Specimen samples were collected from the Wuyi Mountain National Nature Reserve, Fujian Province, China. Colonies of the two new species Melanconiella described herein were isolated from diseased leaves of Loropetalum chinense and Camellia sinensis using standard issue isolation methods (Senanayake et al., 2020; Jiang et al., 2022). Tissue fragments about 25 mm² in total extent were taken from the margin of leaves with typical spot symptoms. These were sterilized by immersion in 75% ethanol solution for 60 s, placed in sterile deionized water for 45 s, transferred to 5% sodium hypochlorite solution for 30 s, and then rinsed three times in sterile deionized water for 60 s. The fragments were dried with sterilized filter paper and then transferred onto PDA plates (PDA medium: deionized water 1,000 mL, potato 200 g, agar 20 g, dextrose 20 g, pH ~7.0, available after sterilization) and incubated at 25°C for 5–7 days (Cai et al., 2009). Growing edges of fungal hyphae were removed to new PDA plates (at least two times) to obtain pure cultures. To promote sporulation and visualize the appearance of colonies, hyphae were inoculated onto the center of PDA prepared with pine needle and synthetic low nutrient agar SNA (SNA medium: deionized water 1,000 mL, KH₂PO₄ 1 g, KNO₃ 1 g, MgSO₄^.7H₂O 0.5 g, KCl 0.5 g, dextrose 0.2 g, sucrose 0.2 g, agar 12 g, available after sterilization) and incubated at 25°C under alternating conditions of 12 h near ultraviolet light and 12 h dark (Cai et al., 2009; Zhang et al., 2023).

Following 7–14 days of incubation, morphological characteristics of the (Melanconiella) isolates were recorded as per previous reports (Cai et al., 2009). Photographs of the colonies were taken at 7 days and 14 days after inoculation using a digital camera (Canon EOS 6D MarkII). Micromorphological characters of conidiomata were observed using a stereomicroscope (Nikon SMZ74), as well as by a compound microscope and by scanning electron microscopy (SEM, Nikon Ni-U; HITACHI SU3500). Measurements of micromorphological structures were determined using Digimizer software. All strains were stored in 10% sterilized glycerin and sterile water at 4°C for detailed studies in the future. The specimens were deposited in the Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), accession numbers given in text. Living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org; Crous et al., 2004).

Genomic DNA was extracted from Melanconiella fungal mycelia grown on PDA for 14 days, using the Fungal DNA Mini Kit (OMEGA-D3390, Feiyang Biological Engineering Corporation, Guangzhou, China) according to the product manual. Nucleotide sequences were obtained from four gene loci including the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), and the translation elongation factor 1-α gene (TEF1-α). These were amplified by primer pairs and the polymerase chain reaction (PCR) programs as described (Table 1).

PCR was performed using a Bio-Rad Thermocycler (California, United States). Amplification reactions were performed in a 25 μL reaction volume which contained 12.5 μL of 2 × Rapid Taq Master Mix (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Sangon, Shanghai, China), and 1 μL of template genomic DNA in the amplifier. These were adjusted with distilled deionized water to a total volume of 25 μL. PCR products were visualized on using 1% agarose gel electrophoresis. Bidirectional (both strand) sequencing was conducted by the Tsingke Company Limited (Fuzhou, China). Consensus sequences were assembled using MEGA 7.0 (Kumar et al., 2016).

To construct the phylogenetic trees for Melanconiella, the sequences generated from the four strains considered in this study, and all available reference sequences were downloaded from GenBank. Multiple sequence alignments for ITS, LSU, RPB2 and TEF1-α were constructed and carried out using the MAFFT v.7.11 online program (http://mafft.cbrc.jp/alignment/server/; Katoh et al., 2019) and corrected manually using MEGA 7.0 (Kumar et al., 2016). Phylogenetic analyzes were based on maximum likelihood (ML) and Bayesian inference (BI) methods.

The ML was run on the CIPRES Science Gatewayportal by RaxML-HPC2 on XSEDE v. 8.2.12 (Miller et al., 2010; Stamatakis, 2014). Bayesian analysis was performed in MrBayes on XSEDE v. 3.2.7a (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003; Ronquist et al., 2012) and the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Posada and Crandall, 1998; Nylander, 2004; Darriba et al., 2012) and included the analyzes (Dong et al., 2020). Four simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 160,000 generations. In addition, a sampling frequency of 100 generations was performed in the test. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees (He et al., 2022). The consensus trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree; Rambaut, 2018) and edited using Adobe Illustrator CS 6.0 (Figure 1). New sequences generated in this study were deposited in GenBank (https://www.ncbi.nlm.nih.gov; Table 2).

Four isolates of Melanconiella were identified as representing two new species based on an analysis of combined ITS, LSU, RPB2, and TEF1-α gene nucleotide sequences, and combined with 47 isolates of Melanconiella along with Melanconis stilbostoma (CBS 121894) as the outgroup taxon (Voglmayr et al., 2012). The dataset had an aligned length of 6,068 characters including gaps (i.e., ITS: 1–1723, LSU: 1724–3,416, RPB2: 3417–4,590, TEF1-α: 4591–6,068). Of these characters, 4,369 were constant, 357 were variable and parsimony-uninformative, and 1,342 were parsimony-informative.

The Bayesian analysis was performed for 160,000 generations, resulting in 3202 total trees, of which 2,402 trees were used to calculate the posterior probabilities. The BI posterior probabilities were plotted on the ML tree. For the ML and BI analyzes, GTR + I + G for ITS, RPB2, TEF1-α, and LSU [Lsetnst = 6, rates = invgamma; Prsetstatefreqpr = Dirichlet (1,1,1,1)], were selected and incorporated into the analysis. Bayesian analysis resulted in an average standard deviation of split frequencies = 0.009565. The topology of the ML tree was consistent with that of the Bayesian tree. Hence, the ML tree is presented (Figure 1).

ML bootstrap support values (≥70%) and Bayesian posterior probability (≥0.90) are shown as the first and second positions, respectively, above the nodes. The 48 strains were assigned to 20 species clades based on the four gene loci phylogeny (Figure 1). The four strains studied herein represented two novel species. The new species Melanconiella loropetali showed a close relationship to M. syzygii (CBS 142095), with good support (ML-BS:86% and BYPP:0.98). The new species M.camelliae (CGMCC3.24889) showed a close relationship to M. syzygii (CBS 142095) and M. loropetali (CGMCC3.24886) with good support (ML-BS:76% and BYPP:0.99).

Melanconiella loropetali T.C. Mu and Jun Z. Qiu, sp. nov. (Figure 2).

MycoBank number: MB848666.

Holotype: CHINA, Fujian Province, Fujian Wuyi Mountain National Nature Reserve, 117°41′19.82″E, 27°44′53.91″N, from diseased leaves of Loropetalum chinense, 7 September 2022, T.C. Mu (holotype HMAS 257907; ex-type living culture CGMCC3.24886).

Etymology: The epithet “loropetali” pertains to the generic name of the host plant Loropetalum chinense.

Description: Leaf spots circular, sunken in the middle, brown or tan, 4–10 mm diam. Conidiomata acervular to pycnidial, erumpent on agar, solitary, globose, black or creamy, 330–410 μm diam, black and cream conidial droplets exuding from the ostioles. Conidiophores hyaline to light brown, smooth, fusiform, subcylindrical, 1–5-septate, branched and septate at the base, 9.2–26.8 × 1.6–3.5 μm. Conidiogenous cells hyaline, smooth, phialidic, subglobular and globular, subcylindrical, 1.8–4.3 × 1.5–3.4 μm. Conidia unicellular, hyaline, smooth, narrowly ellipsoid to fusoid, subcylindrical, thin-walled, (3.3–6.6 × 1.6–3.0 μm, mean ± SD = 4.57 ± 0.75 × 2.17 ± 0.33 μm, L/W ratio = 2.2, n = 30). Sexual morph was not observed.

Culture characteristics: Colonies on PDA flat with feathery margin, aerial mycelium white or dark brick-red, floccose. On PDA surface pale mouse gray to mouse gray, reverse black. On SNA surface and reverse gray in the center and white margin. PDA attaining 18.5–22.8 mm in diameter after 7 days, at 25°C, with a calculated growth rate 2.6–3.2 mm/day. SNA attaining 9.8–13.6 mm in diameter after 7 days, at 25°C, slow growing, calculated growth rate 1.4–1.9 mm/day.

Other specimens examined: CHINA, Fujian Province, Fujian Wuyi Mountain National Nature Reserve, 117°41′19.82″E, 27°44′53.91″N, from diseased leaves of Loropetalum chinense, 7 September 2022, T.C. Mu (paratype HMAS 257908; ex-paratype living culture CGMCC3.24887).

Notes: In this study, a member of the genus Melanconiella was collected from the Hamamelidaceae. Two strains (CGMCC3.24886; CGMCC3.24887) were isolated from diseased leaves of Loropetalum chinense and identified as Melanconiella loropetali sp. nov. Phylogenetic analysis using four genes showed that M. loropetali formed an independent clade (Figure 1) and was phylogenetically distinct from M. syzygii (CBS 142095) with high statistical support (86% ML/0.98 PP, Figure 1). The nucleotide comparison of ITS sequences of M. syzygii revealed 58 bp (58/601 bp, 9.65%) nucleotide differences. The nucleotide comparison of LSU sequences of M. syzygii revealed 14 bp (14/817 bp, 1.71%) nucleotide differences. Morphologically, M. loropetali differs from M. syzygii in having smaller conidia and conidiophores (3.3–6.6 × 1.6–3.0 vs. 8.0–11.0 × 5.0–6.0 μm; 9.2–26.8 × 1.6–3.5 vs. 12.0–30.0 × 4.0–6.0 μm [Crous et al., 2016]). Therefore, we describe this fungus as a new species.

Melanconiella camelliae T.C. Mu and Jun Z. Qiu, sp. nov. (Figure 3).

MycoBank number: MB848667.

Holotype: CHINA, Fujian Province, Fujian Wuyi Mountain National Nature Reserve, 117°41′19.81″E, 27°44′53.92″N, from diseased leaves of Camellia sinensis, 7 September 2022, T.C. Mu (holotype HMAS 257910; ex-type living culture CGMCC3.24889).

Etymology: The epithet “camelliae” refers to the generic name of the host plant Camellia sinensis.

Description: Leaf spots irregular, brown or umber. Conidiomata acervular to pycnidial, solitary, 180–260 μm diam, with cream conidial droplets exuding from the ostioles. Conidiophores, hyaline, smooth, 1–3-septate, mostly straight, cylindrical-clavate, branched at the base, 10.4–18.6 × 1.4–2.1 μm. Conidiogenous cells hyaline, smooth, circular, elliptical, phialidic, 1.5–3.5 × 1.0–1.7 μm. Conidia hyaline, smooth, oblong elliptical and fusoid, multi-guttulate, (4.7–5.9 × 2.0–2.8 μm, mean ± SD = 5.36 ± 0.35 × 2.32 ± 0.23 μm, L/W ratio = 2.3, n = 30). Sexual morph was not observed.

Culture characteristics: Colonies on PDA flat with irregular stripes, aerial mycelium white, cottony. On PDA surface white, reverse yellowish and darker. Colonies on SNA sparse hyphae, slow growing. PDA attaining 26.3–31.9 mm in diameter after 7 days, at 25°C, with a calculated growth rate 3.7–4.5 mm/day. SNA attaining 13.8–18.8 mm in diameter after 7 days, at 25°C, slow growing, calculated growth rate 1.9–2.6 mm/day.

Other specimens examined: CHINA, Fujian Province, Fujian Wuyi Mountain National Nature Reserve, 117°41′19.81″E, 27°44′53.92″N, from diseased leaves of Camellia sinensis, 7 September 2022, T.C. Mu (paratype HMAS 257909; ex-paratype living culture CGMCC3.24888).

Notes: In this study a member of the Melanconiella was collected for the first time from the Theaceae. Two strains (CGMCC3.24888; CGMCC3.24889) were isolated from diseased leaves of Camellia sinensis and identified as Melanconiella camelliae sp. nov. Phylogenetic analysis of four genes showed that M. camelliae formed an independent clade (Figure 1) and was phylogenetically distinct from M. syzygii and M. loropetali with moderate statistical support (76% ML/0.99 PP, Figure 1). The nucleotide comparison of ITS sequences of M. syzygii revealed 116 bp (116/488 bp, 23.77%) nucleotide differences. The nucleotide comparison of LSU sequences of M. syzygii revealed 53 bp (53/821 bp, 6.46%) nucleotide differences. Morphologically, M. camelliae differs from M. syzygii in having smaller conidia and conidiophores (4.7–5.9 × 2.0–2.8 vs. 8.0–11.0 × 5.0–6.0 μm; 10.4–18.6 × 1.4–2.1 vs. 12.0–30.0 × 4.0–6.0 μm [Crous et al., 2016]). Melanconiella camelliae differs from M. loropetali in having smaller conidiophores (10.4–18.6 × 1.4–2.1 vs. 9.2–26.8 × 1.6–3.5 μm). Therefore, we describe this fungus as a new species.

1. Conidiophores septate……………2.

1. Conidiophores aseptate……………4.

2. On Camellia sinensis; conidiophores 1–3-septate; conidial size 4.7–5.9 × 2.0–2.8 μm, L/W ratio = 2.3……………M. camelliae sp. nov.

2. Conidiomata solitary, globose……………3.

3. On Syzygium; conidiophores ampulliform, unbranched; conidiogenous cells integrated, terminal; conidial size 8.0–11.0 × 5.0–6.0 μm……………M. syzygii.

3. On Loropetalum; conidiophores 1–5-septate; conidial size 3.3–6.6 × 1.6–3.0 μm……………M. loropetali sp.nov.

4. Ascospores dark brown; conidia dark brown……………5.

4. Ascospores hyaline; conidia hyaline or dark brown……………7.

5. Ascospores without appendages; conidia pip-shaped, multiguttulate when fresh, with a large guttule when dead; on Betula spp. in the north temperate zone……………M. decorahensis.

5. Conidia usually ellipsoid, guttulate……………6.

6. Perithecia 0.3–0.5 mm diam, up to 20 per stroma; conidia variable in shape, ovoid, obovoid, usually not distinctly pip-shaped; on Carpinus in Europe……………M. spodiaea.

6. Ectostromatic disc inconspicuous and cracked around the margin at maturity; conidia narrowly ellipsoid, 1–3 guttulate; on Corylus mandshurica in China……………M. corylina.

7. On Carpinus……………8.

7. On other hosts……………16.

8. Ascospores with obvious ascospore wall and with monomorphic, (sub) globose to bullet-shaped ascospore cells mostly larger than 7 μm in width; commonly with yellow ectostroma……………9.

8. Ascospores without obvious ascospore wall and with slightly to distinctly dimorphic cells mostly smaller than 7 μm in width; ectostroma gray, brownish, yellowish, pale brown, cream or yellow……………11.

9. Ascospores 16.0–27.5 × 7.5–15.5 μm; with obvious conidiomata containing dark brown conidia; on C. betulus……………M. chrysomelanconium.

9. Ascospores 15.5–22.0 × 6.0–15.0 μm; hyaline conidia……………10.

10. Entostroma crumbly, of subhyaline to yellowish hyphae; conidial size 12.5–19.0 × 4.5–6.0 μm; on C. betulus……………M. chrysodiscosporina.

10. Asci rarely biseriate ascospores; conidial size 11.5–15.5 × 5.5–7.5 μm; on C. orientalis……………M. chrysorientalis.

11.Ascospores were usually more than 5.5 μm in width and 18.5 μm in length……………12.

11. Ascospores were usually less than 5.5 μm in width and 18.5 μm in length……………14.

12. On Carpinus in Europe; ectostromatic discs concave, flat or slightly pustulate and little projecting above the perithecial level, paler to fawn; central column pale yellow to grayish brown……………13.

12. On C. caroliniana in North America; ectostromatic discs well-developed, distinctly projecting, often pulvinate, with circular, angular or ellipsoid outline, brownish, brown, cream or yellow; central column yellow when young, later green to brown; entostroma well-developed, of compacted hyphae, green or brown……………M. echinata.

13. Central column pale, yellowish, pale grayish brown or brownish; conidiogenous cells 10.0–30.0 × 1.5–3.0 μm; on C. betulus……………M. hyperopta.

13. Ascospores 2.5–3.6 × 1.8–2.3 μm; conidia often with one to three larger and few to numerous small guttules; On C. orientalis……………M. hyperopta var. orientalis.

14. Pseudostromata perithecia only rarely projecting, 0.8–1.7 mm diam; conidiogenous cells 10.0–18.0 × 1.5–2.5 μm; on C. betulus in Europe……………M. carpinicola.

14. On C. caroliniana or Betula……………15.

15. Ectostromatic discs distinctly projecting, mostly circular, angular or ellipsoid, often pulvinate, pale yellow to pale brown; or concealed by ostioles; ostioles evenly spaced in the disc; central column yellow, gray or brownish; conidiogenous cells 10.0–25.0 × 2.0–3.0 μm……………M. elegans.

15. Ectostromatic disc mostly circular; ostioles black……………16.

16. Ectostromatic disc mostly irregularly shaped, usually surrounded by irregular edges or flaps of bark, drab, light to dark gray or tanned; ostioles mostly marginal in the disc; central column gray or pale brown; conidial size 8.5–13.0 × 2.5–4.0 μm……………M. ellisii.

16. Ectostromatic disc usually buff to hazel; conidial size 9.5–15.0 × 2.0–5.5 μm; on Betula in China……………M. betulicola.

17. On Corylus; ascospores broadly fusoid; ascospore ends obviously subacute with persistent knob-like hyaline appendages; conidia often with one or two larger and numerous small guttules……………M. flavovirens.

17. On Ostrya; ascospores fusoid; ascospore ends narrowly rounded to subacute, without or with cap-like appendages……………18.

18. Ascospores 20.0–37.5 × 5.0–9.5 μm; conidia hyaline cylindrical to suballantoid; on Ostrya carpinifolia in Europe……………M. meridionalis.

18. Ascospores 14.5–23.0 × 4.0–8.5 μm; conidia dark brown with a light brown equatorial zone, without guttules or granules; on Ostrya virginiana in North America……………M. ostryae.