Abstract

INTRODUCTION

A leaf-spotting fungus common on Metrosideros excelsa (pohutukawa, Myrtaceae) in New Zealand is morphologically similar to a number of leaf-spotting fungi on another myrtaceous host, Eucalyptus. These fungi on Eucalyptus leaves have been placed in a range of genera, including Alysidiella, Blastacervulus, and Staninwardia (Swart 1988, Sutton 1971, Summerell et al. 2006, Cheewangkoon et al. 2012), and collectively the diseases they cause on Eucalyptus have sometimes been referred to as chocolate spot (e.g. Cheewangkoon et al. 2012; Crous et al. 2016).

Cheewangkoon et al. (2012) presented a phylogeny that resolves a clade of closely-related species of Eucalyptus chocolate-spot leaf pathogens that they refer to the genera Blastacervulus, Alysidiella and Aulographina. These taxa form a well-resolved clade, and were placed in the Asterinaceae by Giraldo et al. (2017). Species in the genera Alysidiella and Blastacervulus have a similar morphology. They share acervular conidiomata with dry, powdery masses of conidia, the 0–3-septate conidia having thick, verruculate, brown to pale brown walls, base truncate, the conidiogenous cells with a broad conidiogenous locus, sometimes with annellate thickenings. The species accepted by Summerell et al. (2006) and Cheewangkoon et al. (2012) were distinguished genetically and by differences in conidial size. Crous (2016) later described another genetically similar Eucalyptus leaf spotting pathogen as Blastacervulus eucalyptorum. Aulographina was represented by DNA sequences from the culture CPC 12986 that Cheewangkoon et al. (2012) and Giraldo et al. (2017) accepted as A. eucalypti. Aulographina eucalypti differs morphologically from the other chocolate spot pathogens treated by Cheewangkoon et al. (2012) in forming a sexual morph and a putatively spermatial asexual morph (Swart 1988, Wall & Keane 1984), and in lacking the acervular conidial morph so distinctive of the other species in this clade. Although the sexual morph of A. eucalypti is morphologically typical of Asterinaceae, the lack of an acervular asexual morph means that it would be useful to confirm its genetic characterisation with additional specimens.

Staninwardia was first described by Sutton (1971), based on S. breviuscula, a Eucalyptus-associated fungus from Mauritius that is morphologically similar to the chocolate spot pathogens. Summerell et al. (2006) described a second species, S. suttonii, on Eucalyptus from Australia. Based on DNA sequencing from an ex-type culture of S. suttonii, Quaedvlieg et al. (2014) place Staninwardia in their new family Extremaceae (Capnodiales), genetically distant from the chocolate spot species of Alysidiella and Blastacervulus treated by Summerell et al. (2006) and Cheewangkoon et al. (2012). Another species with similar morphology and associated with similar symptoms on Eucalyptus was described by Sutton (1974) as Leptomelanconium australiense. Crous et al. (2009) recombined it as Teratosphaeria australiensis, and Taylor et al. (2012) selected an epitype for this species, which again proved to be genetically distant to the chocolate spot species of Cheewangkoon et al. (2012).

The New Zealand Metrosideros leaf-spotting pathogen was identified tentatively as Leptomelanconium sp. by McKenzie et al. (1999) and as Staninwardia breviuscula by Gadgil & Dick (2006). A record of S. breviuscula on Metrosideros umbellata may represent the same fungus (Bain 2007), although because of the host difference, this should be confirmed genetically. Based on DNA sequences from a recently obtained culture of the Metrosideros pathogen, we describe it here as a new species of Blastacervulus.

MATERIALS AND METHODS

Conidia from the fresh collection subsequently dried and stored as fungarium specimen PDD 108694 were suspended in streptomycin solution and streaked across a water agar plate. After 24 h germinating conidia were removed and transferred to 2 % Difco potato dextrose agar (PDA) plates. The cultures had a consistent macromorphological appearance and one was later selected and stored as ICMP 21883. DNA was extracted from mycelium from this culture, and ITS and LSU sequences generated following the methods of Johnston & Park (2013). The sequences were aligned with Alysidiella, Blastacervulus and Aulographina ITS and LSU sequences from Cheewangkoon et al. (2012), Blastacervulus eucalyptorum from Crous et al. (2016), B. eucalypti from Cheewangkoon et al. (2009) and additional Asterinaceae LSU sequences selected from sister clades in the Giraldo et al. (2017) phylogeny, with Venturia populina as the outgroup (Table 1). For taxa with both ITS and LSU sequences available, the sequences were concatenated, an alignment carried out using MAFFT v. 1.3.7 as implemented in Geneious R10 (Kearse et al. 2012), and a ML phylogenetic tree generated using PhyML v. 3.2.2 (Guindon et al. 2010) with the GTR model as implemented in Geneious R10, with support values estimated using 1 000 bootstrap replicates.

Dried specimens were rehydrated using 3 % KOH, conidia and conidiogenous cells examined in 3 % KOH in squash mounts, and the excised acervuli sectioned at about 10 µm thickness using a freezing microtome and the sections mounted in lactic acid.

RESULTS

Based on the taxa and genes sampled, there is no clear genetic difference that can be used to distinguish Alysidiella from Blastacervulus (Fig. 1). Based on published descriptions, species in the two genera are also very similar morphologically (Swart 1988, Summerell et al. 2006, Crous et al. 2016). We have chosen to refer our new species to the older genus Blastacervulus.

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Fig. 1.

PhyML maximum likelihood tree based on concatenated ITS and LSU sequences. Bootstrap support values are provided where greater than 90 %. The novel species described here is indicated in bold text.

Blastacervulus metrosideri P.R. Johnst. sp. nov.MycoBank MB829588. Fig. 2.

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Fig. 2.

Blastacervulus metrosideri. A. Immature and mature spots on Metrosideros excelsa leaf. B. Detail of one of the mature spots, with several individual erumpent acervuli. C. Acervulus in vertical section. D. Leaf in vertical section with incipient acervulus, apart from the dark-walled cells of the acervulus, fungal tissue restricted to a single layer of hyaline cells beneath the cuticle. E. Conidiogenous cells. F. Conidiogenous cell and released conidia. A, B, E, F — PDD 116628; C, D — PDD 108694. Scale bars: A = 5 mm; B = 1 mm; C = 100 µm; D = 20 µm; E, F = 10 µm.

Etymology: Refers to the host plant.

Diagnosis: Differs from the type species Blastacervulus eucalypti in host preference and conidial size and shape.

Typus: New Zealand, Auckland, Glen Innes, Auckland University Tamaki Campus, on living leaves of Metrosideros excelsa (Myrtaceae), 5 Oct. 2017, P.R. Johnston (holotype PDD 108694; ex-type culture ICMP 21883).

Leaf spots on upper surface of living leaves, initially red-brown with narrow yellow margin, becoming darker, almost black with age, round, up to 4 mm diam. Discolouration of the spots sometimes extending through to the lower leaf surface. Apart from the cells associated with the acervuli, fungal tissue is sparse within the leaf, forming a narrow plate of hyaline, thin-walled hyphae about 3–5 µm diam between the cuticle and epidermis. Little or no fungal hyphae is present more deeply in the leaf. The colour of the spots is associated with deposition of tannins or other compounds within the intact epidermal and palisade cells of the host. Acervuli develop within the spots on the upper leaf surface, 0.1–0.3 mm diam, upper wall black, breaking open irregularly to expose the black, powdery conidial mass. In vertical section acervuli develop between the cuticle and the epidermal cells, with the upper and lower walls comprising 2–3 rows of angular cells 4–7(–10) µm diam. Conidiogenous cells line the inside of both the upper and lower walls, solitary, 6–8 × 4–5 µm, cylindrical, conidiogenous locus broad, apical, often with several thickened and slightly flaring annellations. Conidia cylindrical, base truncate, apex broadly rounded, 1–3(–9)-septate, 1-septate 8–10 µm long, 2-septate 10–14 (–18) µm long, 3-septate 13–16 (–19) µm long, 4-septate 16–19 µm long, 9-septate up to 33 µm long × 3.5–6 µm wide, walls thickened, dark brown, finely verruculate.

Culture characteristics: Cultures on PDA about 15–25 mm diam after 20 wk. Margin of colony uneven, surface black, convoluted, lumpy and cracked, finely felted, brown pigment diffusing into agar. Cells in the mycelium near the edge of the colony starting to become swollen and to develop thick and dark walls, cells in the older parts of the colony almost all short, broad-cylindric, with walls thick, dark, smooth, hyphae partly disarticulating.

Additional materials examined: New Zealand, Auckland, Leigh, on Metrosideros excelsa, 30 Mar. 1924, E.G. Bollard, (PDD 43314); Te Henga, on M. excelsa, 25 Mar. 1949, J.M. Dingley (PDD 15909); Titirangi Beach, on M. excelsa, 3 Dec. 1963, F.J. Morton & J.D. Read (PDD 30158); Langholm, on M. excelsa, 3 Dec. 1963, F.J. Morton & J.D. Read (PDD 30159); Auckland City, Mt Albert Rd, on M. excelsa, Feb. 1994, P.R. Johnston (PDD 64252); Glen Innes, Auckland University Tamaki Campus, on M. excelsa, 5 Oct. 2016, P.R. Johnston (PDD 108727); Glen Innes, Colin Maiden Park, on M. excelsa, 23 Jan. 2019, P.R. Johnston (PDD 116628); Coromandel: Port Charles, between wharf and Big Sandy Bay, on M. excelsa, 26 Mar. 1989, P.R. Johnston (PDD 55197); Port Charles, on M. excelsa, 28 Mar. 1993, P.R. Johnston (PDD 62168); Port Charles, Little Sandy Bay, on M. excelsa, Nov. 1993, P.R. Johnston & E.M. Gibellini (PDD 64249); Port Charles, Big Sandy Bay, on M. excelsa, 29 Dec. 1993, P.R. Johnston & E.M. Gibellini (PDD 64236); Port Charles, Big Sandy Bay, on M. excelsa, 24 Oct. 1994, P.R. Johnston & E.M. Gibellini (PDD 64251); Northland: Bay of Islands, Black Rocks off Moturoa, north west islet of Crater Rim group, on M. excelsa, 23 Jan. 1990, R.E. Beever (PDD 56841); Westland: Hokitika, Hokitika Hospital, on M. umbellata cultivated plant, 22 May 2007, B.H. Doherty (NZFS 5422).

Notes: Symptoms that match those associated with Blastacervulus metrosideri are very common on Metrosideros excelsa wherever it grows in New Zealand. The literature cited in the Introduction shows that Eucalyptus has several superficially similar leaf-spotting fungi, and more intensive study of the M. excelsa associated fungi may reveal a greater diversity of species than currently recognised. For example, Teratosphaeria spp. were commonly detected from environmental DNA sequences from M. excelsa leaves (unpubl. data), and the symptoms caused by Teratosphaeria australiensis are similar to those associated with B. metrosideri (Sutton 1974, as Leptomelanconium australiense; Taylor et al. 2012). Commonly, the blastacervulus-like spots seen in the field are sterile, making a definitive identification based on morphology impossible.

The single, small specimen on Metrosideros umbellata (NZFS 5422) has markedly paler spots than those on M. excelsa and its acercvuli are smaller. Microscopically, this specimen appears to match those from M. excelsa. Additional specimens are needed to determine whether the macroscopic differences are consistent, and DNA sequences from a specimen on M. umbellata would confirm whether M. metrosideri occurs on more than one species of Metrosideros.

The holotype specimen was selected because a culture and DNA sequences were derived from it, but this specimen is not large. To examine the morphology, particularly nice specimens include PDD 30158 and PDD 116628.

ACKNOWLEDGEMENTS

REFERENCES