Kata kunci: rhizobakteri pemacu pertumbuhan tanaman (PGPR), pelarut fosfat, penghasil IAA, penghasil siderofor, sagu Metroxylon, Rottb. Kata kunci: rhizobakteri pemacu pertumbuhan tanaman (PGPR), pelarut fosfat, produksi IAA, produksi siderofor, sagu Metroxylon, Rottb.
Problem Statement
To build on previous findings, the present study was conducted with the aim of identifying the potential PGPRs native to the rhizosphere of sago palm (M. sagu, Rottb.) at selected locations in Sarawak State, Malaysia. Nevertheless, little research has been conducted regarding PGPR native to the rhizosphere of wild-grown M.sagu.
Objectives
The Outlook on The Status of Global Food Production
Due to the rapidly growing population, the United Nations estimates that global food production will need to grow by at least 60% by 2050 (Konuma, 2018). This scenario indirectly drives up global food demand and ultimately raises fears that the world's cupboards may run bare in the next few decades.
Agriculture in Malaysia
Agrochemicals Practice in Malaysia
Deleterious Effects of Synthetic Fertilizers on Pollution
As a result, the creation of the environment which is harmful to aquatic life due to the reduction of oxygen supply (Chandini et al., 2019). This soil acidity will also result in stunted plant growth as the concentration of toxic ions accumulated in the soil increases (Ilker et al., 2007).
Application of Organic Fertilizer
Fertilizer application has indeed been proven to be an effective approach in terms of increasing crop yield, maintaining soil organic carbon as well as increasing carbon sequestration (Gai et al., 2018). Nevertheless, considering the manure as a reservoir of ARGs and ARBs, there is still potential damage from the effect of manure fertilization of ARGs distribution in soils (Wang et al., 2018).
Application of Biofertilizer
Meanwhile, in the 1980s, research was conducted on the contribution of mycorrhizae and the nitrogen contribution of Azospirillum to oil palm seedlings (Bang et al., 2018). Biofertilizers have historically been used in Malaysia by pioneers such as SOField Agrobio Resources in Sarawak and BIO AG in Pahang.
Potential Application of Plant Growth Promoting Rhizobacteria
According to Ahemad and Kibret, (2014) PGPR can directly and indirectly affect plant growth promotion. On the other hand, indirect growth promotion occurred as PGPR promotes plant development in restricted environments.
Plant Growth Promoting Rhizobacteria
Nitrogen Fixation
Biological nitrogen fixation consists of two types, in which symbiotic nitrogen fixation and non-symbiotic nitrogen fixation. These beneficial plant-microbe interactions were found to be an important criterion in biological nitrogen fixation, which is considered to be beneficial for the development of organic fertilizers (Kuypers et al., 2018).
Ammonia Production
Symbiotic nitrogen fixation has involved members of the Rhizobiaceae family (Dinnage et al., 2019), while various genera such as Pseudomonas, Diazotrophicus, Arthrobacter, Acetobacter, Bacillus, Clostridium and also Azotobacter are commonly associated with non-symbiotic nitrogen fixation (Martins et al. al.). al., 2019). These PGPRs will eventually fix much of the elemental nitrogen that entered the soil under normal settings (Ji et al., 2019).
Phosphate Solubilization
The presence of enzymes, especially phosphatases and phytases released by soil bacteria, catalyzes the mineralization process (Wu et al., 2019). Phosphatases are extracellular enzymes that take organic forms of P as a substrate and convert them to inorganic forms (Ghosh et al., 2018).
Plant Growth Promoting Rhizobacteria as Phytostimulators
Indole Acetic Acid (IAA) Production
In the next step, flavin-containing monooxygenases convert indole-3-pyruvate to IAA (Matthes et al., 2019). In addition, auxins trigger signaling pathways for root epidermal hair cells, leading to root hair growth (Nascimento et al., 2020).
Plant Growth Promoting Rhizobacteria as Biopesticides
Siderophore Production
Siderophores secreted by PGPR remove the iron present in the extracellular environment and form siderophore-iron complexes that are transported into the cell via receptors found in the membrane (Neilands, 1981). Since the iron solubility is limited at higher pH, these conditions mostly occur in soils with neutral to alkaline pH.
Status of PGPR Research
As contrasted with other carbohydrate-producing crops, it is the most fertile (Bintoro et al., 2018). Furthermore, even within closely related taxonomic groups, 16S rDNA sequencing has revealed significant diversity ( Wang et al., 2015 ).
Sampling Sites
Soil Samples Collection
Isolation of Plant Growth Promoting Rhizobacteria (PGPR)
Microbial counts were performed and expressed as colony forming units (CFU) per gram of soil. An independent t-test was then performed to determine the difference in the mean of the isolated microbial population.
Purification and Maintenance of Cultures
Characterization of Diazotrophic Bacteria
Taxonomic Identification of Plant Growth Promoting Rhizobacteria .1 DNA Extraction
Gel Electrophoresis
The size of the amplicons was determined in which a comparison was made with the 1 kb DNA molecular size marker run simultaneously (Thermo Fisher Scientific, USA). The PCR products were then electrophoresed on 1.5% (w/v) agarose gel, which was pre-stained with Ethidium Bromide (EtBr). The size of the amplicons was then determined by comparing each profiled band to the 1 kb molecular DNA marker run simultaneously (Thermo Fisher Scientific, USA).
DNA Purification
The QIAquick column was reinserted into the collection tube after the flow-through was discarded. Approximately 500 μl of QG buffer was added to the QIAquick column and centrifuged at 10,000 rpm for another minute. Approximately 750 μL of Buffer PE was added to the QIAquick column and centrifuged at 10,000 rpm for 1 minute to complete the washing procedure.
In Vitro Screening of Plant Growth Promoting Activities
Preparation of Identified Inoculum
Turbidity of inoculated broth was determined using UVmini-1240 UV-VIS spectrophotometer (Shimadzu, Japan). The turbidity of the bacterial suspension was then adjusted to CFU/ml to standardize the microbial test. To ensure the validity of the experiments, all screening tests were performed simultaneously with the control experiment in natural settings.
Screening of Ammonia Production
A bacterial strain, Pseudomonas aeruginosa, was chosen as a positive control and uninoculated medium served as a negative control.
Analysis of Phosphate Solubilization
Qualitative Analysis of Phosphate Solubilization
Quantitative Analysis of Phosphate Solubilization
Every 3 days, a 5 ml aliquot was withdrawn and centrifuged at 10,000 rpm for 20 minutes.
Screening of Indole Acetic Acid (IAA) Production
Preparation of Standard Curve
The production of IAA was indicated by the development of a pink color of the medium. The intensity of the color is directly proportional to the increase in the concentration of IAA produced (Appendix 3 - page 119). An IAA standard curve was plotted based on the absorbance reading versus IAA concentration (Appendix 4 - page 120).
Evaluation of Siderophore Production
Preparation of Chrome Azurol S (CAS) Agar Plates
Meanwhile, solution III was prepared by dissolving 0.073 g of hexadecyltrimethylammonium bromide, HDTMA (Sigma-Aldrich, USA) in 40 mL of distilled water. The three shares; (i) Minimal medium 9 (MM9) stock salt solution, (ii) 20% (w/v) glucose stock and (iii) casamic acid solution were prepared accordingly:. i) Minimal Media 9 (MM9) salt solution stock was prepared by dissolving 15 g potassium dihydrogen phosphate, KH2PO4 (Merck Millipore, USA), 25 g sodium chloride, NaCl (Merck Millipore, USA) and 50 g ammonium chloride, NH4Cl (Merck Millipore) in 500 mL of distilled water. iii) Casamino Acid solution was prepared by mixing 3 g of Casamino Acid with 27 mL of distilled water. After autoclaving, about 30 mL of sterile casamino acid solution and 10 mL of sterile 20% (w/v) glucose solution were added.
Screening of Siderophore Production by Selected Isolates
PYPE (Sigma-Aldrich, USA) will not dissolve below pH 5, so the pH of the solution was monitored. Sodium hydroxide, NaOH (Merck Milipore, USA) was gradually added after adding PIPES (Sigma-Aldrich, USA) to MM9 Saline to make sure that the pH did not drop to pH of 5. This was followed by the addition of 100 mL of Blue Dye Solution before being aseptically poured into the petri dishes.
Statistical Analysis
Ranking Plant Growth Promoting Traits
Sampling Sites
Isolation and Enumeration of Microbial Population
The total viable count of the rhizobacterium varied from Log10 7.5 x 104 CFU/mL to Log10 1.19 x 105 CFU/mL There was a clear trend between both locations where the microbial population native to soil samples collected from Dalat Division , was higher in comparison to those collected from Kuching Division.
Isolation and Characterization of Putative Diazotrophic Rhizobacteria
Bacterial Identification by 16S rDNA Sequencing
The main group 9 consisted of 26 isolates with the smallest group 5 and group 7 each containing one isolate. The BLASTn phylogenetic analyzes revealed a clear grouping of these 47 isolates into two Phyla: Firmicutes (55.6 %) and Proteobacteria (44.4. At the genus level, these isolates were grouped into four genera: Bacillus (n=4), Pseudomonas ( n= 3), Serratia (n=1) and Staphylococcus (n=1).
Secondary In Vitro Screening of Plant Growth Promoting Traits in Isolates .1 Screening of Ammonia Production
- Qualification of Phosphate Solubilization
- Quantification of Phosphate Solubilization
- Screening of Indole Acetic Acid (IAA) Production
- Screening of Siderophore Production
Based on the solubilization zone, all isolates were selected for secondary screening of phosphate solubilization. The phosphate that was dissolved as well as the changes in pH of Pikovskaya's medium were recorded in Table 4.8 and Table 4.9. The amount of IAA was expressed in µg/mL (Table 4.9) extrapolated from the standard curve of IAA (Appendix 4 - page 121).
Ranking of Plant Growth Promoting Traits
The potential of PGPR as an emerging tool in supporting sustainable agriculture efforts is well recognized. However, due to the impact of climate change on the agricultural sector, this study identified a gap in PGPR research in selecting an effective PGPR that can thrive and make a positive impact in stress-prone environments. Therefore, in this study, the exploitation of beneficial indigenous potential food crops of Sarawak is the isolation and identification of PGPR from Metroxylon sago, Rottb.
Microbial Population in Metroxylon sagu, Rottb
However, it is worth mentioning that the above distinction was only valid on the representative soil samples due to the limitation in the cultivation fraction of the great microbial diversity in the soil. In order to provide conclusions and precise data about the microbial population of the selected habitat, a metagenomic approach can be considered. Nevertheless, the study of microbial population of the sampling sites was not carried out as the main objective of the study was to identify PGPR native to Metroxylon sagu.
Preliminary Selection of PGPR Isolates
Therefore, this study suggested that the differences are probably due to the different media used, as well as the different incubation period that affect the efficiency of microbial growth. This was consistent with Jiang et al. 2017) study in which they stated that plant species and soil properties play a determining role in the diversity and composition of the rhizosphere bacterial community. It is worth noting that this was due to the different nature of the study in which their study was focused on identifying bacterial diversity at four different soil depths.
In Vitro Screening of Identified PGPR
- Ammonia Production by Identified PGPR Isolates
- Phosphate Solubilization by Identified PGPR
- IAA Production by Identified PGPR Isolates
- Siderophore Production by Identified PGPR Isolates
Furthermore, microorganisms involved in the phosphorus cycle through phosphate solubilization and phosphorus fixation present in the soil, in a pH-dependent manner (Bhattacharyya et al., 2020). However, the IAA concentration produced in this study is much lower compared to previous research by Oliveira et al. These were reported by Sasirekha et al. 2012), in which Pseudomonas bacteria observed an increase in IAA production from 80 to 123 μg/mL with an increase in tryptophan addition from 0.1 to 0.5 g/L.
Ranking of Identified PGPR Based on PGP Traits
However, it is worth noting that the literature comparison involved different host plant species and different environmental conditions, which may result in differences in the present results. Meanwhile, the rest of the genera contained those placed at the top of the scale and it was not found consistent in any research related to Metroxylon sagu, Rottb., suggesting a new PGPR finding related to this crop. The similar trend was observed in the current study, where all the identified PGPR isolates had multiple PGP traits.
Conclusion
Recommendations
The role of plant growth promoting rhizobacteria (PGPR) in the sustainable agricultural effect of different gamma irradiations on quantitative and qualitative traits of sesame (Sesamum indicum). Isolation and identification of plant growth-promoting rhizobacteria from sago palm (Metroxylon sagu, Rottb.), Tropical Life Sciences Research. Characterization of plant growth promoting rhizobacteria and fungi associated with rice, mangrove and effluent contaminated soil.
The red marks indicated the sampling site’s location chosen for this study, in Sarawak, Malaysia
Development of pink colour in different IAA concentrations
Standard curve of IAA
Dalat average temperature
Dalat average rainfall
Sago seedlings at sampling sites Kuching
GPS coordinates for each sampling sites
Examples of isolates with different morphology grown on Burks agar
