Basal Stem Rot of Broccoli Caused by Rhizoctonia solani AG-2-1 and AG-4 in Korea Basal Stem Rot of Broccoli Caused by

Basal stem rot symptoms were frequently observed on broccoli plants grown in farmers’ fields located in Gimje and Jeju, Korea during disease surveys carried out in November and December 2019. Incidence of the diseased plants was 1-10% in the fields investigated. Five isolates of Rhizoctonia sp. were obtained from the diseased plants. Of the isolates, two were identified as Rhizoctonia solani AG-2-1 based on the morphological characteristics and anastomosis test, and three were as R. solani AG-4. Two isolates each of R. solani AG-2-1 and AG-4 were tested for pathogenicity on broccoli by artificial inoculation. All the tested isolates induced basal stem rot symptoms on the inoculated plants. The symptoms were similar to those observed in the fields investigated. This is the first report of basal stem rot of broccoli caused by AG-2-1 and AG-4 in Korea.

. Of the Rhizoctonia spp., Rhizoctonia solani Kühn has the most strains and hosts compared to other species. Many anastomosis groups are reported to exist in R. solani and known to have different genetic and pathological characteristics [2]. Anastmosis groups of R. solani isolates are identified by hyphal fusion reaction with tester isolates.
In this study, identification of Rhizoctonia sp. isolates from diseased broccoli plants was carried out based on the morphological characteristics and anastomosis test. In addition, pathogenicity of the isolates was investigated by inoculation test.

Field Survey and Collection of Samples
Broccoli fields were surveyed at locations Gimje and Jeju in Korea in November and December 2019. Three sites were observed in the field, and 100 plants at each site were investigated for disease incidence. The diseased broccoli plants were collected to isolate causal fungus.

Isolation
Nine to 15mm 2 lesion pieces cut from the diseased plant parts were plated on 2% water agar (WA) after surface-sterilizing with 1% sodium hypochlorite solution for one minute. Fungi were isolated from the lesion pieces after one to two days of incubation at 25 ℃. The fungal isolates were transferred to potato dextrose agar (PDA) slants and used for identification and pathogenicity test.

Investigation of Morphology and Anastomosis Test
Rhizoctonia sp. isolates obtained from the diseased plants were cultured on PDA in 9cm-diameter petri dishes at 25 ℃ for 10 days and examined for the morphological characteristics. The Rhizoctonia sp. isolates were tested to classify anastomosis groups using tester isolates of R. solani as previously described [3]. The R.

Pathogenicity Test
Broccoli seeds were sown in circular plastic pots (height, 9cm; 100% relative humidity at room temperature (24-26 ℃) for two days. Then, the inoculated plant pots were removed from the boxes and kept indoors. The virulence of the isolates was rated based on the degree of basal stem rot symptoms five days after inoculation.
The inoculation test was performed in three replicates.

Symptoms and Disease Incidence
Basal stem rot symptoms were frequently observed on broccoli plants grown in the fields investigated. The symptoms began on the plant parts at the soil surface. The diseased plants wilted and blighted in the early stage of the plant growth ( Figures 1A & 1B). The basal stem of the infected plant receded and was thin, discolored, and rotten ( Figure 1C). The diseased plants were retarded in their growth and fell down in the medium stage of the plant growth ( Figure 1D). The basal stems of the diseased plants turned dark brown and rotted ( Figure 1E). The incidence of diseased plants in the fields investigated at two locations in Korea during the disease surveys was 1-10% (Table 1).

Isolation and Identification
Five isolates of Rhizoctonia sp. were obtained from basal stems of the diseased plants. All the isolates were identified as R. solani based on the morphological characteristics as per descriptions from previous studies [1,2]. Of five isolates of R. solani, two were classified as R. solani AG-2-1 by anastomosis test, and three as R.
solani . Anastomosis reactions between the tested isolates and the tester isolates of R. solani AG-2-1 and AG-4 observed by light microscope are shown in Figures 2A & 2B. The colony of R. solani AG-2-1 cultured on PDA showed brown concentric zones ( Figure   2C). The colony of R. solani AG-4 cultured on PDA were whitish light brown, and sclerotia were not produced on the medium ( Figure   2D).  in many crops [4][5][6]. It has been reported that R. solani AG-2-1 and AG-2-2 Ⅵ cause damping-off and foot rot of broccoli in Japan, respectively [7,8]. However, there has been no report of basal stem rot on broccoli caused by R. solani AG-2-1 and AG-4. This is the first report of basal stem rot of broccoli caused by AG-2-1 and AG-4 in Korea.
Anastmosis groups AG-1 through 13 of R. solani have been reported, and some anastomosis groups of R. solani are further divided into subgroups by cultural type, pathogenicity, DNA-DNA hybridization, rDNA-ITS sequence analysis, etc. [9]. It is reported that R. solani AG-4 is divided into subgroups HG-Ⅰ, HG-Ⅱ, and HG-Ⅲ by rDNA-ITS sequence analysis [10] and analysis of fatty acids [11]. The authors identified R. solani AG-4 causing basal stem rot of broccoli based on the morphological characteristics and anastomosis test. It is considered that subgroups of R. solani AG-4 isolates identified in this study could be classified by the aforementioned methods via further study.