Compost and microbial biostimulant applications improve plant growth and soil biological fertility of a grass-based phytostabilization system.

In this work, a grass-based phytoremediation system integrated with an organic amendment and biostimulants was evaluated for remediating contaminated sites. Plant growth and biological fertility were monitored to assess the efficacy of a vegetative cap used as a safety measure to reduce sanitary and environmental risks of industrially contaminated soils and soil-washing sludges. Both matrices were potentially contaminated with Pb and Zn with an ecological risk index from low to moderate. According to potentially toxic elements (PTEs) bioaccessibility tests, the exposure to the released fine particulate matter may cause serious risks to human beings, in particular to children. The grass mixture was well adapted to both the substrates and a low PTEs mobility was detected, thus, reducing the leaching risk to ground water sources. Compost addition augmented significantly nitrogenase reductase (nifH) and ammonia monooxygenase (amoA) gene expression abundance in both substrates. Furthermore, a positive interaction between compost fertilization and a Trichoderma-based biostimulant inoculation was recorded in sludges resulting in a significant stimulation of nitrogen-fixing and ammonia-oxidizing bacteria. The application of compost and biostimulant increased soil fertility and plant growth. Furthermore, there was a slight reduction in PTE bioaccessibility, thus, improving the efficiency of the phytostabilization, limiting the resuspension and dispersion of the health-risk soil particulate.

Root inoculation with Azotobacter chroococcum 76A enhances tomato plants adaptation to salt stress under low N conditions.

BACKGROUND: The emerging roles of rhizobacteria in improving plant nutrition and stress protection have great potential for sustainable use in saline soils. We evaluated the function of the salt-tolerant strain Azotobacter chroococcum 76A as stress protectant in an important horticultural crop, tomato. Specifically we hypothesized that treatment of tomato plants with A. chroococcum 76A could improve plant performance under salinity stress and sub-optimal nutrient regimen. RESULTS: Inoculation of Micro Tom tomato plants with A. chroococcum 76A increased numerous growth parameters and also conferred protective effects under both moderate (50 mM NaCl) and severe (100 mM NaCl) salt stresses. These benefits were mostly observed under reduced nutrient regimen and were less appreciable in optimal nitrogen conditions. Therefore, the efficiency of A. chroococcum 76A was found to be dependent on the nutrient status of the rhizosphere. The expression profiles of LEA genes indicated that A. chroococcum 76A treated plants were more responsive to stress stimuli when compared to untreated controls. However, transcript levels of key nitrogen assimilation genes revealed that the optimal nitrogen regimen, in combination with the strain A. chroococcum 76A, may have saturated plant's ability to assimilate nitrogen. CONCLUSIONS: Roots inoculation with A. chroococcum 76A tomato promoted tomato plant growth, stress tolerance and nutrient assimilation efficiency under moderate and severe salinity. Inoculation with beneficial bacteria such as A. chroococcum 76A may be an ideal solution for low-input systems, where environmental constraints and limited chemical fertilization may affect the potential yield.

The effect of bacterial and archaeal populations on anaerobic process fed with mozzarella cheese whey and buttermilk.

Dairy wastes can be conveniently processed and valorized in a biorefinery value chain since they are abundant, zero-cost and all year round available. For a comprehensive knowledge of the microbial species involved in producing biofuels and valuable intermediates from dairy wastes, the changes in bacterial and archaeal population were evaluated when H2, CH4 and chemical intermediates were produced. Batch anaerobic tests were conducted with a mixture of mozzarella cheese whey and buttermilk as organic substrate, inoculated with 1% and 3% w/v industrial animal manure pellets. The archaeal methanogens concentration increased in the test inoculated at 3% (w/v) when H2 and CH4 production occurred, being 1 log higher than that achieved in the test inoculated at 1% (w/v). Many archaeal species, mostly involved in the production of CH4, were identified by sequencing denaturing gradient gel electrophoresis (DGGE) bands. Methanoculleus, Methanocorpusculum and Methanobrevibacter genera were dominant archaea involved in the anaerobic process for bioenergy production from mozzarella cheese whey and buttermilk mixture.

Bioreactors for lignocellulose conversion into fermentable sugars for production of high added value products.

Lignocellulosic biomasses derived from dedicated crops and agro-industrial residual materials are promising renewable resources for the production of fuels and other added value bioproducts. Due to the tolerance to a wide range of environments, the dedicated crops can be cultivated on marginal lands, avoiding conflict with food production and having beneficial effects on the environment. Besides, the agro-industrial residual materials represent an abundant, available, and cheap source of bioproducts that completely cut out the economical and environmental issues related to the cultivation of energy crops. Different processing steps like pretreatment, hydrolysis and microbial fermentation are needed to convert biomass into added value bioproducts. The reactor configuration, the operative conditions, and the operation mode of the conversion processes are crucial parameters for a high yield and productivity of the biomass bioconversion process. This review summarizes the last progresses in the bioreactor field, with main attention on the new configurations and the agitation systems, for conversion of dedicated energy crops (Arundo donax) and residual materials (corn stover, wheat straw, mesquite wood, agave bagasse, fruit and citrus peel wastes, sunflower seed hull, switchgrass, poplar sawdust, cogon grass, sugarcane bagasse, sunflower seed hull, and poplar wood) into sugars and ethanol. The main novelty of this review is its focus on reactor components and properties.

Chestnut green waste composting for sustainable forest management: Microbiota dynamics and impact on plant disease control.

Making compost from chestnut lignocellulosic waste is a possible sustainable management strategy for forests that employs a high-quality renewable organic resource. Characterization of the microbiota involved in composting is essential to better understand the entire process as well as the properties of the final product. Therefore, this study investigated the microbial communities involved in the composting of chestnut residues obtained from tree cleaning and pruning. The culture-independent approach taken highlighted the fact that the microbiota varied only slightly during the process, with the exception of those of the starting substrate and mature compost. The statistical analysis indicated that most of the bacterial and fungal species in the chestnut compost persisted during composting. The dominant microbial population detected during the process belonged to genera known to degrade recalcitrant lignocellulosic materials. Specifically, we identified fungal genera, such as Penicillium, Fusarium, Cladosporium, Aspergillus and Mucor, and prokaryotic species affiliated with Bacilli, Actinobacteria, Flavobacteria and γ-Proteobacteria. The suppressive properties of compost supplements for the biocontrol of Sclerotinia minor and Rhizoctonia solani were also investigated. Compared to pure substrate, the addition of compost to the peat-based growth substrates resulted in a significant reduction of disease in tomato plants of up to 70 % or 51 % in the presence of Sclerotinia minor or Rhizoctonia solani, respectively. The obtained results were related to the presence of putative bio-control agents and plant growth-promoting rhizobacteria belonging to the genera Azotobacter, Pseudomonas, Stenotrophomonas, Bacillus, Flavobacterium, Streptomyces and Actinomyces in the chestnut compost. The composting of chestnut waste may represent a sustainable agricultural practice for disposing of lignocellulosic waste by transforming it into green waste compost that can be used to improve the fitness of agricultural plants.

The effect of Pleurotus ostreatus arabinofuranosidase and its evolved variant in lignocellulosic biomasses conversion.

The fungal arabinofuranosidase from Pleurotus ostreatus PoAbf recombinantly expressed in Pichia pastoris rPoAbf and its evolved variant rPoAbf F435Y/Y446F were tested for their effectiveness to enhance the enzymatic saccharification of three lignocellulosic biomasses, namely Arundo donax, corn cobs and brewer's spent grains (BSG), after chemical or chemical-physical pretreatment. All the raw materials were subjected to an alkaline pretreatment by soaking in aqueous ammonia solution whilst the biomass from A. donax was also pretreated by steam explosion. The capability of the wild-type and mutant rPoAbf to increase the fermentable sugars recovery was assessed by using these enzymes in combination with different (hemi)cellulolytic activities. These enzymatic mixtures were either entirely of commercial origin or contained the cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli in substitution to the commercial counterparts. The addition of the arabinofuranosidases from P. ostreatus improved the hydrolytic efficiency of the commercial enzymatic cocktails on all the pretreated biomasses. The best results were obtained using the rPoAbf evolved variant and are represented by increases of the xylose recovery up to 56.4%. These data clearly highlight the important role of the accessory hemicellulolytic activities to optimize the xylan bioconversion yields.

Methylobacterium populi VP2: plant growth-promoting bacterium isolated from a highly polluted environment for polycyclic aromatic hydrocarbon (PAH) biodegradation.

The use of microorganisms to accelerate the natural detoxification processes of toxic substances in the soil represents an alternative ecofriendly and low-cost method of environmental remediation compared to harmful incineration and chemical treatments. Fourteen strains able to grow on minimal selective medium with a complex mixture of different classes of xenobiotic compounds as the sole carbon source were isolated from the soil of the ex-industrial site ACNA (Aziende Chimiche Nazionali Associate) in Cengio (Savona, Italy). The best putative degrading isolate, Methylobacterium populi VP2, was identified using a polyphasic approach on the basis of its phenotypic, biochemical, and molecular characterisation. Moreover, this strain also showed multiple plant growth promotion activities: it was able to produce indole-3-acetic acid (IAA) and siderophores, solubilise phosphate, and produce a biofilm in the presence of phenanthrene and alleviate phenanthrene stress in tomato seeds. This is the first report on the simultaneous occurrence of the PAH-degrading ability by Methylobacterium populi and its multiple plant growth-promoting activities. Therefore, the selected indigenous strain, which is naturally present in highly contaminated soils, is good candidate for plant growth promotion and is capable of biodegrading xenobiotic organic compounds to remediate contaminated soil alone and/or soil associated with plants.

Prebiotic content of bread prepared with flour from immature wheat grain and selected dextran-producing lactic acid bacteria.

In the last few years the need to produce food with added value has fueled the search for new ingredients and health-promoting compounds. In particular, to improve the quality of bakery products with distinct nutritional properties, the identification of new raw materials, appropriate technologies, and specific microbial strains is necessary. In this study, different doughs were prepared, with 10% and 20% flour from immature wheat grain blended with type "0 America" wheat flour. Immature flour was obtained from durum wheat grains harvested 1 to 2 weeks after anthesis. Doughs were obtained by both the straight-dough and sourdough processes. Two selected exopolysaccharide-producing strains of lactic acid bacteria (LAB), Leuconostoc lactis A95 and Lactobacillus curvatus 69B2, were used as starters. Immature flour contained 2.21 g/100 g (dry weight) of fructo-oligosaccharides. Twenty percent immature flour in dough resulted in a shorter leavening time (4.23 ± 0.03 h) than with the control and dough with 10% immature flour. The total titratable acidity of sourdough with 20% immature flour was higher (12.75 ± 0.15 ml 0.1 N NaOH) than in the control and sourdough with 10% immature wheat flour (9.20 ml 0.1 N NaOH). Molecular analysis showed that all samples contained three LAB species identified as L. lactis, L. curvatus, and Pediococcus acidilactici. A larger amount of exopolysaccharide was found in sourdough obtained with 20% immature flour (5.33 ± 0.032 g/kg), positively influencing the exopolysaccharide content of the bread prepared by the sourdough process (1.70 ± 0.03 g/kg). The addition of 20% immature flour also led to a greater presence of fructo-oligosaccharides in the bread (900 mg/100 g dry weight), which improved its nutritional characteristics. While bread volume decreased as the concentration of immature wheat flour increased, its mechanical characteristics (stress at a strain of 30%) were the same in all samples obtained with different percentages of fructo-oligosaccharides. These data support the use of immature wheat grain flour, and exopolysaccaride-producing lactic acid bacteria in formulating functional prebiotic baked goods whose nutritional value can be suitably improved.

Valorisation of hydrothermal liquefaction wastewater in agriculture: effects on tobacco plants and rhizosphere microbiota.

Industrial wastewater obtained from hydrothermal liquefaction (HTL-WW) of food wastes for biofuels production could represent a source of crop nutrients since it is characterized by a high amount of organic and inorganic compounds. In the present work, the potential use of HTL-WW as irrigation water for industrial crops was investigated. The composition of the HTL-WW was rich in nitrogen, phosphorus, and potassium with high level of organic carbon. A pot experiment with Nicotiana tabacum L. plants was conducted using diluted wastewater to reduce the concentration of some chemical elements below the official accepted threshold values. Plants were grown in the greenhouse under controlled conditions for 21 days and irrigated with diluted HTL-WW every 24 hours. Soils and plants were sampled every seven days to evaluate, over time, the effect of wastewater irrigation both on soil microbial populations, through high-throughput sequencing, and plant growth parameters, through the measurement of different biometric indices. Metagenomic results highlighted that, in the HTL-WW treated rhizosphere, the microbial populations shifted via their mechanisms of adaptation to the new environmental conditions, establishing a new balance among bacterial and fungal communities. Identification of microbial taxa occurring in the rhizosphere of tobacco plants during the experiment highlighted that the HTL-WW application improved the growth of Micrococcaceae, Nocardiaceae and Nectriaceae, which included key species for denitrification, organic compounds degradation and plant growth promotion. As a result, irrigation with HTL-WW improved the overall performance of tobacco plants which showed higher leaf greenness and increased number of flowers compared to irrigated control plants. Overall, these results demonstrate the potential feasibility of using of HTL-WW in irrigated agriculture.

Fungal Biodeterioration and Preservation of Miniature Artworks.

The study of biodeterioration is an important issue to allow the best conservation and prevent the decay of cultural heritage and artworks. In Naples (Italy), a particular museum (Museodivino) preserves the miniature artworks representing Dante's Divine Comedy and Nativity scenes, executed with organic-based materials in walnut and clay shells. Since they showed putative signs of biodeterioration, the first aim of this study was to verify the presence of microbial colonization. A culture-dependent approach and molecular biology allowed us to isolate and identify the sole fungal strain Aspergillus NCCD (Nativity and Dante's Divine Comedy) belonging to the A. sydowii sub-clade. Based on this result, a sustainable and eco-friendly approach was applied to find a method to preserve the miniature artwork by contrasting the growth of the strain NCCD. Several essential oils used as a natural biocide were tested against Aspergillus strain NCCD belonging to the A. sydowii subclade to determine their potential antimicrobial activity. Results revealed that basil, cloves, fennel, and thyme essential oils exerted antifungal activity, although their effect depended also on the concentration used. Moreover, anoxic treatment and the control of the relative humidity were used in the presence of thyme, in vitro, and in vivo assays to define the impact on fungal growth. No fungal development was detected in vivo in the shells treated with thyme essential oil at high relative humidity after 60 days of incubation at 28 °C. These results highlighted that although relative humidity was the major factor affecting the development of the strain Aspergillus NDDC, the application of thyme in an anaerobic environment is essential in contrasting the fungal growth. Identifying the biodeterioration agent allowed us to plan an eco-friendly, non-destructive approach to be successfully used to guarantee the conditions suitable for conserving miniature artwork.

Inoculation with a microbial consortium increases soil microbial diversity and improves agronomic traits of tomato under water and nitrogen deficiency.

Microbial-based biostimulants, functioning as biotic and abiotic stress protectants and growth enhancers, are becoming increasingly important in agriculture also in the context of climate change. The search for new products that can help reduce chemical inputs under a variety of field conditions is the new challenge. In this study, we tested whether the combination of two microbial growth enhancers with complementary modes of action, Azotobacter chroococcum 76A and Trichoderma afroharzianum T22, could facilitate tomato adaptation to a 30% reduction of optimal water and nitrogen requirements. The microbial inoculum increased tomato yield (+48.5%) under optimal water and nutrient conditions. In addition, the microbial application improved leaf water potential under stress conditions (+9.5%), decreased the overall leaf temperature (-4.6%), and increased shoot fresh weight (+15%), indicating that this consortium could act as a positive regulator of plant water relations under limited water and nitrogen availability. A significant increase in microbial populations in the rhizosphere with applications of A. chroococcum 76A and T. afroharzianum T22 under stress conditions, suggested that these inoculants could enhance soil microbial abundance, including the abundance of native beneficial microorganisms. Sampling time, limited water and nitrogen regimes and microbial inoculations all affected bacterial and fungal populations in the rhizospheric soil. Overall, these results indicated that the selected microbial consortium could function as plant growth enhancer and stress protectant, possibly by triggering adaptation mechanisms via functional changes in the soil microbial diversity and relative abundance.

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture.

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an α-(1→6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30°C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.

Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost.

BACKGROUND: The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. RESULTS: Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose following a Michaelis-Menten kinetics with a KM of 9.13 mg/ml and a vmax of 3469 µM min-1. The enzyme exhibits a half life of around 24 h and 96 h at 60°C and 50°C, respectively and shows a retention of around 80% of activity after 96 h at 40°C. CONCLUSIONS: In this manuscript, we describe the isolation of a new cellulolytic strain, Streptomyces sp. G12, from industrial waste based compost, the identification of the enzymes putatively responsible for its cellulolytic activity, the cloning and the recombinant expression of the gene coding for the Streptomyces sp. G12 cellulase CelStrep, that was characterized showing to exhibit a relevant thermoresistance increasing its potential for cellulose conversion.

Diversity, mechanisms and beneficial features of phosphate-solubilizing Streptomyces in sustainable agriculture: A review.

Currently, the use of phosphate (P) biofertilizers among many bioformulations has attracted a large amount of interest for sustainable agriculture. By acting as growth promoters, members of the Streptomyces genus can positively interact with plants. Several studies have shown the great potential of this bacterial group in supplementing P in a soluble, plant-available form by several mechanisms. Furthermore, some P-solubilizing Streptomyces (PSS) species are known as plant growth-promoting rhizobacteria that are able to promote plant growth through other means, such as increasing the availability of soil nutrients and producing a wide range of antibiotics, phytohormones, bioactive compounds, and secondary metabolites other than antimicrobial compounds. Therefore, the use of PSS with multiple plant growth-promoting activities as an alternative strategy appears to limit the negative impacts of chemical fertilizers in agricultural practices on environmental and human health, and the potential effects of these PSS on enhancing plant fitness and crop yields have been explored. However, compared with studies on the use of other gram-positive bacteria, studies on the use of Streptomyces as P solubilizers are still lacking, and their results are unclear. Although PSS have been reported as potential bioinoculants in both greenhouse and field experiments, no PSS-based biofertilizers have been commercialized to date. In this regard, this review provides an overview mainly of the P solubilization activity of Streptomyces species, including their use as P biofertilizers in competitive agronomic practices and the mechanisms through which they release P by solubilization/mineralization, for both increasing P use efficiency in the soil and plant growth. This review further highlights and discusses the beneficial association of PSS with plants in detail with the latest developments and research to expand the knowledge concerning the use of PSS as P biofertilizers for field applications by exploiting their numerous advantages in improving crop production to meet global food demands.

Characterization in the archaeological excavation site of heterotrophic bacteria and fungi of deteriorated wall painting of Herculaneum in Italy.

Microbiological characterization of frescos in four different locations (Collegio degli Augustali, Casa del Colonnato Tuscanico, Casa dello Scheletro and Casa del Gran Portale) of excavation sites of Herculaneum was carried out. The use of infrared thermography allowed detecting sample points on frescos with greatest moisture not visible to the naked eye, resulting in structural damage. The microclimatic conditions provided perfect habitat for bacteria and fungi, particularly of spore forming and mould. In fact, heterotrophic bacteria were prevalent in all wall paintings monitored (ranging from 18 +/- 2 CFU 100 cm(-2) to 68 +/- 4 CFU 100 cm(-2)), whereas fungi were also detected but at lower levels (ranging from 9 +/- 2 CFU 100 cm(-2) to 45 +/- 3 CFU 100 cm(-2)). Cultural-based method allow us to identify by 16S and 26S rRNA partial sequence analysis heterotrophic microorganisms belonging to different genera of Bacillus and Aspergillus, Penicillium and Fusarium together with the unusual genera as Microascus and Coprinus. By using this approach, Bacillus-related species (B. cereus/B. thuringiensis group, B. simplex/B. muralis group, B. megaterium and B. subtilis) were isolated in all sample points analysed with the exception of the Casa dello Scheletro in which Micrococcus luteus/Arthrobactersp. group and Streptomyces fragilis were found. DGGE analysis of PCR amplified V3 region of rDNA from DNA directly recovered from frescos samples, enabled identification of bacterial species not identified using culturable technology asthose closest related to Microbacterium group, often associated with Brevibacterium, Streptomyces and Stenotrophomonas. Combination of culture-dependent and independent methods provided better microbiology characterization of heterotrophic microbiota present on the surface of ancient frescos of this important archaeological site.

Bioformulations with Beneficial Microbial Consortia, a Bioactive Compound and Plant Biopolymers Modulate Sweet Basil Productivity, Photosynthetic Activity and Metabolites.

Increasing attention is being given to the development of innovative formulations to substitute the use of synthetic chemicals to improve agricultural production and resource use efficiency. Alternatives can include biological products containing beneficial microorganisms and bioactive metabolites able to inhibit plant pathogens, induce systemic resistance and promote plant growth. The efficacy of such bioformulations can be increased by the addition of polymers as adjuvants or carriers. Trichoderma afroharzianum T22, Azotobacter chroococcum 76A and 6-pentyl-α-pyrone (6PP; a Trichoderma secondary metabolite) were administrated singularly or in a consortium, with or without a carboxymethyl cellulose-based biopolymer (BP), and tested on sweet basil (Ocimum basilicum L.) grown in a protected greenhouse. The effect of the treatments on basil yield, photosynthetic activity and secondary metabolites production was assessed. Photosynthetic efficiency was augmented by the applications of the bioformulations. The applications to the rhizosphere with BP + 6PP and BP + T22 + 76A increased the total fresh weight of basil by 26.3% and 23.6%, respectively. Untargeted LC-MS qTOF analysis demonstrated that the plant metabolome was significantly modified by the treatments. Quantification of the profiles for the major phenolic acids indicated that the treatment with the T22 + 76A consortium increased rosmarinic acid content by 110%. The use of innovative bioformulations containing microbes, their metabolites and a biopolymer was found to modulate the cultivation of fresh basil by improving yield and quality, thus providing the opportunity to develop farming systems with minimal impact on the environmental footprint from the agricultural production process.

Diversity of Staphylococcus species strains based on partial kat (catalase) gene sequences and design of a PCR-restriction fragment length polymorphism assay for identification and differentiation of coagulase-positive species (S. aureus, S. delphini, S. hyicus, S. intermedius, S. pseudintermedius, and S. schleiferi subsp. coagulans).

A set of degenerate PCR primers was designed and used to amplify and sequence about 75% of the catalase (kat) gene from each of 49 staphylococcal strains. In some strains of Staphylococcus xylosus, S. saprophyticus, and S. equorum, two catalase genes, katA and katB, were found. A phylogenetic tree was generated and showed diversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequences found in GenBank) representing 26 different species. The topology of this tree showed a distribution of staphylococcal species similar, but not identical, to those reported previously based on 16S rRNA, hsp60, sodA, rpoB, tuf, and gap genes. The kat gene sequences were less conserved than those of 16S rRNA, rpoB, hsp60, and tuf genes and slightly more conserved than those of the gap gene. Therefore, kat gene sequence analysis may provide an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discrete nucleotide polymorphism revealed in this gene could be exploited for rapid, low-cost identification of staphylococcal species through PCR-restriction fragment length polymorphism (RFLP) analysis. In this study, a PCR-RFLP assay performed by using only the TaqI restriction enzyme was successfully developed for rapid unequivocal identification/differentiation, at species and subspecies levels, of coagulase-positive staphylococci (CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference and wild CPS strains isolated from different environments. This reliable, rapid, and low-cost approach (requiring about 6 h from DNA isolation to the achievement of results and <5 Euros for each strain tested) allowed unambiguous identification of all the strains assayed, including the newly described S. delphini and S. pseudintermedius CPS species.

Effectiveness of Plant Beneficial Microbes: Overview of the Methodological Approaches for the Assessment of Root Colonization and Persistence.

Issues concerning the use of harmful chemical fertilizers and pesticides that have large negative impacts on environmental and human health have generated increasing interest in the use of beneficial microorganisms for the development of sustainable agri-food systems. A successful microbial inoculant has to colonize the root system, establish a positive interaction and persist in the environment in competition with native microorganisms living in the soil through rhizocompetence traits. Currently, several approaches based on culture-dependent, microscopic and molecular methods have been developed to follow bioinoculants in the soil and plant surface over time. Although culture-dependent methods are commonly used to estimate the persistence of bioinoculants, it is difficult to differentiate inoculated organisms from native populations based on morphological characteristics. Therefore, these methods should be used complementary to culture-independent approaches. Microscopy-based techniques (bright-field, electron and fluorescence microscopy) allow to obtain a picture of microbial colonization outside and inside plant tissues also at high resolution, but it is not possible to always distinguish living cells from dead cells by direct observation as well as distinguish bioinoculants from indigenous microbial populations living in soils. In addition, the development of metagenomic techniques, including the use of DNA probes, PCR-based methods, next-generation sequencing, whole-genome sequencing and pangenome methods, provides a complementary approach useful to understand plant-soil-microbe interactions. However, to ensure good results in microbiological analysis, the first fundamental prerequisite is correct soil sampling and sample preparation for the different methodological approaches that will be assayed. Here, we provide an overview of the advantages and limitations of the currently used methods and new methodological approaches that could be developed to assess the presence, plant colonization and soil persistence of bioinoculants in the rhizosphere. We further discuss the possibility of integrating multidisciplinary approaches to examine the variations in microbial communities after inoculation and to track the inoculated microbial strains.

Development and Application of Low-Cost and Eco-Sustainable Bio-Stimulant Containing a New Plant Growth-Promoting Strain Kosakonia pseudosacchari TL13.

The use of beneficial microbes as inoculants able to improve fitness, growth and health of plants also in stress conditions is an attractive low-cost and eco-friendly alternative strategy to harmful chemical inputs. Thirteen potential plant growth-promoting bacteria were isolated from the rhizosphere of wheat plants cultivated under drought stress and nitrogen deficiency. Among these, the two isolates TL8 and TL13 showed multiple plant growth promotion activities as production of indole-3-acetic acid (IAA), siderophores, ammonia, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production, the ability to solubilize phosphate as well as exerted antimicrobial activity against plant pathogens as Botrytis spp. and Phytophthora spp. The two selected strains were identified as Kosakonia pseudosacchari by sequencing of 16S rRNA gene. They resulted also tolerant to abiotic stress and were able to efficiently colonize plant roots as observed in vitro assay under fluorescence microscope. Based on the best PGP properties, the strain K. pseudosacchari TL13 was selected to develop a new microbial based formulate. A sustainable and environmentally friendly process for inoculant production was developed using agro-industrial by-products for microbial growth. Moreover, the application of K. pseudosacchari TL13- based formulates in pot experiment improved growth performance of maize plants.

Copper accumulation in agricultural soils: Risks for the food chain and soil microbial populations.

The long-term use of Cu-based fungicides at doses of several kilograms per hectare stimulated a wide debate about the human health and environmental risks of the progressive accumulation of Cu in agricultural soils. Here, the health risks due to copper accumulation in agricultural soils were evaluated with a survey in intensive agricultural land of the Campania region (Italy), aiming to evaluate Cu accumulation in food crops. The health risk due to dietary exposure was estimated by using the Hazard Quotient (HQ), calculated as the ratio between the average daily dose and the reference dose of copper, suggesting that when HQ > 1 there is a potential risk for consumers. According to a survey of soils with a Cu content up to 217 mg kg-1, no foodstuffs showed dietary risks. Nevertheless, the contribution of Cu contained in these foodstuffs to the overall intake of Cu by consumers could increase health risks since such risks must be evaluated on the basis of the whole standard diet by quantifying the Cu content not only in vegetables and fruits but also in other sources, such as cereals, not cultivated in the study area and thus not considered in this paper. The environmental risks due to copper accumulation in agricultural soils were then evaluated with a field experiment in a soil characterized by a very high Cu concentration (up to 1700 mg kg-1), aiming to study the impacts of Cu on native soil microorganisms. The study of the microbiota highlighted that the presence of Cu in soil did not reduce the total richness and diversity of microorganisms, which were not related to increasing concentrations of Cu in the soil. Nevertheless, Cu contamination was found to exert significant selection pressure on the soil microbiota, as shown by beta diversity and correlation analysis between taxa and Cu content.