2.1. Disease Survey Site, Sample Collection and Fungal Isolation

The disease survey was conducted in a one-year-old Eucalyptus breeding experimental plantation in the BeiHai region, GuangXi Province, southern China (21°33′19.8756″ N, 109°42′27.0792″ E) in October, 2018. Ten Eucalyptus genotypes were planted in the experimental plantation. These included six Eucalyptus urophylla × E. grandis hybrid genotypes (CEPT1860–CEPT1865) and four E. urophylla × E. tereticornis hybrid genotypes (CEPT1866–CEPT1869). All ten Eucalyptus genotypes were naturally infected by Calonectria species (Figure 1).

Disease symptoms on multiple Eucalyptus genotypes in one experimental plantation caused by species of Calonectria. (AC): Leaf spot in three E. urophylla × E. grandis hybrid genotypes CEPT1863 (A), CEPT1861 (B), and CEPT1862 (C), the infected leaves of CEPT1861 and CEPT1862 became blighted and dried; (DH): Leaf spot and blight in three E. urophylla × E. tereticornis hybrid genotypes, CEPT1866 (D,E), CEPT1868 (F), and CEPT1869 (G,H).

Diseased leaves with typical symptoms caused by Calonectria species were collected from 13 to 20 trees for each of the ten Eucalyptus hybrid genotypes, depending on the planted areas of each genotype. Soil samples under each sampled diseased tree were also collected. These samples of diseased leaves and soils were transported to the laboratory for isolation, morphological examination, and further molecular research.

To induce Calonectria sporulation, diseased leaves were placed in moist dishes (diameter 70 mm, height 16 mm; tissue paper moistened with sterile water) at room temperature and incubated for 1–3 days. Soil samples were baited with Medicago sativa (alfalfa) germinating seeds using the method described by Crous [23]. Fungal isolates with typical morphological characteristics of Calonectria were isolated from diseased leaves and soil samples. The conidia masses were transferred to 2% (v/v) malt extract agar (MEA) (20 g malt extract powder and 20 g agar powder per liter of water: malt extract powder was obtained from the Beijing Shuangxuan microbial culture medium products factory, Beijing, China; the agar powder was obtained from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) with a sterile needles under stereoscopic microscope and incubated for 3–5 days. To obtain pure cultures, a single hyphal tip from each culture was transferred to 2% MEA plates and incubated at room temperature for 7–10 days. The pure cultures were deposited in the culture collection (CSF) at the China Eucalypt Research Centre (CERC) of the Chinese Academy of Forestry (CAF) in ZhanJiang, GuangDong Province, China.

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