Molecular Identification of Begomovirus Infecting Angled Luffa

Cucurbits is an important commodity group that produces fruits and vegetables in various areas around Indonesia. Cucurbit producing centrals, such as East Java, Central Java, Jakarta, West Java, and Yogyakarta, contribute to approximately 80% of national production in Indonesia (BSCI, 2003 cit. Daryono & Natsuaki, 2009). One of the challenges in cultivating cucurbit crops are plant diseases caused by viruses that result in mosaic symptoms, leaf distortions, stunting, discoloration and malformed fruit (Julijantono, 2006). Lovisolo (1980) discovered various diseases caused by 32 viruses or viroid that infect cucurbits. Many of those viruses are able to infect many plant species from different genera to families (Zitter et al., 1996). Diseases caused by viruses from the Geminiviridae family is a major challenge in increasing production of stable and specialty crops. Begomovirus is one of the most important virus that majorly affects plant health and production (Fauquet & Stanley, 2003; King et al., 2012). Begomovirus owns circular singlestranded DNA (ssDNA) genome that consists of single molecules within the size of 2.7–2.8 kb (monopartite) and mainly double molecules, DNA-A and DNA-B (bipartite) (Vanitharani et al., 2005 cit. Wilisiani et al., 2014). Identification the type of virus and determining the pathogen’s characteristics is an essential step that determines the success of effective, safe, and efficient disease management (Wilisiani et al., 2014). Identification using molecular techniques, such as Polymerase Chain Reaction (PCR), have been vastly used to identify viruses. PCR technique to identify Begomovirus on crops have widely used the Krusty Homer primer that is able to amplify this viruses’ coat protein (Revill et al., 2003). Yellow-curly mosaic virus does not only cause decreases yield, but also affect seed quality. Seeds from infected plants reduce in size and weight. Defected seeds are recognized by the yellow discoloration (Nene, 1973; Varma et al., 1992) and yellow lesions on newly sprouted leaves in nurseries (Nene, 1973). There have been many reports regarding the characteristics of various plant species infected with Begomovirus. However, to our knowledge, there has not been any report on the molecular characteristics of Begomovirus on angled luffa cultivated in Yogyakarta and Central Java. ABSTRACT


INTRODUCTION
Cucurbits is an important commodity group that produces fruits and vegetables in various areas around Indonesia. Cucurbit producing centrals, such as East Java, Central Java, Jakarta, West Java, and Yogyakarta, contribute to approximately 80% of national production in Indonesia (BSCI, 2003 cit. Daryono & Natsuaki, 2009).
One of the challenges in cultivating cucurbit crops are plant diseases caused by viruses that result in mosaic symptoms, leaf distortions, stunting, discoloration and malformed fruit (Julijantono, 2006). Lovisolo (1980) discovered various diseases caused by 32 viruses or viroid that infect cucurbits. Many of those viruses are able to infect many plant species from different genera to families (Zitter et al., 1996). Diseases caused by viruses from the Geminiviridae family is a major challenge in increasing production of stable and specialty crops. Begomovirus is one of the most important virus that majorly affects plant health and production (Fauquet & Stanley, 2003;King et al., 2012).
Begomovirus owns circular singlestranded DNA (ssDNA) genome that consists of single molecules within the size of 2.7-2.8 kb (monopartite) and mainly double molecules, DNA-A and DNA-B (bipartite) (Vanitharani et al., 2005cit. Wilisiani et al., 2014. Identification the type of virus and determining the pathogen's characteristics is an essential step that determines the success of effective, safe, and efficient disease management (Wilisiani et al., 2014). Identification using molecular techniques, such as Polymerase Chain Reaction (PCR), have been vastly used to identify viruses. PCR technique to identify Begomovirus on crops have widely used the Krusty Homer primer that is able to amplify this viruses' coat protein (Revill et al., 2003).
Yellow-curly mosaic virus does not only cause decreases yield, but also affect seed quality. Seeds from infected plants reduce in size and weight. Defected seeds are recognized by the yellow discoloration (Nene, 1973;Varma et al., 1992) and yellow lesions on newly sprouted leaves in nurseries (Nene, 1973).
There have been many reports regarding the characteristics of various plant species infected with Begomovirus. However, to our knowledge, there has not been any report on the molecular characteristics of Begomovirus on angled luffa cultivated in Yogyakarta and Central Java.

Sample Collection and Disease Incidences
Disease incidence of yellow curly virus on angled luffa were observed on sample fields in Sleman, Yogyakarta and Magelang, Central Java. Leaf and seed samples were taken from angled luffa plants with yellow lesions, curly leaves, and stunting symptoms. Leaf samples were then placed in a plastic bag with silica gel and stored in -20°C for further processing.

Molecular Identification of Begomovirus
Plants that showed symptoms of Begomovirus infection, were sampled for further molecular identification using PCR and a universal Begomovirus primer, Krusty-Homer (Revill et al., 2003). Sample of DNA were extracted from angled luffa leaves with yellow curly symptoms using a DNA Geneaid Minikit (Geneaid Biotehc Ltd., Taiwan). As much as 0.05 g of leaf samples were placed on porcelain mortars, 400 µl GP1 buffer and 5 µl RNAse A were added and leaf samples with mentioned solutions were macerated and put into a 1.5 mL tube. Solutions were homogenized. Incubation was done in a waterbath at 60°C for 10 minutes and every 5 minutes tubes' positions were moved to ensure equal temperature. After that, 100 µL GP2 buffer was added, homogenized, and incubated in ice for 3 minutes. Mixtures were moved into 2 mL tubes that were placed in a filter column and centrifuged for 1 minute at 1,000 G. Supernatan was taken and placed in 1.5 mL tubes and GP3 buffer was added by 1.5 times of the supernatan volume. This mixture was then homogenized for 5 seconds. Mixture was placed on GD column into the 2 mL tube, Elution Buffer was warmed at 60°C. The mixture inside the GD column was centrifuged at 10,000 G for 4 minutes. Supernatan was then disposed and 400 µL of W1 buffer was added dan centrifuged at 10,000 G for 3 minutes. Supernatan was disposed and 600 µL of wash buffer was added, centrifuged at 10,000 G for 3 minutes, and repeated twice. Supernatan was disposed and recentrifuged at 10,000 G for 6 minutes until the column matrix was dry. GD columns were placed 1.5 mL tubes and 100 µL of pre-heated elution buffer was added, left for 3 minutes and centrifuged at 10,000 G for 2 minutes. Extracted solutions were stored at -20°C. The DNA amplification used a primer specific Begomovirus Krusty (Forward): 5'CCNM RDGGHTGTGARGGNCC'3; Homer (Reverse): 5'SVDGCRTGVGTRCANGCCAT'3) with a predenaturation stage of 95°C for 1 minute, denaturation 95°C for 14 seconds, annealing stage at 55°C for 15 seconds, elongation at 72°C for 10 seconds, repeated for 35 cycles, and ended with a final elongation stage at 72°C for 5 minutes. The DNA electrophoresis was visualized on 1% agarose gel at 50 V for 50 minutes. Targeted DNA band was 580 bp (Revill et. al., 2003).

Begomovirus Seed-borne Test
The possibility of Begomovirus to be contracted by seeds was tested using a growing on-test. Seeds were obtained from ripe angled luffa fruit that were cleaned. Seeds were placed in petri dishes with wet filter paper to maintain humidity. Seeds that have sprouted were identified using the similar molecular procedures used to test angled luffa leaves

Data Analysis
Sequencing results were analyzed through Basic Local Alignment Search Tool (BLAST) program of the National Center of Biotechnology Information (NCBI) for similarity search that at http://blast.ncbi. nlm.nih.gov/Blast.cgi to obtain species with high homology. Corrections were done and sequences were multiple aligned using the ClustalOmega program, which was accessed through http://www.ebi.ac.uk/tools. Results from multiple alignments were used to create nucleotide homology percentage tables and phylogenetic tree using Molecular Evolutionary Genetical Analysis versi 7 (MEGA7) with a Maximum-Likelihood, 2 parameters Kimura, and 1,000 bootstraps (Kumar et al., 2016).

Disease Incidence and Symptoms on Angled Luffa
Disease incidence observation at both location where Begomovirus infected angled luffa fields imply that geographical conditions of the field affects disease incidence. According to Febria (2015), different geographical conditions of fields significantly affected disease incidences. Disease incidence on angled luffa at Berbah, Sleman with altitude of 109 m asl was 80% while disease incidence at Dukun, Magelang was 100% (Table 1). Disease incidence of yellow curly on angled luffa was also affected by other factors, such as surrounding vegetation, existences of alternative host and this disease's vector. The high disease incidence of yellow curly at Dukun, Magelang was caused by geographical condition of the location due to it being at a medium altitude land, which was an endemic area for Begomovirus (Sulandari et al., 2006), surrounding vegetation of this field included other commodities that were host for Begomovirus, such as chili pepper, eggplants, tomato, various species of weeds and an existing population of its insect vector, Bemisia tabaci Genn. yang tinggi. The dispersion of B. tabaci is affected by abiotic factors, such as the change of average yearly air temperature (Kandito, 2017). Kandito (2017) stated that average yearly air temperature was 19. 3°C-20.1°C between 2009-2015. Observation of yellow curly symptoms on angled luffa that these plants were presumed to be infected by Begomovirus with early infection symptoms to be yellow lesions on leaves, thickening of leaf midribs, and curly leaves ( Figure 1c) and stunning ( Figure  1a). Other than the previous mentioned symptoms, Begomovirus infected plants have malformation on its leaves, fruits, and seeds. These malformations are usually shown by mosaic on leaves (Figure 1c), stunting (Figure 1a), small fruits followed by discoloration and misshaped fruits (Figure 1e), and shrunken and sometimes discolored seeds (Figure 1g). Comparison between healthy and Begomovirus infected plant are shown in Figure 1. ISSN (Giovanni, 2017) Figure 1. Morphological comparison between healthy and malformed angled luffa (Luffa acutangula) plants (a) angled luffa plant with almost all of its body turned yellow (b) healthy angled luffa plant (c) angled luffa leaf with yellow mosaic (d) healthy angled luffa leaf (e) curved angled luffa fruit (f) healthy angled luffa plant (g) shrunken and empty seeds (h) healthy angled luffa seeds (Giovanni, 2017) Virus infection may decrease photosynthetic efficiency by decreasing chlorophyll content in leaves, chlorophyll efficiency, leaf areas, and growth regulating substances (Mushtaq et al., 2014), which will then hinder plant growth. Ariyanti (2012) stated that symptoms that appeared due to virus infection is caused by disruption in nutrition transportation from the source to sink. This later will disrupt plant growth in general and not produce fruit if plants are affected at early stages or during the vegetative stage. If plants are infected during the generative stage may result in smaller fruits with hard textures.

Molecular Identification for Virus Causing Yellow Curly Leaves on Angled Luffa and Seed-borne Test
Molecular identification from samples using the PCR Krusty-Homer Universal Begomovirus primer (Revill et al., 2003) showed positive results from leaf samples while negative results from angled luffa seeds. Leaf samples positively infected by Begomovirus showed ±580bp bands (Figure 2). Krusty-Homer primer is able to amplify nucleotides that encode Begomovirus coat protein, which is a conserve gene from Begomovirus (Revil et al., 2003;Reddy et al., 2005). Thus, that primer can be used in early identification and surveillance method.
Seed samples did not result in these bands; therefore, it implies that seeds did not contain Begomovirus (Figure 3).
Begomovirus that cause yellow curly leaves in Indonesia are not seed-borne, are not able to be transmitted mechanically and can only be transmitted through its vector B. tabaci (Rusli et al., 2000;Sulandari et al., 2006;Sulandari et al., 2001). However, Kil et al. (2012) proved that Tomato yellow leaf curl virus (ToYLCV) was seed-borne and had significant importance of its survival through planting seasons and geographical dispersion. This implies that although there are species of Begomovirus that are seed-borne, the species found on angled luffa in Berbah, Sleman and Dukun, Magelang were not one of them. Begomovirus may not become seed-borne due to seeds that go into dormant stages (Akin, 2006) or viruses not reaching seed embryo causing them to not survive (Matthews, 1970).

Sequence Analysis
The BLAST analysis from the NCBI implied that sequences from GD1 sample (angled luffa infected . PCR product from DNA amplification from root and seed sample after electrophoresis using 1% agarose gel: (1) isolate GB1; (2) isolate GB2; (3) isolate GB3; (4) isolate GD1; (5) isolate GD2; (6) isolate GD3; (M) marker 100bp DNA ladder; negative results, no bands formed (Giovanni, 2017) relationship due to it being in different cluster as. In addition, Tomato leaf curl virus satelite Indonesia (access code AB113651.1) was an outgroup species. Angled luffa Begomovirus isolate GD1 also had high relations with LYMV (access code AF509739.1) due being in the same cluster. Meanwhile Tomato leaf curl virus satelite Indonesia (access code AB113651.1) had relations with BCTV-NM (access code EF501977.1) due being in the same cluster. Different cluster can be implied that isolates between them have distinct relations.
The BLAST analysis showed that GD1 had similarities to Squash leaf curl virus from China isolate MC1 (GenBank EF197940) based on it phylogenetics. Homology percentage between both isolates were taken from Genbank ( Table 2).

CONCLUSION
All samples tested using PCR were positively infected by Begomovirus. Analysis of sequence results of GD1 Begomovirus from angled luffa isolates had homologies closed to SLCV MC1 isolates from China with 97.8% similarity and close relationships with the same isolate. Root samples from angled luffa seedlings detected using PCR shoed negative results implying that this virus is not seed-borne.

ACKNOWLEDGEMENT
This article was written by partly using data from the first author's bachelor thesis that was advised by the second and third author. We would also like to thank the Faculty of Agriculture, Universitas Gadjah Mada for the 2017 Research Grant with contract no. 3172/PN/PG/2017 for funding this study.