Bio-prospecting for endophytes of Brassica : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand
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Disease and insect pests are major limiting factors for crop production worldwide. Farmers are often heavily reliant on synthetic biochemicals and fertilisers to mitigate the negative impact of pests and disease and to increase crop yield. However, the extensive use of chemicals has led to environmental concerns due to contamination of soil and water, human health issues, disturbance of macro and microorganisms balance and the development of resistance by both insects and fungal pathogens. Use of biological control agents including endophytic microorganisms is an alternative control option to combat these problems. Many endophytes are able to provide their host with beneficial traits such as resistance against insect-pests and pathogens and enhance crop performance under abiotic stresses. Although beneficial microorganisms of brassica crops have been discovered, endophytes of wild brassica’s, particularly those associated with the seed, have been ignored. In this study, we screened seed of various brassica species with a worldwide distribution and isolated 131 bacterial and two fungal species. Molecular identification of bacterial isolates indicates that most seed accessions harboured endophytic bacteria belonging to 17 species. Among these isolates, two species, identified as Methylobacterium fujisawaense and Me. phyllosphaerae were dominant and widespread across the majority of accessions sampled, and originated from different species and locations. The inoculation of oilseed rape (Brassica napus) root with these endophytic bacteria significantly increased the fresh weight of the seedlings. The fungal endophyte species identified were Beauveria bassiana and Geomyces pannorum, isolated from two different accession of a wild brassica species (B. rapa). Inoculation of the seeds of three brassica species, B. napus, B. rapa and B. oleracea with these fungal endophytes resulted in infection of below and above ground tissues of inoculated seedlings but colonisation of the next generation seeds/seedlings did not occur. Seed inoculation, foliar application and soil drenching when the plants were grown on non-sterile soil also did not result in plant colonisation. A dual culture test was performed to study the antagonistic effect of these bacterial and fungal endophytes against Leptosphaeria maculans, the causal agent of phoma stem canker in brassica crops. The highest inhibition rate was recorded for Stenotrophomonas rhizophila, Novosphingobium resinovorum, Pseudomonas azotoformans, Plantibacter flavus, Me. fujisawaense and Me. phyllosphaerae which produced a significant inhibition zone indicating the antagonistic ability of these species. The fungal endophytes also suppressed the growth of the pathogen and created an inhibition zone. In planta tests in which the fungal endophytes were inoculated on to seed of a susceptible oilseed rape cultivar were also undertaken. At the cotyledon leaf stage, the leaf was punctured and spore suspension of L. maculans was placed on the wound site. Inoculated seedlings particularly B. bassiana, significantly decreased phoma stem canker disease symptoms on the cotyledon. To our knowledge, this is the first study that screen the fungal and bacterial endophytes of wild brassica species associated with the seeds and demonstrate their beneficial characteristic when inoculated to brassica crops.
Brassica, Diseases and pests, Biological control, Seeds, Endophytes, fungal endophyte, bacterial endophyte, wild brassica species, seed-associated endophyte, oilseed rape, inoculation method, seed inoculation, bioactivity, disease tolerance, plant growth promotion, Beauveria bassiana, Geomyces pannorum, Methylobacterium sp., phoma stem canker, Leptosphaeria maculans