Effects of fruit maturity, wound age, temperature and the duration of wetness periods on infection of apple fruits by conidia of the brown rot fungus, Monilinia ...
Plant Pathology (2000) 49, 201–206
Epidemiology of brown rot (Monilinia fructigena) on apple: infection of fruits by conidia X.-M. Xu and J. D. Robinson Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK
Effects of fruit maturity, wound age, temperature and the duration of wetness periods on infection of apple fruits by conidia of the brown rot fungus, Monilinia fructigena, were studied. Inoculation of fruits on potted apple trees and harvested mature fruits showed that wounding was essential for infection by M. fructigena. On potted trees, there was a significant difference between the susceptibility of cvs Cox and Gala and this difference depended on wound age. The incidence of brown rot was affected greatly by fruit maturity and wound age. Wounds on younger fruits were more resistant to infection than those on older fruits, whilst the older the wound, the more resistant it was to infection. Furthermore, the degree of wound age-related resistance was greater on younger fruits than on older fruits. These relationships were well described by regression models. The effect of the duration of wetness periods was very small: increasing the duration of wetness periods reduced the incidence of brown rot on older wounds. For detached fruits, all those wounded were rotted after inoculation, except for those in two treatments under 208C on fruits with wounds which were 8 days old. The incubation period of the fungus was generally very short. Wound age was the single most important factor influencing the length of the incubation period; the incubation period increased as wound age increased. Keywords: apple, fruit maturity, infection, Monilinia fructigena, wound age
Introduction Brown rot of apple and pear caused by Monilinia fructigena leads to economic losses every year in the orchard and in store (Byrde & Willetts, 1977), with the severity of the losses varying greatly from one year to another, largely as a result of differences in weather. In a badly affected cider apple orchard in the UK 35·5% of fruit was infected by M. fructigena (Burchill & Edney, 1972), whilst in Italy a loss of 7·3% of apple fruit was reported on unsprayed trees of Rome Beauty (Byrde & Willetts, 1977). According to Preece (1967) and Berrie (1989), 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. Currently in the UK, routine postharvest drenching of apple fruit with fungicides reduces storage losses by approximately 50%. In general, apple cultivars are susceptible to M. fructigena (Sharma & Kaul, 1988), and the incidence of brown rot is believed to be associated mainly with wounds on the fruit (Moore, 1950; Byrde & Willetts, 1977), the degree of resistance to infection increasing with wound age (Byrde, 1952). Applying chemicals immediately after wounding significantly reduced incidence of brown rot (Hellmann, 1998). Compared with Accepted 30 September 1999. Q 2000 BSPP
brown rot on stone fruits caused by M. laxa and M. fructicola, knowledge of the epidemiology of M. fructigena on the apple is limited; such knowledge is essential to develop a more rational disease management strategy. The purpose of the present research was to evaluate the effects of environmental and host factors on the infection of apple fruit by conidia of M. fructigena. The specific objectives were to determine: (i) whether wounding is essential for infection by conidia, and (ii) the effects of wound age, fruit maturity, temperature and the duration of wetness on infection by conidia of M. fructigena.
Materials and methods Five experiments were carried out at Horticulture Research International (HRI) ¹ East Malling, Kent, UK, using three apple cultivars (Table 1).
Plant material Experiments 1–3 used fruits attached to potted apple trees of 3 or 4 years old on MM106 rootstocks. Trees were in 30-cm diameter pots (15 L) and grown at HRI ¹ East Malling on sand-beds or in a polythene tunnel. Fungicides were not applied to the trees at any stage. 201
202
X.-M. Xu & J. D. Robinson
Table 1 Summary of experimental details of the five experiments conducted to study infections of apple fruits by conidia of Monilinia fructigena Fruits attached to potted trees
Fruits picked from orchards
Treatment
Expt 1
Expt 2
Expt 3
Expt 4
Expt 5
Cultivars Temperature (8C) Fruit maturity (weeks after full bloom) Wounded (W)/healthy (H) Wound age (days) Duration of wetness (h)
Cox 20 10, 14, 18 W, H 0 (Fresh) 24
Cox, Gala 20 10, 14, 18 W 0, 1, 3, 6 24
Cox 20 18 W 2, 4, 6, 8 0, 2, 8, 24, 48
Cox, Golden Delicious 10, 20 Near mature W, H 0, 1, 2 24
Cox 10, 20 Near mature W 2, 4, 6, 8 8, 24
Total number of treatments Number of replicates
6 4 trees per treatment
24 3 trees
20 3 trees
16 4 boxes
16 2 boxes
Inoculum concentration (conidia per mL)
7·5 × 105
2·5 × 105
2·5 × 105
2·5 × 105
2·5 × 105
Experiments 4 and 5 used healthy mature fruits harvested from trees on M9 rootstocks in orchards planted in 1974 (Expt 4) or trees on MM106 rootstocks planted in 1984 (Expt 5). Pests and diseases were controlled in the orchards with routine spray programmes. Harvested fruits were stored in a cold store (38C) until needed for experimentation.
Inoculum and inoculation Isolates of M. fructigena from apple were subcultured onto plates of potato dextrose agar (PDA) and incubated in the dark at 208C for < 2 weeks. Apple fruits (cv. Golden Delicious) were wounded 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 in desiccators at 75% relative humidity which was maintained by a saturated solution of NaCl. On the day of inoculation, < 2 weeks later, fruits with sporulating lesions were washed with distilled water and a spore suspension was prepared with its spore concentration adjusted as required using a haemocytometer. In all experiments and in inoculum production, fruits were wounded by pushing the tip of a pair of fine forceps into the fruit skin (< 0·5 cm deep). In the experiments wounding was carried out at various times, to create fruits with different ages of wound. Each wound was inoculated with two 10-mL droplets of spore suspension.
Treatments Table 1 gives the summary of treatment factors in each experiment. In Expts 1–3, trees were moved to glasshouse compartments (< 25 m2) with three misting nozzles to maintain surface wetness on the day before the first wound was made. Trees were assigned randomly to treatments and positioned randomly in the compartments. Temperature in the compartments was set at 208C: the actual recorded temperature ranged from 19
to 238C. Only one compartment was used in Expts 1 and 2 and misting was turned off 24 h (i.e. 24 h duration of wetness) after inoculation. Two compartments were needed for Expt 3 because there was not enough space in one compartment for all the experimental trees. Two trees from each treatment, randomly chosen from the three trees, were moved into one compartment and the remaining third tree into the other. To prevent the fruit dropping when moving the trees, trees allocated to shorter wetness periods were placed near the door. In Expts 4 and 5, fruits were placed individually in the cups of polystyrene apple-marketing trays, each tray in an open-top waxed cardboard box holding a maximum of 27 (Expt 4) or 35 (Expt 5) fruits. Treatments were allocated randomly to boxes, which were stacked randomly in each cabinet. In Expt 4, a further four boxes of fruits were maintained at each temperature: two boxes as an unwounded control and the other two as a freshly wounded control. Immediately after inoculation, the fruits were wetted with distilled water using a garden sprayer. The boxes were then enclosed in loosely sealed wet polythene bags to keep fruits wet, and placed in controlled environment (CE) cabinets with the temperature set appropriately.
Disease assessment The inoculated trees were moved out of the compartments to a polythene tunnel at the end of each designated wet period. The inoculated fruits were usually examined twice a week for brown rot lesions and the date on which each rot was first seen was recorded. To prevent the fungus from spreading onto spurs, fruits with brown rot lesions were removed immediately after they were first seen. The boxes of fruits were removed from the polythene bags at the end of each designated wet period and placed back in the cabinet. After the longest wet period was complete, fruits were incubated at 108C in CE cabinets Q 2000 BSPP
Plant Pathology (2000) 49, 201–206
Infection of apples by Monilinia fructigena
(Expt 4) or incubated at 108C in CE cabinets for a week and then at room temperature (< 188C: Expt 5). Fruits were frequently observed for rots; rotted fruits were removed immediately after they were first seen.
Data analysis Logistic regression analysis (Cox & Snell, 1989), which is based on the logit transformation of the proportion, p, of fruits infected, i.e. ln[p/(1 ¹ p)], was used to assess the effects of treatments on the final incidence of inoculated fruits with brown rot lesions. The number of fruits with brown rot lesions, within each tree in Expts 1–3, or within each box in Expts 4 and 5, was assumed to be distributed binomially. Analysis of variance (ANOVA) was used to assess the effects of treatments on the incubation period (logarithmically transformed). The incubation period was estimated as the time from inoculation to the day the lesion was first seen. In Expt 2, there were not enough rots for complete ANOVA for this experiment as a whole. Enough rots for ANOVA were only found within the following two subgroups: the inoculated fruits with fresh wounds and the fruits inoculated 18 weeks after full bloom. Therefore, only one-way ANOVA was applied to each of the two subgroups. Because of the unequal numbers of fruits with brown rot lesions within each treatment combination, an unbalanced factorial ANOVA was applied to Expts 3 and 5. In Expts 1–3, the effects of treatment factors were tested against the variation among trees to assess the significance; in Expts 4 and 5, the effects of treatment factors were tested against the variation among boxes to assess the significance. A regression model was not developed for the incubation period because ANOVA showed that treatment factors explained less than 30% of the total variation for all the experiments.
203
Incidence of brown rot The number of fruits per treatment in Expt 1 ranged from 120 to 280. None of the inoculations of nonwounded fruits resulted in rots. The effect of fruit maturity on infection was significant (P < 0·01). On the first inoculation date (10 weeks after full bloom), about 50% of the inoculated fruits with fresh wounds became infected, whereas inoculation at 14 and 18 weeks after full bloom resulted in more than 97% infection. In Expt 2, the final incidence of brown rot was affected significantly by cultivar, wound age and fruit maturity (P < 0·01) as well as by the interactions between cultivar and wound age (P < 0·05) and between wound age and fruit maturity (P < 0·01). However, the effects of the interactions were much smaller than the main effects. Overall, the incidence for Cox and Gala was 46·8% and 40·6%, respectively, but the difference between the cultivars decreased as wound age increased (Table 2). The incidence increased significantly with fruit maturity and decreased significantly with increasing wound age. In Expt 3, the incidence of brown rot was significantly affected by wound age (P < 0·01), the duration of wet periods (P < 0·05) and their interaction (P < 0·05) (Table 3). The incidence decreased with increasing wound age and with increasing length of the wet periods. The effects of wound age increased with increasing duration of the wet periods. The combined data from Expts 1–3, excluding the data of healthy unwounded fruits in Expt 1 as infection could only occur on wounded fruits, were used to develop models that relate the incidence of brown rot to cultivar, wound age and fruit maturity. The following empirical regression models were chosen based on the goodness of the fit and the residuals. For Gala, the model is: ln½p=ð1 ¹ pÞÿ ¼ ¹ 4:177 þ 0:034M2 √ ¹ 3:694 A þ 0:021MA
Results
(0·2110) (0·0014) (0·2280) (0·0035)
In Expts 1–3, the number of fruits inoculated varied considerably between treatments because of the variation in fruit set among the trees and the number of fruits that dropped during the experiments.
where M and A are fruit maturity (numbers of weeks from full bloom) and wound age (days), respectively. The standard errors of the estimates are shown in parentheses below the corresponding parameters; all the
Table 2 Effects of wound age and fruit maturity on the final incidence (%) of brown rot on apple fruits on potted cvs Gala and Cox trees, inoculated with M. fructigena conidia (Expt 2) Galaa
Coxa
Wound age
10
14
18
10
14
18
Fresh 1 day old 3 days old 6 days old
36.7 (80)b 0.0 (118) 0.9 (106) 0.0 (114)
94.0 (77) 20.0 (92) 4.8 (80) 1.2 (116)
96.3 (69) 100.0 (75) 82.4 (83) 51.0 (68)
50.9 (83) 2.1 (87) 0.0 (72) 0.0 (54)
100.0 (47) 62.6 (109) 5.8 (83) 0.0 (49)
100.0 (57) 99.0 (47) 88.4 (29) 53.2 (30)
a
Fruit maturity at inoculation (numbers of weeks after full bloom). Numbers in the brackets are the total numbers of fruits inoculated.
b
Q 2000 BSPP
Plant Pathology (2000) 49, 201–206
204
X.-M. Xu & J. D. Robinson
Figure 1 The incidence of brown rot on apple fruits caused by M. fructigena in relation to fruit maturity and wound age predicted by the regression model for cv. Cox.
parameter estimates were significant (P < 0·01). For Cox, the constant term in this equation is ¹3·223, not ¹4·177, with the standard error of 0·178. The correlation between fitted and observed incidence was 0·98 for both Gala and Cox. The incidence of brown rot predicted by the equation in relation to fruit maturity and wound age is shown in Fig. 1 for Cox. All the wounded fruits in Expt 4 were rotted by the fungus after inoculation; none of the inoculated healthy fruits was rotted and only two of the 216 uninoculated control fruits were rotted. In most treatment combinations of Expt 5, more than 99% of the inoculated fruits became infected. The only exception was for 8-day-old wounds under 208C where the percentages infected, based on 70 fruits, were 85·7% and 66·7% after an 8-h and a 24-h wet period, respectively.
Length of incubation period For the rots resulting from inoculation of fruits with fresh wounds in Expts 1 and 2 as well as for all the rots in Expt 4, there were no differences between treatments: all the rots developed within 5 days (the first recording date) of the inoculation. The length of the incubation period increased significantly (P < 0·01) with increasing wound age in Expts 2 and 3; however, the actual differences in the length of the incubation period in Expt 2 were small (Table 4). In Expt 2, the effects of cultivar were significant (P < 0·01); the average length of the incubation period was 0·4 days longer on Gala than on Cox. However, the cultivar effects in Expt 4 were not significant. In Expt 5, the length of the incubation period was affected significantly by wound age (P < 0·01), temperature
Table 3 Effects of wound age and the duration of wet periods on the final incidence (%) of brown rot on apple fruits on potted cv. Cox trees, inoculated with M. fructigena conidia (Expt 3) Duration of wetness periods (h) Wound age
0
4
8
24
48
2 days old 4 days old 6 days old 8 days old
90·8 (79)a 97·8 (63) 77·8 (31) 79·3 (47)
100·0 (90) 89·8 (58) 67·4 (46) 88·9 (39)
87·9 (66) 90·0 (72) 61·0 (38) 54·8 (30)
100·0 (66) 85·7 (45) 65·6 (51) 44·2 (19)
94·9 (88) 90·7 (45) 66·1 (43) 49·8 (72)
a
Numbers in the brackets are the total numbers of fruits inoculated.
Q 2000 BSPP
Plant Pathology (2000) 49, 201–206
Infection of apples by Monilinia fructigena
205
Table 4 The average, median and interquartile range (IQR: the number of days from the 25% to the 75% quartiles) of incubation period (days) of brown rot on apple fruits following inoculation with M. fructigena conidia Expt 5 Wound age (days) 0 1 2 3 4 6 8
Expt 2
108C
Expt 3
Average
Median
IQR
4·0 4·2
4 4
0 0
5·2
5
3
5·3
5
3
Median
IQR
Average
Median
IQR
Average
Median
IQR
4·4
4
0
8·1
7
3
5·3
4
3
6·9 7·9 9·6
5 7 7
3 7 7
7·8 8·5 11·3
7 7 10
3 3 5
6·6 7·4 12·0
7 7 10
3 0 11
(P < 0·01) and their interaction (P < 0·05) but not by the duration of the wet periods. The incubation period lengthened with increasing wound age and with decreasing temperature, but the difference between temperatures decreased as wound age increased (Table 4).
Discussion Conidia of M. fructigena were shown in the present study to infect apple fruits only via wounds. Although direct infection of apple fruits by M. fructigena conidia through lenticels has been observed in the laboratory (Sharma & Kaul, 1990), in nature brown rot is believed to result mainly from wound parasitism (Byrde & Willetts, 1977), as confirmed by detailed orchard studies on brown rot of apple and pear (Xu et al., 1998). Younger fruits were less susceptible to infection by M. fructigena conidia even when fresh wounds were inoculated. There are several possible explanations for this. Firstly, the incidence of brown rot has been correlated negatively with acidity and positively with the pH of fruit juice (Sharma & Kaul, 1988). Higher levels of acidity in fruit juice are associated with younger fruits. Secondly, low sugar contents of fruit juice and high respiration rates are considered to be associated with resistance to M. fructigena (Byrde & Willetts, 1977). In younger fruits, sugar levels are generally lower than in older fruits and respiration rates are expected to be higher in response to wounding on young fruits than on old fruits. On stone fruits, nonwounded green fruits of cherry (Brown & Wilcox, 1989), peach and plum (Fourie & Holz, 1987) were less susceptible to the brown rot fungus, M. fructicola. The susceptibility of immature peach fruits to M. fructicola fluctuated with the stage of fruit development (Biggs & Northover, 1988a). The incidence of brown rot caused by M. fructigena decreased with increasing wound age, as observed by Byrde (1952). Similarly, wounds on apple fruits became more resistant to infection by Botrytis cinerea and Penicillium expansum as they aged (Lakshminarayana et al., 1987). This may be due to the wound healing process that leads to the formation of boundary zone Q 2000 BSPP
Average
208C
Plant Pathology (2000) 49, 201–206
tissue and wound periderm (Mullick, 1977; Skene, 1981; Bostock & Stermer, 1989) and/or to the gradual depletion of free nutrients on the wound surface (Srivastova & Walker, 1959; Wade & Cruickshank, 1992). The effect of wound age decreased as fruits matured and this dependence may be because of the effects of fruit maturity on the wound healing and nutrient depletion processes. Immature developing apple fruits have an extensive and rapid wound reaction that results in periderm formation whereas, on mature fruits, this reaction is reduced, resulting in the absence of periderm (Skene, 1981). A better understanding of the age-related mechanisms of the resistance of wounds on fruits to pathogens is needed. The effects of fruit maturity and wound age on infection of apple fruits of potted trees by M. fructigena were well described by regression models. The accuracy and reliability of the models need to be further evaluated in field conditions. The differences between the incidences of brown rot on Cox and Gala might be due to their differences in the development stage at the times of inoculation as well as due to their true differences in susceptibility. The effects of the duration of wet periods on infection were very small. This may be due to two reasons. First, conidia may enter directly into fruits through unhealed wound tissues. Second, moisture on the wound surface may be enough for conidia to germinate and infect. Sporulation of newly infected fruits and infection of wounded fruits by dry conidia do not need free water (X.-M. Xu, unpublished data). It is likely therefore that infection of wounded fruits by M. fructigena does not need rainfall or wetness although rainfall was needed to initiate sporulation on overwintered mummified fruits (Byrde & Willetts, 1977). Generally, incidence of brown rot was negatively correlated with the incubation period. However, residual variation was substantial for the incubation period compared with disease incidence. The incubation period increased with increasing wound age and this is consistent with the explanations of resistance associated with wound age. The effects of temperature on the incubation period also depended on wound age and this probably resulted from interactions between the rate of
206
X.-M. Xu & J. D. Robinson
fungal development and the rates of wound healing and/ or nutrient depletion on wound surfaces. The incubation period was not affected significantly by the duration of the wet period. This is in contrast to the development of brown rot on sweet cherry where increasing lengths of the wet periods reduced the incubation period (Biggs & Northover, 1988b). The incidence of brown rot was shown to be affected greatly by fruit maturity and wound age. In the current study, each fruit was inoculated on two wounds. A fruit was classified as infected irrespective of whether both wounds were infected. Assuming that the infection on the two wounds is independent, the disease incidence (ps) based on a single wound can be estimated from the incidence (pt) reported in the present study. The difference between ps and pt is a nonlinear function of pt (i.e. pt ¹ ps ¼ ps ¹ p2t $ 0), which has a maximum (25%) at ps ¼ 50% and decreases to zero as ps either decreases to zero or increases to 100%. The effects of fruit maturity and wound age on the infection of fruits by conidia were therefore expected to be larger if ps was used instead of pt. This study clearly showed that in order to reduce the incidence of brown rot, it is essential to protect fruits, especially near-mature fruits, from being wounded. Furthermore, careful picking of fruits is warranted to reduce the risk of wounded fruits with incubating lesions being picked and thus acting as a focus of the secondary spread by mycelia in store. Careful postharvest handling is also needed to prevent fruit from being damaged and infected.
Acknowledgements This work was funded by the Ministry of Agriculture, Fisheries and Food (MAFF).
References Berrie AM, 1989. Storage rots of apple and pear in South East England 1980–88: incidence and fungicide resistance. In: Gessler BK, ed. IOBC Bulletin, Vol. II, Integrated Control of Pome Fruit Diseases. Locarno, Switzerland: IOBC, 229–39. Biggs AR, Northover J, 1988a. Early- and late-season susceptibility of peach fruits to Monilinia fructicola. Plant Disease 72, 1070–4. Biggs AR, Northover J, 1988b. Influence of temperature and wetness duration on infection of peach and sweet cherry fruits by Monilinia fructicola. Phytopathology 78, 1352–6. Bostock RM, Stermer BA, 1989. Perspectives on wound healing in resistance to pathogens. Annual Review of Phytopathology 27, 343–71.
Brown SK, Wilcox WF, 1989. Evaluation of cherry genotypes for resistance to fruit infection by Monilinia fructicola (Wint.) Honey. Hortscience 24, 1013–5. Burchill RT, Edney KL, 1972. An assessment of some new treatments for the control of rotting of stored apples. Annals of Applied Biology 72, 249–55. Byrde RJW, 1952. The effect of age of wound and weather on the susceptibility of apple injuries to infection by the brown rot fungus. In: University of Bristol Annual Report of Agriculture and Horticulture Research Station, 1951. Bristol, UK: Long Ashton Research Station, 128–31. Byrde RJW, Willetts HJ, 1977. The Brown Rot Fungi of Fruit. Their Biology and Control. Oxford, UK: Pergamon Press. Cox DR, Snell EJ, 1989. Analysis of Binary Data. London, UK: Chapman & Hall. Fourie JF, Holz G, 1987. Infection and decay of stone fruit by Botrytis cinerea and Monilinia laxa at different stages after anthesis. Phytophylactica 19, 45–6. Hellmann M, 1998. Monilia fructigena ¹ fruit rot in apple after artificial infraction of fruit skin in the summertime. Acta Horticulturae 466, 149–53. Lakshminarayana S, Sommer NF, Polito V, Fortlage RJ, 1987. Development of resistance to infection by Botrytis cinerea and Penicillium expansum in wounds of mature apple fruits. Phytopathology 77, 1674–8. Moore MH, 1950. Brown rot of apples: fungicide trials, and studies of the relative importance of different woundagents. Journal of Horticultural Science 25, 225–34. Mullick DB, 1977. The non-specific nature of defence in bark and wood during wounding, insect, and pathogen attack. Recent Advances in Phytochemistry 11, 395–441. Preece TF, 1967. Losses of Cox’s Orange Pippin apples during refrigerated storage in England, 1961–65. Plant Pathology 16, 176–80. Sharma RL, Kaul JL, 1988. Susceptibility of apples to brown rot in relation to qualitative characters. Indian Phytopathology 41, 410–5. Sharma RL, Kaul JL, 1990. Mode of entry and histopathological changes induced by Monilinia species in apple fruit. Indian Phytopathology 43, 113–5. Skene DS, 1981. Wound healing in apple fruits: the anatomical response of Cox’s Orange Pippin at different stages of development. Journal of Horticultural Science 56, 145–53. Srivastova DN, Walker JC, 1959. Mechanisms of infection of sweet potato roots by Rhizopus stolonifer. Phytopathology 49, 400–6. Wade GC, Cruickshank RH, 1992. Rapid development of resistance of wounds on immature apricot fruit to infection with Monilinia fructicola. Journal of Phytopathology 136, 89–94. Xu X-M, Berrie AM, Harris DC, 1998. Epidemiology of brown rot on apple and pear. Proceedings of the 7th International Congress of Plant Pathology, Edinburgh III, 3.7.52 (Abstract).
Q 2000 BSPP
Plant Pathology (2000) 49, 201–206
