Characterization of Fusarium spp. associated with lupin in central Alberta, Canada

Abstract

Narrow-leaved lupin (Lupinus angustifolius) is being assessed as a potential pulse crop for western Canada. However, root rot caused by Fusarium spp. is a potentially important disease of lupin in the region. The aim of this study was to identify the Fusarium spp. that aggressively attack lupin in central Alberta. Fusarium isolates were recovered from diseased lupin plants in 2005 and 2006. Fusarium avenaceum was the most frequently recovered species, followed by F. oxysporum, F. solani, and F. acuminatum. Inoculation of lupin with F. avenaceum produced severe root rot symptoms in a greenhouse assay, although there was a wide range in aggressiveness among isolates within this species. Although lupin roots were often colonized by other Fusarium species in addition to F. avenaceum, these other species were shown to be non-pathogenic or only weakly pathogenic. Co-inoculation studies indicated that the other Fusarium species that are associated with lupin roots do not contribute to a disease complex, so we conclude that F. avenaceum is the dominant pathogen responsible for root rot on lupin in central Alberta. The host range of F. avenaceum isolates from lupin included a wide range of pulse and oilseed crops grown in the region, so crop rotation is unlikely to have an important impact on management of this potentially destructive pathogen.

Résumé

Le lupin à folioles étroites (Lupinus angustifolius) est actuellement évalué en tant que légumineuse à grain dans l'Ouest canadien. Toutefois, le pourridié causé par Fusarium spp. est une maladie importante dans la région. Le but de cette étude était d'identifier les espèces de Fusarium qui agressent le lupin dans le centre de l'Alberta. En 2005 et 2006, des isolats de Fusarium ont été récupérés de lupins infectés. F. avenaceum était l'espèce la plus souvent récupérée, suivie de F. oxysporum, F. solani et F. acuminatum. L'inoculation du lupin avec F. avenaceum a produit, au cours d'essais en serre, de graves symptômes de pourridié, bien que l'agressivité des isolats au sein de l'espèce variât grandement. Bien que les racines de lupin aient été souvent colonisées par d'autres espèces de Fusarium, à part F. avenaceum, celles-ci se sont avérées non pathogènes ou seulement faiblement pathogènes. Des études sur la co-inoculation ont indiqué que les autres espèces de Fusarium associées aux racines du lupin ne contribuent pas à provoquer un ensemble de maladies. Nous avons alors conclu que F. avenaceum est l'agent pathogène dominant responsable du pourridié chez le lupin dans le centre de l'Alberta. La gamme d'hôtes des isolats de F. avenaceum provenant du lupin incluait une vaste gamme de légumineuses et d'oléagineuses produite dans cette région. Ceci implique que la rotation des cultures n'est pas susceptible d'influencer notablement la gestion de cet agent pathogène potentiellement destructeur.

Keywords:

• F. avenaceum
• Fusarium spp.
• host range
• Lupinus angustifolius
• pathogenicity
• root rot

Mots clés:

• F. avenaceum
• Fusarium spp.
• gamme d'hôtes
• Lupinus angustifolius
• pathogénicité
• pourridié

Introduction

Narrow-leaved lupin (Lupinus angustifolius L.) is a legume crop species native to the Mediterranean basin. It is grown for seed in many parts of the world, especially in western Australia and Europe, and it may have potential as an alternative pulse crop for central Alberta. Lupin produces a high-protein grain that is desirable as a livestock feed (Cox, 1998). It can also serve as a green manure crop, providing a substantial benefit to subsequent crops, due in part to nitrogen fixation (Helgadóttir et al., 2004; Jensen et al., 2004). Narrow-leafed lupin has not been grown commercially in Canada, but has been grown previously in the USA (Putnam, 1993; Prince & Chambliss, 2004). The adoption of lupin as a crop in Alberta could provide farmers with an option to diversify their operations, since field pea is currently the only grain legume grown over large areas of the province (Strydhorst et al., 2008).

Fusarium is a widespread anamorphic fungal genus that contains many aggressive plant pathogens. Many members of the genus are cosmopolitan and occur worldwide in a wide variety of environments (Leslie & Summerell, 2006). However, not all of the species in this genus are pathogens. Many species are saprophytes, colonize the rhizosphere, or interact with plants as endophytes or beneficial organisms (Larkin & Fravel, 1999; Desjardins, 2003).

The main fusarium diseases of lupin are wilts caused by F. oxysporum Schlecht. and root rot (Infantino et al., 2006), caused by a single species or multiple species that form a disease complex (Weimer, 1944; Smiley et al., 2005). The predominant species associated with root rot of lupin in other regions are F. oxysporum and F. avenaceum (Fr.) Sacc. [teleomorph: Gibberella avenacea Cook (Golubev & Kurlovich, 2002)], but other species have also been shown to be able to cause root rot or seedling blight of L. angustifolius L. These include F. culmorum (W.G. Smith) Saccardo, F. poae (Peck) Wollenweber, F. equiseti (Corda) Saccardo, F. solani (Martius) Appel & Wollenweber emend. Snyder & Hansen, F. semitectum Berkeley & Ravenel, F. tricinctum (Corda) Saccardo and F. moliliforme sensu lato (Wollenweber & Reinking, 1935; Weimer, 1944; Nowicki, 1995). In the USA, F. oxysporum, F. solani and F. moniliforme sensu lato are the predominant root rot pathogens of lupin in southern areas (Weimer, 1944), whereas F. oxysporum and F. avenaceum predominate in northern areas, with F. solani, F. moniliforme sensu lato and F. acuminatum being less important (Kalis et al., 1990). In Australia, Fusarium spp. have been reported from lupin, but root rot is not a major problem (Sweetingham, 1989).

The symptoms of root rot on L. angustifolius include pre-emergence damping-off and post-emergence collapse of seedlings, and girdling of the upper tap root, which leads to yellowing, stunting and wilting of older plants (Bateman, 1997; Chang et al., 2005). Initial studies indicated that root rot of lupin in central Alberta is likely predominantly caused by F. avenaceum (Chang et al., 2005), but additional studies are needed to confirm these initial observations. More information is also required for the development of practices to mitigate the occurrence and severity of root rot of lupin if this crop is to achieve its potential in the region.

The main objective of this study was to identify the Fusarium species that are the primary cause of root rot of lupin in central Alberta, and separate them from those that are saprophytes or secondary invaders. A second objective was to assess the host range of the pathogenic species, with a focus on other crops grown in the region.

Materials and methods

Sample collection and identification of Fusarium isolates

During July and August of 2005 and 2006, plants of L. angustifolius ‘Arabella’ with symptoms of root rot were collected from experimental trials located at Barrhead, Tofield, Edmonton, Carstairs and Penhold in 2005, and Edmonton and Westlock in 2006 (Fig. 1). Plants exhibiting wilting, stunted growth or chlorosis were uprooted and placed in paper bags for transport back to the laboratory. The roots were washed under running tap water, cut into pieces ∼2 mm long per side, then surface-sterilized in 0.6% sodium hypochlorite for 1 min and rinsed in sterile deionized H₂O (Hwang et al., 1994). The tissue pieces were air-dried and up to 10 pieces were plated on half-strength potato dextrose agar (½PDA, BD/Difco, Sparks, MD). Stems were cut longitudinally and examined for vascular discolouration. If discolouration was observed, tissue pieces were excised, sterilized in the same manner as root pieces and plated on ½PDA. Cultures were incubated for 3–4 days in the dark at room temperature. Cultures were then subcultured on PDA acidified to pH 5.0 with lactic acid (APDA) or PDA with streptomycin. Isolates were identified to the genus level based on their colony morphology and via microscopic observations, using the key of Barnett & Hunter (1998). In 2005, only one Fusarium isolate was collected per plant. In 2006, whenever multiple Fusarium colonies were isolated from a plant, all were retained for further assessment. Each Fusarium culture was then subcultured from a single conidium or hyphal tip, and maintained on Spezieller Nährstoffarmer Agar (SNA) (1.0 g KH₂PO₄, 1.0 g KNO₃, 0.5 g MgSO₄•7H₂O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose and 20 g agar per 1 L water) at 4 °C (Leslie & Summerell, 2006). In total, 244 Fusarium isolates were obtained, with 116 isolates from 2005 and 128 from 2006. In addition, 50 isolates collected from lupin in 2005 in central Alberta using a similar protocol were also included in the study.

Fig. 1. Location of lupin plots where Fusarium isolates were collected in the Province of Alberta.

Colonies were transferred from SNA to Carnation Leaf Agar (CLA) and PDA and incubated at room temperature under fluorescent light with a 12-h photoperiod for species identification (Leslie & Summerell, 2006). Observations of the cultural and microscopic characteristics of each isolate were recorded 10–14 days later. CLA plates of select isolates were kept for several months to check for slow-forming chlamydospores, to aid in the differentiation of isolates suspected to be either F. avenaceum or F. acuminatum. Isolates were identified using the taxonomic key of Nelson et al. (1983) and the guide of Leslie & Summerell (2006).

Screening for pathogenic Fusarium isolates

The pathogenicity of the Fusarium isolates was tested on lupin ‘Arabella’ and ‘Rose’. These cultivars were selected because their production potential was being assessed concurrently in central Alberta. The majority of isolates were included in the assay. However, where multiple isolates from a single plant (2006 collection) had the same cultural morphology, only one isolate was assessed to represent the group. Inoculum was produced by growing each isolate on PDA in a Petri dish for 13–15 days at room temperature under cool fluorescent lights with a 12-h photoperiod. The colonized medium in each dish was homogenized with 60 mL of sterile deionized water, and was used to inoculate 10 experimental units. Homogenized, non-inoculated PDA served as the control.

The experimental design was a simple factorial (isolate × cultivar) with five replicates arranged in a completely randomized design. Given the large number of isolates, the experiment was divided into two groups of isolates that were assessed separately (Trials 1 and 2). The experiment was repeated with approximately 25% of the isolates (Trial 3), which were selected to represent the range of responses in Trials 1 and 2.

Seeds of each cultivar were surface disinfected in 70% ethanol for 2 min, then in 0.6% sodium hypochlorite for 2 min, rinsed in three changes of deionized water (Hwang et al., 1994) and air-dried. Nursery trays with 6 cm diameter × 13 cm deep cells (ITML Horticultural Products Inc, Brantford, ON) were filled with pasteurized (121 °C for 1 h) soil-less potting mix (ProMix BX, Premier Horticulture, Dorval, QC). The potting mix was compressed and five seeds of one cultivar were placed in each cell (experimental unit). The inoculum of each isolate, described above, was poured over the seeds in 10 cells, five per cultivar. The seeds were then covered with ∼1.5 cm of ProMix. A 12-h photoperiod was provided by high-pressure sodium lights in a greenhouse. The potting mix was watered as required with tap water, and fertilized every 2 wk with a 0.1% solution of N : P : K (20 : 20 : 20).

Seedling emergence was assessed 2 wk after inoculation. Plants were examined weekly for signs of wilt and scored for wilting on a 0–4 scale (Salleh & Owen, 1983). After 5 wk, the plants were removed from their cells, washed and rated for root rot severity on a 0–4 scale (Hwang et al., 1994), where: 0 = healthy; 1 = small light brown lesions on < 25% of the length of the tap root; 2 = brown lesions on 25–49% of the length of the tap root; 3 = brown lesions on 50–74% of the tap root, tap root constricted; 4 = tap root extensively girdled, brown lesions on >75%, limited lateral roots present, plants wilted and stunted or dead. The shoots were cut and placed in paper bags, air-dried for several days and then weighed.

Attempts were made to re-isolate all of the isolates that consistently produced root rot ratings ≥2. The isolates that were successfully recovered were identified to species level and used to inoculate lupin ‘Arabella’ and ‘Rose’ as described previously.

The statistical analyses were conducted using SAS software (version 9.1.3, SAS Institute Inc., Cary, NC). The data from each trial were analysed separately because not all of the isolates were present in each trial. Each dataset was tested for normality using the Kolmogorov–Smirnov test. Within each trial, the isolates were ranked by emergence, root rot severity and shoot weight. Shoot weight was examined using analysis of variance (ANOVA) and Dunnett's t-test (one-tailed test) to identify isolates for which growth of the inoculated plant was lower than the uninoculated control.

Emergence and root rot severity ratings were analysed non-parametrically. Each experimental unit within a trial was ranked and then analysed using PROC MIXED according to the method of Shah & Madden (2004) to generate ANOVA type statistics. There was no interaction between isolate and cultivar, so the data for the two cultivars were pooled for analysis. Isolates were compared with the control using contrasts, and adjustments for multiple comparisons were made using step-down Bonferroni adjustments in PROC MULTTEST to control type I error. To determine if there were differences in the pathogenicity of isolates of F. avenaceum (the predominant pathogen) among locations, shoot weight, emergence and root rot were analysed using PROC MIXED, where F. avenaceum isolates were nested within location and the trials were treated as random effects. Tukey's Honestly Significant Difference (HSD) test at P < 0.05 was used for means separation. The lettered groupings that indicated differences in the least squares means were produced using a macro (Saxton, 1998).

Co-inoculation of lupin with F. avenaceum and other Fusarium spp.

Inoculum of F. avenaceum was produced in a cornmeal sand (CMS) mixture (Holtz et al., 2011). Liquid inoculum of the other Fusarium spp. included in the trial was produced in 250 mL Erlenmeyer flasks containing 150 mL of sterile Czapek-Dox broth (BD/Difco). The broth was inoculated with two 25 mm² pieces of agar colonized by F. oxysporum (isolates 1 and 2, collected from lupin root and stem tissue, respectively), F. solani or F. acuminatum, with two flasks per isolate. Cultures were incubated at room temperature as described previously on an orbital shaker (Lab-Line Instruments Inc, Melrose Park, IL) with continuous agitation set at 150 rpm. After 7 days, F. oxysporum and F. solani cultures were filtered through two layers of cheesecloth, centrifuged in 50 mL tubes for 3 min, then resuspended in sterile deionized water with the spore concentration adjusted to 5 ×10⁵ conidia mL⁻¹. The concentration of F. acuminatum inoculum was adjusted to 2.5 ×10⁵ conidia and hyphal fragments mL⁻¹.

The experiment was set up in a randomized complete block design with eight replicates. Treatments consisted of liquid inoculum of each isolate alone or in combination with F. avenaceum, and a non-inoculated control. CMS colonized by F. avenaceum was mixed with autoclaved Promix BX potting mix to provide an inoculum density of 2 × 10⁴ CFU g⁻¹. Sterile CMS mixed with potting mix at the same rate was used as the control. Potting mix inoculated with F. avenaceum and a non-inoculated control were added to separate plastic pots (450 mL tuffcups, Georgia-Pacific, Dixie Business, Norwalk, CT). Seeds of lupin ‘Arabella’ were surface-disinfected (Robinson et al., 2000) and 10 seeds were added to each pot. Five mL of liquid inoculum of the Fusarium species or the control treatment was pipetted over the seeds in each cup (experimental unit). The control consisted of non-infested potting mix with sterile water pipetted over the seed.

The pots were placed in a greenhouse at 25 ± 4 °C with natural lighting and watered as required for 3 wk. Emergence was recorded at 2 wk after inoculation, and the plants were removed from their pots at 3 wk, the roots were washed and root rot incidence and severity were assessed as described previously, except that dead plants were rated as 5. The shoots were severed from the roots, air-dried, and weighed. Re-isolation from infected roots was conducted as described above. Recovered isolates were identified by colony morphology on PDA and microscopic morphology on CLA. The experiment was repeated once. Plant mortality in treatments with F. avenaceum was higher than expected, so the entire experiment was repeated an additional two times with the F. avenaceum concentration reduced to 5 ×10³ CFU g⁻¹. Fusarium acuminatum was not included in these repetitions. Data were tested for normality using the Kolmogorov–Smirnov test. Data on seedling emergence, shoot dry weight, and root rot incidence and severity were analysed using a mixed model analysis of variance. The inoculation treatments were fixed effects and the repetitions were random effects.

Dual culture assay

The possible antagonism of Fusarium isolates against pathogenic F. avenaceum was tested in vitro in a dual culture assay. Plugs (4-mm diameter) were taken from the edge of colonies growing on SNA. Plugs of F. avenaceum and the other Fusarium spp. were placed 5 cm apart on PDA. Fusarium avenaceum paired with sterile SNA plugs and F. avenaceum paired with F. avenaceum in dual culture served as controls. The experiment was set up in a completely random design with four replicates. The cultures were incubated at room temperature in the dark. After 6 days, the width of the zone of inhibition (ZI) and the inhibition of radial growth (RGI) (Royse & Ries, 1978) were assessed. RGI is defined as:

(1)

where r₁ is the largest radius of the F. avenaceum colony and r₂ is the radius of the F. avenaceum colony measured in the direction of the inoculation site of the other Fusarium isolate or control. Plates were also measured at 12 days to accommodate the slow growth of F. acuminatum. The experiment was repeated once.

The RGI of F. avenaceum and ZI were analysed using a mixed model analysis of variance. The pairings of isolates in dual culture were fixed effects and repetitions were random effects. Differences between treatment means were tested using Tukey–Kramer pairwise comparisons and letter groupings were generated using the macro of Saxton (1998). The repetitions involving high and low concentrations of F. avenaceum were analysed separately.

Table 1.   Fusarium spp. isolated from lupin roots collected in central Alberta in 2005 and 2006

			Edmonton				Westlock
Fusarium spp.^a	Barrhead 2005	Carstairs 2005	2005	2006	Penhold 2005	Tofield 2005	2005	2006	Total (%)
F. acuminatum	1	2	2	12	1	0	1	5	24 (9.8)
F. avenaceum	20	15	14	29	2	2	0	5	87 (36)
F. equiseti	2	0	0	0	0	0	1	2	5 (2.0)
F. oxysporum	10	5	8	22	0	3	0	2	50 (20)
F. redolens	1	0	1	1	0	0	0	1	4 (1.6)
F. solani	3	0	3	20	0	2	1	0	29 (12)
Discolor	2	1	3	9	0	0	0	4	19 (7.8)
Liseola	0	0	0	0	0	0	1	0	1 (0.4)
Sporotrichiella	0	0	1	3	0	0	0	0	4 (1.6)
Other Fusaria	3	2	2	11	0	1	0	2	21 (8.6)
Total (%)	42 (17)	25 (10)	34 (14)	107 (44)	3 (1.2)	8 (3.3)	4 (1.6)	21 (8.6)	244 (100)
^aSection Discolor includes F. crookwellense, F. culmorum, F. graminearum, and F. sambucinum; Section Liseola includes F. moniliforme sensu lato; Section Sporotrichella includes F. chlamydosporum, F. poae, F. sporotrichioides and F. tricinctum.

Host range of F. avenaceum

Seed of alfalfa (Medicago sativa L.) ‘Anchor’, barley (Hordeum vulgare L.) ‘Harrington’ and ‘Vivar’, bean (Phaseolus vulgaris L.) ‘CDC Pintium’, bird's-foot trefoil (Lotus corniculatus L.) ‘Leo’, canola ‘Invigor 52’ (Brassica napus L.) and ‘Hysyn 10’ (B. rapa L.), chickpea (Cicer arietinum L.) ‘Chico’ and ‘Myles’, yellow sweet clover (Melilotus officinalis L.) ‘Yellow Blossom’, fababean (Vicia faba L.) ‘Snowbird’, flax (Linum usitatissimum L.) ‘solin’, lentil (Lens culinaris Medik.) types black, green, and red, lupin (L. angustifolius) ‘Arabella’, oat (Avena sativa L.) ‘Mustang’ (hulled) and LAO 790 (hulless), field pea (Pisum sativum L.) ‘Cutlass’, spring rye (Secale cereale L.) ‘AC Rifle’, soybean (Glycine max (L.) Merr.) ‘Gaillard’, triticale (× triticosecale Wittmack) ‘Pronghorn’ (spring) and ‘Bobcat’ (winter), and wheat (Triticum aestivum L.) ‘AC Vista’ (spring) and ‘Radiant’ (winter) were surface-disinfected and planted at 10 seeds per pot with 10 replicates (pots) per treatment. Milled grain inoculum (Hwang et al., 1994) colonized by several pathogenic F. avenaceum isolates (Chang et al., 2011) was applied to the soil surface. Non-infested ground grain was used as the control. The experiment was arranged as a randomized complete block design. Emergence was recorded at 10 days after planting, and plant height, root length, plant fresh weight and root rot severity (0–9 scale) were recorded 6 wk after planting.

The data were tested for normality prior to analysis. Inoculated and non-inoculated treatments were compared using PROC GLM. Multiple comparisons were conducted using the step-down Bonferroni adjustment in PROC MULTTEST to adjust for the large number of paired comparisons. Differences are significant at P ≤ 0.05 unless otherwise noted.

Results

Identification of Fusarium isolates

Fusarium spp. were recovered from 71% of lupin roots. The other fungi recovered included Rhizopus spp. (37% of roots), Penicillium spp. (19%), Pythium spp. (18%), Botrytis spp. (5.4%), Rhizoctonia spp. (3.6%) and Alternaria spp. (3.6%). More than one fungal genus was recovered from the majority of roots. The predominant Fusarium species at each location in both years was F. avenaceum (36% of isolates), followed by F. oxysporum (20%) (Table 1). The only exception was the Tofield location in 2005, where F. oxysporum was slightly more common (Table 1). Also, F. avenaceum was present in more locations than any other species, being recovered from all sites. Where multiple isolates were collected from individual roots (2006), 43% of the roots were colonized by multiple Fusarium spp., with up to six species recovered per root. In 2006, F. avenaceum was recovered from 60% of the plants, and F. acuminatum and F. oxysporum from 30%. Isolation from stems was also assessed in 2006; of 57 plants, F. oxysporum was isolated from two and F. avenaceum from one plant. All three of these plants also showed root rot symptoms and had other Fusarium spp. colonizing their roots.

Screening for pathogenic Fusarium isolates

In total, 285 isolates (116 from surveys in 2005, 50 additional isolates (mostly F. oxysporum) from 2005 and 119 from surveys in 2006) were tested for pathogenicity on lupin ‘Arabella’ and ‘Rose’. In each experiment, Fusarium isolate and cultivar had an effect on seedling emergence and shoot dry weight (Table 2), and Fusarium isolate had an effect on root rot severity. Isolates maintained the same rank order between runs when present in multiple replicates. There was no isolate × cultivar interaction for any of the response variables. Seedling emergence and final shoot weight were consistently higher for ‘Rose’ compared with ‘Arabella’ (data not shown). Reduction in emergence and shoot weight occurred only with isolates of F. avenaceum and one unidentified Fusarium isolate (Table 3). The majority of F. avenaceum isolates (51%) increased root rot severity, as did a few isolates of F. acuminatum (18%). The pathogenicity of F. avenaceum isolates was readily reproduced in subsequent experiments, whereas only one F. acuminatum, one F. oxysporum and two unidentified isolates were consistently pathogenic (data not shown). The largest range in aggressiveness occurred within F. avenaceum. The most severe root rot and the largest reductions in emergence and shoot weight were always caused by isolates of F. avenaceum (data not shown).

Table 2.  Test statistics for the effects of Fusarium isolate and cultivar on seedling emergence, root rot severity and shoot dry weight of lupin

		Analysis of variance-type statistics ^a								ANOVA statistics
		Emergence				Root rot				Shoot weight
Trial	Effect	dfN	dfD	F	P value	dfN	dfD	F	P value	df	F	P value
1	Isolate (I)	118	856	2.98	< 0.01	112	697	3.43	< 0.01	140	3.90	< 0.01
	Cultivar (C)	1	856	78.4	< 0.01	1	697	1.71	0.19	1	88.7	< 0.01
	C × I	118	856	1.07	0.30	112	697	1.21	0.07	140	0.90	0.78
2	I	124	879	2.51	< 0.01	124	862	2.54	< 0.01	147	2.68	< 0.01
	C	1	879	103	< 0.01	1	862	0.31	0.58	1	184	< 0.01
	C × I	124	879	1.1	0.22	124	862	0.92	0.72	147	1.21	0.05
3	I	57.3	402	5.81	< 0.01	60.8	418	2.13	< 0.01	73	4.0	< 0.01
	C	1	403	7.16	< 0.01	1	418	0	0.99	1	45.0	< 0.01
	C × I	57.3	403	0.69	0.97	60.8	418	0.76	0.92	73	1.0	0.47
^aBased on a mixed model analysis of ranked data: dfN = numerator degrees of freedom, dfD = denominator degrees of freedom.

Table 3.  Pathogenicity of Fusarium isolates on lupin seedlings as assessed based on impact on seedling emergence, and subsequent shoot dry weight and root rot severity

		Isolates (%) adversely affecting:
Fusarium spp.	No. of isolates	Emergence	Shoot weight	Root rot	Pathogenic isolates (%)^a
F. acuminatum	22	0	0	18	18
F. avenaceum	87	14	22	51	56
F. equiseti	6	0	0	0	0
F. oxysporum	86	0	0	3.5	3.5
F. redolens	6	0	0	0	0
F. solani	35	0	0	2.9	2.9
Section Discolor	20	0	0	10	10
Section Liseola	1	0	0	0	0
Section Sporotrichiella	4	0	0	0	0
Other/Unidentified	18	5.6	5.6	11	17
Total	285	4.56	7.02	20.4	22.5
^aIsolates that were pathogenic for one or more of the response variables.

Fusarium avenaceum could be consistently re-isolated from severely diseased plants. When these recovered isolates were inoculated onto lupin seed, the symptoms observed in the initial pathogenicity trials were reproduced. Wilt symptoms were not observed except on plants inoculated with F. avenaceum, which were suffering from root rot (data not shown). There was no effect of the site of origin of the isolates on emergence (P = 0.30), root rot severity (P = 0.29) or shoot dry mass (P = 0.46).

Greenhouse assay, co-inoculation

Inoculation with F. avenaceum (high and low inoculum concentrations) reduced seedling emergence, increased root rot incidence and severity, and reduced final shoot weight in lupin (Table 4). None of the other Fusarium spp. had an effect on any response variable in the absence of F. avenaceum. However, the effect on emergence and shoot weight of F. avenaceum was occasionally reduced slightly by co-inoculation with another Fusarium spp. (Table 4).

At the low inoculum concentration, inoculation with F. avenaceum produced only a small reduction in emergence, and none of the other isolates affected emergence. At the high concentration, F. avenaceum substantially reduced emergence. Co-inoculation with the other isolates numerically reduced the adverse effect of F. avenaceum, but the difference was not significant.

Table 4.  The effect of Fusarium isolates alone and in combination with F. avenaceum on seedling emergence, root rot incidence (%) and severity (0–5), and shoot dry weight at two inoculum concentrations: High (2 × 10⁴ CFU g⁻¹) and Low (5 × 10³ CFU g⁻¹)

Inoculation treatment			Root rot
Soil	Seed	Emergence (%)	Incidence (%)	Severity	Shoot wt. (g)
High concentration
Control	Control	97 ab	11 b	0.1 b	1.17 a
	F. acuminatum	99 a	8 b	0.04 b	1.24 a
	F. oxysporum 1	90 abc	25 b	0.3 b	1.04 a
	F. oxysporum 2	96 ab	25 b	0.3 b	1.21 a
	F. solani	98 ab	29 b	0.2 b	1.25 a
F. avenaceum	Control	13 d	100 a	4.6 a	0.08 b
	F. acuminatum	35 bcd	99 a	4.5 a	0.33 b
	F. oxysporum 1	39 a–d	100 a	4.4 a	0.33 b
	F. oxysporum 2	29 cd	100 a	4.4 a	0.25 b
	F. solani	36 a–d	99 a	4.2 a	0.24 b
Low concentration
Control	Control	96 a	3 b	0.004 b	1.35 a
	F. oxysporum 1	96 a	14 b	0.1 b	1.29 ab
	F. oxysporum 2	97 a	3 b	0.001 b	1.29 ab
	F. solani	98 a	11 b	0.1 b	1.36 a
F. avenaceum	Control	86 b	89 a	2.6 a	0.97 b
	F. oxysporum 1	91 ab	90 a	2.5 a	1.02 b
	F. oxysporum 2	90 ab	88 a	2.3 a	1.08 ab
	F. solani	88 ab	83 a	2.1 a	1.03 b
Means in a column and concentration followed by the same letter are not different (P ≤ 0.05) according to Tukey–Kramer pairwise comparisons.

Table 5.  Inhibition of radial growth (RGI) of Fusarium avenaceum and width of the zone of inhibition (ZI) by other Fusarium spp. isolated from lupin, in dual culture on potato-dextrose agar (PDA) medium

	RGI (%)^a		ZI (mm)^b
Fusarium spp.	6 days	12 days	6 days	12 days
F. acuminatum	6.60 ± 5.59 a	31.80 ± 6.46 ab	10.75 ± 2.38 a	2.13 ± 1.27 a
F. oxysporum 1	7.75 ± 4.40 a	30.21 ± 7.11 ab	3.44 ± 1.27 b	0 b
F. oxysporum 2	7.41 ± 7.09 a	36.79 ± 2.90 a	5.44 ± 10.04 ab	0 b
F. solani	7.35 ± 4.42 a	25.40 ± 14.17 b	2.25 ± 0.85 b	0.13 ± 0.25 b
F. avenaceum	7.71 ± 7.78 a	32.19 ± 4.99 ab	2.63 ± 2.38 b	0 b
Control^c	2.66 ± 2.65 a	1.32 ± 3.26 c	−	−
^aRGI, inhibition of radial growth of F. avenaceum; RGI = [100(r1−r2)/r1], where r1 is the largest radius of the F. avenaceum colony and r2 is the radius of the F. avenaceum colony measured in the direction of the inoculation location of the other Fusarium isolate or control (Royse & Ries, 1978).
^bZI, the zone of inhibition between F. avenaceum and the other Fusarium isolate.
Values represent the mean of eight replicates ± standard deviation of the mean. Means followed by the same letter in each column do not differ based on Tukey–Kramer pairwise comparisons at P ≤ 0.05.
^cControl was a sterile piece of SNA medium.

Table 6.  Effect of inoculation with Fusarium avenaceum on seedling emergence, root rot severity, root length, plant height and fresh weight of 18 crop species

	Emergence		Severity (0–9)		Root length (cm)		Plant height (cm)		Fresh weight (g)
Crop	Control	Inoc.	Control	Inoc.	Control	Inoc.	Control	Inoc.	Control	Inoc.
Alfalfa ‘Anchor’	7.8	0.6 *	0	8.6 *	13.0	0.5 *	7.7	0.2 *	0.95	0.020 *
Bean ‘CDC Pintium’	8.4	8.8	0	0	12.6	12.3	16.1	15.3	9.49	7.95 *
Bird's-foot trefoil	8.8	1.2 *	0	8.5 *	12.9	0.3 *	6.0	0.4 *	0.41	0.009
Chickpea ‘Chico’	7.0	0.3 *	0.2	8.9 *	12.5	0.3 *	15.0	0.2 *	3.84	0.03 *
‘Myles’	8.4	3.8 *	0.11	0.96	16.0	6.0 *	15.5	3.1 *	4.44	0.98 *
Fababean ‘Snowbird’	4.1	1.1 *	2.8	8.7 *	8.1	1.3 *	7.3	0.6 *	5.84	0.31 *
Lentil – black	9.3	0.4 *	0	8.7 *	8.7	0*	10.6	0.4 *	1.29	0.041 *
− green	9.2	0.4 *	0.07	8.9 *	16.6	0.5 *	15.7	0.4 *	3.66	0.053 *
− red	9.8	1.4 *	0.02	8.1 *	16.5	2.2 *	16.0	1.1 *	3.33	0.14 *
Lupin ‘Arabella’	8.2	2.8 *	0.07	7.1 *	14.4	5.6 *	13.1	3.0 *	4.42	1.24 *
Pea ‘Cutlass’	9.3	3.9 *	0.12	0	15.8	7.8 *	13.8	3.1 *	4.93	1.79 *
Soybean ‘Gaillard’	7.8	5.6 *	0	0	12.0	9.3	7.0	4.0	1.85	1.17
Sweet clover	9.3	0.4 *	0	8.7 *	13.9	0.4 *	7.9	0.1 *	0.75	0.012
Barley ‘Harrington’	9.7	7.3 *	0	0	17.7	15.1	43.0	21.0 *	5.99	2.86 *
‘Vivar’	9.3	6.0 *	0	0	17.8	14.3 *	40.0	20.6 *	6.36	3.41 *
Oat – hulled	9.7	9.4	0	0	19.4	17.3	35.6	32.0 *	3.78	2.58 *
− hulless	7.6	3.2 *	0	0	15.5	5.9 *	22.0	8.5 *	2.00	0.70 *
Rye ‘AC Rifle’	7.7	1.0 *	0	0	16.7	2.1 *	22.7	2.3 *	3.86	0.34 *
Triticale – spring	9.4	5.6 *	0	0	21.0	14.0 *	34.7	16.5 *	5.33	2.35 *
− winter	8.8	5.1 *	0	0	17.1	10.1 *	25.1	12.9 *	2.36	0.94 *
Wheat – spring	8.2	1.3 *	0	0	16.3	2.9 *	28.9	3.4 *	2.21	0.37 *
− winter	9.3	5.5 *	0	0	22.2	14.7 *	29.1	14.2 *	2.79	1.42 *
Canola – B. napus	7.6	2.6 *	0	6.9 *	7.0	3.1 *	5.0	1.1 *	2.30	1.33 *
− B. rapa	8.9	0*	4.5	9.0 *	4.1	0*	8.6	0*	2.17	0*
Flax (solin)	7.3	0.4 *	0	8.6 *	8.4	0.4 *	12.3	0.4 *	0.73	0.025
Differences between inoculated and control treatments for a cultivar at P ≤ 0.05 are indicated by an asterisk.

There were no differences in root rot incidence or severity amongst treatments inoculated with F. avenaceum, or amongst those without F. avenaceum. Severe root rot was associated only with inoculation of F. avenaceum (Table 4). Shoot weight was reduced by inoculation with F. avenaceum at both high and low concentrations. At the high F. avenaceum concentration, there were no differences among the treatments that included F. avenaceum and no differences among treatments that did not include F. avenaceum. At the low concentration, inoculation with F. avenaceum (alone or in combination) reduced shoot weight except for co-inoculation with isolate 2 of F. oxysporum. Each of the Fusarium spp. could be re-isolated from the roots of plants onto which they had been inoculated, after inoculation either singly or in combination (data not shown).

Dual culture assay

None of the isolates reduced radial growth of F. avenaceum more than F. avenaceum paired with itself (Table 5). No stable zone of inhibition formed between F. avenaceum and any of the Fusarium isolates tested, so all of the colonies grew together over time. Due to the slow growth of F. acuminatum, a total of 20 days was required to determine that there was no zone of inhibition for this species.

Host range

Inoculation with F. avenaceum reduced seedling emergence in each of the crop species tested, except for bean and hulled oat (Table 6). Plant height was reduced for all of the crops except bean and soybean, and root length was reduced for all of the crops except bean, soybean and the barley ‘Harrington’. Inoculation reduced the fresh weight of all of the crops. Most of the oilseed and legume crops developed severe root rot when inoculated with F. avenaceum, except bean, chickpea ‘Myles’, field pea and soybean. No cereal cultivars developed any root rot symptoms. For the legumes that developed root rot, severity was similar to that of lupin.

Discussion

Fusarium spp. were the dominant species recovered from lupin roots in the collection phase of this study. Within this genus, F. avenaceum was the most commonly isolated species and the only species that was recovered at all locations, with F. oxysporum being the second most commonly recovered species. The identification of isolates as F. avenaceum was confirmed previously using molecular approaches (Holtz et al., 2011). In the northern USA, Fusarium spp. were commonly isolated from lupin (Weimer, 1944; Kalis et al., 1990), and were recovered from ∼97% of diseased lupin roots in Australia (Sweetingham, 1989). Fusarium avenaceum was one of the most commonly recovered species from diseased white lupin in the UK during the winter (Bateman, 1997), and it was also frequently recovered from white lupin in Minnesota (Kalis et al., 1990). An early report indicates that F. oxysporum was recovered frequently from L. angustifolius in the south-eastern USA (Weimer, 1944).

A number of factors may have biased the isolation frequency towards particular species in this study. Isolations were restricted to lupin ‘Arabella’, which was bred for resistance to root rot caused by F. avenaceum and to wilt caused by F. oxysporum (Joernsgaard et al., 2004; Danish Research Centre for Organic Food and Farming (DARCOF), 2005; Kutpsov et al., 2006). Also, the incidence of infection by Fusarium spp. has been reported to vary on resistant and susceptible cultivars of other crops (Luo et al., 1999), so it is possible that a different pattern of species prevalence would have occurred on other cultivars. The lupin seed was treated with Apron Maxx (fludioxonil + metalaxyl-M) fungicide prior to planting, which may have favoured particular fungal species and deterred others. Certain Fusarium species are more tolerant to fludioxonil than others (Munkvold & O'Mara, 2002), and sensitivity of isolates within a species can also vary (Broders et al., 2007). The relatively late collection of samples may have also favoured the isolation of secondary invaders and saprophytes colonizing already diseased plants. In Denmark, the recovery of Fusarium spp. from 8-week-old lupin roots resulted in more non-pathogenic species than did recovery at four weeks (DARCOF, 2005). Also, the cropping history of the field sites, which may be considered atypical relative to commercial fields because they were sown to a wide variety of crop species in research trials, may have influenced the soil microflora.

In the current study, F. avenaceum was the most commonly isolated Fusarium species and caused the most severe root rot symptoms. The results indicated that the various other Fusarium species were not important components of a disease complex on lupin in the region. Therefore, we conclude that F. avenaceum is the dominant root rot pathogen of lupin in central Alberta. In central and eastern Europe, F. avenaceum can cause severe seedling blight and root rot of lupin (Wollenweber & Reinking, 1935; Schneider, 1958; Debelyi et al., 1977).

There was a wide range of aggressiveness on lupin among the isolates of F. avenaceum in the collection. This variation among isolates is similar to results reported for F. avenaceum from lupin in Europe (Schneider, 1958; DARCOF, 2005) and from other pulse crops in Alberta (Hwang et al., 1994). In contrast, lupin ‘Arabella’ and ‘Rose’ exhibited a similar pattern of response to the range of isolates examined. This similarity may be due, at least in part, to the fact that both cultivars have been selected for resistance to F. avenaceum (Kutpsov et al., 2006). Both cultivars also had a similar pattern of root rot reaction in field trials in Alberta (Chang et al., 2011).

Fusarium acuminatum produced low to moderate levels of root rot on lupin in the current study. Although F. acuminatum is associated with diseased lupin (Sweetingham, 1989; Kalis et al., 1990), it is not usually reported as a pathogen of lupins, although it is pathogenic on other legume species (Hancock, 1983; Hwang et al., 1994). The absence of severe seedling blight or root rot symptoms caused by F. oxysporum, F. redolens and F. solani contrasts with reports from northern Europe and the southern USA, where these species are highly aggressive against narrow-leafed lupin (Weimer, 1944; Debelyi et al., 1977; DARCOF, 2005). The results are similar to Australia, however, where F. oxysporum and F. solani are reported to be weakly or non-pathogenic (Sweetingham, 1989).

The absence of wilt diseases caused by F. oxysporum or F. redolens is likely associated with the non-existence of lupin Fusarium wilt in this region. Unlike Fusarium wilts of other legume species such as pea, lentil and chickpea that have a worldwide distribution (Infantino et al., 2006), Fusarium wilt of narrow-leafed lupin only occurs in Europe. It is likely that the F. oxysporum strains isolated from stem tissue (two plants) were non-pathogenic, endophytic strains, given their inability to cause disease in the current study nor in an earlier analysis of their possible involvement in vascular wilt (Holtz, 2008).

Inoculation of lupin with F. avenaceum in combination with other Fusarium species confirmed that only F. avenaceum was capable of reducing seedling emergence and causing severe root rot. Interactions between Fusarium species can alter the progression and severity of disease (Wong et al., 1984; Bateman, 1997; Demirchi et al., 1999; Peters & Grau, 2002). However, no synergistic or inhibitory effects on pathogenicity were observed in the current study. Also, the lack of a significant reduction in mycelial growth or zones of inhibition in the plate confrontation assay indicates that antibiosis did not occur in this study. However, it should be noted that the interactions of only a limited number of isolates were assessed, and differences in reactions among isolates of F. avenaceum have been reported (Maèkinaitë, 2005).

The results of the host range tests support previous reports that F. avenaceum attacks a broad range of the field crops grown on the Canadian prairies (Cormack, 1937; Calman et al., 1986; Hwang et al., 2000, 2006). The host range of F. avenaceum collected from lupin crops in central Alberta is comparable to that reported in European host range studies involving lupins and other legumes (Schneider, 1958; DARCOF, 2005). Also, F. avenaceum from white lupin has been reported to cause root rot on wheat (Satyaparasad et al., 2000). The reduction in emergence of cereal crops without any root rot symptoms in this report is in contrast to many other studies of F. avenaceum, where it caused severe root rot (Schneider, 1958; Celetti et al., 1990). Flax and canola, although highly susceptible to the F. avenaceum strain used in this experiment, do not usually develop severe root rot symptoms (Fernandez, 2007). The wide host range of F. avenaceum on field crops in the Canadian prairies makes it highly probable that field populations of F. avenaceum will remain at levels sufficient to cause damage to lupin between successive lupin crops in most cropping rotations that are employed in the region.

The field sampling, species identification, and pathogenicity studies reported here show that although Fusarium species are frequent colonizers of lupin roots, only F. avenaceum is a strong root pathogen of lupin. It is capable of causing disease alone and is not part of a Fusarium disease complex; moreover, it is not specific to one crop species, but instead infects a wide range of host crops.

Acknowledgements

We thank G. Turnbull, R. Bowness and T. Dubitz for technical assistance and D. Rennie for help in preparing the map of Alberta. We also thank the Alberta Crop Industry Development Fund Ltd. and the Alberta Pulse Growers Commission for funding portions of this study.

Notes

Present address: Field Crop Development Centre, Alberta Agriculture and Rural Development, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada