Evaluation of The Effect of Epiphytic, Endophytic And Rhizosphere Bacteria On Seed Germination And Seedling Characteristics

During 2013 and 2014, in order to investigate the effect of bacteria on the characteristics and growth rate of cotton seedlings, isolated epiphytic, endophytic and rhizospheric bacteria from cotton plants in Golestan province. Based on biochemical and phenotypic tests, amplication and sequencing of 16S rRNA gene, were identied strains. Isolates of Bacillus pumilus MR11, B. pumilus MR12, B. pumilus MR13, B. safensis MR21, B. safensis MR22 and Stenotrophomonas pavanii MR31 identied as superior endophytes and rhizosphere. Also Pseudomonas uorescens, P. syringae and Pantoea annanatis were identied as epiphytic from plants and seeds. These isolates were evaluated to effect on seedling growth characteristics. Based on results, the six-day-old seedlings treated with Bacillus pumilus MR11 and B. pumilus MR12 had the highest root and shoot length and fresh shoot weight. Due to the fact that B. pumilus MR12 and S. pavanii MR31 isolates had the highest percentage and germination rate, so their use as seed treatment can increase the percentage and germination rate of seeds and be effective in reducing the damage caused by seedling diseases. This is the rst report of isolation of B. safensis, B. pumilus and S. pavanii bacteria from rhizosphere and cotton endophytes in Iran.


Introduction
Phylosphere is the surface of the aerial parts of plants and a place for micro-organisms to live. Simple sugars such as glucose, fructose and sucrose are the predominant carbonaceous substances on leaves and stem that pass out of wound areas or secretory pores. In these areas, can be seen the largest population bacteria of phylosphere (Mercier and Lindow 2000). The microbial inhabitants of the phylosphere include different genera of bacteria, lamentous fungi, yeasts, algae, protozoa and nematodes. The bacteria are the most abundant inhabitants of the phylosphere (Andrews and Harris 2000). Plant species and leaf type have a bene cial effect on the number of bacteria of phylosphere. For example, the number of bacteria in the phylosphere of broadleaf plants such as cucumbers and beans is signi cantly higher than that of broadleaf plants with waxy leaves and grasses. On the other hand, the surface of the aerial parts of the plant is subject to rapid and continuous changes in temperature, humidity, ultraviolet rays, relative humidity and the concentration gradient of nutrients (Brencic and Winans 2005).
The rhizosphere is an area of the root that soil microorganisms are very interested in, using root secretions as carbon and energy sources and competing for food and trritory. Bacteria such as Bacillus, Pseudomonas, Azospirillium, Azotobacter, Arthrobacter, Entrobacter and Serratia are among the growthpromoting bacteria (Podile and Kishore 2006). Plant growth-promoting bacteria are part of the integrated disease management program and reduce chemical consumption (Shivalingaiah et al. 2013). These bacteria can stimulate the induction resistance system against a wide range of plant pathogens (Pieterse et al. 2003). The induction of diseases resistance in various crops such as banana, beans, rice, and cucumbers has been reported by Pseudomonas and Bacillus (Harish et al. 2008;Hasan et al. 2010). Most Pseudomonas spp. signi cantly increases plant length, root length, and dry matter, leading to the production of plant buds and roots. Most plant growth-promoting bacteria are able to settle in the roots, especially if inoculated before sowing. The use of plant growth promoting bacteria as a biological control agent is an alternative to the use of chemical fertilizers that have led to environmental pollution (Ali et al. 2010). Pseudomonads are the major group of the plant growth-promoting bacteria, which act by strategies such as the production of plant hormones, stimulating nutrient uptake, and controlling plant pathogens (Minaxi 2010).
Bacillus species were reported as endophytes within different plant tissues. B. megaterium was the most abundant species in the rhizosphere and B. pumilus and B. subtilis were the most abundant species in the soybean phylosphere (Arias et al. 1999). B. endophyticus from the inner tissue of cotton bers (Reva et al, 2002), Erwinia sp., Bacillus sp., B. pumilus, B. brevis, Clavibacter sp. and Xanthomonas sp. isolated as endophytes from roots, stems, buds and bolls (Misaghi and Donndelinger 1990). Enterobacter asburiae was isolated as an endophyte and from the cotton rhizosphere, which showed that this bacterium is able to spread systemically in the plant (Hallmann et al. 1998). P. uorescens caused a signi cant increase in germination and dry weight of cotton in greenhouse conditions (Salaheddin et al. 2010). P. uorescens stimulated seedling growth and increased yield with reduced disease (Safyazov et al. 1995).

Evaluation of the effect of bacteria on seedling growth
The seeds were soaked in 10 6 CFU dilution bacterial suspension for 10 minutes, then 100 seeds were sown in 3 repetitions in Whatman paper and incubated at 25°C with a period of 16 hours of light and 8 hours of darkness. Number of germinated seeds counted on the third and sixth days and 10 seeds randomly selected in each treatment and were measured characteristics such as root and shoot growth length, fresh and dry root weight, stem fresh and dry weight, total fresh and dry weight, amount of root, stem and total tissue water. To obtain the dry weight of stem and root, the samples were placed in an oven at 75°C for 72 hours and then were recorded data (ISTA 2015).
Identi cation of selected bacteria using molecular studies The taxonomic position of the selected bacteria was assessed by determining the sequence of the 16s rRNA gene region. To propagate this region, CinnaGen Company primers were used with sequences F27: 5´-AGAGTTTGATCMTGGCTCAG-3´ and R1492: 5´-TACGGYTACCTTGTTACGACTT-3´ (Lane 1991). For DNA extraction, bacterial isolates were cultured on Nutrient Agar medium for 24 hours after growth at 28°C . Bacterial cells were suspended in sterile distilled water and the turbidity of the suspension was adjusted to 0.1-1.2 units at 600 nm. To each sample was added 0.1 volume of normal potassium hydroxide. The samples were boiled for 10 minutes and then centrifuged at 13,000 rpm for 5 minutes. The top of solution was separated and used as a DNA-containing solution for some genotypic tests (Arabi et al, 2006). To amplify this gene region using PCR, the reaction was performed at a nal volume of 25 µl as follows: The reaction mixture consisted of 2 µl of DNA, 2.5 µl of PCR buffer 1X, 1 µmol of each primer, 0.2 mmol of dNTPs, 1.5 µmol of magnesium chloride and 3 units of Taq polymerase and a water content of 0.95 17 µl. Temporal and temperature conditions included the initial step of denaturation at 94° C for 5 minutes and then 35 separate cycles (including denaturation at 94° C for one minute, anealing at one minute in 55° C and DNA extension at 72° C for 90 seconds) and nally, a nal elongation step at 72° C for 10 minutes (Ausubel et al. 1992). After con rmation of the presence of ampli cation band in 1.5% agarose gel in PCR product, ampli ed samples were sent to Macrogen (South Korea) for puri cation and sequencing.
Bioedit 7.0.9.0 software was used for multiple alignments of the sequences obtained from the studied isolates. Mega5 software was used to compare the similarity and genetic distance of sequences (Felsenstein 1989). Phylogenetic tree was calculated by Neighbor-Joining method and Jukes-Cantor distance matrix with 1000 Bootstrap replications and E. coli were used for gram-negative bacteria and Paenibacillus bracinonensis for gram positive bacteria as an out group in drawing (Jukes and Cantor 1969) and Tree View 1.6.6 (Page 1996) was used to observe and analyze it.

Results And Discussion
Based on the comparison of characteristics means, the six-day-old seedlings of B. pumilus MR11 and B. pumilus MR12 had the highest root and shoot length and shoot fresh weight. They were also superior to other characrteristics (Tables 2 and 3). B. pumilus MR12 and S. pavanii MR31 isolates had the highest percentage and germination rate, so its use as seed treatment can increase the percentage and germination rate of seeds and reduce the amount of seedling diseases. Based on the results of 16S rRNA gene ampli cation and phenotypic characteristics, were identi ed isolates of S. pavanii MR31, B. safensis MR21, B. safensis MR22, B. pumilus MR13, B. pumilus MR12 and B. pumilus MR11 ( Fig. 1 and Table 1).   The numbers in each column, which are in at least one common letter, are statistically grouped together  Availability of data and materials are applicable.
Ethics approval and consent: Dear Editor in chief, we wish to submit a new manuscript entitled "Evaluation of the effect of epiphytic, endophytic and rhizosphere bacteria on seed germination and seedling characteristics" for consideration by the Journal of Cotton Research. We do not con rm that this work is original and has not been published elsewhere nor is it currently under consideration for publication elsewhere.
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