Integration of Transcriptomics and WGCNA to Characterize Trichoderma harzianum-Induced Systemic Resistance in Astragalus mongholicus for Defense against Fusarium solani.

Beneficial fungi of the genus Trichoderma are among the most widespread biocontrol agents that induce a plant's defense response against pathogens. Fusarium solani is one of the main pathogens that can negatively affect Astragalus mongholicus production and quality. To investigate the impact of Trichoderma harzianum on Astragalus mongholicus defense responses to Fusarium solani, A. mongholicus roots under T. harzianum + F. solani (T + F) treatment and F. solani (F) treatment were sampled and subjected to transcriptomic analysis. A differential expression analysis revealed that 6361 differentially expressed genes (DEGs) responded to T. harzianum induction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the 6361 DEGs revealed that the genes significantly clustered into resistance-related pathways, such as the plant-pathogen interaction pathway, phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, isoflavonoid biosynthesis pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction pathway. Pathway analysis revealed that the PR1, formononetin biosynthesis, biochanin A biosynthesis, and CHIB, ROS production, and HSP90 may be upregulated by T. harzianum and play important roles in disease resistance. Our study further revealed that the H2O2 content was significantly increased by T. harzianum induction. Formononetin and biochanin A had the potential to suppress F. solani. Weighted gene coexpression network analysis (WGCNA) revealed one module, including 58 DEGs associated with T. harzianum induction. One core hub gene, RPS25, was found to be upregulated by T. harzianum, SA (salicylic acid) and ETH (ethephon). Overall, our data indicate that T. harzianum can induce induced systemic resistance (ISR) and systemic acquired resistance (SAR) in A. mongholicus. The results of this study lay a foundation for a further understanding of the molecular mechanism by which T. harzianum induces resistance in A. mongholicus.

Evaluation of synergistic effect of entomopathogenic fungi Beauveria bassiana and Lecanicillium lecacii on the mosquito Culex quinquefaciatus.

Vector-borne diseases resulted into several cases of human morbidity and mortality over the years and among them is filariasis, caused by the mosquito Culex quinquefasciatus. Developing novel strategies for mosquito control without jeopardizing the environmental conditions has always been a topic of discussion and research. Integrated Vector Management (IVM) emphasizes a comprehensive approach and use of a range of strategies for vector control. Recent research evaluated the use of two entomopathogenic fungi; Beauveria bassiana and Lecanicillium lecanii in IVM, which can serve as potential organic insecticide for mosquito population control. However, their combined efficacy has not yet been evaluated against mosquito control in prior research and a gap of knowledge is still existing. So, this research was an attempt to bridge up the knowledge gap by (1) Assessing the combined efficacy of Beauveria bassiana and Lecanicillium lecanii on Culex quinquefasciatus (2) To investigate the sub-lethal concentration (LC50) of the combined fungal concentration and (3) To examine the post-mortem effects caused by the combined fungal concentration under Scanning Electron Microscope (SEM). The larval pathogenicity assay was performed on 4th instar C. quinquefasciatus larvae. Individual processed fungal solution of B. bassiana and L. lecanii were procured and to test the combined efficacy, the two solutions were mixed in equal proportions. To evaluate the sub-lethal concentration (LC50), different concentrations of the combined fungal solution were prepared by serial dilations. The mortality was recorded after 24 hours for each concentration. Upon treatment and evaluation, The LC50 values of B. bassiana and L. lecanii were 0.25 x 104 spores/ml and 0.12 x 104 spores/ml respectively and the combined fungal concentration was 0.06 x 103 spores/ml. This clearly indicated that the combined efficacy of the fungi is more significant. Further, SEM analysis revealed morphological deformities and extensive body perforations upon combined fungal treatment. These findings suggested that combining the two fungi can be a more effective way in controlling the population of Culex quinquefasciatus.

A secreted fungal laccase targets the receptor kinase OsSRF3 to inhibit OsBAK1-OsSRF3-mediated immunity in rice.

The identification effector targets and characterization of their functions are crucial for understanding pathogen infection mechanisms and components of plant immunity. Here, we identify the effector UgsL, a ustilaginoidin synthetase with a key role in regulating virulence of the rice false smut fungus Ustilaginoidea virens. Heterologous expression of UgsL in rice (Oryza sativa) enhances plant susceptibility to multiple pathogens, and host-induced gene silencing of UgsL enhances plant resistance to U. virens, indicating that UgsL inhibits rice immunity. UgsL interacts with STRUBBELIG RECEPTOR KINASE 3 (OsSRF3). Genome editing and overexpression of OsSRF3 demonstrate that OsSRF3 plays a pivotal role in the resistance of rice to multiple pathogens. Remarkably, overexpressing OsSRF3 enhances resistance without adversely affecting plant growth or yield. We show that BRASSINOSTEROID RECEPTOR-ASSOCIATED KINASE 1 (OsBAK1) interacts with and phosphorylates OsSRF3 to activate pathogen-triggered immunity, inducing the mitogen-activated protein kinase cascade, a reactive oxygen species burst, callose deposition, and expression of defense-related genes. UgsL interferes with the phosphorylation of OsSRF3 by OsBAK1. Furthermore, UgsL mediates OsSRF3 degradation by facilitating its association with the ubiquitin-26S proteasome. Our results reveal that OsSRF3 positively regulates immunity in rice and that UgsL mediates its degradation, thereby inhibiting the activation of OsBAK1-OsSRF3-mediated immune pathways.

Ustilaginoidea virens, an emerging pathogen of rice: the dynamic interplay between the pathogen virulence strategies and host defense.

MAIN CONCLUSION: The Ustilaginoidea virens -rice pathosystem has been used as a model for flower-infecting fungal pathogens. The molecular biology of the interactions between U. virens and rice, with an emphasis on the attempt to get a deeper comprehension of the false smut fungus's genomes, proteome, host range, and pathogen biology, has been investigated. Meta-QTL analysis was performed to identify potential QTL hotspots for use in marker-assisted breeding. The Rice False Smut (RFS) caused by the fungus Ustilaginoidea virens currently threatens rice cultivators across the globe. RFS infects rice panicles, causing a significant reduction in grain yield. U. virens can also parasitize other hosts though they play only a minor role in its life cycle. Furthermore, because it produces mycotoxins in edible rice grains, it puts both humans and animals at risk of health problems. Although fungicides are used to control the disease, some fungicides have enabled the pathogen to develop resistance, making its management challenging. Several QTLs have been reported but stable gene(s) that confer RFS resistance have not been discovered yet. This review offers a comprehensive overview of the pathogen, its virulence mechanisms, the genome and proteome of U. virens, and its molecular interactions with rice. In addition, information has been compiled on reported resistance QTLs, facilitating the development of a consensus genetic map using meta-QTL analysis for identifying potential QTL hotspots. Finally, this review highlights current developments and trends in U. virens-rice pathosystem research while identifying opportunities for future investigations.

Trichoderma based formulations control the wilt disease of chickpea (Cicer arietinum L.) caused by Fusarium oxysporum f. sp. ciceris, better when inoculated as consortia: findings from pot experiments under field conditions.

Background: Commercial/chemical pesticides are available to control Fusarium wilt of chickpea, but these antifungals have numerous environmental and human health hazards. Amongst various organic alternatives, use of antagonistic fungi like Trichoderma, is the most promising option. Although, Trichoderma spp. are known to control Fusarium wilt in chickpea but there are no reports that indicate the biocontrol efficacy of indigenous Trichoderma spp. against the local pathogen, in relation to environmental conditions. Methods: In the present study, biological control activity of Trichoderma species formulations viz., Trichoderma asperellum, Trichoderma harzianum (strain 1), and Trichoderma harzianum (strain 2), either singly or in the form of consortia, was investigated against Fusarium oxysporum f. sp. ciceris, the cause of Fusarium wilt in chickpea, in multiyear pot trials under open field conditions. The antagonistic effect of Trichoderma spp. was first evaluated in in vitro dual culture experiments. Then the effects of Trichoderma as well as F. oxysporum, were investigated on the morphological parameters, disease incidence (DI), and disease severity (DS) of chickpea plants grown in pots. Results: In dual culture experiments, all the Trichoderma species effectively reduced the mycelial growth of F. oxysporum. T. asperellum, T. harzianum (strain 1), and T. harzianum(strain 2) declined the mycelial growth of F. oxysporumby 37.6%, 40%, and 42%. In open field pot trials, the infestation of F. oxysporum in chickpea plants significantly reduced the morphological growth of chickpea. However, the application of T. asperellum, T. harzianum (strain 1), and T. harzianum (strain 2), either singly or in the form of consortia, significantly overcome the deleterious effects of the pathogen, thereby resulted in lower DI (22.2% and 11.1%) and DS (86% and 92%), and ultimately improved the shoot length, shoot fresh weight and shoot dry weight by 69% and 72%, 67% and 73%, 68% and 75%, during the years 1 and 2, respectively, in comparison with infested control. The present study concludes the usefulness and efficacy of Trichoderma species in controlling wilt disease of chickpea plants under variable weather conditions.

Role of chitinase expression in the virulence of Lecanicillium lecanii against citrus black aphid (Toxoptera aurantii).

Chitinase plays a vital role in the virulence of entomopathogenic fungi (EPF) when it infects host insects. We used gene recombination technology to express chitinase of three strains of Lecanicillium lecanii: Vl6063, V3450, and Vp28. The ORF of ChitVl6063, ChitV3450 and ChitVp28 were inserted into the fungal expression vector pBARGPE-1, which contained strong promoter and terminator, respectively, to construct a chitinase overpressing plasmid, then transformed the wild-type strain with blastospore transformation method. The virulence of the three recombinant strains against Toxoptera aurantii was improved by overproduction of ChitVl6063, ChitV3450, and ChitVp28, as demonstrated by significantly lower 3.43 %, 1.72 %, and 1.23 % fatal doses, respectively, according to an insect bioassay. Similarly, lethal times of recombinants (ChitVl6063, ChitV3450 and ChitVp28) were also decreased up to 29.51 %, 30.46 % and 33.90 %, respectively, compared to the wild-type strains. Improving the expression of chitinase is considered as an effective method for the enhancement of the EPF value. The efficacy could be enhanced using recombinant technology, which provides a prospecting view for future insecticidal applications.

A potent endophytic fungus Purpureocillium lilacinum YZ1 protects against Fusarium infection in field-grown wheat.

Fusarium diseases pose a severe global threat to major cereal crops, particularly wheat. Existing biocontrol strains against Fusarium diseases are believed to primarily rely on antagonistic mechanisms, but not widely used under field conditions. Here, we report an endophytic fungus, Purpureocillium lilacinum YZ1, that shows promise in combating wheat Fusarium diseases. Under glasshouse conditions, YZ1 inoculation increased the survival rate of Fusarium graminearum (Fg)-infected wheat seedlings from 0% to > 60% at the seedling stage, and reduced spikelet infections by 70.8% during anthesis. In field trials, the application of YZ1 resulted in an impressive 89.0% reduction in Fg-susceptible spikelets. While a slight antagonistic effect of YZ1 against Fg was observed on plates, the induction of wheat systemic resistance by YZ1, which is distantly effective, non-specific, and long-lasting, appeared to be a key contributor to YZ1's biocontrol capabilities. Utilizing three imaging methods, we confirmed YZ1 as a potent endophyte capable of rapid colonization of wheat roots, and systematically spreading to the stem and leaves. Integrating dual RNA-Seq, photosynthesis measurements and cell wall visualization supported the link between YZ1's growth-promoting abilities and the activation of wheat systemic resistance. In conclusion, endophytes such as YZ1, which exhibits non-antagonistic mechanisms, hold significant potential for industrial-scale biocontrol applications.

Clavispora lusitaniae: From a saprophytic yeast to an emergent pathogen.

Clavispora lusitaniae has been isolated from different substrates, such as soil, water, fruit, vegetables, plants, and the gastrointestinal tract of animals and humans. However, its importance lies in being isolated from in invasive infections, particularly in pediatric patients with hematologic malignancies. It is an emerging nosocomial pathogen commonly associated with fatal prognosis in immunocompromised hosts. C. lusitaniae has attracted attention in the last decade because of resistance to amphotericin B, 5- flucytosine, and fluconazole. The adaptations of this yeast to the human host may contribute to its pathogenicity. Further study will be needed to understand C. lusitaniae's ability as a potential pathogen. This mini-review highlights the importance of the growing number of invasive disease cases caused by this yeast.

Biostimulant and antagonistic potential of endophytic fungi against fusarium wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici.

Endophytic fungal-based biopesticides are sustainable and ecologically-friendly biocontrol agents of several pests and diseases. However, their potential in managing tomato fusarium wilt disease (FWD) remains unexploited. This study therefore evaluated effectiveness of nine fungal isolates against tomato fusarium wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL) in vitro using dual culture and co-culture assays. The efficacy of three potent endophytes that inhibited the pathogen in vitro was assessed against FWD incidence, severity, and ability to enhance growth and yield of tomatoes in planta. The ability of endophytically-colonized tomato (Solanum lycopersicum L.) plants to systemically defend themselves upon exposure to FOL were also assessed through defence genes expression using qPCR. In vitro assays showed that endophytes inhibited and suppressed FOL mycelial growth better than entomopathogenic fungi (EPF). Endophytes Trichoderma asperellum M2RT4, Hypocrea lixii F3ST1, Trichoderma harzianum KF2R41, and Trichoderma atroviride ICIPE 710 had the highest (68.84-99.61%) suppression and FOL radial growth inhibition rates compared to EPF which exhibited lowest (27.05-40.63%) inhibition rates. Endophytes T. asperellum M2RT4, H. lixii F3ST1 and T. harzianum KF2R41 colonized all tomato plant parts. During the in planta experiment, endophytically-colonized and FOL-infected tomato plants showed significant reduction of FWD incidence and severity compared to non-inoculated plants. In addition, these endophytes contributed to improved growth promotion parameters and yield. Moreover, there was significantly higher expression of tomato defence genes in T. asperellum M2RT4 colonized than in un-inoculated tomato plants. These findings demonstrated that H. lixii F3ST1 and T. asperellum M2RT4 are effective biocontrol agents against FWD and could sustainably mitigate tomato yield losses associated with fusarium wilt.

Regulation of morphogenesis and pathogenicity by OsMep2, OsCph1, and OsPes1 in dimorphic entomopathogenic fungus Ophiocordyceps sinensis (Hypocreales: Ophiocordycipitaceae).

Polarized growth plays a key role in all domains of their biology, including morphogenesis and pathogenicity of filamentous fungi. However, little information is available about the determinants of polarized growth. The fungal Mep2, Pes1, and Cph1 proteins were identified to be involved in the dimorphic transition between yeast and hyphal forms in Candida albicans. In this study, evidence that the dimorphic fungal entomopathogen Ophiocordyceps sinensis Mep2, Pes1, and Cph1 proteins are involved in polarized growth is presented. OsMep2 was significantly upregulated at aerial hyphae and conidia germination stages. OsCph1 was significantly upregulated at aerial hyphae, conidia initiation, and conidia germination stages, and OsPes1 was significantly upregulated at the conidia germination stage. Deletions of OsMep2, OsCph1, and OsPes1 provoked defects in the polarized growth. The abilities of hyphal formation and the yields of blastospores and conidia for the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants were significantly reduced. The conidia yields of the ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were decreased by 69.17%, 60.90%, and 75.82%, respectively. Moreover, the pathogenicity of the ∆ OsMep2, ∆OsCph1, and ∆ OsPes1 mutants against Thitarodes xiaojinensis was significantly reduced. The mummification rate caused by wide type and ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were 36.98% ± 8.52%, 0.31% ± 0.63%, 1.15% ± 1.57%, and 19.69% ± 5.6%, respectively. These results indicated that OsMep2, OsCph1, and OsPes1 are involved in the regulation of hyphal formation, sporulation, and pathogenicity of O. sinensis. This study provided a basis for the understanding of the fungal dimorphic development and improving the efficiency of artificial cultivation of O. sinensis.

Transcriptional Activator UvXlnR Is Required for Conidiation and Pathogenicity of Rice False Smut Fungus Ustilaginoidea virens.

Transcription factors play critical roles in diverse biological processes in fungi. XlnR, identified as a transcriptional activator that regulates the expression of the extracellular xylanase genes in fungi, has not been extensively studied for its function in fungal development and pathogenicity in rice false smut fungus Ustilaginoidea virens. In this study, we characterized UvXlnR in U. virens and established that the full-length, N-terminal, and C-terminal forms have the ability to activate transcription. The study further demonstrated that UvXlnR plays crucial roles in various aspects of U. virens biology. Deletion of UvXlnR affected growth, conidiation, and stress response. UvXlnR mutants also exhibited reduced pathogenicity, which could be partially attributed to the reduced expression of xylanolytic genes and extracellular xylanase activity of U. virens during the infection process. Our results indicate that UvXlnR is involved in regulating growth, conidiation, stress response, and pathogenicity.

Activity of natural occurring entomopathogenic fungi on nymphal and adult stages of Philaenus spumarius.

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia, Italy and the rest of Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P, spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius (≈ 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P, spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis®, while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave way for testing EPF isolates against P, spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.

Rice false smut virulence protein subverts host chitin perception and signaling at lemma and palea for floral infection.

The flower-infecting fungus Ustilaginoidea virens causes rice false smut, which is a severe emerging disease threatening rice (Oryza sativa) production worldwide. False smut not only reduces yield, but more importantly produces toxins on grains, posing a great threat to food safety. U. virens invades spikelets via the gap between the 2 bracts (lemma and palea) enclosing the floret and specifically infects the stamen and pistil. Molecular mechanisms for the U. virens-rice interaction are largely unknown. Here, we demonstrate that rice flowers predominantly employ chitin-triggered immunity against U. virens in the lemma and palea, rather than in the stamen and pistil. We identify a crucial U. virens virulence factor, named UvGH18.1, which carries glycoside hydrolase activity. Mechanistically, UvGH18.1 functions by binding to and hydrolyzing immune elicitor chitin and interacting with the chitin receptor CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (OsCERK1) to impair their chitin-induced dimerization, suppressing host immunity exerted at the lemma and palea for gaining access to the stamen and pistil. Conversely, pretreatment on spikelets with chitin induces a defense response in the lemma and palea, promoting resistance against U. virens. Collectively, our data uncover a mechanism for a U. virens virulence factor and the critical location of the host-pathogen interaction in flowers and provide a potential strategy to control rice false smut disease.

Metagenomic sequencing for detection and identification of the boxwood blight pathogen Calonectria pseudonaviculata.

Pathogen detection and identification are key elements in outbreak control of human, animal, and plant diseases. Since many fungal plant pathogens cause similar symptoms, are difficult to distinguish morphologically, and grow slowly in culture, culture-independent, sequence-based diagnostic methods are desirable. Whole genome metagenomic sequencing has emerged as a promising technique because it can potentially detect any pathogen without culturing and without the need for pathogen-specific probes. However, efficient DNA extraction protocols, computational tools, and sequence databases are required. Here we applied metagenomic sequencing with the Oxford Nanopore Technologies MinION to the detection of the fungus Calonectria pseudonaviculata, the causal agent of boxwood (Buxus spp.) blight disease. Two DNA extraction protocols, several DNA purification kits, and various computational tools were tested. All DNA extraction methods and purification kits provided sufficient quantity and quality of DNA. Several bioinformatics tools for taxonomic identification were found suitable to assign sequencing reads to the pathogen with an extremely low false positive rate. Over 9% of total reads were identified as C. pseudonaviculata in a severely diseased sample and identification at strain-level resolution was approached as the number of sequencing reads was increased. We discuss how metagenomic sequencing could be implemented in routine plant disease diagnostics.

Cyclophilin acts as a ribosome biogenesis factor by chaperoning the ribosomal protein (PlRPS15) in filamentous fungi.

The rapid transport of ribosomal proteins (RPs) into the nucleus and their efficient assembly into pre-ribosomal particles are prerequisites for ribosome biogenesis. Proteins that act as dedicated chaperones for RPs to maintain their stability and facilitate their assembly have not been identified in filamentous fungi. PlCYP5 is a nuclear cyclophilin in the nematophagous fungus Purpureocillium lilacinum, whose expression is up-regulated during abiotic stress and nematode egg-parasitism. Here, we found that PlCYP5 co-translationally interacted with the unassembled small ribosomal subunit protein, PlRPS15 (uS19). PlRPS15 contained an eukaryote-specific N-terminal extension that mediated the interaction with PlCYP5. PlCYP5 increased the solubility of PlRPS15 independent of its catalytic peptide-prolyl isomerase function and supported the integration of PlRPS15 into pre-ribosomes. Consistently, the phenotypes of the PlCYP5 loss-of-function mutant were similar to those of the PlRPS15 knockdown mutant (e.g. growth and ribosome biogenesis defects). PlCYP5 homologs in Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, Sclerotinia sclerotiorum, Botrytis cinerea and Metarhizium anisopliae were identified. Notably, PlCYP5-PlRPS15 homologs from three filamentous fungi interacted with each other but not those from other species. In summary, our data disclosed a unique dedicated chaperone system for RPs by cyclophilin in filamentous fungi.

Advances in the biological control of phytoparasitic nematodes via the use of nematophagous fungi.

Agricultural production is one of most important activities for food supply and demand, that provides a source of raw materials, and generates commercial opportunities for other industries around the world. It may be both positively and negatively affected by climatic and biological factors. Negative biological factors are those caused by viruses, bacteria, or parasites. Given the serious problems posed by phytoparasitic nematodes for farmers, causing crop losses globally every year, the agrochemical industry has developed compounds with the capacity to inhibit their development; however, they can cause the death of other beneficial organisms and their lixiviation can contaminate the water table. On the other hand, the positive biological factors are found in biotechnology, the scientific discipline that develops products, such as nematophagous fungi (of which Purpureocillium lilacinum and Pochonia chlamydosporia have the greatest potential), for the control of pests and/or diseases. The present review focuses on the importance of nematophagous fungi, particularly sedentary endoparasitic nematodes, their research on the development of biological control agents, the mass production of fungi Purpureocillium lilacinum and Pochonia chlamydosporia, and their limited commercialization due to the lack of rigorous methods that enable the anticipation of complex interactions between plant and phytopathogenic agents.

Isolation and Purification of a New Bacillus Subtilis Strain from Deer Dung with Anti-microbial and Anti-cancer Activities.

OBJECTIVE: Bacillus strains are well known for their natural bioactive products that have antimicrobial and/or anti-cancer activities. Many of Bacillus' structurally unique metabolites can combat human diseases, including cancers. However, because Bacillus' metabolites are so abundant, few have been studied extensively enough to fully characterize their chemical constitutions and biological functions. METHODS: In this study, we focused on the isolation and purification of a new Bacillus strain, and determined the effects of its metabolites on bacteria and cancer cells. Our study focused on a new strain of Bacillus isolated from deer dung. Based on BLAST results, this isolate belongs to Bacillus subtilis, and therefore we named the strain Bacillus subtilis NC16. Congo red assay was used to test the cellulase activity. The inhibition zone was measured to test the antimicrobial activity. CCK-8, wound healing and flow cytometry were used to test the anti-cancer activity. RESULTS: Metabolites from Bacillus subtilis NC16 have both antimicrobial and anti-cancer activities. They can both suppress the growth of Trichoderma vride and Staphylococcus aureus, and inhibit the proliferation and promote the apoptosis of non-small cell lung cancer cell lines. CONCLUSION: Our results suggest that Bacillus subtilis NC16 can not only degrade cellulose, but its metabolites may be sources of antibiotics and anti-cancer drugs.

Activation of defense response in common bean against stem rot disease triggered by Trichoderma erinaceum and Trichoderma viride.

White mold and stem rot is a common disease of Phaseolus vulgaris caused by Sclerotinia sclerotiorum. Biological control is a promising alternative for the control of this disease. In the present study, two Trichoderma spp., T. erinaceum and T. viride, and the consortium of both were evaluated as biocontrol agents against sclerotinia stem rot disease. The results revealed that T. erinaceum (NAIMCC-F-02171) and T. viride (NAIMCC-F-02500) when applied alone, significantly suppressed the infection rate of S. sclerotiorum and increased the rate of survival of plants by 74.5%. On the contrary, the combination of both the Trichoderma spp. was found to be more effective in reducing stem rot by 57.2% and increasing the survival of plants by 87.5% when compared to the individual Trichoderma applications. Further, the exogenous supplementation of Trichoderma activated antioxidative machineries, such as peroxidase, polyphenol oxidase, superoxide dismutase, catalase, and ascorbic acid in the plant. Besides, hydrogen peroxide and superoxide-free radical accumulation were also found to be reduced when T. erinaceum and T. viride were used either individually or in combination under the pathogen-challenged condition. Additionally, the photopigments in the bioprimed plants were markedly increased. Moreover, the combined inoculation of the two isolates yielded the highest records of growth parameters (root weight, shoot length, and leaf weight) compared with individual inoculation. Therefore, based on the above results, it was concluded that the combination of T. erinaceum and T. viride can be effectively used as an alternative to control white mold and stem rot caused by S. sclerotiorum.

A novel transcription factor UvCGBP1 regulates development and virulence of rice false smut fungus Ustilaginoidea virens.

Ustilaginoidea virens, causing rice false smut (RFS) is an economically important ascomycetous fungal pathogen distributed in rice-growing regions worldwide. Here, we identified a novel transcription factor UvCGBP1 (Cutinase G-box binding protein) from this fungus, which is unique to ascomycetes. Deletion of UvCGBP1 affected development and virulence of U. virens. A total of 865 downstream target genes of UvCGBP1 was identified using ChIP-seq and the most significant KEGG enriched functional pathway was the MAPK signaling pathway. Approximately 36% of target genes contain the AGGGG (G-box) motif in their promoter. Among the targets, deletion of UvCGBP1 affected transcriptional and translational levels of UvPmk1 and UvSlt2, both of which were important in virulence. ChIP-qPCR, yeast one-hybrid and EMSA confirmed that UvCGBP1 can bind the promoter of UvPmk1 or UvSlt2. Overexpression of UvPmk1 in the ∆UvCGBP1-33 mutant restored partially its virulence and hyphae growth, indicating that UvCGBP1 could function via the MAPK pathway to regulate fungal virulence. Taken together, this study uncovered a novel regulatory mechanism of fungal virulence linking the MAPK pathway mediated by a G-box binding transcription factor, UvCGBP1.

Effects of Trichoderma harzianum on Photosynthetic Characteristics and Fruit Quality of Tomato Plants.

The beneficial role of fungi from the Trichoderma genus and its secondary metabolites in promoting plant growth, uptake and use efficiency of macronutrients and oligo/micro-nutrients, activation of plant secondary metabolism and plant protection from diseases makes it interesting for application in environmentally friendly agriculture. However, the literature data on the effect of Trichoderma inoculation on tomato fruit quality is scarce. Commercially used tomato cultivars were chosen in combination with indigenous Trichodrema species previously characterized on molecular and biochemical level, to investigate the effect of Trichoderma on photosynthetic characteristics and fruit quality of plants grown in organic system of production. Examined cultivars differed in the majority of examined parameters. Response of cultivar Gruzanski zlatni to Trichoderma application was more significant. As a consequence of increased epidermal flavonols and decreased chlorophyll, the nitrogen balance index in leaves has decreased, indicating a shift from primary to secondary metabolism. The quality of its fruit was altered in the sense of increased total flavonoids content, decreased starch, increased Bioaccumulation Index (BI) for Fe and Cr, and decreased BI for heavy metals Ni and Pb. Higher expression of swolenin gene in tomato roots of more responsive tomato cultivar indicates better root colonization, which correlates with observed positive effects of Trichodrema.