1. Sustainability Challenges in Oil Palm Mono-cultivation: Are Microbes the Solution? Khim-Phin,Chong Sustainable Palm Oil Research Unit (SPOR) Universiti Malaysia Sabah Sustainable Palm Oil Research Unit (SPOR)

2. Content of Talk: Oil Palm Success and Challenges: In Brief Fundamental of Microbial in Agriculture Case Studies: -Roles of Microbes in Oil Palm Improvement: -Roles of Microbes in Enhancing Oil Palm Disease Resistant -Microbial Diversity in Oil Palm Soil Conclusions

3. Oil Palm World Palm Oil production increase from 13 to 28% from 1990 to 2011, with increase of exports from 36 to 57% Two key exporting countries: Malaysia (47%) and Indonesia (46%). Account 93% of world palm oil exports Malaysia: 5 Million Ha of Oil Palm. Contributing over 11% of global supply of edible oils and fats with 0.1% of the total global agricultural land area

4. High Land Productivity Oil Palm vs Other Oil Seeds: 11x more than soyabean 10x more than sunflower 7x more than rapeseed In 2011, Malaysia’s Export revenue of oil palm products reached a record high of RM80.4 billion, an increase of 34.5% against RM59.8 billion achieved in 2010.

5. Palm Oil Industry: At Crossroad or Under Real Threat? Workers Socio- Environment Productivity Cost of Production Human Capital

7. Soil Environments Surface and subsurface soils are typically nutrient- poor environments for microbes Rhizosphere is enriched in nutrients as a result of nearby plant activities

8. Rhizosphere Rhizoplane –soil in direct contact with plant root Endophytes –microbes attached to root surface Decreasing moisture Increasing organic C

9. Organic material in rhizosphere Exudates –low molecular weight compounds released from plant cells in a non- metabolic manner (leakage) Secretions –compounds metabolically released from plant cells Lysates –compounds released from moribund cells during autolysis Plant mucilage –plant polysacchrides

10. Some bacteria and blue green algae are able to fix nitrogen from the atmosphere to enrich soil with nitrogen and increase its fertility. These microbes are commonly called biological nitrogen fixers.

11. Rhizobium bacteria is involved in the fixation of nitrogen in leguminous plants (pulses). Rhizobium lives in the root nodules of leguminous plants, such as beans and peas, with which it has a symbiotic relationship. Sometimes nitrogen gets fixed through the action of lightning.

12. Our atmosphere has 78% nitrogen gas. Nitrogen is one of the essential constituents of all living organisms as part of proteins, chlorophyll, nucleic acids and vitamins. The atmospheric nitrogen cannot be taken directly by plants and animals. Certain bacteria and blue green algae present in the soil fix nitrogen from the atmosphere and convert into compounds of nitrogen. Once nitrogen is converted into these usable compounds, it can be utilized by plants from the soil through their root system.

13. Nitrogen is then used for the synthesis of plant proteins and other compounds. Animals feeding on plants get these proteins and other nitrogen compounds. When plants and animals die, bacteria and fungi present in the soil convert the nitrogenous wastes into nitrogenous compounds to be used by plants again. Certain other bacteria convert some part of them to nitrogen gas which goes back into the atmosphere. As a result, the percentage of nitrogen in the atmosphere remains more or less constant.

14. Beneficial root-microbe interactions Atmosphere contains 10 15 tons N 2 gas –Biological nitrogen fixation –Minimum of 70 million tons N fixed/year

17. Vegetative Growth Improvement-Nursery (Johor Estate) Combinations of Bacillus spp, Aspergillus spp & Pseudomonas spp Feb 2010: Seed sowing May 2010: Transplanting to main nursery June 2010: 1 st Application Oct 2010: 2 nd Application Dec 2010: Field transplanting Courtesy of Agrinos Microbes treatedUntreated

18. Observation (Avg)TreatedControl Frond Length (cm)133.8125.6 Leaflet Length (cm)41.836.8 Number of Total Fronds21.220.4 Girth (cm)37.230.4 Root Length (cm)122.8111.4 Seedling Height (cm)154.8140.2 Results show the averages for each parameters measure for microorganisms treated oil palm seedlings compared to control (Kalimantan Nursery, Indonesia) Courtesy of Agrinos

19. Yield-Immature Plantings (Tawau Estate) Nitrogen based fertilizer reduction: 1 st Yr Planting-0% 2 nd Yr Planting-15% Subsequent Yr-25% Yield Improvement (%) 1 st Yr Harvesting-80% 2 nd Yr Harvesting-120% 3 rd Yr Harvesting-49% 4 th Yr Harvesting-17% 5 th Yr Harvesting-11% Courtesy of Agrinos 3.57% bigger girth 10% > LAI

20. BlockDepth(cm)pHExchg. KAvailable P (p.p.p.m) Total Available Organic C (%) Organic N (%) Opt4.5- 5.5 0.30400 20.002.500.25 Treat- Blk 1 0-155.60.38187 20.300.770.13 Treat- Blk 1 15-355.80.17199 11.800.990.18 Cont- Blk 2 0-155.50.66178 6.900.660.12 Cont- Blk 2 15-354.70.65137 5.100.370.09 Soil Analysis Courtesy of Agrinos

21. Yield-Mature Plantings (Sandakan Estate) 1 st application of microbes: 10 years age Yield Improvement (%) 1 st Yr treatment-12% 2 nd Yr treatment-15% 3 rd Yr treatment-25% 4 th Yr treatment-28% *Salinity and sandy problem Courtesy of Agrinos Years of treatment

23. MicrobesErgosterol (Average) Infection percentage GSM percentage Bacillus spp. and Trichoderma spp. 0.19 µg/mL -1 30 % Lactobacillus, Nattobacillus and Yeasts 0.19 µg/mL -1 60 % Bacillus spp, Aspergillus spp. and Pseudomonas spp. Control (Infect without microbes) Control (Healthy) 0.23 µg/mL -1 1.52 µg/mL -1 0 µg/mL -1 100 % 0% 100 % 0% Percentage of Ganoderma infection after two months based on ergosterol (fungal sterol) content and growth on GSM for oil palm seedlings pre-treated with various combination of microbes

24. MicrobesErgosterol (Average) Infection percentage GSM percentage Bacillus spp. and Trichoderma spp. 0.22 µg/mL -1 60 % Lactobacillus, Nattobacillus and Yeasts 0.34 µg/mL -1 50 % Bacillus spp, Aspergillus spp. and Pseudomonas spp. Control (Infect without microbes) Control (Healthy) 0.22 µg/mL -1 2.35 µg/mL -1 0 µg/mL -1 60 % 100% 0% 60 % 100% 0% Percentage of Ganoderma infection after four months based on ergosterol (fungal sterol) content and growth on GSM for oil palm seedlings pre-treated with various combination of microbes

25. Ergosterol HPLC Chromatograms of ergosterol, peak was detected at RT 7-8 min. (A) Ergosterol standard (B) Healthy palm (C) Infected palm. Ergosterol peaks are arrowed. Oil palm roots cultured on Ganoderma Selective Media (GSM) after 5 days of incubation. A: Infected oil palm roots, indicated by Ganoderma growth. B: Uninfected oil palm roots. A A C B B

26. Ganoderma-inoculated oil palm seedling roots treated with different combination of microbes. A: Bacillus sp and Trichoderma sp. B: Bacillus sp., Aspergillus sp. and Pseudomonas sp. C: Lactobacillus, Nattobacillus and Yeasts, D: Control. Bar= 3 cm

27. Oil palm estate soil inoculated with Ganoderma boninense and treated with combinations of Bacillus spp, Aspergillus spp and Pseudomonas spp 1 st App 2 nd App Blue line: control Green line: Microbes treated Courtesy of Agrinos & UPM

28. Hyphal extension of G. boninense in sterile and non sterile soil and frond debris (FD). Colonised wheat grains were used as inoculum source. a: Mycelial grown in FD after 4 days. b: Extension in FD after 10 days. c: Growth in soil after 4 days. d: Hyphal extension in soil after 10 days 2 nd App Cooper, R. (2011) in Sustainable Agriculture: An Insight into Ganoderma

29. Assessment on field application is currently on going. Data collection due mid Nov 2012 2 nd App Courtesy of Martin Kong, One Good Earth

31. Population density of microorganisms in all soil samples collected from oil palm plantation of Sapi Nangoh, Sandakan. Sample Growth medium NA(Bacteria)PDA (Fungi)MEA (Yeast) S13.2 x 10 3 5.0 x 10 2 2.8 x 10 2 S25.1 x 10 3 1.0 x 10 2 1.3 x 10 4 S37.0 x 10 2 -1.5 x 10 4 S47.2 x 10 4 -4.7 x 10 4 S53.8 x 10 3 3.0 x 10 2 7.4 x 10 3 S6-3.0 x 10 2 5.3 x 10 3 S73.0 x 10 6 -1.5 x 10 3 S84.0 x 10 3 -4.8 x 10 3 S92.4 x 10 7 9.0 x 10 2 1.1 x 10 3 S101.6 x 10 3 1.0 x 10 2 2.0 x 10 6 S113.6 x 10 3 -3.6 x 10 6 S127.0 x 10 2 1.0 x 10 2 4.2 x 10 4 S135.0 x 10 6 -1.9 x 10 3 S141.2 x 10 6 -7.9 x 10 3 S154.6 x 10 3 -6.4 x 10 4 S166.7 x 10 5 -7.2 x 10 4 S171.7 x 10 3 1.0 x 10 2 6.3 x 10 2 S185.7 x 10 3 -4.7 x 10 4 S193.2 x 10 3 -3.1 x 10 3 S203.2 x 10 3 1.0 x 10 2 4.7 x 10 3

32. Cont….. Sample Growth medium NA(Bacteria)PDA (Fungi)MEA (Yeast) S212.0 x 10 3 1.0 x 10 2 1.8 x 10 3 S222.0 x 10 5 3.0 x 10 2 9.6 x 10 3 S231.3 x 10 3 1.0 x 10 2 1.6 x 10 3 S243.2 x 10 3 3.0 x 10 2 1.1 x 10 3 S252.2 x 10 4 1.0 x 10 2 8.0 x 10 2 S263.1 x 10 4 1.0 x 10 2 1.6 x 10 3 S273.3 x 10 4 -7.6 x 10 3 S283.1 x 10 4 -2.4 x 10 3 S291.9 x 10 4 -4.8 x 10 3 S303.6 x 10 3 -2.0 x 10 3 S311.6 x 10 3 -2.5 x 10 3 S327.3 x 10 3 8.0 x 10 2 2.7 x 10 3 S335.6 x 10 4 2.0 x 10 2 4.7 x 10 3 S341.5 x 10 3 -4.0 x 10 2 S351.2 x 10 3 -4.9 x 10 3

33. MicrorganismsGenusSpecies BacteriaBacillusB. thuringiensis B. pumilus B. humi B. lichenformis B. albus B. pseudomycoides B. amyloliquefaciens ArthobacterA. woluwensis A. globiformis KytococousK. sedentarius RalstoniaR. picketii ActinomycetesCorynebacteriumC. boris C. mycetoides BrevibacteriumB. epidermis RhodotococcusR. fascians YeastRhodotorulaR. minuta R. graminis R. pustula GuilliermondellaG. selenospora FilobasidiellaF. neoformansbacillisporus FellomycesF. fuzhouensis SporidiobolusS. johnsonii BulleromycesB. albus CryptococcusC. albidus FungiPeniciliumP. solitumwestling P. neoechinulatum CladosporiumC. herbarum FusariumF. tricinctum AcremoniumA. kiliense Identification using Biolog technique

34. GEN III microplate (left) showed purple colour -change pattern due to the carbon utilization by the bacteria after 24 hr of incubation. Based on the colour changes, bacteria was identified as Bacillus pumilus (squared in red) with the probability of 1.0 and 0.764 similarity. Microbes Identification based on Biolog technique

35. Bacterial culture (left) isolated from soil sample which identified as B.pumilus based on Biolog identification technique. Observation of B. pumilus (right) under light microscope using 40x magnification power. Bar=100µm

36. Bacterial population: 10 2 to 10 7 cfu/g of soil No growth was observed from soil sample of S6 on NA. Fungal population: only 10 2 cfu/g of soil. No growth was observed on PDA from soil of S3, S4, S7, S8, S13, S14, S15, S16, S18, S19, S27, S28, S29, S30, S31, S34 and S35. All soil samples (S1-S35) showed the presence of yeasts Yeasts population: 10 2 to 10 6 cfu/g of soil Oil Palm Bacterial populations: 10 5 to 10 8 cfu/g of soil Fungal population: 10 5 to 10 6 cfu/g of soil Yeasts population: 10 3 cfu/g of soil Forest

37. Conclusion

38. Soil microbial ecosystem is complex but combinations of microbes have the ability to enhance Oil Palm nutrient uptake, increase availability of nutrient to Oil Palm and suppress pathogens attack especially Ganoderma Ganoderma is weak competitor with many other microorganisms. We are the one who provides suitable soil condition for them to become the champion! Is extensive use of agrochemicals lead to depletion of microbial population in oil palm soil? Need further investigation More funding is required to accelerate research related to potential indigenous microbes for the betterment/enrichment of agriculture/plantation soil for the future.

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