Dose-response relationships for infection of potato leaves by zoospores of Phytophthora infestans

[ 679 ] Trans. Br. mycol Soc. 49 (4), 679-686 (1966) Printed in Great Britain

DOSE-RESPONSE RELATIONSHIPS FOR INFECTION OF POTATO LEAVES BY ZOOSPORES OF PHYTOPHTHORA INFESTANS By D. H. LAPWOOD

Rothamsted Experimental Station, Harpenden, Herts. AND

R. K. McKEE*

Faculty of Agriculture, Queen's University, Belfast, Northern Ireland (With 2 Text-figures) The relationship between the number of zoospores (dose) of Phytophthora infestans applied as inoculum and the relative infection (response) of the upper surface of leaves of nine varieties differing in field susceptibility to potato blight was studied. The ED 50 values (the numbers of spores required for 50% infection) calculated from probit analysis for varieties with little (Bintje, King Edward, Up-to-Date) or intermediate (Arran Viking, Majestic, Ulster Supreme) field resistance ranged from six to fifteen spores per drop with no clear differences between the groups, whereas the ED 50 of resistant varieties (Libertas, Pimpernel, Zeeburger) ranged from twenty-three to forty-six spores. Differences in ED 50 values between varieties were smaller when the lower leaf surface was inoculated and, with few exceptions, susceptibility to infection was greater than on the upper surface. As the dose decreased, the average generation time (time to sporulate) increased for all varieties, and with the same dose, generation times were longer with resistant than susceptible varieties.

Many years of field observation have shown that potato varieties differ greatly in their resistance to blight (Phytophthora infestans (Mont.) de Bary), and this experience has been used to ascribe to varieties susceptibility ratings. Many attempts have been made to devise laboratory or glasshouse tests to indicate the resistance mechanisms involv.ed and allow potato breeders to select resistant seedlings (van der Zaag, 1959; U maerus, 1960). However, most tests that readily distinguish the extremes of the field susceptibility range, fail to discriminate between less contrasting varieties such as King Edward and Majestic, which differ consistently in the field (Muller, 1953; Lapwood, 1961 c). Differences between such varieties may depend on field characteristics, for example, canopy structure, which cannot be considered in laboratory tests, but perhaps the tests themselves are not sufficiently refined. In the past, estimates of leaf susceptibility have been based on the incidence of infection when detached leaves are sprayed with a standardized inoculum under suitable conditions (Muller, 1953; Lapwood, 1961 c). An extension of this method, in which several strengths of inocula were

* Formerly at John Innes Institute, Bayfordbury, Hertford, Herts.

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Transactions British Mycological Society

applied, was used by Hodgson (1961) to estimate the number of spores required to infect a given proportion of leaf disks; he found that to infect 40 % of inoculated disks fifty spores were required for a susceptible and 400 for a resistant variety. McKee (1964) determined the ED 50 (the number of spores required to infect 50 % of the replicates) by probit analysis of results from inoculated potato tuber disks with a dilution series of inocula; he found that not only the ED 50 but also the rate of infection developed was related to resistance. The serial dilution method has the advantages that susceptibility is determined at several inoculum levels and the results are amenable to statistical analysis. This method was therefore used to estimate the leaf susceptibility of nine varieties covering a range of field susceptibilities, to see whether it would distinguish varietal resistance more satisfactorily than previous methods. MATERIALS AND METHODS

Plants of the varieties Bintje, King Edward and Up-to-Date (field susceptible); Majestic, Ulster Supreme and Arran Viking (moderately resistant); and Libertas, Pimpernel and Zeeburger (resistant) were grown in 17'5 ern. pots of John Innes compost NO.3 in a glasshouse; stems were restricted to one or two per pot. In 1963, enough leaves for an experiment were harvested from each batch of plants on two occasions, namely 6-7 weeks and 9-10 weeks after planting; in 1964, leaves were harvested once from each plant, from positions 7, 8, 9 and 10 (numbering from the base upwards) when the stem had 12-14 leaves. The terminal leaflet and two distal pairs were cut from each detached leaf and the leaflet sample was thoroughly mixed before allocation to the inoculation trays. These trays, 30'5 x 30'5 cm., accommodated forty-eight leaflets in six rows of eight on suspended terylene netting (McKee, 1964). In 1963, two rows of three varieties were included in each tray, one row exposing the upper and the other the lower leaf surface; in 1964, when only the upper surface was inoculated, six varieties were used per tray giving one row of each; a different row order was used for each replicate tray. Spores produced on leaves the previous night, or occasionally on tuber slices, were harvested in glass-distilled water and incubated at 50 C, to liberate zoospores. The suspension was filtered through Whatman's NO.4 paper to remove sporangia, and zoospores were counted with a haemacytometer (Thoma) in I ml. aliquots, mixed with I ml. I: 50,000 iodine solution to immobilize the zoospores. Suspensions with the required zoospore concentrations were then prepared by dilution in glass-distilled water, at 10°, containing I % tuber extract (McKee, 1964). Each leaflet was inoculated with one drop from a hypodermic syringe needle (No. 18), cut short and ground square. The needle delivered about 110 drops per ml. and this factor was used in the dilution calculation to give a dilution series containing approximately 256,64, 16,4 and I zoospore(s) per drop. Every leaflet in a tray received the same inoculation dose and there were six replicate trays per dilution, giving thirty trays per experiment with five dilutions. After inoculation the trays were stacked, spaced with sheets of glass and moistened paper, so that each stack contained one tray of each

Phytophthora infestans. D. H. Lapzoood and R. K. McKee 681 dilution (five trays). Each stack was then wrapped in 'Polythene' (polyethylene film) to give six replicate parcels. The parcels were incubated at ISo, and leaflets were examined daily for sporulation, the criterion used as evidence of successful infection. All the varieties could not be tested in an experiment, but some were used as common standards (M aj estic in 1963; Bintje, Majestic and Pimpernel in 1964) and included in all trials. Analysis of individual experiments, using the loglotransformation for zoospore concentration, indicated that results from corresponding trials could be bulked; however, two trials (one of each group in 1964) gave anomalous results, which were attributed to interference by residues following fumigation with a B.H.C. smoke, and were discarded. INTERPRETATION OF RESULTS

The relationship between the number of propagules (the dose) of a pathogen applied to a host and the incidence of infection (the response) they incite is a function of the virulence of the pathogen and the host resistance, each defined with respect to the other. As both virulence and resistance may be affected by environmental factors, this relationship is complex, but may be simplified under laboratory conditions by arranging that only one of the variables is altered at a time. Thus comparison of the dose-response relationships of several varieties exposed to infection by a single isolate of the pathogen, under uniform conditions, gives a measure of one of the variable factors, the relative varietal resistance to that isolate (Dimond, Horsfall, Heuberger & Stoddard, 1941). Plotting percentage response against dose gives a characteristic sigmoid curve for each variety. Comparison of these curves is easier if the response is transformed to probits and plotted against log. dose (Fig. I), as this gives an approximately linear relationship, so that each curve can be defined by only two parameters specifying its slope and position, and which, in this instance, give a numerical measure of resistance. These parameters can be derived from the experimental results either graphically or by probit analysis (Finney, 1952) and expressed as the constants of the regression equationy = a + bx; alternatively, position may be expressed as the ED So value, the number of zoospores per drop required to infect half the inoculated leaflets (probit 5'0, Fig. I). Varietal lines can be compared in pairs, either with each other or with a standard variety (here Majestic was used), and X2 values calculated for position, slope (parallelism) and linearity (Table 2). A significant (large) X2 value for position or slope indicates that the lines differ in the relevant characteristic. A significant linearity X2 is obtained when the relationship between log. dose and probit response is not linear, which means that the standard error of the ED 50 value is larger than that from the direct calculation shown in the Tables. For simplicity of presentation the 10gIO ED So and standard errors from parallel pro bit analysis are shown in absolute units whereas significance is tested by the t test on the log. values, or by the X2 test, shown in the Tables.

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Transactions British Mycological Society R ESULTS

Effect of leaf surface on dose-response relationship In 1963, various aspec ts of the experime nta l procedure were exa mined in preliminary tri als with th e intention of expressing leaf resistan ce as the mean response from inoc ula ting both upper and lower leaf surface. Three varieties were tested in each expe riment including the control Majestic, and th e bulked results from several experime nts are given as th e ED 50 values in T abl e I. 8
-; v '" :E

7

~

...0

6

..e: "'0 ...

75

~

50

ti

.s..

5

OJ c::: 25 ~ ..:i ~

3 4 16 64 256 [umbers of zoospo res (loglo sca le) F ig. I, Relati onsh ip between spore dose and leaf infection for three va rie ties based on d ata fro m six experiments, 0 Majestic, x Bintje, 0 Pimpern el. ~ indica tes ED 50 va lue ,

T able

I.

The number ofzoospores for 50 % injection (ED 50) upper and lower leaf surfaces ofsix varieties

of

Leaf surface

Variety Bintje M ajestic Arran Viking Zee burger Pimpernel Libertas

No, tri als 2

5 1

I

4 2

Upper

Lower

~ S,E,*

,---------A--..

ED 50

(antilog)

5'o±o'55 7'1±o'50 13'4±2 '54 13' 8 ± 2'3 1 18'4± 1'54 20·6±2·76

ED 50

S,E ,

(antilog)

7'8± o' 90 6'5 ±O '44 6'5± O'95 9'6± 1'39 8'8± o'7 2 25'O± 2'98

x·t 8'1** 1'110'8*** 3' 239'6*** 1'5 2 -

* St udent's t test ca n on ly be app lied to loglo transformed da ta , t For diffe rences b etween leaf surface X2 ( rdf ), -, no t significa nt, ** 0' 0 1, ***0'001 probability.

The upper leaf surface was usually more resista nt th an the lower, requiring more spores for 50 % infection, and differences between va rieties were greater with upper surface inoculations. The X2 values comparing

Phytophthora infestans. D. H. Laptoood and R. K. McKee 683 the positions of the dosage-response curves for upper and lower surfaces showed significant differences for Bintje, Arran Viking and Pimpernel. The upper leaf surface of Arran Viking and Pimpernel was the more resistant, but the lower leaf surface of Bintje; more work would be required to substantiate this interesting difference between varieties. In Table I the varieties are arranged in ED 50 value order for the upper leaf surface, which is closer to the accepted field performance than the values for the lower surface. There seemed little advantage in inoculating both surfaces, which decreased the number of varieties that could be tested, so in later tests only the upper surface was inoculated. Table

2.

Probit analysis ofresults of upper leaf surface inoculations of nine varieties No, trials

Variety Group I King Edward Arran Viking Libertas Majestic (control) Group 2 Up-to-Date Ulster Supreme Zeeburger Majestic (control) Group 1-2 Bintje Majestic Pimpernel

Slope S,E, (lOglO)

ED 50 S,E, (antilog)

·X2

..,

A

r

Position Parallelism Linearity

3 3 3 3

1'22±O'o77 1'34±o'083 1'29±o'085 1'68±o'105

I!'7± 1'23 7,8±o'79 45'5±4'90 5'7±o'4 1

31,6 7'5 221'9

13'1 6,8 8'3

19'5 24'4 20'2

3 3 3 3

1'34±o'083 1'33±o'081 1'19±o'078 1'46±O'092

9'3±o'93 14'5± 1'43 32'7±3'60 6'9±O'4 1

5"1 29'7 114'3

1'1 1'2 5'1

8'2 6'8 7,8

1'55±O'068 7'6±o'49 1'56±O'069 6'3±O'4 1 22'7± 1,62 1'32 ± 0'057 Significance level (P = 0'05) df

4'4

O'O!

4'9

7'4

11'4 12,6 6

6 6 6

178'4 3,8 I

3,8 I

·Parallel probit comparisons with Majestic,

Effect of variery on dose-response relationship In 1964, dose-response assessments were made on nine varieties in two series of trials, Group I and Group 2 (Table 2), each of six varieties (three test varieties with Bintje, Majestic and Pimpernel as controls). Table 2 shows the detailed results of the probit analysis, comparing each variety with Majestic. Table 3 summarizes ED 50 values relative to field susceptibility. All varieties, including the susceptible King Edward, have larger ED 50 values than Majestic, and Arran Viking, which like Majestic is moderately resistant in the field, also has a smaller ED 50 than King Edward; in fact there appears to be no relationship between the ED 50 value and field susceptibility for susceptible and moderately resistant varieties. In contrast, the three varieties most resistant in the field (Table 3) all have much larger ED 50 values than the other varieties.

Transactions British Mycological Society Table 3. The ED 50 values of nine varieties grouped according tofield susceptibility Field susceptibility

,

Suscep tible Bintj e K ing Edward Up-to-Date

(a)

7.6 11'7 9"3

Moderately resistant Maj estic 6' 3 Ulster Supreme 14'5 Arran Viking 7.8

0 - 0 Pimp ern el

(,

(b)

R esistant Libertas Pimpernel Zeeburger

45"5

22' 7 32' 7

0 - 0 Liber t as

D.--D.- Arran Vik ing

x - x Bint je

x -x K ing Edward

0 - 0 Majest ic

C--a Majest ic

41---'--_--1._ _-'--_--1._---1 4

Fig.

2.

16

64 256 1 4 16 64 256 N umbers of zoospo res (Iog l t scale) Relationship between spore number and gen erat ion time . The 5 % fidu cial limits are shown for Pimpernel and Majestic.

Factors affecting generation time In the various trials, the inoculated leaflets were examined daily a nd on each occasion the leaflets with spores were counted. From thes e records th e generation time (time to sporulate) can be estimated for each infected leaflet and a mean generation time calculated for each treatment. The mean generation times plotted ag ainst log. dose for Bintje, M ajestic , Pimpernel (Fig. 2 a) and for Group I varieties, Table 2 (Fig. 2 b) show that generation time lengthened as the dose decreased and seemed to reach a maximal value, possibly corresponding to infection by single zoospores (M cK ee, 1964). However, th e fiducial limits of the estimates for the smaller doses are very wide, as these means were derived from few infected leaflets. Generation times in the resistant Pimpernel wer e longer than in M ajestic and Bintj e (Fig. 2a) ; the resistant Libertas (Fig. 2b) differed still more conspicuously from th e susceptible varieties with which it was compared. V arietal differences showed more consistently at the larger doses with which th e fiducial limits of the estimates of generation tim e were smaller. Grouping by similarity of generation time gives three groups, in order of increasing time: (I) Bintje, King Edward, M ajestic, Up-to-Date; (2) Arran Viking, Ulster Supreme; and (3) Libertas, Pimpernel, Zeeburger. This, except for Majestic, agrees with field susceptibility (T a ble 3) .

Phytophthora infestans. D. H. Lapwood and A. K. McKee 685 DISCUSSION

The ED 50 values of the resistant varieties differed widely from those of varieties rated as field susceptible or moderately resistant, indicating that the method could be used to identify varieties as resistant as Libertas or Zeeburger. Majestic's ED 50 value was smaller than King Edward's, although it is consistently more resistant in the field; also mean generation times in these two varieties differed little (Fig. 2). This suggests that other factors than leaf susceptibility determine the difference in behaviour of these varieties in the field and that, for example, stem and leaf petiole resistance and the numbers of spores produced per area of infected leaf may be more important (Lapwood, 1961 a, b). The chance of a leaf becoming infected in the field depends on many factors, some of which were inoperative in our tests, which were made with detached leaflets of uniform age from glasshouse-grown plants, inoculated with uniform droplets containing zoospores of a single isolate of the fungus, and were incubated at a constant temperature in a humid atmosphere. Lowings & Acha (1959) showed that detaching leaves alters their susceptibility, and Grainger (1956) drew attention to changes in resistance as leaves age. Plants grown in the glasshouse may lose varietal characteristics that influence susceptibility in the field (Knutson, 1962), or the characteristics may be such that they produce differences between plant populations but not between detached leaflets. The use of a single fungus isolate may give misleading results, for Jeffrey, Jinks & Grindle ( I 962) found that strains of P. infestans grew most vigorously on leaves and tubers of the variety from which they were first isolated; unfortunately the origin of the isolate used in our tests is unknown. The size of the inoculation droplet may be important; for with King Edward the ED 50 using 0'01 ml. droplets averaged twelve spores (Table 2), whereas with 0'02 ml. droplets about twenty-five spores were required (McIntosh & Eveling, 1965). The extent to which the droplet spreads over the leaf surface depends on the nature of the leaf surface; on the upper leaf surface the inoculation droplet often spread widely to give a thin film, whereas on the lower surface it remained on a small area. The rate at which droplets dry is influenced by temperature and relative humidity and therefore the constant temperature and high humidity of the test environment would mask differences between varieties in time required for fungal penetration (Umaerus, 1960; Lapwood, 1964). The results showed that for varieties with small or intermediate field resistance the ED 50 values ranged from six to fifteen zoospores per drop, which is the equivalent of one to two sporangia germinating indirectly, whereas resistant varieties required twenty-three to forty-six zoospores, or three to six sporangia, for a similar level of infection. The probability of infection by a single sporangium (eight zoospores) on Majestic is 0'5 but on Pimpernel only 0'2 (Fig. I). However, inoculation with zoospores will not disclose a varietal effect on sporangial germination, if such exists. Generation time, which was affected by varietal resistance and dose, like intensity of sporulation, is important in the epidemiology of a disease.

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We did not measure intensity of sporulation, but general observations suggested that it was related to generation time. There is little evidence that synergistic effects are important in affecting incidence of infection at the doses likely to be found in natural epidemics (van der Plank, 1963), but allowance should be made for the effect of dose on generation time when devising theoretical models for epidemiological analysis.. The method described here is too laborious to be suitable for screening in a breeding programme, although it would select resistant clones. However, it could be used to compare the effect on resistance of leaflet age and position on the plant, the relative pathogenicity of different isolates, effect of cultural conditions and, in general, in the investigation of factors that affect host susceptibility or virulence of the pathogen. We thank Ir J. A. Hogen Esch for supplying seed tubers of the Dutch varieties used, Mrs M. G. Morris and Mr G. J. S. Ross for the probit analyses and Mrs A. Philips for technical assistance. REFERENCES

DIMOND, A. E., HORSFALL, J. G., HEUBERGER, J. W. & STODDARD, E. M. (1941). Role of the dosage-response curve in the evaluation of fungicides. Bull. Conn. agric. Exp, Stn 451,635-667. FINNEY, D. J. (1952). Probit analysis, end ed. 256 pp. Cambridge University Press. GRAINGER, J. (1956). Host nutrition and attack by fungal parasites. Phytopathology 46, 445-45 6. HODGSON, W. A. (1961). Laboratory testing of the potato for partial resistance to Phytophthora infestans. Am. Potato ]. 38, 259-264. JEFFREY, S. I. B., JINKS, J. L. & GRINDLE, M. (1962). Intraracial variation in Phytophthora in.festansand field resistance to potato blight. Genetica 32, 323-338. KNUTSON, K. W. (1962). Studies of the nature of field resistance of the potato to late blight. Am. Potato]. 39, 152-161. LAPWOOD, D. H. (196Ia). Potato haulm resistance to Phytophthora infestans. II. Lesion production and sporulation. Ann. appl. Biol. 49, 316-330. LAPWOOD, D. H. (1961b). Potato haulm resistance to Phytophlhora infestans. III. Lesion distribution and leaf destruction. Ann. app]. Biol. 49, 704-716. LAPWOOD, D. H. (I96IC). Laboratory assessments of the susceptibility of potato haulm to blight (Phytophthora infestans). Eur. Potato ]. 4, 117-128. UPWOOD, D. H. (1964). Haulm and tuber resistance to blight (Phytophthora in.festans). Rep. Rothamsted expo Stn for 1963, p. 112. LOWINGs, P. H. & ACHA, I. G. (1959). Some factors affecting growth of Phytophthora irfestans (Mont.) de Bary. I. P. in.festans on living potato leaves. Trans. Br. mycol. Soc. 42, 491-501. McINTOSH, A. H. & EVELING, D. W. (1965). Tests for aphicides for possible systemic control of potato blight. Eur. Potato ]. 8, 98-103. MCKEE, R. K. (1964). Observations on infection by Phvtophthora infestans. Trans. Br. mycol. Soc. 47, 365-374. MULLER, K. O. (1953). The nature of resistance of the potato plant to blight-Phytophthora infestans. ]. natn. Inst. agric. Bot. 6, 346-360. PLANK, J. E. VAN DER (1963). Plant diseases: epidemics and control, 349 pp. New York and London: Academic Press. UMAERUS, A. V. (1960). Iakttagelser rorande faltresistens mot bladmogel iPhytophthora infestans (Mont.) de By.) hos potatis. Soer. Utsiidesfiir. Tidskr. 70, 59-89. ZAAG, D. E. VAN DER (1959). Some observations on breeding for resistance to Phytophthora in.festans. Eur. Potato ]. 2, 278-286.

(Accepted for publication 8 March 1966)