Orchard recycling improves climate change adaptation and mitigation potential of almond production systems

RESEARCH ARTICLE Orchard recycling improves climate change adaptation and mitigation potential of almond production systems Emad JahanzadID1, Brent A. Holtz2, Cameron A. ZuberID2, David Doll2, Kelsey M. Brewer1, Sean Hogan2, Ame´ lie C. M. Gaudin1* 1 Department of Plant Sciences, University of California, Davis, California, United States of America, 2 University of California Agriculture and Natural Recourses, Davis, California, United States of America * agaudin@ucdavis.edu Abstract There is an urgent need to develop climate smart agroecosystems capable of mitigating cli- mate change and adapting to its effects. In California, high commodity prices and increased frequency of drought have encouraged orchard turnover, providing an opportunity to recycle tree biomass in situ prior to replanting an orchard. Whole orchard recycling (WOR) has potential as a carbon (C) negative cultural practice to build soil C storage, soil health, and orchard productivity. We tested the potential of this practice for long term C sequestration and hypothesized that associated co-benefits to soil health will enhance sustainability and resiliency of almond orchards to water-deficit conditions. We measured soil health metrics and productivity of an almond orchard following grinding and incorporation of woody bio- mass vs. burning of old orchard biomass 9 years after implementation. We also conducted a deficit irrigation trial with control and deficit irrigation (-20%) treatments to quantify shifts in tree water status and resilience. Biomass recycling led to higher yields and substantial

improvement in soil functioning, including nutrient content, aggregation, porosity, and water retention. This practice also sequestered significantly higher levels of C in the topsoil (+5 t ha-1) compared to burning. We measured a 20% increase in irrigation water use efficiency and improved soil and tree water status under stress, suggesting that in situ biomass recy- cling can be considered as a climate smart practice in California irrigated almond systems. Introduction The major global challenge facing agriculture is to increase sustainable food production in a changing climate while simultaneously reducing greenhouse gas emissions. Enhancing soil carbon (C) content of agricultural soils provides enormous potential to help reach these goals as C sequestration mitigates climate change [1,2] but also builds sustainability and resilience by improving plant growth conditions and conservation of natural resources [3,4]. Soil organic C (SOC) is essential for soils to function as living ecosystems that provide and cycle nutrients, protect crops from pests and pathogens, conserve water, and reduce risks of soil erosion, thus PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 1 / 22 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Jahanzad E, Holtz BA, Zuber CA, Doll D, Brewer KM, Hogan S, et al. (2020) Orchard recycling improves climate change adaptation and mitigation potential of almond production systems. PLoS ONE 15(3): e0229588. https://doi.org/ 10.1371/journal.pone.0229588

Editor: Ying Ma, Universidade de Coimbra, PORTUGAL Received: August 16, 2019 Accepted: February 11, 2020 Published: March 27, 2020 Copyright: © 2020 Jahanzad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and Supporting Information files. Funding: ACMG SCB16043 California Department of Food and Agriculture https://www.cdfa.ca.gov/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

minimizing the environmental footprint of agriculture while increasing adaptation to harsh climatic conditions [5]. Ambitious climate mitigation and adaptation policies are increasingly recognizing this potential and incentivizing the development and adoption of innovative man- agement practices that promote cycling and storage of SOC. However, uncertainties remain about the potential of management practices to help meet regional climate smart agriculture and soil health targets. Given the anticipated larger incentives to offset CO2 emissions by investing in increasing agricultural soil C storage, it is essential to estimate the potential for C gains and soil health co-benefits associated with specific management practices as well as the downstream impacts on adaptation to future climate conditions. Biomass recycling is gaining widespread interest as a means to use agriculture and its by- products as a resource for renewable energy production. Grinding and incorporating whole trees into soil during orchard turnover may also provide a unique opportunity to store C in soils and build synergies between mitigation and adaptation goals at large scales [6,7]. This may be of particular significance for drought-prone semi-arid agroecosystems with lower lev- els of SOC and water retention, and with high vulnerability to impending shifts in resource availability [8]. Perennial cropping systems, such as California almonds [Prunus dulcis (Mill.) D. A. Webb], usually have low SOC and are uniquely susceptible to the impacts of a changing

climate, including increased frequency of extreme weather events, constrained water resources, and reduced winter chill hours [9]. These production systems are prominent across the landscape and rapidly increasing with more than 530,000 hectares supplying 80% of global almond demand [10]. High commodity prices, costly and increasingly scarce water inputs, and more stringent air quality regulations have promoted turnover of less productive orchards to new almond plantings and renewed interest in using in situ biomass recycling as a climate adaptation and mitigation tool [11,12]. However, the potential for long-term C sequestration and practical tradeoffs associated with high C input practices such as whole orchard recycling (WOR) remain unclear in semi-arid orchard production systems where production relies heavily on irrigation. We tested the long-term viability of biomass recycling within test plots of a highly produc- tive almond orchard and evaluated its potential benefits for climate change adaptation. We hypothesized that addition of substantial amounts of high organic C woody biomass would lead to long term soil C sequestration and improvements in soil health characteristics with positive impacts on yields and orchard productivity. We also explored the implications for orchard adaptation to future climate conditions by testing how and to what extent potential improvements in soil quality, especially hydraulic properties, would impact tree and yield resistance to an acute water shortage (i.e. deficit irrigation). Although it is often claimed that

healthy soils have the potential to build resilience, this claim has seldom been empirically tested, especially in intensive perennial systems. This work provides novel insights on mitiga- tion potential of this practice and the soil health mediated mechanisms influencing orchard health and productivity. Materials and methods Trial establishment and experimental design This experiment was conducted in an irrigated almond orchard located at the University of California Kearney Agricultural Research and Extension Center in Parlier, CA (36˚35’59.4"N 119˚30’11.7"W) on a Hanford fine sandy loam site. The climate is Mediterranean, with precipi- tation levels below evapotranspiration (ET) requirements during most of the growing season. Long term (68 years) annual rainfall and temperature averages are 285 mm and 17˚C, respec- tively. The soil treatments were established in a complete randomized block design with seven PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 2 / 22

replications in 2008 following termination of a 20-year old peach (Prunus persica Var. Fay Elberta) orchard. The estimated amount of woody biomass returned to the soil, excluding roots, was ~74 t ha-1 comprised of ~50% C. Two soil treatments were established in 2008: i) the grind treatment (WOR), which consisted of whole trees ground and shredded using a land clearing equipment (Ironwolf 700B Slasher, Nobe, OK, USA) and incorporated within the top 15 cm of soil in the tree row. The IronWolf pushed standing trees over in place and ground them on its first forward pass using a 3-meter wide × 1-meter high head. On the second pass, going in reverse, the IronWolf lowered the rotating head into the soil so that the ground trees were incorporated into soil to a depth of 15 cm. The orchard was disked twice (0–15 cm) before berms were re-created down the previous tree-rows with a Disc-Ridger. ii) the burn treatment was established by uprooting and burning the trees and incorporating ashes into the topsoil (0–15 cm) in the tree row. The orchard was replanted in January of 2009 with three almond varieties (Nonpareil, Butte, and Carmel) on Nemaguard rootstock. All measurements were performed on the Nonpareil variety rows because i) they have been historically more intensively measured and monitored over the last nine years and ii) Nonpareil is the most commercially relevant variety in California. Spacing between trees was 5.5 m × 6.7 m and one variety was planted per row. Each plot was composed of 18 trees arranged as 1 row of 6 trees

per variety. One-tree buffer per row was established between the grind and burn treatments. Trees were irrigated daily to match potential evapotranspiration needs using micro sprinklers. Trees were fertilized with urea ammonium nitrate (UAN, 32-0-0) fertilizer at a rate of 100 kg N ha-1 via three fertigation events April through May. Deficit irrigation trial In summer of 2017, a deficit irrigation experiment was implemented on 6 replicated plots on the Nonpareil variety. A split-plot arrangement with three replications was used with two irri- gation treatments as main plot effects and grind and burn treatments as sub-plots across three replicates (n = 12 plots). Irrigation treatments consisted of regular (100% ET) and deficit (80% ET for 28 days) irrigation based on reference crop water use, and the almond crop coefficient (Kc) [13]. The deficit irrigation treatment lasted for 28 days and was cut off at the hull split to avoid hull rot in Nonpareil variety [14,15]. Weekly evapotranspiration values for the reference crop were obtained from the state of California Irrigation Management Information System (CIMIS) station located at the Kearney site and Kc values for mature almond trees were obtained from Sanden [16]. Trees were irrigated using Bowsmith Fan-Jet Micro sprinklers (Bowsmith Inc, Exeter, CA, USA) with a flow rate of 38 to 65 liters per hour placed in the tree row mid-way between trees. The deficit irrigation treatment (80% ET) was imposed using emitters with 20% less output (30 to 52 liter per hour). Pressure regulators (DG-5025, Hen-

drickson Bros, Corona, CA, USA) were installed before the water meters to maintain constant pressure within each row. The deficit irrigation treatment started on June 5, 2017 and was cut off on July 3, 2017 (hull split). The irrigation treatment was applied only to the middle Nonpa- reil variety row. The amount of irrigation water applied to each treatment row was measured with in-line water meters during the experiment. Irrigation water use efficiency (IWUE) (kg m-3) was calculated as kernel yield (kg ha-1) divided by the volume of irrigation water (m3 ha- 1) [15]. Soil sampling and processing Soil samples were collected on the Nonpareil rows from the deficit irrigation trial in spring of 2017, 9 years after incorporation of woody biomass, using AMS regular soil augurs (AMS Inc, American Falls, ID, USA). All samples were collected from the berms between the trees to a PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 3 / 22

depth of 0–15 cm, which corresponds to the woody biomass incorporation depth. Only the middle row trees (Nonpareil) were used for sampling and field measurements and trees at the beginning and at the end of each tree row were not sampled to avoid edge effects. Three root zone samples per plot were taken and composited (n = 12) on the same day to a depth of 0–15 cm for biological analysis. Field-moist soil was sieved to 2 mm and stored at 4˚C prior to assays of microbial biomass and enzyme activities. For soil chemical properties, five soil cores per plot were sampled composited (n = 12) and dried in a forced-air oven at 50˚C prior to analysis. Three separate undisturbed soil cores were also taken from each plot (n = 36, not composited) and stored at 4˚C until processing for aggregate stability and fractionation tests. In addition, five soil cores were collected from each plot (n = 60) for bulk density (BD) measurements. Soil analysis Chemical properties. Soils were analyzed in house at the University of California Divi- sion of Agriculture and Natural Resources Analytical Laboratory. The pH was determined using a saturated paste method [17]. Electrical conductivity (EC) was measured according to the method described by Rhoades [18]. Sodium (Na), Ca, Mg, K, B, S and micronutrients (Zn, Mn, Fe, Cu, and Mo) were measured using Inductively Coupled Plasma Emission Spectros- copy (ICP-AES) [19]. Cation exchange capacity (CEC) was determined based on the method described by Rible and Quick [20]. Soil nitrate, extractable ammonium, and Cl were measured

using Flow Injection Analysis Colorimetry (QuikChem, Lachat Instruments, Milwaukee, WI, USA) [21]. Total N and C were determined using the combustion method (ECS 4010, Costech Analytical Technologies Inc, Valencia, CA; USA). Soil C stock was calculated as the product of soil C concentration, soil sample bulk density, and sampling depth [22–24]. Soil organic mat- ter (SOM) was measured using the Loss-On-Ignition Method [25]. Permanganate Oxidizable C (POxC) was measured using the procedure described by Weil et al. [26]. Physical properties. Soil texture was determined using the hydrometer technique [27]. Soil compaction, defined as resistance, was measured using a soil cone penetrometer (Dickey- John, Auburn, IL, USA). Bulk density (BD) was measured according to Blake and Hartge [28]. Briefly, volume of soil (cm3) was calculated by subtracting volume of rocks from total soil vol- ume. Volume of rocks was determined by submerging the rocks in a graduated cylinder with water and recording the difference before and after submerging (1 mL = 1 cm-3). The below equation was used to calculate BD: Soil BD (g cm-3) = oven dry weight of soil / volume of soil Soil water-stable aggregation and C and N content of different aggregate fractions were measured on undisturbed cores. Large particles were removed through sieving moist soil (8mm) and water-stable aggregates were separated by wet-sieving of a 40g subsample into four aggregate size fractions: >2000 μm (large macroaggregates), 250–2000 μm (small macroaggre-

gates), 53–250 μm (microaggregates), and <53 μm (silt and clay fraction) according to a proto- col modified from Elliot [29]. A vibratory sieve shaker with rainfall simulator (Fritsch Analysette 3 Pro, Pittsboro, NC, USA) was used. Aggregate fractions remaining on each sieve were oven-dried at 50˚C and weighed. Carbon and N content of different aggregate fractions were measured using the combustion method. Mean weight diameter (MWD), a weighted- average index of aggregate stability, was calculated according to the following equation [30]: MWD ¼ P4 i¼1Pi � Si where Si is the average diameter (μm) for particles in that fraction and Pi is the weight percent- age of the fraction in the whole soil. PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 4 / 22

Hydraulic properties and water dynamics. Infiltration rate was obtained by measuring soil saturated hydraulic conductivity (Kfs) with a DualHead Infiltrometer (Decagon Devices, Inc., Pullman, WA, USA). Water-retention characteristics of recycled orchard soils (grind) and control soils (burn) were determined using the evaporation method (HYPROP technique, 250-cm3 intact soil cores) (UMS HYPROP, Decagon Devices, Inc., Pullman, WA, USA) [31]. Soil water content was measured during the deficit irrigation trial at the 22, 45, 76, 106, and 137 cm depths using a neutron probe (CPN model 305 DR, Concord, CA, USA). One access tube was installed in each treatment plot next to a randomly selected Nonpareil trees (n = 12). The location of the access tubes was in-line with the tree row. To calibrate neutron probe read- ings, soil samples were collected from the same soil depths mentioned above upon installation of the access tubes to measure BD and then volumetric water content (%VWC). The actual moisture content of the soil at different depths were then calculated using the calibration curve from the %VWC of the samples and count ratio of slow neutrons (R2-value of 0.87 for all depths) [32]. Soil water content was measured between April 12 and July 24; measurements were made on a weekly basis during June 5 to July 3 and bi-weekly before and after the deficit irrigation experiment. Biological properties. Microbial biomass C and N (MBC and MBN, respectively) were measured on field moist samples using the chloroform fumigation extraction method [33].

Carbon content in the extracts was determined using a TOC analyzer (TOC-V, Shimadzu, Japan). MBC was calculated as the difference between extractable organic C in unfumigated and fumigated samples, divided by a Ke factor of 0.35 [33]. Dissolved N in extracts was mea- sured colorimetrically following the alkaline persulfate oxidation method [34]. MBN was cal- culated by dividing the difference in N content between the fumigated and unfumigated samples divided by 0.68 to account for incomplete N extraction [33]. The activity of various microbial enzymes involved in C and N cycling was measured in soil samples using fluorescence [35]. Targeted enzymes included: β-glucosidase (BG), β-D-cello- biosidase (CB), β-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). Briefly, 2.75 g of field moist soil were homogenized with 91 mL of 50 mM buffer (NaOH) and stirred for one minute. 800 μL of soil slurry were used for each assay along with 200 μL of 4-Methylumbelliferone (MUB) and 7-Amino-4-methylcoumarin (MUC) of assay standards. 200 μL of BG, CB, NAG, and LAP substrates were added to the 96 well plates and then incu- bated in the dark at room temperature for three hours. Plates were then centrifuged for 3 min- utes and fluorescence was read (Synergy™ H4 Hybrid Multi-Mode Microplate Reader, BioTek, Winooski, VT, USA). Soil Health Index (SHI). To calculate the SHI, samples were analyzed for water extract- able organic C (WEOC), water extractable organic N (WEON), and 1-day CO2-C respiration

determined using a digital-color reader (DCR) (www.solvita.com) [37]. These values were then used to calculate a SHI according to Haney [36]: SHI ¼ 1 day CO2 C C : N þ ðWEOC 100 þ WEON 10Þ Plant analysis Yield and leaf analysis. Almond kernel yields (Nonpareil) were measured by mechanical harvest on September 2017. Gross and dry kernel weights (~3% moisture) were measured on subsamples from 6 trees per treatment plot. Twenty almond leaves per tree were sampled in mid-July, dried at 60˚C, ground, sieved through 2-mm mesh and then sent to the University of California Division of Agriculture and Natural Resources (UC ANR) Analytical Laboratory for analysis of accumulated nutrients. Total Kjeldahl Nitrogen (TKN) was determined according to Horneck and Miller [38]. Phosphorus, K, S, Ca, Mg, Zn, Fe, Mn, Cu, B, Cl, Na, Al, and Mo were analyzed using nitric acid/hydrogen peroxide microwave digestion method and ICP-AES [19]. Tree water status. Tree water status was measured weekly using midday stem water potential (SWP) with a pressure chamber (Soilmoisture Equipment, Santa Barbara, CA, USA). Two lower-canopy, fully expanded, shaded leaves were measured after enclosing the leaves in bags made of plastic film and aluminum foil for at least one hour before measuring. Stomatal conductance (mmol m-2s-1) was measured on June 28 at solar noon using a leaf porometer (model SC-1, Decagon Devices, Inc., Pullman, WA, USA) on six randomly chosen leaves per tree in each plot (n = 72). Photosynthetically Active Radiations (PAR) intercepted by the tree

canopy was measured as an indicator of tree growth using a utility vehicle at solar noon (model 610 Mule; Kawasaki Heavy Industries, Tokyo) fitted with a mobile PAR measurement platform [39] on August 25. Aerial thermal images were acquired on June 28 in clear conditions between the hours of 12:00 and 13:00 to minimize effects of shadowing on the images. These images were collected at 70 meters above ground level with a metrically calibrated Zenmuse XT infrared thermal camera, mounted on a DJI Inspire 1 unmanned aerial vehicle. The Zenmuse thermal camera was equipped with a 9Hz FLIR microbolometer sensor, capturing 640 × 512 pixel frames (digi- tal images) from a 640/30 fps from an active feed, with a sensitivity (NEdT) of 50 mK at f/1.0 in the spectral range of 7.5–13.5 μm, and a lens with an field of view of 69˚ × 56˚. The aerial images were later processed in Pix4D Mapper Pro and ArcGIS software applications to pro- duce a 9.2 cm spatial resolution orthomosaic map of the canopy temperature, segmenting each almond tree from surrounding soil pixels within the entire 2.37-acre orchard, to test whether there were significant temperature differences between the irrigation and burn vs. grind treat- ment areas. Statistical analysis Data were analyzed using the PROC MIXED procedure of SAS [40]. Soil and irrigation treat- ments and the interaction of these factors were analyzed as fixed effects, and blocks and the interaction of blocks with main effects as random effects. The assumptions of ANOVA were

tested, and transformations were applied where necessary to achieve normality of residuals. Weekly measurements of SWP and neutron probe readings were analyzed with repeated mea- sure covariance structure [41]. When ANOVA showed significant differences (P � 0.05), com- parisons of means were made using the Tukey’s range test. Principal components analysis (PCA) was conducted using the JMP software [42]. Pearson correlation coefficients were cal- culated using the PROC CORR procedure to determine correlations between almond yield PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 6 / 22

and soil health parameters as well as plant water status measurements [40]. The HYPROP data evaluation software (UMS GmbH) was used to fit the parameters of a model for the soil water retention q(h) and unsaturated hydraulic conductivity K(h) to the evaporation. We tested van Genuchten unimodal [43] and Durner bimodal retention model equations [44,45] and evalu- ated model fit quality with the corrected Akaike Information Criterion (AIC) [46]. Smaller AIC values indicate a better estimation quality of the model. Data were analyzed with the PROC MIXED model to compare soil treatments and means were separated using Tukey’s range test. Results Tree nutrition, yields, and productivity Trees following WOR significantly out-yielded those in the burn treatment nine years after establishment (+19%, 400 kg ha-1; Fig 1A), which led to increased irrigation water use effi- ciency (IWUE) compared to the burn treatment (1.25 kg m-3 vs 1.04 kg m-3; Fig 1C). Higher kernel yields have been consistently observed in the grind treatment since 2014 (S1A Fig). Fur- thermore, the accumulated yields indicated a 15% kernel yield increase in the grind treatment compared to the burn (S1B Fig). Trees grown under WOR accumulated slightly higher leaf N content compared to the burning practice regardless of the irrigation treatments; yet, accumu- lation of other minerals was not significantly affected by the treatments (S1 Table). Soil carbon pools The grind treatment had higher SOM content (+ 0.45%) than the burn treatment nine years

after WOR implementation (P = 0.001; Table 1). A proportional increase was observed in SOC of the grind compared to the burn treatment (Table 1). The total accumulation of soil C in the grind treatment was 5.2 tons per hectare (0–15 cm) higher than in the burn treatment (Table 1). Labile pools of SOC, such as POxC (Table 1), MBC (Table 2), and WEOC (Table 2), also increased relative to the burn treatment (+66, +47, and +26%, respectively). The distribu- tion of C and N within aggregate fractions differed between the soil treatments, with a higher relative proportion and total weight of SOC occluded within large macroaggregates after grinding (35 vs 28% after burning, Fig 2A). Additionally, a larger proportion of TN was bound to minerals in the clay and silt fractions of the grind treatment (+ 34%) than in the burn treat- ment (Fig 2B). Soil chemical properties Soil TN (+23%), Mn (+33%), Cl (+46%), and Na (+24%) contents were significantly greater following WOR implementation relative to the burn experiment (Table 1). Observed increases in TN content of the grind treatment did not result in increased NH4+ nor NO3- contents. No significant differences were observed in other chemical properties, including pH, CEC, and EC (Table 1). Aggregation, compaction, and hydraulic properties As the proportion of stable large macroaggregates increased (Fig 3A), WOR significantly enhanced aggregate stability (MWD) compared to the burn treatment (Fig 3B). Bulk density and compaction of topsoil layers (Table 1) were also significantly reduced under WOR (5 vs

16%). Water retention curves showed that the addition of woody biomass into the orchard soil had a significant effect on water retention in the topsoil. Soils were able to retain higher mois- ture (%VWC) at matric potential from 0 to ~10 kPA. The recycled treatment was able to store PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 7 / 22

PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 8 / 22

30% more VWC at field capacity (FC) (Fig 3C). Water infiltration, measured as hydraulic con- ductivity (Fig 3D), was also significantly improved compared to the burn treatment (0.003 vs 0.001 cm s-1). Soil biology and SHI WOR significantly increased soil MBC by 28% compared to the burn (Table 2). While MBN trended higher (+13%), this increase was not significant (P = 0.41). Soil enzyme potential activ- ity, which indicates potential rates of C and N cycling, was generally higher in the grind soil compared to the burn (Fig 4A–4D). The grind treatment soils had relatively higher levels of soil C cycling enzyme activity, with significant increases for both BG and CB (Fig 4A and 4B). Soil N cycling enzyme activity for NAG was also significantly increased under WOR, but not for LAP despite trending higher (Fig 4C and 4D). Additions of woody biomass into the orchard soil significantly increased estimated soil respiration rates (1-day CO2 evolution; +76%; P � 0.05, Table 2). Because of these changes and shifts in WEOC, the soil health index (SHI) significantly increased from 4.24 in the burn treatment to 6.13 in the grind treatment (Table 2). Principal component analysis and correlations Principal component (PCA) and correlation analysis were used to further identify relation- ships between soil properties across treatments (Fig 5, Table 3). We observed clear clustering of the two management practices and two principal components accounted for 64.3% of the total variance (PC1 and PC2 explaining 45.6% and 18.7% of total variance, respectively) (Fig

5). Soil parameters with high impact on treatment separation (>0.5) were soil C (SOC, SOM), total N, microbial biomass (MBC, MBN), soil enzymes (BG, CB, NAG) and physical properties such as aggregation (MWD), infiltration, and compaction. Total soil C (SOC) was strongly positively correlated with increases in total nitrogen, microbially stored C pools (MBC) and two enzymes (BG, NAG) (Table 3). Improvements in aggregation, infiltrations, soil water con- tent and mitigation of compaction issues also trended with increases in SOC. Fig 1. Effects of soil treatments (a) and soil treatments × irrigation regimes (b, P = 0.25) on kernel yields and irrigation water use efficiency (IWUE). (�) indicate significant difference at P �0.05. https://doi.org/10.1371/journal.pone.0229588.g001 Table 1. Impact of WOR on soil physical and chemical properties (0–15 cm) in 2017. Treatment BD (g cm-3) Cpt (MPa) EC (ds m-1) CEC (meq†) NO3- (mg kg-1) NH4+ (mg kg-1) TN (%) SOC (%) C Stock (T ha-1) SOM (%) POxC (mg kg-1) Grind 1.58 142 0.57 8.28 11.93 1.39 0.069 0.79 18.72 1.52 250 Burn 1.64 165 0.58 7.78 12.66 1.31 0.056 0.55 13.53 1.07 153 P Value 0.05 0.05 0.45 0.28 0.73 0.35 0.05 0.001 0.01 0.001 0.04 Treatment pH Ca Mg K Na Cl B Fe Cu Mn Zn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mg kg-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Grind 6.94 60.52 17.74 11.06 20.38 22.6 0.3 33.23 9.25 9.03 9.69 Burn 7.02 61.12 17.37 11.68 16.49 15.51 0.31 28.01 9.26 6.79 9.64 P Value 0.39 0.48 0.47 0.39 0.03 0.003 0.4 0.11 0.5 0.01 0.47 BD, bulk density; Cpt, compaction; EC, electrical conductivity; CEC, cation exchange capacity († Per 100 g of soil) TN, total nitrogen; SOC, soil organic carbon; SOM, soil organic matter; POxC, permanganate oxidizable carbon https://doi.org/10.1371/journal.pone.0229588.t001 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 9 / 22

Resistance to deficit irrigation We tested the significance of changes in soil health parameters for tree and yield resistance to water shortages. Deficit irrigation resulted in a decrease in soil moisture in both treatments; however, the difference was more pronounced in the burn treatment (Fig 6). We observed a significant interaction between irrigation and soil treatments (P = 0.05), where the burn plots had a lower soil water content than the grind treatments under deficit irrigation, especially in the topsoil and at depth (Fig 6). We observed no significant interaction between soil treatment and irrigation on yields (P = 0.25, Fig 1B). However, stem water potential (SWP) measure- ments showed that trees in the grind plots maintained less negative value than those in the burn treatment (-14.11 vs -16.22 bar, respectively) over the course of the season (Fig 7A). Trees were significantly less water stressed on the last day of the deficit irrigation period in the grind treatment, as shown by significantly less negative SWP and higher stomatal conductance (Fig 7B and 7C). The grind treatment also assisted trees in their recovery post stress (Fig 7B). No changes in plant nutrition, canopy temperature and PAR interception were detected between treatments (S2 Table). Yields and soil health parameters We examined correlations between soil health indicators and tree water status and yields (Table 3). Indicators of soil health improvement were positively correlated with almond yields. Significant positive correlations were observed between yield, soil N and C content (TN, SOC,

and SOM) and C cycling (BG). Among soil physical properties, improvements in infiltration were significantly corrected with higher yields while bulk density and soil compaction trended negatively with yields. Total C content and SOM levels were also positively correlated with some orchard resiliency indicators (Table 3) such as soil water content and tree water status (SWP), where less negative SWP values indicate more favorable plant water status (Table 3). Discussion The purpose of this experiment was to evaluate long term effects of WOR adoption on almond orchard functioning and climate change mitigation and adaptation services. We found that in situ biomass recycling prior to replanting orchards can help sequester a significant amount of C in the soil while improving yields and mitigating the impacts of acute water shortages on tree water stress. Although this practice can be costlier and challenging operationally on the short term, it offers a promising strategy for long term improvement in soil health and reduce risks of crop failure of orchard cropping systems. Biomass recycling provides substantial mitigation potential The adoption of WOR is characterized by the reincorporation of large quantities of high C:N woody biomass (ratio of ~160) into orchard top soils. We found that the recycled orchard soils Table 2. Impact of WOR on microbial parameters and Soil Health Index determinants. Treatment MBN (μg N g soil-1) MBC (μg C g soil-1) Respiration (ppm) WEON (ppm) WEOC (ppm) C:N SHI Grind 7.91 192 57.7 4.98 56.69 11.38 6.13

Burn 6.97 150 32.7 4.58 44.99 9.81 4.24 P value 0.4113 0.0490 0.002 0.065 0.012 0.056 0.019 MBN and MBC, Microbial biomass carbon and nitrogen, respectively; WEON, water extractable organic nitrogen; WEOC, water extractable organic carbon; C:N, carbon to nitrogen ratio of soil; SHI, soil health index. https://doi.org/10.1371/journal.pone.0229588.t002 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 10 / 22

contained greater quantities of stored C compared to soils where residues were burned both when represented as a percentage (per 100g of soil) and total C stock per hectare in the topsoil (t ha-1). Taken over nine years, C storage services provided by this practice exceed the 4o/oo tar- gets set by the international community to significantly mitigate anthropogenic C concentra- tion in the atmosphere [46–48]. Fig 2. Impact of WOR on (a) total carbon and (b) nitrogen content in aggregate fractions. (�) indicate significant differences at P �0.05. https://doi.org/10.1371/journal.pone.0229588.g002 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 11 / 22

WOR increased the formation and stability of large macroaggregates and preserved a larger quantity of intra-aggregate SOC relative to other aggregate fractions. The incorporation of woody biomass may provide sorption points for the formation of aggregate-scale microbial habitats while providing substrate for microbial growth, thereby providing energy and a favor- able environment for particle aggregation and protection of soil C. A significant positive corre- lation between the SOM content, SOC, and aggregate MWD further confirms this trend, which was in accordance with the results reported by Fonte et al. [49], who found that macro- aggregates were a large reservoir of organic C. A large input of plant residues in this system is likely one of the main factors underlying the improved content and stability of macroaggre- gates within the surface soil layer, resulting in more protection of soil C pools and longer turn- over periods of sequestered C. Labile carbon pools (POxC and MBC) also increased after biomass reincorporation. Differ- ences in total SOC content of orchard soils were correlated to MBC, as well as BG and NAG activity potential, indicating that increased microbial biomass and C cycling under WOR had benefits for SOC storage. These results are in accordance with the observations of Bonanomi et al. [50], who found that high enzyme activities were mainly attributable to the relatively higher SOC and to energy sources and nutrients that sustain growth and activity of soil microbes. An increase in mineralization of organic substances from woody biomass decompo-

sition may have in turn contributed to promoting the cementation of soil particles, forming Fig 3. Effect of Grind and Burn treatments on (a) Proportion of soil aggregate fractions, (b) mean weight diameter (MWD), (c) water retention curves, and (d) infiltration rate. (�) indicate significant differences at P �0.05. https://doi.org/10.1371/journal.pone.0229588.g003 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 12 / 22

macroaggregates and physically protected intra-aggregate SOC [51,52]. However, without any sustained C inputs, the C benefits of this practice might decrease over time, especially if smaller fast degrading woodchips are incorporated. Given the potential of this practice for C seques- tration, holistic assessments of C turnover and the C footprint of WOR including emissions from added machinery is essential to more accurately predict the mitigation potential of this practice. Biomass recycling improves soil health metrics In addition to contributing to achieving the long-term objective of limiting the global tempera- ture increase, these changes in soil C were also crucial to improve soil fertility and functioning as a living ecosystem. Large additions of C as woody residues resulted in a higher soil health index and biological activity as represented by significant increases in microbial biomass, potential activity of soil enzymes, and microbial respiration. Decomposition of woody biomass releases substantial amounts of C into soil, where it is subsequently used to fuel microbial metabolism and cell production [53]. CB, an enzyme involved in cellulose hydrolysis, is essen- tial in woody biomass decomposition. BG is a key member of the cellulase class of enzymes and completes the final step during cellulose hydrolysis by converting cellobiose to glucose [54]. Additions of woody biomass, composed of about 40% cellulose by weight, increased CB and BG potential activity under WOR. We found the same significant trend in the potential

Fig 4. Effect of Grind and Burn treatments on (a) Cellulase (CB); (b) β -glucosidase (BG); (c) β-N-acetylglucosaminidase; (NAG), and (d) Leucine Aminopeptidase (LAP) enzyme activities. (�) indicate significant differences at P �0.05. https://doi.org/10.1371/journal.pone.0229588.g004 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 13 / 22

activity of NAG, involved in chitin degradation, that is likely due to an increase in fungal abun- dance [55]. These results suggest that incorporation of woody biomass has a notable positive effect on facilitating biologically active soils, capable of more effectively recycling and storing C and essential nutrients for plants and microbial processes. Soil TN was also higher under WOR and was correlated to increases in microbial C cycling. The relatively high quantity of TN under WOR did not however translate to significant increases in inorganic N but did significantly correlate to increases in MBN. This indicates that competition for soil N may be higher under WOR and that, while more TN is present, a Fig 5. Principal Components Analysis (PCA) of soil health indicators and treatment clusters. BD, bulk density; Cmpt, compaction; Inflt, infiltration; MWD, mean weight diameter; EC, electrical conductivity; TN, total nitrogen; SOM, soil organic matter; SOC, soil organic carbon; POxC, permanganate oxidizable carbon; CB, cellulose; BG; β –glucosidase; NAG; β-N-acetylglucosaminidase. https://doi.org/10.1371/journal.pone.0229588.g005 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 14 / 22

high quantity of it is microbially immobilized. The recycling of woody biomass could lead to increased N needs to sustain tree growth in the first year; however, it appears to increase the retention of N within the orchard system without affecting yields later on. Whether immobi- lized within microbial biomass, assimilated back into orchard biomass (as represented by sig- nificantly higher leaf N content and yield under WOR), or retained through binding to SOC and/or soil mineral surfaces, WOR likely decreases soil N leaching potential. Biomass recycling also improved soil physical properties of importance to growers and effi- cient use of irrigation water. Soil compaction was alleviated, and it was negatively associated with improvements in SOC and BG enzymatic activity, indicating that microbial decomposi- tion of woody inputs and subsequent enhancements in soil C may partially drive reductions in soil compaction. Increased volume of pore space resulting directly from chunky woody bio- mass additions to the topsoil might have also led to better porosity and reduction in compac- tion. Low soil surface compaction is an important component of fertile soils since it facilitates root growth, infiltration, and water and nutrient retention [56,57]. We observed a negative correlation between compaction metrics and soil water infiltration rates. Improvements in infiltration rates were likely attributed to an increase in soil porosity as shown previously [58] and increased preferential flow pathways and rates of hydraulic conduc-

tivity created after decomposition of large woody residues. Infiltration rate also positively cor- related with aggregation, with macroaggregate stability understood as a key attribute impacting soil water movement [59]. In addition to infiltration rates, neutron probe readings and moisture retention curves indicated significant improvements in hydraulic properties. The 30% improvement in water retention at field capacity in the grind treatment could be in part attributed to increased formation and stabilization of soil aggregates associated with C Table 3. Correlation coefficients among almond kernel yield, soil physicochemical, and biological properties as well as tree-water relations. Yield MWD Ift Cpt BD TN SOC SOM POxC MBC MBN BG CB NAG SWP SCt SWC Yield 1.00 MWD 0.62 1.00 Ift 0.74� 0.60� 1.00 Cpt -0.17 -0.30 -0.61� 1.00 BD -0.90� -0.59� -0.83� 0.75� 1.00 TN 0.75� 0.41 0.36 -0.40 -0.81� 1.00 SOC 0.74� 0.43 0.51� -0.43 -0.82� 0.99�� 1.00 SOM 0.83� 0.50� 0.64� -0.40� -0.87� 0.98�� 0.98�� 1.00 POxC 0.46 0.54� 0.48 -0.15 -0.46 0.22 0.23 0.27 1.00 MBC 0.38 0.66 0.76� -0.07 -0.59 0.48 0.52� 0.52� 0.55� 1.00 MBN 0.32 0.42 0.36 -0.15 -0.10 0.69� 0.15 0.19 0.49 0.57� 1.00 BG 0.80� 0.64 0.44 -0.66� -0.62� 0.68� 0.68� 0.74� 0.48 0.14 0.13 1.00 CB 0.08 0.14 0.20 -0.32 -0.22 0.54� 0.53 0.44 0.18 0.18 0.62 0.31 1.00 NAG 0.50 0.57 0.41 -0.75� -0.51 0.72� 0.74� 0.68� 0.51 0.35 0.17 0.77� 0.50 1.00

SWP -0.56 -0.08 0.30 0.17 0.71 -0.16 -0.46 -0.75� -0.06 -0.26 -0.58 -0.28 -0.56 -0.19 1.00 SCt 0.43 0.66 0.22 -0.07 -0.25 0.24 0.27 0.21 0.73 0.64 0.45 0.12 0.18 0.52 -0.64� 1.00 SWC 0.53� 0.72� 0.39 -0.27 -0.34 0.11 0.57� 0.55� 0.79 0.32 0.45 0.39 0.47 0.22 -0.30 0.59 1.00 �Significant at P � 0.05 ��Significant at P � 0.01 Abbreviations: MWD, mean weight diameter; Ift, infiltration; Cpt; compaction; BD, bulk density; TN, total nitrogen; SOC, soil organic carbon; SOM, soil organic matter; POxC, Permanganate-oxidizable C, MBC, microbial biomass carbon; MBN, microbial biomass nitrogen; BG, β -glucosidase; CB, Cellulase; NAG, β-N- acetylglucosaminidase; SWP, stem water potential; SCt, stomatal conductance; SWC, soil water content. https://doi.org/10.1371/journal.pone.0229588.t003 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 15 / 22

additions and potential alteration of pore-size distribution of amended soil [60]. These signifi- cant gains in soil water conservation could help better retain precipitation and water inputs, allowing growers to delay the onset or decrease the frequency of irrigation. Biomass recycling improves crop yield, productivity and water stress resistance Trees following biomass recycling out-yielded those grown in the burn treatment under both irrigation scenarios. Yield benefits were significantly associated with differences in SOC between treatments, attributed to improvements in soil nutrition and moisture retention as well as increased soil biological activity [61,62]. Presumably, long term N supply from mineral- ization of SOM also contributed to higher yields in the grind plots. This was supported by greater accumulation of N in the leaves of grind trees compared to that of burn. Biomass recycling decreased tree water stress as shown by a significantly less negative SWP throughout the course of the trial. The difference in SWP between the treatments was more pronounced when trees were at their most water stressed at hull split. Trees also showed greater recovery post stress after WOR. Stomatal conductance of WOR orchard trees was also significantly higher under the deficit irrigation treatment, and trended higher under regular irrigation. It has been shown that changes in SWP play a major role in short-term stomatal regulation of woody plants [63]. This was confirmed in our study, where a negative correlation

was found between stomatal conductance and SWP. Trees under deficit irrigation conditions Fig 6. Average of neutron probe readings at different soil depths during the deficit irrigation period (6/5-7/3) as affected by soil (grind and burn) and irrigation treatments. https://doi.org/10.1371/journal.pone.0229588.g006 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 16 / 22

also decrease their stomatal conductance in response to a decrease in pre-dawn and mid-day leaf water potentials, in order to avoid substantial loss of moisture through transpiration [64,65]. WOR management has the potential to decrease water limitations of yields and Fig 7. Effect of soil (Burn and Grind) and irrigation treatments on (a) average of weekly stem water potential readings (b) weekly measurements of stem water potential, and (c) stomatal conductance measurement (7/3). (�) indicate significant differences at P �0.05. https://doi.org/10.1371/journal.pone.0229588.g007 PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 17 / 22