Introduction

Camellia oleifera, a widely cultivated oilseed crop, is primarily grown in East and Southeast Asia, including China, Japan, Vietnam, Thailand, the Philippines, India, Brazil, and other regions of South America. In China, the cultivation area has steadily expanded from 3.0 million hectares (45 million mu) in 2008 to 4.53 million hectares (68 million mu) by 2019, resulting in an annual production of approximately 700,000 metric tons of camellia seed oil1. The seeds of C. oleifera are rich in oil, which is extensively utilized for edible oil extraction due to its high content of unsaturated fatty acids and vitamin E, conferring significant nutritional and health benefits. Highly adaptable and drought-tolerant, C. oleifera thrives in hilly and mountainous regions, serving as a vital crop for local economic development and enhancing the livelihoods of agricultural producers. Additionally, this species contributes to soil and water conservation, as well as biodiversity preservation2,3.

The development of Pestalotiopsis taxonomy has been shaped by both morphological and molecular approaches, leading to significant advancements in its identification. Steyaert et al. (1949) classified Pestalotia into three genera: Pestalotia, Pestalotiopsis, and Truncatella. Subsequently, Pestalotia pezizoides was originally described from the leaves and stems of Vitis vinifera collected in Italy. The conidia of this species are characterized by apical appendages with either branched or unbranched filaments, a central section consisting of four to six cells, and pointed hyaline terminal cells4. Building upon this morphological framework, Guba (1956; 1961) emphasized the number of apical appendages as a key criterion for classification, proposing a system to categorize Pestalotia into four-, five-, and six-celled groups5,6. Further refining these classifications, Sutton (1980) refined this classification by considering both the number of cells in the hyphal tissue and the structure of the apical appendage filaments, recognizing Pestalotiopsis as a distinct genus and incorporating Monochaetia and Seiridium within its framework7. With advancements in molecular techniques, DNA-based methods have increasingly been employed for fungal classification. Jeewon et al. (2002, 2004) conducted a phylogenetic analysis of Pestalotiopsis using ITS and identified it as a monophyletic taxon, though they noted that relying on a single gene fragment alone may not provide sufficient accuracy for species identification8,9. Corroborating this, Junior et al. (2006) stated that the internal transcribed spacer (ITS) region of the ribosomal RNA gene is the most commonly used marker for the molecular identification of fungi10. However, given the limitations of single-gene analyses, Hu et al. (2007) recommended using at least two genes for phylogenetic analysis, highlighting that the tub2 gene fragment, when combined with ITS, improves the resolution of species relationships and enhances the efficiency of phylogenetic analysis11.

The taxonomic refinement of Pestalotiopsis has led to the recognition of distinct genera based on morphological and molecular evidence, highlighting the ecological and pathogenic diversity within this group. Maharachchikumbura et al. (2014) proposed the reclassification of Neopestalotiopsis and Pseudopestalotiopsis from the Pestalotiopsis genus, based on both morphological traits and multilocus phylogenetic analysis, which included ITS, tub2, and tef1-α sequences. These newly delineated genera can be distinguished by differences in the colour of their conidial cells and the characteristics of their intermediate conidiogenous cells. Specifically, in Pestalotiopsis, the three intermediate cells exhibit a uniform colour, and the conidiophore is delineated. In contrast, Neopestalotiopsis is characterized by heterochromatic intermediate cells with an indistinct conidiophore. Although Pseudopestalotiopsis shares the conidiophore characteristics of Neopestalotiopsis, its intermediate cells remain uniform in colour12. While the intermediate cells of Pestalotiopsis and Neopestalotiopsis are generally similar in colour, typically brown or olive, Neopestalotiopsis often exhibits heterochromatic intermediate cells, distinguishing it morphologically from its relatives. This genus is commonly encountered in various ecological roles, including as an endophyte, pathogen, or saprophyte13,14,15,16. Moreover, Neopestalotiopsis is a major phytopathogen, responsible for diverse plant diseases that significantly impact crop yield and quality, resulting in substantial economic losses. Notable examples include guava scab in Colombia, root rot, stem rot, and leaf spot in strawberries in Mexico, leaf spot and dieback in eucalyptus in Brazil, tomato fruit rot in Iran, and leaf blight of heart fruit in India17,18,19,20,21.

From August 2023 to May 2024, leaves of Camellia oleifera exhibiting typical symptoms of fungal infection were collected from major cultivation regions in Sichuan Province. Considering the limited focus of previous studies on C. oleifera pathogens in other geographical areas, this study aimed to identify pestalotioid fungi associated with diseases in primary C. oleifera plantations in Sichuan and the pathogenicity of these fungi based on Koch’s postulates.

Materials and methods

Isolation and culture

Between August 2023 and May 2024, a field survey was conducted in a 66.67-hectare Camellia oleifera plantation in Ya’an City, Sichuan Province, China. At least twenty symptomatic samples were collected per site, while additional sampling was performed in areas exhibiting severe infections. Several diseases were identified, including Camellia anthracnose, algal leaf spot, and soft rot. Among them, anthracnose was the most prevalent, as it typically affected entire trees simultaneously, thereby causing substantial damage. After sample collection, the specimens were transported to the laboratory for further analysis. The fungal isolates examined in this study were subsequently preserved in an ultra-low-temperature freezer at the Herbarium of the Sichuan Agricultural University Culture Collection (SICAUCC), Chengdu, China. Meanwhile, the plant samples analyzed in this study were archived in the Herbarium of Sichuan Agricultural University (SICAU), Chengdu, China.

Morphological analysis

Leaf fragments (5 mm × 5 mm) were excised from the lesion margins and healthy areas of the leaves. All tissue fragments were then surface-sterilized with 75% ethanol for 30 s, followed by a 3-min treatment with 10% sodium hypochlorite. The fragments were rinsed 3–5 times with sterile water and subsequently cultured on potato dextrose agar (PDA, prepared by combining 200 g of potato, 1 L of distilled water, 20 g of glucose, and 20 g of agar per litre), and incubated at room temperature (25 ± 1 °C). The morphology of conidiomata and colony characteristics were examined using an NVT-GG dissecting microscope (Shanghai Advanced Optoelectronics Technology Co., Ltd., Shanghai, China). Additionally, more detailed microscopic morphological features were examined using a BX53 microscope (Olympus Corporation, Japan). The diameters of conidiomata, the dimensions of conidiogenous cells, the size of conidia, and other microscopic morphological characteristics were measured using Image Frame Work v.0.9.7, with approximately 20–30 measurements taken for each structure. Finally, Images were manipulated with Adobe Photoshop CC 2019 software (Adobe Systems, San Jose, CA, USA).

DNA extraction and PCR amplification

Genomic DNA was extracted from 26 well-developed fungal colonies following the standard protocol using the Genomic DNA Extraction Kit (Beijing Adler Biotechnology Co., Ltd.). For PCR amplification of the extracted gene fragments, a 25 µL reaction mixture was prepared, consisting of 1 µL of DNA template, 1 µL of forward primer, 1 µL of reverse primer, 12.5 µL of Premix Taq™, and 9.5 µL of distilled water (Table 1). The PCR products were analyzed via 2% agarose gel electrophoresis. Following amplification, the PCR products, along with their respective primers, were submitted to Hangzhou Youkang Biotech Co., Ltd. (Chengdu, China) for sequencing.

Table 1 PCR primers and amplification conditions.
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Genetic and phylogenetic analysis

Sequence data for Pestalotiopsis and Neopestalotiopsis species were retrieved from GenBank and used to construct a multigene phylogenetic tree. Individual locus sequences were first aligned using MAFFT (https://MAFFT.cbrc.jp/alignment/server)25,26. These sequences were then assembled into a consensus sequence with Mesquite software. Phylogenetic trees based on maximum likelihood (ML) were constructed through the CIPRES Science Gateway (https://www.phylo.org), with a bootstrap analysis performed 1000 times; branches were considered significant if the maximum-likelihood bootstrap proportion (MLBS) was ≥ 60%. Bayesian inference (BI) phylogenetic trees were also generated using MrBayes v.3.1.2, with posterior probability (BIPP) values calculated; branches were considered robust if the BIPP was ≥ 0.9. The final phylogenetic tree was visualized with FigTree v1.4.3 software to examine species relationships and evaluate node support27,28,29.

Pathogenicity test

Three pestalotioid fungal isolates were selected for the subsequent pathogenicity test, which involved inoculating detached Camellia oleifera leaves with both spore suspension and mycelial plug. Each isolate was tested in triplicate. Following inoculation, the leaves were placed in a growth chamber at 25 ± 1 °C and 95% relative humidity and continuously monitored for 15 days. Initially, healthy leaves were gently cleaned with sterile water before being surface-sterilized with 75% ethanol. The disposable syringe was sterilized by passing it through a flame under an alcohol lamp, and after cooling, the leaves were evenly scratched on the right side with needles. In the control group, sterile distilled water and sterilized PDA were applied to the wounds, while in the experimental groups, the leaf scars were treated with a spore suspension and a mycelial plug, respectively. For the preparation of spore suspension, the isolated and purified fungal strains were inoculated into potato dextrose liquid medium and incubated on a shaker for 30 to 60 min at 25 °C. The culture broth was then filtered through sterile gauze to remove the mycelium, yielding a spore suspension with a concentration of 1.0 × 106 spores/mL. In cases where some pathogen strains failed to produce spores, the inoculum was prepared from mycelial plugs, which were cut into 4 mm diameter discs using a punch.

Results

Phylogenetic analysis

The ITS, tef1-α, and tub2 gene sequences effectively distinguish between pestalotioid fungal species. In this study, a multigene phylogenetic analysis of Pestalotiopsis species included sequences from 140 taxa, with Neopestalotiopsis protearum CBS 114,178* serving as the outgroup (Fig. 1). The alignment consisted of 2906 total characters, including gaps, with the following distribution: ITS = 726, tef1-α = 1162, and tub2 = 1018. The matrix contained 1359 unique alignment patterns, of which 48.22% were either unidentified characters or gaps. The estimated base frequencies were: A = 0.239, C = 0.293, G = 0.217, and T = 0.251. The substitution rates were as follows: AC = 1.173, AG = 3.271, AT = 1.237, CG = 0.943, CT = 4.021, and GT = 1.000. The gamma distribution shape parameter was α = 0.335, and the tree length was 2.235. The tree with the highest likelihood, generated by RAxML, achieved a final likelihood value of − 20,283.62. Four strains (SICAUCC 23-0160, SICAUCC 23-0161, SICAUCC 23-0162, and SICAUCC 23-0163) formed two well-supported branches (MLBS = 99%, BYPP = 1.00; MLBS = 100%, BYPP = 1.00). After validation through molecular differences and morphological analysis, these strains were identified as two known species within the genus Pestalotiopsis.

The multigene phylogenetic tree for Neopestalotiopsis species included 140 taxa, with Pestalotiopsis trachycarpicola (BJFUCC42) and P. trachycarpicola (BJFUCC42-2) used as the outgroups (Fig. 2). The analysis included a total of 3454 characters (ITS = 1196, tef1-α = 1173, tub2 = 1085), accounting for gaps. The alignment yielded 1097 distinct patterns, with gaps and unresolved characters representing 53.08% of the total alignment. The estimated base frequencies were as follows: A = 0.236, C = 0.271, G = 0.219, and T = 0.274. Substitution rates were calculated as follows: AC = 0.814, AG = 2.392, AT = 1.085, CG = 0.669, CT = 2.338, and GT = 1.000. The gamma distribution shape parameter was α = 0.212, and the tree length was 3.236. The best-scoring RAxML tree reached a final likelihood value of − 15,792.11. Four strains (SICAUCC 24-0003, SICAUCC 24-0004, SICAUCC 24-0005, and SICAUCC 24-0006) formed distinct, well-supported branches (MLBS = 93%, BYPP = 1.00). These strains were identified as a new species within the genus Neopestalotiopsis based on comparisons between genetic and morphological differences.

Fig. 1
figure 1figure 1

The phylogenetic tree was constructed based on the ITS, tub2, and tef1-α gene sequences. Nodes are annotated with maximum-likelihood bootstrap values (MLBS, left) greater than 60% and Bayesian posterior probabilities (BYPP, right) of 0.90 or higher. Strains included in this study are highlighted in bold, and type strains are designated by an asterisk (*).

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Fig. 2
figure 2figure 2

The phylogenetic tree was constructed based on the ITS, tub2, and tef1-α gene sequences. Nodes are annotated with maximum-likelihood bootstrap values (MLBS, left) greater than 60% and Bayesian posterior probabilities (BYPP, right) of 0.90 or higher. Strains included in this study are highlighted in bold, and type strains are designated by an asterisk (*).

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Taxonomy

Pestalotiopsis trachycarpicola Yan M. Zhang & K.D. Hyde [as ‘trachicarpicola’], Cryptog. Mycol. 33(3): 315 (2012) Fig. 3.

Index Fungorum No: IF631319.

Description: The fungus infects the leaves of Camellia oleifera, causing anthracnose. Asexual morph: Conidiomata, black, irregularly globose, buried or surface-adhered, solitary or tufted, 359.31–587.08 μm diam (\(\bar {x}\) = 454.21 μm, n = 30). Conidiophores, thin-walled, transparent, and subcylindrical in shape, frequently merging with conidiophore cells. Conidiogenous cells, cylindrical and hyaline, and have rough-walled surfaces, 1.87–3.13 × 7.96–28.7 μm (\(\bar {x}\) = 2.47 × 19.73 μm, n = 20). Conidia, brown or light brown, spindle-shaped, upright or gently curved, 4-septate, thin-walled, 6–8.08 × 20.54–34.09 μm (\(\bar {x}\) = 7.1 × 29.45 μm, n = 30), bearing appendages; the basal cell, obconical with a flattened base, hyaline or pale brown, and characterized by thin walls, 3.69–8.66 μm (\(\bar {x}\) = 6.27 μm, n = 30), three median cells, pale brown, subcylindrical, and thin-walled; the first median cell, pale brown, 4.42–7.44 μm (\(\bar {x}\) = 5.85 μm, n = 30); the second median cell, pale brown, 4.03–6.8 μm (\(\bar {x}\) = 5.7 μm, n = 30); the third median cell, pale brown or brown, 5.01–7.21 μm (\(\bar {x}\) = 5.95 μm, n = 30); together 15.57–21.22 μm (\(\bar {x}\) = 18.28 μm, n = 30). Apical cells, conical, apex truncate or acute, hyaline, thin-walled, 3.81–7.71 μm (\(\bar {x}\) = 5.78 μm, n = 30). Apical appendages, tubular, transparent, 2–3 (mostly 3), branched, 10.28–22.02 μm (\(\bar {x}\) = 15.69 μm, n = 30); basal appendage, single, centrally located, and unbranched, 3.69–10.51 μm (\(\bar {x}\) = 6.68 μm, n = 30). Sexual morph: not observed.

Culture characteristics: The PDA medium was incubated at 25 ± 1 °C with 95% relative humidity under alternating light and dark cycles. After six days, the culture completely covered the plate surface, with colonies reaching a diameter of 57 mm, corresponding to an average mycelial growth rate of 9.5 mm/day. The colony margin was white, indicating the presence of aerial mycelium, while the reverse side appeared light orange to bright yellow. After approximately 15 days, the colony developed abundant conidial masses, which were goose-yellow to egg-yellow.

Materials examined: China, Sichuan Province, Ya’an City, Babu Township; collected from Camellia oleifera leaves. 5 April 2024, YaqianYan, YYQ202404006 (SICAU 24-0057), living culture SICAUCC 23-0160. Ibid. YYQ2024040066 (SICAU 24-0058), living culture SICAUCC 23-0161.

Note: In the multigene phylogenetic analysis (Fig. 1), the strains from this study (SICAUCC 23-0160 and SICAUCC 23-0161) grouped within a clade that included ex-type strains of Pestalotiopsis species, with significant support (MLBS = 99%, BYPP = 1.00). Considering the minimal genetic divergence between the two strains (SICAUCC 23-0160 and SICAUCC 23-0161) and P. trachycarpicola (BJFUCC42), the morphological characteristics of these strains are consistent with those of P. trachycarpicola, thereby supporting the identification of both strains. P. trachycarpicola (BJFUCC42) was originally isolated from Chinese Yew30, while NTUPPMCC 18-160 was sourced from Ophiocordyceps sp31. , Additionally, MFLU 18-2524 was isolated from Celtis formosana32, and NTUCC 18-004 was derived from Camellia sinensis33. Qi et al. (2021) isolated P. trachycarpicola from Pinus bungeana34. This study reports the first record of this species on a novel host.

Fig. 3
figure 3

Pestalotiopsis trachycarpicola (SICAU 24-0057). (a) Symptoms of leaf disease on Camellia oleifera. (b) Conidiomata developing on infected leaves. (c) Colony morphology on PDA, shown as the bottom view (left) and top view (right). (d) Yellowish conidial masses were observed on colonies after 12 days of incubation on PDA. (e) Conidiogenous cells and developing conidia. (f–k) Mature conidia. Scale bars: (b–d) = 500 μm; (e) = 10 μm; (f–k) = 20 μm.

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Pestalotiopsis hispanica F. Liu, L. Cai & Crous, Stud. Mycol. 92: 362 (2018) [2019] Fig. 4.

Index Fungorum No: IF828379.

Description: The fungus infects the leaves of Camellia oleifera, causing leaf blight. Asexual morph: Conidiomata, brown or dark brown, globose or ellipsoid, semi-buried, solitary or tufted, 439.64–1145.01 μm diam (\(\bar {x}\) = 705.80 μm, n = 20). Conidiophores, clavate to sub-cylindrical in shape, translucent, smooth, and frequently merge with the conidiogenous cells. Conidiogenous cells, elongated, and transparent, ranging from pear-shaped to cylindrical, 1.56–3.59 × 9.42–29.48 μm (\(\bar {x}\) = 2.53 × 19.63 μm, n = 20). Conidia, brown or light brown, fusoid, erect or slightly curved, 4-septate, smooth-walled, lighter diaphragm colour, 4.68–8.49 × 24.39–32.6 μm (\(\bar {x}\) = 7.17–28.96 μm, n = 30), bearing appendages; the basal cell, obconical with a flattened base, transparent, and thin-walled, 3.92–9.68 μm (\(\bar {x}\) = 5.84 μm, n = 30), the three median cells, brown or light brown, with a uniform colouration, ellipsoidal or subterete, thin-walled; the first median cell, 4.09–7.86 μm (\(\bar {x}\) = 6.02 μm, n = 30); the second median cell, 3.75–6.81 μm (\(\bar {x}\) = 5.29 μm, n = 30); the third median cell, 3.87–7.53 μm (\(\bar {x}\) = 5.3 μm, n = 30); together 14.06–19.81 μm (\(\bar {x}\) = 17.04 μm, n = 30). The apical cells, conical in shape, tapering to sharp points, transparent, thin-walled, 3.46–7.28 μm (\(\bar {x}\) = 5.67 μm, n = 30). Apical appendages, transparent, tubular, 2–3 (mostly 2), unbranched, 7.09–20.08 μm (\(\bar {x}\) = 14.74 μm, n = 40); basal appendage, 0–1 (mostly 1), transparent, tubular, centric, slightly curved, unbranched, 1.98–9.17 μm (\(\bar {x}\) = 4.26 μm, n = 30). Sexual morph: not observed.

Culture characteristics: The PDA medium was incubated at 25 ± 1 °C with 95% relative humidity under alternating light and dark cycles. After six days, the colonies expanded to a diameter of 54 mm, corresponding to an average mycelial growth rate of 9 mm/day. The colony surface was white and densely covered with aerial mycelium, while the underside exhibited a dark orange central zone, transitioning to a lighter orange toward the periphery. After approximately 15 days, the colony began to produce black conidial masses.

Materials examined: China, Sichuan Province, Ya’an City, Babu Township; collected from Camellia oleifera leaves. 14 August 2023, Yaqian Yan, YYQ202308017 (SICAU 24-0059), living culture SICAUCC 23-0162. Ibid. YYQ2023080177 (SICAU 24-0060), living culture SICAUCC 23-0163.

Notes: In the multigene phylogenetic analysis (Fig. 1), the two newly isolated strains (SICAUCC 23-0162 and SICAUCC 23-0163) in this study clustered within a clade that included ex-type strains of Pestalotiopsis species, with substantial support rate (MLBS = 100%, BYPP = 1.00). Based on the minimal genetic differences and morphological similarities to P. hispanica (CBS 115391*), the two strains were identified as the species. P. hispanica (CBS 115391*) was initially isolated from Protea species (Proteaceae)35, while P. hispanica (NTUPPMCC 18-162) was isolated from Ophiocordyceps sp31. The strains examined in this study were isolated from Camellia oleifera, representing a new host record for this species.

Fig. 4
figure 4

Pestalotiopsis hispanica (SICAU 24-0059). (a) Symptoms of leaf disease on Camellia oleifera. (b) Conidiomata developing on infected leaves. (c) Colony morphology on PDA, shown as the bottom view (left) and top view (right). (d) Black conidial masses were observed on colonies after 14 days of incubation on PDA. (e,f) Conidiogenous cells and developing conidia. (g–j) Mature conidia. Scale bars: (b–d) = 400 μm; (e–j) = 10 μm.

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Neopestalotiopsis folii Y.Q. Yan, C.L. Yang & Y.G. Liu, sp. nov. Figure 5.

Index Fungorum No: IF900503.

Etymology: The name is derived from the Latin term folium, referring to the site of collection where the fungi grow on leaves.

Holotype: SICAU 24-0069.

Description: The fungus infects the leaves of Camellia oleifera, leading to algal spot disease. Asexual morph: Conidiomata, circular, light to dark brown, globose, solitary or clustered, semi-buried, has a diameter of 447.78–1105.5 μm and often forms a symbiotic association with algae. Conidiophores, ellipsoidal to somewhat cylindrical, translucent, smooth, ringed, indistinguishable, and often fuse with the conidiogenous cells. Conidiogenous cells, typically ampulliform to fusiform in morphology, transparent, 5.35–10.7 × 4.76–8.23 μm (\(\bar {x}\) = 7.18 × 6.57 μm, n = 30). Conidia, fusoid, light brown, erect or slightly curved, 4-septate, 6.86–9.83 × 26.6–29.2 μm (\(\bar {x}\) = 7.74 × 25.51 μm, n = 30), with bearing appendages; the basal cell, obconical with a flat base, semi-translucent, thick-walled, measuring 4.48–7.99 μm in length (\(\bar {x}\) = 6.1 μm, n = 30); three median cells, cylindrical to sub-cylindrical, light brown, uniform in colour, with lightly pigmented septa; the first median cell, from the base, ranges from 4.71 to 7.23 μm (\(\bar {x}\) = 5.76 μm, n = 30); the second median cell, 4.54–7.91 μm (\(\bar {x}\) = 6.18 μm, n = 30); and the third median cell ranges from 5.3 to 7.88 μm (\(\bar {x}\) = 6.37 μm, n = 30); with a total combined length of 14.55–23.02 μm (\(\bar {x}\) = 18.3 μm, n = 30); The apical cells, conical, with either a truncate or acute apex, hyaline, thin-walled, 4.41–7.93 μm (\(\bar {x}\) = 6.01 μm, n = 30). Apical appendages, tubular, hyaline, only three, unbranched, 11.76–32.84 μm (\(\bar {x}\) = 25.24 μm, n = 30); basal appendage, single, centric, unbranched, 5.4–11.14 μm (\(\bar {x}\) = 8.29 μm, n = 30). Sexual morph: not observed.

Culture characteristics: The PDA medium was incubated at 25 ± 1 °C with 95% relative humidity under alternating light and dark conditions. After ten days, the colony diameter reached 60 mm, corresponding to an average mycelial growth rate of 6 mm/day. The colony surface was white and densely populated with aerial mycelium, while the reverse side appeared light orange. After approximately 15 days, the colony began to produce black conidial masses.

Materials examined: China, Sichuan Province, Ya’an City, Babu Township; parasitic on Camellia oleifera leaves, 5 April 2024, YaqianYan, YYQ202404004-1 (SICAU 24-0069, holotype), ex-type living culture SICAUCC 24-0003. Ibid. YYQ202404004-2 (SICAU 24-0070), living culture SICAUCC 24-0004. Ibid. YYQ202404005-1 (SICAU 24-0071), living culture SICAUCC 24-0005. Ibid. YYQ202404005-2 (SICAU 24-0072), living culture SICAUCC 24-0006.

Notes: In the multigene phylogenetic analysis (Fig. 2), the isolates SICAUCC 24-0003 (ex-type strain), SICAUCC 24-0004, SICAUCC 24-0005, and SICAUCC 24-0006 obtained in this study formed a distinct, well-supported clade (MLBS = 93%, BYPP = 1.00) and were closely related to Neopestalotiopsis vheenae (BRIP 72293a*, type strain)36. Compared to N. vheenae (BRIP 72293a*), the ITS region exhibited four nucleotide differences (99.2% identity, 497/501 nucleotides, 0 gaps), the tef1-α region showed fourteen differences (97.2% identity, 491/505 nucleotides, 7 gaps), and the tub2 region had seventeen differences (95.9% identity, 406/423 nucleotides, 7 gaps). In addition, relative to N. vheenae (BRIP 72293a*), SICAUCC 24-0003 exhibited broader conidiogenous cells (4.76–8.23 μm vs. 3–5 μm). Furthermore, SICAUCC 24-0003 developed longer conidia (26.6–29.2 μm vs. 22–26 μm) and longer apical appendages (11.76–32.84 μm vs. 15–25 μm) than N. vheenae (BRIP 72293a*). Based on significant molecular and morphological differences, the strains identified in this study have been classified as novel species.

Fig. 5
figure 5

Neopestalotiopsis folii (SICAU 24-0069, holotype). (a,b) Disease symptoms manifested on Camellia oleifera leaves. (c) Colony morphology on PDA, shown as the top view (left) and bottom view (right) (d) Black conidial masses observed on colonies after 15 days of incubation on PDA. (e,f) Conidiogenous cells and developing conidia. (g–j) Mature conidia. Scale bars: (b) = 500 μm; (d) = 50 μm; (e–j) = 10 μm.

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Pathogenicity analysis

The pathogenicity of the three tested strains—Pestalotiopsis trachycarpicola (SICAUCC 23-0160), P. hispanica (SICAUCC 23-0162), and Neopestalotiopsis folii (SICAUCC 24-0003)—was evaluated using both spore suspension and mycelial plug inoculation methods. Under field conditions, P. trachycarpicola caused severe anthracnose, resulting in extensive lesion formation. P. hispanica induced leaf blight, with lesions > 2 cm in diameter, accompanied by severe yellowing and deformation. N. folii caused infections that affected the entire leaf. However, in the spore suspension inoculation assay, leaves inoculated with SICAUCC 23-0162 exhibited anthracnose symptoms (Fig. 6); while those inoculated with SICAUCC 23-0160 developed lesions with yellow halos around the edges; leaves inoculated with SICAUCC 24-0003 initially showed light brown lesions, which gradually progressed into grey-brown necrotic tissue. On the other hand, in the mycelial plug inoculation assay, leaves inoculated with SICAUCC 23-0162 exhibited necrosis in the central area (Fig. 6); leaves inoculated with SICAUCC 23-0160 displayed distinct anthracnose symptoms; while those inoculated with SICAUCC 24-0003 consistently developed light brown lesions. Sterile distilled water and sterilized PDA served as blank controls, with no disease symptoms observed in the control group. Pathogenicity tests were conducted on three fungal isolates using two distinct inoculation methods. Remarkably, the results demonstrated that SICAUCC 23-0162 exhibited the highest virulence in the spore suspension inoculation assay, while SICAUCC 23-0160 exhibited the highest pathogenicity in the mycelial plug inoculation assay. Furthermore, the pathogenicity of SICAUCC 24-0003 was less pronounced under controlled conditions compared to field conditions, suggesting the potential involvement of other microorganisms, possibly including algae. Thus, further investigation is required to better elucidate these factors and their potential roles in the observed pathogenicity.

Fig. 6
figure 6

Splice-back experiments involving the injection of spore suspension and mycelial plug.

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Discussion

Camellia oleifera is widely affected by various fungal diseases, with pestalotioid fungi emerging as significant pathogens of concern. Several studies have identified diverse species associated with fungal infections in C. oleifera and related plants. Liu et al. (2017) identified and isolated fungi belonging to the genera Neopestalotiopsis, Pestalotiopsis, and Pseudopestalotiopsis from Camellia sinensis and other Camellia species37. Li et al. (2021) characterized multiple Pestalotiopsis and Neopestalotiopsis species infecting C. oleifera across China, particularly in Guangdong, Guangxi, Hainan, Hunan, and Jiangsu provinces38. Tang et al. (2021) identified N. protearum as a novel pathogen causing seed rot in C. oleifera39. Nevertheless, despite these findings, the fungal diversity associated with C. oleifera in Sichuan remains largely unexplored. Addressing this knowledge gap is crucial for enhancing disease management strategies and ensuring the sustainable development of the tea oil industry.

Molecular techniques have been essential for the accurate identification of pestalotioid fungi. Phylogenetic relationships within Pestalotiopsis can be reliably resolved using four optimality criteria: maximum parsimony, weighted parsimony, maximum likelihood, and neighbor joining, with the integration of morphological traits significantly improving species identification accuracy40,41. In particular, multi-gene sequencing has demonstrated superior robustness over single-gene approaches, with ITS, tub2, and tef1-α identified as the most effective markers for distinguishing Pestalotiopsis species42,43. Furthermore, Pestalotiopsis was initially considered a low-virulence pathogen; however, recent reports have demonstrated its ability to cause diseases of varying intensities in a broad spectrum of hosts, including mango, coconut, strawberries, hazelnut, guava, Japanese yew, Dillenia indica, Pinus pinea, and Camellia oleifera, resulting in substantial economic losses to affected agricultural sectors38,44,45,46,47,48,49,50,51. Recent studies continue to expand the known diversity of the genus, with novel species being identified from various hosts through integrated morphological and phylogenetic analyses52,53,54,55. Given the economic importance of C. oleifera, identifying and characterizing pestalotioid fungi on this crop is crucial for effective disease management and sustainable production.

In addition to Pestalotiopsis, the closely related genus Neopestalotiopsis has emerged as an important plant pathogen, exhibiting broad host adaptability and causing significant economic losses. Recent studies have demonstrated that Neopestalotiopsis infects a diverse range of hosts, including strawberries, Areca ipot, and Elaeagnus pungens, with documented occurrences in multiple countries, such as but not limited to China, India, and the Philippines56,57,58,59. Moreover, N. saprophytica has been documented as the etiological agent of leaf spot disease in oil palms, causing necrotic lesions that can progress to extensive defoliation in severe cases and highlighting its impact on woody oilseed crops60. Given the growing acknowledgement of Neopestalotiopsis as an emerging pathogen, further investigations are essential for its accurate identification on C. oleifera.

The rising importance of Pestalotiopsis and Neopestalotiopsis in plant pathology extends beyond disease management, as these fungi also exhibit significant biotechnological potential. A comprehensive understanding of their pathogenic mechanisms is essential for developing effective disease control strategies and exploring their applications in agriculture and industry. Infections caused by these fungi have resulted in substantial economic losses, reducing crop yields and impacting farmer livelihoods. Thus, accurate identification of these pathogens is critical for protecting agricultural economies and preserving ecological stability.

Conclusions

This study characterized eight pestalotioid fungal strains associated with Camellia oleifera using morphological and phylogenetic analyses. Among them, four strains were classified as a novel species, Neopestalotiopsis folii, newly described in this study. The remaining four strains were identified as two newly recorded species, namely Pestalotiopsis trachycarpicola and P. hispanica. Furthermore, Koch’s postulates verified the pathogenicity of three strains, emphasizing their potential threat to C. oleifera. These findings enhance our understanding of fungal diversity in C. oleifera and offer essential insights for developing effective disease management strategies, thereby supporting the sustainable development of the woody oilseed industry.