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Description of Colletotrichum lupini comb. nov. in modern terms

     Institute of Plant Virology, Microbiology, and Biological Safety, Federal Biological Research Center for Agriculture and Forestry, Königin-Luise-Str. 19, 14195 Berlin, Germany

Uta Feiler

     Institute of Horticultural Sciences, Department of Phytomedicine, Humboldt University, Lentzeallee 55-57, 14195 Berlin, Germany

Gregor Hagedorn

     Institute of Plant Virology, Microbiology, and Biological Safety, Federal Biological Research Center for Agriculture and Forestry, Königin-Luise-Str. 19, 14195 Berlin, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Gloeosporium lupini Bondar is transferred to Colletotrichum. The fungus is characterized morphologically and illustrated. The two varieties, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn, comb. nov. var. lupini and Colletotrichum lupini var. setosum Nirenberg, Feiler & Hagedorn var. nov. are described. They are compared with additional Colletotrichum species reported from lupins and other hosts by morphological and physiological methods as well as by RAPD-PCR and DNA-sequencing.

Key words: Colletotrichum spp, DNA-sequences, Gloeosporium spp, Lupinus spp, morphology, RAPD-PCR

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Anthracnose of lupins is an important disease that occurs world-wide. Under wet and warm climatic conditions an infection rate of 0.001% of the seeds may lead to the loss of 30% of the harvest (Thomas et al 1998 ). In the first published report of this disease, Bondar (1912) described the causal organism of anthracnose on white lupins in Brazil as Gloeosporium lupinus. Dearness later found a fungus on lupins in the USA and called it Glomerella lupinicola. Although a description of this species was never published, it was included in the "Host index of the fungi of North America" as Gloeosporium lupinicola Dearness, by Seymour (1929) . Surprisingly neither of the two names was later adopted by plant pathologists working on anthracnose of lupins. When dealing with the causal agent of lupin anthracnose, Weimer (1943, 1951, 1952) referred to the fungus as Colletotrichum gloeosporioides or its teleomorph Glomerella cingulata, and in accordance with Weimer (1943) , Wells and Forbes (1967) and Wells and Bell (1969) used the name of the suspected teleomorph, G. cingulata. Welty (1984) reported C. trifolii from alfalfa and C. fragariae from strawberries as being pathogenic to blue lupins after inoculation. Te Beest (1988) and Weidemann et al (1988) concluded on the basis of positive infection tests that C. gloeosporioides f. sp. aeschynomenes is pathogenic towards some species of lupins. This pathogen of Aeschynomene is now commercially used in the bioherbicide COLLEGO against this weed (Te Beest 1988 ).

In the early 1980s, anthracnose of lupins spread in Europe (France: Gondran et al 1986 , United Kingdom: Reed et al 1996 , Russia: Yakusheva 1996 , Ukraine: Korneichuk 1996 ), as well as in Africa (South Africa: Koch 1997 ), and Oceania (New Zealand: Dick 1994 , western Australia: Sweetingham et al 1995 ). The anthracnose pathogen was identified by most authors as C. gloeosporioides. A few authors, like Reed et al (1996) and Gondran et al (1996) , used C. acutatum for the pathogen or one of its groups, which was a name introduced by Simmonds (1965) for Colletotrichum strains producing slender conidia with both ends pointed. Reports of Colletotrichum species with curved conidia like C. dematium var. minus (published as Vermicularia dematium var. minor by Wollenweber and Hochapfel 1949 ), C. truncatum (Almeida et al 1981 , Weidemann et al 1988 ), and C. capsici (Pring et al 1995 ) are not relevant to this investigation, because the Colletotrichum species causing the anthracnose of lupins that is currently troubling lupin growers on all continents produces straight conidia.

In this study strains isolated from diseased lupin plants or seeds are compared with each other and with members of Colletotrichum species described in the literature on lupins and other hosts. The causal organism of anthracnose of lupins is redescribed in modern terms using morphological, physiological, and molecular data.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Over 100 Colletotrichum strains were isolated at our institute from lupins and other hosts grown in Germany and other European countries, as well as in South and North America. Additional isolates from lupins as well as strains of Colletotrichum species believed to be pathogenic to lupins or other Leguminosae were received from other scientists and culture collections. Many of these strains are preserved in our culture collection (BBA) either in soil vials, freeze-dried, or frozen at -141 C (Table I ).

For DNA isolation 44 strains of Colletotrichum species and related fungi (Table I ) were grown on liquid malt-yeast-peptone medium (MYP). The mycelium was obtained by vacuum filtration and cells were disrupted either by pulverization in a precooled mortar using liquid nitrogen, or using a glass bead beater method (mycelium + 750 µL 1% SDS / 10 mM EDTA + 250 µL 0.5 mm diam glass beads in 1.5 mL screw-cap reaction tubes; agitated in a Retsch beater at highest speed for 30 min). DNA was further isolated and purified according to the method described by Hering (1997) .

RAPD-PCR – Two 10-mer primers (PSM-No 72 (5'-CAG CAC CCA C-3'), and PSM-No 86 (5'-GAT GAC CGC C-3') and two 15-mer primers PSM-No 5 = M13 (5'-GAG GGT GGC GGT TCT-3'), and PSM-No 63 (5'-CAG CAG GTC GAT GCG-3') were used. The DNA was amplified and separated electrophoretically, somewhat modifying the methods used by Hering and Nirenberg (1995) . First 5 µL fungal DNA template (10–20 ng DNA) and 5 µL primer (1µM) were transferred to the reaction tube, then 40 µL master mix consisting of 0.6 U Taq DNA polymerase (Perkin Elmer), 150 µM deoxynucleotide triphosphates dNTP (Promega), 5 µL Ampli-Taq-polymerase buffer (100 mM Tris HCl, ph 8.3, 500 mM KCl, 15 mM MgCl₂, 0.01% gelatine), 3 mM Mg-acetate-tetrahydrate, and 28.88 µL sterile distilled H₂O was added. After overlaying these components with 30 µL light mineral oil the tube was placed in the PCR thermal cycler applying the following program: initial denaturation for 1 min at 95 C; 36 cycles, each consisting of denaturation (45 s at 95 C), primer annealing (1 min at 53 C) and extension (90 s at 75 C); final extension (5 min at 75 C). The product was cooled down to 4 C and stored.

Before electrophoresis, each amplification product was furnished with 5 µL gel loading buffer. The 1% agarose gels were stained with ethidium bromide (0.005%) before solidification. The DNA bands were separated for 1.5 h at 5.5 V/cm. One microgram of the 1 kb DNA ladder was used as a molecular weight marker in the first and last lanes of the gel. The gels were photographed under UV light at 254 nm.

DNA amplification and sequencing – Two regions of the ribosomal DNA were amplified using the primers NS0 (5'-TACCTGGTTGATCCTGCC-3') to NS8 (White et al 1990 ) and NS7a (5'-AAGTTTGAGGCAATAACAGG-3') to NL4a (5'-TCCTTGGTCCGTGTTTCAAG-3'). The length of the fragments was determined on 1% agarose gels. PCR conditions were optimized until all strains yielded a single PCR product, which was purified using 12 µL of Prep-A-Gene matrix (BioRad), according to the manufacturer's instructions. The purified double-stranded PCR products were directly sequenced using Amersham Thermo-Sequenase reactions (6 µL scale) and IRD-labeled primers (generally NS1, NS2, NS3, NS4, NS5, NS7, NS8, ITS5, ITS4, NL1, and NL4; occasionally NS6, ITS1, ITS2, and ITS3; White et al 1990 , O'Donnell 1993 ). The reactions were separated on a LI-COR 4000L automated DNA sequencer using 66 cm gels with 4.3% acrylamide (Amersham RapidGel XL), obtaining an average reading length of 900 bases. The entire product was sequenced bi-directionally. Sequences were assembled and corrected using Sequencher 4.0.5 (Gene Codes Corporation, Ann Arbor, Michigan) and deposited in GenBank (for accession numbers see Table I ). The terminal primers were excluded from the published sequences.

For the outgroup sequence of Plectosphaerella cucumerina (Anamorph: Plectosporium tabacinum = Fusarium tabacinum), a contig was created based on AF176951-AF176953, provided by K. O'Donnell for strain NRRL 20430. The GenBank accessions AJ246154, U17399, and L36639 of other strains of the same species were used to fill the remaining short gaps between the main sequences and to resolve ambiguities. An additional outgroup sequence for Pyricularia grisea (=Magnaporthe grisea) was derived from AB026819.

DNA sequences were aligned using GeneDoc 2.6 (Nicholas and Nicholas 1997 ). Neighbor-joining analyses were performed using TreeCon vers. 1.3b (Van de Peer and De Wachter 1994 ). Distances were calculated under the Kimura two-parameter model with insertions separately accounted for; all bootstrap analyses were performed with 1000 replicates. The bootstrap replicate trees were re-rooted using Pyricularia grisea as an outgroup before the bootstrap consensus was computed. The outgroup for the analysis contained Pyricularia grisea, Plectosphaerella cucumerina, and Volutella ciliata (AJ301966).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Morphology and physiology – Fresh isolates of Colletotrichum from lupins grew well on all media under all conditions used. In addition to conidiomatal conidia the cultures also produced conidia in the aerial mycelium on simple or branched conidiophores that end in phialides. The latter conidia vary greatly in shape and size. Consequently, only conidia from conidiomata were included in this study for morphological examinations. The culture medium and light conditions influenced not only the production of conidiomata, appressoria, and color of the mycelium, but also the shape and size of the conidia. Two varieties of Colletotrichum capable of causing anthracnose on lupins in nature could be distinguished. One is represented by 7 strains originating from Belarus, Bolivia, Canada, Russia, and the Ukraine. The Bolivian isolate represents the oldest strain of Colletotrichum on lupins in our culture collection and is also, to the best of our knowledge, the oldest living anthracnose pathogen of lupins in the world. Therefore we choose this variety as the type. All other isolates of the lupin anthracnose pathogen belong to the other variety.

Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn, comb. nov. var. lupini Figs. 1–4

View larger version (161K): [in this window] [in a new window]    FIGS. 1–4. Conidia of Colletotrichum lupini var. lupini (=group 1); scale bar = 10 µm. Fig. 1 : Conidia from the aerial mycelium (BBA 63879). Fig. 2 : Conidiomatal conidia produced on PDA in the laboratory under natural day-night rhythm at ca 22 C (BBA 70884). Fig. 3 : Conidiomatal conidia produced on SNA under continuous near UV light at 20 C (BBA 63879). Fig. 4 : Conidiomatal conidia produced on SNA in complete darkness at 20 C (BBA 63879)

Gloeosporium lupini Bondar G. 1912 . Tremoco branco e suas molestias. Bol Agric Sao Paolo 13:427–432.

On SNA cultures only with sparse aerial mycelium, and many aerial conidia showing a wide variation in shape and size. On and around filter paper mycelium more vigorous. Under black light conidiomata rarely produced, no setae. Conidiomatal conidia pointed at one end measuring 14.0–14.5–15.0 x 4.3–4.5–4.6 µm. In darkness mycelium floccose, grayish white, with small and larger black spots that may produce rose-orange conidial masses. Reverse on filter paper light gray color with small and somewhat larger black spots. Conidiomatal conidia measure 12.8–14.0–15.1 x 4.6–4.7–4.9 µm. On the bottom of the agar—where it touches the plastic Petri dish—dark brown subglobose appressoria are formed within 2 wk, measuring mostly 3.5–6 µm.

Radial growth rate of culture on PDA at 20 C in complete darkness between 2 and 3 mm per day. Cardinal temperatures: minimum between 5 and 10 C, optimum at 22.5 C, maximum between 30 C and 35 C. Aerial mycelium lanose, ash gray when >2 mm high (1 B2), when short reddish gray (7 B2), reverse of culture in the center earth-colored (5 F2) pale orange (5 A3) towards the margin. Conidia develop abundantly in the aerial mycelium from cylindrical phialides with clearly visible periclinal thickening, on single or branched conidiophores, these conidia are subglobose to cylindrical, with rounded ends, strongly varying in size: 7.0–21.5 x 3.5–7.0 µm. Acervuli developing later, first in the center, then in rings across the colony, with cylindrical conidia, pointed at one end, at the other rounded, measuring mostly (10.0–)13.5–13.8–14.1(–16.0) x (4.2–)4.7–4.9–5.1(–6.0) µm, consistently lacking setae.

NEOTYPE: dried culture of BBA 70884, from Lupinus albus seed in the Ukraine, in B

EX-NEOTYPE: BBA 70884 = CBS 109225

Colletotrichum lupini var. setosum Nirenberg, Feiler & Hagedorn var. nov.

Figs. 5–10

View larger version (132K): [in this window] [in a new window]    FIGS. 5–10. Colletotrichum lupini var. setosum (=group 2); scale bar = 10 µm. Fig. 5 : Acervulus with setae and conidia grown on SNA under continuous NUVL at 20 C (BBA 70554). Fig. 6 : Appressoria grown on SNA in complete darkness at 20 C where the agar touches the bottom of the Petri-dish (wild type). Fig. 7 : Conidia from the aerial mycelium (BBA70051). Fig. 8 : Conidiomatal conidia produced on PDA in the laboratory under natural day-night rhythm at ca 22 C (BBA 70555). Fig. 9 : Conidiomatal conidia produced on SNA under continuous near UV light at 20 C (BBA 70354). Fig. 10 : Conidiomatal conidia produced on SNA in complete darkness at 20 C (BBA 70358)

TYPUS: cultura exsiccata, BBA 70352, ex Lupino albo in Germania, in B

On SNA cultures only with sparse aerial mycelium and few aerial conidia, showing a wide variation in shape and size (Fig. 2 ). On and up to 2 mm around the filter paper mycelium more vigorous. Under black light conidiomata more regularly formed, exhibiting pigmented, rigid and acute setae. Conidiomatal conidia pointed at one end measuring15.4–15.7–16.0 x 4.2–4.4–4.6 µm. In darkness mycelium densely floccose, blackish green, with small and larger black spots consisting of pigmented hyphal aggregations. Reverse on filter paper of same color furnished also with small and somewhat larger black spots. Conidiomatal conidia measure 13.8–14.2–14.6 x 4.5–4.6–4.7 µm. On the bottom of the agar—where it touches the plastic Petri dish—dark brown, subglobose to navicular appressoria form within 2 wk, measuring 4–6 µm if subglobose and 10 x 4–5 µm if navicular.

Radial growth rate of cultures on PDA at 20 C in complete darkness 3 mm per day. Cardinal temperatures: minimum between 5 and 10 C, optimum 25 C, maximum between 30 C and 35 C. Aerial mycelium long, lanose, of grayish beige color, cultures in reverse earth-colored to sepia (4 E3) in the middle and pale yellow (4 A2–3) towards the margin. Those conidia developing in the aerial mycelium from cylindrical phialides, on single or sparingly branched conidiophores were mostly cylindrical and with rounded ends, varying in size: 7.0–18.0 x 3.5–5.0 µm. Conidiomata never or very seldom developing, if so, then singly, with long oval to cylindrical conidia that are pointed at one end and rounded at the other, measuring mostly (7.2–)13.1–14.2–15.3(–20) x (4.1–)4.7–4.9–5.0(–5.6) µm, setae lacking.

TYPE: dried culture of BBA 70352 from Lupinus albus in Germany, in B

EX-TYPE: BBA 70352, CBS 109221

The two varieties of C. lupini differ from the following Gloeosporium or Colletotrichum species reported on lupins: The only specimen of G. lupinicola available was collected by Daubenmire 1946 (preserved in the Mycological Herbarium of the Department of Plant Pathology, Washington State University [WSP] with the accession No 39733) and has curved conidia (Fig. 13 ). Colletotrichum gloeosporioides f. sp. aeschynomenes has cylindrical conidia which are rounded at both ends (Fig. 14 ). Colletotrichum trifolii also has cylindrical conidia that are rounded at both ends but differ in size from the former (Fig. 12 ), and C. acutatum from Fragaria has fusiform conidia when grown in the dark on SNA (compare the shape of these conidia with those of C. lupini: Figs. 4, 10 ). Colletotrichum fragariae, with conidia also pointed only at one end, is the only species that is very similar in morphology to C. lupini, especially to var. setosum. Colletotrichum lupini (both varieties) and C. fragariae differ in their DNA-fingerprints and sequences (compare Fig. 16 , lane 17 to lanes 1–6 and the phylogram, Fig. 17 ).

View larger version (165K): [in this window] [in a new window]    FIGS. 11–15. Colletotrichum species reported to be pathogenic on lupins. Fig. 11 : Setae of C. trifolii (BBA 70709 = CBS 158.53) on SNA in complete darkness at 20 C after 13 d. Fig. 12 : Conidiomatal conidia of BBA 70709 grown in complete darkness at 20 C after 13 d. Fig. 13 : Conidiomatal conidia of C. acutatum (BBA 70093) grown for 25 d on SNA in complete darkness at 20 C. Fig. 14 : Conidiomatal conidia of C. gloeosporioides f. sp. aeschynomenes (BBA 71407) grown for 24 d on SNA in complete darkness at 20 C. Fig. 15 : Conidiomatal conidia of C. lupinicola from leaf of Lupinus laxiflorus (exsiccatum WS No 181877 in the Mycological Herbarium of the Department of Plant Pathology, State College of Washington)

View larger version (88K): [in this window] [in a new window]    FIG. 16. RAPD-PCR banding patterns of species from different hosts. Lane 1–3 = C. lupini var. setosum; lane 4–6 = C. lupini var. lupini; lane 7–19 = strains of different Colletotrichum-species

View larger version (45K): [in this window] [in a new window]    FIG. 17. Phylogenetic analysis of Colletotrichum species and related fungi on lupins and other hosts. The bootstrapped neighbor-joining analysis (1000 replicates) is based on the end of the 18S rDNA, the ITS1 region, the 5.8S rDNA, and the ITS2 region. All bootstrap values above 50 % are shown, although groups with a bootstrap below 75% should be considered hypothetical. Host species in the legumes are printed bold. For abbreviations see Table I

Similar differences exist in strains forming conidia with one end pointed and one end rounded, or both ends rounded. Strains from Syringa (lane 12, BBA 65797) and Salix (lane 13, BBA 70991) have similar RAPD patterns and produce conidia with both ends mostly rounded, but showing a very slight curve. Both isolates have a Glomerella teleomorph (unpubl). A second group with relatively homogenous RAPD banding patterns is formed by strains from Sambucus, Hepatica, Prunus, and Fragaria with conidia pointed only at one end (lanes 14–17: BBA 67435, BBA 70820, BBA 70345, BBA 70342).

The differences in RAPD banding patterns between C. lupini and other Colletotrichum species are most pronounced in lane 18 = C. fuscum (BBA 70535), lane 19 = C. pisi (CBS 107.40), lane 20 = C. truncatum (CBS 129.57) and lane 21 = C. trifolii (BBA 70709), lane 22 = C. lindemuthianum (BBA 71100), lane 23 = G. cf. cingulata (BBA 70048), lane 24 = C. musae (BBA 62471), lane 25 = C. gloeosporioides (BBA 70071). Some of these strains form curved conidia, some straight ones with both ends rounded. The small differences in the banding patterns between the lupin strains and the big differences to the other species support our conclusion that C. lupini is a separate species with two varieties.

DNA sequences – The sequences of the region measuring between 836 and 889 bp did not contain any major inserts. A phylogram based on a bootstrapped neighbor-joining analysis is depicted in Fig. 17 . Most strains from lupins formed a homogeneous group supported by a high bootstrap value of 96%. This group encompasses all strains that can be identified morphologically as C. lupini. The two varieties described above are separated in the phylogenetic analysis, albeit with an insignificant bootstrap support of 64%. The differentiation rests on a single base mutation in the ITS2 region (position 110 from start of ITS2 in both the ex-neotype strains BBA 70884 and BBA 70352).

Strain BBA 71292 from the Azores isolated from lupins does not belong to C. lupini, but is closely related to the group of C. acutatum strains including pathogens on Fragaria and Anemone. The bootstrap support for this C. acutatum group is, however, insufficient (62–65%). This group is the sister group of C. lupini, held together with a significant bootstrap value of 88%. Together with the two strains from Primula and Vaccinium (BBA 70343 and BBA 70339), a monophylum is supported by a bootstrap value of 76%. All species identified as C. acutatum in this group form conidia pointed on both ends (i.e., all sequenced strains within the monophylum with the exception of C. lupini).

Clearly separated from this group is a group of Colletotrichum or Glomerella strains from Sambucus (BBA 67435), Syringa (BBA 65797), Salix (BBA 70991), Hepatica (BBA 70820), and Prunus (BBA 70345), held together with a significant bootstrap value of 93%. The conidia of these strains are mostly pointed at one end.

Colletotrichum strains from legumes other than lupins (like Phaseolus, Trifolium, Pisum, and Medicago) are clearly separated from C. lupini. Among these groups are several species with falcate or curved conidia. Only the terminal groups, e.g., those that contain C. lindemuthianum (CBS 132.57) and C. trifolii (BBA 70709, CBS 158.83), are supported by significant bootstrap values. The relation between these groups and the groups mentioned above is, however, not clearly resolved and not supported by significant bootstrap values.

Strain BBA 71528, isolated from Lupinus polyphyllus in Germany, also does not belong to C. lupini. It produces curved conidia resembling those of strain BBA 70523, and the similarity of the two strains was confirmed by the DNA sequence analysis. The Volutella strain (BBA 70047) from lupins was grouped, as expected, with the outgroup.

Finally, the group containing C. gloeosporioides from Citrus (BBA 70071), C. gloeosporioides f. sp. aeschynomenes (BBA 71407), and C. musae (BBA 62471) is widely separated from all the other Colletotrichum species.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Frequently strains of Glomerella or Colletotrichum species produce two types of conidia: those that are produced in the aerial mycelium and those that are produced in conidiomata. The first ones vary greatly in size but are all of similar shape. They are cylindrical with rounded ends and therefore cannot be used successfully for the differentiation of taxa. The conidiomatal conidia are not produced on all media and under all light conditions and may vary in shape and size with the culture conditions used. Therefore the culture conditions have to be standardized if conidiomatal conidia are used for morphological differentiation of Colletotrichum species. In the case of C. lupini it is recommended to use PDA in the laboratory (natural day-night rhythm at 20 to 22 C) to study macroscopical features, and/or SNA with filter paper at 20 C in complete darkness as well as under continuous black light for microscopical identification.

The basic shape of conidiomatal conidia produced by Colletotrichum species is either curved or cylindrical. The latter type of conidia vary in regard to their ends: both ends are either rounded or pointed, or one end is rounded and one pointed. In order to place a species into the correct group it is best to use conidia grown on SNA in darkness at 20 C. In the case of C. lupini, the conidia are pointed at one end and rounded at the other end. It seems that strains of Colletotrichum species with straight conidia which have one or both ends pointed originate in the temperate zone whereas those with both ends rounded come from the tropics or sub-tropics.

From the RAPD banding patterns and the DNA sequences, we can deduce that many Colletotrichum strains belong either to different species or represent formae speciales. But there are also a few that show the same patterns like seen in Fig. 16 , lane 10, 11, and lanes 14–17. It is very likely that each group represents a different taxon.

Yang and Sweetingham (1998) also studied RAPD groups of Colletotrichum strains. Although these Australian authors were using different sets of RAPD primers, we can conclude that the two RAPD groups reported here correspond to those defined by Yang and Sweetingham (1998) . The Canadian isolate (BBA 71149 = TMC; RAPD group 1) was included in both studies. The fact that all German lupin isolates were placed in RAPD group 2 in both studies further supports the conclusion about the identity of the two groups. Therefore RAPD group 1 is identical with C. lupini var. lupini and RAPD group 2 with C. lupini var. setosum.

BBA 71292 (=Azores 1), a strain isolated from Lupinus albus from the Azores which was also examined by both scientific groups, was singled out by Yang and Sweetingham (1998) according to the RAPD banding patterns as COL-3. This strain shows a close relation to Colletotrichum isolates from strawberry, and the authors concluded that COL-3 isolates are identical with C. acutatum, exhibiting a very weak aggressiveness towards lupins (Yang and Sweetingham 1997, 1998 ). This is supported by our sequence data of the ITS region, where isolate BBA 71292 has a strong similarity with C. acutatum from strawberries (BBA 67866, see Fig. 16 ).

Sherriff et al (1994) studied a Colletotrichum strain from French lupins. They sequenced the ITS2 and the first two domains of the 28S rDNA from strain Lars 163 (=G1, =CBS 507.97, GenBank accession Z18989). The original identification was C. gloeosporioides. Later Bailey et al (1996) identified it as C. acutatum and it is currently identified in GenBank as Glomerella cingulata. The same strain was reported by Yang and Sweetingham (1998) as C. gloeosporioides, VCG 1. Based on this result and the sequence available (only partially overlapping with that of the current study) we conclude that this strain belongs to C. lupini var. lupini. Other sequences from Colletotrichum isolates from lupins were presented by Sreenivasaprasad et al (1994) . However since they presented only 181 base pairs in the ITS1 region, any identification based on these sequences is tentative. The six sequences available may well belong to C. lupini. A differentiation of the two varieties is not possible, since the ITS1 region in both varieties is identical.

Another species, C. trifolii, is also reported to be pathogenic to lupins. This species was first described in the USA as a pathogen on Trifolium and Medicago (Bain and Essary 1906 ). On the basis of morphological characteristics and DNA-sequences, we now conclude that certain strains originally identified as C. trifolii (BBA 63879, by R. Schneider at the BBA; also strain CBS 149.34) do not belong to this species. The only isolate in our study representing C. trifolii is BBA 70709 = CBS 158.83. It fits the morphological description of the authors (Bain and Essary 1906 ), producing comparatively small and wide conidia in conidiomata that have dark-brown pigmented, undulate, blunt setae (Figs. 11, 12 ), and it was isolated from Trifolium in the USA.

The teleomorph of Colletotrichum is Glomerella. Those species that are homothallic often produce the teleomorph more easily in culture than the anamorph. An example is the Glomerella species pathogenic to Hypericum officinalis (unpubl). We assume that others, for example, C. lupini var. setosum, are neither homothallic nor are they able to produce the teleomorph. During our extensive studies in Europe, to where the anthracnose pathogen of lupins probably was introduced, we could never observe the teleomorph. Also in South America, the presumed area of origin, no teleomorph was reported during field collections. Weimer (1952) found a Glomerella teleomorph on leaf lesions of L. angustifolius. It may thus be possible that he either worked with Colletotrichum gloeosporioides s. str., as he reported, or with C. lupini var. lupini, or with C. lupini var. setosum. We do not know for certain yet if any variety of C. lupini produces the perfect state. Since we studied C. lupini only under European conditions, it is possible that the teleomorph may be produced under conditions prevailing in the south of North America.

Appressoria are a good means of differentiating the genus Colletotrichum from other fungal species if grown on SNA in complete darkness. However, only rarely the shape is pronounced enough to be used as a differentiating criterion between species. This is also true in the case of the two C. lupini varieties. It is impossible to see differences in the appressoria between both varieties or between them and species which produce conidia with one end pointed.

The two varieties of C. lupini differ only slightly morphologically and physiologically: The variety lupini produces considerably more conidia in the aerial mycelium, grows on PDA somewhat slower than var. setosum, and has a lower optimum temperature. The variety lupini grows on the same agar usually in concentric rings which are marked by the light-gray to grayish yellow aerial mycelium alternating with the orange conidial masses of the conidiomata, whereas var. setosum produces 4 mm high fluffy, dense, light gray to brownish gray aerial mycelium only. Similar statements can be made for the RAPD banding patterns. In the DNA sequences of the partial 18S rDNA, 5.8S rDNA and the ITS regions 1 and 2, the two varieties differ only in a single base. We consider these differences insufficient to justify species rank for the two entities.

To the best of our knowledge, the anthracnose fungus from lupins was first described by Bondar in 1912 . Although Bondar did not provide a Latin diagnosis, his description of the disease is so precise that there is no doubt that his fungus is identical with the anthracnose fungus on lupins in this study and thus we accepted this name. Bondar also did not mention a type specimen, and we have been unable to locate one. Therefore we placed a dried culture of each variety (as a neotype for the type variety, and a holotype for the new variety) in the herbarium of the Botanical Museum in Berlin (B). The ex-neotype and the ex-holotype cultures are also deposited at the CBS in Baarn (CBS 109225 and CBS 109221).

The two varieties of C. lupini produce anthracnose on lupins under natural conditions. If lupins are inoculated with other Colletotrichum species or specialized forms of this genus, they may get infected and exhibit the same or similar symptoms as reported for C. fragariae, C. trifolii (Welty 1984 ), and C. gloeosporioides f. sp. aeschynomenes (Te Beest 1988 ). But the opposite may happen, as can be shown by the fact that Colletotrichum lupini var. setosum (BBA 70358) was able to infect Bergenia in green house tests under special conditions (Nirenberg and Gerlach 2000 ).

The two varieties of C. lupini very closely resemble other Colletotrichum species which produce conidia that are pointed at one end. It is therefore also necessary to differentiate Colletotrichum isolates on the basis of their culture characteristics: they have to be grown on PDA (Difco) under standardized conditions and compared with representative cultures of C. lupini. Additionally they can be diagnosed by sequencing the ITS region or comparing RAPD banding patterns with those of representative isolates. Although only a single base pair mutation in the ITS2 region differentiates the two varieties, this mutation may be sufficient to develop a specific primer for a PCR-based test. Further studies are necessary to develop this method.

Until the revision of the genus Colletotrichum by von Arx (1957) the anthracnose pathogens of different hosts were described as different species. At least 250 species were grouped together by this author as C. gloeosporioides since he did not find enough morphological evidence to differentiate them. He considered the variation of the isolates in culture too wide. As a consequence, in later years most economically important species were re-established as formae speciales, because of their host specificity. At present two competing systems exist side by side, sometimes interfering even with each other: the taxonomic system governed by the Botanical Code and the ungoverned plant-pathological system dealing with specialized forms and races. Which system is chosen is mostly a matter of opinion. However, it may also be a matter of being able to notice small morphological differences between isolates and to accept them as valuable enough to delimit species. But nowadays mycologists have the assistance of new methods from molecular biology like DNA sequencing and RAPD analyses. We therefore advocate a multidisciplinary approach to the differentiation of fungi. If a fungal entity is based on many supporting data and is meaningful for mankind, it should be given species rank as in the case of C. lupini.

Recently Cisar and TeBeest (1999) have successfully crossed five Colletotrichum isolates from pecan with isolates from five other hosts. This may be an indication that at least some strains of C. gloeosporioides represent specialized forms rather than different species. Within the group of Colletotrichum strains that have conidia with at least one end pointed, we assume that mating studies are most likely to show conspecifity in those strains that exhibit identical or similar RAPD banding patterns or DNA sequences (compare for example Fig. 16 , lanes 14–17 and Fig. 17 , strains BBA 67435, BBA 70345, and BBA 70820). However, the separation of C. lupini from its closest relatives is supported by a bootstrap value of 96, indicating a high reliability. This is similar to the value of 95 separating C. gloeosporioides f. sp. aeschymenes (BBA 71407) and C. gloeosporioides (BBA 70071) from C. musae (BBA 62471). We therefore believe that C. lupini is not conspecific with C. acutatum.

A study of the two varieties of C. lupini in regard to occurrence and spread, etiology, pathogenicity, as well as specialization, will be published in subsequent articles.

	ACKNOWLEDGMENTS

 
The authors thank Ms. Heidrun Anders, Ms. Astrid Hansen, and Ms. Ute Hopf for their excellent technical assistance and W. Gams for providing the Latin diagnosis. The study was supported by a grant to U. Feiler from the German Federal Ministry of Food, Agriculture, and Forestry.

	FOOTNOTES

 
¹ Corresponding author, Email: h.nirenberg{at}bba.de

Accepted for publication August 9, 2001.

	LITERATURE CITED

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