EXPLORING GROWTH ENHANCING RHIZOSPHERIC MICROORGANISMS FOR SILVICULTURE OF NEOLAMARCKIA CADAMBA by ALAN CHUA YEE QUAN A thesis presented in fulfilment of the requirement for the degree of Master of Science by Research at Swinburne University of Technology 2018 Abstract Neolamarckia cadamba (N. cadamba) is an indigenous timber tree that was selected to rehabilitate degraded forest lands in Sarawak. Application of chemical fertilisers is a standard practice to support growth of N. cadamba on degraded forest lands. The negative impacts of chemical fertilisers on environments and human health are always raised up as public concerns. Thus, alternatives like plant growth enhancing microorganisms are introduced to agricultural and silvicultural sectors. Plant growth enhancing microorganisms were widely reported in the studies of agricultural crops but seldom presented in articles of silviculture. N. cadamba was reported to grow naturally in primary and secondary forests. It is possible that these microorganisms that co-exist in the natural rhizospheres of N. cadamba may enhance growth of host plants either by increasing the availabilities of nutrients for root uptake or promoting the root growth. The objectives of this study are to isolate and characterise such plant growth enhancing microorganisms from N. cadamba rhizospheric soils in Sarawak to formulate microbial consortia, and evaluate their plant growth enhancing effects through pot trial using N. cadamba seedlings. The N. cadamba rhizospheric soil samples were collected from Kubah National Park in Matang, Similajau National Park in Bintulu, Sabal Forest Reserve in Simunjan, Semenggoh Nature Reserve in Siburan and a planted forest in Bintulu, Sarawak. From the soil analysis, soil textures in these locations were ranging from sandy clay loam, sandy loam to clay. As for the soil pH, Semenggoh Nature Reserve had the lowest pH of 3.86 and Kubah National Park had the highest pH of 5.46. From these soil samples, 41 strains of plant growth enhancing microorganisms were successfully isolated and screened based on four functional traits namely nitrogen fixation, phosphate solubilisation, potassium solubilisation and plant hormone indole-3- acetic acid (IAA) production using Jensen’s medium, Pikovskaya’s agar, modified Aleksandrov’s medium and tryptophan broth respectively. The isolated strains could render the availabilities of nitrogen, phosphorus and potassium which are essential macronutrients for plants, as well as produce IAA, a hormone generally known to stimulate root growth. These selected strains were genetically identified as Methylobacterium sp., Pseudomonas spp., Stenotrophomonas sp., Streptomyces sp., Streptacidiphilus sp., Lysinibacillus spp., Serratia sp., Bacillus spp., Arthrobacter sp., Metarhizium spp., Phanerochaete sp. and Penicillium sp. Their I efficiencies in nitrogen, phosphate, potassium and IAA production were evaluated using phenate method, vanadomolybdophosphoric acid colorimetric method, atomic absorption spectroscopy (AAS) and Salkowski’s method. After that, four best strains namely Streptomyces N11 (nitrogen fixation), Serratia P8 (phosphate solubilisation), and Bacillus K5 (potassium solubilisation) and Bacillus I6 (IAA production) were selected for further in-vitro experiment. To ascertain plant growth enhancing properties of the microbial strains, a small scale pot experiment was conducted using N. cadamba seedlings in 10 replicates for each of the treatment. Each strain used for formulation of consortia would reach the recommended concentration range of 107-108 cfu/mL. The first consortium labelled as Consortium A contained nitrogen fixing, phosphate solubilising and potassium solubilising strains. The second consortium labelled as Consortium B included all the three strains as in Consortium A but with the addition of IAA producing strain. Besides using per se as treatments in the pot experiment, each consortium was also mixed with chemical fertiliser. The Consortium A-chemical fertiliser and Consortium B- chemical fertiliser were therefore the two integrated treatments in which each consortium was mixed with half regime of chemical fertiliser. The results showed that both consortia exerted significant plant growth enhancing effects on both the shoot and root growths comparing to negative control which was without any fertiliser. Among the consortial treatments, Consortium B containing IAA producing strain showed significantly better shoot and root growth results than Consortium A. Besides that, Consortium A-chemical fertiliser and Consortium B-chemical fertiliser were found to be as effective as full regime chemical fertiliser in promoting the shoot growths. Similar experience from Consortium B, Consortium B-chemical fertiliser showed significant plant growth enhancing effect on root growth to both Consortium A-chemical fertiliser treatment and full regime chemical fertiliser treatment. Consortium A-chemical fertiliser showed on par root growth result to full regime chemical fertiliser treatment. In conclusion, the study showed that indigenous microbial strains of Sarawak could enhance the growth of N. cadamba effectively either in consortial forms or in combination with half regime of chemical fertiliser. The integrated consortium-chemical fertiliser treatments were as good as full regime chemical fertiliser in enhancing the growth of N. cadamba seedlings in this study. As such, through using such microbial strains, the usage of chemical fertiliser could be substituted or reduced for silviculture of N. cadamba. II Acknowledgement First and foremost, I would like to say thank you to my principal coordinating supervisor, Dr Daniel Tan Lee Tung for his generous guidance and support throughout my master project. In addition, I would like to convey my appreciation to my co-supervisors, Associate Professor Peter Morin Nissom and Associate Professor Moritz Müller for their continuous academic supports throughout my master project. I would like to thank Sarawak Forestry Cooperation for their kind support to conduct the research under Memorandum of Understanding (Adjudication No. G01L003313-Q040). I would like to thank Sarawak Biodiversity Centre for their kind assistance and advice. I am grateful to Sarawak Timber Association for funding and assistance for this project under Memorandum of Agreement (Adjudication No. C01K966016-Q040). In addition, I would like to send my appreciation to John Sabang from Seed Bank for his advice in establishing field and nursery activities. Furthermore, I would like to thank Associate Professor Wallace Wong Shung Hui, Associate Professor Ng Sing Muk and Research Consultancy Office Swinburne Sarawak staff members sincerely for their assistances in candidature matters. I would also like to appreciate Science Team from Faculty of Engineering, Computing and Science in Swinburne University of Technology Sarawak Campus for their kind assistances and laboratory supports. Finally, I would like to thank my family members for their continuous support and unbeatable encouragement throughout my master project. In addition, I would like to convey my appreciation to my lab buddies, Chan Wen Loong, Mertensia Kho Kia Chen, Stanley Ho Han Kiat, Wendy Ong Han Sze, Cindy Wee Shui Shui, Phua Ye Li, Nurnajwani Senian, Ang Chung Huap, Fiona Chung Yi Li, Wong Changi, Johnny Tang Jock Lee and Phyllis Jacqueline Nishi for their experience sharing and assistances in conducting experiments. III Declaration I, Alan Chua Yee Quan, candidate of Master of Science by Research from Faculty of Engineering, Computing and Science in Swinburne University of Technology Sarawak Campus hereby declare that my master thesis entitled “Exploring growth enhancing rhizospheric microorganisms for silviculture of Neolamarckia cadamba” is original writing outcome and contains no material or content which has been accepted for the award to the stated candidate of any other degree or diploma studies, except where due references are made in the text of the examinable outcomes; and where the work is based on joint research or publications, the disclosed relative contributions of the respective workers or authors. Alan Chua Yee Quan As the principal coordinating supervisor, I hereby acknowledge and verify that the above mentioned statements are legitimate to the best of my knowledge. Dr Daniel Tan Lee Tung IV Conference presentations Chua, AYQ, Müller, M, Ginjom, IR, Sabang, J, Nissom, PM & Tan, LT 2016, ʻExploring growth enhancing rhizospheric microorganisms for silviculture of Neolamarckia cadambaʼ, 33rd Symposium of the Malaysian Society for Microbiology 2016, Ramada Plaza, Melaka, Malaysia, December 14-17, 2016. V Thesis Outline The thesis contents were distributed in five chapters and listed accordingly as follow: Chapter 1: Introduction and literature review Chapter 1 would include a brief introduction on the importance of this study. The literature review would mention the relevant studies on forest plantations in Sarawak, Neolamarckia cadamba, tropical soil physicochemical properties, forest land degradation, chemical fertilisers and plant growth enhancing microorganisms. Besides these, the aim and objectives of this study would be stated. Chapter 2: Isolation and characterisation of plant growth enhancing microorganisms from Neolamarckia cadamba rhizospheres Chapter 2 would focus on the isolation of plant growth enhancing microorganisms from rhizospheric soil samples of N. cadamba mature trees and seedlings. The isolated strains were screened using selective media