Botryosphaeriaceous fungi comprised plant endophytes, saprobes and parasites mostly associated with woody hosts (Phillips et al. 2013; Ariyawansa et al. 2016; Dissanayake et al. 2016; Slippers et al. 2017). Botryosphaeria Ces. & de Not. was established in 1863 by Cesati & de Notaris based on twelve species including Botryosphaeria dothidea which was designated as the lecotype by Barr (1972). Earlier species of Botryosphaeria had been described mostly based on their sexual morph and host associations, which led to the introduction of a large number of species (Cesati & de Notaris 1863; de Notaris 1863; Saccardo 1877, 1882; Grossenbacher & Duggar 1911; Putterill 1919; Trotter 1928). So far, more than 200 epithets have been included in Index Fungorum (http://www.indexfungorum.org/, Aug., 2018).

The polyphyletic status of Botryosphaeria sensu lato has been well documented with species mostly encompassed in various genera of the Botryosphaeriaceae (Slippers et al. 2004; Crous et al. 2006; Phillips et al. 2008, 2013; Dissanayake et al. 2016). Based on the phylogenetic analysis of five loci (SSU, LSU, ITS, TUB and tef1-α), seven species was recognized within Botryosphaeria s. str. by Phillips et al. (2013), namely B. agaves (Henn.) E.J. Butler, B. corticis (Demaree & Wilcox) Arx & E. Müll., B. dothidea (Moug.) Ces. & De Not., B. fabicerciana (S.F. Chen bis, Pavlic, M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves, B. fusispora Boonmee, J.K. Liu & K.D. Hyde, B. ramosa (Pavlic, T.I. Burgess & M.J. Wingf.) A.J.L. Phillips & A. Alves and B. scharifii Abdollahz., Zare & A.J.L. Phillips. Subsequently, other eight species namely B. auasmontanum F.J.J. Van der Walt, Slippers & G.J. Marais, B. kuwatsukai (Hara) G.Y. Sun & E. Tanaka, B. minutispermatia Ariyaw., K.D. Hyde & Zi Y. Liu, B. pseudoramosa G.Q. Li & S.F. Chen, B. qingyuanensis G.Q. Li & S.F. Chen, B. rosaceae Y.P. Zhou, Y. Zhang ter., B. sinensia Y.P. Zhou, Y. Zhang ter. and B. wangensis G.Q. Li & S.F. Chen were described (Slippers et al. 2014; Xu et al. 2015; Ariyawansa et al. 2016; Zhou et al. 2016, 2017; Li et al. 2018). So far, fifteen species have been included in Botryosphaeria s. str.

Botryosphaeriacous fungi associated with blueberries have been widely documented (Kong et al. 2010; Yu et al. 2012, 2013, 2013; Xu et al. 2015; Xu 2016; Wang et al. 2016; Dou et al. 2017). Three species of Botryosphaeria have been reported from blueberries (Vaccinium spp.), namely B. corticis, B. dothidea and B. vaccinii (Arx & Müller 1954; Barr 1972; Phillips et al. 2006; Wright & Harmon 2010; Yu et al. 2012; Xu et al. 2015). In the course of an ongoing survey of blueberry diseases in China, two new taxa with general characteristics of Botryosphaeria were collected. Molecular characteristics as well as the phylogeny based on combined ITS, LSU, tef1-α and TUB nuDNA sequences support their new status within Botryosphaeria.

Cankered stems of blueberries were collected from Fujian Province, China in 26 February 2018. About 20cm cankered branches were cut for sampling. The wood segments were first surface sterilized (Pavlic et al. 2004), cut about 0.5cm×0.5cm×0.2cm from the junction of disease and health, and then incubated on malt extract agar (MEA). Isolates grown on MEA were kept at ambient temperatures (about 28°C) in the dark. Fungal isolates were deposited in Beijing Forestry University (BJFU) with duplicates in the China General Microbiological Culture Collection Center (CGMCC). Herbarium specimens were deposited in Herbarium Mycologicum Academiae Sinicae (HMAS).

To induce sporulation, colonies resembling Botryosphaeriaceae spp. were selected and transferred to synthetic nutrient-poor agar (SNA) with sterilized pine needles placed onto the medium. Plates were incubated at 28°C under continuous near-UV light for two weeks. Released conidia and squash mounts of pycnidia formed on the pine needles were mounted in water on microscope slides and examined microscopically. Measurements and digital photographs were made using a Nikon Coolpix 995 digital camera connected to a trinocular Leitz Orthoplan microscope and processed with Adobe Photoshop Elements 10 software. Measurements of conidia, paraphyses and conidiogenous cells were made from water mounts.

DNA was extracted from mycelium grown on MEA plates with CTAB plant genome DNA fast extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing, China). The internal transcribed spacer of rDNA (ITS) was amplified and sequenced with primers ITS1 and ITS4 (White et al. 1990). The translation elongation factor-1α (tef1-α) was amplified and sequenced with primers EF1-688F and EF1-1251R (Alves et al. 2008). The β-tubulin gene (TUB) was amplified and sequenced with primers Bt2a and Bt2b (Glass & Donaldson 1995). The 28S large subunit nuDNA (LSU) was amplified and sequenced with primers LR0R and LR5 (Vilgalys & Hester 1990). PCR amplification and sequencing followed the protocol of Zhang et al. (2009).

The combined loci of ITS, LSU, tef1-α and TUB were used to infer the phylogenetic relationships among different species of Botryosphaeria by Maximum parsimony (MP) and MrBayes analyses. Sequences generated were analyzed with other sequences obtained from GenBank (Table 1). Alignments were conducted in MEGA v. 6 (Tamura et al. 2013) and phylogenetic analyses performed in PAUP v. 4.0b10 (Swofford 2002) and MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003). Prior to phylogenetic analysis, ambiguous sequences at the start and the end were deleted and gaps manually adjusted to optimize the alignments. MP was used to conduct heuristic searches as implemented in PAUP with the default options method (Zhang et al. 2008). Analyses were done under different parameters of maximum parsimony criteria as outlined in Zhang et al. (2008). Clade stability was assessed in a bootstrap analysis with 500 replicates, random sequence additions with maxtrees set to 500 and other default parameters as implemented in PAUP. For the MrBayes analysis, the best-fit model of nucleotide evolution (GTR+I+G) was selected by Akaike information criterion (AIC; Posada & Buckley 2004) in MrModeltest v. 2.3. The metropolis-coupled Markov Chain Monte Carlo (MCMCMC) approach was used to calculate posterior probabilities (Huelsenbeck & Ronquist 2005). A preliminary Bayesian inference (BI) analysis using MrBayes software revealed that the Markov Chain Monte Carlo (MCMC; Huelsenbeck & Ronquist 2001) steady state was reached after less than 10 000 generations (the average standard deviation of split frequencies was constantly below 0.01). A conservative burn-in of 100 trees was chosen and a full analysis of 5 000 000 generations was carried out with sampling every 100 generations. Trees were viewed in TREEVIEW. The nucleotide sequences generated in this paper were deposited in GenBank (Table 1). Trees and alignments were deposited in TreeBase (http://purl.org/phylo/treebase/phylows/ study/TB2:S27260?x-access-code=861cea5bca6c572233daa22d34caad22&format=html).

To determine the pathogenicity of newly isolated taxa, four isolates (CGMCC 3.19099, CGMCC 3.19096, CGMCC 3.19097, CGMCC 3.19098) were used to perform the tests of Koch’s postulates. The tests were conducted in semi-shaded greenhouse with 2-yr-old Vaccinium seedlings. Stems for inoculating were surface sterilized with 75% ethanol and a 0.4cm×0.2cm bark was removed with a scalpel. For inoculation, corresponding size of mycelia from the margins of colonies grown on 2% MEA for 5d in the dark were taken and placed into the wounds with the mycelia facing the cambium. Subsequently, the branch was covered with moist sterilized cotton, and covered by masking tape. Three replicates were conducted in different seedlings for each isolate. Sterilized MEA were used as negative controls. After two weeks’ incubation, all inoculated branches (Fig. 1) were cut for data collection and photographing. Pathogenicity was determined by the length of the necrotic lesion. The fungi were re-isolated by cutting small pieces of wood from the edges of the lesions, cultivated in MEA at 28°C, morphologically studied and sequenced again for Koch’s postulates.

Phylogenetic analysis of the combined ITS, LSU, tef1-α and TUB sequence dataset comprising 2 268bp revealed 293 parsimony- informative characters. The outgroup taxon was Neofusicoccum luteum and N. parvum. The heuristic search with random addition of taxa (500 replicates) generated 5 000 most parsimonious trees of 511 steps (CI=0.783, RI= 0.894, RC=0.699, HI=0.217). The phylogenetic tree resulting from the Bayesian analysis using the general time reversible model of DNA evolution (Rodríguez et al. 1990), including estimation of invariable sites and assuming a discrete gamma distribution with six rate categories (GTR+Γ+G), had a topology identical to the MP tree presented. In both analyses (MP and Bayesian) the clade of Botryosphaeria received a high level of support (100 % for MP and 1.00 for BI). The subclade of B. dolichospermatii and B. fujianensis sibling to other species of Botryosphaeria, while B. dolichospermatii closely related to B. sinensia, B. auasmontanum, B. minutispermatia, B. dothidea and B. wangensis, and B. fujianensis to B. fabicerciana and B. fusispora (Fig. 2).

Botryosphaeria dolichospermatii Z.P. Dou, W. He, Y. Zhang, sp. nov. Fig. 3

MycoBank MB826937

Holotype: CHINA, Fujian Province, Nanping, Jianyang District, Huilong, from cankered branch of Vaccinium uliginosum, 26 February 2018, L. Zhao (HMAS 255410): ex-type living culture, CGMCC 3.19096.

Diagnosis: Botryosphaeria dolichospermatii is characterized by its conidia with mucilaginous sheath and obvious curved spermatia.

Etymology: GK. comp., dolichospermatii, referring to the relatively longer spermatia produced in culture.

Description: Sexual morph unknown. Conidiomata pycnidial, stromatic, produced on pine needles on SNA within 2-3wk, solitary, immersed to semi-immersed, iron grey to black, covered with dense mycelium, mostly uniloculate, 140-315μm diam., thick-walled, globose without papilla. Paraphyses not observed. Conidiogenous cells holoblastic, discrete, hyaline, smooth, thin-walled, cylindrical to ampulliform, producing a single conidium at the tip, (8-)9-15×(2-)3-5μm (av.= 11.5×4.1μm, n=30). Conidia fusiform, aseptate, straight, hyaline, thin-walled, smooth, apex slightly sharp, base subtruncate to bluntly rounded, sometimes with a mucous apical sheath, mostly one or two septa formed before germination, sometimes different conidia connected with tubes when germinating, (21-) 22-30(-32)×5-8μm (av. of 50 conidia=24.8× 6.5μm, L/W ratio=3.85, range from 3.14 to 4.8). Spermatia hyaline, aseptate, smooth, oblong to cylindrical, straight to obvious curved, most rounded ends, up to 9×4μm.

Culture characteristics: Colonies on MEA at 28°C in darkness oliveaceous becoming grey with reverse black. Mycelial mat moderately dense, margin irregular. Colonies reaching 53.9mm on MEA after 3d in the dark at 28°C.

Additional specimens examined: CHINA, Fujian Province, Nanping city, Jianyang district, Huilong town, from cankered branch of Vaccinium uliginosum, 26 February 2018, L. Zhao, paratype, HMAS 255411: living culture, CGMCC 3.19097; paratype, HMAS 255412: living culture, CGMCC 3.19098.

Note: Phylogenetically, Botryosphaeria dolichospermatii is distinct from but closely related to B. sinensia, B. auasmontanum, B. minutispermatia, B. dothidea and B. wangensis. Morphologically, the condia of B. dolichospermatii [(21-)22-30(-32)×5-8μm] are larger than those of B. auasmontanum [(8-)8.5-11.5(-13)× (2.5-)3-4(-5)µm] and B. minutispermatia (8-14× 3-4μm) (Slippers et al. 2014; Ariyawansa et al. 2016). The larger and sometimes curved spermatia of B. dolichospermatii (up to 9×4μm) are distinguishable from those of B. dothidea (3-6×1.5-2μm), B. sinensia (4-7× 2-3μm) and B. wangensis (3.5-4.5×1-1.5μm) (Phillips et al. 2013; Zhou et al. 2017; Li et al. 2018). Furthermore, the apical sheath presenting on some conidia of B. dolichospermatii also differs from that of other five species.

Botryosphaeria fujianensis Z.P. Dou, W. He, Y. Zhang sp. nov. Fig. 4

MycoBank MB826938

Holotype: CHINA, Fujian Province, Nanping, Jianyang District, Huilong, from cankered branch of Vaccinium uliginosum, 26 February 2018, L. Zhao (HMAS 255413), ex-type living culture, CGMCC 3.19099.

Diagnosis: Botryosphaeria fujianensis is characterized by its larger conidia dimension, colonies with a circular area, lacking aerial mycelium in the middle, and different tef1-α sequence.

Etymology: in reference to the location where the species was first described.

Description: Sexual morph unknown. Conidiomata pycnidial, stromatic, produced on pine needles on SNA within 1-2wk, solitary, semi-immersed to superficial, iron grey to black, covered with dense mycelium, uniloculate, 140-330μm diam., thick-walled, globose without papilla. Paraphyses hyaline, septate, cylindrical, sometimes tapering towards the apices, arising between the conidiophores and conidiogenous cells, up to 62×4μm. Conidiophores when present hyaline, cylindrical, sometimes branches, smooth, 0-1-septate. Conidiogenous cells holoblastic, discrete, hyaline, smooth, thin-walled, cylindrical to ampulliform, producing a single conidium at the tip, 8-19× 2-4μm (av.=13.1×3.1μm, n=30). Conidia fusiform, aseptate, mostly straight, hyaline, thin-walled, smooth with granular contents, widest in the middle, both ends rounded, base sometimes truncate, mostly forming one to three septa before germination, (20-)21-29 (-31)×7-9μm (av. of 30 conidia=24×7.8μm, L/W ratio=3.08, range from 2.63 to 3.63), aged conidia hyaline, fusiform and becoming 0-3-septate, more shorter and slender, sometimes two aged conidia connected with tube, 19-24×5-7μm (av. of 30 conidia=21.3× 5.6μm, L/W ratio=3.87, range from 3.14 to 4.8). Spermatia hyaline, aseptate, smooth, rod-shaped, straight, most rounded ends, 4-8×2μm.

Culture characteristics: Colonies on MEA at 28°C in darkness oliveaceous becoming grey with reverse black. Abundant aerial mycelium reaching to the lid of Petri dishes, margin irregular with a circular black area lacking aerial mycelium in the middle. Colonies reaching 71.3mm on MEA after 3d in the dark at 28°C.

Additional specimens examined: CHINA, Fujian Province, Nanping city, Jianyang district, Huilong town, from cankered branch of Vaccinium uliginosum, 26 February 2018, L. Zhao, paratype, BJFU180226-3: living culture, BJFUCC180226-3; paratype, BJFU180226-4: living culture, BJFUCC180226-4.

Note: Phylogenetically, Botryosphaeria fujianensis is closely related but distinct from B. fabicerciana and B. fusispora. Morphologically, conidiomata of B. fujianensis (140-330μm) are larger than those of B. fusispora (140-180μm), while smaller than those of B. fabicerciana (346-470μm) (Liu et al. 2012; Phillips et al. 2013). The colonies of B. fujianensis with a circular area, lacking aerial mycelium in the middle differ from those of B. fabicerciana and B. fusispora.

Four isolates were inoculated on the blueberry stems. After two weeks of inoculation, a few black necrotic lesions were observed on the surface of branches while these were not seen in negative controls (Fig. 1). Koch’s postulates were fulfilled by successful re-isolation of pathogen from all the necrotic stems, and the morphological characteristics and DNA sequences of re-isolated pathogen was consistent with those of pathogen isolated from original diseased blueberry stems.