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Tesi etd-03142019-161507

Thesis type
Tesi di laurea magistrale
Preliminary investigation of the expression of a putative cluster containing a Tri5 gene in Trichoderma gamsii T6085
Corso di studi
relatore Prof. Vannacci, Giovanni
Parole chiave
  • Trichoderma gamsii
  • Fusarium graminearum
  • Trichothecenes
  • Tri5
  • qPCR
  • secondary metabolites
  • biocontrol
  • wheat
  • FHB
Data inizio appello
Riassunto analitico
Fusarium Head Blight is one of the most important disease of cereals including wheat, barley, oats, rye, and triticale. It is caused by a wide range of fungi mostly belonging to Fusarium genus, with F. graminearum and F. culmorum as the most important. The disease can cause important economic losses, mostly due to the damage caused on kernels as well as quality reduction due to contamination with mycotoxins such as deoxynivalenol (DON) and its acetyl derivates, 3- and 5-acetyl-deoxynivalenol, and nivalenol (NIV). These fungal secondary metabolites are trichothecenes, a group of mycotoxin extremely harmful for humans and animals, causing hemorrhagic lesions, loss of appetite and vomiting.
There are different approaches to control the disease and to reduce mycotoxins contamination: cultural practices, such as crop rotation, application of fungicides and breeding program to obtain resistant lines are the most important. However, none of these approaches alone are totally effective against the disease. Biological control is today considered an additional valid tool to be implemented in an Integrated Pest Management strategy. Several studies have shown that the use of biocontrol agents such as bacteria and fungi have led to a decrease in the infection and disease rate and in mycotoxin contamination.
Trichoderma genus includes many species commonly present in nature on different substrates. Many isolates are very competitive for space and nutrients against other organisms, included fungal plant pathogens, thanks to their saprotrophic capacity and the ability to antagonize and parasitize other fungi. Trichoderma spp. represent one of the main bioactive principles of commercial biopesticides. In the last decade many studies have been performed concerning the use of an isolate of T. gamsii, T6085, successfully used as biocontrol agent of FHB both in lab and in field. This T. gamsii T6085 is able to inhibit F. graminearum growth, to reduce mycotoxin production and to compete with the pathogen for natural substrates such as wheat straw.
Trichothecenes are produced not only by Fusarium species, but also by isolates belonging to other genera such as Isaria, Microcyclospora, Myrothecium, Pelaster, Spicellum, Stachybotrys, and Trichothecium. In all of these fungi the biosynthetic pathway is similar, with the initial step corresponding to a cyclization of the farnesyl diphosphate to trichodiene by a terpene synthase regulated by the Tri5 gene.
Some studies have shown the presence of the Tri5 gene, functionally associated with a cluster in species of Trichoderma, such as T. brevicompactum and T. arundinaceum, Recently, the research group of the lab where I made my thesis, found a putative cluster containing a Tri5 gene, similar to that responsible for the initial step in the biosynthesis of the trichothecenes, also in T. gamsii T6085.
Since this isolate is under development as potential biocontrol agent of FHB, aim of this work was to preliminarily investigate the expression of the Tri5 gene, and the putative cluster, in this isolate.
Genomic DNA of T. gamsii T6085 was used to validate primers of Tri5 and other genes included in the cluster, previously designed on the basis of the fungal genome.
In order to understand the growth rate of T. gamsii T6085, a test was performed on PDB (potato Dextrose Broth), thus resulting in growth curves where the linear phase was detected. This information was further used to set up a test to evaluate the expression of Tri5 and the genes included in the putative cluster, on two different growth media, PDB and Fries.
RNA extracted after 6 days of incubation was used to analyze the expression of the genes by RT-PCR and, after, by qPCR. Results obtained in the present work, by both semi-quantitative and quantitative PCR gave a preliminary overview of the expression of Tri5 and of the other genes included in the putative cluster that will be of interest for a future evaluation of the safety in the use of T. gamsii T6085 as biocontrol agent.