Kidney bean (Phaseolus vulgaris), also being known as common bean, dry bean or french bean, is one of the most precious and highly nutritious legume crop cultivated and consumed worldwide(Blair et al.,2012; Choudhary et al.,2018) , which is an important edible foods or one of the most economically important vegetable crops in China. It is widely grown in the Heilongjiang Province in China. In July of 2020, leaf spot symptoms were found on the old or new leaves of Kidney bean plants (Phaseolus vulgaris L.) in our experimental fields located in Zhaozhou County(N45°42 '20.16 ", E125°15' 58.63" ), Daqing City, Heilongjiang Province, China. This field had disease incidences of approximately 20%. The leaf spot is conducive to the onset at high temperature and humidity environment, and this disease spreads very quickly after rainy days, therefore it is potentially a large risk for the development of Kidney bean industry. In its early occurrence phase, the infected leaves showed yellowish halo on the leaves, in which the middle mesophyll lost green. Thereafter, the yellow halo turned brown, and the middle leaf tissue of the halo appeared brown, ultimately the whole leaves had many brown spots (Supplementary Figure S1). To isolate the pathogen, diseased tissue (5×5 mm) was excised from the margins of individual lesions from the leaves of diseased plants with typical symptoms, and was disinfected with 75% ethanol for 10s followed by 2% NaClO for 3 min and then washed five to eight times with sterile water. Afterwards, the samples were transferred to potato dextrose agar (PDA) plates and incubated. After 5 to 7 days of incubation at 25°C (Wei et al.,2018), the mycelia were dark green with white margins in obverse and dark in reverse. Conidiophores were light brown with 2 to 4 septa and obclavate, 17.5to 44.0 × 6.5 to 14.5μm, with a short beak, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa, light brown to olive-brown (Supplementary Figure S2). Based on morphological features and sporulation pattern, the pathogen was similar to characteristics described Alternaria alternate (Zhou et al,2014), being identified as A. alternata. To confirm pathogenicity, the isolates were cultured on PCA for 7days to prepare conidial suspensions, then being produced a final concentration of 1×108 spores/ml. Five potted Kidney bean plants were sprayed with conidial suspensions, and five control potted plants were sprayed with sterile distilled water, in which these potted Kidney bean plants were treated after wiping each leaf surface with 75% ethanol and washing each leaf with sterilized distilled water five times. These plants were incubated in an artificial growth chamber at 26 to 28°C with a 12 h light/dark photoperiod, with 85% relative humidity. After 3 days, yellowish halo lesions appeared on the inoculated plants, and pale lesions with distinct dark brownish red borders on Kidney bean leaves were observed after eight days, but no lesions were observed on the control leaves. Pathogenicity tests were repeated three times. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced with primers ITS1/ITS4. BLAST analysis of the sequences showed 100% sequence identity with a pathogenic A. alternata (Fr.) Keissl (Supplementary Figure S3), and the nucleotide sequence of the ITS region was submitted to GenBank under accession MZ951052. In China, there are no detailed records about the causal agent of this disease on Kidney bean in a paper in Chinese. To our knowledge, this is the first confirmed report of leaf spot causing by A. alternata on Kidney bean in China.
Occurrence and Host Range of Ascochyta Phaseolorum in Queensland
