Spore deposition of the ear rot pathogen, Gibberella zeae, inside corn ...

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patterns of viable spore deposition of G. zeae over two years (2002 and 2003) inside corn fields ... Spores were deposited inside corn canopies during every day.
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NOTE Epidemiology / Épidémiologie

Spore deposition of the ear rot pathogen, Gibberella zeae, inside corn canopies David G. Schmale III and Gary C. Bergstrom

Abstract: Understanding the biological and meteorological interactions that direct the movement of airborne propagules of Gibberella zeae inside corn canopies is crucial to the epidemiology of gibberella ear rot. We observed the temporal patterns of viable spore deposition of G. zeae over two years (2002 and 2003) inside corn fields in Aurora, New York, United States. Viable, airborne propagules of G. zeae were collected inside corn canopies on Petri plates containing selective media placed at the level of receptive corn silks. Spores were deposited inside corn canopies during every day or night sampling period spanning the duration of silking in both years. In 2003, more than 91% of the colonies were collected during night-time sampling periods. On average, spore deposition was greater than 50 spores per plate for five sampling periods that occurred only at night. The cumulative exposure of corn silks to airborne spores of G. zeae should be considered in the development of risk models and management strategies for the disease. Key words: spore dispersal, aerobiology, corn silks, fungal spores. Résumé : Pour l’épidémiologie de la fusariose de l’épi du maïs, il est essentiel de comprendre les interactions biologiques et météorologiques qui guident le mouvement des propagules aériennes du Gibberella zeae à l’intérieur du couvert végétal. Nous avons observé le patron temporel du dépôt des spores viables de G. zeae sur deux ans (2002 et 2003) à l’intérieur de champs de maïs à Aurora, New York, États-Unis. Des propagules aériennes viables de G. zeae furent recueillies à l’intérieur du couvert végétal du maïs sur des boîtes de Pétri contenant un milieu sélectif et placées au niveau des soies réceptives. Des spores se déposèrent à l’intérieur du couvert végétal du maïs pendant chaque période d’échantillonnage diurne ou nocturne couvrant la durée de l’apparition des soies lors des deux années. En 2003, plus de 91% des colonies furent obtenues pendant les périodes d’échantillonnage nocturnes. Le dépôt de spores était supérieur à 50 spores par boîte en moyenne au cours de cinq périodes d’échantillonnage, toutes nocturnes. L’exposition cumulative des soies de maïs aux spores aériennes de G. zeae devrait être prise en compte dans le développement de modèles du risque et de stratégies de lutte contre la maladie. Mots clés : dissémination des spores, aérobiologie, soies du maïs, spores fongiques. 595

Introduction corn ear rot / Gibberella zeae / spore deposition

The fungal pathogen Gibberella zeae (Schwein.) Petch (anamorph Fusarium graminearum Schwabe) is the causal agent of gibberella ear rot (GER), a disease of great economic importance to corn crops in Canada and the United States (Sutton 1982; Vigier et al. 1997). Grain infected with GER often contains the mycotoxins deoxynivalenol and zearalenone, threatening human and livestock health (Mirocha and Christensen 1974; Vesonder et al. 1981). Gibberella zeae overwinters in infected residues of corn and small grains (Shaner 2003). Sexual structures (perithecia) of the fungus develop from these residues and forcibly discharge ascospores into the air during the spring Accepted 27 June 2004. D.G. Schmale III1 and G.C. Bergstrom. Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA. 1

Corresponding author (e-mail: [email protected]).

Can. J. Plant Pathol. 26: 591–595 (2004)

Schmale and Bergstrom:

and summer months (Andersen 1948; Schmale and Bergstrom 2003; Sutton 1982). Ascospores (and potentially asexual macroconidia) are thought to be carried by wind to corn ears where they may germinate on emerging corn silks, infecting the developing kernels and rachises (cobs) of susceptible cultivars (Hesseltine and Bothast 1977; Sutton 1982). Symptoms of GER usually start at the tip of the ear and spread toward the base. Signs of the disease include the presence of a pinkish to reddish coloured mycelium on the kernels (Sutton 1982). Artificial inoculations with G. zeae have demonstrated that corn silks provide an effective point of entry for infection of corn ears and the resulting development of GER (Reid et al. 1992, 2000, 2002). However, no published data exist on the incidence of natural inoculum of G. zeae inside corn canopies at ear height during the period of corn silking. Corn ears appear to be the most susceptible to GER within 6 days following silk emergence (Reid et al. 1992, 2002), but knowledge of how and when airborne propagules

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of G. zeae arrive at corn silks to cause GER is limited. Information on the biological and meteorological interactions that direct the movement of G. zeae inside corn canopies is critical to the understanding of the epidemiology of GER and to the development of successful strategies for disease management. In an extensive study of spore deposition of G. zeae over wheat canopies, Schmale et al. (2003) demonstrated that viable propagules of G. zeae are deposited above wheat spikes for periods of weeks contiguous to wheat flowering. Based on these observations, we hypothesized that spores of G. zeae are present inside corn canopies throughout the period of corn silking (the suggested window for infection of corn ears and the resulting development of GER). Furthermore, Schmale et al. (2003) established that airborne propagules of G. zeae settle out of the air and are deposited over wheat canopies predominantly at night. Based on these observations, we hypothesized that airborne propagules of G. zeae are deposited in corn canopies predominantly at night. The specific objectives of this study were: (1) to determine the presence of viable, airborne propagules of G. zeae inside corn canopies at the level of receptive corn silks and (2) to determine the temporal patterns of spore deposition of G. zeae inside corn canopies.

Can. J. Plant Pathol. Vol. 26, 2004 Fig. 1. T- shaped sampling stakes for the collection of spores of Gibberella zeae inside corn canopies. Petri plates containing Fusarium-selective media were used to trap viable propagules of G. zeae at the level of silks in 2002 (A) and at the level of silks and tassels in 2003 (B).

Studies were performed at the Robert B. Musgrave Research Farm in Aurora, New York State, United States. Experiments were conducted in July and August of 2002 and 2003. In 2002, experiments were conducted in three separate corn fields (F, J, and W). In 2003, experiments were conducted in a single corn field (U). Fields F, J, and W were selected based on the absence of cereal crop residues (a potential source of G. zeae), similar planting dates, and simultaneous silk emergence within and among fields. Field U was selected based on the absence of cereal crop residues and simultaneous silk emergence within the field. Rainfall and temperature were recorded every hour at a weather station in Scipioville, New York State (approximately 4 km from the Musgrave Research Farm).

(silks and tassels) contained two plates. The rows and sampling stakes were separated by 15 m. For 2002 experiments, spore collection started at sunrise on 31 July and ended at sunset on 7 August. For 2003 experiments, spore collection started at sunset on 29 July and ended at sunrise on 5 August. Plates were exposed continuously for sampling periods consisting of either sunrise to sunset, or sunset to sunrise. Plates were immediately covered following exposure, gathered, and incubated for 5 to 7 days at room temperature. The number of resulting F. graminearum colonies was counted and recorded. Colonies of several Fusarium spp. were recovered on many of the plates. Only salmon-coloured colonies characteristic of the F. graminearum anamorph were counted and recorded (Schmale et al. 2002). A subset of isolates putatively identified as F. graminearum was transferred to new media and refrigerated in storage for future analyses.

Sampling approach Petri plates containing Fusarium-selective medium (FSM) were used to trap viable propagules of G. zeae inside corn canopies. The FSM consisted of a modified Nash– Snyder formulation, prepared as described by Burgess et al. (1994), with a concentration of neomycin sulfate increased to 0.175 g L–1. In fields F, J, and W in 2002, plates were placed at the level of corn silks, on wooden, T-shaped sampling stakes (Fig. 1, A). In each field, there were six sampling stakes placed in two rows of three stakes, and each stake contained two plates. The rows and sampling stakes were separated by 30 m. In field U in 2003, plates were placed at the level of corn silks and at the level of tassels, on wooden, T- shaped sampling stakes (Fig. 1, B). There were 12 sampling stakes placed in three rows of 4 stakes, and each sampling level

Statistical analyses For 2002, analysis of variance (ANOVA) was used to test for significant differences in colony counts among fields and among sampling periods. For 2003, ANOVA was used to test for significant differences between day and night sampling times, among sampling periods, and between top and bottom sampling positions. Statistical analyses were performed with SAS System for Windows (release 8.02; SAS Institute Inc., Cary, N.C.). Experiments were analyzed with PROC GLM. Model statements included all possible interactions, where appropriate. Significance was evaluated at P < 0.05 for all tests. Data from colony counts were transformed by natural logarithm to verify normality and equal-variance assumptions. Assumptions about the error terms were validated with plots of residuals versus y$ (fitted values) and residuals versus main effects. A normal probability plot of errors was constructed to assess normality.

Materials and methods

Schmale and Bergstrom: corn ear rot / Gibberella zeae / spore deposition

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Fig. 2. Mean spore deposition of Gibberella zeae inside corn canopies at the level of receptive corn silks in 2002 and 2003 in Aurora, New York State. Viable spore deposition was observed over day or night sampling periods. Error bars represent ± standard error of the mean. Rainfall is reported (in millimetres) for sampling periods 5, 6, and 9 in 2003.

Results Viable spores of G. zeae were deposited inside corn canopies at the level of receptive corn silks during every sampling period in both 2002 and 2003 (Fig. 2). No rainfall occurred during the sampling periods in 2002, while rainfall occurred during three sampling periods in 2003 (Fig. 2, sampling periods 5, 6, and 9). Spore deposition in 2002 Deposition at within-field sampling locations varied from 0 to 19 spores. Though spore deposition did not exceed 12 spores, on average, during any of the sampling periods (Fig. 2), deposition varied significantly among 11 sampling periods (P < 0.001) and significantly among the three fields (P = 0.009). The interaction between field and sampling period was also significant (P = 0.007). Tukey’s pairwise comparisons showed that spore deposition in field F was significantly higher than in field J, and that deposition in field W was intermediate. Spore deposition in 2003 Deposition at within-field sampling locations ranged from 0 to 387 spores. Spore deposition was greater than 50

spores, on average, for 5 out of 13 sampling periods (Fig. 2, sampling periods 1, 7, 9, 11, and 13). On average, 74 spores were deposited on individual plates over all night-time sampling periods compared with 12 spores over all day-time sampling periods. Spore deposition varied significantly among 13 sampling periods (P < 0.001). Significantly more colonies were collected at night than during the day (P < 0.001). Spore deposition did not vary significantly between top and bottom sampling plates (Fig. 1, B) (P = 0.4427).

Discussion The present findings provide the first report of the temporal dynamics of spore deposition of G. zeae inside corn canopies. We report the incidence of viable spores of G. zeae inside corn canopies at the level of receptive corn silks. Previous studies, using artificial inoculations of corn silks with aqueous suspensions of G. zeae, have shown that corn silks provide an effective point of entry for the pathogen (Reid et al. 1992, 2000, 2002). Corn ears that had newly emerged silks (6 days old or less) demonstrated enhanced susceptibility to GER (Reid et al. 1992, 2002). We assessed the presence of airborne G. zeae inoculum in a natural environ-

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ment during a period when corn ears are susceptible to GER. In both 2002 and 2003, viable spores of G. zeae were present during every day and night sampling period throughout the duration of corn silking (Fig. 2), regardless of meteorological conditions during the sampling period. Spore deposition varied between years, among fields, and among sampling periods. In 2002, spore deposition did not exceed 12 spores, on average, during any of the sampling periods. In 2003, spore deposition ranged from 3 to 200 spores, on average, per sampling period. The differences in the prevalence of airborne spores during these years may be related to the amount of rainfall surrounding periods of inoculum production. Warm, moist conditions favour the production of perithecia of G. zeae on cereal crop residues, and the resulting discharge of ascospores from these substrates (Andersen 1948; Schmale and Bergstrom 2003; Shaner 2003; Sutton 1982). In 2002, drought conditions (total rainfall, 4.18 mm; mean temperature, 21.33 °C from 1st July to 1st September) did not support a high concentration of airborne inoculum of G. zeae in periods surrounding corn silking. In 2003, wetter conditions (total rainfall, 10.65 mm; mean temperature, 21.22 °C from 1st July to 1st September) favoured a greater airborne inoculum source of G. zeae in the region. The deposition of airborne spores of G. zeae inside corn canopies may be accelerated by rainfall (Aylor 1998; Oke 1987). We recorded precipitation during three of the sampling periods in 2003, and over 25 spores, on average, were deposited during these three events (Fig. 2, sampling periods 5, 6, and 9). Schmale et al. (2003) recorded rainfall during three major spore-deposition events of G. zeae (>50 spores on average) over wheat canopies. Other investigators have also reported major deposition events of airborne spores of G. zeae over wheat spikes during periods of rainfall (Francl et al. 1999; Markell and Francl 2003). The cumulative deposition (by atmospheric settling and rainfall) of spores of G. zeae on corn silks is related to the risk of GER and the resulting contamination with mycotoxins (Reid and Hamilton 1996; Reid et al. 1996). In 2003, more than 91% of the colonies were collected during the night. Seventy-four spores, on average, were deposited on individual plates over all night-time sampling periods compared with 12 spores over all day-time sampling periods. Schmale et al. (2003) demonstrated that airborne propagules of G. zeae settle out of the air and are deposited over wheat canopies predominantly at night. In their study, more than 94% of the spores collected were deposited over wheat canopies at night, with less than 6% deposited during the day. The settling of the atmosphere during the evening hours may explain the high percentage of spores deposited inside corn canopies at night (Oke 1987), whereas a turbulent atmosphere during the daylight hours may account for the low percentage of spores deposited during the day (Aylor 1994, 1998; Oke 1987). Maldonado-Ramirez (2001) demonstrated that ascospores of G. zeae were released from corn substrates in greater amounts during the day than during the night. Spores that are released during the day may have a greater potential for leaving the surface of the ground, escaping the plant canopy, and traveling away from the source (Aylor 1994, 1998;

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Oke 1987). At night, because of atmospheric settling, these spores may settle over plant canopies at a distance from the original source. A number of investigators have trapped spores of G. zeae within and above small grain canopies (de Luna et al. 2002; Fernando et al. 2000; Francl et al. 1999, 2000; MaldonadoRamirez 2001; Maldonado-Ramirez et al. 1999; Markell and Francl 2003; Paulitz 1996; Schmale et al. 2002, 2003). In New York, investigators have used remote-controlled aircraft to trap viable G. zeae spores over agricultural fields, forests, and lakes, during the spring and summer months, under a broad range of environmental conditions (Maldonado-Ramirez 2001; Maldonado-Ramirez et al. 1999; and D.G. Schmale III, E.J. Shields, and G.C. Bergstrom, unpublished results). It is thought that airborne ascospores and macroconidia constitute the principal inoculum for infection of corn ears during silking (Sutton 1982). We found that viable spores of G. zeae were deposited inside corn canopies at the level of corn silks every day and every night throughout corn silking, thus confirming the presence of inoculum for GER infection. But under what circumstances does the natural infection of corn ears occur? Rainfall, humidity, and temperature have been proposed as key factors influencing epidemics of ear rot (Sutton 1982), but the relationships between these factors and the development of infection are not well understood. Tuite et al. (1974) observed a positive correlation between rainfall and the colonization of corn ears over 7 days during silking. Sutton (1982) reported a strong correlation between rainfall and the incidence of zearalenone in corn kernels during periods of silking, but not in periods after silking had occurred. During the period of silk receptivity, moisture may be more critical than the prevalence of inoculum for the infection of corn ears and the resulting development of GER. Risk assessment models for predicting GER epidemics have yet to be developed. Information on the biological and meteorological interactions that direct the movement of G. zeae inside corn canopies is sparse, yet critical to the understanding of the epidemiology of GER. The cumulative exposure of corn silks to airborne spores of G. zeae should be considered in risk models for GER and in the development of future management strategies for the disease.

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