Characterization of endophytic bacteria associated with rose plant (Rosa damascena trigintipeta) during flowering stage and their plant growth promoting traits

Abstract

Little is known about the bacterial communities associated with the rose plants inhabiting dry desert ecosystems. The aim of this study was to isolate and characterize endophytic bacteria from different organs of rose plant. Endophytic bacteria were observed in healthy roots, stems, leaves, and flowers of rose plant, with a significantly higher density in roots, followed by stems, leaves, and petals. A total of 38 bacterial endophytes were isolated and are closely related phylogenetically to Acetobacter, Acinetobacter, Methylococcus, Bacillus, Micrococcus, Planococcus by 16S rRNA sequence analysis. Six endophytic bacteria were found to produce IAA, solubilize Ca₃(PO₄)₂ and produce siderophore. The six endophytic bacteria all had the capacity to produce hydrolytic enzyme such as cellulase, xylanase, pectinase, amylase, protease, lipase, and chitinase, but difference existed among these isolates.

Keywords:

• endophytic bacteria
• IAA
• hydrolytic enzymes
• Rosa damascena trigintipeta

Introduction

Endophytic bacteria are defined as bacteria that colonize healthy plant tissue without causing obvious disease symptoms in host plant (Hallmann et al. 1997). Endophytic bacteria seem to be ubiquitous in most plant species and have been isolated from roots, leaves, and stems, and a few from flowers, fruits, and seeds (Lodewyckx et al. 2002). Endophytic bacteria may accompaniment certain metabolic properties, such as promoting plant growth, controlling soil-borne pathogens, or helping host plant to defeat stress responses to environmental abuse (Mastretta et al. 2006; Taghavi et al. 2007; Ryan et al. 2008). Furthermore, the interactions between plants and bacteria help plants to settle in ecosystem restoration processes (Glick et al. 1995). These interactions may increase the ability of plants to utilize nutrients from the soil by increasing root development, nitrate uptake, or solubilizing phosphorus, and to control soil-borne pathogens (Whipps 2001).

Plant communities in arid habitat are controlled by the interaction between biotic and physico-chemical components of the desert matrix (Read 1998). Interactions with microbes appear crucial in obtaining inorganic nutrients or growth-influencing substances. Despite the important role played by bacterial diversity in such plant communities, little is known on the distribution and abundance of endophytic bacteria in rose plant growing in such habitat. Rose plant has been studied with respect to its endophytic fungi characteristics (Catalina Salgado et al. 2007). However, no study has been reported to the relationships between rose plant and their associated endophytic bacteria. The purpose of this study is to isolate and characterize endophytic bacteria from rose plant collected from rose farm, and to assess isolates plant growth promoting (PGP) traits like production of IAA and siderophore, and phosphate solubilization. This study is the first report on the diversity of culturable endophytic bacteria associated with the rose plant growing in arid habitat. The outcome of this study will form the basis for the selection of endophytic bacteria that can be utilized for the facilitation of plant growth in such habitat.

Materials and methods

Chemicals and medium

All reagents used were of analytical grade and were purchased from Sigma Co. Ltd., USA. Putative endophytic bacterial strains which were isolated from plants and cultured at 28°C in tryptic soy agar (TSA) medium containing of 15 g Trypticase peptone, 5 g phytone peptone, 5 g NaCl, 15 g Agar per liter of water and PDA medium comprising of 200 g potato infusion, 20 g dextrose, 15 g Agar. The pH of the medium was adjusted to 7.2–7.4.

Isolation of endophytic bacteria

Putative endophytic bacterial strains were isolated from surface-sterilized rose plants, collected randomly at flowering stage from rose farm at Taif city, Saudi Arabia. Plant samples were transported in plastic bags in a cooler, stored overnight at 4°C and processed on the following day. Root, stem, leave, and flower of each plant were separately. Endophytic bacteria were isolated after removing epiphytes by surface disinfection using serial washing in 70% ethanol for 3 min, sodium hypochlorite solution (2% available Cl ) for 3 min, and rinsed three times in sterilized distilled water (Barzanti et al. 2007). To confirm that the sterilization process was successful, the aliquots of the sterile distilled water used in the final rinse were set on TSA medium plates. The plates were examined for bacterial growth after incubation at 28°C for 5 days. The root, stem, leaf, and petal tissues were then macerated, respectively, using a sterile mortar and pestle in a small volume of sterile phosphate buffered saline (pH 7.4), with sterile quartz sand being added to improve the wall disruption. Samples (100 µl) of tissue extracts and their different dilutions were plated onto TSA and/or PDA agar medium After incubation at 28°C for 4 days, colonies in varying morphology were picked and repeatedly re-streaked on above media until the colony morphology of each isolate reached homogenous.

Genotypic characterization of selected isolates

Standard tests were performed for identification of the studied strains in accordance with Bergey's Manual of Determinative Bacteriology (1994). Cell form and size, Gram staining, spore formation, motility, colony pigmentation, and production of UV-fluorescent pigments were studied. The strains were also identified by determination of 16S rRNA gene sequences. The genomic DNA of endophytic bacteria was extracted (Govindarajan et al. 2007), and 16S rDNA was amplified in polymerase chain reaction (PCR) using the genomic DNA as template and bacterial universal primers, 27 F (5′-GAGTTTGATCACTGGCTCAG-3′) and 1492 R (5′-TACGGCTACCTTGTTACGACTT-3′) (Byers et al. 1998). Briefly, a 25-µL reaction mixture contained 1.25 U Taq polymerase (Sigma Chemical Co., St. Louis, MO), 0.2 mM dNTPs, 25 mM MgCl₂ (Sigma), 10 pmol of each primer, 2.5 µL of 10× reaction buffer (Sigma), and 1 µg of template DNA. Aliquots of PCR reaction products were electrophoresed in 1% agarose, containing 10 µg·mL⁻¹ ethidium bromide. To know the identity of organism, obtained sequences were compared with nucleotides databases like GenBank (Benson et al. 2009).

Evaluation of plant growth promoting (PGPE) properties of the isolates

ACC deaminase activity

The 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of cell-free extracts was determined by monitoring the amount of a α-ketobutyrate (α KB) generated by the enzymatic hydrolysis of ACC (Saleh and Glick 2001).The absorbance (OD540) was compared with a standard curve of α-ketobutyrate ranging between 0.1 and 1.0 µmol according to (Belimov et al. 2005).

IAA production

IAA production was measured according to Sheng et al. (2008). Briefly, 1 ml cell suspension obtained from bacterial culture grown in MSM medium with l-tryptophan (0.5 mg/ml) was transferred into a tube and mixed vigorously with 2 ml of Salkowski's reagent (Gordon and Weber 1951). A pink color developed after 20 min incubation at room temperature, and the absorbance was obtained at 530 nm. The IAA concentration was determined using a calibration curve of pure IAA as a standard following the linear regression analysis.

Siderophore production

Siderophore secretion by these strains was detected by the ‘universal’ method of Schwyn and Neilands (1987) using blue agar plates containing the dye Chrome azurol S. Orange halos around the colonies on blue agar were indicative of siderophore excretion.

Phosphate solubilizing capacity

The capability of the bacteria to solubilize phosphates was tested by measuring the diameter of the solubilized zone around bacterial colonies grown on a solid medium supplemented with freshly precipitated Ca₃(PO₄)₂ (Puente et al. 2004)

Exoenzyme activity tests

The tests included (1) starch hydrolysis on starch plates (Claus 1988); (2) Lipid hydrolysis using both egg yolk agar (Claus 1988) and Tributyrin agar (Lusty and Doudorof 1966); (3) proteolysis as hydrolysis of skim milk (Claus 1988), and gelatin (Biling 1970); cellulose degradation (Farkas et al. 1985); Pectolysis with either polygalaturonate or sodium polypectate as substrates after the method of Collmer et al. (1988) and chitin hydrolysis as described by Zhou et al. (1999). Bacterial cultures were streaked on the medium and incubated at 30°C for 48 hours. A clearing zone in the medium indicated positive enzyme activity.

Results and discussion

Taif rose (Rosa damascena trigintipetala) are considering one of the best rose quality around the world. This rose grows in the hill mountain outside Taif, Saudi Arabia at an elevation of around 2000 m above sea level. This study focuses on obtaining a comprehensive picture of the endophytic bacterial community of the rose plants growing on a arid habitat.

Frequency, diversity, and identity of endophytic bacteria from rose plant

The endophytic bacterial communities of healthy looking roots, stems, leaves, and flowers of rose plant were assessed in surface disinfested plant parts on cultivation in TSA medium. The result of effectiveness of the surface sterilization protocol was determined. Rinsed water of each sample showed no microbial growth on TSA medium after incubation at 30°C for 15 days, indicating that the epiphytic microbes were completely removed by this surface sterilization procedures. Indeed, the major key to succeed in isolating and studying endophytes is to ensure the sterility of the plant surface (Hallmann et al. 1997). The diversity of isolated endophytic bacteria was also largely dependent on the isolation methods (Das et al. 2007). The number of colony-forming units per gram fresh weight of culturable endophytic bacteria isolated from various plant organs of Rose plant was determined (Table 1). The results showed that CFU value in flower (1.2×10²) is much lower than in leaves (2.4×10³) of Rose plants. In contrast, root (4.6×10⁴) and stems (3.1×10⁴) had higher CFU values than other organs. This distribution pattern, in which lower plant parts harbor higher frequencies of endophytes, confirms and extends results reported previously (Das et al. 2007). Furthermore, our results indicated that TSA supported more of endophytic bacterial growth than PDA as previously reported by Hung and Annapurna (2004).

Table 1. Endophytic bacterial population recovered at flowering stage (cfu g⁻¹ FW).

Host plant	TSA medium				PDA medium
	Root	Stem	Leaves	Flower	Root	Stem	Leaves	Flower
Rose plant	4.6×10^4	4.1×10^4	3.4×10^3	1.8×10^2	4.1×10^4	3.1×10^4	3.1×10^3	1.2×10^2

In general, the CFU value did not differ markedly between roots and stems of rose plants collected from different locations (Table 1). The population densities reported in the present study are comparable to the earlier reports in maize and also agree with population ranges in other crops such as sugar beet (10³–10⁵ CFU/gm fw of roots) (Bugbee et al. 1975) and potato (3.3×10⁵ CFU/gm fw of stem) (De Boer and Copeman 1974).

Isolation, selection, and identification PGPE bacteria

Endophytic bacteria have been found in virtually every plant studied, however, these studies mainly focused on agricultural crops, economically important crops, and some trees (McInroy and Kloepper 1994; Ulrich et al. 2008; Jha and Kumar 2009; Naik et al. 2009). Of these, cotton, potato, rice, wheat, and sweet corn were extensively studied, whereas plant species growing in harsh conditions and having important officinal and industrial values such as Rose plant, which is growing on the crags of high-altitude mountains, have not been investigated with respect to their associated microbial community. Therefore, endophytes associated with rose plants have attracted the attention of investigators (Catalina Salgado et al. 2007). In this study, we found rose plant also harbored an abundance of culturable endophytic bacteria, and varied between roots, stems, leaves, and flowers. Thirty-eight colonies were originally isolated for their different morphological appearance on TSA and/or PDA agar medium. Preliminary characterization of these isolates indicated that studied parts of rose plant contained both Gram-negative and Gram-positive bacteria. To obtain the PGPE, all isolates were qualitative tested for a number of important properties regarding PGP activity (Table 2). Here, six isolates namely TUB1, TUB2, TUB3, TUB4, TUB5, and TUB6 possessed multiple of PGP properties were selected for subsequent studies. On the basis of morphological, biochemical characteristics and comparative analysis of the partial sequence of 16S rRNA gene, the TUB1 (720 pb), TUB2 (720 pb), and TUB3 (720 pb) showed 100% homology with Planococcus sp., Micrococcus sp., Bacillus sp., respectively. However, the TUB3(720 bp), TUB4(720 bp), and TUB6 (720 pb) showed 98.6% homology with Methylococcus sp., Acinetobacter sp., and Acetobacter sp., respectively. The sequence was deposited at GenBank under accession numbers as indicated in Table 2. Zinniel et al. (2003) identified 15 bacterial genera from endophytic bacteria associated with corn and sorghum, among these genera Micrococcus and Bacillus were reported. The previously mentioned bacteria were isolated by our group from aquatic plant Echhornia crassipe (El-Deeb et al. 2006).

Table 2. Plant growth promoting activities of the six endophytic bacteria (each value is a mean±standard deviation of three replicates).

Bacterial strains	Isolate code	Gene Bank accession NO	Plant organs	Siderophore production	ACC deaminase activity (µM α KB mg^−1 h^−1)	Indole production (µg IAA/mL)	Phosphate solubliziation activity (mm)
Planococcus sp.	TUB1	FR718964	Leaves	+	12.2±1.2	10.2±2.4	10.2 ±2.1
Micrococcus sp.	TUB2	FR18965	Petals	+	14.2±1.6	16.2±1.8	7.6 ±0.8
Bacillus sp.	TUB3	FR18966	Stem	+	18.5±1.4	18.6±1,6	12.2±1.2
Methylococcus sp.	TUB4	FR18967	Stem	ND	ND	7.8±0.8	6.4±0.6
Acinetobacter sp.	TUB5	FR18968	Root	+	38.8±3.6	36.6±3.1	28.4±3.1
Acetobacter sp.	TUB6	FR18969	Leaves	+	18.6±2.2	12.3±1.5	12.2±1.2

PGPE of the bacterial isolates

Bacterial endophytes may interact in several ways with host plants to improve their growth through similar mechanisms described for Plant growth promoting rhizospheric bacteria (PGPR), including nitrogen fixation, phosphate solubilization, IAA production and the production of a siderophore (Glick et al. 1995; Rajkumar et al. 2009). This makes sense because most of the bacterial endophytes isolated from various plants can be considered to be facultatively endophytic and are capable of living outside plant tissues as rhizospheric bacteria (Di Fiori and Del Gallo 1995). In the present study, the estimation of IAA in culture filtrate showed that all the six endophytic bacteria isolates had the capacity to produce IAA when the culture medium was supplemented with L-tryptophan, strains TUB3 and TUB5 showed a higher production of IAA (18.6±1.6 and 38.8. ±3.6 µg IAA/mL, respectively) than TUB1, TUB2, TUB4, and TUB6 (10.2±2.2, 16.6±1.8, 7.8±0.8 µg IAA/mL, respectively) (Table 3). Similarly, Strain TUB3 and TUB5 had high levels of ACC deaminase activity than that of TUB1, TUB2, TUB4, and TUB6. Moreover, All the strains displayed the ability of producing siderophore (Costa and Loper 1994). As can be indicated in Table 2, TUB3 and TUB5 were the best ACC deaminase activity and IAA-production strains among these six PGP. In general, the IAA produced by microbe promotes root growth by directly stimulating plant cell elongation or cell division (Glick et al. 1995; Patten and Glick 2002). According to the IAA level, root elongation changes qualitatively. A low level of IAA promotes primary root elongation whereas a high level of IAA stimulates lateral and adventitious root formation but inhibits the primary root growth (Tsavkelova et al. 2007). Thus, the IAA producing PGPE can facilitate plant growth by altering the plant hormonal balance. Phosphorus is known to be one of the major essential mineral nutrients for plants growth. Although sandy soils may have large reserves of total phosphates, but the amounts available to plants is usually a tiny proportion of this total (Stevenson and Cole 1999; Egamberdiyeva and Hflich 2004) due to its chemical fixation and low solubility. Moreover, the low solubility of phosphate lead to plant growth retardation (Halstead et al. 1969). This deficiency can be compensated by bacteria for its inorganic phosphate-solubilizing ability (Zaidi et al. 2006). In present work, the estimation of phosphate solublization in vitro showed that all the six endophytic bacteria isolates had the capacity to solublize phosphate (Table 3) when the solid medium was supplemented with Ca₃(PO₄)₂ (Puente et al. 2004), strain TUB5 showed a high capacity for phosphate solublization than other tested strains. Furthermore, these particular bacteria have more potential as a PGPE strategy in arid region plants than the PGPB used in agriculture.

Table 3. Evaluation of extracellular hydrolytic enzyme activity from the endophytic bacteria isolated from Rose plants.

Isolate code	Bacterial strains	Chitinase	Pectinase	Xylanase	Amylase	Protease	Lipase	Cellulase
TUB1	Planococcus sp.	+	+	−	−	+	−	−
TUB2	Micrococcus sp.	+	+ +	−	−	+ +	+	−
TUB3	Bacillus sp.	+	+ +	+ + +	+ + +	+ + +	−	+ + +
TUB4	Methylococcus sp.	–	+	–	−	+	−	−
TUB5	Acinetobacter sp.	+	+ +	+	−	+	−	+
TUB6	Acetobacter sp.	+	+	−	+	+	−	+
Size of halos formed around bacterial colonies on agar media.
Note: –, implies no halo zone indicates no enzyme activity; +, implies 2 mm diameter of the halo zone indicates enzyme activity; ++, implies 4 mm halo zone indicates enzyme activity; + + +, implies 6 mm diameter of the halo zone indicates enzyme activity.

Evaluation of hydrolytic enzyme activities

The isolated Rose plant endophytic bacteria were evaluated for hydrolytic enzyme activities such as cellulase, xylanase, pectinase, amylase, protease, lipase, and chitinase (Table 3). Among the isolated bacterial strains, especially Bacillus sp. (TUB3), showed maximum number of enzymes activities among tested enzymes activities (Table 3). All groups examined displayed proteolytic activity. Among isolates, Planococcus sp. (TUB1) and Methylococcus sp. (TUB4) had pectinase and protease activity, whereas Planococcus sp. (TUB1) demonstrated only chitinase activity. Among isolates Micrococcus sp., Acinetobacter sp., and Acetobacter sp. (TUB6) possessed chitinase, cellulase, xylanase, pectinase, and protease activity, whereas Micrococcus sp. (TUB2) showed only lipase activity. In general, the hydrolytic enzymes of endophytes appear to be important for the colonization of plant roots (Quadt-Hallmann et al. 1997; Reinhold-Hurek and Hurek 1998; Sakiyama et al. 2001). This hypothesis is supported by the presence of cellulolytic and pectinolytic enzymes produced by numerous endophytic bacteria such as Rhizobium sp. (Al-Mallah et al. 1987). Verma et al. (2001) demonstrated the presence of varying levels of cellulase and pectinase activities in different isolates, possibly affecting their potential for inter/intracellular colonization. In addition, bacteria enter the interior of the root by hydrolyzing wall-bound cellulose, auxin-induced tumors, water flow and wounds, or where the lateral roots branch (Al-Mallah et al. 1987). Endophytic bacteria likely have a signaling mechanism (quorum-sensing) that specifically regulates the amount and timing of enzyme production. Interestingly, plants can perceive these signals from the bacteria and control quorum-regulated bacterial responses (Mathesius et al. 2003; Bauer and Mathesius 2004). It would be interesting to determine if endophytes produce quorum-sensing molecules inside the plants and to study their effects. There could be an exchange of signal molecules among microorganisms inside the plant and between bacteria and the host, though this has not been reported.

Conclusion

Results of our work suggest that Rose plant is naturally associated with a variety of endophytic microorganisms, which have different physiological and biochemical capabilities. In regard to the direct mechanisms, phytohormone production (IAA) was confirmed in many isolates, and we speculate that the bacterial production of these molecules could benefit the plant through induction of some changes in its morphology, especially in roots, or increasing its length to absorb more water or its length to explore the soil in depth specially in harsh environments.

Acknowledgements

This research was supported by the Research Program of Taif University, Saudi Arabia (project 717-431-1).