Potential of efficient and resistant plant growth‐promoting rhizobacteria in lead uptake and plant defence stimulation in Lathyrus sativus under lead stress

• The ability of plant growth‐promoting rhizobacteria (PGPR) to enhance Lathyrus sativus tolerance to lead (Pb) stress was investigated.
• Ten consortia formed by mixing four efficient and Pb‐resistant PGPR strains were assessed for their beneficial effect in improving Pb (0.5 mM) uptake and in inducing the host defence system of L. sativus under hydroponic conditions based on various physiological and biochemical parameters.
• Lead stress significantly decreased shoot (SDW) and root (RDW) dry weight, but PGPR inoculation improved both dry weights, with highest increases in SDW and RDW of plants inoculated with I5 (R. leguminosarum (M5) + P. fluorescens (K23) + Luteibacter sp. + Variovorax sp.) and I9 (R. leguminosarum (M5) + Variovorax sp. + Luteibacter sp. + S. meliloti) by 151% and 94%, respectively. Additionally, inoculation significantly enhanced both chlorophyll and soluble sugar content, mainly in I5 inoculated leaves by 238% and 71%, respectively, despite the fact that Pb decreased these parameters. We also found that PGPR inoculation helps to reduce oxidative damage and enhances antioxidant enzyme activity, phenolic compound biosynthesis, carotenoids and proline content. PGPR inoculation increased Pb uptake in L. sativus, with highest increase in shoots of plants inoculated with I5 and I7, and in roots and nodules of plants inoculated with I1. Moreover, PGPR inoculation enhanced mineral homeostasis for Ca, Cu and Zn under Pb stress, mainly in plants inoculated with I1, I5, I7 and I9.
• Results of our study suggest the potential of efficient and Pb‐resistant PGPR in alleviating harmful effects of metal stress via activation of various defence mechanisms and enhancing Pb uptake that promotes tolerance of L. sativus to Pb stress.

     

ACC deaminase‐containing plant growth‐promoting rhizobacteria protect Papaver somniferum from downy mildew

Aims

Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract.

Methods and Results

A major challenge of using nano‐vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here, we investigated the thermo‐acidophilic archaeal virus SMV1 as a candidate therapeutic nano‐vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (in vitro) and in mice (in vivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labelled SMV1 did not invade or otherwise perturb the human GI tract epithelium.

Conclusion

Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract.

Significance and Impact of the Study

This is the first study evaluating an archaeal virus as a potential therapeutic nanoparticle delivery system and it opens new possibilities for future development of novel nanoplatforms.     

Investigating the mechanisms underlying phytoprotection by plant growth‐promoting rhizobacteria in Spartina densiflora under metal stress

• Pollution of coasts by toxic metals and metalloids is a worldwide problem for which phytoremediation using halophytes and associated microbiomes is becoming relevant. Metal(loid) excess is a constraint for plant establishment and development, and plant growth promoting rhizobacteria (PGPR) mitigate plant stress under these conditions. However, mechanisms underlying this effect remain elusive. The effect of toxic metal(loid)s on activity and gene expression of ROS‐scavenging enzymes in roots of the halophyte Spartina densiflora grown on real polluted sediments in a greenhouse experiment was investigated.
• Sediments of the metal‐polluted joint estuary of Tinto and Odiel rivers and control, unpollutred samples from the Piedras estuary were collected and submitted to ICP‐OES. Seeds of S. densiflora were collected from the polluted Odiel marshes and grown in polluted and unpolluted sediments. Rhizophere biofilm‐forming bacteria were selected based on metal tolerance and inoculated to S. densiflora and grown for 4 months. Fresh or frozen harvested plants were used for enzyme assays and gene expression studies, respectively.
• Metal excess induced SOD (five‐fold increase), whereas CAT and ascorbate peroxidase displayed minor induction (twofold). A twofold increase of TBARs indicated membrane damage. Our results showed that metal‐resistant PGPR (P. agglomerans RSO6 and RSO7 and B. aryabhattai RSO25) contributed to alleviate metal stress, as deduced from lower levels of all antioxidant enzymes to levels below those of non‐exposed plants. The oxidative stress index (OSI) decreased between 50 and 75% upon inoculation.
• The results also evidenced the important role of PAL, involved in secondary metabolism and/or lignin synthesis, as a pathway for metal stress management in this halophyte upon inoculation with appropriate PGPR, since the different inoculation treatments enhanced PAL expression between 3.75‐ and five‐fold. Our data confirm, at the molecular level, the role of PGPR in alleviating metal stress in S. densiflora and evidence the difficulty of working with halophytes for which little genetic information is available.

     

Rapid immuno‐monitoring of inoculant plant growth‐promoting microorganisms

Rapid, specific techniques are essential to monitor the quality of inoculant plant growth‐promoting strains at all stages of manufacture from starter culture to the final product in its carrier medium. In this study, colony immunoblotting was evaluated for the specific detection and enumeration of Citrobacter freundii, one component of a Vietnamese commercial inoculant plant growth‐promoting product used to improve the yield and nutrient efficiency of paddy rice. For quality control of either sterilised or unsterilised carrier media in commercial products colony immunoblotting proved to be a promising tool. Furthermore, it was possible using this technique to measure the survival of this strain in soil and the rhizosphere.     

Effect of salt‐tolerant plant growth‐promoting rhizobacteria on wheat plants and soil health in a saline environment

Salt‐tolerant plant growth‐promoting rhizobacteria (ST‐PGPR) significantly influence the growth and yield of wheat crops in saline soil. Wheat growth improved in pots with inoculation of all nine ST‐PGPR (ECe = 4.3 dS·m^?1; greenhouse experiment), while maximum growth and dry biomass was observed in isolate SU18 Arthrobacter sp.; simultaneously, all ST‐PGPR improved soil health in treated pot soil over controls. In the field experiment, maximum wheat root dry weight and shoot biomass was observed after inoculation with SU44 B. aquimaris, and SU8 B. aquimaris, respectively, after 60 and 90 days. Isolate SU8 B. aquimaris, induced significantly higher proline and total soluble sugar accumulation in wheat, while isolate SU44 B. aquimaris, resulted in higher accumulation of reducing sugars after 60 days. Percentage nitrogen (N), potassium (K) and phosphorus (P) in leaves of wheat increased significantly after inoculation with ST‐PGPR, as compared to un‐inoculated plants. Isolate SU47 B. subtilis showed maximum reduction of sodium (Na) content in wheat leaves of about 23% at both 60 and 90 days after sowing, and produced the best yield of around 17.8% more than the control.     

Role of ethylene and related gene expression in the interaction between strawberry plants and the plant growth‐promoting bacterium Azospirillum brasilense

• Induced systemic resistance (ISR) is one of the indirect mechanisms of growth promotion exerted by plant growth‐promoting bacteria, and can be mediated by ethylene (ET). We assessed ET production and the expression of related genes in the Azospirillum–strawberry plant interaction.
• Ethylene production was evaluated by gas chromatography in plants inoculated or not with A. brasilense REC3. Also, plants were treated with AgNO₃, an inhibitor of ET biosynthesis; with 1‐aminocyclopropane‐1‐carboxylic acid (ACC), a precursor of ET biosynthesis; and with indole acetic acid (IAA). Plant dry biomass and the growth index were determined to assess the growth‐promoting effect of A. brasilense REC3 in strawberry plants. Quantitative real time PCR (qRT‐PCR) was performed to analyse relative expression of the genes Faetr1, Faers1 and Faein4, which encode ET receptors; Factr1 and Faein2, involved in the ET signalling pathway; Faacs1 encoding ACC synthase; Faaco1 encoding ACC oxidase; and Faaux1 and Faami1 for IAA synthesis enzymes.
• Results showed that ET acts as a rapid and transient signal in the first 12 h post‐treatment. A. brasilense REC3‐inoculated plants had a significantly higher growth index compared to control plants. Modulation of the genes Faetr1, Faers1, Faein4, Factr1, Faein2 and Faaco1 indicated activation of ET synthesis and signalling pathways. The up‐regulation of Faaux1 and Faami1 involved in IAA synthesis suggested that inoculation with A. brasilense REC3 induces production of this auxin, modulating ET signalling.
• Ethylene production and up‐regulation of genes associated with ET signalling in strawberry plants inoculated with A. brasilense REC3 support the priming activation characteristic of ISR. This type of resistance and the activation of systemic acquired resistance previously observed in this interaction indicate that both are present in strawberry plants, could act synergistically and increase protection against pathogens.

     

Mitigation of nitrous oxide emissions from acidic soils by Bacillus amyloliquefaciens,a plant growth‐promoting bacterium

Nitrous oxide (N₂O) is a long‐lived greenhouse gas that can result in the alteration of atmospheric chemistry and cause accompanying changes in global climate. To date, many techniques have been used to mitigate the emissions of N₂O from agricultural fields, which represent one of the most important sources of N₂O. In this study, we designed a greenhouse pot experiment and a microcosmic serum bottle incubation experiment using acidic soil from a vegetable farm to study the effects of Bacillus amyloliquefaciens (BA) on plant growth and N₂O emission rates. The addition of BA to the soil promoted plant growth enhanced the soil pH and increased the total nitrogen (TN) contents in the plants. At the same time, it decreased the concentrations of ammonium (NH₄⁺), nitrate (NO₃^?) and TN in the soil. Overall, the addition of BA resulted in a 50% net reduction of N₂O emissions compared with the control. Based on quantitative PCR and the network analysis of DNA sequencing, it was demonstrated that BA partially inhibited the nitrification process through the significant reduction of ammonia oxidizing bacteria. Meanwhile, it enhanced the denitrification process, mainly by increasing the abundance of N₂O‐reducing bacteria in the treatment with BA. The results of our microcosm experiment provided evidence that strongly supported the above findings under more strictly controlled laboratory conditions. Taken together, the results of our study evidently demonstrated that BA has dual effects on the promotion of plant growth and the dramatic reduction of greenhouse emissions, thus suggesting the possibility of screening beneficial microbial organisms from the environment that can promote plant growth and mitigate greenhouse trace gases.     

Biocontrol and plant growth‐promoting activity of rhizobacteria from Chinese fields with contaminated soils

The aim of this study was to inventory the types of plant growth‐promoting rhizobacteria (PGPR) present in the rhizosphere of plants grown in soils contaminated with heavy metals, recalcitrant organics, petroleum sewage or salinity in China. We screened 1223 isolates for antifungal activity and about 24% inhibited Rhizoctonia solani or Sclerotinia sclerotiorum. Twenty‐four strains inhibitory to R. solani, Gaeumannomyces graminis var. tritici and/or S. sclerotiorum and representing the dominant morphotypes were assayed for PGPR activity. Seven strains contained phlD, prnD, pltC or phzF genes and produced the antibiotics 2,4‐diacetylphloroglucinol, pyrrolnitrin, pyoluteorin and phenazines respectively. Six strains contained acdS, which encodes 1‐aminocyclopropane‐1‐carboxylic acid deaminase. Phylogenetic analysis of 16S rDNA and phlD, phzF and acdS genes demonstrated that some strains identified as Pseudomonas were similar to model PGPR strains Pseudomonas protegens Pf‐5, Pseudomonas chlororaphis subsp. aureofaciens 30–84 and P. brassicacearum Q8r1‐96. Pseudomonas protegens‐ and P. chlororaphis‐like strains had the greatest biocontrol activity against Rhizoctonia root rot and take‐all of wheat. Pseudomonas protegens and P. brassicacearum‐like strains showed the greatest promotion of canola growth. Our results indicate that strains from contaminated soils are similar to well‐described PGPR found in agricultural soils worldwide.     

Evaluation of small organic acids present in fast pyrolysis bio‐oil from lignocellulose as feedstocks for bacterial bioconversion

Small organic acids derived from fast pyrolysis of lignocellulosic biomass represent a significant proportion of microbially accessible carbon in bio‐oil. However, using bio‐oil for microbial cultivation is a highly challenging task due to its strong adverse effects on microbial growth as well as its complex composition. In this study, the main small organic acids present in bio‐oil as acetate, formate and propionate were evaluated with respect to their suitability as feedstocks for bacterial growth. For this purpose, the growth behavior of four biotechnological production hosts—Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Corynebacterium glutamicum—was quantified and compared. The bacteria were cultivated on single acids and mixtures of acids in different concentrations and evaluated using common biotechnological efficiency parameters. In addition, cultivation experiments on pretreated fast pyrolysis‐derived bio‐oil fractions were performed with respect to the suitability of the bacterial strains to tolerate inhibitory substances. Results suggest that both P. putida and C. glutamicum metabolize acetate—the major small organic acid generated during fast pyrolysis of lignocellulosic biomass—as sole carbon source over a wide concentration range, are able to grow on mixtures of small organic acids present in bio‐oil and can, to a limited extent, tolerate the highly toxic inhibitory substances within bio‐oil. This work provides an important step in search of suitable bacterial strains for bioconversion of lignocellulosic‐based feedstocks and thus contributes to establishing efficient bioprocesses within a future bioeconomy.     

Role of ethylene signalling in growth and systemic resistance induction by the plant growth‐promoting fungus Penicillium viridicatum in Arabidopsis

The plant growth‐promoting fungi (PGPF) have long been known to improve plant growth and suppress plant diseases. The PGPF Penicillium viridicatum GP15‐1 elicited plant growth and induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. Examination of local and systemic genes indicated that GP15‐1 did not modulate the expression of any of the tested defence‐related marker genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene signalling pathways. Subsequent challenge of GP15‐1‐colonized plants with Pst bacterium primed Arabidopsis plants for enhanced activation of the JA‐inducible Atvsp (vegetative storage protein) gene at a later stage of infection. To assess the contribution of different signalling pathways in GP15‐1‐elicited plant growth and ISR, Arabidopsis genotypes implicated in SA signalling expressing the nahG transgene (NahG) or carrying disruption in NPR1 (npr1), JA signalling (jar1) and ethylene signalling (ein2) were tested. The GP15‐1‐induced plant growth and ISR were fully compromised in an ein2 mutation. Root colonization assay revealed that the inability of the ein2 mutant to express GP15‐1‐induced plant growth and ISR was not associated with reduced root colonization by GP15‐1. In conclusion, our results demonstrate the ethylene signalling pathway is involved in plant growth promotion and ISR elicitation by the PGPF P. viridicatum GP15‐1 in Arabidopsis. These results provide evidence that ethylene signalling has a substantial role in plant growth and disease resistance.     

Elemental composition of strawberry plants inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, assessed with scanning electron microscopy and energy dispersive X‐ray analysis

The elemental composition of strawberry plants (Fragaria ananassa cv. Macarena) inoculated with the plant growth‐promoting bacterium Azospirillum brasilense REC3, and non‐inoculated controls, was studied using scanning electron microscopy (SEM) and energy dispersive X‐ray (EDS) analysis. This allowed simultaneous semi‐quantification of different elements in a small, solid sample. Plants were inoculated and grown hydroponically in 50% or 100% Hoagland solution, corresponding to limited or optimum nutrient medium, respectively. Bacteria‐inoculated plants increased the growth index 45% and 80% compared to controls when grown in 100% and 50% Hoagland solution, respectively. Thus, inoculation with A. brasilense REC3 in a nutrient‐limited medium had the strongest effect in terms of increasing both shoot and root biomass and growth index, as already described for Azospirillum inoculated into nutrient‐poor soils. SEM‐EDS spectra and maps showed the elemental composition and relative distribution of nutrients in strawberry tissues. Leaves contained C, O, N, Na, P, K, Ca and Cu, while roots also had Si and Cl. The organic fraction (C, O and N) accounted for over 96.3% of the total chemical composition; of the mineral fraction, Na had higher accumulation in both leaves and roots. Azospirillum‐inoculated and control plants had similar elemental quantities; however, in bacteria‐inoculated roots, P was significantly increased (34.33%), which constitutes a major benefit for plant nutrition, while Cu content decreased (35.16%).     

Evidence for species‐specific plant responses to soil microbial communities from remnant and degraded land provides promise for restoration

Below‐ground interactions between soil microbial communities and plants play important roles in shaping plant community structure, but are currently poorly understood. Understanding these processes has important practical implications, including for restoration. In this study, we investigated whether soil microbes from remnant areas can aid the restoration of old‐fields, and whether soil microbes from an old‐field encourages further invasive establishment. In a glasshouse experiment, we measured growth and survival of two native grasses (Austrostipa nodosa and Rytidosperma auriculatum) and an invasive grass (Lolium rigidum) grown in sterile soil inoculated with whole soil from three locations: an old‐field, a remnant grassland, and a seed orchard planted with native grasses 7 years ago. Plants grown in sterile, non‐inoculated soil acted as controls. The orchard inoculant was included to test whether soil microbes from an area cultivated with native grasses induced plant responses similar to remnant areas. The remnant treatment resulted in the highest biomass and no mortality for R. auriculatum. All inoculant types increased the biomass of the invasive species equally. The native grass, A. nodosa, was the most sensitive to the addition of inoculum, whereas the invasive L. rigidum suffered very low mortality across all treatments. Overall, mortality was highest in the old‐field treatment at 42.9%. These results give insights into how soil microbes can affect community structure and dynamics, e.g. the high mortality of natives with old‐field inoculant may be one mechanism that allows invasive species to dominate. Poorer performance of native species with the orchard inoculant suggests it would not make a suitable replacement for remnant soil; therefore, more work is needed to understand the requirements of target species and their interactions before this technique can be exploited to maximum benefit.     

Efficient distinction of invasive aquatic plant species from non‐invasive related species using DNA barcoding

Biological invasions are regarded as threats to global biodiversity. Among invasive aliens, a number of plant species belonging to the genera Myriophyllum, Ludwigia and Cabomba, and to the Hydrocharitaceae family pose a particular ecological threat to water bodies. Therefore, one would try to prevent them from entering a country. However, many related species are commercially traded, and distinguishing invasive from non‐invasive species based on morphology alone is often difficult for plants in a vegetative stage. In this regard, DNA barcoding could become a good alternative. In this study, 242 samples belonging to 26 species from 10 genera of aquatic plants were assessed using the chloroplast loci trnH‐psbA, matK and rbcL. Despite testing a large number of primer sets and several PCR protocols, the matK locus could not be amplified or sequenced reliably and therefore was left out of the analysis. Using the other two loci, eight invasive species could be distinguished from their respective related species, a ninth one failed to produce sequences of sufficient quality. Based on the criteria of universal application, high sequence divergence and level of species discrimination, the trnH‐psbA noncoding spacer was the best performing barcode in the aquatic plant species studied. Thus, DNA barcoding may be helpful with enforcing a ban on trade of such invasive species, such as is already in place in the Netherlands. This will become even more so once DNA barcoding would be turned into machinery routinely operable by a nonspecialist in botany and molecular genetics.