Chemical and genetic characterization of lipopeptides from Bacillus velezensis and Paenibacillus ottowii with activity against Fusarium verticillioides.

Introduction: The fungus Fusarium verticillioides significantly threatens maize crops in tropical soils. In light of this, biological control has emerged as a promising strategy to reduce fungicide costs and environmental risks. In this study, we aimed to test the antifungal activity of cell-free supernatant (CFS) from three Bacillus velezensis (CT02, IM14, and LIS05) and one Paenibacillus ottowii (LIS04) against F. verticillioides, thereby contributing to the development of effective biocontrol measures. Methods: The research employed a comprehensive approach. The antifungal activity of the bacterial strains was tested using cell-free supernatant (CFS) from three Bacillus velezensis (CT02, IM14, and LIS05) and one Paenibacillus ottowii (LIS04). The UPLC-MS evaluated the CFS to identify the main bioactive molecules involved in the inhibitory effect on F. verticillioides. Scanning electron microscopy (SEM) was used to assess the impact of CFS on spores and hyphae, and genome sequencing was conducted to identify the genes involved in biological control. These robust methodologies ensure the reliability and validate our findings. Results: The CFS of the four strains demonstrated significant inhibition of fungal growth. The UPLC-MS analysis revealed the presence of lipopeptides with antifungal activity, including surfactin and fengycins A and B expressed by the three strains of Bacillus velezensis and iturin A expressed by strains LIS05 and IM14. For Paenibacillus ottowii, fusaricidins, ABCDE, and five previously unreported lipopeptides were detected. Scanning electron microscopy (SEM) showed that treatments with CFS led to significant distortion and breakage of the F. verticillioides hyphae, in addition to the formation of cavities in the membrane. Genome mining confirmed the presence of genes coding for the lipopeptides identified by UPLC-MS, including the gene for iturin in CTO2. Genomic sequencing revealed that CT02, IM14, and LIS05 belong to different strains of Bacillus velezensis, and LIS04 belongs to Paenibacillus ottowii, a species recently described. Discussion: The four bacterial strains, including three novel strains identified as Bacillus velezensis and one as the recently described species Paenibacillus ottowii, demonstrate significant potential as biocontrol agents for managing fungal disease. This finding underscores the novelty and potential impact of our research.

Inoculation with Bacillus megaterium CNPMS B119 and Bacillus subtilis CNPMS B2084 improve P-acquisition and maize yield in Brazil.

Phosphorus (P) is a critical nutrient for plant growth, yet its uptake is often hindered by soil factors like clay minerals and metal oxides such as aluminum (Al), iron (Fe), and calcium (Ca), which bind P and limit its availability. Phosphate-solubilizing bacteria (PSB) have the unique ability to convert insoluble P into a soluble form, thereby fostering plant growth. This study aimed to assess the efficacy of inoculation of Bacillus megaterium B119 (rhizospheric) and B. subtilis B2084 (endophytic) via seed treatment in enhancing maize yield, grain P content, and enzyme activities across two distinct soil types in field conditions. Additionally, we investigated various mechanisms contributing to plant growth promotion, compatibility with commercial inoculants, and the maize root adhesion profile of these strains. During five crop seasons in two experimental areas in Brazil, Sete Lagoas-MG and Santo Antônio de Goiás-GO, single inoculations with either B119 or B2084 were implemented in three seasons, while a co-inoculation with both strains was applied in two seasons. All treatments received P fertilizer according to plot recommendations, except for control. Both the Bacillus strains exhibited plant growth-promoting properties relevant to P dynamics, including phosphate solubilization and mineralization, production of indole-3-acetic acid (IAA)-like molecules, siderophores, exopolysaccharides (EPS), biofilms, and phosphatases, with no antagonism observed with Azospirillum and Bradyrizhobium. Strain B2084 displayed superior maize root adhesion compared to B119. In field trials, single inoculations with either B119 or B2084 resulted in increased maize grain yield, with relative average productivities of 22 and 16% in Sete Lagoas and 6 and 3% in Santo Antônio de Goiás, respectively. Co-inoculation proved more effective, with an average yield increase of 24% in Sete Lagoas and 11% in Santo Antônio de Goiás compared to the non-inoculated control. Across all seasons, accumulated grain P content correlated with yield, and soil P availability in the rhizosphere increased after co-inoculation in Santo Antônio de Goiás. These findings complement previous research efforts and have led to the validation and registration of the first Brazilian inoculant formulated with Bacillus strains for maize, effectively enhancing and P grain content.

Parameter evaluation for developing phosphate-solubilizing Bacillus inoculants.

Bacterial inoculants have been used in agriculture to improve plant performance. However, laboratory and field requirements must be completed before a candidate can be employed as an inoculant. Therefore, this study aimed to evaluate the parameters for inoculant formulation and the potential of Bacillus subtilis (B70) and B. pumilus (B32) to improve phosphorus availability in maize (Zea mays L.) crops. In vitro experiments assessed the bacterial ability to solubilize and mineralize phosphate, their adherence to roots, and shelf life in cassava starch (CS), carboxymethyl cellulose (CMC), peat, and activated charcoal (AC) stored at 4 °C and room temperature for 6 months. A field experiment evaluated the effectiveness of strains to increase the P availability to plants growing with rock phosphate (RP) and a mixture of RP and triple superphosphate (TS) and their contribution to improving maize yield and P accumulation in grains. The B70 was outstanding in solubilizing RP and phytate mineralization and more stable in carriers and storage conditions than B32. However, root adherence was more noticeable in B32. Among carriers, AC was the most effective for preserving viable cell counts, closely similar to those of the initial inoculum of both strains. Maize productivity using the mixture RPTS was similar for B70 and B32. The best combination was B70 with RP, which improved the maize yield (6532 kg ha-1) and P accumulation in grains (15.95 kg ha-1). Our results indicated that the inoculant formulation with AC carrier and B70 is a feasible strategy for improving phosphorus mobilization in the soil and maize productivity.

Co-inoculation with tropical strains of Azospirillum and Bacillus is more efficient than single inoculation for improving plant growth and nutrient uptake in maize.

Usage of Bacillus and Azospirillum as new eco-friendly microbial consortium inoculants is a promising strategy to increase plant growth and crop yield by improving nutrient availability in agricultural sustainable systems. In this study, we designed a multispecies inoculum containing B. thuringiensis (strain B116), B. subtillis (strain B2084) and Azospirillum sp. (strains A1626 and A2142) to investigate their individual or co-inoculated ability to solubilize and mineralize phosphate, produce indole acetic acid (IAA) and their effect on maize growth promotion in hydroponics and in a non-sterile soil. All strains showed significant IAA production, P mineralization (sodium phytate) and Ca-P, Fe-P (tricalcium phosphate and iron phosphate, respectively) solubilization. In hydroponics, co-inoculation with A1626 x A2142, B2084 x A2142, B2084 x A1626 resulted in higher root total length, total surface area, and surface area of roots with diameter between 0 and 1 mm than other treatments with single inoculant, except B2084. In a greenhouse experiment, maize inoculated with the two Azospirillum strains exhibited enhanced shoot dry weight, shoot P and K content, root dry weight, root N and K content and acid and alkaline phosphatase activities than the other treatments. There was a significant correlation between soil P and P shoot, alkaline phosphatase and P shoot and between acid phosphatase and root dry weight. It may be concluded that co-inoculations are most effective than single inoculants strains, mainly between two selected Azospirillum strains. Thus, they could have synergistic interactions during maize growth, and be useful in the formulation of new inoculants to improve the tropical cropping systems sustainability.

Antifungal activity of bacterial strains from maize silks against Fusarium verticillioides.

Fusarium verticillioides is pathogenic to maize and mycotoxin-producer, causing yield losses, feed and food contamination, and risks to human and animal health. Endophytic (ISD04 and IPR45) and epiphytic (CT02 and IM14) bacteria from maize silks were tested in vitro and greenhouse against F. verticillioides and for hydrolytic enzyme production (cellulase, pectinase, protease, lipase, and chitinase). The strains preliminarily identified as Achromobacter xylosoxidans (ISD04), Pseudomonas aeruginosa (IPR45), and Bacillus velezensis (CT02 and IM14) by 16S gene sequencing. All strains showed antifungal activity in vitro with inhibition values from 58.5 to 100%; they changed hyphae morphology and inhibited the conidial germination by up to 100% (IPR45). The four strains produced at least one enzyme with antifungal activity. The microbiolized seeds reduced the fungal development in stored grains and stalk rot severity in the greenhouse by 72.6% (ISD04). These results highlight the potential of these strains as biocontrol agents against F. verticillioides.