer, and acted on the plasma membrane, changed the integrity and permeability of the cell membrane, leaked cell contents, induced reactive oxygen species (ROS) accumulation, stimulated membrane lipid peroxidation, changed the morphology of the mycelium, and finally caused apoptosis. In addition, in vivo experiments, MeSA significantly delayed the occurrence of soft rot of various fruits and vegetables. The results indicated that MeSA has direct antifungal activity for Rhizopus stolonifer, and proposed its antifungal mechanism from membrane targeting.

Antifungal activities of chitosan nanoparticles against Fusarium pseudensiforme on durian trees

Antifungal nano-biomaterials are gaining interest as an effective strategy for controlling fungal pathogens because of their nontoxicity, biodegradability, biocompatibility, and high activity. The study aimed to present an antifungal agent, squid chitosan nanoparticles (SCNs), against the Fusarium pseudensiforme pathogen in durian trees. Besides, the in vitro antifungal activity of SCNs was compared to other agents, including neem oil and squid pen chitosan. All biomaterials showed a decrease in mycelial growth as their treated concentrations increased. However, at the same concentration of 250 ppm after 8 days of incubation at 28 °C, SCNs exhibited the highest in totally inhibiting the growth of Fusarium pseudensiforme, while the lesion diameters were 2.5 cm (neem oil) and 2.1 cm (chitosan). Besides, the lowest dried biomass was obtained after 8 days of incubation in broth culture at 250 ppm of the SCNs. The findings suggested that SCNs could be used as an effective alternative nano-biomaterial for controlling the Fusarium pseudensiforme pathogen in durian trees.

Reduction of Phytophthora palmivora plant root infection in weak electric fields

The global food security crisis is partly caused by significant crop losses due to pests and pathogens, leading to economic burdens. Phytophthora palmivora, an oomycete pathogen, affects many plantation crops and costs over USD 1 billion each year. Unfortunately, there is currently no prevention plan in place, highlighting the urgent need for an effective solution. P. palmivora produces motile zoospores that respond to weak electric fields. Here, we show that external electric fields can be used to reduce root infection in two plant species. We developed two original essays to study the effects of weak electric fields on the interaction between P. palmivora’s zoospores and roots of Arabidopsis thaliana and Medicago truncatula. In the first configuration, a global artificial electric field is set up to induce ionic currents engulfing the plant roots while, in the second configuration, ionic currents are induced only locally and at a distance from the roots. In both cases, we found that weak ionic currents (250–550 μA) are sufficient to reduce zoospore attachment to Arabidopsis and Medicago roots, without affecting plant health. Moreover, we show that the same configurations decrease P. palmivora mycelial growth in Medicago roots after 24 h. We conclude that ionic currents can reduce more than one stage of P. palmivora root infection in hydroponics. Overall, our findings suggest that weak external electric fields can be used as a sustainable strategy for preventing P. palmivora infection, providing innovative prospects for agricultural crop protection.

Effects of Meyerozyma guilliermondii PJ15 on the biocontrol of Penicillium crustosum causing postharvest decay in Orah and its influence on the microbial diversity of Orah

Abstract Objectives Isolate biocontrol microorganisms that inhibit Penicillium crutosum, and explore their effects on the microbial control and microbial diversity change of Penicillium crustosum causing postharvest decay in Orah. Materials and Methods The biocontrol effect was verified by confrontation experiment. The microbial diversity was analyzed using high-throughput sequencing technology. Metabolomics analysis was performed using LC-MS/MS. Results a strain of Meyerozyma guilliermondii PJ15 that strongly inhibited Penicillium crustosum was isolated from grape surface, which could effectively inhibit the mycelium growth and green spore production of Penicillium crustosum. PJ15 could reduce the sourness and nutrient loss of Orah caused by Penicillium crutosum. Compared with the control, PJ15 treatment increased pH by 11.32%, soluble protein by 154.51%, ascorbic acid by 160.99%, and soluble total sugar by 203.53%. Microbial diversity analysis showed that PJ15 has a relatively small impact on the bacterial composition and diversity on the surface of Orah, but has a significant impact on the fungal composition and diversity. It can increase the fungal diversity of Orah invaded by Penicillium crustosum, increase the density of fungal interaction networks, and form a stronger coexisting survival interaction network. The vaccination of PJ15 downregulated differential metabolites 5,6-Epoxytetraene, which showed a positive correlation with Penicillium crustosum and a negative correlation with PJ15. Conclusions Meyerozyma guilliermondii PJ15 has a strong inhibitory effect on the growth of Penicillium crustosum, and has a significant impact on the fungal composition and diversity on the surface of Orah. It has the potential for biocontrol of Penicillium crustosum causing postharvest decay in Orah.

Dipicolinic acid reduces Epicoccum sorghinum symptoms on maize and inhibits tenuazonic acid biosynthesis.

Epicoccum sorghinum is a pathogenic fungus that causes leaf spot in a wide range of plants, including maize, and synthesizes the mycotoxin tenuazonic acid (TEA), which is carcinogenic. Despite the relevant economic and yield losses caused by E. sorghinum worldwide, methods for the control of this pathogen are lacking. In this work, the efficacy of Bacillus-produced dipicolinic acid (DPA) for control of E. sorghinum was evaluated using in vitro and in vivo assays, and compared with the efficacy of three commercial fungicides, including carbendazim, prochloraz, and thiram. DPA inhibited E. sorghinum mycelial growth, and conidia germination, and produced important alterations in E. sorghinum hyphae. Interestingly, 10 mM DPA reduced TEA biosynthesis by 86.6%. Although DPA rapidly degraded on maize leaves, 10 mM DPA showed higher preventive (67.4% lesion length inhibition) and inhibitory (89.5% lesion length inhibition) efficacies for the control of E. sorghinum on maize leaves compared to the commercial fungicides. Collectively, this study presents the first method for the control of E. sorghinum on maize and demonstrates that DPA application is a suitable approach to inhibit E. sorghinum symptoms in plants and TEA biosynthesis. © 2024 Society of Chemical Industry.

Enhancing saffron (Crocus sativus L.) growth in the Kashmir valley with resilient and widely effective Plant Growth-Promoting Rhizobacteria (PGPR) under field conditions

This study provides a thorough examination of stress-tolerant plant growth-promoting rhizobacteria (PGPR) and their significance in enhancing plant growth and biocontrol activities, and influence on saffron cultivation in the Kashmir Valley. Quantitative analysis of 35 efficient PGPR revealed significant variability in plant growth promotion (PGP) attributes ranging from 60 to 653 µg/ml tri-calcium phosphate (TCP) solubilization, 1548–4311 nM α-ketobutyrate h/mg/protein ACC-deaminase activity, 1.4–74.8 µg/ml IAA-like auxins production, and 94–214 % siderophore production. Molecular fingerprinting disclosed 52 ERIC types that belonged to 25 genera, mainly Bacillus. Bacillus species including B. aryabhattai, B. halotolerans, B. methylotrophicus, B. safensis, B. siamensis and B. simplex dominated the PGPR population, along with Pseudomonas spp., such as P. azotoformans and P. chlororaphis, indicating a selective buildup of PGPR in the saffron rhizosphere. In-vivo and In-vitro dual inoculation test of these widespread PGPR and stress-tolerant also inhibited the mycelial growth of Fusarium species including F. oxysporum, F. oxysporum f. sp. ciceris, and F. proliferatum. The field application of seven selected PGPR strains resulted in significant improvements in saffron productivity, including increased sprouted corms, plant height, stigma length, and stigma fresh and dry weight, which ranged from 0.6-1.8 %, 0.4–0.7 %, 0.3–0.5 %, and 6–19 %, respectively. These strains have the potential to stimulate cormlet growth and ultimately increase saffron production. Therefore, the PGP traits of these stress-tolerant rhizobacteria contributing to nutrients solubilization, auxin biosynthesis, and fungicidal attributes can ameliorate the saffron agricultural practices.

AfSwi6 Regulates the Stress Response, Chlamydospore Production, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys flagrans.

Nematode-trapping (NT) fungi are a major resource for controlling parasitic nematodes. Arthrobotrys flagrans, as a typical NT fungus, can capture nematodes by producing three-dimensional nets. The APSES transcription factor Swi6 plays a vital role in fungal growth and the pathogenicity of pathogens. In this study, we characterized AfSwi6 via gene disruption using the homologous recombinant method and transcriptome sequencing. Knockout of the AfSwi6 gene caused defects in mycelial growth, trap formation and pathogenicity, chlamydospore production, and stress response. Moreover, the transcriptome data indicated that AfSwi6 was related to DNA repair, stress response, and plasma membrane fusion. The result showed that AfSwi6 has a significant effect on trap development and chlamydospore production in A. flagrans.

Pyrolysed maize feedstock utilization in combination with Trichoderma viride against Macrophomina phaseolina

Maize cultivation is under the growing threat of charcoal rot (Macrophomina phaseolina). Chemical control of diseases imparts serious health hazards to humans and the ecosystem. Biochar as an alternative disease management approach has been under consideration of the researchers for some time now. The biochar utilized in this study was derived from maize stalks and cobs. Crystallographic structure, inorganic minerals content and size of maize biochar were analyzed by powder X-ray diffractometer, while scanning electron microscopy revealed rough, irregular, tubular structure of the biochar surface. EDX spectra revealed that the maize biochar composition was dominated by ‘C’ followed by ‘O’. The current study was designed to determine the synergistic effect of maize biochar (MB), and biocontrol agent (BCA) Trichoderma viride as soil amendments on the suppression of M. phaseolina. In vitro bioassays were conducted to check the efficiency of antagonistic effect of Trichoderma spp., in combination with maize biochar. On the basis of maximum mycelial growth inhibition T. viride was selected for a glasshouse experiment. Maize plants were grown in pots containing a mixture of soil with MB at application at the rate of 3 and 6% (v/v) separately, associated with or without T. viride. Treatments amended with 3% MB inoculated with M. phaseolina significantly reduced the percentage disease severity index by 40%. While in the presence of T. viride, 3% MB showed maximum disease suppression and a minimum percentage severity index i.e. 60 and 20%, respectively. Highest nitrogen contents were 18.4 g kg−1 observed in treatment 6% MB, while highest phosphorus and potassium contents were 3.11 and 15.2 g kg−1, respectively in the treatment with 3% MB. Conclusively, the effect of variable concentrations of maize biochar and T. viride as soil amendment was evident on the development of charcoal rot, growth and physiology of maize plants. According to the available literature, our report is the first on the implementation of biochar in synergism with T. viride to suppress the charcoal rot in maize.

Mycelial growth, yield and decomposition capability of white oyster mushroom (Pleurotus florida) grown in low-density polyethylene (LDPE) plastic and lignocellulosic wastes

White Oyster Mushroom (Pleurotus florida) is one of the domesticated mushrooms in the Philippines and known to grow in various biodegradable wastes. Low Density Polyethelene (LDPE) plastic is one of the most common non-biodegradable wastes in the environment which causes pollution. At present, biodegradation process is one of the ways to degrade plastics because of its non-polluting mechanism and cost-effectiveness. Thus, this study aimed to determine if P. florida could grow in LDPE plastic and facilitate its decomposition. Fourteen treatments were used in the study, laid out in Complete Randomized Design, and replicated thrice. The first four (4) treatments were composed of substrates with no LDPE plastics (rice straw, sawdust and cocopeat) while the rests were substrates with certain percentage of LDPE plastics. Observation was done for eight (8) weeks in the PUP Lopez Mushroom Laboratory and the data obtained was analyzed using ANOVA. Result shows that slight decomposition happened in LDPE plastic mixed with lingo-cellulosic wastes. Small spots or dark holes appear in the surface of LDPE plastics observed under microscope which is a sign of decomposition. In terms of mycelial ramification, fruiting bags with LDPE plastics were first to occupy the fruiting bag. There was also a decreased weight of the fruiting bags after 8 weeks. In terms of yield, substrates without LDPE plastics grew mushrooms with the highest weight. However, yield in sawdust and cocopeat substrates were not significantly different with the yield of mushrooms grown in substrates with LDPE plastics.

Evaluation of the Antifungal and Biochemical Activities of Fungicides and Biological Agents against Ginseng Sclerotinia Root Rot Caused by Sclerotinia nivalis.

The objective of this study was to identify effective agents for the prevention and control of ginseng Sclerotinia root rot disease caused by Sclerotinia nivalis. The inhibitory effects of 16 chemical fungicides and 10 biocontrol agents (strains) on mycelial growth and sclerotium formation in S. nivalis were determined using a plate confrontation essay. The results showed that the best chemical agents for inhibiting the mycelial growth and sclerotium formation of S. nivalis were fluconazole and fludioxonil, while Bacillus amyloliquefaciens FS6 and B. subtilis (Kono) were the best biocontrol agents (strains). The results of field trials in 2022 and 2023 showed that the control effects of fluconazole and fludioxonil on ginseng Sclerotinia root rot disease were 90.60-98.16%, and those of the biocontrol agents B. amyloliquefaciens FS6 and B. subtilis (Kono) were 94.80-97.24%, respectively. Chemical agents produced abnormal and twisted mycelia, while the biocontrol agents increased mycelial branching, dilated the mycelium tip, and revealed an abnormal balloon. All of the fungicides decreased the ergosterol content, changed the cell membrane permeability, and increased the protein and nucleic acid permeability. These results suggest that these are potential agents for controlling ginseng Sclerotinia root rot disease, and their biochemical mechanisms of chemical and biocontrol of this disease were demonstrated.

Soybean inoculation with Azospirillum argentinense improves nodulation, grain yield, and alleviates the incidence of Fusarium at field with drought stress

ABSTRACT The efficacy of Azospirillum argentinense REC3, applied by seed inoculation, in enhancing soybean (Glycine max [L.] Merr.) nodulation with native rhizobia, improving grain yield, and protecting against Fusarium fungi was studied. In La Cruz, Tucumán, Argentina, 23 fungal isolates were identified as F. oxysporum (2 strains), F. solani (2), and F. tucumaniae (19). Following soil infestation, only F. tucumaniae was confirmed as a phytopathogen, with REC3 inhibiting its mycelial growth. Greenhouse trials, for a duration of 30 days, showed significant reductions in disease severity (78.2%), root rot (32.3%), and disease incidence (50.9%) in plants susceptible to sudden death syndrome (SDS) when REC3 was applied. Field experiments (2018–2019; January-April) under drought stress conditions, displayed increased nodulation (37.2%) and grain yield (2077 ± 272.8 kg ha−1, p < 0.05) in REC3-treated plants compared to controls (1516 ± 272.8 kg ha−1, p < 0.05). Additionally, a low disease incidence (0.90%) was observed only in the SDS-susceptible soybean variety without REC3 treatment. Overall, the findings showed the significant potential of REC3 inoculation as a biotechnological strategy for enhancing agricultural productivity, managing diseases, and improving crop resilience under challenging environmental conditions. This integrated approach, combining the use of a partially SDS-resistant soybean variety with REC3 inoculation, showed promise in protecting soybean cultivation against SDS and other fungal diseases, thereby ensuring more sustainable and resilient agricultural practices.

Optimization of fermentation temperature and time for production of an antifungal extract from Bacillus amyloliquefaciens B17

Background/Objective. Species of Bacillus are currently gaining interest because its ability to produce secondary metabolites with antifungal properties against various plant pathogenic fungi. The objective of this study was to optimize fermentation temperature and time for antifungal extract production by Bacillus amyloliquefaciens B17 and to verify its activity against plant pathogenic fungi Gilbertella persicaria, Choanephora cucurbitarum, Colletotrichum asianum, and Botrytis cinerea. Materials and Methods. A central composite design (CCD) with two factors and five levels (fermentation temperature: 23.7, 25, 28, 31, and 32.2 °C and fermentation time: 25, 46, 95, 144, and 164. 3 h) was used. Thirteen combinations of temperature and fermentation time were randomly performed. The thirteen crude extracts of B. amyloliquefaciens B17 were obtained from the cell-free fermentation broth by acid precipitation followed by alkaline solubilization. The response variable was the diameter of the inhibition halos generated by placing drops of the different crude extracts onto the medium already inoculated with a suspension of Gilbertella persicaria spores. Results. The optimal conditions for the production of the extract with the greatest antifungal activity in B. amyloliquefaciens B17 were 26.8 °C and 158.6 h. Conclusion. The optimized crude extract from B. amyloliquefaciens B17 exhibited a strong ability to inhibit mycelial growth and spore germination of Gilbertella persicaria, Choanephora cucurbitarum, Colletotrichum asianum, and Botrytis cinerea.

First report of Fusarium luffae causing soybean wilt in the USA.

During the 2023 soybean growing season in South Dakota, we scouted a farmer's field and observed soybean (Glycine max (L.) Merr.) plants with wilting symptoms and blighted leaves. Symptomatic stems and leaves were collected from the field to identify associated pathogens. 0.5 cm2 size leaf and stem segments of the sample were surface sterilized by rinsing with 10% bleach for 5 minutes then dipping in 70% ethanol for one minute, and later placing in deionized sterile water for one minute. The sterilized segments were placed on wet filter paper and incubated under fluorescent light for three days. Fungal growth was observed, and the growing mycelia were transferred to potato dextrose agar plates amended with 50 µg/ml Ampicillin (PDAa). Pure culture of the isolate was obtained using single sporing and transferring on new PDAa plates. A dense aerial mycelial growth showing waxy yellow color with a pale orange tinge on the rear side covered the full plate after seven days of incubation at room temperature under fluorescent lights (Figure S1a and b). Developing macroconidia were falcate, curved, smooth to slightly rough, and hyaline with three-five septa (Figure S1c). For molecular identification, DNA of the recovered isolate was extracted and subjected to multiloci PCR (O'Donnell et al., 2010) to amplify and Sanger sequence the internal transcribed spacers region (ITS) (GenBank accession number PP393518), calmodulin (CAM-PP401978), RNA polymerase II second largest subunit (RPB2-PP401980), and translation elongation factor 1-α gene (TEF1-PP401979). The South Dakota isolate (SLSDF2) was identified as Fusairum luffae on NCBI and Fusarioid polyphasic identification databases with 99.40% similarity to Fusarium luffae strain NRRL31167. A phylogeny was inferred based on concatenated TEF1, RPB2, and CAM sequences to show species relatedness (Figure S3). The characterized isolate SDSLF2 was evaluated for soybean pathogenicity using spray inoculations on detached leaves and V2 stage soybean plants (Figure S2a and b). The conidial suspension was prepared by growing the pathogen on mung bean agar for seven days. 2 ml of conidial suspensions (2.6 × 104 conidia/ml) and mock control (sterilized water with 0.1% Tween-20) was sprayed on the detached leaves and whole plants. The experiment was repeated three times with four replicates in each. In the detached leaf assay, leaves were completely blighted (Figure S2a) within 96 hours. In whole plant assays, after two days of incubation, leaf blighting was visible and progressed with time. Four days post-inoculation, the infected plants showed extensive leaf symptoms, and ultimately defoliation occurred (Figure S2b). No symptoms were observed in mock controls of either of the experiments. The pathogen was reisolated from the infected tissues and its identity was confirmed as F. luffae by CAM sequencing fulfilling Koch's postulates. F. luffae has been reported to associated with soybeans in China (Zhao et al., 2022), however, to our knowledge, this is the first report of F. luffae pathogenic on soybeans in the USA, stressing the need to identify resistance sources to avoid any potential disease epidemic.

Characterization and fungicide sensitivity of Gnomoniopsis fructicola causing Gnomonia Leaf Blotch of strawberry in the Carolinas.

Emerging fungal pathogens have always been an issue of concern in southeastern U.S. strawberry production. In 2023, an unusual outbreak of Gnomonia leaf blotch occurred at one North Carolina (NC) and multiple South Carolina (SC) strawberry farms and marked the first report of its occurrence in SC. Molecular identification and phylogenetic analysis of isolates from multiple locations identified the fungus Gnomoniopsis fructicola as the causal agent. In vitro germination of G. fructicola progressed slowly and remained less than 40% even after 24 h of incubation. Similarly, germ tube growth was slow compared to other pathogens. Slow symptom development on strawberry leaves of young strawberry plants grown in the greenhouse started 5 weeks after inoculation. Once the pathogen established on greenhouse plants, leaf necrosis forming blotches was observed. The baseline sensitivity of G. fructicola isolates to commonly used chemical classes of fungicides was assessed. Propiconazole, cyprodinil, pyraclostrobin, and fludioxonil were highly effective in mycelial growth assays with EC50 values < 0.01 µg/ml. Iprodione and thiophanate-methyl were also effective with EC50 values ranging from 0.05 to 1.38 and 2.01 to 23.96 µg/ml, respectively. Fluopyram and fenhexamid were ineffective with EC50 values >100 µg/ml. Based on conversations with the producers, the disease outbreak was linked to transplants from the same nursery source. This study reports for the first time the presence of Gnomonia leaf blotch in South Carolina and provides valuable insights into chemical management options for G. fructicola.

Dual actions of chloroinconazide on pepper blight in Capsicum annuum: disruption of Phytophthora capsici mycelium and activation of CaCNGC9-mediated SA signaling.

Pepper blight, caused by Phytophthora capsici, is a devastating disease that seriously threatens pepper production worldwide. With the emergence of resistance in P. capsici against conventional fungicides, there is an urgent need to explore novel alternatives for pepper blight management. This study aims to assess the inhibitory effect of chloroinconazide (CHI), a compound synthesized from tryptophan, against pepper blight, and to explore its potential mechanisms of action. The results demonstrated that CHI effectively targeted P. capsici, disrupting its growth and mycelial structure, which resulted in the release of dissolved intracellular substances. Additionally, CHI significantly inhibited the sporangium formation, zoospores release, and zoospores germination, thereby reducing the re-infection of P. capsici. In contrast, the commercial pesticide methylaxyl only inhibited mycelial growth and had limited effect on re-infection, while azoxystrobin inhibited re-infection but had a weak inhibitory effect on mycelial growth. Furthermore, CHI activated the salicylic acid (SA) signaling pathway-mediated immune response to inhibit P. capsici infection in pepper, with this activation being contingent upon cyclic nucleotide-gated ion channel CaCNGC9. CHI exhibited potent dual inhibitory effects on P. capsici by disrupting mycelial structure and activating the CaCNGC9-mediated SA signaling pathway. These dual mechanisms of action suggested that CHI could serve as a promising alternative chemical fungicide for the effective management of pepper blight, offering a new approach to control this devastating disease. Our findings highlighted the potential of CHI as a sustainable and efficient solution to combat the increasing resistance of P. capsici to conventional fungicides, ensuring better crop protection and yield. © 2024 Society of Chemical Industry.

Baseline tebuconazole sensitivity and potential resistant risk in Fusarium graminearum

BackgroundThe Fusarium head blight caused by Fusarium graminearum results in reduced crop yields and the potential for vomitoxin contamination, which poses a risk to both human and livestock health. The primary method of control relies on the application of chemical fungicides.ResultsThe current study found that the tebuconazole sensitivity of 165 F. graminearum isolates collected from the Huang-Huai-Hai region of China between 2019 and 2023 ranged from 0.005 to 2.029 µg/mL, with an average EC50 value of 0.33 ± 0.03 µg/mL. The frequency distribution conformed to a unimodal curve around the mean, and therefore provides a useful reference for monitoring the emergence of tebuconazole resistance in field populations of F. graminearum. No cross-resistance was detected between tebuconazole and other unrelated fungicides such as flutriafol, propiconazole and fluazinam, but there was a clear negative cross-resistance with triazole fungicides including fludioxonil, epoxiconazole, hexaconazole, and metconazole. Analysis of five tebuconazole-resistant mutants produced under laboratory conditions indicated that although the mycelial growth of the mutants were significantly (p < 0.05) reduced, spore production and germination rates could be significantly (p < 0.05) increased. However, pathogenicity tests confirmed a severe fitness cost associated with tebuconazole resistance, as all of the mutants completely loss the ability to infect host tissue. Furthermore, in general the resistant mutants were found to have increased sensitivity to abiotic stress, such as ionic and osmotic stress, though not to Congo red and oxidative stress, to which they were more tolerant. Meanwhile, molecular analysis identified several point mutations in the CYP51 genes of the mutants, which resulted in two substitutions (I281T, and T314A) in the predicted sequence of the FgCYP51A subunit, as well as seven (S195F, Q332V, V333L, L334G, M399T, E507G, and E267G) in the FgCYP51C subunit. In addition, it was also noted that the expression of the CYP51 genes in one of the mutants, which lacked point mutations, was significantly up-regulated in response to tebuconazole treatment.ConclusionsThese results provide useful data that allow for more rational use of tebuconazole in the control of F. graminearum, as well as for more effective monitoring of fungicide resistance in the field.

Isolation of bacteria from Grifola frondosa cultivation on wood logs to find mycelial growth-promoting bacteria.

This study aimed to isolate bacteria that coexist with the edible mushroom Grifola frondosa when it is cultured on wood, and to determine their interactions; in turn, the aim was to find bacteria that stimulate mycelial growth so as to decrease the time required for spawn preparation on potato dextrose agar (PDA). Some Pseudomonas, Dyella, Bacillus, and Priestia spp. isolated from the cultivation surroundings of G. frondosa had a positive effect on the mycelial growth of the fungus in PDA. However, some isolated bacteria had a severe negative effect on the mycelial growth, especially Burkholderia spp. Thus, both mycelial-promoting bacteria and potentially pathogenic bacteria coexist with G. frondosa in cultivation. Enzyme activity assays indicated that some wood-degrading bacteria inhabit the cultivation surroundings of G. frondosa, and these bacteria probably help the fungus to degrade wood (especially cellulose).