Mycelial Growth Research Articles

Exploring the Antifungal Activity of Moroccan Bacterial and Fungal Isolates and a Strobilurin Fungicide in the Control of Cladosporium fulvum, the Causal Agent of Tomato Leaf Mold Disease.

The causal agent of tomato leaf mold, Cladosporium fulvum, is prevalent in greenhouses worldwide, especially under high humidity conditions. Despite its economic impact, studies on antifungal agents targeting C. fulvum remain limited. This study evaluates biocontrol agents (BCAs) as alternatives to chemical controls for managing this disease, alongside the strobilurin fungicide azoxystrobin. From a Moroccan collection of potential BCAs, five bacterial isolates (Alcaligenes faecalis ACBC1, Pantoea agglomerans ACBC2, ACBP1, ACBP2, and Bacillus amyloliquefaciens SF14) and three fungal isolates (Trichoderma spp. OT1, AT2, and BT3) were selected and tested. The in vitro results demonstrated that P. agglomerans isolates reduced mycelial growth by over 60% at 12 days post-inoculation (dpi), while Trichoderma isolates achieved 100% inhibition in just 5 dpi. All bacterial isolates produced volatile organic compounds (VOCs) with mycelial inhibition rates ranging from 38.8% to 57.4%. Likewise, bacterial cell-free filtrates significantly inhibited the pathogen's mycelial growth. Greenhouse tests validated these findings, showing that all the tested isolates were effective in reducing disease incidence and severity. Azoxystrobin effectively impeded C. fulvum growth, particularly in protective treatments. Fourier transform infrared spectroscopy (FTIR) analysis revealed significant biochemical changes in the treated plants, indicating fungal activity. This study provides valuable insights into the efficacy of these BCAs and azoxystrobin, contributing to integrated management strategies for tomato leaf mold disease.

Effect of silver nanochitosan on control of seed-borne pathogens and maintaining seed quality of wheat

Seeds, considered as the foundation of agriculture, are invaded by a broad spectrum of seed-borne pathogens. The current study aimed to control seed-borne fungal pathogens of wheat, Aspergillus flavus and A. niger, by using Ag+ nanochitosan (Ag-NC) for nano-priming of seeds and enhancing seed quality. Nanochitosan (NC) and Ag-NC were synthesized using the gelation method and characterized by UV–vis spectrophotometry, FESEM, EDXS, and HRTEM. NC and Ag-NC showed irregular surface topography with an average particle size of 275 and 325 nm, respectively. Antifungal activity of both the nanoparticles at 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL revealed that Ag-NC at 0.5 mg/mL has completely terminated the mycelial growth of both pathogens. Malonaldehyde content increased to 77.77% in A. flavus and 82.66% in A. niger when exposed to 0.5 mg/mL Ag-NC. High-intensity fluorescence due to oxidative stress was observed in Ag-NC-treated pathogens. Ultra-structural changes in Ag-NC treated pathogenic spores under SEM displayed pronounced membrane damages. Wheat seeds were nano-primed with NC and Ag-NC at 0.5 mg/mL, and fungal load was examined to evaluate the mitigation of pathogenic stress and its effect on seedling growth promotion activity. Ag-NC priming reduced the fungal load and allowed successful seed germination. Ag-NC priming increased the albumin, gliadin, gluten, and glutenin content along with total phenol, reducing sugar and starch levels. Ag-NC priming increased the overall protein levels traced through SDS-PAGE. Seed priming with Ag-NC promotes seed germination, mean germination time, stress tolerance index, vigour, etc. NC and Ag-NC at 0.5 mg/mL showed no cytotoxic effect on the Human Embryonic Kidney (HEK293) cell line that ensures the nanoparticles are non-toxic. Thus, the synthesized nanoparticles exhibit a dual role in antifungal activity and plant growth promotion.

Sustainable management of peanut damping-off and root rot diseases caused by Rhizoctonia solani using environmentally friendly bio-formulations prepared from batch fermentation broth of chitinase-producing Streptomyces cellulosae.

Soil-borne plant diseases represent a severe problem that negatively impacts the production of food crops. Actinobacteria play a vital role in biocontrolling soil-borne fungi. The target of the present study is to test the antagonistic activity of chitinase-producing Streptomyces cellulosae Actino 48 (accession number, MT573878) against Rhizoctonia solani. Subsequently, maximization of Actino 48 production using different fermentation processes in a stirred tank bioreactor. Finally, preparation of bio-friendly formulations prepared from the culture broth of Actino 48 using talc powder (TP) and bentonite in a natural as well as nano forms as carriers. Meanwhile, investigating their activities in reducing the damping-off and root rot diseases of peanut plants, infected by R. solani under greenhouse conditions. Actino 48 was found to be the most significant antagonistic isolate strain at p ≤ 0.05 and showed the highest inhibition percentage of fungal mycelium growth, which reached 97%. The results of scanning electron microscope (SEM) images analysis showed a large reduction in R. solani mycelia mass. Additionally, many aberrations changes and fungal hypha damages were found. Batch fermentation No. 2, which was performed using agitation speed of 200rpm, achieved high chitinase activity of 0.1163 U mL- 1 min- 1 with a yield coefficient of 0.004 U mL- 1 min- 1 chitinase activity/g chitin. Nano-talc formulation of Actino 48 had more a significant effect compared to the other formulations in reducing percentages of damping-off and root rot diseases that equal to 19.05% and 4.76% with reduction percentages of 60% and 80%, respectively. The healthy survival percentage of peanut plants recorded 76.19%. Furthermore, the nano-talc formulation of Actino 48 was sufficient in increasing the dry weight of the peanut plants shoot, root systems, and the total number of peanut pods with increasing percentages of 47.62%, 55.62%, and 38.07%, respectively. The bio-friendly formulations of actinobacteria resulting from this investigation may play an active role in managing soil-borne diseases.

Evaluation of aromatic plants hydrosols on the growth of Trichoderma harzianum

Objective: To evaluate the compatibility of hydrosols from aromatic plants in the growth of T. harzianum. Design/Methodology/Approach: All hydrosols of Foeniculum vulgare, Plectranthus coleoides, Tagetes arenicola, T. coronopifolia, T. erecta, and T. lucida were evaluated in vitro on the mycelium Trichoderma Harzianum. Their growth rates and inhibition percentages were recorded. T. harzianum was inoculated in 50 g of sterilized corn cob enriched with F. vulgare hydrosol (3, 5 and 7%), this mixture was incubated at 28 ±2 °C; spore counting, and viability tests were performed on PDA medium. The Tukey test (p ≤ 0.05) was used for mean comparisons. Results: All hydrosols inhibited in vitro the mycelial growth of T. harzianum at 100%, but at 5% concentration, some hydrosols promoted the growth of the fungus more than the control. The concentration of F. vulgare hydrosol influenced the production and viability of T. harzianum spores in the cob substrate. Study Limitations/Implications: This study provides Information on the use of hydrosol, which are typically considered waste products. Findings/Conclusions: At low concentration, F. vulgare hydrosol can be used to enrich corn cob and promote the growth of T. harzianum.

Effect of Media on the Mycelial Growth of Honey Bee Fungal Pathogen <i>Ascosphaera apis</i> Mating Types

The fungal pathogen Ascosphaera apis significantly impacts the honey bee Apis mellifera by causing larval mortality and reducing colony productivity. Understanding the growth dynamics of A. apis under different media conditions is crucial for advancing research on its biology and pathogenicity. This study evaluates the effect of eight artificial media namely Potato Dextrose Agar (PDA), Carrot Dextrose Agar (CDA), Beetroot Dextrose Agar (BDA), Tender Coconut Water (TCW) media, Sabouraud Dextrose Agar (SDA), Sabouraud Dextrose Agar Yeast (SDAY), Sabouraud Maltose Agar Yeast (SMAY), and Malt Extract Agar (MEA) on the mycelial growth and reproductive structure formation of A. apis's two mating types. Results indicated significant differences in mycelial growth across media. SDA (75.40 mm dia) and SDAY (74.83 mm dia) supported maximum growth for mating type 1 (MAT1) and, SDA (72.72 mm dia) and BDA (68.17 mm dia) yielded the maximum growth for mating type 2 (MAT2). The TCW resulted in the least growth for MAT1 (44.19 mm dia) and MAT2 (41.25 mm dia). The growth patterns showed that media supplemented with sugars generally enhanced A. apis growth, whereas non-sugar media did not. Furthermore, reproductive structures formed faster on PDA and CDA than on other media. The study confirms that media composition, particularly sugar content, critically influences the growth and development of A. apis, providing insights that could help to develop better management strategies for controlling chalkbrood disease in honey bee colonies.

Knit-Mycelium Hybrids: Textile Design Strategies for Biofabrication Systems Based on Mycocrete Injection in 3D Knitted Tubular Formworks

This paper investigates the design potential of monolithically grown Mycelium-based Composites (MBCs), focusing on integrating 3D knitted formworks within the fabrication process. Mycelium has gained attention as a sustainable, biodegradable construction material with insulating and self-healing capabilities. While there have been advancements in MBC, challenges remain regarding geometrical complexity and design potential at scale through in situ practices. Recent interest has emerged in digital fabrication methods and mould creation using textile logic and CNC knitting. However, fabricating mycelium within tubular soft moulds present a lack of stability and uneven growth distribution. The novel technique using injection filling of Mycocrete, a viscous mycelium paste, overcomes this structural disintegration. This method enables the creation of complex designs within a two-staged fabrication process while providing high structural performance. The paper presents three case studies demonstrating the impact of hanging, draping, and internal shape integration as fabrication steps as design tools for geometrical possibilities and diversity of expressions. It examines the relationship between knitted soft preform design and biofabrication parameters in relation to design expression, scale and mycelium growth. The paper concludes by emphasising the significance of 3D knitted formworks in scaling up MBCs in the built environment and its potential for implementing textile design parameters in MBC expressions and geometries. The fabrication steps provide newfound design opportunities, opening possibilities for aesthetical, material-saving and functionally graded design approaches. Further research in this area holds promise for advancing sustainable construction and textile design practices.

An implementation framework for evaluating the biocidal potential of essential oils in controlling Fusarium wilt in spinach: from in vitro to in planta.

Fusarium wilt, caused by Fusarium oxysporum f. sp. spinaciae, causes a significant challenge on vegetative spinach and seed production. Addressing this issue necessitates continuous research focused on innovative treatments and protocols through comprehensive bioassays. Recent studies have highlighted the potential of plant-based compounds in controlling fungal diseases. The present work aims to conduct a series of experiments, encompassing both in vitro and in planta assessments, to investigate the biocontrol capabilities of different essential oils (EOs) at various application rates, with the ultimate goal of reducing the incidence of Fusarium wilt in spinach. The inhibitory effect of four plant EOs (marjoram, thyme, oregano, and tea tree) was initially assessed on the spore germination of five unknown Fusarium strains. The outcomes revealed diverse sensitivities of Fusarium strains to EOs, with thyme exhibiting the broadest inhibition, followed by oregano at the highest concentration (6.66 μL/mL) in most strains. The tested compounds displayed a diverse range of median effective dose (ED50) values (0.69 to 7.53 µL/mL), with thyme and oregano consistently showing lower ED50 values. The direct and indirect inhibitory impact of these compounds on Fusarium mycelial growth ranged from ~14% to ~100%, wherein thyme and oregano consistently exhibiting the highest effectiveness. Following the results of five distinct inoculation approaches and molecular identification, the highly pathogenic strain F-17536 (F. oxysporum f.sp. spinaciae) was chosen for Fusarium wilt assessment in spinach seedlings, employing two promising EO candidates through seed and soil treatments. Our findings indicate that colonized grain (CG) proved to be a convenient and optimal inoculation method for consistent Fusarium wilt assessment under greenhouse conditions. Seed treatments with thyme and oregano EOs consistently resulted in significantly better disease reduction rates, approximately 54% and 36% respectively, compared to soil treatments (P > 0.05). Notably, thyme, applied at 6.66 µL/mL, exhibited a favorable emergence rate (ERI), exceeding seven, in both treatments, emphasizing its potential for effective disease control in spinach seedlings without inducing phytotoxic effects. This study successfully transitions from in vitro to in planta experiments, highlighting the potential incorporation of EOs into integrated disease management for Fusarium wilt in spinach production.

Antifungal activity of Bacillus velezensis and Pseudomonas azotoformans isolated from compost tea against anthracnose (Colletotrichum spp.) on strawberry fruit

AbstractAnthracnose, caused by Colletotrichum spp., is a threat to strawberry production globally. Unlike their chemical counterparts, microbial biofungicides offer a method of postharvest fungal disease control that is safe, sustainable and less affected by pathogen resistance. The present study evaluated the antifungal effects of three bacteria, Bacillus velezensis strains SH1 and SH2 and Pseudomonas azotoformans strain SH3, obtained from sheep manure compost tea. The bacteria or their cell‐free filtrates were tested against Colletotrichum acutatum and Colletotrichum gloeosporioides in bioassays and against strawberry anthracnose. In addition, precipitated or extracted extracellular fractions were tested to determine the effects on membrane permeability of Colletotrichum spp. spores. Confrontation assay results showed all bacteria inhibited mycelial growth, with B. velezensis SH1 and P. azotoformans SH3 being the most effective. All cell‐free filtrates inhibited mycelial growth with B. velezensis SH1 and SH2 resulting in the highest inhibition. The bacteria suppressed anthracnose lesions on strawberry fruit although effective treatments varied by causal mould. B. velezensis SH1 and SH2 significantly permeabilized spore membranes, indicating antibiosis as a possible mode of action. Investigation into antimicrobial compound production found various homologues of the lipopeptides fengycin, iturin and surfactin were produced by B. velezensis SH1 and SH2. Results suggest that lipopeptides produced by B. velezensis strains permeabilize Colletotrichum cell membranes, and that fengycins were the most inhibitory of the lipopeptides against Colletotrichum spp.

Thymol-a plant-based monoterpenoid phenol compound of an essential oil for the management of sheath blight disease of rice.

Sheath blight of rice is a global disease that significantly reduces rice yield. This study reports the antifungal activity of an active compound of essential oil, thymol, at different concentrations against Rhizoctonia solani (strain RS-Gvt). In vitro assay results indicated that thymol concentrations (0.5 mg mL-1 and 0.25 mg mL-1) completely inhibited (100%) the mycelial growth of RS-Gvt (p ≤ 0.01). Microscopic observations of thymol-treated mycelium of RS-Gvt at 0.0312 mg mL-1 and above concentrations, revealed a distorted mycelial morphology with deformed hyphae. Hyphae showed a bead-like appearance, reduction in size, and constriction of the hyphae at uneven points with increased hyphal density often entangling with each other. Further, an on-field experiment was conducted to study the field bio-efficacy of thymol for two consecutive Kharif seasons of 2022 and 2023 using a factorial RCBD design. The disease severity was measured as the percent disease index (PDI), and the results of two seasons were pooled. Pathogen (RS-Gvt) and thymol were inoculated in different combinations/methods as main treatments (M1-M3), and concentrations of thymol (0.0625-1.0 mg ML-1) as sub-treatments. The results indicated that all two factors significantly (P = 0.05) influenced the PDI and grain yield. The pooled data of two seasons indicated a significant difference between the main treatments (M1: RS-Gvt + thymol together; M2: thymol sprayed first followed by RS-Gvt; M3: RS-Gvt first followed by thymol spray) on PDI (53.39-59.67) and grain yield (4.16-4.75 t ha-1). M1 exhibited a lower PDI (53.39) and a higher grain yield (4.75 t ha-1) compared to M2 and M3, indicating a protective mode of action of thymol against sheath blight disease of rice. The sub-treatments have shown significant variation in PDI and grain yield. The PDI and grain yield ranged from 33.70 (at 1 mg mL-1) to 66.21 (at 0.0625 mg mL-1) and 4.18 (at 1 mg mL-1) to 5.26 (at 0.0625 mg mL-1) t ha-1, respectively, among the thymol concentrations. This indicates that increasing concentrations of thymol have negatively influenced the PDI and positively impacted the yield. Therefore, the spray of 1 mg mL-1 of thymol at the potential disease-infection stage is most effective in controlling the sheath blight disease of rice. This study provides an alternative green bioactive compound for controlling the sheath blight disease, and thymol can be included in developing eco-friendly integrated disease management practices. © 2024 Society of Chemical Industry.

Sclerotinia sclerotiorum on bean and potato in Sinaloa: Etiology, epidemiology and alternatives for management

White mold (Sclerotinia sclerotiorum) is the main disease of bean and potato in Sinaloa. In the present review, the symptoms and signs of the disease as well as cultural and morphological characteristics of the teleomorph of the pathogen, its ecology and the epidemiology of the disease are addressed. The implementation of a prediction system which includes the carpogenic germination of the sclerotia and the phenology of both bean and potato for the management of the disease is described. This system considers soil temperature ranging from 13 to 19 °C a at depth of 2.5 cm in the soil and the flowering stage in both bean and potato to do the first spray application of synthetic fungicide to prevent the disease. In vitro studies indicated that Trichoderma harzianum, T. viride and T. atroviride reduced mycelial growth rate of S. sclerotiorum. The same antagonistic species exerted control of white mold under field conditions, where an increment of 40% of yield was observed in the treated plots, with respect to those treated with fungicide fluazinam. Future lines of research focusing on the ecology of the pathogen and management of the disease including the antagonistic fungi in the prediction system are suggested.

Phenolic Compounds Synthesized by Trichoderma longibrachiatum Native to Semi-Arid Areas Show Antifungal Activity against Phytopathogenic Fungi of Horticultural Interest

Fungal diseases are a major threat to the horticultural industry and cause substantial postharvest losses. While secondary metabolites from Trichoderma sp. have been explored for their antifungal properties, limited information exists on the phenolic compounds produced by less studied species like Trichoderma longibrachiatum. In this study, phenolic compounds were extracted from a liquid culture of T. longibrachiatum using various solvents and methods (conventional and ultrasonic-assisted). Phenolic compounds were quantified by spectrophotometry and identified by high-performance liquid chromatography with diode array detection (HPLC-DAD). The antifungal activity against Alternaria alternata and Fusarium oxysporum was determined by mycelial growth inhibition assays, maximum growth rate (µmax) by the Gompertz equation, and spore germination tests. Although no significant differences (p ≥ 0.05) were found between the extraction methods, the type of solvent significantly influenced the phenolic content (p ≤ 0.05). Extraction with 70% ethanol showed the highest content of phenolic compounds and flavonoids. More than eight phenolic compounds were detected. Further, this is the first report of the phenolics ferulic, chlorogenic and p-coumaric acids identification in T. longibrachiatum, along with flavonoids such as epicatechin and quercetin, among others. The 70% ethanolic extracts notably inhibited the mycelial growth of A. alternata and F. oxysporum, reducing their maximum growth rate by 1.5 and 1.4 mm/h, respectively. Furthermore, p-coumaric and ferulic acids significantly inhibited spore germination of both pathogens, with a minimum inhibitory concentration (MIC) of 1.5 mg/mL and a minimum fungicidal concentration (MFC) of 2 mg/mL. These findings demonstrate the potential of T. longibrachiatum and its phenolic compounds as viable alternatives for biological control in horticulture and postharvest disease management.

Kosakonia oryziphila NP19 bacterium acts as a plant growth promoter and biopesticide to suppress blast disease in KDML105 rice

This study demonstrates that root-associated Kosakonia oryziphila NP19, isolated from rice roots, is a promising plant growth-promoting bioagent and biopesticide for combating rice blast caused by Pyricularia oryzae. In vitro experiments were conducted on fresh leaves of Khao Dawk Mali 105 (KDML105) jasmine rice seedlings. The results showed that NP19 effectively inhibited the germination of P. oryzae fungal conidia. Fungal infection was suppressed across three different treatment conditions: rice colonized with NP19 and inoculated by fungal conidia, a mix of NP19 and fungal conidia concurrently inoculated on the leaves, and fungal conidia inoculation first followed by NP19 inoculation after 30 h. Additionally, NP19 reduced fungal mycelial growth by 9.9–53.4%. In pot experiments, NP19 enhanced the activities of peroxidase (POD) and superoxide dismutase (SOD) by 6.1–63.0% and 3.0–67.7%, respectively, indicating a boost in the plant’s defense mechanisms. Compared to the uncolonized control, the NP19-colonized rice had 0.3–24.7% more pigment contents, 4.1% more filled grains per panicle, 26.3% greater filled grain yield, 34.4% higher harvest index, and 10.1% more content of the aroma compound 2-acetyl-1-pyrroline (2AP); for rice colonized with NP19 and infected with P. oryzae, these increases were 0.2–49.2%, 4.6%, 9.1%, 54.4%, and 7.5%, respectively. In field experiments, blast-infected rice that was colonized and/or inoculated with NP19 treatments had 15.1–27.2% more filled grains per panicle, 103.6–119.8% greater filled grain yield, and 18.0–35.8% higher 2AP content. A higher SOD activity (6.9–29.5%) was also observed in the above-mentioned rice than in the blast-infected rice that was not colonized and inoculated with NP19. Following blast infection, NP19 applied to leaves decreased blast lesion progression. Therefore, K. oryziphila NP19 was demonstrated to be a potential candidate for use as a plant growth-promoting bioagent and biopesticide for suppressing rice blast.

Synergistic biocontrol mixture: free-cell extract with β-glucanase activity and chitosan against Thielaviopsis paradoxa, Colletotrichum gloeosporioides, and Rhizopus stolonifer

Biocontrol, which uses antagonistic microorganisms to manage plant diseases, holds promise for reducing postharvest losses but faces challenges due to varied pathogen responses. In response, biocontrol mixtures were proposed to enhance the antagonistic effect. This study evaluates the efficacy of chitosan and extracellular glucanase (EG) from Wickerhamomyces anomalus, both individually and combined, against Thielaviopsis paradoxa, Colletotrichum gloeosporioides, and Rhizopus stolonifer. The maximum inhibition of mycelial growth by chitosan for these fungi was 90.25% at 3 mg/mL, 14.2% at 5 mg/mL, and 56.28% at 3 mg/mL, respectively. Additionally, chitosan significantly inhibited spore germination across all fungi. While W. anomalus cells prevented mycelial filament association, EG alone had minimal impact on mycelial development but inhibited spore germination in a concentration-dependent manner. Combining EG with chitosan enhanced spore germination inhibition, notably achieving 98.18%. The biocontrol mixture effectively protected fruit under laboratory-controlled conditions, suggesting a robust biocontrol strategy for postharvest fungal disease management.

Inhibitory activity and antioomycete mechanism of citral against Phytophthora capsici

The natural terpenoid citral has antifungal activity against multiple fungi, but its bioactivity against oomycetes is unclear. Therefore, this study investigated the antioomycete activity and mechanism of citral against Phytophthora capsici, a highly destructive invasive oomycete. Results showed that citral not only had a great inhibition on the mycelial growth of P. capsici (EC50 = 94.15 mg/L), but also had a significant inhibition on multiple spores, such as sporangia formation, zoospore discharge and zoospore germination. Citral at 4000 mg/L exhibited favorable protective (73.33%) and curative efficacy (55.11%) against pepper Phytophthora blight. Citral significantly damaged the hyphal morphology, disrupted the cell membrane integrity, increased the permeability of cell membrane, and increased the glycerol content in P. capsici. A total of 250 upregulated and 288 downregulated proteins were identified in iTRAQ-based quantitative proteomic analysis. Downregulated proteins were mostly enriched in pathways of ABC transporters, cyanoamino acid metabolism and starch and sucrose metabolism, suggesting an inhibition of citral on transmembrane transporter (e.g., ABC transporters) and pathogenicity (e.g., β-glucosidases) proteins. Upregulated proteins were enriched in biosynthesis of unsaturated fatty acids, pyruvate metabolism and glycolysis/gluconeogenesis, suggesting an activation of citral on energy generation proteins, including acyl-CoA oxidase, D-lactate dehydrogenase, pyruvate kinase, acetyl-CoA synthetase and phosphoenolpyruvate carboxykinase. Biochemical and iTRAQ analysis suggested that cell membrane may be the target of citral in P. capsici.

Histological characterization of downy mildew infection in wild rocket (Diplotaxis tenuifolia)

AbstractWild rocket downy mildew (DM), caused by oomycete Hyaloperonospora sp., is a widely spread disease reducing crop production and quality. New productive wild rocket varieties resistant to DM are crucial to control disease and ensure high quality leaves. A histological characterization of Hyaloperonospora sp. infection was performed in 11 wild rocket (Diplotaxis tenuifolia) accessions with contrasting DM responses (R, resistant; PR, partially resistant; S, susceptible). Samples of infected cotyledons, first and second leaves of 14‐day‐old seedlings were collected at 3, 6, 9, 21 and 24 h post‐inoculation (hpi) and 7 days post‐inoculation (dpi) and stained for appressoria and haustoria observation and tissue necrosis evaluation. Occurrence of appressoria on host surfaces was higher in leaves compared with cotyledons but unrelated with DM resistance response. Haustoria growth in mycelium was delayed in R accessions from 3 hpi, and signs of cell hypersensitivity reaction were observed at 9 hpi. At 24 hpi, necrotic spots limited pathogen growth in resistant accessions, whilst mycelium invaded larger mesophyll areas and produced more haustoria in susceptible ones. At 7 dpi, sporulation was heavy in S, restricted to droplet deposition sites in PR, and did not occur in R hosts. A rapid response of R accessions resulted in slower mycelium growth and longer infection periods. These traits have important agronomic value and should be considered in germplasm selection for breeding programmes aimed at crop protection. A better understanding of the host response to DM infection will allow selection of more suitable wild rocket accessions in future breeding programmes.

Transcription Factor CgSte12 Regulates Pathogenicity by Affecting Appressorium Structural Development in the Anthracnose-Causing Fungus Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is the causal agent of poplar anthracnose, which induces major economic losses and adversely affects the ecosystem services of poplar forests. The appressorium serves as a penetration structure for many pathogenic fungi, including C. gloeosporioides. The production of mucilage and the formation of penetration pegs are critically important for the appressorium-mediated penetration of host tissues. We previously found that CgPmk1 is a key protein involved in appressorium formation, penetration, and pathogenicity. Although CgSte12, which is a transcription factor that functions downstream of CgPmk1, regulates the formation of penetration pegs, its role in C. gloeosporioides appressorium development and pathogenicity has not been elucidated. Here, we developed C. gloeosporioides CgSTE12 mutants and characterized the molecular and cellular functions of CgSTE12. The results showed that mycelial growth and morphology were not affected in the CgSTE12 knockout mutants, which produced normal melanized appressoria. However, these mutants had less mucilage secreted around the appressoria, impaired appressorial cone formation, and the inability to form penetration pores and pegs, which ultimately led to a significant loss of pathogenicity. Our comparative transcriptome analysis revealed that CgSte12 controls the expression of genes involved in appressorium development and function, including genes encoding cutinases, NADPH oxidase, spermine biosynthesis-related proteins, ceramide biosynthesis-related proteins, fatty acid metabolism-related proteins, and glycerophospholipid metabolism-related proteins. Overall, our findings indicate that CgSte12 is a critical regulator of appressorium development and affects C. gloeosporioides pathogenicity by modulating the structural integrity of appressoria.

Amino acid substitutions in succinate dehydrogenase complex conferring resistance to the SDHI fungicide pydiflumetofen in Cochlibolus heterostrophus causing southern corn leaf blight1

Southern corn leaf blight (SCLB) caused by Cochlibolus heterostrophus, is a widespread foliar disease that has a substantial impact on maize yield in the Huanghuaihai region of China. Pydiflumetofen (Pyd), a new succinate dehydrogenase inhibitor (SDHI), has been found as a promising fungicide for the efficient control of SCLB, however, resistance of C. heterostrophus to Pyd has not been studied well. Here, five Pyd-resistant mutants were generated through fungicide adaptation. Sequence alignment analysis revealed that these mutants primarily mutated in ChSdhB and ChSdhD, with 3 genotypes: ChSdhBH277Y, ChSdhBI279T and ChSdhDH133Y, exhibiting two distinct categories of resistance: high resistance (HR) and moderate resistance (MR), which resistance factors (RF) is 214.22 and 44.33-53.67, respectively. These mutants were more pathogenic than the wild-type parental strains, but there was a significant reduction in mycelial growth rate and sporulation in the resistant mutants, indicating a significant fitness cost associated with resistance to Pyd. In addition, this study revealed a positive cross-resistance between Pyd and another SDHI fungicide cyclobutrifluram. However, no cross-resistance was found between Pyd and other classes of fungicide, including prochloraz, fludioxonil, iprodioneand pyraclostrobin. Homology modeling and molecular docking further confirmed that point mutation of ChSdhBH277Y, ChSdhBI279T, and ChSdhDH133Y could reduce binding affinity between Pyd and its target subunits from −74.07, −74.07, −152.52 kcal mol-1 to −3.90, −4.95, −9.93 kcal/mol, respectively. These findings not only provided valuable insights for managing SCLB caused by C. heterostrophus, but also enhanced our understanding of molecular mechanism underlying plant pathogen resistance to Pyd.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

Enhanced mycelium biomass and polysaccharide production in genetically modified Pleurotus ostreatus using agricultural wastes

Edible fungi, healthier for humans and sustainable for the planet, attract unprecedented attention. In the study, the genetically modified Pleurotus ostreatus overexpression phosphoglucomutase (PGM) was constructed. P. ostreatus overexpression PGM (Po::PGM) had 4.96-folds higher expression level of PGM. Po::PGM grew thicker mycelium and more mycelium branches. Additional Ca2+ can inhibit mycelium growth, and cyclic adenosine monophosphate completely inhibited their growth of Po::PGM. Secondly, Overexpression of PGM made P. ostreatus become more sensitive to cell wall disruptors, and caused 12.75 % reduction of β-1, 3-glucan and 40.53 % increase of chitin in cell wall. In submerged fermentation, the mycelia biomass yield and endopolysaccharide (IPS) production of Po::PGM in basic PDB can reach 11.18 g/l and 2.55 g/l, increasing by 20.86 % and 28.79 %, respectively. Whereas exopolysaccharide (EPS) reduced by 3.28 %. After replacing potato and glucose in PDB by wheat bran, mycelia biomass and EPS production of Po::PGM were all improved. The additional lactose in wheat bran did not only furtherly enhance mycelia biomass yield of Po::PGM to 27.78 g/l by 199.03 %, but IPS production also increased by 277.99 % to 6.07 g/l. The results provided us key ideas and important research directions that at least manipulating the PGM gene could obtain high-efficient use of agricultural wastes producing more fungus-based foods.

Aspergillus Cell Surface Structural Analysis and Its Applications to Industrial and Medical Use

The hyphal surface of cells of filamentous fungi is covered with cell wall, which is mainly composed of polysaccharides. Since the cell wall is the first structure to come in contact with the infection host, the environment, and the fungus itself, the elucidation of the cell wall structure and biogenesis is essential for understanding fungal ecology. Among filamentous fungi, the genus Aspergillus is an important group in the industrial, food, and medical fields. It is known that Aspergillus species form hyphal pellets in shake liquid culture. The authors previously found the role of α-1,3-glucan in hyphal aggregation in Aspergillus species. In addition, extracellular polysaccharide galactosaminogalactan contributed to hyphal aggregation as well, and dual disruption of biosynthesis genes of α-1,3-glucan and galactosaminogalactan resulted in complete hyphal dispersion in shake liquid culture. The characteristic of mycelia to form pellets under liquid culture conditions was the main reason why the growth measurement methods used for unicellular organisms could not be applied. We reported that hyphal growth of the dual disruption mutant could be measured by optical density. A real-time plate reader could be used to determine the growth curve of the mycelial growth of the dual disruption mutant. This measurement approach not only provides basic microbiological insights in filamentous fungi, but also has the potential to be applied to high-throughput screening of anti-Aspergillus drugs.