RESUMO
Endophytic microorganisms are found within the tissues of many plants species, with some conferring several benefits to the host plant including resistance to plant diseases. In this study, two putative endophytic fungi that were previously isolated from wild seeds of Brassica, identified as Beauveria bassiana and Pseudogymnoascus pannorum, were inoculated into cultivars of three Brassica species-Brassica napus, Br. rapa and Br. oleracea. Both fungal endophytes were reisolated from above- and below-ground tissues of inoculated plants at four different plant-growth stages, including cotyledon, one-leaf, two-leaf, and four-leaf stages. None of the plants colonised by these fungi exhibited any obvious disease symptoms, indicating the formation of novel mutualistic associations. These novel plant-endophyte associations formed between Brassica plants and Be. bassiana significantly inhibited phoma stem canker, a devastating disease of Brassica crops worldwide, caused by the fungal pathogen Leptosphaeria maculans. The novel association formed with P. pannorum significantly suppressed the amount of disease caused by L. maculans in one out of two experiments. Although biological control is not a new strategy, endophytic fungi with both antiinsect and antifungal activity are a highly conceivable, sustainable option to manage pests and diseases of economically important crops.
RESUMO
BACKGROUND: Plants are commonly colonized by a wide diversity of microbial species and the relationships created can range from mutualistic through to parasitic. Microorganisms that typically form symptomless associations with internal plant tissues are termed endophytes. Endophytes associate with most plant species found in natural and managed ecosystems. They are extremely important plant partners that provide improved stress tolerance to the host compared with plants that lack this symbiosis. Plant domestication has reduced endophyte diversity and therefore the wild relatives of many crop species remain untapped reservoirs of beneficial microbes. Brassica species display immense diversity and consequently provide the greatest assortment of products used by humans from a single plant genus important for agriculture, horticulture, bioremediation, medicine, soil conditioners, composting crops, and in the production of edible and industrial oils. Many endophytes are horizontally transmitted, but some can colonize the plant's reproductive tissues, and this gives these symbionts an efficient mechanism of propagation via plant seed (termed vertical transmission). METHODS: This study surveyed 83 wild and landrace Brassica accessions composed of 14 different species with a worldwide distribution for seed-originating bacterial endophytes. Seed was stringently disinfected, sown within sterile tissue culture pots within a sterile environment and incubated. After approximately 1-month, direct isolation techniques were used to recover bacterial endophytes from roots and shoots of symptomless plants. Bacteria were identified based on the PCR amplification of partial 16S rDNA gene sequences and annotated using the BLASTn program against the NCBI rRNA database. A diversity index was used as a quantitative measure to reflect how many different bacterial species there were in the seed-originating microbial community of the Brassica accessions sampled. RESULTS: Bacterial endophytes were recovered from the majority of the Brassica accessions screened. 16S rDNA gene sequencing identified 19 different bacterial species belonging to three phyla, namely Actinobacteria, Firmicutes and Proteobacteria with the most frequently isolated species being Methylobacterium fujisawaense, Stenotrophomonas rhizophila and Pseudomonas lactis. Methylobacterium was the dominant genus composing 56% of the culturable isolated bacterial community and was common in 77% of accessions possessing culturable bacterial endophytes. Two selected isolates of Methylobacterium significantly promoted plant growth when inoculated into a cultivar of oilseed rape and inhibited the growth of the pathogen Leptosphaeria maculans in dual culture. This is the first report that investigates the seed-originating endophytic microorganisms of wild Brassica species and highlights the Brassica microbiome as a resource for plant growth promoting bacteria and biological control agents.
