Functional and phylogenetic diversity of cultivable rhizobacterial endophytes of sorghum

[Sorghum bicolor (L.) Moench]

A diverse group of bacteria colonize the exo- and endo-rhizospheres of sorghum and play a
critical role in its tolerance to drought and other abiotic stresses. Two hundred and eighty
endophytic bacteria were isolated from the surface-sterilized roots of four sorghum cultivars
that were grown on three soil types at three different phenological stages of growth. The
isolates were subjected to in vitro screening for their plant growth promoting traits. Out of 280
isolates, 70 could produce Indole 3-Acetic Acid (IAA), 28 showed N-fixation, 28 could solubilize
phosphate, 24 had ACC deaminase activity and 13 isolates were able to produce siderophores.
Functional diversity grouping of the isolates indicated one isolate having five PGP traits and
two isolates having four PGP traits; two and 29 isolates having three and two PGP traits,
respectively. Among the thirty-four isolates that possessed multiple PGP traits, 19 and 17
isolates were able to produce significant quantities of IAA in the presence and absence of L-
tryptophan, an inducer. Eight isolates possessed high levels of ACC deaminase activity. PCR
RFLP of the 16Sr RNA gene revealed a distinct clustering and considerable genetic diversity
among these functionally characterized isolates. The 16S rRNA gene based identification of the
isolates of single and multiple PGP traits revealed phylogenetic dominance of Firmicutes;
Acinetobacter, Bacillus, Enterobacter, Geobacillus, Lysinibacillus, Microbacterium,
Ochrobactrum, Paenibacillus and Pseudomonas were the major genera present in the
endorhizosphere of sorghum. Results of this study are constructive in selection of effective
rhizobacterial endophytes or consortia for drought stress alleviation in sorghum.

Introduction

A range of plant associated bacteria that significantly increase vegetative growth and grain
yield of various crops have been isolated and used as bio-fertilizers (James 2000; Andrews et
al. 2003; Kennedy et al. 2004). These include rhizospheric and endophytic bacterial species
that are critical for nutrient cycling, the maintenance of soil fertility and plant fitness.
Endophytic bacteria live within plant tissues without causing visible damage to the host plant,
and, on the contrary, may promote plant growth directly or indirectly through a combination
of mechanisms. Bacterial endophytes have been shown to have neutral, detrimental or
beneficial effects on plants (Hafeez et al. 2006; Rosenblueth and Martnez-Romero 2006;
Compant et al. 2010). In recent years, the application of natural or genetically modified
bacteria to enhance plant productivity under abiotically stressed environments has been
actively considered over plant genetic improvement (Hafeez et al. 2006; Berg 2009; Compant
et al. 2010; Schenk et al. 2012). This approach favours the utilization of plant-associated
bacteria that have evolved a structured and intimate relationship with the plant hosts (Berg
2009; Compant et al. 2010; Schenk et al. 2012).

Plants are endophytically colonized by a wide variety of bacteria belonging to different

phylogenetic groups (Reiter and Sessitsch 2006; Berg 2009; Compant et al. 2010). Most
endophytic bacteria belong to phylum Proteobacteria, and are also widespread in the phyla
Firmicutes, Actinobacteria and Bacteroidetes (Rosenblueth and Martnez-Romero 2006;
ReinholdHurek and Hurek 2011). However, the structural composition of endophytic bacterial
communities depends on the host plant genotype, the plant organ growth stage, stress levels
(Sessitsch et al. 2002; Reiter et al. 2002; Rasche et al. 2006a, b) and the soil type on which the
plant is grown (Conn and Franco 2004). Moreover, some root-endophytic colonizing bacteria
are known for their ability to induce systemic tolerance (IST) mechanisms that makes plant
resilient to various abiotic stresses, such as drought, salinity and metal toxicity (Yang et al.
2009; Dimkpa et al. 2009a; Sukweenadhi et al. 2015).

Endophytic colonization by such bacterial species can accelerate seedling emergence, promote
plant establishment and enhance plant growth/fitness under different adverse environmental
conditions. Bacterial endophytes are believed to elicit plant growth promotion either indirectly
by helping plants acquire nutrients or directly by producing phytohormones such as auxin,
cytokinins or by producing enzyme 1-amino cyclopropane-1-carboxylate (ACC) deaminase
which lowers the ethylene levels under various abiotic stress conditions (Glick et al. 2007;
Compant et al. 2010; Goswami et al. 2014). Some of these are diazotrophic bacteria, which are
able to fix atmospheric nitrogen (James 2000; Ji et al. 2014). Auxin is one of the most vital
phytohormones, primarily due to its pivotal functions in the initial processes of lateral and
adventitious root formation and root elongation. Earlier reports indicate that bacterial
endophytes may also enhance plant auxin synthesis, in addition to their own synthesis, with
different pathways contributing total plant auxin pools which effects root growth (Kloepper et
al. 2004; Yao et al. 2006; Idris et al. 2007). Bacterial endophytes are known to release metal-
chelating substances such as iron-chelating siderophores, which influence the uptake by plants
of various micronutrients, including Fe, Zn, and Cu (Egamberdiyeva 2007; Dimkpa et al. 2008,
2009b).

Besides, siderophores act as determinants of induced systemic resistance against plant

pathogens and or induced systemic tolerance against nutritional stresses; they also play an
important role in endophytic colonization within the plant interior environment (Reinhold-
Hurek and Hurek 2011; Gururani et al. 2013).

Sorghum [Sorghum bicolor (L.) Moench] is the worlds fifth most important cultivated cereal
after wheat, rice, maize and barley, with a global production of 60 million tons (Dicko et al.
2006). It is the dietary staple of more than 500 million people in 30 countries and is the third
most consumed cereal grain in India after rice and wheat (Rao et al. 2010). India is the third
largest producer of grain sorghum in the world with a production of

10.62 million tons during 201314 and almost the entire production of sorghum ([95%) in the
country comes from the semi-arid and arid regions (Rao et al. 2010; GOI 2014). Besides grains,
sorghum stover is an important feed in the livestock sector in India particularly in the dry
seasons when other feed resources are in short supply. Naturally, sorghum is a hardy and
drought tolerant crop by virtue of its own genetic architecture and interactions mainly with its
rhizospheric and endophytic beneficial microbes in its growing environment, which partly
might be contributing to its adaptation and tolerance mechanisms. Based on cultivation
dependent studies, various sorghum rhizospheric associated bacteria have been isolated and
identified (Pedersen et al. 1978; Budi et al. 1999; Zinniel et al. 2002) some of which have
exhibited plant growth promotion (PGP) activities such as nitrogen-fixation by members of the
Enterobacteriaceae related to Klebsiella pneumoniae, Enterobacter cloacae or Erwinia
herbicola (Pedersen et al. 1978), the capacity for biocontrol by Paenibacillus sp. strain B2 and
Pseudomonas spp. (Budi et al. 1999; Funnell-Harris et al. 2013) and mitigation of abiotic stress
(Ali et al. 2009). However, few attempts have been made to identify and characterize bacterial
groups associated within root-endophytic environments of sorghum cultivars, and more
particularly their functional and phylogenetic diversity. Sorghum is among the few resilient
crops that can adapt well to future climate change conditions, particularly the increasing
drought, soil salinity and high temperatures. Hence the present study was designed to isolate,
identify and determine the phylogenetic diversity of root endophytic bacterial populations
from sorghum cultivars cultivated on different soil types and to characterize the plant growth
promotional attributes of such bacterial endophytes.

Materials and methods

Sorghum cultivars, soil types and soil analysis

Seeds of four drought tolerant (rabi season/rain fed) sorghum cultivars viz., Maldandi 35-1,
Selection-3, Phule Anuradha and Phule Maulee were collected from the Sorghum
Improvement Project, Mahatma Pule Krishi Vidyapeeth, Rahuri, India. During rabi season,
seeds of four cultivars were sown in three soil types viz., red soil, black soil in pots and native
murrum soil in field. Prior to sowing of seeds, replicated soil samples were subjected to
analysis of their chemical properties such as pH, EC, organic carbon, available N, P, K and S
(Table 1) using standard protocols.

Sorghum root sampling, sample preparation and isolation of endophytic bacteria

Root samples of the sorghum cultivars were collected during different phenological stages of
growth viz., seedling development, panicle initiation/pre-flowering and
flowering/reproduction stages. Healthy roots were washed with tap water and rinsed in sterile
water. Root samples were surface sterilized by soaking in 70% ethanol for 1 min and then
treated with sodium hypochlorite (35% available chlorine) for 35 min. Samples were then
rinsed thrice by soaking in sterile distilled water (15 min each). As a sterility check, 1 mL from
the final washing was transferred to 9 mL of tryptone soy broth and 100 lL of this solution was

spread on tryptone soy agar (TSA) plates. The root samples corresponding to wash water
aliquots that showed signs of bacterial growth on TSA after a 24 h incubation period were
discarded. Surface sterilized root samples were cut into small pieces with a sterile scalpel and
then ground aseptically in 12 g lots using sterile mortar and pestle in 5-mL of sterile distilled
water. Initial tenfold serial dilutions were also made from 1 g of crushed root tissues and
dilutions were inoculated in TSA agar plates in triplicates and incubated at 28 2 C for 2448
h. Initial endophytic bacterial population size of each cultivar across the growth stages was
obtained based on bacterial colony counts from these TSA plates. Each crushed root tissue
sample was serially diluted and inoculants were transferred and spread on Nutrient agar (NA),
TSA and Kings B agar (KBA) in triplicates and incubated at 28 2 C for 2448 h. After the
incubation period, the endophytic bacterial colonies were transferred to fresh nutrient or
tryptone soy media and incubated at 30 C further for 48 h. The transfer procedure mentioned
above was carried out 34 times to isolate pure endophytic bacterial colonies. The number of
endophytic bacterial isolates obtained/ purified and their characteristics were recorded. The
purified isolates were stored in 15% (v/v) glycerol stocks at -80 C.

In vitro qualitative assessment of PGP traits of purified endophytic bacterial flora

N-fixation

All the purified endophytic bacterial isolates were screened for their ability to grow on N free
medium by streak inoculation of individual cultures on N-free medium (Jensens N-free
medium, HiMedia, India) and incubated at 28 2 C for 4872 h. The presence of mucoid and
slimy growth of bacterial isolates on culture plates was considered/recorded as positive.
Mineral phosphate solubilization

Solubilization of phosphate was determined by spot inoculation of the bacterial isolates on

Pikovskayas agar (HiMedia) (10 lL of 106 CFU mL-1) followed by incubation at 28 2 C for 48
72 h. Bacterial isolates that were surrounded by clearing zones were considered as positive
(Pikovskaya 1948).

Siderophore production

Bacterial isolates were assayed for their ability to produce siderophores on Chrome azurol S
agar medium (Schwyn and Neilands 1987). Chrome azurol S agar plates spot inoculated with
individual bacterial isolates (10 lL of 106 CFU mL-1) and incubated at 28 2 C for 4872 h.
Development of a yellow orange halo zone around the bacterial growth was considered as
positive for siderophore production.

IAA production

Individual isolates were initially screened for their ability to produce IAA on LuriaBertanis
(LB) agar (Brick et al. 1991). Individual bacterial isolates (10 lL of 106 CFU mL-1) were spot
inoculated on LB agar and LB agar medium supplemented with 5 mM of Ltryptophan. The
inoculated plates were overlaid with a sterile nylon membrane. After incubation the colonies
adhering to the nylon membrane were treated with Salkowskis reagent. The development of a
pink colour on the bacterial colonies was considered as indication of IAA production by the
bacterial isolates (Brick et al. 1991).

ACC deaminase activity

Qualitative plate screening for ACC deaminase activity was performed by the standard
protocol (Penrose and Glick 2003). Individual bacterial isolates were initially
inoculatedinnutrientrichTSBmediumandincubatedin a refrigerated incubator shaker (150 rpm)
at 28 2 C for 24 h. Cell pellets were collected by centrifuging 5 mLofcultureat8000 rpmfor5
minandwashedthrice with sterile distilled water. The pellets was resuspended in 1 mL of sterile
distilled water and spot inoculated on DF salts minimal medium (Dworkin and Foster 1958)
supplemented with 3 mM ACC. Negative control plates of DF minimal medium without ACC
and positive control plates i.e. DF minimal medium with(NH4)2SO4 as nitrogen source were
also spot inoculated and incubated at 28 2 C for 34 days. Isolates growing on DF minimal
medium with ACC were visibly compared with the control plates for ACC deaminase
production. The above procedure was repeated thrice to avoid false positives (Govindasamy et
al. 2009).

Quantitative estimation of indole-3-acetic-acid (IAA) production by the endophytic bacteria

Quantitative estimation of IAA production by the isolates was performed (Gordon and Weber
1951) with modifications (Brick et al. 1991). Individual bacterial isolates were inoculated in LB
medium alone (without L-tryptophan) and LB medium supplemented with L-tryptophan (100 lg
mL-1) as a precursor/ inducer of IAA. The supernatant obtained by centrifuging 72 h grown
cultures at 8000 rpm at room temperature (25 C) for 10 min, was mixed with Salkowskis
reagent (50 mL, 35% of perchloric acid, 1 mL 0.5 M FeCl3 solution) in the ratio of 2:1 or 1:1 and
left in dark for 30 min. The pink colour developed was read in a spectrophotometer at 530 nm.
The concentration of IAA produced by the individual bacterial isolates was determined from a
standard curve prepared with IAA (Hi-media, India).
Quantitative estimation of ACC consumption and ACC deaminase enzyme activity of the
endophytic bacteria

All endophytic bacterial isolates were further screened for utilization of ACC as a N-source by
the ACCninhydrin reaction method (Li et al. 2011). Overnight grown cultures in LB broth were
centrifuged at 8000 rpm at room temperature (25 C) for 10 min and bacterial cell pellets were
washed thrice with sterile DF medium. Cell pellets were re-suspended in DF medium
supplemented with ACC (3 mM L-1) medium and incubated at 30 C in incubator shaker at 200
rpm for 24 h. From these each culture, 1 mL of culture fluid was centrifuged at 8000 rpm at
room temperature (25 C) for 10 min and 100 lL of supernatant was diluted to 1 mL with DF
medium. To this, 2 mL of the ninhydrin reagent was mixed in the test tubes and kept in boiling
water bath for 15 min. After keeping the tubes at room temperature for 10 min, optical
density was recorded in the spectrophotometer at 570 nm. Leftover concentration of ACC in
the bacterial grown DF liquid medium was estimated against a standard curve of ACC (Sigma-
Aldrich, USA). The amount of ACC consumption by the individual bacterial isolates was
calculated from the initial ACC concentration of DF medium.

Quantitative assessment of ACC deaminase enzyme activity requires bacterial growth

conditions that favour the induction of ACC deaminase. ACC deaminase enzyme activity was
measured in bacterial extracts prepared in the following manner as described (Penrose and
Glick 2003). The bacterial cells were grown in 5 mL of TSB medium at 28 2 C for

3 days until they reached the mid to late-log phase.

Cell pellets were collected by centrifugation at 8000 rpm for 5 min and washed several times
with DF minimal medium. To induce ACC deaminase activity, cell pellets were resuspended in 2
mL of DF minimal medium supplemented with 3 mM ACC and then incubated at 30 C for 40 h
in shaking condition in an incubator shaker at 200 rpm (Penrose and Glick 2003). The bacteria
were harvested by centrifugation at 80009g for 10 min at 4 C and the cell pellets were washed
twice by 5 mL of 0.1 mol L-1 Tris-HCl, pH 7.6 and the contents were centrifuged at 80009g for

5 min and then resuspended in 1 mL of 0.1 mol L-1 Tris-HCl (pH 7.6). These bacterial contents
were centrifuged at 160009g for 5 min and cell pellet was again suspended in 600 lL of 0.1 mol
L-1 of Tris-HCl (pH 8.5). 30 lL of toluene were added to the above cell suspension and vortexed
at the highest setting for 30 s and 100 lL aliquot of the toluenized cells is set aside and stored
at 4 C for protein assay (Lowry et al. 1951). The remaining toluenized bacterial cell extracts
were used for assay of ACC deaminase activity (Honma and Shimomura 1978). To the 200 lL of
the toluenized cells, 20 lL of 0.5 mol L-1 ACC was added, briefly vortexed and then incubated at
30 C for 15 min. Following the addition of 1 mL of