Appressoria-Producing Sordariomycetes Taxa Associated with Jasminum Species

Appressoria are specialized structures formed by certain phytopathogenic fungi during the early stages of the infection process. Over the years, significant advancements have been made in understanding the formation, types, and functions of appressoria. Besides being formed primarily by fungal pathogens, many studies have reported their occurrence in other life modes such as endophytes, epiphytes, and saprobes. In this study, we observed the formation of appressoria in fungal genera that have been found associated with leaf spots and, interestingly, by a saprobic species. We used morphological descriptions and illustrations, molecular phylogeny, coalescent-based Poisson tree processes (PTP) model, inter- and intra-species genetic distances based on their respective DNA markers, and Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR) to establish a new species (Pseudoplagiostoma jasmini), a Ciliochorella sp., and a new host record (Coniella malaysiana). The Ciliochorella sp. is reported as a saprobe, while Pseudoplagiostoma jasmini and Coniella malaysiana were found to be associated with leaf spots of Jasminum species. All three taxa produce appressoria, and this is the first study that reports the formation of appressoria by a Ciliochorella sp. and a Pseudoplagiostoma sp.


Introduction
Appressoria are infection pegs, mostly produced by pathogenic fungi [1].However, since these structures are also produced by endophytes, epiphytes, and saprobes, Chethana et al. [1] proposed a general definition of appressoria as "specialized cells or adhesion structures produced by fungi from which a penetration peg emerges that pierces or enters the host tissues".Frank [2] discovered appressoria and came up with this term when he isolated the pathogen, Colletotrichum lindemuthanium, that causes diseases of beans.Based on the various shapes and sizes, appressoria can be grouped either as single-celled or multi-cellular/compound structures [3].Single-celled appressoria are sub-divided into proto-appressoria, hyaline, and melanized appressoria.Compound appressoria are further classified as expressoria, infection cushion, and infection plaques [1,3].
Overall, in pathogenesis, appressoria are important for the successful invasion of host plants by certain pathogenic fungi.By attaching to the host, generating turgor pressure, and facilitating penetration, these structures ensure that the pathogen can overcome physical barriers and initiate infection of the plant [1,4].The most frequently observed appressoria among several fungal species are single-celled, occurring mostly at the tip of germ tubes,
The results of the amplification procedure were visualized using gel electrophoresis (1.7% agarose gel) by loading the resulting amplicons and DNA fluorescent loading dye (FluoroDye TM , SMOBIO, Seoul, Republic of Korea) in the sample wells.These amplicons were purified, and DNA was sequenced at SolGent Co. (Daejeon, Republic of Korea).Consensus sequences of the forward and reverse DNA sequence data were produced using SeqMan (DNAStar, Madison, WI, USA).
Maximum likelihood analyses (ML-IQ) were performed in the webserver (https://iqtree.cibiv.univie.ac.at/ accessed on 20 November 2023), by selecting the default parameters and 1000 ultrafast bootstrap replicates [32].Phylogenetic Analysis Using Parsimony (PAUP) v.4.0b10 was used to compute MP analyses [33].A heuristic search option with the addition of 1000 random sequence additions was applied.Maxtrees and bootstrap replicates were set up to 1000.Bayesian inference analysis (MrBayes on XSEDE v.3.2.7a) was performed in the CIPRES Science Gateway v.3.3 [34,35], after implementing MrModeltest to estimate the model of evolution of individual gene regions [36].The partition model for each gene region is given (Table 2).Markov chain Monte Carlo (MCMC) sampling with four Markov chains was used to infer posterior probabilities (PP) for 1,000,000, 5,000,000, and 2,000,000 generations for Ciliochorella, Coniella, and Pseudoplagiostoma, respectively.The tree sample frequencies were set to 100.The first 20% of the total trees were discarded as "burn in" and the remaining 80% was used to calculate posterior probabilities.FigTree v.1.4.4 was used to display the phylogenetic trees [37], and the phylograms were edited and produced in Microsoft PowerPoint (2016).

Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR)
The GCPSR model was applied to scrutinize any significant recombination event that occurred between the new taxon and other phylogenetically closely related species [38], as inferred by a pairwise homoplasy index (Φw) (PHI) test.The analysis was performed in SplitsTree4 by applying the LogDet transformation and splits decomposition options [39,40].The final layout of the splitsTree graphs was produced in Microsoft PowerPoint (2016).

Poisson Tree Processes (PTP)
The coalescent-based PTP model was applied to delineate species in Pseudoplagiostoma.The analysis was computed on the Web Server (https://species.h-its.org/ptp/accessed on 20 November 2023) [41].The model assumes that the process of speciation is marked by a branching event in the evolutionary tree of a group of organisms, which separates the ancestral lineage into two or more new lineages.The model further assumes that the number of lineages in a group evolves according to a Poisson process, with the rate of speciation being proportional to the number of lineages.The PTP analysis was based on the concatenated ITS, 28S, β-tub, and tef-1α regions.Maximum likelihood analysis prior to computing PTP was conducted on the IQ-tree Web Server.Genetic distances were calculated in MEGA-X by applying the Kimura 2-parameter substitution model and selecting the gamma distribution and pairwise deletion options.

Sequence Alignment and Phylogenetic Analyses
The number of strains used in the phylogenetic analyses of each genus is given (Table 3).Phylogenetic analyses from single and combined gene regions support the identification of the new species (Pseudoplagiostoma jasmini), a Ciliochorella sp., and the new host record, Coniella malaysiana.The phylogenetic trees generated from ML-IQ, MP, and BI yielded similar taxonomic placements for our isolates.The tef-1α of Pseudoplagiostoma mangiferae was excluded from the phylogenetic analyses because when we used the BLAST tool for P. mangiferae (accession number: MK084822; 100% identity; 100% query cover; e-value = 0.0), the sequence tallied with Melanconis instead of Pseudoplagiostoma.

Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR)
The LogDet transformation and splits decomposition options were selected while configuring the PHI test.The analysis yielded a threshold over 0.05 (Фw = 1.0) for the Ciliochorella sp., indicating no significant recombination event (Figure 2).

Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR)
The LogDet transformation and splits decomposition options were selected while configuring the PHI test.The analysis yielded a threshold over 0.05 (Φw = 1.0) for the Ciliochorella sp., indicating no significant recombination event (Figure 2).

Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR)
The LogDet transformation and splits decomposition options were selected while configuring the PHI test.The analysis yielded a threshold over 0.05 (Фw = 1.0) for the Ciliochorella sp., indicating no significant recombination event (Figure 2).
posterior probabilities below 0.80.Melanconiella hyperopta (CBS 132231 and CBS 131696) are selected as outgroups.Ex-type and reference strains are in bold, and our isolate is in red.

Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR)
The LogDet transformation and splits decomposition options were selected while configuring the PHI test.The analysis yielded a threshold over 0.05 (Фw = 0.7314) for the new species, Pseudoplagiostoma jasmini, indicating no significant recombination (Figure 5).

Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR)
The LogDet transformation and splits decomposition options were selected while configuring the PHI test.The analysis yielded a threshold over 0.05 (Φw = 0.7314) for the new species, Pseudoplagiostoma jasmini, indicating no significant recombination (Figure 5).

Poisson Tree Processes
The result generated from the PTP analysis (Figure 6) is congruent with the maximum likelihood phylogram that delimits Pseudoplagiostoma jasmini as a new species (Figure 4).Genetic distances of Pseudoplagiostoma jasmini compared with its phylogenetically closely related taxa are summarized in the "note" section under Pseudoplagiostoma in the "Taxonomy" section.

Poisson Tree Processes
The result generated from the PTP analysis (Figure 6) is congruent with the maximum likelihood phylogram that delimits Pseudoplagiostoma jasmini as a new species (Figure 4).Genetic distances of Pseudoplagiostoma jasmini compared with its phylogenetically closely related taxa are summarized in the "note" section under Pseudoplagiostoma in the "Taxonomy" section.

Poisson Tree Processes
The result generated from the PTP analysis (Figure 6) is congruent with the maximum likelihood phylogram that delimits Pseudoplagiostoma jasmini as a new species (Figure 4).Genetic distances of Pseudoplagiostoma jasmini compared with its phylogenetically closely related taxa are summarized in the "note" section under Pseudoplagiostoma in the "Taxonomy" section.This family comprises saprobic, pathogenic, as well as endophytic genera that are commonly characterized by conidia that have appendages at one or both ends.Sporocadaceae has previously been subjected to multiple taxonomic re-evaluations and classifications [42,43].Bartaliniaceae, Discosiaceae, Pestalotiopsidaceae, and Robillardaceae were previously treated as synonyms of Sporocadaceae [43][44][45].This family comprises saprobic, pathogenic, as well as endophytic genera that are commonly characterized by conidia that have appendages at one or both ends.Sporocadaceae has previously been subjected to multiple taxonomic re-evaluations and classifications [42,43].Bartaliniaceae, Discosiaceae, Pestalotiopsidaceae, and Robillardaceae were previously treated as synonyms of Sporocadaceae [43][44][45]  Type species-Ciliochorella mangiferae Syd.Ciliochorella (Sporocadaceae, Amphisphaeriales, Xylariomycetidae) [42,43,46,47] was established by Sydow and Mitter [48].There are ten species in Index Fungorum [18] and nine species in Species Fungorum [49].Among these, only five Ciliochorella species have sequence data for one or more gene loci.Ciliochorella species comprise saprobic taxa that have been reported from India, Japan, South America, and Thailand [42,[50][51][52].Our isolate is also reported in its saprobic mode.
Excluding gaps in our aligned untrimmed dataset, in comparison of the inter-species genetic distance of Ciliochorella sp.(MFLUCC 23-0239) and C. phanericola, a difference of 0.34% was seen across ITS (533 nucleotides), but no difference was observed across 28S (868 nucleotides).We were unable to compare the differences across β-tub as Ciliochorella sp.(MFLUCC 23-0239) lacks sequence data for the gene region.Despite several trials using different amplification conditions, we were unable to obtain sequence data for β-tub.Therefore, coupled with morphological description and multi-locus phylogenetic analyses, a PHI test was also conducted to support the taxonomic placement of our isolate (MFLUCC 23-0239).The PHI test of the combined ITS and 28S yielded a threshold exceeding 0.05 (Φw = 1.0), suggesting that no recombination event has occurred.Nevertheless, despite the PHI test result, we suggest establishing our isolate as Ciliochorella sp.instead of identifying it as a new species due to the lack of sequence data.Further studies focusing on the collection of more Ciliochorella taxa and providing sequence data for protein-coding gene regions (β-tub, Rpb2, tef-1α) will yield better resolution in the phylogenetic trees and contribute to proper species identification (Figure 7).
Based on morphology and multigene phylogenetic analyses, we identify our strain as a new host record of Coniella malaysiana, associated with leaf spots of Jasminum sp. in northern Thailand (Figure 8).
Excluding gaps in our aligned untrimmed dataset, in pairwise nucleotide comparisons of P. jasmini and P. dipterocarpicola (MFLUCC 21-0142), the following differences were observed: 5.76% across ITS (543 nucleotide base pairs, bp), 1.86% across 28S (818 bp), 21.1% across β-tub (448 bp), and 43.7% across tef-1α (164 bp).The inter-species genetic distances (%) grouped according to the PTP result are provided (Table 7).Based on the guidelines of Chethana et al. [20], Jayawardena et al. [21], and Maharachchikumbura et al. [22] for introducing new species, we describe P. jasmini as a new species.Despite its support values (35% ML-IQ, 32% MP, and 0.95 PP), we establish P. jasmini as a new taxon, considering the formation of one or two septa in the conidia, a feature lacking in all other Pseudoplagiostoma species; all Pseudoplagiostoma spp.have aseptate conidia.Besides morphology and multigene phylogenetic analyses, we included GCPSR and PTP analyses as further evidence to support the distinct species status of Pseudoplagiostoma jasmini (Figure 10).

Discussion
In pathology, appressoria are infection structures generated to invade plant tissues [1,4,69,70].Basically, they are penetration pegs [1].Appressoria are not solely confined to fungal pathogens.They also occur in endophytes [71,72], epiphytes [3,73,74], and saprobes [75].In this study, we establish one new species (Pseudoplagiostoma jasmini), a Ciliochorella sp., and a new host record (Coniella malaysiana) that produce single-celled, irregular-shaped, hyaline appressoria.The Ciliochorella sp. is reported from dead leaves as a saprobe, while P. jasmini and C. malaysiana were found associated with leaf spots.In our study, pathogenicity tests were not performed.Therefore, the occurrence of appressoria in C. malaysiana and P. jasmini reveals their pathogenic and possibly endophytic nature.Certain fungi can switch their lifestyles from endophyte to saprobe and become pathogenic under suitable conditions [76].We hypothesize that, under favorable circumstances, C. malaysiana, P. jasmini, and the saprobic Ciliochorella sp. may develop phytopathogenic traits and cause diseases.Given that appressoria are produced by fungi in various life modes, as mentioned above, it is of dire need to record their occurrences and diversity from different hosts.This is the first study that reports the formation of appressoria in a Ciliochorella sp. and a Pseudoplagiostoma sp., but appressoria have previously been observed in Coniella musaiaensis [77].The primary function of appressoria produced by endophytes is to cross from one cell to another [3].For saprobes to obtain their nutrients, a living host is not a requisite.Thus, the formation of an appressorium in saprobic fungi is probably a result of adaptation while in their endophytic life mode [4,[78][79][80].
Species delimitation is essential to developing a proper comprehension of the biology, geography, host-fungal association, and life modes of individual fungal taxa, as well as their respective roles in the ecosystem [20].The taxonomy of certain Pseudoplagiostoma species yielded low support values when constructing the phylograms (ML-IQ, MP, PP).Despite the support values for the placement of P. jasmini, we establish the latter as a novel taxon as there are significant differences in the conidial morphology.All Pseudoplagiostoma taxa, except P. jasmini, have aseptate conidia.Apart from P. jasmini, all other species of Pseudoplagiostoma are cryptic, sharing similar morphologies such as shape, color, and size.Therefore, coupled with morphology and phylogenetic analyses, we employed the Genealogical Concordance Phylogenetic Species Recognition Analysis (GCPSR) to infer the species boundaries in Pseudoplagiostoma [38].Furthermore, we advocate the use of the coalescent-based Poisson tree processes (PTP) model to compare the inter-and intra-species genetic distances in Pseudoplagiostoma [41].
Many Sordariomycetes taxa are demarcated based on ITS, 28S, small subunit (18S, nuclear rDNA), β-tub, tef-1α, and Rpb2 loci [43].Only five Ciliochorella spp., but all Pseudoplagiostoma spp., have molecular data for one or more gene loci.A few Ciliochorella spp.lack sequence data for β-tub.The collection and examination of more Ciliochorella species, with the addition of more gene regions in the phylogenetic analyses, as applied in the analysis and delineation of other Sordariomycetes taxa, would lead to a better phylogenetic resolution and taxonomic placement of each species.Based on high-throughput sequencing, Baldrian et al. [81] suggested that the fungal diversity is around 6.28 million species worldwide but with only 1.08 million published species.A probable reason for the smaller number of Ciliochorella spp.and Pseudoplagiostoma spp.might be because they occur in poorly studied hosts and countries [82].Northern Thailand is rich in fungal biodiversity [82].Undoubtedly, further exploration of the fungal diversity in this area as well as other hotspots worldwide will reveal a higher diversity of these two and other genera [83].

Figure 2 .
Figure 2. Split graph derived from the PHI analysis, generated for Ciliochorella.Our isolate is in red.

Figure 2 .
Figure 2. Split graph derived from the PHI analysis, generated for Ciliochorella.Our isolate is in red.Figure 2. Split graph derived from the PHI analysis, generated for Ciliochorella.Our isolate is in red.

Figure 2 .
Figure 2. Split graph derived from the PHI analysis, generated for Ciliochorella.Our isolate is in red.Figure 2. Split graph derived from the PHI analysis, generated for Ciliochorella.Our isolate is in red.

Figure 3 .
Figure 3. Maximum likelihood phylogram based on the combined ITS, 28S, Rpb2, and tef-1α matrices of Coniella.Bootstrap support values (ML-IQ ≥ 80%) and maximum parsimony (MP ≥ 80%), and Bayesian posterior probabilities (PP ≥ 0.80) are given above the branches or at the nodes as ML-IQ/MP/PP.Hyphen (-) indicates bootstrap support values below 80% for ML-IQ and MP, and posterior probabilities below 0.80.Melanconiella hyperopta (CBS 132231 and CBS 131696) are selected as outgroups.Ex-type and reference strains are in bold, and our isolate is in red.

Figure 5 .
Figure 5. Split graph derived from the PHI analysis, generated for Pseudoplagiostoma.The novel species is in bold red.

Figure 6 .
Figure 6.Results generated from the PTP analysis of Pseudoplagiostoma.The analysis was based on the ML-IQ topologies of the concatenated ITS, 28S, β-tub, and tef-1α matrices.Groups of species are denoted by colored branches, with blue-colored branches indicating that they are different species, and red-colored branches representing different strains of the same species.Numbers near the nodes are posterior probabilities.The new taxon is given in bold red.

Figure 5 .
Figure 5. Split graph derived from the PHI analysis, generated for Pseudoplagiostoma.The novel species is in bold red.

Figure 5 .
Figure 5. Split graph derived from the PHI analysis, generated for Pseudoplagiostoma.The novel species is in bold red.

Figure 6 .
Figure 6.Results generated from the PTP analysis of Pseudoplagiostoma.The analysis was based on the ML-IQ topologies of the concatenated ITS, 28S, β-tub, and tef-1α matrices.Groups of species are denoted by colored branches, with blue-colored branches indicating that they are different species, and red-colored branches representing different strains of the same species.Numbers near the nodes are posterior probabilities.The new taxon is given in bold red.

Figure 6 .
Figure 6.Results generated from the PTP analysis of Pseudoplagiostoma.The analysis was based on the ML-IQ topologies of the concatenated ITS, 28S, β-tub, and tef-1α matrices.Groups of species are denoted by colored branches, with blue-colored branches indicating that they are different species, and red-colored branches representing different strains of the same species.Numbers near the nodes are posterior probabilities.The new taxon is given in bold red.

Table 1 .
GenBank accession numbers of sequences used in the phylogenetic analyses.Ex-type and reference strains are denoted with an '*'.Our isolates are in blue.
N/A: Not applicable.

Table 2 .
Partition model selected for each locus for the Bayesian analyses.

Table 3 .
Total number of characters, ML-IQ, and MP analysis parameters.
L/W: length-to-width ratio.

Table 5 .
Morphological comparison between our strain and the ex-type of Coniella malaysiana.
L/W: Length to width ratio.
L/W: Length-to-width ratio.
N/A: not applicable.