www.nature.com/ismecomms ARTICLE OPEN Phenolic acid-degrading Paraburkholderia prime decomposition in forest soil ✉ Roland C. Wilhelm 1,3 , Christopher M. DeRito2,3, James P. Shapleigh 2, Eugene L. Madsen2,4 and Daniel H. Buckley1 © The Author(s) 2021 Plant-derived phenolic acids are catabolized by soil microorganisms whose activity may enhance the decomposition of soil organic carbon (SOC). We characterized whether phenolic acid-degrading bacteria enhance SOC mineralization in forest soils when primed with 13C-labeled p-hydroxybenzoic acid (pHB). We further tested whether pHB-induced priming could explain differences in SOC content among mono-specific tree plantations in a 70-year-old common garden experiment. pHB addition primed significant losses of SOC (3–13 µmols C g−1 dry wt soil over 7 days) compared to glucose, which reduced mineralization (-3 to -8 µmols C g−1 dry wt soil over 7 days). The principal degraders of pHB were Paraburkholderia and Caballeronia in all plantations regardless of tree species or soil type, with one predominant phylotype (RP11ASV) enriched 23-fold following peak pHB respiration. We isolated and confirmed the phenolic degrading activity of a strain matching this phylotype (RP11T), which encoded numerous oxidative enzymes, including secretion signal-bearing laccase, Dyp-type peroxidase and aryl-alcohol oxidase. Increased relative abundance of RP11ASV corresponded with higher pHB respiration and expression of pHB monooxygenase (pobA), which was inversely proportional to SOC content among plantations. pobA expression proved a responsive measure of priming activity. We found that stimulating phenolic- acid degrading bacteria can prime decomposition and that this activity, corresponding with differences in tree species, is a potential mechanism in SOC cycling in forests. Overall, this study highlights the ecology and function of Paraburkholderia whose associations with plant roots and capacity to degrade phenolics suggest a role for specialized bacteria in the priming effect. ISME Communications (2021) 1:4 ; https://doi.org/10.1038/s43705-021-00009-z INTRODUCTION community, environmental conditions, and soil properties,21–24 Forest soils are rich in plant-derived phenolic acids which with broad differences between coniferous and deciduous represent a sizeable proportion of fast-cycling, low-molecular forests.2,25–27 p-hydroxybenzoic acid (pHB) is one of the most weight soil organic carbon (SOC),1 estimated at between 10 and abundant phenolic acids found in plant litter, tree root exudates, 4000 mg · kg−1 dry organic layer soil.2–5 Concentrations of soil and soil, ranging in concentrations from 0.1 to 50 mg · kg−1 dry wt phenolics fluctuate as a function of plant inputs and phenolic acid- in bulk soil4,5,7,28–30 which can be three to four-fold higher in the degrading activity.4,6–10 The cycling of soil phenolics has the rhizosphere.31 Plant pHB content was found to correlate with the potential to accelerate decomposition according to research strength of priming13 and benzoic acid, an abundant root exudate linking phenolic acids, and phenol oxidase activity, to the soil with similar properties as pHB, can induce priming.15 To compare priming effect.11–16 The priming effect refers to the enhanced differences in pHB sources, and corresponding activity of pHB- decomposition of SOC which can occur when an influx of new C degrading populations, we conducted an experiment using a 70- stimulates microbial activity, which is a fundamental process in year-old common garden with plantations of a coniferous tree terrestrial C-cycling.17,18 The activity of phenolic acid-degrading species (red pine) and two deciduous tree species: one leguminous populations appear to be especially impactful, priming a greater (black locust) and one non-leguminous (sugar maple).32 loss of SOC (per unit biomass) than glucose or cellobiose.14 In this Most phenolic acid-degrading microorganisms isolated from light, the low phenolic content of older accumulations of SOC19,20 soil are fast-growing members of the Beta- and Gammaproteo- may reflect a subsidence in the priming activity fueled by bacteria (Table S1).33–36 Rapid growth may be essential for phenolics. Accordingly, we would expect to find a link between metabolizing phenolics in soil, which quickly adsorb to surfaces phenolic acid-degrading populations, soil priming, and SOC of minerals and organic matter, reducing their bioavailability and accrual in nature. However, this link has not yet been tested and promoting SOC accumulation.31,37–39 Though a culture- the identity and ecology of these populations, and the mechan- independent survey of phenolic acid-degrading microbes has isms behind their priming activity, remain poorly described. not yet been made in forest soils, metagenomic analyses of lignin- The primary sources of phenolic acids to soil are litter leachate, degrading populations implicate Alphaproteobacteria (Rhizobiales, root exudates, and the decomposition products of lignin-rich plant Sphingomonadales and Caulobacterales), Betaproteobacteria residues. The composition of soil phenolics depends on the plant (Burkholderiaceae) and Actinobacteria (Nocardiaceae, Frankiaceae, 1School of Integrative Plant Science, Bradfield Hall, Cornell University, Ithaca, NY, USA. 2Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY, USA. 3These authors ✉ contributed equally: Christopher M. DeRito, Roland C. Wilhelm. 4Deceased August 9th, 2017: Eugene L. Madsen. email: [email protected] Received: 11 November 2020 Revised: 21 February 2021 Accepted: 1 March 2021 R.C. Wilhelm et al. 2 Streptomycetaceae and Microbacteriaceae).40–42 Populations of series (“LnC”; Typic Dystrudepts). Our study examined a total of Alpha- and Betaproteobacteria are abundant during white-rot five combinations of tree species + soil type, which we have decay of wood, where phenolic acid concentrations are high;43,44 termed ‘ecoplots’, that encompassed a gradient in total soil and in the rhizosphere, where roots exude phenolic acids to organic matter: BLMaB (21.9% w/w) > SMMaB (14.5%) > BLLnC 45,46 facilitate plant-microbe interactions. Accordingly, we expect (14.0%) > RPLnC (12.75%) > SMLnC (10.2%). There were no the phenolic-acid degrading populations that mediate priming plantations of RP in MaB soil. Earthworms populations, which will belong to these three bacterial classes, representing a can accelerate decomposition,56,57 did not correspond with trends narrower diversity of taxa than observed during glucose-induced in SOC in our plantations.58 Details on the site and three separate priming.47 field sampling campaigns are provided in the Supplementary The priming effect can be generated by a variety of Methods. The concentration of bioavailable elements in soils was mechanisms that interact in a context-dependent manner.48,49 In determined using the modified Morgan extraction procedure59 by certain cases, priming coincides with an increased oxidative the Cornell Nutrient Analysis Laboratory. Estimates of soil organic potential of soils.11,12,50 Measures of potential oxidative activity matter were made according to the loss-on-ignition method. may therefore serve as useful proxies for priming responses. pHB oxidation is catalyzed by p-hydroxybenzoate 3-monooxygenase pHB-induced soil priming (pobA; EC 1.14.13.2) as part of the peripheral pathways for the Microcosms were prepared with soils from BLLnC,RPLnC and SMLnC degradation of aromatics.51 PobA catalyzes an essential step in the to test whether pHB degrading activity induced priming and to degradation of phenolics, which can be the rate limiting step in identity and link active populations with those observed in the the degradation of certain polyphenolics in soil.52 Furthermore, field experiment. Microcosms were prepared and amended with the expression of pobA is responsive to environmental conditions, the following treatments: (i) water-only control, (ii) 13C-glucose, (iii) evidenced by its upregulation in Burkholderia multivorans grown in 13C-pHB, (iv) 13C-pHB + PobA inhibitor (4-hydroxy-3-iodobenzo- soil versus laboratory media.53 Consequently, the expression of ate) and (v) a PobA inhibitor-only control (overview in Fig. S1). The pobA may provide a proxy for the activity of phenolic acid- inhibition of PobA was performed to measure the direct degrading soil populations and any potential priming effects. contribution of PobA activity to priming. An additional set of In previous research, we have shown that an abundant aromatic microcosms was used to test whether priming could be induced acid in plant root exudates (benzoic acid) can prime decomposi- by artificially increasing the abundance of a phenolic acid- tion mediated by the activity of phenolic-degrading bacteria.15,54 degrading isolate (RP11T), corresponding to main pHB-degrading ASV 1234567890();,: Our present study sought to test the link between phenolic acid- phylotype in field data (RP11 ; details in Supplementary degrading populations, soil priming, and SOC accrual in a 70-year- Methods). Microcosms were prepared using fresh soil collected old common garden experiment32 and the mechanism by which from the upper 5 cm of a 20 × 20 cm sampling plot, which was their metabolism controls soil priming. We tested whether pHB sieved to remove root and litter (2-mm), homogenized, and pre- primes microbial activity that enhances SOC decomposition, and incubated for 10 days. Sieving, homogenization, and pre- whether this phenomenon corresponded to differences in SOC incubation were necessary