Control of Pepper yellow leaf curl Indonesia virus and Its Vector (Bemisia tabaci Genn.) on Chili Plants (Capsicum annum L.) Using Resistant Variety and Insecticide Application

PepYLCIV, the cause of chili yellow leaf curl disease, is currently widespread in Indonesia and cause up to 100% yield loss, transmitted by whitefly, namely Bemisia tabaci. The purpose of the current study was to determine the effect of using chili varieties with different resistance to Geminivirus and different frequencies of insecticides application on B. tabaci population, PepYLCIV incidence and severity on two chili varieties with different resistance to Geminivirus. The experimental design used was a Split Plot Design. The main plots were chili varieties consisting of Baja MC F1 and Pilar F1. Sub-plots were the frequency of insecticides application which consisted of three levels, those are: not applied, applied once a week, and applied twice a week. Parameters observed were B. tabaci population, disease incidence and severity which carried out every week (starting 23 days after planting). The results showed, the use of insecticides and varieties with different resistance to Geminivirus had no effect on suppressing B. tabaci populations during the rainy season. Thus, chili plants didn’t need insecticides application during the rainy season. The use of resistant varieties can reduce the incidence and severity of chili yellow leaf curl disease, but both varieties reacted similarly to B. tabaci.


Introduction
One of the most damaging diseases of chili in Indonesia is chili yellow leaf curl disease caused by Pepper yellow leaf curl Indonesia virus (PepYLCIV) [1].Chili yellow leaf curl disease has become an endemic disease in chili plantations in many parts of Indonesia [2].This disease spreads easily and can cause serious yield losses ranging from 20-100% [3].Chili plants that are infected in the vegetative phase suffer greater losses because their fruiting capacity is substantially reduced.While, infection occurs during the generative growth stage reduces the fruit quality [4].The use of unsuitable varieties makes plants susceptible to chili yellow leaf curl disease and causes a decrease in chili productivity [5].One of the chili varieties that is resistant to Geminivirus is the Baja MC F1 [6].However, there has been no related research on the resistance of these varieties to chili yellow leaf curl disease, especially in South Sulawesi as a region where chilies have a great influence on people's daily lives [7].The use of resistant plant is expected to control diseases in the field and is a safe measure to control plant diseases [8].
PepYLCIV can only be transmitted by grafting and its insect vector, the sweet potato whitefly B. tabaci Genn.(Hemiptera: Aleyrodidae) [3].Bemisia tabaci Genn. is a pest that attacks chili plants from the nymph to adult phase.There is a positive correlation between the vector population and the viral disease incidence in the field.The percentage of plants attacked increase with the increasing number of viruliferous whiteflies [9].Thus, to control the disease, most farmers depend on synthetic insecticide with high frequency to control the vector population.This cultivation practice could inflict negative impacts on workers, consumers, and the environment.However, if the insecticide use is used in accordance with predetermined recommendations and applied based on the economic threshold, it can be incorporated into an integrated pest management strategy for the disease and its vector.One of them is the use of low-risk insecticides in integrated pest control (IPM) programs which can minimize the negative impact of synthetic insecticides [10].
Therefore, the purpose of the current study was to determine the effect of using chili varieties with different resistance to Geminivirus and different frequencies of insecticide applications on PepYLCIV incidence and B. tabaci population.

Methods
The study was carried out in the Experimental Farm, Faculty of Agriculture, Hasanuddin University, Makassar, Indonesia, from June 2021 to February 2022.Pepper yellow leaf curl Indonesia virus and its vector, B. tabaci have been naturally present in the farm for the last five years.

Design and implementation of the experiment
The experiment design used in the current study was a Split Plot Design.The main plot consisted of two chili varieties, namely: Baja MC F1 (resistant to PepYLCIV) and Pilar F1 (susceptible to PepYLCIV).Subplots were the frequency of insecticide application which consisted of three levels, namely: control (not applied), applied once a week, and applied twice a week.The insecticides used were low-risk insecticides with the active ingredients spinoteram and abamectin at the recommended dosage and applied alternately using a knapsack sprayer.The first application was carried out 23 days after transplanting.Each treatment combination was replicated four times and each replication consisted of a plot with three rows wide and 20 m long.Plant spacing used was 70 cm between rows and 50 cm between plants in one row, thus, each plot contained 30 plants.The chili seeds used are commercial seeds.Seedbeds and nurseries were confined in a whitefly-proof cage before they were transplanted in the field.One week before planting, 30 kg/plot chicken manure was applied.The experimental plots were covered with silver plastic mulch.The plants were fertilized using NPK Phonska (15:15:15) equivalent to 250 kg/ha one week after planting and subsequent fertilization was carried out every 10 days.Weeds were controlled manually using a hoe and plants were watered as needed using an overhead springler.

Observation
Observations were conducted to determine the number of whitefly adults, nymphs, and eggs; the incidence and severity of chili yellow leaf curl disease.Observation of whitefly adults, nymphs, and eggs was carried out by randomly taking one upper leaf and one middle leaf from each plant randomly selected in a diagonal pattern of five plants in each plot.The number of whitefly adults was determined by slowly turning the leaves and counting the adult present on the lower surface of the leaves.After that, the leaves were picked up and placed inside of a zip-lock bag and then brought to the laboratory.The leaveas samples were observed on the lower surface of the leaves using an insect dissecting microscope with an area of 1 cm x 1 cm in the center of the leaf to count the number of eggs and nymphs.
Observation of disease incidence and severity was carried out every week from 29 days after planting.Disease incidence is determined using the following equation: where, I : disease incidence (%); a : number of virus-symptomatic plants; b : number of surveyed plants.
Determination of the disease severity using the following equation: where, KP : disease severity (%); ni : number of branches that indicate a certain scale value; vi : certain scale (i = 0-4); N : number of branches observed; Z : highest scale value (4).

Statistical analysis
The data obtained in the current study were analyzed quantitatively using parametric and nonparametric tests (P = 0.05) and then continued using Duncan's test (P = 0.05) if there was a significant difference.Quantitative analysis using SPSS 2016 version 23.0.0.0 (SPSS Inc., Chicago, IL, USA).

Number of whitefly eggs, nymphs, and adults
All insecticide frequency treatments on both susceptible and resistant cultivars to PepYLCIV effectively suppressed B. tabaci populations to the levels of significantly lower than those in the control.However, there was no significant differences among the insecticide treatments in the numbers of eggs, nymphs, and adults (Figure 1).

Disease Incidence of PepYLCIV
PepYLCIV infection on the two tested chili varieties with different resistance to Geminivirus showed symptoms of upward rolling of leaf margins, thickening of leaf veins, yellowing of leaf blades, and curling.Symptoms developed further with the leaves in the middle and the top of the plant became bright yellow, while the leaves on the bottom of the chili plant remained green.In general, in all treatments, including control, the resistant cultivar (Baja F1) had significantly lower PepYLCIV incidence than did the susceptible one (Pilar F1).However, for the same cultivar, there were no significant differences among the application frequencies, including the control (Figure 2).

Disease severity of yellow leaf curl disease
Similar to the disease incidence, in all treatments, including control, the resistant cultivar (Baja F1) had significantly lower PepYLCIV severity than did the susceptible one (Pilar F1).However, for the same cultivar, there were no significant differences among the application frequencies, including the control.Baja MC F1 and Pilar F1 reacted resistant and moderately resistant to the disease (Figure 3).

Discussion
Insecticide application on both PepYLCIV-susceptible and resistant cultivars effectively suppressed B. tabaci populations to the levels of significantly lower than those in the control.However, the insect populations were not significantly different among the insecticide frequencies (Figure 1).Both tested cultivars were equally susceptible to B. tabaci.These results suggested that insecticide application was capable of reducing the vector population that eventually suppressing the PepYLCIV incidence.Vector population is positively correlated with PepYLCIV incidence [9].However, no significant differences in vector population among the application frequencies: 0 (control), once a week, and twice a week.During the study, the vector population was relatively very low, it was probably due to the high rainfall rate occurred during the study.This is in agreement with [11] that whitefly population is negatively correlated with rainfall rate.Thus, during the rainy season, insecticide applications are not necessary.All insecticide frequency treatments, including control, the resistant cultivar (Baja F1) had significantly lower PepYLCIV incidence and severity than did those in the susceptible one (Pilar F1).However, in each cultivar, no significant differences among the application frequencies, including the control, were detected (Figure 2 and Figure 3).Thus, vector populations in all treatments are not significantly different but PepYLCIV incidence and severity in the resistant cultivar were significantly lower than those in the susceptible cultivar.The results confirmed that during the rainy season, resistance against PepYLCIV was more important in reducing the disease infection than the role of insecticide.Even,insecticide use in the rainy season seemed to be unneeded.Another interesting phenomenon found in this experiment is that the incidence of the viral disease steadily increased as the season progressed until the end of the observation, although the vector population very low and decreasing.This finding is also supported by the observation of [12], that there were chili plants showing symptoms of severe yellow leaf curl disease in the field, while there was no B. tabaci present in the field.This finding contradicted to [9] that there is positive correlation between vector population and the spread of the viral disease in the field.This probably due to the infection occurred early in the season when rainfall rate was low and vector population high.The infection continuously developed and expressed symptoms later in the season.

Conclusion
The frequency of insecticide application did not affect the population of stadia B. tabaci in chili cultivation in the field during the rainy season.Vector population was so low that insecticide application was not needed during the rainy season.Therefore, management of the disease and its vector should be focused on the use of resistant varieties, such as Baja F1.

Figure 1 .
Figure 1.The average number of whiteflies eggs, nymphs, and adults in the combination of Baja MC F1 and Pilar F1 with different frequencies of insecticide application during 13 weeks of observation

Figure 3 .
Figure 3. Disease severity of PepYLCIV in all combination's treatments of chili varieties and frequencies of insecticide application in the twelfth week of observation.R = resistant, MR = moderately resistant