Molecular detection of Pepper yellow leaf curl virus, Tomato leaf curl virus, Tomato yellow leaf curl virus, and Mungbean yellow mosaic virus on eggplant, tomato, and pepper at different altitudes in East Java, Indonesia

The genus Begomovirus causes yellow disease is the most damaging and threatening for eggplant, tomato, and pepper production worldwide. Begomoviruses (Family Geminiviridae) are transmitted circulative persistent manner by Bemisia tabaci. This study aims to determine the variation of infection of several species of Begomoviruses in tomato, eggplant, and pepper at different altitudes in East Java. Molecular detection of infection Begomoviruses were discovered using PCR and four pairs of specific primers. Samples were collected at different altitudes in East Java (Kencong, Karangploso, and Pujon). The PCR was successfully applied to detect Pepper yellow leaf curl virus, Tomato leaf curl virus, and Tomato yellow leaf curl virus from tomato, eggplant, and pepper in Kencong. Tomato, eggplant, and pepper from all locations were not infected by Mungbean yellow mosaic virus. Symptoms of Begomovirus infection found in the samples from Kencong were more varied than those found in Karangploso and Pujon. The phylogenetic analysis’ findings revealed that the TYLCV Kencong shared the highest homologies with TYLCKaV pepper and eggplant from Indonesia; PepYLCV Kencong shared with PepYLCV Ageratum conyzoides from Indonesia; and ToLCV Kencong shared with ToLCNDV cucumber from Central Java. The molecular detection has shown that there are different combinations of Begomovirus infection at altitudes.


Introduction
Solanaceae was infected by begomovirus and is no longer a new case of the disease.The first case of Indonesia's yellow leaf curl disease, which is brought on by the begomovirus genus Geminivirus, was discovered in West Java in 1999 [1] which the Pepper Yellow Leaf Curl Virus caused Pepper yellow leaf curl virus (PepYLCV), Tobacco leaf curl virus (TLCV) has infected tobacco in East Java since 1984 [2], in the Lombok island (West Nusa Tenggara) begomovirus infection on pepper was discovered since 2006 [3].Tomato yellow leaf curl virus (TYLCV) infection causes disease incidence to reach 50-1230 (2023) 012117 IOP Publishing doi:10.1088/1755-1315/1230/1/012117 2 70% so it becomes a major obstacle in increasing production especially in Central Java and West Java [4,5].
The PCR detection was only limited to the use of universal or specific primer but not combination by another specific primer in a single plant.Information on the characterization of symptoms and infection caused by multiple begomovirus species in a single plant of Solanaceae has not been widely available at different geographical conditions.Tomato hosts at least 68 begomovirus species, the most ever described for a single plant [21].In 2008, three naturally occurring begomovirus infections associated with soybeans were discovered in Brazil i.e., Okra mottle virus (OMoV), Sida mottle virus (SiMoV), and Bean Golden Mosaic virus (BGMV) [22].The objective of this research is to understand the variations in begomovirus infections and distinctive symptoms that develop on Solanaceae in various geographic contexts.

Samples collection and DNA extraction
Tomato, eggplant, and pepper showing symptoms i.e., mosaic, leaf curly, yellowing, and malformations were collected from November 2015 to February 2016 at East Java i.e., Kediri (Kencong), Malang (Karangploso and Pujon), which represent the low, medium, and high altitudes respectively.The leaves of 20 symptomatic plants were collected using a purposive sample [23].A total of 180 samples, made up of three harvests from each location, were collected and placed to plastic bags and then keep in a deep freezer at -80ºC for DNA extraction.
Total DNA was extracted from leaf tissue using a modified CTAB method by the Molecular Breeding Laboratory (PT.BISI International Tbk).According to [24] total nucleic acids were recovered from about 0.3 g of leaf tissue.The resulting total nucleid acid pellets were suspended in TE buffer pH 8 and stored at 4ºC.Amplification of total DNA by PCR using four pairs of specific primer i.e., PepYLCV-F and PepYLCV-R; TLCV-CPI and TLCV-CPT; TYLCV-F and TYLCV-R; AC2-F and AC2-R (table 1).Amplification using PCR by a master mix composition from New England Biolabs with a 20 µL total volume, the procedure according to [24].PCR products were imaged on a 1.5% agarose gel stained with ethidium bromide (EtBr) in a solution of 1 µL per 100 ml and documented using gel documentation (Kodak Molecular Imaging from UK).

Phylogenetic analysis of PepYLCV, ToLCV, and TYLCV
Analysis of nucleotide sequences was used to determine the homology level among the begomovirus species.Product PCR amplification was done by DNA sequencing at PT. BISI International Tbk using begomovirus specific primers.The nucleotide sequences were used for homology level analysis among begomovirus species that was discovered in GenBank by Blast (Basic Local Alignment Search Tool) in (www.ncbi.nlm.nih.gov).The nucleotide identity matrix was obtained from analysis using the BioEdit (v.7.0.5) tool to obtain sequences resulting from the merging of forward and revers [4], and the phylogenetic tree was constructed using the MEGA X version 10.2.6 based on the Neighbor Joining (NJ) approach.

Variations in symptoms of begomovirus infection on eggplant, tomato, and pepper
Survey of 20 pepper showed symptoms of infected begomovirus at each location (Kencong, Karangploso, and Pujon).Typical symptoms of pepper assosiated begomovirus i.e., leaf mosaic, bleaching, upward cupping, downward cupping, vein binding, crinkle, dwarf, and reduced size of leaves (figure 1a, 1b, 1c).The symptoms caused by begomoviruses for each location showed symptoms that vary greatly, but mosaic and cupping always found [19,28,29].Variations of symptoms by begomoviruses infection in tomato i.e.; mosaic, upward cupping, downward cupping, crinkle, leaf roll, dwarf, and reduced size of leaves (figure 1d, 1e, 1f).Mosaic, upward cupping, leaves are reduced in size, and dwarf were found in all locations.Tomato has a various symptomp in different geographical areas, the symptoms that arise in Kaliurang (high altitude) i.e.; upward cupping, yellowing, and dwarf [30].In eggplant, symptoms of begomoviruses infection i.e.; mosaic, yellowing, vein binding, bleaching, and reduced size of leaves (figure 1g, 1h, 1i).Mosaic symptoms were found in each location, while yellowing symptoms are found in Kencong and Karangploso, and the most varied symptoms were found in Pujon.
An investigation of various symptoms at various altitudes realized that begomoviruses infect peppers, eggplant, and tomato at low altitudes (Kencong) were more variable than others.That then suggests a link among distinct symptoms, locations, and different begomovirus infections.

PepYLCV, ToLCV, TYLCV, and MYMV detection on eggplant, tomato, and pepper using PCR
Leaves tissue of all eggplant, pepper, and tomato at all locations negative for MYMV that meaning they were not the main host.So far, infections by MYMV in Indonesia still has a few specific hosts.Host range of MYMV still very limited than other begomovirus species i.e., Leguminosae, Gramineae, and Compositae.MYMV can be transmitted by B. tabaci not only in Leguminosae including Xanthium strumarium (Compositae), Cosmos bipinnatus, Brachiaria ramosa (Gramineae), and Eclipta alba [31].These results indicated that MYMV appertain under species of begomovirus which still has a few main hosts in Indonesia.Earlier studies, MYMV were found to infect common bean and yard long bean (Leguminosae) in Kencong, Karangploso, and Pujon [24].In this present study, species of begomoviruses were mostly detected on pepper at Kencong (table 2, figure 2) i.e.; PepYLCV, TYLCV, and ToLCV.Based on these PCR results (figure 2, and 3) then PepYLCV were more dominant infected pepper at Kencong compared to TYLCV and ToLCV.The presence of begomoviruses in plant presenced the frequency of disease incidence caused by each infecting begomovirus species.Test plant samples that showed positive for PCR can described the frequency of disease incidence caused by begomovirus infection.By comparing the amount of PCR-positive plants, the frequency of disease incidence was assessed (infected by begomovirus species) and the number of samples of plants amplified [32].The highest frequency of disease incidence occurred in Kencong, Karangploso, and Pujon were pepper and tomato infection by PepYLCV and eggplant infection by TYLCV.Frequency of disease incidence on pepper at high altitude higher than at medium altitude.Environmental factors and the presence of whitefly around pepper in Pujon are more promote of begomovirus infection than in Karangploso.Earlier study [33], vegetation around the main host of begomovirus in pepper affected the presence of detected begomovirus species.In Karanganyar, whitefly (medium altitude) was only detected by PepYLCV.Whereas whitefly infestation around on cucumber at Garut was detected by PepYLCV and MYMV.The highest frequency of disease incidence occurred on tomato in Kencong was caused by ToLCV, in Karangploso caused by PepYLCV, and in Pujon caused by both of PepYLCV and TYLCV (table 3).Eggplant in all locations were infected by TYLCV, thus proving that the species was very dominant in infecting eggplant.
The combination of mixed infections of several species of begomoviruses is most detected on tomato in Kencong i.e.; PepYLCV, TYLCV, and ToLCV then in Pujon and most recently in Karangploso.Mixed infections from several begomovirus species have indeed been found naturally to occuring in the field.Between 1998 and 2009, tomato in Taiwan were detected infected by four species of begomoviruses i.e.; ToLCTWV (Tomato leaf curl Taiwan virus), AYVHuV (Ageratum yellow vein Hualien virus), ToLCHsV (Tomato leaf curl Hsinchu virus), and TYLCTHV (Tomato yellow leaf curl Thailand virus) [32].TYLCV was the most dominant species and often infects eggplant (table 3), it was detected to infect in all locations covered low, medium and high altitude.Pepper, tomato, and eggplant in all locations were also detected to infect TYLCV.

Phylogenetic Analysis of PepYLCV, ToLCV, and TYLCV
Genetic diversity analysis of TYLCV species from eggplant isolates (Kencong) showed a familial relationship with the TYLCKaV pepper from Indonesian (Tomato yellow leaf curl Kanchanaburi virus) isolate with accession code KF446676 and TYLCKaV Indonesian eggplant isolate with accession code KF446662 from the GeneBank database with bootstrap values of 99 in 1000 replications (figure 4a).TYLCV from eggplant isolate (Kencong) differed in clusters from TYLCKaV Laos eggplant isolate and TYLCKaV Thailand eggplant isolate (figure 4a).
The PepYLCV Kencong isolate conveyed the most correlations with the PepYLCV Ageratum conyzoides isolate from Indonesia (figure 4b).The phylogenic tree shows that the homology of the Kencong PepYLCV has a bootstrap value of 96 in 1000 replications (figure 4b).
The homology of ToLCV of tomato isolates from Kencong was compared with some isolates in GeneBank i.e.; ToLCNDV-Central Java Cucumber, ToLCNDV-Bangladesh Tomato, ToLCNDV-Thailand Luffa, ToLCNDV-Thailand Melon, ToLCNDV-Thailand Cucumber, ToLCNDV-Bangladesh Tomato, and ToLCNDV-Pakistan Tomato (figure 4c).The alignment was found that the ToLCV of tomato isolate from Kencong conveyed the most correlations with ToLCNDV from the cucumber isolates from Central Java.The percentage of identity is 99%, with a bootstrap value of 100 in 1000 replications (figure 4c).

Conclusion
Compared to the ones observed in Karangploso and Pujon, the begomovirus infection symptoms from Kencong were more variable.In different altitudes on eggplant, tomato, and pepper at East Java (Kencong, Karangploso, and Pujon) were found infected by the complex begomoviruses (three species in a single plant).Molecular detection indicated that three begomoviruses together infect on eggplant, tomato, and pepper in naturally growing.The combination of infections of more than one species of Begomovirus causes complex symptoms, therefore future studies should discuss factors that cause complex symptoms and a combination of begomovirus species and their correlation of begomoviruses diversity.

Figure 4 .
Figure 4. Phylogenetic relationships (a) Tomato yellow leaf curl virus-Pepper at Kencong; (b) Pepper yellow leaf curl virus-Eggplant at Kencong; (c) Tomato leaf curl virus-Tomato at Kencong with several isolates in GeneBank.Each node shows a bootstrap value.

Table 2 .
Result total of detection begomoviruses on pepper using PCR at Kencong, Karangploso, and Pujon

Table 3 .
Result total of detection begomoviruses on tomato using PCR at Kencong, Karangploso, and