Intraspecific genetic variation in Heterobasidion annosum revealed by amplification of minisatellite DNA

Mycal. Res. 98 (1): 57-63 (1994)

Printed in Great Britain

57

Intraspecific genetic variation in Heterobasidion annosum revealed by amplification of minisatellite DNA

JAN STENLID, JAN-OLOF KARLSSON AND NILS HOGBERG Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden

DNA from 124 strains of Heterobasidion annosum was amplified using the core sequence of the MI3 minisatellite region as primer. The strains were derived from II S-, 8 P- and I F-intersterility group populations originating in Scandinavia, Germany and Italy. Following electrophoresis the banding patterns of all isolates were compared and 23 fragments were scored. Average band-sharing indices (ABS!) were used to compare genetic diversity within and between populations. Genetic similarity of populations decreased with increasing geographical distance in the S group. In both the 5 and P intersterility groups, ABS! values were higher within than among populations (70'3 ± 2-3 S.D. and 66'4 ± 2'2, respectively in the S group and 77'0 ± 2'3 and 75'0 ± 2'4, respectively in the P group) indicating local differentiation. The higher values in the P than in the S group indicate that the P group is less variable. ABSI was much lower between S and P group populations (49'2 ± 2'5) than within either group. This result demonstrates the large divergence between these two intersterility groups. In the F population ABSI was higher (68 ± 3' I) than for the comparisons with the S- (50'1 ± 1'6) and P-groups (44'0 ± 2'4). By using discriminant analysis, the agreement between geographical population affiliation and that inferred from banding patterns of amplified DNA was 20'8% within the S group, and 14'9% within the P group. Chi-square test of the discriminant analysis indicated regional differentiation in the S but not in the P intersterility group. The agreement between methods was 98'4% when strains were classified to intersterility group by pairing tests or amplified DNA profiles. Results are consistent with a high degree of exchange between populations of the same intersterility group but low exchange between intersterility groups.

Heterobasidion annosum (Fr.) Bref, is a serious pathogen causing root and butt rot to conifers all over the northern hemisphere, Several intersterile groups are found in the species; in Europe the S group is confined to Norway spruce, the P group has a broader host range and infects Pinus sylvestris, Picea abies, Betula spp., etc., and the F group infects Abies alba in southern Europe (Korhonen, 1978; Stenlid & Swedjemark, 1988; Capretti et aI., 1990). Several studies are directed to study inter-group variation, in terms of interfertility (Korhonen, 1978; Harrington, Worrall & Rizzo, 1989; CapreHi et al., 1990; Chase & Ullrich, 1990; Stenlid & Karlsson, 1991), pathogenicity (Worrall, Parmeter & Cobb, 1983; Stenlid & Swedjemark, 1988) and isozymes (Karlsson & Stenlid, 1991; Otrosina, Chase & Cobb, 1992). In these studies, partial interfertility between groups can be demonstrated in the laboratory, but biochemical markers indicate that genetic exchange between intersterility groups is rare in nature. However, variation between populations within an intersterility group has not been studied in detail. Repetitive DNA with tandem repeats of a core or consensus sequences (minisatellites) has recently been used to study intraspecific genetic variability in many organisms Geffreys, Wilson & Thein, 1985; Ryskov et aI., 1988; Gilbert et aI., 1990; Schaal, O'Kane & Rogstad, 1991). Minisatellites originally derived from the M13 phage have been used to study variation in plant populations (Schaal et al., 1991). Individuals of Acer negundo collected from within 20 km

exhibited a higher degree of minisatellite similarity than those from a more Widely distributed sample (Nybom & Rogstad, 1990). Similarly, Norway spruce trees from France were more similar to each other in M13 minisatellites than to Swedish trees (Kvarnheden & Engstrom, 1992), Additionally, in outcrossing plants high variation in minisatellites was shown, while selfing plants showed a lower degree (Schaal et al., 1991). In fungi, M13 minisatellites have been used to discriminate between aggressive and non-aggressive isolates of Leptosphaeria maculans (Meyer et al., 1992) and also between species in the Trichoderma aggregate and strains in Penicillium and Aspergillus (Meyer et ai" 1991). PCR amplification of the M13 forward sequencing primer allowed for strain typing in Lentinula edodes (Kwan et al., 1992). Here we present the results of a study on genetic similarity among European populations of H. annosum using PCRamplified DNA. The average band-sharing index (ABSn was used as a measure of genetic similarity and was compared with the geographical distance between populations to detect regional differentiation.

MATERIALS AND METHODS Fungal strains Strains of H. annosum were collected from diseased trees and stumps according to Table 1. All strains within a population

Variation in Heterobasidion annosum DNA

58

Table 1. Origin of strains used in the investigation

Population

Location

Host

SA SB SC SD

Spikkestad, N Spikkestad, N Spikkestad, N Brynge, S Satuna, S Satuna, P Siarii, S Siarii, S Siarii, P Mjiilby, S Ramsasa, S Ramsasa, P Willestrup, DK

P.a. P.a. P.a. P.a. P.a. P.a. P.a. P.a. P.a. P.s. P.a. P.a. P.sit. P.c. Ap. P.a. P.sit. P.s. P.s. P.a. P.c. P.a. Ap. P.a. P.a. P.a. P.a. Aa.

SE PF SG SH PI PK SL PM PN

PO

PP

Ulborg, DK Lovenholm, DK

PQ

RandbiiL DK

SR

Munich, D

SS FT

Asiago, I Toscana, I

Collector and year HS, 1987 HS, 1987 HS,1987 JS, 1985 JS, 1986 JS, 1986 JOK, 1990 GS, 1990 GS,1990 JS, 1986 JS, 1985 JS, 1985 IT, 1992

IT, 1992 IT, 1992 JS, 1990 IT,1992 IT, 1992 IT, 1992 IT, 1992 IT, 1992 KK, 1986 HM, 1988 KK, 1987 KK, 1987

Strain 87-1657/5, -168/1, -176/2, -179/3, -183/3, -184/3, -197/2 87-208/2, -209/3, -210/3, -214/2, -215/2, -219/1, -221/3 87-225/1, -226/3, -227/1, -240/1, -243/3, -245/1 br 88,202,212,228, 231, 237, 304, 331, 371, d sa 33, 48, 94, 131, 139, 159'5, 192, 205, 208 sa 2, 16, 16'4, 16'7, 34 sig 4b, 5b, 19 si 4, 7, 9, 21, 35, 58, 7Ib, 73, 78b, b50c, b63b, b120a si g14, b27b, 45c mj 87, 107, 124 rb 14, 31, 48, 89, 94, 132, 175 rb 28, 90, 162, 199, 245, 257, 282 d 14 d 15 d 16, 17 d 7, 8, 9, 10, II, 12 d 18, 19 dk I d 20,28 d 21, 22, 23, 24, 25, 26, 27 d1 d 2,3 d6 fas 2, 3 fas 15-3, 16-1 fas 6, 9-4, 9-5, 10, 13-4 faf 6-2 mf1, 4, 5-6, 8-5, 9-1

Locations: S, Sweden; N, Norway; D, Germany; DK, Denmark; I, Italy. Host trees: P.a. Picea abies, P.s. Pinus sylves/ris, P.sit. Picea si/chensis, P.c. Pinus con/or/a, Ap. Abies procera, Aa. Abies alba. Collectors: JOK, Jan-Olof Karlsson; KK, Kari Korhonen; HM, Helga Marxmuller; HS, Halvor Solheim; IS, Jan Stenlid; GS, Gunilla Swedjemark; IT, Iben Thomsen. The three Norwegian populations were from three separate locations within 5 km of each other. Population SG was collected from decayed wood in a clear cutting of an old stand, while SH was collected from a diseased 30-year-old stand I km away.

represent distinctive genets in tests of somatic incompatibility (d. Stenlid, 1985). Strains were kept on Hagem agar (HA) at 5 °C until used. Prior to DNA extraction, each strain was grown on 5 HA plates covered with cellophane at 21° for 10 d. The mycelia were then scraped off and ground in a mortar together with liquid nitrogen. FollOWing freeze-drying, DNA was extracted using the method described by Raeda & Broda (1985). Strains were assigned to intersterility group by pairings with homokaryotic tester strains of known intersterility group affiliation (Stenlid & Karlsson, 1991).

peR procedure Amplification of fungal DNA was carried out in reaction lots of 100 1-11 containing 5 ng DNA, 10 mM Tris-HCL 5 mM KC!, 0'001 % gelatin optimized with 2'5 mM MgCl 2 , 200 mM dNTP mix, 200 nM primer and 2'5 U Taq polymerase. We used the core sequence of MI3 minisatellite DNAGAGGGTGGCGGTTCT- as primer (Wieland Meyer, pers. comm.). The cycling parameters were: 45 cycles of denaturing, 93° 20 s, annealing, 48° I min, extension 72° 20 s, final extension 72° 6 min. The annealing temperature was calculated according to Innis et al. (1990) and empirically tested. The amplified products were subjected to electrophoresis on agarose gels, stained with ethidium bromide and visualized

under uv light. Two separate ways of verifying that the results were reprodUcible were used. (i) Six strains were run in three separate amplifications under the same conditions. (ii) Seventyone of the tested strains were run in duplicates in the same amplification. Duplicate samples did not differ in amplified products in any of these controls. To test the sensitivity of the method for DNA concentration differences, a 5 ng DNA sample from strain br 202 was subjected to 10- and 100-fold dilutions. The banding patterns did not differ following amplification, shOWing a low sensitivity to dilution of H. annosum DNA in this concentration interval.

Gel interpretation All interpretations were made from photographic prints. Molecular weight markers were run in triplicate on each gel. Presence and absence of amplified fragments were scored in relation to these. Only clear and distinct bands were included in the analysis.

Statistical analyses The band-sharing index (BSI) was calculated as the number of shared bands (5ab ) divided by the number of bands in strain A (Sa) and strain B (5b ) from the formula 25ab /(5a + 5 b ) (Lynch,

J. Stenlid, I.-O. Karlsson and N, Hogberg

59

Table 2. ABSI matrix of strains from different populations (above the diagonal) and S,D, of ABSI values (below the diagonal), Populations as in Table 1. ABSI is the average of comparisons of individual strain similarities in amplified ONA (see text for details), SA

SB

SC

SO

SE

SG

SH

SL

SR

55

PF

PI

PK

PM

PN

PO

PP

PQ

FT

69

72

68

70

71

66

64

57

43

45

46

50

44

46

42

50

50

70

67

72

70

71

66

59

49

46

50

52

47

48

46

52

47

69 74 2']

65

67

69

68

65

56

49

44

48

50

45

45

44

50

50

68 66 2'1

70

69

68

65

59

45

46

50

52

46

46

44

52

50

70 75

69

67

63

61

46

47

53

55

48

50

46

53

53

],g

]'7

72 72

69

63

66

47

45

53

55

48

50

47

55

47

71

SA

],g

71 73

SB

2"0

2'0

73

68 2'4

SC

2']

2'1

SO

2'0

2'2

SE

2'0

2']

2'2

SG

],g

],g

2'2

SH

2"0

2'1

2']

2']

2'0

],g

],g

68 69

61

61

47

48

51

54

48

49

46

55

50

SL

2"0

2'0

2']

2']

2'0

1'9

2'0

1'9

64 67

61

51

48

56

58

51

52

50

57

51

SR

2'3

2'2

2'7

52

56

59

60

54

57

51

59

59

55

2'2

2'1

2'5

57 55 2-3

48 66

50

56

57

51

54

50

56

45

PF

2'3

2'9

2-5

2'7

67 67

74

70

69

70

67

72

40

PI

2'3

2'5

2'7

74

75

78

72

74

42

PK

2'3

2'5

2-]

78 84 1'6

79

82

75

81

49

PM

2'4

2'5

2-2

]'8

82 75 2-]

76

78

73

78

48

81 85 ]-7

79

78

43

81

80

45

2'1

78 2-4

74 76

42

2-2

46 68 3-]

2'2

2'5 2'2 2'5

2'1

2'2

2'5

2'5

2'5

75

PN

2"4

PO

2"4

2'5 2'5

PP

2'6

2'8

PQ

2"4

2-5

FT

2'6

2'5

2'8

2'1

2'2

2'6

2'2

1-9

2-0

2-0

2'8

2-5 2'9

2'5

2'5

2'5

1990). Average band-sharing index (ABSI) was calculated as 100 x the mean of BSI for all strain comparisons in any two populations (cf. Gilbert et al., 1990), Within population ABSI

is the mean of all BSI for individual strain comparisons within that population, Two populations where all bands are identical for all individual strains have an ABSI of 100, and two populations where no bands are identical in a betweenpopulation comparison have an ABSI of 0_ An unbiased estimation of the standard deviation was defined as 100 x 2BSI(1-BSI)(2-BSI)/Ii(4-BSI), where Ii = mean number of amplified fragments per individual (Lynch. 1990)_ The numbers of BSI included in the within-population ABSI were 294. 167 and 21 for the S, P and F groups, respectively_ For the among-population ABSI, 2171 and 816 BSI were used in the Sand P groups. respectively_ Between-intersterility group ABSI were based on 3080. 420 and 264 individual BSI for the S-P, S-F and P-F comparisons, respectively_ Principal component analysis (PCA) was carried out on the matrix of ABSI (Table 2) of the various population comparisons using the SIRIUS software package_ The results give information on how the various populations relate to each

2'5 2'9

1'9

2'5

2-4

2-0

2'3

2-9

3-2

3']

3']

3'0

other. Populations that are close to each other on the plot are correlated in their ABSIs with other populations, Discriminant analysis was carried out on individual strains using the DISCRIM procedure in Statistical analyses system (SAS) computer program_ A cross-validation of the geographical population affiliation and that inferred from the banding pattern matrix was made (Table 3), Misclassified strains indicate that populations overlap in their banding patterns_ In order to evaluate whether the misclassifications were caused by chance alone a chi-squared test was performed, Expected numbers of strains classified in various populations of the same intersterility group were calculated as follows. The populations were divided into classes of distances from each other; original population. populations less than 100 km away, populations 100-500 km away, 500-1000 km away and more than 1000 km away_ The probability (Pr) of each strain to be classified into a particular distance class is the number of populations in that class (M) divided by the total number of populations (N) in the intersterility group (Pr = MIN), The expected number of strains for a distance class is

60

Variation in Heterobasidion annosum DNA

Table 3, Crossvalidation of population classification based on geographical origin and amplified DNA. Observed values represent the number of strains of the three IS groups that were classified in the same or another geographical population from the banding patterns using discriminant analysis_ 'Expected values' are the summation of probabilities for each strain to be classified into a particular population class by chance alone (see text for calculation details) From:

Into:

IS group (number of strains)

Original population

S (72) Observed Expected

X' P (45) Observed Expected

X'

F (6)

15 7-2 8-45 7 5-6 0-36 6

Populations < 100 km away

Populations 100-500 km away

Populations 500-1000 km away

Populations > 1000km away

Sum of X'

11

9-2 0-35

15 20-0 1-25

13 16-6 0-78

5 18-7 10-04

20-86'"

6 4-6 0-41

25 21-1 0-71

8 13-9 2-49

_a

Not investigated_ '" Significantly different from the expected numbers, P < 0-001.

a

simply the summation of all strain probabilities for that class (Sum Prj.

RESULTS H. annosum DNA was successfully amplified using the MI3 core sequence as primer. Typically, there was a distinct pattern of bands amplified from each individual strain (Figs 1, 2). The average number of amplified fragments was higher for the S intersterility group (9'37 ±O'22 S.E.M.) than for P (7'36 ±0'24) and F (6-33 ± 0'50). The distribution of amplified fragments in the isolates studied is given in Fig. 2. Similarity was lower within S populations (ABSI 70'3 ± 2'0 S.D., weighted mean of intrapopulation values) than within P populations (ABSI 77'0 ± 2'3) (Table 2). Interpopulation differences were greater than the within-population variation for both intersterility groups (66'4 ± 2'2 and 75'0 ± 2'4 for the Sand P group, respectively).

4072-+ 3054-+ 2035-+

16361018517_

Fig. 1. Gel of amplified DNA. Lanes are, from left to right: molecular weight markers, faf 6-2, faf 8-5, faf 9-1, d 10, d 11, d 12, mj 87, mj 124, mj 107, markers, fas 2, fas 3, fas 16-1, 87-208/1, 87-243/2, 87168/1, sib SOc, si 63b, si 78b, and markers. Molecular weights (bp) are indicated on the left-hand side of the gel.

Between-intersterility group comparison revealed greater differences than within intersterility groups both for the comparisons S - P (49'2 ± 2'5) and the distance from both groups to a population of the F intersterility group (ABSI 50'1±1'6 and 44'O±2'4 with Sand P, respectively). The ABSI decreased with geographical distance between populations of the S group (y = 0'701- 4'95x, r2 = 0'44, P = 0'001, Fig, 3). In contrast to what was found for the S group, the geographical trends were not statistically significant from the Scandinavian populations of the P intersterility group, Populations within short distances from each other, for example SA, SB and SC or SG and SH, showed ABSI that were similar within and between populations (Table 2). Population affiliation inferred from banding patterns using discriminant analysis did not completely correlate with the geographical origins of strains (Table 3), with 78% of the strains not being classified in the correct population. Among the misclassified strains there was a tendency for strains to be classified into populations of a geographical origin close to the original population. In the S group the pattern of misclassifications was significantly different from that expected by chance alone (P = 0'001, X2 test), but not in the P group (Table 3). However, the intersterility group affiliations were not conflicting when mating experiment data were compared with banding patterns. That is, in only two cases was a P strain judged to belong to the S intersterility group based on the amplified DNA. The S, P and F intersterility group populations were found in three distinctive clusters in principal component analysis (PCA). PCA indicated that the populations within intersterility groups of geographical origin close to each other also had similar banding patterns (Fig. 4). Interestingly, the S populations from Germany and Italy formed a cluster on their own, away from the Scandinavian S populations (Fig. 4).

61

]. Stenlid. J.-O. Karlsson and N. Hogberg

Pcp Strain

FraSJ"8nt no.

Pcp Strain

~11111111112222

12345678901234567890123

12345678901234567890123

SA SA SA SA SA SA SA

~. 11111111112222

87·1657/5 ‫סס‬oo1001‫סס‬oo10100100111 87-168/1 0‫ס‬0ooooo100010100100111 87-176/2 ‫ס‬0ooooo1100110101001111 87-179/3 0CKKKM)00100010101001111

SR SR SR SR

tas2 tas3 tas15-3 tas16-1

‫סס‬oo100111001111‫סס‬oo100 ‫ס‬0ooooo110001011‫סס‬oo101 00010001 ‫סס‬oo1 01 0‫סס‬oo1 01 0‫סס‬oo101000010100100111

10101001100111110100111

SS SS SS SS SS

tasS 1as9-4 1as9-5 1as10 1as13-4

0100010100101‫סס‬oo100101 010‫סס‬oo110110‫סס‬oo100111 00010111‫סס‬oo10110100101 0001011011101010‫סס‬oo101

PF PF PF PF PF

sa16 5816.4 5&16.7 sa34

0000000000001 011 o00ooo1

PI PI PI

sig14a sib27b si45c

0100011100111011000ooo1

PK mj87 PK mjl07 PK mj124

‫סס‬oo101101101011000ooo1 0‫סס‬oo1110010101100‫סס‬oo1 0‫סס‬oo111‫סס‬oo10110000001

PM PM PM PM PM PM PM

rb28 rboo rb162 rb199 rb245 rb257 rb282

o00ooo1‫סס‬oo010110000001

PN PN PN PN

d14 d15 d16 d17

01000110‫סס‬oo101100‫סס‬oo1 000011110010101000‫סס‬oo1 01‫סס‬oo11‫סס‬oo1011000ooo1 o00ooo10‫סס‬oo10110000001

PO PO PO PO PO PO

d7

0‫סס‬oo110‫סס‬oo101 ‫ס‬0ooooo1 01000111‫סס‬oo1011000ooo1 01000111‫סס‬oo1011000ooo1 ‫סס‬oo1111‫סס‬oo1011000ooo1 0‫סס‬oo1110000101100‫סס‬oo1 0‫סס‬oo110000010110100001

PP PP PP PP PP PP PP PP PP PP PP PP

d18 d19 d20 d21

87-18313

10‫סס‬oo11100111010100111

87-184/3 87-197/2

‫ס‬0ooooo1100011101100111

87-20812

‫סס‬oo1101100010100100111

S8 S8 S8 S8 S8 S8 S8

87-209/3 87-210/3 87-214/2 87-215/2 87-219/1 87-221/3

SC SC SC SC SC SC

87-225/1 87-226/3 87-227/1 87-240/1 87-243/3 87-245/1

SO SO SO SO SO SO SO SO SO SO

br88 br202 br212 br228 br231 br237 br304 br331 br371 brei

SE SE SE SE SE SE SE SE SE

sa33 5848 S894 58131 58139 58159.5 sA192 s8205 sa208

00000101100010100000101

SG SG SG SG SG

sig4b sigSb si910a sig11 sig19

010‫סס‬oo1100010100100111 ‫סס‬oo1001100010100100111 0001111110101‫סס‬oo100111

SH SH SH SH SH SH SH SH SH SH SH SH

Si4 si7 Si9 Si21 Si35 Si58 si71b Si73 si78b sib50c sib63b sib120a

Sl SL SL Sl SL Sl SL

rb14 rD31 rb48 rb89 rb94 rb132 rb175

00000000100010101000111 00011001011010101000111 0‫ס‬0ooooo100110110100111 0001100101001010‫סס‬oo111 ‫סס‬oo1000100010101000111

00011001100010110100111 0‫ס‬0ooooo110110100100101 ‫סס‬oo1000100010100100111 0‫סס‬oo001100010111000100 ‫סס‬oo1001010010100100111 ‫סס‬oo1001100010101000111 0‫ס‬0ooooo111010101000111

‫סס‬oo000010‫סס‬oo110000101

00000001101010100101111

000000111010101‫סס‬oo1111 ‫סס‬oo10011000101‫סס‬oo1111 ~11ooo101oo1oo111 0‫ס‬0ooooo100010100100111

00000001000100101101111 00001001011010100101111 00001001001010100101111 ‫ס‬0ooooo1100010100100111

‫סס‬oo0011100110001000111 0‫סס‬oo101101010100100111

00111001010111000100101

‫סס‬oo1011001110100101111 0010111110001‫ס‬0ooooo110

00110001111110100100111

10011011‫סס‬oo10101000111 00‫סס‬oo01000010100100111

00001011100110010100111 00011011101010001000111 010‫סס‬oo110011010‫סס‬oo111

00000001100110010100111

‫סס‬oo0010100010101000111 010‫סס‬oo110011010‫סס‬oo111 10‫סס‬oo11110110100110111 0‫ס‬0ooooo10001010‫סס‬oo111 ‫סס‬oo1011101111000100111 000‫סס‬oo1110010100100111

00101011100010101000111

0‫סס‬oo111111110100100111 0000101110011010‫סס‬oo111 ‫ס‬0ooooo1100010001000101

00010111101010101000101 00010011000010100101111 ‫סס‬oo110110001010‫סס‬oo101 ‫סס‬oo1001101010101000111

00011111101010101000111 0001‫סס‬oo110010101000101

o00ooo11111010101001111

PO PO PO PO

sa2

dB

d9 d10 dl1 d12

d22

d23 d24 d25 d26 d27

d28

OK1

d1 d2 d3 d6

FT 1a11 FT la14 FT 1af5-6 FT 1a16-2

FT

FT

t818-5

f819-1

Fig. 2. Amplified band scores. Strains are grouped into populations according to Table 1.

‫סס‬oo1011100010011000111

0‫סס‬oo11010101011000ooo1

‫סס‬oo1011101010110101000 ‫סס‬oo101110001011‫ס‬0ooooo ‫סס‬oo111001101011000ooo1 0‫סס‬oo111‫סס‬oo1011000ooo1

0000000000001 011 001 0001

0010011110111011000ooo1

0‫סס‬oo11100101011000ooo1 ‫סס‬oo1111‫סס‬oo1011000ooo1 01000111000010110‫סס‬oo01

00001011000010110000101 000000110010101100‫סס‬oo1

00000110‫סס‬oo1011000ooo1 00000111‫סס‬oo1011‫סס‬oo101 01000110‫סס‬oo1011000ooo1 01001010‫סס‬oo10100000001 01000110‫סס‬oo1010‫סס‬oo001 ‫סס‬oo1111‫סס‬oo1011000ooo1

01001111001010110001001

01000110‫סס‬oo101‫ס‬0ooooo1 0100101000001010‫ס‬0ooooo 0‫סס‬oo110‫סס‬oo1011000ooo1 01‫סס‬oo10‫סס‬oo101000‫סס‬oo1 ‫סס‬oo1111‫סס‬oo1011000ooo1

01‫סס‬oo10‫סס‬oo101000‫סס‬oo1 ‫סס‬oo111110101011000ooo1 0‫סס‬oo11110001011000ooo1 0‫סס‬oo111‫סס‬oo1011000ooo1

10‫סס‬oo1101001010‫סס‬oo100

10000001010010100101100 10‫סס‬oo110000101‫סס‬oo1100 10‫סס‬oo11‫סס‬oo1010‫סס‬oo100 1000ooo1 ‫סס‬oo11 o00ooo100 1000ooo100001010‫סס‬oo100

62

Variation in Heterobasidion annosum DNA 90

80

ti3 ~ 70 60

50 +---.,.....---,r----...,.--..,---.......---, 3000 1000 2000 o

Distance (km) Fig. 3. ABSI for a pair of populations at various geographical distances. ABSI for a population pair was calculated as the mean of all interpopulation comparisons of individual strains within the population pair. 0, comparisons within the S group, +, comparisons within the P group. The line is a regression on the S-group values, y = 0'701- 4'95x, r 2 = 0'44, P = 0'001. The regression on P-group values was not significant.

0·20

0.00

_

SG SBSH SD

I I

SL

SS

~t£C_ s~

~ ~

SR

e

I I 1 I

PQ PM PK

PO

l'N__ PF P),p

I

'<::5 -0·20 o

0..

S o

U -0-40

-0·60 FT

-0·80

'---~

-0·60

~

-0· 30

...-O· 30 Component 1 (69·5%) ~

0·00

.--------' 0·60

Fig. 4. Principal component analysis of the ABSI of the different populations. Populations as in Table 1.

DISCUSSION M13 regions previously used with Southern blotting for fingerprinting in a variety of organisms was successfully amplified in our experiments with Heterobasidion annosum. These and possibly other primers provide us with the tools to follow the regional spread in this serious forest pathogen. The variation observed in the banding patterns was sufficiently detailed to detect genetic differences among populations of the S intersterility group of H. annosum in Europe that were larger in comparisons over long distances than in short distances. The PCA and the discriminant analysis of population affiliation did support the trend over geographical distances

found by comparisons of similarity indices. All three methods could be included in future analyses of regional population structures in H. annosum. By combining measurements of genetic similarity with information on abrupt geographical barriers such as the Alps, the Baltic sea, etc. it would be possible to interpret the spreading capacity of spores of the fungus over large distances in more detail. Direct measures of gene flow include spore catches on various distances from a fruiting body or population. Rishbeth (1959) reported catches of viable spores of H. annosum that had travelled about 320 Ian over open sea. Similarly, Kallio (1970) reported on spore catches in the Gulf of Finland and, because of wind directions, reckoned that the spores had travelled 50-500 Ian from Estonia or Sweden. It is therefore not surprising that the range of distances in the P group in our investigation was not large enough to pick up any trend in similarity indices with geographical distance (Fig. 3). A low percentage of isolates were placed in their original population when classified from the banding patterns. This indicates that a certain amount of overlapping occurs in the genetic structure of the various populations. This also indicates that spores may have entered or even founded populations over large distances. In the S group there was a significant trend for strains to be classified in the original population (Table 3), indicating a local differentiation in this intersterility group. A similar but not significant trend existed also in the P interstility group. In future investigations it would be of interest to include samples from a wider geographical range to examine on what scale differentiation occurs in the two intersterility groups. Traditional methods of measuring gene flow presuppose that populations are at equilibrium with each other (Slatkin, 1987). The time needed to reach this eqUilibrium may be counted in hundreds or even thousands of generations (Slatkin & Barton, 1989; Boileau, Hebert & Schwartz, 1992). However, it is not likely that H. annosum populations are at equilibrium in Europe. Norway spruce has recolonized northern Europe following the last ice age. For example, the first pollen records of Norway spruce in Norway are only about 2000 years old (Huntley & Birks, 1983; Hafsten, 1991). Given that the S group of H. annosum is restricted to growth on that host and that some of the genets of the fungus must be in the range of a hundred years (Stenlid, 1985, 1987; Piri et a/., 1990), it seems that the genetic structures studied are reflections of recent migration rather than steady-state situations. In contrast to Norway spruce, Scots pine has been present in the Scandinavian peninsula for up to 9000 years (Huntley & Birks, 1983; Hafsten, 1991). The P group has had a longer period of time available for its local spread in Scandinavia and hence also to reach eqUilibrium. The P group was less variable in the banding patterns than the S group in local populations also (Fig. 2). This might be a reflection of higher gene flow in the P group. Alternatively, the P group could have been subjected to a genetic bottleneck in its history. The number and sizes of refuges during the last ice age might be important for this. Amplification products could easily be used for intersterility group classification. The degree of misclassified isolates was low (less than 2 %). Pectinolytic isozymes (Karlsson & Stenlid,

J. Stenlid, J.-O. Karlsson and N. Hogberg 1991) as well as a set of different allozymes (Otrosina et al., 1992) did earlier give good correlation with intersterility

groups. Which method to use is a question of cost, availability and labour.

We are grateful to Gunilla Swedjemark Iben Thomsen, Kari Korhonen and Halvor Solheim for providing us with fungal strains used in this study, and to Anders Dahlberg for useful discussions. Financial support was from the Swedish Council for Forestry and Agricultural Sciences, Nordic Council for Forestry Research and the Karl Erik Onnesjo Foundation.

REFERENCES Boileau, M. G., Hebert, P. D. N. & Schwartz, S. S. (1992). Non-equilibrium gene frequency divergence: persistent founder effects in natural populations. Journal of Evolutionary Biology 5, 25-39. Capretti, P., Korhonen, K., Mugnai, L. & Romagnoli, C (1990). An intersterility group of Heterobasidion annosum specialized to Abies alba. European Journal of Forest Pathololgy 20, 231-240. Chase, T. E. & Ullrich, R. C (1990). Genetic basis of biological species in Heterobasidion annosum: Mendelian determinants. Mycologia 82, 67-72. Gilbert, D. A., Lehman, N .. O'Brien, S.). & Wayne, R. K. (1990). Genetic fingerprinting reflects population differentiation in the California channel island fox. Nature 344, 764-767. Hafsten, U. (1991). Granskogens historie i Norge under opprullning. Blyttia 49, 171-181. [In Norwegian with English summary.] Harrington, T. C, Worrall,).). & Rizzo. D. MJ. (1989). Compatibility among host-specialized isolates of Heterobasidion annosum from western North America. Phytopathology 79, 290--296. Huntley, B. & Birks, H. ). B. (1933). An Atlas of Past and Present Pollen Maps of Europe, (}--13,OOO years ago. Cambridge University Press: Cambridge and New York. Innis, M. A., Gelfand, D. H., Sninsky, J. J. & White, J. T. (1990). PCR Protocols. A guide to Methods and Applications. Academic Press: San Diego. Jeffreys, A. J.. Wilson, V. & Thein, S. L. (1985). Hypervariable "minisatellite" regions in human DNA. Nature 314. 67-73. Kallio. T. (1970). Aerial distribution of the root-rot fungus Fornes ann05US (Fr.) Cooke in Finland. Acta Forestalia Fennica 107, 1-55. Karlsson, J.-O. & Stenlid. J. (1990). Pectic enzyme profiles of intersterility groups in Heterobasidion annosum. Mycological Research 95, 531-536. Korhonen, K. (1978). Intersterility groups of Heterobasidion annosum. Communicationes [nslituti Forestalis Fenniae 94, 1-25.

(Accepted 10 June 1993)

63 Kvarnheden, A. & Engstrom, P. (1992). Genetically stable, individual-specific differences in hypervariable DNA in Norway spruce, detected by hybridization to a phage M13 probe. Canadian Journal of Forest Research 22, 117-123. Kwan, H.-S., Chiu, S.-W.. Pang, K.-M. & Cheng, S.-C (1992). Shain typing in Lentinula edodes by polymerase chain reaction. Experimental Mycology 16, 163-166. Lynch, M. (1990). The similarity index and DNA fingerprinting. Molecular Biology and Evolution 7, 473-484. Meyer, W .. Koch, A., Niemann. C, Beyermann, B.. Epplen, ). T. & Bomer, T. (1991). Differentiation of species and strains among filamentous fungi by DNA fingerprinting. Current Genetics 19, 239-242. Meyer, W., Lieckfeldt, E., Wostemeyer, ). & Bomer, T. (1992). DNA fingerprinting for differentiating aggressivity groups of the rape seed pathogen Leptosphaeria maculans. Mycological Research 96, 651-657. Nybom, H. & Rogstad, S. H. (1990). DNA "fingerprints" detect genetic variation in Acer negundo. Plant Systematics and Evolulion 173, 49-56. Otrosina, W. L Chase, T. E. & Cobb, F. W. (1992). A1lozyme differentiation of intersterility groups of Heterobasidion annosum isolated from conifers in the western United States. Phytopathology 82, 540--545. Piri, T., Korhonen, K. & Sairanen, A. (1990). Occurrence of Heterobasidion annosum in pure and mixed spruce stands in southern Finland. Scandinavian Journal of Forest Research 5, 113-125. Raeda, U. & Broda, P. (1985). Rapid preparation of DNA form filamentous fungi. Lellers in Applied Microbiology I, 17-20. Rishbeth,). (1959). Dispersal of Fornes annosus Fr. and Peniophora gigantea (Fr.) Massee. Transactions of the British Mycological Society 42. 243-260. Ryskov, A. P., )incharadze, A. G., Prosnyak, M. I., Ivanov, P. L. & Limborska, S. A. (1938). M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms. FEBS Lellers 232, 388-392. SchaaL B. A., O'Kane, S. L. & Rogstad, S. H. (1991). DNA variation in plant populations. Trends in Ecology and Evolution 6, 329-333. Slatkin, M. (1987). Gene flow and the geographic structure of natural populations. Science 236, 787-792. Slatkin, M. & Barton, N. H. (1989). A comparison of three indirect methods for estimating average levels of gene flow. Evolution 43, 1349-1368. Stenlid,]. (1985). Population structure in Helerobasidion annosum as determined by somatic incompatibility, sexual compatibility, and isoenzyme patterns. Canadian Journal of Botany 63, 2268-2273. Stenlid, J. (1987). Controlling and predicting the spread of Heterobasidion annosum from infected stumps and trees of Picea abies. Scandinavian Journal of Forest Research 2, 187-198. Stenlid, ). & Karlsson, J.-O. (1991). Partial intersterility in Helerobasidion annosum. Mycological Research 95, 1153-1159. Stenlid, J. & Swedjemark, G. (1988). Differential growth of S- and P-isolates of Heterobasidion annosum in Picea abies and Pinus sylvestris. Transactions of the British Mycological Society 90, 209-213. Worrall. ). J.. Parmeter, J. R. & Cobb, F. W. (1983). Host specialization of Heterohasidion annosum. Phytopathology 73, 304-307.