Accepted Manuscript

Temporal patterns of entomopathogenic nematodes in Florida citrus orchards:

Evidence of natural regulation by microorganisms and nematode competitors

Raquel Campos-Herrera, Robin J. Stuart, Ekta Pathak, Fahiem E. El-Borai, Larry W.

Duncan

PII: S0038-0717(18)30360-2
DOI: https://doi.org/10.1016/j.soilbio.2018.10.012
Reference: SBB 7316

To appear in: Soil Biology and Biochemistry

Received Date: 17 June 2018

Revised Date: 20 October 2018
Accepted Date: 20 October 2018

Please cite this article as: Campos-Herrera, R., Stuart, R.J., Pathak, E., El-Borai, F.E., Duncan, L.W.,
Temporal patterns of entomopathogenic nematodes in Florida citrus orchards: Evidence of natural
regulation by microorganisms and nematode competitors, Soil Biology and Biochemistry (2018), doi:
https://doi.org/10.1016/j.soilbio.2018.10.012.

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1 Temporal patterns of entomopathogenic nematodes in Florida citrus orchards: evidence of

2 natural regulation by microorganisms and nematode competitors

1,2,3,
4 Raquel Campos-Herrera Robin J. Stuart 4, Ekta Pathak 1,5, Fahiem E. El-Borai 1,6
, Larry W.

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5 Duncan 1*

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7 Citrus Research and Education Center (CREC), University of Florida (UF), 700 Experiment

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8 Station Road, Fl 33850, USA
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9 Instituto de Ciencias Agrarias, CSIC, Serrano 115 Dpdo, Madrid 28006, Spain

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10 Current address: Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja,

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Gobierno de La Rioja), Finca La Grajera, Carretera de Burgos km 6, 26007 Logroño, Spain
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12 DPI, Florida Department of Agriculture and Consumer Services, Dundee FL–33838 (USA)
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13 Sam Higginbottom Institute of Agriculture, Technology & Sciences, Allahabad, U.P., India
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14 Plant Protection Department, Faculty of Agriculture, Zagazig University, Egypt
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16 * Corresponding author: Larry W. Duncan (lwduncan@ufl.edu)

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17 Citrus Research and Education Center (CREC), University of Florida (UF), 700 Experiment

18 Station Road, Fl 33850, USA

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19 Phone: +1 863 9568821; Fax: +1 863 9564631

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21 To be submitted to:

22 Soil Biology and Biochemistry, IF 4.857 (Q1 in Soil Science, position 2/34)

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25 Abstract

26 The abundance of three entomopathogenic nematode (EPN) species, seven species of

27 nematophagous fungi, free living nematode competitors of EPNs and a bacterial ectoparasite of

28 EPNs were monitored during approximately two years in four Florida citrus orchards. The

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29 objective of the surveys was to identify natural enemies that potentially regulate the temporal

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30 abundance of naturally occurring EPNs by using molecular tools. Nematodes were extracted

31 from two soil depths at ~ monthly intervals, DNA extracted, and target organisms were measured

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32 using qPCR. Potentially causal relationships between the temporal patterns of EPNs and their

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33 natural enemies were assessed primarily with stepwise regression of variables at various time

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lags. Drechslerella dactyloides was the only nematophagous fungus species exhibiting evidence

of top-down regulation, being inversely related to population change of Heterorhabditis indica at

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36 two of three sites. Acrobeloides spp. (EPN competitors) were associated with reduced

37 population growth of Steinernema diaprepesi at two sites and H. indica was associated with
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38 reduced abundance of H. zealandica and S. diaprepesi at one site each. A Paenibacillus sp.
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39 exhibited significant phase-space, predator-prey dynamics at two of three sites with abundant S.

40 diaprepesi, a species-specific host of the bacterium. The initial abundance of both S. diaprepesi
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41 and Paenibacillus sp. explained significant variability in population changes of S. diaprepesi in a

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42 multiple regression model at a lag of three months. Controlled experiments showed that pH is
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43 directly related to adherence of the bacterial endospores to the S. diaprepesi cuticle. Soil pH in

44 these surveys was directly related to the infestation rate (encumbrance) by Paenibacillus and

45 inversely related to the abundance of S. diaprepesi. Vertical distribution patterns reflected

46 potential routes for avoiding competition and predation between species. Nematode-trapping

47 fungi tended to inhabit shallower soil horizons than did several other nematophagous fungal

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48 species and S. diaprepesi and H. indica. The nematophagous fungus Purpureocillium lilacinum

49 was especially abundant in deeper soil horizons and in the flatwoods sites. Depth distribution

50 among EPNs also differed significantly with H. indica > S. diaprepesi > than H. zealandica.

51 These results support soil pH management to conserve the demonstrated services of S.

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52 diaprepesi. They also suggest a need to better understand potential non-target effects of practices

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53 favorable to nematophagous fungi and bactivorous nematode competitors of EPNs.

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55 Key words: Entomopathogenic nematode, Nematophagous fungi, Nematode competitors, Non-

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56 target effects, Paenibacillus, Soil pH

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58 1. Introduction

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60 The possibility of exploiting services of native entomopathogenic nematodes (EPNs)

61 through conservation tactics was proposed to help manage a serious pest of citrus (Stuart et al.,

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62 2008; Campos-Herrera et al., 2010, 2015). Diaprepes abbreviatus is a polyphagous weevil pest

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63 that is widespread in the Caribbean Basin. The insect was first detected in Florida in the mid-

64 1960s and has since spread throughout the citrus industry there and in other localities such as

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65 Texas and California (Lapointe et al., 2007). The adult weevil feeds and oviposits on expanding

66 leaves. Upon hatching, neonate larvae fall to the soil where they develop, feeding on the cortex

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67 of progressively larger roots. Phytophthora spp. readily infect the extensive wounds on the root

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surface, resulting in a serious pest-disease complex (Graham et al., 2003). Moreover, the 2005

69 introduction in Florida of the root-destroying, bacterial disease huanglongbing exacerbated the

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70 challenge posed by D. abbreviatus (Duncan, 2017).

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71 Integrated management of the Diaprepes-Phytophthora complex in Florida employs

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72 insecticides and ovacides, rootstocks resistant to Phytophthora infection (Graham et al. 2003),

73 physical barriers (Duncan, et al., 2009; Bender et al., 2014) and EPNs. Florida growers have
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74 used commercial EPN products for root weevil control since the early 1990s (Dolinski et al.,

75 2012; Duncan, 2017). The initial research to employ commercially available EPNs as a weevil
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76 management tactic revealed that Florida citrus orchards support abundant, species-diverse
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77 communities of native EPNs (Duncan et al., 2003, 2007). Moreover, the infection rates of

78 sentinel weevil larvae by native EPNs varied in two different ecoregions in inverse proportion to

79 the population size of weevils and the attendant damage to citrus in those regions (Duncan et al.,

80 2003; Futch et al., 2005). The inverse relationship implied that discovery of biotic and/or abiotic

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81 soil conditions favoring native EPNs might reveal ways in which soils can be modified to

82 enhance natural biological control of root weevil pests (Campos-Herrera et al., 2015; Duncan et

83 al., 2015). Surveys of citrus orchards and adjacent natural areas which measured soil physical

84 properties and some of the organisms involved in EPN food webs revealed that EPN spatial

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85 patterns at a landscape scale were best explained by soil properties which affect soil water

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86 potential (e.g., groundwater depth, water-holding capacity, clay and organic matter content)

87 (Campos-Herrera et al., 2013, 2016; Pathak et al., 2017). Steinernema diaprepesi and

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88 Heterorhabditis zealandica were strongly associated with drier soil conditions typical of the deep

89 sands that characterize the central ridge ecoregion, whereas the recently named EPN

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90 Steinernema khuongii (Stock et al., 2018) was detected primarily in the shallow, wetter soils of

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the central flatwoods ecoregion. A fourth species, H. indica, was virtually ubiquitous in all citrus

92 orchards surveyed. Controlled studies confirmed that S. diaprepesi and S. khuongii are
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93 differentially adapted to drier and wetter habitats, respectively (El-Borai et al., 2016).
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94 Edaphic properties could also affect EPNs by modulating populations of natural

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95 antagonists. Several studies report density dependent regulation of nematodes by naturally

96 occurring NF in microcosms (e.g., Jaffee et al., 1992; Jaffee and Strong, 2005; El-Borai et al.,
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97 2007), but few studies have examined the role of naturally occurring NF on EPN population flux

98 in the field (Jaffee et al., 1989; Jaffee and Strong 2005). Although Campos-Herrera et al. (2013,
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99 2016) and Pathak et al. (2017) reported positive associations between population density of some
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100 nematophagous fungus species (NF) and EPNs, they found no evidence that these responses

101 varied by ecoregion. Thus, while NF can increase in response to EPN population growth (Jaffee

102 et al., 2003; Jaffee and Strong, 2005; Elborai et al., 2007), their population patterns did not

103 explain the EPN spatial patterns across regions. By contrast, a phoretic Paenibacillus sp.

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104 bacterium that adheres to the cuticle of S. diaprepesi, inhibiting the nematode’s motility (Enright

105 et al., 2003; El-Borai et al., 2005), was found to encumber the nematode more in experimental

106 field plots treated with a novel fertigation regime than in plots managed conventionally

107 (Campos-Herrera et al., 2013, 2014). The novel treatment also reduced the numbers of S.

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108 diaprepesi and increased the numbers of Diaprepes root weevils and the abundance of

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109 Phytophthora nicotianae. The intensive fertigation increased the soil water potential, pH and

110 levels of several nutrients, any of which may have affected the attachment of Paenibacillus sp. to

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111 the cuticle of S. diaprepesi and the abundance of the nematode. A major question is whether the

112 bacterial encumbrance or other factors that varied between intensive and conventional

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113 management affected the nematode spatial patterns in the field.

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Detecting EPN population regulation by natural enemies in nature is challenging for

115 several reasons. Limited motility of soil organisms means that population regulation is most
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116 likely to occur at the micro-level, as the infective juveniles (IJs) emerge in large numbers from
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117 insect cadavers (Jaffee et al., 2007). However, sequentially sampling large numbers of plots at a
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118 micro scale is difficult because of cost and the destructive nature of sampling (Spiridonov et al.,

119 2007; Steel and Ferris, 2016). Most field studies utilize soil samples comprising numerous
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120 composited soil cores from sample areas, hence, encompassing many heterogeneous micro-

121 populations. Moreover, many NF are non-specific predators of nematodes and are primarily
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122 saprophytic (Jaffee et al., 1993); therefore, population flux at the micro-level may have little
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123 effect on the nutrients available to NF at the field plot level. Density-dependent regulation should

124 be relatively easier to detect in species-specific antagonists of nematodes. For example, the

125 genus Pasteuria comprises numerous species-specific bacterial parasites of nematodes, some of

126 which have been reported to exhibit density-dependent regulation of Xiphinema

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127 diversicaudatum, Meloidogyne incognita and Tylenchulus semipenetrans in nature (Ciancio,

128 1995; Ciancio et al., 2016; Timper et al., 2016). Similarly, Paenibacillus sp. only adhered in

129 abundance to S. diaprepesi, with very little attachment to other steinernematids and no reported

130 attachment to nematodes in other genera including Heterorhabditis (El-Borai et al., 2005).

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131 To explore temporal relationships between EPNs, some antagonists of EPNs, and certain

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132 soil climatic and chemical properties, soil samples from beneath citrus trees were collected

133 monthly, from two depths, during 21 months at four sites and an additional 6 months at two of

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134 the sites. The sites were divided equally between the central ridge and the central flatwoods. The

135 objectives of the surveys were to 1) identify biotic or abiotic factors associated with EPN

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136 population flux, 2) identify variables associated with EPN natural enemy population size and

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flux, and 3) determine whether temporal patterns of Paenibacillus sp. and S. diaprepesi are

138 consistent with density-dependent regulation as suggested by Campos-Herrera et al. (2013).

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139 Additionally, the effect of pH on the attachment of Paenibacillus sp. endospores to the cuticle of
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140 infective juvenile S. diaprepesi was studied in the laboratory.

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142 2. Materials and Methods

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144 2.1 Sites, sampling and soil processing

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146 Temporal surveys were conducted in mature citrus orchards at four sites (Table 1). Two

147 sites were located on the Florida Central Ridge ecosystem, one at the UF-IFAS Citrus Research

148 and Education Center (henceforth referred to as ‘CREC’) and the other at the ‘North-40’ off-

149 campus, research orchard (henceforth ‘N40’) owned by CREC. The other two sites were located

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150 in the Central Flatwoods ecoregion in an orchard near Plant City, FL. One site within the orchard

151 was characterized by well-drained soil (henceforth ‘PCD’) and the other, lower elevation site, by

152 poorly drained soil (henceforth ‘PCW’). Soil texture was determined (Waters Agricultural

153 Laboratories, Camilla GA) and depth to groundwater was estimated (USDA, Natural Resource

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154 Conservation Service, https://websoilsurvey.nrcs.usda.gov/app/WebSoilSurvey.aspx)

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155 Ten trees at each site were selected for repeated (~monthly) sampling. On each sampling

156 date, three soil cores (30 cm depth; 2.5 cm dia.) were taken from widely separated points (>1 m)

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157 beneath the canopy of each tree. Each core was separated into two sub-cores based on soil depth:

158 shallow (0-15 cm) and deep (16-30 cm). Each sub-core was then combined with a corresponding

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159 sub-core from each of the other nine trees to produce a total of six composite samples. This

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process resulted in three shallow-horizon samples and three deep-horizon samples from each site

161 each month. Samples were processed the same day they were recovered.
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162 After weighing and then gently mixing soil in each sample, organisms were extracted
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163 differently from two aliquots. The total number of nematodes in 60 cm3 soil was estimated using
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164 modified Baermann funnels. On each date, 24 funnels were incubated at room temperature (22º–

165 25ºC) for 7 days, nematodes were recovered in test tubes, allowed to settle overnight at 4 °C,
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166 after which excess water was aspirated to a final volume of 25 ml. Nematodes in a 5 ml

167 subsample were counted. A second 500 g aliquot was employed to extract EPN and associated
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168 microorganisms to be enumerated by qPCR protocols (Campos-Herrera et al. 2011a, 2014).

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169 Briefly, soil organisms were extracted by sucrose centrifugation (Jenkins, 1964), decanted

170 overnight at 4 °C, transferred to 1.5 mL Eppendorf tubes and stored at –20 ºC until DNA

171 extraction (Campos–Herrera et al., 2011a). DNA in each nematode sample was extracted using

172 the UltraCleanTM Soil DNA Extraction Kit (MoBio) and quality and quantity of duplicate DNA

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173 samples were measured using the Nanodrop System 1000 v.3.3.0 (Thermo Scientific,

174 Wilmington, DE, USA). Portions of the original DNA were stored at –80ºC for further analysis.

175 All the samples were adjusted to the appropriate quantity for EPN or free-living bactivorous
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176 nematodes (FLN) ) or nematophagous fungi and bacteria quantification (10 ng µL–1)

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177 (Campos–Herrera et al., 2011a, 2011b, 2012b; Pathak et al., 2012).

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178 We recovered the fine roots from sieves (25 µm opening) employed during the sucrose

179 centrifugation (500 g fresh soil) and from the remaining soil (2 mm mesh opening), oven dried

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180 (70 °C) and weighed them. Finally, an additional 180 g of fresh soil of each sample was dried

181 and reweighed to measure soil moisture and stored until being processed to determine soil pH

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182 and electrical conductivity (Orion 290A, Thermo Electron Corp.).

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184 2.2. Molecular quantification of soil organisms
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186 Fifteen organisms comprising five EPNs, two phoretic bacteria, one free–living nematode
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187 genus–group (Acrobeloides–group), and seven NF were monitored in this survey using primer-

188 probe sets and standard curves described by Atkins et al. (2005), Campos-Herrera et al. (2011a,
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189 2011b, 2012), Pathak et al. (2012), and Zhang et al (2006) (Supplementary material 1). All the

190 primers and probes were synthesized by Integrated DNA Technologies Inc. (IDT, San Diego,
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191 CA). TaqMan® PCR probes were labeled at the 5’ end with the fluorogenic reporter dye FAM,
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192 and the 3’ end with the quencher Iowa BlackTM FG. The ZEN molecule was included in the

193 middle of the oligonucleotide to provide greater stability (IDT, San Diego, CA). Real–time PCR

194 was performed in optical 96–well reaction plates (USA Scientific, Orlando, FL, USA) on an ABI

195 Prism 7000 (Applied Biosystem). All reactions were performed in a final volume of 20 µL,

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196 containing 10 µL of the TaqMan Master Mix (Applied Biosystem, manufactured by Roche,

197 Branchburg, NJ), and the optimal primers/probe combination adjusted for each nematode, fungi

198 and bacterium (Atkins et al. 2005; Campos–Herrera et al., 2011a, 2011b, 2012b; Pathak et al.,

199 2012; Zhang et al 2006). In all the reactions, Bovine Serum Albumin (BSA) (PROMEGA) was

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200 added at a final concentration of 400 nM to reduce the possible interference of some remaining

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201 humic acids and other soil compounds (Torr et al., 2007). The annealing temperature was

202 dependent on the species analysed and was conducted for a total of 35 cycles with the nematodes

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203 (EPN and FLN), 50 cycles for the NF and 42 cycles for the bacteria (Campos–Herrera et al.,

204 2011a, 2011b, 2012b; Pathak et al., 2012). Positive and negative controls were included in all

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205 runs. The negative control consisted of sterile de–ionized water; the positive control was the

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corresponding standard curve (5 points each). All unknowns and controls were performed in

207 duplicate. All the data generated by real–time PCR protocols followed the MIQE procedure
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208 (Bustin et al., 2009). Data from the standard curves were log (x) transformed and a linear
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209 regression of number of EPNs, quantity of FLN or NF or bacterial dilution on threshold cycle
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210 value (Ct) was performed to estimate the efficiency and accuracy of the qPCR assay. A

211 correction factor based on dilution adjusted all the qPCR results.
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212

213 2.3. Effect of pH on Paenibacillus sp. adherence to nematode cuticle

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215 Infective juvenile (IJs) of S. diaprepesi, reared in Galleria mellonella larvae were rinsed

216 repeatedly and added to Petri dishes with 6-8-wk-old cultures of Paenibacillus sp. with abundant

217 endospores on nutrient agar (NA; Difco, MD, USA). After 72 h, the heavily spore encumbered

218 IJs were recovered by rinsing on 500 mesh sieves m opening) for use in the trials. Buffered

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219 phosphate solutions at pH 5.6, 6.0, 6.5, 7.0 and 7.7 were prepared and diluted to P concentrations

220 of 10M, 50M and 100M. Fifty ml of each pH-molarity combination were added to five, sterile

221 Petri dishes (60 × 15 mm). Approximately 250-300 spore-encumbered S. diaprepesi in 0.5 ml

222 water were pipetted into each plate. The Paenibacillus spore detachment was evaluated after 24 h

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223 by rinsing the IJs on 500 mesh sieves and counting the numbers of IJ with or without spores on

the cuticle using an inverted microscope. This test was repeated 3 times.

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224

225

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226 2.4. Statistical analysis

227 Differences in physical properties between sites were determined by analysis of variance

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228 followed by mean separation using Tukey’s Honestly Significant Difference Test. Temporal

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changes in properties were measured using Pearson’s correlation coefficient.

230 The relationship between soil pH and Paenibacillus spore encumbrance of S. diaprepesi
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231 in the field was measured by Pearson’s correlation coefficient of the 2-year average for both
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232 variables measured in the shallow and deep soil samples at the CREC, N40 and PCD sites. The
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233 PCW site was omitted from the analysis because S. diaprepesi, a species averse to wet soil

234 (Campos-Herrera et al., 2013; El-Borai et al., 2016) was rarely detected at that site during the
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235 two-year survey. Spore encumbrance was calculated by subtracting the log (X+1) S. diaprepesi

236 IJs from the log (X+1) Paenibacillus sp. plasmid copy numbers.
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237 The mean spore encumbrance of S. diaprepesi was plotted against the mean population
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238 density of S. diaprepesi each month at each site to determine whether it conformed with classic

239 Lotka-Volterra phase-space, predator-prey dynamics (Lotka, 1925; Volterra, 1926). The angle

240 vectors for all successive pairs of monthly flux (eg., from June-July-August, July-August-

241 September, etc.) were scored (0 or 1) as moving either in a clockwise (1) or counterclockwise (0)

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242 pattern. A Students t-test was used to determine whether the means of the 23 vectors per site

243 were significantly less (trending counterclockwise) or greater (trending clockwise) than 0.5. The

244 log (X+1) population density each month of S. diaprepesi was also predicted based on multiple

245 regression against log (x+1) population of the previous population density of S. diaprepesi and

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246 Paenibacillus sp. for time lags ranging from one to four months. This model was fitted to pooled

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247 data from the two ridge sites (CREC and N40) because the monthly EPN population densities

248 were highly correlated (r = 0.70; P ≤ 0.001) and the soil properties and climate were very similar

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249 at both sites. A second regression included just the data from PCD, the only flatwoods site with a

250 significant infestation by S. diaprepesi.

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251 Relationships between temporal patterns of the same EPN or NF species at the four sites

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were measured by Spearman’s rank correlation coefficients (rho). Relationships between all NF

253 species pooled across all sites was measured in the same manner. The effects of NF on EPN
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254 population change from one sample date to the next was also measured by this method where the
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255 EPN abundance at time X+1 was compared to each NF species at time X. Stepwise multiple
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256 regression of log (X+1) transformed S. diaprepesi or H. indica at time X+1 against square root

257 transformed NF species at time X was also performed.

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258 Depth distribution of variables was expressed by a proportion P = S/S+D where S = the

259 quantity in the shallow soil horizon and D is the quantity in the deeper horizon. Thus P greater or
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260 less than 0.5 indicates a predominance in the shallow or deeper horizons, respectively. Paired t-
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261 tests were used to determine whether patterns of vertical abundance differed significantly

262 between species or guilds.

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264 3. Results

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265

266 3.1. Regional and seasonal patterns

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268 The soil salinity, as measured by electrical conductivity, increased in both years during

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269 the spring dry season (Fig. 1A). The increase was greater in 2011 than in 2010, in agreement

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270 with the lower amount of springtime rainfall that occurred in the second year (Supplementary

271 data 2). The average soil pH varied between sites (Tukey HSD, F=58.43, df=3,188, P < 0.001)

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272 with the highest values measured at N40 (6.5) followed by CREC (6.2), PCD (5.9) and PCW

273 (5.5) (Fig. 1B). Moreover, whereas pH increased over time at CREC (r = 0.46, n = 48, P = 0.01)

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274 from a mean of 6.0 in 2010 to 6.3 in 2011, it declined (r= -0.52, n = 32, P = 0.002) at PCD

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beginning in September 2010 from a mean of 6.1 in 2010 to 5.7 in 2011. The mean soil moisture

276 measured at each sample date during two years was higher at PCW (11.8%) than at all other sites
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277 and lower at CREC (4.1%) than at all other sites (Tukey’s HSD Test P=0.05; ANOVA DF 3,
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278 572, F = 77.84, P = 0.001, data not shown). Soil moisture at N40 (6.6%) did not differ from that
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279 of PCD (7.1%).

280 The EPN communities at three of the four sites were dominated by H. indica (Fig. 2
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281 inset), in particular for PCW. By contrast, EPNs in the nearly adjacent, but drier, PCD site were

282 almost exclusively S. diaprepesi. The EPN communities at both sites on the central ridge were
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283 also dominatated by H. indica. The species S. khuongi and S. scapterisci were only occassionally
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284 detected, at CREC and PCW for the former species, and at CREC for the latter. The population

285 density of the EPN communities at all sites peaked in May or May-June each year; however, in

286 2011 they exhibited a second growth peak in autumn that produced very high densities at CREC

287 and PCW (Fig. 2).

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288 The influence of abiotic, seasonal variables on the population flux of EPNs was evident

289 in the numerous positive correlations between IJ numbers at the same and different sites

290 (Supplementary data 3, Fig. 3). Populations of S. diaprepesi at the two ridge sites (CREC and

291 N40; r = 0.31, P ≤ 0.001) both grew to their largest population sizes during the first few months

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292 of the survey and then declined to relatively low levels thereafter (Fig 3). By contrast, S.

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293 diaprepesi in the PCD flatwoods site increased during spring-summer in both 2010 and 2011, a

294 pattern that was unrelated to those of the same species on the ridge, but was highly correlated

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295 with those for H. indica at all sites where H. indica dominated. H. zealandica at N40

296 (Supplementary data 3) also exhibited a very similar population pattern to those of S. diaprepesi

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297 at that site (r = 0.25 P ≤ 0.001) and CREC (r = 0.26, P ≤ 0.001).

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The average abundance of Drechslerella dactyloides, Arthrobotrys musiformis and

299 Catenaria sp., did not differ among sites (Fig. 4). Arthrobotrys oligospora was more abundant at
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300 PCD than at PCW or N40 and did not differ at CREC from the other sites. Abundance of G.
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301 gephyropaga was least at PCW and did not differ among the other three sites. H. rhossiliensis
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302 was more abundant at N40 than at the other sites, whereas P. lilacinum was much more abundant

303 in both flatwoods sites than in those on the central ridge.

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304 An apparent effect of climate on NF occurred during springtime 2010 when the relative

305 levels (0-1) of Catenaria sp. were much higher at all sites than at any other time during the
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306 survey (Fig. 4). This period of population growth coincided with warmer, rainier weather than
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307 occurred during spring 2011 (Supplementary data 2). A similar concordance of population

308 growth at all sites occurred for P. lilacinum, which reached its highest levels in July and August

309 2011 on the central ridge and one month later in the flatwoods. A. oligospora exhibited very

310 similar patterns of temporal flux to those of S. diaprepesi at both sites on the central ridge (Figs.

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311 3-4), but not in the flatwoods where it was most abundant in the summer 2010 at PCD and in the

312 winter 2011 at PCW. Gamsylella gephyropaga showed major peaks at both central ridge sites in

313 early autumn 2010 and at both flatwoods sites in early spring 2011. Significant correlations

314 between sites for each species (Supplementary data 4) were frequent for the CREC and N40

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315 central ridge locations (5 of 7 species), but occurred infrequently between the other combinations

of sites. Spearman’s correlations for relationships between temporal patterns of all species were

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317 significant for all combinations of the trapping NF but for none of the others (Supplementary

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318 data 5).

319 Seasonality was most evident for the soil nematode community as a whole and for the

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320 species of Acrobeloides detected by qPCR (Supplementary data 4; Fig. 5). Temporal population

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patterns within both groups were highly correlated among all sites. The total nematode

322 populations tended to grow from winter until summer during both years and sharply decline
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323 either in mid-autumn or mid-to-late-summer, followed by peaks again in early winter in both
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324 years. Concordance of some trends was marked, for example a sharp decline of both the total
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325 nematodes and Acrobeloides spp. at all sites in July, 2011.

326
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327 3.2. Depth distribution

328
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329 Across all sites and samplings, the numbers of nematodes and the amount of DNA
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330 recovered were more than 75% greater in the shallow compared to the deeper soil horizon (Fig.

331 6). The shallow horizon tended to be slightly wetter, more saline and less acidic than the deeper

332 horizon. Citrus fibrous roots were 70% more abundant near the surface. The trapping NF were

333 more abundant in shallow than deeper soil. The endoparasitic Catenaria sp. exhibited no depth

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334 preference, but H. rhossiliensis and P. lilacinus were between 38% and 50% more abundant

335 deeper in the soil than near the surface. H. zealandica was equally abundant along the soil

336 profile, whereas S. diaprepesi and especially H. indica ranged from 18% to 40% more abundant

337 in the deeper soil samples.

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338

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339 3.3 Relationships

340 3.3.1 Interaction among pH, the bacterium Paenibacillus sp. and the nematode S. diaprepesi

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341 Paenibacillus showed a significant (Spearman) correlation only with S. diaprepesi at

342 CREC (n=198, rho=0.39, P<0.001), N40 (n=198, rho=0.15, P=0.04) and PCW (n=144, rho=0.43,

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343 P<0.001). Although the bacterium was also correlated with Acrobeloides spp. and free living

344
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nematodes in data pooled from the three sites, multiple regression against these species and S.

345 diaprepesi revealed that only S. diaprepesi was a significant predictor (R2=0.16, P<0.001).
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346 In laboratory experiments, the number of S. diaprepesi IJs with Paenbacillus sp.
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347 endospores attached to the cuticle was related to higher pH values after the spore-infested
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348 juveniles were soaked in phosphate buffer solutions for 24 hours (Fig. 7). Seventy-five percent of

349 IJs were infested at pH 7.7 compared to just 17% at pH 5.5. The relationship did not vary
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350 between solutions when phosphorous molarities ranging from 10M to 100M were used (Fig. 7

351 inset). A congruent relationship occurred in the field surveys (Fig. 8). The number of
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352 Paenibacillus sp. endospores per S. diaprepesi detected by qPCR in shallow and deep soil
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353 samples from the three sites infested with the nematode was related to the soil solution pH. By

354 contrast, the numbers of S. diaprepesi in those samples were inversely related to soil

355 pH. Beyond these spatial relationships between pH, Paenibacillus sp. and S. diaprepesi,

356 temporal changes in levels of S. diaprepesi at CREC and PCD were inversely related to the

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357 temporal increases and decreases in soil pH at the two sites, respectively. At PCD the population

358 density of S. diaprepesi during spring through winter was 50% greater in 2011 than in 2010

359 (Tukeys HSD, P = 0.05) and at CREC S. diaprepesi declined by half (Tukeys HSD, F = 7.98, df

360 1, 130, P = 0.005) from 10.2 IJs in 2010 to 5.0 IJs in 2011. The average spore encumbrance rate

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361 (Paenibacillus sp. DNA per S. diaprepesi) each month decreased over time at PCD (r = -0.67, n

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362 = 19, P = 0.002), but exhibited no significant trend at CREC.

363 The monthly S. diaprepesi population density and Paenibacillus sp. infestation rates are

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364 shown in Fig. 9A. The monthly changes in Paenibacillus sp. copy number per S. diaprepesi IJ

365 plotted against the monthly change in S. diaprepesi population density, consistently conformed

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366 to the Lotka-Volterra ‘phase space’ model of predator-prey dynamics (Fig. 9B). Successive

367
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vectors of monthly flux for the two variables were more frequently counter-clockwise than

368 clockwise at both N40 (80% of vectors; P ≤ 0.01) and PCD (88% of vectors; P ≤ 0.001). The
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369 period of largest flux for both organisms at CREC was counterclockwise, but overall there was
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370 no significant trend in vector directions (47%; P = ns).

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371 Forty-three percent of S. diaprepesi population variation on the central ridge was

372 explained by the equation Pf = 0.617 + 0.735 Pi – 0.208 Psp, where Pf = log (X+1) S. diaprepesi
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373 at time T, Pi = log (X+1) initial population density of S. diaprepesi at time T-3 (P ≤ 0.001), and

374 Psp. = log (X+1) Paenibacillus sp. at time T-3 (P ≤ .01) (Supplementary data 6). At the PCD
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375 flatwoods site, Paenibacillus sp. was not significant as a predictor of S. diaprepesi population
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376 change.

377

378 3.3.2 Relationships between entomopathogenic nematodes and their other predators and

379 competitors

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380

381 The spatial relationships between EPNs and the other measured organisms at each site

382 showed a number of positive correlations (Supplementary data 7). Acrobeloides spp. were

383 directly related to at least one and often two EPN species at all sites. There were no obvious

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384 competitive inhibitions among EPNs; S. diaprepesi was positively related to H. indica at CREC

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385 and to H. indica and H. zealandica at N40. Positive associations were also occasionally detected

386 between EPNs and all NF species except A. musiformis and G. gephyropaga.

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387 Stepwise multiple regression explained between 4% and 28% of the variation in monthly

388 population flux of EPNs at each site (Table 2). Drechslerella dactyloides was the only NF

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389 species exhibiting evidence of top-down regulation, being inversely related to population change

390
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of H. indica at both N40 and PCW. Acrobeloides spp. were associated with reduced population

391 growth of S. diaprepesi at CREC and PCD and H. indica was associated with reduced abundance
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392 of H. zealandica at N40 and S. diaprepesi at PCD.

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393 The depth distribution patterns varied between sites for most species and guilds (Fig 10).
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394 Nearly 80% of trapping NF were near the surface at CREC, the driest site, whereas a majority of

395 trappers were in deeper soil at the wettest site, PCW with the N40 and PCD sites as most similar
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396 both in terms of moisture and trapping NF by depth. Indeed, the distributions of Catenaria sp.,

397 H. rhossiliensis, and P. lilacinum at N40 and PCD were generally also more similar to each other
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398 than to the patterns of the other two sites. Patterns for S. diaprepesi and H. indica also differed
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399 by sites. Within sites where they co-occurred, S. diaprepesi remained nearer the soil surface than

400 did H. indica at CREC (41% vs 32%, P=0.001) and N40 (53% vs 44%, P=0.05).

401 Heterorhabditis zealandica depth distribution differed from that of H. indica at N40 (57% vs

402 44%, P<0.01), but not that of S. diaprepesi. Across all sites, S. diaprepesi and H. indica tended

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403 to inhabit deeper soil (P<0.001) than did the trapping NF. Heterorhabditis indica were in deeper

404 horizons than Catenaria sp. (P<0.01) and H. zealandica occurred in more shallow soil than did

405 P. lilacinus (P<0.001). Acrobeloides-group species were much more abundant in the shallow

406 horizon in both flatwoods sites (PCD=69%, PCW=73%) compared to the percentages on the

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407 ridge sites (CREC=50%, N40=53%) (data not shown).

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408

409

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410 4. Discussion

411 Comparatively few of the parasites, predators, or competitors of EPNs studied in these

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412 surveys exhibited density-dependent relationships with those nematodes (Stuart et al., 2015). In

413
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one sense this is unsurprising, given the myriad, unmeasured and thus hidden, physical and biotic

414 variables affecting EPNs. But the relative strength of different trophic relationships measured
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415 here may also reflect both the sampling methodology used and differences in host specificity.
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416 Bulked samples of highly patchy EPNs (Stuart and Gaugler, 1994) obfuscate many functional
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417 relationships that require microcosm proximity of nematodes to other organisms. The responses

418 of NF and Acrobeloides spp. when an insect is killed by EPNs occur locally, within hours or
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419 days. Sampling at such a micro-scale (e.g., Jaffee et al., 1989; Steel and Ferris, 2016) would

420 facilitate detecting the impact of these antagonists on EPN abundance, because the nutrients
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421 provided by EPNs and their insect prey are but one of many sources available to NF and
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422 Acrobeloides spp. By contrast, the phoretic reliance of Paenibacillus sp. on S. diaprepesi to

423 obtain nutrients was reported to be species-specific (El-Borai et al., 2005). However, the

424 detection of Paenibacillus sp. in both Florida and Portugal (Campos-Herrera et al., 2016, under

425 review) in the absence of detectable S. diaprepesi, indicates either a wider host range than

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426 actually known or the lack of specificity of the targeted sequences. Nevertheless, consistent,

427 often highly significant correlations between the bacterium and only S. diaprepesi measured in

428 these surveys support specificity of the association. Therefore, conditions favorable to S.

429 diaprepesi should later increase the abundance of Paenibacillus sp., even in bulk samples.

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430 Likewise, if the proportion of Paenibacillus sp. to S. diaprepesi increases in an orchard, it should

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431 be possible to measure a detrimental effect on the future abundance of the nematode as reported

432 previously by Campos-Herrera et al. (2013, 2014).

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433 It is noteworthy that Paenibacillus sp., seemingly the most benign among the variety of

434 EPN antagonists studied in these surveys, was among those most evidently associated with EPN

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435 population dynamics. These bacteria do not prey on EPNs as do NF (Jaffee and Strong, 2005),

436
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nor do they deprive EPNs of nutrients as do Acrobeloides spp. competitors (Campos-Herrera et

437 al., 2012). Rather, Paenibacillus sp. and Paenibacillus nematophilus were shown in laboratory
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438 experiments to impede EPN motility and infectivity when large numbers of spores were attached
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439 to the cuticle (Enright et al., 2005; El-Borai et al., 2005). The relationships between
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440 Paenibacillus sp. and other variables measured in these surveys were determined in a natural

441 condition, not by an experimental manipulation that could demonstrate causation. Nonetheless,
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442 they consistently conformed to a priori expectations for the effects of i) pH on the bacterial

443 infestation rate and ii) the bacterial infestation rate on the abundance of S. diaprepesi. Those
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444 relationships reveal new possibilities for pest control through soil management. Florida citrus
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445 growers are advised to maintain soil pH below 6.5 to reduce bicarbonate stress to the citrus root

446 system (Graham et al., 2012; Johnson, et al., 2017). Results here and in Campos-Herrera et al.,

447 (2013, 2014) indicate that reducing soil pH is likely to reduce spore encumbrance by

448 Paenibacillus sp. and increase the abundance and services of S. diaprepesi, at a 3-month dealy,

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449 in habitats otherwise conducive to the nematode. The endoparasitic Pasteuria sp. was reported to

450 have a similar 2-3 month lag response to population density changes of Tylenchulus

451 semipenetrans and to exhibit predator-prey, phase-space properties much as found here for

452 Paenibacillus sp. and S. diaprepesi (Ciancio et al., 2016).

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453 The NF are a diverse assemblage of fungi that evolved a variety of strategies by which to

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454 prey on nematodes. Jaffee et al. (1993) reviewed earlier work indicating that fungi producing

455 adhesive networks, branches or knobs are less reliant on predation than those producing adhesive

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456 spores or constricting rings (Cooke, 1963; Jansson and Norbring-Hertz, 1979, 1980; Jansson,

457 1982, 1983; Gray, 1987). Bioassays showed that H. rhossiliensis and D. dactyloides exhibited

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458 density-dependent regulation of nematodes, whereas A. oligospora and G. gephyropaga did not.

459
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Field trials also found that adhesive spore forming NF (H. rhossiliensis) exhibited density

460 dependent regulation of nematodes, whereas adhesive net/branch formers (including A.

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461 oligospora and G. gephyropaga) did not (Farrell et al., 2006; Jaffee et al., 1989, 1996, 2005,
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462 2007).
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463 Our survey results appear to conform in part to the NF saprophyte-predator paradigm.

464 The only NF inversely related to EPN population change from one month to the next was the
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465 constricting ring former, D. dactyloides. Moreover, this fungus potentially regulated H. indica at

466 two of the three sites dominated by that EPN but had no significant relationship at any site to the
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467 population flux of S. diaprepesi. Grown on agar, it readily infects S. diaprepesi (personal
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468 observation, Duncan and El-Borai). However, in soil bioassays S. diaprepesi populations were

469 unaffected by any of the three Arthrobotrys species measured in these surveys, whereas D.

470 dactyloides reduced populations of S. khuongi and S. riobrave (El-Borai et al., 2009).

471 Heterorhabditis indica was not tested for susceptibility to D. dactyloides but was susceptible to

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472 A. oligospora and A. musiformis (El-Borai et al., 2009). Additional controlled characterization of

473 responses by populations of S. diaprepesi and H. indica to D. dactyloides are needed, because

474 endemic S. diaprepesi is thought to be superior to H. indica in regulating Diaprepes root weevils

475 (El-Borai et al., 2012; Campos-Herrera et al., 2015). Population growth of both H. zealandica

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476 and S. diaprepesi showed a possible regulation by competition with H. indica; therefore, soil

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477 properties favoring D. dactyloides may conceivably be beneficial to S. diaprepesi, underpinning

478 suppression of Diaprepes root weevil.

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479 Except for D. dactyloides, no NF species adapted to predation (i.e., H. rhossiliensis, C.

480 anguillulae) showed a regulation potential for nematodes. Rather, all NF species except G.

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481 gephyropaga were occasionally or frequently positively related to EPN numbers as reported in

482
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geospatial surveys of Florida orchards (Campos-Herrera et al., 2013). Similar positive

483 relationships between the two guilds have been frequently detected elsewhere (Farrell et al.,
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484 2006; Jaffee et al., 1989, 1996, 2007) and aptly characterized by Jaffee and Strong (2005) as
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485 strong bottom-up, weak top-down interactions. Campos-Herrera et al. (2012) noted that strong
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486 associations measured between NF, Acrobeloides spp. and EPNs supported Linford’s (1937)

487 hypothesis that decomposition of organic matter (e.g., insect cadavers) dramatically increases
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488 first bactivorous nematodes, later followed by an increase of their NF antagonists.

489 The role of free living, bactivorous nematodes (FLN) in regulating natural biological
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490 control by competing for insect cadavers is unclear, but they are among the antagonists most
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491 commonly encountered by EPNs (Duncan et al., 2003, 2007; Campos-Herrera et al., 2012, 2013,

492 2014, 2015a,b, 2018; Jaffuel et al., 2017). The use of qPCR to characterize communities of

493 nematodes emerging from insect cadavers recently revealed the extent to which EPN populations

494 might be regulated by competition in the cadaver. For example, Swiss agricultural soils are

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495 relatively depauperate for EPNs, due partly to competition with Oscheius spp. (Campos-Herrera

496 et al., 2015a,b). Given the necromenic life histories of Pristionchus spp., it should be

497 unsurprising that P. maupasi and P. pacificus were recently reported to be competitors with

498 EPNs in Portugal (Rae et al., 2008; Campos-Herrera et al., under review). EPNs themselves are

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499 excellent scavengers (Sans-Blas et al., 2012). Nevertheless, the augmentation of EPNs in soil

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500 greatly increases the rate at which insect cadavers support FLNs (Duncan et al., 2003, 2007)

501 which do not kill insects; therefore, competition favors FLNs at the expense of EPNs.

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502 Acrobeloides spp. were associated with declining numbers of S. diaprepesi at two sites.

503 Interspecific competition appears to have been especially keen at PCD where both Acrobeloides

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504 spp. and H. indica were inversely related to population growth of S. diaprepesi.

505
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Bactivorous nematodes are the dominant organisms in the nutrient recycling process of

506 decomposition. Many agronomic practices that increase organic matter to improve soil structure
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507 and function rely on these nematodes for nutrient release, but they presumably also favor the
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508 population growth of species adapted to compete with EPNs. The effects of organic matter
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509 augmentation on systems as complex as the soil food webs remains an especially rich area for

510 investigation. For example, Bednarek and Gaugler (1997) speculated that increased EPN activity
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511 in soil amended continually with animal manure was due to increased resources for soil borne

512 insect larvae. Duncan et al. (2007) also reported beneficial effects for EPNs in plots amended
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513 with composted chicken manure. Campos-Herrera et al., (2015) detected more H. zealandica and
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514 recovered fewer Diaprepes weevils in plots amended with pelletized chicken manure, a

515 treatment that also increased Acrobeloides-group nematodes, but without affecting S. diaprepesi.

516 By contrast, a manure amended agricultural system in Switzerland had no measurable effect on

517 EPN, despite increasing microbial biomass (Jaffuel et al., 2016).

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518 The greater depth at which H. indica consistently tended to reside in these orchards

519 compared to S. diaprepesi or H. zealandica is similar to reports for H. bacteriophora in

520 comparison to S. carpocapsae, S. feliae and S. riobrave (Ferguson et al., 1995; Garcia del Pino

521 and Paloma, 1997; Millar and Barbercheck, 2001; Susurluk, 2008). Although vertical niche

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522 separation in the soil profile is a plausible mechanism for EPN species packing within habitats

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523 (e.g., Griffin, 2015), the separation is quantitative and interaction between species remains

524 possible. Significant differences in depth distribution at N40 may not have prevented H. indica

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525 from modulating the growth of H. zealandica. Similarly, Acrobeloides-group may have

526 suppressed S. diaprepesi population growth at both CREC and PCD, despite residing primarily

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527 in different horizons at the latter site.

528
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The reports of habitat or ecoregion specificity exhibited by the EPN studied are

529 consistent with their occurrence in these surveys. Heterorhabditis zealandica, detected only at
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530 N40, was previously found in citrus orchards almost exclusively on the central ridge (Duncan et
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531 al., 2003, 2007; Campos-Herrera et al., 2013), but occasionally from natural areas in flatwoods
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532 (Duncan et al., 2003, 2007; Campos-Herrera et al., 2016). That S. diaprepsi was virtually absent

533 at PCW, dominant at PCD, and abundant at N40 and CREC was unsurprising because it is
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534 adapted to drier soil conditions typical of those in the deep sandy soils of the central ridge

535 (Campos-Herrera et al., 2013; El-Borai et al., 2016). The virtually complete segregation of S.
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536 diaprepsi and H. indica at the Plant City orchard containing the PCD and PCW sites supports
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537 observations by Duncan et al. (2007) that EPN communities tend to form species specific

538 enclaves within sites. Unlike the edaphically homogenous site in Duncan et al. (2007), the

539 different soil moisture properties of PCD and PCW probably reinforced the tendency to

540 segregate almost completely. Purpureocillium lilacinum was the only NF exhibiting a regional

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541 distinction in this survey, being more prevalent in the flatwoods sites as reported previously for

542 both orchards and natural areas in Florida (Pathak et al., 2017). Despite considerable commercial

543 potential of this fungus as a biological control agent for plant parasitic nematodes, we found no

544 indication that it affected EPNs here or in a previous study (Campos-Herrera et al., 2015).

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545 Conclusion

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546 The results of these experiments and surveys provide compelling evidence that

547 Paenibacillus spp. modulate EPN abundance in nature. They support speculation that

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548 horticultural practices harmful to S. diaprepesi, resulted from conditions favoring increased

549 Paenibacillus sp. infestation of the nematode (Campos-Herrera et al., 2014). Controlled

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550 experiments are needed to test the hypothesis that reducing the soil pH in alkaline sites with

551
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naturally occurring S. diaprepesi can increase this EPN species and reduce Diaprepes root

552 weevils. Validation of this potential biocontrol tactic would expand the repertoire of cultural
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553 practices to exploit EPN services in Florida citrus orchards (Ali et al., Duncan et al., 2015).
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554 The inverse relationships between H. indica and population growth of S. diaprepesi and
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555 H. zealandica suggests the intriguing possibility that suppressing certain EPNs might improve

556 biological control by increasing the abundance of more effective EPN species. Heterorhabditis
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557 indica and H. zealandica are less effective than S. diaprepesi and S. khoungi for control of D.

558 abbreviatus and protection of citrus seedlings (El-Borai et al., 2007, 2012). Adopting a strategy
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559 of augmenting natural enemies such as D. dactyloides or improving conditions to favor those
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560 natural enemies, may increase S. diaprepesi at the expense of both H. indica and D. abbreviatus.

561 The physical properties of subterranean habitats sustain a biological complexity that

562 greatly complicates efforts to conserve or enhance natural regulation. The impact of a given

563 practice must hence vary in different soils that support different microbial and mesofaunal

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564 assemblages. A further impediment is the dearth of information about the composition and

565 function of belowground communities. However, the use of molecular tools in this study

566 provided direct evidence of the natural interplay between EPNs, soil properties and food webs.

567 Data suggested practical methods that growers may apply to enhance the performance of

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568 beneficial organisms in their orchard soils.

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569

570 Acknowledgments

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571 This study is supported by a USDA–CSREES Special Grant (TSTAR), and U.S–Egypt

572 Science and Technology Joint Fund (206). RCH was awarded with postdoctoral fellowships by

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573 the Ramón Areces Spanish Foundation and the Seventh Framework Programme of the European

574
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Union (Marie Curie International Outgoing Fellowship for Career Development, FP7–PEOPLE–

575 2009–IOF–252980). Currently, the Government of Spain supports RCH with a Ramon y Cajal
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576 contract award (RYC-2016-19939).

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577
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578 References

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580 Koppenhöfer, A.M., telinski, L.L., 2012. Subterranean, herbivore-induced plant volatile
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849 Stuart, R.J., Barbercheck, M.E., Grewal, P.S., 2015. Entomopathogenic nematodes in the soil
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864 316–323.
865
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Table 1. Localities and soil characteristics of the citrus orchard sites surveyed in this study.
M

Site
D

(n = Soil Sampling
TE

sampling Geographical Soil texture water times: start-

events) coordinates Region %sand:silt:clay depth end

EP

CREC (33) 28.105085, - Central 96.8: 0.4: 2.8 >2.0 Sep 21st 2009-
C

81.715079 Ridge Dec 6th 2011

AC

N40 (33) 28.128401, - Central 96.4: 0.4: 3.2 >2.0 Sep 21st 2009-

81.718458 Ridge Dec 6th 2011

PCD (23) 27.980455, - Interior 94.0: 2.4: 2.8 0.76 Mar 15th

82.061356 Flatwoods 2010-Dec 6th

33
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2011

PCW (23) 27.980455, - Interior 94.0: 2.4: 2.8 0.76 Mar 15th 2010-

82.062778 Flatwoods Dec 6th 2011

866

PT
867

RI
U SC
AN
M
D
TE
C EP
AC

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868

Table 2. Significant variables in multiple regression analysis of entomopathogenic nematodes

(EPN) population density regressed against EPNs and organisms that can prey on or compete

with EPNs during the previous month (i.e., one moth lag). Sampling occurred approximately

PT
monthly during approximately two years at two soil depths at each of the four survey sites.

RI
Steinernema diaprepesi (Sd), Heterorhabditis indica (Hi), H. zealadica (Hz), Acrobeloides group

spp. (Acrob). See Fig. 6 for nematophagous fungi abbreviations.

SC
sd hi hz acrob ad am ao gg ca hr pl R2

U
Crec sd *** ns ns -*** ns ns ns ns ns ns ns 12%

N40 sd * ns ns ns ns AN * ns ns ns ns ns 4%

PCD sd *** -*** ns -*** ns * ns ns ns ns ns 14%

M
Crec hi ns *** ns ns ns ns ns ns ns ** ns 16.8%

N40 hi ns ns ns ns -** ns * ns ns ns ns 6%
D

PCW hi ns *** ns ns -** ns * ns * ns ns 21%

TE

N40 hz ** -** *** ns ns ns ns ns ns ns ns 28%

EP

869

870
C

871
AC

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872 Figure Legends

873 Fig. 1 Soil electrical conductivity and pH during 18-24 months at four survey sites. CREC and
874 N40 sites are on the central ridge ecoregion and PCD and PCW are in the flatwoods. Insets are
875 comparisons of the soil properties in the shallow (black bars) or deeper (gray bars) soil profiles.
876 Error bars are standard errors (SE) of the means.
877 Fig. 2. Population change of infective juvenile entomopathogenicn nematode (EPN)

PT
878 communities comprising Steinernema diaprepesi (Sd), Heterorhabditis indica (Hi) and H.
879 zealandica (Hz) during 21 months at four survey sites. Inset shows the proportion of the total
880 EPN population of each species at each site. Error bars are SE of the means.

RI
881 Fig. 3. Population change of infective juvenile Steinernema diaprepesi (Sd), Heterorhabditis
882 indica (Hi) and H. zealandica (Hz) during 21 months at four survey sites. Error bars are SE of

SC
883 the means.
884 Fig. 4. Average nematophagous fungi infection rates at four sites (left-hand panels), and the
885 seasonal fluxes of the standardized (0-1) infection rates at each site (right-hand panels). Bars

U
886 with different letters indicate significant differences between sites (p=0.05). Error bars are SE of
887

888
the means. AN
Fig. 5. Population changes of total nematodes and Acrobeloides group nematodes during
889 temporal surveys at the four sites. Error bars are SE of the means.
M
890 Fig. 6. Depth distribution of total DNA and soil properties (light gray bars), EPNs (black bars),
891 and potential EPN antagonists (dark gray bars). Abbreviations refer to soil moisture content
892 (MC), electrical conductivity (ec), soil pH (pH), fibrous root mass density (rt), Steinernema
D

893 disprepesi (Sd), Heterorhabditis indica (Hi), H. zealandica (Hz), total free living nematodes
894
TE

(fln), Acrobeloides group nematodes (Acr), Paenibacillus sp. (Psp), Drechslerella dactyloides
895 (Dd), Arthrobotrys musiformis (Am), A. oligospora (Ao), Gamsylella gephyropaga (Gg),
896 Catenaria anguilulae (Ca), Hirsutella rhossiliensis (Hr) and Purpureocillium lilacinum (Pl).
897 Asterisks indicate proportions larger or smaller than 50% (* P=0.05, ** P=0.01, *** P=0.001).
EP

898 Fig. 7. The effect of buffer pH on the adherence of Paenibacillus sp. endospores to the cuticle of
899 Steinernema diaprepesi after 24 hours. Inset shows spore adherence in phosphate buffers of
C

900 different molarity to test whether effect is due to pH or phosphate concentrations. Error bars are
901 SE of the means.
AC

902 Fig. 8. Linear relationship between soil pH and the abundance of Steinernema diaprepesi (top
903 panel) or the encumbrance rate of Paenibacillus sp. (logn [Paenibacillus sp. + 1] – logn [S.
904 diaprepesi + 1]). Symbol abbreviations represent sites (c = CREC, n = N40, d = PCD) and soil
905 depth (deep = d, shallow = s).
906 Fig. 9. Abundance of Steinernema diaprepesi (Sd, left panels, black) and Paenibacillus sp
907 endospore infestation rate (Psp, left panels, white) at three sites with abundant S. diaprepesi and
908 phase-space diagrams showing the sequential direction of monthly flux of Paenibacillus sp.

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909 endospore infestation rate compared to S. diaprepesi (Sd) abundance (right panels) Error bars are
910 SE of the means.
911 Fig. 10. Proportions of organisms in the shallow soil profile (0-15 cm) compared to the total
912 sample (0-30 cm) at four sites. Bars with the same or no letter are not significantly different
913 (p=0.05). Error bars are SE of the means.
914

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915

916

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D
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C EP
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Nematodes with spores

10 M
1.0
50 M

RI
100 M
0.8

U SC
0.6
AN
0.4
M
D

0.2
TE

0.0
EP

5.0 5.5 6.0 6.5 7.0 7.5 8.0

pH
C
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1200 500
Soil EC

400
300
1000

RI
200
100
Soil EC (mV)

800
CREC N40 PCD PCW

SC
600

400

U
200
AN
0
6.5 CREC
Soil pH

8 6.0
N40
M

5.5
5.0
PCD
CREC N40 PCD PCW PCW
Soil pH

7
D
TE

6
EP

5
MA A MJ J A AS O N D J F M AM J J A S O N D

Month
C
AC
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1.0 Trapping fungi a

a 12 Soil moisture

PT
0.8
b 10

Percent dw
bc
0.6 c 8 b
0.4 6 b

RI
4 c
0.2
2
0.0 0

SC
Catenaria anguilulae
Proportion Infection rate in shallow soil

0.8 Steinernema diaprepesi

0.8
0.6

U
0.6 a
0.4 b b
0.4
AN
0.2
Proportion IJs in shallow soil

0.2
0.0
0.0
Hirsutella rhossiliensis
M

0.8 Heterorhabditis indica

a ab 0.8
0.6
ab a
0.6
ab
b
D

0.4
0.4 b
0.2
0.2
TE

0.0
0.0
Purpureocillium lilacinum Heterorhabditis zealandica
0.8 0.8
EP

0.6 0.6
0.4 0.4
C

0.2 0.2
0.0 0.0
AC

CREC N40 PCD PCW CREC N40 PCD PCW

Site Site
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PT
RI
800 1.2
Proportion of total EPNs

Sd
CREC
1.0 Hi
Hz N40

SC
0.8 PCD
0.6
PCW
600
0.4
(500 cm soil)

U
0.2
IJ EPNs

AN
3

0.0
400 CREC N40 PCD PCW
M

200
D
TE

0
M A M J J A S O N D J F M A M J J A S O N D
EP

Month
C
AC
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PT
H. indica
600

RI
CREC
N40
400 PCD
PCW

SC
200

U
H. zealandica
(500 cm soil)

40
AN
IJ EPNs

30
3

20
10
M

0
D

S. diaprepepsi
150
TE

100
50
EP

0
S OND J F MAM J J AS OND J F MAM J J AS OND
C

Month
AC
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PT
RI
U SC
AN
M
D
TE
EP
C
AC
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3000

CREC

RI
2500 N40
Total nematodes

PCD
PCW
(60 cm soil)

2000

SC
3

1500

U
1000 AN
500

0
M

1
Acrobeloides group
(500 cm soil)

0.1
3

TE

0.01
EP

0.001
C

S O N D J F MAM J J A S O N D J F MAM J J A S O N D
AC

Month
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PT
RI
SC
0.8
Proportion in surface horizon

*** ***
***
*** ***
*** *** ns *** ***

U
0.6 ns ns
***
AN
*** *
*
*** ***
0.4
M

0.2
D

0.0
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dna h2o ec pH rt Sd Hi Hz fln Acr Psp Ad Am Ao Gg Ca Hr Pl

Variable
C EP
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RI
SC
0.9

0.8
Proportion IJs with spores

0.7
U
AN
0.6
10 M
M

1.0
0.5 50 M
IJs with spores

100 M
0.8

0.4 0.6
D

0.4

0.3
TE

0.2

0.0
0.2 5.0 5.5 6.0 6.5 7.0 7.5 8.0
pH
EP

0.1
5.0 5.5 6.0 6.5 7.0 7.5 8.0
C

pH
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4.0

PT
3.5
Dd
Ds
Log n S. diaprepesi

RI
3.0
(500 cm soil)

2.5
3

SC
2.0
Cd
1.5 Cs

U
1.0
r = 0.92 Ns
Nc
AN
0.5 P = 0.01
0.0
Paenibacillus encumbrance

r = 0.97
0.5 P = 0.001
Ns
Nc
D

0.0
TE

-0.5 Cs
-1.0 Cd
EP

-1.5 Ds
-2.0 Dd
C

-2.5
AC

5.6 5.8 6.0 6.2 6.4 6.6

Soil pH
 ACCEPTED MANUSCRIPT

6 3 2
A. PCD B. PCD a7
2
5 a22 1
1
0

PT
4 a10

0
a11
a18 a13
-1
a8 a12
3 -1 a9
a14
a15 -2
-2 a16

RI
2 a19
a17

-3
a23
a20
a21
-3
1
-4 a25
-4

SC
a24

logn (Psp + 1) - logn (Sd + 1)

0 -5 -5

logn (Psp + 1) - logn (Sd + 1)

A. CREC B. CREC
5 4 4
Logn (Sd + 1)

a7

U
a6

4
2 2
AN
a5
3
0 a13
a12
a8 0
2 a23
a24
a17
a16
a11
a9
a15a10 a4
a20 a2
a21
-2 a18
M

a19 a1
1
a22
a14
a25
-2
a3

0 -4 -4
B. N40
D

A. N40
4 6
a6
6
TE

3 4
a22
4
a7

2 2 2
a9 a12 a5
a13
EP

a16 a15
1 0 a17a20a10
a24
a11a14
a2a18 a8 0
a25
a23
a19
a4
a21
a3
0 -2 a1 -2
C

-4 -4
SOND J FMAM J J A SOND J FMAM J J A S 0 1 2 3 4 5
AC

Month Logn (Sd+1)

 ACCEPTED MANUSCRIPT

Research highlights

Entomopathogenic nematode (EPN) populations measured for 2 years at 4 sites.

EPN population flux inversely associated free living nematode competitors.

PT
EPN and phoretic bacteria exhibited phase-space, predator prey dynamics.

Soil pH regulated bacteria endospore adherence to nematode cuticle.

RI
U SC
AN
M
D
TE
C EP
AC