Establishment of a quadruplex PCR assay for the molecular detection and quantification of different fungal BCAs

Establishment of a quadruplex probe qPCR assay for molecular detection and quantification of different fungal MBCAs. Abstract: Microbial biological control agents (MBCAs) are administered to crops for biological control of plant diseases, and they work in a variety of ways. Some MBCAs interact with plants inducing resistance or priming plants without interacting directly with the pathogen of interest, whereas others parasitize the pathogen, kill it using toxic compounds or outcompete it for nutrients or space. The genus Trichoderma has been known since at least the 1920s. Until recently, the primary control mechanisms were considered to be direct. Recent discoveries show that Trichoderma spp. impacts on plants, inducing systemic or localized resistance, are also very relevant. These fungi colonize the root epidermis and outer cortical layers, releasing bioactive chemicals that induce the Trichoderma thallus to wall off. Plant transcriptome and proteome are both significantly changed at the same time. As a result, in addition to better resistance, greater plant growth and nutrient uptake also occur. Quantifying MBCAs is important for several reasons, including effectiveness assessment, quality control, dosage optimisation, environmental impact assessment, and regulatory compliance. Accurate quantification ensures potent results, minimizes underdosing and overdosing, and helps to monitor the survival and persistence in the target environment. Quantification also helps to meet regulatory standards and ensures the safety and efficacy of applications A general method used for quantification of MBCA is the plate count method. Here, the microorganism of interest is cultured on a suitable growth medium and the number of developing colonies is counted. However, this method is time consuming and not specific. Using real-time PCR (qPCR) for quantification offers several advantages over the traditional plate counting method. Indeed, qPCR is better in terms of sensitivity, specificity, speed and efficiency, and reduces labor and bias. Besides, qPCR allows for high-throughput analysis and can process a larger number of samples in a shorter time, making it more efficient for large-scale studies. qPCR provides quantitative data by measuring the amplification of the target DNA during each cycle. This allows for accurate determination of the initial amount of microbial DNA present in the sample. In contrast, plate counting methods provide colony-forming unit (CFU) counts, which are an estimation of viable cells. This research aims to establish quantification of four MBCAs, three Trichoderma species (Trichoderma.harzianum, Trichoderma.asperellum, Trichoderma koningiopsis) and Clonostachys rosea f.sp. rosea). The pathogens Fusarium graminearum, F. culmorum, and Sclerotina.sclerotiorum and the crop plants wheat and soybean are used to check the assay for specificity. After culturing of BCAs and pathogens, DNA extraction was performed. DNA extraction of plants was also performed after cultivation. Four species-specific primer-probe combinations for the MBCAs were either taken from the literature or designed using Primer BLAST. Primers were then tested in real time PCR for specificity to the MBCAs. For this, the primer-probe combinations are multiplexed and DNA from all species mentioned above was applied to the qPCR. Standard curves are established for quantification. To test the assay, plants are treated with the MBCAs. DNA was then extracted from the roots of the plant. Real time PCR assay was performed for the quantification and detection of the fungal BCAs. Quadruplex PCR technology is an ideal choice for analyzing large numbers of samples in a shorter time period. It allows for the simultaneous amplification of four different DNAs from MBCAs in a single reaction, saving both time and resources.