Investigating the interactions of endornaviruses with each other and with other viruses in common bean, Phaseolus vulgaris.

BACKGROUND: Plant viruses of the genus Alphaendornavirus are transmitted solely via seed and pollen and generally cause no apparent disease. It has been conjectured that certain plant endornaviruses may confer advantages on their hosts through improved performance (e.g., seed yield) or resilience to abiotic or biotic insult. We recently characterised nine common bean (Phaseolus vulgaris L.) varieties that harboured either Phaseolus vulgaris endornavirus (PvEV1) alone, or PvEV1 in combination with PvEV2 or PvEV1 in combination with PvEV2 and PvEV3. Here, we investigated the interactions of these endornaviruses with each other, and with three infectious pathogenic viruses: cucumber mosaic virus (CMV), bean common mosaic virus (BCMV), and bean common mosaic necrosis virus (BCMNV). RESULTS: In lines harbouring PvEV1, PvEV1 and PvEV2, or PvEV1, PvEV2 plus PvEV3, the levels of PvEV1 and PvEV3 RNA were very similar between lines, although there were variations in PvEV2 RNA accumulation. In plants inoculated with infectious viruses, CMV, BCMV and BCMNV levels varied between lines, but this was most likely due to host genotype differences rather than to the presence or absence of endornaviruses. We tested the effects of endornaviruses on seed production and seedborne transmission of infectious pathogenic viruses but found no consistent relationship between the presence of endornaviruses and seed yield or protection from seedborne transmission of infectious pathogenic viruses. CONCLUSIONS: It was concluded that endornaviruses do not interfere with each other's accumulation. There appears to be no direct synergy or competition between infectious pathogenic viruses and endornaviruses, however, the effects of host genotype may obscure interactions between endornaviruses and infectious viruses. There is no consistent effect of endornaviruses on seed yield or susceptibility to seedborne transmission of other viruses.

Identification and characterization of Phaseolus vulgaris endornavirus 1, 2 and 3 in common bean cultivars of East Africa.

Persistent viruses include members of the family Endornavirus that cause no apparent disease and are transmitted exclusively via seed or pollen. It is speculated that these RNA viruses may be mutualists that enhance plant resilience to biotic and abiotic stresses. Using reverse transcription coupled polymerase chain reactions, we investigated if common bean (Phaseolus vulgaris L.) varieties popular in east Africa were hosts for Phaseolus vulgaris endornavirus (PvEV) 1, 2 or 3. Out of 26 bean varieties examined, four were infected with PvEV1, three were infected with both PvEV1 and PvEV2 and three had infections of all three (PvEV) 1, 2 and 3. Notably, this was the first identification of PvEV3 in common bean from Africa. Using high-throughput sequencing of two east African bean varieties (KK022 and KK072), we confirmed the presence of these viruses and generated their genomes. Intra- and inter-species sequence comparisons of these genomes with comparator sequences from GenBank revealed clear species demarcation. In addition, phylogenetic analyses based on sequences generated from the helicase domains showed that geographical distribution does not correlate to genetic relatedness or the occurrence of endornaviruses. These findings are an important first step towards future investigations to determine if these viruses engender positive effects in common bean, a vital crop in east Africa.

Detection and Identification of a 'Candidatus Liberibacter solanacearum' Species from Ash Tree Infesting Psyllids.

'Candidatus Liberibacter' species are associated with severe, economically important diseases. Nearly all known species are putatively insect transmitted, specifically by psyllids. Detection of 'Ca. Liberibacter' in plants is complicated by their uneven distribution in host plants and largely fastidius nature. The death of black (Fraxinus nigra) and mancana (Fraxinus mandshurica) ash trees in Saskatchewan, Canada has been associated with infestation by the cottony ash psyllid (Psyllopsis discrepans). A combination of conventional PCR amplification and Sanger sequencing of the 16S recombinant DNA was used to detect and identify 'Ca. Liberibacter' in psyllids collected from ash trees in Saskatchewan. BLAST analysis of two 16S sequences that were 1,058 and 1,085 bp long (NTHA 5, GenBank accession number MK942379 and NTHA 6, GenBank accession number MK937570, respectively) revealed they were 99 to 100% similar to a 'Ca. Liberibacter solanacearum' sequence (GenBank accession number KX197200) isolated from the Nearctic psyllid (Bactericera maculipennis) of U.S. provenance. Sequencing the psyllid genes CO1 and Cyt-b confirmed that the psyllids from which the bacterial DNA was isolated were P. discrepans, based on comparisons with sequences in GenBank and BOLD and a reference sample from the United Kingdom. These results provide the first evidence that 'Ca. Liberibacter solanacearum' species are associated with psyllids collected from ash trees and specifically P. discrepans. The recent episodes of dieback of ash in Saskatchewan associated with psyllid feeding are consistent with disease symptoms caused by 'Ca. Liberibacter' pathogens, and this possibility warrants further study.

Three Aphid-Transmitted Viruses Encourage Vector Migration From Infected Common Bean (Phaseolus vulgaris) Plants Through a Combination of Volatile and Surface Cues.

Bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV), and cucumber mosaic virus (CMV) are important pathogens of common bean (Phaseolus vulgaris), a crop vital for food security in sub-Saharan Africa. These viruses are vectored by aphids non-persistently, with virions bound loosely to stylet receptors. These viruses also manipulate aphid-mediated transmission by altering host properties. Virus-induced effects on host-aphid interactions were investigated using choice test (migration) assays, olfactometry, and analysis of insect-perceivable volatile organic compounds (VOCs) using gas chromatography (GC)-coupled mass spectrometry, and GC-coupled electroantennography. When allowed to choose freely between infected and uninfected plants, aphids of the legume specialist species Aphis fabae, and of the generalist species Myzus persicae, were repelled by plants infected with BCMV, BCMNV, or CMV. However, in olfactometer experiments with A. fabae, only the VOCs emitted by BCMNV-infected plants repelled aphids. Although BCMV, BCMNV, and CMV each induced distinctive changes in emission of aphid-perceivable volatiles, all three suppressed emission of an attractant sesquiterpene, α-copaene, suggesting these three different viruses promote migration of virus-bearing aphids in a similar fashion.

Post-COVID-19 Action: Guarding Africa's Crops against Viral Epidemics Requires Research Capacity Building That Unifies a Trio of Transdisciplinary Interventions.

The COVID-19 pandemic has shown that understanding the genomics of a virus, diagnostics and breaking virus transmission is essential in managing viral pandemics. The same lessons can apply for plant viruses. There are plant viruses that have severely disrupted crop production in multiple countries, as recently seen with maize lethal necrosis disease in eastern and southern Africa. High-throughput sequencing (HTS) is needed to detect new viral threats. Equally important is building local capacity to develop the tools required for rapid diagnosis of plant viruses. Most plant viruses are insect-vectored, hence, biological insights on virus transmission are vital in modelling disease spread. Research in Africa in these three areas is in its infancy and disjointed. Despite intense interest, uptake of HTS by African researchers is hampered by infrastructural gaps. The use of whole-genome information to develop field-deployable diagnostics on the continent is virtually inexistent. There is fledgling research into plant-virus-vector interactions to inform modelling of viral transmission. The gains so far have been modest but encouraging, and therefore must be consolidated. For this, I propose the creation of a new Research Centre for Africa. This bold investment is needed to secure the future of Africa's crops from insect-vectored viral diseases.

Modelling and manipulation of aphid-mediated spread of non-persistently transmitted viruses.

Aphids vector many plant viruses in a non-persistent manner i.e., virus particles bind loosely to the insect mouthparts (stylet). This means that acquisition of virus particles from infected plants, and inoculation of uninfected plants by viruliferous aphids, are rapid processes that require only brief probes of the plant's epidermal cells. Virus infection alters plant biochemistry, which causes changes in emission of volatile organic compounds and altered accumulation of nutrients and defence compounds in host tissues. These virus-induced biochemical changes can influence the migration, settling and feeding behaviours of aphids. Working mainly with cucumber mosaic virus and several potyviruses, a number of research groups have noted that in some plants, virus infection engenders resistance to aphid settling (sometimes accompanied by emission of deceptively attractive volatiles, that can lead to exploratory penetration by aphids without settling). However, in certain other hosts, virus infection renders plants more susceptible to aphid colonisation. It has been suggested that induction of resistance to aphid settling encourages transmission of non-persistently transmitted viruses, while induction of susceptibility to settling retards transmission. However, recent mathematical modelling indicates that both virus-induced effects contribute to epidemic development at different scales. We have also investigated at the molecular level the processes leading to induction, by cucumber mosaic virus, of feeding deterrence versus susceptibility to aphid infestation. Both processes involve complex interactions between specific viral proteins and host factors, resulting in manipulation or suppression of the plant's immune networks.

Different Plant Viruses Induce Changes in Feeding Behavior of Specialist and Generalist Aphids on Common Bean That Are Likely to Enhance Virus Transmission.

Bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV), and cucumber mosaic virus (CMV) cause serious epidemics in common bean (Phaseolus vulgaris), a vital food security crop in many low-to-medium income countries, particularly in Sub-Saharan Africa. Aphids transmit these viruses "non-persistently," i.e., virions attach loosely to the insects' stylets. Viruses may manipulate aphid-host interactions to enhance transmission. We used direct observation and electrical penetration graph measurements to see if the three viruses induced similar or distinct changes in feeding behaviors of two aphid species, Aphis fabae and Myzus persicae. Both aphids vector BCMV, BCMNV, and CMV but A. fabae is a legume specialist (the dominant species in bean fields) while M. persicae is a generalist that feeds on and transmits viruses to diverse plant hosts. Aphids of both species commenced probing epidermal cells (behavior optimal for virus acquisition and inoculation) sooner on virus-infected plants than on mock-inoculated plants. Infection with CMV was especially disruptive of phloem feeding by the bean specialist aphid A. fabae. A. fabae also experienced mechanical stylet difficulty when feeding on virus-infected plants, and this was also exacerbated for M. persicae. Overall, feeding on virus-infected host plants by specialist and generalist aphids was affected in different ways but all three viruses induced similar effects on each aphid type. Specifically, non-specialist (M. persicae) aphids encountered increased stylet difficulties on plants infected with BCMV, BCMNV, or CMV, whereas specialist aphids (A. fabae) showed decreased phloem ingestion on infected plants. Probing and stylet pathway activity (which facilitate virus transmission) were not decreased by any of the viruses for either of the aphid species, except in the case of A. fabae on CMV-infected bean, where these activities were increased. Overall, these virus-induced changes in host-aphid interactions are likely to enhance non-persistent virus transmission, and data from this work will be useful in epidemiological modeling of non-persistent vectoring of viruses by aphids.

Metagenomic Analysis of Plant Virus Occurrence in Common Bean (Phaseolus vulgaris) in Central Kenya.

Two closely related potyviruses, bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV), are regarded as major constraints on production of common bean (Phaseolus vulgaris L.) in Eastern and Central Africa, where this crop provides a high proportion of dietary protein as well as other nutritional, agronomic, and economic benefits. Previous studies using antibody-based assays and indicator plants indicated that BCMV and BCMNV are both prevalent in bean fields in the region but these approaches cannot distinguish between these potyviruses or detect other viruses that may threaten the crop. In this study, we utilized next generation shotgun sequencing for a metagenomic examination of viruses present in bean plants growing at two locations in Kenya: the University of Nairobi Research Farm in Nairobi's Kabete district and at sites in Kirinyaga County. RNA was extracted from leaves of bean plants exhibiting apparent viral symptoms and sequenced on the Illumina MiSeq platform. We detected BCMNV, cucumber mosaic virus (CMV), and Phaseolus vulgaris alphaendornaviruses 1 and 2 (PvEV1 and 2), with CMV present in the Kirinyaga samples. The CMV strain detected in this study was most closely related to Asian strains, which suggests that it may be a recent introduction to the region. Surprisingly, and in contrast to previous surveys, BCMV was not detected in plants at either location. Some plants were infected with PvEV1 and 2. The detection of PvEV1 and 2 suggests these seed transmitted viruses may be more prevalent in Eastern African bean germplasm than previously thought.

Viral metagenomics of aphids present in bean and maize plots on mixed-use farms in Kenya reveals the presence of three dicistroviruses including a novel Big Sioux River virus-like dicistrovirus.

BACKGROUND: Aphids are major vectors of plant viruses. Common bean (Phaseolus vulgaris L.) and maize (Zea mays L.) are important crops that are vulnerable to aphid herbivory and aphid-transmitted viruses. In East and Central Africa, common bean is frequently intercropped by smallholder farmers to provide fixed nitrogen for cultivation of starch crops such as maize. We used a PCR-based technique to identify aphids prevalent in smallholder bean farms and next generation sequencing shotgun metagenomics to examine the diversity of viruses present in aphids and in maize leaf samples. Samples were collected from farms in Kenya in a range of agro-ecological zones. RESULTS: Cytochrome oxidase 1 (CO1) gene sequencing showed that Aphis fabae was the sole aphid species present in bean plots in the farms visited. Sequencing of total RNA from aphids using the Illumina platform detected three dicistroviruses. Maize leaf RNA was also analysed. Identification of Aphid lethal paralysis virus (ALPV), Rhopalosiphum padi virus (RhPV), and a novel Big Sioux River virus (BSRV)-like dicistrovirus in aphid and maize samples was confirmed using reverse transcription-polymerase chain reactions and sequencing of amplified DNA products. Phylogenetic, nucleotide and protein sequence analyses of eight ALPV genomes revealed evidence of intra-species recombination, with the data suggesting there may be two ALPV lineages. Analysis of BSRV-like virus genomic RNA sequences revealed features that are consistent with other dicistroviruses and that it is phylogenetically closely related to dicistroviruses of the genus Cripavirus. CONCLUSIONS: The discovery of ALPV and RhPV in aphids and maize further demonstrates the broad occurrence of these dicistroviruses. Dicistroviruses are remarkable in that they use plants as reservoirs that facilitate infection of their insect replicative hosts, such as aphids. This is the first report of these viruses being isolated from either organism. The BSRV-like sequences represent a potentially novel dicistrovirus infecting A. fabae.

Engineering resistance to virus transmission.

Engineering plants for resistance to virus transmission by invertebrate vectors has lagged behind other forms of plant protection. Vectors typically transmit more than one virus. Thus, vector resistance could provide a wider range of protection than defenses directed solely against one virus or virus group. We discuss current knowledge of vector-host-virus interactions, the roles of viral gene products in host and vector manipulation, and the effects of semiochemicals on host-vector interactions, and how this knowledge could be employed to disrupt transmission dynamics. We also discuss how resistance to vectors could be generated through genetic engineering or gene editing or indirectly through use of biocontrol using plant-resident viruses that infect vectors.

Bean Common Mosaic Virus and Bean Common Mosaic Necrosis Virus: Relationships, Biology, and Prospects for Control.

The closely related potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) are major constraints on common bean (Phaseolus vulgaris) production. Crop losses caused by BCMV and BCMNV impact severely not only on commercial scale cultivation of this high-value crop but also on production by smallholder farmers in the developing world, where bean serves as a key source of dietary protein and mineral nutrition. In many parts of the world, progress has been made in combating BCMV through breeding bean varieties possessing the I gene, a dominant gene conferring resistance to most BCMV strains. However, in Africa, and in particular in Central and East Africa, BCMNV is endemic and this presents a serious problem for deployment of the I gene because this virus triggers systemic necrosis (black root disease) in plants possessing this resistance gene. Information on these two important viruses is scattered throughout the literature from 1917 onward, and although reviews on resistance to BCMV and BCMNV exist, there is currently no comprehensive review on the biology and taxonomy of BCMV and BCMNV. In this chapter, we discuss the current state of our knowledge of these two potyviruses including fundamental aspects of classification and phylogeny, molecular biology, host interactions, transmission through seed and by aphid vectors, geographic distribution, as well as current and future prospects for the control of these important viruses.

Mouse cytokine profile skewed towards Th2 in pregnancy during infection with Brucella abortus S19 strain.

The two classes of cytokines Th1 and Th2 determine the type of immune response elicited. The Th2 immune response is associated with successful pregnancy. Brucellosis is an intracellular bacterium that elicits the Th1 response and is known to cause spontaneous abortion in mammalian species. This study sought to determine if Brucella infection causes spontaneous abortion by causing the circulating cytokine profile be Th1 dominant during pregnancy. Forty-eight Swiss white mice were used in this murine model and the S19 strain of Brucella abortus was used in as the infective agent. Pregnant mice in the test group were injected intraperitoneally with 10(5-8) CFU of Brucella and cytokine profile evaluated over the three trimesters of pregnancy. Pregnant mice in the control group were left to go through normal pregnancy and their cytokine profile evaluated over the three trimesters of pregnancy. Cytokines in serum samples were analyzed by Cytometric Bead Array. The data was analyzed using the Paired T- test and p < 0.05 was considered significant. IFN-γ and TNF-α represented the Th1 cytokines while IL-4 and IL-5 represented the Th2 cytokines. None of the mice in the test group had spontaneous abortion. IFN-γ and TNF-α had no significant differences between cytokine levels for infected and uninfected groups in all 3 trimesters of pregnancy. IL-4 levels had significant differences in all three trimesters of pregnancy (t = 13, P = 0.036, 0.0071 and 0.0277). IL-5 levels had significant differences second trimester (t = 14, P = 0.0075). The cytokine profile was robustly Th2. In conclusion, Brucella abortus cannot cause spontaneous abortion by altering the mouse cytokine profile towards Th1 in pregnancy. Elevated IL-4 levels with corresponding suppression of IFN-γ can be used as a marker for successful pregnancy in Brucellosis.