Differentiation of Colletotrichum species responsible for anthracnose of strawberry by arbitrarily primed PCR

Myca/. Res. 99 (4): 501-504 (1995)

501

Printed in Great Britain

Differentiation of Colletotrichum species responsible for anthracnose of strawberry by arbitrarily primed peR

S. FREEMAN 1 ". AND R. 1 2

J. RODRIGUEZ!

Department of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel National Fisheries Research Center, Building 204, Naual Station, Seattle, WA 98115, U.s.A.

A collection of 39 isolates of Colletotrichum acutatum, C. fragariae and C. gloeosporioides, which cause anthracnose on strawberry, was grouped into species based on the arbitrarily primed polymerase chain reaction (ap-PCR). All isolates used had previously been identified according to classical taxonomic morphology. Ap-PCR amplification of genomic DNA using four different primers allowed for reliable differentiation between isolates of C. acutatum, C. fragariae and two genotypes of C. gloeosporioides. Fifteen of the 18 C. acutatum isolates were very similar, although three isolates which produced a red pigment had distinctly different banding patterns. Nearly identical banding patterns were observed for all nine isolates of C. fragariae. The 12 C. gloeosporioides isolates were more diverse and two separate genotypes, Cgl-l (six isolates) and Cgl-Z (five isolates) were distinguished by ap-PCR. An additional isolate did not conform to either the Cgl-l or Cgl-Z genotypes. The utility of ap-PCR compared with other molecular techniques for reliable identification of Colletotrichum isolates pathogenic on strawberry is discussed.

Anthracnose diseases of strawberry are caused by the fungal pathogens Colletotrichum acutatum J. H. Simmonds, C. fragariae A. N. Brooks and C. gloeosporioides (Penz.) Penz. & Sacco in Penz. (teleomorph: Glomerella cingulata [Stoneman] Spauld. & H. Schrenk) (Brooks, 193 I; Howard & Albregts, 1983; Smith & Black, 1990; Howard et aI., 1992). These species cause similar disease symptoms in crowns, leaves, stolons and various parts of strawberry fruit. Although C. fragariae has been found only in the U.S.A. (Farr et aI., 1989; Gunnell & Gubler, 1992), C. acutatum and C. gloeosporioides are distributed worldwide (Dyko & Mordue, 1979). Traditional taxonomy for discriminating isolates of the different Colletotrichum species relies on conidial morphology, presence or absence of setae, presence or absence of the teleomorph G. cingulata and colony colour (Sturgess, 1954; Arx, 1957; Sutton, 1980). C. acutatum is regarded as a distinct species according to morphotaxonomic criteria (Sutton, 1992). However, considerable uncertainty exists regarding the interrelationship between the species C. fragariae and C. gloeosporioides. In the past C. fragariae has been included within C. gloeosporioides (Arx, 1957) whereas a recent study, based on setae and conidial morphology, indicates that the two taxa are not synonymous (Gunnell & Gubler, 1992). Several studies have been conducted to decipher the taxonomic complexity of Colletotrichum species infecting strawberry by utilizing a variety of biochemical and molecular techniques. According to isozyme comparisons and gel electrophoresis of total protein extracts, the pathogens C. gloeosporioides, C. acutatum and C. fragariae have been classified as separate entities (Bonde, Peterson & Maas, 1991). • Corresponding author.

Distribution of the GcpR1 repetitive DNA element from C. lindemuthianum has recently been used for grouping various isolates of Colletotrichum (Rodriguez & Yoder, 1991). Using GcpR1 and two other independent molecular techniques (A + T-rich DNA and ap-PCR) it is possible to differentiate reliably between U.S.A. isolates of C. acutatum, C. fragariae and C. gloeosporioides from strawberry (Freeman, Pham & Rodriguez, 1993). However, in a related study, sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) indicated that isolates of C. fragariae were closely related to strawberry isolates of C. gloeosporioides (Sreenivasaprasad, Brown & Mills, 1992). In this study we analysed strawberry isolates of Colletotrichum spp., which had been thoroughly investigated by morphotaxonomy and reclassified into distinct species (Gunnell & Gubler, 1992). The intent of this investigation was to determine the reliability of using ap-PCR for grouping 39 Colletotrichum isolates into separate species or genotypes in comparison with that done by classical systematics. MATERIALS AND METHODS

Cultures and growth conditions All Colletotrichum isolates used in this study (Table I) had previously been identified by classical taxonomic methods (Gunnell & Gubler, 1992). Isolates of C. acutatum, C. fragariae and C. gloeosporioides were obtained from Dr P. Gunnell, U.c. Davis, CA; Dr C. M. Howard, University of Florida, FL; Dr J. L. Maas, USDA, Beltsville, MD.; Dr R. D. Milholland, North Carolina State University, NC and Dr B. J. Smith, USDA/ARS, Poplarville, MS. Fungal isolates were cultured on a modified Mathur's

502

Ap-PCR of Colletotrichum spp. of strawberry Table 1. Isolates of Collefofrichumspp. used in this study Isolate

Origin

,...

Host plant (part)

1

A

,... ,...

",...,;,",

C. acufafum 0037-1 0081-1 215-1 C-216-1 247-1 305-1 310-1 330-1 341-1 DC-l 8-4e-l 5-4h-l LOF-2 Surecrop-1 Wolf-l 204-1 CF-6-1 Gc-7-1

California (U.S.A.) California (U.S.A.) California (U.s.A.) California (U.s.A.) California (U.s.A.) California (U.S.A.) North Carolina (U.S.A.) Mississippi (U.S.A.) North Carolina (U.S.A.)

C. fragariae 63-1 163-1 251-1 321-1 326-1 B3-2 CF4-1 CFcard-l MS-9-1

Mississippi (U.S.A.) Florida (U.s.A.) Canada Florida (U.s.A.) Florida (U.S.A.) California (U.S.A.) North Carolina (U.s.A) Mississippi (U.s.A.) Mississippi (USA.)

Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry

(crown) (crown) (crown) (root) (crown) (crown) (crown) (crown) (crown)

C. gloeosporioides 231-1 272-1 291-1 303-1 311-1 314-1 315-1 327-1 329-1 336-1 343-1 B5-2

Florida (USA.) Florida (U.S.A.) Michigan (U.S.A.) Tennessee (U.S.A.) Florida (U.s.A.) Canada Nova Scotia Florida (U.s.A.) Florida (U.s.A.) Tennessee (U.s.A.) Florida (U .s.A.) California (U.s.A.)

Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry

(runner) (leaf) (crown) (runner) (runner) (petiole) (crown) (crown) (root) (crown) (leaf) (crown)

California (U.s.A.) Australia Florida (U.s.A.) North Carolina (U.S.A.) Florida (U.S.A.) North Carolina (U.s.A.) Tennessee (USA.)

Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry Strawberry

(crown) (-) (fruit) (fruit) (petiole) (-) (leaf) (fruit) (runner) (crown) (crown) (crown) (fruit) (crown) (fruit) (fruit)

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0.5 Fig. 1. Inter-species band patterns of ap-PCR amplified genomic DNA from isolates of C. acufatum (isolate 0037-1), C. fragariae (isolate 63-1), genotypes Cgl-l (isolate 231-1) and Cgl-2 (isolate 2721) of C. gloeosporioides using primers A (CAG)5' B (GACA)4' C (GACAC)3 and D (TGTC)4' respectively. Lanes M contain DNA markers with sizes in kb.

Grape

medium (MS) (Tu, 1985) (0'1 % yeast extract, 0'1 % Bactopeptone, 1 % sucrose, 0'25 % MgS0 4 . 7H 2 0, 0'27% KH 2P0 4, 2 % agar supplemented with 25 mg Ampicillin 1-1) at 21°C under cool fluorescent illumination. Liquid cultures comprising 100 ml of MS devoid of agar in 250 ml Erlenmeyer flasks were inoculated with five mycelial discs derived from colony margins. The cultures were agitated on a rotary shaker at 150 rpm and maintained at 22° for 3-4 d. Twelve hours before harvesting mycelia, the cultures were fragmented by blending for 10 s at 24000 rpm with a tissue homogenizer (Tekmar).

Fungal DNA preparation Mycelia from liquid cultures were collected by vacuum filtration and Iyophilised until dry. DNA was extracted and purified as previously described (Freeman et al., 1993; Rodriguez, 1993). The DNA was suspended in 0'5 ml TE buffer to an approximate concentration of 200-500 IJg and diluted to a concentration of 10-100 ng for ap-PCR.

2.0 1.0

0.5

2.0

1.0 0.5 Fig. 2. Intra-species band patterns of ap-PCR amplified genomic DNA from isolates of C. acutatum using primers A (CAG)5 and B (GACA)4' Lanes M contain DNA markers with sizes in kb.

Arbitrarily primed peR of genomic DNA Primers were derived from minisatellite or repeat sequences and comprised the following sequences: CAG CAG CAG CAG CAG (Rodriguez & Yoder, 1991), TGTC TGTC TGTC TGTC (Freeman et a/., 1993), GACAC GACAC GACAC (Gupta & Filner, 1991), GACA GACA GACA GACA (Weising et aI., 1989), which have been designated (CAG)5' (TGTC)4' (GACAC)3 and (GACA)4' respectively. PCR reactions were performed in a total volume of 20 IJ!, containing 10-100 ng genomic DNA, 50 mM KC!, 10 mM Tris-HC!, pH 9'0, 0'2 mM dNTP, 1'5 mM MgCl 2, 1 unit Taq polymerase (Promega) and 1 IJm primer. The reactions were incubated in an

S. Freeman and R.

J.

Rodriguez

A

503

B

2.0

(isolate 63-1), and two representatives of different genotypes of C. gloeosporioides (isolate 231-1 of genotype Cgl-1 and isolate 272-1 of genotype Cgl-2) were amplified by ap-PCR using four different oligonucleotide primers. As shown in Fig. 1, the PCR amplified bands of the four representative isolates were distinctly different for all four primers used.

Intraspecies comparison of Colletotrichum isolates by ap-PCR

1.0

0.5 Fig. 3. Intra-species band patterns of ap-PCR amplified genomic DNA from isolates of C. fragariae using primers A (CAG)5 and B (GACA)4. Lanes M contain DNA markers with sizes in kb.

A

2.0

to 0.5

B

2.0

1.0 0.5 Fig. 4. Intra-species band patterns of ap-PCR amplified genomic DNA from isolates of C. gloeosporioides using primers A (CAG)5 and B (GACA)4. Lanes M contain DNA markers with sizes in kb.

Ericornp TwinBlock system, starting with 5 min of denaturation at 95°. This was followed by 30 cycles consisting of 30 s at 95°, 30 s at either 60° [for (CAG)5] or 48° [for (GACA)4' (GACAC)3 and (TGTC)4] and 1'5 min at 72°. Amplification products were separated in 1'8% agarose gels in TAE buffer (Sambrook, Fritsch & Maniatis, 1989). An isolate was chosen to represent a species or genotype when at least three other isolates of that group had identical ap-PCR products. All experiments were repeated at least twice with similar results being obtained.

RESULTS

Interspecies comparison of Colletotrichum isolates by ap-

peR

All isolates used in this study (Table 1) were preViously defined by taxonomic classification according to morphological criteria (Gunnell & Gubler, 1992). Genomic DNA from representatives of C. acutatum (isolate 0037-1), C. jragariae

PCR-amplified bands from 18 isolates of C. acutatum were compared. Fifteen isolates exhibited similar banding patterns, whereas three isolates (204-1, CF-6-1 and Gc-7-1) were distinctly different when using primers (CAG)5 and (GACA)4 (Fig. 2A, B, respectively). The three distinct isolates produced a red pigment in culture and were previously separated from the former 15 isolates according to this phenomenon (Gunnell & Gubler, 1992). The pigmented and non-pigmented C. acutatum isolates were discriminated to the same extent with primers (TGTC)4 and (GACAC)3 (data not shown). Nearly identical banding patterns were observed by apPCR from nine C. jragariae isolates with primers (CAG)5 and (GACA)4 (Fig. 3 A, B, respectively). Similar results were obtained using primers (TGTC)4 and (GACAC)3 (data not shown). Isolates of C. gloeosporioides were more complex and appear to comprise a diverse population. PCR-amplified products from 12 isolates were compared using primers (CAG)5 and (GACA)4 (Fig. 4A, B, respectively). Two distinct genotypes, Cgl-1 and Cgl-2 were distinguished. The band patterns of Cgl-1 (isolates 231-1, 311-1, 314-2, 315-1, 329-1 and 336-1) were very similar and differed from those of Cgl-2 isolates (272-1, 291-1, 327-1, 343-1 and B5-2) (Fig. 4A, B). The Cgl2 isolates were more diverse than the Cgl-l group and exhibited differences in banding patterns with all of the primers. Isolate 303-1, which represents an additional genotype (Cgl-3) had a distinctly different banding pattern than isolates of Cgl-1 and Cgl-2.

DISCUSSION A previous study with a limited number of isolates indicated that isolates of C. jragariae, C. acutatum and C. gloeosporioides infecting strawberry were distinctly different based on nuclear and A+T-rich DNA analyses and ap-PCR (Freeman et a/., 1993). In the current study, we have elaborated on the previous work by expanding the collection of isolates analysed. In addition, all the isolates used in this study were recently discriminated by classical morphotaxonomy which resulted in the re-classification of some isolates (Gunnell & Gubler, 1992). Ap-PCR with four different primers was reliable in grouping the strawberry isolates (Table 1) into distinct species or genotypes (Fig. 1). In most cases, ap-PCR analysis of Colletotrichum isolates corresponded accurately with classical morphotaxonomic identification. Fifteen of the 18 C. acutatum isolates were very similar (Fig. 2). However, three isolates (204-1, CF-6-1 and GC-7-1) which had similar banding patterns among themselves were distinctly different from the others when analysed by ap-PCR. This distinct group within C. acutatum had previously been separated according to their

504

Ap-PCR of Colletotrichum spp. of strawberry ability to produce a red pigment (Gunnell & Gubler, 1992). Isolate Gc-7-1 was also shown to be separate based on nuclear and A + T-rich DNA analyses (Freeman et at 1993). Other red-pigmented isolates have also been reported from a wide variety of hosts including apple, peach, pecan, blueberry and other hosts (Bernstein et al., 1993; CorrelL Rhoads & Guerber, 1993). Perhaps the red-pigmented isolates represent another species of Colletotrichum, or as suggested by Gunnell & Gubler (1992), they may be deSignated as a variety of C. acutatum. In a related study, European isolates of C. acutalum infecting strawberry were discrete from U.S.A. C. aculatum isolates which were isolated from a variety of hosts (Sreenivasaprasad el al., 1992). This either indicates that C. aculalum is a heterogenous group without a specific host range or that isolates previously classified as C. aculalum actually represent multiple genotypes. The C. jragariae isolates had nearly identical banding patterns when analysed by ap-PCR (Fig. 3). Five of these isolates were previously shown by nuclear and A + T-rich DNA analyses to represent a Single genotype (Freeman et al., 1993). However, much uncertainty exists regarding the distinction of C. jragariae as a separate species from C. gloeosporioides. Gunnell & Gubler (1992) confirmed C. fragariae as a separate species from C. gloeosporioides by conidial and setae morphology as opposed to von Arx (1957). Sreenivasaprasad et al. (1992) suggested that C. fragariae may fit within the group species of C. gloeosporioides based on sequence analyses of the conserved regions of rONA. This discrepancy suggests that genotype or species discrimination based on limited sequence analysis may differ significantly from analyses based on diverse phenotypic or genotypic markers. Since 'speciation' involves extensive genotypic differences and hence phenotypic differences, discriminating species, subspecies or genotypes by more genomically dispersed DNA-based genetic markers may be more valid. The species C. gloeosporioides showed considerable heterogeneity based on ap-PCR, nuclear and A + T-rich DNA analyses, and two distinct genotypes, Cgl-1 and Cgl-2, were identified (Fig. 4; Freeman et al., 1993). Sreenivasaprasad et al. (1992) recently analysed isolates 231-1, 311-1 and 315-1 of C. gloeosporioides and found them similar according to rONA and mtDNA. This confirms a previous report and the present study designating these three isolates into a distinct genotype, Cgl-1 (Freeman et al., 1993). Furthermore, isolates 272-1, 2911, 327-1 and 343-1 that were originally identified as C. jragariae, were re-classified within the species C. gloeosporioides based on conidial and setae morphology (Gunnell & Gubler, 1992). These same isolates were confirmed in this study as genotype Cgl-2 of C. gloeosporioides (Fig. 4). Although there has been much confusion regarding the systematics and subsequent identification of Colletotrichum species causing anthracnose of strawberry, ap-PCR analyses were accurate and supported the classical morphotaxonomic studies (Gunnell & Gubler, 1992). Regardless of the taxonomic classification of Colletotrichum species, it is necessary to be able to group isolates into defined genotypes and be aware of their genetic diversity. Due to the fact that strawberry anthracnose may be caused by a complex of Colletotrichum species, the subsequent grouping of distinct genotypes is vital for developing efficient approaches for pathogen control. (Accepted 26 September

1994)

This research was supported in part by postdoctoral fellowship grant SI-0103-89 from BARD (United States-Israel Binational Agricultural Research and Development Fund) awarded to Stanley Freeman. Additional support was also provided to Rusty Rodriguez by a joint DOE, NSF, USDA grant (9237310-7821) and a USDA grant (9200655). REFERENCES 1. A. von (1957). Die Arten der GaHung Colletolrichum Cda. Phytopathologische Zei/schrift 29, 413-468. Bernstein. B.. Zehr, E. I., Dean, R. A. & Shabi. E. (1993). Characteristics of Colletotrichum &om peach, apple. pecan, and other hosts. Phytopathology 83. Arx,

1409. Bonde, M. R., Peterson, G. L. & Maas, 1. L. (1991). Isozyme comparisons for identification of Colletotrichum species pathogenic to strawberry. Phytopathology 81, 1523-1528. Brooks, A. N. (1931). Anthracnose of strawberry caused by Colletotrichum fragariae, n. sp. Phytopathology 21, 739-744. Correll, j. Rhoads, D. D. & Guerber, j. C. (1993). Taxonomic, mtDNA haplotype. VCG. and morphological diversity of Col/etotrichum spp. causing &oit-rot of apples. Phytopathology 83, 1412-1413. Dyko, B. j. & Mordue, ]. E. M, (1979). Colletotrichum acutatum. CMI descriptions of pathogenic fungi and bacteria. No. 630. Commonwealth Mycological Institute: Kew, UK Farr, D. F.. Bills. G. F.. Chamuris, G. P. & Rossman, A. Y. (1989). Fungi on Plants and Plant Products in the United States. American Phytopathological Society Press: St Paul, MN, U.s.A. Freeman, S., Pham, M. & Rodriguez. R.]. (1993). Molecular genotyping of Colletolrichum species based on arbitrarily primed PCR A + T-rich DNA. and nuclear DNA analyses. Experimental Mycology 17, 309--322. Gunnell, P. S. & Gubler, W. D. (1992). Taxonomy and morphology of Colletolrichum species pathogenic to strawberry. Mycologia 84, 157-165. Gupta, M. & Filner, P. (1991). Microsatellites amplify highly polymorphic DNA bands in SPAR of plant DNA. International Socitiy of Plant Molecular Biology. Tucson. AZ. Abstract 1705. Howard, C. M. & A1bregts, E. E. (1983). Black leaf spot phase of strawberry anthracnose caused by Colletolrichum gloeosporioides (= c. fragariae). Plant Disease 67. 1144-1146. Howard. C. M., Maas, j. L., Chancller, C. K. & A1bregts, E. E. (1992). Anthracnose of strawberry caused by the Colletotrichum complex in Florida. Plant Disease 76, 976-981. Rodriguez, R. j. (1993). Polyphosphate present in DNA preparations from the filamentous fungal species of Colletotrichum inhibits restriction endonuclease and other enzymes. Analytical Biochemistry 209, 291-297. Rodriguez, R. j. & Yoder, O. C. (1991). A family of conserved repetitive DNA elements &om the fungal plant pathogen Glomerella dngulata (Colletotrichum lindemuthianum). Experimental Mycology 15, 232-242. Sambrook J.. Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Press: New York. Smith, B. j. & Black. L. L. (1990). Morphological, cultural, and pathogenic variation among Colletotrichum species isolates from strawberry. Plant Disease 74, 69--76. Sreenivasaprasad, S.. Brown, A. E. & Mills, P. R. (1992). DNA sequence variation and interrelationships among Colletotrichum species causing strawberry anthracnose. Physiological and Molecular Plant Pathology 41. 265-281. Sturgess. O. W. (1954). A strawberry ripe &oit rot. Queensland Agricultural Journal 78, 269--270. Sutton, B. C. (1980). The Coelomycetes, Fungi Imperfecti with Pycnidia. Acervuli and Stromata. Commonwealth Mycological Institute: Kew, U.K. Sutton, B. C. (1992). The genus Glomerella and its anamorph Colletolrichum. In Colletotrichum - Biology, Pathology and Conlrol (ed. j. A. Bailey & M. j. jeger), pp. 1-26. CAB International: Wallingford. Tu, j. C. (1985). An improved Mathur's medium for growth, sporulation and germination of spores of Colletolrichum lindemuthianum. Microbios 44. 87-93. Weising, K., Weigand, F., Driesel, A. J.. Kahl, A. j.• Zischer. H. & Epplen. j. T. (1989). Polymorphic simple GATA/GACA repeats in plant genomes. Nucleic Acids Research 17. 10128.

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