pinodes on yield and seed infection of field pea

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1 Effects of timings of inoculation with Mycosphaerella pinodes on yield and seed infection of field pea A. G. Xue, T. D. Warkentin, and E. O. Kenaschuk Agriculture and Agri-Food Canada, Morden Research Centre, Morden, Manitoba, Canada R6M 1Y5. Received 23 September 1996, accepted 7 April Xue, A. G., Warkentin, T. D. and Kenaschuk, E. O Effects of timings of inoculation with Mycosphaerella pinodes on yield and seed infection of field pea. Can. J. Plant Sci. 77: Inoculated field experiments were carried out in 1994 and 1995 to study the effect of the timing of inoculation with Mycosphaerella pinodes (Berk. & Bloxam) Vestergren on disease development, yield reduction and seed infection, in three field pea (Pisum sativum L.) cv. Bohatyr, cv. Scorpio and cv. Triumph. The greatest impact of inoculation on all disease and yield parameters was at the 8 10 node stage in 1994, and at the mid-flowering stage in The lowest impact of inoculation was at the pod swell stage for both years. When inoculated at 8 10 nodes, midflowering and pod swell stages, M. pinodes reduced yield by 31, 24 and 19%, respectively, in 1994 and 33, 43 and 30%, respectively, in The 1000-seed weight was not affected by the timing of inoculation; however, all inoculations reduced seed weight in both years. Plant-to-seed transmission of M. pinodes was affected by the timing of inoculation in 1994, but not in Results of this study suggest that prevention of early infection by M. pinodes will provide the best economic return in a mycosphaerella blight control program on field pea. Key words: Mycosphaerella blight, Mycosphaerella pinodes, field pea, Pisum sativum, yield reduction Xue, A. G., Warkentin, T. D. et Kenaschuk, E. O Effet de Mycosphaerella pinodes sur le rendement et sur le degré d infection des graines du pois de grande culture selon le stade de croissance du pois au moment de l inoculation. Can. J. Plant Sci. 77: Des essais d inoculation au champ étaient réalisés en 1994 et en 1995 pour observer l effet du stade de croissance du pois lors de l ensemencement par Mycosphaerella pinodes (Berk. & Bloxam) Vestergren sur la gravité de la maladie, sur le rendement et sur le degré d infection des grains chez trois cultivars de pois de grande culture (Pisum sativum L.) : Bohatyr, Scorpio et Triumph. L impact le plus fort de l infection sur tous les paramètres de morbidité et de rendement survenait lorsque l inoculation avait lieu en, 1994, au stade 8 10 noeuds et, en 1995, au stade milieu de floraison. L impact le plus faible dans les deux années résultait de l inoculation au stade du gonflement des cosses. Les baisses de rendement dues à M. pinodes étaient, respectivement, de 31, 24 et 19 % en 1994 et de 33, 43 et 30 % en 1995, selon que l ensemencement était fait au stade 8 10 noeuds, milieu de floraison ou gonflement des cosses. L inoculation abaissait le poids de grains les 2 années sans manifester de différence selon le stade de croissance du pois lors de l ensemencement. Le degré de transmission de l agent infectieux de la plante aux grains variait selon le stade de croissance lors de l inoculation en 1994 mais pas en Il se dégage de ces observations que les mesures de lutte les plus rentables contre la brûlure ascochytique du pois de grande culture seront celles qui préviendront le plus l infection précoce de la culture. Mots clés: Brûlure ascochytique, Mycosphaerella pinodes, pois de grande culture, Pisum sativum, réduction du rendement Mycosphaerella blight, caused by Mycosphaerella pinodes, is a serious disease of field pea in western Canada (Warkentin et al. 1995; Xue et al. 1995a,b). Surveys in reported that 100% of the fields were infected by M. pinodes (Kirkham 1992; Xue and Burnett 1994, Xue et al. 1995d). The expansion of field pea production in recent years, and the use of susceptible cultivars introduced from European countries have partly contributed to the wide distribution of this disease and the occurrence of moderate to severe levels of infection. Although mycosphaerella blight can be devastating to field pea production, few studies have been conducted on its impact on yield and seed quality (Wallen 1974; Tivoli et al. 1995), and none in the prairie provinces of Canada. The present study was designed to evaluate how the timing of inoculation with M. pinodes can influence the yield and seed infection in three field pea cultivars that differ in susceptibility to the disease in western Canada. 685 MATERIALS AND METHODS Growth of plants Field studies were conducted at the Agriculture and Agri- Food Canada Research Centre, Morden, in 1994 and Three field pea cultivars, cv. Bohatyr, cv. Scorpio and cv. Triumph were used for the tests. These cultivars were different in susceptibility to mycosphaerella blight in field observations. Experiments were carried out in a split-plot design with four timings of inoculation treatments as the main plots and cultivars as the subplots. Peas were grown in two-row plots in 1994 and four-row plots in 1995 with four replicates each year. Plots were 3.0 m long with 30 cm row spacing and 1.5 m between subplots and 6.0 m between main plots. Plots were seeded on a loam soil 17 May 1994 Abbreviations: AUDPC, area under disease progress curve; TSW, thousand seed weight

2 686 CANADIAN JOURNAL OF PLANT SCIENCE Table 1. Rainfall frequency, monthly total precipitation and mean temperature for Morden, Manitoba, in z No. rain days y Precipitation (mm) Temperature ( C) Month yr mean yr mean May June July August September z Data were collected by Environment Canada weather station at Agriculture and Agri-Food Canada Research Centre in Morden, Manitoba. y Total daily precipitation > 0.1 mm. and on a clay loam soil 10 May 1995 at a seeding rate of 75 seeds m 2. Based on soil test recommendations, no fertilizers were used in 1994 and a blanket fertilizer treatment of 22 kg N and 50 kg P 2 O 5 ha 1 was applied prior to planting in Weeds were controlled using a pre-emergent application of Edge TM herbicide (60% ethalfluralin) on 5 May 1994 and 2 May The volume of herbicide applications were 185 L ha 1 (Hardi nozzle). Plants were harvested at maturity using a small plot combine on 12 September 1994 and 5 September The total seed yield and 1000-seed weight (TSW) were collected when seeds were air dried to 13% seed moisture content. Field infection Plants in three of the four main plots received inoculation treatments. The inoculation was carried out using a single application of M. pinodes spore suspension of 10 5 spores ml 1 plus 0.01% Tween 20. Plants were inoculated at three growth stages, 8 10 node, mid-flowering or pod swell. Dates of inoculation were 20 June, 8 and 25 July in 1994, and 21 June, 3 and 17 July in Control plots were not inoculated but sprayed with the protectant fungicide Bravo 500 (50% chlorothalonil), at 2.0 kg a.i. ha 1, at the time of each inoculation. Similarly, to prevent infection from M. pinodes prior to inoculation treatments, chlorothalonil fungicide was applied once at the 8 10 node stage to plants inoculated at mid-flowering, and twice at both the 8 10 node and mid-flowering stages to plants inoculated at pod swell. For each inoculation, a mixture of equal proportion of three isolates of M. pinodes was used. The three isolates of M. pinodes were collected from field pea in naturally infected fields in Manitoba. For spore production, each isolate was single-spored and grown at 20 C for 14 d on potato dextrose agar with a 14-h photoperiod provided by fluorescent and long-wave ultraviolet lamps. Inoculum was applied to all aboveground portions of plants using a 12-L compressed air sprayer equipped with a single nozzle. Application of the inoculum was done to incipient run-off in the late evening to ensure high humidity for disease development. Fig. 1. Mycosphaerella blight disease progress in plants inoculated with M. pinodes at different growth stages. Each point is the mean of three cultivars and four replicates per cultivar. l 8 10 node; n mid-flowering; s pod swell; * control (uninoculated). Disease Assessment The development of mycosphaerella blight was monitored by visually estimating the disease severity on 10 plants, selected at random, from each plot using a 0 9 scale, described by Xue et al. (1996). Assessments were carried out five times at 2-wk intervals starting at the first inoculation date and ending 3 4 wk after the final date of inoculation. Severity of mycosphaerella blight over time was summarized as the area under the disease progress curve (AUDPC) for each plot using the formula described by Wilcoxson et al. (1975). Critical-time disease severity was assessed, using the same scale described previously, at the pod fill stage which occurred 2 3 wk after the final date of inoculation depending on cultivar maturity. The percentage of seed infection by M. pinodes was assessed about 3 wk after harvest for 100 randomly selected seeds from each plot using a paper towel technique (Xue et al. 1996). Statistical Analysis Data were analyzed using a split-plot design and treatment means were separated by the least significant difference

3 XUE ET AL. EFFECT OF MYCOSPHAERELLA PINODES ON YIELD OF FIELD PEA 687 Table 2. Mean squares from the analysis of variance for effects of Mycosphaerella pinodes inoculation timings (T) and their interaction with cultivar (C) on disease parameters, yield, 1000-seed weight and seed infection of field pea in 1994 and 1995 Source of Degree of AUDPC z Disease severity Yield ( 10 5 ) 1000-seed weight Seed infection variance freedom Replicate T ** 221.1** 4.8** 33.6** 33.5** 28.5** 604.1** 701.6** 891.8** 20.9* Error (Mainplot) C ** 72.0** 14.9** 4.4** 280.9** 180.6** ** ** ** 21.4* T C Error (Subplot) z Area under the disease progress curve. *, **significant at P < 0.05, P < 0.01 levels, respectively. Table 3. Effect of timing of infection by Mycosphaerella pinodes and field pea cultivars on disease parameters, yield, 1000-seed weight and seed infection in 1994 and 1995 Disease severity Yield 1000-seed weight Seed infection (0 9) AUCPC z (kg ha 1 ) (g) (%) Factor Treatment Inoculation timing 8 10 nodes 5.8a 5.0a 19.7a 12.3a 3069c 1775b 173b 271ab 39.8a 6.5a Mid-flowering 5.7a 5.4a 16.7b 13.4a 3360bc 1553b 175b 260b 37.0ab 3.8ab Pod swell 5.5a 3.1b 11.0c 6.6b 3545b 1857b 175b 270ab 33.5b 6.0a Control 4.5b 1.8c 7.5d 4.6c 4306a 2669a 188a 279a 20.3c 2.0b Cultivar Bohatyr 4.8b 3.6b 12.9b 9.0ab 4173a 3129a 167b 251b 22.3b 2.3b Scorpio 6.5a 4.4a 15.4a 11.5a 4486a 1712b 192a 274a 36.6ab 4.6a Triumph 4.8b 3.5b 12.8b 7.3b 2051b 1050b 175b 285a 39.1a 3.4ab z Area under the disease progress curve. a cmeans followed by the same letter in a column under each factor group are not significantly different at P = 0.05 (LSD). (LSD) test at a probability level of Relationships between yield and disease parameters were investigated by correlation analysis (Snedecor and Cochran 1980). Analyses were performed using the SAS Institute, Inc. for personal computers (Cody and Smith 1991). RESULTS AND DISCUSSION Weather conditions varied between the two growing seasons (Table 1). In 1994, monthly temperature and total precipitation were near the 30-yr average. June, July and August were relatively cool and frequent showers provided favorable conditions for rapid development of M. pinodes on plants after each inoculation (Fig. 1). In 1995, rainfall in June, July and August was less frequent compared with 1994 and precipitation was below the 30-yr average in June and July. Temperature in June was slightly higher compared with 1994 and the 30-yr average. As a result, disease development was slower and final disease severity was lower in 1995 than in A heavy rainfall of 199 mm over a 24-h period between 17 and 18 August in 1995 caused crops to die prematurely and as a result the grain yield was lower in 1995 than in Analyses of variance indicated that inoculation timing and cultivar had significant effects on disease parameters, yield, 1000-seed weight and seed infection in both 1994 and 1995, while inoculation timing cultivar interactions were not significant for all the parameters (Table 2). All timings of inoculation produced significantly higher levels of disease and lower yield compared with control plots that were treated with three applications of chlorothalonil fungicide in both 1994 and 1995 (Table 3). The effects of timings of inoculation varied between the 2 yr. In 1994, inoculation at the 8 10 node stage had the greatest impact on all disease and yield parameters, while in 1995, this occurred at the mid-flowering stage. The lowest impact of inoculation was at the pod swell stage for both years. Compared with fungicide treated controls, inoculation at the 8 10 node, midflowering and pod swell stages reduced seed yield by 31, 24 and 19%, respectively, in 1994 and by 33, 43 and 30%, respectively, in These results suggest that measures preventing infection at earlier plant growth stages (before mid-flowering) can be more cost-beneficial than controls at later growth stages. The 1000-seed weight was not affected significantly by the timing of inoculation; however, all inoculations reduced seed weight in both years. Plant-to-seed transmission of M. pinodes was affected significantly by the timing of inoculation in 1994, but not in 1995 due to the low levels of infection (<7%). The lower levels of disease and plant-to-seed transmission of M. pinodes in 1995 than in 1994 may have been due to the less favorable conditions for infection resulting from slightly warmer temperatures and less frequent rainfall. Cultivars responded differentially to inoculation with M. pinodes (Table 3). Bohatyr and Triumph were less susceptible to the disease than Scorpio in both 1994 and This was in agreement with our previous field observations that Scorpio was one of the most susceptible cultivars in Manitoba (Xue et al. 1995c). However, Scorpio had significantly greater yield than Triumph in Bohatyr, had similar levels of disease to Triumph, did not suffer yield

4 688 CANADIAN JOURNAL OF PLANT SCIENCE Table 4. Correlation coefficients relating yield, 1000-seed weight, seed infection, disease severity, and the area under the disease progress curve (AUDPC) in 1994 and 1995 AUDPC Yield 1000-seed weight Seed infection Yield seed weight * Seed infection ** * Disease severity ** 0.981* ** ** *, **significant at P < 0.05 and P < 0.01 levels respectively. The correlation coefficients were calculated using treatment means with two degrees of freedom for both 1994 and reduction as great as Triumph in both years, and had the lowest levels of seed infection in both years. Bohatyr had significantly greater yield than both Scorpio and Triumph in Maximum percent yield reduction for the three cultivars between the 2 yr was 31% for Bohatyr, 46% for Triumph and 54% for Scorpio. These results indicate that Bohatyr possessed the greatest level of tolerance to the disease. It is not clear whether this is due to genotypical difference in tolerance or an effect of genotype by environment interactions. Xue (unpublished data, 1996) has observed evidence of tolerance to M. pinodes infection in some Pisum genotypes. A range of disease severities was generated in this study as a result of the different inoculation timings and fungicide treatments. Based on these data, disease-yield relationships were analyzed using treatment means of the timing inoculations (Table 4). Yield correlated significantly with disease severity, 1000-seed weight and percent seed infection, but not with AUDPC, in However, correlations of yield with these parameters were not significant in The 1000-seed weight correlated significantly with disease severity and seed infection only in 1994, and was not correlated with AUDPC in both years. Seed infection was significantly related to disease severity in 1994 only, and was not correlated significantly with AUDPC. The greater correlation between disease severity and yield than that between AUDPC and yield, suggests that disease severity may be a more useful parameter in quantifying disease-yield loss relationships. In this study mycosphaerella blight reduced grain yield of field pea from 19 to 31% in 1994 and from 35 to 54% in A similar type of experiment was carried out in eastern Canada by Wallen (1974), who compared inoculated and fungicide treated plots. He found up to 50% yield losses due to induced field epidemics caused by M. pinodes. In France, Tivoli et al. (1995) reported crop losses of up to 70% as assessed on single pea plants under field conditions. A low level of mycosphaerella blight infection was observed prior to inoculation in 1994 (Fig. 1). This infection was due to natural inoculum of M. pinodes in the field. Despite three applications of chlorothalonil in control (uninoculated) plots, disease severity was not reduced to zero in each year. The development of the disease in control plots at later growth stages might also be attributed to some interplot interference. Since control plots were not totally free of the disease, the actual yield reductions due to mycosphaerella blight could be greater than those recorded in this study. The most effective method of controlling mycosphaerella blight would be the development of resistant pea cultivars and their use in commercial production. Several lines with partial resistance to the disease have been identified (Xue and Warkentin 1995), and the resistance is being incorporated into field pea cultivars for western Canada. However, breeding mycosphaerella blight resistant field peas is still at an early stage, and all cultivars currently grown in western Canada are susceptible to the disease (Xue, unpublished data, 1996). Fungicide control of mycosphaerella blight of field pea is new in western Canada. Two to three applications of chlorothalonil or benomyl fungicides were reported effective in reducing the disease severity and improving yield in small experimental plots (Warkentin et al. 1995). However, multiple applications of fungicides in a growing season may not be commercially feasible primarily because of the cost of fungicides and their application (Xue et al. 1995a,b). The present study indicated that the time of infection by M. pinodes has important practical implications that must be considered when trying to control the disease with fungicides. Infections before the mid-flowering stage had a greater impact on yield and quality of the crop than infection at later stages. However, it is not possible to predict the expected level of yield reduction based only on the timing of infection without referring to environmental conditions. In 1995, for instance, the lower rainfall frequency during the growing season coinciding with warmer temperatures in June were less favorable conditions for both the subsequent development of mycosphaerella blight following each inoculation and the growth of the plants. As a result, disease severity was lower in 1995 than 1994, and so was the yield potential. There was no significant difference in yield among the timing inoculation treatments in It appears that fungicide treatment may be more beneficial in cool and wet years, when both disease levels and yield potential are high, but it is unlikely to be profitable when conditions are less favorable for either the disease or crop development. ACKNOWLEDGMENTS This study was assisted financially by grants from the Western Grains Research Foundation, Manitoba Pulse Growers Association Inc., Saskatchewan Pulse Crop Development Board, and Alberta Pulse Growers Commission. The technical assistance of H. Tuey, I. Wolfe, A. Sloan and G. Mardli is gratefully acknowledged. Cody, R. P. and Smith, J. K Applied statistics and SAS

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