First Report of Cercospora Leaf Spot Caused by Cercospora beticola on Spinach in Pakistan

Abstract

Spinach (Spinacia oleracea L.) is one of the most important leafy vegetables, cultivated throughout Pakistan. During September to November 2016, five spinach (cultivar ‘Kandiari’) fields (31°36′17″N, 74°34′23″E) were inspected near Lahore city, Pakistan. Leaf spot symptoms on plants were observed in 10 to 30% of inspected fields. Symptoms began as small circular to subcircular light-brown spots with a distinct dark brown margin. In mature more or less sunken lesions, the center was whitish gray to black, owing to sporulation. Microscopic study of the whitish-gray lesions revealed the presence of small and black stromata within the leaf substomatal cavities. The stromata produced loose fascicles of conidiophores, up to 15 conidiophores in a fascicle. Conidiophores were simple, one to four septate, golden brown but subhyaline at the apex, straight, 4.5 to 5 × 20 to 100 µm (n = 50), geniculate, having distinctive spore scars, and unbranched. Conidia were solitary, hyaline, filiform, straight to slightly curved, with obtuse to subacute at the apex and subtruncate bases, multiseptate (one to 18), nonconstricted at the septa, 28 to 230 × 2.5 to 4 µm (n = 50). On the basis of morphological characteristics, the causal organism was identified as Cercospora beticola Sacc. (Chupp 1954; Crous and Braun 2003). To obtain single-spore isolates from samples, conidial suspension from one lesion per sample was inoculated on separate 2% water agar plates and incubated at 24°C. After 24 h, individual germinating conidia were planted on separate potato dextrose agar (PDA) plates and incubated at 24°C for 10 days. Pure cultures of six C. beticola isolates were used for DNA extraction. The partial internal transcribed spacer (ITS) regions of the fungal rDNA and calmodulin (CAL) gene were amplified using the primers ITS1/ITS4 and CAL228F/CAL737R and sequenced. Obtained DNA sequences of the six isolates were identical; therefore, ITS (MH318663) and CAL (MK333232) sequences of a representative isolate (IM084) were submitted to GenBank. The resulting ITS and CAL sequences of IM084 showed 99 to 100% similarity with C. beticola accessions (MH42448, MF681169, JX142819, AY752227, and FJ473438). A pathogenicity test was accomplished by placing PDA discs (0.5 mm²) from a 10-day-old representative fungal culture onto leaves of six 3-week-old potted spinach (cv. Kandiari) and sugar beet (cv. ‘9597’) (three plants of each). For noninoculated controls, three plants of each type were inoculated with noncolonized PDA plugs. After inoculation, plants were kept 48 h in a dew chamber at 25°C and then transferred to the greenhouse under 12/12-h light/dark conditions at 25°C. Lesions developed on fungal inoculated leaves of spinach and sugar beet plants with similar characteristic symptoms as those observed on naturally infected spinach leaves within 10 days after inoculation. Control spinach and sugar beet plants remained asymptomatic. Sporulation of C. beticola was observed in lesions similar to initial symptoms observed in field samples. The pathogen was reisolated from infected leaf lesions and confirmed as C. beticola. Previously, C. beticola causing Cercospora leaf spot has been reported on sugar beet (Khan et al. 2007); however, to our knowledge this is a new record of C. beticola on spinach in Pakistan. Spinach is one of the high demand leafy vegetables, and incidence of this new pathogen can be a significant threat for spinach production.