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Overwintering of Venturia inaequalis in relation to lesion intensity on leaf surfaces, and leaf surface exposed
[ 495 ] Trans. Br. mycol. Soc. 83 (3), 495-500 (1984) Printed in Great Britain
OVERWINTERING OF VENTURIA INAEQUALIS IN RELATION TO LESION INTENSITY ON LEAF SURFACES, AND LEAF SURFACE EXPOSED By M. J. JEGER* East Malling Research Station, Maidstone, Kent ME19 6B], U.K. Apple leaves bearing scab lesions were overwintered under natural conditions on grass. The relationship of pseudothecium development with lesion intensity at leaf-fall was monitored on cvs Cox's Orange Pippin and Lord Lamboume. More pseudothecia were observed on Lamboume than on Cox, and they matured at a faster rate, especially on the abaxial surface. Lesion intensity at leaf-fall had little effect on either numbers of pesudothecia or the rate of maturation although there were higher-order interactions with the surface on which pseudothecia developed. The effects on pseudothecium development of exposing upper or lower surfaces were examined in cv. Bramley's Seedling. Where lesions were on the adaxial surface, more pseudothecia developed there than on the abaxial surface irrespective of which surface was exposed. Where lesions were on the abaxial surface, the largest number of pseudothecia developed on whichever surface was exposed. Ascospore production em"? leaf was related to pseudothecium maturation. Factors influencing the overwintering of Venturia inaequalis (Cke) Wint., as pseudothecia on fallen apple leaves, were proposed by Wilson (1928) and included temperature, moisture, cultivar, time of leaf-fall and time of infection. There have been many studies, under natural and controlled conditions, ofenvironmental influences on the numbers of pseudothecia initiated and their subsequent development to produce mature ascospores in spring (see MacHardy & Ieger, 1983; Jeger & Butt, 1983; and references therein). The influences of cultivar are equally important (Moller, 1980; Jeger, Swait & Butt, 1982) but less studied. The time of leaf-fall influences the number of pseudothecia initiated (Hirst & Stedman, 1962; Burchill, 1968) but environmental conditions at leaf-fall probably have a greater effect (Gadoury & MacHardy, 1982). The influence ofthe age distribution and numbers of lesions on pseudothecial development has not been studied, even by such a gross measure as the proportion of leaf surface scabbed. Early lesions are more common on adaxial (upper) surfaces and are well-defined at leaf-fall, whereas late lesions are often on abaxial (lower) surfaces and more diffuse. Young lesions produce pseudothecia readily, whereas the older do so only at the lesion margins (Wilson, 1928). Pseudothecia can develop with ostioles protruding on either leaf surface, but it is
* Present address: Department of Plant Sciences, Texas A & M University, College Station, TX 77843, U.S.A.
not known whether their formation depends on which surface bears the lesions, or on which surface is exposed. James, Sutton & Grand (1981), on the basis ofan unusual ascocarp development, suggested that there is also a geotropic response. The aims of the present study, complementary to previous studies (Jeger et al., 1982; Jeger & Butt, 1983), were to evaluate the influence of lesion intensity on each leaf surface, and of the leaf surface exposed, on the development of pseudothecia under field conditions. MATERIALS AND METHODS
Influence of lesion intensity Scabbed leaves of cvs Cox's Orange Pippin and Lord Lambourne were collected from an unsprayed orchard at Rocks Farm, East Mailing, on 5 November 1982. Scab intensity was assessed as light (few, discrete lesions), medium (~10% of leaf surface covered by lesions) and heavy (~50 % of leaf surface covered by lesions). Leaves were also classified according to whether one or both surfaces were bearing lesions at a given class of intensity. Those leaves which could not readily be classified, and those with high levels of necrosis, were discarded. The stringent criteria for selection ofleaves meant that abaxial and adaxial surfaces could not be discriminated within the one-surface-scabbed categories. Samples comprising ten leaves/bag were placed in 1 mm mesh nylon bags (Tygan T030) and pegged out in a completely randomized design on
Overwintering of Venturia inaequalis grass on 8 November. The number of replicate bags for each combination of cultivar x intensity class x number of surfaces was usually three, but ranged from one to four depending on the number ofleaves selected. Samples were taken on seven occasions at weekly intervals from 1 February to 21 March 1983. On each occasion, one disk (1 em") was punched from a leaf, wetted for 2 min, and examined under a dissecting microscope. A maximum of 25 pseudothecia were dissected from each leaf surface, using a standard sampling procedure and the contents classified according to the stage of maturation (Jeger et al., 1982). The numbers of pseudothecia were independently counted for correlating with the numbers sampled; counts of pseudothecia alone can be unreliable because other fungal structures may be included inadvertently in the counts. All leaf remains were collected on 24 March, a maximum of 50 disks (1 ern") punched for each treatment combination and ascospore production cm ? leaf assessed as described by Hutton & Burchill (1965). The procedure was repeated on 28 March. Influence of the surface bearing the lesions, and the surface exposed Leaves of Cox's Orange Pippin and Bramley's Seedling were collected from unsprayed orchards at Rocks Farm, East Mailing, and sorted into groups bearing lesions on the abaxial surface only, the adaxial surface only, or both surfaces. In this experiment it was not possible to discriminate intensity classes for a given leaf surface. Two sets of leaves were matched for each category and ro-leaf samples placed in nylon bags. One set of bags was pegged out with the abaxial surface exposed (i.e, facing up), the other set with the adaxial surface exposed, giving a total of six treatment combinations for each cultivar. Samples were taken at z-weekly intervals from 5 January to 15 February 1982, and at weekly intervals from 22 February to 5 April. On each occasion, disks were punched and examined as described for the other experiment and a maximum of 25 pseudothecia classified for each treatment combination. Ascospore production cm"" leaf was assessed on 6 April. RESULTS
There were no trends in numbers of pseudothecia during the assessment period of either experiment, nor interactions between any partitioning of time (linear and quadratic trends, and deviations from these trends) and other factors. Maturation, however, showed a linear trend with time in both experiments (P < 0'01 in lesion intensity expt ;
Table 1. Maturation ofV. inaequalis pseudothecia in relation to time of assessment Lesion intensity expt
Date 1. 14. 21. 28. 7. 14. 21.
ii. 83 ii. 83 ii. 83 ii. 83 iii. 83 iii. 83 iii. 83
S.E.D.
Maturation (0-5) 0'74 0'84 1'03 1'16 1'3 8 1'46 1'7 6 0'36
Surface exposed expt
Date 22. 1. 8. 15. 22. 29. 5.
ii. 82 iii. 82 iii. 82 iii. 82 iii. 82 iii. 82 iv. 82
S.E.D.
Maturation (0-5) 1'25 1'25 1'28 2'26 2'65 2'55 2'93 0,60
P < 0'001 in surface exposed expt) (Table 1). The number of pseudothecia counted independently (y) was related to the numbers sampled for classification of their contents (x) according to the regression equation y = -4·78+1·006x. The slope was not significantly different from unity, which gives some confidence in the sampling procedure used, but the negative intercept indicates a possible bias in searching for pseudothecia at very low densities and the equation is unreliable at high densities because the number of pseudothecia sampled was limited to 25. Despite these qualifications, the numbers of pseudothecia sampled (and known to be of V. inaequalis) were examined in the following analysis. Influence of lesion intensity Lord Lamboume generally exhibited larger mean values than Cox's Orange Pippin for both numbers of pseudothecia and maturation (Table 2). For numbers of pseudothecia, the difference was only significant where pseudothecia developed on the abaxial surface (P < 0'01). Maturation over the assessment period was more advanced where pseudothecia developed on the abaxial surface, but was not affected by whether one or both surfaces bore lesions. There was a significant interaction between cultivar, the number of surfaces bearing lesions, and the surface on which pseudothecia developed (P < 0'05): where both sides bore lesions, there was little difference in maturation on adaxial surfaces when comparing Lord Lamboume and Cox's Orange Pippin. There were significant interactions between lesion intensity, number of surfaces bearing lesions and the surface on which pseudothecia developed (Table 3) that arose when one side only was heavily scabbed: for pseudothecial numbers (P < 0'01), a primary cause was the relatively low value for the adaxial surface, whereas for mean maturation
M.J.Jeger
497
Table 2. Mean maturation (during assessment period) and numbersofpseudothecia ofV. inaequalis in relation to cultivar, the number of surfaces bearing lesions and the surface examined Abundance on:
Maturation on: Surfaces (no.)
Adaxial surface
Abaxial surface
1 2
0'78 0'63
0'96 0,83
1 2
1'50 0'89
1,81 2'16
Cox's Orange Pippin Mean Lord Lamboume
Adaxial surface
Abaxial surface
16'98
16'52
Mean S.E.D.
Horizontal Other
~n
~~
0'31
0'76
Table 3. Mean maturation (during assessment period) and numbers ofV. inaequalis pseudothecia in relation to lesion intensity, the number of surfaces bearing lesions and the surface examined Maturation on: Surfaces (no.)
2
Lesion intensity Low Medium Heavy Low Medium Heavy
Adaxial surface 0,86 1'3 1 1'25 0'67 0'91 0'70
Abaxial surface 0'9 6 0'79 2'42 1'92 1'54 1'02
Abundance on: Adaxial surface
Abaxial surface
17'8 18'3 13'6
17'3 16'3 20'4
17'3 16'7 19'0
19'5 16'9 19'2
S.E,D.
Horizontal Other
0'26 0'38
(P < 0'01), a primary cause was the relatively high value for the abaxial surface. Where both sides bore lesions, the more advanced maturation on abaxial surfaces was most marked at low lesion intensities.
Influence of the surface bearing the lesions and the surface exposed There was considerable decomposition, especially of leaves of Cox's Orange Pippin and of leaves where both surfaces bore lesions. Accordingly, only leaves of Bramley's Seedling with lesions on either the abaxial or the adaxial surface were included in the analyses. Of these leaves, decomposition was more advanced where lesions were on the adaxial surfaces (about one fifth ofthe dry weight) compared with leaves with lesions on the abaxial surface. Results for maturation differed from those of the other experiment, showing more advanced maturation on the adaxial surface than on the abaxial, with
1'45 1'32
a mean value of 2'26 compared to 1'79 (P < 0'05), but it was unaffected by any other factor or interaction except the linear trend with time already noted. There was a significant three-factor interaction between the surface bearing the lesions, the surface exposed, and the surface on which pseudothecia developed (P < 0'05) (Table 4), When the lesions were on the adaxial surface, numbers of pseudothecia were about 50 % higher on the adaxial surface, irrespective of which surface was exposed; however, when lesions were on the abaxial surface, the largest numbers were observed on whichever surface was exposed.
Relationship of ascospore production with pseudothecium maturation Ascospore production cm? leaf was transformed to natural logarithms and plotted against mean
Overwintering of Venturia inaequalis Table 4. Numbers (during assessment period) of v, inaequalis pseudothecia in relation to the surface bearing lesions, the surface exposed and the surface examined Surface exposed ",
Abaxial
Adaxial Adaxial
Surface bearing lesions ." Surface examined Adaxial Abaxial
Abaxial
Adaxial
Abaxial
21'1 14'1
20'9 14'9
12'0 20'7
S.E.D.
Horizontal Other
5000
1000 500 '-
'"
~
'i' E o
100
...'"o0
50
Co
0 '" o «'"
0
,.-
10
,.-
,.-
.e
,.-
,.-
5
0,10
0,50
0·25 Mean maturation
Fig. 1. Mean ascospore production (log scale) of Venturia inaequalis (y), from leaves overwintered in two experiments (0, lesion intensity expt, . , leaf surface exposure expt), in relation to mean maturation (logit transformed) (x), The regression line fitted for these experiments (--) is y = 7'93 (0'83)+2'17 (o-j i )x; where standard errors on the coefficients are shown in parentheses and the variance accounted for is 88 %. Regression lines fitted for experiments with different cultivars, reported by ]eger et al, (1982), are shown as (-----).
maturation (expressed as a proportion of the maximum value 5'0) on a logit scale for each experiment. The latter variable is equivalent to the maturation index proposed by Jeger et al. (1982) when assessments, as here, are equidistant, The regression equation fitted to all data (both expts) is shown in Fig. 1, together with the lines fitted for cultivars in two separate years of the previous study (J eger et al., 1982), indicating the robust nature of the relationship.
DISCUSSION
Epidemics of apple scab result in varied patterns of scab incidence and intensity at leaf-fall (Jeger, 1981). In some cases, scab at leaf-fall may be directly related to infections early the previous spring if subsequent fungicide control or weather has prevented secondary spread. Alternatively, if fungicide control has been relaxed too early and weather is favourable, then the majority of lesions
M.J.Jeger may arise quite late in the season. In either case, a uniform age structure of scab lesions on leaves would result. In most cases, however, and certainly in the unsprayed orchard used to collect leaves for this study, secondary spread from primary infection occurs and results in different disease intensities and age distributions of lesions at leaf-fall. The results suggest that intensity of disease per se is not an important main factor in determining the numbers of pseudothecia initiated, or the rate at which they develop, but may interact strongly with other factors. The influence of lesion age (Wilson, 1928), or rather the age distribution oflesions on the two leaf surfaces, is likely to be more important than disease intensity. It was not possible to distinguish clearly the effects of lesion intensity from those of the lesion-bearing surfaces in these experiments, and hence it is not possible to evaluate the relative contribution of each surface to ascospore inoculum in the spring. Indeed, it is difficult to imagine how this could be achieved without precise inoculation of leaves under controlled conditions; with conidia ofthe opposite mating types applied to each surface, at different concentrations (to obtain a range of intensities) and at different times prior to leaf-fall, perhaps using similar techniques to those described by Ross & Hamlin (1962). Such work would be invaluable in determining whether the higher order interactions found in this study were real or spurious, although controlled environment studies do not always resolve discrepancies found in the field (Jeger & Butt, 1983). Several pieces of evidence suggest that late infections, especially on the abaxial surface, may be of great importance in scab overwintering. Firstly, leaves with early infections on the adaxial surface are likely to fall prematurely at times when environmental conditions, especially temperature, are not favourable for pseudothecium initiation (Jeger & Butt, 1983); secondly, leaves with lesions on adaxial surfaces decompose more rapidly, leaving less of this type of leaf on the orchard floor in the spring; thirdly, when lesions are on the abaxial surface, pseudothecia tend to develop on whichever surface is exposed and this may facilitate discharge depending upon the mobility of leaves on the orchard floor. A further, indirect, reason why late infections on abaxial surfaces are important is that they are much more difficult to observe in conventional disease assessments and the grower or advisor may be lulled into a false sense of security by an apparent absence of scab at leaf-fall. Despite the results when lesions were on the abaxial surface, there was no real evidence for a significant geotropic response (James et al., 1981). Indeed, any' tropism' was marginally in preference
499
for the adaxial rather than abaxial leaf surfaces in Cox's Orange Pippin and Bramley's Seedling, but not in Lord Lambourne. In the preceding study on the effects of weather on scab overwintering (Jeger & Butt, 1983) a complete microscopic record of pseudothecium development was obtained. A sample of 91 mature pseudothecia containing dischargeable ascospores was re-examined: 60 % had their ostioles directed to the adaxial surface, 40 % to the abaxial surface. As the arrangement of leaves within bags in that study was such that half had abaxial, and half had adaxial surfaces exposed, this again confirms the tendency for pseudothecia to develop more frequently on adaxial surfaces, although it was not possible to determine from the slides which surface had originally borne the lesions. This study suggests that the relationship between numbers of pseudothecia and scab intensity at leaf-fall is not particularly useful, at least in field populations of leaves. This is disappointing, given the need to forecast pseudothecium abundance for the management of primary apple scab (MacHardy & Ieger, 1983; Ieger & Butt, 1983). However the robust relationship between ascospore production and pseudothecium maturation found by J eger et al, (1982), which is not explicitly dependent upon abundance of pseudothecia, has been confirmed in a different context. Hence, the potential amount of ascospore inoculum may be forecast, by monitoring or forecasting maturation of pseudothecia, thus simplifying further the conceptual scheme proposed by MacHardy & jeger (1983). I thank K. Martin for clarifying the interpretation of interactions and Dr P. W. Talboys for many helpful comments during the preparation of the manuscript. REFERENCES BURCHILL, R. T. (1968). Field and laboratory studies on the effect of urea on ascospore production of Venturia inaequalis (Cke) Wint. Annals of Applied Biology 62, 297-3 07. GADOURY, D. M. & MAcHARDY, W. E. (1982). Effects of temperature on the development of pseudothecia of Venturia inaequalis. Plant Disease 66, 464-468. HIRST,]. M. & STEDMAN, 0.]. (1962). The epidemiology of apple scab (Venturia inaequalis (Cke) Wint.). III. The supply of ascospores. Annals ofApplied Biology 50, 551-5 67. HUTTON, K. E. & BURCHILL, R. T. (1965). The effects of some fungicides and herbicides on ascospore production of Venturia inaequalis (Cke) Wint. Annals of Applied Biology 56, 279-284. ]AMES, J. R., SUTTON, T. B. & GRAND, L. F. (1981). Unusual ascocarp formation in Venturia inaequalis. Mycologia 73, 564-566.
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Overwintering of Venturia inaequalis
JEGER, M . J. (1981). Disease measurement in a study of apple scab epidemics. Annals of Applied B iology 99, 43-51. JEGER, M . J . & BUTT, D . J . (19 83). Overwintering of Ventu ria inaequalis, the causal agent of apple scab , in relation to weather. Annals of Applied B iology 103, 201-218. JEGER, M . J ., SWAIT, A. A. J. & BUTT, D. J . (1982). Overwintering of Venturia inaequalis, the causal agent of apple scab, on different cultivars. Annals of Applied Biology 100, 91--98. MAcHARDY, W. E. & JEGER, M. J. (1983). Integrating control measures for the management of primary apple
scab, Venturia inaequalis (Cke) Wint. Protection Ecology 5,1 0 3- 12 5. MOLLER, W . J. ( 1980) . Effe ct ofapple cultivar on V enturia inaequalis ascospore emission in California. Plant Disease 64 , 930--931. Ross, R . G . & HAMLIN, S. A. (1962). Production of perithecia of Ve ntu ria inaequalis (C ke) Wint. on sterile apple leaf d iscs . Canadian J ournal of Botany 40, 629-"635. WILSON, E. E. ( 1928) . Studies on the ascigerous stage of Venturia ina equalis in relation to certain factors of the environment. Phytopathology 18, 375-418.
(R eceiv ed f or publication 27 October 1983)
OVERWINTERING OF VENTURIA INAEQUALIS IN RELATION TO LESION INTENSITY ON LEAF SURFACES, AND LEAF SURFACE EXPOSED By M. J. JEGER* East Malling Research Station, Maidstone, Kent ME19 6B], U.K. Apple leaves bearing scab lesions were overwintered under natural conditions on grass. The relationship of pseudothecium development with lesion intensity at leaf-fall was monitored on cvs Cox's Orange Pippin and Lord Lamboume. More pseudothecia were observed on Lamboume than on Cox, and they matured at a faster rate, especially on the abaxial surface. Lesion intensity at leaf-fall had little effect on either numbers of pesudothecia or the rate of maturation although there were higher-order interactions with the surface on which pseudothecia developed. The effects on pseudothecium development of exposing upper or lower surfaces were examined in cv. Bramley's Seedling. Where lesions were on the adaxial surface, more pseudothecia developed there than on the abaxial surface irrespective of which surface was exposed. Where lesions were on the abaxial surface, the largest number of pseudothecia developed on whichever surface was exposed. Ascospore production em"? leaf was related to pseudothecium maturation. Factors influencing the overwintering of Venturia inaequalis (Cke) Wint., as pseudothecia on fallen apple leaves, were proposed by Wilson (1928) and included temperature, moisture, cultivar, time of leaf-fall and time of infection. There have been many studies, under natural and controlled conditions, ofenvironmental influences on the numbers of pseudothecia initiated and their subsequent development to produce mature ascospores in spring (see MacHardy & Ieger, 1983; Jeger & Butt, 1983; and references therein). The influences of cultivar are equally important (Moller, 1980; Jeger, Swait & Butt, 1982) but less studied. The time of leaf-fall influences the number of pseudothecia initiated (Hirst & Stedman, 1962; Burchill, 1968) but environmental conditions at leaf-fall probably have a greater effect (Gadoury & MacHardy, 1982). The influence ofthe age distribution and numbers of lesions on pseudothecial development has not been studied, even by such a gross measure as the proportion of leaf surface scabbed. Early lesions are more common on adaxial (upper) surfaces and are well-defined at leaf-fall, whereas late lesions are often on abaxial (lower) surfaces and more diffuse. Young lesions produce pseudothecia readily, whereas the older do so only at the lesion margins (Wilson, 1928). Pseudothecia can develop with ostioles protruding on either leaf surface, but it is
* Present address: Department of Plant Sciences, Texas A & M University, College Station, TX 77843, U.S.A.
not known whether their formation depends on which surface bears the lesions, or on which surface is exposed. James, Sutton & Grand (1981), on the basis ofan unusual ascocarp development, suggested that there is also a geotropic response. The aims of the present study, complementary to previous studies (Jeger et al., 1982; Jeger & Butt, 1983), were to evaluate the influence of lesion intensity on each leaf surface, and of the leaf surface exposed, on the development of pseudothecia under field conditions. MATERIALS AND METHODS
Influence of lesion intensity Scabbed leaves of cvs Cox's Orange Pippin and Lord Lambourne were collected from an unsprayed orchard at Rocks Farm, East Mailing, on 5 November 1982. Scab intensity was assessed as light (few, discrete lesions), medium (~10% of leaf surface covered by lesions) and heavy (~50 % of leaf surface covered by lesions). Leaves were also classified according to whether one or both surfaces were bearing lesions at a given class of intensity. Those leaves which could not readily be classified, and those with high levels of necrosis, were discarded. The stringent criteria for selection ofleaves meant that abaxial and adaxial surfaces could not be discriminated within the one-surface-scabbed categories. Samples comprising ten leaves/bag were placed in 1 mm mesh nylon bags (Tygan T030) and pegged out in a completely randomized design on
Overwintering of Venturia inaequalis grass on 8 November. The number of replicate bags for each combination of cultivar x intensity class x number of surfaces was usually three, but ranged from one to four depending on the number ofleaves selected. Samples were taken on seven occasions at weekly intervals from 1 February to 21 March 1983. On each occasion, one disk (1 em") was punched from a leaf, wetted for 2 min, and examined under a dissecting microscope. A maximum of 25 pseudothecia were dissected from each leaf surface, using a standard sampling procedure and the contents classified according to the stage of maturation (Jeger et al., 1982). The numbers of pseudothecia were independently counted for correlating with the numbers sampled; counts of pseudothecia alone can be unreliable because other fungal structures may be included inadvertently in the counts. All leaf remains were collected on 24 March, a maximum of 50 disks (1 ern") punched for each treatment combination and ascospore production cm ? leaf assessed as described by Hutton & Burchill (1965). The procedure was repeated on 28 March. Influence of the surface bearing the lesions, and the surface exposed Leaves of Cox's Orange Pippin and Bramley's Seedling were collected from unsprayed orchards at Rocks Farm, East Mailing, and sorted into groups bearing lesions on the abaxial surface only, the adaxial surface only, or both surfaces. In this experiment it was not possible to discriminate intensity classes for a given leaf surface. Two sets of leaves were matched for each category and ro-leaf samples placed in nylon bags. One set of bags was pegged out with the abaxial surface exposed (i.e, facing up), the other set with the adaxial surface exposed, giving a total of six treatment combinations for each cultivar. Samples were taken at z-weekly intervals from 5 January to 15 February 1982, and at weekly intervals from 22 February to 5 April. On each occasion, disks were punched and examined as described for the other experiment and a maximum of 25 pseudothecia classified for each treatment combination. Ascospore production cm"" leaf was assessed on 6 April. RESULTS
There were no trends in numbers of pseudothecia during the assessment period of either experiment, nor interactions between any partitioning of time (linear and quadratic trends, and deviations from these trends) and other factors. Maturation, however, showed a linear trend with time in both experiments (P < 0'01 in lesion intensity expt ;
Table 1. Maturation ofV. inaequalis pseudothecia in relation to time of assessment Lesion intensity expt
Date 1. 14. 21. 28. 7. 14. 21.
ii. 83 ii. 83 ii. 83 ii. 83 iii. 83 iii. 83 iii. 83
S.E.D.
Maturation (0-5) 0'74 0'84 1'03 1'16 1'3 8 1'46 1'7 6 0'36
Surface exposed expt
Date 22. 1. 8. 15. 22. 29. 5.
ii. 82 iii. 82 iii. 82 iii. 82 iii. 82 iii. 82 iv. 82
S.E.D.
Maturation (0-5) 1'25 1'25 1'28 2'26 2'65 2'55 2'93 0,60
P < 0'001 in surface exposed expt) (Table 1). The number of pseudothecia counted independently (y) was related to the numbers sampled for classification of their contents (x) according to the regression equation y = -4·78+1·006x. The slope was not significantly different from unity, which gives some confidence in the sampling procedure used, but the negative intercept indicates a possible bias in searching for pseudothecia at very low densities and the equation is unreliable at high densities because the number of pseudothecia sampled was limited to 25. Despite these qualifications, the numbers of pseudothecia sampled (and known to be of V. inaequalis) were examined in the following analysis. Influence of lesion intensity Lord Lamboume generally exhibited larger mean values than Cox's Orange Pippin for both numbers of pseudothecia and maturation (Table 2). For numbers of pseudothecia, the difference was only significant where pseudothecia developed on the abaxial surface (P < 0'01). Maturation over the assessment period was more advanced where pseudothecia developed on the abaxial surface, but was not affected by whether one or both surfaces bore lesions. There was a significant interaction between cultivar, the number of surfaces bearing lesions, and the surface on which pseudothecia developed (P < 0'05): where both sides bore lesions, there was little difference in maturation on adaxial surfaces when comparing Lord Lamboume and Cox's Orange Pippin. There were significant interactions between lesion intensity, number of surfaces bearing lesions and the surface on which pseudothecia developed (Table 3) that arose when one side only was heavily scabbed: for pseudothecial numbers (P < 0'01), a primary cause was the relatively low value for the adaxial surface, whereas for mean maturation
M.J.Jeger
497
Table 2. Mean maturation (during assessment period) and numbersofpseudothecia ofV. inaequalis in relation to cultivar, the number of surfaces bearing lesions and the surface examined Abundance on:
Maturation on: Surfaces (no.)
Adaxial surface
Abaxial surface
1 2
0'78 0'63
0'96 0,83
1 2
1'50 0'89
1,81 2'16
Cox's Orange Pippin Mean Lord Lamboume
Adaxial surface
Abaxial surface
16'98
16'52
Mean S.E.D.
Horizontal Other
~n
~~
0'31
0'76
Table 3. Mean maturation (during assessment period) and numbers ofV. inaequalis pseudothecia in relation to lesion intensity, the number of surfaces bearing lesions and the surface examined Maturation on: Surfaces (no.)
2
Lesion intensity Low Medium Heavy Low Medium Heavy
Adaxial surface 0,86 1'3 1 1'25 0'67 0'91 0'70
Abaxial surface 0'9 6 0'79 2'42 1'92 1'54 1'02
Abundance on: Adaxial surface
Abaxial surface
17'8 18'3 13'6
17'3 16'3 20'4
17'3 16'7 19'0
19'5 16'9 19'2
S.E,D.
Horizontal Other
0'26 0'38
(P < 0'01), a primary cause was the relatively high value for the abaxial surface. Where both sides bore lesions, the more advanced maturation on abaxial surfaces was most marked at low lesion intensities.
Influence of the surface bearing the lesions and the surface exposed There was considerable decomposition, especially of leaves of Cox's Orange Pippin and of leaves where both surfaces bore lesions. Accordingly, only leaves of Bramley's Seedling with lesions on either the abaxial or the adaxial surface were included in the analyses. Of these leaves, decomposition was more advanced where lesions were on the adaxial surfaces (about one fifth ofthe dry weight) compared with leaves with lesions on the abaxial surface. Results for maturation differed from those of the other experiment, showing more advanced maturation on the adaxial surface than on the abaxial, with
1'45 1'32
a mean value of 2'26 compared to 1'79 (P < 0'05), but it was unaffected by any other factor or interaction except the linear trend with time already noted. There was a significant three-factor interaction between the surface bearing the lesions, the surface exposed, and the surface on which pseudothecia developed (P < 0'05) (Table 4), When the lesions were on the adaxial surface, numbers of pseudothecia were about 50 % higher on the adaxial surface, irrespective of which surface was exposed; however, when lesions were on the abaxial surface, the largest numbers were observed on whichever surface was exposed.
Relationship of ascospore production with pseudothecium maturation Ascospore production cm? leaf was transformed to natural logarithms and plotted against mean
Overwintering of Venturia inaequalis Table 4. Numbers (during assessment period) of v, inaequalis pseudothecia in relation to the surface bearing lesions, the surface exposed and the surface examined Surface exposed ",
Abaxial
Adaxial Adaxial
Surface bearing lesions ." Surface examined Adaxial Abaxial
Abaxial
Adaxial
Abaxial
21'1 14'1
20'9 14'9
12'0 20'7
S.E.D.
Horizontal Other
5000
1000 500 '-
'"
~
'i' E o
100
...'"o0
50
Co
0 '" o «'"
0
,.-
10
,.-
,.-
.e
,.-
,.-
5
0,10
0,50
0·25 Mean maturation
Fig. 1. Mean ascospore production (log scale) of Venturia inaequalis (y), from leaves overwintered in two experiments (0, lesion intensity expt, . , leaf surface exposure expt), in relation to mean maturation (logit transformed) (x), The regression line fitted for these experiments (--) is y = 7'93 (0'83)+2'17 (o-j i )x; where standard errors on the coefficients are shown in parentheses and the variance accounted for is 88 %. Regression lines fitted for experiments with different cultivars, reported by ]eger et al, (1982), are shown as (-----).
maturation (expressed as a proportion of the maximum value 5'0) on a logit scale for each experiment. The latter variable is equivalent to the maturation index proposed by Jeger et al. (1982) when assessments, as here, are equidistant, The regression equation fitted to all data (both expts) is shown in Fig. 1, together with the lines fitted for cultivars in two separate years of the previous study (J eger et al., 1982), indicating the robust nature of the relationship.
DISCUSSION
Epidemics of apple scab result in varied patterns of scab incidence and intensity at leaf-fall (Jeger, 1981). In some cases, scab at leaf-fall may be directly related to infections early the previous spring if subsequent fungicide control or weather has prevented secondary spread. Alternatively, if fungicide control has been relaxed too early and weather is favourable, then the majority of lesions
M.J.Jeger may arise quite late in the season. In either case, a uniform age structure of scab lesions on leaves would result. In most cases, however, and certainly in the unsprayed orchard used to collect leaves for this study, secondary spread from primary infection occurs and results in different disease intensities and age distributions of lesions at leaf-fall. The results suggest that intensity of disease per se is not an important main factor in determining the numbers of pseudothecia initiated, or the rate at which they develop, but may interact strongly with other factors. The influence of lesion age (Wilson, 1928), or rather the age distribution oflesions on the two leaf surfaces, is likely to be more important than disease intensity. It was not possible to distinguish clearly the effects of lesion intensity from those of the lesion-bearing surfaces in these experiments, and hence it is not possible to evaluate the relative contribution of each surface to ascospore inoculum in the spring. Indeed, it is difficult to imagine how this could be achieved without precise inoculation of leaves under controlled conditions; with conidia ofthe opposite mating types applied to each surface, at different concentrations (to obtain a range of intensities) and at different times prior to leaf-fall, perhaps using similar techniques to those described by Ross & Hamlin (1962). Such work would be invaluable in determining whether the higher order interactions found in this study were real or spurious, although controlled environment studies do not always resolve discrepancies found in the field (Jeger & Butt, 1983). Several pieces of evidence suggest that late infections, especially on the abaxial surface, may be of great importance in scab overwintering. Firstly, leaves with early infections on the adaxial surface are likely to fall prematurely at times when environmental conditions, especially temperature, are not favourable for pseudothecium initiation (Jeger & Butt, 1983); secondly, leaves with lesions on adaxial surfaces decompose more rapidly, leaving less of this type of leaf on the orchard floor in the spring; thirdly, when lesions are on the abaxial surface, pseudothecia tend to develop on whichever surface is exposed and this may facilitate discharge depending upon the mobility of leaves on the orchard floor. A further, indirect, reason why late infections on abaxial surfaces are important is that they are much more difficult to observe in conventional disease assessments and the grower or advisor may be lulled into a false sense of security by an apparent absence of scab at leaf-fall. Despite the results when lesions were on the abaxial surface, there was no real evidence for a significant geotropic response (James et al., 1981). Indeed, any' tropism' was marginally in preference
499
for the adaxial rather than abaxial leaf surfaces in Cox's Orange Pippin and Bramley's Seedling, but not in Lord Lambourne. In the preceding study on the effects of weather on scab overwintering (Jeger & Butt, 1983) a complete microscopic record of pseudothecium development was obtained. A sample of 91 mature pseudothecia containing dischargeable ascospores was re-examined: 60 % had their ostioles directed to the adaxial surface, 40 % to the abaxial surface. As the arrangement of leaves within bags in that study was such that half had abaxial, and half had adaxial surfaces exposed, this again confirms the tendency for pseudothecia to develop more frequently on adaxial surfaces, although it was not possible to determine from the slides which surface had originally borne the lesions. This study suggests that the relationship between numbers of pseudothecia and scab intensity at leaf-fall is not particularly useful, at least in field populations of leaves. This is disappointing, given the need to forecast pseudothecium abundance for the management of primary apple scab (MacHardy & Ieger, 1983; Ieger & Butt, 1983). However the robust relationship between ascospore production and pseudothecium maturation found by J eger et al, (1982), which is not explicitly dependent upon abundance of pseudothecia, has been confirmed in a different context. Hence, the potential amount of ascospore inoculum may be forecast, by monitoring or forecasting maturation of pseudothecia, thus simplifying further the conceptual scheme proposed by MacHardy & jeger (1983). I thank K. Martin for clarifying the interpretation of interactions and Dr P. W. Talboys for many helpful comments during the preparation of the manuscript. REFERENCES BURCHILL, R. T. (1968). Field and laboratory studies on the effect of urea on ascospore production of Venturia inaequalis (Cke) Wint. Annals of Applied Biology 62, 297-3 07. GADOURY, D. M. & MAcHARDY, W. E. (1982). Effects of temperature on the development of pseudothecia of Venturia inaequalis. Plant Disease 66, 464-468. HIRST,]. M. & STEDMAN, 0.]. (1962). The epidemiology of apple scab (Venturia inaequalis (Cke) Wint.). III. The supply of ascospores. Annals ofApplied Biology 50, 551-5 67. HUTTON, K. E. & BURCHILL, R. T. (1965). The effects of some fungicides and herbicides on ascospore production of Venturia inaequalis (Cke) Wint. Annals of Applied Biology 56, 279-284. ]AMES, J. R., SUTTON, T. B. & GRAND, L. F. (1981). Unusual ascocarp formation in Venturia inaequalis. Mycologia 73, 564-566.
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Overwintering of Venturia inaequalis
JEGER, M . J. (1981). Disease measurement in a study of apple scab epidemics. Annals of Applied B iology 99, 43-51. JEGER, M . J . & BUTT, D . J . (19 83). Overwintering of Ventu ria inaequalis, the causal agent of apple scab , in relation to weather. Annals of Applied B iology 103, 201-218. JEGER, M . J ., SWAIT, A. A. J. & BUTT, D. J . (1982). Overwintering of Venturia inaequalis, the causal agent of apple scab, on different cultivars. Annals of Applied Biology 100, 91--98. MAcHARDY, W. E. & JEGER, M. J. (1983). Integrating control measures for the management of primary apple
scab, Venturia inaequalis (Cke) Wint. Protection Ecology 5,1 0 3- 12 5. MOLLER, W . J. ( 1980) . Effe ct ofapple cultivar on V enturia inaequalis ascospore emission in California. Plant Disease 64 , 930--931. Ross, R . G . & HAMLIN, S. A. (1962). Production of perithecia of Ve ntu ria inaequalis (C ke) Wint. on sterile apple leaf d iscs . Canadian J ournal of Botany 40, 629-"635. WILSON, E. E. ( 1928) . Studies on the ascigerous stage of Venturia ina equalis in relation to certain factors of the environment. Phytopathology 18, 375-418.
(R eceiv ed f or publication 27 October 1983)