Comparison of nuclear ribosomal DNA sequences from Alternaria species pathogenic to crucifers

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Mycol. Res. 99 (5): 604--614 (1995)

604

Printed in Great Britain

Comparison of nuclear ribosomal DNA sequences from Alternaria species pathogenic to crucifers

CLAUDIA A. JASALAVICW, VICTOR M. MORALES 2 "', LAWRENCE E. PELCHER2 AND GINETTE SEGUIN-SWARTZ 1 1 Agriculture 2

and Agri-Food Canada Research Station, 107 Science Place, Saskatoon, Saskatchewan S7N OX2, Canada National Research Council Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N OW9, Canada

The sequences coding for the nuclear 18s rRNA, S'8s rRNA, and the internal transcribed spacers (ITSI and ITS2) were amplified by the polymerase chain reaction and sequenced for one isolate each of Alternaria brassicae, A. brassicicola, A. raphani, A. alternata and Pleospora herbarum. The S'8s rONA sequences from the four Alternaria species were identical and differed at only one base pair from that of P. herbarum. The internal transcribed spacer sequences, especially ITS1, were very variable in both base composition and length. The 18s rONA sequences were highly conserved, but enough variability was present to distinguish genera clearly. Phylogenetic analysis of the sequence data sets by both parsimony and maximum likelihood methods clearly separated genera and species. All of the Alternaria species were closely related. Pleospora also appeared to be more closely related to Alternaria than to Leptosphaeria.

Several species of the anamorphic genus Alternaria are commonly found on crucifers worldwide. Whereas Alternaria alternata is usually regarded as a saprotroph. on this plant family, the pathogenic A. brassicae, A. brassicicola and A. raphani cause the disease of crucifers known as blackspot. Symptoms of the disease include necrotic lesions surrounded by chlorotic halos on aerial plant parts, (e.g. leaves, stems and siliques), grey to black lesions due to mycelial growth and sporulation under favourable environmental conditions, and discolouration and shrivelling of the seed (Weimer, 1924, 1926; Groves & Skolko, 1944; Wiltshire, 1947). Chlorosis is caused by a host-selective toxin (Buchwaldt & Green, 1992), which in A. brassicae has been identified as destruxin B (Ayer & Pena-Rodriguez, 1987; Bains & Tewari, 1987; Buchwaldt & Jensen, 1991). The three pathogenic Alternaria species infect a number of genera in the Cruciferae with many species of Brassica and Raphanus being very susceptible; A. alternata is commonly isolated from the seed of cruciferous species as well as those of many other plant families (Groves & Skolko, 1944; Buchwaldt & Green, 1992). Alternaria is composed of about 60 species (Rao, 1969; Rossman, Palm & Spielman, 1987), the vast majority of which are plant pathogens and have no known sexual stage. Only five species with small beakless conidia have a reported Pleospora-like teleomorph. Simmons (1986) placed the Pleospora species with Alternaria anamorphs in Lewia. Although some taxonomists accept this (Barr, 1987; Eriksson & Hawksworth, • Present address; Brigham and Women's Hospital, Gastroenterology Division, Thorn Research Building, Room 1310, 20 Shattuck Street. Boston, Massachusetts 02115, U.S.A.

1991), others still consider them Pleospora species (Sivanesan, 1984; Hanlin, 1990). In any case, this association would suggest that Alternaria is related to the Ascomycetes, in particular to the Loculoascomycetes. The taxonomy of Alternaria is based primarily on the morphology and development of conidia and conidiophores, and to a lesser degree on host plant association and colony morphology (Elliott, 1917; Wiltshire, 1933; Simmons, 1967). Although each Alternaria species which occurs on crucifers has a distinct spore morphology, when one considers the entire genus species identification becomes more difficult. Spore characteristics, such as length, width, septation and beak size, may overlap among certain species or vary with cultural conditions, so that knowledge of the host plant association becomes important for identification to the species level. Ribosomal DNA sequences, in particular the 5'85 rONA and flanking internal transcribed spacer regions ITS1 and ITS2, have been used to study the phylogenetic relationships for a number of plant pathogenic fungi. Recently Zambino & Szabo (1993) examined the relationship of strains andformae speciales of many Puccinia species from cereals and other grasses. Morales et al. (1993) have shown that the highly virulent and weakly virulent strains of Leptosphaeria maculans (Desm.) Ces. & De Not. may actually be different species. Based on the ITS1 sequences alone, Carbone & Kohn (1993) found evidence of a sclerotial lineage within the Sclerotiniaceae. The phylogenetic relationship among several Leptosphaeria species has been studied based on the 5'85 rONA and ITS regions as well as the 185 rONA (Morales et al., 1995). The objectives of our study were: (1) to determine regions of ribosomal genes which would distinguish among Alternaria

605

Claudia A. Jasalavich and others Table 1. Fungal isolates used in this study

Allernaria alternata

Isolate

Isolation host

Geographic origin

Source

AA6:1:

Brassica rapa L. ssp. oleifera (Metzg.) Sinsk Brassica rapa ssp. oleifera Brassica oleracea L. ssp. capitata L. Brassica rapa ssp. oleifera Trifolium pratense L.

Alberta, Canada

G. A. Petrie

Alberta, Canada

G. A. Petrie

British Columbia, Canada Saskatchewan, Canada Alberta, Canada

G. A. Petrie

(Fr.) Kiss!.

Alternaria brassicae

AB 11:1:

(Berk.) Sacc.

Allernaria brassicicola

ABc2:1:

(Schwein.) Wiltshire

Alternaria raphani

AR6t

). W. Groves & Skolko

Pleospora herbarum

DAOM 150679

G. A. Petrie

CCFC

(Fr.) Rabenh. • CCFC Canadian Collection of Fungus Cultures.

t AR6 has been deposited with the International Mycological Institute as IMI 354205 and with the International Collection of Micro-organisms as ICMP 11040. :I: These isolates have been deposited with the International Mycological Institute.

species and between Alternaria and related genera, (2) to examine the phylogenetic relationship among species of Alternaria commonly found on crucifers, and (3) to look at the relationship between Alternaria and Pleospora. To address these aims we amplified and sequenced the 5'85 rDNA, the internal transcribed spacers ITS1 and ITS2, and the 185 rDNA.

MATERIALS AND METHODS Fungal isolates and cultural conditions The fungal isolates used in this study are described in Table 1. Spores were stored in 50% (vIv) glycerol at -70°C. Cultures started from glycerol stocks were grown on V8 agar (Petrie, 1969) supplemented with Rose Bengal at 4 IJg ml- 1 and streptomycin sulphate at 100 IJg ml- 1 at 20° under fluorescent lighting (GE Cool White F96GTI2/CW) with an 18 h photoperiod to promote sporulation. For DNA isolation, spores harvested from 10- to 14-d-old plate cultures were germinated for 1-3 d in potato dextrose broth at room temperature and 100 rpm on a gyratory shaker. Mycelia were collected aseptically by vacuum filtration through a Buchner funnel lined with filter paper, washed with four volumes of sterile distilled water, and lyophilized. Freeze-dried mycelia were stored over desiccant at - 20° until use.

DNA isolation DNA was isolated from lyophilized mycelia by a modification of the procedure of Bainbridge et al. (1990). Powdered mycelia were resuspended in a new lysis buffer [50 mM Tris HC!, 450 mM EDTA, pH 8'0, 0'5% (w/v) sodium dodecyl sulphate and 200 IJg ml- 1 proteinase K] and incubated at 65° for 2 h, a protocol adopted to maximize inactivation of nucleases. Samples were extracted twice with cetyltrimethyl-ammonium bromide (Ausubel et a/., 1987) to remove polysaccharides which could interfere with amplification. Subsequent RNase treatment and organic extractions of the aqueous phase, and precipitation of the DNA were as in the original method (Bainbridge et al., 1990). DNA concentrations were estimated from the ethidium bromide fluorescence of samples compared with that of known amounts of uncut II DNA after electrophoresis in an agarose gel.

peR amplification

Nuclear rDNAs and the internal transcribed spacer regions were amplified by the polymerase chain reaction (PCR) from each of the Alternaria and Pleospora isolates described in Table 1. Amplifications were performed in 200 IJI of NE buffer [10 mM KC!, 10 mM (NH4)2S04' 20 mM Tris HCI pH 8'8, 2 mM MgS0 4, 0'1 % TritonX-100], 200 IJM of each deoxyribonucleotide triphosphate, 165 nM of each of the appropriate primers with 100 ng of fungal total DNA and 10 units of Taq polymerase. Each reaction consisted of an initial denaturation step of 2 min at 94°, 35 cycles of amplification, and a final extension of 7 min at 72°. For amplification of the ITS regions and 5'85 rDNA with primers ITS1 and ITS4 (Table 2), each cycle consisted of 30 s at 94° followed by 30 s at 55° for annealing and 2 min at 72° for extension. For amplification of the 185 rDNA with primers NS1 and NS8, each cycle consisted of 30 s at 94° followed by I min at 50° for annealing and 2 min at 72° for extension. PCR amplification products were purified by agarose gel electrophoresis followed by binding to NA-45 membranes, elution and passage through Elutips in accordance with the manufacturer's instructions (Schleicher & Schuell, Keene, NH, U.S.A.).

DNA sequencing Purified PCR products were direct sequenced in an Applied Biosystems (Foster, City, CA, U.S.A.) 370A sequencer by the Taq DyeDeoxi Terminator™ cycle system. The amplification products were sequenced in both directions with the primers listed in Table 2. In cases where the internal sequencing primers of White et al. (1990) failed, new primers were selected with the program PRIMER DESIGNER version 2.0 (Scientific & Educational Software, Stateline, PA, U.S.A., copyright 1991) from the portion of sequence already obtained and synthesized. DNA sequences were edited and assembled with the program CLONE MANAGER version 4.0 (Scientific & Educational Software, Stateline, PA, U.S.A., copyright 1993).

Analysis of DNA sequences DNA sequences were aligned with the program PILEUP from the UWGCG package (Devereux, Haeberli & Smithies, 1984); a

Nuclear rRNAs of Alternaria species on crucifers

606

Table 2. Primers for PCR amplification and sequencing Primer name·

Primer sequencet

Nuclear ITS regions and 5'85 ITSI TCC GTA GGT GAA CCT GCG ITS2 GCT GCG TIC TIC ATC GAT GC ITS3 GCA TCG ATG AAG AAC GCA GC ITS4 TCC TCC GCT TAT TGA TAT GC Nuclear 185 NSI NS2 NS3 NS4M NS5 NS6 NS7 NS8 NS8A NS8C NS9 NS9M NSI0B NSI0M NS11M NS12M

GTA GTC ATA TGC TIG TCT C GGC TGC TGG CAC CAG ACT TGC GCA AGT CTG GTG CCA GCA GCC AGC CTI GCG ACC ATA CTC CC AAC TIA AAG GAA TIG ACG GAA G GCA TCA CAG ACC TGT TAT TGC CTC GAG GCA ATA ACA GGT CTG TGA TGC TCC GCA GGT TCA CCT ACG GA ATG ACA CGC GCT TAC TAG TCA ACG CAT GCT GAT GAC ATC CAA GGA AGG CAG CAG GC TIA CGG ATC GCA TAG CCT AAG GCT ATG CGA TCC GTA CAA GGC CAT GCG ATC CGT AA ATC AGT ATI CAG TIG TCA GA CAA CTG AAT ACT GAT GCC

Position in sequence of Neurospora crassa,*,

Position in sequence of Alternaria altemata,*,

5-23 268-249 249-268 579-560

1-19 244-225 225-244 570-551

20-38 570-550 550-570 1125-1106 1128-1149§ 1434-1411 1411-1434 1785-1766

1-19 551-531 531-551 1108-1089§ 1111-1132§ 1417-1394 1394-1417 1770-1751 1578-1561 1591-1574 390-409 238-255 256-239 257-238§ 859-878 873-856

1576-1593~

1589-1606§ 409-428 np np np 878-897 891-875§

Reference

White White White White

et et et et

al.• al.• al.• al.•

1990 1990 1990 1990

White et al.. 1990 White et al.• 1990 White et al.. 1990 Morales et a/., 1995 White et al.• 1990 White et al.. 1990 White et al.. 1990 White et a/., 1990 This study This study Morales et aI., 1995 This study This study Morales et al.. 1995 This study This study

• Primers whose names end in an odd number are for sequencing in the 5' to 3' direction. Primers whose names end in an even number are for sequencing the complementary strand. t Primers are written 5' to 3'. '*' Numbers refer to positions in the following GenBank accessions: for N. crassa, M13906 (ITS regions and the 5'85 rONA) and X04971 (185 rONA); for A. alternata. U05195 (ITS regions and the 5'85 rONA) and U05194 (185 rONA). § With 1 bp diff. ~ With 2 bp diff.

few further adjustments were made by eye where necessary. Phylogenetic analyses were performed with programs contained in the package PHYLIP version 3.5c (Felsenstein, 1993) and PAUP version 3.1 (Swofford, 1993) on sequence data for the different rDNAs separately and in combination. The two rDNA data sets were: (I) ITS1, 5'85 rDNA and ITS2 sequences, and (2) 185 rDNA sequences; each included previously known DNA sequences from other filamentous Ascomycetes obtained from GenBank. (All GenBank accession numbers for both new and old sequences are given in the legends of Figs I and 2 which show the sequence alignments.) For each rDNA data set phylogenetic trees were constructed by two methods. (I) The most parsimonious tree was determined from the original sequence data set with the program DNAPARS using 10 randomizations of sequence input order and global rearrangement of the tree. Also a heuristic search with randomization of sequence input order was done in PAUP to generate various goodness-of-fit statistics. In order to evaluate the robustness of this tree, 1000 bootstrap data sets were generated by the program SEQBOOT from the original rDNA data set. All of the most parsimonious trees were determined for the bootstrap data sets with DNAPARS using 10 randomizations of sequence input order in each data set and global rearrangement of the tree; the consensus tree was calculated from the results by the program CONSENSE. (2) The best fit maximum likelihood tree was calculated by the program DNAML using 10 randomizations of sequence input order of the original rDNA data set and global rearrangement of the tree. Trees were compared with the Kishino &

Hasegawa test (1989) contained in the USER TREE option of Genetic distances between pairs of species were calculated for the total 2295 bp of sequence data (185 rDNA lTS1, 5'8r rDNA and ITS2) by the program DNADIST using the Kimura two-parameter model.

DNAML.

RESULTS Electrophoresis and direct sequencing confirmed that a single product was amplified in accordance with each peR reaction and that each product corresponded to the expected rDNA. Sequence alignments

The alignment of the DNA sequences of the internal transcribed spacers ITSl and lTS2 and the 5'85 rDNA is shown in Fig. 1. The internal transcribed spacers contained most of the sequence variation with ITSl being more variable than ITS2. The length of lTSl varied more than that of ITS2 among the species and was different for each Alternaria and Pleospora species (Table 3). Also, the lTSl of A. raphani appeared to have a large insertion compared with the other Alternaria species. The base composition of ITSl was more variable than that of ITS2, the variability being mostly at the species level in ITSl and at the genus level in lTS2. Across all species in the alignment shown in Fig. 1, there are a total of 255 variable sites (48'1 %) of which 182 (34'3 %) are phylogenetically informative, out of a possible 530 sites. ITSl contained 159 variable sites (30%) of which 107 (20'2 %) were

Claudia

A.

607

Jasalavich and others

ABc2 AR6 ABll AA6 PH OHU OKU Leroy Unity Ldol ABc2

AR6

ABll AA6 PH OHU OKU Leroy Unity Ldol ABc2

AR6

ABll AA6 PH OHU OKU Leroy Unity Ldol ABc2

AR6

1 11 21 31 41 51 61 71 CAC-AATATG AAAGCGGGCT GGACTCACCT CAGCA----- ---------- GCATCTGCTG TT-GGGGCCA GCCTTGCTGA ... C .. C '1' GTGCA TTGCTTTACG .. G.GC A.C .. ... G.. A A.A .. T.TC G.. ------- ---------- ---------- ----AC.A.G .....•.•.. .. . A G AC.T.TC GG.------- ---------- ---------- ----.TTA . . .. . T. . .GAC. '1' .AC ---------- ---------- ---- •. CGGT GAG .. CT. .. .. '1' .GT .••• .'1' •• TA--.A .. C.C .. AG. GTAG------ ---------- ----AACAAA C.AC.CAGAC .GG .. ATGTC .'1' •• TA--.A .. C.C .. AG. GCAG------ ---------- ----.ACAAA C.GCAT.GGC .GG .. ATGTC .C.AT-.T.C ....• A-- . . • CCGC.T.GA TCAGTG---- ---------G CGGCAGT •• A C.T------- ---------.C.TTC .•. C .G .. GA--T . . . TG ... GGA TTT.GG---- ---------C CTT.GGCT.A C.TTCT.G.C CTT.CCT.TC .CATT.CC.C .. C.G ... GG A.TTCAG.AG '1'--------- ---------- --G.A.T.G. C.GAACT .. C .... GAT .•. 81 91 101 III 121 131 141 151 ATTATTCACC CGTGTC-TTT TGCGTACTTC TTG-TTTCCT TGGTGGGCTC GCCCACCACA AGGACAAACC A-TAAACC-T ................ -

'1'

................ -

. . . . . . . . . . . '1'

.......... ---TA.T ---TA.T TGAT.CTG TGAT.CT CG---. '1'

.A '1' '1'

A A A

-

-

'1'- .."

T-.A

'1''1''1'

AR6

ABll AA6 PH OHU OKU Leroy Unity Ldol ABc2 AR6 ABll AA6 PH OHU OKU Leroy Unity Ldol ABc2 AR6 AB11 AA6 PH OHU OKU Leroy Unity Ldol

C

-CT A.. -CT A.. AT AT AGT T

G.. C G A C A.CA

'1' '1'

'1'

- -A

- •

-.

C C .. A. CA '1'. A CC.T .'1' -A CC . . . T.G -AT '1' -TCC .. TAA .. C.A-T TG A --C.AT TCA .. C.A-ACT TTGTG .. C.T .C.GG. '1'.'1' • • A.CC -'1' '1' '1'

G G TG

-.

161 171 181 191 201 211 221 231 TTTGTAATTG CAATCAGCGT CAGT-AACAA CATAATAATT ACAACTTTCA ACAACGGATC TCTTGGTTCT GGCATCGATG .....••••• A .•..•.•..

... '1' .. '1' •• '1' .. . A.T •. '1' .. '1' '1' . C .. . C G . C •••••.••..• G ••.••••

C ... A.T . . . . . G .. CATAG

.•.• - •.•.• A •••••••••

....•.•.• .. .. A..... .T.AAT.A .. . T.AAT.AC. .... A.CACT ...• A.CA.T TCTGA .. A.T

AT .......• TG .... '1' . ATA.T. ATA.T- ..•• GTA.TA . GTA.T. A . .. ...... '1'.

241 251 261 271 281 291 301 311 AAGAACGCAG CGAAATGCGA TAAGTAGTGT GAATTGCAGA ATTCAGTGAA TCATCGAATC TTTGAACGCA CATTGCGCCC

ABll AA6 PH OHU OKU Leroy Unity Ldol ABc2

A A

-

'1'

TA

.. C . . . . . . .

321 331 341 351 361 371 381 391 TTTGGTATTC CAAAGGGCAT GCCTGTTCGA GCGTCATTTG TACCCTCAAG CTTTGCTTGG TGTTGGGCGT C-TT---GTC

TG TG TG TG TG

C C

c

C C

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '1' .. --- ... .... '1' C '1' .. TTG.CTAC .. .. .. '1' A '1' .. TTG.CCAC .. ............ C '1' .. TTG.TC.---

.. .. .. . ..

. . . . . • . . . . . . . . • . . . . . . . . . . . . T.A ATG.TCTC.G . . . . . . . . .. . T.A TTG .CCGCG.

401 411 421 431 441 451 461 471 TCCAG-TTTG CTGGAGACTC GCCTTAAAGT CATTGGCAGC CGGCCTACTG GTTTCGGAGC GCAGCACAAG TC--GCGCTC . . • • . • • • • • • • • • . • • . . • . . • • • • . • . A •.•.••••..••••••••••...•••.•••••.••••••

.. '1' .. .. T .. C • . '1'------- .AC

A. G. TTG. . . .. . .....•... G.CTG C .. T.G G.. AC GACTG G AC '1' GC.C TC

CA. T----GC CA.T----GC --------CA .G. TTGCGCA .-----

. A . G....•.... A.•...•..• A .. A . A . A .

.. A .••••••

.TATA.-T .. • • •• -'1' •••• .TATA.- ... .... -TA ... ... . AC.T .. .CC. - •••••

.... A .. T ..

• CC. - ••.••

.... A.GT.A .CC.- .....

..-- .. A ... .. . . . . . . . '1' •• '1''1' •• A .. T .'1''1''1' ••• TCT .'1''1''1' ••• TCT •'1''1''1' ••• -c . .'1''1''1' ••• -C . .'1''1''1' ••• -CA

481 491 501 511 521 TCTTCCAGCC AAGGTC-AGC ATCCATAAAG ----CC-TTT TTTCAACTTT A C.... . . C ---- .. '1' .. A - .. '1' AT A '1' '1' ---- .. '1' .. .GAAT '1''1' TG C ACCA.AT .. .AC.G.CAGT T.TA.AGC AT ---- .. '1' A -.G .. .AC.G.TAGT '1'.'1' .. GGC '1' ---- .. TC A - .G .. GTCATG GTT .. TG-.. . C ----A.AC.. . .. A.G .. C. G. TATG . '1''1' •• TG-.. . C ----TTA... .A. T ...• C. C G.T.G. GGT .. TG- .. CC C ----T.CA.A .A.TGG .• C.

Fig. 1. Alignment of the sequences of the 5'8s rONA with the flanking internal transcribed spacers ITS1 and ITS2. This alignment was generated by the multiple alignment program

PILEUP

using a gap weight of 3'0 and a gaplength weight of 0'1 with a small revision by

eye from bp 469 to 497. ITS1 spans from bp 1 to 201; the 5'8s coding region is from bp 202 to 358; and ITS2 is from bp 359 to 529. A hyphen represents a gap and a period represents a base identical to that of the top sequence. The abbreviation for each fungal species and isolate name, the corresponding GenBank accession number, and source of each sequence are as follows: ABc2 for

brassicicoia

isolate ABc2 (U05198. this study); AR6 for

A. raphani

isolate AR6 (U05200, this study); ABU for

A. brassicae

Alternaria

isolate ABU

A. alternata isolate AA6 (U05195, this study); PH for Pleospora herbarum isolate DAOM 150679 Ophiosphaerella herpotricha (U04861; Tisserat, Hulbert & Sauer, 1994); OKU for 0. korrae (U04862; Tisserat et al., 1994); Leroy for Leptosphaeria maculans isolate Leroy (M96384; Morales et al., 1993); Unity for Leptosphaeria maculans isolate Unity (M96383; Morales et al., 1993); Ldol for L. doliolum (U04207, Morales et al., 1995).

(U05253, this study); AA6 for

(U05202, this study); OHU for

Nuclear rRNAs of Alternaria species on crucifers informative, while ITS2 had 91 (17'2%) of which 72 (13'6%) were informative. In contrast, the sequence of the 5'8s rDNA was highly conserved across all the species and contained only five variable sites (0'9%) of which three (0'6%) were informative. This sequence was identical in all four species of Alternaria, and differed in Pleospora herbarum at only one base pair from that of the Alternaria species. The 18s rDNA sequence was also highly conserved. Across all species in the alignment shown in Fig. 2, there are 260 variable sites (14'7%) of which 114 (6'5 %) are phylogenetically informative, out of a possible 1765 sites. Most of the variability in the 18s rDNA sequences appeared to correlate with genus. All 2295 bp of sequence for the 18s rDNA, ITSI, 5'8s rDNA, and ITS2 combined contains a total of 311 variable sites (13'5 %) of which 199 (8'7%) are phylogenetically informative. Aureobasidium pullanans (de Bary) G. Arnaud and Aspergillus fumigatus Fr. were not included in this combined data set because we had only the sequence for the 18s rDNA; Neurospora crassa was also eliminated since it was not used in the analysis of the 530 bp data set.

Phylogenetic analyses of ITS], the 5'8s rDNA and ITS2 aligned sequences (530 hpj One most parsimonious tree, requiring 526 changes (CI = 0'821, HI = 0'179, RI = 0'776), was found for the original data set composed of the 5'8s rDNA and both internal transcribed spacer sequences (Fig. 3). The consensus tree generated by global parsimony of bootstrap replications of this data set had an identical topology to that of the rnost parsimonious tree and indicated that the majority of the branches were favoured at a high level of confidence. All of the Alternaria species formed a clade which was highly significant (98 %). The two Ophiosphaerella species were sister to the Leptosphaeria species. The topology of the best fit maximum likelihood tree was the same, except for the position of P. herbarum. Pleospora herbarum was basal to the Alternaria clade in the bootstrap consensus tree, but was placed inside the Alternaria clade as a sister taxon to A. alternata in the best fit maximum likelihood tree. These two trees were significantly different according to the Kishino & Hasegawa test (1989) (log likelihoods = - 2594'65 and - 2583'93, respectively; S.D. = 10'04).

Phylogenetic analysis of the 18s rDNA aligned sequences (1765 hpj Three equally parsimonious trees, each requiring 33 I changes (CI = 0'879, HI = 0'121, RI = 0'859), were estimated from the original 18s rDNA data set. One of the trees was identical in topology to that of the consensus tree (Fig. 4) generated by global parsimony of bootstrap replications of the data set. The other two trees varied slightly in the branching patterns within the Alternaria clade. The best fit maximum likelihood tree (not shown) contained the same major groups as the consensus tree, but there were slightly different branching patterns of species within the Alternaria clade and also within the Leptosphaeria group. However, according to the Kishino & Hasegawa test (1989) the bootstrap consensus tree and the

608 best fit maximum likelihood tree are not significantly different (log likelihood = -4225'40 and -4231'69, respectively; S.D. = 7'71). Resolution in the consensus tree was fairly robust (87-100%) at the genus level, but weaker among species within a particular genus. The position of Pleospora herbarum was confirmed as being basal to the Alternaria clade. Pleospora herbarum occurred outside of Alternaria in all 1000 trees used to generate the bootstrap consensus tree, and the Kishino & Hasegawa test (1989) showed that trees with P. herbarum placed inside the Alternaria clade were Significantly worse than trees in which P. herbarum was placed basal to the Alternaria clade (log likelihood = -4231'69 and -4272'55, respectively; S.D. = 16'27). Pleospora herbarum is more closely related to Alternaria than to Leptosphaeria.

Genetic distances A matrix of Kimura (1980) genetic distances between pairs of fungal species and estimated from the entire 2295 bp of sequence data is given in Table 4. The Jin & Nei (1990) model, which uses the gamma distribution to estimate the change in the rate of substitution at different sites, gave very similar genetic distances for runs with different coefficients of variation (data not shown), and these values were also very similar to those obtained with the Kimura (1980) model. The genetic distance between pairs of Alternaria species is quite small. The genetic distance between P. herbarum and any of the Alternaria species falls within the same range as that between the two isolates of L. maculans.

DISCUSSION Currently taxonomists consider Pleospora and Leptosphaeria to be in separate loculoascomycete families, the Pleosporaceae and the Leptosphaeriaceae, respectively (Barr, 1987; Eriksson & Hawksworth, 1991), with the main differential criterion being the conidiomatal structure of the anamorphs. Leptosphaeria species have coelomycetous anamorphs, while genera in the Pleosporaceae have hyphomycetous anamorphs (Barr, 1987). The phylogenetic trees (Figs 3, 4) based on rDNA sequences support this taxonomic view. All the Loculoascomycetes included in the analyses, i.e. Leptosphaeria and Pleospora, as well as Alternaria, formed a phyletic group. Within this group there were two major lineages: the Leptosphaeriaceae composed of Leptosphaeria, and the Pleosporaceae composed of Pleospora and Alternaria. The two Ophiosphaerella species, which were included only in the analysis based on the 5'8s rDNA and flanking internal transcribed spacers, fell within the Leptosphaeria group (Fig. 3). Ophiosphaerella korrae (J. C. Walker & A. M. Smith) Shoemaker & C. E. Babc. has been placed in Leptosphaeria (J. C. Walker & A. M. Sm., 1972), while O. herpotricha (Fr.: Fr.) J. c. Walker was previously in Phaeosphaeria (Holm, 1957), a segregate of Leptosphaeria (Holm, 1957). For further discussion of the phylogeny of Leptosphaeria, see Morales et al. (1995). Most species of Alternaria, including the four species used in this study, lack a known sexual stage. Alternaria and

609

Claudia A. Jasalavich and others ABc2 AR6 AA6 ABll PH Ldo1 Leroy Unity Aureo Aspf Neuro ABc 2 AR6 AA6 ABll PH Ldo1 Leroy Unity Aureo Aspf Neuro ABc 2 AR6 AA6 ABll PH Ldo1 Leroy Unity Aureo Aspf Neuro ABc 2 AR6 AA6 ABll PH Ldo1 Leroy Unity Aureo Aspf Neuro ABc2 AR6 AA6 ABll

1 11 21 31 41 51 61 71 ATATGCTTGT CTCAAAGATT AAGCCATGCA TGTCTAAGTA TAAGCAA-TT ATACCGTGAA ACTGCGAATG GCTCATTAAA

..•... • -C . •..• G ••••• .•••.•• T • • • • . • G ••••.

. . . . . . . . . T .•••.••

••

• A .•• • C •••

81 91 101 III 121 131 141 151 TCAGTTATCG TTTATTTGAT AATACCTTAC TACTTGGATA ACCGTGGTAA TTCTAGAGCT AATACATGCT GAAAATCCCG ......... A

•..•.•. . A.

.G . .G . .G ••.••••. .G . .G ......•.

.. . A

•G •••••••.

.. •A •...••••••••••..

. A

C ..• A C ..•.

A ..•. C .•• A

C.T

A •••. C ... A A •.•• C.T .. A •• •• C . . . .

.

161 171 181 191 201 211 221 231 ACTTCGGAAG GGATGTGTTT ATTAGATAAA AAACCAATGC CCTTCGGGGC TTTTTGGTGA TTCATGATAA CTTTACGGAT

. . . . . . . G ..

• .G ••. A •••

... .... C ••

· .G ••. A •••

. . . . . . . C .. . . . . . . . C ••

• .G ••• A ••• · .G ... A ..• • .G ••• A ••.

• • C •••••••

....... C ..

· .G ... A ..•

• .C •..•••.

.. C ....... .CC .••...• • •.•• A ••..•• AA ... A •. .CC .••...• A •••• A • • • . • • • A ••• A ••

.AAC

. . . . . . . T •.

.

. ••.• A • • . • • . • CT .. A ..

241 251 261 271 281 291 301 311 CGCATAGCCT TGCGCTGGCG ACGGTTCATT CAAATTTCTG CCCTATCAAC TTTCGATGGT AAGGTATTGG CTTACCATGG

..... G.... ..... G •••• •••• • G •••• ••••• G .... •••• •G •••• . . . . . G ••••

..... C ..... C •••• •C T ..... C ••••• T

. .. . .

..........

..

•T

. . .

G.A .. G • . . . C •.••.••. G.A .. G • • . . C •••..••• C.. C TG C CAG .

321 331 341 351 361 371 381 391 TTTCAACGGG TAACGGGGAA TTAGGGTTCG ATTCCGGAGA GGGAGCCTGA GAAACGGCTA CCACATCCAA GGAAGGCAGC

PH

Ldo1 Leroy Unity Aureo Aspf Neuro ABc 2 AR6 AA6 ABll PH Ldol Leroy Unity Aureo Aspf Neuro ABc2 AR6 AA6 ABll

.A .GG

.GA

T

A.GG

C

CC

A

. .

.

.T

.

401 411 421 431 441 451 461 471 AGGCGCGCAA ATTACCCAAT CCCGACACGG GGAGGTAGTG ACAATAAATA CTGATACAGG GCTCTTTTGG GTCTTGTAAT

....... G ..

• ... C .....

481 491 501 511 521 531 541 551 TGGAATGAGT ACAATTTAAA CCTCTTAACG AGGAACAATT GGAGGGCAAG TCTGGTGCCA GCAGCCGCGG TAATTCCAGC

PH

Ldo1 Leroy Unity Aureo Aspf Neuro Fig. 2. For caption see p. 612.

.......... T.C ..... C.... T.C .......... T.C

.. . .

Nuclear rRNAs of Alternaria species on crucifers

610

561 571 581 591 601 611 621 631 TCCAATAGCG TATATTAAAG TTGTTGCAGT TAAAAAGCTC GTAGTTGAAA CTTGGGCCTG GCTGGCGGGT CCGCCTCACC

ABc2 AR6 AA6

ABll

PH Ldo1 Leroy Unity Aureo Aspf Neuro

. .C

.

. ..... T ••. . . . . . . T .••

. . . . . . T ••. •..•••..• C ...••.•••• ...••.•.• C ••••.. T ... .•.••...• C •••••.• TC .

..... . AG ..

...... A ... •.••. •A .•• .. .... C . ...... C

.

· .C.T.- ..

641 651 661 671 681 691 701 711 GCGTGCACTC GTCCGGCCGG GCC-TTCCTT CTGAAGAACC TCATGCCCTT CACTGGGCGT GCTGGGGAAT CAGGACTTTT

ABc2 AR6

AA6

ABll

PH Ldol Leroy Unity Aureo

Aspf Neuro

. . . . . T ... T '"

..

•. T •.• T

..... T

..... T

. . .-

.

... -

.

.

T

..

G

. .T .T T ......... .T .T .T T

... G

.

...G

.

• •. G

..

.. ..

-.C

-.C -.C

.. ... T ...... ... GG .. G.. G . .TC C ... A.T G T .. A .• T •.•.•. •• •GG ••••. . G CT .. · -G .....• C ......... G ACTG .. T ... .. . T .. TT.C • .. G ...... G •••••••••••••••• T •• .TC C

721 731 741 751 761 771 781 791 ACTTTGAAAA AATTAGAGTG TTCAAAGCAG GCCTTTGCTC GAATACGTTA GCATGGAATA ATAAAATAGG GCGTGCGTTT

ABc2 AR6

AA6 ABll PH Ldol Leroy Unity Aureo Aspf Neuro ABc2 AR6 AA6 ABll PH Ldo1 Leroy Unity Aureo Aspf Neuro

••••••••••

... G

. . CG

C

••••••••••

••••••••••

••••••••••

C .. • TC. C •.... . A

A A A

G.C G.C G.C

... G

A

T.G ..

., .G •.••.• A•..•. • G •• . .. G•••••• A•••••• G••

••••• . A •••

A ..

A

· .. G · .. G ... G

GTAC ..

801 811 821 831 841 851 861 871 CTATTTTGTT GGTTTCTAGA GACGCCGCAA TGATTAACAG GAACAGTCGG GGGCATCAGT ATTCAGTTGT CAGAGGTGAA .... C .....

......... G AC ..... T.. •••..•••• G !'.C .••.. T ..

......... G AC . . . . . . . . . G AC ......... G AC ......... G AC

....... T .•...• . T ....... T

G G G

T.. T.. ....... T T.. ..... .. T T.. . . . . . . . T

. .... A .... . •..• A ••.. • •••• A ••.• .Y ... A ....

.. . ..

G.T G.T

.. .

G

..

.... G .....

. ••••• C •••

.. ... A....

881 891 901 911 921 931 941 951 ATTCTTGGAT TTACTGAAGA CTAACTACTG CGAAAGCATT TGCCAAGGAT GTTTTCATTA ATCAGTGAAC GAAAGTTAGG

ABc2 AR6

AA6

ABU

.... G.....

PH Ldol Leroy unity Aureo Aspf Neuro ABc2

... T

..

... T

..

... T

.

.• G

.

.•• T

. C .........

.. . T •.•...

. .... G....

961 971 981 991 1001 1011 1021 1031 GGATCGAAGA CGATCAGATA CCGTCGTAGT CTTAACCGTA AACTATGCCG ACTAGGGATC GGGCGATGTT CTTTTTCTGA

AR6

AA6 ABll PH Ldol Leroy Unity Aureo Aspf Neuro ABc 2 AR6 AA6

.................... A.CA .. T .. ............... G TC .A.GA .

. . . . . . . A.. . . . . . . . A ..

.•.... . A .•

.T

A

- .•.

1041 1051 1061 1071 1081 1091 1101 1111 CTCGCTCGGC ACCTTACGAG AAATCAAAGT TTTTGGGTTC TGGGGGGATT ATGGTCGCAA GGCTGAAACT TAAAGAAATT

ABll

PH Ldol Leroy Unity Aureo Aspf Neuro

A .. G

........ G •

........ G.

C .C . C . . T . . . . • . . . . . • • . • T •..••.•. A. G

Fig. 2. For caption see p. 611.

.

........ G • . . . . . . . . G. •••••••• G •

.. C .. CT

G•

611

Claudia A. Jasalavich and others

ABc2

AR6 AA6

1121 1131 1141 1151 1161 1171 1181 1191 GACGGAAGGT CACCACCAOO CGTGGAGCCT GCGGCTTAAT TTGACTCAAC ACGGGGAAAC TCACCAOOTC CAGATGAAAT

ABll

PH Ldol Leroy Unity Aureo Aspf Neuro ABc2

AR6 AA6

••••••••• G

.••..••.. G

••.••...• G

.... CAC ... .... CA ...• . ... CACG ..

•......•• G

•••••••• • G •••••• A ••• •..•.•.• •G G .........

1201 1211 1221 1231 1241 1251 1261 1271 AAGGATTGAC AGATTGAGAG CTCTTTCTTG ATTTTTCAoo TGGTGGTGCA TOOCCGTTCT TAGTTCGTGG GGTGACTTGT ..• A ••.•••

ABll

PH Ldol Leroy Unity Aureo Aspf Neuro

..... G ..... G ..... G ..... G

.... . GTG .. . . C... TG.A ... . CGTG ..

G .........

.. . . ..

A A A A

T •••• T . T . .. T

••••• G •••• A

T T

..... G .... A

. ..

1281 1291 1301 1311 1321 1331 1341 1351 CTGCTTAATT GCGATAACGA GCGAGACCTT ACTCTGCTAA ATAGCCAGGC TAGCTTTOOC TOOTCGCCGG CTTCTTAGAG

ABc2

AR6 AA6

.• A ..•.•. T

ABll

PH Ldol Leroy Unity Aureo Aspf Neuro

A A A A A A

TC . .. .... T ... AC . AC .. AC . ... C CC G T C GGC.-CT . ..... . C.. T CC .. A.. T .. G.. C AC . ..... . C.TA .T A.TA T

.

G. .

1361 1371 1381 1391 1401 1411 1421 1431 AGACTATCAA CTCAAGTTGA TGGAAGTTTG AGGCAATAAC AooTCTGTGA TGCCCTTAGA TGTTCTGGGC CGCACGCGCG

ABc 2

AR6 AA6

ABll

PH Ldol Leroy unity Aureo Aspf Neuro ABc 2

AR6 AA6

G G G

G G

G

oo oo oo GG oo GG

. . . . . . . TT. . . . . . . . TT.

PH Ldol Leroy Unity Aureo Aspf Neuro

AR6 AA6

.

1441 1451 1461 1471 1481 1491 1501 1511 CTACACTGAC AGAGCCAACG AGTTCTTCAC CTTGTTCGAA AGAATTGGGT AATCTTGTTA AACTCTGTCG TGCTGGGGAT

ABll

ABc 2

CC .. CC .. CC .. CC .. CC .. ..... .. GC. C CC ..

.... GC GTC . '" .AC GTC . .... AC GTC . .... AC GTC .. ............... A.TT . ... . CC .. G ••• GG •••••• •• G •••• G ••••• A.A •••• ... . GC ... G .. GTC . • C •.••• G•.•.• A •• --c. .... GC .. G. . . GTCC .

• .. C .. .. G

.. .

1521 1531 1541 1551 1561 1571 1581 1591 AAAGCATTGC AATTATTGCT TTTCAACGAG GAATGCCTAG TAAGCGCGTG TCATCAGCAT GCGTTGATTA CGTCCCTGAC

ABll

PH Ldol Leroy unity Aureo Aspf Neuro ABc 2

AR6 AA6

.G •....... .G •....... .G . .G ••••.••. .G . .G •••..••.

c

..

C

..

C

..

c c c

. .

.. .......... . TAC .. .......... .. G.. A.. A . ... . C AA

G TC .T.CC

. .

T

.

C. C.

• •.•.•. . C.

........ c. ........ c. ........ C.

1601 1611 1621 1631 1641 1651 1661 1671 CTTTGTACAC ACCGCCCGTC GCTACTACCG ATTGAATGGC TCAGTGAGGC CTTCOOACTG GCTCGOOGAG GTTOOCAACG .••.• A ••••

ABll

PH Ldol Leroy Unity Aureo Aspf Neuro Fig. 2. For caption see p. 612.

••••• A ••••

.G •• G

G . .. C T .. A C .. ........................C . •••••.....••••• A . . • • . • . • . . • . . •

.. .......... .

•.....•.•.

.. C.A .•. . A

T.C ......... C.A

GA

C

.

C

.

.

Nuclear rRNAs of Alternaria species on crucifers

ABe 2

AR6 AA6

ABU

PH Ldol Leroy

Unity

Aureo Aspf

Neuro

612

1681 1691 1701 1711 1721 1731 1741 1751 ACCACCCCAA GCCGGAAAGT T-CGCCAAAC TCGGTCATTT AGAGGAAGTA AAAGTCGTAA CAAGGTCTCC GTAGGTGAAC . . . . . . . . . . . . . . . . . . . . . - .. T ...... T . ...... T . .- .. T .....

.... . T.TG. ...... . TG. ..... . T.G. ..... .. AGG . . TC ... AG. ...... . AGG ••••••.•• C

.C .- •. T .- .• T

.-G.T .-G.T .-AT

. . ..

.. . .

..

.. .... T ...

. .•••. T •••

.. c c

.. •••• T ...

.. ..

• .

.... c.....

T ••• T ...

.. T . . . . . . .

1761 ABe 2 CTGCG AR6 AA6 ABU PH

Ldol Leroy Unity

Aureo

•••• A

Neuro

.A ...

Aspf

•••• A

Fig. 2. Alignment of the 185 rONA sequences. This alignment was generated by the multiple alignment program PILEUP using a gap weight of 5'0 and a gaplength weight of 0'1. A hyphen represents a gap and a period represents a base identical to that of the top sequence. The abbreviation for each fungal species and isolate name, the corresponding GenBank accession, and source of each sequence are as follows: ABcl for Alternaria brassicicola isolate ABcl (U05197, this study); AR6 for A. raphani isolate AR6 (U05199, this study); AA6 for A. alternata isolate AA6 (U05194, this study); ABl1 for A. brassicae isolate ABl1 (U05196, this study); PH for Pleospora herbarurn isolate DAOM 150679 (U05201, this study); Ldol for Leptosphaeria doliolurn (U04205; Morales et aI., 1995); Leroy for L. rnaculans isolate Leroy (U04233; Morales et aI., 1995); Unity for L. rnuculans isolate Unity (U04238; Morales et aI., 1995); Aureo for Aureobasidiurn pullulans (M55639; Illingworth et aI., 1991); Aspf for Aspergillus furnigatus (M60300, unpublished); and Neuro for Neurospora crassa (X04971; Sogin, MioHo & Miller, 1986). Table 3. Length of the internal transcribed spacers

0·05

13 6 Alternaria brassicicola 99-4%

Length (bp)

Alternaria alternata Alternaria brassicae Alternaria brassicicola Alternaria raphani Pleospora herbarum Leptosphaeria dolio/um Leptosphaeria maculans (Leroy) Leptosphaeria maculans (Unity) Ophiosphaerella herpotricha Ophiosphaerella korrae Neurospora crassa

ITS1

ITS2

165 166 179 197 175

161 159 158 158 161

175

157

164 179

150 162 167 167 146

164 164 HI5

based on the 185 rDNA sequence data. Simmons (1967) considered the Alternaria, Stemphylium and Ulocladium to be closely related on the basis of conidial development, as they all produce dictyoporospores. Our data corroborate Simmons's view, since P. herbarum has a Stemphylium anamorph, and provide the first molecular evidence that asexual species of Alternaria are indeed related to Pleospora. Therefore, the anamorphic Alternaria should be included in the Pleosporaceae. Wiltshire (1947) considered A. raphani and A. brassicaeto be closely related. It would appear, however, that A. raphani is actually more closely related to A. brassicicola than to A. brassicae based on rDNA sequence data. Alternaria alternata, A. brassicae, A. brassicicola and A. raphani formed a strong clade of very closely related sister taxa. The 185 rDNA resolved two subclades of species within Alternaria at a level of

Alternaria alternata

98%

60 100%

41 85·8%

Alternaria raphani Alternaria brassicae

23

36 99-4%

L--

Pleospora, although distinct genera, are sister taxa (Fig. 4)

22

23

77 ---'---'

49 35

Leptosphaeria maculans (Leroy) Leptosphaeria maculans (Unity) Leptosphaeria doliolurn

Fig. 3. The consensus tree generated by global parsimony bootstrap analysis of the alignment of the 5'85 rONA and the flanking internal transcribed spacer (ITSI and ITS2) sequences shown in Fig. 1. The percentages represent the proportion of 1000 bootstrap replications in which the taxa to the right of the node were placed together by the program DNAPARS with randomization of the sequence input order; the other numbers represent the steps. Branch lengths (drawn in the horizontal dimension only) are the maximum likelihood estimates made by the program DNAML when the user tree was defined as the bootstrap consensus tree. The length of vertical lines has no meaning and was adjusted arbitrarily for ease in labelling termini. Leptosphaeria doliolurn was designated as the outgroup. confidence of 98 % (Fig. 4), based on five informative sites contained within the 300 bp at the 3'-end of the sequence alignment (Fig. 2). Complete resolution of the species of Alternaria was achieved with the ITS sequence data (Figs 1, 3) which were much more variable and contained more

613

Claudia A. ]asalavich and others Alternaria brassicicola

0·01 36 100% 40

100%

27

17

,----="-----

Aureobasidium pullulans 68

50.9%L-----....::..::'---------Aspergillus

fumigatus

'---------"'9-'-1---------Neurospora crassa

Fig. 4. The consensus tree generated by global parsimony bootstrap analysis of the alignment of the 18s rONA sequences shown in Fig. 2. The percentages represent the proportion of 1000 bootstrap replications in which the taxa to the right of the node were placed together by the program DNAPARS with randomization of the sequence input order; the other numbers represent the steps. Branch lengths (drawn in the horizontal dimension only) are the maximum likelihood estimates made by the program DNAML when the user tree was defined as the bootstrap consensus tree. The length of vertical lines has no meaning and was adjusted arbitrarily for ease in labelling termini. Neurospora crassa was designated as the outgroup. Table 4. Genetic distances between pairs of fungal species calculated by the Kimura two-parameter model from the combined sequence data for ITSl, the 5'85 rDNA, ITS2, and the 185 rDNA (2295 bp)

1 2 3 4 5 6 7 8

0'0053 0'0135 0'0131 0'0265 0'0725 0'0813 0'0856

2

3

4

5

6

7

0'0135 0'0126 0'0293 0'0723 0'0816 0'0871

0'0131 0'0238 0'0707 0'0822 0'0881

0'0298 0'0713 0'0797 0'0881

0'0731 0'0791 0'0868

0'0266 0'0552

0'0620

8

I, Alternaria brassicicola; 2, A. raphani; 3, A. alternata; 4, A. brassicae; 5, Pleospora herbarum; 6, Leptosphaeria maculans (Leroy); 7, L. maculans (Unity); 8, L. doliolum.

phylogenetically informative sites than the 18s rONA. The ITS sequence data of Leptosphaeria were even more variable than those of Alternaria (Fig. I). Likewise, the range of genetic distances among the Leptosphaeria species was larger than those seen among the Pleospora and Alternaria species (Table 4). The genetic distances between P. herbarum and any of the four Alternaria species examined were on the same order of magnitude as that between the highly virulent and weakly virulent isolates of L. maculans. These differences suggest that the genus Alternaria encompasses less genetic variability than does the genus Leptosphaeria. This may reflect the differences in size and reproductive strategies of the two genera. Alternaria comprises roughly 60 species (Rao, 1969; Rossman et al., 1987), while Leptosphaeria includes a heterogeneous mixture of about 1689 taxa (Crane & Shearer, 1991) and is very likely polyphyletic. Perhaps the four Alternaria species included in this study have separated from each other only recently in evolutionary time, and so have not yet accumulated

many changes in their rONA sequences. Alternatively, perhaps plant host association may exert a pressure against further divergence. Since all four Alternaria species used in this study were isolated from crucifers, it would be necessary to examine other Alternaria species isolated from plant hosts from different families to answer this question. Along with allowing us to determine the phylogenetic relationships among the Alternaria species, portions of the rONA sequence could have practical uses, e.g. production of diagnostic tools. The variability present in ITS1 could be exploited to design species-specific probes and primers in order to identify rapidly species of Alternaria both in culture and in plant materials. The authors thank Mr Barry Panchuk for automated sequencing of the DNA samples, Mr Don Schwab for synthesis of the oligonucleotide primers and Dr G. Allan Petrie for supplying fungal isolates. This research was partly funded by a Fellowship in a Canadian Government Laboratory to CA.]. This is Agriculture and Agri-Food Canada, Saskatoon Research Station Publication, No. 1112.

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Petrie, G. A (1969). Variability in Leptosphaeria maculans the cause of blackleg in rape. PhD. Thesis. University of Saskatchewan: Saskaloon, Saskatchewan, Canada. Rao, V. G. (1969). The genus Alternaria from India. Nova Hedwigia 17, 219-258.

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Mycologia 59, 67-92. (Accepted 27 September

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