POTENTIAL EFFECT OF RHIZOBACTERIA Streptomyces sp. AND Bacillus sp. to GEMINIVIRUS INFECTION AND RED CHILI ( Capsicum annum L.) QUALITY PRODUCTION

The chili yellow leaf curl disease is one of the biggest obctacle in chili’s production in Indonesia. This diseases only transmitted by whitefly (Bemisia tabaci). One of the disease controlling method is by using Plant Growth Promoting Rhizobacteria (PGPR) to reduce pesticide application for the health and environment. The aim of this research is to find out the effectiveness of Streptomyces sp. and Bacillus sp. in supressing yellow leaf curl disease, plant growth and chili harvest. This research has conducted at the experimental farmer’s field in Harjobinangun, Pakem, Sleman, Special Region of Yogyakarta. Using varieties of chili Twist 42, Streptomyces isolates which from Bacteriology Laboratory collection’s with 10 -8 CFU/ml and Bacillus isolates which is from Mycrobiology Laboratory collection with 10 -8 CFU/ml. The result of this research showed that the treatment of combination between Bacillus sp. and Streptomyces sp. could decrease and supress disease incidence and disease intensity if it compared with others treatment. Parameters of plant growth included plant high, stem diameter, root length, root volume and number of productive branch showed that single of Streptomyces sp. and Bacillus sp. without combination were better than combination and untreated. The single application Streptomyces sp. or Bacillus sp. could increase amount of total number and weight of fruits.


INTRODUCTION
Chili (Capsicum annuum L.) is one of the most important vegetable commodities in Indonesia due to its high economic value. According to BPS (2021) stated that the production of national chili in 2021 is decreasing compare with last year. One of the issues for the chili productivity fluctuation is pest and diseases infection which attacks plants from seedling stage to the harvesting stage of plant.
Yellow leaf curl disease is emerged as a serious constraint to chili production in Indonesia. This disease is caused by Geminivirus (Famili : Geminiviridae) that was reported in Sleman and is known to cause 20-100% losses to the marketable fruits (Sumardiyono et al. 2003). Epidemics of chili yellow leaf curl disease have occurred in several chili growing centers in Central Java, West Java, and the Special Region of Yogyakarta since the early 2000 (Sulandari et al . 2006). Geminivirus causes huge yield losses and a decrease in vegetable crop production (Thakur et al. 2018). The symptoms of this disases was observed in the field, involving yellow mosaic, mottle, and yellowing. Green mosaic on the top of leaf was the early symptom chili pepper infecting viruses, followed by cupping upward or cupping downward and yellowing, plants will become dwarfs when infected with the virus as the seeds form (Selangga & Distihani 2021).
Therefore, proper countermeasures to control disease caused by Geminivirus are needed. Several efforts to reduce Geminivirus infection are planting varieties that are disease resistant. One way is by inducing resistance by using PGPR (Plant Growth Promoting Rhizobacteria) to enhance plant's resistance.
PGPRs are saprophytic bacterial microorganisms that are free-living in the rhizosphere and colonize the root system aggressively (Ramjegathesh et al. 2013). Plant roots with bacteria have a beneficial impact on the development and production of crop plants (Fasusi et al. 2021). The beneficial bacterial species is the PGPR that lives in the soil found around the plant's root, influencing plant development and is profitable health-related. They are agricultural biological resources that induce the plant's growth and fruitfulness. They also motivate resistance in plants, a wide range of vegetation of fruits, vegetables, and various forest trees, to different phytopathogens (Zia et al. 2020). Streptomyces sp. and Bacillus sp. are reported as PGPRs.
Bacillus spp. in controlling plant diseases through several mechanisms such as competition, inducing systemic resistance in plants and excreting extracellular metabolites such as antibiotics, cell wall hydrolases and siderophores (Miljakovic et al. 2020). Plant root application of Bacillus subtilis can triggered ISR against Tobacco Mosaic Virus by activating the signaling defense genes (PR-1a and PR-1b), and regulatory genes (NPR1 and Coi1) indicating salicylic acid signaling pathway activation (Wang et al. 2009). According to Arwiyanto et al. (2009) also reported the results of his research that the antagonistic mechanism of Streptomyces as a biological control agent for bacterial wilt is an antibiosis mechanism by producing antibiotics. Streptomyces can inhibit the virus through a variety of mechanisms, including the activation of plant defense pathways and the production of signaling molecules. Those strains induced the systemic acquired resistance (SAR) to virus infections (Majunatha et al. 2022). The aim of this research is to find out the effectiveness of Streptomyces sp. and Bacillus sp. in supressing yellow leaf curl disease, plant growth and chili harvest.

MATERIALS AND METHODS Study Area
The study was conducted in farmer's field at Cepit, Pakem, Yogyakarta; while other work was done at the Laboratory of Plant Virology, Faculty of Agriculture, Universitas Gadjah Mada. Field study used a Complete Randomized Block Design (RCBD) with three blocks and four treatments, which were Bacillus sp., Streptomyces sp., combination both Bacillus sp. and Streptomyces, and untreated (without using biological control and soaked by pure water). Each block was four treatments and 10 chili plants were taken as samples for observation. The variety of chili seeds used in this study was Twist 42. It was planted with spacing 50 cm in row and 60 cm between rows. The chilli seeds were then soaked in a suspension of each treatments. Previously, the seeds were rinsed using pure water to remove commercial coating. They were single treatment of Bacillus sp., single treatment of Streptomyces sp., combination both of Streptomyces sp. and Bacillus sp. and untreated by pure water for 60 minutes. 10 8 CFU/ml suspension of Bacillus sp., Streptomyces sp., and their combination were applied into each planting hole before planting.

Diseases Severity and Plant Growth Observation
The observed parameters were root length, root volume, total of productive branches, fruit length, diameter fruit, total of fruit, weight of fruit, total of fruit every harvested, and weight of fruit every harvested. Plant height, stem diameter and diseases severity were observed after fifth weeks during nine times. There were divided into 6 times at the vegetative stages on 6, 7, 8, 9, 10, 11 week after planting and 3 times at the generative stages on 12, 19, 23 week after planting. Yellow leaf curl diasease were observed using the following formula (Ismiyatuningsih et al., 2016): : > 5% -25% leaves curl, yellow and begin to swell in the veins 3 : > 25 -50% leaves curl, yellow and the proportion of malformations leaf (10 -25%) 4 : > 50 -75 % leaves curl, yellow and the proportion of malformations leaf (26 -50%) 5 : >75 % leaves curl, yellow and the proportion of malformations leaf (>50%) and the plants are stunted Measuring disease incidence can also be using with a formula :

Data Analysis
The chili crops were harvested at twelves weeks after planting in ten samples. They were harvested and observed every three day times. Chili crops would be divided into five categories; healthy, mild malformation, moderate malformation, severe malformation and rotten. The variables observed were total of fruit and weight of fruit. Proximate test data were analyzed using Analysis of Variance (ANOVA) and Duncan Multiple Range Test (DMRT) with 5% level.

RESULTS AND DISCUSSION
The symptoms were shown for each plant infected with the Begomovirus in the field, but the initial symptoms were shown by yellowing of the leaves or experiencing chlorosis. The severe symptoms of this virus infection are shown by the yellowing leaf curl, cuppings (bowl), leaves malformations and then the plants become stunted. Infection of young plants will make greater yield loss when it was compared to Begomovirus infection at the generative phase ( Figure 1). According to Sulandari (2006) the symptoms of yellow leaf curl disease caused vary greatly depending on the cultivar, virus strain and physical environment. Begomovirus infection in chilies has very distinctive symptoms, namely thickening of the leaf veins, leaf edges curling upwards and bright yellow leaf blades. Even though the pathogen does not kill the plant, in severe symptoms it causes new leaves to grow smaller, flowers fall off and do not produce fruit.
Observation of disease development in a single application of Streptomyces sp., Bacillus sp., a combination of both and controls showed different results. Disease incidence is the ratio of the number of plants infected with the pathogen to the entire plant population. Incidence is indicated by counting the number of unhealthy plants population to the total plants population. While disease severity is the severity of the disease which is calculated using scoring on plants. The results were not significantly different for both the diseases incidence and severity between all the treatments were showed on Figure 2 and Figure  3.   Table 1 the result of diseases incidence at the vegetative stages on 11 th week after planting were shown significantly different among the treatments. The combination of Bacillus sp. and Streptomyces sp. were effective to suppress the development of yellow leaf curl disease in chilli at the initial stage of plantation. But the other results were not significantly different between the treatments both of diseases incidence and severity on 23 rd week after planting. Meanwhile, for the diseases severity was showing good trend for yellow leaf curl suppression. According to Widodo et al. (2023)  Bacillus sp.

Combination of B+S
Untreated expression of symptoms in plants infected with Begomovirus is also influenced by the virus strain and the time of infection. The chili juveniles can experience decay and be unable to produce when contracting the Begomovirus disease. The treatments used tend to inhibit the diseases severity during the generative stages, so the losses of yield could be decreased. In the experimental garden, Bemisia tabaci as vector mostly found at the generative stages Figure 2 and Figure 3. The growth and development of chili plants were vegetative and the generative stages. The vegetative phase was the phase that starts from seed germination, grows into seedlings and characterized by the formation of the first leaves and continued until the first flowering and or fruiting period. In red chili plants, this stage starts from seed germination until the plants form flower primordia. The generative stages is a phase characterized by shorter growth of twigs and nodes, shorter spacing between leaves on plant shoots, and shoot growth stops (Prihmantoro 2005). There are so many factors that can affect the agronomic parameters in chili plant, especially is pathogens. The diseases caused by Begomovirus has an important roles to inhibit the plant growth in chili. So it was necessary to determine the effectiveness of the application the treatment Table 2. Parameters of growth and development on red chilli were observed at the end of plantations on 23 rd week after planting. The result of root volume was significantly different between the treatments Table  2. The root volume was one of indicator to root growth. Bacillus sp. and Streptomyces sp. were reported as PGPR that can stimulate plant growth with producing hormones such as IAA. IAA (Indole-3-Acetic-Acid) is a growth hormone that plays an important role in plant growth and development. Microbes that are able to produce IAA can increase root growth and extension so that the root surface becomes wider and ultimately plants are able to absorb more nutrients from the soil (Vurukonda et al. 2018;Hashem et al. 2019).
Based on Figure 4 the appearance of chilli root were shown different size between the treatments. Root morphology of single treatment of Bacillus sp. was shown the biggest and the longest root hair than others. IAA hormones can stimulate plants to form lateral roots branching and development of root hairs. These lateral roots aim to expand the area of absorption of nutrients by plants, so that the nutritional needs of plants can also be met (Vacheron et al. 2013). According to Khalid et al. (2004) stated that wheat rhizosphere bacteria can produce auxin, this hormone can be used by plants to increase root length by up to 17.3 %. The plant height results on Table 2 were not significantly different between the treatments. But they tend to good trend to increase the growth and development on chilli. The other parameters was stem diameter. For the single treatments both of Bacillus sp. and Streptomyces sp. and their combination were shown that higher score of stem diameter compared to untreated Table 2. The appearance of chilli stem were shown different size between the treatments Figure 4. According to Vurukonda et al. (2018) endophytic Streptomyces sp. can produce a compound that acts as a plant growth promoter, namely auxin, gibberellins, and cytokinins and many more. Besides that, the bacteria Bacillus sp. able to stimulate plant growth because it is known to help plants produce growth hormones such as indoleacetic acid, gibberellic acid, cytokinins, and ethylene in plants (Hashem et al. 2019).
The result of number of productive branch was not significantly different between the treatments. According to Gardner et al. (1991) states that the number of branches in a plant is influenced by genotype and environment. The availability of nutrients results in better plant growth and easier absorption of nutrients so that plants can form optimal branches so that their growth will also be optimal. As previously explained that the use of antagonistic bacteria Bacillus sp. as well as Streptomyces sp. and the combination of the two is a bacterium that is able to support plant growth well so that the formation of branches will also occur optimally formed.  PGPR plays an important role in increasing plant growth, yields and soil fertility (Bhat et al, 2023). Based on Table 3. the fruit length and diameter were not significantly different between the treatments. The optimum fruit length of chilli twist varieties is about 15 -17 cm. The results of fruits length showed smaller than the potential length. Measurement of fruit length and diameter were sorted onto several criteria. They were healthy, mild malformation, moderate malformation and severe malformation Figure 5. The optimum fruit diameter of twist variety range is 0.8 -0.9 cm. The highest average yield of chili fruit diameter was shown on the combination treatment of Bacillus sp. and Streptomyces sp. on the criteria for severe malformation. This severe malformation caused by Begomovirus infection which could be assumed changing formation of the chillies and the fruit would become shorter and tend to be larger diameter. Saleable fruits were categorized as worth selling to consumer included as healthy, mild malformation and moderate malformation. For unsaleable fruits were included as severe malformation and rotten. The number of saleable chilli fruit yield was not significantly different between the treatments Table  4. Nevertheless, there were positive result using antagonistic bacteria for the treatments especially in single treatment of Streptomyces sp. According Ghashash et al. (2022) PGPR application can increase plant height, number of leaves, number of branches, number of flowers, number of fruits and fruit weight. Streptomyces sp. is also one of the bacteria belonging to the Actinomycetes genus which can produce plant growth hormones which can also act as PGPR. The weight of the fruit is largely determined by the number and size of the fruit, the more the number of fruit and the length of the size, the weight will also heavier (Gashash et al. 2022). The heaviest weight of saleable fruits were shown in single treatment of Streptomyces sp., and then following by Bacillus sp., combination of Bacillus sp. and Streptomyces sp. and the last one was untreated. This study was proven that single treatment of Streptomyces sp. and Bacillus sp. was better than combination between Streptomyces sp. and Bacillus sp. These antagonistic bacteria were play role as PGPR which has a mechanism for producing growth hormones so it can increase fruit weight. This is in accordance with Putra's research (2014) which states that the population decline that has occurred can be caused by competition for nutrients and living space so that the growth and development of these bacteria is more limited. One of the very large types of Actinomycetes is Streptomyces spp. The ability of actinomycetes and Bacillus to degrade complex compounds into simple compounds will affect the compatibility of the two bacteria (Putra 2014). It could be assumed that the combination of Streptomyces sp. and Bacillus sp. was not compatible. Treatment using single of Streptomyces sp., Bacillus sp. and combinations could suppress the diseases at the initial stages of chilli plant, but at the generative stages could not suppress yet. They could increase the chilli production and growth except the root volume. It could be the bacterial microbes did not survive in the soil for long time.
There is also variability in the performance of PGPR that may be due to various environmental factors that might affect their growth and proliferation in the plants. These gaps and limitations can be addressed through use of modern approaches and techniques such as nano-encapsulation and micro-encapsulation along with exploring multidisciplinary research that combines applications in biotechnology, nanotechnology, agro biotechnology, chemical engineering and material science and bringing together different ecological and functional biological approaches to provide new formulations and opportunities with immense potential (Gouda et al. 2017). .

CONCLUSION
The treatment of single of Streptomyces sp., Bacillus sp. and combination both of them could suppress the diseases at the initial stages of chilli plant, but at the generative stages could not suppress yet. It could not be increase the chilli production and growth except the root volume. Single of Bacillus sp treatment could increase the root volume in chilli plantation. For another parameters, it tend to good tendency using the treatment compared to untreated.