ISSN 0026-2617, Microbiology, 2017, Vol. 86, No. 2, pp. 257–263. © Pleiades Publishing, Ltd., 2017. Original Russian Text © T.G. Dobrovol’skaya, K.A. Khusnetdinova, N.A. Manucharova, A.V. Golovchenko, 2017, published in Mikrobiologiya, 2017, Vol. 86, No. 2, pp. 247–254. EXPERIMENTAL ARTICLES Structure of Epiphytic Bacterial Communities of Weeds T. G. Dobrovol’skaya*, K. A. Khusnetdinova, N. A. Manucharova, and A. V. Golovchenko Faculty of Soil Science, Lomonosov Moscow State University, Moscow, Russia *e-mail: [email protected] Received June 15, 2016 Abstract⎯Dynamics of the taxonomic structure of epiphytic bacterial communities of the rhizosphere and phyllosphere of seven weed species was studied. The major types of isolated organisms were identified using phenotypic and molecular biological approaches. Dispersion analysis revealed that the ontogenesis stage and plant organ were the factors with the greatest effect on the taxonomic structure of the communities. The dom- inant microorganisms of weeds were similar to those of cultivated plants. The minor components revealed in the spectra of bacterial communities of weeds belonged to poorly studied genera of chemolithotrophic pro- teobacteria. Keywords: epiphytic bacteria, taxonomic structure, weeds, ontogenesis stages, plant organs, ecological niche DOI: 10.1134/S0026261717020072 At the current stage of development of agricultural cal importance and serves as a model object in plant science, the concept of struggling with weed plants is genomics. The taxonomic composition of epiphytic being replaced by a different paradigm, which implies bacterial communities associated with this plant was management of the weed component of agrophyto- close to those described for other weed and medicinal cenoses (Zakharenko, 2000). In the countries of plants. The authors established that bacterial epi- northern Europe, especially in Ireland, a common phytes colonizing the leaves and roots of A. thaliana practice is targeted planting of herbs along field mar- belonged to the genera Pseudomonas, Sphingomonas, gins (Marshall and Moonen, 2002). The variety of Flavobacterium, Massilia, Rhizobim, and Variovorax. A plants that are grown at field margins includes not only research group from Colombia (Kremer et al., 1990) forest species (such as violet or ivy), but also ruderal isolated rhizobacterial communities associated with ones, e.g., creeping thistle or cleavers; they partially seven weed species. Among all the isolated strains, 11 protect the crops from mechanical damage that can occur at the field edge, as well as from wind and dust. to 42% were fluorescent pseudomonads. Other bacte- Moreover, such grass margins serve as habitats for use- ria commonly isolated from the surface of weed roots ful pollinating insects. belonged to the taxa Erwinia herbicola, Alcaligenes spp., and spp. Canadian microbiolo- Bacterial populations of epiphytic complexes of Flavobacterium weed plants are poorly studied. The available publica- gists investigated six weed species as potential sources tions from different groups worldwide that we are of growth-stimulating rhizobacteria in agrocenoses going to discuss below analyzed the genus-level taxo- (Sturz et al., 2001). Many of these plants were also nomic composition of epiphytic bacterial communi- medicinal: spurrey, sawthistle, Italian ryegrass, couch ties associated with weed plants, but did not consider grass. The most commonly isolated bacteria belonged their dynamics in the course of plant development. to the genera Bacillus, Arthrobacter, Stenotro- Mukhtar et al., (2010) identified epiphytic and endo- phomonas, Acinetobacter, and Pseudomonas. These phytic bacteria associated with four weed species: field bacteria stimulated the growth of potato plants, bindweed, sun spurge, pyrethrum, and lamb’s quar- increasing the weight of shoots and roots. Thus, the ters. In contrast to other similar studies, this work did authors cited above believe that bacterial communities not detect any Pseudomonas or Arthrobacter strains of weed plants are worth thorough investigation from among epiphytic bacteria, but identified members of both the ecological and agrobiological positions. the genera Burkholderia, Acidovorax, Enterobacter, Klebsiella, Peptococcus, and Kurthia. A study from the The goal of the present work was to investigate the United States (Bodenhausen et al., 2013) investigated dynamics of the taxonomic structure of epiphytic bac- epiphytic bacterial communities of thale cress (Arabi- terial communities associated with different organs of dopsis thaliana). This unique weed known as an astro- weed plants in the course of their ontogenesis in differ- naut and a miner plant is also of considerable ecologi- ent ecological niches. 257 258 DOBROVOL’SKAYA et al. MATERIAL AND METHODS 5-days-old cultures. The abundance of bacteria was expressed as colony-forming units per 1 g substrate. Research subjects. The study was conducted on the The genus attribution of the isolated bacterial cultures Chashnikovo Educational and Experimental plot of was established based on their morphological, cul- the Lomonosov University Center of Soil Science and tural, and chemotaxonomic traits using the Bergey’s Ecology located in the Solnechnogorsk district of Manual of Systematic Bacteriology and the guidelines Moscow oblast. The study concerned bacterial com- proposed by Lysak et al. (2003). In addition, genus munities associated with weed plants: shepherd’s identification employed diagnostic systems that deter- purse (Capsella bursa-pastoris), spear saltbush (Atri- mine the biochemical traits of bacteria: the ability to plex patula), brittlestem hempnettle (Galeopsis tetra- utilize sodium citrate, sodium malonate, glucose, lac- hit), field sowthistle (Sonchus arvensis), yellow bed- tose, mannitol, sucrose, inositol, sorbitol, arabinose, straw (Galium verum), bittercress (Barbarea vulgaris), or maltose, the production of indol, hydrogen sulfide, and bishop’s weed (Aegopodium podagraria). All these acetylmethylcarbinol (Voges-Proskauer test), the weed plants are apophytes: representatives of the local activity of urease, β-galactosidase, ornithine and flora that have spread from their natural habitats into lysine decarboxylases, arginine dehydrolase, or phe- the territories modified by human agricultural activity nylalanine deaminase. (arable lands and pasture grounds). Taxonomic composition of proteobacteria was The same plants species were collected in the cen- determined by molecular analysis of the 16S rRNA ter and at the edge of the experimental field, as well as genes, which involved isolation of DNA from pure in the neighboring mixed forest. Weed specimens were bacterial cultures, and subsequent amplification and collected at the stages of shooting (June 08, 2013), sequencing of the relevant gene fragments. flowering (July 27, 2013), and seed production (August 25, 2013). The study considered microbial DNA isolation. Total DNA was isolated from phyl- communities isolated from plant leaves, flowers, and losphere and rhizosphere samples, as well as from pure roots, as well as from the underlying soil. microbial cultures using a PowerSoil DNA Isolation Kit (MO BIO, United States) with modifications The soil of the test field was sod-podzolic, well cul- described by Manucharova et al. 2008). tivated, middle loamy, on clay loam mantle seated on PCR primers and DNA sequencing. PCR amplifi- red brown loamy moraine. The soil had the following cation and subsequent sequencing of the 16S rRNA characteristics: pH 6.5; humus content, 4.7%; K2O gene fragments were performed using a universal content, 6.7 mg/100 g soil, and P2O5 content, primer system (Manucharova et al., 2008; Edwards 98 mg/100 g soil. et al., 1989). In the mixed forest predominated by hardwood tree Phylogenetic analysis. Preliminary analysis of the species, the soil was sod-podzolic, loamy, on clay obtained 16S rRNA gene sequences was performed loam mantle seated on fluvioglacial sands. The soil using the software service of the GenBank database had the following characteristics: pH 3.95; humus (http://www.ncbi.nlm.nih.gov/blast). content, 2.02%; K2O content, 0.37 mg/100 g soil; Next, the sequences were aligned and edited using P2O5 content, 4.6 mg/100 g soil. the BioEdit software (http://jwbrown.mbio.ncsu.edu/ Treatment of samples and isolation of bacterial cul- BioEdit/bioedit.html). Phylogenetic trees were con- tures. Weed leaves, roots, and flowers were chopped structed using the neighbor-joining algorithm (NJ) with scissors, and 1-g substrate aliquots, as well as 1-g with the MEGA 4 software. The tree significance was soil samples were placed in flasks with 100 mL sterile assessed by bootstrap support analysis with 1000 alter- water. Samples were shaken on a Vortex to induce native trees (Thompson et al., 1994). The newly microbial desorption, the liquid phase was separated obtained gene sequences were deposited into Gen- by centrifugation (5000 rpm, 5 min), and the abun- Bank under individual accession numbers. dance and composition of the released bacterial com- munities were determined by plating. Bacteria were RESULTS AND DISCUSSION plated in five replicates from 105–108 dilutions on solid glucose-peptone-yeast medium (g/L): peptone, 0.5; Altogether, we analyzed 118 specimens of weed glucose, 0.5; yeast extract, 0.5; casein hydrolysate, 0.5; plants, including leaves, roots, and flowers collected at agar, 10; chalk, 5; water, to 1 L. The medium was sup- different stages of plant development. The abundance plemented