Introduction

Most of the cercosporoid genera with and without connection with mycosphaerella-like sexual morphs belong to Mycosphaerellaceae (Mycosphaerellales, Dothideomycetes, Ascomycota; Abdollahzadeh et al. 2020). They cover about 120 genera within this family (Videira et al. 2017; Crous et al. 2020; Bakhshi et al. 2021; Rajeshkumar et al. 2021; Bakhshi and Braun 2022; Chen et al. 2022; Yadav et al. 2022). The hyphomycetous ramularioid complex includes genera with colourless conidiophores and conidia. Morphologically, the most closely related genera of this complex are Cercosporella Sacc., Pseudocercosporella Deighton and Ramularia Unger. These genera are very difficult to distinguish based on characteristics of conidiophores and conidia using the light microscope. Accordingly, numerous species have been transferred repeatedly from one of these genera to the other. The taxonomic problems related to this complex were extensively discussed in several studies (Hughes 1949; Sutton and Waller 1988; Braun 1990, 1991a, b, 1994a, 1995, 1998; Verkley et al. 2004; Kirschner 2009; Videira et al. 2015, 2016, 2017).

In Ramularia, conidial scars and hila are slightly thickened and darkened while those of Cercosporella are clearly thickened. The ultrastructure of the conidiogenous loci are smooth and shaped as a truncated cone in Cercosporella, while a cladosporium-type circular rim with a central dome is seen in Ramularia (Kirschner 2009; Bensch et al. 2012). Cercosporella produces cup-shaped appressoria which are lacking in Ramularia (Kirschner 2009; Videira et al. 2016). Pseudocercosporella is characterised by unthickened and inconspicuous conidial loci and hila (Deighton 1973; Braun 1995; Frank et al. 2010).

Cercosporella, Hawksworthiana, Neoovularia, Phacellium, Pseudodidymaria, Ramularia and Ramulariopsis have hyaline conidiophores and conspicuous conidial loci. Monodidymaria, Neoramularia and Pseudocercosporella have inconspicuous conidial loci (Videira et al. 2016). The phylogenetic placement of Ramularia and allied genera within the order Capnodiales was established by Videira et al. (2016, 2017) using polyphasic approaches based on multi-locus DNA sequences and morphological and cultural data.

Several studies focus on the diversity of phytopathogenic fungi in India that are related to the Mycosphaerellaceae (Singh et al. 2007, 2008, 2011, 2012, 2013, 2014a, b, 2020a, 2022; Kamal 2010; Kumar et al. 2013, 2014; Awasthi et al. 2015, 2016; Kharwar et al. 2015; Kumar and Singh 2015, 2016; Singh and Kumar 2017; Kushwaha et al. 2020). However, all previous studies have relied exclusively on morphological data, and very few records are supported by cultures and DNA sequence data (Singh et al. 2020b; Verma et al. 2021a, b; Yadav et al. 2021).

During a survey for foliicolous fungi in the Afchand forest of Sagar, M.P., India, in December 2019, a colourless hyphomycete was found on Peristrophe bicalyculata (Retz.) Nees. The same fungus was originally collected from the same locality in 2013 and described as Pseudocercosporella andrographidis Awasthi et al. (Awasthi et al. 2016). Due to lack of phylogenetic analysis, the true generic affinity of P. andrographidis is unclear and unproven. In view of the limitation of using morphological traits for the elucidation of generic affiliations (Videira et al. 2017), phylogenetic examinations of the materials showed that this fungus could not be placed in any of the genera already described in the Mycosphaerellaceae. Therefore, the new genus Neocercosporella is proposed. The recognition of this novel genus segregated closely related genera in the Mycosphaerellaceae and rendered several of these paraphyletic.

Materials and methods

Isolates and morphology

Infected leaves were collected in separate sterilized polyethylene bags and kept in dry paper envelopes and brought to the laboratory along with collection details. Close-up photographs of the infected host parts were taken under a Stereo Zoom Microscope (Magnus: MSZ-TR) with attached camera (CatCam300EF). For light microscopy, fungal structures were transferred from the infected part of leaves and mounted on clear glass slides in a 1:1 mixture of glycerin and lactophenol cotton-blue. Fungal propagules were photographed using an Olympus compound microscope (CH20i-TR) equipped with a Magnus camera (MIPS CMOS). Scanning electron microscopy (SEM) was done with a field emission scanning electron microscope (FEI Nova Nano SEM-450). Detailed observations of morphological characters were carried out at different magnifications through light microscopy (450 × and 1000 ×) and scanning electron microscopy (up to ~ 18 K ×). For SEM micrographs, specimens were coated with gold-paladium using a POLARON Sputter coater (180 s in nitrogen atmosphere of 20 mA, 30 mm distant from the electrode) and examined with a LEO-430 scanning electron microscope. Size ranges of morphological features derived from at least 25 measurements, and 95% confidence intervals were calculated, with the extreme values given in parentheses. The holotype material is deposited in the Ajrekar Mycological Herbarium (AMH), Agharkar Research Institute (ARI), Pune, India, and isotype material is retained in the Mycological Herbarium of the Department of Botany of Banaras Hindu University, Varanasi, U.P., India (MH-BHU).

For the cultivation of samples of Neocercosporella AMH 9671 and AMH 10363, conidia were transferred to Petri dishes containing malt extract agar (2% w/v malt extract, 1.5% w/v agar agar). The dishes were placed at room temperature and diffuse daylight. Because cultures from both specimens grew about 1 mm in 4 week and ceased to grow, a living culture was not deposited.

DNA extraction, PCR and sequencing

For isolation, amplification and sequencing of nuclear DNA, specimens AMH 9671 and AMH 10363 were used. DNA was isolated from mycelia and spores freshly scrapped from the heavily infected surface of collected leaves using a sterile scalpel blade. Approximately 200 mg of wet-weight was transferred to 2-mL microcentrifuge tubes kept in liquid nitrogen for 2 min and then grinded to a fine powder using pestle and mortar. DNA was extracted using Himedia DNA Isolation Kit (HiPurA™ Fungal DNA Purification Kit) following the manufacturers’ protocols. Isolated DNA fragments were visualized by electrophoresis in a 1% agarose gel (w/v) stained with ethidium bromide under a Gel Documentation system (Bio-Rad Universal Hood II) and DNA concentration was quantified by using NanoDrop microvolume spectrophotometers (Thermo Scientific™ NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotometer with Wi-Fi).

The internal transcribed spacer (ITS) region was amplified by using ITS1/ITS4 (White et al. 1990), large subunit nuclear ribosomal DNA (LSU) gene with primers LROR/LR7 (Vilgalys and Hester 1990; Rehner and Samuels 1994) and partial DNA-directed RNA polymerase II subunit (RPB2) with primers RPB2-5F2/RPB2-7cR (Liu et al. 1999; Sung et al. 2007). PCR mixtures included the following ingredients for each 50 µL reaction: 5 µL of template DNA (~ 7 ng/µL), 5 µL PCR buffer containing MgCl2, 1.5 µL of each forward and reverse primer (10 pmol), 1 µL dNTP (10 mM), 0.3 μL Taq DNA polymerase (HiMedia: 5 Unit/μL) and 35.7 µL milli-Q water. The PCRs were carried out in a thermal cycler (Bio-Rad T100™). Conditions for the PCR amplification consisted of an initial denaturation at 95 °C for 5 min; followed by 35 cycles of denaturation at 94 °C for 1 min; annealing at 55, 52 and 54 °C for 1 min and extension at 72 °C for 1 min. The final extension step was done at 72 °C for 8 min. The amplicon was run in 1.2% agarose gel and visualized in a Gel Documentation system (Bio-Rad Universal Hood II) for the product size and purity. The PCR products were purified with FavorPrep™ PCR purification kit. Sequencing was done at AgriGenome Labs Private Ltd., Kerala, by the Sanger sequencing method using BigDye® Terminator v3.1 Cycle sequencing kit and ABI 3100 DNA analyzer with the same primers as for the PCR.

Sequence alignment and phylogenetic analysis

The obtained ITS, LSU and RBP2 sequences from AMH 9671and AMH 10363 were assembled and edited with Chromas v.2.6.6. The manually edited sequences were submitted to NCBI GenBank (Table 1) and were subjected to a megablast search of the NCBI GenBank nucleotide database to retrieve sequences of related strains. Reference sequences were also selected based on sequence availability from relevant published literature (Table 1). From the strains listed in Table 1, only those with the complete dataset of genes were used in the subsequent phylogenetic analyses, with the exception of Cercospora rodmanii (5H-GTOX), Cercosporella pfaffiae (Vic31849) and Sonderhenia sp. (CPC 17710) lacking the RPB2 sequence. Sequence alignments were generated with MUSCLE in MEGA-X v.10.1.8 (Kumar et al. 2018). The alignments were manually checked, improved and concatenated where necessary using BioEdit v.7.0.9 (Hall 1999) and MEGA-X v.10.1.8 (Kumar et al. 2018) and deposited as electronic supplementary materials in TreeBASE, study number 30079.

Table 1 Taxa included in molecular phylogenetic analyses and their GenBank accession numbers. Newly generated sequences are in bold
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The phylogenetic methods used in this study included a Bayesian analysis (BI) performed with MrBayes v.3.2.7 (Ronquist et al. 2012), maximum likelihood (ML) analysis performed with RAxML v.8.2.10 (Stamatakis 2014) and maximum parsimony (MP) analysis performed with PAUP v. 4.0b10 (Swofford 2003). The phylogenetic analyses were individually applied to two datasets as different combinations of genes may result in alternative phylogenetic hypotheses as described by Videira et al. (2017). All trees were rooted with Cylindroseptoria ceratoniae (CBS 477.69). Dataset 1 consisted of a concatenated alignment of LSU and RPB2 sequences, and dataset 2 consisted of concatenated alignments of LSU, RPB2 and ITS sequences from 19 genera currently known to belong in the Mycosphaerellaceae, and from closely related other families.

Model GTR + I + G was selected for BI using a Markov chain Monte Carlo (MCMC) algorithm (Rannala and Yang 1996). The analysis was performed till the standard deviation of split frequency was below 0.01. The first 25% of generated trees representing the burn-in phase were discarded, and the remaining trees were used to calculate posterior probabilities as a majority rule consensus tree. ML analysis was also performed using a GTR model of site substitution, including GAMMA with a proportion of invariant sites (Stamatakis 2014). The ML support values were evaluated with a bootstrapping method of 1000 replicates. For the maximum parsimony analysis, a heuristic search option with 100 random sequence additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm was used. Alignment gaps were treated as fifth character states, and all characters were unordered and of equal weight. Maxtrees were set to 5000, branches of zero length were collapsed, and all multiple, equally most parsimonious trees were saved. The robustness of the most parsimonious trees obtained was evaluated by 1000 bootstrap replications (Hillis and Bull 1993). Descriptive tree statistics for parsimony tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), homoplasy index (HI) and G-fit were calculated. These analyses involved 69 nucleotide sequences.

The trees presented here were obtained with the ML approach. Tree reconstruction, visualization and editing were done using FigTree v.1.4.4 and TreeGraph_2.15.0. The multigene phylograms are shown in Figs. 1 and 2.

Fig. 1
figure 1

Phylogenetic tree resulting from a RAxML analysis of the combined LSU/RPB2 sequence alignment (dataset 1). The Bayesian posterior probabilities (≥ 0.90; BI-PP), maximum likelihood bootstrap support values (≥ 50%; ML-BS) and maximum parsimony bootstrap support values (≥ 50%; MP-BS) are given at the nodes (BI-PP/ML-BS/MP-BS). The newly introduced lineage is represented in bold and novel genera denoted in blue. The family name Mycosphaerellaceae is unabbreviated while others are abbreviated as follows: D = Dissoconiaceae, P = Phaeothecoidiellaceae, S = Schizothyriaceae, T = Teratosphaeriaceae, C = Cladosporiaceae. The tree is rooted to Cylindroseptoria ceratoniae (CBS 477.69)

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Fig. 2
figure 2

Phylogenetic tree resulting from a RAxML analysis of the combined LSU/RPB2/ITS sequence alignment (dataset 2). The Bayesian posterior probabilities (≥ 0.90; BI-PP), maximum likelihood bootstrap support values (≥ 50%; ML-BS) and maximum parsimony bootstrap support values (≥ 50%; MP-BS) are given at the nodes (BI-PP/ML-BS/MP-BS). The newly introduced lineage is represented in bold and novel genera denoted in blue. The family name Mycosphaerellaceae is unabbreviated while others are abbreviated as follows: D = Dissoconiaceae, P = Phaeothecoidiellaceae, S = Schizothyriaceae, T = Teratosphaeriaceae, C = Cladosporiaceae. The tree is rooted to Cylindroseptoria ceratoniae (CBS 477.69)

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Results

The sequences from specimens AMH 9671 and AMH 10363 were 100% identical in each region. The data for the trees conducted in the different analyses are shown in Table 1. Phylogenetic trees obtained from the combined gene analyses are supplied below (Figs. 1 and 2).

Dataset 1 (LSU and RPB2 phylogeny)

This dataset consisted of a concatenated alignment of two loci (LSU, RPB2). The final alignment contained a total of 1439 characters divided in two partitions containing 748 (LSU) and 691 (RPB2) characters, including alignment gaps. Phylogenetic trees generated from Bayesian analyses (BI), maximum likelihood (ML) and maximum parsimony (MP) produced trees with similar overall topology. A best scoring RAxML tree is presented in Fig. 1, with the Likelihood value of − 21,290.719845. The most parsimonious tree was characterized by TL = 6393, CI = 0.297513, RI = 0.576999, RC = 0.171665 and HI = 0.702487, and G-fit is − 491.819875. From the analysed characters, 530 were constant, 78 were variable and parsimony-uninformative, and 831 were parsimony-informative. In this analysis, Cercosporella catenulata (CBS 355.73) and Cercosporella dolichandrae (CBS 138101) are now separated from the Cercosporella clade and are placed in a separate sister branch of Ramulariopsis (Fig. 1). Acervuloseptoria fraxini (CPC36558) and A. ziziphicola (CBS138009) form a paraphyletic group. Acervuloseptoria ziziphicola and N. peristrophes form a statistically supported monophyletic group (BI-PP/ML-BS/MP-BS: 1/100/100).

Dataset 2 (LSU, RPB2 and ITS phylogeny)

The final alignment of this dataset contained a total of 1979 characters divided into three partitions containing 748 (LSU), 691 (RPB2) and 540 (ITS) characters, including alignment gaps. Phylogenetic trees generated from BI, ML and MP analysis had similar overall topology. A best scoring RAxML tree is presented in Fig. 2, with the Likelihood value of − 27,134.491457. The most parsimonious tree was characterized by TL = 7894, CI = 0.321257, RI = 0.575503, RC = 0.184884 and HI = 0.678743, and G-fit is − 615.475663. From the analysed characters, 744 were constant, 181 were variable and parsimony-uninformative, and 1054 were parsimony-informative. The results of analysis of dataset 2 (Fig. 2) fully support the dataset 1 analysis (Fig. 1).

Acervuloseptoria, Cercosporella, Neoacervuloseptoria, Neocercosporella, Neoramulariopsis and Ramulariopsis formed a statistically supported monophyletic group.

Taxonomy

Neoacervuloseptoria Raghv. Singh & Sanjay, gen. nov.

MycoBank MB840502

Etymology: derived from the genus name Acervuloseptoria.

Diagnosis: differs from the genus Acervuloseptoria by its pycnidial conidiomata opening via central ostioles and intermingled among spermatogonia.

Description: (adapted from Crous et al. 2020): plant pathogenic, foliicolous. Conidiomata pycnidial, intermingled among spermatogonia, black, opening via ostiole; wall brown, textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity. Conidiogenous cells subcylindrical to ampulliform, hyaline, smooth, proliferating percurrently and sympodially at apex. Conidia solitary, subcylindrical, hyaline, smooth, granular, straight to curved, apex subobtuse, base truncate with basal marginal frill, septate.

Type species: Neoacervuloseptoria fraxini (Crous & Bulgakov) Raghv. Singh & Sanjay (≡ Acervuloseptoria fraxini Crous & Bulgakov).

Neoacervuloseptoria fraxini (Crous & Bulgakov) Raghv. Singh & Sanjay, comb. nov.

MycoBank MB840503

Basionym: Acervuloseptoria fraxini Crous & Bulgakov, Fungal Syst. Evol. 6: 175 (2020).

Description and illustration: Crous et al. (2020)

Notes: Acervuloseptoria was established with the type species A. ziziphicola Crous & Jol. Roux (Crous et al. 2014). Only three species names are validly accepted in Acervuloseptoria (https://www.mycobank.org, queried 8 December 2021). In Videira et al. (2017), A. ziziphicola (CBS 138009) formed a sister lineage of Cercosporella based on LSU-RPB2 sequence data, while it clustered among the Cercosporella species based on LSU-RPB2-ITS sequence data. In 2020, A. fraxini Crous & Bulgakov (CPC 36558) was inferred as a relative of A. ziziphicola based on LSU-RPB2 sequence data (Crous et al. 2020). According to Crous et al. (2020), A. fraxini does not show morphological similarity with A. ziziphicola but was tentatively maintained in Acervuloseptoria.

In this study, based on both datasets, A. fraxini clustered apart from A. ziziphicola (Figs. 1 and 2). Acervuloseptoria ziziphicola has acervular conidiomata that are black, erumpent and multilocular; their upper layer disintegrates upon maturity (Crous et al. 2014). Conidiomata in A. fraxini are pycnidial (thus, not acervular), have a central ostiole and are intermingled among spermatogonia (Crous et al. 2020). Therefore, a new genus Neoacervuloseptoria is to be introduced for the strain CPC 36558 in the Mycosphaerellaceae. Acervuloseptoria ziziphicola separated as a sister lineage of Neocercosporella with high bootstrap support (BI-PP/ML-BS/MP-BS: 1/100/100) (Figs. 1 and 2). The differences in morphology are significant enough for retaining Acervuloseptoria (a coelomycete) as distinct from Neocercosporella (a hyphomycete). No molecular sequence data is available for A. capensis (G. Winter) Crous (Crous et al. 2015).

Neocercosporella Sanjay & Raghv. Singh, gen. nov. Figs. 3, 4, 5, and 6

Fig. 3
figure 3

Symptoms of infection of Neocercosporella peristrophes on Peristrophe bicalyculata. (a) Initial stage of symptom on upper surface of leaf, (b) initial stage of infection on lower surface of leaf, (c, d) late stage of infection on lower surface of leaves, (e, f) fascicles of conidiophores developed on the surface of leaves. Bars: (a–d) 20 mm, (e) 200 µm, (f) 100 µm

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Fig. 4
figure 4

Microphotographs of Neocercosporella peristrophes (AMH 10363). (a–c) Fascicles of conidiophores, (d–g) conidiophores with conidia, (h) branched conidiophores. Bars: 10 µm

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Fig. 5
figure 5

Microphotographs of Neocercosporella peristrophes (AMH 10363). (a–l) Conidia, (m–o) catenate conidia, (p) germinating conidium. Bars: 10 µm

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Fig. 6
figure 6

Scanning electron microphotographs of Neocercosporella peristrophes (AMH 10363). (a) Initial stage of development of conidiophores through stomata, (b, c) fascicles of conidiophores, (d) polyblastic conidiogenous cell (yellow arrows), (e–g) top view of conidiogenous loci, (h, i) lateral view of conidiogenous loci, (j, k) conidia, (l, m) Hila of conidia. Bars: (a–c) = 10 µm, (d–i) = 1 µm, (j, k) = 10 µm, (l, m) = 1 µm

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MycoBank MB840500

Etymology: derived from the genus name Cercosporella.

Diagnosis: differs from Cercosporella s. str. by its conidiogenous locus, which is conical in shape and having a small, rim-like depression on the top, encircling a small, flat, protuberant-like structure. In Cercosporella, conidiogenous cells are terminal and conidia formed singly, while conidiogenous cells in Neocercosporella are terminal and intercalary, and the conidia are produced at least initially in chains. It also differs from Acervuloseptoria due to its hyphomycetous nature, while the latter is coelomycetous.

Description: Plant pathogenic, foliicolous. Hyphae restricted to intercellular spaces. Colonies hypogenous. Stromata substomatal or subcuticular to erumpent. Conidiophores macronematous, fasciculate, arising from stromata, initially erumping through stomata, later by rupturing epidermis, erect to procumbent, hyaline to light olivaceous, smooth, thin- to thick-walled, unbranched, rarely branched, straight to slightly curved, geniculate at the tip, septate. Conidiogenous cells integrated, terminal and intercalary, polyblastic, sympodial, conidiogenous loci slightly protuberant, thickened and darkened, loci conical having a very small rim-like depression on the top encircling a small flat protuberant-like structure (ultrastructure). Conidia formed singly, rarely catenate, mostly hyaline, rarely light olivaceous, dry, obclavate to obclavate-cylindrical, straight to curved, smooth, thin-walled, euseptate, base obconically truncate to rounded, tip obtuse, hila unthickened, sometimes slightly thickened and darkened.

Type species: Neocercosporella peristrophes (Syd.) Sanjay & Raghv. Singh (≡ Cercosporella peristrophes Syd.)

Notes: Based on a megablast search of NCBI’s GenBank nucleotide database, the closest hits using the ITS sequence had highest similarity to Acervuloseptoria ziziphicola [strain CBS 138009, GenBank NR_156287; identities = 461/484 (95%), 8 gaps (1%)], Cercosporella dolichandrae [strain CBS 138101, GenBank NR_156282; identities = 459/495 (93%), 11 gaps (2%)] and Cercosporella virgaureae [strain CBS 113304, GenBank GU214658; identities = 461/484 (95%), 8 gaps (1%)]. Closest hits using the LSU sequence are Cercosporella virgaureae [strain CBS 113304, GenBank GU214658; identities = 1096/1133 (97%), 6 gap (0%)], Septoria obesa [strain CBS 354.58, GenBank GU214493; identities = 1095/1133 (97%), 6 gap (0%)] and Septoria dysentericae [strain CBS 12328, GenBank GU214699; identities = 1092/1133 (96%), 6 gap (0%)]. Closest hits using the RPB2 sequence had highest similarity to Acervuloseptoria ziziphicola [strain CBS 138009, GenBank MF951425; identities = 815/891 (91%), 0 gaps (0%)], Cercosporella virgaureae [strain CBS 113304, GenBank KX348051; identities = 746/893 (84%), 2 gaps (0%)] and Cercosporella catenulata [strain CBS 355.73, GenBank KX288424; identities = 655/795 (82%), 4 gaps (0%)].

Neocercosporella peristrophes (Syd.) Sanjay & Raghv. Singh, comb. nov. Figs. 3, 4, 5, and 6

MycoBank MB840501

Basionym: Cercosporella peristrophes Syd., Ann. Mycol. 31: 93 (1933).

Synonyms: Cercosporella peristrophes var. microspora N.D. Sharma & R.P. Mishra, J. Indian Bot. Soc. 56: 133 (1977).

Pseudocercosporella andrographidis N. Awasthi, Raghv. Singh & Sh. Kumar, Sydowia 68: 30 (2016), syn. nov.

Description: Infection spots amphiphyllous, white, circular to irregular, 1–10 mm in diam., later covering the entire, necrotic leaf surface. Colonies hypogenous, white, velvety. Mycelium internal. Stromata present, globose to somewhat angular, substomatal or subcuticular to erumpent, hyaline, (9)15–25(35) × (10)15–20(25) µm. Conidiophores macronematous, densely fasciculate, arising from stromata, initially erumping through stomata, later by rupturing epidermis, erect to procumbent, hyaline to light olivaceous, smooth, thin-walled to thick-walled, unbranched, rarely branched, straight to slightly curved, geniculate at the tip, 0–3-euseptate, (10)15–40(53) × (2)3–4(6) µm. Conidiogenous cells integrated, terminal and intercalary, polyblastic, cylindrical, conidiogenous loci slightly protuberant, thickened and darkened, loci conical having small rim-like depression on the top encircling a small, flat, protuberant-like structure (ultrastructure), 1.5–2.0 µm wide. Conidia formed singly, rarely catenate, mostly hyaline, rarely light olivaceous, dry, obclavate to obclavate-cylindrical, straight to curved, smooth, thin-walled, (0)1–6(12)-euseptate, base obconically truncated to rounded, tip obtuse, (18)30–80(117) × (2)3–5(6.5) µm, hila unthickened, sometimes slightly thickened and darkened, 1–2 µm wide.

Materials examined: India, Uttar Pradesh, Allahabad, on leaves of Peristrophe bicalyculata (Retz.) Nees, Nov. 1928, Tandon (holotype HCIO 12215); India, Madhya Pradesh, Sagar, Afchand forest, on living leaves of P. bicalyculata, Sept. 2013, N. Awasthi (epitype designated here AMH 9671, MycoBank MBT10009148, gene sequence GenBank: MZ311866 (ITS), MZ311874 (LSU), OL773683 (RPB2); India, Madhya Pradesh, Sagar, Afchand forest, 23.834030°N 78.746567°E, on living leaves of P. bicalyculata, 01 Dec. 2019, R. Singh (AMH 10363, gene sequence GenBank: ON310831 (ITS), ON310846 (LSU), ON376994 (RPB2).

Notes: Neocercosporella belongs to the Mycosphaerellaceae (Figs. 1 and 2). The type species of this genus was originally described as Pseudocercosporella andrographidis (Awasthi et al. 2016) from the same locality as the epitype. The host of P. andrographidis was mistakenly identified as Andrographis paniculata instead of Peristrophe bicalyculata. The true generic affinity of P. andrographidis was quite unclear and unproven, due to lack of molecular sequence data and lack of discussion of ultrastructure; hence, it was established as a member of Pseudocercosporella, solely based on morphological features (Awasthi et al. 2016). However, the phylogenetic position of P. andrographidis, quite distant from the Pseudocercosporella s. str. clade, does now allow maintaining this species in Pseudocercosporella. We obtained cultures from specimens AMH 9671 and AMH 10363 but, unfortunately, they stopped growing after few subculturing events. DNA sequence data from specimens AMH 9671 and AMH 10363 are identical, cluster together with statistical support (BI-PP/ML-BS/MP-BS: 1/100/100), and could not be placed in any of the genera already described in the Mycosphaerellaceae (Figs. 1 and 2). Hence, it is justified to introduce a new genus for this monotypic lineage, viz., Neocercosporella.

Cercosporella peristrophes, the name of a common cercosporoid hyphomycete on Peristrophe bicalyculata, is the agent of a leaf spot disease and used as type species for Neocercosporella. Cercosporella peristrophes var. microspora, described from India on Peristrophe bicalyculata, is morphologically indistinguishable from Cercosporella peristrophes (Braun 1995).

On the basis of the two datasets, it is confirmed that Cercosporella, Neocercosporella, Pseudocercosporella and Ramularia represent separate clades (Figs. 1 and 2). Cercosporella, Neocercosporella and Ramularia can be easily distinguished, based on the ultrastructure of their conidiogenous loci. Cercosporella has flat conidial loci in the shape of a truncated cone (Kirschner 2009), while Neocercosporella has conical loci with very small rim-like depressions on the top encircling a small, flat and protuberant-like structure. Conidiogenous loci of Ramularia have a raised rim with a central dome (Kirschner 2009) that is cladosporium-like. Cercosporella produces terminal conidiogenous cells forming conidia solitarily, while Neocercosporella produces terminal and intercalary conidiogenous cells and weak catenation of conidia.

Cercospora acanthi Pass., C. peristrophes Thirum. & Govindu and C. peristrophigena Narayan et al. are additional asexual species of the Mycosphaerellaceae reported on Peristrophe bicalyculata (Thirumalachar and Govindu 1953; Narayan et al. 1999; Crous and Braun 2003; Kamal 2010). These species are irrelevant for the new genus since they belong to the genus Cercospora Fresen., which is characterized by having pigmented conidiophores and thickened and darkened conidiogenous loci and hila.

Semipseudocercospora peristrophes-acuminatae (J.M. Yen) J.M. Yen is also reported on Peristrophe acuminata (Yen 1983) and differs from the novel genus due to its dark-olivaceous to dark-brown nature of conidia and conidiophores. The conidiogenous loci are distinctly denticle-like, and the solitary conidia are didymo- to phragmosporous, i.e. not scolecosporous (Videira et al. 2017).

Neoramulariopsis Raghv. Singh & Kushwaha, gen. nov.

MycoBank MB840504

Etymology: derived from the genus name Ramulariopsis.

Diagnosis: differs from Cercosporella due to its highly branched chains of conidia and its phylogenetic position that is closer to Ramulariopsis. The latter differs from Neoramulariopsis in having frequently branched conidiophores with integrated, terminal, intercalary and pleurogenous conidiogenous cells.

Description: (adopted from Crous et al. 2014 and Videira et al. 2016): plant pathogenic, foliicolous. Stromata immersed to erumpent, substomatal, brown, consisting of pseudoparenchymatal cells. Ascomata developing from stromata, with central ostiole; wall multilayers of brown textura angularis. Asci bitunicate, hyaline, smooth, obovoid, stipitate, with minute apical chamber. Ascospores guttulate, septate. Mycelium composed of hyaline, septate, branched hyphae. Conidiophores arising from hyphae or stromata, simple or branched, straight and subcylindrical to flexuous or geniculate, sinuous, septate, hyaline, thin-walled, smooth. Conidiogenous cells integrated, terminal or lateral, hyaline, subcylindrical to geniculate-sinuous, with single to multiple conidiogenous loci, loci truncate, thickened to unthickened, not darkened or very slightly darkened. Conidia hyaline, smooth, formed singly or in branched chains; ramoconidia, intercalary and terminal conidia aseptate or septate, with thickened but not darkened hila.

Type species: Neoramulariopsis unguis-cati (Speg.) Raghv. Singh & Kushwaha (≡ Cercosporella unguis-cati Speg.)

Neoramulariopsis catenulata (Videira & Crous) Raghv. Singh & Kushwaha, comb. nov.

MycoBank MB840505

Basionym: Cercosporella catenulata Videira & Crous, Stud. Mycol. 83: 91 (2016).

Description and illustration: Videira et al. (2016)

Neoramulariopsis unguis‑cati (Speg.) Raghv. Singh & Kushwaha, comb. nov.

MycoBank MB840506

Basionym: Cercosporella unguis-cati Speg. 13: 422–423 (1911).

Synonyms: Pseudocercospora unguis-cati (Speg.) U. Braun, Mycotaxon 51: 49 (1994b).

Cercosporella dolichandrae Crous & den Breeÿen, Persoonia 32: 233 (2014).

Description and illustration: Crous et al. (2014)

Notes: Cercosporella catenulata and C. dolichandrae clustered together with Cercosporella virgaureae (Thüm.) Allesch. (the type species of Cercosporella) and formed a well-defined clade close to Acervuloseptoria and Ramulariopsis in Mycosphaerellaceae (Crous et al. 2014; Videira et al. 2016, 2017). Phylogenetically, Acervuloseptoria is represented by a single-strain lineage that is closely related to Cercosporella and Ramulariopsis (Videira et al. 2017). However, the phylogenetic position of Acervuloseptoria is not yet clear. It clustered near Cercosporella when LSU-RBP2 sequence data were analysed, but among Cercosporella species based on LSU-RBP2-ITS sequence data (Videira et al. 2017). The latter dataset separated C. catenulata and C. dolichandrae from Cercosporella. In the single-gene analysis of either LSU or ITS, Acervuloseptoria clustered outside both the Cercosporella and the Ramulariopsis clades with high support in the LSU (BI, PP = 0.94) but without support in the ITS tree (Videira et al. 2017). Thus, Acervuloseptoria appears as a single-strain lineage sister to both Cercosporella and Ramulariopsis (Videira et al. 2017).

We obtained similar results (Figs. 1 and 2), as reported by Crous et al. (2020). Morphological characters support separating C. catenulata and C. dolichandrae from Cercosporella s. str. as the two species produce branched conidial chains (Crous et al. 2014; Videira et al. 2016), as does Ramulariopsis. Ramulariopsis species have frequently branched conidiophores, terminal and intercalary conidiogenous cells, forming small lateral projections or branchlets usually just below the septa, and cicatrized, thickened and darkened loci (Braun 1998). As Cercosporella catenulata and C. dolichandrae form terminal conidiogenous cells with truncate loci (Crous et al. 2014), it is clear that a new genus, Neoramulariopsis, is required to accommodate these two Cercosporella species and perhaps other cercosporella-like species producing terminal conidiogenous cells with truncate loci.

Silva et al. (2012) reported leaf spots on D. unguis-cati in Brazil caused by Pseudocercospora unguis-cati (Speg.) U. Braun (Braun 1994b). Similar leaf spots on the same host are also caused by Cercosporella dolichandrae (Crous et al. 2014), and questions emerged as to whether Ps. unguis-cati and C. dolichandrae are conspecific. A BLASTn search of the ITS sequence of Ps. unguis-cati (accession no. MW036753) showed a 99.83% identity (604/605 nt) to the type sequence of C. dolichandrae (NR_156282/ KJ869140; Crous et al. 2014). Cercosporella unguis-cati was originally described on D. unguis-cati by Spegazzini (1911) and is morphologically identical to Ps. unguis-cati Silva et al. (2012). The earlier name of Cercosporella unguis-cati Speg. takes priority over Ps. unguis-cati (Speg.) U. Braun and C. dolichandrae Crous & den Breeÿen, which are now considered synonyms (Colmán et al. 2020).

Similarly, Neocercosporella also differs from Ramulariopsis in having hyaline to very light olivaceous, mostly unbranched or rarely branched conidiophores. Neocercosporella does not form conidiogenous cells as small lateral projections or branchlets just below the septa, and its conidia are mostly formed solitary, although they can be rarely catenate. Its conidia are also mostly hyaline, rarely light olivaceous and hila are mostly unthickened or rarely slightly thickened and darkened. The phylogenetic analysis in this study also supports the separation of Neocercosporella from Ramulariopsis.