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Temperature and water activity effects on growth and temporal deoxynivalenol production by two Argentinean strains of Fusarium graminearum on irradiated wheat grain
International Journal of Food Microbiology 106 (2006) 291 – 296 www.elsevier.com/locate/ijfoodmicro
Temperature and water activity effects on growth and temporal deoxynivalenol production by two Argentinean strains of Fusarium graminearum on irradiated wheat grain Maria L. Ramirez a,b, Sofia Chulze b, Naresh Magan a,* a
Applied Mycology Group, Cranfield Biotechnology Centre, Cranfield University, Silsoe, Bedford MK45 4DT, UK b Departamento de Microbiologia e Inmunologia, Universidad Nacional de Rio Cuarto, Cordoba, Argentina Received 11 March 2005; received in revised form 18 July 2005; accepted 2 September 2005
Abstract The objective of this study was to determine the effects of water activity (a W; 0.900 – 0.995), temperature (5, 15, 25 and 30 -C), time of incubation (7 – 49 days) and their interactions on mycelial growth and deoxynivalenol (DON) production on irradiated wheat grain by two strains of Fusarium graminearum isolated from wheat ears in Argentina. Optimal a W levels for growth were in the range 0.950 – 0.995 with a temperature optima of 25 -C. Maximum growth rates were obtained at the highest a W (0.995) and 25 -C for both strains. No growth was observed at 5 -C regardless of the a W levels assayed. Both strains were able to growth at the lowest a W assayed (0.900), although the temperature ranges allowing growth at this minimal a W was 15 – 25 -C. DON was produced the most rapidly (7 days) when incubated at 25 -C and 0.995 a W. All other conditions required 7 – 14 days before DON was produced on grain. Maximum amounts of DON for both strains were produced at the highest a W treatment (0.995) after 6 weeks at 30 -C. The range of DON concentrations varied considerably (5 to 140,000 ng g 1) depending on a W and temperature interaction treatments. Production of DON occurred over a narrower range of a W (0.995 – 0.95) than that for growth (0.995 – 0.90). DON was more rapidly produced at 25 -C but the maximum amount produced was at 30 -C. Two-dimensional profiles of a W temperature were developed from these data to identify areas where conditions indicate a significant risk from DON accumulation. D 2005 Elsevier B.V. All rights reserved. Keywords: Fusarium graminiarum; Mycotoxin; Water activity; Temperature; Growth; Two-dimensional profiles; Wheat grain; Fusarium species; Environmental factors; Trichothecenes; Colonisation
1. Introduction In Argentina, the main pathogen associated with Fusarium head blight (FHB) is Fusarium graminearum Schwabe, whose perfect stage is Gibberella zeae (Schwein) Petch. It has been detected in Argentina since 1928 and during the last 50 years, with 16 FHB epidemics of varying severity caused by F. graminearum occurring in the central-north area (Galich, 1996). In the last outbreak (1993) the highest estimated losses reached 50% in areas of no-till over maize stubble (Dalcero et al., 1997). The extent of the damage was magnified by a considerable loss in trading value of the grain due to low grain weight, presence of scabby grains, and mycotoxin contamina-
* Corresponding author. Tel.: +44 1525 863539; fax: +44 1525 863540. E-mail address: [email protected] (N. Magan). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.09.004
tion (Galich, 1996). During this severe epidemic, deoxynivalenol (DON) was the only toxin reported (Dalcero et al., 1997). DON, a member of the trichothecene group of mycotoxins, is primarily produced by the genus Fusarium (Sutton, 1982). The occurrence of DON in cereal grains is of concern, since this toxin results in feed refusal, vomiting and depressed immune functions in animals (WHO, 2001). Fungal growth and mycotoxin production results from the complex interaction of several factors and, therefore, an understanding of each factor involved is essential to understand the overall process and to predict and prevent mycotoxin development (Chamley et al., 1994). Environmental conditions have a major impact on the fungal growth and play a critical role in epidemiology of FHB and mycotoxin contamination. In addition, mycotoxin production is genetically regulated in response to environmental conditions (Holliger and Ekperigin, 1999). Temperature and water activity (a W) are the primary
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environmental factors that influence growth and mycotoxin production by several Fusarium species (Magan and Lacey, 1984; Marı´n et al., 1995a,b; Marı´n et al., 1996; Bakan et al., 2001; Hope and Magan, 2003). Ramirez et al. (2004a) showed that the type of water stress, whether caused by osmotic or matric forces, also impacts on the activity and colonisation of cereal-based substrates by strains of F. graminearum. Recently, Hope et al. (2005) compared growth and DON production by strains of Fusarium culmorum and F. graminearum from the UK and found that the main statistically significant difference was in relation to temperature effects on DON production. Prevention of mycotoxin contamination of food raw materials is now considered more important than subsequent cure. Thus hazard analysis critical control point (HACCP) approaches are being developed to examine the critical control points (CCPs) at which mycotoxicogenic moulds and mycotoxins may enter a range of food chains (Aldred and Magan, 2004). Accurate information is therefore needed on the impact of key environmental factors such as a W, temperature and their interactions, and on identifying marginal and optimum conditions for growth and toxin production (Sanchis and Magan, 2004). The aim of the present work was to determine the impact of a W, temperature and incubation time on growth and DON production on irradiated wheat grain by two strains of F. graminearum isolated from Argentinean wheat.
2.3. Inoculation, incubation and growth assessment
2. Materials and methods
Rehydrated wheat was place in sterile 9-cm Petri dishes to form a monolayer of grains (? 20 g). Then a 3-mm-diameter agar disk was taken from the margin of a 7-day-old growing colony of each isolate on synthetic nutrient agar (Gerlach and Nirenberg, 1982) at 25 -C and transferred face down to the centre of each plate. To maintain the correct equilibrium relative humidity inside the boxes, Petri plates containing grains of the same a W were enclosed in plastic containers together with two beakers of glycerol – water solution of the same a W as the treatments. Containers were incubated at 5, 15, 25 and 30 -C and the experiment consisted of a fully replicated set of treatments with three replicates per treatment and the whole set carried out twice. Grain layers were checked to ensure that the appropriate target a W (T 0.005) was achieved. Assessment of growth was made daily during the incubation period, with wheat grain cultures being examined using a binocular magnifier ( 10). Two diameters of the growing colonies were measured at right angles to each other until the colony reached the edge of the plate. The radii of the colonies were plotted against time, and a linear regression applied to obtain the growth rate as the slope of the line. Three complete Petri plate cultures per treatment were destructively sampled after 7, 14, 21, 28, 35, 42 or 49 days of incubation, dried at 50 -C for 24 h and stored at 20 -C until DON analysis was carried out.
2.1. Fungal strains
2.4. Deoxynivalenol analysis
Two strains of F. graminearum (RC 17-2 and RC 22-2) isolated from Argentinean wheat were used. Extensive studies have already been carried out with these strains in relation to efficacy of fungicides on growth and DON production (Ramirez et al., 2004b). These strains are deposited in the Universidad Nacional de Rio Cuarto, Argentina collection (RC). Cultures were maintained in 15% glycerol at 80 -C.
The DON analysis was done using a modified version of the procedure originally reported by Cooney et al. (2001). Each sample was finely ground and mixed well. A subsample (15 g) was extracted by mixing with acetonitrile/ methanol (14 : 1; 40 ml), shaken for 2 h and then filtered through Whatman No. 1 filter paper. A syringe was plugged with glass wool and dry-packed with alumina/carbon (20 : 1; 500 mg) to form a mini-cleanup column. A 2 ml aliquot of extract was applied to the column and allowed to drain under gravity and the eluant collected. The column was washed with acetonitrile/methanol/water (80 : 5 : 15; 500 Al), and the combined eluant was evaporated to dryness (N2, 50 -C). The cleaned-up residue was dissolved in methanol/water (5 : 95; 500 Al). The HPLC system consisted of a Gilson modular system with a Gilson UV detector. Chromatographic separations were performed on a Lunai C18 reversed-phase column (100 4.6 mm, 5 Am particle size) connected to a guard column SecurityGuardi (4 3.0 mm) filled with the same phase. The mobile phase consisted of methanol/water (12 : 88, v/v), at a flow rate of 1.5 ml min 1. The detector was set at 220 nm with an attenuation of 0.01 AUFS. Injection volume was 50 Al and the retention time of DON was 800 s. Quantification was relative to external standards of 1 to 4 Ag ml 1 in methanol/water (5 : 95). Recovery rate for replicate spiked blank wheat samples fortified with 0.5, 0.15 and 0.20 Ag g 1 of
2.2. Grain Wheat grain (14.5% moisture content, 1 kg batches) were gamma irradiated (10 – 12 kgray (kGy)) using a Cobalt radiation source (Isotron Ltd, Swindon, U.K.) and stored aseptically at 4 -C. The irradiated grain contained no microbial infection or mycotoxin contamination and had retained germinative capacity of about 75%, although respiration was reduced by about 30% and shoot length by about 65 –70% (Hamer, 1994). The initial a W of the grain was 0.766. Four hundred grams of irradiated wheat were weighed into sterile beakers and rehydrated to the required a W (0.995, 0.97, 0.95, 0.93 and 0.90) by addition of sterile distilled water using a moisture absorption curve. Flasks were subsequently refrigerated at 4 -C for 48 h with periodic shaking to allow absorption and equilibration. Finally, the a W levels were confirmed by using an Aqualab Series 3 water activity meter (Labcell Ltd., Basingstoke, Hants, UK).
M.L. Ramirez et al. / International Journal of Food Microbiology 106 (2006) 291 – 296
14
293
14
F. graminearum RC 17-2
13
0.98
12
12
10
11
0.96
10 9 8 7 5 6
6 0.94 4
4 3 2
2 0 14
0.92 0
F. graminearum RC 22-2
20
25
30
35
Water activity (aW)
Growth rate (mm day-1)
8
12 10 8 6 4
0.90
1
0.88 5
10
15
20
25
30
14
0.98 13
2
12
0.96
0 0
5
10
15
20
25
30
11
35
10 89
Temperature (°C) 0.94
˝
Fig. 1. Effect of a W, 0.90 (0) 0.93 (g), 0.95 (r), 0.97 (), 0.995 ( ), and temperature on growth rate of two strains of F. graminearum on irradiated wheat grains (n = 6).
4
0.92
0.90
1
0.88 5
10
15
3.1. Effect of a W temperature on growth Optimum growth at all a W levels was best at 25 -C with a maximum growth at the highest a W tested (0.995) (Fig. 1). Growth of both strains decreased as water availability of the grain was reduced. The conditions under which the same growth rates occurred were joined to produce contour lines which produce a map of the relative optimum and marginal rates of growth of the F. graminearum strains (see Magan and Lacey, 1984). At 0.93 a W, regardless of the temperature, growth was reduced by a factor of four. Both strains failed to grow at 5 -C at all the a W levels tested. Also, no growth was observed at 0.900 a W and 30 -C during the incubation period (Fig. 2).
20
25
30
Temperature (°C)
2.5. Statistical treatment of results
3. Results and discussion
3
2
DON was 83.2%, with a range 76 –100%. The quantification limit was 5 ng g 1. Where necessary samples were diluted and analysed again.
The linear regression of increase in radius against time (in days) was used to obtain the growth rates (mm day 1) under each set of treatment conditions. The growth rates and deoxynivalenol concentration were then evaluated by analysis of variance (ANOVA) using SigmaStat for Windows Version 2.03 (SPSS Inc.). Statistical significance was judged at the level P < 0.05.
6 7 5
Fig. 2. Contour maps for two strains of F. graminearum in relation to water activity and temperature. The numbers on the contour lines refers to growth rates (mm day 1; n = 6).
The analysis of variance of the effect of single (isolate, a W and temperature) two- and three-way interaction showed that all factors alone and all the interactions were statistically significant in relation to growth rates (Table 1). This is the first study comparing the impact of a W temperature regimes on rate of growth and DON production by Table 1 Analysis of variance of effect of water activity (a W), temperature (T), and different isolates (i), and their interactions on growth of Fusarium graminearum on irradiated wheat grain Source of variation
df a
MSb
Fc
aW T I aW T a W i T i a W T i
4 2 1 8 4 2 8
204.432 243.360 3.199 25.706 8.591 18.648 8.607
3085.893* 3673.502* 48.288* 388.029* 129.684* 281.484* 129.926*
a
Degrees of freedom. Mean square. c F-Snedecor. * Significant at P < 0.001 level. b
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strains of F. graminearum from Argentina when grown on irradiated wheat grain. Both factors affected mycelial extension of the strains and the pattern of effect of environmental factors on the strains was similar. Previous studies on F. graminearum from root and stalk rot of cereals have shown optimum water content and temperature for growth were 0.1 to 2.8 MPa (0.995 – 0.98 a W) at 20 –30 -C changing to 2.8 to 5.5 MPa (0.95 – 0.96 a W) at 35 -C. However, these isolates only grew if the water potential was 8.3 to 11.3 MPa (equivalent to 0.94 –0.92 a W at 25 -C) although incubation times were short (Cook and Christen, 1976; Wearing and Burgess, 1979). Periods of incubation as short as 7 days, as used in this previous study, may fail to detect growth at < 0.92 a W as we have demonstrated in the present study. Recent studies by Hope et al. (2005) suggest that growth of UK strains of F. graminearum do not grow below 0.90 a W although germination may occur at about 0.87 –0.88 a W. Also, Brennan et al. (2003) showed that the optimum temperature for in vitro growth of F. graminearum was 25 -C, although this study was carried out in media in which the a W was not controlled (= 0.995 a W), which makes direct comparisons difficult. Since Fusarium spp. may be present on a substrate for long periods during which a W may change, it is important to know both the optimal a W range for growth and that permitting sub-optimal growth. Nutrient source may also influence the minimum a W for growth (Griffin, 1972) and, consequently, studies on artificial substrates may not accurately reflect capabilities for growth on natural substrates (Magan and Lacey, 1984). The elimination of the natural microflora by irradiation without killing the seed is likely a better way to study the relationship between colonisation patterns and DON production than using artificial or autoclaved substrates (Lacey and Magan, 1991). Studies by Magan et al. (2003) and Cooney et al. (2001) have however demonstrated that the presence of other fungi such as Alternaria, Cladosporium and Micro-
dochium nivale can influence growth and trichothecene production on wheat grain. 3.2. Effect of a W, temperature and incubation time on deoxynivalenol production In general DON concentration increased as a W increased with no significant production at 0.93– 0.90 a W under all temperature levels tested (Table 2). Similarly, no DON was found in samples incubated at 5 -C for both isolates over the incubation period (49 days). Production occurred over a range of temperatures (15 – 30 -C) with optimum at 25 and 30 -C depending on the a W assayed. Maximum DON production was obtained for both strains at 0.995 a W, 30 -C, and 42 days of incubation (135,462 and 98,446 ng g 1). All single factors (a W, temperature and days of incubation) as well as two- and three-way interactions significantly influenced DON production for both strains. There was also a statistically significant difference under optimum conditions between the two strains (data not shown). The data for the two strains were used to develop further contour maps in order to identify the influence of a range of a W and temperature conditions on DON production (Fig. 3). In contrast to the results obtained for growth, these clearly identified 30 -C and 0.995 a W as the optimum conditions for maximum DON production. Previous studies with F. graminearum have also demonstrated the effect of either temperature or a W on DON production but the interactions between these parameters were not investigated (Vesonder et al., 1982; Greenhalgh et al., 1983; Comerio et al., 1999; Ryu and Bullerman, 1999; Martins and Martins, 2002; Lorens et al., 2004). Also, most of the work, except by Comerio et al. (1999), were done on strains of F. graminearum isolated from corn and using corn or rice as a substrate for DON production. The effects of temperature and time have been examined in some previous studies. For
Table 2 Mean deoxynivalenol concentration (ng g 1) on irradiated wheat grain at three water activities (a W) inoculated with two different strains of Fusarium graminearum at 15, 25 and 30 -C after 7, 14, 21, 28, 35, 42 and 49 days of incubation (Tstandard error of mean) Isolate
Incubation Deoxynivalenol (ng g (days) 0.995 a W 15 -C
RC 17-2 7 14 21 28 35 42 49 RC 22-2 7 14 21 28 35 42 49 a
Not detected.
a
nd nd 883 (T224) 523 (T234) 67,306 (T5794) 29,455 (T1980) 875 (T101) nd nd 1179 (T429) 486 (T27) 862 (T184) 348 (T17) nd
1
) 0.97 a W
0.95 a W
25 -C
30 -C
15 -C
25 -C
30 -C
15 -C
25 -C
30 -C
179 (T28) 262 (T36) 401 (T130) 1367 (T300) 1313 (T425) 2017 (T450) 1117 (T205) nd nd 61 (T32) 161 (T27) 865 (T93) 598 (T79) 210 (T62)
nd 448 (T 138) 133 (T 33) 6181 (T374) 10,937 (T419) 135,462 (T2857) 6610 (T570) nd 148 (T3) 757 (T25) 934 (T34) 1292 (T263) 98,446 (T2129) 78,333 (T1666)
nd nd 767 (T 80) 740 (T25) 1276 (T141) 714 (T214) 623 (T20) nd nd 994 (T161) 487 (T53) 492 (T123) 644 (T42) 594 (T24)
43 1538 (T232) 1272 (T370) 1897 (T497) 4367 (T1402) 4110 (T628) 7005 (T364) nd 294 (T74) 214 (T89) 5849 (T226) 16,501 (T 1088) 6345 (T918) 10,072 (T 904)
nd nd 92 (T2) 128 (T 31) 704 (T 66) 208 (T 6) nd nd nd 92 (T6) 101 (T 8) 506 (T 66) 146 (T 10) 7
nd nd nd nd 102 (T9) 146 (T22) 130 (T8) nd nd 34 (T3) 110 (T40) 361 (T43) 352 (T90) 70 (T21)
nd 430 (T 70) 543 (T68) 716 (T20) 1207 (T124) 3584 (T887) 3614 (T406) nd 146 (T81) 105 (T62) 760 (T31) 1473 (T120) 1140 (T234) 4923 (T324)
nd 132 (T16) 28 (T1) 91 (T17) nd nd nd nd nd 162(T69) 414 (T92) 117 (T15) 4277 (T195) 2294 (T302)
M.L. Ramirez et al. / International Journal of Food Microbiology 106 (2006) 291 – 296
30
A 20
0.98
10 0.5
1
2
3
4
5
0.96
0.94
Water activity (aW)
0.92
0.90 10
15
20
25
30
B
40
0.98
80 60
20 10
0.5 3
4
5
2
0.96
1
0.94
0.92
0.90 10
15
20
25
30
Temperature (°C) Fig. 3. Contour maps for two strains of F. graminearum in relation to water activity and temperature. The numbers on the contour lines refers to mean deoxynivalenol concentration (Ag g 1; n = 6).
example, Vesonder et al. (1982) demonstrated that a F. graminearum and a Fusarium roseum (= F. culmorum) strain produced DON optimally at 29 –30 and 25– 26 -C, respectively, on autoclaved cracked moist maize (30% water content = 0.99 a W) after 40 days. They found each strain only produced small amounts of DON at 15 and 20 -C. The minimum temperature for DON production by both species was about 11 -C, depending on time of incubation. Their contour maps were very useful but excluded interaction with water availability. Cycling of temperature can have a significant impact on both DON and NIV production. Studies by Ryu and Bullerman (1999), although using rice cultures, showed that temperature cycling of 15 and 30 -C over a 6-week period resulted in the highest biomass. However, steady-state incubation at 25 -C for about 2 weeks resulted in the highest DON and zearalenone (ZEA) production. There was a correlation between DON and ZEA production, but none between fungal biomass and production of either toxin. This suggests that environmental stress has an important influence on mycotoxin
295
production, often unrelated to total fungal biomass as shown in this study when maximum DON is produced at 30 -C and maximum growth is at 25 -C. Greenhalgh et al. (1983) studied the effects of the initial moisture content before autoclaving, incubation temperature, and time on DON production on rice by F. graminearum isolated from corn. They found that the higher incubation temperature (28 -C) favoured DON formation with the maximum amount being 515 ppm formed after 24 days at an initial moisture content of 40% (approx. 1.00 a W). Lorens et al. (2004) studied the ability of three isolates of F. graminearum to produce DON on autoclaved maize at different a W levels (0.96, 0.97 and 0.98) and at 15, 20, 28 and 32 -C for 21 days. In contrast to the present study, they found that a W did not significantly affect DON production; although temperature did. The highest levels of DON were reached at 28 -C, but no DON was detected at 32 -C and very little was observed at 15 and 20 -C. Martins and Martins (2002) compared the ability of a strain of F. graminearum from maize to produce DON on sterilized cracked corn at 0.97 a W and at 22 and 28 -C for 14 days followed by incubation at 12 and 37 -C for 8 weeks. They found that 22 and 28 -C after 35 days of incubation were the optimum culture conditions for DON production. This is close to the optimum temperature found in the present study. Comerio et al. (1999) studied the influence of a W (0.98, 0.945, 0.925 and 0.90) on DON accumulation at 25- on irradiated wheat grain by one strain of F. graminearum. They obtained the maximum amounts of DON at 0.98 a W. However, only a single temperature was used. In the present study, the knowledge of interacting environmental conditions provides very useful information indicative of the possible risk factor for DON contamination of wheat. The a W and temperature range used in this study simulated those occurring in ripening grain (Magan and Lacey, 1985) and harvested grain in a wet year (19 –30%; 0.90 –0.995 a W). The present study has also demonstrated the contrasting impact of a W, temperature and incubation time on growth and DON production by the two strains examined and that conditions for optimum growth and maximum toxin production are not the same. It has been shown that, among the tested values, the most suitable temperature for DON production is 30 -C. Knowledge of DON production under marginal or sub-optimal temperature and a W conditions for growth can be important since improper storage accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality. The profiles presented in the present study on growth and DON production may provide useful guidelines for assessing whether the weather conditions were conducive to growth and DON contamination during the critical anthesis period when the toxin is most likely to be formed. Acknowledgements Dr. M.L. Ramirez is grateful to Consejo Nacional de Investigaciones Cientificas y Tecnicas de la Republica Argen-
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Lorens, A., Mateo, R., Hinojo, M.J., Valle-Algarra, F.M., Jimenez, M., 2004. Influence of environmental factors on biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops. International Journal of Food Microbiology 94, 43 – 54. Magan, N., Lacey, J., 1984. Water relations of some Fusarium species from infected wheat ears and grain. Transactions of the British Mycological Society 83, 281 – 285. Magan, N., Lacey, J., 1985. The effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain. In: Lacey, J. (Ed.), Cereal Grain Mycotoxins, Fungi and Quality in Drying and Storage. Elsevier, Amsterdam, pp. 77 – 118. Magan, N., Hope, R., Cairns, V., Aldred, D., 2003. Post-harvest fungal ecology: impact of fungal growth and mycotoxin accumulation in stored grain. European Journal of Plant Pathology 109, 723 – 730. Marı´n, S., Sanchis, V., Vin˜as, I., Canela, R., Magan, N., 1995a. Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain. Letters in Applied Microbiology 21, 298 – 301. Marı´n, S., Sanchis, V., Magan, N., 1995b. Water activity, temperature and pH effects on growth of Fusarium moniliforme and Fusarium proliferatum isolates from maize. Canadian Journal of Microbiology 41, 1063 – 1070. Marı´n, S., Sanchis, V., Teixeido, R., Saenz, A.J., Ramos, A.J., Magan, N., 1996. Water activity, temperature relationships and microconidial germination of Fusarium moniliforme and Fusarium proliferatum from maize. Canadian Journal of Microbiology 42, 1045 – 1050. Martins, M.L., Martins, H.M., 2002. Effect of water activity, temperature and incubation time on the simultaneous production of deoxynivalenol and zearalenone in corn (Zea mays) by Fusarium graminearum. Food Chemistry 79, 315 – 318. Ramirez, M.L., Chulze, S., Magan, N., 2004a. Impact of environmental factors and fungicides on growth and deoxynivalenol production by Fusarium graminearum isolates from Argentinian wheat. Crop Protection 23, 117 – 125. Ramirez, M.L., Chulze, S.N., Magan, N., 2004b. Impact of osmotic and matric water stress on germination, growth, mycelial water potentials and endogenous accumulation of sugars and sugar alcohols by Fusarium graminearum. Mycologia 96, 470 – 478. Ryu, D., Bullerman, L.B., 1999. Effect of cycling temperatures on the production of deoxynivalenol and zearalenone by Fusarium graminearum NRRL 5883. Journal of Food Protection 62, 1451 – 1455. Sanchis, V., Magan, N., 2004. Environmental conditions affecting mycotoxins. In: Magan, N., Olsen, M. (Eds.), Mycotoxins in Food: Detection and Control. Woodhead Publishing Ltd, Oxford, pp. 174 – 189. Sutton, J.C., 1982. Epidemeology of wheat blight and maize ear rot caused by Fusarium graminearum. Canadian Journal of Plant Pathology 4, 195 – 209. Vesonder, R.F., Ellis, J.J., Kwolek, W.F., DeMarini, D.J., 1982. Production of vomitoxin on corn by Fusarium graminearum NRRL 5883 and Fusarium roseum NRRL 6101. Applied and Environmental Microbiology 43, 967 – 970. Wearing, A.H., Burgess, L.W., 1979. Water potential and the saprophytic growth of Fusarium roseum ‘graminearum’. Soil Biology and Biochemistry 11, 661 – 667. World Health Organization,, 2001. Deoxynivalenol. WHO Food Additives Series 47, FAO Food and Nutrition Paper 74. International Programme on Chemical Safety, World Health Organization, Geneva, pp. 419 – 556.
Temperature and water activity effects on growth and temporal deoxynivalenol production by two Argentinean strains of Fusarium graminearum on irradiated wheat grain Maria L. Ramirez a,b, Sofia Chulze b, Naresh Magan a,* a
Applied Mycology Group, Cranfield Biotechnology Centre, Cranfield University, Silsoe, Bedford MK45 4DT, UK b Departamento de Microbiologia e Inmunologia, Universidad Nacional de Rio Cuarto, Cordoba, Argentina Received 11 March 2005; received in revised form 18 July 2005; accepted 2 September 2005
Abstract The objective of this study was to determine the effects of water activity (a W; 0.900 – 0.995), temperature (5, 15, 25 and 30 -C), time of incubation (7 – 49 days) and their interactions on mycelial growth and deoxynivalenol (DON) production on irradiated wheat grain by two strains of Fusarium graminearum isolated from wheat ears in Argentina. Optimal a W levels for growth were in the range 0.950 – 0.995 with a temperature optima of 25 -C. Maximum growth rates were obtained at the highest a W (0.995) and 25 -C for both strains. No growth was observed at 5 -C regardless of the a W levels assayed. Both strains were able to growth at the lowest a W assayed (0.900), although the temperature ranges allowing growth at this minimal a W was 15 – 25 -C. DON was produced the most rapidly (7 days) when incubated at 25 -C and 0.995 a W. All other conditions required 7 – 14 days before DON was produced on grain. Maximum amounts of DON for both strains were produced at the highest a W treatment (0.995) after 6 weeks at 30 -C. The range of DON concentrations varied considerably (5 to 140,000 ng g 1) depending on a W and temperature interaction treatments. Production of DON occurred over a narrower range of a W (0.995 – 0.95) than that for growth (0.995 – 0.90). DON was more rapidly produced at 25 -C but the maximum amount produced was at 30 -C. Two-dimensional profiles of a W temperature were developed from these data to identify areas where conditions indicate a significant risk from DON accumulation. D 2005 Elsevier B.V. All rights reserved. Keywords: Fusarium graminiarum; Mycotoxin; Water activity; Temperature; Growth; Two-dimensional profiles; Wheat grain; Fusarium species; Environmental factors; Trichothecenes; Colonisation
1. Introduction In Argentina, the main pathogen associated with Fusarium head blight (FHB) is Fusarium graminearum Schwabe, whose perfect stage is Gibberella zeae (Schwein) Petch. It has been detected in Argentina since 1928 and during the last 50 years, with 16 FHB epidemics of varying severity caused by F. graminearum occurring in the central-north area (Galich, 1996). In the last outbreak (1993) the highest estimated losses reached 50% in areas of no-till over maize stubble (Dalcero et al., 1997). The extent of the damage was magnified by a considerable loss in trading value of the grain due to low grain weight, presence of scabby grains, and mycotoxin contamina-
* Corresponding author. Tel.: +44 1525 863539; fax: +44 1525 863540. E-mail address: [email protected] (N. Magan). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.09.004
tion (Galich, 1996). During this severe epidemic, deoxynivalenol (DON) was the only toxin reported (Dalcero et al., 1997). DON, a member of the trichothecene group of mycotoxins, is primarily produced by the genus Fusarium (Sutton, 1982). The occurrence of DON in cereal grains is of concern, since this toxin results in feed refusal, vomiting and depressed immune functions in animals (WHO, 2001). Fungal growth and mycotoxin production results from the complex interaction of several factors and, therefore, an understanding of each factor involved is essential to understand the overall process and to predict and prevent mycotoxin development (Chamley et al., 1994). Environmental conditions have a major impact on the fungal growth and play a critical role in epidemiology of FHB and mycotoxin contamination. In addition, mycotoxin production is genetically regulated in response to environmental conditions (Holliger and Ekperigin, 1999). Temperature and water activity (a W) are the primary
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environmental factors that influence growth and mycotoxin production by several Fusarium species (Magan and Lacey, 1984; Marı´n et al., 1995a,b; Marı´n et al., 1996; Bakan et al., 2001; Hope and Magan, 2003). Ramirez et al. (2004a) showed that the type of water stress, whether caused by osmotic or matric forces, also impacts on the activity and colonisation of cereal-based substrates by strains of F. graminearum. Recently, Hope et al. (2005) compared growth and DON production by strains of Fusarium culmorum and F. graminearum from the UK and found that the main statistically significant difference was in relation to temperature effects on DON production. Prevention of mycotoxin contamination of food raw materials is now considered more important than subsequent cure. Thus hazard analysis critical control point (HACCP) approaches are being developed to examine the critical control points (CCPs) at which mycotoxicogenic moulds and mycotoxins may enter a range of food chains (Aldred and Magan, 2004). Accurate information is therefore needed on the impact of key environmental factors such as a W, temperature and their interactions, and on identifying marginal and optimum conditions for growth and toxin production (Sanchis and Magan, 2004). The aim of the present work was to determine the impact of a W, temperature and incubation time on growth and DON production on irradiated wheat grain by two strains of F. graminearum isolated from Argentinean wheat.
2.3. Inoculation, incubation and growth assessment
2. Materials and methods
Rehydrated wheat was place in sterile 9-cm Petri dishes to form a monolayer of grains (? 20 g). Then a 3-mm-diameter agar disk was taken from the margin of a 7-day-old growing colony of each isolate on synthetic nutrient agar (Gerlach and Nirenberg, 1982) at 25 -C and transferred face down to the centre of each plate. To maintain the correct equilibrium relative humidity inside the boxes, Petri plates containing grains of the same a W were enclosed in plastic containers together with two beakers of glycerol – water solution of the same a W as the treatments. Containers were incubated at 5, 15, 25 and 30 -C and the experiment consisted of a fully replicated set of treatments with three replicates per treatment and the whole set carried out twice. Grain layers were checked to ensure that the appropriate target a W (T 0.005) was achieved. Assessment of growth was made daily during the incubation period, with wheat grain cultures being examined using a binocular magnifier ( 10). Two diameters of the growing colonies were measured at right angles to each other until the colony reached the edge of the plate. The radii of the colonies were plotted against time, and a linear regression applied to obtain the growth rate as the slope of the line. Three complete Petri plate cultures per treatment were destructively sampled after 7, 14, 21, 28, 35, 42 or 49 days of incubation, dried at 50 -C for 24 h and stored at 20 -C until DON analysis was carried out.
2.1. Fungal strains
2.4. Deoxynivalenol analysis
Two strains of F. graminearum (RC 17-2 and RC 22-2) isolated from Argentinean wheat were used. Extensive studies have already been carried out with these strains in relation to efficacy of fungicides on growth and DON production (Ramirez et al., 2004b). These strains are deposited in the Universidad Nacional de Rio Cuarto, Argentina collection (RC). Cultures were maintained in 15% glycerol at 80 -C.
The DON analysis was done using a modified version of the procedure originally reported by Cooney et al. (2001). Each sample was finely ground and mixed well. A subsample (15 g) was extracted by mixing with acetonitrile/ methanol (14 : 1; 40 ml), shaken for 2 h and then filtered through Whatman No. 1 filter paper. A syringe was plugged with glass wool and dry-packed with alumina/carbon (20 : 1; 500 mg) to form a mini-cleanup column. A 2 ml aliquot of extract was applied to the column and allowed to drain under gravity and the eluant collected. The column was washed with acetonitrile/methanol/water (80 : 5 : 15; 500 Al), and the combined eluant was evaporated to dryness (N2, 50 -C). The cleaned-up residue was dissolved in methanol/water (5 : 95; 500 Al). The HPLC system consisted of a Gilson modular system with a Gilson UV detector. Chromatographic separations were performed on a Lunai C18 reversed-phase column (100 4.6 mm, 5 Am particle size) connected to a guard column SecurityGuardi (4 3.0 mm) filled with the same phase. The mobile phase consisted of methanol/water (12 : 88, v/v), at a flow rate of 1.5 ml min 1. The detector was set at 220 nm with an attenuation of 0.01 AUFS. Injection volume was 50 Al and the retention time of DON was 800 s. Quantification was relative to external standards of 1 to 4 Ag ml 1 in methanol/water (5 : 95). Recovery rate for replicate spiked blank wheat samples fortified with 0.5, 0.15 and 0.20 Ag g 1 of
2.2. Grain Wheat grain (14.5% moisture content, 1 kg batches) were gamma irradiated (10 – 12 kgray (kGy)) using a Cobalt radiation source (Isotron Ltd, Swindon, U.K.) and stored aseptically at 4 -C. The irradiated grain contained no microbial infection or mycotoxin contamination and had retained germinative capacity of about 75%, although respiration was reduced by about 30% and shoot length by about 65 –70% (Hamer, 1994). The initial a W of the grain was 0.766. Four hundred grams of irradiated wheat were weighed into sterile beakers and rehydrated to the required a W (0.995, 0.97, 0.95, 0.93 and 0.90) by addition of sterile distilled water using a moisture absorption curve. Flasks were subsequently refrigerated at 4 -C for 48 h with periodic shaking to allow absorption and equilibration. Finally, the a W levels were confirmed by using an Aqualab Series 3 water activity meter (Labcell Ltd., Basingstoke, Hants, UK).
M.L. Ramirez et al. / International Journal of Food Microbiology 106 (2006) 291 – 296
14
293
14
F. graminearum RC 17-2
13
0.98
12
12
10
11
0.96
10 9 8 7 5 6
6 0.94 4
4 3 2
2 0 14
0.92 0
F. graminearum RC 22-2
20
25
30
35
Water activity (aW)
Growth rate (mm day-1)
8
12 10 8 6 4
0.90
1
0.88 5
10
15
20
25
30
14
0.98 13
2
12
0.96
0 0
5
10
15
20
25
30
11
35
10 89
Temperature (°C) 0.94
˝
Fig. 1. Effect of a W, 0.90 (0) 0.93 (g), 0.95 (r), 0.97 (), 0.995 ( ), and temperature on growth rate of two strains of F. graminearum on irradiated wheat grains (n = 6).
4
0.92
0.90
1
0.88 5
10
15
3.1. Effect of a W temperature on growth Optimum growth at all a W levels was best at 25 -C with a maximum growth at the highest a W tested (0.995) (Fig. 1). Growth of both strains decreased as water availability of the grain was reduced. The conditions under which the same growth rates occurred were joined to produce contour lines which produce a map of the relative optimum and marginal rates of growth of the F. graminearum strains (see Magan and Lacey, 1984). At 0.93 a W, regardless of the temperature, growth was reduced by a factor of four. Both strains failed to grow at 5 -C at all the a W levels tested. Also, no growth was observed at 0.900 a W and 30 -C during the incubation period (Fig. 2).
20
25
30
Temperature (°C)
2.5. Statistical treatment of results
3. Results and discussion
3
2
DON was 83.2%, with a range 76 –100%. The quantification limit was 5 ng g 1. Where necessary samples were diluted and analysed again.
The linear regression of increase in radius against time (in days) was used to obtain the growth rates (mm day 1) under each set of treatment conditions. The growth rates and deoxynivalenol concentration were then evaluated by analysis of variance (ANOVA) using SigmaStat for Windows Version 2.03 (SPSS Inc.). Statistical significance was judged at the level P < 0.05.
6 7 5
Fig. 2. Contour maps for two strains of F. graminearum in relation to water activity and temperature. The numbers on the contour lines refers to growth rates (mm day 1; n = 6).
The analysis of variance of the effect of single (isolate, a W and temperature) two- and three-way interaction showed that all factors alone and all the interactions were statistically significant in relation to growth rates (Table 1). This is the first study comparing the impact of a W temperature regimes on rate of growth and DON production by Table 1 Analysis of variance of effect of water activity (a W), temperature (T), and different isolates (i), and their interactions on growth of Fusarium graminearum on irradiated wheat grain Source of variation
df a
MSb
Fc
aW T I aW T a W i T i a W T i
4 2 1 8 4 2 8
204.432 243.360 3.199 25.706 8.591 18.648 8.607
3085.893* 3673.502* 48.288* 388.029* 129.684* 281.484* 129.926*
a
Degrees of freedom. Mean square. c F-Snedecor. * Significant at P < 0.001 level. b
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strains of F. graminearum from Argentina when grown on irradiated wheat grain. Both factors affected mycelial extension of the strains and the pattern of effect of environmental factors on the strains was similar. Previous studies on F. graminearum from root and stalk rot of cereals have shown optimum water content and temperature for growth were 0.1 to 2.8 MPa (0.995 – 0.98 a W) at 20 –30 -C changing to 2.8 to 5.5 MPa (0.95 – 0.96 a W) at 35 -C. However, these isolates only grew if the water potential was 8.3 to 11.3 MPa (equivalent to 0.94 –0.92 a W at 25 -C) although incubation times were short (Cook and Christen, 1976; Wearing and Burgess, 1979). Periods of incubation as short as 7 days, as used in this previous study, may fail to detect growth at < 0.92 a W as we have demonstrated in the present study. Recent studies by Hope et al. (2005) suggest that growth of UK strains of F. graminearum do not grow below 0.90 a W although germination may occur at about 0.87 –0.88 a W. Also, Brennan et al. (2003) showed that the optimum temperature for in vitro growth of F. graminearum was 25 -C, although this study was carried out in media in which the a W was not controlled (= 0.995 a W), which makes direct comparisons difficult. Since Fusarium spp. may be present on a substrate for long periods during which a W may change, it is important to know both the optimal a W range for growth and that permitting sub-optimal growth. Nutrient source may also influence the minimum a W for growth (Griffin, 1972) and, consequently, studies on artificial substrates may not accurately reflect capabilities for growth on natural substrates (Magan and Lacey, 1984). The elimination of the natural microflora by irradiation without killing the seed is likely a better way to study the relationship between colonisation patterns and DON production than using artificial or autoclaved substrates (Lacey and Magan, 1991). Studies by Magan et al. (2003) and Cooney et al. (2001) have however demonstrated that the presence of other fungi such as Alternaria, Cladosporium and Micro-
dochium nivale can influence growth and trichothecene production on wheat grain. 3.2. Effect of a W, temperature and incubation time on deoxynivalenol production In general DON concentration increased as a W increased with no significant production at 0.93– 0.90 a W under all temperature levels tested (Table 2). Similarly, no DON was found in samples incubated at 5 -C for both isolates over the incubation period (49 days). Production occurred over a range of temperatures (15 – 30 -C) with optimum at 25 and 30 -C depending on the a W assayed. Maximum DON production was obtained for both strains at 0.995 a W, 30 -C, and 42 days of incubation (135,462 and 98,446 ng g 1). All single factors (a W, temperature and days of incubation) as well as two- and three-way interactions significantly influenced DON production for both strains. There was also a statistically significant difference under optimum conditions between the two strains (data not shown). The data for the two strains were used to develop further contour maps in order to identify the influence of a range of a W and temperature conditions on DON production (Fig. 3). In contrast to the results obtained for growth, these clearly identified 30 -C and 0.995 a W as the optimum conditions for maximum DON production. Previous studies with F. graminearum have also demonstrated the effect of either temperature or a W on DON production but the interactions between these parameters were not investigated (Vesonder et al., 1982; Greenhalgh et al., 1983; Comerio et al., 1999; Ryu and Bullerman, 1999; Martins and Martins, 2002; Lorens et al., 2004). Also, most of the work, except by Comerio et al. (1999), were done on strains of F. graminearum isolated from corn and using corn or rice as a substrate for DON production. The effects of temperature and time have been examined in some previous studies. For
Table 2 Mean deoxynivalenol concentration (ng g 1) on irradiated wheat grain at three water activities (a W) inoculated with two different strains of Fusarium graminearum at 15, 25 and 30 -C after 7, 14, 21, 28, 35, 42 and 49 days of incubation (Tstandard error of mean) Isolate
Incubation Deoxynivalenol (ng g (days) 0.995 a W 15 -C
RC 17-2 7 14 21 28 35 42 49 RC 22-2 7 14 21 28 35 42 49 a
Not detected.
a
nd nd 883 (T224) 523 (T234) 67,306 (T5794) 29,455 (T1980) 875 (T101) nd nd 1179 (T429) 486 (T27) 862 (T184) 348 (T17) nd
1
) 0.97 a W
0.95 a W
25 -C
30 -C
15 -C
25 -C
30 -C
15 -C
25 -C
30 -C
179 (T28) 262 (T36) 401 (T130) 1367 (T300) 1313 (T425) 2017 (T450) 1117 (T205) nd nd 61 (T32) 161 (T27) 865 (T93) 598 (T79) 210 (T62)
nd 448 (T 138) 133 (T 33) 6181 (T374) 10,937 (T419) 135,462 (T2857) 6610 (T570) nd 148 (T3) 757 (T25) 934 (T34) 1292 (T263) 98,446 (T2129) 78,333 (T1666)
nd nd 767 (T 80) 740 (T25) 1276 (T141) 714 (T214) 623 (T20) nd nd 994 (T161) 487 (T53) 492 (T123) 644 (T42) 594 (T24)
43 1538 (T232) 1272 (T370) 1897 (T497) 4367 (T1402) 4110 (T628) 7005 (T364) nd 294 (T74) 214 (T89) 5849 (T226) 16,501 (T 1088) 6345 (T918) 10,072 (T 904)
nd nd 92 (T2) 128 (T 31) 704 (T 66) 208 (T 6) nd nd nd 92 (T6) 101 (T 8) 506 (T 66) 146 (T 10) 7
nd nd nd nd 102 (T9) 146 (T22) 130 (T8) nd nd 34 (T3) 110 (T40) 361 (T43) 352 (T90) 70 (T21)
nd 430 (T 70) 543 (T68) 716 (T20) 1207 (T124) 3584 (T887) 3614 (T406) nd 146 (T81) 105 (T62) 760 (T31) 1473 (T120) 1140 (T234) 4923 (T324)
nd 132 (T16) 28 (T1) 91 (T17) nd nd nd nd nd 162(T69) 414 (T92) 117 (T15) 4277 (T195) 2294 (T302)
M.L. Ramirez et al. / International Journal of Food Microbiology 106 (2006) 291 – 296
30
A 20
0.98
10 0.5
1
2
3
4
5
0.96
0.94
Water activity (aW)
0.92
0.90 10
15
20
25
30
B
40
0.98
80 60
20 10
0.5 3
4
5
2
0.96
1
0.94
0.92
0.90 10
15
20
25
30
Temperature (°C) Fig. 3. Contour maps for two strains of F. graminearum in relation to water activity and temperature. The numbers on the contour lines refers to mean deoxynivalenol concentration (Ag g 1; n = 6).
example, Vesonder et al. (1982) demonstrated that a F. graminearum and a Fusarium roseum (= F. culmorum) strain produced DON optimally at 29 –30 and 25– 26 -C, respectively, on autoclaved cracked moist maize (30% water content = 0.99 a W) after 40 days. They found each strain only produced small amounts of DON at 15 and 20 -C. The minimum temperature for DON production by both species was about 11 -C, depending on time of incubation. Their contour maps were very useful but excluded interaction with water availability. Cycling of temperature can have a significant impact on both DON and NIV production. Studies by Ryu and Bullerman (1999), although using rice cultures, showed that temperature cycling of 15 and 30 -C over a 6-week period resulted in the highest biomass. However, steady-state incubation at 25 -C for about 2 weeks resulted in the highest DON and zearalenone (ZEA) production. There was a correlation between DON and ZEA production, but none between fungal biomass and production of either toxin. This suggests that environmental stress has an important influence on mycotoxin
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production, often unrelated to total fungal biomass as shown in this study when maximum DON is produced at 30 -C and maximum growth is at 25 -C. Greenhalgh et al. (1983) studied the effects of the initial moisture content before autoclaving, incubation temperature, and time on DON production on rice by F. graminearum isolated from corn. They found that the higher incubation temperature (28 -C) favoured DON formation with the maximum amount being 515 ppm formed after 24 days at an initial moisture content of 40% (approx. 1.00 a W). Lorens et al. (2004) studied the ability of three isolates of F. graminearum to produce DON on autoclaved maize at different a W levels (0.96, 0.97 and 0.98) and at 15, 20, 28 and 32 -C for 21 days. In contrast to the present study, they found that a W did not significantly affect DON production; although temperature did. The highest levels of DON were reached at 28 -C, but no DON was detected at 32 -C and very little was observed at 15 and 20 -C. Martins and Martins (2002) compared the ability of a strain of F. graminearum from maize to produce DON on sterilized cracked corn at 0.97 a W and at 22 and 28 -C for 14 days followed by incubation at 12 and 37 -C for 8 weeks. They found that 22 and 28 -C after 35 days of incubation were the optimum culture conditions for DON production. This is close to the optimum temperature found in the present study. Comerio et al. (1999) studied the influence of a W (0.98, 0.945, 0.925 and 0.90) on DON accumulation at 25- on irradiated wheat grain by one strain of F. graminearum. They obtained the maximum amounts of DON at 0.98 a W. However, only a single temperature was used. In the present study, the knowledge of interacting environmental conditions provides very useful information indicative of the possible risk factor for DON contamination of wheat. The a W and temperature range used in this study simulated those occurring in ripening grain (Magan and Lacey, 1985) and harvested grain in a wet year (19 –30%; 0.90 –0.995 a W). The present study has also demonstrated the contrasting impact of a W, temperature and incubation time on growth and DON production by the two strains examined and that conditions for optimum growth and maximum toxin production are not the same. It has been shown that, among the tested values, the most suitable temperature for DON production is 30 -C. Knowledge of DON production under marginal or sub-optimal temperature and a W conditions for growth can be important since improper storage accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality. The profiles presented in the present study on growth and DON production may provide useful guidelines for assessing whether the weather conditions were conducive to growth and DON contamination during the critical anthesis period when the toxin is most likely to be formed. Acknowledgements Dr. M.L. Ramirez is grateful to Consejo Nacional de Investigaciones Cientificas y Tecnicas de la Republica Argen-
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