﻿Morphology and phylogeny of Cytospora (Cytosporaceae, Diaporthales) species associated with plant cankers in Tibet, China

﻿Abstract During our biodiversity investigations in Tibet, China, typical Cytospora canker symptoms were observed on branches of hosts Myricariapaniculate, Prunuscerasifera and Sibiraeaangustata. Samples were studied, based on morphological features coupled with multigene phylogenetic analyses of ITS, act, rpb2, tef1 and tub2 sequence data, which revealed two new species (Cytosporamyricicolasp. nov. and C.sibiraeicolasp. nov.) and a known species (C.populina). In addition, Cytosporapopulina is newly discovered on the host Prunuscerasifera and in Tibet.

Cytospora is distributed worldwide and often known to be associated with plant diseases (Monkai et al. 2021;Pan et al. 2021;Lin et al. 2022;Travadon et al. 2022).MycoKeys 104: 51-70 (2024), DOI: 10.3897/mycokeys.104.113567 Jiangrong Li et al.: Two new species of Cytospora from Tibet, China For example, C. chrysosperma is the main canker disease pathogen of polar and willow trees in China (Fan et al. 2014;Wang et al. 2015;Lin et al. 2023); C. kuanchengensis and additional five species are associated with Chinese chestnut cankers (Jiang et al. 2020); Fifteen Cytospora species were identified from destructive canker and dieback pathogens of woody hosts in the USA (Lawrence et al. 2018).
The Tibet Tibetan Autonomous Region is located on the Qinghai-Tibet Plateau, which is known as the third pole of the earth.During our biodiversity investigations in Tibet, typical fruiting bodies of Cytospora were discovered from Myricaria paniculate (Tamaricaceae), Prunus cerasifera (Rosaceae) and Sibiraea angustata (Rosaceae).The aim of the present study was to identify Cytospora species from the three hosts based on morphological features and molecular phylogeny of combined sequence data.

Sample collection, morphology and isolation
Our biodiversity investigations were conducted in Lhasa and Shigatse cities in Tibet Tibetan Autonomous Region, China during 2022 and 2023.Diseased branches of Myricaria paniculate, Prunus cerasifera and Sibiraea angustata were observed and collected, packed in paper bags and returned to the laboratory for morphological study and fungal isolation.
Observation and description of Cytospora species was based on fruiting bodies naturally formed on the host barks.Ascostromata and conidiomata from tree barks were sectioned by hand using a double-edged blade and structures were observed under a dissecting microscope.At least 10 conidiomata/ ascostromata, 10 asci and 50 conidia/ascospores were measured to calculate the mean size and standard deviation.Measurements are reported as maximum and minimum in parentheses and the range representing the mean plus and minus the standard deviation of the number of measurements is given in parentheses.Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope, equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination.
Isolates of Cytospora were obtained by removing the spore masses from the fruiting bodies on to clean PDA plates and incubating at 25 °C until spores germinated.Single germinated spores were then transferred to the new PDA plates and incubated at 25 °C in the dark.The cultures were deposited in the China Forestry Culture Collection Center (CFCC, http://cfcc.caf.ac.cn/) and the specimens in the Herbarium of the Chinese Academy of Forestry (CAF, http://museum.caf.ac.cn/).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh fungal mycelia following the method described by Doyle and Doyle (1990).Polymerase chain reactions (PCR) were conducted to amplify the internal transcribed spacer region rDNA (ITS), the partial actin (act) region, RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1) and the partial beta-tubulin (tub2) gene using primers and conditions listed in Table 1.The PCR products were assayed via electrophoresis in 2% agarose gels.DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminator Kit v.3.1 (Invitrogen, Waltham, MA, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Sequence alignment and phylogenetic analyses
The obtained sequences of ITS, act, rpb2, tef1 and tub2 were assembled using Se-qMan software version 7.1.0(DNASTAR Inc., WI) and subjected to BLASTn search against the GenBank nucleotide database at National Center for Biotechnology Information (NCBI) to identify closely-related sequences.Sequences data of related taxa were obtained from previous publications (Fan et al. 2020;Lin et al. 2023) and downloaded from the GenBank database (Table 2).The sequences were aligned using MAFFT v.7 online web server (http://mafft.cbrc.jp/alignment/server/index.html, Katoh et al. 2019) under default settings.The Maximum Likelihood (ML) phylogenic analysis was run in the CIPRES Science Gateway platform (Miller et al. 2010), using RAxMLHPC2 on the XSEDE (v.8.2.10) tool under the GTR substitution model and 1000 non-parametric bootstrap replicates.Bayesian analysis was performed using MrBayes v. 3.2.6 on XSEDE at the CIPRES Science Gateway with four simultaneous Markov Chain runs for 1,000.000generations.The resulting trees were visualised in FigTree v. 1.4.0 (Rambaut 2012).
Culture characteristics.Colonies on PDA flat, with flocculent aerial mycelium and entire edge, initially white, becoming dark and reaching 90 mm diameter after 10 days at 25 °C, sterile.(Farr 1973;Fan et al. 2015;Gafforov 2017).This fungus is distinguished from the other Cytospora species by its 4-ascospored asci and undiscovered asexual state (Fan et al. 2015).In the present study, we firstly found this fungus causing cankered branches of Prunus cerasifera in Tibet, China.
Culture characteristics.Colonies on PDA flat, with flocculent aerial mycelium and undulate margin, initially white, becoming olivaceous grey and reaching 90 mm diameter after 10 days at 25 °C, sterile.

Discussion
Cytospora is a species-rich genus occurring on various plant hosts (Fotouhifar et al. 2010;Aiello et al. 2019;Jayawardena et al. 2019;Úrbez-Torres et al. 2020;Hanifeh et al. 2022).However, in the third pole of the Earth named Qinghai-Tibet Plateau, canker pathogens, such as Cytospora, have been seldom surveyed previously.In the comprehensive study on the genus Cytospora in China, only one species C. chrysosperma was recorded from Ulmus pumila in Tibet (Fan et al. 2020).Subsequently, Cytospora cotoneastricola and C. tibetensis from Cotoneaster sp. and Cytospora rosicola from Rosa sp. were discovered in Tibet (Pan et al. 2020).The current study introduces two new species named C. myricicola from Myricaria paniculate and C. sibiraeicola from Sibiraea angustata in Tibet, China.In addition, a new host record of C. populina on Prunus cerasifera was discovered.
To our knowledge, Cytospora myricicola is the first species of Cytospora discoved on the host genus Myricaria (Fan et al. 2020).Cytospora sibiraeicola and C. sibiraeae have been recorded from the host species Sibiraea angustata (Liu et al. 2015).Cytospora sibiraeae was described, based only on the sexual morph and is currently impossible to be distinguished from C. sibiraeicola morphologically (Liu et al. 2015).However, these two species occurring on Sibiraea angustata are phylogenetically obviously distinct (Fig. 1).
Species of Cytospora are known as opportunistic pathogens mainly infecting woody hosts and some of the species occur on a wide host range (Adams et al. 2005;Fan et al. 2020).The Cytospora species and their host association have been revealed in this study; however, further studies are required to confirm the fungal pathogenicity.

Figure 1 .
Figure 1.Maximum Likelihood tree generated from combined ITS, act, rpb2, tef1 and tub2 sequence data.Bootstrap support values ≥ 50% and Bayesian posterior probabilities ≥ 0.90 are demonstrated at the branches.Ex-type cultures are marked with (*).

Table 1 .
Primers and PCR protocols.

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