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First Report of Nectria haematococca as the Causal Agent of Canker Rot in Phellodendron amurense in China

Li, S. J., Cui, Y. T., Fang, X. M., He, Q. Q., Zhu, T. H., Qiao, T. M., Han, S.
Plant disease 2018 v.102 no.5 pp. 1033
Citrus, DNA, Fusarium eumartii, Fusarium solani, Phellodendron amurense, alcohols, anamorphs, asci, ascospores, axenic culture, bark, conidia, cortex, culture media, cutting, dead wood, economic valuation, epidermis (plant), fruit products, fungi, genes, internal transcribed spacers, leaves, medicinal plants, medicinal properties, microscopy, mycelium, peptide elongation factors, perithecia, polymerase chain reaction, seedlings, shrubs, sodium hypochlorite, stems, translation (genetics), tree diseases, trees, vegetables, China
Phellodendron amurense Rupr., a rare traditional Chinese medicinal plant, is native to East Asia and is widely cultivated in the northeastern and southwestern parts of China. This tree has considerable pharmacological properties and significant economic value. Since June 2016, a large number of P. amurense trees in Sichuan Province have shown canker symptoms. Approximately 40% of the trees in the area have been seriously damaged. The symptoms and signs of the disease include dimming of the cortex, decay, tissue softening, yellowing and shedding of leaves, dead branches, and orange spherical perithecia on the stems. Many of the trees were dying soon after the apparition of the symptoms. The stems of infected trees were systematically sampled by cutting the epidermis at the junction of infected and healthy area in 4 × 4 mm sections. The samples were surface-sterilized with 3% NaClO and 75% alcohol, each for 30 s before being rinsed three times with sterilized distilled water and then cultured on potato dextrose agar (PDA) (25°C for 3 to 8 days). The colonies were white and grew up to 48 to 76 mm in diameter after 5 days. The mycelia were flocculent, appressed to the culture medium, and produced microconidia, which were ovoid with 0 to 1 septum and 6.3 to 21.2 × 2.7 to 4.9 µm in size. Macroconidia were cylindrical to falcate with 3 to 7 septa and 21.2 to 52.7 × 3.7 to 5.4 µm in size. These conidia were similar to Fusarium solani (Mart.) Sacc. (Barnes 1979; Leslie and Summerell 2006). Fifteen days later, the colonies changed from white to yellowish and then became reddish, and either ceased to grow or grew slowly. Microscopic examination showed many orange spheroid to oblate perithecia on the bark surface, 205 to 250 µm in diameter, containing asci that were clavate and 66.7 to 78.5 × 7.1 to 9.3 µm in size. Each ascus contained eight oval to obovate ascospores, 10.7 to 15.8 × 4.2 to 6.2 µm. The perithecia were quite similar to those described for Nectria haematococca Berk. & Broome (Gai et al. 2012; anamorph F. solani). PCR was performed using DNA extracted from the fungal colony using universal primers (ITS1/ITS4) and then sequenced. Translation elongation factor 1-alpha genes of the fungus were also amplified with EF1T/EF2T primers (Gai et al. 2012). BLAST results in GenBank indicated that the ITS sequences (accession no. MG544175) had 99% similarity to N. haematococca (JN088237) and the EF1-α sequences (MG017494) had 98% similarity to F. solani (JF740841). To complete Koch’s postulates, 10 1-year-old potted P. amurense seedlings were inoculated by scratching the stem epidermis with a piece of sandpaper and applying 50 µl of a microconidial suspension (1 × 10⁶ spores/ml) to the wound with a brush. Axenic culture medium (10 seedlings) and sterilized distilled water (10 seedlings) were used as controls. Fifteen days later, inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The fungus was reisolated from symptomatic stems and showed the same morphological characteristics to the isolates used for inoculating the plants, thus confirming that N. haematococca caused the canker rot of P. amurense. Although N. haematococca usually affects fruits and vegetables, it is also occasionally found in shrubs or trees such as citrus (Yago and Chung 2011), and this is the first report of this fungus causing canker rot in P. amurense trees in China.