Variation in Botryosphaeriaceae from Eucalyptus plantations in YunNan Province in southwestern China across a climatic gradient

The Botryosphaeriaceae accommodates many important pathogens of woody plants, including Eucalyptus. Recently, Botryosphaeriaceae were isolated from diseased plant parts from surveys of Eucalyptus plantations in the YunNan Province, China. The aims of this study were to identify these Botryosphaeriaceae isolates and to evaluate their pathogenicity to Eucalyptus. A total of 166 isolates of Botryosphaeriaceae were obtained from six regions in the YunNan Province, of which 76 were from Eucalyptus urophylla × E. grandis hybrids, 49 from E. globulus trees, and 41 isolates were from other unknown Eucalyptus species or hybrids. Isolates were identified by comparing DNA sequences of the internal transcribed spacer ribosomal RNA locus (ITS), partial translation elongation factor 1-alpha (tef1), β-tubulin 2 (tub2) and DNA-directed RNA polymerase II subunit (rpb2) genes, and combined with their morphological characteristics. Eleven species were identified, including Botryosphaeria fusispora, B. wangensis, Lasiodiplodia pseudotheobromae, Neofusicoccum kwambonambiense, N. parvum, and six novel species described as B. puerensis, N. dianense, N. magniconidium, N. ningerense, N. parviconidium and N. yunnanense. The dominant species across the regions were N. yunnanense, N. parvum and B. wangensis, representing 31.3, 25.3 and 19.9% of the total isolates, respectively. Species diversity and composition changed across the different climatic zones, despite their relatively close geographic proximity and the fact that some of the species have a global distribution. All the Botryosphaeriaceae species were pathogenic to one-year-old plants of an E. urophylla × E. grandis clone and E. globulus seed-derived plants, but showed significant inter- and intra-species variation in aggressiveness amongst isolates. The study provides a foundation for monitoring and management of Botryosphaeriaceae through selection and breeding of Eucalyptus in the YunNan Province of southwestern China.


INTRODUCTION
Eucalyptus species have been widely planted in many countries of the world for wood and fibre needs, mostly due to their rapid growth and adaptability to a variety of ecological conditions (Coppen 2002). In China, with more than 4.5 million hectares of Eucalyptus planted, an important area for Eucalyptus plantation establishment is the YunNan Province (Xie et al. 2017). This province includes seven climatic zones due to variation in altitude. These include a cold highland zone (T1), central temperate zone (T2), southern temperate zone (T3), northern sub-tropical zone (T4), central sub-tropical zone (T5), southern sub-tropical zone (T6) and tropical zone (T7) (Ye 2017). Most Eucalyptus have been planted in the sub-tropical and tropical (T4-T7), central and southern parts of the YunNan Province. The Eucalyptus species planted include large areas of E. urophylla × E. grandis hybrids and E. globulus, and smaller areas of E. nitens and E. smithii (Qi 2002).
Diseases associated with Botryosphaeriaceae have been reported on a variety of woody plants globally Dissanayake et al. 2016;Mehl et al. 2017;Slippers et al. 2017). They usually occur when plants are subjected to environmental stresses, including drought, frost, physical damage and biological stress (Old et al. 2003;Slippers and Wingfield 2007;Manawasinghe et al. 2016). Typical symptoms associated with Botryosphaeriaceae infections include die-back, canker, shoot blight, and fruit rot Slippers et al. 2017; Billones-Baaijens and Savocchia 2019). On Eucalyptus in China, the Botryosphaeriaceae has been associated with stem cankers as well as shoot and twig blights.
The taxonomic status of Botryosphaeriaceae has been substantially revised in recent years and now includes 23 genera and at least 200 species known from culture (Liu et al. 2012;Phillips et al. 2013;Dissanayake et al. 2016;Slippers et al. 2017;Yang et al. 2017;Jayawardena et al. 2019aJayawardena et al. , 2019b. These species include many cryptic taxa and require DNA sequence-based identification, often considering sequence data from multiple loci. Recent studies on the Botryosphaeriaceae from Eucalyptus in China that have been based on DNA sequence data have identified twelve species. These include Botryosphaeria dothidea, B. fabicerciana, B. fusispora, B. pseudoramosa, B. qingyuanensis, Lasiodiplodia brasiliense, L. pseudotheobromae, L. theobromae, Neofusicoccum microconidium, N. parvum, N. ribis sensu lato and N. sinoeucalypti (Yu et al. 2009;Chen et al. 2011;Li et al. 2015Li et al. , 2018. These studies have, however, not included thorough sampling from Eucalyptus in the YunNan Province. During disease surveys in Eucalyptus plantations in the YunNan Province in 2014, typical disease symptoms linked to the Botryosphaeriaceae were observed. The aims of this study were to (1) identify the species of Botryosphaeriaceae isolated from diseased Eucalyptus trees in YunNan Province based on phylogenetic inference combined with morphological characteristics, (2) determine their geographic distribution in different regions of this province, and (3) evaluate their pathogenicity on one-year-old plants of an E. urophylla × E. grandis hybrid clone and E. globulus seed-derived plants.

Sample collection and fungal isolation
Field surveys of Eucalyptus plantations were conducted in YunNan Province of southwestern China during 2014. A large area of these Eucalyptus plantations was severely damaged by disease with symptoms typical of the Botryosphaeriaceae. These symptoms included die-back, leaf and shoot blight, stem and branch canker, and they resulted in tree death in some plantations (Fig. 1).
Stems, branches and twigs from Eucalyptus trees showing typical symptoms of Botryosphaeriaceae infection were collected. Botryosphaeriaceae isolates were obtained as described in Li et al. (2018). All cultures were deposited in the Culture Collection (CSF) of the China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang, GuangDong Province, China. Duplicate cultures were deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa, and representative cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China. The dried specimens were deposited in the mycological fungarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS), Beijing, China.

DNA extraction, PCR amplification and sequencing
Total DNA of each isolate was extracted from the mycelium of 7-day-old cultures using the CTAB method as described in van Burik et al. (1998). RNA from each DNA sample was removed by adding 2 mL RNase A (10 mg/mL) and incubating at 37°C for 1 h. Quality and quantity of the DNA samples were determined using a NanoDrop 2000 Spectrometer (Thermo Fisher Scientific Inc. Waltham, MA, USA), and each DNA sample was diluted to approximately 100 ng/uL with DNase/RNasefree ddH 2 O (Sangon Biotech Co., Ltd., Shanghai, China) for PCR amplification. Three to four loci were amplified, including the internal transcribed spacer (ITS), a part of the translation elongation factor 1-alpha (tef1), a part of the β-tubulin 2 (tub2) and a part of DNA directed RNA polymerase II subunit (rpb2). Details regarding primers, PCR reactions and cycling conditions were as described by Li et al. (2018). Primers were synthesised and PCR products were sequenced by the Beijing Genomics Institute (BGI), GuangZhou, GuangDong Province, China.

Phylogenetic analyses
Sequences of the ITS, tef1 and tub2 regions for all isolates obtained in this study were generated for species identification. Based on these sequences, the initial genotype of each isolate was determined. Representative isolates based on initial genotype characterisation, host and location for each species were selected for sequencing of the rpb2 locus. The final genotypes of the selected isolates were thus determined based on sequence data from four loci. Preliminary identification in this study was performed using Standard Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and available sequences of all species in related genera containing ex-             type isolates were downloaded from the NCBI for phylogenetic analyses. The sequences were aligned using the online version of MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013), with the iterative refinement method (FFT-NS-i setting). The alignments were checked manually and edited in MEGA v.6.0.5 (Tamura et al. 2013). Sequence alignments were deposited in TreeBASE. Maximum likelihood (ML) analyses with 1000 bootstrap replicates were conducted using PhyML v.3.0 (Guindon et al. 2010). The best-fit model of nucleotide substitution for each dataset was determined using jMo-delTest v.2.1.5 (Darriba et al. 2012). Maximum parsimony (MP) trees were generated in PAUP v.1.0b10 (Swofford 2002), using the heuristic search function with tree bisection and reconstruction (TBR) as branch swapping algorithms and 1000 random addition replicates. Gaps were treated as a fifth character and the characters were unordered and given equal weight. MAXTREES were set to 5000, branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated. Bootstrap support values were evaluated using 1000 bootstrap replicates (Hillis and Bull 1993). The phylogenetic analyses for Botryosphaeria were rooted using N. parvum (ATCC 58191), and phylogenetic analyses for Lasiodiplodia and Neofusicoccum were rooted using Botryosphaeria dothidea (CBS 115476) ( Table 2).
The criterion applied to determine species boundaries was based on phylogenetic analyses and sequences comparisons. Thus, species were considered unique when isolate(s) formed a distinct lineage that differentiated them from other isolates in at least two of the three or four individual loci (ITS, tef1 and tub2 for Botryosphaeria; or ITS, tef1, tub2 and rpb2 for Lasiodiplodia and Neofusicoccum). Furthermore, where these groupings were not contradicted at the other loci, and where they had fixed Single Nucleotide Polymorphisms (SNPs) that differentiated them from their phylogenetically closest species.

Morphology
For the description of putatively novel species, microscopic features and colony characteristics were examined. More than one Botryosphaeriaceae species was frequently isolated from the pycnidia on the same Eucalyptus branch, and most of the isolates were obtained from diseased tissues, which were free of fruiting structures. Consequently, isolates were grown on Petri dishes containing 2% water agar (WA) with several doubleautoclaved pine needles on their surface (Smith et al. 1996). These plates were incubated at room temperature under near-ultraviolet light for 4-6 wk. to induce sporulation. Relevant morphological characteristics were examined and recorded using a Zeiss Axio Imager A1 microscope and a Zeiss AxioCam MRc digital camera with Zeiss Axio Vision v.4.8 software (Carl Zeiss Ltd., Oberkochen, Germany). The lengths and widths of 50 conidia per isolate were measured. These are presented as average (mean), standard deviation (SD), minimum (min) and maximum (max) of the conidial measurements are presented as (min-) (mean-SD)-(mean + SD)(−max). The ratio of average length to average width (L/W) for each species was calculated. Morphological descriptions were deposited in MycoBank (www.mycobank.org).
To determine the optimum temperatures for growth of the novel species, a 5-mm-diam plug of agar was cut from the actively growing margin of a 7-day-old colony and placed at the centre of a 90-mm-diam Petri dish containing 2% MEA. Five replicate plates were used for each isolate at each temperature and these were incubated in the dark at temperatures ranging from 5 to 40°C at 5°C intervals. Two diameter measurements, perpendicular to each other, were recorded daily until the fastest growing culture reached the edge of the Petri dish. The average colony diameter for each of the eight temperatures was calculated. Colony colour was determined from 7-day-old cultures grown on 2% MEA at 25°C using the colour charts of Rayner (1970).

Pathogenicity tests
To determine the relative pathogenicity of the species identified in this study, inoculation trials were conducted under natural conditions using potted-trees of an E. urophylla × E. grandis hybrid clone and E. globulus seedderived plants at the South China Experiment Nursery (SCEN), located in ZhanJiang, GuangDong, China. Oneyear-old healthy plants of the E. urophylla × E. grandis clone and E. globulus seed-derived plants, approximately 170 cm high and 2 cm diameter at the root collar, were utilised. For each plant, a 5-mm-diam wound was made on the stem (approximately 30 cm above the root collar) using a cork borer to remove the bark and expose the cambium. Seven-day-old cultures of representative isolates, representing different species of Botryosphaeriaceae incubated at 25°C in the dark, were prepared and mycelial plugs were cut with a 5-mm-diam cork borer from the actively growing margins of these cultures. Mycelial plugs were placed into wounds with the mycelium facing the xylem. The wounds were sealed with masking tape immediately after inoculation to protect them from contamination and desiccation.
Ten trees of each Eucalyptus species were inoculated for each isolate. Negative controls were conducted on ten trees of the E. urophylla × E. grandis hybrid clone or Li et al. IMA Fungus (2020) 11:22 E. globulus seed-derived plants with clean 2% MEA plugs. After one month, lesion lengths were measured and the average lesion length for the control treatments was subtracted from the average length for the fungustreated plants. This measurement reflected the result of the fungal inoculation without including the wound response due to physical damage in the controls. Reisolations were made from the inoculated plants to fulfil Koch's postulates. General Linear Model (GLM) Univariate Analysis (two-way ANOVA) and one-way ANOVA were used to determine the differences in aggressiveness among isolates utilising the programmes SPSS v.20 (IBM Corp 2011) and SAS v.9.3 (SAS Institute Inc 2011), respectively for the two analyses.

Sample collection and fungal isolation
For each sampled tree, between one and five isolates of Botryosphaeriaceae were obtained. A total of 166 Botryosphaeriaceae isolates from 89 Eucalyptus trees were collected from the six regions (ChuXiong, HongHe, KunMing, PuEr, WenShan and YuXi) sampled (Table 1, Fig. 11). Of these, 76 isolates (45.8%) were from E. urophylla × E. grandis, including 23 isolates from 11 trees in the HongHe Region, 25 isolates were from 12 trees in the PuEr Region, 14 isolates from six trees in the Wen-Shan Region and 14 isolates were from nine trees in the YuXi Region. Forty-nine isolates (29.5%) were from E. globulus, including 23 isolates from 18 trees in the ChuXiong Region, 16 isolates from eight trees in the HongHe Region and 10 isolates from four trees in the KunMing Region. Forty-one isolates (24.7%) were from 21 other unknown Eucalyptus species or hybrids in the HongHe Region.

Phylogenetic analyses
The ITS, tef1 and tub2 loci were amplified for all the 166 isolates (Table 1). Subsequently, 82 representative isolates were selected based on these sequences so as to include all the genotypes revealed by these three loci, as well as all the sampling regions and Eucalyptus genotypes. The rpb2 locus was then also sequenced for these 82 isolates (Table 1). The sequence fragments were approximately 520 bp for the ITS, 280 bp for the tef1, 430 bp for the tub2 and 610 bp for the rpb2. The genotype of each isolate was determined based on the four loci, and one or two isolates were then selected for phylogenetic analyses, depending on the number of isolates available for each genotype (Table 1). Based on the BLAST search against the nucleotide database on the NCBI website, three genera (Botryosphaeria, Lasiodiplodia and Neofusicoccum) in the Botryosphaeriaceae were identified. Sequences of ex-type isolates for all species in these genera were downloaded and used in the phylogenetic analyses. The aligned sequences for each locus (ITS, tef1, tub2 and rpb2), as well as the combined sequences of three or four loci (Botryosphaeria: ITS, tef1, tub2; Lasiodiplodia and Neofusicoccum: ITS, tef1, tub2, rpb2) were deposited in TreeBASE (No. S25832). Statistical values for all datasets for ML and MP analyses are presented in Table 3. Isolates obtained in this study were divided into 11 groups (A to K) based on phylogenetic analyses. Single nucleotide polymorphism (SNP) analyses for the novel taxa emerging from this study and their closest sister taxa are presented in Table 4.

Species in Botryosphaeria
Sequence data were not available for rpb2 for ex-type isolates of various Botryosphaeria species (Table 2). The Botryosphaeria isolates clustered in three groups (Group A, Group B and Group C) based on tef1, tub2, rpb2 and combined ITS/tef1/tub2 analyses, and two groups based on ITS analyses, including Group A and where Group B clustered with Group C (Fig. 2).
Isolates in Group A clustered with B. wangensis and B. minutispermatia based on phylogenetic analyses of ITS dataset (Fig. 2a). In the tef1 tree, they clustered with or were closely related to B. wangensis, B. auasmontanum, B. dothidea, B. minutispermatia and B. sinensia (Fig.  2b). In the tub2 tree, they clustered with B. dothidea, B. fabicerciana, B. qingyuanensis, B. rosaceae and B. sinensia, and were closely related to B. wangensis (Fig. 2c). In the rpb2 tree, they clustered with or were closely related to B. wangensis and B. dothidea (Fig. 2d). In the combined ITS/tef1/tub2 tree, these isolates were closely related to B. wangensis (Fig. 2e). Some isolates formed an independent clade based on one of the four individual loci (isolates CSF6173 and CSF6174 in the tef1 tree, isolate CSF6237 in the tef1 tree, and isolate CSF6113 in the rpb2 tree) (Fig. 2b-d); isolates CSF5781 and CSF5878 formed an independent clade based on two loci (tef1 and rpb2 trees) (Fig. 2b, d), while they only had three fixed SNPs (one in each of ITS, tef1 and tub2 loci, respectively) different to the phylogenetically closest species, B. wangensis. Based on the phylogenetic analyses for the different datasets and fixed SNPs difference, isolates in Group A were identified as B. wangensis.
Isolate CSF6052 in Group B clustered with B. fabicerciana, B. fusispora, B. kuwatsukai and B. rosaceae based on the ITS tree (Fig. 2a). This isolate formed an independent clade that was distinct from all known species based on the tef1, tub2, rpb2 and the combined ITS/ tef1/tub2 trees (Fig. 2b-e). There were also 23 fixed SNPs different to its phylogenetically closest species, B. qingyuanensis. Consequently, isolate CSF6052 was recognised as an undescribed species.  Li et al. IMA Fungus (2020) 11:22 Isolates in Group C clustered with B. fusispora, B. fabicerciana, B. kuwatsukai, B. puerensis and B. rosaceae in the ITS tree (Fig. 2a). They were closely related to B. fusispora and B. fabicerciana in the tef1 tree (Fig. 2b) and clustered with B. fusispora in the tub2 tree (Fig. 2c). They clustered with or were close to B. fabicerciana in the rpb2 tree, but could not be compared with B. fusispora because sequence data for this region are not available for that species (Fig. 2d). Based on tef1 data (Fig.  2b), three independent clades emerged accommodating isolates CSF5683, CSF6021 and CSF6056; CSF5871 and CSF5872; and CSF6063 and CSF6178, but they had only three or four fixed SNPs different to their phylogenetically closest species B. fusispora. These isolates in Group C were phylogenetically close to B. fusispora based on ITS, tef1, tub2 and the combined ITS/tef1/tub2 trees (Fig. 2) and they were identified as that species.

Species in Lasiodiplodia
Analyses were conducted for Lasiodiplodia based on sequences for the ITS, tef1, tub2 and rpb2 loci. Based on phylogenetic analyses for these loci and the combined ITS/tef1/tub2/rpb2 datasets, two Lasiodiplodia isolates clustered in one group (Group D) (Fig. 3). These isolates were phylogenetically related to L. pseudotheobromae and various other species based on ITS and tub2 trees (Fig. 3a, c). They were closest L. pseudotheobromae based on tef1 tree (Fig. 3b), and clustered with L. pseudotheobromae based on rpb2 tree (Fig. 3d). The tree based on the combined ITS/tef1/tub2/rpb2 dataset also showed that the two isolates making up Group D were phylogenetically closely related to L. pseudotheobromae and they were treated as that species (Fig. 3e).

Species in Neofusicoccum
The Neofusicoccum isolates resided in seven groups based on ITS, tub2 and the combined ITS/tef1/tub2/ rpb2 datasets (Groups E-K). For the tef1 dataset, there were six groups including Groups E-H, Group I that clustered with Group J and Group K. For the rpb2 dataset, there were six groups including Group E that clustered with Group F and Groups G-K (Fig. 4).
Isolates in Group E were closely related to N. parvum and various other species based on the ITS, tef1 and rpb2 trees (Fig. 4a, b, d) and they also clustered with N. parvum based on the tub2 tree (Fig. 4c). They formed multiple independent clades based on the ITS, tef1, rpb2 and the combined ITS/tef1/tub2/rpb2 trees (Fig. 4a, b, d, e). Based on these analyses of five datasets, isolates in Group E were treated as N. parvum (Fig. 4).
Isolates in Group F were closely related to N. algeriense based on phylogenetic analyses of tef1 dataset (Fig.  4b). They clustered with N. mangiferae and N. parvum in the rpb2 tree (Fig. 4d). Isolates in Group F formed one independent clade that was distinct from all known species based on ITS and tub2 trees, and isolates CSF6034 and CSF6142 (ex-type) formed a distinct lineage in tef1 tree (Fig. 4a-c). In the combined tree, isolates CSF6034 and CSF6142 (ex-type), and other isolates in Group F formed an independent sub-clade (Fig. 4e).
a The number means the difference of two species in four loci, ITS/tef1/tub2/rpb2; "*" represents the sequence is unavailable b "-" represent the sequences between two species were not compared   Seven fixed SNPs also differentiated isolates in Group F from their phylogenetically closest relatives N. algeriense and N. parvum in the ITS, tef1 and tub2 regions, and five fixed SNPs differentiated them from N. italicum in the ITS and tef1 regions (tub2 not available for N. italicum) (Table 4). These isolates were consequently treated as representing a novel species. Isolate CSF6037 in Group G clustered with N. kwambonambiense in the tub2 tree (Fig. 4c). It also clustered with N. kwambonambiense and various other species in the tef1 tree (Fig. 4b), and was most closely related to that species in the ITS, rpb2 and the combined ITS/tef1/ tub2/rpb2 trees (Fig. 4a, d, e). Isolate CSF6037 was consequently identified as N. kwambonambiense.
Isolates in Group H clustered with N. illicii in the ITS tree (Fig. 4a) and with N. hongkongense in the tef1 tree (Fig. 4b). Based on the tub2 and rpb2 trees, these isolates formed an independent clade that was distinct from all known species of Neofusicoccum (Fig.  4c, d). This clade was well supported by high bootstrap values in the tub2 and combined ITS/tef1/tub2/ rpb2 trees (tub2, ML/MP = 87%/87%; ITS/tef1/tub2/ rpb2, ML/MP = 95%/98%) (Fig. 4c, e). There were also ten fixed SNPs differentiating isolates in Group H from their phylogenetically closest species, N. hongkongense (Table 4). Consequently, isolates in Group H were considered to represent a novel species of Neofusicoccum.
Isolates in both Group I and Group J formed a single clade that clustered with N. illicii in the tef1 tree, and isolates in Group I clustered with N. illicii in the tub2 tree (Fig. 4c). But isolates in these two groups formed two independent clades in the ITS and rpb2 trees (Fig. 4a, d), and those in Group J also formed an independent clade in the tub2 tree (Fig. 4c). The two independent clades were supported by high bootstrap values in the combined ITS/tef1/tub2/rpb2 tree (Group I, ML/MP = 99%/98%; Group J, ML/MP = 94%/85%) (Fig. 4e). In addition, there were six fixed SNPs observed between isolates in Group I and Group J (Table 4). Thus, isolates in Group I and Group J were considered to represent two undescribed species of Neofusicoccum. Isolates in Group K clustered with N. microconidium in the ITS tree (Fig. 4a). However, they formed a distinct clade that was separated from all known species in the tef1, tub2, and rpb2 trees (Fig. 4b-d).
These isolates resided in a single clade, which was supported by high bootstrap values in the combined ITS/tef1/tub2/rpb2 tree (ML/MP = 99%/98%) (Fig. 4e). There were also six fixed SNPs observed between isolates in Group K and their phylogenetically closest relative, N. microconidium (Table 4). Consequently, isolates in Group K were considered to represent a novel species.

Morphology and taxonomy
Based on analyses of DNA sequence data, the isolates obtained in the present study clustered in 11 phylogenetic groups of the Botryosphaeriaceae. The culture morphology of all isolates in these groups was morphologically similar to other species of Botryosphaeriaceae, consistent with the fact that this characteristic has little taxonomic significance.
Isolates representing Groups B, F and H-K were identified as novel species based on the phylogenetic analyses. Representative isolates for these groups were selected to induce fruiting structures (Table 1). With the exception of those in Group J (isolates CSF6028 and CSF6030), that did not sporulate, these putatively novel taxa produced only asexual structures. Morphological differences were observed for the phylogenetically distinct species (Table 5) and these have been included in their descriptions. Based primarily on phylogenetic inference but including available morphological characteristics, isolates in Groups B, F, H-K were recognised as representing six previously undescribed species for which names are proposed as follows: Botryosphaeria puerensis G.Q. Li & S.F. Chen, sp. nov.
Host: E. urophylla × E. grandis. Distribution: Currently only known from PuEr Region in YunNan Province, China.
Etymology: Name refers to "Dian", an ancient kingdom of YunNan Province, where the type specimen was collected.
Diagnosis: Based on phylogenetic inference, Neofusicoccum dianense resides in 'N. parvum / N. ribis' complex. It produces the longer conidia than its closest phylogenetic relatives including N. algeriense, N. hongkongense, N. italium, N. parvum, N. yunnanense. The optimal growth temperature of N. dianense also differs from that of N. yunnanense.
Etymology: Name refers to the exceptionally large conidia in this species.
Host: E. urophylla × E. grandis. Distribution: Currently known only from HongHe Region in YunNan Province, China.
Etymology: Name refers to the NingEr County where the fungus was isolated for the first time.
Diagnosis: Neofusicoccum ningerense is closely related to N. magniconidium, but differs from the latter species at two bases in each of the ITS, tub2 and rpb2 loci. The optimal growth temperature for N. ningerense is also different from that of N. magniconidium.
Type Description: Sexual state unknown. Conidiomata-like structures produced on pine needles on WA medium within 4-6 wk., embedded in needle tissue, unilocular . No conidiophores, conidiogenous cells or conidia have been observed.
Host: E. urophylla × E. grandis. Distribution: Currently known only from the PuEr Region in YunNan Province, China.
Notes: Only conidiomata were observed in this fungus, and no other asexual structures were observed. Different methods were used in an attempt to induce sporulation but all of these failed. Neofusicoccum ningerense is phylogenetically closely related to N. magniconidium (Fig. 4). The optimal growth temperature of N. ningerense (30°C) differs from that of N. magniconidium (25°C).
Etymology: Name refers to the small conidia in this fungus.
Diagnosis: Neofusicoccum parviconidium can be distinguished from other Neofusicoccum species by its exceptionally short conidia.
Sampling sites in this study included four distinct climate types. Samples in ChuXiong (Region A), KunMing (Region B) and WenShan (Region F) Regions were from the northern sub-tropical or central sub-tropical zone; samples in HongHe (Region E), PuEr (Region D) and YuXi (Region C) were from the southern sub-tropical or tropical zone. Four species were detected in all four climate types surveyed and these included B. fusispora, B. wangensis, N. parvum and N. yunnanense. The remaining seven species identified in this study were detected in only southern sub-tropical or tropical zone (Fig. 11a, c).

Pathogenicity tests
Based on their ITS, tef1 and tub2 genotypes, thirty-six isolates of the Botryosphaeriaceae in three genera and representing 11 species were selected for inoculation. Typical lesions were observed on inoculated Eucalyptus plants and lesion lengths were recorded one month after inoculation. The results of pathogenicity tests showed that all isolates produced lesions on the test plants, while the controls produced only small zones of wound reaction (Fig. 12, Additional file 1: Figure S1). The inoculated species were re-isolated from the lesions, but never from the negative controls. Consequently, Koch's postulates were fulfilled.
Lesion length data were not normally distributed based on Kolmogorov-Smirnov normality test (P < 0.05). All data were consequently transformed (Kolmogorov-Smirnov normality test, P = 0.2) by conducting a Rank transformation using the statistical package SPSS v. 20.
Results of GLM Univariate Analysis (two-way ANOVA) showed a significant (P = 0.001) interaction effect between isolate and host. The analyses also showed that not all isolates of the same species of Botryosphaeriaceae reacted in the same manner on the tested E. urophylla × E. grandis clone or E. globulus plants. For example, lesions produced by isolate CSF5802 (L. pseudotheobromae) on E. urophylla × E. grandis were significantly longer than those on E. globulus, while the lesion lengths produced by isolate CSF6050 (L. pseudotheobromae) on the two tested Eucalyptus genotypes were not significantly different (P = 0.05). The results also showed that the pathogenicity of isolates of the same species on the two tested Eucalyptus genotypes can be different. For example, lesion lengths produced by isolate CSF5820 (B. wangensis) on E. urophylla × E. grandis and E. globulus were significantly longer than the other isolates of this species (P = 0.05) (Fig. 12). In contrast, lesion lengths produced by all isolates of B. fusispora on both E. urophylla × E. grandis and E. globulus were not significantly different (P = 0.05) from each other (Fig. 12).
For the tested isolates residing in three genera of the Botryosphaeriaceae, the overall data showed that species of Lasiodiplodia were the most aggressive, followed by those in Neofusicoccum (Fig. 12). The overall data also showed that plants of the E. urophylla × E. grandis clone and E. globulus seed-derived plants had similar levels of susceptibility to most of the tested isolates (Fig. 12). The exceptions were for isolates CSF5802 (L.  Analysis of multi-gene phylogenetic concordance has emerged as standard practice for species identification in the Botryosphaeriaceae Chen et al. 2014aChen et al. , 2014bSlippers et al. 2017;Yang et al. 2017;Li et al. 2018;Jayawardena et al. 2019aJayawardena et al. , 2019bPhillips et al. 2019). This approach was also essential in the present study to distinguish between closely related species, where we considered the phylogenetic signal for four loci, including ITS, tef1, tub2 and rpb2. The most common loci used for species delineation in Botryosphaeria are ITS, tef1 and tub2 Chen et al. 2014aChen et al. , 2014bOsorio et al. 2017;Li et al. 2018) and in Lasiodiplodia and Neofusicoccum are ITS, tef1, tub2 and rpb2 (Pavlic et al. 2009a(Pavlic et al. , 2009bSakalidis et al. 2011;Cruywagen et al. 2017;Yang et al. 2017;Li et al. 2018;Phillips et al. 2019). These were also the most informative loci for the genera in this study. However, a limitation lies in the fact that there are numerous species for which sequence data are not available for all of these loci.
The majority of the isolates (67%) obtained in this study were species of Neofusicoccum. Five of these were previously undescribed taxa and these were found in addition to the well-known species N. kwambonambiense and N. parvum. Together with the newly described species, Neofusicoccum now includes 48 species Yang et al. 2017;Jami et al. 2018;Li et al. 2018).
Neofusicoccum yunnanense was isolated from all six regions in the sub-tropical and tropical zones, suggesting that it has a wide distribution in different climatic zones.
In contrast, the other new species of Neofusicoccum (N. dianense, N. magniconidium, N. ningerense and N. parviconidium) were all from the southern sub-tropical or tropical zone that has relatively high average temperatures. Neofusicoccum parvum was isolated in five sampled regions, while N. kwambonambiense was isolated only from PuEr. A previous study has shown that these two species have a wide geographic distribution including areas, with mediterranean and sub-tropical climates worldwide (Sakalidis et al. 2013), and that they have a wide range of hosts (Pavlic et al. 2009a;Phillips et al. 2013;Sakalidis et al. 2013). In China, N. parvum has also been reported from a wide range of hosts including Cupressus funebris (Li et al. 2010), Eriobotrya japonica (Zhai and Zhang 2019), Eucalyptus spp. (Chen et al. 2011), Koelreuteria paniculata (Fang et al. 2019), Hevea brasiliensis  and Juglans regia (Yu et al. 2015) and in these cases, from sub-tropical and tropical zones. Neofusicoccum kwambonambiense was first reported from Syzygium cordatum (Myrtaceae) in South Africa (Pavlic et al. 2009a). The present study represents the first report of this species associated with Eucalyptus and also the Myrtaceae in China.
Two new cryptic species (N. dianense and N. yunnanense) were discovered in the 'N. parvum / N. ribis' complex based on concordance in the phylogenetic analyses of the ITS, tef1, tub2 and rpb2 datasets in this study. Cryptic species are defined as two or more distinct species often treated as a single species because they are at least superficially indistinguishable based on their morphology (Bickford et al. 2007). The use of multi-locus phylogenetic concordance has revealed numerous cryptic species in the Botryosphaeriaceae in recent years Pavlic et al. 2009b;Phillips et al. 2013;Slippers et al. 2014Slippers et al. , 2017Yang et al. 2017). This is especially true in the 'N. parvum / N. ribis' complex, where six cryptic species with similar conidia have been distinguished based on multigene analyses (Pavlic et al. 2009a;Sakalidis et al. 2011;Li et al. 2018). Amongst the three new Neofusicoccum species (N. magniconidium, N. ningerense and N. parviconidium) discovered in the present study and that reside in the 'N. parvum / N. ribis' complex, N. parviconidium, like N. microconidium, have relatively small conidia compared to other species in the genus. Neofusicoccum magniconidium has larger conidia in comparison with those of N. macroclavatum, and it is phylogenetically most closely related to N. macroclavatum, and N. ningerense, the latter of (See figure on previous page.) Fig. 11 Botryosphaeriaceae species detected from Eucalyptus plantations in six regions in YunNan Province. a. Sampling regions across different climatic zones. T1: cold highland zone, T2: central temperate zone, T3: southern temperate zone, T4: northern sub-tropical zone, T5: central sub-tropical zone, T6: southern sub-tropical zone, T7: tropical zone; b. Prevalence of Botryosphaeriaceae species as a percentage of the total isolates in YunNan Province. Different species are represented by numbers with different colours; c. Prevalence of Botryosphaeriaceae species as a percentage of the total isolates in each of the different sampling regions   which failed to produce fruiting structures. These newly described species, together with other species in the 'N. parvum / N. ribis' complex, makes this one of the most widespread 'lineages' in the Botryosphaeriaceae. When our results are consolidated with those from previous studies (Chen et al. 2011;Li et al. 2018), a total of nine species of Neofusicoccum have been identified from Eucalyptus plantations in China. These include N. dianense, N. kwambonambiense, N. magniconidium, N. microconidium, N. ningerense, N. parviconidium, N. parvum, N. sinoeucalypti and N. yunnanense. Seven of these nine species were first described from or are known only from China on Eucalyptus in plantations. The exceptions are N. parvum and N. kwambonambiense (Chen et al. 2011. These results suggest an unusually high diversity of Neofusicoccum species in non-native Eucalyptus plantations in China. They could also imply that many additional Neofusicoccum species could exist in yet unsampled regions of the country. A total of 52 isolates were identified as species of Botryosphaeria, including B. fusispora, B. wangensis and the newly described B. puerensis found in this study. The genus Botryosphaeria was first introduced in 1863 by Cesati & De Notaris, and 143 species were recorded in this genus up to 1997 (Denman et al. 2000). As is true for most groups in the Botryosphaeriaceae, Botryosphaeria has been substantially revised in recent years using a combination of DNA sequence and morphological data. The genus now accommodates 16 species for which clear taxonomic descriptions and DNA sequence data are available Slippers et al. 2014;Xu et al. 2015;Ariyawansa et al. 2016;Zhou et al. 2016Zhou et al. , 2017Li et al. 2018).
Many Botryosphaeria species occur widespread across a broad climatic environment and on diverse hosts. For example, Botryosphaeria fusispora was first described from Entada sp. in Thailand (Chiang Rai, Doi Tung: tropical zone; Liu et al. 2012), and subsequently in the FuJian, GuangDong and GuangXi Provinces in subtropical and tropical zones in China ). In the present study, B. fusispora was isolated in four of six sampled regions in the YunNan Province, indicating that this species has a wide distribution in Eucalyptus plantations in sub-tropical and tropical zones. Botryosphaeria wangensis was known only from Cedrus deodara in the HeNan Province in Central China (temperate zone) previously . In contrast, it was detected in five regions (sub-tropical and tropical zones) in YunNan Province in the present study, suggesting that it can also survive at a broad range of temperatures. Many of the other Botryosphaeria species previously described occur in more temperate climates, but this is clearly not a characteristic of the genus.
The newly described B. puerensis is known from only one isolate. It was clearly separate from all other known species based on phylogenetic analyses of tef1, tub2 and rpb2 datasets. Obvious morphological differences were also observed between B. puerensis and its closest known sister species. While we recognise that it is preferable to describe new species based on more than one isolate or specimen (Seifert and Rossman 2010), we chose to describe this species because it was well defined and this is not unprecedented in studies of the Botryosphaeriaceae (e.g. Slippers et al. 2014;Yang et al. 2017;Zhang et al. 2017).
Lasiodiplodia pseudotheobromae was identified from Eucalyptus plantations in PuEr and HongHe Regions (tropical zone) in YunNan Province. This species has previously been reported from a wide variety of hosts across many different climate zones globally including Brazil (tropical zone) (Netto et al. 2014), China (subtropical and tropical zones) (Zhao et al. 2010;Li et al. 2018), Costa Rica and Suriname (tropical zone) , amongst many others. In China, L. pseudotheobromae was first reported in 2010 (Zhao et al. 2010) and recorded from different plant species more recently (Chen et al. 2011;Dissanayake et al. 2015;Li et al. 2015;Tennakoon et al. 2016;Wu et al. 2019). Collectively, these results suggest that L. pseudotheobromae is one of the most widespread species in the Botryosphaeriaceae globally and it has at least 105 recorded hosts (NCBI Nucleotide Database, 2019). It is a species that might easily be spread amongst regions and can be expected to have an important impact on a wide variety of plant-based industries in a diversity of environments.
Overall, the results of this study suggest that climate influences the distribution of Botryosphaeriaceae, even over relatively small distances (560 km across the widest sampling points in this study). This is despite the obvious adaptability to both hosts and temperature ranges that is reflected in their wide geographic distribution across climates worldwide Slippers et al. 2014). Only three species of Botryosphaeria and one species of Lasiodiplodia were detected in the sub-tropical or tropical zone in YunNan Province, compared to the seven species of Neofusicoccum. A greater number of Botryosphaeriaceae species were detected in the southern sub-tropical or tropical zone (PuEr and HongHe Regions) than northern subtropical or central sub-tropical zone (ChuXiong, Kun-Ming and WenShan Regions), suggesting that climate affects the distribution of species in the Botryosphaeiraceae. Relatively few species were detected from YuXi Region in the sub-tropical or tropical zone, which might have been affected by the lower number of samples collected in this region. Factors that probably affect this species diversity and distribution include climates such as temperature and water, host-associated factors such as species and age of host and the host structures from which isolations are made Velásquez et al. 2018).
All 11 species identified in this study were pathogenic to the E. urophylla × E. grandis hybrid clone and E. globulus seed-derived plants. Some of these species could present threats to the Eucalyptus industry. One isolate of L. pseudotheobromae produced significantly longer lesions than those of other genera of Botryosphaeriaceae on the tested Eucalyptus genotypes, which is consistent with the results of previous studies (Pérez et al. 2010;Chen et al. 2011;Li et al. 2018). With the exception of one isolate, isolates of the Botryosphaeria spp. produced the smallest lesions in the pathogenicity tests; a result similar to that of previous studies . The species of Neofusicoccum were also pathogenic and produced lesions that were generally larger than those associated with the Botryosphaeria species, which is also consistent with the results of previous studies (Mohali et al. 2009;Pérez et al. 2010;Chen et al. 2011;Li et al. 2018). There was also significant variation in aggressiveness between isolates of species, which emphasises that evaluation of pathogenicity linked to Eucalyptus breeding trials should include isolates covering a broad range of aggressiveness.
The present study provides foundational data on the diversity, distribution and pathogenicity of the Botryosphaeriaceae from Eucalyptus plantations in YunNan Province in southwestern China. Together with previous studies (Chen et al. 2011;Li et al. 2015Li et al. , 2018, the results revealed a high level of Botryosphaeriaceae diversity associated with diseased Eucalyptus in the sampled plantations. Special attention should be afforded in future monitoring, to species with wide distributions and high levels of aggressiveness to species of Eucalyptus.

CONCLUSIONS
This study provides important new data regarding on the diversity, distribution and pathogenicity of the Botryosphaeriaceae from Eucalyptus plantations in YunNan Province in southwestern China. Results revealed a high level of Botryosphaeriaceae diversity associated with diseased Eucalyptus in the sampled plantations. Species diversity and composition changed across the different climatic zones, despite their relatively close proximity and the fact that some of the species have a global distribution. All the Botryosphaeriaceae species were pathogenic to tested oneyear-old Eucalyptus plants, but showed significant inter-and intra-species variation in aggressiveness amongst isolates. Future tree disease monitoring should consider Botryosphaeriaceae species with wide distributions and high levels of aggressiveness to species of Eucalyptus. The study also provides a foundation for monitoring and management of Botryosphaeriaceae through selection and breeding of Eucalyptus in the Yun-Nan Province in southwestern China.
Additional file 1: Figure S1. Symptoms observed on E. globulus and E. urophylla × E. grandis one month after inoculation. a, b. lesion produced on E. globulus by isolates (a)