| Summary: | Many <i>Calonectria</i> species are causal agents of diseases on several forestry, agricultural and horticultural crops. <i>Calonectria</i> leaf blight is one of the most important diseases associated with <i>Eucalyptus</i> plantations and nurseries in Asia and South America. Recently, symptoms of leaf rot and leaf blight caused by <i>Calonectria</i> species were observed in a one-year-old <i>Eucalyptus</i> experimental plantation in GuangXi Province, southern China. To better understand the species diversity, mating strategy and pathogenicity of <i>Calonectria</i> species isolated from diseased tissues and soils, diseased leaves and soils under the trees from ten <i>Eucalyptus urophylla</i> hybrid genotypes were collected. Three hundred and sixty-eight <i>Calonectria</i> isolates were obtained from diseased <i>Eucalyptus</i> leaves and soils under these trees, and 245 representative isolates were selected based on the sampling substrates and <i>Eucalyptus</i> genotypes and identified by DNA sequence analyses based on the translation elongation factor 1-alpha (<i>tef1</i>), β-tubulin (<i>tub2</i>), calmodulin (<i>cmdA</i>) and histone H3 (<i>his3</i>) gene regions, as well as a combination of morphological characteristics. These isolates were identified as <i>Calonectria hongkongensis</i> (50.2%), <i>C. pseudoreteaudii</i> (47.4%), <i>C. aconidialis</i> (1.6%), <i>C. reteaudii</i> (0.4%) and <i>C. auriculiformis</i> (0.4%). This is the first report of <i>C. reteaudii</i> and <i>C. auriculiformis</i> occurrence in China. <i>Calonectria pseudoreteaudii</i> was isolated from both <i>Eucalyptus</i> diseased leaves and soils; the other four species were only obtained from soils. <i>MAT1-1-1</i> and <i>MAT1-2-1</i> gene amplification and mating type assignment results showed that <i>C. pseudoreteaudii</i> is heterothallic and an asexual cycle represents the primary reproductive mode, <i>C. reteaudii</i> and <i>C. auriculiformis</i> are likely to be heterothallic and <i>C. hongkongensis</i> and <i>C. aconidialis</i> are homothallic. Based on the genetic diversity comparisons for <i>C. pseudoreteaudii</i> isolates from diseased leaves and soils, we hypothesize that <i>C. pseudoreteaudii</i> in soils was spread from diseased leaves. Both the mycelia plug and conidia suspension inoculations indicated that all five <i>Calonectria</i> species were pathogenic to the two <i>Eucalyptus</i> genotypes tested and the tolerance of the two genotypes differed. It is necessary to understand the ecological niche and epidemiological characteristics of these <i>Calonectria</i> species and to select disease resistant <i>Eucalyptus</i> genotypes in southern China in the future.
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