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

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

based on a protocol described by Haney [36]. Briefly, total N was determined on 2 g dried sub- samples using the combustion method. WEOC and water extractable N were measured on 4 g of soil in slurry after shaking for 10 minutes. Samples were centrifuged for 5 minutes at 3500 rpm, filtered through Whatman 2 V paper, and analyzed for WEOC (TOC analyzer) and water extractable N (Colorimetry). Inorganic NH4-N, and NO3-N concentrations were also determined using colorimetry technique [21]. WEON was calculated by subtracting inorganic N content (NH4-N and NO3-N) from water extractable N [37]. The flush of CO2 in 24 hours following rewetting of dried soil was measured on 40 g sub- samples in 50 ml disposable beakers with four to five 6.35-mm holes drilled in the bottom fit- ted with a glass microfiber filter to prevent soil loss. The beaker and Solvita1 gel paddle were placed in a gas-tight 250-mL glass jar filled with 25 mL of water and a convex bottom to allow for drainage. Capillary action was used to rewet soil samples, which were then incubated at 25˚C, and respired CO2 was trapped over 24 h. The quantity of 1-day CO2-C released was PLOS ONE Whole orchard recycling PLOS ONE | https://doi.org/10.1371/journal.pone.0229588 March 27, 2020 5 / 22