Dataset Information

A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents Trichoderma asperellum and Trichoderma gamsii.

ABSTRACT: Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of Remedier^TM (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were developed. Primers/probe sets were designed on the T. asperellum and T. gamsii rpb2 genes and tested for specificity on a panel of microorganisms commonly associated with grape wood and soil. No differences were observed comparing single- and duplex-qPCR assays on different BCAs, 1 pg of target DNA was detected approximately at C_q = 34. R²-values and the efficiency were always equal to 0.99 and >80%, respectively. The detection limit of the duplex-qPCR assay on artificially inoculated samples was 2 × 10³ and 4 × 10⁴ conidia g^-1 of grape wood tissue and soil, respectively. The methods will be useful to better schedule BCA application in the field and in grapevine nurseries, as well as for investigating the dynamic of BCA populations.

SUBMITTER: Gerin D

PROVIDER: S-EPMC6127317 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents Trichoderma asperellum and Trichoderma gamsii.

Gerin Donato D Pollastro Stefania S Raguseo Celeste C De Miccolis Angelini Rita M RM Faretra Francesco F

Frontiers in microbiology 20180831

Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of RemedierTM (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were dev ...[more]

PMID: 30233545

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Project description:Maize is a crucial cereal crop in China, serving both as a staple food and an essential industrial resource. Northern corn leaf blight (NCLB) is a disease of corn caused by a fungus, Exserohilum turcicum (sexual stage Setosphaeria turcica). This study aimed to assess the biocontrol potential of various Trichoderma strains against Exserohilum turcicum 101 in Jilin, China. Through dual culture tests, the Trichoderma strains were categorized into four groups based on their antagonistic abilities. Eleven Trichoderma strains exhibited strong antagonistic behavior, with comparable or faster growth rates than E. turcicum 101. Microscopic observations confirmed that T. asperellum 576 hyphae effectively encircled E. turcicum 101 hyphae, reinforcing their antagonistic behavior. The production of non-volatile and volatile substances by the Trichoderma strains was evaluated, with T. asperellum 576 showing the highest potency in producing non-volatile and volatile substances, leading to an impressive 80.81% and 65.86% inhibition of E. turcicum 101 growth. Remarkably, co-culture suspensions of T. asperellum 576 + E. turcicum 101 and T. atroviride 393 + E. turcicum 101 exhibited strong antifungal activity. Furthermore, the activities of chitinase, β-1.3-glucanase, and cellulase were evaluated using the 3, 5-dinitrosalicylic acid (DNS) method. T. asperellum 576 + E. turcicum 101 displayed stronger cell wall degradation enzyme activity compared to T. atroviride 393 + E. turcicum 101, with values of 8.34 U/mL, 3.42 U/mL, and 7.75 U/mL, respectively. In greenhouse conditions, the application of a 107 spores/mL conidia suspension of T. asperellum 576 significantly enhanced maize seed germination and plant growth while effectively suppressing E. turcicum 101 infection. Maize seedlings inoculated/treated with both E. turcicum 101 and T. asperellum 576 demonstrated substantial improvements compared to those inoculated solely with E. turcicum 101. The T. asperellum 576 treatment involved a 107 spores/mL conidia suspension applied through a combination of foliar spray and soil drench. These findings highlight T. asperellum 576 as a promising biocontrol candidate against northern leaf blight in maize. Its antagonistic behavior, production of inhibitory compounds, and promotion of plant growth all contribute to its potential as an effective biocontrol agent for disease management.

Project description:Fusarium head blight (FHB) is a major fungal disease that causes severe yield and quality loss in wheat. Biological control can be integrated with other management strategies to control FHB. For this purpose, Trichoderma gamsii strain T6085 is a potential biocontrol agent to limit the infection of F. graminearum and F. culmorum in wheat. However, the possible impacts of T. gamsii T6085 on the broader microbiome associated with the wheat plant are not currently understood. Therefore, we identified bacteria and fungi associated with different wheat tissues, including assessment of their relative abundances and dynamics in response to the application of T6085 and over time, using amplicon sequencing. Residues of the prior year's wheat crop and the current year's wheat spikes were collected at multiple time points, and kernel samples were collected at harvest. DNA was extracted from the collected wheat tissues, and amplicon sequencing was performed to profile microbiomes using 16S v4 rRNA amplicons for bacteria and ITS2 amplicons for fungi. Quantitative PCR was performed to evaluate the absolute abundances of F. graminearum and T. gamsii in different wheat tissues. Disease progression was tracked visually during the growing season, revealing that FHB severity and incidence were significantly reduced when T6085 was applied to wheat spikes at anthesis. However, treatment with T6085 did not lessen the F. graminearum abundance in wheat spikes or kernels. There were substantial changes in F. graminearum abundance over time; in crop residue, pathogen abundance was highest at the initial time point and declined over time, while in wheat spikes, pathogen abundance increased significantly over time. The predominant bacterial taxa in wheat spikes and kernels were Pseudomonas, Enterobacter, and Pantoea, while Alternaria and Fusarium were the dominant fungal groups. Although the microbiome structure changed substantially over time, there were no community-scale rearrangements due to the T6085 treatment. The work suggests several other taxa that could be explored as potential biocontrol agents to integrate with T6085 treatment. However, the timing and the type of T6085 application need to be improved to give more advantages for T6085 to colonize and reduce the F. graminearum inoculum in the field.

Dataset Information

A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents Trichoderma asperellum and Trichoderma gamsii.

Publications

A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents <i>Trichoderma asperellum</i> and <i>Trichoderma gamsii</i>.

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