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Resistance to dothistroma needle blight induced in Pinus radiata by maturity and shade
[ 2°5 ] Trans. Br, mycol. Soc. 59 (2),205-212 (1972) Printed in Great Britain
RESISTANCE TO DOTHISTROMA NEEDLE BLIGHT INDUCED IN PINUS RADIATA BY MATURITY AND SHADE By M. H. IVORY East African Agriculture and Forestry Research Organization, Muguga, Nairobi, Kenya (With
2
Text-figures)
Resistance to Dothistroma needle blight in Pinus radiate is induced by host maturity and is a characteristic of the whole tree. Survey data, collected in Kenya, show that the host plant gradually becomes more blight resistant with increasing age and that the effectiveness of this resistance is related to the amount of rainfall, in that a higher level of resistance is required to control blight in areas where high rainfall favours the spread and development of the disease. Resistance induced by shade treatment is restricted to shaded foliage, and is not transferred throughout the whole tree. It also operates against the fungus after the foliage has been penetrated normally. The resistance induced by shade treatment during the period 5-20 days after inoculation is almost 100 % effective whereas that induced by treatments applied thereafter is progressively less effective. This resistance is thought to be induced by a reduction in the light intensity. Analysis offoliage for phenolic compounds showed no differences consistently associated with shade treatment.
Dothistroma needle blight caused by Scirrhia pini Funk & Parker has been recorded on many Pinus spp. in East Africa (Ivory, 1967, 1968), mainly as the conidial state, Dothistroma septosporum (Dorog.) Morelet var. keniense (Ivory) Morelet, and once as the perfect state, on Pinus clausa (Chapm.) Vasey (IMI 148402). Susceptibility to infection has been related to the host species, the age of the host, and climatic factors such as duration of precipitation, light and temperature (Gibson, Christensen & Munga, 1964; Gibson, Christensen & Dedan, 1967; Ivory, 1968). Similar observations have been made in other parts of the world for D . septosporum var. pini (Hulbary) Morelet (Murray & Batko, 1962; Gilmour, 1967). Ivory (1972) has described the process of infection on young susceptible foliage of Pinus radiata D. Don . by S. pini in East Africa, Observations on the process of infection in the present study are compared with this description. MATERIALS AND METHODS
The effect of needle blight on trees of different ages was determined by visual assessment of foliage attack at several points in each plantation, the number ofpoints depending on the size and accessibility ofeach plantation. Wherever possible, several plantations of the same age at each of several localities were examined.
206
Transactions British Mycological Society
Shade-induced resistance was studied on inoculated branch tips using the technique described by Ivory (1972). However, in the present trials the control branches were incubated inside clear polythene bags and the shaded branches were incubated inside black polythene bags coated with aluminium paint. The process of infection was followed on detached young fully formed needles and branch tips of young shaded trees, and non-shaded trees of different ages using techniques described previously (Ivory, 1972). The trees were all located in arboreta at E.A.A.F.R.O. and Uplands Forest Station, near Nairobi in central Kenya; In detached needle experiments, needles were kept in glass Petri dishes near a window but not exposed to direct sunlight. The shaded treatment was shielded from daylight with aluminium foil. Chemical analyses for phenolic compounds of shaded and unshaded foliage from young P. radiata trees were carried out using modifications of the hot-water extraction technique and the method of two-dimensional paper chromatography described by Hanover & Hoff (1966). In the extraction procedure foliage (15 g fresh weight) was cut into I em lengths and boiled in roo ml water for 6 min, during which the mixture was homogenized for two periods for 30 sec duration. The mixture was filtered and the filtrate clarified by centrifugation. The extract was washed four times with 10 ml portions of diethyl ether and extracted five times with 10 ml aliquots of butanol. The butanol extract was evaporated in a Petri dish in a warm current of air (approx. 35° for 2 h) and the residue dissolved in 2 ml absolute ethanol. Extracts were applied with a micropipette to sheets of Whatman No. I paper (46 x 57 em), Four sheets were equilibrated over a solution of butanol, acetic acid and water (B.A. w.) 6: I : 2 for 24 h and then developed for 18 h in the first direction with the same solution descending from a trough supported by a plastic-coated frame in a sealed polythene bag. The sheets were removed, dried in a B.T.L. chromatography oven for 2 h, replaced in the frame and equilibrated over 2 % acetic acid for 24 h. They were then developed for 3 h in the second direction with 2 % acetic acid, removed, and dried as before. Separated compounds were located firstly by examining the sheets for fluorescent spots in ultraviolet light in the absence and presence of ammonia vapour and then by the application of phenol-detecting reagents to the sheets. Reagents used were diazotized sulphanilic acid (Hanover & Hoff, 1966), potassium permanganate (Pacsu, Mora & Kent, 1949), 2,6-dichloroquinone chloroimide (Bray, Thorpe & White, 1950), ferric chloride (Hathway, 1960), diazotized p-nitroaniline (Bray et al. 1950) and silver nitrate (Smith, 1960). The RF values of the located spots were then measured using an elastic scale. RESULTS
Mature plant resistance Data collected from several forest areas of Kenya in 1966 show that, in each rainfall class, the severity of needle blight is inversely related to the age of the plantation (Table 1), except in very young plantations where the
Resistance to Dothistroma. M. H. Ivory Table
2°7
Relationship between severiry of needle blight, age of plantation and rainfall in Pinus radiata plantations in Kenya
1.
Foliage attack (%) A
Age (yr)
Rainfall > 1650 rom p.a.
Rainfall 1270-1650 rom p.a.
<
Rainfall 1270 mm p.a,
I
15
2
63
3
46
4
5 6-10 11 -15 16-20 21-26
88 88
88
41 10
38
57 42
15
12
50
3
- , No observations.
Table 2. The effect of shade on needle-blight development Nwnber of infected needles ,..----------''---..,..----"""\
Expt. 2 (81 days)
Treatment Control Unshaded Shaded-10 to 0 days -7 to 0 days o to 5 days 5 to 81 days 5 to 100 days Least significant differences (5 %)
44
I 62
30 39
o 32'5
• Plants were inoculated on day o.
disease is still developing. Lack of data for some age classes reflects in some instances changing planting policies and in other instances the write-off, prior to the survey, of plantations because of high mortality caused by needle blight and armillaria root disease. Conidial germination and stomatal pit entry by germ-tubes occurred to the same extent on detached young foliage from 26-year-old and 3-year-old trees. In addition, microscopic examination of cleared and stained needles from zfi-year-old trees revealed that the method of penetration by the fungus is similar to that described earlier (Ivory, 1972) for young trees.
Shade-induced resistance Gibson et al. (1967) showed that shade treatment markedly reduced needle-blight development in field trials. This was confirmed in smallscale experiments using artificially inoculated branch tips of young P. radiata trees. Table 2 shows that shade treatment applied for the periods 5-100 days and 5-81 days after inoculation reduced symptom development to nil, whereas shorter or pre-inoculation shade treatments had no significant effect (5 %). A further more detailed experiment showed that the critical period is from nil to 20 days after inoculation, and that there-
Transactions British Mycological Society
208 300 "0
'" > 0 E ~
on
:a'" '" r: '"
200
"0
'"
U
~
.s 100 '0
... '"
F
..0
a z='
0
50
60
70 80 Time after inoculation (days)
90
100
Fig. I. The effect of shade treatments on needle-blight development. A, Unshaded. B-G, Shaded. B, 0-21 days;C, 0-30 days; D, 0-39 days; E, 21-50 days; F,30-sodays; G, 39-50 days; H, control.
after shade treatment causes progressively less reduction of lesion development (Fig. I). The two experiments together therefore indicate the critical period to be from 5 to 20 days after inoculation. The reduction of blight development in the shaded treatments cannot be attributed to excessive heat as the temperatures inside the inoculation bags were found to be ambient plus 2 0 for the shaded treatment and ambient plus 50 for the control treatment. Observations made in New Zealand by Mr]. W. Gilmour (pers. comm.) suggested that light quality may affect blight development, especially with regard to ultraviolet radiation. The screening effects of soda glass and Pyrex glass on blight development were therefore compared with that caused by thin clear polythene sheeting. In all cases blight lesions developed on approximately 95 % of the inoculated needles, indicating that the treatments had no significant effect. The incidence of conidial germination, stomatal pit entry (Table 3) and the method of germ-tube penetration in darkness were observed to be no different from those (Ivory, 1972) observed under daylight conditions. However, on shaded foliage penetration does not result in the development of blight symptoms (Table 3), although occasionally some lesions appear about 20 days after the foliage is returned to normal conditions. When foliage from shaded and unshaded P. radiata treas was analysed chromatographically 36 compounds were detected, 20 of which reacted with phenol-detecting reagents (Fig. 2). Most of the qualitative differences were in the minor phenolic and non-phenolic constituents, but these were not reliable indicators of resistance or susceptibility. Similarly, quantitative differences of the major components varied as much between replicates as between treatments.
Resistance to Dothistroma. M. H. Ivory
2°9
Table 3. The effect ofdarkness anddaylight on conidial germination and stomatal pit entry by Scirrhia pini, andon symptom development onfoliage ofPinus radiata Foliage in situ
,
Treatment
,
Detached foliage No. of infected needles Conidial Conidial gergerStomatal Stomatal 63 days mination pit entry mination pit entry after (% of total) inoculation (% of total) (%) (%)
4-2 h after inoculation Daylight Darkness 96 h after inoculation Daylight Darkness 120 h after inoculation Daylight Darkness
2 2
6
94-
87
2
o
13
97
II
_Q:)°o
• o
96
\80 (\1'
0"
® o
o
\!.sJ
0
8
@
01'
p
o o _ I'
I'
SrOIS detected in shad ed an d unshad cd fo liage Spots detect ed in shaded fo liage onl y Spots detected in un shadcd fo liage onl y Suspected phen o lic co mpo unds
Ori gin 2 ~~ ac et ic acid
Fig.
2.
Chromatogram of extracts from shaded and unshaded foliage of young P. radiata trees.
210
Transactions British Mycological Society DISCUSSION
Mature plant resistance in P. radiata is conclusively demonstrated by survey data obtained in 1966. These show that in each rainfall class blight attack is inversely related to the age of the tree. The data also suggest that blight resistance is acquired very gradually and that its effectiveness is related to the amount of rainfall. However, as needle blight did not become established in Kenya until about 1960, the data refer to the impact of blight on the trees for the 6 years preceding the survey date and not to the continued impact of blight on the trees from the time of planting. Because of this limitation, no indication of the recovery rate of severely infected young trees can be deduced from these data. Penetration by the fungus occurs normally on young foliage of old trees. The mechanism of resistance must therefore operate against the fungus at some time after penetration of the host tissue, but before visible symptoms of blight are produced. Observations also indicate that mature plant resistance is a characteristic of the tree as a whole, and that it is not restricted to old foliage or to particular branches. It is therefore possibly related to the changes in host physiology which accompany advancing maturity. Mature-plant resistance has been demonstrated in many other hostpathogen relationships (Melander & Craigie, 1927; Gaumann, 1950), but mature-plant susceptibility frequently occurs also (Wade, 1956; Ludwig, Spencer & Unwin, 1960; Paxton & Chamberlain, 1969). Many different mechanisms are responsible for mature-plant resistance but the mechanism is probably similar in closely related plants. This may apply in the matureplant resistance to S. pini shown by P. radiate, P. canariensis C. Smith and P. elliottii Engelm. (Ivory, 1968). The existence of shade-induced resistance, first suggested by Gibson et al. (1964), was confirmed by the present series of experiments. Since significant differences were obtained between shaded and unshaded treatments replicated on different branches of the same tree, this resistance is not translocated within the tree. These experiments also indicated that shade treatments are most effective during the period 5-20 days after inoculation and that shade applied more than 20 days after inoculation causes progressively less effect on blight development. The effect is shortlived in the host because treatment with shade before inoculation had no effect on blight development. Also, occasionally, some blight symptoms developed shortly after the removal of shade treatment. Since the process of infection occurs normally on foliage kept in darkness, the shade-induced resistance mechanism does not operate until the fungus has penetrated the host. This conclusion is supported by results from shade treatments on inoculated branch tips, which show that shade for the first 5 days after inoculation (i.e. the penetration phase) has no effect on blight development. P. radiata is very shade-intolerant. Even light shade gives rise to abnormal shoot elongation and poor development of chlorophyll in new foliage (Gibson et al. 1967). It is therefore reasonable to suppose that shade affects the chemical composition of the foliage with respect to one or more constituents which may stimulate or inhibit fungal development. However,
Resistance to Dothistroma, M. H. Ivory
211
detailed analyses of shaded, compared to unshaded foliage, showed that these substances, if present, are probably not phenolic. Filtration of solar radiation by Pyrex and soda glass has no effect on blight development. Inhibition of blight development in glasshouses noticed in New Zealand (Gilmour, pers. comm.) is therefore probably caused by a reduction in the light intensity resulting from mechanical shades or light-reflecting substances on the glass, rather than from the screening effect of the glass itself. High temperatures under glass may also inhibit disease development by preventing spore germination and germtube growth. However, this phenomenon did not apply in the case reported from New Zealand, where inoculated plants developed blight lesions soon after being taken out into the open, whereas those remaining inside glasshouses did not. Shade-induced resistance has also been reported for other host-pathogen combinations and has been related to light intensity, light quality and photoperiod (Thorold, 1940; Zscheile, 1955; Buxton & Last, 1956; Yirgou & Caldwell, 1963). The mechanism responsible has not been determined in any instance but it has been suggested that it is related in some way to the photosynthetic mechanism of the host (Yirgou & Caldwell, 1963) or to the concentration of hormones in the tissues (Van Andel, 1968). The observations reported in this paper do not conflict with these findings and to some extent support the view that shade-induced resistance may be induced by the same mechanism in many host-pathogen combinations. Other data suggest that different mechanisms give rise to shadeinduced resistance and mature plant resistance in P. radiata, especially as the former appears to be localized and the latter systemic. This work formed part of a Ph.D. thesis submitted to the University of London, and is published with the permission of the Director, East African Agriculture and Forestry Research Organization.
REFERENCES
BRAY, H. G., THORPE, W. V. & WWTE, K. (1950). The fate of certain organic acids and amides in the rabbit. 10. The application of paper chromatography to metabolic studies of hydroxybenzoic acids and amides. Journal of Biochemistry 46, 271-275. BUXTON, E. W. & LAsT, F. T. (1956). Report Rothamsted experimental Station 1955, pp. 100106. GXUMANN, E. (1950). Principles ofplant infection, London: Crosby Lockwood and Son Ltd. GIBSON, I. A. S., CHRISTENSEN, P. S. & DEDAN, J. K. (1967). Further observations in Kenya on a foliage disease of pines caused by Dothistroma pini Hulbary. III. The effect of shade on the incidence of disease in Pinus radiata. Commonwealth Forestry Review 46,239-247. Grasox, I. A. S., CHRISTENSEN, P. S. & MUNGA, F. M. (1964). First observations in Kenya on a foliage disease of pines caused by Dothistroma pini Hulbary. Commonwealth Forestry Review 42,31-48. GILMOUR,J. W. (1967). Distribution and significance of the needle blight of pines caused by Dothistroma pini in New Zealand. Plant Disease Reporter 51, 727-730. HANOVER, J. W. & HOFF, R. J. (1966). A comparison of phenolic constituents of Pinus monticola resistant and susceptible to Cronartium ribicola, Physiologia Plantarum 19, 554-5 62.
212
Transactions British Mycological Society
HATHWAY, D. E. (1960). Plant phenols and tannins. Chromatography and electrophoretic techniques (ed. 1. Smith). Vol. 1. Chromatography, pp. 308-354. London: W. Heinemann. IVORY, M. H. (1967). A new variety of Dothistroma pini in Kenya. Transactions of the British Mycological Society 50, 28g-297. IVORY, M. H. (1968). Reaction of pines in Kenya to attack by Dothistroma pini var, keniensis. East African Agriculture andForestry Journal 33, 236-244. IVORY, M. H. (1972). Infection of Pinusradiata foliage by Scirrhia pini. Transactions of the British Mycological Society. LUDWIG, R. A., SPENCER, E. Y. & UNWIN, C. A. (1960). An antifungal factor from barley of possible significance in disease resistance. Canadian Journal of Botany 38, 21-29. MELANDER, L. W. & CRAIGm, J. H. (1927). Nature of resistance of Berberis spp. to Puccinia graminis. Phytopathology 17, 95-114. MURRAY, J. S. & BATKO, S. (1962). Dothistroma pini (Hul.) - a new disease of pine in G.B. Forestry 35, 57-65. PACSU, E., MORA, T. P. & KENT, P. W. (1949). General method for paper chromatographic analysis of reducing and non-reducing carbohydrates and derivatives. Science 110, 446-447. PAXTON, J. D. & CHAMBERLAIN, D. W. (1967). Phytoalexin production and disease resistance in soybeans as affected by age. Phytopathology 59, 775-777. SMITH, I. (1960). Phenolic acids. Chromatography andelectrophoretic techniques (ed. 1. Smith). Vol. 1. Chromatography, pp. 291-307. London: W. Heinemann. THOROLD, C. A. (1940). Cultivation of bananas under shade for the control of leaf spot disease. Tropical Agriculturist 17, 213-214. VAN ANDEL, O. M. (1968). Shifts in disease resistance induced by growth regulators. Netherlands Journal of Plant Pathology 74 (Suppl. I), 113-120. WADE, G. C. (1956). Investigations of brown rot of apricots caused by Sclerotinia fructicola (Wint.) Rehm. 1. The occurrence of latent infection in fruit. Australian Journal of agricultural Research 7, 504-515. YIRGOU, D. & CALDWELL, R. M. (1963). Stomatal penetration of wheat seedlings by stem and leaf rusts: effect oflight and carbon dioxide. Science 141, 272-273. ZSCHEILE, F. P. (1955)' Some physiological aspects of bunt resistance in wheat. Plant Physiology Lancaster 30, 432-437.
(Accepted for publication 28 February 1972)
RESISTANCE TO DOTHISTROMA NEEDLE BLIGHT INDUCED IN PINUS RADIATA BY MATURITY AND SHADE By M. H. IVORY East African Agriculture and Forestry Research Organization, Muguga, Nairobi, Kenya (With
2
Text-figures)
Resistance to Dothistroma needle blight in Pinus radiate is induced by host maturity and is a characteristic of the whole tree. Survey data, collected in Kenya, show that the host plant gradually becomes more blight resistant with increasing age and that the effectiveness of this resistance is related to the amount of rainfall, in that a higher level of resistance is required to control blight in areas where high rainfall favours the spread and development of the disease. Resistance induced by shade treatment is restricted to shaded foliage, and is not transferred throughout the whole tree. It also operates against the fungus after the foliage has been penetrated normally. The resistance induced by shade treatment during the period 5-20 days after inoculation is almost 100 % effective whereas that induced by treatments applied thereafter is progressively less effective. This resistance is thought to be induced by a reduction in the light intensity. Analysis offoliage for phenolic compounds showed no differences consistently associated with shade treatment.
Dothistroma needle blight caused by Scirrhia pini Funk & Parker has been recorded on many Pinus spp. in East Africa (Ivory, 1967, 1968), mainly as the conidial state, Dothistroma septosporum (Dorog.) Morelet var. keniense (Ivory) Morelet, and once as the perfect state, on Pinus clausa (Chapm.) Vasey (IMI 148402). Susceptibility to infection has been related to the host species, the age of the host, and climatic factors such as duration of precipitation, light and temperature (Gibson, Christensen & Munga, 1964; Gibson, Christensen & Dedan, 1967; Ivory, 1968). Similar observations have been made in other parts of the world for D . septosporum var. pini (Hulbary) Morelet (Murray & Batko, 1962; Gilmour, 1967). Ivory (1972) has described the process of infection on young susceptible foliage of Pinus radiata D. Don . by S. pini in East Africa, Observations on the process of infection in the present study are compared with this description. MATERIALS AND METHODS
The effect of needle blight on trees of different ages was determined by visual assessment of foliage attack at several points in each plantation, the number ofpoints depending on the size and accessibility ofeach plantation. Wherever possible, several plantations of the same age at each of several localities were examined.
206
Transactions British Mycological Society
Shade-induced resistance was studied on inoculated branch tips using the technique described by Ivory (1972). However, in the present trials the control branches were incubated inside clear polythene bags and the shaded branches were incubated inside black polythene bags coated with aluminium paint. The process of infection was followed on detached young fully formed needles and branch tips of young shaded trees, and non-shaded trees of different ages using techniques described previously (Ivory, 1972). The trees were all located in arboreta at E.A.A.F.R.O. and Uplands Forest Station, near Nairobi in central Kenya; In detached needle experiments, needles were kept in glass Petri dishes near a window but not exposed to direct sunlight. The shaded treatment was shielded from daylight with aluminium foil. Chemical analyses for phenolic compounds of shaded and unshaded foliage from young P. radiata trees were carried out using modifications of the hot-water extraction technique and the method of two-dimensional paper chromatography described by Hanover & Hoff (1966). In the extraction procedure foliage (15 g fresh weight) was cut into I em lengths and boiled in roo ml water for 6 min, during which the mixture was homogenized for two periods for 30 sec duration. The mixture was filtered and the filtrate clarified by centrifugation. The extract was washed four times with 10 ml portions of diethyl ether and extracted five times with 10 ml aliquots of butanol. The butanol extract was evaporated in a Petri dish in a warm current of air (approx. 35° for 2 h) and the residue dissolved in 2 ml absolute ethanol. Extracts were applied with a micropipette to sheets of Whatman No. I paper (46 x 57 em), Four sheets were equilibrated over a solution of butanol, acetic acid and water (B.A. w.) 6: I : 2 for 24 h and then developed for 18 h in the first direction with the same solution descending from a trough supported by a plastic-coated frame in a sealed polythene bag. The sheets were removed, dried in a B.T.L. chromatography oven for 2 h, replaced in the frame and equilibrated over 2 % acetic acid for 24 h. They were then developed for 3 h in the second direction with 2 % acetic acid, removed, and dried as before. Separated compounds were located firstly by examining the sheets for fluorescent spots in ultraviolet light in the absence and presence of ammonia vapour and then by the application of phenol-detecting reagents to the sheets. Reagents used were diazotized sulphanilic acid (Hanover & Hoff, 1966), potassium permanganate (Pacsu, Mora & Kent, 1949), 2,6-dichloroquinone chloroimide (Bray, Thorpe & White, 1950), ferric chloride (Hathway, 1960), diazotized p-nitroaniline (Bray et al. 1950) and silver nitrate (Smith, 1960). The RF values of the located spots were then measured using an elastic scale. RESULTS
Mature plant resistance Data collected from several forest areas of Kenya in 1966 show that, in each rainfall class, the severity of needle blight is inversely related to the age of the plantation (Table 1), except in very young plantations where the
Resistance to Dothistroma. M. H. Ivory Table
2°7
Relationship between severiry of needle blight, age of plantation and rainfall in Pinus radiata plantations in Kenya
1.
Foliage attack (%) A
Age (yr)
Rainfall > 1650 rom p.a.
Rainfall 1270-1650 rom p.a.
<
Rainfall 1270 mm p.a,
I
15
2
63
3
46
4
5 6-10 11 -15 16-20 21-26
88 88
88
41 10
38
57 42
15
12
50
3
- , No observations.
Table 2. The effect of shade on needle-blight development Nwnber of infected needles ,..----------''---..,..----"""\
Expt. 2 (81 days)
Treatment Control Unshaded Shaded-10 to 0 days -7 to 0 days o to 5 days 5 to 81 days 5 to 100 days Least significant differences (5 %)
44
I 62
30 39
o 32'5
• Plants were inoculated on day o.
disease is still developing. Lack of data for some age classes reflects in some instances changing planting policies and in other instances the write-off, prior to the survey, of plantations because of high mortality caused by needle blight and armillaria root disease. Conidial germination and stomatal pit entry by germ-tubes occurred to the same extent on detached young foliage from 26-year-old and 3-year-old trees. In addition, microscopic examination of cleared and stained needles from zfi-year-old trees revealed that the method of penetration by the fungus is similar to that described earlier (Ivory, 1972) for young trees.
Shade-induced resistance Gibson et al. (1967) showed that shade treatment markedly reduced needle-blight development in field trials. This was confirmed in smallscale experiments using artificially inoculated branch tips of young P. radiata trees. Table 2 shows that shade treatment applied for the periods 5-100 days and 5-81 days after inoculation reduced symptom development to nil, whereas shorter or pre-inoculation shade treatments had no significant effect (5 %). A further more detailed experiment showed that the critical period is from nil to 20 days after inoculation, and that there-
Transactions British Mycological Society
208 300 "0
'" > 0 E ~
on
:a'" '" r: '"
200
"0
'"
U
~
.s 100 '0
... '"
F
..0
a z='
0
50
60
70 80 Time after inoculation (days)
90
100
Fig. I. The effect of shade treatments on needle-blight development. A, Unshaded. B-G, Shaded. B, 0-21 days;C, 0-30 days; D, 0-39 days; E, 21-50 days; F,30-sodays; G, 39-50 days; H, control.
after shade treatment causes progressively less reduction of lesion development (Fig. I). The two experiments together therefore indicate the critical period to be from 5 to 20 days after inoculation. The reduction of blight development in the shaded treatments cannot be attributed to excessive heat as the temperatures inside the inoculation bags were found to be ambient plus 2 0 for the shaded treatment and ambient plus 50 for the control treatment. Observations made in New Zealand by Mr]. W. Gilmour (pers. comm.) suggested that light quality may affect blight development, especially with regard to ultraviolet radiation. The screening effects of soda glass and Pyrex glass on blight development were therefore compared with that caused by thin clear polythene sheeting. In all cases blight lesions developed on approximately 95 % of the inoculated needles, indicating that the treatments had no significant effect. The incidence of conidial germination, stomatal pit entry (Table 3) and the method of germ-tube penetration in darkness were observed to be no different from those (Ivory, 1972) observed under daylight conditions. However, on shaded foliage penetration does not result in the development of blight symptoms (Table 3), although occasionally some lesions appear about 20 days after the foliage is returned to normal conditions. When foliage from shaded and unshaded P. radiata treas was analysed chromatographically 36 compounds were detected, 20 of which reacted with phenol-detecting reagents (Fig. 2). Most of the qualitative differences were in the minor phenolic and non-phenolic constituents, but these were not reliable indicators of resistance or susceptibility. Similarly, quantitative differences of the major components varied as much between replicates as between treatments.
Resistance to Dothistroma. M. H. Ivory
2°9
Table 3. The effect ofdarkness anddaylight on conidial germination and stomatal pit entry by Scirrhia pini, andon symptom development onfoliage ofPinus radiata Foliage in situ
,
Treatment
,
Detached foliage No. of infected needles Conidial Conidial gergerStomatal Stomatal 63 days mination pit entry mination pit entry after (% of total) inoculation (% of total) (%) (%)
4-2 h after inoculation Daylight Darkness 96 h after inoculation Daylight Darkness 120 h after inoculation Daylight Darkness
2 2
6
94-
87
2
o
13
97
II
_Q:)°o
• o
96
\80 (\1'
0"
® o
o
\!.sJ
0
8
@
01'
p
o o _ I'
I'
SrOIS detected in shad ed an d unshad cd fo liage Spots detect ed in shaded fo liage onl y Spots detected in un shadcd fo liage onl y Suspected phen o lic co mpo unds
Ori gin 2 ~~ ac et ic acid
Fig.
2.
Chromatogram of extracts from shaded and unshaded foliage of young P. radiata trees.
210
Transactions British Mycological Society DISCUSSION
Mature plant resistance in P. radiata is conclusively demonstrated by survey data obtained in 1966. These show that in each rainfall class blight attack is inversely related to the age of the tree. The data also suggest that blight resistance is acquired very gradually and that its effectiveness is related to the amount of rainfall. However, as needle blight did not become established in Kenya until about 1960, the data refer to the impact of blight on the trees for the 6 years preceding the survey date and not to the continued impact of blight on the trees from the time of planting. Because of this limitation, no indication of the recovery rate of severely infected young trees can be deduced from these data. Penetration by the fungus occurs normally on young foliage of old trees. The mechanism of resistance must therefore operate against the fungus at some time after penetration of the host tissue, but before visible symptoms of blight are produced. Observations also indicate that mature plant resistance is a characteristic of the tree as a whole, and that it is not restricted to old foliage or to particular branches. It is therefore possibly related to the changes in host physiology which accompany advancing maturity. Mature-plant resistance has been demonstrated in many other hostpathogen relationships (Melander & Craigie, 1927; Gaumann, 1950), but mature-plant susceptibility frequently occurs also (Wade, 1956; Ludwig, Spencer & Unwin, 1960; Paxton & Chamberlain, 1969). Many different mechanisms are responsible for mature-plant resistance but the mechanism is probably similar in closely related plants. This may apply in the matureplant resistance to S. pini shown by P. radiate, P. canariensis C. Smith and P. elliottii Engelm. (Ivory, 1968). The existence of shade-induced resistance, first suggested by Gibson et al. (1964), was confirmed by the present series of experiments. Since significant differences were obtained between shaded and unshaded treatments replicated on different branches of the same tree, this resistance is not translocated within the tree. These experiments also indicated that shade treatments are most effective during the period 5-20 days after inoculation and that shade applied more than 20 days after inoculation causes progressively less effect on blight development. The effect is shortlived in the host because treatment with shade before inoculation had no effect on blight development. Also, occasionally, some blight symptoms developed shortly after the removal of shade treatment. Since the process of infection occurs normally on foliage kept in darkness, the shade-induced resistance mechanism does not operate until the fungus has penetrated the host. This conclusion is supported by results from shade treatments on inoculated branch tips, which show that shade for the first 5 days after inoculation (i.e. the penetration phase) has no effect on blight development. P. radiata is very shade-intolerant. Even light shade gives rise to abnormal shoot elongation and poor development of chlorophyll in new foliage (Gibson et al. 1967). It is therefore reasonable to suppose that shade affects the chemical composition of the foliage with respect to one or more constituents which may stimulate or inhibit fungal development. However,
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detailed analyses of shaded, compared to unshaded foliage, showed that these substances, if present, are probably not phenolic. Filtration of solar radiation by Pyrex and soda glass has no effect on blight development. Inhibition of blight development in glasshouses noticed in New Zealand (Gilmour, pers. comm.) is therefore probably caused by a reduction in the light intensity resulting from mechanical shades or light-reflecting substances on the glass, rather than from the screening effect of the glass itself. High temperatures under glass may also inhibit disease development by preventing spore germination and germtube growth. However, this phenomenon did not apply in the case reported from New Zealand, where inoculated plants developed blight lesions soon after being taken out into the open, whereas those remaining inside glasshouses did not. Shade-induced resistance has also been reported for other host-pathogen combinations and has been related to light intensity, light quality and photoperiod (Thorold, 1940; Zscheile, 1955; Buxton & Last, 1956; Yirgou & Caldwell, 1963). The mechanism responsible has not been determined in any instance but it has been suggested that it is related in some way to the photosynthetic mechanism of the host (Yirgou & Caldwell, 1963) or to the concentration of hormones in the tissues (Van Andel, 1968). The observations reported in this paper do not conflict with these findings and to some extent support the view that shade-induced resistance may be induced by the same mechanism in many host-pathogen combinations. Other data suggest that different mechanisms give rise to shadeinduced resistance and mature plant resistance in P. radiata, especially as the former appears to be localized and the latter systemic. This work formed part of a Ph.D. thesis submitted to the University of London, and is published with the permission of the Director, East African Agriculture and Forestry Research Organization.
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(Accepted for publication 28 February 1972)