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

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.


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][5][6][7]. Disease symptoms caused by Paramyrothecium may also include stem and crown canker and fruit rot [8][9][10]. Lombard et al. [4] designated an epitype for the generic type Paramyrothecium roridum (≡M. roridum). Paramyrothecium species are distinguished from related Myrothecium

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

Culture Morphology
Diverse culture characters were observed on PDA at room temperature (25-

Culture Morphology
Diverse culture characters were observed on PDA at room temperature (25-

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).

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).

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. 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. 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 vignicola Withee & Cheew., sp. nov. (Figure 4).
Paramyrothecium based on taxonomy (Table 1) and multi-gene phylogeny ( Figure 3). The morphologies of the Paramyrothecium species are described herein.
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.
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.
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.

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).
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.

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.

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.

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. GGT AAC CAA ATC GGT GCT TTC ACC CTC AGT GTA GTG ACC CTT GGC ca. 320 [32] 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

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

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. 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).