Root rot is a common disease in soybean (Glycine max) and field pea (Pisum sativum), which restrain increased production in Canada. Sixty-seven isolates of Rhizoctonia were recovered from various diseased plants in Alberta, Canada along with three isolates from diseased soybean plants in Manitoba, Canada. According to their anastomosis behavior, 23 (32.9%) of the isolates were identified as anastomosis group (AG) 4 (AG4), 7 (10.0%) were AG2-1, 10 (14.3%) were AG2-2, 7 (10.0%) were AG5, 3 (4.3%) were AG-E and the AGs of the remaining 20 (28.6%) isolates could not be determined. Isolates belonging to AG4 produced typical symptoms of stem rot and root rot on seedlings of soybean and pea and were more aggressive than the AG2-1, AG2-2, AG5 and AG-E isolates. Selected isolates of AG4, AG2-1, AG2-2, AG5 and AG-E were to some degree able to infect common crops in Alberta, which included barley, canola, corn, faba bean, flax, lupin, lentil, pea, potato, soybean, and wheat. The genetic variability among these isolates was evaluated using phylogenetic analysis based on the rDNA ITS sequences and inter-simple sequence repeat (ISSR) markers. For the ITS sequence analysis, a neighbour-joining tree was constructed using the PAUP program, which clustered the Rhizoctonia isolates into five groups (Groups I to V). However, no correlation was observed between AGs, locations, aggressiveness or host origins. For the ISSR analysis, 54 polymorphic ISSR patterns were identified, indicating a high level of diversity among the isolates.
Introduction
Soybean [Glycine max (L.) Merr.] and field pea (Pisum sativum L.) are important leguminous crops that are widely grown in Canada (Pesticide Risk Reduction Program 2008; Masuda and Goldsmith 2009; Gabruch and Gietz 2014). Root rot diseases are important constraints to the expansion of soybean and sustainable production of field pea in Alberta, Canada (Hwang and Chang 1989; Gossen et al. 2016; Chang et al. 2018). The root rot pathogens cause damping-off, seedling blight, and root rot of soybean and field pea, which has resulted in large yield losses in Canada (Hwang et al. 2007; Xue et al. 2007; Chang et al. 2018). The root rot pathogen complex includes Rhizoctonia solani Kühn [teleomorph Thanatephorus cucumeris (Frank) Donk], Fusarium spp., Pythium spp., Aphanomyces euteiches Drechsler, and Sclerotinia sclerotiorum (Lib.) De Bary (Xue 2003; Chatterton et al. 2015; Gossen et al. 2016; Zhou et al. 2018).
Among all the pathogens, R. solani is the most frequently isolated fungi from soybean and pea roots with root rot symptoms in western Canada (Hwang et al. 2007; McLaren et al. 2015; Nyandoro et al. 2014; Chang et al. 2018). Rhizoctonia solani has a broad host range that includes pulse crops, canola, cereals, and ornamental crops (Sneh et al. 1996). In the family of Leguminosae, R. solani caused seedling blight and root rot of beans (Phaseolus vulgaris L.) (Engelkes and Windels 1996), chickpea (Cicer arietinum L.) (Hwang et al. 2003), field pea (Xi et al. 1995; Hwang et al. 2007), lupin (Lupinus angustifolius L.) (Zhou et al. 2009), and soybean (Zhao et al. 2005). In southern Alberta, the occurrence of root rot of soybean was documented in all of the 29 fields surveyed in 2014 and 2015, and R. solani was shown to be one of the components of the root rot disease complex (Nyandoro et al. 2015, 2017).
Rhizoctonia solani is a species complex with various morphological and physical characters. Based on the characterization of hyphal fusion of the isolates via somatic incompatibility (i.e., anastomosis grouping, AG), Rhizoctonia spp. can be classified into 14 AGs (AG1 to AG13 and AG-BI) (Sneh et al. 1991; Carling 1996; Cubeta and Vilgalys 1997; Kuninaga et al. 1997; Carling et al. 2002a; Stodart et al. 2007). Rhizoctonia isolates belonging to different AG often have different host ranges and differences in aggressiveness (Carling et al. 2002a). Previously, R. solani AG2-1, AG2-2, AG4, AG5, and Rhizoctonia AG-E have been isolated from soybean and pea, causing seedling blight and root rot (Zhao et al. 2005; Hwang et al. 2007).
To evaluate genetic variability and to characterize R. solani, numerous genetic markers were applied, which included restriction fragment length polymorphisms (RFLP) (Rosewich et al. 1999; Ceresini et al. 2002), random amplified polymorphic DNA (RAPD) (Cubeta et al. 1991; Zhou et al. 2009; Dubey et al. 2012; Mirmajlessi et al. 2012), amplified fragment length polymorphism (AFLP) (Taheri et al. 2007), inter-simple sequence repeats (ISSR) (Sharma et al. 2005; Stodart et al. 2007; Khodayari et al. 2009; Dubey et al. 2012; Zhou et al. 2014), and sequence analysis of internal transcribed spacers (ITS) (Carling et al. 2002b; Justesen et al. 2003; Zhou et al. 2009, 2014; Çebi Kiliçoğlu and Özkoç 2010). ISSR employs a dominant inheritance marker and can generate a great number of highly informative and reproducible alleles, which can be used to rapidly differentiate closely related individuals. ISSR analysis can be highly variable within a species and has high repeatability and stability (Stodart et al. 2007). The ITS region of rDNA, which contains the ITS1 spacer, 5.8S rRNA gene, and the ITS2 spacer, has been used to identify various AGs of R. solani (Boysen et al. 1996; Kuninaga et al. 1997) and evaluate genetic variation within this species (Zhou et al. 2009, 2014).
In the present study, a total of 70 Rhizoctonia isolates were recovered from various pulse and special crops in Alberta and Manitoba. The objectives of the present study were to: (1) characterize the isolates recovered from the infected roots of soybean or pea, (2) determine their pathogenicity to pea and soybean, (3) to evaluate the host range of the major AG types found in Alberta and Manitoba, and (4) to examine the genetic diversity of those isolates and evaluate molecular methods for grouping Rhizoctonia isolates using their ITS sequences and ISSR markers.
Materials and Methods
Isolation of the pathogens
During the summers of 2015 and 2016, disease surveys were conducted across the southern (for soybean fields) and central (for pea isolates fields) regions of Alberta to assess the occurrence and impact of root rot on soybean and pea crops (Nyandoro et al. 2015, 2017). For pathogen isolation, roots of the plants with root rot symptoms were washed under running tap water and cut into small pieces (~1 cm in length) with a sharp knife. The root pieces were placed in a 0.5% sodium hypochlorite solution for 30 s to 1 min to surface-sterilize the tissue, rinsed three times with sterile distilled water, and then placed on sterile paper towels to remove excess water. The surface-sterilized root pieces were transferred into Petri dishes onto water agar medium (WA) amended with 50 μg·mL−1 streptomycin sulfate in a laminar flow hood. The Petri dishes were incubated under laboratory conditions at a temperature of 20 °C ± 2 °C and an 8 h/16 h light and dark period, and monitored daily for the growth of fungal colonies. A total of 70 Rhizoctonia isolates were collected from diseased soybean, pea plants and other plants in those fields, which are listed in Table 1. A hyphal tip of each isolate was transferred to fresh potato–dextrose agar media (PDA) and incubated for 1 wk at room temperature. All the isolates were stored on PDA at 4 °C.
Table 1.
Location of sample collection, host, AGs, and isolate ID of Rhizoctonia isolates.
Identification and anastomosis grouping
The nuclei number of the young hyphal cells, the septa and the branching of the hyphae of Rhizoctonia isolates were examined to identify R. solani (Sneh et al. 1991). Each Rhizoctonia isolate was paired with tester isolates representing AG1, AG2-1, AG2-2, AG3, AG4, AG5, AG6, AG7, AG8 and binucleate Rhizoctonia AG-E following the protocol of Zhou et al. (2009, 2014). Testers were obtained from Department of Agriculture, Food and Nutritional Science, at the University of Alberta. When hyphae of the two isolates touched, slides were removed from the Petri dishes and examined under a microscope. The AG type of Rhizoctonia isolate was determined based on the hyphae fusion between the testing isolate and tester isolate. Two slides were prepared for each isolate under investigation. Anastomosis behaviour was determined at a 400× magnification.
Pathogenicity
All of the 70 Rhizoctonia isolates were tested for pathogenicity on the pea cv. Midas and soybean cv.900Y61 in two separate experiments following the protocols of Zhou et al. (2009, 2014). Plants were grown in 500-mL cups filled with Pro-Mix potting soil (Westgro Horticultural Supplies Inc., Calgary, AB, Canada). To inoculate the soil, Rhizoctonia isolates were grown on PDA plates (90 mm) for 10 d. The cultures with agar were cut into small pieces (~3 mm × 3 mm) with a sharp knife before inoculation. One-half of a plate of a culture with agar was added in replicate cup. Five replicate cups were used for each crop. Five seeds were planted 2 cm deep along with inoculum. Similar number of cups was included as control where the same amount of PDA pieces was added with the seeds. The experiments were laid out in a randomized complete block design in a greenhouse set for a 20/15 °C day/night regime with a 16-h photoperiod. Pots were watered daily. Seedling emergence was recorded 10 d after seeding. At 21 d after seeding, plant height was recorded. The plants were uprooted and root rot severity (0–4) and total dry biomass were recorded (Table 2). Root rot severity was assessed on a scale of 0–4, where; 0 = no lesion, 1 = small brown lesions on <25% of exterior circumference of tap root, 2 = lesions on 25%–49% of tap root, 3 = lesions on 50%–74% and tap root constricted, and 4 = tap root girdled (75%–100%) and plant wilted or dead (Zhou et al. 2009, 2014). The experiment was repeated once under the same conditions in a greenhouse. Data were subjected to analysis of variance using the SAS program and, where appropriate, Duncan’s new multiple range test at P ≤ 0.001 of significance was performed for means comparison (SAS Institute, Inc., NC, USA).
Table 2.
Pathogenicity of Rhizoctonia isolates and their effects on seedling emergence, plant height, plant biomass and root rot severity on soybean and pea.
Host range of Rhizoctonia
Eleven isolates which belonged to R. solani AG2-1, AG2-2, AG4, AG5 and a binucleate Rhizoctonia AG-E were selected for the pathogenicity test following the inoculation protocol described above (Table 3). Prior to inoculation, each Rhizoctonia isolate was grown on sterilized wheat grains for 12 d, air dried and ground, and incorporated at the time of seeding at the rate of 10 mL per pot. Ten plant species, which were in crop rotations systems in western Canada, were tested as potential hosts of barley (Hordeum vulgare L.), canola (Brassica napus L.), corn (Zea mays L.), faba bean (Vicia faba L.), flax (Linum usitatissimum L.), lentil (Lens culinaris L.), lupin (Lupinus angustifolius L.), pea, soybean, and wheat (Triticum aestivum L.) (Table 4). Ten seeds of each plant species (except for 5 seeds of faba bean per cup) were inoculated with Rhizoctonia inoculum grown in a 500-mL cup (except 5-inch square pot for growing potato) filled with Promix potting soil (Westgro Horticultural Supplies Inc., Calgary, Canada). The cups (pots) were arranged following a split-plot design with the host crops in the main plots and the isolates in the sub-plots on the bench in a greenhouse set for a 20/15 °C day/night regime with a 16-h photoperiod. Plants were watered daily and fertilized once a week. Four replicate cups were used for each crop. Plant grown with non-inoculated PDA pieces was served as non-inoculated control. To evaluate the aggressiveness of Rhizoctonia isolate on different crops, seedling emergence, height, and disease severity of individual plant were recorded 10, 15 and 21 d after seeding, respectively. At 21 d after seeding, the plants were uprooted and root rot severity (0–4) and total dry biomass were recorded. Root rot severity was assessed on a scale of 0–4 as described above.
Table 3.
Host range of Rhizoctonia spp. and their effect on root rot severity.
Table 4.
ISSR primers used to detect polymorphisms in Rhizoctonia isolates.
ITS sequencing and analysis
The genomic DNA of all the Rhizoctonia isolates was extracted from the mycelial mats which grown in potato-dextrose broth for 7 d following the method of CTAB (Doyle and Doyle 1987). The DNA concentration of each Rhizoctonia isolate was adjusted to 10–20 ng·μL−1 after estimation with a Thermo Scientific NANODROP™ 1000 Spectrophotometer (Fisher Scientific, Nepean, ON, Canada).
The ITS region was amplified with primers of ITS4 and ITS5 (White et al. 1990) in a 25 μL reaction volume containing PCR Master Mix (Promega, Madison, WI, USA), 0.5 μM of each primer and 10–20 ng of genomic DNA. The PCR amplification in the thermocycler included initial denaturation at 94 °C for 5 min followed by 35 denaturation cycles of 25 s at 94 °C, annealing for 30 s at 58 °C, and extension for 60 s at 72 °C, with a final extension of 10 min at 72 °C. The amplicons were purified using a Wizard® SV Gel and PCR Clean-Up System (Promega) and transformed into the competent Escherichia coli strain JM109 using the EcoRV-digested and T-tailed pGEM®-T EasyVector System I (Promega). Plasmid DNA containing the insert was extracted with a PureYield Plasmid Miniprep System (Promega) and sequenced in the Department of Biological Sciences, University of Alberta.
The ITS sequences were analysed and edited with BioEdit, version 7.2.6.1 (Hall 1999), and then submitted to GenBank (Accession numbers MK084631–MK084700). The 70 ITS sequences and one reference sequence from GenBank (Rhizoctonia zeae KC620581) were aligned using BioEdit. To evaluate the genetic variation among the Rhizoctonia isolates, the best-fit nucleotide substitution model was selected using the program jModelTest 2.1.10 v 20160303 (Darriba et al. 2012). Distance tree was produced with PAUP 4.0b10 software (Swofford 2002) using the neighbour-joining (NJ) approach (Saitou and Nei 1987). The NJ tree was rooted by user-specified outgroup and displayed using the TreeViewX version 0.5.0 (Page 1996). Support for groups in the NJ tree was assessed using a bootstrap analysis with 1000 replicates.
ISSR analysis
A total of 100 ISSR primer sequences were obtained from the Biotechnology Laboratory, University of British Columbia (UBC sets 9) and synthesized by Integrated DNA Technologies Inc. (IDT, Coralville, IA, USA) (Zhou et al. 2014). All 100 primers were screened against a mixture of DNA randomly chosen from the 70 Rhizoctonia isolates from the preliminary experiments. Ten primers that produced 2–10 polymorphic bands were selected for further ISSR PCR procedures with the genomic DNA of the 70 Rhizoctonia isolates (Table 4).
ISSR PCR was performed in a 25 μL reaction volume containing PCR Master Mix (Promega, Madison, WI, USA), 0.5 μM of each primer and 10–20 ng of genomic DNA. Amplification conditions in the thermocycler include a 5-min initial denaturation at 94 °C, followed by 35 cycles of 30 s at 94 °C to denature genomic DNA, 45 s for annealing of the primer at 42 °C, and 90 s at 72 °C for primer extension, with a final extension of 10 min at 72 °C. The PCR products were visualized by gel electrophoresis on 2.5% agarose gels in 1 × TAE buffer, at 110 V for 2 h. Gels were photographed with a Gel Doc™ XR System (BIO-RAD, Columbus OH, USA).
In the ISSR analysis, amplified fragments were scored for the presence (1) or absence (0) of homologous bands (Zhou et al. 2014). Only reproducible bands of strong intensity were included in the analysis. The gene frequency, genetic diversity, and genetic distance of 70 Rhizoctonia isolates were calculated. A dendrogram was also constructed for illustration of the phenetic relationships among these isolates (Zhou et al. 2014). All calculations and the dendrogram were generated using the POPGENE program (Yeh and Boyle 1997).
Results
Anastomosis grouping
According to the anastomosis behavior with Rhizoctonia tester isolates, seven isolates (10.0%) were R. solani AG2-1 (Table 1). One isolate (R98) was isolated from dill roots [Peucedanum graveolens (L.) C.B. Clark] in Bow Island, the other six isolates were isolated from soybean in Brooks (3), Fort Saskachewan (1), Strathcona (1) and Westlock (1). Ten isolates (14.3%) were AG2-2 of which two isolates from dill (1) and bean roots (1) in Bow Island, four isolates from mint (Mentha spicata L.) (1) and soybean (3) in Brooks, one from cauliflower in Leduc, one from Alstroemeria sp. in Spruce Grove, one from lupin (Lupinus angustifolius L.) in Tofield, and one from soybean in Vauxhall. Twenty-three (32.9%) isolates were identified as AG4 of R. solani, which was the dominate AG in this study. All the isolates belonging to AG4 were isolated from the family of Leguminosae (including field pea, lupin and soybean) in various locations except one isolate from Echinacea sp. in Stettler. Another seven (10.0%) isolates were collected from soybean roots and identified as AG5. These isolates were from Brooks (3), Seven Persons (1) and Strathcona (3). Three isolates were identified as binucleate Rhizoctonia AG-E were also identified from fababean in Edmonton and soybean in Westlock. However, the anastomosis groups of a total of 20 isolates remained un-determined based on anastomosis reactions with the tester strains. These isolates were recovered from field pea, soybean and cauliflower at various locations.
Pathogenicity
Pathogenicity tests revealed that out of 70 isolates of Rhizoctonia isolates, 60 isolates were able to infect soybean (DS ≥ 0.5), and 55 isolates were able to infect pea (DS ≥ 0.5) (Table 2). Among the 70 isolates tested on soybean, only six isolates (R24, R55, R56, R92a, R97b and R102) were highly aggressive (DS 3.2–4.0), 32 isolates were moderately aggressive (DS from 1.5 to 2.9), and 22 isolates were weakly aggressive on soybean (DS from 0.5 to 1.5) pathogenic. The other ten isolates were considered as non-aggressive (DS 0.0–0.4). On pea, 30 isolates were highly aggressive (DS from 3.2 to 4.0), 11 isolates were moderately aggressive (DS from 1.5 to 2.9), 13 isolates were weakly aggressive (DS from 0.5 to 1.5), and the other isolates were considered as non-aggressive (DS 0.0–0.5). Generally, the highly aggressive isolates significantly reduced seedling emergence, plant height, root and shoot biomass and increased the disease severity of both pea and soybean compared with the non-inoculated control.
Host range of Rhizoctonia
Ten plant species belonging to four families were infected (Table 3). Plant species belonging to families of Brassicaceae (canola), Graminaceae (barley, corn and wheat), Leguminosae (faba bean, lentil, lupin, pea and soybean) and Linaceae (flax) produced typical symptoms of root rot.
Host range studies indicated that 11 of the Rhizoctonia isolates which belonged to R. solani AG2-1, AG2-2, AG4, AG5 and binucleate Rhizoctonia AG-E aggressively infected almost all the plant species (Table 3). Pre-emergence damping-off, plant height and root and shoot biomass were recorded for the evaluation the aggressiveness (Data not shown). Root rot disease severity (DS) was also recorded at 21 d after inoculation.
Two isolates of AG4 (R24 and R56) were highly aggressive on canola and legume species (DS 3.2–4.0) and moderate aggressive on cereal species (1.0–2.8). The isolates of AG2-1 produced significantly different DS values on some species. The AG2-1 isolate R07 was highly aggressive on both canola cultivars (DS 4.0), moderately aggressive on flax, faba bean, lentil and soybean (DS 1.3–2.8), but less aggressive on the other plant species (0.5–1.4). The other isolate of AG2-1 (R38a) was more aggressive compared with R07 of AG2-1. R38a also was highly aggressive on both cultivars of canola (DS 4.0), faba bean (DS 3.9–4.0), flax (DS 3.3) and one cultivar of lentil (DS 3.1), moderately aggressive on another cultivar of lentil (DS 2.6), soybean (DS 2.0–2.1), lupin (DS 1.4–2.5), pea (1.5–1.7), and one cultivar of barley (DS 1.7), corn (DS 1.6) and weakly aggressive on the other plant species (0.6–1.2). Two isolates of AG2-2 were highly aggressive on canola, flax, faba bean and lentil (DS 2.5–4.0), moderate aggressive on lupin, pea, soybean and barley (DS 1.7–3.2) and weakly aggressive on corn and wheat (DS 0.4–2.1). The isolates that belonged to AG5 varied in aggressiveness on different plant species. R70 of AG5 was highly aggressive on both cultivars of faba bean (DS 3.3–3.5) and moderately aggressive on both cultivars of corn (DS 1.8–1.9), flax (DS 1.8–2.5), lentil (DS 1.5–1.9), lupin (DS 2.1–2.9), pea (DS 1.6–1.8) and soybean (DS 2.4), but weakly aggressive on barley and wheat (DS 0.8–1.7). The other isolate of AG5 (R36) was moderately aggressive on corn cv. ‘Alto’ (DS 2.2), canola cv. ‘1950’ (DS 1.8), flax cv. ‘Prairie Thunder’ (DS 2.3), two cultivars of faba bean (DS 1.6–2.1), and weakly aggressive on the other plant species (DS 0.1–1. 5). Three isolates of Rhizoctonia AG-E were highly aggressive on faba bean, lentil, lupin, pea and soybean (DS 3.2–4.0), moderately aggressive to canola, flax and the cereal crops (DS 1.6–3.7).
Sequence analysis of ITS regions
After alignment with the BioEdit program, the ITS fragments from 70 Rhizoctonia isolates were 690 bp in length. Nucleotide frequencies were A = 0.2736, C = 0.2053, G = 0.1910 and T = 0.3301. After alignment, the data optimal nucleotide substitution model was applied to the data matrix. The best-fit model (TPM2uf+G) was selected using the program jModelTest 2.1.10 v 20160303 (Darriba et al. 2012). Then, a neighbour-joining (NJ) tree was produced from the ITS sequence data, and the isolates were clustered into five groups (Groups I–V, Fig. 1). Group I was composed of two AG2-1 isolates (R01 and R98), three AG2-2 isolates (R90, R104 and R100), six AG5 isolates (R09, R14b, R36, R19, R20 and R70) and one isolate that had undetermined AG grouping (R75), with a bootstrap value of 98. Group II consisted of three AG2-1 isolates (R07, R38a and R89), four AG2-2 isolates (R22, R117, R50 and R42), one AG4 isolate (R57) and three undetermined AG isolates (R124, R127 and R131), with a bootstrap value of 95. Group III was the largest group, which included one AG2-1 isolate (R32), two AG2-2 isolates (R08 and R113), 21 AG4 isolate, one isolate of AG5 (R06), three isolates of AG-E (R92, R97 and R102) and nine undetermined AG isolates with a bootstrap value of 63. Group IV included one AG2-1 isolate (R126), one AG2-2 isolate (R02), one AG4 isolate (R53) and six undetermined AG isolates, with a bootstrap value of 97. Only one undetermined AG isolate (R55) formed a separate clad as Group V.
ISSR analysis
Of the 100 primers tested, ten primers generated reproducible banding patterns and these were selected for ISSR-PCR (Table 4). A total of 64 bands were consistently produced in repeated amplifications using the DNA of the 70 Rhizoctonia isolates as templates. All of those bands were polymorphic. The gene frequency of those 54 loci ranged from 0.0143 to 0.9857, and the genetic diversity was 0.3013 ± 0.1697. The genetic distance of these isolates ranged from 0.0000 (between R02, R09, R11, R89, R91, R42, R71 and R104; and between R21a and R131) to 1.2809 (between R07 and R55). The phenetic relationships among the 70 Rhizoctonia isolates were illustrated in a dendrogram (Fig. 2). The isolates were clustered into four groups (Groups I–IV). Group I was composed of six isolates including two AG2-1 isolates (R01 and R98), two AG2-2 isolates (R100 and R117) and two undetermined AG isolates (R124 and R127). Group II included five AG2-1 isolates (R89, R32, R126, R08 and R07), six AG2-2 isolates, 16 AG4 isolate, six AG5 isolates, three AG-E isolates (R92, R97 and R102) and 12 undetermined AG isolates. In Group III, one isolate (R38a) belonged to AG-2-1, six isolates belonged to AG4 and the other eight were undetermined AG isolates. Group IV included only the undetermined isolate of R55.
Discussion
Rhizoctonia root rot has been reported as a common disease on soybean and pea in many countries and areas (Hwang and Chang 1989; Jones and Belmar 1989; Yang et al. 1990; Muyolo et al. 1993; Naito et al. 1995; Nelson et al. 1996; Fenille et al. 2002; Ajayi-Oyetunde and Bradley 2018). In the present study, a total of 70 Rhizoctonia isolates were recovered and some of the isolates were classified as R. solani AG2-1 (7 isolates), AG2-2 (10), AG4 (23), AG5 (7), binucleate Rhizoctonia AG-E (3), but the AG designation of 20 isolates was difficult to determine. Pathogenicity of all the isolates was tested on soybean and pea separately. The tests revealed that out of 70 Rhizoctonia isolates, 60 isolates were able to infect soybean (DS > 0.50), and 55 isolates were able to infect pea (DS > 0.50).
Rhizoctonia solani AG2-1 has a broad host range that includes lupin, pea, soybean, canola, wheat, turf grass, potato, tomato, and the ornamental plant Matthiola incana (Kataria and Verma 1992; Nicoletti et al. 1999; Hwang et al. 2007; Lehtonen et al. 2009; Zhou et al. 2009, 2014; Broders et al. 2014; Gómez et al. 2015; Sharma-Poudyal et al. 2015). Seven isolates of R. solani AG2-1 were originally collected from dill (1), pea (1) and soybean (5) in the current study, and they were less aggressive on both pea and soybean except one isolate originally from soybean (R32), which was highly aggressive to both soybean and pea.
Rhizoctonia solani AG2-2 also has been reported to have a broad host range that includes beans, lupin, pea, soybean, canola, turf grass, potato, and sugar beet (Kataria and Verma 1992; Engelkes and Windels 1996; Aoyagi et al. 1998; Lehtonen et al. 2009; Zhou et al. 2009, 2014). Ten isolates of R. solani AG2-2 in the current study were originally obtained from Alstroemeria (1), bean (1), cauliflower (1), dill (1), lupin (1), mint (1) and soybean (4). All the isolates were non-pathogenic or weakly pathogenic to soybean and pea except R08 (from soybean) and R90 (from Alstroemeria), which were highly and moderate aggressively on pea, respectively.
Rhizoctonia solani AG4 was reported to infect leguminous crops like pea, chickpea, lupin, and soybean (Hwang et al. 2003, 2007; Mathew et al. 2012; Sharma-Poudyal et al. 2015). It also has been recovered from canola, potato, and wheat (Mathew et al. 2012; Woodhall et al. 2012; Zhou et al. 2014; Muzhinji et al. 2015). Twenty-three isolates of R. solani were identified as AG4 making it the dominate AG in the current study. Most isolates were originally collected from leguminous crops including lupin (4), pea (3), and soybean (15). One isolate from Echinacea (R86) was recovered and identified as AG4. All the isolates except R57 (from soybean) and R86 (from Echinacea) were highly aggressive on pea and moderately aggressive on soybean.
Rhizoctonia solani AG5 were reported as pathogens of couch grass [Elymus repens (L.) Gould] (Woodhall and Lees 2004), stem canker of buckwheat (Fagopyrum esculentum Moench) in Maine, USA (Zhang et al. 2016) and potato (Solanum tuberosum L.) in UK (Yang and Wu 2012; Muzhinji et al. 2015), wheat (Triticum aestivum L.) in the UK, Turkey, Canada, and USA (Woodhall et al. 2012; Broders et al. 2014), soybean in Canada and USA (Nelson et al. 1996; Zhao et al. 2005). In fact, R. solani AG5 was most frequently detected in the soil samples from Europe (Goll et al. 2014). In Canada, R. solani AG5 isolates were isolated from soybean in Ontario (Zhao et al. 2005) and from wheat in Alberta and Manitoba (Broders et al. 2014). In the current study, seven isolates of AG5 were originally isolated from soybean in Alberta. These isolates could infect, but were weakly aggressive on soybean and pea except for one isolate (R06), which was highly aggressive on pea. This appears to be the first report of AG5 from soybean in Alberta.
Three isolates identified as the binucleate Rhizoctonia AG-E were recovered from soybean (R92a) and faba bean (R97b and R102) in the current study. All these isolates were highly aggressive on pea and soybean. AG-E had been reported to infect bean (Demirci and Döken 1995), soybean (Ploetz et al. 1985; Erper et al. 2011), but was considered to be moderately or weakly pathogenic.
In the current study, there were 20 Rhizoctonia isolates for which the AG designation could not be determined. These isolates were originally collected from cauliflower, pea, and soybean, whose aggressiveness on soybean and pea also varied. Four isolates (R10, R15, R55 and R151) were highly or moderately aggressive on soybean. The other isolates were non-pathogenic or weakly pathogenic on soybean. When tested on pea, five isolates (R10, R15, R55, R148 and R151) were highly aggressive, five isolates (R12, R14a, R27a, R77 and R88) appeared to be non-pathogenic and the others were weakly or moderately aggressive.
Generally, in Alberta, R. solani most commonly occurs as AG4, AG2-1, AG2-2 in pulse crops (Hwang et al. 2003; Chang et al. 2005; Zhou et al. 2009), wheat, and canola (Broders et al. 2014; Zhou et al. 2014), AG5 in wheat (Broders et al. 2014) and AG9 in soil samples (Yang et al. 1996). Also, in pulse crops, AG4 isolates was the dominate AG and highly aggressive on host plants in Alberta (Hwang et al. 2003). The findings of the current study support that AG4 was the dominate AG infecting leguminous crops and causing severe seedling blight and root rot disease.
From the host range study, isolates belonging to AG4 and AG-E were more aggressive than the isolates of other AGs. Isolates belonging to AG2-1 and AG2-2 were moderately aggressive on most host plant species and highly aggressive on canola and faba bean. Isolates of AG5 displayed the lowest aggressiveness to all the plant species except R70, which was highly aggressive on faba bean and moderately aggressive on soybean. From the eleven plant species, canola, faba bean, and flax were more susceptible while potato and wheat were highly resistant to the Rhizoctonia isolates.
To evaluate the genetic variation among all the Rhizoctonia isolates, sequencing and phylogenetic analysis of the ITS regions and the ISSR were used in the current study. Sequencing of ITS regions has been shown to be a reliable method to divide isolates of R. solani into distinct groups, corresponding to the different anastomosis groups (Boysen et al. 1996; Kuninaga et al. 1997; Carling et al. 2002b). Lehtonen et al. (2008) reported that differences in the ITS regions were sufficiently large to reliably differentiate the AGs, but the region could not be used to detect differences between isolates within the same AG. Based on the ITS sequences, all the isolates were clustered to five groups. Unfortunately, there was no delineation between AGs and the clades based on the sequence analysis. Because ISSR markers produced greater polymorphisms with greater reproducibility than RAPD (Parsons et al. 1997; Chowdhury et al. 2002), ISSR was used to analyze the genetic diversity of R. solani and binucleate Rhizoctonia AG-E (Dubey et al. 2012; Zheng et al. 2013; Shu et al. 2014; Zhou et al. 2014). Based on the ISSR analysis in the current study, four groups could be recognized, but there was no correlation between the AGs and the clades.
In summary, Rhizotonia was important pathogen, which restrains the production of soybean and pea in Alberta and Manitoba. AG4 was the dominate anastomosis group, followed by AG2-2, AG2-1, AG5 and AG-E, but the AG of 20 isolates could not be determined. Isolates belonged to AG4 were more aggressive on soybean and pea. The selected isolates belonging to AG4, AG2-1, AG2-2 and AG5 were able to infect common crops in Alberta which including barley, canola, corn, faba bean, flax, lupin, lentil, pea, potato, soybean and wheat. AG4 isolates were more highly aggressive than the isolates of the other AGs. There was no correlation between AGs, location and host origin based on the ITS and ISSR analysis.
Acknowledgements
This research was funded by the Government of Canada through the Agri-Flex Program, Growing Forward 2, and Pest Management and Surveillance Implementation (Pulse Science Cluster of Agriculture and Agri-Food Canada), and by the Saskatchewan Pulse Growers and Manitoba Pulse Growers Associations. We would also like to acknowledge the technical support provided by Heting Fu.
