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The myo-inositol-1-phosphate synthase (MIPS) catalyses the biosynthesis of myo-inositol, an important sugar that regulates various physiological and biochemical processes in plants. Here, we provide evidence that host (SlMIPS1) and pathogen (Rs_MIPS) myo-inositol-1-phosphate synthase (MIPS) genes are required for successful infection of Rhizoctonia solani, a devastating necrotrophic fungal pathogen, in tomato. Silencing of either SlMIPS1 or Rs_MIPS prevented disease, whereas an exogenous spray of myo-inositol enhanced disease severity. SlMIPS1 was upregulated upon R. solani infection, and potentially promoted source-to-sink transition, induced SWEET gene expression, and facilitated sugar availability in the infected tissues. In addition, salicylic acid (SA)-jasmonic acid homeostasis was altered and SA-mediated defence was suppressed; therefore, disease was promoted. On the other hand, silencing of SlMIPS1 limited sugar availability and induced SA-mediated defence to prevent R. solani infection. Virus-induced gene silencing of NPR1, a key gene in SA signalling, rendered SlMIPS1-silenced tomato lines susceptible to infection. These analyses suggest that induction of SA-mediated defence imparts disease tolerance in SlMIPS1-silenced tomato lines. In addition, we present evidence that SlMIPS1 and SA negatively regulate each other to modulate the defence response. SA treatment reduced SlMIPS1 expression and myo-inositol content in tomato, whereas myo-inositol treatment prevented SA-mediated defence. We emphasize that downregulation of host/pathogen MIPS can be an important strategy for controlling diseases caused by R. solani in agriculturally important crops., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δ 2 H), carbon (δ 13 C), and nitrogen (δ 15 N) stable isotope ratios are usually adopted. However, previous studies have not fully accounted for exchangeable hydrogens, which could affect these evaluations. Here, we performed standard δ 13 C, δ 15 N, and δ 2 H analyses on bulk samples. Additionally, we conducted δ 2 H analysis on α-cellulose and cellulose nitrate samples to investigate whether the heterogeneity of exchangeable hydrogens among plant species influences the assessment of nutritional modes. The δ 2 H of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δ 13 C, its δ 2 H was higher than those of the autotrophs. Notably, among all response variables, δ 15 N and δ 2 H exhibited high abilities for discriminating the nutritional modes of rhizoctonia-associated orchids. These results indicate that a time-efficient bulk sample analysis is an effective method for evaluating plant nutritional modes, as the heterogeneity of exchangeable hydrogens does not significantly impact the estimation. Using δ 15 N and δ 2 H benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Background: 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., Aim and Objectives: 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., Results: 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 200 rpm, 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., Conclusion: The bio-friendly formulations of actinobacteria resulting from this investigation may play an active role in managing soil-borne diseases., (© 2024. The Author(s).)

A pathogen arriving on a host typically encounters a diverse community of microbes that can shape priority effects, other within-host interactions and infection outcomes. In plants, environmental nutrients can drive trade-offs between host growth and defence and can mediate interactions between co-infecting pathogens. Nutrients may thus alter the outcome of pathogen priority effects for the host, but this possibility has received little experimental investigation. To disentangle the relationship between nutrient availability and co-infection dynamics, we factorially manipulated the nutrient availability and order of arrival of two foliar fungal pathogens ( Rhizoctonia solani and Colletotrichum cereale ) on the grass tall fescue ( Lolium arundinaceum ) and tracked disease outcomes. Nutrient addition did not influence infection rates, infection severity or plant biomass. Colletotrichum cereale facilitated R. solani , increasing its infection rate regardless of their order of inoculation. Additionally, simultaneous and C. cereale -first inoculations decreased plant growth and-in plants that did not receive nutrient addition-increased leaf nitrogen concentrations compared to uninoculated plants. These effects were partially, but not completely, explained by the duration and severity of pathogen infections. This study highlights the importance of understanding the intricate associations between the order of pathogen arrival, host nutrient availability and host defence to better predict infection outcomes.

Common bean (Phaseolus vulgaris L.) is an essential food staple and source of income for small-holder farmers across Africa. However, yields are greatly threatened by fungal diseases like root rot induced by Rhizoctonia solani. This study aimed to evaluate an integrated approach utilizing vermicompost tea (VCT) and antagonistic microbes for effective and sustainable management of R. solani root rot in common beans. Fourteen fungal strains were first isolated from infected common bean plants collected across three Egyptian governorates, with R. solani being the most virulent isolate with 50% dominance. Subsequently, the antagonistic potential of vermicompost tea (VCT), Serratia sp., and Trichoderma sp. was assessed against this destructive pathogen. Combinations of 10% VCT and the biocontrol agent isolates displayed potent inhibition of R. solani growth in vitro, prompting in planta testing. Under greenhouse conditions, integrated applications of 5 or 10% VCT with Serratia marcescens, Trichoderma harzianum, or effective microorganisms (EM1) afforded up to 95% protection against pre- and post-emergence damping-off induced by R. solani in common bean cv. Giza 6. Similarly, under field conditions, combining VCT with EM1 (VCT + EM1) or Trichoderma harzianum (VCT + Trichoderma harzianum) substantially suppressed disease severity by 65.6% and 64.34%, respectively, relative to untreated plants. These treatments also elicited defense enzyme activity and distinctly improved growth parameters including 136.68% and 132.49% increases in pod weight per plant over control plants. GC-MS profiling of Trichoderma harzianum, Serratia marcescens, and vermicompost tea (VCT) extracts revealed unique compounds dominated by cyclic pregnane, fatty acid methyl esters, linoleic acid derivatives, and free fatty acids like oleic, palmitic, and stearic acids with confirmed biocontrol and plant growth-promoting activities. The results verify VCT-mediated delivery of synergistic microbial consortia as a sustainable platform for integrated management of debilitating soil-borne diseases, enhancing productivity and incomes for smallholder bean farmers through regeneration of soil health. Further large-scale validation can pave the adoption of this climate-resilient approach for securing food and nutrition security., (© 2024. The Author(s).)

Co-incubation of plant growth promoting rhizobacteria (PGPRs) have been proposed as a potential alternative to pesticides for controlling fungal pathogens in crops, but their synergism mechanisms are not yet fully understood. In this study, combined use of Bacillus subtilis SL44 and Enterobacter hormaechei Wu15 could decrease the density of Colletotrichum gloeosporioides and Rhizoctonia solani and enhance the growth of beneficial bacteria on the mycelial surface, thereby mitigating disease severity. Meanwhile, PGPR application led to a reorganization of the rhizosphere microbial community through modulating its metabolites, such as extracellular polymeric substances and chitinase. These metabolites demonstrated positive effects on attracting and enhancing conventional periphery bacteria, inhibiting fungal pathogens and promoting soil health effectively. The improvement in the microbial community structure altered the trophic mode of soil fungal communities, effectively decreasing the proportion of saprotrophic soil and reducing fungal plant diseases. Certain combinations of PGPR have the potential to serve as precise instruments for managing plant pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Plants typically activate distinct defense pathways against various pathogens. Heightened resistance to one pathogen often coincides with increased susceptibility to another pathogen. However, the underlying molecular basis of this antagonistic response remains unclear. Here, we demonstrate that mutants defective in the transcription factor ETHYLENE-INSENSITIVE 3-LIKE 2 (OsEIL2) exhibited enhanced resistance to the biotrophic bacterial pathogen Xanthomonas oryzae pv oryzae and to the hemibiotrophic fungal pathogen Magnaporthe oryzae, but enhanced susceptibility to the necrotrophic fungal pathogen Rhizoctonia solani. Furthermore, necrotroph-induced OsEIL2 binds to the promoter of OsWRKY67 with high affinity, leading to the upregulation of salicylic acid (SA)/jasmonic acid (JA) pathway genes and increased SA/JA levels, ultimately resulting in enhanced resistance. However, biotroph- and hemibiotroph-induced OsEIL2 targets OsERF083, resulting in the inhibition of SA/JA pathway genes and decreased SA/JA levels, ultimately leading to reduced resistance. Our findings unveil a previously uncharacterized defense mechanism wherein two distinct transcriptional regulatory modules differentially mediate immunity against pathogens with different lifestyles through the transcriptional reprogramming of phytohormone pathway genes., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Rhizoctonia solani is a fungal pathogen that causes significant losses in agricultural production. Because of its rapid transmission and broad host range, the exploration of genes involved in defense responses to the infection of R. solani has become an important task. Here, we performed a time-course RNA-Seq experiment to explore crucial genes or pathways involved in host responses to R. solani AG3-TB infection at 6, 12, 24, 36, 48, and 72 hours post inoculation (hpi). GO and KEGG enrichment analysis revealed that most DEGs were enriched in the basal metabolism pathways, including carbohydrate metabolic processes and the biosynthesis of amino acids. Moreover, catalase (CAT) and superoxide dismutase (SOD) were up-regulated, and transcription factors (TFs) such as WRKY, AP2, and MYB were increased significantly compared to the control (0 hpi). Silencing of WRKY70 and catalase-3 exhibited elevated susceptibility to the fungal infection. To summarize, the TFs WRKY70 and WRKY75, genes involved in jasmonic acid (JA), salicylic acid (SA), and brassinosteroids (BR) signaling pathways, and defense-related enzymes may play crucial roles in the host responses to R. solani AG3-TB infection., (© 2024 Scandinavian Plant Physiology Society.)

Background: Apple Replant Disease (ARD) is common in major apple-growing regions worldwide, but the role of rhizosphere microbiota in conferring ARD resistance and promoting plant growth remains unclear., Results: In this study, a synthetic microbial community (SynCom) was developed to enhance apple plant growth and combat apple pathogens. Eight unique bacteria selected via microbial culture were used to construct the antagonistic synthetic community, which was then inoculated into apple seedlings in greenhouse experiments. Changes in the rhizomicroflora and the growth of aboveground plants were monitored. The eight strains, belonging to the genera Bacillus and Streptomyces, have the ability to antagonize pathogens such as Fusarium oxysporum, Rhizoctonia solani, Botryosphaeria ribis, and Physalospora piricola. Additionally, these eight strains can stably colonize in apple rhizosphere and some of them can produce siderophores, ACC deaminase, and IAA. Greenhouse experiments with Malus hupehensis Rehd indicated that SynCom promotes plant growth (5.23%) and increases the nutrient content of the soil, including soil organic matter (9.25%) and available K (1.99%), P (7.89%), and N (0.19%), and increases bacterial richness and the relative abundance of potentially beneficial bacteria. SynCom also increased the stability of the rhizosphere microbial community, the assembly of which was dominated by deterministic processes (|β NTI| > 2)., Conclusions: Our results provide insights into the contribution of the microbiome to pathogen inhibition and host growth. The formulation and manipulation of similar SynComs may be a beneficial strategy for promoting plant growth and controlling soil-borne disease., (© 2024. The Author(s).)

Rhizoctonia solani, the causal agent of banded leaf and sheath blight (BL&SB), poses a significant threat to maize and various crops globally. The increasing concerns surrounding the environmental and health impacts of chemical fungicides have encouraged intensified concern in the development of biological control agents (BCAs) as eco-friendly alternatives. In this study, we explored the potential of 22 rhizobacteria strains (AS1-AS22) isolates, recovered from the grasslands of the Pithoragarh region in the Central Himalayas, as effective BCAs against BL&SB disease. Among these strains, two Pseudomonas isolates, AS19 and AS21, exhibited pronounced inhibition of fungal mycelium growth in vitro, with respective inhibition rates of 57.04% and 54.15% in cell cultures and 66.56% and 65.60% in cell-free culture filtrates. Additionally, both strains demonstrated effective suppression of sclerotium growth. The strains AS19 and AS21 were identified as Pseudomonas sp. by 16S rDNA phylogeny and deposited under accession numbers NAIMCC-B-02303 and NAIMCC-B-02304, respectively. Further investigations revealed the mechanisms of action of AS19 and AS21, demonstrating their ability to induce systemic resistance (ISR) and exhibit broad-spectrum antifungal activity against Alternaria triticina, Bipolaris sorokiniana, Rhizoctonia maydis, and Fusarium oxysporum f. sp. lentis. Pot trials demonstrated significant reductions in BL&SB disease incidence (DI) following foliar applications of AS19 and AS21, with reductions ranging from 25 to 38.33% compared to control treatments. Scanning electron microscopy revealed substantial degradation of fungal mycelium by the strains, accompanied by the production of hydrolytic enzymes. These findings suggest the potential of Pseudomonas strains AS19 and AS21 as promising BCAs against BL&SB and other fungal pathogens. However, further field trials are warranted to validate their efficacy under natural conditions and elucidate the specific bacterial metabolites responsible for inducing systemic resistance. This study contributes to the advancement of sustainable disease management strategies and emphasizes the potential of Pseudomonas strains AS19 and AS21 in combating BL&SB and other fungal diseases affecting agricultural crops., (© 2024. The Author(s).)

Pepper southern blight, caused by Sclerotium rolfsii, is a devastating soil-borne disease resulting in significant loss to pepper, Capsicum annuum L. production. Here, we isolated an antagonistic bacterial strain XQ-29 with antifungal activity against S. rolfsii from rhizospheric soil of pepper. Combining the morphological and biochemical characteristics with the 16S rDNA sequencing, XQ-29 was identified as Streptomyces griseoaurantiacus. It exhibited an inhibition of 96.83% against S. rolfsii and displayed significant inhibitory effects on Botrytis cinerea, Phytophthora capsica and Rhizoctonia solani. Furthermore, XQ-29 significantly reduced the pepper southern blight by 100% and 70.42% during seedling and growth stages, respectively. The antifungal mechanism involved altering the mycelial morphology, disrupting cell wall and membrane integrity, accompanied by accumulation of reactive oxygen species and lipid peroxidation in S. rolfsii mycelia. Furthermore, XQ-29 promoted growth and stimulated resistance of pepper plants by increasing defense-related enzyme activities and upregulating defense-related genes. Correspondingly, XQ-29 harbors numerous functional biosynthesis gene clusters in its genome, including those for siderophores and melanin production. The metabolic constituents present in the ethyl acetate extracts, which exhibited an EC 50 value of 85.48 ± 1.62 μg/mL, were identified using LC-MS. Overall, XQ-29 demonstrates significant potential as a biocontrol agent against southern blight disease., Competing Interests: Declaration of competing interest No conflict of interest exits., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris [Frank] Donk) is an aggressive soilborne pathogen with a wide host range that survives saprophytically between crops, presenting a challenge for organic vegetable farmers who lack effective management tools. A 2-year field experiment was conducted at two organic farms to compare anaerobic soil disinfestation (ASD) and worm-cured compost (vermicompost) to manage bottom rot caused by R. solani subspecies AG1-IB in field-grown organic lettuce ( Lactuca sativa ). At each farm, four replicate plots of seven treatments were arranged in a randomized complete block design. Randomization was restricted by grouping treatments to evaluate ASD, and treatments to evaluate vermicompost in starter plugs. ASD experiment treatments were three different ASD carbon sources that are commonly used and widely available to local farmers in Vermont: compost, cover crop residues, and poultry manure fertilizer, as well as a tarped control. Vermicompost experimental treatments were vermicompost compared with two types of controls: a commercial biocontrol product (RootShield PLUS + G), and unamended (untarped control). This study demonstrated that the ASD method is achievable in a field setting on Vermont farms. However, neither ASD nor vermicompost produced significant disease suppression or resulted in higher marketable yields than standard growing practices. Given the laborious nature of ASD, it is likely more appropriate in a greenhouse setting with high-value crops that could especially benefit from being grown in plastic tarped beds (e.g., tomatoes and strawberries). This study is the first known attempt of field-implemented ASD for soil pathogen control in the northeastern United States., Competing Interests: The author(s) declare no conflict of interest.

Key Message: We highlight the emerging role of the R. solani novel lipase domain effector AGLIP1 in suppressing pattern-triggered immunity and inducing plant cell death. The dynamic interplay between plants and Rhizoctonia solani constitutes a multifaceted struggle for survival and dominance. Within this complex dynamic, R. solani has evolved virulence mechanisms by secreting effectors that disrupt plants' first line of defense. A newly discovered effector, AGLIP1 in R. solani, plays a pivotal role in inducing plant cell death and subverting immune responses. AGLIP1, a protein containing a signal peptide and a lipase domain, involves complex formation in the intercellular space, followed by translocation to the plant cytoplasm, where it induces cell death (CD) and suppresses defense gene regulation. This study provides valuable insights into the intricate molecular interactions between plants and necrotrophic fungi, underscoring the imperative for further exploration in this field., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study investigated the effects of Rhizoctonia solani J.G. Kühn infestation on the volatile organic compound (VOC) emissions and biochemical composition of ten cultivars of chrysanthemum (Chrysanthemum × morifolium /Ramat./ Hemsl.) to bring new insights for future disease management strategies and the development of resistant chrysanthemum cultivars. The chrysanthemum plants were propagated vegetatively and cultivated in a greenhouse under semi-controlled conditions. VOCs emitted by the plants were collected using a specialized system and analyzed by gas chromatography/mass spectrometry. Biochemical analyses of the leaves were performed, including the extraction and quantification of chlorophylls, carotenoids, and phenolic compounds. The emission of VOCs varied among the cultivars, with some cultivars producing a wider range of VOCs compared to others. The analysis of the VOC emissions from control plants revealed differences in both their quality and quantity among the tested cultivars. R. solani infection influenced the VOC emissions, with different cultivars exhibiting varying responses to the infection. Statistical analyses confirmed the significant effects of cultivar, collection time, and their interaction on the VOCs. Correlation analyses revealed positive relationships between certain pairs of VOCs. The results show significant differences in the biochemical composition among the cultivars, with variations in chlorophyll, carotenoids, and phenolic compounds content. Interestingly, R. solani soil and leaf infestation decreased the content of carotenoids in chrysanthemums. Plants subjected to soil infestation were characterized with the highest content of phenolics. This study unveils alterations in the volatile and biochemical responses of chrysanthemum plants to R. solani infestation, which can contribute to the development of strategies for disease management and the improvement of chrysanthemum cultivars with enhanced resistance to R. solani., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Piesik et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chitosan is an environmentally friendly natural substance that is used in crop disease management as an alternative to chemical pesticides. A significant issue restricting output in Egypt is root rot, which is a disease, caused by Rhizoctonia solani. Therefore, a greenhouse experiment was conducted to assess the effects of R. solani on 60-day-old tomato plants under fungal infection and to determine the antifungal activity of chitosan and Rizolax T fungicide against the pathogenic fungus. The findings demonstrated that 4 g/L of chitosan seed application completely obstructed the radial mycelial growth of R. solani and decreased the disease severity. Pathogenic infection significantly decreased morphological characteristics and total chlorophyll but significantly increased carotenoid, total thiol, non-protein thiol, protein thiol, antioxidant enzymes, oxidative stress, total phenolic, total flavonoid, and isoflavone compared to healthy plants. Tomato plants treated with chitosan exhibited lower rates of oxidative stress, but higher levels of all previously mentioned parameters compared to untreated infected plants. The number and molecular mass of protein banding patterns varied in all treated tomato plants as compared to the healthy control. There are 42 bands in the treatments, and their polymorphism rate is 69.55%. Moreover, the number and density of α- and β-esterase, and peroxidase isozymes in treated tomato plants exhibited varied responses. Moreover, in treated and control plants, chitosan treatment raised the expression levels of phenylalanine ammonia-lyase, pathogenesis-related protein-1, β-1,3-glucanases and chitinase. In conclusions, chitosan reduces R. solani infection by controlling the biochemical and molecular mechanisms in tomato plants during infection., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Aerial blight, caused by the fungus Rhizoctonia solani anastomosis group (AG) 1-IA, is an economically important soybean disease in the mid-Southern United States. Management has relied on fungicide applications during the season, but there is an increasing prevalence of resistance to commonly used strobilurin fungicides and an urgent need to identify soybean varieties resistant to aerial blight. Because the patchy distribution of the pathogen complicates field variety screening, the present study aimed to develop a greenhouse screening protocol to identify soybean varieties resistant to aerial blight. For this, 88 pathogen isolates were collected from commercial fields and research farms across five Louisiana parishes, and 77% were confirmed to be R. solani AG1-IA. Three polymorphic codominant microsatellite markers were used to explore the genetic diversity of 43 R. solani AG1-IA isolates, which showed high genetic diversity, with 35 haplotypes in total and only two haplotypes common to two other locations. Six genetically diverse isolates were chosen and characterized for their virulence and fungicide sensitivity. The isolate AC2 was identified as the most virulent and was resistant to both active ingredients, azoxystrobin and pyraclostrobin, tested. The six isolates were used in greenhouse variety screening trials using a millet inoculation protocol. Of the 31 varieties screened, only Armor 48-D25 was classified as moderately resistant, and plant height to the first node influenced final disease severity. The study provides short-term solutions for growers to choose less susceptible varieties for planting and lays the foundation to characterize host resistance against this important soybean pathogen., Competing Interests: The author(s) declare no conflict of interest.

Anaerobic soil disinfestation (ASD) is a nonchemical soil treatment where an easily decomposable carbon source is incorporated into soil, which is then irrigated to saturation and tarped to create anaerobic conditions, which prompts shifts in the soil microbiota from aerobes to anaerobes. ASD has been tested successfully for soilborne disease management in a variety of cropping systems but has not been sufficiently investigated in ornamentals. In this study, ASD was evaluated in soil-based and soilless substrates commonly used in specialty cut flower production using two model pathosystems: Rhizoctonia solani - Zinnia elegans and Phytophthora drechsleri - Gerbera jamesonii . Each substrate was mixed with pathogen-infested vermiculite and amended with either wheat bran, tomato pomace, or soybean meal as the carbon source. Amended substrates were incubated at 25°C for 4 weeks and used as growing substrates for the two crops mentioned above, which were monitored weekly for disease development for up to 5 weeks posttransplant. Additional experiments tested the effect of plant age and inoculum concentration in the substrate on ASD efficacy. Results showed that ASD has the potential to be deployed successfully for the control of Rhizoctonia stem rot in both substrates. Conversely, ASD was not effective at controlling Phytophthora crown rot on gerbera daisy in any of the experiments conducted in this study. More research is needed to understand the influence of carbon amendments, inoculum thresholds, and environmental conditions on ASD efficacy., Competing Interests: The author(s) declare no conflict of interest.

Rice sheath blight (ShB) is a devastating disease that severely threatens rice production worldwide. Induction of cell death represents a key step during infection by the ShB pathogen Rhizoctonia solani. Nonetheless, the underlying mechanisms remain largely unclear. In the present study, we identified a rice transcription factor, OsERF65, that negatively regulates resistance to ShB by suppressing cell death. OsERF65 was significantly upregulated by R. solani infection in susceptible cultivar Lemont and was highly expressed in the leaf sheath. Overexpression of OsERF65 (OsERF65OE) decreased rice resistance, while the knockout mutant (oserf65) exhibited significantly increased resistance against ShB. The transcriptome assay revealed that OsERF65 repressed the expression of peroxidase genes after R. solani infection. The antioxidative enzyme activity was significantly increased in oserf65 plants but reduced in OsERF65OE plants. Consistently, hydrogen peroxide content was apparently reduced in oserf65 plants but accumulated in OsERF65OE plants. OsERF65 directly bound to the GCC box in the promoter regions of four peroxidase genes and suppressed their transcription, reducing the ability to scavenge reactive oxygen species (ROS). The oserf65 mutant exhibited a slight decrease in plant height but increased grain yield. Overall, our results revealed an undocumented role of OsERF65 that acts as a crucial regulator of rice resistance to R. solani and a potential target for improving both ShB resistance and rice yield., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Environmental pollution due to the improper use of the chemical fungicides represents a vital ecological problem, which affects human and animal health, as well as the microbial biodiversity and abundance in the soil. In this study, an endophytic fungus Aspergillus oryzae YRA3, isolated from the wild plant Atractylis carduus (Forssk.) C.Chr, was tested for its biocontrol activity against Rhizoctonia root rot of sorghum. The antagonistic potential of A. oryzae YRA3 was tested against Rhizoctonia solani in vitro. A full inhibition in the growth of R. solani was recorded indicating a strong antagonistic potential for this endophyte. To investigate the chemical composition of its metabolites, GC/MS analysis was used and thirty-two compounds in its culture filtrate were identified. Among these metabolites, some compounds with an antifungal background were detected including palmitic acid, 2-heptanone, and 2,3-butanediol. To these antifungal metabolites the antagonistic activity of A. oryzae YRA3 can be attributed. In the greenhouse experiment, treating of the infected sorghum plants with A. oryzae YRA3 significantly reduced severity of the Rhizoctonia root rot by 73.4%. An upregulation of the defensive genes (JERF3), (POD) and (CHI II) was recorded in sorghum roots when were inoculated with A. oryzae YRA3. In addition, an increment in the activity of peroxidase and polyphenol oxidase, as well as the total phenolic content in the sorghum roots was also recorded. Furthermore, the results obtained from the greenhouse experiment revealed a growth-promoting effect for inoculating the sorghum plants with A. oryzae YRA3. It can be concluded that A. oryzae YRA3 can be a probable biological agent to control this disease in sorghum. However, its evaluation under field conditions is highly needed in the future studies., (© 2023. The Author(s).)

A decline in common bean production and the ineffectiveness of synthetic chemical products in managing plant pathogens has led to exploiting Kenyan soda lakes as an alternative search for biocontrol agents. This study aimed to identify phylogenetically Bacillus spp. from Lake Magadi and their antagonistic activity against Rhizoctonia solani under in vitro and in vivo conditions. The 16 S ribosomal RNA (rRNA) subunit sequences of six bacterial strains isolated from Lake Magadi showed diversity similar to the Bacillus genus; Bacillus velezensis, Bacillus subtilis, and Bacillus pumilus. In vitro, antagonism showed varied mycelium inhibition rates of fungi in the coculture method. Enzymatic assays showed the varied ability of isolates to produce phosphatase, pectinase, chitinase, protease, indole-3-acetic acid (IAA), and hydrogen cyanide (HCD). The in vivo assay showed M09 (B. velezensis) with the lowest root mortality and incidence of postemergence wilt. Pre-emergence wilt incidence was recorded as lowest in M10 (B. subtilis). Isolate M10 had the highest phenylalanine ammonia-lyase (PAL) for defense enzymes, while polyphenol oxidase (PPO) and peroxidase were recorded as highest in M09. For the phenolic content, M10 recorded the highest phenolic content. In conclusion, Lake Magadi harbors Bacillus spp, which can be used as a potential biocontrol of R. solani., (© 2023 Wiley-VCH GmbH.)

Rice sheath blight (ShB) caused by Rhizoctonia solani is one of the most serious diseases that threatens rice ( Oryza sativa ) production. However, the mechanisms of defense against ShB in rice remain largely unknown. In this study, we identified that the expression levels of β- glucanase ( OsBGL ) family genes sensitively respond to infection by R. solani , and OsBGL s positively regulate rice resistance to ShB. In addition, OsBGL2 colocalized with AtPDCB1 at the plasmodesmata (PD) and limited the PD permeability. The level of callose accumulation in osbgls mutants and overexpressors was examined, and OsBGL s were found contribute to callose accumulation. Taken together, these data suggest that OsBGL s can regulate the deposition of callose at the PD to reduce its permeability to defend itself against ShB. Through the identification of these genes and the elucidation of their functions, this research fills the gap in the mechanism of PD permeability in rice ShB resistance.

Wheat sharp eyespot is a serious disease caused by the phytopathogens Rhizoctonia cerealis and R. solani . Some species in the genus Streptomyces have been identified as potential biocontrol agents against phytopathogens. In this investigation, the physiological, biochemical, phylogenetic, and genomic characteristics of strain HU2014 indicate that it is a novel Streptomyces sp. most closely related to Streptomyces albireticuli . Strain HU2014 exhibited strong antifungal activity against R. cerealis G11 and R. solani YL-3. Ultraperformance liquid chromatography-mass spectrometry on the four extracts from the extracellular filtrate of strain HU2014 identified 10 chemical constituents in the Natural Products Atlas with high match levels (more than 90%). In an antifungal efficiency test on wheat sharp eyespot, two extracts significantly reduced the lesion areas on bean leaves infected by R. solani YL-3. The drenching of wheat in pots with spore suspension of strain HU2014 demonstrated a control efficiency of 65.1% against R. cerealis G11 (compared with 66.9% when treated by a 30% hymexazol aqueous solution). Additionally, in vitro and pot experiments demonstrated that strain HU2014 can produce indoleacetic acid, siderophores, extracellular enzymes, and solubilized phosphate, and it can promote plant growth. We conclude that strain HU2014 could be a valuable microbial resource for growth promotion of wheat and biological control of wheat sharp eyespot.

Sheath blight (ShB) severely threatens rice cultivation and production; however, the molecular mechanism of rice defence against ShB remains unclear. Screening of transposon Ds insertion mutants identified that Calcineurin B-like protein-interacting protein kinase 31 (CIPK31) mutants were more susceptible to ShB, while CIPK31 overexpressors (OX) were less susceptible. Sequence analysis indicated two haplotypes of CIPK31: Hap_1, with significantly higher CIPK31 expression, was less sensitive to ShB than the Hap_2 lines. Further analyses showed that the NAF domain of CIPK31 interacted with the EF-hand motif of respiratory burst oxidase homologue (RBOHA) to inhibit RBOHA-induced H 2 O 2 production, and RBOHA RNAi plants were more susceptible to ShB. These data suggested that the CIPK31-mediated increase in resistance is not associated with RBOHA. Interestingly, the study also found that CIPK31 interacted with catalase C (CatC); cipk31 mutants accumulated less H 2 O 2 while CIPK31 OX accumulated more H 2 O 2 compared to the wild-type control. Further analysis showed the interaction of the catalase domain of CatC with the NAF domain of CIPK31 by which CIPK31 inhibits CatC activity to accumulate more H 2 O 2 ., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

MicroRNAs (miRNAs) are a class of conserved small RNA with a length of 21-24 nucleotides in eukaryotes, which are involved in development and defense responses against biotic and abiotic stresses. By RNA-seq, Osa-miR444b.2 was identified to be induced after Rhizoctonia solani ( R. solani ) infection. In order to clarify the function of Osa-miR444b.2 responding to R. solani infection in rice, transgenic lines over-expressing and knocking out Osa-miR444b.2 were generated in the background of susceptible cultivar Xu3 and resistant cultivar YSBR1, respectively. Over-expressing Osa-miR444b.2 resulted in compromised resistance to R. solani . In contrast, the knocking out Osa-miR444b.2 lines exhibited improved resistance to R. solani . Furthermore, knocking out Osa-miR444b.2 resulted in increased height, tillers, smaller panicle, and decreased 1000-grain weight and primary branches. However, the transgenic lines over-expressing Osa-miR444b.2 showed decreased primary branches and tillers, but increased panicle length. These results indicated that Osa-miR444b.2 was also involved in regulating the agronomic traits in rice. The RNA-seq assay revealed that Osa-miR444b.2 mainly regulated the resistance to rice sheath blight disease by affecting the expression of plant hormone signaling pathways-related genes such as ET and IAA, and transcription factors such as WRKYs and F-boxes. Together, our results suggest that Osa-miR444b.2 negatively mediated the resistance to R. solani in rice, which will contribute to the cultivation of sheath blight resistant varieties.

Main Conclusion: Screening for resistance in 40 potato genotypes to Rhizoctonia solani AG-3PT-stem-canker, antioxidant enzymes activity as well as total phenol compounds were documented. Rhizoctonia solani AG-3PT-stem-canker is one of the most devastating diseases that leads to severe economic losses in potatoes, Solanum tuberosum globally. Crop management and eugenic practices, especially the use of resistance can be effective in reducing the disease incidence. However, the information about potato-R. Solani interaction is still limited. This study explored screening for resistance in forty potato genotypes to R. solani, analyzing biomass growth parameters (BGPs), as well as antioxidant enzymes activity of which peroxidase/peroxide-reductases (POXs), superoxide dismutase (SOD), polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU) and total phenol compounds (TPCs) were taken into account. In addition, we analyzed up-regulation of two gene markers (PR-1 and Osmotin), using reverse transcription quantitative PCR (RT-qPCR). For which, the resistant 'Savalan', partially resistant 'Agria', partially susceptible 'Sagita' and susceptible 'Pashandi' were selected to explore the trails in their roots and leaves over the time courses of 1, 2 and 3-weeks post inoculation (wpi) following inoculation. Cluster analysis divided potatoes into four distinct groups, based on disease severity scales (0-100%) significance. The BGPs, shoot and root length, fresh and dry weight, and root volume were also significantly higher in infected potatoes compared to non-inoculated controls. Antioxidant enzymes activity also indicated the highest increased levels for POX (fourfold at 3wpi), CAT (1.5-fold at 3wpi), SOD (6.8-fold at 1wpi), and PAL (2.7-fold at 3wpi) in the resistant genotype, 'Savalan', whereas the highest activity was recorded in TPC (twofold at 1 wpi), PPO (threefold at 3wpi), and GLU (2.3-fold at 1wpi) in partially resistant genotypes. Although the defense-related enzymatic activities were sharply elevated in the resistant and partially resistant genotypes following inoculation, no significant correlations were between the activity trends of the related enzymes. The two related gene markers also showed comprehensive transcriptional responses up to 3.4-fold, predominantly in resistant genotypes. Surprisingly, the PR-1 gene marker, basically resistant to Wilting agent Verticillium dahlia was overexpressed in resistant 'Savalan' and 'Agria' against R. solani AG3-PT. Similar results were obtained on Osmotin gene marker resistant to late-blight P. infestans, and early-blight Alternaria solani that similarly modulates immunity against R. solani. Furthermore, there was a significant correlation between resistance, enzyme activity, and gene expression in the aforesaid cultivars. Studying the physiological metabolic pathways of antioxidant enzymes activity appears to be an important direction in research to elucidate resistance to R. solani in potatoes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

Aim: To understand the mechanism of necrosis incited by a host-selective phytotoxin designated as Rhizoctonia solani toxin (RST) identified to be a potential pathogenic factor of R. solani AG1 IA, causing sheath blight (ShB) of rice., Methods and Results: The metabolomic changes induced by the phytotoxic metabolite in a ShB susceptible rice cultivar were elucidated by gas chromatography-mass spectrometry analysis and compared with that of the pathogen to identify rice metabolites targeted by the phytotoxin. The profiles of about 29 metabolites with various physiological roles in rice plants have been identified worldwide. Unsupervised and supervised multivariate chemometrics (principal component analysis and partial least squares-discriminant analysis) and cluster (Heat maps) analyses were used to compare the metabolites obtained from chemical profiles of the treatments with sterile distilled water (SDW) control. The results indicated that the rice plant expressed more metabolites in response to the pathogen than the phytotoxin and was lowest in SDW control. The key metabolites expressed in rice in response to the treatments were investigated by the variable importance in projection (VIP) analysis using p < 0.05 VIP >15. The analysis identified 7 and 11 upregulating metabolites in the phytotoxin and the pathogen treatments, respectively, compared to the untreated control. Among the phytotoxin-treated and the pathogen inoculated samples, the phytotoxin-treated sample recorded upregulation of six metabolites, whereas nine metabolites were upregulated in the pathogen-inoculated samples. These upregulating metabolites are speculated for the necrotic symptoms characteristic to both the phytotoxin and pathogen. In this analysis, hexadecanoic acid and dotriacontane were highly expressed metabolites specific to the phytotoxin and pathogen-treated samples, respectively. Besides upregulation, the metabolites also have a VIP score of >1.5 and hence fulfilled the criteria of classifying them as reliable potential biomarkers. In the pathway analysis, hexadecanoic acid and dotriacontane were identified to be involved in several important biosynthetic pathways of rice, such as the biosynthesis of saturated fatty acid and unsaturated fatty acids cutin, suberin and wax., Conclusions: The study concludes that though certain metabolites induced by the phytotoxin in the susceptible variety during necrosis shares with that of the pathogen, the identification of metabolites specific to the phytotoxin in comparison to the pathogenic and SDW controls indicated that the phytotoxin modulates the host metabolism differently and hence can be a potential pathogenicity factor of the ShB fungus., Significance and Impact of the Study: Due to lack of knowledge on the pathway genes of RST and in the absence of an ShB-resistant variety, understanding differentially expressed metabolic changes induced in the susceptible variety by the phytotoxin in comparison to that of the pathogenic and uninoculated controls enables us to identify the key metabolite changes during the ShB infection. Such metabolomic changes can further be used to infer gene functions for exploitation in ShB control., (© 2022 Society for Applied Microbiology.)

Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of cereal crops including wheat. Several metalloproteases have been implicated in pathogenic virulence, but little is known about whole-genome metalloproteases in R. cerealis . In this study, a total of 116 metalloproteases-encoding genes were identified and characterized from the R. cerealis Rc207 genome. The gene expression profiles and phylogenetic relationship of 11 MEP36/fungalysin metalloproteases were examined during the fungal infection to wheat, and function of an upregulated secretory MEP36 named RcFL1 was validated. Of 11 MEP36 family metalloproteases, ten, except RcFL5, were predicted to be secreted proteins and nine encoding genes, but not RcFL5 and RcFL2 , were expressed during the R. cerealis infection process. Phylogenetic analysis suggested that MEP36 metalloproteases in R. cerealis were closely related to those of Rhizoctonia solani but were remote to those of Bipolaris sorokiniana , Fusarium graminearum , F. pseudograminearum , and Pyricularia oryzae . Expression of RcFL1 was significantly upregulated during the infection process and induced plant cell death in wheat to promote the virulence of the pathogen. The MEP36 domain was necessary for the activities of RcFL1. Furthermore, RcFL1 could repress the expression of wheat genes coding for the chitin elicitor receptor kinase TaCERK1 and chitinases. These results suggest that this MEP36 metalloprotease RcFL1 may function as a virulence factor of R. cerealis through inhibiting host chitin-triggered immunity and chitinases. This study provides insights on pathogenic mechanisms of R. cerealis . RcFL1 likely is an important gene resource for improving resistance of wheat to R. cerealis through host-induced gene silencing strategy.

Sheath blight (ShB) caused by Rhizoctonia solani is a major disease of rice, seriously affecting yield; however, the molecular defense mechanism against ShB remains unclear. A previous transcriptome analysis of rice identified that R. solani inoculation significantly induced MDPK . Genetic studies using MDPK RNAi and overexpressing plants identified that MDPK positively regulates ShB resistance. This MDPK protein was found localized in the endoplasmic reticulum (ER) and Golgi apparatus. Yeast one-hybrid assay, electrophoresis mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) showed that the intermediate domain proteins IDD12, IDD13, and IDD14 bind to the MDPK promoter. Moreover, IDD14 was found to interact with IDD12 and IDD13 to form a transcription complex to activate MDPK expression. The three IDDs demonstrated an additive effect on MDPK activation. Further genetic studies showed that the IDD13 and IDD14 single mutants were more susceptible to ShB but not IDD12 , while IDD12 , IDD13 , and IDD14 overexpressing plants were less susceptible than the wild-type plants. The IDD12 , IDD13 , and IDD14 mutants also proved the additive effect of the three IDDs on MDPK expression, which regulates ShB resistance in rice. Notably, MDPK overexpression maintained normal yield levels in rice. Thus, our study proves that IDD12, IDD13, and IDD14 activate MDPK to enhance ShB resistance in rice. These results improve our knowledge of rice defense mechanisms and provide a valuable marker for resistance breeding.

Some green orchids obtain carbon from both mycobionts and photosynthesis at the adult stage. Intriguingly, these orchids can produce albino and, in rare cases, variegated phenotypes. Here, we studied a Platanthera hondoensis population with green, variegated, and albino individuals. Although its closely related Platanthera species are usually associated with non-ectomycorrhizal rhizoctonias, and several studies have failed to find evidence of trophic plasticity in rhizoctonia-associated orchids, variegated and albino P. hondoensis must possess a higher fungal dependency than green P. hondoensis. Therefore, we investigated whether (i) P. hondoensis is associated with non-ectomycorrhizal rhizoctonias and (ii) the degree of mycoheterotrophy (using 13C abundance as a proxy) correlates with the foliar chlorophyll concentration. High-throughput DNA sequencing revealed that all P. hondoensis phenotypes were dominantly associated with a rhizoctonia from Ceratobasidiaceae belonging to a clade distinct from recognized ectomycorrhizal clades. Regression analysis revealed a positive linear relationship between foliar chlorophyll concentration and the degree of mycoheterotrophy. This study strongly suggests that rhizoctonia-associated P. hondoensis can dynamically adjust fungal exploitation in response to photosynthetic carbon levels. Since rhizoctonia is the most common orchid mycorrhizal partner, trophic plasticity may be a widespread adaptive trait in green orchids., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Sheath blight is an important disease caused by Rhizoctonia cerealis that affects wheat yields worldwide. No wheat varieties have been identified with high resistance or immunity to sheath blight. Understanding the sheath blight resistance mechanism is essential for controlling this disease. In this study, we investigated the response of wheat to Rhizoctonia cerealis infection by analyzing the cytological changes and transcriptomes of common wheat 7182 with moderate sensitivity to sheath blight and H83 with moderate resistance., Results: The cytological observation showed that the growth of Rhizoctonia cerealis on the surface and its expansion inside the leaf sheath tissue were more rapid in the susceptible material. According to the transcriptome sequencing results, a total of 88685 genes were identified in both materials, including 20156 differentially expressed genes (DEGs) of which 12087 was upregulated genes and 8069 was downregulated genes. At 36 h post-inoculation, compared with the uninfected control, 11498 DEGs were identified in resistant materials, with 5064 downregulated genes and 6434 upregulated genes, and 13058 genes were detected in susceptible materials, with 6759 downregulated genes and 6299 upregulated genes. At 72 h post-inoculation, compared with the uninfected control, 6578 DEGs were detected in resistant materials, with 2991 downregulated genes and 3587 upregulated genes, and 7324 genes were detected in susceptible materials, with 4119 downregulated genes and 3205 upregulated genes. Functional annotation and enrichment analysis showed that the main pathways enriched for the DEGs included biosynthesis of secondary metabolites, carbon metabolism, plant hormone signal transduction, and plant-pathogen interaction. In particular, phenylpropane biosynthesis pathway is specifically activated in resistant variety H83 after infection. Many DEGs also belonged to the MYB, AP2, NAC, and WRKY transcription factor families., Conclusions: Thus, we suggest that the normal functioning of plant signaling pathways and differences in the expression of key genes and transcription factors in some important metabolic pathways may be important for defending wheat against sheath blight. These findings may facilitate further exploration of the sheath blight resistance mechanism in wheat and the cloning of related genes., (© 2022. The Author(s).)

The sharp eyespot, mainly caused by the soil-borne fungus Rhizoctonia cerealis , is a devastating disease endangering production of wheat ( Triticum aestivum ). Multi-Antimicrobial Extrusion (MATE) family genes are widely distributed in plant species, but little is known about MATE functions in wheat disease resistance. In this study, we identified TaPIMA1 , a pathogen-induced MATE gene in wheat, from RNA-seq data. TaPIMA1 expression was induced by Rhizoctonia cerealis and was higher in sharp eyespot-resistant wheat genotypes than in susceptible wheat genotypes. Molecular biology assays showed that TaPIMA1 belonged to the MATE family, and the expressed protein could distribute in the cytoplasm and plasma membrane. Virus-Induced Gene Silencing plus disease assessment indicated that knock-down of TaPIMA1 impaired resistance of wheat to sharp eyespot and down-regulated the expression of defense genes ( Defensin , PR10 , PR1.2 , and Chitinase3 ). Furthermore, TaPIMA1 was rapidly induced by exogenous H 2 O 2 and jasmonate (JA) treatments, which also promoted the expression of pathogenesis-related genes. These results suggested that TaPIMA1 might positively regulate the defense against R. cerealis by up-regulating the expression of defense-associated genes in H 2 O 2 and JA signal pathways. This study sheds light on the role of MATE transporter in wheat defense to Rhizoctonia cerealis and provides a potential gene for improving wheat resistance against sharp eyespot.

Background: Phytopathogenic microorganisms are the main cause of plant diseases, generating significant economic losses for the agricultural and food supply chain. Cherry tomatoes (Solanum lycopersicum var. cerasiforme) are very perishable plants and highly demanding in the use of pesticides; therefore, alternative solutions such as biosurfactants have aroused as a potent substituent. The main objective of the present study was to investigate the antimicrobial activity of sophorolipids against the phytopathogens Botrytis cinerea, Sclerotium rolfsii, Rhizoctonia solani and Pythium ultimum., Results: The biosurfactant inhibited the mycelial growth in vitro with a minimum concentration of 2 mg mL -1 . The application of sophorolipids at 1, 2 and 4 mg mL -1 in detached leaves of tomato before the inoculation of the fungus B. cinerea was the best treatment, reducing leaf necrosis by up to 76.90%. The use of sophorolipids for washing tomato fruits before the inoculation of B. cinerea was able to inhibit the development of gray mold by up to 96.27%., Conclusion: The results for tomato leaves and fruits revealed that the biosurfactant acts more effectively when used preventively. Sophorolipids are stable molecules that show promising action for the potential replacement of pesticides in the field and the post-harvest process against the main tomato phytopathogens. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Sheath blight (ShB) is one of the most common diseases in rice that significantly affects yield production. However, the underlying mechanisms of rice defense remain largely unknown. Our previous transcriptome analysis identified that infection with Rhizoctonia solani significantly induced the expression level of SWEET2a, a member of the SWEET sugar transporter. The sweet2a genome-editing mutants were less susceptible to ShB. Further yeast-one hybrid, ChIP, and transient assays demonstrated that WRKY53 binds to the SWEET2a promoter to activate its expression. WRKY53 is a key brassinosteroid (BR) signaling transcription factor. Similar to the BR receptor gene BRI1 and biosynthetic gene D2 mutants, the WRKY53 mutant and overexpressor were less and more susceptible to ShB compared to wild-type, respectively. Inoculation with R. solani induced expression of BRI1, D2, and WRKY53, but inhibited MPK6 (a BR-signaling regulator) activity. Also, MPK6 is known to phosphorylate WRKY53 to enhance its transcription activation activity. Transient assay results indicated that co-expression of MPK6 and WRKY53 enhanced WRKY53 trans-activation activity to SWEET2a. Furthermore, expression of WRKY53 SD (the active phosphorylated forms of WRKY53) but not WRKY53 SA (the inactive phosphorylated forms of WRKY53), enhanced WRKY53-mediated activation of SWEET2a compared to expression of WRKY53 alone. Taken together, our analyses showed that R. solani infection may activate BR signaling to induce SWEET2a expression via WRKY53 through negative regulation of ShB resistance in rice., Competing Interests: Declaration of competing interest The authors declare no known competing financial interests or no personal relationships that could have appeared to influence the work reported in this paper. None of the data presented in this manuscript have been published or are under consideration for publication elsewhere., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dry root rot ( Rhizoctonia bataticola ) is an important disease of lentils ( Lens culinaris Medik.).To gain an insight into the molecular aspects of host-pathogen interactions, the RNA-seq approach was used in lentils following inoculation with R. bataticola . The RNA-Seq has generated >450 million high-quality reads (HQRs) and nearly 96.97% were properly aligned to the reference genome. Very high similarity in FPKM (fragments per kilobase of exon per million mapped fragments) values ( R > 0.9) among biological replicates showed the consistency of the RNA-Seq results. The study revealed various DEGs (differentially expressed genes) that were associated with changes in phenolic compounds, transcription factors (TFs), antioxidants, receptor kinases, hormone signals which corresponded to the cell wall modification enzymes, defense-related metabolites, and jasmonic acid (JA)/ethylene (ET) pathways. Gene ontology (GO) categorization also showed similar kinds of significantly enriched similar GO terms. Interestingly, of the total unigenes (42,606), 12,648 got assembled and showed significant hit with Rhizoctonia species. String analysis also revealed the role of various disease responsive proteins viz., LRR family proteins, LRR-RLKs, protein kinases, etc. in the host-pathogen interaction. Insilico validation analysis was performed using Genevestigator ® and DEGs belonging to six major defense-response groups viz., defense-related enzymes, disease responsive genes, hormones, kinases, PR (pathogenesis related) proteins, and TFs were validated. For the first time some key miRNA targets viz. miR156, miR159, miR167, miR169, and miR482 were identified from the studied transcriptome, which may have some vital role in Rhizoctonia -based responses in lentils. The study has revealed the molecular mechanisms of the lentil/ R. bataticola interactions and also provided a theoretical approach for the development of lentil genotypes resistant to R. bataticola .

The concern regarding the emergence of phytopathogens strains which are resistant to conventional agrochemicals has given support to the search for alternatives on the use of chemical pesticides in agriculture. In this context, microorganisms are considered as promising sources of useful natural compounds and actinobacteria are particularly relevant since they are known to produce several bioactive metabolites. The objective of this work was to investigate the production of secondary metabolites with antifungal activity by a strain of the actinobacteria Streptomyces lunalinharesii (A54A) under axenic conditions and in co-cultivation with the phytopathogen Rhizoctonia solani. Tests to evaluate antifungal activity of the extracts indicated the presence of diffusable molecules capable of inhibiting the growth of R. solani produced by S. lunalinharesii, especially when in the presence of the fungus during fermentation. Metabolomic analyzes allowed the putative annotation of the bioactive compounds desferrioxamine E and anisomycin, in addition to the evaluation of the metabolic profile of the isolate when grown in axenic mode and in co-cultivation, while statistical analyzes enabled the comparison of such profiles and the identification of metabolites produced in greater relative quantities in the elicitation condition. Such methodologies provided the selection of unknown features with high bioactive potential for dereplication, and several metabolites of S. lunalinharesii possibly represent novel compounds., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Xylanase secreted by Trichoderma asperellum ACCC30536 can stimulate the systemic resistance of host plants against pathogenic fungi. Following T. asperellum conidia co-culture with Populus davidiana × P. alba var. pyramidalis Louche (PdPap) seedlings, the expression of xylanases TasXyn29.4 and TasXyn24.2 in T. asperellum were upregulated, peaking at 12 h, by 106 (2 6.74 ) and 10.1 (2 3.34 )-fold compared with the control, respectively. However, the expression of TasXyn24.4 and TasXyn24.0 was not detected. When recombinant xylanases rTasXyn29.4 and rTasXyn24.2 were heterologously expressed in Pichia pastoris GS115, their activities reached 18.9 IU/mL and 20.4 IU/mL, respectively. In PdPap seedlings induced by rTasXyn29.4 and rTasXyn24.2, the auxin and jasmonic acid signaling pathways were activated to promote growth and enhance resistance against pathogens. PdPap seedlings treated with both xylanases showed increased methyl jasmonate contents at 12 hpi, reaching 122 % (127 μg/g) compared with the control. However, neither of the xylanases could induce the salicylic acid signaling pathway in PdPap seedlings. Meanwhile, both xylanases could enhance the antioxidant ability of PdPap seedlings by improving their catalase activity. Both xylanases significantly induced systemic resistance of PdPap seedlings against Alternaria alternata, Rhizoctonia solani, and Fusarium oxysporum. However, the xylanases could only be sensed by the roots of the PdPap seedlings, not the leaves. In summary, rTasXyn29.4 and rTasXyn24.2 from T. asperellum ACCC30536 promoted growth and induced systemic resistance of PdPap seedlings, which endowed the PdPap seedlings broad-spectrum resistance to phytopathogens., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Enhancing soil suppressiveness against plant pathogens or pests is a promising alternative strategy to chemical pesticides. Organic amendments have been shown to reduce crop diseases and pests, with chitin products the most efficient against fungal pathogens. To study which characteristics of organic products are correlated with disease suppression, an experiment was designed in which 10 types of organic amendments with different physicochemical properties were tested against the soilborne pathogen Rhizoctonia solani in sugar beet seedlings. Organic amendments rich in keratin or chitin reduced Rhizoctonia solani disease symptoms in sugar beet plants. The bacterial and fungal microbial communities in amended soils were distinct from the microbial communities in nonamended soil, as well as those in soils that received other nonsuppressive treatments. The Rhizoctonia -suppressive amended soils were rich in saprophytic bacteria and fungi that are known for their keratinolytic and chitinolytic properties (i.e., Oxalobacteraceae and Mortierellaceae ). The microbial community in keratin- and chitin-amended soils was associated with higher zinc, copper, and selenium, respectively. IMPORTANCE Our results highlight the importance of soil microorganisms in plant disease suppression and the possibility to steer soil microbial community composition by applying organic amendments to the soil., (Copyright © 2021 Andreo-Jimenez et al.)

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants' molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants' comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (-Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani . Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term "response to abiotic stimulus" was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.

Proper management of agricultural disease is important to ensure sustainable food security. Staple food crops like rice, wheat, cereals, and other cash crops hold great export value for countries. Ensuring proper supply is critical; hence any biotic or abiotic factors contributing to the shortfall in yield of these crops should be alleviated. Rhizoctonia solani is a major biotic factor that results in yield losses in many agriculturally important crops. This paper focuses on genome informatics of our Malaysian Draft R. solani AG1-IA, and the comparative genomics (inter- and intra- AG) with four AGs including China AG1-IA (AG1-IA&#95;KB317705.1), AG1-IB, AG3, and AG8. The genomic content of repeat elements, transposable elements (TEs), syntenic genomic blocks, functions of protein-coding genes as well as core orthologous genic information that underlies R. solani 's pathogenicity strategy were investigated. Our analyses show that all studied AGs have low content and varying profiles of TEs. All AGs were dominant for Class I TE, much like other basidiomycete pathogens. All AGs demonstrate dominance in Glycoside Hydrolase protein-coding gene assignments suggesting its importance in infiltration and infection of host. Our profiling also provides a basis for further investigation on lack of correlation observed between number of pathogenicity and enzyme-related genes with host range. Despite being grouped within the same AG with China AG1-IA, our Draft AG1-IA exhibits differences in terms of protein-coding gene proportions and classifications. This implies that strains from similar AG do not necessarily have to retain similar proportions and classification of TE but must have the necessary arsenal to enable successful infiltration and colonization of host. In a larger perspective, all the studied AGs essentially share core genes that are generally involved in adhesion, penetration, and host colonization. However, the different infiltration strategies will depend on the level of host resilience where this is clearly exhibited by the gene sets encoded for the process of infiltration, infection, and protection from host.

Elucidation of the inhibitory effects of humic substances (HSs) on phytopathogenic fungi and the underlying molecular mechanisms are highly important for improved biocontrol. In this study, we investigated the growth suppression, morphological characteristics, transcriptomic sequence, and radical signals of Rhizoctonia solani following HS addition (50 mg/L). Through mycelial cultured experiment, mycelia growth of R. solani had been suppressed with HS addition, and the inhibition rate was 24.88 ± 0.11% compared to the control. Field emission-scanning electron microscopy showed increased and superimposed branching mycelial growth, with a shriveled appearance. RNA samples of R. solani cultured with or without HSs were both extracted to examine the sequence on molecular level by Illumina HiSeq sequencing platform. RNA sequencing analysis revealed 175 differentially expressed genes (DEGs; 111 upregulated and 64 downregulated) between the HSs treatment and control. The upregulated unigenes were annotated and significantly enriched to three molecular processes: vitamin B6 metabolism, ABC transporters, and glutathione metabolism, while the downregulated unigenes were annotated to carbohydrate metabolism, but not significantly enriched. Real time-quantitative polymerase chain reaction analysis showed that the unigenes related to hexokinase, glucose-6-phosphate isomerase, glutathione synthase, and glutathione reductase were significantly decreased (by 60.03%, 70.70%, 60.33%, and 57.59%, respectively), while those related to glutathione S-transferase were significantly increased (2.66-fold). The electron paramagnetic resonance spectra showed that HSs induced increased the intensity of radical signals of R. solani in a cultured system increased by 59.56% compared to CK (without HSs addition). Network analysis based on DEGs expression and the chemical structure of HSs revealed that the carbonyl moiety in HSs formed the most links with nodes of the DEGs (sum of the links of positive and negative effects = 70), implicating this structure as the active fraction responsible for the inhibitory effect. This study provides molecular and chemical evidence of the biofungicidal activity of HSs with the potential for practical application., (Copyright © 2020. Published by Elsevier Inc.)

Aims: Arbuscular mycorrhizal fungi (AMF) are symbiotic partners of many invasive plants, however, it is still unclear how AMF contribute to traits that are important for the successful invasion of their host and how environmental factors, such as nutrient conditions, influence this. This study was to explore the effects of Glomus versiforme (GV) and Glomus mosseae (GM) on the growth and disease resistance of the invasive plant Wedelia trilobata under different nutrient conditions., Methods and Results: We found that GV and GM had higher root colonization rates resulting in faster W. trilobata growth under both low-N and low-P nutrient conditions compared to the normal condition. Also, the colonization of W. trilobata by GV significantly reduced the infection area of the pathogenic fungus Rhizoctonia solani under low-N conditions., Conclusions: These results demonstrated that AMF can promote the growth and pathogenic defence of W. trilobata in a nutrient-poor environment, which might contribute to their successful invasion into certain type of habitats., Significance and Impact of the Study: In this study, we report for the first time that AMF can promote growth and disease resistance of W. trilobata under nutrient-poor environment, which contribute to a better understanding of plant invasion., (© 2019 The Society for Applied Microbiology.)

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components-plant, pathogen, and environment-involved in the disease triangle. Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is one of the most beneficial traits of plant growth promoting (PGP) rhizobacteria responsible for protecting the plants from detrimental effects of abiotic and biotic stress. The strain S3 with ACC deaminase activity (724.56 nmol α-ketobutyrate mg -1 protein hr -1 ) was isolated from rhizospheric soil of turmeric (Curcuma longa), a medicinal plant, growing in Motihari district of Indian state, Bihar. The halotolerant strain S3, exhibited optimum growth at 8% (w/v) NaCl. It also exhibited multiple PGP traits such as indole acetic acid production (37.71 μg mL -1 ), phosphate solubilization (69.68 mg L -1 ), siderophore, hydrocyanic acid (HCN) and ammonia production as well as revealed antagonism against Rhizoctonia solani. The potential of isolated strain to alleviate salinity stress in tomato plants was investigated through pots trials by inoculating strain S3 through-seed bacterization, soil drenching, root dipping as well as seed treatment + soil drenching. The strain S3 inoculated through seed treatment and soil drenching method led to improved morphological attributes (root/shoot length, root/shoot fresh weight and root/shoot dry weight), photosynthetic pigment content, increased accumulation of osmolytes (proline and total soluble sugar), enhanced activities of antioxidants (Catalase and Peroxidase) and phenolic content in salt stressed tomato plants. The biochemical characterisation, FAMEs analysis and 16S rRNA gene sequencing revealed that strain S3 belongs to the genus Pseudomonas. The overall findings of the study revealed that Pseudomonas sp. strain S3 can be explored as an effective plant growth promoter which stimulate growth and improve resilience in tomato plants under saline condition.

Rhizoctonia solani is a soil-borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3-1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession-specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3-1 and Tek-3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY-dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.

A total of 120 rhizobacteria were isolated from seven different tea estates of Darjeeling, West Bengal, India. Based on a functional screening of in vitro plant growth-promoting (PGP) activities, thirty potential rhizobacterial isolates were selected for in-planta evaluation of PGP activities in rice and maize crops. All the thirty rhizobacterial isolates were identified using partial 16S rRNA gene sequencing. Out of thirty rhizobacteria, sixteen (53.3%) isolates belong to genus Bacillus, five (16.6%) represent genus Staphylococcus, three (10%) represent genus Ochrobactrum, and one (3.3%) isolate each belongs to genera Pseudomonas, Lysinibacillus, Micrococcus, Leifsonia, Exiguobacterium, and Arthrobacter. Treatment of rice and maize seedlings with these thirty rhizobacterial isolates resulted in growth promotion. Besides, rhizobacterial treatment in rice triggered enzymatic [ascorbate peroxidase (APX), catalase (CAT), chitinase, and phenylalanine ammonia-lyase (PAL)], and non-enzymatic [proline and polyphenolics] antioxidative defense reactions indicating their possible role in the reduction of reactive oxygen species (ROS) burden and thereby priming of plants towards stress mitigation. To understand such a possibility, we tested the effect of rhizobacterial consortia on biotic stress tolerance of rice against necrotrophic fungi, Rhizoctonia solani AG1-IA. Our results indicated that the pretreatment with rhizobacterial consortia increased resistance of the rice plants towards the common foliar pathogen like R. solani AG1-IA. This study supports the idea of the application of plant growth-promoting rhizobacterial consortia in sustainable crop practice through the management of biotic stress under field conditions.

The domain of unknown function 26 (DUF26), harboring a conserved cysteine-rich motif (C-X8-C-X2-C), is unique to land plants. Several cysteine-rich repeat proteins (CRRs), belonging to DUF26-containing proteins, have been implicated in the defense against fungal pathogens in ginkgo, cotton, and maize. However, little is known about the functional roles of CRRs in the important staple crop wheat ( Triticum aestivum ). In this study, we identified a wheat CRR-encoding gene TaCRR1 through transcriptomic analysis, and dissected the defense role of TaCRR1 against the soil-borne fungi Rhizoctonia cerealis and Bipolaris sorokiniana , causal pathogens of destructive wheat diseases. TaCRR1 transcription was up-regulated in wheat towards B. Sorokiniana or R. cerealis infection. The deduced TaCRR1 protein contained a signal peptide and two DUF26 domains. Heterologously-expressed TaCRR1 protein markedly inhibited the mycelia growth of B. sorokiniana and R. cerealis . Furthermore, the silencing of TaCRR1 both impaired host resistance to B. sorokiniana and R . cerealis and repressed the expression of several pathogenesis-related genes in wheat. These results suggest that the TaCRR1 positively participated in wheat defense against both B. sorokiniana and R. cerealis through its antifungal activity and modulating expression of pathogenesis-related genes. Thus, TaCRR1 is a candidate gene for improving wheat resistance to B. sorokiniana and R. cerealis .

Damping-off disease in seeds and young seedlings in agricultural crops is a major fungal disease that limits the agriculture production. Frequent use of synthetic fungicides against damping-off diseases is known to hamper the environmental balance. Thus, an alternative approach needs to be explored for the management of such economically important fungal diseases. In the present study, simple, economically feasible chitosan-coupled copper nanoparticles (Ch-CuNPs) were synthesized and demonstrated antifungal activity against damping-off disease causing phytopathogens, Rhizoctonia solani and Pythium aphanidermatum. Physico-chemical studies confirmed the size, shape, surface charge, element confirmation and mono-dispersed nature of Ch-CuNPs. In vitro efficacy studies revealed up to 98% mycelial growth inhibition at 0.1% Ch-CuNPs. An extracellular conductivity study of the mycelium showed cellular content leakage within 12 h of treatment. Further, plant toxicity study against chili, cowpea and tomato plants; showed that ≤0.2% NPs were safe under greenhouse conditions. NPs also exhibited growth-promoting activity with chili seeds, by overcoming the limited germination rate of susceptible seeds. Overall, the present study emphasizes the benefits of synthesized Ch-CuNPs on agricultural crops as fungicide and growth-promoter, as well as a safe alternative to pesticides in order to avoid hazardous effect on the environment., Competing Interests: Declaration of competing interest All contributing authors declare no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The soil-borne pathogen Rhizoctonia solani infects a broad range of plants worldwide and is responsible for significant crop losses. Rhizoctonia solani AG3-PT attacks germinating potato sprouts underground while molecular responses during interaction are unknown. To gain insights into processes induced in the fungus especially at early stage of interaction, transcriptional activity was compared between growth of mycelium in liquid culture and the growing fungus in interaction with potato sprouts using RNA-sequencing. Genes coding for enzymes with diverse hydrolase activities were strongly differentially expressed, however with remarkably dissimilar time response. While at 3 dpi, expression of genes coding for peptidases was predominantly induced, strongest induction was found for genes encoding hydrolases acting on cell wall components at 8 dpi. Several genes with unknown function were also differentially expressed, thus assuming putative roles as effectors to support host colonization. In summary, the presented analysis characterizes the necrotrophic lifestyle of R. solani AG3-PT during early interaction with its host.