Corresponding author: Xinlei Fan (
Academic editor: C. Sharma Bhunjun
The
Lin L, Bai Y, Pan M, Tian C, Fan X (2023) Morphology and molecular analyses reveal three new species of Botryosphaeriales isolated from diseased plant branches in China. MycoKeys 97: 1–19.
The
With the modern taxonomic approaches applying, more than 30 novel species have been identified in the last five years (
Fresh specimens (woody branches and twigs with canker or dieback symptoms) were randomly collected in Beijing and Yunnan Province from the summer of 2021 to the autumn of 2022. The specimens were packed in kraft paper bags and transferred to the laboratory for fungal isolation following
Morphological observations were conducted based on conidiomata produced on infected plant tissues. The conidiomata were manually sectioned using a double-edged blade and examined under a dissecting microscope for macroscopic and microscopic characterization, while conidiomata structure and size were imaged with a Leica stereomicroscope (M205) (Leica Microsystems, Wetzlar, Germany). Conidia and other microstructures were selected randomly for observation using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon digital sight DSRi2 high-definition colour camera with differential interference contrast (
The fresh mycelium from
Genes used in this study with PCR primers and optimal annealing temperature.
Locus | PCR primers | PCR: thermal cycles: (Annealing temp. in bold) | Genus |
---|---|---|---|
ITS | ITS1/ITS4 | (95 °C: 30 s, |
|
LSU | LR0R/LR5 | (95 °C: 45 s, |
|
EF1-728F/EF1-986R | (95 °C: 15 s, |
||
|
Bt2a/Bt2b | (95 °C: 30 s, |
|
T1/Bt2b |
The sequences obtained in this study were supplemented with additional sequences obtained from GenBank (Suppl. material
Maximum Likelihood (
The BLAST results indicated that the 13 isolates in this study resided in
Substitution models used for Bayesian analyses in this study.
Analyses | Number of ingroup sequences | outgroup | Substitution models used for Bayesian analyses/Number of characters with gaps | |||
---|---|---|---|---|---|---|
ITS | LSU | tef1 | tub2 | |||
24 | SYM+G /553 | – | GTR+G /417 | – | ||
66 | GTR+I+G /494 | – | GTR+G /322 | GTR+I+G /448 | ||
36 | GTR+I /488 | HKY+I /562 | HKY+G/299 | – |
Five isolates clustered into two phylogenetic groups for the individual genes (ITS and
Phylogram of
Three isolates clustered in two clades for the individual genes (ITS,
Phylogram of
Five isolates clustered into three clades for the individual genes (ITS, LSU, and
Phylogram of
Based on DNA sequences and morphology, seven species belonging to three genera were identified. Of these,
See
China, Yunnan Province, Kunming City, Panlong District, Jinma County, Bailongsi Town,
Named after the collection site of the type specimen, Yanqing District in Beijing City.
Conidiomata pycnidial, immersed to semi immersed, erumpent from bark surface, multilocular, 650–1500 μm in diam. Disc straw to greenish olivaceous, circular to ovoid, 350–650 μm in diam, with one central ostiole per disc. Ostioles inconspicuous, sometimes covered below disc by lighter entostroma, 100–300 µm in diam. Locules multiple, irregularly arranged, subdivided frequently by invaginations with common walls. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, phialidic, 6.0–13.5 × 2.0–3.0 μm (av. ± S.D. = 10.7 ± 2.0 × 2.5 ± 0.2 μm). Paraphyses present, hyaline, smooth-walled, septate, unbranched, 26.5–37.5 × 2.0–3.0 μm (av. ± S.D. = 32.0 ± 3.5 × 2.4 ± 0.3 μm). Conidia aseptate, smooth, ellipsoid to subcylindrical, brown when mature, 16.0–21.5 × 6.0–9.5 μm (av. ± S.D. = 18.5 ± 1.3 × 7.7 ± 0.7 μm). Sexual morph not observed.
Colonies on
China, Beijing City, Yanqing District, Yeyahu National Wetland Park,
In the multi-gene analyses,
See
China, Yunnan Province, Diqing Tibetan Autonomous Prefecture, Shangri-La City, Sanba County, East Ring Road,
Named after the collection site of the type specimen, Baihuashan Natural Scenic Area in Beijing City.
Conidiomata pycnidial, superficial or immersed, separate, ovoid, 350–500 µm in diam, occasionally aggregated into botryose clusters. Disc black, 200–300 µm in diam. Ostioles single, central, papillate. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, holoblastic, cylindrical to subcylindrical or broadly lageniform, 7.5–16.0 × 3.5–6.5 μm (av. ± S.D. = 11.7 ± 2.2 × 4.6 ± 0.7 μm). Conidia1-septate, hazel to blackish brown, mostly truncate at the base and constricted at the septum or with a thickening at the base of the septum, moderately thick-walled, ovoid or oblong to ellipsoidal, 22.5–35.0 × 11.0–19.0 μm (av. ± S.D. = 27.9 ± 2.9 × 14.3 ± 2.2 μm).
Colonies on
China, Beijing City, Mentougou District, Qingshui County, Baihuashan Natural Scenic Area,
The isolates
See
China, Beijing City, Mentougou District, Qingshui County, Baihuashan Natural Scenic Area,
Named after the host genus,
Conidiomata pycnidial, scattered, subglobose to globose, erumpent, exuding faint yellow translucent conidial droplets from central ostioles, unilocular, 150–250 μm diam. Disc black, 80–200 µm in diam. Ostioles single, central, papillate, 21–35 µm. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, thin-walled, cylindrical, holoblastic, phialidic, proliferating internally with visible periclinal thickening, 5.5–14.0 × 2.5–4.0 μm (av. ± S.D. = 10.2 ± 2.5 × 3.2 ± 0.4 μm). Conidia initially hyaline, oval, both ends broadly rounded, aseptate, rarely becoming 1-septate, 23.0–31.0 × 9.5–12.5 μm (av. ± S.D. = 26.2 ± 2.5 × 10.8 ± 0.8 μm).
Colonies on
China, Beijing City, Haidian District, National Botanic Gardens,
See
China, Beijing City, Yanqing District, Zhangshanying County,
In this study, a total of 13 isolates are identified as seven species of
The fungi of
In this study, both
This research was funded by the National Natural Science Foundation of China (32101533), National Science and Technology Fundamental Resources Investigation Program of China (2021FY100900). We are grateful to Xiaohong Liang, Jing Han (the Experimental Teaching Centre, College of Forestry, Beijing Forestry University) for providing installed scientific equipment through the whole process. Lu Lin is grateful for the assistance of Ziqiang Wu (Southwest Forestry University) and Min Lin during the specimen collection, also Yixuan Li and Aoli Jia (Beijing Forestry University) during this study. Xinlei Fan would like to acknowledge the support of strain preservation of Chungen Piao and Minwei Guo (China Forestry Culture Collection Centre, Chinese Academy of Forestry, Beijing).
Strains used in the molecular analyses in this study
table (Excel spreadsheet)