Canadian Journal of Forest Research
Long-term effect of inoculating lodgepole pine seedlings with plant growth-promoting bacteria originating from disturbed gravel mining ecosystem
Journal: Canadian Journal of Forest Research
Manuscript ID cjfr-2020-0333.R1
Manuscript Type: Article
Date Submitted by the 09-Sep-2020 Author:
Complete List of Authors: Padda, Kiran Preet; The University of British Columbia, Forest and Conservation Sciences Puri, Akshit; The University of British Columbia, Forest and Conservation Sciences Draft Chanway, Christopher; The University of British Columbia, Forest and Conservation Sciences
Pinus, Pseudomonas, Gravel mining, Lodgepole pine, Keyword: ACC deaminase
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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1 Long-term effect of inoculating lodgepole pine seedlings with plant growth-
2 promoting bacteria originating from disturbed gravel mining ecosystem
3 Kiran Preet Padda1, 2*, Akshit Puri1, 2 and Chris P Chanway1, 2
4 1 Faculty of Land and Food Systems, The University of British Columbia, 3041 – 2424 Main Mall,
5 Vancouver, BC V6T 1Z4, Canada
6 2 Faculty of Forestry, The University of British Columbia, 3041 – 2424 Main Mall, Vancouver, BC V6T
7 1Z4, Canada
8 *Corresponding author ([email protected]) Draft
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9 Abstract
10 Gravel mining is prevalent in forest landscapes of Canada, typically resulting in complete loss of vegetation
11 and topsoil. Despite such extreme disturbance, lodgepole pine (Pinus contorta var. latifolia) trees are
12 thriving at unreclaimed gravel pits located in central-interior British Columbia, possibly due, at least in
13 part, to the association of pine trees with their endophytic bacteria. Testing this possibility, several bacterial
14 strains were previously isolated from pine trees growing at these pits, of which 14 were identified as
15 effective nitrogen-fixers. In this study, we evaluated the inoculation effect of these 14 strains on lodgepole
16 pine growth under nitrogen-poor conditions. Each strain colonized the rhizosphere and internal tissues of
17 pine seedlings and significantly enhanced their length (24–65%) and biomass (100–300%), 18 months after
18 sowing and inoculation. Notably, three Pseudomonas strains increased pine seedling length by 1.6-fold and 19 biomass by 4-fold. Most strains also demonstratedDraft substantial potential to promote plant growth via 20 phosphorus solubilization, siderophore production, 1-aminocyclopropane-1-carboxylic acid deaminase
21 activity, indole-3-acetic acid production, lytic enzyme activity and catalase activity. Our results suggest that
22 such effective bacteria could be sustaining pine growth on bare gravel, indicating a possible ecological
23 association that may explain natural tree regeneration in such a disturbed ecosystem.
24 Keywords: Pinus, Pseudomonas, Gravel mining, Lodgepole pine, ACC deaminase
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25 Introduction
26 Natural and anthropogenic land disturbances, such as wildfires, resource mining and land-use changes,
27 induce severe soil degradation and represent major forms of environmental stress for re-establishing plant
28 communities (Small and Degenhardt 2018). In particular, the forest landscapes around North America,
29 including Canada, are severely altered during gravel mining – a resource widely used in construction
30 activities (LeMay 1999). Restoration or reclamation of forest landscapes after gravel extraction present
31 substantial challenges because these mining operations typically leave large disturbed areas that lack natural
32 vegetation cover and the medium for plant growth (i.e. topsoil). In other words, the bare gravel substrate
33 left after mining mimics primary succession conditions (LeMay 1999). In order to effectively reclaim such
34 disturbed ecosystems, it is crucial to re-establish land-form complexity and natural soil type, which would 35 otherwise develop only over long periods ofDraft time (Macdonald et al. 2015). Thus, efficient, sustainable and 36 cost-effective reclamation technologies need to be investigated and implemented to restore soil and native
37 vegetation. One possible strategy is the use of plant-beneficial microbes that can help restore natural plant
38 communities and ecosystem functioning (de-Bashan et al. 2012; Glick 2012).
39 Plant growth-promoting bacteria (PGPB) are bacterial strains isolated from diverse environments
40 (such as the rhizosphere, endosphere or bulk soil) with the potential to positively influence plant growth
41 and yield (de-Bashan et al. 2012). Several studies have revealed that in both natural and managed
42 ecosystems, rhizospheric and endophytic (living asymptomatically within plant tissues) bacteria can confer
43 considerable benefits to their host plants via one or more plant growth-promoting mechanisms. These
44 include facilitating nutrient acquisition via nitrogen-fixation, phosphorus solubilization and siderophore
45 production; modulating plant hormone levels via indole-3-acetic acid (IAA) production and 1-
46 aminocyclopropane-1-carboxylicacid (ACC) deaminase activity; and reducing the inhibitory effects of
47 phytopathogens via lytic enzyme production (Chaiharn and Lumyong 2009; Glick 2012; Khan et al. 2015;
48 Kandel et al. 2017; Padda et al. 2017a, b; Puri et al. 2020c, d). In disturbed environments, where plants
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49 encounter growth limiting conditions, PGPB may employ one or more of these strategies to effectively
50 overcome such limitations and support plant growth. Though under-studied, recent research has provided
51 convincing evidence that PGPB can significantly influence the growth of boreal forest trees in nutrient-
52 poor ecosystems affected by severe biotic and abiotic stresses (reviewed by Puri et al. 2017a; Witzell and
53 Martín 2018). Substantial growth-enhancement by PGPB has been reported in limber pine (Pinus flexilis)
54 (Moyes et al. 2016), lodgepole pine (Pinus contorta var. latifolia) (Yang et al. 2016; Tang et al. 2017),
55 Engelmann spruce (Picea engelmannii) (Carrell and Frank 2014), hybrid white spruce (Picea glauca x
56 engelmannii) (Puri et al. 2018a) and Douglas-fir (Pseudotsuga menziesii) (Aghai et al. 2019) growing under
57 degraded soil environments.
58 Gravel mining pits located in the Sub-Boreal Pine-Spruce dry-cool (SBPSdc) biogeoclimatic zone 59 of British Columbia (BC), Canada – characterizedDraft by the absence of topsoil and forest floor as well as the 60 prevalence of gravelly parent material (glacial till) and harsh climatic conditions (cold, dry winters and
61 cool, dry summers) – signify one of the most nutrient-poor and disturbed environments where trees have
62 been observed to grow (Steen and Coupé 1997; Chapman and Paul 2012). Apparently unaffected by such
63 disturbances, lodgepole pine trees are naturally regenerating at these gravel-dominated sites with tissue
64 nitrogen content and growth rates comparable to pine trees growing at nearby undisturbed forest sites with
65 intact topsoil (Chapman and Paul 2012). Tree height, leader length and root collar diameter of lodgepole
66 pine trees at both disturbed and undisturbed sites were found to be similar whereas a significant disparity
67 in soil nutrient status was reported, with soil nitrogen levels being six-fold lower at the gravel sites
68 (Chapman and Paul 2012). These findings raise an intriguing question – how do lodgepole pine trees grow
69 on bare gravel with no topsoil and extremely limited plant-available nutrients, particularly nitrogen? To
70 address this question, we previously sampled young lodgepole pine trees from two gravel mining pits
71 located in the SBPSdc zone of BC and isolated 77 potential nitrogen-fixing bacteria from the internal tissues
72 of pine trees (Padda et al. 2018). Of these, 14 bacteria were identified as effective nitrogen-fixers on the
73 basis of acetylene reduction assay and 15N isotope dilution assay (Padda et al. 2019). In the current study,
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74 our primary objective was to evaluate the longer term response of inoculation of these 14 bacteria on the
75 growth of lodgepole pine seedlings under extremely nitrogen-poor soil conditions with future implications
76 of selecting highly-effective bacteria for field testing at gravel mining pits. A secondary objective was to
77 investigate the potential ability of these bacteria to support plant growth via phosphorus solubilization,
78 siderophore production, ACC deaminase activity, IAA production, lytic enzyme activity and catalase
79 activity.
80 Materials and methods
81 Fourteen bacterial strains tested in this study were originally isolated from needle, stem and root tissues of
82 lodgepole pine trees growing at gravel mining pits located in the SBPSdc biogeoclimatic zone of BC, 83 Canada – namely, Anah pit (52°09’42.7’ N,Draft 123°10’24.3’ W, 1132 m a.s.l.) and Skulow pit (52°18’54.1’ 84 N, 121°53’39.3’ W, 1064 m a.s.l.) (Padda et al. 2018). For the greenhouse experiment, antibiotic-resistant
85 derivatives of these strains were raised by growing each strain on combined carbon medium (CCM) (Rennie
86 1981) amended with an antibiotic agent (200 mg L-1 of rifamycin) in order to assess plant colonization by
87 these strains after inoculation (Bal and Chanway 2012). Previous studies have indicated that antibiotic-
88 resistance mutation does not affect the plant growth-promoting abilities of endophytic bacteria (Shishido et
89 al. 1995; Bal and Chanway 2012; Puri et al. 2020b).
90 Experiment 1 – Greenhouse study
91 Each bacterial strain was evaluated for its ability to: (i) colonize rhizosphere and internal tissues of
92 lodgepole pine seedlings after inoculation, and (ii) promote lodgepole pine seedling growth under nitrogen-
93 poor conditions in an 18-month long greenhouse study. Non-inoculated seedlings were treated as controls,
94 which resulted in 15 treatments (14 bacteria-inoculated and 1 control).
95 Seedling growth assay
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96 For each strain, bacterial inoculum was prepared by thawing the frozen cultures and streaking a loopful
97 onto CCM agar amended with rifamycin (200 mgL-1) and incubating at 30°C for 48 h. After the colonies
98 grew, a loopful of each strain was inoculated into fresh CCM broth amended with rifamycin (200 mg L-1)
99 and incubated for 48 h at 30°C in a rotary shaker (150 rpm). Bacterial cells were harvested by centrifugation
100 (3000x g, 30 min), washed twice with sterile phosphate buffer saline (PBS) solution (pH 7.4) and re-
101 suspended in the same buffer to a density of 106 cfu mL-1 (Puri et al. 2015).
102 Lodgepole pine seeds were obtained from the British Columbia Ministry of Forests Tree Seed
103 Center, Surrey, BC, Canada and originated from forest sites located near Williams Lake, BC (52° 27′ N
104 latitude, 123° 20′ W longitude, elevation 1188 m, SBPSdc zone). Seeds were surface-sterilized by
105 immersion in 30% hydrogen peroxide for 90 s, followed by three 30 s rinses in sterile distilled water. The 106 effectiveness of the surface sterilization wasDraft confirmed by imprinting sterilized seed on tryptic soy agar 107 (TSA) and then checking for microbial contamination 48 h later (Puri et al. 2016a, b). Seeds found to be
108 free of surface contamination were placed in sterile cheesecloth bags containing moist autoclaved sand and
109 stratified for 28 d at 4°C. Stratified seeds were again imprinted on TSA plates for 48 h to confirm the
110 absence of surface contamination. Ten randomly selected surface-sterilized stratified seeds were crushed
111 and imprinted on TSA plates amended with rifamycin (200 mg L-1) for 48 h to confirm the absence of
112 internal seed contamination by any of the 14 bacterial strains prior to inoculation.
113 Seedling growth assays were performed in Ray Leach Cone-tainers (height: 210 mm, diameter: 38
114 mm) filled to 67% capacity with a sterile soil growth medium (69% w/w silica sand; 29% w/w Turface
115 (illite clay); 2% w/w CaCO3). Each Cone-tainer was fertilized with limited amounts (20 mL) of nutrient
-1 116 solution (Chanway et al. 1988) containing (g L ): Ca(NO3)2 (0.0576), Na2FeEDTA (0.02), KH2PO4 (0.14),
117 MgSO4 (0.49), H3BO3 (0.001), MnCI2.4H2O (0.001), ZnSO4.7H2O (0.001); CuSO4.5H2O (0.0001) and
118 NaMoO4.2H2O (0.001). Three stratified pine seeds were aseptically sown in each Cone-tainer and covered
119 with 5 mm of moist autoclaved silica sand. Immediately after sowing, 5 mL bacterial suspension of each
120 strain was pipetted directly over the seeds into Cone-tainers designated for each treatment. Non-inoculated
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121 control seeds received 5 mL of sterile PBS containing no bacteria. The Cone-tainers were placed in 98 cell-
122 trays in the University of British Columbia Plant Services Centre greenhouse under a 16 h photoperiod with
123 an intensity of at least 300 μmol s-1 m-2. In each Cone-tainer, seedlings were thinned to contain the single
124 largest germinant once emergence was complete. Seedlings were watered as required with sterile distilled
125 water during the 18-month long growth trial. Nutrient solution (20 mL) was provided once every month,
126 but without Ca(NO3)2 in order to grow the seedlings under similar nitrogen-poor conditions as reported at
127 the gravel mining pits (Chapman and Paul 2012). Tray positions were randomized weekly to reduce
128 positional effects during the trial.
129 Rhizospheric and endophytic colonization
130 Rhizospheric colonization by bacteria was assessed by harvesting five randomly selected seedlings from
131 each treatment at the end of the growth trialDraft (18 months after inoculation). Loosely adhering growth media
132 was removed from roots of each selected seedling with gentle shaking. Roots were separated from shoots,
133 placed in sterile Falcon tubes (50 mL; BD Biosciences, California, USA) containing 10 mL sterile PBS and
134 vortexed at 1000 rpm for 1 min. Serial dilutions were performed, and aliquots of 100 µL were plated on
135 CCM agar amended with rifamycin (200 mg L-1) and cycloheximide (100 mg L-1). Colonies were counted
136 after incubating the plates at 30°C for 7 days. Root dry weight was measured after oven-drying the roots at
137 65°C for 48 h. Rhizospheric bacterial populations were then calculated as cfu per gram of dry root tissue.
138 To evaluate endophytic colonization by each bacterial strain in needle, stem and root tissues, five
139 randomly selected seedlings were harvested per treatment at the end of the growth trial (18 months after
140 inoculation). Seedlings were surface-sterilized by rinsing in 0.6 % (w/v) sodium hypochlorite for 5 min,
141 washed three times in sterile distilled water and imprinted on TSA plates for 24 h to check for surface
142 contamination (Padda et al. 2016a, b). Samples of root, stem, and leaf tissues were triturated separately in
143 1 mL of sterile PBS using a sterilized mortar and pestle. Serial dilutions of the triturated tissue suspensions
144 were performed, and 100 µL aliquot of each dilution was plated onto CCM agar amended with 100 mg L-1
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145 cycloheximide (to suppress fungal growth) and 200 mg L-1 rifamycin (to inhibit the growth of other
146 bacteria). Plates were incubated for 7 days at 30°C and endophytic bacterial colonies were counted as cfu
147 per gram of fresh plant tissue.
148 Effect of inoculation on seedling growth
149 The longer term impact of different bacterial treatments on the growth and development of lodgepole pine
150 seedlings was evaluated after 18 months of inoculation. Ten randomly selected pine seedlings from each
151 treatment were removed from Cone-tainers to measure seedling length and biomass. Loosely adhering
152 growth-media particles were removed by washing the seedlings under running water before measuring their
153 length. Seedlings were then oven-dried at 65°C for 48 h to determine their biomass (dry weight).
154 Experiment 2 – Mechanisms of plant Draftgrowth-promotion
155 ACC deaminase activity of each strain was evaluated via two assays using the methodology outlined by
156 Penrose and Glick (2003): (i) an 훼-ketobutyrate quantification assay to determine the amount of 훼-
157 ketobutyrate produced when the enzyme ACC deaminase cleaves ACC (precursor of plant ethylene) and
158 (ii) a gnotobiotic root elongation assay to assess the effect of ACC deaminase producing bacteria on the
159 root-growth of ethylene-sensitive plants, canola and tomato. IAA production by each bacterial strain was
160 examined following the protocol described by Glickmann and Dessaux (1995).
161 The phosphate solubilizing ability of the bacterial strains was determined using Pikovskaya’s
162 (PVK) growth medium (Pikovskaya 1948) containing 0.5% tri-calcium phosphate as the inorganic
163 phosphate source. Qualitative assessment of phosphate solubilization ability was conducted using the
164 methodology explained by Kandel et al. (2017). Phosphate solubilization index (ratio of halo + colony
165 diameter to the colony diameter) was calculated for bacterial strains capable of producing a distinct clear
166 halo as a zone of solubilization. Quantitative assessment of phosphate solubilization was carried out
167 following the procedure outlined by Chaiharn and Lumyong (2009). Bacterial strains were also evaluated
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168 for their ability to mineralize (hydrolyze) sodium phytate (organic form of phosphorus) using phytase
169 screening medium (PSM) (Kerovuo et al. 1998). Qualitative phytase activity was estimated using PSM agar
170 plates and expressed as the solubilization index. Quantitative phytase activity was determined according to
171 the method described by Yanke et al. (1998). One unit (U) of phytase was defined as the amount of enzyme
172 required to liberate 1 nmol of inorganic phosphorus per minute under the given assay conditions and
173 expressed per mL of the original culture. To assess the ability of the bacterial strains to chelate iron, a
174 siderophore production test was performed as described in Louden et al. (2011) using blue-colored chrome
175 azurol S (CAS) agar plates. The area of color conversion from blue to orange around each bacterial colony
176 indicated siderophore production (Kandel et al. 2017).
177 All bacterial strains were assessed for the production of cell wall degrading (lytic) enzymes – 178 cellulase, chitinase, -1,3-glucanase and proteaseDraft – using plate and quantitative assays. For evaluation of 179 cellulase enzyme activity of bacterial strains, casein–yeast extract (CYE) agar plates amended with 1%
180 sodium carboxymethylcellulose were used following the protocol described by Yang et al. (2017). Chitinase
181 enzyme activity was tested on a chitin medium agar plate containing colloidal chitin (8 g L-1) (Sahoo et al.
182 1999). -1,3-glucanase enzyme activity was determined using -1,3-glucan agar plates (containing 5 g L-1
183 laminarin as the carbon source) (Cattelan et al. 1999). Protease enzyme activity of each bacterial strain was
184 determined using CYE agar plates amended with 7% skimmed milk powder (Padda et al. 2017a). The clear
185 zone surrounding bacterial colonies indicated positive cellulase, chitinase, -1,3-glucanase and protease
186 activity and the zone of clearance for each activity was estimated as: (diameter of clear zone + colony) –
187 (diameter of colony). Cellulase, chitinase and -1,3-glucanase activities were assessed quantitatively by
188 measuring the release of reducing sugars using carboxymethylcellulose, colloidal chitin and laminarin,
189 respectively, as substrates and glucose as the standard (Chaiharn and Lumyong 2009). One unit (U) of
190 cellulase, chitinase or -1,3-glucanase activity was defined as the amount of enzyme resulting in the release
191 of 1 μmol of glucose equivalent from carboxymethylcellulose, colloidal chitin or laminarin per minute
192 under the assay conditions. Protease activity was determined by measuring the release of reducing sugars
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193 by using azocasein as the substrate and tyrosine as the standard (Chaiharn and Lumyong 2009). One unit
194 of protease enzyme activity was defined as the amount of the enzyme resulting in the release of 1 μmol of
195 tyrosine equivalent from azocasein per minute under assay conditions. Catalase activity by each bacterial
196 strain was evaluated by mixing a loopful of fresh bacterial culture with 50 μL of 3% (v/v) hydrogen peroxide
197 on a clean glass slide and incubating at room temperature for 1 min (Padda et al. 2017a). The evolution of
198 oxygen (development of gas bubbles) was recorded as a positive catalase reaction.
199 Statistical analyses
200 To evaluate the treatment effects of each bacterial strain on the growth of lodgepole pine seedlings in the
201 greenhouse study, a completely randomized design was used. Analysis of variance (ANOVA) including F-
202 test and Student’s t-test were performed to determine significant differences between treatment means for
203 seedling length and biomass (n=10 seedlingsDraft per treatment). Using ANOVA (F-test and t-test), the means
204 of different bacterial treatments were also compared for: (i) all quantitative assays viz. ACC deaminase
205 activity, IAA production, inorganic phosphorus solubilization, organic phosphorus mineralization, cellulase
206 activity, chitinase activity, -1,3-glucanase activity and protease activity (n=3 per treatment for each
207 quantitative assay); (ii) siderophore production assay (n=3 per treatment); and (iii) gnotobiotic root
208 elongation assay (n=7 seedlings per treatment for each plant species). The statistical package, SAS
209 University Edition (SAS Institute Inc., Cary, NC, USA), was used to perform statistical analyses (훼 = 0.05).
210 Results
211 Experiment 1 – Greenhouse study
212 After 18 months of inoculation, each bacterial strain colonized the rhizosphere of pine seedlings with
213 population sizes of 104–106 cfu g-1 dry root weight (Table 1). Strains AN1r and AR1r colonized the pine
214 rhizosphere most densely, closely followed by strain SN1r. All bacterial strains were able to successfully
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215 colonize internal root tissues of lodgepole pine seedlings, with population sizes varying between 104 and
216 107 cfu g-1 fresh tissue weight (Table 1). The internal root population of strain AN1r (1.01 x 107 cfu g-1)
217 was highest among all strains, followed by strains AR1r (8.80 x 106 cfu g-1) and SN1r (5.15 x 106 cfu g-1).
218 Ten bacterial strains were detected inside stem tissues of pine seedlings, with strains AN1r, AR1r and SN1r
219 forming the highest population densities (5.81–9.94 x 105 cfu g-1). Needle colonization in pine seedlings
220 was observed for strains AS1r, AN1r, AN2r, AR1r, SN1r and SN2r, with population densities ranging from
221 102 to 104 cfu g-1 fresh tissue weight (Table 1). Among these six strains, the needle population sizes of
222 strains AN1r and SN1r were highest (6.14 x 104 cfu g-1 and 9.21 x 104 cfu g-1, respectively). All in all,
223 strains AN1r, AR1r and SN1r were the most effective plant tissue colonizers, with aggregated endophytic
224 and rhizosphere population densities of 106–107 cfu g-1. No bacterial colonies were detected in the internal
225 plant tissues and rhizosphere of the control seedlings. Draft 226 Lodgepole pine growth (length and biomass) was significantly enhanced due to bacterial
227 inoculation under nitrogen-poor conditions after 18 months (Fig. 1). A substantial increase (up to 1.6-fold)
228 in seedling length was observed for all bacteria-inoculated treatments over the non-inoculated control
229 treatment (Fig. 1a). Specifically, inoculation with strains AN1r, AR1r and SN1r, resulted in > 60% increase
230 in the length of pine seedlings, performing significantly better than half of the bacterial treatments. Similar
231 to seedling length, bacterial inoculation considerably enhanced the biomass of pine seedlings by
232 accumulating > 2-fold higher biomass than the control seedlings (Fig. 1b). Notably, AR1r-, SN1r- and
233 AN1r-inoculated seedlings accumulated 300%, 270% and 250% greater biomass than the control seedlings,
234 respectively. These three bacterial treatments also outperformed all other bacterial treatments by
235 accumulating up to 100% greater biomass than other strains. Furthermore, it was determined that both
236 length and biomass had strong correlations with endophytic colonization (aggregated for all tissue types) (r
237 = 0.85 – 0.95; p < 0.0001) as well as rhizospheric colonization (r = 0.86 – 0.94; p < 0.0001) (Fig. 2).
238 Experiment 2 – Mechanisms of plant growth-promotion
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239 ACC deaminase activity of bacterial strains tested in vitro via 훼-ketobutyrate production ranged from 8.33
240 to 119 nmol h-1 mg-1 protein (Fig. 3a). Strain AN1r produced the highest levels of 훼-ketobutyrate (up to 14-
241 fold in comparison to other bacteria), followed by strains SN1r, AR1r and SN2r. In the gnotobiotic root
242 elongation assay, bacterial inoculation considerably increased the primary root length of canola and tomato
243 seedlings (up to 230% and 300%, respectively) (Figs. 4 and S1). Notably, strains AN1r and SN1r enhanced
244 the root length of canola seedlings by 3-fold and tomato seedlings by 4-fold, performing significantly better
245 than all other bacterial treatments except AR1r. The in vitro 훼-ketobutyrate production was observed to
246 have a very strong correlation with canola root length (r = 0.96; p < 0.0001) as well as tomato root length
247 (r = 0.91; p < 0.0001) (Fig. 5). The amount of IAA produced by different bacterial strains when
248 supplemented with L-tryptophan varied between 11.7 – 40.3 μg mL-1 (Fig. 3b). The highest-level of IAA
249 production was detected in strains AS1r, AN1r, AN4r, AR1r and SN1r, all belonging to the genus
250 Pseudomonas. In particular, strains AN1r andDraft AN4r produced up to 3.5-fold greater amounts of IAA in
251 vitro compared to the nine other strains.
252 Several bacterial strains tested positive for their ability to solubilize inorganic phosphorus,
253 mineralize organic phosphorus and sequester iron (via siderophore production). Inorganic phosphorus
254 solubilization was confirmed for 11 strains, of which, five strains (AN1r, AR1r, SS2r, SN1r and SR1r)
255 solubilized significantly greater amounts of tri-calcium phosphate than the others (solubilization index ≥
256 2.20; quantitative phosphate solubilization ≥ 100 μg mL-1) (Table 2). No phosphate solubilization was
257 observed for strains AN2r, SN2r and SR2r. Ten of the 14 bacterial strains hydrolyzed phytate, with
258 solubilization indices ranging from 1.14 – 3.14 and quantitative phytase activity ranging from 25.3 – 77.2
259 U mL-1 (Table 2). In particular, the pseudomonads AN1r, AR1r and SN1r were the best-performing strains,
260 demonstrating up to 3-fold, 2.5-fold and 2.6-fold higher phytase activity, respectively, in comparison to all
261 other strains. No phytase activity was detected in strains AN2r, AN3r, SS3r and SN2r. The highest
262 efficiency for phosphate solubilization and phytate hydrolyzation was observed for strain AN1r, which
263 outperformed all other bacterial strains in both qualitative (plate-based) and quantitative (broth-based)
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264 assays. Siderophore production was confirmed for 10 bacterial strains in vitro by measuring the area of
265 orange halo contiguous with colony growth (Fig. 3c). The largest orange halo area was observed for strains
266 AN1r, AR1r and SN1r (1.87 – 2.58 cm2).
267 All bacterial strains except SS3r tested positive for the ability to produce at least one of the lytic
268 enzymes in both qualitative and quantitative assays (Table 3). Cellulase enzyme activity was confirmed for
269 11 bacterial strains in the plate assay, with the highest zone of clearance (15–25 mm) observed for strains
270 AS1r, AR1r, SS2r and SN1r. A similar trend was observed in the quantitative assay, where bacterial strains
271 produced between 0.23 and 1.03 U mL-1 of cellulase (Table 3). In particular, strains AS1r, AR1r and SN1r
272 showed the highest cellulase activity, outperforming all other bacterial strains. More than half of the
273 bacterial strains exhibited chitinase enzyme activity, with AR1r dissolving the most colloidal chitin in both 274 plate (15–25 mm zone of clearance) and quantitativeDraft (0.93 0.04 U mL-1) assays (Table 3). Seven bacterial 275 strains were tested positive for synthesis of -1,3-glucanase (Table 3). Of these, the highest -1,3-glucanase
276 activity was detected in strains AN1r and AR1r – zone of clearance (15–25 mm) in the plate assay and
277 0.81–0.90 U mL-1 produced in the quantitative assay. Substantial protease enzyme activity was confirmed
278 for nine strains, which showed positive results in the plate assay and dissolved significant amounts of
279 azocasein (23.6–76.1 U mL-1). The highest proteolytic activity was observed in strains AS1r and AR1r,
280 followed by strains SN1r, AN1r, SS2r and AN5r (Table 3). Overall, for all cell wall degrading enzymes, a
281 good association between the zone of clearance on agar plates and the estimated level of enzyme production
282 in liquid medium was observed. Notably, strains AS1r, AN1r, AR1r and SN1r exhibited positive results for
283 all four enzyme activities. Catalase enzyme activity was detected in 10 strains, of which strains AN1r, AN4r
284 and SN1r showed highest activity (Table 3).
285 Discussion
286 In this study, we examined the efficacy of plant growth-promoting bacteria naturally associating with
287 lodgepole pine trees on nutrient-poor, unreclaimed gravel mining pits, where survival and growth of trees
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288 should be marginal, but is robust (Chapman and Paul 2012). An 18-month long greenhouse study was
289 conducted to evaluate the effects of bacterial inoculation on the growth of lodgepole pine seedlings under
290 extremely nitrogen-poor edaphic conditions. Bacterial inoculation significantly enhanced the growth
291 (length >1.2-fold, biomass >2-fold) of lodgepole pine seedlings in comparison to the control treatment (Fig.
292 1), suggesting that each bacterial strain tested in this study has considerable potential to promote host-tree
293 growth under nitrogen stress. Aghai et al. (2019) and Khan et al. (2015) reported similar results for
294 Salicaceae endophytes, originally isolated from nutrient-poor disturbed environments where inoculation
295 with these endophytes substantially improved the growth and survival-rate of Douglas-fir and western red
296 cedar (Thuja plicata) under greenhouse and field conditions. The highest growth promotion was observed
297 for P. graminis AN1r, P. migulae AR1r and P. lini SN1r – interestingly all pseudomonads – a genus well-
298 known to colonize diverse ecological niches and promote plant growth through a variety of mechanisms
299 including nitrogen-fixation, biocontrol ofDraft pathogens, phosphorus solubilization and phytohormone
300 secretion (Miller et al. 2008; Puri et al. 2017b). Previously, P. graminis, P. migulae and P. lini strains have
301 been reported to stimulate the length and biomass of poplar, hybrid white spruce and wheat (Triticum
302 aestivum L.) (Knoth et al. 2014; Ehsan et al. 2016; Puri et al. 2020a). Furthermore, since bacterial consortia
303 containing diverse Pseudomonas strains have been reported to perform significantly better in promoting
304 plant growth compared to a single Pseudomonas strain (Hu et al. 2017), we propose that the Pseudomonas
305 strains tested in this study should be further evaluated as a consortium to determine their combined potential
306 to sustain tree growth, if any.
307 In addition to growth promotion, all bacterial strains were able to colonize the rhizosphere and
308 internal tissues of lodgepole pine seedlings, with the highest population sizes detected for strains AN1r,
309 AR1r and SN1r (106–107 cfu g-1) (Table 1). Several previous studies have reported similar bacterial
310 population sizes in coniferous trees (Yang et al. 2016; Puri et al. 2020a, b). Our results support the notion
311 proposed by Compant et al. (2010) that effective colonization by a bacterial inoculant is a prerequisite for
312 successful plant growth promotion. This concept is further supported by the strong correlation of endophytic
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313 and rhizospheric colonization with seedling growth (length and biomass) (Fig. 2), signifying that colonizing
314 potential of a bacterial strain could have a direct impact on its growth-promotion ability. Since both
315 endophytic and rhizospheric population densities had an equally strong correlation with seedling length (r
316 = 0.85 or 0.86; P < 0.0001) and biomass (r = 0.95 or 0.94; P < 0.0001), it is possible that both types of
317 colonization might have had a synergistic effect on plant growth. However, further in situ analyses need to
318 be done to investigate the role played by endophytic versus rhizospheric bacterial populations in improving
319 plant growth.
320 Since previous studies have suggested that nitrogen-fixing bacteria have the potential to stimulate
321 plant growth through an array of mechanisms other than nitrogen fixation (Piccoli et al. 2011; Khan et al.
322 2015; Kandel et al. 2017), we examined our bacterial strains for multiple plant growth-promoting traits. 323 Our results indicate that these strains have theDraft potential to influence plant growth through a wide range of 324 mechanisms in addition to the originally postulated nitrogen fixation, viz., converting inaccessible plant
325 nutrients to bioavailable forms (phosphorus and iron), altering phytohormone levels (IAA and ethylene)
326 and protecting host plants against biotic and abiotic stresses. Our research outcomes support previous
327 findings, where endophytic PGPB isolated from trees growing in disturbed environments have been
328 reported to enhance the growth, fitness, and adaptation of their hosts to stressful environmental conditions
329 in a comprehensive manner (Kandel et al. 2017; Frank 2018; Puri et al. 2018b).
330 Significant ACC deaminase activity was detected in most of the bacterial strains as indicated by
331 the production of 훼-ketobutyrate (Fig. 3a). When subjected to various biotic and abiotic stresses, plants
332 synthesize unusual levels of the plant hormone ethylene, also called ‘stress ethylene’, that can have
333 detrimental effects on plant growth. Under such conditions, ACC deaminase synthesized by bacteria lower
334 the stress ethylene levels by converting ACC, the immediate precursor of plant ethylene, to ammonia and
335 훼-ketobutyrate (Glick 2012). Since ethylene is a key regulator of the plant tissue colonization by bacteria,
336 the ability of ACC deaminase to reduce the ethylene concentration has also been linked to enhanced
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337 bacterial colonization (Frank 2018). Subsequently, in the gnotobiotic assay, bacterial inoculation via seed
338 coating resulted in considerable root elongation of the ethylene-sensitive model plants, canola and tomato
339 (Figs. 4 and S1). Even though some ethylene production is essential to stimulate seed germination,
340 continually elevated plant ethylene levels following germination may obstruct root growth (Esashi 1991).
341 It has been suggested that ACC deaminase producing bacteria establish a sink for plant ACC after
342 inoculation, which consequently lowers plant ethylene levels, thereby enhancing initial root growth and
343 development (Glick 2012). A strong correlation was observed between in vitro (훼-ketobutyrate production)
344 and in vivo ACC deaminase activity for both canola (r = 0.96; P < 0.0001) and tomato (r = 0.91; P < 0.0001)
345 (Fig. 5), suggesting that the greater the amount of ACC converted to 훼-ketobutyrate, rather than stress
346 ethylene, the greater the primary root length. This can be clearly seen in the P. graminis AN1r, P. lini SN1r
347 and P. migulae AR1r bacterial treatments. In addition, Penrose and Glick (2003) implied that lower in vitro
348 훼-ketobutyrate production (< 20 nmol h-1 mgDraft-1) generally has little to no effect on root growth, which proved
349 to be true for bacterial strains SS3r and SR1r (Fig. 5).
350 Each bacterial strain tested positive for IAA production in the presence of L-tryptophan, an amino
351 acid commonly found in root exudates. Biosynthesis of phytohormones like IAA is considered to be one of
352 the key plant growth-promoting mechanisms due to its direct effect on plant cell proliferation, stem and
353 root development, and seed germination (Glick 2012). IAA-producing bacteria have been reported to form
354 a mutually beneficial association with the host plant by converting the host metabolite L-tryptophan to a
355 useful plant growth hormone IAA (Pilet and Saugy 1987). Five Pseudomonas strains (AS1r, AN1r, AN4r,
356 AR1r and SN1r) produced significantly greater amounts of IAA than the other bacterial isolates (Fig. 3b),
357 which is consistent with previous reports of IAA synthesis by endophytic Pseudomonas strains isolated
358 from agricultural and forest ecosystems (Patten and Glick 2002; Kandel et al. 2017).
359 Solubilization and mineralization of phosphorus is considered to be an important trait of PGPB due
360 to the limited bioavailability of phosphorus in soil. Based on our results, we infer that most of the bacterial
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361 strains were able to solubilize considerable amounts of inorganic (tri-calcium phosphate) and organic
362 (sodium phytate) phosphorus (Table 2). Pseudomonas graminis AN1r, Pseudomonas lini SN1r and
363 Pseudomonas migulae AR1r solubilized the highest amounts of both inorganic (> 120 µg mL-1) and organic
364 (> 65 U mL-1) forms of phosphorus with solubilization indices of 2.5 or higher (Table 2). These findings
365 are comparable with the amounts of phosphorus solubilized by endophytic strains of Douglas-fir (Khan et
366 al. 2015), Himalayan pine (Pinus wallichiana) (Hayat et al. 2017), hybrid white spruce (Puri et al. 2020c)
367 and poplar (Populus trichocarpa) (Kandel et al. 2017). The solubilization of inorganic phosphorus occurs
368 when pH is lowered either through the production of organic acids by bacteria such as gluconic, citric, lactic
369 and oxalic acid or through release of protons (Rodríguez and Fraga 1999). The mineralization of organic
370 phosphorus occurs through the synthesis of various phosphatases such as phytase that hydrolyze organic
371 forms of phosphate compounds and release the inorganic phosphorus for plant uptake (Rodríguez and Fraga
372 1999). A positive correlation (r = 0.84; PDraft < 0.0001) was observed between solubilization of inorganic
373 phosphorus and mineralization of organic phosphorus by our bacterial strains which supports the hypothesis
374 proposed by Tao et al. (2008) that bacterial strains with strong phosphate solubilization ability also have a
375 strong mineralization ability. Iron sequestration through siderophore production was confirmed for 10 of
376 the 14 bacterial strains (Fig. 3c). Highest in vitro siderophore production was observed for P. graminis
377 strain AN1r, followed by P. migulae AR1r and P. lini SN1r. Due to the sparingly soluble nature of ferric
378 iron, the predominant form of iron in soil, bacteria can synthesize low-molecular weight siderophores with
379 an exceptionally high affinity for ferric iron to mobilize and facilitate iron uptake by plants (Loper and
380 Buyer 1991). Similar findings for siderophore production have been reported for Salicaceae endophytes
381 isolated from nutrient-poor environments (Khan et al. 2015; Kandel et al. 2017). In addition to their
382 potential role in acquiring iron, siderophores can also prevent the proliferation of fungal pathogens by
383 scavenging iron available in soil (Loaces et al. 2011). Because of this dual role, siderophore production by
384 PGPB plays an even more important role when plants are subjected to stressful environments (Glick 2012).
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385 The majority of the bacterial strains tested positive for the ability to degrade cell walls by producing
386 at least one of the lytic enzymes – cellulase (degrades cellulose), protease (degrades cell wall proteins),
387 chitinase (degrades chitin) and -1,3-glucanase (degrades glucan). Synthesis of lytic enzymes has been
388 reported to assist PGPB in establishing endophytic populations in plants along with playing a key role in
389 the biocontrol of plant pathogen populations (Compant et al. 2005; Won et al. 2019). Bacterial strains P.
390 migulae AR1r, P. lini SN1r and Pseudomonas mandelii AS1r exhibited significantly high-levels of
391 cellulolytic activity (Table 3), indicating that these strains may have the potential to break β-1,4-glycosidic
392 linkages in cellulose, a polysaccharide present in cell walls of plants and microorganisms, including
393 phytopathogens (Lynd et al. 2002). In this sense, cellulose-degradation might be playing a role in plant
394 colonization by bacteria, as evident from the large endophytic populations formed by these strains in
395 lodgepole pine seedlings (Table 1). Compant et al. (2005) also reported that Burkholderia sp. strain PsJN,
396 an endophytic PGPB with strong biocontrolDraft ability, was able to heavily colonize the internal tissues of
397 grapevine (Vitis vinifera) following the secretion of cellulase. Of all bacterial treatments, P. migulae AR1r
398 and P. mandelli AS1r showed considerable protease enzyme activity by dissolving significantly high
399 amounts of azocasein in both plate and quantitative assays (Table 3). Similar results for proteolytic activity
400 were reported by Chaiharn and Lumyong (2009) and Puri et al. (2020c, d). About half of the bacterial strains
401 tested positive for chitinase and β-1,3-glucanase activity, with P. graminis AN1r and P. migulae AR1r
402 displaying highest activity for both lytic enzymes (0.80 – 0.93 U mL-1) (Table 3). This is consistent with
403 the findings of Won et al. (2019) where comparable enzyme activity was observed for a Bacillus
404 licheniformis strain isolated from disturbed soils. Chitin and β-glucan occur widely in the exoskeleton of
405 arthropods and cell walls of fungi, therefore bacterial production of chitinase and β-1,3-glucanase may play
406 a role in protecting the plant from diverse phytopathogens (Jadhav et al. 2017). Catalase activity was also
407 observed in 10 bacterial strains (Table 3). Considering that levels of reactive oxygen species (ROS) in plants
408 are increased significantly under environmental stress, production of catalase indicates that these strains
409 could help promote stress tolerance in host plants by lowering their ROS levels (reviewed by Frank 2018).
410 The synthesis of catalase could also assist bacteria in colonizing plant tissues because they need protection
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411 from plant defense compounds such as ROS to survive inside plants (Frank 2018). Thus, the high colonizing
412 potential of P. graminis AN1r and P. lini SN1r observed in the greenhouse study may be linked to their
413 high catalase activity (Tables 1 and 3).
414 Using a bonitur scale, similar to that described by Krechel et al. (2002), bacterial strains tested in
415 this study were ranked based on the results of different plant growth-promoting mechanisms. In this scale,
416 points were given to bacterial strains for each in vitro plant growth-promoting trait examined in this study,
417 with a maximum possible score of 30 points (10 traits x 3 maximum points per trait) (scoring explained in
418 Fig. 6a). Based on the ranking, it was observed that the top eight strains belonged to the genus Pseudomonas
419 (Fig. 6b), highlighting that Pseudomonas may be playing a crucial role in supporting pine tree growth and
420 survival at gravel mining pits. Strains P. graminis AN1r, P. migulae AR1r and P. lini SN1r showed 421 substantially higher activity in all 10 plant Draftgrowth-promoting assays compared to the other strains (AN1r 422 and AR1r bonitur score = 28 points; SN1r bonitur score = 26 points) (Fig. 6b). In addition, these three
423 strains showed the greatest ability to colonize and enhance the growth of lodgepole pine seedlings under
424 nitrogen-poor conditions in the greenhouse study. Their high colonization ability may be attributed to their
425 significant ability to secrete multiple cell wall degrading enzymes, suppress plant ethylene levels (via ACC
426 deaminase activity) and lower ROS content (via catalase activity) (Tables 1 and 3 and Fig. 3a). In a previous
427 study, these three strains were also observed to fix significant amounts of nitrogen from the atmosphere
428 (~50%) in pine seedlings (Padda et al. 2019). Therefore, the pine seedling length and biomass stimulated
429 by these three strains in the greenhouse study could be attributed to their nitrogen-fixing ability. However,
430 other mechanisms including phosphorus solubilization, IAA production and ACC deaminase activity may
431 have contributed to the enhanced growth of pine seedlings, but this needs to be further evaluated in planta.
432 To summarize, P. graminis AN1r, P. migulae AR1r and P. lini SN1r inoculation had a significant
433 positive effect on the growth of lodgepole pine under extreme nitrogen stress. Results of this study highlight
434 the potential of these strains to be used as bioinoculants in association with lodgepole pine trees to
435 rehabilitate disturbed ecosystems, but further long-term testing in the field is necessary before their role in
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436 pine growth can be ascertained. If effective, such bacteria with multiple plant growth-promoting abilities
437 may sustain the tree growth for years and could act as an environment-friendly and cost-effective solution
438 to reclaim degraded surface mining sites. In addition, since lodgepole pine trees have been reported to
439 associate with ectomycorrhizal fungi in natural temperate and boreal forests (Shishido et al. 1996a, b; Paul
440 et al. 2006, 2013), their role in supporting pine growth in such disturbed ecosystems needs to be considered
441 as well. Therefore, in future field studies, these bacterial strains should be further evaluated in association
442 with ectomycorrhizal fungi for their possible coherent role in sustaining the growth of lodgepole pine trees
443 on these gravel mining sites.
444 Acknowledgements
445 This work was supported by the Natural SciencesDraft and Engineering Research Council of Canada (NSERC) 446 Discovery Grant (RGPIN 41832–13) to CPC. KP received funding from Hugo E Meilicke Memorial
447 Fellowship (University of British Columbia (UBC) award no. 527) and Mary and David Macaree
448 Fellowship (UBC award no. 4848). AP was supported by Li Tze Fong Memorial Fellowship (UBC award
449 no. 4895), Four-year Doctoral Fellowship (UBC award no. 6456) and Mary and David Macaree Fellowship
450 (UBC award no. 4848).
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603 Shishido, M., Loeb, B.M., and Chanway, C.P. 1995. External and internal root colonization of lodgepole 604 pine seedlings by two growth-promoting Bacillus strains originated from different root microsites. Can. J. 605 Microbiol., 41(8): 707–713.
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608 Shishido, M., Petersen, D.J., Massicotte, H.B., and Chanway, C.P. 1996b. Pine and spruce seedling 609 growth and mycorrhizal infection after inoculation with plant growth promoting Pseudomonas strains. 610 FEMS Microbiol. Ecol., 21(2): 109–119.
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622 Witzell, J., and Martín, J.A. 2018. Endophytes and Forest Health. In Endophytes of Forest Trees. Edited 623 by A. Pirttilä, A. Frank. Springer, Cham. pp. 261–282.
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635 Figure Captions
636 Fig. 1. (a) Seedling length and (b) biomass of lodgepole pine seedlings (mean and standard error; n=10 637 seedlings per treatment) inoculated with different bacterial strains measured 18 months after inoculation. 638 Bars with different letters are significantly different (P < 0.05).
639 Fig. 2. Correlation of (a) endophytic (aggregated for all tissue types) and (b) rhizospheric population 640 densities with length and biomass of lodgepole pine seedlings, 18 months after inoculation (P < 0.0001).
641 Fig. 3. (a) ACC deaminase activity of each bacterial strain measured as the amount of 훼-ketobutyrate 642 produced per mg protein per hour, (b) IAA production by each bacterial strain in the presence of L- 643 tryptophan, (c) Siderophore production by each bacterial strain estimated via the area of orange halo (cm2) 644 detected on blue CAS agar plates. Triplicate samples were used for each bacterial strain. Bars with different 645 letters are significantly different (P < 0.05).
646 Fig. 4. Primary root length of (a) canola and (b) tomato seedlings (mean and standard error; n=7 seedlings 647 per treatment) inoculated with different bacterial strains in the gnotobiotic root elongation assay to assess 648 in vivo ACC deaminase activity. Bars with Draftdifferent letters are significantly different (P < 0.05). 649 Fig. 5. Correlation between in vitro and in vivo ACC deaminase activity of lodgepole pine bacterial strains 650 (P < 0.0001). In vitro ACC deaminase activity was determined as the nmol of 훼-ketobutyrate produced per 651 mg protein per hour. In vivo ACC deaminase activity was determined as the primary root length of canola 652 and tomato seedlings inoculated with different bacterial strains.
653 Fig. 6. (a) Point distribution for different plant growth-promoting traits of bacterial strains on a bonitur 654 scale (maximum points = 3 per trait; minimum points = 0 per trait). (b) Ranking and score of all bacterial 655 strains based on their results for different in vitro plant growth-promotion assays using a bonitur scale 656 (maximum possible points = 30). Bacterial strains have been arranged on the x-axis from left to right based 657 on their ranking.
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Tables
Table 1. Mean bacterial densities in the lodgepole pine tissues (root, stem and needle) and rhizosphere expressed as colony forming unit (cfu) per gram fresh weight or dry root weight, 18 months after inoculation (n = 5 per bacterial strain for endophytic and rhizospheric colonization).
Bacterial strains Root population Stem population Needle population Rhizospheric population (cfu g-1 fresh weight) (cfu g-1 fresh weight) (cfu g-1 fresh weight) (cfu g-1 dry root weight)
Pseudomonas mandelii AS1r 8.21 x 105 7.13 x 104 6.32 x 102 5.25 x 104 Pseudomonas graminis AN1r 1.01 x 107 5.81 x 105 6.14 x 104 1.37 x 106 Rathayibacter tanaceti AN2r 2.23 x 105 3.84 x 105 8.67 x 102 3.97 x 104 Frigoribacterium endophyticum AN3r 3.15 x 105 1.03 x 105 – 8.92 x 104 Pseudomonas rhizosphaerae AN4r 9.21 x 104 3.63 x 103 – 4.62 x 104 Pseudomonas frederiksbergensis AN5r 7.43 x 104 Draft– – 1.04 x 105 Pseudomonas migulae AR1r 8.80 x 106 9.94 x 105 4.28 x 102 1.10 x 106 Pseudomonas mandelii SS1r 7.32 x 104 6.92 x 104 – 8.12 x 104 Pseudomonas frederiksbergensis SS2r 6.71 x 104 – – 8.47 x 104 Herbiconiux solani SS3r 1.11 x 104 – – 1.05 x 104 Pseudomonas lini SN1r 5.98 x 106 7.23 x 105 9.21 x 104 9.91 x 105 Flavobacterium aquidurense SN2r 8.77 x 105 1.11 x 105 2.57 x 103 1.32 x 105 Caballeronia sordidicola SR1r 2.26 x 104 – – 1.45 x 104 Rhizobium herbae SR2r 4.46 x 105 9.74 x 104 – 9.18 x 104
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Table 2. In vitro phosphate solubilization and phytate hydrolyzation by bacterial strains originally isolated from lodgepole pine trees growing at gravel mining sites.
Bacterial strains Phosphate solubilization Phytate hydrolyzation Solubilization index Quantitative assay Solubilization index Quantitative assay (µg mL-1) (U mL-1)
Pseudomonas mandelii AS1r 1.60 77.2 2.12*b 1.73 32.8 1.75a
Pseudomonas graminis AN1r 3.20 161 3.29e 3.14 77.2 3.10d Rathayibacter tanaceti AN2r – – – –
Frigoribacterium endophyticum AN3r 1.25 46.7 1.75a – –
Pseudomonas rhizosphaerae AN4r 1.07 46.2 2.55a 2.10 55.8 2.73bc
Pseudomonas frederiksbergensis AN5r 1.80 89.1 1.61bc 1.90 50.7 1.39b
Pseudomonas migulae AR1r 2.50Draft121 2.54d 2.42 64.7 2.36c
Pseudomonas mandelii SS1r 1.33 55.5 2.46a 1.63 29.5 1.08a
Pseudomonas frederiksbergensis SS2r 2.27 101 3.73c 2.00 47.3 1.61b
Herbiconiux solani SS3r 1.13 48.6 2.49a – –
Pseudomonas lini SN1r 2.53 126 5.55d 3.00 65.7 1.79c Flavobacterium aquidurense SN2r – – – –
Caballeronia sordidicola SR1r 2.20 98.2 4.62c 2.00 50.4 2.94b
Rhizobium herbae SR2r – – 1.14 25.3 1.77a
Phytate hydrolyzation: One unit (U) of phytase represents the amount of enzyme required to liberate 1 nmol of inorganic phosphorus per minute under the assay conditions. *Mean standard error (n = 3 per strain); values with different letters are significantly different (P < 0.05)
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Table 3. Cell wall degrading (lytic) and catalase enzyme activities of lodgepole pine bacterial strains.
Bacterial Cellulase activity Chitinase activity -1,3-glucanase activity Protease activity Catalase strains activity Plate Quantitative Plate Quantitative Plate Quantitative Plate Quantitative assay assay assay assay assay assay assay assay (U mL-1) (U mL-1) (U mL-1) (U mL-1)
AS1r +++ 0.94 0.01*e ++ 0.72 0.06b + 0.16 0.01a +++ 69.2 2.06e ++
AN1r ++ 0.66 0.04cd ++ 0.80 0.05bc +++ 0.90 0.02c ++ 52.4 1.73cd +++
AN2r – – – – – – + 23.6 1.67a +
AN3r + 0.19 0.01a – – – – – – +
AN4r ++ 0.53 0.02bc – – + 0.22 0.02a – – +++
AN5r ++ 0.61 0.02bcd + 0.33 0.03a – – ++ 47.5 3.15c –
e Draftc c e AR1r +++ 1.03 0.07 +++ 0.93 0.04 +++ 0.81 0.02 +++ 76.1 3.77 ++
SS1r ++ 0.47 0.01b – – – – + 27.8 1.43ab +
SS2r +++ 0.71 0.01d – – – – ++ 49.3 2.05cd + SS3r – – – – – – – – ++
SN1r +++ 0.95 0.02e ++ 0.74 0.01b ++ 0.54 0.02b ++ 58.4 2.24d +++
SN2r + 0.27 0.03a + 0.36 0.01a + 0.19 0.01a – – –
SR1r + 0.23 0.02a + 0.28 0.02a – – – – –
SR2r – – + 0.31 0.03a + 0.46 0.01b + 34.3 2.06b –
Plate assay: +++ (high activity: 15–25 mm zone of clearance); ++ (medium activity: 5–15 mm zone of clearance); + (low activity: <5 mm zone of clearance); – (no zone of clearance detected) Quantitative assay: One unit (U) of cellulase, chitinase and -1,3-glucanase activity represents the amount of enzyme resulting in the release of 1 μmol of glucose equivalent from carboxymethylcellulose, colloidal chitin and laminarin, respectively, per minute under the assay conditions. One unit (U) of protease activity represents the amount of the enzyme resulting in the release of 1 μmol of tyrosine equivalent from azocasein per minute under assay conditions. *Mean standard error (n = 3 per strain); values with different letters are significantly different (P < 0.05)
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Fig. 1.
(a) Seedling length: Lodgepole pine
50 de de e
40 bcd bcd bcd cde bcd bc bc bc bc b bc
30 a
20 Seedling length (cm) Seedling length
10
0 AS1r AN1r AN2r AN3r AN4r AN5r AR1r SS1r SS2r SS3r SN1r SN2r SR1r SR2r Control Draft
(b) Seedling biomass: Lodgepole pine
180 c c c 150
120 b b b b b b b b b 90 b b
Seedling biomass (mg) Seedling biomass 60 a
30
0 AS1r AN1r AN2r AN3r AN4r AN5r AR1r SS1r SS2r SS3r SN1r SN2r SR1r SR2r Control
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Fig. 2.
(a) Seedling biomass (mg) Seedling length (cm)
180
160
140 r = 0.95 120
100
80
60 r = 0.85 40
20
0 0.0E+00 3.0E+06 6.0E+06 9.0E+06 1.2E+07 EndophyticDraft colonization (cfu g-1) (b)
180
160
140 r = 0.94 120
100
80
60 r = 0.86
40
20
0 0.0E+00 5.0E+05 1.0E+06 1.5E+06 Rhizospheric colonization (cfu g-1)
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Fig. 3.
(a) ACC deaminase activity (b) IAA production
AS1r bc AS1r b AN1r e AN1r bc AN2r a AN2r a AN3r b AN3r a AN4r b AN4r c AN5r a AN5r a AR1r d AR1r b SS1r b SS1r a SS2r b SS2r a SS3r a SS3r a SN1r de SN1r b SN2r cd SN2r a SR1r a SR1r a SR2r a SR2r a
0 20 40 60 80 100 120 140 0 10 20 30 40 50 nmol -ketobutyrate h-1 mg-1 protein Draft IAA produced (μg ml-1)
(c) Siderophore production
AS1r de AN1r e AN2r a AN3r a AN4r ab AN5r bcd AR1r de SS1r bc SS2r ab SS3r ab SN1r cde SN2r ab SR1r a SR2r a
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Area of orange halo (cm2)
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Fig. 4.
(a) ACC Gnotobiotic Assay: Canola
14 f 12 f ef 10 e e de cde 8 cde abcd bcd abcd 6 abc ab ab a 4 Primary root length (cm) Primary root length 2
0 SS1r SS2r SS3r SR1r SR2r AS1r SN1r SN2r AR1r AN1r AN2r AN3r AN4r AN5r Control Draft (b) ACC Gnotobiotic Assay: Tomato
14 f ef 12 de cd 10 cd cd bc bc 8 bc bc bc b 6
4 a a a Primary root length (cm) Primary root length
2
0 SS1r SS2r SS3r SR1r SR2r AS1r SN1r SN2r AR1r AN1r AN2r AN3r AN4r AN5r Control
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Fig. 5.
14
12 r = 0.96 10 r = 0.91 8 Canola Tomato 6 Correlation (Canola)
Primary root length (cm) Primary Correlation (Tomato) 4
2
0 0.00 25.00 50.00 75.00 100.00 125.00 ACC deaminase activity (nmol -ketobutyrate h-1 mg-1 protein) Draft
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Fig. 6.
Draft
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