Antifungal activity and genomic characterization of the biocontrol agent Bacillus velezensis CMRP 4489.

The development of bio-based products has increased in recent years, and species of the Bacillus genus have been widely used for product development due to their elevated production of antimicrobial molecules and resistance to extreme environmental conditions through endospore formation. In this context, the antifungal potential of Bacillus velezensis CMRP 4489 was investigated using in silico predictions of secondary metabolites in its genome and in vitro tests against the following phytopathogenic fungi: Sclerotinia sclerotiorum, Macrophomina phaseolina, and Botrytis cinerea. The in-silico predictions indicated that CMRP 4489 possesses several Biosynthetic Gene Clusters (BGCs) capable of producing molecules with antifungal properties and other non-identified BGCs. The in vitro assay results evidenced strong antifungal activity, inhibiting more than 60% of the tested fungi, and the isolate's molecules were stable under diverse physicochemical conditions. The in vitro assay evidenced significant antifungal activity, deformation of the hyphal structure in SS, biofilm formation capacity, and swarming motility. In the colonization assay, we observed attachment, colonization, and net-shaped biofilm formation, with the strain transitioning from the seeds to nearby structures. Therefore, CMRP 4489 showed to be a potential biocontrol agent against various diseases with agronomic importance and can be used under adverse environmental conditions.

In vitro control of phytopathogenic fungi and damping-off of tomato by Bacillus velezensis LABIM40 (CMRP 4489) / Controle in vitro de fungos fitopatogênicos e tombamento de mudas de tomateiro por Bacillus velezensis LABIM40 (CMRP 4489)

The in vitro antagonistic activity of Bacillus velezensis LABIM40 (strain CMRP 4489) was assessed against Alternaria linariae, Botryotinia squamosa, Colletotrichum lindemuthianum, Gibberella zeae, and Rhizoctonia solani. An experiment was conducted using treated seeds under growth chamber conditions to determine the impact of various LABIM40 formulations on tomato seedling growth and the biocontrol of damping-off caused by R. solani. The treatments included the use of LABIM40 cell suspension, LABIM40 cell-free supernatant (CFS), 10 times concentrated CFS (10× CFS), commercial products based on Bacillus amyloliquefaciens (CP_1) and Bacillus subtilis (CP_2), and water. The effects of these products were assessed on tomato seedlings grown in sterile substrate or substrate inoculated with R. solani. In a dual culture test, B. velezensis LABIM40 inhibited the mycelial growth of the aforementioned fungal pathogens by 46.6%, 67.4%, 64.7%, 49.0%, and 54.4%, respectively. The minimum inhibitory concentration against each fungus was determined using varying concentrations of CFS in potato dextrose agar medium, followed by a regression analysis of mycelial growth inhibition. Except for A. linariae, the logarithmic model provided the best fit in all cases. Tomato seedlings from seeds treated with 10× CFS in inoculated substrate exhibited a survival rate 57% higher than that exhibited by the control treatment. However, no growth promotion was observed in tomato plants from seeds treated with LABIM40 cells or its CFS metabolites. In summary, these findings highlight the antagonistic activity of B. velezensis LABIM40 against A. linariae, B. squamosa, C. lindemuthianum, G. zeae, and R. solani, as demonstrated by dual culture and CFS diffusion tests. This suggests its potential as a biocontrol agent for damping-off in tomatoes.(AU)

O antagonismo do Bacillus velezensis LABIM40 (cepa CMRP 4489) foi avaliado in vitro contra Alternaria linariae, Botryotinia squamosa, Colletotrichum lindemuthianum, Gibberella zeae e Rhizoctonia solani. Com o objetivo de avaliar o efeito de diferentes formulações de LABIM40 no crescimento de mudas de tomateiro e no biocontrole do tombamento causado por R. solani, foi conduzido um experimento com sementes tratadas em condições de câmara de crescimento. Os tratamentos foram suspensão de células de LABIM40, LABIM40 SLC, SLC 10 vezes concentrado (10xSLC), produtos comerciais à base de Bacillus amyloliquefaciens (PC_1) e Bacillus subtilis (PC_2) e água. Os efeitos do produto foram avaliados em mudas de tomate cultivadas em substrato estéril e em substrato inoculado com R. solani. No teste de cultura dupla, B. velezensis LABIM40 inibiu em 46,6, 67,4, 64,7, 49,0 e 54,4% o crescimento micelial desses fungos fitopatogênicos, respectivamente. A concentração inibitória mínima para cada fungo foi determinada por diferentes concentrações de sobrenadante livre de células (SLC) em meio de batata dextrose ágar (PDA), seguido de análise de regressão da inibição do crescimento micelial. Com exceção de A. linariae, em todos os casos o modelo com melhor ajuste foi o logarítmico. A sobrevivência de mudas de tomate de sementes tratadas com 10xSLC em substrato inoculado foi 57% maior do que o tratamento controle. Não foi observada promoção de crescimento em plantas de tomate oriundas de sementes tratadas com células de LABIM40 ou metabólitos de seu SLC. Coletivamente, esses resultados destacam a atividade antagonista de B. velezensis LABIM40 contra A. linariae, B. squamosa, C. lindemuthianum, G. zeae e R. solani verificada por cultura dupla e testes de difusão de sobrenadante livre de células e seu potencial como agente de biocontrole do tombamento do tomateiro.(AU)

Complete Genome Sequence of Bacillus velezensis LABIM40, an Effective Antagonist of Fungal Plant Pathogens.

Bacillus velezensis strain LABIM40 holds high potential for biological control of plant pathogens. Its complete genome contains one chromosome of 3,972,310 bp with 3,777 DNA coding sequences and displays 33 gene clusters potentially involved in the suppression of fungal pathogens.