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Journal of Fungi
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15 December 2021

Morphology, Phylogeny, and Pathogenicity of Pestalotioid Species on Camellia oleifera in China

,
and
1
Key Laboratory for Non-Wood Forest Cultivation and Conservation of the Ministry of Education, Central South University of Forestry and Technology, Changsha 410004, China
2
Key Laboratory of National Forestry and Grassland Administration for Control of Diseases and Pests of South Plantation, Central South University of Forestry and Technology, Changsha 410004, China
3
Hunan Provincial Key Laboratory for Control of Forest Diseases and Pests, Central South University of Forestry and Technology, Changsha 410004, China
4
Hunan Provincial Key Laboratory for Forestry Biotechnology, Central South University of Forestry and Technology, Changsha 410004, China
J. Fungi2021, 7(12), 1080;https://doi.org/10.3390/jof7121080
This article belongs to the Special Issue Polyphasic Identification of Fungi
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Abstract

Tea-oil tree (Camellia oleifera) is an important edible oil woody plant with a planting area of over 3,800,000 hectares in southern China. Pestalotioid fungi are associated with a wide variety of plants worldwide along with endophytes, pathogens, and saprobes. In this study, symptomatic leaves of C. oleifera were collected from Guangdong, Guangxi, Hainan, Hunan, and Jiangsu Provinces and pestalotioid fungi are characterized based on combined sequence data analyses of internal transcribed spacer (ITS), beta tubulin (tub2), and translation elongation factor 1-alpha (tef-1α) coupled with morphological characteristics. As a result, seven species were confirmed, of which five species are described as new viz. N. camelliae-oleiferae, P. camelliae-oleiferae, P. hunanensis, P. nanjingensis, P. nanningensis, while the other two are reported as known species, viz., N. cubana and N. iberica. Pathogenicity assays showed that all species except for P. nanjingensis developed brown lesions on healthy leaves and P. camelliae-oleiferae showed stronger virulence.

1. Introduction

Tea-oil tree (Camellia oleifera Abel.) is a unique woody edible oil species in China, mainly distributed in the Qinling-Huaihe River area. It has a long history of cultivation and utilization for more than 2300 years since ancient China [1]. Statistical data for 2014 indicated that these plantations comprise over 3,800,000 hectares and produce 518,000 tons of edible oil (State-owned Forest Farms and Nurseries Station, State Forestry Administration of China, 2016). Camellia oil, obtained from C. oleifera seeds, is rich in unsaturated fatty acids and unique flavors, and has become a rising high-quality edible vegetable oil in China [2]. Thus, the development of the C. oleifera industry is of great significance for the national economy and poverty alleviation of local farmers in China.
The expanding cultivation of C. oleifera over the last several decades has also attracted increasing attention from plant pathologists to infectious diseases on this crop. Anthracnose disease caused by Colletotrichum species is one of the foremost diseases in southern China, which can infect leaves and fruits of C. oleifera, causing up to a 40% fruit drop and up to 40% camellia seeds loss [3]. Several studies have focused on the diversity and the pathogenicity of fungi in this special habitat [3,4,5]. However, relatively little is known about the taxonomy, genetic diversity, and pathogenicity of pestalotioid species on C. oleifera.
Pestalotioid species represent a cosmopolitan group of fungi occupying diverse ecological behavior as plant pathogens, endophytes, or saprobes, and are widely distributed throughout tropical and temperate regions [6,7,8]. However, species identification in this genus remains a major challenge because of overlapping conidial measurements [6,7,9,10]. Maharachchikumbura et al. [8] segregated Neopestalotiopsis and Pseudopestalotiopsis from Pestalotiopsis, based on conidial pigment color, conidiophores and multi-locus phylogenetic analyses. Neopestalotiopsis can be easily distinguished from Pseudopestalotiopsis and Pestalotiopsis by its versicolorous median cells [8]. Pseudopestalotiopsis differs from Pestalotiopsis by having three darker median cells and knobbed apical appendages [8]. Many novel species were introduced into this group during recent years through a polyphasic approaches together with morphology [11,12,13,14,15,16,17,18,19,20,21]. This study aimed to identify the pestalotioid fungi associated with Camellia oleifera in China based on both morphological characters and molecular phylogeny.

2. Materials and Methods

2.1. Sample Collection and Isolation

The isolates in this study were collected from Camellia oleifera with irregular, brownish-grey lesions on leaves, and accounted for 25% of the surveyed leaves. Samples were obtained from the main tea-oil camellia production fields in Guangdong, Guangxi, Hainan, Hunan, and Jiangsu Provinces in 2020. Small sections (3 × 3 mm) were cut from the margins of infected tissues, and surface-sterilized in 75% ethanol for 30 s, then sterilized in 5% (vol/vol) sodium hypochlorite for 1 min, followed by three rinses with sterilized water and finally dried on sterilized filter paper. The sections were then plated onto PDA plates and incubated at 25 °C. Fungal growth was examined daily for up to 7 d. Isolates were then transferred aseptically to fresh PDA and purified by single-spore culturing. All fungal isolates were placed on PDA slants and stored at 4 °C. Specimens and isolates of the new species have been deposited in the Central South University of Forestry and Technology Culture Collection (CSUFTCC).

2.2. Morphological and Cultural Characterization

Colony characteristics of cultures on potato dextrose agar (PDA) medium were recorded after 7 d incubation at 25 °C. Fungal morphology was recorded from colonies grown in the dark for 14 d at 25 °C on PDA. The morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at ×1000 magnification were determined for each isolate using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature, and illustrations of taxonomic novelties are deposited in MycoBank [22].

2.3. DNA Extraction, PCR Amplification, and Sequencing

Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a CTAB [cetyltrimethylammonium bromide] method [23]. For PCR amplifications of phylogenetic markers, three different primer pairs were used [19]. The PCR conditions were: an initial denaturation step of 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 50 s at 48 °C (ITS), 54 °C (tef-1α), or 55 °C (tub2), and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, Waltham, MA, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

2.4. Phylogenetic Analyses

The quality of our amplified nucleotide sequences was checked and combined by SeqMan v.7.1.0 and reference sequences (Table 1) were retrieved from the National Center for Biotechnology Information (NCBI), according to recent publications of the genus [19,20,21]. Sequences were aligned using MAFFT v. 6 [24] and manually corrected using Bioedit 7.0.9.0 [25]. Phylogenetic analyses were carried out with maximum likelihood analysis (ML), which was performed at the CIPRES web portal [26], 1000 rapid bootstrap replicates were run with GTRGAMMA model of nucleotide evolution. Bayesian inference analysis (BI) was performed in MrBayes v. 3.2.0 [27,28]. The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 [29] under the Akaike Information Criterion. GTR + I model was selected a best-fit model for the ITS (Neopestalotiopsis), HKY + I + G was selected as the best-fit model for the ITS (Pestalotiopsis), GTR + I + G model was selected as the best-fit model for the β-tubulin, HKY + G was selected as the best-fit model for the tef-1α. Phylogenetic trees were viewed in FigTree v1.4. The names of the isolates from the present study are marked in blue in the trees. Maximum likelihood bootstrap support values ≥50% (BT) and Bayesian posterior probabilities ≥0.90 (PP) are given at the nodes, respectively. Alignment and trees were deposited in TreeBASE (submission ID: S29114 and S29115).
Table 1. Isolates and GenBank accession numbers of sequences used in this study.

2.5. Pathogenicity Testing

Young and healthy leaves of Camellia oleifera were collected from trees growing in the greenhouse. The leaves were washed with tap water, then submerged in 70% ethanol for 2 min, and finally rinsed in sterilized water twice. The petioles of leaves were wrapped with damp cotton wool and the leaves were placed into petri dishes, three leaves per dish. One piercing wounds of each leaf were made in the mid-region forming a tiny little dot using a sterilized needle. Three drops of 6 μL spore suspension (106 conidia/mL) were individually placed directly onto the leaf upper surfaces. For the control group, 6 μL of sterilized water was used. Each set of three leaves per petri dish was incubated with a different isolate. The petri dishes were placed inside a plastic box and the leaves incubated at 25 °C with humidity and 12/12 h fluorescent light/dark cycle. After 5 d, the leaves were examined for symptom development, and the diameter of diseased spot was measured.

3. Results

3.1. Phylogenetic Analyses

The first sequence datasets for the ITS, tef-1α and tub2, were analyzed in combination to infer the interspecific relationships within Neopestalotiopsis. The combined species phylogeny of the Neopestalotiopsis isolates consisted of 105 sequences, including the outgroup Pestalotiopsis trachicarpicola (culture OP068). A total of 1389 characters including gaps (479 for ITS, 498 for tef-1α, and 412 for tub2) were included in the phylogenetic analysis. Similar tree topologies were obtained by ML and BI methods, and the best scoring ML tree is shown in Figure 1. ML bootstrap values and BI posterior probabilities (MLBS/BIPP) are given at nodes of the phylogram (Figure 1). The phylogenetic tree inferred from the concatenated alignment resolved the ten Neopestalotiopsis isolates from symptomatic leaves of Camellia oleifera into four well-supported monophyletic clades that represent one novel species, one undetermined species and two known species of Neopestalotiopsis (Figure 1).
Figure 1. Phylogram generated from RAxML analysis based on combined ITS, tef-1α and tub2 sequence data of Neopestalotiopsis isolates. The tree was rooted to Pestalotiopsis trachicarpicola (OP068). The scale bar indicates 0.04 nucleotide changes per site. Isolates from this study are marked in red and the identified species is marked in yellow. Ex-type strains are labeled with *.
The second sequence datasets for the ITS, tef-1α and tub2 were analyzed in combination to infer the interspecific relationships within Pestalotiopsis. The combined species phylogeny of the Pestalotiopsis isolates consisted of 129 sequences, including the outgroup Neopestalotiopsis magna (culture MFLUCC 12-652). A total of 1557 characters including gaps (515 for ITS, 537 for tef-1α, and 505 for tub2) were included in the phylogenetic analysis. Similar tree topologies were obtained by ML and BI methods, and the best scoring ML tree is shown in Figure 2. ML bootstrap values and BI posterior probabilities (MLBS/BIPP) are given at nodes of the phylogram (Figure 2). The phylogenetic tree inferred from the concatenated alignment resolved the 12 Pestalotiopsis isolates from symptomatic leaves of Camellia oleifera into four well-supported monophyletic clades that represent four novel species of Pestalotiopsis (Figure 2).
Figure 2. Phylogram generated from RAxML analysis based on combined ITS, tef-1α and tub2 sequence data of Pestalotiopsis isolates. The tree was rooted to Neopestalotiopsis magna (MFLUCC 12-652). The scale bar indicates 0.04 nucleotide changes per site. Isolates from this study are marked in red and the identified species is marked in yellow. Ex-type strains are labeled with *.

3.2. Taxonomy

Neopestalotiopsis camelliae-oleiferae Q. Yang & H. Li, sp. nov. (Figure 3).
Figure 3. Neopestalotiopsis camelliae-oleiferae (CSUFTCC81). (a) Conidioma formed on PDA, (b) conidiogenous cells, and (c–g) conidia. Scale bars: (a) = 1 mm, (b–g) = 10 μm.
MycoBank: MB841476.
Etymology: Named after the host species, Camellia oleifera.
Holotype: CSUFT081.
Description:Conidiomata acervular in culture on PDA, globose, 300–800 μm diam., solitary or aggregated in clusters, exuding black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells ampulliform, hyaline, smooth, annelidic. Conidia fusiform to clavate, straight or slightly curved, 22.5–24(−26.5) × (7–)8.5–10 μm, 4-septate; basal cell conical, 3.5–4.5 μm, hyaline or sometimes pale brown, smooth, thin-walled; with a single appendage filiform, unbranched, centric, (4.5–)6–8(−9) μm long; three median cells doliiform, 14–16(−18) μm long, smooth, versicoloured, septa darker than the rest of the cell (second cell from base pale brown, 4.5–5.5 μm long; third cell medium to dark brown, 5–5.5(−6.5) μm long; fourth cell medium to dark brown, 4.5–6 μm long); apical cell conical, 2.5–4.5 μm long, hyaline, smooth, thin-walled; with 2–3 apical tubular appendages unbranched, filiform, (13.5–)15.5–18.5(−20.5) μm long. Sexual morph not observed.
Culture characteristics: Colonies on PDA reaching 55 mm diameter after seven days at 25 °C. Colonies filamentous to circular, with dense aerial mycelium on surface, fruiting bodies black.
Material examined: CHINA, Jiangsu Province, Nanjing City, from leaf spots of Camellia oleifera, 25 Oct. 2020, H. Li (CSUFT081, holotype); ex-type living culture CSUFTCC81, living culture CSUFTCC82.
Notes:Neopestalotiopsis camelliae-oleiferae was collected from symptomatic leaves of C. oleifera in Jiangsu Province, China. Two isolates (CSUFTCC81 and CSUFTCC82) representing N. camelliae-oleiferae clustered in a well-support clade (ML/BI = 100/1). Neopestalotiopsis camelliae-oleiferae was sister to a clade containing N. longiappendiculata and N. vacciniicola. N. camelliae-oleiferae can be distinguished from N. longiappendiculata based on ITS, tef-1α and tub2 loci (3/449 in ITS, 3/450 in tef-1α , and 6/404 in tub2, no gaps). Morphologically, N. camelliae-oleiferae differs from N. longiappendiculata by wider conidia (8.5–10 vs. 7–7.8 μm); from N. vacciniicola by shorter apical tubular appendages (15.5–18.5 vs. 25.7–30.2 μm) [20]. Therefore, the collection in the present study is designated as a new species.
Neopestalotiopsis cubana Maharachch, K.D. Hyde & Crous, in Maharachchikumbura, Hyde, Groenewald, Xu & Crous, Stud. Mycol. 79: 138 (2014) (Figure 4).
Figure 4. Neopestalotiopsis cubana (CSUFTCC37). (a) Conidiomata formed on PDA, (b) conidiogenous cells, and (c–f) conidia. Scale bars: (a) = 500 μm, (b–f) = 10 μm.
Description:Conidiomata acervular in culture on PDA, globose, 800–1350 μm diam., solitary or aggregated in clusters, exuding black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells ampulliform to cylindrical, hyaline, smooth, annelidic. Conidia fusoid to ellipsoidal, straight or slightly curved, (19.5–)21–25(−26.5) × (5.5–)6.5–8 μm, 4-septate; basal cell conical, 3.5–4.5 μm, hyaline or sometimes pale brown, smooth, thin-walled; with a single appendage filiform, unbranched, centric, 3–5.5 μm long; three median cells doliiform, 13.5–15(−16) μm long, smooth, versicoloured, septa darker than the rest of the cell (second cell from base pale brown, 3.5–5.5 μm long; third cell medium to dark brown, 4–5 μm long; fourth cell medium to dark brown, 3.5–4.5 μm long); apical cell conical, 3.5–4.5 μm long, hyaline, smooth, thin-walled; with 2–3 apical tubular appendages, unbranched, filiform, (21–)24–29(−31) μm long. Sexual morph not observed.
Culture characteristics: Colonies on PDA reaching 70 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, aerial mycelium on surface flat or raised, pycnidia abundant, fruiting bodies black.
Material examined: CHINA, Hainan Province, Chengmai County, from leaf spots of Camellia oleifera, 9 Nov. 2020, H. Li (CSUFT042); living cultures CSUFTCC37 and CSUFTCC42.
Notes:Neopestalotiopsis cubana was originally described from leaf litter in Cuba [8]. In the present study, two isolates from leaves of symptomatic C. oleifera were congruent with N. cubana based on morphology and DNA sequences data (Figure 1). We therefore describe N. cubana as a known species for this clade.
Neopestalotiopsis iberica E. Diogo, M.H. Bragança & A.J.L. Phillips, in Diogo, Gonçalves, Silva, Valente, Bragança & Phillips, Mycol. Progr. 20(11): 1449 (2021) (Figure 5).
Figure 5. Neopestalotiopsis iberica (CSUFTCC91). (a) Conidiomata formed on PDA, (b) conidiogenous cells, and (c–f) conidia. Scale bars: (a) = 1 mm, (b–f) = 10 μm.
Description:Conidiomata acervular in culture on PDA, globose, 600–1500 μm diameter, solitary or aggregated in clusters, exuding black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells ampulliform, hyaline, smooth, annelidic. Conidia fusiform to ellipsoidal, straight or slightly curved, (21.5–)22.5–24(−26.5) × 7–9(−10.5) μm, 4-septate; basal cell conical, 3.5–4.5 μm, hyaline or sometimes pale brown, smooth, thin-walled; with a single appendage filiform, unbranched, centric, 2.5–4 μm long; three median cells doliiform, 12.5–14.5(−15.5) μm long, smooth, versicoloured, septa darker than the rest of the cell (second cell from base pale brown, 4.5–5 μm long; third cell medium to dark brown, 4.5–5.5(−6) μm long; fourth cell medium to dark brown, 4.5–5.5 μm long); apical cell conical, 2.5–4 μm long, hyaline, smooth, thin-walled; with 2–3 apical tubular appendages, unbranched, filiform, 24–26(−29.5) μm long. Sexual morph not observed.
Culture characteristics: Colonies on PDA reaching 70 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, aerial mycelium on surface flat or raised, with filiform margin, fluffy, fruiting bodies black.
Material examined: CHINA, Jiangsu Province, Nanjing City, from leaf spots of Camellia oleifera, 25 Oct. 2020, H. Li (CSUFT091); living cultures LHNJ91, LHNJ92, and LHNJ93.
Notes:Neopestalotiopsis iberica was originally described from leaves and stems of Eucalyptus globulus in Portugal [30]. In the present study, three isolates from leaves of symptomatic C. oleifera were congruent with N. iberica based on morphology and DNA sequences data (Figure 1). We therefore describe N. iberica as a known species for this clade.
Pestalotiopsis camelliae-oleiferae Q. Yang & H. Li, sp. nov. (Figure 6).
Figure 6. Pestalotiopsis camelliae-oleiferae (CSUFTCC08). (a) Conidioma formed on PDA, (b) conidiogenous cells, and (c–g) conidia. Scale bars: (a) = 1 mm, (b–g) = 10 μm.
MycoBank: MB841478.
Etymology: Named after the host species, Camellia oleifera.
Holotype: CSUFT008.
Description:Conidiomata acervular in culture on PDA, globose, 1.0–2.6 mm diameter, solitary or aggregated in clusters, exuding black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells discrete or integrated, cylindrical to subcylindrical, hyaline, smooth. Conidia fusoid, ellipsoid, straight or slightly curved, (19.5–)21.5–23(−25) × (5–)6–7 μm, 4-septate; basal cell conic to obconic with a truncate base, 3.5–5.5 μm, hyaline, smooth, thin-walled; with a single appendage filiform, unbranched, centric, 2.5–4.5 μm long; three median cells doliiform, 12.5–14 μm long, smooth, concolorous, brown, septa darker than the rest of the cell (second cell from base 4–4.5 μm long; third cell 4.5–5 μm long; fourth cell 3.5–4.5 μm long); apical cell conical, 2.5–4(−4.5) μm long, hyaline, smooth, thin-walled; with 2–3 apical tubular appendages, unbranched, filiform, (11–)12.5–14.5(−16) μm long. Sexual morph not observed.
Culture characteristics: Colonies on PDA reaching 70 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, with white sparse mycelium, fruiting bodies black.
Material examined: CHINA, Hunan Province, Changsha City, from leaf spots of Camellia oleifera, 30 Aug. 2020, H. Li (CSUFT008, holotype); ex-type living culture CSUFTCC08, living cultures CSUFTCC09 and CSUFTCC10.
Notes:Pestalotiopsis camelliae-oleiferae was sister to P. biciliata in a well-supported clade (ML/BI = 100/1) (Figure 2). Pestalotiopsis camelliae-oleiferae can be distinguished from P. biciliata based on ITS, tef-1α and tub2 loci (4/500 in ITS, 1/473 intef-1α , and 6/443 in tub2, no gaps). Morphologically, P. camelliae-oleiferae differs from P. biciliata by shorter conidia (21.5–23 vs. 22–28 μm) [8]. Therefore, the collection in the present study is designated as a new species.
Pestalotiopsis hunanensis Q. Yang & H. Li, sp. nov. (Figure 7).
Figure 7. Pestalotiopsis hunanensis (CSUFTCC15). (a) Conidioma formed on PDA, (b) conidiogenous cells, and (c–g) conidia. Scale bars: (a) = 1 mm, (b–g) = 10 μm.
MycoBank: MB841480.
Etymology: In reference to the Hunan Province, from where the fungus was first collected.
Holotype: CSUFT015.
Description:Conidiomata acervular in culture on PDA, globose, 500–1000 μm diameter, solitary or aggregated in clusters, exuding black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells discrete or integrated, cylindrical to subcylindrical, hyaline, smooth, annelidic. Conidia fusoid, ellipsoid, straight or slightly curved, (20.5–)23–25(−26.5) × (7–)9–10.5 μm, 4-septate; basal cell conic to obconic with a truncate base, 4–5.5 μm, hyaline, smooth, thin-walled; with a single appendage filiform, unbranched, centric, 3–3.5 μm long; three median cells doliiform, (14–)15–18 μm long, smooth, concolorous, brown, septa darker than the rest of the cell (second cell from base 4–5 μm long; third cell 5–6.5 μm long; fourth cell 4.5–5.5 μm long); apical cell conical, 2.5–3 μm long, hyaline, smooth, thin-walled; with 2–3 apical tubular appendages, unbranched, filiform, (13.5–)15–22(−26.5) μm long. Sexual morph not observed.
Culture characteristics: Colonies on PDA reaching 50 mm diameter after seven days at 25 °C. Colonies filamentous to circular, with sparse aerial mycelium, fruiting bodies black.
Material examined: CHINA, Hunan Province, Xiangtan City, from leaf spots of Camellia oleifera, 7 Nov. 2020, H. Li (CSUFT015, holotype); ex-type living culture CSUFTCC15, living cultures CSUFTCC18 and CSUFTCC19.
Notes:Pestalotiopsis hunanensis was sister to P. rosae in a well-supported clade (ML/BI = 100/1) (Figure 2). Pestalotiopsis hunanensis can be distinguished from P. rosea based on ITS, tef-1α and tub2 loci (6/501 in ITS, 13/475 in tef-1α, and 7/446 in tub2, 12 gaps). Morphologically, P. hunanensis differs from P. rosae by lager conidia (23–25 × 9–10.5 vs. 17.5–21.8 × 5.7–7 μm) [6]. Therefore, the collection in the present study is designated as a new species.
Pestalotiopsis nanjingensis Q. Yang & H. Li, sp. nov. (Figure 8).
Figure 8. Pestalotiopsis nanjingensis (CSUFTCC16). (a) Conidioma formed on PDA, (b) conidiogenous cells, and (c–f) conidia. Scale bars: (a) = 1 mm, (b–f) = 10 μm.
MycoBank: MB841481.
Etymology: In reference to the Nanjing City, from where the fungus was first collected.
Holotype: CSUFT016.
Description:Conidiomata acervular in culture on PDA, globose, 1000–1600 μm diameter, solitary or aggregated in clusters, exuding black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells discrete or integrated, cylindrical to subcylindrical, hyaline, smooth, annelidic. Conidia fusoid, ellipsoid, straight or slightly curved, (19.5–)22–25 × (4.5–)5–6.5 μm, 4-septate; basal cell conic to obconic with a truncate base, 4.5–5 μm, hyaline, smooth, thin-walled; with a single appendage filiform, unbranched, centric, 2.5–3.5 μm long; three median cells doliiform, 13–14.5(−16) μm long, smooth, concolorous, brown, septa darker than the rest of the cell (second cell from base 4.5–5.5 μm long; third cell 4.5–5.5 μm long; fourth cell 3.5–4.5 μm long); apical cell conical, 3.5–4 μm long, hyaline, smooth, thin-walled; with two apical tubular appendages, unbranched, filiform, (11–)13.5–18(−20) μm long. Sexual morph not observed.
Culture characteristics: Colonies on PDA reaching 60 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, aerial mycelium on surface flat, fruiting bodies black.
Material examined: CHINA, Jiangsu Province, Nanjing city, from leaf spots of Camellia oleifera, 25 Oct. 2020, H. Li (CSUFT016, holotype); ex-type living culture CSUFTCC 16, living cultures CSUFTCC04 and CSUFTCC20.
Notes:Pestalotiopsis nanjingensis was sister to P. neolitseae in a well-supported clade (ML/BI = 100/1) (Figure 2). Pestalotiopsis nanjingensis can be distinguished from P. neolitseae based on ITS, tef-1α and tub2 loci (2/500 in ITS, 26/472 in tef-1α, and 2/442 in tub2, 5 gaps). Morphologically, P. nanjingensis differs from P. neolitseae by longer conidia (22–25 vs. 18–21 μm) and apical appendages (13.5–18 vs. 10–15 μm) [15]. Therefore, the collection in the present study is designated as a new species.
Pestalotiopsis nanningensis Q. Yang & H. Li, sp. nov. (Figure 9).
Figure 9. Pestalotiopsis nanningensis (CSUFTCC10). (a) Conidioma formed on PDA, (b) conidiogenous cells, and (c–f) conidia. Scale bars: (a) = 500 μm, (b–f) = 10 μm.
MycoBank: MB841479.
Etymology: In reference to the Nanning City, from where the fungus was first collected.
Holotype: CSUFT011.
Description:Conidiomata acervular in culture on PDA, globose, 750–1200 μm diameter, solitary or aggregated in clusters, exuding black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells discrete or integrated, cylindrical to subcylindrical, hyaline, smooth, annelidic. Conidia fusoid, ellipsoid, straight or slightly curved, (22–)24–26.5 × (6–)7–8(−9) μm, 4-septate; basal cell conical, 4.5–6 μm, hyaline, smooth, thin-walled; with a single appendage filiform, unbranched, centric, 4.5–6.5 μm long; three median cells doliiform, 13.5–15(−17) μm long, smooth, concolorous, brown, septa darker than the rest of the cell (second cell from base 4.5–5.5 μm long; third cell 5–6 μm long; fourth cell 4–5 μm long); apical cell conical, 3.5–4.5 μm long, hyaline, smooth, thin-walled; with 2–3 apical tubular appendages, unbranched, filiform, (13.5–)18–22.5(−26.5) μm long. Sexual morph not observed.
Culture characteristics: Colonies on PDA reaching 80 mm diameter after seven days at 25 °C. Colonies filamentous to circular, medium dense, white aerial mycelium on surface flat or raised.
Material examined: CHINA, Guangxi Province, Nanning City, from leaf spots of Camellia oleifera, 20 Oct. 2020, H. Li (CSUFT011, holotype); ex-type living culture CSUFTCC11, living cultures CSUFTCC12 and CSUFTCC13.
Notes: Pestalotiopsis nanningensis was sister to P. formosana in a well-supported clade (ML/BI = 100/1) (Figure 2). Pestalotiopsis nanningensis can be distinguished from P. formosana based on ITS and tef-1α loci (4/500 in ITS, 2/472 in tef-1α , and 1/442 in tub2, no gaps). Morphologically, P. nanningensis differs from P. formosana by lager conidia (24–26.5 × 7–8 vs. 18–22 × 6–7 μm) and longer apical appendages (18–22.5 vs. 11–16 μm) [15]. Therefore, the collection in the present study is designated as a new species.

3.3. Pathogenicity Assay

After five days, for the pathogenicity tests, N. camelliae-oleiferae, N. cubana, N. iberica Neopestalotiopsis sp.1, P. camelliae-oleiferae, P. hunanensis , and P. nanningensis developed brown lesions on wounded leaves (right), whereas the controls showed no symptoms (left). Neopestalotiopsis sp.1 had the highest virulence, while P. nanjingensis did not cause obvious symptoms (Figure 10). Koch’s postulates were fulfilled by reisolating the same fungi and verifying its colony and morphological characters.
Figure 10. Pathogenicity of eight pestalotioid species from tea-oil leaves. (a) Induced symptoms on tea-oil leaves after 5 days. (b). The virulence of the isolates was evaluated by measuring the diameters of the necrotic lesions on infected tea-oil leaves 5 days after wounding.

4. Discussion

In this study, an investigation of C. oleifera diseases in China was carried out and Camellia leaf disease caused by pestalotioid fungi was observed as a common disease. Identification of our collections was conducted, based on isolates from symptomatic leaves of C. oleifera using three combined loci (ITS, tef-1α and tub2), as well as morphological characteristics. It includes N. cubana, N. iberica, as well as five new species named N. camelliae-oleiferae, P. camelliae-oleiferae, P. hunanensis, P. nanjingensis, and P. nanningensis.
The expanding cultivation of C. oleifera over the last several decades has attracted increasing attention from plant pathologists to infectious diseases on this crop. Therein, pestalotioid species are more frequently regarded as endophytes or latent pathogens causing diseases only on specific situations [4,6,12,63,64]. Understanding the diversity of pestalotioid species and the genetic variation within pathogen populations could help in developing sustainable disease management strategies.
Pestalotioid fungi (Pestalotiopsidaceae, Sordariomycetes) are species-rich asexual taxa, which are common pathogens that cause a variety of diseases, including leaf spots, shoot dieback, fruit rots and various post-harvest diseases [6,8,15,19,20,46,65]. As many peatalotioid species have overlapping morphological traits, sequence data is essential to resolve these three genera and introduce new species [8]. Combined gene sequence of ITS, tef-1α, and tub2 can provide a better resolution for Pestalotiopsis and Pseudopestalotiopsis. However, more genes are needed to provide better resolution and support in Neopestalotiopsis. Furthermore, this is the first systematic report of Neopestalotiopsis and Pestalotiopsis fungi associated with Camellia oleifera in China, which indicates that there may be a high undescribed diversity of fungi in this host.
Pathogenicity tests of eight pestalotioid species from Camellia oleifera showed that all species except for P. nanjingensis were capable of infecting wounded leaves. Neopestalotiopsis sp.1 and P. camelliae-oleiferae showed stronger virulence, with lesion diameters ranged from 14.7 to 17.8 mm on leaves of the Neopestalotiopsis sp.1 isolate (CSUFTCC61) and 13.5 to 15.5 mm on leaves of the P. camelliae-oleiferae isolate (CSUFTCC08). All pathogenicity tests were performed with a single C. camellia cultivar. Since different C. oleifera cultivars may have different resistance to pestalotioid species, more cultivars of C. oleifera should be studied for the variation of their resistance to pestalotioid pathogens. During the tests, the symptoms vary considerably with factors, such as relative humidity, temperature, and the inoculum concentration. In the future, field conditions with natural inoculum should be conducted rather than just in vitro artificial inoculation.

5. Conclusions

Seven peatalotioid species (two known species and five new species) were described and illustrated. This is the first systematic report of Neopestalotiopsis and Pestalotiopsis fungi associated with Camellia oleifera in China. The pathogenicity of these species on leaves were examined and showed that there were significant differences in the pathogenicity.

Author Contributions

Experiments, L.L.; Writing—original draft preparation, Q.Y.; Writing—review and editing, Q.Y. and H.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the introduction of talent research start-up fund project of CSUFT, grant number 2019YJ025 and the Research Foundation of Education Bureau of Hunan Province, grant number 19B608.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.

Acknowledgments

We are grateful for the assistance of Yuanhao He and Linxue Cao.

Conflicts of Interest

The authors declare no conflict of interest.

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