Baseline Sensitivity of Fusarium Species Associated with Fusarium Diseases to Metconazole, Triticonazole, and Thiabendazole Fungicides

Abstract

Baseline sensitivity was determined using in vitro radial growth assay for Fusarium oxysporum, Fusarium graminearum, and a novel uncharacterized Fusarium sp. nov. from sugarbeet, and F. sambucinum from potato, to metconazole, triticonazole, and thiabendazole. All the isolates from sugarbeet and a thiabendazole-sensitive F. sambucinum isolate were sensitive to the tested fungicides. A thiabendazole-resistant isolate of F. sambucinum was resistant to thiabendazole but sensitive to the other two fungicides. The EC50 values of F. oxysporum, F. graminearum, and F. sp. nov. for triticonazole were 0.51 μg ml-1, 2.15 μg ml-1, and 0.04 μg ml-1, respectively. The EC50 values of F. oxysporum, F. graminearum, and F. sp. nov. for metconazole were 0.04 μg ml-1, 0.03 μg ml-1, and 0.02 μg ml-1, respectively. The EC50 values of F. oxysporum, F. graminearum, and F. sp. nov. for thiabendazole were 0.57 μg ml-1, 0.54 μg ml-1, and 0.64 μg ml-1, respectively. Generally, a higher concentration of triticonazole and thiabendazole compared to metconazole were required to reduce colony growth by 50%. The low EC50 values of metconazole, triticonazole, and thiabendazole for the Fusarium species tested suggest that they are potential candidates for control of Fusarium diseases of sugarbeet. Research is needed to determine the 24 Journal of Sugar Beet Research Vol. 47 Nos. 1 & 2 effectiveness of these fungicides for control of Fusarium diseases under field conditions. Additional key words: Beta vulgaris, benzimidazole, EC50, triazole. M and North Dakota ranked first and second in sugarbeet production in the United States during 2008 (USDA ERS, 2009). The acreage planted in the bi-state area was 312,417 ha, that represented 58% of the total United States crop. The fungal diseases, Cercospora leaf spot, Aphanomyces root rot, and Rhizoctonia crown and root rot, are the most damaging diseases for the sugarbeet industry in this region. In 2002, another fungal disease, Fusarium yellows, was reported in Minnesota and North Dakota (Khan et al., 2003). Fusarium yellows is becoming a serious problem particularly for growers in the Moorhead factory district (Windels et al., 2005). Pathogenicity studies (Burlakoti, 2007) confirmed that F. oxysporum, F. graminearum, and novel, previously undescribed Fusarium species, hereafter referred to as F. sp. nov (Rivera et al., 2008) recovered from diseased sugarbeet with foliar symptoms similar to Fusarium yellows, from fields in west-central Minnesota (Sabin, Fossum, and Georgetown), caused foliar yellowing and vascular discoloration of sugarbeet seedlings in greenhouse conditions. Fusarium diseases of sugarbeet have been reported from Nebraska (Bockstahler, 1940), Wyoming and Minnesota (Hanson, 2006a), Michigan (Hanson, 2006b), Colorado (Hanson and Jacobsen, 2006; Stewart, 1931), Texas, (Harveson and Rush, 1997; Ruppel, 1991; Rush and Martyn, 1991), Oregon (Rush and Martyn, 1991), North Dakota and Minnesota (Windels et al., 2005; Khan et al., 2003), India, Belgium, Germany, and the Netherlands (Duffus and Ruppel, 1993). Fusarium diseases on sugarbeet causes yield loss due to poor plant population, stunted growth, and increased impurities in extracted juice (Bosch and Mirocha, 1992; Harveson and Rush, 1997; Schneider and Whitney, 1986; Windels et al., 2005). The use of resistant cultivars is an effective means to manage many diseases of sugarbeet (Biancardi, 2005; Bugbee and Cole, 1979). Screening for cultivars resistant to Fusarium diseases in Minnesota and North Dakota started in 2005 (Niehaus, 2006). Effective host-plant resistance to Fusarium diseases of sugarbeet have not been identified (Francis and Luterbacher, 2003). No commercial sugarbeet cultivar is immune to Fusarium diseases and most cultivars have at least some susceptibility (Biancardi, 2005). In this context, fungicides should be considered as a tactic to control the disease. Currently, triazole fungicides provide the most effective control Jan. July 2010 Baseline Sensitivity 25 of Fusarium species (Mesterhazy et al., 2003). Triazoles have provided control of Fusarium head blight of small grains, dollar spot and other diseases of turfgrass in the United States for many years (Koller, 1988; Loss et al., 2005). Haidukowski et al. (2005) reported that efficacy of triazole fungicides at controlling Fusarium head blight depended upon the time of application and control ranged from 25% to 89% in field conditions. Thiabendazole belongs to the benzimidazole class of fungicide. Thiabendazole binds the beta tubulin gene and inhibits fungal growth (Davidse, 1986). Benzimidazole has provided control of Fusarium species on seedlings, and tubers of a variety of crops (Davidse, 1986; Hanson et al., 1996). For example, soil drenching by benomyl reduced foliar and internal xylem-browning symptoms of Fusarium wilt of tomato caused by F. oxysporum f. sp. lycopersici (Erwin, 1973). Thiabendazole provided effective control against Fusarium wilt in potato caused by several Fusarium species (Secor et al., 1992). However, prolonged usage of thiabendazole resulted in resistance to thiabendazole in some isolates of F. sambucinum, F. solani, F. oxysporum, F. acuminatum, and F. culmorum from potato (Hanson et al., 1996). In vitro sensitivity of Fusarium species associated with sugarbeet to the triazoles, metconazole and triticonazole, and thiabendazole have not been determined. It would be useful to determine sensitivity of Fusarium species associated with Fusarium diseases of sugarbeet to triazoles and thiabendazole to evaluate their potential for field use. The objective of this study was to determine the baseline sensitivity of F. oxysporum, F. graminearum, and F. sp. nov. (Rivera et al., 2008) associated with sugarbeet in Minnesota and North Dakota to metconazole, triticonazole, and thiabendazole. MATERIALS AND METHODS Isolate collection and growing condition. Ninety-eight isolates [50 Fusarium sp. nov., 18 Fusarium oxysporum, and 30 Fusarium graminearum] were collected in Minnesota from Fusarium-diseased sugarbeet in 2005, and twenty five Fusarium oxysporum isolates were collected in 2006. A thiabendazole-resistant isolate (420-1c) and thiabendazole-sensitive (413-1a) isolate of Fusarium sambucinum from potato was used as a check in the thiabendazole-sensitivity in-vitro assay and also was grown on metconazole and triticonazole amended plates as described by Estrada (2007). Each isolate was transferred to two petri dishes containing freshly prepared half strength potato dextrose agar (HPDA) and incubated at 25 to 26°C for five days under 24 h fluorescent light. HPDA was prepared by autoclaving 100 26 Journal of Sugar Beet Research Vol. 47 Nos. 1 & 2 g of potato in 1 liter of distilled water for 20 min. followed by straining through cheesecloth to remove the water. The potato extract was mixed with 10 g dextrose and 10 g agar and autoclaved for 20 min. Fungicide sensitivity in vitro assay. The sensitivity of each isolate to thiabendazole (technical grade, Merck and Co., Rahway, NJ), metconazole, and triticonazole (commercial, BASF, Raleigh, NC) was determined by comparing the radial growth of each isolate on fungicide amended media to growth on nonamended media. Thiabendazole was dissolved in dimethyl sulfoxide (DMSO) due to greater solubility and to control bacterial growth (Kawchuk et al., 1994). Metconazole and triticonazole were dissolved in sterile distilled water to obtain stock solution of 100μg mL-1 which was further diluted to 10, 1, 0.1, and 0.01μg mL-1. Each concentration of each fungicide was incorporated into autoclaved media. The effect of the fungicide on mycelial growth in vitro was determined on HPDA media amended with 0, 0.01, 0.1, 1, 10, and 100 μg mL-1 of fungicide. A 5-mm diameter mycelial plug from the margin of a five day old actively growing culture of each isolate was inverted and transferred to the center of petri dishes (90 mm) with the fungicide amended media and the non-amended media. Isolate sensitivity to fungicides was assessed by measuring colony diameter of mycelial growth after 6 days of incubation at room temperature in the dark for F. graminearum and after seven days of incubation for F. oxysporum and F. sp. nov. Two perpendicular measurements of colony diameter, excluding the original plug diameter (5 mm), were obtained from each plate. Isolates were replicated twice with two plates per replication at each fungicide concentration. The experiment was repeated. The diameter of each colony on fungicide amended medium relative to the diameter of the colony on non-amended medium was recorded. The relative growth reduction percentage for each fungicide concentration was calculated as follows; [100-(diameter on fungicide amended medium/diameter on nonamended medium)*100]. EC50 values of each isolate were calculated by determining the effective fungicide concentration that inhibited mycelial growth by 50%. We considered isolates as sensitive if their EC50 values were equal to or less than 10 μg ml-1, and resistant if their EC50 values were greater than 10 μg ml-1 (Kawchuk et al. 1994). Data analysis. The effective fungicide dose that inhibited radial growth by 50% (EC50) was determined for each isolate by using the general linear modeling procedure in Statistical Analysis System (SAS Institute, Cary, Jan. July 2010 Baseline Sensitivity 27 NC). A homogeneous test was conducted for the repeated experiments based on calculated F values and Bartlett chi-square values before combining the data from the repeated experiment.