Solid-State Fermentation: Applications and Future Perspectives for Biostimulant and Biopesticides Production.

With the expansion of the green products market and the worldwide policies and strategies directed toward a green revolution and ecological transition, the demand for innovative approaches is always on the rise. Among the sustainable agricultural approaches, microbial-based products are emerging over time as effective and feasible alternatives to agrochemicals. However, the production, formulation, and commercialization of some products can be challenging. Among the main challenges are the industrial production processes that ensure the quality of the product and its cost on the market. In the context of a circular economy, solid-state fermentation (SSF) might represent a smart approach to obtaining valuable products from waste and by-products. SSF enables the growth of various microorganisms on solid surfaces in the absence or near absence of free-flowing water. It is a valuable and practical method and is used in the food, pharmaceutical, energy, and chemical industries. Nevertheless, the application of this technology in the production of formulations useful in agriculture is still limited. This review summarizes the literature dealing with SSF agricultural applications and the future perspective of its use in sustainable agriculture. The survey showed good potential for SSF to produce biostimulants and biopesticides useful in agriculture.

Role of Polyamines in the Response to Salt Stress of Tomato.

Plants irrigated with saline solutions undergo osmotic and oxidative stresses, which affect their growth, photosynthetic activity and yield. Therefore, the use of saline water for irrigation, in addition to the increasing soil salinity, is one of the major threats to crop productivity worldwide. Plant tolerance to stressful conditions can be improved using different strategies, i.e., seed priming and acclimation, which elicit morphological and biochemical responses to overcome stress. In this work, we evaluated the combined effect of priming and acclimation on salt stress response of a tomato cultivar (Solanum lycopersicum L.), very sensitive to salinity. Chemical priming of seeds was performed by treating seeds with polyamines (PAs): 2.5 mM putrescine (PUT), 2.5 mM spermine (SPM) and 2.5 mM spermidine (SPD). Germinated seeds of primed and non-primed (controls) were sown in non-saline soil. The acclimation consisted of irrigating the seedlings for 2 weeks with tap water, followed by irrigation with saline and non-saline water for 4 weeks. At the end of the growth period, morphological, physiological and biochemical parameters were determined. The positive effects of combined treatments were evident, when primed plants were compared to non-primed, grown under the same conditions. Priming with PAs improved tolerance to salt stress, reduced the negative effects of salinity on growth, improved membrane integrity, and increased photosynthetic pigments, proline and enzymatic and non-enzymatic antioxidant responses in all salt-exposed plants. These results may open new perspectives and strategies to increase tolerance to salt stress in sensitive species, such as tomato.

Biosynthesis and characterization of nanoparticles, its advantages, various aspects and risk assessment to maintain the sustainable agriculture: Emerging technology in modern era science.

The current review aims to gain knowledge on the biosynthesis and characterization of nanoparticles (NPs), their multifactorial role, and emerging trends of NPs utilization in modern science, particularly in sustainable agriculture, for increased yield to solve the food problem in the coming era. However, it is well known that an environment-friendly resource is in excessive demand, and green chemistry is an advanced and rising resource in exploring eco-friendly processes. Plant extracts or other resources can be utilized to synthesize different types of NPS. Hence NPs can be synthesized by organic or inorganic molecules. Inorganic molecules are hydrophilic, biocompatible, and highly steady compared to organic types. NPs occur in numerous chemical conformations ranging from amphiphilic molecules to metal oxides, from artificial polymers to bulky biomolecules. NPs structures can be examined by different approaches, i.e., Raman spectroscopy, optical spectroscopy, X-ray fluorescence, and solid-state NMR. Nano-agrochemical is a unification of nanotechnology and agro-chemicals, which has brought about the manufacture of nano-fertilizers, nano-pesticides, nano-herbicides, nano-insecticides, and nano-fungicides. NPs can also be utilized as an antimicrobial solution, but the mode of action for antibacterial NPs is poorly understood. Presently known mechanisms comprise the induction of oxidative stress, the release of metal ions, and non-oxidative stress. Multiple modes of action towards microbes would be needed in a similar bacterial cell for antibacterial resistance to develop. Finally, we visualize multidisciplinary cooperative methods will be essential to fill the information gap in nano-agrochemicals and drive toward the usage of green NPs in agriculture and plant science study.

Preliminary indication of the role of AHL-dependent quorum sensing systems in calcium carbonate precipitation in Gram-negative bacteria.

Numerous microbial species participate in precipitation of carbonates in various natural environments, including soils, geological formations, freshwater biofilms and oceans. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and adaptive aspects remain poorly known. Many Gram-negative bacteria use cell-to-cell communication systems relying on N-acylhomoserine lactone (AHLs) signal molecules to express certain phenotypic traits in a density-dependent manner, a phenomenon referred as to quorum-sensing (QS). In this work, bacterial isolates collected from cave and rhizosphere soil were analyzed to study the occurrence of the AHL-mediated QS in bacterial calcium carbonate (CaCO3) precipitation. To test the production of AHLs signal molecules, we cross-streaked Gram-negative calcifying strains, selected among the environmental strains studied, with the AHL-negative mutant Chromobacterium subtsugae strain CV026. Only Burkholderia ambifaria LMG 11351 was able to restore violacein production in CV026 among the tested strains. The constructed AHL-negative mutant of B. ambifaria LMG 11351 could not precipitate CaCO3 on B-4 agar. Scanning Electron Microscopy (SEM) analysis on CaCO3 crystals obtained in vitro shows crystals of different morphologies, calcified biofilms and bacteria in close contact with the precipitated crystals. In the inner layers of the bioliths deposited by B. ambifaria LMG 11351, a stream-like organization of the Burkholderia imprints was not detected by SEM. Our data provide preliminary evidence that the activation of AHL-regulated genes may be a prerequisite for in vitro bacterial carbonatogenesis, in some cases, confirming the specific role of bacteria as CaCO3 precipitating agents. We enhance the understanding of bacterial CaCO3 biomineralization and its potential biotechnology implications for QS-based strategies to enhance or decrease CaCO3 precipitation through specific bacterial processes. The AHL-negative mutant of B. ambifaria LMG 11351 (a well-known plant growth-promoting bacterium) could also be used to study plant-bacteria interactions. The adaptive role of bacterial CaCO3 biomineralization was also discussed.

Exploring structure, microbiota, and metagenome functions of epigean and hypogean black deposits by microscopic, molecular and bioinformatic approaches.

This study revealed how Bacteria and Archaea communities and their metabolic functions differed between two groups of black deposits identified in gorge and cave environments. Scanning electron microscopy coupled with energy dispersive spectroscopy was used to analyse the presence of microbial biosignatures and the elemental composition of samples. Metabarcoding of the V3-V4 regions of 16S rRNA was used to investigate Bacteria and Archaea communities. Based on 16S rRNA sequencing results, PICRUSt software was used to predict metagenome functions. Micrographs showed that samples presented microbial biosignatures and microanalyses highlighted Mn concretions and layers on Al-Si surfaces. The 16S rRNA metabarcoding alpha-diversity metrics showed similar Simpson's and Shannon indices and different values of the Chao-1 index. The amplicon sequence variants (ASVs) analysis at the different taxonomic levels showed a diverse genera composition. However, the communities of all samples shared the presence of uncultured ASVs belonging to the Gemmatales family (Phylogenesis: Gemmataceae; Planctomycetes; Planctomycetota; Bacteria). The predicted metagenome functions analysis revealed diverse metabolic profiles of the Cave and Gorge groups. Genes coding for essential Mn metabolism were present in all samples. Overall, the findings on structure, microbiota, and predicted metagenome functions showed a similar microbial contribution to epigean and hypogean black deposits Mn metabolism.

Polyhydroxybutyrate-producing cyanobacteria from lampenflora: The case study of the "Stiffe" caves in Italy.

This study aimed to estimate the green formation lampenflora of "Stiffe" caves in order to evaluate their suitability as an isolation source of cyanobacteria useful for the production of polyhydroxyalkanoates (PHAs). The cave system was chosen as the sampling site due to its touristic use and the presence of high-impact illuminations. The biofilms and the mats of the illuminated walls were sampled. Samples were investigated by 16S rRNA gene analysis and culturable cyanobacteria isolation. The isolated strains were then screened for the production of PHAs under typical culturing and nutritional starvation. Cultures were checked for PHA accumulation, poly-ß-hydroxybutyrate (PHB) presence (infrared spectroscopy), and pigment production. The 16S rRNA gene metabarcoding. Highlighted a considerable extent of the pressure exerted by anthropogenic activities. However, the isolation yielded eleven cyanobacteria isolates with good PHA (mainly PHB)-producing abilities and interesting pigment production rates (chlorophyll a and carotenoids). Under normal conditions (BG110), the accumulation abilities ranged from 266 to 1,152 ng mg dry biomass-1. The optimization of bioprocesses through nutritional starvation resulted in a 2.5-fold increase. Fourier transform infrared (FTIR) studies established the occurrence of PHB within PHAs extracted by cyanobacteria isolates. The comparison of results with standard strains underlined good production rates. For C2 and C8 strains, PHA accumulation rates under starvation were higher than Azospirillum brasilense and similar to Synechocystis cf. salina 192. This study broadened the knowledge of the microbial communities of mats and biofilms on the lightened walls of the caves. These findings suggested that these structures, which are common in tourist caves, could be used to isolate valuable strains before remediation measures are adopted.

The "Infernaccio" Gorges: Microbial Diversity of Black Deposits and Isolation of Manganese-Solubilizing Bacteria.

The present study explored the microbial diversity of black deposits found in the "Infernaccio" gorge. X-ray Powdered Diffraction (XRPD) was used to investigate the crystallinity of the samples and to identify the minerals. Scanning electron microscope and energy-dispersive X-ray spectroscopy (SEM-EDS) were used to detect the bacterial imprints, analyze microbe-mineral interactions, and highlight the chemical element distribution in the black deposits. 16S rRNA gene metabarcoding allowed the study of Archaea and Bacteria communities. Mn-oxide-solubilizing isolates were also obtained and characterized by culturable and molecular approaches. The multidisciplinary approach showed the occurrence of deposits composed of birnessite, diopside, halloysite, and leucite. Numerous bacterial imprints confirmed the role of microorganisms in forming these deposits. The Bacteria and Archaea communities associated with these deposits and runoff waters are dynamic and shaped by seasonal changes. The uncultured and unknown taxa are the most common and abundant. These amplicon sequence variants (ASVs) were mainly assigned to Proteobacteria and Bacteroidetes phyla. Six isolates showed interesting Mn solubilization abilities under microaerophilic conditions. Molecular characterization associated isolates to Brevibacterium, Bacillus, Neobacillus, and Rhodococcus genera. The findings enriched our knowledge of geomicrobiological aspects of one of the Earth's hidden habitats. The study also unveiled the potential of this environment as an isolation source of biotechnologically relevant bacteria.

Actinomycetes from Caves: An Overview of Their Diversity, Biotechnological Properties, and Insights for Their Use in Soil Environments.

The environmental conditions of caves shape microbiota. Within caves' microbial communities, actinomycetes are among the most abundant bacteria. Cave actinomycetes have gained increasing attention during the last decades due to novel bioactive compounds with antibacterial, antioxidant and anticancer activities. However, their potential role in soil environments is still unknown. This review summarises the literature dealing with actinomycetes from caves, underlining for the first time their potential roles in soil environments. We provide an overview of their diversity and biotechnological properties, underling their potential role in soil environments applications. The contribution of caves' actinomycetes in soil fertility and bioremediation and crops biostimulation and biocontrol are discussed. The survey on the literature show that several actinomycetes genera are present in cave ecosystems, mainly Streptomyces, Micromonospora, and Nocardiopsis. Among caves' actinomycetes, Streptomyces is the most studied genus due to its ubiquity, survival capabilities, and metabolic versatility. Despite actinomycetes' outstanding capabilities and versatility, we still have inadequate information regarding cave actinomycetes distribution, population dynamics, biogeochemical processes, and metabolisms. Research on cave actinomycetes needs to be encouraged, especially concerning environmental soil applications to improve soil fertility and health and to antagonise phytopathogens.

Fusarium Oxysporum f. sp. Cannabis Isolated from Cannabis Sativa L.: In Vitro and In Planta Biocontrol by a Plant Growth Promoting-Bacteria Consortium.

Industrial hemp (Cannabis sativa L.) is a multipurpose plant used in several fields. Several phytopathogens attack hemp crops. Fusarium oxysporum is a common fungal pathogen that causes wilt disease in nurseries and in field cultivation and causes high losses. In the present study, a pathogenic strain belonging to F. oxysporum f. sp. cannabis was isolated from a plant showing Fusarium wilt. After isolation, identification was conducted based on morphological and molecular characterizations and pathogenicity tests. Selected plant growth-promoting bacteria with interesting biocontrol properties-Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Burkholderia ambifaria-were tested against this pathogen. In vitro antagonistic activity was determined by the dual culture method. Effective strains (in vitro inhibition > of 50%) G. diazotrophicus, H. seropedicae and B. ambifaria were combined in a consortium and screened for in planta antagonistic activity in pre-emergence (before germination) and post-emergence (after germination). The consortium counteracted Fusarium infection both in pre-emergence and post-emergence. Our preliminary results show that the selected consortium could be further investigated as an effective biocontrol agent for the management of this pathogen.

Biocontrol of Soil-Borne Pathogens of Solanum lycopersicum L. and Daucus carota L. by Plant Growth-Promoting Actinomycetes: In Vitro and In Planta Antagonistic Activity.

Biotic stress caused by pathogenic microorganisms leads to damage in crops. Tomato and carrot are among the most important vegetables cultivated worldwide. These plants are attacked by several pathogens, affecting their growth and productivity. Fourteen plant growth-promoting actinomycetes (PGPA) were screened for their in vitro biocontrol activity against Solanum lycopersicum and Daucus carota microbial phytopathogens. Their antifungal activity was evaluated against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) and Rhizoctonia solani (RHS). Antibacterial activity was evaluated against Pseudomonas syringae, Pseudomonas corrugata, Pseudomonas syringae pv. actinidiae, and Pectobacterium carotovorum subsp. carotovorum. Strains that showed good in vitro results were further investigated in vitro (cell-free supernatants activity, scanning electron microscope observations of fungal inhibition). The consortium of the most active PGPA was then utilized as biocontrol agents in planta experiments on S. lycopersicum and D. carota. The Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 strains showed the best in vitro biocontrol activities. The diffusible and volatile compounds and cell-free supernatants of these strains showed both antifungal (in vitro inhibition up to 85%, hyphal desegregation and fungicidal properties) and antibacterial activity (in vitro inhibition >25 mm and bactericidal properties). Their consortium was also able to counteract the infection symptoms of microbial phytopathogens during in planta experiments, improving plant status. The results obtained highlight the efficacy of the selected actinomycetes strains as biocontrol agents of S. lycopersicum and D. carota.

Allium cepa L. Inoculation with a Consortium of Plant Growth-Promoting Bacteria: Effects on Plants, Soil, and the Autochthonous Microbial Community.

The present work was aimed at investigating the effects of a four bacterial strain consortium-Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, and Burkholderia ambifaria-on Allium cepa L. and on soil health. The bacterial consortium was inoculated on seeds of two different onion varieties; inoculated and Control seeds (treated with autoclaved inoculum) were sown in an open-field and followed until harvest. Plant growth development parameters, as well as soil physico-chemical and molecular profiles (DNA extraction and 16S community sequencing on the Mi-Seq Illumina platform), were investigated. The results showed a positive influence of bacterial application on plant growth, with increased plant height (+18%), total chlorophylls (+42%), crop yields (+13%), and bulb dry matter (+3%) with respect to the Control. The differences between Control and treatments were also underlined in the bulb extracts in terms of total phenolic contents (+25%) and antioxidant activities (+20%). Soil fertility and microbial community structure and diversity were also positively affected by the bacterial inoculum. At harvest, the soil with the presence of the bacterial consortium showed an increase in total organic carbon, organic matter, and available phosphorus, as well as higher concentrations of nutrients than the Control. The ecological indexes calculated from the molecular profiles showed that community diversity was positively affected by the bacterial treatment. The present work showed the effective use of plant growth-promoting bacteria as a valid fertilization strategy to improve yield in productive landscapes whilst safeguarding soil biodiversity.

In vitro and in planta antagonistic effects of plant growth-promoting rhizobacteria consortium against soilborne plant pathogens of Solanum tuberosum and Solanum lycopersicum.

Potatoes (Solanum tuberosum L.) and tomatoes (Solanum lycopersicum L.), among the main crops belonging to the Solanaceae family, are attacked by several pathogens. Among them Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani are very common and cause significant losses. Four plant growth-promoting rhizobacteria, Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Burkholderia ambifaria were tested against these pathogens. In vitro antagonistic activities of single strains were assessed through dual culture plates. Strains showing antagonistic activity (G. diazotrophicus, H. seropedicae and B. ambifaria) were combined and, after an in vitro confirmation, the consortium was applied on S. lycopersicum and S. tuberosum in a greenhouse pot experiment. The bioprotection was assessed in pre-emergence (infection before germination) and post-emergence (infection after germination). The consortium was able to successfully counteract the infection of both F. oxysporum and R. solani, allowing a regular development of plants. The biocontrol of the fungal pathogens was highlighted both in pre-emergence and post-emergence conditions. This selected consortium could be a valid alternative to agrochemicals and could be exploited as biocontrol agent to counteract losses due to these pathogenic fungi.

Dynamical model for sympatric speciation in an ecological niche.

The speciation phenomenon is the process used by the evolution to allow populations to become distinct species. The speciation is the primary cause of the complexity of the ecological network. Sympatric speciation concerns the rise of a new species from a surviving ancestral species while both continue to inhabit the same ecological niche or geographical region. In sympatric speciation, reproductive isolation evolves within a population in an ecological niche without the aid of geographic barriers. Different models have been proposed for alternative modes of sympatric speciation. The most popular was first put forward by John Maynard Smith in 1966 who suggested that in a given population homozygous individuals may, under particular environmental conditions, have a greater fitness than those with alleles heterozygous for a certain trait, eventually leading to speciation in the population. In this framework we assume an effective description of the speciation process based on a dynamical model for the populations in an ecological system. Our basic assumption is the existence of an ancestral population in an ecological niche that can express two phenotypes. In presence of certain environmental conditions one of the phenotypes has the propensity to separate from the original population in the reproduction process. Then new individuals may give rise to a new species in the ecosystem realizing a sympatric speciation. Due to the finite resources in the niche the populations are continuously competing each other's, and their numerousness fluctuates according to the changes of the environmental conditions. The effect of natural selection is introduced in the model by stochastic perturbations, that decrease the reproduction rate of the populations in the niche. We show some the dynamical properties of the system and we prove the existence of a threshold values in the environmental stress in order to observe the speciation process. We also discuss some biological implications of the model and the validation problem using empirical data.

Selection of a Very Active Microbial Community for the Coupled Treatment of Tetramethylammonium Hydroxide and Photoresist in Aqueous Solutions.

Aerobic treatment of wastewater containing Tetramethylammonium hydroxide (TMAH) and photoresist was investigated using a lab scale reactor inoculated with activated sludge coming from urban wastewater treatment that never received TMAH before. The consumption of TMAH was monitored by liquid ion chromatography. Biodiversity indices were calculated from Denaturing Gradient Gel Electrophoresis (DGGE) bands distribution and used to estimate changes in community composition related to adaptation to the new feeding compound. The first week of adaptation was crucial, and it was analyzed in detail: many organisms died, and the microbial community suffered a great shock. TMAH levels remained constant through the first four days, and then suddenly dropped to undetectable, and at the same time NH4⁺ increased. When the community showed complete adaptation, predominant groups of bacteria were obtained by the Illumina sequencing of 16s rDNA amplicons, to provide insights on ecology of the adapted community, focusing on the main actors of TMAH abatement. Richness of species (Rr) peaks suggest that the development of TMAH-consuming bacteria leads to persistent consortia that maintain toxicity resistance over time. This showed adaptation and changes of the population to the different feeding conditions, and it opens new perspectives in the in situ treatment of these important residues of industrial processes without relying on external processing plants.

A study of chlorinated solvent contamination of the aquifers of an industrial area in central Italy: a possibility of bioremediation.

Perchloroethene, trichloroethene, and other chlorinated solvents are widespread groundwater pollutants. They form dense non-aqueous phase liquids that sink through permeable groundwater aquifers until non-permeable zone is reached. In Italy, there are many situations of serious contamination of groundwater that might compromise their use in industry, agriculture, private, as the critical case of a Central Italy valley located in the province of Teramo ("Val Vibrata"), characterized by a significant chlorinated solvents contamination. Data from the various monitoring campaigns that have taken place over time were collected, and new samplings were carried out, resulting in a complete database. The data matrix was processed with a multivariate statistic analysis (in particular principal component analysis, PCA) and was then imported into geographic information system (GIS), to obtain a model of the contamination. A microcosm anaerobic study was utilized to assess the potential for in situ natural or enhanced bioremediation. Most of the microcosms were positive for dechlorination, particularly those inoculated with a mineral medium. This indicate the presence of an active native dechlorinating population in the subsurface, probably inhibited by co-contaminants in the groundwater, or more likely by the absence or lack of nutritional factors. Among the tested electron donors (i.e., yeast extract, lactate, and butyrate) lactate and butyrate enhanced dechlorination of chlorinated compounds. PCA and GIS studies allowed delimiting the contamination; the microcosm study helped to identify the conditions to promote the bioremediation of the area.

In vitro and in vivo inoculation of four endophytic bacteria on Lycopersicon esculentum.

Four bacteria selected on the basis of their capability of fixing atmospheric nitrogen, stimulating plant-growth, and protecting the host plant from pathogens - Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Burkholderia ambifaria - were inoculated on tomato seeds either singularly, in couple and in a four bacteria mixer. Aim of this research was to evaluate: (1) effect of single and mixed cultures on the inoculated plant - plant growth, dry weight, root length and surface, number of leaves, among others; (2) colonization and interactions of the bacteria inside the host plant; (3) localization inside the host of single bacterial strains marked with the gusA reporter gene. The results obtained indicate that all selected microbial strains have colonized Lycopersicon esculentum but in a different way, depending on the single species. A. brasilense, G. diazotrophicus inoculated in vitro singularly and together were the best plant colonizers. In vivo essays, instead, B. ambifaria and the four-bacteria mixer gave the best results. It was possible to localize both A. brasilense and H. seropedicae inside the plant by the gusA reporter gene. The bacterial strains occur along the root axis from the apical zone until to the basal stem, on the shoot from the base up to the leaves. The four bacteria actively colonize tomato seeds and establish an endophytic community inside the plant. This review gives new information about colonization processes, in particular how bacteria interact with plants and whether they are likely to establish themselves in the plant environment after field application as biofertilizers or biocontrol agents.

Calcium carbonate mineralization: involvement of extracellular polymeric materials isolated from calcifying bacteria.

This study highlights the role of specific outer bacterial structures, such as the glycocalix, in calcium carbonate crystallization in vitro. We describe the formation of calcite crystals by extracellular polymeric materials, such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. Organic matrices were isolated from calcifying bacteria grown on synthetic medium--in the presence or absence of calcium ions--and their effect on calcite precipitation was assessed. Scanning electron microscopy observations and energy dispersive X-ray spectrometry analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystal formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the biofilm production and protein profile of extracellular polymeric materials. Proteins of CPS fractions with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. This higher level of protein synthesis could be related to the active process of bioprecipitation.

Extended phenotype of an mreB-like mutant in Azospirillum brasilense.

Tn5 mutagenesis was used to generate an Azospirillum brasilense SPF94 mutant. Genetic analysis of this mutant revealed that a homologue of the mreB gene, which controls cell shape in Bacillus subtilis and Escherichia coli, was inactivated. The cell-surface properties of the mutant were different from those of the parental strain. The mutant colonies were highly fluorescent when grown on plates containing Calcofluor White. Light and electron microscopy revealed that the mutant cells were round and had thicker capsules than the spiral parental strain. The mutants contained up to ten times more capsule protein than the parental strain, but lacked a 40 kDa protein that is abundant in the parental strain. The phenotype of the isolated mutant resembled that of the cyst-like differentiated forms of Azospirillum, suggesting that the mreB homologue could be involved in differentiation.