Colonization capacity and monitoring of the biocontrol agent Pseudomonas sp. IMBG163 on wheat roots

The effect of Pseudomonas sp. IMBG163 on wheat seedlings (Triticum aestivum L. cv. Katyusha) was studied. This strain has been marked with the gus reporter gene in order to examine a colonization pattern and to monitor its maintenance on the plant roots. The strain IMBG163 significantly enhanced all biometric parameters measured: dry weighty height of shoots, length of roots. Co-culture of IMBG163 with the plant growth promoting bacterium Paenibacillus sp. IMBG156 did enhance the PGP effects. Detection of marked experimental bacterium on/into the plant have being performed with histochemical GUS assay and did not reveal the endophytic pattern of colonization For monitoring of IMBGJ63 in the rhizosphere, a culture-independent method, ARDRA, has been used. A combination of conventional and molecular techniques showed IMBG163 to be effective and persistent colonizer of the wheat roots.

Introduction.Saprophytic pseudomonads are common root-colonizing bacteria that can improve plant growth or health.Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere.Current environmental criteria designed to improve crop and forest productivity, together with the development of a sustainable agriculture, and the intended reduction of the use of fertilizers and pesticides have produced an ongoing increase in scientific interest and practical use of the plant growth promoting bacteria and biocontrol agents as inocula [1 ].These effects are due mostly to a non-infective interaction of root-associated bacteria with plants.However, in some cases, the use of bacteria in agriculture has no success expected, since the environment can appear to be different and hostile to some bacteria.Therefore, before the deli berate use of a beneficial bacterium as an inoculum, it is necessary to know some key parameters such as root colonization capacity, location, and degree of persistence of the inoculum [2].The strain used in this work has already shown its antagonistic capacity [3].
Nevertheless, there is a paucity of data about conditions under which the inoculum strain has to compete with a wide variety of soil microorganisms.It is a well-established fact that Pseudomonas, being introduced into the microcosm, is able to enter into an unculturable state due to different reasons and cannot be cultivated by current techniques like the traditional agar plating [4,5].This limits the use fulness of colony counts.As a consequence, doubt has been raised that the results obtained seem not to be representative enough for the actual processes in nature.
On the other hand, there is an increasing concern about the risk of using this group of bacteria in the processes such as biological control since some species are pathogens and monitoring Pseudomonas is an essential element of inoculation programs [6, 7].Among the methods that have been suggested for monitoring bacteria in environmental samples are those based on usage of nucleic acids [8][9][10].
The aim of this work was to study the coloniza tion capacity, persistence, and colonization pattern the wheat rhizosphere and endosphere of Pseudo monas sp.IMBG 163, using both conventional and molecular methods.
Materials and Methods.The bacterial strains used in this study -Pseudomonas sp.IMBG 163, Pseudomonas aureofaciens IMBG164, Klebsiella oxytoca IMBG26, Paenibacillus sp.IMBG156 -are de posited in the collection of Institute of Molecular Biology and Genetics of NASU, Pantoea agglomerans IMV56 was kindly provided by Prof. R. Gvozdyak.The strains were grown on nutrient broth medium LB [111, except Paenibacillus sp., that used minimal MZ medium [12].Pseudomonas sp.IMBG163 was tested for production of indole acetic acid (IAA), using the method described in [13].Siderophore production was detected by growing the culture on KB [14].Phosphate solubilization capacity [15] was tested on KB supplemented with calcium phosphate.Plants were maintained under natural light in controlled conditions (25/20 °С, 14/10 h light/dark,) in sand (a monoculture) and zeolite (Sokyrnytzya) (a bacterial assemblage) in a culture chamber of our design [16].
Ten wheat germinated seeds were inoculated by suspension of 10 b colony-forming units (CFU).At the end of the experiment (30 days after inoculation) all plants were harvested and the following data were collected: dry weight, height of shoots, length of roots.To estimate the external root colonization, root sections were vortexed in 0.9 % NaCl, and serial dilutions were plated on LB and KB media supple mented with rifampicin (50 jug/ml) or streptomycin (100 jug/ml) when needed.To estimate the interior root colonization, root samples were surface disin fected with several incubations in Belizna (Kyiv) for 2 min alternated with rinsing in 0.9 % NaCl for 2 min; the surface disinfection parameters were opti mized prior to the experimentation with control of surface contamination.The conjugation between bac teria was performed on LB agar medium plates, and the plasmid pCAM140, conferring mTn5SsgusA20, was used as a vehicle of the gus gene [17].Detection of bacteria on/into the plant and estimation of survival of experimental bacteria in the rhizosphere was performed with histochemical GUS assay [17].DNA was isolated from the bacterial culture and plant tissues with kits from «МоВіо Inc.» (USA).Pseudomonas-specific PCR amplifications were done as prescribed in [9].445 bp amplicons of the 16S rDNA were cleaved with Sail («Fermentas», Lithuania).
The experimental scheme was repeated three times.Statistical analysis was performed using Sigma Plot 8.0 software.Standard deviations were calculated for each data point.
Results and Discussion.Metabolic capacity of Pseudomonas sp.IMBG163.The auxin production of strain was not detected after growth in LB medium, however, this strain was able to mobilize iron from chelating agents of medium KB.The cleared halo of 2-3 cm has been observed around colonies.The strain was able also to solubilise calcium phosphate, forming the 2.5 cm halo around the colony.Although the mechanisms by which IMBG 163 promotes plant growth are not yet fully understood, it is clear that this may be siderophore production and phosphate mobilizing that is in agreement with well established data [18].
Colonization capacity.Pseudomonads are often unable to establish population in significant number on plant roots, including wheat, and turn nonculturable [4, [19][20][21].In this connection, Raaijmakers and Weller postulate that particular rhizobacterial genotypes have evolved a preference for colonization of specific crops [22].Certain genotypes of pseu domonads well colonize wheat, including genetically modified strains, and establish high rhizosphere po pulation densities up to 10 7 CFU/g of root [23][24][25][26].Our results show that strain IMBG 163 is also able to colonize the wheat root, grown in mineral substrate of low bioavailability.There were no striking differences in the total number of CFU among different sampling times within a 30 day period, and the number of bacteria isolated from 1 g of fresh root was (0.94-1.3) 10 5 CFU.We did not also reveal a decrease in the population size or unculturable state of the strain when it was used as monoculture in aseptic condi tions.Van Elsas et al. [8 ], studying the colonization of wheat roots by different strains of Pseudomonas, found that the highest number of bacteria was obtai ned 4 weeks after inoculation, decreasing afterwards.
In the wheat rhizosphere inoculated with the rationally assembled consortium of plant growth pro moting rhizobacteria, the strain IMBG 163 was quite competitive on the background of beneficial bacteria (fig.1).It was not eliminated from the bacterial community in the plant rhizosphere.Moreover, the strain raised 20-fold population being used in concert with the mentioned bacteria.Our data exhibited high colonization capacity of Pseudomonas sp.IMBG 163 in the wheat rhizosphere grown in soil of low bioavaila bility in contrast to the data of Hase et al. [4] and Bjorklof et al. [21 ], who found that the inoculant strain on the roots of plants grown in the low fertility soil was mostly nonculturable cells.
We did not observe the endophytic colonization of wheat, although certain species of pseudomonads are known as endorhizosphere colonizers [27][28][29][30].The bacterial cells entered into root hair, however, they did not spread in plant tissue.
Effects of the inoculated bacterium on the wheat growth.As shown in the table, the strain increased all the biometrical parameters measured, and this corre lated with the earlier data [31 ].Since the strain was shown to mobilize iron and phosphorus, the improved shoot growth could be the final result of improved nutrient uptake and plant nutrition.
The dual-culture assays in which two strains were grown in a liquid medium and later were used for the inoculation of wheat seeds showed that two unrelated bacteria could stimulate the wheat growth and biomass accumulation (table).This is in agree ment with the recent data on interrelation of two unrelated Pseudomonas in the wheat rhizosphere [32].Further experiments will show whether the bacteria due to this interpopulation communication in the rhizosphere stimulate each other in the expression of traits beneficial for plant growth or it is a cumulative effect of two bacteria.
In conclusion, these results show that the strain IMBG 163 has good colonization capacity in the rhizo sphere of wheat.However, it is necessary not only to provide the right bacterium, but also the correct techniques to check the fate of the inoculum in order to establish the most suitable way to use the micro organisms in agriculture.

Biometrical data on effects of inoculation with plant growth promoting bacteria Pseudomonas sp. IMBG 163 on growth parameters of the 30-day wheat seedlings
Monitoring the strain was carried out by a culture-independent method in order to detect the bacterium in a case when it entered in unculturable state under some unfavorable conditions.We used a PCR method targeting 16S ribosomal DNA for spe cific detection of Pseudomonas DNA in both plant and substratum DNA.A pair of primers, the universal 9-27 and specific for Pseudomonas DNA, PSM, creates amplicon of 445 bp [9].The restriction enzyme Sail generates three fragments of around 75, 120 and 250 bp specific for Pseudomonas sp.IMBG163 (fig.2).Periodical samplings and amp lifications showed presence of the bacterium DNA in total DNA isolated from the plant roots.

Fig. 1 .Fig. 2 .
Fig. 1.Colonization capacity of Pseudomonas sp.IMBG 163 as a member of bacterial assemblage in the wheat rhizosphere: 2 weeks after a planting (7 -Klebsiella oxytoca\ 2 -Paenibacillus sp.; 3 -Pseudomonas sp.; 4 -Pantoea agglomerans; 5 -Pseudomonas aureofaciens) represents standard deviation.Treatment is different from the control at p = 0.05 as determined by Student's Mest.Values followed by the same letter in a column are not significantly different.