DISCUSSION
Diagnosis of plant diseases can generally be divided into 2 stages based on the process and method of implementation, the first was observations in the field and the second was further molecular observations in the laboratory. The initial stage is carried out by observing disease symptoms, namely deformities or changes in plant characteristics. If visually a disease is still difficult to distinguish, further observations are carried out in the laboratory by taking samples of diseased plant parts (which show early symptoms of disease).
The lack of visual observation methods for diagnosis is that the observer cannot confirm what type of specific symptoms are infecting the plant because several diseases show similar, even complex, symptoms and signs, so it is necessary to carry out further confirmation in the laboratory through a molecular analysis process.
As part of diagnosing plant diseases caused by viruses, it is usually recommended to observe the signs and symptoms as well as testing the host range of the virus by re- inoculating either mechanically or with the help of an insect vector. Apart from understanding the typical symptoms caused by various types of plants, host range testing also functions to determine the level of infectivity and spread of viruses through observing plant reactions in various species s (Hull, 2009). Host range testing in the study was carried out using sap from pumpkin leaf samples infected with SLCCNV which was then inoculated against Cucurbitaceae: squash, cucumber, melon, and bitter gourd (Momordica charantia);
Solanaceae: chili pepper and tomato; Fabaceae: yardlong bean (Vigna unguiculata subsp.
sesquipedalis), and Amaranthaceae: Chenopodium amaranticolor. After more than four months of observation, the plants did not show any signs and symptoms in the form of systemic mosaic and chlorosis so they could be said to be negative, which means the virus was not transmitted mechanically.
From the results obtained, tomato, chili and pumpkin samples showed positive results when molecular analysis was carried out using the PCR method with the universal primer Begomovirus by forming a single 580 bp band on agarose. The inability of Begomovirus to be transmitted through mechanical inoculation is thought to be closely related to the nature of the virus which can only replicate in the phloem of the plant systemically (Vuorinen et al, 2011). Begomovirus transmission generally relies heavily on insect vectors such as Bemicia tabaci to move from one plant to another easily (Ganefianti et al. 2008). The limitations can happen not caused by low competence during transport in mesophyll tissue, but rather are the result of the virus inability to invade cells that are not phloem (Wege & Pohl, 2007).
Begomovirus itself is a DNA virus from the Geminiviridae family of viruses which infects many horticultural commodities in Indonesia, such as Tomato yellow leaf curl virus (TYLCV), Pepper yellow leaf curl virus (PepYLCV), and Mungbean yellow mosaic India virus (MYMIV) (Nurlita et al., 2015). Genom of begomovirus consist of two ssDNA molecules (circular) which is around 2.6 kb, as representative of DNA-A and DNA-B (Fiallo -Olive et al., 2021).
Based on molecular analysis, even though the chili samples (OR924280) and tomatoes (OR924279) are in the same group and share a common basal node, in the phylogenetic analysis, the chili samples are in different subgroups or branches. The tomato sample had similarities to the PepYLCIV isolate from Bali which both infected tomato plants.
Apart from knowing the existence of kinship relationships, the results of analysis using phylogenetic trees can also be used to understand whether there is evolution occurring between species (Staton. 2015). The formation of subgroups within one large group in a branch of the phylogenetic tree shows that there is quite complex genetic differentiation which causes samples to be non-identical or different based on their host plant. Viruses originating from or having the same host plant can show greater genetic similarity than viruses originating from different host plants (Dolja & Koonin. 2011). In addition, the phylogenetic tree analysis in this study was based on the sequence results from the AV1 gene from Begomovirus which codes for the coat protein. This gene is quite important in viral pathogenicity and plays a role in the virus life cycle, including interactions between the virus and its host (Subiastuti et al., 2019).
Classification of the viral genome based on the sequence of amino acid and nucleotide base pairs was carried out using the Sequence Damarcation Tool (SDT), showing the results that the PepYLCIV test samples from chilies (OR924280) and tomatoes (OR924279) had the highest similarity to LC051113. This is indicated by the highest pairways results for amino acids (aa) in the range of 98.4 – 99.5% and nucleotide base pairs (nt): 95.8 – 96.7%.
Meanwhile, the SLCCNV pumpkin sample from pumpkin (OR924278) had the highest similarity to three isolates from Malaysia (MW248685, MW248687, and MW248689). This is indicated by the highest pairways results for amino acids (aa) in the range of 98.4 – 99.5%
and nucleotide base pairs (nt): 97.3 – 97.8%. According to Silva (2022), amino acids and nucleotides have an important role in virus classification, this is because they are generally used to measure the percentage similarity of proteins that function during replication and capsid formation.
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