Draft genome sequence of the cyanobacterium Sphaerospermopsis aphanizomenoides BCCUSP55 from the Brazilian semiarid region reveals potential for anti-cancer applications.

Sphaerospermopsis aphanizomenoides is a filamentous nitrogen-fixing and bloom-forming cyanobacterium, which biomass can fertilize natural water with nutrients, especially through nitrogen fixation. The Sphaerospermopsis aphanizomenoides strain BCCUSP55 was previously isolated from a water supply reservoir in the Brazilian semiarid region, and its draft genome assembly coupled with the gene contents are reported here. The obtained BCCUSP55 draft genome comprised 254 scaffolds with a genome size estimated of 6,096,273 bp. In addition, it has 5250 predicted coding sequences (CDS) and the G + C content is 38.5%. Further, the BCCUSP55 draft genome presented the putative nocuolin A gene complete cluster, a natural oxadiazine that triggers apoptosis in human cancer cells. Thus, our results contribute to extend the knowledge on the genus Sphaerospermopsis and reveal its biotechnological potential.

The auxin-producing Bacillus thuringiensis RZ2MS9 promotes the growth and modifies the root architecture of tomato (Solanum lycopersicum cv. Micro-Tom).

Strains of Bacillus thuringiensis (Bt) are commonly commercialized as bioinoculants for insect pest control, but their benefits go beyond their insecticidal property: they can act as plant growth-promoters. Auxins play a major role in the plant growth promotion. However, the mechanism of auxin production by the Bacilli group, and more specifically by Bt strains, is unclear. In previous work, the plant growth-promoting rhizobacterium (PGPR) B. thuringiensis strain RZ2MS9 increased the corn roots. This drew our attention to the strain's auxin production trait, earlier detected in vitro. Here, we demonstrate that in its genome, RZ2MS9 harbours the complete set of genes required in two pathways that are used for Indole acetic acid (IAA) production. We also detected that the strain produces almost five times more IAA during the stationary phase. The bacterial application increased the shoot dry weight of the Micro-Tom (MT) tomato by 24%. The application also modified MT root architecture, with an increase of 26% in the average lateral root length and inhibition of the axial root. At the cellular level, RZ2MS9-treated MT plants presented elongated root cortical cells with intensified mitotic activity. Altogether, these are the best characterized auxin-associated phenotypes. Besides that, no growth alteration was detected in the auxin-insensitive diageotropic (dgt) plants either with or without the RZ2MS9 inoculation. Our results suggest that auxins play an important role in the ability of B. thuringiensis RZ2MS9 to promote MT growth and provide a better understanding of the auxin production mechanism by a Bt strain.

On the genetic architecture in a public tropical maize panel of the symbiosis between corn and plant growth-promoting bacteria aiming to improve plant resilience.

Exploring the symbiosis between plants and plant growth-promoting bacteria (PGPB) is a new challenge for sustainable agriculture. Even though many works have reported the beneficial effects of PGPB in increasing plant resilience for several stresses, its potential is not yet widely explored. One of the many reasons is the differential symbiosis performance depending on the host genotype. This opens doors to plant breeding programs to explore the genetic variability and develop new cultivars with higher responses to PGPB interaction and, therefore, have higher resilience to stress. Hence, we aimed to study the genetic architecture of the symbiosis between PGPB and tropical maize germplasm, using a public association panel and its impact on plant resilience. Our findings reveal that the synthetic PGPB population can modulate and impact root architecture traits and improve resilience to nitrogen stress, and 37 regions were significant for controlling the symbiosis between PGPB and tropical maize. In addition, we found two overlapping SNPs in the GWAS analysis indicating strong candidates for further investigations. Furthermore, genomic prediction analysis with genomic relationship matrix computed using only significant SNPs obtained from GWAS analysis substantially increased the predictive ability for several traits endorsing the importance of these genomic regions for the response of PGPB. Finally, the public tropical panel reveals a significant genetic variability to the symbiosis with the PGPB and can be a source of alleles to improve plant resilience. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01257-6.

Bacterial communities associated with anthracnose symptomatic and asymptomatic leaves of guarana, an endogenous tropical crop, and their pathogen antagonistic effects.

Plants are colonized by diverse microorganisms that can substantially impact their health and growth. Understanding bacterial diversity and the relationships between bacteria and phytopathogens may be key to finding effective biocontrol agents. We evaluated the bacterial community associated with anthracnose symptomatic and asymptomatic leaves of guarana, a typical tropical crop. Bacterial communities were assessed through culture-independent techniques based on extensive 16S rRNA sequencing, and cultured bacterial strains were evaluated for their ability to inhibit the growth of Colletotrichum sp. as well as for enzyme and siderophore production. The culture-independent method revealed that Proteobacteria was the most abundant phylum, but many sequences were unclassified. The emergence of anthracnose disease did not significantly affect the bacterial community, but the abundance of the genera Acinetobacter, Pseudomonas and Klebsiella were significantly higher in the symptomatic leaves. In vitro growth of Colletotrichum sp. was inhibited by 11.38% of the cultured bacterial strains, and bacteria with the highest inhibition rates were isolated from symptomatic leaves, while asymptomatic leaves hosted significantly more bacteria that produced amylase and polygalacturonase. The bacterial isolate Bacillus sp. EpD2-5 demonstrated the highest inhibition rate against Colletotrichum sp., whereas the isolates EpD2-12 and FD5-12 from the same genus also had high inhibition rates. These isolates were also able to produce several hydrolytic enzymes and siderophores, indicating that they may be good candidates for the biocontrol of anthracnose. Our work demonstrated the importance of using a polyphasic approach to study microbial communities from plant diseases, and future work should focus on elucidating the roles of culture-independent bacterial communities in guarana anthracnose disease.

Biocontrol of Botrytis cinerea and Calonectria gracilis by eucalypts growth promoters Bacillus spp.

The clonal Eucalyptus plants are commonly obtained by vegetative propagation under a protected environment. This system improves the Botrytis cinerea and Calonectria spp infection on the young eucalypts plantings, resulting gray mold and cutting rot respectively. Currently, the unique available control method is based on chemicals. As alternative, novel methods to manage plant diseases, endophytic microorganisms could be an interesting alternative. Thus, we aimed to evaluate endophytic Bacillus isolated from eucalypts as a biocontrol agent against Botrytis cinerea and Calonectria gracilis, important fungal pathogens in the greenhouse, using clonal plantlets of E. urograndis. Eight endophytic strains of Bacillus, previously described as eucalyptus growth promoters, were evaluated in vitro and in vivo against Botrytis cinerea and Calonectria gracilis. The diffusible metabolites assay showed the potential of endophytic Bacillus to decrease the growth of both pathogens. Differences in the susceptibility of the pathogens to bacterial volatile metabolites were observed, B. cinerea showed more susceptible than Calonectria gracilis. In vivo assays, Bacillus amyloliquefaciens EUCB 10 demonstrated better overall reductions in these diseases. Based on the results obtained from the in vitro and in vivo analyses, we suggest that the endophytic B. amyloliquefaciens strain EUCB 10 constitutes a promising biocontrol agent against B. cinerea and Calonectria gracilis. Furthermore, this is the first reporting of B. amyloliquefaciens previously describe as plant growth promoter and also as potential control agent of B. cinerea and Calonectria gracilis to eucalyptus.

Endophytic cultivable bacterial community obtained from the Paullinia cupana seed in Amazonas and Bahia regions and its antagonistic effects against Colletotrichum gloeosporioides.

Guarana (Paullinia cupana var. sorbilis) is a plant from the Amazonas region with socio-economic importance. However, guarana production has been increasingly affected by unfavorable conditions resulting from anthracnose, caused by the Colletotrichum fungal genus, which primarily affects mainly the Amazonas region. The aim of the present study was to isolate bacterial endophytes from the seeds of guarana plants obtained from Amazonas region and the Northeast state of Bahia, a region where this disease is not a problem for guarana plantations. The number of bacterial Colony Forming Units (CFU/g seeds) was 2.4 × 10(4) from the Bahia and 2.9 × 10(4) from the Amazonas region. One hundred and two isolated bacteria were evaluated in vitro against the phytopathogenic strain Colletotrichum gloeosporioides L1. These isolates were also analyzed for the enzymatic production of amylase, cellulase, protease, pectinase, lipase and esterase. Approximately 15% of isolates, showing high antagonistic activity, and the production of at least one enzyme were identified through the partial sequencing of 16S rDNA. The genus Bacillus was the most frequently observed, followed by Paenibacillus, Ochrobactrum, Microbacterium and Stenotrophomonas. Proteolytic activity was observed in 24 isolates followed by amylolytic, pectinolytic and cellulolytic activities. No esterase and lipase production was detected. Most of the isolates, showing antagonistic effects against C. gloeosporioides and high enzymatic activities, were isolated from the anthracnose-affected region. A biocontrol method using the endophytes from guarana seeds could be applied in the future, as these bacteria are vertically transferred to guarana seedlings.

An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.

Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism.

Genetic Variability in Puccinia psidii Populations as Revealed by PCR-DGGE and T-RFLP Markers.

Eucalyptus rust caused by Puccinia psidii is responsible for losses of approximately 20% of young Eucalyptus plants, depending on the environmental conditions and the geographic location. Despite its economic importance, there are few studies describing the genetic variability in P. psidii populations that infect different host plants. In the present study, we evaluated the ribosomal DNA internal transcribed spacer region (rDNA-ITS) using polymerase chain reaction denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism to assess the genetic variability in P. psidii populations infecting different Eucalyptus spp. and hybrids, as well as guava, jabuticaba, and syzygium. These culture-independent methods were efficient in differentiating populations based on the host species from which they were collected. In general, the results from both techniques showed that the populations collected from guava, jabuticaba, and syzygium were different from and had a greater level of diversity than the Eucalyptus rust populations. The sequencing of cloned rDNA-ITS fragments confirmed that the vast majority of the profiles generated were from P. psidii. This analysis also revealed interesting single-nucleotide polymorphisms. Therefore, these culture-independent methods are suitable for the rapid assessment of genetic variability within and between populations of this biotrophic fungus on a variety of host species and could be a tool to study the evolution of this pathogen and its interactions with host plants.

Accessing the specialized metabolome of actinobacteria from the bulk soil of Paullinia cupana Mart. on the Brazilian Amazon: a promising source of bioactive compounds against soybean phytopathogens.

The Amazon rainforest, an incredibly biodiverse ecosystem, has been increasingly vulnerable to deforestation. Despite its undeniable importance and potential, the Amazonian microbiome has historically received limited study, particularly in relation to its unique arsenal of specialized metabolites. Therefore, in this study our aim was to assess the metabolic diversity and the antifungal activity of actinobacterial strains isolated from the bulk soil of Paullinia cupana, a native crop, in the Brazilian Amazon Rainforest. Extracts from 24 strains were subjected to UPLC-MS/MS analysis using an integrative approach that relied on the Chemical Structural and Compositional Similarity (CSCS) metric, GNPS molecular networking, and in silico dereplication tools. This procedure allowed the comprehensive understanding of the chemical space encompassed by these actinobacteria, which consists of features belonging to known bioactive metabolite classes and several unannotated molecular families. Among the evaluated strains, five isolates exhibited bioactivity against a panel of soybean fungal phytopathogens (Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). A focused inspection led to the annotation of pepstatins, oligomycins, hydroxamate siderophores and dorrigocins as metabolites produced by these bioactive strains, with potentially unknown compounds also comprising their metabolomes. This study introduces a pragmatic protocol grounded in established and readily available tools for the annotation of metabolites and the prioritization of strains to optimize further isolation of specialized metabolites. Conclusively, we demonstrate the relevance of the Amazonian actinobacteria as sources for bioactive metabolites useful for agriculture. We also emphasize the importance of preserving this biome and conducting more in-depth studies on its microbiota.

Meta-omics integration approach reveals the effect of soil native microbiome diversity in the performance of inoculant Azospirillum brasilense.

Plant growth promoting bacteria (PGPB) have been used as integrative inputs to minimize the use of chemical fertilizers. However, a holistic comprehension about PGPB-plant-microbiome interactions is still incipient. Furthermore, the interaction among PGPB and the holobiont (host-microbiome association) represent a new frontier to plant breeding programs. We aimed to characterize maize bulk soil and rhizosphere microbiomes in irradiated soil (IS) and a native soil (NS) microbial community gradient (dilution-to-extinction) with Azospirillum brasilense Ab-V5, a PGPB commercial inoculant. Our hypothesis was that plant growth promotion efficiency is a result of PGPB niche occupation and persistence according to the holobiont conditions. The effects of Ab-V5 and NS microbial communities were evaluated in microcosms by a combined approach of microbiomics (species-specific qPCR, 16S rRNA metataxonomics and metagenomics) and plant phenomics (conventional and high-throughput methods). Our results revealed a weak maize growth promoting effect of Ab-V5 inoculation in undiluted NS, contrasting the positive effects of NS dilutions 10-3, 10-6, 10-9 and IS with Ab-V5. Alpha diversity in NS + Ab-V5 soil samples was higher than in all other treatments in a time course of 25 days after sowing (DAS). At 15 DAS, alpha diversity indexes were different between NS and IS, but similar in all NS dilutions in rhizospheric samples. These differences were not persistent at 25 DAS, demonstrating a stabilization process in the rhizobiomes. In NS 10-3 +Ab-V5 and NS 10-6 Ab-V5, Ab-V5 persisted in the maize rhizosphere until 15 DAS in higher abundances compared to NS. In NS + Ab-V5, abundance of six taxa were positively correlated with response to (a)biotic stresses in plant-soil interface. Genes involved in bacterial metabolism of riboses and amino acids, and cresol degradation were abundant on NS 10-3 + Ab-V5, indicating that these pathways can contribute to plant growth promotion and might be a result of Ab-V5 performance as a microbial recruiter of beneficial functions to the plant. Our results demonstrated the effects of holobiont on Ab-V5 performance. The meta-omics integration supported by plant phenomics opens new perspectives to better understanding of inoculants-holobiont interaction and for developing better strategies for optimization in the use of microbial products.

Draft Genome Sequence of the Plant Growth Promoter and Biocontrol Agent Bacillus velezensis Strain BIB0110.

Here, we report the draft genome sequence of Bacillus velezensis strain BIB0110, a broad-range biocontrol agent isolated from cultivated eucalyptus in Brazil. The genome has a size of 4.19 Mbp, with a GC content of 45.87%, and it was assembled into 32 scaffolds.

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.

Mechanisms of Mucor sp. CM3 isolated from the aquatic macrophyte Eichhornia crassipes (Mart.) Solms to increase cadmium bioremediation.

Bioremediation of toxic metals is a feasible and low-cost remediation tool to reduce metal contamination. Plant-fungus interactions can improve this technique. Eichhornia crassipes (Mart.) Solms is a macrophyte reported to bioremediate contaminated water. Thus, the present study aimed to isolate endophytic fungi from E. crassipes, select a highly cadmium (Cd) tolerant isolate and evaluate its bioremediation potential. This was evaluated by (1) the fungus tolerance and capacity to accumulate Cd; (2) Cd effects on cell morphology (using SEM and TEM) and on the fungal antioxidant defense system, as well as (3) the effect on model plant Solanum lycopersicum L. cultivar Calabash Rouge, inoculated with the endophyte fungus and exposed to Cd. Our results selected the endophyte Mucor sp. CM3, which was able to tolerate up to 1000 g/L of Cd and to accumulate 900 mg of Cd/g of biomass. Significant changes in Mucor sp. CM3 morphology were observed when exposed to high Cd concentrations, retaining this metal both in its cytoplasm and in its cell wall, which may be linked to detoxification and metal sequestration mechanisms related to the formation of Cd-GSH complexes. In addition, Cd stress induced the activation of all tested antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) - in this endophytic fungus. Moreover, when inoculated in tomato plants, this fungus promoted plant growth (in treatments without Cd) and induced an increased metal translocation to plant shoot, showing its potential to increase metal bioremediation. Therefore, this study indicates that the isolated endophyte Mucor sp. CM3 can be applied as a tool in different plant conditions, improving plant bioremediation and reducing the environmental damage caused by Cd, while also promoting plant growth in the absence of contaminants.

In Planta Study Localizes an Effector Candidate from Austropuccinia psidii Strain MF-1 to the Nucleus and Demonstrates In Vitro Cuticular Wax-Dependent Differential Expression.

Austropuccinia psidii is a biotrophic fungus that causes myrtle rust. First described in Brazil, it has since spread to become a globally important pathogen that infects more than 480 myrtaceous species. One of the most important commercial crops affected by A. psidii is eucalypt, a widely grown forestry tree. The A. psidii-Eucalyptus spp. interaction is poorly understood, but pathogenesis is likely driven by pathogen-secreted effector molecules. Here, we identified and characterized a total of 255 virulence effector candidates using a genome assembly of A. psidii strain MF-1, which was recovered from Eucalyptus grandis in Brazil. We show that the expression of seven effector candidate genes is modulated by cell wax from leaves sourced from resistant and susceptible hosts. Two effector candidates with different subcellular localization predictions, and with specific gene expression profiles, were transiently expressed with GFP-fusions in Nicotiana benthamiana leaves. Interestingly, we observed the accumulation of an effector candidate, Ap28303, which was upregulated under cell wax from rust susceptible E. grandis and described as a peptidase inhibitor I9 domain-containing protein in the nucleus. This was in accordance with in silico analyses. Few studies have characterized nuclear effectors. Our findings open new perspectives on the study of A. psidii-Eucalyptus interactions by providing a potential entry point to understand how the pathogen manipulates its hosts in modulating physiology, structure, or function with effector proteins.

Endophytic Methylobacterium extorquens expresses a heterologous ß-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa.

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.

Opposite Sides of Pantoea agglomerans and Its Associated Commercial Outlook.

Multifaceted microorganisms such as the bacterium Pantoea colonize a wide range of habitats and can exhibit both beneficial and harmful behaviors, which provide new insights into microbial ecology. In the agricultural context, several strains of Pantoea spp. can promote plant growth through direct or indirect mechanisms. Members of this genus contribute to plant growth mainly by increasing the supply of nitrogen, solubilizing ammonia and inorganic phosphate, and producing phytohormones (e.g., auxins). Several other studies have shown the potential of strains of Pantoea spp. to induce systemic resistance and protection against pests and pathogenic microorganisms in cultivated plants. Strains of the species Pantoea agglomerans deserve attention as a pest and phytopathogen control agent. Several of them also possess a biotechnological potential for therapeutic purposes (e.g., immunomodulators) and are implicated in human infections. Thus, the differentiation between the harmful and beneficial strains of P. agglomerans is mandatory to apply this bacterium safely as a biofertilizer or biocontroller. This review specifically evaluates the potential of the strain-associated features of P. agglomerans for bioprospecting and agricultural applications through its biological versatility as well as clarifying its potential animal and human health risks from a genomic point of view.

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5.

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved ß-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

Gene Knockout of Beneficial Plant-associated Bacillus spp. Using the CRISPR-Cas9 Double Plasmid System.

Bacillus spp. have great agricultural potential as a plant growth promoter and biocontrol agent. However, little is known concerning the bacterial molecular basis for the improvement of plant fitness. Thus, it is highly desirable to develop techniques that can contribute to the elucidation of the genetic basis for the mechanisms involved in beneficial bacterium-plant interactions. In this context, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 is a powerful tool based on programmable molecular scissors that perform precise incisions in any DNA sequence. CRISPR-Cas9 can alter gene sequences and constitutes a cutting-edge tool to elucidate the role and function of bacterial genes associated with the benefits of plant interactions. The method described here uses a feasible CRISPR-Cas9 system in a double plasmid, one plasmid harboring the Cas9 endonuclease and the other the sgRNA, to promote gene knockout/editing in the Bacillus genus. This approach favors high efficiency in generating mutants for one or more genes in continuous or multiplex editing. Additionally, due to its universality, it can be applied to genera other than Bacillus.

Fast Screening of Bacteria for Plant Growth Promoting Traits.

Plant Growth Promoting Bacteria (PGPB) are a group of beneficial microorganisms that can positively influence plant fitness and development by improving nutrient acquisition, influencing global plant hormone levels (direct effect), or by reducing the detrimental effects of various pathogens on plant development (indirect effect). The use of PGPB in agriculture as formulated bioinoculants is a potential approach to reduce the negative environmental impacts caused by the continuous application of chemical fertilizers and pesticides. The evaluation of a great number of bacteria in the laboratory for key traits involved in the improvement of plant fitness is a suitable strategy to find prospective candidates for bioinoculants. This chapter presents the main methods described in the literature to quickly screen potential candidates from a bacterial collection to directly and indirectly promote the plant 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.