Genomic sequence data of bacterial isolates from pistachio trees and other woody plants in California are inconsistent with a role of Rhodococcus as the causative agent of Pistachio Bushy Top Syndrome

Pistachio Bushy Top Syndrome (PBTS) is a serious problem for pistachio growers in the western U.S. but the cause of this disorder remains controversial. Recently, it was proposed that the Rhodococcus species, R. fascians and R. corynebacterioides caused PBTS outbreaks in 2011 and 2015. To investigate the association of Rhodococcus spp with PBTS in California’s pistachio growing region, Rhodococcus-like isolates were collected from diverse hosts and environments, including pistachio nurseries and orchards. Whole genome sequence analysis of 231 isolates revealed their evolutionary relationships and identified six Rhodococcus species. Combined with data on geography and host of origin, the data reveal that Rhodococcus generally, and R. fascians specifically, is ubiquitous in nature, frequently occurring in both symptomatic and asymptomatic pistachio trees and on other woody and native species. Core gene and SNP-based phylogenies, and pan-genome analyses differentiate R. fascians into distinct genotypes. Although we found examples of common genotypes shared between nurseries and orchards, the observed patterns are most consistent with an environmental source of strains and do not support a scenario where individual nurseries are point sources of Rhodococcus. Moreover, none of the collected strains harbored known virulence genes, calling into question the role of these common, environmental bacteria in causing PBTS.

Enzyme fatigue limits the detoxification of aflatoxin by Rhodococcus species

Bacterial detoxification of mycotoxins has the potential to offer a low-cost solution to ensure that feed and food commodities contaminated by fungal growth become safe to consume. Among bacteria, Rhodococcus species are of particular interest because they can be metabolically versatile, non-pathogenic, and environment-friendly. However, the native response of Rhodococcus environmental isolates appears inadequate for current detoxification needs. By analyzing the detoxification of aflatoxin by two Rhodococcus species: R. pyridinivorans and R. erythropolis, we examine important features of the dynamics that could guide future optimization of bacterial detoxification. Our results for Rhodococcus species suggest that detoxification happens through a regulated process of secreting extracellular enzymes. We show that enzyme fatigue in the presence of the toxin determines the lifetime of the enzyme and limits the overall detoxification performance of these species. Additionally, we show that the regulation of enzyme production can be both species- and environment-dependent. Overall, our quantitative approach reveals that enzyme fatigue is a major determinant of overall detoxification and needs to be accounted for in assessing the performance of detoxification by live cells or cell-free filtrates.

Responses to Ecopollutants and Pathogenization Risks of Saprotrophic Rhodococcus Species

Under conditions of increasing environmental pollution, true saprophytes are capable of changing their survival strategies and demonstrating certain pathogenicity factors. Actinobacteria of the genus Rhodococcus, typical soil and aquatic biotope inhabitants, are characterized by high ecological plasticity and a wide range of oxidized organic substrates, including hydrocarbons and their derivatives. Their cell adaptations, such as the ability of adhering and colonizing surfaces, a complex life cycle, formation of resting cells and capsule-like structures, diauxotrophy, and a rigid cell wall, developed against the negative effects of anthropogenic pollutants are discussed and the risks of possible pathogenization of free-living saprotrophic Rhodococcus species are proposed. Due to universal adaptation features, Rhodococcus species are among the candidates, if further anthropogenic pressure increases, to move into the group of potentially pathogenic organisms with “unprofessional” parasitism, and to join an expanding list of infectious agents as facultative or occasional parasites.

Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

Sequence analysis of 16S rDNA, gyrB and alkB genes of plant-associated Rhodococcus species from Tunisia

The genus Rhodococcus contains several species with agricultural, biotechnological and ecological importance. Within this genus, many phyllosphere, rhizosphere and endosphere strains are plant growth promoting bacteria, whereas strains designated as R. fascians are plant pathogens. In this study, we isolated 47 Rhodococcus strains from a range of herbaceous and woody plant species. Phylogenetic analysis based on 16S rDNA, gyrB and alkB genes was used to compare our strains with type strains of Rhodococcus . For most of our strains, sequence similarity of the 16S rDNA, gyrB and alkB regions to type strains ranged from 98–100 %. Results of the concatenated gene sequence comparisons identified 18 strains of R. fascians and three strains of R. kroppenstedtii . The remaining strains were unclassified, and may represent novel species of Rhodococcus . Phylogenetic analysis based on gyrB sequences provided a more precise classification of our strains to species level than 16S rDNA sequences, whereas analysis of alkB sequences was unable to identify strains with orange-coloured colonies to species level.

Characterization of a New Rhamnolipid Biosurfactant Complex from Pseudomonas Isolate DYNA270

The chemical and physical properties of extracellular rhamnolipid synthesized by a nonfluorescent Pseudomonas species soil isolate, identified as DYNA270, is described, along with characteristics of rhamnolipid production under varying growth conditions and substrates. The biosurfactant is determined to be an anionic, extracellular glycolipid consisting of two major components, the rhamnopyranoside β-1-3-hydroxydecanoyl-3-hydroxydecanoic acid (GU-6) and rhamnopyranosyl β→β2-rhamnopyranoside-β1-3-hydroxydecanoyl-3-hydroxydecanoic acid (GL-2), of molecular weight 504 and 649 daltons, respectively. These glycolipids are produced in a stoichiometric ratio of 1:3, respectively. The purified rhamnolipid mixture exhibits a critical micelle concentration of 20 mg/L, minimum surface (air/water interface) tension of 22 mN/m, and minimum interfacial tension values of 0.005 mN/m (against hexane). The pH optimum, critical micelle concentration, and effective alkane carbon number were established for Pseudomonas species DYNA270 and compared to those of rhamnolipid produced by Pseudomonas aeruginosa PG201. Significant differences are documented in the physical properties of extracellular rhamnolipids derived from these two microorganisms. The surface properties of this rhamnolipid are unique in that ultra-low surface and interfacial tension values are present in both purified rhamnolipid and culture broth containing the rhamnolipid complex (GU6 and GL2). We are not aware of prior studies reporting surface activity values this low for rhamnolipids. An exception is noted for an extracellular trehalose glycolipid produced by Rhodococcus species H13-A, which measured 0.00005 mN/m in the presence of the co-agent pentanol (Singer et al. 1990). Similar CMC values of 20 mg/L have been reported for rhamnolipids, a few being recorded as 5–10 mg/L for Pseudomonas species DSM2874 (Lang et al. 1984).

Catabolic Fingerprinting and Diversity of Bacteria in Mollic Gleysol Contaminated with Petroleum Substances

This study focused on the determination of both catabolic and genetic fingerprinting of bacteria inhabiting soil contaminated with car fuels. A surface layer (0–20 cm) of Mollic Gleysol was used for the experiment and was contaminated with car fuels—unleaded 95-octane petrol and diesel at a dose of 15 g per 10 g of soil. The experiment lasted 42 days and was performed at 20 °C. The metabolic potential of soil bacterial communities was evaluated using the Biolog EcoPlate system. The results demonstrated that petroleum substances influenced the structure of the microbial populations and their catabolic activity. The Arthrobacter, Paenibacillus, and Pseudomonas genera were found in diesel-contaminated soil, whilst Bacillus and Microbacterium were found in petrol-contaminated soil. Rhodococcus species were identified in both variants of impurities, suggesting the widest capability of car fuel degradation by this bacterial genus. The contamination with unleaded 95-octane petrol caused rapid inhibition of the metabolic activity of soil bacteria in contrast to the diesel treatment, where high metabolic activity of bacteria was observed until the end of the incubation period. Higher toxicity of petrol in comparison with diesel car fuel was evidenced.