New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER Research Paper Inoculation with microorganisms of Lolium perenne L.: evaluation of plant growth parameters and endophytic colonization of roots Francesca Gaggı̀a, Loredana Baffoni, Diana Di Gioia, Mattia Accorsi, Sara Bosi, Ilaria Marotti, Bruno Biavati and Giovanni Dinelli Department of Agricultural Sciences, Alma Mater Studiorum-University of Bologna, v.le Fanin 44, Bologna 40127, Italy Turfgrasses are not only designed for recreation activities, but they also provide beneficial environmental effects and positively influence the human wellness. Their major problems are predisposition to tearing out and microbial diseases. The aim of this study was to investigate whether the inoculation of microorganisms can be effective to improve plant growth and root development of perennial ryegrass, to evaluate new sustainable practice for green preservation. A microorganism-based commercial product was used to amend hydroponically grown Lolium perenne L. and results compared with the use of the same filtered product, a phytohormone solution and an untreated control. Plants were grown for five weeks, shoots cut and measured at one-week interval and, at the end, roots were measured for length and weight. Shoot resistance to tearing out was also tested. Moreover, the main microbial groups present in the product were characterized and the microbial profile of sand and root samples was investigated by PCR-DGGE. The plants treated with the product showed an increased resistance to tearing out with respect to other treatments and roots were longer with respect to the control. Microbial analyses of the product evidenced bacterial and yeast species with plant growth promoting activity, such as Stenothrophomonas maltophilia, Candida utilis and several Lactobacillus species. Some Lactobacillus strains were also found to be able to colonize plant roots. In conclusion, the treatment with microorganisms has a great potential for the maintenance and increased performance of turfgrass surfaces. Introduction Turfgrasses are the primary plant covers for football fields, golf courses, home lawns, parks, and roadsides and are typically thought of for recreation and aesthetic value. However, they also provide a valuable environmental service by preventing soil erosion from wind and rain and improving soil absorption of water [1]. The turfgrass industry has grown rapidly since the 1970s when lands were developed to accommodate an expanding suburban population and to meet the increasing request of sport surfaces. Unfortunately, high standards for aesthetics and playability, necessarily rely on preventive chemical treatments (e.g. fertilizers, Corresponding author Baffoni, L. (loredana.baffoni@unibo.it) herbicides, fungicides). Some cultural practices can promote, for example, nitrogen availability but simultaneously increase susceptibility to diseases, such as Phytium blight and brown patch [2]. A new concept on turfgrass management is required to avoid chemical treatment abuses as well as a biodiversity reduction. Bio-control and other sustainable intervention measures are currently being studied in turf systems to improve turfgrass management in an environmental sustainable way [3]. The concept of plant–microbe interaction to promote plant growth and to combat diseases dates back in the 1980s [4] and, recently, a renewed interest has been dedicated to the use of microbial inoculants [5]. A wide range of bacteria and fungi has been the subject of considerable studies and strains belonging to 1871-6784/$ - see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2013.04.006 www.elsevier.com/locate/nbt 695 RESEARCH PAPER Research Paper different genera have been isolated both from the rhizosphere and inside plant tissues. Numerous authors reported the plant growth promoting abilities of such microorganisms as well as their protective role against plant pathogens. The improved cycling of nutrients and minerals such as nitrogen, phosphate, indole acetic acid (IAA) production [6], the production of siderophores [7], stomatal regulation and modification of root morphology are among the beneficial mechanisms associated with their presence. Additionally, these microorganisms can produce secondary metabolites including antibiotics, antifungal, antiviral, insecticidal and immuno-suppressant agents [8]. Diseases of fungal, bacterial, viral origin and in some instances even damages caused by insects and nematodes can be reduced following inoculation with bacterial/ fungal endophytes [9–12]. This work is focused on the effect of inoculation of a commercial product containing a mixture of microorganisms on hydroponically grown perennial ryegrass (Lolium perenne L.), in comparison with the filtered product, a plant hormone-rich (HRM) solution and an untreated control. Perennial ryegrass is a cool-season turfgrass species that is widely used on home lawns, sports fields, and golf courses due to its rapid establishment and excellent trampling tolerance. The objective of this study was to collect data on aboveground and underground biomass to evaluate the efficacy of the bio-inoculum; a special attention was given to root development and resistance to tearing out, representing a crucial issue for the maintenance of lawns and sport fields. Moreover, a combination of microbiological and molecular analyses has been applied to characterize and quantify the main microbial groups declared in the label (lactobacilli, yeasts and aerobic bacteria). PCR-DGGE analyses were also performed to investigate differences in the microbial profile of sand and root samples and the endophytic ability of inoculated microorganisms. Materials and methods Seed source and plant growth condition Perennial ryegrass (L. perenne L.) seeds used in this study were provided by Fratelli Ingegnoli (Milan, Italy). An aggregate hydroponic system with complete recycling of nutrient solution was used. Seeds were sown in pots of 10 cm diameter  12 cm height, filled with a bottom layer of 2 cm of pumice stone and a sand layer on top. The nutrient solution was prepared according to Hoagland and Arnon [13] and diluted 1:1 (v/v) with distilled water. Plant water uptake was weekly compensated with fresh nutrient solution. The re-circulating nutrient solution was automatically pumped by a drip irrigation system in daily irrigations. Plants were placed in a growth chamber set at 248C and 70% relative humidity (RH) during the day, and 208C and 50% RH in night conditions. Light was supplemented to a 12-hour photoperiod with artificial illumination at 550 mmol photons m 2 s 1. Treatments and growth measurements The efficacy of the commercial product EM11 (Punto EM, Sanremo, Italy), composed of a mixture of microorganisms, water and molasses, was evaluated. The experimental plan was a completely randomized design with four treatments, each consisting of eight pots placed in a random position on a shelf in the growth chamber. Treatments consist of distilled water (control – CTR), activated EM11 (EM), 696 www.elsevier.com/locate/nbt New Biotechnology  Volume 30, Number 6  September 2013 filtered/activated EM11 (F-EM) and a commercial plant hormone-rich solution (HRM) containing gibberellic acid (GA3) (1.65 g/l) and naphthalene acetic acid (NAA) (3.30 g/l) (Fast-Speed Top, Agrisystem, Lamezia Terme, Italy). Before using the product, an activation step is required, which consists in the addition of 5% (v/v) EM11 and 5% (v/v) molasses in water followed by incubation at 35  18C for five days. To prepare the filtered solution, the activated product was filtered through a 0.20 mm nylon filter (Millipore). Both activated and filtered solutions were diluted 1:500 (v/v) in water, HRM was diluted 1:2000 (v/v), and used in the irrigation system. During the course of the experiment, plants were cut 2 cm above the soil level with a cutting interval of one week, for five weeks. After each collection, the shoots (stems + leaves) were measured (cm) and weighed (fresh and dry weight); total shoot length (SL), total shoot fresh (SFW) and dry weight (SDW) were determined as sum of all measurements. At the end of the experiment, roots were sampled, washed and measured for length (RL), fresh (RFW) and dry weight (RDW). Finally, shoot resistance to tearing out (TR) was tested by a dynamometer (PCE Instrument, Lucca, Italy). The method employed consisted of measuring the load (g) necessary to tear out a fresh shoot sample. Enumeration, isolation, purification and maintenance of microorganisms Viable cell counts were carried out to determine the bacterial concentration of the product before and after the activation step. 10 ml of the two solutions were suspended in 90 ml sterile water, and serial dilutions plated on selected media. Lactobacilli were enumerated by inoculation on de Man, Rogosa and Sharpe Broth (MRS, Merck, Darmstadt, Germany) containing 0.2% (w/v) sorbic acid (Sigma–Aldrich, Milan, Italy) and 0.1% (w/v) cycloheximide (Sigma–Aldrich) to inhibit growth of yeasts, and incubated anaerobically (2–3 days, 308C). Aerobic mesophilic bacteria were enumerated by inoculation on Nutrient Agar (NA, Merck) containing 0.1% (w/v) cycloheximide and incubated for 3 days at 308C. Yeasts were enumerated by inoculation on Sabouraud Dextrose Agar (SDA, Merck) containing 0.1% (w/v) chloramphenicol (Sigma– Aldrich) after 3–5 day incubation at 258C. Each analysis was conducted in triplicate. Following incubation the number of colony forming units (cfu)/ml was recorded and means and standard deviations were calculated. An average of 25 colonies from the activated product were selected from each medium, re-streaked and purified for further characterization. For long-term storage purified isolates were stored at 808C. Phenotypic characterization of lactobacilli, aerobic and yeasts isolates Presumptive lactobacilli isolates were characterized on the basis of cell morphology, Gram staining, catalase and oxidase reactions. The carbohydrate fermentation profiles were determined using the commercial API1 50CH test (BioMérieux, Marcy l‘Etoile, France), followed by APILAB software (version 5.1) analyses. Aerobic bacteria were also studied considering cell morphology, Gram staining, catalase and oxidase reactions. Concerning yeasts, cells grown for 3–5 days on Sabouraud Dextrose Broth (SDB, Merck) were identified using the commercial API1 C-AUX test (BioMérieux). New Biotechnology  Volume 30, Number 6  September 2013 Cells were harvested from 2 ml of overnight cultures and used for chromosomal DNA extraction, using the Wizard1 Genomic DNA Purification Kit (Promega, Madison, USA). Genus-specific PCR was performed according to Walter et al. [14] to confirm the affiliation to the genus Lactobacillus. Molecular biology-based grouping of lactobacilli and aerobic isolates was performed by ERIC-PCR with primers ERIC-1 (50 ATGTAAGCTCCTGGGGATTCAC-30 ) and ERICII (50 -AAGTAAGTGACTGGGGTGAGCG-30 ) [15]. After electrophoresis (2% w/v agarose gel at 50 V for four hours), gels were stained with ethidium bromide and visualized with the gel documentation system Gel DocTM XR (Bio-Rad, Hercules, CA, USA). Images were elaborated with ImageLab software (Bio-Rad) and a binary matrix has been constructed. Mega 5.1 was employed to obtain the phylogenetic trees based on neighbor-joining method [16]. DNA extraction For PCR-DGGE analysis, bacterial DNA was extracted from sand and root samples of the four treatments, and from the activated product. Root samples were previously surface-sterilized with consecutive washes in ethanol 70%, water, NaOCl (2%) and water (3 times) [17], snap frozen in liquid nitrogen and then ground by mortar and pestle. Metagenomic DNA from approximately 250 mg sand and 100 mg of roots was extracted using the PowerSoil DNA kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions with some adjustments. In particular, 5 ml of mutanolysin (100 U/ml, Sigma–Aldrich) and 195 ml of lysozyme (50 mg/ml, Sigma–Aldrich) were added to the soil and root powder in the bead solution supplied with the kit. The suspension was then incubated at 378C on a rotary shaker for two hours, before chemical (SDS-containing solution) and mechanical (bead beating on vortex at maximum speed for 10 min) cell lysis. DNA was eluted in 100 ml of TE buffer pH 8.0. Bacterial DNA was extracted from the activated product, according to Iacumin et al. [18]. The purity and concentration of extracted DNA were determined by measuring the ratio of the absorbance at 260 and 280 nm (Infinite1 200 PRO NanoQuant, Tecan, Mannedorf, Switzerland). The DNA was stored at 208C. 16S rRNA gene PCR-DGGE analysis For bacterial PCR-DGGE analysis on sand and root samples, PCR amplification of 16S rDNA was performed with universal primers 357f with GC clamp (50 -CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCTACGGGAGGCAGCAG-30 ) and 907r (50 -CCGTCAATTCCTTTGAGTTT-30 ) [19]. A further PCRDGGE targeting LAB (Lactic Acid Bacteria) and using primers Lac1f (50 -AGCAGTAGGGAATCTTCCA-30 ) and Lac2r with GC clamp (50 CGCCCGGGGCGCGCCCCGGGCGGCCCGGGGGCACCGG-30 ) [14], was performed on: (i) metagenomic DNA extracted from roots; (ii) activated product; and (iii) lactobacilli isolates. The use of these primers generates a PCR fragment of about 600 bp and 340 bp respectively, suitable for a subsequent DGGE analysis. All reactions for PCR-DGGE were carried out in a 50 ml volume containing 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystem), 5 ml of 10 PCR Gold Buffer (Applied Biosystem), 200 mM of each deoxynucleotide triphosphate (Fermentas GmbH, St. Leon-Rot, Germany), 1.50 mM MgCl2 (Fermentas), 0.45 mM of each primer (MWG), 2.5% (w/v) bovine serum albumin (Fermentas), 4 ml DNA template, and sterile MilliQ water. The PCR with universal primers was performed under the following thermocycling program: 5 min initial denaturation at 958C; 35 cycles of 958C for 30 s, 558C for 60 s, 728C for 40 s; followed by a final elongation step of 728C for 7 min. PCR with primers Lac1f and Lac2r-GC was carried out according to Walter et al. [14]. The size and amount of the PCR products were estimated by analyzing 2 ml samples by 1.5% agarose gel (w/v) electrophoresis and ethidium bromide staining. The DGGE analysis was basically performed as first described by Muyzer et al. [20], using a DCode System apparatus (Bio-Rad). Polyacrylamide gels [7% (w/v) acrylamide:bisacrylamide (37.5:1) (Bio-Rad)] in 1 Tris-Acetate-EDTA (TAE) buffer were prepared using a Bio-Rad Gradient Delivery System (Model 475, Bio-Rad), using solutions containing 35–55% denaturant [100% denaturant corresponds to 7 M urea (Sigma–Aldrich) and 40% (v/v) formamide (Sigma–Aldrich)]. The electrophoresis was run at 55 V for 16 hours at 608C. Gels were stained in a solution of 1 SYBR-Green (Sigma– Aldrich) in 1 TAE for 20 min and their images captured in UV transillumination with Gel DocTM XR apparatus (Bio-Rad). Selected dominant bands were cut from the gel with a sterile scalpel and DNA was eluted by incubating the gel fragments for 16 hours in 50 ml of sterile deionized water at 48C. 2 ml of the solution were then used as template to re-amplify the band fragments using the same primers without the GC-clamp and the same PCR conditions. Sequence analysis of 16S rDNA of pure cultures and DGGE bands On the basis of phenotypic and genotypic investigations, representative isolates (lactobacilli and aerobic bacteria) were selected and the 16S rDNA amplification performed with universal primers 27f and 1492r [21]. The amplified 16S rDNA were then purified (PCR clean-up; Macherey-Nagel GmbH & Co. KG, Germany) and sequenced (Eurofins MWG Operon, Ebersberg, Germany). Concerning PCR-DGGE bands, the obtained amplicons were sequenced with primer 907r and Lac1f. Sequence chromatograms were edited and analyzed using the software programs Finch TV version 1.4.0 (Geospiza Inc., Seattle, WA, USA) and obtained sequences were subjected to taxon classification using RDP classifier, an available tool at the RDP-II website (http://rdp.cme. msu.edu/classifier/classifier.jsp). Moreover, SeqMatch search was used to find the closest match for each 16S rRNA fragment (http:// rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp) [22]. Statistical analysis The statistical software CoSTAT Version 6.002 (CoHort Software, Monterey, California, USA) was used for analyses of variance. Comparisons between the means were conducted using the Student–Newman–Keuls test. Significant differences (P < 0.05) among treatments were marked with different letters. Results and discussion Growth responses and physiological features The effect of inoculation of the EM product on hydroponically grown L. perenne L. was evaluated and compared to the use of the same filtered product, the HRM solution and an untreated control. www.elsevier.com/locate/nbt 697 Research Paper Genotypic characterization of lactobacilli and aerobic isolates RESEARCH PAPER New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER TABLE 1 Effects of different amendments (Control = distilled water; EM = activated EM1W, F-EM = filtered EM1W, HRM = phytohormone mixture) on total shoot fresh (SFW) and dry (SDW) weight, total shoot length (SL), root fresh (RFW) and dry (RDW) weight, root length (RL) and tearing out resistance (TR) of Lolium perenne L. hydroponically grown under controlled conditions. Data are expressed as mean values  SD (n = 8). Mean values in the same row with common letters are not significantly different (P < 0.05) Treatment Control EM EM-F HRM SFW (g) 10.3  0.9 (c) 13.8  1.6 (b) 13.8  1.5 (b) 17.8  2.0 (a) SDW (g) 1.62  0.24 (b) 1.85  0.31 (b) 1.86  0.17 (b) 2.77  0.88 (a) SDW/SFW 0.16  0.02 (a) 0.13  0.01 (b) 0.14  0.01 (b) 0.16  0.04 (a) SL (cm) 48.5  1.8 (d) 54.3  1.7 (c) 56.6  2.6 (b) 58.4  2.2(a) Research Paper RFW (g) 7.9  1.4 (ab) 6.0  2.4 (c) 6.6  1.6 (ab) 8.3  2.1 (a) RDW (g) 1.28  0.28 (a) 0.86  0.52 (b) 0.86  0.23 (b) 1.02  0.43 (ab) RDW/RFW 0.17  0.04 (a) 0.14  0.03 (b) 0.13  0.02 (b) 0.12  0.03 (b) RL (cm) 14.2  1.4 (c) 19.2  2.8 (b) 19.6  3.0 (b) 24.6  1.3 (a) 133.5  12.1 (d) 191.5  16.8 (a) 162.5  23.5 (b) 149.0  18.7 (c) TR (g) The HRM treatment induced a significant increase of total shoot (leaves + culm) length, fresh and dry weight with respect to the untreated control (Table 1). Even if to a lesser extent, a positive effect on EM and F-EM concerning aboveground ryegrass growth was also observed. Data suggested that the inoculated microorganisms stimulated the plant cell extension growth, as evidenced by the significant decrease of the ratio between shoot dry and fresh weight. By contrast, HRM did not cause significant perturbation of the ratio between shoot dry and fresh weight. With respect to underground biomass, the investigated amendments did not significantly increase fresh and dry root weight; in particular a decrease of root weight was observed in EM and F-EM. However, the significant decrease of the ratio between root dry and fresh weight of EM, F-EM and HRM suggested a stimulation of the root extension growth. Moreover, ryegrass plants treated with HRM, EM and F-EM exhibited longer root system than untreated plants. EM treatments and F-EM induced a considerable increase of root system length around 35%, while HMR approximately doubled the length of ryegrass root system with respect to the untreated plants. These data are in agreement with published results reporting that some phytohormones, such as IAA, could promote not only shoot development but also root elongation [23,24]. Because of their morpho-physiological effects on plant shoot and root, EM and HMR significantly improved the ryegrass resistance to tearing out (Table 1). The best performance was observed for plants treated with EM, while F-EM and HRM induced less evident effects on tearing out resistance (11 and 21% increase of TR index, respectively). Considering the overall results, EM treatment best fits with the scope of the experiment. Microbiological counts and strain identification Lactobacilli, total aerobic bacteria and yeasts were counted on MRS agar, SDA and NA, respectively. The counts obtained from concentrated and activated product samples were comparable, evidencing that the activation step revitalized the inoculum but it did not increase the bacterial number. Lactobacilli were the most represented group of microorganisms, being at levels of 106 cfu/ml, while total aerobic bacteria and yeasts were counted at 105 cfu/ml and 104 cfu/ml, respectively. 698 www.elsevier.com/locate/nbt All the 25 isolates (referred to LA1–LA25) obtained from MRS agar were Gram positive, catalase negative, oxidase negative and had a rod shape at the microscopic analysis. Genus-specific PCR confirmed for all the isolates the affiliation to the Lactferobacillus genus. Most of the isolates fermented L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, melibiose, gluconate and 5cheto-gluconate. Six of the isolates (LA1, LA4, LA5, LA13, LA14, LA20) fermented only galactose, glucose, fructose after five days incubation. Six strains (LA6, LA9, LA15, LA17, LA22, LA25) could also grow on mannitol, maltose, melezitose, raffinose and D-arabitol; in addition LA6 and LA25 ferment melibiose and sucrose. A group of six isolates was positive to arabinose, ribose, D-xylose, galactose, glucose, fructose and gluconate. The remaining seven strains ferment L-arabinose, ribose, galactose, glucose, fructose, gluconate and 5-ketogluconate and hydrolyze esculin; five of them were also positive to a-methyl-D-glucoside, cellobiose, maltose, melibiose and raffinose, while the other two to sucrose and xylitol. Further, the isolates were genotypically grouped using ERIC-PCR. Cluster analysis showed that they could be divided into six groups (Fig. 1), which almost corresponded to the fermentation patterns obtained. On the basis of fermentation and genotypic results, a representative isolate from each group (LA1, LA3, LA6, LA7, LA9 and LA12) was selected for sequencing of the 16S rRNA gene. Five of the six clusters contained microorganisms identified as: Lactobacillus buchneri, Lactobacillus parafarraginis and Lactobacillus diolivorans (Table 2). These species belong to the L. buchneri group which is mostly linked to food and silage fermentation [25]. For one cluster, whose members were found to ferment only three sugars, it was not possible to obtain a clear taxonomic identification but only a similarity of 97% to an uncultured Lactobacillus spp. Further analyses are required to better characterize these strains. Twenty isolates from NA were Gram negative, catalase positive, oxidase negative and the microscopic analysis showed the presence of motile rods (100%). The remaining 5 were Gram positive, catalase positive, oxidase negative and the microscopic analysis showed the presence of rod shaped cells (100%). The genotypic grouping and the sequencing results allowed the selection of five groups (Fig. 2). As for lactobacilli, a representative isolate from each group (A1–A5) was selected for sequencing of the 16S rRNA New Biotechnology  Volume 30, Number 6  September 2013 Research Paper RESEARCH PAPER FIGURE 1 Dendrogram obtained by cluster analysis of ERIC-PCR fingerprints of Lactobacillus strains isolated from the activated product. gene. Strains belonged to Stenotrophomonas maltophilia (ten isolates, three different strains), Microbacterium trichotecenolyticum (seven isolates) and Escherichia coli (eight isolates) (Table 2). Twenty-five yeasts isolated from SDA were identified by API20C AUX1 as Candida utilis (15 isolates) and Saccharomyces cerevisiae (ten isolates). FIGURE 2 Dendrogram obtained by cluster analysis of ERIC-PCR fingerprints of aerobic strains isolated from the activated product. The use of lactobacilli as beneficial bacteria for plant growth is not widespread, although their presence inside plants has been sometimes detected [26–28]. Considering their efficacy as protective and probiotic cultures in human, animal, food and feed [29,30], their use in the agronomic field is rapidly increasing. In vitro studies on their strong activity against plant pathogenic TABLE 2 Strains isolated from the activated product Isolatedstrain bp La1 1371 La3 809 JX426086 Accession # Closest match Accession # S_ab score DGGE bandsa DGGE profile Uncultured compost bacterium FS423 FN667288 0.972 8 F-EM, EM Lactobacillus buchneri 482A HM162413 0.995 p7 EM1W p5, 3 EM, EM1W La6 1439 JX426087 Lactobacillus parafarraginis (T) NRIC 0677 AB262734 1.000 La7 808 JX426088 Lactobacillus buchneri TB-H34 AB425940 1.000 La9 1390 JX426089 Lactobacillus parafarraginis (T) NRIC 0677 AB262734 1.000 La12 A1 1441 1382 JX426090 JX426091 Lactobacillus diolivorans NM60-7 Stenotrophomonas maltophilia BAC3155 HM218272 HM355743 0.996 0.999 A2 1386 JX426092 Stenotrophomonas maltophilia BAC3155 HM355743 1.000 A3 1395 JX426093 Stenotrophomonas maltophilia BAC2024 HM355615 0.999 A4 1385 JX426094 Escherichia coli S88 CU928161 0.996 A5 1370 JX426095 Microbacterium trichothecenolyticum M23047 HM032796 0.994 a DGGE bands in Fig. 4. www.elsevier.com/locate/nbt 699 Research Paper www.elsevier.com/locate/nbt RESEARCH PAPER 700 TABLE 3 Best-match identification of phylotypes (Seqmatch tool, from Ribosomal Database Project-II) of excised DGGE bands obtained with universal primers Phylogenetic group Closest match Accession # S_ab score Closest described bacterium Accession # S_ab score DGGE profilea 1 Lewinella (28%) Uncultured bacterium T2-68 JF502990 0.888 Lewinella marina (T) MKG-38 AB301495 0.605 All sand profiles 2 Hydrogenophaga (100%) Hydrogenophaga pseudoflava (T) ATCC 33668 Hydrogenophaga bisanensis (T) K102 AF078770 EF532793 0.990 0.990 3 Fibrobacter (55%) Uncultured Fibrobacteres bacterium clone AUVE_12B11 EF651500 0.972 Fibrobacter intestinalis NR9 AJ496284 0.499 All sand profiles 4 Vogesella (100%) Vogesella sp. SK-2 AM689950 1.000 Vogesella indigofera (T) ATCC 19706 AM989119 0.97 All sand profiles 5 Polaromonas (97%) Polaromonas aquatica (T) CCUG 39402 AM039830 0.975 All sand profiles 6 Lactobacillus (99%) Lactobacillus zeae (T) ATCC15820 Lactobacillus casei (T) ATCC 393 Lactobacillus paracasei (T) JCM 8130 D86516 AF469172 D79212 1.000; 1.000; 1.000 sEM, sHRM 7 Acidovorax (50%) Uncultured bacterium DR128 JF429172 0.978 Variovorax sp. Z0-YC6806 GQ369074 0.943 all sand profiles 8 Flavobacterium (100%) Flavobacterium sp. WB 2.1-30 AM167558 1.000 Flavobacterium psychrolimnae (T) LMG 22018 AJ585428 0.939 rF-EM 9 Leptospira (100%) Leptospira biflexa (T) ATCC 23582 Leptospira wolbachii (T) ATCC 43284 AY631876 AY631879 1.000; 1.000 10 Emticicia (55%) Cytophaga sp. GPl-11 AJ456975 0.703 Emticicia sp. IMCC1731 DQ664246 0.615 rF-EM 11 Niastella (100%) Niastella sp. RHYL-67 EU917053 0.992 Niastella populi (T) THYL-44 EU877262 0.953 rEM, rHRM 12 Ohtaekwangia (90%) Bacteroidetes bacterium Mo-0.2plat-K3 AJ622888 0.944 Ohtaekwangia koreensis (T) 3B-2 Ohtaekwangia kribbensis (T) 10AO GU117702 GU117703 0.645; 0.645 rEM 13 Ohtaekwangia (100%) Ohtaekwangia kribbensis (T) 10AO GU117703 0.942 all root profiles 14 Hydrogenophaga (100%) Hydrogenophaga pseudoflava (T) ATCC 33668 Hydrogenophaga bisanensis (T) K102 AF078770 EF532793 0.985; 0.985 rEM 15 Fibrobacteres (44%) uncultured Fibrobacteres bacterium clone AUVE_12B11 EF651500 0.972 16 Chloroplast DGGE band DGGE profiles of Fig. 3. rF-EM Fibrobacter intestinalis NR9 AJ496284 0.495 rEM, rHRM, rCTR All root profiles New Biotechnology  Volume 30, Number 6  September 2013 a sF-EM, sEM, sCTR New Biotechnology  Volume 30, Number 6  September 2013 commercial crops (maize, sugar cane and wheat) producing antifungal metabolites against several soil-borne pathogens [42] and several phytohormones such as IAA, gibberellic acid and abscissic acid [43]. Moreover, its outstanding nitrogen-fixing capacity and degradation of several organic pollutants show high biotechnological potential for use in commercial inoculants and bioremediation application [44,45]. M. trichotecenolytcum has been isolated as endophyte from several plant species [46,47]. Strains belonging to Microbacterium spp. were found to produce IAA and promote orchid seed germination [48]. Concerning E. coli, which is mostly associated with gut microbiota and infection, some non-pathogenic strains are used as probiotics because of the production of interesting antimicrobial compounds [49]. Characterization of bacterial communities by PCR-DGGE Genomic DNA from sand and surface-sterilized root samples was used to evaluate the impact of the different amendments (CTR, EM, F-EM and HRM) on their microbial profile using PCR-DGGE. Moreover, it was interesting to verify if in the EM treatment the inoculated lactobacilli, being the most representative group, could be detected at root level and thus becoming endophytes. Results, using bacterial universal primers, are shown in Fig. 3. Sixteen PCR-DGGE bands have been identified by sequencing (Table 3). PCR-DGGE analysis of bacterial community in sand samples (Fig. 3a) revealed that all profiles were comparable, thus indicating that detected microorganisms probably derive from sand, water or seeds. TABLE 4 Best-match identification of phylotypes (Seqmatch tool, from Ribosomal Database Project-II) of excised DGGE bands obtained with Lactobacillus spp. specific primers DGGE band Phylogenetic group Closest match Accession # S_ab score DGGE profilea 1 Lactobacillus (100%) Lactobacillus pentosus Taj-Apis 359 HM027643 0.926 EM 2 Lactobacillus (100%) Lactobacillus sanfranciscensis LMG 16002 EU350220 0.985 EM 3 Lactobacillus (100%) Lactobacillus parafarraginis (T) NRIC 0677 AB262734 0.990 EM, EM1W 4 Exiguobacterium (100%) Exiguobacterium sp. MH70 EU182898 1.000 F-EM, EM 5 Lactobacillus (100%) Lactobacillus sanfranciscensis LMG 16002 EU350220 0.970 EM 6 Lactobacillus (100%) Lactobacillus sanfranciscensis TMW 1.1304 CP002461 0.977 EM 7 Lactobacillus (100%) Lactobacillus sanfranciscensis LS1 DQ875745 1.000 EM 8 Lactobacillus (100%) Uncultured compost bacterium FS423 FN667288 1.000 F-EM, EM p1 Lactobacillus (100%) Lactobacillus diolivorans (T) JKD6 Lactobacillus farraginis (T) NRIC 0676 AF264701 AB262731 1.000 1.000 EM1W p2 Lactobacillus (99%) Lactobacillus parafarraginis (T) NRIC 0677 AB262734 0.966 EM1W p3 Lactobacillus (100%) Lactobacillus diolivorans (T) JKD6 Lactobacillus farraginis (T) NRIC 0676 AF264701 AB262731 1.000 1.000 EM1W p4 Lactobacillus (100%) Lactobacillus parafarraginis (T) NRIC 0677 AB262734 1.000 EM1W p5 Lactobacillus (100%) Lactobacillus parafarraginis (T) NRIC 0677 AB262734 0.980 EM, EM1W p6 Lactobacillus (100%) Lactobacillus parafarraginis (T) NRIC 0677 AB262734 1.000 EM1W p7 Lactobacillus (100%) Lactobacillus buchneri (T) JCM1115 AB205055 1.000 EM1W p8 Lactobacillus (100%) Lactobacillus diolivorans (T) JKD6 Lactobacillus farraginis (T) NRIC 0676 AF264701 AB262731 1.000 1.000 EM1W p9 Lactobacillus (98%) Lactobacillus zeae (T) ATCC15820 Lactobacillus casei (T) ATCC 393 Lactobacillus paracasei (T) JCM 8130 D86516 AF469172 D79212 1.000; 1.000; 1.000 EM1W a DGGE bands in Fig. 4. www.elsevier.com/locate/nbt 701 Research Paper fungi [31,32] are promising for the application of lactobacilli in real field condition. As in other ecosystems (gut, fermented foods, among others), they are expected to improve mineral availability for plants in the rhizosphere through the production of enzymes and organic acids [33], and control pathogen diffusion with the production of several compounds. Species isolated in this study (L. buchneri, L. parafarraginis and L. diolivorans) and those identified with PCR-DGGE (L. pentosus and L. sanfranciscensis, Table 4) are often reported in the literature to improve the stability of silages, thanks to the production of a wide range of metabolites inhibiting moulds and fungi [34,35]. Moreover, they are frequently isolated from sourdough, giving to sourdough bread an extended shelf-life and improved microbial safety [36]. Up to now, only a few studies have reported the use of lactobacilli as bio-inoculants together with other bacterial species [37–39]. Concerning yeasts, they are common resident of soil and rhizosphere and they have also been isolated inside plants tissues. Several studies reported their role as plant growth promoters and soil-borne fungal antagonists [8,40]. Moreover, it has been documented the beneficial association of lactobacilli and yeasts in other ecosystems. In sourdough, when Lactobacillus plantarum is associated with yeasts (S. cerevisiae or S. exiguus) cell yield and lactic acid production increase [41]. A higher propionate formation was observed in sourdough co-fermented with L. buchneri and L. diolivorans and exhibited increased antifungal properties [34]. The three aerobic species isolated are listed in Table 2. S. maltophilia has been found in strict association with plant hosts; it was also described as a growth promoter microorganism of some RESEARCH PAPER New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER Research Paper FIGURE 4 FIGURE 3 DGGE bands obtained amplifying metagenomic DNA with universal primers from sand and roots. (a) Sand samples, (b) root samples. CTR, control; HRM, treatment with hormone rich solution; F-EM, treatment with filtered/ activated product; EM, treatment with activated product. Concerning root profiles (Fig. 3b), PCR-DGGE analysis revealed a considerable microbial community complexity in the EM and FEM treatments. Unfortunately, the biggest band corresponded to chloroplast DNA and the preferential amplification of this DNA could have reduced the yield of amplification of microbial DNA, which clearly has given rise to fainter bands, difficult to be excised. Bands 14 and 15 showed high sequence similarity to bands 2 and 3 of sand profiles (Fig. 3 and Table 3). However, in contrast to sand, root profiles showed a peculiar band pattern in each treatment condition, except for band number 13, present in all samples. Sequencing revealed a close similarity to Ohtaekwangia kribbensisT (GeneBank accession no. GU117703), firstly isolated from sand [50]. Sequences from bands 8–10 were closely related to Flavobacterium sp. WB 2.1-30, Leptospira spp. and Cytophaga sp. GP 1-11 (Table 3) and were present only in F-EM. These microorganisms have been frequently isolated from fresh- and hardwater. The EM treatment has two typical bands: number 12, closely related to a Bacteroidetes bacterium recovered from water (GeneBank accession no. AJ622888), and number 14, which can be ascribed to Hydrogenophaga spp., a root endophyte of several crops [51]. Niastella spp., frequently isolated from soil [52] has been detected in EM and HRM profiles (band number 11). 702 www.elsevier.com/locate/nbt DGGE bands obtained amplifying DNA with Lactobacillus spp. specific primers from root samples, activated EM1W and pure cultures (LA1, LA3, LA9, LA12). CTR, control; HRM, treatment with hormone rich solution; F-EM, treatment with filtered/activated product; EM, treatment with activated product; EM1W, the activated product. Overall, PCR-DGGE analysis revealed the presence of a large variety of microorganisms colonizing the rhizosphere and roots, and most of them have been already reported as endophytes in different plants [53–55]. To better investigate the presence of lactobacilli in root samples, specific primers targeting LAB were used in an additional PCR-DGGE. Fig. 4 shows PCR-DGGE pattern of DNA extracted from root samples of the 4 treatments, the activated product and Lactobacillus isolates (Lactobacillus sp. LA1, L. buchneri LA3, L. parafarraginis LA9 and L. diolivorans LA12). The analysis of sequenced bands (Table 4) evidenced the root colonization ability of the following species: (i) L. parafarraginis LA 9 (bands p5 and 3); (ii) Lactobacillus spp. LA1 (band 8); (iii) L. pentosus (band 1); (iv) L. sanfranciscensis (band 2, 5, 6, 7). Surprisingly, bands ascribed to Lactobacillus spp. LA1, L. pentosus and L. sanfranciscensis, in the EM profile, were absent in the PCRDGGE profile of the product. It can be hypothesized that the activation step led to a selective growth of some species whereas, after inoculation, an in situ enrichment of other phylotypes gives rise to a different microbial population. This can explain the different profiles obtained for the activated product and EM treatment. By contrast, the lack of recovery of some species by plating (e.g. L. pentosus and L. sanfranciscensis) may reflect the different sensitivity of diverse species to plate culturing techniques when a mixed culture is analyzed. On the whole, the presence of several lactobacilli in the EM treatment, compared to the CTR and HRM, showed the ability of these microorganisms to colonize root tissues and become plant endophytes. Conclusion The treatment of hydroponically grown L. perenne L. with a commercial solution of microorganisms has led to an increased resistance to tearing out, as well as to an increased root length, which are particularly favorable features for any essence of a recreational or sport turfgrass. Identification of isolates clearly RESEARCH PAPER showed the presence of species reported in literature as having plant growth promoting activity and antifungal action. The PCRDGGE analysis targeting LAB evidenced the colonization ability of four species derived from the product; this is the first time that the endophytic behavior of inoculated lactobacilli has been assessed. The inoculation of microorganisms may contribute to the beneficial morpho-physiological effects on plant shoots and roots. This may lead to the conclusion that bio-inoculants have a great potential for the maintenance and increased performance of turfgrass surfaces. Further on field investigations are necessary to confirm the effect of this low-impact technology aimed at developing a more sustainable environmental management of lawn and sport fields. References [1] Waltz C, McCullough P, Hudson W, Braman K, Martinez A. Turfgrass pest control recommendations for professionals. GA: Georgia Turfgrass Association; 2010. [2] David W, Held DW, Potter DA. Prospects for managing turfgrass pests with reduced chemical inputs. Annual Review of Entomology 2012;57:329–54. [3] Cisar JL. Managing turf sustainably. In: Proceedings of the 4th International Crop Science Congress ‘New directions for a diverse planet’; 2004. p. 1–6, www.cropscience.org.au. [4] Paau AS. Formulations useful in applying beneficial microorganisms to seeds. Trends in Biotechnology 1998;6:276–9. [5] Berg G. Plant–microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture. Applied Microbiology and Biotechnology 2009;84:11–8. [6] Lee S, Flores-Encarnacion M, Contreras-Zentella M, Garcia-Flores L, Escamilla JE, Kennedy C. Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome C biogenesis genes. Journal of Bacteriology 2004;186:5384–91. [7] Costa JM, Loper JE. Characterization of siderophore production by the biological-control agent Enterobacter cloacae. Molecular Plant–Microbe Interactions 1994;7:440–8. [8] Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN. Bacterial endophytes: recent developments and applications. FEMS Microbiology Letters 2008;27:1–9. [9] Sturz AV, Matheson BG. Populations of endophytic bacteria which influence host-resistance to Erwinia-induced bacterial soft rot in potato tubers. Plant and Soil 1996;184:265–71. [10] Kerry BR. Rhizosphere interactions and the exploitation of microbial agents for the biological control of plant–parasitic nematodes. Annual Review of Phytopathology 2000;38:423–41. [11] Ping L, Boland W. Signals from the underground: bacterial volatiles promote growth in Arabidopsis. Trends in Plant Science 2004;9:263–6. [12] Berg G, Hallmann J. Control of plant pathogenic fungi with bacterial endophytes. In: Schulz BJE, Boyle CJC, Sieber TN, editors. Microbial root endophytes. Berlin: Springer-Verlag; 2006. p. 53–69. [13] Hoagland DR, Arnon DI. The water-culture method for growing plants without soil. California Agriculture Experiment Station 1950;347:1–32. [14] Walter J, Hertel C, Tannock GW, Lis CM, Munro K, Hammes WP. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Applied and Environment Microbiology 2001;67:2578–85. [15] Ventura M, Zink R. Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis. FEMS Microbiology Letters 2002;217:141–54. [16] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5. Molecular evolutionary genetics analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution 2011;28:2731–9. [17] Hardoim PR, Andreote FD, Reinhold-Hurek B, Sessitsch A, van Overbeek LS, van Elsas JD. Rice root-associated bacteria: insights into community structures across 10 cultivars. FEMS Microbiology Ecology 2011;77:154–64. [18] Iacumin L, Cecchini F, Manzano M, Osualdini M, Boscolo D, Orlic S, Comi G. Description of the microflora of sourdoughs by culture-dependent and cultureindependent methods. Food Microbiology 2009;26:128–35. [19] Sass AM, Sass H, Coolen MJ, Cypionka H, Overmann J. Microbial communities in the chemocline of a hypersaline deep-sea basin (Urania basin, Mediterranean Sea). Applied and Environment Microbiology 2001;67:5392–402. [20] Muyzer G, De Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environment Microbiology 1993;59:695–700. [21] Lane DJ. 16S/23S rRNA sequencing. Nucleic acid techniques. In: Stackebrandt E, Goodfellow M, editors. Bacterial systematics. New York: John Wiley & Sons; 1991. p. 115–75. [22] Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Research 2009;37:141–5. [23] Pilet PE, Saugy M. Effect of applied and endogenous IAA on maize root growth. Planta 1985;164:254–8. [24] Pilet PE, Saugy M. Effect on root growth of endogenous and applied IAA and ABA. Plant Physiology 1987;83:33–8. [25] Pot B, Tsakalidou E. Taxonomy and metabolism of Lactobacillus. In: Ljungh A, Wadstrom T, editors. Lactobacillus molecular biology: from genomics to probiotics. Norfolk, UK: Caister Academic Press; 2005. p. 3–58. [26] Mundt OJ, Hammer JL. Lactobacilli on plants. Applied Microbiology 1968;16:1326–30. [27] Jacobs MJ, Bugbee WM, Gabrielson DA. Enumeration, location, and characterization of endophytic bacteria within sugar beet roots. Canadian Journal of Botany 1985;63:1362–5. [28] Hallmann J. Biologische Bekämpfung pflanzenparasitärer Nematoden mit antagonistischen Bakterien. Mitt Biol Bundesanst Land Forstwirtsch 2003;392:1–128. [29] Gaggia F, Di Gioia D, Baffoni L, Biavati B. The role of protective and probiotic cultures in food and feed and their impact in food safety. Trends in Food Science Technology 2011;22:58–66. [30] Gaggı̀a F, Mattarelli P, Biavati B. Probiotics and prebiotics in animal feeding for safe food production. International Journal of Food Microbiology 2010;141: 15–28. [31] Wang H, Yan Y, Wang J, Zhang H, Qi W. Production and characterization of antifungal compounds produced by Lactobacillus plantarum IMAU10014. PLoS ONE 2012;7:294–352. [32] Wang H, Yan H, Shin J, Huang L, Zhang H, Wei Qi W. Activity against plant pathogenic fungi of Lactobacillus plantarum IMAU10014 isolated from Xinjiang koumiss in China. Annals in Microbiology 2011;61:879–85. [33] Lopez HW, Krespine V, Guy C, Messager A, Demigne C, Remesy C. Prolonged fermentation of whole wheat sourdough reduces phytate level and increases soluble magnesium. Journal of Agricultural and Food Chemistry 2001;49: 2657–62. [34] Holzer M, Mayrhuber E, Danner H, Braun R. The role of Lactobacillus buchneri in forage preservation. Trends in Biotechnology 2003;21:282–7. [35] Zhang C, Brandt MJ, Schwab C, Gänzle MG. Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough. Food Microbiology 2010;27:390–5. [36] Corsetti A, Settanni L. Lactobacilli in sourdough fermentation. Food Research International 2007;40:539–58. [37] Hu C, Qi Y. Long-term effective microorganisms application promote growth and increase yields and nutrition of wheat in China. European Journal of Agronomy 2013;46:63–7. [38] Javaid A, Bajwa R. Field evaluation of effective microorganisms (EM) application for growth, nodulation, and nutrition of mung bean. Turkish Journal of Agriculture 2011;35:443–52. [39] Khaliq A, Abbasi MK, Hussain T. Effect of integrated use of organic and inorganic nutrient sources with effective microorganisms (EM) on seed cotton yield in Pakistan. Bioresource Technology 2006;97:967–72. [40] El-Tarabily KA, Sivasithamparam K. Potential of yeasts as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. Mycoscience 2006;47:25–35. [41] Gobbetti M. The sourdough microflora: interactions of lactic acid bacteria and yeasts. Trends in Food Science Technology 1998;9:267–74. [42] Mehnaz S, Kowalik T, Reynolds B, Lazarovits G. Growth promoting effects of corn (Zea mays L.) bacterial isolates under greenhouse and field conditions. Soil Biology and Biochemistry 2010;42:1848–56. [43] Naz I, Bano A. Assessment of phytohormones producing capacity of Stenotrophomonas malthopilia and its interaction with Zea mays L. Pakistanian Journal of Botany 2012;44:465–9. www.elsevier.com/locate/nbt 703 Research Paper New Biotechnology  Volume 30, Number 6  September 2013 RESEARCH PAPER Research Paper [44] Seo JS, Keum YS, Li QX. Bacterial degradation of aromatic compounds. International Journal of Environmental Research and Public Health 2009;6:278–309. [45] Cardoso JD, Hungria M, Andrade DS. Polyphasic approach for the characterization of rhizobial symbionts effective in fixing N(2) with common bean (Phaseolus vulgaris L.). Applied Microbiology and Biotechnology 2012;93:2035–49. [46] Zaspel I, Ulrich A, Boine B, Stauber T. Occurrence of culturable bacteria living in micropropagated black locust cultures (Robinia pseudoacacia L.). European Journal of Horticultural Science 2008;73:231–5. [47] Shinohara H, Yoshida S, Enya J, Watanabe Y, Tsukiboshi T, Negishi H, Tsushima S. Culturable bacterial communities on leaf sheaths and panicles of rice plants in Japan. Folia Microbiologica (Praha) 2011;56:505–17. [48] Tsavkelova EA, Cherdyntseva TA, Klimova SY, Shestakov AI, Botina SG, Netrusov AI. Orchid-associated bacteria produce indole-3-acetic acid, promote seed germination, and increase their microbial yield in response to exogenous auxin. Archives of Microbiology 2007;188:655–64. [49] Setia A, Bhandari SK, House JD, Nyachoti CM, Krause DO. Development and in vitro evaluation of an Escherichia coli probiotic able to inhibit the growth of pathogenic Escherichia coli K88. Journal of Animal Science 2009;87: 2005–12. 704 www.elsevier.com/locate/nbt New Biotechnology  Volume 30, Number 6  September 2013 [50] Yoon JH, Kang SJ, Lee SY, Lee JS, Sooyeon Park S. Ohtaekwangia koreensis gen. nov., sp. nov. and Ohtaekwangia kribbensis sp. nov., isolated from marine sand, deep-branching members of the phylum Bacteroidetes. International Journal of Systematic and Evolutionary Microbiology 2011;61:1066–72. [51] Mano H, Morisaki H. Endophytic bacteria in the rice plant. Microbes Environment 2008;23:109–17. [52] Weon HY, Kim BY, Yoo SH, Lee SY, Kwon SW, Go SJ, Stackebrandt E. Niastella koreensis gen. nov., sp. nov. and Niastella yeongjuensis sp. nov., novel members of the phylum Bacteroidetes, isolated from soil cultivated with Korean ginseng. International Journal of Systematic and Evolutionary Microbiology 2006;56: 1777–82. [53] Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, van der Lelie D. Endophytic bacteria and their potential applications. Critical Reviews in Plant Science 2002;21:583–606. [54] Diallo S, Crépin A, Barbey C, Orange N, Burini JF, Latour X. Mechanisms and recent advances in biological control mediated through the potato rhizosphere. FEMS Microbiology Ecology 2011;75:351–64. [55] Hardoim PR, Hardoim CC, van Overbeek LS, van Elsas JD. Dynamics of seedborne rice endophytes on early plant growth stages. PLoS ONE 2012;7:1–13.