STUDIES IN MYCOLOGY 55: 65–74. 2006. Characterisation of Phomopsis spp. associated with die-back of rooibos (Aspalathus linearis) in South Africa Johan C. Janse van Rensburg1, Sandra C. Lamprecht1*, Johannes Z. Groenewald2, Lisa A. Castlebury3 and Pedro W. Crous2 1ARC Plant Protection Research Institute, P. Bag X5017, Stellenbosch, 7599; 2Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box 85167, NL 3508 AD, Utrecht, The Netherlands; 3USDA-ARS, Systematic Botany & Mycology Laboratory, Rm 304, Bldg 011A, Beltsville, MD 20705, U.S.A. *Correspondence: Sandra Lamprecht, lamprechts@arc.agric.za Abstract: Die-back of rooibos (Aspalathus linearis) causes substantial losses in commercial Aspalathus plantations in South Africa. In the past, the disease has been attributed to Phomopsis phaseoli (teleomorph: Diaporthe phaseolorum). Isolates obtained from diseased plants, however, were highly variable with regard to morphology and pathogenicity. The aim of the present study was thus to identify the Phomopsis species associated with die-back of rooibos. Isolates were subjected to DNA sequence comparisons of the internal transcribed spacer region (ITS1, 5.8S, ITS2) and partial sequences of the translation elongation factor-1 alpha gene. Furthermore, isolates were also compared in glasshouse inoculation trials on 8-mo-old potted plants to evaluate their pathogenicity. Five species were identified, of which D. aspalathi (formerly identified as D. phaseolorum or D. phaseolorum var. meridionalis) proved to be the most virulent, followed by D. ambigua, Phomopsis theicola, one species of Libertella and Phomopsis, respectively, and a newly described species, P. cuppatea. A description is also provided for D. ambigua based on a newly designated epitype specimen. Taxonomic novelties: Diaporthe aspalathi Janse van Rensburg, Castlebury & Crous stat. et nom. nov., Phomopsis cuppatea Janse van Rensburg, Lamprecht & Crous sp. nov. Key words: Elongation factor 1-alpha gene, Diaporthe, endophytes, ITS, pathogenicity, Phomopsis die-back, systematics. INTRODUCTION Rooibos (Aspalathus linearis) is a leguminous shrub that is indigenous to the Western Cape Province of South Africa, and used for the production of rooibos tea. A serious die-back disease of plants in the Clanwilliam area was first observed in 1977 and officially reported in the scientific literature by Smit & Knox-Davies (1989a, b), who identified the causal organism as Phomopsis phaseoli (Desm.) Sacc. [teleomorph: Diaporthe phaseolorum (Cooke & Ellis) Sacc.]. Since die-back of rooibos was originally reported, it has developed into a disease of considerable economic importance, affecting up to 89 % of plants in 3-yr-old plantations (Lamprecht et al., unpubl. data). The genus Phomopsis (Sacc.) Bubák contains a large number of cosmopolitan plant pathogens, many of which incite blights, cankers, die-backs, rots, spots, and wilts in a wide assortment of plants of economic importance (Kulik 1984, Uecker 1988). Phomopsis diseases usually manifest themselves in the production of characteristic symptoms, some of which can culminate in the death of the host plant (Kulik 1984). In rooibos, symptoms manifest themselves as a dieback of harvested branches, with pycnidia forming on dead tissue, and a characteristic internal discoloration of infected branches. Eventually this leads to death of the host plant, after which perithecia form just below the soil surface (Fig. 1). Contrary to earlier reports (Smit & Knox-Davies 1989a, b), preliminary surveys and pathogenicity studies revealed the Phomopsis isolates associated with the disease to be highly variable with regards to morphology and virulence, indicating the possible existence of more than one species. To develop a sustainable die-back management programme for the rooibos industry, it was necessary to determine which species were involved in this disease complex and which of these were the most important pathogens. The aim of the present study was to characterise the Phomopsis/Diaporthe spp. associated with die-back symptoms of rooibos bushes. This was done by generating DNA sequence data of the ITS region and partial translation elongation factor1 alpha (TEF1 or EF1-α) gene and analysing these data with morphological and cultural observations. A further aim was to conduct pathogenicity studies with the various species identified and to determine which of these were the most virulent pathogens involved with the die-back disease of Aspalathus. MATERIALS AND METHODS Isolates Symptomatic plants were collected throughout the rooibos-producing area ranging from Citrusdal in the south to Nieuwoudtville in the north. Isolations were made from surface-disinfected host tissue onto Petri dishes containing 2 % potato-dextrose agar (PDA; Difco, Becton Dickinson, Sparks, MD, U.S.A.). A total of 28 Phomopsis isolates representing the different morphological groups recognised on PDA were selected for further molecular characterisation. The origin of isolates, as well as plant parts from which they were isolated, are listed in Table 1. Reference strains were deposited in the Centraalbureau voor Schimmelcultures in Utrecht, the Netherlands (CBS). 65 JANSE VAN RENSBURG ET AL. Fig. 1. Aspalathus linearis plants with Phomopsis die-back. A. Healthy plants under cultivation. B. Plants after harvest. C. Aspalathus bush with die-back symptoms. D–E. Stem cankers. F. Pycnidial formation on dead stem tissue. G. Formation of perithecia on stems just below the soil surface. Table 1. Phomopsis and Diaporthe isolates from South Africa used in this study. Species Strain no.1 Farm, area Rainfall2 Plant part Lesion length Collector (cm)3 D. ambigua D. aspalathi 66 GenBank numbers (EF, ITS)4 CBS 117167; CPC 5414; R86AM Taaibosdam, Gifberg High Crown 3.44d–g J.C. Janse van Rensburg DQ286237, DQ286263 CBS 117170; R59O Vaalkrans, Nardouwsberg Low Branch 5.63b J.C. Janse van Rensburg DQ286238, DQ286264 CBS 117371; CPC 5421; R350U Uitsig, AgterPakhuis Low Branch 4.83b–d J.C. Janse van Rensburg DQ286239, DQ286265 CBS 117372; CPC 5411; 10040K Clanwilliam High Root 2.17g–k S.C. Lamprecht DQ286240, DQ286266 CBS 117373; CPC 5427; R408AP Langebergpunt, Clanwilliam High Root 0.69j–l J.C. Janse van Rensburg DQ286241, DQ286267 CBS 117374; CPC 5418; R165AI Karnemelksvlei, Citrusdal High Crown 5.36b–c J.C. Janse van Rensburg DQ286242, DQ286268 CPC 5409; 9963K Clanwilliam High Crown 4.00b–e S.C. Lamprecht DQ286243, DQ286269 CPC 5412; R66I Taaiboskraal, Agter-Pakhuis Low Root 0.61j–l J.C. Janse van Rensburg DQ286244, DQ286270 CPC 5413; R78U Nardouw, Nardouwsberg Low Crown 0.94i–l J.C. Janse van Rensburg DQ286245, DQ286271 CPC 5419; R337AR Vaalkrans, Nardouwsberg Low Branch 3.06d–h J.C. Janse van Rensburg DQ286246, DQ286272 CPC 5423; R366A Taaibosdam, Gifberg High Branch 2.25e–j J.C. Janse van Rensburg DQ286247, DQ286273 CPC 5425; R379S Vondeling, Nardouwsberg Low Branch 2.25e–j J.C. Janse van Rensburg DQ286248, DQ286274 CBS 117168; CPC 5420; R338E Vaalkrans, Nardouwsberg Low Crown 10.00a J.C. Janse van Rensburg AY339353, AY339321 CBS 117169; CPC 5428; R412AY Langebergpunt, Clanwilliam High Branch 10.00a J.C. Janse van Rensburg DQ286249, DQ286275 CBS 117500; CPC 5408; 9940AF Clanwilliam High Crown 9.67a S.C. Lamprecht DQ286250, DQ286276 CPC 5410; 9996D Clanwilliam High Crown 10.00a S.C. Lamprecht DQ286251, DQ286277 PHOMOPSIS ON ASPALATHUS Table 1. (Continued). Species Strain no.1 Farm, area Rainfall2 Plant part Lesion length Collector (cm)3 GenBank numbers (EF, ITS)4 CPC 5430; R425B Koelfontein, Clanwilliam High Branch 10.00a J.C. Janse van Rensburg DQ286252, DQ286278 CBS 117163; CPC 5426; R380Z Vondeling, Nardouwsberg Low Branch 1.81g–k J.C. Janse van Rensburg DQ286253, DQ286279 CBS 117164; CPC 5429; R424T Koelfontein, Clanwilliam High Crown 2.64e–i J.C. Janse van Rensburg DQ286254, DQ286280 R686I Snorkfontein, Gifberg High Root 0.72j–l J.C. Janse van Rensburg DQ286255, DQ286281 R699H Pendoringkraal, VanRhynsdorp Low Root 0.78j–l J.C. Janse van Rensburg DQ286256, DQ286282 CBS 117499; CPC 5431; R433R Kossak se werf, Clanwilliam High Branch 1.19i–l J.C. Janse van Rensburg AY339354, AY339322 CPC 5416; R162AO Berg-en-dal, Citrusdal High Crown 3.94b–f J.C. Janse van Rensburg DQ286257, DQ286283 R164AN Karnemelksvlei, Citrusdal High Branch 1.61h–l J.C. Janse van Rensburg DQ286258, DQ286284 Phomopsis sp. 9 CBS 117165; CPC 5417; R162L Berg-en-dal, Citrusdal High Crown 0.42k–l J.C. Janse van Rensburg DQ286259, DQ286285 P. theicola CBS 117166; CPC 5415; R120AB Boskloof, Niewoudtville High Branch 4.75b–d J.C. Janse van Rensburg DQ286260, DQ286286 CBS 117501; CPC 5422; R353AH Uitsig, AgterPakhuis Low Branch 5.39b–c J.C. Janse van Rensburg DQ286261, DQ286287 CPC 5424; R374AP Snorkfontein, Gifberg High Branch 3.72c–f J.C. Janse van Rensburg DQ286262, DQ286288 Libertella sp. P. cuppatea Control 1CBS: 2Low Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC & R: Culture collection of Pedro Crous, housed at CBS. = 180–200 mm/yr; High = 250–400 mm/yr. 3Values 4EF: 0.00l in a column followed by the same letter do not differ significantly (P = 0.05). partial elongation factor 1-alpha gene; ITS: internal transcribed spacer region. Sequence Analysis Mycelium was grown on PDA plates and isolated using the protocol of Lee & Taylor (1990). PCR amplification and sequencing of the ITS rDNA, as well as partial EF1-α gene introns and exons, were performed as described by Van Niekerk et al. (2004). Newly generated sequences have been deposited in GenBank (Table 1) and the alignment in TreeBASE (S1506, M2708). Sequences were manually aligned using Sequence Alignment Editor v. 2.0a11 (Rambaut 2002). Additional sequences were obtained from GenBank and added to the alignment. In the phylogenetic trees, downloaded sequences are labelled with GenBank accession numbers; newly generated sequences are indicated with strain numbers. Two datasets were created and analysed using PAUP v. 4.0b10 (Swofford 2002) as described by Van Niekerk et al. (2004). Taxonomy Strains were grown under continuous near-ultraviolet light (400–315 nm) (Sylvania Blacklight-Blue, Osram Nederland B.V., Alphen aan den Rijn, The Netherlands) at 25 °C. Media used were PDA, and 2 % water agar containing pieces of autoclaved Aspalathus twigs using 9 cm diam Petri dishes. Growth rates and colony diameters of cultures incubated in darkness were measured on PDA. Structures were mounted in lactic acid, and 30 measurements at × 1000 magnification were made of each structure. The 95 % confidence levels were determined, and the extremes of spore measurements given in parentheses. Images were taken from slides mounted in lactic acid. Macroscopic characters of colonies were described after 14 d using the colour charts of Rayner (1970). Pathogenicity The 28 isolates used in the molecular studies were also used to conduct the pathogenicity trials. Rooibos plants were cultivated for 8 mo in 18 cm diam pots in a pasteurised sand : soil : perlite medium (1 : 1 : 1) (3 plants per pot). Plants were maintained in a glasshouse at 25 ºC (night) and 30 ºC (day) temperature, and watered three times a week. Nitrosol (Fleuron) (Universal selected services, Braamfontein, S.A.) fertiliser was applied every second week at 200 mL/pot. Colonised PDA agar plugs (5 mm diam) of the respective isolates were used to inoculate plants (three pots per isolate, with three plants per pot). Plant stems were trimmed to a uniform length of 20 cm. A cut was made 10 cm from the top of the main stem of a rooibos seedling, and an 67 JANSE VAN RENSBURG ET AL. 10 changes Cylindrocladiella peruviana AY793459 Phomopsis sp. 8 AY485743 Phomopsis sp. 7 AY485742 87 CBS 117166 100 CBS 187.27 AF230762 AF230759 Phomopsis theicola 58 AY485724 63 52 CPC 5424 CBS 117501 Phomopsis sp. 4 AY485726 Phomopsis quercina AJ293877 87 Phomopsis magnoliae AY622995 56 AY485732 100 98 AY485734 Phomopsis sp. 6 AF317563 Phomopsis vaccinii 72 AF317557 Phomopsis sp. 5 AY485727 100 98 AF102997 AF230755 Phomopsis amygdali 100 AF230760 51 AF230765 Diaporthe perjuncta AF000567 Diaporthe caulivola 93 AF000563 Diaporthe phaseolus var. caulivora AJ312360 AF001016 CBS 117500 CBS 117169 98 CPC 5430 CPC 5410 Diaporthe aspalathi CBS 117168 85 AF001015 95 AF000564 AF000566 100 AY485765 AF230751 62 AY485775 Phomopsis viticola 60 AY485757 59 AF230763 100 R164AN Phomopsis cuppatea CPC 5416 66 CBS 117499 98 CBS 117165 Phomopsis sp. 9 88 AJ312366 AJ312348 Diaporthe phaseolorum U11323 & U11373 100 AF000207 Phomopsis longicolla U97658 AF000211 Phomopsis sp. 3 AY485725 CBS 117167 CPC 5419 CPC 5409 CBS 117170 CBS 117374 CPC 5425 66 100 CBS 117373 Diaporthe ambigua CPC 5412 CPC 5413 CBS 117371 CPC 5423 CBS 117372 AJ458389 71 64 AF230768 AF230767 AJ312363 100 AJ312364 AJ312358 Diaporthe helianthi AY705842 72 AJ312354 100 Phomopsis columnaris AF439625 Phomopsis sclerotioides AF439626 100 CBS 117164 R686I 100 64 CBS 117163 Libertella sp. R699H 96 Eutypella vitis AJ302466 Eutypella leprosa AJ302463 Fig. 2. One of 176 equally parsimonious trees obtained from a heuristic search with 10 random addition replicates of the ITS sequence alignment. The scale bar shows ten changes and bootstrap support values from 1000 replicates are shown at the nodes. Thickened lines indicate branches present in the strict consensus tree and isolates obtained from Aspalathus linearis are shown in bold print. The tree was rooted to Cylindrocladiella peruviana. 68 PHOMOPSIS ON ASPALATHUS RESULTS agar plug inserted into the cut, and sealed with Parafilm. Three months after inoculation, plants were evaluated for disease symptoms, and lesion length measured. Survival of plants was also recorded. Re-isolations were made from plant material with disease symptoms onto PDA amended with 0.02 % novostreptomycin. A standard one-way analysis of variance was performed on these data using SAS statistical software v. 6.08 (SAS Institute, Cary, NC). The Shapiro-Wilk test was performed to test for normality (Shapiro & Wilk 1965). There was no evidence against normality and the original data were analysed. Student’s t-least significant differences were calculated at the 5 % level to compare ranked means. Sequence Analysis Approximately 510 and 340 bases were sequenced for ITS and EF1-α, respectively. As EF1-α sequences were not available for the taxa for which ITS sequences could be downloaded from GenBank, a tree was generated for all of them using only ITS sequences (Fig. 2). A partition homogeneity test did not indicate significant incongruence between the two genes (P = 0.7630) and the two genes were combined into a single alignment for isolates with both gene sequences (Fig. 3). Using different outgroups did not change the clades presented in Figs 2–3; nor did analyses using 1000 Cylindrocladiella peruviana 100 CBS 117166 CBS 187.27 Phomopsis theicola CPC 5424 51 CBS 117501 Diaporthe phaseolorum FAU458 65 69 Phomopsis sp. 9 CBS 117165 100 R164AN 100 CPC 5416 Phomopsis cuppatea CBS 117499 CBS 117167 98 CBS 117170 CBS 117373 CPC 5413 CBS 117372 100 CPC 5409 100 CBS 117374 Diaporthe ambigua CBS 117371 CPC 5419 79 CPC 5425 CPC 5412 CPC 5423 CBS 117169 10 changes 100 CBS 117168 CBS 117500 62 Diaporthe aspalathi CPC 5430 CPC 5410 CBS 117164 100 62 R686I CBS 117163 Libertella sp. R699H Fig. 3. One of eight most parsimonious trees obtained from a heuristic search with 10 random addition replicates of the combined ITS and EF1-α alignment. The scale bar shows ten changes; bootstrap support values from 1000 replicates are shown at the nodes. Thickened lines indicate the strict consensus branches and type strains are shown in bold print. The tree was rooted to Cylindrocladiella peruviana (ITS: AY793459; EF: AY725736). 69 JANSE VAN RENSBURG ET AL. random taxon additions change the number of trees found or the scores calculated (data not shown). The ITS data matrix contains 78 taxa (including the outgroup) and 353 positions including alignment gaps (the sequence of the 5.8S rDNA gene of Diaporthe phaseolorum strain FAU458 was not available on GenBank and this region was therefore excluded from the analysis). Only the sequences of the Eutypella and Libertella species were variable in this excluded region. Of these characters, 191 are parsimony-informative, 49 are variable and parsimony-uninformative, and 113 are constant. Neighbour-joining analysis using substitution models representative of three different assumptions (uncorrected “p”, Kimura-2-parameter and HKY85) on the sequence data yielded trees with similar topology and bootstrap values. Parsimony analysis of the alignment yielded 176 equally parsimonious trees (Fig. 2; TL = 698 steps; CI = 0.626; RI = 0.896; RC = 0.561), most of which differed only in the order of taxa within terminal clades. The same terminal clades were found in both the parsimony and neighbour-joining trees, although the order of branching was not always congruent (data not shown). Isolates from Aspalathus linearis are present in six clades (Fig. 2). The first clade (100 % bootstrap support) is identified as P. theicola Curzi, containing sequences from the ex-type strain CBS 187.27. The second clade (98 % bootstrap support) is identified as D. aspalathi sp. nov. and contains isolates from A. linearis, as well as isolates from Glycine max. The third and fourth clade containing Aspalathus linearis isolates are described as P. cuppatea sp. nov., and Phomopsis sp. 9, with bootstrap support values of 100 % and 88 %, respectively. Two sequences obtained from GenBank from Helianthus annuus isolates group together with the A. linearis isolate in the Phomopsis sp. 9 clade. Aspalathus linearis isolates in the final two clades belong to Diaporthe ambigua (containing GenBank sequence AF230767 of the ex-epitype; 100 % bootstrap support) and a Libertella sp. (100 % bootstrap support). The Libertella Desm. species groups with 100 % bootstrap support with sequences of Eutypella (Nitschke) Sacc. species obtained from GenBank. The combined data matrix contains 31 taxa (including the outgroup) with 755 positions including alignment gaps. Of these characters, 438 are parsimony-informative, 142 are variable and parsimonyuninformative, and 175 are constant. Parsimony analysis of the alignment yielded eight most parsimonious trees (TL = 1196 steps; CI = 0.823; RI = 0.931; RC = 0.766), one of which is shown in Fig. 3. The same six species that were identified in the ITS tree (Fig. 2) were found in the analysis of the combined dataset. In all cases where multiple isolates are available, the bootstrap support for the species is 100 %. Neighbour-joining analysis using three substitution models (uncorrected “p”, Kimura 2parameter and HKY85) on the sequence data yielded trees with similar topology and bootstrap values, except for the position of the Libertella Desm. sp. clade which was placed as a sister clade to D. aspalathi sp. nov. in the Kimura 2-parameter (57 % bootstrap support) and HKY85 (59 % bootstrap support) analyses (data not shown). The trees obtained using neighbour-joining 70 differed from those obtained using parsimony only with respect to the branching of the deeper nodes, which are not supported by bootstrap analysis (data not shown). Taxonomy Diaporthe ambigua Nitschke, in Nitschke, Pyrenomycetum Germanicum: 311. 1867. Fig. 4. Anamorph: Phomopsis ambigua (Sacc.) Traverso, Fl. Ital. Cryptog., Pars 1: Fungi. Pyrenomycetae. Xylariaceae, Valsaceae, Ceratostomataceae 2(1): 266. 1906. ≡ Phoma ambigua Sacc. Grevillea 2: 91. 1880. Perithecia globose, solitary to aggregated, up to 500 µm diam. Perithecial neck dark brown to black, subcylindrical, smooth, tapering towards the apex, up to 1000 µm long, 250 µm wide at the base, 70 µm wide at the apex; ostiole red-brown, obtusely rounded. Asci unitunicate, cylindrical–clavate with a refractive apical ring, 8-spored, biseriate, 50–60(–65) × 7–8(–9) µm. Paraphyses constricted at the septa, unbranched, tapering towards the apex with a rounded tip, extending above the asci, up to 150 µm long, and up to 7 µm wide at the base, and 3–4 µm wide at the apex. Ascospores hyaline, smooth, fusoid–ellipsoidal, widest just above the septum, tapering towards both ends, medianly septate, constricted at the septum at maturity, with 1–2 guttules per cell, (12–)13–15 × (3–)3.5–4 µm. Pycnidia formed on PDA and on Aspalathus twigs. Conidiophores subcylindrical, branched below or unbranched, 0–1septate, 15–45 × 2–3 µm. Alpha-conidia ellipsoidal, biguttulate, with an obtuse apex, tapering to an obtuse or bluntly rounded base with a visible scar, 6–7(–8) × 2(–3) µm, corresponding to the dimensions reported for the anamorph (Uecker 1988). Beta-conidia not seen. Description based on CBS 114015 = CPC 2657; cultures homothallic. Cultural characteristics: Colonies on OA olivaceousblack, spreading with patches of white, with sparse aerial mycelium and cream conidial masses; colonies on PDA flat, spreading, with sparse, white, dense aerial mycelium; surface with solid patches of olivaceousblack in the central part; outer region dirty-white to cream; aerial mycelium sparse, consisting of a dense layer of dirty white to cream mycelium; reverse with solid, iron-grey patches in the central part, also with isolated patches in the outer region, surrounded by cream areas. Specimens examined: Germany, Nordrhein-Westfalen, Landkreis Unna, on Pyrus communis, Th. Nitschke, Aug. 1866, holotype in B. South Africa, on Pyrus communis, S. Denman, CBS H-19685, epitype designated here, culture ex-epitype CBS 114015 (= AF230767). Notes: As shown in the present study, the host range of D. ambigua is wider than originally suspected by Nitschke (1867), but not as extreme as stated by Wehmeyer (1933). The type specimen is depauperate, containing perithecia of a Pleospora sp. and Togninia minima, and some remnants of Diaporthe ambigua. A few ascospores were observed, 12–15 × 3.5–4 µm, that were constricted at the median septum, and guttulate. Diaporthe ambigua was originally described PHOMOPSIS ON ASPALATHUS Fig. 4. Diaporthe ambigua (epitype). A. Pycnidia forming on Aspalathus stems in culture. B–C. Conidiophores. D. Alpha-conidia. E. Perithecia. F–G. Asci with ascospores. Scale bars: A, E = 70 µm, B = 4 µm, C–D, F–G = 2 µm. from cankers on pear in Germany (Nitschke 1867), and the name was subsequently used for the organism causing cankers on apples, pears and plums in South Africa (Smit et al. 1996). (6–)7–8(–9) × (2–)2.5(–3) µm. Beta- and gammaconidia absent. Description based on CBS 117169; cultures homothallic. ≡ Diaporthe phaseolorum var. meridionalis F.A. Fernández, Mycologia 88: 438. 1996. [non D. meridionalis Sacc., Syll. Fung. I: 638. 1878]. Cultural characteristics: On OA flat, spreading with sparse to no aerial mycelium; surface with irregular patches of pale white to cream and olivaceous-grey, with sparse strands of pale white aerial mycelium; on PDA flat, spreading, with sparse to no aerial mycelium; surface smoke-grey; reverse smoke-grey to olivaceousgrey. Etymology: Named after Aspalathus, on which it causes a prominent die-back disease. Specimen examined: South Africa, Western Cape Province, Clanwilliam, Langebergpunt, on Aspalathus linearus, J. Janse Van Rensburg, CBS H-19686, culture CBS 117169. Perithecia globose, solitary, scattered to aggregated, up to 500 µm wide. Perithecial neck black, cylindrical, mostly smooth, but tapering near the apex, up to 800–1000(–2000) µm long, 150 µm wide at the base, 90 µm wide at the apex; ostiole widening once spores discharge, 90–130 µm wide. Asci unitunicate, cylindrical with a refractive apical ring, 8-spored, biseriate, 52–55(–60) × 7–8(–10) µm. Paraphyses septate, unbranched, tapering towards the apex with a rounded tip, extending above the asci, up to 110 µm long, and up to 8 µm wide. Ascospores hyaline, smooth, fusoid, widest at the septum, tapering towards both ends, medianly septate, not constricted at the septum, with 1–2 guttules per cell, (12–)13–15(–16) × 3(–3.5) µm. Pycnidia formed on PDA and on Aspalathus twigs. Alpha-conidia biguttulate, fusoid with obtuse ends, Notes: Isolates (see Table 1) readily produce perithecia on PDA and on Aspalathus twigs. Diaporthe phaseolorum var. meridionalis, which was described as causing soybean stem canker in the South-eastern U.S.A. (Fernández & Hanlin 1996), is not closely related to D. phaseolorum as earlier expected. Although morphologically similar, this species clusters apart from the reference strain of D. phaseolorum (Figs 2–3). Diaporthe phaseolorum var. meridionalis is also the main causal organism of canker and die-back of rooibos, and not D. phaseolorum as reported earlier (Smit & Knox-Davies 1989a, b). The name D. meridionalis Sacc. (1878) is preoccupied, and represents a species similar to D. eres Nitschke (Wehmeyer 1933), and hence a new name, D. aspalathi is proposed here for the species pathogenic to Aspalathus and soybean. Diaporthe aspalathi Janse van Rensburg, Castlebury & Crous, nom. et. stat. nov. MycoBank MB500803. Fig. 5. 71 JANSE VAN RENSBURG ET AL. Fig. 5. Diaporthe aspalathi (CBS 117169). A–B. Perithecia. C–F. Asci and ascospores. G. Conidia. Scale bars: A = 150 µm, B = 70 µm, C = 3 µm. Phomopsis cuppatea Janse van Rensburg, Lamprecht & Crous, sp. nov. MycoBank MB500804. Fig. 6. aerial mycelium; surface smoke-grey to pale olivaceousgrey; reverse smoke-grey. Etymology: Named after the primary use of the host substrate, which is to make “rooibos” tea. Specimen examined: South Africa, Western Cape Province, Clanwilliam, Kossakse werf, on Aspalathus linearis, J. Janse van Rensburg, CBS H-19687, culture CBS 117499. Conidiophora cylindrica, 1–3-septata, 30–80 × 3–5 µm. Cellulae conidiogenae rectae vel curvatae, in collare modice distensum ad 3 μm longum exeuntes, exigue periclinaliter inspissatae, 10–35 x 1.5– 2 μm. Alpha-conidia fusoidea–ellipsoidea, sursum hebeter rotundata, ad basim obtusa vel subtruncata, bi- vel multiguttulata, (10–)12–13(– 14) × (3–)4(–5) µm. Phomopsis sp. 9 Cultural characteristics: Colonies on OA flat, spreading with sparse dirty-white aerial mycelium; surface and reverse with diffuse patches of fuscous-black and dirtywhite; colonies on PDA flat, spreading, with sparse, dirty-white aerial mycelium at the edge of the dish; surface and reverse having a translucent to ochreous central part; outer region umber. Description based on CBS 117165. Pycnidia eustromatic, black, scattered or aggregated, globose to conical, convulated to unilocular, singly ostiolate, up to 400 µm wide; pycnidial wall consisting of brown, thick-walled cells of textura angularis; conidial mass globose, pale-luteous to cream. Conidiophores cylindrical, noticeably flexuous and tall, well-developed, branched above or below, 1–3-septate, 30–80 × 3–5 µm. Conidiogenous cells straight to curved, tapering slightly towards the apex, collarettes slightly flaring, up to 3 µm long, with minute periclinal thickening, 10–35 × 1.5–2 µm. Alpha-conidia fusoid–ellipsoidal, apex bluntly rounded, base obtuse to subtruncate, bi- to multiguttulate, (10–)12–13(–14) × (3–)4(–5) µm; betaand gamma-conidia not observed. Description based on CBS 117499. Notes: When CPC 5417 was deposited in the CBS collection as 117165, it was sterile, and thus could not be named in the present study. Connecting to the numbering system used by Van Niekerk et al. (2005), it is thus referred to as Phomopsis sp. 9. Two ITS sequences obtained from GenBank represent strains isolated from Helianthus annuus, and grouped together with this isolate, proving that this species may have a wider host range than just Aspalathus. Cultural characteristics: Colonies on OA flat, spreading, with sparse, dirty white aerial mycelium; surface with irregular patches of olivaceous-black and pale olivaceous-grey; on PDA flat, spreading, with sparse Pathogenicity In inoculation experiments, the longest lesions were observed for isolates of D. aspalathi, which proved to be the most virulent species. Significantly shorter 72 PHOMOPSIS ON ASPALATHUS Fig. 6. Phomopsis cuppatea (holotype). A–B. Conidiophores. C–D. Conidia. Scale bar = 4 µm. lesions were observed for isolates of the other taxa tested (P = 0.05) (Table 1). Three months after inoculation, 95.56 % of plants inoculated with isolates belonging to D. aspalathi were dead, followed by 25.71 % of plants inoculated with P. theicola, and 16.67 % of plants inoculated with the Libertella sp. Only 14.14 % of plants inoculated with isolates of D. ambigua died. The new species, P. cuppatea and Phomopsis sp. 9, proved to be the least virulent, and in both cases only 8.33 % of the inoculated plants died. All inoculated taxa could be successfully re-isolated from inoculated plants, which in many cases ended up with dead tissue being covered in pycnidia and perithecia. None of the controls died, or showed any disease symptoms. DISCUSSION Contrary to earlier reports which were based on morphological observations alone (Smit & Knox-Davies 1989a, b), the current study has revealed that up to five Phomopsis spp. are involved with die-back of rooibos bushes, while a sixth Phomopsis-like taxon proved to be better accommodated in Libertella, which clustered among Eutypella teleomorphs. Mostert et al. (2001) isolated a species of Phomopsis from grapevines, which also proved to be present on Protea and Pyrus in countries such as Australia, Portugal and South Africa. As no name could be attributed to this species it was eventually referred to as Taxon 3. The same species was again encountered in the Phomopsis study on grapevines by Van Niekerk et al. (2005), where 15 species were distinguished, and taxon 3 was referred to as Phomopsis sp. 1. In the current study we finally managed to identify this species, as its ITS DNA sequence is identical to that of the ex-type strain of P. theicola Curzi (CBS 187.27), which was originally described from Camellia sinensis in Italy (Uecker 1988). This species obviously has a wide host range and distribution, which once again underlines the difficulties mycologists encounter when trying to identify species of Phomopsis. Several isolates which also formed a Diaporthe state in culture, proved to be identical to D. phaseolorum var. meridionalis based on morphology and sequence data. Although this pathogen was originally identified as D. phaseolorum by Smit & Knox-Davies (1989a, b), the reference strain available to us of D. phaseolorum (Figs 3–4) (treated as authentic by F.A. Uecker and preserved at BPI), clustered apart from the Aspalathus pathogen. Furthermore, as D. phaseolorum var. meridionalis is clearly not a variety of D. phaseolorum, and as the name D. meridionalis is already preoccupied, a new name is proposed for this pathogen as D. aspalathi. Another species which proved to be very common on Aspalathus matched GenBank sequences for Diaporthe ambigua. Diaporthe ambigua was originally described from branches of Pyrus communis from Germany, and was later associated with a Diaporthe canker of apple, pear and plum rootstocks in South Africa (Smit et al. 1996). To reduce any further confusion surrounding this name, we have thus chosen to designate an epitype specimen and ex-epitype culture in the present study, from which DNA sequence data are derived. Two new Phomopsis spp. were also encountered during the current study, namely P. cuppatea, and Phomopsis sp. 9. As the culture of the latter proved to be sterile, further collections are required before its taxonomy can be resolved. Several isolates of a Libertella sp. were also isolated from Aspalathus during the present study. Although Libertella is an anamorph of Eutypella and Eutypa Tul. & C. Tul. and this taxon grouped with Eutypella spp. known from GenBank sequence data, no teleomorph was ever observed on host material or induced in culture. Lesions and pycnidia formed faster downwards than upwards on stems of inoculated plants. Previous reports stated that after infection of blueberry twigs, hyphae of Phomopsis spp. move to the stem cortex (Daykin & Milholland 1990). Movement through the stem cortex takes place through the intercellular spaces, and intracellularly through the parenchyma of the outer cortex. Only after the cortex has been completely colonised, does the fungus invade the vascular tissue and pith (Daykin & Milholland, 1990). Phomopsis infection of sunflower follows the same pattern where, after penetration in the host, infection hyphae invade the intercellular spaces in the cortex (Muntañola-Cvetković 73 JANSE VAN RENSBURG ET AL. et al. 1981). Xylem elements are invaded, but affected less than the phloem and parenchyma tissues which disintegrate completely (Muntañola-Cvetković et al. 1981). Daykin & Milholland (1990) suggested that the formation of vast numbers of tyloses inside the xylem in advance of infection together with phloem plugging due to gums and hyphae, causes the lesions associated with die-back. From these masses of hyphae pycnidia are initiated (Muntañola-Cvetković et al. 1981). Tyloses form in xylem vessels of most plants under various conditions of stress and during invasion by a pathogen (Agrios 2004). Formation of these structures is an attempt by the plant to close off invaded cells to limit fungal movement in the plant (Sinclair et al. 1987). This shutting down of infected vascular tissues reduces the flow of water from the roots upward. At this point, reduced water flow and toxins often result in external symptoms (Sinclair et al. 1987). In rooibos these external symptoms are twig die-back, reduction in biomass and eventually plant death. Although the pathogenicity data obtained in the present study are still preliminary, and need to be confirmed in the field, they clearly show that D. aspalathi was the most virulent taxon, producing the longest stem lesions and also causing the most plant death. In contrast, the Libertella sp. hardly caused any tissue discoloration. The latter could be due to the fact that symptoms were rated after 3 mo, and that species from the Eutypa/Eutypella complex, as observed in grapevines, generally take much longer for symptom expression. Further studies will be necessary, however, to fully resolve the phylogenetic status of the various other Phomopsis spp. associated with die-back of Aspalathus, which in this study appeared to be of less importance than D. aspalathi. REFERENCES rd Agrios GN (2004). Plant Pathology. 3 ed. Elsevier Academic Press, Netherlands. Daykin ME, Milholland RD (1990). Histopathology of blueberry twig blight caused by Phomopsis vaccinii. Phytopathology 80: 736– 740. Kulik MM (1984). Symptomless infection, persistence, and production of pycnidia in host and non-host plants by Phomopsis batatae, Phomopsis phaseoli, and Phomopsis sojae, and the taxonomic implications. Mycologia 76: 274–291. Lee SB, Taylor JW (1990). Isolation of DNA from fungal mycelia and single spores. In: PCR protocols: a guide to methods and applications. (Innis MA, Gelfand DA, Sninsky JJ, White TJ, eds). Academic Press, San Diego, CA, U.S.A.: 282–287. Mostert L, Crous PW, Kang J-C, Phillips AJL (2001). Species of Phomopsis and a Libertella sp. occurring on grapevines with specific reference to South Africa: morphological, cultural, molecular and pathological characterization. Mycologia 93: 146– 167. Muntañola-Cvetković M, Mihaljčević M, Petrov M (1981). On the identity of the causative agent of a serious Phomopsis-Diaporthe disease in sunflower plants. Nova Hedwigia 34: 417–435. Niekerk JM van, Groenewald JZ, Farr DF, Fourie PH, Halleen F, Crous PW (2005). Reassessment of Phomopsis species on grapevines. Australasian Plant Pathology 34: 27–39. Niekerk JM van, Groenewald JZ, Verkley GJ, Fourie PH, Wingfield MJ, Crous PW (2004). Systematic reappraisal of Coniella and Pilidiella, with specific reference to species occurring on Eucalyptus and Vitis in South Africa. Mycological Research 108: 74 283–303. Rambaut A (2002). Sequence Alignment Editor. Version 2.0. Department of Zoology, University of Oxford, Oxford. Rayner RW (1970). A mycological colour chart. CMI and British Mycological Society. Kew, Surrey, England. Shapiro SS, Wilk MB (1965). An analysis of variance test for normality (complete samples). Biometrika 52: 591–611. Sinclair WA, Lyon HH, Johnson WJ (1987). Diseases of Trees and Shrubs. Cornell University Press. U.S.A. Smit WA, Knox-Davies PS (1989a). Die-back of rooibos tea caused by Diaporthe phaseolorum. Phytophylactica 21: 183–188. Smit WA, Knox-Davies PS (1989b). Comparison of Diaporthe phaseolorum isolates from rooibos tea, Aspalathus linearis. Phytophylactica 21: 301–306. Smit WA, Viljoen CD, Wingfield BD, Wingfield MJ, Calitz FJ (1996). A new canker disease of apple, pear, and plum rootstocks caused by Diaporthe ambigua in South Africa. Plant Disease 80: 1331– 1335. Swofford DL (2002). PAUP 4.0b10: Phylogenetic analysis using parsimony. Sinauer Associates, Sunderland, MA, U.S.A. Uecker FA (1988). A world list of Phomopsis names with notes on nomenclature, morphology and biology. Mycologia Memoir 13, 1–231.