First Report of Root Rot Caused by Calonectria montana on Sugar Beet in Heilongjiang Province, China.

Sugar beet (Beta vulgaris L.) is an important crop that has significant economic value in northern regions of China, especially in Heilongjiang Province. In October 2019, root rot was discovered on the sugar beet cultivar HDW09 in Hulan (126°64' E, 46°00' N), Heilongjiang Province, China. Typical symptoms included lesions on root tissues, which were initially small and dark brownish, then gradually turned into irregular shapes and black in color. As the disease progressed, the extent of necrosis penetrated from external layers into inner tissue. Root tissues suffered from severe decay, which resembles symptoms of several previously reported root rot diseases of sugar beet(Harveson 2006). To identify the pathogen, pieces of the transition zones (3-5mm) between asymptomatic and symptomatic tissues were surface sterilized for 15 seconds in 1% NaClO, rinsed twice with sterilized distilled water， plated on corn meal agar supplemented with penicillin G (50 mg/L), and incubated at 25 ± 2°C in the dark. Isolates belonging to a Calonectria sp. were recovered and purified using the hyphal tipping technique. Four isolates including A1, A5, A6, and A7 were used for morphological characterization and identification by DNA sequencing. The seven-day-old colonies on malt extract agar produced buff and wooly aerial mycelia. They were sienna to umber in color. Chlamydospores and microsclerotia were produced abundantly throughout the medium. For further identification, the isolates were cultured on potato dextrose agar (PDA) at room temperature(25°C) under near-ultraviolet light irradiation. Macroconidiophores comprised of a stipe, a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle. Stipe extension were septate, straight to flexuous, 61-117 μm long, 2-4 μm wide at the apical septum, terminating in a sphaeropedunculate vesicle, 5-9 μm diam. Conidiogenous apparatus were 31-177 μm long, and 16-110 μm wide(n=30). Primary branches of conidiogenous apparatus were aseptate or 1-septate, 16-51 × 3-7 µm; secondary branches aseptate, 6-31 × 2-7 µm; tertiary branches aseptate, 8-19 × 2-6 µm, each terminal branch producing 1-6 phialides. Conidia cylindrical were rounded at both ends, straight, 32-53 × 3-5 µm, (mean = 47 × 4 µm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime(n=100). Partial sequences of calmodulin (Carbone et al. 1999), histone H3, the translation elongation factor 1-alpha, and beta-tubulin 2 (Crous et al. 2004) genes of the four isolates were obtained and deposited into GenBank under accession numbers MW118652 to MW118667. BLAST results showed that the calmodulin, histone H3, the translation elongation factor 1-alpha and beta-tubulin 2 sequences of A1, A5, A6 and A7 were highly identical to the sequences of Ca. montana strain CERC 8957 MF527082.1 (CAL) (99-100%), CERC 8930 MF527061.1 (HIS) ( 98-99%), HSP4 MN356465.1 (EF1-alpha) (100%) and HSP4 MN356460.1 (tub2) (98-99%), respectively. A phylogenetic tree using the maximum likelihood algorithm and sequences of the four concatenated genes was reconstructed in RAxML and revealed that the four isolates clustered in the clade of Ca. montana. The pathogen was identified as Ca. montana based upon these morphological and molecular traits(Liu et al. 2017; Stepniewska et al. 2020). Ten eight-week-old sugar beet plants without root rot symptoms were selected for pathogenicity test. The roots near the ground were carefully cleaned with hands. One mycelial plug (5 mm in diameter) from a seven-day-old colony of isolate A1 were used to inoculate each sugar beet root. Ten plants inoculated with plugs of noncolonized PDA served as the control plants. Pathogenicity tests were repeated three times. Plants were incubated under greenhouse conditions at 25 ± 2°C and watered when the surface soil appeared dry. All inoculated plants showed symptoms that resembled those in the field after 30 days, while the control plants remained healthy. The symptomatic tissues were plated in corn meal agar for 7 days at 25°C. Ca. montana was reisolated from 100% of the inoculated tissues, and identification was confirmed by molecular sequencing, validating Koch's postulates. This is the first report of Ca. montana in China causing root rot on sugar beet. The study suggests its broader host range and wider geographical distribution than ever known and lays a basis for further monitoring and managing this important pathogen.