Structure of Epiphytic Bacterial Communities of Weeds T

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 dominant 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 proteobacteria.

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 communities 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 with 50 mg nistatin to inhibit fungal of epiphytic bacteria colonizing plant leaves and roots growth. The cultures were grown for 7 to 10 days at ranged from 106 to 109 CFU/g. Moreover, the bacte- room temperature. After counting the total number of rial abundance tended to increase by 2‒3 orders of colonies, they were analyzed by light microscopy with magnitude at the stages of flowering and seed produc- phase contrast using a MikMed-2 microscope tion, especially on leaves. The density of bacterial (LOMO, Russia). The dominant bacteria obtained populations on weed flowers was close to the density on solid medium were isolated as pure cultures. Mor- of leaf populations, 106‒108 CFU/g. The abundance phological traits were analyzed in early (24 h) and 3- to of bacteria in soil samples was constantly lower,

MICROBIOLOGY Vol. 86 No. 2 2017 STRUCTURE OF EPIPHYTIC BACTERIAL 259

100 Leaves

Bacillus Pantoe CytophagaAdvenella AzospirillumParacoccus Arthrobacter Myxococcales RhodococcusMicrococcusPseudomonas Sphingobacterium Stenotrophpmonas

100 Roots

Bacillus Cytophaga Advenella ArthrobacterMyxococcales Streptomyces Pseudomonas Sphingobacterium

Fig. 1. Frequency of domination of different bacterial taxa on the leaves and roots of weed plants, %.

105‒107 CFU/g. We obtained a collection of epiphytic lize cellobiose. It should be noted that the genus bacteria that comprised 590 strains. Based on the phe- Sphingobacterium, which was described by Yabuuchi notypic and molecular biological data, 16 taxa could et al. (1983), differs from Flavobacterium by the pres- be identified. ence of sphingophospholipids. These genera include eccrisotrophic bacteria, which utilize various plant Taxonomic composition of epiphytic bacterial com- exometabolites: sugars, organic acids, or amino acids, munities. Phyllosphere and rhizosphere communities as substrates. differed in the frequency of domination of bacterial taxa (Fig. 1). The leaf communities of all weed plants Members of the genus Arthrobacter are soil actino- were most commonly dominated by bacteria of three bacteria. They reach the above-ground plant organs genera: Sphingobacterium, Arthrobacter, and with soil particles, not only those coming from the Cytophaga. The genera Sphingobacterium and roots, but also those brought by the wind, atmospheric Cytophaga belong to different classes of the same phy- dust, or precipitation. For a long time, nothing was lum, Bacteroidetes. Whereas members of Cytophaga known about their possible survival in the phyllo- are typical cellulolytics, representatives of Sphingobac- sphere. However, a recent work showed that four terium degrade starch, and only a few species can uti- Arthrobacter species could actively proliferate on

MICROBIOLOGY Vol. 86 No. 2 2017 260 DOBROVOL’SKAYA et al.

Table 1. Factors affecting the relative abundance of different bacterial groups in the samples studied Variation by factor grades* Degrees of freedom Variance Fisher’s test Significance Proteobacteria 1 2 1035.34 157.934 <0.001 2 1 456.33 69.610 3 Not significant 4 2 132.56 20.222 Residual 72 6.56 Cellulose degraders (cytophagas + myxobacteria) 1 2 3560.9 83.458 <0.001 2 1 3125.6 73.255 3 1 485.6 11.380 4 2 3043.4 71.331 Residual 72 42.7 Gram-positive bacteria (actinobacteria + bacilli) 1 2 1537.9 45.869 <0.001 2 1 1134.3 33.830 3 1 616.3 18.383 4 2 1805.4 53.848 Residual 72 33.5 * 1, stage of plant development (shooting, flowering, seed production); 2, plant organ (leaves, roots); 3, microniche inhabited (field, field edge, forest); 4, plant species. apple-tree leaves by utilizing phenol, chlorophenol, facultative chemolithotrophs capable of oxidizing and phenanthrene compounds that are present in the tetrathionates and thiosulfate. Communities associ- atmosphere as anthropogenic pollutants, in particular, ated with forest weeds also contained bacteria of the due to pesticide application (Sheublin and Leveau, genus Stenotrophomonas, close to Pseudomonas 2012). In this context, the authors discussed the maltophila (class Gammaproteobacteria). potential role of arthrobacteria in the remediation of both soils and plants from environmental pollutants. All samples obtained from the weeds collected in the middle of the cultivated field contained only mem- The weed root communities were frequently domi- bers of the genus Sphingobacterium, which belongs to nated by the same bacteria as found on leaves (Arthro- the phylum Bacteroidetes, class Bacteroidia, family bacter and Sphingobacterium), as well as by active Bacteroidaceae. Based on their phenotypic traits, we hydrolytics, although belonging to other taxa than identified the isolated bacteria as members of the those found in the phyllosphere: mainly bacilli (genus genus Flavobacterium, also of the phylum Bacteroide- Bacillus) and myxobacteria (order Myxococcales). tes. The leaves and flowers of the same weed species Dynamics of the structure of epiphytic bacterial were colonized by different bacteria (Fig. 4). For communities. The objectives of this study were not lim- example, while the epiphytic community of bittercress ited to determining the general structure of bacterial leaves included over 60% of cytophages, the flower communities associated with weed plants and the total community was dominated by facultative anaerobes of number of bacterial taxa in them. The most challeng- the genus Pantoea (previously Erwinia herbicola). ing was to follow the dynamics of the taxonomic struc- Sphingobacteria and rhodococci were present as ture of epiphytic bacterial communities associated minor members of the community. with different plant organs in the course of develop- The minor proteobacterial components of the ment of different weed species. We were also taking communities are worth special consideration (Table into account the differences between the ecological 2). These are bacteria of the genera Advenella, Tetra- niches occupied by plants in question: forest, the edge, thiobacter, and Stenotrophomonas, which were isolated or the middle of the field. The vast body of data on the only from the weeds of forest habitats. The first two taxonomic composition of bacterial communities was genera belong to the family Alcaligenaceae of the order analyzed using 4-way analysis of variance. The results Burkholderiales. Members of Tetrathiobacter, later (Table 1) showed that the principal factor affecting the reclassified as the genus Advenella, were described as taxonomic composition of epiphytic bacterial com-

MICROBIOLOGY Vol. 86 No. 2 2017 STRUCTURE OF EPIPHYTIC BACTERIAL 261

Table 2. Taxonomic composition of proteobacteria associated with weed plants as determined by molecular genetic analysis Plant species Plant organ Collection period Microniche Bacterial subclass Bacterial genus Spear saltbush Leaves Autumn Forest Betaproteobacteria Tetrathiobacter kashmirensis* Shepherd’s purse Leaves Autumn Forest Betaproteobacteria Advenella kashmirensis Shepherd’s purse Roots Summer, flowering Forest Betaproteobacteria Advenella kashmirensis Hempnettle Leaves Spring, shootig Forest Gammaproteobacteria Stenotrophomonas sp. Bittercress Flowers Summer, flowering Field edge Gammaproteobacteria Pantoea sp. * Currently, bacteria Tetrathiobacter kashmirensis have been reclassified as Advenella kashmirensis (Thompson et al., 1994). munities was the stage of plant development, followed resentatives of Sphingobacterium. At the seed produc- by the plant organ and the ecological niche (in the tion stage, the monodominant group were spore- order of decreasing significance). The effect of plant forming bacteria of the genus Bacillus, which are typi- species was not significant; in other words, plants of cal hydrolytics. different species were associated with basically similar Thus, both the phyllosphere and the rhizosphere bacterial communities. bacterial communities of sowthistle exhibited major The taxonomic structure of bacterial communities changes in their taxonomic and ecotrophic structure. in the course of sowthistle development provides a In particular, eccrisotrophic bacteria present at the good example of sharp changes (Fig. 2). At the shoot- flowering stages were replaced by hydrolytic bacteria ing stage, leave communities were dominated by at the stage of seed production, i.e., with the aging of arthrobacters and cellulolytic cytophagas and myxo- plant tissues. bacteria. At the flowering stage, the single dominant To sum up, our study showed that the development group was the genus Sphingobacterium, while the bac- of weed plants is associated with changes in both the terial taxa detected on the shoots were present as taxonomic and the ecotrophic structure of epiphytic minor components of the community. Finally, at the bacterial complexes. In particular, eccrisotrophic bac- seed production stage, the dominant species changed teria inhabiting young plants are gradually replaced by again: sphingobacteria were replaced by cellulose- hydrolytic bacteria and actinobacteria in the course of degrading bacteria of the order Myxococcales, and bac- plant tissue aging. Based on the calculated frequency teria of the genus Cellulomonas appeared as new mem- of domination of different bacterial taxa on the leaves bers of the community. and roots of plants studied, it was shown that phyllo- Next, let us consider the dynamics of the taxo- sphere and rhizosphere communities were dominated nomic structure of the epiphytic bacterial community by different bacterial groups. of sowthistle roots (Fig. 3). At the shooting stage, two The distinction was especially clear in the compo- dominant groups typical for soil communities were sition of hydrolytic bacteria: the phyllosphere was detected: Streptomyces and Arthrobacter. At the flower- dominated by cytophagas, and the rhizosphere, by ing stage, the rhizosphere community was dominated myxobacteria and bacilli. It should be noted that the by the same bacteria as the phyllosphere complex: rep- spectrum of dominant groups was very similar to that

Shooting Flowering Seed production

Arthrobacter Sphingobacterium

Cytophaga Myxococcales Cellulomonas Pseudomonas

Fig. 2. Taxonomic structure of bacterial communities of sowthistle phyllosphere in the course of plant ontogenesis, %.

MICROBIOLOGY Vol. 86 No. 2 2017 262 DOBROVOL’SKAYA et al.

Shooting Flowering Seed production

Arthrobacter Cellulomonas

Rhodococcus Myxococcales

Bacillus Micrococcus

Streptomyces Sphingobacterium

Cytophaga Pseudomonas

Fig. 3. Taxonomic structure of bacterial communities of sowthistle rhizosphere in the course of plant ontogenesis, %.

Leaves Flowers

Arthrobacter Pantoe

Rhodococcus Myxococcales

Cytophaga Micrococcus

Streptomyces

Fig. 4. Taxonomic structure of bacterial communities of the bittercress phyllosphere in the flowering period (leaves vs flowers), %. we described previously for cultivated crops (Dobro- species of chemolithotrophic bacteria of the genera vol’skaya et al., 2016). Interesting data were obtained Tetratiobacter and Advenella capable of oxidizing by molecular biological identification of minor com- tetrathionates and thiosulfate, as well as members of ponents of bacterial communities associated with the genus Stenotrophomonas close to Pseudomonas plants collected in the forest habitat. We detected rare maltophila.

MICROBIOLOGY Vol. 86 No. 2 2017 STRUCTURE OF EPIPHYTIC BACTERIAL 263

ACKNOWLEDGMENTS Manucharova, N.A., Vlasenko, N.A., Zenova, G.M., Dobrovol’skaya, T.G., and Stepanov, A.L., Methodologi- This work was financially supported by the Russian cal aspects of assessing chitin utilization by soil microorgan- Science Foundation (grant no. 14-50-00029, 50%) isms, Biol. Bull., 2008, vol. 35, no. 5, pp. 549–554. and the Russian Foundation for Basic Research (proj- Marshall, E.J.P. and Moonen, A.C. Field margins in north- ect no. 15-29-02499, 50%). ern Europe: their functions and interactions with agriculture, Agriculture, Ecosystems, Environment, 2003, vol. 89, no. 1–2, pp. 5–21. REFERENCES Mukhtar, I., Khokhar, I., Mushtag, S., and Ali, A., Diver- Bergey’s Manual of Systematic Bacteriology, vol.1–2, sity of epiphytic and endophytic microorganisms in some Holt, J.G., Ed., Baltimore: Williams and Wilkins, 1986. dominant weeds, Pak. J. Weed Sci. Res., 2010, vol. 16, pp. 287–297. Bodenhausen, N., Horton, M.W., and Bergelson J., Bacte- rial communities associated with the leaves and the roots of Sheublin, T.R. and Leveau, J.H.J., Isolation of Arthrobacter Arabidopsis thaliana, PLoS One, 2013, vol. 8, no. 2, pp. 1–9. species from the phyllosphere and demonstration of their epiphytic fitness, Microbiology Open, 2013, vol. 2, no. 1, Dobrovol’skaya, T.G., Khusnetdinova, K.A., Manucha- pp. 205–213. rova, N.A., and Balabko, P.N., The structure and functions Sturz, A.V., Matheson, B.G., Arsenault, W., Kimpinski, J., of bacterial communities in an agrocenosis, Euras. Soil Sci., and Christie B.R., Weeds as a source of plant growth pro- 2016, vol. 49, no. 1, pp. 70–76. moting rhizobacteria in agricultural soils, Can. J. Micro- Edwards, U., Rogall, T., Bloeker, H., Ende, M.D., and biol., 2001, vol. 47, pp. 1013–1024. Boeettge, E.C., Isolation and direct complete nucleotide Thompson, J.D., Higgins, D.G., and Gibson, T.J., determination of entire genes, characterization of gene cod- Improving the sensitivity of progressive multiple sequence ing for 16S ribosomal RNA, Nucl. Acids Res., 1989, vol. 17, alignment through sequence weighting, positions-specific pp. 7843–7853. gap penalties and weight matrixchoice, Nucl. Acids Res., Gibello, A., Vela, A.I., Martin, M., Bara-Caracciiolo, A., 1994, vol. 22, pp. 4673–4680. Grenni, P., and Fernangez-Garayzarball, J.F., Reclassifi- Yabuuchi, E., Kanenko, T., Yano, I., Moss, C.W., and Miy- cation of the members of the genus Tetrathiobacter Ghosh oshi, N., Sphingobacterium gen. nov., Sphingobacterium et al. 2005 to the genus Advenella Coenye et al. 2005, Int. J. spiritivorum comb. nov., Sphingobacterium mizutae sp. nov., Syst. Evol. Microbiol., 2009, vol. 59, pp. 1914–1918. and Flavobacterium indologenes sp. nov.: glucose-nonfer- Kremer, R.J., Begonia, M.F.T., Stanley, L., and menting gram-negative rods in CDC groups IIK-2 and IIb, Lanham, E.T., Characterization of rhizobacteria associated Int. J. Syst. Bacteriol., 1983, vol. 33, pp. 580–598. with weed seedlings, Appl. Environ. Microbiol., 1990, Zakharenko, A.V., Teoreticheskie osnovy upravleniya sornym vol. 56, pp. 1649–1655. kmoponentom agrpfitotsenoza v sistemakh zemledeliya (The- Lysak, L.V., Dobrovol’skaya, T.G., and Skvortsova, I.N., oretical Basics of Controlling the Weed Component of Metody otsenki bakterial’nogo raznoobraziya pochv i identi- Agrophytocenoses in Agricultural Systems), Moscow: 2000, fikatsii pochvennykh bakterii (Methods for Assessment of Soil MSKhA. Bacterial Diversity and Identification of Soil Bacteria), Mos- cow: MAKS, 2003. Translated by D.Timchenko

SPELL: 1. OK

MICROBIOLOGY Vol. 86 No. 2 2017