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Keywords:

  • ecological determinants;
  • germination rates;
  • growth rate;
  • lag phase;
  • temperature;
  • water activity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

Aims: This study compares the effect of temperature (4–37°C) and water activity (aw: 0·99–0·87) and their interactions on the germination rates, lag times prior to germination and mycelial growth ‘in vitro’ of Penicillium digitatum, P. italicum and Geotrichum candidum, the main postharvest pathogens affecting citrus fruits.

Methods and Results: Germination and growth were markedly influenced by temperature and aw. Generally, lag times were longer and germination and growth rates were slower when conditions of temperature and aw were far from optimum. All the studied species were able to germinate over a range of 4–30°C at 0·995 aw, although in non-optimal conditions P. digitatum only reached 40–60% of germinated conidia. At low temperatures, P. italicum germinated and grew faster than P. digitatum and G. candidum, particularly at 0·95 aw. Penicillium italicum was also able to germinate and grow in the driest studied conditions (0·87 aw), while G. candidum did not germinate under 0·95 aw.

Conclusions: Knowledge of the ecological requirements of these fungi is important in order to understand their behaviour in natural situations and to predict fungal spoilage on citrus fruits.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

Green and blue mould rots, caused by Penicillium digitatum (Pers.: Fr.) Sacc. and P. italicum Wehmer, respectively, are probably the most common postharvest diseases affecting citrus fruit. Sour rot, caused by Geotrichum candidum Link, is not a disease of major industry-wide importance, but it should not be underestimated because initial infections are easily overgrown by other moulds (Smoot and Johnson 1983).

In all cases, these pathogens may attack the fruit on the tree, in the packinghouse, in transit, in storage and in the market. During these stages, citrus fruits can be affected by different environmental conditions that could favour the development of one pathogen to the detriment of the others. Thus, blue mould is the major postharvest diseases on fruits stored at cold temperatures while green mould reaches 60–80% of decay caused by Penicillium genera on fruits stored under ambient conditions (Tuset 1987). On the other hand, the incidence of fruit decay caused by G. candidum increases during wet seasons (Smoot and Johnson 1983) and when harvesting occurs after abundant rainfall (Tuset 1987).

The risk of deterioration and the methods that could be used for their control depend on knowledge of the ecological requirements of these fungi and their interactions with other microorganisms (Lacey 1989). Water availability (water activity, aw) and temperature are the most important abiotic parameters determining the potential for spore germination and growth of propagules on the fruit surface (Magan and Lacey 1988).

Recent investigations carried out with common cereal fungi (Marín et al. 1996, 1998) showed how different environmental conditions can affect the activity of Fusarium moniliforme, F. proliferatum and some Aspergillus and Penicillium spp. on spoilage of feeds and foods. These data allow predictive modelling of fungal behaviour to be developed in order to manipulate these environmental conditions as a means of controlling grain spoilage.

Knowledge of the effect of temperature and water activity alone and their interactions on germination and growth of P. digitatum, P. italicum and G. candidum is limited. The maximum growth for P. digitatum occurred between 20 and 25°C and at 1·00 aw, but it was able to grow in the range 6–37°C and no growth was observed under 0·90 aw (Hocking and Pitt 1979; Lacey 1989). Penicillium italicum is able to germinate at lower temperatures than P. digitatum, and even at 0°C as previously reported Wyatt and Parish (1995) on orange juice serum agar (OJSA). On the other hand, G. candidum grows optimally at 30°C on both agar medium and fruit (Kassim and Khan 1996). However, these studies were always limited to one condition, one temperature or one aw, and no information is available on the water activity and temperature interactions on spore germination and mycelial growth for isolates of P. digitatum, P. italicum and G. candidum.

The objective of this study was to determine the effect of aw and temperature and their interactions on germination rates, lag times for germination and growth rates of P. digitatum, P. italicum and G. candidum in in vitro tests.

Isolates

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

Penicillium digitatum strain PDM-1, P. italicum PIM-1 and G. candidum GCTA were isolated from decayed citrus fruits. These isolates were the most aggressive in our collection (Pathology Laboratory, UdL-IRTA, Lleida, Catalonia, Spain).

Medium

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

The basic medium used was orange serum agar (OSA) with a pH of 5·5. The water activity of this basal medium was 0·995 aw. This aw was modified by the addition of known amounts of the non-ionic solute, glycerol, in order to obtain aw levels of 0·95, 0·90 and 0·87. The aw of all media was determined with an Aqua Lab Water Meter (Decagon Devices, Inc., Washington, DC, USA).

Germination studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

Fungi were grown on potato dextrose agar (PDA) medium for 7–12 days at 25°C to obtain heavily sporulating cultures. Spores were suspended in sterile distilled water containing a drop of a wetting agent per litre (Tween-80). Stock spore suspensions (1 ml) were added to 9 ml sterile water previously modified with glycerol to give 10 ml of solution at the required water availability. The final water activity of the treatments was 0·995 (unmodified), 0·95, 0·90 and 0·87 aw and the final spore concentration was adjusted to 1 × 106 spores ml−1 by haemocytometer.

A 0·1-ml aliquot of the spore suspensions was pipetted onto OSA plates of the same aw and spread with a surface sterilized bent glass rod as quickly and carefully as possible. Petri dishes of the same aw treatment were enclosed in polyethylene bags and incubated at 4, 10, 25, 30 and 37°C. Experiments were carried out with three replicate Petri plates per treatment. Preliminary experiments showed that this method gave consistent and repeatable results (data not shown).

Periodically, depending on the treatment, three agar discs (5 mm diameter) were aseptically removed from each replicate plate using a cork borer, placed on a slide and examined microscopically. Fifty single spores per disc (150/replicate; 450/treatment) were examined. Spores were considered to have germinated when the germ-tube was equal to or longer than the diameter of the spore (Marín et al. 1996). The experiments were carried out for a maximum of 30 days.

Growth studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

Actively growing 7–12-day-old colonies of the isolates grown on the PDA medium were used for all experiments. A 10 μl aliquot of the spore suspensions adjusted to 1 × 106 spores ml−1 was inoculated in the centre of each Petri plate. Petri plates of the same aw and temperature were placed in polyethylene boxes and incubated at 4, 10, 25, 30 and 37°C. The aw studied were 0·995 (unmodified), 0·95, 0·90 and 0·87. In all cases, the experiments were carried out with three replicates per treatment.

The Petri plates were examined daily, or as necessary, and the radius of the growing colonies was measured in two directions at right angles to each other (Marín et al. 1996). Measurements were carried out for a maximum of 60 days.

Statistical treatment of the results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

For the germination studies the variable measured was the percentage germination at different aw/temperatures against time. The percentage germination at each aw/temperature condition was plotted against time, and a nonlinear regression was used to estimate the following two parameters at the 95% confidence level: maximum germination rate (h−1) and lag phase (h). The Gompertz model was used as the fitting equation (Zwietering et al. 1990).

  • image

where A is the asymptotic value at which the germination rate becomes constant (100% in most cases), μm, maximum specific germination rate (h−1) given by the slope of the line when spores germinate exponentially; δ, lag phase (h); t, time (h).

For the growth studies the radial growth rate (mm day−1) at each aw and temperature treatment was obtained from the slopes of the linear regression of the linear parts of the temporal growth curves.

Effect of temperature on germination. The three studied isolates were able to germinate in the temperature range studied at unmodified aw (Fig. 1), except at 37°C, at which no germination was observed for any of the tested fungi.

image

Figure 1. Effect of temperature on the germination of Penicillium digitatum, P. italicum and Geotrichum candidum at 0·99 aw on orange serum agar (OSA) medium. Temperature levels are 4°C (○), 10°C (▵), 25°C (□) and 30°C (•)

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At 25°C P. digitatum, P. italicum and G. candidum reached 100% germinated spores within 15 h of inoculation and germination was delayed and slowed down when temperature decreased to 10 and 4°C. Germination delay also occurred at 30°C, except for G. candidum in which no differences were observed between germination at 25°C and 30°C.

P. italicum and G. candidum were able to get 100% spores germinated at all temperatures and aw studied. However,P. digitatum only reached 40–60% conidia germinated when temperature and aw conditions were different from unmodified aw and 25°C (data not shown).

The minimum water activity at which germination occurred varied with temperature (Table 1). Thus, P. digitatum was able to germinate at 0·90 aw at 25°C, but at 10 and 30°C it only germinated above 0·95 aw. In contrast, P. italicum is the most adapted fungus in low aw and temperature conditions because it was able to germinate at 0·87 aw even at 10°C. On the other hand, G. candidum did not manage to germinate even at its optimum temperature under 0·95 aw during the 30-day time period of our experiments.

Table 1.  Minimum water activity for germination/growth found at different temperature levels for the isolates of Penicillium digitatum,P. italicum and Geotrichum candidum on OSA medium. Germination is considered to take place when a 10% germination is achieved, and growth when a growth rate higher than 0·1 mm day−1 is achieved
 Temperature (°C)
 410253037
  1. NG /NG, No germination/no growth, OSA, orange serum agar.

P. digitatum0·99/0·990·95/0·950·90/0·900·95/0·95NG/NG
P. italicum0·95/0·950·87/0·900·87/0·870·90/0·90NG/NG
G. candidum0·99/NG0·95/0·990·95/0·950·95/0·95NG/NG

Effect of aw and temperature on lag phases before germination. Lag phases prior to germination increased when temperature varied from optimum to marginal conditions, regardless of aw for all three fungi studied. Generally, lag phases were longer at low aw and differences were more evident as temperatures were lower than the optimum (Fig. 2). The profiles generally suggest shorter lag phases in conditions close to the optimum temperature and aw levels than in other conditions for the three studied fungi.

image

Figure 2. Effect of water activity and temperature on the lag phase before germination of Penicillium digitatum, P. italicum and Geotrichum candidum on OSA medium. Water activity levels are 0·99 (○), 0·95 (▵), 0·90 (□) and 0·87 (•). Error bars show s.e. of estimated parameters

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At unmodified awP. digitatum lag phase increased by 10 h when the temperature was reduced from 25 to 10°C and increased by 80 h when the temperature decreased from 10 to 4°C. Similar values were presented for P. italicum at the same temperatures. In contrast, G. candidum began germination before P. digitatum and P. italicum at all temperatures studied and unmodified aw. Within the first 4 h, arthrospores of G. candidum were able to initiate the germination process at both 25 and 30°C.

Reducing aw to 0·95 involved a lag phase increment of around 3 h for P. digitatum and P. italicum and 1 h forG. candidum at 25°C. However, at lower temperatures,G. candidum extended its lag phases more than P. digitatum and P. italicum.

Although at 0·90 awP. digitatum was able to germinate at 25°C, its lag phase was more than twice that of P. italicum in the same conditions (78 and 28 h, respectively).

Under the driest studied conditions (0·87 aw) germination only occurred at 25–10°C for P. italicum, with lag phases of around 50 and 400 h, respectively. No germination was achieved with G. candidum and P. digitatum at 0·87 aw.

Effect of aw and temperature on germination rate. In general, when temperature varied from optimum to marginal values a decrease in germination rates was observed for the three studied fungi. A similar profile was observed when aw was reduced, regardless of temperature.

Maximum germination rates occurred at 25°C for P. digitatum and P. italicum, regardless of aw, and at 25–30°C for G. candidum (Fig. 3). These values were around 30 h−1 for the three studied fungi, so they reached the maximum percentage of germination in 3–4 h after the start of the process.

image

Figure 3. Effect of water activity and temperature on the germination rate of Penicillium digitatum, P. italicum and Geotrichum candidum on OSA medium. Water activity levels are 0·99 (○), 0·95 (▵), 0·90 (□) and 0·87 (•). Error bars show s.e. of estimated parameters

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At unmodified aw, a reduction from 25 to 10°C resulted in a germination decrease of more than 20 h−1 for the three isolates tested. However, in the same situation but at 0·95 or 0·90 aw, the reduction was <10 h−1. At 10 and 4°C P. italicum germinated faster than the other two studied fungi at any aw.

At 30°C P. digitatum and P. italicum showed a similar germination rate at both 0·995 aw and 0·95 aw, thus differing from G. candidum which showed a germination that was over three times higher at 0·995 aw than at 0·95 aw.

Effect of aw and temperature on mycelial growth rate. As in the germination results, P. digitatum and P. italicum showed maximum growth at 25°C and unmodified aw medium with growth rates of around 3·5 mm day−1 and at 25–30°C for G. candidum with more than 4 mm day−1 (Fig. 4). Generally, growth rates were reduced when temperatures varied from optimum to the rest of the studied conditions, regardless of aw, except for P. digitatum which at 0·95 aw was able to grow faster at 30°C than at 25°C.

image

Figure 4. Effect of water activity and temperature on the growth rate of Penicillium digitatum, P. italicum and Geotrichum candidum on OSA medium. Water activity levels are 0·99 (○), 0·95 (▵), 0·90 (□) and 0·87 (•). Error bars show s.e. of estimated parameters

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Reducing aw at different steady-state temperatures also decreased growth rates for the three fungi studied, with the exception of P. digitatum at 30°C and P. italicum at all temperatures tested. For the former, a reduction from 0·99 to 0·95 aw at 30°C involved an increase in the growth rate from 0·5 to 3 mm day−1. Penicillium italicum achieved similar or even greater growth rates at 0·95 aw than in the unmodified aw medium for all temperatures at which growth occurred.

Geotrichum candidum was more drastically affected by reduction of aw than P. digitatum and P. italicum. Thus, decreasing the aw level from 0·99 to 0·95 aw involved an ca. 10-fold reduction in growth rate at both 25 and 30°C. No growth of G. candidum was observed below 0·95 aw.

At 4 and 10°C and 0·95 awP. italicum was the fungus that grew faster, with growth rates of 0·5–1·3 mm day−1 respectively, while no growth occurred on P. digitatum and G. candidum.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

This study has shown that germination of spores and mycelial growth of the isolates of P. digitatum, P. italicum and G. candidum are markedly influenced by temperature, aw and their interactions.

The optimum temperature for germination and growth for P. digitatum was 25°C, as has previously been reported by Lacey (1989). On the other hand, P. digitatum was able to germinate and grow in the range 4–30°C, and no growth or germination was observed at 37°C (at 0·995 aw). These results differ from those obtained by Lacey (1989), which established the growth range for P. digitatum between 6 and 37°C. These differences could be because of the culture medium used and the isolate studied. This study showed that the maximum germination for P. digitatum was lower than 100% germinated spores (40–60% depending on the treatment) at temperatures different from 25°C and aw lower than 0·995 aw. A possible explanation of this phenomenon could be mycostasis. This has been envisaged 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-susceptible fungi have adopted such a high-risk system, while others have evolved dormancy-activation systems to achieve the same end (Cooke and Whipps 1993).

Similar to P. digitatum, P. italicum germinated and grew optimally at 25°C and also germinated at all temperatures studied, except at 37°C. The minimum temperature at which P. italicum growth occurred was not determined in this study because it was not tested at temperatures lower than 4°C, but Wyatt and Parish (1995) found that P. italicum was able to grow at 0°C on OJSA medium.

In contrast, G. candidum germinated and grew optimally at 25 and 30°C. This result has previously been reported by Kassim and Khan (1996) on both agar medium and fruit. Geotrichum candidum did not grow at 37°C, and although it germinated at 4°C it was not able to grow at this temperature. In general, the range of aw conditions over which germination occurred was wider than that for growth, as has previously been reported in other Penicillium species grown on maize (Marín et al. 1998). The reasons cannot be fully explained, but in such a situation there is usually an extended lag phase, and development often fails to proceed further than the germ tube stage (Magan 1988). According to the water activity requirements, G. candidum showed a marked dependence because it was not able to germinate under 0·95 aw. This could explain why sour rot is more of a problem during and after prolonged wet seasons (Smoot and Johnson 1983) and when harvesting is performed on over-mature fruit or after abundant rainfall (Tuset 1987). Geotrichum candidum does not develop on active decay lesion unless the peel has a relatively high water content and the inoculated fruit are held in a water-saturated atmosphere (Baudoin and Eckert 1982).

Lag times, like germination rates, were markedly influenced by temperature and aw conditions. Generally, germination was slower and lag phases were longer when the conditions were far from optimum. At 0·995 aw lag times were shorter for G. candidum, and its germination and growth rates were higher than those of P. digitatum and P. italicum at 25–30°C. According to these results, on fruits kept under ambient conditions G. candidum must be the most important disease, because it germinated before and grew faster than P. digitatum and P. italicum. In contrast, the incidence of G. candidum on fruits is lower than 2–3% decayed fruits (Tuset 1987). This controversy could suggest that aw in the injury was not 0·99 but less and G. candidum is very susceptible to low aw as has been demonstrated in this study.

Penicillium digitatum and P. italicum showed lag times of around 19 h at 10°C. These results contrast with the ones obtained by Wyatt and Parish (1995), who determined that lag times for these fungi on OJSA at low temperatures (0, 3, 7 and 10°C) were around 28 h. The variability could be the result of differences between tested isolates and the culture medium used.

In germination and growth studies, P. italicum was the best adapted fungus at low conditions of temperature and aw. It was even able to grow at 0·87 aw but the mycelium appearance was not the habitual and germination eruptive by means of a plug of aggregated hyphae. This could be because very low aw may induce aberrant behaviours of germ tubes, which are abnormally short, broad and vacuolated (Anikster 1988). The dominance of P. italicum to grow at low temperatures, as germination does, was not so clear at 0·995 aw. In contrast, considering the aw of the injury to be less < 0·995 could explain why blue mould is the predominant disease in cold-stored citrus fruits (Tuset 1987).

In conclusion, the study of the impact of aw and temperature on germination and growth in vitro for these isolates has provided detailed knowledge on the ecological requirements of these species for colonizing and infecting the surface of citrus fruits. However, extrapolation of laboratory findings to natural situations is dangerous, because environmental conditions can fluctuate simultaneously, and other factors such as pH, antifungal compounds of the peel, essential oils, etc. could vary the behaviour of these species. Moreover, fungi rarely occur in a monospecific culture on the surface of the fruit but interact with others and this competitiveness may alter the predominant species. For these reasons, further studies are needed in order to determine competitive abilities when these fungi interact with each other both in vitro and over the citrus surface.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References

The authors are grateful to the Catalan Government for its financial support (Departament d'Universitats, Recerca i Societat de la Informació).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Isolates
  6. Medium
  7. Germination studies
  8. Growth studies
  9. Statistical treatment of the results
  10. Results
  11. Germination studies
  12. Growth studies
  13. Discussion
  14. Acknowledgements
  15. References
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  • Kassim, M.Y. and Khan, S. (1996) Effect of temperature on growth of Penicillium digitatum and Geotrichum candidum on agar and on citrus fruit and chemical control of post-harvest rot caused by the two fungi. Journal of King Saud University Science 8, 3337.
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