Effect of temperature and water activity on in vitro
germination of Monilinia spp. C. Casals, I. Vin as, R. Torres, C. Griera and J. Usall IRTA, UdL-IRTA Centre, XaRTA-Postharvest, Lleida, Catalonia, Spain Introduction Monilinia spp. are the most important cause of postharvest decay in peaches [Prunus persica (L.) Batsch.] and nectar- ines [Prunus persica var. nectarine (Ait) Maxim.] world- wide. Brown rot of stone and pome fruit is caused by Monilinia laxa (Aderh et Rulh) Honey, Monilinia fructigena Honey in Whetzel and Monilinia fructicola (Wint.) Honey (Byrde and Willetts 1977). In the European Mediterranean areas, brown rot of stone fruit is caused by the fungi M. laxa and M. fructigena (De Cal and Melgarejo 1999). In contrast, M. fructicola causes brown rot in India, Japan, Republic of Korea, Oceania and many areas of North and South America and is included in the A2 list of quarantine organisms for Europe [organisms present in the European and Mediterranean organization for plant protection region (EPPO), but contained, under ofcial control] (Bosshard et al. 2006). Brown rot postharvest losses in the European Mediterranean areas are typically more severe than preharvest losses and occur during storage and trans- port, in some cases even affecting fruit at the processing stage (Hong et al. 1997; Larena et al. 2005). When condi- tions are favourable for disease development, postharvest losses are typically more severe reaching values of 8090% in some cases (Hong et al. 1997; Hong and Michailides 1998). However, the most important cause of postharvest diseases in peaches and nectarines in Spain is M. laxa followed by M. fructigena (isolated from 10% to 15% of Keywords ecological determinants, germination percentage, lag phase, Monilinia fructicola, Monilinia fructigena, Monilinia laxa. Correspondence Josep Usall Rodie , IRTA, Centre UdL-IRTA, XaRTA-Postharvest, 191, Rovira Roure Avenue, 25198-Lleida, Catalonia, Spain. E-mail: josep.usall@irta.cat 2008 2101: received 9 December 2008, revised and accepted 6 May 2009 doi:10.1111/j.1365-2672.2009.04402.x Abstract Aims: This study evaluated the effect of temperature (038C) and water activ- ity (a w : 087099) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa, Monilinia fructicola and Monilinia fructigena. Methods and Results: More than 80% of viable conidia germinated at 25C and 099 a w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa. There was no germination at 38C, and all three Monilinia spp. germinated at 0C. At the lowest a w (087), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 090 a w , conidia were only able to germinate at 15, 25 and 30C for the three species studied, except for M. fructicola at 15C. In contrast, at 095, 097 and 099 a w , germina- tion occurred at all studied temperatures less 38C. Generally, the lag phase was longer at low levels of a w (090095), and differences were more evident as temperatures were far from the optimum (05C). Conclusions: Germination and lag phase period were markedly inuenced by temperature and a w , and in general when conditions of temperature and a w were suboptimal, the lag phase was longer and the percentage of germination was lower. Signicance and Impact of the Study: Knowledge of the germination require- ments of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp. Journal of Applied Microbiology ISSN 1364-5072 2009 The Authors Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 4754 47 fruit affected by brown rot) (Larena et al. 2005). Posthar- vest losses on pome fruit caused by M. fructigena are also usually low around 152% (Van Leeuwen et al. 2000). Chemical control applied at preharvest is the main method used to prevent fruit rot. However, preharvest controls are often reported as not being efcient (Larena et al. 2005). Moreover, the disease cannot be controlled by postharvest treatments too, as no chemical is allowed on picked stone fruit in the European Union. Conidia are widely recognized as the most important inoculum of brown rot fungi, and the risk of deterioration and the methods that could be used for their control depend on the knowledge of the ecological requirements of these conidia and their interactions with other micro-organisms (Lacey 1989). Thus, determining the fac- tors that affect the survival of this fungus may help in understanding the epidemiology of brown rot and the development of disease management strategies (Tian and Bertolini 1999; Hong et al. 2000; Van Leeuwen et al. 2002). Several studies have dealt with the biology of the conidia in eld and in in vitro conditions (Tamm and Fluckiger 1993; Xu and Robinson 2000; Xu et al. 2001). However, for the Monilinia spp. affecting stone fruit (M. laxa, M. fructicola and M. fructigena), no information on the comparison of the three species is available regarding the ecological determinants in a wide range of conditions. Temperature and water availability (water activity: a w ) are the most important abiotic parameters determining the potential for conidia germination and the growth of propagules on the fruit surface (Magan and Lacey 1988), being a w the same value of relative humidity (RH) under equilibrium conditions. Although many efforts have been made to determinate the germination conditions for Monilinia spp., the knowledge of the effect of temperature and a w over a wide range is limited. Weaver (1950) reported that M. fructicola conidia needed between 2 and 25 h to germinate at 25C on potato dextrose agar (PDA) medium. In other studies, was reported the effect of temperature on the germination of M. fructicola ascospores, where after 24 h of incubation at 15, 20 and 25C, 100% of them had been germinated (Hong and Michailides 1998). For M. laxa, the maximum conidial germination occurred between 15 and 25C exposed at 100% of RH and up to 88% was reported over a wide temperature range (530C) (Tamm and Fluckiger 1993). Moreover, it was observed that the conidia of M. laxa germinated even at )4C (Tian and Bertolini 1999). The maximum conidia germination rate for M. fructigena occurred in the range 2325C in free water; and this species was able to germinate over a wide temper- ature range (330C). In contrast, germination observed below 97% of RH was rare (Xu et al. 2001). In order to plan rational disease-control managements is necessary to study the conidial germination of the pathogen in different environmental conditions. Therefore, the aim of this study was to determine and compare the effect of temperature and a w on germination percentages and lag times for germination for M. laxa, M. fructigena and M. fructicola in in vitro tests. Material and methods Isolates Isolates of M. laxa (CPML1), M. fructigena (CPMG1) and M. fructicola (CPMC1) were from the collection of the Pathology Unit, UdL-IRTA Center of Lleida, Catalonia, classied at Department of Plant Protection, INIA, Madrid, Spain. They were originally isolated from decayed fruits from commercial orchards and were main- tained on PDA (39 g l )1 ; Biokar Diagnostics, Beauvais, France) Petri dishes amended with acetone (1%; J.T. Baker, Deventer, Holland) and stored at 4C in darkness. Medium The basic medium used was PDA at pH of 56. The a w of this basal medium was 099. This a w was modied by the addition of known amounts of the nonionic solute, glyc- erol, in order to obtain a w levels of 097, 095, 090 and 087. The a w of all media was checked with a a w meter (AquaLab, Pullman, WA, USA). Inoculum preparation Isolates were subcultured onto new PDA Petri dishes and incubated in darkness at 25C for c. 2 weeks before fruit inoculation. To obtain heavy sporulation of Monilinia spp., each isolate strain was inoculated on freshly har- vested peach or nectarine. For M. laxa and M. fructicola, peach fruit were wounded with a scalped and a mycelial plug cultured on PDA was inserted into each wound. Fruit were then incubated at 25C and 85% RH in dark- ness for 57 days for M. fructicola and 710 days for M. laxa. Low sporulation on fresh fruit by M. fructigena was overcome by inoculating canned peach halves with a mycelial plug of M. fructigena that was cut from the region of an actively growing region. The canned peach halves were then placed into a sterile glass container and incubated at 25C and 85% RH in darkness for 57 days. After the incubation, conidia from sporulated fruit area were suspended in 5 ml of sterile distilled water that was amended with one drop of wetting agent per litre (Tween-80), previously modied with glycerol to the required a w treatment. The nal a w of the conidial Effect of temperature and water activity on Monilinia spp. C. Casals et al. 48 Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 4754 2009 The Authors suspensions were 087, 090, 095, 097 and 099. The nal conidial concentration was adjusted to 15 10 5 coni- dia ml )1 using a haemocytometer. Germination study To carry out the germination study, 10 ll droplets of the conidia suspensions were placed around PDA Petri dishes with the same a w . Then, Petri dishes with the same a w were unclosed in polyethylene bags and incubated at 0, 5, 15, 25, 30, 35 and 38C. Experiments were carried out with three replicate Petri dishes per treatment. After each incubation period, three agar discs (5 mm diameter) coincided with each of the placed drops were removed from each replicate using a cork borer. At each sampling time, discs from the same temperature and a w were placed into a sterile empty Petri dish, and conidia germination was immediately stopped by adding 1 ml of ethanol (EtOH) 999% onto a (60 mm diameter) lter paper placed into the Petri dish. Then, Petri dishes were closed and stored at 4C until microscopic examination. Fifty single conidia per disc (150 per replicate; 450 per treat- ment) were examined. Conidia were considered germi- nated when the germ tube was equal to or longer than the smallest diameter of conidia. The experiments were carried out for a maximum of 30 days. The experiment was repeated twice. For the germination studies, the variable measured was the percentage of germination at different temperatures and a w over time. The lag phase period for each tem- perature and a w treatment was considered to have ended when the percentage of germinated conidia was exceeded 10%. Results Effect of temperature on germination The three species of Monilinia studied were able to germinate over a wide temperature range (035C) at 35C 0 20 40 60 80 100 Time (h) G e r m i n a t i o n
( % ) G e r m i n a t i o n
( % ) 0 20 40 60 80 100 30C Time (h) 15C 0 20 40 60 80 100 Time (h) G e r m i n a t i o n
( % ) 5C 0 20 40 60 80 100 0 1 2 3 4 5 Time (days) G e r m i n a t i o n
( % ) 0C 0 20 40 60 80 100 0 3 6 9 12 15 Time (days) G e r m i n a t i o n
( % ) 25C 0 20 40 60 80 100 0 12 24 36 48 60 72 0 12 24 36 48 60 72 0 12 24 36 96 144 192 240 0 12 24 96 144 192 240 Time (h) G e r m i n a t i o n
( % ) Figure 1 Effect of temperature and water activity on germination percentage of Monilinia laxa. Water activity levels are ( ) 099; ( ) 097; ( ) 095; ( ) 090 and ( ) 087. Values are the mean of three replicates and 150 conidia per replicate. Vertical bars are the standard error. C. Casals et al. Effect of temperature and water activity on Monilinia spp. 2009 The Authors Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 4754 49 099 a w (Figs 13), but no germination occurred at 38C for any of the tested isolates. The optimum temperature for germination after 4 h of incubation was in the range 1530C for the studied species (Fig. 4). Isolates of M. fructicola and M. fructigena reached 8595% germina- tion after 2 h of incubation at 25C and 099 a w while M. laxa needed 4 h (Figs 13). When temperature increased to 35C at 099 a w , the percentage of germinated conidia reached 76% (average of M. laxa, M. fruticola and M. fructigena) within the rst 8 h of incubation (Figs 13). All studied species had similar behaviour of germination at 5C and 099 a w having c. 62% and 82% of germination after 24 and 48 h, respectively (Figs 13,5). At 0C, the percentage of germination after 24 h of incubation was around 5% for the studied species (Figs 13). However, after 48 h, the percentage of germination reached was 30%, 71% and 78% for M. laxa, M. fructigena and M. fructicola respectively (Figs 13, 5). Effect of temperature and a w interactions on germination In general, when temperature varied from optimum conditions to marginal values, a decrease in germination percentage and an increase in the length of the lag phase were observed for the studied species. A similar pattern was observed when a w was reduced, regardless of temper- ature (Figs 13). The minimum a w at which germination begins varied with temperature; and only at 15C was related to the studied species (Table 1). Low levels of a w (087) and high temperatures (38C) prevented conidia germination by all three species almost until 30 days. Moreover, none of the Monilinia spp. were able to germinate when incubation conditions were 0, 5 and 35C and 090 a w . Monilinia fructicola was the only Monilinia spp. that was not able to germinate at 15C and 090 a w . Generally, the lag phase was longer at low levels of a w (090095), and differences were more evident as 15C 30C 0 20 40 60 80 100 Time (h) G e r m i n a t i o n
( % ) 35C 0 20 40 60 80 100 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Time (h) 0 12 24 36 48 60 72 Time (h) 0 12 24 36 48 60 72 Time (h) G e r m i n a t i o n
( % ) 25C 0 20 40 60 80 100 G e r m i n a t i o n
( % ) 0 20 40 60 80 100 G e r m i n a t i o n
( % ) 0 20 40 60 80 100 G e r m i n a t i o n
( % ) 5C 0 1 2 3 4 5 Time (days) 0 20 40 60 80 100 G e r m i n a t i o n
( % ) Time (days) 0C 0 3 6 9 12 15 Figure 2 Effect of temperature and water activity on germination percentage of Monilinia fructicola. Water activity levels are ( ) 099; ( ) 097; ( ) 095; ( ) 090 and ( ) 087. Values are the mean of three replicates and 150 conidia per replicate. Vertical bars are the standard error. Effect of temperature and water activity on Monilinia spp. C. Casals et al. 50 Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 4754 2009 The Authors temperatures were far from the optimum (05C) (Table 1). At the optimal temperature of 25C, the duration of the lag phase was less inuenced by a w compared with the other temperatures studied. Thus, for M. fructicola and M. fructigena, the duration of the lag phase was not increased by reducing a w from 099 to 097 a w and for M. laxa was increased by only 2 h. Moreover, further reducing the a w to 095, the duration of the lag phase only increased to 4 h for M. fructicola and M. fructigena and to 8 h for M. laxa (Table 1). In general, the duration of the lag phase of the three Monilinia spp. studied showed similar patterns of response to temperature and a w . However, the lag phase for M. laxa tended to be equal to or longer than the lag phases for M. fructicola and M. fructigena. The percentage and rate of germination tended to decrease as a w decreased from 099 to 090. At 5C, the percentage of germination after 48 h of incubation, at 099 097, 095 a w was 82%, 69% and 8% respectively (mean of the three Monilinia spp.). After 72 h of incubation at 5C, the percentage of germination at 099 a w had a similar proles to that at 097 a w (c. 80%) for the three studied species of Monilinia spp. In contrast, at 5C and 095 a w , the percentage of germination after 72 h of incubation was lower compared with 099 and 097 a w and depended on the Monilinia spp. studied. Thus, for M. laxa and M. fructigena, it was on average 39% while for M. fructicola was 88%. At 0C and 099 a w , germination after 48 h of incubation occurred, but it depended on the Monilinia spp. studied. For M. fructigena and M. fructicola, the percentage of germination was c. 75% and was nearly double that for M. laxa (c. 30%). In contrast, after 72 h of incubation, the percentage of germination showed similar proles for the three species studied being around 15% and 82% testing 099 and 097 a w respectively. Discussion Prior to this study, there were only partial reports about the germination of the three Monilinia species. However, 35C 0 20 40 60 80 100 Time (h) G e r m i n a t i o n
( % ) 30C 25C 5C Time (days) Time (days) 0C 0 3 6 9 12 15 15C 0 12 24 36 48 60 72 0 20 40 60 80 100 Time (h) G e r m i n a t i o n
( % ) 0 20 40 60 80 100 G e r m i n a t i o n
( % ) 0 20 40 60 80 100 G e r m i n a t i o n
( % ) 0 0 1 2 3 4 5 12 24 36 48 60 72 0 20 40 60 80 100 Time (h) G e r m i n a t i o n
( % ) 0 12 24 20 0 40 60 80 100 Time (h) G e r m i n a t i o n
( % ) 0 12 24 48 96 144 192 240 36 48 60 72 Figure 3 Effect of temperature and water activity on germination percentage of Monilinia fructigena. Water activity levels are ( ) 099; ( ) 097; ( ) 095; ( ) 090 and ( ) 087. Values are the mean of three replicates and 150 conidia per replicate. Vertical bars are the standard error. C. Casals et al. Effect of temperature and water activity on Monilinia spp. 2009 The Authors Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 4754 51 to our knowledge, this is the rst time that the effect of temperature and a w on germination has been studied for all three species over a wide range of temperature and a w . The results of this study have shown that germination of conidia is markedly inuenced by the interaction of temperature and a w . The optimum temperature for germination of M. laxa conidia at 099 a w was over a wide range (1530C), and the species was even able to germinate at suboptimal temperatures (035C), but at 38C no germination was observed. Tian and Bertolini (1999) also observed germination of M. laxa at )4C, but in our work we studied temperatures only up to 0C because this is the lowest temperature at which stone fruit are stored after harvest. In our experiments, when tem- peratures were far from the optimum (0, 5 and 35C), the driest condition in which germination was observed was 095 a w , while at the optimum range (15 25C) ger- mination occurred even at 090 a w . The dry exposure led to reduced germination, and in addition the germination process was considerably delayed. Nevertheless, these results suggest that M. laxa could have the potential to germinate in absence of free water in the host tissues. Experiments of Tamm and Fluckiger (1993) tested a narrower range of RH (88100%) and also suggested that the absence of free water is a limiting but not an excluding factor in the infection process. Monilinia fructicola also had a wide optimum tempera- ture range (1530C) for germination and germinated over all temperatures studied (035C), except at 38C. Weaver (1950) also observed germination in the tested range from 5 to 30C. But these results showed a narrower optimum range of temperature for germination (1525C). The effect of temperature on the ability of M. fructicola conidia to germinate is consistent with previous reports that showed that temperature and duration of wetness are considered to be the most important factors that affect infection by M. fructicola (Luo and Michailides 2001a,b, 2003). 40 60 80 100 0 20 0 10 20 30 40 G e r m i n a t i o n
( % ) Temperature (C) Figure 4 Effect of temperature at 099 water activity on germination percentage of ( ) Monilinia laxa; ( ) Monilinia fructicola and ( ) Monilinia fructigena, after 4 h of inoculation. Values are the mean of three replicates and 150 conidia per replicate. Vertical bars are the standard error. 80 100 0 20 40 60 0 1 2 3 4 5 G e r m i n a t i o n
( % ) Temperature (C) Figure 5 Effect of temperature at 099 water activity on germination percentage of ( ) Monilinia laxa, ( ) Monilinia fructicola and ( ) Monilinia fructigena, after 48 h of inoculation. Values are the mean of three replicates and 150 conidia per replicate. Vertical bars are the standard error. Table 1 Time (in h) before germination begins (lag phase period) at different temperatures (T a ) and water activities (a w ) for each isolate of Monilinia laxa, Monilinia fructicola and Monilinia fructigena T a (C) a w M. laxa M. fructicola M. fructigena 099 097 095 090 087 099 097 095 090 087 099 097 095 090 087 38 * * * * * * * * * * * * * * * 35 2 4 4 * * 2 4 24 * * 4 4 8 * * 30 4 4 12 120 * 2 2 4 72 * 2 4 12 48 * 25 2 4 8 48 * 2 2 4 48 * 2 2 4 72 * 15 2 8 12 264 * 4 4 8 * * 4 4 8 96 * 5 24 48 72 * * 24 48 48 * * 24 48 72 * * 0 48 120 168 * * 48 72 120 * * 24 72 168 * * We consider germination begins if more than 10% of conidia were germinated. *Germination was not observed after 30 days. Effect of temperature and water activity on Monilinia spp. C. Casals et al. 52 Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 4754 2009 The Authors Furthermore, Biggs and Northover (1988) observed that the optimum temperature for peach infection was 12 h of wetness in the range 22525C, which is also within the range presented in this work. The optimum germination range was within 1530C for M. fructigena. However, a narrower range of optimal temperature (2325C) was reported by Xu et al. (2001). That difference between studies could be because of the fact that Xu et al. (2001) used a different model to determine the optimum range of temperature. Our results showed that conidia can germinate over a wide range of tempera- tures, at least from 0 to 35C. Moreover, depending on the temperature tested, M. fructigena can germinate from 090 to 099 a w . In contrast, Xu et al. (2001) determined that conidia only germinated under near saturation humidity (97% RH). The difference to our results could be because of the isolate used or the solute used to reduce a w in the medium: we used glycerol and they used NaCl solute. Marin et al. (1995) reported for some species of Fusarium spp. that, at low a w , growth rates were higher on glycerol-amended medium compared with NaCl or glucose-modied medium. Although our results showed a clear effect of a w on conidia germination in in vitro studies, Xu and Robinson (2000) suggested that the effects of the duration of wet periods on infection were very less, unless the conidia enter directly into fruits through unhealed wound tissues and moisture on the wound surface was sufcient for conidia to germinate and infect. The results of this study showed that the maximum germination for the Monilinia species studied was generally lower than 100% even at optimal germination condition. A possible explanation of this phenomenon could be mycostasis. This has been investigated as a mechanism by which propagules are protected from spontaneous germination in the absence of potentially colonisable substrata, such protection being at the expense of debilitation (Lockwood 1988). This behaviour seems to be an intrinsic mechanism of each species, and no information is available about why mycostasis suscep- tible fungi have adopted such a high-risk system, while others have evolved dormancy activation systems to achieve the same end (Cooke and Whipps 1993). Our study showed that lag times were markedly inu- enced by temperature and a w conditions. Similar proles for M. fructicola, M. laxa and M. fructigena were observed by Weaver (1950), Tamm and Fluckiger (1993) and Xu et al. (2001) respectively. Our results, frequently showed that reducing a w from 099 to 097, lag phase period did not increase or increased less than reducing a w from 097 to 090. In our report when a w was 099 at 25C, more than 20% of conidia for M. laxa and 85% for M. fructicola and M. fructigena were already germinated after 2 h of incubation. In contrast, Xu et al. (2001) results showed that the percentage of germination for M. fructigena was only around 60%. Our results showed that, at 0C and 099 a w , no germinated conidia for all studied species were observed before 1 day of incubation but more than 80% of conidia were germinated after 6 days also at 097 a w ; this showed their potential infection in peaches at this cold-stored conditions. Similar results were observed by Tian and Bertolini (1999). However, testing M. laxa at 5C and after 24 h of incubation detected a little difference in comparison with Tian and Bertolini (1999) results. Thus, at 5C, we observed that only around 60% of conidia were already germinated while they found up to 90%. 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Casals et al. 54 Journal compilation 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 4754 2009 The Authors