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Article

Potential Novel Plant Growth Promoting Rhizobacteria for Bio-Organic Fertilizer Production in the Oil Palm (Elaeis guineensis Jacq.) in Malaysia

by
Shih Hao Tony Peng
1,2,*,
Kheng Hoy Chee
2,
Halimi Mohd Saud
3,
Mohd Rafii Yusop
4,5 and
Geok Hun Tan
1,*
1
Department Land Management, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2
All Cosmos Bio-Tech Holding Corporation, PLO650, Jalan Keluli, Pasir Gudang Industrial Estate, Pasir Gudang 81700, Johor, Malaysia
3
Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
4
Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
5
Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2023, 13(12), 7105; https://doi.org/10.3390/app13127105
Submission received: 10 May 2023 / Revised: 11 June 2023 / Accepted: 12 June 2023 / Published: 14 June 2023
(This article belongs to the Special Issue Agroecosystems: From Soil Environment to Plant Nutrition)
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Versions Notes

Abstract

:
The present study aimed to characterize the potential plant growth-promoting rhizobacteria (PGPR) based on biochemical tests based on eight bacterial isolates, and to identify potential PGPR based on the 16S rRNA sequencing molecular method. Eight potential PGPR strains (UPMC1166, UPMC1168, UPMC1254, UPMC1376, UPMC1389, UPMC1393, UPMC703 and UPMC704) isolated from the soils in the oil palm (Elaeis guineensis) estates across Malaysia were selected because of their most PGPR activities. They were screened for nitrogen fixation, phosphate and potassium solubilization, and production of indole-3-acetic acid (IAA). All isolates showed the ability to grow between pH 2 to 9 and survive from 2 to 15% (w/v) of the salt medium. Among the isolated PGPRs, four PGPRs (UPMC1166, UPMC1168, UPMC1254 and UPMC1389) showed the ability to fix nitrogen and had the potential to produce IAA. Furthermore, two PGPRs (UPMC1393 and UPMC1376) demonstrated the ability to solubilize phosphate, while three PGPRs (UPMC703, UPMC704, and UPMC1393) showed the ability to solubilize potassium. Therefore, all the above eight PGPR isolates can benefit the oil palm cultivation industry. The molecular identification based on 16S rRNA gene sequence revealed that UPMC1166 was identified as Bacillus methylotrophicus; UPMC1168 as B. siamensis; UPMC1254 as B. subtilis; UPMC1389 as B. albus; UPMC1376 as Lactobacillus plantarum; UPMC1393 as B. marisflavi; UPMC703 as Burkhoderiaanthina and UPMC704 as B. metallica. These novel strains can be further investigated for their viability and effectiveness for bio-organic fertilizer production and application in the immature stage of oil palm growth.

1. Introduction

Today, oil palm (Elaeis guineensis Jacq.) has become a critical commercial crop in Malaysia [1]. Malaysia accounted for 25.8% and 34.3% of the world’s palm oil output and exports in 2020, respectively, according to the Malaysian Palm Oil Council [2]. Nonetheless, it is hypothesized that Malaysia’s oil palm output might be impacted by climate change [3]. About one-third (75.17 million tonnes) of the world’s total oils and fats produced in 2017 were from 19.04 million hectares of palm oil, mostly contributed by Malaysia and Indonesia [1]. From 2020 to 2021, the output of palm oil reached around 72.27 million metric tonnes, with the Southeast Asian countries (Indonesia and Malaysia) as the major global exporters of palm oil [4].
Basal stem rot incidence in oil palms was reported to significantly decrease due to the chemical treatment such as fungicides [5]. Yet, because they inhibit the formation of beneficial microbes, these biocidal chemical compound fungicides are not eco-friendly and can be highly toxic to the plantation ecosystem [6]. Concerns about the detrimental effects of chemical fertilizers have prompted new studies, such as biofertilizers, as free-living nitrogen-fixing bacteria into ecological crop management strategies have been carried out [7]. Therefore, the alternative fertilizer, such as a combination with bio-based, will be more useful in the long run [1].
Due to the high nutritional needs and high expense of chemical fertilizers for oil palm trees, producers have been looking for lower-priced options that allow for more efficient nutrient utilization. As a result, agricultural experts are looking at biofertilizers, which are cost-effective [8,9,10]. Biofertilizers are fertilizers that contain living microorganisms that boost soil microbial activity. They are renewable low-cost nutrient sources that can substitute for chemical fertilizers in an eco-friendly manner [11,12]. Biofertilizers are plant-nourishing organic fertilizers that are simple to make and can be applied feasibly in the oil palm plantation area [13]. Compared to the control tea plants. Pendey et al. [14] found inoculants of Pseudomonas corrugate and Bacillus subtilis could increase host plant survival by 88% and 100%, respectively.
The inoculation of chosen diazotrophic rhizobacteria into the tissues of oil palm during in vitro micropropagation would allow for premature relationships between the host and the bacteria. These positive relationships would allow the host plants to better adjust to environmental conditions/stresses so as to have a better survivorship [15,16]. Furthermore, because the diazotroph can fix nitrogen in situ, the bacterized plants require very little nitrogen fertilizer [16,17]. Finally, diazotrophs generate plant growth hormones that will help in roots’ development and the overall plants’ growth [18]). Therefore, the plant growth-promoting rhizobacteria (PGPR) are the important and agronomically helpful soil microbiota [19] that would be colonizing in the plant’s roots and consequently stimulate the plant’s growth [20]. The PGPR (in the rhizosphere of plants) would promote their hosts’ growth and development of the plants. The root system acts as a massive anchor for nutrient and water intake and a chemical factory for beneficial microorganisms, rhizobia, mycorrhizae, endophytes, and PGPRs, as well as parasitic connections with other plants [21]. The PGPR can decrease the requirement for inorganic fertilizer, particularly for nitrogen and phosphorus, and increasing the nutrient release needed by oil palm [22]. The use of PGPR added in the biofertilizers for oil palm growths has been reported in the literature [23,24,25,26,27].
The PGPRs, colonizing inside the root surface, would express plant-growth-promoting and protective functions, and stimulate plant development [28]. PGPRs are also directly associated with N2 fixation, growth hormones, phosphate solubilization, and siderophore formation [29]. They can also eliminate phototoxic chemicals produced by harmful microbes [30]. N2-fixing and P-solubilizing bacteria are critical in plant nutrition because they increase P absorption [31]. PGPR can also aid plant growth by delivering soluble phosphate, which is transformed from insoluble mineral phosphates by acidification, or by mobilizing other essential nutrients [32,33]. As a result, PGPRs may be employed to improve the nutrient availability, plant growth, and yields of the oil palm [34]. The use of Hendersonia PGPR had been proven effective against Ganoderma disease in the oil palm seedlings under a field-based study [35]. The early linkages would aid seedling adaptability to environmental conditions/stresses and increase survivorship [15,16]. Previous research has shown that PGPR inoculation at an early stage of plant growth can clearly improve the survivorship of the host plants [14].
Kloepper et al. [36] also discovered the ability of PGPR to promote plant growth as well as protect plants from pathogens. Many phosphate-solubilizing bacteria, such Pseudomonas [37], Aspergillus and Penicillium [38], and Burkholderia [39], have been reported as PGPR biofertilizers in the oil palm cultivation for promoting the oil palm growth. Consequently, a sustainable yield could be achieved. P-solubilizing biofertilizers have been proven cost-effective and environmentally friendly in sustainable agricultural production [40]. PGPR holds the greatest agricultural potential since it provides an appealing alternative to conventional chemical fertilizers [19]. The quest for PGPR and research into their mechanisms of action is accelerating as efforts are made to commercialize them as biofertilizers [41]. Azri et al. [42] investigated the plant–microbe interaction between the oil palm and Bacillus salmalaya strain 139SI at the seedling stage of the oil palm.
This study focused on converting oil palm plantations from the use of pure chemical fertilizer to bio-chemical fertilizer. These contains certain beneficial microbes or PGPRs that can promote and maintain the soil’s natural oil palm ecosystem. Thus, this study aims to characterize potential PGPRs based on biochemical tests of eight bacterial isolates, and identify these potential PGPRs based on 16S rRNA sequencing.

2. Materials and Methods

2.1. Sources of PGPR

From the previous studies by Tan and Nazaruddin [43] and Mansor et al. [44], potential PGPRs were reactivated and revived for bio-chemical fertilizer production. These bacterial isolates were freeze-dried for long-term preservation. These eight PGPRs were isolated from the soils in the oil palm estates across Malaysia. They included UPMC1166, UPMC1168, UPMC1254, UPMC1389, UPMC1376, UPMC1393, UPMC703 and UPMC704. They were chosen in this study due to some functionality, potential of being PGPR, and being aerobic bacterial types [43,44]. All of these potential PGPRs can grow well under growth temperature conditions between 35 and 37 °C except for UPMC1389 (30–35 °C) [43,44]. They were collected from the culture collection site at Microbial Culture Collection Universiti Putra Malaysia (UPM). Certain biochemical tests were carried out to test the PGPR abilities.

2.2. Morphological and Biochemical Characterization of PGPR

Bacterial isolates from the stock cultures in the nutrient agar slant were inoculated into the nutrient broth (NB) The nutrient broth consisted of 13 g of nutrient broth powder (beef extract 1.0 g; peptone 5.0 g; yeast extract 2.0 g; sodium chloride 5.0 g) in 1 L of distilled water, with a final pH 6.8 ± 0.2 at 25 °C. Later, they were incubated for 20 h at temperatures between 35 and 37 °C. A dilution series of the broth inoculum was distributed onto nutrient agar plates and incubated at temperatures between 35–37 °C for 1 to 3 days. The nutrient agar consisted of 28 g of nutrient agar powder (peptone 5.0 g; beef extract 3.0 g; sodium chloride 8.0 g; agar 15.0 g) in 1 L of distilled water. The colony morphology was observed using a 20× magnifier lens, and colony size, elevation, margin, and form were recorded and compared.

2.2.1. Gram Stain

The Gram stain method (Merck, Germany) was performed based on the instructions provided by the manufacturer to differentiate Gram-negative and Gram-positive PGPRs. Isolates were cultured on nutrient agar (Merck, Germany) and incubated for 72 h. Each isolate was observed for its colony characteristics and was Gram-stained every 24 h of incubation. The Gram-stained slides were examined under a light microscope (Leica, Germany).

2.2.2. Catalase Test

A catalase test was performed, as mentioned by Reiner [45]. A small quantity of culture was collected from a 24-h-old colony and smeared on a microscope glass slide. Then, 40–50 µL of 3% hydrogen peroxide (H2O2) (Merck, Germany) was dropped. The test was performed using three replicates for each isolate. The formation of gas bubble revealed a positive reaction, while the absence of a bubble showed a negative response [45]. Most Bacillus species gave positive results due to the presence of catalase [46].

2.2.3. pH and Salinity

All isolates (UPMC1166, UPMC1168, UPMC1254, UPMC1389, UPMC1376, UPMC1393, UPMC703 and UPMC704) were screened to determine the ability of growth under extreme conditions such as pH and salinity. For these screening procedures, Brahmbhatt and Modi’s method [47] was used with some modifications. Three replicates for each isolate were performed.
(a)
pH
Nutrient broths were prepared with pH adjusted to pH 2, 4, 6, 7, 8, and 9. The 1N NaOH and 1N HCl were used to adjust the pH levels. Bacteria were inoculated into the broths. They were incubated at 35–37 °C for up to 48 h. Bacterial growth was checked every 24 h by observing the presence of turbidity in the broth and comparing it with the non-cultured broth. The broth was also streaked on a plate to reconfirm the bacterial presence by observing the growth of colonies.
(b)
Salinity
Nutrient agar plates were prepared with salt (NaCl) concentrations (w/v) of 2, 5, 10 and 15%. The ability of bacteria to survive under saline conditions was checked by streaking the bacterial culture onto the saline agar plates and incubating at 35–37 °C for 24–48 h. The presence of bacterial colonies was observed every 24 h.

2.2.4. Functionality Tests

For functionality tests, isolates were revived and re-cultured to form a single colony. Several tests were carried out to identify the beneficial bacteria which can produce indole acetic acids (IAA), and fix nitrogen, solubilize phosphorus and potassium.
(a)
Nitrogen fixation ability
For nitrogen fixation ability, nitrogen fixation bacteria (NFB) medium was prepared [48]. The composition (g/L) of the medium was: MgSO4·7H2O (0.20); DL-malic acids (5.00); KOH (4.00); NaCl (0.10); K2HPO4 (0.50); and CaCl2 (0.02). Another component (ml/L) included was: Trace elements solution (2.00); Fe-EDTA (4.00); 5% alcoholic solution of Bromothymol Blue (2.00); and vitamin solution (1.00). For trace element composition (mg/200 mL distilled water), they included MnSO4·H2O (235); NaMoO4 (200); ZnSO4·7H2O (24); H3BO3 (280); CuSO4·5H2O (8). For the vitamin solution, they included biotin (10 mg/100 mL distilled water) and pyridoxine (20 mg/100 mL distilled water). Finally, all these components were combined and the pH of the solutions was maintained at 6.8 with NaOH before sterilization. NFB media-containing petri dishes were divided into four segments to simulate the replications. The replications were done by each quarter of the agar plates getting a separate inoculum disc.
One loopful of bacterial stock cultures from nutrient agar slants was transferred into 10 mL of sterile nutrient broth in a universal bottle. Later, these bottles were incubated for 18 h at 35–37 °C in an incubator shaker with a controlled temperature. A sterile plaque disc from filter papers was inserted into the bacterial inoculum for 30 min before being transferred into the NFB medium plate. The plate was incubated for 24 h. The nitrogen contents were measured using micro-Kjeldahl method [48].
(b)
Phosphate solubilization ability
Pikovskaya agar medium was employed to test the ability of the bacteria to solubilize phosphorus [49]. The medium was cooked using standard compositions (g/L) as follows: (NH4)2SO4 (0.50); glucose (10.0); Ca3(PO4)2 (5.00); MgSO4·7H2O (0.10); NaCl (0.20); KCl (0.20); FeSO4·7H2O (0.002); yeast extract (0.50), MnSO4·H2O (0.02); and agar (15.0). These components were mixed and sterilized before being poured into a sterilized petri dish. Petri dishes containing Pikovskaya agar medium were segmented into four segments representing the replications. One loopful of bacterial stock cultures from nutrient agar slants was transferred into 10 mL of sterile nutrient broth in a universal bottle. The bottle was incubated for 18 h at 35–37 °C in an incubator shaker with a controlled temperature. A sterile plague disc from filter papers was inserted into the bacterial inoculum for 30 min before being transferred into the Pikovskaya agar medium plate. The plate was incubated for 24 to 168 h. The ability to solubilize phosphorus was shown by a clear halo zone formation due to the organic acid production from the bacteria that were solubilizing tricalcium phosphates in the media. The presence of a clear halo zone was determined and measured every 24 h using a caliper by deducting the colony diameter from the total diameter [49].
(c)
Potassium solubilization ability
The chosen isolates were cultured on a modified Aleksandrov agar medium in order to test the potassium solubilizing ability [50]. The composition of Aleksandrov agar medium in (g/L) was: glucose (5.00); FeCl3·6H2O (0.01); MgSO4·7H2O (0.50); CaCO3 (0.10); Ca (H2PO4)2 (2.00); agar (20.0); and potassium aluminium silicate (muscovite mica) (3.00). This component was mixed and sterilized before being poured into a sterilized petri dish. Petri dishes containing Aleksandrov agar media were segmented into four segments representing the replications. One loopful of bacterial stock cultures from nutrient agar slants was transferred into 10 mL of sterile nutrient broth in a universal bottle. Later, the bottles were incubated for 18 h at 35–37 °C in an incubator shaker with a controlled temperature. A sterile plague disc from filter papers was inserted into the bacterial inoculum for 30 min before being transferred into the Aleksandrov agar medium plate. The plates were incubated for 24 to 240 h. The formation of a clear halo zone around the colony was an indication of the ability to solubilize muscovite mica as a source of an insoluble form of potassium. The appearance of a clear halo zone was determined and measured every 24 h using a caliper by deducting the colony diameter from the total diameter.
(d)
Phytohormone production
The colorimetric approach was used to determine the production of IAA [51]. The isolates were shaken for 24 h after being cultured in nutrient broth. Along with 5.0 mL L-Tryptophan as an IAA indicator, 1.0 mL of bacterial culture was moved to a new nutrient broth. The control was nutrient broth without any bacterial isolates. A total of 1.5 mL of bacterial control was moved to a new sterile Eppendorf tube and later centrifuged for 7 min at 7000× g rpm. Afterwards, 1.00 mL supernatant from bacterial isolates was collected and they were mixed with 2 mL of Salkowasky reagent (2% of 0.5 M FeCl3 in 35% perchloric acid). These solutions were left for 25 min to see the pink color development, indicating IAA production. A spectrophotometer at 535 nm was used to determine the absorbance values for each isolate. Absorbance values were compared using the standard curve to obtain the IAA concentrations. In the present study, the standard curve was prepared using pure IAA stock with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL of IAA. The supernatants of un-inoculated test tubes were used as control.

2.3. Genomic DNA Extraction

By using the modified protocol proposed by Sambrook et al. [52], bacterial genomic DNA extraction was performed. The bacteria were cultured for 18 to 24 h at 35–37 °C. Afterwards, the culture was centrifugated, and the pellets acquired were resuspended in 0.5 mL of lysis solution 8.0 M urea and 0.5 mL of 10% (w/v) SDS (Merck, Germany), 10.0 mM Tris-HCL (Merck, Germany), and 0.3 M NaCl (Merck, Germany). The suspension was incubated at 37 °C for 20 min. The DNA was extracted by adding 2 volumes of phenol and mixed well using a vortex mixer for 10 min. The mixture was centrifugated at 9750× g (Thermo Sorvall Legend Micro, Germany) for 10 min. The liquid at the aqueous phase was aliquoted into a new tube. The same volume of chloroform and isoamyl alcohol was added. They were mixed well and later centrifugated at 9750× g for 5 min.
By adding 2.5 volumes of 100% ethanol (Merck, Germany) and 1/10 volumes of 3 M sodium acetate (pH 5.2) buffer (Merck, Germany), the aqueous phase containing genomic DNA was precipitated. The combination was incubated at −20 °C for 1 h and then centrifugated at 9750× g for 5 min. Afterwards, the pellets containing DNA were rinsed with 70% (v/v) ethanol (Merck, Germany). To dissolve the dried DNA pellet, a combination of 1.0 mM EDTA (pH 8.0) (Merck, Germany), and TE buffer 10.0 mM Tris-HCl (pH 7.5), was used. Later, the isolated DNA was stored at −20 °C.
The reaction mixture used in this study is indicated in Table 1. Polymerase Chain Reaction (PCR) was performed based on the manufacturer’s instructions (Lucigen, WI, USA) for the amplification of 16S rRNA sequence of all isolates (UPMC 1166, UPMC 1168, UPMC 1254, UPMC 1389, UPMC 1376, UPMC 1393, UPMC 703 and UPMC 704). The PCR was conducted using a thermal cycler (peqSTAR, Germany) with the thermal cycling conditions following the order of (a) pre-heat (94 °C for 2 min), (b) denaturation (35 cycles at 94 °C for 30 s), (c) annealing (52 °C for 30 s), (d) extension (72 °C for 1 min), and (e) final elongation (72 °C for 10 min). Later, the amplified 16S rRNA was kept at 4 °C before electrophoretic analysis.
Gel electrophoresis was used to analyze the amplified DNA samples. The preferred protocol of Brahmbhatt and Modi [47] was used with some modifications. One hundred mL of 1% (w/v) agarose gel was made by dissolving the agarose powder in 1 × TBE buffer and 5 µL of GelREdTM (Biotium, Fremont, CA, USA) nucleic acid gel stain was added. The solution was heated in a microwave for 2–3 min and mixed well by swirling. The agarose gel was poured into the gel tray before the comb well placement. The solution was left to solidify for 30 min. The comb was removed after the gel had solidified. The gel tray was placed in the buffer tank and immersed with 1 × TBE for a depth of 2 to 5 mm. For the sample loading process, 2 µL of Bromophenol blue loading dye (Lucigen, USA) was mixed with 5–7 µL of amplified genomic DNA and 3–5 µL of PCR products. Then, 10 µL of 1 kb DNA ladder (Lucigen, USA) was used as the molecular weight marker. Then, 75 to 80 Volts (V) of electricity was applied for the gel electrophoresis analysis. For viewing and recording purposes, the gel was then positioned on the molecular imager Gel Doc (Bio Rad, USA) system.
HiYieldTM Gel/PCR DNA Mini Kit (Real Biotech, Taiwan) was used to purify the PCR product [53]. The agarose gel with the PCR products was removed until 300 mg and was then transferred into a 1.5 mL microcentrifuge tube. Later, the DF buffer was supplemented to it. The First BASE Laboratories Sdn Bhd service was employed to analyze the purified PCR product for the sequencing analysis. By using ClustalW and BioEdit 7.2.4 software, the sequences were derived from the forward and reverse primers of PCR.
A comparison between the aligned partial 16S rRNA gene sequence with genes from the Basic Local Alignment Search Tool (BLAST) (NCBI Genbank) was performed.

2.4. Statistical Analysis

The overall statistics of the present data were obtained using IBM SPSS Statistics (Version 28.0.0.0 (190), 2021). The mean values of statistical significance were obtained using Duncan’s Multiple Range Test.

3. Results and Discussion

3.1. Morphological and Biochemical Characterization of PGPR

Based on the observation, all isolates showed creamy white pigmentation. The UPMC1166, UPMC1254 and UPMC1389 strains had irregular shapes: UPMC1168 had a filamentous shape, while the rest (UPMC1376, UPMC1393, UPMC703 and UPMC704) had a circular colony shape. Colonies of UPMC1166 showed raised elevation, undulate margin, bumpy with a shiny appearance, and stained as Gram-positive bacterium with a rod shape. UPMC1168 also showed a unique morphology, with a bumpy and shiny appearance in the middle but dull at the colony’s edge. The UPMC1168 also contained a Gram-positive bacterium with a rod shape. The colonies for UPMC1254, UPMC1389, UPMC1376, UPMC1393, UPMC703 and UPMC704 were also raised in elevation, with undulate margin, bumpy and shiny in appearance, and stained as Gram-positive rod-shaped bacterium, except for UPMC1376, UPMC1393 and UPMC704, which had a coccus shape (Table 2).
The present colony characterization for UPMC1166 was supported by Mansor et al. [44], who found that the UPMC1166 was a Gram-positive bacterium with a single or in-pair Bacilli arrangement and a rod shape after 24, 48, and 72 h of incubation at 30 °C. The colonies ranged from 3 to 5 mm in size after 24 h of incubation. After 48 h of incubation, it extended to 5 to 7 mm. During the 48 h observation period, the isolate produced endospores. Creamy white coloring, increased elevation, bumpy and shiny appearance, uneven form, translucent and sticky structure and undulate edge, were all characteristics of UPMC1166 colonies. UPMC1166 produced bubbles when H2O2 was applied, showing that it was catalase positive. The morphological properties of UPMC1166, such as Gram-positive bacteria, catalase positivity, rod-shaped cells, and the presence of one endospore in a single cell, matched those of Bacillus species [45]. The form of colonies shows a wide range of variation in within and between Bacillus species, most likely due to the medium component of another incubation environment [46]. The BLAST results revealed that UPMC1166 was intimately connected to Bacillus atrophaeus, B. subtilis, B. siamensis, B. amyloliquefaciens, B. vallismortis, and B. mojavensis, based on molecular identification using 16S rRNA Gene Sequence, because they shared pairwise sequence similarities [46].
All of the isolates were discovered to be rod shaped and tiny in size. Sugar fermentation, oxidase test, urea, TSI, and other biochemical assays were used to identify the isolate. Rahmoune et al. [33] used the L. fusiformis strain found in soils. According to Rahmoune et al. [33], a vast group of Gram-positive aerobic (facultatively anaerobic), Bacillus genus, and endospore-forming bacteria are widely distributed in the environment. Bacilli can use a variety of simple organic compounds, including sugars, amino acids, carbohydrates, and organic acids. Kumari et al. [53] identified Bacillus, Pseudomonas, and Acinetobacter based on the biochemical and morphological characteristics of the PGPR isolates.

3.2. Physiological Ability and Functionality Study

All isolates show growth ability at pH of 2 to 9 and can survive between 2 and 15% (w/v) of the salt medium (Table 3). Salinity is a severe abiotic stress that restricts crop development and productivity. The soil’s high alkalinity and exchangeable salt levels harm crop output and productivity. According to Kumar et al. [20], Bacillus sp. CL3, B. cereus CL7, and Burkholderia thailandensis CL4 could tolerate salinity up to 5% NaCl, but B. subti lis CL1, Pseudomonas fluorescens CL12, and P. putida CL9 had the highest salt tolerance (6% NaCl). Kumar et al. [20] reported that P. fluorescens, P. putida, and B. subtilis, had the highest salt tolerance, while Agrobacterium tumifaciens had the lowest (1% NaCl). Rashid et al. [54] found almost identical results for A. tumifaciens (0.50–1.00% NaCl), P. fluorescens (4% NaCl), and Bacillus sp. (3.50% NaCl), while Zhao et al. [55] found salinity tolerance in Burkholderia sp. (2–5% NaCl). Bacillus thalindensis was also found to withstand 5% NaCl [20]. Gayathri et al. [56] reported that Bacillus sp. isolated from peaty environments had a high salinity level of 10% NaCl. As a result, all of the used isolates were equivalent to and within the salinity ranges described in various bacteria species, although salinity tolerance was species-specifically dependent.
The rhizosphere is a critically important zone for microbial proliferations because of the abundance of root exudates. Most rhizobacteria observed belonged to one of three proteobacterial phyla, which colonized the Gram-negative microbial population [57].

3.2.1. Nitrogen Fixation Ability

Table 4 and Figure 1 show the positive interaction of bacteria which can fix nitrogen. Based on the result, four PGPRs (UPMC1166, UPMC1168, UPMC1254 and UPMC1389) showed a positive interaction where it turns the agar medium’s green color into blue as an indicator of the nitrogen fixation ability.
Bacillus sp., Pantoea sp., Klebsiella sp., Stenotrophomonas sp. and Burkholderia sp. were among the nitrogen-fixing bacteria (NFB) reported by Toyota [58]. When these NFB isolates were cocultured with polymer-degrading bacteria in this medium, their nitrogen-fixing activity skyrocketed. According to Toyota [58], complex microbial interactions may contribute to nitrogen nutrition in the sago palm by boosting in situ nitrogen fixation. Biological nitrogen fixation (BNF) is a natural mechanism for plants acquiring nitrogen. Replacing BNF inputs for predominantly fossil energy N-fertilizers can decrease production costs and reduce dependence on fossil energy sources [59,60].
Azospirillum brasilense, Herbaspirillum seropedicae, and A. amazonense have been found colonizing these plants [61]. These bacteria can be found in the fruit’s endosperm, roots, stems, and leaves. A novel Herbaspirillum species was most likely present in these palm trees (leaves, stems, and roots) [59]. It has been reported that oil palm plantlets which had been previously inoculated with Azospirillum brasilense and A. lipoferum could display faster root and shoot growths when compared to non-inoculated plantlets [62,63]. A variety of diazotrophic bacteria have been discovered in the rhizosphere of oil palm trees, indicating that they may benefit from BNF [64]. However, Carvalho et al. [65] reported no significant variations in the diazotrophic bacteria isolated from the roots and leaves of oil palm plants grown in the nitrogen fertilizer applied soil with that without. Another key PGPR feature that promotes plant growth indirectly is ammonia generation [66]. Malleswari and Bagyanarayana [67] and Bumunang and Babalola [68] both reported ammonia generation by all of the tested rhizobacterial isolates.

3.2.2. Phosphate Solubilization Ability

UPMC1393 and UPMC1376 showed a clearing zone as an indicator of the ability to solubilize phosphate (Figure 2). Vessey [41] found phosphate-solubilizing rhizobacteria to be one of the finest alternatives to inorganic phosphate fertilizers for improving plant productivity and development since phosphorus has been identified as one of the most critical nutrients for plant growth [69]. According to Modi and Patel [70], the best isolates as phosphate solubilizers were F372 (15 mg/L), which was associated with Saccharum officinarum, followed by ESB4 (14 mg/L). Bhardwaj et al. [71] obtained similar consistent phosphate solubilization findings in both agar and broth assays.
Three highly efficient phosphate-solubilizing bacteria, Pseudomonas fluorescens [72], Burkholderia gladioli and Penicillium aculeatum [73], have been identified as potential biofertilizers for oil palm trees. The effects of phosphate-solubilizing bacteria on commercial plant growth have also been reported by Bakhshandeh et al. [74] who investigated the ability of three phosphate-solubilizing bacteria (Rahnella aquatilis (KM977991), Pantoea ananatis (KM977993), and Enterobacter sp. (KM977992) to release potassium (K) from mica to understand their impact on rice plant growth at an early initial stage of development.

3.2.3. Potassium Solubilization Ability

UPMC703, UPMC704 and UPMC1393 showed a halo zone in the agar medium, indicating solubilization of insoluble muscovite mica phosphate (Ca(H2PO4)2) (Figure 3).
In the plants, the allocation of non-exchangeable to the exchangeable potassiums was aided by microbial activities [74]. According to Angraini et al. [75], potassium-solubilizing bacteria (B. cepacia) were a potential biofertilizer for providing K content to plants cultivated in limestone quarry reclamation areas.

3.2.4. Phytohormone Production

UPMC1166, UPMC1168, UPMC1254 and UPMC1389 have the potential to produce IAA (Table 3). These four isolates exhibited a positive interaction by the development of pink color (Figure 4).
IAA synthesis is a key plant growth-boosting feature in PGPRs and a signal molecule in plant regulations [76]. Auxin regulates various activities in tissue culture, including tropic responses to gravity and light, organ patterning, shoot architecture, general root, and vascular growth [70]. A higher auxin level weakens plant defensive mechanisms, enhancing responses of long-term (cell division) and short-term (cell elongation) in plants, and granting easier colonization in the plant cells.
According to Dar et al. [76], Bacillus cereus strains WI 41 and WI 36 produced the highest IAA (30 µg/mL), followed by M. yunnanensis WI 60 (28 µg IAA/mL). Dar et al. [76] found that numerous Paenibacillus sp. and Bacillus sp. created IAA in Luria Bertani broth, which agreed with Beneduzi et al. [77]. In ideal growth conditions, Kaur and Sharma [78] and Khin et al. [79] reported IAA production by rhizobacteria in the range of 53.1 to 71.1 ppm, but Husen [80] reported IAA synthesis in bacteria in the range of 2.09 to 33.28 µmol/mL. Shobha and Kumudini [81] reported that Bacillus isolates produced IAA in levels ranging from 35 to 217 µg/mL. Four major factors (cultural circumstances, the organism involved, substrate availability and growth stage) influence the IAA generation in different PGPR species and strains [82].
Plant-associated bacteria generate phytohormones (including IAA, gibberellins, and cytokinin) that could promote faster plant growth and have improved defensive mechanisms against abiotic and biotic stressors [83]. Modi and Patel [70] isolated and characterized the PGPRs associated with Saccharum officinarum. According to Modi and Patel [70], the PGPR isolates could stimulate plant development indirectly or indirectly. According to the data, F372 had the most phosphate solubilization strain (15 mg/L), while F271 had the highest IAA production (63 mg/L). As a result, they proposed that using specific PGPR isolates as inoculants could be advantageous to sugarcane farming.
According to Kumar et al. [20], all nine bacterial isolates produced ammonia, solubilized tri-calcium phosphate, and IAA on Pikoskaya’s nutrient agar petri plates. Except for Agrobacterium tumifaciens CL5, all strains showed resistance to salt up to 4% NaCl (1% NaCl). Phosphate solubilization, and IAA production have also been reported in A. tumifaciens, Burkholderia sp., Bacillus sp. and Pseudomonas sp. by Zhao et al. [55], Pseudomonas sp. and Burkholderia by Laslo et al. [84], and in Azotobacter chroococcum by Glick [85].
In general, rhizobacteria release IAA, which increases lengths and root surface areas. This facilitates the absorption of nutrients from the soils. Additionally, rhizobacterial IAA weakens the cell walls of the plants so as to produce a surge in root exudation, and this could provide extra nutrient levels to sustain rhizosphere bacteria development [86]. In the oil palm study, the three major nutrients (potassium, phosphorus and nitrogen) were the important elements in vegetative growth and production. However, the application of added PGPR with chemical fertilizer could improve the soil nutrient conditions and help disease control [87,88].

3.3. Identification of Potential Bacteria

Figure 5 shows the 16S rRNA PCR product separated by 1% (w/v) agarose gel. The size of the amplified 16S rRNA was approximately 1500 bp.
According to Piotrowska-Seget et al. [89], sizable areas of soils have been metal-contaminated due to the extensive use of chemical fertilizers containing essential minerals. Areas close to industrial activities typically have poor soil quality, such as low pH, adverse carbon-to-nitrogen ratios, low organic carbon content, and low essential macronutrients [87,88,90]. The use of PGPRs complemented with the chemical fertilizers could enhance the soil nutrients and control plant diseases in a more sustainable and eco-friendly way [87].

4. Conclusions

In summary, the present study revealed that UPMC1166, UPMC1168, UPMC1254 and UPMC1389 have the potential to produce IAA. All the isolates (UPMC1166, UPMC1168, UPMC1254, UPMC1389, UPMC1376, UPMC1393, UPMC703 and UPMC704) were proven to have the capability of nitrogen-fixing, phosphate and potassium solubilising, and IAA production. Most interestingly, the present study also concluded that UPMC1166 was identified as B. methylotrophicus; UPMC1168 as B. siamensis; UPMC1254 as B. subtilis; UPMC1389 as B. albus; UPMC1376 as L. plantarum; UPMC1393 as B. marisflavi; UPMC703 as B. anthina and UPMC704 as B. metallica. The potential strains identified are novel and they can be proposed in making novel bio-organic fertilizer production in combination with chemical fertilizer at the immature stage of the oil palms under field conditions. Therefore, a trial plot field experimental study is needed to confirm the effectiveness and success rate under the oil palm plantation condition.

Author Contributions

Conceptualization, S.H.T.P. and G.H.T.; methodology and validation, S.H.T.P. and G.H.T.; formal analysis, S.H.T.P. and G.H.T.; investigation, S.H.T.P.; resources, S.H.T.P.; data curation, K.H.C., H.M.S. and M.R.Y.; writing—original draft preparation, S.H.T.P.; writing—review and editing, K.H.C., H.M.S., M.R.Y. and G.H.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research received funding from All Cosmos Bio-Tech Holding Corporation.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank the reviewers for providing the constructive comments.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Nitrogen fixation ability of (a) UPMC1166 and (b) UPMC 1168. Example of changes in media color from pale green to blue is an indicator nitrogen fixation process which was carried out on bacteria with the increase in pH by the formation of ammonia and nitrates. Note: Arrows show the color changes from green to blue.
Figure 1. Nitrogen fixation ability of (a) UPMC1166 and (b) UPMC 1168. Example of changes in media color from pale green to blue is an indicator nitrogen fixation process which was carried out on bacteria with the increase in pH by the formation of ammonia and nitrates. Note: Arrows show the color changes from green to blue.
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Figure 2. Phosphate solubilizing ability of (a) UPMC 1393 and (b) UPMC 1376. The formation of a clear halo zone is due to the production of organic acid by bacteria that shows potential to solubilize tri-calcium phosphate in Pikovskaya’s agar media. Note: Arrows show clear zones.
Figure 2. Phosphate solubilizing ability of (a) UPMC 1393 and (b) UPMC 1376. The formation of a clear halo zone is due to the production of organic acid by bacteria that shows potential to solubilize tri-calcium phosphate in Pikovskaya’s agar media. Note: Arrows show clear zones.
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Figure 3. Potassium solubilizing ability of (a) UPMC703, (b) UPMC704 and (c) UPMC1393 as indicated by the halo zone on modified Aleksandrov agar medium.
Figure 3. Potassium solubilizing ability of (a) UPMC703, (b) UPMC704 and (c) UPMC1393 as indicated by the halo zone on modified Aleksandrov agar medium.
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Figure 4. Bacteria with ability to produce phytohormone. These four isolates showed the positive reaction by formation of pink color after interaction with Salkowasky reagent.
Figure 4. Bacteria with ability to produce phytohormone. These four isolates showed the positive reaction by formation of pink color after interaction with Salkowasky reagent.
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Figure 5. Electrophoresis gel image of PCR products. Marker: 1 kb marker. A 3 μL volume of the sample was loaded in each well. The target sequences were approximately 1500 bp. The percentage of gel was 1%. The forward primer was 5′ GAG TTT GAT CCT GC TCA G 3′ and the reverse primer was 5′ GTT ACC TTG TTA CGA CTT 3′ (BioSune Biotechnology Co., Ltd., China). Note1: Lane 1: empty; Lane 2: UPMC1166; Lane 3: UPMC1168; Lane 4: UPMC1254 (red box due to low density as compared to the other lanes); Lane 5: UPMC1389; Lane 6: UPMC1376; Lane 7: UPMC1393; Lane 8: Empty; Lane 9: UPMC703; Lane 10: UPMC704. Note2: Gel electrophoresis was conducted to separate amplified DNA fragments of different lengths. The length of DNA fragments affects the speed of DNA migration through the gel pores. The stability of biological molecules is achieved with the help of the buffer that functions as a conductor as well as controls the pH. The DNA ladder containing known fragment sizes was employed to assess the length of the amplified DNA sample [52]. All the isolates (UPMC1166, UPMC1168, UPMC1254, UPMC 1389, UPMC1376, UPMC1393, UPMC703 and UPMC704) were sent to Apical Scientific for bacteria identification and are shown in Table 5.
Figure 5. Electrophoresis gel image of PCR products. Marker: 1 kb marker. A 3 μL volume of the sample was loaded in each well. The target sequences were approximately 1500 bp. The percentage of gel was 1%. The forward primer was 5′ GAG TTT GAT CCT GC TCA G 3′ and the reverse primer was 5′ GTT ACC TTG TTA CGA CTT 3′ (BioSune Biotechnology Co., Ltd., China). Note1: Lane 1: empty; Lane 2: UPMC1166; Lane 3: UPMC1168; Lane 4: UPMC1254 (red box due to low density as compared to the other lanes); Lane 5: UPMC1389; Lane 6: UPMC1376; Lane 7: UPMC1393; Lane 8: Empty; Lane 9: UPMC703; Lane 10: UPMC704. Note2: Gel electrophoresis was conducted to separate amplified DNA fragments of different lengths. The length of DNA fragments affects the speed of DNA migration through the gel pores. The stability of biological molecules is achieved with the help of the buffer that functions as a conductor as well as controls the pH. The DNA ladder containing known fragment sizes was employed to assess the length of the amplified DNA sample [52]. All the isolates (UPMC1166, UPMC1168, UPMC1254, UPMC 1389, UPMC1376, UPMC1393, UPMC703 and UPMC704) were sent to Apical Scientific for bacteria identification and are shown in Table 5.
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Table
Table 1. Master mix of PCR reaction.
Table 1. Master mix of PCR reaction.
ComponentVolume per
Reaction
5 U/mL Taq Polymerase (Lucigen, USA)0.50 µL
100 pmol/μL universal primers (Forward: 5′ GAG TTT GAT CCT GCT CAG 3′; BioSune Biotechnology Co., Ltd., China)0.25 µL
Deionized water140 µL
2.5 mM dNTP mix, PCR Grade (Lucigen, USA)2.00 µL
100 pmol/μL universal primers (Reverse: 5′ GTT ACC TTG TTA CGA CTT 3′, BioSune Biotechnology Co., Ltd., China)0.25 µL
10 × Reaction buffer containing 15 mM MgCl2 (Lucigen, USA)2.50 µL
Genomic DNA as the template (50–200 ng)5.00 µL
Notes: Total volume of 25 µL per reaction.
Table
Table 2. Colony characterization after 24 h of incubation.
Table 2. Colony characterization after 24 h of incubation.
IsolatesShapePigmentationMarginFormElevationAppearance
UPMC1166Rod-shapedcreamy whiteundulateIrregularConvexbumpy, shiny
UPMC1254Rod-shapedcreamy whiteundulateIrregularConvexbumpy, shiny
UPMC1168Rod-shapedcreamy whiteundulatefilamentousUmbonatebumpy and shiny in the middle, dull in the side
UPMC1389coccus shapedcreamy whiteEntireCircularConvexbumpy, shiny
UPMC703Rod-shapedcreamy whiteEntireCircularConvexbumpy, shiny
UPMC1376coccus shapedcreamy whiteEntireCircularConvexbumpy, shiny
UPMC1393Rod-shapedcreamy whiteundulateIrregularConvexbumpy, shiny
UPMC704coccus shapedcreamy whiteEntireCircularConvexbumpy, shiny
Note: All isolates exhibited positive Gram stain reactions.
Table
Table 3. Physiological ability and functionality study of eight PGPR isolates.
Table 3. Physiological ability and functionality study of eight PGPR isolates.
CharacteristicsGrowth in NaCl (%)Catalase TestIndole Acetic Acid (IAA)
Production		Growth at pH
251015246789
UPMC 1166++++++++++++
UPMC 1168++++++++++++
UPMC 1254++++++++++++
UPMC 1389++++++++++++
UPMC 1376++++---+++++
UPMC 1393++++---+++++
UPMC 703++++---+++++
UPMC 704+++++-++++++
Table
Table 4. Nitrogen fixation ability, phosphate solubilizing ability and potassium solubilizing ability of eight PGPR isolates.
Table 4. Nitrogen fixation ability, phosphate solubilizing ability and potassium solubilizing ability of eight PGPR isolates.
CharacteristicsNitrogen Fixation Ability (Diameter in cm)Phosphate Solubilizing Ability (Diameter in cm)Potassium Solubilizing Ability (Diameter in cm)
UPMC 1166+(4.50 ± 1.91 a)−(0.00)−(0.00)
UPMC 1168+(4.25 ± 1.71 a)−(0.00)−(0.00)
UPMC 1254+(2.50 ± 0.58 a)−(0.00)−(0.00)
UPMC 1389+(3.25 ± 3.20 a)−(0.00)+(4.75 ± 1.50 a)
UPMC 1376−(0.00)+(3.00 ± 1.16 a)−(0.00)
UPMC 1393−(0.00)+(3.25 ± 0.96 a)−(0.00)
UPMC 703−(0.00)−(0.00)+(3.75 ± 2.50 a)
UPMC 704−(0.00)−(0.00)+(4.25 ± 1.5 a)
Notes: “−” indicates absence of growth; “+” indicates slow growth. The test was carried out using three replicates for each isolate. Column means followed by the same letter indicate insignificant (p > 0.05) difference using Duncan’s Multiple Range Test.
Table
Table 5. Identification of potential plant growth-promoting rhizobacteria (PGPR).
Table 5. Identification of potential plant growth-promoting rhizobacteria (PGPR).
IsolatesIDSimilarity (%)
UPMC 1166Bacillus methylotrophicus99.0%
UPMC 1168Bacillus siamensis99.0%
UPMC 1254Bacillus subtilis99.9%
UPMC 1389Bacillus albus99.0%
UPMC 1376Lactobacillus plantarum100%
UPMC 1393Bacillus marisflavi98.8%
UPMC 703Burkholderia anthina99.4%
UPMC 704Burkholderia metallica98.9%
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Peng, S.H.T.; Chee, K.H.; Saud, H.M.; Yusop, M.R.; Tan, G.H. Potential Novel Plant Growth Promoting Rhizobacteria for Bio-Organic Fertilizer Production in the Oil Palm (Elaeis guineensis Jacq.) in Malaysia. Appl. Sci. 2023, 13, 7105. https://doi.org/10.3390/app13127105

AMA Style

Peng SHT, Chee KH, Saud HM, Yusop MR, Tan GH. Potential Novel Plant Growth Promoting Rhizobacteria for Bio-Organic Fertilizer Production in the Oil Palm (Elaeis guineensis Jacq.) in Malaysia. Applied Sciences. 2023; 13(12):7105. https://doi.org/10.3390/app13127105

Chicago/Turabian Style

Peng, Shih Hao Tony, Kheng Hoy Chee, Halimi Mohd Saud, Mohd Rafii Yusop, and Geok Hun Tan. 2023. "Potential Novel Plant Growth Promoting Rhizobacteria for Bio-Organic Fertilizer Production in the Oil Palm (Elaeis guineensis Jacq.) in Malaysia" Applied Sciences 13, no. 12: 7105. https://doi.org/10.3390/app13127105

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