Isolation and characterization of a β-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene

Gene, 124 (1993) 121-125 0 1993 Elsevier Science Publishers

B.V. All rights reserved.

121

0378-l 119/93/$06.00

GENE 06889

Isolation and characterization

of a P-tubulin-encoding Colletotrichum gloeosporioides f. sp. aeschynomene

gene from

(Fungal pathogen; recombinant DNA; codon usage; nucleotide sequence; BTub; TUB)

T. L. Buhr and M. B. Dickman Department of Plant Pathology, University of Nebraska. Lincoln, NE 68583-0722, Received by J.A. Gorman:

17 March

1992; Revised/Accepted:

USA

15 June; 20 August/22

August

1992; Received at publishers:

15 October

1992

SUMMARY

Colletotrichum gloeosporioides f. sp. aeschynomene (C.g.a.) is a fungal pathogen of legumes and is used as a commercial mycoherbicide for rice and soybeans. As an initial study to potentially improve the utility of this fungus and develop a gene transfer system, a l%tubulin @Tub)-encoding gene (TUBI) was isolated, cloned and sequenced. The coding sequence and deduced amino acid sequence of the C.g.a. TUB1 gene was highly homologous to the TUB1 gene of Colletotrichum graminicola. Southern hybridizations, using the C.g.a. TUB1 and C. graminicola TUB2 genes as probes, suggest that C.g.a. contains two TUB genes. Variation in both the restriction pattern and the number of TUB genes present in different formae specialis of C. gloeosporioides was evident. These observations are relevant for assessing relationships among formae specialis of C. gloeosporioides.

INTRODUCTION

The haploid, phytopathogenic fungus, C. gloeosporioides (Penz.) Sacc. f. sp. aeschynomene (C.g.a.), is the causal agent of anthracnose of Aeschynomene oirginica, commonly known as northern jointvetch (Daniel et al., 1973). C.g.a. is commercially marketed in some southern states as a mycoherbicide, COLLEGO, for biocontrol of northern jointvetch in rice and soybean fields. Northern jointvetch produces seeds which are difficult to separate Correspondence to: Dr. M. B. Dickman, ogy, 406 Plant Science Hall, University 68583-0722, USA. Tel. (402) 472-2849; MDICKMAN(&crcvms.unl.edu Abbreviations:

aa, amino

Department

of Plant

Pathol-

of Nebraska, Lincoln, NE Fax (402) 472-2853; e-mail

acid(s); A., Aspergillus; Be, benomyl;

bp, base

pair(s); C., Colletotrichum; C.g.a., C. gloeosporioides f. sp. aeschynomene; E., Escherichia; Exo III, E. coli exonuclease III; f. sp., forma specialis; ff., formae; kb, kilobase or 1000 bp; N., Neurospora; nt, nucleotide(s); S., Saccharomyces; SDS, sodium dodecyl sulfate; SSPE, 0.15 M NaCI/lO mM NaH,P04.H,0/l mM EDTA pH 7.4; gTub, 8-tubulin; TUB, gene(s) encoding gTub; wt, wild type.

from rice and if present, reduces its market value. Hostspecific infection by C.g.a. decreases competition of northern jointvetch with these crops. To increase the utility of C.g.a. as a biocontrol agent, it would be desirable to create a fungicide-resistant strain. Presumably, such a strain would be pathogenic on northern jointvetch but would be unaffected by the presence of the fungicide. If C.g.a. were resistant to a fungicide, the fungicide could be applied to control other fungal pathogens without inhibiting C.g.a. A clone of the fungicideresistance gene would be useful to monitor strains possessing this phenotype in population studies and for tracking the organism. Benomyl (Be) is a commonly used, effective fungicide (active ingredient: methyl-2-benzimidazole carbamate). Substantial evidence indicates that Be inhibits fungi via specific binding to the microtubule subunit, BTub (Osmani and Oakley, 1991). The majority of fungi, including S. cereuisiae (Neff et al., 1983), Candida albicans (Smith et al., 1988), and Neurospora crassa (Orbach et al., 1986) contain a single highly conserved gene for PTub, desig-

122 nated TUBZ. However, Aspergillus nidulans (May et al., 1987) and C. graminicolu (Panaccione and .Hanau, 1990) each contain a second, divergent gene encoding STub, designated tube and TUBl, respectively. The majority of mutations which confer Be resistance (reviewed by Leroux, 1991) have been shown to reside in PTub-encoding genes. Be-resistant TUB2 alleles from N. crassa and A. nidulans have been used as dominant selectable markers for transformation (Osmani and Oakley, 1991). A divergent TUB gene (TUBI) from a Be-resistant strain of C. gru~inico~~has also been used to transform the wt fungus to Be resistance (Panaccione et al., 1988). This study was initiated to isolate a BTub-encoding gene from wt C.g.a. as a preliminary step for development of a transformation system in this fungus. Since C.g.a. and C. graminicola are members of the same genus and both are phytopathogens, we reasoned that an analagous TUB1 gene from C.g.u. might be the most promising candidate. We report the nt sequence of a TUB gene and present evidence that C.g.u. contains at least two divergent TUB genes.

EXPERIMENTAL AND DISCUSSION

(a) Cloning A genomic library of wt C.g.u. was screened as described by Sambrook et al. (1989) with pCG7, which contains the C, grum~nico~u TUBI gene (Panaccione et al., 1988). Plaque hybridization revealed several strongly hybridizing clones. Three clones were isolated for further analysis and each contained similar restriction fragments which hybridized to pCG7 (data not shown). Digestion with Hind111 revealed a single hybridizing 2.9-kb fragment, which was subcloned into pUCll9 and designated pHWTTUB1 (Fig. 1). When probed under stringent conditions with pCGTUB2, which contains the TUB2 gene from C. gruminicolu (Panaccione and Hanau, 1990), none of the subclones revealed hybridizing fragments. Therefore, each phage clone appeared to contain a sequence with homology only to C. graminicolu TUBl.

(b) Sequence analysis The strategy for sequencing pHWTTUB1 is shown in Fig. 1. Sequential digestion of pHWTTUB1 with ExoIII produced a set of overlapping clones which were sequenced by dideoxynucleotide chain-termination (Sanger et al., 1977). The coding strand sequence was determined from these clones. Based on this sequence, primers complementary to the strand shown in Fig. 2 were synthesized, and used to sequence the bottom strand of pHWTTUI31. Comparison of the deduced aa sequence with C. grumin~colu STubs indicated that the subclone encoded a STub but was missing some of the 5’ coding region. An overlapping 2.8-kb Sal1 fragment from one of the phage clones was then subcloned (designated pSWTTUB1) and used to sequence the 5’ end of the TUB1 gene (Fig. 1). Two overlapping clones were sequenced after digestion of pSWTTUB1 with ExoIII. Complementary primers were synthesized and used to generate sequence for the second strand. To obtain a clone with a complete TUB1 gene, a 2.5kb NcoI-PstI fragment from one of the phage clones was subcloned into similarly digested pUC 120 (designated pNPWTTUB 1). Sequence alignment of the coding region with the C. grum~nico~a TUB1 showed 74.9% nt identity with C.g.a. TUB1 and strongly suggested that the entire gene had been cloned. Alignment of the coding region from C.g.a. TUB1 with the C. gruminicola TUB2 and N. crassa tub-2 genes showed 70.0% and 49.4% nt identity, respectively. From the high sequence homology between C.g.u. and C. gruminico~a TUB1 genes we inferred probable coding regions and intron splice junctions (Fig. 2). These introns have not yet been experimentally verified. However, all intron/exon borders are in good agreement with consensus splice sites (Ballance, 1986). When these ‘introns’ are spliced, one 1335 bp (445 aa) open reading frame is observed. Thus, the C.g.a. TUB1 gene appears to contain three introns concentrated in the 5’ region of the gene (Fig. 2). The C. graminicolu TUB1 and TUB2 genes (Panaccione and Hanau, 1990) and the N. crassa tub-2 gene

A

Fig. I. Restriction map of C.g.n. TUBI and flanking regions. Map coordinates are in kb. The relative position of the ATG and TAG codons are indicated below the map. pHWTTUB1 contains a Hind111 insert. The pSWTTUB1 clone contains a Sal1 insert and the pNPW~UB1 clone contains a NcoI-PstI insert. Arrows indicate the direction and extent of TUBI regions that were sequenced. The four arrows to the left of the 5’ Hind111 site indicate sequence obtained from pSWTTUB1. Restriction sites are designated as follows: A, ApaI;H, HindHI; N, NcoI; P, PstI; S, SalI; Sm, SmaI; St, StyI; x, XhoI.

123 MAGCGACWUGGGCGCCMCCGCATCGCCC~llTGTMTACAGGGTCCACC~CT~~TGTTGCGGCGT~CT~~TGGCCTGTCC~CCACG~~CA~TGTACC GCACGTGUCTCUGGCTTCTTeAAMCGGATAGGCAGTCGCTTACCGGGTGCAGG~lCCGTTT~~~CCGTGTCTGTGGTCTGGGCCCCTCCG~GTTTGGGCC~CGGCGG AGGGGCCGAGCAGTCCGWCGT~GGGGGTAGGCATTTTCGGCGTTM~GGCGTGTTGTTTCGGCMTGGTACTT~T~TGGT~TATGTCCCTG~TACG~~T~~C ACTGGTTCTGTGTACCCCCTTTCCATCATTTCAGTCMGCGTTCCTGCCTTCATC ATG CGT GAG ATT GTAAGTGTTGCGTCGTCATTGTCGTAAGCTGGGAAAAGTGTGAGTAAG Met Arg Glu Ilc CCTTACTCACGAATGTCATCAACAG ATC CAC CTC CAG ACC GGT CM TGT GTAAGTCGCTGTCTGTCCTMTTGATCACTTTATT~GGCTTGATACAG GGC MC Ile His Leu Gln Thr Gly Gln Cys Gly Asn CM GTC CGA ACT CCC TTT TG GTATGTCCCCTMATGTTCCCCTGAGACCCAA~ACCGAATCACCGMCAG G CAG ACA ATC CAT CAT GAA CAT CCC CTG Gln Vet Gly Thr Ala Phe Tr p Gln Thr Ilc His His Glu His Gly Leu WC CAC GAT GGT TAT TTC CGC GGC GM TCC ACG CM CAG TCA WC CCC CTG AGC GTC TIC TTC CCC GAA CCC TCC MC AAC AM TAC GTC Asp Wis Asp Gly Tyr Phc Arg Gly Glu SW Thr Gln GLn Ser Asp Arg Leu Ser Val Tyr Phc Ale Glu Ala SW Am Am Lys Tyr Val CCC CCC CCC GTG CTC GTG GAC CTC GAG CCG GCC ACA ATG GAC GCG ATC CCC TGC CCC CCC CTG CCC MC TTC TTC CGT CCC GAT MC ATG Pro Arg Ale Vat Leu Vet Asp Leu Glu Pro Ala Thr Met Asp Ale Ile Arg Scr Gly Pro Leu Gly Am Phe Phe Arg Pro Asp Am Met GTT WC GGC CAG TCC CCC GCG CCC MT MC TGG GCC MC CCC CAC TAT ACC GAA GGC CCC GAA CTG GTG GAC CM GTC CTG GAC GTC GTG Val His Gly Gln Scr Gly Ala Gly Asn Am Trp Ala Lys Gly His Tyr Thr Glu Gly Ala Glu Leu Vat Asp Gln Val Leu Asp Val Val CGG CCC GAG CCC GAG ACG TGC GAT TCC TTG CAG GGC TTC CAG ATC ACG CAC TCG CTG CCC CCC CGA ACG GGC TCG CCC ATG GGC ACC CTG Arg Arg Glu Ala Glu Thr Cys Asp Ser Leu Gln Gly Phe Gin Ile Thr His Ser Leu Gly Gly Gly Thr Gly Ser Gly net Gly Thr Leu CTC ATC CCC AAG GTG AGA GAA GAG TTC CCG GAC CCC ATG ATG GCT ACT TTT TCG GTT CTG CCC TCG CCC AM GTG TCG GAG GTC GTC GTT Lw Ile Ala Lys Val Arg Glu Glu Phe Pro Asp Arg Met Met Ala Thr Phe Ser Val Leu Pro Ser Pro Lys Vat Ser Gtu Val Val Val GAG CCA TAC MT GCG ACG CTA TCG GTC CAT CM CTT GTC GAA MC AGC GAC GAG ACG TTT TGT ATC GAT MC GAA GCA TTG TIC CAT ATC Glu Pro Tyr Am Ala Thr Leu Ser Val His Gln Lw Val Glu Am Ser Asp Glu Thr Phe Cys Ile Asp Am Glu Ala Leu Tyr Asp Ile TGT CCC CCC ACG CTG AAG CAG CCC CAT CCT TCG TAT GGG GAT CTG MT MC CTG GTA TCG AGG GTG ATG TCC CCC CTG ACG ACG CGA TTC Cys Arg Arg Thr Leu Lys Gln Ala His Pro SW Tyr Gly Asp Leu Am Lys Leu Vel Ser Arg Vsl Met Ser Gly Lw Thr Thr Gly Phe GCG TTC CCC GGG CAG CTG AAT CCC CAT CTG AGG MG TTG CCC GTG MT CTT GTG CCA TTT CCT AGA TTG CAT TTC TTC ACG GTT CCC TTC Arg Phe Pro Gly Gln Leu Am Ala Asp Leu Arg Lys Leu Ala Val Am Leu Val Pro Phe Pro Arg Leu His Phe Phe Thr Vat Gly Phe CCC CCG TTG ACA ACT CCC GCG GCG TAC CAG MT CTC CCC GTG CCC GAG TTG ACG CAG CAG ATG TTT CAT CCC MC MC GTT ATG TCG GCG Ale Pro Leu Thr Thr Ala Ala Ala Tyr Gln Asn Leu Gly Val Ala Glu Leu Thr Gin Gln Met Phe Asp Pro Lys Am Val Met Ser Ala TCA GAT TTC CCC MC GGG CCC TTC TTG ACT TGC TCT GCG ATC TAC CCC GGA MC GTG TCG ACC AAG CAG ATT GAG GAG CAG ATT CGG CCC Ser Asp Phe Arg Am Gly Arg Phe Leu Thr Cys Ser Ala Ile Tyr Arg Gly Lys Vat SW Thr Lys Gln Ile Glu Glu Gin Ile Arg Gly GTG CAG CCC MC MC TCG GCA TAC TTC GTG GM TGG ATT CCC AAC AAT GTG CM ACT GCG CAC TGT TCT ATC CCA CCT GTT CGA ATG AAT Vet Gln Ala Lys Asn Ser Ala Tyr Phe Val Glu Trp Ile Pro Am Am Val Gln Thr Ala His Cys Ser Ile Pro Pro Vat Gly Met Asn GCT TCC TCG ACT TTC ATC CGA MT TCA ACG CCC ATT CAG GAC ATT TCC AGG AGG GTT GGG GAC CAG TTT AGC GTC ATG TTT CCC AGG MC Ala Ser Ser Thr Phe Ile Gly Am Ser Thr Ala Ile Gln Asp Ile Phe Arg Arg Val Gly Asp Gln Phe Ser Vat Met Phc Arg Arg Lys GCT TTC TTG CAT TGG TAC ACT GGG GM CCC ATG CAT GAG ATG GAG TTC ACG GAG CCC GAG TCC MC ATG MT WIT CTC GTG TCA GAA TAT Ala Phe Leu His Trp Tyr Thr Gly GLu Gly Met Asp Glu net Glu Phe Thr Glu Ala Glu Ser Asn Met Am Asp Leu Val Ser Glu Tyr CAG CAG TAC CAG GAT CCC GGT ATG CAT GAT GAC GAA CCC GM GM GCA TAT GAG GAA GAG GAG CCC GTG GAG GAG TAG ATGGATTTCATGTGA Gin Gln Tyr Gln Asp Ale Gly Met Asp Asp Asp Glu Ala Glu Glu Ala Tyr Glu Glu Glu Glu Pro Val Glu Glu End TAATCTCACAGCTTTTATATCGAAACATCTCAGTACAGGAGTAGTTTTCCCTCATMTTTCATCGGCAGTACGCATGTMCTCCATTGC~GTCAG~TATG~CTCT~GTATCCCAG MCTCCTCTTCCTCCTCTTCCTCTTCGCTMGCACCACCACTGTAGACTCGATGCCGAGATCTTCAAGMGTTCCTCATATTGGGCCMGCGCTCCCGACTCTGGGCCGCAGCCACTTCG GCTGGCGTACATCTCTGACCCCAGATTGACTCGG~TATGACTGAMAACGGTCTGGACTCCGCATMGGACTCGAGCCGGCCT

Fig. 2. The nt sequence G&Bank

accession

of C.g.a.

TUBI

and deduced

aa sequence.

Internal

conserved

sequences

within

introns

are underlined.

-296 -176 -56 60 168 271 361 451 541 631 721 811 901 991 lOI31 1171 1261 1351 1441 1534 1654 1774 1859

This sequence

has

No. M90977.

(Orbach et al., 1986) contain identically positioned introns but each of these genes contain three additional introns. Furthermore, the introns in C.g.a. TUBZ contain internal conserved sequences (Fig. 2), which are present in other fungal introns (Orbach et al., 1986). The coding region had a G+ C content of 55.1%, and showed biased codon usage. Although 57 of the 61 sense codons were used, there was a strong preference for codons ending in G or C (71.3%). Codon bias in C.g.a. TUBZ is similar to that of C. graminicola TUBI, but less pronounced than that of C. graminicola TUB2 (Panaccione and Hanau, 1990), N. crassa tub-2 (Orbach et a!., 1986) or A. nidulans benA (May et al., 1987) genes. Eukaryotic transcription signals were not immediately apparent. A possible GC box begins at nt - 174 and a possible TATA box begins at nt -96 (Fig. 2). However, caution must be observed since many fungal genes including the C. graminicola TUB2 (Panaccione and Hanau, 1990), N. crassa tub-2 (Orbach et al., 1986) and A. nidulans benA and tubC (May et al., 1987) genes do not have typical eukaryotic transcription signals. Similarly the 3’ end does not show the AATAAA polyadenylation signal described in higher eukaryotes. This is generally not highly conserved in filamentous fungi (Ballance, 1986). Alignment of the deduced aa sequence indicated that the C.g.a. Tub1 protein had 87.4% amino acid identity to C. graminicola Tub1 protein, 78.5% identity to C. graminicola Tub2 protein, and 77.8% identity to N. crassa Tub2 protein (Fig. 3).

GTP binds to tubulin heterodimers and hydrolyzes during Tub polymerization (Jacobs et al., 1974). Photoaffinity labelling indicated that GTP binds to aa residues 60-69 of the BTub subunit (Linse and Mandelkow, 1988). Fig. 3 shows that aa residues 60-69 are conserved among the BTub indicated. Three other conserved regions are also likely involved in GTP binding (Cameron et al., 1990). Fig. 3 also shows the high degrees of conservation for the putative phosphate (aa 145-151) ribose (aa 183-186) and nucleic acid (aa 245-249)-binding sites. (c) Southern hybridizations Southern hybridizations strongly suggest the presence of two TUB genes in C.g.a. (Fig. 4 A, lane 1; 4 B, lane l), with one gene being highly homologous to C. graminicola TUB1 and another gene being highly homologous to C. graminicola TUBZ. Using pNPWTTUB1 as a probe, two Hind111 fragments of approximately 2.9 kb (3’ end of TUBI) and 10 kb (5’ end of TUBZ) from total wt DNA hybridized. Using pCGTUB2 as a probe, a single, Hind111 fragment of approximately 16 kb hybridized. Surprisingly, C.g.a. TUB1 and C. graminicola TUB2 DNA probes revealed marked differences in both restriction patterns and the number of TUB genes among ff. sp. of C. gloeosporioides (Fig. 4,A and B). Two ff sp., vicia sativa (lane 2) and malus (lane 5), contain similarly sized DNA fragments that hybridized only to the TUB2 probe and appear not to contain sequence with homology to the TUB1 gene. Three ff sp., aeschynomene (lane l), par-

124 CgeTUBl

NREIIHLPT~CCNGVGTAFVQTINHEHGLDWDGYFRGESTWSDRLSWFAEASNNKYVPRAVLVDLEPATIY)AIRSGPLGNFFRPDN~

CgTtJ81

G

a

SDE

v

E

cg n/82

V

I A

N SG

SN WY

T EL LE M

Nctub-2

V

I A

SC

AS WN

T EL LE RN

CgeTUBI

1

AKP

N N

G G

A

@TUB2

F

G

C

A

s I

Nctub-2

F

G

C

A

s I

QaTUBf

VA VA

FPL

II

I

Co TU82 I

Nctub-2

180 V Y

V

DT A

DT

270

II

M

LSN

H

A

v

CL

S

n

N

n

LSN

II

A

v VSL

S

n

N

APLTT--AAAYPNLGVAELTPGMFDPKNVMSASDFRNGRFLTCSAIYRGKVSTKOIEEGIRGVOAKNSAYFVEUIPNNVQTAHCSIPPVG S--S

C#TU81

SFS

I

L

D

THF

N

v

GAIKN

N

360 I

V

K

Cg TUB2

SRG HSFRAVS

P

NA

Y

F

AMDVDNN

N

S

L

R

Nctub-2

SRG HHFRAVS

P

NA

Y

F

NEVDHN

N

S

L

R

CgsTU87

MNASSTFIGNSTAlDDIFRRVGDGFSVMFRRKAFLHUYTGEG~EMEFTEAESNHNDLVSEYGGYDDAGMDDDEAEEAYEEEEPVEE*

CgTU8f

LDV

EYG

YED APAE

l

Cg TUB2

LKR

V

EL K

E

TA

V-

E E

YEDDAPLE

V”

Nctub-2

LKM

V

EL K I E

TA

V-

E E

YE

E”

NS

F F

A

EP~LSVHPLVENSDETFCIDNEALYDICRRTLKPAHPSYGDLNKLVSRVllSGLTTGFRFPC~LRKLAVNLVPFPRLHFFTVGF

CgTU81

CgeTU87

G G

VHGPSGAGNNUAKGWYTEGAELM)aVLDWRREAETU)SLOGFOITHSLGGGTG~IAIREEFPDRMATFSVLPSPKVSEVVV Y

CoTUBf

90 D FPL

L

APLEG

Fig. 3. Comparison of deduced aa sequences from C. gloeosporioides 1: sp. aeschynomene (Cga), C. graminicola (Cg) (Panaccione and Hanau, 1990) and N. crassa (NC) (Orbach et al., 1986) PTubs. Those aa common to all PTubs were omitted and dashes were introduced to maintain maximum aa homology.

Putative

(A> I

regions

that bind GTP

are underlined.

TUBI 2

3

4

5

Fig. 4. Southern hybridizations with different ff. sp. of CoUetotrichum gloeosporioides. (Panel A) Southern blot of HindHI-digested total DNA (I ug) from f. sp. aeschynomene (lane l), f. sp. vicia satiua (lane 2), f. sp. parthenocissus (lane 3), f. sp. ludwigia decurrens (lane 4), and f. sp. malus (lane 5) probed with pNPWTTUB1 (Fig. 1). Markers are in kb (left margin). (Panel B) Southern blot of HindIII-digested total DNA (1 ug) probed with pCGTUB2 (Panaccione and Hanau, 1990). Lanes are the same as described in panel A. Methods: DNA was isolated by the method of Panaccione et al., 1988. Digested DNA samples were electrophoresed on 0.7% agarose gels and transferred to nitrocellulose (Schleicher & Schuell) by the method of Southern, as described by Sambrook et al. (1989). DNA probes for hybridization were radio-labelled by nick translation (Rigby et al., 1977) to a specific activity of IO8 cpm/ug of DNA. Hybridizations were incubated at 42°C for 16-20 h in the presence of 50% formamide/S x Denhardt’s solution/S x SSPE/O.I% SDS/l00 ug denatured salmon sperm DNA per ml. Filters were washed three times in 6 x SSPE/O. 1% SDS, at room temp for 5 min each, and then 2 times in 0.2 x SSPE/O.I % SDS, at 65°C for 1 h each. Autoradiography was performed at - 70°C with Kodak X-OMAT film.

thenocissus (lane 3), and ludwigia decurrens (lane 4), hybridized with both probes, suggesting that each contains two TUB genes. C. g. f. sp. ludwigia decurrens differs considerably from C.g.a. in restriction patterns of both TUB

genes, but C.g.a. and C. g. f. sp. parthenocissus appear to be closely related. The larger DNA fragment hybridizing to TUBZ, and the single DNA fragment hybridizing to TUB2 off. sp. parthenocissus (lane 3) comigrate with similarly sized fragments from f. sp. aeschynomene. The 0.8kb difference in the 3’ end of the TUB1 genes from f. sp. aeschynomene (2.9-kb fragment; Fig. 4A, lane 1) and f. sp. parthenocissus (2.1-kb fragment; Fig. 4A, lane 3) could reflect an additional Hind111 site, or a deletion in the flanking 3’ region. Our data indicate that C.g.a. is more similar to C. graminicola than to some other C. gloeosporioides ff. sp. with respect to the TUB gene family, suggesting that C.g.a. is more closely related to C. graminicola than to some other members in the gloeosporioides species. While it is possible that these isolates contain TUB genes highly divergent from C.g.a. TUBl, it is unusual that members of the same species are different in genome organization of highly conserved genes such as TUB. Based on these results, we question the taxonomic criteria for the gloeosporioides species designation, and suggest taxonomical relationships be re-examined by other means (e.g., ribosomal DNA, mitochondrial DNA, vegetative compatibility). Strengthening this assessment of the genetic relatedness of these so-called formae specialis is the fact that vicia sativa and malus do not cross with C.g.a., whereas ludwigia decurrens does (parthenocissus has not been tested) (Cisar et al., 1991). Thus, by classical genetic criteria for a species designation, C. g. f. sp. vicia sativa and C. g. f. sp. malus are not the same biological species as C.g.a. It is noteworthy that TUB genes may be useful in the examination of genetic relatedness among fungal species.

125 (d) Conclusions (I) C.g.a. contains at least two divergent

Linse, K. and

TUB genes.

(2) TUB genes can serve as a nuclear marker for studies of genetic relatedness among Colletotrichum species.

Mandelkow,

E.M.: The GTP-binding

peptide

of S-tu-

bulin. J. Biol. Chem. 263 (1988) 15205-15210. May, G.S., Tsang, M.L.-S., Smith, H., Fidel, S. and Morris, N.R.: Aspergillus nidtdans Btubulin genes are unusually divergent. Gene 55 (1987) 231-243. Neff, N.F., Thomas, J.H., Grisafi, P. and Botstein, D.: Isolation of the B-tubulin gene from yeast and demonstration of its essential function

ACKNOWLEDGEMENTS

We thank Cindy Stryker for excellent technical assistance. This research was funded in part by the Leva B. and Elda R. Walker. and the U.S. Harkson Funds.

Ballance, D.J.: Sequences important fungi. Yeast 2 (1986) 229-236. L.E., Hutsul,

for gene expression

J.-A., Thorlacius,

L. and LeJohn,

in filamentous H.B.: Cloning

and analysis of B-tubulin gene from a protoctist. J. Biol. Chem. 265 (1990) 15245-15252. Cisar, C.R., TeBeest, D.O. and Spiegel, F.W.:Characterization of progeny

from

sexual

crosses

gloeosparioides with different (1991) 1239. Daniel, J.T., Templeton,

between

strains

host specificities.

of

Colletotrichum

Phytopathology

in rice with an endemic

fungal disease.

tides. Phytoma

des champignons

434 (1991) 20-26.

phytopathogenes

binding aux fongi-

Fungi.

Academic

M. and

Hanau,

Press,

NY,

Panaccione, 8-tubulin 1633170.

D.G. and Hanau, genes from

Rigby, P.W.J., Dieckman,

R.M.: Characterization

Cokrotrichum

1991,

R.M.: Colletotrichum

graminicola transformed with homologous and heterologous omyl-resistant genes retains expected pathogenicity to corn. Plant-Microbe Interact. 1 (1988) 113-120.

benMol.

of two divergent

graminicola. Gene

86 (1990)

M., Rhodes, C. and Berg, P.: Labelling

deoxy-

ribonucleic acid to high specific activity in oitro by nick translation with DNA polymerase I. J. Mol. Biol. 113 (1977) 237-251. Sambrook,

Jacobs, M., Smith, H. and Taylor, E.W.: Tubulin: nucleotide and enzymatic activity. J. Mol. Biol. 89 (1974) 4555468. P.: Resistance

81

G.E., Smith Jr., R.J. and Fox, W.T.: Biological

control of northern jointvetch Weed Sci. 21 (1973) 303-307.

Leroux,

Osmani, S.A. and Oakley, B.R.: Cell cycle and tubulin mutations in filamentous fungi. In: Bennet, J.W. and Lasure, L.L., (Eds.), More Gene Manipulations in pp. 107-125. Panaccione, D.G., McKiernan,

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Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 54635467. Smith, H.A., Allaudeen, J.A.: Isolation

H.S., Whitman,

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gene from Candida