Understanding Trichoderma bio-inoculants in the root ecosystem of Pinus Radiata seedlings

The overall aim of this study was to understand better the ecological behaviour of Trichoderma bio-inoculants in the root system of commercially grown Pinus radiata seedlings. Three Trichoderma isolates, representing three different species of the genus, were selected initially to investigate associations between rhizosphere competence and plant growth enhancement parameters. Improvements in various plant health and growth factors were confirmed for T. hamatum LU592 in a large-scale experiment under commercial conditions. Application of LU592 increased seedling shoot height and the number of seedlings meeting market specifications by up to 9.5% and 5%, respectively. Strong rhizosphere colonisation and the ability to penetrate the roots were also demonstrated. At the end of the experiment, the Trichoderma population was found to be up to 10 times higher compared to the untreated control. LU592 was recovered as the predominant isolate within a diverse indigenous Trichoderma population in the root system of 20-week old P. radiata seedlings. No stimulation of plant performance was detected for the other two isolates T. atroviride LU132 or T. harzianum LU686. Both isolates were early rhizosphere colonisers, with populations declining after 12 weeks. Two different Trichoderma application methods were investigated. Trichoderma spores were introduced either as a seed coat or a spray application. The inoculum rates between seed coat and spray application differed by a factor of 10, however, no differences in the effect on plant health and growth were observed between application methods. The spray application promoted the establishment of the isolates in the root zone with increased Trichoderma populations for LU132, LU686 and LU592 by 1.4, 2.1 and 3.4 times, respectively, compared with the seed coat. Two marker genes, expressing the green fluorescent protein (GFP) and the hygromycin B phosphotransferase (hph), were successfully inserted into the genome of T. hamatum LU592. Subsequent physiological comparisons with the wild type combined with recovery and visualisation in non-sterile potting mix identified LU592/C as a suitable transformant for ecological studies. Using this marked strain, rhizosphere competence and root penetration ability were confirmed, but LU592/C was shown to perform poorly as a saprophyte. A moderate rather than a high inoculum concentration resulted in strongest establishment of LU592/C in the P. radiata root zone. Spatio-temporal population dynamics were directly related to root development. The quantification of total propagules did not always correlate with the biological activity of LU592/C. For instance, low levels of mycelia were detected in the bulk potting mix, despite reasonable total colony forming unit (cfu) concentrations of >10³ cfu/g dry potting mix. By contrast, the proportion of the total cfu as mycelia was 44% around the emerging seedling radicle at a total cfu level of <102 cfu/g dry potting mix. The results suggest that assessments of root penetration and mycelia proportion should be used as additional indicators of fungal activity to supplement traditional enumeration techniques. This research highlighted that plant performance assessments need to be associated with comprehensive ecological studies to successfully identify beneficial Trichoderma-based bio-inoculants. Implications of the outcomes of this study are discussed and future recommendations made. The research presented here has significantly increased the understanding of the ecological behaviour of Trichoderma bio-inoculants in the root ecosystem of P. radiata and will contribute to the body of literature on fungal ecology.