Introduction

The woody plant family Proteaceae is an important component of the South African cut flower industry (Littlejohn 2001; Crous et al. 2004a). Three genera are mainly cultivated for the export market in South Africa, namely Leucadendron, Leucospermum and Protea (Coetzee and Littlejohn 2001). Initially, these flowers were harvested from naturally occurring plants, but an increase in international demand has led to the establishment of commercial plantations employing various propagation techniques (Littlejohn 2001; Crous et al. 2004a).

Although Proteaceae are generally difficult to propagate, seed and vegetative propagation are used extensively for commercial purposes (Malan 1992; Coetzee and Littlejohn 2001). Seed propagation is an inexpensive and easy method to establish seedlings, whereas vegetative cuttings allows for the establishment of genotypic uniform plants in large quantities (Crous et al. 2004a). Both seed and vegetative propagation methods are limited in the nursery by diseases associated with fungal infection (Benic 1986). Fungi cause most of the nursery diseases known on Proteaceae and include foliar, stem and root diseases (Benic 1986; Crous et al. 2004a). Among the most important diseases are damping-off, root and collar rot, seedling blight, cutting dieback and scab (Benic 1986). Adequate control of these pathogens relies on sound management strategies in nurseries, which in turn requires knowledge of their disease development and spread.

Development of diseases in cuttings can be attributed to several factors during production. The optimal environmental requirements for rooting of Proteaceae cuttings (Malan 1992; Crous et al. 2004a) are similar to those for fungal growth and infection. Close spacing of plants combined with regular watering and heavy use of fertilizers provides a microclimate between the plants that is ideal for fungal pathogens. Wounding caused during cutting production or handling further facilitates infection.

During a survey conducted on Proteaceae propagation material in nurseries by Benic (1986), nursery diseases were observed to be caused by several soil and seed-borne fungi. Some of these pathogens were subsequently studied in more detail, namely Botrytis (Denman 1999), Colletotrichum (Lubbe et al. 2004, 2006), Fusarium (Swart et al. 1999; Lubbe et al. 2008), Phomopsis (Mostert et al. 2001a, b; Crous et al. 2011b), Phoma (Crous et al. 2011a, b), Rhizoctonia (Crous et al. 2004a) and the oomycete genus Phytophthora (Swart and Denman 2000). Calonectria has also been associated with cutting end rot of Proteaceae in nurseries in Australia and South Africa (Forsberg 1993; Porter et al. 1996; Crous 2002), which is not surprising, as it has been reported from several other horticultural and forestry crops propagated by vegetative cuttings (Lombard et al. 2010a, b).

Recently disease symptoms typical of black foot rot (including root, collar, stem and crown rot), were observed on vegetative cuttings of Protea and Leucospermum in a nursery near Stanford, Western Cape Province, South Africa. Similar symptoms were also observed on plants in a recently planted plantation, where the cuttings originated from this specific nursery. Further investigations resulted in the consistent isolation of many Ilyonectria isolates, and hence the aim of this study was to identify them to species. Furthermore, pathogenicity trials were conducted to test Koch’s Postulates on a subset of these isolates to determine if they were the causal agents of the disease observed in the nursery and plantation.

Materials and methods

Isolations

Vegetative cuttings of Protea and Leucospermum displaying symptoms of black foot rot (including root, collar, stem and crown rot) (Fig. 1) were collected from a nursery and plantation near Stanford, Western Cape Province, South Africa. The plant material was surface disinfected by submerging in 70 % ethanol for 1 min and then rinsing in sterile, distilled water. Pieces of tissue from the lesion margins of discoloured basal and collar regions were plated onto 2 % (w/v) potato-dextrose agar (PDA; Crous et al. 2009). Hyphal tips from mycelial growth that developed on PDA, were transferred to divided Petri dishes containing PDA in one half of the dish and carnation leaf agar (CLA; Fisher et al. 1982) on the other. Plates were incubated under near-ultraviolet and cool white light with a 12 h photoperiod for 3 week. Preliminary identifications to genus level were made using microscopy. For each isolate, single conidial cultures were prepared on PDA and representative isolates deposited in the working collection of Pedro Crous (CPC) and the culture collection of the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands.

Fig. 1
figure 1

Proteaceae cuttings (a–f) and pathogenicity tests (g–h). a Newly established Leucospermum cuttings. b Older Protea cuttings. c Cutting showing external symptoms of cutting end rot. d Internal discolouration of cutting end region. e Field planted Protea cuttings showing symptoms of decline. f Internal discolouration of root collar of field-planted Protea cutting. g Protea ‘Sylvia’ cutting inoculated with Ilyonectria protearum (CBS 132811) (right) and control plant (left). h Protea ‘Sylvia’ cutting inoculated with Ilyonectria vredehoekensis (CBS 132814) (right) and control plant (left)

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DNA phylogeny

Total genomic DNA was isolated from 7-day-old single-conidial isolates grown on PDA at 24 °C, using the UltraClean™ Microbial DNA isolation kits (Mo Bio Laboratories, Inc., California, USA) according to the manufacturer’s protocol. Partial gene sequences were determined for β-tubulin (BTUB), histone H3 (HIS3), internal transcribed spacer region (ITS), and translation elongation factor 1-α (TEF-1α) using the primers and protocols described by Cabral et al. (2012a, c).

To ensure the integrity of the sequences, the amplicons were sequenced in both directions with the same primer pairs used for amplification. Sequences generated were added to other sequences obtained from GenBank and subsequent alignments were generated using MAFFT v. 6 (Katoh and Toh 2010), and manually corrected where necessary.

Congruency of the sequence datasets for the separate loci were determined using tree topologies of 70 % reciprocal Neighbour-Joining (NJ) bootstrap trees with Maximum Likelihood distances that were compared visually to identify conflicts in tree topologies between partitions (Mason-Gamer and Kellogg 1996; Gueidan et al. 2007). Molecular evolution models for the separate gene regions were determined in Modeltest v. 3.7 (Posada and Crandall 1998) and bootstrap analyses were run for 10 000 replicates.

PAUP (Phylogenetic Analysis Using Parsimony; Swofford 2003) was used to analyse the DNA sequence dataset. Phylogenetic relationships were estimated by heuristic searches with 1 000 random addition sequences and tree bisection-reconnection was used, with the branch swapping option set on ‘best trees’ only. All characters were weighted equally and alignment gaps were treated as missing data. Measures calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistence index (RC). Bootstrap analysis (Hillis and Bull 1993) was based on 1 000 replications.

A second phylogenetic analysis using a Markov Chain Monte Carlo (MCMC) algorithm was done to generate trees with Bayesian probabilities in MrBayes v. 3.1.1 (Ronquist and Huelsenbeck 2003). Nucleotide substitution models were determined using MrModeltest (Nylander 2004) for each gene region and included in the analyses. Two analyses of four MCMC chains were run from random trees for one million generations and sampled every 100 generations. All runs converged on the same likelihood score and tree topology, and therefore the first 1 000 trees were discarded as the burn-in phase of each analysis and posterior probabilities determined from the remaining trees.

The phylogenetic analyses included 51 partial gene sequences for each gene region representing closely related Ilyonectria spp. (Table 1). Campylocarpon faciculare (CBS 112613) and C. pseudofasiculare (CBS 112679) were used as outgroup taxa (Cabral et al. 2012a, c) in both parsimony and Bayesian analyses. All novel sequences were deposited in GenBank. The alignments of the individual loci, including the 70 % reciprocal NJ bootstrap trees generated were deposited in TreeBASE as S13657.

Table 1 Ilyonectria isolates used in this study
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Morphology

For morphological identification of the isolates, single-conidial cultures were grown on synthetic nutrient-poor agar (SNA; Nirenberg 1981) with two 1 cm2 sterile filter paper pieces, PDA and oatmeal agar (OA; Crous et al. 2009) as described by Cabral et al. (2012a). Inoculated plates were incubated at 24 °C and examined after 1–3 weeks. Gross morphological characteristics were determined by mounting fungal structures in 85 % clear lactic acid and 30 measurements at ×1 000 magnification were made for each isolate of the conidiophores, conidia and chlamydospores using a Zeiss Axioscope 2 microscope with interference contrast (DIC) illumination. The 95 % confidence levels were determined and extremes of conidial measurements are given in parentheses. For other structures, only extremes are presented. Colony characteristics were noted after 14 days of growth on PDA and OA at 24 °C and colony colours determined using the colour charts of Rayner (1970). Sexual compatibility was determined following the protocol provided by Cabral et al. (2012a). Descriptions, nomenclature and illustrations were deposited in MycoBank (Crous et al. 2004b).

Pathogenicity tests

Koch’s postulates were tested on 4-month-old rooted cuttings of Protea ‘Sylvia’. The millet seed inoculation method developed by Strauss and Labuschagne (1995) was used. The millet seed was soaked in distilled water for 12 h (250 g seed/125 mL in 1 L Schott bottles), and then autoclaved for 20 min at 120 °C for two consecutive days. The bottles were shaken before autoclaving, as well as each time after autoclaving. The test isolates (Table 1) were plated onto PDA and were incubated for 14 d at 25 °C under near-ultraviolet and cool white light with a 12 h photoperiod (Chaverri et al. 2011). Mycelial discs (5 mm diam) of the different isolates were made and 10 mycelial plugs were transferred to each respective bottle. The bottles were incubated in the dark at 25 °C for 10 days and shaken every third day to ensure thorough colonisation. The rooted cuttings were transplanted into plastic pots containing growth medium (Seedling mix, Hygrotech, South Africa) and 2 % wt/wt colonised millet seed. Control plants were inoculated with sterilised millet seed. Twelve isolates (CBS 132807, 132808, 132809, 132810, 132811, 132812, CPC 20690, 20692, 20694, 20698, 20706, 20709) were used as inoculum and were inoculated separately using six plants per isolate. The potted plants were incubated in the laboratory at 25 ± 2 °C for 4 weeks during which time they received only water. Isolations were made from every plant 20 days after inoculation. Tissue from the root and crown areas were plated onto PDA and incubated at 22 °C for 4 weeks.

Results

DNA phylogeny

Amplicons of approximately 650 bases were determined for BTUB and ITS, 500 bases for HIS3 and 750 bases for TEF-1α. The phylogenetic analysis included 49 ingroup taxa, with C. fasciculare (CBS 112613) and C. pseudofasciculare (CBS 112679) as outgroup taxa. Comparisons of the 70 % reciprocal bootstrap NJ tree topologies of the individual loci showed no conflict in branching pattern and branch support and therefore the sequence datasets were combined. The resulting dataset of 2 099 characters, including alignment gaps, consisted of 595 parsimony-informative, 1 399 constant and 105 parsimony-uninformative characters. Analysis of the combined dataset yielded 18 trees (TL = 1412; CI = 0.674; RI = 0.901; RC = 0.608), of which the first is presented in Fig. 2. For the Bayesian analysis, a HKY + I + G model was selected for BTUB and TEF-1α, GTR + I + G for HIS3, and SYM + I + G for ITS which was incorporated into the analysis. The Bayesian consensus tree confirmed both the tree topology and bootstrap support of the strict consensus tree obtained with maximum-parsimony.

Fig. 2
figure 2

One of 18 most parsimonious trees obtained from a heuristic search with 1 000 random addition sequences of the combined sequences of β-tubulin, histone H3, internal transcribed spacer region and translation elongation factor 1α sequence alignments of Ilyonectria spp. Scale bar shows 10 changes and bootstrap support values (bold) and Bayesian posterior probability values are indicated at the nodes. Red lines indicate bootstrap support values of 100 and posterior probability values of 1.00. Thickened lines indicate branches in the strict consensus tree and the consensus tree of the Bayesian analyses. The tree was rooted to Campylocarpon fasciculare (CBS 112613) and C. pseudofasciculare (CBS 112679)

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In the tree (Fig. 2) the isolates obtained from the Proteaceae cuttings grouped into several well-supported clades. Isolate CPC 20709 clustered in the I. macrodidyma (ex-type CBS 112615) clade, whereas isolate CPC 20694 clustered in the I. torresensis (ex-type CBS 129086) clade. Isolates CBS 132809 and CBS 132810 grouped together in a clade (bootstrap value (BP) = 100; posterior probability value (PP) = 1.00) close but separate from I. cyclaminicola (ex-type CBS 302.93) and isolates CBS 132811 and CBS 132812, which also clustered together (BP = 100; PP = 1.00), thereby representing two distinct new phylogenetic species. The remaining isolates clustered into two distinct well-supported clades (both BP = 100; PP = 1.00) closely related to I. venezuelensis (ex-type CBS 102032), with each clade representing a novel phylogenetic species.

Taxonomy

Morphological observations supported by phylogenetic inference revealed that isolate CPC 20709 represents I. macrodidyma and isolate CPC 20694 represents I. torresensis. Based on phylogenetic inference and morphological comparisons, the remaining isolates obtained from the Proteaceae cuttings were confirmed to represent four novel taxa. Following the approach of Chaverri et al. (2011) and Cabral et al. (2012a, c) these taxa are named in the genus Ilyonectria. Sexual compatibility tests failed to induce perithecia within or among any of the species studied.

Ilyonectria capensis L. Lombard & Crous, sp. nov. (Fig. 3)

Fig. 3
figure 3

Ilyonectria capensis (ex-type CBS 132815). a–b Solitary conidiophores. c–d Sporodochia. e Microconidia with displaced hilum (arrows). f 1-septate microconidium (white arrow) and 1-septate macroconidium (black arrow). g–h Macroconidia with displaced hilum (arrows). i Chlamydospores. Scale bars a = 10 μm (apply to b and d); c = 50 μm; e = 10 μm (apply to g–i)

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MycoBank MB800707

Etymology: Name refers to the Western Cape Province, South Africa, where the fungus was isolated.

Conidiophores solitary or forming dense sporodochia. Solitary conidiophores arising laterally or terminally from aerial mycelium, unbranched or sparsely branched with up to three phialides, 1–3-septate, 56–96 μm long; phialides monophialidic, cylindrical, tapering towards the apex, 10–51 μm long, 2–3 μm wide at the base, 3 μm at the widest point, 1.5–2.5 μm near the aperture. Sporodochia consist of a pulvinate mass of short conidiophores; phialides cylindrical, tapering towards the apex, 21–28 μm long, 3–4 μm wide at the base, 2–3 μm wide at the apex. Macroconidia formed by both types of conidiophores, forming flat domes of slimy masses, 1–3-septate, straight, cylindrical, with both ends obtusely rounded, base sometimes with a visible, centrally located to laterally displaced hilum; 1-septate macroconidia, (22–)24–32(−36) × (5–)5.5–6.5(−7) μm (av. 28 × 6 μm); 2-septate macroconidia, 29–35(−40) × 5–7(−8) μm (av. 32 × 6 μm); and 3-septate macroconidia, (33–)34.5–39.5(−43) × 6–8 μm (av. 37 × 7 μm). Microconidia abundant, aseptate to 1-septate, with a minutely or clearly laterally displaced hilum; aseptate microconidia ovoid to fusiform, (5–)7.5–10.5(−12) × (3–)3.5–4.5(−5) μm (av. 9 × 4 μm); 1-septate microconidia fusiform to ellipsoidal to ovoid, (12–)13.5–16.5(−19) × (3–)3.5–4.5(−6) μm (av. 15 × 4 μm); microconidia formed in heads on solitary conidiophores or as masses on sporodochia. Chlamydospores only observed on PDA and OA with none forming on SNA; globose to subglobose, 12–17 μm diam, thick-walled, intercalary, hyaline becoming brown with age.

Culture characteristics: Mycelium cottony with moderate density. On OA surface and reverse sienna to umber. On PDA surface and reverse luteous to sienna.

Holotype: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on roots of Protea sp. (cutting), Dec 2011, C.M. Bezuidenhout, (CBS H-20979; ex-type culture CBS 132815 = CPC 20695).

Additional culture examined: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on roots of Protea sp. (cutting), Dec 2011, C.M. Bezuidenhout (CBS 132816 = CPC 20700).

Host and distribution: Protea sp. (Western Cape Province, South Africa).

Notes: Based on the phylogenetic inference in this study, I. venezuelensis is the closest previously identified (Cabral et al. 2012a) phylogenetic neighbour to I. capensis (Fig. 2). The solitary conidiophores of I. capensis (56–96 μm) are shorter than those of I. venezuelensis (35–200 μm; Cabral et al. 2012a) and the phialides of I. capensis (10–51 μm long) are also shorter than those of I. venezuelensis (40–60 μm; Cabral et al. 2012a).

Ilyonectria leucospermi L. Lombard & Crous, sp. nov. (Fig. 4)

Fig. 4
figure 4

Ilyonectria leucospermi (ex-type CBS 132809). a–b Solitary conidiophores. c–d Microconidia with displaced hilum (arrows). e–f Macroconidia with displaced hilum (arrows). g Chlamydospores. Scale bars a = 10 μm (apply to b); e = 10 μm (apply to d–f); g = 10 μm (apply to k–l)

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MycoBank MB800708

Etymology: Name refers to the host, Leucospermum, from which the holotype was isolated.

Conidiophores solitary; no sporodochia observed. Solitary conidiophores arising laterally or terminally from aerial mycelium, unbranched with up to two phialides, 1–3-septate, 65–179 μm long; phialides monophialidic, cylindrical, tapering towards the apex, 26–58 μm long, 3–5 μm wide at the base, 4–5 μm at the widest point, 1–3 μm near the aperture, forming both macro- and microcondia. Macroconidia 1–3(−4)-septate, straight, cylindrical, with both ends obtusely rounded, base sometimes with a visible, centrally located to laterally displaced hilum; 1-septate macroconidia, (17–)20–26(−28) × (4–)5–7 μm (av. 23 × 6 μm); 2-septate macroconidia, (29–)31–37(−38) × (4–)5–7(−8) μm (av. 34 × 7 μm); and 3-septate macroconidia, (30–)31–37(−41) × (5–)6–8 μm (av. 34 × 7 μm). Microconidia abundant, aseptate to 1-septate, with a minutely or clearly laterally displaced hilum; aseptate microconidia oval to fusiform to globose, (7–)9–13(−16) × 3–5(−7) μm (av. 11 × 4 μm); 1-septate microconidia fusiform to ellipsoidal to ovoid, (14–)15–21(−25) × (3–)4–6 μm (av. 18 × 5 μm); microconidia formed in heads on solitary conidiophores. Chlamydospores formed on PDA, OA and SNA; globose to subglobose, 8–20 μm diam, thick-walled, intercalary, hyaline becoming brown with age.

Culture characteristics: Mycelium cottony with moderate density. On OA surface and reverse sienna to umber. On PDA surface and reverse luteous to sienna.

Holotype: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on stem of Leucospermum sp. (cutting), Dec 2011, C.M. Bezuidenhout, (CBS H-20980; ex-type culture CBS 132809 = CPC 20701).

Additional culture examined: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on crown of Protea sp. (cutting), Dec 2011, C.M. Bezuidenhout (CBS 132810 = CPC 20703).

Hosts and distribution: Leucospermum sp. & Protea sp. (Western Cape Province, South Africa).

Notes: Morphologically, I. leucospermi is very similar to I. cyclaminicola, but can be distinguished by having longer (65–179 μm), solitary conidiophores compared to I. cyclaminicola (60–120 μm; Cabral et al. 2012a). Both the 2-septate ((29–)31–37(−38) × (4–)5–7(−8) μm (av. 34 × 7 μm)) and 3-septate ((30–)31–37(−41) ×(5–)6–8 μm (av. 34 × 7 μm)) macroconidia of I. leucospermi are slightly larger than those of I. cyclaminicola ((23.8–)24–28.4(−29.8) × (5–)5.5–7.3(−8) μm (av. 26.2 × 6.4 μm) and (25.3–)26.9–31.9(−33.6) × (5.8–)5.9–6.5(−6.9) μm (av. 29.4 × 6.2 μm), respectively; Cabral et al. 2012a). Both aseptate and 1-septate microconidia of I. leucospermi ((7–)9–13(−16) × 3–5(−7) μm (av. 11 × 4 μm)and (14–)15–21(−25) × (3–)4–6 μm (av. 18 × 5 μm), respectively) are also larger than those of I. cyclaminicola ((3.9–)7.6–8.9(−12.9) × (2.2–)3.6–3.9(−5.4) μm (av. 8 × 4 μm) and (11.5–)13.8–15.2(−17.5) × (3.7–)4.6–4.9(−5.5) μm (av. 15 × 5 μm), respectively; Cabral et al. 2012a). Furthermore, both isolates representing I. leucospermi failed to form sporodochia after 8 weeks incubation at 24 °C under continuous uv light.

Ilyonectria protearum L. Lombard & Crous, sp. nov. (Fig. 5)

Fig. 5
figure 5

Ilyonectria protearum (ex-type CBS 132811). a–b Solitary conidiophores. c–d Sporodochia. e–f Microconidia with displaced hilum (arrows). g–h Macroconidia with displaced hilum (arrows). i Chlamydospores. Scale bars a = 10 μm; (apply to b–d); e = 10 μm (apply to f–i)

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MycoBank MB800709

Etymology: Name refers to the plant host genus, Protea, from which this fungus was isolated.

Conidiophores solitary or forming dense sporodochia. Solitary conidiophores arising laterally or terminally from aerial mycelium, unbranched or sparsely branched with up to three phialides, 1–3-septate, 62–143 μm long; phialides monophialidic, cylindrical, tapering towards the apex, 22–47 μm long, 2–5 μm wide at the base, 3–5 μm at the widest point, 1–3 μm near the aperture. Sporodochia consist of a pulvinate mass of short conidiophores; phialides cylindrical, tapering towards the apex, 9–23 μm long, 2–4 μm wide at the base, 1–3 μm wide at the apex. Macroconidia predominant, formed by both types of conidiophores, forming flat domes of slimy masses, 1–3-septate, straight, cylindrical, with both ends obtusely rounded, base sometimes with a visible, centrally located to laterally displaced hilum; 1-septate macroconidia, (19–)21–27(−30) × (4–)5–7 μm (av. 24× 6 μm); 2-septate macroconidia, (18–)26–34(−37) × (4–)6–8 μm (av. 30 × 7 μm); and 3-septate macroconidia, (29–)32–38(−42) × (5–)7–9 μm (av. 35 ×8 μm). Microconidia aseptate to 1-septate, with a minutely or clearly laterally displaced hilum; aseptate microconidia oval to ovoid to fusiform, (7–)10–14(−15) × 3–5(−6) μm (av. 12 × 4 μm); 1-septate microconidia fusiform to ellipsoidal to ovoid, (11–)14–18(−22) × (3–)4–6(−7) μm (av. 17 × 5 μm); microconidia formed in heads on solitary conidiophores or as masses on sporodochia. Chlamydospores observed on PDA, OA and SNA; globose to subglobose, 11–18 μm diam, thick-walled, intercalary or solitary, hyaline, becoming brown with age.

Culture characteristics: Mycelium cottony with moderate density. On OA surface sienna and reverse luteous to sienna. On PDA surface and reverse luteous to sienna.

Holotype: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on roots of Protea sp. (cutting), Dec 2011, C.M. Bezuidenhout, (CBS H-20981; ex-type culture CBS 132811 = CPC 20707).

Additional culture examined: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on roots of Protea sp., Dec 2011, C.M. Bezuidenhout (CBS 132812 = CPC 20711).

Host and distribution: Protea sp. (Western Cape Province, South Africa).

Notes: Ilyonectria protearum closely resembles I. cyclaminicola and I. leucospermi in morphology, but can be distinguished by having solitary conidiophores (62–143 μm), that are shorter than those of I. leucospermi (65–179 μm), but longer than those of I. cyclaminicola (60–120 μm; Cabral et al. 2012a). Isolates of I. protearum also readily formed sporodochia within 5 week, which was not observed for I. leucospermi.

Ilyonectria vredehoekensis L. Lombard & Crous, sp. nov. (Fig. 6)

Fig. 6
figure 6

Ilyonectria vredehoekensis (ex-type CBS 132807) a–b Solitary conidiophores. c–d Sporodochia. e Microconidia. f Macroconidia with displaced hilum (arrows). g Chlamydospores. Scale bars a = 10 (apply to b–d); e = 10 μm (apply to f–g)

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MycoBank MB800710

Etymology: Name refers to Vredehoek Farm, where this fungus was isolated.

Conidiophores solitary or forming dense sporodochia. Solitary conidiophores arising laterally or terminally from aerial mycelium, unbranched or sparsely branched with up to three phialides, 1–3-septate, 55–168 μm long; phialides monophialidic, cylindrical, tapering towards the apex, 18–36 μm long, 3–5 μm wide at the base, 3–5 μm at the widest point, 2–4 μm near the aperture. Sporodochia consist of a pulvinate mass of short conidiophores; phialides cylindrical, tapering towards the apex, 16–29 μm long, 3–5 μm wide at the base, 2–4 μm wide at the apex. Macroconidia predominant, formed by both types of conidiophores, forming flat domes of slimy masses, 1–3-septate, straight, cylindrical, with both ends obtusely rounded, base sometimes with a visible, centrally located to laterally displaced hilum; 1-septate macroconidia, (21–)24–28(−30) × 5–7(−8) μm (av. 26 × 6 μm); 2-septate macroconidia, (24–)26–30(−31) × (5–)6–8 μm (av. 28 × 7 μm); and 3-septate macroconidia, (27–)30–34(−37) × 5–7 μm (av. 32 × 6 μm). Microconidia aseptate to 1-septate, with a minutely or clearly laterally displaced hilum; aseptate microconidia oval to fusiform to globose, (4–)6–8(−10) × 3–4 μm (av. 7 × 3 μm); 1-septate microconidia fusiform to ellipsoidal to ovoid, (11–)12–17(−19) × 4–6 μm (av. 15 × 3 μm); microconidia formed in heads on solitary conidiophores or as masses on sporodochia. Chlamydospores observed on PDA, OA and SNA; globose to subglobose, 8–14 μm diam, thick-walled, intercalary, hyaline becoming brown with age.

Culture characteristics: Mycelium cottony with moderate density. On OA surface and reverse sienna to umber. On PDA surface and reverse sienna to umber.

Holotype: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on roots of Protea sp. (cutting), Dec 2011, C.M. Bezuidenhout, (CBS H-20982; ex-type culture CBS 132807 = CPC 20699).

Additional culture examined: SOUTH AFRICA, Western Cape Province, Stanford, Vredehoek Farm, on roots of Protea sp. (cutting), Dec 2011, C.M. Bezuidenhout, (CBS 132808 = CPC 20697).

Host and distribution: Protea sp. (Western Cape Province, South Africa).

Notes: Ilyonectria vredehoekensis is closely related to I. capensis and I. venezuelensis based on the phylogenetic inference results obtained in this study. The simple conidiophores of I. vredehoekensis (55–168 μm) are intermediate between those of I. capensis (56–96 μm) and I. venezuelensis (35–200 μm; Cabral et al. 2012a), distinguishing it from both these species. Furthermore, the 2-septate macroconidia ((24–)26–30(−31) × (5–)6–8 μm (av. 28 × 7 μm)) and aseptate microconidia ((4–)6–8(−10) × 3–4 μm (av. 7 × 3 μm)) of this fungus are slightly smaller than those of I. capensis (29–35(−40) × 5–7(−8) μm (av. 32 × 6 μm) and (5–)7.5–10.5(−12) × (3–)3.5–4.5(−5) μm (av. 9 × 4 μm), respectively) and I. venezuelensis ((25–)26.3–37.4(−44) × (5.9–)6–6.6(−7) μm (av. 32 × 6 μm) and (5–)8.4–10.5(−13) × (3–)3.3–3.7(−4) μm (av. 10 × 4 μm), respectively; Cabral et al. 2012a). Isolates CBS 132813 and CBS 132814 could only be distinguished from the other two isolates representing I. vredehoekensis (CBS 132807 and CBS 132808) based on TEF-1α sequence data. Although there are some morphological differences between isolates CBS 132813 and CBS 132814, and isolates CBS 132807 and CBS 132808, we refrain from naming them as distinct until more isolates are obtained, and more loci are sequenced to support this distinction. Isolates CBS 132813 and CBS 132814 have slightly larger 2-septate ((26–)29–35(−39) × (6–)7–9(−10) μm (av. 32 × 8 μm)) and 3-septate ((28–)31–37(−39) × (7–)8–10 μm (av. 34 × 9 μm)) macroconidia and their simple conidiophores are also slightly shorter (55–122 μm).

Pathogenicity tests

During the pathogenicity trial, symptoms associated with Ilyonectria black foot rot (stem blackening, wilting and leaf blight) developed 10–20 days after inoculation. Dissection of the stems of infected plants revealed internal necrosis progressing from the base of the plant upwards. Isolate CPC 20709 (I. macrodidyma; known from grapevines) and CBS 132807 + CBS 132808 (I. vredehoekensis) were clearly pathogenic, and were successfully re-isolated from inoculated plants (Fig. 1). No Ilyonectria sp. was isolated from any of the control plants. Back isolations from symptomatic plants yielded only the inoculated Ilyonectria species.

Discussion

This study emerged from a collection of Ilyonectria isolates from Proteaceae cuttings displaying black foot rot symptoms observed in a nursery and plantation, planted with cuttings originating from the same nursery, in the Western Cape Province of South Africa. These isolates were shown to represent six species which included I. macrodidyma, I torresensis, and four new species that have been described as I. capensis, I. leucospermi, I. protearum and I. vredehoekensis. This study represents the first record of this group of fungi on Protea and Leucospermum in a Proteaceae nursery.

The genus Ilyonectria was introduced by Chaverri et al. (2011) to accommodate fungi with Cylindrocarpon-like anamorphs belonging to the “C. destructans” complex based on Booth’s Group 3 (Booth 1966) with I. radicicola as the type species. This genus can be distinguished from its sister genus, Neonectria, and other closely related genera, Rugonectria and Thelonectria, based on its soil-borne nature and ability to produce both microconidia and chlamydospores (Booth 1966; Cabral et al. 2012a, c; Chaverri et al. 2011). Globally, species of Ilyonectria are regarded as important pathogens of various economically important plant hosts (Samuels and Brayford 1990; Seifert et al. 2003; Halleen et al. 2004, 2006; Cabral et al. 2012a, b, c) responsible for disease symptoms such as root rots and general decline. Using a multi-gene DNA analysis supported by morphological characters, Cabral et al. (2012a) was able to delineate 12 new taxa in the I. radicicola complex, previously known as the “C. destructans” complex. Following this study, Cabral et al. (2012c) were able to identify a further six monophyletic lineages in the I. macrodidyma complex associated with black foot disease of grapevine, four of which could be described as novel species.

Both I. macrodidyma and I. torresensis have previously been reported from South Africa associated with black foot disease of grapevine (Halleen et al. 2004; Cabral et al. 2012c). However, this study represents the first report of both fungi on Protea spp. associated with black foot rot of nursery cuttings. Pathogenicity tests applied in this study showed that both Ilyonectria spp. are capable of causing black foot rot of Protea spp., inducing similar disease symptoms observed in the nursery. However, the full extent of their importance as pathogens on Proteaceae will require further investigation.

The introduction of I. capensis, I. leucospermi, I. protearum and I. vredehoekensis in this study adds four new species to the I. radicicola complex. With the exception of I. leucospermi, which was also isolated from Leucospermum, all four species were isolated from Protea cuttings. All four Ilyonectria species were also capable of causing disease on inoculated Protea cuttings.

As far as we could establish, this study represents the first report of black foot rot disease of Proteaceae cuttings. The only other known record on Proteaceae is that of Summerell et al. (1990), who reported crown and stem canker of Telopea speciosissima (waratha) seedlings and field plants associated with an Ilyonectria sp. (as Cylindrocarpon destructans). Black foot rot, however, has been well documented on grapevines, both in nurseries and vineyards (Halleen et al. 2003, 2004, 2006; Cabral et al. 2012b, c). Halleen et al. (2003) proposed that infection of grapevine cuttings mainly occur in the nursery, with the potting medium as likely inoculum source (Rego et al. 2001). Therefore, the potting medium used for rooting of Proteaceae cuttings could play a significant role as inoculum source, as planting material is collected from plantations, where black foot rot has not yet been observed. Planting of infected, rooted Proteaceae cuttings in plantations, could therefore introduce these Ilyonectria species into plantation soils, resulting in losses of mature plants. The use of clean potting medium for the rooting of Proteaceae cuttings is therefore essential to prevent dissemination of these plant pathogens to plantations, as limited control measures are presently available to control the disease (Halleen et al. 2006; Cabral et al. 2012b).