*Article* **Identification and Pathogenicity of** *Paramyrothecium* **Species Associated with Leaf Spot Disease in Northern Thailand**

**Patchareeya Withee <sup>1</sup> , Sukanya Haituk <sup>1</sup> , Chanokned Senwanna <sup>1</sup> , Anuruddha Karunarathna <sup>1</sup> , Nisachon Tamakaew <sup>1</sup> , Parichad Pakdeeniti <sup>1</sup> , Nakarin Suwannarach 2,3 , Jaturong Kumla 2,3 , Piyawan Suttiprapan 1,4 , Paul W. J. Taylor <sup>5</sup> , Milan C. Samarakoon 1,\* and Ratchadawan Cheewangkoon 1,2,4,\***


**Abstract:** Species of *Paramyrothecium* that are reported as plant pathogens and cause leaf spot or leaf blight have been reported on many commercial crops worldwide. In 2019, during a survey of fungi causing leaf spots on plants in Chiang Mai and Mae Hong Son provinces, northern Thailand, 16 isolates from 14 host species across nine plant families were collected. A new species *Paramyrothecium vignicola* sp. nov. was identified based on morphology and concatenated (ITS, *cmdA*, *rpb2*, and *tub2*) phylogeny. Further, *P. breviseta* and *P. foliicola* represented novel geographic records to Thailand, while *P. eichhorniae* represented a novel host record (*Psophocarpus* sp., *Centrosema* sp., *Aristolochia* sp.). These species were confirmed to be the causal agents of the leaf spot disease through pathogenicity assay. Furthermore, cross pathogenicity tests on *Coffea arabica* L., *Commelina benghalensis* L., *Glycine max* (L.) Merr., and *Dieffenbachia seguine* (Jacq.) Schott revealed multiple host ranges for these pathogens. Further research is required into the host–pathogen relationship of *Paramyrothecium* species that cause leaf spot and their management. Biotic and abiotic stresses caused by climate change may affect plant health and disease susceptibility. Hence, proper identification and monitoring of fungal communities in the environment are important to understand emerging diseases and for implementation of disease management strategies.

**Keywords:** climate change; diversity; food security; multi-gene phylogeny; new species; plant pathology; taxonomy

## **1. Introduction**

Plant diseases have a high impact on food security [1] and fungi play a major role in plant diseases [2]. Foliar fungal pathogens severely affect the yield and health of commercial crops [3]. Leaf spots are an early indicator of foliar diseases and may initially occur on the adaxial leaf surfaces and then appear on the abaxial leaf surface.

*Paramyrothecium* species have been frequently identified to cause leaf spot and blight disease on a wide range of vegetables, ornamental plants, and economic crops [4–7]. Disease symptoms caused by *Paramyrothecium* may also include stem and crown canker and fruit rot [8–10]. Lombard et al. [4] designated an epitype for the generic type *Paramyrothecium roridum* (≡*M. roridum*). *Paramyrothecium* species are distinguished from related *Myrothecium*

**Citation:** Withee, P.; Haituk, S.; Senwanna, C.; Karunarathna, A.; Tamakaew, N.; Pakdeeniti, P.; Suwannarach, N.; Kumla, J.; Suttiprapan, P.; Taylor, P.W.J.; et al. Identification and Pathogenicity of *Paramyrothecium* Species Associated with Leaf Spot Disease in Northern Thailand. *Plants* **2022**, *11*, 1445. https://doi.org/10.3390/ plants11111445

Academic Editor: Alessandro Vitale

Received: 17 May 2022 Accepted: 26 May 2022 Published: 29 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sensu stricto and other myrothecium-like genera by the presence of 1–3 septate, thin-walled setae surrounding the sporodochia. Currently, there are 19 species listed in Index Fungorum (http://www.indexfungorum.org/; accessed on 14 April 2022).

*Paramyrothecium roridum* and *P. foliicola* are well-known pathogens that cause leaf spot or leaf blight and have been reported on many commercial crops and a wide range of hosts, such as soybean, strawberry, and muskmelon [5,8,9]. Rennberger and Keinath [11] isolated *P. foliicola* and *P. humicola* from watermelon and two other cucurbits and confirmed their pathogenicity on watermelons, tomatoes, and southern peas. Aumentado and Balendres [12] reported *P. foliicola* causing crater rot in eggplant and 45 plant species from 21 plant families and were tested for the pathogenicity on detached fruit or leaf assays. Furthermore, *P. foliicola* is pathogenic to cucumber seedlings and watermelon, causing stem canker [13]. Due to the lignicolous nature of the *Paramyrothecium*, they are being used as bio-pesticides for the control of weeds and insects [14,15]. Interestingly, several important secondary metabolites or toxins found in *Paramyrothecium* include trichothecenes macrolides such as roridin, verrucarin, and mytoxin B, which are important for some medicinal and biotechnological applications [16–18].

In Thailand, only *P. eichhorniae* has been reported and this was identified as the cause of the leaf blight disease of water hyacinth [19]. The diversity of *Paramyrothecium* species in Thailand is unknown. As a result, surveys and additional research on the distribution of *Paramyrothecium* in Thailand is required. The objective of this study was to identify and describe *Paramyrothecium* spp. from northern Thailand and assess their pathogenicity across a broad range of potential host plant species.

#### **2. Results**

#### *2.1. Symptoms*

Leaf spots varying in size and shape, depending on the host, were most visible on the upper surface. The leaf spots consisted of small brown spots or necrotic lesions with a dark border, while in older lesions, small sporodochia were visible (Figure 1f,n,o). Necrotic lesions appeared dark gray or black on *Centrosema* sp., *Coccinia grandis*, *Oroxylum indicum*, *Solanum virginianum*, *Tectona grandis*, *Vigna mungo*, *Vigna* sp., and *V. unguiculata* (Figure 1a,d,e,g,h–k,m), and surrounded by a prominent yellow halo on *Lablab purpureus*, *Psophocarpus* sp., and *Spilanthes* sp. (Figure 1b,c,l). Lesions on *Aristolochia* sp., *Coffea arabica*, and *Commelina benghalensis* consisted of light to dark brown concentric rings with a target-like appearance, and small sporodochia that appeared on lower and upper surfaces (Figure 1f,n,o).

**Figure 1.** Symptoms on different hosts caused by *Paramyrothecium* (**left**) and sporodochia on the host surface (**right**); (**a**) *Solanum virginianum*; (**b**) *Lablab purpureus*; (**c**) *Psophocarpus* sp.; (**d**,**i**) *Vigna* sp.; (**e**) *Coccinia grandis*; (**f**) *Commelina benghalensis*; (**g**) *Tectona grandis*; (**h**) *Vigna mungo*; (**j**) *Vigna unguiculata*; (**k**) *Oroxylum indicum*; (**l**) *Spilanthes* sp.; (**m**) *Centrosema* sp.; (**n**) *Aristolochia* sp.; (**o**) *Coffea arabica*. Scale bars: (**c**,**g**,j) = 1 mm; (**b**,**d**–**f**,**l**) = 2 mm; (**a**,**i**,**k**) = 4 mm; (**h**,**m**) = 5 mm; (**o**) = 6 mm; (**n**) = 1 cm. *2.2. Culture Morphology* **Figure 1.** Symptoms on different hosts caused by *Paramyrothecium* (**left**) and sporodochia on the host surface (**right**); (**a**) *Solanum virginianum*; (**b**) *Lablab purpureus*; (**c**) *Psophocarpus* sp.; (**d**,**i**) *Vigna* sp.; (**e**) *Coccinia grandis*; (**f**) *Commelina benghalensis*; (**g**) *Tectona grandis*; (**h**) *Vigna mungo*; (**j**) *Vigna unguiculata*; (**k**) *Oroxylum indicum*; (**l**) *Spilanthes* sp.; (**m**) *Centrosema* sp.; (**n**) *Aristolochia* sp.; (**o**) *Coffea arabica*. Scale bars: (**c**,**g**,j) = 1 mm; (**b**,**d**–**f**,**l**) = 2 mm; (**a**,**i**,**k**) = 4 mm; (**h**,**m**) = 5 mm; (**o**) = 6 mm; (**n**) = 1 cm.

#### Diverse culture characters were observed on PDA at room temperature (25–30 °C) *2.2. Culture Morphology*

(Figure 2). Eleven isolates of *Paramyrothecium* sp. (SDBR-CMU374, SDBR-CMU375, SDBR-CMU376, SDBR-CMU377, SDBR-CMU378, SDBR-CMU379, SDBR-CMU380, SDBR-CMU382, SDBR-CMU387, SDBR-CMU388, and SDBR-CMU389) (Figure 2a–j,l) formed whitish colonies with entire to slightly undulated margins, radial or in concentric rings with sporodochia, covered with slimy olivaceous green to black conidial masses, while the other four isolates (SDBR-CMU383, SDBR-CMU384, SDBR-CMU385, and SDBR-CMU386) (Figure 2m–p) formed abundant white aerial mycelium with sporodochia forming on the stroma and surface of the medium, covered by slimy olivaceous green to black conidial masses. Isolate SDBR-CMU381 (Figure 2k) produced exudates with brown pigment into the medium. Diverse culture characters were observed on PDA at room temperature (25–30 ◦C) (Figure 2). Eleven isolates of *Paramyrothecium* sp. (SDBR-CMU374, SDBR-CMU375, SDBR-CMU376, SDBR-CMU377, SDBR-CMU378, SDBR-CMU379, SDBR-CMU380, SDBR-CMU382, SDBR-CMU387, SDBR-CMU388, and SDBR-CMU389) (Figure 2a–j,l) formed whitish colonies with entire to slightly undulated margins, radial or in concentric rings with sporodochia, covered with slimy olivaceous green to black conidial masses, while the other four isolates (SDBR-CMU383, SDBR-CMU384, SDBR-CMU385, and SDBR-CMU386) (Figure 2m–p) formed abundant white aerial mycelium with sporodochia forming on the stroma and surface of the medium, covered by slimy olivaceous green to black conidial masses. Isolate SDBR-CMU381 (Figure 2k) produced exudates with brown pigment into the medium.

**Figure 2.** Colonies of *Paramyrothecium* species on PDA after 15 days at 25–30 °C. **Figure 2.** Colonies of *Paramyrothecium* species on PDA after 15 days at 25–30 ◦C.

#### *2.3. Phylogenetic Analysis 2.3. Phylogenetic Analysis*

The phylogenetic tree topologies of the ML and BI analyses for concatenated ITS, *cmdA*, *rpb2*, and *tub2* were similar. Hence, a phylogenetic tree from ML analyses is used to represent the results of both ML and BI analyses. The dataset comprised 53 taxa with 1760 characters (ITS: 1–542; *cmdA*: 543–824; *rpb2*: 825–1548; *tub2*: 1549–1760), including gaps. The GTR+G+I model was the best-fit model for all loci. The best scoring likelihood tree was selected on the basis of the ML analysis, with a final ML optimization likelihood value of *−*8176.4871, as shown in Figure 3. Sixteen new isolates were clustered into four distinct clades in *Paramyrothecium* (see the notes). The phylogenetic tree topologies of the ML and BI analyses for concatenated ITS, *cmdA*, *rpb2*, and *tub2* were similar. Hence, a phylogenetic tree from ML analyses is used to represent the results of both ML and BI analyses. The dataset comprised 53 taxa with 1760 characters (ITS: 1–542; *cmdA*: 543–824; *rpb2*: 825–1548; *tub2*: 1549–1760), including gaps. The GTR+G+I model was the best-fit model for all loci. The best scoring likelihood tree was selected on the basis of the ML analysis, with a final ML optimization likelihood value of −8176.4871, as shown in Figure 3. Sixteen new isolates were clustered into four distinct clades in *Paramyrothecium* (see the notes).

## *2.4. Taxonomy*

Isolates from symptomatic living leaves of different hosts were recognized under *Paramyrothecium* based on taxonomy (Table 1) and multi-gene phylogeny (Figure 3). The morphologies of the *Paramyrothecium* species are described herein.

**Figure 3.** Phylogram generated from maximum likelihood analysis based on combined ITS, *cmdA*, *rpb2*, and *tub2* sequenced data. Fifty-three strains are included in the combined sequence analyses, which comprise 1760 characters with gaps. Single gene analyses were also performed, and topology and clade stability were compared from combined gene analyses. *Striaticonidium cinctum* (CBS 932.69), *S. humicola* (CBS 388.97), and *S. synnematum* (CBS 479.85) are used as the outgroup taxa. The best scoring RAxML tree with a final likelihood value of −8176.4871 is presented. The matrix had 524 distinct alignment patterns. Estimated base frequencies were as follows; A = 0.2266, C = 0.2915, G = 0.2681, T = 0.2138; substitution rates AC = 1.1215, AG = 5.1556, AT = 1.0792, CG = 1.2292, CT = 11.1203, GT = 1.0000; gamma distribution shape parameter α = 0.3855. The bootstrap support (≥50%) of ML and the posterior probability values (≥0.9) of BI analyses are indicated above or below the respective branches. The fungal isolates from this study are indicated in red. The type species are indicated in bold. **Figure 3.** Phylogram generated from maximum likelihood analysis based on combined ITS, *cmdA*, *rpb2*, and *tub2* sequenced data. Fifty-three strains are included in the combined sequence analyses, which comprise 1760 characters with gaps. Single gene analyses were also performed, and topology and clade stability were compared from combined gene analyses. *Striaticonidium cinctum* (CBS 932.69), *S. humicola* (CBS 388.97), and *S. synnematum* (CBS 479.85) are used as the outgroup taxa. The best scoring RAxML tree with a final likelihood value of −8176.4871 is presented. The matrix had 524 distinct alignment patterns. Estimated base frequencies were as follows; A = 0.2266, C = 0.2915, G = 0.2681, T = 0.2138; substitution rates AC = 1.1215, AG = 5.1556, AT = 1.0792, CG = 1.2292, CT = 11.1203, GT = 1.0000; gamma distribution shape parameter α = 0.3855. The bootstrap support (≥50%) of ML and the posterior probability values (≥0.9) of BI analyses are indicated above or below the respective branches. The fungal isolates from this study are indicated in red. The type species are indicated in bold.



*Paramyrothecium acadiense*

*P. eichhorniae Eichhornia* 

*P. foeniculicola Foeniculum* 

*Tussilago* 

*vulgare*

*Plants* **2022**, *11*, x FOR PEER REVIEW 6 of 20

morphologies of the *Paramyrothecium* species are described herein.

*2.4. Taxonomy*

collected.

Mycobank: MB 843763.

*Paramyrothecium vignicola* Withee & Cheew., sp. nov. (Figure 4). Holotype: SDBR–CMU376.

*Paramyrothecium vignicola* Withee & Cheew., sp. nov. (Figure 4).

Isolates from symptomatic living leaves of different hosts were recognized under *Paramyrothecium* based on taxonomy (Table 1) and multi-gene phylogeny (Figure 3). The

Etymology: Name reflects the host genus Vigna, from which the species was

**Figure 4.** *Paramyrothecium vignicola* (CRC4-H, holotype); (**a**) leaf spot of *Vigna* sp.; (**b**) sporodochia on leaf; (**c**) sporodochial conidiomata on PDA; (**d**,**e**) conidiophores and conidiogenous cells; (**f**,**g**) conidiogenous cells; (**h**) setae; (**i**) conidia. Scale bars: (**b**,**c**) = 1 mm; (**d**–**h**) = 10 µm; (**i**) = 5 µm. **Figure 4.** *Paramyrothecium vignicola* (CRC4-H, holotype); (**a**) leaf spot of *Vigna* sp.; (**b**) sporodochia on leaf; (**c**) sporodochial conidiomata on PDA; (**d**,**e**) conidiophores and conidiogenous cells; (**f**,**g**) conidiogenous cells; (**h**) setae; (**i**) conidia. Scale bars: (**b**,**c**) = 1 mm; (**d**–**h**) = 10 µm; (**i**) = 5 µm.

**Table 1.** Synopsis of *Paramyrothecium* type species. Mycobank: MB 843763.

**Species Host Location Conidiophores (µm) Conidiogenous Cells (µm) Conidia (µm) Setae (µm) References** Etymology: Name reflects the host genus Vigna, from which the species was collected. Holotype: SDBR–CMU376.

*farfara* Canada <sup>9</sup>–14 × 2–2.5 – 0–1-septate, 5.5– 16.5 × 1.5–2.5 – [20] *P. breviseta* unknown India 6–9 × 2–4 6–11 × 1–2 aseptate, 4–5 × 1–2 1–3-septate, 25–40 × 2–3 [4] *P. cupuliforme* Soil Namibia 15–25 × 2–4 4–11 × 1–3 aseptate, 6–8 × 1–2 1–3-septate, 45–90 × 2–3 [4] *crassipes* Thailand <sup>15</sup>–40 × 2–<sup>3</sup> (8–)11–17(–20) × 2–<sup>3</sup> aseptate,5– 6.5 × 1.5–2.5 1–3-septate, 40–120 × 2–3 [19] Netherlands 7–17 × 2–3 6–16 × 1–2 aseptate, 5–7 × 1–2 – [4] *P. foliicola* Decaying leaf Brazil 15–25 × 2–3 8–14 × 1–2 aseptate, 5–6 × 1–2 1–3-septate, 60–100 × 2–3 [4] Description: Sexual morph: unknown. Asexual morph: *Conidiomata* sporodochial, stromatic, superficial, cupulate, scattered or gregarious, oval or irregular in outline, (60–)90–300(–385) µm diam, (70–)140–180(–200) µm deep, with a white to creamy setose fringe surrounding an olivaceous green agglutinated slimy mass of conidia. *Stroma* poorly developed, hyaline. *Setae* arising from the stroma thin-walled, hyaline, 3–8-septate, straight becoming sinuous above the apical septum, 80–155 µm long, 2–3 µm wide, tapering to an acutely rounded apex. *Conidiophores* arising from the basal stroma, consisting of a stipe and a penicillately branched conidiogenous apparatus; stipes unbranched, hyaline sometimes covered by a green mucoid layer, septate, smooth, 40–60 × 2–3 µm; primary branches aseptate, unbranched, smooth, 10–26 × 2–3 µm (*x* = 18 × 3 µm, *n* = 20); secondary branches aseptate, unbranched, smooth, 10–17 × 2–3 µm (*x* = 13 × 3 µm, *n* = 20); terminating in a whorl of 3–6 conidiogenous cells; conidiogenous cells phialidic, cylindrical to subcylindrical, hyaline, smooth, straight to slightly curved, 11–16 × 1–3 µm (*x* = 13 × 2 µm, *n* = 20), with conspicuous collarettes and periclinal thickenings. *Conidia* aseptate, hyaline, smooth, cylindrical to ellipsoidal, 5–7 × 1–3 µm (*x* = 6 × 2 µm, *n* = 20), rounded at both ends.

Culture characteristics: Colonies on PDA, dense, circular, flattened, slightly raised, floccose, white aerial mycelium, radiating with concentric ring of sporodochia forming, covered by slimy olivaceous green to black conidial masses.

Material examined: Thailand, Mae Hong Son Province, on living leaf of *Vigna* sp. (*Fabaceae*), 11 September 2019, N. Tamakaew, CRC4-H (holotype), ex-type living culture SDBR-CMU376; *ibid.*, on living leaf of *Solanum virginianum* (*Solanaceae*), 11 September 2019,

N. Tamakaew, CRC1-H, living culture SDBR-CMU389; *ibid.*, on living leaf of *Lablab purpureus* (*Fabaceae*), 11 September 2019, N. Tamakaew, CRC2-H, living culture SDBR-CMU374; *ibid.*, on living leaf of *Coccinia grandis* (*Cucurbitaceae*), CRC6-H, living culture SDBR-CMU377; Chiang Mai province, on living leaf of *Commelina benghalensis* (*Commelinaceae*), 20 November 2019, P. Withee, CRC14-H, living culture SDBR-CMU381; *ibid.*, on living leaf of *Vigna mungo* (*Fabaceae*), 5 December 2019, N. Tamakaew, CRC144-H, living culture SDBR-CMU384; *ibid.*, on living leaf of *Vigna* sp. (*Fabaceae*), CRC145-H, living culture SDBR-CMU385; *ibid.*, on living leaf of *Vigna unguiculata* (*Fabaceae*), 10 February 2020, P. Withee, CRC146-H, living culture SDBR–CMU386.

Notes: Based on ITS, *cmdA*, *rpb2* and *tub2* phylogeny (Figure 3) and *cmdA* and *tub2* (data not shown), *Paramyrothecium foliicola* formed two distinct clades. The clade with *Paramyrothecium foliicola* type (CBS 113121) was treated as the *Paramyrothecium* sensu stricto. Eight of the new strains clustered with eight previously described *Paramyrothecium* strains (as *P*. *foliicola*) and formed a well-supported clade (100% BS/1.00 PP) (*Paramyrothecium* sensu lato) closely related to *P*. *eichhorniae* and *P*. *foliicola* (Figure 3). Based on morphology and phylogeny, we introduce a new species to accommodate taxa in *P. foliicola* sensu lato. *Paramyrothecium vignicola* differs from *P. eichhorniae* and *P*. *foliicola* with longer setae (up to 155 µm vs. up to 120 µm and up to 100 µm). The conidia of *P. vignicola* (5–7 × 1–3 µm) are slightly larger than those of *P*. *eichhorniae* (5–6.5 × 1.5–2.5 µm) [20] and *P*. *foliicola* (5–6 × 1–2 µm) [4]. *Paramyrothecium vignicola* differs from other *Paramyrothecium* species by its 3–8-septate, thin-walled setae surrounding the sporodochia. In BLAST searches of NCBI GenBank, the closest matches of the sequences are *Paramyrothecium*: *P*. *foliicola* (CBS 11321) with 98.98% similarity in ITS sequence, 93.89% similarity in *cmdA*. *P*. *vignicola*, 96.32% in *tub2* with *P*. *foliicola* (CBS 11321). Based on phylogenetic evidence and morphological differences, *P*. *vignicola* is a new species.

*Paramyrothecium breviseta* L. Lombard & Crous, in Lombard et al., Persoonia 36: 207 (2016) (Figure 5). *Plants* **2022**, *11*, x FOR PEER REVIEW 9 of 20

**Figure 5.** *Paramyrothecium breviseta* (CRC13-H); (**a**,**b**) leaf spot of *Coffea arabica*; (**c**) sporodochia on leaf; (**d**) conidiophores and conidiogenous cells; (**e**–**g**) conidiogenous cells; (**h**) setae; (**i**) conidia. Scale bars: (**d**) = 20 µm; (**e**–**h**) = 10 µm; (**i**) = 5 µm. **Figure 5.** *Paramyrothecium breviseta* (CRC13-H); (**a**,**b**) leaf spot of *Coffea arabica*; (**c**) sporodochia on leaf; (**d**) conidiophores and conidiogenous cells; (**e**–**g**) conidiogenous cells; (**h**) setae; (**i**) conidia. Scale bars: (**d**) = 20 µm; (**e**–**h**) = 10 µm; (**i**) = 5 µm.

Culture characteristics: Colonies on PDA, dense, circular, flattened, slightly raised, floccose, white aerial mycelium, radiating with concentric ring of sporodochia forming,

20 November 2019, R. Cheewangkoon and P. Withee, CRC13-H, living culture SDBR-

Notes: Phylogenetically, SDBR-CMU387 and SDBR-CMU388 formed a wellsupported clade closely related to *Paramyrothecium breviseta* L. Lombard & Crous (Figure 2). *Paramyrothecium breviseta* was collected on an unknown substrate in India [4] and in this study, we collected *P. breviseta* from *Coffea arabica* (*Rubiaceae*) in Chiang Mai Province. The morphology of the fresh specimen is similar to that described by Lombard et al. [4], but the conidia (5–7 × 1–2 vs*.* 4–5 × 1–2 µm) and setae (25–120 × 2–3 vs*.* 25–40 × 2–3 µm) are longer. However, this is the first hostreport of leaf spot causing *P*. *breviseta* on *C. arabica*

*Paramyrothecium eichhorniae* J. Unartngam, A. Unartngam & U. Pinruan, in

Description: Sexual morph: unknown. Asexual morph: *Conidiomata* sporodochial, stromatic, superficial, cupulate, scattered or gregarious, oval or irregular in outline, (60– )70–250(–500) µm diam, (60–)70–270(−370) μm deep, with a white setose fringe surrounding an olivaceous green to dark green slimy mass of conidia. *Setae* arising from the stroma thin-walled, hyaline, 1–5-septate, straight to flexuous, 60–120 μm long, 2–3 μm wide, tapering to an acutely rounded apex. *Conidiophores* arising from the basal stroma, consisting of a stipe and a penicillately branched conidiogenous apparatus; stipes

CMU387; *ibid.*, CRC12-H, living culture SDBR-CMU388.

Pinruan et al., Mycobiology 50: 17 (2022) (Figure 6).

in Thailand.

Description: Sexual morph: unknown. Asexual morph: *Conidiomata* sporodochial, stromatic, cupulate, superficial, scattered or rarely gregarious, oval or irregular in outline, 135–790 µm diam, 9–15 µm deep, with a white setose fringe surrounding an olivaceous green to black agglutinated slimy mass of conidia. *Setae* arising from the stroma thinwalled, hyaline, 1–5-septate, straight to flexuous, 25–120 µm long, 2–3 µm wide, tapering to an acutely rounded apex. *Conidiophores* arising from the basal stroma, consisting of a stipe and a penicillately branched conidiogenous apparatus; stipes unbranched, hyaline, septate, smooth, 6–9 × 2–4 µm; primary branches aseptate, unbranched, smooth, 12–24 × 3–4 µm (*x* = 18 × 3 µm, *n* = 20); secondary branches aseptate, unbranched, smooth, 10–17 × 2–4 µm (*x* = 12 × 3 µm, *n* = 20); terminating in a whorl of 3–6 conidiogenous cells; conidiogenous cells phialidic, cylindrical to subcylindrical, hyaline, smooth, straight to slightly curved, 6–11 × 1–2 µm (*x* = 9 × 2 µm, *n* = 20), with conspicuous collarettes and periclinal thickenings. *Conidia* aseptate, hyaline, smooth, cylindrical to ellipsoidal, 5–7 × 1–2 µm (*x* = 6 × 2 µm, *n* = 20), rounded at both ends.

Culture characteristics: Colonies on PDA, dense, circular, flattened, slightly raised, floccose, white aerial mycelium, radiating with concentric ring of sporodochia forming, covered by slimy olivaceous green to black conidial masses.

Material examined: Thailand, Chiang Mai, on living leaf of *Coffea arabica* (*Rubiaceae*), 20 November 2019, R. Cheewangkoon and P. Withee, CRC13-H, living culture SDBR-CMU387; *ibid.*, CRC12-H, living culture SDBR-CMU388.

Notes: Phylogenetically, SDBR-CMU387 and SDBR-CMU388 formed a well-supported clade closely related to *Paramyrothecium breviseta* L. Lombard & Crous (Figure 2). *Paramyrothecium breviseta* was collected on an unknown substrate in India [4] and in this study, we collected *P. breviseta* from *Coffea arabica* (*Rubiaceae*) in Chiang Mai Province. The morphology of the fresh specimen is similar to that described by Lombard et al. [4], but the conidia (5–7 × 1–2 vs. 4–5 × 1–2 µm) and setae (25–120 × 2–3 vs. 25–40 × 2–3 µm) are longer. However, this is the first host report of leaf spot causing *P*. *breviseta* on *C. arabica* in Thailand.

*Paramyrothecium eichhorniae* J. Unartngam, A. Unartngam & U. Pinruan, in Pinruan et al., Mycobiology 50: 17 (2022) (Figure 6).

Description: Sexual morph: unknown. Asexual morph: *Conidiomata* sporodochial, stromatic, superficial, cupulate, scattered or gregarious, oval or irregular in outline, (60–)70–250(–500) µm diam, (60–)70–270(−370) µm deep, with a white setose fringe surrounding an olivaceous green to dark green slimy mass of conidia. *Setae* arising from the stroma thin-walled, hyaline, 1–5-septate, straight to flexuous, 60–120 µm long, 2–3 µm wide, tapering to an acutely rounded apex. *Conidiophores* arising from the basal stroma, consisting of a stipe and a penicillately branched conidiogenous apparatus; stipes unbranched, hyaline, septate, smooth, 15–40 × 2–3 µm; primary branches aseptate, unbranched, smooth, 10–17 × 2–3 µm (*x* = 12 × 3 µm, *n* = 20); secondary branches aseptate, unbranched, smooth, 7–14 × 2–3 µm (*x* = 10 × 3 µm, *n* = 20); terminating in a whorl of 3–6 conidiogenous cells; conidiogenous cells phialidic, cylindrical to subcylindrical, hyaline, smooth, straight to slightly curved, 11–17 × 2–3 µm (*x* = 14 × 2 µm, *n* = 20), with conspicuous collarettes and periclinal thickenings. *Conidia* aseptate, hyaline, smooth, cylindrical to ellipsoidal, 5–7 × 1–2 µm (*x* = 6 × 2 µm, *n* = 20), rounded at both ends.

Culture characteristics: Colonies on PDA, entire to slightly undulated margins, with sporodochia forming on the surface of the medium, covered by slimy olivaceous green to black conidial masses.

**Figure 6.** *Paramyrothecium eichhorniae* (CRC143); (**a**) leaf spot of *Aristolochia* sp.; (**b**) sporodochia on leaf; (**c**) sporodochial conidiomata on PDA; (**d**) sporodochia; (**e**,**f**) conidiogenous cells; (**g**) setae; (**h**) conidia. Scale bars: (**b**,**c**) = 1 mm; (**d**,**g**) = 20 µm; (**e**,**f**) = 10 µm; (**h**) = 5 µm. **Figure 6.** *Paramyrothecium eichhorniae* (CRC143); (**a**) leaf spot of *Aristolochia* sp.; (**b**) sporodochia on leaf; (**c**) sporodochial conidiomata on PDA; (**d**) sporodochia; (**e**,**f**) conidiogenous cells; (**g**) setae; (**h**) conidia. Scale bars: (**b**,**c**) = 1 mm; (**d**,**g**) = 20 µm; (**e**,**f**) = 10 µm; (**h**) = 5 µm.

unbranched, hyaline, septate, smooth, 15–40 × 2–3 µm; primary branches aseptate, unbranched, smooth, 10–17× 2–3 µm ( = 12 × 3 µm, *n* = 20); secondary branches aseptate, unbranched, smooth, 7–14 × 2–3 µm ( = 10 × 3 µm, *n* = 20); terminating in a whorl of 3–6 conidiogenous cells; conidiogenous cells phialidic, cylindrical to subcylindrical, hyaline, smooth, straight to slightly curved, 11–17 × 2–3 µm ( = 14 × 2 µm, *n* = 20), with conspicuous collarettes and periclinal thickenings. *Conidia* aseptate, hyaline, smooth, cylindrical to ellipsoidal, 5–7 × 1–2 µm ( = 6 × 2 µm, *n* = 20), rounded at both ends.

Culture characteristics: Colonies on PDA, entire to slightly undulated margins, with sporodochia forming on the surface of the medium, covered by slimy olivaceous green to black conidial masses. Material examined: Thailand, Mae Hong Son Province, on living leaf of *Psophocarpus* sp. (*Fabaceae*), 11 September 2019, N. Tamakaew, CRC3-H, living culture SDBR-CMU375; *ibid.*, on living leaf of *Oroxylum indicum* (*Bignoniaceae*), 11 September 2019, N. Tamakaew, CRC8-H, living culture SDBR-CMU378; *ibid.*, on living leaf of *Spilanthes* sp. (*Asteraceae*), 11 Material examined: Thailand, Mae Hong Son Province, on living leaf of *Psophocarpus* sp. (*Fabaceae*), 11 September 2019, N. Tamakaew, CRC3-H, living culture SDBR-CMU375; *ibid.*, on living leaf of *Oroxylum indicum* (*Bignoniaceae*), 11 September 2019, N. Tamakaew, CRC8-H, living culture SDBR-CMU378; *ibid.*, on living leaf of *Spilanthes* sp. (*Asteraceae*), 11 September 2019, N. Tamakaew, CRC148-H, living culture SDBR-CMU379; *ibid.*, on living leaf of *Centrosema* sp. (*Fabaceae*), 11 September 2019, N. Tamakaew, CRC11-H, living culture SDBR-CMU380; Chiang Mai province, on living leaf of *Aristolochia* sp. (*Aristolochiaceae*), January 2020, P. Suttiprapan, CRC143-H, living culture SDBR-CMU383.

September 2019, N. Tamakaew, CRC148-H, living culture SDBR-CMU379; *ibid.*, on living leaf of *Centrosema* sp. (*Fabaceae*), 11 September 2019, N. Tamakaew, CRC11-H, living culture SDBR-CMU380; Chiang Mai province, on living leaf of *Aristolochia* sp. (*Aristolochiaceae*), January 2020, P. Suttiprapan, CRC143-H, living culture SDBR-CMU383. Note: Based on multigene phylogeny, five isolates in this study clustered with *Paramyrothecium eichhorniae*, which was associated with water hyacinth (*Eichhornia crassipes*) and recently described from Thailand [10]. Morphologically, the conidiogenous cells of our collections are similar to those of the holotype of *P. eichhorniae*. However, the Note: Based on multigene phylogeny, five isolates in this study clustered with *Paramyrothecium eichhorniae*, which was associated with water hyacinth (*Eichhornia crassipes*) and recently described from Thailand [10]. Morphologically, the conidiogenous cells of our collections are similar to those of the holotype of *P. eichhorniae*. However, the conidia of *P. eichhorniae* in this study are thinner than reported by Pinruan et al. [19] (5–7 × 1–2 µm vs. 5–6.5 × 1.5–2.5 µm) and have more septa in setae than the holotype (1–5 vs. 1–3 septate). This is the first report of *P. eichhorniae* on *Psophocarpus* sp., *Centrosema* sp., and *Aristolochia* sp. from Thailand.

conidia of *P. eichhorniae* in this study are thinner than reported by Pinruan et al. [19] (5–7 × 1–2 µm vs. 5–6.5 × 1.5–2.5 µm) and have more septa in setae than the holotype (1–5 vs*. Paramyrothecium foliicola* L. Lombard & Crous, in Lombard et al., Persoonia 36: 209 (2016) (Figure 7).

1–3 septate). This is the first report of *P. eichhorniae* on *Psophocarpus* sp., *Centrosema* sp., and *Aristolochia* sp. from Thailand. Description: Sexual morph: unknown. Asexual morph: *Conidiomata* sporodochial, stromatic, superficial, cupulate, scattered or gregarious, oval or irregular in outline, (60–)100–170(–245) µm diam, (70–)140–165(–200) µm deep, with a white to creamy setose fringe surrounding an olivaceous green agglutinated slimy mass of conidia. *Stroma* poorly developed, hyaline. *Setae* arising from the stroma thin-walled, hyaline, 1–4(–8)-septate, straight becoming sinuous above the apical septum, 35–175 µm long, 2–3 µm wide, tapering to an acutely rounded apex. *Conidiophores* arising from the basal stroma, consisting of a stipe

and a penicillately branched conidiogenous apparatus; stipes unbranched, hyaline sometimes covered by a green mucoid layer, septate, smooth, 20–75 × 2–4 µm; primary branches aseptate, unbranched, smooth, (10–)17–21(–26) × 2–3(–4) µm (*x* = 15 × 3 µm, *n* = 20); secondary branches aseptate, unbranched, smooth, (7–)9–17(–19) × 2–3(–4) µm (*x* = 14 × 3 µm, *n* = 20); terminating in a whorl of 3–6 conidiogenous cells; conidiogenous cells phialidic, cylindrical to subcylindrical, hyaline, smooth, straight to slightly curved, 10–17 × 1–3 µm (*x* = 13 × 2 µm, *n* = 20), with conspicuous collarettes and periclinal thickenings. Conidia aseptate, hyaline, smooth, cylindrical to ellipsoidal, 5–8 × 1–3 µm (*x* = 7 × 2 µm, *n* = 20), rounded at both ends. *Plants* **2022**, *11*, x FOR PEER REVIEW 11 of 20 *Paramyrothecium foliicola* L. Lombard & Crous, in Lombard et al*.*, Persoonia 36: 209 (2016) (Figure 7).

**Figure 7.** *Paramyrothecium foliicola* (CRC15); (**a**) sporodochia on leaves of *Tectona grandis* (**b**) sporodochial conidiomata on PDA; (**c**,**d**) sporodochia (**e**,**f**) conidiogenous cells; (**g**) setae; (**h**) condia. Scale bars: (**a**) = 500 µm; (**b**) = 1 mm; (**c**) = 30 µm; (**d**,**g**) = 20 µm; (**h**) = 5 µm. **Figure 7.** *Paramyrothecium foliicola* (CRC15); (**a**) sporodochia on leaves of *Tectona grandis* (**b**) sporodochial conidiomata on PDA; (**c**,**d**) sporodochia (**e**,**f**) conidiogenous cells; (**g**) setae; (**h**) condia. Scale bars: (**a**) = 500 µm; (**b**) = 1 mm; (**c**) = 30 µm; (**d**,**g**) = 20 µm; (**h**) = 5 µm.

Description: Sexual morph: unknown. Asexual morph: *Conidiomata* sporodochial, stromatic, superficial, cupulate, scattered or gregarious, oval or irregular in outline, (60– )100–170(–245) µm diam, (70–)140–165(–200) µm deep, with a white to creamy setose Culture characteristics: Colonies on PDA, abundant white aerial mycelium with sporodochia forming on the aerial mycelium and surface of the medium, covered by slimy olivaceous green to black conidial masses.

fringe surrounding an olivaceous green agglutinated slimy mass of conidia. *Stroma* poorly developed, hyaline. *Setae* arising from the stroma thin-walled, hyaline, 1–4(–8)-septate, Materials examined: Thailand, Chiang Mai, on living leaf of *Tectona grandis* (*Lamiaceae*), 20 November 2019, P. Withee, CRC15-H, living culture SDBR-CMU382.

straight becoming sinuous above the apical septum, 35–175 μm long, 2–3 μm wide, tapering to an acutely rounded apex. *Conidiophores* arising from the basal stroma, consisting of a stipe and a penicillately branched conidiogenous apparatus; stipes unbranched, hyaline sometimes covered by a green mucoid layer, septate, smooth, 20–75 × 2–4 µm; primary branches aseptate, unbranched, smooth, (10–)17–21(–26) × 2–3(–4) µm ( = 15 × 3 µm, *n* = 20); secondary branches aseptate, unbranched, smooth, (7–)9–17(–19) × 2–3(–4) µm ( = 14 × 3 µm, *n* = 20); terminating in a whorl of 3–6 conidiogenous cells; Notes: Based on our phylogenetic analysis (Figure 3), SDBR-CMU382 isolates were clustered with *Paramyrothecium foliicola*. The morphology of our collection (CRC15-H) is similar to that of *P. foliicola* described by Lombard et al. [4]. However, our collection has longer conidiophores (20–75 × 2–4 vs. 15–25 × 2–3 µm) and more septa in setae (1–4(–8) vs. 1–3 septate), conidiogenous cells (10–17 × 1–3 vs. 8–14 × 1–2 µm) and conidia (5–8 × 1–3 vs. 5–6 × 1–2 µm). This may be due to distribution, environment, and morphological variability within the species. This is the first report of *P. foliicola* from *Tectona grandis* in Thailand.

conidiogenous cells phialidic, cylindrical to subcylindrical, hyaline, smooth, straight to slightly curved, 10–17 × 1–3 µm ( = 13 × 2 µm, *n* = 20), with conspicuous collarettes and periclinal thickenings. Conidia aseptate, hyaline, smooth, cylindrical to ellipsoidal, 5–8 ×

Culture characteristics: Colonies on PDA, abundant white aerial mycelium with

Materials examined: Thailand, Chiang Mai, on living leaf of *Tectona grandis*

Notes: Based on our phylogenetic analysis (Figure 3), SDBR-CMU382 isolates were clustered with *Paramyrothecium foliicola*. The morphology of our collection (CRC15-H) is

(*Lamiaceae*), 20 November 2019, P. Withee, CRC15-H, living culture SDBR-CMU382.

1–3 µm ( = 7 × 2 µm, *n* = 20), rounded at both ends.

#### *2.5. Pathogenicity Test and Cross Pathogenicity*

Koch's postulates confirmed that all the fungal isolates were able to cause disease in unwounded leaves of *Commelina benghalensis* and *Glycine max* (Figure 8b,c). The SDBR– CMU383 isolate infected all inoculated plants and was highly aggressive on most, except for *C. benghalensis*. No infection was observed in the unwounded inoculation of *Coffea arabica* and *Dieffenbachia seguine* (Figure 8a,d). Leaves receiving sterilized distilled water remained healthy. The fungi were re-isolated from the diseased leaf tissues in each experiment, and each isolated fungus was identical to the inoculated fungus. Further, Koch's postulates confirmed that all isolates of *Paramyrothecium vignicola*, *P. breviseta*, *P. eichhorniae*, and *P. foliicola* were pathogenic to their original host plants. Cross pathogenicity tests showed that all isolates infected inoculated (wounded) *C. arabica*, *C. benghalensis*, *G. max*, and *D. seguine* leaves (Table 2). The symptoms showed light to dark brown and irregular to round lesions, which had scattered olive-colured sporodochia and dark exudates of spore masses (Figure 8). *Plants* **2022**, *11*, x FOR PEER REVIEW 13 of 20

**Figure 8***.* Pathogenicity test (**a**,**b**) and cross pathogenicity (**c**,**d**); Control (left); (**a**) *Paramyrothecium brevista* on *Coffea arabica*; (**b**) *P*. *vignicola* on *Commelina benghalensis*; (**c**) *P. vignicola* on *Glycine max*; (**d**) *P. vignicola* on *Dieffenbachia seguine*; (w) wound and (uw) unwound. Scale bars: (**a**–**c**) = 1 cm; (**d**) = 6 cm. **Figure 8.** Pathogenicity test (**a**,**b**) and cross pathogenicity (**c**,**d**); Control (left); (**a**) *Paramyrothecium brevista* on *Coffea arabica*; (**b**) *P*. *vignicola* on *Commelina benghalensis*; (**c**) *P. vignicola* on *Glycine max*; (**d**) *P. vignicola* on *Dieffenbachia seguine*; (w) wound and (uw) unwound. Scale bars: (**a**–**c**) = 1 cm; (**d**) = 6 cm.

The new species *Paramyrothecium vignicola* was described using morphology and multi-gene phylogeny and the host range included *Solanum virginianum* (*Solanaceae*),

The pathogenicity assays showed that *P. vignicola*, *P. breviseta*, *P. eichhorniae*, and *P. foliicola* isolated from different hosts from different locations in northern Thailand can all cause leaf spot disease on different host families, including *Rubiaceae*, *Fabaceae*, *Commelinaceae*, and *Araceae*. However, the disease severity was related to the plant species and inoculation methods, where *Paramyrothecium* spp. could not cause disease in *Coffea arabica* and *Dieffenbachia seguine* without wounding. Wounding involves the breakage of the plant's first barrier of defense; cuticle and epidermal cells. The tissue then becomes more susceptible to the pathogens. Some species cannot infect non-wounded leaves, hence they are weakly aggressive on these hosts [15]. On the other hand, *Commelina benghalensis* and *Glycine max* were susceptible to all isolates. These results are similar to those of Rennberger and Keinath [11] and Aumentado and Balendres [12], in which

using ITS, *cmdA*, *rpb2*, and *tub2* sequence data clearly identified *P. eichhorniae*, *P. vignicola*, *P. breviseta*, and *P. foliicola* as distinct species within *Paramyrothecium*. Further, multi-gene

phylogeny precisely demonstrated the species delineation of *Paramyrothecium*.

**3. Discussion**


**Table 2.** Pathogenicity test and cross pathogenicity of *Paramyrothecium* species on original hosts and other plant species.

Note: (-) No symptoms (+) Symptoms (w) wound and (uw) unwound.; Superscript "T" indicates type species.

#### **3. Discussion**

The new species *Paramyrothecium vignicola* was described using morphology and multi-gene phylogeny and the host range included *Solanum virginianum* (*Solanaceae*), *Lablab purpureus* (*Fabaceae*), *Coccinia grandis* (*Cucurbitaceae*), *Commelina benghalensis* (*Commelinaceae*), *Vigna* sp., *V. mungo*, and *V. unguiculata* (*Fabaceae*). Multi-gene phylogeny using ITS, *cmdA*, *rpb2*, and *tub2* sequence data clearly identified *P. eichhorniae*, *P. vignicola*, *P. breviseta*, and *P. foliicola* as distinct species within *Paramyrothecium*. Further, multi-gene phylogeny precisely demonstrated the species delineation of *Paramyrothecium*.

The pathogenicity assays showed that *P. vignicola*, *P. breviseta*, *P. eichhorniae*, and *P. foliicola* isolated from different hosts from different locations in northern Thailand can all cause leaf spot disease on different host families, including *Rubiaceae*, *Fabaceae*, *Commelinaceae*, and *Araceae*. However, the disease severity was related to the plant species and inoculation methods, where *Paramyrothecium* spp. could not cause disease in *Coffea arabica* and *Dieffenbachia seguine* without wounding. Wounding involves the breakage of the plant's first barrier of defense; cuticle and epidermal cells. The tissue then becomes more susceptible to the pathogens. Some species cannot infect non-wounded leaves, hence they are weakly aggressive on these hosts [15]. On the other hand, *Commelina benghalensis* and *Glycine max* were susceptible to all isolates. These results are similar to those of Rennberger and Keinath [11] and Aumentado and Balendres [12], in which *Parammyrothecium* species were able to infect original and non-original hosts within the same family (host shift ability) and different families (host jump ability).

For species diversity and distribution, more gene studies and more reference sequences are needed to resolve the species boundaries of *Paramyrothecium*. Field inspections are needed to confirm the importance of this pathogen and prove that diseases associated with *Paramyrothecium* species are threats to economic crops in Thailand. The information on the spread of related species to new areas is necessary as climate change may enable saprotrophic fungi to switch their nutritional mode across a wider host range, even if an area is predicted to be at risk from an introduced pathogen. It may be the case that few of the susceptible host species are present in this predicted area [26], so for the risk to be realized, climate change should also favor the migration of susceptible species or increase the susceptibility of the resident hosts.

*Paramyrothecium* leaf spot occurs in commercially important plants (*Coffea arabica*, *Tectona grandis*, *Vigna mungo*, and *V. unguiculata*) as well as on non-commercial plants (*Aristolochia* sp., *Centrosema* sp., *Coccinia grandis*, *Commelina benghalensis*, *Lablab purpureus*, *Oroxylum indicum*, *Psophocarpus* sp., *Solanum virginianum*, *Spilanthes* sp., and *Vigna* sp.). In cross pathogenicity assays, all the isolates from host plants could induce the disease on non-original hosts. *Paramyrothecium* species can stay in non-commercial plants, and they can infect commercially important crops. Hence, *Paramyrothecium* leaf spot disease has the potential to be an emerging fungal disease in Thailand. Thus, more research on *Paramyrothecium* is required for epidemiology studies and management strategies in agriculture, horticulture, and plantation forestry.

#### **4. Materials and Methods**

#### *4.1. Sample Collection*

Symptomatic plant leaves were collected from fields or forests in different locations in northern Thailand. The name of the host, location, and collection dates were recorded. Specimens were taken to the lab, and infected leaves were examined directly using the stereo microscope (Zeiss Stemi 305) to observe the fungal structures (sporodochia). Symptomatic leaves without fungal structures were also incubated in moist chambers (Petri dishes containing moist filter paper). Leaves were inspected daily for Paramyrothecium-like fungi.

#### *4.2. Fungal Isolation and Taxonomic Description*

Fungal structures on leaf samples were mounted in lactic acid and photographed under a light microscope (Axiovision Zeiss Scope-A1). Measurements were made with the Tarosoft (R) Image Frame Work program (Tarosoft, Bangkok, Thailand). The fungi were isolated using the single spore isolation technique [27]. Cultures were plated onto fresh PDA and incubated at 25–30 ◦C in daylight to promote sporulation. Cultural characteristics were observed after 14 days. The specimens were deposited in the fungal collection library at the Department of Entomology and Plant Pathology (CRC), Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand. Pure fungal isolates were deposited in the Culture Collection of the Sustainable Development of Biological Resources Laboratory (SDBR), Faculty of Science, Chiang Mai University, Chiang Mai, Thailand.

#### *4.3. DNA Extraction, Amplification, and Analyses*

Fungal mycelia were grown on PDA at 25–30◦C for 7 days and DNA was extracted by using the DNA Extraction Mini Kit (FAVORGEN, Ping-Tung, Taiwan) following the manufacturer's instructions. DNA amplifications were performed by polymerase chain reaction (PCR). The relevant primer pairs used in this study are listed in Table 3.


**Table 3.** Gene regions and primer sequences used in this study.

The quality of PCR amplification was confirmed on 1% agarose gel electrophoresis and viewed under ultraviolet light, and the sizes of amplicons were determined against a HyperLadderTM I molecular marker (BIOLINE). Further purification of PCR products was performed using the PCR Clean-up Gel Extraction NucleoSpin **®** Gel and PCR Clean-Up Kit (Macherey-Nagel, Düren, Germany). The purified PCR fragments were sent to the 1st Base Company (Kembangan, Selangor, Malaysia). The obtained nucleotide sequences were deposited in GenBank.

Sequences were assembled using SeqMan 5.00 and the closely related taxa for newly generated sequences were selected from GenBank® based on BLAST searches of the NCBI nucleotide database (http://blast.ncbi.nlm.nih.gov/; accessed on 4 March 2022). The reference nucleotide sequences of representative genera in *Stachybotriaceae* are in Table 4. The individual gene sequences were initially aligned by MAFFT version 7 [33] (http: //mafft.cbrc.jp/align-ment/server/; accessed on 4 March 2022) and improved manually where necessary in BioEdit v.7.0.9.1 [34]. The final alignment of the combined multigene dataset was analyzed and inferred the phylogenetic trees based on maximum likelihood (ML) and Bayesian inference (BI) analyses. The ML analyses were carried out on RAxML-HPC2 on XSEDE (v. 8.2.8) [35,36] via the CIPRES Science Gateway platform [37]. Maximum likelihood bootstrap values (BS) equal or greater than 50% are defined above each node. The BI analyses were performed by MrBayes on XSEDE, MrBayes 3.2.6 [38] via the CIPRES Science Gateway. Bayesian posterior probabilities (PP) [39,40] were determined by Markov Chain Monte Carlo Sampling (BMCMC). Six simultaneous Markov chains were run from random trees for 2,000,000 generations, and trees were sampled every 100th generation. The run was stopped when the standard deviation of split frequencies was reached at less than 0.01. The first 20% of generated trees representing the burn-in phase of the analysis were discarded, and the remaining trees were used for calculating PP in the majority rule consensus tree. The Bayesian posterior probabilities (BYPP) equal to or greater than 0.9 are defined above the nodes. The phylogenetic tree was visualized in FigTree v.1.4.3 [41] and edited in Adobe Illustrator CC 2021 version 23.0.3.585 and Adobe Photoshop CS6 version 13.0. (Adobe Systems, New York, USA).

#### *4.4. Pathogenicity Tests and Cross Pathogenicity*

Koch's postulates were used to confirm the pathogenicity of all the isolates on their original hosts. Cross pathogenicity of all the isolates was performed in healthy leaves of selected economically important plants in northern Thailand, including *Coffea arabica* (*Rubiaceae*) and *Glycine max* (*Fabaceae*) and widespread herbaceous plants including *Commelina benghalensis* (*Commelinaceae*) and *Dieffenbachia seguine* (*Araceae*). Healthy leaves were surface disinfected with 70% ethanol, washed two times with sterile distilled water, and air-dried under laminar flow. Conidial suspensions (10<sup>6</sup> conidia/mL) were prepared for all fungal isolates in sterile distilled water. The conidia (10 µL of spore suspension) were placed on the upper surface of the leaves. In addition, the leaves were also wounded before inoculation. The upper epidermis was wounded approximately 2 cm from the mid-vein by pricking with a sterile needle to about 1 mm depth. Three wounds were made for each leaf, vertically on each side of the mid-vein. Control leaves received drops of sterile distilled water. All inoculated leaves were placed in a moist chamber at 25–30 ◦C under daylight condition. After 7 days, symptoms were recorded, compared, and confirmed with the original morphology and molecular relationships.

*Plants* **2022**, *11*, 1445


**Table 4.** Taxa used in the phylogenetic analyses and their corresponding GenBank numbers.






Laboratory, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand; TBRC: Thailand Bioresource Research Center, Thailand. Species obtained in this study are in bold.

Superscript "T" indicates type species and "–" represents the absence of sequence data in GenBank.

#### **5. Conclusions**

Leaf spots caused by *Paramyrothecium* spp. were isolated from commercially important plants (*Coffea arabica*, *Tectona grandis*, *Vigna mungo*, and *V. unguiculata*), and non-commercial plants (*Aristolochia* sp., *Centrosema* sp., *Coccinia grandis*, *Commelina benghalensis*, *Lablab purpureus*, *Oroxylum indicum*, *Psophocarpus* sp., *Solanum virginianum*, *Spilanthes* sp., and *Vigna* sp.) in northern Thailand. Based on morphology and concatenated (ITS, *cmdA*, *rpb2*, and *tub2*) phylogeny, *P. vignicola*, *P. breviseta, P. eichhorniae*, and *P. foliicola* were identified. The pathogenicity of each isolate was proven using Koch's postulates. The pathogenicity assay revealed that all the isolates can cause the leaf spot disease. Interestingly, cross pathogenicity assay proved the ability of all 16 isolates to cause the disease on a wide range of hosts.

**Author Contributions:** Conceptualization, R.C. and M.C.S.; methodology, P.W., S.H., N.T., C.S., A.K., P.S. and P.P.; validation, R.C., M.C.S., N.S., J.K. and P.W.J.T.; investigation, P.W., S.H., N.T., C.S., A.K. and P.P.; writing—original draft preparation, P.W., S.H. and C.S.; writing—review and editing, all authors; supervision, R.C.; funding acquisition, R.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project was supported by Fundamental Fund 2022 (FF65/002), Chiang Mai University.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Publicly available datasets were analyzed in this study. These data can be found here: https://www.ncbi.nlm.nih.gov/ (access on 30 June 2022).

**Acknowledgments:** This research work was partially supported by Graduate School Chiang Mai University, TA/RA Scholarship, and Chiang Mai University. We thank Hiran A. Ariyawansa for his valuable suggestions and help.

**Conflicts of Interest:** The authors declare no conflict of interest.

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