1 Bacterial community diversity in the rhizosphere of nickel 2 hyperaccumulator plant species from Borneo Island (Malaysia) 3 4 Séverine Lopez,1 Antony van der Ent,1,2 Sukaibin Sumail,3 John B. Sugau,3 Matsain Mohd 5 Buang,4 Zarina Amin,5 Guillaume Echevarria,1,2 Jean Louis Morel1 and Emile Benizri1 6 7 1Université de Lorraine, INRAE, Laboratoire Sols et Environnement, 54000, Nancy, France. 8 9 2Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of 10 Queensland, St Lucia, 4072, QLD, Australia. 11 12 3Sabah Parks, Kota Kinabalu, Sabah, Malaysia. 13 14 4Forest Research Centre, Sabah Forestry Department, Sandakan, Sabah, Malaysia. 15 16 5Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, 17 Malaysia. 18 19 20 SUMMARY 21 The Island of Borneo is a major biodiversity hotspot, and in the Malaysian state of Sabah, 22 ultramafic soils are extensive and home to more than 31 endemic nickel hyperaccumulator 23 plants. The aim of this study was to characterize the structure and the diversity of the 24 rhizosphere bacterial communities of several of these nickel hyperaccumulator plants and 25 factors that affect these bacterial communities in Sabah. The most abundant phyla were 26 Proteobacteria, Acidobacteria and Actinobacteria. At family level, Burkholderiaceae and 27 Xanthobacteraceae (Proteobacteria phylum) were the most abundant families in the 28 hyperaccumulator rhizospheres. Redundancy analysis based on soil chemical analyses and 29 relative abundances of the major bacterial phyla showed that abiotic factors of the studied sites 30 drove the bacterial diversity. For all R. aff. bengalensis rhizosphere soil samples, irrespective 31 of studied site, the bacterial diversity was similar. Moreover, the Saprospiraceae family showed 32 a high representativeness in the R. aff. bengalensis rhizosphere soils and was linked with the 33 nickel availability in soils. The ability of R. aff. bengalensis to concentrate nickel in its 34 rhizosphere appears to be the major factor driving the rhizobacterial community diversity unlike 35 for other hyperaccumulator species. 36 37 38 INTRODUCTION 39 Ultramafic soils cover 3% of the terrestrial surface (Guillot and Hattori, 2013; Echevarria, 40 2018). The largest areas are found in Cuba, New Caledonia, Philippines, Indonesia, Turkey and 41 Malaysia (van der Ent, 2015). Due to the nature of the ultramafic bedrock, they naturally contain 42 high metal concentrations, particularly nickel (Ni), cobalt (Co), chromium (Cr) and manganese 43 (Mn; Proctor and Woodell, 1975). They are also characterized by high concentrations of iron 44 (Fe) and magnesium (Mg), with a low calcium/magnesium ratio (Harrison and Rajakaruna, 45 2011). In addition, they have low levels of macronutrients such as nitrogen (N), phosphorus (P) 46 and potassium (K; Shallari et al., 1998; Boyd and Jaffré, 2009) and a low water holding capacity 47 (Kay et al., 2011). In the state of Sabah in Malaysia (Northern tip of Borneo Island), ultramafic 48 soils are extensive and cover an area of approximatively 3500 km2 (Proctor et al., 1988). This 49 region has a great floristic richness with about 8000 vascular plant species, including more than 50 5000 species inventoried in the Kinabalu Park (Beaman, 2005, van der Ent et al., 2015a). The 51 park was established in 1964 as the first fully protected area in Sabah, and has an area of 754 52 km2 around Mount Kinabalu, the highest mountain on the island of Borneo (4095 m a.s.l.). 53 54 Because of their extreme properties, ultramafic soils support a particular flora with high rates 55 of endemism which have evolved both morphological and physiological adaptations and allow 56 it to survive in these environments. Within this flora, hyperaccumulator plants have the ability 57 to concentrate metals in their aerial parts, whatever the concentration found in the soil (Baker, 58 1981). There are about 700 taxa of plants that are known to hyperaccumulate one or more 59 metals, and around 90% of these accumulate Ni (van der Ent et al., 2013; Pollard et al., 2014; 60 Galey et al., 2017; Reeves et al., 2017; van der Ent et al., 2017; Kidd et al., 2018; Nkrumah et 61 al., 2019). In the case of Ni hyperaccumulator plants, the threshold of Ni accumulation is at 62 least 1000 mg Ni kg-1 of dry weight in aerial parts (0.1% of plant biomass; Brooks et al., 1977). 63 Nickel hyperaccumulator plants occurring in Sabah mainly belong to the botanical families of 64 Phyllanthaceae, Rubiaceae, Salicaceae and Violaceae (Reeves, 2006). The known Ni 65 hyperaccumulators of this region are Rinorea aff. bengalensis and R. aff. javanica (Violaceae; 66 Brooks et al., 1977; Brooks and Wither, 1977), Phyllanthus balgooyi and Phyllanthus 67 rufuschaneyi (Phyllanthaceae; Baker et al., 1992; Hoffmann et al., 2003; MesjaszPrzybylowicz 68 et al., 2016; Bouman et al., 2018), Dichapetalum gelonioides (Dichapetalaceae; Baker et al., 69 1992; Nkrumah et al., 2018b), Psychotria sarmentosa (Rubiaceae; Reeves, 2003) and Shorea 70 tenuiramulosa (Dipterocarpaceae; Proctor et al., 1989). Since prospecting campaigns conducted 71 in Kinabalu Park, new nickel hyperaccumulator species have been discovered such as Actephila 72 alanbakeri (Phyllanthaceae; van der Ent et al., 2015a) in Kinabalu Park or Antidesma montis- 73 silam (Phyllanthaceae; Nkrumah et al., 2018c) at Mount Silam. 74 75 Due to the high concentrations of certain elements (Ni, Mg), ultramafic soils harbour specific 76 microbial communities (Schipper and Lee, 2004; Rajkumar et al., 2009; Branco, 2010; Lopez 77 et al., 2019a, 2019b). It has been shown that the presence of these elements can reduce microbial 78 biomass (Wang et al., 2008), affect microbial enzymatic activities (Kuperman and Carreiro, 79 1997; Kandeler et al., 2000) and consequently can alter the mineralization of soil organic matter 80 (Epelde et al., 2012). Based on highthroughput Illumina sequencing approaches, recent studies 81 have revealed that Proteobacteria, Actinobacteria and Acidobacteria are the major phyla in the 82 rhizosphere of Ni hyperaccumulator plants growing on ultramafic soils (Venter et al., 2018; 83 Visioli et al., 2019; Lopez et al., 2019a, 2019b). These phyla are commonly encountered as the 84 predominant taxa in agricultural, polluted or ultramafic soils (Janssen, 2006; Rastogi et al., 85 2010). Unlike other environments, Ni hyperaccumulator plant rhizospheres typically have a 86 high representation of the Chloroflexi phylum (Lopez et al., 2017). 87 88 The Island of Borneo is known to be one of the major hotspots of biodiversity for plants and 89 vertebrates (Myers et al., 2000), however there is a paucity of data available on the soil bacterial 90 diversity present in this region (Tripathi et al., 2012; Lee-Cruz et al., 2013). There is currently 91 no known study on the rhizobacterial diversity of Ni hyperaccumulator plants from Borneo 92 Island. The rhizobacterial communities can, on the one hand, provide mineral elements 93 necessary for the plant and, on the other hand, improve their resistance to stress such as those 94 induced by metals (Lebeau et al., 2008). Moreover, the bacteria can also affect mobility and the 95 availability of metals present in the soil, favouring or not their transfer from the rhizosphere to 96 the plant and therefore phytoextraction (Kidd et al., 2009; Sessitsch et al., 2013). The aims of 97 this study were to characterize the structure and the diversity of the rhizosphere bacterial 98 communities of Ni hyperaccumulator plants, and to highlight the physicochemical factors that 99 may influence the composition of these bacterial communities. 100 101 RESULTS 102 103 Study area and sample collection 104 During fieldwork, 52 Ni hyperaccumulator plants and their associated rhizosphere soil were 105 sampled from 10 different ultramafic sites (Table 1 and Fig. 1) around Mount Kinabalu Park. 106 The samples were collected associated with the following species: Flacourtia kinabaluensis 107 Sleumer (Salicaceae), Mischocarpus sundaicus Blume (Sapindaceae), Phyllanthus balgooyi 108 Petra Hoffm. & A.J.M. Baker, P. rufuschaneyi Welzen, R.W. Bouman & Ent (Phyllanthaceae), 109 Psychotria sarmentosa Blume (Rubiaceae), Rinorea aff. bengalensis (Wall.) Kuntze, R. aff. 110 javanica Kuntze, Rinorea sp. (Violaceae) and Walsura pinnata Hassk. (Meliaceae). The highest 111 plant diversity was found at sites 06 and 10, with six plant species harvested. Site 01, with the 112 highest elevation (1410 m a.s. l.) has only one species: P. balgooyi. Flacourtia kinabaluensis 113 and M. sundaicus showed a better geographical repartition (found in 6 of the 10 sites), followed 114 by P. balgooyi and P. sarmentosa (5 sites), R. aff. javanica (4 sites), R. aff. bengalensis, the 115 undetermined Rinorea sp. and W. pinnata (3 sites) and finally P. rufuschaneyi (2 sites). 116 117 Chemical analyses of rhizosphere soil samples from each site 118 Abiotic soil parameters were measured as seen in Tables 2–4. The mean pH ranged from 6.0 119 (Sites 01 and 05) to 7.4 (Site 07) with an average pH of 6.6 (Table 2). The C/N ratio of the 120 rhizosphere soils showed significant differences (P < 0.05) depending on the site. Sites 01 and 121 05 had statistically the highest C/N ratio (21 and 20 respectively) in comparison to sites 02, 03, 122 06, 07 and 09 (lower than 14). Due to its rhizosphere origin, the soil had an organic C content 123 corresponding to an average of 96% of the total C. The elements that were most extracted by 124 DTPA in the rhizosphere soils were Ni (193 μg g-1), Fe (140 μg g-1) and Mn (102 μg g-1) as 125 seen in Table 3. There were no significant differences for DTPA-extractable Ni and Zn among 126 the different rhizosphere soil samples. Although Cr was present in soil samples (total Cr), this 127 element was poorly available in the rhizosphere soils (<1 μg g-1). While site 09 had the highest 128 Co DTPA-extractable (29 μg g-1), Site 01 had the highest Cu (14 μg g-1) and Pb (5.8 μg g-1) 129 DTPA-extractable.