Introduction
Chilli (Capsicum annuum L.) plant belongs to the family Solanaceae (chromosome number, 2n = 2x = 24) is one of the most cultivated spices and vegetable crops (Siddappa et al., Reference Siddappa, Ravindra and Shashikanth2019; Thakur et al., Reference Thakur, Jindal, Sharma and Dhaliwal2019; Sindhusha and Rawat, Reference Sindhusha and Rawat2020). Chilli has been cultivated on more than 2.0 million hectares and the annual global production for chilli is about 36 million tonnes. Asian region itself contributes to almost 24.6 million tonnes of hectares with 1.3 million cultivated lands (FAO STAT, 2021). Chilli hybrids are very popular among farmers around the world due to their high yielding ability and other quality characteristics. The development of hybrid varieties allows to combine desired and most important traits from two selected parents like disease resistance (Sahid et al., Reference Sahid, Syukur and Maharijaya2020; Anilkumar, Reference Anilkumar2021).
Fresh fruit yield in chilli is affected by genetic and environmental factors (Meena et al., Reference Meena, Dhaliwal and Jindal2020). Further, chilli genotypes are often susceptible to many diseases and it results in low productivity of chilli cultivation (Kothari et al., Reference Kothari, Joshi, Kachhwaha and Ochoa-Alejo2010). The yield of chilli fruit is affected by anthracnose, a devastating fungal disease, worldwide. Anthracnose disease has been identified as one of the major constraints in chilli production reported globally (Chunying et al., Reference Chunying, Sheng, Zheng, Alain and Li2015; Ananthan et al., Reference Ananthan, Subhash and Longvah2018; Mishra et al., Reference Mishra, Rout and Joshi2019a; Zhao et al., Reference Zhao, Liu, Zhang, Cao, Yu, Ma, Zhang, Wang, Gao and Wang2020). Application of fungicides and integrated disease management to control this disease is not a long-lasting and sustainable solution (Chunying et al., Reference Chunying, Sheng, Zheng, Alain and Li2015). Development of anthracnose disease-resistant chilli varieties is the most economical and environmentally friendly method to control this disease (Mishra et al., Reference Mishra, Rout and Joshi2019a). In the process of development of anthracnose disease-resistant chilli varieties, only the phenotypic evaluation is not enough. Confirmation that resistant varieties carry the resistant gene using the molecular markers provides a more scientific base to phenotypic observations (Zhao et al., Reference Zhao, Liu, Zhang, Cao, Yu, Ma, Zhang, Wang, Gao and Wang2020).
According to Kim et al., (Reference Kim, Yoon, Do and Park2008) and Lee et al., (Reference Lee, Do and Yoon2011), the pattern of inheritance of anthracnose disease resistance is very complex and it varies with the Colletotrichum isolate and source of resistance. Among these Colletotrichum species, Colletotrichum truncatum is the most prevalent species in major chilli growing areas resulting in a huge decline in the quality and quantity of the harvest (Noor and Zakaria, Reference Noor and Zakaria2018; Silva et al., Reference Silva, Groenewald, Crous, Ades, Nasruddin, Mongkolporn and Taylor2019; Welideniya et al., Reference Welideniya, Rienzie, Wickramaarachchi and Aruggoda2019). Based on previous studies, resistance against C. truncatum in C. annuum L. is controlled by single dominant gene (Park et al., Reference Park, Kim and Lee1990; Ridzuan, Reference Ridzuan2018; Mishra et al., Reference Mishra, Rout and Joshi2019a, Reference Mishra, Rout, Mohanty and Joshi2019b). In contrast, Mashuk et al., (Reference Mashuk, Khumpeng, Wasee, Taylor and Mongkolporn2009) reported that resistance against C. truncatum in Capsicum Chinense is controlled by recessive genes.
In the process of developing anthracnose disease-resistant chilli hybrids, both phenotypic selection and genotypic selection are equally important. Phenotypic selection can be practised as a field experiment or laboratory experiment (Garg et al., Reference Garg, Kumar, Kumar, Loganathan, Saha, Kumar, Rai and Roy2013). Various molecular markers are utilized in genotypic selection (Srivastava and Mangal, Reference Srivastava and Mangal2019). Molecular markers such as Amplified Fragment Length Polymorphic markers, Sequence Characterized Amplified Region, Cleaved Amplified Polymorphic Sequence, Simple Sequence Repeats (SSR) associated with anthracnose disease resistance in chilli have been identified by many researchers (Voorrips et al., Reference Voorrips, Finkers and Sanjaya2004; Lee et al., Reference Lee, Do and Yoon2011; Ying et al., Reference Ying, Li, Hai, Alain, Hao and Xi2015; Suwor et al., Reference Suwor, Sanitchon, Thummabenjapone, Kumar and Techawongstien2017; Mishra et al., Reference Mishra, Rout, Mohanty and Joshi2019b). These identified molecular markers can be utilized to breed the cultivars with resistance to anthracnose disease (Lee et al., Reference Lee, Do and Yoon2011). Among the identified markers, SSR markers are widely applied in plant breeding. These markers are highly polymorphic, multiallelic and monolocus. Therefore SSR markers are applied by the researchers to increase the efficiency of chilli breeding against anthracnose disease (Nanda, et al., Reference Nanda, Mohan Rao, Ramesh, Hittalmani and Prathibha2016;.Suwor et al., Reference Suwor, Sanitchon, Thummabenjapone, Kumar and Techawongstien2017; Ly et al., Reference Ly, Truong and Nguyen2020). According to the study conducted by Ridzuan, (Reference Ridzuan2018) using the resistant genotypes of C. annuum L, AVPP0805 and AVPP9813 developed by the Asian Vegetable Research and Development Centre, Taiwan, SSR markers, HpmsE 082 and HpmsE 051 were linked markers for anthracnose disease caused by the C. truncatum. Therefore, there is a possibility to identify C. truncatum resistant parent lines of C. annuum L further using these markers.
Homozygous parent lines are used as parents in the production of hybrid varieties. Qualities of the hybrid varieties depend on these parent lines (Shuro, Reference Shuro2017). Efforts have been taken to develop chilli hybrids against anthracnose using the parent lines with anthracnose disease resistance (Ridzuan, Reference Ridzuan2018). Herath et al. (Reference Herath, Rafii, Smil, Jaafar and Ramlee2022) have identified four resistant parents of C. annuum L against C. truncatum. Chilli hybrids developed using these parents have applied to this study with the purpose of identification of new chilli hybrids resistant to anthracnose disease caused by the C. truncatum by the phenotypic and genotypic selection.
Materials and methods
Fourteen single cross chilli hybrids (Table 1) were developed using four resistant parents (MICH PL CA 2018/3, MICH PL CA 2018/20, MICH PL CA 2018/21 and MICH PL 35) against C. truncatum, previously identified through the fruit inoculation of C. truncatum and three susceptible parents (MICH PL CC 2018/33, MICH PL 21, MICH PL CC 2018/17) previously identified through the fruit inoculation of C. truncatum (Herath et al., Reference Herath, Rafii, Smil, Jaafar and Ramlee2022). These 14 single cross chilli hybrids, their parental inbred lines and two commercial varieties (SJ2- 461, Kulai 907) as check varieties were used for this study (Table 1). The experiment was conducted in the glasshouse facility belonging to the Faculty of Agriculture, University Putra Malaysia (UPM) in two locations (Field 10 and Field 15). The experiment was conducted from August 2020 to December 2020. At the beginning of August 2020, seeds of chilli genotypes were sown in 50-cell seed trays filled with peat moss. Polybags (35 × 35 cm) were filled with a 4.5 kg soil mixture of 1:1 compost and topsoil to transplant the seedlings.
Table 1. Chilli hybrids evaluated at two locations (Field 10 and Field 15)
After one month, seedlings were transplanted in the poly bags inside plant houses at Field 10 (GPS location 2058’54.0”N latitude and 101042’53.8”E longitude) and Field 15 (GPS location 2098’33.4”N latitude and 101072’49.2”E longitude) under the randomized complete block design with three replicates with the spacing of 60 cm between rows and 45 cm within the row. Each replicate contained three plants per treatment. Chemical fertilizer application was done following recommendations of the Malaysian Agricultural Research and Development Institute (MARDI) (MARDI, 1997). As a fertilizer, 18 g of N, 3 g of P and 15 g of K were applied per each plant in total as one basal dressing and three top dressings. Sufficient irrigation was supplied throughout the study period.
Five red ripened fruits (40–45 days after flowering) from each treatment, and replicate were harvested separately from the chilli plants at the glasshouses at two locations (Field 10 and Field 15) for the fruit inoculation as two different experiments. Anthracnose disease severity assessment was conducted with the randomized complete block design with three replicates. C. truncatum was isolated using chilli fruits from three chilli cultivated field with severe anthracnose infestation and confirmed through molecular identification (gene bank accession numbers; MT995064 and MW030430) (Herath et al., Reference Herath, Rafii, Smil, Jaafar and Ramlee2022). Nine days old C. truncatum cultures collected and confirmed by molecular level were incubated at room temperature (28°C–30°C) were used to prepare the conidial suspension of C. truncatum. Fruit inoculation with 1 μl conidial suspension of C. truncatum was done following Montri et al. (Reference Montri, Pathom and Ten2009) using a Micro injector (micro syringe model 1705 TLL with a dispenser, PB 600-1Hamilton). The concentration of the conidial suspension was adjusted as 5 × 105 conidia⋅mL−1 using a haemocytometer (Marienfeld, Germany). Inoculated fruits were incubated in plastic boxes (13 cm × 13 cm × 7 cm), on four layers of white tissue moistened with 10 ml of sterilized distilled water. Data were collected on lesion size and fruit size after nine days of fruit inoculation.
After assessing 14 SSR markers and two STS markers, two polymorphic markers HpmsE 051 and HpmsE 082 were identified for genotypic selection. Total genomic DNA of chilli hybrids and parental inbred lines were extracted following Cetyltrimethyl Ammonium Bromide method according to Doyle and Doyle (Reference Doyle and Doyle1987). The polymerase chain reaction (PCR) included 7.5 μl of PCR master mix (1st base ex10 2X PCR master mix), 1 μl of template DNA, 1 μl of each primer. The reaction was adjusted to 15 μl with nuclease-free water. Amplification was performed using T100 Thermal Cycler (Bio-Rad, USA) following 3 min of initial denaturation, 30 s at 95°C of denaturation, 30 s at 55°C of annealing, 1minute at 72°C of extension, 10 min at 72°C for final elongation and cooled down to 4°C. Gel electrophoresis was done using 2% agarose gel in TBE buffer and set to run at 90 V for 60 min. Agarose gel was visualized under Gel DocTM XR with molecular image software (Bio-Rad, USA).
Data analysis
Per cent lesion size relative to the overall size of the fruit ([lesion area/fruit area]*100) was estimated and anthracnose severity score was given according to 0–9 scale described by Montri et al., (Reference Montri, Pathom and Ten2009). Statistical Analysis Software (SAS) version 9.4 were used for the data analysis. Arcsin square root transformation was done for the data before analysis since data were not normally distributed. Data were checked for normality using the Shapiro–Wilk test. Mean separation for disease severity data was done by Duncan Multiple Range Test by using the transformed data after confirming the significant difference among genotypes (hybrids, parents and check varieties) for anthracnose disease by the analysis of variance. Under the molecular marker analysis, individuals that are similar to the amplified product size of parental inbred lines were categorized as homozygous [resistant (R) or susceptible (S)] and the individuals that showed resemblances with both parental inbred lines were classified as heterozygous.
Results
Table 2 showed the percentage mean of anthracnose disease severity (%) and resistance level of chilli hybrids, parents and check varieties harvested from the glasshouses at Field 10 and Field 15. Figure 1 showed the lesion development on chilli hybrid fruits and commercial varieties after 9 days of fruit inoculation. Fruits of chilli hybrids, H1, H2, H3, H4, H6, H7, H8, H9, H11 and H12 harvested from the glasshouses at Field 10 and Field 15 showed <2% of disease severity at both green mature fruit and red ripened fruit stage. From the total of 14 developed chilli hybrids, 10 showed a resistant response against anthracnose disease. According to Montri et al., (Reference Montri, Pathom and Ten2009), <2% of anthracnose disease severity denotes the resistance against the disease. Therefore, these hybrids could be grouped as resistant chilli hybrids.
Table 2. Percentage means of anthracnose disease severity against Colletotrichum truncatum and resistant level of chilli hybrids, parents and commercial varieties
Within the column, the means followed by the same letters are not significantly different at p = 0.05. Anthracnose disease severity as disease score of 0 – highly resistant (HR), 1- resistant (R) 1-2% ,3- moderately resistant (MR)>2-5% -,5- moderately susceptible (MS) >5-15% ,7- susceptible (S) >15-25%, 9 - highly susceptible (HS) >25%.
Figure 1. Anthracnose lesions produced after 9 days of inoculation on chilli hybrids.
Among the parents, MICH PL CA 2018/3, MICH PL CA 2018/20, MICH PL CA 2018/21 and MICH PL 35 that were included as resistant parents the in crossing programme exhibited resistant response at both fruit stages with <2% of anthracnose disease severity in case of chilli fruits harvested from both Field 10 and Field 15. All the other developed hybrids (H5, H10, H13, H14) and parents (MICH PL CC 2018/33, MICH PL 21, MICH PL CC 2018/17) exhibited >15% or >25% of disease severity. According to Montri et al., (Reference Montri, Pathom and Ten2009) >15% and >25% of anthracnose disease severity indicate the susceptible and highly susceptible responses respectively.
Commercial imported chilli hybrid, SJ2-461 had > 28% of disease severity at both fruit stages in the case of Field 10 and Filed 15 indicating the highly susceptible nature of this hybrid to the anthracnose disease caused by the C. truncatum. Similarly, local open-pollinated commercial chilli variety, Kulai 907 exhibited >26% of disease severity in the case of the chilli fruits harvested at both Field 10 and Filed 15. Therefore, Kulai 907 was a highly susceptible genotype for the anthracnose disease caused by the C. truncatum.
When considering the screening of chilli hybrids using the markers, HpmsE 051 and HpmsE 082 (Fig. 2 and Fig. 3), 10 hybrids were showed heterozygous nature for anthracnose disease resistance. Those hybrids were, H1, H2, H3, H4, H6, H7, H8, H9, H11 and H12. Amplified product size of the other four hybrids, H5, H10, H13, H14 were similar to the amplified product size of susceptible parental inbred lines and it indicated that these hybrids were susceptible to the anthracnose disease.
Figure 2. Screening of developed 14 chilli hybrids using marker HpmsE 051(262 bp) (R: resistant, S: susceptible, P7: MICH PL CA 2018/3 (R), P24: MICH PL CC 2018/33 (S), P12: MICH PL CA 2018/20 (R), P31: MICH PL 21 (S), P13: MICH PL CA 2018/21 (R), P19: MICH PL CC 2018/17 (S), P30: MICH PL 35 (R), H1 (P24 × P7), H2 (P24 × P12), H3 (P24 × P13), H4 (P24 × P30), H5 (P24 × P19), H6 (P31 × P7), H7 (P31 × P12), H8 (P31 × P13), H9 (P31 × P30), H10 (P31 × P19), H11 (P12 × P24), H12 (P12 × P19), H13 (P24 × P31), H14 (P31 × P24).
Figure 3. Screening of developed 14 chilli hybrids using marker, HpmsE 082 (232 bp). (R: resistant, S: susceptible, P7: MICH PL CA 2018/3 (R), P24: MICH PL CC 2018/33 (S), P12: MICH PL CA 2018/20 (R), P31: MICH PL 21 (S), P13: MICH PL CA 2018/21 (R), P19: MICH PL CC 2018/17 (S), P30: MICH PL 35 (R), H1 (P24 × P7), H2 (P24 × P12), H3 (P24 × P13), H4 (P24 × P30), H5 (P24 × P19), H6 (P31 × P7), H7 (P31 × P12), H8 (P31 × P13), H9 (P31 × P30), H10 (P31 × P19), H11 (P12 × P24), H12 (P12 × P19), H13 (P24 × P31), H14 (P31 × P24).
When comparing both phenotypic and genotypic data (Table 3) hybrids, H1, H2, H3, H4, H6, H7, H8, H9, H11 and H12 that showed heterozygous nature for anthracnose disease under the markers HpmsE 051 and HpmsE 082 were resistant to anthracnose disease caused by the C. truncatum based on the phenotypic evaluation. When one parent is resistant to C. truncatum in a cross, resulting hybrids were resistant to anthracnose disease caused by the C. truncatum. When both parents were susceptible to anthracnose disease caused by the C. truncatum, resulting hybrids were susceptible to the disease (H5, H10, H13 and H14).
Table 3. Comparison of phenotypic and genotypic data
R, resistant; MS, moderately susceptible; S, susceptible; HS, highly susceptible.
Discussion
Based on the phenotypic and genotypic data, new chilli hybrids (H1, H2, H3, H4, H6, H7, H8, H9, H11 and H12) developed using resistant breeding lines could be isolated as anthracnose disease-resistant chilli hybrids that carry the resistant gene. Further testing of these hybrids is needed to check the yield performance to isolate the potential hybrids for commercial cultivation with anthracnose disease-resistant character. In addition, these hybrids could be utilized to develop second cycle inbred lines that could be utilized as parents in the process of new anthracnose disease-resistant variety development. Even though, effort on the development of anthracnose disease-resistant varieties is very limited according to the available literature, this study provides information on the possibility of the development of anthracnose disease-resistant hybrids. Dominance nature of disease resistance of C. annuum against C. truncatum was observed in this study, because, when one parent is resistant to C. truncatum, resulting hybrid was anthracnose disease resistant.
However, based on the resistant germplasm of chilli, researchers have reported different findings regarding the inheritance of anthracnose disease resistance from past to present (1990–2021). Park, Kim and Lee (Reference Park, Kim and Lee1990) found that inheritance of resistance to C. truncatum is controlled by a partial dominance gene in the C. annuum chilli accession, Chungryong. According to Lin et al., (2002), C. annuum breeding line, 83–168 was resistant to C. truncatum and inheritance of resistance was controlled by a single dominant gene. Ridzuan, (Reference Ridzuan2018) observed the dominant gene action in the inheritance of anthracnose disease caused by the C. truncatum by the evaluation of F2 segregation population resulted through the self-pollination of a cross between a resistant parent and susceptible parents. According to the study conducted using the C. annuum species, ‘Punjab Lal’ – Resistant parent × ‘Arka Lohit’- susceptible parent found that monogenic dominant gene is responsible for the anthracnose disease caused by C. truncatum (Mishra et al., Reference Mishra, Rout and Joshi2019a). These findings are in conformity with our study. In contrast to these findings, Kim et al., (Reference Kim, Yoon, Do and Park2008) found that local Korean variety, Daepoong-cho belongs to the species, C. annuum, exhibited resistance to C. truncatum and further studies conducted by them revealed that this resistance is controlled by a single recessive gene. A study was conducted using C. chinense accession, PBC 932 and observed that three recessive genes namely, co 1, co 2 and co 3 were responsible for resistance to C. truncatum during the seedling, mature green fruit and red ripen fruit stages (Mashuk, et al., Reference Mashuk, Khumpeng, Wasee, Taylor and Mongkolporn2009). Even though anthracnose disease is a devastating fungal disease in chilli, still it has difficult to find the responsible genes conferring disease resistance (Son et al., Reference Son, Kim, Lee, Oh, Choi, Do and Yoon2021).
As observed by this study, markers, HpmsE 051 and HpmsE 082 were good polymorphic markers to isolate resistant genotypes of C. annuum (parents and hybrids) for the anthracnose disease caused by the C. truncatum for anthracnose disease-resistant breeding of chilli. Yi et al., (Reference Yi, Lee, Lee, Choi and Kim2006), developed SSR markers based chilli linkage map and reported that, two markers, HpmsE 082 and HpmsE 051 were located on chromosome number 9 in the chilli genome. It implied that gene/genes located on chromosome number 9 are responsible for resistance against anthracnose disease caused by the C. truncatum.
Conclusion
Based on the phenotypic and genotypic data, chilli hybrids, H1, H2, H3, H4, H6, H7, H8, H9, H11 and H12 were identified as resistant chilli hybrids against anthracnose disease caused by the C. truncatum whereas hybrids, H5, H10, H13 and H14 were susceptible to anthracnose disease. Molecular markers, HpmsE 051 and HpmsE 082 validated in this study could be utilized as polymorphic markers to isolate resistant genotypes of C. annuum in the process of anthracnose disease-resistant variety development against C. truncatum.
Acknowledgements
The authors wish to thank Sri Lanka Council for Agriculture Research Policy for granting the PhD scholarship to H.M.S.N. Herath.
Competing interest
None.
