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Australasian Plant Pathology, 2009, 38, 505–513

Three new Phaeoacremonium species on grapevines in New Zealand A. B. Graham A, P. R. Johnston B,C and B. S. Weir A,B A

Corbans Viticulture, 8 Bristol Road, Whenuapai, Waitakere 0618, New Zealand. Landcare Research, Private Bag 92170, Auckland 1142, New Zealand. C Corresponding author. Email: [email protected] B

Abstract. Three new species of Phaeoacremonium (Pm.) found in discoloured wood of grapevine are described and named Pm. armeniacum, Pm. globosum and Pm. occidentale. Phaeomoniella chlamydospora was isolated from the same vines, but no other Phaeoacremonium spp. were present. Phaeoacremonium spp. have been associated with Petri and esca diseases in grapevine. In pathogenicity trials, the new Phaeoacremonium spp. consistently caused brown discolouration in the inoculated wood. All species caused mortality of cv. 101–14 rootstock cuttings, 22–60% of cuttings surviving 10 weeks after inoculation. Rootstock cv. 5C was less susceptible, with 80–100% of cuttings surviving. The three new species of Phaeoacremonium are genetically distinct from all those previously described and also show subtle morphological differences in the structure and size of the phialides and conidia. Phylogenetic analysis of b-tubulin and actin genes showed that the new species Pm. globosum and Pm. armeniacum are closely related to Pm. argentinense from Argentina, while Pm. occidentale is more closely related to Pm. mortoniae from vineyards in the northern hemisphere. The new species were isolated from rootstock imported into New Zealand ~25 years ago from California. They are not closely related to species known from grape in California, and no conclusion can be made about whether they were imported in the recent past on infected plants, or if they are indigenous to New Zealand. Many Phaeoacremonium species have a broad host range, and more intensive surveys of the native New Zealand flora and vineyards are needed before their origin can be determined. Additional keywords: Togninia, Vitis vinifera.

Introduction Phaeoacremonium W. Gams, Crous & M.J. Wingf. (teleomorph Togninia Berl.) is a member of the Diaporthales. The genus comprises 28 species (Mostert et al. 2006; Damm et al. 2008), many of which are found associated with discoloured vascular tissue of woody plants. The esca and Petri diseases of grapevines are often associated with Phaeoacremonium (Pm.) species together with a complex of other fungi, including Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingf. & L. Mugnai) Crous & W. Gams (Pa. chlamydospora), and basidiomycetes such as Fomitiporia spp., Stereum hirsutum (Willd.) Pers. and Inonotus sp. (Mugnai et al. 1999; Ridgway et al. 2005; Edwards et al. 2006; Mostert et al. 2006). The role of each of the various species in the development of the vascular diseases of grape is poorly understood. Surveys of fungi inhabiting grape wood in New Zealand have shown Pa. chlamydospora to be common in streaked vascular tissue (Reed-Graham 2006; M. V. Jaspers, pers. comm.; M. A. Manning, pers. comm.). Ridgway et al. (2005) reported Pm. aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai, one of the most widespread grape-inhabiting Phaeoacremonium species, from New Zealand but this report was not supported by a voucher culture, and the species has not been found in more recent surveys of declining vines (ReedGraham 2006; M. Jaspers, pers. comm.; M. Manning, pers.  Australasian Plant Pathology Society 2009

comm.). The only other report of Phaeoacremonium from New Zealand is Pm. novae-zealandiae L. Mostert, W. Gams & Crous, which has been isolated from Pinus radiata, Cupressus macrocarpa, and Desmoschoenus spiralis (Mostert et al. 2006). Réblová et al. (2004) described the Togninia-like species Togniniella acerosa Réblová, L. Mostert, W. Gams & Crous from dead, fallen wood in native forests in New Zealand. This fungus was distinguished from Togninia on the basis of large subunit and small subunit rDNA sequences and its Phaeoacremonium-like anamorph was given the new genus Phaeocrella Réblová, L. Mostert, W. Gams, Crous (Réblová et al. 2004). Here we describe three new species of Phaeoacremonium from discoloured vascular tissue from grapevine rootstock wood from Auckland, New Zealand. They are distinguished from the 28 species described to date (Damm et al. 2008) by morphology in culture and b-tubulin (TUB) and actin (ACT) gene sequences. The pathogenicity of these new species towards two varieties of grapevine rootstock varieties was investigated. Materials and methods Fungal isolations Twenty grapevine rootstock cordons were examined from a small block of Vitis berlandieri  V. riparia cv. 5C rootstock vines. The vines were externally healthy but internally showed 10.1071/AP09035

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vascular discolouration typical of that associated with Petri disease. These vines were originally imported from California ~25 years ago. For each cordon, trunks were sliced in cross section five times and isolations made from discoloured woody tissue. Pieces of discoloured tissue (2 mm3), freshly exposed by slicing the surface of the wood with a sterile scalpel, were excised and without surface sterilisation placed on malt extract agar (MEA, 2% malt extract, Becton, Dickenson and Co., Franklin Lakes, NJ, USA; 1.5% standard agar, Germantown Co., Manukau, New Zealand) amended with 0.1% yeast extract (Difco, Detroit, MI, USA), 0.1 g/L tetracycline (SigmaAldrich, St Louis, MO, USA), and 0.1 g/mL Benlate (active ingredient benomyl – DuPont, Wilmington, DE, USA). These were grown at ~20C under a 12-h light/dark regime and examined for growth of fungi periodically over 2 weeks. Colonies were subcultured onto MEA and identified by morphology or by their internal transcribed spacer (ITS) sequences, as described below. The Phaeoacremonium species were described from culture using the methods of Mostert et al. (2003). Cultures were grown on MEA, oatmeal agar (OA, Difco) and potato dextrose agar (PDA, Difco) in the dark at 25C, with four replicates of each. Growth rates were recorded and colony descriptions made after 14 days. Colour codes follow Kornerup and Wanscher (1963). Microscopic features of the hyphae, conidia, conidiophores and conidiogenous cells, from cultures on MEA were photographed and described in lactic acid on glass slides, terminology following Mostert et al. (2006). Living cultures were deposited in the International Collection of Microorganisms from Plants (ICMP, Landcare Research, Auckland, New Zealand), and are maintained in this collection in an inactively inert state over liquid nitrogen, and dried agar plates of the cultures were deposited in the New Zealand Fungal Herbarium (PDD). DNA extraction, PCR amplification and sequencing DNA was extracted from fungal cultures and inoculated vines with a REDExtract-N-Amp plant PCR kit (Sigma-Aldrich). Three genes [ITS, TUB and ACT] were PCR amplified according to the protocols of Mostert et al. (2006). PCR amplifications were performed with FastStart polymerase (Roche Applied Sciences, Indianapolis, IN, US) and an Applied Biosystems 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). For ITS, the PCR primers were ITS-1 (TCC GTA GGT GAA CCT GCG G) and ITS-4 (TCC TCC GCT TAT TGA TAT GC) (White et al. 1990) the cycle conditions were: 95C – 4 min, then 35 cycles of 95C – 45 s, 49C – 45 s, 72C – 60 s, then 72C for 7 min. For ACT, the PCR primers were ACT-512F (ATG TGC AAG GCC GGT TTC GC), and ACT-783R (TAC GAG TCC TTC TGG CCC AT) (Carbone and Kohn 1999). The cycle conditions were: 96C – 5 min, then 35 cycles of 94C – 30 s, 52C – 30 s, 72C – 80 s, then 72C for 7 min. For TUB, the primers were T1 (AAC ATG CGT GAG ATT GTA AGT) (O’Donnell and Cigelnik 1997), and Bt2b (ACC CTC AGT GTA GTG ACC CTT GGC) (Glass and Donaldson 1995). The cycle conditions were: 96C – 5 min, then 36 cycles of 94C – 30 s, 58C – 30 s, 72C – 80 s, then 72C for 7 min. PCR products were purified with a Qiagen MinElute 96 UF plate (Qiagen, Germantown, MD, US). Purified products were

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cycle sequenced using the appropriate primers and BigDye terminator cycle sequencing chemistry 3.1 (Applied Biosystems). Sequences were obtained in both directions with an ABI 3100 Prism genetic analyser (Applied Biosystems) and were assembled and edited with Sequencher 4.5 (Gene Codes Corp., Ann Arbor, MI, USA). Phylogenetic analysis Phylogenetic analysis was performed on DNA sequences obtained from the novel Phaeoacremonium isolates together with reference sequences of other Phaeoacremonium species obtained from Mostert et al. (2006) and Damm et al. (2008). TUB and ACT sequence alignments were constructed with Prankster (Loytynoja and Goldman 2005) then concatenated. Multiple methods were used to construct phylogenetic trees. For DNA maximum likelihood (ML) trees the appropriate parameters were selected with Modeltest version 3.7 (Posada and Crondall 1998). The Akaike Information Criterion test was used to determine which model best fitted the sequence data. The HKY + I + G model of DNA evolution was selected. The model parameters (base frequencies, proportion of invariant sites, gamma distribution shape parameter, and substitution rate matrix) were then specified in PAUP* 4.0b10 (Swofford 2002) to build phylograms with tree-bisection-reconnection heuristics, and 30 replicates run. ML bootstraps were performed with PhyML (Guindon et al. 2005). DNA sequences were also analysed under Bayesian inference using MrBayes 3.12 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). For these analyses, exact specification of model parameters was not possible. The equivalent of the GTR + I + G model was used with a ‘flat’ Dirichlet distribution (all distribution parameters set to 1), as the prior probability for both the stationary state frequencies and the substitution rates. Separate unlinked partitions were created for each gene. The analysis was run for 10-million generations (sampling every 1000), with the first 25% discarded as ‘burn in’. Pathogenicity trials Pathogenicity of the new Phaeoacremonium species and Pa. chlamydospora was tested. Dense suspensions of conidia (~1  107 conidia/mL) were prepared by flooding the surface of 21-day-old MEA cultures with sterile water, and gently rubbing the surface with a sterile rod. The pathogenicity of four isolates of Pm. globosum (ICMP 16987, 16988, 17038, 17039), and a single isolate each of Pm. occidentale (ICMP 17037), Pm. armeniacum (ICMP 17421), and Pa. chlamydospora (ICMP 15813) were tested. A 20-ml aliquot of each suspension was transferred to MEA plates and incubated at 25C for 16 days to check the viability of the inoculum. Dormant rootstock cuttings of two rootstock cultivars (V. berlandieri  V. riparia cv. 5C and V. rupestris  V. riparia cv. 101–14) were grafted with Sauvignon Blanc clone 1 budwood. For each isolate, groups of 20 grafted 5C and 101–14 cuttings were inoculated directly into the graft wound with 20 mL of inoculum using a fresh autoclaved pipette tip for each inoculation. Control cuttings were grafted at the same time, and were either uninoculated or inoculated with sterile water. Grafted rootstocks were waxed and placed in the

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callusing room for 3 weeks, after which time they were removed from callusing media. At the time they were removed from the callusing media, isolations were made from xylem 1–2 cm below the inoculation site of six of the grafted cuttings from each group, using MEA amended with tetracycline and Benlate as previously described. The fungi isolated were identified by morphology or by sequencing of the ITS region. For each group of inoculated plants, five of the remaining grafted cuttings were planted in pots and propagated in a glasshouse, and the rest were planted in the field. Survival of the vines was assessed 10 weeks after inoculation, and for those still alive, external and internal symptoms were noted and the presence of the inoculated species was checked in a single vine for each isolate, using the t-restriction fragment length polymorphism method described by Weir and Graham (2008). Statistical analysis of data from the pathogenicity trials was conducted by comparing two sample proportions with Chi-square analysis (Moore and McCabe 1996). Results Fungal isolations from mature 5C rootstock vines Botryosphaeria parva, B. lutea, and Phomopsis viticola were isolated from wedge-shaped areas of necrosis near old pruning wounds. Several basidiomycetes were isolated from necrotic portions of discoloured wood including species of Baeospora (GenBank EU770252, voucher ICMP 16979), Sistotrema (GenBank EU770230, voucher ICMP 17490), and Trichosporon (GenBank EU770241, voucher ICMP 16990). Pa. chlamydospora was common in all vines sampled, being consistently isolated from small, black patches of wood containing a tar-like substance (Fig. 1). The Phaeoacremonium species were isolated from brown, grey-brown and orange-brown areas of the wood towards the centre of the trunk (Fig. 1a, b). Pm. globosum and Pm. armeniacum were each found in two vines, Pm. occidentale in a single vine.

(a)

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Taxonomy Phaeoacremonium armeniacum A.B. Graham, P.R. Johnst. & B. Weir sp. nov. (Figs 2a, 3) MYCOBANK # 513299. Holotypus: New Zealand, Auckland, Whenuapai, on V. berlandieri  V. riparia hybrid rootstock, vascular staining of living wood, A. Graham ES68, 21 March 2007, ICMP 17421 (PDD 93586 is a dried subculture from holotype). In mycelio aerio hyphae singulae vel ad 5 fasciculatae, plerumque hyalinae, leaves. Conidiophora nulla. Phialides 9.5–17  2–3 mm, terminales vel laterales, praecipue typi I vel typi II, cylindricae vel elongato-ampulliformes. Conidia 6.5–9 (–12)  1.5–2.5 mm, oblongo-ellipsoidea, hyalinae, 0–septatae; in agaro 6.5–9 (–12)  1.5–2.5 mm, plerumque allantoidea. Etymology: armeniacus = apricot coloured; refers to the pigmentation of the infected wood from which this species was isolated. Hyphae smooth, hyaline to brown, sometimes aggregated into strands up to ~5 hyphae wide. Strands of grouped hyphae sometimes arranged in small coils, with conidiogenous cells held on the outside of these coils. Conidiogenous cells terminal and lateral, mostly monophialidic, rarely with 2–3 conidiogenous cells held on a one-celled, short-cylindric conidiophore. Conidiogenous cells 9.5–17  2–3 mm, hyaline, Type I and Type II (sensu Mostert et al. 2006), subcylindrical to elongateampulliform, often slightly swollen at base, not constricted; conidiogenous locus with non-flaring collarette ~1–1.5 mm long and wide. Conidia 4.0–5.5  1.5–2 (–2.5) mm, oblongelliptic, straight or irregularly curved, ends broadly rounded, 0–septate, hyaline. Conidia on agar surface 6.5–9 (–12)  1.5–2.5 mm, cylindric, curved, allantoid, 0–septate, hyaline. Cultural characteristics: Colonies on MEA 30–32 mm diam. after 16 days, aerial mycelium cottony, uniform in height, pale greyish-green (28B2), with scattered, irregular pinkish patches, margin entire. PDA 18–19 mm diam. after 16 days, aerial mycelium cottony, sparse towards margin, greyish-red (7B2),

(b)

Fig. 1. Cross sections through grapevine cordons of mature Vitis berlandieri  V. riparia cv. 5C rootstock vines: (a) grey-brown discolouration associated with Phaeoacremonium occidentale, dark brown discolouration associated with Pm. globosum, and black spots of Phaeomoniella chlamydospora; (b) orange-brown rot of Pm. armeniacum together with numerous Pa. chlamydospora infections.

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white to margin, agar surface undulate with radiate ridges, in reverse reddish, margin entire. OA 20–21 mm diam. after 16 days, aerial mycelium cottony, in ropey upright strands, pale bluish grey (20C2) with irregular greyish red patches, yellow in reverse, yellow pigment diffusing across agar, margin slightly and irregularly scalloped.

(a)

Phaeoacremonium globosum A.B. Graham, P.R. Johnst. & B. Weir sp. nov. (Figs 2b, 4)

(b)

(c)

Fig. 2. Fourteen-day-old cultures on (from left to right) malt extract agar, potato dextrose agar and oatmeal agar: (a) Phaeoacremonium armeniacum [International Collection of Microorganisms from Plants (ICMP) 17421]; (b) Pm. globosum (ICMP 16988); and (c) Pm. occidentale (ICMP 17037).

(a)

(e)

(b)

(f )

MYCOBANK # 513300. Holotypus: New Zealand, Auckland, Whenuapai, on V. berlandieri  V. riparia, vascular staining of living wood, B. Weir ES63, 16 Jan. 2007, ICMP 16988 (PDD 92917 is a dried subculture from holotype). In mycelio aerio hyphae singulae vel ad 10 fasciculatae, brunneae vel hyalinae, laeves vel verrucosae. Conidiophora plerumque ramosa. Phialides (6.5–) 8–12  2–3 mm, terminales vel laterales, praecipue typi II, ampulliformes vel subuliformes, interdum fundo tumido. Conidia 4.5–6.5  1.5–2 mm, oblongoellipsoidea, hyalinae, 0–septatae; in agaro 5–7.5  1.5–2.5 mm, plerumque allantoidea. Etymology: globosus; refers to the distinct, globose shape to the base of many of the conidiogenous cells. Hyphae smooth or rarely with warts, hyaline to brown, brown hyphae often with verrucose walls, hyphae sometimes aggregated into strands up to ~10 hyphae wide. Conidiophores irregularly and often copiously branched, comprising cells 2–5 mm long. (c)

(d)

(g)

(h)

Fig. 3. Phaeoacremonium armeniacum [International Collection of Microorganisms from Plants (ICMP) 17421] on malt extract agar. (a) conidiogenous cells mostly solitary; (b) conidiogenous cells with prominent collarette; (c) hyphae sometimes forming coils, conidiogenous cells held on outside of coil; (d ) conidiogenous cells rarely held on short conidiophore; (e) conidia from aerial mycelium; ( f ) conidia from agar surface; (g) smooth-walled hyphae; and (h) hyphae rarely with finely encrusted walls. Bars = 20 mm (a); 10 mm (b–h).

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(b)

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Fig. 4. Phaeoacremonium globosum [International Collection of Microorganisms from Plants (ICMP) 17038] on malt extract agar. (a) and (b) conidiophores and conidiogenous cells formed on mycelial strand; (c) ampulliform to subulate conidiogenous cells on conidiophores; (d) and (e) conidiogenous cells with swollen, globose bases; ( f ) conidia; (g) coarsely encrusted hyphae. Bars = 20 mm (a, b); 10 mm (c–g).

Conidiogenous cells (6.5–) 8–12  2–3 mm, pale brown to hyaline, mostly Type II (sensu Mostert et al. 2006), variable in shape, some ampulliform to subulate, slightly constricted at base, others with swollen, almost globose base, and in this case the adjacent cells of the conidiophore similarly swollen and constricted at septa; conidiogenous locus with non-flaring collarette ~1–1.5 mm long and wide, often with lateral conidiogenous locus on a short, narrow branch below septa on conidiophore. Conidia 4.5–6.5  1.5–2 mm, oblong-elliptic, straight to slightly curved, ends rounded, 0–septate, hyaline. Conidia on agar surface 5–7.5  1.5–2.5 mm, often curved, allantoid, cells within conidiophores and conidiogenous cells longer than in aerial mycelium, 12–20 mm long, mixture of Type II and Type III phialides. Cultural characteristics: Colonies on MEA 28–34 mm diam. after 16 days, aerial mycelium almost lacking, colony surface flat, dark brown (9F7) in patches towards the centre, margin entire, yellow pigmentation diffusing from edge of colony across plate. PDA 20–25 mm diam. after 16 days, aerial mycelium sparse, arranged in small upright tufts, agar surface undulate with radiate

ridges, in reverse greyish-yellow (4B5), darker and more orange towards margin (5C8), bright yellow pigment diffusing across plate, margin scalloped. OA 23–33 mm diam. after 16 days, aerial mycelium cottony, in ropey upright strands, grey with a hint of yellow (2C2), yellow in reverse, yellow pigment diffusing across plate, margin scalloped. Notes: All isolates were crossed following the methods of Mostert et al. (2006), but no teleomorph was formed. Other specimens examined: New Zealand: Auckland: Whenuapai, on V. berlandieri  V. riparia hybrid rootstock, vascular staining of living wood, B. Weir ES62, 16 Jan. 2007, ICMP 16987 (dried culture PDD 92918); ibid. B. Weir ES66, 16 Jan. 2007, ICMP 17039 (dried culture PDD 92919); ibid. B. Weir ES67, 16 Jan. 2007, ICMP 17038 (dried culture PDD 92920).

Phaeoacremonium occidentale A.B. Graham, P.R. Johnst. & B. Weir, sp. nov. (Figs 2c, 5) MYCOBANK # 513301. Holotypus: New Zealand, Auckland, Whenuapai, on V. berlandieri  V. riparia hybrid rootstock, vascular staining

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(a)

(d)

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Fig. 5. Phaeoacremonium occidentale [International Collection of Microorganisms from Plants (ICMP) 17037] on malt extract agar. (a) and (b) conidiogenous cells; (c) hyphae with coarse warts; (d) and (e) detail of conidiogenous cells, mostly solitary, sometimes on short conidiophores; ( f ) conidia from aerial mycelium. Bars = 20 mm (a, b); 10 mm (c–f ).

of living wood, A. Graham ES76, 21 Mar. 2007, ICMP 17037 (PDD 92921 is a dried suculture derived from holotype). In mycelio aerio hyphae singulae vel ad 10–15 fasciculatae, plerumque hyalinae, laeves. Conidiophora interdum ramosa. Phialides (4–) 10.5–17  2–3 mm, terminales vel laterales, praecipue typi III, elongato-ampulliformes. Conidia 4–5  2 mm, oblongo-ellipsoidea, hyalinae, 0-septatae; in agaro 4.5–9.5  1.5–2 mm, plerumque allantoidea. Etymology: occidentalis = west; refers to the type locality, the west of Auckland having a long history of growing vines and making wine. Hyphae mostly smooth, rarely with warts, sometimes aggregated into strands of 10–15 hyphae, walls mostly hyaline, sometimes pale brown when aggregated. Conidiophores sparingly branched or lacking, comprising cells 10–15 mm long. Conidiogenous cells (4–)10.5–17  2–3 mm, wall smooth, hyaline, mostly Type III, a few Type I (sensu Mostert et al. 2006), mostly elongate-ampulliform, a few subulate and constricted at basal septum; conidiogenous locus with nonflaring collarette 2–2.5 mm long, 1.5 mm wide, occasionally polyphialidic with a lateral as well as an apical conidiogenous locus, occasionally with a lateral conidiogenous locus on a short, narrow branch below septa on conidiophore. Conidia 4–5  2 mm, oblong-elliptic to elliptic, ends rounded, 0-septate, hyaline. Conidia on agar surface 4.5–9.5  1.5–2 mm, cylindric, curved, 0–septate, hyaline. Cultural characteristics: Colonies on MEA 48–49 mm diam. after 16 days, aerial mycelium almost lacking, colony surface flat,

greyish yellow (3B3), margin entire, slight yellow pigmentation diffusing into agar. PDA 30–40 mm diam. after 16 days, aerial mycelium sparse, low white, tufted, agar surface smooth, small irregular lumps towards centre, no distinct pigments in reverse, margin slightly scalloped. OA 24 mm diam. after 16 days, aerial mycelium sparse, white, in short, cottony tufts, agar surface smooth, yellow pigmentation in reverse, margin entire. Molecular analyses GenBank reference numbers to sequences generated are provided in Table 1. The ITS region was inadequate for differentiation of Phaeoacremonium species, and not used in the analyses. Sequences for the protein-coding genes ACT and TUB were Table 1. GenBank accession numbers for the internal transcribed spacer (ITS), actin and b-tubulin sequences of Phaeoacremonium isolates. Type strain indicated byT ICMP, International Collection of Microorganisms from Plants ICMP no. 16987 16988 17038 17039 17037 17421

Species

ITS

Actin

b-tubulin

Pm. globosum
 Pm. globosumT 
 Pm. globosum
 Pm. globosum Pm. occidentaleT Pm. armeniacumT

EU770228 EU770229 EU770225 EU770227 EU770226 EU770224

EU595459 EU595466 EU595465 EU595462 EU595460 EU595463

EU596527 EU596525 EU596521 EU596522 EU596524 EU596526

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field trials (Table 2). The survival of these vines was not significantly different between isolates (P > 0.05), but was significantly different from the controls (P  0.01). There was 100% survival of the cv. 5C grafted vines when inoculated with either Pm. occidentale, Pm. armeniacum or Pa. chlamydospora. For all of the Phaeoacremonium and Pa. chlamydospora isolates, longitudinal sections of inoculated stems showed brown streaking of the xylem particularly below the inoculation site. Externally, there was visible stunting of shoots in approximately half of the cv. 5C vines inoculated with Pm. globosum and Pm. occidentale. There were no visible external symptoms in vines inoculated with the other two species, or in the controls. The cv. 101–14 grafted rootstock vines showed a greater incidence of disease symptoms than the cv. 5C when inoculated with any of the Phaeoacremonium and

concatenated for analysis and gave a well resolved tree (Fig. 6). The phylogenetic tree shows excellent support for the named species with all the posterior probabilities of the clades containing the new taxa at 1.00, the somewhat cryptic morphological distinctiveness of the new species being clearly supported phylogenetically. Pathogenicity trials All inoculum suspensions were viable at the time of inoculation, and each inoculated species was successfully reisolated from most of the samples taken from vines 3 weeks after inoculation and again in the vines sampled after the field trial. The survival of the grafted cv. 5C grafted cuttings inoculated with the each of the four isolates of Pm. globosum ranged from 80 to 100% in the glasshouse trial and from 89 to 100% in the

Pm. globosum ICMP17038 Pm. globosum ICMP17039 1

Pm. globosum ICMP16988 1

T

Pm. globosum ICMP16987

1

Pm. armeniacum ICMP17421T T. argentinensis CBS777.83 Pm. mortoniae CBS101585

1 1

Pm. occidentale ICMP17037

1

T. novae-zealandiae CBS110156

0.65

1

T

T. africana STE-U 6177 1

Pm. prunicolum STE-U 5967 T. griseo-olivacea STE-U 5966 1

T. viticola CBS101738 1

Pm. angustius CBS114992

0.99

T. austroafricana CBS112949

1

Pm. theobromatis CBS111586

0.96

T. minima CBS246.91 1

Pm. iranianum CBS101357 Pm. scolyti CBS113597 0.97 1

1

Pm. griseorubrum CBS111657 Pm. amstelodamense CBS110627

0.79

Pm. australiense CBS113589 1

1

Pm. subulatum CBS113584 Pm. tardigrescens CBS110573

1

Pm. alvesii CBS110034 1

1

T. rubrigena CBS498.94 T. parasitica CBS860.73 Pm. inflatipes CBS391.71

1

1

T. vibratilis CBS 117115 0.84

Pm. venezuelense CBS651.85 1 0.7

Pm. fuscum STE-U 5969 Pm. sphinctrophorum CBS337.90 0.99

T. krajdenii CBS109479 Wuestneia molokaiensis CBS114877 Pleurostomophora richardsiae CBS270.33

0.05

Fig. 6. A 50% majority rule consensus Bayesian phylogenetic tree showing the phylogenetic tree obtained using concatenated b-tubulin and actin sequences. Strains in bold were sequenced in this analysis, all other data from Mostert et al. (2006), and Damm et al. (2008). Pm. – Phaeoacremonium, T. – Togninia. Type strain indicated by T.

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Table 2. SurvivalA of 10-week-old grafted cv. 5C and cv. 101–14 rootstock vines grown in the glasshouse and in the field after inoculation with Phaeoacremonium (Pm.) species and Phaeomoniella chlamydospora ICMP, International Collection of Microorganisms from Plants Fungus (isolate)

Pm. globosum (ICMP 16987) Pm. globosum (ICMP 16988) Pm. globosum (ICMP 17039) Pm. globosum (ICMP 17038) Pm. occidentale (ICMP 17037) Pm. armeniacum (ICMP 17421) Phaeomoniella chlamydospora (ICMP 15813) Uninoculated control 1 Uninoculated control 2 Uninoculated control 3 Uninoculated control 4 Water inoculated control A

cv. 5C cv. 101–14 rootstock rootstock Glasshouse Field Glasshouse Field 80 100 80 80 100 100 100

89 89 100 89 89 100 100

60 60 40 60 60 60 0

45 45 22 33 45 33 0

100 100 100 100 100

89 100 100 100 100

80 – – – –

100 – – – –

Survival of cv. 5C rootstock vines inoculated with Pm. globosum significantly less than the controls (P  0.01). Survival of cv. 5C rootstock vines inoculated with Pm. occidentale, Pm. armeniacum and Phaeomoniella chlamydospora, respectively, not significantly less than controls (P > 0.05). Survival of cv. 101–14 rootstock vines significantly less than controls (P  0.01) for each of the fungi inoculated.

Pa. chlamydospora isolates (Table 2). Survival of grafted vines ranged from 40 to 60% in the glasshouse trial and from 22 to 45% for the field trial, which for each of the fungi tested was significantly less than survival in the controls (P  0.01). None of the cv. 101–14 vines inoculated with Pa. chlamydospora survived. Internal symptoms adjacent to the site of inoculation with all of the Phaeoacremonium isolates comprised brown streaking in the xylem. The internal symptoms of brown streaking caused by this fungus were similar to those of the cv. 5C, and there was inhibition of callus growth at the graft. External symptoms in cv. 101–14 included stunted shoot and root initiation, and incomplete callus formation at the graft union, resulting in weak, easily broken grafts. Discussion Phaeoacremonium species occur as part of a disease complex with Pa. chlamydospora causing Petri disease in younger vines and with several basidiomycete species causing esca in older vines (Mugnai et al. 1999; Edwards et al. 2006; Fischer 2006). The 25-year-old rootstock cordons from which the new Phaeoacremonium spp. were isolated did not show the ‘tiger stripe’ pattern of chlorosis typical of esca, and symptoms of Petri disease were restricted to vascular staining. Pm. globosum and Pm. armeniacum were found in localised regions of dark brown rot and orange-brown rot in the heart of 2 of the 20 vines surveyed. Pm. occidentale was isolated from a single vine from a wider band of grey-brown discolouration in the wood tissue and with no associated softening of the wood. In contrast, Pa. chlamydospora was present in all vines surveyed. The heart rots in vines with esca are typically caused by basidiomycetes. None of the genera most commonly reported

from other parts of the world as causing esca, Fomitiporia, Phellinus and Inonotus (Mugnai et al. 1999; Edwards et al. 2006; Fischer 2006), were found in the vines surveyed during this study, and none have been reported from grape in New Zealand. Of the basidiomycete genera we isolated, some species of Baeospora and Sistotrema have been reported to cause white and brown rots, respectively (Berg and Verhoef 1998; Kim et al. 2005), but have not previously been reported from grapevines. None of the vines we sampled showed foliar symptoms of esca, and the effect of these basidiomycetes on the health of the vines may well be relatively benign. The reduced survival rate of inoculated vines in this study showed that cv. 101–14 rootstock is more susceptible to the Phaeoacremonium spp. and Pa. chlamydospora than cv. 5C rootstock. Pm. aleophilum and Pa. chlamydospora can be spread to other vines through pruning wounds (Eskalen et al. 2007) and Pa. chlamydospora can be transmitted to young vines through the use of canes from infected rootstock as grafting material (Whiteman et al. 2007). This suggests that these new Phaeoacremonium spp. have the potential to increase the mortality of young vines grafted with cv. 101–14. It is not known how these new Phaeoacremonium species affect mature cv. 101–14 vines. The new Phaeoacremonium spp. were limited to a small block of mature cv. 5C rootstock vines. These vines were cultivated from rootstock imported from California 25 years ago, with the possibility that the species described in this paper may have been introduced at the same time. However, the Phaeoacremonium spp. reported here are not phylogenitically close to the seven species reported from grapevine material in the USA (Mostert et al. 2006). The new species described here are present in a single clade (Fig. 6). This clade also contains one other New Zealand species, Pm. novae-zealandiae. The new species Pm. globosum and Pm. armeniacum are closely related to Pm. argentinense from Argentina. Pm. occidentale is more closely related to Pm. mortoniae from vineyards in the northern hemisphere, with both these species having a sister relationship to another New Zealand species, Pm. novae-zealandiae. The biology of Pm. novae-zealandiae is poorly understood, being reported from exotic conifers and a native sedge (Réblová et al. 2004). Other members of this clade are Togninia africana and Pm. prunicolum, both from Prunus in South Africa. No clear conclusion can be made if the novel species described here are originate in New Zealand or were introduced in the recent past. Many Phaeoacremonium species have a broad host range (Mostert et al. 2006) and some grape-inhabiting species have been found on other hosts in adjacent areas (Rooney-Latham et al. 2005). Broader surveys of native New Zealand flora and vineyards are thus needed before the origin of these new species can be determined.

Acknowledgements We thank Mike Manning of HortResearch and Marlene Jaspers from Lincoln University for their information from surveys of fungal diseases of grapevines in New Zealand. Peter Johnston was funded in part by the New Zealand Foundation for Research, Science and Technology through the Defining New Zealand’s Land Biota OBI and Bevan Weir was funded by Corbans Viticulture.

New Phaeoacremonium species in New Zealand

Australasian Plant Pathology

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Manuscript received 13 October 2008, accepted 20 May 2009

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