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Groundnut ringspot virus (GRSV) is a tospovirus species transmitted in a circulative propagative manner by several thrips species. Traditionally, GRSV is detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR), so this paper describes a rapid diagnostic system for the reliable detection of GRSV in plants and individual thrips by real-time RT-PCR. Watermelon GRSV infected leaves and fruits, and individual thrips (Frankliniella schultzei) collected from flowers on infected watermelons were used in the test. We designed a GRSV TaqMan assay to efficiently detect GRSV in all samples. The method had high specificity and could distinguish GRSV from the other tospovirus species, like Tomato spotted wilt virus (TSWV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV). The test will help us to collect epidemiological data for GRSV in plants and thrips worldwide. [ABSTRACT FROM AUTHOR]

Fusarium langsethiae is an European species, while Fusarium sibiricum is mainly distributed in northern Asia. Morphological characters alone do not allow a clear separation of F. langsethiae from F. sibiricum. The long TG repeat in the ribosomal IGS region is the only DNA sequence that has been used to design the species-specific primer pair CNL 12/PulvIGSr for the identification of F. sibiricum isolates. Another way to identify F. sibiricum is to use a combination of F. sporotrichioides- and F. langsethiae-specific primer pairs. The aim of this study was to find new ways for identifying and discriminating F. langsethiae from F. sibiricum. The paper also reports the first record of F. sibiricum from Iran, outside northern Asia and Norway, and the first isolation of F. langsethiae, an European pathogen, from western Siberia. Thus, the distribution of F. sibiricum and F. langsethiae may be much larger in Eurasia than what is currently known. [ABSTRACT FROM AUTHOR]

Virus-like symptoms on fig tree leaves are a common occurrence worldwide and has mostly been attributed to fig mosaic disease (FMD). Even though only fig mosaic virus (FMV) has been shown to cause FMD, many other viruses have been identified in diseased fig trees. In 2021, total RNA was extracted from fig leaf samples displaying symptoms of mosaic and chlorotic mottling and was subjected to high-throughput sequencing (HTS) to construct the first virome profile of a South African fig tree. Bioinformatic analyses identified FMV, fig leaf mottle-associated virus 1 (FLMaV1), fig leaf mottle-associated virus 2 (FLMaV2), fig latent virus 1 (FLV1), fig badnavirus 1 (FBV1) and grapevine badnavirus 1 (GBV1) in the data. Reverse transcription polymerase chain reaction (RT-PCR) was conducted, for these viruses, on 24 additional fig leaf samples collected in the Western Cape. FBV1, GBV1, FMV, FLMaV1, FLV1, FLMaV2, and fig fleck-associated virus (FFkaV) were detected in 100%, 96%, 92%, 54%, 46%, 21%, and 12.5% of the samples, respectively. This is the first report on the presence of FMV, FLMaV2, FLV1, FFkaV, FBV1 and GBV1 in South Africa and offers a preliminary insight into the virus status of fig trees in the country. [ABSTRACT FROM AUTHOR]

Potato viruses including Potato aucuba mosaic virus (PAMV), Potato leafroll virus (PLRV), Potato virus M (PVM), Potato virus S (PVS) and Potato virus X (PVX) are well known viruses infecting potato and can cause serious economic losses. In this study, a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed and standardized for simultaneous detection of PAMV, PLRV, PVM, PVS and PVX with elongation factor 1-α (ef1α) as plant internal control. At least three primer pairs were designed within the conserved regions of genomic sequences of five potato viruses and ef1α of potato for single and/or multiplex RT-PCR. The reaction conditions were initially optimized by selecting primer pairs and standardized the individual RT-PCR. Then, one pair of primer for each virus including ef1α was selected for multiplex RT-PCR producing distinct fragments of 305, 452, 408, 363, 565, 803 bp, representing PAMV, PLRV, PVM, PVS, PVX and ef1α, respectively. All the primers were designed based on specificity and compatibility and the reliability was confirmed by cloning and sequencing followed by BLAST analysis. The sensitivity of single as well as multiplex RT-PCR was evaluated. The assay could detect all five viruses including ef1α even at total RNA dilutions of 10-3 and total cDNA dilution of 10-7. The multiplex RT-PCR assay was then used to test virus infections from field samples of potato (leaves and tubers) collected from different states of India along with plant internal control. This multiplex RT-PCR was equally enough in detecting mixed infection of five viruses in foliage as well as tubers. The results indicate the robustness and reliability of the assay for virus indexing of mother stocks for five viruses simultaneously in potato seed production and will be useful for seed certification programme, breeding and epidemiological studies. [ABSTRACT FROM AUTHOR]

The genetic diversity of potyviruses infecting potato and vegetables in Iraq was determined by reverse transcription polymerase chain reaction (RT-PCR) targeting the 3´ terminal part of the genome. RT-PCR products were cloned and sequenced. Sequence analysis confirmed the occurrence of potyviruses, namely Bean yellow mosaic virus (BYMV) in broad bean, Potato virus Y (PVY) in potato and tomato, and Zucchini yellow mosaic virus (ZYMV) in zucchini squash. BYMV sequences showed maximum nucleotide sequence identity (97.2-98.7%) with isolates from Australia and Japan. Most PVY sequences were very closely related (ca. 99% nucleotide sequence identity) to PVYO: N isolates from USA and Canada, whereas other sequences showed nucleotide identities (98.5-98.7%) with PVYNTN isolates from Poland, UK and Syria. ZYMV sequences showed maximum nucleotide identity (97.9%) with an isolate from Israel. The low genetic diversity amongst Iraqi potyviruses, alongside their high sequence identities with potyviruses from other countries, suggests a relatively recent introduction [ABSTRACT FROM AUTHOR]

Capsicum chlorosis virus (CaCV), which belongs to the Watermelon silver mottle virus (WSMoV) serogroup of the genus Tospovirus, can seriously harm economically important crops. A CaCV strain from Phalaenopsis orchids (CaCV-pha) was identified by RT-PCR. The amino acid sequence of the CaCV-pha nucleocapsid protein (NP) shared 92.0% to 97.1% identity with comparable proteins of other CaCV isolates, with which it was phylogentically related. Since the identification of CaCV-infected crops requires the availability of sensitive diagnostic tools, a prokaryotic expression system was used to obtain a recombinant CaCV-pha NP for antiserum production. The CaCV-pha NP gene was subcloned into the pET-28a (+) vector and transformed into Escherichia coli Rosetta (DE3) cells for expression. After purification, the recombinant protein was injected into rabbits to generate a polyclonal antiserum which, in Western blot tests reacted strongly with the recombinant protein used as antigen. The availability of an anti-CaCV-NP serum will facilitates field surveys and further research on CaCV. [ABSTRACT FROM AUTHOR]

Potato spindle tuber viroid (PSTVd) was the first species of the genus Pospiviroid detected in naturally infected symptomless ornamental plants (Solanum jasminoides) in 2006. Since then, other pospiviroid species were found in several naturally infected solanaceous ornamentals. The combination of latent infection, the widespread occurrence and the possibility of transmission to edible solanaceous crops (tomato and potato) has made the development of accurate and reliable molecular diagnostic detection methods a high priority. The aim of this study was the development of a genus-specific RT-PCR protocol, using universal pospiviroid primers, which would allow also a species-specific identification through restriction fragment length polymorphism (RFLP) analysis. The primers were designed to amplify the majority of the pospiviroid genome (ca. 80%) and the identification of each species using appropriate restriction endonuclease enzymes (AluI and Sau96I). The method was evaluated for specificity using healthy and/or non-target viroid-infected plants and for sensitivity using serial dilutions of total RNA extracted from plants infected by different pospiviroid species. The reliability of the method was successfully ascertained during a survey when numerous plants of the families Solanaceae and Asteraceae were assayed for single and mixed infection of pospiviroid species. [ABSTRACT FROM AUTHOR]

Virus infections are a major constraint to cultivation of black pepper (Piper nigrum L.), a perennial vegetatively propagated spice crop. Real-time PCR and real-time RTP-CR were developed to detect Piper yellow mottle virus (PYMoV) and Cucumber mosaic virus (CMV), respectively, in black pepper plants. These detection assays were up to 1000 times more sensitive than the conventional PCR/RT-PCR assays. Field samples from different pepper-growing regions in India were used to validate the two detection assays, which identified more infected plants than conventional PCR. Our results indicate that real-time PCR should be the method of choice for screening mother plants for PYMoV and CMV. [ABSTRACT FROM AUTHOR]

Citrus yellow vein clearing virus (CYVCV), the putative causal agent of yellow vein clearing disease of citrus, is transmissible by grafting and is spread by aphids. However, whether CYVCV is seed-transmitted is not well documented. In this study, seed transmission of CYVCV was investigated in Daidai, Carrizo citrange and Rough lemon. CYVCV was detected in the endotesta, cotyledons and embryonic axis of seeds collected from the fruits of CYVCV-infected plants by qRT-PCR. Furthermore, the relative expression values of CYVCV were significantly higher in infected leaves than in the cotyledons or embryonic axis of seeds. Although viral RNA was found in few samples of radicles of germinating seed, none of the seedlings showed any symptoms for one year and were RT-PCR negative for CYVCV. The present study indicated that CYVCV could be localized in the seed tissues, but was not transmitted to the progeny. Absence of seed transmission may be consequent to the low CYVCV titre in embryo tissue. [ABSTRACT FROM AUTHOR]