Environmental and Experimental Biology (2018) 16: 177–183 Original Paper DOI: 10.22364/eeb.16.17 Characteristics of an endophytic microbial consortium and its impact on rhizosphere microbiota of barley

Dagnija Vecstaudža1, Māris Seņkovs2, Vizma Nikolajeva2*, Olga Mutere1

1Institute of Microbiology and Biotechnology, University of Latvia, Jelgavas 1, Riga LV-1004, Latvia 2Faculty of Biology, University of Latvia, Jelgavas 1, Riga LV-1004, Latvia

*Corresponding author, E-mail: [email protected]

Abstract

Biological fertilizers are being developed on the basis of plant growth promoting microorganisms isolated from the plant rhizosphere, phyllosphere and endosphere. The aim of this study was to determine influence of a consortium of plant endophytes on rhizosphere microbiota in a greenhouse vegetation experiment with barley. Fifty-five bacterial and fungal strains were isolated from leaves, stems and roots of oilseed rape and barley. Twentty five of them did not inhibit seed germination. Most of them belonged to the genera Serratia, Enterobacter, Brevibacillus and Pseudomonas. Individual isolates demonstrated phosphate solubilization ability, nitrification potential and urease activity. Impact of the multi-strain microbial consortium on cultivable microbiota of rhizosphere was estimated in a 19-day greenhouse experiment with barley. Concentration of phosphate solubilizing and cellulolytic microorganisms in the rhizosphere was below the detection limit. The consortium increased the total number of bacterial colony-forming units but did not affect the number of actinobacterial and fungal colony-forming units. Interactions between species in the multi-strain consortium and between introduced and indigenous populations are discussed.

Key words: endophyte, microbial consortium, nitrification, phosphate solubilization, rhizosphere, urease. Abbreviations: CFU, colony-forming units; IAA, indole-3-acetic acid; P, phosphate; PSI, phosphate solubilising index.

Introduction reported as growth promoting. In particular, stable colonization ability as well as regulation of the microbiota Biological fertilizers contain microorganisms and improve in the rhizosphere, with inhibition of Fusarium solani, was plant growth and development (Herrmann, Lesueur shown in the pot experiments with Streptomyces spp. and 2013). Microorganisms can be isolated from soil and the tomato plants (He et al. 2015). Ma et al. (2017) reported plant rhizosphere (Bhardwaj, Ansari 2014). Incorporation a 37% decrease of root knot nematode index and growth of mycorrhizal fungi, phyllosphere microorganisms and stimulating effect in tomato plants after inoculation endophytes into biofertilizer formulations has attracted by Streptomyces spp. Representatives of Azospirillum, great attention in development of alternative fertilizers Bacillus, Enterobacter, Klebsiella, and Pseudomonas spp. are for improving plant growth and soil quality (Schulz, Boyle considered to contribute plant growth by production of 2006). osmolytes, polysaccharides and phytohormones, by action Endophytes originate from microbial communities of of specific enzymes, stress adaptation abilities and positive the rhizosphere and phylloplane (Hallmann et al. 1997). interactions with other microbial communities (Nadeem et Studies have shown that endophytic cultivable strains al. 2016). isolated from surface sterilized roots, stems or leaves A study of endophytic mycobiota in grasses in terms have promising plant growth-promoting traits that can of richness and distribution reported 77 and 79 fungal be introduced in biofertilizers (Qin et al. 2015). Plant- endophyte isolates from leaves and roots of Holcus lanatus, associated bacteria can be screened in vitro for multiple and only 22 were common for both organs (Márquez et al. plant growth-promoting activities like production of 2010). It was shown that plant species-specific selection of indole-3-acetic acid (IAA), ammonia, siderophore and endophytes is driven by functional rather than phylogenetic phosphate (P) solubilization (Ahmad et al. 2008). traits (Wemheuer et al. 2017). Thus, efficiency of developed There is evidence that the soil microorganisms respond biofertilizers depends on the plant species, origin and differently to inputs of inorganic and organic fertilizers, physiological characteristics of microorganisms, as well as leading to the change of bacterial and fungal composition on a broad spectrum of environmental factors. (Wang et al. 2017). Response of higher plants to introduction Many biofertilizers consist of one microbial strain. In of beneficial microorganisms into the rhizosphere was such a case, all the effects can be easily attributed to the

Environmental and Experimental Biology ISSN 2255-9582 177 D. Vecstaudža, M. Seņkovs, V. Nikolajeva, O. Mutere particular species. Coinoculation would combine different Characterization of the activity of plant endophytes mechanisms and effects. Combination of strains and species Isolated microorganisms were selected for development can produce additive or synergic effect (Marimuthu et al. of a biofertilizer by testing their influence on seed 2002), but also can cause competitive processes (Probanza germination. Suspensions of microorganisms (a loopful et al. 2002) that reduce or prevent growth enhancement of a fresh colony of isolate mixed with 50 mL of sterile and, ultimately, the impact can be unpredictable (reviewed water) was added to seeds of radish, alfalfa, cress and barley in Trabelsi, Mhamdi 2013). Plant growth might not to liquid, which minimally covered the seeds. Three seeds necessarily be affected by direct action but may be related from every species separately in three repetitions (nine to positive and negative interactions between introduced seeds in total) were incubated in 10 mL containers for 7 and indigenous microbial populations. There are too many days at room temperature in the dark. Isolates that did not unknowns to assess the health of the soil and soil microbial inhibit germination in comparison with control without community in each specific case (reviewed in Das, Varma microorganisms, were selected for further testing (data not 2011). shown). The aim of this study was investigation of influence of a Ability of microorganisms to affect plant growth was consortium of plant endophytes on rhizosphere microbiota. tested by measuring their phosphate solubilizing index Endophytic microorganisms were isolated, identified and (PSI) on agar medium (Promwee et al. 2014) and their characterized with emphasis on nitrification potential, nitrification potential and urease activity (American urease activity and P solubilizing activity. Previously, it was Public Health Association 1992). Isolate suspensions found that the created consortium did not have significant were incubated in reaction mixture for 48 h at 37 °C for effect on shoot and root length of barley (Vecstaudza et al. determination of nitrification potential and urease activity. 2017). In this study, influence of a microbial consortium Protein concentration of microbial suspensions was on rhizosphere microbiota was estimated in a greenhouse measured (Bradford 1976) to characterize the concentration vegetation experiment with barley. of microorganisms. Specific nitrification potential and urease activity were expressed in relative units by dividing Materials and methods + the concentration of N-NH4 in isolate suspensions with optical density of suspensions obtained in the Bradford Isolation and identification of plant endophytes protein assay. Endophytic microorganisms were isolated from oilseed rape (Brassica napus L.) and barley (Hordeum vulgare L.) Testing of consortium impact on rhizosphere microbiota which were grown in Dobele municipality (Latvia). Plants Twenty-five isolates which were obtained from all studied were grown for 75 days in loamy soil. Microorganisms were organs of rape and barley, and did not inhibit seed isolated from leaves, stems and roots of the plants. Samples germination, were selected for the vegetation experiment were washed in distilled water three times and surface- and mixed together. The vegetation experiment was sterilized by dipping in 70% ethanol for 30 s followed by conducted in 60 mL volume seed trays with 50 g of loamy repeated rinsing with sterile water. Sterile 0.02 M potassium soil and barley cv. `Austris` in a greenhouse for 19 days as phosphate buffer (pH 7.4) was added to the samples, which described previously (Vecstaudza et al. 2017). One mL of were then ground (Zinniel et al. 2002) and added to 50 mL each microorganism isolate was mixed with sterile water tubes with malt extract broth (Biofile, Italy) or nutrient to achieve 2.7 × 1010 colony-forming units (CFU) mL–1 for broth (Bio-Rad, France). The volume of the medium was bacteria and 1.2 × 107 CFU mL–1 for fungi. The microbial 10 times larger than the sample volume. Samples were suspension was mixed with the soil. The experiment was incubated at 37 °C and 100 rpm for 24 h. Decimal dilutions done in triplicate. At the end of the experiment, average of samples were prepared and spread plated on nutrient samples of rhizosphere soil consisting of three subsamples agar (Scharlab, Spain) plates for isolation of bacteria or were collected. Decimal dilutions of average samples were on Malt extract agar (Lab M, UK) for isolation of fungi. prepared and plated on five media: nutrient agar (Scharlab, Nutrient agar plates were incubated at 37 °C for 24 h and Spain) for total number of cultivable bacteria, Rose Bengal Malt extract agar plates were incubated at 24 °C for 7 days. agar with chloramphenicol (Biolife, Italy) for fungi, NBRIP –1 –1 After incubation morphologically different colonies were agar (glucose 10 g L , MgCl2 × 6H2O 5 g L , Ca3(PO4)2 5 g –1 –1 –1 plated separately. Isolates were preserved in 4 °C and re- L , MgSO4 × 7H2O 0.25 g L , KCl 0.2 g L , (NH4)2SO4 0.1 g plated monthly. L–1; agar 15 g L–1) (Nautiyal 1999) for P solubilising bacteria, Microorganisms were identified using microscopic Actinomycete isolation agar (Sigma-Aldrich, Germany) for –1 appearance, Gram staining, the oxidase test, BD BBL™ actinobacteria and CMC agar (KH2PO4 1.0 g L , MgSO4 –1 –1 –1 Crystal™ Identification Systems (Becton & Dickinson, × 7H2O 0.5 g L , NaCl 0.5 g L , FeSO4 × 7H2O 0.01 g L , –1 –1 USA) for bacteria and the identification system API® ID MnSO4 × H2O 0.01 g L , NH4NO3 0.3 g L , carboxymethyl 32C (Biomerieux, France) for yeasts. Genera of filamentous cellulose sodium salt 10.0 g L–1, agar 12.0 g L–1) (Samira et fungi were determined according to micromorphological al. 2011) for cellulolytic microorganisms. Nutrient agar and features (Kiffer, Morelet 2000). Actinomycete agar plates were incubated at 37 °C for 24 h, 178 Impact of endophytic microorganisms on rhizosphere microbiota

Rose Bengal agar at 24 °C for 48 h, CMC plates at 37 °C for and many of them are known to promote plant growth. 5 days and NBRIP at 28 °C for two weeks. The number of In our study, bacteria and fungi that did not inhibit CFU was determined at the end of an incubation period. germination had such plant growth affecting properties like Cellulolytic activity was determined by the Teather and solubilization of inorganic insoluble P and nitrification and Wood (1982) method. urease activity. All isolates were able to form colonies on NBRIP Statistical analysis agar, while only 11 isolates from six genera (Enterobacter, Microsoft Excel was used for calculating average values and Lactococcus, Pantoea, Serratia, Cutaneotrichosporon and standard deviations. Program R was used for data statistical Wickerhamomyces) showed clear lysis zones (Fig. 1). Two processing with a level of significance 0.05. isolates had the highest PSI, i.e. Lactococcus lactis ssp. hordniae and Pantoea agglomerans. Phosphorus is one of Results and discussion the major growth-limiting nutrient elements for plants in any ecosystem although it is abundant in soils in different Fifty-five isolates were obtained from plant material forms of usually insoluble inorganic P (Gyaneshwar et al. and tested for their effect on plant seed germination. 2002). The ability to solubilize P was characterized using Germination of radish, alfalfa, cress and barley was PSI. Formation of halo zones around colonies or tricalcium estimated in suspensions with the isolates. Twenty-five P is commonly used for screening of P-solubilizing isolates did not inhibit plant germination after 7-day microorganisms. It has been shown that PSI is likely incubation. These microorganisms were (number of not a reliable indicator for determining the ability of strains shown in brackets): bacteria Brevibacillus brevis microorganisms to solubilize P. Microorganisms that do (3), Enterobacter cancerogenus (1), Enterobacter cloacae (1), not produce lysis zone on agar plates often can solubilize Enterobacter sp. (2), Kytococcus sedentarius (1), Lactococcus P in liquid medium (Nautiyal 1999). Also Spagnoletti et al. lactis ssp. hordniae (1), Micrococcus luteus (1), Pantoea (2017) found differences between solid and liquid medium agglomerans (1), Pseudomonas putida (2), Pseudomonas in a study with dark septate endophytic fungi. Chen et al. sp. (1), Serratia liquefaciens (5) and Stenotrophomonas (2014) showed that a reliable indicator for microorganism maltophilia (2), filamentous fungi Mucor sp. (2) and yeasts ability to solubilise P is the colony diameter and size of Cutaneotrichosporon mucoides (1) and Wickerhamomyces cells. Their observations explain why the P concentration in anomalus (1). Many isolates of the same species differed liquid broth does not always correlate with PSI or diameter in physiological characteristics and thus belonged to of the lysis zone. Isolates with small cells form relatively different biotypes. These 25 isolates were further tested as small colonies, but are able to solubilize a significant a consortium. amount of P in liquid broth due to a high cell surface Many microorganisms produce phytotoxic volatile area-volume ratio. The ability to solubilize P is generally and/or soluble compounds and compete with seeds for associated with production of organic acids, which results oxygen and other compounds, and as a result, inhibit in the acidification of media (Vyas, Gulati 2009). Bacteria seed germination (Sarlistyaningsih et al. 1996; Weise et al. from the genera Acinetobacter, Burkholderia, Enterobacter, 2013). Other microorganisms do not inhibit germination Exiguobacterium, Pantoea, Pseudomonas etc. have been

Fig. 1. Mean PSI of microbial genera in NBRIP medium. Values are mean ± standard deviation of four replicates from each strain.

179 D. Vecstaudža, M. Seņkovs, V. Nikolajeva, O. Mutere

Fig. 2. Mean specific activity of nitrification potential of microbial genera. Suspensions were incubated in reaction mixture for 48 h at 37 °C. Values are mean ± standard deviation of four replicates from each strain. recognized as active P-solubilizers (Collavino et al. 2010; of one Mucor sp., while both our Mucor isolates showed high Oteino et al. 2015). Lactococcus and Pantoea as well as urease activity. Mucor spp. can produce urease and other Enterobacter, Serratia and yeasts isolated in this study, extracellular enzymes (Thompson, Eribo 1984). Ureolytic showed relatively high P-solubilization ability. However, species (from genera Sporosarcina, Bacillus, Lysinibacillus, solubilization of inorganic P in vitro is not always associated Arthrobacter, Brevibacterium etc.) are widely distributed with promotion of plant growth (Collavino et al. 2010). in soil (Burbank et al. 2012). It was previously found that Furthermore, this feature may be advantageous to plants the consortium used in the present study significantly only under deficiency of bioavailable or dissolved P. diminished urease activity in the soil and did not have any Nitrification potential (Fig. 2) varied in microbial impact on the nitrification potential of soil (Vecstaudza et suspensions from 29.2 relative units for Mucor spp. to 379.7 al. 2017). It should be noted that urease and nitrification are for Lactococcus lactis ssp. hordniae. All isolates showed not always considered as plant growth-promoting. Urea is a urease activity (Fig. 3). Specific activity of urease varied from widely used nitrogen fertilizer but in most soils it is rapidly 29.3 units for Enterobacter spp. to 288.0 units for Mucor spp. converted to carbon dioxide and ammonia and volatilized In our experiments, nitrification activity characterized the (Choi et al. 2007). Urease also contributes to the production ability of the isolates to perform heterotrophic nitrification. of nitrous oxide and dinitrogen gas and leaching of soil Nitrification was observed for all isolates, with the exception nitrate, and leads to reduced availability of nitrogen to

+ –1 –1 Fig. 3. Mean specific activity of urease (N-NH4 mg L OD620 protein ) of microbial genera. 48 h incubation was carried out in reaction mixture at 37 °C. Values are mean ± standard deviation of four replicates from each strain.

180 Impact of endophytic microorganisms on rhizosphere microbiota

from three German regions and came to the conclusion that soil pH is the best predictor for bacterial community structure, diversity and function. Zhao et al. (2005) showed that biofertilizers containing plant growth promoting microorganisms influenced the diversity of fungal community by promoting development of cellulolytic fungi. In our study, concentration of P solubilizing and cellulolytic microorganisms in the rhizosphere at the end of the vegetation experiment was too low to be detected, even though particular components of the consortium were capable of solubilizing P. Molecular testing should be used in future to clarify the effect of the microbial consortium(s) Fig. 4. Amount of CFU of microorganisms in the barley on the composition of microbial communities in the rhizosphere after 19-day vegetation experiment. *, significant rhizosphere. difference with control P( < 0.05). Acknowledgements plants and environmental damage (Smith et al. 2015). 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Received 14 July 2018; received in revised form 15 August 2018; accepted 25 September 2018

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