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Epidemiology / Épidémiologie
Frequency of isolation of species of Diaporthe and Phomopsis from soybean plants in Ontario and benefits of seed treatments A.G. Xue, M.J. Morrison, E. Cober, T.R. Anderson, S. Rioux, G.R. Ablett, I. Rajcan, R. Hall, and J.X. Zhang
Abstract: Diaporthe–Phomopsis complex, caused by Diaporthe phaseolorum var. caulivora, D. phaseolorum var. sojae, and Phomopsis longicolla, is an important disease complex of soybean (Glycine max) in Canada. To determine the predominant species of causal fungi in Ontario, 2700 pieces of stem tissue, 2700 pieces of pod tissue, and 9000 seeds from diseased plants were collected at nine locations each year from 2002 through 2004. In total, 17 280 isolates of the three pathogens were obtained from these plant parts over the 3 years, of which 73% were from stems, 23% from pods, and only 3% from seeds. Phomopsis longicolla was the predominant species (41% of isolates), followed by D. phaseolorum var. caulivora (37%) and D. phaseolorum var. sojae (22%). In total, 19 seed treatments consisting of various combinations of 10 formulated fungicides and two bioagents were evaluated at two sites each year from 2002 through 2004. Significant increases (P ≤ 0.05) in emergence and yield were observed for 8 and 2 of 11 seed treatments in 2002, 7 and none of 19 treatments in 2003, and all of 18 treatments in 2004, respectively. All 19 treatments in 2003 and 13 of 18 treatments in 2004 reduced root-rot severity. These treatments increased emergence by 2%–10% and yield by 7%–19%, and reduced root-rot severity by 20%–79%. ACM941 (Clonostachys rosea) + Apron Maxx RTA® (fludioxonil plus metalaxyl) was the only treatment that increased yield in 2 of the 3 years. Other treatments increased yield only in a single year. The results of this study provide evidence that fludioxonil, difenoconazole, triazolinthion, and trifloxystrobin protect soybean from seed-borne D. phaseolorum var. caulivora, D. phaseolorum var. sojae, and P. longicolla and increase plant emergence and yield, and that the effectiveness of these fungicides may be enhanced when they are used in combination with the bioagents.
fungicide
Key words: soybean, Glycine max, Diaporthe phaseolorum var. caulivora, Diaporthe phaseolorum var. sojae, Phomopsis longicolla, seed treatment. Xue et al.: soybean / Diaporthe and Phomopsis / seed treatment / 364Résumé : Au Canada, l’association Diaporthe–Phomopsis, constituée du Diaporthe phaseolorum var. caulivora, du D. phaseolorum var. sojae et du Phomopsis longicolla, est importante association de maladies sur le soja (Glycine max). À chaque année, entre 2002 et 2004, 2700 tissus pédonculaires, 2700 tissus de gousses et 9000 graines provenant de plantes malades furent récoltés à neuf endroits afin de déterminer l’identité des espèces prédominantes parmi les champignons causaux en Ontario. En 3 ans, un total de 17 280 isolats des agents pathogènes furent obtenus de ces parties de plante; de ceux-ci, 73 % provenaient de pédoncules, 23 % de gousses et seulement 3 % de graines. Le P. longicolla fut l’espèce prédominante (41 % des isolats), suivi du D. phaseolorum var. caulivora (37 %) et du D. phaseolorum var. sojae (22 %). De 2002 à 2004, un total de 19 traitements de semences, soit diverses combinaisons de 10 formulations de fongicides et de deux agents de lutte biologique, furent évalués à deux endroits à chaque année. Des augmentations significatives (P ≤ 0,05) de la levée et du rendement furent observées pour 8 et 2 des 11 traitements de semences en 2002, pour 7 et aucun des 19 traitements en 2003, et pour tous les 18 traitements en 2004,
Accepted 28 September 2007. A.G. Xue,1 M.J. Morrison, E. Cober, and J.X. Zhang. Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada. T.R. Anderson. Harrow Research Centre, Agriculture and Agri-Food Canada, 2585 County Road 20 East, Harrow, ON N0R 1G0, Canada. S. Rioux. Centre de recherche sur les grains (CEROM), 2700 Einstein, Ste-Foy, QC G1P 3W8, Canada. G.R. Ablett. Ridgetown College, University of Guelph, Ridgetown, ON N0P 2C0, Canada. I. Rajcan. Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada. R. Hall. Department of Environmental Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. 1
Corresponding author (e-mail:
[email protected]).
Can. J. Plant Pathol. 29: 354–364 (2007)
Xue et al.: soybean / Diaporthe and Phomopsis / seed treatment / fungicide
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respectivement. Les 19 traitements en 2003 et 13 des 18 traitements en 2004 ont réduit l’intensité de la pourriture des racines. Ces traitements ont augmenté la levée de 2 % à 10 % et le rendement de 7 % à 19 %, et ont réduit l’intensité de pourriture des racines de 20 % à 79 %. Le traitement ACM941 (Clonostachys rosea) + Apron Maxx RTA® (fludioxonil plus métalaxyle) fut le seul qui augmenta le rendement lors de 2 des 3 années. D’autres traitements ont augmenté le rendement lors d’une seule année. Les résultats de la présente étude prouvent que le fludioxonil, le difénoconazole, le triazolinthion et la trifloxystrobine protègent le soja contre les D. phaseolorum var. caulivora, D. phaseolorum var. sojae et P. longicolla séminicoles et augmentent la levée et le rendement, et que l’efficacité de ces fongicides peut être améliorée lorsqu’ils sont combinés à des agents de lutte biologique. Mots-clés : soja, Glycine max, Diaporthe phaseolorum var. caulivora, Diaporthe phaseolorum var. sojae, Phomopsis longicolla, traitement de semences.
Introduction Diaporthe phaseolorum (Cooke & Ellis) Sacc. var. caulivora Athow &. Cald., Diaporthe phaseolorum var. sojae (Lehman) Wehm., and Phomopsis longicolla Hobbs are an important group of fungi causing stem canker, pod and stem blight, and phomopsis seed decay of soybean (Glycine max (L.) Merr.) (Sinclair 1999a, 1999b). These diseases are commonly referred to as the Diaporthe– Phomopsis (D–P) complex and are responsible for significant losses in yield and quality in many soybean-producing countries (Backman et al. 1985; Hall and Xue 1995; Hepperly and Sinclair 1978; Pioli et al. 1997; Wrather et al. 1997, 2001a, 2001b). Worldwide, it was estimated that in 1994, stem canker, pod and stem blight, and phomopsis seed decay caused yield losses of 1 900 000, 265 000, and 186 000 t, respectively (Fernandez et al. 1999; Kulik and Sinclair 1999a, 1999b). Collectively, these diseases cause more losses in soybean than any other fungal disease in the world (Sinclair 1999a, 1999b). Diaporthe and Phomopsis species can attack soybean at all stages of plant development, but symptoms are usually more apparent later in the growing season during reproduction and ripening, favoured by warm wet weather and delayed harvest (Balducchi and McGee 1987; McGee 1986a; Rupe and Ferriss 1987; Rupe et al. 1999; Sinclair 1991; Spilker et al. 1981; Tekrony et al. 1984). Although it is commonly reported in the literature that D. phaseolorum var. caulivora is the pathogen for stem canker, D. phaseolorum var. sojae for pod and stem blight, and P. longicolla for phomopsis seed decay (Sinclair 1999a, 1999b), all three fungi may be isolated from diseased tissue or infested seed (Hall and Xue 1995; Holland and Abney 1988; Kmetz et al. 1974, 1978; Sinclair 1991, 1999b). The fungi survive the winter as dormant mycelia within infected seed or in plant debris (Baird et al. 1997; Garzonio and McGee 1983). During the growing season, fruiting bodies (pycnidia and perithecia) are produced on overwintered plant debris (McGee 1983). Pycnidia are also produced on stems, petioles, and pods of diseased plants during the current growing season. Conidia formed in pycnidia and ascospores formed in perithecia are released and spread to plants by wind and rain splash. These spores serve as initial inoculum for stem canker and pod and stem blight and account for short-distance dissemination of the pathogens (Garzonio and McGee 1983; McGee 1983). Pods are the main pathway for seed infection (McGee 1986b; Ploper et
al. 1992; Roy and Ratnayake 1997; Rupe and Ferriss 1987). Infected seed may become the primary source of inoculum in fields free of these diseases and account for long-distance dissemination of these fungi (McGee and Biddle 1987; McGee et al. 1980; Zorrilla et al. 1994). The diseases caused by species of Diaporthe and Phomopsis have been reported over a period of more than 40 years in Ontario (Wallen 1960) and have occasionally led to an inadequate supply of high-quality soybean seed for sowing (Anderson 1985; Hall and Xue 1995; Wallen and Seaman 1963). Recent surveys suggest that the distribution and severity of these diseases have increased (Anderson and Tenuta 2001). A single infected seed can yield one or any combination of D. phaseolorum var. caulivora, D. phaseolorum var. sojae, and P. longicolla (Anderson 1985; Hall and Xue 1995). Frequencies of isolation of these fungi from infected soybean stems and pods in Ontario are not known and may differ from frequencies of isolation from infested seed. Knowledge of the relative incidence of these fungi in soybean plants and seed in Ontario is important for disease-control programs, particularly the development of soybean cultivars with improved resistance to these pathogens. Resistance to species of Diaporthe and Phomopsis has not been knowingly incorporated into soybean cultivars in Ontario, and disease management is currently limited to the use of fungicide seed treatments (Ontario Ministry of Agriculture and Food 2003). Anchor® (carbathiin plus thiram), Captan® (captan), and Vitaflo 280® (carbathiin plus thiram) have been the only three seed-treatment products available for controlling D–P complex in Ontario since the late 1980s. Although the efficacy of these fungicides has not been questioned, information on new and alternative treatments is lacking. There has been little research effort to evaluate and develop bioagents as seed treatments for organic agriculture in Ontario. The objectives of this study were to determine the predominant species of Diaporthe and Phomopsis occurring in soybean plants and seeds in Ontario and examine the benefits of fungicidal seed treatments and biocontrol agents applied alone or combined.
Materials and methods Isolation of Diaporthe and Phomopsis species Ten samples of plants infected with species of Diaporthe and Phomopsis were collected from each of nine locations
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representing the five crop heat unit (CHU) areas (2400, 2600, 2800, 3100, and 3400) of soybean production in Ontario at the mature-plant stage each year from 2002 through 2004. Each sample consisted of 6–10 diseased plants selected at random from three or four replicated plots of a susceptible soybean cultivar in the Ontario Soybean Performance Trials at each location. Seed of each sample was hand-threshed and samples of infected stem and pod tissues were obtained by cutting the infected areas into approximately 0.5 cm long pieces. To determine the frequency of isolation of the pathogens, 30 pieces each of stem and pod and 100 seeds were randomly selected, surface-sterilized in 1% NaOCl for 90 s, rinsed in sterile distilled water, and plated on potato dextrose agar acidified to pH 4.0 with lactic acid in 9 cm diameter Petri dishes. These plant parts were plated at five pieces or seeds per Petri dish. The dishes were incubated in darkness for 7 d at 23 °C and then exposed to a 16 h light : 8 h dark photoperiod provided by fluorescent and long-wave-length ultraviolet lamps at ambient room temperature (23–25 °C) for a further 14–21 d. In total, 2700 pieces each of stem and pod tissues and 9000 seeds were tested each year and putative colonies of Diaporthe and Phomopsis species growing from the plant parts were identified on the basis of the sexual-reproduction structures and morphological characteristics of the colonies and spores as described by Hartman et al. (1999) and McGee (1992). Evaluation of seed treatments Soybean seed treatment trials were conducted at two sites each year on the Central Experimental Farm, Ottawa, Ontario (45°23′N, 75°43′W), from 2002 through 2004. In each year, the two field sites were within 2 km of each other but differed in soil type. Site I was on a Grandby sandy loam (Humic Gleysol) in 2002, Farmington loam (Melanic Brunisol) in 2003, and North Gower clay (Humic Gleysol) in 2004. Site II was on an Uplands sand (Humo-Ferric Podzol) in 2002, Rubicon sandy loam (Humo-Ferric Podzol) in 2003, and Rubicon sandy loam (Humo-Ferric Podzol) in 2004. Both sites were under conventional tillage in a 3 year soybean/corn/cereal (wheat, barley, or oat) rotation typical of central and eastern Ontario. Seed of ‘Orleans’ was used in 2002, ‘Pro270’ in 2003, and ‘OAC Bayfield’ in 2004. The seed was obtained from fields naturally affected by D–P complex in a previous year. The percentages of seed-borne infection by Diaporthe and Phomopsis species were 12.3%, 11.3%, and 15.0% for ‘Orleans’, ‘Pro270’, and ‘OAC Bayfield’, respectively, based on 300 seeds randomly selected from each cultivar. In total, 19 seed treatments consisting of various combinations of 10 formulated fungicides and 2 bioagents were evaluated (Table 1), of which 10 treatments were included in all 3 years and 9 in 2 years. Choice of treatments used each year was based on the collaborator’s requirements, lack of effectiveness in previous trials, and availability. Of these fungicides and bioagents, Vitaflo 280, Allegiance®, Vitavax FL®, Jau 6476®, and TFS-Metalaxyl RTU® were provided by the Gustafson Partnership, Calgary, Alberta, and Apron Maxx RTA®, Apron Maxx RFC®, Maxim 480 FS®, Dividend XL RTA®, and Cell-Tech® Soybean (Bradyrhizobium japonicum) were provided by Syngenta Crop Protection Canada Inc., Guelph, Ontario. The bioagent
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ACM941 (Clonostachys rosea) was selected in previous research (Xue 2003). Seed treatments were applied according to the manufacturer’s specifications at standard recommended rates using a Seedburo BLT/D treater (Seedburo Equipment Co., Chicago, Ill.). The uniformly treated seed was spread in a thin layer on clean paper to air-dry overnight and stored in an open paper bag until it was planted, usually within 24–48 h. Untreated seed was used as control. Experiments were arranged in a randomized complete block design with four replications. Plots were seeded at a rate of 300 seeds per plot between 16 May and 1 June each year and consisted of six rows, 5.0 m long, with 0.22 m row spacing and 0.5 m between plots. The plots were fertilized according to soil-test recommendations and treated with applications of appropriate herbicides for effective weed control according to standard management practices (Ontario Ministry of Agriculture and Food 2003; Ontario Ministry of Agriculture, Food, and Rural Affairs 2002). Emerging seedlings were counted in the entire plot 4 weeks after planting. Percent emergence was calculated for each plot by dividing the total number of seedlings by the 300 seeds sown per plot. Approximately 15–20 seedlings from a 50 cm length of a border row in each plot were carefully removed 5 weeks after emergence to assess rootrot severity on a scale of 0–5, where 0 = no visible lesions on the lower stem and taproot, seedling well developed; 1 = slight necrosis or few small lesions on the lower stem and taproot,
