Introduction

Grey mould caused by Botrytis species is one of the most prevalent and devastating plant diseases globally (Elad et al. 2016a). Botrytis cinerea, the lectotype of Botrytis, can infect more than 200 plant species worldwide (Hahn et al. 2014). Under suitable weather conditions (humid and cool), grey mould can infect at any growth stage in susceptible plants (Davidson et al. 2004; Elad et al. 2004; Zhang 2006). The disease emerges as small water-soaked spots on leaves and stems that merge into large necrotic lesions covered with grey conidia and conidiophores, causing stem, leaf, and fruit rot (Zhang 2006; Williamson et al. 2007; Carisse 2016).

To meet increasing demands for pulp and paper, plantation forestry based on fast-growing trees, especially species of Eucalyptus, has expanded rapidly in South China during the past three decades (Zhou and Wingfield 2011). Currently, approximately 4.5 million ha of Eucalyptus plantations have been established in South China (Chen and Chen 2013). During the last two decades, however, several disease problems have emerged, either in plantations or nurseries. Diseases on Eucalyptus plantations include stem canker/wilt caused by species of Botryosphaeria, Lasiodiplodia, and Neofusicoccum in Botryosphaeriaceae (Chen et al. 2011a), Ceratocystis (Chen et al. 2013; Liu et al. 2015), Chrysoporthe (Chen et al. 2010), and Teratosphaeria (Chen et al. 2011b); leaf blight/spot caused by species of Calonectria (Lombard et al. 2010; Chen et al. 2011c), Mycosphaerella (Burgess et al. 2007), Quambalaria (Zhou et al. 2007), and Teratosphaeria (Burgess et al. 2006); and bacterial wilt associated with Ralstonia solanacearum (Old et al. 2003). In Eucalyptus nurseries, isolates of the genera Botrytis and Calonectria have been found (Lombard et al. 2010, 2015; Chen et al. 2011c; Li 2012). Diseases associated with Calonectria species include cutting rot and leaf blight (Lombard et al. 2010; Chen et al. 2011c). Several species of Calonectria have been isolated from diseased Eucalyptus seedlings in South China (Lombard et al. 2010; Chen et al. 2011c), and pathogenicity testing has revealed that Calonectria species are aggressive pathogens of two important E. urophylla × E. grandis clones extensively cultivated in nurseries in South China (Chen et al. 2011c). In Eucalyptus, Botrytis species cause stem wilt and leaf rot (Zaldúa and Sanfuentes 2010; Liao et al. 2013; Muñoz and Campos 2013). Only one species of Botrytis has been reported from Eucalyptus in China, symptoms of grey mould were observed on leaves of Eucalyptus trees, the pathogen’s identification was based solely on morphological characteristics (Li 2012; Liao et al. 2013).

Recently, grey mould was observed on E. urophylla × E. grandis seedlings in one nursery in GuangDong Province in South China. A typical manifestation of Botrytis infection was observed on the leaves and stems of seedlings. The objectives of this study were to identify those fungal isolates based on sequence comparisons and morphological characteristics and to test its pathogenicity on three E. urophylla × E. grandis clones.

Materials and methods

Sample collection and isolation

In March 2015, leaves and stems exhibiting symptoms of grey mould were collected from Eucalyptus urophylla × Eucalyptus grandis seedlings at the South China Experiment Nursery of the China Eucalypt Research Centre (CERC), Chinese Academy of Forestry, located in ZhanJiang, GuangDong Province, China. The diseased seedlings were collected from multiple sites in the nursery and placed in individual plastic bags and transported to the laboratory for isolation.

Conidial masses were transferred under a stereomicroscope AxioCam Stemi 2000C (Carl Zeiss, Germany) directly from the symptomatic tissues to 2 % malt extract agar (20 g malt extract powder and 20 g agar powder per liter water: malt extract powder from Beijing Shuangxuan microbial culture medium products factory, Beijing, China; agar powder from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) (MEA). After incubation at 20 °C for 24–48 h, single germinating conidia from the conidial masses were transferred onto fresh MEA plates and incubated at 20 °C in darkness for 12 days to obtain pure cultures. The cultures were deposited in the CERC culture collection. Representative isolates were deposited at the China Forestry Culture Collection Center (CFCC), Beijing, China. Representative isolates were stored in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China. The specimens (fungal pure cultures) were deposited in the Collection of Central South Forestry Fungi of China (CSFF), GuangDong Province, China.

DNA extraction, polymerase chain reaction (PCR) amplification and sequencing

Fungal isolates grew on MEA for 7 days at 20 °C, after which mycelia were scraped from the culture surface with a sterilized scalpel and transferred to 2.0 mL Eppendorf tubes. Genomic DNA was extracted using the “Extraction method 5: grinding and CTAB,” described by Van Burik et al. (1998). The extracted DNA was dissolved in 30 μL TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH 8.0) and quantified using a Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

A 1 μL aliquot of genomic DNA was added as the template to each PCR reaction [TopTaqTM Master Mix 25 μL (Qiagen Inc., Hilden, Germany); forward primer: 1 μL, 10 μM (Invitrogen, Shanghai, China); reverse primer: 1 μL, 10 μM (Invitrogen, Shanghai, China); RNase-Free H2O 22 μL (Qiagen Inc., Hilden, Germany)] to amplify the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA and the partial DNA sequences of five nuclear genes, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2), and necrosis and ethylene-inducing proteins (NEP1 and NEP2). The ITS region was amplified with primers ITS1 and ITS4 (White et al. 1990). Primer pairs G3PDHfor/G3PDHrev, HSP60for/HSP60rev, RPB2for/RPB2rev, NEP1for/NEP1revB, NEP2forD/NEP2revD were used to amplify fragments of the G3PDH, HSP60, RPB2, NEP1, and NEP2 genes, respectively (Staats et al. 2005, 2007; Grant-Downton et al. 2014; Lorenzini and Zapparoli 2014; Ferrada et al. 2016). The amplifications were performed in 50 μL reaction volumes on an MJ Mini Cycler (BIO-RAD, Hercules, CA, USA) under the conditions described by Staats et al. (2005, 2007). The PCR amplification products were separated by 1 % agarose gel electrophoresis and visualized with SYBR Safe DNA gel stain (Thermo Fisher Scientific).

The PCR products were sequenced in both directions using the same primers as described above by the Beijing Genomics Institute, Guangzhou, China. After assembling with MEGA v. 6.0 (Tamura et al. 2007), sequences of the isolates obtained in this study were deposited in GenBank (Table 1).

Table 1 Isolates used for phylogenetic studies and pathogenicity tests in this study
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Phylogenetic analysis

Sequences of the ex-type strains of Botrytis species and other published strains closely related to the Botrytis isolates sequenced in this study were downloaded from NCBI (http://www.ncbi.nlm.nih.gov) and used for phylogenetic analysis (Table 1). Sequences of the Botrytis isolates collected in this study and those from NCBI were aligned using the online version of MAFFT (http://mafft.cbrc.jp/alignment/server) with the interactive refinement method (FFT-NS-i) setting. The alignments were further edited manually in MEGA v. 6.0 and deposited in TreeBASE (http://treebase.org).

Phylogenetic analyses were conducted on both individual ITS, G3PDH, HSP60, RPB2, NEP1, and NEP2 sequence datasets, but also with combined datasets for two to five gene regions, depending on the Botrytis species/strains availability. Two methods were used for phylogenetic analyses. Maximum parsimony (MP) analyses were performed using PAUP v. 4.0 b10 (Swofford 2003) and maximum likelihood (ML) analyses were conducted with PhyML v. 3.0 (Guindon and Gascuel 2003).

For MP analyses, gaps were treated as a fifth character, and characters were unordered and of equal weight with 1000 random addition replicates. A partition homogeneity test (PHT) using PAUP v. 4.0 b10 (Swofford 2003) was conducted to determine whether data for the two to five genes could be combined. The most parsimonious trees were obtained using the heuristic search option with stepwise addition, tree bisection, and reconstruction branch swapping. MAXTREES was set to 5,000 and zero-length branches were collapsed. A bootstrap analysis (50 % majority rule, 1,000 replicates) was carried out to determine statistical support for internal nodes in trees. Tree length (TL), consistency index (CI), retention index (RI) and homoplasy index (HI) were used to assess phylogenetic trees (Hillis and Huelsenbeck 1992).

For ML analyses, the best nucleotide substitution model was established with jModeltest v. 2.1.5 (Posada 2008). In PhyML, the maximum number of retained trees was set to 1,000, and nodal support was determined by non-parametric bootstrapping with 1,000 replicates. For both MP and ML analyses, the phylogenetic trees were viewed using MEGA v. 6.0.

Morphology

Representative isolates identified by DNA sequence comparisons and from different Eucalyptus seedlings were selected to determine morphological characteristics. Mycelial agar plugs (6 mm diameter) of selected isolates were transferred using a cork borer onto 90 mm Petri dishes containing MEA and potato dextrose agar (200 g diced potato, 20 g dextrose and 20 g agar powder per liter water: dextrose from Shantou Xilong Chemical Co., Ltd., Guangdong, China; agar powder from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) (PDA) and incubated at 20 °C in the dark to induce sclerotia formation for five replicates of each isolate. Sclerotia were examined microscopically (AxioCam Stemi 2000C stereomicroscope; AxioCam ERc 5S digital camera, Carl Zeiss Ltd., Munchen, Germany). Mycelial agar plugs (6 mm diam) were transferred onto water agar (20 g agar power per liter water, agar powder from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) (WA) with sterilized Eucalyptus leaves placed on the agar surface and incubated at 20 °C under continuous near-fluorescent light to induce sporulation. Conidiophores and conidia that emerged on the surface of the Eucalyptus leaves were mounted in one drop of 85 % lactic acid on glass slides and examined under an Axio Imager A1 microscope (Carl Zeiss Ltd., Munchen, Germany) and an AxioCam ERc 5S digital camera with Zeiss Axio Vision Rel. 4.8 software (Carl Zeiss Ltd., Munchen, Germany).

The size dimensions of 25–50 sclerotia, conidiophores and conidia of each isolate were measured. Minimum, maximum, and average (mean) values were determined and presented in the taxonomic description as follows: [minimum – (average – standard deviation)] – [(average + standard deviation) – maximum]. Ratios of average length to average width were also calculated.

Culture characteristics of the Botrytis isolates were determined using cultures grown in 90 mm Petri dishes containing MEA for 3–12 days. To determine the effect of temperature on mycelial growth rate of the representative isolates, mycelial agar plugs (6 mm diam) were transferred from these cultures to new MEA and PDA Petri dishes and incubated in darkness under different temperatures ranging from 5 °C to 40 °C at 5 °C intervals. Five replicates of each isolate (CERC7160, CERC7163, CERC7170, and CERC7208) were incubated at each temperature. Colony diameters were measured at the 24 h intervals up to 48 h (for 2 days), and the data were used to calculate growth rates. The average growth rate was calculated for each of the eight temperatures and the experiment was repeated once.

Pathogenicity tests

To identify whether the Botrytis strains isolated from E. urophylla × E. grandis seedlings were pathogenic to different Eucalyptus clones, isolates (CERC7160, CERC7163, CERC7170, and CERC7208) were used in pathogenicity tests against three different genotypes of E. urophylla × E. grandis clones (CEPT-11, CEPT-12, and CEPT-13). The CEPT-11 is the clone from which Botrytis was originally isolated, this clone was used for pathogenicity tests to fulfil Koch’s postulates. The three Eucalyptus clones were planted in the South China Experiment Nursery in ZhanJiang.

Young healthy leaves were collected, surface-sterilized in 5 % bleach for 2 min, and then rinsed three times with sterilized water. Mycelial agar plugs (6 mm diam) were transferred from 2-day-old MEA cultures and placed face down on the detached leaflets. Ten detached leaves of each Eucalyptus clone were inoculated with each of the representative isolates. Additional ten detached leaves from each Eucalyptus clone were inoculated with sterile MEA plugs as negative controls. All inoculated leaves were placed in sealed plastic boxes, the bases of which were lined with moist pads to maintain humidity. These boxes were kept in a moist chamber at 25 °C. After 1 week, lesions on each leaf were measured, and the experiment was repeated once. Re-isolations were made by cutting small pieces (0.04 mm2) of leaf tissue from the diseased/wounded edges and transferring them to MEA at 25 °C. Re-isolations were made from all leaves inoculated as controls and from four randomly selected leaves per isolate. The lesion lengths were analyzed with a one-way analysis of variance (ANOVA) in SPSS Statistics v. 20 (SPSS 2011). Duncan’s multiple range test was applied, and F-values with P < 0.05 were considered significant.

Results

Isolation

Fungal strains were isolated from four E. urophylla × E. grandis seedlings exhibiting grey mould symptoms, the sclerotia and conidia of these strains consistent with the typical morphological characteristics of Botrytis species. Four isolates (CERC7160, CERC7163, CERC7170, and CERC7208) collected from four seedlings in one nursery were selected for further study.

Phylogenetic analysis

To obtain preliminary identification, the ITS, G3PDH, HSP60, RPB2, NEP1, and NEP2 sequences of the four Chinese Botrytis isolates were subjected to BLAST searches using the NCBI nucleotide database. Among the four Chinese isolates, differences were not observed among the G3PDH, HSP60, RPB2, NEP1, and NEP2 sequences, although a single nucleotide polymorphism was observed in the ITS sequences (genotype 1: CERC7160 and CERC7163; genotype 2: CERC7170 and CERC7208). The PHT comparing the G3PDH/HSP60/RPB2, NEP1/NEP2, G3PDH/HSP60/RPB2/NEP1/NEP2 gene datasets generated the P values as 0.250, 0.333, 0.250, respectively, which indicated that no significant conflict existed between these datasets. These datasets were consequently combined and subjected to phylogenetic analyses. The sequence alignments for ITS (52 taxa, 450 characters), G3PDH (76 taxa, 887 characters), HSP60 (82 taxa, 980 characters), RPB2 (72 taxa, 1095 characters), NEP1 (52 taxa, 742 characters), NEP2 (53 taxa, 802 characters), and the combined G3PDH/HSP60/RPB2 (72 taxa, 2960 characters), NEP1/NEP2 (39 taxa, 1579 characters), G3PDH/HSP60/RPB2/NEP1/NEP2 (37 taxa, 4519 characters) were deposited in TreeBASE (TreeBASE No. 18759).

Phylogenetic analyses of individual ITS, G3PDH, HSP60, RPB2, NEP1, NEP2, and combined sequence datasets were conducted using both MP and ML methods. The number of parsimony informative characters, the statistical values for phylogenetic trees of MP analyses, and the parameters for best-fit substitution models of ML analyses are shown in Table 2. Although the relative positions of individual Botrytis species differed slightly between MP and ML trees, the overall topologies were similar.

Table 2 Statistics resulting from phylogenetic analyses
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In the tree based on ITS sequences (Fig. 1), the Botrytis species were separated into two main phylogenetic groups (Fig. 1), strains of some Botrytis species were grouped in the same phylogenetic clade in each of the two phylogenetic groups. For example, in Phylogenetic Group 1, B. sinoviticola (ex-type strain GBC-3-2b) and B. pelargonii (ex-type strain CBS 497.50) were grouped together, in Phylogenetic Group 2, B. fabiopsis (ex-type strain BC-2), B. ficariarum (ex-type strain CBS 176.63), B. caroliniana (ex-type strain CB15), and B. deweyae (ex-type strain CBS 134649) were in the same clade (Fig. 1). The four Chinese Botrytis isolates collected in this study from Eucalyptus seedlings reside in Phylogenetic Group 1, and were subdivided into two phylogenetic clades (clade 1: CERC7170, CERC7208; clade 2: CERC7160, CERC7163) (Fig. 1). Similar to other Botrytis isolates collected from Eucalyptus species in other countries (Brazil: isolates B01G1 and B6; Mexico: isolate G1; Spain: isolate 4879), which were also grouped in Phylogenetic Group 1, the ITS sequences alone did not provide sufficient resolution to distinguish different Botrytis species (Fig. 1).

Fig. 1
figure 1

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of DNA sequence data of the ITS region. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. Sclerotinia sclerotiorum (isolate No. F113458) was the outgroup

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In the trees based on G3PDH sequences (Fig. 2), B. cinerea, B. pelargonii and the four Chinese isolates (CERC7160, CERC7163, CERC7170, and CERC7208) were indistinguishable. In the HSP60 tree, the Chinese isolates were grouped in one separate phylogenetic clade, closely related to B. pelargonii, B. cinerea and “Botrytis Group S”, a novel clade in Leroch et al. (2013) (Fig. 3). In the RPB2 tree, the Chinese isolates were closest to B. fabae, but formed a single separate phylogenetic clade (Fig. 4). In the NEP1 tree, the Chinese isolates were separated from other phylogenetically closely related species, including B. cinerea and B. pelargonii (Fig. 5). In the NEP2 tree, the four Chinese isolates and six isolates of “Botrytis Group S” (Leroch et al. 2013) formed one group which is phylogenetically different from B. cinerea and B. pelargonii, this is supported by bootstrap values of 85 %/83 % (ML/MP), and the four Chinese isolates formed a clade separated from the six isolates of “Botrytis Group S” (Fig. 6).

Fig. 2
figure 2

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of G3PDH gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. Sclerotinia sclerotiorum (isolate No. 484) was the outgroup

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Fig. 3
figure 3

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of HSP60 gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. Sclerotinia sclerotiorum (isolate No. 484) was the outgroup

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Fig. 4
figure 4

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of RPB2 gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. Sclerotinia sclerotiorum (isolate No. 484) was the outgroup

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Fig. 5
figure 5

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of NEP1 gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. The tree was un-rooted

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Fig. 6
figure 6

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of NEP2 gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. The tree was un-rooted

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Phylogenetic analyses of each of the combined datasets of G3PDH/HSP60/RPB2, NEP1/NEP2, and G3PDH/HSP60/RPB2/NEP1/NEP2 indicated that the Chinese isolates were more closely related to B. cinerea and B. pelargonii than B. fabae (Figs. 7, 8, and 9), and formed an independent clade distinct from B. cinerea, B. fabae and B. pelargonii (ML/MP bootstrap values: G3PDH/HSP60/RPB2, 82 %/62 %; NEP1/NEP2, 100 %/98 %; G3PDH/HSP60/RPB2/NEP1/NEP2, 100 %/99 %).

Fig. 7
figure 7

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of combination of G3PDH, HSP60, and RPB2 gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. Sclerotinia sclerotiorum (isolate No. 484) was the outgroup

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Fig. 8
figure 8

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of combination of NEP1 and NEP2 gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. The tree was un-rooted

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Fig. 9
figure 9

Phylogenetic tree of Botrytis species based on maximum likelihood (ML) analysis of combination of G3PDH, HSP60, RPB2, NEP1, and NEP2 gene sequences. Isolates highlighted in bold were sequenced in this study. ML and maximum parsimony (MP) bootstrap values (ML/MP) are shown above branches, with bootstrap values below 60 % marked with an *, and absent analysis values are marked with -. T refers to ex-type isolates. The tree was un-rooted

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In the individual HSP60, RPB2, NEP1, and NEP2 trees and in the trees based on combined sequences of G3PDH/HSP60/RPB2, NEP1/NEP2, and G3PDH/HSP60/RPB2/NEP1/NEP2, the four Chinese isolates clustered together and formed an independent clade distinct from other previously described Botrytis species. The results of these phylogenetic analyses indicate that the Chinese isolates represent a distinct, undescribed species.

The phylogenetic analyses indicated that the Chinese isolates were most closely related to B. cinerea and B. pelargonii, while they could be distinguished from B. cinerea and B. pelargonii using single nucleotide polymorphism (SNP) analyses for each of HSP60, RPB2, NEP1, and NEP2 gene regions sequenced (Tables 3 and 4). The Chinese group could be separated from B. cinerea and B. pelargonii by nine and 12 unique SNPs for the four gene regions (Tables 3 and 4) .

Table 3 Summary of polymorphic nucleotides found within the HSP60, RPB2, NEP1, and NEP2 gene regions generated for the phylogenetic groups of B. eucalypti, B. cinerea, and B. pelargonii
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Table 4 Number of unique alleles in B. eucalypti, B. cinerea, and B. pelargonii
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Taxonomy

The four Chinese Botrytis isolates collected from E. urophylla × E. grandis seedlings produced sclerotia on MEA, but not on PDA and formed conidiophores and conidia on MEA and WA medium within 2 weeks. Sexual structures were not observed.

The size of sclerotia, conidiophores and conidia were used to distinguish different species of Botrytis. The morphological characteristics of sclerotia, conidiophores and conidia were very similar between Chinese Botrytis species and other phylogenetically closely related species that have been characterized previously, including B. cinerea and B. pelargonii, while the conidia of Chinese Botrytis species (up to 8.5 μm) are narrower than that of B. cinerea (up to 12.0 μm) and wider than B. pelargonii (up to 6.5 μm) (Table 5).

Table 5 Morphological characteristics of B. eucalypti, B. cinerea, and B. pelargonii
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Based on the phylogenetic analyses and morphological characterization, the fungus isolated from diseased Eucalyptus seedlings was identified as an undescribed species of Botrytis. The new species is described as follows:

Botrytis eucalypti Q.L. Liu & S.F. Chen, sp. nov. (Fig. 10)

Fig. 10
figure 10

Morphological characteristics of Botrytis eucalypti. A. Colony of B. eucalypti on MEA after 3 days at 20 °C; B, C. Colony of B. eucalypti on MEA after 12 days at 25 °C and 20 °C, respectively; D. Sclerotia produced on the medium; E, F. Sporulation of B. eucalypti on the medium and a Eucalyptus leaf at 20 °C, respectively; G, H. Conidiophores; I. Conidia. Bars: A, B, C = 1 cm; D, E = 500 μm; F = 200 μm; G, H = 20 μm; I = 5 μm

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MycoBank MB817103

Etymology: “eucalypti” refers to the plant genus Eucalyptus, the host from which this fungus was first isolated.

Description: On MEA and PDA at 10, 15, 20, 25, and 30 °C, the mycelial growth rate and colony morphology is similar, no growth at 5 or 35 °C. On MEA at 20 °C in the dark, after 3 days colony surface white, mycelium fluffy (Fig. 10a), after 12 days mycelium became buff brown (Fig. 10b). On PDA, no sclerotia were produced, on MEA at 20 °C, sclerotia formed on the surface of the medium after 12 days, small, grey to black, irregular to spherical, scattered or aggregated (Fig. 10c and d), (0.9–)1.0–1.5(–1.9) × (0.8–)0.9–1.4(–1.8) mm. Sclerotia could also formed on the surface of detached Eucalyptus leaf tissues on WA (inoculated with B. eucalypti). Conidiophores, conidiogenous cells and conidia were produced on MEA and Eucalyptus leaves on WA within 2 weeks (Fig. 10e and f), but not on PDA media. Conidiophores, conidiogenous cells and conidia formed on MEA and WA were similar. Conidiophores erect, septate, branched at the top, brown, (558–)894–1574(–1887) × (11–)13–15(–17) μm. Conidiogenous cells inflated and swollen at the apex, (8–)9–11(–13) × (8–)10–14(–16) μm (average 10.3 × 11.7 μm). Conidia three dimensional, botryoidal, obovate to elliptical, unicellular, hyaline to pale brown, (6.5–)8.0–11.0(–12.5) × (4.0–)5.5–7.0(–8.5) μm (average 9.6 × 6.3 μm; length/width = 1.5) (Fig. 10i, Table 5).

Growth characteristics: Colonies on MEA after 48 h at 10, 15, 20, 25, and 30 °C reaching 25, 26, 56, 49, and 12 mm, respectively. Optimal growth at 20 °C. No growth at 5, 35, or 40 °C.

Sexual morph: Not observed.

Habitat: Leaves and stems of Eucalyptus seedlings in nurseries.

Host and distribution: Eucalyptus urophylla × E. grandis, GuangDong Province, China.

Typification: CHINA. GuangDong Province, ZhanJiang Region, SuiXi County, LingBei Town, South China Experiment Nursery, 21°16′0″N, 110°05′14″E, on leaves of a E. urophylla × E. grandis seedling, 2 March 2015, S.F. Chen & Q.L. Liu, a Eucalyptus leaf sample on the surface of WA medium inoculated with isolate CERC7170 was dried and deposited as Herb. CSFF2013 (HOLOTYPE), culture EX-TYPE CERC7170 = CFCC51324 = CGMCC3.18028.

Additional specimens: CHINA. GuangDong Province, ZhanJiang Region, SuiXi County, LingBei Town, South China Experiment Nursery, 21°16′0″N, 110°05′14″E, on leaves of a E. urophylla × E. grandis seedling, 27 February 2015, S.F. Chen & Q.L. Liu, a Eucalyptus leaf sample on the surface of WA medium inoculated with isolate CERC7160 was dried and deposited as Herb. CSFF2014, culture CERC7160 = CFCC51322, culture CERC7163 = CFCC51323; CHINA. GuangDong Province, ZhanJiang Region, SuiXi County, LingBei Town, South China Experiment Nursery, 21°16′03″N, 110°05′14″E, on leaves of a E. urophylla × E. grandis seedling, 6 March 2015, S.F. Chen & Q.L. Liu, a Eucalyptus leaf sample on the surface of WA medium inoculated with isolate CERC7208 was dried and deposited as Herb. CSFF2015, culture CERC7208 = CFCC51325 = CGMCC3.18029.

Notes: Botrytis eucalypti is phylogenetically closely related to B. cinerea and B. pelargonii. However, morphological differences exist among these three species, namely the conidia of B. eucalypti (width up to 8.5 μm) are wider than B. pelargonii (width up to 6.5 μm), but narrower than B. cinerea (width up to 12 μm) (Table 5). Botrytis eucalypti differed from B. cinerea in culture mycelial growth rate (the growth information of B. pelargonii is not available), on PDA at 5 °C, B. cinerea grew more than 3 mm/d, but B. eucalypti did not grow; on PDA at 10, 15, and 25 °C, B. eucalypti grew slower than B. cinerea (Fig. 11).

Fig. 11
figure 11

Effect of temperature on mycelial radial growth of two species, Botrytis eucalypti (on MEA and PDA) and B. cinerea (on PDA) in the dark. Each value represents the average of 5 replicates

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Pathogenicity tests

All four tested B. eucalypti isolates (CERC7160, CERC7163, CERC7170, and CERC7208) produced lesions on detached leaves collected from three Eucalyptus clones after 1 week, while no disease symptoms and only wounds were observed on leaves treated with the negative controls (Fig. 12). Mean comparison tests showed that the lesions produced by the tested isolates were all significantly longer (P < 0.05) than the wounds caused by the negative controls (Fig. 13). An ANOVA revealed significant differences among the three Eucalyptus clones in susceptibility to the tested isolates (P < 0.05). Not all the B. eucalypti isolates reacted in the same manner to the tested Eucalyptus clones. For example, lesions produced by isolate CERC7160 on Eucalyptus clones CEPT-11 and CEPT-12 were significantly longer than those on CEPT-13, whereas lesions produced by isolate CERC7163 on CEPT-11 and CEPT-12 were significantly shorter than those on CEPT-13 (Fig. 13). Lesions produced by isolates CERC7170 and CERC7208 on Eucalyptus clones CEPT-11 were significantly longer than those on CEPT-13 and CEPT-12, and lesions produced by CERC7208 on three Eucalyptus clones are significantly longer than CERC7170. The inoculated isolates were re-isolated from the lesions, while no Botrytis species was isolated from the negative controls.

Fig. 12
figure 12

Symptoms of Botrytis eucalypti (CERC7160) inoculated on detached leaves of three Eucalyptus clones compared with responses of negative controls. A–C. Lesions produced by isolate CERC7160 on leaves of Eucalyptus clones CEPT-11, CEPT-12, and CEPT-13, respectively. D–F. Respective negative controls of Eucalyptus clones CEPT-11, CEPT-12, and CEPT-13 were marked by the absence of lesions. Bars: A–F = 10 mm

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Fig. 13
figure 13

Column chart showing average lesion lengths (mm) resulting from inoculation trials on detached leaves of three Eucalyptus clones (CEPT-11, CEPT-12, and CEPT-13). Four isolates of B. eucalypti were used. Vertical bars represent the standard error of means. Different letters above the bars indicate treatments that were statistically significantly different (P = 0.05)

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Discussion

In this study, a previously undescribed species of Botrytis was identified and described. This species was isolated from diseased leaves of Eucalyptus seedlings in South China and named B. eucalypti. Identification of this fungus as a novel species is supported by phylogenetic analyses, morphological characteristics, and culture growth rates. Pathogenicity tests demonstrated that B. eucalypti is pathogenic to all three tested Eucalyptus grandis clones.

DNA sequence comparison of G3PDH, HSP60, RPB2, NEP1, and NEP2 gene regions has recently become an effective and increasingly important method for species identification of Botrytis (Zhang et al. 2010a; Li et al. 2012; Khan et al. 2013; Leroch et al. 2013; Grant-Downton et al, 2014; Lorenzini and Zapparoli 2014; Zhou et al. 2014; Ferrada et al. 2016; Saito et al. 2016). In this study, the Chinese Botrytis isolates were identified as a new species, the conclusions of which are supported by phylogenetic analyses of each of HSP60, RPB2, NEP1, and NEP2 gene sequences, as well as by the combined sequences of G3PDH/HSP60/RPB2, NEP1/NEP2, and G3PDH/HSP60/RPB2/NEP1/NEP2. Consistent with previous findings, ITS sequences could not provide sufficient resolution for the identification and differentiation of Botrytis species (Zhang et al. 2010a; Li et al. 2012; Ferrada et al. 2016; Saito et al. 2016). Morphological differences of conidial size were also observed among B. eucalypti and its phylogenetically closest relatives, including B. cinerea and B. pelargonii (Zhang et al. 2010a, b, 2016; Li et al. 2012; Zhou et al. 2014; Ferrada et al. 2016; Saito et al. 2016).

Except for B. cinerea, which infects over 200 eudicot hosts in various families (MacFarlane 1968; Williamson et al. 2007), and B. fabae, which can infect some species in genera Lens, Phaseolus, Pisum, and Vicia of the Fabaceae (Jarvis 1977; Zhang 2006), most species of Botrytis are considered specialists with a narrow host range, infecting only one or a few closely related species within the same plant genus (Mansfield 1980; Staats et al. 2005; Elad et al. 2016b). In regard to the three species phylogenetically closest to B. eucalypti, isolates of B. cinerea and B. fabae have been obtained from hosts in multiple genera, whereas B. pelargonii has only been isolated from Pelargonium species (Zhang 2006; Saito et al. 2016).

Prior to this study, the only Botrytis species reported to infect Eucalyptus has been B. cinerea (Old et al. 2003; Pérez-Vera et al. 2005; Muñoz and Campos 2013; Sánchez Márquez et al. 2011; Sbravatti Júnior et al. 2013; Elad et al. 2016b). Grey mould caused by B. cinerea is one of the most severe diseases in Eucalyptus nurseries worldwide (Zaldúa and Sanfuentes 2010; Muñoz and Campos 2013; Caires et al. 2015). Before this study, only one species of Botrytis, B. cinerea was identified from Eucalyptus leaves solely based on morphological characteristics (Liao et al. 2013). In this study, B. eucalypti was discovered on naturally infecting Eucalyptus seedlings. Morphological differences exists between the Chinese B. cinerea and B. eucalypti isolated from Eucalyptus, for example, the conidia of B. cinerea (9.0–16.0 × 6.0–10.0 μm) are larger than that of B. eucalypti (6.6–12.7 × 4.2–8.4 μm) (Liao et al. 2013).

The pathogenicity tests conducted in this study revealed that B. eucalypti is pathogenic to all three tested Eucalyptus clones and it is capable of producing necrotic lesions on Eucalyptus leaves within a short time. This fungus has the potential to cause serious disease in Eucalyptus seedlings in nurseries and may be distributed to other nurseries in South China. Careful monitoring is needed to detect the spread of this fungus to other regions. Extensive epidemiological studies should be conducted to help control grey mould caused by B. eucalypti on Eucalyptus seedlings.