bioRxiv preprint doi: https://doi.org/10.1101/351916; this version posted June 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Potential PGPR properties of cellulolytic, nitrogen- 2 fixing, and phosphate-solubilizing bacteria of a 3 rehabilitated tropical forest soil 4 Amelia Tang1¶*, Ahmed Osumanu Haruna123¶, Nik Muhamad Ab. Majid3¶ 5 1 Faculty of Agriculture and Food Sciences, Universiti Putra Malaysia Bintulu Campus, 6 Sarawak, Malaysia 7 2 Institute of Tropical Agriculture and Food Security (ITAFoS), Universiti Putra 8 Malaysia, Serdang, Selangor, Malaysia 9 3 Institute of Tropical Forestry and Forest Products (INTROP), Universiti Putra 10 Malaysia, Selangor, Malaysia 11 12 *Corresponding author 13 E-mail: [email protected] (AT) 14 ¶ These authors contributed equally to this work. 15 16 17 18 19 1 bioRxiv preprint doi: https://doi.org/10.1101/351916; this version posted June 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 20 Abstract 21 In the midst of major soil degradation and erosion faced by tropical ecosystems, 22 rehabilitated forests are established to avoid further deterioration of forest land. In this context, 23 cellulolytic, nitrogen-fixing (N-fixing), and phosphate-solubilizing bacteria are very important 24 functional groups in regulating the elemental cycle and plant nutrition, hence replenishing the 25 nutrient content in forest soil. As other potential plant growth-promoting (PGP) rhizobacteria, 26 these functional bacteria could have cross-functional abilities or beneficial traits that are 27 essential for plants and improve their growths. This study was conducted to isolate, identify, and 28 characterize selected PGP properties of these 3 functional groups of bacteria from tropical 29 rehabilitated forest soils at Universiti Putra Malaysia Bintulu Sarawak Campus, Malaysia. 30 Isolated cellulolytic, N-fixing and phosphate-solubilizing bacteria were characterized for 31 respective functional activities, biochemical properties, molecularly identified, and assessed for 32 PGP assays based on seed germination and indole-3-acetic acid (IAA) production. Out of 15 33 identified bacterial isolates exhibiting beneficial phenotypic traits, a third belong to genus 34 Burkholderia and a fifth to Stenotrophomonas sp. with both genera consisting of members from 35 two different functional groups. Among the tested bacterial strains, isolate Serratia 36 nematodiphila C46d, Burkholderia nodosa NB1, and Burkholderia cepacia PC8 showed 37 outstanding cellulase, N-fixing, and phosphate-solubilizing activities, respectively. The results 38 of the experiments confirmed the multiple PGP traits of selected bacterial isolates based on 39 respective high functional activities, root, shoot lengths, and seedling vigour improvements 40 when bacterized on mung bean seeds, as well as presented some significant IAA productions. 41 The results of this study indicated that these functional bacterial strains could potentially be 42 included in future biotechnological screenings to produce beneficial synergistic effects via their 43 versatile properties on improving soil fertility and possible crop growth stimulation. 44 Keywords: rehabilitated tropical forest soil; cellulolytic bacteria; N-fixing bacteria; phosphate- 45 solubilizing bacteria; PGP traits 2 bioRxiv preprint doi: https://doi.org/10.1101/351916; this version posted June 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 46 Introduction 47 Albeit vast in carbon and biodiversity, tropical ecosystems are facing major 48 deforestations, leading to widespread soil erosion which garnered much recent attention 49 [1-3]. As soil erosion aggravates, the soil loss overrides soil establishment, which 50 results in diminishing soil resources and the ecosystem supports they render [4-5], in 51 particularly manifested as low productivity and farming sustainability on the degraded 52 soils [6]. Planted forest or rehabilitation efforts are undertaken to avoid further 53 deterioration of forest land. 54 Bacteria has large biodiversity composition in soils, and thus, is major 55 participant of soil principal processes that regulate whole terrestrial ecosystems 56 operations, by means of completing nutrient, and geochemical cycles, including C,N,S, 57 and P [7-9]. In plant litter mass, 20-30% portion are constituted as cellulose, making the 58 latter as most abundant biopolymer [10]. Decomposition of cellulose has been 59 established to be primarily accountable by soil microorganisms, which assimilate the 60 simple sugars resulting from the reduction process of the complex polysaccharides [11- 61 12]. Despite the general ruling capabilities of fungi over bacteria in cellulose 62 decomposition in soils [13-14], these capabilities are also reserved to some, albeit 63 phylogenetically different taxa of bacteria [11]. Previous reports have emphasized 64 bacterial taxa which were recently discovered to possess cellulolytic capabilities, in 65 which they were formerly not known to reduce cellulose [15-16]. To this end, the study 66 of cellulolytic bacterial diversity in tropical soils is warranted. 67 Nitrogen needs by plant are supported by means of organic compound 68 degradation, atmospheric deposition of N, and biological nitrogen-fixation (BNF), 69 whereby BNF supplies 97% of natural N reserve [17] via Bacteria and Archaea [18-19]. 70 The N allocation in tropical ecosystems contributed by N-fixing microorganisms 3 bioRxiv preprint doi: https://doi.org/10.1101/351916; this version posted June 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 71 (diazotrophs), prevailing mostly in free-living condition, could range from 12.2 to 36.1 72 kg ha-1 year-1 [20]. Considering the importance of free-living diazotrophic communities 73 to ecosystem processes, there is a need to broaden our knowledge on their diversity in 74 rehabilitated tropical forests. This is essential because there is dearth of information of 75 this kind. 76 In most tropical soils which are usually very acidic, P is one of the most 77 commonly deficient macronutrients, owing to the prevalence of typically high Al and/or 78 Fe level, in which P is fixed as insoluble mineral complexes, causing unavailability of P 79 for root uptake [21]. Apart from chemical treatments, microorganisms have also been 80 applied over the years to improve nutrient availability, especially P in soils, as well as 81 alleviating Al toxicity [22]. Furthermore, tropical acid soils support only limited acid- 82 tolerant plant species and microbes. In soils, phosphate-solubilizers are predominated 83 by bacteria over fungi at 1-50% and 0.1-0.5%, of total population, respectively [23]. 84 Bacterial strains consisting of Pseudomonas, Bacillus, and Rhizobium have been cited to 85 be the dominant phosphate-solubilizers [24]. In addition, Burkholderia strains and 86 species which occur in broadly different natural niches have been frequently reported as 87 plant-associated bacteria, albeit their prevalence as free-living in the rhizosphere, as 88 well as epiphytic or endophytic, obligate endosymbionts, or phytopathogens [25]. The 89 phosphate-solubilizing bacteria could release substantial amount of organic acids crucial 90 for fixing Al via a chelation process, thus reducing Al toxicity to plant roots in the 91 highly weathered soils [26]. 92 As other potential PGPR, these functional bacteria could have cross-functional 93 abilities or beneficial traits, apart from their primary functional ability such as the 94 production of PGP phytohormones, polysaccharides, and organic acids that are essential 95 for plant growth and development [22]. In addition, IAA which is known to stimulate 4 bioRxiv preprint doi: https://doi.org/10.1101/351916; this version posted June 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 96 shoot elongation and/or in particularly root structures by those IAA produced by the 97 bacteria [27], could result in more efficient elemental nutrient acquisition by host plants, 98 leading to higher plant growth and development. To be specific, adoption of functional 99 bacterial strains with multiple functional and PGP traits in crop production would not 100 only promote sustainably increased crop productivity, the practice also offers an option 101 for minimizing costs related to chemical fertilizers application, while at the same time 102 reducing the pollution risks from rampant application of chemical fertilizers. 103 However, the isolation and characterization of these particular three types of 104 potential functional bacteria from tropical soils of rehabilitated forest remain