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Inactivation of conidia of Botrytis cinerea and Monilinia fructigena using UV-C and heat treatment
International Journal of Food Microbiology 74 (2002) 27 – 35 www.elsevier.com/locate/ijfoodmicro
Inactivation of conidia of Botrytis cinerea and Monilinia fructigena using UV-C and heat treatment D. Marquenie a,*, J. Lammertyn a, A.H. Geeraerd b, C. Soontjens c, J.F. Van Impe b, B.M. Nicolaı¨ a, C.W. Michiels c a
Laboratory/Flanders Centre of Postharvest Technology, Katholieke Universiteit Leuven, W. de Croylaan 42, B-3001 Leuven, Belgium b BioTeC-Bioprocess Technology and Control, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium c Laboratory of Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium Received 24 January 2001; received in revised form 20 August 2001; accepted 4 October 2001
Abstract The effect of UV-C (k = 254 nm) and heat treatment was investigated on the inactivation of conidia of Botrytis cinerea and Monilinia fructigena, two major postharvest spoilage fungi of strawberries and cherries, respectively. Both fungi were grown at 21 C in the dark and conidia were isolated after 1 week by washing the mycelium with a mild detergent solution. After filtration and resuspension in phosphate buffer to a titer of 105 to 106 cfu/ml, the conidia were subjected to different treatments. The applied UV-C doses varied from 0.01 to 1.50 J/cm2, and the conditions for the thermal treatment were 3, 5, 10, 15 and 20 min at temperatures ranging from 35 to 48 C. Both techniques were applied individually and in combination. Spore inactivation increased with increasing intensity of single treatments. No surviving spores of B. cinerea were observed after 15 min at 45 C or an UV-C treatment of 1.00 J/cm2. M. fructigena was more sensitive and a thermal treatment of 3 min at 45 C or an UV-C treatment of 0.50 J/cm2 resulted in complete spore inactivation. Combination of both techniques reduced the required intensity of the treatment for inactivation of both fungi. The order of the applications had a significant effect on the degree of inactivation. The inactivation of B. cinerea conidia was greater when the heat treatment came first, and for M. fructigena, most inactivation was achieved when the heat treatment was preceded with an UV-C irradiation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Inactivation; UV-C; Heat; Botrytis cinerea; Monilinia fructigena
1. Introduction Fungal development on fruit and vegetables during storage and transport is a major cause of postharvest losses. Important fungi are Botrytis cinerea (many fruit and vegetable species) and Monilinia fructigena (stone * Corresponding author. Tel.: +32-16-32-2376; fax: +32-16-322955. E-mail address: [email protected] (D. Marquenie).
and pip fruit). A common procedure for reducing postharvest rot is the use of chemicals. However, many fungal strains are developing resistance to the most widely used fungicides, and a growing aversion is developing among consumers against the use of chemicals in fresh produce. As a result, the legislation in most countries is becoming more severe. This has led to an increasing need for alternative techniques for fruit disinfection, which do not leave any residue. Thermal treatment and ultraviolet irradiation have been proposed as possible methods for surface dis-
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infection of small fruits (Phillips and Barkai-Golan, 1991; Islam et al., 1998; De Cal and Melgarejo, 1999). In vitro studies have shown that vegetative cells and conidia of most fungi are inactivated when exposed to a temperature of 60 C for 5 to 10 min (Baker and Smith, 1969; Agrios, 1988; Civello et al., 1997). However, for surface decontamination of fresh fruit, the thermal treatment should be less severe to avoid loss of turgor and quality in general. Typically, a temperature below 55 C is applied during a relatively short time (several minutes). For strawberries, Garcı´a et al. (1995) used a treatment of 15 min at 44 to 48 C. These mild temperatures are effective, suggesting that either the fungi are less heat resistant on the surface of the fruit than in the buffers used in laboratory experiments, or that a heat shock response is induced in the fruit which increases resistance against fungi. The decontaminating properties of UV-C light are well known, but the potential of ultraviolet light for surface disinfection of fruit and vegetables has been studied only recently (Aylor and Sanogo, 1997; Ranganna et al., 1997; Islam et al., 1998; Stevens et al., 1998). In addition to its lethal effect on the fungal pathogen (Nigro et al., 1998; Shaffer et al., 1999), low doses of UV-C were also reported to induce a physiological reaction in the exposed product, inhibiting the fungal development (Stevens et al., 1998). The objective of this study was to investigate the inactivation properties of mild thermal treatments and exposure to UV-C light on conidia of B. cinerea and M. fructigena. Different time – temperature conditions and UV-C doses were screened, both individually and in combination. Combinations of treatments sometimes act synergistically, which decreases the required intensity of the individual treatments to achieve a certain inactivation level. Synergistic effects have been observed when combining a thermal treatment with anoxia (Bussel et al., 1970) or g-irradiation (Sommer et al., 1966). So far, the combined effect of heat and UV-C has not been reported.
2. Materials and methods 2.1. Production of conidia The fungal cultures used for this study were B. cinerea MUCL 18864 obtained from the BCCM2/
MUCL collection (Mycothe`que de l’Universite´ catholique de Louvain, Louvain-la-Neuve, Belgium) and M. fructigena CBS 101499 from the CBS collection (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands). A suspension of sporulating fungal material in 25% glycerol was stored at 80 C as a stock culture and was used to start new cultures. The fungi were grown at 21 C in the dark on potato dextrose agar (PDA, Oxoid, Hampshire, UK) for B. cinerea or V8 agar for M. fructigena. These media were found to support good growth and sporulation for both fungi. After 7 days, the cultures were subcultured by transferring mycelium fragments to fresh nutrient medium. Seven days later, the conidia were harvested by washing the surface of the plate with 10 ml detergent solution (3 g/l Tween-20). This suspension was filtered through a column with a plug of glass wool to remove large mycelium fragments and then centrifuged to replace the detergent by a volume of 10 mM phosphate buffer (pH 7.2) so that a concentration of 105 to 106 spores ml 1 was obtained. Spore titers were determined microscopically using a Thoma counting chamber and spores were used immediately for the inactivation experiments. 2.2. Heat treatment To investigate the effect of a thermal treatment on the inactivation of B. cinerea and M. fructigena conidia, spore suspensions were exposed to different temperature –time combinations. The temperatures were 40, 43, 45 and 48 C for B. cinerea. For the preliminary experiments with M. fructigena, the same conditions as for B. cinerea were used. Since no surviving spores were observed at 48 C, the temperature range was reduced and smaller temperature intervals were chosen (39, 41, 43 and 45 C). The duration of the treatment was 3, 5, 10 or 15 min. Inactivation was determined by surface plating treated and control (untreated) spores. For the thermal treatment, 100 ml of spore suspension was transferred into a glass capillary (1.55 mm diameter, 150 mm length). Both ends were sealed in a flame, leaving a sufficient air column to avoid heating of the spore suspension, and the capillary was immersed at a preset temperature in a circulating water bath (Grant Instruments, Cambridge, UK). Immediately after treatment, the capillaries were cooled to 20 C in water, aseptically opened and the suspension was
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serially diluted and plated onto the counting medium, respectively, rose bengal agar (RBA, Oxoid) for B. cinerea, and malt extract agar (MEA, Oxoid) for M. fructigena. These media were chosen because they resulted in the formation of discrete colonies that could readily be counted. The plates were incubated at 25 C during 5 days and colonies were counted.
violet light had no significant influence on spore inactivation. The values used in this series of experiments were based on the results obtained for the individual methods. The temperature ranges chosen for these combined treatments were 40 to 48 C for B. cinerea and 35 to 43 C for M. fructigena, and the three UV levels, 0.00, 0.05 and 0.10 J/cm2.
2.3. UV-C treatment
2.5. Data analysis
The UV radiation used for these experiments is the short-wave band or UV-C, running from 180 to 280 nm with a peak at 254 nm (the other parts being UV-B and UV-A running, respectively, from 280 to 320 nm and from 320 to 380 nm). The UV treatment cabinet used in this work (Bio-Link, Vilber Lourmat, France) was equipped with five fluorescent lamps of 8 W each (k = 254 nm), and a radiometer that permanently measured the emitted UV energy, allowing the user to program energy levels. The effect of UV treatment on spore viability and the evolution of the inactivation as function of the UV dose was studied by exposing conidia to varying UV-C doses, ranging from 0.01 to 1.50 J/cm2 (corresponding with a treatment duration of 4 to 600 s). For simple UV treatment, the spore suspension was serially diluted, 100 ml solution was plated on RBA (B. cinerea) or MEA (M. fructigena) and exposed to the different UV-C doses. The distance between the lamps and the nutrient plates was 14 cm. The plates were incubated at 25 C during 5 days and colonies were counted. In a preliminary experiment, the possible influence of an UV-illumination on the used nutrient medium and the subsequent effect on spore development was investigated. No differences between RBA and PDA for B. cinerea and between MEA and V8 agar for M. fructigena were observed, so RBA and MEA were used for the experiments.
Spore inactivation was expressed using a logarithmic reduction factor log N/No, with No the number of developing spores before treatment and N the number of developing spores after treatment. The data from the combination experiments were analysed using analysis of variance (ANOVA). This technique has the advantage that there is no assumption required about the nature of the statistical relation between the dependent (spore inactivation) and the independent variables (temperature, treatment duration, UV dose, treatment sequence), and that the independent variables in ANOVA models may be qualitative (Neter et al., 1990). All main effects and interaction effects were tested with an F test statistic at a 5% significance level. All statistical analyses in this report were performed using the SAS software, version 6.12 (SAS Institute, Cary, NC.).
2.4. Combined treatments In these experiments, the techniques were combined in both sequences to examine possible interactions. When UV illumination preceded heating of the conidia, 3 ml suspension was first transferred onto a watchglass for UV treatment, and then transferred in glass capillaries for the thermal treatment. In the reverse order, the procedures were followed as described previously. The way of exposing the conidia suspension to the ultra-
2.6. Inactivation modelling To describe the thermal inactivation of the conidia as a function of time, a classical first order inactivation model was used: dN ¼ kN dt
ð1Þ
where N is the number of surviving spores after treatment [cfu/ml], t is the time [min] and k is an inactivation rate [1/min]. The inactivation can in an analogous way be described with the D value [min], this is the Thermal Death Time (TDT) model (Bigelow, 1921). The D value gives the time required to decrease the number of surviving conidiospores with one logarithmic unit at a constant temperature. The D value is related to the k value by the equation D=(ln 10)/k. The z value (C) is the temperature increase required to have a 10-fold decrease of the D value. This
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relationship between the D and z values is described by this equation: D ¼ Dref 10ðTref T Þ=z
ð2Þ
The relation between the inactivation rate k and the temperature T was described with the Arrhenius equation: k ¼ kref e
Ea R
1 1 Tref T
ð3Þ
In this equation, Ea is the Arrhenius activation energy [J/mol], R is the universal gas constant [J/ mol K] and kref is the inactivation rate [1/min] at the reference temperature Tref [K]. At a constant temperature, the solution of Eq. (1) is given by: N ln ¼ kt ð4Þ No The inactivation by means of UV-C radiation was also modelled with first-order kinetics, but with the UV dose as independent variable. The dimensions of the corresponding D value are J/cm2. The estimation of the parameters Ea and kref was carried out using non-linear regression of Eqs. (3) and (4) to the inactivation data. The Marquardt method was used for the non-linear least square iterative computing (SAS 6.12., SAS Institute, Cary, NC.).
3. Results 3.1. Heat treatment Spore inactivation increased with increasing temperature and treatment duration, and could be described using the first-order inactivation model combined with the Arrhenius equation. The reference temperature was 43 C. For B. cinerea, the activation energy Ea was 407 kJ/mol and the z value was 4.65 C. The D values for
Fig. 1. Inactivation of conidia of B. cinerea as a function of temperature (C) and treatment duration (min). Legend: model predictions ( – : 40 C, . . .: 43 C, _._: 45 C and —: 48 C), and observed values ( 6: 40 C, : 43 C, r: 45 C and : 48 C).
the different temperatures are given in Table 1. The observed values and the predicted values are plotted in Fig. 1. No surviving spores were observed after 15 min at 45 C and all treatments at 48 C. In the case of M. fructigena, the Arrhenius activation energy Ea was 443 kJ/mol, and the z value was 4.17 C. The D values for the different temperatures are given in Table 2. The observed and the fitted values for M. fructigena are presented in Fig. 2. Conidia of M. fructigena were more sensitive to heat than those of B. cinerea: the D values were lower for the same temperature (e.g. for B. cinerea, D43 C = 6.78 min and for M. fructigena, D43 C = 2.49 min). Complete spore inactivation was already achieved upon treatment at 43 C during 15 min and during 5 min at 45 C. 3.2. UV-C treatment The number of surviving conidia decreased loglinearly with increasing UV-C dose. As such, spore
Table 1 The D values for B. cinerea at different temperatures
Table 2 The D values for M. fructigena at different temperatures
Temperature (C)
D value (min)
Temperature (C)
D value (min)
40 43 45 48
29.959 6.782 2.559 0.607
39 41 43 45
21.697 7.302 2.492 0.862
D. Marquenie et al. / International Journal of Food Microbiology 74 (2002) 27–35
Fig. 2. Inactivation of conidia of M. fructigena as a function of temperature (C) and treatment duration (min). Legend: model predictions ( – : 39 C, . . .: 41 C, _._: 43 C and —: 45 C), and observed values (6: 39 C, : 41 C, r: 43 C and : 45 C).
inactivation could be described using only the firstorder model, since the inactivation rate seemed to be constant for the different UV-C doses. For B. cinerea, the D value was 0.026 J/cm2, and for M. fructigena, this D value was 0.016 J/cm2. The observed and the fitted inactivation data for both fungi are given in Fig. 3. In this figure, values lying beneath the detection limit are not represented, since they do not add any significant information for the description of the inactivation process. Furthermore, these values might suggest the presence of a resistant subpopulation causing a tailing effect, and this is most probably not the case. As was the case for heat, M. fructigena was also more sensitive to UV illumination than B. cinerea: the doses at which no surviving spores were found were 0.50 and 1.00 J/cm2, respectively. The lower D value for M. fructigena reflects that the treatment time needed to achieve a reduction in number of surviving conidia of one logarithmic unit is shorter than for B. cinerea (using the same UV-C dose). For this reason, smaller intervals were chosen between 0.01 and 0.05 J/cm2.
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interactions, an ANOVA was applied. For B. cinerea, all main effects (temperature, duration, UV dose and sequence) and two interaction effects (temperature duration, sequence UV dose) were found to be significant. Since the parameters ‘‘temperature’’ and ‘‘duration’’ are linked to the same treatment, the interaction between both is obvious. The interaction between the parameters ‘‘UV dose’’ and ‘‘sequence’’ can be explained as a change in inactivation efficiency of UV upon inversion of the treatment sequence. An increase in UV-C dose will cause a larger inactivation when the thermal treatment precedes the UV treatment. The remaining second-order interaction terms and all higher-order interaction terms had no significant effect. A graphical representation of the different temperature – time combinations required for spore inactivation for the three UV-C doses is given in Fig. 4. In this figure, the three graphs on the left side represent the inactivation when the UV treatment comes first. The temperatures needed for a certain degree of inactivation at UV = 0.00 J/cm2 (the results for the single temperature treatment) are higher than for the combinations with 0.05 and 0.10 J/cm2. The highest UV dose that was used in these combinations was 10 times lower than the dose required for inactivation when only UV-C was used.
3.3. Combined treatments To investigate the importance of the different parameters involved (temperature, treatment duration, UV dose and treatment sequence) and their possible
Fig. 3. Inactivation of conidiospores of B. cinerea and M. fructigena as a function of UV-dose (J/cm2). Legend: model predictions ( – ) and observed values (6: B. cinerea, : M. fructigena).
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Fig. 4. Effect of a combined heat and UV treatment on inactivation of conidiospores of B. cinerea. The lines give time – temperature combinations with equal inactivation (no inactivation: white, complete inactivation: black), each line represents a change in inactivation of 12.5%. The temperatures are ranging from 40 to 48 C. For each UV-dose (0.00, 0.05 and 0.10 J/cm2), both treatment sequences are represented, while for UV-dose 0.00 J/cm2, the sequence is not relevant.
The influence of the same parameters was also studied for M. fructigena. Each parameter had a significant effect on inactivation since all main effects were retained in the ANOVA results. Only for the parameter ‘‘sequence’’, the main effect was not significant, but since this parameter was present in two interaction terms (sequence temperature, sequence UV dose temperature), the main effect had to be conserved due to the hierarchical principle (Verbeke, 1997). The interaction between temperature and treatment duration is again present, together with the second- and third-order interactions with the parameter ‘‘UV dose’’ (UV dose temperature, UV dose duration, UV dose temperature duration). The effects of the treatment sequence on, respec-
tively, the UV and the thermal treatments are described by the second-order interaction terms sequence UV dose and sequence temperature. Also, a third-order interaction between these three parameters is present. For M. fructigena, the largest inactivation was obtained when the UV treatment comes first. The resulting inactivation is represented as a function of time and temperature for each UV-C dose in Fig. 5. Total inactivation was achieved faster or at lower temperature when heat treatment was preceded by an UV-C treatment. The treatment duration had a clear effect when no UV was used, but this influence was reduced in the other cases. A possible explanation for this is that a thermal treatment with a low duration
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Fig. 5. Effect of a combined heat and UV treatment on inactivation of conidiospores of M. fructigena. The lines give time – temperature combinations with equal inactivation (no inactivation: white, complete inactivation: black), each line represents a change in inactivation of 12.5%. The temperatures are ranging from 35 to 43 C. For each UV-dose (0.00, 0.05 and 0.10 J/cm2), both treatment sequences are represented, while for UV-dose 0.00 J/cm2, the sequence is not relevant.
already has a great inactivation effect when combined with UV-C.
4. Discussion It has been reported that a 5 log unit inactivation of M. fructigena conidia was achieved by a thermal treatment in water at 52 C during 2 min (Baker and Smith, 1969). The Arrhenius activation energy values calculated in the present research for B. cinerea and M. fructigena (resp. 407 and 443 kJ/mol) are analogous to values obtained for other fungi like Colletotrichum gloeosoporiodes and Guignardia psidii (resp. 447 and 418 kJ/mol) (Chan et al., 1996). In our own
work, complete inactivation was achieved at lower temperatures (45 and 48 C), but the duration of the treatment was longer (resp. 15 and 5 min). UV-C has been reported to change the permeability of the cell, with leakage of electrolytes, amino acids and carbohydrates, and polysaccharide accumulation as a consequence (Nigro et al., 1998). De Cal and Melgarejo (1999) reported also the inhibition of mycelial growth of Monilinia spp. after long-wave (320 –380 nm) UV exposure. In our experiments, no surviving spores were observed after the exposure of a suspension of 105 – 106 cfu/ml of M. fructigena to a dose of 1.00 J/cm2. This UV level was reduced to 0.10 J/cm2 when combined with thermal treatment. Using lower doses is advantageous because it reduces the possible damage to treated
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food products. Relatively low doses of UV-C can also induce hormesis in diverse horticultural products, a physiological reaction leading to the induction of an increased resistance to pathogens and, in this way, reduce the development of storage rots (Stevens et al., 1998). It is important, however, that the development of infection hyphae as a result of negative phototropic reaction of conidial germtubes is not promoted (Islam et al., 1998). So low doses can be used in combination with other methods provided that all spores are inactivated as a result of the second treatment. Combination of a thermal treatment with a second method was described by different authors. Bussel et al. (1970) combined heat and anoxia (absence of oxygen) to inactivate spores of M. fructicola and Sommer et al. (1966) used g-irradiation and heat for inactivation of Rhizopus stolonifer and B. cinerea. The sequence of the treatments influenced the inactivation of the microorganisms. When anoxia preceded heat, the observed total inactivation was lower than expected in case of an additive effect, and Bussel et al. (1970) concluded that anoxia rendered the spores less sensitive to subsequent heat treatment. These data are comparable with the results Sommer et al. (1966) obtained for B. cinerea. The inactivation was higher when heat treatment was followed by g-irradiation than vice-versa, leading to the conclusion that a thermal treatment increased the susceptibility for irradiation. A comparable reaction was observed in this work for B. cinerea, with an increased inactivation when UV irradiation was preceded by the thermal treatment. On the other hand, the inactivation of R. stolonifer was greater when the spores were irradiated prior to the heat treatment. These results agree with our observations on conidia of M. fructigena, where a greater inactivation is obtained when the UV treatment comes first. An UV irradiation followed by a thermal treatment at 41 C resulted in the complete inactivation of M. cinerea spores, whereas a higher temperature was necessary in the reverse order. For B. cinerea, 3 min at 45 C followed by an exposure to 0.05 J/cm2 results in complete inactivation, while in the reverse sequence, a temperature of 48 C was needed.
5. Conclusion Heat treatment and UV-C irradiation were used for the inactivation of conidia of B. cinerea and M.
fructigena, both individually and in combination. The conidia of M. fructigena were more sensitive to heat and UV-C treatment than those of B. cinerea. Combining different methods was appropriate to reduce the intensity of the treatment needed to achieve the inactivation of conidia. The effect of treatment sequence and intensity depended on the organism. The highest inactivation was obtained when the heat treatment preceded the UV-C treatment for B. cinerea, and when the heat treatment followed an UV treatment for M. fructigena. Acknowledgements This research is supported by the Ministry of Small Enterprises, Traders and Agriculture, Directorate of Research and Development (Project S-5856). The research at the Flanders Centre of Postharvest Technology is supported by the Flemish Government. References Agrios, G.N., 1988. Plant Pathology, 3rd edn. Academic Press, San Diego, CA. Aylor, D.E., Sanogo, S., 1997. Germinability of Venturia inaequalis conidia exposed to sunlight. Phytopathology 87, 628 – 633. Baker, K.F., Smith, W.L., 1969. Heat-induced ultrastructural changes in germinating spores of Rhizopus stolonifer and Monilinia fructicola. Phytopathology 60, 869 – 874. Bigelow, W.D., 1921. The logarithmic nature of thermal death time curves. J. Infect. Dis. 29, 528 – 536. Bussel, J., Miranda, M., Sommer, N.F., 1970. Response of Monilinia fructicola conidia to individual and combined treatments of anoxia and heat. Phytopathology 61, 61 – 64. Chan, H.T., Nishijima, K.A., Taniguchi, M.H., Linse, E.S., 1996. Thermal death kinetics of some common postharvest pathogens of papaya. HortScience 31, 998 – 1002. Civello, P.M., Martı´nez, G.A., Chaves, A.R., An˜o´n, M.C., 1997. Heat treatments delay ripening and postharvest decay of strawberry fruit. J. Agric. Food Chem. 45, 4589 – 4594. De Cal, A., Melgarejo, P., 1999. Effects of long-wave UV light on Monilinia growth and identification of species. Plant Dis. 83, 62 – 65. Garcı´a, J.M., Aquilera, C., Albi, M.A., 1995. Postharvest heat treatment on Spanish strawberry. J. Agric. Food Chem. 43, 1489 – 1492. Islam, S.Z., Honda, Y., Sonhaji, M., 1998. Phototropism of conidial germ tubes of Botrytis cinerea and its implication in plant infection processes. Plant Dis. 82, 850 – 856. Neter, J., Wasserman, W., Kutner, M.H., 1990. Applied Linear Statistical Models. Regression, Analysis of Variance, and Experimental Design. Irwin, Boston, MA.
D. Marquenie et al. / International Journal of Food Microbiology 74 (2002) 27–35 Nigro, F., Ippolito, A., Lima, G., 1998. Use of UV-C light to reduce Botrytis storage rot of table grape. Postharvest Biol. Technol. 13, 171 – 181. Phillips, D.J., Barkai-Golan, R., 1991. Postharvest heat treatment of fresh fruits and vegetables for decay control. Plant Dis. 75, 1085 – 1089. Ranganna, B., Kushalappa, A.C., Raghavan, G.S.V., 1997. Ultraviolet irradiance to control dry rot and soft rot of potato in storage. Can. J. Plant Pathol. 19, 30 – 35. Shaffer, J.J., Jacobsen, L.M., Schrader, J.O., Lee, K.W., Martin, E.L., Kokjohn, T.A., 1999. Characterization of Pseudomonas aeruginosa bacteriophage UNL-1, a bacterial virus with a novel
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uv-A-inducible DNA damage reactivation phenotype. Appl. Environ. Microbiol. 65, 2606 – 2613. Sommer, N.F., Fortlage, R.J., Buckley, M., Maxie, E.C., 1966. Radiation-heat synergism for inactivation of market disease fungi of stone fruit. Phytopathology 57, 428 – 433. Stevens, C., Khan, V.A., Lu, L.Y., Wilson, C.L., Pusey, P.L., Kabwe, M.K., Igwegbe, E.C.K., Chalutz, E., Droby, S., 1998. The germicidal and hormetic effect of UV-C light on reducing brown rot and yeast microflora of peaches. Crop Prot. 17, 75 – 84. Verbeke, G., 1997. Linear mixed models for longitudinal data. In: Verbeke, G., Molenberghs, G. (Eds.), Linear Mixed Models in Practice. Springer, New York, pp. 63 – 154.
Inactivation of conidia of Botrytis cinerea and Monilinia fructigena using UV-C and heat treatment D. Marquenie a,*, J. Lammertyn a, A.H. Geeraerd b, C. Soontjens c, J.F. Van Impe b, B.M. Nicolaı¨ a, C.W. Michiels c a
Laboratory/Flanders Centre of Postharvest Technology, Katholieke Universiteit Leuven, W. de Croylaan 42, B-3001 Leuven, Belgium b BioTeC-Bioprocess Technology and Control, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium c Laboratory of Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium Received 24 January 2001; received in revised form 20 August 2001; accepted 4 October 2001
Abstract The effect of UV-C (k = 254 nm) and heat treatment was investigated on the inactivation of conidia of Botrytis cinerea and Monilinia fructigena, two major postharvest spoilage fungi of strawberries and cherries, respectively. Both fungi were grown at 21 C in the dark and conidia were isolated after 1 week by washing the mycelium with a mild detergent solution. After filtration and resuspension in phosphate buffer to a titer of 105 to 106 cfu/ml, the conidia were subjected to different treatments. The applied UV-C doses varied from 0.01 to 1.50 J/cm2, and the conditions for the thermal treatment were 3, 5, 10, 15 and 20 min at temperatures ranging from 35 to 48 C. Both techniques were applied individually and in combination. Spore inactivation increased with increasing intensity of single treatments. No surviving spores of B. cinerea were observed after 15 min at 45 C or an UV-C treatment of 1.00 J/cm2. M. fructigena was more sensitive and a thermal treatment of 3 min at 45 C or an UV-C treatment of 0.50 J/cm2 resulted in complete spore inactivation. Combination of both techniques reduced the required intensity of the treatment for inactivation of both fungi. The order of the applications had a significant effect on the degree of inactivation. The inactivation of B. cinerea conidia was greater when the heat treatment came first, and for M. fructigena, most inactivation was achieved when the heat treatment was preceded with an UV-C irradiation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Inactivation; UV-C; Heat; Botrytis cinerea; Monilinia fructigena
1. Introduction Fungal development on fruit and vegetables during storage and transport is a major cause of postharvest losses. Important fungi are Botrytis cinerea (many fruit and vegetable species) and Monilinia fructigena (stone * Corresponding author. Tel.: +32-16-32-2376; fax: +32-16-322955. E-mail address: [email protected] (D. Marquenie).
and pip fruit). A common procedure for reducing postharvest rot is the use of chemicals. However, many fungal strains are developing resistance to the most widely used fungicides, and a growing aversion is developing among consumers against the use of chemicals in fresh produce. As a result, the legislation in most countries is becoming more severe. This has led to an increasing need for alternative techniques for fruit disinfection, which do not leave any residue. Thermal treatment and ultraviolet irradiation have been proposed as possible methods for surface dis-
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 7 1 9 - X
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infection of small fruits (Phillips and Barkai-Golan, 1991; Islam et al., 1998; De Cal and Melgarejo, 1999). In vitro studies have shown that vegetative cells and conidia of most fungi are inactivated when exposed to a temperature of 60 C for 5 to 10 min (Baker and Smith, 1969; Agrios, 1988; Civello et al., 1997). However, for surface decontamination of fresh fruit, the thermal treatment should be less severe to avoid loss of turgor and quality in general. Typically, a temperature below 55 C is applied during a relatively short time (several minutes). For strawberries, Garcı´a et al. (1995) used a treatment of 15 min at 44 to 48 C. These mild temperatures are effective, suggesting that either the fungi are less heat resistant on the surface of the fruit than in the buffers used in laboratory experiments, or that a heat shock response is induced in the fruit which increases resistance against fungi. The decontaminating properties of UV-C light are well known, but the potential of ultraviolet light for surface disinfection of fruit and vegetables has been studied only recently (Aylor and Sanogo, 1997; Ranganna et al., 1997; Islam et al., 1998; Stevens et al., 1998). In addition to its lethal effect on the fungal pathogen (Nigro et al., 1998; Shaffer et al., 1999), low doses of UV-C were also reported to induce a physiological reaction in the exposed product, inhibiting the fungal development (Stevens et al., 1998). The objective of this study was to investigate the inactivation properties of mild thermal treatments and exposure to UV-C light on conidia of B. cinerea and M. fructigena. Different time – temperature conditions and UV-C doses were screened, both individually and in combination. Combinations of treatments sometimes act synergistically, which decreases the required intensity of the individual treatments to achieve a certain inactivation level. Synergistic effects have been observed when combining a thermal treatment with anoxia (Bussel et al., 1970) or g-irradiation (Sommer et al., 1966). So far, the combined effect of heat and UV-C has not been reported.
2. Materials and methods 2.1. Production of conidia The fungal cultures used for this study were B. cinerea MUCL 18864 obtained from the BCCM2/
MUCL collection (Mycothe`que de l’Universite´ catholique de Louvain, Louvain-la-Neuve, Belgium) and M. fructigena CBS 101499 from the CBS collection (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands). A suspension of sporulating fungal material in 25% glycerol was stored at 80 C as a stock culture and was used to start new cultures. The fungi were grown at 21 C in the dark on potato dextrose agar (PDA, Oxoid, Hampshire, UK) for B. cinerea or V8 agar for M. fructigena. These media were found to support good growth and sporulation for both fungi. After 7 days, the cultures were subcultured by transferring mycelium fragments to fresh nutrient medium. Seven days later, the conidia were harvested by washing the surface of the plate with 10 ml detergent solution (3 g/l Tween-20). This suspension was filtered through a column with a plug of glass wool to remove large mycelium fragments and then centrifuged to replace the detergent by a volume of 10 mM phosphate buffer (pH 7.2) so that a concentration of 105 to 106 spores ml 1 was obtained. Spore titers were determined microscopically using a Thoma counting chamber and spores were used immediately for the inactivation experiments. 2.2. Heat treatment To investigate the effect of a thermal treatment on the inactivation of B. cinerea and M. fructigena conidia, spore suspensions were exposed to different temperature –time combinations. The temperatures were 40, 43, 45 and 48 C for B. cinerea. For the preliminary experiments with M. fructigena, the same conditions as for B. cinerea were used. Since no surviving spores were observed at 48 C, the temperature range was reduced and smaller temperature intervals were chosen (39, 41, 43 and 45 C). The duration of the treatment was 3, 5, 10 or 15 min. Inactivation was determined by surface plating treated and control (untreated) spores. For the thermal treatment, 100 ml of spore suspension was transferred into a glass capillary (1.55 mm diameter, 150 mm length). Both ends were sealed in a flame, leaving a sufficient air column to avoid heating of the spore suspension, and the capillary was immersed at a preset temperature in a circulating water bath (Grant Instruments, Cambridge, UK). Immediately after treatment, the capillaries were cooled to 20 C in water, aseptically opened and the suspension was
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serially diluted and plated onto the counting medium, respectively, rose bengal agar (RBA, Oxoid) for B. cinerea, and malt extract agar (MEA, Oxoid) for M. fructigena. These media were chosen because they resulted in the formation of discrete colonies that could readily be counted. The plates were incubated at 25 C during 5 days and colonies were counted.
violet light had no significant influence on spore inactivation. The values used in this series of experiments were based on the results obtained for the individual methods. The temperature ranges chosen for these combined treatments were 40 to 48 C for B. cinerea and 35 to 43 C for M. fructigena, and the three UV levels, 0.00, 0.05 and 0.10 J/cm2.
2.3. UV-C treatment
2.5. Data analysis
The UV radiation used for these experiments is the short-wave band or UV-C, running from 180 to 280 nm with a peak at 254 nm (the other parts being UV-B and UV-A running, respectively, from 280 to 320 nm and from 320 to 380 nm). The UV treatment cabinet used in this work (Bio-Link, Vilber Lourmat, France) was equipped with five fluorescent lamps of 8 W each (k = 254 nm), and a radiometer that permanently measured the emitted UV energy, allowing the user to program energy levels. The effect of UV treatment on spore viability and the evolution of the inactivation as function of the UV dose was studied by exposing conidia to varying UV-C doses, ranging from 0.01 to 1.50 J/cm2 (corresponding with a treatment duration of 4 to 600 s). For simple UV treatment, the spore suspension was serially diluted, 100 ml solution was plated on RBA (B. cinerea) or MEA (M. fructigena) and exposed to the different UV-C doses. The distance between the lamps and the nutrient plates was 14 cm. The plates were incubated at 25 C during 5 days and colonies were counted. In a preliminary experiment, the possible influence of an UV-illumination on the used nutrient medium and the subsequent effect on spore development was investigated. No differences between RBA and PDA for B. cinerea and between MEA and V8 agar for M. fructigena were observed, so RBA and MEA were used for the experiments.
Spore inactivation was expressed using a logarithmic reduction factor log N/No, with No the number of developing spores before treatment and N the number of developing spores after treatment. The data from the combination experiments were analysed using analysis of variance (ANOVA). This technique has the advantage that there is no assumption required about the nature of the statistical relation between the dependent (spore inactivation) and the independent variables (temperature, treatment duration, UV dose, treatment sequence), and that the independent variables in ANOVA models may be qualitative (Neter et al., 1990). All main effects and interaction effects were tested with an F test statistic at a 5% significance level. All statistical analyses in this report were performed using the SAS software, version 6.12 (SAS Institute, Cary, NC.).
2.4. Combined treatments In these experiments, the techniques were combined in both sequences to examine possible interactions. When UV illumination preceded heating of the conidia, 3 ml suspension was first transferred onto a watchglass for UV treatment, and then transferred in glass capillaries for the thermal treatment. In the reverse order, the procedures were followed as described previously. The way of exposing the conidia suspension to the ultra-
2.6. Inactivation modelling To describe the thermal inactivation of the conidia as a function of time, a classical first order inactivation model was used: dN ¼ kN dt
ð1Þ
where N is the number of surviving spores after treatment [cfu/ml], t is the time [min] and k is an inactivation rate [1/min]. The inactivation can in an analogous way be described with the D value [min], this is the Thermal Death Time (TDT) model (Bigelow, 1921). The D value gives the time required to decrease the number of surviving conidiospores with one logarithmic unit at a constant temperature. The D value is related to the k value by the equation D=(ln 10)/k. The z value (C) is the temperature increase required to have a 10-fold decrease of the D value. This
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relationship between the D and z values is described by this equation: D ¼ Dref 10ðTref T Þ=z
ð2Þ
The relation between the inactivation rate k and the temperature T was described with the Arrhenius equation: k ¼ kref e
Ea R
1 1 Tref T
ð3Þ
In this equation, Ea is the Arrhenius activation energy [J/mol], R is the universal gas constant [J/ mol K] and kref is the inactivation rate [1/min] at the reference temperature Tref [K]. At a constant temperature, the solution of Eq. (1) is given by: N ln ¼ kt ð4Þ No The inactivation by means of UV-C radiation was also modelled with first-order kinetics, but with the UV dose as independent variable. The dimensions of the corresponding D value are J/cm2. The estimation of the parameters Ea and kref was carried out using non-linear regression of Eqs. (3) and (4) to the inactivation data. The Marquardt method was used for the non-linear least square iterative computing (SAS 6.12., SAS Institute, Cary, NC.).
3. Results 3.1. Heat treatment Spore inactivation increased with increasing temperature and treatment duration, and could be described using the first-order inactivation model combined with the Arrhenius equation. The reference temperature was 43 C. For B. cinerea, the activation energy Ea was 407 kJ/mol and the z value was 4.65 C. The D values for
Fig. 1. Inactivation of conidia of B. cinerea as a function of temperature (C) and treatment duration (min). Legend: model predictions ( – : 40 C, . . .: 43 C, _._: 45 C and —: 48 C), and observed values ( 6: 40 C, : 43 C, r: 45 C and : 48 C).
the different temperatures are given in Table 1. The observed values and the predicted values are plotted in Fig. 1. No surviving spores were observed after 15 min at 45 C and all treatments at 48 C. In the case of M. fructigena, the Arrhenius activation energy Ea was 443 kJ/mol, and the z value was 4.17 C. The D values for the different temperatures are given in Table 2. The observed and the fitted values for M. fructigena are presented in Fig. 2. Conidia of M. fructigena were more sensitive to heat than those of B. cinerea: the D values were lower for the same temperature (e.g. for B. cinerea, D43 C = 6.78 min and for M. fructigena, D43 C = 2.49 min). Complete spore inactivation was already achieved upon treatment at 43 C during 15 min and during 5 min at 45 C. 3.2. UV-C treatment The number of surviving conidia decreased loglinearly with increasing UV-C dose. As such, spore
Table 1 The D values for B. cinerea at different temperatures
Table 2 The D values for M. fructigena at different temperatures
Temperature (C)
D value (min)
Temperature (C)
D value (min)
40 43 45 48
29.959 6.782 2.559 0.607
39 41 43 45
21.697 7.302 2.492 0.862
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Fig. 2. Inactivation of conidia of M. fructigena as a function of temperature (C) and treatment duration (min). Legend: model predictions ( – : 39 C, . . .: 41 C, _._: 43 C and —: 45 C), and observed values (6: 39 C, : 41 C, r: 43 C and : 45 C).
inactivation could be described using only the firstorder model, since the inactivation rate seemed to be constant for the different UV-C doses. For B. cinerea, the D value was 0.026 J/cm2, and for M. fructigena, this D value was 0.016 J/cm2. The observed and the fitted inactivation data for both fungi are given in Fig. 3. In this figure, values lying beneath the detection limit are not represented, since they do not add any significant information for the description of the inactivation process. Furthermore, these values might suggest the presence of a resistant subpopulation causing a tailing effect, and this is most probably not the case. As was the case for heat, M. fructigena was also more sensitive to UV illumination than B. cinerea: the doses at which no surviving spores were found were 0.50 and 1.00 J/cm2, respectively. The lower D value for M. fructigena reflects that the treatment time needed to achieve a reduction in number of surviving conidia of one logarithmic unit is shorter than for B. cinerea (using the same UV-C dose). For this reason, smaller intervals were chosen between 0.01 and 0.05 J/cm2.
31
interactions, an ANOVA was applied. For B. cinerea, all main effects (temperature, duration, UV dose and sequence) and two interaction effects (temperature duration, sequence UV dose) were found to be significant. Since the parameters ‘‘temperature’’ and ‘‘duration’’ are linked to the same treatment, the interaction between both is obvious. The interaction between the parameters ‘‘UV dose’’ and ‘‘sequence’’ can be explained as a change in inactivation efficiency of UV upon inversion of the treatment sequence. An increase in UV-C dose will cause a larger inactivation when the thermal treatment precedes the UV treatment. The remaining second-order interaction terms and all higher-order interaction terms had no significant effect. A graphical representation of the different temperature – time combinations required for spore inactivation for the three UV-C doses is given in Fig. 4. In this figure, the three graphs on the left side represent the inactivation when the UV treatment comes first. The temperatures needed for a certain degree of inactivation at UV = 0.00 J/cm2 (the results for the single temperature treatment) are higher than for the combinations with 0.05 and 0.10 J/cm2. The highest UV dose that was used in these combinations was 10 times lower than the dose required for inactivation when only UV-C was used.
3.3. Combined treatments To investigate the importance of the different parameters involved (temperature, treatment duration, UV dose and treatment sequence) and their possible
Fig. 3. Inactivation of conidiospores of B. cinerea and M. fructigena as a function of UV-dose (J/cm2). Legend: model predictions ( – ) and observed values (6: B. cinerea, : M. fructigena).
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Fig. 4. Effect of a combined heat and UV treatment on inactivation of conidiospores of B. cinerea. The lines give time – temperature combinations with equal inactivation (no inactivation: white, complete inactivation: black), each line represents a change in inactivation of 12.5%. The temperatures are ranging from 40 to 48 C. For each UV-dose (0.00, 0.05 and 0.10 J/cm2), both treatment sequences are represented, while for UV-dose 0.00 J/cm2, the sequence is not relevant.
The influence of the same parameters was also studied for M. fructigena. Each parameter had a significant effect on inactivation since all main effects were retained in the ANOVA results. Only for the parameter ‘‘sequence’’, the main effect was not significant, but since this parameter was present in two interaction terms (sequence temperature, sequence UV dose temperature), the main effect had to be conserved due to the hierarchical principle (Verbeke, 1997). The interaction between temperature and treatment duration is again present, together with the second- and third-order interactions with the parameter ‘‘UV dose’’ (UV dose temperature, UV dose duration, UV dose temperature duration). The effects of the treatment sequence on, respec-
tively, the UV and the thermal treatments are described by the second-order interaction terms sequence UV dose and sequence temperature. Also, a third-order interaction between these three parameters is present. For M. fructigena, the largest inactivation was obtained when the UV treatment comes first. The resulting inactivation is represented as a function of time and temperature for each UV-C dose in Fig. 5. Total inactivation was achieved faster or at lower temperature when heat treatment was preceded by an UV-C treatment. The treatment duration had a clear effect when no UV was used, but this influence was reduced in the other cases. A possible explanation for this is that a thermal treatment with a low duration
D. Marquenie et al. / International Journal of Food Microbiology 74 (2002) 27–35
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Fig. 5. Effect of a combined heat and UV treatment on inactivation of conidiospores of M. fructigena. The lines give time – temperature combinations with equal inactivation (no inactivation: white, complete inactivation: black), each line represents a change in inactivation of 12.5%. The temperatures are ranging from 35 to 43 C. For each UV-dose (0.00, 0.05 and 0.10 J/cm2), both treatment sequences are represented, while for UV-dose 0.00 J/cm2, the sequence is not relevant.
already has a great inactivation effect when combined with UV-C.
4. Discussion It has been reported that a 5 log unit inactivation of M. fructigena conidia was achieved by a thermal treatment in water at 52 C during 2 min (Baker and Smith, 1969). The Arrhenius activation energy values calculated in the present research for B. cinerea and M. fructigena (resp. 407 and 443 kJ/mol) are analogous to values obtained for other fungi like Colletotrichum gloeosoporiodes and Guignardia psidii (resp. 447 and 418 kJ/mol) (Chan et al., 1996). In our own
work, complete inactivation was achieved at lower temperatures (45 and 48 C), but the duration of the treatment was longer (resp. 15 and 5 min). UV-C has been reported to change the permeability of the cell, with leakage of electrolytes, amino acids and carbohydrates, and polysaccharide accumulation as a consequence (Nigro et al., 1998). De Cal and Melgarejo (1999) reported also the inhibition of mycelial growth of Monilinia spp. after long-wave (320 –380 nm) UV exposure. In our experiments, no surviving spores were observed after the exposure of a suspension of 105 – 106 cfu/ml of M. fructigena to a dose of 1.00 J/cm2. This UV level was reduced to 0.10 J/cm2 when combined with thermal treatment. Using lower doses is advantageous because it reduces the possible damage to treated
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food products. Relatively low doses of UV-C can also induce hormesis in diverse horticultural products, a physiological reaction leading to the induction of an increased resistance to pathogens and, in this way, reduce the development of storage rots (Stevens et al., 1998). It is important, however, that the development of infection hyphae as a result of negative phototropic reaction of conidial germtubes is not promoted (Islam et al., 1998). So low doses can be used in combination with other methods provided that all spores are inactivated as a result of the second treatment. Combination of a thermal treatment with a second method was described by different authors. Bussel et al. (1970) combined heat and anoxia (absence of oxygen) to inactivate spores of M. fructicola and Sommer et al. (1966) used g-irradiation and heat for inactivation of Rhizopus stolonifer and B. cinerea. The sequence of the treatments influenced the inactivation of the microorganisms. When anoxia preceded heat, the observed total inactivation was lower than expected in case of an additive effect, and Bussel et al. (1970) concluded that anoxia rendered the spores less sensitive to subsequent heat treatment. These data are comparable with the results Sommer et al. (1966) obtained for B. cinerea. The inactivation was higher when heat treatment was followed by g-irradiation than vice-versa, leading to the conclusion that a thermal treatment increased the susceptibility for irradiation. A comparable reaction was observed in this work for B. cinerea, with an increased inactivation when UV irradiation was preceded by the thermal treatment. On the other hand, the inactivation of R. stolonifer was greater when the spores were irradiated prior to the heat treatment. These results agree with our observations on conidia of M. fructigena, where a greater inactivation is obtained when the UV treatment comes first. An UV irradiation followed by a thermal treatment at 41 C resulted in the complete inactivation of M. cinerea spores, whereas a higher temperature was necessary in the reverse order. For B. cinerea, 3 min at 45 C followed by an exposure to 0.05 J/cm2 results in complete inactivation, while in the reverse sequence, a temperature of 48 C was needed.
5. Conclusion Heat treatment and UV-C irradiation were used for the inactivation of conidia of B. cinerea and M.
fructigena, both individually and in combination. The conidia of M. fructigena were more sensitive to heat and UV-C treatment than those of B. cinerea. Combining different methods was appropriate to reduce the intensity of the treatment needed to achieve the inactivation of conidia. The effect of treatment sequence and intensity depended on the organism. The highest inactivation was obtained when the heat treatment preceded the UV-C treatment for B. cinerea, and when the heat treatment followed an UV treatment for M. fructigena. Acknowledgements This research is supported by the Ministry of Small Enterprises, Traders and Agriculture, Directorate of Research and Development (Project S-5856). The research at the Flanders Centre of Postharvest Technology is supported by the Flemish Government. References Agrios, G.N., 1988. Plant Pathology, 3rd edn. Academic Press, San Diego, CA. Aylor, D.E., Sanogo, S., 1997. Germinability of Venturia inaequalis conidia exposed to sunlight. Phytopathology 87, 628 – 633. Baker, K.F., Smith, W.L., 1969. Heat-induced ultrastructural changes in germinating spores of Rhizopus stolonifer and Monilinia fructicola. Phytopathology 60, 869 – 874. Bigelow, W.D., 1921. The logarithmic nature of thermal death time curves. J. Infect. Dis. 29, 528 – 536. Bussel, J., Miranda, M., Sommer, N.F., 1970. Response of Monilinia fructicola conidia to individual and combined treatments of anoxia and heat. Phytopathology 61, 61 – 64. Chan, H.T., Nishijima, K.A., Taniguchi, M.H., Linse, E.S., 1996. Thermal death kinetics of some common postharvest pathogens of papaya. HortScience 31, 998 – 1002. Civello, P.M., Martı´nez, G.A., Chaves, A.R., An˜o´n, M.C., 1997. Heat treatments delay ripening and postharvest decay of strawberry fruit. J. Agric. Food Chem. 45, 4589 – 4594. De Cal, A., Melgarejo, P., 1999. Effects of long-wave UV light on Monilinia growth and identification of species. Plant Dis. 83, 62 – 65. Garcı´a, J.M., Aquilera, C., Albi, M.A., 1995. Postharvest heat treatment on Spanish strawberry. J. Agric. Food Chem. 43, 1489 – 1492. Islam, S.Z., Honda, Y., Sonhaji, M., 1998. Phototropism of conidial germ tubes of Botrytis cinerea and its implication in plant infection processes. Plant Dis. 82, 850 – 856. Neter, J., Wasserman, W., Kutner, M.H., 1990. Applied Linear Statistical Models. Regression, Analysis of Variance, and Experimental Design. Irwin, Boston, MA.
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uv-A-inducible DNA damage reactivation phenotype. Appl. Environ. Microbiol. 65, 2606 – 2613. Sommer, N.F., Fortlage, R.J., Buckley, M., Maxie, E.C., 1966. Radiation-heat synergism for inactivation of market disease fungi of stone fruit. Phytopathology 57, 428 – 433. Stevens, C., Khan, V.A., Lu, L.Y., Wilson, C.L., Pusey, P.L., Kabwe, M.K., Igwegbe, E.C.K., Chalutz, E., Droby, S., 1998. The germicidal and hormetic effect of UV-C light on reducing brown rot and yeast microflora of peaches. Crop Prot. 17, 75 – 84. Verbeke, G., 1997. Linear mixed models for longitudinal data. In: Verbeke, G., Molenberghs, G. (Eds.), Linear Mixed Models in Practice. Springer, New York, pp. 63 – 154.