Endophytic bacteria with plant growth promoting abilities from Ophioglossum reticulatum L.

Endophytic bacteria colonizing the internal tissues of plants are known to improve plant growth by a wide variety of mechanisms. This study envisages the isolation and evaluation of plant growth promoting attributes of bacterial endophytes in perennial fern Ophioglossum reticulatum L. A total of 20 phenotypically distinguishable bacterial endophytes were isolated from surface sterilized leaf lamina, petiole, rhizome and spike of O. reticulatum L. The Shannon-Weaver diversity index showed that the rhizome (1.54) harbor more diverse types of endophytic bacteria than in its petiole, leaf lamina and spike. The isolated endophytes were characterized on the basis of micromorphological and physio-biochemical characters and tentatively assigned to the genus Bacillus, Pseudomonas and Staphylococcus. The isolates showed distinct variations in their enzymatic activities, sugar fermentation and antibiotic sensitivity profile. A number of endophytic isolates showed plant growth promoting activities like production of indole-3-acetic acid (IAA) and siderophore, growth in nitrogen-free medium and solubilization of phosphate. Time course of growth and IAA production by the potent isolate Bacillus OPR 7 have been determined. Exploitation of such plant growth promoting endophytes appears to be one of the best options in increasing biomass yield and improving plant fitness and productivity.


Introduction
All plants in nature harbor a diverse community of endophytic bacteria that colonize the internal tissues of the plant without imposing any negative impact on their host [1]. They have been isolated from roots, leaves, stems, flowers, fruits, and seeds from various plants [2] and found to play a pivotal role in plant growth enhancement. Production of phytohormones, solubilization of inorganic phosphate, sequestration of iron by siderophore, nitrogen fixation, etc. are the different ways by which endophytic bacteria stimulate plant growth [3,4]. Such endophytic bacteria with plant growth promoting characters have been reported from different plants [5,6]. These include species of Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Alcaligenes, Arthrobacter, Burkholderia, Bacillus and Serratia [7]. Enterobacter spp. like E. sakazakii and E. agglomerans from soybean [8]; E. cloacae from citrus and maize [9,10] and E. asburiae from sweet potato [11] have been reported to possess multiple plant growth promoting activities. Planococcus sp., Micrococcus sp., Bacillus sp., Methylococcus sp., Acinetobacter sp. and Acetobacter sp. endophytic to Rosa damascena trigintipeta were found to produce indole-3-acetic acid (IAA), solubilize calcium phosphate and produce siderophore [12].
In recent years, much attention has been focused on the natural methods of crop production for moving towards agriculturally and environmentally sustainable development. The use of bacterial endophytes as bio-fertilizers for improving crop production is gaining importance among agronomists and environmentalists as they would significantly reduce chemical input into the environment [13,14]. The bacterial strain, Bacillus sp. SLS18 has been found to promote the biomass production of sweet sorghum [14], while the growth of poplar tree was increased by 60% after inoculation with different endophytic strains [15]. Both mycorrhizal fungi and bacterial endophyte have also been shown to enhance biomass production in switch grass [16,17].
Ophioglossum reticulatum L. (Ophioglossaceae) is a small terrestrial erect fern, pantropical in distribution and is differentiated into a sub-terranean rhizome with roots and a single leaf bearing a simple, stalked, cylindrical sporangiferous spike with two rows of embedded sporangia. Out of the 40 species so far known, in India it is represented only by 12 species. However, unsustainable utilization is causing serious threat to the survival of a number of species. From economic view points young leaves are commonly used as salad or vegetable. Similarly, decoction of leaves and rhizomes are also used topically on boils, wounds and as an anti-inflammatory medicine. Ophioglossum spp. have been reported to be colonized by various species of vesicular arbuscular mycorrhizal (VAM) fungi, like Endogone microcarpa, Enterophospora sp., Gigaspora sp., Glomus epigaeum, G. macrocarpum and G. occulatum [18]. Such mycorrhizal association has been shown to improve plant health, disease resistance and drought tolerance. However, bacteria endophytic to O. reticulatum L. with plant growth promoting potential have not yet been reported. The aim of the present study was to isolate the endophytic bacteria from the surface sterilized leaf lamina, petiole, rhizome and spike of O. reticulatum L., characterize them to determine their taxonomic identity and to determine and evaluate their plant growth promoting activities.

Collection of plant materials
Ophioglossum reticulatum L. (family Ophioglossaceae) plants with healthy leaves and mature spike were collected from Darjeeling hills, West Bengal (277' N and 882' E, 6710' above sea level) during August-September, 2015-2016. Plants along with soil were collected in polythene bags, brought to the laboratory and stored at 4 °C until used for the isolation of bacterial endophytes.

Isolation and characterization of endophytes
Bacterial endophytes were isolated from the leaf lamina, petiole, rhizome and spike of healthy O. reticulatum L. The collected plant parts were first washed thoroughly under running tap water and transferred to sterile glass bottles for surface sterilization. The samples were sterilized by consecutive immersion in 70% ethanol (2-3 min), 0.5% sodium hypochlorite (5-10 min) and again with 70% ethanol for 30 sec. After washing for several times in sterile distilled water, the samples were cut into 2 mm sections and plated aseptically on previously prepared tryptic soy agar, glycerol asparagine agar and R2A agar plates for isolation of bacteria. The plates were then incubated at 32 °C for 2-4 days and observed for growth of the bacterial colonies surrounding the leaf lamina, petiole, rhizome and spike sections. Morphologically distinguishable bacterial colonies growing out of the plant segments were isolated in pure form by dilution streaking and maintained by regular sub-culturing on the same media. Bacterial strains were characterized and identified following micromorphological and physio-biochemical analysis following standard protocols.

Diversity of endophytes
Based on the total number of samples plated and the number of samples yielding isolates, the colonization frequency and the isolation rate were calculated. Colonization frequency of the bacterial endophytes was calculated as the total number of plant segments yielding the bacteria divided by the total number of segments incubated. Isolation rate was determined as the number of bacterial isolates obtained from the plant samples divided by the total number of samples incubated. The Shannon-Weaver diversity index was calculated as H = -Σ Pi ln Pi, where Pi is the species abundance.

Indole-3-acetic acid production
The ability of the endophytes to produce indole-3-acetic acid (IAA) was determined following Salkowski colorimetric assay. Isolates were grown in tryptophan broth at 32 °C for 5 days and the culture filtrate was separated by centrifugation at 10,000 × g for 10 min. To 1 ml of the culture filtrate, 3 ml of Salkowski's reagent and 2 ml of distilled water was added. After an incubation of 30 min in dark, the tubes were observed for the development of pink colour. The OD was measured at 540 nm using a Systronics Photoelectric Colorimeter 112 and the quantity of the IAA produced was estimated from the standard curve prepared in the same way with authentic IAA from Sigma (USA).

Phosphate solubilization
Ability of the bacterial endophytes for solubilizing insoluble phosphate was determined on Pikovskaya's medium supplemented with calcium triphosphate. The isolates were inoculated onto Pikovskaya agar and incubated for 5-7 days at 32 °C. The presence of halo zone around the bacterial colony was considered as an indicator for positive mineral phosphate solubilization. Solubilization index was calculated according to the ratio of the halo diameter to the colony diameter.

Growth in nitrogen-free medium
Overnight grown bacterial endophytes were washed thoroughly in sterile normal saline and inoculated in Norris nitrogen-free medium. The ability of the isolates to fix atmospheric nitrogen was indicated by their growth in N 2 -free medium.

Siderophore production
Production of siderophore by the endophytic bacterial isolates was tested qualitatively using chrome azurol S (CAS) agar following the protocol of Alexander and Zuberer [19]. The CAS agar, a mixture of four solutions, Solution 1 (Fe-CAS indicator solution), Solution 2 (PIPES buffer), Solution 3 (glucose, mannitol and trace elements) and Solution 4 (casamino acid) was prepared and sterilized separately before mixing. This mixture (Fe-CAS dye complex) yielded blue to dark green colour. The bacterial isolates were grown on it at 32 °C for 96 h. Orange halos around the colonies indicated siderophore production.

Diversity of bacterial endophytes
Surface sterilized segments of leaf lamina, petiole, rhizome and spike of Ophioglossum reticulatum L. incubated on tryptic soy agar, glycerol asparagine agar and R2A agar plates showed growth of morphologically distinguishable bacterial colonies surrounding the segments after 48-96 h of incubation at 32 °C ( Figure 1). Avoiding the repetitive strains, a total of 20 phenotypically distinguishable bacterial endophytes were isolated in pure form from 497 segments (202 leaf lamina, 60 petiole, 179 rhizome and 56 spike) of O. reticulatum L. Out of these 20 isolates, maximum (8) were derived from the rhizome and was followed by the leaf lamina (5), petiole (4), and the spike (3) tissues ( Table 1). The colonization frequency was recorded to be low in petiole (35%) and spike (58.92%) samples as compared to leaf lamina (61.38%) and rhizome (87.7%). The isolation rate was poor in leaf lamina (0.02) but increased gradually in rhizome (0.04), spike (0.05) and petiole (0.06) samples. The Shannon-Weaver diversity index showed that the rhizome (1.54) of O. reticulatum L. harbor more diverse types of endophytic bacteria than in its petiole (1.05), leaf lamina (1.01) and spike (0.98).

Characterization and identification of the endophytic isolates
The bacterial endophytes of O. reticulatum L. were primarily characterized on the basis of micromorphological (Table 2) and physio-biochemical (Table 3) characters. Out of the 20 isolates 13 were Gram-positive (12 rods and a coccus) and 7 were Gram-negative rods. Almost all the isolates showed motility except OPL 4, OPP 4 and OPS 1. Endospore formation was observed in all the Gram-positive rods. Enzyme profile of the endophytes showed that while all the bacteria produced catalase, 95% of them produced caseinase and 85% produced gelatinase. Production of amylase (60%), inulinase (65%), PHB depolymerase (55%), lipase, CM cellulase and nitrate reductase (50%) were not uncommon. Glucose was utilized by all the isolates, while majority of the isolates could ferment glucose, sucrose and fructose ( Table 4).

Antibiotic sensitivity profile
Antibiotic sensitivity pattern of the endophytic bacterial isolates was determined by discdiffusion method against six different antibiotics (penicillin G, streptomycin, sulphatriad, tetracycline, ampicillin and chloramphenicol). The bacterial endophytes from leaf lamina, petiole, rhizome and spike tissues of O. reticulatum L. were all sensitive to chloramphenicol. One rhizome endophyte, OPR 2 was sensitive to all 6 antibiotics tested. Most of the isolates were also sensitive to tetracycline and resistant to ampicillin and penicillin G ( Table 5).

Evaluation of plant growth promoting traits
When grown in tryptophan broth, the endophytic bacterial isolates showed the production of IAA as revealed by the development of pink colour on treatment with Salkowski's reagent (Table 6). Among the 20 isolates, 11 showed IAA production. The concentration of IAA ranged between 5 µg/ml to 39.79 µg/ml. Isolate OPR 7 was the best producer (39.79 µg/ml) followed by OPR 6 (10 µg/ml).  The ability of the endophytic bacterial isolates to solubilize insoluble phosphate was detected in 9 isolates as revealed by the formation of clear zone surrounding the growth of the isolates on Pikovskaya's medium. Isolates OPR 7 and OPS 3 showed comparatively higher phosphate solubilizing index (Figure 2). Out of the 20 isolates inoculated in Norris N 2 -free glucose medium, majority of them could grow in the absence of nitrogen in the medium except OPL 1, OPL 3, OPL 5, OPR 3 and OPS 1. Qualitative siderophore assay showed that the isolates OPL 3 and OPR 4 were good producers of siderophore whereas OPL 4, OPP 1, OPR 1, OPR 7 and OPR 8 were moderate producers (Table 7).

Time course of growth and production of IAA
Isolate Bacillus OPR 7, the best IAA producing isolate was chosen for time course study of growth and IAA production in tryptophan broth under batch culture. Production of IAA was initiated in the exponential phase of growth and reached its maximum after 96 h of growth during which it produced nearly 40 µg/ml of IAA (Figure 3).

Discussion
It has been seen in recent years that plant growth promoting endophytes have the ability to colonize the interior tissues of the host plant and further build a beneficial symbiotic association with their host plants to improve host plant growth [21,22]. In this study we isolated a total of 20 morphologically distinguishable endophytic bacteria from different parts of O. reticulatum L. (Figure 1).The colonization frequency was recorded to be highest in the rhizome and the Shannon-Weaver diversity index also indicated the presence of most diverse types of endophytic bacteria in the rhizome of O. reticulatum L. (Table 1). The endophytes so far isolated and characterized were tentatively assigned to the genera Bacillus, Pseudomonas and Staphylococcus (Table 2). Although during the present study, the identity of the bacterial endophytes could not be determined at the species level, which requires an indepth 16S rRNA sequence analysis. Bacillus sp. has been reported as an endophyte of Polygonum cuspidatum [23], tomato [24] and Aquilaria sp. [25]. Similarly, Bacillus sp. and Pseudomonas sp. have been extensively reported to colonize the roots of many crop plants [26] and found to induce the growth in green gram plants [27]. Species of Staphylococcus as endophytes are also not uncommon [28].
Hydrolytic enzymes of endophytes in general appear to be important for the colonization of plant roots [29,30]. Bacterial endophytes of O. reticulatum L. in this study were found to produce a variety of hydrolytic enzymes such as catalase, caseinase, gelatinase, PHB depolymerase, amylase and inulinase (Table 3). The presence of nitrate reductase in some of the isolates suggests that they play a role in the nitrogen cycle, thereby having agricultural and environmental implications.
Majority of the isolates could also ferment glucose, sucrose and fructose and were resistant to cell wall inhibiting antibiotics ampicillin and penicillin G (Table 4 and 5). The isolates also possessed multiple plant growth promoting traits such as IAA production, phosphate solubilization, growth in N 2 -free medium and production of siderophore. More than 50% of the endophytic isolates showed IAA production (in the presence of tryptophan) with isolate Bacillus OPR 7 being the best producer (Table 6). IAA is reported to increase root size and spreading, resulting in greater nutrient absorption from the soil [31]. Reports of IAA production by plant associated bacteria are not uncommon. Pseudomonas stutzeri isolated from Echinacea sp. produces 18.8 μg/ml of IAA [32], Methylobacterium sp. from red and white clover produces 6-13.3 μg/ml IAA [33] and Bacillus thuringiensis produces 1.53-9.71 μg/ml IAA [34].
It is known that improved phosphorous nutrition enhances the overall growth of the plants and help in root development [35]. Nearly 50% of isolates exhibited the phosphate solubilizing activity by forming clear zones (Figure 2). Bacillus sp., Pseudomonas sp., Serratia sp. and Enterobacter sp. are reported to solubilize the insoluble phosphate compounds and assist in plant growth [36,37]. Siderophore production was recorded in 40% of the isolates (Table 7), which are likely to play an important role in the acquisition of nutrients such as iron availability to the plant [38]. Majority of the isolates (15 out of 20 isolates) were able to grow in N 2 -free medium ( Table 7) indicating their ability to fix atmospheric nitrogen. For time course study of growth and IAA production the isolate Bacillus OPR 7 was chosen and the maximum production was observed after 4 days of incubation ( Figure 3). Experiments related to exploration of more bacterial traits for growth promotion are required to further strengthen the findings and application in plant growth and development in a sustainable manner.

Conclusion
Endophytic bacterial isolates was found to be associated with leaf lamina, petiole, spike and rhizome of Ophioglossum reticulatum L. The endophytes produced several hydrolytic enzymes of commercial importance and also possessed plant growth promoting traits. To our knowledge this is the first report addressing the exploration of the diversity of the endophytic bacteria from O. reticulatum L. and their evaluation of plant growth promoting traits. These potent endophytic bacteria, either singly or in combination could be developed as an eco-friendly biofertilizer for growth and development of many important plant species including O. reticulatum L.