Morphological and phylogenetic analyzes reveal two new species of Melanconiella from Fujian Province, China

Introduction Species of Melanconiella include a diverse array of plant pathogens as well as endophytic fungi. Members of this genus have been frequently collected from the family Betulaceae (birches) in Europe and North America. Little, however, if known concerning the distribution of Melanconiella and/or their potential as pathogens of other plant hosts. Methods Fungi were noted and isolated from diseased leaves of Loropetalum chinense (Chinese fringe flower) and Camellia sinensis (tea) in Fujian Province, China. Genomic DNA was extracted from fungal isolates and the nucleotide sequences of four loci were determined and sued to construct phylogenetic trees. Morphological characteristics of fungal structures were determined via microscopic analyses. Results Four strains and two new species of Melanconiella were isolated from infected leaves of L. chinense and C. sinensis in Fujian Province, China. Based on morphology and a multi-gene phylogeny of 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), Melanconiellaloropetali sp. nov. and Melanconiellacamelliae sp. nov. were identified and described herein. Detailed descriptions, illustrations, and a key to the known species of Melanconiella are provided. Discussion These data identify new species of Melanconiella, expanding the potential range and distribution of these dark septate fungi. The developed keys provide a reference source for further characterization of these fungi.


Introduction
Species of Melanconiella (Melanconiellaceae, Diaporthales) are important fungal wood pathogens and endophytes in Europe and North America (Du et al., 2017).Based on the darkcolored ascospore characteristics of the fungus, the genus Melanconiella was established and typified as M. spodiaea, with numerous revisions and adjustments occurring over the years (Saccardo, 1882;Voglmayr et al., 2012).These fungi belong within the broader category of dark Mu et al. 10.3389/fmicb.2023.1229705Frontiers in Microbiology 02 frontiersin.orgseptate fungi, that includes the dark septate root endophytes (DSE) which are conidial Ascomycetes (Jumpponen and Trappe, 1998;Ruotsalainen et al., 2022).DSE have been isolated from over 600 plant species, and have been distributed into >320 genera, occurring from the tropics to arctic and alpine habitats.These fungi comprise a heterogeneous group overlapping with soil, saprotrophic rhizoplaneinhabiting, mycorrhizal, and obligate/facultatively pathogenic fungi within ecosystems and in terms of their biology.Of note, many DSE have been examined in particular due to their effects on plant resistances to a wide range of biotic and abiotic stress (Santos et al., 2021).Within the past 30 years, 21 species in the genus Melanconiella have been recorded (Voglmayr et al., 2012).Wehmeyer (1926) indicated that most ascospores are arranged in a single row and only one type of conidia are needed to distinguish between Melanconiella and Melanconis.However, Wehmeyer (1941) considered Melanconiella to be a synonym of Melanconis, based on new classification standards.This viewpoint was supported by most scholars and led to confusion between Melanconiella and Melanconis (Voglmayr et al., 2012).
Since the development of modern genetic methods, molecular phylogenetic analysis has been applied to the taxonomy of Melanconiella.Based on the nucleotide sequence of the 28S large subunit of nuclear ribosomal RNA (LSU), the genus Melanconiella was separated from Melanconidaceae by Castlebury et al. (2002).Based on a multi-gene phylogeny (ITS, LSU, RPB2 and TEF1-α), Voglmayr et al. (2012) confirmed that Melanconiella was a special branch of Melanconis and suggested 13 accepted species.The genus Melanconiella was later accommodated into the Melanconiellaceae (Senanayake et al., 2017).A new species (M.syzygii) from diseased leaves of Syzygium sp. was added to Melanconiella by Crous et al. (2016).Du et al. (2017) reported a new species (M.cornuta) from Cornus controversa in Shaanxi Province.However, on the basis of phylogenetic analysis of combined ITS, LSU, CAL, RPB2 and TEF1-α sequence data, Fan et al. (2018) suggested that M.cornuta should be transferred from Melanconiella to Sheathospora.Meanwhile, two additional species (M.betulicola and M. corylina) were added to Melanconiella (Fan et al., 2018).
In the present study, four specimens of Melanconiella were collected from diseased leaves of Loropetalum chinense and Camellia sinensis in Fujian Province, China.Here, we sought to: i. Determine/extend the host range and geographical distribution of Melanconiella; ii. Report new species Melanconiella loropetali sp.nov.and Melanconiella camelliae sp.nov.with detailed descriptions and illustrations; iii.Compare these new species with other species in the genus Melanconiella; and iv.Provide a key to all known species of Melanconiella.

Fungal isolates and morphology
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 mm 2 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, KH 2 PO 4 1 g, KNO 3 1 g, MgSO 4 . 7H 2 O 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).

DNA extraction and amplification
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).

Phylogenetic analysis
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 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).
Etymology: The epithet "loropetali" pertains to the generic name of the host plant Loropetalum chinense.
Etymology: The epithet "camelliae" refers to the generic name of the host plant Camellia sinensis.
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 26.3-31.9mm in diameter after 7 days, at 25°C, with a calculated growth rate 3.7-4.5 mm/day.SNA attaining 13.8-18.8mm in diameter after 7 days, at 25°C, slow growing, calculated growth rate 1.9-2.6 mm/day.

FIGURE 1
FIGURE 1 Phylogram of Melanconiella based on combined ITS, LSU, RPB2 and TEF1-α genes, with Melanconis stilbostoma (CBS 121894) as outgroup.The ML and BI bootstrap support values above 70% and 0.90 BYPP are shown at the first and second position, respectively.Strains marked with "T" are ex-type or ex-epitype.Strains from this study are shown in red.The scale bar at the left-bottom represents 0.02 substitutions per site.

TABLE 1
Gene regions and PCR primers and programs used in this study.

TABLE 2
Species and GenBank accession numbers of DNA sequences used in this study, with new sequences indicated in bold.