First Report of Colletotrichum truncatum Causing Anthracnose of Mouse Garlic (Allium angulosum) in India

Abstract

Mouse garlic (Allium angulosum L.) is economically important for its use as a spice, vegetable, and ornamental plant. During the monsoon (June to September) of 2015 and 2016, A. angulosum plants with symptoms of anthracnose were observed in the wild allium repository at ICAR-Directorate of Onion and Garlic Research, Rajgurunagar (18°84ʹ31″N, 73°88ʹ50″E), Pune, India. Disease severity was up to 30 to 45% during the two consecutive years. Initially, tips of older leaves showed prominent, white, sunken spots that converted to light brown necrotic lesions causing die-back of the whole leaf. On necrotic lesions, small, black fruiting bodies (acervuli) developed in concentric rings. As the disease progressed, plants dried completely. Three isolates (AAC-1, AAC-2, AAC-3) were collected from infected leaves of different plants. For isolation, infected leaves were cut into small pieces (5 to 10 mm²) and immersed in 1% NaClO solution for 2 min, rinsed three times in sterilized distilled water, and dried on sterilized filter paper. Infected pieces were then plated on oatmeal agar (OMA) for 4 days at 25 ± 2°C with a 12-h photoperiod. Purified isolates showing colony growth changed from grayish to dark gray with salmon color conidial masses at the center. After 7 days incubation on OMA, colonies from single spores had an average colony diameter of 59.2 mm. Microscopically, conidia were hyaline, single-celled, slightly curved with tapered tips and truncate bases, measuring (n = 20) 18.6 to 23.3 × 1.9 to 2.6 μm, originated from an acervular conidiomata delimited with dark brown, septate, straight, pointed setae measuring 72.5 to 126.2 × 3.1 to 5.8 μm. Appressoria formed in culture were light brown, single or in groups, lobate or round, and ranged from 7.5 to 20.5 × 3.9 to 7.3 μm. Morphological characteristics were consistent with Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore (Damm et al. 2009). For molecular characterization of three isolates, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and a partial sequence of the actin (ACT) gene were amplified with the primer pairs ITS1/ITS4, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (Damm et al. 2009). The ITS sequence (KY611807) and GAPDH gene (MF282477) of these isolates shared 99% identity with C. truncatum from soybean (JQ9336249) and Capsicum annum (KY435779), respectively. Similarly, the ACT gene (MF582476) shared 98% identity with C. truncatum from Cynanchum paniculatum (KF488581). Koch’s postulates of all the three isolates were completed on 45-day-old seedlings. Five seedlings were sprayed with conidia suspension (10⁶ conidia/ml) prepared from 15-day-old cultures. Control plants were sprayed with sterilized water. Plants were kept separately in a growth chamber at 25°C, 85% relative humidity, and 12-h photoperiod. Anthracnose (light brown necrotic patches) developed 5 days after inoculation; control plants remained symptomless. The fungus was successfully reisolated from symptomatic leaves and confirmed by morphological characters and molecular identification. The same experiment was repeated the following year. Results obtained on morphological characters, molecular data, and pathogenicity were matched with C. truncatum. Earlier, a severe outbreak of onion anthracnose due to C. truncatum and C. siamense was reported from southwest India (Chowdappa et al. 2015). Similarly, C. truncatum and C. theobromicola causing anthracnose of Welsh onion (A. fistulosum) was documented from Brazil (Matos et al. 2017). Identification of the cause of mouse garlic anthracnose now allows appropriate management measures. To our knowledge, this is the first report of C. truncatum causing anthracnose on A. angulosum in the world.