Nothophoma spp. causing leaf blight of ancient Platycladus orientalis

Abstract

Ancient Platycladus orientalis holds significant ecological, landscape, historical, and cultural value. In northern China, leaf blight has significantly impacted the growth and ornamental value of ancient P. orientalis. In this study, 26 blight leaf samples of ancient P. orientalis were collected in Beijing, China. Phylogenetic analysis based on the concatenated internal transcribed spacer (ITS), β-tubulin (tub2), and RNA polymerase second largest subunit (rpb2) DNA sequence data combined with fungal morphological characteristics revealed three taxa of Nothophoma, i.e. N. platycladus, N. spiraeae, and N. juglandis. Of which, N. platycladus is a species new to science. Koch’s postulates indicated that all these three species of Nothophoma could cause leaf blight of P. orientalis.

Background

Ancient trees are ecologically significant groups that play important roles in supporting the natural community structure and dynamics (Liu et al. 2019). They can enhance ecosystem services, including maintaining water balance, promoting carbon sequestration, and improving air quality as well as having high landscape, historical and cultural value (Blicharska and Mikusiński 2014; Lindenmayer and Laurance 2017; Wan et al. 2020). The Platycladus orientalis (Platycladus, Cupressaceae), an evergreen tree with significant medicinal value, is native to China and North Korea (Li et al. 2023). There are 39,408 ancient trees in Beijing, with ancient P. orientalis accounting for 19% (Chen 2010). The ancient P. orientalis, symbolizing auspiciousness and longevity due to its robust vitality and long lifespan, is commonly found in prominent historical and cultural sites such as palaces, parks, and temples in China (Huang et al. 2021).

Leaf blight is the most common disease of P. orientalis in China, significantly impacting plant growth and landscape aesthetics (Zheng et al. 2022). Early to 1922, P. orientalis leaf blight caused by Alternaria pruni was identified in China (Zhu 1922). Subsequently, Keithia thujina was recognized as the causal agent of the P. orientalis leaf blight in Xiangshan Park, Beijing (Zhao 1978). Monochaetia sp. was reported to cause P. orientalis leaf blight in Hanwangshan Forest Farm, Yang County, Shaanxi Province (Jiang et al. 1984). Dai et al. (1992) discovered that Chloroscypha platycladus caused leaf blight of P. orientalis in the hilly areas of southern China. The study on leaf blight of ancient P. orientalis in Mausoleum of Yellow Emperor of Shaanxi showed that Alternaria alternata and Pestalotiopsis paeoniicola were the causal agent in this area (Li et al. 2021). Due to the over-mature stage of ancient trees, their physiological functions decline, stress resistance decreases, and the probability of disease infection significantly increases (Chen 2014). This phenomenon is particularly noticeable in ancient P. orientalis. However, the study on ancient P. orientalis disease is still quite scarce.

We recently investigated the diseases of ancient P. orientalis. During the investigation of the ancient P. orientalis in Beijing, approximately half of the plants were suffered from leaf blight. In this study, blight leaves of ancient P. orientalis were collected from four scenic areas in Beijing, and 20 fungal isolates were obtained. The aim of this study is to 1) identify the fungal isolates based on morphological characteristics and multigene phylogenetic analysis, and 2) evaluate their pathogenicity by applying Koch’s postulates.

Results

Phylogenetic analysis

The isolates of Nothophoma (including seven isolates: CGMCC3.27066, CGMCC3.27067, CGMCC3.27068, CGMCC3.27069, CGMCC3.27070, CGMCC3.27071, and CGMCC3.27072) from ancient P. orientalis blight leaves were identified as a new species and two known species based on an analysis of concatenated ITS, tub2, and rpb2 sequence dataset composed of 39 isolates of Nothophoma species and Phoma herbarum (CBS 615.75) as an outgroup taxon (Table 1). A total of 1491 characters with 179 parsimony-informative characters were obtained in the phylogenetic analysis. The heuristic search with random addition of taxa (1000 replicates) generated 5000 most parsimonious trees (Length = 492, Consistency index = 0.628, Retention index = 0.781, Rescaled consistency = 0.491, and Homoplasy index = 0.372). Through three analyses (Maximum likelihood ML, Bayesian inference BI, and Maximum parsimony MP), this new species (the strains CGMCC3.27069, CGMCC3.27070, and CGMCC3.27071) was introduced based on the molecular data, indicating that it is a distinct clade with well support (ML/BI/MP = 83/1/82) and it is closely related to Nothophoma pruni (Fig. 1). The strains CGMCC3.27072 and CGMCC3.27066 were co-clustered into the clade of Nothophoma spiraeae according to the multi-locus phylogeny. The strains CGMCC3.27067 and CGMCC3.27068 belong to Nothophoma juglandis by the multi-locus phylogeny assays. Furthermore, only the tree shape of ML is presented here, with ML, BI, and MP values plotted on the branches.

Table 1 Isolates and GenBank accession numbers used in this study^a

Fig. 1

Phylogenetic tree of Maximum Likelihood analyses of Nothophoma based on combined ITS, tub2, and rpb2 genes. Designated outgroup taxa is Phoma herbarum (CBS 615.75). RAxML bootstrap support values (ML ≥ 75%), Bayesian posterior probability (PP ≥ 0.95), and Maximum parsimony bootstrap support values (MP ≥ 75%) are shown in nodes (ML/PP/MP). The scale bar shows 0.02 changes

Taxonomy

Nothophoma platycladus Y. Zhang ter & N. Jiao, sp. nov. (Fig. 2).

Fig. 2

Morphology of Nothophoma platycladus (CGMCC3.27070). a, b Colonies and reverse sides after seven days incubation on MEA. c, d Pycnidia were produced on the colony surface. e Conidiogenous cells. f, g Released conidia. Scale bars: 650 μm for c; Scale bars: 285 μm for d; Scale bars: 10 μm for e–g

Mycobank No: 853425.

Etymology: In reference to the genus of the host name, Platycladus.

Description: Sexual morph was not observed. Asexual morph was developed on malt extract agar (MEA). Conidiomata pycnidial, semi-immersed or superficial on the agar surface, spherical to irregular, mostly aggregated, solitary or confluent, glabrous, thick-walled, with hypha around the pycnidial, 301–545 × 236–456 μm (mean SD = 438 ± 84 × 331 ± 66 μm, n = 20), dark brown, gradually with age turning black, eventually forming small black spots (Fig. 2c). Ostioles single, central, papillate or elongated to a neck, aperture 43–74 μm (mean SD = 56 ± 14 μm, n = 20) (Fig. 2d). Pycnidial wall pseudoparenchymatous, composed of isodiametric cells, 3–6 layers, 22–46 μm thick, outer wall 2–3 layers, and pigmented. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to doliiform, 4.0–6.5 × 2.5–4.5 μm (mean SD = 5.0 ± 1.5 × 3.5 ± 1.0 μm, n = 20) (Fig. 2e). Conidia hyaline but incidentally olivaceous buff, ovoid or ellipsoidal, smooth and thin-walled, aseptate, 4.7–6.4 × 2.8–4.3 μm (mean SD = 5.2 ± 0.5 × 3.7 ± 0.4 μm, n = 50), sometimes with some very small guttules (Fig. 2f, g).

Culture characteristics: Colonies on MEA, 42 to 48 mm in diameter after 7 days at 26°C, colony edges are regular, nearly round, aerial mycelium flat, dark brown to black in center, and have white mycelium on the edges, reversing pale black in center, and darkened gradually after 10 days. Colonies were dense and fluffy, with abundant pycnidia, irregular distribution on the surface of the medium, which produce conidial matrix drop (Fig. 2a, b).

Additional specimens examined: China, Beijing, Chaoyang District, Ritan Park, from blight leaf of ancient Platycladus orientalis, 20 September 2023, Y. Zhang and N. Jiao (holotype HMAS 352975; ex-type living culture CGMCC3.27069). China, Beijing, Chaoyang District, Ritan Park, from blight leaf of ancient P. orientalis, 20 September 2023, Y. Zhang and N. Jiao (Paratype HMAS 352976; living culture CGMCC3.27070). China, Beijing, Xicheng District, Beihai Park, from blight leaf of ancient P. orientalis, 25 September 2023, Y. Zhang and N. Jiao (Paratype HMAS 352977; living culture CGMCC3.27071).

Notes: On the phylogram, Nothophoma platycladus is closely related but sibling to Nothophoma brennandiae, Nothophoma spiraeae, Nothophoma quercina, Nothophoma juglandis, and Nothophoma pruni (Fig. 1). Morphologically, the conidia of N. platycladus have a darker color and smaller aspect ratio compared to those of N. pruni (5.2 × 3.7; L/W = 1.4 vs. 6.0 × 3.3; L/W = 1.8 μm) (Fig. 2f, g). Morphological characteristics and multi-locus phylogenetic analysis indicated that N. platycladus is a novel species of Nothophoma. Nothophoma multilocularis without available DNA sequence was eliminated from the phylogenetic tree (http://www.indexfungorum.org.asp, April 11, 2024). N. platycladus can be clearly distinguished from Nothophoma multilocularis in terms of the conidium dimension (4.7–6.4 × 2.7–4.3 vs. 9–20 × 3–4 µm) (Fig. 2f, g) (Abdel-Wahab et al. 2017).

Nothophoma spiraeae L. X. Zhang, T. Yin, M. Pan, C. M. Tian, and X. L. Fan, Phytotaxa 430:150, (2020) (Fig. 3).

Fig. 3

Morphology of Nothophoma spiraeae (CGMCC3.27072). a, b Colonies and reverse sides after seven days incubation on MEA. c, d Pycnidia produced on the colony surface. e Conidiogenous cells. f, g Released conidia. Scale bars: 650 μm for c, d; Scale bars: 10 μm for e–g

Description: Sexual morph was not observed. Asexual morph: Pycnidia is solitary or aggregated, globose to subglobose, glabrous, olivaceous buff, superficial on or semi-immersed in agar (Fig. 3a, b), Conidia are hyaline, occasionally olive, ovoid, smooth, thin-walled, aseptate, 5.0–7.5 × 2.8–4.8 μm (mean SD = 6.3 ± 1.0 × 3.9 ± 0.5 μm, n = 50) (Fig. 3f, g).

Culture characteristics: Colonies on MEA, 52 to 59 mm in diameter after 7 days at 26°C, the cultures initially appeared hazel and flat, with a thick texture in the center and a thin texture surrounding it after 3 days. Of 7 to 10 days, they were gradually darkened. The colonies were dense and fluffy, showing abundant pycnidia irregularly distributed on the medium's surface, along with the production of a creamy white conidial matrix drop (Fig. 3a, b).

Additional specimens examined: China, Beijing, Dongcheng District, Zhongshan Park, from blight leaf of ancient Platycladus orientalis, 17 September 2023, Y. Zhang and N. Jiao (ZSCB7.1; living culture CGMCC3.27072). China, Beijing, Changping District, Ming Tombs, from blight leaf of ancient P. orientalis, 26 September 2023, Y. Zhang and N. Jiao (CLCB5.3; living culture CGMCC3.27066).

Notes: Nothophoma spiraeae was first reported as a plant pathogen causing canker disease from Spiraea salicifolia in Beijing, China (Zhang et al. 2020). In this study, two strains (CGMCC3.27066 and CGMCC3.27072) are clustered to the N. spiraeae clade in the combined phylogenetic tree (Fig. 1). Morphologically, the strains were similar to N. spiraeae revealed by conidia morphology (5.0–7.5 × 2.8–4.8 vs. 5.0–6.5 × 3.5–4.0 μm) (Fig. 3f, g). Therefore, we defined the isolated strains as N. spiraeae.

Nothophoma juglandis L. L. Zhao, W. Sun, L. Zhang, Y. Q. Yin, Y. Q. Xie, and Y. Zhang, Plant Disease, (2024) (Fig. 4).

Fig. 4

Morphology of Nothophoma juglandis (CGMCC3.27068). a, b Colonies and reverse sides after seven days incubation on MEA. c, d Pycnidia produced on the colony surface. e Conidiogenous cells. f, g Released conidia. Scale bars: 650 μm for c, d; Scale bars: 10 μm for e–g

Description: Conidiomata was pycnidial, semi-immersed or superficial on the agar surface, mostly aggregated, solitary or confluent. Pycnidia with age become black, appearing as small black spots, mostly globose to irregular, pale brown, dark brown near the ostioles, glabrous, thick-walled (Fig. 4a, b). Conidia are hyaline but incidentally olivaceous buff, ovoid, and blong to ellipsoidal, smooth and thin-walled, aseptate, 4.5–7.5 × 4.0–5.5 μm (mean SD = 5.0 ± 0.4 × 4.5 ± 0.5 μm, n = 50), sometimes with some very small guttules (Fig. 4f, g).

Culture characteristics: Colonies on MEA, 50 to 54 mm in diameter after 7 days at 26°C, margin regular. The aerial mycelium was flat, dark brown to black. The back side was pale brown to black, gradually darkening at 7 to 10 days. The colonies appeared dense and fluffy, with abundant pycnidia and irregular distribution on the medium's surface, producing a conidial matrix drop (Fig. 4a, b).

Additional specimens examined: China, Beijing, Changping District, Ming Tombs, from blight leaf of ancient Platycladus orientalis, 26 September 2023, Y. Zhang and N. Jiao (CLCB2.3; living culture CGMCC3.27067). China, Beijing, Changping District, Ming Tombs, from blight leaf of ancient P. orientalis, 26 September 2023, Y. Zhang and N. Jiao (CLCB2.4; living culture CGMCC3.27068).

Notes: Nothophoma juglandis was first isolated from the branches of heart rot diseased walnut in Beijing, China (Zhao et al., 2024). In this study, two strains (CGMCC3.27067 and CGMCC3.27068) are clustered to the N. juglandis clade in the combined phylogenetic tree (Fig. 1). There is almost no difference in morphology and size of conidia between our strains and type specimen (4.5–7.5 × 4.0–5.5 vs. 5.0–8.0 × 4.0–6.0 μm) (Fig. 4f, g). Therefore, we identified the isolated strains as N. juglandis.

Pathogenicity test

There is no distinction in the symptoms of leaves infected by three Nothophoma species collected in the wild, and all are shown as symptoms in Fig. 5a. Three species of Nothophoma were performed for the pathogenicity testing on P. orientalis leaves. One week later, leaves inoculated with Nothophoma isolates have developed a yellow or brown necrotic blight at the inoculation site, with lesions gradually spreading towards the edge of the leaves (Fig. 5b–h). Pathological symptoms are similar to leaf blight spots collected in the wild, while all controls remained healthy (Fig. 5i, j). The pathogenicity assessment indicated that the length of lesions on leaves inoculated with mycelial plugs was significantly longer than that on un-inoculated leaves, and all tested strains exhibited similar pathogenic levels (Table 2). Koch’s postulates were performed by successful pathogen re-isolation from all the necrotic areas of leaves. The morphology and DNA sequences of these new isolates were consistent with the initial inoculation.

Fig. 5

Pathogenicity test of seven Nothophoma strains on Platycladus orientalis leaves. a (i, ii) Blight leaves collected in the wild. b (I, II)–h (I, II) Symptoms of P. orientalis leaves one week after inoculation with seven strains of Nothophoma (CGMCC3.27069, CGMCC3.27070, CGMCC3.27071, CGMCC3.27072, CGMCC3.27066, CGMCC3.27067, CGMCC3.27068 in order). i–j (I, II) control. Scale bars: 2 mm for I, i; Scale bars: 0.4 mm for ii; Scale bars: 1 mm for II

Table 2 Pathogenicity assessment of seven Nothophoma strains one week after inoculation on Platycladus orientalis leaves

Discussion

The genus Nothophoma was introduced by Chen et al. (2015) based on an independent evolutionary branch formed from five “Phoma” species (Phoma anigozanthi, Phoma arachidis-hypogaeae, Phoma infossa, Phoma quercina, and Phoma gossypiicol) in the Didymellaceae. Morphologically, Nothophoma was characterized that conidiomata pycnidial were immersed to superficial, solitary or confluent, and spherical to irregular. The pycnidial wall pseudoparenchymatous was composed of 3–6-layered isodiametric cells. The conidiogenous cells were phialidic, hyaline, smooth, ampulliform to doliiform, and producing aseptate conidia (Chen et al. 2015). The sub-immersed, solitary conidiomata, phialidicconidiogenous cells are hyaline, smooth, ampulliform to doliiform. The aseptate conidia point all these three isolated species to Nothrophoma.

Some species of Nothophoma have weak host specificity and can infect multiple plants and cause diseases. For example, Nothophoma quercina can cause leaf or branch diseases in various plants, including Anacardiaceae, Fagaceae, Garryaceae, Oleaceae, Rhamnaceae, Rosaceae, Rhamnaceae, and Ulmaceae (Hou et al. 2020; Wang et al. 2023). This species has been reported to cause leaf blight of Magnolia coco, and leaf spot of Phellodendron amurense, Aucuba japonica, and Juglans regia (Jiao et al. 2017; Lv et al. 2020; Zeng et al. 2021; Wang et al. 2023). In addition to angiosperms, N. quercina can also cause leaf blight in gymnosperms. A recent study on the dieback and decline of coniferous in Tbilisi (capital of Georgia) revealed that N. quercina caused leaf blight and branch dieback of local urban coniferous plantations, including P. orientalis (Danelia et al. 2021). This result proves that Nothphoma can infect the plants of Cupressaceae.

Based on the combined morphology with ITS, 18S rDNA, and β-tubulin phylogenetic analysis, Alternaria alternata and Pestalotiopsis paeoniicola were identified as the causal agents of leaf blight on ancient P. orientalis at the Mausoleum of the Yellow Emperor in Shaanxi Province. These pathogens can infect ancient P. orientalis either individually or in combination (Li et al. 2021). The diverse pathogenic fungi that infect the leaves of ancient P. orientalis are varied and different pathogens may act synergistically to produce complex pathogenic mechanisms. Revealing the pathogenic species of ancient P. orientalis leaf blight is a prerequisite for control. In this study, all these three species of Nothophoma, namely N. platycladus, N. spiraeae, and N. juglandis, cause leaf blight of ancient P. orientalis in Beijing, China, which is the first report of a species of Nothophoma causing disease on ancient cypresses.

Conclusion

In this study, we combined morphological and phylogenetic analysis to identify three Nothophoma species isolated from blight leaves of ancient P. orientalis in Beijing, namely N. platycladus, N. spiraeae, and N. juglandis. Pathogenicity testing revealed that all three species are the causal agents of leaf blight, with N. platycladus being recognized as a species new to science. The ancient P. orientalis leaf blight, caused by this three Nothophoma species mentioned above, was reported for the first time globally. Further studies require the collection of a large number of leaf samples to study the diversity of pathogenic fungi that can cause leaf blight of ancient P. orientalis.

Method

Sample collection and fungal isolation

A total of 26 blight leaf samples were collected from four locations in Beijing, China, in September, 2023 (11 samples from Zhongshan Park, 6 samples from Ritan Park, 5 samples from Beihai Park, and 4 samples from the Ming Tombs). Plant tissues (0.5 × 0.5 × 0.2 cm) were cut from the disease-healthy transition zone, then were surface sterilized with 75% ethanol for 30 s, rinsed 3 times with sterile distilled water, dried on sterilized filter paper and incubated on MEA for fungal strains (Soltani and Moghaddam 2014; Zhao et al. 2019). The petri dishes were incubated in the dark at 26°C until fungal colonies were observed. Pure cultures were obtained by hyphal tips from the margin of the suspected Nothophoma colonies, which were subcultured on fresh MEA and maintained at 26°C. A total of 20 Nothophoma strains were obtained. Based on colony features, conidial morphology, and rpb2 gene sequences, seven strains representing different tentative species and different locations were selected for further study (Table 1).

Morphological studies

To evaluate the colony characteristics, mycelial plugs in 8 mm diameter were transferred from the growing edges of 7-day-old colonies in new MEA dishes and incubated at 26°C under dark conditions (Liu et al. 2017). Following a week of incubation, black pycnidia were subsequently observed. Next, 1 mL of sterile water were added onto the face of each plate to release conidia from the pycnidia, and then collected for further measurements (Zou et al. 2021). The color, structure, and size of pycnidia were photographed, and at least 30 conidia were randomly selected for length and width measurement using a microscope (Nikon Eclipse E600) (Zhang et al. 2023). Fungal isolates and specimens were deposited at Beijing Forestry University (BJFU), with duplicates at the China General Microbiological Culture Collection Center (CGMCC) and the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS).

DNA extraction, PCR amplification, and sequencing

DNA was extracted from mycelia grown on MEA plates with CTAB plant genome DNA fast extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing, China). The ITS region was amplified with the primers ITS1 and ITS4 (White et al. 1990), the tub2 region with primers Bt2a and Bt2b (Glass and Donaldson 1995), and the rpb2 region with primers rpb2-5F and rpb2-7cR (Liu et al. 1999). PCR amplification and sequencing followed the protocol of Zhang et al. (2009). PCR amplicons were purified and sequenced at BGI Tech Solutions (Beijing Liuhe) Co., Limited (Beijing, China).

Sequence alignment and phylogenetic analysis

DNA sequences of concatenated ITS, rpb2, and tub2 loci were analyzed to investigate the phylogenetic relationships among Nothophoma species with DNA sequences available from GenBank (http://www.ncbi.nlm.nih.gov/genbank/), as well as the sequences generated herein (Table 1). Multiple sequence alignment was performed by the MAFFT v.7.110 (http://mafft.cbrc.jp/alignment/server/). Ambiguous sequences at the start and the end were deleted and manually adjusted using BioEdit (Dania et al. 2021).

Maximum likelihood, Bayesian inference, and Maximum parsimony are used for the multi-locus analyses. Maximum likelihood analyses were constructed on the RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014) using the GTR + GAMMA model with 1000 bootstrap replicates. Bayesian phylogenetic analysis was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.2.6 (Ronquist et al. 2012). Four MCMC chains were run from random trees for 2,000,000 generations and trees were sampled by each 1000th generation. The first 25% of the trees of MCMC sampling were discarded as burn-in and posterior probabilities (PP) were determined from the remaining trees. Maximum parsimony was conducted with heuristic searches as implemented in PAUP* v. 4.0b10 with the default options method (Swofford 2002). Ambiguous regions in the alignment were excluded and gaps were treated as missing data. Clade stability was evaluated in a bootstrap analysis with 1000 replicates with the Maxtrees set to 1000 and other default parameters implemented in PAUP* (Hillis and Bull 1993). Other measurements calculated parsimony scores including consistency index (CI), retention index (RI), rescaled consistency (RC), and homoplasy index (HI). The phylogenetic trees were plotted using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited using Adobe Illustrator CC2020 (Adobe Systems Inc., USA). New sequences generated in this study were deposited in GenBank (submission ID: 14366474, 2816058, and 2816060).

Pathogenicity test

Three identified species of Nothophoma, including their type and authentic isolates (seven selected isolates in total), were tested for their pathogenicity on detached living leaves of P. orientalis. Fresh and healthy leaves were collected from P. orientalis in Ritan Park on October 18, 2023. For pathogenicity testing, the leaves were washed with sterilized water and then surface sterilized with 75% ethanol for 1 min. Subsequently, mycelial plugs in 5 mm diameter cut from 7-day pure cultures of the isolates grown on MEA, and placed on the micro-wounds of leaves created with a sterile needle. Controls were treated with plugs cut from uninoculated MEA (Gomzhina and Gannibal 2023). Three leaves were included in per group, and the experiments were repeated three times. The inoculated leaves were incubated in sterile petri dishes under light at 12/12 h photoperiod, 26/22°C, as well as 80% relative humidity. Pathogenicity was determined by observing and measuring lesion length of the leaves after one week using stereomicroscope (OLYMPUS SZX7 and Nikon SMZ800N). Fungal isolates were re-isolated from the infected tissue, and morphological characterization and DNA sequence comparisons were conducted to fulfill Koch’s postulates. Mean comparisons were conducted using Tukey’s honest significant difference (HSD) test (α = 0.05) in R (Version 3.2.2, R Inc. Auckland, NZL).