Biocontrol Agent Clonostachys Rosea Research Articles

Sterol regulatory element-binding proteins mediate intrinsic fungicide tolerance and antagonism in the fungal biocontrol agent Clonostachys rosea IK726

Sterol regulatory element-binding proteins (SREBPs) are transcription factors governing various biological processes in fungi, including virulence and fungicide tolerance, by regulating ergosterol biosynthesis and homeostasis. While studied in model fungal species, their role in fungal species used for biocontrol remains elusive. This study delves into the biological and regulatory function of SREBPs in the fungal biocontrol agent (BCA) Clonostachys rosea IK726, with a specific focus on fungicide tolerance and antagonism. Clonostachys rosea genome contains two SREBP coding genes (sre1 and sre2) with distinct characteristics. Deletion of sre1 resulted in mutant strains with pleiotropic phenotypes, including reduced C. rosea growth on medium supplemented with prothioconazole and boscalid fungicides, hypoxia mimicking agent CoCl2 and cell wall stressor SDS, and altered antagonistic abilities against Botrytis cinerea and Rhizoctonia solani. However, Δsre2 strains showed no significant effect. Consistent with the gene deletion results, overexpression of sre1 in Saccharomyces cerevisiae enhanced tolerance to prothioconazole. The functional differentiation between SRE1 and SRE2 was elucidated by the yeast-two-hybridization assay, which showed an interaction between SREBP cleavage-activating protein (SCAP) and SRE1 but not between SRE2 and SCAP. Transcriptome analysis of the Δsre1 strain unveiled SRE1-mediated expression regulation of genes involved in lipid metabolism, respiration, and xenobiotic tolerance. Notably, genes coding for antimicrobial compounds chitinases and polyketide synthases were downregulated, aligning with the altered antagonism phenotype. This study uncovers the role of SREBPs in fungal BCAs, providing insights for C. rosea IK726 application into integrated pest management strategies.

New N-acylated aminoalkanoic acids from tea roots derived biocontrol agent Clonostachys rosea 15020

Four new N-acylated aminoalkanoic acids, namely clonoroseins E−H (1−4), together with three previously identified analogs, clonoroseins A, B, and D (5−7), were identified from the endophytic fungus Clonostachys rosea strain 15020 (CR15020), using Feature-based Molecular Networking (FBMN). The elucidation of their chemical structures, including their absolute configurations, was achieved through spectroscopic analysis combined with quantum chemical calculations. Bioinformatics analyses suggested that an iterative type I HR-PKS (CrsE) generates the polyketide side chain of these clonoroseins. Furthermore, a downstream adenylate-forming enzyme of the PKS (CrsD) was suspected to function as an amide synthetase. CrsD potentially facilitates the transformation of the polyketide moiety into an acyl-AMP intermediate, followed by nucleophilic substitution with either β-alanine or γ-aminobutyric acid to produce amide derivatives. These findings significantly expand our understanding of PKS-related products originating from C. rosea and also underscore the powerful application of FBMN analytical methods in characterization of new compounds.

A Three-Way Transcriptomic Interaction Study of a Biocontrol Agent (Clonostachys rosea), a Fungal Pathogen (Helminthosporium solani), and a Potato Host (Solanum tuberosum).

Helminthosporium solani causes silver scurf, which affects the quality of potato. The biocontrol agent Clonostachys rosea greatly limited the severity of silver scurf symptoms and amount of H. solani genomic DNA in laboratory experiments. Transcriptomic analysis during interaction showed that H. solani gene expression was highly reduced when coinoculated with the biocontrol agent C. rosea, whereas gene expression of C. rosea was clearly boosted as a response to the pathogen. The most notable upregulated C. rosea genes were those encoding proteins involved in cellular response to oxidative stress, proteases, G-protein signaling, and the methyltransferase LaeA. The most notable potato response to both fungi was downregulation of defense-related genes and mitogen-activated protein kinase kinase kinases. At a later stage, this shifted, and most potato defense genes were turned on, especially those involved in terpenoid biosynthesis when H. solani was present. Some biocontrol-activated defense-related genes in potato were upregulated during early interaction with C. rosea alone that were not triggered by H. solani alone. Our results indicate that the reductions of silver scurf using C. rosea are probably due to a combination of mechanisms, including mycoparasitism, biocontrol-activated stimulation of plant defense mechanisms, microbial competition for nutrients, space, and antibiosis.

UV-B radiation reduces biocontrol ability of Clonostachys rosea against Botrytis cinerea

ABSTRACTThere is a lack of information comparing UV-B radiation conidial sensitivity of the biocontrol agent Clonostachys rosea (Cr) and its target pathogen, Botrytis cinerea (Bc). We investigated the interactions in vitro and on strawberry leaf discs between previously selected Cr and Bc strains tolerant to UV-B radiation. Strawberry leaf discs inoculated with Bc, Cr, or combinations of both fungi were exposed to UV-B doses (2.9, 5.9, and 8.9 kJ m−2). Incidence and sporulation of both fungi were evaluated, and the Area Under Incidence Progress Curve (AUIPC) and Area Under Sporulation Progress Curve (AUSPC) were calculated. AUIPC and AUSPC of Cr on leaf discs were negatively correlated to increased UV-B. When inoculated alone on leaf discs, Bc was not affected by UV-B, but when inoculated with Cr the incidence and sporulation of Bc were positively correlated to UV-B radiation dose. In the absence of UV-B, Cr reduced incidence and sporulation of Bc. However, the ability of Cr to control Bc was reduced by 20% to 50% with increasing UV-B radiation. Increasing the applied concentration of Cr conidia 10-fold partially overcame the deleterious effects of UV-B on the ability of the biocontrol agent to reduce Bc sporulation in strawberry leaves. The selection of antagonists must fulfil many requirements; besides being active against the specific targeted plant pathogens, they must be cost-effective and have ecological characteristics suitable for the desired use conditions. We suggest that UV-B exposure must be taken into account during the development of bio-fungicides based on Cr.

Relationship between environmental conditions, carbon utilisation patterns and Niche Overlap Indices of the mycotoxigenic species Fusarium verticillioides and the biocontrol agent Clonostachys rosea

Abstract Recently, it was shown that a strain of the fungal antagonist Clonostachys rosea 016 was able to inhibit fumonisin B1 mycotoxin production by Fusarium verticillioides FV1 when using different ratios of spores of each species in vitro. The objectives of the present work were therefore to: (a) compare the nutritional utilisation patterns and rates of uptake of key C-sources in maize by the antagonist C. rosea 016 and that by the pathogen F. verticillioides FV1; (b) examine their Niche Overlap Indices (NOI) under different interacting environmental conditions; and (c) evaluate whether the rate of utilisation of key maize C-sources influenced the competitiveness of either species using the Bioscreen®. It was found that water potential (Ψ) × temperature interactions had significant impacts on C-source utilisation patterns by C. rosea 016 and the pathogen. The NOIs, based on the utilisation of the C-sources by each strain divided by those utilised in common, showed that the antagonist and the pathogen occupied similar niches at −0.70 MPa Ψ+30 °C and −2.8 MPa Ψ+25 °C. Under the other conditions tested, they appeared to occupy separate niches suggesting niche exclusion. Temporal C-source utilisation patterns were then compared under different Ψ × temperature treatments. This showed that the dominant maize-based C-sources utilised by the pathogen and the antagonist were different. The pathogen F. verticillioides FV1 utilised carbohydrates rapidly followed by amino acids and then one fatty acid, palmitic acid. The antagonist C. rosea 016 utilised both carbohydrates and amino acids at a similar rate but more slowly than the pathogen. There were also differences in the utilisation of some individual amino acids and carbohydrates which might explain the occupation of different niches under some interacting environmental conditions. These findings are discussed in the context of why some competitors are able to inhibit mycotoxin production while others cannot.

Spore production of Clonostachys rosea in a new solid-state fermentation reactor.

A new solid-state fermentation (SSF) reactor was developed for the spore production of biocontrol agent Clonostachys rosea. The greatest spore production in the reactor, 3.36 × 10(10) spores g DM(-1) occurred with mixings, which was about 10 times greater than that in traditional tray reactor. The reactor provides about two times sporulation surface area for spore formation. Moisture content of the medium was adjusted to meet the spore production by changing the surface porosity. Two mixings were carried out during cultivation to make the medium loose, which resulted in a mass of new sporulation surface. The fermentation period shortened from 14-15 to 10-11 days. It is suggested that the new reactor has great potential in the mass production of spores of C. rosea and other fungal biocontrol agents.

Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea.

BackgroundFilamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent Clonostachys rosea in order to understand their potential roles in biocontrol mechanisms.ResultsBased on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in C. rosea. Gene expression analysis showed basal expression of Hyd1, Hyd2 and Hyd3 in all conditions tested with the exception of induced Hyd1 expression in conidiating mycelium. Interestingly, up-regulation of Hyd1, Hyd2 and Hyd3 was found during C. rosea self interaction compared to interactions with the fungal plant pathogens Botrytis cinerea or Fusarium graminearum in dual culture assays. Phenotypic analysis of C. rosea deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using in vitro dual culture and detached leaf assays showed that the mutant strains were more aggressive towards B. cinerea, F. graminearum or Rhizoctonia solani, and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore, in vitro Arabidopsis thaliana root colonization assays revealed reduced root colonization ability of the ΔHyd3 strain, but not for the ΔHyd1 strain. Furthermore, enhanced root colonization ability for the ΔHyd1ΔHyd3 strain was found in comparison to WT.ConclusionsThese results show a role for hydrophobins in conidial hydrophobicity, control of conidial germination under stress conditions, and in root colonization in C. rosea. However, functional studies of Hyd2 remains to be performed in order to fully assess the role of hydrophobins in C. rosea.

Colonization of geranium foliage by Clonostachys rosea f. catenulata, a biological control agent of botrytis grey mould

The ecological requirements for the colonization of geranium leaves by the biocontrol agent Clonostachys rosea f. catenulata strain J1446 were investigated. Although this biocontrol agent is a soil-inhabiting fungus, treatment of geranium foliage with the agent can reduce grey mould caused by Botrytis cinerea in the greenhouse. To characterize the extent of foliar colonization, a GUS-transformed isolate of C. rosea f. catenulata was applied to foliage of two geranium cultivars, Pelargonium × hortorum and Pelargonium × domesticum . Population levels of C. rosea f. catenulata were found to be highest on senescent leaves and stems, followed by fully expanded leaves, and lowest on newly emerged leaves of both cultivars. Optimum temperature for leaf and petiole colonization was 20–25 °C for both cultivars. The biocontrol agent required at least 12 h of continuous leaf wetness to achieve maximum population densities on the leaves and stems of both cultivars. On whole plants, colonization was significantly higher on wounded leaves, stems, and senescing leaves compared with that on nonwounded leaves, stems, and mature leaves, respectively. GUS staining indicated that the fungus preferentially colonized the wound sites of leaves and the cut portions of stems. Results indicate that this biocontrol agent can successfully colonize the foliage of geraniums, thus demonstrating the endophytic ability of C. rosea f. catenulata in both root and foliar tissues.

Effects of compost amendment and the biocontrol agent clonostachys rosea on the development of charcoal rot (macrophomina phaseolina) on cowpea

Macrophomina phaseolina is a destructive pathogen causing charcoal rot of cowpea and other crops in the semi- arid areas of the Sahel (north-west Africa). Chemical management is not feasible in conditions of subsistence farming, and the plurivorous nature of the fungus limits the effectiveness of some cultural methods. This study aimed at identifying the effects of composting on the survival of M. phaseolina and of soil application of compost alone or in combination with the biocontrol agent Clonostachys rosea on inoculum density of M. phaseolina and on cowpea production. Inside the compost heap with diseased cowpea residues, the temperature reached 52 to 60°C and completely destroyed M. phaseolina microsclerotia. Addition of compost to planting holes significantly suppressed charcoal rot disease. Among the doses tested 6 tonnes of compost alone or supplemented with 50 kg NPK ha-1 resulted in 28-45% lower Area-Under- the-Disease-Progress-Curves (AUDPC) and 43-66% higher cowpea production. The addition of compost combined with C. rosea in the planting holes reduced the AUDPC up to 4-fold and increased the grain yield 2-5- fold. The best treatment was a mixture of two C. rosea isolates and the compost.

An N-acetyl-β- d-glucosaminidase gene, cr-nag1, from the biocontrol agent Clonostachys rosea is up-regulated in antagonistic interactions with Fusarium culmorum

Clonostachys rosea is a widely distributed fungus that often acts as a parasite on other soil fungi. This fungus has also been reported as a potential parasite against nematodes and insects. The antagonistic activity is thought to be correlated with the secretion of cell wall-degrading enzymes, including chitinases. In this work, we identified and characterized an N-acetyl-β- d-glucosaminidase-encoding gene, cr-nag1, belonging to glycosyl hydrolase family 20, from the C. rosea strain IK726 using a degenerated primer strategy designed from conserved motifs. The complete gene, including its promoter region, was obtained by genomic walking. Southern analysis showed that cr-nag1 is present as a single copy gene in C. rosea. Phylogenetically, cr-nag1 showed the highest similarity to N-acetyl-β- d-glucosaminidase genes from other mycoparasitic fungi. Enzymatic assays and RT-PCR showed that the NAGase activity of C. rosea is specifically repressed in medium containing a high glucose content and is expressed in media containing chitin or Fusarium culmorum cell walls as sole carbon sources. Macroscopic and microscopic observations indicated that the mycelial growth of F. culmorum and Pythium ultimum were inhibited during interactions with C. rosea. High expression of cr-nag1 was found in interactions between C. rosea and F. culmorum, whereas the expression of cr-nag1 in interactions between C. rosea and P. ultimum was similar to the control. This indicates that although C. rosea secretes chitin-hydrolysing agents in order to target the cell wall of F. culmorum, it seems to use another strategy for controlling the development of the oomycete P. ultimum.

Induction of PR proteins and resistance by the biocontrol agent Clonostachys rosea in wheat plants infected with Fusarium culmorum

Clonostachys rosea (CR) is a common worldwide saprophyte with destructive effect against several plant pathogenic fungi showing antagonistic features against a wide variety of pathogens. We recently isolated a strain of C. rosea, named CR47, from wheat crown infected with Fusarium culmorum (FC); this strain proved to be effective against Fusarium seed borne diseases of cereals under field condition. In this paper the function of C. rosea applied as seed treatment on wheat seedling growth was investigated. In addition, we investigated the expression pattern of peroxidases and chitinases as well as PR4 proteins following both CR treatments of seeds and FC infection and also in the three-component system pathogen–antagonist–wheat. Several chitinase isoforms were induced by CR-treatment both in coleoptiles and roots, whereas some peroxidase isoforms were induced only in the presence of both antagonist and pathogen. In the latter case, it seems that CR-treatment by itself promotes plant growth and reduces the peroxidase expression, while enhances some chitinase isoforms probably involved in cell wall disruption. Moreover, both the antagonist and the pathogen studied induced PR4 protein expression, which probably exerts its role on the invading microorganisms by a translation-inhibitory process that could be ascribed to their ribonuclease activity.

Soil inoculation with the biocontrol agent Clonostachys rosea and the mycorrhizal fungus Glomus intraradices results in mutual inhibition, plant growth promotion and alteration of soil microbial communities

Interactions between the biocontrol fungus Clonostachys rosea IK 726 and a tomato/ Glomus intraradices BEG87 symbiosis were examined with and without wheat bran, which served as a food base for C. rosea. In soil without wheat bran amendment, inoculation with C. rosea increased plant growth and altered shoot nutrient content resulting in an increase and decrease in P and N content, respectively. Inoculation with G. intraradices had no effect on plant growth, but increased the shoot P content. Dual inoculation with G. intraradices and C. rosea followed the pattern of C. rosea in terms of plant growth and nutrient content. Wheat bran amendment resulted in marked plant growth depressions, which were counteracted by both inoculants and dual inoculation increased plant growth synergistically. Amendment with wheat bran increased the population density of C. rosea and reduced mycorrhizal fungus colonisation of roots. The inoculants were mutually inhibitory, which was shown by a reduction in root colonisation with G. intraradices in treatments with C. rosea and a reduction in colony-forming units (cfu) of C. rosea in treatments with G. intraradices, irrespective of wheat bran amendment. Moreover, both inoculants markedly influenced soil microbial communities examined with biomarker fatty acids. Inoculation with G. intraradices increased most groups of microorganisms irrespective of wheat bran amendment, whereas the influence of C. rosea on other soil microorganisms was affected by wheat bran amendment. Overall, inoculation with C. rosea increased and decreased most groups of microorganisms without and with wheat bran amendment, respectively. In conclusion, despite mutual inhibition between the two inoculants this interaction did not impair their observed plant growth promotion. Both inoculants also markedly influenced other soil microorganisms, which should be further studied in relation to their plant growth-promoting features.

Relationships of microclimatic variables to colonization of rose debris by Botrytis cinerea and the biocontrol agent Clonostachys rosea

Biological control of Botrytis cinerea by Clonostachys rosea is an alternative to chemical control of rose Botrytis blight in greenhouses. Environmental conditions affect the colonization of senescing and dead tissues by both fungi. The contribution of microclimatic variables to debris colonization/sporulation by both fungi was estimated by path coefficient analysis. We monitored daily values of: maximum, average, and minimum temperatures (T max, T ave, and T min), and relative humidity (RHmax, RHave, and RHmin); accumulated rainfall; vapour pressure deficit average; hours with RH > 90% (RH90); and average temperature during RH90 (T ave90). Association of variables accumulated between the first and seventh day before sampling explained colonization/sporulation variation: R 2=0.81–0.86 for B. cinerea and 0.91–0.96 for C. rosea. RHmax and RH90 were the main factors that directly favoured colonization/sporulation of both fungi. Colonization/sporulation negatively correlated with RHmin, T min, and T ave for B. cinerea and T min, T ave, and T ave90 for C. rosea. The antagonist can suppress B. cinerea colonization/sporulation on rose debris under a wide range of environmental conditions.

Two-step liquid/solid state scaled-up production of Clonostachys rosea

Economic mass production is a crucial stage in the development of biocontrol agents. This paper compares different solid substrates with and without preceding liquid culture for efficacy and economics in the scaled-up production of the mycoparasitic biocontrol agent Clonostachys rosea. In a basic liquid medium (BLM) containing (in g l −1 ): molasses (80), neopeptone (10) and yeast extract (2), Clonostachys rosea produced 10 7 conidia ml −1 after 4 d in agitated culture. Spore viability was 99% and did not differ from that of conidia derived from potato dextrose agar plates. Guata, an inert polyester fibre, coated with BLM and cassava starch (30 g l −1 ), was the best solid matrix yielding 10 9 conidia g −1 after 20 d. On a volume basis after extraction and on a production unit basis, Guata was in a similar range to Biodac®, a granular cellulose complex, which had also been enriched with BLM, producing up to 10 7 conidia ml −1 or over 10 10 conidia per 23 × 28 cm polypropylene bag, the production unit in which all solid matrices were incubated. Rice was the lowest-yielding solid matrix regardless of quality. Chlamydospores were not observed in liquid or on solid media. All conidia produced on solid matrices had a viability >90%. Conidia produced on Biodac had a higher viability (95.6%) than spores produced on Guata (93.1%) after 24 h on potato-dextrose plates. However, production on Biodac was more variable and negatively affected by prefermentation in liquid culture, whereas production on Guata and rice was unaffected by the origin of the starting inoculum. Guata of medium thickness (1.27 cm) was superior to thicker (2.54 cm) or thinner (0.64 cm) sheets with respect to both yield per gram and per production unit. Although prefermentation neither improved yield nor reduced cost, it increased throughput by a factor five and may be advantageous where fermentor and/or incubator space is limited. Biodac and Guata were the most economical solid matrices; inoculum to treat one hectare (3 × 10 12 conidia) cost US $ 27, whereas rice-based production cost US $ 613 under Costa Rican conditions. Liquid culture alone would be fast but too costly: US $ 1078 ha −1 . Experiments to further reduce costs in the Guata system are suggested.

Influence of preinoculation light intensity on development and interactions of Botrytis cinerea and Clonostachys rosea in tomato leaves

Tomato plants produced in pots in a greenhouse were kept in low intensities of photosynthetically active radiation (400-700 nm at means of 384, 308, 135, and 14 μmol·m–2·s–1) during 16-h photoperiods for 0, 5, 10, or 15 days. Immediately after treatment, leaflets of the fourth and fifth leaves were detached and inoculated with Botrytis cinerea Pers.:Fr. (106 conidia/mL), the biocontrol agent Clonostachys rosea (Link:Fr.) Schroers, Samuels, Siefert and W. Gams (107 conidia/mL), or both fungi (same final concentrations). Germination of each fungus was estimated after leaflets were incubated at high humidity for 24 h, and sporulation was measured after further incubation on paraquat-chloramphenicol agar medium. When B. cinerea was inoculated alone, germination incidence, germ tube length, and percent leaf area with conidiophores of the pathogen each generally increased as preinoculation light intensity was decreased and number of days in reduced light intensity was increased. Similar response patterns were found for C. rosea when inoculated alone, except that germination, high on untreated leaves, was affected by duration but not intensity of light treatment. On coinoculated leaves, germination and germ tube length of B. cinerea increased with duration of low-intensity light treatments, but no conidiophores were produced. Germination and germ tube length of C. rosea were each higher when the agent was coinoculated than when inoculated alone, but conidiophore production was similar. Germination, germ tube length, and conidiophore production of B. cinerea, inoculated alone, correlated with electrolyte leakage from the leaves (r = 0.78, 0.67, and 0.82, respectively). Respective values for C. rosea were r = 0.65, 0.68, and 0.67. Collectively, the data indicate that low-intensity light predisposed the leaves to development of B. cinerea and C. rosea on the phylloplane, and to sporulation of the two fungi, after leaves were killed on paraquat-chloramphenicol agar, but did not affect ability of C. rosea to control spore production of B. cinerea. A hypothesis that increased nutrient leakage from leaf cells mediated effects of low-intensity light was supported by the correlations of electrolyte leakage with development of B. cinerea and C. rosea.