Phenotypic, genomic and in planta characterization of Bacillus sensu lato for their phosphorus biofertilization and plant growth promotion features in soybean.

Bacillus sensu lato were screened for their capacity to mineralize organic phosphorus (P) and promote plant growth, improving nitrogen (N) and P nutrition of soybean. Isolates were identified through Type Strain Genome Server (TYGS) and Average Nucleotide Identity (ANI). ILBB95, ILBB510 and ILBB592 were identified as Priestia megaterium, ILBB139 as Bacillus wiedmannii, ILBB44 as a member of a sister clade of B. pumilus, ILBB15 as Peribacillus butanolivorans and ILBB64 as Lysinibacillus sp. These strains were evaluated for their capacity to mineralize sodium phytate as organic P and solubilize inorganic P in liquid medium. These assays ranked ILBB15 and ILBB64 with the highest orthophosphate production from phytate. Rhizocompetence and plant growth promotion traits were evaluated in vitro and in silico. Finally, plant bioassays were conducted to assess the effect of the co-inoculation with rhizobial inoculants on nodulation, N and P nutrition. These bioassays showed that B. pumilus, ILBB44 and P. megaterium ILBB95 increased P-uptake in plants on the poor substrate of sand:vermiculite and also on a more fertile mix. Priestia megaterium ILBB592 increased nodulation and N content in plants on the sand:vermiculite:peat mixture. Peribacillus butanolivorans ILBB15 reduced plant growth and nutrition on both substrates. Genomes of ILBB95 and ILBB592 were characterized by genes related with plant growth and biofertilization, whereas ILBB15 was differentiated by genes related to bioremediation. Priestia megaterium ILBB592 is considered as nodule-enhancing rhizobacteria and together with ILBB95, can be envisaged as prospective PGPR with the capacity to exert positive effects on N and P nutrition of soybean plants.

Beauveria bassiana transcriptomics reveal virulence-associated shifts during insect lipid assimilation.

Insect cuticular lipids, especially epicuticular hydrocarbons (CHC), have a significant role in insect ecology and interactions with other organisms, including fungi. The CHC composition of a specific insect species may influence the outcome of the interaction with a specific fungal strain. Some insects, such as Piezodorus guildinii, have low susceptibility towards fungal infections seemingly due to their CHC composition. The entomopathogenic fungus Beauveria bassiana can assimilate CHC and incorporate them as building blocks via cytochrome P450 monooxygenases (CYPs). However, little is known about other enzymes that promote the degradation/assimilation of these cuticular components. In this study, we performed a transcriptomic analysis to evaluate the in vitro response of two virulence-contrasting B. bassiana strains when grown on three different P. guildinii CHC sources. We found a different expression profile of virulence-related genes, as well as different GO and KEGG parameters enriched at 4 days post-inoculation, which could help account for the intrinsic virulence and for an alkane-priming virulence enhancement effect. The hypovirulent strain predominantly showed higher expression of cuticle penetration genes, including chitinases, proteases, and CYPs, with GO term categories of "heme binding," "monooxygenase activity," and "peroxisome" pathways enriched. The hypervirulent strain showed higher expression of cell wall remodeling and cell cycle genes, and cuticle adhesion and a distinct set of CYPs, with GO categories of "DNA-binding transcription factor activity" and KEGG pathways corresponding to "meiosis-yeast" and "cell cycle" enriched. These results suggest a delay and alternate routes in pathogenicity-related metabolism in the hypovirulent strain in comparison with the hypervirulent strain. KEY POINTS: •Transcriptomics of two B. bassiana strains grown in P. guildinii cuticular components •Virulence-related genes correlated with virulence enhancement towards P. guildinii •Differentially expressed genes, GOs and KEGGs showed different metabolic timelines associated with virulence.

Bioprospecting the Antibiofilm and Antimicrobial Activity of Soil and Insect Gut Bacteria.

Antimicrobial resistance is a growing concern in public health and current research shows an important role for bacterial biofilms in recurrent or chronic infections. New strategies, therefore, are necessary to overcome antimicrobial resistance, through the development of new therapies that could alter or inhibit biofilm formation. In this sense, antibiofilm natural products are very promising. In this work, a bioprospection of antimicrobial and antibiofilm extracts from Uruguayan soil bacteria and insect gut bacteria was carried out. Extracts from extracellular broths were tested for their ability to inhibit planktonic cell growth and biofilm formation. Genomic analysis of Bacillus cereus ILBB55 was carried out. All extracts were able to inhibit the growth of, at least, one microorganism and several extracts showed MICs lower than 500 µg mL-1 against microorganisms of clinical relevance (Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter cloacae). Among the extracts evaluated for biofilm inhibition only ILBB55, from B. cereus, was able to inhibit, S. aureus (99%) and P. aeruginosa (62%) biofilms. Genomic analysis of this strain showed gene clusters similar to other clusters that code for known antimicrobial compounds. Our study revealed that extracts from soil bacteria and insect gut bacteria, especially from B. cereus ILBB55, could be potential candidates for drug discovery to treat infectious diseases and inhibit S. aureus and P. aeruginosa biofilms.

Alkane-priming of Beauveria bassiana strains to improve biocontrol of the redbanded stink bug Piezodorus guildinii and the bronze bug Thaumastocoris peregrinus.

Insect epicuticle hydrocarbons (CHC) are known to be important determinants in the susceptibility degree of insects to fungal entomopathogens. Five Beauveria bassiana (Balsamo) Vuillemin (Hypocreales; Clavicipitaceae) strains were phenotypically analyzed regarding their response to CHC nutrition and their pathogenicity and virulence towards high fungal-susceptible Thaumastocoris peregrinus (Carpintero and Dellapé) (Heteroptera: Thaumastocoridae) and low fungal-susceptible Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), which are important hemipteran pests in eucalyptus and soybean plantations, respectively. Two of these strains, which were the most (ILB308) and the least (ILB299) virulent to P. guildinii, were also evaluated at gene expression level after growth on n-pentadecane, a P. guildinii epicuticular hydrocarbon. Beauveria bassiana hypervirulent strain ILB308 showed the lowest growth on most evaluated CHC media. However, this strain distinctively induced most of the analyzed genes involved in CHC assimilation, cuticle degradation and stress tolerance. Virulence towards low susceptibility P. guildinii was enhanced in both hypervirulent ILB308 and hypovirulent ILB299 strains after growth on n-pentadecane as the sole carbon source, whereas virulence enhancement towards high susceptibility T. peregrinus was only observed in the hypervirulent strain. Virulence enhancement towards P. guildinii could be mostly explained by a priming effect produced by CHC on the induction of some genes related to hydrocarbon assimilation in ILB299 and ILB308, such as cytochrome P450 genes (BbCyp52g11 and BbCyp52x1), together with adhesion and stress tolerance genes, such as hydrophobin (Bbhyd2) and catalase (Bbcatc) and glutathione peroxidase (Bbgpx), respectively.

Epicuticular hydrocarbons of the redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae): sexual dimorphism and alterations in insects collected in insecticide-treated soybean crops.

BACKGROUND: The redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae) is one of the most important species affecting soybean crops in southern South America. Capillary gas chromatography coupled to mass spectrometry was used to characterize the epicuticular hydrocarbon profiles of field-collected insects, and to identify differences in their composition between fifth-instar nymphs and adults, males and females, and between bugs collected in insecticide-treated and insecticide-free soybean crops. RESULTS: Straight chain saturated n-C27 and n-C29, and monomethyl and dimethyl chains of C31 and C33 were the most abundant compounds. A group of volatile hydrocarbons with n-C13 and n-C15 as the predominant compounds were also detected. The hydrocarbon pattern was different between nymphs and adults, either males or females. Heneicosene was almost exclusively detected in adult males and was the most important component to differentiate between both sexes, followed by tricosadiene. The total hydrocarbon amount was significantly higher in nymphs, males and females collected in insecticide-treated fields compared with insects obtained from untreated fields. CONCLUSION: Differences were found in the epicuticular hydrocarbon pattern among nymphs and adults, as well as sexual dimorphism in adult stink bugs. Interestingly, an alteration was also found in the hydrocarbon profile of insects collected in insecticide-treated soybean crops and its relevance is discussed within a pest management context.

Control of damping-off in tomato seedlings exerted by Serratia spp. strains and identification of inhibitory bacterial volatiles in vitro.

Serratia marcescens can be a plant growth promoting bacteria (PGPB) and an opportunistic human and plant pathogen. We have identified and characterized strains of related species of Serratia and evaluated their biological control of damping-off of tomato seeds caused by Pythium cryptoirregulare. Serratia ureilytica, S. bockelmannii and S. nevei were identified by phylogenetic analysis of partial gyrB gene sequence and average nucleotide identity (ANI). Tomato seeds inoculated with S. ureilytica ILBB 145 showed higher germination percentage and reduced damping-off in greenhouse experiment resembling a commercial operation, and volatiles produced by this strain caused the nearly complete inhibition in vitro of P. cryptoirregulare. Analysis of volatile organic compounds (VOCs) showed that ILBB 145 produced dimethyl disulfide (DMDS), which can partially account for this inhibition. Serratia bockelmannii ILBB 162 performance against damping-off was intermediate and the inhibition of P. cryptoirregulare in vitro was lower and explained by volatile and diffusible metabolites. Both strains augmented DMDS production in the presence of P. cryptoirregulare, suggesting this compound may play a role in the context of interspecific competition. Serratia nevei ILBB 219 showed the lowest inhibition of P. cryptoirregulare in vitro, no DMDS production, and no biocontrol in planta. Draft genomes of the three strains were annotated and individual genes and biosynthesis gene clusters were identified in relation with the observed phenotypes. We report S. ureilytica - a low risk species- with activity as a biological control agent and DMDS produced by this bacterial species putatively involved in seed and seedling protection against P. cryptoirregulare.

Pangenome of Serratia marcescens strains from nosocomial and environmental origins reveals different populations and the links between them.

Serratia marcescens is a Gram-negative bacterial species that can be found in a wide range of environments like soil, water and plant surfaces, while it is also known as an opportunistic human pathogen in hospitals and as a plant growth promoting bacteria (PGPR) in crops. We have used a pangenome-based approach, based on publicly available genomes, to apply whole genome multilocus sequence type schemes to assess whether there is an association between source and genotype, aiming at differentiating between isolates from nosocomial sources and the environment, and between strains reported as PGPR from other environmental strains. Most genomes from a nosocomial setting and environmental origin could be assigned to the proposed nosocomial or environmental MLSTs, which is indicative of an association between source and genotype. The fact that a few genomes from a nosocomial source showed an environmental MLST suggests that a minority of nosocomial strains have recently derived from the environment. PGPR strains were assigned to different environmental types and clades but only one clade comprised strains accumulating a low number of known virulence and antibiotic resistance determinants and was exclusively from environmental sources. This clade is envisaged as a group of promissory MLSTs for selecting prospective PGPR strains.

Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must.

Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic--malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41™ was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated.

Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must

Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic -malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41TM was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated.