Bacterization Effect of Culture Containing 1-aminocyclopropane-1-carboxylic Acid Deaminase Activity Implicated for Plant Development

ACC deaminase regulates the production of ethylene level of plants by metabolizing 1-aminocyclopropane-1-carboxylate (ACC) into α-ketobutyric acid and ammonia. It was observed that two important phyllospheric bacteria of Spinach (Spinacia oleracea) which are Paenibacillus polymyxa SNKp6 and Bacillus thuringiensis SNKr10 exhibit significant ACC deaminase activity which can help plant growth under stressful conditions like heavy metal contaminated sites. This growth activity was studied by in vitro seed germination of Vigna radiata plant. In the present study, we concluded that these two important bacterial strains can be helpful for plants to grow under stressful conditions and also help in their growth. Original Research Article Sharma and Saharan; BMRJ, 16(1): 1-10, 2016; Article no.BMRJ.27135 2


INTRODUCTION
Phyllospheric bacteria are those bacteria lie on surface of plants leaves. These bacteria play important role for plants by forming mutual interaction with them. The phyllosphere is a very important biologically active interface between the aerial parts of the plant and the air. It precedes the fixation of carbon dioxide, release of molecular oxide and thus facilitates primary productivity. Microorganisms on leaf surfaces are said to be extremophiles as they are able to grow/tolerate high temperature from 40-55°C, Ultraviolet radiation and cool temperatures upto 5-10°C [1]. Many bacteria like Methylobacterium and Pseudomonas are found in phyllosphere and play useful role [2]. The microbial communities of leaves include different genera of algae, bacteria, filamentous fungi, yeasts and less frequently protozoa and nematodes. The plant growth promoting bacteria (PGPB) are those useful bacteria that helps the enhancement in plant's growth and yield [3], antimicrobial activity against phytopathogens (through HCN, siderophore antifungal activity), ammonia, biological nitrogen fixation and also by ACC deaminase activity. The plant growth promoting bacteria has been shown to be useful in many aspects of plant development and their response toward stress [4]. It plays important role as epinasty, leaf abscission, senescence and ripening of fruits [5]. Ethylene is produced mainly by two enzymes including ACC oxidase, ACC synthase. ACC is a precursor of ethylene in plant which is hydrolysed by ACC deaminase that help in growth and development of plants. 1-aminocyclopropane-1carboxylate synthase (ACC synthase) is the enzyme that catalyzes ethylene synthesis by conversion of s-adenosyl methionine to 1aminocyclopropane-1-carboxylate and ethylene synthesis involves the conversion of ACC, to ethylene with the help of ACC oxidase enzyme. The production of these enzymes by plants help the plants to grow under stress conditions during flooding, drought, presence of organic toxicants, metals, salt and flower wilting etc. [6]. Various plant growth promoting bacteria helps in resisting the plant during stressful conditions as P. syringae that is an epiphytic bacteria helping in reducing the plants ability to supercool conditions and avoid damage of plants from ice formation on their surface [7]. They binds to the seed coat, assimilate to its surface and then bacterium hydrolyzes ACC into α-ketobutyrate and ammonia by using ACC deaminase, thus the level of ethylene lowered in developing plants [8]. These ACC deaminase genes thus regulate the ethylene level in plants and play important role in optimum growth even under stressed conditions.
Our resent investigation involves the study of important bacteria from medicinally important spinach plant's phyllosphere having properties to resist plants in stress environment with the help of ACC deaminase activity.

Sample Collection
Spinach leaves were collected from different regions of Haryana (Chika, Karnal, Kurukshetra and Shahabad) region and from Punjab (Kapiyal and Sangrur) in India. Fresh leaves plucked and stored in sterile propylene bags and analysed for isolation process within 24 hours (h) [9].

Isolation of Phyllospheric Bacteria
The fresh leaves were washed properly using sterile distilled water and vortexed for 15-20 minutes (min.). Serial dilution of samples were prepared in range of 10 -1 -10 -5 and 100 µL of diluted samples were spreaded on respective media plates. Further, the plates were incubated at 35-37°C for 24-48 h. The isolates so obtained were preserved in 50% glycerol containing respective slants at 4°C [9]. The bacterial isolates were examined for their morphological features including colony size, elevation, shape, surface, colour and pigmentation etc. [10].

Screening of Bacterial Isolates for Plant Growth Promoting Attributes
The screening of isolated bacterial isolates for PGP attributes were done by examining their response towards phosphate solubilization, ammonia production, IAA production and In vitro biological nitrogen fixation. In phosphate solubilization, the bacterial isolates were observed on pikovskaya's agar media containing Bromothymol blue dye. The cultures were incubated at 35-37°C for 3-5 days [11]. The cultures were observed for zone of solubilization which indicated positive results. The ammonia production was confirmed by inoculating the respective fresh bacterial culture in 10 mL peptone water and incubated at 30-37ºC for 48-72 h. Nessler's reagent (0.5 mL) was added and allowed to settle at room temperature (temp.). The appearance of brown colour indicated the positive test for ammonia production. The faint yellow was taken as weak ammonia producer and deep brown colour as strong ammonia producer [12].
Similarly, IAA production was confirmed by inoculating the respective bacterial isolate in Luria broth containing L-tryptophan (0.1%) and incubated at 35-37°C for 48-72 h. After incubation the culture broth was centrifuged at 10,000 rpm for 10 min. Two drops of ophosphoric acid were added into 2 mL of supernatant along with 4 mL Salkowski reagent (50 mL, 35% of perchloric acid, 1 mL of 0.5 m FeCl 3 solution). It was observed for the development of pink colour within 2 h at room temperature [13]. In vitro BNF was observed by streaking the bacterial culture on nitrogen free Jensen's media along with addition of bromothymol blue (BTB) stain as an indicator dye and incubated at 35-37ºC for 24-48 h. Yellow coloured zone around the colonies indicated the positive result of in vitro BNF [14].

ACC enzyme obtaining from bacterial cells
Bacterial cells prepared in minimal salt media were suspended in 1 mL of 0.1M Tris-HCl (pH 7.6), and transferred to 1.5 mL microcentrifuge tube. The contents of the 1.5 mL microcentrifuge tube were then centrifuged at 14,000 rpm for 5 min. and the supernatant was removed. The pellet was suspended in 600 µL of 0.1M Tris-HCl (pH 8.5). 300 µL of toluene was added to the cell suspension and vortexed at the highest setting for 30 sec. The toluenized cell suspension was immediately assayed for ACC-deaminase activity [15].

ACC-deaminase activity assay for bacterial samples
Two hundred microlitres of the toluenized cells were placed in a fresh 1.5 mL microcentrifuge tube; 20 µL of 0.5M ACC was added to the suspension, vortexed for short time, and then incubated at 30°C for 15 min. It was followed by addition of 1.0 mL of 0.56M HCl, the mixture was vortexed and centrifuged for 5 min. at 13,500 rpm at room temp. One mL of the supernatant was vortexed together with 800 µL of 0.56M HCl. After that, 300 µL of the 2,4dinitrophenylhydrazine reagent (0.2% 2,4dinitrophenylhydrazine in 2M HCl) was added to the glass tube, the contents were vortexed and then incubated at 30°C for 30 min. Following the addition and mixing of 2.0 mL of 2M NaOH, the absorbance of the mixture was measured at 540 nm [15,16]. ACC deaminase enzyme conc. was calculated by the formula shown as [16].
Units/mL = µmole of α ketobutyrate x reaction volume Sample vol x reaction time x vol assayed µmole of α ketobutyrate = µmole of α ketobutyrate equivalents released as per graph.
Reaction volume = total volume (in ml) of assay.
Sample vol = volume (in ml) of enzyme sample used.
Reaction time = time (in minutes) of reaction incubation.
Vol assayed = volume of reaction product (in ml) used in OD determination.

Stress Response Shown by Heavy Metal Resistance Activity
The selected bacterial isolates were tested for their resistance to heavy metals by agar dilution method [17].

In vitro Seed Germination Assay
Seeds of useful plant like Vigna radiata (mung bean) seeds were collected and surface sterilized by soaking in sodium hypochlorite solution for 2-3 min. and washed with sterile distilled water for three times to remove the sodium hypochlorite solution [18]. Then seeds were allowed to inoculate with bacterial culture (10 8 cfu/mL) for 1-2 h, kept in 1% soft agar plates and then incubated for 3-5 days in the dark at the 30°C of seed germination. The seeds germination was evaluated and untreated seeds were taken as control. Germination efficiency along with seedling height, root/shoot length, wet/dry weight, % seed germination and vigor index was also analysed after incubation time and calculated by the formula shown as.

Statistical Analysis
Statistical analysis of all the data represented was done using SPSS version 16.0 lnc.

RESULTS AND DISCUSSION
The useful bacteria from spinach plants were isolated and studied for the important properties. These all results are discussed below:

Isolation
A total of 200 bacterial isolates were obtained using serial dilution technique on Nutrient Agar media. These bacterial isolates were collected on the basis of colony colour, elevation, size, shape and pigmentation, etc. The morphological study of 15 bacterial isolates having proficient plant growth promoting activities are discussed in Table 1. Our result outcomes of PGPB bacterial isolation from spinach phyllosphere was supported by findings of Verma et al. [19] who isolated total 89 bacteria from wheat phyllosphere and studied the bacterial diversity and their PGP attribute.

Screening for PGP Attributes
The 15 bacterial isolates out of 200 isolated colonies showed plant growth promoting activities like Paenibacillus polymyxa SNKp6 and Bacillus thuringiensis SNKr10 showed maximum phosphate solubilization, ammonia production, IAA production etc. after 48 h of incubation at 37°C are as shown in Table 2.
The biological nitrogen fixation ability of our isolated phyllospheric bacterial isolates was favoured by the findings of Fürnkranz et al. [20]. They reported that γ-proteobacteria and cyanobacteria (Nostoc sp.) helps in improving the plant growth through biological nitrogen fixation in Grias cauliflora, Carludovica drudei and Costus laevis plants. Taurian et al. [21] also reported in favour of our findings by stating that form 37 bacterial isolates some phyllospheric bacteria help in promoting Arachis hypogaea L. plant growth by performing certain PGP activities like phosphate solubilization and IAA production etc. Similarly findings of Verma et al. [22] also favoured our result out comes by reporting that various methylotrophic bacteria isolated from wheat (Triticum aestivum) phyllosphere also help to promote the plants growth by IAA, phosphate solubilization and ammonia production.
We selected SNKp6 and SNKr10 bacterial isolates for the study of ACC deaminase as well as some other properties that are discussed below. The gram staining of SNKp6 and SNKr10 revealed that these as gram positive and rod shaped bacteria. The 16S rRNA sequence of these two selected bacterial isolates were also obtained and submitted to the NCBI. The Accession number received were KU569965 for Paenibacillus polymyxa SNKp6 and KU569966 for Bacillus thuringiensis SNKr10.

Calculation of ACC Deaminase Activity
In the present study, ACC deaminase activity was implemented by inoculating the selected bacterial isolates namely Paenibacillus polymyxa SNKp6 and Bacillus thuringiensis SNKr10 on modified DF broth medium and deaminase enzyme assay was done after an incubation period of 24 h at 30°C using above described method by taking O.D. at 540 nm. We observed that SNKp6 produced significant amount of αketobutyrate and ACC deaminase enzyme activity in comparison to SNKr10 (Fig. 1).

Heavy Metal Resistance Activity
Soil contamination with heavy metals is very serious problem as it causes various serious health problems by enter through food chains [23]. These two bacterial isolates i.e.  4 and HgCl 2 respectively and 50µg mL -1 conc. of ZnSO 4 . Paenibacillus polymyxa stain SNKp6 also showed its maximum growth and resistance against 25 µg mL -1 of Pb, ZnSO 4 and HgCl 2 and upto 100 µg mL -1 of K 2 Cr 2 O 7 and CuSO 4 . Along with this, we also observed that these bacterial strains showed their resistance towards these heavy metals upto 400 µg mL -1 except for HgCl 2 . These results are discussed in Table 3; Fig. 2 by taking O.D. value at 600 nm. Our result outcomes are favoured by findings of Pereira et al. [24] as they reported that Rhodococcus erythropolis EC 34, Achromobacter sp. 1AP2 and Microbacterium sp. 3ZP2 increased clover biomass maximally at 250 mg/kg of zinc contaminated soil therefore useful for phytoremediation of polluted soils. One of the other PGPB named Micrococcus sp. TISTR2221 also showed its stimulatory effects on the growth of Zea mays L. seedlings under toxic cadmium conditions [25] and therefore comparable to our findings.

In vitro Seed Germination Assay
In present study, we observed that isolates Paenibacillus polymyxa stain SNKp6 and Bacillus thuringiensis SNKr10 has shown significant plant growth activities when checked their effect on germination of V. radiata (Fig. 3). These bacterial treatment to seeds had shown a stimulatory effect on all seed germination parameters i.e. vigor index, germination rate, seedling height, wet/dry weight and root/shoot length as compared to the control. We observed that progressively 100% seed germination when seeds of V. radiata were treated with SNKr10 followed by SNKp6 having 98.33% seed germination while in control seed it was only 70.22%. Seedling height was also found to be significant in case of SNKp6 followed by SNKr10 in comparison to control (Fig. 4). The root and shoot length of V. radiata plants was also observed to be maximum in case of SNKp6 (11.97 cm; 12.9 cm) and less in SNKr10 treated seedlings in comparison to SNKp6 while little root/shoot length was observed in untreated control (0.3 cm; 0.8 cm) (Fig. 5). The highest increase in wet and dry weight of V. radiata of seeds was in case of isolate SNKp6 less in seeds treated with SNKr10 (Fig. 6). The significant increase in seedling vigor index was also found in SNKp6 treated V. radiata seedlings followed by SNKr10 as compared to the control after 5 days of germination at 30°C (Fig. 7). Similar improvement in growth parameters by PGPB is also reported by Kefela et al. [26]. They reported that Bradyrhizobium japonicum IRAT FA3, Paenibacillus polymyxa and Bacillus licheniformis promote the seed germination rate up to 145% higher than the untreated control. Along with this, Zhao et al.
[27] also reported that Burkholderia phytofirmans strain PsJN helps to increase the biomass and growth by improving the root and shoot growth of several plant species as in potatoes (Solanum tuberosum L.) by inducing various phytohormones and are also in favour of our result outcomes. Therefore, our bacterial effect on all these growth parameters of V. radiata was also observed to be significant as that of earlier reports of PGPB by researchers.

CONCLUSION
On the basis of our result outcomes, we can conclude that Paenibacillus polymyxa SNKp6 and Bacillus thuringiensis SNKr10 bacterial strain have the capability to grow under stress condition due to ACC deaminase activity. They have also shown the ability to grow in high conc. of heavy metals therefor; these can be useful for the sustainable agriculture by helping plants to resist and grow in such type of stressful conditions.