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Influence of light intensity on reactions of certain poplar cultivars to races of Melampsora larici-populina
[ 375 ] Trans. Br. mycol. Soc. 77 (2) 375-380 (1981)
Primed in Great Britain
INFLUENCE OF LIGHT INTENSITY ON REACTIONS OF CERTAIN POPLAR CUL TIVARS TO RACES OF MELAMPSORA LARICI-POPULINA By M. CHANDRASHEKAR AND W. A. HEATHER Department of Forestry, The Australian National University, Canberra, ACT, Australia 2600 The resistance offour cultivars of poplar, of varying compatibility to four races of Melampsora larici-populina, was sensitive to the light intensity of incubation. Disease severity, assessed on two parameters (incubation period and uredinial numbers) was lower at 1000 pE m~2 S-1 than at 100 or 250 pE m- 2 s- 1light intensity. The major components: light, race, cultivar, and all the second and third order interactions between these were highly significant (P < 0'001). Thus, light intensity of incubation is very significant in determining the susceptibility of these cultivars of Populus to the races of M. larici-populina. In natural ecosystems such a differential interaction of race/cultivar with the light intensity should reinforce the stability in this host-pathogen relationship. The response of cultivars of poplars to races of Melampsora larici-populina Kleb. is sensitive to temperature. Resistance in 'congenial cultivars' was higher when cultivar /race combinations were incubated at 25°, rather than at 12° or 20° C (Chandrashekar & Heather, 1980a). Further, resistance in such cultivars to leaf rust was higher when the plants were cultured on a high (28/20°, day /night) than on lower pre-inoculation temperature regime (20/10°) (Chandrashekar & Heather, 1980b). In these cultivar /race interactions leaf rust intensity was lowest with the combination of high pre- and high post-inoculation temperature than in any other coupling of such variables. In these experiments, the first, second, third and fourth order interactions between cultivars, races, preand post-inoculation temperatures were all highly significant (P <0'001). Germination and germ-tube growth of urediniospores of M. larici-populina are reduced when incubated at 10000 rather than at 1000 or 5000 lx ; with inhibition of germination at 20000 lx (Omar, 1978). However, Spiers (1978) observed that germination of urediniospores of M. larici-populina and M. medusae Thum., and infection of leaf disks of poplar by these organisms, were unaffected by light intensity (level unspecified) when compared to continuous darkness. Reports on the effect of light intensity on susceptibility of cereal hosts to rusts are conflicting, with increased light intensity causing reduced (Gassner, 1927; Johnson, 1931), increased (Hart & Zaleski, 1935) and no effect (Melander, 1935) on host resistance. The importance of quantitative type resistance
to leaf rust in poplar cultivars and its sensitivity to temperature has been discussed in relation to the establishment of poplar groves (Heather & Sharma, 1977; Chandrashekar & Heather, 1980a, b, c). In this paper the effect of another environmental variable, light intensity, on the reactions of 'congenial cultivars' of poplar to races of M. larici-populina is reported. The epidemiological and ecological implications of effects of light on leaf rust in poplar are discussed. MATERIALS AND METHODS
Four races of M.larici-populina, designated A, B, D, E, and four cultivars of Populus spp., P. x euramericana (Dode) Guinier cvs. '1-488 " '1-214' and '65/27' and P. nigra L. cv. 'evergreen', with which they demonstrated varying degrees of compatibility (Chandrashekar & Heather, 1980c), were used in the experiment. The individual races were purified and multiplied as described previously (Chandrashekar & Heather, 1980c) to produce adequate quantities of urediniospores. Replicate cultivars were cultured in a rust free, growth cabinet (temperature 20 ± 1° and 16 h photoperiod). Leaf production on shoots of similar age of each cultivar was recorded to enable collection of leaves of comparable maturity, thus ensuring uniform disease expression of subsamples within the cultivar (Sharma, Heather & Winer, 1980). Banks of cool fluorescent tubes (Phillips, TL 20W/ 84) were used to obtain post-inoculation light intensities of 100, 250 and 1000 micro Einsteins per square metre per second (jlE m~2 S-1). The
Light intensity and poplar rust inoculation, incubation and observations on leaf disks followed the procedure of Chandrashekar & Heather (1980c) . Fifteen leaf disks (1'76 ern") were cut from leaf samples of each cultivar and inoculated separately with 5 mg of urediniospores of each race of M.larici-populina in a settling tower, floated on 10 p.p.m, giberellic acid solution in Petri dishes and incubated at 20°, with a light intensity of 100, 250 or 1000 JiE m ? S-1 on a 16 h photoperiod. Due to space limitations of the tower, inoculation of leaf disks of cultivars with the races was repeated to obtain sufficient disks for incubationat each light level. Preliminaryexperiments had demonstrated that, providing certain precautions were followed, the mean spore load deposited per unit area in successive inoculations in the tower did not differ significantly (Chandrashekar & Heather, unpubl.). The experiment was terminated on day 14 after inoculation when the number of uredinia per leaf disk in all treatments had ceased to increase. Disease severity was assessed using the following parameters. (1) Incubation period (days) from inoculation to fleck production (IPF). (Flecks, localized chlorotic areas, were the initial symptoms of successful infection and were formed 2-3 days prior to uredinia.) (2) Uredinia per leaf disk (ULD) were assessed daily from their first appearence until day 14. The results were tested for homoscedasticity and normality (N eter & Wasserman, 1974) using a GLIM program (Nelder, 1975) and subjected to analysis of variance usingthesub-programANOVA of SPSS (Nie et al., 1975 ) . For each light intensity, the mean ULD at each daily observation was grouped separately by race across the four culti vars, and plotted against time after first appearance of uredinia until day 14. Since there was a difference in incubation period to flecking (IPF) between light levels, the day of first appearance of uredinia at each light intensity was treated as day one in these graphs. This adjustment permitted the fitting of the cubic curve
Yi
= /30 +/31xi +/32xi2+/33xi3 +Ei
to the data using the GLIM program. Within a race, comparisons were made pairwise between curves developed at different light levels using the methods described by Sharma et at. (1980 ). RESULTS
The disease severity induced by the races in the cultivars is sensitive to the light intensity of incubation (Tables 1, 2). Incubation period (IPF) was directly, and the numbers of uredinia (ULD) inversely, correlated with increasing light intensity
over the range 100-1000 JiE m- 2 S-1 (T ables 1,2). For the light regimes of 100 and 250 JiE m- 2 S-1 the ranking of races for relative aggressiveness on IPF and ULD is reasonably comparable, however at 1000 JiE m- 2 S-1 the relative ranking of races depends on the parameter of disease employed (Table 1). In a comparison between light intensities the relative aggressiveness of each race is dependent on the light intensity of incubation. Thus A which is the most aggressive race at 100, is relatively less aggressive at 1000 JiE m- 2 S-I. There is also a reversal in relative aggressiveness of race D (the most aggressive at 250 and the least aggressive at 100 JiE m- 2 S-l). The relative aggressiveness of race B is not greatly affected by the light intensity, however E, which is intermediate in its aggressiveness at 100 and 1000, is the least aggressive race at 250 JiE m- 2 S-I. The relative ranking of cultivars for resistance within the light intensities of 100 and 250 JiE m- 2 S-l is also independent of the parameter (IPF of ULD) used, but again at 1000 JiE m- 2 S-1 ranking varies with the parameter employed. When disease severity is compared between cultivars at different light intensities the relative resistance is light intensity dependent. P. x euramericana '1488,' which is the most resistant cultivar at 250 is generally relatively less resistant at 100 and 1000 JiE m - 2 S-1 (T able 2). Similarly P. nigra' evergreen' which is the most resistant cultivar at 100, is the most susceptible at 250 JiE m- 2 S-I, i.e, complete reversal of relative resistance. Cultivars P. x euramericana '1-214' and' 65 /27' are intermediate in relative resistance at all levels of light intensity. Analysis of variance of the results for IPF and ULD (T able 3) emphasizes the importance of the light intensity regime of incubation in determining the susceptibility of a cultivar or the aggressiveness of a race. The major components of variance; light regimes, races and cultivars, their second and third order interactions all differ significantly at P < 0 '001. If the interactive variances are added to the residual variance, the variances due to the major components are still very highly significant. For both parameters of the disease, the variance due to light intensity is very much greater than that due to cultivars or races. Similarly the variances of the second order interactions involving light intensity are greater than that for the race x cultivar interaction (Table 3). The cumulative disease progress curves in terms of uredinia per leaf disk per day, for each race across all the cultivars at three light intensities, are plotted in Fig. 1. The curves for race D (mean of four cultivars) incubated at 100 and 250 JiE m - 2 S-1 and those for race E, at 250 and 1000 JiE m- 2 S-1 do not differ
M. Chandrashekar and W. A. Heather
377
Table 1. Mean aggressiveness of four races of M. larici-populina, on four' congenial cultioars' of poplar as measured by two disease parameters, at three light intensity levels Light intensity CuE m- 2 S-1) 100
25°
1000
r-~
r-~
r-~
Race A B
D E Mean
IPF
ULD
IPF
ULD
IPF
ULD
5'00* 5'52 6'37 5'75 5'66
77"45 28,65 23'77 32'18
6'96 6,64 6'44 7'93 6'99
11,66 17'02 20'00 2'3 8
8'34 7"42 7,64
2'05
12'77
7'69
40'79
BS 3'85 2'5 1 4'19
rss
IPF - Incubation period to flecking (days). ULD - Uredinia per leaf disk at 14 days. * Mean of 15 replicates. Table
2.
Mean resistance of four' congenial cultivars' of poplar to four races of M. larici-populina, as measured by two parameters of disease severity at three light intensities
Light intensity CuE m- 2 S-1) '-
Cultivar
100
P. x euramericana '1-488 ' '1-214' '65/27'
P. nigra cv. 'evergreen' Mean
1000
25°
IPF
ULD
IPF
ULD
IPF
ULD
5'25*
32'00
7'68
5'34
8'02
1'3 8
5'25 5'7 0
7'09 6'63 6'57
9'54 9'27 26'57
8'12
6"44
63'47 36'92 29'7 6
7'34
3'62 3'68 7'68
5'66
40'46
6'99
12'77
7,68
4'09
7'23
IPF - Incubation period to flecking (days). ULD - Uredinia per leaf disk at 14 days. * Mean of 15 replicates. Table 3. Analysis of variance of two parameters of disease severity, resulting from the interaction of four cultivars of poplar and four races of M. laricipopulina at three light intensities Variance ,------"-~
Source of variation Light Race Cultivar Light x race Light x cultivar Race x cultivar Light x race x cultivar Residual Total
d.f.
IPF
ULD (x 103)
2 3 3 6 6 9 17
193'05 7'19 4'12 22'9 8 22'23 7'87 1'74
77'4 2 10'49 4,83 13'29 6'15 1'23 1'°5
576 622
0,62
0'23
1,84
0'75
All variance ratios are significant at P < 0'001.
significantly (P < 0'05). For each of the remaining comparisons between curves the difference is either very highly (P < 0'001) or highly (P < 0'01) significant. Thus, in a particular race/cultivar combination the pronounced effect of light on disease severity is not an artifact of the time at which the latter is assessed. DISSCUSSION
Intensities of sunlight in fully exposed areas in Canberra throughout the year vary from 502000,uE m- 2 S-1, however, a maximum intensity of 2000,uE m- 2 S-1 is common in the late spring summer months, i.e. during the period when the leaf rust due to M. larici-populina is most prevalent. Variation in disease severity due to differences in light intensity, over the range of lOO-1000,uE m -2 s-I, is much more significant than that due to
378
Light intensity andpoplar rust 80 70
Race A
Race B a'
60 50 40 30 20 is ...J
2-
~
10
'5
'Ol
~
0
....
8
9
10
I I 12 13 14
0
8
9
10 I I
12 13 14
"0Ol
'5
".... ::l
'0
....
80
"
70
::l
60
.D
E
Z
Race D
Race E
50 40 30
p'
~
20 10 0
8
Lr-tjj; 9 10 II 12 13 14
L-....,
0
L: 8
9
10
II
12 13 14
Days aft er inoc ulation
Fig. 1. Cumulative number of uredinia per leaf disk, induced by four races of M. larici-populina, averaged acrossfour' congenial' cultivars of Populus spp., incubated at light intensities of 100 ( - .- ) , 250 (-e-) and 1000 ( - .,. - ) ItE m- 2 S -I . (Same letter, same superscript - NS ; Different letter, same superscript - P < 0 '01 ; different letter, different superscript - P < 0'001.)
races or cultivars used in the present experiment (Table 3). For both parameters of disease severity, cultivar resistance was positively correlated (Table 2) with increasing light intensity while aggressiveness of the races is negatively correlated (Table 3) with this variable. The inhibitory effect of high light intensity on germination of urediniospores and on germ-tube growth in M .larici-populina (Omar, 1978) is probably a partial explanation for these observations. The present results conflict with those of Spiers (1978) which indicated that light (unspecified), in contrast to complete darkness, had no effect on germination of urediniospores of two species of Melampsora, or on infection of leaf disks of Populus spp. by these organi sms. Possibl y the light intensity emplo yed in those experiments explains this conflict. High light intensity caused
reduced infection of wheat by stem rust (Puccinia graminis Pers. f. sp. tritici Erikss. & Henn.) (Hart & Zaleski, 1935), while a light intensity of 900 ft ca. inhibited formation of infection structures of this organism on artificial substrates (Ernge, 1958). Similarly high light intensity reduced the germination of urediniospores of P. graminis f.sp, tritici (Givan & Bromfield, 1964a ), and germination (Eyal & Peturson, 1967) and appressorial formation (Givan & Bromfield, 1964b) in those of P. recondita Rob. ex Desm, However, in contrast Gassner (1927) and Johnson (1931) reported increased, while Melander (1935) observed no effect on, susceptibility of wheat to stem rust with increased light intensity. At light intensities of 100 and 250/lE m ? S-1 the ranking of the races for relative aggressiveness
M. Chandrashekar and W. A. Heather (Table 1) or cultivars for their relative resistance (Table 2), is independent of the disease parameter employed. Further at these light intensities there is a good inverse correlation between IPF and ULD; this agrees with the relationship observed between these parameters in leaf rust of barley (Parlevliet, 1975). However at 1000,uE m " S-l relative aggressiveness of races (Table 1) or resistance of cultivars (Table 2) depends on the parameter employed for the ranking. In addition the expected inverse relationship between IPF and ULD is less pronounced at this light intensity than at 100 or 250,uE m- 2 S-l. Possibly high light intensity affects differentially the processes leading to these forms of disease expression. A similar probable differential effect of temperature of incubation on such processes has been proposed previously (Chandrashekar & Heather, 1980a). In a comparison between light regimes the order of the ranking of races for aggressiveness, or cultivars for resistance, is dependent on the light intensity of incubation. The differential nature of the overall cultivar/race/light intensity interaction is confirmed by the very high level of significance (P < 0'001) of the second and third order interactions, for both IPF and ULD, in the ANOVA (Table 3). Thus, this host/pathogen/environment system demonstrates quantitative interaction in some instances with, in others without, reversal (Scott et al., 1979). The significant differences between the slopes of the curves for cultivar /race combinations incubated at three light intensities (Fig. 1) suggest that such differential interactions occur throughout the period of uredinial production. The sensitivity to light intensity of aggressiveness in the races of M. larici-populina and of resistance among cultivars of Populus spp. has both epidemiological and ecological significance. Mean IPF is reduced by ca. two days and mean ULD reduced by ca. ten-fold when the race/cultivar combinations are incubated at a light intensity of 1000 rather than 100,uE m- 2 S-l. Increased IPF will lengthen the period for a monocycle in an epidemic and this together with reduced ULD, should slow the rate of disease progress (Van der Plank, 1968) in a high compared with a low light intensity. It has been demonstrated that high temperature has the potential for a similar effect on disease progress (Chandrashekar & Heather, 1980a). Thus, in areas west of the Great Dividing Range in eastern Australia, high light intensity and high temperatures could act additively to reduce the rate of the disease progress when leaf rust in poplar is recorded initially in mid-summer. Low rates of disease increase have been noted in these areas under these conditions (Heather, unpubl.).
379
Endemic pathogens rarely cause epidemic disease in natural ecosystems. It has been proposed that variability in the racial composition of pathogen populations, resulting from the diversity of host genotypes in such systems, is a partial explanation for this phenomenon (Browning, 1974). The differential interactions of the race /cultivar combinations and light intensity demonstrated in the present results, and those reported previously with temperature (Chandrashekar & Heather, 1980a), would reinforce the diversity of racial composition in natural ecosystems. Such an interactive host/pathogen/environment system, -possibly indicative of a long period of co-existence of host and pathogen, should be particularly robust and thus free of epidemic disease except in unusual circumstances. This agrees with the observations in natural poplar/leaf rust ecosystems of Europe and North America.
REFERENCES BROWNING, J. A. (1974).Relevance of knowledge about natural ecosystems to development of pest management programs for agro-ecosystems, Proceedings of the American Phytopathological Society 1, 191-199. CHANDRASHEKAR, M. & HEATHER, W. A. (198oa). Temperature sensitivity of reactions of Populus spp. to races of Melampsora larici-populina Kleb. Phytopathology (in press). CHANDRASHEKAR, M. & HEATHER, W. A. (1980b). The effect of pre- and post-inoculation temperature on resistance in certain clones of poplar to races of Melampsora larici-populina Kleb. Euphytica (in press). CHANDr.ASHEKAR, M. & HEATHER, W. A. (198oc). Reactions of poplar clones to physiologic races of Melampsora larici-populina Kleb. Euphytica 29, 401407· EMGE, R. G. (1958).The influence of light and temperature on the formation of infection-structures of Puccinia graminis var. tritici on artificial substrates. Phytopathology 44, 649-652. EYAL, Z. & PETURSON, J. L. (1967). Uredospore production of five races of Puccinia recondita Rob. ex Desm. as affected by light and temperature. Canadian Journal of Botany 45, 537-539. GASSNER, G. (1927). Die Frage der Rostanfalligkeit als ernahrungsphysiologisches Problem. Angewandte Botanik 9, 531-541. GIVAN, C. V. & BROMFIELD, K. R. (1964a). Light inhibition of uredospore germination in Puccinia graminis var, tritici. Phytopathology 54, 382-384. GIVAN, C. V. & BROMFIELD, K. R. (1964b). Light inhibition of uredospore germination in Puccinia recondita. Phytopathology 54, 116-117. HART, H. & ZALESKI, K. (1935). The effect of light intensity and temperature on infection of Hope wheat by Puccinia graminis tritici. Phytopathology 25, 1°41-1066.
380
Light intensity and poplar rus:
HEATHER, W. A. & SHARMA, J. K. (1977). Some aspects of poplar rust research in Australia. Australian Forestry 40, 28-43. JOHNSON, T. (1931). A study of the effect of environmental factors on the physiologic forms of Puccinia graminis tritici. Dominion of Canada Department of Agriculture Bulletin (New Series) 140, 1-76. MELANDER, L. W. (1935). Effect of temperature and light on development of the uredial stages of Puccinia graminis, Journal of Agricultural Research 50, 861880. NELDER, J. A. (1975). General Linear Interactive modelling. Pub. Royal Statistical Society. GUM Manual release 2. NETER, J. & WASSERMAN, W. A. (1974). Applied Linear Statistical Models. London: R. D. Irwin. NIE, H. H., HULL, C. H., JENKINS, J. G., STEINGRENNER, K. & BENT, D. H. (1975). Statistical Package for the Social Sciences (znd ed.), New York: McGraw-Hill. OMAR, M. B. (1978). Aspects of pre- and post-penetration phenomena of Melampsora larici-populina Kleb. Ph.D. thesis, The Australian National University.
PARLEVLIET, J. E. (1975). Partial resistance of barley to leaf rust, Puccinia hordei I. Effect of cultivar and development stage on latent period. Euphytica 24, 21-27. SCOTT, P. R., JOHNSON, R., WOLFE, M. S., LOWE, H. J. B. & BENNET, F. G. A. (1979). Host specificity in cereal parasites in relation to their control. Plant Breeding Institute, Trumpington, Cambridge, Annual Report, 27-62. SHARMA, J. K., HEATHER, W. A. & WINER, P. (1980). Effect of leaf maturity and shoot age of poplar clones on susceptibility to Melampsora larici-populina. Phytopathology 70, 548-554. SPIERS, A. G. (1978). Effects of light, temperature and relative humidity on germination of urediniospores of, and infection of poplars by, Melampsora laricipopulina and M. medusae. New Zealand Journal of Science 21, 393-400. VAN DER PLANK, J. E. (1968). Disease Resistance in Plants. New York/London: Academic press.
(Received for publication 4 October 1980)
Primed in Great Britain
INFLUENCE OF LIGHT INTENSITY ON REACTIONS OF CERTAIN POPLAR CUL TIVARS TO RACES OF MELAMPSORA LARICI-POPULINA By M. CHANDRASHEKAR AND W. A. HEATHER Department of Forestry, The Australian National University, Canberra, ACT, Australia 2600 The resistance offour cultivars of poplar, of varying compatibility to four races of Melampsora larici-populina, was sensitive to the light intensity of incubation. Disease severity, assessed on two parameters (incubation period and uredinial numbers) was lower at 1000 pE m~2 S-1 than at 100 or 250 pE m- 2 s- 1light intensity. The major components: light, race, cultivar, and all the second and third order interactions between these were highly significant (P < 0'001). Thus, light intensity of incubation is very significant in determining the susceptibility of these cultivars of Populus to the races of M. larici-populina. In natural ecosystems such a differential interaction of race/cultivar with the light intensity should reinforce the stability in this host-pathogen relationship. The response of cultivars of poplars to races of Melampsora larici-populina Kleb. is sensitive to temperature. Resistance in 'congenial cultivars' was higher when cultivar /race combinations were incubated at 25°, rather than at 12° or 20° C (Chandrashekar & Heather, 1980a). Further, resistance in such cultivars to leaf rust was higher when the plants were cultured on a high (28/20°, day /night) than on lower pre-inoculation temperature regime (20/10°) (Chandrashekar & Heather, 1980b). In these cultivar /race interactions leaf rust intensity was lowest with the combination of high pre- and high post-inoculation temperature than in any other coupling of such variables. In these experiments, the first, second, third and fourth order interactions between cultivars, races, preand post-inoculation temperatures were all highly significant (P <0'001). Germination and germ-tube growth of urediniospores of M. larici-populina are reduced when incubated at 10000 rather than at 1000 or 5000 lx ; with inhibition of germination at 20000 lx (Omar, 1978). However, Spiers (1978) observed that germination of urediniospores of M. larici-populina and M. medusae Thum., and infection of leaf disks of poplar by these organisms, were unaffected by light intensity (level unspecified) when compared to continuous darkness. Reports on the effect of light intensity on susceptibility of cereal hosts to rusts are conflicting, with increased light intensity causing reduced (Gassner, 1927; Johnson, 1931), increased (Hart & Zaleski, 1935) and no effect (Melander, 1935) on host resistance. The importance of quantitative type resistance
to leaf rust in poplar cultivars and its sensitivity to temperature has been discussed in relation to the establishment of poplar groves (Heather & Sharma, 1977; Chandrashekar & Heather, 1980a, b, c). In this paper the effect of another environmental variable, light intensity, on the reactions of 'congenial cultivars' of poplar to races of M. larici-populina is reported. The epidemiological and ecological implications of effects of light on leaf rust in poplar are discussed. MATERIALS AND METHODS
Four races of M.larici-populina, designated A, B, D, E, and four cultivars of Populus spp., P. x euramericana (Dode) Guinier cvs. '1-488 " '1-214' and '65/27' and P. nigra L. cv. 'evergreen', with which they demonstrated varying degrees of compatibility (Chandrashekar & Heather, 1980c), were used in the experiment. The individual races were purified and multiplied as described previously (Chandrashekar & Heather, 1980c) to produce adequate quantities of urediniospores. Replicate cultivars were cultured in a rust free, growth cabinet (temperature 20 ± 1° and 16 h photoperiod). Leaf production on shoots of similar age of each cultivar was recorded to enable collection of leaves of comparable maturity, thus ensuring uniform disease expression of subsamples within the cultivar (Sharma, Heather & Winer, 1980). Banks of cool fluorescent tubes (Phillips, TL 20W/ 84) were used to obtain post-inoculation light intensities of 100, 250 and 1000 micro Einsteins per square metre per second (jlE m~2 S-1). The
Light intensity and poplar rust inoculation, incubation and observations on leaf disks followed the procedure of Chandrashekar & Heather (1980c) . Fifteen leaf disks (1'76 ern") were cut from leaf samples of each cultivar and inoculated separately with 5 mg of urediniospores of each race of M.larici-populina in a settling tower, floated on 10 p.p.m, giberellic acid solution in Petri dishes and incubated at 20°, with a light intensity of 100, 250 or 1000 JiE m ? S-1 on a 16 h photoperiod. Due to space limitations of the tower, inoculation of leaf disks of cultivars with the races was repeated to obtain sufficient disks for incubationat each light level. Preliminaryexperiments had demonstrated that, providing certain precautions were followed, the mean spore load deposited per unit area in successive inoculations in the tower did not differ significantly (Chandrashekar & Heather, unpubl.). The experiment was terminated on day 14 after inoculation when the number of uredinia per leaf disk in all treatments had ceased to increase. Disease severity was assessed using the following parameters. (1) Incubation period (days) from inoculation to fleck production (IPF). (Flecks, localized chlorotic areas, were the initial symptoms of successful infection and were formed 2-3 days prior to uredinia.) (2) Uredinia per leaf disk (ULD) were assessed daily from their first appearence until day 14. The results were tested for homoscedasticity and normality (N eter & Wasserman, 1974) using a GLIM program (Nelder, 1975) and subjected to analysis of variance usingthesub-programANOVA of SPSS (Nie et al., 1975 ) . For each light intensity, the mean ULD at each daily observation was grouped separately by race across the four culti vars, and plotted against time after first appearance of uredinia until day 14. Since there was a difference in incubation period to flecking (IPF) between light levels, the day of first appearance of uredinia at each light intensity was treated as day one in these graphs. This adjustment permitted the fitting of the cubic curve
Yi
= /30 +/31xi +/32xi2+/33xi3 +Ei
to the data using the GLIM program. Within a race, comparisons were made pairwise between curves developed at different light levels using the methods described by Sharma et at. (1980 ). RESULTS
The disease severity induced by the races in the cultivars is sensitive to the light intensity of incubation (Tables 1, 2). Incubation period (IPF) was directly, and the numbers of uredinia (ULD) inversely, correlated with increasing light intensity
over the range 100-1000 JiE m- 2 S-1 (T ables 1,2). For the light regimes of 100 and 250 JiE m- 2 S-1 the ranking of races for relative aggressiveness on IPF and ULD is reasonably comparable, however at 1000 JiE m- 2 S-1 the relative ranking of races depends on the parameter of disease employed (Table 1). In a comparison between light intensities the relative aggressiveness of each race is dependent on the light intensity of incubation. Thus A which is the most aggressive race at 100, is relatively less aggressive at 1000 JiE m- 2 S-I. There is also a reversal in relative aggressiveness of race D (the most aggressive at 250 and the least aggressive at 100 JiE m- 2 S-l). The relative aggressiveness of race B is not greatly affected by the light intensity, however E, which is intermediate in its aggressiveness at 100 and 1000, is the least aggressive race at 250 JiE m- 2 S-I. The relative ranking of cultivars for resistance within the light intensities of 100 and 250 JiE m- 2 S-l is also independent of the parameter (IPF of ULD) used, but again at 1000 JiE m- 2 S-1 ranking varies with the parameter employed. When disease severity is compared between cultivars at different light intensities the relative resistance is light intensity dependent. P. x euramericana '1488,' which is the most resistant cultivar at 250 is generally relatively less resistant at 100 and 1000 JiE m - 2 S-1 (T able 2). Similarly P. nigra' evergreen' which is the most resistant cultivar at 100, is the most susceptible at 250 JiE m- 2 S-I, i.e, complete reversal of relative resistance. Cultivars P. x euramericana '1-214' and' 65 /27' are intermediate in relative resistance at all levels of light intensity. Analysis of variance of the results for IPF and ULD (T able 3) emphasizes the importance of the light intensity regime of incubation in determining the susceptibility of a cultivar or the aggressiveness of a race. The major components of variance; light regimes, races and cultivars, their second and third order interactions all differ significantly at P < 0 '001. If the interactive variances are added to the residual variance, the variances due to the major components are still very highly significant. For both parameters of the disease, the variance due to light intensity is very much greater than that due to cultivars or races. Similarly the variances of the second order interactions involving light intensity are greater than that for the race x cultivar interaction (Table 3). The cumulative disease progress curves in terms of uredinia per leaf disk per day, for each race across all the cultivars at three light intensities, are plotted in Fig. 1. The curves for race D (mean of four cultivars) incubated at 100 and 250 JiE m - 2 S-1 and those for race E, at 250 and 1000 JiE m- 2 S-1 do not differ
M. Chandrashekar and W. A. Heather
377
Table 1. Mean aggressiveness of four races of M. larici-populina, on four' congenial cultioars' of poplar as measured by two disease parameters, at three light intensity levels Light intensity CuE m- 2 S-1) 100
25°
1000
r-~
r-~
r-~
Race A B
D E Mean
IPF
ULD
IPF
ULD
IPF
ULD
5'00* 5'52 6'37 5'75 5'66
77"45 28,65 23'77 32'18
6'96 6,64 6'44 7'93 6'99
11,66 17'02 20'00 2'3 8
8'34 7"42 7,64
2'05
12'77
7'69
40'79
BS 3'85 2'5 1 4'19
rss
IPF - Incubation period to flecking (days). ULD - Uredinia per leaf disk at 14 days. * Mean of 15 replicates. Table
2.
Mean resistance of four' congenial cultivars' of poplar to four races of M. larici-populina, as measured by two parameters of disease severity at three light intensities
Light intensity CuE m- 2 S-1) '-
Cultivar
100
P. x euramericana '1-488 ' '1-214' '65/27'
P. nigra cv. 'evergreen' Mean
1000
25°
IPF
ULD
IPF
ULD
IPF
ULD
5'25*
32'00
7'68
5'34
8'02
1'3 8
5'25 5'7 0
7'09 6'63 6'57
9'54 9'27 26'57
8'12
6"44
63'47 36'92 29'7 6
7'34
3'62 3'68 7'68
5'66
40'46
6'99
12'77
7,68
4'09
7'23
IPF - Incubation period to flecking (days). ULD - Uredinia per leaf disk at 14 days. * Mean of 15 replicates. Table 3. Analysis of variance of two parameters of disease severity, resulting from the interaction of four cultivars of poplar and four races of M. laricipopulina at three light intensities Variance ,------"-~
Source of variation Light Race Cultivar Light x race Light x cultivar Race x cultivar Light x race x cultivar Residual Total
d.f.
IPF
ULD (x 103)
2 3 3 6 6 9 17
193'05 7'19 4'12 22'9 8 22'23 7'87 1'74
77'4 2 10'49 4,83 13'29 6'15 1'23 1'°5
576 622
0,62
0'23
1,84
0'75
All variance ratios are significant at P < 0'001.
significantly (P < 0'05). For each of the remaining comparisons between curves the difference is either very highly (P < 0'001) or highly (P < 0'01) significant. Thus, in a particular race/cultivar combination the pronounced effect of light on disease severity is not an artifact of the time at which the latter is assessed. DISSCUSSION
Intensities of sunlight in fully exposed areas in Canberra throughout the year vary from 502000,uE m- 2 S-1, however, a maximum intensity of 2000,uE m- 2 S-1 is common in the late spring summer months, i.e. during the period when the leaf rust due to M. larici-populina is most prevalent. Variation in disease severity due to differences in light intensity, over the range of lOO-1000,uE m -2 s-I, is much more significant than that due to
378
Light intensity andpoplar rust 80 70
Race A
Race B a'
60 50 40 30 20 is ...J
2-
~
10
'5
'Ol
~
0
....
8
9
10
I I 12 13 14
0
8
9
10 I I
12 13 14
"0Ol
'5
".... ::l
'0
....
80
"
70
::l
60
.D
E
Z
Race D
Race E
50 40 30
p'
~
20 10 0
8
Lr-tjj; 9 10 II 12 13 14
L-....,
0
L: 8
9
10
II
12 13 14
Days aft er inoc ulation
Fig. 1. Cumulative number of uredinia per leaf disk, induced by four races of M. larici-populina, averaged acrossfour' congenial' cultivars of Populus spp., incubated at light intensities of 100 ( - .- ) , 250 (-e-) and 1000 ( - .,. - ) ItE m- 2 S -I . (Same letter, same superscript - NS ; Different letter, same superscript - P < 0 '01 ; different letter, different superscript - P < 0'001.)
races or cultivars used in the present experiment (Table 3). For both parameters of disease severity, cultivar resistance was positively correlated (Table 2) with increasing light intensity while aggressiveness of the races is negatively correlated (Table 3) with this variable. The inhibitory effect of high light intensity on germination of urediniospores and on germ-tube growth in M .larici-populina (Omar, 1978) is probably a partial explanation for these observations. The present results conflict with those of Spiers (1978) which indicated that light (unspecified), in contrast to complete darkness, had no effect on germination of urediniospores of two species of Melampsora, or on infection of leaf disks of Populus spp. by these organi sms. Possibl y the light intensity emplo yed in those experiments explains this conflict. High light intensity caused
reduced infection of wheat by stem rust (Puccinia graminis Pers. f. sp. tritici Erikss. & Henn.) (Hart & Zaleski, 1935), while a light intensity of 900 ft ca. inhibited formation of infection structures of this organism on artificial substrates (Ernge, 1958). Similarly high light intensity reduced the germination of urediniospores of P. graminis f.sp, tritici (Givan & Bromfield, 1964a ), and germination (Eyal & Peturson, 1967) and appressorial formation (Givan & Bromfield, 1964b) in those of P. recondita Rob. ex Desm, However, in contrast Gassner (1927) and Johnson (1931) reported increased, while Melander (1935) observed no effect on, susceptibility of wheat to stem rust with increased light intensity. At light intensities of 100 and 250/lE m ? S-1 the ranking of the races for relative aggressiveness
M. Chandrashekar and W. A. Heather (Table 1) or cultivars for their relative resistance (Table 2), is independent of the disease parameter employed. Further at these light intensities there is a good inverse correlation between IPF and ULD; this agrees with the relationship observed between these parameters in leaf rust of barley (Parlevliet, 1975). However at 1000,uE m " S-l relative aggressiveness of races (Table 1) or resistance of cultivars (Table 2) depends on the parameter employed for the ranking. In addition the expected inverse relationship between IPF and ULD is less pronounced at this light intensity than at 100 or 250,uE m- 2 S-l. Possibly high light intensity affects differentially the processes leading to these forms of disease expression. A similar probable differential effect of temperature of incubation on such processes has been proposed previously (Chandrashekar & Heather, 1980a). In a comparison between light regimes the order of the ranking of races for aggressiveness, or cultivars for resistance, is dependent on the light intensity of incubation. The differential nature of the overall cultivar/race/light intensity interaction is confirmed by the very high level of significance (P < 0'001) of the second and third order interactions, for both IPF and ULD, in the ANOVA (Table 3). Thus, this host/pathogen/environment system demonstrates quantitative interaction in some instances with, in others without, reversal (Scott et al., 1979). The significant differences between the slopes of the curves for cultivar /race combinations incubated at three light intensities (Fig. 1) suggest that such differential interactions occur throughout the period of uredinial production. The sensitivity to light intensity of aggressiveness in the races of M. larici-populina and of resistance among cultivars of Populus spp. has both epidemiological and ecological significance. Mean IPF is reduced by ca. two days and mean ULD reduced by ca. ten-fold when the race/cultivar combinations are incubated at a light intensity of 1000 rather than 100,uE m- 2 S-l. Increased IPF will lengthen the period for a monocycle in an epidemic and this together with reduced ULD, should slow the rate of disease progress (Van der Plank, 1968) in a high compared with a low light intensity. It has been demonstrated that high temperature has the potential for a similar effect on disease progress (Chandrashekar & Heather, 1980a). Thus, in areas west of the Great Dividing Range in eastern Australia, high light intensity and high temperatures could act additively to reduce the rate of the disease progress when leaf rust in poplar is recorded initially in mid-summer. Low rates of disease increase have been noted in these areas under these conditions (Heather, unpubl.).
379
Endemic pathogens rarely cause epidemic disease in natural ecosystems. It has been proposed that variability in the racial composition of pathogen populations, resulting from the diversity of host genotypes in such systems, is a partial explanation for this phenomenon (Browning, 1974). The differential interactions of the race /cultivar combinations and light intensity demonstrated in the present results, and those reported previously with temperature (Chandrashekar & Heather, 1980a), would reinforce the diversity of racial composition in natural ecosystems. Such an interactive host/pathogen/environment system, -possibly indicative of a long period of co-existence of host and pathogen, should be particularly robust and thus free of epidemic disease except in unusual circumstances. This agrees with the observations in natural poplar/leaf rust ecosystems of Europe and North America.
REFERENCES BROWNING, J. A. (1974).Relevance of knowledge about natural ecosystems to development of pest management programs for agro-ecosystems, Proceedings of the American Phytopathological Society 1, 191-199. CHANDRASHEKAR, M. & HEATHER, W. A. (198oa). Temperature sensitivity of reactions of Populus spp. to races of Melampsora larici-populina Kleb. Phytopathology (in press). CHANDRASHEKAR, M. & HEATHER, W. A. (1980b). The effect of pre- and post-inoculation temperature on resistance in certain clones of poplar to races of Melampsora larici-populina Kleb. Euphytica (in press). CHANDr.ASHEKAR, M. & HEATHER, W. A. (198oc). Reactions of poplar clones to physiologic races of Melampsora larici-populina Kleb. Euphytica 29, 401407· EMGE, R. G. (1958).The influence of light and temperature on the formation of infection-structures of Puccinia graminis var. tritici on artificial substrates. Phytopathology 44, 649-652. EYAL, Z. & PETURSON, J. L. (1967). Uredospore production of five races of Puccinia recondita Rob. ex Desm. as affected by light and temperature. Canadian Journal of Botany 45, 537-539. GASSNER, G. (1927). Die Frage der Rostanfalligkeit als ernahrungsphysiologisches Problem. Angewandte Botanik 9, 531-541. GIVAN, C. V. & BROMFIELD, K. R. (1964a). Light inhibition of uredospore germination in Puccinia graminis var, tritici. Phytopathology 54, 382-384. GIVAN, C. V. & BROMFIELD, K. R. (1964b). Light inhibition of uredospore germination in Puccinia recondita. Phytopathology 54, 116-117. HART, H. & ZALESKI, K. (1935). The effect of light intensity and temperature on infection of Hope wheat by Puccinia graminis tritici. Phytopathology 25, 1°41-1066.
380
Light intensity and poplar rus:
HEATHER, W. A. & SHARMA, J. K. (1977). Some aspects of poplar rust research in Australia. Australian Forestry 40, 28-43. JOHNSON, T. (1931). A study of the effect of environmental factors on the physiologic forms of Puccinia graminis tritici. Dominion of Canada Department of Agriculture Bulletin (New Series) 140, 1-76. MELANDER, L. W. (1935). Effect of temperature and light on development of the uredial stages of Puccinia graminis, Journal of Agricultural Research 50, 861880. NELDER, J. A. (1975). General Linear Interactive modelling. Pub. Royal Statistical Society. GUM Manual release 2. NETER, J. & WASSERMAN, W. A. (1974). Applied Linear Statistical Models. London: R. D. Irwin. NIE, H. H., HULL, C. H., JENKINS, J. G., STEINGRENNER, K. & BENT, D. H. (1975). Statistical Package for the Social Sciences (znd ed.), New York: McGraw-Hill. OMAR, M. B. (1978). Aspects of pre- and post-penetration phenomena of Melampsora larici-populina Kleb. Ph.D. thesis, The Australian National University.
PARLEVLIET, J. E. (1975). Partial resistance of barley to leaf rust, Puccinia hordei I. Effect of cultivar and development stage on latent period. Euphytica 24, 21-27. SCOTT, P. R., JOHNSON, R., WOLFE, M. S., LOWE, H. J. B. & BENNET, F. G. A. (1979). Host specificity in cereal parasites in relation to their control. Plant Breeding Institute, Trumpington, Cambridge, Annual Report, 27-62. SHARMA, J. K., HEATHER, W. A. & WINER, P. (1980). Effect of leaf maturity and shoot age of poplar clones on susceptibility to Melampsora larici-populina. Phytopathology 70, 548-554. SPIERS, A. G. (1978). Effects of light, temperature and relative humidity on germination of urediniospores of, and infection of poplars by, Melampsora laricipopulina and M. medusae. New Zealand Journal of Science 21, 393-400. VAN DER PLANK, J. E. (1968). Disease Resistance in Plants. New York/London: Academic press.
(Received for publication 4 October 1980)