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[Audio] Impact of fertilizer addition on the structure and function of bacterial communities in oil palm soil and root analysed using shotgun metagenomics Zakiah Anis Binti Ahmad Nawawi (GS62962) Supervisor : Dr. Amalia Binti Mohd Hashim.

[Audio] Overview of Malaysia's Palm Oil Industry Global Production and Exports Malaysia is the second-largest palm oil producer globally, accounting for 25.8% of global production and 34.3% of global exports in 2020 (Ahmad Kushairi, 2017; MPOC, 2022). Economic Contribution Palm oil contributes 2.5% to Malaysia's GDP and 37.9% to its agricultural sector, employing millions, especially in rural areas (Ngan et al., 2022). Environmental Challenges The palm oil industry faces environmental issues, including water pollution and concerns about excessive fertilizer usage (Awang Ali et al., 2011; Salamat et al., 2021; Teo et al., 2010)..

[Audio] The Benefits of Palm Oil. healthy yield Average Oil Yield (tonne/hectare) palm Oil & palm kernel oil 3.49 sustainable palm oil global use. Palm Kernel Oil (from seed) in Malaysia for Palm Oil (from mesocarp) sunflower Oil rapeseed oil 0.80 0.78 soybean Oil Source: Oil World Database (Mar•ch, 2021) healthy consugnption Malaysian Palm Oil rich in Vitamin E Tocotrienols suitable for vegans healthy cooking Malaysian Palm Oil imparts crispy & crunchy texture to fried food neutral taste, retains natural flavour of food suitable for various food applications Excellent for Frying 255 •c stable at high temperature mseo free of trans fat and cholesterol non-GMO helps in the absorption of vitamins less smoke, spatter, and foam less gurnmy residues on walls and utensils.

[Audio] PROBLEM STATEMENTS Insufficient understanding of bacterial species, functions, and interactions within oil palm root niches, as well as their response to fertilization..

[Audio] Fertilizer addition will alter bacterial community structure and diversity in oil palm niches (bulk soil, rhizosphere, rhizoplane, endosphere) Fertilizer addition will increase the abundance of functional structures related to plant nutrient uptake within the bacterial community across oil palm niches Fertilizer will induce more complex bacterial co-occurrence networks across oil palm niches Potential biofertilizer candidates for oil palms with distinct mechanisms of action that enhance nutrient uptake in plants will be identified and proposed. RESEARCH HYPOTHESES.

[Audio] RESEARCH OBJECTIVES To investigate the impact of fertilizer addition on soil and foliar properties, bacterial community structure, specifically the bacteria that play a role in plant nutrient uptake, and diversity across niches To examine the impact of fertilizer addition on the functional structure related to plant nutrient uptake of the bacterial community across niches. To inspect the impact of fertilizer on the bacterial cooccurrence networks across niches. To identify the potential biofertilizers and their mechanisms of action in facilitating nutrient uptake..

[Audio] EFFECTS OF FERTILIZER ADDITION IN OIL PALM CROPS Pros of Fertilizer Addition Enhanced Growth Improved Soil Health Increased Productivity Urea and rice husk charcoal organic fertilizers promote early seedling growth and development, leading to increased plant height, leaf area, and diameter (Harahap et al., 2022). Nitrogen, phosphorus, and potassium (NPK) compound fertilizers stimulate the growth of beneficial soil microbes, enhancing soil and plant health (Ding et al., 2023). Drip irrigation with slow-release NPK fertilizer improves oil palm seedling growth, stem diameter, and leaf area, contributing to higher productivity (Sukmawan et al., 2021). Silicon fertilizer increase plant resistance to pest (Duangpan et al. 2022).

[Audio] Cons of Fertilizer Addition Soil Degradation Environmental Impact Excessive use of chemical fertilizers can reduce soil nitrogen and organic carbon content, potentially disrupting the soil ecosystem (Salamat et al., 2021). Improper application of chemical fertilizers may lead to nutrient leaching and runoff, causing water pollution (S.Kumar et al., 2022). Risk of Overapplication Inaccurate dosage or frequency of fertilizer application can lead to nutrient imbalances, nutrient toxicity, and reduced crop yields (Gu et al. 2021)..

[Audio] CHALLENGES FACED BY THE OIL PALM INDUSTRY Raw Material Shortages Environmental Issues High costs.

[Audio] SOLUTIONS AND INITIATIVES Sustainable Practices Eco-Friendly Fertilizers Implementing eco-friendly fertilizers is a sustainable approach to farming practices. It helps in reducing the environmental impact and promotes healthier soil. Reduced Fertilizer Application By reducing the application of fertilizers, farmers can minimize the risk of soil and water contamination, contributing to a more sustainable agricultural ecosystem. Research Investment Innovative Fertilization Methods Investing in research for innovative fertilization methods can lead to sustainable advancements in the industry, ensuring long-term environmental and agricultural benefits..

[Audio] PLANT MICROBIOME PLANT-ROOT NICHES Definitions A diverse community of microorganisms coexists near a plant (Aamir et al., 2021). Plant Recruits Their Microbiomes through substrate-driven selection in rhizosphere to provide an extra line of protection against pathogens (Berendsen et al, 2012; Berg et al., 2006)..

[Audio] Exploring Metagenomics Taxa found in the oil palm crops Roles Microbiomes Analysis methods References Novosphingobium, Pelomonas, Rhodoblastus, Sphingomonas and Zoogloea Isoprene-degrading bacteria Bacterial microbiome in soil and phyllosphere Amplicon sequencing (Carrión et al., 2020) Agaricomycetes, Glomeromycetes, and Lecanoromycetes Arbuscular mycorrhizal fung Fungal microbiome in root, rhizosphere, soil Amplicon sequencing (Kirkman et al., 2022) Rubrobacter (Phylum: Actinobacteria), AKYG587 (Phylum: Planctomycetes), and JdFR-76 (Phylum: Calditrichaeota) Fungal antagonist Soil microbiome of basal stem rot oil palm Amplicon sequencing (Goh, Zoqratt, Goh, Ayub, & Ting, 2020) Pseudomonas Staphylococcus Serratia, Burkholderia, Yersinia, and Salmonella Unknown Palm oil mill effluent microbiome Metagenomics (Parman, Mat Isa, Benbelgacem, Noorbatcha, & Salleh, 2019) Micrococcus luteus 101PB, Stenotrophomonas maltophilia 102PB, Bacillus cereus 103PB, Providencia vermicola 104PB, Klebsiella pneumoniae 105PB, Bacillus subtilis 106PB, Aspergillus fumigatus 107PF, Aspergillus nomius 108PF, Aspergillus niger 109PF and Meyerozyma guilliermondii 110PF Oil degrading bacteria Palm oil mill effluent microbiome Amplicon sequencing (Bala, Lalung, Al-gheethi, & Hossain, 2018) Humibacter, Microbacterium, Mycobacterium, 1921-2, HSB OF53-F07, Mucilaginibacter Unknown Bulk soil, rhizosphere, and rhizoplane Metagenomics (Ding et al., 2023) Metagenomics involves comprehensive genetic material analysis in diverse environments, providing insights into microbial communities and their abundance (Fanning et al., 2017). In Malaysia, the bacterial communities of oil palm are relatively poorly studies, despite the country has a large oil palm cultivation area. RESEARCH GAP Table 2.1: Previous metagenomics studies on the oil palm microbiome in Malaysia..

[Audio] OVERALL WORKFLOW Meganized data of 24 samples UF BS/R/P/E F Bacterial Community Structure Relative abundance analysis using MicrobiomeAnalyst V2.0 Functional Community Structure CCA analysis of soil and foliar physicochemical properties vs functional community structure using Galaxy pipeline Network Analysis Correlation analysis using Metagenonet Comparison of bacterial community structure between unfertilized and fertilized treatments using NetConfer Alpha and beta diversity analysis using MicrobiomeAnalyst V2.0 CCA analysis of soil and foliar physicochemical properties vs bacterial community structure using Galaxy pipeline Differentially abundant taxa analysis using STAMP Differentially abundant taxa with LDA score > 2 analysis, LefSe using MicrobiomeAnalyst V2.0 Metabolic phenotypes analysis using METAGENanalyst Fold change analysis of metabolic phenotypes using METAGENanalyst Differentially abundant GO functions analysis using STAMP Alpha and beta diversity analysis using MicrobiomeAnalyst V2.0 Enrichment analysis of GoTerm (generalized GO functions) using REVIGO Network similarity analysis, Venn Diagram of number of nodes and edges using NetConfer Cluster analysis using NetConfer Potential biofertilizer candidates Differentially abundant KO functions using STAMP Enrichment of KEGG modules analysis using KEGG reconstruct Bacterial involvement in KEGG modules analysis using MEGAN.

[Audio] OBJ.1. To investigate physicochemical properties, bacterial community structure, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane and endosphere of unfertilized and fertilized oil palm. IMPACT OF SOIL & FOLIAR PHYSIOCHEMICAL PROPERTIES ON BACTERIAL COMMUNITY COMPOSITION ACROSS TREATMENTS AND NICHES: CCA PLOTS CCA plot of unfertilized and fertilized treatment in the bulk soil CCA plot of unfertilized and fertilized treatment in the rhizosphere Total variation of bacterial community composition explained by environmental variables: 75.29% Total variation of bacterial community composition explained by environmental variables: 84.32%,.

[Audio] CCA plot of unfertilized and fertilized treatment in the rhizoplane CCA plot of unfertilized and fertilized treatment in the endosphere Total variation of bacterial community composition explained by environmental variables : 85.01% Total variation of bacterial community composition explained by environmental variables : 78.53%.

[Audio] Research question 1: How do soil and foliar physiochemical properties influence bacterial community composition in unfertilized and fertilized oil palm cultivation? Results: Important factors that influence the composition: exchangeable calcium, foliar nitrogen, foliar dry weight, and total phosphorus levels. These changes in soil and foliar physicochemical properties led to a distinct separation of bacterial composition in the CCA plots.

[Audio] Research question 2: How does bacterial community composition vary across different niches in unfertilized and fertilized oil palm cultivation? Results: Bacterial community composition varies significantly across different niches in unfertilized and fertilized oil palm cultivation. CCA analysis shows a strong relationship between bacterial community structure and environmental variables across bulk soil, rhizosphere, and rhizoplane, except in endosphere. Endosphere: less affected by changes in the soil and foliar physiochemical properties..

[Audio] IMPACT OF SOIL & FOLIAR PHYSIOCHEMICAL PROPERTIES ON BACTERIAL COMMUNITY COMPOSITION ACROSS TREATMENTS AND NICHES: MANTEL TEST Research question: How do soil and foliar physiochemical properties affect bacterial community composition in unfertilized and fertilized oil palm cultivation across different niches, as determined by Mantel test analysis? Results: Total phosphorus affects bacteria in all niches. Niches Environmental variables r p Bulk soil Foliar nitrogen* .8019 .0055 Total phosphorus* .7963 .0083 Exchangeable calcium .7086 .0722 Foliar dry weight .5636 .0763 Rhizosphere Foliar nitrogen* .8780 .0222 Total phosphorus* .8904 .0083 Exchangeable calcium .7588 .0902 Foliar dry weight* .7953 .0319 Rhizoplane Foliar nitrogen* .7095 .0236 Total phosphorus* .9290 .0083 Exchangeable calcium .5317 .0902 Foliar dry weight* .5905 .0375 Endosphere Foliar nitrogen .3420 .0736 Total phosphorus* .6936 .0083 Exchangeable calcium* .4177 .0277 Foliar dry weight* .3610 .0416.

[Audio] IMPACT OF SOIL & FOLIAR PHYSIOCHEMICAL PROPERTIES ON BACTERIAL COMPOSITION IN UNFERTILIZED AND FERTILIZED OIL PALM ACROSS DIFFERENT NICHES: MANTEL TEST Discussion: The Mantel test confirmed the addition of fertilizer affected the soil and foliar properties and bacterial composition, specifically the total phosphorus levels in the soil of all niches. Previous study on the impact of total phosphorus on bacterial composition: Phosphorus impacts bacterial population, changing the abundance of functional degrading and copiotrophic bacteria, and shifting from oligotrophic to copiotrophic bacteria (Cheng et al., 2020). Nitrifiers and denitrifiers are most affected, leading to increased nitrogen retention and nitrous oxide emission rates (Mehnaz, Keitel, & Dijkstra, 2019)..

[Audio] IMPACT OF SOIL & FOLIAR PHYSIOCHEMICAL PROPERTIES ON BACTERIAL COMMUNITY COMPOSITION ACROSS TREATMENTS AND NICHES Conclusion: The application of NPK fertilizer led to a significant increase in total phosphorus, which in turn altered the oil palm bacterial community composition in all niches..

[Audio] OBJ.1. To investigate physicochemical properties, bacterial community structure, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane and endosphere of unfertilized and fertilized oil palm. RICHNESS OF THE BACTERIAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Research question: Does the application of fertilizers increase the richness of bacterial communities across niches? Results: Mann-Whitney test showed no significant difference in Shannon diversity between fertilized and unfertilized oil palm. However, the results revealed an increase in Shannon diversity in the bulk soil and rhizosphere niches. Conversely, a decrease in Shannon diversity was observed in the rhizoplane and endosphere niches. (BS) (E) (R) (P) p-value: 1; [Mann-Whitney] statistic: 4 p-value: 0.1; [Mann-Whitney] statistic: 0 p-value: 0.7; [Mann-Whitney] statistic: 6 p-value: 0.4; [Mann-Whitney] statistic: 7 Fig.1. Shannon index (p>0.05) of the bacterial communities of unfertilized and fertilized oil palm across niches.The index values are graphically presented by box plots, each of which represents the interquartile range, whereas the line inside the box represents the median..

[Audio] OBJ.1. To investigate physicochemical properties, bacterial community structure, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane and endosphere of unfertilized and fertilized oil palm. RICHNESS OF THE BACTERIAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Discussion: Fertilizer use caused soil acidification and decreased bacterial diversity (Bledsoe et al., 2020; A. L. Wu et al., 2021), but our study found an increase in bacterial diversity in bulk soil and rhizosphere due to improved soil fertility. However, the increase in diversity may not be long-term, as suggested by decreased diversity in rhizoplane and endosphere. Further research with longer experimental durations is needed to fully understand the effects of fertilizer use on bacterial diversity in oil palm..

[Audio] RICHNESS OF THE BACTERIAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Conclusion: Fertilizer use did not result in a significant difference in Shannon diversity, but it did cause an increase in diversity in bulk soil and rhizosphere, and a decrease in diversity in rhizoplane and endosphere niches. Further research is needed with longer experimental durations to fully understand the effects of fertilizer use on soil bacterial diversity in oil palm..

[Audio] OBJ.1. To investigate physicochemical properties, bacterial community structure, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane and endosphere of unfertilized and fertilized oil palm. DIVERSITY OF THE BACTERIAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Research question: Does the application of fertilizer result in a diverse bacterial community structure in the oil palm ecosystem? Results: While no significant shift was observed in the beta diversity between unfertilized and fertilized oil palm, clear separation was observed in the rhizosphere and rhizoplane. These findings suggest that fertilization may impact the bacterial diversity in specific niches of oil palm. . (E) (P) (BS) (R) [ANOSIM] R: 0.25926; p-value < 0.1 [ANOSIM] R: 0.44444; p-value < 0.2 [ANOSIM] R: 1; p-value < 0.1 [ANOSIM] R: 1; p-value < 0.1 Fig 3. Principal coordinate analysis (PCoA) plot based on Bray–Curtis distances (p>0.05).

[Audio] DIVERSITY OF THE BACTERIAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Discussion: The rhizosphere and rhizoplane are crucial for microbial activity, contributing up to 40% of total dry matter (Lynch & Whipps, 1990). The fertilization treatment had a noticeable impact on bacterial community structure in rhizosphere and rhizoplane, but not in bulk soil. This is in line with the "rhizosphere effect“ (Berendsen et al., 2012; Berg et al., 2006), where root exudates increase microbial biomass and activity in the rhizosphere. No significant differences observed in the Shannon and beta diversity analysis may be due to limited replicates, highlighting the need for increased replicates in future studies to improve reliability and accuracy of results..

[Audio] Conclusion: No significant difference was observed in beta diversity between fertilized and unfertilized oil palms across all niches. However, clear separation was observed in the bacterial community structure of the rhizosphere and rhizoplane, indicating that the fertilization treatment had an impact on the bacterial community structure in these niches..

[Audio] IMPACT OF FERTILIZER ADDITION ON OIL PALM BACTERIAL COMMUNITY COMPOSITION ACROSS DIFFERENT NICHEs Hypothesis: The addition of fertilizer to oil palm soil will result in a significant increase in the relative abundance of bacterial taxa associated with plant nutrient uptake, as compared to unfertilized soil..

[Audio] OBJ.1. To investigate physicochemical properties, bacterial community structure, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane and endosphere of unfertilized and fertilized oil palm. IMPACT OF FERTILIZER ADDITION ON OIL PALM BACTERIAL COMMUNITY COMPOSITION ACROSS DIFFERENT NICHES Research question: How does the application of fertilizer impact the relative abundance of bacterial species in different niches? Results: Fertilizer application impacts relative abundance of bacterial species in different niches: Top 3 dominant phyla in oil palm samples were Proteobacteria, Actinobacteria, and Acidobacteria. Chloroflexi and Acidobacteria presence decreased in the endosphere of fertilized soil. Tenericutes present in endosphere of both fertilized and unfertilized soil. in F BS, R, P in F.

[Audio] Discussion: Top 3 phyla in oil palm: Proteobacteria, Actinobacteria, Acidobacteria, accounting for >70% of bacterial communities. Similar findings observed in previous studies on agricultural soils (Cui et al., 2018; Hegyi et al., 2021). Chloroflexi and Acidobacteria decrease in abundance and are absent in the endosphere of fertilized soil. This trend has also been observed in previous studies that investigated the effect of straw and biochar incorporation with chemical fertilizer on maize crops (Du et al., 2022). According to Naumova et al. (2022), possible reasons for the absence of Chloroflexi and Acidobacteria in the endosphere include : competition with more successful root colonizers, exclusion by the host plant's immune system, the presence of strains that favor Tenericutes in the root endosphere show negative interactions with other community components (Marasco et al., 2018). Acholeplasma genus of Tenericutes is exclusively found in contaminated soil's root endosphere (Gao et al., 2021). Tenericutes lack a cell wall and can colonize the gut of animals, insects, and plants (Mitter et al., 2017). These findings suggest that Tenericutes are resilient microorganisms that can tolerate various conditions and colonize plant endosphere, which may have significant implications for plant health..

[Audio] IMPACT OF FERTILIZER ADDITION: DIFFERENTIAL ABUNDANT TAXA OF UNFERTILIZED & FERTILIZED OIL PALM ACROSS DIFFERENT NICHES Differential abundant taxa (phylum level) of unfertilized and fertilized oil palm in bulk soil Research question: Which taxa significantly increase under fertilizer addition? Results: Nitrospirae in bulk soil & Proteobacteria in rhizosphere significantly increased with fertilizer application. Differential abundant taxa (phylum level) of unfertilized and fertilized oil palm in rhizosphere Differential abundant taxa (phylum level) of unfertilized and fertilized oil palm in rhizoplane Differential abundant taxa (phylum level) of unfertilized and fertilized oil palm in endosphere.

[Audio] POTENTIAL BIOMARKER OF UNFERTILIZED & FERTILIZED OIL PALM ACROSS DIFFERENT NICHES Differential abundant taxa of unfertilized and fertilized oil palm in rhizosphere (LDA score = 4) Differential abundant taxa of unfertilized and fertilized oil palm in rhizoplane (LDA score = 4) Differential abundant taxa of unfertilized and fertilized oil palm in bulk soil (LDA score = 2) No significant taxa were found, and thus, no potential biomarkers were discovered in the endosphere..

[Audio] Discussion on significantly enriched taxa in fertilized oil palm with a fold increase of at least two, across niches:.

[Audio] BACTERIAL BIOMARKER OF UNFERTILIZED & FERTILIZED OIL PALM ACROSS DIFFERENT NICHES Discussion on significantly enriched taxa in unfertilized oil palm with a fold increase of at least two, across niches:.

[Audio] IMPACT OF FERTILIZER ADDITION ON THE BACTERIAL COMMUNITY COMPOSITION OF UNFERTILIZED & FERTILIZED OIL PALM ACROSS DIFFERENT NICHES Discussion: Our previous findings (soil physicochemical analysis) predicted a shift in bacterial composition towards copiotrophy, an increase in diazotroph bacteria, and an increase in functional degrading bacteria following fertilizer application, based on previous studies (Cheng et al., 2020; Mehnaz, Keitel, & Dijkstra, 2019) . In our current study, we observed a significant increase in Proteobacteria (Copiotroph) and Nitrospirae (diazotroph) , as well as a four-fold increase in Sphingomonas, Nocardioides, and Pandorea (functional degrading bacteria) in the fertilized soil, confirming our previous predictions..

[Audio] IMPACT OF FERTILIZER ADDITION ON THE BACTERIAL COMMUNITY COMPOSITION OF UNFERTILIZED & FERTILIZED OIL PALM ACROSS DIFFERENT NICHES Discussion: The rhizosphere is a microbial hotspot due to the release of carbon compounds via rhizodeposition, selectively promoting certain bacteria and inhibiting others (Paterson et al., 2007). Fertilizer addition led to a four-fold increase in Nocardioides and Burkholderia in the rhizosphere, promoting the selection of fungal antagonist Nocardioides and plant growth-promoting Burkholderia (Lazcano et al., 2021). This increase in beneficial bacteria may enhance oil palm plant health and productivity, as indicated by a significant increase in plant dry weight in this study. Burkholderia and Nocardiodes were also four-fold more abundant in the rhizoplane, suggesting that these beneficial bacteria were recruited by the plant from the surrounding soil. Unfertilized soil showed increased abundance of Verrucomicrobia and Bacteroidetes in the rhizosphere and rhizoplane, which promote plant growth and fight fungal pathogens (Lazcano et al., 2021). This highlights the importance of plant-microbe interactions for plant health, especially in nutrient-limited environments..

[Audio] IMPACT OF FERTILIZER ADDITION ON THE BACTERIAL COMMUNITY COMPOSITION OF UNFERTILIZED & FERTILIZED OIL PALM ACROSS DIFFERENT NICHES Our hypothesis: The addition of fertilizer to oil palm soil will result in a significant increase in the relative abundance of bacterial taxa associated with plant nutrient uptake, as compared to unfertilized soil. Conclusion Hypothesis was confirmed by the results. Adding fertilizer to oil palm soil increases relative abundance of bacterial taxa associated with plant nutrient uptake compared to unfertilized soil. in F.

[Audio] RICHNESS OF THE functional COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Hypothesis: The richness of the functional community structure of fertilized oil palm will be higher than that of unfertilized oil palm across niches..

[Audio] OBJ.2. To investigate functional community structure of GO functions, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane, and endosphere of unfertilized and fertilized oil palm. RICHNESS OF THE FUNCTIONAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Research question: Does the application of fertilizers increase the richness of functional communities across niches? Results: Mann-Whitney test showed no significant difference in Shannon diversity between fertilized and unfertilized oil palm. However, the results revealed an increase in Shannon diversity in the rhizosphere. (BS) (E) (R) (P) p-value: 0.1; [Mann-Whitney] statistic: 0 p-value: 0.2; [Mann- Whitney] statistic: 1 p-value: 1; [Mann- Whitney] statistic: 4 6 p-value: 0.7; [Mann-Whitney] statistic: 6 Fig.2. Shannon index (p>0.05) of the bacterial communities of control and fertilized oil palm across niches.The index values are graphically presented by box plots, each of which represents the interquartile range, whereas the line inside the box represents the median..

[Audio] OBJ.1. To investigate physicochemical properties, bacterial community structure, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane and endosphere of unfertilized and fertilized oil palm. RICHNESS OF THE FUNCTIONAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Discussion: On-going.

[Audio] OBJ.2. To investigate functional community structure of GO functions, alpha and beta diversity in the bulk soil, rhizosphere, rhizoplane, and endosphere of unfertilized and fertilized oil palm. DIVERSITY OF THE FUNCTIONAL COMMUNITY STRUCTURE OF FERTILIZED AND UNFERTILIZED OIL PALM ACROSS NICHES Research question: Can fertilization induce a significant shift in the functional community structure of oil palm, and is this shift consistent across different niches? Results: While no significant shift was observed in the beta diversity between unfertilized and fertilized oil palm, clear separation was observed in the rhizosphere and rhizoplane. . (E) (P) (BS) (R) [ANOSIM] R: 0.44444; p-value < 0.2 [ANOSIM] R: 0.55556; p-value < 0.1 [ANOSIM] R: 1; p-value < 0.1 [ANOSIM] R: 0.074074; p-value < 0.5 Fig 4. Principal coordinate analysis (PCoA) plot based on Bray–Curtis distances (p>0.05).

[Audio] ENRICHED FUNCTIONAL COMMUNITY COMPOSITIONS IN UNFERTILIZED AND FERTILIZED OIL PALM (SEMANTIC PLOTS) a. Unfertilized and fertilized bulk soil: b. Unfertilized and fertilized rhizosphere: RF BSUF BSF RUF Generation of precursor metabolites & energy Translation DNA&RNA metabolic process Proteolysis Cell adhesion Response to stress due to chemical RNA metabolic process Pathogenesis Parameters were set: P- value: < 0.05 No. of Hits: More sig Higher hits.

[Audio] a. Unfertilized and fertilized rhizoplane: b. Unfertilized and fertilized endosphere: PUF PF EUF EF RNA & small molecule metabolic process Transport Carbohydrate metabolic process Phosphorelay signal transduction system Photosynthesis Proteolysis Generation of precursor metabolites & energy Parameters were set: P- value: < 0.05 No. of Hits: More sig Higher hits.

[Audio] OBJ.3. To investigate the bacterial cooccurrence networks (at genus level) in bulk soil, rhizosphere, rhizoplane, and endosphere of the unfertilized and fertilized oil palm. EFFECTS OF FERTILIZER ADDITON ON BACTERIAL COOCCURRENCE NETWORKS Example: Cluster 1 Cluster 2 Cluster 3 Absent a. Bulk soil b. Rhizosphere Fertilizer addition increased important bacterial key players (Cluster 1) in all niches (Cluster 1 = Bacterial with the highest degree of interactions). d. Endosphere Parameters were set: Algorithm: Spearman (reshuffle) Iteration: 500 p – value: < .05 (significant) r – value: .8 (strong) c. Rhizoplane.

[Audio] OBJ.4. To propose potential biofertilizer candidates and their mechanism of actions in improving plant health and development. ENRICHED FUCTIONS (KEGG MODULE) AND CONTRIBUTED BACTERIA IN UNFERTILIZED AND FERTILIZED OIL PALM Unfertilized bulk soil: Fertilized bulk soil: Cofactor and vitamin metabolism: One carbon pool by folate Nitrogen Metabolism: Assimilatory nitrate reduction Candidatus Sulfotelmatobacter kueseliae Candidatus Sulfotelmatomonas gaucii Kibdelosporangium aridum Planctomycetia bacterium 21-64-5 Terracidiphilus gabretensis Acidobacteria bacterium 13_1_40CM_56_16 Acidobacteria bacterium 13_1_40CM_2_64_6 Gammaproteobacteria bacterium 13_2_20CM_66_19 Actinospica robiniae Ktedonobacter racemifer Ktedonobacter (Cluster 2) Candidatus Sulfotelmatobacter(Cluster 3) Occallatibacter savannae Occallatibacter savannae, Thermostaphylospora chromogena, and Planctomycetia bacterium 21-64-5 (r= 0.9429, p= 0.01) (r= 0.9429, p= 0.01) Gemmataceae (Cluster 2) Bacterial secondary metabolite: Ditryptophenaline (fungal compound) Legend: Cross-linked the contributed bacteria with bacterial clusters found in network analysis Paraburkholderia tropica (Cluster 1) Enriched functions (almost complete KEGG module) Contributed bacteria Enriched functions (complete KEGG module) Potential biofertilizer candidates.

[Audio] ENRICHED FUCTIONS (KEGG MODULE) AND CONTRIBUTED BACTERIA IN UNFERTILIZED AND FERTILIZED OIL PALM Unfertilized rhizosphere: Fertilized rhizosphere: Cofactor & vitamin: Thiamine salvage pathway Carbapenem resistance Opitutus (PGPR) (Cluster 2) Ralstonia insidiosa, Ralstonia sp. A12, and Ralstonia sp. NFACC01 Bordetella sp FB 8, Burkholderia dabaoshanensis, Thiomonas sp FD 6 and Pandorea thioxydans Thiomonas (Cluster 1) Pandorea (Cluster 3) (r= - 0.9, p= 0.001) Unique OTU Cofactor: Nicotine degradation, pyrrolidine pathway Bradyrhizobium erythrophlei, Ktedonobacter racemifer, and Occalatibacter savannae. Cofactor and vitamin metabolism: One carbon pool by folate Ktedonobacter Bradyrhizobium(Cluster 2) Occallatibacter savannae Thiomonas sp. FB-6 Candidatus Sulfotelmatomonas gaucii Terracidiphilus gabretensis Burkholderia dabaoshanensis Bordetella sp. FB-8 Candidatus Sulfotelmatobacter kueseliae Silvibacterium bohemicum Gammaproteobacteria bacterium 13_2_20CM_66_19 Acidobacteriaceae bacterium URHE0068 Thiomonas, Bulkholderia and Leifsonia (Cluster 1) Bacterial secondary metabolite: Ditryptophenaline (fungal compound) Pedosphaera (Cluster 1) Ktedonobacter, Thermogemmatispora (Cluster 3) Ktedonobacter racemifer, Alphaproteobacteria bacterium 64 11, Pedosphaera parvula, Occallatibacter savannae, Rhodospirillales bacterium URHD0088, and Thermogemmatispora carboxidivorans..

[Audio] SUMMARY: BACTERIAL SPECIES AND THEIR ASSOCIATED FUNCTIONS BS UF Occallatibacter savannae (Nitrogen metabolism) F 1. Ktedonobacter racemifer (Cofactor (carbon pool) metabolism) Candidatus Sulfotelmatobacter kueseliae (Cofactor (carbon pool) metabolism) R UF 1. Opitutus (Pathogen defense; Ralstonia) 2. Bradyrhizobium erythrophlei (Cofactor metabolism) 3. Ktedonobacter racemifer (Cofactor metabolism, secondary metabolite) 4. Occalatibacter savannae (Cofactor metabolism, secondary metabollte) 5. Pedosphaera parvula (Secondary metabolite) 6. Thermogemmatispora carboxidivorans (Secondary metabolite) F 1. Bordetella sp FB 8 (Cofactor metabolism) 2. Burkholderia dabaoshanensis (Cofactor metabolism (thiamine and carbon pool) 3. Thiomonas sp FD 6 (Cofactor metabolism (thiamine and carbon pool) 4. Pandorea thioxydans (Cofactor metabolism)) Leifsonia xyli Cofactor metabolism) LCA algorithm: 95% similarity Highlighted species= performed multiple functions.

[Audio] DISCUSSION No complete or almost complete KEGG modules with functions associated to plant nutrient uptake (e.g: nitrogen metabolism and biosynthesis of cofactors, vitamins, and bacterial secondary metabolites) or signature module (Carbapanem resistance) was found in the rhizoplane and endosphere. Joint analysis of co-occurrence networks and enriched KEGG functions revealed what happened in the unfertilized bacterial community and how they defended themselves from pathogen attacks (evidence: enriched in carbapenem resistance produced by Ralstonia), devised their own strategy to deal with low nutrient availability (enriched in nitrogen assimilation functions), and collaborated to produce cofactors and secondary metabolites. Our findings suggested that bacteria with the highest interactions (cluster 1 in network analysis) perform important functions and contribute significantly to the environment. If we must select the most suitable bacterial candidate as biofertilizer, we will select according to the following order; because they can perform multiple functions. Thiomonas sp. FD 6 (cluster 1, unique OTU) Burkholderia dabaoshanensis (cluster 1) Ktedonobacter racemifer (cluster 2) Occallatibacter savannae.

[Audio] OVERALL CONCLUSION Our research hypotheses: H1: The addition of fertilizer to soil will have a positive effect on the structure of oil palm bacterial communities, specifically by increasing the abundance of bacteria with functions related to nutrient uptake. H2: The rhizosphere will show the greatest response among these compartments, optimizing its role as a filtering site for the recruitment of beneficial bacteria involved in plant nutrient uptake. Conclusion: H1: Hypothesis was confirmed by the research. The addition of fertilizer to the soil was found to have a positive effect on the structure of bacterial communities in oil palm, with an increase in bacterial taxa associated with plant nutrient uptake. H2: Hypothesis was confirmed by the research. The rhizosphere was found to show the greatest response among plant root compartments, which optimizes its role as a filtering site for the recruitment of beneficial bacteria involved in plant nutrient uptake..

[Audio] RECOMMENDATIONS This research sheds light on the interactions between plants and microorganisms in the presence and absence of fertilizer. This vital information may be utilized to develop an efficient biofertilizer. Beneficial bacteria with multiple functional capability screened from both unfertilized and fertilized environment are the best options for biofertilizer formulation. Furthermore, I recommend that the fertilized rhizosphere's keystone taxa (Azospirillium; the one with the highest degree of interaction in cluster 1) be chosen because it interacted the most with beneficial bacteria, including our potential biofertilizer candidates (Thiomonas sp. FD 6 and Burkholderia dabaoshanensis), and its absence will result in rapid shifts in bacterial community composition..

[Audio] GANTT CHART Year 2021 2022 2023 Project activities J F M A M J J A S O N D J F M A M J J J A S O N D S1 S2 1. Project implementation plan write up 2. Taxonomic composition and diversity analysis 3. Functional structure and diversity based on KEGG pathway and GO analysis 4. Co-occurrence network analysis 5. Identifying potential biofertilizer candidates 11 Final report Year 2021 2022 2023 Project (Milestone) J F M A M J J A S O N D J F M A M J J A S O N D S1 S2 1. Complete project implementation plan write up  2. Complete taxonomic composition and diversity analysis  3. Complete functional structure and diversity based on KEGG pathway and GO analysis  4. Complete co-occurrence network analysis  5. Complete identifying potential biofertilizer candidates  6. Complete final report  7. Project completion .