First record of Penicilliopsis clavariiformis Solms. on seeds of Diospyros ferrea (Willd.) Bakh. from the Northern Western Ghats, India

Abstract

Penicilliopsis clavariiformis Solms. is a rare filamentous fungus previously reported from various woody substrates and decaying plant matter. In this study, we report the first time host association of P. clavariiformis on seeds of Diospyros ferrea (Willd.) Bakh., collected from the Manoli Reserve Forest in the Northern Western Ghats, India. The fungus was isolated using standard seed mycobiota screening methods and identified based on both morphological features such as distinctive clavarioid ascomata, septate conidiophores, and cyanophilic conidia and molecular characterization of the internal transcribed spacer (ITS) rDNA region. The ITS sequence showed > 99% similarity with authenticated P. clavariiformis sequences in GenBank. This is the first confirmed report of P. clavariiformis from seeds of Diospyros ferrea, expanding its known ecological and substrate range. The finding highlights the underexplored fungal diversity associated with forest seeds and emphasizes the need for targeted mycological surveys in biodiversity hotspots like the Western Ghats. Our results contribute to the growing knowledge of seed-associated fungi and provide a baseline for future ecological and pathological investigations involving this species.

Introduction

Penicilliopsis clavariiformis is a distinctive ascomycete fungus known for its unusual clavarioid ascomata and notable ecological adaptations. Initially reported from the fruits and seeds of Diospyros paniculata in Kerala, India, its identification has been confirmed through detailed morphological and molecular analyses¹. A rare halophilic species, P. clavariiformis demonstrates a high tolerance to saline conditions, thriving in environments with up to 10% NaCl and showing optimal growth at 24 °C². This salt tolerance is linked to its ability to shift in carbon metabolism from glycolysis to the hexose monophosphate (HMP) shunt serves as an adaptive mechanism that maintains cellular function during stress conditions. Enhanced expression of stress-related genes (Hsp98, Hsp60, RHO, HTB) further supports its halotolerance. Morphologically, it is characterized by biverticillate penicilli and oval to ellipsoidal conidia, as observed through scanning electron microscopy. The species was first discovered in a sacred grove, reflecting its niche in ecologically rich and often underexplored habitats².

The genus Penicilliopsis, belonging to the family Aspergillaceae (previously Trichocomaceae), is a tropical group of fungi recognized for its distinct synnemata and varied conidial morphology, including cylindrical to clavate phialides and a range of spore shapes. Although currently comprising a small number of described species, some members, such as P. clavariiformis, are notable for producing bioactive mycotoxins with toxicological relevance and potential therapeutic applications, including anti-HIV properties³. P. clavariiformis has been found on fruits and seeds of Diospyros species, including D. paniculata and D. crumenata, and is associated with seed rot disease, causing significant ecological and economic impact. The fungal pathogen Penicilliopsis clavariiformis causes significant seed damage in Diospyros crumenata, a critically endangered tree with high timber and nutritional fruit value, highlighting the need for holistic seed disease management⁴.

Diospyros ferrea, a medium-sized evergreen tree from the family Ebenaceae, is widely distributed across tropical and warm temperate regions, including the Western Ghats, coastal areas, the Deccan Plateau, and dry evergreen forests of India. Known locally in Tamil as “Kattu Thuvarai” and “Irumbuli,” it has traditionally been used as a famine food⁵. The genus Diospyros is highly important for its dense ebony wood, used usually in high-end furniture and musical tools, and for its medicinal properties. Bioactive compounds such as naphthoquinones and triterpenoids found in these species have shown promise in treating infectious diseases^6,7. Despite its economic and ecological importance, D. ferrea faces threats from fungal pathogens, particularly cercosporoid ascomycetes like Pseudocercospora spp., which exhibit cryptic speciation and complicate disease management⁸.

Diospyros ferrea differs from other Diospyros species by its evergreen habit, thick leathery glossy leaves, very hard and dense wood, smooth grey-black bark, and larger globose to ovoid fruits with persistent calyx lobes⁵. It holds notable phytochemical and medicinal significance. Traditionally, its roots have been used to treat ailments such as dyspepsia and inflammation, a practice supported by the presence of bioactive compounds identified through various solvent extraction techniques. Studies have revealed that ethanol extracts contain the highest concentration of phytochemicals, including flavonoids, terpenoids, alkaloids, and tannins, underscoring the importance of selecting appropriate solvents for maximizing extraction efficiency⁹. Medicinally, leaf extracts of D. ferrea exhibit strong anti-inflammatory, antibacterial, and antioxidant properties, aligning with their traditional use in wound healing¹⁰. Additionally, several Diospyros species have demonstrated anticancer potential, with compounds shown to inhibit tumor progress and promote programmed cell death in cancerous cells¹¹. While these findings highlight the therapeutic promise of D. ferrea. The study of P. clavariiformis not only underscores its unique physiological traits but also highlights the need for continued exploration of fungal biodiversity, particularly in extreme or specialized ecosystems where novel species and adaptations are likely to be found.

Materials and methods

Field collection and fungal isolation

Fruit body samples of Penicilliopsis clavariiformis were collected from the fruits and seeds of Diospyros ferrea during October 2021 and September–October 2024 in the Manoli Reserve Forest, Maharashtra, India (16.92296°N, 73.79556°E). Geographical locations have been marked in Fig. 1. The samples were transported to the Research Laboratory of Mycology and Plant Pathology at Shivaji University, Kolhapur, in sterile plastic bags. The specimens were surface-sterilized by method of Petrini et al.¹². Infected tissue sections from the fruits and seeds were aseptically blotted dry, cut into tiny pieces, and inoculated onto PDA (Potato Dextrose Agar) enriched with streptomycin. The cultures were incubated at 28 ± 2 °C for a period of 8 days, following which pure fungal colonies were isolated and stored at 4 °C for further examination.

Fig. 1.

Geographical map showing the collection site of Penicilliopsis clavariiformis from the seed samples of Diospyros ferrea in the Northern Western Ghats, Maharashtra, India. The map was created using QGIS Geographic Information System (Version 3.30) developed by the QGIS Development Team (https://qgis.org).

To ensure compliance with good taxonomic practice, a voucher specimen of the host plant (Diospyros ferrea) has been deposited in the SUK Herbarium (Shivaji University, Kolhapur, MH, India) under voucher number ABM006. The fungal voucher specimen of Penicilliopsis clavariiformis obtained from seeds of Diospyros ferrea has been deposited in the Agharkar Research Institute, Mycology and Plant Pathology Department, Pune, MH, India under accession number AMH: 10820.

Microscopic examination

Microscopic features of the fungal isolates were examined using cotton blue and lactophenol-stained preparations. Freshly mounted slides from pure cultures were observed under a LEICA DM2000 compound microscope at 1000× magnification, and images were captured for documentation. Spores were measured to determine their dimensions, including the length, width, and the spore quotient (Q), which is the length-to-width ratio. The average spore quotient (Qm) was also calculated.

Genomic DNA isolation and ITS region amplification

Genomic DNA was isolated from a fungal culture designated as sample CL using standard CTAB-based methods¹³. PCR amplification was carried out in a 25 µL reaction volume containing approximately 20 ng of genomic DNA, using fungal-specific primers ITS1 and ITS4 to target the internal transcribed spacer (ITS) region¹⁴. The amplified PCR products were resolved using a 1.5% agarose gel electrophoresis set in 1.0% TBE buffer to verify successful amplification.

Sequencing and species confirmation

Amplified PCR products were purified using a commercial DNA purification kit and subsequently sequenced with the BigDye Terminator Cycle Sequencing Ready Reaction Kit. Sequencing was performed on an Applied Biosystems ABI3500 DNA Sequencer to generate high-quality nucleotide data. The obtained ITS sequence was analyzed for quality and compared with reference sequences using the BLAST algorithm against the NCBI GenBank database. A phylogenetic tree was constructed based on the ITS sequence data to determine the evolutionary relationship of the identified fungus with related taxa.

The evolutionary relationships among 37 fungal taxa were inferred using the Neighbor-Joining (NJ) method as described by Saitou and Nei¹⁵. The optimal phylogenetic tree obtained had a total branch length of 0.131. The bootstrap test with 1000 replicates was performed to assess the statistical support for the clustering of taxa,bootstrap values are shown above the branches¹⁶.

The evolutionary distances were computed using the Maximum Composite Likelihood method¹⁷, and are in the units of the number of base substitutions per site. The proportion of sites where at least 1 unambiguous base is present in at least 1 sequence for each descendent clade is shown next to each internal node in the tree. The analytical procedure encompassed 37 nucleotide sequences. The pairwise deletion option was applied to all ambiguous positions for each sequence pair resulting in a final data set comprising 507 positions.

All evolutionary analyses were conducted in MEGA version 12¹⁸, utilizing up to four parallel computing threads for enhanced computational efficiency.

Results

Taxonomy

The fruiting bodies reached heights of up to 6–9 cm and measured 1–3 mm in width (As shown in Fig. 2g,h). They appeared upright to slightly curved and ranged from simple, unbranched forms to occasionally branched structures (Fig. 2). In branched specimens, terminal branches display irregular patterns. The structures tapered towards the apex, were smooth-surfaced, and solid rather than fragile. They emitted an unpleasant odor, and the spore print was distinctly yellow. Fresh specimens exhibited a vivid yellow coloration, which faded to a dull brown upon drying.

Fig. 2.

(a) A habitat of Diospyros ferrea; (b–d) Fruits of D. ferrea; (e–h) Ascomata of Penicilliopsis clavariiformis on Fruit and Seeds of D. ferrea.

The conidiophores measured between 6–7 µm in width and were pale brown. They exhibited distinct ring-like septations, with each segment often swollen at both ends. Their surface looks smooth, and the walls were moderately thick, about 0.5–1 µm. Short branches (ramuli), arising in groups of conidiophores. Metulae hyaline to pale brown. Phialides were cylindrical and hyaline. Conidia (n = 20) ranged in size from 6–8 × 4–5 µm, with a Q-value of 1.5–1.6 and a mean Qm of 1.55. They were sub globose to ellipsoid, eguttulate, hyaline, densely granular, and had smooth to slightly ornamented surfaces. Conidial walls were about 1 µm thick and showed cyanophilic reactions in cotton blue (Fig. 3).

Fig. 3.

(a) Penicilliopsis clavariiformis on D. ferrea; (b–d) Pure culture of P. clavariiformis on PDA: (b) initial colony morphology; (c) development of clavarioid mycelial structures; (d) developed, dense clavarioid colonies; (e,f). The conidiophore of P. clavariiformis exhibits characteristic ramuli, metulae, and cylindrical phialides; (g) Conidia of P. clavariiformis.

Molecular profiling and phylogenetic analysis

The Neighbour-Joining phylogenetic analysis based on ITS sequences clearly supports the identification of our isolate PX136227 (Penicilliopsis clavariiformis voucher AMH 10820). In the reconstructed tree, PX136227 forms a strongly supported clade (bootstrap values 96–98%) with previously authenticated sequences of Penicilliopsis clavariiformis, including AF033391 (Penicilliopsis clavariiformis strain NRRL 2482), EF669714 (Penicilliopsis clavariiformis isolate NRRL 2482), JN864881 (Penicilliopsis clavariiformis strain AP), MK459552 (Penicilliopsis clavariiformis voucher KP144), and PP218122 (Penicilliopsis clavariiformis isolate KFRIMCC 718). The close clustering of PX136227 with these reference strains indicates a high degree of sequence similarity and confirms that the present isolate belongs to the same species. Importantly, all Penicilliopsis accessions form a distinct monophyletic group clearly separated from the surrounding Penicillium species cluster, reaffirming the taxonomic validity and phylogenetic distinctness of the genus Penicilliopsis within Aspergillaceae. The high bootstrap support across nodes further strengthens the robustness of this placement (Fig. 4).

Fig. 4.

Phylogenetic tree constructed using the Neighbor-Joining method based on ITS rDNA sequences, showing the evolutionary relationship of Penicilliopsis clavariiformis with related fungal taxa. Bootstrap values (1000 replicates) are shown at the branch nodes. The tree was generated using MEGA12 (Version 12.0.11).

Discussion

Species of the genus Penicilliopsis Solms are predominantly found in tropical regions and are commonly associated with seeds instead of soil. As of 2024, the genus comprises seventeen recognized species⁴. Penicilliopsis clavariiformis has been reported from various Diospyros hosts, including D. crassiflora³, and D. macrophylla¹⁹, D. crumenata⁴, while, occurrence of Penicilliopsis pseudocordyceps, a newly identified species from Diospyros discolor seeds was reported by Hsieh and Ju²⁰. There are no confirmed reports of Penicilliopsis clavariiformis occurring on host plants outside the genus Diospyros. All documented occurrences so far, indicate a host-specific association. This suggests a potential co-evolutionary relationship or ecological specialization, though more research is needed to verify whether this exclusivity is due to host preference.

These fungi exhibit a specialized ecological preference for seeds, and are infrequently encountered in soil, suggesting a narrow ecological niche. Phylogenetically, Penicilliopsis shows affinity with genera such as Talaromyces and Penicillium but can be distinguished by features like stipitate ascomata and unique reproductive structures. However, comprehensive molecular studies remain limited, highlighting the need for further phylogenetic analysis to clarify relationships within the group³.

The report of Penicilliopsis clavariiformis from Diospyros paniculata¹, extends the known host range of this species, which appears to have a specific ecological association with members of the genus Diospyros. Morphological and molecular comparisons confirm its distinction from related taxa such as P. africana and P. bambusae, based on synnemata color, conidial dimensions, and host specificity. The observed yellow pigmentation, attributed to penicilliopsin, along with its halotolerant properties, supports earlier findings on its ecological adaptability and potential bioactivity^1,3. While Penicilliopsis is within the class Eurotiomycetes (family Aspergillaceae), most clavarioid fungi belong to different taxonomic groups, such as Basidiomycota (e.g., Clavariaceae). This places Penicilliopsis as a morphologically convergent but phylogenetically distinct group compared to typical clavarioid fungi^1,21.

The present specimen shares key morphological traits with P. clavariiformis from Diospyros paniculata¹, such as pale brown conidiophores with distinct septations, cylindrical hyaline phialides, and subglobose to ellipsoid, cyanophilic conidia with granular contents. These features align closely with those reported from Kerala. Compared to P. Africana²², which also has stipitate stromata and verticillate conidiophores, the absence of tentaculate synnemata and larger conidia in the present sample help distinguish it. This record of Penicilliopsis clavariiformis on Diospyros ferrea expands its known host range within the Diospyros genus and highlights its potential role as a specialized, seed-inhabiting pathogen. Its consistent association with seed tissues across multiple hosts suggests ecological adaptability and possible implications for seed viability and regeneration. Given the ecological importance of Diospyros species in the Western Ghats, this finding underscores the need for continued seed health monitoring and further investigation into the biology, host specificity, and ecological impact of P. clavariiformis.

Conclusion

This study reports the occurrence of Penicilliopsis clavariiformis on the seeds of Diospyros ferrea from the Manoli Reserve Forests in the Northern Western Ghats of India. Identification was supported by distinct morphological features—such as clavarioid structures, ring-septate conidiophores, and cyanophilic conidia—combined with ITS sequence analysis, which showed high similarity to authenticated P. clavariiformis strains. Phylogenetic analysis further confirmed the placement of the present isolate within the Penicilliopsis clade, with strong bootstrap support and close grouping with reference sequences. This record adds to existing knowledge of the species’ association with Diospyros seeds and contributes to ongoing efforts to document fungal diversity in the region. The findings underscore the value of integrating morphological and molecular data in fungal identification and highlight the need for additional studies to better understand the ecological role and host associations of P. clavariiformis.

Author contributions

P.H.B. and A.B.M.: collection, methodology, investigation, data curation, formal analysis, and writing-original draft. K.V.S.: data curation, and formal analysis. S.S.K.: collection, data curation, and formal analysis. M.Y.B. and S.K.S.: supervision, investigation support, validation, and resources. S.S.K.: supervision and validation. M.S.N.: supervision, investigation, resources, writing-review & editing.

Data availability

The datasets generated and/or analysed during the current study are available in the NCBI GenBank repository, https://www.ncbi.nlm.nih.gov/nuccore/PX136227.1?report=GenBank, under accession number PX136227.

Declarations

Competing interests

The authors declare no competing interests.