Melanconis-like fungi are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae. A new Melanconis-like genus of Pseudomelanconidaceae was discovered on branches of Chinese chestnut (Castanea mollissima) in southern China, which was confirmed by both morphology and phylogenetic analysis of combined ITS, LSU, tef1a and rpb2 sequences. The new genus Micromelanconis is characterized by two types of conidia from natural substrate and manual media of PDA, respectively. Conidia from Chinese chestnut branches are pale brown, ellipsoid, multiguttulate, aseptate with hyaline sheath. While conidia from PDA plates are pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, and also with hyaline sheath. All Pseudomelanconidaceae species were only reported on tree branches in China until now. More interesting taxa may be discovered if detailed surveys on tree-inhabiting fungi are carried out in East Asia in the future.

Castanea mollissima, Diaporthales, DNA phylogeny, Melanconis, systematics

Diaporthales, a species-rich order within Sordariomycetes of Ascomycota, is characterized by perithecia with elongate beaks, often forming within stromatic tissues, deliquescent paraphyses, and asci that have a refractive apical annulus (Barr 1978; Rossman et al. 2007; Senanayake et al. 2017, 2018; Fan et al. 2018a; Jiang et al. 2020a). Species of this order inhabit a variety of substrates, including plants, soil, even living animal tissues (Barr 1978; Castlebury et al. 2002; Sogonov et al. 2008; Yang et al. 2020). Most of them are pathogens associated with plant diseases, and the rest are endophytes in healthy plants or saprobes on dead tissues (Crous et al. 2012a; Chen et al. 2016; Norphanphoun et al. 2018; Jiang et al. 2019d; Xavier et al. 2019; Zhu et al. 2020; Yang et al. 2021). Some diaporthalean fungi cause severe forest diseases, so gained attention in forest pathological studies in recent years. For example, Cryphonectria parasitica (Cryphonectriaceae) causes chestnut blight worldwide (Rigling and Prospero 2018; Jiang et al. 2019b); Cytospora chrysosperma (Cytosporaceae) causes common polar and willow cankers in China (Fan et al. 2020); Gnomoniopsis smithogilvyi (Gnomoniaceae) results in European chestnut fruit rot and branch canker (Shuttleworth et al. 2016; Shuttleworth and Guest 2017; Jiang and Tian 2019; Jiang et al. 2020b).

Diaporthales is well classified into families based on morphological and phylogenetic studies (Voglmayr and Jaklitsch 2014; Norphanphoun et al. 2016; Voglmayr et al. 2017; Fan et al. 2018a; Senanayake et al. 2018; Yang et al. 2018a), and up to 32 families were accepted in the order Diaporthales (Jiang et al. 2021). Specimens can be identified to specific level by morphological characters, such as transversely distoseptate brown conidia of Coryneum (Jiang et al. 2018b, 2019c; Senwanna et al. 2018); allantoid ascospores and conidia of Cytospora (Fan et al. 2020); two-guttulate fusiform conidia of Diaporthe-like taxa (Fan et al. 2018a; Yang et al. 2018a, b); stromatic tissues turning to purple in 3% KOH of Cryphonectriaceae species (Chen et al. 2013, 2018); dark acervular conidiomata with conspicuous central column of Melanconis-like taxa (Fan et al. 2016; Jaklitsch and Voglmayr 2020).

Melanconis-like taxa are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae, which are four morphologically similar clades in distinct phylogenetic clades within this order (Fan et al. 2018b). Species of these four families are usually discovered on branches of Betulaceae, Juglandaceae and Fagaceae, but they are not strong pathogens (Wehmeyer 1937; Du et al. 2017; Voglmayr et al. 2019).

Castanea, commonly known as chestnut trees, is a worldwide genus containing several economic species. Chinese chestnut (C. mollissima), is widely cultivated in most of the provinces in China. Previous studies have revealed that seven families (Coryneaceae, Cryphonectriaceae, Cytosporaceae, Diaporthaceae, Erythrogloeaceae, Gnomoniaceae and Pseudomelanconidaceae) of Diaporthales have been reported on branches of Castanea. Coryneum castaneicola, C. gigasporum and C. suttonii of Coryneaceae were reported on dead and diseased Castanea mollissima branches (Jiang et al. 2018b). Aurantiosacculus castaneae, Cryphonectria neoparasitica, C. parasitica and Endothia chinensis of Cryphonectriaceae were confirmed to be Castanea mollissima canker pathogens (Jiang et al. 2019b). Cytospora ceratospermopsis, C. kuanchengensis, C. leucostoma, C. myrtagena, C. schulzeri and C. xinglongensis of Cytosporaceae were reported to be associated with Castanea mollissima branch cankers (Jiang et al. 2020c). Diaporthe eres of Diaporthaceae was discovered on dead branches of Castanea mollissima in Beijing (Yang 2018). Dendrostoma aurorae, D. castaneae, D. castaneicola, D. chinense, D. parasiticum, D. shaanxiense and D. shandongense of Erythrogloeaceae were associated with Castanea mollissima stem, branch and twig cankers (Jiang et al. 2019a). Gnomoniopsis chinensis of Gnomoniaceae caused severe stem and branch cankers only in Hebei Province (Jiang and Tian 2019; Jiang et al. 2020b). Neopseudomelanconis castaneae of Pseudomelanconidaceae was discovered on Castanea mollissima branches in Shaanxi Province (Jiang et al. 2018a).

In the present study, investigations were conducted in Castanea mollissima plantations in Hunan Province of south China. Two Melanconis-like specimens were collected on a cultivated chestnut tree. The aim of the present study was to identify the fresh collections and confirm their phylogenetic positions.

The ITS, LSU, tef1a and rpb2, and combined data matrices contained 624, 867, 513, 865, and 2869 characters with gaps, respectively. The alignment comprised 92 strains, with Nakataea oryzae (CBS 243.76) and Pyricularia grisea (Ina168) from Magnaporthales as outgroup taxa. The ML analysis yielded a tree with a ln likelihood value of –45806.266577 and the following model parameters: alpha = 0.298226, Π(A) = 0.241173, Π(C) = 0.258552, Π(G) = 0.275145, and Π(T) = 0.225130. For BI analyses, the general time reversible model, additionally assuming a proportion of invariant sites with gamma-distributed substitution rates of the remaining sites (GTR+I+G), was determined to be the best for the ITS, LSU, and tef1a loci by MrModeltest, whereas the most appropriate model for the rpb2 locus was the Tamura-Nei model, additionally assuming a proportion of invariant sites with gamma-distributed substitution rates of the remaining sites (TrN+I+G). The phylogeny resulting from the RAxML maximum likelihood analysis of the combined gene sequence data is shown in Fig. 1. Overall, the topologies obtained from the different phylogenetic analyses were similar, and the best scoring RAxML tree is illustrated here. The bootstrap support values above 50% of maximum likelihood analysis (ML) and Bayesian posterior probability scores (≥0.90) are noted at the nodes.

The Diaporthales separates into 32 clades, representing 32 families, and the new isolates were clustered with a well-supported clade (ML/BI = 100/1) in Pseudomelanconidaceae. The two new isolates were different from any known genera in Pseudomelanconidaceae, and represented a new genus (Fig. 1).

Figure 1. 

Phylogram of Diaporthales from a maximum likelihood analysis based on combined ITS, LSU, tef1a and rpb2. Values above the branches indicate maximum likelihood bootstrap (left, ML BP ≥ 50%) and Bayesian probabilities (right, BI PP ≥ 0.90). The tree is rooted with Nakataea oryzae (CBS 243.76) and Pyricularia grisea (Ina168). New species proposed in the current study is in blue and the ex-type strains are marked with *.

Figure 1. 

Continued.

Taxonomy

Micromelanconis C.M. Tian & N. Jiang, gen. nov.

MycoBank No: 838927

Etymology

Name derived from micro- and the genus name Melanconis.

Type species

Micromelanconis kaihuiae C.M. Tian & N. Jiang.

Description

Sexual morph: not observed. Asexual morph: Conidiomata acervular, conspicuous, immersed in host bark to erumpent, covered by brown to blackish exuding conidial masses at maturity. Central column beneath the disc more or less conical. Conidiophores unbranched, aseptate, cylindrical, pale brown, smooth-walled. Conidiogenous cells annellidic, occasionally with distinct annellations and collarettes. Conidia hyaline when immature, becoming pale brown, ellipsoid, multiguttulate, aseptate, with hyaline sheath. Conidiomata formed on PDA after three weeks, randomly distributed, and black. Conidiophores unbranched, septate, cylindrical, pale brown, smooth-walled. Conidiogenous cells annellidic. Conidia pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, with hyaline sheath.

Notes

Micromelanconis is the third genus after Neopseudomelanconis and Pseudomelanconis in the family Pseudomelanconidaceae (Fig. 1). Micromelanconis is united in this family based on the Melanconis-like conidiomata, and pale brown conidia with conspicuous hyaline sheath. Micromelanconis produces two types of conidia from natural branches and manual media respectively, which differs from Neopseudomelanconis and Pseudomelanconis (Fan et al. 2018a; Jiang et al. 2018a). Additionally, Neopseudomelanconis is characterized by its septate conidia (Jiang et al. 2018a).

Micromelanconis kaihuiae C.M. Tian & N. Jiang, sp. nov.

MycoBank No: 838928

Figures 2, 3

Etymology

Named after Kaihui Yang, a Chinese heroine; Kaihui is also the name of the town where holotype was collected.

Figure 2. 

Morphology of Micromelanconis kaihuiae on branches of Castanea mollissima (BJFC-S1831) A, B habit of conidiomata on a branch C transverse section of conidiomata D longitudinal section through conidiomata E conidiogenous cells attached with conidia F, G conidia. Scale bars: 100 μm (C, D); 10 μm (E–G).

Description

Sexual morph: not observed. Asexual morph: Conidiomata acervular, 350–800 μm diam., conspicuous, immersed in host bark to erumpent, covered by brown to blackish exuding conidial masses at maturity. Central column beneath the disc more or less conical. Conidiophores unbranched, aseptate, cylindrical, pale brown, smooth-walled. Conidiogenous cells annellidic, occasionally with distinct annellations and collarettes, 12.4–47.1 × 1.2–3.8 μm. Conidia hyaline when immature, becoming pale brown, ellipsoid, multiguttulate, aseptate, 7.6–10.3 × 3.1–4.1 μm, L/W = 2–3.2, with hyaline sheath, 1 μm.

Figure 3. 

Morphology of Micromelanconis kaihuiae on the PDA plate (CFCC 54572) A colony on PDA B habit of conidiomata formed on PDA C, D conidiogenous cells attached with conidia E, F conidia. Scale bars: 10 μm (C–F).

Culture characters

Colony on PDA at 25 °C irregular, grey olivaceous, margin becoming diffuse, aerial hyphae short, dense, surface becoming imbricate, growth limited and ceasing after two weeks. Conidiomata formed after three weeks, randomly distributed, black. Conidiophores unbranched, septate, cylindrical, pale brown, smooth-walled. Conidiogenous cells annellidic, 9.1–18.5 × 2.5–5.3 μm. Conidia pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, 10.4–13.5 × 4–5 μm, L/W = 2.3–3.3, with hyaline sheath, 1.5 μm.

Specimens examined

China, Hunan Province, Changsha City, Changsha County, Kaihui Town, chestnut plantation, 40°24'32.16"N, 117°28'56.24"E, 262 m asl, on stems and branches of Castanea mollissima, Tian Chengming and Ning Jiang, 10 November 2020 (BJFC-S1831, holotype; ex-type culture, CFCC 54572 = KH5-3). Ibid. (BJFC-S1832, KH5-4).

Notes

Micromelanconis kaihuiae on Castanea mollissima (Fagaceae, Fagales) is phylogenetically close to Neopseudomelanconis castaneae on Castanea mollissima and Pseudomelanconis caryae on Carya cathayensis (Juglandaceae, Juglandales) (Fig. 1). All these three species are discovered on tree branches in China, and share similar morphological characters in having pale brown conidia with conspicuous hyaline sheath. Micromelanconis kaihuiae and Neopseudomelanconis castaneae even share the same host. However, they can be easily distinguished based on conidia shape, color and overall size of conidia (M. kaihuiae, pale brown, ellipsoid and aseptate conidia, 7.6–10.3 × 3.1–4.1 μm; pale brown, long dumbbell-shaped and aseptate conidia, 10.4–13.5 × 4–5 μm vs. N. castaneae, brown, ellipsoid to oblong and septate conidia, 18–21.5 × 4.8–7 μm vs. P. caryae, pale brown, ellipsoid to oblong and aseptate conidia, 12.5–16 × 4–5 μm) (Fan et al. 2018a; Jiang et al. 2018a). Furthermore, M. kaihuiae is separated from N. castaneae by 51/490 bp (10.4%) differences in ITS and 12/563 bp (2.1%) differences in LSU, and from P. caryae by 56/490 bp (11.4%) differences in ITS and 6/563 bp (1.1%) differences in LSU.

Diaporthales is a well-studied order based on integrated approaches of morphology and phylogeny in recent years (Castlebury et al. 2002; Rossman et al. 2007; Voglmayr and Jaklitsch 2014; Alvarez et al. 2016; Senanayake et al. 2017, 2018; Voglmayr et al. 2017; Braun et al. 2018; Fan et al. 2018a; Jiang et al. 2020a). Thirty-two accepted families are monophyletic and supported by morphological characters; four of them contain Melanconis-like fungi, namely Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae (Fan et al. 2018a). The Melanconis-like fungi were similar in their asexual morph, but well-separated in the phylogeny and their hosts (Voglmayr et al. 2012, 2017, 2019; Fan et al. 2018a, b; Jaklitsch and Voglmayr 2020). In the present study, a new genus and species were clustered in the family Pseudomelanconidaceae (Fig. 1), and differed from the other Melanconis-like genera by its long dumbbell-shaped conidia formed on PDA plates.

Hosts are useful taxonomic information in some families of Diaporthales, such as Coryneaceae, Cryphonectriaceae, Erythrogloeaceae and Gnomoniaceae (Voglmayr et al. 2012; Jaklitsch and Voglmayr 2019; Roux et al. 2020; Wang et al. 2020; Yang et al. 2020). Hosts are important to separate Melanconis-like genera, Juglanconis inhabit Juglans and Pterocarya of Juglandaceae, Melanconiella and Melanconis occur only on the plant family Betulaceae (Voglmayr et al. 2012, 2017, 2019; Fan et al. 2018b; Jaklitsch and Voglmayr 2020). Melanconis species are discovered only on Alnus and Betula, while Melanconiella occurs in the subfamily Coryloideae with the exception of M. betulae and M. decorahensis on Betula (Voglmayr et al. 2012; Du et al. 2017; Fan et al. 2018a). Species of Pseudomelanconidaceae inhabit Carya of Juglandaceae, and Castanea of Fagaceae (Fan et al. 2018a; Jiang et al. 2021). More interesting Melanconis-like may be revealed by more detailed surveys on tree-inhabiting fungi in the future.

This study is financed by the National Natural Science Foundation of China (Project No.: 31670647). We are grateful to Chungen Piao and Minwei Guo (China Forestry Culture Collection Center, Chinese Academy of Forestry, Beijing) for support of strain preservation during this study.