The key role of indole-3-acetic acid biosynthesis by Bacillus thuringiensis RZ2MS9 in promoting maize growth revealed by the ipdC gene knockout mediated by the CRISPR-Cas9 system.

The bacterial biosynthesis of indole-3-acetic acid (IAA) is often related to the beneficial effects of plant growth-promoting rhizobacteria (PGPR) on plant development. In PGPR belonging to the Bacillus genus, the synthesis of IAA may occur through different metabolic pathways that are still poorly understood. B. thuringiensis (Bt) is well known for its insecticidal properties; however, its beneficial features are not limited to pest control. Our group has been studed the beneficial effects of Bt strain RZ2MS9 as growth promoter in a range of plant crops, including soybean, tomato, and maize. We recently demonstrated that bacterial IAA biosynthesis plays an important role in the ability of RZ2MS9 to benefit plant development. However, the molecular involved mechanisms in the IAA biosynthesis by this bacterium in the beneficial interaction with plants remain unclear. Here, we investigated the genetic basis of IAA biosynthesis by RZ2MS9. We knocked out the ipdC gene, involved in IAA biosynthesis via the tryptophan-dependent IPyA pathway, using the CRISPR-Cas9 system. Our results showed that, by disrupting the IPyA pathway, the amount of IAA synthesized by the mutant RZ2MS9 (ΔipdC) in the presence of tryptophan drops 57%. The gene knockout did not affect the bacterial growth, but it did affect its ability to colonize maize. Moreover, deactivating the ipdC gene in RZ2MS9 significantly reduces its ability to promote maize growth. ΔipdC performed worse than RZ2MS9 in almost all evaluated plant parameters, including total root length, projected root area, lateral roots, aerial part dry matter, and germination speed index. Therefore, we demonstrated that tryptophan-dependent IAA biosynthesis via the IPyA pathway by RZ2MS9 is strongly influenced by the ipdC gene. Furthermore, IAA biosynthesis by RZ2MS9 is a major mechanism used by this PGPR to promote maize growth.

Revealing the high variability on nonconserved core and mobile elements of Austropuccinia psidii and other rust mitochondrial genomes.

Mitochondrial genomes are highly conserved in many fungal groups, and they can help characterize the phylogenetic relationships and evolutionary biology of plant pathogenic fungi. Rust fungi are among the most devastating diseases for economically important crops around the world. Here, we report the complete sequence and annotation of the mitochondrial genome of Austropuccinia psidii (syn. Puccinia psidii), the causal agent of myrtle rust. We performed a phylogenomic analysis including the complete mitochondrial sequences from other rust fungi. The genome composed of 93.299 bp has 73 predicted genes, 33 of which encoded nonconserved proteins (ncORFs), representing almost 45% of all predicted genes. A. psidii mtDNA is one of the largest rust mtDNA sequenced to date, most likely due to the abundance of ncORFs. Among them, 33% were within intronic regions of diverse intron groups. Mobile genetic elements invading intron sequences may have played significant roles in size but not shaping of the rust mitochondrial genome structure. The mtDNAs from rust fungi are highly syntenic. Phylogenetic inferences with 14 concatenated mitochondrial proteins encoded by the core genes placed A. psidii according to phylogenetic analysis based on 18S rDNA. Interestingly, cox1, the gene with the greatest number of introns, provided phylogenies not congruent with the core set. For the first time, we identified the proteins encoded by three A. psidii ncORFs using proteomics analyses. Also, the orf208 encoded a transmembrane protein repressed during in vitro morphogenesis. To the best of our knowledge, we presented the first report of a complete mtDNA sequence of a member of the family Sphaerophragmiacea.

Scorpionicidal activity of secondary metabolites from Paecilomyces sp. CMAA1686 against Tityus serrulatus.

INTRODUCTION: Urban pests pose enormous risks to human health. Control initiatives are carried out in regions of high infestation and incidence of accidents caused by scorpions OBJECTIVE: In this study, we aimed to analyze the anti-scorpionic activity of fungal isolates obtained from a cemetery in Brazil. MATERIALS AND METHODS: A total of thirteen fungi were subjected to a bioassay test against Tityus serrulatus, and the two isolates with the highest scorpionicidal activity were selected for molecular identification through sequencing of the ITS DNA hypervariable region and large-scale cultivation on liquid medium for secondary metabolite extraction. The crude extracts were partitioned by solid-phase extraction, and the resulting purified extracts were tested for anti-scorpionic activity. The extracts from one of the isolates presented better results and were submitted to UPLC-MS/MS. The metabolomics data were submitted to GNPS website for Molecular Networking and MASST searches. We also performed a MolNetEnhancer analysis to identify the chemical classes of the molecules found in the samples. RESULTS: The most promising fungal isolate was identified as Paecilomyces sp. CMAA1686 which has 98% of similarity to Paecilomyces formosus. The sub-fractions C and D had the best activity against the scorpions (54 and 32% mortality, respectively). Molecular Networking and MolNetEnhancer revealed a range of molecular classes in our extracts that are known to include bioactive metabolites from Paecilomyces species. CONCLUSIONS: The scorpionicidal activity of Paecilomyces sp. CMAA1686 and its secondary metabolites may provide new alternative compounds for biological and chemical control of scorpions from the species T. serrulatus. Paecilomyces sp. CMAA1686 is an isolate that has great potential for isolation of secondary metabolites.

The bacterial and fungal communities associated with Anthurium ssp. leaves: Insights into plant endemism and microbe association.

It is known that different plant species select specific microbes to live inside their tissues in a process determined by the host genotype, phenotype and geographic location, which can introduce discussion on plant endemism and the assembly of specific microbial communities. Herein, we report the results of an investigation relating the geographic distribution of plant species and the composition of microbial communities associated with plant hosts. The bacterial and fungal community associated with Anthurium plant leaves was mapped to assess the diversity and ecology of the endophytic community associated with Anthurium spp. collected on islands and on the Brazilian mainland. Twenty-six Anthurium specimens were surveyed, distributed throughout the São Paulo state coastline, including Alcatrazes Island, some coastal islands and distinct mainland environments. Bacterial and fungal endophytes were obtained from the leaves of A. alcatrazense, A. loefgrenii, A. penthaphyllum, A. urvellianum and A. intermedium and subjected to massive bacterial 16S rRNA and fungal ITS sequencing. The results indicated that A. alcatrazense, endemic to Alcatrazes Island, hosted a specific bacterial community structure, while its fungal community was similar to that of Anthurium species from other locations. Betaproteobacteria showed a high differential occurrence in A. alcatrazense. Some groups of fungi were found mainly inhabiting A. loefgrenii plants. While Alphaproteobacteria, Gammaproteobacteria, Actinobacteria and Sordariomycetes, Dothiodeomycetes and Tremellomycetes composed the core microbial community among Anthurium plants. The results suggest crucial role for the bacterial communities to endemic plants, while endophytic fungal diversity is less specifically distributed among endemic and nonendemic plant species.

Rehydration of dry corn preserves the desirable bacterial community during ensiling.

This study evaluated the rehydration approach of mature corn grains as an alternative for high-moisture corn grain silage production in distinct corn hybrids, storage period, cultivation locations and kernel maturity at plant harvest. High-moisture corn was used as a control. The dry matter content and pH of the silage were measured, and the bacterial community associated with corn grains pre- and post-ensiling was also assessed through 16S rRNA high-throughput sequencing. The decrease in pH value was directly linked to an ecological microbial succession of Enterobacteriales and Actinomycetales to Lactobacillales in the silage at 120 days after storage, either in rehydrated or high-moisture corn. These results were similar for both maize production locations and hybrids tested. Finally, the similarity between the ensiling processes including rehydrated corn and the high-moisture corn grain silages proves the reliability of the rehydration approach as an alternative for the maintenance of a successful bacterial community structure and composition capable of producing high-quality silages from dent and flint corn hybrids in tropical conditions.

Shifts in the bacterial community of saliva give insights on the relationship between obesity and oral microbiota in adolescents.

The current study aimed at the determination of the impact of obesity on the salivary microbiome in adolescents. Sixty subjects ranging 14-17 years old were enrolled (obese: n = 30-50% females, and normal weight: n = 30-50% females). Stimulated saliva was collected for denaturing gradient gel electrophoresis (DGGE) band patterns and massive 16S rRNA gene sequencing using the Ion Torrent platform. Overall, data analysis revealed that male subjects harbored a higher diverse salivary microbiome, defined by a significant higher richness (32.48 versus 26.74) and diversity (3.36 versus 3.20), higher Simpson values (0.96 versus 0.95) and distinct bacterial community structure considering either sex or condition (p < 0.05). Bacterial community fingerprinting analysis in human saliva showed a positive correlation with increased body mass index (BMI) in adolescents. Veillonella, Haemophilus and Prevotella occurrence was found to be affected by BMI, whereas Neisseria and Rothia occurrence was significantly impacted by sex in obese subjects. Our findings suggest that male and female adolescents may harbor a naturally distinct salivary microbiota and that obesity may specifically have an impact on their oral bacterial community. The potential dysbiotic oral microbiome in obese adolescents raises new insights on the etiology and prevention of future conditions in these populations.

Respiration induced by substrate and bacteria diversity after application of diuron, hexazinone, and sulfometuron-methyl alone and in mixture.

After application, herbicides often reach the soil and affect non-target soil microorganisms, decreasing their population, diversity or affecting metabolic activity. Therefore, laboratory studies were performed to evaluate the effects of diuron, hexazinone and sulfometuron-methyl alone and mixed upon carbon transformation by soil microorganisms in clayey and sandy soils and the effect on bacterial diversity and structure. Control treatment without herbicide application was also performed. Sub-samples from the control and herbicide treatments (10 g - in triplicate) were collected before herbicide application and 7, 14, 28 and 42 days after treatment (DAT), then 1 mL of 14C-glucose solution was applied. The released 14CO2 was trapped in 2 M NaOH solution and the radioactivity was analyzed by liquid scintillation counting (LSC), 12 h after glucose application. The effect of herbicides on bacterial diversity was evaluated by T-RFLP. The experiment was conducted in a complete randomized design. Hexazinone did not affect 14CO2 evolution. Diuron showed a greater 14CO2 evolution in sandy and clayey soil, while sulfometuron-methyl led to an increase in sandy soil, at 42 DAT. A greater evolution of carbon was observed in the treatment with herbicide mixture in sandy soil, compared with the same treatment in clayey soil or control. However, the herbicide mixture application did not affect the soil biological activity measured by the respiration rate induced by substrate. On the other hand, the herbicide mixtures affected the bacterial diversity in both soils, being the strongest effect to diuron and sulfometuron-methyl in clayey soil and hexazinone in sandy soil.

Sewage Sludge Microbial Structures and Relations to Their Sources, Treatments, and Chemical Attributes.

Sewage sludges generation and their disposal have become one of the greatest challenges of the 21st century. They have great microbial diversity that may impact wastewater treatment plant (WWTP) efficiency and soil quality whether used as fertilizers. Therefore, this research aimed to characterize microbial community diversity and structure of 19 sewage sludges from São Paulo, Brazil, as well as to draw their relations to sludge sources [domestic and mixed (domestic+industrial)], biological treatments (redox conditions and liming), and chemical attributes, using molecular biology as a tool. All sludges revealed high bacterial diversity, but their sources and redox operating conditions as well as liming did not consistently affect bacterial community structures. Proteobacteria was the dominant phylum followed by Bacteroidetes and Firmicutes; whereas Clostridium was the dominant genus followed by Treponema, Propionibacterium, Syntrophus, and Desulfobulbus. The sludge samples could be clustered into six groups (C1 to C6) according their microbial structure similarities. Very high pH (≥11.9) was the main sludge attribute segregating C6, that presented very distinct microbial structure from the others. Its most dominant genera were Propionibacterium > > Comamonas > Brevundimonas > Methylobacterium ∼Stenotrophomonas ∼Cloacibacterium. The other clusters' dominant genera were Clostridium > > Treponema > Desulfobulbus ∼Syntrophus. Moreover, high Fe and S were important modulators of microbial structure in certain sludges undertaking anaerobic treatment and having relatively low N-Kj, B, and P contents (C5). However, high N-Kj, B, P, and low Fe and Al contents were typical of domestic, unlimed, and aerobically treated sludges (C1). In general, heavy metals had little impact on microbial community structure of the sludges. However, our sludges shared a common core of 77 bacteria, mostly Clostridium, Treponema, Syntrophus, and Comamonas. They should dictate microbial functioning within WWTPs, except by SS12 and SS13.

The depleted mineralization of the fungicide chlorothalonil derived from loss in soil microbial diversity.

There are lack of studies regarding the effects of microbial diversity on specific soil functions, such as pesticides degradation. This study evaluated the role of bacterial community diversity and biochar on chlorothalonil (CTN) degradation, using 'dilution to extinction' approach, PCR-DGGE/16S rRNA gene technique, and radiorespirometry (14C-CTN). Biochar and microbial community dilution affected structure of the microbial community. In spite of that, CTN mineralization was slow, but dissipation was very fast (D50 < 1.0 d) due to immediate chemical degradation and formation of non-extractable (bound) residues. However, any depletion on soil microbial diversity strongly affected CTN mineralization, suggesting that this function is related to less abundant but specific microbial groups (CTN degraders) or to soil microbial diversity. The extent of these effects will strongly depend on the compound nature (recalcitrance) and soil matrix/substrate (bioavailability). It can be corroborated by the fact that biochar affected CTN sorption, its bioavailability, and subsequently its mineralization rate in the NS. These data indicate a strong relationship between soil microbial diversity and pesticide degradation, which is an acting form to mitigate xenobiotics accumulation in the environment.

Shifts in the bacterial community composition along deep soil profiles in monospecific and mixed stands of Eucalyptus grandis and Acacia mangium.

Our knowledge of the rhizosphere bacterial communities in deep soils and the role of Eucalyptus and Acacia on the structure of these communities remains very limited. In this study, we targeted the bacterial community along a depth profile (0 to 800 cm) and compared community structure in monospecific or mixed plantations of Acacia mangium and Eucalyptus grandis. We applied quantitative PCR (qPCR) and sequence the V6 region of the 16S rRNA gene to characterize composition of bacterial communities. We identified a decrease in bacterial abundance with soil depth, and differences in community patterns between monospecific and mixed cultivations. Sequence analysis indicated a prevalent effect of soil depth on bacterial communities in the mixed plant cultivation system, and a remarkable differentiation of bacterial communities in areas solely cultivated with Eucalyptus. The groups most influenced by soil depth were Proteobacteria and Acidobacteria (more frequent in samples between 0 and 300 cm). The predominant bacterial groups differentially displayed in the monospecific stands of Eucalyptus were Firmicutes and Proteobacteria. Our results suggest that the addition of an N2-fixing tree in a monospecific cultivation system modulates bacterial community composition even at a great depth. We conclude that co-cultivation systems may represent a key strategy to improve soil resources and to establish more sustainable cultivation of Eucalyptus in Brazil.

Changes in bacterial community after application of three different herbicides.

The native soil microbiota is very important to maintain the quality of that environment, but with the intensive use of agrochemicals, changes in microbial biomass and formation of large quantities of toxic waste were observed in soil, groundwater and surface water. Thereby, the goal of this study was to evaluate if the selective pressure exerted by the presence of the herbicides atrazine, diuron and 2,4-D changes the bacterial community structure of an agricultural soil, using denaturing gradient gel electrophoresis technique. According to PERMANOVA analysis, a greater effect of the herbicide persistence time in the soil, the effect of the herbicide class and the effect of interaction between these two factors (persistence time and herbicide class) were observed. In conclusion, the results showed that the selective pressure exerted by the presence of these herbicides altered the composition of the local microbiota, being atrazine and diuron that most significantly affected the bacterial community in soil, and the herbicide 2,4-D was the one that less altered the microbial community and that bacterial community was reestablished first.

Bacterial communities involved in sulfur transformations in wastewater treatment plants.

The main sulfate-reducing (SRB) and sulfur-oxidizing bacteria (SOB) in six wastewater treatment plants (WWTPs) located at southern Brazil were described based on high-throughput sequencing of the 16S rDNA. Specific taxa of SRB and SOB were correlated with some abiotic factors, such as the source of the wastewater, oxygen content, sample type, and physical chemical attributes of these WWTPs. When the 22 families of SRB and SOB were clustered together, the samples presented a striking distribution, demonstrating grouping patterns according to the sample type. For SOB, the most abundant families were Spirochaetaceae, Chromatiaceae, Helicobacteriaceae, Rhodospirillaceae, and Neisseriaceae, whereas, for SRB, were Syntrophaceae, Desulfobacteraceae, Nitrospiraceae, and Desulfovibriaceae. The structure and composition of the major families related to the sulfur cycle were also influenced by six chemical attributes (sulfur, potassium, zinc, manganese, phosphorus, and nitrogen). Sulfur was the chemical attribute that most influenced the variation of bacterial communities in the WWTPs (λ = 0.14, p = 0.008). The OTUs affiliated to Syntrophus showed the highest response to the increase of total sulfur. All these findings can contribute to improve the understanding in relation to the sulfur-oxidizing and sulfate-reducing communities in WWTPs aiming to reduce H2S emissions.

Endophytic bacterial communities associated with two explant sources of Eucalyptus benthamii Maiden & Cambage.

Micropropagation has been applied in the recovery and rejuvenation of adult trees, which is achieved by various subcultures in the multiplication phase. This strategy has brought questions about the endophytic microbiota associated with these plants along its manipulation. Therefore, the aim of this study was to evaluate the composition of the endophytic bacterial communities associated with two explants sources [the canopy branches (CB) and the trunk base of the tree (TB)] under prolonged in vitro cultivation. In addition we analyzed the bacterial community dynamic along the subcultures in different micropropagation phases. Bacterial DNA was extracted from samples of mini-stumps (in vivo) from CB and TB and in micro-stumps produced by in vitro cultivations of these explants sources--both originated from one single matrix plant of Eucalyptus benthamii. In vitro establishment occurred in two dates and the evaluation of endophytic bacterial communities was made in vivo and in vitro samples (on 10th, 13th and 16th subcultures), when elongated shoots and roots were analyzed. Analysis was performed by PCR-DGGE based on the V6 region of ribosomal gene 16S rDNA. Bands profiles showed differences in communities between in vivo and in vitro samples, and also distinctions of communities assessed in the subcultures, elongated and rooted samples. Distinctions in the composition of endophytic bacterial communities were greater in CB micro-stumps. These results indicate a differential colonization of explants by endophytic bacteria, with predominance of common (ever-present) endophytes in TB samples and casual, here named opportunistic, in CB samples.