Materials and Methods
Disease rating & resistance response
Perilla (Perilla frutescens L.), originally from Southeast Asia, is an annual herbaceous plant under the family Lamiaceae, and the second-biggest upland crop in Korea. Nowadays, it is widely distributed in many regions of the world (Lee and Ohnishi, 2003). In 2017, perilla seed production was 50,738 metric tons with cultivation area covering approximately 43,352 ha (KOSIS, 2018). It has been used as an antioxidant, and a traditional herbal medicine for treating various diseases such as, anxiety, depression, cough, mild seasonal allergic rhino conjunctivities, intoxication, tumor, and some intestinal disorders (Assefa et al., 2018; Makino et al., 2003; Yang et al., 2012). In Korea, perilla used as oil, as an aromatic vegetable, in sushi, salads, pickles and mainly consumed with meat. Hence, the perilla cultivar has been developed with regard to either extracting oil or harvesting fresh leaves. Perilla oil is one of the richest omega-3 fatty acid sources among the edible seed oils (Asif, 2011; Eckert et al., 2010). Linolenic acid occupies 63.1% of the overall fatty acid in perilla oil. Numerous functional components contained in perilla have been reported to show anti-inflammatory response, inhibition for α-glucosidase, and antioxidative activity (Banno et al., 2014; Ha et al., 2012; James et al., 2000). Furthermore, α- linolenic acid, the main fatty acid in perilla seed, has been reported by various studies to reduce the risk of cardiovascular diseases and affect the change of brain lipid composition and recognition ability (Guixiang et al., 2004; Kim et al., 2010; Lee et al., 2017; Zhang et al., 2014).
In Korea, various diseases such as leaf spot, gray mold, anthracnose, Sclerotinia rot, rust, downy mildew, stem blight and Phytophthora blight have been found in perilla (Cho and Moon, 1994; Choi et al., 2009; Kim et al., 2001; Lee et al., 2009; Maeng et al., 2009; Moon et al., 1998; The Korean Society of Plant Pathology, 2009; Yun et al., 2007). Among them, Sclerotinia rot is one of the most devastating fungal diseases that decrease the yield of perilla significantly. Sclerotinia rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is a ubiquitous necrotrophic fungal pathogen capable of infecting about 408 plant species among 75 families (Boland and Hall, 1994). The pathogen is recognized by the fluffy white mycelium and black sclerotia that develops on the surface of lesions (Bolton et al., 2006).
Various authors have reported various inoculation techniques for screening of Sclerotinia rot in different crops such as pea, soybean, oilseed rape, dry bean and also perilla, by using cut stem, detached leaf methods, cut petiole inoculation, spray- mycelium & drop-mycelium, cotyledon inoculation, soil drenching (Afroz et al., 2019; Chen and Wang 2005; Grau and Bissonette 1974; Kull, 2003; Miorini et al., 2018; Del Rio et al., 2001; Vuong et al., 2004). Based on our previous study, the detached leaf method is a simple and rapid disease inoculation method for screening of Sclerotinia rot in perilla (Afroz et al., 2019).
Plant genetic resources (PGR) represent a wealth of genetic diversity, part of which is of potential value for breeding better crop plants (Frankel, 1977). For instance, landraces or crop wild relatives, modern cultivars, breeding lines and close or distant relatives may bear valuable genes for disease resistance, yield-improving properties, or quality- related traits. Therefore, the success of breeding programs for disease resistance always depends on the availability of wide range of genetic resources. To understand the genetic basis of quantitative resistance, various researches have carried out for quantitative trait locus (QTL) mapping using monogenic and/or polygenic depending on the plant species, derived normally from crosses between a partially resistant parent and a susceptible parent (Wu et al., 2016; Yin et al., 2010; Zhao and Meng, 2003; Zhao et al., 2006). Quantitative trait loci (QTL) mapping technique for polygenic resistance is used to identify loci related to S. sclerotiorum resistance in different crop species like soybean, common bean, sunflower and B. napus, but there is no published data in its application in perilla so far (Chen, 2007; Godoy et al., 2005; Hartman et al., 2000; Kim and Diers, 2000). The objective of the present study was to determine the differential responses of perilla germplasm in Korea, by detached leaf method to identify the source of resistance and to evaluate the expression and relationship of resistances to Sclerotinia rot.
Materials and Methods
Five hundred and forty four Korean origin perilla accessions (including 400 landraces, 29 cultivars, 24 breeding lines, and 1 relative wild type, and 90 unknown) were obtained from National Institute of Crop Science, and Jeonju, Republic of Korea, to identify the resistance of those perilla germplasms against Sclerotinia rot caused by Sclerotinia sclerotiorum. This experiment was conducted in a greenhouse and growth chamber of the National Agrobiodiversity Center (NAC), National Institute of Agricultural Science, and Jeonju, Republic of Korea. Experiments were done at the seedling stage when plant had five to six leaves. The characteristics of perilla accessions information were provided by Germplasm Management System of National Agrobiodiversity Center, National Institute of Agricultural Science, RDA, and Jeonju, Republic of Korea.
Sclerotinia sclerotiorum isolate (KACC40457) was obtained from the Korean Agricultural Culture Collection (KACC) and confirmed the pathogenicity to perilla leaves using detached leaf method. S. sclerotiorum was sub-cultured and maintained at 25℃ on Potato Dextrose Agar (PDA) in culture room.
Detached leaf method was used for this experiment. A single mycelial agar plug (size 7 ㎟ after growing at 25℃ for 7 days) was cut from the margin of PDA with actively growing mycelial colony and was placed mycelial-side down at 1/3 point on the main leaf vein of detached leaf for S. sclerotiorum; at front side. For the sake of getting in touch with the leaf surface, the plug was slightly compressed. The leaves were inoculated and kept in a plastic box with moistened paper towel to maintain humidity and incubated at 25℃ in dark condition. Three replications, each consisting of fifteen leaves, were used in this experiment.
Disease rating & resistance response
Necrotic and water soaked lesions appeared after two days of inoculation. At 7 days after inoculation, lesions diameters were measured using a linear ruler. The resistance response was measured according to Naher et al., 2018. In addition, the resistance levels were represented based on resistance ratio (%) based on the following categories: >90% = highly resistance (HR); 80.0 to 90% = resistance (R); 70.0 to 79.9% = moderately resistance (MR); 50.0 to 69.9% = moderately susceptible (MS); 30.0 to 49.9% = susceptible (S); <30% = highly susceptible (HS). Resistance ratio (%) was calculated by the following formula.
Resistance ratio (%) = (No. of leaves showed below 1 ㎝ of lesion size/ No. of total evaluated leaves) × 100
The results of resistance ratio (%) and resistant response of perilla accessions were as presented in Table 1, 2 and Fig. 1. Among the 544 accessions, two were highly resistant, five were resistant, five were moderately resistant, 16 were moderately susceptible, 31 were susceptible, and 485 were highly susceptible against S. sclerotiorum. Out of 400 landrace perilla accessions, only two were moderately resistant (IT 220624, IT178655) with a resistance ratio of 70.0%, eight were moderately susceptible, 15 were susceptible, and 375 were highly susceptible. All 24 breeding lines were highly sensitive. Out of 29 cultivars, two were susceptible, and 27 were highly susceptible. Also, one relative perilla accession was susceptible. Out of 90 unknown accessions, two were highly resistant (IT226504, IT226533) with a resistance ratio of 100%, five were resistant (IT226561, IT226532, IT226526, IT226441 and IT226589) with a resistance ratio of 80.0 to 86.7%, five were moderately resistant (IT226525, IT226640, IT226568, IT220624 and IT178655) with a resistance ratio of 70.0 to 76.9%, eight were moderately susceptible, 13 were susceptible, and 59 were highly susceptible.
Table 1. Resistant response of perilla accessions against Sclerotinia rot caused by Sclerotinia sclerotiorum
|Type of Perilla accessions||Number of. accessions||Resistant responsez|
Table 2. Perilla accessions that showed resistance ratio (%) & resistant response against Sclerotinia sclerotiorum
y(No. of plants showed below 1㎝ of lesion size/No. of total evaluated plants) × 100.
xHR=highly resistance (>90% of resistance ratio); R=resistance (80.0 to 90% of resistance ratio); MR=moderately resistance (70.0 to 79.9% of resistance ratio); MS=moderately susceptible (50.0 to 69.9% of resistance ratio), S=susceptible (30.0 to 49.9% resistance ratio); HS=highly susceptible (<30%=resistance ratio).
The Morphological characteristics of seven accessions, which showed high resistance to sclerotinia rot, were as presented in Table 3. All accessions were planted on 30th May, 2017 and flowering occurred on 04 September, 2017 and all were green adaxial leaf color (except IT226526- pale green). Trichom density of all accessions was medium and leaf shapes were cordate. Four accessions (IT226533, IT226532, IT226526, IT226541) produced purplish green abaxial leaf color, whereas, IT226504, IT226561, and IT226589 produced green, purple, and pale green abaxial leaf color, respectively. The leaf length ranged from (13 to 17.25 ㎝) while the leaf width varied between (9.60 and 13.10 ㎝).
Table 3. Morphological characteristics of 7 accessions which showed highly resistance to resistance of Sclerotinia rot provided by Germplasm Management System of National Agrobiodiversity Center, National Institute of Agricultural Science, RDA, Jeonju, Jellabuk-do, Rep. of Korea
In the present study, 544 perilla accessions were assessed in vitro against Sclerotinia rot caused by S. sclerotiorum using detached leaf method. Diseases due to S. sclerotiorum have traditionally been difficult to manage (Bolton et al., 2006). Breeding for Sclerotinia stem rot resistance is complicated by polygenic resistance alleles, with several likely controlling structural disease avoidance phenotypes, like plant height, and others controlling physiological resistance mechanisms, and also complex genetic and environmental interactions. Breeding initiatives have mainly focused on increasing yield, then attempting to incorporate disease resistance traits. Therefore, molecular breeding is pursued as a significant approach for controlling sclerotinia diseases. Actually, breeding for S. sclerotiorum resistant cultivars using conventional method is difficult since no immune or highly resistant germplasm is available from genetic resources like landrace, wild or relatives etc (Liu et al., 2005). The present study found phenotypically two highly resistant (IT226504, IT226533), five were resistant (IT226561, IT226532, IT226526, IT226441 and IT226589), five moderately resistant (IT226525, IT226640, IT226568, IT220624 and IT178655) perilla germplasm against S. sclerotiorum from unknown accessions. The results of this study with morphological characteristics of perilla flowering time, leaf shape cordate, adaxial and abaxial leaf colors being pale green to purple are similar to the results of Kim et al., 2011; Ma and Lee, 2017; Woo et al., 2016. Identification of genetic variation is important for long-term achievements of breeding programs and maximizes the use of germplasm resources (Mwangi et al., 2019). The findings in this study can play a significant role to find out resistant breeding line & quantitative trait loci (QTL) against S. sclerotiorum for perilla.
Due to their purplish color, perilla leaves look attractive and containing high health beneficial anthocyanin content, most people like to consume. The present study highlighted in vitro screening of perilla germplasm resistant against Sclerotinia sclerotiorum that causes Sclerotinia rot using detached leaf method. The study also revealed that various levels of resistance to Sclerotinia rot exist in perilla germplasm collections. Out of 544 perilla accessions, two were highly resistant, five were resistant, five were moderately resistant, 16 were moderately susceptible, 31 were susceptible, and 485 were highly susceptible. As this study’s experiment was conducted in seedling stage, it is recommended to conduct the experiment at different growth stage in experimental field agro-ecological conditions. Breeders could use the resistant germplasm as a source of resistance for the development of resistant cultivars.