• Mycoparasitism
• Biocontrol
• Transcriptomic
• Sclerotia
• Trichoderma

The replacement or reduction of chemical inputs in modern agriculture can be achieved through biological control strategies targeted to the reduction of the pathogen inoculum or activity by or through one or more antagonist organisms. Trichoderma harzianum has long been recognized as biocontrol agent and plant growth promoter, therefore it is successfully used as biopesticide in greenhouse and field applications. The ability of Trichoderma harzianum to suppress plant diseases caused by phytopathogenic fungi has long been known and the antagonistic properties have been related to mechanisms of action such as the production of antibiotics and hydrolytic enzymes, competition and mycoparasitism. This latter has special relevance when the prey is a plant pathogen such as Sclerotinia sclerotiorum that produces resting structures known as sclerotia. These structures have a strong resistance both to chemical and biological degradation and permit the fungi to survive in the absence of a host.
Aim of this study was to preliminary explore gene expression during mycoparasitic interaction between Trichoderma harzianum (T6776) and Sclerotinia sclerotiorum sclerotia by using RNA-seq approach and RT-PCR. The differential genes expression was evaluated in the early stages of mycoparasitic interaction at 12h, 52h, 72h under two different in vitro growth conditions: Trichoderma harzianum T6776 grown up Sclerotinia sclerotiorum sclerotia or on Potato Dextrose Agar (PDA), this last as control.
Different sclerotia colonization experiments with T6776 nutrient-activated conidia were carried out but obtained data have not resulted in reliable and reproducible results. Therefore, the biological system has been modified, and T6776 mycelium was used to colonize sclerotia on PDA plate. T6776 spore suspensions were used to inoculate PDA plates; after 3 days growth, T6776 mycelia plugs were transferred on new PDA plates covered with nylon disks and incubated for additional 36h. Then Sclerotinia sclerotiorum sclerotia previously sterilized were inoculated on each T6776 actively growing colony and the cultures were incubated again. The experiment consists of three biological replicates for each thesis, each biological replicate formed by three technical replicates, each consisting of three plates with fifteen sclerotia in each one. As a control, Trichoderma harzianum T6776 was grown on PDA plates covered with nylon disks. After 12h, 52h and 72h of incubation, fungal tissues (colonized sclerotia or mycelium) were harvested and immediately frozen in liquid nitrogen and stored at -80°C. The timing (12h, 52h and 72h) was previously defined on the base of results of T6776-sclerotia colonization assays.
RNA was extracted from macerated frozen fungal tissues using Plant/Fungi Total RNA purification Kit: the RNA quantity was assessed using spectrophotometric analysis. Samples were retrotranscripted and normalized starting quantities of total RNA were then used to prepare 12 separate barcoded RNA-seq libraries that were sequenced on a single lane on an Illumina HiSeq2000 sequencer. The sequencing data will be analysed with suitable bioinformatics tools for the gene expression analysis. In addition to the whole transcriptome sequencing, part of the extracted RNA has been used to investigate the expression of T6776 genes, that are known to be potentially involved in the early stages of mycoparasitism, using PCR approaches.
The present study provides insights into the mechanisms of gene expression involved in early stages of this mycoparasitic interaction, supported also by the availability of the sequenced and annotated genome of T6776.