Efficacy and mechanisms of bacterial biocontrol agents against <i>Leptosphaeria maculans</i> (Desm.) causing blackleg disease of canola (<i>Brassica napus L</i>.)

Blackleg is a destructive and economically important disease of canola (Brassica napus L.) in Australia and other canola cultivation regions such as Europe and Canada. The disease is caused by the fungal pathogen, Leptosphaeria maculans (Desm.) Ces &amp; De Not (anamorph Phoma lingam Tode: Fr. /Desm). In Australia, the presence of this pathogen resulted in an epidemic in 2003 due to the breakdown of host resistance. Changes in farming practices and the intensified cultivation of canola has also increased the amount of stubble present in the field, therefore potentially leading to increased sexual reproduction of L. maculans and ascospore showers on canola plants. The use of fungicides has increased over the years, and this is raising environmental concerns. Biological control of plant pathogens is a potentially sustainable and environmentally friendly disease management strategy. Biocontrol products based on species of Bacillus have proven to be effective against a variety of pathogens in the field. Additionally, Bacillus spp. are also known for their persistence in harsh environmental conditions. Therefore, this study aimed to isolate and screen Bacillus species with antagonistic efficacy towards L. maculans, and to understand the underlying mechanisms of biocontrol.An extensive collection of rhizospheric and phyllospheric bacteria (532) was accessed and spore forming bacteria were identified by heat shock method, with 148 of these surviving the treatment and determined to be likely Bacillus spp. Screening of antagonistic capability towards L. maculans identified 19 potential biocontrol agents (BCAs) with the greatest inhibition of mycelial growth (≥80%), which were then characterised via sequencing of the 16S rRNA gene. Identifications were further confirmed using multigene sequence analysis based on Gyr A, Che A and an antiporter gene, Tet-b. From this, nine highly antagonistic and closely related Bacillus strains were identified to species level and phylogenetic analysis was carried out for comparative purposes. Volatile organic compounds (VOCs) produced by these nine strains showed a differential reaction against a set of 14 isolates of L. maculans with known virulence. VOCs of Bacillus amyloliquefaciens strain B89 and Bacillus velezensis strain B94 demonstrated comparatively high inhibition potential in comparison to other Bacillus strains. The VOCs of these strains were also effective in in planta closed container assays. SPME-GC-MS was used for the detection and comparative analysis of VOCs produced by B89 and B94 individually and in the presence of L. maculans. Most of the detected volatiles belonged to fatty acid derivatives, aldehydes, esters, furans, ketones, lactones and benzoids and L. maculans was able to modulate the profile of bacterial VOCs.Detection of antibiotic production by a particular bacterium is important in determining its capability to be an effective BCA. Biosynthetic genes belonging to different operons within Bacillus species genomes, and which were previously reported to be involved in the synthesis of lipopeptides or other secondary metabolites, were selected to screen a group of Bacillus species. Polymerase chain reaction analysis was performed for the detection of eight genes related to lipopeptide production and two genes related to the biosynthesis of chitinase and β, 1-3 glucanase. B. velezensis (B94) demonstrated the presence of eight genes whereas, B. amyloliquefaciens (B89) harboured seven genes of interest. MALDI-TOF-MS analysis of whole cell surface extracts of B89 and B94 confirmed that these strains were able to produce different analogues of surfactin, iturin A, iturin C, bacillomycin, fengycin and mycosubtilin. The antagonistic potential of these strains was mainly dependent on the non- volatile/diffusible compounds as compared to the VOCs, and the presence of L. maculans changed the profile of detected lipopeptides in B89 and B94.An analysis of crude cultural filtrates of B. amyloliquefaciens (B89) and B. velezensis (B94) against mycelial growth and conidial viability determined that these cultural filtrates have high antagonistic potential against L. maculans. Further to this, in planta assays using Brassica napus (AV-Garnet) determined that these filtrates can promote lateral root growth. When the roots of canola seedlings were exposed to the bacterial cultural filtrates, protection against infection by L. maculans was recorded for detached leaves, indicating a systemic protective effect. However, the application of Bacillus strains and L. maculans on separate cotyledons demonstrated no protective or inhibitory effect on the pathogen. The use of rifampicin (Rif) mutants established that B89 and B94 are able to colonise B. napus seedlings and that they have the potential to be endophytic, therefore providing benefits to the host. In planta assays at seedling stage showed complete inhibition of L. maculans after the prior application of B89 and B94.In conclusion, B. amyloliquefaciens (B89) and B. velezensis (B94) show promise as BCAs of L. maculans in canola. Furthermore, the secondary metabolites produced by these Bacillus strains are strain specific and they are able to antagonise L. maculans using multiple modes of action.

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