Microfungi Associated with Peach Branch Diseases in China

Peach (Prunus persica L.) is one of the most important and oldest stone fruits grown in China. Even though P. persica is one of the most commonly grown stone fruits in China, little is known about the biodiversity of microfungi associated with peach branch diseases. In the present study, samples were collected from a wide range of peach growing areas in China, and fungal pathogens associated with peach branch diseases were isolated. In total, 85 isolates were obtained and further classified into nine genera and 10 species. Most of the isolates belonged to Botryosphaeriaceae (46), including Botryosphaeria, Diplodia, Neofusicoccum, Phaeobotryon, and Lasiodiplodia species; Ascochyta, Didymella, and Nothophoma species representing Didymellaceae were also identified. Herein, we introduce Ascochyta prunus and Lasiodiplodia pruni as novel species. In addition, we report the first records of Nothophoma pruni, Neofusicoccum occulatum, and Phaeobotryon rhois on peach worldwide, and Didymella glomerata, Nothophoma quercina, and Phaeoacremonium scolyti are the first records from China. This research is the first comprehensive investigation to explore the microfungi associated with peach branch disease in China. Future studies are necessary to understand the pathogenicity and disease epidemiology of these identified species.


Introduction
Peach (Prunus persica L.) belongs to the family Rosaceae and is an important stone fruit that contains vitamins, flavonols, sugars, and catechins [1].Peaches are rich in dietary fibres that provide health benefits [2].They are consumed as fresh fruits and in processed foods such as jam and beverages.Prunus species were first domesticated and cultivated in northwestern China [3] and originated in China as long ago as 3300-2500 BC according to archaeological evidence [4].According to the Food and Agricultural Organization 2019-United Nations (FAOSTAT), China is the top peach-producing country, with a production of 15.8 million tons in 2019, which accounted for 57% of the global production.
In China, the genetic diversity of peach is high, comprising more than 396 peach cultivars [5].Peach cultivation areas in China are mainly located from the subtropical southern to the northern region and range from warm to cold and dry [6].Peach trees are perennial plants that can grow up to 21 feet.Although the lifespan of peach plants

Sample Collection and Fungal Isolation
Diseased trunk, branch, and twig samples were collected from 20 peach orchards in 2020 and 2022 in Beijing, Hebei Province, Gansu Province, Liaoning Province, Guizhou Province, Sichuan Province, Yunnan Province, and Anhui Province in China.The disease samples were mainly collected from plants with typical dieback symptoms, such as gummosis, canker, twig canker spots, and shoot blight (Figure 1).The samples were cut into 0.5 × 0.5 cm pieces, surface sterilized for 1 min in 75% ethanol, rinsed for 2 min in distilled water, and blotted dry on sterilized filter paper.Then, the samples were transferred onto potato dextrose agar (PDA; 200 g potato, 20 g dextrose, and 20 g agar per L) plates and incubated at 25 °C to obtain pure cultures.Pure cultures were obtained via both single-tip isolation and single-spore isolation.The purified isolates were preserved on PDA slants at 4 °C [16].

Morphological and Cultural Characterization
The isolates were incubated on PDA at 25 • C or on malt extract agar (MEA; 30 g malt extract, 5 g mycological peptone, and 15 g agar/L), on oatmeal agar (OA; 40 g oatmeal and 5 g agar/L) under near-ultraviolet (UV) light (12 h light/12 h dark) when it was necessary, or on pine needle agar (PNA) [17] to induce sporulation.Colony diameters were measured after 5-7 days of incubation, and the culture characteristics were determined after 14 days [18].Colony colours were recorded based on the colour charts of Rayner [19].The micromorphological structures of mature conidiomata, conidia, and conidiogenous cells were studied on PDA, OA, and MEA [20,21].Observations were conducted with an Axio-Cam 506 colour Imager Z2 photographic microscope (Carl Zeiss Microscopy, Oberkochen, Germany).Morphological features such as conidial length, width, and size were measured (at least 30/40 per isolate) with a ZEN Pro 2012 (Carl Zeiss Microscopy).The structure of the mature pycnidial wall was observed using microtome sections of 6-10 µm in thickness, which were prepared with a Leica CM 1950 freezing microtome (Leica Biosystems, Nussloch, Germany) and mounted in lactic acid [20][21][22].All pure cultures obtained in this study were deposited in the culture collection of the Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences (JZB), China.All herbarium material including holotypes of novel species were deposited in the herbarium of the Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences (JZBH), China, as dry cultures.

DNA Extraction, PCR Amplification, and Sequencing
Fresh mycelia were scraped from the strains on PDA plates that were grown for seven days at 25 • C and collected in 1.5 mL centrifuge tubes.Genomic DNA was extracted using a TIANcombi DNA Lyse&Det PCR Kit (TIANGEN Biotech Co., Ltd., Beijing, China).Polymerase chain reaction (PCR) was carried out using selected genes and primers (Tables 1 and 2).The 25 µL volume of each PCR mixture included 12.5 µL of 2× Taq PCR MasterMix (Beijing Bomede Gene Technology Co., Ltd., Beijing, China), 10.5 µL of ddH 2 O, 0.5 µL each of forward and reverse primer (Sangon Biotech, Shanghai, China), and 1 µL of DNA template.The thermal cycler conditions were as follows: initial denaturation for 3 min at 95 • C; 34 cycles of denaturation for 30 s at 95 • C, annealing for 30 s at 58 • C (the internal transcribed spacer region [ITS]), 56 • C (β-tubulin [tub2]; actin [act]), 54 • C (RNA polymerase II second largest subunit [rpb2]; partial translation elongation factor 1-alpha[tef1]), or 52 • C (28S large subunit of nuclear ribosomal RNA [LSU]); elongation at 72 • C; and a final extension for 10 min at 72 • C. The PCR products were assessed using agarose gel electrophoresis after staining with ethidium bromide and sequenced at Beijing Qingke Biotechnology Co., Ltd.(Beijing, China).

Phylogenetic Analyses
For all the isolates obtained in this study, sequence quality was assured by checking the chromatograms using BioEdit 7.0.9.0.All sequences were subjected to BLAST searches in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLASTn) v. 2.15.0 (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for preliminary identification of isolates.Based on the BLAST results, we identified our isolates as belonging to nine genera, Ascochyta, Didymella, Nothophoma, Botryosphaeria, Diplodia, Neofusicoccum, Phaeobotryon, Lasiodiplodia, and Phaeoacremonium.Reference sequences for phylogenetic analyses were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/), following the recently updated taxonomic literature (Supplementary Table S1).The sequence dataset of each genus was aligned with MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/).BioEdit 7.0.9.0 was used to improve the alignment manually when necessary, such as trimming.
The ML analyses were performed with RAxML-HPC2 on XSEDE (8.2.12) [27,28] on the CIPRES Science Gateway platform [30] with 1000 nonparametric bootstrapping replicates, and the GTR + GAMMA was the nucleotide evolution model.Bayesian inference (BI) was performed in MrBayes v.3.2.7a [29] on the XSEDE tool.The evolution model was tested by using jModelTest2 on XSEDE in the CIPRES Gateway.MrBayes analyses were run for 1,000,000 generations, sampling the trees at every 100th generation.From the 10,000 trees obtained, the first 2000 representing the burn-in phase were discarded.The remaining 8000 trees were used to calculate posterior probabilities in the majority rule consensus tree.
In PAUP, tree stability was evaluated by 1000 bootstrap replications.Branches of zero length were collapsed, and all multiple most parsimonious trees were saved.Parameters, including tree-length (TL), consistency index (CI), retention index (RI), relative consistency index (RC), and homoplasy index (HI) were calculated.Differences between the trees inferred under different optimality criteria were evaluated using Kishino-Hasegawa tests (KHT).Phylogenetic trees were visualized in FigTree v1. 4. The names of the isolates from the present study are marked in red in the trees.ML and MP bootstrap support values greater than 50% (BT) and Bayesian posterior probabilities (PPs) greater than 0.70 are given at the nodes.
Ascochyta is a prominent genus that encompasses not only pathogens but also saprophytic and endophytic fungi that exist on a wide range of substrates [43][44][45][46][47].The combined dataset of LSU, ITS, rpb2, and tub2 ingroup isolates from 15 species consisted of 2276 characters (891 for LSU, 490 for ITS, 596 for rpb2, and 299 for tub2), including alignment gaps.Based on the results of jModel test for BI, TrN + I was determined to be the best model for the LSU dataset, TIM2ef + I was determined to be the best model for the ITS dataset, TIM3 + I + G was determined to be the best model for the rpb2 dataset, and TIM3 + G was determined to be the best model for the tub2 dataset.Didymella aeria (CGMCC 3.18353) and Didymella sinensis (CGMCC 3.18348) were used as outgroup taxa.The best-scoring ML tree with a final likelihood value of −5834.327678 is shown in Figure 2. The matrix had 281 distinct alignment patterns, with 12.84% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.239085, C = 0.240356, G = 0.275806, T = 0.244754; substitution rates, AC = 1.072413,AG = 4.489167, AT = 1.459809,CG = 0.739866, CT = 13.212760, and GT = 1.000000; and gamma distribution shape parameter α = 0.020000.According to the results of the multilocus phylogenetic analysis, eight isolates from P. persica in this study were clustered into an independent branch with 92% bootstrap support and 1.0 BYPP (Figure 2).
2. The matrix had 281 distinct alignment patterns, with 12.84% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.239085, C = 0.240356, G = 0.275806, T = 0.244754; substitution rates, AC = 1.072413,AG = 4.489167, AT = 1.459809,CG = 0.739866, CT = 13.212760, and GT = 1.000000; and gamma distribution shape parameter α = 0.020000.According to the results of the multilocus phylogenetic analysis, eight isolates from P. persica in this study were clustered into an independent branch with 92% bootstrap support and 1.0 BYPP (Figure 2).Conidia 3.9-6.5 × 2.2-3.5 µm (av.= 5.5 × 2.9 µm, n = 50), greatly variable in shape and size, oblong, ovoid, or broad ellipsoidal, smooth and thin-walled, aseptate.Culture characteristics-Colonies on OA were 80-81 mm in diameter after 7 days, margin regular, covered by floccose aerial mycelia, dense, white; reverse black.Colonies grown on MEA had an 84-85 mm diameter after 7 days; margin regular, aerial mycelia sparse, flattened, light grey to white, with some radial line near the centre, reverse concolourous.Colonies on PDA were similar to those on OA but somewhat slower growing, with a 74-76 mm diameter after 7 days, covered by floccose aerial mycelia that were whiter and denser than those on OA, reverse olivaceous (Figure 3).
Notes-In the phylogenetic analysis of the present study, eight isolates from Prunus developed a distinct lineage from other known Ascochyta species with 100% ML, 99% MP bootstrap, and 1.00 BYPP values.Phylogenetically, our isolates showed close affinity to A. pisi, but they can be distinguished by their conidial length, whereas our isolates developed smaller conidia (3.9-6.5 µm) than A. pisi (7-16 µm, CBS 122785; 10-16 µm, CBS 122751) [21,48].The nucleotide differences between JZB380109 and A. pisi Didymella was established by Saccardo in 1880, with the description of Didymella exigua [42].These species are plant pathogens and saprobes on a wide range of hosts [42].
In the present study, six isolates were identified as belonging to Didymella.The combined dataset of LSU, ITS, rpb2, and tub2 with 13 species as ingroup consisted of 2239 characters (854 for LSU, 488 for ITS, 597 for rpb2, and 300 for tub2, including alignment gaps).TrN + I was determined to be the best model for the LSU dataset, TIM2ef + I was determined to be the best model for the ITS dataset, TrN + G was determined to be the best model for the rpb2 dataset, and TrN + I was determined to be the best model for the tub2 dataset.Ascochyta boeremae (CBS 373.84) and Ascochyta fabae (CBS 524.77) were used as outgroup taxa.
The best-scoring ML tree with a final likelihood value of −5647.047275 is shown in Figure 4.The matrix had 242 distinct alignment patterns, with 1.88% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies: A = 0.236386, C = 0.243959, G = 0.279575, and T = 0.240080; substitution rates: AC = 1.269086,AG = 4.594322, AT = 1.024640,CG = 0.692657, CT = 14.061872, and GT = 1.000000; and gamma distribution shape parameter α = 0.020000.According to the results of the phylogenetic analyses of this study, our strains were clustered together with Didymella glomerata, with 98% ML and 1.00 BYPP values (Figure 4).
The best-scoring ML tree with a final likelihood value of −5647.047275 is shown in Figure 4.The matrix had 242 distinct alignment patterns, with 1.88% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies: A = 0.236386, C = 0.243959, G = 0.279575, and T = 0.240080; substitution rates: AC = 1.269086,AG = 4.594322, AT = 1.024640,CG = 0.692657, CT = 14.061872, and GT = 1.000000; and gamma distribution shape parameter α = 0.020000.According to the results of the phylogenetic analyses of this study, our strains were clustered together with Didymella glomerata, with 98% ML and 1.00 BYPP values (Figure 4).This genus was described by Chen et al. [21] and typified with Nothophoma infossa.There are 23 accepted species in this genus (Index Fungorum 2023).For the taxonomic treatments of this genus, we followed Keirnan et al. [53].The combined dataset of LSU, ITS, rpb2, and tub2 ingroup isolates from seven species consisted of 2228 characters (848 for LSU, 485 for ITS, 596 for rpb2, and 299 for tub2, including alignment gaps).TrN was determined to be the best model for the LSU dataset, K80 was determined to be the best model for the ITS dataset, TIM3 + G was the best model for rpb2, and TrN + G was the best model for tub2.Didymella protuberans (CBS 391.93) and Didymella protuberans (CBS Culture characteristics-Colonies on OA, 55-57 mm in diameter after 7 days, margin regular, smoky grey to grey olivaceous, white near the margin, covered by fluffy, dense, white to grey aerial mycelia; reverse concolourous and white near the margin.Colonies on MEA, 68-69 mm in diameter after 7 days, margin regular, covered by floccose, white and greenish olivaceous aerial mycelia, reverse concolourous, white near the margin.Colonies on PDA, 76-79 mm in diameter after 7 days, similar to those on MEA but somewhat faster growing and sparser, reverse concolourous, white margin narrower than those on OA and MEA.
Notes-Six isolates from twig spot and gummosis trunk of peach (Prunus persica L.) in this study were phylogenetically related to Didymella glomerata (Figure 4).Didymella glomerata, known to cause diseases in dicots and conifers, is generally found in the rhizosphere flora but has recently been identified as a cause of stem canker in peach trees, damping off and root necrosis in fennel, and stem rot in coriander [49][50][51][52].This is the first report of this fungus on Prunus persica in China.
Nothophoma Qian Chen & L. Cai, Stud.Mycol.82: 212 (2015).This genus was described by Chen et al. [21] and typified with Nothophoma infossa.There are 23 accepted species in this genus (Index Fungorum 2023).For the taxonomic treatments of this genus, we followed Keirnan et al. [53].The combined dataset of LSU, ITS, rpb2, and tub2 ingroup isolates from seven species consisted of 2228 characters (848 for LSU, 485 for ITS, 596 for rpb2, and 299 for tub2, including alignment gaps).TrN was determined to be the best model for the LSU dataset, K80 was determined to be the best model for the ITS dataset, TIM3 + G was the best model for rpb2, and TrN + G was the best model for tub2.Didymella protuberans (CBS 391.93) and Didymella protuberans (CBS 381.96) were used as outgroup taxa.
The best-scoring ML tree with a final likelihood value of −4754.240240 is given in Figure 6.The matrix had 180 distinct alignment patterns, with 6.46% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.237873, C = 0.242414, G = 0.279069, and T = 0.240643; substitution rates, AC = 1.232725 and AG = 3.271098; AT = 1.025485;CG = 0.670080; CT = 11.394298; and GT = 1.000000; and gamma distribution shape parameter α = 0.020000 (Figure 6).
Culture characteristics-Colonies on OA 57-60 mm in diameter after 7 days with regular margins.Aerial mycelium white, floccose to woolly.Immersed mycelium greygreen olivaceous to deep olivaceous near the colony centre and grey near the margin; reverse concolourous.Colonies on MEA 36-41 mm in diameter after 7 days, margin regular.Aerial mycelia covering the whole colony, compact, white to pale grey; reverse concentric circles of different colours, orange to yellow.Colonies on PDA, 60-66 mm in diameter after 7 days, aerial mycelium sparse, white to grey-green; reverse deep brown, grey near the margin.

Nothophoma quercina
Culture characteristics-Colonies on OA were 61-69 mm in diameter after 7 days with regular margins.Aerial mycelium white, floccose to woolly.Immersed mycelium grey-green olivaceous to light olivaceous near the colony centre and white near the margin; reverse concolourous.Colonies on MEA were 55-75 mm in diameter after 7 days, margins regular.Aerial mycelia covering the whole colony were compact, white to pale grey, with some radially furrowed zones; reverse concentric circles of different colours, orange to yellow and light yellow near the margin.Colonies on PDA were 65-68 mm in diameter after 7 days, margins regular, covered by floccose, white and greenish olivaceous aerial mycelia, reverse concolourous, light green near the margin.
Notes-In this study, 11 isolates obtained from shoot blight and gummosis trunk of peach (Prunus persica) were phylogenetically closely related to Nothophoma quercina (Figure 6).Morphologically, our isolates share the same characteristics as given in the type species description [21].Nothophoma quercina has been reported as the main pathogen causing branch blight [55,56].This is the first report of this fungus infecting the host Prunus persica in China.
Botryosphaeriaceae includes diverse pathogenic members that are classified as plant opportunistic fungal pathogens [57,58].Species of Botryosphaeriaceae cause gummosis and shoot blight disease in peach [59][60][61][62][63].These species are also important pathogens of grapevines and are associated with a variety of diseases [64].Additionally, more than 20 species of Botryosphaeriaceae have been reported to cause Botryosphaeria dieback [65].For taxonomic treatments, we followed Hongsanan et al. [24] and Wu et al. [ For the taxonomic treatment of this genus, we followed Zhang et al. [67].The combined dataset of ITS, tef1, and tub2 ingroup isolates from nine species consisted of 1436 characters (611 for ITS, 362 for tef1, and 463 for tub2, including alignment gaps).TrN + G was determined to be the best model for the ITS dataset, TPM2uf + I was the best model for the tef1 dataset, and TIM3 + G was the best model for the tub2 dataset.Diplodia corticola (CBS 112546) and Diplodia corticola (CBS 112549) were used as outgroup taxa.
The best-scoring ML tree with a final likelihood value of −3605.559999 is shown in Figure 9.The matrix had 327 distinct alignment patterns, 16.87% of which were undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.216286, C = 0.301284, G = 0.257502, and T = 0.224928; substitution rates, AC = 1.420115,AG = 2.115530, AT = 1.012019,CG = 1.284585,CT = 3.876955, and GT = 1.000000; and gamma distribution shape parameter α = 0.319888 (Figure 9).Diplodia Fr., Ann.Sci.Nat., Bot.Sér. 2, 1: 302 (1834).For the taxonomic treatment of this genus, we followed Zhang et al. [67].The combined dataset of ITS, tef1, and tub2 ingroup isolates of 13 species consisted of 1261 characters (540 for ITS, 300 for tef1, and 421 for tub2, including alignment gaps).TPM3 + I + G was determined to be the best model for ITS, TrN + G was determined to be the best model for tef1, and TrN + I + G was the best model for the tub2 dataset.Lasiodiplodia theobromae (CBS 164.96) was used as the outgroup taxon.
The best-scoring ML tree with a final likelihood value of −3415.055016 is given in Figure 11.The matrix had 270 distinct alignment patterns, with 7.54% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.200294, C = 0.314841, G = 0.255463, and T = 0.229402; substitution rates, AC = 0.879909, AG = 2.534595, AT = 1.049732,CG = 1.056783,CT = 5.084602, and GT = 1.000000; and gamma distribution shape parameter α = 0.104584 (Figure 11).Note-In the phylogenetic analysis of the present study, 15 isolates from branch canker and gummosis trunk of peach from four provinces in China were clustered together with Botryosphaeria dothidea (Figure 9).Further, these isolates were morphologically similar to those given in the type species description.Botryosphaeria dothidea is an opportunistic pathogen with a wide host range [68].It has been reported to cause shoot blight [63], and it is also related to gummosis-causing agents [62].
For the taxonomic treatment of this genus, we followed Zhang et al. [67].The combined dataset of ITS, tef1, and tub2 ingroup isolates of 13 species consisted of 1261 characters (540 for ITS, 300 for tef1, and 421 for tub2, including alignment gaps).TPM3 + I + G was determined to be the best model for ITS, TrN + G was determined to be the best model for tef1, and TrN + I + G was the best model for the tub2 dataset.Lasiodiplodia theobromae (CBS 164.96) was used as the outgroup taxon.
The best-scoring ML tree with a final likelihood value of −3415.055016 is given in Figure 11.The matrix had 270 distinct alignment patterns, with 7.54% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.200294, C = 0.314841, G = 0.255463, and T = 0.229402; substitution rates, AC = 0.879909, AG = 2.534595, AT = 1.049732,CG = 1.056783,CT = 5.084602, and GT = 1.000000; and gamma distribution shape parameter α = 0.104584 (Figure 11).Notes-In the present study, we examined the morphology and phylogeny of samples of peach twig spots from China and identified these isolates as Diplodia seriata (Figure 11).Diplodia seriata (syn.B. obtusa) has been reported in many countries and is recognized as an important pathogen of stone, pome, and soft fruit trees, causing cankers, leaf spots, and black fruit rot [69][70][71][72].Neofusicoccum was introduced by Crous et al. [73] as a species that is morphologically similar to but phylogenetically distinct from Botryosphaeria and thus could no longer be included in that genus.For the taxonomic treatment of this genus, we followed Zhang et al. [67].The combined dataset of ITS, tef1, and tub2 included 24 ingroup isolates from 11 species and consisted of 1406 characters 542 for ITS, 441 for tef1, and 423 for tub2, including alignment gaps.TIM1 + I was determined to be the best model for the ITS dataset, HKY + G was determined to be the best model for the tef1 dataset, and TrN + G was determined to be the best model for the tub2 dataset.Botryosphaeria dothidea (CBS 115476) was used as the outgroup taxon.
The best-scoring ML tree with a final likelihood value of −2311.055412 is given in Figure 13.The matrix had 101 distinct alignment patterns, with 8.20% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.204892, C = 0.316100, G = 0.266872, and T = 0.212136; substitution rates, AC = 0.804456, AG = 7.315164, AT = 3.072031, CG = 1.357888,CT = 9.437879, and GT = 1.000000; and gamma distribution shape parameter α = 1.010866 (Figure 13).Culture characteristics-Colonies on PDA had fluffy aerial mycelia with irregular margins, appressed moderately dense mycelial mats and smoky grey to dark olivaceous, covering the dish after 5 days at 25 • C in the dark.
Notes-In the present study, we examined the morphology and phylogeny of samples of peach twig spots from China and identified these isolates as Diplodia seriata (Figure 11).Diplodia seriata (syn.B. obtusa) has been reported in many countries and is recognized as an important pathogen of stone, pome, and soft fruit trees, causing cankers, leaf spots, and black fruit rot [69][70][71][72].
Neofusicoccum was introduced by Crous et al. [73] as a species that is morphologically similar to but phylogenetically distinct from Botryosphaeria and thus could no longer be included in that genus.For the taxonomic treatment of this genus, we followed Zhang et al. [67].The combined dataset of ITS, tef1, and tub2 included 24 ingroup isolates from 11 species and consisted of 1406 characters 542 for ITS, 441 for tef1, and 423 for tub2, including alignment gaps.TIM1 + I was determined to be the best model for the ITS dataset, HKY + G was determined to be the best model for the tef1 dataset, and TrN + G was determined to be the best model for the tub2 dataset.Botryosphaeria dothidea (CBS 115476) was used as the outgroup taxon.
(CBS128008).Morphologically, our isolates have similar characteristics to the Ne.occulatum type species [74], thus we identified our isolates as Ne.occulatum.Neofusicoccum occulatum was reported as the pathogen causing shoot blight in Platycladus orientalis [74].In the present study, we presented the morphology and phylogeny of peach twig canker samples from China and identified these isolates as Neofusicoccum occulatum (Figures 13  and 14).Phaeobotryon was introduced by Theiss.and Syd. to accommodate Dothidae cercidis as Phaeobotryon cercidis and the species which are phylogenetically and morphologically distinguished from the other genera in Botryosphaeriaceae [57,75].For the taxonomic treatment of this genus, we followed Zhang et al. [67].The combined dataset of ITS, LSU, and tef1 from 21 ingroup isolates of seven species consisted of 1272 characters (449 for ITS, 558 for LSU, and 265 for tef1, including alignment gaps).TIM1ef + I was determined to be the best model for the ITS dataset, TrN + I for the LSU dataset, and HKY + G was determined to be the best model for the tef1 dataset.Barriopsis iraniana (CBS 124698) was used as the outgroup taxon.
The best-scoring ML tree with a final likelihood value of −2724.623212 is given in Figure 15.The matrix had 144 distinct alignment patterns, 19.13% of which were undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.225969, C = 0.265270, G = 0.277126, and T = 0.231634; substitution rates, AC = 0.866717, AG = 2.192592, AT = 0.532373, CG = 0.670797, CT = 5.651745, and GT = 1.000000; and gamma distribution shape parameter α = 0.836999 (Figure 15).Notes-In the phylogenetic analysis of the present study, three isolates obtained from Prunus clustered together with the Neofusicoccum occulatum type species (CBS128008).Morphologically, our isolates have similar characteristics to the Ne.occulatum type species [74], thus we identified our isolates as Ne.occulatum.Neofusicoccum occulatum was reported as the pathogen causing shoot blight in Platycladus orientalis [74].In the present study, we presented the morphology and phylogeny of peach twig canker samples from China and identified these isolates as Neofusicoccum occulatum (Figures 13 and 14).
Phaeobotryon was introduced by Theiss.and Syd. to accommodate Dothidae cercidis as Phaeobotryon cercidis and the species which are phylogenetically and morphologically distinguished from the other genera in Botryosphaeriaceae [57,75].For the taxonomic treatment of this genus, we followed Zhang et al. [67].The combined dataset of ITS, LSU, and tef1 from 21 ingroup isolates of seven species consisted of 1272 characters (449 for ITS, 558 for LSU, and 265 for tef1, including alignment gaps).TIM1ef + I was determined to be the best model for the ITS dataset, TrN + I for the LSU dataset, and HKY + G was determined to be the best model for the tef1 dataset.Barriopsis iraniana (CBS 124698) was used as the outgroup taxon.
The best-scoring ML tree with a final likelihood value of −2724.623212 is given in Figure 15 16).
Culture characteristics-The colonies were originally white and produced dark green to black pigments after they had been incubated for 7-10 days.The texture was felty with an appressed mycelial mat and fluffy aerial mycelia near the centre, with regular edges.Colonies reached the 90 mm diameter of a Petri dish after 5 days in the dark at 25 °C.
Culture characteristics-The colonies were originally white and produced dark green to black pigments after they had been incubated for 7-10 days.The texture was felty with an appressed mycelial mat and fluffy aerial mycelia near the centre, with regular edges.Colonies reached the 90 mm diameter of a Petri dish after 5 days in the dark at 25 • C.
Notes-In the present study, we examined the morphology and phylogeny of peach twig canker samples from China and identified these isolates as Phaeobotryon rhois (Figure 16).Phaeobotryon rhois is known to cause canker and dieback disease in Rhus typhina in China [76].This is the first report of P. rhois being associated with twig canker disease in peach.
Lasiodiplodia Ellis & Everh., Bot.Gaz.21: 92 (1896).Lasiodiplodia species are cosmopolitan and have an extensive host and geographical range.They are pathogenic on economically important fruit crops [57].For the taxonomic treatment of this genus, we followed Zhang et al. [67] and Xia et al. [77].The combined ITS, tef1, and tub2 dataset of 79 ingroup strains from 43 species consisted of 1158 characters, (415 for ITS, 332 for tef1, and 411 for tub2, including alignment gaps).TVM + I was determined to be the best model for the ITS dataset, HKY + I + G was determined to be the best model for the TEF dataset, and TrN + I was determined to be the best model for the tub2 dataset.Diplodia seriata (CBS 112555) and Diplodia mutila (CMW 7060) were used as the outgroup taxon.
The best-scoring ML tree with a final likelihood value of −5119.098465 is given in Figure 17.The matrix had 357 distinct alignment patterns, 13.73% of which were undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.209134, C = 0.307908, G = 0.255624, and T = 0.227335; substitution rates, AC = 1.006140,AG = 3.800814, AT = 1.344431,CG = 0.991903, CT = 5.144516, and GT = 1.000000; and gamma distribution shape parameter α = 0.798124 (Figure 17).
Togniniaceae Réblová, L. Phaeoacremonium comprises common pathogens that cause stem and branch diseases in a wide range of woody hosts [78].In 2021, P. minimum was first reported as a pathogen causing esca disease in China [79].In this study, we followed Ye et al. [23] for taxonomic treatments.
The combined dataset of act and tub2 contained 25 ingroup isolates from 12 species and consisted of 880 characters (259 for act and 621 for tub2, including alignment gaps).HKY + G was determined to be the best model for the act dataset, and TPM2uf + G was the best model for the tub2 dataset.Pleurostomophora richardsiae (CBS 270.33) was used as the outgroup taxon.The best-scoring ML tree with a final likelihood value of −4698.584463 is given in Figure 19.The matrix had 391 distinct alignment patterns, with 6.55% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.204162, C = 0.311249, G = 0.238989, and T = 0.245600; substitution rates, AC = 1.158313,AG = 4.407917, AT = 1.364022,CG = 0.995655, CT = 4.976526, and GT = 1.000000; and gamma distribution shape parameter α = 0.505328 (Figure 19). the outgroup taxon.The best-scoring ML tree with a final likelihood value of −4698.584463 is given in Figure 19.The matrix had 391 distinct alignment patterns, with 6.55% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.204162, C = 0.311249, G = 0.238989, and T = 0.245600; substitution rates, AC = 1.158313,AG = 4.407917, AT = 1.364022,CG = 0.995655, CT = 4.976526, and GT = 1.000000; and gamma distribution shape parameter α = 0.505328 (Figure 19).Notes-Three isolates obtained from gummosis trunk disease of peach (Prunus persica) were phylogenetically closely related to P. scolyti.P. scolyti is also known as V. vinifera in South Africa [80,81].This fungus had the broadest host range and was found on the Prunus species sampled [82].
Culture characteristics-colonies on PDA were flat, felty to woolly, with irregular edges at a radius of 10-12 mm after 9 days at 25 • C; after 9 days, the cells became pinkish white to hyaline/translucent.
Notes-Three isolates obtained from gummosis trunk disease of peach (Prunus persica) were phylogenetically closely related to P. scolyti.P. scolyti is also known as V. vinifera in South Africa [80,81].This fungus had the broadest host range and was on the Prunus species sampled [82].

Discussion
China is the world's largest producer of peach.The peach orchards in China are commonly affected by cankers, leaf spots, and fruit rot diseases.However, compared to fruit rot and other fruit diseases, trunk disease is usually disregarded even though trunk diseases directly affect the lifespan of the plant.In the present study, we focused on trunk, branch, and twig-inhabiting fungi, and 85 isolates were obtained from diseased peach trunks, branches, and twigs.The isolates were identified as 10 fungal species belonging to nine genera using phenotypic characteristics and a multilocus phylogeny.Among them, two species were identified as new, three species were reported on peach in China for the first time, and four species were reported on peach for the first time worldwide.Among these isolates, Didymellaceae and Botryosphaeriaceae were the most common taxa, and Didymellaceae (42.4%) and Botryosphaeriaceae (54.1%) accounted for more than 90% of the total isolates.
Didymellaceae is a species-rich family that features a diverse range of fungi that showcase global distribution patterns.Additionally, many of these fungi are economically important plant pathogens [42].We isolated and identified species belonging to three Didymellaceae genera, and Nothophoma was the most frequently isolated genus.Species belonging to this genus are pathogens, endophytes, and saprobes on economically important crops and forest trees [54].Nothophoma quercina (syn.Phoma fungicola) was the main pathogen causing branch blight [55,56].This fungus produces abundant pycnidia on plant residues, which can subsequently become the primary source of infection.Under favourable conditions, such as cloudy and cool weather (moisture above 70% and temperature between 20 and 25 • C), the fungus can immediately asexually reproduce and kill the host [83].Nothophoma pruni has been reported to be a saprobe on diseased leaves of Prunus avium [54].In this study, we first isolated Nothophoma pruni from twig spot and gummosis trunk samples of peach from around the world.N. quercina was first isolated from shoot blight and gummosis trunk samples of peach in China.
Ascochyta was introduced by Libert in 1830, with A. pisi described as a type species [84].Some of the species have been reported as plant pathogens; Ascochyta syringae causes Ascochyta blight of lilac (Syringa vulgaris) in America, Australia, and Europe [46], and it has been isolated mostly from soil [47].In the present study, A. prunus was isolated from twig canker and branch canker samples of P. persica as a novel species.Ascochyta prunus was distinguished by its conidial length compared to A. pisi (CBS 122785) and A. pisi (CBS 122751).
Didymella glomerata (former name Phoma glomerata) is a globally distributed soil fungus that has been isolated from various plants (more than 100 host plant genera).Generally, it is considered a secondary invasive or opportunistic pathogen [18].Didymella glomerata is associated with stem canker of peach, damping off, and root necrosis in fennel and stem rot of coriander [49][50][51][52].It has also been reported to be a mycoparasite of powdery mildew [85].Didymella glomerata as P. glomerata has been recorded as an endophytic fungus from Korean pine (Pinus koraiensis) leaves [86].It has also been associated with the pea "Ascochyta blight complex" in Australia [87].In this study, we first isolated D. glomerata from twig spot and gummosis trunk samples from peach worldwide.
Botryosphaeriaceae harbours a collection of fungi that exhibit considerable diversity in terms of morphology; these fungi include endophytic, pathogenic, and saprobic variants that primarily affect woody plants.The frequency with which these fungi are involved with plant diseases is substantial.Most species of Botryosphaeria are considered latent plant pathogens that cause dieback, cankers, gummosis, leaf spots, or fruit rot on many woody plants, including pear, grape, mango, olive, eucalyptus, maple, oak, and almond [57].These species are important pathogens of peach and are associated with a series of diseases, including gummosis [59][60][61] and shoot blight [63].Botryosphaeria dothidea is one of the most common species of Botryosphaeriaceae and has been reported in hundreds of plant species worldwide [57].According to a previous study, B. dothidea causes perennial cankers in peach tree trunks, branches, and shoots [57].Gummosis is a common disease of peach that was first observed in the 1970s in Fort Valley, GA, and the causal agent was first identified as B. dothidea [59].A subsequent report revealed that B. dothidea, B. rhodina, and B. obtusa cause peach tree gummosis in Georgia [60].Chen [61] first reported the occurrence of B. dothidea causing gummosis of peach trees in China.Wang reported that Lasiodiplodia theobromae or Diplodia seriata also cause peach tree gummosis in China [62].In the present study, we isolated B. dothidea from samples collected from four provinces in China, which presented branch canker and gummosis trunk symptoms.
Diplodia is a common pathogen on a wide range of hosts.Diplodia seriata is associated with olive plants in Tunisia [69] and Croatia [70], and in Uruguay, it was isolated from grapevine [71], apple [72], and peach [88].Diplodia mutila was reported as a new record for olive in Uruguay.Previously, this species was isolated only from pear [89].In the present study, we isolated D. seriata from peach twig spot samples.Neofusicoccum occulatum was reported as the pathogen causing shoot blight in Platycladus orientalis [74].Ma et al. [90] first reported that N. occulatum was associated with Dendrobium chrysanthum.Neofusicoccum occulatum was reported as a new record on olives worldwide [89].It was also described in Australia to affect Eucalyptus and Wollemia nobilis [91] and subsequently found in blueberry [92].In this study, we isolated N. occulatum from twig canker of peach.Phaeobotryon was introduced by Theissen & Sydow [75].Phaeobotryon rhois was previously reported as a pathogen associated with cankers on Rhus typhinain in northwestern China [76].Zhu et al. reported this species from peach on Mount Dongling, Beijing, China [93].In addition, Phaeobotryon rhois has been reported from various hosts including Dioscoreanipponica, Platycladus orientalis, and Rhamnus davurica [94].In this study, we extended its host range to peach.
Lasiodiplodia is commonly associated with diseases of agricultural and forestry crops and has a wide global distribution.This genus is typified by L. theobromae.In the present study, we introduce a novel species closely related to L. acacia.Zhang et al. [67] reported L. acacie, which was isolated for the first time from Acacia sp.Identification and characterization of Lasiodiplodia species have become challenging recently.Previous studies have mentioned that morphology and phylogeny play an important role in species delineation [37,77].In addition, some studies have proposed that Lasiodiplodia species might have host specificity as well [77].However, pathogenicity assays are required to understand the relationship of the novel species with the gummosis disease in peach.
Phaeoacremonium is associated with stunted growth and dieback in various woody hosts.Damm et al. [82] isolated Phaeoacremonium species from necrotic woody tissue of Prunus spp.(plum, peach, nectarine, and apricot) from the growing areas of different stone fruits in South Africa.In this study, we isolated P. scolyti from peach with gummosis trunk disease.
Based on the results above, it is evident that peach trunk disease is a complex disease that might be caused by different fungal species.These fungal taxa may have varying impacts on peach.However, future studies are required to understand the infection mechanisms and co-infection of these species that lead to peach trunk disease in China.A similar observation has been reported for grapevine woody pathogens.Kraus [95] reported that grapevine wood is a highly complex habitat, with the simultaneous presence of plant pathogens and beneficial, potentially protective fungi.Pathogenic fungi can shift their biotrophic mode from pathogenic to saprotrophic and can become active again under favourable conditions, thus serving as the primary source of inoculation within a vineyard [96].Therefore, it is important to conduct pathogenicity tests to discern the roles of saprotrophs, endophytes, and pathogens and to investigate the interactions between different communities.Rather than focusing on a single peach branch disease, our research included a comprehensive examination of the various fungi responsible for causing the main symptoms of peach branch diseases throughout China in recent years.More comprehensive field investigations and pathogenicity tests will be implemented in the future.
Overall, early detection and development of management strategies for the correct species identification are important in plant pathology [97].The present study allowed us to gain a better understanding of the fungal communities associated with peach branch diseases and their roles in the ecosystem.Our results revealed that there was a high diversity of fungi associated with peach branch diseases, with each disease type being dominated by a specific set of fungal species.Additionally, our study revealed that some fungi were present in multiple disease types, suggesting that they may play a role in the progression of multiple diseases.These findings highlight the intricate and complex nature of fungal communities associated with peach branch diseases and emphasize the need for further investigation to fully understand the ecological roles and interactions of fungal communities in peach orchard ecosystems.

Figure 4 .
Figure 4. Maximum likelihood (ML) phylogram reconstructed from the combined sequences of LSU, ITS, rpb2, and tub2 of Didymella isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/PP/MP BS.The scale bar represents the expected number of changes

Figure 4 . 34 Figure 5 .
Figure 4. Maximum likelihood (ML) phylogram reconstructed from the combined sequences of LSU, ITS, rpb2, and tub2 of Didymella isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/PP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Ascochyta fabae (CBS 524.77) and Ascochyta boeremae (CBS 373.84).Isolates from this study are marked in red, and the type specimens are indicated in bold.J. Fungi 2024, 10, x FOR PEER REVIEW 10 of 34

Figure 6 .
Figure 6.Maximum likelihood (ML) phylogram reconstructed from the combined sequences of LSU, ITS, rpb2, and tub2 of Nothophoma isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/PP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Didymella protuberans (CBS 391.93) and Didymella protuberans (CBS 381.96).Isolates from this study are marked in red, and the type specimens are indicated in bold.

Figure 9 .
Figure 9. Maximum likelihood (ML) phylogram reconstructed from the combined ITS, tef1, and tub2 sequences of Botryosphaeria isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/BPP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Diplodia corticola (CBS 112546) and Diplodia corticola (CBS 112549).Isolates from this study are marked in red.and the type specimens are indicated in bold.

Figure 12 .
Figure 12.Diplodia seriata (JZB310241) (A,B) Colony on PDA (front and reverse); (C) Appearance of conidiomata on PDA; (D,E) Conidiogenous cells; (F,G) Conidia; Scale bars: (D-F) = 10 µm and (G) = 5 µm.Neofusicoccum Crous, Slippers & A.J.L. Phillips, Stud.Mycol.55: 247 (2006).Neofusicoccum was introduced by Crous et al.[73] as a species that is morphologically similar to but phylogenetically distinct from Botryosphaeria and thus could no longer be included in that genus.For the taxonomic treatment of this genus, we followed Zhang et al.[67].The combined dataset of ITS, tef1, and tub2 included 24 ingroup isolates from 11 species and consisted of 1406 characters 542 for ITS, 441 for tef1, and 423 for tub2, including alignment gaps.TIM1 + I was determined to be the best model for the ITS dataset, HKY + G was determined to be the best model for the tef1 dataset, and TrN + G was determined to be the best model for the tub2 dataset.Botryosphaeria dothidea (CBS 115476) was used as the outgroup taxon.The best-scoring ML tree with a final likelihood value of −2311.055412 is given in Figure13.The matrix had 101 distinct alignment patterns, with 8.20% undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.204892, C = 0.316100, G = 0.266872, and T = 0.212136; substitution rates, AC = 0.804456, AG = 7.315164, AT = 3.072031, CG = 1.357888,CT = 9.437879, and GT = 1.000000; and gamma distribution shape parameter α = 1.010866 (Figure13).

Figure 12 .
Figure 12.Diplodia seriata (JZB310241) (A,B) Colony on PDA (front and reverse); (C) Appearance of conidiomata on PDA; (D,E) Conidiogenous cells; (F,G) Conidia; Scale bars: (D-F) = 10 µm and (G) = 5 µm.MycoBank number: MB180468.Associated with twig spots on Prunus persica.Sexual morph: not observed.Asexual morph: Conidiomata pycnidial, solitary, globose to ovoid, dark brown to black, embedded, semi-immersed to superficial.Conidiogenous cells hyaline, smooth, thin-walled, and discrete, producing a single conidia at the tip, proliferating internally and giving rise to periclinal thickening or proliferating concurrently, forming 2-3 annellations, 8-15 × 3-6 µm (av.= 11.7 × 4.6 µm, n = 30).Conidia hyaline, thin-walled, smooth, aseptate, ovoid, apex subobtuse, becoming brown when mature, 19-28 × 9-12 µm (av.= 23.2 × 10.4 µm, n = 50; L/W = 2.2).Culture characteristics-Colonies on PDA had fluffy aerial mycelia with irregular margins, appressed moderately dense mycelial mats and smoky grey to dark olivaceous, covering the dish after 5 days at 25 • C in the dark.Material examined-Pinggu district, Beijing municipality, China, from twig spots on Prunus persica, April 2020.Y Zhou (living culture JZB310240-JZB31043).Notes-In the present study, we examined the morphology and phylogeny of samples of peach twig spots from China and identified these isolates as Diplodia seriata (Figure11).Diplodia seriata (syn.B. obtusa) has been reported in many countries and is recognized as an important pathogen of stone, pome, and soft fruit trees, causing cankers, leaf spots, and black fruit rot[69][70][71][72].Neofusicoccum Crous, Slippers & A.J.L. Phillips, Stud.Mycol.55: 247 (2006).Neofusicoccum was introduced by Crous et al.[73] as a species that is morphologically similar to but phylogenetically distinct from Botryosphaeria and thus could no longer be included in that genus.For the taxonomic treatment of this genus, we followed Zhang et al.[67].The combined dataset of ITS, tef1, and tub2 included 24 ingroup isolates from 11 species and consisted of 1406 characters 542 for ITS, 441 for tef1, and 423 for tub2, including alignment gaps.TIM1 + I was determined to be the best model for the ITS dataset, HKY + G was determined to be the best model for the tef1 dataset, and TrN + G was determined to be the best model for the tub2 dataset.Botryosphaeria dothidea (CBS 115476) was used as the outgroup taxon.The best-scoring ML tree with a final likelihood value of −2311.055412 is given in Figure13.The matrix had 101 distinct alignment patterns, with 8.20% undetermined characters or gaps.The parameters for the model of the combined dataset were as

Figure 14 .
Figure 14.Neofusicoccum occulatum (JZB3120010) (A,B) Colony on PDA (front and reverse); (C,D) appearance of conidiomata on PDA; (E) conidia developing on conidiogenous cells; (F,G) conidia; Scale bars: (E) = 20 µm; (F) = 5 µm; and (G) = 10 µm.Phaeobotryon Theiss.& Syd., Annales Mycologici 13 (3-4): 664 (1915).Phaeobotryon was introduced by Theiss.and Syd. to accommodate Dothidae cercidis as Phaeobotryon cercidis and the species which are phylogenetically and morphologically distinguished from the other genera in Botryosphaeriaceae[57,75]. For the taxonomic treatment of this genus, we followed Zhang et al.[67].The combined dataset of ITS, LSU, and tef1 from 21 ingroup isolates of seven species consisted of 1272 characters (449 for ITS, 558 for LSU, and 265 for tef1, including alignment gaps).TIM1ef + I was determined to be the best model for the ITS dataset, TrN + I for the LSU dataset, and HKY + G was determined to be the best model for the tef1 dataset.Barriopsis iraniana (CBS 124698) was used as the outgroup taxon.The best-scoring ML tree with a final likelihood value of −2724.623212 is given in Figure15.The matrix had 144 distinct alignment patterns, 19.13% of which were undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.225969, C = 0.265270, G = 0.277126, and T = 0.231634; substitution rates, AC = 0.866717, AG = 2.192592, AT = 0.532373, CG = 0.670797, CT = 5.651745, and GT = 1.000000; and gamma distribution shape parameter α = 0.836999 (Figure15).

Figure 14 .
Figure 14.Neofusicoccum occulatum (JZB3120010) (A,B) Colony on PDA (front and reverse); (C,D) appearance of conidiomata on PDA; (E) conidia developing on conidiogenous cells; (F,G) conidia; Scale bars: (E) = 20 µm; (F) = 5 µm; and = 10 µm.Culture characteristics-Colony on PDA superficial, grey, fluffy, reverse dark brown to black, and colonies covering the 90 mm diameter Petri dish were incubated for 5 days in the dark at 25 • C. Material examined-Changping district, Beijing municipality, China, from the twig canker of Prunus persica, Aug. 2021.Y Zhou (living cultures JZB3600010-JZB3600012).Notes-In the phylogenetic analysis of the present study, three isolates obtained from Prunus clustered together with the Neofusicoccum occulatum type species (CBS128008).Morphologically, our isolates have similar characteristics to the Ne.occulatum type species[74], thus we identified our isolates as Ne.occulatum.Neofusicoccum occulatum was reported as the pathogen causing shoot blight in Platycladus orientalis[74].In the present study, we presented the morphology and phylogeny of peach twig canker samples from China and identified these isolates as Neofusicoccum occulatum (Figures13 and 14).Phaeobotryon Theiss.& Syd., Annales Mycologici 13 (3-4): 664 (1915).Phaeobotryon was introduced by Theiss.and Syd. to accommodate Dothidae cercidis as Phaeobotryon cercidis and the species which are phylogenetically and morphologically distinguished from the other genera in Botryosphaeriaceae[57,75]. For the taxonomic treatment of this genus, we followed Zhang et al.[67].The combined dataset of ITS, LSU, and tef1 from 21 ingroup isolates of seven species consisted of 1272 characters (449 for ITS, 558 for LSU, and 265 for tef1, including alignment gaps).TIM1ef + I was determined to be the best model for the ITS dataset, TrN + I for the LSU dataset, and HKY + G was determined to be the best model for the tef1 dataset.Barriopsis iraniana (CBS 124698) was used as the outgroup taxon.The best-scoring ML tree with a final likelihood value of −2724.623212 is given in Figure15.The matrix had 144 distinct alignment patterns, 19.13% of which were undetermined characters or gaps.The parameters for the model of the combined dataset were as follows: estimated base frequencies, A = 0.225969, C = 0.265270, G = 0.277126, and T = 0.231634; substitution rates, AC = 0.866717, AG = 2.192592, AT = 0.532373, CG = 0.670797, CT = 5.651745, and GT = 1.000000; and gamma distribution shape parameter α = 0.836999 (Figure15).Phaeobotryon rhois C.M. Tian, X.L. Fan & K.D. Hyde, Phytotaxa 205(2): 95 (2015) (Figure16).

Figure 15 .
Figure 15.Maximum likelihood (ML) phylogram reconstructed from the combined sequences of ITS, LSU, and tef1 of Phaeobotryon isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/BPP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Barriopsis iraniana CBS 124698.Isolates from this study are marked in red, and the type specimens are indicated in bold.Phaeobotryon rhois C.M. Tian, X.L. Fan & K.D. Hyde, Phytotaxa 205(2): 95 (2015) (Figure16).MycoBank number: MB 811599; Facesoffungi number: FoF 00596.Associated with twig canker of Prunus persica Sexual morph: not observed.Asexual morph: Conidiomata pycnidial, produced on PDA, solitary, globose to ovoid, dark brown to black, 274.73-1155 µm.Conidiogenous cells discrete, hyaline, cylindrical to lageniform, 6-18 × 3-7 µm (av.= 13.3 × 4.3 µm, n = 30).Conidia ellipsoid to oblong or subcylindrical or obovoid, smooth to verruculose, moderately thick-walled, guttulate, ends rounded, initial hyaline, aseptate, becoming brown, 1-septate when mature, 18-29 × 10-16 µm (av.= 25 × 13 µm, n = 50, L/W = 1.9).Culture characteristics-The colonies were originally white and produced dark green to black pigments after they had been incubated for 7-10 days.The texture was felty with an appressed mycelial mat and fluffy aerial mycelia near the centre, with regular edges.Colonies reached the 90 mm diameter of a Petri dish after 5 days in the dark at 25 °C.Material examined-Pinggu district, Beijing municipality, China, from the twig canker of Prunus persica, Aug. 2021.Y Zhou, DL Ma, and ZZ He, living cultures JZB3600007-JZB3600009.Notes-In the present study, we examined the morphology and phylogeny of peach twig canker samples from China and identified these isolates as Phaeobotryon rhois (Figure16).Phaeobotryon rhois is known to cause canker and dieback disease in Rhus typhina in China[76].This is the first report of P. rhois being associated with twig canker disease in peach.

Figure 15 .
Figure 15.Maximum likelihood (ML) phylogram reconstructed from the combined sequences of ITS, LSU, and tef1 of Phaeobotryon isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/BPP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Barriopsis iraniana CBS 124698.Isolates from this study are marked in red, and the type specimens are indicated in bold.

Figure 17 .
Figure 17.Maximum likelihood (ML) phylogram reconstructed from the combined sequences of ITS, tef1, and tub2 of Lasiodiplodia isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/BPP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Neodeightonia phoenicum CBS 122528.Isolates from this study are marked in red, and the type specimens are indicated in bold.

Table 1 .
Gene regions and related primers used for molecular analysis.

Table 2 .
Selected genes and primers for PCR amplification of each genus.

Ascochyta prunus Figure 2. Maximum
likelihood (ML) phylogram reconstructed from the combined sequences of LSU, ITS, rpb2, and tub2 of Ascochyta species.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/PP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Didymella sinensis (CGMCC 3.18348) and Didymella aeria (CGMCC 3.18353).The novel species proposed are indicated in red font, and the type specimens are indicated in bold.
Qian Chen & L. Cai. in Qian Chen and L. Cai, Stud.Mycology 82: 213 (2015) Figure 8. Maximum likelihood (ML) phylogram reconstructed from the combined sequences of LSU, ITS, rpb2, and tub2 of Nothophoma isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/PP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Didymella protuberans (CBS 391.93) and Didymella protuberans (CBS 381.96).Isolates from this study are marked in red, and the type specimens are indicated in bold.

Lasiodiplodia rubropurpurea CBS 118739T Lasiodiplodia vitis CBS 124060T Diplodia seriata CBS 112555T Lasiodiplodia avicenniae CBS 139670T Lasiodiplodia gravistriata CMM 4564T Lasiodiplodia gravistriata CMM 4565 Lasiodiplodia cinnamomi CFCC 51997T Lasiodiplodia vaccinii CGMCC 3.19022T Lasiodiplodia venezuelensis CBS 118739T Lasiodiplodia margaritacea CBS 122519T
Maximum likelihood (ML) phylogram reconstructed from the combined sequences of ITS, tef1, and tub2 of Lasiodiplodia isolates.Bootstrap support values for ML and maximum parsimony (MP) greater than 50% and Bayesian posterior probabilities greater than 0.70 are indicated above the branches as ML BS/BPP/MP BS.The scale bar represents the expected number of changes per site.The tree is rooted with Diplodia seriata (CBS 112555) and Diplodia mutila (CMW 7060).Isolates from this study are marked in red, and the type specimens are indicated in bold.