1. Introduction
The
Capsicum L. (Solanaceae) represents a diverse plant group contains a large number of cultivated species as well as wild species that are grown for their fruits, and are an important vegetable consumed throughout the world. Approximately, 25
Capsicum species have been cultivated extensively [
1] and being used as food flavoring, pharmaceutical ingredient, coloring agent and in many other innovative ways [
2]. Systemically, the genus
Capsicum was classified by its flower, fruit structure and chromosome number [
3]. Similarly,
Capsicum species have been divided into three complexes such as,
C. annuum,
C. baccatum and
C. pubescens complexes based on cytogenetics and cross fertility [
4]. However, the wild ancestor of
Capsicum species remains unclear; due to some wild species have a predominant chromosome numbers [
5,
6]. Wild species of
Capsicum are important sources of genetic diversity and reservoirs of genes for breeding programs of cultivated pepper [
7]. Hence, conservation of wild genotypes and screening for novel allele is an increasing priority to make modern cultivars which gradually substitutes the landraces to increase productivity [
8].
Anthracnose, is one of the serious fungal disease in pepper fruit caused by
Colletotrichum spp. such as
C. acutatum,
C. capsici, and
C. gloeosporioides, leads to significant yield losses worldwide [
9]. However, in Korea the
C. acutatum species complex is a most significant causal pathogen of the disease, which infects both immature and mature pepper fruits [
10]. Typical anthracnose symptoms on pepper fruit includes sunken necrotic tissues, with concentric rings of acervuli which reduce fruit quality [
11]. In general, the anthracnose disease was controlled by using chemical fungicides which might have negative impact on human health and pollute the environment. Biocontrol agents such as
Bacillus sp. and its putative catalase may be useful to protect pepper from anthracnose [
12]. However, the development of resistant cultivars is the best long-term strategy to control the disease, and so it is a very important goal for pepper breeders. There is still little information available about the interactions between the host and the causal pathogens of pepper anthracnose [
9].
Breeding for anthracnose resistance began in the early 1990s, involving some
Capsicum species such as,
C. annuum,
C. frutescens, and
C. baccatum with potential resistance traits. It suggested that the
C. baccatum germplasm contained higher levels of resistance to anthracnose, which may prove useful as genetic resources for anthracnose resistance [
13]. Marker-assisted selection is a significant tool for the breeding of pepper. Anthracnose resistance is controlled by a major resistance locus and STS marker (CaR12.2M1-CAPS) was developed [
14,
15]. The introgression of the resistance gene from
C. baccatum to
C. annuum is difficult. For example, PBC80 was introduced into
C. annuum through a tri-species cross by using
C. chinense as an intermediate host [
16]. New crosses have been created to combine a good source of disease resistance, such as the
C. chinense germplasm selection PBC932, with elite Indonesian OP varieties, primarily “Jatilaba”, “TitSuper” and “KR-B” (“Keriting” from Bogor). In Korea, resistant varieties, “AR legend”, that crossed from
C. baccatum to
C. annuum with embryo rescue had been developed since 2014. There were several studies focused on the introgression of anthracnose resistance into
C. annuum to develop new varieties [
17,
18].
Genetic resources with excellent disease resistance are an important prerequisite for the development of elite varieties [
19]. Various studies have been reported for evaluation of
C. acutaum resistance in pepper genetic resources [
13,
20,
21,
22]. Similarly different methods such as, anthracnose inoculation method, wounding and non-wounding inoculation method have been reported [
23]. Non-wounding inoculation could evaluate resistance to anthracnose for cuticular wax defense of the fruit. Anthracnose development as negatively related with fruit developmental stage. As well-developed fruits had more cuticular wax than less developed fruits, the cuticular wax layers of pepper fruits may play a significant role in fruit infection by
C. gloeosporioides isolate KG13 [
24].
Phenotypic and genotypic characterization of a resistance gene (AVPP0207) located on chromosome P5 of
C. annuum (progressive line derived from PBC932) was reported against two anthracnose isolates of
C. acutatum and
C. truncatum [
25]. The fine mapping of a major anthracnose resistance QTL AnRGO5 in
C. chinense ‘PBC932′ was also reported [
26]. However, the resistance genes in
C. chinense and
C. baccatum were differentially expressed at different fruit maturity stages. Alternatively, some recent research reported that the inheritance of anthracnose resistance is controlled by recessive genes [
27]. The finding of the study revealed that, in mature green fruit, the resistance gene is the recessive gene co1, while in ripe fruit and seedlings, the recessive genes co2 and co3, respectively, are responsible for anthracnose resistance. Mahasuk et al. found that the resistance at the ripe red fruit and mature green stages is controlled by a single dominant and single recessive gene, respectively, between an intraspecific cross derived from
C. baccatum PBC1422 and PBC80 [
28].
Sources of anthracnose resistance in
C. chinense L. and
C. baccatum Jacq. have been reported in Asia and used as parents in breeding programs [
29]. In Korea, some studies searching for anthracnose resistance sources have been performed [
13,
21,
30]. However, screening for inheritance of anthracnose resistance in the wild as well as the domesticated
Capsicum species against
Colletotrichum are still lacking, particularly for
C. scovillei (formerly known as
C. acutatum). The aim of this study was to find anthracnose resistant genetic resources and make these materials available for breeding purposes.
4. Discussion
In the recent years, pepper anthracnose disease is becoming a major threat to Korean pepper production [
34]. Evaluation of germplasm resources to find different resistance traits is an effective control method for Anthracnose (
Colletotrichum spp.) disease resistance [
14], which has high level of genetic diversity with different species and strains in the Korean regions [
34]. Similarly, selection of plants carrying resistance genes are prerequisite for breeding studies. In pepper, molecular markers linked with anthracnose resistance genes have been identified and used in breeding programs [
19]. Mainly two pepper cultivars sources are known to play a role in Anthracnose (
Colletotrichum spp.) disease resistance [
30]. The Korean genebank preserves about 6700 peppers collected from countries around the world. This study was conducted to determine the degree of resistance through non-wound and wound inoculation of
C. acutatum on pepper fruit, and the selected resources were validated with molecular marker.
Based on the inoculation experiment, 261 resistant pepper genetic resources were selected successfully. In non-wound inoculation,
C. baccatum and
C. chinense,
C. chacoense and
C. frutescens were showed highly resistant to disease infection as reported previously [
13,
21]. The results of wound inoculation showed that the resistance resources were significantly distributed in the
C. baccatum species when compared with other resources (
Table 3). The
C. baccatum species is known to exhibit stable resistance to various
Colletotrichum spp. including
C. capsici, C. gloeosporioides, C. acutatum species [
28,
35].
However, in wound inoculation, all the 215 tested accessions were developed anthracnose symptoms. Hence based on the inoculation experiments, wound inoculation method showed efficient for resistance evaluation, in which 12 resources with disease incidence rate of 0–25% were selected. The results showed there were some accessions with high incidence rates in wound inoculation, where they appeared strong resistance to non-wound inoculation. This is because the pepper anthracnose unable invades cell wall cuticle, resistance by initial defense such as cuticle layer and cell wall of plants [
24,
36]. It involves dynamic changes in the epidermis of the plant during pathogen infection, the crosstalk of various hormonal signaling pathways and cuticles for plant cell wall and plant disease resistance, and the major biochemical, molecular and cellular mechanisms responsible for the role of the cuticle during plant-pathogen interactions [
37].
Plants recognize the attachment of pathogens and respond very quickly by inducing the innate immunity with microbe/pathogen associated molecular pattern [
37]. DAMP, a product of pathogen-infected plant degradation, such as cutin monomers and cell wall oligosaccharides, also serves as a signal to activate plant defense against pathogens [
38,
39]. For instance, tomato fruit cuticle was changed in response to infection with the fungal pathogen
C. gloeosporioides, and fruit cuticle biosynthesis was upregulated during appressorium formation even before penetration [
40]. Similarly, during infection of citrus by
C. acutatum, epidermal cells responded to pathogens by increasing lipid synthesis and deposition of cuticles and cell wall-related compounds, which eventually altered the cuticle structure [
41]. The
C. gloeosporioides induced the methyl jasmonate esterase was reported [
42]. Jasmonates (JAs) has been demonstrated to be involved in plant resistance to pathogens by activating pathogenic related (PR) proteins such as PR-1, PR-3, and PR-8 [
43]. Some PR genes were activated, including genes encoding pathogenesis-related protein 1 and a second pathogenicity-related protein [
42].
Based on these findings, pepper resources that appear to be resistant in non-wound inoculation but to be susceptible in wound inoculation could have developed cuticles or defense signaling by JAs. However, wound inoculation method skipped up the step of the cuticle’s defense mechanism, it would not be able to show resistance if there were no
C. acutatum resistance genes inside the pepper genetic resources. The 12 resources that appeared as resistant resources in wound and non-wound inoculation are likely to have two defense mechanisms. First, the cuticles are used to defend against it, and the second is the resistance genes. Anthracnose resistance is controlled by a major resistance locus [
15] and resistance to
C. capsici in ‘PBC932’ was found to be controlled by a single recessive gene [
34]. Resistance to
C. acutatum derived from
C. chinense ‘PBC932’ was stated to be controlled by two complementary dominant genes in green fruit, but by two recessive genes in red fruit [
44]. Zhao et al. (2020) narrowed down the interval of a QTL AnRGO5 conferring resistance with fine-mapping analyses [
26]. Based on these findings, it can be seen that the major genes or QTL involved in
Capsicum anthracnose are present according to a specific pathogen and
Capsicum species.
As a result of testing with CA12g19240 marker and CaR12.2M1-CAPS marker from selected accessions (
Table 4), the markers for
C. baccatum and
C. annuum were well matched, but
C. chinense and
C. frutescens were susceptible although their phenotype to
C. acutatum were resistant. As CA12g19240 marker and CaR12.2M1-CAPS marker were developed between
C. baccatum and
C. annuum, two makers could not distinguish resistance to
C. acutatum in
C. chinense and
C. frutescens. Recently, a marker using PBC932 was developed [
26]. However, it has not yet been applied to
C. chinense genetic resource, it is expected that
C. chinense can be used to determine resistance using a marker derived from PBC932. The CAPS and HRM markers have been reported to detect the intra- and interspecies variation and genotypic discrimination of different species [
45]. Similarly, in this study CAPS and HRM markers have successfully identified the diversity between the
Capsicum species.
The
Capsicum species has been studied using morphological as well as with molecular markers [
46]. The genetic similarity information can complement phenotypic information in the development of breeding populations [
47]. Thus, morphological characterization is an important step in the classification of germplasm. Previously, Luitel et al. reported wide variation in the pepper fruit characters of a core collection [
48]. Similarly, in this study the fruit characteristic analysis revealed that the selected pepper fruits vary in size, shape, color and even in their sugar content as the pepper germplasm collected from different countries. Since each country has different pepper preferences, selecting various anthracnose resistant genetic resources will help international pepper breeding.