First report of Stemphylium eturmiunum causing Stemphylium blight on Vicia faba in Canada

Abstract

Faba bean is an important, protein-rich pulse crop grown worldwide, with increasing acreage in Canada in the last 50 years. Stemphylium blight can cause substantial yield losses due to defoliation of the plant. In 2022, high incidence levels of Stemphylium blight were observed in research plots at Rosthern and Melfort, SK. Based on morphological characteristics, DNA barcoding, and virulence testing, S. eturmiunum was confirmed as the causal pathogen of these Stemphylium blight lesions. This is the first report of S. eturmiunum infection causing Stemphylium blight on faba bean in Canada. Based on the severity of symptoms it can cause, S. eturmiunum has the potential to pose a serious threat to faba bean production.

Résumé

La féverole est une importante légumineuse riche en protéines cultivée dans le monde entier, dont la superficie a augmenté au Canada au cours des 50 dernières années. La brûlure du Stemphylium peut entraîner des pertes de rendement substantielles en raison de la défoliation de la plante. En 2022, des niveaux d’incidence élevés de Stemphylium blight ont été observés dans des parcelles de recherche à Rosthern et Melfort, SK. Sur la base des caractéristiques morphologiques, du codage à barres de l’ADN et des tests de virulence, S. eturmiunum a été confirmé comme l’agent pathogène responsable de ces lésions de mildiou du Stemphylium. Il s’agit du premier signalement d’une infection par S. eturmiunum causant la brûlure du Stemphylium sur la féverole au Canada. Sur la base de la sévérité des symptômes qu’il peut causer, S. eturmiunum a le potentiel de constituer une menace sérieuse pour la production de féveroles.

Keywords:

• Botrytis spp.
• chocolate spot
• faba bean
• Lens culinaris
• lentil

Mots clés:

• Botrytis spp.
• féverole
• Lens culinaris
• lentille
• tache de chocolat

Introduction

Vicia faba L. (faba bean), also known as broad bean, horse bean and field bean, is an important atmospheric nitrogen fixing legume from the Fabaceae grown throughout the world (Duc 1997; Barłóg et al. 2018). In the 1960s, European germplasm was introduced to western Canada. Breeding efforts and commercial production began in the 1970s, with fluctuations in seeded acreage occurring to the present day (Anonymous 2022). Faba bean is well adapted to the Canadian prairies and is an attractive crop for producers for its nitrogen fixing capabilities, and most varieties have high resistance to Aphanomyces root rot (Lizarazo et al. 2015).

Stemphylium blight caused by Stemphylium spp. has been reported on faba bean in Saskatchewan and is assumed to be caused by Stemphylium botryosum Wallroth, the causal pathogen of Stemphylium blight of lentil (Lens culinaris Medik.) (Caudillo-Ruiz et al. 2017; Woudenberg et al. 2017). Stemphylium blight of faba bean can also be caused by Stemphylium vesicarium (Sheikh et al. 2015). In lentil, Stemphylium blight causes small pinhead lesions that coalesce and may lead to the complete defoliation of the plant, resulting in large yield losses (Mwakutuya and Banniza 2010). Vaghefi et al. (2020) described and demonstrated, for the first time, that a third species, S. eturmiunum, was a highly virulent pathogen of faba bean in Australia.

Recent field surveys revealed disease symptoms that were similar to those of chocolate spot caused by Botrytis spp. (Fig. 1). In August 2022, large reddish-grey lesions with red margins and slight chlorosis around the lesions, appeared in multiple research plots in the rural municipality of Rosthern and south of the City of Melfort, Saskatchewan, where a high incidence (Incidence = # of plots showing symptoms/# of plots) of more than 50% was observed. The disease caused defoliation of plants of faba bean line CDC 1310–5. At the time, lesions from symptomatic plants were subject to DNA barcoding based on the cytochrome b coding region and primers previously described by Graf et al. (2016) that revealed the sole presence of Stemphylium species. The objectives here were to confirm the presence of Stemphylium spp., identify the species of isolates, and verify through re-inoculation of faba bean plants that the isolates were the cause of the observed lesions (Koch’s postulates).

Fig. 1 (Colour online) Field samples of Stemphylium blight on faba bean leaves caused by Stemphylium eturmiunum at Rosthern (A) and Melfort (B), Saskatchewan.

Materials and methods

Isolation and identification of the pathogen

Lesions were excised from symptomatic leaves and sterilized using a 10% bleach solution for 60 s followed by one rinse in sterile distilled water. They were then plated onto PDA amended with 0.01% chloramphenicol. Developing colonies were transferred to a fresh PDA plate and single-spored to obtain pure cultures. Cultures were grown under standard laboratory conditions with continuous light at ambient temperatures (20 ± 2°C) for 14 days. Three isolates (SB170, SB171 and SB172) were purified from samples collected south of Melfort (52°42'45.0”N 106°14'45”W) and one isolate (SB173) from samples originating from the rural municipality of Rosthern (52°49’47”N 104°35’46’W). The colony of each purified isolate was inspected for colour and mycelial growth habit after 14 days of incubation. The length and width of 30 conidia were measured at 40× with a compound microscope using Zeiss software (Carl Zeiss Microscopy LLC., NY, USA).

Plugs of each isolate were placed into sterile Erlenmeyer flasks filled with 50 mL of glucose yeast media broth (1 g NH₄H₂PO₄, 0.2 g KCl, 0.2 g MgSO₄·7 H₂O, 10 g glucose, 5 g yeast extract, 1.0 mL copper solution [0.5 g CuSO₄·5 H₂O, 100 mL distilled water], 1.0 mL zinc solution [1 g ZnSO₄·7 H₂O, 100 mL distilled water], 1000 mL deionized water) and were incubated on a shaker (Labtech, Korea) at 24°C and 110 rpm for 48–72 h. Mycelia were harvested and filtered through autoclaved miracloth and placed into 2.0 mL Eppendorf tubes. These cultures were lyophilized in a freeze drier for 72 h and stored at −80°C, after which DNA was extracted using the Edward’s method with modifications (Edwards et al. 1991). Genomic DNA from all isolates were subjected to end-point polymerase chain reactions with primer pairs V9G/ITS4, GPD1/GPD2 and CalDF1/CalDR2 to obtain partial sequences of the internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2 (ITS region), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and partial sequences of the calmodulin (cmdA) gene, respectively, as described by Woudenberg et al. (2017). Bands were excised and purified using the Monarch® DNA Gel Extraction Kit (New England Biolabs, MA, USA) and sent to Eurofins Genomics (Eurofins Genomics LLC, KY, USA) for Sanger sequencing. Sequence data were aligned and single contigs were constructed using DNAbaser. Alignment of contigs against type sequences deposited in GenBank by Woudenberg et al. (2017) was performed in MEGA11.

Pathogenicity trials

Four seeds of susceptible faba bean line CDC 1310–5 were grown for each replicate in 10 cm pots for 21 days under standard greenhouse conditions with a 16 h photoperiod and day:night temperatures of 22:20°C. Plants were fertilized weekly after emergence and watered as needed. Prior to inoculation, pots were thinned to three plants and staked. Inoculum was prepared by growing each isolate (SB170–SB173) on V8PDA media (70 mL V8 juice (Cambell Soup Company, NJ, USA), 15 g PDA, 15 g agar, 3 g CaCO₃) and scraping mature conidia from the colony. The suspension was adjusted to a concentration of 1.0 × 10⁵ conidia mL⁻¹ and approximately 5 mL were applied to each plant using an airbrush. In addition to the four isolates, both non-inoculated and water-sprayed plants were included as controls. Plants were incubated at 90 ± 5% relative humidity for 48 h followed by another 12 days on a misting bench with 3 s of misting every 10 min. Disease severity was assessed 14 days after inoculations with a 0 to 10 scale based on 10% incremental increases in lesion surface area. A completely randomized design with four replicates was designed with three subsamples per replicate. The experiment was performed twice for all four Stemphylium isolates to demonstrate reproducibility.

Reisolation of the pathogen

Symptomatic leaves from each isolate were removed from plants upon assessment and surface sterilized as previously described. The lesions were excised and placed onto PDA as previously described and monitored for fungal growth. Cultures were compared to those used as inoculum to fulfill Koch’s postulates.

Results and discussion

In vitro, cultures produced whitish-grey mycelium that turned dark brown as the colony matured. The mycelium grew close to the medium and did not develop aerial hyphae during maturation. Mature conidia were subject to microscopy and ranged in width from 17.3 to 25.5 µm and lengths from 18.7 to 32.8 µm (n = 30). Conidia were muriform, highly melanized, and produced two to four transverse and one longitudinal septa. Simmons (2001) described the conidia of in vitro grown S. eturmiunum with similar dimensions and similar numbers of septa as observed here.

Aligned sequence data for each isolate resulted in contigs for all three genes that had identity matches and query coverage of 100% to those deposited by Woudenberg et al. (2017) for the type isolate of S. eturmiunum CBS 109845. Sequences have been deposited in GenBank (Table 1).

Table 1. Isolate ID and GenBank accession numbers for sequences of the internal transcribed spacer 1, 5.8S ribosomal RNA gene, internal transcribed spacer 2 (ITS region), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, and calmodulin (cmdA) gene of Stemphylium eturmiunum isolates cultures included in experiments.

	Locus
Isolate/Strain	ITS Region	GAPDH	cmdA
CBS 109 845	KU850541.1	KU850689.1	KU850831.1
SB170	OR500223	OR504760	OR504756
SB171	OR500224	OR504761	OR504757
SB172	OR500225	OR504762	OR504758
SB173	OR500226	OR504763	OR504759

Disease symptoms on faba bean line CDC 1310–5 were first observed 72 h after inoculation, and final disease severity was, on average, 55, 89, 82, and 84% for S. eturmiunum isolates SB170, SB171, SB172, and SB173, respectively (n = 8). Plants from both controls were asymptomatic (DS = 0%, n = 8). Disease symptoms manifested as large grey lesions with red margins and slight chlorosis, along with defoliation of leaves from lower nodes (Fig. 2). These symptoms are similar to those observed in the field and as described by Vaghefi et al. (2020), who described similar symptoms of Stemphylium blight on faba bean caused by S. eturmiunum. Symptomatic leaves were surface sterilized as previously described and all isolates were reisolated from lesions of leaves onto which they had been inoculated. There were no other pathogens present during re-isolation which confirms that all four S. eturmiunum isolates were pathogenic and highly virulent on faba bean.

Fig. 2 (Colour online) Leaf samples from pathogenicity trials of Stemphylium blight on CDC 1310–5 faba bean leaves caused by Stemphylium eturmiunum isolates SB170 (A), SB171 (B), SB172 (C), and SB173 (D).

Stemphylium blight detection appears to have become more common in pulse crops with high recovery rates reported from New South Wales in 2016 (Vaghefi et al. 2020) and from Saskatchewan in 2022 (current report). While S. eturmiunum was previously shown to cause high levels of disease on faba bean in pathogenicity tests under controlled conditions (Vaghefi et al. 2020) this is the first report of S. eturmiunum causing disease under field conditions in Saskatchewan, Canada. Some research on Stemphylium blight on lentil was previously conducted to explore conducive environmental conditions for the causal species, S. botryosum (Mwakutuya and Banniza 2010), a pathogen species of minor or no importance on faba bean, as shown here. Research is currently underway to study the effects of temperature and leaf wetness on the development of S. eturmiunum on faba bean. Due to the increase in seeded acreage of faba bean in Canada and the increasing interest in the crop as a source of alternative, plant-based protein, Stemphylium blight caused by S. eturmiunum could be a potential threat to the industry and may be, or become, as important as chocolate spot. Further research is warranted to determine the prevalence of S. eturmiunum on faba bean grown in the Prairies and the overall impact of Stemphylium blight caused by S. eturmiunum on yield in commercial faba bean production. This information is needed to inform faba bean breeding programs as to whether resistance breeding to this pathogen is needed.

Acknowledgments

The researchers acknowledge that this study was conducted on Treaty 6 territory and the homeland of the Metis, and that isolates were collected from Treaty 6 territory. We pay our respect to the First Nations and Métis ancestors of this place and reaffirm our relationship with one another. Special thanks to Meagan Carroll for her technical assistance. We gratefully acknowledge funding from the Saskatchewan Ministry of Agriculture, Western Grains Research Foundation and Saskatchewan Pulse Growers.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Government of Saskatchewan [20200193]; Saskatchewan Pulse Growers [AGR2104]; Western Grains Research Foundation [AGR2136].