ASSESSMENT OF ENDOPHYTIC BACTERIA FOR GROWTH PROMOTION IN CHICKPEA

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1 ASSESSMENT OF ENDOPHYTIC BACTERIA FOR GROWTH PROMOTION IN CHICKPEA Priyanka and * Leelawati Department of Microbiology, CCS Haryana Agricultural University, Hisar, Haryana, India * Author for Correspondence ABSTRACT The aim of present research was to increase the growth of chickpea by using bacterial endophytes isolated from the nodules of chickpea. Eight non rhizobial and three rhizobial bacterial endophytes from the nodules of chickpea were isolated using Tryptone soya agar and Yeast extract mannitol agar medium and screened for plant growth promoting activities viz. Indole acetic acid production, phosphate solubilisation and siderophore production. Among eight nonrhizobial and three rhizobial endophytic bacteria, two nonrhizobial isolates (HE-5 and HE-7) and one rhizobial isolate (HM-2) showed effective plant growth promoting traits viz. Indole acetic acid production, phosphate solubilization and siderophore production. Inoculation of non rhizobial endophyte to chickpea seeds along with Rhizobium HM-2 strain showed increased plant growth and grain yield as compared to control under pot house condition suggesting that the nodule endophytes can be used for plant growth promotion. Key Words: Chickpea, Endophytes, Growth Promoting Traits and Rhizobium INTRODUCTION As the world population increases, the problem of food security arises. This means that the increase of agricultural production has to meet the need of the fast growing population (Roger et al., 1994). The challenges faced by the world are how to feed the increasing populations where there is little food. The insufficient food level of population has driven farmers to change their agricultural behavior which leads to the use of chemical fertilizer in order to meet the human demand. The purpose for applying chemical fertilizer to agriculture land was to promote high yield of crop production (Crawford et al., 2006). This application has led to serious environmental problems such as deterioration of soil quality and health, rivers and ground water pollution, and emergence of resistant pathogens (Delmer, 2005). These concerns about environmental health and safety have led to increased restrictions on a variety of chemical fertilizers (Josephine, 2005). Knowing and understanding the negative effects of chemical fertilizers in agriculture, novel technology using the application of endophytic bacteria associated with plants, may help to sustain productivity and improve plant growth. Endophytic bacteria live inside the plant tissues and do not cause visible damage or morphological changes to their hosts. Rhizobia are the endophytes which establish a symbiotic relationship with legumes and results in the root nodule development. Occurrence of bacteria other than rhizobia in root nodule was first reported by Beijerinck and Van Delden (1902). In the last few decades, endophytic bacteria have attracted more and more attention as novel resources in the biocontrol of plant diseases and in the promotion of plant growth. They can benefit the host plants in a variety of ways, such as producing IAA (Indole acetic acid), fixing nitrogen, solubilizing phosphates, producing siderophores, suppressing phytopathogens by competition in the invasion sites and by secreting antibiotic compounds (Ryan et al., 2008). This group of bacteria is considered as an environmentally friendly alternative solution of reducing the use of chemicals fertilizers in agriculture sector (Goswami et al., 2014). Chickpea is generally consumed as a seed food, being a good source of protein and other essential human nutrients. Young chickpea leaves are also cooked and eaten as green vegetable in certain parts of the world and could be a useful source of dietary nutrients, especially in malnourished populations nutritionally; chickpea has a high protein digestibility and is richer in phosphorus and calcium than other pulses. Because of its higher fat content and better fiber digestibility, chickpea holds great promise as a protein and calorie source for animal feed for both ruminants and nonruminants. Therefore it is necessary to feed the increasing population by using sustainable agriculture practices such as using the bacterial endophytes for growth promotion. So the aim of this research was to increase the growth of chickpea by using bacterial endophytes isolated from the nodules of chickpea. MATERIALS AND METHODS Collection and Isolation of Endophytic Bacteria For the isolation of bacterial endophytes, the nodules were collected from the roots of chickpea (Cicer arietinum L.) grown in Hisar, Haryana. Nodules were surface-sterilized using 70% ethanol and 0.1% HgCl 2 and repeatedly washed with sterile water. Sterile nodules were crushed in a sterilized Petri dish with the help of sterilized glass rod 25

2 in one ml sterilized distilled water and the resulting suspension was streaked on Yeast extract mannitol agar (YEMA) and Typtone Soy agar (TSA) plates for the isolation of rhizobial and nonrhizobial endophytes and plates were incubated in BOD incubator at 28±2 C. After h, the colonies were picked and purified by single colony streaking on the YEMA and TSA plates and maintained at 4 0 C in refrigerator. Evaluation of bacterial endophytes for Plant Growth Promoting (PGP) Traits Indole Acetic Acid (IAA) Production Bacterial endophytes were tested for their ability to produce IAA under liquid culture. The bacterial cultures were inoculated in tryptone soya broth supplemented with 100 µg ml -1 DL- tryptophan and were incubated at 30 0 C for 72 h. Indole acetic acid was determined in the culture supernatant by adding Salkowski reagent (Gorden and Weber 1951). Two ml of Salkowski reagent was added to 2 ml of culture supernatant, mixed and allowed to stand for 30 min for the development of pink colour and colour intensity was estimated at 500 nm using spectrophotometer against a reagent blank (Fig. 1). Indole acetic acid (100µg ml -1 ) was used as a standard and results were expressed as µg IAA produced ml -1 of culture supernatant. Figure 1: Indole acetic acid production activity of endophytes using Salkowski reagent Figure 2: Phosphate solubilization activity of endophytes on Pikovskya s agar plates Figure 3: Siderophore production activity of endophytes on Chrome Azurol S plates Phosphate Solubilization Phosphate solubilization ability of the endophytes was determined by spotting of cultures on Pikovskaya s agar plates (Pikovskya 1948). Plates were incubated at 30 o C for one week and the appearance of clear halo zone around the colonies indicated solubilization of inorganic phosphate by bacteria (Fig. 2). The halo size produced by the respective bacteria was calculated according to the formula: Solubilization Index= zone diameter (cm) - colony diameter (cm) / colony diameter (cm) Siderophore production All the endophyic bacterial isolates were screened for siderophore production activity using universal chemical assay of Schwyn and Neilands (1987) on Chrome azurol S (CAS) agar plates. Five μl of log phase grown culture of each endophyte was spotted on the CAS plates and incubated at 28+2ºC for 5-7 days. The presence of iron chelator (siderophore) was indicated by the decolourization of the blue-coloured ferric dye complex, resulting in yelloworange halo zones around the colonies (Fig. 3). Inoculation of selected endophytes for growth promotion of chickpea under pot culture conditions Selected endophytic isolates were assessed for growth promotion of chickpea grown in November 2014 under pot culuter conditions. Five kg of sandy soil was taken in earthern pots. Seeds of chickpea variety HC-5 were surface sterilized using 0.1% mercuric chloride and alcohol. Three replicates of each treatment were kept and seeds were inoculated with endophytes alone as well as coinoculated with Rhizobium. One control was also kept without any bacterial inoculation. After germination, three plants in each pot were maintained. Pots were irrigated on alternate day or as and when required. At 50% flowering stage observation of nodule number, nodule fresh weight, nodule dry weight, root dry weight and shoot dry weight were taken and yield data was taken at maturity. RESULTS AND DISCUSSION Isolation of Endophytic Bacteria A total of eight non rhizobial and three rhizobial endophytic bacteria were isolated and designated as HE-1, HE-2, HE-3, HE-4, HE-5, HE-6, HE-7, HE-8 for nonrhizobial bacteria and HM-1, HM-2, and HM-3 for rhizobia. The endophytes were studied for their morphological characters such as colony color and appearance. The colonies were 26

3 white, cream or yellow in color with rough, shiny or gummy appearance (Table 1). Kumar et al., (2013) isolated endophytes from roots and nodules of various crops and found that nodule endophytes were more diverse than root endophytes. Determination of Plant Growth Promoting (PGP) Traits Indole acetic acid production Production of phytohormone (IAA) is an important mechanism of plant growth promotion by endophytic bacteria. This hormone promotes the growth of roots. All the eight endophytic bacterial isolates were screened for quantitative production of IAA after 5 days of growth. Seven out of eleven endophytic isolates showed positive IAA production ranging from 0.69 µg ml -1 to10.04 µg ml -1 (Table 2). Uma Maheswari et al., (2013) isolated endophytic bacteria from various tropical grain legume crops and reported the production of IAA ranging between 0.12 µg ml -1 to 6.46 µg ml -1. Table 1: Morphological characters of bacterial endophytes isolated from chickpea nodules Bacterial Isolate Colony morphology HE-1 White, rough, flat HE-2 Cream, shiny, flat HE-3 Cream, rough, flat HE-4 Cream, rough,elevated HE-5 Cream, rough, flat HE-6 White, shiny, elevated HE-7 Yellow, shiny,elevated HE-8 Cream, shiny, elevated HM-1 HM-2 HM-3 Table 2: Screening of chickpea nodule endophytes based on plant growth promoting traits Isolates IAA production (µg/ml) Phosphate solubilization(s.i.) Siderophore production HE HE-2 _ HE-3 _ HE-4 _ HE HE-6 _ HE HE HM _ HM HM Table 3: Effect of inoculation of bacterial endophytes on nodulation, plant growth and grain yield of chickpea Treatment Nodule number Nodule dry Root dry weight Shoot dry weight Grain weight per plant weight per plant per plant (g) per plant (g) per plant (g) (g) Uninoculated Endophyte (HE-5) Rhizobium (HM-2) HE-5+HM C.D. N/A

4 Phosphate solubilisation Solubilisation of insoluble phosphates in soil help plants for better uptake of phosphorous from soil. The ability of some microorganisms to covert insoluble P to an accessible form, like orthophosphate, is an important trait in plant growth promoting bacteria for increasing plant yield. The results showed that out of eleven endophytic isolates eight showed transparent halo zone with solubilizing efficiency ranging from (Table 2). Saini et al., (2015) isolated 76 endophytes from roots and nodules of chickpea and reported that 41.7% endophytic bacteria from roots and 73.6% from nodules of chickpea were solubilising phosphate. Siderophore production Siderophores are low molecular weight compounds with high iron (III) chelating affinity and are responsible for the solubilisation and transport of iron (III) into bacterial cell. Iron is an essential mineral and its sequestration by specific endophytic bacterial siderophores can make it available to the plants under iron limiting conditions. The result showed that out of eleven endophytes seven showed detectable siderophore production on CAS plates (Table 2). Similarly Matsuoka et al., (2013) isolated endophytic Bacillus sp., Streptomyces luteogriseus, and Pseudomonas flourescens from Carex kobomugi roots, which exihibited siderophore production activity under Fe limiting conditions. Evaluation of bacterial endophytes for growth promotion of chickpea under pot culture conditions The non rhizobial bacterial endophyte HE-5 and rhizobial endophyte HM-2 were selected for pot house experiment because of three growth promoting traits viz. Indole acetic acid production, phosphate solubilisation and siderophore production. Inoculation of HE-5 or HM-2 alone to chickpea showed increase in nodulation and plant growth yield when compared with uninoculated control (Table 3). Coinoculation of endophyte HE-5 with Rhizobium strain HM- 2, significantly increased plant growth and grain yield when compared with inoculation of HE-5 or HM-2 individually suggesting that rhizobial as well as nonrhizobial endophytes have cumulative effect on plant growh due to various plant growth promoting traits. Tariq et al., (2012) reported that co-inoculation of non-rhizobial bacteria with Bradyrhizobium sp. MN-S on mung bean significantly improved nodulation and grain yield compared with Bradyrhizobium sp. MN-S alone inoculation. CONCLUSION The present study showed the presence of endophytic non-rhizobia in chickpea nodules. The above findings suggest that inoculation of HE-7 with Rhizobium HM-2 on chickpea could be beneficial for plant growth promotion. REFERENCES Beijerinck MW & Van Delden A (1902). Uber die Assimilation des freien stickstoffs durch Bakerien. Centrabl Bakt Abt II, Crawford EW, Jayne TS & Kelly VA (2006). Alternative Approaches for Promoting Fertilizer Use in Africa. Washington, DC: Agriculture and Rural Development, Delmer DP (2005). For small-scale African farmers, we are trying strategies to optimize yield under conditions of stress and low inputs. Proceeding of the National Academic Science, Gordon SA & Weber RP (1951). Colorimeteric estimation of indole acetic acid. Plant Physiology, Goswami D, Dhandhukia P, Patela P & Thakkera JN (2014). Screening of PGPR from saline desert of Kutch: Growth promotion in Arachis hypogea by Bacillus licheniformis A2. Microbiological Research, Josephine W (2005). Understanding the Overuse of Chemical Fertilizer in China a Synthesis of Historic Trends, Recent Studies, and Field Experiences. St. Paul, Minnesota, USA: Macalester University. Kumar V, Pathak DV, Dudeja SS, Saini R, Giri R, Narula S & Anand RC (2013). Legume nodule endophytes more diverse than endophytes from roots of legumes or non legumes in soils of Haryana, India. Journal of Microbiology and Biotechnology, 3(3) Matsuoka H, Akiyama M, Kobayashi K & Yamaji K (2013). Fe and P solubilisation under limiting conditions by bacteria isolated from Carex kobomugi roots at the Hasaki coast. Current Microbiology, 66(3) Pikovskya RI (1948). Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Microbiology, Roger PA, Simpson I, Oficial R, Ardales S & Jimenez R (1994). Effects of pesticides on soil and water microflora and mesofauna in wetland ricefields: A summary A 3 of current knowledge and extrapolation to temperate 1 environments. Australian Journal of Experimental Agriculture, Ryan RP, Germaine K, Franks A, Ryan DJ and Dowling DN (2008). Bacterial endophytes: Recent developments and applications. FEMS Microbiology Letters,

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