Colletotrichum squamosae W.A.S. Vieira, V. Doyle and M.P.S. Câmara, sp. nov.

Index Fungorum number: IF 902395; Mycobank number: MB 902395; Facesoffungi number: FoF 1606; Fig. 1

Etymology – The specific epithet refers to the host Annona squamosa from where the ex-type was isolated.

Holotype – VIC49401

Associated with foliar anthracnose of Annona squamosa Sexual morph: not observed. Asexual morph: Conidiogenous cells hyaline, smooth-walled, cylindrical to ampulliform, often extending to form a new conidiogenous locus. Conidiophores 7.5–20.7 μm (av. 13.2 ± 3.7 μm) length on PDA, 8.4–35.5 μm (av. 16.8 ± 6.7 μm) on CMA, hyaline, smooth-walled to verruculose, aseptate, unbranched, Conidia on PDA 9.9–11.7 × 2.9–3.9 μm (av. 10.5 ± 0.6 × 3.4 ± 0.2 μm), length/width ratio 2.6–3.7 (av. 3.1± 0.3), CMA 9.1–12.9 × 2.6–3.8 μm (av. 10.6±0.8 × 3.2±0.3 μm), length/width ratio 2.7–4.5 (av. 5.1 ± 0.1), hyaline, one-celled, smooth-walled, cylindrical with rounded ends, contents appearing granular. Appressoria in slide cultures 3.9–7.5 × 3.1–5.4 μm (av. 6.1 ± 0.9 × 4.4 ± 0.5 μm), length/width ratio 0.8–2.4 (av. 1.4±0.3), single, medium to dark brown, smooth-walled, clavate, irregularly shaped or rarely ovoid, solitary or in small groups. Sclerotia, chlamydospores, and setae not observed.

Culture Characters – Colonies on PDA were saffron with chestnut centre in the obverse view and salmon with a brown vinaceous centre in the reverse, aerial mycelium sparse, growth rate at 25 °C 5–5.3 mm day−1 (average 5.1±0.1 mm day−1).

Material examined – Brazil, SÃO PAULO: Itapetininga, from anthracnose lesions on Annona squamosa leaves, Jun 2017, W.A.S. Vieira. VIC49401(holotype, preserved in a metabolically inactive state). Ex-type LM04, COAD3520.

Known hosts – Annona squamosa (present study), Anacardim occidentale, Carica spp. and Licania tomentosa (Lisboa et al. 2018; Veloso et al. 2018; Vieira et al. 2022).

GenBank numbers – apn2/mat-igs: OP946694, apn2: OP946695, cal: OP946696; gap2-igs: OP946697; gapdh: OP946698; GS: OP946699; tub2: OP946700.

Notes – The multilocus analyses reveal a major clade containing the Colletotrichum isolate from Annona squamosa and isolates previously identified as C. australianum and C. queenslandicum (Fig. 2). The first clade represents the species C. australianum, which includes its ex-type (UMC002) and isolates previously identified as C. queenslandicum from various hosts in Australiaandis supported by97% ML bootstrap and 0.96 BYPP support. The second clade, with maximum support in both multilocus analyses, is comprised of the ex-type of C. queenslandicum (ICMP1778) and isolate CPC17123. Finally, the third clade contains isolate LM04 from A. squamosa and isolates associated with different hosts from Brazil which were previously identified as C. queenslandicum by Veloso et al. (2018) and Lisboa et al. (2018). This strongly supported clade (ML-BS 97%; BPP 1.0), introduced as the new species Colletotrichum squamosae, did not contain an ex-type of any previously described species. APN2/MAT-IGS is the best marker to discriminate between these three species. The cal sequence places C. australianum and C. queenslandicum in a polytomous clade but separates C. squamosae from those species. Colletotrichum squamosae isolates were split among two distantly related clades in GAPDH and GS trees (polyphyletic), but these clades did not contain any isolate of C. australianum or C. queenslandicum. This issue was previously reported in GS topology for several Colletotrichcum species by Silva et al. (2012) and is first reported here for GAPDH. Although those markers are among the most informative in identifying species within the C. gloeosporioides species complex (Liu et al. 2015a, b; Vieira et al. 2017, 2020), they negatively affect species delimitation when concordance criteria are employed. The tub2 has no ability to separate C. australianum, C. queenslandicum and C. squamosae. It was not possible to evaluate APN2 and GAP2-IGS due
to the absence of sequences for C. australianum and C. queenslandicum. All isolates from Australia (Weir et al. 2012; Wang et al. 2021) were distributed among C. queenslandicum and C. australianum, while isolates from Brazil (Lisboa et al. 2018; Veloso et al. 2018; Vieira et al. 2022) were re-assigned to C. squamosae. This result indicates that these species may have a distinct geographical distribution. Morphological features show that C. squamosae conida on PDA are smaller than C. australianum (14.1–14.5×4.5–4.7) and C. queenslandicum (14.5–16.5×4.5–5). Colletotrichum squamosae appressorial shape is mostly irregular, while C. australianum and C. queenslandicum produce globose appressoria.

Figure 1 – Colletotrichum squamosae (COAD3520, holotype). a–b. Cultures on PDA, 7 d growth, from above (a) and below (b). c–d. Conidiophores and conidiogenous cells from PDA (c) and CMA (d). e–f. Conida from PDA (e) and CMA (f). g–j. Appressoria. Scale bars: C–F=10 µm; G–J=5 µm

Figure 2 – Maximum likelihood (ML) tree of the Colletotrichum gloeosporioides species complex inferred on IQ-TREE from a concatenated alignment of apn2, apn2/mat-igs, cal, gapdh, gap2-igs, gs and tub2. Significant supports for ML (SH-alrt bootstrap≥80) are shown above the nodes. Thickened branches indicate significant support from Bayesian inference analysis (posterior probability≥0.95). The tree was rooted at the midpoint. Ex-type isolates are highlighted in bold. New isolates are highlighted in blue bold. The scale bar indicates the average number of substitutions per site