Thermal and salt stress effects on the survival of plant growth-promoting bacteria Azospirillum brasilense in inoculants for maize cultivation.

BACKGROUND: The plant growth-promoting bacteria (PGPB) Azospirillum brasilense is widely used as an inoculant for important grass crops, providing numerous benefits to the plants. However, one limitation to develop viable commercial inoculants is the control of PGPB survival, requiring strategies that guarantee their survival during handling and field application. The application of sublethal stress appears to be a promising strategy to increase bacterial cells tolerance to adverse environmental conditions since previous stress induces the activation of physiological protection in bacterial cell. In this work, we evaluated the effects of thermal and salt stresses on the survival of inoculant containing A. brasilense Ab-V5 and Ab-V6 strains and we monitored A. brasilense viability in inoculated maize roots after stress treatment of inoculant. RESULTS: Thermal stress application (> 35 °C) in isolated cultures for both strains, as well as salt stress [sodium chloride (NaCl) concentrations > 0.3 mol L-1 ], resulted in growth rate decline. The A. brasilense enumeration in maize roots obtained by propidium monoazide quantitative polymerase chain reaction (PMA-qPCR), for inoculated maize seedlings grown in vitro for 7 days, showed that there is an increased number of viable cells after the salt stress treatment, indicating that A. brasilense Ab-V5 and Ab-V6 strains are able to adapt to salt stress (0.3 mol L-1 NaCl) growth conditions. CONCLUSION: Azospirillum brasilense Ab-V5 and Ab-V6 strains had potential for osmoadaptation and salt stress, resulting in increased cell survival after inoculation in maize plants. © 2024 Society of Chemical Industry.

Effects of sublethal stress application on the survival of bacterial inoculants: a systematic review.

The use of commercial bacterial inoculants formulated with plant-growth promoting bacteria (PGPB) in agriculture has shown significant prominence in recent years due to growth-promotion benefits provided to plants through different mechanisms. However, the survival and viability of bacterial cells in inoculants are affected during use and may decrease their effectiveness. Physiological adaptation strategies have attracted attention to solve the viability problem. This review aims to provide an overview of research on selecting sublethal stress strategies to increase the effectiveness of bacterial inoculants. The searches were performed in November 2021 using Web of Science, Scopus, PubMed, and Proquest databases. The keywords "nitrogen-fixing bacteria", "plant growth-promoting rhizobacteria", "azospirillum", "pseudomonas", "rhizobium", "stress pre-conditioning", "adaptation", "metabolic physiological adaptation", "cellular adaptation", "increasing survival", "protective agent" and "protective strategy" were used in the searches. A total of 2573 publications were found, and 34 studies were selected for a deeper study of the subject. Based on the studies analysis, gaps and potential applications related to sublethal stress were identified. The most used strategies included osmotic, thermal, oxidative, and nutritional stress, and the primary cell response mechanism to stress was the accumulation of osmolytes, phytohormones, and exopolysaccharides (EPS). Under sublethal stress, the inoculant survival showed positive increments after lyophilization, desiccation, and long-term storage processes. The effectiveness of inoculant-plants interaction also had positive increments after sublethal stress, improving plant development, disease control, and tolerance to environmental stresses compared to unappealed inoculants.

Distribution of Genes Related to Probiotic Effects Across Lacticaseibacillus rhamnosus Revealed by Population Structure.

The Gram-positive Lacticaseibacillus rhamnosus has been broadly reported as capable of exerting beneficial health effects. Bacterial genomic diversity may promote niche specialization, thus creating subpatterns within populations. As L. rhamnosus advantageous effects have been widely reported at strain level and few is known regarding the distribution of beneficial genes among L. rhamnosus strains, we investigated all publicly available genomes of Lactobacillus and Lacticaseibacillus genera to study the pangenome and general population structure of L. rhamnosus. Core genome multilocus sequence typing detected eight L. rhamnosus phylogroups (PG1 to PG8). L. rhamnosus harbors an open pangenome; PG1, PG3, PG4, and PG5 exhibited highly conserved gene distribution patterns. Genes significantly associated to the PG1, which comprises L. rhamnosus GG, are mainly phage-related. The adhesion operon spaCBA-srtC1 was found in 44 (24.7%) genomes; however, considering only the PG1, the prevalence was of 65%. In PG2 the spaCBA-srtC1 prevalence was of 43%. Nevertheless, both human and milk-derived strains harbored this operon. Further, two main types of bacteriocin clusters were found (Bact1 and Bact2). Bact1 predictions indicate the presence of garQ, encoding the class II bacteriocin garvieacin Q, that is mainly present in the closely related PG8A and a PG2 subcluster. PG2 harbors two distinct subclusters, harboring either spaCBA-srtC1 or Bact1. Our findings provide novel insights on the distribution of biotechnological relevant genes across L. rhamnosus population, uncovering intra-species patterns that may bring forth the development of more efficient probiotic products.

Comparative Genomics Reveal the High Conservation and Scarce Distribution of Nitrogen Fixation nif Genes in the Plant-Associated Genus Herbaspirillum.

The genus Herbaspirillum gained the spotlight due to the several reports of diazotrophic strains and promising results in plant-growth field assays. However, as diversity exploration of Herbaspirillum species gained momentum, it became clearer that the plant beneficial lifestyle was not the only form of ecological interaction in this genus, due to reports of phytopathogenesis and nosocomial infections. Here we performed a deep search across all publicly available Herbaspirillum genomes. Using a robust core genome phylogeny, we have found that all described species are well delineated, being the only exception H. aquaticum and H. huttiense clade. We also uncovered that the nif genes are only highly prevalent in H. rubrisubalbicans; however, irrespective to the species, all nif genes share the same gene arrangement with high protein identity, and are present in only two main types, in inverted strands. By means of a NifHDKENB phylogenetic tree, we have further revealed that the Herbaspirillum nif sequences may have been acquired from the same last common ancestor belonging to the Nitrosomonadales order.

Inoculation of Herbaspirillum seropedicae strain SmR1 increases biomass in maize roots DKB 390 variety in the early stages of plant development.

Herbaspirillum seropedicae is a plant growth-promoting bacteria isolated from diverse plant species. In this work, the main objective was to investigate the efficiency of H. seropedicae strain SmR1 in colonizing and increasing maize growth (DKB 390 variety) in the early stages of development under greenhouse conditions. Inoculation with H. seropedicae resulted in 19.43 % (regarding High and Low N controls) and 10.51% (regarding Low N control) in mean of increase of root biomass, for 1st and 2nd greenhouse experiments, respectively, mainly in the initial stages of plant development, at 21 days after emergence (DAE). Quantification of H. seropedicae in roots and leaves was performed by quantitative PCR. H. seropedicae was detected only in maize inoculated roots by qPCR, and a slight decrease in DNA copy number g-1 of fresh root weight was observed from 7 to 21 DAE, suggesting that there was initial effective colonization on maize plants. H. seropedicae strain SmR1 efficiently increased maize root biomass exhibiting its potential to be used as inoculant in agricultures systems.

High-added value co-products obtained from pecan nut (Carya illinoinensis) using a green extraction technology.

Abstract: The aqueous (AF) and solid (SF) fractions obtained as co-products in the aqueous extraction of pecan nut oil assisted by Alcalase® were evaluated. In the AF, the degree of protein hydrolysis (DH) and the electrophoretic profile of protein hydrolysates, phenolic compounds, and antioxidant capacity (reducing potential of the hydrophilic compounds, RPHC, 2,2-diphenyl-1-picrylhydrazyl, DPPH; and inhibition of lipid peroxidation) were determined. The proximate composition and microstructure were evaluated in SF. The results indicated a DH of 3.9%. The sample treated with the enzyme (ET) showed a molecular weight of proteins lower than 15 kDa. The ET showed higher content of phenolics (726.3 mg GAE/100 g) and antioxidant capacity higher than the sample without enzymatic treatment. The SF showed a residual lipid content rich in oleic and linoleic acids. Furthermore, changes in the proximate composition and the microstructure were observed. The results indicate the potentiality of hydrolyzed fractions for application in food.

Bifidobacterium animalis ssp. lactis BB-12 enumeration by quantitative PCR assay in microcapsules with full-fat goat milk and inulin-type fructans.

The current study was conducted to develop a quantitative polymerase chain reaction (qPCR) assay for Bifidobacterium animalis ssp. lactis BB-12 quantification in microcapsules matrix with full-fat goat milk and inulin-type fructans. DNA was isolated from milk, feed solutions (before spray drying) and microcapsules (after spray drying) using DNAzol. Two primer pairs targeting Bal-23S or Tuf sequences were evaluated by qPCR. The qPCR efficiency was higher (89.5%) using the Tuf primers than Bal-23S primers (84.8%). Tuf primer pair was able to selectively detect B. animalis ssp. lactis BB-12. After, quantification of bifidobacteria in the microcapsules matrix by Tuf qPCR assay was compared to conventional enumeration by plate counting. The analysis of probiotic feed solutions and microcapsules showed higher (P < 0.05) bacterial enumeration determined by Tuf qPCR assay compared to those obtained by plate counting. This qPCR assay was considered a rapid and sensitive alternative for the quantification of B. animalis ssp. lactis BB-12 in probiotic microcapsules compared to plate counting.

Azospirillum brasilense viable cells enumeration using propidium monoazide-quantitative PCR.

Azospirillum brasilense is a plant growth promoting bacteria used as an inoculant in diverse crops. Accurate analytical methods are required to enumerate viable cells in inoculant formulations or in planta. We developed a quantitative polymerase chain reaction (qPCR) assay associated to propidium monoazide (PMA) to evaluate the cell viability of A. brasilense in inoculant and in maize roots. A. brasilense was grown in culture medium and was exposed to 50 â„ƒ. Maize roots were grown in vitro and harvested 7 days after inoculation. Quantification was performed by qPCR, PMA-qPCR, and plate counting. Standard curves efficiency values ranged from 85 to 99%. The limit of detection was 104 CFU per gram of fresh root. Enumeration obtained in maize roots by qPCR where higher than enumeration by PMA-qPCR and by plate counting. PMA-qPCR assay was efficient in quantifying inoculant viable cells and provides reliable results in a quickly and accurately way compared to culture-dependent methods.

Extraction of antifreeze proteins from cold acclimated leaves of Drimys angustifolia and their application to star fruit (Averrhoa carambola) freezing.

Antifreeze proteins (AFPs) have the ability to modify ice crystal growth and thus there is great interest in identifying new sources of these compounds. All extracts of cold acclimated leaves of Drimys angustifolia, obtained using different buffers, inhibited recrystallization and they presented similar SDS-PAGE profiles, with bands close to 20 and 37 kDa. The extract obtained using Tris-HCl/DDT buffer (pH 8) was used in the pre-treatment of frozen star fruit (Averrhoa carambola) by immersion or vacuum infiltration. The treatments did not affect the titratable acidity, pH, soluble solids, ascorbic acid content and colour. However, only star fruits that were vacuum infiltrated with AFPs retained their drip loss constant after 15 days. Moreover, with this treatment the star fruit firmness was maintained on thawing after 60 days of storage. The vacuum infiltration of Drimys angustifolia AFPs into the star fruit allowed an initial cryoprotection, indicating that the application of AFPs can increase the quality of frozen star fruit.

Herbaspirillum seropedicae promotes maize growth but fails to control the maize leaf anthracnose.

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium and a plant growth promoting bacteria. Colletotrichum graminicola causes the anthracnose, one of the most destructive maize diseases worldwide. The main objective of this work was to evaluate the effects of H. seropedicae SmR1 strain on the plant growth and leaf anthracnose of maize plants grown in substrate amended or not amended with humic substances. In the first assay, plants were pre-treated with H. seropedicae and inoculated with C. graminicola at 7, 14 and 21 days after treatment (DAT). In the second assay, plants were treated with H. seropedicae, grown in substrate amended with humic substances and inoculated at 3 and 7 DAT. The anthracnose severity was assessed by measurement of necrotic and chlorotic leaf area, and bacteria were quantified in leaves by quantitative PCR. H. seropedicae did not affect the disease severity in maize leaves, although it efficiently colonized the leaf tissues and it promoted maize leaf growth. Humic substances improved H. seropedicae colonization in maize.

Quantification of Lactobacillus paracasei viable cells in probiotic yoghurt by propidium monoazide combined with quantitative PCR.

Propidium monoazide (PMA) coupled with qPCR has been successfully used for specific quantification of viable bacteria cells in diverse matrices food. The present study aimed to develop PMA-qPCR assay for quantification of Lactobacillus paracasei viable cells in probiotic yoghurt. L. paracasei grown in culture medium was submitted to heat treatment at 60°C for different periods of time and probiotic yoghurt containing L. paracasei were prepared and stored at 4°C for 30days. The viable cells were quantified using qPCR and PMA-qPCR assays targeting tuf gene and also by plate counting. Standard curves were prepared and mean efficiency obtained was 94% and 96% (R2>0.98) to L. paracasei in culture medium and probiotic yoghurt stored one day, respectively. The limit of detection (LOD) for both samples was 104 genome copies, corresponding to 32.1pg of DNA. For viable cells quantification, standard curves Cq versus log CFU were plotted using mean CFU by plate counting of L. paracasei grown in culture medium and probiotic yoghurt. Results obtained for L. paracasei heat-treated cells were concordant by PMA-qPCR and plate count, CFU decreased as the heat treatment time increased, while qPCR count remained constant. L. paracasei enumerations obtained by qPCR for probiotic yoghurt stored one day and 30days were higher than enumerations by PMA-qPCR for the same samples. The plate count values were similar to CFU values obtained by PMA-qPCR. These results showed that PMA-qPCR is a powerful approach compared with culture-dependent methods for quantification of L. paracasei viable cells in yoghurt. PMA-qPCR allowed reliable obtained results much faster than plate counting.

Comparison of Grain Proteome Profiles of Four Brazilian Common Bean (Phaseolus vulgaris L.) Cultivars.

Common bean (Phaseolus vulgaris L.) is a source of proteins for about one billion people worldwide. In Brazil, 'BRS Sublime', 'BRS Vereda', 'BRS Esteio', and 'BRS Estilo' cultivars were developed by Embrapa to offer high yield to farmers and excellent quality to final consumers. In this work, grain proteomes of these common bean cultivars were compared based on two-dimensional gel electrophoresis (2-DE) and tandem mass spectrometry (MS/MS). Principal component analysis (PCA) was applied to compare 349 matched spots in these cultivars proteomes, and all cultivars were clearly separated in PCA plot. Thirty-two differentially accumulated proteins were identified by MS. Storage proteins such as phaseolins, legumins, and lectins were the most abundant, and novel proteins were also identified. We have built a useful platform that could be used to analyze other Brazilian cultivars and genotypes of common beans.

Proteome comparison for discrimination between honeydew and floral honeys from botanical species Mimosa scabrella Bentham by principal component analysis.

BACKGROUND: Due to the increasing valuation and appreciation of honeydew honey in many European countries and also to existing contamination among different types of honeys, authentication is an important aspect of quality control with regard to guaranteeing the origin in terms of source (honeydew or floral) and needs to be determined. Furthermore, proteins are minor components of the honey, despite the importance of their physiological effects, and can differ according to the source of the honey. In this context, the aims of this study were to carry out protein extraction from honeydew and floral honeys and to discriminate these honeys from the same botanical species, Mimosa scabrella Bentham, through proteome comparison using two-dimensional gel electrophoresis and principal component analysis. RESULTS: The results showed that the proteome profile and principal component analysis can be a useful tool for discrimination between these types of honey using matched proteins (45 matched spots). Also, the proteome profile showed 160 protein spots in honeydew honey and 84 spots in the floral honey. CONCLUSION: The protein profile can be a differential characteristic of this type of honey, in view of the importance of proteins as bioactive compounds in honey. © 2017 Society of Chemical Industry.

Tuber proteome comparison of five potato varieties by principal component analysis.

BACKGROUND: Data analysis of omics data should be performed by multivariate analysis such as principal component analysis (PCA). The way data are clustered in PCA is of major importance to develop some classification systems based on multivariate analysis, such as soft independent modeling of class analogy (SIMCA). In a previous study a one-class classifier based on SIMCA was built using microarray data from a set of potatoes. The PCA grouped the transcriptomic data according to varieties. The present work aimed to use PCA to verify the clustering of the proteomic profiles for the same potato varieties. RESULTS: Proteomic profiles of five potato varieties (Biogold, Fontane, Innovator, Lady Rosetta and Maris Piper) were evaluated by two-dimensional gel electrophoresis (2-DE) performed on two immobilized pH gradient (IPG) strip lengths, 13 and 24 cm, both under pH range 4-7. For each strip length, two gels were prepared from each variety; in total there were ten gels per analysis. For 13 cm strips, 199-320 spots were detected per gel, and for 24 cm strips, 365-684 spots. CONCLUSION: All four PCAs performed with these datasets presented clear grouping of samples according to the varieties. The data presented here showed that PCA was applicable for proteomic analysis of potato and was able to separate the samples by varieties. © 2016 Society of Chemical Industry.

Expressed proteins of Herbaspirillum seropedicae in maize (DKB240) roots-bacteria interaction revealed using proteomics.

Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.

Safety assessment of plant varieties using transcriptomics profiling and a one-class classifier.

An important part of the current hazard identification of novel plant varieties is comparative targeted analysis of the novel and reference varieties. Comparative analysis will become much more informative with unbiased analytical approaches, e.g. omics profiling. Data analysis estimating the similarity of new varieties to a reference baseline class of known safe varieties would subsequently greatly facilitate hazard identification. Further biological and eventually toxicological analysis would then only be necessary for varieties that fall outside this reference class. For this purpose, a one-class classifier tool was explored to assess and classify transcriptome profiles of potato (Solanum tuberosum) varieties in a model study. Profiles of six different varieties, two locations of growth, two year of harvest and including biological and technical replication were used to build the model. Two scenarios were applied representing evaluation of a 'different' variety and a 'similar' variety. Within the model higher class distances resulted for the 'different' test set compared with the 'similar' test set. The present study may contribute to a more global hazard identification of novel plant varieties.

Genetic mapping of semi-polar metabolites in pepper fruits (Capsicum sp.): towards unravelling the molecular regulation of flavonoid quantitative trait loci.

Untargeted LCMS profiling of semi-polar metabolites followed by metabolite quantitative trait locus (mQTL) analysis was performed in ripe pepper fruits of 113 F2 plants derived from a cross between Capsicum annuum AC1979 (no. 19) and Capsicum chinense No. 4661 Selection (no. 18). The parental accessions were selected based on their variation in fruit morphological characteristics and fruit content of some target phytonutrients. Clear segregation of fruit colour and fruit metabolite profiles was observed in the F2 population. The F2 plants formed three clusters based on their metabolite profiles. Of the total of 542 metabolites, 52 could be annotated, including a range of flavonoids, such as flavone C-glycosides, flavonol O-glycosides and naringenin chalcone, as well as several phenylpropanoids, a capsaicin analogue, fatty acid derivatives and amino acid derivatives. Interval mapping revealed 279 mQTLs in total. Two mQTL hotspots were found on chromosome 9. These two chromosomal regions regulated the relative levels of 35 and 103 metabolites, respectively. Analysis also revealed an mQTL for a capsaicin analogue, located on chromosome 7. Confirmation of flavonoid mQTLs using a set of six flavonoid candidate gene markers and their corresponding expression data (expression QTLs) indicated the Ca-MYB12 transcription factor gene on chromosome 1 and the gene encoding flavone synthase (FS-2) on chromosome 6 as likely causative genes determining the variation in naringenin chalcone and flavone C-glycosides, respectively, in this population. The combination of large-scale metabolite profiling and QTL analysis provided valuable insight into the genomic regions and genes important for the production of (secondary) metabolites in pepper fruit. This will impact breeding strategies aimed at optimising the content of specific metabolites in pepper fruit.

Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing.

The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

Real-time PCR quantification of the plant growth promoting bacteria Herbaspirillum seropedicae strain SmR1 in maize roots.

The plant growth promoting bacteria Herbaspirillum seropedicae SmR1 is an endophytic diazotroph found in several economically important crops. Considering that methods to monitor the plant-bacteria interaction are required, our objective was to develop a real-time PCR method for quantification of PGPB H. seropedicae in the rhizosphere of maize seedlings. Primer pairs were designed, and their specificity was verified using DNA from 12 different bacterial species. Ten standard curves of qPCR assay using HERBAS1 primers and tenfold serial dilutions of H. seropedicae SmR1 DNA were performed, and PCR efficiency of 91 % and correlation coefficient of 0.99 were obtained. H. seropedicae SmR1 limit of detection was 10(1) copies (corresponding to 60.3 fg of bacterial DNA). qPCR assay using HERBAS1 was used to detect and quantify H. seropedicae strain SmR1 in inoculated maize roots, cultivated in vitro and in pots, harvested 1, 4, 7, and 10 days after inoculation. The estimated bacterial DNA copy number per gram of root was in the range 10(7)-10(9) for plants grown in vitro and it was around 10(6) for plants grown in pots. Primer pair HERBAS1 was able to quantify H. seropedicae SmR1, and this assay can be useful for monitoring plant-bacteria interaction.

A high-throughput method for GMO multi-detection using a microfluidic dynamic array.

The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.