Diaporthe species (Sordariomycetes, Diaporthales) causing walnut blight and dieback in China

Lili Zhao; Lin Zhang; Yi Ding; Ming Li; Ying Zhang

doi:10.3897/mycokeys.122.158807

Abstract

English walnut (Juglans regia L.) is widely cultivated in China due to its economic value and nutritional benefits. Walnut stem blight and dieback is one of the most severe diseases affecting walnut productivity and quality in the country. To clarify the pathogens responsible for walnut stem disease, a comprehensive nationwide survey was conducted. From 276 walnut stem blight and dieback samples collected across seven provinces in China, 292 isolates of Diaporthe spp. were obtained. Both morphological characteristics and phylogenetic analyses based on partial ITS, cal, his3, tef1-α, and tub2 loci were used for fungal identification. Seven species of Diaporthe were identified, including one novel species, D. yunnana. Diaporthe species were most abundant in subtropical southwest China, less common in the temperate north, and absent in Xinjiang. Koch’s postulates confirmed that all seven Diaporthe species could cause blight and dieback on walnut branches, with pathogenicity varying significantly among the species. D. eres and D. rostrata were the most virulent, followed by D. sackstonii, D. amygdali, D. citrichinensis, and D. yunnana, while D. psoraleae-pinnatae was the least aggressive. This is the first report of D. citrichinensis, D. psoraleae-pinnatae, and D. sackstonii occurring on J. regia.

Key words:

Distribution, novel species, pathogenicity, Sordariomycetes, taxonomy, walnut disease

Introduction

English walnut (Juglans regia L.) is a nut tree species with high nutritional and economic value. Walnut trees are extensively cultivated worldwide, especially in Europe, Asia, and various regions of America (Zhang and Tao 2025). In China, walnut cultivation has a history of more than two thousand years, and China is the largest walnut-producing country in the world, accounting for approximately 48% of global production (Shen et al. 2021; Ting et al. 2023). According to the latest China Forestry and Grassland Statistical Yearbook, China produced nearly 5.93 million tons from over 35.67 million hectares in 2022, with the primary production areas located in Yunnan, Xinjiang, Sichuan, and Shaanxi (Zhang and Tao 2025).

Diaporthe (Diaporthaceae, Diaporthales) was typified by Diaporthe eres Nitschke (Nitschke 1870). Both generic names, Diaporthe and Phomopsis, were regularly used. Based on the concept of “one fungus, one name,” Diaporthe, being the older generic name, has priority over Phomopsis (Rossman et al. 2015). Morphologically, Diaporthe is characterized by ostiolate conidiomata, cylindrical phialides, and three types (alpha, beta, and gamma) of conidia (Gomes et al. 2013). Previously, species of Diaporthe were identified based on host association, morphology, and cultural characteristics, which led to a large number of species being assigned within the genus (Uecker 1988). Recently, multi-locus phylogenetic analyses have been extensively used for identifying Diaporthe species (Guo et al. 2020; Norphanphoun et al. 2022). Norphanphoun et al. (2022) divided the genus into 13 species complexes based on well-supported clades that showed consistent placements in both combined and single-gene trees. However, to facilitate species identification, Dissanayake et al. (2024) reclassified the genus into seven distinct sections. To date, over 1,300 epithets of Diaporthe have been listed in Index Fungorum (http://www.indexfungorum.org/; accessed March 2025).

Diaporthe species occur as plant pathogens, endophytes, or saprobes on a wide range of hosts, such as Alnus nepalensis, citrus, grapevine, sunflower, Citrus spp., Rosa spp., Heliconia metallica, Heterostemma grandiflorum, as well as in marine and polluted water environments (Gomes et al. 2013; Yang et al. 2018; Sun et al. 2021; Calabon et al. 2023; Dela Cruz et al. 2025; Li et al. 2025). As plant pathogens, Diaporthe spp. can cause a variety of plant diseases, such as root and fruit rots, dieback, stem cankers, leaf spots, blights, seed decay, stem-end rot, shoot blight, branch dieback, and gummosis in various host plants (Gomes et al. 2013; Norphanphoun et al. 2022; Hilário and Gonçalves 2023; López-Moral et al. 2023; Jia et al. 2024; Li et al. 2025). To date, several species of Diaporthe have been identified as the main causal agents of walnut disease. For instance, D. australafricana, D. cynaroidis, D. eres, D. neotheicola, and D. rhusicola have been reported causing dieback of walnut branches and shoots in Chile, southern Spain, California, and the Moravia region of the Czech Republic (Chen et al. 2014; Eichmeier et al. 2020; López-Moral et al. 2020, 2023; Luna et al. 2020). Diaporthe ampelina, D. chamaeropisrhusicola, D. eres, and D. novem have been reported to cause walnut canker in the United States (Luna et al. 2023). In China, several species of Diaporthe have been reported as causal agents of walnut disease in various regions (Fan et al. 2018; Meng et al. 2018; Wang et al. 2022; Jia et al. 2024). For instance, D. actinidiicola has been reported causing branch canker or dieback in many orchards in Henan Province (Cao et al. 2023). Diaporthe dejiangensis, D. juglandigena, D. tongrensis, and D. hypericin were isolated from J. regia in Guizhou Province (Wang et al. 2022). Diaporthe amygdali has been identified as the causal agent of walnut twig canker in Shandong Province (Meng et al. 2018). Three species—D. eres, D. rostrata, and D. tibetensis—have been reported causing canker disease of walnut in Gansu, Beijing, and Tibet (Fan et al. 2015, 2018). Jia et al. (2024) reported that six species, including D. chaotianensis, D. gammata, D. olivacea, D. tibetensis, D. shangluoensis, and D. shangrilaensis, cause walnut branch disease in Shaanxi, Sichuan, and Yunnan provinces.

Walnut branch disease is a serious problem in China. The infection typically starts on the shoots and then spreads along entire branches. It generally causes discolored areas on the bark, and removing the bark reveals tissues that range from brown to black, eventually leading to the death of the entire branch (López-Moral et al. 2020) (Fig. 1). To clarify the causal agents of walnut branch blight and dieback in China, a screening survey was conducted from 2020 to 2024. The aims of this study were to (i) identify the Diaporthe taxa isolated from blighted and dieback branches of walnut, (ii) evaluate the diversity and prevalence of Diaporthe associated with walnut in China, and (iii) determine their pathogenicity on walnut.

Figure 1.

Typical symptoms of walnut blight and dieback on branches caused by Diaporthe spp. Scale bars: 1 cm (A–H).

Methods

Sample collection and fungal isolation

From 2020 to 2024, a total of 276 samples were collected from diseased or dead branches in walnut plantations across seven sites in China: Beijing, Hebei, Gansu, Shandong, Shanxi, Yunnan, and Xinjiang. Wood fragments (0.5 × 0.5 × 0.2 cm³) were aseptically cut from the margin of disease lesions, surface-sterilized with 75% ethanol for 30 seconds, rinsed three times with sterile distilled water, and incubated on Petri dishes containing 2% malt extract agar (MEA) (Zhao et al. 2024). The Petri dishes were incubated in the dark at 28 °C until colonies appeared. Pure cultures were obtained by transferring hyphal tips from the margins of suspected Diaporthe colonies onto fresh MEA and incubated in the dark at 28 °C.

Morphological characterization

Fungal colonies were initially identified based on morphological characteristics, including colony appearance, conidiomata, conidiogenous cells, and conidia. Colony diameters were measured at 28 °C in darkness on PDA and MEA after 7 days. Colony colors were determined according to Rayner (1970). Additionally, the shapes, colors, and sizes of conidiogenous cells, conidia, and conidiophores were observed under a Nikon Eclipse E600 microscope, and 30–50 conidia were measured to determine their size. Fungal isolates and specimens were deposited at Beijing Forestry University, with duplicates stored at the China General Microbiological Culture Collection Center (CGMCC).

DNA extraction, PCR amplification, and sequencing

DNA was extracted from fungal mycelia grown on MEA plates using a CTAB plant genome DNA fast extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). The internal transcribed spacer (ITS) region of ribosomal DNA was amplified and sequenced using primers ITS1 and ITS4 (White et al. 1990); the β-tubulin gene (tub2) using primers Bt2a and Bt2b (Glass and Donaldson 1995); the translation elongation factor 1-α gene (tef1-α) using primers EF1-728F and EF1-986R (Carbone and Kohn 1999); the calmodulin gene (cal) using primers CAL-228F and CAL-737R (Carbone and Kohn 1999); and the histone H3 gene (his3) using primers CYLH-3F and H3-1b (Glass and Donaldson 1995). PCR amplification and sequencing followed the protocol of Guo et al. (2020). PCR products were purified and sequenced by BGI Tech Solutions (Beijing Liuhe) Co., Limited (Beijing, China).

Phylogenetic analysis

DNA sequences from concatenated ITS, cal, his3, tef1-α, and tub2 loci were analyzed to investigate the phylogenetic relationships among Diaporthe species, using both newly generated sequences and reference sequences retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) (Suppl. material 1). Sequences were aligned using MAFFT v.7 (Katoh et al. 2019) and edited manually using MEGA v.6.0 (Tamura et al. 2013). Gaps were adjusted manually to optimize the alignment.

Phylogenetic analyses of Diaporthe followed the sections proposed by Dissanayake et al. (2024). Maximum likelihood (ML), Bayesian inference (BI), and maximum parsimony (MP) analyses were performed. ML analyses were conducted using RAxML-HPC BlackBox v.8.2.10 (Stamatakis 2014) with the GTR+GAMMA model. MP analysis based on the combined dataset was conducted in PAUP* v.4.0b10 using default settings (Swofford 2002). Ambiguous regions in the alignment were excluded, and gaps were treated as missing data. Clade stability was assessed using bootstrap analysis with 1,000 replicates, with maxtrees set to 1,000 and other default parameters (Hillis and Bull 1993). Parsimony scores calculated included consistency index (CI), rescaled consistency index (RC), homoplasy index (HI), and retention index (RI). Bayesian phylogenetic analysis was conducted using MrBayes v.3.2.5 (Ronquist et al. 2012). The best-fit model of nucleotide substitution was selected using the Akaike information criterion (AIC) in MrModeltest v.2.3 (Posada and Buckley 2004). Four Markov Chain Monte Carlo (MCMC) chains were run, with trees sampled every 1,000 generations. Trees were visualized using TreeView v.1.6.6 (Page 1996) and edited in Adobe Illustrator CC2020 (Adobe Systems Inc., USA).

Prevalence

To determine the prevalence of Diaporthe species obtained in this study, the isolation rate (R^I) was calculated for each species with the formula R^I% = (N^s/N^t) × 100, where N^s was the number of isolates from the same species and N^t is the total number of isolates from each sample-collected site (Fu et al. 2019).

The Shannon–Wiener index was used to estimate species diversity at each sampling site, using R version 4.1.2.

Pathogenicity testing

The Diaporthe isolates obtained in this study were used for pathogenicity testing. Isolates of all species were incubated on MEA plates for 7 days prior to inoculation. The test was performed on lignified, 2-year-old detached walnut branches. The branches were washed, surface-sterilized with 75% ethanol for 1 minute, and the bark surface of each disinfected branch was punctured 20 times with a sterilized inoculating needle within a 10-mm region to a depth of 2 mm (Zhao et al. 2024). An 8-mm-diameter mycelial plug taken from the edge of a fresh colony was placed onto each wounded site. The inoculated area was covered with parafilm. Five replicate branches were inoculated for each isolate, and additional branches were inoculated with fresh MEA agar plugs as controls. All inoculated branches were sealed with parafilm at their ends to prevent desiccation. Pathogenicity was determined by measuring lesion length after three weeks. The data were subjected to analysis of variance (ANOVA), and mean comparisons were conducted using Tukey’s honest significant difference (HSD) test (α = 0.05) in R version 4.1.2.

To fulfill Koch’s postulates, fragments of infected tissue were plated on MEA to re-isolate the fungal isolates, which were identified based on morphological characteristics and DNA sequences.

Result

Fungal isolation

From 2020 to 2024, 276 samples of diseased or dead walnut branches and trunks were collected from seven sites in China. A total of 292 strains of Diaporthe were isolated from these samples, including 103 strains from Beijing, 18 from Gansu, 30 from Hebei, 35 from Shandong, 48 from Shanxi, and 58 from Yunnan, while no strains were obtained from Xinjiang. The occurrence of Diaporthe species is shown in Table 2.

Phylogenetic analyses

Multi-locus phylogenetic analyses were performed using concatenated ITS, cal, his3, tef1-α, and tub2 sequences. The Diaporthe isolates formed branches representing seven species on the phylogenetic trees, belonging to Section Betulicola, Section Eres, Section Sojae, Section Rudis, and Section Psoraleae-pinnatae (Figs 2–5).

For Section Betulicola, the concatenated ITS, cal, his3, tef1-α, and tub2 dataset (2,348 characters, with 515 parsimony-informative characters) from 38 ingroup isolates was used for phylogenetic analysis. The outgroup taxon was D. amygdali (CBS 126679). The best RAxML tree, with a final likelihood value of –11053.482380, is presented in Fig. 2. RAxML analysis yielded 895 distinct alignment patterns and 11.24% undetermined characters or gaps. For BI analysis, four simultaneous Markov chains were run for 5,100,000 generations. The final average standard deviation of split frequencies was 0.009993. For MP, the heuristic search with random addition of taxa (1,000 replicates) generated 5,000 most parsimonious trees (CI = 0.675, RI = 0.813, RC = 0.549, HI = 0.325). Six isolates clustered in two clades corresponding to D. rostrata (four isolates) and D. yunnana (two isolates) (Fig. 2).

Figure 2.

Maximum likelihood (ML) tree generated from sequence analysis of the concatenated ITS, cal, his3, tef1-α, and tub2 gene dataset of Section Betulicola. RAxML bootstrap support values (ML ≥ 50%), Bayesian posterior probability (PP ≥ 0.70), and maximum parsimony bootstrap support values (MP ≥ 50%) are shown at the nodes (ML/PP/MP).

For Section Eres, the concatenated ITS, cal, his3, tef1-α, and tub2 dataset (2,262 characters, with 485 parsimony-informative characters) from 67 ingroup isolates was used for phylogenetic analysis. The outgroup taxon was D. amygdali (CBS 126679). The best RAxML tree, with a final likelihood value of –13844.510016, is presented in Fig. 3. RAxML analysis yielded 973 distinct alignment patterns and 16.18% undetermined characters or gaps. For BI analysis, four simultaneous Markov chains were run for 2,000,000 generations. The final average standard deviation of split frequencies was 0.009935. The heuristic search with random addition of taxa (1,000 replicates) generated 5,000 most parsimonious trees (CI = 0.556, RI = 0.706, RC = 0.393, HI = 0.444). Ten isolates clustered in two clades corresponding to D. eres (eight isolates) and D. citrichinensis (two isolates) (Fig. 3).

Figure 3.

Maximum likelihood (ML) tree generated from sequence analysis of the concatenated ITS, cal, his3, tef1-α, and tub2 gene dataset of Section Eres. RAxML bootstrap support values (ML ≥ 50%), Bayesian posterior probability (PP ≥ 0.70), and maximum parsimony bootstrap support values (MP ≥ 50%) are shown at the nodes (ML/PP/MP).

For Sections Rudis and Psoraleae-pinnatae, the concatenated ITS, cal, his3, tef1-α, and tub2 dataset (2,249 characters, with 628 parsimony-informative characters) from 42 ingroup isolates was used for phylogenetic analysis. The outgroup taxon was D. corylina (CBS 121124). The best RAxML tree, with a final likelihood value of –12244.597254, is presented in Fig. 4. RAxML analysis yielded 1,009 distinct alignment patterns and 17.85% undetermined characters or gaps. For BI analysis, four simultaneous Markov chains were run for 1,500,000 generations. The final average standard deviation of split frequencies was 0.009051. The heuristic search with random addition of taxa (1,000 replicates) generated 5,000 most parsimonious trees (CI = 0.718, RI = 0.911, RC = 0.654, HI = 0.282). Five isolates clustered in two clades corresponding to D. amygdali (two isolates) and D. psoraleae-pinnatae (three isolates) (Fig. 4).

Figure 4.

Maximum likelihood (ML) tree generated from sequence analysis of the concatenated ITS, cal, his3, tef1-α, and tub2 gene dataset of Section Rudis and Psoraleae-pinnatae. RAxML bootstrap support values (ML ≥ 50%), Bayesian posterior probability (PP ≥ 0.70), and maximum parsimony bootstrap support values (MP ≥ 50%) are shown at the nodes (ML/PP/MP).

For Section Sojae, the concatenated ITS, cal, his3, tef1-α, and tub2 dataset (2,408 characters, with 984 parsimony-informative characters) from 104 ingroup isolates was used for phylogenetic analysis. The outgroup taxon was D. corylina (CBS 121124). The best RAxML tree, with a final likelihood value of –32296.521228, is presented in Fig. 5. RAxML analysis yielded 1,484 distinct alignment patterns and 25.65% undetermined characters or gaps. For BI analysis, four simultaneous Markov chains were run for 7,500,000 generations. The final average standard deviation of split frequencies was 0.009634. The heuristic search with random addition of taxa (1,000 replicates) generated 5,000 most parsimonious trees (CI = 0.374, RI = 0.693, RC = 0.259, HI = 0.626). Three isolates clustered in one clade corresponding to D. sackstonii (three isolates) (Fig. 5).

Figure 5.

Maximum likelihood (ML) tree generated from sequence analysis of the concatenated ITS, cal, his3, tef1-α, and tub2 gene dataset of Section Sojae. RAxML bootstrap support values (ML ≥ 50%), Bayesian posterior probability (PP ≥ 0.70), and maximum parsimony bootstrap support values (MP ≥ 50%) are shown at the nodes (ML/PP/MP).

Taxonomy

Diaporthe amygdali (Delacr.) Udayanga, Crous, K.D. Hyde, Fungal Divers 56: 166 (2012)

Suppl. material 2

Specimens examined.

China • Yunnan Province, Kunming City, from diseased branches of J. regia., Y. Ding, M. Li and L.L. Zhao, 23 February 2024 (YN-6, culture CGMCC3.27752; YN-2, culture CGMCC3.28500).

Notes.

Diaporthe amygdali was first described as Fusicoccum amygdali Delacr., causing cankers on almonds in France (Delacroix 1905). Udayanga et al. (2012) assigned F. amygdali to Diaporthe as D. amygdali. Dissanayake et al. (2024) verify D. amygdali is a single species rather than a species complex. Phylogenetically, two isolates clustered together with the ex-type isolates of D. amygdali CBS 126679 with middle statistical (62%/0.87/62%) (Fig. 4); the base pair similarity shows 98.9% (464/469) on cal, 99.3% (449/452) on his, 99% (519/524) on ITS, 99.4% (327/329) on tef1-α, and 99.8% (469/470) on tub2 compared to the ex-type of D. amygdali. Based on sequence data and morphology, these isolates were confirmed to belong to D. amygdali (Table 1). Thus far, D. amygdali has been reported to cause walnut branch disease in Southern Spain and Shandong province in China (Meng et al. 2018, López-Moral et al. 2020). In this study, D. amygdali were isolated from Yunnan Province.

Table 1.

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Morphological characteristics of known Diaporthe species.

Species	Conidiomata	Conidiophores	Conidiogenous cells	Alpha conidia	Beta conidia	Gamma conidia
D. amygdali	Pycnidial globose or irregular, solitary or aggregated, wrapped in hyphae embedded colony surface, white to brown.	hyaline, subcylindrical, densely aggregated, 7.0–18.5 × 1.5–4.0 μm.	phialidic, hyaline, cylindrical, straight or slightly curved, 1.5–2.5 μm, tapered towards the apex.	Not observed.	hyaline, aseptate, filiform, curved, tapering towards end, 23.5–35.0 × 1.0–2.0 μm.	Not observed.
D. citrichinensis	Pycnidial irregular, solitary or aggregated, brown to dark brown	cylindrical, hyaline, densely aggregated, 6.0–10.5 × 1.0–3.0 μm.	phialidic, cylindrical, 5.0–11.0 × 1.5–2.5 μm, tapered towards the apex	aseptate, fusoid with obtuse ends, hyaline, biguttulate 6.0–10.0 × 1.5–3.0 μm.	filiform, hyaline, aseptate, slightly curved at one end and both ends rounded, 32.0–44.0 × 1.0–2.0 μm.	Not observed.
D. eres	Pycnidial solitary or aggregated, with yellowish or white translucent conidial drops exuded from the ostioles.	hyaline, smooth, unbranched, ampulliform, straight to sinuous, 9.0–16 × 2.0–3.0 μm.	phialidic, cylindrical, terminal, slightly, 5.0–1.5 μm diam tapering towards the apex	aseptate, hyaline, smooth, ovate to ellipsoidal, biguttulate, 6.0–9.0 × 2.5–3.0 μm.	aseptate, hyaline, smooth, fusiform to hooked, 23.0–32.0 × 1.0–2.0 μm.	Not observed.
D. psoraleae-pinnatae	Pycnidial irregular shape, solitary or aggregated, white or yellowish translucent conidial drops exuded from the ostioles.	hyaline, smooth, densely aggregated, ampulliform, 9.5–23.0 × 2–6 μm.	phialidic, hyaline, terminal, cylindrical, straight or slightly curved, 6.0–19.0 × 1.5–2.5 μm, tapered towards the apex.	aseptate, fusiform, biguttulate, apex subobtuse, 7.5–10.5 × 2.0–3.0 μm.	hyaline, aseptate, guttulate, filiform, curved, 32.0–48.0 × 1.0–2.5 μm.	Not observed.
D. rostrata	Pycnidial solitary or aggregated, wrapped in hyphae embedded on colony surface, with yellowish translucent conidial drops exuded from the ostioles.	hyaline, smooth, densely aggregated, 12.0–21.0 × 2–4.5 μm.	Conidiogenous cells phialidic, hyaline, terminal, cylindrical, straight, 5.5–8.5 × 2.0–3.5 μm.	hyaline, smooth, aseptate, fusiform to oval, biguttulate or multi-guttulate, 7.0–10.0 × 4.0–5.0 μm.	Not observed.	hyaline, guttulate, smooth, aseptate, with only one acute end, 10.5–13.0 × 2.5–4.0 µm.
D. sackstonii	Pycnidial globose, solitary or aggregated, wrapped in hyphae embedded on MEA colony surface, white to brown.	hyaline, smooth, unbranched, densely aggregated, ampulliform, 12.5–37 × 2.0–4.0 μm.	Conidiogenous cells phialidic, hyaline, terminal, cylindrical, straight, 7.5–17 × 1.5–2.5 μm, tapered towards the apex.	hyaline, aseptate, fusiform to oval, obtuse at both ends, 5.5–7.5 × 2.5–3.5 μm.	hyaline, aseptate, multi-guttulate, filiform, curved, tapering towards both ends, 27.0–39.0 × 1.0–2.5 μm.	Not observed.

Table 2.

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Number of isolates collected for each Diaporthe species identified and sites investigated in this study.

	Beijing	Gansu	Hebei	Shandong	Shanxi	Yunnan	Xinjiang	Total
D. amygdali	-	-	-	-	-	5	-	5 (1.7%)
D. citrichinensis	-	-	-	-	-	15	-	15 (5.1%)
D. eres	81	17	10	31	32	26	-	197 (67.5%)
D. psoraleae-pinnatae	8	-	-	-	-	-	-	8 (2.7%)
D. rostrata	7	1	20	2	16	6	-	52 (17.8%)
D. sackstonii	7	-	-	-	-	-	-	7 (2.4%)
D. yunnana	-	-	-	2	-	6	-	8 (2.7%)
Total	103	18	30	35	48	58	0
Shannon-Wiener index	0.75	0.21	0.64	0.43	0.64	1.40	0

Diaporthe citrichinensis F. Huang, K.D. Hyde & H.Y. Li, Fungal Divers 61: 247 (2013)

Suppl. material 3

Specimens examined.

China • Yunnan Province, Kunming City, from diseased branches of J. regia, Y. Ding, M. Li and L.L. Zhao, 23 February 2024 (YN-7, culture CGMCC3.27759; YN-26, culture CGMCC3.27753).

Notes.

Diaporthe citrichinensis was first described from decaying wood of Citrus unshiu in China (Huang et al. 2013). Dissanayake et al. (2024) treated Diaporthe acerigena, D. albosinensis, D. coryli, D. fraxinicola, D. tibetensis, and D. ukurunduensis as the synonyms of D. citrichinensis. Phylogenetically, two isolates clustered together with D. citrichinensis with high support (100%/1/100%) (Fig. 3); the base pair similarity shows 100% (471/471) on cal, 97.7% (434/444) on his, 100% (530/530) on ITS, 100% (333/333) on tef1-α, and 100% (480/480) on tub2 compared to the ex-type of D. citrichinensis. Morphologically, alpha and beta conidia are similar to D. citrichinensis (6.0–10.0 × 1.5–3.0 vs. 5.5–9 × 1.5–2.5 μm) and (32–44 × 1–2 vs. 27.5–40 × 1–1.5 μm) (Table 1) (Huang et al. 2013). In this study, D. citrichinensis was collected from the walnut plantation of Yunnan. This is the first report of D. citrichinensis occurring on J. regia.

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

Suppl. material 4

Specimens examined.

China • Beijing City, from diseased branches of J. regia., Y. Zhang, L.L. Zhao and L. Zhang, 14 December 2021 (2021-JF-6, culture CGMCC3.28277; CGMCC 3.28281; 2021-JF-10, culture CGMCC3.28282, CGMCC3.28284). • Shanxi Province, Jiaokou City, from diseased branches of J. regia, Y. Ding, M. Li and L.L. Zhao, 28 February (JK-4, culture CGMCC3.28276). • Yunnan Province, Kunming City, from diseased branches of J. regia, Y. Ding, M. Li and L.L. Zhao, 23 February 2024 (YN-23, culture CGMCC3.28290). • Shandong Province, Liaocheng City, from diseased branches of J. regia, Y. Ding, M. Li and L.L. Zhao, 5 February 2024 (LC-12, culture CGMCC 3.28280). • Hebei Province, Chengde City, from diseased branches of J. regia, Y. Ding, M. Li and L.L. Zhao, 13 February 2024 (CD-3, culture CGMCC3.28298).

Notes.

Diaporthe eres was first described by Nitschke (1870) and collected from Ulmus sp. in Germany. It has a wide distribution and a broad host range as a pathogen, endophyte, or saprobe, and can cause a variety of plant diseases (Udayanga et al. 2014). Hilário et al. (2021b) and Dissanayake et al. (2024) identified the D. eres complex as a single species, D. eres. Phylogenetically, eight isolates clustered within D. eres (Fig. 3). Therefore, these isolates were confirmed to belong to D. eres, based on sequence data and morphology (Table 1). In this study, more than half of the isolates (189, 67.5%) belong to D. eres, which is nationally distributed in Beijing, Gansu, Hebei, Shandong, Shanxi, and Yunnan, causing walnut branch diseases.

Diaporthe psoraleae-pinnatae Crous & M.J. Wingf., Persoonia 31: 205 (2013)

Suppl. material 5

Specimens examined.

China • Beijing, Changping District, Heishanzhai Village, from branches of J. regia, Y. Zhang, L.L. Zhao and L. Zhang, 26 August 2022 (HSZ-1, culture CGMCC3.28292; HSZ-5, culture CGMCC3.28293, CGMCC3.28296).

Notes.

Diaporthe psoraleae-pinnatae was first described from dieback branches of Psoralea pinnata in South Africa (Crous et al. 2013). Recently, the entire section Psoraleae-pinnatae has been identified as a single species and named Diaporthe psoraleae-pinnatae, with D. aquatica, D. bauhiniae, D. ellipsospora, D. incomplete, D. jinxiu, D. rhoina, D. shaanxiensis, and D. varians all treated as its synonyms (Dissanayake et al. 2024). Phylogenetically, three isolates clustered together with D. psoraleae-pinnatae (Fig. 4); the base pair similarity shows 100% (469/469) on cal, 97.7% (452/452) on his, 97% (508/524) on ITS, 100% (329/329) on tef1-α, and 100% (470/470) on tub2 compared to the ex-type of D. psoraleae-pinnatae. Morphologically, conidiogenous cells and alpha conidia are similar to the ex-type isolate of D. psoraleae-pinnatae (6–19 × 1.5–2.5 vs. 8–15 × 2–3 μm) and (7.5–10.5 × 2.0–3.0 vs. 9–10 × 2.5–3 μm) (Table 1) (Crous et al. 2013). In this study, D. psoraleae-pinnatae was collected from Beijing. This is the first report of D. psoraleae-pinnatae occurring on J. regia.

Diaporthe rostrata C.M.Tian, X.L.Fan & K.D.Hyde, Mycol. Progr. 14: 82 (2015)

Suppl. material 6

Material examined.

China • Beijing City, Haidian District, from diseased branches of J. regia, M. Li, L.L. Zhao and L. Zhang, 19 October 2020 (JF-11, ex-type culture CGMCC 3.28283); Hebei Province, Chengde City, from diseased branches of J. regia, M. Li and L.L. Zhao, 13 February 2024 (CD-22, culture CGMCC3.27755); Shanxi Province, Jiaokou City, from diseased branches of J. regia, Y. Ding, M. Li and L.L. Zhao, 28 February 2024 (JK-14-2, culture CGMCC3.27757; JK-16-2, culture CGMCC3.27760).

Notes.

Diaporthe rostrata was first described from Juglans mandshurica in Gansu Province, China (Fan et al. 2015). Dissanayake et al. (2024) compared morphological details and phylogenetic analysis, treating D. juglandicola as the synonym of D. rostrata. Phylogenetically, four isolates clustered together with D. rostrata with high support (100%/1/100%) (Fig. 2); the base pair similarity shows 99.6% (455/457) on cal, 98.2% (439/447) on his, 100% (550/550) on ITS, 99.2% (357/360) on tef1-α, and 100% (473/473) on tub2 compared to the ex-type of D. rostrata. Morphologically, the culture characteristics and alpha conidia are consistent with the description of D. rostrata (Table 1) (Fan et al. 2015). In this study, D. rostrata was collected from the walnut plantations of Beijing, Gansu, Hebei, Shandong, Shanxi, and Yunnan provinces.

Diaporthe sackstonii R.G. Shivas, S.M. Thomps. & Y.P. Tan, Persoonia 35: 46 (2015)

Suppl. material 7

Material examined.

China • Beijing City, Haidian District, JiuFeng forest farm, from branches of J. regia, M. Li, L.L. Zhao and L. Zhang, 26 August 2022 (2022-JF-34, culture CGMCC3.28287, CGMCC3.28295, CGMCC3.28297).

Notes.

Diaporthe sackstonii was first described from Helianthus annuus in Australia (Thompson et al. 2015). Pereira and Phillips (2024) treated D. caryae, D. machili, D. juglandigena, and D. orixae as the synonyms of D. sackstonii. Phylogenetically, three isolates clustered together with D. sackstonii (Fig. 5); the base pair similarity shows 97.5% (466/478) on his, 99.2% (506/510) on ITS, 98.1% (370/377) on tef1-α, and 98.6% (488/495) on tub2 compared to the ex-type of D. sackstonii. Morphologically, the alpha conidia are similar to D. sackstonii (5–7.5 × 2.5–3.5 vs. 6–7 × 2–2.5) (Table 1) (Thompson et al. 2015). In this study, D. sackstonii was collected from the walnut plantation of Beijing. This collection is the first report of D. sackstonii occurring on J. regia.

Diaporthe yunnana Y. Zhang ter & L.L. Zhao, sp. nov.

MycoBank No: MycoBank No: 854968

Fig. 6

Etymology.

Named after the place, Yunnan, where the fungus was abundantly found.

Description.

Sexual morph : not observed. Asexual morph: Conidiomata pycnidial, produced on PDA, globose or irregular, solitary, dark brown to black, 290–810 μm diam. Conidiophores hyaline, smooth, densely aggregated, 12–20.5 × 1.5–3 μm; Conidiogenous cells phialidic, hyaline, terminal, cylindrical, 5.5–10 × 1.5–2.5 μm diam, tapered towards the apex. Alpha conidia hyaline, aseptate, ellipsoid to cylindrical, obtuse at both ends, multi-guttulate, 6–10.5 × 2–3 μm (mean ± SD = 8.5 ± 1.0 × 2.8 ± 0.2 μm, n = 30). Beta conidia hyaline, aseptate, filiform, curved, tapering towards both ends, multi-guttulate, 25.5–42 × 1–1.7 μm (mean ± SD = 34.5 ± 3.6 × 1.4 ± 0.2 μm, n = 30). Gamma conidia infrequent, hyaline, aseptate, botuliform, tapering towards both ends, multi-guttulate, 12.5–18 × 2–2.5 μm (mean ± SD = 14.0 ± 1.5 × 1.9 ± 0.1 μm, n = 30).

Figure 6.

Morphological characteristics of Diaporthe yunnana. A, B. Colonies and reverse after 7 days on PDA; C, D. Colonies and reverse after 7 days on MEA; E. Conidiomata; F, G. Conidiophores and conidiogenous cells; H. Alpha conidia; I. Beta conidia; J, K. Alpha, beta, and gamma conidia. Scale bars: 500 μm (E, F); 10 μm (G–K).

Culture characteristics.

On PDA, colony at first flat with white felty mycelium, becoming brown in the center, flourishing at center of colony, reverse white to brown. On MEA, white on surface, reverse white to dark brown. Colonies cover the Petri dish diameter on PDA and reach 57 mm in diameter on MEA.

Material examined.

China • Yunnan Province, Kunming City, from diseased branches of J. regia, Y. Ding, M. Li and L.L. Zhao, 23 February 2024 (holotype YN-12, ex-type culture CGMCC3.27754; other culture CGMCC3.27756).

Notes.

Multi-locus phylogenetic analysis indicated that Diaporthe yunnana formed a moderately supported subclade with D. gammata (77%/0.79/74%) (Fig. 2). Morphologically, D. yunnana can be readily distinguishable from D. gammata by its shorter beta conidia (25.5–42 × 1–1.7 vs. 29–48.5 × 1–2 μm) and smaller-size gamma conidia (12.4–18.2 × 1.8–2.3 vs. 16–31.5 × 1.5–4 μm) (Xiao et al. 2023). Based on nucleotide base comparison, D. yunnana can be distinguished from D. gammata by base differences as follows: 13/548 bp for ITS (2.37%), 18/440 bp for his3 (4.08%), 23/360 bp for tef1-α (6.39%), and 8/486 bp for tub2 (1.65%) (Xiao et al. 2023).

Prevalence

Prevalence analysis revealed that Diaporthe eres was the dominant species (67.5%), followed by D. rostrata, D. citrichinensis, D. psoraleae-pinnatae, D. yunnana, D. sackstonii, and D. amygdali. Among them, D. eres was the most prevalent species in Beijing, Gansu, Shandong, Shanxi, and Yunnan, while D. rostrata was dominant in Hebei (Table 2, Fig. 7).

Figure 7.

Map of China indicating locations where walnut trees were sampled and the species of Diaporthe obtained from each site. The seven species of Diaporthe are indicated.

Further analysis of Diaporthe species prevalence across the sampling areas showed that fewer species were identified in the northern regions with a temperate monsoon climate, whereas greater diversity was observed in the southwestern regions with a subtropical monsoon climate. In the northwestern regions with a temperate continental climate, Diaporthe species were not isolated (Table 2, Fig. 7).

Pathogenicity testing

All Diaporthe species tested in this study were pathogenic on walnut branches. Brown lesions appeared at the inoculation sites three weeks after inoculation, while no symptoms were observed in the control treatment (Fig. 8). The average lengths of the necrotic lesions caused by D. eres (29.6 ± 4.9 mm) and D. rostrata (29.2 ± 5.5 mm) were significantly greater than those caused by D. amygdali (21.3 ± 2.9 mm), D. citrichinensis (18.0 ± 0.7 mm), D. psoraleae-pinnatae (16.2 ± 2.4 mm), and D. yunnana (18.0 ± 2.4 mm). There was no significant difference among D. eres, D. rostrata, and D. sackstonii. The lesion length caused by D. sackstonii (25.0 ± 2.3 mm) was significantly greater than that caused by D. psoraleae-pinnatae. No significant differences were observed among D. amygdali, D. citrichinensis, D. psoraleae-pinnatae, and D. yunnana (Fig. 9).

Figure 8.

Symptoms of seven Diaporthe species inoculated on walnut branches after three weeks. A. Inoculated D. amygdali; B. Inoculated D. citrichinensis; C. Inoculated D. eres; D. Inoculated D. psoraleae-pinnatae; E. Inoculated D. rostrata; F. Inoculated D. sackstonii; G. Inoculated D. yunnana; H. CK. Scale bars: 1 cm (A–H).

Figure 9.

Lesion length caused by Diaporthe species inoculated on walnut detached stems. Columns represent the mean values of five replicate branches. A vertical bar with different letters indicates significantly different (p < 0.05).

Discussion

Among the Diaporthe species isolated from walnut, D. eres was the most prevalent in this study, comprising 67.5% of all Diaporthe isolates. Similar results were reported by Fan et al. (2018), who collected infected walnut branches in Beijing, Gansu, Henan, Ningxia, Sichuan, and Tibet, showing that most strains were D. eres, occurring in five provinces. Guo et al. (2020) also reported that D. eres was the most prevalent species causing pear shoot canker in China, accounting for 54.7% of all Diaporthe isolates. Moreover, D. eres was identified as the most prominent species associated with grapevine dieback in China, representing 37.5% of total isolates (Manawasinghe et al. 2019). Diaporthe citri, however, was the dominant species associated with Citrus in southern China (Xiao et al. 2023), possibly indicating that Diaporthe species composition varies across different host plants.

Climate types largely influenced the species diversity of Diaporthe. Yunnan Province, characterized by a subtropical monsoon climate, exhibited the highest species diversity, followed by Beijing, Shanxi, Hebei, Shandong, and Gansu, which are mainly temperate monsoon regions. No Diaporthe species were found in Xinjiang, which has a temperate continental climate. Jia et al. (2024) isolated six species from twigs of Juglans regia in Shaanxi, Sichuan, and Yunnan. Of these, five species were collected from Sichuan and Yunnan, which are both characterized by a subtropical monsoon climate. Similar findings were reported by Guo et al. (2020), who observed higher species diversity in the southern Yangtze River region (subtropical monsoon climate) compared to the northern region (temperate monsoon climate). However, no Diaporthe species were found in Gansu, Shanxi, and Xinjiang, likely due to the predominantly temperate continental climate in these areas (Guo et al. 2020). Xiao et al. (2023) collected citrus disease samples in southern China and reported significant Diaporthe diversity, particularly in Hunan Province. This may be because the southern climate is humid and warm—conditions suitable for the survival and prevalence of Diaporthe species—whereas drought and extremely low temperatures prevail in northern and northwestern China, especially in Xinjiang, making these regions unsuitable for Diaporthe.

Pathogenicity tests showed that all seven species retrieved in this study were causal agents of walnut branch blight and dieback, producing dark brown necrosis at the inoculation sites. However, pathogenicity varied significantly among species. Diaporthe eres, D. rostrata, and D. sackstonii exhibited the greatest severity on walnut branches, followed by D. amygdali, D. citrichinensis, and D. yunnana, while D. psoraleae-pinnatae was the least aggressive. A similar result was obtained by Jia et al. (2024), who reported that six species were associated with walnut branch and twig cankers. Their pathogenicity tests revealed that all six species could cause disease on walnut, with D. shangrilaensis, D. olivacea, and D. shangluoensis being more aggressive, followed by D. chaotianensis and D. tibetensis, while D. gammata was the least aggressive. Previous studies have demonstrated that the pathogenicity of Diaporthe species varies on a single host (Manawasinghe et al. 2019; Guo et al. 2020; Hilário et al. 2021a; Xiao et al. 2023). For instance, 19 Diaporthe species were reported to be associated with pear shoot canker in China, and Koch’s postulates confirmed that all were pathogenic. Among them, D. chongqingensis, D. fusicola, and D. eres were highly aggressive (Guo et al. 2020). Xiao et al. (2023) reported 36 Diaporthe species associated with citrus disease; the pathogenicity test revealed that D. citri produced the longest lesions, followed by D. tectonae, D. hubeiensis, D. sexualispora, and D. citriasiana. Eight Diaporthe species were reported in association with grapevine dieback, and D. gulyae was the most aggressive taxon, followed by D. eres and D. unshiuensis (Manawasinghe et al. 2019). In Portugal, Hilário et al. (2021a) found that D. eres and D. amygdali were the most virulent species associated with blueberry twig blight and dieback. These results indicate that the pathogenicity of the same species can vary depending on the host. For instance, D. eres was highly aggressive on walnut, pear, and blueberry, but considerably less so on citrus.

Conclusion

In conclusion, this study presents the Diaporthe species associated with branch diseases of walnuts in China. A total of seven species, including one novel species, were identified, all of which were confirmed as causal agents of walnut branch blight and dieback. This study also revealed the diversity and geographical distribution of Diaporthe spp. associated with walnut. The findings provide valuable insight into the ecology and pathogenicity of Diaporthe spp. involved in walnut blight and dieback.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Use of AI

No use of AI was reported.

Funding

This work was supported by the National Natural Science Foundation of China (General Program) under grant numbers 31971658, 31770015, and 31370063. Project Implementation Cost for the National Integrated Survey of Natural Resources (No: DD20230471)

Author contributions

YZ designed the research; YZ and ML revised the manuscript; YD, ML, LLZ, and LZ performed the research; LLZ wrote the manuscript. All authors read and approved the final version of the manuscript.

Author ORCIDs

Lili Zhao https://orcid.org/0000-0003-1451-3301

Lin Zhang https://orcid.org/0009-0002-6325-1440

Ying Zhang https://orcid.org/0000-0001-8817-6032

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Lili Zhao and Lin Zhang contributed equally to this work.

Supplementary materials

Supplementary material 1

GenBank accession numbers of isolates included in this study