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