Genomic insights and biocontrol potential of ten bacterial strains from the tomato core microbiome.

Introduction: Despite their adverse environmental effects, modern agriculture relies heavily on agrochemicals to manage diseases and pests and enhance plant growth and productivity. Some of these functions could instead be fulfilled by endophytes from the plant microbiota, which have diverse activities beneficial for plant growth and health. Methods: We therefore used a microbiome-guided top-down approach to select ten bacterial strains from different taxa in the core microbiome of tomato plants in the production chain for evaluation as potential bioinoculants. High-quality genomes for each strain were obtained using Oxford Nanopore long-read and Illumina short-read sequencing, enabling the dissection of their genetic makeup to identify phyto-beneficial traits. Results: Bacterial strains included both taxa commonly used as biofertilizers and biocontrol agents (i.e. Pseudomonas and Bacillus) as well as the less studied genera Leclercia, Chryseobacterium, Glutamicibacter, and Paenarthorbacter. When inoculated in the tomato rhizosphere, these strains promoted plant growth and reduced the severity of Fusarium Crown and Root Rot and Bacterial Spot infections. Genome analysis yielded a comprehensive inventory of genes from each strain related to processes including colonization, biofertilization, phytohormones, and plant signaling. Traits directly relevant to fertilization including phosphate solubilization and acquisition of nitrogen and iron were also identified. Moreover, the strains carried several functional genes putatively involved in abiotic stress alleviation and biotic stress management, traits that indirectly foster plant health and growth. Discussion: This study employs a top-down approach to identify new plant growth-promoting rhizobacteria (PGPRs), offering an alternative to the conventional bottom-up strategy. This method goes beyond the traditional screening of the strains and thus can expand the range of potential bioinoculants available for market application, paving the way to the use of new still underexplored genera.

Global citrus root microbiota unravels assembly cues and core members.

Introduction: Citrus is one of the most important fruit crops worldwide, and the root-associated microbiota can have a profound impact on tree health and growth. Methods: In a collaborative effort, the International Citrus Microbiome Consortium investigated the global citrus root microbiota with samples collected from nine citrus-producing countries across six continents. We analyzed 16S rDNA and ITS2 amplicon sequencing data to identify predominant prokaryotic and fungal taxa in citrus root samples. Comparative analyses were conducted between root-associated microbial communities and those from the corresponding rhizosphere and bulk soil samples. Additionally, genotype-based group-wise comparisons were performed to assess the impact of citrus genotype on root microbiota composition. Results: Ten predominant prokaryotic phyla, containing nine bacterial phyla including Proteobacteria, Actinobacteria, Acidobacteria, and Bacteroidetes and one archaeal phylum (Thaumarchaeota), and multiple fungal phyla including Ascomycota and Basidiomycota were identified in the citrus root samples. Compared with the microbial communities from the corresponding rhizosphere and bulk soil samples from the same trees, the prokaryotic and fungal communities in the roots exhibited lower diversity and complexity but greater modularity compared to those in the rhizosphere. In total, 30 root-enriched and 150 root-depleted genera in bacterial community were identified, whereas 21 fungal genera were enriched, and 147 fungal genera were depleted in the root niche compared with the rhizosphere. The citrus genotype significantly affected the root prokaryotic and fungal communities. In addition, we have identified the core root prokaryotic genera comprising Acidibacter, Allorhizobium, Bradyrhizobium, Chitinophaga, Cupriavidus, Devosia, Dongia, Niastella, Pseudomonas, Sphingobium, Steroidobacter and Streptomyces, and the core fungal genera including Acrocalymma, Cladosporium, Fusarium, Gibberella, Mortierella, Neocosmospora and Volutella. The potential functions of these core genera of root microbiota were predicted. Conclusion: Overall, this study provides new insights into the assembly of microbial communities and identifies core members of citrus root microbiota across a wide geographic range. The findings offer valuable information for manipulating root microbiota to enhance plant growth and health.

Soil Microbial Communities in Lemon Orchards Affected by Citrus Mal Secco Disease.

Mal secco is a vascular disease of citrus caused by the mitosporic fungus Plenodomus tracheiphilus. Soil containing infected plant material constitutes an inoculum source for root infections. In this study, the soil bacterial and fungal communities of five lemon orchards located in Syracuse Province (Sicily, Italy) affected by mal secco were analyzed. Soil samples were collected under lemon tree canopies and subjected to total genomic DNA extraction. The fungal DNA was detected through qPCR in all orchards, with variable concentrations. Bacterial and fungal communities were profiled using 16S and ITS amplicon-based high-throughput sequencing, respectively. According to our results, the relative abundances of the most represented bacterial phyla (e.g., Proteobacteria, Actinobacteriota, Acidobacteriota) changed across the orchards, while in the fungal community, the phylum Ascomycota was dominant, with Basidiomycota and Mortierellomycota abundances fluctuating. On the whole, ß diversity analysis showed significant variation in the composition of the soil microbial communities across the orchards. This result was confirmed by the analysis of the core community (taxa present at ≥ 75% of total samples), where putative beneficial bacteria resulted in significantly enriched fungus-infected soil samples, suggesting complex microbial interactions. Our findings shed light on the composition and diversity of the soil microbiome in lemon orchards with the occurrence of mal secco infections.

Biocontrol efficacy of Pseudomonas mediterranea PVCT 3C against Plenodomus tracheiphilus: In vitro and in planta mechanisms at early disease stages.

In this study, we investigated the biocontrol activity of the P. mediterranea strain PVCT 3C against Mal secco, a severe disease of citrus caused by the vascular fungus Plenodomus tracheiphilus. In vitro, bacterial diffusible compounds, volatile organic compounds and culture filtrates produced by PVCT 3C reduced the mycelial growth and conidial germination of P. tracheiphilus, also affecting the mycelial pigmentation. The application of bacterial suspensions by leaf-spraying before the inoculation with the pathogen on plants of the highly susceptible species sour orange and lemon led to an overall reduction in incidence and disease index, above all during the early disease stage. PVCT 3C genome was subjected to whole-genome shotgun sequencing to study the molecular mechanisms of action of this strain. In silico annotation of biosynthetic gene clusters for secondary metabolites revealed the presence of numerous clusters encoding antimicrobial compounds (e.g. cyclic lipopeptides, hydrogen cyanide, siderophores) and candidate novel products. During the asymptomatic disease phase (seven days post-inoculation), bacterial treatments interfered with the expression of different fungal genes, as assessed with an NGS and de novo assembly RNA-seq approach. These results suggest that P. mediterranea PVCT 3C or its secondary metabolites may offer a potential effective and sustainable alternative to contain P. tracheiphilus infections via integrated management.

Draft genome sequence of Xanthomonas arboricola pv. pruni PVCT 262.1 isolated from Prunus dulcis in italy.

Here, we report the draft genome sequence of Xanthomonas arboricola pv. pruni strain PVCT 262.1, isolated from almond (Prunus dulcis) leaves affected by bacterial spots in Italy in 2020. Genome size is 5,076,418 bp and G+C content is 65.44%. A total of 4,096 protein-coding genes and 92 RNAs are predicted.

Hub Genes and Pathways Related to Lemon (Citrus limon) Leaf Response to Plenodomus tracheiphilus Infection and Influenced by Pseudomonas mediterranea Biocontrol Activity.

The lemon industry in the Mediterranean basin is strongly threatened by "mal secco" disease (MSD) caused by the fungus Plenodomus tracheiphlilus. Leaf pretreatments with Pseudomonas mediterranea 3C have been proposed as innovative tools for eco-sustainable interventions aimed at controlling the disease. In this study, by exploiting the results of previously performed RNAseq analysis, WCGNA was conducted among gene expression patterns in both inoculated (Pt) and pretreated and fungus-inoculated lemon plants (Citrus limon L.) (3CPt), and two indicators of fungal infection, i.e., the amount of fungus DNA measured in planta and the disease index (DI). The aims of this work were (a) to identify gene modules significantly associated with those traits, (b) to construct co-expression networks related to mal secco disease; (c) to define the effect and action mechanisms of P. mediterranea by comparing the networks. The results led to the identification of nine hub genes in the networks, with three of them belonging to receptor-like kinases (RLK), such as HERK1, CLAVATA1 and LRR, which play crucial roles in plant-pathogen interaction. Moreover, the comparison between networks indicated that the expression of those receptors is not induced in the presence of P. mediterranea, suggesting how powerful WCGNA is in discovering crucial genes that must undergo further investigation and be eventually knocked out.

Assessing Copper-Alternative Products for the Control of Pre- and Postharvest Citrus Anthracnose.

Citrus production is worldwide threatened by Colletotrichum spp., causal agents of pre- and postharvest anthracnose. The recent limitation on the use of copper-based antimicrobials, due to its demonstrated noxious effect on the environment, makes the control of this pathogen difficult. Thus, alternative products able to reduce/phase out copper in organic citrus farming are needed. In this study, the efficacy of 11 commercial alternative products were evaluated in vitro, in growth chamber, in open field and in postharvest environments. In vitro, mineral fertilizers, basic substances, essential oils, plant defense stimulators and biological control agents were able to inhibit the mycelial growth with variable efficacy. On artificially infected citrus fruit, almost all tested products significantly reduced disease incidence and severity, but with lower efficacy than copper. The efficacy of mineral fertilizers-based Kiram and Vitibiosap 458 Plus, citrus essential oil-based Prev-Am Plus and chitosan-based Biorend was confirmed in open field trials, in naturally infected citrus fruits. In these trials Biorend was the best alternative product, significantly reducing disease incidence (71% DI reduction) with better results than copper (47.5%). Field treatments reduced the incidence and severity of the disease in postharvest conditions, especially in fruits field-treated three times. Overall, selected products tested in open field can represent a good alternative to copper compounds in the view of future limitation of its use.

Soil and Soilless Tomato Cultivation Promote Different Microbial Communities That Provide New Models for Future Crop Interventions.

The cultivation of soilless tomato in greenhouses has increased considerably, but little is known about the assembly of the root microbiome compared to plants grown in soil. To obtain such information, we constructed an assay in which we traced the bacterial and fungal communities by amplicon-based metagenomics during the cultivation chain from nursery to greenhouse. In the greenhouse, the plants were transplanted either into agricultural soil or into coconut fiber bags (soilless). At the phylum level, bacterial and fungal communities were primarily constituted in all microhabitats by Proteobacteria and Ascomycota, respectively. The results showed that the tomato rhizosphere microbiome was shaped by the substrate or soil in which the plants were grown. The microbiome was different particularly in terms of the bacterial communities. In agriculture, enrichment has been observed in putative biological control bacteria of the genera Pseudomonas and Bacillus and in potential phytopathogenic fungi. Overall, the study describes the different shaping of microbial communities in the two cultivation methods.

Epiphytic and endophytic microorganisms associated to different cultivar of tomato fruits in greenhouse environment and characterization of beneficial bacterial strains for the control of post-harvest tomato pathogens.

Several bacterial and fungal diseases affect greenhouse-grown tomato crops, causing severe annual yield losses worldwide. The need to reduce chemical compound applications has encouraged the search of alternative approach for the control of tomato diseases, including the use of biological control agents. The presence of total and beneficial microbial populations was investigated on the surface and in the pulp of seven cultivars of tomato fruit coming from eleven greenhouses in the Pachino district (south-east of Sicily), recognized by the European Community with the "Protected Geographical Indication" label. Principal component analysis (PCA) showed that epiphytic and endophytic microbial populations clustered into groups according to the areas of origin. Approximately 240 tomato fruit-associated bacterial isolates were selected and a high percentage of them showed antagonistic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, Xanthomonas euvesicatoria pv. perforans, Botrytis cinerea and Alternaria alternata. Analysis of the 16S rRNA gene sequences revealed a predominance of bacteria in Bacillus and Pseudomonas genera, followed by Citrobacter and Enterobacter. The presence of these genera differed according to the geographical areas of tomato samples, whereas their antagonistic capabilities varied according to the five tomato pathogens. The in vitro effectiveness of eight representative bacterial strains belonging to Pseudomonas, Bacillus and Enterobacter genera was confirmed in in vivo assays, carried out on tomato fruit artificially inoculated with A. alternata and B. cinerea. Altogether, these results revealed differences in population density of native microbiota of "Pomodoro di Pachino" fruits and allowed the selection of antagonistic bacterial strains that can be applied in field and in postharvest conditions as alternatives to chemical compounds.

Pathotyping Citrus Ornamental Relatives with Xanthomonas citri pv. citri and X. citri pv. aurantifolii Refines Our Understanding of Their Susceptibility to These Pathogens.

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca) are causal agents of Citrus Bacterial Canker (CBC), a devastating disease that severely affects citrus plants. They are harmful organisms not reported in Europe or the Mediterranean Basin. Host plants are in the Rutaceae family, including the genera Citrus, Poncirus, and Fortunella, and their hybrids. In addition, other genera of ornamental interest are reported as susceptible, but results are not uniform and sometimes incongruent. We evaluated the susceptibility of 32 ornamental accessions of the Rutaceae family belonging to the genera Citrus, Fortunella, Atalantia, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Casimiroa, Calodendrum, and Aegle, and three hybrids to seven strains of Xcc and Xca. Pathotyping evaluation was assessed by scoring the symptomatic reactions on detached leaves. High variability in symptoms and bacterial population was shown among the different strains in the different hosts, indicative of complex host-pathogen interactions. The results are mostly consistent with past findings, with the few discrepancies probably due to our more complete experimental approach using multiple strains of the pathogen and multiple hosts. Our work supports the need to regulate non-citrus Rutaceae plant introductions into areas, like the EU and Mediterranean, that are currently free of this economically important pathogen.

Plant Growth-Promoting Activity of Pseudomonas aeruginosa FG106 and Its Ability to Act as a Biocontrol Agent against Potato, Tomato and Taro Pathogens.

P. aeruginosa strain FG106 was isolated from the rhizosphere of tomato plants and identified through morphological analysis, 16S rRNA gene sequencing, and whole-genome sequencing. In vitro and in vivo experiments demonstrated that this strain could control several pathogens on tomato, potato, taro, and strawberry. Volatile and non-volatile metabolites produced by the strain are known to adversely affect the tested pathogens. FG106 showed clear antagonism against Alternaria alternata, Botrytis cinerea, Clavibacter michiganensis subsp. michiganensis, Phytophthora colocasiae, P. infestans, Rhizoctonia solani, and Xanthomonas euvesicatoria pv. perforans. FG106 produced proteases and lipases while also inducing high phosphate solubilization, producing siderophores, ammonia, indole acetic acid (IAA), and hydrogen cyanide (HCN) and forming biofilms that promote plant growth and facilitate biocontrol. Genome mining approaches showed that this strain harbors genes related to biocontrol and growth promotion. These results suggest that this bacterial strain provides good protection against pathogens of several agriculturally important plants via direct and indirect modes of action and could thus be a valuable bio-control agent.

Endophytic Bacterial Isolates From Halophytes Demonstrate Phytopathogen Biocontrol and Plant Growth Promotion Under High Salinity.

Halophytic endophytes potentially contribute to the host's adaptation to adverse environments, improving its tolerance against various biotic and abiotic stresses. Here, we identified the culturable endophytic bacteria of three crop wild relative (CWR) halophytes: Cakile maritima, Matthiola tricuspidata, and Crithmum maritimum. In the present study, the potential of these isolates to improve crop adaptations to various stresses was investigated, using both in vitro and in-planta approaches. Endophytic isolates were identified by their 16S rRNA gene sequence and evaluated for their ability to: grow in vitro in high levels of NaCl; inhibit the growth of the economically important phytopathogens Verticillium dahliae, Ralstonia solanacearum, and Clavibacter michiganensis and the human pathogen Aspergillus fumigatus; provide salt tolerance in-planta; and provide growth promoting effect in-planta. Genomes of selected isolates were sequenced. In total, 115 endophytic isolates were identified. At least 16 isolates demonstrated growth under increased salinity, plant growth promotion and phytopathogen antagonistic activity. Three showed in-planta suppression of Verticillium growth. Furthermore, representatives of three novel species were identified: two Pseudomonas species and one Arthrobacter. This study provides proof-of-concept that the endophytes from CWR halophytes can be used as "bio-inoculants," for the enhancement of growth and stress tolerance in crops, including the high-salinity stress.

Trends in Molecular Diagnosis and Diversity Studies for Phytosanitary Regulated Xanthomonas.

Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non-specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular-based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis.

Bioprospecting of Beneficial Bacteria Traits Associated With Tomato Root in Greenhouse Environment Reveals That Sampling Sites Impact More Than the Root Compartment.

Tomato is subject to several diseases that affect both field- and greenhouse-grown crops. To select cost-effective potential biocontrol agents, we used laboratory throughput screening to identify bacterial strains with versatile characteristics suitable for multipurpose uses. The natural diversity of tomato root-associated bacterial communities was bioprospected under a real-world environment represented by an intensive tomato cultivation area characterized by extraseasonal productions in the greenhouse. Approximately 400 tomato root-associated bacterial isolates, in majority Gram-negative bacteria, were isolated from three compartments: the soil close to the root surface (rhizosphere, R), the root surface (rhizoplane, RP), and the root interior (endorhizosphere, E). A total of 33% of the isolates produced siderophores and were able to solubilize phosphates and grow on NA with 8% NaCl. A total of 30% of the root-associated bacteria showed antagonistic activity against all the tomato pathogens tested, i.e., Clavibacter michiganesis pv. michiganensis, Pseudomonas syringae pv. tomato, Pseudomonas corrugata and Xanthomonas euvesicatoria pv. perforans, and Fusarium oxysporum f. sp. lycopersici. We found that the sampling site rather than the root compartment of isolation influenced bacterial composition in terms of analyzed phenotype. This was demonstrated through a diversity analysis including general characteristics and PGPR traits, as well as biocontrol activity in vitro. Analysis of 16S rRNA gene (rDNA) sequencing of 77 culturable endophytic bacteria that shared multiple beneficial activity revealed a predominance of bacteria in Bacillales, Enterobacteriales, and Pseudomonadales. Their in vitro antagonistic activity showed that Bacillus species were significantly more active than the isolates in the other taxonomic group. In planta activity against phytopathogenic bacteria of a subset of Bacillus and Pseudomonas isolates was also assessed.

Fungal Infection Induces Anthocyanin Biosynthesis and Changes in DNA Methylation Configuration of Blood Orange [Citrus sinensis L. (Osbeck)].

The biosynthesis of sweet orange anthocyanins is triggered by several environmental factors such as low temperature. Much less is known about the effect of biotic stress on anthocyanin production in sweet orange, although in other species anthocyanins are often indicated as "defense molecules". In this work, citrus fruits were inoculated with Penicillium digitatum, the causal agent of green mold, and the amount of anthocyanins and the expression of genes related to their biosynthesis was monitored by RT-real time PCR after 3 and 5 days from inoculation (DPI). Moreover, the status of cytosine methylation of DFR and RUBY promoter regions was investigated by McrBC digestion followed in real-time. Our results highlight that fungal infection induces anthocyanin production by activating the expression of several genes in the biosynthetic pathway. The induction of gene expression is accompanied by maintenance of high levels of methylation at the DFR and RUBY promoters in the inoculated fruits, thus suggesting that DNA methylation is not a repressive mark of anthocyanin related gene expression in sweet orange subjected to biotic stress. Finally, by measuring the expression levels of the Citrus DNA demethylase genes, we found that none of them is up-regulated in response to fungal infection, this result being in accordance with the observed maintenance of high-level DFR and Ruby promoter regions methylation.

Production of Polyhydroxyalkanoates and Extracellular Products Using Pseudomonas Corrugata and P. Mediterranea: A Review.

Some strains of Pseudomonas corrugata (Pco) and P. mediterranea (Pme) efficiently synthesize medium-chain-length polyhydroxyalkanoates elastomers (mcl-PHA) and extracellular products on related and unrelated carbon sources. Yield and composition are dependent on the strain, carbon source, fermentation process, and any additives. Selected Pco strains produce amorphous and sticky mcl-PHA, whereas strains of Pme produce, on high grade and partially refined biodiesel glycerol, a distinctive filmable PHA, very different from the conventional microbial mcl-PHA, suitable for making blends with polylactide acid. However, the yields still need to be improved and production costs lowered. An integrated process has been developed to recover intracellular mcl-PHA and extracellular bioactive molecules. Transcriptional regulation studies during PHA production contribute to understanding the metabolic potential of Pco and Pme strains. Data available suggest that pha biosynthesis genes and their regulations will be helpful to develop new, integrated strategies for cost-effective production.

The structure and function of the global citrus rhizosphere microbiome.

Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.

The LuxR Regulators PcoR and RfiA Co-regulate Antimicrobial Peptide and Alginate Production in Pseudomonas corrugata.

Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR-RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.