A reverse transcription-cross-priming amplification method with lateral flow dipstick assay for the rapid detection of Bean pod mottle virus

Bean pod mottle virus (BPMV) is a destructive virus that causes serious economic losses in many countries every year, highlighting the importance of its effective detection. In this study, we developed a fast reverse transcription-cross-priming amplification (RT-CPA) coupled with lateral flow dipstick (LFD) diagnostic method for BPMV detection. The RT-CPA-LFD assay that targets the coat protein gene of BPMV was highly specific against diagnosing four other common viruses transmitted by soybean seeds, i.e., Southern bean mosaic virus (SBMV), Tomato ringspot virus (ToRSV), Arabis mosaic virus (ArMV), and Tobacco ringspot virus (TRSV). The sensitivities of the real-time fluorescent RT-CPA and the RT-CPA-LFD assay were at least 50 pg/μl and 500 pg/μl, respectively. Despite a compromise in the limit of detection of the RT-CPA method compared with TaqMan-MGB real-time RT-PCR, our results demonstrated a notably better performance in the detection of field samples of BPMV-infested soybean seeds. With the advantages of efficiency and convenience by visual determination, the RT-CPA-LFD assay presents a potential application for the rapid and accurate detection of BPMV in routine tests.

Natural Infection of Cichorium intybus (Asteraceae) by Groundnut Ringspot Virus (Genus Orthotospovirus) Isolates in Brazil

Leaf chicory (Cichorium intybus L.) is a nutritionally rich vegetable used in regional cuisine in Brazil. Plants of C. intybus displaying symptoms (viz. chlorotic and necrotic ringspots, mosaic, and leaf deformation) similar to that induced by orthotospoviruses (genus Orthotospovirus, family Tospoviridae) were observed in three fields (≈ 0.2 ha each) in Gama County, in the Federal District, Brazil, from September 2016 to January 2020 in plants of the cultivars ‘Folha-Larga’ and ‘Spadona’ (Fig. 1). Incidence of symptomatic plants was nearly 10% in each field. Transmission electron microscopic examination of thin sections from symptomatic leaf samples showed typical membrane-bounded orthotospovirus particles within cisternae of spongy parenchymal cells (Fig 2). Two individual leaf samples per field were collected and submitted to dot enzyme-linked immunosorbent assay with polyclonal antisera against N protein of tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV). Symptomatic samples strongly reacted only against GRSV antibodies. Total RNA was extracted (Trizol®, Sigma) from all six samples and used as template in RT-PCR assays. The primer J13 (5’-CCCGGATCCAGAGCAAT-3’) was employed for cDNA synthesis using M-MLV reverse transcriptase. PCR assays were done with the primer pair BR60/BR65 (Eiras et al., 2001) to obtain ≈ 500 bp fragment of untranslated region and partial N gene in the S RNA segment from each sample. Purified RT-PCR products of two randomly selected individual samples were directly sequenced (GenBank MW467981 and MZ126602) and their BLASTn analyses displayed 99 to 100% nucleotide identity to GRSV isolates previously reported infecting C. endivia L. in Brazil (Jorge et al., 2021). Our analyses combining N protein serology and N-gene sequencing (both directed to the S RNA segment) allowed us to confirm the GRSV infection of C. intybus, but the potential reassortant nature of these isolates (Webster et al., 2015; Silva et al., 2019) are unknown since their M RNA segments were not characterized. Individual leaf extracts (in phosphate buffer, pH 7.0) of the sequenced isolates were mechanically inoculated onto ten seedlings of two C. intybus cultivars (‘Folha Larga’ and ‘Pão-de-Açúcar’) and three plants each of the indicator hosts Capsicum chinense PI 159236, Nicandra physalodes; Nicotiana rustica; Datura stramonium; and tomato cv. Santa Clara. Systemic chlorotic and necrotic ringspots, mosaic, and leaf deformation developed in the indicator hosts and infection by GRSV was confirmed via serological assays 20 days after inoculation. However, no symptoms and no serological reaction to GRSV antibodies were observed on the C. intybus cultivars even after two successive mechanical inoculations. This transmission failure might be due to factors such as the requirement of the thrips vector(s), physicochemical barriers in the foliage or the presence of non-mechanically transmissible helper agent(s) necessary to ensure GRSV infection of C. intybus. The natural infection of C. intybus by a not fully characterized orthotospovirus (mostly likely TSWV) has been observed since 1938 in Brazil (Kitajima, 2020). Our report of GRSV infecting C. intybus is thus confirming previous speculations that similar symptoms in this vegetable crop were induced by orthotospovirus infection in Brazil. References: Eiras, M. et al. 2001. Fitopatol. Bras. 26: 170. Jorge, T. S. et al. 2021. Plant Dis. 105: 714. Kitajima, E.W. 2020. Biota Neotrop. 20: e2019932. Silva, J. M. F. et al. 2019. Viruses 11: 187. Webster, C.G. et al. 2015. Phytopathology 105: 388.

Features of the strawberry field repository formation

For the successful functioning of a breeding and nursery center of scientific and practical work with fruit and small fruit crops, an important task is to create repositories, including thosein the field. A field repository is a plant gene bank based in accordance with international standards on planting material that is free from dangerous pathogens, including viruses, representing tested for productivity typical plants.For the purpose of a comparative study of promising varieties, hybrids and clones-candidates for original plants, a field repository and mother plantation of strawberries clones and varieties have been created on the territory of the Federal Horticultural Research Center for Breeding, Agrotechnology and Nursery.As a result of research in 2015-2020, 386 high-yielding strawberry plants were selected and tested for the main harmful viruses using diagnostic kits from “Loewe” firm (Germany). The prevalence of harmful Arabis mosaic virus (ArMV), Raspberry ringspot virus (RpRSV), Tomato black ring virus (TBRV), Strawberry latent ringspot virus (SLRSV), Cucumber mosaic virus (CMV) in strawberry plantations depended on the area cultivation, varietal composition of plantings and ranged from 31 to 69 %. The prevalence of viruses RpRSV (up to 36 %), TBRV (up to 31 %) and CMV (up to 22 %) was established. The high efficiency of dry-air thermotherapy for the recovery of strawberries with the number of virus-free intact plants of 56 % has been shown.A genebank of "candidates for original plants" has been formed from 234 strawberry plants of 39 varieties and hybrids, which, after confirming their status by PCR, will be transferred to the category of "original plants".

Genetic variability and evolutionary dynamics of atypical Papaya ringspot virus infecting Papaya

Papaya ringspot virus biotype-P is a detrimental pathogen of economically important papaya and cucurbits worldwide. The mutation prone feature of this virus perhaps accounts for its geographical dissemination. In this study, investigations of the atypical PRSV-P strain was conducted based on phylogenetic, recombination and genetic differentiation analyses considering of it’s likely spread across India and Bangladesh. Full length genomic sequences of 38 PRSV isolates and 35 CP gene sequences were subjected to recombination analysis. A total of 61 recombination events were detected in aligned complete PRSV genome sequences. 3 events were detected in complete genome of PRSV strain PK whereas one was in its CP gene sequence. The PRSV-PK appeared to be recombinant of a major parent from Bangladesh. However, the genetic differentiation based on full length genomic sequences revealed less frequent gene flow between virus PRSV-PK and the population from America, India, Colombia, other Asian Countries and Australia. Whereas, frequent gene flow exists between Pakistan and Bangladesh virus populations. These results provided evidence correlating geographical position and genetic distances. We speculate that the genetic variations and evolutionary dynamics of this virus may challenge the resistance developed in papaya against PRSV and give rise to virus lineage because of its atypical emergence where geographic spread is already occurring.

Effects of Actinidia yellowing ringspot virus on the yield and quality of kiwifruit

China is the origin and distribution center of kiwifruit, as well as the country with the largest cultivated area and output of kiwifruit. A previous study found that a new kiwifruit virus, Actinidia yellowing ringspot virus (AYRSpV), has been detected in kiwifruit samples with yellowed leaves. The incidence of this virus was high in kiwifruit plantings in Shaanxi Province. To determine the symptoms of this viral infection and the effects of this virus on the yield and quality of kiwifruits, we measured leaf chlorophyll levels and the fruit yield, total sugar, total acid and dry matter contents of ‘Hayward’ kiwifruits grafted with AYRSpV-infected scions. The results showed that after AYRSpV infection, symptoms including chlorotic ringspots were mainly observed in the spring and gradually recovered with high summer temperatures. A few of the leaves that did not recover showed symptoms of albinism, which lasted until the leaves fell. We found that AYRSpV infection could reduce the chlorophyll content of ‘Hayward’ kiwifruit by 74.61-76.64%, the fruit yield by 14.50-24.10%, the sugar to acid ratio by 50.09-50.57%, and the fruit dry matter content by 1.67-1.78%. Our results showed that AYRSpV infection could significantly affect the yield and quality of ‘Hayward’ kiwifruit.