Characterization and pathogenicity of Rhizoctonia spp. from field crops in Canada

Abstract

A survey for the presence of Rhizoctonia species on barley, bean, canola, corn, pea, soybean and wheat in 170 fields in Canada was completed during 2009–2011. Rhizoctonia solani was recovered from 52 (31%) of the 170 fields surveyed. A total of 191 isolates of R. solani belonging to anastomosis groups (AG) AG 2-1, AG 2-2, AG 4, AG 5, AG 9 and AG 11 were recovered. Fifty-two binucleate isolates of Rhizoctonia spp. were also recovered from 35 (21%) of the crops surveyed. In growth room pathogenicity studies, isolates belonging to AG 2-1 were pathogenic primarily on canola while isolates of other anastomosis groups tended to have wider host ranges. Symptoms included pre- and post-emergence damping off, crown rot and, to a lesser extent, root rot. The majority of binucleate isolates were not pathogenic or only had low aggressiveness on the hosts tested. There was considerable variation in host range among isolates of R. solani within individual AGs, and isolates recovered from asymptomatic seedlings of some hosts were pathogenic to other hosts often used in the same rotation. Collectively, the results are important for documenting the widespread occurrence of R. solani in seedlings of field crops in Canada, for characterizing variation in AGs and host range, and for identifying effective crop sequences used in crop rotations.

Résumé

Une étude visant à détecter la présence d’espèces de Rhizoctonia chez l’orge, les haricots, le canola, le maïs, les pois, le soya et le blé a été menée dans 170 champs au Canada de 2009 à 2011. Rhizoctonia solani a été prélevé dans 52 (31%) des champs échantillonnés. En tout, 191 isolats de R. solani appartenant aux catégories d’anastomoses (AG) AG 2-1, AG 2-2, AG 4, AG 5, AG 9 et AG 11 ont été trouvés. Cinquante-deux isolats binucléés de Rhizoctonia spp. ont également été prélevés chez 35 (21%) des plantes cultivées échantillonnées. Au cours d’études de pathogénicité menées en chambre de croissance, des isolats appartenant à AG 2-1 se sont avérés virulents à l’égard du canola tout particulièrement, tandis que les isolats des autres catégories d’anastomoses semblaient posséder des gammes d’hôtes plus étendues. Les symptômes incluaient la fonte des semis, prélevée et postlevée, ainsi que la pourriture du collet et, dans une moindre mesure, le pourridié des racines. La majorité des isolats binucléés n’étaient pas virulents ou n’étaient que faiblement pathogènes à l’égard des hôtes testés. Les isolats de R. solani affichaient une grande variation quant à la gamme d’hôtes, et ce, au sein d’une même catégorie d’anastomoses, et des isolats prélevés sur des semis asymptomatiques de certains hôtes étaient virulents à l’égard d’autres hôtes souvent utilisés dans le cadre de la même rotation. Collectivement, les résultats obtenus sont importants en ce qui a trait à la description de la dispersion à très grande échelle de R. solani dans les semis des cultures commerciales au Canada, principalement pour caractériser les variations qui existent dans les catégories d’anastomoses et dans les gammes d’hôtes ainsi que pour établir des séquences culturales efficaces à utiliser dans les rotations.

Keywords:

• anastomosis group
• binucleate
• crop rotation
• host range
• pathogenicity
• Rhizoctonia solani
• Rhizoctonia spp.

Mots clés:

• binucléé
• catégorie d’anastomoses
• gamme d’hôtes
• pathogénicité
• Rhizoctonia spp
• Rhizoctonia solani
• rotation des cultures

Introduction

Plant pathogenic Rhizoctonia species occur worldwide and are among the soilborne fungal plant pathogen complex that cause damping off and root and crown rots of numerous important agricultural and horticultural crops. Within this genus, the multinucleate anamorphic species complex Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris (A.B. Frank) Donk.) is comprised of genetically diverse isolates that are characterized by intraspecific anastomosis groups (AGs). These AGs have varying levels of host specificity, virulence and cultural morphology (Ogoshi 1987; Sneh et al. 1996), and are used for intraspecific classification of isolates. Currently, there are 14 recognized AGs (AG 1–AG 13 and AGBI) (Carling et al. 2002a, 2002b) but several AGs have been further subdivided into subgroups based on one or more biochemical, genetic or pathogenic characteristics (Sneh et al. 1996).

In Canada, R. solani is a widely distributed and important plant pathogen that is associated with seed rot, damping-off, seedling blight, stem rot and/or root rot of numerous field crops, including bean, canola, chickpea, corn, lentil, pea and soybean. Disease severity on these crops can vary widely and individual AGs within R. solani are associated with varying levels of host specificity. For example, isolates of R. solani that cause disease in bean are primarily AG 4 and AG 2-2 (Schwartz et al. 2005) although other AGs have been reported on bean (Erpera et al. 2011). On canola and oilseed rape in western Canada, isolates of R. solani are primarily AG 2-1 and AG 4 (Kaminski & Verma 1985; Gugel et al. 1987; Yitbarek et al. 1987; Verma 1996), but low numbers of AG 2-2 and binucleate isolates of R. solani have also been reported (Yitbarek et al. 1987). Seedling blight and crown and brace root rot of corn are primarily associated with isolates of AG 2-2 (Sumner & Minton 1989; White 1999) but other AGs have been detected in other regions or cropping systems, such as intercropped vegetable production in New York (Ohkura et al. 2009). On field pea, isolates of R. solani are primarily assigned to AG 4 (Kraft & Pfleger 2001) but AG 2-1 and AG 5 also occur (Xue 2003; Hwang et al. 2007; Mathew et al. 2012). On lentil, isolates of R. solani cause damping-off, seedling blight and root rot, and are assigned to AG 4 (Wang et al. 2006). On soybean, R. solani causes seed rot, damping-off, seedling blight, and root and stem rot, associated primarily with isolates belonging to AG-4 but some isolates within AG 2-2, AG 5 and AG 7 also cause root rot (Hartman et al. 1999; Zhao et al. 2005). Rhizoctonia solani is not considered an important pathogen of wheat in Canada (Menzies & Gilbert 2003) although it can be severe in Australia, parts of Europe and in the Pacific Northwest of the United States where disease is caused primarily by isolates of AG 8 (Bockus et al. 2010). There is evidence that Rhizoctonia spp. may be more important in wheat in the central wheat-growing regions of North America than currently thought as characteristic symptoms (Strausbaugh et al. 2004) and the frequent recovery of isolates of Rhizoctonia spp. (Fernandez & Jefferson 2004) have been reported. Broders et al. (2014) recently suggested that wheat may be harbouring isolates of AG 2-1 and allowing inoculum levels to remain high enough to cause disease on successive canola crops.

Binucleate isolates of Rhizoctonia spp. are also commonly reported from soil, plant rhizospheres and roots, and represent another diverse group of pathogenic, symbiotic and saprophytic taxa. Many of these isolates are associated with a Ceratobasidium teleomorph (González García et al. 2006). Binucleate isolates of Rhizoctonia spp. are known to be pathogens of various crops (Sneh et al. 1991) but can also play important roles as mycorrhizal endophytes of orchids and as biological control agents of other pathogenic Rhizoctonia spp. (Vilgalys & Cubeta 1994; Herr 1995; Sneh et al. 1996). As with the R. solani complex, there is considerable genetic variability within the binucleate Rhizoctonia isolates, and at least 26 groups have been described based on anastomosis behaviour (Vilgalys & Cubeta 1994; González García et al. 2006).

The implementation of conservation tillage practices in recent decades has greatly benefitted crop production through protection of soil, conservation of soil moisture, and improvement of soil quality. In general, these benefits are associated with increases in the amount of crop residues on, and in, the soil, but the amount and distribution of residue within the soil can vary according to the type and orientation of crop residues present (Sumner et al. 1981; Smika & Unger 1986; Lafond et al. 1996; Bockus & Shroyer 1998). Conservation tillage can also influence the prevalence and severity of foliar and soilborne plant diseases as these practices can alter the soil habitat, including the amount, distribution and decomposition of crop residues where pathogens can proliferate and/or overwinter. Rhizoctonia solani is associated with survival in crop residues, and disease can be favoured by undisturbed soils associated with conservation tillage or reduced crop rotation (Sumner et al. 1981; Bailey 1996; Bockus & Shroyer 1998). In addition, conservation tillage can contribute to lower soil temperatures and higher soil moisture at seeding, factors which contribute to increased pre- and post-emergence damping off by R. solani. Survival of many plant pathogens is also influenced by crop rotation, and there has been a trend in many regions towards reduced crop rotations where, for example, two-year cropping sequences of wheat-canola in western Canada or wheat-soybean in eastern Canada are now common. Crop rotations that incorporate plants that are less susceptible to specific pathogens can result in a decline in populations of the pathogen due to natural mortality and the antagonistic activities of other soil microorganisms (Williams & Schmitthenner 1962; Fry 1982). Changes in production practices such as these can also result in changes in the presence and population structure of R. solani and AGs within cropping systems. Surveys of the occurrence or distribution of R. solani commonly report on the presence of isolates associated with infested soils or diseased plants, and often also report on the identification of specific AG groups among these isolates (e.g. Tewoldemedhin et al. 2006; Goll et al. 2014). However, relatively few studies have compared the potential variation in pathogenicity or host range within individual AGs. Knowledge of such variation among AGs is important in identifying effective disease management practices.

The objectives of this research were to assess the distribution and prevalence of Rhizoctonia spp. in field crops in western and central Canada over a 3-year period, and to characterize recovered isolates of R. solani for anastomosis groupings and pathogenicity on selected field crops.

Materials and methods

Sampling and isolation

During 2009–2011, selected fields were surveyed in Alberta, Saskatchewan, Manitoba, Ontario and Quebec (Table 1). Canola, corn, pea, soybean, wheat and white bean crops were sampled in May, June and July. Winter wheat crops were sampled in Ontario in November and December 2009. The majority of crops were sampled at the seedling stage but some crops were sampled at later stages of plant development. A diamond-shaped pattern of 50 m per side was used for sampling. Twenty seedlings were collected per field, with one seedling sampled every 10 m around each side of the diamond. If plants with symptoms of disease were observed during sampling, additional seedlings were collected from these diseased area(s). Seedlings were stored at 4°C until being processed. Prior to isolation, seedlings were rinsed in cool water, upper stems and leaves were removed, and remaining crowns and roots were then surface-disinfested for 30 s in 70% ethanol followed by 90 s in 0.6% sodium hypochlorite. Tissues were rinsed, blotted dry, then plated onto water agar (WA, Difco Laboratories, Detroit, MI). Isolates of Rhizoctonia spp. were identified using hyphal and colony characteristics (Sneh et al. 1991), and transferred to potato dextrose agar (PDA, Difco) acidified with lactic acid (APDA) and stored on slants at 7°C.

Table 1. Province, crop, number of fields surveyed, number of fields with Rhizoctonia spp., and anastomosis groups (AG) of recovered R. solani isolates.

			No. fields with Rhizoctonia		R. solani
Province	Crop	No. fields Surveyed	R. solani	Binucleate	AGs
Alberta	Barley	4	0	0	–
	Canola	13	6	0	2-1
	Pea	1	1	0	2-1
	Wheat	10	6	2	2-1, 4, 5, 9
Saskatchewan	Barley	1	0	0	–
	Canola	9	8	0	2-1, 9, 11
	Pea	1	1	0	2-1
	Wheat	3	2	0	2-1, 9
Manitoba	Canola	6	4	0	2-1
	Soybean	1	0	0	–
	Wheat	12	7	3	2-1, 4, 5
Ontario	Corn	3	0	2	–
	Soybean	80	11	20	2-2, 5
	Winter Wheat	12	0	4	–
	Bean	4	3	1	2-2
Quebec	Soybean	10	3	3	2-2
Total		170	52	35

Characterization of isolates

Hyphae of isolates of Rhizoctonia spp. were stained with trypan blue (0.5% in lactophenol) and examined microscopically for the number of nuclei per cell to determine if the isolates were binucleate or multinucleate. Multinucleate isolates were tentatively considered to be R. solani. These identifications were confirmed for a subset of 128 isolates from western Canada using PCR-derived internal transcribed spacer (ITS) sequences (Broders et al. 2014) and the phylogenetic relationships determined.

To determine the AGs of the recovered multinucleate R. solani isolates, an agar plug was taken from the actively growing margin of an isolate of unknown AG and plated 2–3 cm apart from an isolate of known AG on WA. Tester isolates were available for AG 1, AG 2-1, AG 2-2, AG 3, AG 4, AG 5, AG 6, AG 8 and AG 9 (Dr Sheau-Fan Hwang, Alberta Agriculture and Rural Development). Isolates were considered to be in the same AG when anastomosis was observed a minimum of five times using a microscope.

Pathogenicity testing

Rye (Secale cereale) seed colonized with individual isolates of R. solani was used as inoculum in pathogenicity tests. Equal volumes of water and untreated rye seed were autoclaved for 1 h in bags with a gas exchange patch (#14 Patch PP, Western Biologicals Ltd, Aldergrove, BC). After cooling, agar plugs cut from one-half of a 9-cm-diameter colony of individual isolates were mixed into the rye and left to colonize at room temperature for 7–10 days. The colonized rye was then dried, ground in a grain grinder (http://www.grainmill.com, FGM Enterprises, Inc., Daytona Beach, FL), and stored at 6–10°C. Inoculum was mixed into soil-less potting mix (LC1 Mix, Sun Gro Horticulture, AB) at 5 g per L and used to fill 15 pots (12 × 12 × 11 cm). Uncolonized, ground rye was used as a control treatment. For isolates recovered from western Canada, three pots each of canola (Brassica napus L., ‘1841’), lentil (Lens culinaris Medikus, ‘CDC Plato’), pea (Pisum sativum L., ‘Agassiz’), soybean (Glycine max (L.) Merr, ‘S14-A7’), and wheat (Triticum aestivum L., ‘Norwell’) were planted. For isolates recovered from Ontario and Quebec, lentil was replaced with corn (Zea mays L., ‘N39Z’). Five seeds per pot were planted for corn, lentil, pea and soybean, and 10 seeds for canola and wheat. Following planting, pots were incubated in plastic bags for 5–7 days to promote disease development. Pots were maintained in a growth room at 19–22°C with a 16:8 h light:dark period. Plants were harvested 28 days after planting and rated for disease severity on a scale of 0–10 (e.g. 0 = no symptoms, 10 = dead seedlings; ratings of 2 and 7 correspond to 11–20 and 61–70% of root and/or crown with symptoms, respectively) (Tu & Park 1993; Peña et al. 2013). In total, 25 pathogenicity trials were conducted over a 3-year period, and each trial had 3–11 randomly selected isolates and one non-infested control treatment per tested host. One hundred and ninety-one isolates of R. solani and 52 binucleate isolates were tested. Isolates that had a higher disease severity than the corresponding control treatment for each host in each trial were considered to be pathogenic on that host.

Statistical analyses

Disease severity data from each pathogenicity trial were analysed using SAS software (SAS 9.3 SAS Institute Inc., Cary, NC). The ordinal data did not have a normal distribution based on a normality test using PROC UNIVARIATE; therefore, the results were analysed using the non-parametric methodology of Brunner et al. (2002) as described by Shah & Madden (2004). Preliminary analyses indicated that there were significant differences among the non-infested control treatments of the individual trials and, therefore, results from individual trials could not be combined. Two-way analyses of variance were performed to compare the disease severity of individual isolates on selected hosts in comparison to their corresponding non-infested control treatments. PROC RANK was used to obtain mid-ranks followed by PROC MIXED with option anovaf method = mivque0 to calculate the test statistics and significance levels. Confidence intervals were calculated using the LD_CI macro (Brunner et al. 2002).

Results

Sampling and isolation

A total of 170 fields were surveyed for Rhizoctonia spp. in Alberta, Saskatchewan, Manitoba, Ontario and Quebec and 243 isolates of Rhizoctonia spp. were recovered. Multinucleate isolates of R. solani originated from 52 fields (31%) and binucleate isolates of Rhizoctonia spp. were recovered from 35 fields (21%) (Table 1). The R. solani isolates totalled 191, with 76% of isolates originating from crowns of plants and 24% from roots (Table 2). A total of 52 binucleate isolates were recovered, with 85% of isolates originating from crowns of plants and 15% from roots.

Table 2. Number of isolates of Rhizoctonia spp. recovered and source of isolates listed by anastomosis group (AG).

		Source of Isolate
AG	Number of Isolates	Roots (%)	Crown (%)
2-1	94	18	82
2-2	47	38	62
4	15	7	93
5	18	28	72
9	3	0	100
11	1	0	100
Unknown	13	31	69
Binucleate	52	15	85
Total	243	22	78

Anastomosis groups

Among the 191 isolates of R. solani recovered, representatives of AG 2-1 (95 isolates), AG 2-2 (47), AG 4 (14), AG 5 (18), AG 9 (3) and AG 11 (1) were identified (Table 1). The AGs for 18 isolates were not determined using the tester isolates, but ITS sequences revealed 5 AGs in a separate study by Broders et al. (2014) (Table 2). Isolates of AG 2-1, AG 4, AG 5 and AG 9 were found in Alberta, AG 2-1, AG 9 and AG 11 in Saskatchewan, AG 2-1, AG 4 and AG 5 in Manitoba, AG 2-2 and AG 5 in Ontario and AG 2-2 in Quebec. Isolates from AG 2-1 were recovered from canola, pea and wheat, AG 2-2 from soybean and bean, AG 4 from wheat and AG 5 from soybean and wheat. Anastomosis groups were not determined for binucleate isolates.

Pathogenicity testing

There was considerable variation in host range among isolates of Rhizoctonia spp., and among isolates within individual AGs of R. solani. Of the 191 isolates of R. solani, 98% caused significantly higher median disease severity in comparison to non-infested control treatments and all were pathogenic on canola, 82% on corn (61 isolates tested), 65% on lentil (130 isolates tested), 62% on pea, 58% on soybean and 62% on wheat (Tables 3 and S1–S5). Of the 52 binucleate isolates that were recovered, 61.5% (32 isolates) had median disease severities higher than the corresponding non-infested control treatment on at least one host. Within individual hosts, 25% of the isolates caused significantly higher median disease severities and were considered pathogenic on canola, 18% on corn (44 isolates tested), 13% on lentil (8 isolates tested), 13% on pea, 23% on soybean and 21% on wheat (Tables 3 and S6).

Table 3. Isolates of Rhizoctonia spp. pathogenic on hosts in growth room pathogenicity assay listed by anastomosis group.

AG	No. of isolates	Isolates pathogenic^a on host plant (%)
		Canola	Corn	Lentil	Pea	Soybean	Wheat
2-1	94	100	–	56	36	28	29
2-2	47	100	83	–	91	98	100
4	15	100	–	100	100	93	93
5	18	94	100^b	93^b	78	72	89
9	3	67	–	0	0	0	67
11	1	100	–	100	100	100	100
Unknown	13	92	73^c	50^c	85	85	92
Binucleate	52	25	18^d	13^d	13	23	21
Total	243	83	55	62	51	51	53

^a Significantly greater disease severity compared with non-infested control.

^b Three isolates from central Canada were tested on corn, 15 isolates from western Canada were tested on lentil.

^c Eleven isolates from central Canada were tested on corn, two isolates from western Canada were tested on lentil.

^d Forty-four isolates from central Canada were tested on corn, eight isolates from western Canada were tested on lentil.

There was considerable variation among isolates within an AG in pathogenicity on the tested hosts. Overall, all isolates of R. solani in AG 2-1, AG 2-2, AG 4, AG 5, AG 9 and AG 11 were considered pathogenic on canola based on the results of the growth room assay (Tables S1–S5), except for three isolates classified within each of AG 5 (Table S4), AG 9 and an unidentified AG (Table S5). However, pathogenicity among isolates within these AGs on other hosts was more variable. For example, pathogenicity in AG 2-1 isolates on lentil, pea, soybean and wheat ranged from 0 (16 of 94 isolates) to infection of all four hosts that were tested (3 isolates) (Table S1). All isolates in AG 2-2 were considered pathogenic on canola, and most isolates (38 of 47 isolates) were also pathogenic on corn, pea, soybean and wheat (Table S2). The remaining 9 of these 47 isolates were pathogenic on two (4 isolates) or three (5 isolates) of the tested hosts. Most isolates in AG 4 were pathogenic on all five hosts tested, except for one isolate that was not considered pathogenic on wheat (Table S3). Most isolates in AG 5 were pathogenic on canola (16 of 18 isolates) but had variable pathogenicity on lentil (14 of 15 isolates), pea (14 of 18 isolates), soybean (13 of 18 isolates) and wheat (16 of 18 isolates) (Table S4). Isolates categorized within AG 9 and AG 11 and isolates from unidentified AGs, were pathogenic on one to five of the canola, corn, lentil, pea, soybean and wheat hosts that were tested (Table S5). All isolates within AG 9 and AG 11, and isolates from unidentified AGs, were pathogenic on wheat, except for individual isolates of AG-9 and one isolate from an unidentified AG.

Disease symptoms

In addition to quantitative differences in disease severity, there were qualitative differences among isolates and AGs of Rhizoctonia spp. regarding development of disease symptoms. The majority of isolates of R. solani caused more damage to the crowns of inoculated plants, and less damage was observed on roots but there were exceptions to this trend. Overall, canola was the most susceptible host to R. solani, and most canola seedlings died due to pre-emergence damping-off within 4 weeks of being sown into Rhizoctonia-infested soil-less potting medium. Seedlings that did emerge later typically died due to post-emergence damping-off or had wire stem symptoms associated with R. solani. However, some plants in pots infested with inoculum of R. solani AG 5 did not exhibit these symptoms. Pre-emergence damping-off was also common with lentil and pea but plants that did emerge would often die of post-emergence damping-off due to rotting of the hypocotyl. As many as six successive shoots developed from individual lentil or pea plants, with each shoot being damped-off due to rotting of the hypocotyl. Stunting was also observed. In soybean, hypocotyl rot and stunting were the most common symptoms observed, while pre- or post-emergence damping-off was not common. Corn and wheat were not as susceptible to R. solani as other hosts that were tested. The most common symptom included seedling stunting and browning or rot of subcrown internodes was also observed. These plants would often die if the subcrown internode was rotted through before adventitious roots formed at soil level. On soybean, most isolates of R. solani primarily caused damage to the crowns of inoculated plants, and less damage was observed on roots. However, several isolates of AG 2-2 from soybean in Ontario caused more root than crown damage.

Discussion

This study extensively characterized Rhizoctonia populations associated with selected canola, pea, soybean, wheat, and bean crops across central and western Canada, and examined variability in pathogenicity of isolates on up to five selected hosts. Collectively, these results reflect the existence of a diverse population of isolates of Rhizoctonia spp., AGs and pathogenicity among the hosts and regions.

Overall, there was a relatively high proportion of fields that contained Rhizoctonia spp. i.e. 51% of the 170 canola, pea, soybean, wheat and bean fields sampled in Alberta, Saskatchewan, Manitoba, Ontario and Quebec. Isolates of R. solani were recovered from 31% of these fields, indicating the prevalence of this pathogen among fields with a history of cultivating field crops. This is not unexpected, given the well documented occurrence of R. solani as a facultative parasite in diverse soils around the world (Sneh et al. 1991). There was a higher proportion of fields in which multinucleate isolates (e.g. R. solani, 191 isolates) were recovered compared with binucleate isolates (e.g. Rhizoctonia spp., 52 isolates). This observation is consistent with other studies (Kaminski & Verma 1985; Ogoshi et al. 1990; Yang et al. 1994).

Using tester isolates, 6 AGs were identified among 174 of 191 (91%) isolates of R. solani, including AG 2-1, AG 2-2, AG 4, AG 5, AG 9 and AG 11. These AG determinations were mostly consistent with phylogenetic comparisons of ITS sequences (Broders et al. 2014) where individual AGs belonged to distinct phylogenetic clades. However, seven isolates in AG 2-1 formed a unique clade within this AG, and several variations were observed among isolates classified as AG 2-1, AG 4 and AG 5 based on the ability to form hyphal fusions with the AG5 tester isolate. The two phylogenetic clades within AG 2-1 may represent distinct lineages with the ability to exchange genetic material but perhaps only rarely (Broders et al. 2014), and indicate the higher sensitivity of ITS sequence analysis to identify variation among isolates of R. solani. This has been reported previously (Tewoldemedhin et al. 2006; Ohkura et al. 2009). There were regional distributions in the AGs between western and central Canada, with isolates of AG 2-1, AG 4, AG 9 and AG 11 only found in western Canada, and isolates of AG 2-2 only found in central Canada. AG 5 was the only AG found in both western and central Canada on wheat and soybean, respectively.

There were 13 isolates of R. solani that could not be characterized into AGs based on the tester isolates available. Seven of these isolates were included in the subset of isolates from western Canada by Broders et al. (2014). ITS sequence analysis showed that two of these isolates were grouped with individual AGs (AG 2-1, AG 5) even though they did not fuse with the corresponding tester strain after repeated attempts. Three additional isolates were shown to cluster with AG 9, and one isolate with AG 11. These results illustrate the sensitivity that ITS sequences can add to studying variation among isolates of Rhizoctonia spp., as has been reported in other studies (e.g. Tewoldemedhin et al. 2006; Ohkura et al. 2009), particularly when tester isolates are not available or when there are difficulties in observing anastomoses among isolates.

Ohkura et al. (2009) reported that some plant species were susceptible to specific AGs while others were susceptible to a wide range of AGs. Our results showed that almost all tested isolates from all AGs were pathogenic on canola but, in general, isolates belonging to AG 2-1 were more restricted in their host range to canola than isolates from other AGs, which tended to have wider host ranges. Similar results were described by Tewoldemedhin et al. (2006) in South Africa, where isolates of AG 2-1, AG 2-2, AG 3, AG 4 and AG 11 were recovered from a field crop rotational trial including alfalfa, barley, canola, clover, lupin, medic and wheat. In pathogenicity tests on 14-day-old seedlings with a small number of isolates, AG 2-2 and AG 4 were the most virulent on all crops and AG 2-1 was highly virulent on canola (Tewoldemedhin et al. 2006). Some AGs were shown to be pathogenic but were never recovered from these plants in the field (Tewoldemedhin et al. 2006). In our study, pathogenicity trials indicated that some hosts were susceptible to specific isolates but R. solani was seldom recovered from these hosts in the field.

The majority of binucleate isolates of Rhizoctonia spp. were pathogenic on at least one host, but among those that were pathogenic, the majority had relatively low median disease severities. One isolate, 11-081 3R, from soybean in Ontario had medium to high disease severities on canola, pea and wheat. Twenty-five per cent of the binucleate isolates caused disease in canola, 18% in corn, 13% in lentil, 18% in pea, 23% in soybean and 21% in wheat. Tewoldemedhin et al. (2006) similarly found the majority of binucleate Rhizoctonia isolates caused little or no disease on the hosts tested. Khangura et al. (1999) reported that 15% of binucleate isolates were weakly to moderately pathogenic on canola, and Zhao et al. (2005) reported that binucleate isolates caused disease on soybean but were not as aggressive as isolates from most AGs of R. solani. These results are consistent with the view that the ecological role of binucleate isolates of Rhizoctonia spp. can also include symbiotic and saprophytic relationships (Sneh et al. 1996; Malcolm et al. 2013).

There was considerable variation in pathogenicity among isolates from most of the AGs included in this study. For example, all isolates of AG 2-1 caused disease on canola but pathogenicity on lentil, pea, soybean and wheat varied from none to all of the hosts. Similar variation in pathogenicity among isolates within the same AG has been reported (Zhao et al. 2005; Ohkura et al. 2009). Such variation is an important consideration when selecting crop rotations to reduce the severity of Rhizoctonia diseases. Heterogeneity in restriction digest patterns of the ITS region of isolates of AG 2-1 from Belgium and the Netherlands (Schneider et al. 1997; Pannecoucque & Höfte 2009), and in ITS sequence patterns from isolates from western Canada (Broders et al. 2014) has been demonstrated. Cloning of ITS regions from selected isolates of AG 2-2 showed up to nine unique rDNA ITS patterns within these isolates, as well as heterogeneity in ITS regions in selected isolates of AG 1, AG 3 and AG 4 (Pannecoucque & Höfte 2009). Broders et al. (2014) also reported that isolates of AG 2-2 can contain at least four independent ITS sequences and concluded that a number of distinct lineages or species make up the R. solani complex. Since isolates of R. solani are typically multinucleate and heterokaryotic, additional study is needed to ascertain the origin of these observed ITS variations.

In this study, R. solani was isolated from non-symptomatic seedlings and could cause disease under experimental conditions on other hosts often used in the same crop rotation. For example, two isolates of AG 2-1 from non-symptomatic pea seedlings did not cause disease on pea in the pathogenicity trials. However, one of these isolates was pathogenic on canola and the other isolate was pathogenic on canola and lentil. Other isolates of AG 2-1 from non-symptomatic wheat seedlings were pathogenic on canola only but others were pathogenic on canola, lentil and pea. These results suggest that pea and wheat plants may act as asymptomatic hosts and harbour AG 2-1 inoculum between canola crops. In New York, vegetable-corn rotations are no longer suppressing diseases caused by R. solani, and isolates from corn were pathogenic on vegetable and vice versa (Ohkura et al. 2009). Increased awareness of variations amongst isolates in pathogenicity and host range is important to develop disease management strategies.

Acknowledgements

The authors would like to thank Ms Laura Barbison for technical assistance, Dr Sheau-Fan Hwang, Alberta Agriculture and Rural Development, for providing tester isolates of R. solani, and Dr Larry V. Madden, The Ohio State University, for assistance with the non-parametric statistical analyses.

Supplemental material

Supplemental data for this article can be accessed online here: http://dx.doi.org/10.1080/07060661.2016.1199596

Additional information

Funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada [Discovery Grant]; Syngenta Canada Inc [Research Grant].