INTRODUCTION

Agricultural productivity faces a multitude of challenges among which, pests and pathogens are major factors that can significantly affect both quality and yield of crops during growth as well post-harvest, making them significant elements in modern day agriculture (Savary et al. 2019). While pests are eukaryotic organisms such as insects and worms, plant pathogens are usually microorganisms which could be fungi, bacteria or viruses. Among them, bacterial pathogens can cause several plant-detrimental diseases, including spots, abscesses and rotting of several parts of the plant leading to plant death. The pectobacteria causing bacterial soft rot are phytopathogens with a broad host range and affect several economically importance crop plants. The major pectobacteria that cause soft rot belong to genera Pectobacterium and Dickeya, and are well documented to cause necrotrophic rotting of roots (Davidsson et al. 2013).

The bacterium P. carotovorum causing soft rot has been considered as one of the top ten plant pathogenic bacteria due to huge economic losses resulting from their infection (Mansfield et al. 2012). It was originally classified as Erwinia carotovora and was known to be geographically globally distributed (Mansfield et al. 2012). The recognized hosts of the pathogen include Kimchi cabbage, cabbage, radish, carrot, potato, and lettuce, etc., which are widely cultivated across China, Japan, and South Korea (Leite et al. 2014;Kim et al. 2016;Meng et al. 2017;Jeon et al. 2018;Liu et al. 2019a;Afroz et al. 2021). The phytopathogen P. carotovorum subsp. carotovorum causes soft rot in plants by degrading plant tissues. Mutagenic approaches to understand its pathogenicity have confirmed the role of its extracellular plant cell wall-degrading enzymes (PCWDE, exoenzymes) including pectatelyases (Pel), polygalacturonases (PGs), proteases (Prt), and cellulases (Cel) (Lee et al. 2013). The pathogen primarily causes damage of the cell membrane and thereby leading to leakage of electrolytes, extensive tissue maceration, rotting, and subsequent plant death (Davidsson et al. 2013). This ultimately results in losses in marketable yield in the field and also during transit, and storage.

The plant family Brassicaceae is one of the largest plant families that naturally inhabit several parts of Asia, especially Korea (Kim et al. 2019;Lee et al. 2020). Members of this family are economically important and their production often affected by diseases and pests leading to huge agricultural losses (Ko et al. 2021a, 2021b) Among them, Kimchi cabbage (Brassica rapa subsp. pekinensis) is a plant that have been historically used in the culinary processes in parts of Asia and are widely grown and consumed in Korea, China, and Taiwan (Lee et al. 2020). The plants occur both as annual and biennial forms as headed or loose-headed types and are economically important crops (Fordham and Hadley 2003). The current study was aimed at screening of the Kimchi cabbage genetic resources collected and stored at the National Agrobiodiversity Center, Rural development Administration, Korea to identify any resistant accessions against soft rot causing P. carotovorum supsp. carotovorum for their characterization which can serve as a potential resource for future studies aimed at understanding the plant immune responses and gaps in the available knowledge in molecular basis of disease resistance.

MATERIALS AND METHODS

The germplasms for screening against bacterial soft rot were obtained from National Agrobiodiversity Center, RDA Genebank. 682 accessions were screened over a period of 3 years from 2020 to 2022. The strain Pectobacterium carotovorum subsp. carotovorum (KACC 21701) was obtained from Korean Agricultural Culture Collection (KACC) and used for infection of the Kimchi cabbage germplasms.

Twenty-five healthy Kimchi cabbage seedlings at the four-leaf stage were used during experiments. For inoculation of 14 days old seedlings, a fresh batch of the strain P. carotovorum subsp. carotovorum KACC 21701 was cultured on tryptic soy agar (TSA) at 28°C followed by transfer to tryptic soy broth where it was grown for 24 h at 150 RPM, 28°C. Cells were then centrifuged at 4,000 rpm for 10 mins and the harvested cells were adjusted for a final O.D. of 0.1. For infecting the seedlings, the bacterial suspension was mixed with mineral oil at a ratio of 4 : 1 and added on the surface of the soil (Chung et al. 2003). The cultivars including Wangmatbaechu, Seoulbaechu and CR-Kiyoshi were used as control. The infected seedlings were then transferred to plant growth chamber maintained at 30°C, 90% relative humidity (RH), no light for one day after which the conditions were changed to 30°C, 80% relative humidity and 12 h light/dark cycle for six more days. The disease index (DI) of at least ten infected plants was examined and recorded at the end of seven days post infection. The values of disease index were determined according to the severity of the disease and are explained in Figure 1. Seedlings with DI=0 were considered highly resistant, 0.1 to 1.0 to be resistant, 1.1 to 2.0 moderately resistant, 2.1 to 3.0 to be moderately susceptible 3.1-4.0 were considered susceptible.

For the replication experiments of the resistance to KACC 21701, the primary accessions during initial screening with a DI value of less than or equal to two were selected and the experiments repeated. The replication experiments were repeated twice independently to find any consistency to primary screening data. The results obtained from initial screening and replication testes were analyzed using Microsoft Excel (2016) and SigmaPlot ver. 15.0.

RESULTS AND DISCUSSION

Six hundred and eighty-two Kimchi cabbage germplasms were selected between 2020 and 2022 (253, 212, and 217 accessions, respectively) for screening of resistance against soft rot caused by P. carotovorum subsp. carotovorum. Disease index was recorded for a minimum of at least 10 seedlings in all the 682 germplasms screened (Table 1). These studied germplasms included landraces and cultivars as well as unassigned germplasms with origins from Asia, Australia, Americas, Russia, Europe and Africa (Table 1; Fig. 2). Among them about 50% of the germplasm were originating from Korea, followed by China (14.8%), Taiwan (13.6%) and Japan (11%) (Fig. 2).

Exponential increase in food production around the world in the past five decades at the expense of environmental degradation has led to several problems including deficiency in particular nutrients, reduced biodiversity and even emergence of susceptibility to diseases at a large scale (Pathirana and Carimi 2022). Genebanks that conserve plant genetic resources play a major role in preserving this depleting biodiversity as well as play a complementary role between currently practiced agriculture and nature. In this context, proper documentation of the various properties of the conserved germplasms in a GenBank is vital as to understand the value of a particular bioresource for breeding and research (Weise et al. 2020). Studies to elucidate such properties of plant genetic resources are necessary and continuous diversification of available data followed by structured documentation and information sharing are essential for successful functioning of genetic resource conservatories (Weise et al. 2020;Pathirana and Carimi 2022). To supply the data available on Kimchi cabbage accessions at the National Agrobiodiversity Center, Rural Development Administration, the present study involved largescale screening and replication experiments of resistance to KACC 21701 in randomly chosen Kimchi cabbage genetic resource.

Screening of germplasms from South Korea for resistance to bacterial soft rot disease has been earlier reported in Kimchi cabbage (Chung et al. 2003). However, the report of Chung and coworkers only involved screening of only forty-three cultivars or line of Kimchi cabbage. With a DI range of 0-3, they observed four out of forty-three cultivars to exhibit DI<1 which they reported to be resistant to KACC 21701. In our screening of Kimchi cabbage germplasms, we recorded DI values at a range of 0 to 4 (Fig. 1). The six hundred and eighty-two germplasms in the current study displayed different degrees of resistance to KACC 21701 observable by their varying DI values and only one accession IT120033 (Pin Chong 1) indicated DI<1 (Table 2; Fig. 3A).

As explained above, the present study differs from Chung et al. (2003) by the total number of germplasms studied and the range of DI values recorded. Therefore, their suggestion of cultivars exhibiting DI<1 to be resistant to KACC 21701 cannot be strictly followed. During our studies, overall, we found a total of one germplasm to have less than DI 1, twenty-nine germplasms to have DI at a range of 1.01-2.00, ninety-nine germplasms to have DI at a range of 2.01-3.00 and five hundred and fifty-three germplasms to have DI range of 3.01-4.00 (Fig. 3A). This distribution of various levels of susceptibility to KACC 21701 was found to occur in landraces, cultivars as well as unassigned germplasms. For our replication studies, among the 682 germplasms showing various levels of susceptibility to KACC 21701, we choose 30 accessions that exhibited a DI value of less than or equal to 2. These accessions consisted of predominantly landraces (23) followed by unassigned (4) and cultivars (3) of Kimchi cabbage. Landraces are understood to be better suited for their environment and in the present study seven of the accessions 23 germplasms with DI≤2 have originated from Korea (KOR) followed by 6 from Taiwan (TWN) and 5 from Hong Kong (HKG) (Fig. 3B). Infection by soft rot causing P. carotovorum has been well studied and reported from members of Brassicaceae, Solanaceae and Cucurbitaceae (Meng et al. 2017;Lee et al. 2018, 2020;Afroz et al. 2021). Previous studies conducted in Korea involved screening of commercial cultivars of radish and Kimchi cabbage whereas the current study included Korean landraces and unassigned germplasms in addition to cultivars.

Our initial screening of the accessions resulted in thirty accessions of Kimchi cabbage to exhibit DI≤2.0 (Figs. 3A and 4). Also, these germplasms were found to have high ratio of resistant individuals to KACC 21701 (Table 2). These 30 germplasms contributed to 5.5% of landraces (23 of 418), 2.3% in case of cultivars (3 of 133) and 3.1% in case of unassigned germplasms (4 of 131) (Fig. 3A) and had their origins as KOR (23%) followed by TWN (20%), HKG (17%), JPN (17%), CHN (7%), USA (7%), NLD (3%), MUS (3%) and IDN (3%) (Table 2; Fig. 3B). Cultivated varieties and hybrids have been generally proved to be competitively superior compared to their wild relatives in terms of biomass and resistance to microbial diseases (Schröder and Prasse 2013;Okoń et al. 2021). However, in the current study landrace germplasms indicated significantly high numbers of disease resistance compared to cultivated or unassigned varieties.

In the replication experiments, it was observed that all of the selected thirty accessions were affected by KACC 21701 at the end of seven days (Table 3). Among them, five of the germplasms consisting of four landraces (IT102883, IT120036, IT120044, and IT120048) and one cultivar (IT187919) were observed to be moderately susceptible to KACC 21701. We observed that one of our controls CR Kiyoshi which was earlier reported to be moderately resistant to soft rot causing P. carotovorum (Lee et al. 2020) but was severely affected in this study (Fig. 4). This could be attributed to the difference in the pathogenic Pectobacterium strains (KACC 21701 used in the current study vs KACC 10225) which indicates high pathogenicity of the current strain as well as difference in the methods employed. In the earlier study where CR Kiyoshi was found to be moderately resistant, the age of seedlings was much higher (24-days old seedling vs 14 days old seedlings in the present study) and methods of infection were different (transient dipping vs soil inoculation of pathogen). These factors combined could have produced such results in our studies. We identified five germplasms among the thirty to be moderately susceptible to KACC 21701 and exhibiting high levels of variance in disease resistance (Table 3). Taking into consideration that the moderately resistant CR Kiyoshi control seedlings were completely dead (Fig. 4) during our experiments, it can be understood that our strain of choice (KACC 21701) and experimental conditions led to high rates of infection and disease in the studied Kimchi cabbage genetic resources. Moreover, presence of individual seedlings with no symptoms of KACC 21701 infection can still be observed in the thirty germplasms from the percentage ratio of resistant individuals (Table 3).

In screening-based studies such as the present one, despite the fact that all the seedlings tested originated from same batch of seeds of a germplasm, few of the seedlings survive the infection whereas others perish. Although this is a common phenomenon accounting for the deviancy of disease index (DI) values in screening-based studies, it fascinating to see how few seedlings stand out of the homogeneity. A recent study on resistance of Solanum chilense germplasms to fungal pathogen Phytophthora infestans has established that the relative changes in resistance phenotypes that occur among individuals were mainly determined by the plant genotype (Kahlon et al. 2021). Although the seedlings during the screening studies were raised from a single batch of seeds collected from a specific germplasm, they may not genotypically identical as they are collected from plants grown ex-situ which are vulnerable to pollination by wild plants or other relatives. In addition to the genetic characteristic, ecological aspects have also been reported to cause such results in case of fungal pathogens studied on field scale (Davelos et al. 1996). Though such bias in in-situ studies is diminutive, minute differences in age of the plant, small changes in environmental conditions and spontaneous changes in plants can also be suggested to contribute to the resistance or susceptibility of the seedlings.

Generally, glucosinolates and lignin biosysnthesis has been reportedly increased in Kimchi cabbage cultivars resistant to soft rot causing P. carotovorum which might help the plant to play a defensive role against the pathogen (Liu et al. 2019b). Also, the pathogen-pattern triggered immu- nity (PTI) has been reported to be strongly triggered in resistant mutant lines, leading to expression of defense response genes including calcium dependent protein kinases (CDPK), calcium binding proteins (CaBP), respiratory burst oxidase homologs (RBOH) and mitogen activated protein kinases (MAPK3 and MAPK4) (Liu et al. 2019b). Overexpression of a polygalacturonase-inhibiting protein gene BrPGIP2 has been observed to play a significant role in resistance of Kimchi cabbage against soft rot causing Pectobacterium (Hwang et al. 2010). Though the molecular mechanisms involved in the pathogenicity of P. carotovorum and resistance in plants has been studied, elucidation of the molecular details involved in signaling networks in plant cells is essential to understand the necrotrophic nature of the phytopathogen (Davidsson et al. 2013). While this of interest to scientists, breeders may be interested to develop soft rot resistant cultivars with desired traits that would economically benefit farmers from Korea at a large scale. In the current study, the studied germplasm exhibited various degrees of resistance or susceptibility to the phytopathogen P. carotovorum and we identified five germplasms to be moderately susceptible to its infection. This data was documented and amended to already available passport information on these 682 germplasms currently conserved at RDA Genebank, the National Agrobiodiversity Center.