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Diaporthe species causing shoot dieback of Acer (maple) in Henan Province, China

BMC Microbiology volume 24, Article number: 356 (2024) Cite this article

Abstract

Background

Maple is an important ornamental plant in China. With the increasing use of maple trees in landscaping, a symptom of shoot dieback has been observed in Henan province, China.

Results

In this study, 28 Diaporthe isolates were obtained from symptomatic shoots of maple trees between 2020 and 2023. Phylogenetic analyses based on five loci (ITS, TEF, CAL, HIS and TUB) coupled with morphology of 12 representative isolates identified three known species (D. eres, D. pescicola and D. spinosa) and one new species, namely D. pseudoacerina sp. nov. Koch’s postulates confirmed that all these species were pathogenic. Additionally, D. pseudoacerina was able to infect China wingnut (Pterocarya stenoptera), pear (Pyrus sp.), and black locust (Robinia pseudoacacia). This study marks the first report of Diaporthe spinosa and D. pescicola pathogens infecting maple trees.

Conclusions

These findings enhance the existing knowledge of the taxonomy and host diversity of Diaporthe species as, while also providing valuable information for managing of maple shoot dieback in Henan Province, China.

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Introduction

Maple (Acer species) belong to the Aceraceae family, and is widely distributed throughout the world, primarily native to the northern temperate regions of Asia, Europe, and the Americas. As it has a large crown, beautiful shape, high ornamental value, strong environmental adaptability and rapid growth, it has been widely cultivated in urban greening, especially in northern China. Additionally, some maple species provide benefits for some industries, such as medicine, and food. For example, the boxelder maple (Acer negundo) nectar is a valuable food source for honey bees, and the wood can be used for furniture, while its bark fibers can also be utilized to produce paper [1]. The leaves of the purpleblow maple (A. truncatum) are used to produce health-promoting tea and traditional medicines for cerebrovascular diseases and angina pectoris [2] due to their substantial content of tannins, flavonoids, and chlorogenic acid [3]. However, diseases affecting maple trees have increased significantly. Various symptoms and their corresponding pathogens have been reported, such as Inonotus rickii associate with canker rot [4], Verticillium dahliae [5] and Fusarium solani [6] related to wilt, Rhytisma acerinum [7] and Diaporthe foliicola causing leaf spot [8], Diaporthe eres leading to shoot blight [9].

The genus Diaporthe (Phomopsis), established by Nitschke [10], with Diaporthe eres as the type species. The members of Diaporthe are important endophytes, saprobes and pathogens [11,12,13]. Diaporthe species are widely dispersed and can infect a variety of plant hosts, i.e., pear (Pyrus) [14], apple (Malus) [15], soybean (Glycine max) [16] and ornamental plants like whitebark pine (Pinus bungeana) [17], elderberry (Sambucus williamsii) [18], leading to significant losses. Currently, the taxonomy of Diaporthe species has been mostly resolved by multigene phylogenetic analyses including rDNA internal transcribed spacer (ITS1, 5.8 S, ITS2), translation elongation factor 1α (TEF), β-tubulin (TUB), histone (HIS) and cal-modulin (CAL) gene regions [14, 19]. Dissanayake et al. [20] and Norphanphoun et al. [21] classified Diaporthe into 13 species and 15 species complexes based on the five-locus dataset (ITS, TEF, TUB, CAL, and HIS). Genealogical Concordance Phylogenetic Species Recognition (GCPSR), depends on comparing individual gene genealogies to identify incongruences, and it has proven especially useful in defining species boundaries in fungi that have similar morphologically characteristics [22, 23]. Hilário et al. [24] employed the GCPSR to delineate the species boundaries in the D. eres complex. Monkai et al. [25] utilized pairwise homoplasy index (PHI) analysis to strongly support the establishment of novel species. Accurate identification of Diaporthe species requires phylogeny combined with morphological characteristics [26, 27].

Maple is an important ornamental plant in Henan, China, playing a significant role in urban greening and garden landscape. Despite this, the symptoms of shoot canker and shoot dieback are becoming more severe, and knowledge of the pathogen remains limited. Therefore, the objectives of this study were: (1) to investigate the incidence and symptoms of the disease on maple in Henan Province; (2) to characterize the recovered Diaporthe associated with infected maple exhibiting shoot dieback through morpho-molecular investigations; and (3) to assess the pathogenicity of the species involved and the biological characteristics of novel species.

Materials and methods

Sampling and isolation

From 2020 to 2023, 63 samples with symptoms of shoot blight and dieback were collected from eight regions in Henan Province (including Kaifeng, Luoyang, Nanyang, Pingdingshan, Sanmenxia, Shangqiu, Xinyang, Zhengzhou), China.

Diseased tissue samples from stems, shoots and branches were photographed and recorded. Five tissues (0.5 × 0.5 cm) from the margin of the necrotic lesions were cut. These tissues were surface sterilized for 45 s in sodium hypochlorite solution (NaClO), followed by soaking in 75% ethanol for 45 s, then immersed in sterile water and rinsed three times [28]. Subsequently, the excised tissues were cultured on potato dextrose agar (PDA: extract of 300 g/L boiled potato, 20 g/L glucose monohydrate, 15 g/L agar, and distilled water) in the dark at 25 °C. Pure isolates were obtained by picking the hyphal tips from the margins of the resulting colonies and transferring to new PDA plates, which were then and incubated as previously described. All isolates were preserved in 25% glycerol at − 40 °C for future use. Type specimens of the new species identified in this study were stored in the Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS) and the ex-types were deposited in the China General Microbiological Culture Collection Centre (CGMCC), Beijing, China.

DNA extraction, PCR amplification and sequencing

Using mycelia from 4-day-old cultures, the genomic DNA of each isolate was extracted by cetyltrimethylammonium bromide (CTAB) method [29]. The PCR amplification of partial sequences of the rDNA-ITS region (ITS), β-tubulin protein (TUB), translation elongation factor (TEF), calmodulin (CAL) and histone H3 (HIS) regions was performed using primers ITS1/ITS4 [30], Bt2a/Bt2b [31], EF1-728 F/ EF1-986R [32], CAL-228 F/CAL-737R [32] and CYL-H3F/H3-1b [19, 31], respectively. All amplification reactions were performed at a total volume of 20 µL mixture containing 7 µL ddH2O, 10 µL 2 × NG PCR MasterMix (NG001S, HLingene Co., Ltd., Shanghai, China), 1 µL of each primer (10 µM) and 1 µL DNA template (100 µg/mL). PCR amplification conditions were a pre-denaturation at 95 °C for 5 min; followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at a suitable temperature for 30 s (52 °C for TEF,54 °C for CAL, 56 °C for ITS, 57 °C for HIS and 60 °C for TUB) and extension at 72 °C for 30 s; finally, extension at 72 °C for 10 min. The amplification products were purified and sequenced by Sangon Biotech, Co., Ltd. (Shanghai, China). The acquired sequences were examined using DNAMAN (v. 9.0; Lynnon Biosoft), and deposited to GenBank (Table 1).

Table 1 GenBank accession numbers for partial sequences of genes from Diaporthe species from Henan province included in the phylogenetic analysis
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Phylogenetic analyses

The sequences generated in this study were compared with the nucleotide database of GenBank (NCBI) to determine the closest genetic relationships. Alignments of different gene regions, including sequences obtained from this study (Table 1) and sequences downloaded from GenBank (Table 2) were initially performed using the MAFFT v. 7 online server [33] (http://mafft.cbrc.jp/alignment/server/index.html) and manually adjusted alignment in MEGA v. 7.0 [34].

Table 2 Taxa used in this study and their GenBank accession numbers
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Phylogenetic analyses were conducted based on concatenated sequences of the five loci (ITS, TUB, TEF, CAL and HIS). For the Bayesian inference (BI) analysis, MrModeltest v. 2.3 [35] recommended the best nucleotide substitutions for each partition: SYM + I + G for ITS, HKY + I + G for CAL, GTR + I + G for HIS, HKY + I + G for TEF, and HKY + I + G for TUB. BI analysis was performed using MrBayes v. 3.2.7 [36]. Two analyses of four Markov chain Monte Carlo (MCMC) were run using 7.99 × 106 generation random trees. In the analysis, samples were collected at intervals of 1000 generations. The sampling process was terminated when the mean standard deviation of the splitting frequency dropped below 0.01. To ensure accurate results, the initial 25% of trees generated in each analysis were considered as burn-in and the remaining trees were then summarized to calculate the posterior probability (PP) of each single-family branch. Furthermore, IQtree v. 1.6.8 was used to conduct a maximum-likelihood (ML) analysis. The analysis employed a GTR site substitution model. A bootstrapping (BS) method with 1000 replicates [37] was used to evaluated branch support. The resulting phylogenetic trees were visualized and modified using FigTree v. 1.4.2 [38]. The alignments and phylogenetic trees were deposited in TreeBASE (Study TB2: S31373).

For the phylogenetically closely related but indeterminate species, the sequence analysis was performed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model by the pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. [39]. To determine the recombination of related species in the phylogeny, PHI tests were performed in SplitsTree v. 4 [40] using five locus concatenated dataset. If the pairwise homoplasy index is below 0.05, it indicates significant recombination in the concatenated dataset [41].

Morphological analyses

The representative isolates were transferred to PDA, oat agar medium (OA: extract of 20 g/L boiled Oatmeal, 15 g/L agar, and distilled water), malt extract agar (MEA: 40 g/L Malt extract powder, 15 g/L agar, and distilled water), synthetic low nutrient agar (SNA: extract of 1 g/L Monopotassium phosphate, 1 g/L Potassium nitrate, 0.5 g/L Magnesium sulfate heptahydrateand, 0.5 g/L Potassium chloride, 0.2 g/L Glucose, 0.2 g/L Sucrose, distilled water), fennel rod medium (15 g/L agar, and distilled water, three fennel rods in each plate) [42] and alfalfa rod medium (15 g/L agar, and distilled water, three alfalfa rods in each plate) [43] and incubated at 25 °C with a 14/10 h fluorescent light/dark cycle. Colony colours were rated according to Rayner (1970) and colony characteristics such as texture and density were noted [44]. The growth rate of each isolate was determined, and the colony diameter was measured daily for 2 − 3 d. The shapes, colors and sizes of sporocarps, conidia, conidiophores, were observed and recorded under a compound microscope (Carl Zeiss Ltd.), and the size of 50 conidia was measured.

Pathogenicity and host range

Pathogenicity tests were conducted by inoculating colonized PDA discs of representative isolates on detached 1-year-old twigs of Acer sp. invitro and invivo. For invitro inoculation, one representative isolate for each species was inoculated on plant shoots measuring 10.0 to 15.0 mm in diam, these shoots were surface disinfected with 75% ethanol and wounded between two of the adjacent buds with a punch (5 mm diameter). Mycelial plugs (5 mm diameter) were excised from the colony margins of cultures grown on PDA at 25 °C for 3 days and inoculated into the wound site on each shoot. Non-colonized PDA was used as a negative control. All inoculation sites were covered with strips of plastic film to maintain high humidity and incubated in plastic containers covered with a plastic film at 25 °C for 7–21 d in alternating light and dark at 12/12 h. Branch inoculation was repeated three times for each isolate and the experiment was repeated two times. The lesion length was measured and recorded at 7 d and 14 d. For invivo inoculation, the resulting wounds were inoculated as before, and covered with strips of plastic film to maintain high humidity for 2 d, then the film was removed. Non-colonized PDA plugs were used in parallel as controls. Each isolate was inoculated by three shoots and the entire experiment was repeated twice. After 21 d, the symptoms were observed and recorded. Re-isolation was also conducted from the infected tissue to fulfil Koch’s postulates.

Host ranges of the new species were determined using detached shoots (10–12 cm) of eight horticultural plants (include: camphor tree (Camphora officinarum), hawthorn (Crataegus pinnatifida), Euonymus maackii, oleander (Nerium oleander), China wingnut (Pterocarya stenoptera), pear (Pyrus sp.), black locust (Robinia pseudoacacia), magnolia (Yulania denudata)) as common as maple trees in Henan province. Branches were inoculated as described above and incubated at 25 °C in plastic containers covered with a plastic film. Lesion lengths were measured and photographed at 7 days post inoculation (dpi) with three replicates for each isolate.

Physiological characteristic determination

To evaluate the biological characteristics of the novel species, the optimal temperature, pH, carbon source and nitrogen source was determined. Temperatures ranged from 5 to 40 °C with 5 °C intervals, and pH ranged from 5 to 11 with 1 interval. Sucrose in Czapek medium (1 g/L Dipotassium phosphate, 0.5 g/L magnesium sulfate heptahydrate, 0.5 g/L potassium chloride, 30 g/L sucrose, 3 g/L sodium nitrate, 0.01 g iron (2+) sulfate (anhydrous) heptahydrate, 15 g/L agar, distilled water) was replaced with different carbon sources (glucose, lactose, maltose, fructose and soluble starch) respectively, and sodium nitrate in Czapek medium was replaced with different nitrogen sources (ammonium nitrate, ammonium sulphate, urea, glycine and peptone) respectively.

Statistical analysis

Statistical analysis was performed with SPSS Statistics 21.0 by one-way analysis of variance and means were compared using Duncan ‘s test at a significance level of α = 0.05. (Each dataset comprises three replicates.) Graphs were prepared using Origin 2022 software.

Results

Field surveys and fungal isolation

The symptoms of the disease were consistently observed on 1-year-old shoots. The lesions were initially brown (Fig. 1A) and expanded into a brownish-black (Fig. 1B), tacking on a fusiform shaped or irregular shape. Later, the lesions cracked and produced small black protuberant spots (pycnidia) (Fig. 1C–E). As the lesions continued to spread and merged with the surrounding shoots, the infected shoots or branches along with their attached leaves and buds were killed (Fig. 1F).

Fig. 1
figure 1

Typical canker symptoms on shoots of maple in the field. White arrow points to small black protuberant spots (pycnidia)

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A total of 63 samples (shoots, branches and twigs) were collected from eight cities (including Kaifeng, Luoyang, Nanyang, Pingdingshan, Sanmenxia, Shangqiu, Xinyang and Zhengzhou) of Henan Province (Fig. 2). Twenty-eight isolates with typical morphological characteristics of Diaporthe were recovered. Among them, five isolates were from Pingdingshan, four isolates were from Sanmenxia, three isolates were from Shangqiu, four isolates were from Xinyang, and 12 isolates were from Zhengzhou.

Fig. 2
figure 2

Sketch map of sampling location of stem samples of maple in Henan province, China

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Phylogenetic analysis

Twelve isolates from maple were subjected to multilocus phylogenetic analyses with concatenated ITS, TEF, CAL, HIS and TUB sequences together with 106 reference isolates, with Cytospora disciformis (CBS 116827) being selected as the outgroup (Table 2). In the phylogenetic tree, a total of 1747 characters (368 for ITS, 348 for CAL, 407 for HIS, 278 for TEF and 388 for TUB) including gaps were included in the multilocus dataset. 12 isolates were assigned to two species complexes including D. eres complex (6 isolates) and D. arecae complex (6 isolates) (Fig. 3). In the D. arecae species complex, six isolates clustered into three clades corresponding to D. pescicola (ZZFS7 and ZZFS8), D. spinosa (ZZFS11 and ZZFS12), whereas 2 isolates (XYFS1 and XYFS2) formed distinct clades with a highly supported values (1.00/100), which were identified as novel species and named D. pseudoacerina. In the D. eres species complex, six isolates all clustered together with D. eres species (Fig. 3).

Fig. 3
figure 3

Inference Phylogenetic tree of Diaporthe spp. resulting from Bayesian analyses using the combination of ITS, TEF, CAL, HIS and TUB genes alignments. The species Cytospora disciformis (CBS 116827) was selected as an outgroup. Bootstrap support values ≥ 0.90 for Bayesian posterior probabilities and ≥ 60% for ML were shown at the nodes (BI/ML). The asterisk symbol (*) represents full support (1/100). Ex-type strains were emphasized in bold. Colored blocks indicate clades containing isolates from Acer spp. in this study. The scale bar indicates 0.04 expected changes per site

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The GCPSR concept was used to assess species boundaries. The PHI test revealed no substantial recombination (Φw = 1.0) between D. pseudoacerina (XYFS1 and XYFS2) and its closely related taxon D. acuta (Fig. 4).

Fig. 4
figure 4

The PHI test of D. pseudoacerina with its closely related taxa using both LogDet transformation and splits decomposition

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Taxonomy

Based on the morphology and phylogeny, 12 isolates were assigned to four species, including three known species and one newly described species. All species studied in culture are characterized below.

Diaporthe eres Nitschke, Pyrenomyc. Germ. 2: 245. (1870) (Fig. 5).

Fig. 5
figure 5

Morphology of Diaporthe eres (ZZFS2). (a–b) Front and reverse colony on PDA plate; (c–d) Front and reverse colony on MEA plate; (c–f) Front and reverse colony on OA plate; (g–h) conidiomata; (i–k) alpha conidia; (l) gamma conidia; (m) alpha conidia and gamma conidia; (n–o) alpha conidia and beta conidia. Scale bars: (g–h) = 500 μm, (i–o) = 10 μm

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Materials examined: CHINA, Henan Province, Zhengzhou City, from shoot die-back of Acer sp., 28 April 2022, W. K. Gao, culture ZZFS1- ZZFS6, ZZFS9 and ZZFS10. CHINA, Henan Province, Pingdingshan City, from shoot dieback of Acer sp., 13 May 2023, W. K. Gao, isolate PDSFS1-PDSFS4. CHINA, Henan Province, Sanmenxia City, from branch dieback of Acer sp., 8 July 2023, W. K. Gao, culture SMXFS1-SMXF4. CHINA, Henan Province, Shangqiu City, from shoot dieback of Acer sp., 11 June 2023, W. K. Gao, culture SQFS1 and SQFS2. CHINA, Henan Province, Xinyang City, from shoot dieback of Acer sp., 28 June 2021, W. K. Gao, culture XYFS3 and XYFS4.

Notes: Diaporthe eres (the type species of Diaporthe) was first described from Ulmus sp. in Germany by Nitschke (1870) and was reported from a wide host range and can cause a variety of plant diseases [45]. In this study, 22 isolates were identified as this species. This is a new host record for D. eres from Acer sp. in China. Diaporthe eres is the main pathogen causing the maple shoot dieback based on our results.

Diaporthe pescicola Dissanayake et al., Mycosphere 8: 542. (2017) (Fig. 6).

Fig. 6
figure 6

Morphology of Diaporthe pescicola (ZZFS8). (a–b) Front and reverse colony on PDA plate; (c–d) Front and reverse colony on MEA plate; (e) conidiomata; (f) paraphyses; (g) alpha conidia, beta conidia and gamma conidia; (h) alpha conidia. Scale bars: (e) = 500 μm, (f) = 20 μm, (g–h) = 10 μm

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Materials examined: CHINA, Henan Province, Zhengzhou City, from shoot canker of Acer truncatum, 28 April 2022, W. K. Gao, culture ZZFS7 and ZZFS8, new host record.

Description: Sexual morph: not observed. Asexual morph: observed on PDA. Pycnidial conidiomata irregular, solitary or aggregated, exposed on the PDA surface, dark brown to black, 668–1269 μm diam. Alpha conidia hyaline, aseptate, fusiform, 4.5–8.0 × 1.5–2.5 μm, mean ± SD = 6.2 ± 0.7 × 2.0 ± 0.2 μm, L/W ratio = 3.1 (n = 50). Beta conidia hyaline, aseptate, filiform, flexuous to J-shaped, tapering towards both ends, 16.5–27.0 × 1.0–2.0 μm, mean ± SD = 21.2 ± 3.0 × 1.5 ± 0.2 μm, L/W ratio = 14.1 (n = 50). Gamma conidia hyaline, aseptate, rod, both ends are column, 11.5–12.0 × 1.5–2.0 μm, mean ± SD = 11.7 ± 1.0 × 1.7 ± 0.2 μm, L/W ratio = 6.9 (n = 5).

Culture characteristics: Colonies on PDA with flocculent mycelium, concentric annular distribution, mycelium sparse in the center, reverse ochreous colored. Colony diam 5.43 mm in 3 d at 25 °C. On MEA with flocculent scattered distribution, reverse salmon colored in the center and edge white.

Note: Diaporthe pescicola was first described from diseased shoots of Prunus persica in Hubei province, China [46]. Diaporthe pescicola is related to the leaf spot of Kerria japonica and grapevine dieback in previous reports [47, 48]. In this study, two isolates (ZZFS7, ZZFS8) formed a clade with D. pescicola (MFLUCC 16–0105) with high bootstrap support (ML/BI = 100/1) (Fig. 3). Alpha conidia are smaller (4.5–8.0 × 1.5–2.5 vs. 6–8.5 × 2–3 μm) and beta conidia are shorter (16.5–27.0 × 1.0–2.0 vs. 18–37 × 1–1.5 μm) than those of D. pescicola (MFLUCC 16–0105) [46]. Therefore, we consider our strain (ZZFS7, ZZFS8) as D. pescicola and as a new host record from Acer truncatum in China.

Diaporthe pseudoacerina Y. S, Guo, M. Zhang and W. K. Gao sp. nov. (Fig. 7)

Fig. 7
figure 7

Morphology of Diaporthe pseudoacerina (CGMCC 3.25234, holotype: HMAS 352665). (a–b) Front and reverse colony on PDA plate; (c–d) Front and reverse colony on MEA plate; (e–f) conidiomata; (g) paraphyses; (h) beta conidia; (i–j) alpha conidia; (k) gamma conidia; (l) beta conidia. (m) gamma conidia. Scale bars: (f) = 500 μm, (g–m) = 10 μm

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Mycobank number: MB851338.

Etymology: The name reflects the host (Acer) from which the fungus was isolated.

Holotype: HMAS 352,665.

Materials examined: CHINA, Henan Province, Xinyang City, from shoot canker of Acer buergerianum, 28 June 2021, W. K. Gao, culture ex-type CGMCC3.25234; ibid., culture XYFS1.

Description. Sexual morph not observed. Asexual morph on fennel stems. Pycnidial conidiomata globose, solitary or aggregated, exposed on the alfalfa stems surface, dark brown to black, 289–428 μm diam. Conidiophores 8.1–18.3 × 1.2–2.6 μm, cylindrical, hyaline, densely aggregated, slightly tapering towards the apex, sometimes slightly curved. Conidiogenous cells phialidic, hyaline, terminal cylindrical, 10.3–14.0 × 1.7–3.3 μm, tapered towards the apex. Alpha conidia hyaline, aseptate, fusiform or oval, 5.5–9.0 × 2.0–3.0 μm, mean ± SD = 7.1 ± 0.6 × 2.3 ± 0.2 μm, L/W ratio = 3.1 (n = 50). Beta conidia hyaline, aseptate, crochetage, 22.0–30.0 × 1.0–2.5 μm, mean ± SD = 26.3 ± 6.0 × 1.5 ± 0.3 μm, L/W ratio = 18.2 (n = 50). Gamma conidia hyaline, aseptate, rod, 15.0–17.0 × 1.5, mean ± SD = 15.6 ± 0.8 × 1.4 ± 0.0 μm, L/W ratio = 3.3 (n = 3).

Culture characteristics: Colonies on PDA with flocculent mycelium, concentric annular distribution, mycelium sparse in the center and edge dense. Colony diam 6.07 mm in 3 d at 25 °C. On MEA with concentric wheel pattern distribution, reverse apricot pigment accumulation, edge mycelium sparse.

Notes: Diaporthe pseudoacerina forms an independent clade in the D. arecae species complex and is phylogenetically distinct from D. acuta in a well-supported clade (ML/BI = 100/1) (Fig. 3). Diaporthe pseudoacerina can be distinguished from D. acuta (CGMCC 3.19600) based on the nucleotide differences in ITS (31 out of 546), TEF (3 out of 311), CAL (28 out of 422) and HIS (9 out of 418). Morphologically, D. pseudoacerina shows shorter and narrower alpha conidia (5.5–9.0 × 2.0–3.0 vs. 6–8.5 × 2–3 μm) than those of D. acuta (CGMCC 3.19600). Beta and gamma conidia were observed in D. pseudoacerina, but was not observed in D. acuta (CGMCC 3.19600) [14]. Despite it, a new description of beta conidia and gamma conidia was reported in 2023. Compared to D. acuta, D. pseudoacerina has wider alpha conidia (7.1 ± 0.6 × 2.3 ± 0.2 vs. 6.5 ± 0.6 × 2.2 ± 0.2 μm, L/W ratio = 3.1 (n = 50)), shorter beta conidia (26.3 ± 6.0 × 1.5 ± 0.3 vs. 31.0 ± 3.5 × 1.0 ± 0.1 μm, L/W ratio = 18.2 (n = 50)), and longer gamma conidia (15.6 ± 0.8 × 1.4 ± 0.0 vs. 12.4 ± 1.2 × 1.4 ± 0.1 μm, L/W ratio = 3.3 (n = 3)) [49].

Diaporthe spinosa Guo et al., Persoonia 45: 154 (2020) (Fig. 8).

Fig. 8
figure 8

Morphology of Diaporthe spinosa (ZZFS11). (a–b) Front and reverse colony on PDA plate; (c–d) Front and reverse colony on MEA plate; (e) conidiomata; (f) paraphyses; (g–h) alpha conidia; (i–j) beta conidia; (k) gamma conidia. Scale bars: (e) = 500 μm, (f–k) = 10 μm

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Materials examined: China, Henan Province, Zhengzhou City, from shoot canker of Acer palmatum, 15 April 2022, W. K. Gao, culture ZZFS11 and ZZFS12, new host record.

Description: Sexual morph: not observed. Asexual morph: observed on SNA. Pycnidial conidiomata irregular, solitary, dark brown to black, 670–834 μm diam. Alpha conidia hyaline, aseptate, fusiform or oval, 5.5–8.5 × 2.0–2.5 μm, mean ± SD = 7.0 ± 0.6 × 2.3 ± 0.2 μm, L/W ratio = 3.1 (n = 50). Beta conidia hyaline, aseptate, crochetage, 18.0–31.0 × 1.0–2.0 μm, mean ± SD = 25.2 ± 2.6 × 1.5 ± 0.2 μm, L/W ratio = 17.1 (n = 50). Gamma co-nidia hyaline, aseptate, rod, both ends are fusiform, 11.0–15.5 × 1.5–2.0 μm, mean ± SD = 13.2 ± 1.7 × 1.8 ± 0.1 μm, L/W ratio = 7.4 (n = 5).

Note: Diaporthe spinosa was first reported on branches of Pyrus pyrifolia cv. Cuiguan in China, Jiangsu province, Nanjing city [14]. In this study, two isolates (ZZFS11, ZZFS12) were identified as this species. This is the first report of D. spinosa responsible for maple shoot dieback, and the gamma conidia of D. spinosa were first recorded in this study.

Pathogenicity and host range analysis

One representative isolate from each species (D. eres: ZZFS2, D. spinosa: ZZFS11, D. pescicola: ZZFS7, D. pseudoacerina: XYFS2) was selected for pathogenicity test in vitro and in vivo to prove Koch’s postulates. For in vitro inoculation, all isolates caused brownish-black lesions on the inoculated branches, with no spread lesions in the control group except for scalding (Fig. 9a). The lesion lengths varied significantly among different species, D. spinosa caused largest lesions length (41.7 ± 4.2 mm), and the remaining three species induced smaller lesions length (5.3–6.3 mm) (Fig. 9b).

Fig. 9
figure 9

Symptoms and lesion lengths induced by inoculation of wounded Acer truncatum. shoots in vitro. (a) Inoculation symptom at 7 dpi; (b) Mean lesions lengths from three replicates of branches measured at 7 dpi. Different letters over the bars indicate a significant difference at the α = 0.05 level

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For in vivo inoculation, four isolates started to produce reddish or brown lesions on maple shoots at 7 dpi (Fig. 10a). In the control group, no symptoms were observed. The epidermis around the lesions of D. spinosa and D. pescicola turned yellow at 14 dpi, but the xylem was not infected after the epidermis was cut off. There was no significant difference in lesion length between different isolates, which were 5.0–6.0 mm (Fig. 10b).

Fig. 10
figure 10

Symptoms and lesion lengths induced by inoculation of wounded Acer truncatum shoots in vivo. (a) Inoculation symptom at 21dpi; (b) Mean lesions lengths from three replicates of branches measured at 21 dpi. Different letters over the bars indicate a significant difference at the α = 0.05 level

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Eight common horticultural plants were selected for determine the host range of the new species D. pseudoacerina. At 7 dpi, D. pseudoacerina caused black or brown lesions on China wingnut, pear and black locust, but did not cause lesions on other hosts (Fig. 11a). The pathogenicity of D. pseudoacerina was strongest on China wingnut, significantly higher than on pear and black locust (Fig. 11b).

Fig. 11
figure 11

Symptoms and lesion lengths on wounded eight different horticultural plant shoots at 7 dpi induced by mycelia plugs of Diaporthe pseudoacerina (CGMCC 3.25234). (a) Inoculation symptom at 7 dpi; (b) Mean lesions lengths from three replicates of branches measured at 7 dpi. Different letters over the bars indicate a significant difference at the α = 0.05 level

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Physiological characteristics determination

The physiological characteristics of D. pseudoacerina were measured. The mycelium of D. pseudoacerina could grow on PDA at temperatures ranging from 10 °C to 35 °C, with an optimal temperature of 30 °C (Fig. 12a). The mycelium of D. pseudoacerina could grow on PDA with the pH value ranged from 5 to 11, and there was no significant difference in the mycelial growth rate at different pH (Fig. 12b). On the Czapek medium with six different nitrogen sources, D. pseudoacerina mycelium could grow. The quickest-growing media was the Czapek medium with ammonium sulphate as the nitrogen source, followed by yeast powder, in which urea as the nitrogen source significantly inhibited the growth of D. pseudoacerina (Fig. 12c). On the Czapek medium with six different carbon sources, the fastest growing media was the Czapek medium with soluble starch as the carbon source, followed by glucose, in which maltose as the carbon source significantly inhibited the growth of D. pseudoacerina (Fig. 12d).

Fig. 12
figure 12

Biological characteristics of D. pseudoacerina (CGMCC 3.25234). (a) Growth rate with different temperatures; (b) Growth rate with different pH; (c) Growth rate with different nitrogen source; (d) Growth rate with different carbon source. Different letters over the bars indicate a significant difference at the α = 0.05 level

Full size image

Discussion

Diaporthe species have a wide host range, but there have been few reports on their impact on maple trees. In this study, phylogenetic analyses based on five combined loci (ITS, TEF, CAL, HIS, and TUB), along with morphology and the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) principle revealed four Diaporthe species (D. eres, D. pescicola, D. pseudoacerina and D. spinosa) associated with maple shoot dieback in Henan province, China. Among these, a novel species D. pseudoacerina was described. These species are responsible for maple shoot dieback, confirmed by Koch’s postulates. This is the first report of D. spinosa and D. pescicola infecting maple trees and causing maple shoot dieback.

Diaporthe species are responsible for causing severe diseases such as dieback, leaf spots, fruit rot, seed decay and branch canker [50,51,52,53,54,55,56,57,58,59,60]. Recently, shoot canker has been identified on various horticultural plants, including sunflower [61] and rose [62]. On maple trees, 26 species of Diaporthe have been reported in previous studies.: D. acericola, D. acerigena, D. acerina, D. acuta, D. albocincta, D. aspalathi, D. cercidis, D. congener, D. dubia, D. fallaciosa, D. hypoxyloides, D. hystricula, D. inaequalis, D. microstroma, D. moriokaensis, D. niessliana, D. ontarieusis, D. petrakiana, D. phaseolorum, D. pustulata, D. robusta, D. subaquila, D. subcongrua, D. ukurunduensis, D. varians and D. zopfii [63]. However, only few species have available sequence data for reference, specifically D. acericola, D. acerigena, D. acerina, D. acuta, D. cercidis and D. ukurunduensis. Additionally, there are few studies on the pathogenicity of Diaporthe species on maple trees. It has been reported that Diaporthe eres is related to Japanese maple shoot blight [9]. In this study, another three additional species were included: D. pescicola, D. pseudoacerina and D. spinosa, all of which were associated with maple shoot dieback.

D. eres is the type species of Diaporthe, with a diverse range of hosts and widespread distribution [64] In China, Diaporthe has rich species diversity, among which D. eres is the dominant species with the highest isolation ratio [65]. This study also showed that D. eres (28 isolates, 78.6% of the total isolates) was the dominant species associated with maple shoot dieback. But the pathogenicity of D. eres on maple branches was not as strong as on other host branches [66, 67]. D. eres may have host preference, and studies have reported that D. eres can parasitize in host plants as an endophyte, and did not cause harm to host plants, and may even be used as a biocontrol agent [68].

Different Diaporthe species may be associated with the species of maple and sampled areas. We collected three maple species (Acer buergerianum, Acer pictum and Acer rubrum). D. eres could be isolated from all three species of maple, D. pseudoacerina was only found on Acer buergerianum, D. pescicola was only found on Acer pictum, and D. spinosa was only found on Acer rubrum. The samples came from five cities. Only D. eres can be isolated from the samples of five cities. D. spinosa and D. pescicola were only from Zhengzhou, while D. pseudoacerina was only from Xinyang. This indicates that there are significant differences in host range and regional distribution among different species of Diaporthe.

Diaporthe pseudoacerina, was shown to be distinct from other species in the D. arecae species complex based on its morphology and phylogeny. Diaporthe acuta, which is most closely related to D. pseudoacerina, was first reported on Pyrus pyrifolia and maple in 2023 [49]. There were basepair differences between D. pseudoacerina and D. acuta in several gene regions (Similarity: ITS: 93.7%; TEF: 98.86%; CAL: 93.7%; HIS: 98.0%). We further distinguished between D. pseudoacerina and D. acuta using Genealogical Concordance Phylogenetic Species Recognition (GCPSR) principle. The PHI test revealed no substantial recombination (Φw = 1.0) between D. pseudoacerina (XYFS1 and XYFS2) and its closely related taxon D. acuta. Morphologically, three forms of conidia of D. pseudoacerina were observed. D. acuta only has alpha conidia in 2020 [14], but a new description of beta conidia and gamma conidia was reported in 2023 [49]. Although the taxonomy of Diaporthe is dominated by molecular data, morphology is also indispensable in the identification of fungal species.

Diaporthe are important endophytes, saprobes and pathogens. To verify that the isolates are the pathogen, we determined the pathogenicity of our isolates on 1-year-old twigs of maple in vitro and in vivo. These results showed that they are all pathogenic and responsible for maple shoot dieback by fulfilling Koch’s postulates. It is worth noting that these isolates showed significantly different virulence related to species and host conditions. For example, the virulence of D. spinosa isolates were significantly higher than other species on maple in vitro inoculation, but in vivo inoculation, D. spinosa, like other species, showed weaker pathogenicity with no significant difference between them. Additionally, we determined the host range of the new species D. pseudoacerina. The results showed that D. pseudoacerina can infect horticultural crops of different families, such as China wingnut (Pterocarya stenoptera), pear (Pyrus sp.) and Robinia pseudoacacia. Hence, with the increasing trend of urban greening, Diaporthe species poses a greater threat to horticultural crops. This study elucidated the pathogen of maple shoot dieback in Henan Province and described a new species D. pseudoacerina, which enriched the knowledge of maple shoot dieback and provided guidance for its prevention and control.

Data availability

All data and material are available upon request to correspondence author. The datasets generated or analyzed during the current study are available in the National Center for Biotechnology Information (NCBI) repository (https://www.ncbi.nlm.nih.gov, accessed on 15 November 2023), and were assigned the accession numbers that list in Tables 1 and 2. Additionally, the accession numbers of the taxa newly described in this study (Diaporthe pseudoacerina CGMCC 3.25234: ITS, OR225658; TEF, OR239198; TUB, OR239202; CAL, OR239200; HIS, OR239204 and Diaporthe pseudoacerina XYFS1: ITS, OR225657; TEF, OR239197; TUB, OR239201; CAL, OR239199; HIS, OR239203) are shown in bold in Table 1.

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Funding

This research was funded by Natural Science Foundation of Henan (24230042048), Science and Technology Planning Project of Henan Province of China (242102111081), Henan Agricultural University of Young Talent Program (30500960) and the open fund of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF202103).

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  1. College of Plant Protection, Institute of Fungi, Henan Agricultural University, Zhengzhou, 450002, China

    Wenkai Gao, Jiayuan Cao, Yuxu Xie, Xiuyuan Sun, Qingzhou Ma, Yuehua Geng, Chao Xu, Yashuang Guo & Meng Zhang

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  1. Wenkai Gao
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Contributions

W.K.G., Y.S.G. and M.Z. conceived and designed the study. W.K.G. wrote the main manuscript text, W.K.G. and J.Y.C. performed experiments and prepared Figs. 1, 2, 3, 4, 5, 6, 7, 11 and 12. X.Y.S., Q.Z.M. and Y.X.X. prepared Figs. 8, 9 and 10. Y.H.G. and C.X. provided software support. Y.S.G. and M.Z. revised manuscript and provided funding support. All authors contributed to the study and approved this submission.

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Gao, W., Cao, J., Xie, Y. et al. Diaporthe species causing shoot dieback of Acer (maple) in Henan Province, China. BMC Microbiol 24, 356 (2024). https://doi.org/10.1186/s12866-024-03501-3

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