Phylogenetic analyses
The combined LSU, ITS, tef1-α, and rpb2 dataset comprised of 201 taxa from Nectriaceae, with Geejayessia [G. atrofusca (CBS 125482; NRRL 22316), G. celtidicola (CBS 125502), G. cicatricum (CBS 125550), G. cicatricum (CBS 125552)] as outgroup taxa (Table 1). The analyzed dataset, after trimming, comprised a total 3,163 characters including gaps (LSU = 815 bp, ITS = 1066 bp, TEF1-α = 602 bp, RPB2 = 680 bp) with 1,407 distinct alignment patterns and 32.60% proportion of gaps and completely undetermined characters. The ML analysis for the combined dataset provided the best scoring tree (Fig. 1) with a final ML optimization likelihood value of -33795.848267 (ln). Parameters for the GTR+I+G model of the combined LSU, ITS, tef1-α, and rpb2 dataset are as follows: estimated base frequencies; A = 0.241529, C = 0.269280, G = 0.265706, T = 0.223486; substitution rates AC = 1.650028, AG = 3.631450, AT = 1.934861, CG = 0.900810, CT = 7.653185, GT = 1.000000; gamma distribution shape parameter α = 0.276755. The Bayesian analysis indicated the average standard deviation of split frequencies at the end of total MCMC generations is 0.009230. Phylogenetic analyses of the combined data matrix resulted in well-resolved clades (Fig. 1). The tree topologies resulting from maximum likelihood (ML) and Bayesian posterior probabilities (BYPP) analyses were congruent.
In the phylogenetic analyses (Fig. 1), two novel species, N. mangrovei (MFLUCC 17-0253, MFLUCC 17-0257) and N. ceriopis (MFLUCC 17-0259), were sister taxa with good bootstrap support (83% ML, 1.00 BYPP), and closely related to N. macrospora and N. ferruginea (73% MP, 1.00 BYPP). GCSPR result of multi-locus gene of N. ceriopis and N. mangrovei, and related species shows no significant recombination happened (Fig. 2).
[Insert Figs.1 & 2]
Taxonomy
Sordariomycetes O.E. Erikss. & Winka, Myconet 1(1): 10 (1997)
Hypocreales Lindau, in Engler & Prantl, Nat. Pflanzenfam., Teil. I (Leipzig) 1(1): 343 (1897)
Nectriaceae Tul. & C. Tul. [as 'Nectriei'], Select. Fung. Carpol. (Paris) 3: 3 (1865)
Neocosmospora E.F. Sm., Bull. U.S. Department of Agriculture 17: 45 (1899)
Type species: Neocosmospora vasinfecta E.F. Sm.
Neocosmospora ceriopis Klomchit, M.S. Calabon, S. Brooks sp. nov. (Fig. 3)
Index Fungorum number: IF**; Faces of Fungi number: FoF 12541
Etymology: The epithet reflects the genus name of the host plant Ceriops decandra, from which the specimens were collected.
Holotype: MFLU 22–****
Colonies growing on PDA, reaching 40-55 mm diam in 14 d at 24 °C, colony circular, white, flat, entire, velvety to cottony, with or without white to straw concentric rings; reverse white to pale straw.
Conidiophores on aerial mycelium straight, smooth, and thin-walled, simple or branched several times verticillately, bearing terminal or lateral, single monophialides; phialides 35.0-73 × 2.2-4.5 µm (x̄ = 52.5 × 3.0 µm, n = 50) subulate, subcylindrical, to acicular, smooth- and thin-walled, conidiogenous loci with inconspicuous periclinal thickening and non-flared, minute collarettes; aerial conidia of two types: microconidia 6.60-14.5 × 2.7-5.5 µm (x̄ = 10.4 × 4.1 µm, n = 50) oval, obovoidal to ellipsoidal, mostly straight to slightly curved, often with a flattened base, aseptate, hyaline, smooth- and thin-walled, clustering in false heads at tip of monophialides; macroconidia up to 55 µm, falcate, rarely straight with moderately curved ends, tapering toward base, septation sometimes indistinct or with thin transverse septa; apical cell of equal length or slightly longer than adjacent cell, blunt to slightly hooked with rounded apex; basal cell inconspicuously to moderately notched, 4-septate, hyaline, smooth- and thick-walled. Sporodochia not observed. Chlamydospores 9.0-21.5 × 5.5-10 µm (x̄ = 13.7 × 7.7 µm, n = 30) abundantly formed, globose to subglobose, smooth- to verruculose and thick-walled, terminal or intercalary in hyphae or conidia, solitary, in chains or in clusters.
Material examined: THAILAND, Ranong Province, The Ranong Biosphere Reserve, on branches of Ceriops decandra, 6 December 2016, Chada Norphanphoun, NG03b (MFLU 22–****, holotype), ex-type living culture MFLUCC 17-0259.
GenBank accession numbers: LSU = ON833276, ITS = MT928794, TEF1-α = **
Notes: In the present study, MFLUCC 17-0253 and MFLUCC 17-0257, showed no morphological distinctions, and pairwise nucleotide comparisons of ITS sequence data showed no nucleotide differences but 8 bp differences (1.29%, 622 bp) in TEF1- α sequence data. This indicates that the two isolates are strains of N. ceriopis (Jeewon and Hyde 2016). The multi-locus phylogenetic analysis shows that N. ceriopis is sister taxon to N. mangrovei, with high bootstrap support (83% ML, 1.00 BYPP) (Fig. 1). In pairwise nucleotide comparisons of N. mangrovei with the sister taxon N. ceriopis, there is a nucleotide difference of 2.81% (14 bp) and 2.32% (14 bp) in ITS (of 499 nucleotides altogether) and TEF1-α (of 603 nucleotides altogether), respectively. Morphologically, Neocosmospora ceriopis has a larger microconidium (6.60-14.5 × 2.7-5.5 µm vs. 4.5-8.0 × 1.5-3.5 µm) than N. mangrovei. Neocosmospora ceriopis also shared the same subclade with N. macrospora and N. ferruginea (73% MP, 1.00 BYPP). Morphologically, N. ceriopis is distinct from N. macrospora by having longer phialides (35.0-73 × 2.2-4.5 µm vs. 19-67 × 2-5 µm) and larger chlamydospores (9.0–21.5 × 5.5-10 µm vs. 5-8.5 × 4.5-8 µm) (Sandoval-Denis et al. 2018). Neocosmospora ceriopis can be distinguished from N. ferruginea by the size of phialides [35.0-73 × 2.2-4.5 µm vs. (29.5-)31-40(-45) × 2.5-3.5 µm], conidia [6.60-14.5 × 2.7-5.5 µm vs. (4 -)4.5-11(-20) × (1.5-)2-4(-60 µm], and chlamydospores (9.0-21.5 × 5.5-10 µm vs. 8-10 µm diam) (Sandoval-Denis et al. 2019).
[Insert Fig. 3]
Neocosmospora mangrovei Klomchit; M.S. Calabon; S. Brooks sp. nov. (Fig. 4)
Index Fungorum number: IF**; Faces of Fungi number: FoF 12542
Etymology: Named after the fungal habitat from mangrove habitat, where the fungus found.
Holotype: MFLU 22–****
Colonies growing on PDA, reaching 40-55 mm diam in 14 days at 24 °C, colony circular, white, flat, entire to filiform, velvety; reverse white to pale straw.
Conidiophores on aerial mycelium straight, smooth, and thin-walled, simple, terminal, single monophialides; phialides 36.5-74.0 × 2.5-5.5 µm (x̄ = 60.8 × 3.8 µm, n = 30), subulate to acicular, smooth and thin-walled, conidiogenous loci with inconspicuous periclinal thickening and non-flared, minute collarettes; aerial conidia of two types: microconidia 4.5-8.0 × 1.5-3.5 µm (x̄ = 6.3 × 2.7 µm, n = 50), subglobose, mostly ellipsoidal, straight to slightly curved, often with a flattened base, aseptate, hyaline, smooth- and thin-walled, clustering in false heads at tip of monophialides; macroconidia 39-45 × 4.5-6.0 µm (x̄ = 42.8 × 5.2 µm, n = 10), falcate, straight to slightly dorsiventrally curved, apical cell blunt and slightly curved; basal cell inconspicuously to moderately notched, 4-5-septate, predominantly 4-septate, hyaline, smooth- and thick-walled. Sporodochia not observed. Chlamydospores 11-20 × 5-10 µm (x̄ = 15.6 × 7.4 µm, n = 50), abundantly formed, globose to subglobose, smooth- to verruculose and thick-walled, terminal or intercalary in hyphae or conidia, solitary or in chains.
Material examined: THAILAND, Ranong Province, The Ranong Biosphere Reserve, on branches of Rhizophora apiculata and R. mucronata, 7 December 2016, Chada Norphanphoun, NG27b (MFLU 22-****, holotype), ex-type living culture MFLUCC 17-0253; NG37b (MFLU 22-****, paratype), ex-paratype living culture MFLUCC 17-0257.
GenBank accession numbers: MFLUCC 17-0253: LSU = **, ITS = MT928792, TEF1-α = **; MFLUCC 17-0257: LSU = ON833274, ON833275; ITS = MT928793; TEF1-α = **; RPB2 = **
Notes: The combined ITS-LSU-RPB2-TEF1-α sequence data showed that Neocosmospora mangrovei is sister taxon to N. ceriopis with strong statistical support. Neocosmospora mangrovei also shared the same subclade with N. macrospora and N. ferruginea (73% MP, 1.00 BYPP). Morphologically, N. mangrovei is distinct from N. macrospora by having longer phialides (36.5-74.0 × 2.5-5.5 µm vs. 19-67 × 2-5 µm) and larger chlamydospores (11-20 × 5-10 µm vs. 5-8.5 × 4.5-8 µm) (Sandoval-Denis et al. 2018). Neocosmospora mangrovei can be distinguished from N. ferruginea by the size of phialides [36.5-74.0 × 2.5-5.5 µm vs. (29.5-)31-40(-45) × 2.5-3.5 µm], microconidia [4.5-8.0 × 1.5-3.5 µm vs. (4 -)4.5-11(-20) × (1.5-)2-4(-6) µm], and chlamydospores (11-20 × 5-10 µm vs. 8-10 µm diam) (Sandoval-Denis et al. 2019).
[Insert Fig.4]
Antagonistic activity against anthracnose disease
Among the three new strains, N. mangrovei MFLUCC 17-0253 and N. ceriopis MFLUCC 17-0259 exhibited antifungal activity against fungi C. truncatum, MFLUCC CG-0064 and C. acutatum, MFLUCC CC-0036, respectively. Based on the dual culture assay, N. ceriopis MFLUCC 17-0259 presented slightly higher activity against C. acutatum, MFLUCC CC-0036 (46.5+7.47%) compared to N. mangrovei MFLUCC 17-0253 with fungi C. truncatum, MFLUCC CG-0064 (21-25+6.22%).
In contrast, to the dual culture assay, the poisoned food assay, spore germination, and leave inoculation demonstrated that at the same concentration fungal crude extract from N. mangrovei MFLUCC 17-0253 is potent toward fungi C. truncatum, MFLUCC CG-0064 more than fungal crude extract of N. ceriopis MFLUCC 17-0259 against C. acutatum, MFLUCC CC-0036 (Table 2; Fig. 5). For instance, fungal crude extract from N. mangrovei MFLUCC 17-0253 at a concentration of 0.9 mg/mL and 2.7 mg/mL was able to completely inhibit the growth of fungi C. truncatum, MFLUCC CG-0064 in poisoned food assay and leave inoculation, respectively (Fig. 5). Even though fungal crude extract of N. ceriopis MFLUCC 17-0259 did not present vigorous activity toward pathogen as presented in a fungal crude extract of N. mangrovei MFLUCC 17-0253 but with slightly higher concentration, it could effectively inhibit spore germination and hypha growth of C. acutatum, MFLUCC CC-0036. For instance, inhibition of hypha expansion on chili leaves after incubation with 2.7 mg/mL of fungal crude extract from N. ceriopis MFLUCC 17-0259 was high as 92.82% (Fig 6; Table 2). Compared to a commercially available fungicide used in this study, fungal crude extract of N. mangrovei MFLUCC 17-0253 exhibited more potent antifungal activity throughout the in-vitro assays and in-vivo assay, while the fungal crude extract's antifungal activity of fungal crude extract of N. ceriopis MFLUCC 17-0259 was slightly lower than Captan (Table 2).
[Insert Figs. 5 & 6]
Antifungal compounds
The crude extract of N. ceriopis MFLUCC 17-0259 was further investigated to identify secondary metabolites produced from this fungus and responsible for this antifungal activity. Fungal extraction (2.0 g) from N. ceriopis MFLUCC 17-0259 was subjected to column chromatography (CC) to yield three subfractions (A-C), and Sephadex LH-20 CC purified all subfractions to afford compound 1-3 (2FU-259, 11FU-259, 12FU-259). Three compounds were subject to disc diffusion assay, and only one compound presented an inhibition zone (Fig. 7a), and only one compound exhibited antifungal activity toward anthracnose pathogen. Structure elucidation by NMR revealed the structure of the compound that exhibited antifungal activity toward anthracnose pathogen as 2,3-hydro-5-hydroxy-8-methoxy-2,4-dimethylnapthol-[1,2-b]-furan-6,9-dione (Fig. 7b).
[Insert Fig. 7]