Isolation and Identification of Plant Growth Promoting Rhizobacteria from Sago Palm (Metroxylon sagu, Rottb.)

Plant growth promoting rhizobacteria (PGPR) are strains of naturally occurring soil bacteria that live in close vicinity to the plant’s rhizosphere region which possess the capability to augment host growth. This study was conducted to isolate and identify potential PGPR isolates indigenous to Metroxylon sagu, Rottb. rhizosphere. These potential isolates were characterised based on their beneficial plant growth promoting (PGP) properties and identified by molecular analysis via 16S rDNA sequencing. A total of 18 isolates were successfully isolated, out of which five isolates were tested, and designated as (S1A, S2B, S3A, S3C and S42). Among the five isolates, two isolates (S2B and S3C) were found to produce high levels of indole-3-acetic acid (2.96 μg/mL and 10.31 μg/mL), able to fix nitrogen and show significant activity in phosphate solubilisation. The analysis of their sequences via National Center for Biotechnology Information (NCBI) suggested their close identity towards Lysinibacillus sphaericus and Bacillus thuringiensis. It can be concluded that the isolated PGPR possesses beneficial PGP attributes. It can be implied that the isolated PGPR are potential to be used as inoculant biofertilisers, beneficial for Metroxylon sagu, Rottb. growth. Hence, further studies need to be done to evaluate the effectiveness of the beneficial microbes towards sago seedlings growth, under pot experiment.


INTRODUCTION
Sago palm (Metroxylon sagu, Rottb.) is one of typical underutilised indigenous food crops found in Asia and Pacific Region (Konuma et al. 2012). M. sagu is most productive plant in comparison with other carbohydrate-producing crops (Bintoro et al. 2018). Sago industry in Malaysia, specifically in Sarawak is well established and becoming one of reliable industry contributing to export revenue. Ming et al. (2018) stated that Malaysia is the third main sago producer in the world, after Indonesia and Papua New Guinea. In Malaysia, the sago-planting area concentrated in Sarawak, covered approximately 19,702 ha, cultivated in wild and semi-wild conditions (Amin et al. 2019). Despite of not being the world's sago producer, Sarawak reported to be one of the world's sole sago exporter. Annually, the state has exported more than 40,000 tons to different countries, including to Peninsular Malaysia, Singapore, Taiwan and Japan (Amin et al. 2019). This number is expected to rise as there is an increment in starch production as well as in export value, at 15% to 20% every year (Amin et al. 2019).
Despite all the positive attributes brought by M. sagu, there is rising concerns, whereby upon high biomass production of M. sagu, the soil nutrients might deplete over the years. Thus, in order to promote productivity, an emergence in wide application of chemical inputs such as fertilisers, herbicides, fungicides and insecticides, was observed in agriculture (Gou et al. 2020).
Nevertheless, extended use of these products has often brought adverse effects on the complex system of biogeochemical cycles of the soil system (Steinsham et al. 2004). This was supported by Gou et al. (2020), at which, though the enormous application of chemical fertilisers benefited countries in providing large amounts of foods, the extended use however has damaged the ecosystem, human health as well as living environments. Moreover, apart generates negative environmental effects, the surplus use of these chemical inputs is costly (Bhargava et al. 2017;Chandini et al. 2019). Hence, the challenge is to promote more environmental-friendly agricultural approach for sustainable agriculture.
In regard of this sense, a wide number of studies have been and still being conducted, focusing on plant growth promoting rhizobacteria as a potential bioinoculant of fertilisers, herbicides and fungicides (Adesemoye et al. 2008). Shaikh et al. (2016), in the study also stated a continuous research in searching for eco-friendly management of plant disease along with promotion of plant growth is conducted. Besides, according to Adedeji et al. (2020), a strategy without having long term consequences towards the ecosystem, for a sustainable and robust crop production, could be focusing on the utilisation of earth microbe. Such an integrated approach will help in sustaining soil health and productivity and thus there is a need to advocate the biofertiliser approach among the farming community.
As described by Backer et al. (2018), microbes assist in plant nutrient acquisition act via various mechanisms included nitrogen fixation, phosphate solubilisation, as well as production of siderophore and hydrogen cyanide (HCN). Thus, manipulating these microbial activities has a great potential in providing crop's nutritional requirements. Therefore, the major emphasis is being stressed on the exploitation of the biodiversity of plant growth promoting rhizobacteria (PGPR) such as nitrogen fixers and as phosphate solubilisers (Bagyaraj & Aparna 2009).
PGPR which also termed as plant health promoting rhizobacteria (PHPR) or nodule promoting rhizobacteria (NPR) are associated with rhizosphere, an important soil ecological environment for plant-microbe interactions (Hayat et al. 2010). Tang et al. (2020) also described PGPR possess beneficial traits or cross functional abilities which essential in improving plant's growth. It capable of promoting the plant growth through multiple mechanisms of actions; either directly, by production of substances which promote plant growth and increase the nutrient availability, or indirectly, by suppressing the plant pathogens in the rhizosphere (Ribeiro & Cardoso 2012;Glick 1995). These PGPR traits, hence, were expected to be a helpful in producing bioinoculant of biofertiliser.
Although the studies related to PGPR are relatively advanced in agricultural systems, research on these bacterial groups in Palmae crops such as sago palm (M. sagu, Rottb.) still requires much further study. Hence, the objective of this study is to isolate and identify the native PGPR of sago palm for the contribution of beneficial information for potential bioinoculant of biofertiliser development.

Isolation of Potential Plant Growth Promoting Rhizobacteria
The isolation of potential diazotrophic bacteria was done by using serial dilution spread plate (Deora et al. 2006;Islama et al. 2007) on Nutrient Agar (Merck Millipore, UK). Approximately 10 g of soil samples were transferred to 250 mL conical flask containing 90 mL of sterile phosphate buffer saline solution (Amresco, USA) and were shaken by orbital shaker (New Brunswick Scientific, USA) at 120 rpm for 30 min. The homogenates samples were diluted up to 10 −3 dilution on nutrient agar and incubated at 28 ± 2°C for 24 h. The population of bacteria was expressed as colony forming units (CFU) and single colony of each isolates, then were subculture several times on the same agar media to obtain pure PGPR bacterial cultures.

Nitrogen fixation
The nitrogenase activity of the isolates was determined by the growth on nitrogenfree medium, the Burks Agar (HiMedia, India) according to Burk (1930). Pure bacterial colonies were streaked on the Burks Agar and incubated for 5-7 days at 28 ± 2°C. The appearance of the bacterial colonies indicated a positive test.

Phosphate solubilisation
Screening of phosphate solubilising activity by all isolates was done on Pikovskaya agar, PVK (HiMedia, India), containing 0.5% Ca 3 (PO 4 ) 2 as P source (Pikovskaya 1948). About 30 mL of optimum grown bacteria culture (OD 0.08-1.0 at 600 nm wavelength) was inoculated on double layered sterilised filter paper. The filter paper then was placed on the PVK. The inoculated PVK plates were incubated at 28 ± 2°C for 7 days. Only isolates surrounded by clear halos were considered as phosphate solubilisers. Their phosphate solubilising efficiency and solubilising index was calculated using following formula: where, Z = Solubilisation zone (cm) C = Colony diameter (cm)

Production of Indole Acetic Acid (IAA)
Indole acetic acid production was quantitatively measured according to Gordon and Weber (1951). Bacterial cultures were grown in test tubes, each containing 5 mL nutrient broth (Merck Millipore, UK) amended with 0.1% (w/v) tryptophan, incubated at 28 ± 2°C for 24 h. Nutrient broth was used instead of yeast extract mannitol medium (YEM) as described in Gordon and Weber (1951) method. Then, the cultures medium was centrifuged at 10,000 rpm for 10 min. The total of 1 mL supernatant was mixed with 2 mL of Salkowski reagent. Tubes were incubated in dark at room temperature for 25 min. The development of pink colour indicates high production of IAA and the intensity of pink colour was read at 530 nm wavelength.
The concentration of IAA produced, then, was extrapolated from the standard curve (Gordon & Weber 1951).

Identification of Bacteria Using 16s rRNA Gene Partial Sequences
Isolates DNA was extracted by boil cell method as described by Freschi et al. (2005). The 16s rRNA gene amplification was performed by using universal primer 356F (5′ ACWCCTACGGGWGGCWGC) and 1064R (5′ AYCTCACGR CACGAGCTGAC). The PCR amplifications were performed with 50 µL PCR Reaction Master Mix containing 10 μL of 5× Green GoTaq ® Flexi buffer, 6 μL of 25 mM MgCl 2 , 3 μL of 10 mM dNTPs, 8 μL of sterile distilled water, 20 μL sample of 20-40 ng DNA, 1 μL of GoTaq® Flexi DNA polymerase, 1 μL of each forward primer and reverse primer. This PCR amplification then performed by thermal cycler (SensQuest, Germany) at an initial denaturation temperature 95°C for 5 min, 35 cycles of; denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 160 s followed by a final extension at 72°C for 5 min (Winsley et al. 2012). Then, each reaction was analysed by agarose gel electrophoresis in 1.0% (w/v) agarose gel and ethidium bromide (Promega, USA) staining. The electrophoresis conditions were done at 90 V with 200 mA for 30 min before visualised under UV transilluminator (Maestrogen, TW). The purification of DNA was performed using QIAquick® Gel Extraction Kit (Qiagen, Germany) following the QIAquick Gel Extraction Kit Protocol (Kathleen et al. 2014). The purified DNA fragment, then were sent to 1st Base Laboratory Sdn. Bhd. Malaysia for sequencing purposes. The sequences obtained, then were subjected to Basic Local Alignment Search Tool (BLAST) analysis and nucleotide sequence similarities were determined with the aid of National Center for Biotechnology Information (NCBI) database.

RESULTS
A total of 17 isolates were successfully isolated from the rhizosphere soils of M. sagu, Rottb. of Dalat and Kuching area (Table 1). As shown in Table 1, the highest number of potential PGPR isolates was located at Sungai Ugui, Dalat, followed by Sago Research Plot, Kuching, Sungai Nunau, Dalat and Intermediate of Sungai Taap and Sungai Petah (Dalat) with a total number of 7, 5, 3 and 2 isolates, respectively. Results also showed that the sampling sites with wild sago palm history namely Sungai Ugui, Dalat, have higher numbers of isolates from total of isolates (41%) compared to new sago palm cultivation sites at Sago Research Plot, Kuching, Sarawak (29%).

Morphological Characterisation of Bacterial Isolates
These five isolates were subjected to morphological and biochemical characterisation prior to in vitro screening of PGP traits (Table 2).  Table 2, all isolates were Gram-positive, whereby they appeared in purple colour with either bacilli or cocci characteristic. The morphological of the bacterial isolates were also determined, at which all isolates appeared in round shape while exhibited various range of sizes and colony colours. In terms of catalase activity, all isolates were found to react positively for catalase assay.

In Vitro Screening of Bacterial Isolates for PGP Traits
About five isolates of these bacteria isolates showed positive tests for all screening tests of PGP traits (Table 3).  Table 3, a total of 5 isolates (31.3%) possessed multiple beneficial PGP traits; as the nitrogen fixer, phosphate solubiliser and IAA producer.
In terms of nitrogen fixation test, all five isolates showed positive reaction with the presence of growth of each isolate on nitrogen free media, Burks agar.
Meanwhile, the phosphate solubilising efficacy of each isolates was directly proportional to the solubilisation zone on Pikovskaya agar after incubation for 5 to 6 days at 28 ± 2°C. The five isolates were shown to response positively, at which S13 isolates solubilised the highest amount of phosphate, followed by S3A isolate, S42 isolate, S2B isolate and S3C isolate, with phosphate solubilising index value of 4.50, 2.67, 2.50, 2.33 and 2.33, respectively.

Identification of Bacteria Using 16s rRNA Gene Partial Sequences
As shown in Table 4, BLAST searches against the NCBI nucleotide database revealed the isolates close relationship to known plant-associated bacteria, genera Bacillus and Lysinibacillus.

DISCUSSION
PGPR does involve as a factor contributing to growth promotion, crop protection as well as in the improvement of M. sagu, Rottb. soil health. As rhizosphere soil live in close association with the roots, it will benefit the growth of M. sagu, Rottb, by direct secretion of plant growth hormones and plant stimulators in their vicinity. Thus, the isolation and identification of the potential PGPR from M. sagu, Rottb. grown in Dalat and Kuching area was decided. A total of 17 isolates were successfully isolated from four soil samples. However, out of all isolates, only five isolates were chosen to further the molecular characterisation, based on their ability to meet the PGPR traits. All isolates were found to be Gram-positive, which indicated by the purple stained after Gram staining and upon observation under compound light microscope. Isolate S13 and S3A which were collected from Sungai Nunau and Sungai Ugui appeared to be Gram-positive cocci, while the remaining three isolates were Gram-positive bacilli. The same research concerning the same field of interest was conducted in the Philippines by Labrador et al. (2014), reported to obtain mostly Gram-negative bacilli isolates, which differed to the isolates isolated in this study. This is might due to the great diverse of the bacteria divisions.
All isolates were found to react positively with nitrogen fixing screening test, as all isolates were found to be growing well on nitrogen free medium, the Burks Agar. The results obtained, thus, corresponded to the diazotrophic characteristic of PGPR, whereby its capability in fixing atmospheric nitrogen. The Burks Agar was designed in such a way that it is lacking in nitrogen source, but the carbohydrate source as well as inorganic salts was remained. Thus, when being introduced on the medium, these isolates were expected to be capable in utilising the atmospheric nitrogen for their cell protein synthesis purposes, which imitate the actual phenomenon undergone by PGPR. The resulted cell protein then, being mineralised into the soil once the senescence of cells occurs. Hence, it will contribute towards the nitrogen availability of the M. sagu, Rottb.
Next to their ability in nitrogen fixing, the isolates have also been found to solubilise phosphate in vitro. The isolate S13 was found to solubilise the highest amount of phosphate through the largest halo zone formation (µ ≈ 2.1 cm) on the Pikovskaya Agar. This medium was modified in such a way that the insoluble tricalcium phosphate is present, imitating the natural phenomenon faced by most of the plants. The tricalcium phosphate will be converted into soluble form once phosphate solubilising bacteria introduced into the medium. This resulted in the formation of clear halo zones. This beneficial to M. sagu, Rottb. development as the application of phosphate fertiliser could be reduced up to 50% without any significant reduction on the yield. This is so because, the rhizosphere could assimilate the soluble phosphate, which eventually preventing the adsorption and fixation in the soil, thereby, supplying the phosphates to the crops. Therefore, these phosphate-dissolving bacteria play a part in correcting phosphorus deficiency of the crop plants (Subba 1977).
IAA is one of the members of phytohormones group, considered as important auxin in plant growth promotion. Out of all isolates tested for IAA production, only two isolates, S2B and S3C were recorded to give significant higher value in phosphate solubilising index and solubilising efficacy. Earlier studies reported that, most of the IAA producing strains are Gram negative, which is differed to the findings obtained in this study (Lindow et al. 1998;Datta & Basu 2000). However, similarly, a study by Wahyudi et al. (2011) found that few Gram-positive strains belong to Bacillus strain are known to produce IAA. These are supported by Nacoon et al. (2020), whereby Bacillus genera has found to capable in producing high amount of IAA. As in this study, the isolate S3C, characterised as Gram-positive bacilli was found to be the most efficient IAA producer.
The ability of isolate S3C to use L-tryptophan supplemented into the medium is one of important factor that influence the isolates in producing IAA. This was supported by Ghosh et al. (2015), which found that bacteria preferred L-tryptophan for growth and IAA production. It also found that with an increase production of tryptophan, the production of IAA increases proportionally. Wahyudi et al. (2011) in his study also stated that tryptophan is the main precursor in IAA biosynthesis via the indole pyruvic pathway (IPA) pathway.
Upon the amplification of 16S rRNA sequences using 356F and 1064R on S3C isolates, it was identified that it belongs to Bacillus thuringiensis with 99% identity value. Previous research by Qi (2016) has similarly reported the same strain, at which Bacillus thuringiensis found to be an effective bio insecticide in tomato. In the study, it was found that seed germination and shoot elongation was observed upon treating tomato seeds with the bacterial culture filtrate and bacterial suspension, indicates B. thuringiensis as effective PGPR.
On the other hand, S2B was identified as Lysinibacillus sphaericus strain LSR1, with 99% identity value. A study on rice-associated Bacillus, by Shrestha et al. (2016) found that some strains closest to Lysinibacillus sp. found to be one of potential bio control agents. These are due to their ability to supress disease development of sheath blight and bacterial panicle blight in rice. However, in the study, it was stated as well that these closest strains of Lysinibacillus sp. did not show visible antagonistic activities, which remains to be tested in further research. Vendan et al. (2010), on the other hand, found Lysinibacillus sp. to be one of the most positive rhizobia in having plant growth promoting traits, indicates their roles in growth promotion of ginseng.

CONCLUSION
Throughout this study, it could be concluded that the plant growth promotion among the isolates were attributed to their individual competencies. The potential rhizobacteria was screened for their plant growth promoting properties and it turned out that two isolates (S3C, S2B) found to be the most effective isolates and were identified as Bacillus thuringiensis and Lysinibacillus sphaericus. The current study effort is towards aiding research on potential biofertiliser agent for economically important palm, particularly sago palm, which used as a plantation crop in Sarawak, Malaysia for starch production. Thus, to evaluate the influence brought by the most promising bacterial strains found in this study, a pot experiment on the sago seedlings should be further conducted.