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Species of Diaporthe (Diaporthaceae, Diaporthales) associated with Alnus nepalensis leaf spot and branch canker diseases in Xizang, China
expand article infoJieting Li‡§, Yi Li§‡, Jiangrong Li‡§, Ning Jiang|
‡ Xizang Agricultural and Animal Husbandry University, Linzhi, China
§ National Forest Ecosystem Observation & Research Station of Linzhi Xizang, Linzhi, China
| Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, China

Corresponding author: Jiangrong Li ( jrong06@xza.edu.cn )

Corresponding author: Ning Jiang ( n.jiang@caf.ac.cn )

Academic editor: Nattawut Boonyuen
© 2025 Jieting Li, Yi Li, Jiangrong Li, Ning Jiang.
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: Li J, Li Y, Li J, Jiang N (2025) Species of Diaporthe (Diaporthaceae, Diaporthales) associated with Alnus nepalensis leaf spot and branch canker diseases in Xizang, China. MycoKeys 116: 185-204. https://doi.org/10.3897/mycokeys.116.142750
Open Access

Abstract

Alnus nepalensis is an important tree species in the Himalayas with significant ecological and economic roles. During disease surveys in Xizang, China, we observed leaf spot and branch canker symptoms on this tree. Fungal isolates associated with these diseases were collected and identified based on morphological characteristics and phylogenetic analysis of ITS, cal, his3, tef1, and tub2 sequences. As a result, Diaporthe alnicola sp. nov. and D. amygdali were identified from the leaf spots, while D. linzhiensis was identified to be associated with the cankered branches. This study identifies pathogenic species from alder trees, providing a foundation for future disease management and forest health research.

Key words:

Alder, molecular phylogeny, novel taxa, plant disease, Sordariomycetes, taxonomy

Introduction

Alnus nepalensis (Nepalese alder) is a tree species of significant ecological and economic importance, particularly in the temperate and subtropical regions of the Himalayas, including Xizang, Nepal, and northern India (Sharma et al. 1998; Xia et al. 2023). This plant fulfills a crucial role in maintaining the ecological balance of forest ecosystems (Tobita et al. 2016; Sen et al. 2022). Beyond its ecological importance, A. nepalensis also has considerable economic value (Saxena et al. 2016). Given its critical role in both ecosystem function and local economies, any threats to A. nepalensis populations, such as leaf spot and canker diseases, could have severe consequences for forest health.

Diaporthe is a pathogenic fungal genus in the Diaporthaceae (Diaporthales, Sordariomycetes, Ascomycota) (Udayanga et al. 2012; Dissanayake et al. 2017; Jiang et al. 2025). Species of this genus are commonly associated with plant diseases, acting as pathogens, endophytes, or saprobes (Dissanayake et al. 2020; Dong et al. 2021; Jiang et al. 2021). For example, D. hsinchuensis and five other species have been shown to cause leaf spots on Camellia sinensis (Ariyawansa et al. 2021). Several other Diaporthe species were shown to be associated with branch canker, dieback, and stem blight diseases (Guarnaccia et al. 2020; Guo et al. 2020; Bai et al. 2023). Additionally, D. biconispora and 15 other species from this genus were described as endophytes in Citrus (Huang et al. 2015).

Diaporthe is a species-rich genus with nearly 1,300 epithets listed in Index Fungorum (https://www.indexfungorum.org/). Over the past decade, many new species of this genus have been described based on both morphological characteristics and molecular phylogeny (Udayanga et al. 2014, 2015; Guarnaccia and Crous 2017; Yang et al. 2017, 2020, 2021; Fan et al. 2018; Manawasinghe et al. 2019; Huang et al. 2021; Sun et al. 2021; Cao et al. 2022; Lambert et al. 2023; Zhu et al. 2023, 2024; Liu et al. 2024). However, the species concepts of several taxa have been re-evaluated in recent years using the genealogical concordance phylogenetic species recognition (GCPSR) principle and coalescence-based models, such as the General Mixed Yule-Coalescent (GMYC) and Poisson Tree Processes (PTP). These re-evaluations have led to the synonymization of several species (Hilário et al. 2021a, 2021b; Dissanayake et al. 2024). For example, recent studies demonstrated that what was once thought to be a complex of nine species (Diaporthe amygdali species complex) is actually a single species (Hilário et al. 2021a, 2021b).

Dissanayake et al. (2024) divided the genus Diaporthe into seven sections and 15 species complexes based on phylogenetic analysis of all available type isolates of this genus, such as section Rudis and the D. virgiliae species complex. This classification has simplified phylogenetic analysis during species identification. In the present study, a survey of alder diseases was conducted in Xizang, China, with the aim of identifying the fungal species associated with leaf spots and branch cankers through a combination of morphological and molecular approaches.

Materials and methods

Sample collection, isolation, and morphology

Disease investigations were conducted from June to October in 2024 in Bayi District and Bomi County, Linzhi City, Xizang, China. Branch canker and leaf spot symptoms were observed, with canker being relatively rare and leaf spots more commonly encountered (Fig. 1). Infected branches exhibited sunken, discolored lesions, along with the presence of conidiomata of the fungal pathogen. Diseased leaves displayed small, rounded, or irregularly shaped spots, characterized by dark brown margins. Branch and leaf samples were collected and placed in paper envelopes for further analysis.

Figure 1. 

A, B sampling site C–F leaf spot symptoms of Alnus nepalensis.

Sample branches and leaves were washed with sterile water and dried using refined absorbent cotton. Tissue fragments (5 × 5 mm) from both healthy and diseased samples were cut with a sterilized surgical knife, then immersed in 75% alcohol for 1 min, subsequently washed three times for 30 seconds each in sterile water, and dried with refined absorbent cotton. These tissue fragments were then transferred to the surface of Potato Dextrose Agar (PDA) plates. Hyphal tips grown from the tissue fragments on PDA were observed under a stereomicroscope (Discovery v8, Zeiss, Oberkochen, Germany). The fragments were then subcultured onto fresh PDA plates to obtain pure cultures. Type specimens were deposited in the herbarium of the Chinese Academy of Forestry (CAF), and ex-type isolates were stored in the China Forestry Culture Collection Center (CFCC, https://cfcc.caf.ac.cn/).

Cultures were grown on PDA, malt extract agar (MEA), and synthetic nutrient agar (SNA) plates for observation. Conidiomata formed on the culture plates and branches were studied. The conidiomata were carefully sectioned using a double-edged blade, and fungal structures were observed under a Zeiss Discovery v8 stereomicroscope. Conidiophores, conidiogenous cells, and conidia were further examined and photographed using an Olympus BX51 microscope (Tokyo, Japan).

Phylogenetic analyses

The genomic DNA of the Diaporthe isolates obtained in this study was extracted from young colonies grown on PDA plates following the protocol of Doyle and Doyle (1990). The internal transcribed spacer (ITS) region of rDNA, along with fragments of the calmodulin (cal), histone H3 (his3), translation elongation factor 1-alpha (tef1), and partial beta-tubulin (tub2) genes, was amplified using the primers and protocols outlined in Table 1. The PCR products were subjected to electrophoresis on 2% agarose gels for analysis, followed by sequencing using the same primers as those employed in the PCR amplification. The sequencing service was provided by Ruibo Xingke Biotechnology Co., Ltd. (Beijing, China).

Table 1.
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Primers and PCR protocols.

Gene Regions Primers PCR conditions References
ITS ITS1/ITS4 95 °C for 4 min, 35 cycles of 94 °C for 45 s, 48 °C for 1 min, and 72 °C for 2 min, 72 °C for 10 min White et al. 1990
cal CAL228F/CAL737R 95 °C for 4 min, 35 cycles of 94 °C for 45 s, 54 °C for 1 min, and 72 °C for 2 min, 72 °C for 10 min Carbone and Kohn 1999
his3 CYLH3F/H3-1b 95 °C for 5 min, 35 cycles of 95 °C for 1 min, 57 °C, 1.25 min, and 72 °C for 2 min, 72 °C for 10 min Crous et al. 2004; Glass and Donaldson 1995
tef1 EF1-728F/EF1-986R 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 54 °C for 50 s, and 72 °C for 2 min, 72 °C for 10 min Carbone and Kohn 1999
tub2 T1(Bt2a)/Bt2b 95 °C for 4 min, 35 cycles of 94 °C for 45 s, 54 °C for 1 min, and 72 °C for 2 min, 72 °C for 10 min Glass and Donaldson 1995; O’Donnell and Cigelnik 1997

The ITS, cal, his3, tef1, and tub2 gene sequences obtained in this study were queried against the GenBank nucleotide database located at the National Center for Biotechnology Information (NCBI) to identify closely related sequences and determine the associated species. Sequence data for related taxa were retrieved from Dissanayake et al. (2024) and downloaded from NCBI (Table 2). The sequences were aligned using the MAFFT v.7 online server (http://mafft.cbrc.jp/alignment/server/index.html, Katoh et al. 2019) with default settings.

Table 2.
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GenBank accession numbers used in the phylogenetic analyses.

Species Strain GenBank accession numbers References
ITS tef1 tub2 cal his3
Diaporthe acaciigena CBS 129521 KC343005 KC343731 KC343973 KC343247 KC343489 Gomes et al. 2013
D. alnicola CFCC 70997* PQ636515 PQ635059 PQ635065 PQ635047 PQ635053 In this study
D. alnicola CFCC 70998* PQ636516 PQ635060 PQ635066 PQ635048 PQ635054 In this study
D. amygdali CBS 126679 KC343022 KC343748 KC343990 KC343264 KC343506 Gomes et al. 2013
D. amygdali CBS 111811 KC343019 KC343745 KC343987 KC343261 KC343503 Gomes et al. 2013
D. amygdali CBS 115620 KC343020 KC343746 KC343988 KC343262 KC343504 Gomes et al. 2013
D. amygdali CBS 120840 KC343021 KC343747 KC343989 KC343263 KC343505 Gomes et al. 2013
D. amygdali syn. D. chongqingensis CGMCC 3.19603 MK626916 MK654866 MK691321 MK691209 MK726257 Guo et al. 2020
D. amygdali syn. D. chongqingensis PSCG 435 MK626916 MK654866 MK691321 MK691209 MK726257 Guo et al. 2020
D. amygdali syn. D. chongqingensis PSCG 436 MK626917 MK654867 MK691322 MK691208 MK726256 Guo et al. 2020
D. amygdali syn. D. chongqingensis PSCG 436-2 MK626917 MK654867 MK691322 MK691208 MK726256 Guo et al. 2020
D. amygdali syn. D. fusicola CGMCC 3.17087 KF576281 KF576256 KF576305 KF576233 NA Gao et al. 2015
D. amygdali syn. D. fusicola CGMCC 3.17088 KF576263 KF576238 KF576287 KF576221 NA Gao et al. 2015
D. amygdali syn. D. garethjonesii MFLUCC 12-0542 KT459423 KT459457 KT459441 KT459470 NA Gao et al. 2015
D. amygdali syn. D. kadsurae CFCC 52586 MH121521 MH121563 MH121600 MH121439 MH121479 Yang et al. 2018
D. amygdali syn. D. kadsurae CFCC 52587 MH121522 MH121564 MH121601 MH121440 MH121480 Yang et al. 2018
D. amygdali syn. D. mediterranea CBS 146754 MT007496 MT006996 MT006693 MT006768 MT007102 León et al. 2020
D. amygdali syn. D. ovoicicola CGMCC 3.17092 KF576264 KF576239 KF576288 KF576222 NA Gao et al. 2015
D. amygdali syn. D. ovoicicola CGMCC 3.17093 KF576265 KF576240 KF576289 KF576223 NA Gao et al. 2015
D. amygdali syn. D. ovoicicola CGMCC 3.17094 KF576266 KF576241 KF576290 KF576224 NA Gao et al. 2015
D. amygdali syn. D. ovoicicola ACJY62 MW578711 MW597404 MW598141 MW598161 MW598183 Gao et al. 2015
D. amygdali syn. D. sterilis CBS 136969 KJ160579 KJ160611 KJ160528 KJ160548 MF418350 Lombard et al. 2014
D. amygdali syn. D. sterilis CPC 20580 KJ160582 KJ160614 KJ160531 KJ160551 NA Lombard et al. 2014
D. amygdali syn. D. ternstroemia CGMCC 3.15183 KC153098 KC153089 NA NA NA Gao et al. 2014
D. amygdali syn. D. ternstroemia CGMCC 3.15184 KC153099 KC153090 NA NA NA Gao et al. 2014
D. amygdali CFCC 70999 PQ636517 PQ635061 PQ635067 PQ635049 PQ635055 In this study
D. amygdali Q3B PQ636518 PQ635062 PQ635068 PQ635050 PQ635056 In this study
D. araucanorum CBS 145285 MN509711 MN509733 MN509722 NA NA Zapata et al. 2020
D. araucanorum CBS 145283 MN509709 MN509731 MN509720 NA NA Zapata et al. 2020
D. beckhausii CBS 138.27 KC343041 KC343767 KC344009 KC343283 KC343525 Gomes et al. 2013
D. benedicti BPI 893190 KM669929 KM669785 NA KM669862 Lawrence et al. 2015
D. breviconidiophora CGMCC 3.24298 OP056725 OP150564 OP150641 OP150718 OP150794 Dissanayake et al. 2024
D. breviconidiophora GZCC 22-0030 OP056725 OP150564 OP150641 OP150718 OP150794 Dissanayake et al. 2024
D. cassines CPC 21916 KF777155 KF777244 NA NA NA Crous et al. 2013
D. celticola CFCC 53074 MK573948 MK574623 MK574643 MK574587 MK574603 Cao et al. 2022
D. celticola CFCC 53075 MK573949 MK574624 MK574644 MK574588 MK574604 Cao et al. 2022
D. crousii CAA823 MK792311 MK828081 MK837932 MK883835 MK871450 Hilário et al. 2020
D. crousii CAA820 MK792300 MK828072 MK837923 MK883828 MK871441 Hilário et al. 2020
D. eres AR5193 KJ210529 KJ210550 KJ420799 KJ434999 KJ420850 Udayanga et al. 2014
D. eres DLR12a KJ210518 KJ210542 KJ420783 KJ434996 KJ420833 Udayanga et al. 2014
D. foikelawen CBS 145289 MN509713 MN509735 MN509724 NA NA Zapata et al. 2020
D. foikelawen CBS 145287 MN509714 MN509736 MN509725 NA NA Zapata et al. 2020
D. grandifori SAUCC 194.84 MT822612 MT855924 MT855809 MT855691 MT855580 Sun et al. 2021
D. guizhouensis GZCC 20-0338 OM060254 OL961761 OL961762 OL961763 NA Bhunjun et al. 2022
D. guizhouensis GZCC 22-0027 OP056683 OP150522 OP150600 OP150679 OP150754 Bhunjun et al. 2022
D. guizhouensis GZCC 22-0045 OP056684 OP150523 OP150601 OP150680 OP150755 Bhunjun et al. 2022
D. heterophyllae CBS 143769 MG600222 MG600224 MG600226 MG600218 MG600220 Marín-Felix et al. 2019
D. heveae B23 KR812219 NA NA NA NA Dos Santos et al. 2015
D. linzhiensis CFCC 71057* PQ636519 PQ635063 PQ635069 PQ635051 PQ635057 In this study
D. linzhiensis N266C* PQ636520 PQ635064 PQ635070 PQ635052 PQ635058 In this study
D. nothofagi BRIP 54801 JX862530 JX862536 KF170922 NA NA Tan et al. 2013
D. obtusifoliae CBS 143449 MG386072 NA NA NA MG386137 Crous et al. 2017
D. ocoteae CPC 26217 KX228293 NA KX228388 NA NA Crous et al. 2013
D. penetriteum LC3353 KP714505 KP714517 KP714529 NA KP714493 Gao et al. 2016
D. pustulata CBS 109742 KC343185 KC343911 KC344153 KC343427 KC343669 Gomes et al. 2013
D. pustulata CBS 109784 KC343187 KC343913 KC344155 KC343429 KC343671 Gomes et al. 2013
D. rudis AR3422 KC843331 KC843090 KC843177 KC843146 NA Udayanga et al. 2014
D. rudis AR3654 KC843338 KC843097 KC843184 KC843153 NA Udayanga et al. 2014
D. rudis DA244 KC843334 KC843093 KC843180 KC843149 NA Udayanga et al. 2014
D. rudis ICMP 16419 KC145904 KC145976 NA NA NA Udayanga et al. 2014
D. rudis ICMP 7025 KC145885 KC145995 NA NA NA Udayanga et al. 2014
D. rudis CBS 113201 MH862916 KC343960 KC344202 KC343476 KC343718 Vu et al. 2019
D. rudis syn. D. australafricana CBS 111886 KC343038 KC343764 KC344006 KC343280 KC343522 Gomes et al. 2013
D. rudis syn. D. australafricana CBS 113487 KC343039 KC343765 KC344007 KC343281 KC343523 Gomes et al. 2013
D. rudis syn. D. cynaroidis CBS 122676 KC343058 KC343784 KC344026 KC343300 KC343542 Gomes et al. 2013
D. rudis syn. D. patagonica CBS 145291 MN509717 MN509739 MN509728 NA NA Zapata et al. 2020
D. rudis syn. D. patagonica CBS 145755 MN509718 MN509740 MN509729 NA NA Zapata et al. 2020
D. rudis syn. D. salicicola BRIP 54825 JX862531 JX862537 KF170923 NA NA Tan et al. 2013
D. rudis syn. D. subcylindrospora KUMCC 17-0151 MG746629 MG746630 MG746631 NA NA Hyde et al. 2018
D. shennongjiaensis CNUCC 201905 MN216229 MN224672 MN227012 MN224551 MN224559 Zhou and Hou 2019
D. shennongjiaensis CNUCC 201906 MN216228 MN224673 MN227013 MN224552 MN224561 Zhou and Hou 2019
D. silvicola CFCC 54191 MZ727041 MZ816347 MZ753491 MZ753472 MZ753481 Jiang et al. 2021
D. silvicola M79 MZ727042 MZ816348 MZ753492 MZ753473 MZ753482 Jiang et al. 2021
D. torilicola MFLUCC 17-1051 KY964212 KY964168 KY964096 KY964127 Dissanayake et al. 2017
D. toxica CBS 534.93 KC343220 KC343946 KC344188 KC343462 KC343704 Gomes et al. 2013
D. toxica CBS 546.93 KC343222 KC343948 KC344190 KC343464 KC343706 Gomes et al. 2013
D. virgiliae CMW 40755 KP247573 NA KP247582 NA NA Machingambi et al. 2015
D. virgiliae CMW 40748 KP247566 NA KP247575 NA NA Machingambi et al. 2015
D. zaofenghuang CGMCC 3.20271 MW477883 MW480871 MW480875 MW480867 MW480863 Wang et al. 2021
D. zaofenghuang TZFH3 MW477884 MW480872 MW480876 MW480868 MW480864 Wang et al. 2021

Note: “NA” indicates unavailable sequences; sequences produced in the current study are in bold, and * means ex-type strains from new species in this study.

The isolates described in this study were shown to belong to the Diaporthe Section Rudis and the D. virgiliae species complex, respectively. Maximum likelihood (ML) phylogenetic analysis was conducted using the CIPRES Science Gateway platform (Miller et al. 2010), with RAxMLHPC2 on XSEDE (v. 8.2.10) under the GTR substitution model and 1000 non-parametric bootstrap replicates. Bayesian analysis was performed with MrBayes v. 3.2.6, utilizing four simultaneous Markov chain runs for 1,000,000 generations. The resulting trees were visualized using FigTree v. 1.4.0 (Rambaut 2012).

The pairwise homoplasy index test was employed to confirm the new species status using SplitsTree v.4.16.1 (Huson and Bryant 2006). Incongruence among the ITS-cal-his3-tef1-tub2 genealogies was used as a criterion to identify hypothesized “species” and infer the occurrence of sexual recombination (Bruen et al. 2006). Results of the Фw-statistic below a 0.05 threshold (p-value < 0.05) indicated significant recombination. A phylogenetic network based on the combined dataset of five loci was constructed using the NeighborNet algorithm to assess the impact of recombination.

Results

Phylogenetic analyses

For the analysis of Diaporthe Section Rudis, the combined dataset of ITS, cal, his3, tef1, and tub2 comprised 67 strains, with D. eres (AR5193 and DLR12a) used as the outgroup taxa. The final alignment included 2,691 characters (ITS: 451, cal: 702, his3: 410, tef1: 596, tub2: 532), including gaps. The final ML optimization likelihood value of the best RAxML tree was -17019.93, and the matrix contained 1,257 distinct alignment patterns, with 32.15% undetermined characters or gaps. The estimated base frequencies were A = 0.216951, C = 0.313266, G = 0.235799, T = 0.233984; substitution rates were AC = 1.028567, AG = 3.157223, AT = 1.223911, CG = 0.822997, CT = 4.362405, GT = 1.0; and the gamma distribution shape parameter α = 0.386104. Both the RAxML and Bayesian analyses produced similar tree topologies, which were consistent with those of previous studies (Norphanphoun et al. 2022; Dissanayake et al. 2024). Isolates from this study (CFCC 70999 and Q3B) clustered together with other Diaporthe amygdali strains, showing strong support (Fig. 2), thus confirming their identification as D. amygdali.

Figure 2. 

Maximum likelihood tree of Diaporthe Section Rudis generated from combined ITS, cal, his3, tef1, and tub2 sequence data. Bootstrap support values ≥ 50% and Bayesian posterior probabilities ≥ 0.90 are demonstrated at the branches. Isolates from the present study are indicated in blue.

In the Diaporthe virgiliae species complex, the combined dataset of ITS, cal, his3, tef1, and tub2 included 13 strains, with D. shennongjiaensis (CUNCC 201905 and CUNCC 201906) as the outgroup taxa. The final alignment contained 2,598 characters (ITS: 593, cal: 421, his3: 466, tef1: 331, tub2: 787), including gaps. The final ML optimization likelihood value of the best RAxML tree was -5834.44, and the matrix had 346 distinct alignment patterns, with 20.11% undetermined characters or gaps. The estimated base frequencies were A = 0.212455, C = 0.329026, G = 0.238268, T = 0.220251; substitution rates were AC = 1.111868, AG = 2.843163, AT = 1.775735, CG = 0.816784, CT = 3.662621, GT = 1.0; and the gamma distribution shape parameter α = 0.047755. Both RAxML and Bayesian analyses produced similar tree topologies, which closely matched those of prior publications (Norphanphoun et al. 2022; Dissanayake et al. 2024). Four isolates from this study formed two new clades distinct from any lineage and are hence accommodated as two novel species: D. alnicola (CFCC 70997 and CFCC 70998) and D. linzhiensis (CFCC 71057 and N266C).

The network relationships within the D. virgiliae species complex are depicted in Fig. 4, indicating no significant recombination based on the PHI test (p = 0.9624). Furthermore, based on the relative distances between species and the structure of the phylogenetic network, isolates within the D. virgiliae complex represent seven different species.

Taxonomy

Diaporthe alnicola Ning Jiang, sp. nov.

MycoBank No: 856742
Fig. 5

Etymology.

“Alni” refers to the host genus Alnus, and “-cola” means inhabiting.

Description.

Associated with leaf spot disease of Alnus nepalensis. Teleomorph: Undetermined. Anamorph: Conidiomata formed on PDA pycnidial, scattered, erumpent, pulvinate to subglobose, dark brown, 150–350 μm diam. Conidiophores indistinct, usually reduced to conidiogenous cells. Conidiogenous cells cylindrical, attenuate towards the apex, hyaline, phialidic, 9.5–33 × 2–3 μm. Alpha conidia aseptate, hyaline, smooth, guttulate, cylindrical, straight, base truncate, (6–)6.5–7(–7.5) × (2–)2.5–3(–3.5) μm (x̄ = 6.8 × 2.6 μm, n = 50), L/W = 2–3.4. Beta conidia aseptate, hyaline, smooth, guttulate, filiform, tapering towards both ends, curved, (13–)14.5–22(–24) × 1.5–2.5 μm (x̄ = 18.3 × 2.1 μm, n = 50), L/W = 5.9–12.5. Gamma conidia not observed.

Culture characteristics.

Colonies on PDA at 25 °C are spreading, flocculent, forming abundant aerial mycelium and an undulate margin, initially white, turning mouse gray and reaching a diameter of 90 mm after 10 d, developing dark brown conidiomata with orange conidial masses after 20 d. Colonies on MEA at 25 °C are flat, spreading, feathery, with a smooth entire margin, white, reaching a diameter of 90 mm after 15 d, sterile. Colonies on SNA at 25 °C are flat, spreading with a smooth entire margin, white, reaching 90 mm in diameter after 20 d, developing dark brown conidiomata with orange conidial masses after 30 d.

Materials examined.

China • Xizang Autonomous Region (Tibet), Linzhi City, Bayi District, Pailong Town, 30°4'22"N, 95°8'2"E, 2192 m, from leaf spots of Alnus nepalensis, 9 Jul. 2024, Ning Jiang, Jieting Li & Haoyin Zhang (holotype CAF800100, ex-paratype cultures CFCC 70997 and CFCC 70998).

Notes.

Diaporthe alnicola, identified from leaf spots on Alnus nepalensis in this study, is phylogenetically closely related to D. virgiliae, which originates from the rot root of Virgilia oroboides in South Africa (Fig. 3). Morphologically, D. alnicola is similar to D. virgiliae in terms of the size of alpha and beta conidia (alpha conidia: 6.5–7 × 2.5–3 μm in D. alnicola vs. 5.2–8 × 1.1–3.5 μm in D. virgiliae; beta conidia: 14.5–22 × 1.5–2.5 μm in D. alnicola vs. 17.1–25.4 × 1–1.8 μm in D. virgiliae). However, they can be distinguished by the size of their conidiogenous cells (9.5–33 × 2–3 μm in D. alnicola vs. 12.3–21.3 × 0.7–1.5 μm in D. virgiliae) (Machingambi et al. 2015). Furthermore, D. alnicola differs from D. virgiliae at the nucleotide level (ITS, 11/432; tub2, 7/743).

Figure 3. 

Maximum likelihood tree of the Diaporthe virgiliae species complex generated from combined ITS, cal, his3, tef1, and tub2 sequence data. Bootstrap support values ≥ 50% and Bayesian posterior probabilities ≥ 0.90 are demonstrated at the branches. Isolates from the present study are indicated in blue.

Figure 4. 

Phylogenetic network from concatenated data (ITS, cal, his3, tef1, and tub2) representing the structure of the Diaporthe virgiliae species complex, based on LogDet transformation and the NeighborNet algorithm, inferred by SplitsTree (p = 0.9624). The scale bar represents the expected number of substitutions per nucleotide position.

Figure 5. 

Morphology of Diaporthe alnicola A colony on PDA after 15 d B Colony on MEA after 15 d C colony on SNA after 15 d D conidioma formed on PDA E, F conidiogenous cells G–K alpha and beta conidia. Scale bars: 500 µm (D); 10 µm (E–K).

Diaporthe amygdali (Delacr.) Udayanga, Crous & K.D. Hyde, Fungal Diversity 56(1): 166. 2012

Fig. 6

Description.

Associated with leaf spot disease of Alnus nepalensis. Teleomorph: Undetermined. Anamorph: Conidiomata formed on PDA pycnidial, scattered, erumpent, subglobose, dark brown, 700–2250 μm diam. Conidiophores indistinct, usually reduced to conidiogenous cells. Conidiogenous cells cylindrical, attenuate towards the apex, hyaline, phialidic, 16.5–34 × 1.5–3 μm. Alpha conidia not observed. Beta conidia aseptate, hyaline, smooth, guttulate, filiform, tapering towards both ends, straight or slightly curved, (27.5–)30–35(–40.5) × 1.5–2 μm (x̄ = 32.6 × 1.6 μm, n = 50), L/W = 15.8–23.1. Gamma conidia not observed.

Figure 6. 

Morphology of Diaporthe amygdali A colony on PDA after 15 d B colony on MEA after 15 d C colony on SNA after 15 d D conidioma formed on PDA E conidiogenous cells E–I beta conidia. Scale bars: 800 µm (D); 10 µm (E–K).

Culture characteristics.

Colonies on PDA at 25 °C are flocculent, forming concentric zones with undulate margins, initially white, turning pale brownish, and reaching a diameter of 90 mm after 10 d, developing dark brown conidiomata with white conidial masses after 25 d. Colonies on MEA at 25 °C are flat, spreading, with a smooth entire margin, white, reaching a diameter of 80 mm after 20 d, sterile. Colonies on SNA at 25 °C are flat, spreading with a feathery margin, white, reaching 80 mm in diameter after 20 d, sterile.

Materials examined.

China • Xizang Autonomous Region (Tibet), Linzhi City, Bayi District, Pailong Town, 30°4'22"N, 95°8'2"E, 2192 m, from leaf spots of Alnus nepalensis, 9 Jul. 2024, Ning Jiang, Jieting Li & Haoyin Zhang (cultures CFCC 70999 and Q3B).

Notes.

The species concept of Diaporthe amygdali has been revised in recent studies using phylogenetic analysis, GCPSR, and coalescence-based models (Hilário et al. 2021b; Dissanayake et al. 2024). Currently, D. amygdali is considered synonymous with D. chongqingensis, D. fusicola, D. garethjonesii, D. kadsurae, D. mediterranea, D. ovoicicola, D. sterilis, and D. ternstroemia (Hilário et al. 2021b; Dissanayake et al. 2024). This fungus is widely distributed, inhabiting a range of plant hosts, including Acer spp., Camellia sinensis, Lithocarpus glabra, Prunus dulcis, Prunus persica, Prunus salicina, Pyrus pyrifolia, Ternstroemia gymnanthera, Vaccinium corymbosum, and Vitis vinifera (Hilário et al. 2021b). In this study, two isolates from leaf spots of Alnus nepalensis clustered with strains of D. amygdali with high support values (Fig. 2). Therefore, these two isolates were identified as D. amygdali, which led us to describe Alnus nepalensis as a new host for this fungus.

Diaporthe linzhiensis Ning Jiang, sp. nov.

MycoBank No: 856743
Fig. 7

Etymology.

Named after the collection site of the type specimen, Linzhi City.

Figure 7. 

Morphology of Diaporthe linzhiensis A, B conidiomata formed on twigs of Alnus nepalensis C transverse section through a conidioma D longitudinal section through a conidioma E colony on PDA after 15 d F colony on MEA after 15 d G colony on SNA after 15 d H, I conidiogenous cells J, K beta conidia. Scale bars: 500 µm (B–D); 20 µm (H–K).

Description.

Associated with branch canker disease of Alnus nepalensis. Teleomorph: Undetermined. Anamorph: Conidiomata pycnidial, immersed in bark, scattered, erumpent through the bark surface, conical, with a solitary locule, 300–500 μm diam., 250–400 μm high. Conidiophores reduced to conidiogenous cells. Conidiogenous cells cylindrical, attenuate towards the apex, hyaline, phialidic, straight or slightly curved, 5.5–16 × 1.5–3 μm. Alpha conidia not observed. Beta conidia aseptate, hyaline, smooth, guttulate, filiform, tapering towards both ends, straight or slightly curved, (23.5–)24.5–29(–30) × 1.5–2 μm (x̄ = 26.6 × 1.8 μm, n = 50), L/W = 12.4–19.4. Gamma conidia not observed.

Culture characteristics.

Colonies on PDA at 25 °C are spreading, flocculent, forming abundant aerial mycelium and concentric zones with an undulate margin, initially white, turning pale luteous, and reaching a diameter of 90 mm after 10 d, sterile. Colonies on MEA at 25 °C are flat, spreading, with a smooth entire margin, white, reaching a diameter of 60 mm after 20 d, sterile. Colonies on SNA at 25 °C are flat, spreading, forming concentric zones with undulate margins, white, reaching 80 mm in diameter after 20 d, sterile.

Materials examined.

China • Xizang Autonomous Region (Tibet), Linzhi City, Bomi County, Tongmai Town, 30°5'53"N, 95°3'49"E, 2055 m, from branches of Alnus nepalensis, 9 Jul. 2024, Ning Jiang, Jieting Li & Haoyin Zhang (holotype CAF800101, ex-paratype cultures CFCC 71057 and N266C).

Notes.

Diaporthe linzhiensis is phylogenetically closely related to D. alnicola, D. heterophyllae, and D. virgiliae (Fig. 2). Both D. linzhiensis and D. alnicola infect Alnus nepalensis in China, while D. heterophyllae is found on Acacia heterophylla in France, and D. virgiliae inhabits Virgilia oroboides in South Africa (Machingambi et al. 2015; Marín-Felix et al. 2019). Morphologically, D. linzhiensis shares a similar conidiogenous cell size with D. alnicola and D. heterophyllae, which is wider than that of D. virgiliae (5.5–16 × 1.5–3 μm in D. linzhiensis vs. 9.5–33 × 2–3 μm in D. alnicola vs. 6–9 × 1–2 μm in D. heterophyllae vs. 12.3–21.3 × 0.7–1.5 μm in D. virgiliae). Additionally, D. linzhiensis has longer beta conidia compared to the other species (24.5–29 × 1.5–2 μm in D. linzhiensis vs. 14.5–22 × 1.5–2.5 μm in D. alnicola vs. 17–24 × 1–2 μm in D. heterophyllae vs. 17.1–25.4 × 1–1.8 μm in D. virgiliae) (Machingambi et al. 2015; Marín-Felix et al. 2019). At the nucleotide level, D. linzhiensis also differs from D. alnicola (ITS, 23/547; cal, 2/382; his3, 6/469; tef1, 4/349; tub2, 6/778), D. heterophyllae (ITS, 28/560; cal, 11/420; his3, 6/447; tef1, 12/326; tub2, 3/406), and D. virgiliae (ITS, 16/434; tub2, 7/743).

Discussion

This study enhances the understanding of Diaporthe species on alder by revealing two previously undescribed species and a new host association, viz. Diaporthe alnicola sp. nov., D. linzhiensis sp. nov., and D. amygdali on Alnus nepalensis. Diaporthe is a morphologically distinct genus characterized by the production of alpha, beta, and gamma conidia. The alpha conidia are typically aseptate, hyaline, guttulate, and cylindrical to fusiform, while the beta conidia are aseptate, hyaline, and filiform (Farr et al. 2002; Udayanga et al. 2015; Guarnaccia and Crous 2017; Manawasinghe et al. 2019; Huang et al. 2021; Sun et al. 2021; Lambert et al. 2023). However, species within the genus usually share the same host genera and are morphologically similar, often exhibiting overlapping sizes of conidia or ascospores. As a result, it is relatively easy to identify specimens at the generic level, but more challenging to distinguish them at the species level (Yang et al. 2020, 2021; Zhu et al. 2023, 2024; Liu et al. 2024). In this study, we present novel findings from Xizang, China, which indicate the potential existence of numerous undescribed species in unexplored or minimally investigated regions worldwide.

Diaporthe alnicola and D. amygdali are here reported to be associated with leaf spot disease of Alnus nepalensis, which is a common disease in Linzhi, Xizang, China. Among these pathogens, D. alnicola is a novel species and may be the primary pathogen associated with A. nepalensis. In contrast, D. amygdali is a generalist fungus that infects a wide range of plant hosts, including Acer spp., Camellia sinensis, Lithocarpus glabra, Prunus dulcis, Pr. persica, Pr. salicina, Pyrus pyrifolia, Ternstroemia gymnanthera, Vaccinium corymbosum, and Vitis vinifera (Hilário et al. 2021b). This suggests that D. amygdali may be a secondary pathogen to A. nepalensis. For successful disease management, it will be of paramount importance, albeit challenging, to effectively interrupt the infection cycle of A. nepalensis maintained by the occurrence of leaf spots caused by and due to the broad host range of D. amygdali. Therefore, future investigations need to focus on identifying other hosts of D. amygdali in Linzhi City.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was funded by the Science and Technology Project of Nyingchi City, Tibet Autonomous Region (SYQ2024-14), the Key Laboratory of Forest Ecology in Xizang Plateau (Xizang Agricultural and Animal Husbandry University), Ministry of Education, Grant numbers XZAJYBSYS-202401 and XZAJYBSYS-202404, the Science and Technology Project of the Department of Science and Technology of Tibet Autonomous Region (XZ202301JD0001G), and the National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (NMRC-2024-7).

Author contributions

Conceptualization: JTL, JRL, NJ. Methodology: JRL, NJ. Formal analysis: JTL, YL. Investigation: JTL, JRL, NJ. Data curation: JTL, JRL, NJ. Writing-original draft: JTL. Writing-review and editing: JRL, NJ. Visualization: NJ.

Author ORCIDs

Jieting Li https://orcid.org/0009-0001-8984-7261

Yi Li https://orcid.org/0009-0004-0656-9799

Jiangrong Li https://orcid.org/0000-0002-6679-5227

Ning Jiang https://orcid.org/0000-0002-9656-8500

Data availability

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

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