Reactions of several chili cultivars against Colletotrichum capsici and Colletotrichum gloeosporioides

Anthracnose disease caused by Colletotrichum spp. is one of the most damaging chili diseases. The purpose of the current study was to determine the pathogenicity of two important pathogens of chili anthracnose disease, C. gloeosporioides (Penz) Penz. & Sacc. and C. capsici E.J. Butler & Bisby, on several chili cultivars in laboratory. The experimental design used was a completely randomized design in factorial. The first factor was chili cultivar: Gandewa, Lado, Laris, Pilar, Provost, Bhaskara, and Bara; and the second factor was inoculation method: wounding and non-wounding the fruits. The treated fruits were then incubated in room temperature. Symptom severity was assessed 48 h after the inoculation and every 24 h thereafter for a total of four observations. The results showed that for non-wounding inoculation, all cultivars were highly resistant against C. gloeosporioides and C. capsici with no symptom, except Gandewa was moderately susceptible to C. gloeosporioides. However, for the wounding inoculation, all cultivars were infected by both C. gloeosporioides and C. capsici with varying severity levels. Lado and Pilar were moderately resistant to C. gloeosporioides. For cultivar reaction against C. capsici, Gandewa, Lado, Laris, and Provost were moderately resistant, and the other cultivars were moderately susceptible.


Introduction
Chili (Capsicum spp.) is one the most important vegetable crops worldwide.In 2019, Indonesia produces 1,214,419 tons red chili (Capsicum annum L.) harvested from 133,436 ha of plantation, and 1,374,217 tons of chili pepper (C.frutescens L.) harvested from 166,943 ha of plantation [1].In managing their crops, chili growers constantly face with plant disease as an important limiting factor in their endeavors.Anthracnose disease caused by fungi, Colletotrichum spp. is a devastating disease of chili in Indonesia that can cause yield loss from 25 to 100% [2,3].In addition, the disease can also reduce the product quality, including reduction of phenol, capsaisin, and oleoresin contents of the fruit by 16-69%, 20-60%, and17-55%, respectively [4].Several species of Colletotrichum have been reported infecting chili fruits in Indonesia, Colletotrichum gloeosporioides (Penz) Penz.& Sacc., C. capsici E.J. Butler & Bisby, and C. acutatum J.H. Simmonds [5].
Significant losses to crop due to the infection of Colletotrichum spp.occurs mainly when the fruits are infected by the fungus.Chili growers depends on fungicides to control the anthracnose disease on 1230 (2023) 012115 IOP Publishing doi:10.1088/1755-1315/1230/1/012115 2 chili and they tend to use fungicide excessively [6].This practice increases input costs and can negatively impact non-target organisms and the environment.Besides that, fungicide use is usually less effective when the environmental conditions are suitable for the anthracnose pathogen to develop [9].
To reduce the use of synthetic fungicide, alternative management approaches that are effective yet safe to the consumers, non-target organisms, and the environment should be sought.The use of resistant plants is effective, practical, economical, and safe measures for controlling plant diseases.However, several cultivars are still unknown how they react to different species of Colletotrichum.Therefore, the purpose of the current study was to elucidate the reactions of seven chili cultivars against C. capsici and C. gloeosporioides.

Materials and Methods
Colletotrichum capsici and C. gloeosporioides isolates were originally obtained from anthracnoseinfected chili fruits in Gowa and Maros Regencies, respectively, after identified their morphological characteristics.The isolates were re-cultured separately in Petri dishes containing PDA in room temperature (28 ± 2 0 C) under 12 h of darkness and 12 h of light.Ten days after the cultures were initiated, the conidia were harvested by gently scraping from the culture dishes using a spatula and then filtering it through a double layers of muslin cloth.The conidia were then suspended in sterilized water and the concentration was adjusted to 10 6 conidia per ml.For each fungal species, the treatments were arranged in a completely randomized factorial design with three replications of one fruit each.The first factor was chili cultivar: Gandewa, Lado, Laris, Pilar, Provost, Bhaskara, and Bara.The second factor was inoculation methods: wounding the fruit and nonwounding the fruit.
Red fruits of the seven chili cultivars were harvested from the Experiment Farm, Faculty of Agriculture, Hasanuddin University, Makassar.The surface of the chili fruits was sterilized using 1% sodium hypochlorite solution for three minutes and then flushed with sterile water three times for three minutes each.For the non-wounding inoculation, 10 µL (10 6  conidia/ml) of the conidial suspension was dropped on the middle part of each tested fruit.While for the wounding inoculation method [7], the middle part of the fruits was pricked with a needle about 1 mm deep before 10 µL (10 6  conidia/ml) of the conidial suspension was dropped on the wound.The treated fruits were then placed on top of wet paper towel inside of a sterile plastic box to maintain high humidity ambient.The boxes were kept in room temperature (28 ± 2 0 C) under 12 h of darkness and 12 h of light.Every 24 h.Disease severity was determined using the following equation [7]: Disease severity levels were scored and categorized as follows: highly resistant (HR) = no lesion, resistant (R) = 1-2%, moderately resistant (MR) = > 2-5%, moderately susceptible (MS) = > 5-15%, susceptible (S) = > 15 -25%, and highly susceptible (HS) = > 25%.Disease severities were transformed using a √x transformation before being subjected to ANOVA (P = 0.05).If ANOVA detected a significant difference amongst the treatments, then the treatment means were separated using Tukey test at 0.05.

Results
There were significant differences among treatment combinations (cultivar x inoculation method) in severity of anthracnose symptoms caused by C. gloesporioides for each observation period (Table 1).When the fruits were inoculated using an un-wounding method, all cultivars had symptom severity of 0%, except Gandewa showed anthracnose symptoms starting at 74 h after inoculation (1.59%) and at the end of the assay (144 h) the severity was 3.87%.However, when the fruits were inoculated using wounding method, all cultivars were infected by C. gloesporioides with varying symptom severities.The highest symptom severity occurred on Bhaskara which was consistently significantly higher than the other treatments throughout the study.This was followed by Bara and Gandewa.The lowest severity was found on Lado and Pilar, which were significantly lower than the other treatments.There were significant differences among treatment combinations (cultivar vs inoculation method) in severity of anthracnose symptoms caused by C. capsici for each observation period (Table 2).When the fruits were inoculated using an un-wounding method, all cultivars had symptom severity of 0%.However, when the fruits were inoculated using wounding method, all cultivars were infected by C. gloesporioides with varying symptom severities.The highest symptom severity occurred on Bara and Pilar, which were consistently significantly higher than the other treatments throughout the study.This was followed by Bhaskara, Lado, Laris, and Gandewa.The lowest severity was found on Provost, which were significantly lower than the other treatments throughout the experiment.Table 3 shows that when fruits were inoculated using non-wounding method, Gandewa was infected and reacted moderately susceptible (MS) to C. gloesporoides but the other cultivars were highly resistant (HR) against the pathogen.However, when fruits were inoculated with C. gloesporoides using the wounding method, two cultivars reacted moderately resistant: Lado and Pilar; two cultivars reacted moderately susceptible: Laris and Provost; and three cultivars reacted highly susceptible: Bhaskara,  Bara., and Gandewa.In contrast, for all cultivars, none of the non-wounded fruits was infected by C. capsici but all cultivars were infected by the pathogen when wounding inoculation was used.Four cultivars reacted moderately resistant: Gandewa, Lado, Laris, and Provost; three cultivars reacted moderately susceptible: Pilar, Bhaskara, and Bara; and none reacted highly susceptible to C. capsici.Disease severities caused by C. gloeosporioides and C. capsici are different on different cultivars (Fig. 1).On Pilar, C. capsici inflicted significantly more severe symptom than did C. gloeosporioides; However, on Bhaskara, C. gloeosporioides caused significantly higher symptom severity than did C. capsici.For each cultivar of Gandewa, Lado F1, Laris, Provos, and Bara, both C. gloeosporioides and C. capsici caused comparable amount of symptom severities.

Discussion
Our results showed that when plants were inoculated using non-wounding method, only Gandewa expressed anthracnose symptoms with moderately susceptible reaction to C. gloeosporioides, while the other cultivars did not show any symptoms and reacted highly resistant to the pathogen.Similarly, none of the cultivars tested showed symptoms when they were inoculated with C. capsici using non-wounding method.The results suggested that the cuticle and epidermis of the chili fruits acted as an effective mechanical barrier to prevent infection to occur.This agrees with [8] reported that resistant chili cultivars against C. gloeosporioides had thicker cuticular layer compared to the susceptible ones.When the physical barrier was broken by pricking the cuticular and epidermal layers, however, all cultivars were infected by both species of Colletotrichum but with different levels of severity.This suggested that the tested cultivars could have different levels of chemical resistance.Resistant chili cultivars against C. capsici produce higher total content of orthodihydroxy phenols than the susceptible cultivars.In addition, higher activity of the enzymes of peroxidase, polyphenol oxidase, and phenyl ammonia lyase occurred in resistant than susceptible genotypes [9].
In general C. gloeosporioides is more virulent than C. capsici to the cultivars tested in this experiment.C. gloeosporioides was capable of infecting Gandewa even when the fruits were not wounded.Besides that, three cultivars are highly susceptible to C. gloeosporioides: Gandewa, Bhaskara, and Bara; while none of the cultivars was highly susceptible to C. capsici (Table 3).In the Description of Chili Cultivars [10], Bara has field resistance against anthracnose disease but in our study the cultivar was highly susceptible to C. gloeosporioides and moderately susceptible to C. capsici when the fruits were wounded.This indicated that mechanical/structural resistance is stronger than chemical resistance in this cultivar against the anthracnose pathogens.While, Lado is listed as a tolerant cultivar to anthracnose [11,12], in our study when fruits were wounded, the cultivar was moderately resistant.This indicated that this cultivar probably has two mechanisms working against the pathogens: structural and chemical resistances.
Different cultivars reacted differently to both species of the pathogen.Bhaskara is more susceptible to C. gloeosporioides than C. capsici, on the other hand, Pilar is more susceptible to C. capsici than C. gloeosporioides.The other cultivars had comparable reactions to both pathogen species (Fig. 1).
Implications of our findings to the management of anthracnose disease on chili are: 1) proper identification of the pathogen species is very important.By knowing the precise Colletotrichum species that is predominant in an area, then appropriate cultivars could be planted to avoid significant damages caused by the anthracnose disease; 2) since infection can occur before harvest and continue in the storage, anthracnose damages can be reduced by preventing fruit wounds during the harvest and produce handling.

Conclusion
Structural barriers -cuticular and epidermal layers -apparently played an important role in the resistant reaction of cultivars tested against C. gloeosporioides and C. capsici.In general, chili cultivars tested were highly resistant against the pathogen when inoculated using non-wounding method.However, when inoculated using wounding method, all cultivars were infected with different levels of severity.

Table 1 .
Severity of anthracnose disease symptoms caused by C. gloesporoides on seven pepper cultivars with wounding and non-wounding inoculation methods.
Numbers followed by different letters are not significantly different (P ˃ 0.05).Means were separated using Tukey's test at 0.05 level.

Table 2 .
Severity of anthracnose disease caused by C. capsici on seven pepper cultivars with wounding and non-wounding inoculation methods.

Table 3 .
Cultivar reactions to anthracnose disease cause by C. gloeosporioides and C. capsici with wounding and non-wounding inoculation methods.