Evaluation of Seed Transmission Rates of Wheat Streak Mosaic Virus in Mechanically Inoculated Winter and Spring Wheat Cultivars in Montana.

Wheat streak mosaic disease is caused by wheat streak mosaic virus (WSMV) and two other viruses and persistently limits wheat yields in the Great Plains region of the United States. Seed transmission of viruses is an important consideration in international movement and is important epidemiologically. Seed transmission of WSMV in wheat was first reported from Australia in 2005, but there is little data from United States cultivars on the rate of seed transmission. In 2018, mechanically inoculated winter and spring wheat cultivars were evaluated in Montana. We found differences in WSMV seed transmission rates between winter and spring wheat, with average transmission rates in spring wheat (3.1%) being five times higher compared to winter wheat (0.6%). Seed transmission rates in spring wheat were twice as high as the highest previously reported transmission rate for individual genotypes, 1.5%. The results from this study provide a strong argument for increasing the current testing of seed for breeding purposes prior to international movement when WSMV has been observed and caution against using grain from WSMV-infected fields as seed source because it can heighten the risk of wheat streak mosaic outbreaks.

Comparison of Models for Quantification of Tomato Brown Rugose Fruit Virus Based on a Bioassay Using a Local Lesion Host.

Considering the availability of serological and molecular biological methods, the bioassay has been paled into insignificance, although it is the only experimental method that can be used to demonstrate the infectivity of a virus. We compared goodness-of-fit and predictability power of five models for the quantification of tomato brown rugose fruit virus (ToBRFV) based on local lesion assays: the Kleczkowski model, Furumoto and Mickey models I and II, the Gokhale and Bald model (growth curve model), and the modified Poisson model. For this purpose, mechanical inoculations onto Nicotiana tabacum L. cv. Xanthi nc and N. glutionosa L. with defined virus concentrations were first performed with half-leaf randomization in a Latin square design. Subsequently, models were implemented using Python software and fitted to the number of local lesions. All models could fit to the data for quantifying ToBRFV based on local lesions, among which the modified Poisson model had the best prediction of virus concentration in spike samples based on local lesions, although data of individual indicator plants showed variations. More accurate modeling was obtained from the test plant N. glutinosa than from N. tabacum cv. Xanthi nc. The position of the half-leaves on the test plants had no significant effect on the number of local lesions.

Mixed infection of an emaravirus, a crinivirus, and a begomovirus in Pueraria lobata (Willd) Ohwi.

Pueraria lobata (Willd) (Pueraria montana var. lobata (Willd.) Maesen & S. M. Almeida ex Sanjappa & Predeep) is an important herbal medicine used in many countries. In P. lobata plants showing symptoms of mosaic, yellow spots, and mottling, mixed infection of new viruses provisionally named Pueraria lobata-associated emaravirus (PloAEV, genus Emaravirus), Pueraria lobata-associated crinivirus (PloACV, genus Crinivirus), and isolate CQ of the previously reported kudzu mosaic virus (KuMV-CQ, genus Begomovirus) was confirmed through high-throughput sequencing. PloAEV has five RNA segments, encoding a putative RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid protein, movement protein, and P5, respectively. PloACV has two RNA segments, encoding 11 putative proteins. Only PloAEV could be mechanically transmitted from mixed infected symptomatic kudzu to Nicotiana benthamiana plants. All three viruses were detected in 35 symptomatic samples collected from five different growing areas, whereas no viruses were detected in 21 non-symptomatic plants, suggesting a high association between these three viruses. Thus, this study provides new knowledge on the diversity and molecular characteristics of viruses in P. lobata plants affected by the viral disease.

Biological and Genetic Characterization of Physostegia Chlorotic Mottle Virus in Europe Based on Host Range, Location, and Time.

Application of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Grafting to Manage Infections of the Emerging Tomato Leaf Curl New Delhi Virus in Cucurbits.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging begomovirus (Geminiviridae family) listed in the EPPO Alert List 2, present in the Mediterranean area and in Italy, where it was reported in 2015 in Sicilian courgette. The virus is widespread in cucurbits where it causes up to 100% production losses. In 2018, ToLCNDV was isolated in Apulia (southern Italy) in commercial fields of zucchini squash and since then its recurrent outbreaks generated justified concern among growers. Thus, a sustainable and environmentally friendly approach must be adopted. Genetic resistances have been identified in Cucurbita moschata and Luffa cylindrica but, compared to genetic resistance, grafting could provide a faster and more flexible solution because the graft wounding induces tolerance rather than resistance against airborne virus infection. Compared to tolerance, the up-regulation of resistance genes requires energy resources mobilized at the expense of primary metabolism, plant growth, and development. Results of screening among twenty-one local cucurbit cvs. ecotypes and accessions to evaluate tolerance levels against rub-inoculation of ToLCNDV led to the identification of potential rootstocks to attain suitable levels of tolerance against the virus in commercial cucurbit varieties. Cucurbit plants were challenged by a ToLCNDV isolated in Apulia denoted ToLCNDV-Le and evaluated for disease symptoms development and viral DNA accumulation up to 28 days after inoculation. On the basis of disease symptoms developed, plants were classified as tolerant, moderately tolerant, moderately susceptible, and susceptible. Cucumis melo cv. Barattiere did not show any detectable disease symptoms and very low levels of viral DNA accumulation was recorded; thus, it was used as rootstock for some of the remaining cucurbit genotypes that were used as scions. The tolerance trait was transmitted to the otherwise susceptible and moderately susceptible cucurbit genotypes grafted onto the cv. Barattiere. The results of this study suggest practical implications of the approach described.

Detection and Characterization of Viroids via Biological Assays on Herbaceous Hosts.

The characterization of the elusive disease agent of the potato spindle tuber disease, potato spindle tuber viroid (PSTVd), was aided by the ability to obtain large amounts of infected tomato tissue in a simple bioassay where PSTVd was easily mechanically transmissible to an alternate herbaceous host in which it thrived and produced dramatic symptoms in a relatively short period (Diener, Viroids. Handbook of plant virus infections: comparative diagnosis. Elsevier/North-Holland, Amsterdam, pp 913-934, 1981; Diener, Virology 45:411-428, 1971; Raymer and O'Brien, Am Pot J, 39:401-408, 1962). Reactions in the primary, or secondary, herbaceous indicator host can range from asymptomatic to severe depending upon the viroid strain, host species, and environmental conditions and can provide evidence of a viroid infection, but do not permit identification of the viroid in question. Further characterization by molecular hybridization, RT-PCR, and sequence analysis is used to determine the etiology of the disease agent. In this chapter, methods are described for mechanical inoculation of viroids to herbaceous hosts to determine the viroid nature of diseases and the experimental host range of the viroid or to shorten the time required for obtaining relatively large amounts of viroid for subsequent purification and characterization.

Resistant Sources and Genetic Control of Resistance to ToLCNDV in Cucumber.

Tomato leaf curl New Delhi virus (ToLCNDV) is a severe threat for cucurbit production worldwide. Resistance has been reported in several crops, but at present, there are no described accessions with resistance to ToLCNDV in cucumber (Cucumis sativus). C. sativus var. sativus accessions were mechanically inoculated with ToLCNDV and screened for resistance, by scoring symptom severity, tissue printing, and PCR (conventional and quantitative). Severe symptoms and high load of viral DNA were found in plants of a nuclear collection of Spanish landraces and in accessions of C. sativus from different geographical origins. Three Indian accessions (CGN23089, CGN23423, and CGN23633) were highly resistant to the mechanical inoculation, as well as all plants of their progenies obtained by selfing. To study the inheritance of the resistance to ToLCNDV, plants of the CGN23089 accession were crossed with the susceptible accession BGV011742, and F1 hybrids were used to construct segregating populations (F2 and backcrosses), which were mechanically inoculated and evaluated for symptom development and viral load by qPCR. The analysis of the genetic control fit with a recessive monogenic inheritance model, and after genotyping with SNPs distributed along the C. sativus genome, a QTL associated with ToLCNDV resistance was identified in chromosome 2 of cucumber.

Delineating the elusive BaMMV resistance gene rym15 in barley by medium-resolution mapping.

Barley mild mosaic virus (BaMMV), transmitted by the soil-borne protist Polymyxa graminis, has a serious impact on winter barley production. Previously, the BaMMV resistance gene rym15 was mapped on chromosome 6HS, but the order of flanking markers was non-collinear between different maps. To resolve the position of the flanking markers and to enable map-based cloning of rym15, two medium-resolution mapping populations Igri (susceptible) × Chikurin Ibaraki 1 (resistant) (I × C) and Chikurin Ibaraki 1 × Uschi (susceptible) (C × U), consisting of 342 and 180 F2 plants, respectively, were developed. Efficiency of the mechanical inoculation of susceptible standards varied from 87.5 to 100% and in F2 populations from 90.56 to 93.23%. Phenotyping of F2 plants and corresponding F3 families revealed segregation ratios of 250 s:92r (I × C, χ2 = 0.659) and 140 s:40r (C × U, χ2 = 0.741), suggesting the presence of a single recessive resistance gene. After screening the parents with the 50 K Infinium chip and anchoring corresponding SNPs to the barley reference genome, 8 KASP assays were developed and used to remap the gene. Newly constructed maps revealed a collinear order of markers, thereby allowing the identification of high throughput flanking markers. This study demonstrates how construction of medium-resolution mapping populations in combination with robust phenotyping can efficiently resolve conflicting marker ordering and reduce the size of the target interval. In the reference genome era and genome-wide genotyping era, medium-resolution mapping will help accelerate candidate gene identification for traits where phenotyping is difficult. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01270-9.

Development of a Virus-Induced Gene Silencing System for Dioecious Coccinia grandis.

Coccinia grandis is an interesting model system to understand dioecy in Cucurbitaceae family. Recent transcriptomics and proteomics studies carried out to understand the sex expression in C. grandis have resulted in identification of many candidate sex-biased genes. In absence of an efficient genetic transformation protocol for C. grandis, virus-induced gene silencing (VIGS) would be a powerful tool to enable gene functional analysis. In current study, we explored the apple latent spherical virus (ALSV) for gene knockdown in C. grandis. The viral infection was achieved through mechanical inoculation of ALSV-infected Chenopodium quinoa leaf extract onto the cotyledons of C. grandis. ALSV-VIGS mediated knockdown of CgPDS gene was successfully achieved in C. grandis by mechanical inoculation method resulting in characteristic photobleaching. Subsequently, we developed agroinfiltration compatible vectors for direct infection of C. grandis and shortened the time-frame by skipping viral propagation in C. quinoa. Typical yellow-leaf phenotype was observed in C. grandis plants agroinfiltrated with ALSV-CgSU constructs, indicating robust silencing of CgSU gene. In addition, we improved the infection efficiency of ALSV by co-infiltration of P19 viral silencing suppressor. These results suggest that ALSV-VIGS is suitable for characterization of gene function in dioecious C. grandis and it can help us understand the mechanism of sex expression.

Molecular and Biological Characterization of a New Strawberry Cytorhabdovirus.

Virus diseases of strawberry present several complex problems. More than 25 viruses have been described in the genus Fragaria thus far. Here, we describe a novel rhabdovirus, tentatively named strawberry virus 1 (StrV-1), that infects F.ananassa and F.vesca plants. Genomic sequences of three distinct StrV-1 genotypes co-infecting a single F.ananassa host were obtained using combined Illumina and Ion Proton high-throughput sequencing. StrV-1 was transmitted to herbaceous plants via Aphisfabae and A.ruborum, further mechanically transmitted to Nicotianaoccidentalis 37B and sub-inoculated to N.benthamiana, N.benthamiana DCL2/4i, N.occidentalis 37B, and Physalisfloridana plants. Irregular chlorotic sectors on leaf blades and the multiplication of calyx leaves seem to be the diagnostic symptoms for StrV-1 on indexed F.vesca clones. StrV-1 was detected in asymptomatic grafted plants and in 49 out of 159 field strawberry samples via RT-PCR followed by Sanger sequencing. The bacilliform shape of the virions, which have a cytoplasm-limited distribution, their size, and phylogenetic relationships support the assignment of StrV-1 to a distinct species of the genus Cytorhabdovirus. Acyrthosiphonmalvae, A.fabae, and A.ruborum were shown to transmit StrV-1 under experimental conditions.

Phenotyping Biological Properties of CTV Isolates.

The protocol described is intended to be used alongside molecular methods in order to reveal the relationship between the genome sequence and the biological properties of a single isolate of Citrus tristeza virus complex (CTV). It enables the phenotypic profile of the isolates to be defined and to infer the associated tristeza diseases (decline, seedling yellows, or stem pitting), to assess their aggressiveness or potential cross protectiveness (if any), and to monitor their movement into the host plants and the transmissibility by aphids.

First report of Cassava brown streak viruses on wild plant species in Mozambique.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the main constraint to cassava (Manihot esculenta Crantz) production in Mozambique. Using RT-PCR to amplify partial coat protein nucleotide sequences, we detected for the first time the occurrence of CBSV in two non-cassava perennial wild plant species: Zanha africana (Radlk.) Exell. and Trichodesma zeylanicum (Burm.f.) R.Br., that occur widely within and near cassava fields in Nampula, Zambezia, Niassa and Cabo Delgado provinces. In addition, we also detected CBSV and UCBSV in Manihot carthaginensis subsp. glaziovii (Müell-Arg.) Allem., a wild cassava relative. These findings were verified in biological assays through mechanical inoculation of CBSV to T. zeylanicum, albeit at low rates of infection. Phylogenetic analysis clustered the CBSV isolates from the non-cassava plant species with those from cultivated cassava, with high sequence homology among CBSV (91.0-99.6%) and with UCBSV (84-92%) isolates. These results provide definitive evidence of a wider host range for CBSV and UCBSV in Mozambique, indicating that these viruses are not restricted to cultivated cassava. Our findings are key to understanding the epidemiology of CBSD and will aid in the development of sustainable management strategies for the disease.

Isolation of Rice Stripe Virus Preparation from Viruliferous Small Brown Planthoppers and Mechanic Inoculation on Rice.

Tenuiviruses can infect the plants of the family Poaceae, and cause serious loss of crops, particularly rice and maize, in South-Eastern Asian countries. Tenuiviruses usually depend on insect vectors for their transmission and cannot be transmitted between plants through wounds or abrasions. Rice stripe virus (RSV), a typical member of tenuiviruses, is efficiently transmitted by the small brown planthopper Laodelphax striatellus in a persistent-propagative manner to cause rice stripe disease. Here we presented a convenient method, the midrib micro-injection, to mechanically inoculate insect-derived RSV into rice leaves for conducting pathogenicity assay on rice plants.

Different pathogenicities of Rice stripe virus from the insect vector and from viruliferous plants.

Persistent plant viruses usually depend on insects for their transmission; they cannot be transmitted between plants or through mechanical inoculation. However, the mechanism by which persistent viruses become pathogenic in insect vectors remains unknown. In this study, we used Rice stripe virus (RSV), its insect vector Laodelphax striatellus and host plant (Oryza sativa) to explore how persistent viruses acquire pathogenicity from insect vectors. RSV acquired phytopathogenicity in both the alimentary tract and the salivary gland of L. striatellus. We mechanically inoculated RSV into rice O. sativa leaves through midrib microinjection. Insect-derived RSV induced a typical stripe symptom, whereas plant-derived RSV only produced chlorosis in rice leaves. Insect-derived RSV had higher expression of genes rdrp, ns2, nsvc2, sp and nsvc4 than plant-derived RSV, and the latter had higher expression of genes cp and ns3 than the former in rice leaves. Different from plant-derived RSV, insect-derived RSV damaged grana stacks within the chloroplast and inhibited photosynthesis by suppressing the photosystem II subunit psbp. This study not only presented a convenient method to mechanically inoculate RSV into plants, but also provided insights into the different pathogenic mechanisms of RSV from the insect vector and from viruliferous plants.

Distribution of Xylella fastidiosa in Sycamore Associated with Low Temperature and Host Resistance.

Experiments were conducted in the field and laboratory to determine effects of low temperatures on Xylella fastidiosa populations in American sycamore. Roots and shoots from naturally infected trees at two locations were collected monthly. Sap extracted from the samples was tested by enzyme-linked immunosorbent assay for presence of X. fastidosa and was diluted and plated on periwinkle wilt medium to determine populations of viable bacteria. Cumulative rainfall and hours below temperature thresholds (-5 to 10°C) were recorded at each site. Bacterial populations in shoots were negatively correlated with cumulative hours below -5°C air temperature (r= -0.96). In roots, bacterial populations were only weakly correlated with cumulative hours below soil temperature thresholds (-0.61 < r <-0.25). Bacterial populations were not correlated with monthly rainfall. In the laboratory, resistant and susceptible sycamore trees were inoculated with X. fastidiosa and held in the dark at 5°C or 22°C. After 12 weeks, inoculated stem sections were collected and sap was extracted and tested as described previously. Stems that tested positive for X. fastidiosa were divided into additional samples and tested as described above. Results of the laboratory study indicated no significant effects of low-temperature treatment (5°C) or host resistance on viable bacteria. Bacterial detection frequency and population size were greatest near the inoculation point and the primary direction of early bacterial spread was acropetal.