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,

Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2013, 3 (3):83-92 (http://scholarsresearchlibrary.com/archive.html)

ISSN : 2231 –3168

CODEN (USA) : JMBRB4

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Legume nodule endophytes more diverse than endophytes from roots of legumes or non legumes in soils of Haryana, India

Vishal Kumar, Dharam Vir Pathak, Surjit Singh Dudej a, Ranjana Saini, Rupa Giri, Shifa

Narula and Ramesh Chander Anand

Department of Microbiology, CCS Haryana Agricultural University, Hisar, Haryana, India *Regional Research Station, CCS Haryana Agricultural University, Bawal, Haryana, India

_____________________________________________________________________________________________ ABSTRACT A total of 136 nodule and 90 root endophytic bacterial isolates were obtained from roots and nodules of legumes and roots of non legumes. Higher number of Gram positive bacteria was present in legume nodules than in its roots. In legume roots 47.8% and in nodules 56 % of bacterial endophytes were solubilizing P. Similarly the number of ammonia and organic acid producers in legumes nodules were higher than roots. Selected 143 endophytes were used to determine molecular diversity by RFLP of PCR amplified 16S rDNA. Seventeen bacterial genotypes in nodules, 7 bacterial genotypes in legume roots and 6 bacterial genotypes in non legume roots were present. Based on the presence all the beneficial traits, three isolates were selected for identification. In chickpea nodules Bacillus subtilis strain CNE 215; in chickpea roots Bacillus licheniformis strain CRE 1 was present. Upon determining the partial sequence of 16S rRNA gene the isolate from wheat root showed more than 98% similarity to Bacillus flexus, so this was identified as Bacillus flexus strain WRE 20. Key words: Roots, Nodules, Endophytes, Molecular Diversity, Legume, Non legumes _____________________________________________________________________________________________

INTRODUCTION

Endophytic bacteria colonize the internal tissue of the plants but do not show any external sign of infection or adverse effect on infected host plants and avail the benefit of the host. It seems that the bacteria best adapted for living inside plants are naturally selected. The soil particularly the rhizosphere is an important source of root endophytes [1, 2]. The population density of endophytes is highly variable, depending mainly on the bacterial species and host genotypes but also on the host developmental stage, inoculum density, and environmental conditions. Endophytic bacteria have been isolated from different parts of the plants and mainly from roots of legumes and non legumes and nodules of legume plants [3-5]. Different endophytes have been identified from different plant tissues. Endophytic bacteria in a single plant host comprises of several genera and species. There is a huge diversity among the endophytic bacteria in different legumes. Molecular diversity of bacterial endophytes varies from genera to genera and species to species of different plants and even in different tissues of a single plant. Plant-associated bacteria play key roles in their hosts’ adaptation to changing environments in various ecosystems. These interactions between plants and beneficial bacteria can significantly affect general plant health and soil quality. Endophytic bacteria are believed to elicit plant growth promotion in one of two ways: either indirectly by helping plants acquire nutrients, e.g. via nitrogen fixation, phosphate solubilization or iron chelation by preventing pathogen infections via antifungal or antibacterial agents,

Vishal Kumar et al J. Microbiol. Biotech. Res., 2013, 3 (3):83-92 ______________________________________________________________________________


by outcompeting pathogens for nutrients by siderophore production, or by establishing the plant’s systemic resistance; or directly by producing phytohormones such as auxin or cytokinin [6], or by producing the enzyme 1-aminocyclopropane-1-carboxylate deaminase, which lowers plant ethylene levels [7]. Plant growth promoting bacteria increases nodulation and growth in a wide variety of legumes and most of the tested strains are plant growth promoting rhizobacteria. However endophytic bacteria have recently drawn particular attention as a group of potential plant growth promoters [8-14]. Bacterial endophytes and plant-associated bacteria and communities has been reviewed recently [4, 15], but no attempts has made to develop bio inoculants from these endophytes from roots and nodules of legumes or even from roots of non legumes and particularly from Indian subcontinent. To have a better understanding of bacterial enophytes, the aim of the present study was to isolate the bacterial endophytes from roots of legumes and non legumes and nodules of legumes to characterize on the basis of various beneficial traits and molecular diversity, so as to explore the possibility of development of biofertilizers for enhancing crop productivity of legumes and non legumes and sustaining the agriculture.

MATERIALS AND METHODS

Isolation of endophytes from roots and nodules Root samples of legumes, chickpea (Cicer arietinum), field pea (Pisum sativum), and lucerne (Medicago sativa) and non-legumes wheat (Triticum aestivum) and oat (Avena sativa) and nodules of chickpea and field pea being grown under CCS, Haryana Agricultural University, Hisar farm were collected. To isolate endophytes the roots and nodules were surface sterilized by using 0.2% mercuric chloride and ethanol and then rinsed with sterile water four to six times [16]. Sterilized roots and nodules were used to isolate the edophytic bacteria. Roots were crushed for isolation of endophytes whereas a cut was made in the nodules with a sterilized knife and then a loop full of root and nodule sap was streaked on separate Tryptone Soya Agar (TSA) medium plates [17]. The plates were incubated at 28+20C for 5-6 days. The endophytic bacterial isolates were picked up from the plates and were re-streaked for purification purpose and endophytic bacteria were maintained on TSA slants under refrigerated conditions. In this study, 90 endophytic isolates from roots and 136 from nodules were made. Simultaneously uncrushed sterilized roots and nodules were also kept on TSA medium plates to ensure proper surface sterilization of roots and nodule samples so as to ensure the isolation of endophytic bacteria. Endophytic isolates were designated as CRE from chickpea root; PRE from field pea root; LRE from lucerne root; CNE from chickpea nodule; PNE from field pea nodule; WRE from wheat root and ORE from oat roots. Morphological diversity of root and nodule endophytes All the 226 bacterial endophytic isolates, 51 from legume roots; 39 from non legume roots and 136 from nodules were observed for colony morphology like colony color, colony size, colony texture and gum production. Cells were stained with Gram’s and spore stains and stained cells were examined under Geytnor’s digital microscope at 100x for recording different observations. Functional diversity of root and nodule endophytes In all the 226 endophytic isolates, presence of beneficial traits like P solubilization, ammonia and organic acid production was assessed to know the functional diversity. All the bacterial endophytes were screened for P solubilization activity in plates using Pikovskaya medium [18]. Isolates were spotted on Pikovskaya’s medium plates in triplicate and depending upon the diameter of solubilization zone formed after 5 days of incubation at 28±2ºC, different endophytes were categorized. Similarly bacterial isolates were tested for the production of ammonia in peptone water. Freshly grown cultures were inoculated in 10 mL peptone water in each tube and incubated for 4 to 5 days at 28±2ºC. Nessler’s reagent (1 mL) was added in each tube. The development of colour from yellow to brownish orange was a positive test for ammonia production [19] and on the basis of intensity of colour developed; the endophytes were categorized into different categories. Just qualitative assessment of organic acid production by the bacterial isolates was determined by methyl red test [20]. Bacterial endophytes were inoculated in methyl red broth and incubated for 5 days at 28±2ºC and drop in pH was indicated by methyl red dye. Isolates having the ability to produce organic acid gave orange to bright red colour, while yellow colour indicated a negative reaction. On the basis of intensity of colour developed, the endophytes were categorized into different categories.



Molecular diversity of roots and nodules endophytes In total 143 selected endophytes including 24 from legume roots, 100 from nodules and 19 from non legume roots were used to study molecular diversity. All the bacterial endophytes were grown in 25 mL of Tryptone soya broth and log phase cells were harvested and total genomic DNA was isolated by standard phenol-chloroform extraction method [21, 22]. Finally the DNA was quantified and stored at –20 °C. Amplification of 16S rDNA of root and nodule endophytes was carried out using bacterial DNA as templates with primers fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rD1 (5′-AAGGAGGTGATCC AGCCGCA-3′) [23]. PCR amplification was carried out by modifying the protocol as described earlier in 23.5 µL volume per reaction [24]. Reaction mixture for each 100.5 µL of reaction mixture included 73.0 µL of milli pore water; 10.0 µL of PCR buffer (10X); 1.5 µL of MgCl2 (25 mM); 1.0 µL of dNTP mixture (10 mM); 2.0 µL of each of primers fD1 (100 ng µL–1) and rD1 (100 ng µL–1); 1.0 µL of taq DNA polymerase (3U µL–1) and 10.0 µL of template DNA (50 ng µL–1 approx.). The conditions for PCR included initial denaturation at 94°C for 3 min; denaturation at 94°C for 45 sec; annealing at 50°C for 40 sec.; extension at 72°C for 1 min and final extension at 72°C for 10 min with 40 repeating cycles. Three restriction enzymes Hae III, RsaI and Hinf I were used for the digestion of amplified 16S rDNA PCR product. Digestion mixture consisted of 6 µL of milli pore water; 2 µL of Y+/Tango buffer (2X); 1 µL of enzyme (3 U µL–1) along with 9 µL of 16S rDNA amplified product and samples were held on constant temperature of 370C for 12 h in thermal cycler. The restriction fragments were separated by electrophoresis at 100 V for 1.5–2.25 h in 1.5% (w/v) agarose gels in TBE buffer (0.089 M Tris-borate; 0.002 M EDTA; 0.089 M boric acid). The gels were stained with ethidium bromide (1 mg mL–1) and photographed under UV illumination with Gel Doc (DNR Bio-Imaging Systems). The PCR product profiles were converted into two dimensional binary matrices. The lanes were compared by reading horizontally across the gel. Depending upon the presence or absence of a particular band 0–1 matrix was prepared. Restriction patterns of three endonucleases were combined for cluster analysis. Similarity matrices were constructed following SimQual Coefficient and analysed by UPGMA (unweighted pair grouping with mathematic average) cluster analysis using biostatistical analysis programme NTSYS-PC programme 2.1 of Exeter Softwares USA [25]. Identification of the root and nodules endophytes To identify endophytes from legume and non legumes, one isolate from each source was selected. Two isolates CNE 215 isolated from chickpea nodules and CRE 1 isolated from chickpea roots were identified as detailed earlier [26]. Another endophytic isolate WRE 20 from wheat roots was identified during the present study. The products of 16S rDNA amplification were purified and got sequenced from Bangalore Genei Pvt Ltd. There were two replicas of PCR products of isolate WRE 20. One was sequenced with the forward primer and the other with the reverse primer. Sequence data was analyzed by comparison to 16SrRNA genes in the Genbank database. The nearest relatives of the organism were obtained by BLAST searches [27]. BLAST algorithm was used to produce a tree from given distances (or dissimilarities) between pair of sequences and alignments using Fast Minimum Evolution and Neighbor Method [28, 29]. BLAST computes a pair wise alignment between a query and the database sequences searched

RESULTS

A total of 90 root and 136 nodule endophytic bacterial isolates were obtained from surface sterilized roots of three legumes chickpea, field pea and lucerne; and two non legumes wheat and oats and nodules of two legumes chickpea and field pea (Table I). Upon authentication under sterilized conditions all were found to be true endophytic. The frequency of total number of endophytic isolates made from roots was much less as compared to nodules and further number of different bacterial isolates appearing on a plate from roots was much low as compared to nodules. The type of colonies of all the root and nodule endophytes varied from gummy to non gummy, flat to raised, rough to smooth, colony size varied from small to medium and from medium to very large and have different colors - brownish cream, pinkish white, milky white, cream, straw and dirty yellow. Cell size varied from minute to small and small to medium, cell shape also varied from cocci to rod. Out of 51 bacteria isolates made from legume roots, 35 isolates were found to be Gram positive and 16 were Gram negative (Table I). Similarly in case of 136 isolates made from nodules, 112 isolates were Gram positive and 24 was Gram negative. Out of 39 bacterial isolates made from non legume wheat and oat roots, 17 isolates were found to be Gram positive while 22 were Gram negative. Irrespective from the source, almost all the gram positive bacteria were forming spores and position of spore was terminal, in few cells even central spore also visible (Table I).



Table I: Morphological diversity of legume nodule endophytes and endophytes from roots of legumes and non legumes

Plant tissue used for endophytes isolation Total isolates Gm +ve Gm -ve Spore formers

Legumes Nodules 136 112 24 102 Roots 51 35 16 34

Non legumes Roots 39 17 22 16 Total 226 164 62 152

Table II: Presence of beneficial traits in legume nodule endophytes and endophytes from roots of legumes and non legumes

Endophytes isolated from High activity showing bacterial endophytes Percentage of isolates

Phosphate solubilization

Legumes Nodules CNE215, CNE216, CNE286, PNE15, PNE24 56.0 Roots CRE1, CRE3, CRE13, PRE1,PRE4 56.9

Non legumes Roots ORE35, WRE10, WRE20 35.9 Ammonia production

Legumes Nodules CNE215, CNE76, CNE80, PNE15, PNE91 81.8 Roots CRE12, PRE12, PRE16, LRE2, LRE3 74.5

Non legumes Roots ORE18, ORE31, WRE3 , WRE12, WRE20 59.0 Organic acid production

Legumes Nodules CNE215, CNE79, CNE217, PNE17 PNE27 25.1 Roots CRE1, CRE8, PRE4 21.6

Non legumes Roots ORE7, ORE27 17.9

a b c d

Fig I: Phosphate solubilization by endophytic bacteria isolated from oat roots (a); chickpea nodules(b); field pea nodules (c) and chickpea roots(d)

Phosphate solubilization is an important beneficial character of the endophytes to make the availability of phosphorus to plants. Therefore, all the bacterial root and nodule endophytes were screened for the presence of phosphate solubilization activity on Pikovskaya’s medium. Depending upon extent of P solubilization, the isolates were categorised as very good, good, moderate, low and very low solubilizers of phosphate so as to compare the different isolates (Fig I). Out of 226 endophytic isolates, 47.8% of root and 56 % of nodule isolates showed phosphate solubilization (Table 2). Further the number of phosphate solubilizing isolates was more in legume roots (56.9%) than non-legume roots (35.9%) and was highest from field pea roots (73.3%) and lowest number of phosphate solubilizing isolates was in oat roots (20%). The amount of P solubilization was higher in isolates from chickpea and oat roots. The good phosphate solubilizers from legume roots and nodules were CRE1 and CNE 215 and from non-legume roots were WRE10, WRE20 and ORE35. Among all these endophytes isolates ORE 35 was the highest P solubilizers (Fig 1a), followed by CNE 215 (Fig Ib), PNE 15 (Fig Ic) and CRE 3(Fig Id). Ammonia is an important metabolite produced by the endophytes as it helps in fulfilling the requirement of nitrogen for the plants. So, all the bacterial endophytes were screened for ammonia production activity in peptone water by adding Nessler’s reagent. Depending upon the quantity of ammonia produced, the isolates were categorised into five categories viz. very good, good, moderate, low and very low producers of ammonia based on the intensity of the color. Out of 226 endophytic isolates 67.8% of the isolates were ammonia producers (Table II). The number of ammonia producing endophytic bacteria in legumes nodules was highest (81.8%), followed by legume roots (74.5%) and non-legumes roots (59.0%). Isolates made from chickpea, field pea roots and nodules; lucerne roots and wheat roots showed very good ammonia production, however isolates made from oat roots showed lowest ammonia production.



Similarity Coefficient0.66 0.75 0.83 0.92 1.00

CRE14AMW

CRE5 CRE7 PRE12 PRE13A PRE14A PRE16 PRE1 LRE2 CRE12 CRE13 CRE6 CRE9 CRE14B PRE8A PRE15 PRE8B CRE10 CRE14A CRE8 PRE4 LRE7 LRE3 LRE4A LRE9

Fig II: UPGMA (unweighted pair grouping with mathematic average) dendrogram showing similarity between RFLP of PCR amplified

16S rDNA of bacterial endophytes from legume roots


WRE5AMW

WRE3

WRE10

WRE12

WRE13

ORE18

ORE31

WRE17

WRE5A

WRE4

WRE7

ORE2

ORE5A

ORE35

ORE4C

ORE5B

ORE34

ORE3A

WRE15

ORE6B

Fig III. UPGMA (unweighted pair grouping with mathe matic average) dendrogram showing similarity between RFLP of PCR amplified

16S rDNA of bacterial endophytes from non legume roots




CRE14AMW

CRE5 CRE7 PRE12 PRE13A PRE14A PRE16 WRE10 WRE3 PRE1 WRE13 ORE18 ORE31 LRE2 WRE5A WRE17 CRE12 CRE13 CRE6 CRE9 CRE14B PRE8A PRE15 PRE8B ORE2 ORE5A ORE35 ORE4C ORE5B ORE34 ORE3A WRE4 WRE7 CRE10 CRE14A WRE15 CRE8 PRE4 LRE7 ORE6B WRE12 LRE3 LRE4A LRE9

Fig IV: UPGMA (unweighted pair grouping with mathematic average) dendrogram showing similarity between RFLP of PCR amplified

16S rDNA of bacterial endophytes from legume and non legume roots A number of organic acids are reported to be produced by the bacterial endophytes like indol acetic acid, gibberellic acid, jasmonic acid and abscisic acid etc. These organic acids support plant growth by solubilization of minerals and chelation of metals and by root growth promotion. Therefore, all the bacterial endophytes were screened for organic acid production by using methyl red dye depending upon the quantity of the organic acid produced, these isolates were categorised as very good, good, moderate, low and very low producers of organic acid according to the level of color (orange to dark red). This test showed that only 20% of bacterial endophytes were found to produce organic acid (Table II). The number of organic acid producers in both legume and non-legume roots were 21.6 and 17.9%, whereas 25.1% of isolates from nodules showed organic acid production. Field pea nodule isolates were better organic acid producer (31.7%) as compared to chickpea nodule isolates (18.4%). Similarly highest number of isolates from pea roots (33.3%) showed organic acid production. Comparatively good organic acid producers from legume roots and nodules were CRE1 and CNE 215 and from non-legume roots were ORE7 and ORE27. To detect the similarity and dissimilarity among the selected 143 endophytes, the RFLP pattern of amplified 16S rDNA was compared. Since it remains highly conserved during the evolutionary history. Genomic DNA of 43 root endophytes made from legume and non legume roots and 100 isolates from nodules was extracted. After amplification of genomic DNA, digestion by three restriction endonucleases Hae III, RsaI and Hinf I resulted in 4 to 22 bands and banding pattern of all the endophytic isolates showed the presence of 39 polymorphic bands. The reproducibility of each PCR amplification profile was confirmed by repeating the amplification at least three times. RFLP analysis based dendrogram of similarity coefficients between 24 isolates from legume roots formed seven major groups at 80% similarity coefficient (Fig II). Considering each group as one bacterial biotype or genotype, overall seven bacterial genotypes in legume roots were present. In major group I, 10 isolates from all the three legumes chickpea, pea and lucerne were there - CRE5, CRE7, PRE12, PRE13A, PRE14A, PRE16, PRE1, LRE2, CRE12 and CRE13. Similarly, in major group II, 11 isolates CRE6, CRE9, CRE14B, PRE8A, PRE15, PRE8B, CRE10, CRE14A, CRE8, PRE4, and LRE7 from all the three legumes were clustered. Dendrogram of similarity coefficients between different 100 chickpea and field pea nodule isolates showed the presence of large variability in nodules. All the nodule endophytes formed 17 clusters at 80% level of similarity. Isolates from chickpea and field pea formed entirely separate as well as intermixed clusters (dandrograme not shown



as number of isolates was much higher than the range). In case of non-legumes, all the 19 isolates from wheat and oat roots formed six major groups at 80% similarity coefficient (Fig III) indicating the presence of six bacterial genotypes.

Fig V : Blast algorithm tree using Fast Minimum Evolution based on alignment of 16S rRNA gene sequences, showing the relationships

of Bacillus flexus strain WRE 20 with other related species of Bacillus. Distance 0.0001 between sequence used for tree generation predicts expected fraction of base substitutions per site given the fraction of mismatched bases in the aligned region.

Again clustering of all the 43 root isolates showed the presence of 10 major groups at at 80% level of similarity coefficient (Fig IV). Thereby, indicating the presence of ten bacterial genotypes in legume and non-legume roots which were able to enter in the roots of all the five crops. There were two major groups, in major group I, 17 isolates from chickpea (CRE5, CRE7, CRE12 and CRE13), pea (PRE12, PRE13A, PRE14A, PRE16 and PRE1), lucerne (LRE2), wheat (WRE3, WRE5A, WRE10, WRE13 and WRE17) and oat (ORE18 and ORE31) were present. Seventeen isolates were clustered to form major group II, which includes the isolates CRE6, CRE9, CRE14B, PRE8A, PRE15, PRE8B, ORE2, ORE5A, ORE35, ORE4C, ORE5B, ORE34, ORE3A, WRE4, WRE7, CRE10 and CRE14A. Based on the presence all the beneficial traits and molecular clustering three isolates CNE 215 isolated from chickpea nodules, CRE 1 isolated from chickpea roots and WRE 20 from wheat roots were selected. Two isolates CNE 215 and CRE 1 were identified earlier as Bacillus subtilis strain CNE 215 and Bacillus licheniformis strain CRE 1. Further isolate WRE 20 was identified by sequencing of amplified 16S rDNA. About 1200 bp fragment of 16S rRNA gene was amplified by PCR. Sequence of 16S rRNA gene was compared with the available sequences of all the organisms. Wheat root endophyte WRE 20 showed more than 98% similarity with Bacillus flexus strain Q 62 and also with Geobacillus stearothermophilus strain WCH 1 partial sequence of 16S rRNA gene. This isolate



belonged to Phylum Firmicutes and alignment tree of Bacillus flexus WRE 20 alongwith other related genera is given in Fig V.

DISCUSSION

During isolation of endophytic bacteria from root or nodules, legumes or non legumes, the complete batch of isolates was discarded if some growth around roots or nodules was observed on TSA plates. This step was taken to ensure that the growth of surface organisms is avoided and only endophytic bacteria are isolated. In case of isolation of endophytes, when root sap was streaked on TSA plates comparatively 4-5 different types of bacteria were observed while nodule sap showed 10-12 different types of bacteria on the TSA plates. Further in chickpea nodules number of isolates was more as compared to field pea nodules. Out of 226 isolates 72.5% were Gram positive and majority of them were spore formers indicating mainly the presence of Gram positive endophytic genera in legume and non legume roots and nodules. Similarly, other workers reported a large variation in morphology, colour and size of the endophytic isolates from Lespedeza sp.[30] and from soybean nodules [31]. However, contrary to this 84.6% of Gram negative and 15.4% Gram positive bacteria from Lespedeza sp were reported [30], though in non legume roots (wheat and oats) predominance of Gram negative bacteria (56.4%) was observed. In northern India, actually temperature range is very high -3 to 48˚C and under these temperature conditions it is quite possible that Gram positive bacteria and particularly spore formers are the better survivors and better colonizers of roots and nodules. Therefore, more number of Gram positive spore formers was observed in roots and nodules of legumes. Though more number of Gram positive isolates in Glycine max tissues and gram negative in Glycine soja has been reported [31] thereby indicating that apart from environmental conditions, host is also an important factor in determining the population of Gram positive and Gram negative bacteria in legumes. To select better strains for use as inoculants, all bacterial endophytes were screened for the presence of beneficial traits. Large functional diversity among the different endophytic isolates from root, nodules, legumes and non legumes was observed. Phosphate solubilization helps plants for easy uptake of phosphate from soil and in the present study an overall 47.8% bacterial root and 56 % nodule endophytes were phosphate solubilizers. Comparatively more isolates from legume roots (56.9%) than non-legume roots (35.9%) phosphate solubilizers. Similarly, phosphate solubilization activity have been reported in endophytes isolated from alfalfa (Medicago sativa L.), Prosopis strombulifera, Lespedeza sp. and Mammillaria fraileana and total percentage of phosphate solubilizers ranged from 0 to 62% [17, 30, 32-34]. Ammonia production is another important beneficial trait exhibited by the endophytes and other rhizospheric organisms. The number of ammonia producing endophytic bacteria in legumes nodules were highest (81.8%), followed by legume roots (74.5%) and non-legumes roots (59.0%). But only three isolates from alfalfa nodules were reported to produce ammonia [32]. How ammonia production is helping the plants is not known but probably is acting as nitrogen source. Another beneficial trait is organic acid production. Organic acids play role in chelation and mineralization and many phytohormones are also found in the form of organic acid which are known to be produced by endophytic bacteria [35]. Comparatively, more number of isolates (25.1%) from nodules was found to produce organic acid than both legume (21.6%) and non-legume (17.9%) roots. Similarly, other workers elsewhere reported the production of organic acids by endophytes from different crops [33, 36]. In contrast to this, none of the isolate was observed to produce organic acid from the nodules of alfalfa [32]. Considering, each cluster representing an endophytic bacterial genotype, thereby in the university farm soils, 17 genotypes were present in nodules, 7 genotypes in legume roots, 6 in non legume roots and in total 10 genotypes were present in both legume and non legume roots. Similarly, 14 genotypes have been reported to be associated with red clover plant nodules [8] (Sturz et al. 1997); 15 bacterial genera were reported from the nodules of different legumes [37] (Zakhia et al. 2006); six taxonomical genera from soyabean nodules [38]; six phylogenitic genera from the nodules of Tephrosia [39]; 13 bacterial genera of non rhizobial endophytes isolated from the nodules of Sphaerophysa salsula [40] and nine genera of non-rhizobial endophytes were found in the nodules of Astragalus terraccianoi [41] have been reported elsewhere. Comparatively less number of endophytic genotypes in root tissue was observed. But elsewhere higher diversity has been reported with 52 operational taxonomic units in rice roots [42], 17 different genera in roots of coastal sand dune plants [43], 16 operational taxonomic units in Mexican husk tomato roots [44], 36 bacterial genera in roots of Medicago truncatula plants [45]. The results indicate wide variation of number and type of bacterial endophytic association with different tissues and legumes.



Based on the presence of multiple benefical traits, the three endophytic isolates were selected for further identification. The identity of two isolates was reported earlier [26]. All the three isolates were Firmicutes. Bacillus subtilis strain CNE 215 was present in chickpea nodules, Bacillus licheniformis strain CRE 1 was present in chickpea roots and Bacillus flexus strain WRE 20 was present in wheat roots. The results indicated that the bacterial specie entering in the plant tissue are dependant upon the type and availability of nutrients in a tissue, their abundance in the soil and environmental conditions prevailing in that region. The species of this genus are known to generate spores under adverse conditions which are encountered in this region of India [46]. Further host specificity with endophytes was reported in Phaseolus vulgaris and in chickpea [26, 47]. It can be concluded from the study that the number of bacterial genera entering in the plant tissue are dependant upon the type and availability of nutrients in a tissue, their abundance in the soil and environmental conditions. In nodules more diverse genera were present as compared to root endophytes of legumes or non legumes. Further one or another beneficial traits was present in most of the endophytes irrespective of the plant tissue from which it was isolated though their effectivity varied. It seems probable that in roots or nodules all the bacteria are complimenting the rhizospheric bacteria to help in more nutrient mobilization. Further most efficient and predominant type of endophyte which would be able to colonize and infect all types of tissues and enters the roots as well as nodules shall enhance the crop productivity.

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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,