Rhytidhysteron juglandis Y. Zhang

Zhao, Li-Li, Zhang, Lin, Jiao, Ning, Li, Ming & Zhang, Ying, 2025, Branch blight and dieback of walnut (Juglans regia L.) caused by Rhytidhysteron juglandis sp. nov. and its endophytic biocontrol agent Granulobasidium vellereum, Phytotaxa 705 (1), pp. 19-34 : 26-29

Rhytidhysteron juglandis Y. Zhang ter & L.L. Zhao, sp. nov. ( Fig. 3 )

MycoBank: MB 849639

Etymology: —from “ Juglans ”, in reference to the host genus.

The typical symptoms of the diseases were branch blight or dieback with brown necrotic lesions appearing on the twigs and branches. The infection typically occurred on one side of the branches and subsequently extended along entire branches. Mycelium composed of branched, septate, smooth, hyaline, thin-walled hyphae, 1.5–4 µm wide, brown, sometimes aggregated in tightly packed, brown to dark brown in mass, up to 8 µm wide. Conidiophores and conidia not produced on malt extract agar (MEA), potato dextrose agar (PDA), oatmeal agar (OA) or synthetic nutrient-poor agar (SNA).

Culture characteristics: —Colonies reached 66 mm diameter on MEA and 67.3 mm diameter on PDA after seven days in darkness at 28 ℃. The colony is soft, circular, texture velutinous to slightly floccose, with smooth margin, dark gray to pale gray in center, and grayish in reverse.

Material examined: — CHINA, Beijing City, Haidian District, from dieback or blight branches of Juglans regia L., 25 June 2022, M. Li , L. L. Zhao and L. Zhang (holotype: 2022JF-2; ex-type: CGMCC 3.25223 ; other living cultures: CGMCC 3.25221 , CGMCC 3.25222 ) .

Notes: —No sexual or asexual sporulation of R. juglandis was observed on either host or colonies in this study. Attempt of inducing sporulation has been conducted by incubating colonies of R. juglandis on MEA, PDA, SNA and OA plate and incubated for 7 days, which was eventually unsuccessful. Based on concatenated ITS, LSU, and tef 1-α DNA sequences, R. juglandis formed a separate clade that was sibling to other species of the genus Rhytidhysteron and closely related to R. xiaokongense ( ML / MP /PP = 82%/66%/0.99) ( Fig. 2 ). Rhytidhysteron xiaokongense was originally isolated from dead wood of Prunus sp. in Yunnan and was found diplodia-like asexual morph ( Ren et al. 2022). In addition, Rhytidhysteron juglandis ( CGMCC 129041) differs from R. xiaokongense ( KUMCC 20-0158 and KUMCC 20-0160) by unique fixed alleles in three loci based on alignments of the separate loci deposited in the tree, by 28 bp in ITS (5.2 %), 10 bp in LSU (1.2 %) and 17 bp in tef 1-α (1.9 %). ITS position: 1( T), 3( T), 5( T), 6( C), 9( G), 15( T), 16( C), 18( G), 19( C), 20( G), 21( T), 22( C), 25( C), 26( T), 28( G), 29( C), 32( G), 36( T), 37( T), 38( A), 39( G), 40( A), 42( A), 44( T), 51( C), 54( A), 176( G), 354( G); tef-α position: 94( A), 99( A), 215( A), 238( T), 263( G), 266( T), 295( C), 310( T), 331( C), 493( C), 503( G), 541( C), 600( A), 607( T), 745( T), 844( T), 853( T); LSU position: 64( T), 97( C), 173( T), 379( C), 382( T), 417( G), 455( G), 496( T), 501( C), 664( C).

Pathogenicity test

Rhytidhysteron juglandis caused lesions on both detached, or on planta branches of walnut. The inoculated sites turned dark brown, and the mean length of the necrotic lesions was 3.68 ± 0.38 cm three weeks after inoculation on detached branches and 1.65 ± 0.36 cm one month after inoculation on planta. No necrotic lesions were observed on negative controls ( Fig. 1 ). Koch’s postulates were satisfied by successful reisolation of R. juglandis from the necrotic lesions of the branches inoculated with the pathogen.

Antagonistic assay

The mycelial growth of R. juglandis was significantly suppressed by the presence of G. vellereum ( Figs. 4 , 5 ). Comparing with all other treatments, the most significant suppression occurred when the colony of G. vellereum inoculated 3 days ahead, and the colony of R. juglandis stopped growing after 9 days’ incubation ( Figs. 4 , 5 ). By simultaneously incubating G. vellereum and R. juglandis ( G. v. / R. j.), the colony growth rate of R. juglandis was significantly lower than the treatment of inoculating R. juglandis 3 days ahead of G. vellereum ( R. j. + G. v.), or inoculating R. juglandis only ( R. j.) ( Figs. 4B 1 to B 5, 5). By inoculating R. juglandis 3 days ahead of G. vellereum ( R. j. + G. v.), the colony growth rate of R. juglandis was significantly lower than the treatment of inoculating R. juglandis only ( R. j.) ( Figs. 4C 1 to C 5, 5). After 14 days’ incubation, a clear inhibition line occurred between the colonies of G. vellereum and N. juglandis in the treatment of R. juglandis incubated 3 days ahead of G. vellereum ( Fig. 4C 3 ). After 20 days’ incubation, the mycelia of G. vellereum overgrew the colony of R. juglandis until covered the whole petri dish in the treatments of the two fungal species incubated simultaneously, or G. vellereum inoculated 3 days ahead of R. juglandis ( Fig. 4 ).

Of all the treatments, the longest lesion length occurred on the branches inoculated with the mycelia of R. juglandis alone. By inoculating G. vellereum 3 days ahead of R. juglandis ( G. v. + R. j.) on walnut branches in planta, the lesion length caused by R. juglandis were significantly shorter than inoculating G. vellereum 3 days after R. juglandis ( R. j. + G. v.) and inoculating R. juglandis alone ( Fig. 6 ). The lesion length in the treatment of simultaneously inoculating G. vellereum and R. juglandis ( G. v. / R. j.) was significantly shorter than the treatment inoculating R. juglandis alone. There was, however, no significant difference between the treatment simultaneously inoculating G. vellereum and R. juglandis ( G. v. / R. j.) and inoculating G. vellereum 3 days ahead of R. juglandis or inoculating G. vellereum 3 days after of R. juglandis ( Fig. 6 ).

Figures

FIGURE 1. Typical symptoms of walnut branch blight and dieback caused by Rhytidhysteron juglandis. A, B. Symptoms of branch blight and dieback of walnut in the field. C. Death of the branches of the walnut in the field. D. Symptoms 3 weeks after inoculating with mycelia of R. juglandis on detached branches. E. Control on detached branches. F. Symptoms one month after inoculating with mycelia of R. juglandis in planta. G. Control in planta. Scale bars: D-G = 1 cm.

FIGURE 2. Maximum Likelihood tree generated from sequence analysis of the concatenated ITS, LSU, and tef1-α gene dataset of Rhytidhysteron species. The designated outgroup taxon was Gloniopsis calami. RAxML bootstrap support values (ML ≥ 50%), Bayesian posterior probabilities (PP ≥ 0.6) and Maximum parsimony (MP) support values (MP ≥ 50%) are shown at the nodes (ML/MP/PP). The species characterized in this study are in bold.

FIGURE 3. Morphology of Rhytidhysteron juglandis.A, B. Colony surface and reverse side after 7 days incubation on PDA. C, D. Colony surface and reverse after 7 days incubation on MEA. E, F. Mycelium of Rhytidhysteron juglandis. Scale bars: E, F = 10 µm.

FIGURE 4. Antagonistic assay of Granulobasidium vellereum on the growth of Rhytidhysteron juglandis on MEA. A. G. vellereum incubated 3 days ahead of R. juglandis. B. G. vellereum and R. juglandis incubated simultaneously. C. G. vellereum incubated 3 days after R. juglandis. D. R. juglandis incubated alone. A1, B1, C1, D1, incubated for 7 days. A2, B2, C2, D2, incubated for 10 days. A3, B3, C3, D3, incubated for 14 days.A4, B4, C4, D4, incubated for 20 days. A5, B5, C5, D5, incubated for 30 days.

FIGURE 5. The colony semidiameter of Rhytidhysteron juglandis in dual cultures on MEA after 7 days. G. v. + R. j.: Granulobasidium vellereum was incubated 3 days ahead of R. juglandis. G. v. / R. j.: G. vellereum and R. juglandis were incubated simultaneously. R. j. + G. v.: G. vellereum was incubated 3 days after R. juglandis. R. j.: R. juglandis was incubated alone. Vertical bars indicate standard errors (honest significant difference tests, P <0.05). Each point represents the mean of five replicate, and error bars with different letters represent the significant difference of four treatments according to honest significant difference (HSD) test at the P <0.05

FIGURE 6. Effect of Granulobasidium vellereum on the disease severity of R. juglandis on walnut branches in planta. G. v. + R. j.: G. vellereum was inoculated 3 days ahead of R. juglandis. G. v. / R. j.: G. vellereum and R. juglandis were inoculated simultaneously. R. j. + G. v.: G. vellereum was inoculated 3 days after R. juglandis. R. j.: R. juglandis was inoculated alone. CK: sterilized MEA was inoculated. Vertical bar with different letters represents significantly different on walnut branches, according to honest significant difference (HSD) test at the P <0.05 level.