Exploration and Collection of Cucumber Mosaic Virus Isolates of Horticultural Plants From Bali

Information regarding the existence and biological character of CMV isolates from several horticultural plants from Bali is still limited. The results of this research on information are very much needed in the further research and development of science and technology, due to it is known that CMV isolates from different hosts can cause different symptoms of infection or specific symptoms in certain plants. Based on these problems, this research was conducted through exploration, collection, and biological testing of CMV isolates found in several horticultural plants in Bali. Therefore, the existence and biological characteristics of each isolate were known. The results showed that all isolates obtained from the field, as many as 36 isolates, only 18 isolates showed symptoms of N. glutinousa. The isolates showed symptoms in N. glutinosa plants were isolates from cucumber plants 4 isolates, eggplant 3 isolates, pumpkin 1 isolate, 3 chili isolates, 2 isolates tomatoes, 2 beans isolates, and watermelons 3 isolates. The types of symptoms that appeared varied, i.e., 10 symptomatic mosaic isolates, 4 malformed isolates, and 4 necrotic symptomatic isolates. Detection of I-ELISA using CMV antiserum succeeded in getting 6 positive CMV isolates, i.e., isolates derived from cucumber plants (CMV-MB isolates), eggplant (CMV-TRB isolates) watermelon (CMV-SB isolates), chili (CMV-CB isolates), pumpkin (CMV-LB isolates), and tomatoes (CMV-TB isolates). The results of biological testing of CMV isolates from Bali, mosaic symptoms occurred in the indicator plants Nicotiana glutinosa, Capsicum annuum (big chili), Cucumis sativus (cucumber), Lycopersum esculentum (tomato), and Cucurbita moschata (pumpkin). Severe mosaic symptoms only occurred in Cucumis sativus (cucumber) plants. In Vigna sinensis (long bean) infection CMV-Bali isolates only cause symptoms of malformation and even cause no symptoms.


Introduction
Cucumber Mosaic Virus (CMV) is a species of virus originating from genus Cucumovirus, family Bromoviridae (Mochizuki and Ohki, 2012). It is round shape with 30 nm diameter, has four types of nucleic acids, which is a single-threaded RNA (Palukaitis et al., 1992;Fauquet et al., 2005). CMV is also one of the mosaic disease-causing viruses found in Cucurbitaceae plants. CMV can infect 1200 species in more than 100 families of vegetable and horticulture plants. These characteristics make CMV a wrong plant virus is economically important in commercial plants (Palukaitis et al., 1992). Important economic plants can be infected by CMV are big chili, cayenne pepper, paprika, tomato, cucumber, melon, banana, eggplant, chrysanthemum, lilies, and a number of other ornamental plants (Agrios, 2005).
CMV has a wide host range, its spread in all countries and strains with different biological properties have been reported from various places. CMV has many strains and differences between strains depend on the host type, the symptoms produced, the transmission nature, and other characteristics (Francki et al., 1979;Agrios 2005). The disease symptoms due to CMV infection vary depending on the host species or CMV strain. Therefore, it appears to be a character of the host reaction to CMV infection. Pandawani et al., (2017), in cucumber plants (Cucumis sativus) and other Cucurbitaceae members, CMV infection causes mosaic and dwarf symptoms and reduces fruit quantity and quality. In tomato plants (Lycopersicon esculentum), CMV infections cause mosaic, dwarf, fern leaf reduction (McNab et al., 1983) symptoms. In Capsicum annuum, CMV infections cause mosaic symptoms, blistering, fruit malformations, and together with other viruses (TMV, TEV, PVY, and CVMV) cause symptoms of complex viral infections (curly symptoms).

Exploration of host samples infected viruses
Virus exploration is conducted on several types of symptomatic horticultural plants infected with the virus. Samples were taken from 9 regencies in Bali, included Jembrana, Buleleng, Tabanan, Badung, Gianyar, Bangli, Klungkung, Karangasem, and Denpasar City. Host plants suspected of being infected with CMV were collected and taken at least three samples from one type of plant in each location. To obtain a variety of isolates with more samples collected were samples that showed different symptoms or virus-specific symptoms, namely mosaic, blistering, necrotic leaves/fruit, and leaf malformations.

Propagation of CMV isolates from different hosts
Propagation of isolates is conducted through mechanical inoculation of symptomatic host saplings infected with the virus. Mechanical viral inoculation was conducted 1 gram of CMV inoculum plant leaves were crushed in sterile mortal by adding 10 ml of phosphate/phosphate buffer (1:10 b/v) containing 1% β-mercaptoethanol (0, 01 M, pH 7.0). The sap yan is formed immediately inoculated on N. glutinosa plant which is on the surface of the first leaf which has been fully opened, the leaves of which have been sprinkled with carborundum 600 mesh first. The plants that have been inoculated are kept in insect vectors that are resistant to symptoms of the disease. The same symptomatic plants on the CMV isolate source plant in the field were then selected, for further purification of CMV isolates.

Purification of CMV isolates
Virus purification was conducted through biological testing to select CMV isolates. The plants needed to obtain pure CMV isolates were Nicotiana glutinosa, Cucumis sativus, and Chenopodium amaranticolor. The purification method of CMV isolates was carried out as follows: a sample from the field that had been propagated in N. glutinosa (the result of point 2.2) was infected with C. sativus in the cotyledon section, and then infected with C. amaranticolor (repeated three times). Furthermore, the virus obtained was re-inoculated into N. glutinosa and C. sativus plants. Furthermore, the obtained isolates were maintained on N. glutinosa and to ensure that CMV isolates were obtained, serological testing of isolates with the Indirect Enzyme-linked immunosorbent assay was performed (I-ELISA) using serum anti-CMV (AGDIA, USA).

CMV isolates collection through the biological test on indicator plants
Indicator plants used to study the symptoms of infection by all isolates obtained were plants: C. annuum (big chili), Cucumis sativus (cucumber), L. esculentum (tomato), Solanum melongena (green eggplant), Vigna sinensis (long beans), and Cucurbita moschata (pumpkin). All CMV positive isolates were inoculated on several indicator plants. The differences in isolates are expected to be identified based on differences in symptoms in indicator plants so that CMV isolates collection from several hosts was obtained.

Collection of plants infected with viruses in the field
Isolates collection, as well as symptoms observations that occur in plants, suspected being infected with CMV have been conducted and data obtained are shown in Table 1. Isolates were obtained from the field as many as 36 isolates namely derived from cucumber plants (5 isolates), big chili (6 isolates), tomatoes (4 isolates), long beans (4 isolates), watermelon (4 isolates), pumpkin (4 isolates) and melons (4 isolates).

Purification results of CMV isolates
The plants are from the field suspected being infected with the virus then purified/conducted purification of the virus with biological techniques to separate CMV from other viruses, using specific indicator plants. All isolates obtained from the field were as many as 36 isolates, after purification only 18 isolates showed symptoms in N. glutinousa plants, 18 other isolates did not show symptoms. Isolates who showed symptoms were 10 mosaic symptom isolates, 4 isolates from leaf malformations and 4 isolates from necrotic symptoms. The isolates showing symptoms of N. glutinosa were isolates from cucumber plants 4 isolates, eggplant 3 isolates, pumpkin 1 isolate, chili 3 isolates, tomatoes 2 isolates, beans 2 isolates, and watermelons 3 isolates. The incubation period in N. glutinosa plants ranged from 11 days to 15 days after inoculation (Table 2). Table 2 Type of symptoms in N. glutinosa indicator plants infected with CMV isolates from different hosts CMV infects systemically in many plants. Older organ or tissue plants that develop before being infected with the virus are usually not affected by the virus presence, but the young tissue or cells that develop after being infected with the virus are very affected and generally show acute symptoms. Virus symptoms will increase several days after the infection, then decrease to a certain extent or until the plant dies.

Serology test results of CMV isolates
The variation in symptoms caused by CMV will be very difficult to identify only based on the symptoms. In addition, it is also difficult to distinguish other CMV from Cucumovirus isolates (such as Alfalfa mosaic virus, Tomato aspermy virus, and Peanut stunt virus). To further ensure, the virus obtained is CMV, the isolates that have been successfully purified are then tested serologically using I-ELISA test. The isolated sample was expressed positively if the results of the absorbance measurements at 405 nm wavelength with ELISA reader (Table 3) had a value twice as large as the negative control value (Matthews, 2002 (Table 4).
Other isolates also showed symptoms of N. glutinosa appeared to be from CMV I-ELISA test result is not positive.

Indicators plant symptoms on the biological test of CMV isolates
The symptoms result from infection with six CMV isolates from Bali on several important indicator plants are highly varied and some are asymptomatic. It appears varying from 6 days after inoculation to 13 days after inoculation with severe mosaic, mosaic, local malformations, and necrotic symptoms (  In Vigna sinensis (long bean) infection CMV-Bali isolates did not cause mosaic symptoms, only caused symptoms of malformations (due to infection with CMV-MB and CMV-TB isolates) and even CMV isolates from Bali, namely CMV-TRB isolates, CMV-SB, CMV-CB, and CMV-LB did not cause symptoms.

Conclusion
1. Exploration of all isolates from the field as many as 36 isolates, and only 18 isolates showed symptoms in N. glutinousa plants. The isolates showed symptoms in N. glutinosa plants were isolates from cucumber plants 4 isolates, eggplant 3 isolates, pumpkin 1 isolate, 3 chili isolates, 2 isolate tomatoes, 2 bean isolates, and 3 watermelon isolates. The symptoms types appear vary, 10 of which are symptomatic mosaic, 4 are symptomatic malformations, and 4 necrotic symptomatic isolates.