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Characterization of Colletotrichum species associated with diseases of Proteaceae

     ARC Fynbos Unit, P. Bag X1, Elsenburg 7607, South Africa, and Department of Plant Pathology, University of Stellenbosch, P. Bag X1, Matieland 7602, South Africa

Sandra Denman

     Forest Research Station, Alice Holt Lodge, Farnham, Surrey, G10 4LH, United Kingdom

Paul F. Cannon

     CABI Bioscience, Bakeham Lane, Egham, Surrey, TW20 9TY, United Kingdom

J.Z. (Ewald) Groenewald

     Centraalbureau voor Schimmelcultures (CBS), Uppsalalaan 8, CT Utrecht, The Netherlands

Sandra C. Lamprecht

     Department of Plant Pathology, University of Stellenbosch, P. Bag X1, Matieland 7602, South Africa

Pedro W. Crous ¹

     Centraalbureau voor Schimmelcultures (CBS), Uppsalalaan 8, CT Utrecht, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 

Colletotrichum spp. are known to occur on and cause diseases of Proteaceae, but their identities are confused and poorly understood. The aim of the present study thus was to identify accurately the Colletotrichum spp. associated with diseases of cultivated Proteaceae. Colletotrichum spp. associated with proteaceous hosts growing in various parts of the world were identified based on morphology, sequence data of the internal transcribed spacer region (ITS-1, ITS-2), the 5.8S gene, and partial sequences of the ß-tubulin gene. Four species of Colletotrichum were found to be associated with Proteaceae. Colletotrichum gloeosporioides, a cosmopolitan species known to occur on numerous hosts, was isolated from Protea cynaroides cultivated in South Africa and Zimbabwe, and from a Leucospermum sp. in Portugal. A recently described species, C. boninense was associated with Zimbabwean and Australian Proteaceae but also occurred on a Eucalyptus sp. in South Africa. This represents a major geographical and host extension for the species and a description of the African strains is provided. Colletotrichum crassipes was represented by a single isolate obtained from a Dryandra plant in Madeira. Colletotrichum acutatum was isolated from Protea and Leucadendron in South Africa as well as from other hosts occurring elsewhere. A pathologically distinct population of this species was found to occur on Hakea in South Africa. This population is described as C. acutatum f. sp. hakeae, and its relationship with other strains of C. acutatum is discussed. Contrary to earlier literature reports linking C. gloeosporioides to anthracnose of Proteaceae, the present study has shown that several distinct species of Colletotrichum are associated with different diseases of this crop, which has serious implications for quarantine and disease control practices.

Key words: ß-tubulin, Colletotrichum acutatum, C. acutatum f. sp. hakeae, C. boninense, C. crassipes, C. gloeosporioides, Glomerella acutata, G. cingulata, ITS, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Members of the plant family Proteaceae are indigenous to Australia, South Africa, Central America, South America, southeastern Asia and the southwestern Pacific Islands (Rebelo 1995). Some members of the Proteaceae are valuable commercially and sought after as cut flowers. Certain species increasingly are being cultivated, and global trade in fresh cut-flower proteas and germplasm is growing. Many species of South African Proteaceae are cultivated in Australia, the Azores, Canary Islands, Chile, Israel, Madeira, New Zealand, Portugal, Spain, USA (California, Hawaii) and Zimbabwe. Some Australian Proteaceae (e.g. species of Banksia L.f. and Telopea R.Br.) similarly are cultivated in countries other than Australia (Crous et al 2000).

One of the factors limiting commercial production of Proteaceae is damage caused by pests and diseases (Knox-Davies 1981, Wright and Saunderson 1995, Crous et al 2004). Some pathogens cause significant losses in the field and in nurseries. Others damage the appearance of blooms, and although they are not debilitating pathogens they are considered important for aesthetic reasons. Furthermore, many pathogens associated with members of the Proteaceae are regarded as actionable quarantine organisms and can result in rejection of consignments at the point of import due to contravention of phytosanitary regulations (Crous et al 2000, Taylor 2001).

Among the most devastating fungal pathogens of Proteaceae are Colletotrichum spp., causing seedling damping off, shepherd’s crook (anthracnose), pruning wound die-back, leaf lesions and stem dieback (Knox-Davies 1981, Knox-Davies et al 1986, von Broembsen 1989, Crous et al 2004). Disease occurrence in cultivated fields tends to be sporadic and is mediated by climatic conditions suitable for disease development and high inoculum levels. Successful infection of Proteaceae is favored by moderate (20–25 C) temperatures and humid conditions (Forsberg 1993). Young tissues are most affected, often displaying the shepherd’s crook symptom or leaf necrosis. Nursery conditions often are conducive to disease development, and young plant material is especially susceptible to infection. Thus, losses in nurseries occur annually.

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. is the only Colletotrichum species reported to date to infect members of the Proteaceae. This pathogen has been recorded from most areas where Proteaceae are cultivated. Proteaceae hosts include Banksia, Grevillea R.Br. ex Knight, Hakea Schrad. & J.C.Wendl., Leucospermum R.Br., Leucadendron R.Br., Protea L., Serruria Salisb., and Telopea (Greenhalgh 1981, Morris 1982, Benic 1986, Knox-Davies et al 1986, von Broembsen 1989, Forsberg 1993, Taylor 2001, Moura and Rodrigues 2001).

Trends in gross morphological characteristics of isolates recently obtained from species of the Proteaceae suggested that more than one species of Colletotrichum might occur on this host family. However, the identification of Colletotrichum spp. based on morphological features has been beset by confusion since earliest times. The main impediments to identification are the culture medium and light conditions that influence the production of conidiomata, and the variation in the color of the mycelia and the shape and size of the conidia (Sutton 1980, Nirenberg et al 2002). Preliminary molecular data supported the hypothesis that several species of Colletotrichum could be pathogens of Proteaceae. The aim of the present study thus was to identify the Colletotrichum spp. associated with diseases of Proteaceae cultivated in different parts of the world.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Isolates. – Forty-eight isolates were examined during this study, as well as their hosts and origins (TABLE I). For comparison, reference strains of several well known species of Colletotrichum were included. Isolates were obtained from these sources: the University of Stellenbosch culture collection (STE-U), the culture collection of the Biocontrol Unit of the Plant Protection Research Institute, Agricultural Research Council in South Africa; CABI Bioscience (IMI) in the UK; the University of Arkansas Department of Plant Pathology; and from infected plant material sampled at various nurseries in the western Cape of South Africa. The sampled nursery material was surface disinfested in 1% sodium hypochlorite for 2 min, 70% ethanol for 1 min and rinsed in distilled water. Infected tissues were plated onto 2% potato-dextrose agar (PDA, Biolab, Midrand, South Africa) amended with 0.04 g/L streptomycin.

Both the ITS and ß-tubulin sequences were assembled with Sequence Alignment Editor version 2.0a11 (Rambaut 2002), from which a consensus sequence was created. These sequences together with retrievals from GenBank were aligned with Clustal W (Thompson et al 1994). Manual improvement of the final alignment based on visual inspection was made where necessary. Sequences of Botryosphaeria ribis Grossenb. & Duggar, Botryosphaeria parva Pennycook & Samuels and Botryosphaeria dothidea (Moug. : Fr.) Ces. & de Not were used as outgroups for both the ITS and ß-tubulin data. Neighbor joining analysis was performed with PAUP* version 4.0b10 (Swofford 2000) on the separate and combined datasets using the Kimura-2-parameter substitution model. Alignment gaps were treated as missing character states, and all characters were unordered and of equal weight. The resulting tree was evaluated with 1000 bootstrap replications to test the clade stability. Resulting trees were printed with TreeView version 1.6.6 (Page 1996). A partition homogeneity test (Farris et al 1994) was conducted in PAUP (Swofford 2000) to examine the possibility of a joint analysis of the different datasets.

Morphology. – Isolates were incubated at 25 C under near-ultraviolet (NUV) light with 12 h light/dark cycles. Cultures were transferred to PDA, carnation leaf agar (CLA) (Fisher et al 1982), and synthetic nutrient-poor agar (SNA) containing filter paper (Gams et al 1998) to stimulate sporulation and facilitate identification. Morphological observations were made from structures mounted in lactic acid. The 95% confidence intervals of conidial measurements were derived from at least 30 observations at 1000x magnification. Slide cultures (Riddell 1950) were made to stimulate the production of appresoria. Reference cultures were established from single-conidium isolates obtained from CLA plates. Cultures of each isolate were maintained on McCartney bottles containing either PDA or malt-extract agar (MEA) and sterile paraffin oil. Cultures are maintained in the culture collection of the Department of Plant Pathology at the University of Stellenbosch (STE-U) in South Africa, at CABI Bioscience (IMI) in the UK and the Centraalbureau voor Schimmelcultures (CBS) in the Netherlands.

Cultural studies. – Six isolates of C. boninense as well as C. acutatum f. sp. hakeae were selected for cultural studies. Colony colors were described from isolates incubated at 25 C under NUV light for 10 d according to the designations of Rayner (1970). Growth rates and cardinal temperature requirements for growth were determined for isolates plated onto PDA in 90 mm Petri dishes and incubated in the dark for 7 d at seven temperature regimes, 5–35 C at 5 degree intervals. Three plates were used for each isolate at each temperature. Radial mycelial growth was measured for each plate and the mean calculated at each temperature to determine the growth rates for each species.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Phylogenetic analysis. – For ITS sequences, approximately 550 bases were determined for the 48 isolates (TABLE I) and added to the alignment. The manually adjusted alignment of the ITS nucleotide sequences contained 87 taxa and 542 characters including alignment gaps (data not shown). Approximately 700 bases of the ß-tubulin gene were determined for the isolates and added to the alignment. The manually adjusted alignment of the ß-tubulin nucleotide sequences contained 50 taxa and 455 characters including alignment gaps (data not shown). Because the use of outgroups with ß-tubulin sequences generated by a different primer combination resulted in shorter sequence lengths, the complete sequences generated for the Colletotrichum isolates in this study could not be used for the phylogenetic analysis.

The result of the partition homogeneity test (P = 0.006, where P 0.05 was taken as significantly incongruent) indicated that it was not possible to combine the different datasets, which therefore were analyzed separately. New sequences were deposited in GenBank (TABLE I) and the alignments in TreeBASE (SN1583).

The phylogram obtained from ITS data delimited three clades concerning Colletotrichum species associated with Proteaceae (FIG. 1). The first clade had 100% support and included the ex-type strain of Colletotrichum acutatum J.H. Simmonds (STE-U 5292) as well as GenBank sequences of C. lupini (Bondar) Nirenberg, Feiler & Hagedorn (AJ301975 [GenBank] , AJ301968 [GenBank] ). Within this clade, four well supported groups were observed: the first group (76% support) contained the C. acutatum ex-type strain as well as three isolates from South African Protea (STE-U 5122, 4460, 4448), the forma specialis from Hakea (STE-U 4469, 4462, 4465, 4461, 4463, 4468, 4470, 4467, 4471, 4466) and Pinus (STE-U 162, 164, 160); the second group (96% support) contained an isolate from apple (STE-U 5287) and a C. acutatum sequence from GenBank (AF207793 [GenBank] ); the third group (65% support) contained an isolate from Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. (STE-U 5303), South African Proteaceae isolates (STE-U 4457, 4452, 4459, 4456, 4458), as well as three C. acutatum sequences obtained from GenBank (AF411765 [GenBank] , AF081292 [GenBank] , AF090853 [GenBank] ); and the fourth group (96% support) contained two C. lupini sequences from GenBank (AJ301975 [GenBank] , AJ301968 [GenBank] ). The second clade (69% support) was identified as C. gloeosporioides. The Proteaceae isolates in this clade originated from Portugal (STE-U 4450), South Africa (STE-U 4454 and 4455) and Zimbabwe (STE-U 2291). This clade also contained isolates of C. kahawae J.M. Waller & Bridge (STE-U 5295), Glomerella cingulata (Stonem.) Spauld & H. Schrenk (STE-U 5291, AF411769 [GenBank] , AF411774 [GenBank] , AF411764 [GenBank] ), C. gloeosporioides (AJ311882 [GenBank] , STE-U 5297, AJ311883 [GenBank] ) and a single isolate from Vitis vinifera L. (STE-U 4453). Two strains of C. gloeosporioides from the type host Citrus (STE-U 5297 and STE-U 4295) formed a well supported group within this clade (95% support), as did sequences obtained from GenBank (AF411774 [GenBank] , AJ311882 [GenBank] , AF411764 [GenBank] ) and isolates STE-U 4453 and 5291 (83% support). Colletotrichum crassipes (Speg.) Arx (STE-U 5302), a GenBank sequence of supposedly Glomerella cingulata (AF411775 [GenBank] ), and one isolate obtained from Dryandra R.Br. in Madeira (STE-U 4445) formed a well supported (86% support) clade sister of the second clade.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Phylogram obtained from a neighbor joining analysis of the ITS1, 5.8S rDNA and ITS2 sequence data of Colletotrichum isolates from Proteaceae. The tree was rooted to three Botryosphaeria species. Branch support is based on 1000 bootstrap replicates and is shown at the nodes. The bar represents 0.1 substitutions per site.

The phylogram obtained from the ß-tubulin data (FIG. 2) showed the same three major clades as observed in the ITS phylogram. A well supported C. acutatum clade emerged (Clade 1: 100% support), but no support was obtained for groups containing the Hakea and Pinus isolates (Group 1). However, the third C. acutatum group observed in the ITS tree was supported (Group 2: 80% support) in the ß-tubulin tree, with the isolates from Proteaceae (STE-U 4458, 4456, 4452, 4459, 4457) forming a subgroup with a 100% support. The C. gloeosporioides clade also was well supported (Clade 2: 100% support), and showed the same topology as the ITS clade. The two strains of C. gloeosporioides from Citrus (STE-U 5297 and STE-U 4295) also formed a group within this clade (99% support). As with the ITS tree, C. crassipes STE-U 5302 and an isolate from Dryandra (STE-U 4445) formed a clade (100% support) sister of this one. The third well supported clade (100% support) contained the isolate from Eucalyptus from South Africa (STE-U 194) and the Proteaceae isolates (STE-U 2290, 2289, 3000, 2998) of C. boninense.

View larger version (43K): [in this window] [in a new window]   FIG. 2. Phylogram obtained from a neighbor joining analysis of the alignment of sequences from part of the ß-tubulin gene of Colletotrichum isolates from Proteaceae. The tree was rooted to three Botryosphaeria species. Branch support is based on 1000 bootstrap replicates and is shown at the nodes. The bar represents 0.1 substitutions per site.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

Colletotrichum acutatum f. sp. hakea FIGS. 3–5

View larger version (47K): [in this window] [in a new window]   FIG. 3. Colletotrichum acutatum f. sp. hakeae (STE-U 4461). Setae, conidiophores, conidia and appresoria. Bar = 10 µm.

View larger version (81K): [in this window] [in a new window]   FIGS. 4–6. Conidiophores and conidia of Colletotrichum spp. 4–5. Colletotrichum acutatum f. sp. hakeae (STE-U 4461). 6. Colletotrichum boninense (STE-U 194). Bar = 10 µm.

Colletotrichum boninense J. Moriwaki, Toy. Sato & T. Tsukiboshi, Mycoscience 44: 48, 2003. FIGS. 6–7

View larger version (54K): [in this window] [in a new window]   FIG. 7. Colletotrichum boninense (STE-U 194). Setae, conidiophores, conidia and appresoria. Bar = 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
We made an attempt to characterize and distinguish the Colletotrichum species associated with species of Proteaceae. Because morphological identification of Colletotrichum spp. is hampered by phenotypic variation (Nirenberg et al 2002), it was essential to link the morphological descriptions to molecular data. Although C. gloeosporioides is the only Colletotrichum species reported to infect Proteaceae, preliminary data led us to suspect that more than one species could be involved; and this suspicion was confirmed in our study.

Four species of Colletotrichum (C. acutatum, C. boninense, C. crassipes, C. gloeosporioides) and a forma specialis (C. acutatum f. sp. hakeae) were found to be associated with diseased Proteaceae. No obvious correlation could be observed between host specificity and symptom type among the species recognized, with the exception of C. acutatum f. sp. hakeae from Hakea, a host to which these isolates appear to be highly specific (Morris 1982). Of these taxa, C. boninense and C. acutatum f. sp. hakeae are described fully and illustrated, while the other species await treatment elsewhere, along with a designation of epitype specimens and cultures.

Colletotrichum acutatum is known to have a wide host range and geographic distribution (Dyko and Mordue 1979), and our data also confirm that it occurs on species of Protea, Leucadendron and Leucospermum in South Africa. This is the first report of C. acutatum on Proteaceae, a host family on which it appears to be a serious pathogen. Various subgroups were delineated within the C. acutatum clade, which correlate with previous findings (Lardner et al 1999, Johnston and Jones 1997). The characterization of the population from Hakea is of special importance to South Africa, because it is used as a biological control agent of Hakea (Morris 1982). The latter plant originates in Australia but is considered a noxious weed in South Africa that is spreading through the indigenous fynbos vegetation. The biocontrol agent is sold as a specific strain of the "C. gloeosporioides" complex (Morris 1982).

Colletotrichum gloeosporioides was confirmed from Protea cynaroides (L.) L. growing in South Africa and Zimbabwe and from a Leucospermum sp. in Portugal, but it also has been reported to occur on other Proteaceae elsewhere (Greenhalgh 1981, Benic 1986, Knox-Davies et al 1986, von Broembsen 1989, Forsberg 1993, Taylor 2001, Moura and Rodrigues 2001). In view of the data presented here, previous reports of this species must be treated with circumspection. A relatively unknown species, C. boninense was found to be associated with Zimbabwean and Australian Proteaceae but also occurred on a Eucalyptus sp. in South Africa. This species until recently was treated as part of C. gloeosporioides complex (Moriwaki et al 2003).

Colletotrichum crassipes was represented by a single isolate obtained from a Dryandra plant in Madeira. A more comprehensive study of the Colletotrichum spp. occurring on Proteaceae in Australia, Madeira and Zimbabwe would be required to reveal the importance and distribution of C. crassipes and especially C. boninense, which until now has been reported only from Japan (Moriwaki et al 2003). The pathogenicity of these species to Proteaceae is being evaluated. Once pathogenicity and more representative global distribution data are available, a re-assessment of the phytosanitary significance of these species can be made.

	ACKNOWLEDGMENTS

 
The authors acknowledge the European Union for financial support. We also would like to thank Dr Jim Correll (Department of Plant Pathology, 217 Plant Science Building, University of Arkansas, Fayetteville, AR 72701) who supplied some of the cultures used in this study.

	FOOTNOTES

 
Accepted for publication June 19, 2004.

¹ Corresponding author. E-mail: crous{at}cbs.knaw.nl

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