Identification and Classification of Tomato Leaf Diseases Using Machine Learning Techniques

Tomato is cultivated in all countries of the world in fields, glasshouses etc. China, India, USA, Turkey, Egypt, Iran, Italy, Spain and Brazil are the important countries which are cultivating tomatoes. It is most commonly and widely cultivated in India. India is one of the countries in harvesting tomatoes. Tomato is a vital vegetable yield with respect to both income and food. Tomatoes are for the most part summer crops, yet it tends to improve steadily. Naturally, it contains A and C of vitamins which also acts as an antioxidant to prevent cancerous cells. Since the organic product contains novel features, the demand remains the same. A significant and unique feature with high nutrients gains the importance in tomatoes cultivation. Challenges towards the cultivation of tomato made us to plan for an automated machine to detect infection and to increase the productivity. This system automatically detects the infected parts and classify the types of disease which occur on the leaf like early blight, bacterial wilt, Leaf Spot, tomato mosaic virus, septoria leaf spot, leaf curl virus, and tomato spotted wilt disease using gradient anisotropic diffusion filter for pre-processing and then features are extracted using GLCM from the pre-processed leaf

First report of a secovirus associated with mountain celery chlorotic spot disease in Heilongjiang, China

Mountain celery (Heracleum moellendorffii Hance), an edible perennial herb of Northeast Asia, is sporadically cultivated as a vegetable crop or for medicinal purposes in Northeast China and Korea [1]. In July 2019, a small field of mountain celeries showing chlorotic spots was found in Wangkui, Heilongjiang, China. A small-RNA (sRNA) library was constructed with equal amounts of leaf tissues of a diseased mountain celery and a tomato sample showing mottling symptom from a nearby field using the TruSeq small RNA library preparation kit (Illumina). The library was sequenced by the HiSeq 4000 sequencer at Lianchuan Biotechnology Co., Ltd (Hangzhou, China). After trimming adaptor sequences and discarding low quality reads by Cutadapt [2], the remaining 6,949,946 reads of 17 to 27 nucleotides (nt) were de novo assembled as described [3]. The resulting 395 contigs were searched against the GenBank viral sequence database using the BLASTn and BLASTx algorithms. Twenty-three contigs showed high nt sequence similarities (89-100%) to the genomic sequence of tomato mosaic virus (ToMV). The deduced amino acid (aa) sequences of thirty contigs had 22-96% aa sequence identities to viruses in the family Secoviridae, e.g., surrounding non-legume associated secovirus (snLaSV) and lettuce secovirus 1 (LSV-1). No contig homologous to the genomic sequences of other plant viruses was identified. Total RNAs were extracted from the mountain celery and tomato separately and reverse transcribed into cDNAs by random hexamer plus Oligo-dT(18) using the Super® IV Reverse Transcriptase (Invitrogen, Shanghai, China). Polymerase chain reactions (PCR) showed that the secovirus was derived from the mountain celery, whereas the tomato was infected by ToMV. The genome of this secovirus was determined by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). Amplicons were cloned and Sanger sequenced with at least three independent clones per amplicon. Sequences were assembled by the SeqMan Pro 7.1.0 in the Lasergene (DNASTAR, Madison, WI). The genome of this virus is composed of two RNAs of 6,616 and 5,356 nt (excluding the polyadenylic acid tails) (GenBank accession nos. MW143070 and MW143071, respectively). The thirty contigs assembled from sRNAs could be mapped to the genome. Pairwise sequence analyses showed that RNA1 and RNA2 and their encoded polyproteins shared the highest nt (82.7% and 82.2%) and aa (93.4% and 91.8%) sequence identities with the respective RNAs (GenBank accession nos. MN412739 and MN412740) and their encoded polyproteins of snLaSV [4]. In the phylogenetic trees, this virus sequence clustered with snLaSV and LSV-1 in a separate branch neighboring viruses of the subgenus Stramovirus or Satsumavirus in the genus Sadwavirus. These results suggest that this virus is an isolate of the unclassified snLaSV and was referred as snLaSV-CHN. RT-PCR with primers SecoR1-3700F and SecoR1-5100R confirmed the presence of snLaSV-CHN in other mountain celeries (11 of 23 tested) showing chlorotic spots symptoms but not in healthy ones from the same field. To the best of our knowledge, this is the first report of snLaSV infecting mountain celery in China and a more orthodox name, mountain celery chlorotic spot virus (MCCSV), is tentatively proposed. Our findings provide a better insight of the distribution and host range of this virus and further surveys are necessary to determine its incidence and damage in mountain celery. Funding: This study is financial supported by the Program for the Scientific Activities of Selected Returned Overseas Professionals in Heilongjiang Province (Grant No. 2018QD0002) and the China National Funds for Excellent Young Scientists (Grant No. 32022071). References Son, H. J. 2020. Food Sci Nutr. 9:514. Martin, M. 2011. EMBnet J. 17:10. Che, X., et al. 2020. Plant Dis. 104: 3085. Gaafar, Y. Z. A., et al. 2020. Front Microbiol. 11: 583242.

First Report of Tomato Brown Rugose Fruit Virus Infecting Tomato Crop in Saudi Arabia

Tomato (Solanum lycopersicum L.) is the most economically important member of family Solanaceae and cultivated worldwide and one of the most important crops in Saudi Arabia. The aim of this study is screening of the most common viruses in Riyadh region and identified the presence of tomato brown rugose fruit virus (ToBRFV) in Saudi Arabia. In January 2021, unusual fruit and leaf symptoms were observed in several greenhouses cultivating tomatoes commercially in Riyadh Region, Saudi Arabia. Fruit symptoms showed irregular brown spots, deformation, and yellowing spots which render the fruits non-marketable, while the leaf symptoms included mottling, mosaic with dark green wrinkled and narrowing. These plants presented the symptoms similar to those described in other studies (Salem et al., 2015, Luria et al., 2017). A total 45 Symptomatic leaf samples were collected and tested serologically against suspected important tomato viruses including: tomato chlorosis virus, tomato spotted wilt virus, tomato yellow leaf curl virus, tomato chlorotic spot virus, tomato aspermy virus, tomato bushy stunt virus, tomato black ring virus, tomato ringspot virus, tomato mosaic virus, pepino mosaic virus and ToBRFV using Enzyme linked immunosorbent assay (ELISA) test (LOEWE®, Biochemica, Germany), according to the manufacturers' instructions. The obtained results showed that 84.4% (38/45) of symptomatic tomato samples were infected with at least one of the detected viruses. The obtained results showed that 55.5% (25/45) of symptomatic tomato samples were found positive to ToBRFV, three out of 25 samples (12%) were singly infected, however 22 out of 45 (48.8%) had mixed infection between ToBRFV and with at least one of tested viruses. A sample with a single infection of ToBRFV was mechanically inoculated into different host range including: Chenopodium amaranticolor, C. quinoa, C. album, C. glaucum, Nicotiana glutinosa, N. benthamiana, N. tabacum, N. occidentalis, Gomphrena globosa, Datura stramonium, Solanum lycopersicum, S. nigrum, petunia hybrida and symptoms were observed weekly and the systemic presence of the ToBRFV was confirmed by RT-PCR and partial nucleotide sequence. A Total RNA was extracted from DAS-ELISA positive samples using Thermo Scientific GeneJET Plant RNA Purification Mini Kit. Reverse transcription-Polymerase chain reaction (RT-PCR) was carried out using specific primers F-3666 (5´-ATGGTACGAACGGCGGCAG-3´) and R-4718 (5´-CAATCCTTGATGTG TTTAGCAC-3´) which amplified a fragment of 1052 bp of Open Reading Frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp). (Luria et al. 2017). RT-PCR products were analyzed using 1.5 % agarose gel electrophoresis. RT-PCR products were sequenced in both directions by Macrogen Inc. Seoul, South Korea. Partial nucleotide sequences obtained from selected samples were submitted to GenBank and assigned the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities ranged between 98.99 % to 99.50 % among them and 98.87-99.87 % identity with ToBRFV isolates from Palestine (MK881101 and MN013187), Turkey (MK888980, MT118666, MN065184, and MT107885), United Kingdom (MN182533), Egypt (MN882030 and MN882031), Jordan (KT383474), USA (MT002973), Mexico (MK273183 and MK273190), Canada (MN549395) and Netherlands (MN882017, MN882018, MN882042, MN882023, MN882024, and MN882045). To our knowledge, this is the first report of occurrence of ToBRFV infecting tomato in Saudi Arabia which suggests its likely introduction by commercial seeds from countries reported this virus and spread in greenhouses through mechanical means. The author(s) declare no conflict of interest. Keywords: Tomato brown rugose fruit virus, tomato, ELISA, RT-PCR, Saudi Arabia References: Luria N, et al., 2017. PLoS ONE 12(1): 1-19. Salem N, et al., 2015. Archives of Virology 161(2): 503-506. Fig. 1. Symptoms caused by ToBRFV showing irregular brown spots, deformation, yellowing spots on fruits (A, B, C) and bubbling and mottling, mosaic with dark green wrinkled and narrowing on leaf (D).

Physicochemical and Toxicity Investigation of Chitosan-based dsRNA Nanocarrier Formation

Technologies involving the use of double-stranded RNA (dsRNA) to elicit RNA interference (RNAi) in pest control have emerged as an alternative to traditional pesticides. RNAi can mediate natural cell protection being a promising tool to provide prompt responses in plant defense against pathogens. The present study is focused on the physicochemical characterization of formed dsRNA-loaded nanoparticles as a result of chitosan-dsRNA ionic interactions. Additionally, a preliminary investigation was conducted of the in-vitro toxicity of loaded nanoparticles in lettuce and human red blood cells. dsRNA molecules, homologous to partial phytopathogenic tomato mosaic virus (ToMV) sequence, were used as a model. The main groups involved in the chitosan-dsRNA ionic coupling were identified by Fourier-transform infrared spectroscopy, and the stability of formed nanoparticles was accessed by dynamic light scattering, electrophoresis, and thermal analyses. The chitosan showed a higher ability to bind to dsRNA at low charge ratios (N/P = 1), ruled by positively charged chitosan methyl groups and negatively charged phosphate groups from the RNA backbone, resulting in small nanoparticles (73.25 nm size) at low polydispersity (0.25). The toxic assays of these particles, on lettuce seeds and in human erythrocytes, revealed very low toxicity demonstrating their safety as a platform, thereby holding potential use as biodefensive for crop protection.

Screening of Solanum (sections Lycopersicon and Juglandifolia) germplasm for reactions to the tomato brown rugose fruit virus (ToBRFV)

The reaction of 636 Solanum (sections Lycopersicon and Juglandifolia) accessions were evaluated under greenhouse conditions after mechanical inoculation with a Jordanian isolate of the new tobamovirus tomato brown rugose fruit virus (ToBRFV). Local and systemic infections were assayed by symptoms evaluation and virus detection via biotests and RT-PCR. All cultivated tomatoes (Solanum lycopersicum) and the great majority of wild tomato accessions proved susceptible to ToBRFV. They showed a wide range of symptoms (mosaic, leaf deformations, mottling, shoestring, and stunting). Twenty-six accessions representing S. lycopersicum var. cerasiforme, S. pimpinellifolium, S. habrochaites, and S. chilense were tolerant. High levels of resistance have been demonstrated in three accessions of S. ochrantum, a close relative to wild tomatoes (member of the sect. Juglandifolia) not only to ToBRFV but also to the tobamoviruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). After mechanical inoculation, the three tobamoviruses could be detected only in inoculated leaves in the accessions LA2160, LA2162, and LA 2166, which remained symptomless. However, two other S. ochrantum accessions PI 473,498 and PI 230,519 reacted unusually. They were demonstrated highly resistant to TMV and ToMV, but proved transiently susceptible to ToBRFV showing mild systemic mosaic followed by total recovery from symptoms and the virus.

Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus

In recent years, tomato mottle mosaic virus (ToMMV) has become one of the most important viral pathogens affecting solanaceous crop production in Yunnan, Hainan, and Shandong provinces of China, often causing huge yield reductions. To provide farmers and vegetable industry with reliable and easy-to-use ToMMV detection methods, we immunized BALB/c mice with purified ToMMV and obtained six hybridoma cell lines (i.e., 2D6, 9C12, 26A10, 3A4, 23A4 and 17B11) that secrete anti-ToMMV monoclonal antibodies (MAbs) through the hybridoma technology. Using these MAbs as the detection antibody, we developed three serological assays: antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue print enzyme-linked immunosorbent assay (tissue print-ELISA) for ToMMV detection. Our test results showed that these three newly developed serological methods can be used to specifically detect ToMMV infection in plant samples, but not tobacco mosaic virus, tomato mosaic virus, cucumber green mottle mosaic virus and cucumber mosaic virus. Sensitivity analyses further showed that ACP-ELISA and dot-ELISA can be used to detect ToMMV infection in plant crude extracts diluted at 1:81,920 and 1:40,960 (weight/volume, g/mL), respectively. Surprisingly, the detection limit of the developed dot-ELISA was 26 times higher than that of traditional RT-PCR. Using field-collected plant samples, we have demonstrated that these three new serological methods are accurate and easy-to-use for large-scale detection of ToMMV in fields.

Detection of Parietaria Mottle Virus by RT-qPCR: An Emerging Virus Native of Mediterranean Area That Undermine Tomato and Pepper Production in Southern Italy

Parietaria mottle virus (PMoV) is considered an emerging virus in many countries of the Mediterranean basin, especially on tomato and pepper crops. Symptoms on tomato leaves and fruits can be easily confused with those induced by cucumber mosaic virus (CMV) with necrogenic satellite RNA (CMV-satRNA), tomato spotted wilt virus (TSWV) or tomato mosaic virus (ToMV). Mixed infection of these viruses has been also reported in some tomato cultivars, with an increase in the complexity of the symptoms and severity of the disease. Although a specific serum and riboprobes have been produced, nowadays no sensitive diagnostic methods are available for the rapid PMoV detection. Here, we have developed a RT-qPCR assay with the aim to establish a more sensitive and specific method for PMoV detection. Specific primers and TaqMan probe were designed and in silico tested with all PMoV isolates available in GenBank. Moreover, this method was evaluated on tomato naturally infected samples from Sicily region (Italy). Results obtained showed that the RT-qPCR assay developed in this work is extremely sensitive, in fact, it is able to detect as few as 10 PMoV RNA copies in tomato total RNA; moreover, it will be a particularly valuable tool for early detection of PMoV. Furthermore, the analyzes on field samples show how this pathogen is increasingly present in tomato crops in the last years, helping to undermine the Italian horticultural sector.

Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Treatment of vegetable seeds with hot water for disease control

Гидротермическая обработка семян представляет собой жизнеспособную альтернативу химической обработке для уничтожения патогенов. Задача гидротермической обработки – установить наилучшее сочетание времени и температуры, которое сокращает выживаемость патогенов при минимальном разрушающем воздействии на семена. Основное предположение, оправдывающее гидротермическую обработку к снижению влияния или полному уничтожению патогенов, заключается в том, что патоген-мишень более чувствителен к высокотемпературному стрессу, чем семена. Целевые патогены микроорганизмов – в основном грибы, вирусы и бактерии, находящиеся на поверхности и внутри семян. Цель исследования – провести обзор термотерапии горячей водой для освобождения семян овощных культур от фитопатогенов. В статье проанализирована доступная информация о влиянии гидротермической обработки семян на рост, заболеваемость и урожайность овощных культур. В обзор включены статьи из различных баз данных, таких как Google Scholar, PubMed, Science Direct, SciFinder, Web of Science, РИНЦ и др., использованы онлайн-источники (Research Gate, Национальный центр биотехнологической информации (NCBI), Springer Nature Open Access, Wiley Online Library и др.). Обобщена протестированная гидротермическая обработка для освобождения семян овощных культур от фитопатогенов. Удовлетворительный контроль получен в отношении нескольких бактериальных болезней на овощных культурах, в основном вызываемых родами Clavibacter, Xanthomonas и Pseudomonas. Однако гидротермическую обработку труднее использовать на крупных семенах бобовых культур, таких как горох, фасоль или соя, потому что часто всхожесть значительно снижается еще до того, как бактерии будут полностью уничтожены. Обработка горячей водой эффективна против грибных патогенов Alternaria, Phoma, Septoria, Stemphylium, Verticillium, Cladosporium, передаваемых через семена. Показана высокая эффективность обработки семян овощных культур горячей водой против вируса табачной мозаики, вируса огуречной мозаики, вируса мозаики томата, двойного стрика томата, вируса мозаики салата и вируса мягкой крапчатости перца. Гидротермическую обработку семян необходимо выполнять в строгом соответствии с регламентом по времени и температуре, ее лучше всего проводить с помощью термостатируемых водяных бань. Hot water seed thermotherapy is a viable alternative to chemical control of pathogens. The challenge for thermotherapy is to find the best combination of time and temperature that maximizes the reduction in pathogen survival while minimizing damage to the seeds. The main assumption justifying a thermotherapeutic approach to pathogen control is that the target pathogen is more sensitive to high temperature stress than seeds. Target pathogens are mainly fungi, viruses and bacteria. The aim of the study is to review the hot water thermotherapy for the release of vegetable seeds from phytopathogens. This article analyzes the available information on the effect of heat treatment of seeds on the growth, morbidity and productivity of vegetable crops. The review includes articles from various databases, such as Google Scholar, PubMed, Science Direct, SciFinder, Web of Science, RSCI, etc., and uses online sources (Research Gate, National Center for Biotechnology Information (NCBI), Springer Nature Open Access, Wiley Online Library, etc.). The tested thermotherapy for the release of vegetable seeds from phytopathogens is generalized. Satisfactory control was obtained for several bacterial diseases in vegetable crops, mainly caused by the genera Clavibacter, Xanthomonas and Pseudomonas. However, thermotherapy is more difficult to use on large legume seeds such as peas, beans or soybeans, because a significant reduction in germination is often achieved before the bacteria are completely destroyed. Hot water thermotherapy is effective against fungal pathogens Alternaria, Phoma, Septoria, Stemphylium, Verticillium, Cladosporium, transmitted through seeds. The high efficiency of treatment of vegetable seeds with hot water against tobacco mosaic virus, cucumber mosaic virus, tomato mosaic virus, tomato double streak, lettuce mosaic virus and pepper mottling virus is shown to be highly effective. Heat treatment of seeds should be carried out in strict accordance with the time and temperature regulations, and it is best carried out using thermostatically controlled water baths.