Search Results (22)

Potyviruses (family Potyviridae, genus Potyvirus), including emerging ones, pose a growing threat to cucurbit production. This study presents the first virome analysis of severely symptomatic cucurbits in continental Croatia, combining high-throughput sequencing (HTS) and RT-PCR diagnostics. Zucchini, cucumber, and butternut squash plants with severe virus-like symptoms sampled in 2021–2022 were found to consistently host a complex of potyviruses, including watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), and Moroccan watermelon mosaic virus (MWMV)—the latter being newly reported in Croatia and representing likely its northernmost detection in Europe. Phylogenetic analysis classified WMV isolates as emerging strains of subgroup EM3 and ZYMV as subgroup A1, consistent with European lineages. Croatian MWMV isolates formed a distinct subclade within the Mediterranean group, raising questions about its diversification trajectory. The findings highlight the expanding range of MWMV and underscore the value of HTS for early detection of emerging threats. These results have critical implications for cucurbit disease management, indicating the need to re-evaluate resistance claims in commercial cultivars and implement stricter phytosanitary surveillance in Croatia. The potential role of climate change in facilitating virus spread via aphid vectors is discussed, warranting further risk assessment and international monitoring efforts. Full article

Squash leaf curl China virus (SLCCNV) belongs to the species Begomovirus cucurbitachinaense in the genus Begomovirus and can infect some Cucurbitaceae crops except for watermelon (Citrullus lanatus). In this study, watermelon plants showing symptoms typical to begomovirus infection in field were observed in Zhejiang Province of China, and SLCCNV presence was identified through PCR and next-generation sequencing (NGS). The pairwise sequence identity of the DNA-A genome shows that SLCCNV watermelon isolate belongs to the SLCCNV/CN strain and shares 96% nucleotide identity with the previously sequenced SLCCNV/CN. An infectious clone of SLCCNV watermelon isolate was constructed using the tandem repeat fragment method. Through agrobacterium-mediated inoculation, the clone could induce systemic infection with typical symptoms in watermelon, melon (Cucumis melo), squash (Cucurbita pepo), pumpkin (Cucurbita maxima), wax gourd (Benicasa hispida), cucumber (Cucumis sativus), and N. benthamiana. It was further demonstrated that the progeny virions derived from the cloned watermelon isolate could be transmitted by whitefly rather than the sap. To the best of our knowledge, this is the first report of a natural infection of SLCCNV on watermelon in China, and the first complete report on the molecular characteristics and pathogenicity of watermelon-infecting SLCCNV in the world. Full article

Viruses are a major pathogen challenging the sustainable production of cucurbits worldwide. Pumpkin and winter squash showed severe virus-like symptoms during the fall of 2022 and 2023 in Georgia, USA. Symptomatic leaves were collected from the field and processed for small RNA sequencing for virus identification using high-throughput sequencing (HTS). HTS analysis revealed the presence of two aphid-transmitted viruses (ATVs), zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV), along with three whitefly-transmitted viruses, cucurbit chlorotic yellows virus, cucurbit yellow stunting disorder virus, and cucurbit leaf crumple virus. The results of our study suggest a significant shift in ATV’s abundance in these two crops between 2022 and 2023. According to the qPCR data in the fall of 2022, pumpkins experience an incidence of 56.25% and 31.25% of PRSV and ZYMV, respectively. Similarly, winter squash shows an incidence of 50% and 32.14% of PRSV and ZYMV, respectively. Mixed infection of both viruses was also observed in these two crops. In 2023, we observed a predominance of ZYMV in pumpkin and winter squash (61.25% and 42.50%, respectively). However, PRSV was not detected in pumpkins, and it was detected at a negligible level (0.62%) in winter squash using qPCR. Phylogenetic analysis of ZYMV-encoded coat protein (CP) and helper component-protease (HC-Pro) from Georgia suggests a close relationship with the European isolates. Conversely, PRSV-encoded CP and NIa-VPg show a more diverse evolutionary history. Overall, this research will provide valuable insights into the dynamics of ZYMV and PRSV in pumpkin and winter squash crops within the southeastern United States. Full article

This study presents a one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) method for the simultaneous detection of multiple viruses affecting melon crops. Viruses such as Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), Squash mosaic virus (SqMV), Tobacco mosaic virus (TMV), Papaya ring spot virus (PRSV), and Melon yellow spot virus (MYSV) pose a great threat to melons. The mixed infection of these viruses is the most common observation in the melon-growing fields. In this study, we surveyed northern Xingjiang (Altay, Changji, Wujiaqu, Urumqi, Turpan, and Hami) and southern Xingjiang (Aksu, Bayingolin, Kashgar, and Hotan) locations in Xinjiang province and developed a one-step multiplex RT-PCR to detect these melon viruses. The detection limits of this multiplex PCR were 10³ copies/μL for ZYMV and MYSV and 10² copies/μL for WMV, SqMV, PRSV, CMV, and TMV. The detection results in the field showed 242 samples were infected by one or more viruses. The multiplex RT-PCR protocol demonstrated rapid, simultaneous, and relatively effective detection of viruses such as WMV, CMV, ZYMV, SqMV, TMV, PRSV, and MYSV. The technique is designed to identify these melon viruses in a single reaction, enhancing diagnostic efficiency and reducing costs, thus serving as a reference for muskmelon anti-virus breeding in Xinjiang. Full article

Rehmannia glutinosa, a crucial medicinal plant native to China, is extensively cultivated across East Asia. We used high-throughput sequencing to identify viruses infecting R. glutinosa with mosaic, leaf yellowing, and necrotic symptoms. A novel Torradovirus, which we tentatively named “Rehmannia torradovirus virus” (ReTV), was identified. The complete sequences were obtained through reverse-transcription polymerase chain reaction (RT-PCR), 5′ and 3′ rapid amplification of cDNA ends, and Sanger sequencing. The amino acid sequence alignment between the ReTV-52 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 51.3–73.3% and 37.1–68.1%, respectively. Meanwhile, the amino acid sequence alignment between the ReTV-8 isolate and known Torradovirus species in the Pro-Pol and coat protein regions were 52.7–72.8% and 36.8–67.5%, respectively. The sequence analysis classified ten ReTV strains into two variants. The ReTV-52 genome has two RNA segments of 6939 and 4569 nucleotides, while that of ReTV-8 consists of two RNA segments containing 6889 and 4662 nucleotides. Sequence comparisons and phylogenetic analysis showed ReTV strains clustered within the Torradovirus, exhibiting the closet relation to the squash chlorotic leaf spot virus. The RT-PCR results showed a 100% ReTV detection rate in all 60 R. glutinosa samples. Therefore, ReTV should be classified as a novel Torradovirus species. ReTV is potentially dangerous to R. glutinosa, and necessitating monitoring this virus in the field. Full article

To cultivate excellent lily germplasms, an interspecific hybrid (LC×SQ-01) was successfully obtained by using a cut-style pollination method in which the rare wild lily Lilium callosum was used as the female parent and the cut flower L. longiflorum ‘Snow Queen’ was used as the male parent. The morphological features of LC×SQ-01 included height, leaf length, and width, which were observed to be between those of the parents in the tissue-cultured seedlings. The height and leaf length of LC×SQ-01 were more similar to those of the male parent, and the width was between the widths of the parents for field-generated plants. The epidermal cell length and the guard cell and stoma sizes were between those of both parents in tissue-cultured and field-generated plants. In addition, the shapes of the epidermal cells and anticlinal wall in LC×SQ-01 were more analogous to those in the male parent, while the stoma morphology was different from that of both parents. Fourteen pairs of polymorphic SSR primers were identified in both parents, and the validity of LC×SQ-01 was demonstrated by PCR amplification using five pairs of SSR primers. Flow cytometry and root tip squashing assays revealed that LC×SQ-01 was a diploid plant, similar to its parents. Furthermore, the LC×SQ-01 hybrid was more resistant to B. cinerea than its parents, and it also showed much greater peroxidase (POD) and catalase (CAT) activity than the parents. These results lay a foundation for breeding a new high-resistance and ornamental lily variety. Full article

In August 2020, the unusual appearance of crown gall symptoms was observed on the tobacco plants (hybrid PVH2310) grown in fields in the Golubinci (Srem district, Serbia) locality. The causal agent isolated from galls located on tobacco roots formed circular, convex, and glistening light blue colonies, and then dark to olive-green-colored bacterial colonies on a semi-selective D1 medium. Molecular analysis based on multiplex PCR and multi-locus sequence analysis (MLSA) using concatenated sequences of the atpD, dnaK, glnA, and rpoB genes as well as 16S rRNA identified Serbian tobacco isolates such as Agrobacterium tumefaciens (biovar 1). Two duplex PCR methods confirmed the presence of the virD2 and virC genes in tobacco isolates. Pathogenicity tests performed on carrot discs and squash fruits resulted in tumor/gall formation after 12 to 16 days post inoculation, respectively. Pathogenicity was also confirmed on tobacco plants, where isolates caused tumor development 21−25 days after inoculation. API 50 CH generated results regarding the biochemical features of the Serbian tobacco isolates. As A. tumefaciens (biovar 1) as a cause of tobacco crown gall has previously been documented solely in Japan, there is presently no data on its wider occurrence. Therefore, this first detailed investigation of A. tumefaciens isolated from naturally infected tobacco in Serbia will contribute to a better understanding of it at the global level. Full article

Microalgae are recognized for their versatility in providing renewable energy, biopharmaceuticals, and nutraceuticals, attributed to their sustainable, renewable, and cost-effective nature. Genetic engineering has proven highly effective in enhancing microalgae production. PCR-based genotyping is the primary method for screening genetically transformed microalgae cells. Recently, we developed a novel PCR method, namely Squash-PCR, and employed it for the molecular analysis of industrially important fungi and yeasts. In this study, we successfully implemented the Squash-PCR technique in 12 industrially significant algae species. This approach offers a quick and reliable means of obtaining DNA templates directly from squashed algal cells, eliminating the need for time-consuming and labor-intensive cultivation and genomic DNA extraction steps. Our results demonstrate the effectiveness of Squash-PCR in detecting and characterizing target genes of interest in 12 different algae species. Overall, this study establishes the Squash-PCR method as a valuable tool for molecular studies in algae, enabling researchers to rapidly screen and manipulate genetic traits in diverse algal species. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 10² genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2–3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions. Full article

The incidence and severity of begomovirus diseases have been increasing around the world recently, and the ridge gourd [Luffa acutangula (Roxb.) L.] is the latest example of a crop that has become highly susceptible to the outbreak of the tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in India. Accurate diagnosis of causal agents is important in designing disease management strategies. In this study the coat protein (CP) gene from a ToLCNDV-Rg ridge gourd isolate was used to produce polyclonal antibodies (ToLCNDV-Rg-CP-PAb) in a rabbit. The antibodies successfully detected a 30.5 kDa ToLCNDV-Rg-CP in extracts of symptomatic ridge gourd leaf samples by several assays, such as Western Blotting (WB), Dot Immuno Binding Assay (DIBA), Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA), Immuno Capture Polymerase Chain Reaction (IC-PCR), and Immuno Capture Loop-Mediated Isothermal Amplification (IC-LAMP) assays. However, none of the negative samples tested positive in either of the detection methods. Among all the methods tested, the immunocapture assay, IC-LAMP, was the most sensitive in detecting ToLCNDV-Rg. Furthermore, antibodies generated in this study also detected other commonly occurring begomoviruses in South India, such as tomato leaf curl Palampur virus and squash leaf curl China virus in cucurbits. Together, ToLCNDV-Rg-CP-PAb can be used for detecting at least three species of begomoviruses infecting cucurbits. The obtained antibodies will contribute to monitoring disease outbreaks in multiple crops. Full article

Cucurbits are important economic crops worldwide. However, the cucurbit leaf curl disease (CuLCD), caused by whitefly-transmitted begomoviruses constrains their production. In Southeast Asia, three major begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV), Squash leaf curl China virus (SLCCNV) and Squash leaf curl Philippines virus (SLCuPV) are associated with CuLCD. SLCuPV and SLCCNV were identified in Luzon, the Philippines. Here, the genetic diversity and geographic distribution of CuLCD-associated begomoviruses in the Philippines were studied based on 103 begomovirus detected out of 249 cucurbit samples collected from 60 locations throughout the country in 2018 and 2019. The presence of SLCCNV and SLCuPV throughout the Philippines were confirmed by begomovirus PCR detection and viral DNA sequence analysis. SLCuPV was determined as a predominant CuLCD-associated begomovirus and grouped into two strains. Interestingly, SLCCNV was detected in pumpkin and bottle gourd without associated viral DNA-B and mixed-infected with SLCuPV. Furthermore, the pathogenicity of selected isolates of SLCCNV and SLCuPV was confirmed. The results provide virus genetic diversity associated with CuLCD for further disease management, especially in developing the disease-resistant cultivars in the Philippines as well as Southeast Asia. Full article

To study the host range of Rose rosette virus (RRV), we employed crude sap inoculum extracted from RRV-infected roses and the RRV infectious clone. We inoculated plants from the families Solanaceae, Cucurbitaceae, Leguminosae, Malvaceae, Amaranthaceae, and Brassicaceae. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect RRV in the inoculated plants throughout their growth stages. Interestingly, RRV was detected in the newly developed leaves of tomato, pepper, tobacco, cucumber, squash, zucchini, pumpkin, pea, peanut, soybean, spinach, okra, and Chenopodium spp. The speed of upward advancement of RRV within infected plants was variable between plants as it took two to three weeks for some plant species and up to five weeks in other plant species to emerge in the newest leaves. No severe symptoms were detected on most of the inoculated plants. Chenopodium spp., spinach, cucumber and Nicotiana rustica exhibited either chlorotic or necrotic lesions with variable shapes and patterns on the systemically infected leaves. Double membrane-bound particles of 80–120 nm in diameter were detected by transmission electron microscopy in the infected tissues of cucumber, pepper, and N. benthamiana plants. This finding infers the validity of mechanical inoculation for RRV on a wide range of plants that would serve as potential natural reservoirs. Full article

The protozoan Sarcocystis bertrami (syn. Sarcocystis fayeri) infects horses and has dogs as definitive hosts. Herein we aimed to detect S. bertrami in Brazilian horses destined for human consumption and to determine the frequency of infection in the examined animals. Muscle fragments from 51 horses were collected in a slaughterhouse in Bahia State during three different seasons of the year. Samples from six tissues from each animal were prepared for macroscopic and microscopic evaluation, using tissue grinding, squash and histology. Sarcocystis sp. was observed in 100% of the examined horses. Selected samples were processed for transmission electron microscopy (TEM). Species identification was confirmed using a PCR targeted to the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Histological examination revealed sarcocysts with variable sizes and shapes, and dispersed within the muscle fibers. When observed by TEM, the sarcocyst wall was wavy and covered by an electrodense layer. The villar protrusions were digitiform and bent. To our knowledge, this study is the first morphological and molecular confirmation of S. bertrami in horses in Brazil and South America. Full article

The identification of alternate hosts that can act as virus inoculum sources and vector reservoirs in the landscape is critical to understanding virus epidemics. Cotton leafroll dwarf virus (CLRDV) is a serious pathogen in cotton production and is transmitted by the cotton/melon aphid, Aphis gossypii, in a persistent, circulative, and non-propagative manner. CLRDV was first reported in the United States in Alabama in 2017, and thereafter in several cotton-producing states. CLRDV has since established itself in the southeastern United States. The role of alternate hosts in CLRDV establishment is not clear. Fourteen common plant species in the landscape, including crops, weeds, and ornamentals (cotton, hollyhock, marshmallow, country mallow, abutilon, arrowleaf sida, okra, hibiscus, squash, chickpea, evening primrose, henbit, Palmer amaranth, and prickly sida) were tested as potential alternate hosts of CLRDV along with an experimental host (Nicotiana benthamiana) via aphid-mediated transmission assays. CLRDV was detected following inoculation in hibiscus, okra, N. benthamiana, Palmer amaranth, and prickly sida by RT-PCR, but not in the others. CLRDV accumulation determined by RT-qPCR was the highest in N. benthamiana compared with cotton and other hosts. However, aphids feeding on CLRDV-infected prickly sida, hibiscus, and okra alone were able to acquire CLRDV and back-transmit it to non-infected cotton seedlings. Additionally, some of the alternate CLRDV hosts supported aphid development on par with cotton. However, in a few instances, aphid fitness was reduced when compared with cotton. Overall, this study demonstrated that plant hosts in the agricultural landscape can serve as CLRDV inoculum sources and as aphid reservoirs and could possibly play a role in the reoccurring epidemics of CLRDV in the southeastern United States. Full article

The chickpea chlorotic dwarf virus (CpCDV) (from the genus Mastrevirus and the family Geminiviridae) and tomato leaf curl New Delhi virus (ToLCNDV) (from the genus Begomovirus and the family Geminiviridae) represent an important threat to different crops worldwide, as they are emerging viruses in the Asian continent, were introduced to the Mediterranean region in 2012, and were then reported in Morocco in 2017 and 2018. The present study addresses the dispersion of the CpCDV and the ToLCNDV and evaluates the genetic diversity of the Moroccan isolates of both viruses. A total of 1333 symptomatic leaf plant samples were analyzed by PCR. The study has reported the detection of the ToLCNDV in melon and tomato, as well as the CpCDV in squash crops for the first time in Morocco. Blast analysis of selected representative isolates showed a 97–99% nucleotide identity with the ToLCNDV and the CpCDV infecting different crops in the Mediterranean region. Phylogenetic analysis showed low variability among the Moroccan isolates for the ToLCNDV compared to the Spanish and Italian isolates, whereas the CpCDV strains were variable regarding strains reported in Tunisia and Egypt. Recombination analysis showed the presence of the ToLCNDV recombinant strains with variable parents. The spread of both geminiviruses represents a threat to different crop production, requiring the development of crop protection and management strategies. To prevent viral outbreaks, restrictive phytosanitary measures and the development of resistance strategies are also necessary. Full article