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Research Article
New species and records of Phaeobotryon (Botryosphaeriales, Botryosphaeriaceae) from Larix in China
expand article infoYeting Zhu‡, Yingmei Liang‡, Cheng Peng‡
‡ Beijing Forestry University, Beijing, China

Corresponding author: Yingmei Liang ( liangym@bjfu.edu.cn )

Academic editor: Danushka Sandaruwan Tennakoon
© 2025 Yeting Zhu, Yingmei Liang, Cheng Peng.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhu Y, Liang Y, Peng C (2025) New species and records of Phaeobotryon (Botryosphaeriales, Botryosphaeriaceae) from Larix in China. MycoKeys 112: 1-15. https://doi.org/10.3897/mycokeys.112.139053
Open Access

Abstract

During the fungal investigations of Larix hosts in China, ten isolates of Phaeobotryon were obtained from dead and dying branches. Morphological characteristics and phylogenetic analyses of combined ITS, LSU, and tef1-α loci revealed the presence of two new species, P. laricinum and P. longiparaphysium, as well as two new host records for P. aplosporum and P. rhois from L. olgensis. In this study, we provide descriptions and illustrations of these species, thereby enriching the diversity within the Phaeobotryon taxa.

Key words

Ascomycota, molecular phylogeny, morphology, taxonomy

Introduction

Phaeobotryon is a monophyletic genus typed with P. cercidis, belonging to Dothideomycetes, Botryosphaeriales, Botryosphaeriaceae (Phillips et al. 2008; Liu et al. 2012). This genus was established by Theissen and Sydow (1915) to classify Dothidea cercidis, which produces light brown, two-septate ascospores. Subsequently, Phaeobotryon was considered a synonym of Botryosphaeria based on the morphology of the sexual morph (Von Arx and Müller 1954, 1975). However, more recent morphological and phylogenetic data indicated that Phaeobotryon constitutes a distinct lineage within the Botryosphaeriaceae, characterized by the 2-septate, brown ascospores with an apiculus at each end (Phillips et al. 2008, 2013; Liu et al. 2012; Zhang et al. 2021).

As of now, 16 epithets of Phaeobotryon have been listed in Index Fungorum (www.indexfungorum.org; accessed on 10 October 2024). Of these, P. disruptum, P. euganeum, and P. visci have been removed from Phaeobotryon and reassigned to their respective genera (Von Arx and Müller 1954; Barr 2001). Consequently, 13 species are accepted within the genus, of which P. cercidis and P. quercicola lack available DNA sequence data. Species of Phaeobotryon have primarily been discovered in Asia, Europe, and North America. Members of this genus exhibit a wide range of hosts, encompassing 24 genera, including three genera of gymnosperms: Calocedrus, Juniperus, and Platycladus (Phillips et al. 2005, 2008; Abdollahzadeh et al. 2009; Fan et al. 2015; Daranagama et al. 2016; Zhu et al. 2018; Pan et al. 2019; Wijayawardene et al. 2021; Zhang et al. 2021; Hattori and Masuya 2023; Lin et al. 2023; Peng et al. 2023; Wu et al. 2024; Zhou et al. 2024). Until now, Phaeobotryon has not been reported to inhabit Larix.

The number of Phaeobotryon species has increased rapidly in recent years, with nine of the 13 species published since 2015, seven of which have been described in China. This trend suggests that the genus exhibits significant diversity within the country. During our investigation of fungal diseases affecting Larix in China, four Phaeobotryon species were collected from L. gmelinii, L. olgensis, and L. gmelinii var. principis-rupprechtii in Heilongjiang, Jilin, and Hebei Provinces, respectively. This study used phylogenetic analysis and morphological comparisons to describe new species and document new host records, thereby enriching the fungal taxa within Phaeobotryon.

Materials and methods

Fungal isolation

Fresh specimens were collected from Larix gmelinii, L. olgensis, and L. gmelinii var. principis-rupprechtii in Heilongjiang, Jilin, and Hebei Provinces, respectively. The specimens were carefully packed in kraft paper bags and transported to the laboratory for fungal isolation. Isolates were obtained using the single spore isolation method as described by Chomnunti et al. (2014). Following incubation at 25 °C, single germinating conidia were transferred to fresh plates of PDA. The cultures have been deposited in the China Forestry Culture Collection Center (CFCC), while the specimens are stored in the Museum of Beijing Forestry University (BJFC).

Morphology

Morphological observations were conducted on conidiomata produced on infected plant tissues. The conidiomata were manually sectioned using a double-edged blade and examined under a dissecting microscope for both macroscopic and microscopic characterization. The structure and size of the conidiomata were imaged with a Leica stereomicroscope (M205) (Leica Microsystems, Wetzlar, Germany). Additionally, conidia and other microstructures were randomly selected for observation using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan), which was equipped with a Nikon digital sight DSRi2 high-definition color camera featuring differential interference contrast (DIC). More than 50 conidia were measured per species, and 30 measurements were taken of other morphological structures. Colony characteristics, including color and texture on PDA at 25 °C, were observed and recorded over 14 days. The colony colors were determined based on the color charts of Rayner (1970).

DNA extraction, amplification, and sequencing

DNA was extracted using the modified CTAB method (Doyle and Doyle 1990) and stored at -20 °C. To confirm species identity, the internal transcribed spacer (ITS) region of all isolates was sequenced. The resulting sequences were subsequently compared with those in GenBank using the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Following genus-level confirmation, additional loci, including the nuclear ribosomal large subunit (LSU) and partial translation elongation factor 1-alpha (tef1-α), were amplified. The PCR mixture for all regions comprised 1 µL of DNA template, 1 µL of each 10 µM primer, 10 µL of T5 Super PCR Mix (which contains Taq polymerase, dNTP, and Mg2+; Beijing TisingKe Biotech Co., Ltd., Beijing, China), and 7 µL of sterile water. The primers and PCR conditions are detailed in Table 1. PCR products were electrophoresed in a 1% agarose gel and subsequently sequenced by Beijing TisingKe Biotech Co., Ltd. (Beijing, China). The forward and reverse reads were edited and assembled using Seqman v.7.1.0 software.

Table 1.
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Loci, PCR primers, protocols, and references used in this study.

Locus Primers Thermal cycles Reference
ITS ITS1/ITS4 (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles White et al. (1990)
LSU LR0R/LR5 (95 °C: 45 s, 55 °C: 45 s, 72 °C: 1 min) × 35 cycles Vilgalys and Hester (1990), Rehner and Samuels (1994)
tef1-α EF1-728F/EF1-986R(EF1-1567R) (95 °C: 15 s, 55 °C: 20 s, 72 °C: 1 min) × 35 cycles Carbone and Kohn (1999), Rehner and Buckley (2005)

Phylogenetic analyses

All sequences generated in this study were submitted to GenBank, and reference sequences from known species were downloaded from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov) to construct the phylogenetic analysis (Table 2). The individual datasets for each gene region were aligned separately using MAFFT v. 6.0 (Katoh and Standley 2013) and subsequently trimmed at both terminal ends in MEGA v. 6.0 (Tamura et al. 2013). MEGA v. 6 software was utilized to align and edit the sequences, with Lasiodiplodia theobromae (CBS 164.96) selected as the outgroup. Multi-gene phylogenetic analyses employing maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) were conducted using PAUP v. 4.0b10, raxmlGUI v. 1.5b1, and MrBayes v. 3.1.2 software, respectively. The resulting phylogenetic tree was visualized using Figtree v. 1.4.0 and modified with AI CS v. 5. The support rate of MP and ML analysis was greater than 50%, and the posterior probability of BI analysis was greater than 0.9 as presented in the tree.

Table 2.
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Taxa used for molecular phylogenetic analyses and their GenBank accession numbers. (T) = ex-type strains. Isolates in this study are shown in bold.

Species Strain Host Origin GenBank accession numbers
ITS LSU tef1-α
Alanphillipsia aloeicola CBS 138896 = CPC 23674T Aloe sp. South Africa Kp004444 Kp004472 Mt592027
A. aloeigena CBS 136408 = CPC 21286T Aloe melanocantha South Africa Kf777137 Kf777193 –
A. aloes CBS 136410 = CPC 21298T Aloe dichotoma South Africa Kf777138 Kf777194 –
A. aloetica CBS 136409 = CPC 21110, 21109T Aloe sp. South Africa Kf777139 Kf777195 Mt592028
A. euphorbiae CBS 136411 = CPC 21629, 21628T Euphorbia sp. South Africa Kf777140 Kf777196 Mt592029
Barriopsis archontophoenicis MFLUCC 14-1164T Archontophoenix alexandrae Thailand Kx235306 Kx235307 –
B. iraniana CBS 124698 = IRAN 1448CT Mangifera indica Iran Fj919663 Kf766318 Fj919652
B. iraniana CBS 124699 = IRAN 1449C Olea sp. Iran Fj919665 Kx464241 Fj919654
B. stevensiana CBS 174.26T Citrus sp. Cuba Eu673330 Dq377857 Eu673296
B. tectonae CBS 137786 = MFLUCC 12-0381 = CMW 40687T Tectona grandis Thailand Kj556515 Mh878606 Kj556516
B. thailandica MFLUCC 14-1190 = KUMCC 16-0185T Tectona grandis Thailand Ky115675 – Ky115676
Lasiodiplodia theobromae CBS 164.96T Fruit along coral reef coast Papua Ay640255 Eu673253 Ku696383
Oblongocollomyces variabilis CBS 121774 = CMW 25419 = CAMS 1174T Acacia karroo Namibia Eu101312 Kx464536 Eu101357
O. variabilis CBS 121775 = CMW 25421 = CAMS 1176 Aplosporella karoo Namibia Eu101314 Mt587319 Eu101359
O. variabilis CBS 121776 = CMW 25422 = CAMS 1177 Acacia mellifera South Afri Eu101326 Kx464537 Eu101371
Phaeobotryon aplosporum CFCC 53773 Syzygium aromaticum China Mn215835 Mn215870 Mn205995
P. aplosporum CFCC 53774 Syzygium aromaticum China Mn215836 Mn215871 Mn205996
P. aplosporum CFCC 53775T Rhus typhina China Mn215837 Mn215872 –
P. aplosporum CFCC 53776 Rhus typhina China Mn215838 Mn215873 Mn205997
P. aplosporum CFCC 70810 Larix olgensis China Pp960186 Pp960196 Pq046939
P. aplosporum CFCC 70811 Larix olgensis China Pp960187 Pp960197 Pq046940
P. cupressi CBS 124700 = IRAN 1455CT Cupressus semipervirens Iran Fj919672 Kx464538 Fj919661
P. cupressi CBS 124701 = IRAN 1458C Cupressus semipervirens Iran Fj919671 Kx464539 Fj919660
P. cupressi IRAN 1454C Cupressus semipervirens Iran Fj919673 – Fj919662
P. fraxini CFCC 70762T Fraxinus chinensis China Pp188527 Pp177348 –
P. fraxini CFCC 70763 Fraxinus chinensis China Pp188528 Pp177349 –
P. juniperi JU 001T Juniperus formosana China Op941637 Op941644 Op948218
P. juniperi JU 005 Juniperus formosana China Op941638 Op941645 Op948219
P. juniperi JU 007 Juniperus formosana China Op941639 Op941646 Op948220
P. laricinum CFCC 70804 Larix olgensis China Pp960188 Pp960198 Pq046941
P. laricinum CFCC 70805T Larix olgensis China Pp960189 Pp960199 Pq046942
P. laricinum CFCC 70806 Larix gmelinii China Pp960190 Pp960200 Pq046943
P. longiparaphysium CFCC 70807T Larix gmelinii var. principis-rupprechtii China Pp960193 Pp960203 Pq046946
P. longiparaphysium CFCC 70808 Larix gmelinii var. principis-rupprechtii China Pp960194 Pp960204 Pq046947
P. longiparaphysium CFCC 70809 Larix olgensis China Pp960195 Pp960205 Pq046948
P. mamane CBS 122980 = CPC 12440T Sophora chrysophylla USA Eu673332 Eu673248 Eu673298
P. mamane CPC 12442 Sophora chrysophylla USA Eu673333 Dq377899 Eu673299
P. mamane CPC 12443 Sophora chrysophylla USA Eu673334 Eu673249 Eu673300
P. negundinis CAA 797 Acer negundo Russia Kx061513 – Kx061507
P. negundinis CAA 798 Ligustrum vulgare Russia Kx061514 – Kx061508
P. negundinis CAA 799 Forsythia intermedia Russia Kx061515 – Kx061509
P. negundinis CPC 33388 Dead stem Ukraine Mt587543 Mt587324 Mt592277
P. negundinis CPC 34752 Acer negundo Ukraine Mt587544 Mt587325 Mt592278
P. negundinis MFLUCC 15-0436T Acer negundo Russia Ku820970 – Ku853997
P. platycladi CFCC 58799T Platycladus orientalis China Oq651172 Oq652543 Oq692930
P. platycladi CFCC 58800 Platycladus orientalis China Oq651173 Oq652544 Oq692931
P. rhoinum CFCC 52449 Rhus typhina China Mh133923 Mh133940 Mh133957
P. rhoinum CFCC 52450T Rhus typhina China Mh133924 Mh133941 Mh133958
P. rhoinum CFCC 52451 Rhus typhina China Mh133925 Mh133942 Mh133959
P. rhois CFCC 89662 = CCTCC AF2014017T Rhus typhina China Km030584 Km030591 Km030598
P. rhois CFCC 89663 = CCTCC AF2014016 Rhus typhina China Km030585 Km030592 Km030599
P. rhois CFCC 58679 Populus alba var. pyramidalis China Oq651171 Oq652542 Oq692929
P. rhois CFCC 52448 Rhus typhina China Mh133922 Mh133939 Mh133956
P. rhois CFCC 53777 Platycladus orientalis China Mn215839 Mn215874
P. rhois CFCC 53779 Rhamnus dahuricus China Mn215841 Mn215876 Mn205999
P. rhois CFCC 53780 Dioscorea nipponica China Mn215842 Mn215877 Mn206000
P. rhois CFCC 70812 Larix olgensis China Pp960191 Pp960201 Pq046944
P. rhois CFCC 70813 Larix olgensis China Pp960192 Pp960202 Pq046945
P. spiraeae CFCC 53925T Spiraea salicifolia China Om049420 Om049432 –
P. spiraeae CFCC 53926 Spiraea salicifolia China Om049421 Om049433 –
P. spiraeae CFCC 53927 Spiraea salicifolia China Om049422 Om049434 –
P. ulmi 94-13 Ulmus pumila USA Af243398 – –
P. ulmi CBS 114123 = UPSC 2552 Ulmus glabra Sweden Mt587539 Mt587320 Mt592273
P. ulmi CBS 138854 = CPC 24264T Ulmus leavis Germany Mt587540 Mt587321 Mt592274
P. ulmi CBS 123.30 = ATCC 24443 = DSM 2491 = MUCL 10057 Ulmus sp. USA Kx464232 Dq377861 Kx464766
P. ulmi CBS 174.63 Ulmus glabra Finland Mt587541 Mt587322 Mt592275
P. ulmi CMH 299 House dust USA Kf800390 – –
P. ulmi PB 11f Ulmus glabra Poland Mk134682 – –
Sphaeropsis citrigena ICMP 16812T Citrus sinensis New Zealand Eu673328 Eu673246 Eu673294
S. citrigena ICMP 16818 Citrus sinensis New Zealand Eu673329 Eu673247 Eu673295
S. eucalypticola CBS 133993 = MFLUCC 11-0579 = CPC 21560 = BT 021T Eucalyptus sp. Thailand Jx646802 Jx646819 Jx646867
S. eucalypticola MFLUCC 11-0654 Eucalyptus sp. Thailand Jx646803 Jx646820 Jx646868
S. porosa CBS 110496 = CPC 5132 = JM 29 = STE-U 5132T Vitis vinifera South Africa Ay343379 Dq377894 Ay343340
S. porosa CBS 110574 = STE-U 5046 Vitis vinifera South Africa Ay343378 Dq377895 Ay343339
S. visci CBS 100163 = 12273 Viscum album Luxembourg Eu673324 Dq377870 Eu673292
S. visci CBS 122526 = CAP 350T Viscum album Ukraine Eu673326 Kx464550 –
S. visci CBS 122527 = CAP 349 Viscum album Ukraine Eu673327 Kx464551 Kx464776
S. visci CBS 186.97 Viscum album Germany Eu673325 Dq377868 Eu673293
S. visci CPC 33386 Dead leaf Ukraine Mt587557 Mt587326 Mt592305
S. visci CPC 35421 Viscum album Germany Mt587558 Mt587327 –
S. visci CPC 35525 Eucalyptus grandis Australia Mt587559 Mt587328 Mt592306

Results

Phylogenetic analysis

The gene loci of ITS, LSU, and tef1-α were combined and analyzed to infer the phylogenetic placement of our isolates in the genus Phaeobotryon. The dataset includes 81 sequences; of these, Lasiodiplodia theobromae (CBS 164.96) was set as the outgroup taxon. The combined dataset after alignment consisted of 1,744 characters, including 508 characters in ITS, 757 characters in LSU, and 469 characters in tef1-α gaps that were included in the phylogenetic analysis. In the alignment, 1,346 characters are constant, 120 variable characters are parsimony-uninformative, and 120 characters are parsimony-informative. In ML analysis based on the combined gene dataset, the matrix had 488 distinct alignment patterns. Estimated base frequencies are as follows: A = 0.226764, C = 0.257594, G = 0.287105, T = 0.228538; substitution rates: AC = 1.111156, AG = 2.606477, AT = 0.720936, CG = 1.166284, CT = 5.223120, GT = 1.000000. Trees from Bayesian analyses and MP were identical to that of the ML tree shown (Fig. 1). Isolates CFCC 70804, CFCC 70805, and CFCC 70806, as well as isolates CFCC 70807, CFCC 70808, and CFCC 70809, are clustered into separate lineages and are designated as two new species. Isolates CFCC 70810 and CFCC 70811 are grouped with P. aplosporum, while isolates CFCC 70812 and CFCC 70813 are grouped with P. rhois, thus designated as species P. aplosporum and P. rhois, respectively.

Figure 1. 

Phylogenetic tree inferred from ML analysis based on combined ITS, LSU, and tef1-α sequence data of Phaeobotryon isolates. The tree was rooted in Lasiodiplodia theobromae (CBS 164.96). The MP, ML (≥ 50%), and BI (≥ 0.9) bootstrap values are given at nodes (MP/ML/BI). Isolates from this study are marked in blue, ex-type strains are marked in bold, and new species are in a colored font.

Taxonomy

Phaeobotryon laricinum Y.T. Zhu & Y.M. Liang, sp. nov.

MycoBank No: 854522
Fig. 2

Etymology

Named after the host genus on which it was collected, Larix.

Descriptions

Sexual morph : Not observed. Asexual morph: Conidiomata pycnidial, scattered, immersed, or semi-immersed to erumpent from bark surface, globose to ovoid, unilocular, 365–820 µm diam. Disc black, 215–360 µm in diam. Ostioles single, central, 35–75 µm. Conidiophores reduced to conidiogenous cells. Paraphyses present, hyaline, thin-walled, arising from the conidiogenous layer, extending above the level of developing conidia, tip rounded, aseptate, up to 60.5 × 2.5 µm. Conidiogenous cells hyaline, smooth, thin-walled, holoblastic, cylindrical, phialidic, proliferating internally with visible periclinal thickening, 11.0–41.0 × 1.0–3.5 µm. Conidia initially hyaline, becoming brown with age, dark brown, aseptate, smooth with granular contents, guttulate, thick-walled, oblong to cylindrical, straight, both ends broadly rounded, 27.5–37.0 × 10.0–18.0 µm (av. ± S.D. = 32.2 ± 2.08 × 14.01 ± 1.77 µm), L/W = 2.3 ± 0.3.

Figure 2. 

Phaeobotryon laricinum (BJFC-S2370) A habit of conidiomata on twig B, D longitudinal section through a conidioma C, E transverse section of a conidioma F–K conidiogenous cells and conidia L conidia M colony on PDA after 14 days. Scale bars: 500 µm (A–C); 50 µm (D, E); 10 µm (F–L);

Culture characteristics

Colonies on PDA flat, spreading, with flocculent mycelium and uneven edges, initially white, gradually turning greenish-grey from center, finally becoming black, covering 40–50 mm after 7 days at 25 °C.

Materials examined

China • Jilin Province, Yanbian Korean Autonomous Prefecture, Yanji City, Maoershan National Forest (42°51'12.96"N, 129°28'24.06"E), alt. 297 m, on branches of Larix olgensis, 7, Sept, 2022, C. Peng, X.Y. Zhang (holotype BJFC-S2370, ex-holotype culture CFCC 70805; isotype BJFC-2371, ex-isotype culture CFCC 70806); China • Heilongjiang Province, Greater Khingan Mountains, Tahe County, Qixiashan Mountains (52°20'32.96"N, 124°41'48.27"E), alt. 456 m, on branches of Larix gmelinii, 10, Sept, 2021, R. Wang, W.T. Yu (BJFC-S2369, living culture CFCC 70804).

Notes

Phaeobotryon currently comprises 13 species, all of which have reported asexual morphs except for P. cercidis (Phillips et al. 2005, 2008; Abdollahzadeh et al. 2009; Fan et al. 2015; Daranagama et al. 2016; Zhu et al. 2018; Pan et al. 2019; Wijayawardene et al. 2021; Zhang et al. 2021; Lin et al. 2023; Peng et al. 2023; Wu et al. 2024). However, P. cercidis has been reported on Cercis canadensis in the USA (Phillips et al. 2008), revealing differences from P. laricinum in terms of both geographic region (China) and host (Larix). The new species can be distinguished from other known species based on conidial characteristics (Table 3). Specifically, P. laricinum conidia are aseptate and can be differentiated from other species in the genus, except for P. negundinis, P. quercicola, and P. spiraeae. Furthermore, they can be distinguished by conidial color (dark brown) from P. quercicola (hyaline). Additionally, the conidial size of P. laricinum (27.5–37 × 10–18 μm) is significantly larger than that of both P. negundinis (16–24.5 × 7.9–11.5 μm) and P. spiraeae (21–28.5 × 8.5–13.5 μm). Moreover, P. laricinum (L/W = 2.3 ± 0.3) can be distinguished by its larger conidial L/W ratio when compared to the new species P. longiparaphysium (L/W = 1.7 ± 0.2) (Table 3). Phylogenetically, P. laricinum is distinct from other Phaeobotryon species, which are grouped within a separate clade that receives high support (MP/ML/BI = 100/100/1) (Fig. 1). Therefore, P. laricinum is introduced as a novel species.

Table 3.
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Conidia comparison of species in Phaeobotryon (new species in bold).

Species Septation colour size (μm) Reference
Phaeobotryon aplosporum aseptate dark brick 15–21.5 × 5.5–7 Pan et al. 2019
P. cercidis No record No record No record Phillips et al. 2008
P. cupressi 1-septate brown 19.8–30 × 10.2–17, L/W = 2 ± 0.3 Abdollahzadeh et al. 2009
P. fraxini 1-septate brownish yellow to dark brown 13.0–20.0 × 7.0–10.0 Wu et al. 2024
P. juniperi 1-septate dark brown 23–28.5 × 11.5–14 Peng et al. 2023
P. laricinum aseptate dark brown 27.5–37 × 10–18, L/W = 2.3 ± 0.3 This study
P. longiparaphysium aseptate dark brown 24–36.5 × 15–20.5, L/W = 1.7 ± 0.2 This study
P. mamane 1(–2)-septate brown 30–43 × 12–16 Phillips et al. 2008, 2013
P. negundinis aseptate dark brown 16–24.5 × 7.9–11.5 Daranagama et al. 2016
P. platycladi aseptate, rarely becoming 1-septate initially hyaline 23.0–31.0 × 9.5–12.5 Lin et al. 2023
P. quercicola aseptate hyaline 24–38 × 11–21.2, L/W = 2.1 Phillips et al. 2005, 2008
P. rhoinum 1-septate brown 18.5–21.5 × 7–9 Zhu et al. 2018
P. rhois 1-septate brown 19–25 × 10–12 Fan et al. 2015
P. spiraeae aseptate dark brown 21–28.5 × 8.5–13.5 Wijayawardene et al. 2021
P. ulmi 1-septate brown 26–34.5 × 15–20 Zhang et al. 2021

Phaeobotryon longiparaphysium Y.T. Zhu & Y.M. Liang, sp. nov.

MycoBank No: 854523
Fig. 3

Etymology

Named after the long paraphyses of conidiomata.

Descriptions

Sexual morph : Not observed. Asexual morph: Conidiomata pycnidial, scattered, immersed or semi-immersed, globose to ovoid, unilocular, 280–550 µm diam. Disc black, 180–330 µm in diam. Ostioles single, central, 65–115 µm. Paraphyses present, hyaline, thin-walled, arising from the conidiogenous layer, extending above the level of developing conidia, tip rounded, aseptate, up to 74.5 × 2.5 µm. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, thin-walled, holoblastic, cylindrical, phialidic, proliferating internally with visible periclinal thickening, 9.5–29.0 × 1.0–4.0 µm. Conidia initially hyaline, becoming brown with age, dark brown, thick-walled, oval, with obtuse or gradually acute apex, rounded, gradually acute base, aseptate, 24.0–36.5 × 15.0–20.5 µm (av. ± S.D. = 31.69 ± 2.86 × 18.34 ± 1.01 µm), L/W = 1.7 ± 0.2.

Figure 3. 

Phaeobotryon longiparaphysium (BJFC-S2372) A habit of conidiomata on twig B, E longitudinal section through a conidioma C, F transverse section of a conidioma D, G–J conidiogenous cells and conidia K conidia L colony on PDA after 14 days. Scale bars: 500 µm (A–C); 50 µm (E, F); 10 µm (D, G–K).

Culture characteristics

Colonies on PDA with aerial mycelium, thick and fluffy at the edge, margin with undulate and irregular, initially white, gradually turning brown, finally becoming black, covering 40–50 mm after 7 days at 25 °C.

Materials examined

China • Hebei Province, Chengde City, Saihanba Forest Farm (42°28'37.08"N, 117°25'45.49"E), alt. 1657 m, on branches of Larix gmelinii var. principis-rupprechtii, 10, Jul. 2023, C.M. Tian, C. Peng, S.J. Li, Y. Yuan, M.W. Zhang (holotype BJFC-S2372, ex-holotype cultures CFCC 70807, CFCC 70808). China • Jilin Province, Yanbian Korean Autonomous Prefecture, Yanji City, Maoershan National Forest (42°51'12.96"N, 129°28'24.06"E), alt. 297 m, on branches of Larix olgensis, 7, Sept, 2022, C. Peng, X.Y. Zhang (BJFC-S2373, living culture CFCC 70809).

Notes

Phaeobotryon longiparaphysium formed a distinct clade (MP/ML/BI = 97/99/1) in the multi-locus analyses and is sister to P. laricinum (Fig. 1). These two species can be distinguished based on the ITS, LSU, and tef1-α loci, with P. longiparaphysium exhibiting 29 bp differences from P. laricinum (5/535 in ITS, 5/780 in LSU, and 19/291 in tef1-α). Furthermore, P. longiparaphysium (L/W = 1.7 ± 0.2) can be distinguished from P. laricinum (L/W = 2.3 ± 0.3) by its smaller conidial L/W ratio (Table 3).

Phaeobotryon aplosporum M. Pan & X.L. Fan, Mycol. Prog. 18(11): 1356 (2019)

Descriptions

See Pan et al. 2019.

Materials examined

China • Jilin Province, Yanbian Korean Autonomous Prefecture, Yanji City, Maoershan National Forest (42°51'12.96"N, 129°28'24.06"E), alt. 297 m, on branches of Larix olgensis, 7, Sept, 2022, C. Peng, X.Y. Zhang (BJFC-S2375, living culture CFCC 70810, CFCC 70811).

Notes

Phaeobotryon aplosporum was first identified in Rhus typhina and Syzygium aromaticum (Pan et al. 2019). Since then, this species has also been reported in Juglans mandshurica (Lin et al. 2023), Wisteria floribunda, Malus sp., and Kerria japonica (Hattori and Masuya 2023). In this study, we observed the asexual morph of P. aplosporum (Fig. 4), which features unilocular conidiomata that differ from previously published descriptions for other hosts. It is known from previous literature reports that P. quercicola exhibits both unilocular conidiomata and multilocular conidiomata (Phillips et al. 2008), and P. cupressi mostly unilocular conidiomata on pine needles and mostly multilocular conidiomata on Populus twigs (Abdollahzadeh et al. 2009). This suggests that both unilocular and multilocular conidiomata may coexist within the same species in this genus. Additionally, the other characteristics of P. aplosporum in L. olgensis we observed (Fig. 4) are consistent with those noted from other hosts. Phylogenetically, the isolates CFCC 70810 and CFCC 70811 clustered within a clade with P. aplosporum, demonstrating high statistical support (MP/ML/BI = 90/98/0.99) (Fig. 1). Therefore, the isolates CFCC 70810 and CFCC 70811 are identified as P. aplosporum. This study extends the host range of P. aplosporum to include L. olgensis.

Figure 4. 

Phaeobotryon aplosporum (BJFC-S2375) A habit of conidiomata on twig B longitudinal section through a conidioma C transverse section of a conidioma D, E conidiogenous cells and conidia F conidia G colony on PDA after 7 days. Scale bars: 500 µm (A); 50 µm (B, C); 10 µm (D–F).

Phaeobotryon rhois C.M. Tian, X.L. Fan & K.D. Hyde, Phytotaxa 205(2): 95 (2015)

Descriptions

See Fan et al. 2015.

Materials examined

China • Jilin Province, Yanbian Korean Autonomous Prefecture, Yanji City, Maoershan National Forest (42°51'12.96"N, 129°28'24.06"E), alt. 297 m, on branches of Larix olgensis, 7, Sept, 2022, C. Peng, X.Y. Zhang (BJFC-S2374, living culture CFCC 70812, CFCC70813).

Notes

Phaeobotryon rhois was first discovered and reported on Rhus typhina (Fan et al. 2015). This species has also been isolated from diseased branches of the hosts Dioscorea nipponica, Platycladus orientalis, and Rhamnus davurica (Pan et al. 2019), as well as from Populus alba var. pyramidalis (Lin et al. 2023). Morphologically, its asexual morph (Fig. 5) aligns with the description provided by Fan et al. (2015). Phylogenetically, the isolates CFCC 70812 and CFCC 70813 clustered within a clade alongside P. rhois, demonstrating high statistical support (MP/ML/BI = 96/99/0.99) (Fig. 1). The current study expands its host range to include L. olgensis.

Figure 5. 

Phaeobotryon rhois (BJFC-S2374) A habit of conidiomata on twig B longitudinal section through a conidioma C transverse section of a conidioma D–G conidiogenous cells and conidia H conidia I colony on PDA after 7 days. Scale bars: 500 µm (A); 50 µm (B, C); 10 µm (D–H).

Discussion

This paper describes and illustrates four species of Phaeobotryon from China. These species included two new species, namely P. laricinum and P. longiparaphysium, and two new host records, P. aplosporum and P. rhois from L. olgensis. This is the first time that this genus has been discovered from Larix.

Phillips et al. (2008) summarized the characteristics of Phaeobotryon as 2-septate, brown ascospores with an apiculus at each end. However, among 13 species of Phaeobotryon, only P. mamane and P. quercicola have both sexual and asexual morphs described so far, while only the sexual morphs of P. cupressi are documented. The remaining ten species have only asexual morphs described (Phillips et al. 2005, 2008; Abdollahzadeh et al. 2009; Fan et al. 2015; Daranagama et al. 2016; Zhu et al. 2018; Pan et al. 2019; Wijayawardene et al. 2021; Zhang et al. 2021; Lin et al. 2023; Peng et al. 2023; Wu et al. 2024). Therefore, recent papers have distinguished between species of Phaeobotryon by asexual morphological characteristics, such as the conidial septation and conidial size (Daranagama et al. 2016; Zhu et al. 2018; Pan et al. 2019; Zhang et al. 2021; Peng et al. 2023; Wu et al. 2024). However, there are many similarities in conidial morphology, with 1(–2) septate or aseptate conidia, similar pigmentation variations, and some species have overlapping conidial sizes. For example, P. juniperi and P. platycladi have overlapping sizes of conidia (Table 3). So, the combination of morphology and phylogenetics is essential for further clarifying the affinities between species in academic research.

Numerous reports have documented the presence of Phaeobotryon on diseased plants (Zhu et al. 2018; Wijayawardene et al. 2021; Zhang et al. 2021; Lin et al. 2023; Peng et al. 2023; Wu et al. 2024), with P. negundinis identified as a causal agent of branch blight in Malus spp. (Evgeny and Walid 2023). However, some studies do not include inoculation experiments necessary to definitively establish the pathogenicity of these species. Similarly, the four Phaeobotryon species described in this paper were found on dead and dying Larix branches; their pathogenicity, however, requires confirmation through further inoculation experiments. Future research should focus on elucidating the pathogenicity of Phaeobotryon while exploring the diversity of its species.

Acknowledgments

We are grateful to Yong Li (China Forestry Culture Collection Center, Chinese Academy of Forestry, Beijing) for support of strain preservation during this study.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study was funded by the National Key Research and Development Program of China (Project No.: 2021YFD1400300).

Author contributions

Yingmei Liang and Yeting Zhu conceived and designed the project. Yingmei Liang and Cheng Peng collected specimens, and Yeting Zhu and Cheng Peng identified isolations. Yingmei Liang and Yeting Zhu performed phylogenetic analyses, and Yeting Zhu curated these data. Yeting Zhu drafted the manuscript. Yeting Zhu, Yingmei Liang, and Cheng Peng revised the manuscript. All authors read and approved the final manuscript.

Author ORCIDs

Yeting Zhu https://orcid.org/0009-0004-6613-1561

Yingmei Liang https://orcid.org/0000-0002-1690-5512

Cheng Peng https://orcid.org/0009-0005-9619-8246

Data availability

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

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