First Report of Stem Rot of Sunflower Broomrape (Orobanche cumana) Caused by Sclerotinia minor Jagger in Inner Mongolia, China

Abstract

Broomrape (Orobanche cumana Wallr.) is a parasitic seed plant that has become a serious threat to the sunflower production in China. In August 2016, diseased samples of damp rot at basal stem of sunflower broomrape were collected in sunflower fields of Gonghecheng and Wulatai, Inner Mongolia, China. The average disease incidence was approximately 30%. Tiny, black, round sclerotia (diameter ranged from 0.30 to 0.99 mm) were observed on the infected stems of broomrapes, but they were much smaller than sclerotia produced by Sclerotinia sclerotiorum. Among 22 samples, five diseased samples were chosen randomly for pathogen isolation and identification. Tiny sclerotia were surface sterilized in 75% alcohol for 10 s, followed by 0.1% NaClO for 3 min, and washed three times with sterile water. The sclerotia were dried on the sterilized filter paper and then placed on potato dextrose agar (PDA) and incubated at 24°C in dark for 3 days. The emerging mycelia were hyphal-tipped three times and subcultured on a PDA plate. Plenty of tiny, dark sclerotia formed after 6 days of culture. The morphological characteristics of sclerotia is similar to those of S. minor Jagger (Kohn 1979). To confirm the fungal species, total genomic DNA was extracted. The internal transcribed spacer (ITS) of nuclear rDNA was amplified using the primer pair ITS1/ITS4 (White et al. 1990). PCR products were submitted for sequencing and aligned in GenBank. All five identified isolates had 100% identity with those of S. minor isolated from Doellingeria scabra (accession no. KY707828.1). One of the ITS sequences named as SZW-1 was deposited in GenBank and assigned with accession number MF471348. To demonstrate the pathogenicity of isolates, seeds of broomrape (10 mg) collected from Siziwangqi, Wulanchabu city, were mixed with 100 g of potting mix (2:1 field soil/vermiculite) in a 10 × 10 cm plastic pot for planting sunflower. Sixty seeds of sunflower (variety LD5009, Kaifurui Seeds Company) were planted in 12 pots, with five seeds in each pot. All pots were kept in a greenhouse at 20 to 25°C and 40% relative humidity for broomrape parasite. Forty-five days later (around the R1 stage of sunflower), the stem of broomrape above the soil line (around 8 to 10 cm in height) was used for pathogen inoculation. A 9-mm-diameter PDA plug was cut from the margin of mycelial culture and placed at the basal stem of broomrapes (approximately 3 to 5 cm above the soil line). Six broomrape stems were inoculated for each isolate, and three stems were inoculated with plain PDA plugs as a control. All PDA plugs were wrapped with Parafilm to maintain moisture. The initiation of stem rot of broomrape was observed at 2 days postinoculation (dpi); gradually the rotted area expanded on the stem longitudinally, and white mycelia and tiny black sclerotia were observed 5 dpi; and the stems collapsed at 7 dpi. No symptoms were observed on the control broomrapes. S. minor was recovered from all inoculated plants with tiny sclerotia but not from the control plants. Although S. minor causing white mold in sunflower has been reported in Australia, Bulgaria, Canada, Chile, Kenya, New Zealand, Spain, the United States, and China (Farr and Rossman 2014; Li et al. 2016), and sclerotinia rot caused by S. sclerotiorum on broomrape was also reported in Xinjiang region of China (Ding et al. 2012), to our knowledge, this is the first report of S. minor-caused stem rot on sunflower broomrape in China. This finding may expand our knowledge on both S. minor and O. cumana.