Euphytica

REACTIONS OF POPLAR CLONES PHYSIOLOGIC RACES OF MELAMPSORA POPULINA KLEB. M. CHANDRASHEKAR Department

of Forestry,

Australian Received

29 (1980)

401407

TO LARICI-

and W. A. HEATHER National 2 October

University,

Canberra,

Australia

2600

1979

INDEX WORDS

Populus tegrated

spp., leaf rust of poplar, Melampsora larici-populina, aggressiveness concept of resistance, physiologic specialization, race-specific, vertical

of races, gene-for-gene, resistance.

in-

SUMMARY

Four mono-uredospore isolates of Melampsora larici-populina KLEB.produced qualitatively distinct reactions on four half-sib clones of Populus deltoides MARSH. and thus were recognisable as races. Two of the races could be distinguished equally readily by the distinctness of their reactions (three quantitative parameters) on four compatible clones of Populus spp. The pronounced race specific reactions of the clones of P. deltoides, allied to the less distinct, but nevertheless race specific reactions, of the compatible clones are consistent with an integrated rather than a disjunctive concept of resistance in Populus spp. to M. Iaricipopulina.

INTRODUCTION

Conventionally, studies on physiologic specialization in the fungi have employed the qualitative (Sensu, occurrence of discrete classes of host reaction on a scale of susceptibility/resistance) response in a host-parasite interaction to detect races (GASSNER& STRAIB,1932; STACKMAN, 1947; and MANNERS, 1950). Thus definition of races of pathogens are based generally on the reactions of a set of host biotypes (differential cultivars). In contrast, differing but non-discrete degrees of compatibility of host cultivars to fungal isolates, as portions of an overall spectrum of susceptibility, have rarely been employed to distinguish physiologic forms of fungi. However CLIFFORD & CLOTHIER (1974) used the latter criterion to distinguish the isolates of Pucciniu hordei OTTH., of varying cultivar origin, by quantitative reactions on cultivars of barley and termed it ‘Physiologic specialization with non-hypersensitive resistance’. Physiologic races in Melampsora larici-populina KLEB. were reported first by VAN VLOTEN (1949), and more recently SHARMA & HEATHER (1976), investigating monouredosorial isolates of leaf rust of poplar, demonstrated the presence of at least five races of M. larici-populina and six in M. medusae THUM. in the Canberra area of Australia. MILLER (1977) employing mono-uredospore isolates of M. lurici-populina obtained from SHARMA & HEATHER (pers. comm.) demonstrated the occurrence of quantitatively differential resistance in certain clones of Popufus spp. This paper reports the recognition of races in M. Zarici-populina both on qualitative and quantitative response of the hosts. Thus aggressiveness (VAN DERPLANK, 1968), as 401

M. CHANDRASHEKAR

AND

W. A. HEATHER

distinct from virulence, of the pathogen can be used in certain instances to distinguish races in the organism. Additionally, the possible genetic relationships of the hostparasite interaction and its implication in the epidemiology of poplar rust are discussed. MATERIALSANDMETHODS

Four mono-uredospore

isolates of hf. lurici-populina, selected from the collection of by inoculating the abaxial surface of individual, detached leaves of the susceptible clone, P. x euramericana ‘I-488’ with single uredospores and repeating the inoculations with the harvested uredospores of each isolate. Hopefully this uniform procedure avoided potential, host-induced changes in pathogen genotype during multiplication. Leaves of similar age from shoots of comparable maturity (SHARMA et al., 1980), obtained from clonal cuttings in a rust-free glasshouse (16 h photoperiod, 15-20°C diurnal temperature) were used in subsequent inoculation experiments. Leaf discs (1.76 cm”) were punched with a cork borer from these leaves, previously surface sterilized (0.35 % Sodium hypochlorite, for 2 min) and subsequently washed in three changes of distilled water. Inoculation and incubation of leaf discs followed the procedure of SHARMA & HEATHER (1976). Fifteen leaf discs of each clone were placed, abaxial side uppermost, on a moist filter paper on the base of a spore settling tower and inoculated with 5 mg of uredospores of a particular race. After 5 min the discs were removed and floated on gibberellic acid (10 ppm) in plastic Petriplates. The dishes were incubated under fluorescent light (1000 lux, 16 h photoperiod) at 20 + 1“C for 15 days. The investigation involved two experiments. Initially discs cut from four clones (actually half-sibs of Populus deltoides MARSH.), were inoculated with four individual mono-uredospore isolates of the rust (Table 1). This experiment was assessed on a combined qualitative/quantitative scale (SHARMA & HEATHER, 1977). Where appropriate, the mean number of uredosori per leaf disc was calculated from assessment of uredosorial numbers on each of the 15 individual leaf discs. The isolates were identified by the essentially qualitative reaction of the clones, i.e. it was possible hereafter to refer to these isolates as ‘races’ (Table 1). In the second experiment leaf discs from four clones viz. P. x euramericana ‘I-488’ and ‘I-214’, spontaneous hybrids of P. deltoides and P. nigra L. in the PO Valley of northern Italy, P x euramericana ‘65127’ a controlled cross of a P. deltoides (0) of Texas origin and P. nigra ‘evergreen’ (3) made in Australia (WILLING, pers. comm.), and P. nigra ‘evergreen’ a somatic mutant of Chilean origin (PRYOR & WILLING, 1965) were inoculated with each of the four races distinguished in the first experiment. All four clones were known to demonstrate compatible reactions with M. larici-populina and are referred to hereafter as ‘congenial clones’. Disease severity in this experiment was assessed on the following parameters. SHARMA & HEATHER (1976) were multiplied

1. Incubation period (days) from inoculation to fleck production (IPF). Flecks, localised chlorotic areas, were the initial symptoms of successful infection and were formed 2-3 days prior to uredinia. 402

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LEAF

RUST

OF POPLAR

2. Mean number of uredinia developed per leaf disc at 15 days (mean of 15 individual leaf discs) (ULD). 3. Mean number of urediniospores produced per mm2 of leaf disc at 15 days (USM). 1979.)

(SHARMA&HEATHER,

The results of experiment two, tested for normality 1974) using a GLIM program, were analysis with the aid of the following sub-program social sciences (NIE et al., 1975), ANOVA (analysis

WASSERMAN,

and homoscedasticity (NETER & subjected to statistical computer of the statistical package for the of variance).

RESULTS

The four races could be distinguished by the disease reaction of one to three clones of P. deltoides. The reactions immune, necrotic and/or resistant (Table 1) were qualitatively distinct and reproducible. Table 1. Reaction populina.

of four

Clone

clones

of P. deltoides

to four

mono-uredospore

isolates

of M. lurici-

Race

P. deltoides

’ Host I N R MS s SS

half-sib

‘l-2’ ‘l-9’ ‘7-10 ‘l-l 3’

reactions (immune) (hypersensitive) (resistant) (moderately susceptible) (susceptible) (highly susceptible)

A

B

D

E

RN’ RN I RN

R RN RN RN

RN I R RN

R RN RN R

Number of uredinia/leaf Nil Nil, necrotic lesions l-25 26-50 SlLlOO > 101

disc

In contrast the reactions of the ‘congenial clones’ were quantitative ranging from ‘moderately susceptible’ to susceptible on an arbitrary scale (Table 2), additionally the races differed in aggressiveness (Table 2). A ranking of clones, based on three parameters, in order of resistance showed reasonable uniformity (Table 2) with P. nigra ‘evergreen’ the most resistant and P x euramericana ‘I-2 14’ the most susceptible. Similarly in a ranking of the races in order of aggressiveness race A was always the most aggressive and D the least aggressive with B and E as intermediates (Table 2). The races can be ranked, on increasing aggressiveness, as D, E, B and A on all the three parameters, despite occasional aberrations and although the mean values for the various disease parameters do not always differ significantly between races (Table 2). Euphytica

29 (1980)

403

period

to flecking;

f 273.04

1475.80

USM

‘Disease parameters: IPF ~ Incubation ‘Mean of 15 observations. 3 Mean of 60 observations. “S.E. of mean.

0.004 17.16

ULD

725.80

0.52 6.61

Uredinia

USM

- Uredimospores

6.37 + 0.21 23.77 4.11 f 399.30 + 34.77

6.80 18.40 364.70 6.67 17.73 333.85 6.00 23.46 404.45 6.00 35.47 494.15

D

races of M. larici-populina,

per leaf disc;

by four

f 151.20

5.52 f 28.65 +

5.00 27.46 713.25 7.07 13.06 320.60 5.00 28.67 828.00 5.00 45.40 1041.25

induced

5.00* 83.07 1678.00 5.00 64.20 1242.70 5.00 40.66 861.80 5.00 121.86 2125.70

parameters,

B

5.003 & 77.45 f

P x euramericana ‘I-214

P x euramericana ‘I-488’

P. nigra ‘evergreen’

disease

A

Races

by three

IPF ULD

as measured

Mean

response,

IPF’ ULD USM IPF ULD USM IPF ULD USM IPF ULD USM

2. Mean

P. x euramericana ‘65121’

Clone

Table

‘congenial

per mm2.

5.75 + 0.48 32.18 7.07 f 601.50 f 76.50

6.00 18.73 391.20 7.00 24.06 133.85 5.00 35.20 588.25 5.00 50.73 692.70

E

on four

5.703 36.92 786.80 6.44 29.76 657.75 5.25 32.00 670.65 5.25 64.47 1087.20

+ f f + + f f k f f f +

of poplar.

Mean

clones’

0.444 15.53 307.45 0.49 11.70 217.28 0.25 3.76 107.61 0.25 19.75 363.77

m Y i ;

5

! 9

.s: 0 F 2 F 2

LEAF

RUST

OF POPLAR

Table 3. Analysis of variance of parameters of disease severity resulting from interaction of four ‘congenial clones of poplar’ and four races of M. larici-populina. Source

d.f.

Clone Race Clone x race Residual Total

3 3 9 224 239

Variance’ IPF

ULD

17.70 18.34 4.82 0.121 0.747

11521.66 32033.78 2725.52 423.89 1046.64

USM’ 19944.18 107165.11 10059.96 1322.25 10939.99

‘All the variance ratios are significant at P < 0.001. ‘Residual and total d.f. were 32 and 47 respectively.

The summary of the ANOVA of the results (Table 3) demonstrates that races and clones differed very significantly (P < 0.001) in their aggressiveness and resistance respectively while the interaction term was highly significant (P < 0.001) for all the three parameters. The latter indicates that the relative disease severity between clones depends on the pathogen race employed in the inoculation. Similarly the relative aggressiveness between races depends on the clone employed. For the three parameters the variance in the ANOVA due to races was greater than that due to clones possibly indicating a closer genetic relationship between clones than between races. DISCUSSION

The four mono-uredospore isolates of M. larici-populina could be distinguished as individual races by the qualitatively distinct reactions of from one to three clones of P. deltoides. These clones were propagations of half-sib seedlings, thus the differences in disease expression result probably from variation in the resistance genotype of the fertilizing pollen. The relationship of the individual ‘congenial clones’ in experiment two to the particular P. deltoides clones in experiment one is unknown however two general points are of interest. Although the four races of mono-uredospore origin were all multiplied on the same clone i.e., P. x euramericana ‘I-488’, this clone was rarely the most susceptible on any disease parameter to the four races i.e., there is no positive evidence of rapid, host-induced changes in race genotypes. In addition P. x euramericana X5/27’, a hybrid of P. deltoides and P. nigra ‘evergreen’, was usually more susceptible than P. nigra ‘evergreen’ (Table 2). While the reactions of the P. deltoides clones would be regarded as demonstrating a distinct form of resistance e.g. vertical resistance those of the ‘congenial clones’ appear as portions of a continuous spectrum from resistance to susceptibility. Thus while races A and D can be as readily distinguished by their quantitative reactions on the ‘congenial clones’ as by their qualitative reactions on the P. deltoides clones, the reactions of the ‘congenial clones’ to races B and E do not permit the distinction of the latter. This observation is in general agreement with that of PARLEVLIET (1978) for polygenic resistance in some barley varieties to isolates of P. hordei. Testing the reactions of races and ‘congenial clones’ may allow the elimination of Euphytica

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M. CHANDRASHEKAR AND W. A. HEATHER

certain clones, e.g. P x euramericana ‘I-214’ as the worst affected clone, from consideration for extensive planting, i.e., this can be a valuable negative screening process. Unfortunately the limited host/race combination employed in most experiments do not permit useful positive selection of resistant clones. There is a further potential hazard in such attempted selection, e.g. P. nigra ‘evergreen’ was ranked the most resistant of the four ‘congenial clones’ but only to three of the four races (Table 2). The ANOVA (Table 3) demonstrates that the race x clone interaction variance is highly significant in this experiment. As suggested elsewhere (HEATHER et al., 1980) this could result in a positive selection pressure by the chosen clone on the population of pathogen races and thus, following establishment of monoclonal plantings, result in epidemic disease. The resistance in P. deltoides is obviously race specific. The significant interaction of races and ‘congenial clones’ (Table 3) indicates a degree of race specificity also in this relationship. These observations are consistent with an integrated rather than a disjunctive concept of resistance in poplar to M. Zaricipopuha. This viewpoint agrees in principle with the integrated model of PARLEVLIET & ZADOKS (1977). The genetic basis of resistance in Populus spp. to leaf rust is unknown. The qualitative type reactions of the P. deltoides clones to races of M. larici-populina are suggestive of a gene-for-gene relationship. However, it is difficult to hypothesise the development of such a relationship between a host and a parasite which do not co-exist in nature. The causal organisms of leaf rust in poplar (M. larici-populina and M. medusae of European and North American origin respectively) do not cause epidemic disease in natural stands of their respective hosts. Possibly, as suggested by NELSON (1978) in another context, the pronounced race specific reactions of the P. deltoides clones to M. Zarici-populina indicate the ‘Newness’ of this host-pathogen combination. The less obvious race specific reactions of the ‘congenial clones’ to M. larici-populina may result from long periods of co-evolution of the pathogen and P. nigra in Europe. REFERENCES CLIFFORD,B. C. & R. B. CLOTHER,1974. Physiologic specialization of Pucciniu hordei on barley hosts with non-hypersensitive resistance. Trans. Br. Mycol. Sot. 63: 421-430. GASSNER,G. & W. STRAIB, 1932. Die Bestimmung der biologische Rassen des Weizengelbrostes (Puccinia glumarum f. sp. tritici (SCHMIDAT)ERIKSS.u. HEEN.) Arb. Biol. Abt. (Anst.-Reichanst.), Berl. 20: 141. HEATHER,W. A., J. K. SHARMA& A. G. MILLER, 1980. Physiologic specialization in Melampsora laricipopulina KLEB. on clones of poplar demonstrating partial resistance to leaf rust. Aust. For. Res. (Submitted for publication.) MANNERS, J. G., 1950. Studies on physiologic specialization of yellow rust (Puccinia glumarum (SCHM.) ERIKSS.and HENN. ) in Great Britain Annals of Applied Biology 37: 187-214. MILLER, A. G., 1977. Physiologic specialization in Melumpsora lurici-populina KLEB. Honours thesis, Department of Forestry, ANU. NELSON, R. R., 1978. Genetics of horizontal resistance to plant diseases. Ann. Rev. Phytopathol. 16: 359-378. NETER,L. & W. WASSERMAN,1974. Applied linear statistical models. R. D. Irwin, Inc. London. pp. 834. NIE, H. N., C. H. HULL, J. G. JENKINS,K. STEINBRENNER & D. H. BENT, 1975. Statistical package of social sciences, 2nd ed. McGraw-Hill, New York, pp. 675. PARLEVLIET,J. E., 1978. Race specific aspects of polygenic resistance of barley to leaf rust, Puccinia hordei. Neth. J. Pl. Path. 84: 121-126. PARLEVLIET,J. E. & J. C. ZADOKS, 1977. The integrated concept of disease resistance; a new view including

406

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LEAF RUST OF POPLAR horizontal and vertical resistance in plants. Euphytica 26: 5-21. PRYOR,L. D. & P. R. WILLING, 1965. The development of poplar clones suited to low latitudes. Silv. Genet. 14: 123-127. SHARMA,J. K. & W. A. HEATHER,1976. Physiologic specialisation in poplar leaf rusts Melampsora medusae THUM. and M. laricipopulina KLEB. in Australia. Proc. XIX Session FAO/IPC Working Party on Poplar Diseases. France 1976. SHARMA,J. K. & W. A. HEATHER,1977. Infection of Populus alba var. hickeliana by Melampsora medusae THUM. Eur. J. For. Path. 7: 119-124. SHARMA,J. K. & W. A. HEATHER,1979. A method for determining density of uredospores of Melampsora larici-populina KLEB. Trans. Br. Mycol. Sot. 72: 479482. SHARMA,J. K., W. A. HEATHER& P. WINER, 1980. Effect of leaf maturity and shoot age ofclones of Populus sp. on susceptibility to Melampsora larici-populina. Phytopathology, (accepted for publication). STACKMAN,E. C., 1947. The nature and importance of physiologic specialization in phytopathogenic fungi. Science, N.S. 105 : 627. VAN DERPLANK, J. E., 1968. Disease resistance in plants. Academic Press. New York/London. pp. 206. VAN VLOTEN,H., 1949. Hybridization experiments with races of Melampsora larici-populina KLEB. Tijdschr. Pl. Ziekt. 55: 196-209.

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