Dot Immunobinding Assay Research Articles

Preparation and Application of Polyclonal Antibodies for the Rapid Detection of Actinidia Chlorotic Ringspot-Associated Virus

Actinidia chlorotic ringspot-associated virus (AcCRaV, Emaravirus actinidiae) is prevalent in Chinese kiwifruit, leading to substantial yield reduction. The intricate nature of symptoms presents diagnostic challenges, underscoring the necessity for a rapid and accurate detection method that facilitates effective control. In this investigation, AcCRaV isolates from key kiwi-producing regions in Sichuan province were collected and analyzed, with representative strains chosen as experimental materials. Primers targeting the nucleoprotein gene of AcCRaV were designed, and their codon usage was optimized to enhance performance. Various serological methods utilizing polyclonal antibodies were developed, including ELISA, dot immunobinding assay, and AcCRaV-specific gold immunochromatographic bands (AcCRaV-GICS). Field samples exhibited high specificity and sensitivity when tested using these methods. Furthermore, the results obtained from a large number of field samples are consistent with those derived from RT-PCR analysis, further validating the applicability of our approach. A detection method capable of handling a large volume of field samples infected with AcCRaV is currently lacking; thus, our system construction provides an important reference for addressing this gap.

Bio-Orthogonally Redirected Bispecific Lentiviral Vectors for In Vivo CAR-T Cell Generation

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a remarkably efficacious treatment modality in recent years for refractory and relapsed hematopoietic malignancies. However, the exorbitant cost and intricate manufacturing procedure of ex vivo CAR-T cell generation have impeded its broader application. In vivo induced CAR-T therapy offers the potential to circumvent cumbersome manufacturing logistics, thereby providing novel insights into the field of CAR-T treatment. In this study, we have developed a bio-orthogonally redirected bispecific lentiviral vector platform for the reprogramming of circulating T lymphocytes into CAR-T cells and subsequent elimination of tumor cells in vivo. Methods: To engineer dual-targeting lentiviral vectors (D-LVs), the packing cell lines 293T was labelled with azide groups by glycometabolic bio-orthogonal chemistry (designated N 3-293T) firstly, and a CD3 promoter (CD3p) was fused to CAR transfer plasmid. Secondly, azide groups modified LVs were further surface engineered with anti-CD3 antibody (OKT3) via click chemistry. The conjunction of OKT3 and was analyzed using dot immunobinding assay, confocal microscopy and the in vitro transduction efficiency was evaluated using flow cytometry. To demonstrate the ability of targeted transduction and specific cytotoxicity in vivo, D-LVs were intravenously infused into humanized NOD-scid-IL2Rγnull (huNSG) mice engrafted with Nalm6-luc cells. Subsequently, the tumor burden was monitored using a noninvasive bioluminescence imaging system and the in-vivo CD19 -CAR-T cells existence were detected by flow cytometry. Results: By displaying OKT3 on the single lentiviral surface and fused CD3p into the key construct, we could achieve targeted delivery of CD19-CAR genes to T cells both in vitro and in vivo. Conclusion: These results demonstrate the great potential applications of this engineered lentiviral system as a new strategy for inducing CAR-T immunotherapy in vivo and a promising approach for leading personalized treatment.

Incidence of main viruses infecting local garlic in Java, Indonesia

Virus infection is one of the challenges in garlic production due to it perpetuates from one generation to the next and its infection caused huge yield reduction. There was still few information regarding virus status on Indonesian local garlic cultivars. This study was aimed to detect four major viruses infecting local garlic in Indonesia, they were members of genus Potyvirus (Onion yellow dwarf virus/OYDV, Leek yellow stripe virus/LYSV), and Carlavirus (Garlic common laten virus/ GCLV and Shallot latent virus/SLV). Garlic samples were obtained from IPB University collection and field survey in Tegal and Karanganyar (Central Java Province). Dot immuno-binding assay (DIBA) was done for initial virus indexing on non-commercial and commercial cultivars. Reverse transcription polymerase chain reaction (RT-PCR) using four specific primers was done to detect virus on commercial cultivars. DIBA from leaf samples showed that virus incidence of OYDV was relatively higher (92.3 to 100%) than GCLV and SLV (84.6 to 100%) from all tested cultivars. On average, ‘Lumbu Hijau’ has the lowest level of virus titter (severity) than other cultivars. The virus incidence of both bulbil and single clover was similar (97 – 100%) while virus titter of OYDV, GCLV, and SLV on bulbil was the lowest than other propagation materials. Detection by RT-PCR from two commercial cultivars showed that ‘Lumbu Hijau’ has less virus incidence than ‘Jawa Lama’. LYSV, OYDV, GCLV were detected on both cultivars but SLV was not found. Further virus indexing using larger number of samples and involving more virus targets needs to be done.

Epidemiological Survey and Risk Factor Analysis of 14 Potential Pathogens in Golden Snub-Nosed Monkeys at Shennongjia National Nature Reserve, China

Golden snub-nosed monkeys (Rhinopithecus roxellanae) belong to Class A, the highest level of endangered primate species. Exploring the infection status of potential pathogens in golden snub-nosed monkeys is important for controlling associated diseases and protecting this species. The objective of this study was to investigate the seroprevalence for a number of potential pathogens and the prevalence of fecal adenovirus and rotavirus. A total of 283 fecal samples were collected from 100 golden snub-nosed monkeys in December 2014, June 2015, and January 2016; 26 blood samples were collected from 26 monkeys in June 2014, June 2015, January 2016 and November 2016 at Shennongjia National Reserve in Hubei, China. The infection of 11 potential viral diseases was examined serologically using an Indirect Enzyme-linked Immunosorbent Assay (iELISA) and Dot Immunobinding Assays (DIA), while the whole blood IFN-γ in vitro release assay was used to test tuberculosis (TB). In addition, fecal Adenovirus and Rotavirus were detected using Polymerase Chain Reaction (PCR). As a result, the Macacine herpesvirus-1 (MaHV-1), Golden snub-nosed monkey cytomegalovirus (GsmCMV), Simian foamy virus (SFV) and Hepatitis A virus (HAV) were detected with the seroprevalence of 57.7% (95% CI: 36.9, 76.6), 38.5% (95% CI: 20.2, 59.4), 26.9% (95% CI: 11.6, 47.8), and 7.7% (95% CI: 0.0, 84.2), respectively. Two fecal samples tested positive for Adenovirus (ADV) by PCR, with a prevalence of 0.7% (95% CI: 0.2, 2.5), and further, the amplification products were sequenced. Phylogenetic analysis revealed that they belonged to the HADV-G group. However, other pathogens, such as Coxsackievirus (CV), Measles virus (MeV), Rotavirus (RV), Simian immunodeficiency virus (SIV), Simian type D retroviruses (SRV), Simian-T-cell lymphotropic virus type 1 (STLV-1), Simian varicella virus (SVV), Simian virus 40 (SV40) and Mycobacterium tuberculosis complex (TB) were negative in all samples. In addition, a risk factor analysis indicated that the seroprevalence of MaHV-1 infection was significantly associated with old age (≥4 years). These results have important implications for understanding the health status and conservation of the endangered golden snub-nosed monkey population at Shennongjia Nature Reserve.

Dot Immunobinding Assay for the Rapid Serodetection of Scedosporium/Lomentospora in Cystic Fibrosis Patients.

The detection of Scedosporium/Lomentospora is still based on non-standardized low-sensitivity culture procedures. This fact is particularly worrying in patients with cystic fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because a poor and delayed diagnosis can worsen the prognosis of the disease. To contribute to the discovery of new diagnostic strategies, a rapid serological dot immunobinding assay (DIA) that allows the detection of serum IgG against Scedosporium/Lomentospora in less than 15 min was developed. A crude protein extract from the conidia and hyphae of Scedosporium boydii was employed as a fungal antigen. The DIA was evaluated using 303 CF serum samples (162 patients) grouped according to the detection of Scedosporium/Lomentospora in the respiratory sample by culture, obtaining a sensitivity and specificity of 90.48% and 79.30%, respectively; positive and negative predictive values of 54.81% and 96.77%, and an efficiency of 81.72%. The clinical factors associated with the results were also studied using a univariate and a multivariate analysis, which showed that Scedosporium/Lomentospora positive sputum, elevated anti-Aspergillus serum IgG and chronic Pseudomonas aeruginosa infection were significantly associated with a positive result in DIA, while Staphylococcus aureus positive sputum showed a negative association. In conclusion, the test developed can offer a complementary, rapid, simple and sensitive method to contribute to the diagnosis of Scedosporium/Lomentospora in patients with CF.

Detecting Tomato Leaf Curl New Delhi Virus Causing Ridge Gourd Yellow Mosaic Disease, and Other Begomoviruses by Antibody-Based Methods.

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.

Generation of specific antibody against recombinant major coat protein of Beet necrotic yellow vein virus and its application for detection of rhizomania disease

Beet necrotic yellow vein virus (BNYVV) is one of the main contributors to economic losses in sugar beet production. The present study generated a polyclonal antibody that detects BNYVV. The conserved genomic region of all BNYVV isolates gene encoding the viral major coat protein (BNYVV-CP) was amplified, cloned, sequenced, and expressed in E. coli strain BL21 (DE3). The expressed protein was purified under native conditions by affinity chromatography. The purified BNYVV CP was used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-CP-IgG detected the BNYVV-CP recombinant protein and BNYVV in infected sugar beet by indirect-ELISA, dot immunobinding assay, and western blot analysis blotting. The serological assays strongly proved the sensitivity and specificity of anti-BNYVV-CP-IgG against BNYVV. These results suggest that the generated anti-BNYVV-CP-IgG polyclonal antibodies can be used as reliable and quick means for the BNYVV virus detection under field conditions.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Pepper mild mottle virus infection in cayenne and sweet pepper in Indonesia

This is a report of the natural occurrence of Capsicum frutescens (cayenne pepper) and Capsicum annuum L. (sweet pepper) infection with pepper mild mottle virus (PMMoV) in Indonesia, which was detected serologically via dot-immunobinding assay and confirmed by RT-PCR of the coat-protein–encoding gene and DNA sequencing. Phylogenetically, the cayenne and sweet-pepper isolates were closely related to Capsicum spp. isolates from Turkey, Italy and Korea, with 99.1%–99.5% and 100% identity at the nucleotide and amino acid levels, respectively.

Advanced and Commonly Used Serological Techniques for Detection and Diagnosis of Plant Virus: Review

Plant diseases caused by viruses are the most menace to sustainable agriculture, leading to numerous billion dollars in losses annually. Proper and accurate detection of plant viruses is always the key to developing appropriate solutions to manage the economic losses caused by them. Advances in technology have simplified the tools available to detect and diagnose viruses at a time when control measures should be used. Currently, nucleic acid and serological based diagnostic methods are widely used for plant viral identifications. Serological techniques which based on antibody and antigen reaction is broadly used for the detection of plant viruses because of its simplicity, adaptability and sensitivity. A large number of advanced serological tests are available such as enzyme-linked immunosorbent assay (ELISA), dot immunobinding assay, tissue blotting immunoassay and Lateral flow assay. Various ELISA varieties such as double-antibody sandwich- ELISA, triple-antibody sandwich-ELISA, and antigen-coated-ELISA are widely used in the detection and testing of plant viruses. Dot immunobinding assay performed on a nitrocellulose that the plant extracts are spotted onto a membrane while tissue blotting immunoassay is an easy method that the crude sap from plants is directly blotted and used for detection through immunology. Lateral flow assay is the latest and the only serological method used for the detection of viruses without the aid of laboratory and skilled personnel. It is an onsite and quick assay that can be performed by anyone and suitable for the detection of viruses that occur in high concentrations in plants. Keywords : Antibody, Antigen, ELISA, Plant virus, Detection, Serology DOI: 10.7176/ALST/91-02 Publication date: January 31 st 2022

Preliminary Study of Viruses Infecting Strawberry (Fragaria spp.) in the Midsouthern United States

Strawberry is an important economic crop in the midsouthern United States; however, limited information is known about the viruses present in strawberry fields in Arkansas, Missouri, Oklahoma, and Texas. The purpose of this study was to determine the presence of particular viruses infecting strawberry crops in these four states. A total of 816 strawberry leaf samples were randomly collected from growers’ fields during the 2014 and 2016 growing seasons and tested by dot-immunobinding assay against antisera of eight available viruses: apple mosaic virus (ApMV), arabis mosaic virus (ArMV), raspberry bushy dwarf virus (RBDV), raspberry ringspot virus, strawberry mild yellow edge virus (SMYEV), tobacco necrosis virus, tomato black ring virus, and tomato ringspot virus (ToRSV). Of the eight viruses, ApMV, RBDV, and SMYEV had the highest infection rates in 2014, whereas ApMV, ArMV, ToRSV and SMYEV were the most prevalent viruses in 2016. Mixed infection of more than one virus was very common in samples collected from all four states. Our preliminary results showed that the distribution of virus relative frequencies differed significantly among seven sample locations across the states within a sample year, but we could not show statistical differences among the years based on our sample sizes. Mixed infections in strawberries were higher than expected in 2014 and lower than expected in 2016 based on viruses being independent infections. Overall, the occurrence of the viruses in strawberry crops could have important potential impacts for strawberry production in the midsouthern states.

Seed-transmission of Cowpea mild mottle virus on several varieties of soybean in Indonesia

Abstract. Sutrawati M, Hidayat SH, Suastika G, Sukarno BPW, Nurmansyah A. 2021. Seed-transmission of Cowpea mild mottle virus on several varieties of soybean in Indonesia. Biodiversitas 22: 4182-4185. Seeds and infected plants play important role as source of disease in the field for seed-transmitted virus, such as Cowpea mild mottle virus (CPMMV). Research was conducted to determine seed transmission nature of CPMMV on 10 soybean varieties based on growing on test method and dot immunobinding assay to confirm CPMMV infection. Field experiment was conducted to evaluate the efficiency of seed-transmitted CPMMV as the source of initial inoculum in the field. Soybean var. ‘Anjasmoro’ from 3 cultivation areas (Cianjur, Bogor, and Cirebon) was used for field experiment. Seed transmission of CPMMV was confirmed on soybean var. ‘Detam 2’, ‘Detam 3’, ‘Malika’, ‘Anjasmoro’, and ‘Argomulyo’; but was not found on ‘Detam 1’, ‘Detam 4’, ‘Wilis’, ‘Grobogan’, and ‘Dena 1’. The infection of CPMMV did not show symptom, either on the seedcoat and the unifoliolate leaves. Infection rate of CPMMV on seeds were relatively high, ranged between 27 to 86%. Disease incidence on var. ‘Anjasmoro’ from Cianjur, Cirebon, and Bogor varied from 32.9 to 75% and 57.9 to 81.3% in screenhouse and field experiment, respectivelly.

High Prevalence of Three Potyviruses Infecting Cucurbits in Oklahoma and Phylogenetic Analysis of Cucurbit Aphid-Borne Yellows Virus Isolated from Pumpkins.

Field information about viruses infecting crops is fundamental for understanding the severity of the effects they cause in plants. To determine the status of cucurbit viruses, surveys were conducted for three consecutive years (2016–2018) in different agricultural districts of Oklahoma. A total of 1331 leaf samples from >90 fields were randomly collected from both symptomatic and asymptomatic cucurbit plants across 11 counties. All samples were tested with the dot-immunobinding assay (DIBA) against the antisera of 10 known viruses. Samples infected with papaya ringspot virus (PRSV-W), watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), and cucurbit aphid-borne-yellows virus (CABYV) were also tested by RT-PCR. Of the 10 viruses, PRSV-W was the most widespread, with an overall prevalence of 59.1%, present in all 11 counties, followed by ZYMV (27.6%), in 10 counties, and WMV (20.7%), in seven counties, while the remaining viruses were present sporadically with low incidence. Approximately 42% of the infected samples were positive, with more than one virus indicating a high proportion of mixed infections. CABYV was detected for the first time in Oklahoma, and the phylogenetic analysis of the first complete genome sequence of a CABYV isolate (BL-4) from the US showed a close relationship with Asian isolates.

Prevalence and associated risk factors of Helicobacter pylori infection in the Wuwei cohort of north-western China.

To evaluate the prevalence of Helicobacter pylori infection and risk factors and to serotype the strains in Wuwei, located in north-western China, which has a high incidence of gastric cancer. Helicobacter pylori infection was analysed in 21291 adults by 14 C-urea breath test, and H.pylori antibody were detected in 9183 serum samples by latex immunoturbidimetric method. The correlation of H.pylori infection with demographic-economic, lifestyle factors and medical history among the participants was determined by questionnaire. The antibodies against H.pylori urease, VacA and CagA in serum were determined by dot immunobinding assay. The infection rate of H.pylori was 53.0%, and 90.1% of strains were type I strains. The H.pylori infection rate was higher among farmers (OR=1.34, 95% CI: 1.19-1.50) and individuals who had a junior high school or higher education level (OR=1.10, 95% CI: 1.06-1.15), and was lower in older individuals (OR=0.86, 95% CI: 0.83-0.90), individuals with high income (OR=0.93, 95% CI: 0.90-0.95), individuals with a habit of eating quickly (OR=0.93, 95% CI: 0.87-0.99) and individuals who consumed more fruit and vegetables (OR=0.90, 95% CI: 0.85-0.95). Individuals with history of cholecystitis/cholecystolithiasis, hypertension and asthma were negatively correlated with H.pylori infection (P<0.05). The prevalence of H.pylori infection is high in Wuwei. The major prevalent strain is type I strain. Age, education, occupation, household income, consumption of fruit and vegetables, and habit of eating quickly are independent risk factors for H.pylori infection, which is also associated with individuals with a history of extragastric diseases.

Detection of major viruses infecting shallot and molecular characterization of Onion yellow dwarf virus from several locations in Indonesia

Abstract. Harti H, Hidayat SH, Sobir, Wiyono S. 2020. Detection of major viruses infecting shallot and molecular characterization of Onion yellow dwarf virus from several locations in Indonesia. Biodiversitas 21: 1697-1701. Research was conducted to identify main viruses infecting shallot in several regions in Indonesia and to further characterize genetic variation of Onion yellow dwarf virus (OYDV). Field survey was conducted in Central Java (Brebes), East Java (Probolinggo), West Sumatera (Alahan Panjang), West Nusa Tenggara (Bima), and South Sulawesi (Enrekang). Virus detection from field samples was conducted by dot immunobinding assay. This detection confirmed that infection of OYDV, Shallot yellow stunt virus (SYSV), Shallot latent virus (SLV), and Garlic common latent virus (GarCLV) has occurred in all field with incidence ranged from 20 to 93.5%, 2 to 93%, 21.5 to 80%, and 2 to 80.5%, respectively. The specific primers of Nib gene successfully amplified DNA fragments of OYDV from all locations. Sequencing of DNA fragments revealed that the amplified product was 351 bp. Sequence analysis indicated that the present OYDV isolates from Indonesia shared homology from 82 to 95%; and they had homology from 81 to 95% with OYDV isolates from other countries. The similarity of OYDV isolates from different geographical locations reflected the movements of seed bulbs among and within countries. The phylogenetic tree also revealed that OYDV isolates from different countries did not group together indicating their diverse origin.

Sensitivity of serological and polymerase chain reaction methods for detection of viruses in Allium spp.

Infection of Onion yellow dwarf virus (OYDV), Shallot yellow stripe virus (SYSV), and Garlic common latent virus (GCLV) has been reported on Allium spp. in Indonesia. Serology methods using enzyme-linked immunosorbent assay (ELISA), and dot immunobinding assay (DIBA) is a common method to detect viruses, while polymerase chain reaction (PCR) is popular as a molecular method to detect plant viruses nowadays. This research was conducted to assess the sensitivity of 3 detection methods, i.e. ELISA, DIBA and PCR for detecting viruses in Allium spp. Sensitivity level of each method was evaluated by diluting plant extract and antibody for ELISA and DIBA, and cDNA template for PCR. Result of this research showed that DIBA was able to detect OYDV, GCLV, and SYSV with plant extract dilution in the range of 10−1 to 10−2, and antibody dilution from 1:300 to 1:1000. ELISA seems to be more sensitive than DIBA because it was able to detect the same virus with plant extract dilution in the range of 10−1, 10−3, and 10−5, and antibody dilution from 1:300 to 1:1000. RT-PCR method was the most sensitive method compared to ELISA and DIBA because it has the highest accuracy, specification and sensitivity level. Specific DNA fragment was amplified using universal primers for Potyvirus and Carlavirus up to 10−3 and 10−4 dilution factor of cDNA respectively. Moreover, amplification using specific primers of OYDV, SYSV, and GCLV is more sensitive than using universal primers, because up to 10−5 dilution factor of cDNA was able to amplify virus target.

Elimination of Garlic common latent virus from garlic through meristem culture and thermotherapy

Virus infection on garlic (Allium sativum) can be accumulated through vegetative propagation and may cause yield losses. It was evidenced in this study that infection of GCLV on garlic seed cv ‘Tawangmangu Baru’ reached 100% based on detection using DIBA (Dotimmuno binding assay). This research was initiated to obtain an elimination method of GCLV from garlic seed bulbs of cv ‘Tawangmangu Baru’. Tissue culture-based method was used in this research involving isolation of 1.0 mm meristem from micro shoots followed by thermotherapy at 25 °C, 28 °C, 31 °C, and heterogeneous temperatures for 4 wk. Virus indexing was conducted using RT-PCR on micro shoots that grown after treatment. The results showed that meristem culture and thermotherapy did not affect the growth of micro shoots based on assessment on the percentage of surviving explants, plant height, and the number of leaves. Infection of GCLV was still detected on micro shoots after thermotherapy, i.e. 64% to 91%. Virus infection on garlic cv ‘Tawangmangu Baru’ was not totally eliminated by meristem culture and thermotherapy. Factors affecting this result will be discussed in this paper as well as methodology improvement to produce virus-free garlic with high multiplication rate of explants.

Complete Genome Characterization and Coat Protein Genealogy of Isolates of Maize dwarf mosaic virus from Johnsongrass and Maize in Oklahoma and Missouri.

Maize dwarf mosaic virus (MDMV) significantly affects maize production worldwide, including the United States. This study describes the distribution and biological and molecular characterization of MDMV isolates from Johnsongrass and maize. A total of 262 samples (symptomatic = 214, asymptomatic = 48) were collected in Oklahoma and Missouri during 2016, 2017, and 2019 growing seasons. Based on a dot-immunobinding assay (DIBA), the average incidence of maize dwarf mosaic disease varied from ∼71% (79/111) in 2016, ∼76% (81/106) in 2017, and 62% (28/45) in 2019. Sixty-five DIBA-positive samples for MDMV were further confirmed by RT-PCR, and the complete coat protein (CP) gene was cloned and sequenced. Phylogenetic analysis of 132 isolates (This study = 65; GenBank = 67) revealed two main groups (G1 and G2) of MDMV isolates. All 65 MDMV isolates contained a 39-nucleotide insertion in the N-terminal region of CP genes and clustered in G1 which were different from the isolates in G2, without 39-nucleotide insertion. The first complete genome (9,563 nucleotides) of a MDMV isolate (Bixby1) from Johnsongrass was sequenced, which was distantly related to eight previously reported MDMV isolates from maize. The dN/dS ratio showed mostly purifying selection on each of cistrons except 6K1 being subjected to the diversifying selection. Further analyses revealed three putative recombination events between MDMV-Bixby1 and MDMV isolates from other countries. The successful mechanical and aphid transmission of MDMV-Bixby1 onto maize cultivars was achieved. Altogether, this information showed that Johnsongrass harbors genetically diverse MDMV isolates, which could pose a threat to cultivated crops such as maize and sorghum.

Immunocapture reverse transcription polymerase chain reaction for detection of sugarcane streak mosaic virus

Detection of plant viruses can be done by protein or nucleic acid approaches. The immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) method is a combination of the two approaches. Research was carried out to develop and validate IC-RT- PCR based-detection method for SCSMV, which can be applied for the sugarcane seed indexing program to support the national government’s goal for sugar self-sufficiency. Evaluation of the IC-RT-PCR method was conducted using 5 field samples. Conventional PCR and serological methods, i.e. dot immunobinding assay (DIBA) and enzyme-linked immunosorbent assay (ELISA) was also performed in the same time. All field samples gave a positive reaction to SCSMV antibodies in the DIBA and ELISA methods with the intensity of the reaction varying from low to high. SCSMV was still detected on plant extract up to 104 dilution by ELISA and DIBA. Specific DNA fragments were successfully amplified from 2 field samples using the conventional PCR method; whereas the IC-RT-PCR method was successfully amplified all field samples. Optimization test showed that the IC-RT-PCR method was able to detect SCSMV from plant extract up to 10−10 dilutions. IC-RT-PCR method is more sensitive than conventional PCR and might be recommended for the indexing method to produce high-quality virus-free sugarcane seed.

First report of cucurbit aphid-borne yellows virus on cucumber in Java, Indonesia

Chlorosis and yellowing with green-vein banding on older leaves were observed in surveys of cultivated cucumber fields in 2016 in Java, Indonesia. Initial dot immuno-binding assays detected seven different viruses varying in frequency among over 500 samples from 27 locations. Cucurbit aphid-borne yellows virus (CABYV) was detected at a frequency approaching 100% in four provinces in Java. The coat protein nucleotide and amino acid sequences of four isolates had highest identity with that of CABYV isolate Aileu in East Timor. This is the first report of CABYV infection of cucumber in Indonesia.