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First Report of Colletotrichum lupini Causing Anthracnose on Lupin in China

Zou, M. F., Yan, M. F., Liu, B., Wang, Y. X., Xiong, G. H., Jiang, J. X.
Plant disease 2019 v.103 no.4 pp. 767
Colletotrichum, DNA, DNA primers, Lupinus polyphyllus, agricultural colleges, anthracnose, conidia, culture media, death, ethanol, fungi, genes, green manures, growth chambers, hyphae, internal transcribed spacers, mercuric chloride, pathogenicity, pathogens, petioles, polymerase chain reaction, relative humidity, sequence analysis, stems, wilting, Australia, China, Europe, North America, South America
Lupin (Lupinus polyphyllus Lindl. in Fabaceae) is a perennial herb that was recently introduced to China, mainly as an ornamental and green manure crop. In April 2018, anthracnose symptoms were observed with approximately 70% incidence (percentage of plants affected) in the lupin cultivation base of Jiangxi Agricultural University in Nanchang, Jiangxi Province, China. Diseased plants exhibited twisting stems and petioles with elliptical, dark brown, and sunken lesions, which were often completely girdled by these lesions, resulting in wilting and death of the leaves. Symptoms on leaf blades appeared as circular, brown lesions with dark brown borders. To isolate the pathogen, small pieces cut from margins of the lesions were surface sterilized in 70% ethanol for 10 s followed by 0.1% HgCl₂ for 1 min. After being washed three times with aseptic water, the pieces were placed onto acidified potato dextrose agar plates and incubated at 28°C. Pure cultures were obtained by subculturing hyphal tips. Colonies were grayish white to pale gray and grew at an average rate of 8.2 mm/day. After 15 days of incubation, orange-colored masses of conidia were observed at the center of the colony. Conidia were single-celled, colorless, oval, with one rounded end and one pointed end, and measured 10.5 to 16 × 3 to 6 μm. These morphological characteristics were similar to those described for Colletotrichum lupini (Nirenberg et al. 2002). Genomic DNA of the representative isolate (YSD2) was extracted with the Ezup Column Fungi Genomic DNA Purification Kit (Shanghai Sangon Biotech Co.) according to the manufacturer’s protocol, and the ITS-rDNA region and RPB2 gene were amplified with the primers ITS1/ITS4 and rRPB2-5F/rRPB2-414R (Quaedvlieg et al. 2011; White et al. 1990), respectively. PCR products were sequenced, and the sequences were deposited in GenBank with accession numbers MH532966 for ITS-rDNA and MH532968 for RPB2 gene. BLAST analysis showed 99% homology with the ITS-rDNA sequence of C. lupini (AJ301968) and 100% homology with the RPB2 gene sequence of C. lupini (JN985517). To test pathogenicity, five healthy lupin plants were sprayed with conidial suspension (1 × 10⁵ conidia/ml) of isolate YSD2, and the same number of healthy lupin plants were sprayed with sterilized water as a control. All inoculated and control plants were incubated in a growth chamber at 28°C and 95% relative humidity with a 12-h photoperiod. After 25 days, all inoculated plants developed symptoms similar to those observed in the field, whereas the control plants remained symptomless. The original isolate was successfully reisolated from all inoculated plants and confirmed by ITS and RPB2 gene analysis, fulfilling Koch’s postulates. Lupin anthracnose is known to occur in Europe, North America, South America, and Australia, and it seriously affects the production and quality of lupin (Talhinhas et al. 2016). As far as we know, this is the first report of C. lupini causing anthracnose of lupin in China.