Understanding the Etiology of Chilli Fruit Rot Disease in Tamil Nadu

Chillies were cultivated all over the world for their vibrant colour, spicy flavour and medicinal properties (Rahman et al., 2011). It is a spice crop of India and plays a significant role in the Indian economy. Although India is a global leader in chilli production (FAOSTAT, 2020), unfortunately its productivity is negatively impacted by more than forty fungal diseases (Rangaswami, 1979). Fruit rot is one of the most threatening pre and postharvest disease of chilli, posing a significant challenge to its profitable cultivation in all the chilli producing regions of the world (Than et al., 2008). It has been reported to be caused by complex pathogens including various species of Colletotrichum, Fusarium and  Alternaria alternata (Machenahalli et al., 2014; Parey et al., 2013). These pathogens extensively damage the fruits and significantly lower the quality, appearance, yield and marketability of the fruits (Suresha et al., 2012).
       
Though there are 24 different species of Colletotrichum documented to cause fruit rot worldwide (Mongkolporn and Taylor, 2018), in India, it is predominantly caused by C. acutatum (Simmonds), C. capsici (Syd.) Butler and Bisby, C. gloeosporioides (Penz) Penz. and Sacc. (Saxena et al., 2016). Fruit rot of chilli is also caused by various species of Fusarium (Zhu et al., 2021; Datar and Ghule, 1985 and Parey et al., 2013). Under congenial environmental conditions, Colletotrichum species complicated fruit rot could result in 80 per cent yield loss (Katoch et al., 2017).  The substantial financial losses due to fruit rot disease is a result of seed borne nature, (Mishra, 1988) pre- and postharvest infection and the complexity of fungal pathogens.
       
Thorough identification of pathogens and accurate characterization of species are pre-requisite to understand the epidemiology of the disease and to establish more effective disease management approaches (Deyol et al., 2015). Thus, studies were conducted to determine the state of chilli fruit rot incidence in different districts of Tamil Nadu and to identify the fungal pathogens associated with fruit rot symptoms using the combination of morpho- cultural and morphological features.

All the experiments were conducted during 2020-2021 at the Department of Plant Pathology, Tamil Nadu Agriculture University, Coimbatore, India.
 
Survey for the incidence of chilli fruit rot disease
 
During 2020-2021, roving surveys were conducted to assess the severity of chilli fruit rot disease in various chilli growing regions of Tamil Nadu, India. Two taluks were selected in each district and in each taluk, four villages were selected and in each village two fields were randomly selected for assessing the disease severity at fruiting stage. One hundred fruits were randomly collected by walking across the field from south-west to north-east corner and disease severity was assessed according to the disease score chart proposed by Montri et al., (2009) (Table 1) and the percentage disease index (PDI) was computed (Wheeler, 1969).
 

 
Isolation of fruit rot pathogens
 
Pathogens were isolated from the infected fruits collected during the survey by tissue segment method. The diseased portion of the fruits were surface sterilized with 1% NaOCl for 1-2 min and rinsed thrice with repeated changes of sterile distilled water (Pappachan et al., 2020). The surface sterilized tissues were inoculated onto sterile PDA medium supplemented with streptomycin sulphate (0.03 g/l) and incubated for 5 days at 28±2°C. Purified cultures were maintained on PDA slants at 25°C and used for further studies.
 
Pathogenicity test and virulence assessment
 
The pathogen cultures were tested for its pathogenicity on chilli fruits by detached fruit assay (De Silva et al., 2017). Fresh healthy unripened fruits (hybrid Ganga) were disinfected in 2% sodium hypochloride solution for 2 minutes followed by 70% ethanol for 30 sec and washed thrice with sterile distilled water. Surface disinfected fruits were inoculated with mycelial agar plugs of each individual isolate by pin-pricking chilli fruit pericarp with a sterile needle to a depth of one mm. Fruits inoculated with plain agar plugs was maintained as control. The Petri dishes were incubated in growth chamber at 25°C and 95-98% relative humidity for 5-7 days. The experiment was repeated twice with three fruits per isolate. In order to validate Koch’s postulates, pathogens were re-isolated from the infected fruits as mentioned previously. The pathogen identity was confirmed by morpho-molecular characteristics. Disease severity on inoculated fruits were assessed 7 days after inoculation (Montri et al., 2009).
 
Identification of fruit rot pathogens
 
The pathogens were identified based on the morpho-molecular characteristics, pathogenicity and by comparison with authentic description given by Booth (1971) and Liu et al., (2016). The cultural characteristics of pathogen isolates such as  colony color, growth pattern and sporulation were studied on PDA medium (Sharma et al., 2014). Semi-permanent slides were made from 10-day-old colonies and the conidial characteristics of various isolates were documented using a phase contrast microscope (Leica DM2000 and DM2000 LED). Three highly virulent isolates viz., TD1, TC2 and TS1 which caused maximum fruit rot infection were confirmed at molecular level by Polymerase chain reaction (PCR) using ITS 1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS 4 (5'TCCTCCGCTTATTGATATGC-3') primer pairs (White et al., 1990). The PCR amplicons were sequenced by Sanger dideoxy method in Biokart India Pvt. Ltd, Bangalore, India. The sequences were submitted to the NCBI GenBank to obtain the accession numbers.
 
Statistical analysis
 
The data were statistically analyzed following standard methods (Gomez and Gomez 1984) and the significant difference between the treatments was found out by Least Significant Difference (LSD) at 5 per cent in the AGRES. The data showing percentages were transformed in to arc sine values.

Survey for the incidence of chilli fruit rot disease
 
The survey revealed that fruit rot disease was prevalent in all the surveyed locations with varied levels of incidence ranging from 44.75 to 72.59 per cent. The disease severity was maximum in Dharmapuri (72.59 per cent) followed by Namakkal (61.88 per cent) and lowest in Nagapattinam district (40.49 per cent) (Table 2). During the survey it was observed that symptoms were observed not only on red ripe fruits but also on green fruits (Fig 1). Present results were in accordance with the findings of Raj and Christopher, (2009) who reported that fruit rot is one of the most devastating disease of chilli with yield loss ranging from 20 to 70 per cent in Tamil Nadu. The highest disease severity may be due to the conducive environmental conditions, cultivation of chilli year after year without crop rotation and susceptibility of cultivar grown in those areas and varying degrees of virulence (Shilpa and Mesta, 2017).
 

 

 
Pathogenicity on chilli fruits
 
In the detached fruit assay typical fruit rot lesions were developed in the fruits inoculated with fungal isolates whereas plain agar plug inoculated fruits remain asymptomatic (control).  The fungal isolates re-isolated from the injected fruits had identical morphologies to the original isolates, proving Koch’s postulates. Among the ten isolates, TD1, TC2 and TS1 isolates obtained from Dharmapuri, Coimbatore and Salem district were found to be highly virulent in causing maximum fruit rot incidence compared to other isolates (Table 3). All the isolates induced fruit rot symptoms in chilli fruits and C. scovillei was found to be highly pathogenic. This is in concurrence with the findings of Liu et al., (2016) who stated that C. scovillei was the most virulent species of acutatum complex affecting Capsicum spp. Further, Than et al., (2008) stated that C. acutatum was a highly virulent species capable of infecting the wound-resistant Capsicum chinense PBC 932. Current findings of Fusarium sp as the casual organism of chilli fruit rot is consistent with the previous reports on the association of Fusarium solani (Mart.) Sacc. (Datar and Ghule, 1985), F. incarnatum (Zhu et al., 2021), Fusarium oxysporum (Yang et al., 2009), Fusarium moniliforme and Fusarium pallidoroseum (Parey et al., 2013) with fruit rot disease.
 
Morpho-molecular characterization of fruit rot pathogens
 
Growth rate of fungal colonies, conidial shape and size of a pathogen were the significant characters for distinguishing among C. gloeosporioides, C. truncatum and C. acutatum (Than et al., 2008). According to morpho-cultural characteristics, ten isolates were classified into three morphological groups (Table 4). Each group exhibited distinct morphology on potato dextrose agar (PDA) medium 10 days after incubation. Isolates from Group 1 produced white colonies which gradually become light brown on upper side and dark brown on reverse side (Fig 2). The mycelial colonies of Group 2 isolates varied from white to pale orange to pale grey. The isolates belonging to Group 3 produced pale grey to dark grey to black cottony colonies. Group 1 and group 3 isolates grew faster while Group 2 isolates recorded medium to sluggish growth. Isolates belonging to group 1 reached full growth in 90 mm Petri plates within seven to ten days, whereas group 3 isolates required approximately eight to ten days and group 2 isolates required approximately twelve to eighteen days. Previous studies have shown that C. acutatum can be differentiated from C. gloeosporioides based on its slower growth rate (Simmonds, 1965). Hence, slow growth of C. scovillei conformed to the characteristics of the C. acutatum complex.
 

 

       
The isolates also reveal substantial variations in their conidial shape. The isolates belonging to Group 1 produced spherical to oval/ellipsoidal microconidia and straight or curved macroconidia. Isolates of Group 2 and 3 produced fusiform and falcate shaped conidia respectively (Fig 3). On the basis of their conidial characteristics, Group 1 isolates correspond to Fusarium sp, Group 2 isolates correspond to C. acutatum complex and Group 3 isolates match the description of C. truncatum. Hence, on the basis of phenotypic and cultural characteristics, the isolates under group 1 were identified as Fusarium sp and group 2 and 3 isolates were identified as C. scovillei and C. truncatum respectively. C. scovillei was reported to be virulent yet slow-growing. Liu et al., (2016) also characterized C. gloeosporioides complex, C. truncatum isolates and C. acutatum isolates in pepper based on the morphological and molecular characters. Mallik et al., (2021) also identified F. solani as casual organism of fruit rot disease of sweet pepper based on morphological features.  The sequences of highly virulent isolates viz., TD1, TC2 and TS1 were provided with accession numbers ON182069, ON178661 and ON178658 respectively which further confirms the identity of the pathogen as C. scovillei.
 

Hence, the fungal pathogens associated with fruit rot disease were identified as Fusarium sp., C. scovillei and C. truncatum based on morpho-cultural characters and further confirmed at molecular level and by pathogenicity tests. Hence, this finding could be the basis for establishing more effective disease management approaches to combat this complex natured infection in chilli cultivation.

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