bioRxiv preprint doi: https://doi.org/10.1101/516575; this version posted January 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Fungal Communities in Soybean Cyst Nematode-Infested Soil under 2 Long Term Corn and Soybean Monoculture and Crop Rotation 3 4 Noah Strom1, Weiming Hu2, Deepak Haarith3, Senyu Chen4, Kathryn Bushley1* 5 6 1Department of Plant and Microbial Biology, University of Minnesota, Saint Paul, MN, USA; 7 2Entomology and Nematology Department, University of Florida, Gainesville, FL, USA; 8 3Department of Plant Pathology, University of Minnesota, Saint Paul, MN, USA. 9 4Southern Research and Outreach Center, University of Minnesota, Waseca, MN, USA; 10 *Correspondence: 11 Kathryn Bushley 12 [email protected] 13 14 Abstract 15 Corn (Zea mays) and soybean (Glycine max) production forms an integral part of economies 16 worldwide, but yields are limited by biotic and abiotic factors associated with short rotations and long- 17 term monocultures. In this study, a long-term rotation study site with corn and soybean planted in 18 annual rotation, five-year rotation, and long-term monoculture was utilized to examine the relationships 19 between crop sequences, soil fungal communities, soybean cyst nematode (SCN, Heterodera glycines) 20 densities, soil properties, and crop yields. High throughput sequencing of the ITS1 region of fungal 21 rDNA revealed that soil fungal community structure varied significantly by crop sequence, with fungal 22 communities under five consecutive years of monoculture becoming progressively similar to 23 communities in long-term monoculture plots associated with their respective crop hosts. Total alpha 24 diversity was greater under corn, but patterns of diversity and relative abundance of specific functional 25 groups differed by crop host, with more pathotrophs proliferating under soybean and more saprotrophs 26 and symbiotrophs proliferating under corn. Soil phosphorus (P) varied significantly by crop sequence, 27 with lower levels of P corresponding with relative abundance of Glomerales, Paraglomerales, and 28 Sebacinales and higher levels of P corresponding with relative abundance of Mortierellales. Soil 29 density of the SCN was positively correlated with relative abundance and diversity of nematode- 30 trapping fungi and with relative abundance of many potential nematode egg parasites. These results 31 suggest several possible explanations for the improved yields associated with crop rotation, including 32 decreased pathogen pressure, modification of soil properties, and increased diversity of soil fungal 33 communities. Future research should investigate the potential of nematode-trapping fungi to regulate 34 SCN densities and examine the relationships between soil P and specific arbuscular mycorrhizal and 35 mortierellalean fungi associated with corn and soybean hosts. bioRxiv preprint doi: https://doi.org/10.1101/516575; this version posted January 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 36 Keywords: Crop rotation, monoculture, soybean cyst nematode, nematophagous fungi, soil 37 phosphorus, mycorrhizae, metabarcoding 38 1. INTRODUCTION 39 Corn (Zea mays) and soybean (Glycine max) production forms an integral part of the economies of 40 multiple nations in both the eastern and western hemisphere (Hartman et al., 2011; Meade et al., 2016) 41 and plays a major role in the food security of developing nations (Hartman et al., 2011; Shiferaw et al., 42 2011). In the United States in 2016, corn and soybean accounted for 53% of total acreage planted to 43 principal crops and 49% of principal crop production (U.S. Department of Agriculture National 44 Agricultural Statistics Service, 2017). However, both crops suffer yield penalties under continuous 45 monoculture. Corn yield is thought to be limited primarily by levels of soil nitrogen (N), whereas the 46 main factor thought to limit soybean yield is disease pressure from plant pathogens, especially the 47 soybean cyst nematode (SCN, Heterodera glycines) (Gentry et al., 2013; Seifert et al., 2017). Other 48 soil properties, especially soil phosphorus (P), may be involved in yield declines of both crops (Bender 49 et al., 2015; Xin et al., 2017). Soil fungi, including arbuscular mycorrhizal fungi (AMF), interact with 50 plants, plant pathogens, and soil properties in complex ways that may affect crop yields (Francl and 51 Dropkin, 1985; Johnson et al., 1992; Tylka et al., 1991). 52 The SCN is responsible for the largest soybean yield declines, averaging 3.5 million metric tons 53 in yield losses in the U.S., annually (Koenning and Wrather, 2010). While crop rotation to corn is the 54 most widely adopted practice to reduce SCN populations, even five years of continuous corn cropping 55 cannot eliminate this pathogen (Porter et al., 2001). With few available sources of genetic resistance 56 to SCN (Liu et al., 2017) and the phasing out of chemical nematicides due to environmental and human 57 toxicity (Warnock et al., 2017), biocontrol of SCN using nematophagous fungi is being investigated as 58 an alternative to manage this pathogen (Chen and Dickson, 2012). Understanding how these fungi 59 respond to common corn-soybean crop sequences may help identify fungal taxa that could serve as 60 biocontrol agents. 61 Methods for characterizing soil fungal communities have undergone significant advances in 62 recent years. Early studies used denaturing gradient gel electrophoresis (DGGE) to demonstrate 63 seasonal shifts in bacterial and fungal communities in plant rhizospheres and to a lesser extent in bulk 64 soils (Gomes et al., 2003; Smalla et al., 2001; Smit et al., 2001), but this technique is known to 65 underestimate species diversity (Gomes et al., 2003). In the last decade, high throughput amplicon 66 sequencing has facilitated more complete characterization of agricultural soil microbial communities 67 than earlier culture-based and molecular approaches (Lindahl et al., 2013). Many of these studies have 68 demonstrated the effects of continuous monoculture cropping on the relative abundance of plant 69 pathogens (Bai et al., 2015; Liu et al., 2014b; Wu et al., 2016; Xiong et al., 2014), while others have 70 described the dynamics of beneficial microbes, including AMF (Martínez-García et al., 2015; Senés- 71 Guerrero and Schüßler, 2016) and antagonists of plant pathogens (Hamid et al., 2017; Xu et al., 2012), 72 including those with hypothesized roles in SCN-suppressive soils (Hu et al., 2017). High throughput 73 amplicon sequencing has also been used to examine the effects of tillage (Hu et al., 2017; Smith et al., 74 2016), organic farming practices (Hartmann et al., 2014), fertilizer use (Lin et al., 2012; Ramirez et al., 75 2010), crop rotation (Bainard et al., 2017), land-use, and soil properties (Lauber et al., 2008) on soil 76 microbial communities. 77 In addition to their effects on soil microbial communities, cropping sequences are known to 78 affect soil physical and chemical properties that could impact yield. For example, rotation of corn to 79 soybean has been shown to increase bioavailable N, which improves corn yield (Peterson and Varvel, bioRxiv preprint doi: https://doi.org/10.1101/516575; this version posted January 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 80 1989). Likewise, rotation of soybean to corn has been shown to improve soil aggregate stability and 81 water holding capacity and to increase soil levels of organic matter, P, K, Mg, and Ca, properties that 82 are associated with increased soybean yield (Perez-brandan et al., 2014). However, another study 83 found that soil P was higher under continuous soybean monoculture compared to continuous corn 84 monoculture at two sites in Minnesota, including our study site (Johnson et al., 1991). AMF, which 85 are canonically thought to enhance plant growth through the uptake and transport of soil P (Parniske, 86 2008), undergo community shifts related to corn-soybean crop sequences (Johnson et al., 1991). It has 87 been hypothesized that the AMF communities associated with corn and soybean becomes less 88 mutualistic under continuous monoculture of each host, potentially contributing to yield declines 89 (Johnson et al., 1992). 90 The population density of nematophagous fungi may also be affected by cropping sequences. 91 For example, the nematode-endoparasitic fungus, Hirsutella rhossiliensis, which is thought to have a 92 density-dependent relationship with SCN (Jaffee et al., 1992), was more commonly isolated from SCN 93 juveniles during soybean cropping years with higher SCN density (Chen and Reese, 1999). Likewise, 94 the SCN egg parasites, Metacordyceps chlamydosporia and Purpureocillium lilacinum, were shown to 95 increase in relative abundance in the soybean rhizosphere and in SCN cysts over continuous soybean 96 monoculture in fields in northeastern China (Hamid et al., 2017; Song et al., 2016a). Nematode- 97 trapping fungi Dactylellina ellipsospora (syn. Monacrosporium ellipsosporum) and Arthrobotrys 98 dactyloides (Syn: Drechslerella dactyloides) have been shown to have a density-dependent relationship 99 with the root knot nematode (Meloidogyne javanica) under greenhouse conditions