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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
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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
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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
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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
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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 scarce.
105 Thus, the aims of this study were to: 1) isolate and identify cellulolytic, N-fixing, and
106 phosphate-solubilizing bacteria from a rehabilitated tropical forest soil at Universiti
107 Putra Malaysia Bintulu Sarawak Campus, Malaysia, and 2) characterize cellulolytic, N-
108 fixing, and phosphate-solubilizing bacteria for their respective functional activities, such
109 as IAA production, early plant growth promotion, some phenotypic, and biochemical
110 properties.
111
112 Materials and Methods
113 Soil sampling
114 Soil samples were collected from a rehabilitated forest site at Universiti Putra
115 Malaysia Bintulu Sarawak Campus through random sampling design using aseptic
116 techniques near rhizospheric zones of planted trees. Soil samples were collected at 0-10
117 cm depth using an auger. Prior to soil sampling, any litter at the surface was manually
118 removed [28]. The plastic bags with soil samples were transported to the laboratory at
119 Universiti Putra Malaysia Bintulu Sarawak Campus for analysis. The soil samples were
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120 homogenized or mixed thoroughly to make composite (consolidated) samples, and
121 surface organic materials, litter, roots, rocks, and macro fauna were removed. Soil
122 samples were kept in tightly sealed plastic bags and stored at 4 °C (for less than 3 days
123 before microbial analysis) to keep the soil moist, and to preserve biological properties.
124
125 Isolation of free-living nitrogen-fixing bacteria
126 Ten-fold serial dilution technique was used to determine the CFU number of the
127 culturable N cycle functional bacteria. Bacterial colonies were enumerated on N-free
128 malate (Nfb) medium [29] with some modifications containing per litre: 0.4 g of
129 KH2PO4, 0.1 g of K2HPO4, 0.2 g of MgSO4.7H2O, 0.1 g of NaCl, 0.02 g of CaCl2, 0.01
130 g of FeCl3, 0.002 g of MoO4Na.2H2O, 5.0 g of sodium malate, 5 mL of bromothymol
131 blue with 0.5% alcohol solution and 15.0 mL of agar. The whole media content were
132 adjusted to pH 7 before being sterilized in autoclave (121 oC for 20 minutes).
133 Approximately 20 mL of media was placed in each petri dish before being incubated at
o 134 30 C after triplicate inoculation. The colonies that fix N2 alkalinized the culture
135 medium (blue) due to ammonia production. Some colonies exhibiting large and instant
136 blue halo on the medium were isolated, purified on new Nfb media, sub cultured on
137 agar slants, and stored at 4 oC for further studies.
138
139 Isolation of phosphate-solubilizing bacteria
140 Cells and spores were detached from soil surface by shaking at 150 rpm a
141 mixture of 5 g of fresh soil and 100 mL of sterile distilled water for 24 hr. Supernatant
142 was ten-fold serially diluted in sterile distilled water and 0.1 mL aliquots in triplicate
143 were spread over Petri dishes containing differential growth media for phosphate-
144 solubilizing bacteria. This isolation technique is based on the formation of halos around
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145 the colonies of bacteria capable to solubilize a calcium phosphate insoluble compound
146 [30-31]. The inoculated plates were incubated at 30 °C. Culture media employed to
147 differentially isolate phosphate-solubilizing bacterial strains consisted of variations of a
148 chemically defined medium [32-33] with some modifications. All media contained per
-1 -1 149 litre: 50 mL of a salts solution (MgCl2·6H2O, 100 g L ; MgSO4·7H2O, 5 g L ; KCl, 4 g
-1 -1 150 L ; (NH4)2SO4, 2 g L ), 10 g of sucrose as C source, and 2.5 g of tricalcium phosphate,
-1 151 Ca3(PO4)2, as an insoluble source of phosphate. Six mL L of a pH indicator dye
152 (bromophenol blue, 0.4% ethanol solution) was added to the media [30]. For
153 solidification, 20 g L-1 of agar was added and pH of medium was adjusted to 7.
154 Phosphate-solubilizing bacterial colonies exhibiting large and instant halos on the media
155 were isolated, further purified on new media, sub cultured on agar slants, and stored at 4
156 oC for further studies.
157
158 Isolation of cellulolytic bacteria
159 This procedure was modified from those as described by Hendricks et al. [34].
160 The cellulolytic bacteria was enumerated by triplicate spread plate inoculation onto
161 cellulose-Congo red agar with 10-fold sterile tap water dilutions of soil, and incubated
o 162 at 30 C. The cellulose-Congo red agar consisted of 0.50 g of K2HPO4, 0.25 g of
163 MgSO4, 1.88 g of cellulose microgranular powder, 0.20 g of Congo red, 5.00 g of agar,
164 2.00 g of gelatin, 100 mL of soil extract, and 900 mL of tap water (used to provide
165 essential trace elements for soil bacteria), with pH adjusted to 7. Following incubation,
166 the colonies exhibiting zones of clearing were counted. Some colonies exhibiting large
167 and instant zones of clearing were isolated, further purified on the same media, sub
168 cultured on agar slants, and stored at 4 oC for further studies.
169
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170 Plate assays for cellulose-hydrolyzing, nitrogen-fixing, and
171 phosphate-solubilizing activities
172 Plate assay for cellulase activity by cellulolytic isolates
173 Based on clear zones formation on cellulose-Congo red agar as modified from
174 Hendricks et al. [34], cellulolytic bacteria were isolated and evaluated for their
175 cellulose-hydrolyzing index values using same medium with following composition:
-1 -1 -1 176 K2HPO4, 0.50 g L ; MgSO4, 0.25 g L ; cellulose powder, 1.88 g L ; Congo red, 0.20 g
177 L-1; agar, 15.00 g L-1; gelatine, 2.00 g L-1; 100 mL of soil extract, and 900 mL of tap
178 water to supply essential trace elements. The medium was maintained at pH 7.
179 Following incubation at 30 °C for over 10 day-period, cellulolytic isolates were
180 assessed for their selective abilities index values based on calculation of ratio of total
181 diameter (colony + halo or clearing zone around colonies) and colony diameter [35].
182
183 Plate assay for nitrogen-fixation activity by nitrogen-fixing isolates
184 Nitrogen-fixing bacteria were cultured on N-free malate (Nfb) medium modified
185 from Döbereiner and Day [29] and incubated at 30 ºC. Bacterial colonies with blue halo
186 formation were isolated and characterized using the same media, after which blue halo
-1 187 index values were calculated. The Nfb medium consisted of: KH2PO4, 0.4 g L ;
-1 -1 -1 -1 188 K2HPO4, 0.1 g L ; MgSO4.7H2O, 0.2 g L ; NaCl, 0.1 g L ; CaCl2, 0.02 g L ; FeCl3,
-1 -1 -1 -1 189 0.01 g L ; Mo.O4Na.2H2O, 0.002 g L ; sodium malate, 5 g L ; agar, 15 g L ;
190 bromothymol blue (0.5% in alcohol solution), 5 mL L-1. The medium was adjusted to
191 pH 7. Following incubation at 30 °C for over 10 day-period, all N-fixing isolates were
192 assessed for their selective abilities index values based on calculation of ratio of total
193 diameter (colony + halo or clearing zone around colonies) and colony diameter [35].
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194 Plate assay for phosphate-solubilization activity by phosphate-
195 solubilizing isolates
196 Phosphate-solubilizing bacterial isolation technique is based on the formation of
197 clear halos around the colonies capable to solubilize calcium phosphate [30-31]. Media
198 containing insoluble phosphate [32-33] with some modifications were used to isolate
199 and assess the phosphate-solubilizing bacterial activities. Its composition was: 50 mL L-
1 -1 -1 -1 200 of a salts solution (MgCl2·6H2O, 100 g L ; MgSO4·7H2O, 5 g L ; KCl, 4 g L ;
-1 -1 -1 -1 201 (NH4)2SO4, 2 g L ); agar, 15 g L ; 10 g L of sucrose as C source, and 2.5 g L of
202 tricalcium phosphate, Ca3(PO4)2, as an insoluble source of phosphate, and content was
203 adjusted to pH 7 before being autoclaved. Six mL L-1 of a pH indicator dye
204 (bromophenol blue, 0.4% ethanol solution) was also added to the media [30]. Following
205 incubation at 30 °C for over 10 day-period, phosphate-solubilizing bacterial isolates
206 were assessed for their selective abilities index values based on calculation of ratio of
207 total diameter (colony + halo or clearing zone around colonies) and colony diameter
208 [35].
209
210 Quantitative assays for functional activities
211 Quantitative assay for cellulase activity
212 For quantitative determination of cellulase activity, cellulolytic isolates were
213 previously inoculated in nutrient broth, shaken at 120 rpm at 30 °C for 16 hr and size of
6 7 214 the culture as preinoculum for cellulase assay was fixed to OD600=0.5 (10 to 10 cfu
215 mL-1). One mL of each isolate preinoculum was seeded in 40 mL cellulose broth as
216 basal medium containing cellulose as sole C source. The composition of cellulose broth
-1 -1 -1 217 per litre was: 5.0 g L cellulose microgranular, 2.5 g L NaNO3, 1.0 g L KH2PO4, 0.6
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-1 -1 -1 -1 -1 218 g L MgSO4·7H2O, 0.1 g L NaCl, 0.1 g L CaCl2·6H2O, 0.01 g L FeCl3, 2.0 g L
219 gelatin, and 0.1 g L-1 yeast extract with pH 7 adjusted using diluted NaOH as modified
220 from Lu et al. [36]. Inoculated broth was incubated at 30 °C under agitation at 180 rpm
221 and CMCase activity was analyzed for up to 5 days. For CMCase activity, 0.5 mL
222 culture filtrate from broth as crude enzyme source, or supernatant cellulase, also
223 specified as CMCase, was incubated with 1 mL of 1% carboxymethylcellulose in 0.1 M
224 citrate buffer, pH 5 at 40 °C in agitation for 30 min. The amount of reducing sugars
225 (glucose) was measured using Somogyi-Nelson method [37-38] at 520 nm. One unit of
226 enzyme activity (IU mL-1) is equivalent to the amount of enzyme per mL culture filtrate
227 that liberates 1 µg of glucose per minute [39].
228
229 Quantitative assay for in vitro biological nitrogen-fixation activity
230 The procedure for quantitative estimation of BNF activity was modified from
231 those described by Soares et al. [40]. Each N-fixing isolate was grown as a preinoculum
232 in nutrient broth under agitation at 120 rpm at 30 oC for 16 hr. Optical density was used
6 7 233 to adjust the inoculum size [OD600= 0.5 is equivalent to 10 to 10 colony forming units
234 (cfu) mL-1]. Inoculum was transferred (100 µL) to new media, NFb broth (containing
235 similar composition as Nfb medium except without agar). The inoculated media were
o 236 incubated at 30 C under constant agitation for 72 hr for N2 fixation quantification. A
237 control medium was also included by using 100 µL nutrient broth as inoculant. The
238 amount of N2 fixation was measured by sulfur digestion and distillation with NaOH 10
239 mol L-1, as described by Bremner and Keeney [41] and done in triplicates. The samples
240 were digested with 2 mL concentrated H2SO4 (d = 1.84), 1 mL H2O2 30% and 0.7 g
o 241 digestion mixture (100 g Na2SO4 + 10 g CuSO4.5H2O + 1 g selenium) at 350–375 C.
242 The products of digestion were distillated with 10 mL NaOH 10 mol L-1, and the
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243 ammonia trapped in 10 mL of 2% boric acid was measured by titration with H2SO4
244 0.0025 mol L-1.
245
246 Quantitative assay for inorganic phosphate-solubilization activity
247 Quantitative estimation of inorganic phosphate-solubilization was done with
248 some modifications from Delvasto et al. [42]. Preinoculum cultures were previously
249 prepared by inoculating phosphate-solubilizing isolate in nutrient broth under agitation
6 7 250 at 120 rpm at 30 °C for 16 hr. Inoculum size was adjusted to OD600=0.5 (10 to 10 cfu
251 mL-1). Prior to the assay, preinoculum cultures were washed 3 times and resuspended
252 using saline solution 0.85% NaCl, and one mL isolate suspension was inoculated into
253 100 mL liquid medium containing insoluble calcium phosphate as in solid medium,
254 except without agar. Inoculated broth was incubated at 30 °C under agitation at 180 rpm
255 for up to 5 days. Saline solution without inoculant in the broth was left as control.
256 Soluble P content in culture filtrates was determined by the molybdenum blue
257 method [43]. Appropriate amount of aliquots from samples containing 1-50 µg mL-1 P
258 were pipetted into 50 mL volumetric flasks containing approximately 15 mL distilled
259 water and 8 mL ascorbic acid antimony sulfomolybdo working reagent (as color
260 developing solution to form blue color complex in the present of P from samples and/or
261 standard solutions), swirled to mix, and filled to the mark with distilled water. A series
262 of standard P solutions (KH2PO4) aliquots were pipetted into a set of volumetric flasks
263 containing the same amount of color developing solutions to make up the range of
264 expected P concentrations in the culture filtrates, swirled to mix, and made up to the
265 mark with distilled water. A blank solution equal to the aliquot size of sample was also
266 included to each flask of standard solutions. A standard curve was plotted using UV-vis
267 spectrophotometer (Unico) at 882 nm. The absorbance of samples were measured by
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268 means of the standard curve using the same wavelength and converted into P
269 concentrations. Values of soluble P are expressed as µg mL-1. The final pH of liquid
270 medium was measured using a pH meter. All values of pH and P concentrations in
271 samples were measured in triplicates.
272
273 Morphological and physiological characterization of selected
274 isolates
275 Selected isolates were each studied for morphological properties, which
276 included color, size, and colony characteristics such as form, margin, and elevation on
277 nutrient agar. The spore staining procedure, done according to Schaeffer-Fulton method
278 and standard Gram staining procedure were conducted as described by Cappuccino and
279 Sherman [44], both with some modification on fixation, whereby bacterial smears were
280 fixed using methanol, without heat fixation. For each procedure, the smears were finally
281 examined under oil immersion at total magnification power of 1000 times. The
282 morphology of each bacterial colony such as the color, shape, and arrangement of the
283 stained cells were recorded. Any presence of endospores for each studied bacterial
284 isolate was also noted.
285 The biochemical tests consisting of several enzymatic, physiological activities,
286 or differential growth tests were carried out. Citrate utilization was determined on
287 Simmons citrate agar; hydrogen sulfide production, along with lactose and glucose
288 fermentation were determined on Kligler Iron Agar; Gram negative strains and lactose
289 fermentation were detected using Mac Conkey agar; gelatinase production, starch
290 hydrolysis, casein proteolysis, lipase production, and indole production were each
291 determined using nutrient gelatine, starch agar, skim milk agar, Tween 80 agar and
292 BD’s Indole Reagent Droppers (modified Kovacs’ reagent) via standard tube method,
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293 respectively. Catalase test was done using 3% hydrogen peroxide solution. Motility test
294 was conducted on Biolife’s Motility Medium whereas nitrate reduction test was done
295 using the test kit by Fluka. The aforementioned physiological and biochemical test
296 results were processed and compared with the results of systematic bacteriology
297 identification according to Bergey’s Manual of Determinative Bacteriology and
298 Bergey’s Manual of Systematic Bacteriology [45-46].
299
300 Molecular method for bacterial identification
301 Deoxyribonucleic acid extraction
302 Bacterial genomic DNA was extracted using sodium dodecyl sulfate (SDS)
303 method. Bacterial inoculum from streaked culture plate overnight was recultured
304 overnight in 1.5 mL nutrient broth in capped tubes with one control (broth without
305 inoculum) in the water bath shaker at 30 oC. The 1.5 mL cell suspension was
306 centrifuged at 13040 g for 3 min at 4 oC. After removing the supernatant, the cells were
307 resuspended or washed with 180 µL TE buffer (10 mM Tris, 1mM EDTA, pH 8.0) by
308 vortex-mixing and incubated on ice for 5 min. Seventy five µL of 2% SDS solution was
309 added, followed by invert-mixing step to lyse the bacterial cells and incubation on ice
310 for 5-10 min. Then 250 µL of 3 M potassium acetate was added, followed by gentle
311 invert-mixing, and incubation on ice for 5 min. The sample was subsequently
312 centrifuged at 13040 g for 5 min at 4 oC, with special attention on the cells pellet
313 formation at the bottom of the tube. The supernatant containing bacterial DNA was
314 carefully transferred to a clean 1.5 mL tube on ice. Absolute ethanol was then added at
315 approximately two times the volume of the supernatant on the same tube, invert-mixed,
316 and followed by centrifugation at 12000 g for 10 min at 4 oC.
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317 Later, supernatant was discarded and 1 mL of 75% ethanol was added to the
318 DNA pellet for washing step. DNA pellet was resuspended by pipette-mixing the
319 suspension in repeated gentle motion, followed by centrifugation at 12000 g for 10 min
320 at 4 oC. The supernatant was discarded with extra care taken at this stage so as not to
321 disrupt the DNA pellet while at the same time ensuring the entire removal of ethanol
322 residues from the pellet and within the tube itself. The pellet was air-dried for about 10
323 to 20 min, till no ethanol residue is visible. Finally, 20 µL of TE buffer (10 mM Tris,
324 1mM EDTA, pH 8.0) was added, pipette-mixed gently, and stored at -20 oC.
325
326 Polymerase chain reaction amplification of extracted DNA
327 Amplification of DNA extraction products was performed in a final volume of
328 25 µL each. For each reaction, 1.25 µL 0.5 µM for each of both primers (16S-F and
329 16S-R1), 2.5 µL 25mM MgCl2, 2.5 µL 10X reaction buffer [(NH4)2SO4], 0.5 µL
330 working stock dNTPs or dNTP mix combined to give a final concentration of 0.2 mM
331 each, 0.25 µL of Taq DNA Polymerase (5 U/µL), 15.75 µL of sterilized distilled water
332 and 1 µL of template DNA. Primers 16S-F and 16S-R1 had the sequences of 5’-
333 AAGAGTTTGATCATGGCTCA-3’ and 5’-
334 TAAGGAGGTGATCCAACCGCAGGTTC-3’, respectively. The PCR was performed
335 in a thermal cycler (Bioer XP Cycler) using cycling conditions consisting of an initial
336 denaturation at 95 oC for 3 min, followed by 35 cycles consisting of denaturation at 94
337 oC for 30 s, annealing at 50 oC for 30 s, and extension at 72 oC for 1 min. A final
338 extension was performed at 72 oC for 5 min. A blank that contained all the components
339 of the reaction mixture without the DNA template was included as a control. The PCR
340 products were analyzed by running on 1% agarose gel electrophoresis.
341
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342 Agarose gel electrophoresis
343 A 1% (w/v) agarose gel was prepared by mixing 0.25 g agarose and 25 mL of
344 1X TAE buffer. The solution was heated gently in a microwave oven until the agarose
345 was fully dissolved. The gel was allowed to cool to the touch before being poured to the
346 gel cask with the well comb in place. The gel was allowed to solidify for 20-30 minutes
347 before removing the comb and mould barriers, placed onto electrophoresis tank, and 1X
348 TAE buffer solution was poured evenly into the electrophoresis tank until it just
349 submerged the surface of the gel. The samples were each prepared by combining 5 µL
350 of PCR products with 3 µL of 6x loading dye and 1 µL of EZ Vision dye before being
351 loaded into the performed wells. One µL of DNA marker or ladder bearing 1.5 kb size
352 was mixed well with 2 µL of 6x loading dye, 1 µL of EZ Vision dye, and 5 µL of 1x
353 TAE buffer solution before being loaded into assigned well. The gel was then run at 90
354 V for about an hour. The EZ Vision dye-stained fragments were visualized and
355 photographed with UV light in a gel imager (AlphaInnotech Fluorchem). Any PCR
356 bands obtained for the specific isolates were recorded and the gel image was captured.
357 Once PCR bands were sighted on the gel image, preparations of the samples were done
358 which included PCR scale-up to 50 µL per reaction, and purification of PCR products
359 prior to sending them out for outsourced sequencing service.
360
361 Purification of PCR products for sequencing
362 Direct precipitation is a general method used for purification of PCR products.
363 Fifty µL of PCR product was transferred to a 1.5 mL tube. Sodium acetate (pH 8) at 0.1
364 volume or equivalent to 4 µL was added into the tube, followed by absolute ethanol
365 (99%) at 2.5 volume or equivalent to 100 µL. The tube was inverted a few times for
366 homogeneous mixing and kept in ice for 30 min. The tube was centrifuged at 13,040 g
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367 for 15 min at 4 oC. Supernatant was decanted and 200 µL of 75% ethanol was added
368 into the tube, followed by vortex-mixing for 1 min. The tube was centrifuged at 13,040
369 g for 5 min at 4 oC. The pellet was air-dried until no traces of ethanol could be seen.
370 Fifteen µL of sterile distilled water was added to resuspend the pellet by tapping. The
371 concentrated PCR product was short-spun and ready to be sent for sequencing. The
372 quantification or estimation of the concentration of the concentrated PCR products was
373 done by using the gel imager software (FluorChem 5500) which was within the range of
374 5 to 100 ng per µL.
375
376 Sequencing of 16S rDNA
377 Determination of the 16S rDNA nucleotide sequences was done by transferring
378 the purified PCR products containing the concentrated amplified DNA, along with
379 primer R2 to First BASE Laboratories Sdn Bhd, a life sciences-based research company
380 located in Peninsular Malaysia.
381
382 Identification of bacterial sequences
383 The sequences obtained for selected bacterial isolates were scanned manually
384 for any errors on the bases interpretation provided by the base sequencing service which
385 consisted primarily of adenine (A), guanine (G), cytosine (C), and thymine (T) bases.
386 The sequence bases were analyzed using Sequence Scanner Software v1.0 which was
387 registered under Applied Biosystems®. Each of the corrected sequences was searched
388 on BLAST website which compared nucleotide or protein sequences to sequence
389 databases and calculated statistical significance of matches. The BLAST website was
390 available at http://blast.ncbi.nlm.nih.gov/Blast.cgi. The closest matches of top-scoring
391 sequences from the BLAST databases were used to presume the identity or closest
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392 identities of the particular bacteria. In the case of multiple identities produced based on
393 sequence matches alone, biochemical properties offered phenotypic information
394 necessary for further comparison in order to finalize each isolate to only one most
395 presumed identity.
396
397 Plant growth-promoting assays
398 Germination assays of maize and green gram seeds
399 Phytotoxic assay for 15 selected bacterial inoculants on green gram seeds were
400 done via germination check of both root and shoot of the germinating seeds prior to pot
401 experiment. Seeds of green gram (Vigna radiata) were obtained from a local market.
402 Prior to the assay, the seeds were sorted for uniformity of size, and all visibly damaged
403 seeds were discarded. The remaining seeds were washed with tap water and surface
404 sterilized in 0.1% sodium hypochlorite solution for 5 min before rinsing at least three
405 times with sterile distilled water to thoroughly remove sterilants. The seeds were then
406 soaked in broth containing 16 hr cultured bacterial isolates according to treatments for 2
407 hr at 30 °C. The seed treatments consisted of 15 selected bacterial isolates. Two control
408 treatments were included whereby first control was designated as seeds treated with
409 sterile distilled water, and another control treatment with sterile nutrient broth.
410 The treated green gram seeds were placed on petri dishes containing sterilized
411 moist sheets of Whatman filter paper per treatment. Each treatment was based on 100
412 seeds of each plant type. The seeds were incubated for up to 7 days at about 30 °C and
413 moisture content of growth support was maintained with an equal volume of autoclaved
414 distilled water on daily basis. The number of germinated green gram seeds was firstly
415 counted on third and fourth day of incubation, respectively, and followed by second
416 counting and measurement of shoots and roots or radicle length for both plants on
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417 seventh day. Percentage of germinated seeds and vigor index were calculated. Vigor
418 index was calculated as the sum of mean shoot and root length multiplied by percentage
419 of germination [47]. All experimental sets and apparatus used were sterilized where
420 necessary.
421
422 Indole-3-acetic acid production assay
423 Some selected bacterial isolates were subjected to in vitro colorimetric
424 determination of IAA production using Salkowski’s reagent (2% of 0.5 M FeCl3 in 35%
425 perchloric acid) as modified from Asghar et al. [48]. Based on seed germination test
426 results, 7 bacterial isolates were selected for IAA production assay. Each selected
427 isolate was grown to exponential growth phase (OD ≥ 1.0) within 16 hr to be used as
6 7 428 pre-inoculum for the bioassay. The inoculum was adjusted to OD600=0.5 (10 to 10 cfu
429 mL-1) before inoculation at 500 µL into 50 mL of nutrient broth without addition of
430 tryptophan and incubated at 30 °C under agitation at 100 rpm for up to 48 hr. Non
431 inoculated broth was included as control. Each culture was centrifuged at 10,000 rpm
432 for 10 min at 4 °C.
433 One millilitre of culture supernatant was mixed with 2 mL of Salkowski’s
434 reagent in a test tube and kept in the dark for 20-25 min before measuring absorbance
435 using a UV-Vis spectrophotometer at 535 nm. The assay for each isolate was conducted
436 in triplicates. Prior to samples measurement, a series of IAA standards prepared within
437 the range of 0 to 100 µg mL-1 reacted with Salkowski’s reagent were measured to
438 construct a standard linear graph as a reference to the samples’ IAA concentration
439 values. Any appearance of reddish to pinkish color in the solution indicated IAA
440 presence in the cultured medium and depending on the color intensity of the test
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441 solutions, coloring responses were classified into two; pink type (pale pink to deep
442 pink), and red type (dark red to red).
443
444 Data analysis of bioassays 445 Analysis of Variance (ANOVA) was used to test for significance differences of
446 measurements of each bioassay whereas Tukey’s Test was employed for the
447 significance difference among different treatments at p ≥ 0.05. The Statistical Analysis
448 System (SAS Ver. 9.2) was used for the analysis.
449
450 Results and Discussion
451 Functional activity assays of respective isolates
452 In this study, 14 out of 62 cellulolytic, 12 out of 39 N-fixing, and 8 out of 36
453 phosphate-solubilizing bacterial isolates obtained from rhizospheres of both planted and
454 indigenous tree species at the rehabilitated forest were screened based on their
455 functional activities on their respective selective media and distinct morphology for
456 further characterization. Among these 3 functional groups, phosphate-solubilizing group
457 had the least number of characterized isolates due to the fact that phosphate-solubilizing
458 bacterial isolates tend to lose their ability after successive transfers or subcultures on
459 agar medium as reported previously [49]. Diazotrophic isolates also encountered similar
460 phenomenon whereby upon successive cultivation, some isolates failed to develop blue
461 halo or unable to grow on N-free media. da Silva et al. [50] has reported that a
462 diazotrophic isolate lost its characteristic pellicle growth upon successive cultures
463 which made its nitrogenase activity assessment not possible. However, none of the
464 cellulolytic isolates lost their functional ability.
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465 Cellulolytic microorganisms play major role in the synthesis of cellulases crucial
466 for the breakdown of complex polysaccharides into simple sugars. There was no
467 specific correlation between cellulose-hydrolyzing index and cellulase (CMCase)
468 activity for the 14 tested cellulolytic strains (Table 1).
469 Table 1. Cellulose-hydrolyzing index and cellulase (CMCase) activities (U mL-1 470 cell-free culture broth after 5 days at 30 °C, 180 rpm, and medium pH 7) of 471 cellulolytic isolates.
Activity Index Cellulase Activity (U mL-1) Isolates Mean value ± S.E. C14 5.25bc ± 1.50 0.016f ± 0.0015 C39d 5.10bc ± 0.44 0.015f ± 0.0017 C41d 5.29bc ± 0.88 0.078b ± 0.0012 C42d 8.52ab ± 1.93 0.077b ± 0.0007 C46d 10.41a ± 0.43 0.089a ± 0.0009 C22b 10.31a ± 0.62 0.035e ± 0.0006 C34c 4.57c ± 3.24 0.047d ± 0.0010 C45d 5.93bc ± 1.23 0.059c ± 0.0012 C4 3.59c ± 0.14 0.079b ± 0.0015 C25b 3.70c ± 0.79 0.078b ± 0.0012 C29b 5.57bc ± 0.70 0.080b ± 0.0007 C40d 4.63c ± 0.61 0.077b ± 0.0007 C43d 4.60c± 2.26 0.078b ± 0.0009 CB15 5.54bc ± 0.36 0.060c ± 0.0010 472 S.E. is standard error of the mean. 473 Means with same letter are not significantly different at p ≥ 0.05 within columns using 474 Tukey’s test. 475
476 Among the 14 cellulolytic isolates assessed, isolates C46d and C42d consistently
477 showed higher cellulolytic activity based on both cellulose-hydrolyzing index (10.41
478 and 8.52, respectively), and cellulase activity (0.089 U mL-1 and 0.077 U mL-1,
479 respectively). The lowest cellulose-hydrolyzing index and cellulose activity were in the
480 range of 1.33-3.73 U mL-1 and 0.015-0.016 U mL-1, respectively. The cellulase
481 (CMCase) activities of the cellulolytic isolates in this present study were comparable to
482 those of bacterial strains isolated from soil at different culture conditions as reported by
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483 Sethi et al. [51]. The cellulase (CMCase) activity demonstrated by C46d was especially
484 higher than those reported for Bacillus amyloliquefaciens SS35 [52] and Bacillus
485 pumilus EB3 [53] at 0.079 U mL-1.
486 For nitrogenase activity assessment, there was positive relationship (r=0.63, p ≤
487 0.05) between N-fixing index values and BNF amount. Among the 12 diazotrophic
488 isolates evaluated, NB1 exhibited the most active N-fixing activity as demonstrated by
489 the consistent highest means of both activity index and nitrogenase activity
490 quantification at 10.82 and 2.21 µg N mL-1 day-1, respectively (Table 2).
491 Table 2. Nitrogen-fixation index and biological nitrogen-fixation quantification of 492 diazotrophic isolates.
Activity Index Quantitative activity (µg N mL-1 day-1) Isolate Mean value ± S.E.
NB1 10.82a ± 0.26 2.21a ± 0.34 NB4 1.84e ± 0.05 1.11cd ± 0.27 NB5 2.03e ± 0.06 0.65cd ± 0.00 NB7 1.97e ± 0.05 1.23bc ± 0.24 NB8 1.80e ± 0.05 0.47d ± 0.00 NC9 4.63c ± 0.12 0.84cd ± 0.00 NB10 2.35e ± 0.32 1.01cd ± 0.19 NC11 1.95e ± 0.04 0.44d ± 0.10 NB11 1.88e ± 0.07 1.87ab ± 0.34 N20c 5.92b ± 0.46 1.24bc ± 0.30 N27d 3.61d ± 0.21 0.73cd ± 0.00 N29d 1.94e ± 0.08 0.60cd ± 0.00 493 S.E. is standard error of the mean. 494 Means with same letter are not significantly different at p ≥ 0.05 within columns using 495 Tukey’s test. 496
497 NB11 also presented comparably high N-fixing activity at 1.87 µg N mL-1 day-1
498 although producing fairly small halo on plate assay, yielding mean index value of 1.88.
499 Strain N20c demonstrated second highest mean N-fixing index (5.92) but at fairly
500 moderate amount of N-fixing activity (1.24 µg N mL-1 day-1). The lowest N-fixing
501 activity was recorded in the range values of 0.34-1.38 µg N mL-1 day-1 by the rest of
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502 assessed isolates. Plate assays revealed lowest N-fixing index values in the range of
503 1.75-2.67. Although there was lack of reports on N-fixing activity (index) evaluation
504 based on plate assay, the quantitative values were within range of other reported
505 diazotrophic strains [40]. The ability of diazotrophic bacteria to convert atmospheric N2
506 into ammonia, the available form of N to be used by living organisms for biosynthesis is
507 undeniably of vital importance to soil system especially through plants either through
508 direct influence on plant growth, and indirectly through production of phytohormones
509 that boost plant growth [50, 54].
510 Being one of the most limited but important plant growth nutrient, P mainly
511 occurs in insoluble forms, whereby phosphate-solubilizing rhizobacteria plays a crucial
512 role in converting it to available forms [55]. Among the 8 phosphate-solubilizing
513 strains, PB3 and PB5 showed superior phosphate-solubilization index values in plate
514 assay (4.21 and 4.10, respectively) whereas in enzymatic assay, the highest activity was
515 demonstrated by isolate PC8 (307.7 µg mL-1) (Table 3).
516 Table 3. Phosphate-solubilization index and quantitative phosphate-solubilization 517 estimation based on soluble P by phosphate-solubilizing isolates.
Quantitative Phosphate-solubilizing phosphate- Final index solubilization (Soluble mean pH Isolate P in µg mL-1) in broth Mean value ± S.E. culture
PB5 4.21a ± 0.22 96.00d ± 2.65 4.27 PC1 2.38cd ± 0.15 9.14f ± 0.75 4.93 PB7 3.18b ± 0.23 159.00c ± 1.53 4.15 PB3 4.10a ± 0.23 209.17b ± 8.97 4.11 PC6 1.85ef ± 0.05 31.53e ± 0.39 4.63 PC8 2.74bc ± 0.22 307.67a ± 8.41 4.04 PB11 1.66f ± 0.08 3.25f ± 0.52 4.57 PB6 2.19de ± 0.24 12.67f ± 0.55 4.20 518 S.E. is standard error of the mean. 519 Means with same letter are not significantly different at p ≥ 0.05 within columns using 520 Tukey’s test. 521
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522 The amount of phosphate-solubilization index and activities with final broth
523 culture pH reported in this present study is comparable to the range reported by Pandey
524 et al. [56], Gulati et al. [57], Song et al. [58]. Isolate PC8 showed higher phosphate-
525 solubilization activity (307.67 µg mL-1) as compared with Pseudomonas putida (B0) at
526 maximum activity of 247 µg mL-1 [56]. There was no significant relationship observed
527 between phosphate-solubilizing index and soluble phosphate. Similar observations had
528 been previously reported by Nautiyal [31] and Chang and Yang [59]. Nautiyal [31]
529 stated the inability of phosphate-solubilizing Pseudomonas aerogenes and
530 Pseudomonas sp.1 to produce halo on solid media but could solubilize substantial
531 amount of P in different type of broth cultures. Nonetheless, there was a significant
532 inverse correlation (r=-0.74, p ≤ 0.05) between soluble phosphate and pH of culture
533 broth, and this observation corroborates previous findings [60-61]. The pH of broth
534 declined to as low 4.04 as observed in PC8 activity from initial pH 7. This observation
535 is consistent with earlier finding where there was a drop in pH due to bacterial activity
536 is accompanied with mineral phosphate-solubilization and increment of its activity
537 efficiency [56]. In their study on various organic acids secretion by 10 bacteria and 3
538 fungal strains, Rashid et al. [62] reported lowering of pH as part of the mechanism of
539 microbial phosphate-solubilization.
540
541 Cultural, biochemical, and molecular identification of selected
542 functional bacterial isolates
543 The isolation of bacteria near the root zones of vegetations thriving in the
544 rehabilitated forest generally lead to the recovery of putative beneficial rhizospheric or
545 even free-living functional bacteria. In this present study, growth in the selective media
546 proved to be a successful strategy to select bacteria based on their phenotypic activities
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547 on respective media. From the isolates assessed for their functional activities, 5 isolates
548 of each functional group were selected to be further characterized on the basis of
549 distinct colony morphology and high functional activities. After being selected based on
550 these combined criteria, the isolates were grouped by morphological similarities and
551 phenotypic characteristics.
552 On the basis of morphological characteristics of the 15 selected isolates depicted
553 in Table 4, they exhibited varying properties. It was observed that the majority or 13
554 isolates exhibited round colonies in configuration or form; 10 colonies were convex
555 whereas the rest shown raised elevations; 14 colonies were opaque in density; colonies
556 of all of the isolates were almost creamy or yellowish in colour; 13 showed Gram
557 negative reactions; all isolates were rods or short rods in single or in pairs arrangement;
558 13 without endospore production; and they were mostly motile as exhibited by 11
559 isolates. The following morphological properties presented more diverse characteristics:
560 colony margin was represented by 8 entires, 3 undulates and serrates, respectively, and
561 1 lobate; colony size was represented by 6 moderates, 5 smalls, 3 larges and 1 pinpoint;
562 and the shapes of endospores produced by 2 isolates were of rods and coccus,
563 respectively.
564
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565 Table 4. Morphological characteristics of selected functional isolates.
Colony Isolate morphology CB15 C22d C29b C41d C46d NB1 NB10 NB11 N27d NC9 PB3 PB5 PB6 PB7 PC8
Configuration Irregular Round Round Irregula Round Round Round Round Round Round Round Round Round Round Round / Form r Undulat Entire Entire Serrate Entire Entire Undulat Undulate Entire Entire Entire Serrate Serrate Entire Lobate Margin e e
Raised Convex Convex Raised Conve Convex Raised Convex Convex Convex Convex Raised Raised Convex Convex Elevation x Opaque Opaque Opaque Opaque Opaqu Opaque Opaque Opaque Opaque Opaque Opaque Opaque Transparen Opaque Opaque Density e t Yellowis Yellowis Pigments Cream Cream Yellow Cream Cream Cream Cream Cream h cream Cream h cream Cream Cream Cream Cream Gram’s + ------+ - - reaction Rods Rods Rods Short Short Short Short Rods Short Short rods Rods Rods Short rods Rods Short rods Shape rods rods rods rods rods Large Moderat Small Large Small Pinpoin Large Moderat Small Moderate Moderate Moderate Moderate Small Small Size e t e Single Single Single/pair Single Single Single Single Single Single Single/pair Single Single/pair Single/pair Single/pair Single/pair Arrangement s s s s s s Endospore + ------+ ------production Endospore Rods ------Coccus ------shape Motility + + + - + - + - - + + + + + + 566 +, positive result; -, negative result.
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567 The isolates were primarily identified based on molecular method, and in cases
568 of multiple identities produced based on BLAST generated database, morphological
569 (Table 4) and biochemical (Table 5) properties facilitated in the downsizing of
570 identification task.
571 Table 5. Biochemical properties of selected functional bacterial isolates.
Isolate Catalase MCA KIA SA SCA SMA T80A NG Indole NRT CB15 + ˗ Glu ferm +, Lacferm - ˗ ˗ + + + ˗ + C22d + + Glu ferm +, Lacferm - ˗ + + + + ˗ + C29b + + Glu ferm -, Lacferm - ˗ + + + + ˗ + C41d + ˗ Glu ferm +, Lacferm - ˗ ˗ + ˗ + ˗ + C46d + + Glu ferm +, Lacferm - ˗ + + + + ˗ + NB1 + + Glu ferm -, Lacferm - ˗ + ˗ + ˗ ˗ + NB10 + ˗ Glu ferm +, Lacferm - + + + + + ˗ ˗ NB11 + ˗ Glu ferm -, Lacferm + + ˗ ˗ + ˗ ˗ + N27d + + Glu ferm -, Lacferm - ˗ + + ˗ + ˗ + NC9 + + Glu ferm +, Lacferm - ˗ + + + + ˗ + PB3 + + Glu ferm -, Lacferm - ˗ + + + + ˗ + PB5 + ˗ Glu ferm -, Lacferm - + + + ˗ + ˗ + PB6 + ˗ Glu ferm -, Lacferm - + + + ˗ + ˗ + PB7 + + Glu ferm -, Lacferm - ˗ + + + + ˗ + PC8 + + Glu ferm -, Lacferm - ˗ + + + + ˗ + 572 +, positive result; -, negative result; MCA, Mac Conkey agar assay; KIA, Kligler Iron 573 Agar assay; Glu ferm, Glucose fermentation; Lacferm, Lactose fermentation; SA, 574 Starch agar assay; SCA, Simmons citrate agar; SMA, Skim milk agar; T80A, Tween 80 575 Agar assay; NG, Nutrient gelatine assay; Indole, Indole production assay; NRT, Nitrate 576 reduction test assay. 577
578 In Table 6, the selected cellulolytic isolates in this present study were identified
579 as Serratia nematodiphila strain SP6 (C46d), Serratia marcescens subspecies sakuensis
580 isolate PSB23 (C22b), Stenotrophomonas maltophilia strain KNUC605 (C29b),
581 Bacillus thuringiensis (Bt) (CB15), and Stenotrophomonas sp. Ellin162 (C41d). The N-
582 fixing isolates were consisted of Burkholderia nodosa strain Br3470 (NB1), Delftia
583 lacustris strain 332 (NC9), Stenotrophomonas sp. strain SeaH-As3w (N27d), Ralstonia
584 sp. strain MCT1 (NB10), and Cupriavidus basilensis strain CCUG 49340T (NB11). The
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585 phosphate-solubilizing isolates were represented by Burkholderia cepacia strain 5.5B
586 (PB5), Burkholderia cepacia strain ATCC 35254 (PB3), Burkholderia uboniae (PB7),
587 Burkholderia cepacia strain 2EJ5 (PC8), and Gluconacetobacter diazotrophicus PAl 5
588 (PB6).
589 Table 6. Identities of cellulolytic, N-fixing, and phosphate-solubilizing bacterial 590 isolates based on 16S rDNA analysis and BLAST databases.
Similarity Accession Phenotype Isolate Closest Identity (%) No. Serratia nematodiphila strain 98 C46d JN315887.1 SP6 (324/329) Serratia marcescens subspecies 97 C22b HQ242736.1 sakuensis isolate PSB23 (578/594) Stenotrophomonas maltophilia 99 Cellulolytic C29b HM047520.1 strain KNUC605 (945/949) 99 CB15 Bacillus thuringiensis FR846529.1 (1018/1021) 100 C41d Stenotrophomonas sp. Ellin162 AF409004.1 (989/989) Burkholderia nodosa strain 98 NB1 AM284972.1 Br3470 (943/967) 99 NC9 Delftia lacustris strain 332 EU888308.1 (1009/1014) Stenotrophomonas sp. SeaH- 100 N-fixing N27d FJ607350.1 As3w (1029/1029) 99 NB10 Ralstonia sp. MCT1 DQ232889.1 (999/1007) Cupriavidus basilensis strain 99 NB11 FN597608.1 CCUG 49340T (961/969) 99 PB5 Burkholderia cepacia strain 5.5B FJ652617.1 (874/885) Burkholderia cepacia strain 99 PB3 AY741346.1 ATCC 35254 (990/993) Phosphate- 99 PB7 Burkholderia uboniae AB030584.1 solubilizing (993/997) 99 PC8 Burkholderia cepacia strain 2EJ5 GQ383907.1 (1008/1016) Gluconacetobacter 97 PB6 CP001189.1 diazotrophicus PAl 5 (778/801) 591
592 These identities were matched with BLAST databases with the range of 97 to
593 100% base sequence similarities (Table 6). It was observed that identification of
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594 bacterial isolates over the three phenotypic groups revealed the predominance of Gram-
595 negative over Gram-positive bacteria in the rehabilitated forest soils. The Gram-positive
596 bacteria belong to only one taxonomic grouping, Firmicutes, represented by the genus
597 Bacillus, whereas Gram-negative bacteria included Alpha-Proteobacteria with genus
598 Gluconacetobacter; Beta-Proteobacteria represented by genera Burkholderia, Delftia,
599 Ralstonia, and/or Cupriavidus; and Gamma-Proteobacteria by genera Serratia and
600 Stenotrophomonas.
601 From the overall identification results of each functional group of isolates,
602 bacteria from genus Burkholderia seemed to predominate in these tropical forest soils at
603 one third of total identified isolates with distinguishing phosphate-solubilizing and N-
604 fixing capabilities over other genera. Stenotrophomonas genus ranked second in
605 abundance to Burkholderia. Burkholderia genus is a well-known classification of
606 bacteria wherein its members possess vast versatility in niches and nutrition regulation
607 such as N-fixing and phosphate-solubilization, as well as possessing numerous PGP
608 attributes. Burkholderia species occur in the natural environment, even though some
609 strains are also found in clinical samples, especially the B. cepacia complex species
610 from patients with cystic fibrosis [63].
611 Based on our 16S rDNA sequences analysis results, the top 4 isolates with
612 highest phosphate-solubilizing and N-fixing activities belong to genus Burkholderia, in
613 which 3 of them were most closely related to B. cepacia (99% similarity) and B. nodosa
614 (98% similarity), respectively. Serratia marcescens (of close similarity by C22b at
615 97%) or Serratia sp. (with C46d closely matched to Serratia nematodiphila by 98%) are
616 known to have both cellulolytic and phosphate-solubilizing abilities [64-65]. Similar to
617 the results of this present study, Stenotrophomonas spp. have been reported previously
618 to possess both N-fixing [66] and cellulose-hydrolyzing abilities [67].
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619 Stenotrophomonas species commonly prevail in nature, whereby S. maltophilia and S.
620 rhizophila are reported to occur as rhizospheric and endophytic bacteria accountable for
621 beneficial plant interactions [67]. The N-fixing isolates in this present study displayed
622 higher diversity in terms of the species, as well as activities.
623 Diazotrophic isolate N27d revealed 100% matching identity with
624 Stenotrophomonas sp. (strain SeaH-As3w) and it is believed to have cellolulytic
625 potential similar to the C41d and C29b isolates. Previously, many studies have reported
626 bacterial belonging to genus Burkholderia especially B. cepacia strains as having
627 biocontrol ability against many phyto-pathogenic fungi such as Fusarium sp., Pythium
628 aphanidermatum, Pythium ultimum, Phytophthora capsici, Aspergillus flavus, Botrytis
629 cinerea and Rhizoctonia solani [68-72] as part of its main contributing PGP factors.
630 The phosphate-solubilizing bacteria showed the lowest diversity that only three
631 species identities from two genera were recorded among the five identified isolates.
632 Isolates PB3, PB5, PB7, and PC8 were closely matched (99% similarity) to
633 Burkholderia sp. These isolates together with the N-fixing NB1 might potentially carry
634 both the N-fixing and phosphate-solubilizing activities [73-74]. Diazotrophic isolate
635 Burkholderia nodosa NB1 along with Burkholderia mimosarum, have been the latest
636 classified novel species with N-fixing capacity and legume-nodulating bacteria that
637 establish effective symbioses with legumes of Mimosa species [75-76]. All
638 Burkholderia strains in this present study demonstrated variability in cultural and
639 biochemical properties, which is typical for members of this genus.
640 The ability of Burkholderia strains to solubilize inorganic phosphates by various
641 studies [77-80] have been well documented and nevertheless, a beneficial property in
642 plant- growth promotion. Among the Burkholderia strains isolated in this study, this
643 trait seemed most prevalent. Few Burkholderia strains are known for other PGP ability,
29 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.
644 which is to fix N2, either as free-living bacteria [81], or even nodulating ones that also
645 fix N2 on media including Burkholderia nodosa strain as reported by da Silva et al. [82].
646 The genus Delftia (of close similarity by NC9 to Delftia lacustris at 99%)
647 belongs to a newly grouped genus closely linked to Comamonas, in which Delftia
648 terephthalate has been reported as a diazotrophic PGPR capable of degrading
649 terephthalate, as well as antagonizing rice blast and blight causing Xanthomonas oryzae,
650 Rhizoctonia solani, and Pyricularia oryzae [83-84]. One of the diazotrophic bacterium
651 IHB B 4037 showing closely matched identity with Delftia lacustris isolated from
652 rhizosphere of tea plant in India showed the highest nitrogenase activity in a study
653 conducted by Gulati et al. [85]. Delftia tsuruhatensis was also revealed to be a
654 diazotroph with biological control ability against several plant pathogens especially the
655 three main rice pathogens in the likes of Xanthomonas oryzae pv. Oryzae, Rhizoctonia
656 solani, and Pyricularia oryzae Cavara [86]. In summary, the variability among the
657 bacterial strains of the same genera pointed to the fact that bacterial phenotypic
658 properties are influenced by genetic, as well as environmental factors [87].
659
660 Seed germination, shoot, and root elongation assays
661 Seed germination, shoot, and root elongation assays were done on 15 selected
662 functional isolates on Green Gram seeds (Tables 7 - 9) to represent an attribute of early
663 plant growth promotion activity by the isolates. After a week of growth, Green Gram
664 roots originating from seeds treated with all selected bacterial strains exhibited different
665 effects on root lengths, shoot lengths, and seedling vigor indexes compared with two
666 controls, treated with distilled water and nutrient broth, respectively.
30 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.
667 Table 7. Effects of selected cellulolytic bacterial inoculation on early growth stage of green gram.
Mean number of Mean number of Mean length of Mean length of % germinated Vigor index Treatment seed germinated seed germinated shoots (cm) radicle (cm) seeds (Mean ± S.E.) (3rd day) (7th day) (Mean ± S.E.) (Mean ± S.E.)
C22b 50.0 50.0 12.84a ± 0.06 5.16ab ± 0.04 100 1799.50a ± 2.50 CB15 50.0 50.0 12.38a ± 0.45 5.47ab ± 0.75 100 1785.00ab ± 30.00 C41d 50.0 50.0 11.75a ± 0.03 5.67a ± 0.13 100 1741.50ab ± 10.50 C46d 50.0 50.0 11.73a ± 0.23 5.28ab ± 0.53 100 1700.00ab ± 30.00 C29b 50.0 50.0 11.81a ± 0.35 5.04ab ± 0.40 100 1684.50b ± 5.50 Control (Nutrient 50.0 50.0 9.26b ± 0.18 3.36bc ± 0.02 100 1261.00c ± 19.00 broth) Control 49.5 49.5 7.91b ± 0.18 2.87c ± 0.10 99 1067.00d ± 17.00 (dH2O) 668 Data are presented as mean ± standard error (S.E.) of duplicates of 50 seedlings for each replicate per treatment for root elongation assay and 669 seedling vigor index. The roots emerging from seeds failing to germinate after 2 days were previously marked and were not measured. The 670 seedling vigor index (SVI) was calculated using formula: SVI = % of germination x seedling length (root length + shoot length). In the same 671 column, significant differences according to Tukey’s test at p ≤ 0.05 level are indicated by different letters. 672
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673 Table 8. Effects of selected N-fixing bacterial inoculation on early growth stage of green gram. Mean number of Mean number of Mean length of Mean length of % Vigor index Treatment seed germinated seed germinated shoots (cm) radicle (cm) germinated (Mean ± S.E.) (3rd day) (7th day) (Mean ± S.E.) (Mean ± S.E.) seeds NB1 50.0 50.0 11.27a ± 0.06 3.84ab ± 0.02 100 1510.00a ± 4.00 NC9 50.0 50.0 11.00a ± 0.32 4.10a ± 0.34 100 1510.00a ± 66.00 NB10 50.0 50.0 10.81ab ± 0.15 3.89ab ± 0.24 100 1469.00a ± 9.00 NB11 50.0 50.0 10.43ab ± 0.27 4.09a ± 0.26 100 1451.50ab ± 52.50 N27d 50.0 50.0 10.31ab ± 0.61 3.68ab ± 0.19 100 1399.00ab ± 42.00 Control (Nutrient 50.0 50.0 9.26bc ± 0.18 3.36ab ± 0.02 100 1261.00bc ± 19.00 broth) Control 49.5 49.5 7.91c ± 0.18 2.87b ± 0.10 99 1067.00c ± 17.00 (dH2O) 674 Data are presented as mean ± standard error (S.E.) of duplicates of 50 seedlings for each replicate per treatment for root elongation assay and 675 seedling vigor index. The roots emerging from seeds failing to germinate after 2 days were previously marked and were not measured. The 676 seedling vigor index (SVI) was calculated using formula: SVI = % of germination x seedling length (root length + shoot length). In the same 677 column, significant differences according to Tukey’s Test at p ≤ 0.05 level are indicated by different letters. 678 679 680 681 682 683
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684 Table 9. Effects of selected phosphate-solubilizing bacterial inoculation on early growth stage of green gram.
Mean number Mean number Mean length of Mean length of of seed of seed % germinated Vigor index Treatment shoots (cm) radicle (cm) germinated (3rd germinated (7th seeds (Mean ± S.E.) (Mean ± S.E.) (Mean ± S.E.) day) day) PB3 50.0 50.0 12.25a ± 0.08 5.14a ± 0.02 100 1738.50a ± 9.50 PB7 49.5 50.0 12.08ab ± 0.40 4.89ab ± 0.33 100 1696.00ab ± 7.00 PB5 50.0 50.0 11.78abc ± 0.21 4.61ab ± 0.35 100 1638.50b ± 14.50 PB6 50.0 50.0 10.97bc ± 0.06 4.18abc ± 0.31 100 1514.50c ± 25.50 PC8 50.0 50.0 10.71c ± 0.18 3.73bcd ± 0.04 100 1443.00c ± 14.00 Control (Nutrient 50.0 50.0 9.26d ± 0.18 3.36cd ± 0.02 100 1261.00d ± 19.00 broth) Control 49.5 49.5 7.91e ± 0.18 2.87d ± 0.10 99 1067.00e ± 17.00 (dH2O) 685 Data are presented as mean ± standard error (S.E.) of duplicates of 50 seedlings for each replicate per treatment for root elongation assay and 686 seedling vigor index. The roots emerging from seeds failing to germinate after 2 days were previously marked and were not measured. The 687 seedling vigor index (SVI) was calculated using formula: SVI = % of germination x seedling length (root length + shoot length). In the same 688 column, significant differences according to Tukey’s test at p ≤ 0.05 level are indicated by different letters.
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689 As shown in Table 7, inoculation with cellulolytic isolate C41d
690 (Stenotrophomonas sp.) resulted in significantly longer root lengths compared to both
691 control treatments, but was not significantly different from the rest of cellulolytic
692 isolates. On the other hand, the treatments with all selected cellulolytic strains resulted
693 in significantly higher shoot lengths compared to both controls but not different with
694 one another, which ranged from 11.46 to 12.9 cm. In terms of SVI, all cellulolytic
695 strains exhibited significantly higher values than both controls, with isolate C22b
696 (Serratia marcescens subsp. sakuensis) presented outstanding values but was not
697 different with isolates CB15, C41d, and C46d (Bt, Stenotrophomonas sp., and Serratia
698 nematodiphila) which ranged from 1670 to 1815.
699 In Table 8, the treatments with N-fixing isolates NC9 (Delftia lacustris) and
700 NB11 (Cupriavidus basilensis) showed higher root lengths and shoot lengths occurring
701 in the range of 3.76 cm to 4.44 cm and 10.68 cm to 11.33 cm, respectively, whereas SVI
702 values for isolates NB1, NC9, and NB10 (Burkholderia nodosa, Delftia lacustris, and
703 Ralstonia sp.) revealed significantly higher SVI values than both controls, ranging from
704 1444 to 1576, but were not significantly different from the rest of N-fixing strains.
705 Isolates PB3, PB5, and PB7 (Burkholderia cepacia, Burkholderia cepacia, and
706 Burkholderia uboniae) resulted in the longest root lengths among the phosphate-
707 solubilizing strains in the range of 4.26 cm to 5.16 cm and SVI in the range of 1624 to
708 1748 using these three isolates (Table 9). Significantly higher shoot lengths however,
709 were achieved with phosphate-solubilizing isolates than both controls, ranging from
710 10.53 cm to 12.33 cm. Isolate PB3 had the most outstanding effect on shoot length, but
711 not different from those of isolates PB5 and PB7, among other phosphate-solubilizing
712 bacterial strains.
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713 A previous study by Satya et al. [88] confirmed similar observation on
714 Burkholderia sp. as their application using Burkholderia sp. strain TNAU-1
715 demonstrated improvement in root and shoot lengths, as well as increased seedling
716 vigour on mung bean. According to Egamberdieva et al. [89], inoculation of one of the
717 salt tolerant rhizobacterial strains, Stenotrophomonas rhizophila ep-17 resulted in
718 improved dry weight of cucumber by up to 68% in various salt concentrations as high as
719 10 dSm-1 in comparison to non-inoculated plants exposed to salt stress.
720 Hameeda et al. [90] demonstrated that one of phosphate-solubilizing bacterial
721 strains in the study, Serratia marcescens designated as EB 67 tested on maize growth by
722 paper towel method was able to produce higher root and plumule lengths, plant weight
723 (43%), and seed vigor index, whereas another strain also included in the study, Serratia
724 sp. designated as EB75 resulted in increased root length, plant weight by 23%, and seed
725 vigor index over uninoculated control.
726 Kang et al. [91] reported that inoculation of a novel cold stress resistant
727 rhizobacteria Serratia nematodiphila PEJ1011 on pepper plants resulted in the latter’s
728 significantly improved growth by means of both increased root and shoot lengths, and
729 fresh weight of the peppers on both normal and cold temperature as compared with
730 controls under normal and cold temperature, respectively. Most previous studies on Bt
731 reported it as a safe and effective biological control agent for the suppression of a broad
732 type of insect pests [92-95] due to its ability to produce parasporal crystals consisting of
733 protein molecules called delta endotoxins which are toxic to insect pests [96]. Besides
734 its apparent biological control attribute, current study by Khan et al. [97] also reported
735 plant growth promotion via increase in plant growth parameters on mung bean seeds
736 coated with various Bt strains used in the study. In their study, all the tested strains
737 improved shoot weight, with strain BT-64 in particular, was reported to have resulted in
35 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.
738 97%, 42%, 46%, and 71% increment in shoot weight, shoot length, root length, and root
739 weight, respectively over control.
740 Apart from having high nitrogenase activity strain from rhizospheric soil of tea
741 plants [85], Jin et al. [98] found that Delftia lacustris isolated from rhizospheric soils of
742 tobacco to be one of strongly antagonistic bacteria against pathogen Phytophthora
743 nicotianae which could have potentially helped promoting growth of tobacco crop.
744 According to a study by Yrjȁlȁ et al. [99], Cupriavidus basilensis of Burkholderiaceae
745 family, and especially strains of the genus Cupriavidus have been shown to be isolated
746 from hybrid aspen root in mostly pristine soil as compared to rhizospheric soils
747 contaminated with polyaromatic hydrocarbon (PAH) constituents.
748 On the other hand, the addition of PAH mixture (anthracene, phenanthrene, and
749 pyrene) resulted in the apparent shift in the diversity of uncultured Burkholderia in
750 rhizosphere of hybrid aspen and lowest fraction (five out of 38 sequences) of this genus
751 was found in pristine rhizosphere. Their study indicated the main difference in terms of
752 soil diversity that could influence the abundance between both genera. Cupriavidus
753 basilensis designated HMF14 has also been recently identified as an important furan-
754 degrading bacterium, and was isolated by employing an elegant assay based on the
755 germination of lettuce seeds [100]. Furanic compounds as toxic fermentation inhibitors
756 are derived from lignocellulose hydrolysates in which occurrences of the former in high
757 amount could pose carcinogenicity effects to humans [100].
758 Cupriavidus (synonym Ralstonia) is closely linked to Burkholderia, but
759 compared to the latter genus it consists of very limited N-fixing species [101]. Both
760 genera, in particular consisting of Burkholderia spp. and Cupriavidus taiwanensis have
761 been reported to be N-fixing symbionts of legume Mimosa with the ability to nodulate
762 the latter [102-104]. Some strains of Burkholderia have been known as Beta-rhizobia
36 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.
763 which could exclusively nodulate large legume of genus Mimosa (Mimosoideae) [101].
764 These legume-nodulating burkholderias are not closely related to phytopathogenic or
765 pathogenic group of burkholderias including the Burkholderia cepacia complex, but
766 instead are closely linked to PGPR, mostly diazotrophic, endophytic environmental
767 Burkholderia species and strains [105].
768 Burkholderia nodosa designated as LMG 23741 has also been isolated from
769 acidic soils of Amazon which was a nodulating N-fixing bacterium [82], as compared to
770 the strain in this present study which did not nodulate, but was a free-living or
771 rhizospheric N fixer. Both strains are known to be absent in ability to solubilize
772 inorganic phosphates. The strain reported by da Silva et al. [82] also did not possess
773 anti-fungal characteristic. Besides the N-fixing isolate Ralstonia sp. characterized in
774 this present study, Ralstonia taiwanensis is the first N-fixing beta-proteobacterium
775 strain having the ability to nodulate the roots of Mimosa from which it was isolated
776 [106]. However, some Ralstonia spp. are environmental variant microorganisms and
777 there are also phytopathogenic strains such as Ralstonia solanacearum that cause
778 bacterial wilt occurrence on a broad range of crops [107].
779 Burkholderia cepacia designated as LMG 1222 by da Silva et al. [82] was also
780 reported as a non N-fixing bacteria isolated from acidic Amazon soils which could
781 solubilize inorganic phosphate, as well as possessing anti-fungal property. However,
782 there is lack of literature on beneficial properties of Burkholderia uboniae and hence,
783 the results of this present study could contribute primary evaluation on the potential of
784 this particular strain on early plant growth promotion. Many studies reported bacterial
785 belonging to genus Burkholderia especially B. cepacia strains as having biocontrol
786 ability against many phyto-pathogenic fungi such as Fusarium sp., Pythium
787 aphanidermatum, Pythium ultimum, Phytophthora capsici, Aspergillus flavus, Botrytis
37 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.
788 cinerea, and Rhizoctonia solani [68-72] as part of its main contributing PGP factors.
789 Gluconacetobacter diazotrophicus is one of diazotrophic species reported to form
790 anomalous symbiotic relationship with plants by thriving on the root system surface as
791 rhizobacteria, whereas a number of strains are known to infect plant tissues as
792 endophytic bacteria and fix N2, resulting in enhanced plant growth [108-111].
793
794 Indole-3-acetic acid assay
795 On the basis of respective phenotypic functional activities, Green Gram seed
796 germination, and root elongation assay results, 7 bacterial isolates were selected for
797 IAA production assay (Table 10).
798 Table 10. Indole-3-acetic acid production by selected functional bacterial isolates.
IAA production Phenotype Isolate (µg mL-1) Mean ± S.E. Stenotrophomonas maltophilia C29b 12.38a ± 1.48 Cellulolytic Serratia nematodiphila C46d 11.39ab ± 1.68 Bacillus thuringiensis CB15 0.98c ± 0.16 Burkholderia cepacia PC8 8.45ab ± 0.28 Phosphate- Burkholderia cepacia PB3 7.81b ± 0.11 solubilizing Gluconacetobacter diazotrophicus 7.51b ± 0.17 PB6 Nitrogen-fixing Burkholderia nodosa NB1 0.43c ± 0.45 799 S.E. is the standard error of the mean value. 800 Significant differences according to Tukey’s test at p ≤ 0.05 level are indicated by 801 different letters. 802
803 All the 7 selected strains produced IAA without supplementation of L-
804 tryptophan, from N-fixing isolate Burkholderia nodosa NB1 to most outstanding IAA
805 production by cellulolytic isolates Stenotrophomonas maltophilia C29b and Serratia
806 nematodiphila C46d, and phosphate-solubilizing isolate Burkholderia cepacia PC8
38 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.
807 ranging from 8.17 to 13.86 µg mL-1. The IAA produced by the cellulolytic, non N-
808 fixing Stenotrophomonas maltophilia strain in this present study, C29b was at 12.38 µg
809 mL-1 with absence of tryptophan. Stenotrophomonas maltophilia has been previously
810 reported as diazotrophic with IAA production of 2.6 mg mL-1 [112], 112.8 µg mL-1 for
811 S. maltophilia PM-1, and 139.2 µg mL-1 in S. maltophilia PM-26 [113] with L-
812 tryptophan as supplementation.
813 Serratia nematodiphila C46d strain in this present study was found to synthesize
814 IAA at 11.39 µg mL-1. Previously, Serratia nematodiphila designated NII-0928 isolated
815 from Western ghat forest soil was found to possess multiple PGP attributes including
816 phosphate-solubilization and IAA production at higher concentration, at 76.6 µg mL-1
817 and 58.9 µg mL-1, respectively [114]. As stated previously, the variability among the
818 bacterial strains of the same genera points to the fact that bacterial phenotypic properties
819 are influenced by genetic and environmental factors [87]. Serratia nematodiphila
820 LRE07 has also been reported to be one of the heavy metals-resistant endophytic
821 bacteria isolated from Solanum nigrum L. planted in metal-polluted soil which
822 improved plant growth and Cd extraction from soil as phytoremediation activity [115].
823 Bacillus thuringiensis (Bt) strain CB15 in this present study yielded IAA amount within
824 the stated range as reported by Vidal-Quist et al. [116], whereby all of 44 Bt strains
825 screened in their studies were reported to produce IAA in a range of 0.12 to 6.59 µg
826 mL-1. The IAA production range is comparable to the findings of Raddadi et al. [117].
827 Besides being well known for having biocontrol activity against insects [118],
828 phytopathogens including fungi [119-120], and bacteria [121], Bt has the potential to
829 promote plant growth via production of phytohormones, such as auxins, or by
830 regulating levels of plant ethylene [117, 122], stimulation of nodulation in legumes by
831 some strains and hence, Rhizobium-based N2 fixation [123-124], improvement by other
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832 strains of mineral nutrients (such as Fe and P), recovery by the plant [117], and promote
833 protection of plants from abiotic stresses [125].
834 Burkholderia cepacia strains PC8 and PB3 evaluated in this present study
835 without addition of tryptophan produced IAA at 8.45 and 7.81 µg mL-1, respectively.
836 Bevivino et al. [126] reported that rhizospheric strains of Burkholderia cepacia PHP7
837 and TVV75 were found to produce IAA at approximately 0.6 and 0.4 µg mL-1,
838 respectively, both of which were far lacking by at least 14-fold, even with addition of L-
839 tryptophan, in comparison to strains in this present study. This phenomenon could
840 indicate that the B. cepacia strains were actively involved in IAA synthesis in pure
841 culture [127].
842 Phosphate-solubilizer and non N-fixing Gluconacetobacter diazotrophicus strain
843 PB6 produced IAA at 7.51 µg mL-1 without precursor tryptophan, which was higher
844 than those reported previously and improved shoot length and vigor index of green
845 gram than uninoculated controls. Anitha and Thangaraju [128] reported an endophytic
846 diazotroph Gluconacetobacter diazotrophicus strain in their study to have exhibited
847 nitrogenase activity, phosphate-solubilization, and IAA production with and without
848 tryptophan at 4.74 µg mL-1 and 1.4 µg mL-1, respectively. The strain in their study could
849 also colonize roots of paddy in the growth solution under aseptic hydroponic condition.
850 The researchers further stated that inoculation of rice seedlings with the strain also
851 resulted in improved shoot length, root length, and plant biomass than uninoculated
852 control.
853 Despite the least amount of IAA produced by Burkholderia nodosa strain NB1
854 at 0.43 µg mL-1, this is the first results on IAA production ever reported for non-
855 nodulating N-fixing Burkholderia nodosa strain. In fact, there is dearth of reports
856 concerning the beneficial PGP traits of Burkholderia nodosa with exception of it being
40 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.
857 reported as N-fixing symbiont of nodulating legumes by being isolated from N-fixing
858 root nodules of legumes Mimosa bimucronata and Mimosa scabrella [129], and its
859 apparent ability as biocontrol agent against bacterial wilt of tomato, as well as Fusarium
860 wilt of tomato and spinach with recorded suppression of disease index by 33-79%
861 [130].
862 The presence of an appropriate precursor such as L-tryptophan may mediate the
863 production of auxins by some microorganisms [131]. The influence of auxins on plant
864 seedlings relies upon their concentrations. For instance, high concentration may
865 suppress growth, whereas low concentration may produce stimulative effects on growth
866 [132]. The absence of tryptophan precursor in IAA production has also been previously
867 observed in other bacterium, Azotobacter brasilense in a mechanism known as a
868 tryptophan-independent pathway which was suggested during experiments using
869 labelled tryptophan [133].
870 Despite this fact, there is still absence of clear genetic or biochemical proof to be
871 established to consolidate this pathway [134]. According to Spaepen and Vanderleyden
872 [134], occurrence of some IAA production despite inactivation of a single pathway on
873 some knockout studies revealed redundancy of IAA biosynthetic pathways in some
874 studied microorganisms, indicating that several pathways do exist and operating in a
875 single microorganism. Nevertheless, the ability to secrete IAA without absolute
876 dependence on tryptophan as the main precursor proved to be an added advantage over
877 tryptophan dependent microbes in plant rhizospheres to produce beneficial associative
878 effects with plant roots to enhance plant growth.
879
880
881
41 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.
882 Conclusions
883 Out of the 15 identified, bacterial isolates possess beneficial phenotypic traits, a
884 third belong to genus Burkholderia and a fifth to Stenotrophomonas sp. with both
885 genera consisting of members from two different functional groups. Among the tested
886 bacterial strains, isolate C46d, NB1, and PC8 showed outstanding cellulase, N-fixing,
887 and phosphate-solubilizing activities, respectively. The results of the experiments
888 confirmed the multiple PGP traits of the selected bacterial isolates based on their
889 respective high functional activities, root, shoot lengths, and seedling vigour
890 improvements when bacterized on mung bean seeds, as well as presented some
891 significant IAA productions. All these functional bacterial strains could potentially
892 produce beneficial synergistic effects via their versatile properties on improving soil
893 fertility and possible plant growth stimulation in beneficial interactions. Further
894 characterizations of these potent strains will be helpful in elucidating their potential uses
895 in biotechnological applications.
896
897 Acknowledgements
898 Many thanks are extended to the colleagues and staff of Universiti Putra
899 Malaysia Bintulu Sarawak Campus and Department of Agriculture, Kuching, Sarawak
900 for their valuable cooperation and support. Contribution of the late Dr. Wong Sing King
901 was also greatly appreciated for his advice on microbiological aspects of the study.
902
903
904
905
42 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.
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