First Report of Phytophthora nicotianae Causing Stem Canker of Catalpa bungei (Chinese Catalpa) in China.

Abstract

Chinese catalpa, Catalpa bungei C.A. Mey is native to China and has been widely cultivated as an important tree species for timber and ornamental purposes (Tao et al. 2019). The properties and high durability of the wood can resist the damage caused by microorganisms and insects (Xiao Y et al. 2019). In September 2020, stem cankers were observed in 5-year-old and 3-year-old C. bungei in a pilot experiment field covering 16-hectare area in Shuyang city (Jiangsu province, China) and in a nursery in Binhai city (Jiangsu Province, China), respectively. The disease incidence in both locations was about 1% to 3%. The typical disease symptoms include small to large, dark-brown and irregular-sunken canker around and along the stem under 2 meters from the stem base. The phloem and xylem of the symptomatic stem were dark brown and the xylem had larger necrosis than the phloem. The cross section of the diseased stem was partially died. The symptomatic stem were collected in both locations for pathogen isolation. In total, seven purified isolates from the diseased samples were obtained using potato dextrose agar (PDA) following standard isolation protocol (Huang et al. 2019). In order to determine the pathogenicity, 3-year-old Chinese catalpa seedlings were artificially inoculated with each of the seven isolates in April 2021. After removing the bark of the stem by a sterilized punch (diameter 6mm), an agar plug (diameter 6mm) pre-colonized by the isolate was inoculated to the stem and the inoculation point was sealed with parafilm. The agar plug without pre-colonization was used as control. Six tree seedlings were inoculated for each isolate. Ten days after inoculation, only the treatment with isolate QS.1 showed obvious discoloration around the inoculation point. One month after inoculation, the canker around the inoculation point was formed (3.4 cm ± 1.0 cm) and spread to the xylem, similar to the symptoms observed in the field. Isolate QS.1 was re-isolated successfully from the inoculated stem based on morphological characters, confirming the Koch's postulates and QS.1 as the causal pathogen. The isolate QS.1 formed white colonies with abundant aerial mycelia on V8 juice agar and produced a large amount of persistent and papillary ovoid sporangia with size of 22 ~ 45μm (average 31μm) × 18 ~ 39μm (average 23μm) in 10% aqueous solution of V8. The spore was spherical with thick-wall and diameter of 24 ± 3.9μm. The morphology of QS.1 is similar to that of Phytophthora nicotianae. The genomic DNA of representative isolate QS.1 was extracted from mycelium by a modified CTAB method (Murray et al. 1980). The rDNA internal transcribed spacer (ITS) region, β-tubulin and EF1-α genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), BTub_F1/TUBUR1 (L. et al. 2004) and EF1A_for/EF1A_rev (Blair et al. 2008), respectively. The BLAST results of these sequences (Accession No. MZ646302, MZ672116, and MZ675589, respectively) showed 99%, 100% and 100% identity with sequences of P. nicotianae (Accession No. KJ494902, KY205750, and MH359041), respectively. Based on the morphological characteristics and DNA analysis, isolate QS.1 was identified as P. nicotianae. To our best knowledge, this is the first report of P. nicotianae causing stem canker on Chinese Catalpa. This disease may pose potential threat on Catalpa due to the increase in Catalpa planting for economic and ecological purposes in China.