Haplohelminthosporium calami Konta & K.D. Hyde, sp. nov.

MycoBank number: MB 557874; Index Fungorum number: IF 557874; Facesoffungi number: FoF 09170; Figure 3

Etymology: Referring to the genus of palm trees Calamus L.

Holotype: MFLU 20-0520.

Saprobic on living leaves and petioles of Calamus sp. On living leaves, small spots, circular to irregular, yellow in the beginning, later becoming red-brown surrounded by yellow. Colonies on natural substrate forming black patches on the upper leaf, petiole surfaces. Sexual morph: Undetermined. Asexual morph: Mycelium mostly immersed, on the surface forming small stroma-like aggregations of red brown pseudoparenchymatous stromal cells (7–)10–14(–20) µm (x = 12 µm). Conidiophores (110–)140–175(–215) x (4–)5–7(–8) µm (x = 160 x 6 µm, n = 50), wide at the base and apex, macronematous, mononematous, arising singly or fasciculate from the stroma cells, erect, simple, unbranched, straight, curved and swollen at the apex, thick-walled, cylindrical, smooth, bulbous at base, hyaline in the middle, brown to red-brown at 1–2-cells above the base, pale brown to red brown at the last cell of the apex, (3–)4–5(–6) septa. Conidiogenous cells monotretic, terminal, determinate, cylindrical, with well-defined small noncicatrized pores at the apex, wide and yellow-brown at the apex. Conidia (55–)70–100(–120) x (13–)17–20(–23) µm (x = 80   20 µm, n = 60), one on each conidiophore, obpyriform to lageniform, straight or curved, smooth, olive-brown, (3–)4–6(–7)-distoseptate, with a dark scar at the base.

Culture characteristics: Culture on PDA, colony yellow-gray-brown at the center, turning dull creamy white toward to margin, smooth, dense, zonate at the margin (Figure 3X). Material examined: THAILAND, Krabi Province, on living leaves and petioles of Calamus sp. (Arecaceae), 14 December 2015, Sirinapa Konta, KHNPR-2 (MFLU 20-0520, holotype); ex-type living culture, MFLUCC 18-0074.

Notes: BLAST search of the ITS sequence of the newly described strain (Haplohelminthosporium calami) shows 88.89% similarity with Helminthosporium juglandinum (L118), the LSU sequence shows 98.75% similarity with H. aquaticum (MFLUCC 15-0357), and the SSU sequence shows 99.52% similarity with H. quercinum (L90). Based on ITS phylogenetic analysis, Haplohelminthosporium calami formed a single branch at the basal clades of Helminthosporiella and Helminthosporium (Figure 1A), while based on LSU analysis, Hap. calami clustered together with H. juglandinum (L97), H. endiandrae (CBS 138902, MH878637), and Hel. stilbacea with no strong statistical support for both analyses. The phylogenetic results of the combined dataset indicated that Hap. calami clustered with H. endiandrae (CBS 138902, MH878637) without strong bootstrap support (Figure 2). Comparison of base pair differences between LSU loci for isolates of Hap. calami strains MFLUCC 18-0074 and H. endiandrae strains CBS 138,902 (KP004478; Ex-type from the holotype, and MH878637; sister strain) including gaps showed 1.74% (15/861 bp) differences, and the position of each base pair difference is shown in Table 3. Other H. endiandrae strains (AKMR1, CBS 138902; ex-type from the holotype, and SM61) grouped together in Helminthosporium, as the other strains have an ITS region, but the H. endiandrae (CBS 138902, MH878637) strain that grouped with our new collection lacks the ITS region. Therefore, we compared the morphology of these two species and found that Hap. calami differs from H. endiandrae with respect to its smaller conidiophores ((110–)140–175(–215) × (4–)5–7(–8) vs. 200–300x 5–7 µm), number of conidiophore septa ((3–)4–5(–6) vs. 8–16 septa), larger conidia ((55–)70–100(–120)x(13–)17–20(–23) vs. (35–)37–45(–57)x(7–)8(–9) µm), solitary conidium per conidiophore, and higher number of distoseptate ((3–)4–6(–7)-distoseptate vs. 3(–4)-distoseptate). The results show the placement of Haplohelminthosporium calami within Massarinaceae, and that this species is distinct from other known species. Therefore, we introduce Hap. Calami as a new species based on both morphological and phylogenetic data.

Table 3. Polymorphic nucleotides from sequence data of the LSU loci for isolates of Haplohelminthosporium calami MFLUCC 18-0074 and Helminthosporium endiandrae CBS 138,902 (KP004478, MH878637).

Species Strain                                                LSU

 

6 34 74 270 400 412 419 427 480 484 490 491 524 644 843
Haplohelminthosporium calami(this study) MFLUCC

18-0074

A A T T T C C A C A T T T G
Helminthosporium endiandrae(Ex-type from the holotype) CBS 138,902

(KP004478)

C C C C C T T C T T G C G G
H. endiandrae (sister strain in Figures 1B and 2) CBS 138,902

(MH878637)

C A C C C C T T C T T G C G

Figure 1. Comparison of the topology of Maximum likelihood majority rule consensus tree for the analyses of some selected Corynesporaceae, Massarinaceae, and Perioconiaceae isolates. (A) Phylogenetic tree of the dataset for ITS sequence data. (B) Phylogenetic tree of the dataset for LSU sequence data. Bootstrap support values for maximum likelihood (ML) equal to or higher than 50%, and Bayesian Posterior Probabilities (BYPP) equal to or greater than 0.90 are given above each branch. Novel taxa are in blue. Ex-type strains are in bold.  The tree is rooted to Cyclothyriella rubronotata strains TR, TR9 (Cyclothyriellaceae).

Figure 2. Maximum likelihood majority rule consensus tree for the analyses of Massarinaceae and sister family Perioconiaceae isolates based on a dataset of combined ITS, LSU, SSU, and tef1-α sequence data. Bootstrap support values for maximum likelihood (ML) equal to or higher than 50%, and Bayesian posterior probabilities (BYPP) equal to or greater than 0.90 are given above each branch. Novel taxa are in blue. Ex-type strains are in bold. The tree is rooted to Cyclothyriella rubronotata strains TR, TR9 (Cyclothyriellaceae).

Figure 3. Haplohelminthosporium calami (MFLU 20-0520, holotype) (A) The forest in Krabi Province. (BE) Fresh and herbarium palm samples. (F,G) Colonies on living leaf. (HL) Conidiophores. (MU) Conidia. (V,W) Germinated conidia. (X) Culture on PDA. (Y) Conidiophore and conidia on culture. (Z) Conidiogenesis. (AA) Conidiophores. (AB,AC) Conidia. Scale bars: C, E =2 cm, H–W, Y–AC = 50 µm.