Phenotypic and Genomic Difference among Four Botryosphaeria Pathogens in Chinese Hickory Trunk Canker

Botryosphaeria species are amongst the most widespread and important canker and dieback pathogens of trees worldwide, with B. dothidea as one of the most common Botryosphaeria species. However, the information related to the widespread incidence and aggressiveness of B. dothidea among various Botryosphaeria species causing trunk cankers is still poorly investigated. In this study, the metabolic phenotypic diversity and genomic differences of four Chinese hickory canker-related Botryosphaeria pathogens, including B. dothidea, B. qingyuanensis, B. fabicerciana, and B. corticis, were systematically studied to address the competitive fitness of B. dothidea. Large-scale screening of physiologic traits using a phenotypic MicroArray/OmniLog system (PMs) found B. dothidea has a broader spectrum of nitrogen source and greater tolerance toward osmotic pressure (sodium benzoate) and alkali stress among Botryosphaeria species. Moreover, the annotation of B. dothidea species-specific genomic information via a comparative genomics analysis found 143 B. dothidea species-specific genes that not only provides crucial cues in the prediction of B. dothidea species-specific function but also give a basis for the development of a B. dothidea molecular identification method. A species-specific primer set Bd_11F/Bd_11R has been designed based on the sequence of B. dothidea species-specific gene jg11 for the accurate identification of B. dothidea in disease diagnoses. Overall, this study deepens the understanding in the widespread incidence and aggressiveness of B. dothidea among various Botryosphaeria species, providing valuable clues to assist in trunk cankers management.


Origin and Collection of Botryosphaeria Species
The four Botryosphaeria species, including B. dothidea strain BDLA16−7, B. corticis strain BCTK16−35, B. fabicerciana strain BFLG18−2, and B. qingyuanensis BQTK16−30, used in phenotypic characterization and genomic analysis were collected from Chinese hickory tree (cultivar of linan) in Linan, Zhejiang province of China. The four stains have been identified via phylogenetic analyses of ITS, β-TUBLIN, and EF gene sequences combined with morphological analyses in our previous study [5], and their raw sequence data and the whole genome sequence data reported are available at Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/), and the Genome Warehouse (GWH, https://ngdc. cncb.ac.cn/gwh), respectively, in National Genomics Data Center, China National Center for Bioinformation (CNCB-NGDC Members and Partners, 2021) [28], under Bioproject PRJCA005744. The B. dothidea stains used for the accuracy and efficiency detection of species molecular identification method, including one sequenced strain BDLA16−7 [28], 89 identified B. dothidea isolated from diseased Chinese hickory in our previous study [5], and one identified B. dothidea stain associated with citrus branch diseases isolated by Xiao et al. [29]. For each strain used in this study, the single-conidium isolates were stored on potato dextrose agar (PDA; 200 g L −1 potato boiled for half an hour and strained, 20 g L −1 glucose, 12 g L −1 agar) slants at 4 • C.

Phenotypic Characterization of Botryosphaeria Species
Large-scale screening of physiologic trait of four Botryosphaeria species including B. dothidea strain BDLA16−7, B. corticis strain BCTK16−35, B. fabicerciana strain BFLG18−2, B. qingyuanensis BQTK16−30 was conducted using a phenotypic MicroArray/OmniLog system according to the Biolog standard procedure [15,16]. The concentration of the spore suspension of each tested Botryosphaeria species was adjusted to 1 × 10 6 conidia/mL before metabolic plate tests. The conidia suspension of each tested Botryosphaeria species was prepared as mentioned in our previous study [5]. Briefly, spores were collected from the 8 to 10 d old colonies grown on PDA plates by pouring 2 to 3 mL of sterile water onto plates, filtering through duplicated gauze, and counting spores with a hemocytometer. Metabolic plates were used to assess the metabolic pathways of nitrogen utilization (PM 3), biosynthetic pathways (PM 5), osmotic pressure (PM 9) and ionic strength and pH (PM 10). To each well of the PM plates, 100 µL of a cell suspension was added with 62% transmittance. The metabolic phenotype test plate was placed in the Biolog system incubator at 28 • C for 96 h. Phenotypic data were recorded every 15 min by the OmniLog 2.4 system capturing digital images of the microarrays and storing turbidity values. Data were then analyzed using Kinetic and Parametric software (Biolog). Phenotypes were estimated according to the area of each well under the kinetic curve of dye formation. The experiment was repeated three times. Heat maps of phenotype analysis was conducted with the software of HemI (Heatmap IIIlustrator, version 1.0).

Comparison of Genomic Differences among Botryosphaeria Species
Orthologous gene clusters analysis was conducted by OrthoFinder v2.5.4 using all of sequences from the four Botryosphaeria species with defalt parameters. Those genes coding unclusted proteins and proteins in species-specific clusters were selected as candidate B. dothidea species-specific genes. These genes were further search against with genome sequences of other three Botryosphaeria species by NCBI-BLASTn, and genes were absent (0 hits) in all the other three Botryosphaeria species were retained as B. dothidea speciesspecific genes.

B. dothidea Rapid Identification Method with Species-Specific Primers
To rapidly identification of B. dothidea, the species-specific genes with length around 1000 bp (listed in the Table S3) were chose for species-specific primer design using Primer3 online web service (https://primer3.org/). For each specific primer the following parameters were checked: primer length, primer melting temperature, GC content, GC clamp, primer secondary structures (hairpins, self-dimer, and cross dimer), repeats, runs, 3 end stability, avoid template secondary structure, optimum annealing temperature, primer pair tm mismatch calculation. In addition, all species-specific primers were further tested for specificity using web-based Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi) to avoid the cross reactivity with the other three Botryosphaeria species. Then primer sets that have no double-ended match with the other three Botryosphaeria species in silico analysis were applied to PCR assays for the screen of the best primers with highest amplification efficiency and specificity.
For the preparation of genomic DNA used in PCR assays, the four Botryosphaeria species were incubated on PDA plates at 25 • C in the dark. After mycelia grew to cover nearly two-thirds of the PDA plate surfaces, the cultures were transferred to a mortar and ground with liquid nitrogen. The resultant powder was placed in a 2 mL centrifuge tube and the mycelial DNA from each isolate was extracted using a Genomic DNA Kit (Sangon Biotech Co., Ltd., Shanghai) according to the manufacturer's instructions.
The PCR was performed in an Applied Biosystems Veriti Thermal Cycler in 50 µL PCR reaction systems comprising 25 µL of 2 × Rapid Taq master mix (Vazyme, P222-01, Nanjing, China), 1 µL of fungal DNA template, 2 µL of 10 µmol· liter −1 upstream and downstream primers, and 20 µL of ddH 2 O. PCR reaction conditions included 94 • C for 5 min for denaturation, followed by 35 cycles of 94 • C for 30 s, annealing 57 • C for 30 s, and extension at 72 • C for 45 s, with a final extension at 72 • C for 10 min. Agarose gel electrophoresis (1%) stained with ethidium bromide was then used to verify successful PCR amplification. The experiment was repeated three times.
Using the PM 9 plate, phenotypes of the four Botryosphaeria species under various osmotic stresses were tested. As shown in Figure 3, all the four fungi showed active metabolism in utilizing up to 6% potassium chloride (plate PM 9, wells D1 to D4), up to 5% sodium sulfate (plate PM 9, wells D5 to D8), up to 6% sodium, up to 20% ethylene glycol (plate PM 9, wells D9 to D12), up to 6% sodium formate (plate PM 9, wells E1 to E6), up to 12% sodium lactate (plate PM 9, wells F1 to F12), and up to 100 mM sodium nitrite (plate PM 9, wells H1 to H12), while demonstrating significantly attenuated metabolism under the stresses of urea (≥6%, plate PM 9, wells E11 to E12). Interestingly, B. dothidea was found to be capable of utilizing up to 50 mM sodium benzoate (plate PM 9, wells G5 to G6), while the other three could only utilize up to 20 mM sodium benzoate ( Figure 3 and Table S1), indicating the possibility that the greater tolerance toward sodium benzoate may account for the widespread incidence and aggressiveness of B. dothidea.
The PM 10 plate assessing the tolerance of pH variation found that though all four Botryosphaeria species were able to survive under pH variation ranged from 3.5 to 10 (plate PM 10, wells A1-A12), and B. dothidea, as well as B. qingyuanensis, was more tolerant to strong alkali ( Figure 4 and Table S1). Notably, they were able to maintain active metabolism of multiple nitrogen sources under pH 9.5, for example L-aspartic acid (plate PM 10, well E5), L-glutamic acid (plate PM 10, well E6), L-histidine (plate PM 10, well E9), and Lisoleucine (plate PM 10, well E10), while B. fabicerciana and B. corticis cannot ( Figure 4 and Table S1), indicating a stronger pH regulation capability in B. dothidea and B. qingyuanensis.     to 12% sodium lactate (plate PM 9, wells F1 to F12), and up to 100 mM sodium nitrite (plate PM 9, wells H1 to H12), while demonstrating significantly attenuated metabolism under the stresses of urea (≥6%, plate PM 9, wells E11 to E12). Interestingly, B. dothidea was found to be capable of utilizing up to 50 mM sodium benzoate (plate PM 9, wells G5 to G6), while the other three could only utilize up to 20 mM sodium benzoate ( Figure 3 and Table S1), indicating the possibility that the greater tolerance toward sodium benzoate may account for the widespread incidence and aggressiveness of B. dothidea. The PM 10 plate assessing the tolerance of pH variation found that though all four Botryosphaeria species were able to survive under pH variation ranged from 3.5 to 10 (plate PM 10, wells A1-A12), and B. dothidea, as well as B. qingyuanensis, was more tolerant to strong alkali ( Figure 4 and Table S1). Notably, they were able to maintain active metabolism of multiple nitrogen sources under pH 9.5, for example L-aspartic acid (plate PM 10, well E5), L-glutamic acid (plate PM 10, well E6), L-histidine (plate PM 10, well E9), and Lisoleucine (plate PM 10, well E10), while B. fabicerciana and B. corticis cannot (Figure 4 and Table S1), indicating a stronger pH regulation capability in B. dothidea and B. qingyuanensis.

Comparison of Genomic Differences among Botryosphaeria Species
According to the NCBI-BLASTn results (gene versus genome, cut at ≥ 95% identity, with 0 hits), we revealed 437 (versus B. corticis), 440 (versus B. fabicerciana) and 185 (versus B. qingyuanensis)B. dothidea specific protein coding genes (Table S2). Combine the above unique gene list, B. dothidea contained 143 species-specific genes absent in the other three Botryosphaeria species (Table S2). Further analysis of the proteins coded by B. dothidea species-specific genes via the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways The legend of color code from blue to green, and red shades represent low, moderate, and high metabolic rate under the corresponding pH stresses, respectively.

Comparison of Genomic Differences among Botryosphaeria Species
According to the NCBI-BLASTn results (gene versus genome, cut at ≥ 95% identity, with 0 hits), we revealed 437 (versus B. corticis), 440 (versus B. fabicerciana) and 185 (versus B. qingyuanensis) B. dothidea specific protein coding genes (Table S2). Combine the above unique gene list, B. dothidea contained 143 species-specific genes absent in the other three Botryosphaeria species (Table S2). Further analysis of the proteins coded by B. dothidea speciesspecific genes via the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (https: //www.genome.jp/tools/kofamkoala/) online service found that only 7 proteins were assigned the corresponding KEGG Orthologs (KOs) ( Table 2), which may be responsible for the species-specific functions. Additionally, among 7 KEGG annotated proteins, one protein (jg10977) was associated with the calcium permeable stress-gated cation channel, which may have potential roles in the specific osmotic stress tolerance of B. dothidea mentioned above.

Design and Application of Primers for B. dothidea Molecular Identification
As showed in Figure 5, the screened primer set Bd_11F (5 -TCCAACGACGAGCA ATCC-3 ) and Bd_11R (5 -TGTGCCCTGAGGCGGTAT-3 ) designed based on the sequence of B. dothidea species-specific gene jg11 was found to give an amplicon of 695 bp for B. dothidea strains, including one sequenced strain BDLA16−7 [28], 89 identified B. dothidea isolated from diseased Chinese hickory in our previous study [5], and one identified B. dothidea stain associated with citrus branch diseases [29], but not for B. qingyuanensis, B. fabicerciana, and B. corticis. Taken together, these results showed that sequence of above strain B. dothidea specific genes not only give a predictive information of speciesspecific function annotation (Table 2) but also provide a basis for the development of B. dothidea molecular identification method. Additionally, the species-specific primer set Bd_11F/Bd_11R was able to carry out the accurate identification of B. dothidea with high efficiency ( Figure 5B).

Discussion
Botryosphaeria species are amongst the most widespread and important canker and dieback pathogens of trees worldwide [6,7], with B. dothidea as one of the most common Botryosphaeria species [4][5][6]12]. In Chinese hickory trees cankers disease, four Botryosphaeria species, including B. dothidea, B. qingyuanensis, B. fabicerciana, and B. corticis, were identified as Chinese hickory canker-causing agents [5]. Consistently, B. dothidea was also the dominant species due to its highest isolation frequency and strongest virulence [5]. However, the information related to the widespread incidence and aggressiveness of B. dothidea among various Botryosphaeria species causing trunk cankers is poorly understood [4,5,12]. In this study, the metabolic phenotypic diversity of four Chinese hickory canker-causing Botryosphaeria pathogens was systematically studied to address the competitive fitness of B. dothidea via PM 3 (nitrogen utilization), PM 5 (biosynthetic pathways), PM 9 (osmotic pressure), and PM 10 (ionic strength and pH). For nitrogen source metabolization, the highest utilization ratio (96.8%) was found in B. dothidea, followed by B. fabicerciana (89.5%), B. qingyuanensis (88.4%) and B. corticis (86.3%) (Figure 1, Table 1 and Table S1). The efficient utilization of available nitrogen is highly associated with the ability of organism to survive under different environmental situations [30]. For instance, the capability of the methylotrophic yeast Candida boidinii Ramirez to adapt a change in the major available nitrogen source from nitrate to methylamine during the host plant aging was crucial for yeast survival on the leaf environment [18,31]. In addition, the atmospheric N 2 fixing endosymbiosis in Ustilago maydis (DC.) Corda, a plant parasitic fungus causing corn smut disease [32], is also a flexible process important for the adaptation of the fungus to survive and grown in media lacking nitrogen [19,33]. Intriguingly, our previous study found that B. dothidea was the most frequently isolated species followed by B. fabicerciana, B. qingyuanensis, and B. cortices [5], which is consistent with the above nitrogen source utilization ratio of the four Botryosphaeria species. These results indicated a possible positive correlation between nitrogen utilization capability and fungal environmental fitness, and the broader spectrum of nitrogen source utilized by B. dothidea may be responsible for its competitive fitness among Botryosphaeria species. However, more researches should be performed to verify this hypothesis in the next study.
Osmotic stress has detrimental effects on microbial survival [22,34,35]. The PM 9 plate testing the phenotypes of the four Botryosphaeria species under various osmotic stresses found that B. dothidea could utilize up to 50 mM sodium benzoate (plate PM 9, wells G5 to G6), while the other three could only utilize up to 20 mM sodium benzoate ( Figure 3 and Table S1). Sodium benzoate has been reported to as useful agent with antimicrobial properties that able to inhibit the growth of many microbes, including Pseudomonas aeruginosa (Schroeter) Migula [36], Penicillium commune Thom [37], E. coli, Staphylococcus aureus Rosenbach, Saccharomyces cerevisiae Meyen ex E.C. Hansen [38], and Candida albicans (Robin) Berkhout [39], indicating the possibility that the greater tolerance toward sodium benzoate caused osmotic stress may account for the environmental fitness of B. dothidea. Additionally, the comparative genomics analysis between B. dothidea and the other three Botryosphaeria species found 143 B. dothidea species-specific genes, and further analysis of the above species-specific genes via the KEGG pathways found jg10977 has a predicated role in the calcium permeable stress-gated cation channel. This channel exists in eukaryotic cells from yeasts to animals and plants, and acts as a primary regulator of the initial responses to osmotic pressure [40,41]. Thus, investigating the potential contribution of jg10977 in the specific osmotic stress tolerance of B. dothidea mentioned above could be a valuable degree to further explore the mechanism involved in the wide prevalence and aggressiveness of B. dothidea.
High pH imposes severe stress on the fungal cell, including difficulties in the acquisition of nutrients or reduced availability of essential elements, such as iron or copper [42,43]. Appropriate responses to ambient pH govern fungal virulence during the infection [44]. For example, Sclerotinia sclerotiorum (Libert) de Bary can secrete organic acids acidifying the environment and ensure the proper deployment of cell wall-degrading enzymes (CWDEs) and other pathogenicity factors that function at an optimal pH for their activity during the infection [45]. Additionally, the dedicated Pal/Rim signaling pathway that functions in the response to alkaline pH was shown to be essential for infection in a number of fungal pathogens [42,46], such as C. albicans [47], Fusarium oxysporum Schlechtendahl [48], and Aspergillus fumigatus Fresenius [49]. In this study, PM 10 plate test assessing the tolerance of pH variation found that B. dothidea and B. qingyuanensis were more tolerant to strong alkali among four Botryosphaeria species (Figure 4 and Table S1), indicating a better ability to sense and respond to alkali environment. Significantly, the virulence test in our previous study found the B. dothidea was the most virulent, closely followed by B. qingyuanensis, then B. fabicerciana and B. cortices [5]. Consider the close relation between pH adaptation and fungal virulence [42,44,45], we speculated that the variety in pH regulation capability may be responsible for the virulence differentiation among four Botryosphaeria species.
In the past, the identification of the B. dothidea associated with Chinese hickory trunk cankers mainly relied on morphological identification [5]. In 2019, we developed a quantitative loop-mediated isothermal amplification (q-LAMP) technique to quantitatively monitor B. dothidea in environmental samples based on the different sites of the β-TUBLIN sequence between B. dothidea and other fungi commonly found on Chinese hickory [3]. In 2011, we used the combination of morphological identification and multi-locus phylogenetic analysis of ITS, β-tublin, and EF gene sequences for the accurate distinction of B. dothidea [5]. However, this technique is laborious, expensive, and requires time and knowledge of phylogenetic analysis for identifying the species. Alternatively, the identification of fungal pathogens through a conventional polymerase chain reaction (PCR) using species-specific primers provide an accurate, reproducible, and rapid identification of the species and has been widely used, particularly for economically important plant pathogens [50,51]. In this context, using a comparative genomics analysis, we identified 143 B. dothidea speciesspecific genes that are not conserved among species (Table S2), and provided a basis for the development of the B. dothidea molecular identification method. The screened primer set Bd_11F (5 -TCCAACGACGAGCAATCC-3 ) and Bd_11R (5 -TGTGCCCTGAGGCGGTAT-3 ) designed based on the sequence of B. dothidea species-specific gene jg11 was found to be used in the accurate identification of B. dothidea with efficiency and high efficiency ( Figure 5B), which is of significant importance for B. dothidea-associated cankers diagnosis, prevention, and control.
Collectively, using the large-scale screening of physiologic traits of four Botryosphaeria species associated with Chinese hickory cankers, we found B. dothidea has a broader spectrum of nitrogen sources and a greater tolerance toward osmotic pressure (sodium benzoate) and alkali stress among Botryosphaeria species. Moreover, the annotation of B. dothidea species-specific genomic information via a comparative genomics analysis not only gives a basis for the development of the B. dothidea molecular identification method but also provides crucial cues in the prediction of B. dothidea species-specific function that account for its high prevalence and wide distribution. However, the speculation of the mechanism involved in the wide prevalence and aggressiveness of B. dothidea in this study still require further study to verify.