Competition among Sclerotinia sclerotiorum

Competition among Sclerotinia sclerotiorum genotypes within canola stems1

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Hunan Normal University on 06/03/13 For personal use only.

A.D. Maltby and J.D. Mihail

Abstract: Populations of Sclerotinia sclerotioru~nare often composed of multiple genotypes. In examining 35 naturally infected canola plants, 29 supported reproduction (i.e., sclerotium formation) by a single S. sc1erotio1-umgenotype, as defined by the mycelial compatibility test. Only six plants supported reproduction by two genotypes. To test the hypothesis that infrequent multiple genotype infections were due to differences in virulence o r competitive ability among isolates, four greenhouse experiments were conducted in which four isolates, representing three genotypes, were used in pairwise coinoculations of canola. There were no differences among the isolates in four virulence parameters. Mean reduction in sclerotial mass produced by a coinoculated isolate was calculated by comparison with the mean sclerotial mass of that isolate in the absence of competition, and used as the measure of competition. In all experiments, at least half of the coinoculation treatments resulted in reduced fungal reproduction for one or both of the coinoculated isolates, providing evidence of competitive differences. Generally, the magnitude of reproduction reduction was the same for each isolate in the pair. However, the magnitude was nonreciprocal when the more competitive isolate was given an advantage of early temporal arrival or spatial placement of inoculum at the lower position on the stem. Competitive differences among S. sclerotiorum isolates affecting reproduction represent one possible mechanism to explain temporal shifts in genotype frequencies. Key words: canola, competition, population structure, Sclerotinia sclerotior~tm,virulence. R6sum6 : Les populations du Sclerotinia sclerotioru~izsont souvent constituees de plusieurs genotypes. Tel que difini par le test de compatibilite mycelienne, sur 35 plants de canola naturellement infectees observees, 29 supportaient la reproduction (i.e., formation de scltrotes) d'un seul genotype du S. sclerotiorum. Seulement six plants supportaient la reproduction de deux gCnotypes. Afin de verifier I'hypothkse que les infections peu frCquentes par des genotypes multiples seraient dues 1 des differences d e virulence ou de compatibilitt entre isolats, quatre experiences ont ttC conduites en serre, dans lesquelles quatre isolats reprtsentatifs de trois gtnotypes ont Ctt utilists dans des essais de co-inoculation en paires, sur du canola. Aucune difftrence n'a etC observee entre les quatre isolats relativement B quatre paramktres de virulence. La rtduction moyenne de la masse de scltrotes produits par un isolat co-inocule a CtC calculCe en la comparant avec la masse moyenne de scltrotes d e cet isolat en absence de compttition, et utiliste comme mesure de la competition. Dans toutes les exptriences, au moins la moitik des traitements ont conduit B une rtduction d e la reproduction fongique, chez les deux ou un seul des isolats co-inoculCs, ce qui dernontre que la competition induit des differences. En gentral, l'ordre de grandeur de la rtduction de la reproduction est le m&me pour chaque isolat de la paire. Cependant, cette ordre de grandeur n'est plus reciproque lorsqu'on donne B I'isolat le plus cornpetitif une longueur d'avance dans le temps, ou dans I'espace en I'inoculant au bas de la tige. Les difftrences compCtitives observees entre les isolats du S. sclerotioru~izet affectant sa reproduction, reprtsentent un mCcanisme possible pour expliquer les dCcalages dans le temps de la frCquence des gtnotypes. Mots clPs : canola, competition, structure des populations, Sclerotiizia sclerotiorum, virulence. [Traduit par la redaction]

Introduction In environments where there is genotypic diversity within a pathogen population, it is reasonable to expect that individual hosts be ''ionized more than One pathogen genotype. Such multiple infections have been demonstrated for various plant -pathogen systems including Aspergillusflavus infecting cotton ( l ) , Cryphorzectria parasitica infecting chestnut (2), Received March 4 , 1996.

I

Fusariutn spp. infecting maize (3), Fusarium moniliforme infecting asparagus (4), and Mycosphaerella gratninicola infecting wheat ( 5 ) . The phenomenon of multiple genotypes colonizing a host is not unique to plants, as it has also been demonstrated in humans infected with the hepatitis C virus (6). Sclerotinia sclerotiorum (Lib.) de Bary is a saprophytic, necrotrophic fungal plant pathogen with a worldwide distribution (7, 8). Among the economically important hosts are alfalfa, bean, canola, lettuce, potato, soybean, sunflower, and tomato (7. 8). Populations of S. sclerotiorutn are highly . - diverse, irrespective of the geographic scale of examination. For example, high genotypic diversity has been documented across the central provinces of Canada (9). Genotypic diversity has also been demonstrated for populations in canola fields in Canada (9- 11) and central Missouri (12). ~

A.D. Maltby and J.D. Mihaif.' Department of Plant Pathology, University of Missouri, Columbia, MO 65211, U.S.A.

'

Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 12445. Author to whom all correspondence should be addressed.

Can. I. Bot. 75: 462-468 (1997)

,

,

O 1997 NRC Canada


Maltby and Mihail

In central Missouri, infection of canola is initiated by ascospores colonizing petals (12). After abscission, infected canola petals may fall into leaf axils where, under conditions of sufficient moisture, the fungus penetrates the canola stem and typical stem decay results. Our preliminary examination of infected canola stems in 1993 revealed that infection by multiple genotypes was rare (12). One explanation for the low frequency of multiple infections was a low probability of infection, as the disease frequency was between 3 and 6 % . Alternatively, differences in virulence or resource utilization among the infecting genotypes may have resulted in competitive elimination of one genotype by another. Suppression of sclerotium production of the less competitive genotype would have reduced the apparent frequency of multiple genotypes colonizing a single plant. An understanding of the mechanism underlying the low frequency of infection by multiple genotypes might contribute to refinement of disease prediction models based on the incidence of petal infection (13) which implicitly assume that pathogen genotypes do not vary in virulence or competitive ability. If the population diversity of the fungus was low, such genotypic differences might be of little concern for successful disease prediction. For S. sclerotiorum populations with high genotypic diversity (9 - 12), however, the diversity could affect successful disease prediction and management (9, 10). A more competitive pathogen genotype is not necessarily a more virulent genotype. The virulence of a pathogen genotype, "the measure of pathogenicity" (14), is determined independently of any other pathogen genotype, whereas the competitive ability of a genotype can be determined only on the basis of its interaction with a second genotype. The virulence and competitive ability of a pathogen may or may not be related. For example, if inocula of two pathogen genotypes arrive simultaneously at a host, the competitiveness of one genotype may depend upon its ability to quickly infect and colonize the host, and thus may be closely related to its virulence. If there were a temporal separation in the arrival of the two genotypes, however, the first genotype to arrive and infect the host plant might have a competitive advantage over genotypes that arrive and infect subsequently. Within a single host, competition between genotypes might result in reduced reproduction by one or both genotypes as a result of exploitative competition or interference competition. Exploitative competition occurs when one individual depletes a limited resource without restricting another individual's access to the resource (15). Interference competition occurs when one individual reduces another individual's access to a limited resource (16). Although these terms were developed originally by ecologists to describe interactions among larger organisms, they can be applied to fungi (15) for which both types of competition can occur simultaneously (16). If one fungal genotype is consistently a superior competitor, it may reduce the frequency of other genotypes in the population. In a population of plant pathogenic fungi, such a change in genotype frequency might be interpreted as the initial development of host specificity when the frequency changes were actually due to intraspecific competition. In this study, we determined the number of S. sclerotiorum genotypes successfully reproducing within naturally infected canola plants using mycelial compatibility (17) to define genotypes. We then selected three S. sclerotiorum genotypes,

represented by four isolates, for comparison of virulence and competitive abilities. In four greenhouse experiments, we examined various combinations of temporal and spatial introduction of inocula of .paired isolates on the successful reproduction of each isolate, as measured by the mass of sclerotia produced.

Materials and methods Naturally infected, symptomatic plants were collected from experimental canola plots (approx. 40 x 15 m) located in central Missouri at the Agronomy Research Center (ARC) and the Horticulture Research Center (HRC), both managed by the University of Missouri. Six plants were collected on 8 June 1993 from HRC, and a total of 35 plants were collected from three different plots at ARC on 21 and 23 June 1993. Plants with more than one Sclerotinia stem rot lesion or with broken stems were not collected. Total stem length was measured. Longitudinal incisions were made in each stem, and the locations of all sclerotia (i.e., in the stem, on the stem surface, or in the roots) and the distance between each sclerotium and the root crown were noted. All collected sclerotia were surface sterilized for 5 min in 70% ethanol and placed on potato dextrose agar (PDA; Difco, Detroit, Mich.; 39 g . L-I) amended with 20 mg . L-' Terrachlora (75% PCNB as a wettable powder; Uniroyal Chemical, Dayton, Ohio) and 250 mg . L-I streptomycin sulfate (Sigma Chemical Co., St. Louis Mo.). After 10 days incubation at 20°C, pure cultures were established from the hyphae emerging from each sclerotium. The number of S. sclerotiorum mycelial compatibility groups (MCGs) within each of the 35 naturally infected plants was determined by confronting each isolate from the plant with one arbitrarily selected isolate from the same plant, designated the tester isolate. The mycelial confrontation technique was modified from Kohn et al. (17). A sclerotium of each isolate was placed at one edge of a 60 mm diameter Petri dish containing PDA and incubated at 20°C for 3-5 days. When the mycelial growth had extended 30 mm, a 3 x 3 x 5 mm plug was taken from within 1 cm of the growing margin of the colony and placed on the edge of a 60 mm diameter petri dish containing modified Patterson's medium (MPM) (17), which was kept in a dark incubator at 20°C. Three or 4 days later, a 3 X 3 X 5 mm plug, taken from within 1 cm of the growing margin, was placed on a second Petri dish containing MPM and a plug from the tester isolate was added. The plugs of the two isolates were placed 2 cm apart near the center of the plate. These mycelial confrontations were placed in a dark incubator for 4-6 days. Most confrontations could be scored by the fourth day. When the paired isolates represented the same MCG, the two colonies grew together without a visible border between them. However, when the two isolates represented different MCGs, there was a visible border between them characterized by abundant aerial myceliun~and a dark stripe resulting from concentration of the dye by the hyphal tips (17). If all isolates from a plant were identical to the tester isolate, we concluded that only one MCG had successfully reproduced within the plant. However, if any isolates-were different from the tester isolate, then all nonidentical isolates were confronted in all possible pairwise combinations, to determine the number of MCGs that had reproduced within the plant.

Greenhouse competition experiments Four isolates of S. sclerotiorum were selected for use in greenhouse competition studies to represent some of the sclerotium deposition patterns observed in naturally infected plants. Isolate A was found within a stem, whereas the remaining sclerotia in the stem of that plant were of a different MCG. Isolate B colonized a second plant, and no other MCG was found colonizing that second plant. Finally, O 1997 NRC Canada

Can. J. Bot. Vol. 75. 1997 Table 1. Parameters used in four greenhouse experiments to investigate competitive differences among three Sclerotiniu sclerotior~ttngenotypes, represented by paired isolates in coinoculations of canola.

Exp. No. 1

Inocula arrival" Space -


Diff 2

-

Same 4

-

Same -

-

Diff 3

Time

Diff -

Same

-

-

Diff Same

Same or diff Same

Inocula type"

Total plantsc

Single Coinoc Single Coinoc Single Coinoc Single Coinoc Coinoc

20 25 20 25 20 25 20 30 10

No. of plants supporting reproduction byd 1 isolate 18 2 19 3 20 7 16 9 3

2 isolates

Boundary'

-

-

23 20

16 8

-

-

15

1

-

-

18 5

13 2

"The order in which two inocula were applied to plants in the several coinoculation treatments. Same and diff, indicate that the two inocula were spatially and (or) temporally coincident or different, respectively. "Inoculations using one (single) or two (coinoc) isolates. 'The number of plants in the indicated inoculation treatment for all replicates. There were five replicate plants per treatment in experiments 1-3 and 10 replicate plants per treatment in experiment 4. "Reproduction denotes sclerotium formation. "The number of plants supporting reproduction by two isolates for which there was no intermixing of sclerotia of the two genotypes within the canola stem.

isolates C and D were two different isolates of the same MCG recovered from a third plant. In all competition experiments, canola seeds were planted in 10 cm diameter clay pots at a rate of three per pot. After the first true leaves were produced, seedlings were thinned to 1 per pot. The variety Westar was chosen as it is a spring-seeded variety and does not require a cold shock to induce flowering. At 7 weeks after seeding, plants were inoculated using a technique modified from Marciano et al. (18). Autoclaved wheat kernels were placed on 4-day-old cultures of S. sclerotiorutt~growing on PDA. After 4 additional days of incubation at 20°C, all wheat kernels were visibly colonized by fungal mycelium. Arbitrarily selected leaf axils were used as inoculation sites. Each inoculation site was rinsed with 70% ethanol, followed by a distilled-water rinse. Two colonized wheat kernels were placed in the axil, and the inoculum was held in place with parafilm wrapped around the leaf axil and stem. For the next 3 days, each inoculation site was moistened with distilled water twice daily. 'The parafilm and inoculum remnants were removed on the 4th day after inoculation. Two control treatments were employed, one in which autoclaved wheat kernels were placed in leaf axils, and a second in which plants received no treatment. For experiments 1 -3, inoculation treatments were each isolate alone (four treatments), coinoculation of paired isolates (i.e., A and B, A and C , A and D, B and C , B and D; five treatments), and the two control treatments. In the first three experiments, each inoculation treatment was represented by five replicate plants. Experiments were arranged as randomized complete blocks. Because an obvious light gradient existed in the greenhouse, plants representing the two control treatments were placed in one block that was the most shaded, whereas the remaining five blocks contained one replicate plant of each of the coinoculation treatments. The first three greenhouse experiments were designed so that a different potentially competitive scenario was examined in each. In the first experiment (Table I), the influence of spatial separation of inocula was examined. Inocula of the two isolates were applied to leaf axils separated by no more than 7 cm and as nearly vertically aligned as permitted by canola phyllotaxis. Inocula of the two isolates were introduced on the same day.

In the second experiment (Table I), inocula of the two isolates were both spatially separated on the canola stem and temporally separated in arrival time by 3 days. The spatial arrangement of inoculum was as described for the first experiment. For all replicates, the inoculum introduced first was placed lower on the stem than inoculum of the second isolate. In the third experiment (Table I), inocula of both isolates were introduced in the same leaf axil at the same time. To confirm the results of the first three experiments, a fourth experiment was conducted which included all competitive scenarios from the first three experiments (Table 1). Further, a fourth potentially competitive scenario was added which was a variation on the spatial and temporal separation of inocula. In this case, the inoculum introduced first was placed higher on the stem than the inoculum of the second isolate which was introduced 3 days later. The fourth experiment differed from the first three experiments in that only two isolates (i.e., isolates A and D) were used. The number of replicate plants in each inoculation treatment was increased from 5 to 10. For all four experiments, 11 days after the first inoculation, or 14 days for those plants receiving temporally separated inocula, soil and plants were allowed to dry to promote sclerotial maturation. After 5 days, sclerotia that were developing on the outside of the stem were dry enough to collect with little damage to the sclerotia. The entire aerial portion of the plant and 5 cm of the taproot were harvested for dissection. Total stem length, total length of the disease lesion, and diameter of the stem at the top of the lesion, at midlesion, and at the base of the lesion were measured. Longitudinal incisions were made in each plant, and the positions and mass of sclerotia were determined as described previously. The MCG identification technique was used to determine the number of sclerotia produced by each isolate in all coinoculated plants from all four experiments.

Data analysis In each experiment, comparisons of isolate virulence were made among plants that were inoculated with a single isolate. The four virulence estimates examined were (i) length of external lesion (i.e., the length of stem tissue bleached by the pathogen), (ii) length of

O

1997 NRC Canada


Maltby and Mihail internal stem necrosis (i.e., the length of pith macerated), (iii) proportion of the stem colonized, and (iv) volume of tissue macerated. The proportion of the stem colonized was calculated by dividing the length of the necrotic portion of the stem by total stem length. To calculate volume of tissue macerated, we assumed that the necrotic stem region was a cylinder with a diameter equal to the average diameter of the necrotic portion of the stem. The reproduction of the four isolates (i.e., sclerotium formation) was compared using three metrics: (I) number of sclerotia per plant, (2) total sclerotial mass per plant, and (3) mean mass per sclerotium. The total mass of sclerotia produced by an isolate in a plant was the primary metric for detection of competitive differences among isolates. For plants inoculated with isolates A, B, C, or D alone, the terms A,, B,, C,, and D,, respectively, indicate the mean mass of sclerotia produced per plant in one experiment. The mass of sclerotia produced by an isolate in a coinoculated plant was compared with the mean sclerotial mass of the corresponding isolate in singly inoculated plants (i.e., in the absence of competition). The reduction in sclerotial mass as a consequence of coinoculation was calculated. Consider, for example, one plant coinoculated with isolates A and B. The terms ABi and BAi denote the total mass of sclerotia of isolates A and B, respectively, for plant i. The two measures of competition calculated were the difference between the sclerotial mass of isolate A or B in the coinoculated plant and the mean sclerotial mass per plant of isolate A or B in the singly inoculated plants, as given in [ l ] and [2]:

The means AAB and ABA were calculated using all replicate plants in the coinoculation treatment. Similarly, mean reduction in reproduction (i.e., sclerotial mass) was calculated for all pairs of coinoculated isolates in each of the four experiments. The first null hypothesis tested was that coinoculation of two S. scleroriorurn isolates had no effect on the reproduction of either isolate. Returning to our example of coinoculation with isolates A and B, this null hypothesis, H,, is given by 131: [3] AA, = AB, = 0 For each coinoculation treatment in each experiment, a similar null hypothesis was tested using a Student's t statistic (19). Rejection of the first null hypothesis supported the conclusion that there was competition between the isolates in coinoculated plants. Where evidence of competition between paired isolates was detected (i.e., the first null hypothesis was rejected), we examined the degree to which competition affected sclerotium production by each isolate. For the coinoculation treatment with isolates A and B, this second null hypothesis is given by equation [4], which states that the magnitude of reproduction reduction was the same for isolates A and B:

This null hypothesis was also tested by means of the Student's r statistic (19) for each pair of isolates in all coinoculation treatments in each experiment. Acceptance of the second null hypothesis provided evidence that the two isolates were equally competitive.

Results Our examination of naturally infected canola plants revealed that as many as 27 S. sclerotioium sclerotia could be produced within a single stem. Plants collected from HRC contained 1- 19 sclerotia (mean f SE = 10.5 2.87, n = 6), whereas those collected from the ARC contained 1-27 sclerotia (mean SE = 12.7 1.37, n = 35). We assessed the num-

*

*

+

ber of genotypes which had successfully reproduced (i.e., produced sclerotia) within each stem using MCG as the indicator of genotype. Only 1 of the 6 plants collected from HRC and 6 of the 35 plants collected from ARC were colonized by two S. sclerotioruin genotypes. No more than two genotypes were detected in any plant.

Comparison of isolate virulence and reproduction Four isolates from naturally infected plants, representing three MCGs, were selected for evaluation of competitive differences within canola stems in four greenhouse experiments. As a part of each experiment, differences in isolate virulence were assessed using plants inoculated with a single genotype. No differences among isolates were found in any experiment when external lesion length, proportion of plant stem colonized, or volume of tissue macerated were used as measures of virulence. Only in experiment 2 (Table 1) was there a difference between isolates B and C with respect to length of internal stem necrosis. In this case, plants inoculated with isolate C had greater internal stem necrosis (F3. 15 = 3.62, P < 0.05; means SE for isolates B and C were 3 1.1 f 1.75 and 22.2 1.89 cm, respectively). There were no differences in any experiment among the four isolates in any of the three measures of reproduction. Thus, there was little a priori reason to expect competitive differences among the isolates.

*

*

Simultaneous, spatially separated inoculation In the first greenhouse experiment, in which coinoculated plants received simultaneous, spatially separated inocula, 23 of 25 successfully coinoculated plants supported reproduction by both isolates (Table 1). In these plants, the sclerotia were arranged within the plant without intermixing of sclerotia of different isolates. The boundary between sclerotia of the two isolates was not a visible structure within the stem, but was determined by MCG testing. All sclerotia above the border were produced by the isolate inoculated higher on the plant, whereas all those below the boundary were produced by the isolate inoculated lower on the plant, regardless of the combination of isolates used. In four of the five coinoculation treatments, there was a significant reduction in reproduction for at least one of the isolates (Table 2). Reproduction was significantly reduced for both isolates where isolates A and D or B and D were coinoculated. In fact, the greatest reduction in reproduction was observed for these two coinoculation treatments (i.e., ADA = -0.47 g, AD, = -0.5 g; Table 2). Thus, competitive effects were detected for all pairs of coinoculated isolates, except isolates B and C , where reproduction did not differ from that in plants inoculated with isolates B or C alone (Table 2). The magnitude of competitive effects was generally reciprocal (i.e., reproduction of both isolates was reduced to the same degree). Only in the case of coinoculation of isolates A and B were the competitive effects nonreciprocal. In this case, reproduction by isolate A was reduced less by competition than was that of isolate B (Table 2). Spatially and temporally separated inoculation In the second experiment, where there was both spatial and temporal separation of inocula, the sclerotia in 3 of the 20 successfully coinoculated plants were exclusively of one isoO 1997 NRC Canada

466

Can. J . Bot. Vol. 75, 1997

Table 2. Mean sclerotial mass reduction when pairs of Sclerotiniu sclerotiorum isolates were coinoculated under three competitive scenarios. -

Exp. no. 1

Inocula arrival" Space

Time

Isolate

Diff

Same

A B C

D


2

Diff

Diff

A B C

D 3

Same

Same

A B C

D

Mass" (g)

0.680 0.776 0.580 0.813 0.356 0.305 0.331 0.272 0.339 0.138 0.267 0.248

Coinoculated isolates A and B

A and C

A and D

-0. 14c (0.066) -0.39"' (0.048)

-0.18" (0.049)

-0.33" (0.054)

-

-

-

-

-0.29" (0.079)

-

-0.21 (0.1 10)

-

-0.23 (0.182) -0.13 (0.173)

-

-

-

-0.50" (0.115)

-0.22" (0.060) -0.13 (0.070)

-0.18" (0.056)

-0.47" (0.128) -0.15'

-

-

-0.1 1 (0.061)

-

-0.14" (0.024)

-

-0.16 (0.050) -0.17" (0.049)

-

-

-0.47'' -0.23" (0.045)

-

-0.12 (0.065)

-0.21'' (0.072) -0.05 (0.049)

-0.21" (0.055)

-

-0.19" (0.047) -

-

A

B and C

B and D

-

-

-

-

-

-

-0.05" (0.034)

-

-0.03 (0.055) 0.0 (0.185)

-0.22" (0.037)

-

A

-

-0.23"'

(0.046)

Note: Tabular values are the mean reduction in sclerotial mass (g) produced per plant when an isolate was coinoculated with a second isolate. For example, in the first experiment, reproduction of isolate A was reduced by 0.14 g (i.e., U , = -0.14 g) when coinoculated with isolate B. In the same coinoculation treatment, the mean sclerotial mass per plant produced by isolate B was reduced by 0.39 g (i.e., AB, = -0.39). For this coinoculation treatment, the mean sclerotial mass per plant produced by isolates A and B, when inoculated alone, was 0.68 and 0.776 g, respectively. Standard error is given in parentheses. "The order in which two inocula were applied to plants in the several coinoculation treatments. Same and diff, indicate that the two inocula were spatially and (or) temporally coincident or different, respectively. 'The mean mass of sclerotia produced per plant by the indicated isolate when inoculated singly (i.e., in the absence of a competing isolate). 'Reciprocal reductions in sclerotial mass were statistically different. For example, in experiment I , AA, f AB,,. "Reduction in sclerotial mass was significantly less than zero as evaluated by Student's r test statistic ( P < 0.05). ''As only one plant in the A and D coinoculation treatment supported reproduction by both isolates, it was not possible to determine a standard error and further statistical comparisons were precluded.

late, indicating that the coinoculated isolate failed to establish itself in the plant (Table 1). Assessing successful dual inoculation in this experiment was more difficult than for the first experiment, because the lesion from the inoculum placed lower on the stem sometimes overgrew the second inoculation point before it could be positively determined that the second isolate had infected the plant. In eight of the plants in which both isolates successfully reproduced, there was a boundary with complete segregation of the sclerotia above and below the point where the isolates apparently met in the plant (Table 1). In three of the five coinoculation treatments, reproduction of at least one isolate was significantly reduced (Table 2, coinoculations A and B, A and C, and B and C). In all three cases, the reduction of reproduction was of the same magnitude for both isolates in the coinoculation treatment. Coinoculations of isolates A and D resulted in one or both isolates failing to penetrate the stem for four of five plants. Thus, a statistical comparison of competitive effects could not be made.

Simultaneous and spatially coincident inoculation Where coinoculated isolates were introduced simultaneously on the same leaf axil, it was not possible to determine whether both isolates successfully infected the plant. The patterns of deposition of sclerotia within and on the plant were more complex than in experiments where inocula were spatially or temporally separated. In only one of the coinoculated plants was there any evidence of a border with partitioning of the host by the isolates (Table 1). In that plant, the border between the two isolates was unrelated to the point of inoculation, as all sclerotia were deposited below the inoculation point. In the coinoculation treatments, reductions in sclerotial

mass had a broader range of values compared with the first two experiments (Table 2). In four of the five coinoculation treatments, reproduction of at least one isolate was significantly reduced (Table 2). Reproduction was significantly reduced for both isolates where isolates A and C or A and D were coinoculated (Table 2). Only in the case of coinoculation with isolates B and D was the magnitude of reproduction depression nonreciprocal (i.e., ABD = -0.05 g versus AD, = -0.23 g, Table 2). The effect of coinoculation upon S. sclerotiorum reproduction was examined in more detail in a fourth greenhouse experiment in which increased replication permitted more refined examination of the three competitive scenarios (Tables 1 and 3). In this experiment, only isolates A and D were used. Specifically, when inocula were both spatially and temporally separated, we examined the influence of position on the stem (Tables 1 and 3). We subdivided this treatment so that when each isolate was the first inoculum introduced it was placed low on the stem in one subtreatment and high on the stem in the second subtreatment (Table 3). Of the 23 plants that- supported reproduction by both isolates A and D, 15 exhibited distinct boundaries between the sclerotia of the two isolates as described for the first three greenhouse experiments (Table 1). For all coinoculation treatments, reproduction of both isolates A and D was reduced compared with reproduction in plants singly inoculated (Table 3). Further, the effect of competition was greater on isolate D in several coinoculation treatments (Table 3). Specifically, in the three coinoculation treatments where isolate D was placed lower on the stem, the competitive effects on isolate D were greater (i.e., AD, < AA,, Table 3). O I997 NRC Canada

Maltby and Mihail

Table 3. Mean sclerotial mass reduction when Sclerotinia sclerotiorurr~isolates A and D were coinoculated under three competitive scenarios. Inocula arrival"

Isolate placement"

Space

Time

Low

Diff

Same

A

D


Diff

Diff

Same

Same

First -

A

A

D

D D

A

Reproduction reductionr (g)

D

A

-

-

Note: The magnitude of reproduction depression was compared for isolates A and D by comparing M,, and bq, with the Student's r test statistic. using degrees of freedom. P denotes the probability of obtaining a more extreme value of t by chance alone. "The order in which two inocula were applied to plants in coinoculation treatments. Same and diff, indicate that the two inocula were spatially and (or) temporally coincident or different, respectively. "Identifies which of the two isolates was placed "low" on the stem and which was given the temporal advantage of "first" placement on the stem. "Values are the mean reduction in sclerotial mass (g) produced per plant when isolates A and D were coinoculated. For example, when inocula were spatially and temporally coincident, reproduction of isolate A was reduced by 0.064 g (i.e., AA, = -0.064 g). In the same coinoculation treatment the mean sclerotial mass per plant produced by isolate D was reduced by 0.152 g (i.e., AQ, = -0.152). All values of M, and All, were significantly negative ( P < 0.05). Standard error is given in parentheses.

Further, when the inocula were spatially and temporally coincident, the effects of competition on isolate D were greater (Table 3).

Discussion Using four parameters to assess virulence and three measures of fungal reproduction, we found no differences among the three S. sclerotiorum genotypes used in the greenhouse experiments. Thus, there was no a priori reason to expect competitive differences among the genotypes when paired isolates were used to coinoculate canola stems. These results are consistent with recent evaluations of the isozyme profiles for both polygalacturonase (PG) and pectinmethylesterase (PME) from 35 isolates of S. sclerotiorum representing 15 clones (20). Although two isozyme banding pattern phenotypes were found for PG, these were not consistently associated with differences in virulence on canola (20). In all four experiments, at least half of the coinoculation treatments resulted in significantly reduced fungal reproduction as measured by sclerotial mass. These results demonstrated competitive interactions between coinoculated isolates in each of the scenarios examined. In most instances, the magnitude of reproduction depression was the same for each isolate in the coinoculated pair. In the first three experiments, there were only two coinoculation treatments for which the magnitudes of competitive effects were nonreciprocal. In both instances, the two inocula were temporally coincident, suggesting that spatial placement of inoculum strongly affected the outcome of the competitive interaction. In the fourth experiment, we examined spatial placement of inocula, more closely focusing on two isolates. Results of this experiment indicated that the more competitive isolate (i.e., isolate A) was less affected by competition when placed

at the inoculation site lower on the stem. The advantage conferred by this position was most likely due to the greater stem diameter and thus to the greater nutrient reserves available to support fungal reproduction. In this experiment spatial position appeared to be more important than temporal advantage for the outcome of the competition between coinoculated isolates. Specifically, reproduction of isolate A was less affected by competition than was that of isolate D when isolate A had the advantage of lower stem position and isolate D had the temporal advantage of 3 days. The results of our experiments with S. sclerotiorum are consistent with a previous report of competitive differences among genotypes of Colletotrich~irngloeosporioides f.sp. aeschynornene (2 1). Competitive differences among Colletotrichum gloeosporioides genotypes were observed using a wild-type and a mutant strain which produced smaller lesions (2 1). The effect of competition over numerous pathogen generations was examined in the greenhouse, and temporal shifts in genotype frequencies were documented (21). If competition among genotypes occurs commonly in nature, it would be a mechanism by which gene frequencies could change in populations of plant-pathogenic fungi without host plant selection. Changes in genotype frequency have been demonstrated within growing seasons for S. sclerotiorurn populations in Canadian canola fields (10). Within-host competition among S. sclerotiorum genotypes should be considered one possible explanation for those observations. Extrapolation from competitive differences observed in our greenhouse studies to equivalent differences in the field requires a recognition of the smaller stature of greenhousegrown plants. The advantage which accrued to isolates infecting lower on the stem in the greenhouse may not exist in the field due to the larger stem diameter. The potential elimination of the spatial advantage would accentuate the temporal advantage of the first infecting isolate. O 1997 NRC Canada


Can. J . Bot. Vol. 75. 1997

Because both exploitation and interference competition can occur at the same time (16), it was not always possible to classify the competition mechanism operating in our experiments. Exploitation competition occurred whenever one isolate had a temporal advantage, consuming the host resource before the arrival of the second isolate. Where inocula were spatially separated, the competition was initially exploitative. However, as the two isolates approached one another in the stem o r when the second isolate was introduced, it was not possible to confirm the competition mechanism. The sclerotial deposition patterns, however, can provide some information as to the type of competition that occurred. Simultaneous, spatially coincident inoculations yielded various complex sclerotial deposition patterns within canola stems. It was common to find a series of sclerotia from one isolate, with some sclerotia of the coinoculated isolate interspersed within the series. W e believe that such a pattern of sclerotial deposition could have arisen from exploitative competition, as the second isolate invaded sections of the stem that the first isolate had not yet invaded, and vice versa. Other sclerotial deposition patterns were suggestive of interference competition, with one isolate "walling off" the other isolate, thereby limiting the resources the second isolate could obtain. In future studies, by destructively sampling the plants as the infection progresses, it should be possible to see how these patterns develop and thereby deduce the competition mechanism involved.

6. 7. 8. 9. 10.

11.

12.

13.

Acknowledgments

14.

T h e authors are grateful for the technical assistance of A . Aldenderfer and S.J. Taylor, and numerous discussions on the assessment of competition with J.T. English. This research was supported, in part, by the University of Missouri Research Board grant No. 94-042.

15.

References 1. Bayman, P., and Cotty, P.J. 1991. Vegetative compatibility and genetic diversity in the Aspergillusflnvus population of a single field. Can. J. Bot. 69: 1707- 1711. 2. Anagnostakis, S.L., and Kranz, J. 1987. Population dynamics of Cryphonecrria parasitica in a mixed-hardwood forest. Phytopathology, 77: 739-741. 3. Kedera, C.J., Leslie, J.F., and Claflin, L.E. 1994. Genetic diversity of Fusariutn section Liseola (Gibberellafidikuroi) in individual maize stalks. Phytopathology, 84: 603-607. 4. La Mondia, J.A., and Elmer, W.H. 1989. Pathogenicity and vegetative compatibility among isolates of Fusariurn oxysporum and Fusariurn monilifortne colonizing asparagus tissues. Can. J. Bot. 67: 2420-2424. 5. McDonald, B.A., and Martinez, J.P. 1990. DNA restriction

16.

17. 18. 19. 20. 21.

fragment length polymorphisms among Myco.splzaerella graminicola (anamorph Seproria tritici) isolates collected from a single wheat field. Phytopathology, 80: 1368- 1373. Mueller, H.M., Pfaff, E., Goeser, T., and Theilmann, L. 1993. Genetic variability of German hepatitis C virus isolates. J. Med. Virol. 40: 291 -306. Farr, D.F., Bills, G .F., Chamuris, G.P., and Rossman, A.Y. 1989. Fungi on plants and plant products in the United States. APS Press, St. Paul, Minn. Purdy, L.H. 1979. Sclerorinia scleroriorurn: history, diseases and symptomatology, host range, geographic distribution, and impact. Phytopathology, 69: 875 -880. Kohli, Y., Morrall, R.A.A., Anderson, J.B., and Kohn, L.M. 1992. Local and trans-Canadian clonal distribution of Sclerorinia scleroriorurn on canola. Phytopathology, 82: 875 - 880. Kohli, Y., Brunner, L.J., Yoell, H., Milgroom, M.G., Anderson, J .B., Morrall, R.A.A., and Kohn, L.M. 1995. Clonal dispersal and spatial mixing in populations of the plant pathogenic fungus, Sclerotirlia scleroriorurn. Mol. Ecol. 4: 69-77. Kohn, L.M., Stasovski, E., Carbone, I., Royer, J., and Anderson, J.B. 1991. Mycelial incompatibility and molecular markers identify genetic variability in field populations of Sclerorinia sclerotior~im.Phy topathology , 81: 480 -485. Maltby, A. D. 1995. Sclerorinia scleroriorurn on canola in Missouri: population diversity and competition among fungal genotypes. Ph.D. dissertation, Department of Plant Pathology, University of Missouri, Columbia, Mo. Turkington, T.K., Morrall, R.A.A., and Gugel, R.K. 1991. Use of petal infestation to forecast sclerotinia stem rot of canola: evaluation of early bloom sampling, 1985-90. Can. J. Plant Pathol. 13: 50-59. Whetzel, H.H. 1929. The terminology of phytopathology. Proc. Int. Congr. Plant Sci. 2: 1204-1215. Lockwood. J.L. 1992. Exploitation competition. In The fungal community: its organization and role in the ecosystem. 2nd ed. Edited by G.C. Carroll and D.T. Wicklow. Marcell Dekker, Inc., New York. pp. 243-263. Wicklow, D.T. 1992. Interference competition. 61 The fungal community: its organization and role in the ecosystem. 2nd ed. Edited by G.C. Carroll and D.T. Wicklow. Marcell Dekker, Inc., New York. pp. 265-274. Kohn, L.M., Carbone, I., and Anderson, J.B. 1990. Mycelial interactions in Sclerotirlia scleroriorurn. Exp. Mycol. 14: 255-267. Marciano, P., Di Lenna, P., and Magro, P. 1982. Polygalacturonase isoenzymes produced by Sclerorinia sclerotiorutn in vivo and in vitro. Physiol. Plant Pathol. 20: 201 -212. Sokal, R.R., and Rohlf, F.J. 1995. Biometry. 3rd ed. W.H. Freeman and Co., New York. Errampalli, D., and Kohn, L.M. 1995. Comparison of pectic zymograms produced by different clones of Scleroritzia scleroriorurn in culture. Phytopathology, 85: 292-298. Yang, X.B., and TeBeest, D.O. 1995. Competitiveness of mutant and wild-type isolates of Colletotrichutngloeosporioides f.sp. aeschynorner~eon northern jointvetch. Phytopathology, 85: 705-710.

O 1997 NRC

Canada