Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production

A bacterial strain IMBG1S6 producing exopolysaccharide (EPS) was isolated from siliceous rock and identified as a Paenibacillus species by partial sequencing its 16S rDNA. Paenibacillus sp. IMBG156 was used in a novel technology for inoculant production based on co-cultivating this bacterium with any bacterium of choice. Paenibacillus sp. provides in situ the bacterial cells of the inoculant with EPS, a carrier, and most likely with a source of carbon and energy. The partner bacterium designates a type of inoculant (biopesticide or biofertiliser). The strain IMBG156 does not destroy the signaling system of Gram-negative partners, based on acylated homoserine lactones, stimulates plant growth, and is rather competitive in the plant rhizosphere and soil. A prototype of the inoculant based on dual-culture Paenibacillus sp. IMBG156 — Pseudomonas sp. IMBG163 exhibits a noticeably longer shelf life than monoculture of Pseudomonas sp. IMBGI63.

Introduction.The increasing concern on agrochemicals hazard to health and economical problems have promoted fundamental research in the area of alter native agriculture and in search for new agrobiotechnologies.Recently, tremendous efforts have been invested in studies of molecular mechanisms of plant defense [1][2][3].
Defense-signaling components have been dis covered, and new, effective and sustainable alter natives to pesticides proposed.However, at the pre sent time a sustainable strategy of crop defense still relies on usage of microbial biological agents able to induce system resistance in crop plants.The microbial inoculants based on the competitive, beneficial for the plant bacteria are considered as a reasonable alter native to agrochemicals [4,5].It is critical for the inoculant development that the inoculant product was in a formulation not only to deliver an adequate bacterial population but also to have enough product shelf life.Nowadays, the practical formulations are in use to prolong survival of Gram-negative bacteria [6][7][8][9][10][11], however, the usage of both mineral and organic carriers for bacteria makes inoculants more expensive.In our previous research we used the exopolysaccharide (EPS) mucilan produced by B. mucilaginosus B-4901 as an inexpensive carrier in the series of inoculants KLEPS (KLityna (a cell, Ukr.) and EPS) to prolong Gram-negative bacteria survival [12].The inoculants enhanced crop production on poor soils and exhibited enough shelf life.In spite of advantages, the technology of KLEPS development needed a separate stage of EPS manufacture, which complicated the procedure and raised the price of the inoculant.The objectives of this study were to isolate slime-producing bacteria, following the idea to use Culture conditions.The media for bacterial gro wth were: KB [13] and LB [14] used for all strains of bacteria, except for Paenibacillus sp.being cultured in the medium MZ [12].Zeolite (10 g/1) was added to LB when needed.
Determination of EPS content.Cells of a two-day culture were centrifuged at 10,000 g for 30 min, EPS was extracted from supernatant by 2 v of ethanol and dried at 37 °C until stable weight was obtained.
Bacteria isolation procedures were performed us ing the samples of zeolite collected from Sokyrnytzya (Transkarpatian region) and fragments of silica rocks originated from Khmelnitsky region.In separate ex periments 1 g of zeolite or silica rocks (fraction of 5 mm) was incubated in MZ for 48 h at 30 "C.The accumulating cultures were diluted serially and spre ad on selective MZ medium.Slime colonies were collected, bacteria were purified, and identified accor ding to N. Krasilnikov [15].
Total DNA isolation was performed as recom mended in [16].

Co-cultivating of partners was performed in MZ.
After 30 h of co-cultivating, the serial dilutions were made for evaluation of population size.The partner cultures were detected on LB or KB agar, Paeni bacillus sp.-on MZ plates.
Assay for production of signaling molecules.Bac terial strains (separately and in pairs with Paenibacil lus sp.) were tested in cross-feeding assays for acylated homoserine lactones (AHLs), using the indi cator strain A tumefaciens A136 as recommended in [18].
Antibacterial activity was tested in vitro on Petri dishes by the standard agar-diffusion assay, using two layer agar with the upper layer of an indicator culture soft agar (0.4 %).
Detection of the acetylene reductase (nitrogenase) activity (ARA) was performed according to [19].The ARA of K. oxytoca IMBG26 was detected with the gas chromatograph Tzvet (Cheh Republic) in 14 ml flasks where the bacterial culture was grown in an N-free medium supplemented with sucrose or EPS (final content 1.5 and 1.0 %, respectively) in the presence of 10 % acetylene within 16 h at 28 °C.were vortexed in 0.9 % NaCl, and serial dilutions were plated on selective media LB, KB, MZ supple mented with rifampicin (50 /ig/ml) when needed to discriminate between bacteria.
Statistical analysis of results.The data on bio metrical parameters of wheat and bacteria survival are means from three replications.Statistical analysis was performed using SigmaPlot 8.0 software.Standard deviations were calculated for each data point.
Nucleotide sequence accession number.The se quence generated in this study has been deposited in the GenBank database under accession number AY645946.
Results and Discussion.Phenotypic characteris tics.Two isolates (from zeolite and silica rock, designed IMBG156 and IMBG157) were characterized as aerobic Gram-positive rods of (0.2-0.5) • 2.0 fim which formed spores of 0.5-0.7 /nm.The spore position was preferentially central but terminal lo cation was rarely observed.The optimal temperature of growth was 28 °C, but they grew well in a range of 10-45 "C.On the agar MZ medium they created transparent slime colonies of 10.0-13.0mm diameter and produced 10.0-13.0g EPS per 1 1 of liquid medium.The isolates consumed carbohydrates, ge nerated acids, and did not utilize amino acids as C and N sources.Bacteria hydrolyzed starch and could not grow anaerobically with nitrate as a respiratory substrate.We did not manage to determine the taxonomic position of the isolates with Bergy's deter minative manual.To clarify their systematic position, it was practical to analyze the phylogenetic marker gene, rrn, encoding RNA of small subunit of ribosome (16S).did not cause a phytotoxic effect and increased wheat shoot height as shown in Table 1.Being applied together with Pseudomonas sp.IMBG163 for seed inoculation, it promoted plant growth more efficiently.
In the rhizosphere of wheat inoculated by a rationally assembled consortium of plant growth promoting rhizobacteria (Pseudomonas sp.IMBG163, P. aureo faciens IMBG288, K. oxytoca IMBG26), the strain IMBG156 was quite competitive on background of beneficial bacteria (Fig. 1).

Co-cultivation of Paenibacillus sp. IMBG156 with bacteria of interest.
With the idea to keep bacteria alive in the gel produced by bacteria within cocultivation, Paenibacillus sp. and chosen Gram-ne gative bacteria were cultured by pairs.The results of experiments on co-cultivating showed practically the absence of restrictions in picking up partners for Paenibacillus sp.(Table 2).The partners gained a population size of log 9-10 CFU/ml.The critical factors in the one-step procedure of prototype inoculant manufacture were the size of population of a strain that determined a sort of inoculant, and also the concentration of EPS produced by Paenibacillus sp.In the case of co-cultivating IMBG 156 together with K. oxytoca IMBG26 or Pseudomonas sp.IMBG163, the culture gel contained 1.4±0.05E+9or 2.5±0.09E+9CFU/ml of a bacterium-partner, res pectively, and not less than 20.0 g/1 of EPS.
A term of the inoculant shelf life is important when the inoculant is based on Gram-negative bac teria.We showed that survival of Pseudomonas sp.IMBG 163 was prolonged in the dual culture with Paenibacillus sp. in comparison with the monoculture (Fig. 2).A better survival of IMBG 163 was also observed in a minimal medium supplemented with zeolite.The results showed that the strain IMBG 163 preserved the population size at the level of log 9 CFU/ml in both dual culture and monoculture grown in the presence of zeolite within 2 months.On the contrary, the population size of IMBG 163 de clined from log 10 to log 7 CFU/ml without coinoculation by Paenibacillus sp.IMBG156 or addition of the mineral to the medium.This clearly de monstrated that both Paenibacillus and zeolite sup ported survival of Pseudomonas sp.IMBG163.K. oxytoca IMBG26 was able to grow and reduce ace tylene (518 nM C 2 H 4 /h flask) in the nitrogen-free medium where 1.0 % EPS (derived from Paeni bacillus sp.IMBG156) was used as a carbon source, and this suggested that EPS produced by Paeni bacillus had the potential to support growth and activity of the partner.

Detection of antibacterial activity in dual cultures.
Signaling systems play a role in bacteria-bacteria and plant-bacteria communications [20].Antibacterial ac tivity of some pseudomonads is controlled with AHLs [21,22].It is well known that Gram-positive bacteria are able to destroy AHLs of Gram-negative neighbors [23 ].To compose dual bacterial pairs, it is important to know that quorum signaling is not impaired by co-cultivation.Bacteria used in experiments on cocultivation were tested in cross-feeding assays for AHL 4 _ 14 detection earlier [24 ].Few of them produced AHLs (Pseudomonas sp.IMBG163, Pseudomonas sp.IMBG168 and P. auerofaciens IMBG288), and no difference was observed between mono-and dual Pseudomonas-Paenibacillus cultures with respect to AHL production.The results represented in Table 3 showed inhibition of pathogenic bacteria by both monocultures of pseudomonads and appropriate dual Pseudomonas-Paenibacillus cultures.This may mean that strain Paenibacillus sp.IMBG156 did not impair AHLs produced by a partner and indirectly demon strated integrity of AHLs in dual cultures.
Paenibacillus sp.IMBG156 has been selected as a bacterium-nurse for the dual-culture technology of inoculant development, first of all, due to production of large amounts of EPS.The strain IMBG156 provided the living cells of a bacterium-partner, the second species of two-component consortium, with a carbon source and apparently caused better survival of the latter.In this study IMBG156 displayed commensal interactions in the pairs with other bac terial strains and synergistic positive impact on the plant.These results are consistent with those ob tained for other bacteria acting synergistically on the plant development [25,26].IMBG156 was quite competitive in the plant rhizosphere bacterial com munity and in the soil, in contrast to the known data on the gradual replacement of Paenibacillus by Pseu domonas [27].These additional beneficial features make the strain rather promising for application for seed inoculation in programs of plant health care and soil remediation in company with biocontrol bacteria.
The inoculants, containing both Paenibacillus sp.IMBG156 and a partner bacterium, can be stored for a relatively long period of time in the presence of large amounts of EPS produced in situ without preservatives and conventional carriers.This finding is based on a concept of keeping bacteria alive under storage of the dual culture in a gel and may be explained, first of all, by the fact that growing any Gram-negative bacterium with Paenibacillus sp.IMBG156 results in stimulation of EPS production which can serve both as a carbon and energy source for the bacterium-partner.In case when EPS serves as a carrier, the organisms appear to establish struc tured populations where cells are not aggregated.Under this condition the bacteria are positioned in a heterogeneous environment with gradients of nutri ents and waste products as a consequence of diffusion and mass transport processes, and it is therefore to be expected that this heterogeneity is reflected in the physiology of the individual cells and better survival.EPS keeps up water and nutritional regime, and therefore bacterial cells are physiologically active under storage, in contrast to a dry KLEPS formu lation where bacteria were dormant.The dual-culture technology based on co-cultivating the bacterium Paenibacillus sp.IMBG156 and any bacterium of choice is simpler and less expensive compared to the previous technology [12].

- 1 Fig 1 .Fig. 2 .
Fig 1.Average size of populations of bacterial strains associated with wheat roots: two weeks after inoculated seed planting

Error represents standard deviation. Treatment is different from the control at p -0.05 as determined by Student's f-test. Values followed by the same letter in a column are not significantly different.
plants were left non-inoculated.The plants were maintained under natural light at 20 °C in a growth chamber.The plants were watered once per two days.At the end of the experiment (14 days after ino culation) all plants were harvested and external root colonization was examined.Root sections of 100 mg Note.