Ecological studies of the bio-inoculant Trichoderma hamatum LU592 in the root system of Pinus radiata

The plant health- and growth-promoting biological inoculant (bio-inoculant) Trichoderma hamatum LU592 was transformed with the constitutively expressed green fluorescent protein (gfp) and hygromycin B resistance (hph) genes to specifically monitor the isolate in the root system of Pinus radiata within a strong indigenous Trichoderma population. A modified dilution plating technique was developed to allow the determination of the mycelia proportion of total propagule levels. LU592 was shown to colonize the rhizosphere most effectively when 105 spores per pot were applied compared with inoculum concentrations of 103 and 107 spores per pot. LU592 extended its zone of activity beyond the rhizosphere to at least 1 cm away from the root surface. A positive relationship was shown between P. radiata root maturation and the spatial and temporal proliferation of LU592 in the root system. A steep increase in mycelia levels and proportion of penetrated root segments was observed after 12 weeks. This study reinforces the value of genetic markers for use in ecological studies of filamentous fungi. However, despite isolate-specific recovery of the introduced isolate, it was shown that total propagule counts do not always correlate with the amount of viable mycelium present in the root system. Therefore, it is proposed that the differentiation of mycelia from spores and root penetration is used as more accurate measures of fungal activity