Potency of Yeasts to Promote Induced Resistance on Chili Plant ( Capsicum annuum L . ) against Yellow Leaf Curl Disease

Gemini virus, the cause of pepper yellow leaf curl virus disease (PYLCV), is a pathogen that cause the low production of chili. The disease was reported to spread in the center of chili production in Indonesia, and caused production loss up to 100% (Hidayat et al., 2006; Adilah et al., 2014). PYLCV is disseminated by Bemisia tabaci, the vector. The rapid population growth of Bemisia tabaci makes it becomes a potential virus disseminator (Adilah et al., 2014). The farmers usually control the vector by insecticide application. Induced resistance on plants may be utilized as an alternative in controlling PYLCV. This method is effective and save to the environment. The induced resistance on plants can be activated by biotic as well as abiotic agents (Kloepper et al., 1992). The plant response of induced resistance will be detected after the pathogen infection (Olivera et al., 2016), involves elicitors that initiate and increase the biosynthesis of certain compounds (Namdeo, 2007). The elicitors induce the production of secondary metabolites by activation of secondary path in response to stress and pathogen infection. Elicitor in plant defense mechanism is an effective strategy to increase production of secondary metabolites (Sharma et al., 2009). Elicitor can further results in plant defense response, initiated by the formation of Reactive Oxygen Species (ROS) (Habibullah et al., 2018). Antagonistic microbes are reported to be able to initiate induced resistance in plants. Yeasts can also function as inducer of plant resistance. Some yeasts were reported to induce plant resis tance, Saccharomyces cerevisiae was one of them. S. cerevisiae was reported to induce resistance against downy mildew on wheat by increasing the production ABSTRACT


INTRODUCTION
Gemini virus, the cause of pepper yellow leaf curl virus disease (PYLCV), is a pathogen that cause the low production of chili. The disease was reported to spread in the center of chili production in Indonesia, and caused production loss up to 100% (Hidayat et al., 2006;Adilah et al., 2014).
PYLCV is disseminated by Bemisia tabaci, the vector. The rapid population growth of Bemisia tabaci makes it becomes a potential virus disseminator (Adilah et al., 2014). The farmers usually control the vector by insecticide application.
Induced resistance on plants may be utilized as an alternative in controlling PYLCV. This method is effective and save to the environment. The induced resistance on plants can be activated by biotic as well as abiotic agents (Kloepper et al., 1992). The plant response of induced resistance will be detected after the pathogen infection (Olivera et al., 2016), involves elicitors that initiate and increase the biosynthesis of certain compounds (Namdeo, 2007). The elicitors induce the production of secondary metabolites by activation of secondary path in response to stress and pathogen infection. Elicitor in plant defense mechanism is an effective strategy to increase production of secondary metabolites (Sharma et al., 2009). Elicitor can further results in plant defense response, initiated by the formation of Reactive Oxygen Species (ROS) (Habibullah et al., 2018).
Antagonistic microbes are reported to be able to initiate induced resistance in plants. Yeasts can also function as inducer of plant resistance. Some yeasts were reported to induce plant resistance, Saccharomyces cerevisiae was one of them. S. cerevisiae was reported to induce resistance against downy mildew on wheat by increasing the production of phytoalexin (Walters et al., 2005). El- Mougy et al. (2013) reported that S. cerevisiae at 10×10 10 cfu/ml decreased the disease intensities of downy mildew and powdery mildew on tomatoes, pepper, and cucumber. The decrease was correlated with the increase of peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, ß-1,3-glucanase and chitinase activities, both locally and systemically (El-Mougy et al., 2013). Shan et al. (2014) reported that yeast XL-1 isolate increased the production of plant resistance enzymes such as polyphenol oxidase, SOD, and β-1, 3-glucanase on melon.
Rhodotorula sp. was also reported to systemically induce potato plant resistance to potato virus Y (PVY) (Al-Ani et al., 2013). As there was no direct contact between PVY and Rhodotorula sp., the resistance manifested in the plant against the virus could be attributed to some form of induced systemic resistance. Pichia membranefaciens induced the peach resistance to Penicillium expansum (Chan et al., 2007), Metschnikowia fructicola induced the citrus resistance to Penicillium digitatum (Hershkovitz et al., 2012). Debaryomices hanseni decreased the symptoms of Ulocladium chartarum infection, the cause of papaya rot, by more than 75% (Sharman et al., 2011). Furthermore, Candida saitoana at 10 8 cells/ml controlled Botrytis cinerea on apples by 50-70% on day 7 after yeast application (El Ghaout et al., 2003). The systemic resistance to B. cinerea was increased with the increase of chitinase and β-1,3-glucanase activity in systemically protected tissue (El Ghaout et al., 2003).
Rhodotorula minuta (isolate of Dmg16BEP) and Candida tropicalis (isolate of Lm6BE) are yeasts isolated from chili fruit and they were proven to control anthracnose on chili by direct antagonism mechanism (Hartati, 2016). Those yeasts suppressed anthracnose by hyper parasitism mechanism, formation of volatile compounds and formation of ACC deaminase enzyme. R. minuta was also reported to initiate induced resistance on chili plants against anthracnose pathogen by increasing peroxidase enzyme activity, and C. tropicalis was able to increase the chili plant growth (Hartati et al., 2019a;2019b). The ability of those yeasts in inducing the plant resistance to the PYLCV is not known yet. This study was objected to evaluate the potencies of R. minuta (Dmg16BEP) and C. tropicalis (Lm6BE) as induced resistance agents on chili plants to yellow leaf curl virus.

MATERIALS AND METHODS
The experiment was carried out at the Laboratory of Plant Protection Biotechnology and Green House of the Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Padjadjaran. It was arranged in the randomized complete block design. The treatments were 2 yeast species of R. minuta (isolate of Dmg16BEP) and C. tropicalis (isolate of Lm6BE) and a control, and 3 different inoculation times at 3, 7, and 10 days after transplanting (dat), as follow: A. R. minuta, virus inoculation at 3 dat, B. R. minuta, virus inoculation at 7 dat, C. R. minuta, virus inoculation at 10 dat, D. C. tropicalis, virus inoculation at 3 dat, E. C. tropicalis, virus inoculation at 7 dat, F. C. tropicalis, virus inoculation at 10 dat, G. control, virus inoculation at 3 dat, H. control, virus inoculation at 7 dat, I. control, virus inoculation at 10 dat. The treatments were replicated 3 times. The number of plants per replication was 10. The yeast isolates were applied two times. First the chili seeds were soaked in the yeast suspensions before planting, second the suspensions were poured into the media at transplanting. The virus was inoculated according to the treatments, which were at 3, 7, or 10 dat. The data were analyzed by Analysis of Variance (ANOVA) using SPSS Version 24.0 for windows. If there were significant differences, the data were then analyzed by Duncan multiple range test at 5%.

Preparation of Yeast Cultures and Suspensions.
R. minuta and C. tropicalis were first isolated from chili fruit (Hartati, 2016). Two isolates were chosen for this study, i.e. R. minuta isolate of Dmg16BEP and C. tropicalis isolate of Lm6BE. The yeasts were sub cultured on PDA and incubated at room temperature. The suspensions were made by adding 10 ml sterile distilled water on the cultures in the Petri dish, and serially diluted to 10 -8 . The yeasts population densities were counted by using hemocytometer, up to10 8 cells/ml.

Rearing of B. tabaci and Preparation of Yellow Leaf Curl Virus Inoculum
The insect was reared on chili plant in iron frame cages covered with cloth, in the glasshouse. The virus was obtained from diseased chili plants cultivated in Jatinangor. To multiply the virus inoculum, nymphs of B. tabaci, the vector, were used to transmit the virus from the diseased plants onto healthy chili plants. 35

Preparation of Chili Plant Growth Media
The seed growth media was a mixture of soil and chicken manure 2:1. The chili seeds were planted in pot-tray 54 cm × 28 cm × 4.2 cm. The seedlings were transplanted on the 21 st day onto polybags 18 × 16 cm. The transplanting media was the mixture of soil and chicken manure 15,000 kg/ha, and SP-36 150 kg/ha. Urea 200 kg/ha, ZA 450 kg/ha and KCl 150 kg/ha were applied 3 times on 3, 6 and 9 weeks after transplanting (wat).

Yeast Treatments
The yeast treatment was initiated with soaking the chili seeds in the suspensions of R. minuta and C. tropicalis at 10 8 cells/ml for 60 minutes, and then the seeds were wrapped in sterile wet tissue paper and incubated for 12 hours. The treated seeds were planted in the pot-tray. On the 21 st day, the seedlings were transplanted and the yeast suspensions of R. minuta and C. tropicalis were reapplied by pouring 10 ml of each yeast suspension per plant onto the media. After the treatments, the chili plants were inoculated with the virus by using the vector, B. tabaci, at 3, 7, and 10 dat.

Inoculation of Pepper Yellow Leaf Curl Virus
The PYLCV was inoculated by using B. tabaci. Nymphs of B. tabaci were placed and fed on diseased chili leaves, the acquisition was set for 24 hours. To inoculate the virus, those nymphs were placed and fed on healthy chili plants at 3, 7, and 10 dat, according to the treatments. The inoculation period was 24 hrs, 5 nymphs per plant.

Incubation Period, Disease Severity, and Disease Incidence
The effect of the yeasts application on virus inoculated chili plants was assessed by incubation period, disease severity, and disease incidence. Incubation period was measured since the day of PYLCV inoculation until the first day the disease symptom was visible. Disease severity and disease incidence was assessed on 14, 21, and 28 dat. The scoring of yellow leaf curl disease on chili was adapted from Trisno et al. (2010) (Table 1). Disease severity was calculated by the formula (Cooke, 2006): N = Total sample number n = Number of sample with the same score Y = The highest score y = Examined score Disease incidence was calculated by the formula (Cooke, 2006):

Ability to Cause Induced Resistance
The ability to cause the induced resistance was calculated by using the formulae as follow (Trisno et al., 2010): a. Effectiveness in reducing disease incidence EiP = Effectiveness in reducing the disease incidence Dt = Disease incidence on plants treated with the yeasts Dc = Disease incidence on control plants (control +) b. Effectiveness in reducing disease severity EiS = Effectiveness in reducing the disease severity Dts = Disease severity on plants treated with the yeasts Dcs = Disease severity on control plants (control +)

Peroxidase, Phenylalanine ammonia lyase (PAL) and Salicylic acid (SA) Analysis
Analysis of peroxidase, PAL and SA were carried out in the Central Laboratory of Universitas Padjadjaran. Peroxidase activity was analyzed by the modified procedure of Simons & Ross (1970), PAL activity was analyzed by the procedure of Lisker et al. (1983), and SA accumulation was analyzed by high performance liquid chromatography (HPLC) modified from the method of Tenhaken & Rubel (1997).

Effect of Yeasts on Incubation Period
The time needed by plants to response to induction is different, and the response will be expressed after the challenge by pathogen infection. The time of the plant response can be estimated by inoculating the pathogen at different times. Induced resistance treatments by R. minuta at virus inoculation at 3, 7 and 10 dat were able to delay the incubation period compared to control, but C. tropicalis could delay the incubation period only on virus inoculation at 7 dat. R. minuta was able to delay the incubation period by 0.67-10 days, but C. tropicalis only delayed the incubation by 2 days (Table 2). Rusli et al. (1999) reported that the incubation period of yellow leaf curl virus, transmitted by vector B. tabaci, without induced resistance treatment was shorter (10-15 days). The induction treatments were able to delay the symptom development of the yellow leaf curl virus caused by Gemini virus. Gunaeni et al. (2015) reported that the incubation of yellow leaf curl virus was 21 days after inoculation on various induced resistance treatments.
The symptom of yellow leaf curl virus on chili plants was first detected on younger leaves or buds as yellow spot around the leaf vein. The spots developed, and extended into vein clearing, concave and curly leaf with slight mosaic or yellowing. The symptoms progressed and became yellowing on all young leaves and buds, some with green mosaic, concave and curly leaves, smaller and thicker leaves. The disease symptoms on yeast treated chili plants were the same as the symptoms on control plants with no yeast treatment.

Effect of Yeasts on Disease Incidence and Severity
Application of R. minuta and C. tropicalis did not affect the disease incidence (Table 3). However, compared to control, although they were not significantly different, lower disease incidences on treatments were observed at 21 and 28 dat. At 28 dat, the decrease was on treatment with C. tropicalis inoculated at 3 and 10 dat, and treatment of R. minuta inoculated at 10 dat. The disease incidence on the treatment of C. tropicalis, virus inoculation at 3 dat, on the 21 st day was 23,33%, and on the 28 th was 33,33%. These were lower than control which were 33,33% on the 21 st and 60% on the 28 th day, although statistically they were not different. There was only disease incidence on treatment of C. tropicalis,   virus inoculation at 3 dat, which was different to control, virus inoculation at 10 dat, on the 21 st day ( Table 3). The disease incidences on treatments of R. minuta and C. tropicalis with virus inoculation on the same dat were not different to control. The effect of treatments on the yellow leaf curl disease severity was only detected at 21 dat. There were differences between treatments of R. minuta inoculated at 3 dat and C. tropicalis inoculated at 7 dat compared to control (Table 4). The lowest disease severity (5.83 %) was caused by treatment of R. minuta inoculated at 3 dat. The best response to the treatments was shown by those plants inoculated at 3 and 7 dat. When an inducer promotes plant resistant to disease, the plant will need some times to activate the resistance genes. The resistance expression was shown when pathogen started to infect (Ouchi, 1983). Different plants with different inducers need certain time to activate the resistance genes. However, in this study, the yellow leaf curl disease severities on the same virus inoculation times were not different to control, both by treatment of R. minuta and C. tropicalis.

Effect of Yeasts on the Effectiveness of the Disease Incidence and Disease Severity Suppressions
Application of the yeasts did not affect the yellow leaf curl disease incidence at 14 days after virus inoculation, at all inoculation times. Based on the effectiveness value of disease incidence on chili plants, at 14 days after virus inoculation, there were three treatments that increased the chili plant resistance to Gemini virus, i.e. treatments of R. minuta virus inoculation at 7 dat, R. minuta virus inoculation at 10 dat, and C. tropicalis virus inoculation at 7 dat, with the effectiveness values of 50-70% (Table 5). However, at 21 to 28 dat, the suppressions were less than 50%, which meant the treatments were not effective (Table 5).
Based on the effectiveness value of disease severity on chili plants, treatments of R. minuta, virus inoculation at 7 dat and treatment of C. tropicalis,   virus inoculation at 7 and 10 dat suppressed the disease severity at 14 dat (Table 6). The highest suppression of disease severity was caused by the treatment of R. minuta, virus inoculation at 7 dat (70%). At 21 and 28 dat, the treatments caused less than 50% effectiveness of disease severity suppression. However, in this experiment, the disease incidences and disease severities on yeast treatments and control were not different.

Effect of Yeasts Treatments on Activities of Peroxidase and PAL, and Accumulation of Salicylic Acid
Results of the enzyme analysis showed that treatments of R. minuta and C. tropicalis, with virus inoculation at 7 dat, increased the peroxidase activity compared to control ( Table 7). The peroxidase activity was increased at 0.5 and 2.5 minutes of incubations. Treatment with R. minuta resulted in higher peroxidase activity than treatment with C. tropicalis. However, the increase of peroxidase activity was not followed by the decrease of disease severity (Table 4). The increase of PAL activity was observed on the treatment with R. minuta. PAL activity was in accordance with the concentration of PAL. The higher the PAL concentration, the higher the PAL activity. C. tropicalis did not increase the PAL activity (Table 7). This was in line with the effectiveness value of disease severity suppression and the extended incubation period on treatment of R. minuta.
In induced resistance, the enzyme activities in the path of certain metabolites are increased, and some enzymes such as chitinase, β-1,3-glucanase, peroxidase, pathogenesis related (PR) proteins are also increased (Park & Kloepper, 2000). Synthesis of these proteins was regulated at the mRNA (Park & Kloepper, 2000). R. minuta and C. tropicalis increased the activity of plant resistance enzymes on 7 dat. However, the increase of peroxidase and PAL activities in this experiment only delayed the disease incubation period for 0.67-10 days and 2 days on the treatments of R. minuta and C. tropicalis respectively (Table 2).
Some studies on the time period of the production of enzymes related to plant resistance showed that at 7 dat the enzymes activities were increased. Some yeasts such as Candida saitoana, Cryptococcus laurentii, and S. cerevisiae were reported to promote systemic induced resistance by increasing the activities of some enzymes related to plant resistance Table 6. Effectiveness of disease severity suppression Notes: dat = day after transplanting; EiS = 1-(Dts/Dcs) × 100 %; X = average. such as chitinolitic enzymes, β-1,3-glucanase, peroxidase, polyphenoloxidase, phenylalanine amonia-lyase at 7 dat (El Ghaouth et al., 2003;Yao & Tian, 2005;El-Mougy et al., 2013;Shan et al., 2014). Analysis of salicylic acid was performed by using HPLC, it revealed that the retention periods between treatments were almost the same with the salicylic acid standard (2.125 minutes). This showed that the plant samples contained salicylic acid. The accumulation of salicylic acid was increased on chili plants treated with R. minuta and C. tropicalis with virus inoculation at 7 dat. Induction resistance by R. minuta and C. tropicalis caused high accumulation of salicylic acid content compared to control (Table 8). This showed that the treatment of induced resistance by R. minuta and C. tropicalis, virus inoculation at 7 dat, increased the accumulation of salicylic acid. The increase of salicylic acid at 7 and 10 dat was also reported to suppress CMV infection (Taufik et al., 2010). In this study, although salicylic acid accumulation was increased, but the disease severity was not decreased.
Salicylic acid is important in the plant defense mechanism against pathogen. It was reported in some studies that salicylic acid had the roles in increasing the plant resistance to virus infection (Taufik et al., 2010;Faizah et al., 2012;Gunaeni et al., 2015). Accumulation of salicylic acid is a fast reaction of plant to defense against virus infection by mobilization of secondary metabolites (Faizah et al., 2012). Indirectly, it inhibits virus movement in the plant vascular tissues, so the disease symptoms were delayed (Naylor et al., 1998). The accumulation of salicylic acid in this study caused the delay on the incubation period of the yellow leaf curl virus disease. Salicylic acid has a role as signal transduction that activate resistance genes against various pathogens systemically, so that the incubation period is extended (Murphy et al., 2001;Gautam & Stein, 2011;Narusaka et al., 2015).
The increase of peroxidase and PAL activities, and salicylic accumulation showed the role of elicitor in signal formation and transduction. The elicitor is received by the receptor in plant. In this experiment, the yeast cells and their metabolites act as the elicitors. It is in accordance with Walters et al. (2005) that the active recognition process between yeast cells and plant tissues was caused by the yeast that actively secreted elicitor. Oligosaccharide fragments derived from the polysaccharide of yeast cell wall were proven to act as elicitors that actively promote induced resistance in the host plants (Talarczyk et al., 2002;Walters et al., 2005;Sharma et al., 2009).
The increase of peroxidase activity from 2590.80 units to 6870.93 units (0.5 minute) and from 577.367 units to 1131.300 units (2.5 minutes), PAL activity from 16.059 to 17.911 A290/mg, and accumulation of salicylic acid from 2.785 to 6.263 ppm in this experiment did not reduce the disease incidence and severity. The increase of peroxidase and PAL activities, and accumulation of salicylic acid caused by the induction treatment of R. minuta and C. tropicalis only extend the incubation period. This condition was assumingly caused by the inadequate concentration of the enzymes and the salicylic acid accumulation to support the plant in combatting the virus infection, the yeasts were applied only by soaking the chili seeds and pouring the suspensions into the media on transplanting, and the severe virus infection compared to the ability of the yeasts in increasing the concentration of the enzymes and the salicylic acid accumulation. It was recommended to reapply the yeast suspension on chili plants at certain age, and the observation on the older chili plant.

CONCLUSIONS
The resistance induction on chili plant by R. minuta and C. tropicalis delayed the incubation period of yellow leaf curl virus disease for 0.67-10 days and 2 days respectively. The treatments of R. minuta and C. tropicalis on chili plant did not decrease the disease incidence and severity of yellow leaf curl virus disease. However,the treatments of R. minuta and C. tropicalis on chili plant increased the peroxidase activity by 2.3-2.7 times higher than control, at 0.5 minute incubation (2590.80 units to 6870.933 units), and increased the salicylic acid accumulation 2.2-2.5 times higher than control (from 2.785 to 6.982 ppm). Resistance induction treatments of R. minuta increased the activity of PAL (the difference was 1.852 A290/mg, i.e. from 16.059 to 17.911 A290/mg).

ACKNOWLEDGEMENT
We would like to thank the Directorate of Research, Community Service and Innovation of Universitas Padjadjaran for funding the research through an Internal Research Grant of Universitas Padjadjaran 2018.