Comprehensive Analysis of the Apple Rhizobiome As Influenced by Different T Brassica Seed Meals and Rootstocks in the Same Soil/Plant System ⁎ Tracey S

Applied Soil Ecology 157 (2021) 103766

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Applied Soil Ecology

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Comprehensive analysis of the apple rhizobiome as influenced by different T Brassica seed meals and rootstocks in the same soil/plant system ⁎ Tracey S. Someraa, , Shiri Freilichb, Mark Mazzolaa,c a United States Department of Agriculture-Agricultural Research Service Tree Fruit Research Lab, 1104 N. Western Ave., Wenatchee, WA 98801, United States of America b Agricultural Research Organization (ARO) and The Volcani Center, Institute of Plant Sciences, Ramat Yishay, Israel c Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland 7600, South Africa

ARTICLE INFO ABSTRACT

Keywords: Replant disease refers to the poor growth of trees when attempting to establish the same or related species on old Apple replant disease orchard sites. The use of pre-plant Brassicaceae seed meal (SM) soil amendments in combination with apple Brassica replant disease-tolerant rootstock genotypes has been shown to be a promising strategy for the control of apple Rootstock genotype replant disease (ARD). However, optimizing microorganism-driven protection of apple roots from infection by Oomycete multiple soil-borne pathogens requires a more comprehensive understanding of how “effective” vs. “ineffective” Brassicaceae seed meal × rootstock genotype disease control systems modulate the composition of rhizosphere microbial communities. In particular, the community of oomycetes associated with the apple rhizosphere remains relatively unexplored compared with bacteria and fungi. To address these issues, we sequenced the root associated bacterial, fungal, and oomycete communities of apple replant disease tolerant (G.210) and susceptible (M.26) rootstocks when grown in an orchard replant soil amended with different Brassicaceae seed meal formulations (Brassica juncea + Sinapis alba, B. juncea, and Brassica napus) previously shown to provide varying levels of replant disease control. Multiple microbial components were associated with observed growth differences between “effective” and “ineffective” disease control systems including the absolute abundance of Ilyonectria/Cylindrocarpon in fine root tissue. Amplicon sequencing provided a more detailed picture ofthege- netic diversity of oomycete groups in the apple rhizosphere than previously appreciated, and highlighted the variability in oomycete community structure between different rootstock × seed meal disease control systems. In Brassica juncea + Sinapis alba SM-structured rhizospheres, the ARD-tolerant rootstock (G.210) harbored higher relative abundances of Peronosporales with reduced potential to infect apple roots and incite replant disease (such as Peronospora destructor and P. acanthicum), whereas the Peronosporales community associated with the sensitive rootstock (M.26) was dominated by the ARD-specific pathogen Phytophthora cactorum. In addition, Brassica juncea + Sinapis alba SM-structured microbiomes were characterized by numerous bacterial and fungal taxa with the potential for biocontrol, biodegradation and bioremediation. Taken together, these results support the hypothesis that particular Brassicaceae SM soil amendments not only provide “effective” disease control, but also promote microbiomes which are likely to contribute to long-term orchard soil health in many other ways. Overall, this comprehensive analysis highlights the significance of the rootstock × seed meal interaction on bacterial, fungal, and oomycete communities within the apple rhizosphere of “effective” vs. “ineffective” disease control systems and the potential influence of these elements on the dynamics ofapple replant disease.

1. Introduction disease (ARD) is primarily due to plant-induced changes in the soil microbiota leading to a build-up of multiple soil-borne pathogens over Replant disease refers to the poor growth of trees when attempting time. In many apple-growing regions of the world, including South to establish the same or related species on old orchard sites (replant Africa, Italy, Australia, New York and Washington State, the causative sites) and is known to largely affect nut as well as stone and pome fruit agents of the disease complex typically include the root lesion nema- trees, including many members of the Rosaceae family. Apple replant tode Pratylenchus spp. in addition to multiple root-associated fungal

⁎ Corresponding author. E-mail address: [email protected] (T.S. Somera). https://doi.org/10.1016/j.apsoil.2020.103766 Received 29 June 2020; Received in revised form 25 August 2020; Accepted 27 August 2020 Available online 18 September 2020 0929-1393/ © 2020 Published by Elsevier B.V. T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766

(Rhizoctonia solani, Ilyonectria/Cylindrocarpon) and oomycete (Pythium, activity of isothiocyanates. Brassicaceae seed meals are complex carbon Phytophthora) pathogens (Dullahide et al., 1994; Manici et al., 2013; substrates that require specific metabolic pathways for effective utili- Mazzola, 1998; Tewoldemedhin et al., 2011a, 2011b). Economic losses zation. As a result of these two features, the BjSa SM formulation results due to reduced tree growth and survival during the first four years of in transformation of the fungal and bacterial rhizosphere communities orchard replanting can run anywhere from US$40,000–$150,000 per and ultimately possesses functional features providing long-term con- acre over a 10-year period (Hewavitharana et al., 2019). Widespread trol of ARD pathogens (Mazzola et al., 2015; Wang and Mazzola, 2019a, use of soil fumigation in Washington State using broad-spectrum che- 2019b; Weerakoon et al., 2012). In field studies, BjSa SM-induced micals (e.g. methyl bromide) as a replant management strategy began modifications to the rhizosphere microbiome persisted over multiple in the early 1990s (Willett et al., 1994). During the last few decades growing seasons and were associated with prolonged pathogen sup- however, global efforts to phase-out methyl bromide (Schafer, 1999) pression (Mazzola et al., 2015; Wang and Mazzola, 2019a). and more recent regulatory actions to limit use of other fumigants (e.g. Integration of seed meal treatments with specific rootstock geno- 1,3-dichloropropene, chloropicrin) have reduced the economic or types known to possess ARD tolerance may be integral to the devel- practical long-term viability of utilizing this method for control of soil- opment of alternative ARD management strategies (Wang and Mazzola, borne pests and pathogens in certain geographic regions. In addition, 2019a). In general, apple rootstocks from the Geneva series have sup- such an approach is not compatible with the expanding production of ported lower populations of Pythium spp. and P. penetrans than Malling organic tree fruits in response to the increasing consumer demand, series rootstocks (Isutsa and Merwin, 2000; Mazzola, 2009; Wang and particularly in Europe and North America. One promising alternative Mazzola, 2019a, 2019b). The rootstock G.210 was specifically bred for strategy to chemical fumigation, suitable for use in conventional and resistance to crown and root rot caused by Phytophthora cactorum organic production systems, is the use of pre-plant Brassicaceae seed (Cummins et al., 2013). In a recent transcriptome study, when chal- meal (SM) soil amendments in combination with apple replant disease lenged with Pythium ultimum, host defense responses were initiated (ARD)-tolerant rootstock genotypes. These types of “effective” disease faster and stronger in the ARD-tolerant rootstock G.935 than in the control systems have repeatedly been shown to provide increased plant susceptible rootstock Bud.9 (Zhu et al., 2016). Another important as- productivity at or above the level attained through the traditional pect of ARD-tolerance is the finding that the tolerant rootstock G.210 practice of soil fumigation (Mazzola, 2009; Mazzola et al., 2015; Wang develops a more extensive fine-root system that is more readily shed and Mazzola, 2019a). and regenerated than in the susceptible rootstock M.26 (Atucha et al., Members of the Brassicaceae family produce an array of glucosino- 2014; Emmett et al., 2014). late compounds which vary both qualitatively and quantitatively by Root exudates which vary in a genotype-dependent manner also species and even by plant cultivar (Brown and Morra, 1997). In re- influence the structure and function of rhizosphere microbial commu- sponse to tissue disruption, and in the presence of water, glucosinolates nities (Leisso et al., 2018). The microorganisms immediately sur- are hydrolyzed by the enzyme myrosinase to result in multiple che- rounding the root play important roles in the growth and ecological mistries, including isothiocyanates, with potential to inhibit various fitness of their plant host. Although soil type has been reported tobea plant pathogenic organisms. Glucosinolates are present in highest major factor determining composition of rhizosphere fungal and bac- concentration in the seed (Popova and Morra, 2014) and thus, the seed terial communities (Chang et al., 2019; Qin et al., 2019; Singh et al., meal generated during the oil extraction process are a superior mate- 2007), the ability of SM amendments to enrich for microbial groups rial, relative to Brassicaceae green manures, as a soil amendment em- with activity against known ARD pathogens has been documented in ployed for the control of soil-borne plant diseases. many different orchard soil-types throughout Washington state Brassicaceae seed meal soil amendments provide disease control (Mazzola et al., 2015; Wang and Mazzola, 2019a; Weerakoon et al., through various biological and chemical mechanisms, which vary de- 2012). That said, optimizing microorganism-driven protection of apple pending on the seed meal source and target pathogen (Mazzola and roots from infection by specific pathogens requires a better under- Brown, 2010). For example, the dominant glucosinolate in Brassica standing of how different seed meal amendments modulate composi- juncea (allyl glucosinolate) yields production of allyl-isothiocyanate tion of rhizosphere communities. Previous trials examined the effect of (AITC) in response to hydrolysis, a compound which leads to suppres- individual seed meals or seed meal formulations applied independently sion of apple root infection by Pythium spp. and Rhizoctonia solani on the resulting composition of the rhizosphere microbiome (Mazzola anastomosis group 5 (AG-5) but not Phytophthora (Mazzola, 2009; et al., 2015; Wang and Mazzola, 2019a; Weerakoon et al., 2012). Mazzola and Brown, 2010; Mazzola and Zhao, 2010). In comparison, However, there has been no in-depth, comprehensive analysis of these Brassica napus and Sinapis alba SM amendments have been shown to communities as influenced by “effective” vs. “ineffective” seed meal suppress root infection by Rhizoctonia spp. but stimulate Pythium formulations in the same soil/plant cultivation system. The rationale for (Cohen et al., 2005; Mazzola and Brown, 2010). In addition, all SM this study is based on previous experiments in which Geneva rootstocks tested generally have a temporary suppressive effect on P. penetrans used in combination with the BjSa SM formulation provided effective which is thought to be partly due to the production of nematistatic control of the ARD pathogen complex, while other rootstock × Brassica ammonia (NH3); although, B. juncea was shown to be superior to B. SM combinations were less effective. napus or S. alba SM in suppressing P. penetrans root infection due to the A secondary aim of this study was to characterize the spectrum of production of nematicidal AITC (Mazzola et al., 2007). oomycetes colonizing apple roots in these systems. In comparison to As noted, Brassica seed meal amendments are not equally suppres- bacteria and fungi, the community of oomycetes associated with the sive to all pathogens (Mazzola, 2009). When seed meals from an in- apple rhizosphere remains poorly explored. Although many oomycete dividual plant species were used as a soil amendment, the treatment species are important pathogens of agriculturally important crops, re- failed to control disease or enhance tree growth to the same level as latively few oomycete-specific community level studies have been attained with soil fumigation (Mazzola and Brown, 2010). However, published (Arcate et al., 2006; Tilston et al., 2018). There is consider- the use of specific composite Brassicaceae seed meal formulations pro- able diversity in virulence of individual Pythium and Phytophthora vided adequate or superior control of the pathogen complex inciting species associated with apple and it is well known that the diverse ARD, with one of the most effective SM formulations being a1:1 complex of pathogenic oomycete species differs among orchard soils combination of B. juncea and S. alba (BjSa) (Mazzola et al., 2015; Wang (Mazzola et al., 2002). As stated above, B. napus and S. alba SM and Mazzola, 2019a, 2019b; Weerakoon et al., 2012). This seed meal amendments have consistently stimulated orchard soil Pythium spp. formulation not only suppresses multiple components of the pathogen populations, while B. juncea has been shown to suppress apple root complex, but also disrupts the microbial community in the bulk soil. infection by Pythium but not Phytophthora. A more comprehensive un- Modification of the soil microbiome is not simply due to the short-term derstanding of the rhizosphere microbiome associated with these

2 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766 different rootstock × Brassica SM combinations may help us further around roots. Plants were grown in the greenhouse at 22 °C and a 16- optimize microorganism-driven protection of apple roots and enable hour photoperiod. Ambient light was supplemented using LED lights. more sustainable management of potential losses caused by specific Photosynthetically active radiation (PAR, 400–700 nm) at the level of ARD pathogens. the rootstock canopy was between 100 and 250 μmol s−1 m−2. The effect of soil treatments on rootstock growth was assessed by measuring the increase in initial rootstock weight, root volume, and trunk dia- 2. Methods meter over the 4-month duration of the experiment. Immediately after planting, the main stem of each rootstock was marked with latex paint 2.1. Soil and Brassica seed meals and two diameter measurements were taken in perpendicular directions using an electronic caliper to obtain a mean trunk diameter. This The soil used in this study was obtained from a commercial (GC) measurement was repeated again prior to harvest and the increase in orchard in Manson, WA, USA (latitude 47° 53′ 05″ N, longitude 120° 09′ trunk diameter was calculated. Root volume was measured by sub- 30″ W). The site had been managed using conventional practices at the mersing roots in water and measuring the volume of water displaced in time of soil collection. Chelan gravelly sandy loam (3–4% organic mL. Terminal leader shoot length (cm) was also measured at harvest. matter, pH = 7.0) is the dominant soil type at the GC orchard (Mazzola, No additional nutrients were supplied to the trees during the experi- 1998). This orchard was established in 1991 on a site previously ment. During the course of the experiment Avid 0.15 EC Miticide/In- planted to apple. Pathogen composition at the GC orchard site was secticide (1× in March) and Procure 480SC Fungicide + Bonide Sys- previously characterized and found to include all the primary agents of temic (1× in April and 2× June) were applied to foliage of rootstocks ARD; the fungal complex was dominated by C. destructans and R. solani to control spider mites, powdery mildew, and whiteflies, respectively. AG-5 (Mazzola, 1998). Bulk soil was collected in late autumn (October 2017), when nematode populations were expected to be returning to 2.3. Collection of soil samples for DNA extraction and nutrient analysis the soil as fine roots decline (Shurtleff and Averre, ).2000 The seed meals used in this study were obtained from Brassica napus cv. Athena, A roughly 10 g bulk soil sample was collected from each pot Brassica juncea cv. Pacific Gold, and Sinapis alba cv. IdaGold (Mazzola (10–15 cm deep) prior to SM amendment (T0) and at 1-week (T1), 3- and Brown, 2010). Seed meals varied in glucosinolate profile and weeks (T2), and 8-weeks (T3) post SM-amendment. Bulk soil samples content as described in Mazzola et al. (2007). All seed meal treatments were then stored at 4 °C until further processing. DNA was extracted were then passed through a 1mm2 sieve and portioned into individual from 5 g bulk soil samples using the DNeasy Power Max Soil Kit bags for addition to soil (Table 1). Treatments also included a no- (Qiagen, Valencia, CA). All extractions were completed within two treatment control and a pasteurized control (as a proxy for soil fumi- weeks of bulk soil sample collection. Rhizosphere soil samples were also gation). Pasteurized controls consisted of GC soil that had been heated collected at harvest (T3). After plants were removed from pots and to 80 °C for 7 h on two successive days, with 12 h in between each shaken to remove loosely adhering soil, soil firmly attached to the roots heating session. The B. napus seed meal amendment, the two different was collected from multiple locations along the root using sterile levels of B. juncea seed meal, and the 1:1 blend of BjSa SM were applied tweezers and a scoopula. DNA was extracted according to the manu- individually to 2.5 L of soil and incorporated into the soil by hand facturer's instructions from 0.25 g of rhizosphere soil per plant using the mixing. After SM application, the soil was immediately placed in a 3 L DNeasy PowerSoil Kit (Qiagen). pot, 300 mL of sterile DI water was added to the soil surface, and the pot was double-bagged in gas-impermeable bags to enhance retention 2.4. Terminal-restriction fragment length polymorphism analysis of volatile glucosinolate hydrolysis products in the soil profile (Bitran Liquid-Tight Specimen Bags, Com-Pac International, Carbondale, IL). T-RFLP analysis was used as a tool to rapidly and qualitatively Pots were removed from bags after one week and placed on a green- compare fungal and bacterial community structure across the different house bench arranged in a complete randomized block design con- treatments prior to planting. Labeled primers for fungal ITS (ITS1F-D4/ sisting of 10 replicates per treatment (5 replicates × 2 genotypes) with ITS4-D3) and bacterial 16S rRNA (799F-D4/1391R-D3) genes were 10 pots per block (60 pots total). used as previously described (Weerakoon et al., 2012). For bacterial community fingerprinting, PCR cycling conditions were: 94 °Cfor 2.2. Rootstocks used and assessment of plant growth 5 min, and 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, followed by extension at 72 °C for 7 min. Bacterial PCR products were Prior to planting, Geneva 210 (G.210) and Malling 26 (M.26) digested using 10 U of the restriction enzyme HpaII (1uL) (New England rootstock bundles (0.635 cm trunk diameter, TRECO, Woodburn, OR) Biolabs, Ipswich, MA). All other methods for bacterial and fungal T- were kept in cold storage and roots were covered with moist wood RFLP were identical. Fragments were separated using a Beckman shavings. Rootstocks were planted 8-weeks after seed meal amendment Coulter GenomeLab GeXP Genetic Analysis System with the same in March of 2018. Certain Geneva series rootstocks have demonstrated conditions as described in Weerakoon et al., 2012. T-RFLP profiles were tolerance to apple replant disease in field trials conducted in New York analyzed and fragments were binned with a 1 nt margin using the AFLP and Washington (Robinson et al., 2014). In comparison, M.26 is con- tool in the fragment analysis software. Dice similarity coefficients were sidered to be more susceptible to ARD (Webster et al., 2000). For each calculated for the microbial community profiles using the Paleontolo- treatment, 5 trees of each genotype were planted 8-weeks post SM gical Statistics freeware package (PAST v. 3.2.5) (Hammer et al., 2001). amendment. After planting, all pots were watered to fill in air spaces 2.5. 16S/ITS sequencing Table 1 Seed meal application rates employed in this study and the translation to tons/ DNA extracted from rhizosphere soil samples as noted above was ha. eluted in a final volume of 100 μl elution buffer and stored at−80°C. Soil amendment g per pot tons/ha Based on previous studies, BjSa SM amendment is considered to be the most “effective” SM amendment for ARD control, while the non-AITC Brassica napus (Canola) 7.9 4.4 producing B. napus SM is typically less effective due to its stimulatory Brassica juncea-High 7.9 4.4 effect on Pythium spp. Therefore, among the different treatments in- B. juncea-Low 3.95 2.2 B. juncea + Sinapis alba (BjSa) 7.9 4.4 cluded in the experiment only BjSa SM, B. napus SM, and no treatment control (NTC) treatments were selected for 16S rRNA and fungal ITS

3 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766 amplicon sequencing. Oomycete community structure was assessed in rank-based approach was used to identify taxa that were significantly BjHigh as well as BjSa, B. napus SM, and the no treatment control. different between treatment groups (Wagner et al., 2011). All p-values Bacterial amplicon libraries were generated using the primer pair 515f were computed using a threshold of 0.05 (Explicet v2.10.5). Sig- and 806r for the V4 region of the 16S rRNA gene (Caporaso et al., nificance tests aimed at determining the effect of amendment typeon 2011). Fungal amplicon libraries were generated using the ITS1f/ITS4r rhizosphere bacterial community composition as well as assessing primer pair. Bacterial 16S rRNA and fungal ITS regions were sequenced consistency between samples within the same rootstock × treatment on an Ion Torrent S5 XL (20,000 reads per sample) using the bacterial system were calculated in Graphpad Prism version 7.05. Separation of tag-encoded FLX amplicon pyrosequencing (bTEFAP®) method (Dowd community structure was also explored using ordination methods in et al., 2008). On average, this platform generated 300 and 500 bp PAST version 2.17. Principle coordinate analysis (PCoA) was performed single-end reads for bacterial and fungal amplicons respectively. Long- to best accommodate the presence of zeroes in the relative abundance read sequencing technology (PacBio Seqeul, 5000 reads per sample) data, and the non-parametric 1-way analysis of similarity (ANOSIM) was used to amplify a much longer ITS region (~600–1000 bp) for the function was used to test for significant differences in community oomycete data set. Oomycete amplicon libraries for the internal tran- composition. scribed spacer 2 (ITS2) region were generated using the universal oomycete primer set ITS1Oo (5′-GGAAGGATCATTACCACAC) and ITS4bt 2.7. Nematode abundance in roots (5′-TCCTCCGCTTATTGATATGC) (Riit et al., 2018, 2016). Sequence data from all analyses were processed by the sequencing facility (Mo- Upon harvest, root densities of Pratylenchus penetrans were de- lecular Research, Shallowater, TX, USA) using the following pipeline: termined for all trees in BjSa, BjHigh, BjLow, B. napus, NTC and pas- sequences were edited to remove barcodes and primers, sequences < teurized treatments (Table S1). For each tree, roots were rinsed under a 150 bp and those containing ambiguous base calls and homopolymer stream of tap water, blotted dry and a 0.5 gram sample was placed into runs exceeding 6 bp were removed, sequences were quality filtered a 125-mL Erlenmeyer flask containing 80 mL of sterile distilled water. using a maximum expected error threshold of 1.0 and dereplicated, Flasks were placed on a shaker at 140 rpm and incubated at room dereplicated sequences were then denoised and unique sequences temperature for 4 days. Nematodes were extracted by passing the sus- identified with sequencing or PCR point errors were removed, chimera pension through a 0.37 um mesh sieve and backwashing into a counting detection and removal was performed. Processed reads were then dish. P. penetrans were identified based upon morphological features clustered to form operational taxonomic units (OTUs) at 97% similarity and counted using a compound light microscope at 40× magnification. and taxonomy was assigned by aligning final OTUs against a manually curated, up-to-date database derived from RDPII (http://rdp.cme.msu. edu) and NCBI (ncbi.nlm.nih.gov) using BLASTn. Prior to analysis of 2.8. Quantification of fungal and oomycete pathogens in apple roots byq- OTU read counts, data was edited to remove singletons and doubletons PCR as well as non-related OTUs. All eukaryotic and archaeal OTUs were removed from the bacterial 16S rRNA dataset, OTUs matching King- DNA was extracted from a 25 mg fine root tissue using the Qiagen doms Plantae, Bacteria, and Archaea were removed from fungal ITS Dneasy PlantPro Kit (Qiagen). Prior to DNA extraction, root tissue was datasets, and all non-oomycete OTUs were removed from the oomycete ground using liquid nitrogen. Q-PCR was conducted using a dataset. StepOnePlus Real-Time PCR System (Applied Biosystems, Warrington, UK) with three technical replicates per sample. For quantification of total fungi, Ilyonectria/Cylindrocarpon species, and Rhizoctonia solani, 2.6. Analysis of microbial diversity and community composition the primer pairs NSI 1/5.8S (Reardon et al., 2013), YT2F/CylR (Tewoldemedhin et al., 2011c), and Rhsp1/ITS4B (Mazzola et al., Assessment of microbial community profiles and microbial diversity 2020) were used, respectively. Each 10 μl reaction consisted of 1.0 μl of as influenced by soil treatment was evaluated using Explicet software a 1:5 dilution of root DNA extract, 3.0 μl PowerUp SYBRGreen Master package version 2.10.5 (Robertson et al., 2013). Rarefaction curves Mix (Applied Biosystems, Warrington, UK), Ambion Nuclease-Free H2O were generated and used to identify samples which were under sampled (ThermoFisher, USA), and either 0.05, 0.07, or 0.1 μl of each primer (in which the rarefaction curves may cross the other samples if the [100 μM] for total fungi, Ilyonectria/Cylindrocarpon and R. solani re- sampling effort was increased). These low-depth samples were excluded spectively. Standard curves were prepared using purified genomic DNA from further analysis and are listed in Table 2. Rarefaction curves of all from C. destructans (#60-1189; isolated from CV orchard soil, Orondo, samples included in the analysis are shown in Fig. S2. Alpha diversity WA) and R. solani AG-8 (#2-43; isolated from wheat field soil in Lind, was measured with the Chao1 index. Samples were normalized by WA) diluted from 100 to 0.01 pg/μl. Amplification of total fungal DNA taking the relative abundance for each OTU as the proportion of all was conducted as in Reardon et al. (2013) with the following mod- sequences (tags) in a sample after all singleton and doubleton OTUs had ifications: 95 °C for 10 min, (95 °C - 30 s, 58 °C - 30 s,72°C- been discarded. The Two-Part Test using a non-parametric Wilcoxon 30 s) × 40 cycles. Quantification of Ilyonectria/Cylindrocarpon and R.

Table 2 Next-generation DNA sequencing related metrics for bacterial, fungal and oomycete amplicon libraries.

Sequencing metric Bacteria Fungi Oomycetes

# samples included in analyses 29 29 33 # nonsingleton/doubleton OTUsa 1434 (754) 755 (464) 60 (11) # amplicon sequencing reads 1,708,178 1,163,431 224,108 Sequencing depth range 30,766–78,408 17,447–122,381 481–27,232 Average library size (# reads) 58, 903 40,118 6,791 # reads to reach asymptoteb ~30,000 ~20,000 < 1000 Samples excluded from analysisc BjSa G210.5 NTC M26.3 G.210 BjSa.3 M26 BjSa.1

a Parentheses indicate number of groups at genus-level. b Genus and species level abundance curves generated using Good's coverage index in Explicet v. 2.10.5. c Samples were excluded from the analysis when read counts were so low that the rarefaction curves may have crossed other samples if the sampling effort was increased. BjSa = B. juncea + S. alba seed meal (4.4 t ha−1), NTC = no treatment control.

4 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766 solani DNA was conducted using same conditions described above with plant health may rely more on the presence of species with particular annealing temperatures of 61 °C and 59 °C, respectively. Amplification functional characteristics than overall number of species present of the oomycete pathogens Phytophthora cactorum and Pythium ultimum (Mazzola et al., 2015). was conducted using the primer sets Yph1f/Yph2r (Tewoldemedhin et al., 2011b), and ULT1f/ULT4r (Mazzola et al., 2020). The same re- 3.3. BjSa SM treatments resulted in distinct bacterial, fungal, and oomycete action conditions for both primer sets were used as described for P. communities ultimum in Mazzola et al. (2020). Standard curves were prepared using purified DNA from P. ultimum (#60–1205, obtained from the Monterey Differences in community structure related to treatment-type and Bay Academy, Watsonville, CA) and P. cactorum (#60-1190; isolated rootstock genotype were further explored using ordination methods. from GC orchard soil located near Manson, WA) diluted from 100 to Samples clustered by soil treatment-type for both fungal and bacterial 0.01 pg/μl. All root DNA extracts were quantified on a Qubit 2.0 rhizosphere communities (Fig. 3A and C, respectively), with significant fluorometer using the Invitrogen Qubit dsDNA HS Assay Kit (Thermo- differences between all treatment combinations: NTC vs. BjSa (one-way Fisher, USA) and q-PCR results were normalized to total DNA input. ANOSIM; p = 0.0001bacteria and fungi), NTC vs. B. napus (p = 0.0002bacteria; p = 0.0001fungi), and BjSa vs. B. napus 3. Results and discussion (p = 0.0001bacteria and fungi). Bacterial communities in BjSa SM- amended soil were also more clearly separated from the NTC than those 3.1. SM amendments shift microbiome structure in bulk soil prior to planting of B. napus SM-amended soil along PC1 (Fig. 3C). This finding indicates that the composition of bacterial communities was more strongly in- Based upon T-RFLP analysis, seed meal treatments shifted fungal fluenced by BjSa than by B. napus SM. In comparison, PCoA plots of and bacterial community composition relative to the NTC (Fig. S1A and fungal community shifts showed BjSa and B. napus as being relatively B, respectively). For both fungi and bacteria, community structure in equidistant from the NTC (Fig. 3A). soils amended with B. juncea at 4.4 t ha−1 or the BjSa SM formulation Although oomycete communities retained similar species diversity were most differentiated from that detected in the NTC soil. Inthe regardless of treatment, the influence of treatment on oomycete species context of this experiment, it was important to detail these shifts prior composition is shown in Fig. S3. Significant differences relative to the to planting as rhizosphere colonizers likely originate from the sur- NTC were identified for all treatments: p = 0.0002 for NTC vs. BjSa, rounding bulk soil. p = 0.0001 for NTC vs. BjHigh, and p = 0.0054 for NTC vs. B. napus. Similarity indices based only on presence/absence data were also sen- 3.2. Reduced α-diversity in bacterial and fungal but not oomycete SM- sitive to differences among oomycete rhizosphere communities grown structured rhizobiomes in BjSa SM vs. B. napus SM (Dice, p = 0.008).

As stated above, BjSa and B. napus SM treatments have previously 3.4. Rootstock genotype also shapes the rhizosphere microbiome been shown to induce “effective” and “ineffective” levels of replant disease suppression, respectively. Thus, BjSa SM, B. napus SM and NTC The influence of rootstock genotype on rhizosphere community treatments were selected for NGS sequencing of bacterial, fungal and composition is typically masked by SM-induced changes (Wang and oomycete communities inhabiting the rhizosphere. As B. juncea SM has Mazzola, 2019a). Rootstock genotype had a significant effect on bac- been shown to suppress Pythium but not Phytophthora spp., oomycete terial (1-way ANOSIM; p = 0.047 species-level, p = 0.035 genus-level) community composition was also assessed in the BjHigh treatment. For but not fungal or oomycete community structure at the genus and bacteria, fungi and oomycetes, 1434, 755, and 60 different non- species-level, respectively (Fig. 3B and D). In Fig. 3D, clustering beha- singleton/nondoubleton OTUs (< 97% similarity) were identified vior is partly structured by rootstock genotype, in which G.210 (green) across all rhizosphere soil samples, respectively (Table 2). Species-level and M.26 (blue) samples generally appear above and below 0 along accumulation curves for bacterial and fungal samples were close to PC2, respectively. In a previous study (Wang and Mazzola, 2019a), NTC reaching a plateau by 30,000 and 20,000 reads, respectively. By com- fungal communities associated with M.9 were found to be significantly parison, oomycete richness generally began to asymptote at sampling different from those of MM.106, G.41, and G.210 in GC orchard soil depths < 1000 reads, indicating that oomycete taxonomic diversity in using T-RFLP analysis. Here, the lack of a detectable genotype-signal in the apple rhizosphere is much lower than that of fungi and bacteria the phylogenetic composition of fungal community may be related to (Fig. S2). This information will be valuable for estimating the sequen- differences in rootstocks used, analysis techniques, and/or the number cing depth (and cost) required to obtain meaningful results on the of samples included in the analysis. In the present study, the effect of taxonomic structure of root-associated oomycete communities in the rootstock genotype on bacterial community composition was only sig- future. nificant when all three treatments were combined providing alarger The Chao 1 index was used to estimate the effect of seed meal number of samples (i.e. for purely statistical reasons). treatment on the richness of the rhizobiome and was calculated for all microbial samples at the species and genus level (Fig. 1). For both 3.5. BjSa SM-structured rhizobiomes are rich in biocontrol and bacteria and fungi, BjSa SM, but not B. napus SM soil amendment, in- bioremediation potential duced a significant reduction in the number of unique taxa relative to that of the NTC. There was also a significant reduction in bacterial The Two-Part test identified a number of potentially beneficial richness in BjSa SM relative to B. napus SM treatments. This loss of bacterial taxa including Bacillus (Firmicutes), Arthrobacter and Lentzea taxonomic diversity in BjSa SM-treated samples is in accordance with (Actinobacteria), and Flavisolibacter (Bacteroidetes) that were sig- other studies and is most likely due to production of the toxic iso- nificantly enriched with at least a 2-fold increase in median relative thiocyanate AITC (Lin et al., 2000; Mazzola et al., 2015; Wang and abundance relative to the NTC in both BjSa and B. napus SM-treatments Mazzola, 2019a). For both bacteria and fungi, species diversity re- (Fig. S4A). Members of the genus Bacillus, which were tentatively flected genus diversity (Fig. 1). Oomycete α-diversity was assessed in 4 identified at the species level included B. vireti and B. badius. In a recent different treatments: BjHigh SM, BjSa SM, B. napus SM and NTC. Oo- field trial, B. vireti and B. badius were also found to be elevated in SM- mycete richness was not significantly influenced by treatment. structured rhizospheres of apple tress cultivated in a different soil type The impact of BjSa SM treatments on rhizosphere bacterial and (Wang and Mazzola, 2019a). B. badius is known for a number of bio- fungal taxonomic diversity did not appear to negatively affect plant degradative traits including penicillin G acylase and azoreductase growth (Fig. 2), supporting the hypothesis that microbiome-mediated production as well as anthracene degradation (Misal et al., 2011;

5 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766

A. B. C.

Bacteria_Genus Fungi_Genus Oomycete_Genus 650 300 b 8 b ab b a 6 600 250

a 4

550 200 2 # of unique taxa # of unique taxa #ofuniquetaxa

500 150 0

a s C a s C h a s C S g j T jS T i jS pu T B apu N B apu N jH B a N B B. n B. n B. n Treatment Treatment Treatment

Bacteria_Species Fungi_Species Oomycete_Species

1150 450 b 25 b ab b 1100 400 20 a a 1050 350 15

1000 300 10 #ofuniquetaxa #ofuniquetaxa #ofuniquetaxa 950 250 5

900 200 0

a s C a s C h a s C S g S j T jS pu T i j T B apu N B a N jH B apu N B n B. n B. n B. Treatment Treatment Treatment

Fig. 1. Effect of soil treatment on taxon richness detected in apple rhizosphere soil. Box-and-whisker plots showing (A) bacterial, (B) fungal, and (C) oomyceteChao1 richness. Boxes represent the interquartile ranges (upper line is the 75% quantile, and the lower line is the 25% quantile), lines inside the boxes are the medians, and whiskers span the high and the low range. The analyses were conducted across both G.210 and M.26 rootstock genotypes, and each treatment contains 8–10 replicates. Genus and species-level assessments are shown above and below, respectively. Letter groups indicate significant differences (p < 0.05) between treatments as indicated by the Kruskal-Wallis test followed by Dunn's multiple comparison test.

A. B.

G.210 M.26 bc 2.0 2.5 b b b b

ab 2.0 1.5 ab abc a ac ac 1.5 1.0 1.0 a 0.5 0.5

0.0 0.0 C w s t C w s t Increase in Trunk Diameter (cm) T u Increase in Trunk Diameter (cm) T gh u s N .Lo High +S.a. Pas N .Lo Hi +S.a. Pa j j. . nap j j. . nap B. .j B. .j B. B B. B. B B. Treatment Treatment

Fig. 2. Effect of soil treatment on increase in apple rootstock trunk diameter. Panels A and B show data for G.210 and M.26, respectively. Rootstocks weregrownin GC orchard replant soil for over a 15-week period. NTC = no treatment control, B.j. Low = 2.2 t ha−1 Brassica juncea seed meal (SM), B.j. High = 4.4 t ha−1 Brassica juncea SM, B.j. + S.a. = 4.4 t ha−1 B. juncea + Sinapis alba SM (1:1), and B. napus = 4.4 t ha−1 Brassica napus SM, Past = pasteurized control. Each treatment × genotype combination contains 4–5 replicates with individual values shown. Error bar show one standard deviation from the mean (line at center). Letter groups indicate significant differences (p < 0.05) between treatment means as indicated by ordinary one-way ANOVA followed by Tukey's multiple comparisontest.

6 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766

Fig. 3. Effect of different soil treatments and apple rootstock genotype on rhizosphere bacterial and fungal community composition. Rhizosphere soil wascollected4- months after planting rootstocks into GC replant orchard soil (or 6 months post-seed meal amendment). Ordination of microbiomes was conducted by principal coordinate analysis of operational taxonomic unit data using the Bray-Curtis dissimilarity coefficient. Fungal (A and B) and bacterial (C and D) communities are shown at the genus-level. In panels A and C, colors represent the different treatments: blue = 4.4 tha−1 B. juncea + Sinapis alba seed meal (SM), green = 4.4 t ha−1 Brassica napus SM, black = no treatment control. The amount of variation captured in each axis is shown. Convex hulls enclose all samples derived from the same soil treatments. In panels B and D, colors represent the different apple rootstock genotypes: green = G.210, light blue = M.26. (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of this article.)

Othman et al., 2012; Rajendhran and Gunasekaran, 2007). In both seed oligophenolica. S. wittichii is known to metabolize a broad array of ha- meal treatments the Arthrobacter population consisted of OTUs with logenated aromatic compounds, many of which have become ubiqui- closest matches to Arthrobacter ramosus and an unidentified Arthrobacter tous pollutants including: polypolychlorinated biphenyls (PCBs) (Seah spp. A variety of beneficial activities have been described in A. ramosus et al., 2007), toxic diaryl ethers (Hong et al., 2002; Nam et al., 2006), strains, including the ability to bioaccumulate and detoxify several and nitrodiphenyl ether herbicides (Young et al., 2008). S. oligopheno- heavy metals, produce trehalose (an osmoprotectant), and secrete ex- lica can degrade certain phenolic acids: ferulic acid, p-hydroxybenzoic tracellular proteases which may play a role in biological control of plant acid, p-coumaric acid and vanillic acid (Ohta et al., 2004). Selective pathogens (Bafana et al., 2010; Nilegaonkar et al., 2002; Yamamoto enrichment of rhizosphere bacterial taxa possessing putative de- et al., 2001). Members of the genus Lentzia and Flavisolibacter are gradative capabilities (e.g. Arthrobacter, Rhodococcus, Sphingomonas) known for the production of chitinolytic enzymes and other bioactive has also been shown in other soil types amended with B. juncea or BjSa compounds (Huynh et al., 2019; Rosenberg, 2014). SM regardless of rootstock-genotype (Mazzola et al., 2015; Wang and Prominent bacterial genera which distinguished BjSa-structured Mazzola, 2019a; Weerakoon et al., 2012). rhizospheres from those of NTC and B. napus SM included taxa from a One factor underlying the unique rhizobiome composition gener- number of potentially beneficial Proteobacteria (e.g. Sphingomonas, ated by BjSa soil amendments may be the distinct chemistries produced Rhizomicrobium, Fusobacterium, Dokdonella, Luteibacter, by this seed meal formulation. Although BjSa and B. napus SM contain Phenylobacterium and Kaistobacter spp.) (Fig. S4A). The ability to de- similar amounts of carbon, nitrogen, sulfur, and phosphorous, they grade complex carbon compounds, including xenobiotics, and to tol- have very different glucosinolate contents and profiles, and corre- erate heavy metals are functional attributes common to many of these spondingly distinct biologically active glucosinolate hydrolysis pro- taxa including Sphingomonas, Phenylobacterium, Kaistobacter ducts. Consistent with our findings, a recent study identified significant (Kamaludeen and Ramasamy, 2008; Kasemodel et al., 2019), and increases in the relative abundance of Bacillus, Sphingomonas, and Dokdonella (Palma et al., 2018). BjSa SM-specific sphingomonads in- Streptomyces following AITC fumigation of soil (Zhu et al., 2020). Mi- cluded OTUs with closest matches to Sphingomonas wittichii and S. crobes with the ability to not only tolerate volatile ITCs (e.g. AITC), but

7 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766 also to degrade and/or utilize a specific mixture of glucosinolate-related B. amyloliquefaciens, an antifungal lipopeptide complex produced by a compounds as substrates for growth would be expected to flourish in variety of Bacillus species (Zihalirwa Kulimushi et al., 2017). response to BjSa SM soil amendment but not in response to B. napus It is also notable that the proportion of fungi belonging to the genus (containing a much lower glucosinolate content). The consistent en- Arthrobotrys increased in both SM treatments by ~8-fold relative to the richment of bacterial communities high in biodegradative and bior- NTC, although this increase was not significant according to the Two- emediation potential by BjSa SM is especially relevant to plant health in Part Test (Fig. S5). Typically, nitrogen is the limiting growth factor for agricultural soils where there may be heavy metal contamination (i.e. many predatory fungi (including Arthrobotrys) which attack other or- lead-arsenate) and/or intensive use of pesticides and herbicides (e.g. ganisms as sources of nitrogen to supplement a primarily carbohydrate aromatic-ring containing compounds like Atrazine and DDT). (woody) diet (Barron, 2003). Seed meals produced from B. juncea, S. When the fungal community was analyzed, 25 and 72 genera be- alba, and B. napus are all especially rich in nitrogen (N ~ 6%) (Cohen longing to 10 and 20 different classes were identified as being sig- et al., 2005), and it is possible that seed-meal derived nitrogen helps nificantly different between controls and B. napus or BjSa SM treatment, promote the proliferation of Arthrobotrys spp. in the rhizosphere. A respectively. In contrast to PCoA analysis (conducted at the genus- recent study also provided strong evidence that Arthrobotrys is AITC- level), this result indicated that BjSa SM affected a more diverse set of tolerant (Zhu et al., 2020). As mentioned earlier, all seed meals have a fungal taxa than B. napus. Curiously, the summed median relative temporary suppressive effect on nematodes (Cohen et al., 2005); how- percentages of these fungal genera comprised roughly 40% of the total ever, once the inhibitory effect of seed meal goes away (e.g. the next population in both SM-types. Thus, both SM treatments appear to have season) elevated numbers of Arthrobotrys spp. in the apple rhizosphere influenced a similar share of the fungal community, albeit different may directly reduce host infection intensity and facilitate continued groups of taxa. suppression of lesion nematodes, a phenomenon which has been ob- A handful of potentially beneficial fungi, including Humicola, served in multiple orchard replant trials conducted with BjSa SM Fusarium, and Rhizomucor (largely R. variabilis) were significantly en- (Mazzola et al., 2015; Wang and Mazzola, 2019a). Thus, this fungal riched in both SM-types relative to the NTC (Fig. S4B). These taxa were group may be an important component of SM-structured, ARD-sup- largely composed of species tentatively designated as H. grisea, F. oxy- pressive rhizospheres. sporum, and R. variabilis, respectively. F. oxysporum is commonly asso- Finally, SM-structured rhizosphere communities were also char- ciated with apple roots in orchards and is considered to be non- acterized by microbial taxa involved in sulfur cycling including pathogenic towards apple (Manici et al., 2013; Mazzola, 1998). Sinorhizobium, Halothiobacillus, and Edaphobacter spp. (Fig. S4A). In Evidence also exists that F. oxysporum may even play a suppressive role another field study, sulfur-oxidizing bacteria involved in the oxidation in specific apple replant disease (Sneh et al., 1977). Some studies have of elemental sulfur to plant available sulfate, were found to be sig- found Fusarium spp. in general to be AITC-tolerant (Hu et al., 2015; nificantly more abundant in the rhizosphere of apple cultivated inBjSa Ishimoto et al., 2000); however, previous studies have shown B. juncea SM-amended soil after two field growing seasons (Mazzola et al., 2015). SM and AITC to inhibit vegetative growth of F. oxysporum Total sulfur content is similar among the seed meals used in this study (Hewavitharana et al., 2014; Smolinska et al., 2003) although this (~1.6%); however, B. napus SM has a much lower glucosinolate content fungus may survive in the soil in the form of chlamydospores. In this (25 μmol g−1) than that of B. juncea and S. alba (170 μmol g−1)(Wang study, the occurrence of Fusarium spp. was most strongly associated and Mazzola, 2019a). Glucosinolates are composed of a thiohydrox- with B. napus SM (Fig. S4B). Interestingly, when all samples were in- imate-O-sulfonate group linked to glucose, and an alkyl, aralkyl, or cluded (n = 28) the relative abundance of F. oxysporum was sig- indolyl side chain (Barba et al., 2016). Thus, the higher glucosinolate nificantly correlated with increases in trunk diameter, although 1out- content in BjSa SM may promote microbial community shifts which lier in which F. oxysporum was 16% of the relative abundance (G.210, favor specific S-uptake and or utilization mechanisms. B. napus-5) was excluded from the analysis (Spearman correlation, Fungal taxa that were significantly enriched in BjSa but not B. napus r = 0.61, p = 0.0005). These data are in contrast to studies which have SM treatments relative to the NTC included Sclerotinia (namely S. implied a role for Fusarium species as contributing to the development homoeocarpa), Chaetomium, Geomyces, and arbuscular mycorrhizal of ARD based on elevated abundance of F. oxysporum in orchard replant fungi (AMF) belonging to the genera Paraglomus and Rhizophagus (Fig. soils (Wang et al., 2018). However, in the same study, other fungi de- S4B). Interestingly, the grass pathogen S. homoeocarpa is known for monstrating similar increased density in ARD soils were not corre- production of oxalic acid. Oxalic acid has been shown to play a role in spondingly identified as causal agents of the disease and no assessment regulating the activities of defense-related compounds. For example, was conducted as to the pathogenicity of these fungi towards apple. As when applied to pear fruit and rice plants, oxalic acid enhanced re- such, the implied role for F. oxysporum as a causal agent of apple replant sistance to post-harvest pathogens and induced resistance to R. solani, disease cannot be supported by studies conducted to date. In light of respectively (Jayaraj et al., 2010; Tian et al., 2006). Chaetomium spp. this, the extent of intraspecific variation in F. oxysporum is high and the have been shown to increase specifically in response to AITC (Hu et al., implications for pathogenicity remain relatively unexplored (Appel and 2015; Zhu et al., 2020) and have been enriched by B. juncea-containing Gordon, 1995; Manici et al., 2017). SM in multiple orchard soil types around central Washington (Mazzola Humicola was also significantly enriched in both SM treatments, but et al., 2015; Wang and Mazzola, 2019a). This genus includes several was more strongly associated with BjSa SM than B. napus SM (Fig. S4B). metabolically gifted members (e.g. C. globosum, C. nigricolor) also pos- A number of studies have shown that Humicola spp. may be stimulated sessing the potential to control ARD pathogens. In particular, C. glo- by AITC (Hu et al., 2015; Zhu et al., 2020). H. fuscoatra has been shown bosum has been shown to inhibit the growth of the ARD-specific oo- to parasitize the oospores of P. cactorum and P. megasperma (Sneh et al., mycete Pythium ultimum (Di Pietro et al., 1992). Tentatively named 1977). H. grisea is known for its cellulolytic ability as well as its ability species within the genus Glomeromycota that were preferentially asso- to produce chitinase and may be an important group of beneficial fungi ciated with roots in BjSa-amended soil included Paraglomus brasilianum capable of suppressing both ARD fungal and oomycete pathogens and Rhizophagus irregularis spp. Within the BjSa × G.210 SM-associated (White and Downing, 1953). Consistent with this hypothesis, the re- fungal community Paraglomus brasilianum was among the most promi- lative abundance of Humicola species significantly correlated with root nent species overall (18.5% relative abundance). Therefore, BjSa (but volume in NTC treatments (Spearman: r = 0.7, p-value = 0.04, n = 9). not B. napus SM) appeared to increase the degree of root colonization Rhizomucor spp. (also more strongly associated with BjSa SM than B. and drive shifts in the AMF community. Interestingly, the relative napus SM) are known for their bioaccumulation and biotransformation abundance of Paraglomus was significantly negatively correlated with potential in soil (Singh et al., 2015). R. variabilis may also play a role in an increase in trunk diameter in BjSa SM (Spearman correlation, biocontrol as it has been shown to stimulate the secretion of fengycin by r = −0.79BjSa, p = 0.008, n = 9), suggesting that plant host-derived

8 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766 sugars and carbohydrates on which AMF depend may have resulted in Table 3 less energy being allocated to woody tissue. Typically, this cost is off-set Bacterial taxa exhibiting differentiating relative abundance between rootstock by a number of benefits to the tree including filtering out heavy metals, genotypes across all treatments. nitrogen and phosphate acquisition, and suppression of root pathogens Class Genus Mean relative Fold p-Valuea (Javaid, 2011; Tamayo et al., 2014; Xu et al., 2008). These may all be abundance (%) changeb important aspects of effective seed meal × genotype disease control systems. It is also interesting to note that while mean relative abun- G.210 M.26 dance of Glomeromycota increased from 12.8% in NTC to 21.7% in BjSa Gammaproteobacteria Pseudomonas 1.63 4.53 2.8 0.002 SM treatments, Glomeromycota species-level OTU richness was reduced Alphaproteobacteria Novosphingobium 0.49 1.12 2.3 0.003 in BjSa SM treatments. This result is consistent with field studies in Gammaproteobacteria Spongiibacter 0.02 0.09 4.5 0.022 which there was a reduction in the diversity of Glomeromycota in the Alphaproteobacteria Rhizobium 1.01 1.48 1.5 0.027 Alphaproteobacteria Acidocella 0.01 0.04 4 0.033 rhizosphere of trees cultivated in BjSa SM-amended soil relative to both Alphaproteobacteria Labrys 0.21 0.27 1.3 0.034 NTC and fumigated plots (Mazzola et al., 2015; Wang and Mazzola, Actinobacteria Arthrobacter 0.5 0.96 1.9 0.043 2019a). This suggests that many different AMF species may be sensitive Alphaproteobacteria Rhodoplanes 5.1 4.28 1.2 0.017 to the chemistries generated in BjSa SM-amended soil and/or differ in Alphaproteobacteria Methylosinus 0.69 0.54 1.3 0.027 Planctomycetia Blastopirellula 0.21 0.16 1.3 0.043 their ability to access nutrients from SM-associated substrates. To- gether, these results demonstrate that BjSa-SM promotes apple rhizo- a Results generated in Explicet v. 2.10.5 using the Two-Part Test at the biomes with increased potential for a combination of beneficial traits genus-level (p ≤ 0.05). including biocontrol and biodegradation/bioremediation. b Fold change refers to the ratio of the mean relative abundance of taxa In comparison, notable fungal taxa identified by the t-test as being “enriched” in one genotype with respect the other. specifically associated with B. napus but not BjSa SM-structured rhizo- + spheres included Trichoderma and the oleaginous yeasts Cryptococcus living Rhizobium excrete a large portion of fixed2 N gas as NH4 (O'Gara and Trichosporon (Fig. S4B). Proliferation of native populations of and Shanmugam, 1976). In G.210, bacterial taxa typically involved in Trichoderma/Hypocrea spp. typically occurs in B. juncea SM-amended nitrogen cycling belonging to the order Rhizobiales including Rhodo- orchard soils (Wang and Mazzola, 2019b; Weerakoon et al., 2012). planes spp. (denitrifying bacteria) and Methylosinus spp. (methanotroph, − These fungi become less prominent elements of the community as soil NO2 and N2O production) (Yoshinari, 1985) were identified as being AITC concentrations are reduced over time (Mazzola et al., 2015). significantly enriched relative to M.26 (Table 3). However, B. napus-mediated stimulation of Trichoderma is unrelated to the release of AITC. Cryptococcus was relatively high-ranking in all samples (1–4%NTC and 1–8%BjSa), but became overwhelmingly domi- 3.7. Higher fungal loads in the susceptible rootstock genotype nant in B. napus SM-amended samples. B. napus SM has been used as a nutrient supplement for the large-scale production of microbial oil from Two-factor ANOVA was used in conjunction with Tukey's multiple oleaginous yeasts including Cryptococcus spp. (Leiva-Candia et al., comparisons test to determine how fungal density was affected by 2015). In addition, yeasts are often highly efficient sporulators which treatment and genotype (normality tests and QQ plots confirmed the may result in an over representation of this group in populations de- assumption that residuals were sampled from a Gaussian distribution). scribed using DNA-based methods. This highlights a shortcoming of the The effect of treatment was only statistically significant in M.26, with amplicon sequencing approach: the assumption that there is a corre- root-associated fungal DNA higher in all treatments relative to the lation between numbers of sequence reads and the mycelial biomass pasteurized control (data not shown). The null hypothesis of no inter- present in the samples. action between treatment and genotype could not be rejected (two-way ANOVA; p = 0.06), indicating that differences between the treatments 3.6. Potentially beneficial cultivar-specific components of the apple were consistent for G.210 and M.26. However, total fungal loads were rhizobiome significantly higher in the roots of M.26 relative to G.210 forBjSa, BjHigh, and BjLow SM treatments (p = 0.01 for all treatments; Fig. 4). To further explore rootstock genotype-specific differences in bacterial community composition as shown along PC2 in Fig. 3D (16.7% of the variation), the Two-Part Test was used to identify taxa that were significantly different between all G.210 and M.26 samples (G.210 n = 14; M.26 n = 15). At the genus-level, Novosphingobium spp. and Pseudomonas spp. were both identified as being significantly enriched in M.26 regardless of soil treatment (Table 3). Pseudomonas spp. are not only known for their ability to improve iron and phosphorous bioavailability (Mosa et al., 2016) but also for their antagonism against specific ARD pathogens (Gu and Mazzola, 2003; Mazzola, 1999). However, the biocontrol capacity of the resident fluorescent pseudomonad population is highly dependent on strain-level diversity and the expression of particular biosynthetic gene clusters contained therein (e.g. 2,4-diacetylphloroglucinol production) (Mazzola et al., 2004). In this study, there was no effect of treatment on Pseudomonad community Fig. 4. Total fungal DNA detected in apple root tissue by qPCR according to structure within M.26, indicating that Pseudomonads are unlikely to be treatment and genotype. Treatments designated with an asterisk differ sig- enriched or reduced relative to NTC in soils amended with Brassicaceae nificantly in fungal DNA content between genotypes (Mann-Whitney Test, SM, a finding which is consistent with a number of other studies p < 0.05). Box-and-whisker plots show the minimum, the 25th percentile, the (Madhavi Gopireddy et al., 2019; Mazzola et al., 2015; Wang and median, the 75th percentile, and the maximum values. NTC = no treatment Mazzola, 2019a). In addition, many Rhizobium spp. were also sig- control, B.j. Low = 2.2 t ha−1 Brassica juncea seed meal (SM), B.j. −1 −1 nificantly enriched in M.26. Rhizobium do not induce formation of no- High = 4.4 t ha Brassica juncea SM, B.j. + S.a. = 4.4 t ha B. −1 dules on apples; however, there is evidence that nodulation factors can juncea + Sinapis alba SM (1:1), and B. napus = 4.4 t ha Brassica napus SM, be perceived by nonhost plants (Staehelin et al., 1994) and that free- Past = pasteurized control.

9 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766

A. B.

10 1

spp. B.napus spp. DNA BjHigh a a 0.1 BjLow a a 1 ab ab BjSa ab b b NTC 0.01 Past relative abundance (%)

0.1 (normalized to DNA input) Ilyonectria/Cylindrocarpon 0.001 G.210 M.26 G210 M26 Treatment Ilyonectria/Cylindrocarpon Treatment

Fig. 5. Abundance of Ilyonectria/Cylindrocarpon spp. in rhizosphere soil of G.210 and M.26 apple rootstocks. Mean relative (A) and mean absolute (B) abundance of Ilyonectria/Cylindrocarpon spp. in apple root tissue detected by ITS amplicon sequencing and qPCR, respectively. Treatments designated with the same letter do not differ significantly. Error bars represent the standard deviation among replicates. In both A and B, Two-factor ANOVA was used in conjunction with Tukey'smultiple comparisons test on log transformed data. NTC = no treatment control, BjLow = 2.2 t ha−1 Brassica juncea SM, BjHigh = 4.4 t ha−1 Brassica juncea SM, BjSa = 4.4 t ha−1 B. juncea + Sinapis alba SM (1:1), and B. napus = 4.4 t ha−1 Brassica napus SM, Past = pasteurized control.

Although not significant, fungal loads showed similar trends forNTC previously, the Geneva rootstocks are generally more tolerant to ARD and B. napus treatments. Total fungal loads were almost identical be- than rootstocks in the Malling series. This result is consistent with a tween the two genotypes in the pasteurized control treatments, sug- study in which the first order roots of M.26 contained higher con- gesting that higher fungal loads associated with the susceptible root- centrations of Ilyonectria/Cylindrocarpon spp. than those of G.210 at the stock may be reflective of higher levels of pathogen establishment inthe final harvest (Emmett et al., 2014). roots, rather than rootstock-specific differences in total fungal carrying These two apple rootstock genotypes also reacted differently to in- capacity. fection by Ilyonectria/Cylindrocarpon spp. depending on the treatment; Exposure to AITC has been shown to restrict hyphal growth of both significant differences were detected between means for Pasteurized vs. R. solani AG-5 and Cylindrocarpon destructans (Wang and Mazzola, BjHigh SM (p = 0.03), and Pasteurized vs. NTC (p = 0.01) in M.26. 2019b). In another study, relative abundance of Ilyonectria robusta/ Thus, Ilyonectria/Cylindrocarpon populations appear to have been sti- Cylindrocarpon was significantly reduced in the rhizosphere of Gala/ mulated by BjHigh SM in M.26 but not in G.210. Higher root loads of M.26 and Gala/G.41 trees cultivated in BjSa SM treated soil relative to this fungal group in M.26 × BjHigh SM and NTC treatments may have the no treatment control at the end of the second growing season (Wang partly contributed to the observed reduced trunk diameter in BjHigh and Mazzola, 2019a). In the current study, the mean relative abundance SM treatment relative to the “effective” BjSa SM treatment (Fig. 2B). It of Ilyonectria/Cylindrocarpon spp. in the rhizosphere was only sig- is also worth noting that in the pasteurized control treatments, mean nificantly reduced in BjSa SM amended soils as compared to NTCwhen abundance of Ilyonectria/Cylindrocarpon spp. was 5× higher in M.26 G.210 was used as the rootstock (Fig. 5A). A significant negative cor- than in G.210 roots (Fig. 5B), raising the possibility that this pathogen relation was identified between trunk diameter and the relative abun- was elevated in M.26 rootstocks prior to planting. dance of Ilyonectria/Cylindrocarpon spp. (primarily I. macrodidyma: Melt curve analysis for specific amplification of R. solani DNA in- 0.15%, I. robusta: 0.09%, and an unidentified species: 0.34%) in NTC dicated that R. solani was present at detectable levels in 4/5 and 2/5 treatments (but not BjSa or B. napus SM), when data for both genotypes pasteurized control samples for M.26 and G.210, respectively. R. solani was included (Spearman correlation: r = −0.78, p = 0.01, n = 9). was only detected in 1 G.210 × NTC sample. The higher proportion of This result suggests that Ilyonectria/Cylindrocarpon was an active com- R. solani-positive samples in pasteurized control soils coupled with the ponent of the pathogen complex in the unamended GC replant soil used failure to detect R. solani in root samples grown in GC soil suggests that in this experiment. this fungus may have been present in rootstocks, but did not become The relative abundance of R. solani present in rhizosphere soil was established under the experimental conditions. Competitive associa- significantly higher in BjSa SM treatments relative to the NTC (Kruskal- tions between pathogens may shift the disease complex towards those Wallis, Dunn's multiple comparisons test: p = 0.009). However, R. that are better adapted to the current conditions. One caveat of both of solani (anastomosis group unknown) relative abundance was fairly low these approaches (ITS amplicon sequencing and qPCR) is that the regardless of treatment (0.01–0.06%), indicating that R. solani was presence of the virulent anastomosis groups (AG-5 and AG-6) cannot be unlikely to be a major source of inoculum for the root and a less im- confirmed. Therefore, it is possible that the species detected mayhave portant component of the replant pathogen complex in this particular consisted of non-pathogenic strains. In addition, although q-PCR am- study. plification is one of the best available tools to quantify microbial targets, it is not a perfect proxy for the absolute number of specific gene 3.8. Apple rootstock genotypes react differently to infection by Ilyonectria/ targets present in a sample. Detection depends on many things in- Cylindrocarpon spp. depending on treatment cluding the quality and quantity of the DNA as well as the sensitivity of the primer pair to inhibitory substances. Soil and root samples com- Absolute abundance of fungal pathogens present in root tissue was monly contain inhibitory substances such as phenolics and humic acids also assessed in all 6 treatments using qPCR. Results for Ilyonectria/ which can be co-extracted with nucleic acids and lead to variability. Cylindrocarpon spp. indicated the null hypothesis of no interaction be- One way around this is to dilute the DNA; however, this can reduce tween treatment and genotype could not be rejected (two-way ANOVA; reaction effectiveness and the ability to detect genes found in relatively p = 0.31; Fig. 5B); however, the effects of treatment and of genotype low abundance. were statistically significant (p = 0.0035 and p = 0.0003, respectively). M.26 consistently harbored significantly higher populations of Ilyonectria/Cylindrocarpon spp. than the G.210 cultivar. As stated

10 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766

Fig. 6. Oomycete relative abundance in the apple rhizosphere as affected by soil treatment and rootstock genotype. Oomycete taxa representing ≥1% in relative abundance of the entire community in any treatment are included and are shown by their re- spective color. All other oomycete taxa are shown in grey. BjHigh = Brassica juncea seed meal (SM) soil amendment (4.4 t ha−1), BjSa = B. juncea + Sinapis alba SM (4.4 t ha−1), Can = Brassica napus SM (4.4 t ha−1), NTC = no treatment control.

3.9. SM-type and apple rootstock genotype jointly shape oomycete (Peronosporales) in BjSa SM treatments was driven by different species community structure depending on genotype. In M.26, this increase was driven by

Phytophthora cactorum (42.4%BjSa vs. 4.6%NTC), a primary agent of As stated above, B. napus and S. alba SM amendments have been Phytophthora crown rot of apple as well as a causal agent of apple re- shown to stimulate Pythium. In contrast, Pythium species are known to plant disease. In G.210, P. cactorum was not enriched relative to the be sensitive to AITC and B. juncea SM-amended soil is particularly ef- NTC (1.1%BjSa vs. 1.8%NTC), although increases in the relative abun- fective at suppressing apple root infection by Pythium but not dances of Phytophthora pseudosyringae (2.0%BjSa vs. 0.2%NTC), and an Phytophthora spp. (Weerakoon et al., 2012). Therefore, in addition to unidentified Phytophthora (1.2%BjSa vs. 0.01%NTC) were observed in BjSa SM, B. napus SM and NTC treatments, oomycete amplicon libraries G.210 × BjSa treatments. P. pseudosyringae has been reported to cause were also generated for BjHigh SM treatments. Apple rootstock rhizo- fruit rot in apple in controlled inoculations and root and collar rot on sphere oomycete assemblages represented 60 different species from 5 certain trees within the beech family (Jung et al., 2003). However, orders: Lagenidiales, Leptomitales, Peronosporales, Pythiales and Sapro- these oomycetes are not considered to be root pathogens of apple and legniales (Fig. S6). Regardless of treatment, members of the order Py- their function within the rhizosphere habitats of apple remains un- thiales largely dominated with Pythium species composing between 35 known. and 99% of the total oomycete reads depending on the sample, a result The increase in relative abundance of Peronosporales in which is consistent with other oomycete community studies of rhizo- G.210 × BjSa was primarily due to elevated level of Peronospora de- sphere soil (Arcate et al., 2006). In total, the rhizosphere oomycete structor (10.6%BjSa vs. 0.01%NTC). P. destructor is known to be a foliar community was composed of 28 and 15 different Pythium and Phy- pathogen of onion and has never been reported on apple (Hildebrand tophthora species in this study, respectively. and Sutton, 1984). It is also noteworthy that several Peronosporales are As stated above, significant differences in oomycete community parasitic on Brassicaceae and it is possible that colonization of SM structure were identified between all three SM treatments relative to substrates in soil may have been a factor related to increase of Per- the NTC. Oomycete community assemblages were not separated sig- onospora destructor in the rhizosphere of G.210. The rootstock-specific nificantly by seed meal type; however, taxa characterizing specific SM- changes in the diversity of Peronosporales in BjSa-structured rhizo- treatments were identified using the Two Part Test. At the genus level, spheres may be explained by prior selection of G.210 for resistance to P. Phytophthora was significantly enriched in BjSa relative to B. napus SM cactorum in the rootstock breeding process (Cummins et al., 2013). while Pythium was significantly enriched in B. napus relative to BjSa SM. Regardless of treatment, the M.26 rhizosphere supported higher levels In both rootstock genotypes, the mean relative abundance of Pythium of Phytophthora cactorum relative to G.210. Thus, it is likely that BjSa species in the rhizosphere was highest in B. napus SM treatments and SM stimulated P. cactorum levels in soil, but rootstock effects led to lowest in BjSa SM treatments. This result is in accordance with previous pathogen control in the G.210 rhizosphere. In a previous field study SM-based studies (Cohen et al., 2005; Mazzola, 2009; Mazzola et al., conducted in a different replant orchard soil, B. juncea-amended SM 2007). At the species level, P. echinulatum was more strongly associated was found to stimulate root infection of “Gala”/M.26 apple trees by with B. napus SM while the relative abundance of P. acanthicum, a Phytophthora cambivora (Mazzola and Brown, 2010). Although P. cam- mycoparasite of other Pythium species (Ali-Shtayeh and Saleh, 1999), bivora was not identified in any of the rhizosphere samples collected was consistently higher in B. juncea-containing SM treatments (as in- from this study, these findings also illustrate how SM-induced disease dicated by the Two-Part Test). control is influenced by apple rootstock genotype and underscore the The increase in the relative abundance of Phytophthora spp. importance of rootstock genotype in these disease control systems.

11 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766

Fig. 6 highlights the complexity of the rootstock × seed meal in- Absolute abundance of the ARD-specific oomycete pathogens P. teraction on oomycete community structure. These results may reflect cactorum and P. ultimum present in fine root tissue was also assessed in differences in rootstock-related tolerance to individual Pythium species all treatments using qPCR. P. cactorum was present at detectable levels as well as differential sensitivity to isothiocyanates derived from sini- in only 18/58 samples tested (13 in M.26; 5 in G.210). Interestingly, P. grin (e.g. B. juncea) and/or sinalbin-containing Brassica species cactorum was detected in 5/5 and 0/5 of M.26 and G.210 plants grown (Cordero et al., 2019; Wang and Mazzola, 2019b). For example, it was in pasteurized soil, respectively. These results indicate a higher degree previously observed that growth of P. heterothallicum was significantly of susceptibility of M.26 to P. cactorum. The number of root samples lower than that observed for P. attrantheridium and P. irregulare after testing positive for P. cactorum in NTC and SM-based systems was too exposure to AITC (Mazzola, 2009). low to draw further conclusions. In comparison, P. ultimum was de- Although > 40% of the rhizosphere-associated oomycete commu- tected in only 12 root tissue samples (6 in M.26; 6 in G.210), none of nity consisted of Phytophthora cactorum in the BjSa × M.26 system, the which were pasteurized controls. Although both of these pathogens high relative abundance of this oomycete did not appear to impact the were detected in rhizosphere soil, it is important to note that Pythium resulting growth characteristics of M.26 rootstocks during the experi- infected root hairs and fine root tissue is rapidly degraded and sus- mental period utilized in this study. For example, in M.26 BjSa SM ceptible to tissue loss during plant harvest. Such an outcome may have resulted in a significant increase in trunk diameter relative to BjHigh contributed to reduced detection levels of the pathogen. SM even though P. cactorum relative abundance was 4× lower in BjHigh treatments (Figs. 2B, 6). As mentioned above, rhizosphere relative abundance is not necessarily a good proxy for root or crown in- 4. Conclusions fection that limits plant growth. In addition, the short duration of the growth period as well as the environmental conditions may help ex- The rhizosphere microbiome represents one of the most dynamic plain why more significant growth limitation was not observed in the and complex environmental niches on the planet, and establishing re- M.26 × BjSa SM system. Disease development in response to this pa- lationships between multiple microbial communities, disease control, thogen generally occurs in poorly drained soils after prolonged and plant productivity is a difficult task. This study is the first time flooding, conditions that were not realized in this study. M.26 hasbeen bacterial, fungal, and oomycete communities in the rhizosphere have shown to be highly susceptible to root infection by native populations been simultaneously examined as influenced by different Brassica seed of Pythium, whereas Geneva series rootstocks are generally less sus- meals in the same soil/plant system. In accordance with other studies ceptible (Mazzola, 2009). The larger trunk diameter achieved with mentioned above, seed-meal amendment type is a major structuring M.26 × BjSa SM relative to M.26 × BjHigh SM may have been driven force broadly affecting the composition and diversity of all of these by the observed suppression in relative abundance of Pythium spp. In microbial components in the apple rhizosphere. addition, M.26 × BjSa SM treatments were shown to have lower ab- The effect of plant genotype on rhizobacterial community compo- solute abundances of Ilyonectria/Cylindrocarpon in the roots than sition was also clearly demonstrated in this study. The results suggested M.26 × BjHigh (Fig. 5). Previous studies have documented synergistic that even in SM-amended systems different apple cultivars selectively interactions between Pythium and Cylindrocarpon in inciting infection of recruit and promote the growth of unique microbes on their roots to apple (Braun, 1991; Tewoldemedhin et al., 2011b). some extent. Interestingly, many of these taxa were associated with the In many systems, the oomycete community was dominated by a ability to improve nutrient availability. single pathogenic species (e.g. P. cactorum in M.26 × BjSa SM, P. het- Amplicon sequencing provided a much more detailed picture of the erothallicum in NTC treatments, and P. irregulare in G.210 × BjHigh genetic diversity of specific oomycete groups than previously appre- SM). An unknown Pythium sp. was the most dominant member of the ciated. Regardless of treatment type, the susceptible rootstock harbored oomycete community in NTC soil regardless of rootstock genotype, as higher levels of fungal and oomycete species with the potential to infect well as the G.210 × B. napus and M.26 × BjHigh SM treatments. The apple roots. In many ways, the collective potential of such a diverse relative abundance of this Pythium sp. was significantly correlated with oomycete complex to influence disease severity still remains unknown. reduced trunk diameter of M.26 but not G.210 when data from all 4 soil Further oomycete-specific community level studies including research treatments were analyzed together (Pearson: r = −0.63, p- on the potential for synergism/antagonism among potentially patho- value = 0.006, n = 17). Thus, differential rootstock susceptibility to genic and non-pathogenic oomycetes is likely to improve our under- this unknown Pythium species may be another factor related to why standing of replant disease as well as many other soil-borne diseases. BjHigh was “effective” in G.210 relative to the NTC but “ineffective” in Finally, this study added to the body of evidence indicating that in M.26, emphasizing the concerted action of multiple fungal and oomy- addition to the control of replant disease pathogens, BjSa SM-structured cete pathogens in the dynamics of apple replant disease. microbiomes may contribute to long-term orchard health in many other When all samples were included, a nearly perfect inverse correlation ways. A number of potentially beneficial bacterial (e.g. Bacillus, was identified between the relative abundance of Pythiales and Sphingomonas) and fungal taxa (e.g. Arthrobotrys, Humicola) were sig- Phytophthora (Spearman rank correlation; r = −0.909, nificantly enriched in both BjSa and B. napus SM. However, the “ef- p = 2.7 × 10−13, n = 33 XY pairs), indicating that the population fective” SM (BjSa) amplified a greater diversity of taxa with potential dynamics of these two dominant oomycete groups are tightly linked ecosystem benefits including nutrient cycling, biodegradation and and likely to be antagonistic. This finding is not entirely unexpected, bioremediation. These attributes of Brassica SM- structured orchard soil considering that Pythium and Phytophthora are competing for the same microbiomes are relatively underappreciated and may have important host. Contrasting seasonal patterns have been shown to influence the implications for agricultural sustainability. Future work will include the relative dominance of Rhizoctonia vs. Pythium spp. (Mazzola et al., use of metagenomics to examine the functional genes present in these 2020). Replacement of ARD-specific pathogenic oomycetes (e.g. P. seed meal × genotype disease control systems. The integration of heterothallicum, P. irregulare, P. ultimum, P. cactorum) with those having multiple NGS data sets from different orchard locations will also be a reduced potential to infect apple roots and incite replant disease (e.g. important for gaining a more robust understanding of the taxonomic Peronospora destructor, P. acanthicum) is likely to help reduce ARD se- and functional patterns which consistently develop in the rhizosphere verity. However, many oomycete species have been shown to limit when apple trees are cultivated in BjSa SM amended soil. growth of apple and the significance of replacing one species with an- Supplementary data to this article can be found online at https:// other is often unclear (Mazzola et al., 2002). This study clearly high- doi.org/10.1016/j.apsoil.2020.103766. lights the need for more detailed knowledge of the oomycete diversity when studying disease control.

12 T.S. Somera, et al. Applied Soil Ecology 157 (2021) 103766

Data statement Rhizoctonia root rot. Soil Biol. Biochem. 37, 1215–1227. https://doi.org/10.1016/j. soilbio.2004.11.027. Cordero, F.T.A., Rodríguez-Arcos, R., Jiménez-Araujo, A., Guillén-Bejarano, R., Basallote, The datasets generated and analyzed during the current study are M.J., Barrau, C., 2019. Inhibitory effect of the glucosinolate–myrosinase system on available in the Figshare repository (doi:https://doi.org/10.6084/m9. Phytophthora cinnamomi and Pythium spiculum. Plant Prot. Sci. 55 (2019), 93–101. figshare.12269303.v1) and include unedited 16S rRNA and ITS (fungi https://doi.org/10.17221/98/2018-PPS. Cummins, J., Aldwinckle, H., Robinson, T., Fazio, G., 2013. Apple Tree Rootstock Named and oomycete) OTU read count tables as well as rootstock growth data. ‘G.210.’ USPP23337P3. Any other data generated or analyzed during this study are included in Di Pietro, A., Gut-Rella, M., Pachlatko, J., Schwinn, F., 1992. 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