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.

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.

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.

Bt Soybean Cry1Ac Does Not Affect Development, Reproduction, or Feeding Behavior of Red-Banded Stink Bug Piezodorus guildinii (Hemiptera: Pentatomidae).

Bt soybean cultivation is increasing worldwide. The Cry1Ac protein expressed in Bt soybean efficiently controls several lepidopteran pests. The stink bug, Piezodorus guildinii (Westwood), a major pest for soybean in the Americas, is not controlled by Bt crops, although possible sub-lethal effects may occur. Even if there were no negative effects for sting bug, ingesting toxins could affect its bio-controllers. We tested through ELISA detection if P. guildinii ingests Cry1Ac from Bt soybean and possible effects on its development, reproduction, survival, and feeding behavior. Biological traits were evaluated under controlled conditions of nymphs and adults feeding on pods of near-isogenic cultivars DM5958iPRO (Bt) and DM59i (non-Bt). Feeding behavior was recorded using an AC-DC electropenetrography (EPG) device. Results indicated that P. guildinii ingested the Cry1Ac protein; however, nymphal period and accumulated survival percentage did not differ between cultivars. Feeding on Bt soybean pods did not affect fecundity (i.e., number of egg masses and eggs/female) nor egg viability. Different feeding behaviors were only detected on the pathway phase (stylet penetration into plant tissue), which was more pronounced in the Bt cultivar. However, the total duration of the feeding activities on seeds was numerically higher (ca. 2X) on Bt plants compared to non-Bt. This is the first study to demonstrate that P. guildinii does ingest the Cry1Ac protein and excrete it without being absorbed, probably explaining the lack of direct adverse effects on its biological parameters. EPG could indicate that Bt soybean plants might be less palatable than non-Bt to red-banded stink bug.

Abundance and phylogenetic distribution of eight key enzymes of the phosphorus biogeochemical cycle in grassland soils.

Grassland biomes provide valuable ecosystem services, including nutrient cycling. Organic phosphorus (Po) represents more than half of the total P in soils. Soil microorganisms release organic P through enzymatic processes, with alkaline phosphatases, acid phosphatases and phytases being the key P enzymes involved in the cycling of organic P. This study analysed 74 soil metagenomes from 17 different grassland biomes worldwide to evaluate the distribution and abundance of eight key P enzymes (PhoD, PhoX, PhoA, Nsap-A, Nsap-B, Nsap-C, BPP and CPhy) and their relationship with environmental factors. Our analyses showed that alkaline phosphatase phoD was the dataset's most abundant P-enzyme encoding genes, with a wide phylogenetic distribution. Followed by the acid phosphatases Nsap-A and Nsap-C showed similar abundance but a different distribution in their respective phylogenetic trees. Multivariate analyses revealed that pH, Tmax , SOC and soil moisture were associated with the abundance and diversity of all genes studied. PhoD and phoX genes strongly correlated with SOC and clay, and the phoX gene was more common in soils with low to medium SOC and neutral pH. In particular, P-enzyme genes tended to respond in a positively correlated manner among them, suggesting a complex relationship of abundance and diversity among them.

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.

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.

Soil structure, nutrient status and water holding capacity shape Uruguayan grassland prokaryotic communities.

Soil microbial communities play critical roles in maintaining natural ecosystems such as the Campos biome grasslands of southern South America. These grasslands are characterized by a high diversity of soils, low available phosphorus (P) and limited water holding capacity. This work aimed to describe prokaryotic communities associated with different soil types and to examine the relationship among these soil communities, the parent material and the soil nutrient status. Five Uruguayan soils with different parent material and nutrient status, under natural grasslands, were compared. The structure and diversity of prokaryotic communities were characterized by sequencing 16S rRNA gene amplicons. Proteobacteria, Actinobacteria, Firmicutes,Verrucomicrobia, Acidobacteria, Planctomycetes and Chloroflexi were the predominant phyla. Ordination based on several distance measures was able to discriminate clearly between communities associated with different soil types. Edge-PCA phylogeny-sensitive ordination and differential relative abundance analyses identified Archaea and the bacterial phyla Firmicutes, Acidobacteria, Actinobacteria and Verrucomicrobia as those with significant differences among soil types. Canonical analysis of principal coordinates identified porosity, clay content, available P, soil organic carbon and water holding capacity as the main variables contributing to determine the characteristic prokaryotic communities of each soil type.

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.

Fluorescent pseudomonads as biocontrol agents for sustainable agricultural systems.

The highly diverse genus Pseudomonas contains very effective biocontrol agents that can increase plant growth and improve plant health. Biocontrol characteristics, however, are strain-dependent and cannot be clearly linked to phylogenetic variation. Isolate screening remains essential to find suitable strains, which can be done by testing large local collections for disease suppression and plant-growth promotion exemplified in a case study on forage legumes in Uruguay or by targeted screening for Pseudomonas spp. which produce desirable secondary metabolites, as demonstrated in a case study on cocoyam in Cameroon. In both case studies, access to reference strains from public and private collections was essential for identification, phylogenetic studies and metabolite characterization.

The biodiversity of beneficial microbe-host mutualism: the case of rhizobia.

Symbiotic nitrogen fixation is the main route for sustainable input of nitrogen into ecosystems. Nitrogen fixation in agriculture can be improved by inoculation of legume crops with suitable rhizobia. Knowledge of the biodiversity of rhizobia and of local populations is important for the design of successful inoculation strategies. Soybeans are major nitrogen-fixing crops in many parts of the world. Bradyrhizobial inoculants for soybean are very diverse, yet classification and characterization of strains have long been difficult. Recent genetic characterization methods permit more reliable identification and will improve our knowledge of local populations. Forage legumes form another group of agronomically important legumes. Research and extension policies valorizing rhizobial germplasm diversity and preservation, farmer training for proper inoculant use and legal enforcement of commercial inoculant quality have proved a successful approach to promoting the use of forage legumes while enhancing biological N(2) fixation. It is worth noting that taxonomically important strains may not necessarily be important reference strains for other uses such as legume inoculation and genomics due to specialization of the different fields. This article points out both current knowledge and gaps remaining to be filled for further interaction and improvement of a rhizobial commons.