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 die-back 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).

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 (more ...)

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 factor-1 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.

Table 1. Phomopsis and Diaporthe isolates from South Africa used in this study.

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).

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 (more ...)

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 (more ...)

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 parsimony-uninformative, 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 2-parameter 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 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).

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 (more ...)

• 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, 0–1-septate, 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 olivaceous-black, 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 olivaceous-black 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 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).

Diaporthe aspalathi Janse van Rensburg, Castlebury & Crous, nom. et. stat. nov. MycoBank MB500803. Fig. 5.

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.

• Diaporthe phaseolorum var. meridionalis F.A. Fernández, Mycologia 88: 438. 1996. [non D. meridionalis Sacc., Syll. Fung. I: 638. 1878].

Etymology: Named after Aspalathus, on which it causes a prominent die-back disease.

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, (6–)7–8(–9) × (2–)2.5(–3) μm. Beta- and gamma-conidia absent. Description based on CBS 117169; cultures homothallic.

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 olivaceous-grey.

Specimen examined: South Africa, Western Cape Province, Clanwilliam, Langebergpunt, on Aspalathus linearus, J. Janse Van Rensburg, CBS H-19686, culture CBS 117169.

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.

Phomopsis cuppatea Janse van Rensburg, Lamprecht & Crous, sp. nov. MycoBank MB500804. Fig. 6.

Fig. 6. Phomopsis cuppatea (holotype). A–B. Conidiophores. C–D. Conidia. Scale bar = 4 μm.

Etymology: Named after the primary use of the host substrate, which is to make “rooibos” tea.

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.

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; beta- and gamma-conidia not observed. Description based on CBS 117499.

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 aerial mycelium; surface smoke-grey to pale olivaceous-grey; reverse smoke-grey.

Specimen examined: South Africa, Western Cape Province, Clanwilliam, Kossakse werf, on Aspalathus linearis, J. Janse van Rensburg, CBS H-19687, culture 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.

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.

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 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.