# Soil pH influences the structure of virus communities at local and global scales

Sungeun Lee, Jackson Sorensen, Robin Walker, Joanne Emerson, Graeme W. Nicol, Christina Hazard

► To cite this version:

Sungeun Lee, Jackson Sorensen, Robin Walker, Joanne Emerson, Graeme W. Nicol, et al., Soil pH influences the structure of virus communities at local and global scales. Soil Biology and Biochemistry, 2022, 166 (2), pp.108569. 10.1016/j.soilbio.2022.108569. hal-04075705

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1 Article type: Short Communication

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3 Soil pH influences the structure of virus communities at local and global scales

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5 Sungeun Lee¹, Jackson W. Sorensen², Robin L. Walker³, Joanne B. Emerson²,

6 Graeme W. Nicol¹, Christina Hazard¹

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8 1. Environmental Microbial Genomics Group, Laboratoire Ampère, École Centrale de

9 Lyon, CNRS UMR 5005, Université de Lyon, Écully, 69134, France

10 2. Department of Plant Pathology, University of California, Davis, One Shields Avenue,

11 Davis, 95616, California, USA

12 3. School of Rural Land Use, Scotland's Rural College, Craibstone Estate, Aberdeen,

13 AB21 9YA, Scotland, United Kingdom

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15 Corresponding author

16 Email: christina.hazard@ec-lyon.fr

17 Tel: +33 4 72 18 60 91

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27 Abstract

28 The influence of biotic and abiotic factors on viral communities across environmental
29 gradients in soil is relatively unknown. While soil pH strongly influences microbial
30 community structure, it is unclear whether there is a similar influence on soil viruses.

31 In this study, prokaryotic and viral communities were characterized in soils from a long-
32 term pH-manipulated soil gradient (pH 4.5 and 7.5), and viral populations also
33 compared to those of other soils ranging in pH (4.0-7.5). Viral communities were
34 significantly influenced by pH at the local scale with 99% of viral operational taxonomic
35 units restricted to pH 4.5 or 7.5 soil only. Analysis of viromes from six other European
36 and North American soil systems demonstrated that a selection of viral clusters from
37 acidic and neutral pH soils were more associated with those from the local gradient
38 pH 4.5 or 7.5 soils, respectively. While direct pH effects on virion integrity and indirect
39 selection via host composition were not distinguished, the results reveal that soil pH
40 is a factor in structuring viral communities at local and global scales.

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42 key words: viruses; prokaryotes; community structure; pH

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## Main

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Viruses play a major role in controlling the abundance, structure and evolution of microbial communities through cell lysis and the release of nutrients and modulation of host cell metabolism (Pratama et al., 2018). In soils, viruses are diverse (Emerson et al., 2018), abundant (Williamson et al., 2017), and have potential to impact nutrient cycling and other ecosystem functions (Emerson et al., 2018; Kuzyakov and Mason-Jones, 2018; Trubl et al., 2018). However, fundamental knowledge gaps in soil viral ecology include a basic understanding of the biotic and abiotic drivers of viral communities.

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Soil bacterial and viral community dynamics co-vary, with the susceptibility of hosts to infection from individual viruses varying over time (Vos et al., 2009) or viral community shifts occurring as an indirect result of nutrient input altering host community structure (Srinivasiah et al., 2015). While host communities are likely the strongest factor for defining viral community structures, the host range of viruses may also play a role in defining whether their community dynamics vary to the same extent over physicochemical gradients (de Jonge et al., 2019). Closely related host populations may be adapted to growth under different conditions, resulting in niche differentiation and contrasting distribution across an ecological gradient. However, it is unclear whether narrow or broad host ranges of their associated viruses reduce the relative variation in virus community structure compared to prokaryotes. In addition, changes in soil physicochemical characteristics may also directly impact the physical integrity and dispersal of viruses (Trubl et al., 2016). As soil pH is a major determinant of prokaryotic community composition at local and global scales (e.g. Bahram et al., 2018; Bartram et al., 2014; Griffiths et al., 2011; Lammel et al., 2018; Lauber et al.,
76 2009), it is possible that virus communities may also exhibit pH-influenced community
77 structures.

78 Soil samples were taken from the extreme ends of an extensively characterized
79 contiguous soil pH gradient (pH 4.5 to 7.5) where total carbon, nitrogen and organic
80 matter do not change significantly across the gradient (Nicol et al., 2008; Bartram et
81 al., 2014). At this site, prokaryotic communities have been shown to vary in response
82 to pH (Nicol et al., 2008; Bartram et al., 2014), and distinct virus populations infecting
83 methylotrophic communities associated with contrasting soil pH have recently been
84 observed (Lee et al., 2021). Both non-targeted total community metagenome and
85 virus-targeted virome libraries were prepared as previously described (Lee et al.,
86 2021), and the assembled predicted viral contigs obtained were clustered into 1,910
87 viral operational taxonomic units (vOTUs) (Table S1), and prokaryotic 16S rRNA gene
88 fragments were extracted and classified into 2,312 OTUs (Supplementary Methods).
89 Sequencing depth was sufficient to capture vOTU richness, although further
90 sequencing and sampling may have increased 16S rRNA OTU recovery (Fig. S1).

91 Of the metagenomic reads, 22.7% were taxonomically defined, with
92 Actinobacteria and Proteobacteria dominating in both pH soils (Fig. 1a). Similar to
93 other soil viral studies, only a small proportion of the prokaryotic viral community (7.0%
94 of vOTUs) was taxonomically defined. A total of 29.1% of vOTUs were linked to
95 predicted hosts, with the majority for both pH soils belonging to Actinobacteria and
96 Proteobacteria (Fig. 1a, Table S2).

97 Analysis of individual 16S rRNA OTUs and vOTUs demonstrated distinct
98 structures between pH 4.5 and 7.5 soils for both prokaryotic and viral communities
99 (Fig. 1b). Specifically, 38.6% of OTUs (pH 4.5 OTUs, 263; pH 7.5 OTUs, 630) and
100 99.0% of the vOTUs (pH 4.5 vOTUs, 524; pH 7.5 vOTUs, 1,361) were found in only
one soil pH. Prokaryote and viral (virome) alpha-diversity were significantly greater in pH 7.5 soil (Table S3). Although absorption of viruses to soil organic particles can decrease with increasing pH (Sobsey et al., 1980), the effect of soil pH biasing measurements of alpha-diversity may be small as organic matter content does not change significantly across the gradient and a neutral pH buffer was used for extracting viral particles from both soils, with no significant difference in the yields of virome DNA.

While viromes produced 73x more assembled viral contigs than metagenomes, read mapping of individual reads to all vOTUs demonstrated that both approaches produced distinct viral community profiles between soils (Fig. 1b). Decreasing the breadth (length of contig covered by mapped reads) threshold (<75%) for defining vOTU detection disproportionately increased vOTU detection in metagenomes compared to viromes (Fig. S2). This suggests that the appropriate breadth thresholds for detection may be different in viromes compared to total metagenomes, with a cut-off ≥75% potentially too conservative for the total metagenomes. However, care should be taken before reducing breadth threshold in other datasets that do not have paired viromes to corroborate viral detection.

Soil viromes from the gradient soils were compared with those from six other ecosystems varying in pH, soil type, land use and location and where viral contigs were predicted using the same tools and standards (Table S4) (Emerson et al., 2018; ter Horst et al., 2021; Santos-Medellin et al., 2021). Using gene-sharing network analysis (Jang et al., 2019), the number of clusters containing vOTUs from these additional soils and the local gradient (pH 4.5 only, pH 7.5 only or both) were determined (Fig. 2, Table S5). On average, 31% of clusters were shared (range 1-62%) in pairwise comparisons between all soils (Fig. S3). A clear trend was also
observed in comparison with the gradient soils (Fig. 2b) with shared viral clusters in
acidic and neutral pH soils more associated with those from the local gradient pH 4.5
or 7.5 soils, respectively. While this demonstrates that certain viral genes were
restricted to acidic vs neutral pH soils, no candidate genes were identified as
potentially conferring host adaptation to a particular pH range, with the majority
encoding uncharacterized hypothetical proteins. In addition, the relative proportion of
virus-encoded putative auxiliary metabolic genes for different metabolic functions was
similar between soils with no trends associated with pH (Fig. S4).

If individual viruses can infect multiple host populations at different soil pH,
there would be a potential for virus community structures to be less distinct over an
ecological gradient. However, analysis of samples from a continuous pH gradient
demonstrated that contrasting soil pH results in the selection for virus community
structures that are at least as distinct as prokaryote host community structures. While
a relatively limited number of sites for analysis of viromes in different soil systems and
that these analyses do not separate direct effects of pH on virion integrity from indirect
effects of host composition, they demonstrate that, as with prokaryote communities,
soil pH correlates with distinct patterns of virus community structures.

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144 Data availability

Metagenome sequence reads are deposited in NCBI's GenBank under BioProject
accession nos. PRJNA621436-PRJNA621447. Metagenome draft assemblies are
accessible through the JGI Genome Portal (DOI: 10.25585/1487501). Assembled
metagenome-derived 16S rRNA gene sequences are available at ftp://ftp-adn.ec-
lyon.fr/. Metagenome sequence reads from 'Agriculture 2' site are available through
NCBI BioProject PRJNA767554.
151 Acknowledgments

152 This work was funded by an AXA Research Chair awarded to GWN. The sequencing
153 data were generated under JGI Community Science Program proposal 503702
154 awarded to GWN and CH. The sequencing data from the soil pH gradient was
155 produced by the U.S. Department of Energy Joint Genome Institute, a DOE Office of
156 Science User Facility, which is supported by the Office of Science of the U.S.
157 Department of Energy under Contract No. DE-AC02-05CH11231. The pH gradient
158 experiment is funded through the Scottish Government RESAS 2016-2021 program.
159 The authors would like to thank Dr. Laurent Pouilloux for assistance with the Newton
160 high performance computing cluster at École Centrale de Lyon. 'Agriculture 2' site data
161 collection and analysis was predominantly funded by the UC Davis College of
162 Agricultural and Environmental Sciences and Department of Plant Pathology (new lab
163 start-up to JBE) and also in part by USDA National Institute of Food and Agriculture
164 (NIFA) Hatch project number CA-D-PPA-2464-H and USDA NIFA grant number 2021-
165 67013-34815-0 to JBE. The authors thank Dr. Laura Zinke for collecting and
166 processing the 'Agriculture 2' site samples.

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168 Competing Interests

169 The authors declare no competing interests.

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171 References

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273 **Figure Legends**

**Fig. 1:** Taxonomic composition and community structure of prokaryotic and virus communities in pH 4.5 and 7.5 replicate soil samples taken from the ends of a contiguous pH gradient. a) Relative abundance of taxonomically-defined prokaryotes, taxonomically-defined viruses and the predicted hosts of viruses. For prokaryotes, reads from metagenomes were annotated at the phylum level using the NCBI nr database. Numbers in parenthesis denote the number of mapped reads:total reads analyzed. Viral contigs ≥10 kb were taxonomically defined at the family level based on gene-sharing network analysis (Jang et al., 2019). Host prediction of viruses was determined by using the gene-sharing network, and gene homology analysis (Al-Shayeb et al., 2020). Numbers in parenthesis denote the number of annotated:total reads or contigs for each sample, and plots display the relative proportion of annotated reads only (i.e. annotated reads of prokaryotes or reads mapped to annotated viral contigs). b) Normalized relative abundance of individual 16S rRNA OTUs and vOTUs in soil samples determined by read-mapping. Only vOTUs where reads were mapped with ≥1x coverage over 75% contig breadth were included. Ordinations show the principal coordinate analysis of Bray-Curtis dissimilarities derived from relative abundance tables. For virus communities, reads from both viromes (V) and metagenomes (M) were analyzed for each sample. Details of all methods used are provided in Supplementary Information.

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**Fig. 2:** Network analysis describing linkages of Gradient 4.5 and 7.5 vOTUs with six sets of soils samples ranging in pH from 4.0 to 7.5 from Europe and North America (Table S4). a) Gene sharing network of vOTUs showing viral clusters containing ≥25 vOTUs. b) Relative abundance of clusters from each soil that contain vOTUs shared
298 with those from gradient pH 4.5 (blue) and gradient pH 7.5 soil (red) or both (grey).

299 Numbers in parenthesis denote total number and percentage of shared viral clusters

300 with Gradient soils. Details of all methods used are provided in Supplementary

301 Information.
<figure><img src="image_1.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>Figure 2</figcaption></figure>