First Report of Leaf and Stem Spot on Common Bean Caused by Stemphylium lycopersici in China

Abstract

Common bean (Phaseolus vulgaris L.) is one of the most economically important vegetable crops in China. In October 2018, leaf and stem spot symptoms were observed on P. vulgaris in Pozhuang County, Anqiu City, Shandong Province, China. The disease incidence ranged from 15 to 20%, causing large economic losses to growers. Lesions were initially small (1 to 2 mm in diameter), scattered, reddish-brown spots on leaves and stems. As the disease progressed, lesions expanded and merged, resulting in withering of the whole leaves and gradually drying of the whole plant. To isolate the pathogen, diseased tissue (5 × 5 mm) was cut from the margins of lesions and surface disinfected with 70% ethanol for 15 s followed by 0.1% mercuric dichloride for 2 min and then rinsed four to five times with sterile distilled water. The specimens were then transferred to potato dextrose agar plates and incubated at 28°C for 5 or 7 days. Nineteen fungal isolates with similar morphological characteristics were obtained. The colony of G9RS, a representative isolate, was white to brownish in obverse and deep red in reverse with a vivid red mycelial pigmentation that diffused into the culture medium. The conidia produced on potato carrot agar (PCA) as the standard medium were solitary, oblong, constricted at the main transverse septum, and measured 36.4 ± 7.3 × 16.5 ± 2.6 μm (n = 50). For molecular characterization, genomic DNA was extracted by CTAB (Niu et al. 2010), and sequences of the internal transcribed spacer (ITS) regions (White et al. 1990) and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes were amplified from isolates using the primer pairs ITS1/ITS4 and gpdf/gpdr (Câmara et al. 2002), respectively. The ITS and GPD sequences of isolate G9RS were submitted to GenBank (MN386223 and MN393479, respectively). BLASTn analysis of the sequences showed 99% identity with Stemphylium lycopersici (KX858848 and KR911809). Phylogenetic analysis (MEGA 7) using the neighbor-joining algorithm showed that the current isolate was clustered with S. lycopersici isolates (NO 425 and EGS46-001). Based on morphological and molecular characteristics, the isolate G9RS was identified as S. lycopersici. A pathogenicity test was conducted on intact plants according to Koch’s postulates. Conidia were collected from 21-day-old PCA cultures grown at 28°C under black light blue irradiation (365 nm) with a 12-h light/dark cycle, suspended in sterilized distilled water at 1.0 × 10⁵ conidia/ml, and the conidial suspension applied to ten 50-day-old bean plants (3 ml/plant). Ten plants inoculated with sterile distilled water served as controls. All samples were incubated in a growth chamber at 28°C. After 8 to 10 days, inoculated plants developed symptoms similar to original diseased plants in the field, whereas control plants remained symptomless. The same fungus was successfully reisolated from inoculated leaves and reidentified based on morphology and molecular evidence. The assay was conducted twice. Although S. lycopersici has been reported to cause leaf spot symptoms on other hosts, such as lettuce (Liu et al. 2019), potato (Yang et al. 2018), eggplant (Yang et al. 2017), Sansevieria (Kee et al. 2018), and pepino (Nasehi et al. 2016), to our knowledge, this is the first report of leaf and stem spot on common bean in China. Because the common bean is one of the most intensively planted crops in Shandong, this pathogen could have serious economic impact and may warrant development of integrated management strategies.