Shamil Validov

High incidence of plant growth-stimulating bacteria associated with the rhizosphere of wheat grown on salinated soil in Uzbekistan.

Soil salinization is increasing steadily in many parts of the world and causes major problems for plant productivity. Under these stress conditions, root-associated beneficial bacteria can help improve plant growth and nutrition. In this study, salt-tolerant bacteria from the rhizosphere of Uzbek wheat with potentially beneficial traits were isolated and characterized. Eight strains which initially positively affect the growth of wheat plants in vitro were investigated in detail. All eight strains are salt tolerant and have some of the following plant growth-beneficial properties: production of auxin, HCN, lipase or protease and wheat growth promotion. Using sequencing of part of the 16S rDNA, the eight new isolates were identified as Acinetobacter (two strains), Pseudomonas aeruginosa, Staphylococcus saprophyticus, Bacillus cereus, Enterobacter hormaechei, Pantoae agglomerans and Alcaligenes faecalis. All these strains are potential human pathogens. Possible reasons for why these bacteria present in the rhizosphere and establish there are discussed.

Genetic tools for tagging Gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies.

Live-cell imaging techniques are essential to gain a better understanding of microbial functioning in natural systems, for example in biofilms. Autofluorescent proteins, such as the green fluorescent protein (GFP) and the red fluorescent protein (DsRed), are valuable tools for studying microbial communities in their natural environment. Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants were created such as mCherry. In this study, we developed genetic tools for labeling Gram-negative bacteria in order to visualize them in vitro and in their natural environment without the necessity of antibiotic pressure for maintenance. mcherry was cloned into two broad host-range cloning vectors and a pBK-miniTn7 transposon under the constitutive expression of the tac promoter. The applicability of the different constructs was shown in Escherichia coli, various Pseudomonas spp. and Edwardsiella tarda. The expression of mcherry was qualitatively analyzed by fluorescence microscopy and quantified by fluorometry. The suitability of the constructs for visualizing microbial communities was shown for biofilms formed on glass and tomato roots. In addition, it is shown that mCherry in combination with GFP is a suitable marker for studying mixed microbial communities.

Bacteria able to control foot and root rot and to promote growth of cucumber in salinated soils

The aim of the present work was to test known bacterial plant growth-promoting strains for their ability to promote cucumber plant growth in salinated soil and to improve cucumber fruit yield by protecting these plants against soil-borne pathogens. Fifty-two plant-beneficial bacterial strains were evaluated for their ability to protect plants against cucumber foot and root rot after bacterization of the seeds and infestation of salinated soil with the isolated Fusarium solani pathogen. Based on the results of initial screenings, five efficient strains were selected, namely Serratia plymuthica RR-2-5-10, Stenotrophomonas rhizophila e-p10, Pseudomonas fluorescens SPB2145, Pseudomonas extremorientalis TSAU20, and P. fluorescens PCL1751. All five strains are salt tolerant since they grow well in a medium to which 3% NaCl was added. Infestation of the soil with F. solani resulted in an increase of the percentage of diseased plants from 17 to 54. Priming of seedlings with the five selected bacterial strains reduced this proportion to as low as 10%. In addition, in the absence of an added pathogen, all five strains showed a significant stimulatory effect on cucumber plant growth, increasing the dry weight of whole cucumber plants up to 62% in comparison to the non-bacterized control. The strains also increased cucumber fruit yield in greenhouse varying from 9% to 32%. We conclude that seed priming with the selected microbes is a very promising approach for improving horticulture in salinated soils. Moreover, allochthonous strains isolated from non-salinated soil, from a moderate or even cold climate, and from other plants than cucumber, functioned as well as autochthonous strains as cucumber-beneficial bacteria in salinated Uzbek soils. These results show that these plant-beneficial strains are robust and they strongly suggest they can also be used successfully in case the climate gets warmer and the soils will become more salinated. Finally, the mechanisms by which they may exert their plant-beneficial action are discussed.

Selection of bacteria able to control Fusarium oxysporum f. sp. radicis-lycopersici in stonewool substrate

Aims:  Tomato foot and root rot (TFRR), caused by Fusariumoxysporum f. sp. radicis‐lycopersici (Forl), is an economically important disease of tomato. The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive root‐colonizing bacteria from total rhizosphere soil samples.

Characterization of Bacillus subtilis HC8, a novel plant‐beneficial endophytic strain from giant hogweed

Thirty endophytic bacteria were isolated from various plant species growing near Saint‐Petersburg, Russia. Based on a screening for various traits, including plant‐beneficial properties and DNA fragment patterns, potential siblings were removed. The remaining isolates were taxonomically identified using 16S rDNA sequences and potential human and plant pathogens were removed. The remaining strains were tested for their ability to promote radish root growth and to protect tomato plants against tomato foot and root rot. One strain, Bacillus subtilis HC8, isolated from the giant hogweed Heracleum sosnowskyi Manden, significantly promoted plant growth and protected tomato against tomato foot and root rot. Metabolites possibly responsible for these plant‐beneficial properties were identified as the hormone gibberellin and (lipo)peptide antibiotics respectively. The antibiotic properties of strain HC8 are similar to those of the commercially available plant‐beneficial strain Bacillus amyloliquefaciens FZB42. However, thin layer chromatography profiles of the two strains differ. It is speculated that endophytes such as B. subtilis HC8 contribute to the fast growth of giant hogweed.

Monitoring of pathogenic and non-pathogenic Fusarium oxysporum strains during tomato plant infection.

Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on agricultural and ornamental plants, is important for predicting disease outbreaks. Since both pathogenic and non‐pathogenic strains of Fox are ubiquitous and are able to colonize plant roots, detection of Fox DNA in plant material is not the ultimate proof of an ongoing infection which would cause damage to the plant. We followed the colonization of tomato plants by strains Fox f. sp. radicis‐lycopersici ZUM2407 (a tomato foot and root rot pathogen), Fox f. sp. radicis‐cucumerinum V03‐2g (a cucumber root rot pathogen) and Fox Fo47 (a well‐known non‐pathogenic biocontrol strain). We determined fungal DNA concentrations in tomato plantlets by quantitative PCR (qPCR) with primers complementary to the intergenic spacer region (IGS) of these three Fox strains. Two weeks after inoculation of tomato seedlings with these Fox strains, the DNA concentration of Forl ZUM2407 was five times higher than that of the non‐compatible pathogen Forc V03‐2g and 10 times higher than that of Fo47. In 3‐week‐old plantlets the concentration of Forl ZUM2407 DNA was at least 10 times higher than those of the other strains. The fungal DNA concentration, as determined by qPCR, appeared to be in good agreement with data of the score of visible symptoms of tomato foot and root rot obtained 3 weeks after inoculation of tomato with Forl ZUM2407. Our results show that targeting of the multicopy ribosomal operon results in a highly sensitive qPCR reaction for the detection of Fox DNA. Since formae speciales of Fox cannot be distinguished by comparison of ribosomal operons, detection of Fox DNA is not evidence of plant infection by a compatible pathogen. Nevertheless, the observed difference in levels of plant colonization between pathogenic and non‐pathogenic strains strongly suggests that a concentration of Fox DNA in plant material above the threshold level of 0.005% is due to proliferation of pathogenic Fox.

Is l-arabinose important for the endophytic lifestyle of Pseudomonas spp.?

Twenty endophytic bacteria were isolated from surface-sterilized stems and roots of cucumber plants. After removal of potential siblings and human pathogens, the remaining seven strains were identified based on their 16S rDNA as Pseudomonas fluorescens (2 strains) and P. putida (5 strains). Three strains, namely P.fluorescens CS1, P. fluorescens CR2 and P. putida CR3, were able to suppress tomato foot and root rot (TFRR). Special attention was paid to the characterization of the BIOLOG carbon oxidation profiles of the isolated pseudomonads in order to identify nutrients which might be important for their endophytic lifestyle. Comparative analysis of the profiles of these seven strains with those of seven rhizospheric Pseudomonas spp. revealed that endophytes were able to oxidize l-arabinose and 2,3-butanediol significantly more often than the rhizospheric group. An independent growth experiment performed in tubes using l-arabinose and 2,3-butanediol as sole carbon sources showed the same results as seen using BIOLOG for l-arabinose, but not for 2,3-butanediol. Since l-arabinose is one of the most abundant sugars in xylem of cucumber plants and was not detected in their rhizosphere, our data suggest that utilization of l-arabinose might be a trait contributing to the endophytic lifestyle of the isolated Pseudomonas endophytes.