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Effect of time of harvest on the incidence of Fusarium spp. in kernels of silage corn

  • Allen G. Xue,
  • Baoluo L. Ma,
  • Randall M. Clear and
  • Bernard J. Vigier

…more information

  • Allen G. Xue
    Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa (Ontario), Canada K1A 0C6

  • Baoluo L. Ma
    Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa (Ontario), Canada K1A 0C6

  • Randall M. Clear
    Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, (Manitoba), Canada R3C 3G8

  • Bernard J. Vigier
    Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa (Ontario), Canada K1A 0C6
    vigierbj@agr.gc.ca

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Introduction

Silage corn (Zea mays L.) is the most commonly grown forage crop fed to livestock as a complement to the hay silage system in eastern Ontario and western Quebec. Silage corn is commonly harvested at 50% of milkline in the kernel development stage, when the whole plant moisture content is at 62-70% and the grain mass accounts for up to 50% of the total silage dry matter (OMAFRA 2002). Milkline is defined as the interface between the liquid and solid portion of the kernel, separating the solid from the liquid endosperm contained in a corn kernel. Although silage yield may continue to increase with advancing maturity, feed quality will decrease. Also, there is a risk of mold growth during the ensiling process and of accumulation of mycotoxins produced by Fusarium spp., especially deoxynivalenol (DON, vomitoxin) and zearalenone that are undesirable in livestock feed (Abramson 1998; Placinta et al. 1999). In late September and early October, when corn silage is harvested in eastern Ontario and western Quebec, weather conditions are often warm with frequent showers. The wet weather can delay the silage corn from being harvested at the optimum time. In addition, the moist conditions accentuate the development of fusarium ear rot and the production of mycotoxins in the kernel (Miller et al. 1995; Reid et al. 1996a, 1996b, 1999; Sutton 1982; Vigier et al. 1997).

In previous researches, more than ten Fusarium spp. were associated with grain corn in Ontario (Neish et al. 1983; Vigier et al. 1997). Of these Fusarium species, F. subglutinans (Wollenweb. & Reinking) Nelson, Toussoun & Marasas, F. graminearum Schwabe, F. verticilloides (Sacc.) Nirenberg (older synonym of F. moniliforme), F. proliferatum (Matsushima) Nirenberg and F. sporotrichioides Sherb. were the most frequently isolated. There has not been any study of the kernel microflora in silage corn in Ontario to determine whether or not Fusarium spp. and their relative frequencies are the same as those from grain corn. Preliminary results of this research have already been published (Xue et al. 2004), and the objective of this research was to investigate the occurrence and prevalence of Fusarium spp. in several silage corn hybrids according to the effect of time of harvest.

Materials and Methods

Field trials were conducted on a clay loam soil at the Greenbelt Research Farm, Eastern Cereal and Oilseed Research Centre (ECORC), Agriculture and Agri-Food Canada (AAFC), Ottawa, Ontario, in 2001 and 2002. The trials were part of a larger study to characterize physiological traits associated with high silage yield and digestibility. Four silage corn hybrids, NK BRAND Enerfeast 1 and PIONEER 37M81, representing the early silage maturity group, and MAIZEX Leafy 4 and MYCOGEN TMF94, representing the late maturity group, were used each year (Table 1). The experiments were arranged in a split-plot design with four replicates, where hybrids were the main plots and time of harvest the sub-plots. Main plots were 14-m long, consisting of eight rows with 76-cm row spacing. Each main plot was divided into three 4-m long sub-plots that were harvested at different times. Trials were initiated in the third week of May each year at a seeding rate of 66 690 plants ha-1. Plots received P and K at rates recommended by a soil test (OMAFRA 1999a) plus 110 kg N ha-1 broadcast and incorporated prior to seeding. Appropriate herbicides for effective weed control were used according to standard management practices (OMAFRA 1999b).

Table 1

Harvest dates for silage corn hybrids in 2001 and 2002

Harvest dates for silage corn hybrids in 2001 and 2002

-> See the list of tables

Each year, kernels were harvested either at the 75, 50 or 25% of the milkline stages of kernel development, in conjunction with biweekly silage harvest. The kernel developmental stages were determined by closely monitoring phenological development and were designated as such when at least 50% of the plants within a plot reached a specific stage. The dates of harvest for each hybrid at the three kernel developmental stages are listed in Table 1. The primary ears of the central three plants from each row were handpicked from each sub-plot, husked and air dried at room temperature. After drying, the ears were shelled and the seeds bulked by sub-plot and harvest date for isolation of kernel-borne Fusarium spp.

A sub-sample of 100 kernels per sub-plot at each harvest date was taken at random to determine the level of infection by Fusarium spp. The kernels were surface disinfected for 3 min in 0.5% NaOCl, rinsed three times with sterile distilled water and drained on sterile filter paper. Five kernels were placed in each of 20 Petri dishes containing modified potato dextrose agar (mPDA) (10 g dextrose per liter, which is 50% of the label rate) amended with 20 ppm streptomycin sulfate. Dishes were placed under a fluorescent light set of mixed UV light and artificial daylight, on a 12-h light/dark cycle for 7-14 d at 22-25°C. Fusarium spp. were detected directly from mPDA or, if necessary, subcultured onto carnation leaf agar (CLA) medium for identification. The fungi were identified by microscopic examination using taxonomic keys for Fusarium spp. described by Nelson et al. (1983), or from morphological illustrations by Samson et al. (2000) and Watanabe (1994).

Incidence of the five most common and total Fusarium spp. was subjected to analyses of variance. Replicate x time of harvest was used as the error term to test the main effect of time of harvest, and replicate x hybrid was the error term to test the main effect of hybrid. The other terms were tested by the residual error. Where treatment effects were significant, treatment means were separated by Fisher’s least significant difference test at a probability level of P ≤ 0.05. Analyses were performed using SAS/STAT (SAS Institute Inc., Cary, NC). Weather information for the experiment site was obtained from the Environment Canada weather station located within 1 km of the field trials.

Results and Discussion

A total of 3329 isolates representing 11 Fusarium spp. were recovered from the 9600 kernels sampled in 2001 and 2002 (Table 2). Among the 11 Fusarium spp. identified, F. subglutinans was the dominant species and recovered from 36.8 and 20.7% of the kernels in 2001 and 2002, respectively. The next most frequent species isolated were F. oxysporum Schlechtend.: FR. (2.6%), F. graminearum (2.5%), F. proliferatum (0.3%) and F. sporotrichioides (0.2%). The remaining six species, F. avenaceum (Fr.:Fr.) Sacc., F. crookwellense Burgess, Nelson & Toussoun, F. culmorum (Wm.G. Sm.) Sacc., F. equiseti (Corda) Sacc., F. poae (Peck) Wollenweb. and F. solani (Mart.) Sacc., were recovered only from < 0.1 % of the kernels. The extent and variation in occurrence of Fusarium spp. in silage corn observed in the present study are similar to those recorded for grain corn in Ontario by Neish et al. (1983) and Vigier et al. (1997), except that F. verticilloides was not detected.

Table 2

Frequency of Fusarium spp. isolated from kernels of silage corn in 2001 and 2002

Frequency of Fusarium spp. isolated from kernels of silage corn in 2001 and 2002

a Number of infected kernels out of 4800.

-> See the list of tables

Analyses of variance indicated that the time of harvest, corn hybrid and year had significant effects on the incidence of the causal agents of fusarium ear and kernel rot (F. subglutinans) and Gibberella ear rot (F. graminearum), as well as total Fusarium spp., but not on the incidence of the other species (Table 3). The time of harvest x hybrid interaction was not significant for any of the species in either year. When the kernels were harvested at 75, 50 and 25% of milkline, the incidence of F. subglutinans increased from 20.9 to 26.7 and to 38.7%, respectively; that of F. graminearum increased from 1.7 to 2.9 and to 3.1%; and for the total of the five main Fusarium species it increased from 28.7 to 32.2 and to 42.3%. The corn hybrids differed in the incidence of those Fusarium spp., but the genotypic variability was not affected significantly by time of harvest and year. Hybrid NK BRAND Enerfeast1 had an incidence of kernel-borne F. subglutinans, F. graminearum and total Fusarium spp. of 16.0, 0.9 and 17.3%, respectively; those percentages were significantly lower than those of MYCOGEN TMF94 (38.0, 6.3 and 51.9%) but not significantly different from the other hybrids. The genotypic variation observed in kernel-borne F. subglutinans and F. graminearum confirms that breeding for resistance is possible.

Incidence of F. subglutinans, F. graminearum and total Fusarium spp. varied greatly in the frequency of recovery between the 2 yr. Incidence of F. graminearum was significantly lower in 2001 than in 2002. However, incidence of F. subglutinans and total Fusarium spp. was significantly greater in 2001 than in 2002 (Table 3). These variations in the incidence of Fusarium spp. were likely related to the weather conditions prevailing at the testing site prior to and during harvesting each year. Temperatures were higher and there were more days with total daily precipitation above 1.0 mm during the harvest interval in 2001 compared with 2002 (Fig. 1). The warmer and wetter conditions in 2001 likely contributed to the higher incidence of F. subglutinans and total Fusarium spp. It is also possible that the various Fusarium spp. were competitors in colonizing the same kernel throughout or at different stages of plant development.

Figure 1

Daily air temperature and precipitation from September 1 to October 31 at the Greenbelt Research Farm, Ottawa, Ontario, in 2001 and 2002.

Daily air temperature and precipitation from September 1 to October 31 at the Greenbelt Research Farm, Ottawa, Ontario, in 2001 and 2002.

Data recorded at the Environment Canada weather station < 1 km from the field trials.

-> See the list of figures

Table 3

Effect of time of harvest, hybrid and year on the incidence of Fusarium spp. in kernels of silage corn in 2001 and 2002

Effect of time of harvest, hybrid and year on the incidence of Fusarium spp. in kernels of silage corn in 2001 and 2002

a Fg = F. graminearum; Fo = F. oxysporum; Fpr = F. proliferatum; Fsp = F. sporotrichioides; Fsu = F. subglutinans.

b Treatment means were separated by Fisher's least significant difference test at a probability level of P ≤ 0.05.

-> See the list of tables

It was apparent that F. graminearum, the causal agent of Gibberella ear rot, was much less common than previously reported in grain corn in Ontario (Neish et al. 1983). The relatively warm and dry weather conditions prevailing during June and July of 2001 and 2002 were less favourable to the endemic presence of this pathogen in cereals in eastern Ontario (Xue et al. 2003). As a result, little inoculum was available for infection in corn. This result is in agreement with previous findings by Reid et al. (1995) and Vigier et al. (1997) indicating that lower F. graminearum infection levels are observed during dry years in eastern Canada.

In this study, F. subglutinans was shown to be the predominant kernel-borne fungus in silage corn and its incidence increased with delayed harvesting time. Fusarium subglutinans was also frequently isolated from ear rot infected kernels in Ontario (Vigier et al. 1997) and from corn in eastern Australia (Francis and Burgess 1977). Despite its frequent occurrence in corn, it is not known when and how this fungus infects the kernels. The higher incidence at later dates of harvest means that F. subglutinans may have a wider window to infect corn kernels than the 10-15 d period during the silking stage recognized for F. graminearum and F. verticillioides (Stewart et al. 1998). It is also possible that F. subglutinans may be a secondary colonizer as suggested by Vigier et al. (1997). Additional studies are needed to examine the role of different environments on the occurrence and frequency of the various kernel-borne Fusarium spp. and other fungi in silage corn in Ontario.

Acknowledgements

We thank D.T. Chi, F. Sabo, Y. Chen, T. Potter, D. Balchin, B. Wilson and K. Conty for their technical assistance. This research was partially supported by Glenn Seed Inc. and AAFC Matching Investment Initiative.

Appendices