Diplodia mutila, Fr.

Pereira, Diana S. & Phillips, Alan J. L., 2023, Botryosphaeriaceae on palms-a new species of Neodeightonia, N. chamaeropicola, and new records from diseased foliage of ornamental palms in Portugal, Phytotaxa 627 (1), pp. 1921-1935 : 1921-1935

Diplodia mutila (Fr.: Fr.) Fr., Summa Vegetabilium Scandinaviae 2: 417 (1849), MycoBank MB201741

( Figure 8 )

Type: FRANCE, Ardenne, Sedan, on bark of Populus nigra ( Salicaceae ), date unknown, Montagne (isotype K 99664). PORTUGAL, Beira Litoral, Aveiro, Populus alba ( Salicaceae ), 2012, A. Alves (epitype designated by Alves et al. (2014), LISE 96136, culture ex-epitype CBS 136014). ENGLAND, Saltash, on bark of Malus sp. , 22 Aug 1935, N.E. Stevens (lectotype designated by Alves et al. (2014), BPI 599153).

Sexual morph and asexual morph reported. See Phillips et al. (2013) and Alves et al. (2014) for illustrations and descriptions.

Isolate CDP 0088. Sexual morph: Undetermined. Asexual morph: Conidiomata on palm leaf pieces in culture pycnidial, globose, non-stromatic, uniloculate, dark brown to black, solitary or aggregated, immersed in the host becoming partially erumpent when mature, occasionally semi-immersed or superficial, densely covered by greyish mycelial hairs, exuding a creamy, whitish mucoid mass or cirrus of conidia. Conidiogenous cells lining the pycnidial cavity, hyaline, smooth- and thin-walled, simple, indeterminate, cylindrical, occasionally swollen at the base, rarely ampulliform, straight or flexuous, aseptate, occasionally 1-septate, enteroblastic, proliferating at the same level giving rise to periclinal thickenings, occasionally proliferating percurrently giving rise to 1–2 annelations, rarely more, 8.9– 16.55 × 2.67–5.79 μm, 95 % confidence limits = 11.02–12.27 × 3.64–4.18 μm (mean ± SD = 11.65 ± 1.75 × 3.91 ± 0.76 μm, n = 30). Conidia oblong to obovoid, broadly rounded ends, smooth- and thick-walled, hyaline and aseptate, with granular contents, eguttulate, few becoming pale brown to brown and 1–2-septate with age, 21.21–26.85(–32.79) × 9.85–13.96(–16.17) μm, 95 % confidence limits = 23.44–24.98 × 11.92–12.78 μm (mean ± SD = 24.21 ± 2.15 × 12.35 ± 1.20 μm), mean ± SD conidium length/width ratio = 1.97 ± 0.18 (n = 30).

Culture characteristics: Colonies on 1/2 PDA, reaching 85 mm diam. after 7 d at 20 ℃ in darkness. Surface flat, raised towards the centre, with dense effuse aerial mycelium, with filamentous, entire margin, circular shape, olivaceous, becoming greyish to brownish towards the centre, opaque. Reverse greyish, becoming dark-brown to blackish towards the centre. Turning entirely black (surface) and dark bluish to black (reverse) after about 2 w. No diffusible pigment.

Material examined: PORTUGAL, Lisbon, Alvalade, Campo Grande, on foliar lesions of leaflets of Phoenix dactylifera ( Arecaceae ), 17 October 2018, Telma P.N.G. da Costa (specimen HDP 048), living culture CDP 0088 (ITS sequence OQ996219, tef1 sequence OR233679).

Hosts: Although up to 60 hosts are listed for Diplodia mutila by Farr & Rossman (2023), since the clarification of the genus concept it became clear that many of the earlier reports of this fungus could be misidentifications ( Alves et al. 2004, Phillips et al. 2013). Most recent reports confirm at least 29 genera in 20 families, including Anacardiaceae ( Pistacia vera ), Aquifoliaceae ( Ilex sp. ), Araucariaceae ( Araucaria araucana ), Arecaceae ( Phoenix dactylifera ), Cupressaceae ( Chamaecyparis lawsoniana, Cha. obtuse, Cupressus arizonica , Cu. sempervirens , Sequoia sempervirens , Thuja plicata ), Ebenaceae ( Diospyros kaki ), Fagaceae ( Quercus rubra , Q. suber ), Hippocastanaceae ( Aesculus hippocastanum ), Juglandaceae ( Juglans regia ), Lauraceae ( Persea americana ), Lythraceae ( Punica granatum ), Oleaceae ( Fraxinus excelsior , F. ornus , Olea europaea ), Pinaceae ( Abies bracteate, Cedrus atlantica , Pinus halepensis ), Pittosporaceae ( Pittosporum tobira ), Rhamnaceae ( Ziziphus jujuba ), Rosaceae ( Amelanchier alnifolia , Malus domestica , M. pumila , Prunus armeniaca , Pru. cerasus , Pru. dulcis , Pru. laurocerasus , Pru. persica , Pru. salicina , Pyrus communis ), Rutaceae ( Citrus limon , Ci. sinensis ), Salicaceae ( Populus alba , Po. nigra , Po. tremula ), Taxaceae ( Taxus baccata ) and Vitaceae ( Vitis vinifera ) ( Farr & Rossman 2023).

Distribution: Algeria, Argentina, Australia, Canada (British Columbia), Chile, France, Germany, Hungary, Iran, Italy, Montenegro, Netherlands, New Zealand, Poland, Portugal, Serbia, South Africa, Spain, United Kingdom ( England), Uruguay and USA (California, Wisconsin) ( Farr & Rossman 2023).

Notes: Based on phylogenetic analyses of the combined ITS- tef1 dataset, strain CDP 0088 clustered with the ex-epitype strain and other strains of Diplodia mutila with high PP value ( Figure 2 ). Sequence comparisons with the ex-epitype of D. mutila (CBS 136014) for ITS and tef1 showed 100 % sequence similarity for both loci. Morphologically, CDP 0088 isolated in this study is similar to the epitype of D. mutila from Populus alba in Portugal ( Alves et al. 2014). Both produce pycnidial conidiomata with oblong, thick-walled, hyaline, aseptate conidia, that may become brown and 1-septate with age. Conidial dimensions of CDP 0088 are very similar to those of the ex-epitype of D. mutila (CBS 136014) (mean = 24.21 × 12.35 μm versus 25.4 × 13.4 μm, respectively) ( Alves et al. 2014) ( Figure 8 ). Thus, based on these morpho-molecular analyses, strain CDP 0088 is here reported as D. mutila . Diplodia mutila have been previously reported on Arecaceae , namely on Phoenix dactylifera in Iran ( Mohammadi 2014) and USA (California) ( Alves et al. 2006, as Botryosphaeria stevensii ). In the present study, D. mutila is reported on P. dactylifera in Portugal ( Table 5). Alvarez-Loayza et al. (2008) reported this species on Iriatea deltoidei in Peru, but no molecular data was associated with the report, which was identified as D. mutila based on “growth characteristics and morphology of pycnidia and conidia”. Since morphology is inadequate to define genera or identify species in Botryosphaeriaceae ( Phillips et al. 2013, Slippers et al. 2013, 2014, 2017), the validity of this report is yet to be determined. Taylor & Hyde (2003) also recorded D. mutila on Arecaceae hosts, including Archontophoenix alexandrae in Malaysia and Trachycarpus fortunei in China and Switzerland, but again, identifications were based solely on morphology and thus the validity of these reports is yet to be determined. The isolate of D. mutila studied was recorded from foliar lesions of P. dactylifera , but pathogenicity has not been tested.

Figures

FIGURE 2. Phylogenetic tree of Diplodia generated from BA inference based on combined ITS and tef1 sequences. ML bootstrap support values (ML-BS ≥ 50 %) and Bayesian posterior probabilities (PP ≥ 0.85) are shown at the nodes. Strains with type status are indicated in bold font, the isolate from this study is presented in brown typeface and species are delimited with coloured blocks. The scale bar represents the expected number of nucleotide changes per site. The tree was rooted to Lasiodiplodia tropica (CGMCC 3.18477) and L. theobromae (CBS 164.96).

FIGURE 8. Diplodia mutila (CDP 0088). A. Conidiomata formed on palm leaf piece. B, C. Conidiogenous layer. D–I. Conidiogenous cells. J. Hyaline, aseptate conidia. K. Hyaline, aseptate and pigmented, 1-septate conidia. Scale bars: A = 250 μm, B, C, J, K = 10 μm, D–I = 2.5 μm.