Epidemiology of sclerotinia rot of carrot caused by Sclerotinia sclerotiorum

Transcription

1 245 Epidemiology / Épidémiologie Epidemiology of sclerotinia rot of carrot caused by Sclerotinia sclerotiorum C. Kora, M.R. McDonald, and G.J. Boland Abstract: The epidemiology of sclerotinia rot of carrot was investigated on carrot Cellobunch during 1999 and 2000 in Ontario. Apothecia were first detected in the crop in early August to mid-september, after the carrot canopy closed and after 7 11 days with soil matric potentials between 0.1 and 0.4 bars and soil temperatures between 14 and 23 C. Ascospores were first detected in mid-july to mid-august, usually before apothecia were observed in the crop, and after 7 12 days with soil matric potentials between 0.1 and 0.3 bars and air temperatures between 15 and 21 C. The numbers of apothecia and ascospores were positively correlated with soil matric potential. Preharvest epidemics started in mid-august to mid-september, after the closure and lodging of the canopy, after the appearance of senescing leaves on the soil and ascospores in the crop, and after rain had initiated h per day of leaf surface wetness. Disease incidence was negatively correlated with air and soil temperatures. Postharvest epidemics in storage followed preharvest epidemics in the field, but not all preharvest epidemics resulted in disease in storage. It is suggested that severe epidemics of sclerotinia rot of carrot can occur in storage when disease in the field progresses rapidly and is associated with soil matric potentials of 0.2 bars and leaf wetness of 14 h per day, particularly close to harvest. The information revealed in this study contributes to the development of inoculum and disease prediction systems and improved management of sclerotinia rot of carrot. Key words: Sclerotinia sclerotiorum, Daucus carota, carrot disease, watery soft rot, cottony rot, epidemiology, phenology. Résumé : En 1999 et en 2000, l épidémiologie de la pourriture à sclérotes de la carotte fut étudiée sur le Cellobunch en Ontario. Des apothécies furent d abord détectées dans la culture du début août jusqu à la mi-septembre, après que le couvert végétal se fut refermé et après 7à11jours de potentiels capillaires du sol entre 0,1 et 0,4 bars et de températures du sol entre 14 et 23 C. Des ascospores furent d abord détectées de la mi-juillet à la mi-août, habituellement avant que les apothécies ne soient observées dans la culture et après 7à12jours de potentiels capillaires du sol entre 0,1 et 0,3 bars et des températures de l air entre 15 et 21 C. Les nombres d apothécies et d ascospores furent positivement corrélés avec le potentiel capillaire du sol. Les épidémies avant récolte commencèrent de la mi-août à la mi-septembre après la fermeture et la verse du couvert, après l apparition de feuilles sénescentes sur le sol et d ascospores dans la culture et après que la pluie eut provoqué 12 à 24 h d humectation des feuilles par jour. La fréquence de la maladie fut négativement corrélée avec les températures de l air et du sol. À l entreposage, des épidémies postrécolte ont suivi les épidémies avant récolte au champ, mais toutes les épidémies avant récolte n ont pas été suivies de maladies d entreposage. En somme, de graves épidémies de pourriture à sclérotes de la carotte peuvent se produire lors de l entreposage lorsque la maladie au champ progresse rapidement et est associée à des potentiels capillaires du sol de 0,2 bars ou plus et des humectations de 14 h ou plus par jour, particulièrement à l approche de la récolte. L information obtenue par la présente étude contribue au développement de systèmes de prédiction de l inoculum et des maladies et d une lutte améliorée contre la pourriture à sclérotes de la carotte. Mots clés : Sclerotinia sclerotiorum, Daucus carota, maladie de la carotte, pourriture humide, pourriture blanche, épidémiologie, phénologie. Accepted 10 January C. Kora and M.R. McDonald. Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada. G.J. Boland. 1 Department of Environmental Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. 1 Corresponding author ( gboland@uoguelph.ca). Kora et al.: sclerotinia rot of carrot / epidemiology / inoculum production 258 Introduction Sclerotinia rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is an economically important disease of carrots (Daucus carota L.) and is characterized by the preharvest epidemic occurring in the field and the postharvest epidemic occurring in storage (Kora et al. 2003). The damage caused by the disease is particularly important in temperate regions where carrots undergo long-term storage. In Can- Can. J. Plant Pathol. 27: (2005)

2 246 Can. J. Plant Pathol. Vol. 27, 2005 ada, sclerotinia rot of carrot (SRC) occurs in carrotproducing areas across all provinces (Conners 1967) and losses of up to 30% and 50% have been reported in stored crops (Anonymous 1970; Finlayson et al. 1989). About 57% of carrots in Ontario are grown in the organic soils of Bradford and District Marshes, where infestation by S. sclerotiorum is widespread, and 50% of the crop is stored for up to 8 months (Anonymous 2002; Fraser and Chaput 1998). In the past two decades, losses caused by SRC in this area have increased because of continuous inoculum build-up in soil, insufficient rotation (e.g., 2 year) with nonhost crops (e.g., onion), and lack of effective disease control measures (McDonald et al. 2001). Sclerotinia sclerotiorum persists on or in soil as sclerotia originating from previous epidemics in the same field or introduced through agricultural activities such as tillage, irrigation, manure fertilization, and contaminated seeds (Adams and Ayres 1979; Schwartz and Steadman 1978). Sclerotia can germinate myceliogenically to produce mycelia or carpogenically to produce apothecia (Adams and Ayres 1979). Previous studies under controlled conditions demonstrated that the number of buried sclerotia that germinated carpogenically in carrot crops increased when moisture was maintained constantly at soil capacity (Couper 2001). In the field, occurrence of apothecia in these studies coincided with closed carrot canopies and prolonged rain or irrigation. Ascospores released by apothecia are the primary inoculum for diseases incited by S. sclerotiorum in the aerial parts of most hosts (Abawi and Grogan 1975, 1979; Cline and Jacobsen 1983; Morrall and Dueck 1982). In carrot crops, both mycelia (Finlayson et al. 1989) and ascospores (Geary 1978) were considered important in initiating preharvest epidemics of SRC. However, there have been no reports on the prevalence and epidemiological importance of these types of inoculum in the field. Ascospores require an exogenous source of nutrients, such as senescing flower tissues, and free water to infect healthy plant tissues (Lumsden 1979). In carrot crops, these conditions most likely occur on shaded senescing leaves located in the lower canopy or those in contact with soil where moisture levels may be more conducive for infection. Geary (1978) demonstrated that foliar-applied ascospores initiated disease after carrot plants had reached the 7- to 8-leaf stage and had at least one senescing leaf at the time of inoculation. In the field, occurrence of disease was associated with poorly drained areas, high plant densities, and vigorous, lodged canopies. These results indicate that carrots become susceptible to infection by S. sclerotiorum when foliage starts to senesce and the senescing leaves collapse onto soil. Therefore, the presence of primary inoculum during this susceptible stage in the phenology of carrot appears to be important for the initiation of SRC epidemics. Postharvest infection of carrot roots originates mainly from infected foliage in the field and occurs via the crown (Finlayson et al. 1989; Geary 1978). Mycelium arising from infected roots introduced from the field can spread to adjacent healthy roots and develop into expanding disease foci in storage. Several aspects of host parasite interaction and epidemiology of S. sclerotiorum on carrots have been previously investigated and reviewed (Couper 2001; Finlayson et al. 1989; Geary 1978; Kora et al. 2003). However, there is no report that relates the occurrence of natural sources of inoculum, environmental conditions, phenological development of carrots, and epidemics of SRC in the field and storage. The limited availability of epidemiological information has restricted efforts to construct prediction models for SRC. Further elucidation of epidemiological characteristics is necessary for the development of effective disease prediction systems and improved management of SRC. The objectives of this study were to characterize epidemics of SRC in field and storage, to identify critical microclimate, pathogen, and crop factors that influence the development of disease, and describe the relationships among these factors. Materials and methods Establishment of field sites One trial was established in experimental carrot crops of the Muck Crops Research Station and one in commercial carrot crops in Bradford Marsh, Ontario (44 15 N, W) in 1999 and All four trials were established on organic (muck) soil (60% organic matter; ph ) naturally infested with S. sclerotiorum and with a history of SRC. Carrot Cellobunch was used in all trials. Experimental crops (13.7 m 36 m) were direct seeded using a precision seeder ( seeds/m) on raised beds 86 cm apart centre-to-centre on 28 May 1999 and 31 May In each bed, three rows of carrots were sown 5 cm apart. The crops were maintained following standard management practices (Anonymous 2000), except that no fungicides were applied to the crop. Commercial crops (25 m 40 m) were direct seeded (45 48 seeds/m) on raised beds 72 cm apart centre-to-centre on 15 May 1999 and 15 May 2000, and established about 30 m from the edge of the field. In each bed, two rows of carrot seeds were sown 7 cm apart. The crops were maintained by the grower following standard management practices (Anonymous 2000), including applications of Benlate (benomyl 50% a.i., DuPont Canada Inc., Mississauga, Ontario, Canada). All crop, pathogen, and disease assessments were conducted in predesignated sampling areas. The sampling pattern in each site was a square with sides of about 20 m in the experimental crops and 24 m in the commercial crops. Eight sample areas about 10 and 12 m apart, respectively, were established in the midpoints of each side and in each angle of the square. Each sample area consisted of a2m long 0.86 m wide (experimental) or 2 m long 0.72 m wide (commercial) section of carrot bed that extended between the middle of one furrow to the middle of the adjacent furrow. A furrow was defined as the area extending between the middles of two adjacent carrot beds. Crop variables examined in relation to SRC epidemics included selected phenological stages of carrot, such as canopy closure and foliar senescence, and canopy lodging. In addition to calendar dates, the occurrence of these stages was described in relation to the number of days after seeding (DAS) to facilitate comparison among crops seeded at variable dates. Canopy closure and foliar senescence were monitored in each site at weekly intervals from the beginning of July until harvest. Canopy coverage was rated visually to estimate the percentage of soil covered by the carrot

3 Kora et al.: sclerotinia rot of carrot / epidemiology / inoculum production 247 foliage. The canopy was defined as closed when foliage of adjacent rows touched and soil was no longer visible. Foliar senescence was rated by counting the number of senescing leaves per plant collapsed on the soil. Counts were conducted on two to three plants per sampling area and the mean number of senescing leaves per plant was determined for each sampling date. Canopy lodging was selected as a variable of canopy architecture and was measured in weekly intervals from mid- July until harvest. Lodging represented the number and degree of contact of healthy carrot leaves with the surface of soil because of bending over the furrow. Canopy lodging was measured using a severity scale with four classes (0, no lodging, with all leaves in an upright position; 1, 1 3 leaves/plant contacting the soil; 2, 4 5 leaves/plant laying on the soil; 3, >5 leaves/plant laying on the soil, excluding collapsed senescing leaves). Assessment of microclimate Ambient air temperature (AT) and relative humidity (RH), rainfall, leaf surface wetness duration (LWD) on the uppermost, exposed leaves, and shaded leaves under the canopy, soil temperature (ST), and soil moisture were recorded from 8 July to 15 October in each trial. Unless otherwise noted, all sensors used to measure microclimate variables were from Campbell Scientific, Inc., Logan, Utah. Atmospheric variables, including AT, RH, and rainfall, were measured at a height of 10 cm above the carrot canopy. Air temperature and RH were measured using a thermistor probe (model HMP 35C). Rainfall was measured in increments of 0.25 mm using a tipping bucket rain gauge (model TE525). Leaf wetness was estimated using flat electronic impedance grid sensors (model 237) coated with light green latex paint (Gillespie and Kidd 1978). Leaf wetness sensors were mounted on wooden stakes positioned at a 45 angle at the canopy surface and under the canopy, 50 and 10 cm above the soil surface, respectively. Soil temperature was measured using a thermistor probe (model 107B) buried horizontally 5 cm deep in the soil. In 1999, soil moisture was measured twice a week by gravimetric determinations (Liddell 1992) at 5 cm soil depth. Percent gravimetric water content was then converted to percent volumetric water content using the specific bulk density of the soil (e.g., 0.2 g/cm 3 ) determined for each site (Kora 2003). In 2000, soil moisture was measured continuously using a ThetaProbe type ML2x sensor (Delta-T Devices Ltd. Cambridge, UK) attached to a micrologger. The ThetaProbe was buried horizontally in soil so that the centre of the cylinder was about 5 cm below the surface and measured volumetric water content of soil between the rods (Anonymous 1999). Outputs of percent volumetric soil water obtained from the ThetaProbe and the gravimetric method were converted to soil matric potential (SMP) from a standard soil water retention curve generated specifically for each site (Kora 2003) using the pressure plate technique (Richards 1965). Sensors were connected to a micrologger (model CR 21X) programmed to sample at 1-min intervals and record averaged or summed (rain gauge) readings over 15-min intervals. Monitoring instrumentation was calibrated according to the recommendations of the manufacturer each year prior to installation. In storage, AT and RH were measured in the proximity of stored carrots inside the bin using HOBO PRO RH/TEMP loggers (Onset Computer Co., Bourne, Massachusetts) programmed to record readings every 30 min. Air temperature and RH readings were averaged over the entire storage trial period. Assessment of occurrence of S. sclerotiorum Occurrence of apothecia and airborne ascospores were monitored at each site twice a week from 15 July to 15 October. The total number of apothecia present in each sample area was counted and the mean number of apothecia per square metre in each site was used as an estimate of the population of apothecia on respective sampling dates. Ascospores were monitored using 90-mm diameter Petri dishes filled with sclerotinia semi-selective medium as spore traps (Steadman et al. 1994; Steadman and O Keefe 1998). One open Petri dish containing 20 ml of medium was exposed in each sampling area on every sampling date from 1000 to The dishes were placed on top of the carrot bed under the canopy at an angle facing southwest, which was in an upwind position to the direction of prevailing winds in the area. Exposed dishes were incubated at 20±1 Cinthedark and evaluated after 3 to 5 days. Individual colony forming units (CFU) were assumed to develop from germinated airborne ascospores and were identified by the colony morphology and the typical yellow halo surrounding each colony. The presence of S. sclerotiorum on dishes was always confirmed by the subsequent formation of sclerotia. The CFU were counted for each dish and the mean number of CFU for each site was used as an estimate of the population of ascospores on respective sampling dates. Assessment of disease The incidence of sclerotinia rot on carrot foliage was evaluated at weekly intervals as the percentage of plants in the sample area that had at least one diseased leaf. Diseased tissues were sampled regularly during the course of epidemics to confirm the presence of S. sclerotiorum. Pieces of carrot leaves and petioles were surface disinfested for 45 s in 70% ethanol, 1 min in 0.6% sodium hypochlorite, rinsed twice in sterile water, and plated onto sclerotinia semiselective medium. The presence of S. sclerotiorum was identified as described previously. Preharvest epidemics were presented as temporal progress of disease incidence on every sampling date. The development of SRC in storage was evaluated by assessing disease incidence and severity of harvested roots at monthly intervals for 6 months. A sample of 180 carrot roots was collected from each sample area at harvest (e.g., mid-october to early November). The topped, unwashed roots of each sample were randomly divided into six subsamples of 30 roots each and placed in separate polyethylene net bags. Carrot bags were individually stored in plastic containers in temperature-controlled Filacell (forced-air cooling system) storage at 1 ± 0.5 C and 85 ± 1% RH (commercial storage) or 2±1 Cand95±2%RH(experimental storage). One carrot subsample was assessed each month for every sample area. Disease incidence represented the percentage of roots in the sample with at least one lesion of SRC. Disease severity represented an estimate of the

4 248 Can. J. Plant Pathol. Vol. 27, 2005 disease index (D index ), calculated using a severity scale consisting of five classes ranging from 0 to 4. Carrots were assigned to the severity classes based on the area of each root covered by lesions as follows: 0, 0%; 1, 1% 25%; 2, 26% 50%; 3, 51% 75%; 4, 76% 100%. Severity values were converted to an index of 0 to 100 using the following equation (Kobriger and Hagedorn 1983): (severity class no. roots in class) D index = 100 (total no. of roots highest class No.) Postharvest epidemics were presented as temporal progress curves of cumulative disease incidence and cumulative disease severity index over the storage trial period. Statistical analyses Independent variables were derived from the measurements of each microclimate factor averaged or summed over selected periods coinciding with pathogen and disease sampling dates. These variables included daily mean, minimum, and maximum AT and ST, daily mean RH, and daily mean SMP averaged over 3-, 2-, and 1-week periods, and cumulative rainfall during 3-, 2-, and 1-week periods, preceding the sampling dates. The weekly mean SMP, calculated as the mean of the SMP measurements on the day of sampling and 1 week prior to sampling (Hunter et al. 1984), and cumulative days with LWD of >15 and >20 h on exposed and shaded leaves, respectively, during the week preceding sampling dates were also tested. Means of SMP in 1999 were calculated from daily gravimetric measurements recorded twice a week. Dependent variables included means of weekly observations of the number of apothecia, number of deposited ascospores, and incidence of foliar SRC. A Pearson correlation analysis was used to examine correlations among the variables. Correlation analyses were performed individually, on data from each site and year, and collectively, on data combined across sites and years. Analyses for the number of apothecia or ascospores as the dependent variables were based on data from crops where the pathogen was observed. Analyses of disease incidence as the dependent variable were based on data from crops where disease was observed. The purpose of this selective analysis of combined data was to identify the critical microclimate factors that were most associated with the development of apothecia, ascospores, and disease in carrot crops. Statistical analyses were performed using the correlation (Proc Corr) procedure of SAS for Windows (SAS Institute Inc., Cary, North Carolina). Results Stages of phenological and architectural development of carrot crops occurred at wide ranges of carrot age and varied across crops and years. In the experimental sites with wide (86 cm) bed spacing, crop canopies closed on 16 August (80 DAS) in 1999 and 28 July (58 DAS) in 2000 (Figs. 1 and 3). In the commercial sites with narrow (72 cm) bed spacing, crop canopies closed on 8 July (54 DAS) in 1999 and on 2 August (79 DAS) in 2000 (Figs. 2 and 4). Canopy closure usually occurred when crops were at the 8- to 9-leaf stage. Senescing leaves in the experimental sites started to collapse on the soil on 26 August (90 DAS) in 1999 and on 12 July (42 DAS) in In commercial sites, senescing leaves collapsed on 12 August (89 DAS) in 1999 and on 31 July (77 DAS) in The collapse of senescing leaves started when crops were at the 6- to 10-leaf stage. Subsequently, the number of senescing leaves accumulated in the furrow increased by one leaf every 7 9 days and a maximum of 5 8 senescing leaves per plant were recorded by late September to early October. The canopy started to lodge between 2 August (63 DAS) and 30 August (94 DAS), when the crops were at the 8- to 11-leaf stage (Figs. 1 4). Mean severity of canopy lodging across sites ranged from 1 to 2.6, and maximum severity was reached within days after the first lodging was observed. Carrots were harvested from mid-october to early November (153 to 161 DAS). Disease development Water-soaked, dark olive green lesions associated with tufts of white mycelia of S. sclerotiorum were observed initially on older senescing leaves and petioles of the lower carrot canopy that had collapsed on the soil. Mycelia arising from diseased tissues advanced basipetally toward the rosette of the infected plant covering entire petioles. However, few carrot plants were observed with symptomatic diseased crowns or collapsed foliage. In addition, mycelium from diseased leaves spread to adjacent uninfected plant tissue through direct colonization of leaves bending over the furrow or senescing foliar debris accumulated along the furrow. At an advanced stage, diseased petioles exhibited a bleached appearance that was most evident during dry weather conditions. Sclerotia formed externally, embedded in the mycelium, or internally, within the pith of diseased petioles and were dislodged on the soil when infected tissues became desiccated. Lesions on stored roots developed initially in the crown region as localized softened tissue and white mycelial tufts erupting through the cuticle. Mycelium from infected carrots spread by contact to adjacent roots, forming enlarging foci of infection. Lesions caused by this secondary spread of the pathogen occurred anywhere on neighbouring roots and initially appeared as circular water-soaked, discoloured spots. At a later stage, expanding lesions developed into a watery soft rot covered by rapidly growing white mycelium and superficial sclerotia embedded in the mycelium. Epidemiology of preharvest epidemics Disease in the field occurred in three of four carrot crops investigated during 1999 and 2000 (Figs. 1 4). In the commercial crop in 1999, disease was first observed on 18 September and incidence increased from 8% to 41% in 14 days. A maximum incidence of 62% was recorded at harvest, on 9 October. In the commercial crop in 2000, disease was first observed on 6 September and incidence increased from 9% to 17% 14 days later. A maximum incidence of 25% was recorded at harvest, on 11 October. In the experimental crop in 2000, disease was first observed on 18 August and incidence increased from 1% to 10% in 14 days. A maximum incidence of 38% was recorded at harvest, on 13 October. Apothecia were observed in three of four sites and were first detected on 4 August in the 2000 experimental crop, on 12 August in the 1999 commercial crop, and on 15 Septem-

5 Kora et al.: sclerotinia rot of carrot / epidemiology / inoculum production 249 Fig. 1. Daily mean air temperature (T), relative humidity (RH), leaf wetness duration (LWD) above and under the canopy, rainfall, soil T, soil matric potential (data points below 3 bars are not shown), canopy cover, mean number of apothecia, mean number of colony forming units (CFU) of ascospores deposited on semi-selective medium, lodging severity, number of senescing leaves per plant in contact with soil, and incidence of foliar disease in the experimental carrot crop in Bradford Marsh, Ontario, from 9 July to 15 October *, records of ascospores deposition were not collected because of continuous rainfall during observation period.

6 250 Can. J. Plant Pathol. Vol. 27, 2005 Fig. 2. Daily mean air temperature (T), relative humidity (RH), leaf wetness duration (LWD) above and under the canopy, rainfall, soil T, soil matric potential (data points below 3 bars are not shown), canopy cover, mean number of apothecia, mean number of colony forming units (CFU) of ascospores deposited on semi-selective medium, lodging severity, number of senescing leaves per plant in contact with soil, and incidence of foliar disease in the commercial carrot crop in Bradford Marsh, Ontario, from 9 July to 10 October *, records of ascospores deposition were not collected because of continuous rainfall during observation period. ber in the 1999 experimental crop (Figs. 1 3). First detection of apothecia always occurred after the crop canopy had closed and after 1 3 senescing leaves had collapsed on the soil. After the first detection, apothecia were continuously or intermittently observed for periods ranging from 1 to 6 weeks, sometimes until close to harvest. The number of apothecia varied across sites and years, ranging from 0 in the 2000 commercial crop to a maximum of 1.5 apothecia/m 2 recorded on 25 August in the 2000 experimental crop. The mean number of apothecia observed across the seasons ranged from 0.2 to 0.4 apothecia/m 2 and was relatively highest during the first 3 weeks following the first detection. The first appearance of apothecia was preceded by periods of 7 11 days of increased SMP ranging from 0.1 to 0.4 bars, with the shorter periods usually associated with higher SMP. Soil matric potential increased as a result of mm of rainfall occurring over the 10- to 12-day periods preceding the emergence of apothecia. Daily ST during the 2-week period preceding the emergence of apothecia ranged from 14 to 23 C, with a mean of 19 C. Subsequent development of apothecia was associated with mean SMP and ST ranging from 0.1 to 6.5 bars and 12 to 17 C, respectively. Ascospores were detected in all four sites starting on 18 July in 2000 commercial and experimental crops, 9 August in the 1999 commercial crop, and 16 August in the 1999 experimental crop at mean densities ranging from 0.6 to 7.5 CFU per dish (Figs. 1 4). The first appearance of ascospores usually occurred prior to or at closure of the canopy, before apothecia were first observed in the crop, and before senescing leaves started to collapse on the soil. After the first detection, ascospores were continuously or intermittently observed for periods ranging from 3 to 7 weeks at mean yearly densities ranging from 10.7 to 20 CFU per dish. Typically, higher numbers of ascospores were associated with the detection of apothecia in the crop, particularly during the 2 4 weeks following the first observation of apothecia. Maximum numbers of ascospores (e.g., CFU per dish) were recorded 7 12 days after apothecia were first detected in the 1999 commercial and 2000 experimental crops, or 10 days after the peak number of apothecia was observed in the 1999 experimental crop. Increased abundance of ascospores occurred when there was an accumulation of two to three senescing leaves per plant collapsed on soil and lodging severity of the canopy was 1. Initial detection of ascospores was preceded by periods of 7 12 days with increased SMP ranging from 0.05 to 0.3 bars, with shorter preceding periods usually being associated with higher SMP. Soil matric potential increased as a result of mm of rainfall occurring prior to the detection of ascospores. Daily mean AT during the periods preceding the first detection of ascospores ranged from 14 to 21 C. Ascospores were not detected or were captured in low numbers when prevailing SMP was below 0.4 bars and daily maximum AT was above 25 C but were trapped again after moist and cooler conditions persisted for at least one week. During periods of ascospore presence, mean SMP ranged from 0.2 to 0.3 bars and mean AT ranged from 15 to 19 C. Disease was first detected on 18 August in the 2000 experimental crop, 6 September in the 2000 commercial crop, and 18 September in the 1999 commercial crop (Figs. 2 4). From the first detection, disease was observed in the crop for periods ranging from 3 to 7 weeks, usually symptomatic until harvest. Disease always occurred after senescing leaves had collapsed on the soil and after the canopy had started to lodge. At the initial detection of disease, there was an accumulation of at least four senescing leaves per plant collapsed on the soil and severity of canopy lodging ranged from 2 to 3. In addition, disease always appeared after apothecia and (or) ascospores were detected in the crop. The periods from the first detection of ascospores after senescing leaves collapsed on soil to the first observation of disease ranged from 31 to 37 days. The periods when the occurrence of ascospores coincided with the presence of senescing leaves on the soil extended from 3 to 7 weeks. Disease was initially detected after periods of 3 11 days with prolonged LWD under the canopy ranging from 12 to 24 h per day, mean SMP of 0.1 to 0.9 bars, and mean AT from 9 to 22 C. Moisture in the crop increased as a result of up to 72 mm of rainfall during the week preceding disease detection. Usually, higher LWD, higher rainfall, and lower AT were associated with shorter preceding periods. During the epidemics, mean LWD, mean SMP, and total rainfall ranged from 14 to 16 h per day, 0.2 to 0.5 bars, and 26 to 140 mm, respectively, with larger increases in the incidence of foliar disease (e.g., 27% per week in the 1999 commercial crop) associated with higher moisture. Epidemiology of postharvest epidemics Epidemics in storage occurred in two of four carrot root crops (Fig. 5). Disease developed in roots that were harvested from the commercial crops and stored in the commercial refrigerated storage. In , disease was observed within the first month of storage at an incidence of 2% and a severity index of 0.4. The cumulative disease incidence increased to 15% and 38% and the cumulative severity index increased to 4.2 and 14.9 after 3 and 6 months in storage, respectively. In , disease was first observed within the third month of storage at an incidence of 0.4% and a severity index of 0.1. Cumulative disease incidence increased to 8%, and cumulative severity increased to 2.6 after 6 months in storage. The postharvest epidemic in the 1999 commercial crop followed a 3-week-long preharvest epidemic with 63% incidence of foliar disease at harvest (Fig. 2). In 2000, the postharvest epidemic followed a 5-week-long preharvest epidemic with 25% incidence of foliar disease at harvest (Fig. 4). The carrot roots were asymptomatic when initially placed in storage. During the preharvest epidemic in 1999,

7 Kora et al.: sclerotinia rot of carrot / epidemiology / inoculum production 251

8 252 Can. J. Plant Pathol. Vol. 27, 2005 Fig. 3. Daily mean air temperature (T), relative humidity (RH), leaf wetness duration (LWD) above and under the canopy, rainfall, soil T, soil matric potential (data points below 3 bars are not shown), canopy cover, mean number of apothecia, mean number of colony forming units (CFU) of ascospores deposited on semi-selective medium, lodging severity, number of senescing leaves per plant in contact with soil, and incidence of foliar disease in the experimental carrot crop in Bradford Marsh, Ontario, from 8 July to 15 October *, mycelium was not observed on diseased tissues, and disease rating was not conclusive. the frequency of days with rainfall was 50%, SMP was consistently in the range of 0.1 to 0.2 bars, and prevailing LWD within the canopy was above 15 h per day. During the preharvest epidemic in 2000, the frequency of days with rainfall was 37%, there were two 6-day periods with no rainfall, and SMP ranged from 0.4 to 1.1 bars. During the week preceding harvest, there were 8.9 mm of rainfall that increased the SMP to 0.07 bars in 1999, but there was no rainfall in 2000 and the mean SMP was 0.4 bars. Air temperature and RH in the proximity of stored carrot samples ranged from 1.1 ± 0.5 to 1.7 ± 0.2 C and 85% ± 1% to 88% ± 1% in 1999 to 2000, respectively. Over the 6-month period, the monthly increase of cumulative disease incidence ranged from 3.7% to 13.2% in 1999 and 0.4% to 6.7% in The monthly increase of cumulative disease severity during the same period ranged from 0.9 to 2.9 in 1999 and from 0.1 to 2.4 in The greatest increase in disease incidence and severity in storage occurred during the third and the sixth months in 1999 and during the sixth month in Disease in storage was not observed in carrots harvested from the 2000 experimental crop although carrots originated from an 8-week-long preharvest epidemic with 37% incidence of foliar disease at harvest. During the preharvest epidemic, the frequency of days with rainfall was 41%, and there were two periods of over 10 days with mm of rain and SMP in the range of 0.5 to > 15 bars. Similarly, during the week preceding harvest, there was 0.25 mm of rain and mean SMP was 0.5 bars. Influence of microclimate on apothecia, ascospore, and disease development In general, the development of apothecia was correlated with AT, ST, and SMP in the 1999 experimental crop, and occasionally with ST when data were combined across crops. Correlations were more consistent across ST and SMP. Occurrence of apothecia in the 1999 experimental crop was negatively correlated with the daily mean, minimum, and maximum ST over the 3-, 2-, and 1-week periods preceding sampling (Table 1). Daily mean and maximum ST yielded the highest coefficients of correlation in all assessed intervals. In addition, occurrence of apothecia in the 1999 experimental crop was positively correlated with daily mean SMP over 3-, 2-, and 1-week periods, and the weekly mean SMP preceding sampling (Table 1). No correlations were observed between ST and SMP and apothecia at the other sites. Development of ascospores was most consistently correlated with SMP in individual and combined crops. Occurrence of ascospores was positively correlated with daily mean SMP over 3-, 2-, and 1-week periods, and with the mean SMP in the week preceding sampling (Table 1). Disease correlated consistently with AT and ST in all individual and combined crops, and occasionally with SMP in the 1999 experimental crop. In all crops, disease incidence was negatively correlated with daily mean, minimum, and maximum AT and ST during the 3-, 2-, and 1-week periods preceding sampling (Table 1). Higher correlation coefficients were obtained for daily maximum AT and ST in most intervals examined (Table 1). Relative humidity and rainfall did not correlate with the number of apothecia, number of deposited ascospores, or disease incidence. Similarly, LWD did not correlate with the disease incidence. Therefore, these data were not reported. Discussion This is the first study that characterizes the epidemics of sclerotinia rot and aspects of the aerobiology of naturally occurring S. sclerotiorum in carrot crops. Phenological and architectural development of carrots, environmental factors, development of S. sclerotiorum, and epidemics of SRC were interrelated in consistent chronological patterns. Preharvest epidemics started as early as mid-august and usually persisted until harvest. Disease occurred after the canopy closed, after senescing leaves collapsed on the soil, after ascospores were detected in the crop, and after prolonged periods of leaf wetness and high soil moisture. These results indicate that presence of senescing leaves, ascospores, and free surface moisture are the most important prerequisites for the occurrence of SRC epidemics in the field. This study provides useful information for improving the management of this disease through the development of prediction systems and timed applications of control practices. Development of SRC was related to phenological and architectural attributes of carrots, specifically foliar senescence and position of leaves within the canopy. Disease first appeared on senescing leaves in contact with soil and disease incidence was higher in crops that had earlier senescence, higher number of accumulated senescing leaves on the soil, and higher lodging severity. Therefore, plant-to-soil and plant-to-plant contacts are deemed important for the initiation of epidemics and the secondary spread of the pathogen, respectively. These results are consistent with previous studies that reported a relationship between senescence of plant tissues and lodged canopies with susceptibility to S. sclerotiorum and epidemics of sclerotinia diseases in carrots (Geary 1978; Couper 2001) and other crops (Abawi and Grogan 1979; Boland and Hall 1988; Cline and Jacobsen 1983; Morrall and Dueck 1982; Schwartz and Steadman 1978). In this study, senescing leaves started to collapse when carrots had 6 10 leaves and progressively accumulated in the furrow until harvest. The increase of susceptible tissue within the canopy of aging carrots may lead to rapid development of SRC if inoculum is present and microclimate conditions are favourable. The present study was conducted using carrot Cellobunch and results may be applicable to other carrot cultivars. However, little information is available on differences in the developmental pattern of

9 Kora et al.: sclerotinia rot of carrot / epidemiology / inoculum production 253 senescence and lodging of leaves among cultivars that would affect the development of SRC. The timing of architectural and phenological stages that were significant for the epidemiology of SRC varied widely in relation to carrot age (DAS) across fields and years. The occurrence of foliar senescence and canopy lodging varied

10 254 Can. J. Plant Pathol. Vol. 27, 2005 Fig. 4. Daily mean air temperature (T), relative humidity (RH), leaf wetness duration (LWD) above and under the canopy, rainfall, soil T, soil matric potential (data points below 3 bars are not shown), canopy cover, mean number of apothecia, mean number of colony forming units (CFU) of ascospores deposited on semi-selective medium, lodging severity, number of senescing leaves per plant in contact with soil, and incidence of foliar disease in the commercial carrot crop in Bradford Marsh, Ontario, from 8 July to 15 October 2000.

11 Kora et al.: sclerotinia rot of carrot / epidemiology / inoculum production 255 Fig. 5. Cumulative disease incidence and cumulative disease severity index of sclerotinia rot of carrots during commercial storage in 1999 and Disease incidence represents the percentage of roots in the sample with at least one lesion. The disease severity index was estimated based on the area of lesion coverage on each diseased root: 0, 0%; 1, 1% 25%; 2, 26% 50%; 3, 51% 75%; 4, 76% 100%. from 3 to 5 weeks among crops. In contrast, canopy closure occurred more consistently in relation to growth of carrot (e.g., at the 8- to 9-leaf stage). These results suggest that age is not an effective indicator for timing the occurrence of foliar senescence, canopy closure, lodging, and by inference, the development of SRC in carrot. The timing of these stages may be a function of several factors other than age, such as cultivar, nutrient and irrigation regimen, soil type, plant density and row spacing, weather suitability, and damage from foliar diseases. Growth of carrot has been primarily described as a function of yield components, such as size and weight of photosynthetic or storage organs (Strandberg 2001), and phenological models that define the developmental stages of the crop are not available. Models of carrot development that incorporate foliar senescence, canopy closure, and lodging may be a useful tool for predicting SRC epidemics and improving the management of the disease. The occurrence of SRC was always associated with the presence of ascospores, suggesting that ascospores were the primary inoculum responsible for initiating disease on carrot foliage. In each epidemic, the highest number of ascospores was observed during a 3-week period when 1 3 senescing leaves and 1 3 healthy leaves were in contact with soil. The time interval from the first detection of ascospores after senescing leaves collapsed on the soil to the first observation of disease depended on environmental conditions and was shorter when moist conditions prevailed. In addition, disease at harvest was high when there were long periods where ascospores coincided with the susceptible stage of carrots. These results are consistent with previous findings that demonstrate the significance of ascospores for initiating epidemics of sclerotinia diseases in carrots (Geary 1978; Couper 2001) and other crops (Abawi and Grogan 1975; Boland and Hall 1987, 1988; Cline and Jacobsen 1983; Hunter et al. 1984; Morrall and Dueck 1982). Therefore, knowledge of carrot development stages and presence of S. sclerotiorum in carrot crops is essential for determining the occurrence of SRC and timing the application of control practices. Moisture in the crop was important for the start and subsequent development of disease. Preharvest epidemics occurred after high levels of LWD and SMP below the canopy persisted for 2 10 days. Finlayson et al. (1989) reported that 11 days of continuous leaf wetness were required for an ascospore suspension sprayed on the foliage to produce foliar and root disease under controlled environment. In the field, these conditions are unlikely to occur on either uppermost or lower leaves of the canopy that are in an upright position. The development of disease mainly on lower leaves in contact with soil indicates that soil moisture and humidity under the canopy are important sources of the moisture required for infection to occur. A closed canopy can contribute to maintaining extended periods of high soil moisture and high humidity below the crop by reducing evaporation and air circulation (Abawi and Grogan 1979). The effect of closed canopies in buffering fluctuating microclimate variables by increasing LWD and RH, and reducing AT and ST has been reported in carrots (Kora 2003; Kora et al. 2005) and bean crops (Blad et al. 1978; Caesar and Pearson 1983; Deshpande et al. 1995; Weiss et al. 1980). The wetness duration on senescing leaves in contact with soil was not investigated in this study. However, measurement of LWD of bottom carrot leaves and soil moisture were deemed to provide an adequate estimate of free moisture available in the crop. Postharvest epidemics in storage followed preharvest epidemics in the field. In 2 of 3 sites, relatively high cumulative incidence of root disease in storage (e.g., 38%) was associated with high rates of disease increase in the field (e.g., mean of 16% per week) and high incidence of foliar disease at harvest (e.g., 62%). In one site, the preharvest epidemic did not result in disease in storage, probably because disease progress in the field was suppressed by intermittent periods of hot dry weather. These results suggest that severe epidemics of SRC can occur in storage when disease in the field progresses rapidly and is associated with continuous periods of SMP greater than 0.3 bars and LWD greater than 14 h per day, particularly close to harvest. Apothecia were detected after the crop canopies were closed and SMP of 0.1 to 0.4 bars persisted for 7 11 days. These soil moisture levels were in the range favourable for apothecia production reported previously (Morrall 1977; Teo and Morrall 1985; Clarkson et al. 2001; Couper 2001). In the present study, periods of wet weather

12 256 Can. J. Plant Pathol. Vol. 27, 2005 Table 1. Correlations among the number of apothecia (APO), number of colony forming units (CFU) of deposited ascospores, and incidence of sclerotinia rot of carrot (SRC) versus air temperature (AT), soil temperature (ST), and soil matric potential (SMP) in individual and combined experimental (e) and commercial (c) carrot crops in Bradford Marsh, Ontario, in 1999 and Coefficient of correlation APO CFU SRC Combined b Variable a 1999e 1999c 2000e 1999e 1999c 2000e 2000c 1999c 2000e 2000c APO CFU SRC AT in preceding weeks Daily mean 3 week 0.71 NS NS NS NS NS NS 0.91* * NS NS 0.76* Daily minimum 3 week 0.85* NS NS NS NS NS NS 0.91* * NS NS 0.62* Daily maximum 3 week NS NS NS NS 0.71 NS NS 0.90* * NS NS 0.82* Daily mean 2 week 0.71 NS NS NS NS NS NS 0.86* * NS NS 0.74* Daily minimum 2 week 0.83* NS NS NS NS NS NS 0.88* NS 0.78* NS NS 0.65* Daily maximum 2 week NS NS NS NS NS NS NS 0.88* * NS NS 0.75* Daily mean 1 week 0.71 NS NS NS NS NS NS 0.86* * NS NS 0.74* Daily minimum 1 week 0.82* NS NS NS NS NS NS 0.86* NS 0.73* NS NS 0.69* Daily maximum 1 week NS NS NS 0.64 NS NS NS 0.90* * NS NS 0.67* ST in preceding weeks Daily mean 3 week 0.80* NS NS NS NS NS NS 0.92* 0.78* 0.82* NS NS 0.80* Daily minimum 3 week 0.74 NS NS NS NS NS NS 0.83* * NS NS 0.68* Daily maximum 3 week 0.89* NS NS NS NS NS NS 0.95* 0.82* 0.86* NS NS 0.80* ST in preceding weeks Daily mean 2 week 0.80* NS NS NS NS NS NS 0.90* * NS NS 0.79* Daily minimum 2 week 0.77* NS NS NS NS NS NS 0.81* * NS NS 0.66* Daily maximum 2 week 0.86* NS NS NS NS NS NS 0.92* 0.84* 0.84* 0.4 NS 0.81* Daily mean 1 week 0.81* NS NS 0.74 NS NS NS 0.90* * NS NS 0.78* Daily minimum 1 week 0.76 NS NS 0.67 NS NS NS 0.78* * NS NS 0.64* Daily maximum 1 week 0.82* NS NS 0.75 NS NS NS 0.90* 0.83* 0.87* 0.4 NS 0.82* SMP in preceding weeks Daily mean 3 week 0.79* NS NS NS * 0.81* NS NS NS 0.56* NS Daily mean 2 week 0.74 NS NS NS NS * NS NS NS 0.48* NS Daily mean 1 week 0.73 NS NS 0.78 NS NS NS 0.67 NS NS NS 0.43* NS Weekly mean c 0.79* NS NS 0.69 NS NS NS NS NS 0.43* NS Note: Values are correlation coefficients statistically significant at P = 0.05; *, statistically significant at P = 0.01; NS, not significant. a Variables are daily mean values averaged over 3, 2, and 1 week periods preceding sampling dates, respectively. b Data were combined to include only sites and years where apothecia (N = 30), ascospores (N = 46), and disease (N = 34) were observed within the crop, respectively. c Mean of two weekly measurements of soil matric potential prior to sampling, calculated from readings on the day of sampling and the week prior.

13 Kora et al.: sclerotinia rot of carrot / epidemiology / inoculum production 257 interrupted periods of dry weather throughout the entire growing season, but apothecia appeared only after rainfall occurred when the canopy was closed. Mean daily ST prior to, and during subsequent production of apothecia in this study, was consistent with earlier observations of optimum temperature for apothecia production (15 20 C) (Abawi and Grogan 1975; Clarkson et al. 2001; Coley-Smith and Cooke 1971). Microclimatic conditions within carrot crops following canopy closure appeared to enhance the development of apothecia as reported in rapeseed (Morrall and Dueck 1982), soybean (Boland and Hall 1988), and bean crops (Boland and Hall 1987; Caesar and Pearson 1983; Schwartz and Steadman 1978; Weiss et al. 1980). These results suggest that canopy closure may be used as a threshold to start monitoring for the emergence of apothecia in carrot crops where canopy closure is common. At each site, ascospores were detected before apothecia were found in the crop and, in one site, ascospores were present although apothecia were not found. Ascospores from external sources, such as infested areas outside the field, may have been present prior to ascospore production within the crop, as reported in bean (Abawi and Grogan 1975; Boland and Hall 1987). Another possibility is that apothecia may have been present in the crop, but their detection was obstructed by the sampling method or the sample size used in this study. Externally produced ascospores were not important in initiating epidemics of white mold in bean when they did not coincide with the susceptible stage of the crop (Boland and Hall 1987), but they were significant for epidemics of sclerotinia stem rot of rapeseed (Gugel and Morrall 1986; Morrall and Dueck 1982). In the current study, the abundance of ascospores was usually low (e.g., <10 ascospores per dish) prior to detection of apothecia and increased substantially when apothecia were present. In addition, ascospores detected prior to observing apothecia occurred up to 13 days before the start of the susceptible stage of carrots and, therefore, were considered to have a minimal impact on disease development. In contrast, apothecia and increased abundance of ascospores in the crop always coincided with the susceptible stage of carrot. Ascospores from apothecia within the crop are thus most likely responsible for initiating important epidemics of SRC in Bradford Marsh. However, development of disease in crops where no apothecia were found in this study, and similar observations in the United Kingdom (Geary 1978), indicate that external inoculum may initiate epidemics if present during the susceptible stage of carrots. The effect of physical environment on the abundance of ascospores within the crop was consistent across epidemics. Detection of ascospores was usually associated with SMP ranging from 0.1 to 0.3 bars and AT from 15 to 21 C. Similarly, in bean crops, ascospores appeared when SMP higher than 0.3 bars persisted for at least 1 2 weeks (Hunter et al. 1984). The results of the present study are also consistent with the findings of Caesar and Pearson (1983), who reported that mean AT 21 C significantly increased ascospore mortality. Maximum AT of 25 C or higher were particularly detrimental and may constitute an important limiting factor for ascospore survival. However, ascospores can escape the detrimental effects of high T and ultraviolet radiation if located on sheltered lower leaves under the buffering effect of the closed canopy (Caesar and Pearson 1983). Development of S. sclerotiorum and SRC epidemics appeared to be sporadic in time and space, but microclimate and architectural requirements for pathogen and disease development were consistent. These results indicate the need to develop inoculum and (or) disease prediction systems for improving the timing of application and the efficacy of control measures. Ascospores appeared to be a more accurate indicator of pathogen presence in the crop than apothecia because they were more consistent and accounted for both external and internal inoculum. Therefore, we conclude that the occurrence of ascospores is the most important pathogen variable to measure, or predict, and include in systems for predicting disease. The development of apothecia and ascospores was most consistently associated with SMP; therefore, SMP was proposed as a critical microclimate variable to be incorporated in models for predicting the occurrence of inoculum in carrot crops. Development of disease was associated with AT and ST; therefore, these variables should be considered in future studies as predictors of the initiation of SRC epidemics. Chronological relationships documented in the current study identified useful crop variables for monitoring in relation to the occurrence of S. sclerotiorum and epidemics of SRC. Monitoring for apothecia and ascospores is recommended to begin within 1 2 weeks of continuous SMP above 0.3 bars following canopy closure. Monitoring for foliar senescence and lodging to determine the susceptible stage of carrots is recommended to start at 90% 95% of canopy cover. Monitoring for disease should start when 70% 80% of carrot plants have at least two collapsed senescing leaves and 1 3 healthy leaves lodged on the soil. Field trials are currently underway to assess the predictive accuracy of these pathogen, microclimate, and crop variables, and the preliminary prediction models developed using the information obtained from this study. Acknowledgements Financial support provided by the Natural Sciences and Engineering Research Council of Canada (NSERC), Ontario Ministry of Agriculture and Food (OMAF), Ontario Fruit and Vegetable Growers Association (OFVGA), and Bradford and District Vegetable Growers Association (B&DVGA) is gratefully acknowledged. We also thank carrot growers Dave and Ray Horlings, and the staff of Muck Crops Research Station, Bradford Marsh, Ontario for providing the field sites and storage facilities to conduct this research. Special thanks to Kevin Vander Kooi for his technical assistance. References Abawi, G.S., and Grogan, R.G Source of primary inoculum and effects of temperature and moisture on infection of beans by Whetzelinia sclerotiorum. Phytopathology, 65: Abawi, G.S., and Grogan, R.G Epidemiology of diseases caused by Sclerotinia species. Phytopathology, 69: Adams, P.B., and Ayres, W.A Ecology of Sclerotinia species. Phytopathology, 69: Anonymous Carrot. Can. Plant Dis. Surv. 50: 20.