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Distribution and abundance of Aphanomyces euteiches in agricultural soils: effect of land use type, soil properties, and crop management practices
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Distribution and abundance of Aphanomyces euteiches in agricultural soils: effect of land use type, soil properties, and crop management practices Erin M. Karppinena, Josephine Paymenta, Syama Chattertonb, Jillian D. Bainarda, ⁎ Michelle Hubbarda, Yantai Gana,1, Luke D. Bainarda, a b
Swift Current Research and Development Centre, Agriculture and Agri-Food Canada, 1 Airport Road, Swift Current, SK S9H 3X2, Canada Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Oomycete Root rot Pea Lentil Rangeland Quantitative PCR
Aphanomyces euteiches is a soil-borne pathogen that causes root rot in susceptible pulse crops. Although widespread across Canada, A. euteiches was not reported in Saskatchewan until 2012. The distribution of this pathogen across the Canadian prairies is unknown and it is unclear whether it is native to the region. The objective of this study was to conduct a survey of A. euteiches across the major agricultural soils of Saskatchewan, Canada to determine its distribution and to assess the relative importance of factors related to its distribution and possible spread across the province. Soil samples were collected from lentil and pea fields and their associated roadside ditches, as well as from native and tame rangelands. Soil samples were analyzed using a quantitative polymerase chain reaction (qPCR) assay to detect and quantify the presence of A. euteiches. In addition, root rot severity was assessed on affected pea and lentil plants, and the physical and chemical properties of the soils were measured. A. euteiches was present across all land use (i.e., annual cropland, roadside ditches, and rangeland) and soil types in Saskatchewan. A. euteiches abundance was highest in samples collected from annually cropped fields. Within annual cropland, A. euteiches abundance was higher in pea fields than lentil fields. Soil moisture, total and organic carbon, and total nitrogen were positively correlated to A. euteiches abundance in field and roadside soils. Based on this survey, A. euteiches is wide-spread across all the major agricultural regions of Saskatchewan and is not limited to specific soil or land use types. The frequent detection of A. euteiches in native rangeland indicates it is likely native to Saskatchewan. The significant relationship between A. euteiches gene abundance and disease severity in pea fields indicates that the qPCR assay may be a useful indicator for predicting the potential for certain agricultural soils to cause A. euteiches root rot in pea crops.
1. Introduction Pulses, such as peas and lentils, are an increasingly important crop for agricultural producers and have supported the diversification of crop rotations across the Canadian prairies. Pulse crops are grown for many reasons, including their relative tolerance to heat and drought (Miller et al., 2002), and their ability to increase the amount of total available nitrogen in the soil (Zhou et al., 2017), which can improve the yields of subsequent crops (Niu et al., 2017). However, increased frequency of pulses in crop rotations has been linked to decreased fungal diversity and build-up of pathotrophs in the soil (Bainard et al., 2017; Niu et al., 2018). As pulses continue to be incorporated into relatively short crop rotations, root rot has become a limiting factor in their production (Wu et al., 2018). In particular, root rot caused by the
oomycete pathogen Aphanomyces euteiches is becoming a major disease in field peas and lentils grown on the Canadian Prairies (Wu et al., 2018). The disease symptoms vary, but generally include honey-colored lesions on the root, which eventually collapse and cause the plant to yellow and wither (Gangneux et al., 2014). The effects of the disease are often exacerbated as A. euteiches weakens the plant, allowing opportunistic pathogens to also invade the root (Willsey et al., 2018). In the soil, A. euteiches oospores can persist for many years without the presence of host crops, and at high levels can remain hazardous to susceptible species for up to ten years (Pfender and Hagedorn, 1983; Willsey et al., 2018). Managing A. euteiches is difficult due to a lack of approved seed treatments, effective conventional control measures, and availability of resistant cultivars (Wu et al., 2019; Gaulin et al., 2007). Currently, the
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Corresponding author. E-mail address: [email protected] (L.D. Bainard). 1 Retired Researcher. https://doi.org/10.1016/j.apsoil.2019.103470 Received 20 June 2019; Received in revised form 6 December 2019; Accepted 10 December 2019 0929-1393/ Crown Copyright © 2019 Published by Elsevier B.V. All rights reserved.
Please cite this article as: Erin M. Karppinen, et al., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.103470
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collected from 48 native and tame (seeded) rangeland fields using the same sampling strategy as the annually cropped fields. The plant species composition in the native and tame rangelands varied across the regions, but were primarily composed of grass and legume mixtures. At each sampling point across all land uses, a soil core was collected to a depth of 10 cm with a diameter of 5–7 cm. Soil samples were kept in coolers with ice packs at the time of sampling and then kept in cold storage (−20 °C) until shipping. Location, plant species, general topography, soil type, tillage, and current and previous crop were recorded for all samples. Soil samples were shipped on ice to the Swift Current Research and Development Center in Swift Current, Saskatchewan, Canada for processing. Soils were homogenized and a subsample was stored at −80 °C for quantitative polymerase chain reaction (qPCR) analysis. The remaining soil from each sample was sieved to 2 mm and air-dried for chemical and physical analyses.
only effective methods of controlling A. euteiches root rot are to lengthen crop rotations and avoid seeding susceptible pulse crops in infected soils (Wu et al., 2019; Gaulin et al., 2007). Other possible control methods include ensuring proper drainage, avoiding clay-heavy soils, using conservation tillage methods (Sturz et al., 1997), and minimizing soil compaction (Gossen et al., 2016). Climatic conditions, crop rotations, and cultural practices (Banniza et al., 2013; Slinkard et al., 1994; Tu, 1992), along with other physical and chemical soil properties, may influence the abundance of A. euteiches oospores in agricultural soils. A. euteiches has been found world-wide, including in the United States, France, Scandinavia, New Zealand and Australia (Allen et al., 1987; Didelot and Chaillet, 1995; Dreschsler and Jones, 1925; Levenfors, 2003; Levenfors et al., 2003; Manning and Menzies, 1980; Papavizas and Ayers, 1974; Persson et al., 1997; Sundheim, 1972; Wicker et al., 2001; Zitnick-Anderson and Pasche, 2016). Given the global distribution of A. euteiches, and its documented presence in areas of the USA that neighbour Saskatchewan, it is not surprising that this organism is also found in Saskatchewan. The wide-spread distribution of A. euteiches makes identification of a geographic source for the pathogen elusive; however, phylogenetic clustering of A. eutieches linking crop host and geographical origin have been documented (Le May et al., 2018; Levenfors and Fatehi, 2004). There have been reports of A. euteiches in Canada since 1948 (Canadian Plant Diesease Survey, 1948), but this pathogen was only recently confirmed in pea and lentil fields across northwest and southwest Saskatchewan in 2012 (Banniza et al., 2013). A. euteiches was likely active in Saskatchewan prior to 2012, but was finally detected due to wetter than average conditions combined with advances in molecular techniques (Banniza et al., 2013; Willsey et al., 2018). Increased pea and lentil production is also likely to have led to a build-up of A. euteiches inoculum in Saskatchewan soils. Recent field surveys in pea and lentil fields in Alberta, Saskatchewan, and Manitoba have revealed the presence and potentially widespread distribution of A. euteiches across the Canadian prairie region (Chatterton et al., 2019; Esmaeili Taheri et al., 2017). Although A. euteiches root rot has been confirmed in Saskatchewan, the distribution of this pathogen across the agricultural regions of the province is unknown and it is unclear whether it is native to the region. This study utilized a large scale survey across the major agricultural regions and soil types of Saskatchewan, including native rangeland that has never been broken, to gain a better understanding of the distribution of A. euteiches. The specific objectives of this study were to (i) determine the effect of land use types (annual cropland, roadside ditches, and rangeland), soil types (Brown, Dark Brown, Black, and Dark Gray Chernozems), and crop management practices (current crop, previous crop, and tillage) on A. euteiches abundance, (ii) identify soil properties that are correlated with A. euteiches abundance, and (iii) determine if A. euteiches abundance in soil is correlated with root rot severity in pea and lentil crops.
2.2. Soil chemical and physical analyses A range of chemical and physical characteristics were quantified for each soil sample. Soil pH and electrical conductivity (EC) were determined using water saturation paste (Hendershot et al., 2008) and paste extracts (Miller and Curtin, 2008). Soil moisture was assessed using the gravimetric method. The dry combustion method was used to determine total C, total N, and organic C (after acidification with HCl) using an Elementar vario MICRO cube elemental analyzer (Schumacher, 2002). Nitrate, phosphate (Olsen P), and potassium concentrations were determined using sodium bicarbonate extractions followed by colorimetric analysis using a Technicon Autoanalyzer (Gentry and Willis, 1988; Hamm et al., 1970). Sulphate concentration was determined using 0.01 mol L−1 CaCl2 extracts followed by colorimetric analysis (Hamm et al., 1973). To determine soil texture, the hydrometer method was used to assess particle size distribution (Gee and Bauder, 1979). 2.3. Molecular analysis Genomic DNA was extracted from 0.3 g of soil using the DNeasy PowerSoil Kit (QIAgen) using a QIAcube automated system (QIAgen). DNA concentration was quantified using a Qubit 2.0 Fluorometer (Invitrogen). DNA extracts were used to detect the presence and quantify the abundance of A. euteiches in soil samples using the qPCR assay developed by Willsey et al. (2018). This qPCR assay is highly specific to A. euteiches, except for the amplification of the non-target species Aphanomyces cladogamus, which had a higher threshold cycle (Ct) value (31.2 vs. 25.8) compared to A. euteiches (Willsey et al., 2018). The primers (Ae1.2_ITS1) and probe (see Supplementary Table S1 for sequences) target the rRNA internal transcribed spacer (ITS1) region of A. euteiches. For qPCR gene amplification, 2 μL of DNA template was added to a 20 μL final volume mixture containing 10 μL of PrimeTime® Gene Expression Master Mix 2× (Integrated DNA Technologies), 5.5 μL of nuclease free water, 1 μL of each 10 μM primer (Integrated DNA Technologies), and 0.5 μL of probe (Integrated DNA Technologies). Reactions were run on the Rotor-Gene Q real-time PCR cycler (QIAgen) using the Rotor-Disc 100 (QIAgen) format. The qPCR amplification conditions were as follows: initial denaturing for 3 min at 95 °C, then 40 cycles of 15 s denaturing at 95 °C and 1 min annealing/extension at 60 °C, followed by a melt curve analysis. A standard curve prepared in duplicate, ranging in concentration from 101 to 107 gene copies μL−1, was used to quantify samples. Each sample was amplified in duplicate and no template controls were included with each run. The mean R2 value for the standard curve across all the qPCR assays was 0.993 and they had an efficiency of 98.2%.
2. Materials and methods 2.1. Sample collection and processing Soil samples were collected throughout the major land use types (annual cropland, roadside ditches, and rangeland) and soil types (Brown, Dark Brown, Gray, Dark Gray, and Black Chernozems) across all the major agricultural regions of Saskatchewan, Canada in 2016. Samples were collected from 142 annually cropped fields that were either seeded to peas (77 fields) or lentils (65 fields) and 135 of their associated adjacent roadside ditches. Samples were taken after first flowering of the crop (late June and early July). In every annually cropped field, five soil samples were collected, each 50 m apart in a ‘W’ pattern. At each roadside, three samples were collected at 50 m intervals. Roadside ditch samples provided a measure to understand whether pulse crops have increased the abundance of A. euteiches in agricultural fields relative to uncropped areas. Soil samples were also
2.4. Disease severity index Root rot severity was scored on all pea and lentil plants. The disease 2
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abundance in roadside samples was also positively correlated (P < 0.05) with all of these factors with the exception of NO3-N, although these factors had a weaker association with A. euteiches abundance compared to the annual crop samples. For example, soil moisture was correlated with A. euteiches abundance in both annual crop and roadside soils but had a stronger association in annual field soils (r = 0.43 in field soils and r = 0.24 in roadside soils). A. euteiches abundance was negatively correlated (P < 0.05) with sand content in annual field soils, and soil pH in roadside soils. A negative correlation between A. euteiches abundance and total N in tame rangeland was the only significant relationship observed for any of the soil chemical or physical properties measured in rangeland samples. The means and standard errors of chemical and physical soil properties measured in different land use types are provided in Supplementary Table S2.
severity index was based on a 1 to 7 scale (e.g. 1 = low, 7 = severe) and involved a visual assessment of symptoms such as root discoloration and reduction of root size (Bilgi et al., 2008). This method is a general assessment of root rot, and encompasses similar symptoms from other pathogens such as Fusarium species (Gossen et al., 2016). 2.5. Statistical analysis Analysis of variance (ANOVA) was used to test the effects of land use type, soil type, and their interaction on A. euteiches (Ae1.2_ITS1) abundance. ANOVA was also used to assess the effects of different crop management practices in annually cropped fields, which included the effect of current crop (pea vs. lentil), previous crop (canola vs. cereal), and tillage (cultivation vs. direct seeding). ANOVA was also used to test the effect of native vs. tame rangeland on A. euteiches abundance. Data was tested for assumptions of normality and homogeneity of variance and log transformed if necessary, and type III sums of squares was used for unbalanced data sets. Means separation was performed using Tukey's HSD multiple comparisons test. Linear regression analysis was used to examine the relationship between (i) A. euteiches abundance in soil and disease severity index, and (ii) A. euteiches abundance in annual cropland soils and their associated roadsides. Pearson correlation was used to examine the relationship between A. euteiches abundance and soil chemical and physical properties. All statistical analyses were conducted and visualized in R v. 3.5.1 (R Core Team, 2017).
3.4. Disease severity Pea fields had a significantly (P < 0.001) higher root rot disease severity index (DSI) compared to lentil fields (pea field DSI = 3.63 ± 0.17 s.e., lentil field DSI = 2.93 ± 0.11 s.e.). In addition, the abundance of A. euteiches had a significant positive relationship with DSI in pea fields, but no significant relationship was observed in lentil fields (Fig. 4). 4. Discussion
3. Results
The wide distribution and geographic spread of A. euteiches across both annually cropped fields and rangeland suggests that this microorganism was present in Saskatchewan prior to its detection in 2012. As noted by Banniza et al. (2013), the particularly wet conditions in 2012 and the preceding years likely favored the development and detection of A. euteiches root rot, along with the advent of improved detection methods. Our results also support the widespread detection of A. euteiches in pea and lentil fields across the Canadian prairie region where these pulse crops are grown (Chatterton et al., 2019). In annual fields, the increased planting of susceptible pulse crops has likely increased the prevalence and inoculum levels of this crop pathogen in the soil. The positive identification of A. euteiches in native and tame rangelands also indicates that other native and cultivated legumes other than pea and lentil can act as hosts for this microorganism. Alfalfa is a known host for A. euteiches (Delwiche et al., 1987; Vandemark et al., 2002; Wicker et al., 2001), as are other tame forages such as red clover (Trifolium pratense) and white clover (Trifolium repens) (Grau et al., 1991). Common native perennial legumes in Saskatchewan rangeland have also been identified as hosts for A. euteiches in the United States, including white prairie clover (Dalea candida), purple prairie clover (Dalea purpurea), and Canadian milk vetch (Astragalus canadensis), which led the authors to suggest that A. euteiches might be an indigenous pathogen (Malvick et al., 2009). The limited relationship with soil properties in rangeland suggests that the distribution and abundance of A. euteiches is likely dependent on plant composition and presence of susceptible plant hosts. This study revealed that A. euteiches is not limited to any agricultural region or soil type in Saskatchewan because it was consistently detected across all the major soil types and agricultural land use types in the province. However, we did find that land use type is a significant factor affecting the abundance of A. euteiches in soil. Not surprisingly, annual field soils containing highly susceptible host crops (pea and lentil) had the highest A. euteiches abundance. Although A. euteiches abundance was significantly lower in roadside soils when compared to field soils, A. euteiches abundance was positively correlated between the two land use types. A. euteiches lacks a mechanism for long-distance dispersal (Grünwald and Hoheisel, 2006); therefore, host species, either native or transferred from cultivated fields, were likely present in roadside ditches. Lower abundance of A. euteiches in rangeland soil compared to the annually cropped fields is not surprising, considering
3.1. A. euteiches distribution across land use and soil types A. euteiches (Ae1.2_ITS1) was detected in all land use types (lentil and pea fields, roadside ditches, native and tame rangeland) and soil types (Brown, Dark Brown, Gray, Dark Gray, and Black) included in this study (Fig. 1). Land use type was a significant factor affecting A. euteiches abundance (Table 1). A. euteiches abundance was significantly higher in annually cropped fields than in adjacent roadsides or in rangeland, while there was no difference between roadsides and rangeland (Fig. 2). A. euteiches abundance in annual fields and their associated roadsides was positively correlated (Fig. 3, R2 = 0.294). Soil type and the soil type by land use type interaction had no significant effect on the abundance of A. euteiches (Table 1). These results indicated that A. euteiches is widespread across the agricultural regions of Saskatchewan and is not limited to annually cropped fields or specific soil types, as it was commonly detected in both native and tame rangeland and all soil types. 3.2. Crop management practices In annual fields, the current crop (i.e., pea vs. lentil) had a significant effect on the abundance of A. euteiches. Pea crops had a significantly higher A. euteiches abundance than lentil crops (Supplementary Fig. S1). None of the other crop management practices (i.e., previous crop = oilseed vs. cereal; tillage = direct seeding vs. cultivation) in annual fields had a significant effect on A. euteiches abundance (Table 1). The management of rangeland (native vs. tame) did have a significant effect on the abundance of A. euteiches as higher levels were detected in native compared to tame pastures (Supplementary Fig. S2). 3.3. Soil properties The abundance of A. euteiches was correlated with several chemical and physical soil properties that varied by land use type (Table 2). In annually cropped field samples, A. euteiches abundance was positively correlated (P < 0.05) with NO3−-N, PO43−-P, total and organic carbon, total nitrogen, soil moisture, and clay content. A. euteiches 3
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log Ae1.2_ITS copies g-1 soil
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log Ae1.2_ITS copies g-1 soil
(caption on next page)
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Fig. 1. Distribution and abundance of Aphanomyces euteiches (log10 Ae1.2_ITS1 copies g−1 dry soil) across agricultural land use (field, rangeland) and soil (brown, dark brown, gray, dark gray, black) types of Saskatchewan. Field: n = 142; Rangeland: n = 48. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 Effect of land use type, soil type and crop management practices on Aphanomyces euteiches gene abundance in agricultural soil. Crop management practices were tested independently within their respective land use types. Factor
df
F value
Pr (> F)
Land use type Soil type Land use type ∗ soil type Field Field vs. roadside Crop (pea vs. lentil) Previous crop (canola vs. cereal) Tillage (cultivation vs. direct seeding) Rangeland Native vs. tame
2 3 6
20.1 2.13 1.35
< 0.0001 0.096 0.234
1 1 1 1
36.8 9.15 0.25 0.74
< 0.0001 0.003 0.62 0.39
1
4.48
0.040
Table 2 Pearson correlation coefficient (r) values between Aphanomyces euteiches abundance (log10 Ae1.2_ITS1 copies g−1 dry soil) and soil chemical and physical properties. Bold text indicates significance at P < 0.05.
log10 Ae1.2_ITS1 copies g-1 dry soil
a
Factor
Field (n = 142)
Roadside (n = 135)
Native (n = 14)
Tame (n = 29)
SO42−-S K NO3−-N PO43−-P Total C Organic C Total N Soil moisture pH EC Sand Clay Silt
−0.06 0.07 0.20 0.23 0.37 0.37 0.40 0.43 −0.13 0.06 −0.25 0.28 −0.05
−0.02 0.16 0.14 0.18 0.23 0.32 0.31 0.24 −0.19 −0.15 −0.05 0.09 −0.14
−0.18 0.45 0.24 0.41 −0.22 0.00 −0.26 0.15 0.02 −0.29 −0.06 −0.03 0.14
0.04 −0.32 0.19 −0.16 −0.22 −0.25 −0.49 −0.23 0.34 0.20 0.24 −0.13 −0.12
n = number of samples; EC = electrical conductivity.
Field
b
b
Roadside
Rangeland
log10 Ae1.2_ITS1 copies g-1 dry field soil
Fig. 2. Aphanomyces euteiches abundance (log10 Ae1.2_ITS1 copies g−1 dry soil) in field, roadside and rangeland samples. Bars are means (Field: n = 142; Roadside: n = 135; Rangeland: n = 47) with standard errors. Different letters indicate significant differences between land use type (ANOVA, df = 2, P < 0.0001).
P < 0.0001 Adj. R2 = 0.294
6
4
2
0 0
6 2 4 log10 Ae1.2_ITS1 copies g-1 dry roadside soil Fig. 4. Relationship between Aphanomyces euteiches (log10 Ae1.2_ITS1 copies g−1 dry soil) abundance in field soil and disease severity index of (a) pea and (b) lentil plants. Ae1.2_ITS1 abundance was quantified with quantitative polymerase chain reaction (qPCR) assays and disease severity index was manually scored on affected plants.
Fig. 3. Relationship between Aphanomyces euteiches (log10 Ae1.2_ITS1 copies g−1 dry soil) abundance in fields and their associated roadsides.
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have been particularly relevant in annual fields where we observed a significant positive relationship between A. euteiches abundance and disease severity in peas. Future research on how A. euteiches abundance changes over the growing season and relates to risk of disease development in subsequent years could facilitate more accurate determination of the risk of planting a susceptible pulse crop in a given field. The higher susceptibility of peas to A. euteiches than lentils is in accordance with reports that emphasize the prevalence and severity of the disease to pea crops in particular (Chatterton et al., 2019; Basu et al., 1973; Tu, 1992). Although partial resistance has been reported in some pea (Wu et al., 2019; Conner et al., 2013) and lentil (Bazghaleh et al., 2018) cultivars, there are no cultivars completely resistant to A. euteiches. In addition to pea and lentil, other legumes including alfalfa, snap bean, vetch, clover, sweet clover, and numerous weed species can be host to A. euteiches (Papavizas and Ayers, 1974). Other crop management practices (i.e., tillage and previous crop) that were expected to influence A. euteiches abundance were not significant factors. For instance, tillage practices that cause soil compaction are expected to increase soil moisture and increase A. euteiches root rot (Wu et al., 2018), but tillage type was not significant in this study. In regards to the impact of previously seeded crops, rotations that incorporate cruciferous plants (e.g. Brassica napus, canola) have been used to reduce disease severity in A. euteiches infested soils (Chan and Close, 1987). However, brassica species may differ in their ability to fulfill this function. Brassica juncea (brown mustard) and Sinapis alba (white mustard) differ in the capacity of their associated glucosinolates and their break-down products, isothiocyanates, to inhibit A. euteiches in vitro (Hossain et al., 2014) or to reduce Aphanomyces root rot in pot studies (Hossain et al., 2015). Thus, the capacity of canola varieties with varying glucosinolate content merits further investigation. As previous crop was not a significant factor in our study, excluding pea and, to a lesser extent, lentil, from crop rotations in fields with high inoculum potential or confirmed disease continues to be the recommended crop management practice to control A. euteiches root rot. Along with A. euteiches, the qPCR primers and probe used in the study have the potential to amplify A. cladogamus DNA (Willsey et al., 2018). A. cladogamus, is a soil-borne oomycete plant pathogen, capable of causing disease on spinach and sugar beets (Larsson, 1994), as well as pansies (Drechsler, 1954), tomato, eggplant and pepper (McKeen, 1952). The prevalence of A. cladogamus in Saskatchewan is not well characterized, but there is some evidence that it is present in the Canadian prairie region (Esmaeili Taheri et al., 2017). However, a recent field study conducted across nine sites in Saskatchewan using amplicon metagenomic sequencing of oomycete communities found that A. cladogamus was rarely detected (i.e., < 5% samples and 0.1% of total reads) of the soil samples (Bainard et al. unpublished data). A high proportion of the Ae1.2_ITS1 copies we detected in our samples were likely from A. euteiches because the high Ct value for A. cladogamus using pure culture DNA reported by Willsey et al. (2018) represents a difference in copy numbers of approximately two orders of magnitude and was approaching the detection limit for this qPCR assay. Further research is required to determine if A. cladogamus is found throughout Saskatchewan and whether the Ae1.2_ITS1 qPCR assay successfully amplifies this microorganism in soil samples. Despite its recent discovery in Saskatchewan, this study demonstrated that A. euteiches is widespread across the province, land use and soil types, and supports the hypothesis that A. euteiches was present prior to its discovery in 2012. In addition, the frequent detection in native rangeland soil indicates that A. euteiches is likely indigenous to this region. The more frequent use of pulse crops combined with higher than average levels of precipitation in the region have likely increased inoculum levels in annually cropped field soils. Overall, this study highlights that pulse crop root rot issues will persist or expand in the future due to the widespread distribution of this pathogen throughout the agricultural regions in Saskatchewan.
that native and tame rangeland host plant species occur in polyculture rather than monoculture. A diverse mixture of host crop species would be expected to vary in disease inoculum potential and virulence (Moussart et al., 2013; Grau et al., 1991). Interestingly, given the previously-documented link between finetextured soil and increased Aphanomyces root rot risk (Allmaras et al., 2003; Fritz et al., 1995; Gossen et al., 2016; Kraft et al., 1990; Papavizas and Ayers, 1974; Pfender and Hagedorn, 1983), soil type was not a significant factor affecting the abundance and distribution of A. euteiches in Saskatchewan. This supports the results from previous root rot field surveys conducted between 2015 and 2017 in Saskatchewan, which also found no effect of soil type on the frequency of pea fields that tested positive for A. euteiches (Chatterton et al., 2019). Soil moisture increases across the province in a southwest to northeast gradient and roughly corresponds with the different soil types. Based on the positive correlation between soil moisture and A. euteiches abundance, a significant relationship with soil type was also expected. However, above-average levels of precipitation across the province during the 2016 growing season (Chatterton et al., 2019) may have provided generally favourable conditions for A. euteiches root rot development and limited the effect of soil type on the abundance of this pathogen. These results suggest that land use type and presence of susceptible plant hosts has a greater impact on A. euteiches inoculum potential than abiotic soil properties. There were four common soil abiotic factors (soil moisture, total N, total and organic C) that positively correlated with A. euteiches abundance in field and roadside samples, but soil moisture had the strongest relationship with A. euteiches abundance. Higher moisture levels facilitate the spread of A. euteiches as it encourages the development of sporangia, and the released zoospores are flagellated and will move through moisture films around roots (Wu et al., 2018). Organic carbon content has also been shown to aid in soil water retention, which would enhance zoospore movement (Rawls et al., 2003). In past studies, A. euteiches was predominantly found in heavy clay soils or poorly drained areas with increased soil moisture (Persson and Olsson, 2000; Kraft et al., 1990; Papavizas and Ayers, 1974; Pfender and Hagedorn, 1983). In both annual field and roadside soils, A. euteiches abundance was negatively correlated with sand content and positively correlated with clay content, but this relationship was only significant in annual field soils. Soil texture is directly related to soil moisture (Gupta and Larson, 1979); therefore, it is not surprising this relationship was observed. Nitrogen fertilization has been reported to reduce root rot caused by A. euteiches (Papavizas and Lewis, 1971) by modifying the morphology of pea roots so they inhibit pathogen colonization (Hossain et al., 2015), but our results show a positive relationship between A. euteiches abundance and NO3−-N and total N in field soils. However, we did detect a significant negative relationship between A. euteiches abundance and total N in tame rangeland. Some of the variability between our results and previous studies may be related to the techniques used to measure disease severity or inoculum potential. Recent advances in molecular techniques have allowed accurate and efficient quantification of A. euteiches inoculum levels in soil (Willsey et al., 2018; Gangneux et al., 2014) while traditional techniques have consisted of microscopic observations and/or greenhouse experiments (Moussart et al., 2013; Papavizas and Lewis, 1971). In this study, the significant factors affecting A. euteiches abundance differed between land use type, which also highlights the complexity of identifying a single factor (or combination of factors) that could be used to control or anticipate inoculum potential across a range of agricultural soils. There was a significant difference in root rot severity and A. euteiches abundance between annual fields with peas compared to lentils. These results suggest that peas are more susceptible to root rot or colonization by A. euteiches, and/or promote higher growth of the pathogen compared to lentils. Amplification of A. euteiches likely represented more than just the oospores (e.g., mycelia and zoospores) in the soil since samples were collected mid-growing season. This may 6
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Declaration of competing interest
ammonium in soil extracts. Commun. Soil Sci. Plant Anal. 19, 721–737. Gossen, B.D., Conner, R.L., Chang, K.-F., Pasche, J.S., McLaren, D.L., Henriquez, M.A., Chatterton, S., Hwang, S.-F., 2016. Identifying and managing root rot of pulses on the Northern Great Plains. Plant Pathol. 100, 1965–1978. Grau, C.R., Muehlchen, A.M., Tofte, J.E., Smith, R.R., 1991. Variability in virulence of Aphanomyces euteiches. Plant Dis. 75, 1153–1156. Grünwald, N.J., Hoheisel, G.-A., 2006. Heirarchical analysis of diversity, selfing, and genetic differentiation in populations of the oomycete Aphanomyces euteiches. Phytopathology 96, 1134–1141. Gupta, S.C., Larson, W.E., 1979. Estimating soil water retention characteristics from particle size distribution, organic matter percent, and bulk density. Water Resour. Res. 15, 1633–1635. Hamm, J.W., Radford, F.G., Halstead, E.H., 1970. The simultaneous determination of nitrogen, phosphorus, and potassium in sodium bicarbonate extracts of soils. In: Advances in Automatic Analysis. Industrial Analysis, Vol. II. Technicon International Congress. Futura Publishing Co, Mount Kisco, NY, pp. 65–69. Hamm, J.W., Bettany, J.R., Halstead, E.H., 1973. A soil test for sulphur and interpretative criteria for Saskatchewan. Commun. Soil Sci. Plan. 4, 219–231. Hendershot, W.H., Lalande, H., Duquette, M., 2008. Ion exchange and exchangeable cations. In: Carter, M.R., Gregorich, E.G. (Eds.), Soil Sampling and Methods of Analysis. Canadian Society of Soil Science, Boca Raton, FL, pp. 197–206. Hossain, S., Bergkvist, G., Berglund, K., Glinwood, R., Kabouw, P., Martensson, A., Persson, P., 2014. Concentration- and time-dependent effects of isothiocyanates produced from Brassicaceae shoot tissues on the pea root rot pathogen Aphanomyces euteiches. J. Agric. Food Chem. 62, 4584–4591. Hossain, S., Bergkvist, G., Glinwood, R., Berglund, K., Martensson, A., Hallin, S., Persson, P., 2015. Brassicaceae cover crops reduce Aphanomyces pea root rot without suppressing genetic potential of microbial nitrogen cycling. Plant Soil 392, 227–238. Kraft, J.M., Marcinkowska, J., Muehlbauer, J., 1990. Detection of Aphanomyces euteiches in field soil from northern Idaho by a wet-sieving/baiting technique. Plant Dis. 74, 716–718. Larsson, M., 1994. Pathogenicity, morphology and isozyme variability among isolates of Aphanomyces spp. from weeds and various crop plants. Mycol. Res. 98, 231–240. Le May, C., Onfroy, C., Moussart, A., Andrivon, D., Baranger, A., Pilet-Nayal, M.L., Vandemark, G., 2018. Genetic structure of Aphanomyces euteiches populations sampled from United States and France pea nurseries. Eur. J. Plant Pathol. 150, 275–286. Levenfors, J.P., 2003. Soil-Borne Pathogens in Intensive Legume Cropping - Aphanomyces spp. and Root Rots (Doctoral thesis). Swedish University of Agricultural Sciences, Uppsala, Sweden. Levenfors, J.P., Fatehi, J., 2004. Molecular characterization of Aphanomyces species associated with legumes. Mycol. Res. 108, 682–689. Levenfors, J.P., Wikstrom, M., Persson, L., Gerhardson, B., 2003. Pathogenicity of Aphanomyces spp. from different leguminous crops in Sweden. Eur. J. Plant Pathol. 109, 535–543. Malvick, D.K., Grünwald, N.J., Dyer, A.T., 2009. Population structure, races, and host range of Aphanomyces euteiches from alfalfa production fields in the central USA. Eur. J. Plant Pathol. 123, 171–182. Manning, M.A., Menzies, S.A., 1980. Root rot of peas in New Zealand caused by Aphanomyces euteiches. N. Z. J. Agric. Res. 23, 263–265. McKeen, C.D., 1952. Aphanomyces cladogamus Drech., a cause of damping-off in peppers and certain other vegetables. Can. J. Bot. 30, 701–709. Miller, J.J., Curtin, D., 2008. Electrical conductivity and soluble ions. In: Carter, M.R., Gregorich, E.G. (Eds.), Soil Sampling and Methods of Analysis. Canadian Society of Soil Science, Boca Raton, FL, pp. 161–171. Miller, P.R., McConkey, B.G., Clayton, G.W., Brandt, S.A., Staricka, J.A., Johnston, A.M., Lafond, G.P., Schatz, B.G., Baltensperger, D.D., Niell, K.E., 2002. Pulse crop adaptation in the Northern Great Plains. Agron. J. 94, 261–272. Moussart, A., Even, M.N., Lesné, A., Tivoli, B., 2013. Successive legumes tested in a greenhouse crop rotation experiment modify the inoculum potential of soils naturally infested by Aphanomyces euteiches. Plant Pathol. 62, 545–551. Niu, Y., Bainard, L.D., Bandara, M., Hamel, C., Gan, Y., 2017. Soil residual water and nutrients explain about 30% of the rotational effect in 4-yr pulse-intensified rotation systems. Can. J. Plant Sci. 97, 852–864. Niu, Y., Bainard, L.D., Hossain, Z., May, W.E., Hamel, C., Gan, Y., 2018. Intensified pulse rotations buildup pea rhizosphere pathogens in cereal and pulse based cropping systems. Front. Microbiol. 9, 1909. Papavizas, G.C., Ayers, W.A., 1974. Aphanomyces species and their root diseases in pea and sugar beet. In: U.S. Department of Agriculture Technical Bulletin. 1484. pp. 158. Papavizas, G.C., Lewis, J.A., 1971. Effect of amendments and fungicides on Aphanomyces root rot of peas. Phytopathology 61, 215–220. Persson, L., Olsson, S., 2000. Abiotic characteristics of soils suppressive to Aphanomyces root rot. Soil Biol. Biochem. 32, 1141–1150. Persson, L., Bodker, L., Larsson-Wikstrom, M., 1997. Prevalence and pathogenicity of foot and root rot pathogens of pea in southern Scandinavia. Plant Dis. 81, 171–174. Pfender, W.F., Hagedorn, D.J., 1983. Disease progress and yield loss in Aphanomyces root rot of peas. Phytopathology 73, 1109–1113. R Core Team, 2017. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Rawls, W.J., Pachepsky, Y.A., Ritchie, J.C., Sobecki, T.M., Bloodworth, H., 2003. Effect of soil organic carbon on soil water retention. Geoderma 116, 61–76. Schumacher, B.A., 2002. Methods for the Determination of Total Organic Carbon (TOC) in Soils and Sediments. Ecological Risk Assessment Support Center, US Environmental Protection Agency. Slinkard, A.E., Bascur, G., Hernandez-Bravo, G., 1994. Biotic and abiotic stresses of cool season food legumes in the western hemisphere. In: Murhlbauer, F.J., Kaiser, W.J. (Eds.), Expanding the Production and Use of Cool Season Food Legumes. Kluwer
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This project was funded by Saskatchewan Pulse Growers. We gratefully thank the assistance provided by Cam Kenny and technical staff and students at the Swift Current Research and Development Centre and all of the volunteers (especially the Saskatchewan Ministry of Agriculture) that helped with the field sampling. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2019.103470. References Allen, R.N., Letham, D.B., Akehurst, A.A., Say, M.M., 1987. Aphanomyces root rot of bean at Valla, New South Wales. Australas. Plant Pathol. 6, 82–84. Allmaras, R.R., Fritz, V.A., Pfleger, F.L., Copeland, S.M., 2003. Impaired internal drainage and Aphanomyces euteiches root rot of pea caused by soil compaction in a fine-textured soil. Soil Tillage Res. 70, 41–52. Bainard, L.D., Navarro-Borrell, A., Hamel, C., Braun, K., Hanson, K., Gan, Y., 2017. Increasing the frequency of pulses in crop rotations reduces soil fungal diversity and increases the proportion of fungal pathotrophs in a semiarid agroecosystem. Agric. Ecosyst. Environ. 240, 206–214. Banniza, S., Bhadauria, V., Peluola, C.O., Armstrong-Cho, C., Morrall, R.A.A., 2013. First report of Aphanomyces euteiches in Saskatchewan. Can. Plant Dis. Surv. 93, 163–164. Basu, P.K., Crête, R., Donaldson, A.G., Gourley, C.O., Haas, J.H., Harper, F.R., Lawrence, C.H., Seaman, W.L., Toms, H.N.W., Wong, S.I., Zimmer, R.C., 1973. Prevalence and severity of diseases processing peas in Canada, 1970-71. Can. Plant Dis. Surv. 53, 49–57. Bazghaleh, N., Prashar, P., Purves, R.W., Vandenberg, A., 2018. Polyphenolic composition of lentil roots in response to infection by Aphanomyces euteiches. Front. Plant Sci. 9, 1131. Bilgi, V.N., Bradley, C.A., Khot, S.D., Grafton, K.F., Rasmussen, J.B., 2008. Response of dry bean genotypes to Fusarium root rot, caused by Fusarium solani f. sp. phaseoli, under field and controlled conditions. Plant Dis. 92, 1197–1200. Canadian Plant Diesease Survey, 1948. Diseases of vegetable and field crops. In: Conners, I.L., Savile, D.B.O. (Eds.), Twenty-eighth Annual Report of the Canadian Plant Disease Survey. Canadian Phytopathological Society, Ottawa, Canada, pp. 36–72. Chan, M.K.Y., Close, R.C., 1987. Aphanomyces root rot of peas 3. Control by the use of cruciferous amendments. New Zeal. J. Agr. Res. 30, 225–233. Chatterton, S., Harding, M.W., Bowness, R., McLaren, D.L., Banniza, S., Gossen, B.D., 2019. Importance and causal agents of root rot on field pea and lentil on the Canadian prairies, 2014–2017. Can. J. Plant Pathol. 41, 98–114. Conner, R.L., Chang, K.F., Hwang, S.F., Warkentin, T.D., McRae, K.B., 2013. Assessment of tolerance for reducing yield losses in field pea caused by Aphanomyces root rot. Can. J. Plant Sci. 93, 473–482. Delwiche, P.A., Grau, C.R., Holub, E.B., Perry, J.B., 1987. Characterization of Aphanomyces euteiches isolates recovered from alfalfa in Wisconsin. Plant Dis. 71, 155–161. Didelot, D., Chaillet, I., 1995. Relevance and interest of root disease prediction tests for pea crop in France. In: 2nd Eur Conf Grain Legumes—Improving Production and Utilisation of Grain Legumes. Drechsler, C., 1954. Association of Aphanomyces cladogamus with severe root rot of pansies. Sydowia 8, 334–342. Dreschsler, C., Jones, F.R., 1925. Boot rot of peas in the United States caused by Aphanomyces euteiches (n. sp.). J. Agric. Res. 30, 293–325. Esmaeili Taheri, A., Chatterton, S., Gossen, B.D., McLaren, D.L., 2017. Metagenomic analysis of oomycete communities from the rhizosphere of field pea on the Canadian prairies. Can. J. Microbiol. 63, 758–768. Fritz, V.A., Allmaras, R.R., Pfleger, F.L., Davis, D.W., 1995. Oat residue and soil compaction influences on common root rot (Aphanomyes euteiches) of peas in a fine-textured soil. Plant Soil 235–244. Gangneux, C., Cannesan, M., Bressan, M., Castel, L., Moussart, A., Vicré-Gibouin, M., Driouich, A., Trinsoutrot-Gattin, I., Laval, K., 2014. A sensitive assay for rapid detection and quantification of Aphanomyces euteiches in soil. Phytopathology 104, 1137–1147. Gaulin, E., Jacquet, C., Bottin, A., Dumans, B., 2007. Root rot disease of legumes caused by Aphanomyces euteiches. Mol. Plant Pathol. 8, 539–548. Gee, G.W., Bauder, J.W., 1979. Particle size analysis by hydrometer: a simplified method for routine textural analysis and a sensitivity test of measurement parameters. Soil Sci. Soc. Am. J. 43, 1004–1007. Gentry, C.E., Willis, R.B., 1988. Improved method for automated determination of
7
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E.M. Karppinen, et al.
between the pea root rot pathogens Aphanomyces euteiches and Fusarium spp. using a multiplex qPCR assay. Plant Pathol. 67, 1912–1923. Wu, L., Chang, K.F., Conner, R.L., Strelkov, S., Fredua-Agyeman, R., Hwang, S.F., Feindel, D., 2018. Aphanomyces euteiches: a threat to Canadian field pea production. Engineering 4, 542–551. Wu, L., Chang, K.F., Hwang, S.F., Conner, R., Fredua-Agyeman, R., Feindel, D., Strelkov, S.E., 2019. Evaluation of host resistance and fungicide application as tools for the management of root rot of field pea caused by Aphanomyces euteiches. The Crop Journal 7, 38–48. Zhou, Y., Zhu, H., Fu, S., Yao, Q., 2017. Variation in soil microbial community structure associated with different legume species is greater than that associated with different grass species. Front. Microbiol. 8, 1007. Zitnick-Anderson, K., Pasche, J.S., 2016. First report of Aphanomyces root rot caused by Aphanomyces euteiches on field pea in North Dakota. Plant Dis. 100, 522.
Academic Publishers, The Netherlands, pp. 195–203. Sturz, A.V., Carter, M.R., Johnston, H.W., 1997. A review of plant disease, pathogen interactions and microbial antagonism under conservation tillage in temperate humid agriculture. Soil Till. Res. 41, 169–189. Sundheim, L., 1972. Physiologic specialization in Aphanomyces euteiches. Physiol. Plant Pathol. 2, 301–306. Tu, J.C., 1992. Management of root rot diseases of peas, beans and tomatoes. Can. J. Plant Pathol. 14, 92–99. Vandemark, G.J., Barker, B.M., Gritsenko, M., 2002. Quantifying Aphanomyces euteiches in alfalfa with a fluorescent polymerase chain reaction assay. Phytopathology 92, 265–272. Wicker, E., Hullé, M., Rouxel, F., 2001. Pathogenic characteristics of isolates of Aphanomyces euteiches from pea in France. Plant Pathol. 50, 433–442. Willsey, T.L., Chatterton, S., Heynen, M., Erickson, A., 2018. Detection of interactions
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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Distribution and abundance of Aphanomyces euteiches in agricultural soils: effect of land use type, soil properties, and crop management practices Erin M. Karppinena, Josephine Paymenta, Syama Chattertonb, Jillian D. Bainarda, ⁎ Michelle Hubbarda, Yantai Gana,1, Luke D. Bainarda, a b
Swift Current Research and Development Centre, Agriculture and Agri-Food Canada, 1 Airport Road, Swift Current, SK S9H 3X2, Canada Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB T1J 4B1, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Oomycete Root rot Pea Lentil Rangeland Quantitative PCR
Aphanomyces euteiches is a soil-borne pathogen that causes root rot in susceptible pulse crops. Although widespread across Canada, A. euteiches was not reported in Saskatchewan until 2012. The distribution of this pathogen across the Canadian prairies is unknown and it is unclear whether it is native to the region. The objective of this study was to conduct a survey of A. euteiches across the major agricultural soils of Saskatchewan, Canada to determine its distribution and to assess the relative importance of factors related to its distribution and possible spread across the province. Soil samples were collected from lentil and pea fields and their associated roadside ditches, as well as from native and tame rangelands. Soil samples were analyzed using a quantitative polymerase chain reaction (qPCR) assay to detect and quantify the presence of A. euteiches. In addition, root rot severity was assessed on affected pea and lentil plants, and the physical and chemical properties of the soils were measured. A. euteiches was present across all land use (i.e., annual cropland, roadside ditches, and rangeland) and soil types in Saskatchewan. A. euteiches abundance was highest in samples collected from annually cropped fields. Within annual cropland, A. euteiches abundance was higher in pea fields than lentil fields. Soil moisture, total and organic carbon, and total nitrogen were positively correlated to A. euteiches abundance in field and roadside soils. Based on this survey, A. euteiches is wide-spread across all the major agricultural regions of Saskatchewan and is not limited to specific soil or land use types. The frequent detection of A. euteiches in native rangeland indicates it is likely native to Saskatchewan. The significant relationship between A. euteiches gene abundance and disease severity in pea fields indicates that the qPCR assay may be a useful indicator for predicting the potential for certain agricultural soils to cause A. euteiches root rot in pea crops.
1. Introduction Pulses, such as peas and lentils, are an increasingly important crop for agricultural producers and have supported the diversification of crop rotations across the Canadian prairies. Pulse crops are grown for many reasons, including their relative tolerance to heat and drought (Miller et al., 2002), and their ability to increase the amount of total available nitrogen in the soil (Zhou et al., 2017), which can improve the yields of subsequent crops (Niu et al., 2017). However, increased frequency of pulses in crop rotations has been linked to decreased fungal diversity and build-up of pathotrophs in the soil (Bainard et al., 2017; Niu et al., 2018). As pulses continue to be incorporated into relatively short crop rotations, root rot has become a limiting factor in their production (Wu et al., 2018). In particular, root rot caused by the
oomycete pathogen Aphanomyces euteiches is becoming a major disease in field peas and lentils grown on the Canadian Prairies (Wu et al., 2018). The disease symptoms vary, but generally include honey-colored lesions on the root, which eventually collapse and cause the plant to yellow and wither (Gangneux et al., 2014). The effects of the disease are often exacerbated as A. euteiches weakens the plant, allowing opportunistic pathogens to also invade the root (Willsey et al., 2018). In the soil, A. euteiches oospores can persist for many years without the presence of host crops, and at high levels can remain hazardous to susceptible species for up to ten years (Pfender and Hagedorn, 1983; Willsey et al., 2018). Managing A. euteiches is difficult due to a lack of approved seed treatments, effective conventional control measures, and availability of resistant cultivars (Wu et al., 2019; Gaulin et al., 2007). Currently, the
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Corresponding author. E-mail address: [email protected] (L.D. Bainard). 1 Retired Researcher. https://doi.org/10.1016/j.apsoil.2019.103470 Received 20 June 2019; Received in revised form 6 December 2019; Accepted 10 December 2019 0929-1393/ Crown Copyright © 2019 Published by Elsevier B.V. All rights reserved.
Please cite this article as: Erin M. Karppinen, et al., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.103470
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collected from 48 native and tame (seeded) rangeland fields using the same sampling strategy as the annually cropped fields. The plant species composition in the native and tame rangelands varied across the regions, but were primarily composed of grass and legume mixtures. At each sampling point across all land uses, a soil core was collected to a depth of 10 cm with a diameter of 5–7 cm. Soil samples were kept in coolers with ice packs at the time of sampling and then kept in cold storage (−20 °C) until shipping. Location, plant species, general topography, soil type, tillage, and current and previous crop were recorded for all samples. Soil samples were shipped on ice to the Swift Current Research and Development Center in Swift Current, Saskatchewan, Canada for processing. Soils were homogenized and a subsample was stored at −80 °C for quantitative polymerase chain reaction (qPCR) analysis. The remaining soil from each sample was sieved to 2 mm and air-dried for chemical and physical analyses.
only effective methods of controlling A. euteiches root rot are to lengthen crop rotations and avoid seeding susceptible pulse crops in infected soils (Wu et al., 2019; Gaulin et al., 2007). Other possible control methods include ensuring proper drainage, avoiding clay-heavy soils, using conservation tillage methods (Sturz et al., 1997), and minimizing soil compaction (Gossen et al., 2016). Climatic conditions, crop rotations, and cultural practices (Banniza et al., 2013; Slinkard et al., 1994; Tu, 1992), along with other physical and chemical soil properties, may influence the abundance of A. euteiches oospores in agricultural soils. A. euteiches has been found world-wide, including in the United States, France, Scandinavia, New Zealand and Australia (Allen et al., 1987; Didelot and Chaillet, 1995; Dreschsler and Jones, 1925; Levenfors, 2003; Levenfors et al., 2003; Manning and Menzies, 1980; Papavizas and Ayers, 1974; Persson et al., 1997; Sundheim, 1972; Wicker et al., 2001; Zitnick-Anderson and Pasche, 2016). Given the global distribution of A. euteiches, and its documented presence in areas of the USA that neighbour Saskatchewan, it is not surprising that this organism is also found in Saskatchewan. The wide-spread distribution of A. euteiches makes identification of a geographic source for the pathogen elusive; however, phylogenetic clustering of A. eutieches linking crop host and geographical origin have been documented (Le May et al., 2018; Levenfors and Fatehi, 2004). There have been reports of A. euteiches in Canada since 1948 (Canadian Plant Diesease Survey, 1948), but this pathogen was only recently confirmed in pea and lentil fields across northwest and southwest Saskatchewan in 2012 (Banniza et al., 2013). A. euteiches was likely active in Saskatchewan prior to 2012, but was finally detected due to wetter than average conditions combined with advances in molecular techniques (Banniza et al., 2013; Willsey et al., 2018). Increased pea and lentil production is also likely to have led to a build-up of A. euteiches inoculum in Saskatchewan soils. Recent field surveys in pea and lentil fields in Alberta, Saskatchewan, and Manitoba have revealed the presence and potentially widespread distribution of A. euteiches across the Canadian prairie region (Chatterton et al., 2019; Esmaeili Taheri et al., 2017). Although A. euteiches root rot has been confirmed in Saskatchewan, the distribution of this pathogen across the agricultural regions of the province is unknown and it is unclear whether it is native to the region. This study utilized a large scale survey across the major agricultural regions and soil types of Saskatchewan, including native rangeland that has never been broken, to gain a better understanding of the distribution of A. euteiches. The specific objectives of this study were to (i) determine the effect of land use types (annual cropland, roadside ditches, and rangeland), soil types (Brown, Dark Brown, Black, and Dark Gray Chernozems), and crop management practices (current crop, previous crop, and tillage) on A. euteiches abundance, (ii) identify soil properties that are correlated with A. euteiches abundance, and (iii) determine if A. euteiches abundance in soil is correlated with root rot severity in pea and lentil crops.
2.2. Soil chemical and physical analyses A range of chemical and physical characteristics were quantified for each soil sample. Soil pH and electrical conductivity (EC) were determined using water saturation paste (Hendershot et al., 2008) and paste extracts (Miller and Curtin, 2008). Soil moisture was assessed using the gravimetric method. The dry combustion method was used to determine total C, total N, and organic C (after acidification with HCl) using an Elementar vario MICRO cube elemental analyzer (Schumacher, 2002). Nitrate, phosphate (Olsen P), and potassium concentrations were determined using sodium bicarbonate extractions followed by colorimetric analysis using a Technicon Autoanalyzer (Gentry and Willis, 1988; Hamm et al., 1970). Sulphate concentration was determined using 0.01 mol L−1 CaCl2 extracts followed by colorimetric analysis (Hamm et al., 1973). To determine soil texture, the hydrometer method was used to assess particle size distribution (Gee and Bauder, 1979). 2.3. Molecular analysis Genomic DNA was extracted from 0.3 g of soil using the DNeasy PowerSoil Kit (QIAgen) using a QIAcube automated system (QIAgen). DNA concentration was quantified using a Qubit 2.0 Fluorometer (Invitrogen). DNA extracts were used to detect the presence and quantify the abundance of A. euteiches in soil samples using the qPCR assay developed by Willsey et al. (2018). This qPCR assay is highly specific to A. euteiches, except for the amplification of the non-target species Aphanomyces cladogamus, which had a higher threshold cycle (Ct) value (31.2 vs. 25.8) compared to A. euteiches (Willsey et al., 2018). The primers (Ae1.2_ITS1) and probe (see Supplementary Table S1 for sequences) target the rRNA internal transcribed spacer (ITS1) region of A. euteiches. For qPCR gene amplification, 2 μL of DNA template was added to a 20 μL final volume mixture containing 10 μL of PrimeTime® Gene Expression Master Mix 2× (Integrated DNA Technologies), 5.5 μL of nuclease free water, 1 μL of each 10 μM primer (Integrated DNA Technologies), and 0.5 μL of probe (Integrated DNA Technologies). Reactions were run on the Rotor-Gene Q real-time PCR cycler (QIAgen) using the Rotor-Disc 100 (QIAgen) format. The qPCR amplification conditions were as follows: initial denaturing for 3 min at 95 °C, then 40 cycles of 15 s denaturing at 95 °C and 1 min annealing/extension at 60 °C, followed by a melt curve analysis. A standard curve prepared in duplicate, ranging in concentration from 101 to 107 gene copies μL−1, was used to quantify samples. Each sample was amplified in duplicate and no template controls were included with each run. The mean R2 value for the standard curve across all the qPCR assays was 0.993 and they had an efficiency of 98.2%.
2. Materials and methods 2.1. Sample collection and processing Soil samples were collected throughout the major land use types (annual cropland, roadside ditches, and rangeland) and soil types (Brown, Dark Brown, Gray, Dark Gray, and Black Chernozems) across all the major agricultural regions of Saskatchewan, Canada in 2016. Samples were collected from 142 annually cropped fields that were either seeded to peas (77 fields) or lentils (65 fields) and 135 of their associated adjacent roadside ditches. Samples were taken after first flowering of the crop (late June and early July). In every annually cropped field, five soil samples were collected, each 50 m apart in a ‘W’ pattern. At each roadside, three samples were collected at 50 m intervals. Roadside ditch samples provided a measure to understand whether pulse crops have increased the abundance of A. euteiches in agricultural fields relative to uncropped areas. Soil samples were also
2.4. Disease severity index Root rot severity was scored on all pea and lentil plants. The disease 2
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abundance in roadside samples was also positively correlated (P < 0.05) with all of these factors with the exception of NO3-N, although these factors had a weaker association with A. euteiches abundance compared to the annual crop samples. For example, soil moisture was correlated with A. euteiches abundance in both annual crop and roadside soils but had a stronger association in annual field soils (r = 0.43 in field soils and r = 0.24 in roadside soils). A. euteiches abundance was negatively correlated (P < 0.05) with sand content in annual field soils, and soil pH in roadside soils. A negative correlation between A. euteiches abundance and total N in tame rangeland was the only significant relationship observed for any of the soil chemical or physical properties measured in rangeland samples. The means and standard errors of chemical and physical soil properties measured in different land use types are provided in Supplementary Table S2.
severity index was based on a 1 to 7 scale (e.g. 1 = low, 7 = severe) and involved a visual assessment of symptoms such as root discoloration and reduction of root size (Bilgi et al., 2008). This method is a general assessment of root rot, and encompasses similar symptoms from other pathogens such as Fusarium species (Gossen et al., 2016). 2.5. Statistical analysis Analysis of variance (ANOVA) was used to test the effects of land use type, soil type, and their interaction on A. euteiches (Ae1.2_ITS1) abundance. ANOVA was also used to assess the effects of different crop management practices in annually cropped fields, which included the effect of current crop (pea vs. lentil), previous crop (canola vs. cereal), and tillage (cultivation vs. direct seeding). ANOVA was also used to test the effect of native vs. tame rangeland on A. euteiches abundance. Data was tested for assumptions of normality and homogeneity of variance and log transformed if necessary, and type III sums of squares was used for unbalanced data sets. Means separation was performed using Tukey's HSD multiple comparisons test. Linear regression analysis was used to examine the relationship between (i) A. euteiches abundance in soil and disease severity index, and (ii) A. euteiches abundance in annual cropland soils and their associated roadsides. Pearson correlation was used to examine the relationship between A. euteiches abundance and soil chemical and physical properties. All statistical analyses were conducted and visualized in R v. 3.5.1 (R Core Team, 2017).
3.4. Disease severity Pea fields had a significantly (P < 0.001) higher root rot disease severity index (DSI) compared to lentil fields (pea field DSI = 3.63 ± 0.17 s.e., lentil field DSI = 2.93 ± 0.11 s.e.). In addition, the abundance of A. euteiches had a significant positive relationship with DSI in pea fields, but no significant relationship was observed in lentil fields (Fig. 4). 4. Discussion
3. Results
The wide distribution and geographic spread of A. euteiches across both annually cropped fields and rangeland suggests that this microorganism was present in Saskatchewan prior to its detection in 2012. As noted by Banniza et al. (2013), the particularly wet conditions in 2012 and the preceding years likely favored the development and detection of A. euteiches root rot, along with the advent of improved detection methods. Our results also support the widespread detection of A. euteiches in pea and lentil fields across the Canadian prairie region where these pulse crops are grown (Chatterton et al., 2019). In annual fields, the increased planting of susceptible pulse crops has likely increased the prevalence and inoculum levels of this crop pathogen in the soil. The positive identification of A. euteiches in native and tame rangelands also indicates that other native and cultivated legumes other than pea and lentil can act as hosts for this microorganism. Alfalfa is a known host for A. euteiches (Delwiche et al., 1987; Vandemark et al., 2002; Wicker et al., 2001), as are other tame forages such as red clover (Trifolium pratense) and white clover (Trifolium repens) (Grau et al., 1991). Common native perennial legumes in Saskatchewan rangeland have also been identified as hosts for A. euteiches in the United States, including white prairie clover (Dalea candida), purple prairie clover (Dalea purpurea), and Canadian milk vetch (Astragalus canadensis), which led the authors to suggest that A. euteiches might be an indigenous pathogen (Malvick et al., 2009). The limited relationship with soil properties in rangeland suggests that the distribution and abundance of A. euteiches is likely dependent on plant composition and presence of susceptible plant hosts. This study revealed that A. euteiches is not limited to any agricultural region or soil type in Saskatchewan because it was consistently detected across all the major soil types and agricultural land use types in the province. However, we did find that land use type is a significant factor affecting the abundance of A. euteiches in soil. Not surprisingly, annual field soils containing highly susceptible host crops (pea and lentil) had the highest A. euteiches abundance. Although A. euteiches abundance was significantly lower in roadside soils when compared to field soils, A. euteiches abundance was positively correlated between the two land use types. A. euteiches lacks a mechanism for long-distance dispersal (Grünwald and Hoheisel, 2006); therefore, host species, either native or transferred from cultivated fields, were likely present in roadside ditches. Lower abundance of A. euteiches in rangeland soil compared to the annually cropped fields is not surprising, considering
3.1. A. euteiches distribution across land use and soil types A. euteiches (Ae1.2_ITS1) was detected in all land use types (lentil and pea fields, roadside ditches, native and tame rangeland) and soil types (Brown, Dark Brown, Gray, Dark Gray, and Black) included in this study (Fig. 1). Land use type was a significant factor affecting A. euteiches abundance (Table 1). A. euteiches abundance was significantly higher in annually cropped fields than in adjacent roadsides or in rangeland, while there was no difference between roadsides and rangeland (Fig. 2). A. euteiches abundance in annual fields and their associated roadsides was positively correlated (Fig. 3, R2 = 0.294). Soil type and the soil type by land use type interaction had no significant effect on the abundance of A. euteiches (Table 1). These results indicated that A. euteiches is widespread across the agricultural regions of Saskatchewan and is not limited to annually cropped fields or specific soil types, as it was commonly detected in both native and tame rangeland and all soil types. 3.2. Crop management practices In annual fields, the current crop (i.e., pea vs. lentil) had a significant effect on the abundance of A. euteiches. Pea crops had a significantly higher A. euteiches abundance than lentil crops (Supplementary Fig. S1). None of the other crop management practices (i.e., previous crop = oilseed vs. cereal; tillage = direct seeding vs. cultivation) in annual fields had a significant effect on A. euteiches abundance (Table 1). The management of rangeland (native vs. tame) did have a significant effect on the abundance of A. euteiches as higher levels were detected in native compared to tame pastures (Supplementary Fig. S2). 3.3. Soil properties The abundance of A. euteiches was correlated with several chemical and physical soil properties that varied by land use type (Table 2). In annually cropped field samples, A. euteiches abundance was positively correlated (P < 0.05) with NO3−-N, PO43−-P, total and organic carbon, total nitrogen, soil moisture, and clay content. A. euteiches 3
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log Ae1.2_ITS copies g-1 soil
4
log Ae1.2_ITS copies g-1 soil
(caption on next page)
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Fig. 1. Distribution and abundance of Aphanomyces euteiches (log10 Ae1.2_ITS1 copies g−1 dry soil) across agricultural land use (field, rangeland) and soil (brown, dark brown, gray, dark gray, black) types of Saskatchewan. Field: n = 142; Rangeland: n = 48. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 Effect of land use type, soil type and crop management practices on Aphanomyces euteiches gene abundance in agricultural soil. Crop management practices were tested independently within their respective land use types. Factor
df
F value
Pr (> F)
Land use type Soil type Land use type ∗ soil type Field Field vs. roadside Crop (pea vs. lentil) Previous crop (canola vs. cereal) Tillage (cultivation vs. direct seeding) Rangeland Native vs. tame
2 3 6
20.1 2.13 1.35
< 0.0001 0.096 0.234
1 1 1 1
36.8 9.15 0.25 0.74
< 0.0001 0.003 0.62 0.39
1
4.48
0.040
Table 2 Pearson correlation coefficient (r) values between Aphanomyces euteiches abundance (log10 Ae1.2_ITS1 copies g−1 dry soil) and soil chemical and physical properties. Bold text indicates significance at P < 0.05.
log10 Ae1.2_ITS1 copies g-1 dry soil
a
Factor
Field (n = 142)
Roadside (n = 135)
Native (n = 14)
Tame (n = 29)
SO42−-S K NO3−-N PO43−-P Total C Organic C Total N Soil moisture pH EC Sand Clay Silt
−0.06 0.07 0.20 0.23 0.37 0.37 0.40 0.43 −0.13 0.06 −0.25 0.28 −0.05
−0.02 0.16 0.14 0.18 0.23 0.32 0.31 0.24 −0.19 −0.15 −0.05 0.09 −0.14
−0.18 0.45 0.24 0.41 −0.22 0.00 −0.26 0.15 0.02 −0.29 −0.06 −0.03 0.14
0.04 −0.32 0.19 −0.16 −0.22 −0.25 −0.49 −0.23 0.34 0.20 0.24 −0.13 −0.12
n = number of samples; EC = electrical conductivity.
Field
b
b
Roadside
Rangeland
log10 Ae1.2_ITS1 copies g-1 dry field soil
Fig. 2. Aphanomyces euteiches abundance (log10 Ae1.2_ITS1 copies g−1 dry soil) in field, roadside and rangeland samples. Bars are means (Field: n = 142; Roadside: n = 135; Rangeland: n = 47) with standard errors. Different letters indicate significant differences between land use type (ANOVA, df = 2, P < 0.0001).
P < 0.0001 Adj. R2 = 0.294
6
4
2
0 0
6 2 4 log10 Ae1.2_ITS1 copies g-1 dry roadside soil Fig. 4. Relationship between Aphanomyces euteiches (log10 Ae1.2_ITS1 copies g−1 dry soil) abundance in field soil and disease severity index of (a) pea and (b) lentil plants. Ae1.2_ITS1 abundance was quantified with quantitative polymerase chain reaction (qPCR) assays and disease severity index was manually scored on affected plants.
Fig. 3. Relationship between Aphanomyces euteiches (log10 Ae1.2_ITS1 copies g−1 dry soil) abundance in fields and their associated roadsides.
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have been particularly relevant in annual fields where we observed a significant positive relationship between A. euteiches abundance and disease severity in peas. Future research on how A. euteiches abundance changes over the growing season and relates to risk of disease development in subsequent years could facilitate more accurate determination of the risk of planting a susceptible pulse crop in a given field. The higher susceptibility of peas to A. euteiches than lentils is in accordance with reports that emphasize the prevalence and severity of the disease to pea crops in particular (Chatterton et al., 2019; Basu et al., 1973; Tu, 1992). Although partial resistance has been reported in some pea (Wu et al., 2019; Conner et al., 2013) and lentil (Bazghaleh et al., 2018) cultivars, there are no cultivars completely resistant to A. euteiches. In addition to pea and lentil, other legumes including alfalfa, snap bean, vetch, clover, sweet clover, and numerous weed species can be host to A. euteiches (Papavizas and Ayers, 1974). Other crop management practices (i.e., tillage and previous crop) that were expected to influence A. euteiches abundance were not significant factors. For instance, tillage practices that cause soil compaction are expected to increase soil moisture and increase A. euteiches root rot (Wu et al., 2018), but tillage type was not significant in this study. In regards to the impact of previously seeded crops, rotations that incorporate cruciferous plants (e.g. Brassica napus, canola) have been used to reduce disease severity in A. euteiches infested soils (Chan and Close, 1987). However, brassica species may differ in their ability to fulfill this function. Brassica juncea (brown mustard) and Sinapis alba (white mustard) differ in the capacity of their associated glucosinolates and their break-down products, isothiocyanates, to inhibit A. euteiches in vitro (Hossain et al., 2014) or to reduce Aphanomyces root rot in pot studies (Hossain et al., 2015). Thus, the capacity of canola varieties with varying glucosinolate content merits further investigation. As previous crop was not a significant factor in our study, excluding pea and, to a lesser extent, lentil, from crop rotations in fields with high inoculum potential or confirmed disease continues to be the recommended crop management practice to control A. euteiches root rot. Along with A. euteiches, the qPCR primers and probe used in the study have the potential to amplify A. cladogamus DNA (Willsey et al., 2018). A. cladogamus, is a soil-borne oomycete plant pathogen, capable of causing disease on spinach and sugar beets (Larsson, 1994), as well as pansies (Drechsler, 1954), tomato, eggplant and pepper (McKeen, 1952). The prevalence of A. cladogamus in Saskatchewan is not well characterized, but there is some evidence that it is present in the Canadian prairie region (Esmaeili Taheri et al., 2017). However, a recent field study conducted across nine sites in Saskatchewan using amplicon metagenomic sequencing of oomycete communities found that A. cladogamus was rarely detected (i.e., < 5% samples and 0.1% of total reads) of the soil samples (Bainard et al. unpublished data). A high proportion of the Ae1.2_ITS1 copies we detected in our samples were likely from A. euteiches because the high Ct value for A. cladogamus using pure culture DNA reported by Willsey et al. (2018) represents a difference in copy numbers of approximately two orders of magnitude and was approaching the detection limit for this qPCR assay. Further research is required to determine if A. cladogamus is found throughout Saskatchewan and whether the Ae1.2_ITS1 qPCR assay successfully amplifies this microorganism in soil samples. Despite its recent discovery in Saskatchewan, this study demonstrated that A. euteiches is widespread across the province, land use and soil types, and supports the hypothesis that A. euteiches was present prior to its discovery in 2012. In addition, the frequent detection in native rangeland soil indicates that A. euteiches is likely indigenous to this region. The more frequent use of pulse crops combined with higher than average levels of precipitation in the region have likely increased inoculum levels in annually cropped field soils. Overall, this study highlights that pulse crop root rot issues will persist or expand in the future due to the widespread distribution of this pathogen throughout the agricultural regions in Saskatchewan.
that native and tame rangeland host plant species occur in polyculture rather than monoculture. A diverse mixture of host crop species would be expected to vary in disease inoculum potential and virulence (Moussart et al., 2013; Grau et al., 1991). Interestingly, given the previously-documented link between finetextured soil and increased Aphanomyces root rot risk (Allmaras et al., 2003; Fritz et al., 1995; Gossen et al., 2016; Kraft et al., 1990; Papavizas and Ayers, 1974; Pfender and Hagedorn, 1983), soil type was not a significant factor affecting the abundance and distribution of A. euteiches in Saskatchewan. This supports the results from previous root rot field surveys conducted between 2015 and 2017 in Saskatchewan, which also found no effect of soil type on the frequency of pea fields that tested positive for A. euteiches (Chatterton et al., 2019). Soil moisture increases across the province in a southwest to northeast gradient and roughly corresponds with the different soil types. Based on the positive correlation between soil moisture and A. euteiches abundance, a significant relationship with soil type was also expected. However, above-average levels of precipitation across the province during the 2016 growing season (Chatterton et al., 2019) may have provided generally favourable conditions for A. euteiches root rot development and limited the effect of soil type on the abundance of this pathogen. These results suggest that land use type and presence of susceptible plant hosts has a greater impact on A. euteiches inoculum potential than abiotic soil properties. There were four common soil abiotic factors (soil moisture, total N, total and organic C) that positively correlated with A. euteiches abundance in field and roadside samples, but soil moisture had the strongest relationship with A. euteiches abundance. Higher moisture levels facilitate the spread of A. euteiches as it encourages the development of sporangia, and the released zoospores are flagellated and will move through moisture films around roots (Wu et al., 2018). Organic carbon content has also been shown to aid in soil water retention, which would enhance zoospore movement (Rawls et al., 2003). In past studies, A. euteiches was predominantly found in heavy clay soils or poorly drained areas with increased soil moisture (Persson and Olsson, 2000; Kraft et al., 1990; Papavizas and Ayers, 1974; Pfender and Hagedorn, 1983). In both annual field and roadside soils, A. euteiches abundance was negatively correlated with sand content and positively correlated with clay content, but this relationship was only significant in annual field soils. Soil texture is directly related to soil moisture (Gupta and Larson, 1979); therefore, it is not surprising this relationship was observed. Nitrogen fertilization has been reported to reduce root rot caused by A. euteiches (Papavizas and Lewis, 1971) by modifying the morphology of pea roots so they inhibit pathogen colonization (Hossain et al., 2015), but our results show a positive relationship between A. euteiches abundance and NO3−-N and total N in field soils. However, we did detect a significant negative relationship between A. euteiches abundance and total N in tame rangeland. Some of the variability between our results and previous studies may be related to the techniques used to measure disease severity or inoculum potential. Recent advances in molecular techniques have allowed accurate and efficient quantification of A. euteiches inoculum levels in soil (Willsey et al., 2018; Gangneux et al., 2014) while traditional techniques have consisted of microscopic observations and/or greenhouse experiments (Moussart et al., 2013; Papavizas and Lewis, 1971). In this study, the significant factors affecting A. euteiches abundance differed between land use type, which also highlights the complexity of identifying a single factor (or combination of factors) that could be used to control or anticipate inoculum potential across a range of agricultural soils. There was a significant difference in root rot severity and A. euteiches abundance between annual fields with peas compared to lentils. These results suggest that peas are more susceptible to root rot or colonization by A. euteiches, and/or promote higher growth of the pathogen compared to lentils. Amplification of A. euteiches likely represented more than just the oospores (e.g., mycelia and zoospores) in the soil since samples were collected mid-growing season. This may 6
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Declaration of competing interest
ammonium in soil extracts. Commun. Soil Sci. Plant Anal. 19, 721–737. Gossen, B.D., Conner, R.L., Chang, K.-F., Pasche, J.S., McLaren, D.L., Henriquez, M.A., Chatterton, S., Hwang, S.-F., 2016. Identifying and managing root rot of pulses on the Northern Great Plains. Plant Pathol. 100, 1965–1978. Grau, C.R., Muehlchen, A.M., Tofte, J.E., Smith, R.R., 1991. Variability in virulence of Aphanomyces euteiches. Plant Dis. 75, 1153–1156. Grünwald, N.J., Hoheisel, G.-A., 2006. Heirarchical analysis of diversity, selfing, and genetic differentiation in populations of the oomycete Aphanomyces euteiches. Phytopathology 96, 1134–1141. Gupta, S.C., Larson, W.E., 1979. Estimating soil water retention characteristics from particle size distribution, organic matter percent, and bulk density. Water Resour. Res. 15, 1633–1635. Hamm, J.W., Radford, F.G., Halstead, E.H., 1970. The simultaneous determination of nitrogen, phosphorus, and potassium in sodium bicarbonate extracts of soils. In: Advances in Automatic Analysis. Industrial Analysis, Vol. II. Technicon International Congress. Futura Publishing Co, Mount Kisco, NY, pp. 65–69. Hamm, J.W., Bettany, J.R., Halstead, E.H., 1973. A soil test for sulphur and interpretative criteria for Saskatchewan. Commun. Soil Sci. Plan. 4, 219–231. Hendershot, W.H., Lalande, H., Duquette, M., 2008. Ion exchange and exchangeable cations. In: Carter, M.R., Gregorich, E.G. (Eds.), Soil Sampling and Methods of Analysis. Canadian Society of Soil Science, Boca Raton, FL, pp. 197–206. Hossain, S., Bergkvist, G., Berglund, K., Glinwood, R., Kabouw, P., Martensson, A., Persson, P., 2014. Concentration- and time-dependent effects of isothiocyanates produced from Brassicaceae shoot tissues on the pea root rot pathogen Aphanomyces euteiches. J. Agric. Food Chem. 62, 4584–4591. Hossain, S., Bergkvist, G., Glinwood, R., Berglund, K., Martensson, A., Hallin, S., Persson, P., 2015. Brassicaceae cover crops reduce Aphanomyces pea root rot without suppressing genetic potential of microbial nitrogen cycling. Plant Soil 392, 227–238. Kraft, J.M., Marcinkowska, J., Muehlbauer, J., 1990. Detection of Aphanomyces euteiches in field soil from northern Idaho by a wet-sieving/baiting technique. Plant Dis. 74, 716–718. Larsson, M., 1994. Pathogenicity, morphology and isozyme variability among isolates of Aphanomyces spp. from weeds and various crop plants. Mycol. Res. 98, 231–240. Le May, C., Onfroy, C., Moussart, A., Andrivon, D., Baranger, A., Pilet-Nayal, M.L., Vandemark, G., 2018. Genetic structure of Aphanomyces euteiches populations sampled from United States and France pea nurseries. Eur. J. Plant Pathol. 150, 275–286. Levenfors, J.P., 2003. Soil-Borne Pathogens in Intensive Legume Cropping - Aphanomyces spp. and Root Rots (Doctoral thesis). Swedish University of Agricultural Sciences, Uppsala, Sweden. Levenfors, J.P., Fatehi, J., 2004. Molecular characterization of Aphanomyces species associated with legumes. Mycol. Res. 108, 682–689. Levenfors, J.P., Wikstrom, M., Persson, L., Gerhardson, B., 2003. Pathogenicity of Aphanomyces spp. from different leguminous crops in Sweden. Eur. J. Plant Pathol. 109, 535–543. Malvick, D.K., Grünwald, N.J., Dyer, A.T., 2009. Population structure, races, and host range of Aphanomyces euteiches from alfalfa production fields in the central USA. Eur. J. Plant Pathol. 123, 171–182. Manning, M.A., Menzies, S.A., 1980. Root rot of peas in New Zealand caused by Aphanomyces euteiches. N. Z. J. Agric. Res. 23, 263–265. McKeen, C.D., 1952. Aphanomyces cladogamus Drech., a cause of damping-off in peppers and certain other vegetables. Can. J. Bot. 30, 701–709. Miller, J.J., Curtin, D., 2008. Electrical conductivity and soluble ions. In: Carter, M.R., Gregorich, E.G. (Eds.), Soil Sampling and Methods of Analysis. Canadian Society of Soil Science, Boca Raton, FL, pp. 161–171. Miller, P.R., McConkey, B.G., Clayton, G.W., Brandt, S.A., Staricka, J.A., Johnston, A.M., Lafond, G.P., Schatz, B.G., Baltensperger, D.D., Niell, K.E., 2002. Pulse crop adaptation in the Northern Great Plains. Agron. J. 94, 261–272. Moussart, A., Even, M.N., Lesné, A., Tivoli, B., 2013. Successive legumes tested in a greenhouse crop rotation experiment modify the inoculum potential of soils naturally infested by Aphanomyces euteiches. Plant Pathol. 62, 545–551. Niu, Y., Bainard, L.D., Bandara, M., Hamel, C., Gan, Y., 2017. Soil residual water and nutrients explain about 30% of the rotational effect in 4-yr pulse-intensified rotation systems. Can. J. Plant Sci. 97, 852–864. Niu, Y., Bainard, L.D., Hossain, Z., May, W.E., Hamel, C., Gan, Y., 2018. Intensified pulse rotations buildup pea rhizosphere pathogens in cereal and pulse based cropping systems. Front. Microbiol. 9, 1909. Papavizas, G.C., Ayers, W.A., 1974. Aphanomyces species and their root diseases in pea and sugar beet. In: U.S. Department of Agriculture Technical Bulletin. 1484. pp. 158. Papavizas, G.C., Lewis, J.A., 1971. Effect of amendments and fungicides on Aphanomyces root rot of peas. Phytopathology 61, 215–220. Persson, L., Olsson, S., 2000. Abiotic characteristics of soils suppressive to Aphanomyces root rot. Soil Biol. Biochem. 32, 1141–1150. Persson, L., Bodker, L., Larsson-Wikstrom, M., 1997. Prevalence and pathogenicity of foot and root rot pathogens of pea in southern Scandinavia. Plant Dis. 81, 171–174. Pfender, W.F., Hagedorn, D.J., 1983. Disease progress and yield loss in Aphanomyces root rot of peas. Phytopathology 73, 1109–1113. R Core Team, 2017. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Rawls, W.J., Pachepsky, Y.A., Ritchie, J.C., Sobecki, T.M., Bloodworth, H., 2003. Effect of soil organic carbon on soil water retention. Geoderma 116, 61–76. Schumacher, B.A., 2002. Methods for the Determination of Total Organic Carbon (TOC) in Soils and Sediments. Ecological Risk Assessment Support Center, US Environmental Protection Agency. Slinkard, A.E., Bascur, G., Hernandez-Bravo, G., 1994. Biotic and abiotic stresses of cool season food legumes in the western hemisphere. In: Murhlbauer, F.J., Kaiser, W.J. (Eds.), Expanding the Production and Use of Cool Season Food Legumes. Kluwer
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This project was funded by Saskatchewan Pulse Growers. We gratefully thank the assistance provided by Cam Kenny and technical staff and students at the Swift Current Research and Development Centre and all of the volunteers (especially the Saskatchewan Ministry of Agriculture) that helped with the field sampling. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2019.103470. References Allen, R.N., Letham, D.B., Akehurst, A.A., Say, M.M., 1987. Aphanomyces root rot of bean at Valla, New South Wales. Australas. Plant Pathol. 6, 82–84. Allmaras, R.R., Fritz, V.A., Pfleger, F.L., Copeland, S.M., 2003. Impaired internal drainage and Aphanomyces euteiches root rot of pea caused by soil compaction in a fine-textured soil. Soil Tillage Res. 70, 41–52. Bainard, L.D., Navarro-Borrell, A., Hamel, C., Braun, K., Hanson, K., Gan, Y., 2017. Increasing the frequency of pulses in crop rotations reduces soil fungal diversity and increases the proportion of fungal pathotrophs in a semiarid agroecosystem. Agric. Ecosyst. Environ. 240, 206–214. Banniza, S., Bhadauria, V., Peluola, C.O., Armstrong-Cho, C., Morrall, R.A.A., 2013. First report of Aphanomyces euteiches in Saskatchewan. Can. Plant Dis. Surv. 93, 163–164. Basu, P.K., Crête, R., Donaldson, A.G., Gourley, C.O., Haas, J.H., Harper, F.R., Lawrence, C.H., Seaman, W.L., Toms, H.N.W., Wong, S.I., Zimmer, R.C., 1973. Prevalence and severity of diseases processing peas in Canada, 1970-71. Can. Plant Dis. Surv. 53, 49–57. Bazghaleh, N., Prashar, P., Purves, R.W., Vandenberg, A., 2018. Polyphenolic composition of lentil roots in response to infection by Aphanomyces euteiches. Front. Plant Sci. 9, 1131. Bilgi, V.N., Bradley, C.A., Khot, S.D., Grafton, K.F., Rasmussen, J.B., 2008. Response of dry bean genotypes to Fusarium root rot, caused by Fusarium solani f. sp. phaseoli, under field and controlled conditions. Plant Dis. 92, 1197–1200. Canadian Plant Diesease Survey, 1948. Diseases of vegetable and field crops. In: Conners, I.L., Savile, D.B.O. (Eds.), Twenty-eighth Annual Report of the Canadian Plant Disease Survey. Canadian Phytopathological Society, Ottawa, Canada, pp. 36–72. Chan, M.K.Y., Close, R.C., 1987. Aphanomyces root rot of peas 3. Control by the use of cruciferous amendments. New Zeal. J. Agr. Res. 30, 225–233. Chatterton, S., Harding, M.W., Bowness, R., McLaren, D.L., Banniza, S., Gossen, B.D., 2019. Importance and causal agents of root rot on field pea and lentil on the Canadian prairies, 2014–2017. Can. J. Plant Pathol. 41, 98–114. Conner, R.L., Chang, K.F., Hwang, S.F., Warkentin, T.D., McRae, K.B., 2013. Assessment of tolerance for reducing yield losses in field pea caused by Aphanomyces root rot. Can. J. Plant Sci. 93, 473–482. Delwiche, P.A., Grau, C.R., Holub, E.B., Perry, J.B., 1987. Characterization of Aphanomyces euteiches isolates recovered from alfalfa in Wisconsin. Plant Dis. 71, 155–161. Didelot, D., Chaillet, I., 1995. Relevance and interest of root disease prediction tests for pea crop in France. In: 2nd Eur Conf Grain Legumes—Improving Production and Utilisation of Grain Legumes. Drechsler, C., 1954. Association of Aphanomyces cladogamus with severe root rot of pansies. Sydowia 8, 334–342. Dreschsler, C., Jones, F.R., 1925. Boot rot of peas in the United States caused by Aphanomyces euteiches (n. sp.). J. Agric. Res. 30, 293–325. Esmaeili Taheri, A., Chatterton, S., Gossen, B.D., McLaren, D.L., 2017. Metagenomic analysis of oomycete communities from the rhizosphere of field pea on the Canadian prairies. Can. J. Microbiol. 63, 758–768. Fritz, V.A., Allmaras, R.R., Pfleger, F.L., Davis, D.W., 1995. Oat residue and soil compaction influences on common root rot (Aphanomyes euteiches) of peas in a fine-textured soil. Plant Soil 235–244. Gangneux, C., Cannesan, M., Bressan, M., Castel, L., Moussart, A., Vicré-Gibouin, M., Driouich, A., Trinsoutrot-Gattin, I., Laval, K., 2014. A sensitive assay for rapid detection and quantification of Aphanomyces euteiches in soil. Phytopathology 104, 1137–1147. Gaulin, E., Jacquet, C., Bottin, A., Dumans, B., 2007. Root rot disease of legumes caused by Aphanomyces euteiches. Mol. Plant Pathol. 8, 539–548. Gee, G.W., Bauder, J.W., 1979. Particle size analysis by hydrometer: a simplified method for routine textural analysis and a sensitivity test of measurement parameters. Soil Sci. Soc. Am. J. 43, 1004–1007. Gentry, C.E., Willis, R.B., 1988. Improved method for automated determination of
7
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E.M. Karppinen, et al.
between the pea root rot pathogens Aphanomyces euteiches and Fusarium spp. using a multiplex qPCR assay. Plant Pathol. 67, 1912–1923. Wu, L., Chang, K.F., Conner, R.L., Strelkov, S., Fredua-Agyeman, R., Hwang, S.F., Feindel, D., 2018. Aphanomyces euteiches: a threat to Canadian field pea production. Engineering 4, 542–551. Wu, L., Chang, K.F., Hwang, S.F., Conner, R., Fredua-Agyeman, R., Feindel, D., Strelkov, S.E., 2019. Evaluation of host resistance and fungicide application as tools for the management of root rot of field pea caused by Aphanomyces euteiches. The Crop Journal 7, 38–48. Zhou, Y., Zhu, H., Fu, S., Yao, Q., 2017. Variation in soil microbial community structure associated with different legume species is greater than that associated with different grass species. Front. Microbiol. 8, 1007. Zitnick-Anderson, K., Pasche, J.S., 2016. First report of Aphanomyces root rot caused by Aphanomyces euteiches on field pea in North Dakota. Plant Dis. 100, 522.
Academic Publishers, The Netherlands, pp. 195–203. Sturz, A.V., Carter, M.R., Johnston, H.W., 1997. A review of plant disease, pathogen interactions and microbial antagonism under conservation tillage in temperate humid agriculture. Soil Till. Res. 41, 169–189. Sundheim, L., 1972. Physiologic specialization in Aphanomyces euteiches. Physiol. Plant Pathol. 2, 301–306. Tu, J.C., 1992. Management of root rot diseases of peas, beans and tomatoes. Can. J. Plant Pathol. 14, 92–99. Vandemark, G.J., Barker, B.M., Gritsenko, M., 2002. Quantifying Aphanomyces euteiches in alfalfa with a fluorescent polymerase chain reaction assay. Phytopathology 92, 265–272. Wicker, E., Hullé, M., Rouxel, F., 2001. Pathogenic characteristics of isolates of Aphanomyces euteiches from pea in France. Plant Pathol. 50, 433–442. Willsey, T.L., Chatterton, S., Heynen, M., Erickson, A., 2018. Detection of interactions
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