Plant Pathology (2001) 50, 561±568
Effects of temperature and relative humidity on conidial germination and viability, colonization and sporulation of Monilinia fructigena X.-M. Xua*², L. Guerinb and J. D. Robinsona a
Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK; and bENITA de Bordeaux, 1 cours du GeÂneÂral de Gaulle, 33 175 Gradignan BP 201 cedex, France
Experiments were conducted to investigate the effects of temperature and relative humidity (RH) on the in vitro germination and viability of conidia of the apple brown rot fungus (Monilinia fructigena), and on colonization and sporulation on detached fruits by M. fructigena. Conidia only germinated under near-saturation humidity ($ 97% RH) and the rate of germination initially increased with temperature to a maximum at < 23±258C and then decreased. Conidia germinated rapidly ± more than 70% of viable conidia had germinated within 2 h at 20 and 258C. The rate of colonization on detached fruits increased log-linearly with increasing temperature. Sporulation on detached fruits was not observed at 5 or 258C; sporulation appeared to be unaffected by either temperature (10± 208C) or RH (45±98%) once infection was established. Detached conidia remained viable for a long period of time, up to 20 days, the longest assessment time in this study, depending on storage temperature (10 or 208C) and RH (45 or 85%). Temperature appeared to be more important than RH in affecting conidial viability. Low temperature and high RH resulted in reduced loss of conidial viability. Storage at 108C and 85% RH for up to 20 days appeared not to affect conidial viability. These results indicate that environmental conditions during the main UK growing seasons are unlikely to be limiting factors for the development of brown rot on apple. Keywords: apple, Monilinia fructigena, mortality, sporulation
Introduction Brown rot of apple and pear, caused by Monilinia fructigena, causes economic losses both in the orchard and in store (Byrde & Willetts, 1977), with the severity of the losses varying greatly from one year to another. In a badly affected cider apple orchard in the UK, 35´5% of fruit was infected by M. fructigena (Burchill & Edney, 1972). The disease causes losses of up to 5% of apple fruit in store in the UK, the extent of the losses varying with individual orchards (Preece, 1967; Berrie, 1989). Currently in the UK, routine postharvest drenching of apple fruit with fungicides reduces storage losses by < 50%. Little is known about the epidemiology of brown rot on apple in comparison to brown rot on stone fruit caused by M. fructicola and M. laxa (Corbin, 1962; Corbin et al., 1968; Biggs & Northover, 1988; Tamm & Fluckiger, 1993; Northover & Cerkauskas, 1994; Northover & Biggs, 1995; Michailides & Morgan, 1997). In general, *To whom correspondence should be addressed.
apple cultivars are susceptible to M. fructigena (Sharma & Kaul, 1988) and the incidence of brown rot is associated with wounds on the fruit (Moore, 1950; Byrde & Willetts, 1977; Xu et al., 1998; Xu & Robinson, 2000). The degree of resistance to infection increases with increasing wound age (Byrde, 1952; Xu & Robinson, 2000) and decreases with increasing fruit maturity (Xu & Robinson, 2000). Knowledge of the basic biology of the pathogen, such as germination and sporulation in relation to temperature and humidity, is useful for developing a more rational strategy of managing this disease; this may include determining the need for postharvest treatment or the storage potential of the fruit. The purpose of the present research was to evaluate the effects of temperature and relative humidity (RH) on the germination and mortality (or viability) of M. fructigena conidia in vitro and on the colonization of detached apple fruits and subsequent sporulation.
Materials and methods
²E-mail:
[email protected]
Inoculum production
Accepted 28 April 2001.
Isolates of M. fructigena from apple were subcultured
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onto plates of potato dextrose agar (PDA) and incubated in the dark at 208C for < 2 weeks. Apple fruits (cv. Golden Delicious) were wounded with a scalpel and a mycelium plug from the PDA plates was inserted into each wound; each fruit had two wounds on opposite sides. The fruits were then incubated at 208C under a 16/8 h light/dark regime in desiccators at 75% RH, which was maintained by a saturated solution of NaCl (Winston & Bates, 1960). About 2 weeks later, fruits with sporulating lesions were washed with distilled water and a conidial suspension was prepared with the concentration of conidia adjusted as required using counts from a haemocytometer. For studies on conidial viability, 20 fruits were inoculated as described above and placed into plastic boxes instead of desiccators because of the large number of fruits. Thoroughly wetted blue tissues were placed on the bottom of each box before fruits were put in. The boxes were then covered with loose-fitting lids and incubated at 208C under a 16/8 h light/dark regime. About 2 weeks later, conidia were collected dry using a cyclone spore collector (Tervet et al., 1951) to suck conidia into a plastic tube.
Effects of temperature and relative humidity on germination of conidia The agar dish equilibration technique, described by Harris et al. (1970) and modified by Alderman & Beute (1986), was used to control the RH inside the sealed agar plates, this being related to the NaCl molality according to the value given by Lang (1967). The levels of RH tested were 100, 99, 97 and 95%. These were obtained by amending the agar with 0, 0´3, 0´9 and 1´5 m NaCl, respectively. In addition, germination of conidia in free water was also included as a control for each temperature. There were seven temperatures: 5, 10, 12´5, 15, 17´5, 20 and 278C. Therefore, there were a total of 35 treatments. Two glass slides were placed on the lid of a Petri dish containing < 30 mL of water agar amended with the appropriate amount of NaCl to achieve the desired RH. Two 10 m L droplets of conidial suspension were placed separately on each slide by means of a micropipettor and then air-dried. Each plate was then sealed with parafilm and placed upside down in incubators set to the appropriate temperatures. Germination was recorded at 4 and 24 h after conidia were placed on the slides; at each assessment time, there were two plates for each treatment. A drop of cotton blue in lactophenol was placed on each inoculum droplet to stop germination and preserve conidia. Percentage germination was estimated by examining 200 conidia on each inoculum droplet. The effect of temperature on conidial germination was further studied on PDA. Germination was tested at six temperatures ± 3, 10, 15, 20, 25 and 308C ± and assessed 2, 6 and 24 h after inoculating the plates. For each temperature there were three plates and each plate
was seeded with three individual 10 m L droplets of conidial suspension, well separated in the plate. Plates were sealed with parafilm and immediately placed into incubators set to the appropriate temperatures. At each assessment time, germination was estimated by observing 100 conidia from each droplet of inoculum in each plate, thus giving a total of nine counts per treatment at each time. Immediately after counting, plates were placed back into the incubators. The experiment was repeated once.
Effect of temperature on colonization of fruits and subsequent sporulation Initially, colonization of apple fruits was studied at four relative humidities (45, 75, 85 and 98%) over five temperatures (5, 10, 15, 20 and 258C) using desiccators. The RH inside desiccators was controlled using saturated salt solutions: K2CO3 (45%), NaCl (75%), KCl (85%) and K2SO4 (98%); the RH achieved by these solutions varied slightly from with temperature (Winston & Bates, 1960). For each temperature/RH combination, there was one desiccator (with five apples), containing 250 mL of an appropriate saturated salt solution with excess of the solid phase of the salt to maintain the desired RH. Each fruit (cv. Golden Delicious) was wounded and a mycelium plug from the PDA plates was inserted into each wound; each fruit had two wounds on opposite sides. The length and width of each resulting rot were measured 6 days after inoculation for 15, 20 and 258C and 14 days after inoculation for 5 and 108C. The effect of temperature on colonization of fruits was further investigated at 75% RH only. Again, five temperatures were used (5, 10, 15, 20 and 258C), with one desiccator per temperature. Inoculation and recording procedures were as described above. This experiment was repeated once. In the first run, there were five fruits per desiccator; in the second, there were three fruits per desiccator. The effect of temperature on production of conidia was also assessed in the same experiment. Two weeks after mycelium was first observed on the fruit surfaces at each temperature, fruits were individually washed with distilled water, a conidial suspension was made and its volume was measured. The total number of conidia per fruit was then estimated using a haemocytometer. In the previous experiment, sporulation was confounded with colonization. Further experiments were conducted to eliminate/reduce this confounding effect. Only two temperatures (10 and 208C) at four levels of RH (45, 75, 85 and 98%) were tested, giving a total of eight treatments. Fruits were inoculated as described above, placed on wet blue tissues inside plastic boxes and incubated at 208C under a 16/8 h light/dark regime. After 1 week, when the fungus had colonized the fruit surface completely and some fruiting bodies could already be seen, three fruits were randomly chosen and placed into each desiccator for each temperature/ Q 2001 BSPP
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RH combination. The total number of conidia on each fruit was estimated 8 days later, as described above.
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most, at three time-points, no attempt was made to fit temporal models to the germination data. Models were fitted only to individual data sets obtained at each assessment time to relate the overall germination to temperature. Similarly, regression models were fitted to relate colonization and sporulation of fruits to temperature. In fitting models, temperature was scaled down by a factor of 10 to avoid small parameter estimates. For the data describing the effect of storage temperature and RH on the viability of conidia, there were no systematic significant correlations between temporal observations, even though there were a few significant correlations. Therefore, a split-plot analysis was used to assess the effect of storage temperature and RH on the viability of conidia, with time as the subplot factor. Conidial viability over time, in relation to temperature and RH, differed greatly between the three repeats of the experiment, and individual experiments were thus analysed separately. All statistical analyses were done using the Genstat program (Payne et al., 1993).
Effect of storage temperature and humidity on the viability of detached conidia Four combinations of temperature and RH were studied: 10 and 208C, at 45 and 85% RH, one combination per desiccator. Inside each desiccator, there was a microtube rack. Conidia were collected dry as described above. Small numbers of collected conidia were then transferred from the collection tube to each microtube on the rack. Desiccators were placed into incubators set to appropriate temperatures. Germination of the stored conidia was assessed at several intervals after collection. At each assessment time, two microtubes were randomly taken out from each treatment. Sterilized distilled water (100 m L) was immediately added to each microtube and the microtube was then shaken to disperse the conidia. Three 10 m L droplets of conidial suspension were placed separately onto each of two PDA plates for each microtube. Germination was estimated by observing 100 conidia per inoculum droplet after 6±8 h incubation at 208C; thus, there were, in total, 12 germination estimates per treatment at each time. The experiment was repeated twice. In the first run, germination was assessed eight times: after 1, 2, 3, 4, 6, 10, 13 and 20 days of storage. However, in the two repeats, it was only assessed six times: after 1, 2, 3, 6, 10 and 14 days of storage. In addition, germination was estimated at the beginning of each experiment, immediately after conidia were collected from the fruits.
Results Effects of temperature and humidity on germination in vitro On slides, conidial germination was very rapid; for most temperatures, germination after 4 h was already comparable to that at 24 h for the wet and 100% RH treatments (Table 1). anova showed that the main effects of temperature and RH were significant (P , 0´01) for both assessments. Most variation in percentage germination was due to RH, which accounted for < 71 and 79% of the total variation on the logit scale for the 4- and 24-h assessments, respectively, compared with the 12 and 7%, respectively, accounted for by temperature. The interaction between temperature and RH was only significant (P , 0´01) for germination at 4 h, accounting for about 12% of the total variation, and was mainly due to the lower than expected germination for combinations of 158C/free water, 12´58C/100% RH and 108C/ 100% RH (Table 1). Overall, germination increased
Data analysis Analysis of variance (anova) was used to assess the effects of temperature and RH on conidial germination and viability in vitro, and colonization and sporulation on fruits. All percentage germination data were logittransformed (Ln[p/(1 2 p)]), and numbers of conidia and the rate of colonization on fruits were transformed using natural logarithms before anova and modelfitting. Since germination in vitro was only assessed, at
Table 1 Average percentage in vitro germination of Monilinia fructigena conidia (based on 1600 conidia) at various combinations of temperature and humidity, assessed after 4 and 24 h. Relative humidity inside sealed agar plates was controlled by amending the agar with the appropriate amount of NaCl Temperature (8C) 5 10 12´5 15 17´5 20 27
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4h
24 h
Wet
100%
99%
97%
95%RH
Wet
100%
99%
97%
95% RH
7´3 38´6 36´0 23´3 39´5 42´1 37´1
0´4 8´6 16´3 32´2 43´3 42´8 30´0
0´6 0´1 4´0 5´0 15´8 7´6 11´0
0´0 0´1 0´0 0´6 0´1 0´3 4´0
0 0 0 0 0 0 0
22´3 48´8 36´2 39´9 30´4 36´8 39´9
10´9 28´9 37´6 33´8 32´0 39´8 44´4
10´7 15´0 18´9 18´8 17´3 19´8 26´4
3´8 7´4 10´8 17´6 5´0 10´1 9´7
0´0 0´6 0´5 0´8 0´5 0´4 2´3
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Figure 1 Relationship of average percentage germination of Monilinia fructigena conidia on PDA media to temperature. The symbols W, A and K represent germination at assessment times of 2, 6 and 24 h, respectively; the solid and dotted lines are the fitted models for 2-h (equation 1 in text) and 6-h (equation 2 in text) data, respectively.
with increasing temperature and RH. At 4 h, average percentage germination was 32´6, 24´8, 6´4, 0´7 and 0% for free water, 100, 99, 97 and 95% RH, respectively; after 24 h, it was 36´3, 32´5, 18´1, 9´2 and 0´7% for free water, 100, 99, 97 and 95% RH, respectively. After 24 h, the percentage germination was similar at all temperatures except 58C (Table 1). There were clearly large differences in average percentage germination on PDA media between temperatures at the three assessment times (Fig. 1), averaged over the repeats as there were little differences between the repeats. Percentage germination varied progressively less between temperatures as the assessment time increased. anova showed that about 95, 78 and 55% of the total variation in germination on the logit scale was due to temperature for the 2-, 6- and 24-h assessments, respectively. Percentage germination increased with increasing temperature to a maximum at 258C and then declined at 308C (Fig. 1). At 20 and 258C, about 48 and 55%, respectively, of conidia had already germinated only 2 h after seeding the plates, whereas no conidia germinated at 3 or 308C by 2 h. After 6 h, the percentage germination at 10±258C was already close to that observed at 24 h. Most conidia germinated during the 2- to 6-h period at 108C, whilst at 38C, most conidia germinated in the period from 6 to 24 h postseeding. The final percentage germination (35%) at 308C was only about half that observed at 15±258C. For germination at 2 h, the following nonlinear model fitted the data well, accounting for about 94% of the total variation on the logit scale:
Figure 2 The rate (natural logarithm transformed) of Monilinia fructigena colonizing apple fruit in relation to (a) temperature and relative humidity, and (b) temperature at 75% RH. The symbols W, X and K represent the three data sets and the solid line is the fitted model (equation 3 in text).
temperature; standard errors of parameter estimates are in the brackets below the corresponding parameter. This model estimated an optimum temperature for germination of < 238C (Fig. 1). For germination at 6 h, the following nonlinear model fitted the data satisfactorily, accounting for about 72% of the total variation on the logit scale: logit
g 24´09 1 3´93T 2 0´33T 3 (0´243)
(0´234)
2
(0´022)
However, this model appeared to underestimate the optimum temperature for germination (Fig. 1). Data obtained at 24 h could not be fitted to any simple model.
1
Effects of temperature on colonization of apple fruits
where g is the percentage germination and T is the
In the experiment where both RH and temperature were included, temperature had much greater effects than RH on colonization (Fig. 2a). anova showed that
logit
g 27´8 1
1´97 2 0´63T3´39T (0´470)
(0´319)
(0´098) (0´196)
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Figure 3 Histogram of numbers of Monilinia fructigena conidia produced on detached apple fruits over five temperatures at 75% RH; no conidia were observed at 5 and 258C. In this experiment, colonization of fruit was confounded with the subsequent sporulation.
temperature accounted for about 75% of the total variation, compared with 8 and 10% by RH and the RH/temperature interaction, respectively. Overall, the rate of colonization decreased with increasing RH. However, the main effect of RH was not significant when tested against the interaction term. The significant (P , 0´05) interaction was mainly due to the much lower than expected colonization rate at 158C and 98% RH. Colonization rate generally increased log-linearly with temperature. This log-linear relationship with temperature was also observed when colonization of apple fruits was studied over the same temperature range but only at 75% RH (Fig. 2b). The following linear model accounted for about 84% of the total variation and fitted the combined data at 75% RH well: ln
x 21´12 1 0´12T (0´137)
3
(0´008)
where x is the colonization rate (cm2 day21).
Effects of temperature and humidity on sporulation on apple fruits In the experiment where colonization and sporulation were confounded, no conidia were detected after 30 days at 58C or 20 days at 258C, even though the fruits at 258C were completely colonized. There were large variations between fruits within each treatment. Overall, numbers of conidia produced per fruit increased significantly with increasing temperature, and the overall pattern between the two experiments was similar (Fig. 3). However, more conidia were produced in the second experiment than in the first and the increase with temperature appeared to be greater (Fig. 3). In the experiments where initial colonization and Q 2001 BSPP
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Figure 4 Conidial germination over time in relation to storage temperature and relative humidity (a, b and c are experiments repeated twice).
spore initiation were in the same environment, there were no significant differences in the number of conidia produced between treatments (Table 2). The average number of conidia produced appeared to be greater at 108C than at 208C; however, the difference was not significant because of large fruit-to-fruit variation within each treatment.
Effects of storage temperature and humidity on the viability of conidia The relationship between the temporal variation in
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Table 2 Average number (£ 103) of conidia of Monilinia fructigena produced per fruit under various combinations of temperature and relative humidity. In this experiment, colonization and sporulation were not confounded initially Temperature (8C) 10 20
Relative humidity 45%
76%
85%
98%
617 199
328 279
368 336
386 303
conidial viability, temperature and RH differed considerably between the three repeated experiments (Fig. 4). In the first experiment, there was a large reduction, from 52 to 23%, in conidial germination following a 24-h period at 45% RH (Fig. 4a). Thereafter, the reduction in percentage germination was slower at 108C than at 208C. For conidia stored at 85% RH, there was no reduction in germination after 4 days. However, after 10 days the germination of conidia stored at 208C decreased steeply to 10%, then decreased gradually to zero by 20 days. For conidia stored at 108C and 85% RH, there were virtually no reductions in germination ability. anova showed that variation in germination between tubes was due almost entirely to temperature (42% of variation in the data) and RH (57% of variation in the data); effects which were both significant at 1%. About 54% of variation in germination within tubes was due to the length of time for which conidia were stored under the treatment conditions; most of the time effect (
