Characterization of epiphytic bacteria isolated from chickpea (Cicer arietinum L.) nodules

Twenty eight isolates associated with the nodule surface of two chickpea cultivars, that is, reverted non-nodulating (ICC4993NN(R)) and normal chickpea (HC5) cultivars were isolated. All of them were found to be Gram positive. The isolates NE81, NE82 from cv ICC4993NN(R) and NE1043 from cv HC5 inhibited the growth of isolates NE84, NE44, NE45, NE32, NE16-1, NE32, NE84, NE44, NE19, NE26 and NE14, NE53, NE16-2, NE18, NE1043, respectively. However, some of these nodule epiphytic bacteria promoted the growth of mesorhizobial isolates from chickpea. The co-inoculation of these nodule epiphytes with mesorhizobial isolate resulted in significant increase in nodulation and shoot dry weight in chickpea as compared to the inoculation only with nodule epiphytes.


INTRODUCTION
Rhizosphere is microbe and nutrient rich environment where the roots exude water and other compounds, thereby providing favourable environment for the growth of microorganisms.Rhizospheric microorganisms have different types of interactions with plant roots and nodules in the rhizosphere (Pinton et al., 2001;Werner, 2004;Hayat et al., 2010).These plant-microbe interactions influence the plant growth.Bacteria are predominantly found in the root rhizosphere and are commonly termed rhizobacteria (Antoun and Kloepper, 2001) but can also be referred to as root epiphytes.These root epiphytes can have beneficial, damaging or harmless effects on plant growth.In the last few decades, a large number of bacteria such as Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus and Serratia have been reported to enhance plant growth (Kloepper et al., 1989;Okon and Labandera-Gonzalez, 1994;Glick, 1995).A total of 150 bacterial isolates belonging to Bacillus, Pseudomonas, Azotobacter and Rhizobium were reported from different rhizospheric soil of chick pea in the vicinity of Allahabad, India (Joseph et al., 2007).
Significant increase in all the plant growth parameters were observed in maize and chickpea plants inoculated with Pantoea agglomerans (Mishra et al., 2011).Harmful rhizobacteria affect the growth of plants by producing metabolites like phytotoxins or phytohormones or by having competition for the nutrients (Nehl et al., 1996;Sturz and Christie, 2003;Kloepper, 2003).These rhizospheric bacteria also exhibit biocontrol activities.Two bacterial strains, Pseudomonas sp. and Rhizobium sp.obtained from the rhizosphere of chickpea have unique ability to inhibit Fusarium oxysporum f.sp.ciceri, Rhizoctonia bataticola and Pythium sp.under in vitro conditions (Nautiyal,1997).Strong antagonistic activity was shown against three races of F. oxysporum f. sp.ciceri by 24 bacterial isolates and Pseudomonas chlororaphis.The extent of growth inhibition was influenced both by the bacterial isolates and the race of pathogen (Landa et al., 1997).A chitinase *Corresponding author.E-mail: poojapavit@gmail.com.Tel: +91-9812028424.
producing strain Lysinibacillus fusiformis was isolated from chickpea rhizosphere.It exhibited in vitro antifungal activity against a wide range of fungal plant pathogens and several plant growth promoting rhizobacteria (PGPR) activities yet exact mechanisms are not known (Singh et al., 2012).However, environmental conditions, soil conditions, growth of host plants or host genotype may also have some effect.
Leguminous plants have symbiotic association with the nitrogen fixing bacteria in their roots nodules.These bacteria are capable of converting atmospheric nitrogen into ammonia which is then incorporated into plants in the form of amino acids.ICRISAT, Hyderabad, India developed a non-nodulating variety of chickpea to estimate the N 2 fixation purely by plants (Rupela, 1994).These non-nodulating selections reverted back to nodulating selections only in certain locations particular at Hisar, Haryana, India (Dudeja et al., 1997).Now pure lines of reverted lines of chickpea from non-nodulating to nodulating ones are available at Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana, India.The nodules of this revertant non-nodulating chickpea cultivar (ICC4993NN(R)) as well as normal chickpea cultivar (HC5) were used to study the epiphytic bacteria associated with them, to have an insight into their effect on host plant.The present investigation was undertaken with the objective of isolating and comparing the different characteristics of epiphytic bacteria associated with the nodules of reverted non-nodulating (ICC4993NN(R)) and normal chickpea (HC5) cultivars.

Isolation of epiphytic bacteria associated with nodules of reverted and normal chickpea cultivars
Reverted non nodulating lines of cv ICC4993NN(R) showing nodulation and normal nodulating cv HC5 were grown under field conditions at research farm of Chaudhary Charan Singh Haryana Agricultural University, Hisar, India, and were then uprooted on the 60 th day of growth.The healthy nodules formed by non-nodulating line and other normally nodulated cultivars were removed and used for isolation.The nodules were mildly surface sterilized with 0.1% HgCl 2 and kept on Tryptone Soya Agar (TSA) plates as sterilization with 0.2% HgCl 2 resulted into complete sterilization of nodules (Vincent, 1970).Growth around these partially sterilized nodules was observed and isolates were picked up from the plates.

Study of morphological characters of nodule epiphytes of reverted and normal chickpea cultivars
The different morphological characters such as growth, colony colour, appearance, morphology, gum production of all the isolated nodule epiphytes were observed.Gram staining of nodule epiphytes was done and cell shape whether cocci or rods were observed under the microscope.These were further used to study their beneficial and inhibitory effects on the growth of mesorhizobial isolates and on each other, respectively.The inhibitory effect was observed by zone of inhibition test.A TSA plate was inoculated with a lawn of a bacterial culture suspension.The test organism was placed in the well.After incubation for 24 h, the plate was examined to check the inhibition of growth of test organism.The beneficial effect was observed by paired test.The test organism and the isolated mesorhizobia from the nodules of chickpea were streaked on the same plate.Following incubation, these plates were observed after 24 h.Those nodule epiphytes which were showing positive effect on the growth of mesorhizobia were further selected for pot experiment.

Effectiveness of nodule epiphytes
The pot experiment was conducted using 10 nodule epiphytes selected on the basis of their growth promotion activity in paired test.Nodule epiphytic isolates (NE32, NE292-1, NE45, NE81, NE12, NE13, NE82, NE16-1, NE53 and NE42) were inoculated separately in TSA broth and the mesorhizobial isolate (NN95) was inoculated in yeast extract mannitol (YEM) broth and incubated on rotary shaker at 28±2°C in an incubator.River sand was thoroughly washed with acid followed by 6-7 washings with water and was sterilized in oven at 180°C for one hour in trays.The sand was put in cups and nitrogen free nutrient solution was added and cups were covered with paper, held in position with the help of a thread.These test assemblies were sterilized in autoclave at 15 lb/in for one hour.Few empty cups were also sterilized in autoclave at 15 lb/in for 20 min and were used whenever needed to support the cups containing sand.In one set of experiment, germinated seedlings of chickpea were transferred to sterilized cups containing sand, together with 1-2 mL broth of nodule epiphytes only and control was kept without any inoculum.In another set of experiment, the germinated seedlings were placed in the test assemblies and co-inoculated with 1-2 mL broth of nodule epiphytes and 1-2 mL broth of chickpea mesorhizobial isolate and control was inoculated only with Mesorhizobium.Then, these nodulation test assemblies were kept in green house and watered daily with sterilized Sloger's nitrogen free watering solution.After 55 days of growth, plants were recovered and analyzed for nodulation and other parameters.For determining dry weight of shoot and root, samples were dried in an oven at 80°C for 48 h or till the constant weight was observed (Somasegaran and Hoben, 1994).

RESULTS
The surface sterilization methods were evaluated for standardization of isolation process of epiphytes.The nodules when, mildly surface sterilized with 0.1% HgCl 2 and placed on TSA plates showed growth around these partially sterilized nodules (Figure1a).However, with 0.2% HgCl 2 (Figure1b) there was complete sterilization of nodules, as no epiphytic growth was observed.
In total, 28 Gram positive nodule epiphytes were obtained from both cultivars (Table 1).They showed large variation in colony colour ranging from white, cream, pale white, light brown, yellow to red.The cell shape also varied from cocci, small rods to long rods.Some isolates produced gum while others did not.All the isolates were assessed for their growth inhibition activity against other isolates.The isolates NE81, NE82 (ICC4993NN(R)) and NE1043 (HC5) showed zone of inhibition against other isolates.The isolate NE81 showed zone of inhi-bition against isolate NE84, NE44, NE45, NE32 and NE16-1 (Figure 2a and b) and NE82 showed inhibition against NE32, NE84, NE44, NE19 and NE26 (Figure 3a and b).Table 1.Morphological characters of rhizospheric bacterial epiphytes from chickpea nodules.zone (Figure 5).Out of 28, 10 nodule epiphytes showed the growth promotion activity towards mesorhizobial isolate NN95.The growth promotion activity of two isolates NE 82 and NE 81 is depicted in Figure 6a and b, respectively.The germinated chickpea seeds were inoculated with nodule epiphytes alone as well as co-inoculated with mesorhizobia and nodule epiphytes.After 55 days of growth, plants were uprooted for nodulation, root and shoot biomass (Figures 7 to 9).Nodule epiphytes when inoculated alone showed no nodulation or nodulation less then five nodules per plant except the isolate NE16-1.When chickpea seeds were co-inoculated with Mesorhizobium and nodule epiphytes, there was significant increase in nodulation.The results showed that there was no nodulation in control but when inoculated with only nodule epiphytes nodulation ranged from 0 to 7 nodules per plant.Mesorhizobial isolate alone resulted in 15 nodules per plant.The nodulation ranged from 14 to 34 nodules per plant when plants were co-inoculated with Mesorhizobium and nodule epiphytes.Highest nodulation was observed in the case of plants co-inoculated with NE292-1 and Mesorhizobium (34 nodules per plant) followed by NE81 and Mesorhizobium (31 nodules per plant) and NE 32 and Mesorhizobium (27 nodules per plant).Shoot dry weight of control plants was 189 mg/plant, whereas it varied from 407 to 965 mg/plant in the case of nodule epiphytes from cv.ICC4993NN(R) and HC5.Highest shoot dry weight was observed in plants inoculated with NE12 followed by NE13, NE81 and NE32.Shoot dry weight varied from 623 to 1320 mg/plant in the case of plants co-inoculated with Mesorhizobium and nodule epiphytes.The increase in shoot dry weight was highly significant when seeds were co-inoculated with isolates NE12, NE16-1, NE82 and Mesorhizobium (P<0.01).Four isolates, NE292-1, NE42, NE53 and NE81 showed significant increase in shoot dry weight when they were co-inoculated with Mesorhizobium (P<0.05).Similarly, there was also a significant increase in root fresh weight when compared with the control (0.91 g/plant).Root fresh weight was 1.66 g/plant when seeds were inoculated alone with Mesorhizobium (P<0.05).It ranged from 2.1 to 3.07 g/plant in the case of nodule epiphytes from both cultivars and 2.61 to 3.88 g/plant in the case of co-inoculation with nodule epiphytes and mesorhizobial isolate.The highest root fresh weight was observed in the case of plants co-inoculated with Mesorhizobium and NE81.The significant increase in root fresh weight was observed only when seeds were co-inoculated with NE45 and Mesorhizobium (P value < 0.05) and insignificant when inoculated along with other nodule epiphytes.

DISCUSSION
Twenty eight nodule epiphytes associated with nodules of the two chickpea cultivars exhibited variation in colony characteristics.A large variation in the colony morphology of isolates from nodules of soybean and Lespedeza sp. has also been reported (Hung et al., 2007;Palaniappan et al., 2010).However, majority (93.4%) of the isolates from soybean nodules were Gram positive whereas 84.6% isolates from Lespedeza sp. were Gram negative.In the present study, all the isolates identified from the surface of chickpea nodules were Gram positive.This is probably because the geographical location (Hisar), has a wide temperature range (-2 to 47°C) and with these adverse temperature variations, it is quite possible that Gram positive bacteria, and particularly spore formers are the better survivors.
Out of 28 isolates, only three isolates (NE81, NE82 and NE1043) showed zone of inhibition against different isolates.These nodule epiphytes may therefore indirectly help the plants by inhibiting the growth of other harmful microorganisms.Bacteria associated with the surface of roots and nodules have been shown to have beneficial effects on plants.Bacillus species has been found to promote the growth of large number of plants and effective in the biological control of many plant microbial diseases (de Freitas et al., 1997;Kokalis-Burelle et al., 2002).In the present study, the isolate NE81 was found to solubilise the media components, suggesting its role in nutrient solubilisation in the soil.Several reports have also indicated that the rhizobacteria promote the plant growth by increasing the availability of nutrients for the plant in the rhizosphere (Glick, 1995;Vessey, 2003).These bacteria help in solubilisation of unavailable forms of nutri-ents, siderophore production and ammonia production (Stevenson and Cole, 1999).
A significant increase in the nodulation of chickpea plants was observed when they were co-inoculated with Mesorhizobium and nodule epiphytes as compared to nodule epiphytes alone.The co-inoculation of NE 292-1 and Mesorhizobium led to maximum nodulation whereas Mesorhizobium and NE 53 resulted in significant increase in shoot dry weight in chickpea.These observations show that Mesorhizobium along with nodule epiphytes have beneficial effect on the plant growth.Similarly, different workers have also reported the use of co-inoculants in nodule stimulation and plant growth promotion of different leguminous crops.A significant increase in nodule weight and shoot biomass in chickpea was reported when coinoculated with Mesorhizobium and Pseudomonas (Goel et al., 2000;Sindhu et al., 2002b).The co-inoculation of Rhizobium with Pseudomonas striata or Bacillus polymyxa increased nodulation, nitrogenase activity, nitrogen and phosphate uptake in blackgram (Prasad and Chandra, 2003;Gunasekaran et al., 2004).The inoculation of pigeonpea and mungbean seeds with multiple co-inoculants consisting of Rhizobium, Azotobacter, Azospirillum, Pseudomonas and Bacillus produced maximum nodule biomass, plant biomass and total soil N in the case of pigeonpea and mungbean hosts (Suneja et al., 2007).
Our study suggests that the nodule epiphytes play a significant role in promoting plant growth either by inhibiting the pathogenic microorganisms or by producing some factors which stimulate the interaction of mesorhizobia with plant roots.These can further be used as coinoculants together with Mesorhizobium to enhance the plant growth.However, their effectiveness on the crop should be assessed under field conditions also.

Figure 5 .Figure 6 .Figure 7 .
Figure 5. Medium components solubilisation by one of the bacterial epiphyte NE81 isolated from the surface of chickpea nodules.

Figure 8 .
Figure 8.Effect of nodule epiphytes together with mesorhizobial inoculation on root biomass of chickpea under sterilized conditions.

Figure 9 .
Figure 9.Effect of nodule epiphytes together with mesorhizobial inoculation on shoot biomass of chickpea under sterilized conditions.