Introduction
Anthracnose is a devastating chilli disease caused by Colletotrichum spp. that has resulted in up to 50% yield losses owing to pre- and post-harvest fruit rot in India (Pakdeevaraporn et al., Reference Pakdeevaraporn, Wasee, Taylor and Mongkolporn2005). India is the largest exporter of chilli and its products (Geetha and Selvarani, Reference Geetha and Selvarani2017), however, anthracnose disease causes an estimated annual loss of 29.5%, corresponding to 491.67 million US$ (Garg et al., Reference Garg, Loganathan, Saha, Roy, Kharwar, Upadhyay, Dubey and Raguwanshi2014). In India, primarily three important species, namely, C. capsici, C. gleosporoides and C. acutatum have been reported to be linked with the disease as solo or as disease complex (Than et al., Reference Than, Jeewon, Hyde, Pongsupasamit, Mongkolporn and Taylor2008a; Saxena et al., Reference Saxena, Raghuwanshi, Gupta and Singh2016). Among these, C. capsici is causing major damage at the ripening stage of the plant (Ranathunge et al., Reference Ranathunge, Mongkolporn, Ford and Taylor2012; Saxena et al., Reference Saxena, Raghuvanshi and Singh2014) while, C. gloeosporioides have been found to be more abundant in developing infections of young and mature green fruits (Hong and Hwang, Reference Hong and Hwang1998; Kim et al., Reference Kim, Oh and Yang1999). Anthracnose results in seedling rot during the initial stages of plant growth, while later die back and has fruit rot disease. In severe epidemic conditions, sunken dark necrotic lesions with acervuli are commonly formed on infected chilli fruits (Katoch et al., Reference Katoch, Sharma and Sharma2017). Acervuli formed in the lesions can competitively serve as the source of initial infectious inoculum in addition to the asexual fruiting bodies; Colletotrichum is also known to be occurring for internally and/or externally seed-borne in nature. To compete with the standards of the global chilli market, the management of anthracnose disease is very important. In this regard, farmers are mainly and unintentionally dependent on chemical fungicides to control anthracnose in the field which can leave accountable pesticide residue in chilli fruit usually harmful/toxic for consumers. However, as people become more aware of the benefits of eating pesticide-free food on a regular basis, demand for organic agri-products has risen, In the case of chilli, this can only be achieved by using anthracnose resistant cultivars. Because of the harmful consequences of chemical pesticides on the environment and human health, an integrated approach has already been adopted around the world. Host resistance is the most reliable and environmentally friendly strategy among the several approaches to integrated disease management. Therefore, the present study is an attempt to screen chilli landraces collected from India's northeastern region for anthracnose resistance. Some genetic resources resistant to anthracnose in chilli have been independently reported from different countries and regions (Park et al., Reference Park, Park, Lee and Ko1987; Kim et al., Reference Kim, Park and Lee1989; Hong and Hwang, Reference Hong and Hwang1998; Pae et al., Reference Pae, Yoon and Lee1998; AVRDC, 1999; Yoon and Park, Reference Yoon and Park2001; Begum et al., Reference Begum, Narjinary, Anand and Nath2015; Katediya et al., Reference Katediya, Jaiman and Acharya2019). In particular, some lines of C. baccatum and C. chinense showed strong resistance to the pathogen, and pathogen inoculation resulted in no or limited lesions on the chilli fruits. ‘PBC80’ and ‘PBC81’ reported as resistant resources in C. baccatum germplasm (AVRDC 1999; Yoon et al., Reference Yoon, Yang, Lee, Ahn and Park2004) and ‘PBC932’ is a resistant resource in C. chinense germplasm (AVRDC 2003a, 2003b; Pakdeevaraporn et al., Reference Pakdeevaraporn, Wasee, Taylor and Mongkolporn2005). However, to date, no strong resistance has been found in Capsicum annuum, which is the only species grown worldwide. Hence, further research is needed to identify more effective resistance sources that can be used in breeding programs.
Bhut Jolokia (King chilli or Ghost chilli) is a well-known landrace from the northeastern region of India which has been domesticated for a long time by the local communities and is grown for its high pungent fruits. A study done at ICAR-Indian Institute of Vegetable Research, Varanasi, India has shown that Bhut Jolokia has significant resistance against anthracnose (Garg et al., Reference Garg, Loganathan, Saha, Roy, Kharwar, Upadhyay, Dubey and Raguwanshi2014). In some other reports also mentioned that Bhut Jolokia chilli lines carry resistant against anthracnose (Garg et al., Reference Garg, Kumar, Kumar, Loganathan, Saha, Kumar, Rai and Roy2013; Mishra et al., Reference Mishra, Rout and Joshi2018). Hence keeping in mind the facts of losses caused by anthracnose and the presence of resistance in the lines of Bhut Jolokia, we have collected Bhut Jolokia landraces from different locations of The northeastern region of India and screened for resistance against C. capsici and C. gloeosporioides through a field and artificial screening.
Material and methods
Plant material
Twenty-four Bhut Jolokia landraces were collected in the month of September 2015 and January 2016 from Assam and Nagaland states of the northeastern region of India (Table S1) and subsequently screened for anthracnose in the next cropping seasons under both in vitro and in vivo conditions at poly-house facility/experimental field of TERI GRAM, Gwal Pahari, Gurugram, Haryana. Seven different Capsicum annum cultivars namely ‘Pusa Sadabahar’, ‘Pusa Jwala’, ‘Kashi Anmol’, ‘IC-570408’, ‘California Wonder’, ‘R line’ and ‘Punjab Lal’ that have already been reported for possessing resistance against other chilli diseases were also screened against anthracnose.
Inoculum preparation
For the inoculation of chilli germplasm, the cultures of Colletotrichum capsici (ITCC No. 6078) and C. gloeosporiodes (ITCC No. 6270) were procured from Indian type culture collection (ITCC), Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi and were maintained on potato dextrose agar medium at 25 °C under 12 h fluorescent light/12 h dark in BOD incubation chamber for 15 days. Conidial suspension of C. capsici and C. gloeosporioides was prepared separately for each strain in sterilized distilled water by harvesting acervuli/conidia from freshly sporulating cultures (15 days old). Inoculum load was adjusted to 5 × 105conidia per ml using a haemocytometer under a compound microscope and diluted accordingly. Tween-80 were added @ 2% and mixed with prepared conidial suspension and was further used as standard inoculum for carrying out phenotypic studies on chilli landraces and cultivars.
Artificial inoculation
Artificial screening of chilli landraces and genotypes were done under controlled conditions using C. capsici and C. gloeosporioides cultures (Fig. 1). Three to five mature fruits of each genotype were harvested and surface sterilized with 0.1% mercuric chloride (HgCl2) for 2 min. followed by two washings with sterile distilled water. The washed fruits were kept on sterile filter paper for drying. Thereafter fruits were inoculated following a superficial minor injury on fruit at two points with 5 μl conidial suspension of 5 × 105 spore concentration at each point. The inoculated fruits were placed on layers of sterile moistened paper towels and kept in acrylic boxes. The boxes were tightly sealed with plastic bags to maintain more than 90% humidity and kept in dark at 20–25 °C for 48 h. The boxes were then removed from plastic bags and incubated at 25 ± 1 °C with a 12 h dark/light cycle in a small moist chamber [relative humidity (RH) more than 90%]. The lesion length (LL) and/or diameter was recorded 9 days after inoculation and the disease reaction against C. capsici and C. gloeosporioides was assessed using an empirical scale based on lesion diameter or length (Hartman and Wang, Reference Hartman and Wang1992; Garg et al., Reference Garg, Loganathan, Saha, Roy, Kharwar, Upadhyay, Dubey and Raguwanshi2014). According to this scale, accessions were categorized into symptomless (SL, no lesion), highly resistant (HR, 1.0–4.9 mm), moderately resistant (MR, 5.0–9.9 mm), moderately susceptible (MS, 10.0–19.9) and highly susceptible (HS, >20.0 mm).
Fig. 1. In vitro screening of chilli germplasm against Colletotrichum capsici. (a) CC0165 (b) CC0181 (c) CC0183 (d) CC0191 (e) CC0193 (f) CC0164 (g) Pusa Jawala (h) Punjab Lal (i) Pusa Sadabahar (J) IC- 570408.
Field screening
Thirty-one chilli cultivars were grown in the experimental field at TERI GRAM, Gual Pahari, Gurugram, Haryana, in three replications with 10 plants per replication. Conidial suspension of C. capsici with the same concentration was evenly sprayed using an atomizer on 70 days old chilli plants bearing green and red fruits. Un-inoculated plants (three) of each germplasm line were kept as control/check. Disease severity was recorded 9 days after inoculation and per cent disease index (PDI) was calculated by dividing the sum of individual scores with the total number of observations, multiplied by the maximum scale and expressed in percentage (Garg et al., Reference Garg, Loganathan, Saha, Roy, Kharwar, Upadhyay, Dubey and Raguwanshi2014). On the basis of PDI values, genotypes were categorized in six disease reactions: symptomless (SL, 0), resistant (R, 0.1–10), moderately resistant (MR, 10.1–25), MS (MS, 25.1–50), susceptible (S, 50.1–75) and HS (HS, 75.1–100) (Garg et al., Reference Garg, Loganathan, Saha, Roy, Kharwar, Upadhyay, Dubey and Raguwanshi2014).
Statistical analysis
Field screening was done by conducting an experiment in randomized block design for two cropping seasons. The obtained data were subjected to a one-way analysis of variance (ANOVA). The comparative analysis of each chilli germplasm was done at 0.05 and 0.01 per cent level of significance using OPSTAT online Agriculture Data Analysis software (Sheoran et al., Reference Sheoran, Tonk, Kaushik, Hasija, Pannu, Hooda and Hasija1998). Likewise, the data obtained in in vitro screening was also statistically analysed with a completely randomized design (CRD).
Results
During in-vitro assessment of chilli genotypes against both Colletotrichum species (C. capsici and C. gloeosporioides), ‘California wonder’ was found to be a HS while, ‘Punjab Lal’, ‘R-line’ and CC0182 were MS (Tables 1 and 2). During in vitro studies, twenty-six genotypes showed resistant reactions to C. capsici, while, twenty-seven genotypes were resistant against C. gloeosporioides. The chilli germplasms (CC0164, CC0165, CC0186, CC0189, CC0192, CC0202, CC0206, CC0209, CC0217 and CC0218) were found to be highly resistant against both pathogens (C. capsici and C. gloeosporioides) under in vitro conditions. Along with these lines, CC0210 and IC-570408 chilli germplasms also showed a highly resistant reaction against C. gloeosporioides (Tables 1 and 2). Likewise, the disease reaction of 31 chilli indigenous lines have also been screened under field conditions that revealed the majority of the Bhut Jolokia genotypes were resistant to the disease. Eight lines were found to be highly resistant viz.,-CC0164, CC0165, CC0191, CC0192, CC0202, CC0206, CC0209, CC0218; 12 lines were resistant viz.,-CC0154, CC0179, CC0181, CC0183, CC0186, CC0189, CC0193, CC0198, CC0205, CC0210, CC0213, CC0217 and six lines viz. CC0157, CC0161, CC0173, CC0182, ‘Kashi Anmol’ and ‘Punjab Lal’ were moderately resistant to C. capsici infection (Table 3). The rest of the five genotypes were MS (‘IC- 570408’, ‘California Wonder’ and ‘R-line’), and susceptible (‘Pusa Sadabahar’ and ‘Pusa Jwala’) (Table 3).
Table 1. In vitro screening of chilli germplasm against Colletotrichum capsici
HR, Highly resistant; MR, Moderately resistant; MS, Moderately susceptible; HS, Highly susceptible.
a Lesion area.
b Lesion length.
c Disease reaction.
Table 2. In vitro screening of chilli germplasm against Colletotrichum gloeosporioides
HR, Highly resistant; MR, Moderately resistant; MS, Moderately susceptible; HS, Highly susceptible.
a Lesion area.
b Lesion length.
c Disease reaction.
Table 3. Field screening of chilli accessions using Colletotrichum capsici
a PDI, Plant disease index.
b Disease reaction.
SL, Symptomless; R, Resistant; MR, Moderately resistant; MS, Moderately susceptible; S, Susceptible.
Analysis of variance for chilliaccessions screened against chilli anthracnose
F tab. (at 0.05) = 1.65; F tab. (at 0.01) = 2.03; *, **Significance at P = 0.05 and P = 0.01 respectively.
The combined analysis of data obtained by experiments conducted under in field and in vitro conditions revealed such finding as maximum germplasm that are symptomless during field screening were also found to have a high level of resistance against anthracnose under controlled epiphytotic environmental conditions also. Statistically, the disease severity as per lesion length was significantly different among various genotypes.
Discussion
Among different Colletotrichum species C. capsici and C. gloeosporioides have been reported from Indonesia, Taiwan, Vietnam, Papua New Guinea and India are the major cause of anthracnose disease in chilli (Park and Kim, Reference Park and Kim1992; Johnston and Jones, Reference Johnston and Jones1997; Don et al., Reference Don, Van, Phuong, Kieu, Oh and Kim2007; Selvakumar, Reference Selvakumar2007; Than et al., Reference Than, Shivas, Jeewon, Pongsupasamit, Marney, Taylor and Hyde2008b). For the management of anthracnose disease two major strategies are used commonly that includes the use of chemical fungicide or developing strong resistant cultivars. Assessment of pesticide residue revealed that 61% of fresh vegetable samples including green chilli showed the presence of pesticide traces above maximum residue limits (MRLs) in chilli samples tested from Pakistan (Latif et al., Reference Latif, Sherazi and Bhanger2011). Hence, it can be postulated that the chilli landraces having long-lasting resistance could be a great resource for the development of anthracnose resistant cultivar. Bhut Jolokia chilli also contains potent landraces cultivated traditionally since years at Brahmaputra flood plain of Assam, Nagaland, Manipur and other parts of the northeastern region of India (Roy, Reference Roy2016) considered to be a hybrid of C. frutescens and C. chinense (Bosland and Baral, Reference Bosland and Baral2007), later on traced out as distinct species (C. assamicum) (Purkayastha et al., Reference Purkayastha, Alam, Gogoi and Singh2012). Earlier reports based on anthracnose screening of different Bhut Jolokia lines have already shown the presence of resistance (Montri et al., Reference Montri, Taylor and Mongkolporn2009; Garg et al., Reference Garg, Kumar, Kumar, Loganathan, Saha, Kumar, Rai and Roy2013; Mishra et al., Reference Mishra, Rout and Joshi2018). However, the diversity within Bhut Jolokia germplasm regarding anthracnose resistance has not been explored yet by any researchers. In the present study, we have collected twenty-four Bhut Jolokia landraces from different geographical locations of the northeastern region of India and screened against two pathogenic species of Colletotrichum (C. capsici and C. gloeosporioides). Maximum of Bhut Jolokia lines were found to be positive for the existence of resistance genes against anthracnose, however, we have also observed a significant variation in anthracnose resistance among different lines of Bhut Jolokia, it is hypothesized that such effect may arise due to differences in pungency level, as a correlation between pungency and anthracnose resistance has been reported earlier by Azad (Reference Azad1991). Similarly, Tenaya et al. (Reference Tenaya, Setiamihardja and Natasasmita2001) also reported that a higher level of capsaicin content in cultivated red chilli (C. annuum) also linked with a greater level of anthracnose resistance. In addition to this, there are reports present as well in which various anthracnose resistant lines (other than Bhut Jolokia) have also been identified (Prasath and Ponnuswami, Reference Prasath and Ponnuswami2008; Babu et al., Reference Babu, Pandravada, Rao, Anitha, Chakrabarty and Varaprasad2011). In the current study, 31 genotypes were screened under in vitro and in vivo conditions among which 32.0 and 38.71% genotypes screened under controlled conditions showed highly resistant reaction against both C. capsici and C. gloeosporioides, respectively. Whereas under field conditions, 25.81% lines were found highly resistant, 38.71% lines resistant and 19.35% lines moderately resistant to C. capsici. Among 24 Bhut Jolokia lines 11 showed highly resistant, 11 showed moderately resistant and only two in the MS category out of fruit puncture method of C. capsici. The most resistant Bhut Jolokia line was CC0192 with 1.58 mm average lesion length followed by CC0202 1.71 mm and CC0164 with 1.79 mm. Almost similar results were recorded with C. gloeosporioides inoculation where 11 Bhut Jolokia lines showed highly resistant, 11 showed moderately resistant and only two in MS where three most resistant Bhut Jolokia line was CC0192 1.39 mm followed by CC0202 1.55 mm and CC0164 with 1.77 mm of lesion length. None of the Bhut Jolokia lines showed HS reaction only C. annuum cultivars displayed HS and MS reactions. California wonder was found as a HS variety with LL 22.76 mm followed by Punjab Lal and R line with 10.23 and 12.89 mm of lesion length, respectively. Infield screening of Bhut Jolokia lines, eight genotypes were found symptomless, 12 resistant and four in moderately resistant categories. Pusa Sadabahar and Pusa Jawala were found susceptible with 64.67, 50.67% PDI, respectively, followed by California Wonder and IC-570408 with 48.67% PDI. A similar finding has also been reported by Garg et al., Reference Garg, Kumar, Kumar, Loganathan, Saha, Kumar, Rai and Roy2013; Mishra et al., Reference Mishra, Rout and Joshi2018. On the basis of the present findings, it is clear that the Bhut Jolokia lines carry resistant genes against Colletotrichum species. Future attempts could be made towards the crossing of Bhut Jolokia lines as donor parents with commercial cultivars of chilli to develop anthracnose resistant varieties. The only contradiction was found with Garg et al. (Reference Garg, Kumar, Kumar, Loganathan, Saha, Kumar, Rai and Roy2013) reported ‘Punjab Lal’ as an anthracnose resistant genotype but in our study, it was found to be MS. Variation in resistance from resistant to the MS reaction of ‘Punjab Lal’ under in vitro conditions, might be because of change in pathogen virulence or diversity or because of different pathogenic strains. Also, ‘IC- 570408’ line (C. annuum) reported as immune to anthracnose caused by C. capsici (Singh et al., Reference Singh, Kumar, Gupta, Pradheep and Rajasekharan2018) was found MS in our field screening against C. capsici. The virulence shift and resistance breakdown can be tackled by the utilization of various resistant genotypes in resistance breeding and gene pyramiding programs. These obtained results provided us information on the reaction of a few Bhut Jolokia landraces against anthracnose and also an idea that these identified resistant Bhut Jolokia landraces may be harbouring new R-genes against C. capsici and C. gloeosporioides which are yet to be identified. Based on the findings of the present study, it could also be concluded that the genotypes collected from states of the northeastern region of India have resistance against anthracnose disease and in future 20 Bhut Jolokia lines (CC0164, CC0165, CC0191, CC0192, CC0202, CC0206, CC0209, CC0218, CC0154, CC0179, CC0181, CC0183, CC0186, CC0189, CC0193, CC0198, CC0205, CC0210, CC0213 and CC0217) would likely to brought under chilli breeding programs for crossing with commercial cultivars. Moreover, Bhut Jolokia landraces are cross-compatible with C. annuum genotypes. This generated information can be effectively utilized in chilli breeding programs against anthracnose and various other stress factors.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262121000666.
Acknowledgements
The first author is grateful to Science and Engineering Research Board (SERB), India, for providing financial support under the Core Research Grant scheme (File No: CRG/2019/004797) (date 07.02.2020). The authors are also thankful to farmers in India's northeastern region for contributing to the valuable chilli landraces used in this study.
