Morphology and Phylogeny Reveal Three New Species of Cytospora Associated with Tree Cankers in China

Cytospora (Cytosporaceae, Diaporthales) is a fungal genus that usually inhabits plants as endophytes, saprobes, as well as pathogens. Species of this genus are characterized by possessing allantoid hyaline conidia and ascospores. Samples with typical Cytospora canker symptoms on Prunus davidiana, P. padus and Salix sp. were collected in Tibet and Xinjiang, China. Species were identified using both morphological and molecular approaches of combined loci of internal transcribed spacer region rDNA (ITS), the partial actin (act) region, RNA polymerase II second largest subunit (rpb2), the translation elongation factor 1-alpha (tef1) gene and the partial be-ta-tubulin (tub2) gene. Six isolates in the present study formed three distinct clades from previously known species. Cytospora hejingensis sp. nov. from Salix sp., C. jilongensis sp. nov. from P. davidiana and C. kunsensis from P. padus were proposed herein. The current study improves the understanding of species concept in Cytospora.

Species of Cytospora were primarily identified and distinguished by their morphology and host [5][6][7].However, recent studies employing molecular phylogeny revealed many cryptic species with similar morphology on the same host of known species of this genus [11][12][13][14][15].For example, up to 28 Cytospora species were discovered from Eucalyptus spp. in South Africa with the help of DNA sequence evidence [2], eight from willow (Salix spp.) trees in China [16], six from Castanea mollissima in China [17], six from Populus hosts in China [18] and six from apple trees in Iran [19].The taxonomy of Cytospora is currently more dependent on combined evidence of DNA sequence data, morphological features and ecology than species morphology and host associations [1,20].
Several species of Cytospora are reported to cause plant diseases including canker, wilt and dieback [21][22][23][24].For example, C. carpobroti causes Carpobrotus edulis wilt disease in South Africa [21]; C. oleicola and C. olivarum are pathogenic to olives in the USA [22]; C. parasitica results in apple cankers in China [23]; and Cytospora pistaciae causes dieback and canker disease of pistachios in Italy [24].There are still many cryptic species of Cytospora pathogenic to plants waiting for description.
In the present study, Cytospora canker symptoms were found from different tree hosts named Prunus davidiana, P. padus and Salix sp. in Tibet and Xinjiang, China.The aims of the present study were to identify the casual agents of the lesions, to introduce and describe new Cytospora species using both molecular and morphological approaches, and to discuss the species differences based on newly collected specimens.

Specimens and Strains
Investigations to collect fungal specimens were conducted in Tibet and Xinjiang during 2021 and 2022.During the surveys, dead and dying twigs and branches of tree hosts were checked manually, and then twigs and branches with obvious fungal fruiting bodies were recorded and collected.Samples were packed in paper bags and posted back for isolation.
Ascomata on branches of Prunus padus and Salix sp., and conidiomata on branches of P. davidiana were sectioned using sterile blades, and mucoid spore masses were removed and placed onto the surface of potato dextrose agar (PDA; potato, 200 g; glucose, 20 g; agar, 20 g; distilled water, to complete 1000 mL) media using sterile insect needles.Then, plates were incubated at 25 • C in darkness until spores germinated.Pieces of mycelium were cut and removed and placed onto a new PDA plate under a stereomicroscope to obtain the pure strains.Specimens and isolates were preserved in the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/ (accessed on 2 January 2024)).

Morphological Observations
The Cytospora species observations were based on ascomata and conidiomata naturally formed on twigs and branches of Prunus davidiana, P. padus and Salix sp.The sexual and asexual fruiting bodies were sectioned using sterile blades and photographed using the Leica stereomicroscope (M205) (Leica Microsystems, Wetzlar, Germany).The asci, ascospores, conidiophores, conidiogenous cells and conidia were measured and photographed by a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan).The colony characteristics were observed and recorded on PDA plates at 25 • C in darkness.

Molecular Phylogeny
Sequences obtained in the present study were preliminarily identified by the BLAST search to confirm their classification.The referenced sequences of Cytospora were collected from recent publications (Table 1) and downloaded [1,24,25].Strain CBS 160.32 (species Diaporthe vaccinii) was selected as the outgroup taxon.The five individual loci ITS, act, rpb2, tef1 and tub2 were aligned using MAFFT v. 6.0 and manually adjusted using MEGA v. 6.0 [31,32].Then, five loci were combined and analyzed based on maximum likelihood (ML) and Bayes methods in the CIPRES Science Gateway platform [33].The GTR substitution model was employed and 1000 non-parametric bootstrap replicates were set for ML phylogenic analysis.Four simultaneous Markov Chain runs for 1,000,000 generations were set during Bayesian analysis.The resulting trees were visualized in FigTree v. 1.4.0 and edited using Adobe Illustrator 2020.

Discussion
In the present study, samples of Cytospora with fruiting bodies were collected from Xinjiang and Tibet, and identified based on both morphological and phylogenetical approaches of combined ITS, act, rpb2, tef1 and tub2 loci.We proposed three new species, i.e., Cytospora hejingensis sp.nov.from Salix sp., C. jilongensis sp.nov.from P. davidiana and C. kunsensis from P. padus.
Of the new species introduced in the current study, two taxa (C.jilongensis and C. kunsensis) were isolated from the plant genus Prunus.Hence, a total of nine species of Cytospora were found in host genus Prunus, where the previous seven species are C. cinnamomea, C. erumpens, C. japonica, C. leucostoma, C. olivacea, C. populinopsis and C. pruni-mume [14].C. kunsensis is distinguished from C. populinopsis in eight-spored asci, and these two species are only known in sexual morph [1].The other seven species are known in asexual species with similar conidial morphology but different sequence data of ITS, act, rpb2, tef1 and tub2 loci.The example of Cytospora species from Prunus implies that DNA sequence data are necessary to separate species during pathogen identifications.
Another example is the Cytospora species from the host genus Salix.Until now, over 10 species of Cytospora were discovered from the host genus Salix, including one species Cytospora hejingensis introduced in the current study [35].Most of them are confirmed to be pathogens associated with canker diseases [35].The new species from the present study needs a pathogenicity test to evaluate its virulence to willow trees in the future.
In the traditional classification and identification of species in Cytospora, spore morphology and host information are the most important evidence to identify Cytospora species [5][6][7].However, by using the molecular data, many cryptic species with the same hosts and similar spore morphology were recently revealed [1,14,15,35].The molecular classification system for Cytospora based on morphology, phylogeny and host information is more scientific than that mainly based on morphology before.

Figure 1 .
Figure 1.Phylogram of Cytospora resulting from a maximum likelihood analysis, based on a combined matrix of ITS, act, rpb2, tef1 and tub2.Numbers above the branches indicate ML bootstraps (left, ML BS ≥ 50%) and Bayesian posterior probabilities (right, BPP ≥ 0.90).The tree is rooted with Diaporthe vaccinii (CBS 160.32).Isolates obtained from the present study are marked in blue.

Figure 2 .Figure 1 .
Figure 1.Phylogram of Cytospora resulting from a maximum likelihood analysis, based on a combined matrix of ITS, act, rpb2, tef1 and tub2.Numbers above the branches indicate ML bootstraps (left, ML BS ≥ 50%) and Bayesian posterior probabilities (right, BPP ≥ 0.90).The tree is rooted with Diaporthe vaccinii (CBS 160.32).Isolates obtained from the present study are marked in blue.

Table 1 .
Strains and their GenBank accession numbers used in this study.