Plant Pathogen Rhizoctonia Solani Research Articles

Peribacillus frigoritolerans T7-IITJ, a potential biofertilizer, induces plant growth-promoting genes of Arabidopsis thaliana.

This study aimed to isolate plant growth and drought tolerance-promoting bacteria from the nutrient-poor rhizosphere soil of Thar desert plants and unravel their molecular mechanisms of plant growth promotion. Among our rhizobacterial isolates, Enterobacter cloacae C1P-IITJ, Kalamiella piersonii J4-IITJ, and Peribacillus frigoritolerans T7-IITJ, significantly enhanced root and shoot growth (4-5-fold) in Arabidopsis thaliana under PEG-induced drought stress. Whole genome sequencing and biochemical analyses of the non-pathogenic bacterium T7-IITJ revealed its plant growth-promoting traits, viz., solubilization of phosphate (40-73µg/ml), iron (24±0.58mm halo on chrome azurol S media), and nitrate (1.58±0.01µg/ml nitrite), along with production of exopolysaccharides (125±20µg/ml) and auxin-like compounds (42.6±0.05µg/ml). Transcriptome analysis of A. thaliana inoculated with T7-IITJ and exposure to drought revealed the induction of 445 plant genes (log2fold-change>1, FDR<0.05) for photosynthesis, auxin and jasmonate signalling, nutrient uptake, redox homeostasis, and secondary metabolite biosynthesis pathways related to beneficial bacteria-plant interaction, but repression of 503 genes (log2fold-change < -1) including many stress-responsive genes. T7-IITJ enhanced proline 2.5-fold, chlorophyll 2.5-2.8-fold, iron 2-fold, phosphate 1.6-fold, and nitrogen 4-fold, and reduced reactive oxygen species 2-4.7-fold in plant tissues under drought. T7-IITJ also improved the germination and seedling growth of Tephrosia purpurea, Triticum aestivum, and Setaria italica under drought and inhibited the growth of two plant pathogenic fungi, Fusarium oxysporum, and Rhizoctonia solani. P. frigoritolerans T7-IITJ is a potent biofertilizer that regulates plant genes to promote growth and drought tolerance.

Bacillus subtilis and Bacillus amyloliquefaciens Mix Suppresses Rhizoctonia Disease and Improves Rhizosphere Microbiome, Growth and Yield of Potato (Solanum tuberosum L.).

Black scurf and stem canker caused by Rhizoctonia solani is a significant disease problem of potatoes. Currently, chemical methods are the primary means of controlling this pathogen. This study sought to explore an alternative approach by harnessing the biocontrol potential of a bacterial mix of Bacillus subtilis and Bacillus amyloliquefaciens against black scurf, and to determine their effect on rhizosphere microorganisms of soil microbiota. This study showed that these bacteria demonstrate antagonistic activity against Rhizoctonia solani. Reduced damage to potato plants during the growing season in Siberia was observed. The index of disease development decreased from 40.9% to 12.0%. The treatment of tubers with this mix of bacteria also led to a change in the composition of the rhizosphere microbiota (according to CFU, 16S and ITS sequencing). This effect was accompanied by a positive change in plant physiological parameters (spectrophotometric analysis). The concentration of chlorophyll in potatoes with the bacterial mix treatment increased by 1.3 fold (p ≤ 0.001), and of carotenoids by 1.2 fold (p ≤ 0.01) compared with the control. After bacterial mix treatment, the length of the aerial parts of plants was 1.3 fold higher (p ≤ 0.001), and the number of stems 1.4 fold higher (p ≤ 0.05). The yield of potatoes was increased by 8.2 t/ha, while the large tuber fraction was increased by 16% (p ≤ 0.05). The bacteria mix of Bacillus subtilis and Bacillus amyloliquefaciens suppressed the plant pathogenic fungus Rhizoctonia solani, and simultaneously enhanced the physiological parameters of potato plants. This treatment can be used to enhance the yield/quality of potato tubers under field conditions.

Mycoparasitism related targets of Tmk1 indicate stimulating regulatory functions of this MAP kinase in Trichoderma atroviride

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress.

Dissecting genomic regions and underlying sheath blight resistance traits in rice (Oryza sativa L.) using a genome-wide association study.

The productivity of rice is greatly affected by the infection of the plant pathogenic fungus Rhizoctonia solani, which causes a significant grain yield reduction globally. There exist a limited number of rice accessions that are available to develop sheath blight resistance (ShB). Our objective was to identify a good source of the ShB resistance, understand the heritability, and trait interactions, and identify the genomic regions for ShB resistance traits by genome-wide association studies (GWAS). In the present study, a set of 330 traditional landraces and improved rice varieties were evaluated for ShB resistance and created a core panel of 192 accessions used in the GWAS. This panel provides a more considerable amount of genetic variance and found a significant phenotypic variation among the panel of rice accessions for all the agro-morphological and disease-resistance traits over the seasons. The infection rate of ShB and disease reaction were calculated as percent disease index (PDI) and area under the disease progress curve (AUDPC). The correlation analysis showed a significant positive association between PDIs and AUPDC and a negative association between PDI and plant height, flag leaf length, and grain yield. The panel was genotyped with 133 SSR microsatellite markers, resulting in a genome coverage of 314.83 Mb, and the average distance between markers is 2.53 Mb. By employing GLM and MLM (Q + K) models, 30 marker-trait associations (MTAs) were identified with targeted traits over the seasons. Among these QTLs, eight were found to be novel and located on 2, 4, 8, 10, and 12 chromosomes, which explained the phenotypic variation ranging from 5% to 15%. With the GWAS approach, six candidate genes were identified. Os05t0566400, Os08t0155900, and Os09t0567300 were found to be associated with defense mechanisms against ShB. These findings provided insights into the novel donors of IC283139, IC 277248, Sivappuchithirai Kar, and Bowalia. The promising genomic regions on 10 of 12 chromosomes associated with ShB would be useful in developing rice varieties with durable disease resistance.

Evaluation of some bioactive compounds of &lt;i&gt;Azadirachta indica&lt;/i&gt; extracts and its application as safe fungicide against different plant pathogenic fungi

Extracts of leaves, seeds and twigs of Azadirachta indica was carried out using Soxhlet extraction technique. These extracts were subjected to percent yield, in vitro phytochemical screening and antifungal activities (minimum fungicidal concentration (MFC), minimum inhibitory concentration (MIC), zone of inhibition and spore germination assay) against plant pathogenic fungi Cladosporium fulvum, Colletotrichum coccodes, Fusarium oxysporum and Rhizoctonia solani. The Soxhlet extractive solvents were methanol, ethanol, chloroform, ethyl acetate and n–hexane. The maximum extractive yield were calculated in ethanol leaves (26±1.5%), methanol leaves (22±01%), n–hexane seeds (32±0.8%) and ethanol seeds (24±01%), while twigs calculated minimum extractive yield. The phytochemicals screening findings confirmed the occurrence of terpenoids, phenols, flavonoids, glycosides, alkaloids, tannins and saponins. The antifungal activities observed that leaves and seeds extracts of neem might cause the inhibition of mycelium and spore germination of tested fungi, though the antifungal activities rate of under study fungal strains diverse with dissimilar concentration and nature of extracts. However the entire concentrations of extract and crude extract especially the methanol and ethanol extracts of neem seeds and leaves reduce the plant pathogenic fungi growth at noteworthy rate. It was concluded from our results that neem leaves and seeds extracts were effectual as antifungal agents against the entire under study fungal strains however the most sensitive fungi were Rhizoctonia solani and Fusarium oxysporum. The findings acquired from this study revealed that chemical constituents from neem seeds and leaves may be formulated as latent agrochemical fungicides.

Molecular Identification of Juglans Regia Endophyte LTL-G3, Its Antifungal Potential and Bioactive Substances.

Endophyte is one of the potential biocontrol agents for inhibiting plant pathogens. However, the mechanisms and characteristics involved in the inhibition of different phytopathogenic fungi by endophytes, especially walnut endophytes, are still largely unknown. The present study aimed to identify the walnut endophytic fungus LTL-G3 from a genetic point of view, assess the strain's antifungal activity, and determine the bioactivities of the substances it produces against plant pathogens. The homologous sequence of strain LTL-G3 was examined, and typical strains of the Trichoderma virens group were used to build NJ phylogenetic trees and analyze the taxonomic position of the strain. The biocontrol agent's antagonistic potential for many plant pathogenic fungi. By using silica gel G chromatography, the active components of the strain were separated and purified. The active components were identified using GC-MS and NMR. The strain LTL-G3 was identified as Trichoderma virens. Its fermentation and secondary metabolite extracts had a broad spectrum and strong inhibitory effect on the spread of six plant pathogens (Botrytis cinerea, Fusarium graminearum, Gloeosporium fructigenum, Phytophthora capsici, Rhizoctonia solani, and Valsa mali) evaluated, of which, its inhibition rate against Valsa mali reached 76.6% (fermentation extract) and 100% (ethyl acetate and n-butanol extracts). On silica gel G chromatography, bioactive compounds were divided into 6 fractions and 7 sub-fractions. Fr.2-2 was the sub-fraction that showed the greatest inhibitory against V. mali, as an inhibition percentage of 89.36% in 1 mg. mL-1. Fifteen key inhibitory chemicals identified using GC-MS. By examining the NMR data, the chemical make-up of the precipitated white solid was identified. The inhibition rate against V. mali increased by over 95% at a dosage of 1 mg. mL-1, indicating a significant linear association between compound A and that rate. The strain LTL-G3 can be applied as an efficient biological control agent against V. mali, and its highly inhibitive secondary metabolites provide the mechanism for this action.

Improvement of antagonism and fungicides tolerance in Trichoderma harzianum and T.viride local isolates by Ultra-Violet irradiation

Among 22 Iraqi Trichoderma harzianum and T.viride isolates, T3,T5,D5,S1 and S6 related to T. harzianum showed a higher antagonistic activity against the plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. These isolates were selected for improvement their biocontrol activity by usi&#x0D; ng uv-irradiation. The optimum exposure time to ultraviolet irradiation is 30 min, in which, the percentage of kill was 98-99% .After UV-irradiation treatment (Quartz lamps 30W at 240-280 nm, peak 254nm, distance from the source was 20 cm), antagonistic capability against R. solani and F. oxysporum was improved in mutants MT3,MT5,MS1 and MS6, in which antagonistic reaction score were 3.6,3.9,3.2 and 3.6 against F. oxysporum and 3.7,4,3.4 and 4 against R. solani compared to 3.2, 2.8, 2.6 and 2.8 against F. oxysporum and 2.7, 2.9,3.1 and 3 against R. solani in the wild types T3, T5, S1 and S6,respectively. The growth of T. harzianum isolates (estimated as colony diameter) was significantly increased in all T. harzianum mutants except MT3 and MD5 mutants, CMCase activity was increased from 13.2, 16.2, 10 and 15.8 U/ml / mg protein in the wild isolates T3, T5, S1 and S6 to 18.6, 20.3, 17.7 and 22.5 U/ml / mg protein in the mutants MT3, MT5, MS1 and MS6, respectively. Chitinase activity was also increased in these mutants to 88.4,132.3,86.1 and 136.7 U/ml / mg protein, compared to 82.7, 93.2, 74.5 and 103.2 U/ml / mg protein in their wild, respectively. As comparison between wild types and mutants, the maximum growth was 5.2 and 4.8 cm (as colonies diameters) by MT5 in present of 25% of the recommended field rate of Topsin and Benomyl, compared to 3.3 and 2.2 cm in the wild type T5, respectively, followed by MS6,in which, the maximum growth in this mutant was 4.8 and 4.2 cm in the same concentration of these two fungicides, compared to 3.4 and 2.5 cm in the wild type S6, respectively. According to these results, the mutants MT5 and MS6 developed from UV-irradiation treatment, showed as promising T. harzianum mutants through their antagonistic activity (at mycoparasitism level) , fast growth, higher CMCase and chitinase activity and growth in 25% of the recommended field rate of Elsa , 25 and 50% of the recommended field rate of Diathen and Mizab and all concentrations of Topsin and Benomyl with complete inhibition of pathogenic fungi F. oxysporum and R. solani.

Defense Surveillance System at the Interface: Response of Rice Towards Rhizoctonia solani During Sheath Blight Infection.

Sheath blight of rice caused by necrotrophic plant pathogen Rhizoctonia solani is one of the most common fungal diseases of rice leading to significant yield loss. Among the defense responses exhibited by the host plants towards fungal infections, those functional within the apoplast contribute significantly. Here, we have studied apoplastic defense response of rice towards R. solani during sheath blight infection. The transcriptome of R. solani-infected rice plants was compared with that of uninfected rice, to identify the set of defense genes that undergo differential expression and code for proteins with a predicted N-terminal signal peptide. Significant changes in the stress-responsive, molecular signal perception, protein modification, and metabolic process pathways represented by a group of differentially expressed genes were observed. Our data also revealed two secreted protease inhibitors from rice that exhibit increased expression during R. solani infection and induce disease resistance when expressed in Nicotiana benthamiana. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A potential role of salicylic acid in the evolutionary behavior of Trichoderma as a plant pathogen: from Marchantia polymorpha to Arabidopsis thaliana

Main conclusionRecognition of the interaction of Trichoderma during the evolution of land plants plays a potential key role in the development of the salicylic acid defense pathway and the establishment of a mutualistic relationship.Marchantia polymorpha is a common liverwort considered in recent years as a model plant for evolutionary studies on plant–microorganism interactions. Despite the lack of research, remarkable results have been reported regarding the understanding of metabolic and evolutionary processes of beneficial and/or harmful interactions, owing to a better understanding of the origin and evolution of different plant defense pathways. In this study, we have carried out work on the direct and indirect interactions (exudates and volatiles) of M. polymorpha with different species of the fungal genus Trichoderma. These interactions showed different outcomes, including resistance or even growth promotion and disease. We have analyzed the level of tissue colonization and defense-related gene expression. Furthermore, we have used the pteridophyte Dryopteris affinis and the angiosperm Arabidopsis thaliana, as subsequent steps in plant evolution, together with the plant pathogen Rhizoctonia solani as a control of plant pathogenicity. Trichoderma virens, T. brevicompactum and T. hamatum are pathogens of M. polymorpha, while exudates of T. asperellum are harmful to the plant. The analysis of the expression of several defense genes in M. polymorpha and A. thaliana showed that there is a correlation of the transcriptional activation of SA-related genes with resistance or susceptibility of M. polymorpha to Trichoderma. Moreover, exogenous SA provides resistance to the virulent Trichoderma species. This beneficial fungus may have had an evolutionary period of interaction with plants in which it behaved as a plant pathogen until plants developed a defense system to limit its colonization through a defense response mediated by SA.

GC-MS Profiling, in vitro Antioxidant and Antimicrobial Activities of Kaempferia parviflora Wall. ex. Baker Rhizome Extract

Bakground: Kaempferia parviflora (zingiberaceae family) rhizomes are used for various pharmacological applications. The present study is aimed for putative identification of phyto-constituents in the chloroform extract of the rhizome of Kaempferia parviflora and to correlate with bioactivities like antioxidant and anti-microbial/fungal activities. Methods: Rhizome extract was prepared using Soxhlet extractor and undergone the phytoconstituents analysis of it by gas chromatography–mass spectrometry (GC-MS). Results: Identified twenty-five compounds, out of which 4H-1-Benzopyran- 4-0ne,5,7-dimethoxy-2-phenyl and Hydroxyurea showed the maximum and minimum relative abundance of 76.4% and 0.002% respectively. By agar well diffusion method, the extract (25 μg/ml) exhibited highest antibacterial potential against Micrococcus luteus (MTCC 106) (gram-positive bacteria) and anti-fungal potential against plant pathogen Rhizoctonia solani (MTCC 4633). The extract also showed the potent scavenging activities of 2,2-diphenyl-1-picryl-hydrazine (DPPH) and 2,2′-Azino-bis (3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) radicals with IC50 values of 3.0 ± 0.1 mg/ml and 19.4 ± 1.2 μg/ml respectively. Subsequently, lipid peroxidation assay was also used for estimation of antioxidant activity of this extract. The extract (20 μg/ml) inhibited lipid peroxidation by 80.0 ± 4.5%. Conclusion: The outcomes of present study provide scope for isolation of bioactive compounds from Kaempferia parviflora for medicinal application.

Plant-associated Pseudomonas aeruginosa strains harbour multiple virulence traits critical for human infection.

Introduction. Pseudomonas aeruginosa causes fatal infections in immunocompromised individuals and patients with pulmonary disorders.Gap Statement. Agricultural ecosystems are the vast reservoirs of this dreaded pathogen. However, there are limited attempts to analyse the pathogenicity of P. aeruginosa strains associated with edible plants.Aim. This study aims to (i) elucidate the virulence attributes of P. aeruginosa strains isolated from the rhizosphere and endophytic niches of cucumber, tomato, eggplant and chili;and (ii) compare these phenotypes with that of previously characterized clinical isolates.Methodology. Crystal-violet microtitre assay, swarm plate experiment, gravimetric quantification and sheep blood lysis were performed to estimate the biofilm formation, swarming motility, rhamnolipid production and haemolytic activity, respectively, of P. aeruginosa strains. In addition, their pathogenicity was also assessed based on their ability to antagonize plant pathogens (Xanthomonas oryzae, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum) and kill a select nematode (Caenorhabditis elegans).Results. Nearly 80 % of the plant-associated strains produced rhamnolipid and exhibited at least one type of lytic activity (haemolysis, proteolysis and lipolysis). Almost 50 % of these strains formed significant levels of biofilm and exhibited swarming motility. The agricultural strains showed significantly higher and lower virulence against the bacterial and fungal pathogens, respectively, compared to the clinical strains. In C. elegans, a maximum of 40 and 100% mortality were induced by the agricultural and clinical strains, respectively.Conclusion. This investigation shows that P. aeruginosa in edible plants isolated directly from the farm express virulence and pathogenicity. Furthermore, clinical and agricultural P. aeruginosa strains antagonized the tested fungal phytopathogens, Pythium aphanidermatum, Rhizoctonia solani and Fusarium oxysporum. Thus, we recommend using these fungi as simple eukaryotic model systems to test P. aeruginosa pathogenicity.

ACTIVITY OF THYME ESSENTIAL OILS AGAINST PLANT PATHOGENIC FUNGI AND ITS POTENTIAL USE AS SEED DISINFECTANT

One potential application of essential oils as low-risk biopesticides is as seeds disinfectant. The main goal of this study was to test the activity of thyme essential oil on four strains of seed/soilborne plant pathogenic fungi, Fusarium graminearum, Botrytis allii, Rhizoctonia solani, and Pythium ultimum, and to evaluate the effect of the inhibitory doses on plant seed germination. The direct application of thyme essential oil on seeds demonstrates a lack of selectivity. The main effects were related to radicle architecture and development of the primary and lateral roots, both in monocotyl, sorghum (Sorghum saccharatum), and in the dicotyls garden cress (Lepidium sativum) and white mustard (Sinapis alba). One potential solution to the lack of selectivity of direct application of the essential oils is biofumigation, i.e., application by volatilization in the rhizosphere area. Our paper raises this issue of compatibility between the necessary doses to inhibit the plant pathogenic fungi and the effects on plant seeds. Formulation of essential oils as slow-release nanoformulation could reduce phytotoxicity while maintaining and even enhancing the activity against plant pathogens.

Regulation of phenazine-1-carboxamide production by quorum sensing in type strains of Pseudomonas chlororaphis subsp. chlororaphis and Pseudomonas chlororaphis subsp. piscium

Quorum sensing is a population density-dependent gene regulation mechanism. N-Acyl-l-homoserine lactone (AHL) has been identified as a signal compound in quorum sensing in gram-negative bacteria. Phenazine derivatives are bacterial secondary metabolites known for their broad-spectrum antifungal activity. Pseudomonas chlororaphis has been demonstrated to be a biocontrol strain, and most of its species can produce phenazine derivatives under AHL-mediated quorum sensing. Although P.chlororaphis is divided into four subspecies, the relationship between phenazine production and quorum sensing has not been investigated in two of the subspecies, P.chlororaphis subsp. chlororaphis and piscium. Two luxI/luxR homolog gene sets, phzI and phzR and csaI and csaR, were found in the complete genome sequences of the type strains of P.chlororaphis subsp. chlororaphis JCM 2778T and P.chlororaphis subsp. piscium DSM 21509T. Two major AHLs, N-(3-hydroxyhexanoyl)-l-homoserine lactone and N-(3-hydroxyoctanoyl)-l-homoserine lactone, were detected in JCM 2778 and DSM 21509 samples. PhzI synthesized all AHLs; however, CsaI could not perform AHL biosynthesis in JCM 2778 and DSM 21509. In both strains, disruption of the phzI caused complete disappearance of phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production; however, disruption of csaI did not induce significant changes in PCA and PCN production. Phenazine derivatives produced by JCM 2778 and DSM 21509 under quorum sensing are crucial for the control of the plant pathogenic fungi, Rhizoctonia solani, Fusarium graminearum, and Fusarium nirenbergiae. These results demonstrated that PhzI/PhzR quorum-sensing system play an important role in production of phenazine derivatives and biocontrol activity.

Optimization of silver nanoparticle biosynthesis by entomopathogenic fungi and assays of their antimicrobial and antifungal properties

Entomopathogenic fungi produce extracellular enzymes to facilitate host infection, and these can also reduce metal ions to produce nanoparticles. In the present study, three isolates of Beauveria bassiana (JS1, JS2 and KA75) and one isolate of Metarhizium anisopliae (TT1) were evaluated for their ability to biosynthesize silver nanoparticles (AgNPs). In general, the best yields and smallest NP sizes were obtained at 60 °C and pH 7.0. Nanoparticle properties were studied using UV–visible spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy. Biosynthesized AgNPs ranged from 23 to 101 nm across, the smallest being produced by KA75, and the largest by TT1. UV–visible spectroscopy confirmed peak absorption of AgNPs in the range of 420–454 nm. AgNP antibacterial activity was highest against the gram-negative bacteria Pectobacterium carotovorum and Erwinia amylovora, and lower against the gram-positive Bacillus sp. AF1. JS1-AgNPs caused the greatest growth restriction of P. carotovorum at a concentration of 75 µL/mL at lower OD600 (0.25). Smaller AgNPs generally had better antifungal activities against B. bassiana, M. anisopliae, and the plant-pathogenic Rhizoctonia solani. Complete inhibition of vegetative growth of the JS2 fungus was obtained with TT1-synthesized AgNPs at 15 µL/mL, a control level similar to half the field rate of benomyl. Generally, fungal sporulation was more inhibited than vegetative growth, and all AgNPs showed good compatibility with the fungi at low concentrations. We conclude that AgNPs mycosynthesized by these entomopathogens have promising antibacterial and antifungal properties with potential for various applications.

MAPK Cascades Mediating Biocontrol Activity of Trichoderma brevicrassum Strain TC967.

Trichoderma brevicrassum strain TC967 is a novel biological control agent (BCA) against the plant pathogen Rhizoctonia solani and promotes plant growth. Mitogen-activated protein kinase (MAPK) cascades are involved in a variety of physiological functions of Trichoderma, but functions of each MAPK in regulating biocontrol have not been characterized in a single Trichoderma strain. In this study, we assembled and annotated the genome of strain TC967 and identified its three MAPK gene sequences. Functions of Fus3-, Slt2-, and Hog1-MAPK in strain TC967 were dissected. All three MAPKs were involved in hyphal growth. Hog1-MAPK was essential for conidiation and tolerance to saline/osmotic stress. Both Fus3- and Slt2-MAPK deletion mutants reduced cell-wall integrity (CWI) and increased the activities of chitinase and protease. The growth of Rhizoctoniasolani was further inhibited by volatile organic compounds (VOCs) and secondary metabolites produced by Fus3- and Slt2-MAPK deletion mutants, respectively. Biocontrol assays demonstrated that Fus3- and Slt2-MAPK deletion mutants were considerably more effective in disease control than the wild-type strain. RNA-seq analysis revealed that MAPK collectively played a major role in regulating biocontrol-related gene expressions, including the genes in charge of secondary metabolism, fungal cell wall-degrading enzymes (FCWDEs), and small secreted cysteine-rich proteins (SSCPs).

Synthesis of benzothiazole-appended bis-triazole-based structural isomers with promising antifungal activity against Rhizoctonia solani.

In order to explore new antifungal agrochemicals, we reported the synthesis of two series 5a–f, 6 and 7a–f, 8 of benzothiazole-appended bis-triazole derivative-based structural isomers using a molecular hybridization approach. The synthesized compounds were tested for fungal growth inhibition against the plant pathogen Rhizoctonia solani. All the synthesized compounds showed excellent antifungal activity in their minimum concentrations (10–0.62 μM). Among all the synthetics, compounds 5b (ED50: 2.33 μM), 5f (ED50: 0.96 μM), and 7f (ED50: 1.48 μM) exerted a superior inhibitory effect in comparison to the commercially available fungicide, hexaconazole (ED50: 2.44 μM). The binding interactions of the active compounds 5f, 7f, 6, and 8 within the active site of the sterol 14α-demethylase enzyme were studied with the help of molecular docking studies. The studies revealed that these hybrid pharmacophores could be used as an important intermediate to demonstrate new structural isomer-based fungicides.

Histidine kinase two-component response regulators Ssk1, Skn7 and Rim15 differentially control growth, developmental and volatile organic compounds emissions as stress responses in Trichoderma atroviride.

The Skn7, Ssk1 and Rim15 proteins are response regulators involved in osmotic, oxidative and nutritional stress in fungi. In order to verify the involvement of these genes in Trichoderma atroviride IMI206040's growth, conidiation, direct antagonism against plant pathogens (Rhizoctonia solani and Sclerotinia sclerotiorum), production of volatile organic compounds (VOCs) with fungistatic effect, and interaction with plants (growth promotion), single mutants were generated, and the phenotypic patterns were analysed in comparison to the wild-type (wt) strain. The mutants were submitted to osmotic, oxidative, membrane and cell wall stress conditions in vitro. The Δskn7 and Δrim15 mutants did not show either significant differences at morphological level, or marked decreases in mycelial growth and conidiation in relation to wt, whereas Δssk1 had altered phenotypes in most conditions tested. The plant-growth promotion of Arabidopsis thaliana seedlings induced by VOCs was not quantitatively modified by any of the mutants in relation to the wt strain, although possible differences in secondary root hairs was noticed for Δrim15. The fungistatic activity was significantly altered for Δssk1 and Δrim15. Overall, the Δssk1 strain showed remarkable morphological differences, with decrease in mycelial growth and conidiation, being also affected in the antagonistic capacity against plant pathogens. The impacts demonstrated by the deletion of ssk1 suggest this gene has a relevant participation in the signalling response to different stresses in T. atroviride and in the interactive metabolism with phytopathogens and plants. On the other hand, unlike other fungal models, Skn7 did not appear to have a critical participation in the above-mentioned processes; Rim15 seemed to confirm its involvement in modulating cellular responses to nutritional status, although with a possible cross-talk with other cellular processes. Our results suggest that Ssk1 likely plays a key regulatory role, not only in basic metabolisms of T. atroviride, but also in biocontrol-related characteristics.

BIOCONTROL ACTIVITY OF PHOSPHATESOLUBILIZING FUNGIISOLATED FROM RHIZOSPHERE SOIL OF MARATHWADA, INDIA

The phosphate solubility and biocontrol activity of fungi isolated from rhizosphere soil of Marathwada were studied in the present study. Fungi were isolated from rhizosphere soil and all the 11 fungal isolates showed good phosphate solubilizing ability. The potent phosphate solubilising isolates Aspergillus niger (PQ9), Trichodermaspp (PQ36) and Penicillium spp (PQ19) showed significant zone of solubilization with 34, 31 to 30 mm respectively after 48 hours of incubation. All potent phosphate solubilising fungal isolates were tested for in vitro antagonistic activity against bacterial plant pathogen Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Rhizopus oryzae and Macrophomina phaseolina. The study therefore proposed that these fungal species have strong phosphate solubilizing properties and the isolated fungal strains in present investigation indicates broad spectrum activity against tested fungal phytopathogen. Therefore, the fungal species can be used for excellent crop productivity as a biofertilizer and as biocontrol agent.

Tomato seed biopriming with water extracts from Anabaena minutissima, Ecklonia maxima and Jania adhaerens as a new agro-ecological option against Rhizoctonia solani

In recent years, the use of synthetic pesticides in agriculture has been restricted for environmental pollution issues. Alternatives to chemicals for plant disease control are highly recommended by the recent EU legislation. We tested tomato seed treatment with water extracts from Anabaena minutissima, Ecklonia maxima, and Jania adhaerens for their biocontrol activity against the fungal plant pathogen Rhizoctonia solani. Algae were characterized into their contents in macro and microelements and into their functional groups by using FT-IR spectroscopy. The extracts were applied at 0.0, 2.5, 5.0, and 10.0 mg/mL concentrations on tomato seeds against the pathogen, in in vitro experiments and under greenhouse conditions. To estimate the efficiency of treatment in priming plant defence response, plant chitinase activity was measured and the different distribution of functional groups of roots was determined by FT-IR spectroscopy. Increases of germination and seedling dry weight for treated seeds without pathogen challenge were observed. The extracts reduced disease severity and increased seedling dry weight both in in vitro and greenhouse experiments at all concentrations. All extracts also increased stem seedling calibre under greenhouse conditions. The plant chitinase activity was increased by all extracts. The aromatic rings assigned to lignin changed with the treatment. We concluded that, although our experiments were based on a small scale, algae and cyanobacteria water extracts could provide a potential tool for the R. solani control on tomato plants, by contributing to the reduction of synthetic product input in the environment.

Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF377 reveals a member of "Fusagraviridae" with changes in antifungal activity of the host fungus.

Trichoderma atroviride is a common fungus found in various ecosystems that shows mycoparasitic ability on other fungi. A novel dsRNA virus was isolated from T. atroviride NFCF377 strain and its molecular features were analyzed. The viral genome consists of a single segmented double-stranded RNA and is 9,584 bp in length, with two discontinuous open reading frames (ORF1 and ORF2). A mycoviral structural protein and an RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively, between which is found a canonical shifty heptameric signal motif (AAAAAAC) followed by an RNA pseudoknot. Analysis of sequence similarity and phylogeny showed that it is closely related to members of the proposed family "Fusagraviridae", with a highest similarity to the Trichoderma atroviride mycovirus 1 (TaMV1). Although the sequence similarity of deduced amino acid to TaMV1 was evident, sequence deviations were distinctive at untranslated regions (UTRs) due to the extended size. Thus, we inferred this dsRNA to be a different strain of Trichoderma atroviride mycovirus 1 (TaMV1-NFCF377). Electron microscopy image exhibited an icosahedral viral particle of 40 nm diameter. Virus-cured isogenic isolates were generated and no differences in growth rate, colony morphology, or conidia production were observed between virus-infected and virus-cured strains. However, culture filtrates of TaMV1-NFCF377-infected strain showed enhanced antifungal activity against the plant pathogen Rhizoctonia solani but not to edible mushroom Pleurotus ostreatus. These results suggested that TaMV1-NFCF377 affected the metabolism of the fungal host to potentiate antifungal compounds against a plant pahogen, but this enhanced antifungal activity appeared to be species-specific.