Abstract
Pythium root rot is widely distributed in major soybean (Glycine max) production areas throughout the world. There are many species of Pythium described on soybean and other crops, although not all species are pathogenic on these crops. The objectives of this study were to isolate and identify Pythium isolates obtained from field soils across Illinois and evaluate their pathogenicity on soybean seedlings. Soil samples were collected from 12 corn-soybean rotation fields in six Illinois counties. All isolates of Pythium were recovered through a baiting technique, identified to the species taxon using morphological and molecular techniques, and evaluated using an in vitro pathogenicity assay on soybean seedlings. Twenty-seven species of Pythium were identified, and P. cryptoirregulare, P. irregulare, P. sylvaticum, P. ultimum var. sporangiiferum and P. ultimum var. ultimum were highly pathogenic on soybean seedlings.
Résumé
On trouve le pourridié pythien des racines dans toutes les principales régions productrices de soja (Glycine max) de la planète. De nombreuses espèces de Pythium sont associées au soja et à d'autres cultures, bien que toutes ces espèces ne soient pas pathogènes. Les objectifs poursuivis par cette étude étaient d'isoler, de déterminer et d'évaluer, sur des semis de soja, la pathogénicité d'isolats de Pythium collectés dans des échantillons de sol provenant de multiples sites de l'Illinois. Des échantillons de sol provenant de champs utilisés pour la rotation maïs-soja ont été collectés à 12 endroits dans 6 comtés de l'État. Tous les isolats de Pythium ont été obtenus par appâtage, identifiés au taxon de l'espèce à l'aide de techniques morphologiques et moléculaires et évalués par des tests de pathogénicité in vitro sur des semis de soja. Vingt-sept espèces de Pythium ont été identifiées et, parmi celles-ci, P. cryptoirregulare, P. irregulare, P. sylvaticum, P. ultimum var. sporangiiferum et P. ultimum var. ultimum se sont révélés hautement pathogènes à l'égard des semis de soja.
Introduction
There are more than 180 species that belong to the oomycete genus Pythium Pringsh. (van der Plaats-Niterink, Citation1981). Many of these species are phytopathogenic and cause damage to important crop plants throughout the world (Hendrix & Campbell, Citation1973). In soybean, Glycine max (L.) Merr., Pythium spp. are frequently associated with seed and seedling diseases. Pythium spp. are often associated with poor stands when soybean is planted in moist and cold soils, causing pre- or post-emergence damping-off during the early vegetative stages and root rot during advanced growth stages (Hartman et al., Citation1999). Economically important losses have been documented, mainly from infection of seedlings, resulting in serious stand reductions. Significant yield losses occur when the pathogen kills plants and causes numerous, large skips in rows or where plants have been killed in larger areas of the field (Rizvi & Yang, Citation1996).
Through 2008, at least 14 different species of Pythium have been reported to be pathogenic to soybean, including P. aphanidermatum (Edson) Fitzp., P. attrantheridium Allain-Boule & Levesque, P. debaryanum R. Hess, P. deliense Meurs., P. dissotocum Drechsler, P. echinulatum V. D. Mathews, P. helicoides Drechsler, P. inflatum V. D. Mathews, P. irregular Buisman, P. myriotylum Drechsler, P. spinosum Sawada, P. sylvaticum W.A. Campb. & F. F. Hendrix, P. torulosum Coker & F. Patt. and P. ultimum (P. ultimum var. sporangiiferum Drechsler and P. ultimum var. ultimum Trow) (Farr & Rossman, Citation2011). Many of these species have recently been proposed for name changes into several new genera (Uzuhashi et al., Citation2010). In Illinois, only P. debaryanum has been reported on soybean to date (Anonymous, Citation1960).
Most Pythium species are easily isolated from soil by using either dilution plating, direct plating of diseased seedlings, or baiting from soil slurries or aquatic samples (Singh & Mitchell, Citation1961; All-Shtayeh et al., Citation1986; Weiland, Citation2011). Following the description of a selective medium for isolation of Pythium spp. (Singh & Mitchell, Citation1961), various media have been developed for enrichment and detection of Pythium spp., including a modified Pythium selective medium named NARF (nystatin + ampicillin + rifampicin + fluazinam agar), which was shown to be comparable to PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar) when using the soil-dilution plating technique to quantify naturally occurring Pythium spp. from different types of soil (Morita & Tojo, Citation2007).
Despite the ease of recovery, the subsequent identification of Pythium to species is more challenging. Difficulty in correctly identifying Pythium spp. hampers attempts to determine how many different species are present in a field, which species are the more abundant, and which are the most pathogenic. The reliable identification of Pythium species is also important for clarifying their host range, geographical distribution, for diagnosis of disease, and for development of management strategies including host resistance. The morphological identification of Pythium species has been based mainly on the sizes and shapes of the oogonia, oospore, antheridia and sporangia (van der Plaats-Niterink, Citation1981; Dick, Citation1990). But as new Pythium species are described, the limitations of the available morphological characteristics in encapsulating all present-day knowledge of the genus become increasingly evident.
Many molecular approaches have been employed to detect and identify pure cultures of oomycetes, including restriction fragment length polymorphisms of nuclear and mitochondrial DNAs, PCR analysis of internal transcribed spacer (ITS) regions of nuclear ribosomal DNA, randomly amplified polymorphic DNA PCR assays, and DNA hybridization probes (Lévesque et al., Citation1998; Ristaino et al., Citation1998; Cooke et al., Citation2000; Lévesque & De Cock Citation2004; Martin et al., Citation2004; Tambong et al., Citation2006). The ITS region has been used extensively for studies in identification and detection of Pythium species, primarily because the PCR primers universally amplify these highly variable regions present in a broad spectrum of taxa, including oomycetes (White et al., Citation1990). The objectives of our study were to isolate and identify Pythium isolates obtained from field soils at multiple sites within Illinois, and to evaluate their pathogenicity on soybean.
Materials and methods
Isolation and identification
Soil samples were collected from 12 corn-soybean rotation fields in six Illinois counties (). Soil from field numbers 1 and 2 were classified as a silt, 3–11 a silt loam, and field 12 a silty clay loam. The crop in each of the 12 fields in 2010 when the soil samples were taken was soybean, except field 12, which was planted to corn. Previous cropping before 2010 and attributes such as slope or elevation of the field were not recorded. From each bulk sample, 3 g of soil was placed in a 300 mL flask with 200 mL distilled water. The flasks were placed on a gyratory shaker set at 150 rpm for 3–5 h at 22 °C. The shaker was then turned off and 50 soybean ‘Williams’ leaf discs (1 cm2) per flask were floated on the surface for 3–7 days in darkness. Leaf discs with lesions were placed on water agar (WA) plates (15 g bacto agar L−1; Becton, Dickinson and Company, Spark, MD) and incubated for 2 days at 22 °C in darkness. Following microscopic examination of resulting cultures, isolates with characteristic coenocytic hyphae were transferred as hyphal tips to NARF selective medium (Morita & Tojo, Citation2007) composed of 17 g corn-meal agar (CMA) (Becton Sigma-Aldrich, St. Louis, MO), 15 g bacto agar, 50 mg nystatin (Sigma-Aldrich; dissolved in 1 mL ethanol), 250 mg ampicillin (Sigma-Aldrich), 10 mg rifampicin (Sigma-Aldrich; dissolved in 1 mL of dimethyl sulfoxide), 1 mg fluazinam (Sigma-Aldrich), and 1 L deionized water. The antibiotics and fungicide were added after the CMA basal medium (with agar) had been autoclaved and cooled to 50 °C, and the solution was mixed thoroughly before pouring 20 mL of medium into 9 cm diameter Petri plates. Once cooled, the medium was stored at 13 °C to 15 °C in darkness and used within 1 to 3 days. Isolates were identified by observing morphological characteristics of oogonia, antheridia, sporangia, and zoospores and comparing those observations to standard keys (Waterhouse, Citation1967; van der Plaats-Niterink, Citation1981).
Table 1. Number of isolates of each Pythium species recovered from Illinois soils from corn–soybean rotation fields (12 locations)
DNA extraction, polymerase chain reaction and sequence analysis
The internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA from at least one representative isolate of each putative species was sequenced to either confirm identification or assist in further identification. Each isolate was grown on NARF for 4 days and one 1.2 cm plug was stored at −20 °C until used to extract DNA. The Fast DNA Spin Kit (MP Biomedicals Inc., Solon, OH) was used to extract DNA according to the manufacturer's instructions, with further purification using the E.Z.N.A. MicroElute DNA Clean Up Kit (Omega Bio-Tek Inc., Norcross, GA) and final elution in 35 μL of 10 mM Tris, pH 8.5. DNAs were stored at −20 °C.
Universal eukaryotic primers UN-UP18S42 (5'-CGTAACAAGGTTTCCGTAGGTGAAC-3') and UN-LO28S22 (5'-GTTTCTTTTCCTCCGCTTATTGATATG-3') were used to generate the sequencing template by amplifying the ITS1, 5.8S gene and ITS2 of the nuclear ribosomal DNA (White et al., Citation1990). PCR amplification was performed using the Phusion™ High-Fidelity PCR Kit (New England Biolabs, Ipswich, MA), and the supplied Phusion high fidelity buffer. Each 50 μL reaction included 200 μM of each dNTP, 1 Unit of Phusion DNA polymerase, 500 nM of each primer and 5 μL of template DNA. Amplification was carried out in 0.2-mL thin-walled microtubes in a PTC-100 Programmable Thermal Controller (MJ Research Inc., Watertown, MA). The thermal profile was an initial denaturation at 98 °C for 30 s, followed by 33 cycles of: 10 s at 98 °C, 30 s at 57 °C, 45 s at 72 °C; and completed with an extension at 72 °C for 10 min. Amplification products were evaluated by electrophoresis of an aliquot on a 1% agarose gel to confirm the presence of a single band of the expected size, and the remaining material was purified using the E.Z.N.A. MicroElute Cycle Pure Kit (Omega Bio-Tek, Inc.).
Purified DNA product was bidirectionally sequenced by the DNA Core Sequencing Facility of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign using the same primers (UN-UP18S42 and UN-LO28S22) that generated the amplicons. Sequence data were compared with known sequences deposited in the National Center for Biotechnology Information (NCBI) non-redundant database to confirm morphological identification and assist with identification of isolates which did not produce all morphological traits.
Pathogenicity assays
The isolates were tested for pathogenicity on soybean seedlings ‘Williams’. Seeds were surface sterilized in 70% ethanol for 1 min and then in 15% commercial bleach (5.25% sodium hypochlorite) for 20 min with occasional shaking, and rinsed with sterile water at least three times for 3 min each time. Seeds were then placed on PDA medium for 2–3 days (10 seeds per plate) at 22 °C, with a 16 h photoperiod, and subsequently visually assessed for contamination. Each Pythium isolate was grown on CMA for 3 days, and then a 5-mm diameter plug was transferred to the centre of a 9-cm diameter Petri plate containing 15 mL of 2% WA. Five clean, 2–3-day-old soybean seedlings were evenly distributed around the plug in the plate. Plates were incubated at 22 °C in the dark for 3 days and then for 5–7 days at 22 °C, under a 16-h photoperiod.
Each isolate was scored for pathogenicity initially based on the following rating scale: 0 = seedlings with no symptoms of infection; 1 = seedlings with a few black/brown lesions on the fibrous roots; 2 = seedlings with coalesced lesions and reduced fibrous root growth; 3 = seedlings with significantly impaired taproot growth and black/brown lesions; and 4 = seedlings completely killed. For purposes of pathogenicity analysis, ratings of 0 and 1 were considered non-pathogenic while ratings of 2, 3, and 4 were pathogenic. There were five seedlings evaluated for each isolate in each experiment. This was conducted three times to make a total of 15 seedlings evaluated for each isolate.
Results
Pythium spp. isolation and identification
A total of 186 isolates of Pythium were recovered from 12 corn-soybean rotation fields in six Illinois counties. These isolates represent 27 species () and were identified by their morphological characteristics with confirmation by sequence analysis of the ITS region (). The species identified were P. acanthophoron (Sideris), P. acrogynum (Y.N. Yu), P. adhaerens Sparrow, P. aphanidermatum, P. attrantheridium, P. catenulatum V.D. Mathews, P. cryptoirregulare Garzón, Yánez & G.W. Moorman, P. diclinum Tokun., P. dissotocum, P. heterothallicum W.A. Campb. & F.F. Hendrix, P. hypogynum (Middleton), P. inflatum, P. irregulare, P. litorale Nechw., P. longisporangium B. Paul, P. lutarium Ali-Shtayeh, P. middletoni Sparrow, P. oopapillum Bala, de Cock & Lévesque, P. orthogonon Ahrens, P. perplexum H. Kouyeas & Theoh., P. porphyrae M. Takah. & M. Sasaki, P. pyrilobum Vaartaja, P. sterilum Belbahri & Lefort, P. sylvaticum, P. torulosum, P. ultimum (including P. ultimum var. sporangiiferum and P. ultimum var. ultimum), and P. vexans de Bary. The species most frequently recovered were P. oopapillum and P. diclinum (), representing 12.4 and 9.1% (23 and 17 isolates) of the isolates collected, respectively. Analysis of the ITS sequences showed a high degree of similarity of sequences, over 99%, to reference GenBank accessions ().
Table 2. Pythium species identified from Illinois soils, their isolate designation, GenBank accession numbers and similarity to published sequences
Pathogenicity assays
Of the 2790 inoculations, 79% were rated 0 or 1, while 21% were rated 2, 3, or 4 (). Isolates of P. ultimum produced disease ratings of 3 and 4, while isolates of other species like P. aphanidermatum produced disease ratings of 0 and 1. In addition to P. ultimum, species of P. cryptoirregulare, P. irregulare and P. sylvaticum were highly pathogenic on soybean seedlings and all had ratings higher than 2.
Table 3. Evaluation of 186 isolates representing 27 species of Pythium on soybean seedlings using a Petri plate assay and rated using a 0–4 scale
Discussion
There are many species of Pythium described on soybean and other crops, although not all species are pathogenic on these crops. Pythium spp. that infect soybean are soil inhabitants and can grow on plant debris saprophytically or survive as oospores in soil for many years. Most Pythium species are easily isolated by soil-dilution plating, direct culture from diseased seedlings, or by one of several baiting methods. We used soybean leaves as bait to isolate Pythium from the soil, which could have favoured species that form zoospores, although controlled experiments are needed to verify this. In all, 186 isolates of Pythium were recovered. We identified 27 species of Pythium and tested their pathogenicity on soybean seedlings; P. cryptoirregulare, P. irregulare, P. sylvaticum, P. ultimum var. sporangiiferum and P. ultimum var. ultimum were highly pathogenic on soybean seedlings. Although only P. debaryanum has been reported on soybean in Illinois (Anonymous, Citation1960), this species has not been recognized in over 40 years and the earlier literature may have been referring to P. irregular or P. ultimum instead of P. debaryanum. The recovery of pathogenic species of Pythium on soybean was similar to what was isolated from soybean in Arkansas with the exception of P. aphanidermatum and P. vexans, which were not pathogenic in our tests, and P. sylvaticum, which was not found in Arkansas (Kirkpatrick et al., Citation2006). Neither our study, nor the one in Arkansas (Kirkpatrick et al., Citation2006) reported P. splendens, which was reported to be highly pathogenic on soybean in Ohio (Dorrance et al., Citation2004), indicating the occurrence of potential regional differences in Pythium species attacking soybean.
The Petri plate pathogenicity assay is similar to that used previously to evaluate pathogenicity and aggressiveness of Pythium spp. on alfalfa (Alteir & Thies, Citation1995), and on corn and soybean (Dorrance et al., Citation2004). Many species of Pythium have been reported as pathogens of soybean, but this is the first report of P. cryptoirregulare, P. irregulare, P. sylvaticum, P. ultimum var. sporangiiferum and P. ultimum var. ultimum from Illinois. Specifically, P. cryptoirregulare, which was delineated from P. irregulare complex (Garzón et al., Citation2007), showed greater level of disease on soybean seedlings than P. irregulare. Most of the species we isolated were non-pathogenic on soybean seedlings. This may be because our isolates were obtained from soil rather than from diseased soybean tissues. Although not tested in our study, some of these species are likely to attack other hosts.
Genetic resistance to P. aphanidermatum has been reported in the soybean cultivar ‘Archer’; this resistance was shown to be controlled by a single dominant resistance gene to P. aphanidermatum (Rosso et al., Citation2008). In addition, ‘Archer’ was reported to be resistant to P. irregulare, P. ultimum and P. vexans (Bates et al., Citation2008), although the genetics of the resistance in ‘Archer’ to these other species has not been reported. Even though soybean resistance to P. aphanidermatum has been described, it does not appear to be utilized in commercial soybean cultivars. In the absence of genetic resistance, other factors like soil drainage, selective planting times, crop history, soil amendments and the use of fungicide seed treatments play a role in the management of Pythium spp. on soybean (Hartman et al., Citation1999; Tamm et al., Citation2010). Further losses may be avoided with deployment of cultivars with resistance to multiple Pythium spp.
Acknowledgements
We thank the Illinois Soybean Association for partial financial support of this research.
Notes
Trade and manufacturers' names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.
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