Morphology and Molecular Phylogenetic Characterization of Novel Tar Spot Disease-Causing Fungi on Fabaceae Trees in Thailand

Abstract

Tar spot diseases have a huge impact on various plants by reducing the photosynthesis surface and allowing secondary severe infections on the host. Phyllachoraceae species causing tar spots infect both monocot and dicot plants and are known as obligate parasites. In the current study, two different tar spot disease symptoms were observed on Pterocarpus macrocarpus and Dalbergia sp. in northern Thailand. The phylogeny of a combined matrix of LSU, SSU, and ITS and morphology revealed that both causal species of tar spot diseases belong to the genus Neophyllachora. Furthermore, these results showed that these are novel species of the genus Neophyllachora, N. pterocarpi-macrocarpae on P. macrocarpus and N. dalbergiae on Dalbergia sp. with high bootstrap supports.

1. Introduction

Tar spot diseases are caused by species belonging to Phyllachoraceae and Rhytismataceae, including Phyllachora spp., Neophyllachora spp., and Rhytisma spp. Of them, Rhytisma spp. (Rhytismataceae) causes tar spots mainly on perennial plants in the Northern Hemisphere while species from Phyllachora and Neophyllachora cause tar spot disease on a variety of monocot and dicot plant hosts. Tar spot symptoms caused by these two families show slight differences in disease morphology. Rhytisma-like species form large black stromata on living leaves with one or several immersed apothecial ascomata, erumpent through the outer layers and exposing the hymenium [1]. In contrast, species in Phyllachoraceae form immersed clypeate pseudostroma in leaf tissues, varying from a subcuticular or intra-epidermal to a generalized infection of the entire section of the mesophyll. This forms characteristic slightly raised, semi-circular, dark brown to black lustrous spots. These raised melanized structures named stromata/pseudostroma are commonly referred to as tar spots [2,3].

The members of Phyllachoraceae cause tar spots on the leaves, stems, and fruits [4] and are well-known as obligate parasites. Phyllachoraceae was established by Theissen and Sydow [5] with Phyllachora as the type genus. It accommodates more than 1000 species worldwide and consists of 54 genera [4,5]. Many species in Phyllachoraceae are considered important plant pathogens, causing diseases that severely affect agricultural food crops [6], forest plants and ornamental plants [7,8], and grasses [9]. However, species in Neophyllachora are usually recognized as minor pathogens but can often facilitate secondary infections by severe pathogens [10].

Initially, the taxonomic classification of Phyllachoraceae was performed mainly based on the morphological characteristics and differences of host plants. Based on this hypothesis, most species are assumed to be host-specific. However, with the development of a polyphasic approach through molecular phylogeny of multiple loci, morphology, and considering the hosts and their ecological niches, the taxonomic position of Phyllachoraceae started to be resolved [10]. Proper taxonomic clarification supports accurate diagnosis and the understanding of disease dynamics [11]. For example, although Phyllachora maydis causes devastating tar spot diseases on corn [12], it is recognized as a species complex composed of several closely related species, having different host ranges [13]. Hence, it is crucial to apply a polyphasic approach in diagnosing diseases and identifying the Phyllachoraceae species.

The genus Neophyllachora Dayar. & K.D. Hyd was introduced by Dayarathne et al. [10], designating N. myrciae as the type. Neophyllachora myrciae, N. myrciariae, N. cerradensis, N. subcircinans, and N. truncatispora were introduced as novel combinations through the reexamination of morphology and molecular phylogeny. Neophyllachora differs from Phyllachora with subepidermal to intra-epidermal stromata with less deep mesophyll invasion and clavate asci, whereas Phyllachora consists mostly of epidermal clypeus. Neophyllachora species have been previously reported on the plant family Moraceae from the Far East and Southeast Asia [14,15], and Myrtaceae from South America [10].

During a random collection visit in the forest areas of Chiang Mai and Lampun provinces in Northern Thailand, tar-spot-like symptoms were observed on plants in Fabaceae (Pterocarpus macrocarpus and Dalbergia sp.). Pterocarpus macrocarpus is widely grown for timber and Dalbergia species are commonly found in deciduous forests in northern Thailand. Dalbergia species are capable of withstanding frequent fires and becoming increasingly common in degraded areas where their ability to fix atmospheric nitrogen gives them a competitive advantage [16]. Therefore, Dalbergia species play a crucial role in Southeast Asian forests. In this study, two novel species of Neophyllachora and the role of Fabaceae hosts in the speciation are discussed.

2. Materials and Methods

2.1. Sample Collection, Morphological Study, and Herbarium Deposit

Symptomatic leaves of tar spot diseases on Pterocarpus macrocarpus (Fabaceae) and Dalbergia sp. (Fabaceae) were collected during random collection visits conducted in 2024 in Chiang Mai and Lamphun Provinces, Thailand. The specimens were transported to the laboratory in plastic bags and examined under 20×–40× magnification using a Zeiss Stemi 305 stereo microscope (Zeiss, Jena, Germany). Ascomata were studied by preparing hand-cut sections, which were mounted in lactic acid and ddH₂O water. Microscopic features, such as ascomata, peridium, paraphyses, asci, and ascospores, were observed using a Zeiss Axiovision Scope-A1 microscope (Zeiss, Jena, Germany) equipped with a Canon EOS 6D digital camera (Canon, Tokyo, Japan). Measurements were taken using the Tarosoft Image Frame Work software version 0.9.7 (Tarosoft, Bangkok, Thailand), and photographic plates were prepared with Adobe Photoshop CC 2014 version 15 (Adobe Systems, San Jose, CA, USA). The specimens were deposited in the collection of the Department of Entomology and Plant Pathology (CDEP), Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand. Index Fungorum numbers were registered as outlined in Index Fungorum [17].

2.2. DNA Extraction, PCR Amplification, and Sequencing

Total genomic DNA was extracted directly from the stromata using the DNA Extraction Mini Kit (FAVORGEN, Ping-Tung, Taiwan) according to the manufacturer’s protocol. The extracted DNA was then amplified using polymerase chain reaction (PCR). Three loci, large subunit rRNA (LSU), internal transcribed spacer region (ITS), and small subunit rRNA (SSU) were targeted for amplification [7,10]. The LSU gene was amplified using the primers LR0R and LR5 [18], the ITS region with the primers ITS1F and ITS4 [19,20], and the SSU region with the primers NS1 and NS4 [19]. Each amplification reaction was conducted in a 25 μL volume, consisting of 12.5 μL of master mix (Quick Taq HS DyeMix, TOYOBO, Osaka, Japan), 1 µL of each forward and reverse primer (Macrogen Inc., Seoul, Republic of Korea), 9.5 µL of PCR-grade water, and 1 µL of DNA template. The PCR thermal cycling conditions for the amplification of LSU, ITS, and SSU were as follows: 30 cycles of denaturation at 98 °C for 10 s, annealing at 56 °C for 10 s, and extension at 72 °C for 10 s (ITS and SSU) or 20 s (LSU), with a final extension at 72 °C for 1 min. The PCR products were visualized on a 1% agarose gel and sequenced using Sanger sequencing by First Base Company (Kembangan, Malaysia). The resulting nucleotide sequences were deposited in GenBank (accession numbers listed in

Table 1. Taxa table for phylogenetic analysis. Culture collection numbers with ^“T” designate the type.

Species	Sample Code	LSU	SSU	ITS
Camarotella costaricensis	MM-21	KX430490	KX451851	KX451900
Coccodiella calatheae	MP5133T	MF460370	MF460376	MF460366
Coccodiella melastomatum	CMU78543		U78543
Coccodiella miconiae	ppMP1342	KX430506	KX451871	MF460365
Coccodiella miconiicola	SO-15	MF460374	MF460380	MF460369
Coccodiella toledoi	MM-165	KX430488	KX451865	KX451917
Neophyllachora cerradensis	UB21823	—	—	KC683470
Neophyllachora cerradensis	UB21908T	—	—	KC683471
Neophyllachora dalbergiae	CDEP-50T	PV241510	—	PV241504
Neophyllachora dalbergiae	CDEP-51	PV241511	—	PV241505
Neophyllachora fici	MFLU 19-2702	—	—	MW114384
Neophyllachora fici	NCYU 19-0061	—	—	MW114385
Neophyllachora fici	NCYU 19-0326	—	—	MW114386
Neophyllachora myrciae	UB21292	—	—	KC683463
Neophyllachora myrciae	UB22192	—	—	KC683476
Neophyllachora myrciariae	UB21781T	—	—	KC683469
Neophyllachora pterocarpi-macrocarpae	CDEP-52T	PV241512	PV241165	PV241506
Neophyllachora pterocarpi-macrocarpae	CDEP-53	PV241513	PV241166	PV241507
Neophyllachora pterocarpi-macrocarpae	CDEP-54	PV241514	PV241167	PV241508
Neophyllachora pterocarpi-macrocarpae	CDEP-55	PV241515	PV241168	PV241509
Neophyllachora subcircinans	UB09748	—	—	KC683441
Neophyllachora subcircinans	UB21347	—	—	KC683466
Neophyllachora subcircinans	UB21747	—	KC902622	KC683467
Neophyllachora truncatispora	UB14083	—	KC902614	KC683448
Neophyllachora religiosa	MFLU 23-0258	—	—	OQ821004
Phyllachora arthraxonis	MHYAU:072	MG269803	—	MG269749
Phyllachora arundinellae	MHYAU:108	MG269815	—	MG269761
Phyllachora capillipediicola	MHYAU 20089	MG356698	—	KY498084
Phyllachora chloridis	MFLU 15-0173T	MF197499	MF197505	KY594026
Phyllachora chloridis-virgatae	MHYAU 20136	MG356685	—	KY498122
Phyllachora chongzhouensis	SICAU 24-0044	PP785312	PP785323	PP785301
Phyllachora chrysopogonicola	MFLU 16-2096T	MF372146	—	MF372145
Phyllachora cynodonticola	MFLU 16-2977T	MF197501	MF197507	KY594024
Phyllachora cynodontis	MHYAU:20043	KY498081	—	KY471329
Phyllachora dendrocalami-hamiltoniicola	MHYAU 221	MK614118	—	—
Phyllachora dendrocalami-membranacei	MHYAU 220	MK614117	—	MK614102
Phyllachora flaccidudis	IFRD9445T	ON072101	ON072097	ON075524
Phyllachora graminis	SICAU 24-0051	PP785306	PP785317	PP785295
Phyllachora heterocladae	MFLU 18-1221T	MK296472	MK296468	MK305902
Phyllachora huiliensis	SICAU 24-0048	PP785308	PP785319	PP785297
Phyllachora imperatae	MHYAU:014	MG269800	—	MG269746
Phyllachora indosasae	MHYAU 125	MG195662	—	MG195637
Phyllachora isachnicola	MHYAU:179T	MH018563	—	MH018561
Phyllachora jiaensis	IFRD9448T	ON075440	ON072100	ON075527
Phyllachora keralensis	MHYAU:20082	MG269792	—	KY498106
Phyllachora maydis	BPI 893231	—	—	KU184459
Phyllachora maydis	BPI 910560	—	—	MG881846
Phyllachora miscanthi	SICAU 24-0050	PP785305	PP785316	PP785294
Phyllachora neidongensis	SICAU 24-0046	PP785314	PP785325	PP785303
Phyllachora panicicola	MFLU 16-2979T	MF197503	MF197504	KY594028
Phyllachora pogonatheri	MHYAU:071	MG269802	—	MG269748
Phyllachora pomigena	CBS 193.33	MH866860	—	MH855409
Phyllachora qualeae	UB 21159	—	—	KU682781
Phyllachora sandiensis	IFRD9446T	ON075528	ON072098	ON075525
Phyllachora sinobambusae	MHYAU 085	MG195655	—	MG195630
Phyllachora sphaerocaryi	MHYAU 178T	MK614114	—	MK614100
Phyllachora thysanolaenae	MFLU 16-2071	—	MF372147	—
Phyllachora virgataes	IFRD9447T	ON075439	ON072099	ON075526
Phyllachora yushaniae-falcatiauritae	MHYAU 123	MG195656	—	MG195631
Phyllachora yushaniae-polytrichae	MHYAU 122	MG195657	MH992455	MG195632
Polystigma pusillum	MM-19	KX430489	KX451850	KX451899
Telimena bicincta	MM-108	KX430473	KX451857	KX451906
Telimena bicincta	MM 133	KX430478	KX451861	KX451910

Abbreviations: MFLUCC = Mae Fah Luang University Culture Collection; CBS = Westerdijk Institute, Utrecht, The Netherlands; CDEP = Culture Collection of Department of Entomology and Plant Pathology; IFRD = International Fungal Research & Development Centre; MHYAU = Mycological Herbarium of the Yunnan Agricultural University; SICAU = Herbarium of Sichuan Agricultural University, Chengdu, China; NCYU = National Chiayi University Herbarium; UB = Department of Botany at the University of Brasilia Herbarium; BPI = U.S. National Fungus Collections.

).

2.3. Sequence Alignment and Phylogenetic Analyses

The DNA sequences from Sanger sequencing were analyzed and contig sequences were prepared using SeqMan software (SeqMan NGen^®. Version 5.0). The sequences of other Neophyllachora and Phyllachoraceae species were retrieved from NCBI GenBank. A data matrix composed of 63 taxa belonging to Phyllachoraceae, six strains obtained in the current study, and two outgroup species, Telimena bicincta MM 108 and Telimena bicincta MM 133, was analyzed. The datasets were prepared for LSU, SSU, and ITS. Each dataset was aligned with MAFFT V.7.036 (http://mafft.cbrc.jp/alignment/server/, accessed on 5 November 2024) [21] and edited manually where necessary using BioEdit v. 7.2 [22]. Data in FASTA format were converted to PHYLIP format using the Alignment Transformation Environment online (https://sing.ei.uvigo.es/ALTER/, accessed on 5 November 2024) [23]. The RAxML maximum likelihood phylogeny was performed using the CIPRES Science Gateway portal (https://www.phylo.org, accessed on 12 January 2025) [24], using RAxML-HPC2 on ACCESS (8.2.12)—Phylogenetic tree inference, using maximum likelihood/rapid bootstrapping run on XSEDE, and employed a GTR+I+G model of nucleotide substitution with 1000 bootstrap replications for each gene to check any incongruences in the datasets. The datasets without incongruences were concatenated in the order of LSU, SSU, and ITS. Multilocus phylogenetic analyses were performed for maximum likelihood (ML). The best-scoring ML tree was used to represent the phylogenetic tree and the resulting tree was visualized in FigTree v.1.4.3 [25] and edited using Adobe Illustrator CC 2019.

3. Results

3.1. Phylogenetic Analysis

A concatenated LSU, SSU, and ITS dataset comprising 2879 characters (LSU: 889, SSU: 1119, ITS: 819) was analyzed. Maximum likelihood (ML) phylogenetic analysis using the dataset generated the best-scoring tree with a final ML optimization likelihood value of -26897.804397. The estimated base frequencies for the GTR+I+G model of the combined dataset were A–0.252985, C–0.233243, G–0.278171, and T–0.235601. The substitution rates were AC–1.200579, AG–2.174439, AT–1.437903, CG–0.713782, CT–4.521910, and GT–1.000000. Further, the proportion of invariable sites was I–0.140638 and the gamma distribution shape parameter was 0.537932. The matrix had 1578 distinct alignment patterns, with 44.23% of undetermined characters or gaps. The best-scoring ML tree is given in Figure 1.

3.2. Taxonomy

Neophyllachora dalbergiae Haituk, Karunarathna & Cheewangkoon sp. nov. Figure 2.

Etymology: Derived from the host plant genus.

Index Fungorum number: IF903460.

Holotype: Thailand, Lumphun Province (18°38′17″ N 99°2′20″ E), on living leaves of Dalbergia sp. (Fabaceae), 28 March 2024, Sukanya Haituk, CDEP-50.

Parasitic on Dalbergia sp. Leaf symptoms visible as tar spots, amphigenous, scattered, small, black with yellow halo. Pseudostromata 1–3 mm diam., circular, black, intra-epidermal, scattered, prominent, multilocular, epiphyllous, glabrous, shiny. Sexual morph: Ascomata perithecial, globose to sub-globose, solitary or aggregated, immersed in pseudostromata occupying parenchymatous mycelial tissues, 509–620 × 272–329 µm ($\overline{\mathrm{x}}$ = 573 × 340 µm, n = 20), ostiolate; ostiole conspicuous, periphysate; peridium 25–35 µm thick, dark brown to black, wider at the lateral than the basal part, compactly arranged strongly melanized cells; paraphyses 1.1–2.3 µm wide, numerous, persistent, filiform, aseptate, branched. Asci 8-spored, unitunicate, persistent, cylindrical to clavate, short to medium pedicellate, with walls uniform in thickness, not specially thickened at apex, 90–100 × 19–25 µm ($\overline{\mathrm{x}}$ = 96 × 21 µm, n = 20). Ascospores overlapping, uni- or biseriate, hyaline, globose to elliptical, guttulate, smooth to rough, often with mucilage sheath, 13–17 × 11–13 µm ($\overline{\mathrm{x}}$ = 15.8 × 11.9 µm, n = 30). Asexual morph: Not observed.

Additional material examined: Thailand, Lumphun Province (18°38′17″ N 99°2′20″ E), on living leaves of Dalbergia sp. (Fabaceae), 28 March 2024, Sukanya Haituk, CDEP-51.

Notes: The disease symptoms and the typical tar spot appearance of Neophyllachora dalbergiae indicate Neophyllachora within Phyllachoraceae. The present species demonstrates closer morphological similarity towards N. fici. Both species show globose ascospores (

Table 2. Synopsis of Neophyllachora species.

	Neophyllachora Species									Phyllachora s.l.
Morphological Characteristics	N. cerradensis	N. fici	N. myrciae	N. myrciariae	N. subcircinans	N. truncatispora	N. religiosa	N. dalbergiae	N. pterocarpi-macrocarpae	P. pterocarpi
Pseudostromata (mm in diam)	2–4	2–3	3–6	0.5–1.5	1–4	2–5	2–3	1–3	2–3	0.75–2
Shape of Ascomata	Ampulliform to Globose	Globose to Subglobose	Globose to Ampulliform	Ampulliform	Ampulliform	Globose to Ovoid	Globose to Subglobose	Globose to sub globose	Globose to Ampulliform	Rotundate to illegular
Position of Ascomata	Occasionally coalescing	Epiphyllous	Occasionally coalescent	Immersed in the pseudostromata	Immersed in the pseudostromatic tissue	Immersed in pseudostroma	Epiphyllous	Epiphyllous	Epiphyllous	Epiphyllous
Peridium thickness (µm)	17–21	20–25	-	19–24	15–21	-	15–40	25–35	22–40	-
Size of Ascomata (µm)	260–362 × 168–264	150–300 × 200–400	205–485 × 148–207	161–432 × 126–267	329–417 × 193–275	160–200 × 100–350	150–300 × 200–400	272–329 × 509–620	213–337 × 456–497	150–200
Size of Asci (µm)	69–92 × 17–28	90–100 × 15–19	89–117 × 13–19	63–90 × 11–15	73–100 × 12–19	69–117 × 14–25	55–185 × 11–26	90–100 × 19–25	81–106 × 19–29	45–80 × 15–25
Shape of Asci	Fusoid	Cylindrical to Fusiform	Fusoid	Clavate–Fusoid	Cylindrical	-	Cylindrical to Fusiform	Cylindrical to clavate	Cylindrical to clavate	Cylindrical clavate to clavate
Width of Paraphyses (µm)	1.6–3	1.5–2.5	2.5–4.5	2–3	2–3.5 µm	2.5–5	1.5–2.5	1.1–2.3	2.8–5.0	-
Septate of Paraphyses	Septate	Aseptate	Septate	Septate	Septate	Septate	Septate	Aseptate	Septate	-
Size of Ascospores (µm)	15–22 × 6–9	12–13 × 10–11	14–18 × 5–7	14–19 × 5–8	11–16 × 7–9	18–26 × 7–8	8–15 × 5–12	13–17 × 11–13	18–21 × 9–12	14–18 × 8–11
Shape of Ascospores	Elliptic–Oblong	Globose to Subglobose	Lunate	Elliptical	Oblong to Ellipsoid	Sublunate to Fusoid	Globose to elliptical	Globose to elliptical with a central concave depression	Globose, ovoid, ellipsoid, pyriform, fusiform, amygdaliform	Ellipsoid
Color of Ascospores	Hyaline	Hyaline	Hyaline	Hyaline	Hyaline to Light olivaceous	Hyaline	Hyaline to Light olivaceous	Hyaline	Hyaline	Hyaline
Ascospores arrangement	Biseriate	1–2 seriate	Biseriate to multi-seriate	Obliquely biseriate	Mostly uniseriate, sometimes with biseriate	-	Mostly uniseriate, sometimes with biseriate	Obliquely uni- or biseriate	Irregularly biseriate	1–2 seriate
Ascospores wall	Covered by a thin gelatinous sheath	Absent	Thin-walled	Covered by a thin gelatinous sheath	Thin wall surrounded by a gelatinous sheath	Wall thickenings at both acute ends	Covered by a thick gelatinous sheath	Covered by a gelatinous sheath, smooth to rough	Covered by a gelatinous sheath, smooth to rough	-
Guttules	Microguttulate cytoplasm	-	-	Irregularly guttulate	Centrally guttulate	-	Irregularly guttulate	Guttulate	Guttulate	-
Asexual morph	Coelomycete	Unknown	Coelomycete	Unknown	Unknown	Coelomycete	Unknown	Unknown	Unknown	-
Host	Leaves of Myrcia torta	Leaves of Ficus septica	Myrcia sp.	Leaves of Myrciaria delicatula	Psidium sp.	Leaves of Myrcia camapuanensis	Leaves of Ficus religiosa	Dalbergia sp.	Pterocarpus macrocarpus	Pterocarpus angolensis
Distribution	Brazil	Taiwan	Brazil	Brazil	Brazil	Brazil	Thailand	Thailand	Thailand	South Africa
References	[3]	[14]	[3]	[3]	[3]	[3]	[15]	Present study	Present study	[26]

). Contrastingly, N. dalbergiae shows more globose to subglobose spores without central concave depression and a gelatinous sheath, which is absent in N. fici. Moreover, the asci of N. fici are fusoid with a narrow apex, while N. dalbergiae is fusoid and club-shaped with a broader apex. Further, N. dalbergiae differs from N. subcircinans and N. cerradensis, having globose to subglobose spores, due to its unique globose shape. Multi-locus phylogeny using a matrix of concatenated LSU, SSU, and ITS shows N. dalbergiae is located in a basal clade with N. pterocarpi-macrocarpae within Neophyllachora with 100% RAxML statistical support (Figure 1).

Neophyllachora pterocarpi-macrocarpae Haituk, Karunarathna & Cheewangkoon sp. nov. Figure 3.

Etymology: Derived from the host plant species.

Index Fungorum number: IF903461.

Holotype: Thailand, Chiang Mai Province (18°52′11.6″ N 98°56′25.0″ E), on living leaves of Pterocarpus macrocarpus Kurz (Fabaceae), 20 December 2023, Sukanya Haituk, CDEP-52.

Parasitic on Pterocarpus macrocarpus. Leaf symptoms visible as tar spots, small, circular to irregular, distinct, black with a narrow halo at the upper leaf surface, indistinct, pale greenish-yellow at the lower leaf surface. Pseudostromata 2–3 mm diam., black, various in shape, elongated, irregular, discrete, sparse, rarely, coalescent, glabrous, shiny, intraepidermal to subepidermal, mainly epiphyllous, multilocular, occasionally amphigenous, rarely scattered whole leaf surface. Sexual morph: Ascomata perithecial, globose to ampulliform, solitary or aggregated, immersed in pseudostromata occupying parenchymatous mycelial tissues, 456–497 × 213–337 µm ($\overline{\mathrm{x}}$ = 477 × 271 µm, n = 20), ostiolate; ostiole conspicuous, periphysate; peridium 22–40 µm thick, dark brown to black, wider at the lateral than the basal part, compactly arranged strongly melanized cells; paraphyses 2.8–5.0 µm wide, numerous, persistent, filiform, septate, branched. Asci 8-spored, unitunicate, persistent, cylindrical to clavate, short to medium pedicellate, with walls uniform in thickness, not specially thickened at apex, 81–106 × 19–29 µm ($\overline{\mathrm{x}}$ = 92 × 23 µm, n = 20). Ascospores overlapping, irregularly biseriate, hyaline, various in shape, globose, ovoid, ellipsoid, pyriform, fusiform, amygdaliform, guttulate, sometimes concave at the center, smooth to rough, surrounded by a gelatinous sheath, 18–21 × 9–12 µm ($\overline{\mathrm{x}}$ = 20.6 × 10.3 µm, n = 30). Asexual morph: Not observed.

Additional materials examined: Thailand, Chiang Mai Province (18°52′11.6″ N 98°56′25.0″ E), on living leaves of Pterocarpus macrocarpus (Fabaceae), 20 December 2023, Sukanya Haituk, CDEP-53.

Notes: Neophyllachora pterocarpi-macrocarpae was collected from Pterocarpus macrocarpus. On the plant genus Pterocarpus, Phyllachora pterocarpi Syd. & P. Syd. is previously known [26]. N. pterocarpi-macrocarpae is easily distinguishable from P. pterocarpi in the larger ascomata, asci, and ascospores (Table 2). Further, N. pterocarpi-macrocarpae differs from other Neophyllachora species in having various shapes of ascospores (Table 2). Additionally, phylogenetic analysis indicates that N. pterocarpi-macrocarpae is phylogenetically a sister to N. dalbergiae (Figure 1).

4. Discussion

Species in Phyllachoraceae are non-culturable obligate parasites [6]. The majority of species in Phyllachoraceae were introduced mainly based only on morphology. In the study by Dayarathne et al. [10] introducing Neophyllachora, they used taxonomy, molecular phylogeny of SSU, LSU, and ITS, and the host specificity towards Myrtaceae as the distinctive characteristics of this genus. However, later studies by Tennakoon et al. [14] and Literatus et al. [15] introduced Neophyllachora species from Moraceae, and our study introduces two novel Neophyllachora species from Fabaceae. Therefore, unlike Phyllachora, where most species are only reported from hosts of the Poaceae family, Neophyllachora can infect various plant hosts across different plant families.

In this study, two species of Neophyllachora, N. dalbergiae and N. pterocarpi-macrocarpae, are described based on morphology, phylogeny, and host associations. Interestingly, Neophyllachora shows a relatively higher morphological diversity of ascospore and conidia than that of other Phyllacholaceae fungi. For example, Neophyllachora myrciae forms unique lunate ascospores and both ellipsoidal and falcate conidia [3], and contrastingly, N. fici and N. dalbergiae have globose ascospores. Furthermore, ascospores of N. pterocarpi-macrocarpae are oblong to ellipsoid, N. truncatispora is sublunate to fusoid, and N. myrciariae is elliptical. These substantial morphological differences and their respective host associations help in separating the species within Neophyllachora. The updated key to Neophyllachora is provided for further clarification.

Key to species of Neophyllachora updated from Literatus et al. [15]

1.

Parasitic on Dalbergia Ficus, Myrcia, Myrciaria, and Pterocarpus species..................................................................................................................................................2

1′.

Parasitic on Psidium species, ascospores thin-walled, short-ellipsoidal covered with thin-walled gelatinous sheath..............................................................................N. subcircinans

2.

Parasitic on Dalbergia, Ficus, Myrcia, and Pterocarpus species..............................................3

2′.

Parasitic on Myrciaria species, clavate-fusoid asci...........................................N. myrciariae

3.

Parasitic on Dalbergia, Ficus, Myrcia and Pterocarpus, species..............................................4

3′.

Parasitic on Myrcia species, lunate-reniform to half-moon shape ascospores with thick walled ....................................................................................................................N. truncatispora

4.

Parasitic on Myrcia species…………………………………………….……………………..5

4′.

Parasitic on Dalbergia, Ficus and Pterocarpus, species…………….…………..……………6

5.

Elliptic-Oblong microguttulate ascospores...................................................... N. cerradensis

5′.

Thin walledLunate ascospores …… ..................................................................... N. myrciae

6.

Parasitic on Ficus species………………………………………………………….………….7

6′.

Parasitic on Dalbergia and Pterocarpus………………………………………………………8

7.

Parasitic on Ficus species, globose to ellipsoidal ascospores with gelatinous ........N. religiosa

7′.

Parasitic on Ficus species, globose to subglobose ascospores without gelatinous sheath..................................................................................................................................... N. fici

8.

Parasitic on Dalbergia species………………………….………………………. N. dalbergiae

8′.

Parasitic on Pterocarpus species…………………….…………….N. pterocarpi-macrocarpae

Species belonging to Phyllachoraceae infect a wide range of host families such as Arecaceae, Fabaceae, Juncaceae, Marantaceae, Melastomataceae, Myrtaceae, Poaceae, Vochysiaceae, and other monocot and dicot plants in tropic and temperate regions. As they are unculturable in artificial media, the host range is difficult to confirm by inoculation tests. Furthermore, overlapping morphologies within Phyllachoraceae cause taxonomic controversies. Therefore, the diagnosis of the causal species and identification faces a number of difficulties, impeding progress. Hence, it is important to identify Phyllachoraceae species by the polyphasic approach based on the morphology, host plant, and phylogeny, as mentioned above.

The seven species of Neophyllachora described in previous studies were associated with only two plant families. These are Myrtaceae, which originated in Gondwana and spread to the southern hemisphere [27], and Moraceae, which originated in Eurasia and spread to the southern hemisphere [28]. Regarding the associated plant hosts of the newly described two species of Neophyllachora from this study, Fabaceae originated around the Tethys Seaway in the early Tertiary period [29,30].

Referring to the phylogenetic tree of Neophyllachora, we can observe that a common ancestral strain acquired parasitism to one of these plant families and jumped to other plant families. The member in this genus might be speciated with the divergence of host plants on each continent. These are Myrtaceae plants for N. cerradensis, N. myrciae, N. myrciariae, N. subcircinans, and N. truncatispora in South America; Moraceae plants for N. fici and N. religiosa; and Fabaceae plants for N. dalbergiae and N. pterocarpi-macrocarpae in Asia. The divergence of obligate parasites on those herbaceous and/or broadleaf tree host plants is known in the case of powdery mildew and their relative plant hosts [31,32]. On the other hand, the co-speciation of the fungal species occurring on Gondwanan plants was denied [33]. Neophyllachora might be a good example for revealing the coevolution of fungi and their host plants. However, further in-depth studies with a larger number of specimens are required to prove this assumption.

5. Conclusions

In this study, two new species, N. pterocarpi-macrocarpae and N. dalbergiae in Neophyllachora, are introduced with a polyphasic approach from Pterocarpus macrocarpus and Dalbergia sp., respectively. Our study expands the host range of Neophyllachora to include an additional plant family other than Myrtaceae and Moraceae. This is the first time Neophyllachora has been reported from Fabaceae. The current reports on the collection of Neophyllachora species are limited geographically to South America and East Asia. The results from our study together with Literatus et al. [15] broaden the geographic distribution of Neophyllachora within Southeast Asia (Thailand).