SYSTEMATIC LITERATURE REVIEW (SLR): DISEASE DETECTION IN MELONS USING DIGITAL IMAGE PROCESSING

Systematic Literature Review (SLR) is a technique used in this study which is used to study techniques for identifying leaf diseases using digital images as a basis for obtaining an understanding of disease identification techniques in melon leaves with digital images. Based on data from the Central Statistics Agency for the last 3 years from 2017-2019, melon production has increased considerably. Melon production data in 2017 was 92.43 tons, in 2018 was 118,708 and in 2019, overall melon production was 122,105 tons collected from 34 provinces in Indonesia. The problem that is often encountered in melon cultivation is the presence of plant pests that can harm and not maximize the yields of farmers. Several viruses cause mosaic disease that infects Cucurbitaceae plants, namely Cucumber aphid borne yellows virus (CABYV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV), Squash mosaic virus (SqMV), Squash leaf curl virus (SLCV), Watermelon mosaic virus (WMV). Information technology has now developed to be able to manage digital image data to identify problems faced by farmers. Several classification methods that can be used to answer problems include SVM, Artificial Neural Network, Decision Tree, Convolutional Neural Network.

Identification of the Begomoviruses Squash Leaf Curl Virus and Watermelon Chlorotic Stunt Virus in Various Plant Samples in North America

Geminiviruses are a group of plant-infecting viruses with single-stranded DNA genomes. Within this family, viruses in the genus Begomovirus are known to have a worldwide distribution causing a range of severe diseases in a multitude of dicotyledonous plant species. Begomoviruses are transmitted by the whitefly Bemisia tabaci, and their ssDNA genomes can be either monopartite or bipartite. As part of a viral survey, various plants including those in the families Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Malvaceae, Oleaceae and Solanaceae were sampled and screened for begomoviruses using both a high-throughput sequencing and a begomovirus-specific primer pair approach. Based on the sequences derived using these approaches, the full-length genome of various begomoviruses were amplified from plants using abutting primers. Squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WCSV) were identified in Cactaceae (n = 25), Solanaceae (n = 7), Cucurbitaceae (n = 2) and Lamiaceae (n = 1) samples. WCSV is an Old World bipartite begomovirus that has only recently been discovered infecting watermelons in the Americas. Our discovery of WCSV in the USA is the first indication that it has reached this country and indicates that this virus might be widespread throughout North America. Phylogenetic analysis suggests WCSV was introduced to the New World twice. The detection of begomoviruses in cactus plants suggests possible spillover events from agricultural areas into native vegetation. Since WCSV and SLCV have previously been found in mixed infections, pseudo-recombination infection experiments were conducted. We demonstrate that WCSV DNA-B is successfully trans-replicated by SLCV DNA-A despite very low degree of similarity between the replication-associated iterative sequences present in their common region, an essential feature for binding of the replication associated protein. This study highlights the importance of viral surveys for the detection of spillover events into native vegetation, but also suggests the need for more surveillance of WCSV in the USA, as this virus is a serious threat to watermelon cultivation in the Middle East.

Cucurbit leaf crumple virus (CuLCrV) associated to watermelon disease in Campeche state, Mexico

An annual recurrent disease causing yield reduction in cultivated watermelon (Citrullus lanatus) was documented by the growers in different farms of Campeche state, Mexico. In April 2019 and March 2020 open field grown watermelon plants showed symptoms such as leaf curling, crumpling, and leaf basal or apical necrosis (Figure S1), with an incidence ranging from 30 up to 80%. These plants also presented high populations of whitefly, especially in the most affected fields. In order to identify the causal agent of the disease, a total of 22 symptomatic watermelon plants were collected in four locations from Campeche state. Total nucleic acids (DNA and RNA) were extracted from these leaf samples. Initially, RT-PCR analysis was performed with specific primers (Table S1) for cucurbit-infecting Crinivirus transmitted by whitefly but the expected size PCR product for those viruses was not amplified in any of these samples. To investigate the presence of cucurbit-infecting begomoviruses, PCR was performed by using specific primers for those begomoviruses reported in Mexico and north/central America including Squash leaf curl virus (SLCV), Watermelon chlorotic stunt virus (WmCSV), Melon chlorotic leaf curl virus (MCLCuV), and Cucurbit leaf crumple virus (CuLCrV) (Table S1). Only the expected amplicon size of ~1089 bp for CuLCrV was amplified from DNA extracts from all 22 watermelon samples, suggesting a single cucurbit-associated virus. The putative complete genome of the CuLCrV Campeche isolate was amplified by circular DNA enrichment using a Rolling Circle Amplification (RCA) procedure from two representative samples, followed by enzymatic digestion using BamHI, EcoRI, KpnI, and SacI enzymes (Inoue-Nagata et al., 2004). Expected linearized full-length viral components (~2.7 kb) were obtained with EcoRI and SacI, and both products, from one selected sample, were cloned in to pGreen0029 vector and were fully sequenced. Sequence analysis of the EcoRI clone, designated as LV2019Camp_A (deposited in GenBank accession no. MW273384) revealed the highest identity of 97.52% to CuLCrV DNA-A isolate Baja California Sur isolate (GeneBank accession no. MN625831.1), whereas the KpnI clone, designated as LV2019Camp_B (deposited in GenBank accession no. MW273385), shared 94.87% identity with DNA B of CuLCrV isolate Arizona (GeneBank accession no. AF327559.1). Subsequently, CuLCrV isolate Campeche-derived agroinfectious clone, was obtained by constructing a partial dimeric tandem repeat of both DNA-A and DNA-B components (Bang et al., 2014). Twelve watermelon plants were agroinfiltrated with the infectious clone at the fourth true leaf stage, resulting in symptomatic plants (11/12) exhibiting leaf yellowing, curling, and crumpling 15 days after agroinfiltrated (Figure S1), and CuLCrV infection was confirmed by PCR specific detection using DNA extract from non-inoculated leaves. Previously CuLCrV has been detected in the USA (Arizona, Texas, California, Florida, South Carolina, and Georgia), and north Mexico (Coahuila) infecting cucurbits including squash, cucumber, cantaloupe, pumpkin, and watermelon (Brown et al., 2000., Keinath et al., 2018), in both single and mixed infection with other whitefly transmitted RNA viruses (CYSDV, genera Crinivirus), and DNA viruses (SLCV, genera Begomovirus) (Kuo et al., 2007). To our knowledge, this is the first report of CuLCrV infecting a cucurbit crop in the Campeche state from the Yucatán peninsula, in Mexico.

First report of begomoviruses infecting Cucumis sativus L. in North America and identification of a proposed new begomovirus species

Background Members of the Begomovirus genus are phytopathogens that infect dicotyledonous plants, producing economic losses in tropical and subtropical regions. To date, only seven species of begomoviruses (BGVs) infecting cucumber have been described. Most cucumber infections were reported in South Asia. In the Americas, begomoviral infections affecting cucumber are scarce; just one report of begomovirus has been described in South America. The presence of whitefly and typical symptoms of viral infections observed in a cucumber field in Colima, Mexico, suggested that plants in this field were affected by BGVs. Methods To identify the BGVs infecting cucumber, we performed a high-throughput sequencing and compared the assembled contigs against the GenBank nucleic acid sequence database. To confirm the presence of viruses in cucumber samples, we performed a PCR detection using specific oligonucleotides. We cloned and sequenced by Sanger method the complete genome of a potential new begomovirus. Begomovirus species demarcation was performed according to the International Committee on Taxonomy of Viruses. The evolutionary relationship of the new virus was inferred using phylogenetic and recombination analyses. Results We identified five species of begomovirus infecting plants in a field. None of these have been previously reported infecting cucumber. One of the five species of viruses here reported is a new begomovirus species. Cucumber chlorotic leaf virus, the new species, is a bipartite begomovirus that has distinctive features of viruses belonging to the squash leaf curl virus clade. Conclusions The findings here described represent the first report of begomoviral infection affecting cucumber plants in North America. Previous to this report, only seven begomovirus species have been reported in the world, here we found five species infecting cucumber plants in a small sample suggesting that cucumber is vulnerable to BGVs. One of these viruses is a new species of begomovirus which is the first begomovirus originally isolated from the cucumber. The findings of this report could help to develop strategies to fight the begomoviral infections that affect cucumber crops.

Molecular Characterization of Begomovirus on Cucumber in Java

A survey on several cucumber cultivation areas in West Java, Central Java, Yogyakarta, and East Java found many plants showing typical Begomovirus symptoms such as yellow mosaic, cupping, and vein banding. This study was aimed to determine disease frequency, detection and molecular characterization of the causal virus of those symptoms on cucumber in Java. Sampling was conducted by purposive sampling by collecting 50 symptomatic plants from each location in West Java (Indramayu, Subang, and Bogor), Central Java (Brebes and Klaten), Yogyakarta (Kulon Progo), and East Java (Nganjuk, Kediri, and Tulungagung). The detection and disease frequency was determined based on DIBA test using a specific antiserum of Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl virus (SLCV). The identification of nucleic acid was conducted by PCR using specific primer of ToLCNDV and SLCV, DNA cloning, and sequencing. The results of serological detection showed the disease frequency of ToLCNDV and SLCV ranged from 92.77-100% and 78.33-93.3%, respectively. PCR using specific primer of ToLCNDV successfully amplified the coat protein gene at a size of 600 bp from all samples. Homology nucleotide and amino acid sequences among ToLCNDV Java isolate ranging from 95.6-99.2% and 99.7-100%. ToLCNDV isolates Java had highest nucleotide and amino acid sequences similarity with cucumber isolate from Klaten, Indonesia (AB613825) ranging from 96.1-98.1% and 99.7-100%, and was considered as “Indonesia” strain. SLCV not amplified on all samples by PCR using specific primer, indicating it might not present yet on cucumber in Java.

A Lineage of Begomoviruses Encode Rep and AC4 Proteins of Enigmatic Ancestry: Hints on the Evolution of Geminiviruses in the New World

The begomoviruses (BGVs) are plant pathogens that evolved in the Old World during the Cretaceous and arrived to the New World (NW) in the Cenozoic era. A subgroup of NW BGVs, the “Squash leaf curl virus (SLCV) lineage” (S-Lin), includes viruses with unique characteristics. To get clues on the evolutionary origin of this lineage, a search for divergent members was undertaken. Four novel BGVs were characterized, including one that is basal to the group. Comparative analyses led to discover a ~670 bp genome module that is nearly exclusive of this lineage, encompassing the replication origin, the AC4 gene, and 480 bp of the Rep gene. A similar DNA module was found in two curtoviruses, hence suggesting that the S-Lin ancestor acquired its distinctive genomic segment by recombination with a curtovirus. This hypothesis was definitely disproved by an in-depth sequence analysis. The search for homologs of S-Lin Rep uncover the common origin of Rep proteins encoded by diverse Geminiviridae genera and viral “fossils” integrated at plant genomes. In contrast, no homolog of S-Lin Rep was found in public databases. Consequently, it was concluded that the SLCV clade ancestor evolved by a recombination event between a primitive NW BGV and a virus from a hitherto unknown lineage.

Molecular characterization and phylogenetic analysis of a Squash leaf curl virus isolate from Baja California Sur, Mexico

Background The begomovirus, squash leaf curl virus (SLCuV) is one of the causal agents of squash leaf curl (SLC) disease, which is among the most destructive diseases of cucurbit crops in tropical, subtropical, and semiarid regions worldwide. This disease was originally reported in the American continent with subsequent spread to the Mediterranean basin. Up to now, SLCuV has only been detected by PCR in Mexico. This study provides the first complete sequence of a Mexican SLCuV isolate from Baja California Sur (BCS). In addition, the genome of the virus was characterized, establishing its phylogenetic relationship with other SLCuV isolates. Methods The full genome (DNA-A and DNA-B) was amplified by rolling circle amplification, cloned and sequenced and the open reading frames (ORF) were annotated. Virus identification was performed according to the International Committee on Taxonomy of Viruses (ICTV) criteria for begomovirus species demarcation. To infer evolutionary relationship with other SLCuV isolates, phylogenetic and recombination analyses were performed. Results The SLCuV-[MX-BCS-La Paz-16] genome (DNA-A and DNA-B) had 99% identity with SLCuV reference genomes. The phylogenetic analysis showed that SLCuV-[MX-BCS-La Paz-16] is closely related to SLCuV isolates from the Middle East (Egypt, Israel, Palestine and Lebanon). No evidence of interspecific recombination was determined and iterons were 100% identical in all isolates in the SLCuV clade. Conclusions SLCuV-[MX-BCS-La Paz-16] showed low genetic variability in its genome, which could be due to a local adaptation process (isolate environment), suggesting that SLCuV isolates from the Middle East could have derived from the southwestern United States of America (USA) and northwestern Mexico.