Chiang Mai Journal of Science

Phylogenetic and Morphological Characterization of Spegazzinia Species from Oryza sativa L. in Thailand

1. INTRODUCTION

     Spegazzinia, a genus of fungi belonging to the family Didymosphaeriaceae, was first introduced by Saccardo [1], with S. ornata designated as the type species. Initially classified in the family Apiosporaceae within class Sordariomycetes [2], molecular phylogenetic studies later reclassified Spegazzinia under Didymosphaeriaceae [3]. This genus is particularly noted for its pleomorphic characteristics, which include the production of both hyphomycete and teleomorph forms, although the latter form has rarely been documented [4]. A total of 35 records (33 species with two varieties) of Spegazzinia are listed in Index Fungorum [5].
     The morphological diversity of Spegazzinia is highlighted by its characteristic production of two conidial types: type "α," which consists of 4–8 subglobose, very dark cells with long spines; and type "β," typically subspherical or broadly ellipsoid, often flattened in one plane, and characterized by being cruciately septate or muriform, usually pale brown and smooth, with shorter spines or lobes [6,7]. Such pleomorphic traits suggest ecological adaptations and varying reproductive strategies that warrant further investigation.
     Species of Spegazzinia are primarily found associated with a variety of plant materials, particularly dead leaves, leading to their consideration as potential plant pathogens or important decomposers in terrestrial ecosystems. Despite increasing knowledge surrounding this genus, many species remain poorly documented in relation to their host interactions and geographical distribution, especially in tropical regions [8–10].
     Rice (Oryza sativa L.) is one of the world's most significant cereal crops, cultivated in diverse environments and serving as a staple food for a vast portion of the global population. The fungal communities inhabiting rice fields are critical to crop health, as they influence both yield and quality. While studies on rice fungal diseases have been extensive, focusing on the identification of various fungal pathogens, the current knowledge is largely based on morphological characteristics alone. Over 300 fungal species associated with rice have been reported; however, many of these have not been confirmed through molecular sequence data, which is essential for accurate taxonomic placements [11].
     The rich biodiversity presents an excellent opportunity to explore underreported fungal species associated with rice in Thailand. This study aims to identify and characterize Spegazzinia species from rice collected in Chiang Rai Province, Thailand, through detailed morphological assessments and phylogenetic analyses. The findings will not only enhance the knowledge of the diversity of Spegazzinia, but also provide insights into the ecological roles these fungi may play in rice cultivation and overall agricultural sustainability.

2. MATERIALS AND METHODS

2.1 Sample Collection, Isolation, and Morphological Examination
     Dead leaves of Oryza sativa with black fungal fruiting bodies were collected from Chiang Rai Province, northern Thailand, and the necessary information was recorded [12]. Specimens were brought in plastic zip lock bags for incubation and examination in the mycology laboratory of the Center of Excellence in Fungal Research. Senanayake et al. [13] was followed for morphological study and single spore isolation. Morphological characteristics were examined using a stereomicroscope (Motic SMZ-171, Wetzlar, Germany). The micro-characteristics of the fungus were observed and photographed using a Nikon camera series DS-Ri2 connected to a Nikon ECLIPSE Ni-U microscope (New York, USA). All microscopic structures were measured using the Image Framework program v.0.9.0.7, and images were processed in Adobe Photoshop CS6 (Adobe Systems, San Jose, USA).
     Specimens were deposited in the Herbarium of Mae Fah Luang University (MFLU), Chiang Rai Province, Thailand, while living cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC).

2.2 DNA Extraction, PCR Amplification, and Sequencing
     Genomic DNA was extracted from 3-week-old pure cultures using a DNA Extraction Kit-BSC14S1 (BioFlux, Hangzhou, China) following the manufacturer’s instructions. The genomic DNA was stored at 4°C and subjected to polymerase chain reaction (PCR) to amplify partial gene regions using corresponding primers. The PCR was carried out using the following primers: the partial large subunit nuclear rDNA (LSU) with primer pairs LR0R/LR5 [16], the internal transcribed spacer (ITS) gene was amplified with primer pairs ITS4/ITS5 [17], SSU gene was amplified using primers NS1 and NS4 [17] and the translation elongation factor 1-alpha gene (tef1-α) with primers 983F/2218R [18].
     The PCR thermal cycle program for LSU, ITS, SSU and tef1-α amplifications were as follows: Initial denaturation 95°C for 5 min. Followed by 35 cycles, denaturation at 95°C for 30 s, annealing at 55°C for 50 s, elongation at 72°C for 90 s. Final extension at 72°C for 10 min [9]. PCR products were checked in 1% agarose gels and sequenced using the same primers by Sangon Biotech Co., Kunming, China for sequencing.

2.3 Phylogenetic Analyses
     Both reverse and forward sequences were combined using SeqMan and subjected to BLASTn in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the most similar taxa. Sequences of Spegazzinia were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/) according to recent publications [8–10]. The additional sequences of four genes of LSU, ITS, SSU, and tef1-α included in the analyses were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/) and are listed in Table 1. Single gene sequence alignment was generated with the MAFFT v.7 online program (http://mafft.cbrc.jp/alignment/server/, 22 June 2025) [19] and trimmed using trimAl v 1.2 [20]. Multiple genes were concatenated by Sequence Matrix. The FASTA alignment formats were changed to PHYLIP and NEXUS formats by Aliview 2.11. Multigene phylogenetic analyses were constructed from maximum likelihood (ML) and Bayesian inference (BI) analyses. Maximum likelihood analysis was done by the online RAxML-HPC v.8 on XSEDE Teragrid on CIPRES Science Gateway V. 3.3 (https://www.phylo.org, 22 January 2025) using the GTRGAMMA substitution model with 1,000 bootstrap replicates [21]. The final tree was selected from suboptimal trees from each run by comparing likelihood scores.
     Bayesian inference analysis was performed using MrBayes v. 3.2 on the XSEDE tool on the CIPRES portal [22]. The models were selected as GTR+I+G for LSU, SSU, ITS, and tef1-α gene regions based on the best-fit model for BI analysis which was estimated using MrModeltest v. 2.2 [23]. Posterior probabilities (PP) [24,25] were defined by the Bayesian Markov chain Monte Carlo (BMCMC) sampling method in MrBayes v.3.0b4 [26]. Two parallel runs were conducted using the default settings, six simultaneous Markov chains were run for 5,000,000 generations, and trees were sampled every 500th generation. Phylogenetic trees were visualized with FigTree v1.4.4 [27], and layouts were carried out with Adobe Illustrator CS5 v. 16.0.0. and Adobe Photoshop 2021 software (Adobe Systems, California, USA). All newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/).

3. RESULTS

3.1 Phylogenetic Analyses
     The combined LSU, ITS, SSU, and tef1-α dataset comprised 27 strains, including two newly sequenced strains. Kalmusia spartii (MFLUCC 14-0560) was used as the outgroup taxon. The tree topology of the RAxML analysis was similar to the Bayesian inference analysis. Multiple genes were concatenated, comprising 3,337 nucleotide characters. The RAxML analysis of the combined dataset yielded the best scoring tree (Fig. 1) with a final ML optimization likelihood value of -10662.614158. The matrix had 656 distinct alignment patterns, with 23.76% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.235998, C = 0.257768, G = 0.277136, T = 0.229098; substitution rates: AC = 1.598042, AG = 2.023990, AT = 1.449332, CG = 1.226201, CT = 5.480007, GT = 1.000000; gamma distribution shape parameter α = 0.086971. Bootstrap support values for ML equal to or greater than 60% and BYPP equal to or greater than 0.90 are given above the nodes.
     Phylogenetic analyses showed that our isolates were placed within Spegazzinia. The newly obtained isolate S. deightonii (MFLUCC 23-0133) clustered with the strains of S. deightonii (yone 212, yone 66, MFLUCC 20-0002, and MFLUCC 23-0133), while S. tessarthra (MFLUCC 23-0132) clustered with four strains of S. tessarthra (SH 287, 12S, MFLUCC 17-2249, and MFLUCC 18-1624) with 100% ML and 1.00 PP statistical support.

3.2 Taxonomy
Spegazzinia deightonii (S. Hughes) Subram., J. Indian Bot. Soc. 35: 78 (1956) (Figure 2)
     Index Fungorum number: IF 306062, Facesoffungi Number: FoF 07238
     Saprobic on dead leaves of Oryza sativa. Sexual morph: Not observed. Asexual morph: Hyphomycetous. Sporodochia powdery, dark, dense, dry. Conidiophores elongated or short and give rise to two types of α and β conidia. Conidiophores of α conidia, (15–)25–155(–217) × 2–2.5 μm (x̅ = 90 × 2 μm, n = 20), erect or flexuous, unbranched, hyaline to brown. Conidiophores of β conidia hyaline to brown at maturity, very short, erect and slightly bent, 6.5–15 × 2–4 μm (x̅ = 10 × 3 μm, n = 15). Conidiogenous cells basauxic, forming a single, terminal holoblastic conidium at the apex of conidiophore. Conidia two types: α conidia stellate, 20–30 × 15–30 μm (x̅ = 25 × 23.5 μm, n = 15), solitary, globose to variously shaped, with short spines, 4–8-celled, deeply constricted at the septa. β Conidia disc-shaped, hyaline to dark brown, 8-celled, 20–30 × 15–25 μm (x̅ = 25 × 20 μm, n = 20), with short spines, frequently with attached conidiogenous cells.

     Culture characteristics: Conidia germinating on PDA within 12 h. Colonies growing on PDA, reaching 30 mm diam. after seven days at 25°C, raised, moderately dense, undulate margin; form above creamy at the margin, white at the middle, grey at the center; from below, white to light yellow at the margin, brown at the center.
     Material examined: THAILAND, Chiang Rai Province, Nang Lae Subdistrict, on dead leaves of O. sativa, 10 November 2020, X.G. Tian, (r6-7 = MFLU 23-0172), living culture MFLUCC 23-0133.
     Known hosts and distribution: On Andropogon, Axonopus, Borassus, Dioscorea, Euchlaena, Oryza, Pennisetum, Phragmites, Rottboellia, Saccharum, Tripsacum, Triticum, Vetiveria, and Zea from Cuba, Ghana, New Guinea, Nigeria, Puerto Rico, and Sierra-Leone [6]; On Arundo donax in Japan [3]; On Musa sp. in Thailand [28]; on Oryza sativa in Thailand (this study).
     GenBank numbers: MFLUCC 23-0133: LSU = OR438818, ITS = OR438347, SSU = OR458339.
     Notes: In the multi-loci phylogenetic analyses, our strain (MFLUCC 23-0133) clustered with S. deightonii strains (Figure 1). Morphologically, our strain shares similar morphology with S. deightonii (MFLUCC 20-0002) in having long or short two types of conidiophores, with two types of conidia, conidia α solitary, 4–8-celled, deeply constricted at the septa with short spines, and conidia β disc-shaped, hyaline to dark brown, 8-celled, with short spines [28]. Our strain also shares a similar size range of conidiophores of α conidia (25–155 × 2–2.5 μm vs. 48–120 × 1–2 μm), and longer size range of conidiophores of β conidia (6.5–15 × 2–4 μm vs. 1.6–2 × 2.5–3 μm); α conidia (20–30 × 15–30 μm vs. 18–28 × 17–29 μm) and larger size range of β conidia (20–30 × 15–25 μm vs. 16–21 × 11–14 μm) [28]. The nucleotide comparisons showed that our strains (MFLUCC 23-0133) is different from S. deightonii in ITS, LSU, SSU, and tef1-α by 1bp, 0 bp, and 1 bp, respectively. Thus, we identified new strains as S. deightonii based on phylogenetic analyses and morphological characteristics.

Spegazzinia tessarthra (Berk. & Curl.) Sacc., Syllo. Furg. IV: 758. (1886) (Figure 3)
     Index Fungorum number: IF 219777; Facesoffungi Number: 08241
     Saprobic on dead leaves of Oryza sativa. Sexual morph: Not observed. Asexual morph: Hyphomycetous, sporodochia dark, colonies on natural substratum punctiform, dense, dry, powdery, velvety. Conidiophores macronematous, mononematous, arising singly, mostly flexuous, pale brown to brown, of two types: conidiophores with α conidia (50–)70–165(–201) × 2–3 μm (x̅ = 119 × 2.5 μm, n = 25); with β conidia 4–10(–12) μm (x̅ = 7 μm, n = 15) long. Conidiogenous cells basauxic, integrated, terminal, narrowly cylindrical. Conidia solitary, two types, α stellate-shaped and β disc-shaped, 4-celled, crossed-septate. α stellate-shaped conidia 20–35 μm diam. (x̅ = 27 µm, n = 25), ovoid to globose, brown to dark brown, conspicuously spinulate, deeply constricted at the septa, with spines measuring up to 2–4 μm long; β disc-shaped conidia 15–20 × 10–20 μm (x̅ = 18.5 × 15 µm, n = 25), long, ovoid to globose, green to dark green, cruciately septate, constricted at septa, smooth, flat from side view.
     Culture characteristics: Conidia germinating on PDA within 12 h. Colonies on PDA, flat, circular, with irregular edges; from above, white at the margin and center, pale gray in the middle, from below, white at the margin, pale gray to white at the middle, light brown at the center.
     Material examined: THAILAND, Chiang Rai Province, Nang Lae Subdistrict, on dead leaves of O. sativa, 10 November 2020, X.G. Tian, r6-6 = MFLU 23-0174, living culture MFLUCC 23-0132.
     GenBank numbers: MFLUCC 23-0132: LSU = OR438820, ITS = OR4383489, SSU = OR458341, tef1-α = OR500313.
     Known hosts and distribution: On Ananas, Borassus, Cassine, Cenchrus, Citrus, Cynodon, Heteropogon, Lantana, Lycopersicon, Mangifera, Musa, Panicum, Pennisetum, Phoenix, Saccharum, Sorghum, Theobroma, Triticum, Zea and isolated from soil in Australia, Ghana, India, Kenya, Malaya, New Guinea, Sierra Leone, Sudan, Tanzania, Trinidad, Uganda, U.S.A., Venezuela, and Zambia [6]; on Acacia auriculiformis in Thailand [29]; on leaves of Andropogon gryllus in Italy [1]; on Balsa wood in Japan [29]; on leaves of Brachypodium sp. in Iran [29]; on lichen Heterodermia flabellate in India [30]; on dead leaves of O. sativa (this study).
     Notes: In the multi-locus phylogenetic analyses, our strain (MFLUCC 23-0132) clustered within S. tessarthra with 100% ML and 1.00 PP statistical support (Fig. 1). Morphologically, our strain is similar to S. tessarthra in having long or short two types of conidiophores and two types of conidia [30]. However, our strain (MFLUCC 23-0132) has brown to dark brown conidia α and light olive to dark olive β conidia, while S. tessarthra has pale to dark brown α and β conidia. Our strain also shares a similar size of conidiophores with S. tessarthra (70–165 μm vs. up to 180 μm) [6,30]. Nucleotide comparisons revealed that our strain (MFLUCC 23-0132) differs from S. tessarthra by 0.49% in the LSU gene (4/823) and 1.59% in tef1-α (14/883). Although, there was 1.59% differences in tef1-α between our strain with S. tessarthra. However, these single-gene differences do not define species boundaries for fungi, as such variation often falls within the range of intraspecific polymorphism. Importantly, phylogenetic analysis revealed that our strain (MFLUCC 23-0132) clusters with S. tessarthra without any distinct separation (Fig. 1), reflecting their close evolutionary relationship. Combined with consistent morphological characteristics, we therefore identify MFLUCC 23-0132 as S. tessarthra, representing a new host record on O. sativa.

4. DISCUSSION

     The identification of two new records of Spegazzinia species from O. sativa represents a significant advancement in understanding this fungal genus, particularly within the context of Southeast Asian biodiversity. To date, a total of 33 Spegazzinia species (including two varieties) have been documented globally, and among these, only Spegazzinia camelliae, S. musae, S. deightonii, S. neosundara, S. radermacherae, and S. tessarthra have been reported in Thailand [4,14,28,29,31]. Our addition of the two new records from rice expands the known diversity of Spegazzinia in Thailand, highlighting that the genus remains understudied in the region especially in agricultural ecosystems.
     Morphological examinations revealed distinct characteristics of the new record, showcasing the diversity within the genus and emphasizing pleomorphism as a key feature of Spegazzinia species [7,32]. Notably, our observations align with established morphological criteria, particularly the presence of two distinct types of conidia, indicating potential ecological adaptations and varying reproductive strategies.
     Phylogenetic analyses further clarify the relationships of these new isolates within the existing framework of Spegazzinia, offering molecular data to aid in resolving taxonomic uncertainties. Employing multiple genetic markers, including LSU, ITS, SSU, and tef1-α, enhances the robustness of our phylogenetic conclusions and supports future research aimed at unraveling the evolutionary relationships among related genera within the Didymosphaeriaceae family.
     The introduction of these new collections underscores the necessity for ongoing exploration of fungal diversity in agricultural systems. As rice is a critical cereal crop worldwide, understanding the fungal communities associated with rice cultivation is crucial for managing plant health and enhancing agricultural productivity. Future research should assess the ecological roles and potential pathogenicity of these newly identified Spegazzinia species, which may have significant implications for rice cultivation practices and overall agricultural sustainability.

5. CONCLUSIONS

    This study expands the known diversity of Spegazzinia and highlights the importance of investigating the ecological and economic significance of this genus. It reinforces the necessity of conserving fungal biodiversity as a vital component of agricultural health and sustainability, paving the way for further studies in this intriguing field. 

ACKNOWLEDGEMENTS

     Xing-Guo Tian thanks the Science and Technology Foundation of Guizhou Province (Qian Ke He Pingtai ZSYS [2025] 029) and the Science and Technology Program of Guizhou Province [No. Qian Ke He Jichu QN (2025) 215]. Dan-Feng Bao would like to thank the Postdoctoral Fellowship Program of CPSF under Grant Number GZC20240346 and Guizhou Provincial Basic Research Program (Natural Science) [No. QianKeHe Basic -ZK (2025) general program 666]. SCK, and ST thank the Yunnan Revitalization Talents Support Plan (High-End Foreign Experts Program) and the Key Laboratory of Yunnan Provincial Department of Education of the Deep-Time Evolution on Biodiversity from the Origin of the Pearl River for their support. SCK also thanks the National Natural Science Foundation of China (No. 32260004) for the support.

AUTHOR CONTRIBUTIONS

Xing-Guo Tian and Yong-Zhong Lu: Conceptualization, Methodology, Resources, Project administration. Xing-Guo Tian and Dan-Feng Bao: Data curation, Writing - Original draft preparation. Dan-Feng Bao: Visualization, Investigation. Samantha C. Karunarathna and Saowaluck Tibpromma: Supervision, Funding acquisition. Jia-Jun Han: Software, Validation. Samantha C. Karunarathna and Saowaluck Tibpromma: Formal analysis, Writing- Reviewing and Editing.

CONFLICT OF INTEREST STATEMENT

     The authors declare that they hold no competing interests.

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Addition to Paraleptosphaeria species (Leptosphaeriaceae, Pleosporales): Paraleptosphaeria senecionis sp. nov., associated with Asteraceae
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