Identification of Pythium spp. from the rhizosphere of soybeans in Ontario, Canada

Abstract

Production of soybean in short-season areas of Canada is affected by Pythium species causing seed rot, seedling damping-off and root rot under the cool, moist conditions prevalent early in the growing season. To determine which Pythium species infect soybean plants under these conditions, soil samples were collected in 2010, 2011 and 2012 from soybean fields in Ontario where symptoms of damping-off or root rot were observed. Pythium spp. were isolated from soil using a seed baiting approach and selective media. PCR amplification and sequencing of the internal transcribed spacer of the nuclear ribosomal DNA allowed for identification of six different Pythium species (out of a total of 104 strains). Pythium ultimum var. ultimum (56.7%) and P. sylvaticum (29.8%) were the most frequently isolated species over all sampling years. Other species isolated included P. dissotocum (6.7%), P. ultimum var. sporangiiferum (3.8%), P. irregulare (1.9%), and P. hypogynum (1.0%).

Résumé

La culture du soja dans les régions du Canada où la saison de croissance est courte est affectée par des espèces de Pythium causant la pourriture des semences, la fonte des semis et la pourriture racinaire chez les plantes adultes au début de la saison de croissance. Dans le but de déterminer l’identité des espèces de Pythium infectant le soja cultivé sous conditions locales, des échantillons de sols furent prélevés en Ontario en 2010, 2011 et 2012, dans des champs de soja où des symptômes de fonte des semis ou de pourriture racinaire étaient visibles. Les espèces de Pythium spp. furent isolées des échantillons de sol à l’aide d’une technique d’appâtage avec des semences et de l’utilisation de milieux sélectifs. L’amplification par PCR et le séquençage de la région intercalaire transcrite de l’ADN ribosomal nucléaire ont permis l’identification de six espèces différentes de Pythium (à partir d’un total de 104 souches). P. ultimum var. ultimum (56.7%) et P. sylvaticum (29.8%) furent les espèces les plus fréquemment isolées durant le total de trois années d’échantillonage. Les espèces P. dissotocum (6.7%), P. ultimum var. sporangiiferum (3.8%), P. irregulare (1.9%), and P. hypogynum (1.0%) étaient aussi représentées.

Keywords:

• damping-off
• DNA barcoding
• Pythium spp., root rot
• soybean [Glycine max]
• soilborne pathogens

Mots clés:

• Pythium spp
• soja [Glycine max]
• code barre génétique
• pourriture racinaire
• fonte des semis
• agents pathogènes telluriques

Introduction

Soybean [Glycine max (L.) Merr.] production in Canada was made possible by the development of varieties adapted to the local climatic conditions, starting in the 1920s (Dorff 2007). The germplasm initially used, mostly from Asia, was photoperiod-sensitive and only used as green forage as it did not mature under Canadian climatic conditions (Beversdorf et al. 1995). A programme was initiated in 1943 to develop varieties specifically for southwestern Ontario due to the region’s long and warm growing season (Dorff 2007). The incorporation of Swedish germplasm with early maturity and cold tolerance (Voldeng et al. 1997) allowed for the development of short-season cultivars, such as the ‘Maple’ series released by Agriculture and Agri-Food Canada (AAFC) beginning in the 1970s (notably ‘Maple Arrow’ in 1976), which permitted expansion of the soybean growing area into eastern Ontario (Dorff 2007). Between 1976 and 2011, the area of soybean production increased from 152 910 to 997 497 ha in Ontario, from 240 to 277 144 ha in Québec and from 153 793 to 1 601 653 ha in all of Canada (Statistics Canada 2012). Concurrently, Cober & Voldeng (2012) estimate the number of short-season soybean cultivars included in annual cultivar trials increased 36-fold between 1971 and 2000. Despite this encouraging trend, some factors limit the expansion of soybean production in short-season production areas of Canada. Soilborne diseases such as seed rot and seedling damping-off caused by Pythium spp. are a limiting factor in cool, moist conditions (Broders et al. 2009) that are prevalent during the early season in these areas.

Numerous Pythium species affect a wide range of agricultural crops, causing seed rot, seedling damping-off and root rot on adult plants (Hendrix & Campbell 1973). Up to 14 species have been reported to infect soybeans (Jiang et al. 2012). In Canada, P. sylvaticum W.A. Campb. & F.F. Hendrix has been isolated from soybean fields (Barasubiye et al. 2005). Many studies evaluating pathogens of soybean in Canada and the USA refer only to Pythium spp., rather than identifying the individual species (Anderson 1987; Rizvi & Yang 1996). In earlier studies, this may have been due in part to the difficulty in determining species from the observation of morphological characters. More recently, molecular techniques such as sequencing of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA have allowed the taxonomy and phylogeny of the Pythium genus (Lévesque & De Cock 2004) and of oomycetes (Robideau et al. 2011) to be revisited. The ITS can thus easily be used as a DNA barcode to quickly and accurately identify oomycete species because reliable reference sequences for most known species are available in the GenBank sequence database.

In order to minimize the damage caused by Pythium spp. to soybean early in the season in short-season production areas, fungicides have been used as seed treatments, but the protection they offer is of short duration. There is an obvious need for short season varieties of soybean resistant to Pythium for effective management of this disease (Agriculture and Agri-Food Canada 2006). To make it possible for plant breeders to develop such varieties, the species causing disease under these conditions and in different locales must be identified and used for screening of resistant germplasm. While some studies report on Pythium spp. associated with soybean damping-off and root rot in the USA (e.g. Rizvi & Yang 1996; Zhang et al. 1998; Dorrance et al. 2004; Broders et al. 2007; Jiang et al. 2012), no extensive recent surveys have been made to determine which Pythium spp. cause disease on soybean plants in short-season soybean growing areas of Canada. The objective of this study was to identify Pythium species causing seedling damping-off and root rot in Ontario, where most Canadian soybean is grown.

Materials and methods

Field sampling

Soil samples were collected across Ontario from 60 soybean fields in 2010, 65 fields in 2011 and 84 fields in 2012. Fields were selected for sampling based on the presence of seedling damping-off or root rot symptoms. In each field, a bulk sample of approximately 2 kg of soil was collected from at least three random sites and soil was taken to a soil depth of 15 cm within the rhizosphere of severely affected plants using a hand trowel. Samples were collected during the second and the third week of June each year when plants were at the first and the second trifoliate stage of development. The soil samples were air-dried and stored at 3°C before being used for pathogen isolation.

Isolation of Pythium species

Pythium spp. were isolated from sampled soils using a protocol loosely based on the method described by Watanabe (1989) as cited by Zhang et al. (1998). Ten soybean seeds of the susceptible soybean cultivars ‘Nattosan’ or ‘Ox20-8’ were surface sterilized with 1% sodium hypochlorite (Javex bleach, Clorox Canada, Brampton, ON) for 2 min and subsequently rinsed in sterile water before planting in 15 cm pots containing approximately 500 g of sampled soil that had been previously flooded with water for 24 h. The pots were incubated in the dark in a growth cabinet set to 15°C for 16 h and 10°C for 8 h, and watered as needed to keep the soil saturated. After 5 days, the seeds were removed from the soil, washed for 1 min under tap water and placed in Erlenmeyer flasks containing 50 mL of sterile distilled water. The flasks were then placed on a shaker at 300 rpm for 5 min, after which the seeds were removed, washed under running water for 1 min, air dried, and placed onto plates of selective medium PDA-PBNNCP (39 g L⁻¹ potato dextrose agar amended with 0.054 g L⁻¹ pentachloronitrobenzene, 0.03 g L⁻¹ beta-sitosterol, 0.005 g L⁻¹ benomyl, 0.01 g L⁻¹ neomycin sulphate, 0.1 g L⁻¹ chloramphenicol, 0.01 g L⁻¹ Delvocid salt (50% pimaricin)) at the rate of two seeds per plate. The plates were then incubated at room temperature (22°C) for 1–2 days. Pythium colonies that grew out of the seeds were transferred to 1.5% water agar (WA) and incubated in the dark for 24–48 h at room temperature. Single hyphal tips were transferred to PDA agar plates under a Nikon SMZ1500 dissecting microscope (Nikon Corporation, Tokyo, Japan). Cultures were then grown in the dark at room temperature for up to one week. Strains were archived for long-term storage on V8 agar slants overlaid with mineral oil and stored at 15°C.

DNA extraction

The microLYSIS® kit (Microzone Limited, Haywards Heath, UK) was used to extract DNA from aerial mycelium of Pythium spp. cultures on PDA plates. The equivalent of approximately 1 µL of mycelium was collected from the margin of the fungal colony using a sterile 10 µL pipette tip and, following the manufacturer’s protocol, mixed with 19 µL of microLYSIS 1× buffer in a 0.2 mL PCR tube. This mixture was incubated in a thermal cycler with the following programme: 65°C for 5 min, 96°C for 2 min, 65°C for 4 min, 96°C for 1 min, 65°C for 1 min, 96°C for 30 s then held at 20°C. After incubation, a centrifugation step was added: 10 min at 10,000 × g, and the supernatant was transferred to a clean 0.2 mL tube. The crude DNA preparation was stored at −20°C for future use, and 1 µL of this preparation was used as a template in PCR reactions.

PCR amplification of ITS sequences

PCR amplification of ITS sequences was performed generally as described by Robideau et al. (2011), with slight modifications to cycling parameters. Universal primers for the internal transcribed spacer region of eukaryotes UNUP18S42 (5′-CGTAACAAGGTTTCCGTAGGTGAAC-3′) and UNLO28S22 (5′-GTTTCTTTTCCTCCGCTTATTGATATG-3′) (Bakkeren et al. 2000) were used to amplify a portion of the 18S ribosomal RNA (rRNA) region, all of ITS1, the 5.8S rRNA region, ITS2, and a portion of the 28S region rRNA. Primers were synthesized by Sigma-Aldrich Canada Co. (Oakville, Ontario). The Titanium Taq PCR kit (Clontech Laboratories Inc., Mountain View, CA, USA) was used to prepare a PCR mix (10 µL reaction volume) containing 1× Titanium buffer (containing 3.5 mm MgCl₂), 0.1 mm dNTPs, 0.08 µm each primer, 0.05 U µL⁻¹ Titanium Taq DNA Polymerase. PCR was carried out in a Multigene thermal cycler (Labnet International, Woodbridge, NJ, USA) with the following programme: initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 1 min, 68°C for 1 min, 72°C for 2 min and a final extension at 72°C for 8 min.

Quantification and size determination of amplicons

One microlitre of PCR product was quantified using a BioDrop µLite spectrophotometer (BioDrop, Cambridge, UK). Three microlitres were loaded, along with 0.75 µL 5× loading buffer, on a 1% agarose gel in Tris-borate-EDTA (TBE; 0.089 m Tris base, 0.089 boric acid, 0.002 m EDTA, pH 8.0) buffer (Sambrook & Russell 2001). Gels were stained using ethidium bromide, GelGreen^TM or GelRed^TM (Biotium, Hayward, CA, USA) after migration and visualized using an Infinity VX2 gel documentation station (Vilbert Lourmat, Marne-la-Vallée, France).

Purification and sequencing of amplicons

Following manufacturer’s protocol, 5 µL of PCR products were treated with 2 µL of USB® ExoSAP-IT® PCR cleanup reagent (Affymetrix, High Wycombe, UK) and incubated in a thermal cycler at 37°C for 15 min, then 80°C for 15 min. One microlitre of purified amplicon was used per sequencing reaction. These were performed as described by Robideau et al. (2011) with Big Dye Terminator version 3.1 (Applied Biosystems, Foster City, CA) in a reaction volume of 10 µL, with Big Dye Seq Mix diluted 1 : 8 with Seq buffer (final concentration: 0.875 sequencing buffer, 5% trehalose, 0.125× Big Dye Seq Mix and 0.16 µm primer (either UNUP18S42 or UNLO28S22). Reactions were incubated in a Multigene thermal cycler with the following programme: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 50°C for 15 s, and 60°C for 2 min. Samples were then purified using ethanol precipitation and run on a 16-capillary ABI 3130xl Avant sequencer. Sequence results were reviewed and edited using ABI Sequence Scanner version 2.0 software. Sequence data was compiled in a single FASTA file used to query the National Centre for Biotechnology Information’s GenBank database using the Basic Local Alignment Search Tool (BLAST) algorithm (Altschul et al. 1990) in order to identify species. To avoid identification bias due to potential misidentified sequences in GenBank, the highest score(s) corresponding to sequences deposited by Lévesque & De Cock (2004) or Robideau et al. (2011) was (were) used for identification purposes. After identification, a subset of representative strains was deposited with the Canadian Collection of Fungal Cultures (AAFC, Ottawa, ON).

Results and discussion

A total of 341 isolates, tentatively identified as Pythium species by microscopic examination, were isolated from the soil samples collected from 108 fields. Seventy-six isolates were recovered on selective media from 25 fields in 2010, 175 were from 43 fields in 2011, and 90 were from 40 fields in 2012. Overall, Pythium species were isolated from approximately half (108/209) of the fields sampled. It is possible that the selective medium used may not have allowed for recovery of all Pythium species (Hendrix & Campbell 1973), or that root rot symptoms observed on soybean plants in these fields were caused by other pathogens, such as Phytophthora sojae Kaufm. & Gerd., Fusarium spp. or Rhizoctonia solani Kühn (Hartman et al. 1999). Of the 341 cultures, ITS regions were amplified and sequenced for 104, and identified as belonging to six Pythium species (Table 1). Pythium ultimum var. ultimum Trow (56.7%) and P. sylvaticum (29.8%) were the most frequently isolated species over three sampling years. Other species isolated included P. dissotocum (6.7%), P. ultimum var. sporangiiferum Drechsler (3.8%), P. irregulare Buisman (1.9%) and P. hypogynum Middleton (1.0%). Representative sequences were deposited in GenBank, and similarity to published sequences for selected isolates per species is shown in Table 2.

Table 1. Pythium species identification of isolates recovered from soil samples collected from soybean fields in Ontario, by sampling year.

	Year
	2010		2011		2012		Total
Pythium species (clade^1)	No.	%	No.	%	No.	%	No.	%
P. ultimum var. ultimum (clade F)	8	57.1	18	60.0	33	51.5	59	56.7
P. sylvaticum (clade F)	0	0.0	9	30.0	22	39.4	31	29.8
P. dissotocum (clade B)	5	35.7	0	0.0	2	3.0	7	6.7
P. ultimum var. sporangiiferum (clade F)	0	0	2	6.7	2	3.0	4	3.8
P. irregulare (clade F)	0	0.0	1	3.3	1	1.5	2	1.9
P. hypogynum (clade E)	1	7.1	0	0.0	0	0.0	1	1.0
Total	14	100.0	30	100.0	60	100.0	104	100.0

¹Based on ITS sequences in Robideau et al. (2011).

Table 2. Sampling information and species identification for select Pythium strains isolated from soil samples collected from soybean fields in Ontario, their isolate designation, GenBank accession numbers and similarity to published sequences.

				GenBank accession
Species	Sampling year	Sampling location	DAOM	New	Comparison	Similarity^1 (%)
P. ultimum var. ultimum	2012	Norfolk	242833	KJ192355	AY598657.2	100
	2012	Ottawa	242834	KJ192354	HQ643886.1	99
	2011	Mosborough	242835	KJ192353	HQ643888.1	99
	2010	NR	Not available (NA)^2	KJ192356	HQ643914.1	98
	2012	Ottawa	242836	KJ192357	HQ643919.1	98
	2011	Ottawa	242837	KJ192352	HQ643942.1	99
P. sylvaticum	2012	Finch	242826	KJ192349	AY598645.2	99
	2011	Fullarton	242827	KJ192348	AY598645.2	99
	2011	Ottawa	242828	KJ192347	AY598645.2	99
	2012	Carlsbad Springs	242829	KJ192350	AY598645.2	98
P. dissotocum	2012	Ottawa	NA	KJ192362	AY598634.2	99
	2010	Not recorded (NR)	NA	KJ192363	AY598634.2	98
P. ultimum var. sporangiiferum	2011	Tecumseh	242832	KJ192358	HQ643918.1	97
	2012	Blenheim	242830	KJ192359	HQ643918.1	97
	2012	Alvinston	242831	KJ192360	HQ643918.1	98
P. irregulare	2012	Ridgetown	242825	KJ192351	AY598702.2	99
P. hypogynum	2010	NR	242824	KJ192361	AY598693.2	98

¹Based on sequence sizes ranging from 546 to 974 base pairs.

²Isolates were not viable when retrieved from storage on V8 agar slants overlaid with mineral oil stored at 15°C.

Previous studies have reported the identification of Pythium species infecting soybean based on molecular techniques. Jiang et al. (2012) isolated 186 Pythium strains belonging to 28 species from soils from 11 soybean fields and one corn field with a history of corn-soybean rotation in Illinois. The species most frequently recovered were P. oopapillum Bala, de Cock & Lévesque (12% of isolates) and P. diclinum Tokun. (9%), although P. irregulare (8%), P. dissotocum (5%) and P. sylvaticum (5%), P. ultimum var. ultimum (2%) and P. ultimum var. sporangiiferum (2%) were also identified. Broders et al. (2007) identified 11 species and two morphological groups from 124 Pythium isolates from diseased corn and soybean seedlings sampled from 42 fields with reports of emergence problems in Idaho. The most frequently isolated species from soybean were P. ultimum var. ultimum (22%) and P. ultimum var. sporangiiferum (22%), followed by P. sylvaticum (8%), P. irregulare (8%), P. helicoides Drechsler (8%), P. echinulatum V.D. Matthews (8%), morphological group 3 (8%). While the differences in the representation and identification of species isolated may reflect the distribution and abundance of Pythium species in the sampled soils as influenced by soil type and locality, differences in the selective media used to isolate these species also possibly contributed to these findings. The influence of the selective media used on the Pythium species recovered has been discussed by Hendrix & Campbell (1973).

ITS sequencing is a powerful tool to identify oomycete species, but it has its limitations in differentiating closely related species. For instance, P. dissotocum, P. coloratum, P. lutarium Ali-Shtayeh and P. marinum Sparrow have identical ITS sequences, and that of P. diclinum differs from these by 1 bp only. These species were included in subclade B2 by Lévesque & De Cock (2004) and were listed as possible synonyms. Pythium sp. ‘group F’, Pythium aff. dictyosporum and Pythium aff. diclinum are grouped with these species in the phylogenetic tree presented (in the supporting information) by Robideau et al. (2011). These species also were strong matches to the species identified as P. dissotocum in this study, the species that would take precedence if the other species with the same ITS sequence are synonyms. According to Robideau et al. (2011), ITS sequences alone cannot differentiate between P. sylvaticum and P. terrestris although sequencing of other loci, such as the cytochrome c oxidase subunit I (COI), which they propose as a complementary barcode to the ITS, would allow differentiation between these two species. Other Pythium species remain indistinguishable by either loci, e.g. P. irregulare from P. cryptoirregulare Garzón, Yánez & G.W. Moorman and P. cylindrosporum B. Paul, and P. acrogynum Y.N. Yu from P. hypogynum. The results presented in Tables 1 and 2 are based on the strongest matches in GenBank. It has been suggested that P. irregulare (Barr et al. 1997) and related species and P. ultimum var. ultimum (Barr et al. 1996) and related species form apparent species complexes based on ITS and COI sequences (Robideau et al. 2011). The identification of four strains as P. ultimum var. sporangiiferum was based on new multigene data on the phylogeny of P. ultimum, which suggests that the ITS clade with the ex-type of P. ultimum var. sporangiiferum is a valid variety or could even be a different species (Eggertson et al. 2012).

Overall, this study has shown that six different Pythium species were isolated from soybean disease-conducive soil samples from Ontario. Among the identified isolates, P. ultimum var. ultimum and P. sylvaticum were the most prevalent species of Pythium in these soils. Results from this study may assist in the development of soybean cultivars resistant to one or multiple aggressive and prevalent Pythium spp.

Acknowledgements

The authors would like to thank J. Chapados (ECORC, AAFC, Ottawa) for her help with the sequencing reactions, and A. Nagasawa and Z. R. Djama (ECORC, AAFC, Ottawa) for technical assistance. This work was supported by the Ontario Soybean Growers (now part of Grain Farmers of Ontario) [S2012ID01] and the Manitoba Pulse Growers Association Inc.