Buddhist pine (Podocarpus macrophyllus [Thunb.] D. Don) is a long-lived tree species that is widely cultivated for landscaping in subtropical regions of China (Luo et al., 2020). In addition to its aesthetic characteristics, Buddhist pine is also recognized for the pharmacological uses of its leaves (Wang et al., 2017). Owing to its ornamental and economic value, Buddhist pine has promising development prospects. However, in recent years, fungal diseases of Buddhist pine have become increasingly serious and restrictive to its cultivation and industrial development.

Pestalotiopsis-like genera form a species-rich fungal group with appendage-bearing conidia in family Sporocadaceae (Chen et al., 2018; Liu et al., 2019). Pestalotiopsis spp. were previously considered opportunistic or weak phytopathogens that could cause little damage to ornamental plants (Pirone, 1978). However, in recent years, abundant studies have confirmed that species of Pestalotiopsis are responsible for diseases such as grey blight, leaf blight, canker, dieback and various postharvest diseases, resulting in a substantial reduction in commercial production (Akinsanmi et al., 2017; Chen et al., 2018; Liu et al., 2017; Maharachchikumbura et al., 2012; Silva et al., 2020).

The classification of Pestalotiopsis species was traditionally limited to a host-based taxonomic system and conidial characters (Hu et al., 2007; Jeewon et al., 2003). However, the morphological features excessively overlapped between species, which restricted research on this type of fungus. Maharachchikumbura et al. (2012) identified the internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF), and β-tubulin (TUB) genes as the best genetic markers for distinguishing taxa in this group; together with conidial morphology, these markers provide a precise tool for distinguishing Pestalotiopsis fungi at the species level (Chen et al., 2018; Maharachchikumbura et al., 2014; Zhang et al., 2021).

In March 2019, during a disease investigation in Quanjiao County, Anhui Province, a severely harmful Podocarpus leaf blight was found, with an incidence rate of over 60%. The purpose of this study was to identify the pathogen causing Podocarpus leaf blight by combining morphological and molecular biological methods.

Buddhist pine leaves with typical symptoms were sampled at Nanping Mountain Forest Park in Quanjiao County (118°16′22"N, 32°5′7"E), Anhui Province, China. Leaf Sects. (4 cm2) were excised from lesion margins, surface sterilized as described by Zheng et al. (2020a), and placed onto potato dextrose agar (PDA) plates amended with 100 μg/ml ampicillin. The samples were then incubated at 25 °C in darkness. The hyphal tip from emerging mycelium was subsequently transferred to a new PDA plate, and pure culture was obtained by monosporic isolation.

Seven single-spore isolates were selected for morphological characterization.

Cultural and conidial characteristics were recorded seven days post inoculation (dpi). The morphological features of conidia were recorded according to the standard described by Maharachchikumbura et al. (2014). Fifty measurements per structure were taken under a ZEISS microscope (Carl Zeiss, Germany) at 100 × magnification.

Total DNA of all isolates was extracted using the cetyltrimethylammonium bromide (CTAB) method according to Wang et al. (2020). DNA concentrations were quantified and manually adjusted to 100 ng/μl for polymerase chain reaction (PCR) using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). Primer pairs ITS1/ITS4 (White et al., 1990), T1 (O'Donnell & Cigelnik, 1997)/Bt-2b (Glass & Donaldson, 1995), and EF1-728F (Carbone & Kohn, 1999)/EF-2 (O'Donnell et al., 1998) were utilized to amplify the ITS region and portions of the TUB and TEF genes, respectively. The PCR assays were run in a volume of 50 μl as described by Zheng et al., (2020a, b). Amplifications were performed on an Eppendorf Nexus Thermal Cycler (Eppendorf) using the following amplification conditions: initial denaturation of 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s; 55 °C for ITS, 55 °C for TUB, and 52 °C for TEF for 30 s; 72 °C for 45 s; and a final extension step at 72 °C for 10 min. Amplicons were subjected to sequencing by Shanghai Jie Li Biological Technology Company (Shanghai, China), using both forward and reverse primers for each locus. All sequences obtained in this study were deposited into GenBank (Table 1).

Table 1 Descriptions and sequence accession numbers used in the phylogenetic study
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Reference Pestalotiopsis spp. Sequences, including type specimens (ex-epitype or ex-holotype culture), were retrieved from GenBank following Liu et al. (2017), Maharachchikumbura et al. (2014) and Tsai et al. (2020), respectively. Neopestalotiopsis saprophytica (MFLUCC 12–0282) was included as the outgroup. Multiple sequence alignments were performed using BioEdit v. 7.0.5 (http://www.mbio.ncsu.edu/bioedit/page2.html) and then checked visually and improved manually to obtain the maximal aligned sets of sequences. Gaps were regarded as a fifth character, which had equal weight in the alignment.

The phylogenetic analysis for the concatenated dataset (ITS + TEF + TUB) were conducted under the maximum likelihood (ML) and Bayesian inference (BI) criteria, using MEGA 7 (Kumar et al., 2016) and MrBayes 3.2.6 (Ronquist et al., 2012), respectively. Maximum likelihood (ML) analyses were conducted using the general time-reversible model with nearest-neighbour interchange (NNI) method, and clade stability was determined from 1,000 bootstrap replicates (Kumar et al., 2016). MEGA v.7 was used to infer the best model of nucleotide substitution according to the corrected Akaike information criterion (AICc) for the ML analyses. For BI analysis, two analyses in parallel were performed with four Markov chains, evaluating 50 million generations, with samples taken every 1000th generation. Posterior probabilities (PP) were calculated after discarding the first 25% of generations as burn-in. Bootstrap values ≥ 70 and posterior probabilities ≥ 0.99 were taken as evidence for support of the branches (Huelsenbeck & Rannala, 2004).

The seven isolates used for morphological characterization were evaluated to determine their pathogenicity.

Two pathogenicity assays of each isolate were conducted using 1-year-old potted seedlings of Buddhist pine with either conidial suspensions or mycelial plugs. All isolates were cultured on PDA at 25 °C as mentioned above, and a spore suspension was obtained by rushing 30-day-old cultures with sterile distilled water and adjusting the concentration to 105 conidia/ml with a haemocytometer.

Prior to inoculation, Buddhist pine leaves were surface-sterilized with alcohol (75%), rinsed in sterile water, and air-dried. One wound was made in the middle of the Buddhist pine leaves by a red-hot needle (insect pin, 0.56 mm in diameter). Subsequently, a mycelial disk (5 mm in diameter) taken from a 4-day-old culture or a 10 μl drop of conidial suspension was placed on each wound. Leaves inoculated with PDA plugs without mycelium or sterile water were employed as controls. The inoculated seedlings were then placed in a climate chamber at 25 °C with high relative humidity (> 90%) under a 12 h photoperiod. Ten leaves per isolate were tested for each treatment (one leaf was randomly selected on each plant). The experiment was repeated twice. The lesion length in each treatment was determined after 20 days of inoculation. Statistical analysis was conducted by one-way variance (ANOVA) and means were compared by using Tukey’s honest significant difference test (P = 0.05) in IBM SPSS Statistics software (V22.0, SPSS Inc., Chicago, IL, USA). The pathogen was reisolated from the inoculated symptomatic leaves, and its identity was confirmed through morphological and molecular tools, as previously described, to fulfil Koch’s postulates.

In March 2019, foliar blight was commonly observed on Buddhist pine at Nanping Mountain Forest Park (Fig. 1). The symptoms of the disease included dark brown lesions on the upper part of leaves at an early stage that later turned grey or brown, with numerous black acervuli scattered on dead tissues (Fig. 1C), and the whole branch was withered in severe cases (Fig. 1).

Fig. 1
figure 1

Symptoms of leaf blight disease on Buddhist pine. A Diseased plant in the field. B Diseased shoots of Buddhist pine. C Acervuli on dead tissues, including a transverse section view. Bar = 200 μm for the middle image, and bar = 20 μm for the right image

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All seven single-spore isolates collected from symptomatic samples exhibited similar morphologies. The colonies were white during early growth and turned a pale honey colour later. Noticeably, the hyphae were flocculent-like with concentric aerial rings in each colony (Fig. 2A). After 2–3 weeks, black fruiting bodies (conidiomata) were present (Fig. 2B). The conidiophores were subcylindrical, hyaline, unbranched and often reduced to conidiogenous cells (Fig. 2C). The conidiogenous cells were hyaline, clavate or subcylindrical, and ampulliform. The conidia were single, fusoid, medium to deep brown, 4-septate, straight to slightly curved, and 19.2–28.2 × 7.1–9.5 μm (av. ± SD = 22.9 ± 1.7 × 7.8 ± 0.6 μm); the basal cell was conic, verruculose, hyaline, and 2.5–5.8 μm long; the three median cells were cylindrical or doliiform, 12.7–17.1 μm (av. ± SD = 14.6 ± 1.0 μm) long, and pale brown to brown; the apical cell was conic, hyaline, and 2.8–4.8 μm long, with 2–3 tubular apical appendages (mostly 3), unbranched, 13.7–28.4 μm (av. ± SD = 19.3 ± 3.1 μm) long; and the basal appendage was centric, unbranched, and 3.5–9.5 μm long (Fig. 2D). These features matched those in previous descriptions of the Pestalotiopsis genus (Liu et al., 2017).

Fig. 2
figure 2

Colony morphology and conidial development of P. lushanensis. A, Top and reverse view of a colony. B, Conidiomata generated on PDA 20 days after incubation. Bar = 500 μm. C, Conidiophore (indicated by an arrow) and conidiogenous cells. Bar = 10 μm. D, Conidia. Bar = 10 μm

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The sequences of the seven isolates and the reference sequences downloaded from GenBank are listed in Table 1. The analysis of the combined dataset (ITS + TEF + TUB) of our isolates compared to sequences of 74 Pestalotiopsis species allowed us to allocate these isolates to molecular groups of known Pestalotiopsis species. The alignment of the three-locus concatenated dataset (ITS + TUB + TEF) consisted of 1445 characters (nucleotides and gaps), with 797 constant sites, 439 parsimony-informative sites and 209 parsimony-uninformative sites. The best-fit model of nucleotide substitution in each dataset was deduced according to AICc (HKY + G + I for ITS, K2 + G + I for TEF, and HKY + G + I for TUB and the concatenated dataset).

The multigene phylogeny revealed that the seven isolates in this study, including the type specimen LC4344, clustered as a strongly supported monophyletic group (94 bootstrap value; 0.999 posterior probabilities) and were clearly distinguished from the other Pestalotiopsis species (Fig. 3). Based on the molecular analysis and morphological characteristics, the seven isolates in this study were identified as Pestalotiopsis lushanensis F. Liu & L. Cai (Liu et al., 2017). P. lushanensis isolate NB3-3 has been deposited in China Forestry Culture Collection Centre (CFCC 55,875).

Fig. 3
figure 3

Phylogenetic tree constructed from the concatenated sequences of the ITS, TUB and TEF regions/genes using the maximum likelihood (ML) and Bayesian inference (BI) method. Thickened branches indicate branches also present in theBayesian tree with > 0.99 posterior probabilities. The ML bootstrap support values (> 70%) are displayed at the nodes. Ex-type or other authoritative specimens are indicated in bold. Neopestalotiopsis saprophytica strain MFLUCC 12–0282 was usedas an outgroup

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The results of the pathogenicity tests showed that all seven P. lushanensis isolates were pathogenic on the leaves of Buddhist pine seedlings, regardless of whether mycelial disks or conidial suspensions were used as inocula (Fig. 4), and the symptoms induced were identical to those that occurred in the field. The disease symptoms began as tiny spots around the inoculated site after three days, and then, these spots enlarged to form extensive areas of necrosis 10 days after inoculation, changing in colour from dark brown to taupe. Finally, black acervuli developed on the surface of these lesions. Although all P. lushanensis isolates were pathogenic to Buddhist pine leaves, they showed some differences in virulence. Isolate NB3-3 exhibited the highest virulence, producing typical lesions 10.0 ± 1.8 mm in length when using mycelial disks as inocula and 8.2 ± 1.9 mm in length when using conidial suspensions as inocula (Table 2). The control seedlings inoculated with sterile water or noncolonized PDA disks remained asymptomatic throughout the experiment (Fig. 4). The fungi were reisolated from inoculated symptomatic leaves at 100% frequency, and their gene sequences and phenotypic features were identical to those originally determined.

Fig. 4
figure 4

Pathogenicity test of P. lushanensis using a mycelial disk or conidial suspension as the inoculum

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Table 2 Pathogenicity of P. lushanensis isolates on Buddhist pine 20 days after inoculation
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Several Pestalotiopsis spp. have been reported to be related to leaf blight disease on Buddhist pine. Among them, Pestalotiopsis podocarpi isolated from diseased Buddhist pine leaves was determined to be the causal agent in Guizhou Province (Sang et al., 2006), and Pestalotiopsis virgamla was reported to cause tip blight disease in Guangxi Province (Su et al., 2015). Based on morphology and virulence tests together with multigene sequence data, this study hasverified that P. lushanensis is capable of causing leaf blight on Buddhist pine in China and will serve as the basis for further studies towards improved management strategies.

Species in the Pestalotiopsis genus have gone largely misidentified when using only phenotypic features or single-gene data, since colony characteristics (e.g., colour, pigment, texture, and growth rate) tend to be variable in Pestalotiopsis spp. (Keith et al., 2006). In addition, the DNA sequences of Pestalotiopsis species deposited in GenBank might have been incorrectly named in the past (Maharachchikumbura et al., 2011). A previous study pointed out that the ITS region is less informative than the TUB gene for distinguishing species in this genus (Hu et al., 2007). Wang et al. (2019), Shu et al. (2020) and Silva et al. (2020) demonstrated that multilocus phylogenetic analysis (ITS + TUB + TEF) provided better species resolution than using either marker alone. In this study, the phylogenetic tree derived from a combination of ITS, TUB and TEF sequences revealed that all seven isolates were clustered in the P. lushanensis clade with a 94 bootstrap support value and 0.999 posterior probabilities.

Regardless of whether a conidial suspension or mycelial disk was used, all seven P. lushanensis isolates were pathogenic to wounded Buddhist pine leaves. In contrast, symptoms were not usually induced on non-wounded plants (data not shown), indicating that wounding may be a necessary condition for the development of symptoms. Similar phenomena were observed in previous studies (Chen et al., 2020; Wang et al., 2019). Pestalotiopsis spp. may invade plants through wounds with the help of insects, mechanical injury or some unknown factors to induce serious diseases (Bai et al., 2015). Further studies are needed to focus on the origin of the causal agent and disease cycle. Developing integrated strategies for disease management in the field is also a top priority.