In May 2020, twenty symptomatic Chinese yew specimens were collected from the Guangxi Province. Small sections (5 × 5 mm) were cut from the margins of the infected needles and stems, surface-sterilised in 75% ethanol for 30 s, sterilised in 3% (vol/vol) sodium hypochlorite for 1 min, followed by three rinses in sterile distilled water and finally dried on sterilised filter paper (Rubini et al. 2005; Li et al. 2021b). The sections were plated on PDA plates and incubated at 25 °C. The isolates were aseptically transferred to fresh PDA and purified using a single-spore culture. The holotype specimens in this study are deposited in the Museum of Beijing Forestry University (Herbarium Code: BJFC). The pure fungal cultures in this study are deposited in China Forestry Culture Collection Center (Acronym: CFCC).

The colony characteristics (colour and texture) of each isolate on PDA were observed after 7 d incubation at 25 °C and the morphological characters of conidiophores, conidiogenous cells and conidia of each isolate on PDA were observed after 14 d incubation at 25 °C (Li et al. 2021b). The size was determined by measuring the length and width of 50 randomly chosen conidia using an Olympus SZX2-FOF Light microscope (Tokyo, Japan) according to the method described by Shu et al. (2020). Descriptions, nomenclature and illustrations of taxonomic novelties have been deposited in MycoBank (https://www.mycobank.org/).

Genomic DNA was extracted from 7 day-old colonies grown on PDA using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). Three loci were amplified: the internal transcribed spacer region of ribosomal DNA (ITS) with primers ITS1/ITS4 (White et al. 1990); beta-tubulin (tub2) with primers Bt2a/Bt2b (Glass and Donaldson 1995) and the translation elongation factor 1-alpha gene (tef-1α) with EF1-688F/EF1-1251R (Alves et al. 2008). The PCR mixture consisted of 10 μl TopTaq Master Mix, 7 μl nuclease-free H₂O, 1 μl of each primer and 1 μl DNA samples were made up to the final volume of 20 μl. Primer sequences and reaction conditions were shown in Suppl. material 1. The PCR products were sent for sequencing at Beijing Tsingke Biotech Company, Ltd., Beijing, China. The DNA sequences have been deposited in GenBank (Suppl. material 2).

The taxa used in the analyses were obtained from the sequence data of Pestalotiopsis and Neopestalotiopsis downloaded from GenBank (Suppl. material 2). Sequence alignments were performed using MAFFT v.7 (mafft.cbrc.jp/alignment/server) (Katoh and Standley 2013). Phylogenetic analyses, based on a combined dataset of ITS, tub2 and tef-1α sequence data, were performed with Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). MP analysis was performed with PAUP 4.0b using a heuristic search algorithm (1,000 random-addition sequences) with tree bisection and reconnection (TBR) branch swapping. MaxTrees were set to 5,000, branches of zero length were collapsed and all equally parsimonious trees were saved (Swofford 2003). ML analysis was performed using RAxML-HPC BlackBox v. 8.2.12, with 1000 rapid bootstrap replicate runs using the GTRGAMMA model of nucleotide evolution (Stamatakis 2014). For BI analysis, the best-fit evolutionary models for each partitioned locus were deduced on the AIC (ITS: GTR+I+G, tub2: HKY+I+G and tef-1α: TrN+I+G) which were estimated in MrModelTest v. 2.3. The BI analysis was performed using MrBayes v.3.1.2, with a Markov Chain Monte Carlo (MCMC) algorithm running from random trees for 1,000,000 generations. The resulting trees were edited using FigTree v.1.4.2 (Rambaut 2016) and Adobe Illustrator CS5.

Five Pestalotioid species were selected to evaluate the effects of temperature, light duration and carbon source on mycelial growth. Mycelial plugs of each species were taken from the colony margins of 3-day-old cultures and transferred to fresh plates (20 ml of culture medium, pH 6). The effects of temperature were tested in the range 19 to 31 °C with a 3 °C gradient on PDA and incubated in dark. To monitor the effects of light duration, the isolates were tested under three diel light cycles (24 h dark, 12 h light/12 h dark and 24 h light per day) on PDA and incubated at 25 °C. The effects of the carbon source were tested using sucrose, maltose and dextrose with the same carbon content. Three replicates were used for the temperature and light duration tests and two replicates were used for the carbon test. The colony diameters were measured daily.

Healthy needles were collected from five-year old and healthy Chinese yew grown in a greenhouse. The needles were washed with tap water, submerged in 70% ethanol for 2 min and rinsed twice with sterile water (Li et al. 2021b). Clean dry needles were placed on Petri dishes with moist filter paper underneath. Puncture wounds were made in the middle of each needle using a sterilised needle. The wounded needles were inoculated with mycelial PDA plugs (3 mm in diameter) and spore suspensions (18 µl, 10⁶ conidia/ml) of five Pestalotioid isolates, respectively. Three replicates were used for the mycelial PDA plug test and 10 replicates were used for the spore suspension test. Lesion length was recorded daily.

A field survey was conducted from April to October 2020 in a forest farm in northern Guangxi, China, where more than 500 Chinese yew trees were maintained. Needle spot disease in Chinese yew causes serious damage, with an incidence of 58– 70% in plants. The spots on the needles were initially small, brown to black and oval to irregular. Subsequently, they gradually expand and finally coalesce, forming large black spots. In severe cases, lesions can develop in large portions of a single needle. Over time, the heavily infected leaves dried and died. Lesions were also visible on the stems and the spots on the stems darkened and became necrotic (Fig. 1).

Thirty-six isolates were obtained from the diseased needles and stems of Chinese yew and 18 isolates of Pestalotioid fungi were identified based on their culture characteristics and conidial morphology. Eight isolates were grouped into Neopestalotiopsis species with versicolorous median cells and ten isolates were identified as Pestalotiopsis with lightly pigmented concolorous median cells. Colonies of most Pestalotioid stains were initially whitish and later greyish or yellow on PDA. Conidia varied from 14.5 to 25.0 µm mean length and 3.5 to 8.0 µm mean width. The apical appendages showed the largest variation in size, with a mean length of 5.0 to 23.0 µm. The number of apical appendages varied between two and four, with three being the most common. Basal appendages were hyaline, straight or slightly curved and varied from 1.0 to 5.0 µm mean length.

The phylogenetic tree (Pestalotiopsis), based on the concatenated sequences of ITS, tub2 and tef-1α, comprised 121 ingroups and one outgroup, Pseudopestalotiopsis cocos (CBS 272.29). A total of 1,496 characters including gaps (401 for ITS, 313 for tub2 and 860 for tef-1α) were included in the phylogenetic analysis. Similar tree topologies were obtained using the MP, ML and BI methods and the best-scoring MP tree obtained from a heuristic search with 1000 random taxon additions is presented (Fig. 2). The phylogenetic tree placed the six Pestalotiopsis isolates into three well-supported monophyletic clades, representing two novel and one known species. Two of our isolates (CFCC59981 and CFCC59982), described as Pestalotiopsis multicolor, formed monophyletic groups with high support values (100% MP and ML, 1.00 BI). Two isolates (CFCC59976 and CFCC59978), identified as Pestalotiopsis taxicola, constituted an independent and strongly-supported subclade (100% MP and ML, 1.00 BI), sharing close affinity with Pestalotiopsis unicolor and Pestalotiopsis jiangxiensis. Two strains (BJFUCC42 and BJFUCC42-2) clustered with Pestalotiopsis trachycarpicola and were, therefore, described as known species.

The phylogenetic tree (Neopestalotiopsis), based on the concatenated gene sequences of ITS, tub2 and tef-1α, comprised 95 ingroups and one outgroup, Pestalotiopsis diversiseta (MFLUCC 12-0287). A total of 1, 386 characters including gaps (411 for ITS, 393 for tub2 and 582 for tef-1α) were included in the phylogenetic analysis. Similar tree topologies were obtained using the MP, ML and BI methods and the best-scoring MP tree obtained from a heuristic search with 1000 random taxon additions is presented (Fig. 3). Two of our isolates (CFCC59989 and CFCC59990) clustered together and nested in a clade containing two different Neopestalotiopsis sp. 2 isolates (CFCC 54340 and ZX22B). In contrast, two isolates (CFCC59985 and CFCC59986), grouped Neopestalotiopsis rhapidis (Fig. 3).

The effects of temperature on the growth of the five Pestalotioid species were shown in Fig. 9. When grown on PDA in the dark, all isolates grew at a temperature range of 19 to 31 °C, with optimum growth between 22 °C and 28 °C. The growth rate was drastically reduced below 22 °C and started to decline above 28 °C. The regression analysis showed the optimum temperatures for P. trachycarpicola, P. taxicola, P. multicolor, Neopestalotiopsis sp. 3 and Neopestalotiopsis sp. 4 were 25 °C, 24–26 °C, 22–25 °C, 25–28 °C and 25 °C, respectively. Within four days, the diameter of the majority of the isolates was approximately 90 mm, which is the diameter of the PDA plate.

Light duration had no effect on the mycelial growth of the two Pestalotioid species (P. trachycarpicola and P. muticolor) and the best growth of the three Pestalotioid fungi (P. taxicola, P. multicolor and Neopestalotiopsis sp. 4) was observed under continuous light (Suppl. material 4). The growth rate was highest for P. taxicola in 24 h light, which reached 66.4 mm diameter after four days. The growth rate was lowest for P. trachycarpicola in 12 h light/12 h dark, which reached only 55.5 mm in diameter after four days.

Five Pestalotioid species grew on all three tested carbon sources (Suppl. material 5). Maltose utilisation was the least efficient and the medium with dextrose showed comparatively high growth of the three tested fungi (P. trachycarpicola, P. multicolor and Neopestalotiopsis sp. 3). For P. taxicola, the utilisation of dextrose appeared to be higher than that of sucrose.

The pathogenicity of two Neopestalotiopsis isolates (Neopestalotiopsis sp. 3 and Neopestalotiopsis sp. 4) and three Pestalotiopsis isolates (P. trachycarpicola, P. taxicola and P. multicolor) was tested by inoculating detached healthy needles according to Keith et al. (2003). Dark brown lesions and a necrotic zone, which resembled the symptoms that occurred in the field, were observed on the needles 20 days after inoculation with mycelium plugs of Neopestalotiopsis sp. 3, which did not occur in the control inoculated with agar media or with the other four Pestalotioid species (Fig. 10). Neopestalotiopsis sp. 3 was re-isolated from the infected needles and was confirmed to be the same as the inoculated pathogen through morphological and phylogenetic analyses. However, none of the five isolates produced typical symptoms in the wounded needles inoculated with the spore suspension (Fig. 10). The results revealed that only Neopestalotiopsis sp. 3 is pathogenic to Chinese yew needles.