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 <i>Pinus</i>, <i>Pseudomonas</i>, Gravel mining, Lodgepole pine, Keyword: ACC deaminase Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/cjfr-pubs Page 1 of 35 Canadian Journal of Forest Research 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 1 https://mc06.manuscriptcentral.com/cjfr-pubs Canadian Journal of Forest Research Page 2 of 35 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 2 https://mc06.manuscriptcentral.com/cjfr-pubs Page 3 of 35 Canadian Journal of Forest Research 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 3 https://mc06.manuscriptcentral.com/cjfr-pubs Canadian Journal of Forest Research Page 4 of 35 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, 4 https://mc06.manuscriptcentral.com/cjfr-pubs Page 5 of 35 Canadian Journal of Forest Research 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)