Early detection of plant virus infection using multispectral imaging and spatial-spectral machine learning.

Cassava brown streak disease (CBSD) is an emerging viral disease that can greatly reduce cassava productivity, while causing only mild aerial symptoms that develop late in infection. Early detection of CBSD enables better crop management and intervention. Current techniques require laboratory equipment and are labour intensive and often inaccurate. We have developed a handheld active multispectral imaging (A-MSI) device combined with machine learning for early detection of CBSD in real-time. The principal benefits of A-MSI over passive MSI and conventional camera systems are improved spectral signal-to-noise ratio and temporal repeatability. Information fusion techniques further combine spectral and spatial information to reliably identify features that distinguish healthy cassava from plants with CBSD as early as 28 days post inoculation on a susceptible and a tolerant cultivar. Application of the device has the potential to increase farmers' access to healthy planting materials and reduce losses due to CBSD in Africa. It can also be adapted for sensing other biotic and abiotic stresses in real-world situations where plants are exposed to multiple pest, pathogen and environmental stresses.

Identification of novel QTL contributing to barley yellow mosaic resistance in wild barley (Hordeum vulgare spp. spontaneum).

BACKGROUND: Barley yellow mosaic disease (BYMD) caused by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) seriously threatens the production of winter barley. Cultivating and promoting varieties that carry disease-resistant genes is one of the most powerful ways to minimize the disease's effect on yield. However, as the BYMD virus mutates rapidly, resistance conferred by the two cloned R genes to the virus had been overcome by new virus strains. There is an urgent need for novel resistance genes in barley that convey sustainable resistance to newly emerging virus strains causing BYMD. RESULTS: A doubled haploid (DH) population derived from a cross of SRY01 (BYMD resistant wild barley) and Gairdner (BYMD susceptible barley cultivar) was used to explore for QTL of resistance to BYMD in barley. A total of six quantitative trait loci (qRYM-1H, qRYM-2Ha, qRYM-2Hb, qRYM-3H, qRYM-5H, and qRYM-7H) related to BYMD resistance were detected, which were located on chromosomes 1H, 2H, 3H, 5H, and 7H. Both qRYM-1H and qRYM-2Ha were detected in all environments. qRYM-1H was found to be overlapped with rym7, a known R gene to the disease, whereas qRYM-2Ha is a novel QTL on chromosome 2H originated from SRY01, explaining phenotypic variation from 9.8 to 17.8%. The closely linked InDel markers for qRYM-2Ha were developed which could be used for marker-assisted selection in barley breeding. qRYM-2Hb and qRYM-3H were stable QTL for specific resistance to Yancheng and Yangzhou virus strains, respectively. qRYM-5H and qRYM-7H identified in Yangzhou were originated from Gairdner. CONCLUSIONS: Our work is focusing on a virus disease (barley yellow mosaic) of barley. It is the first report on BYMD-resistant QTL from wild barley accessions. One novel major QTL (qRYM-2Ha) for the resistance was detected. The consistently detected new genes will potentially serve as novel sources for achieving pre-breeding barley materials with resistance to BYMD.

PAL1 gene of the phenylpropanoid pathway increases resistance to the Cassava brown streak virus in cassava.

BACKGROUND: The phenylalanine ammonia lyase genes play crucial role in plant response to biotic and abiotic stresses. In this study, we characterized the role of PAL genes in increasing resistance to the Cassava brown streak virus that causes the economically important cassava brown streak disease (CBSD) on cassava in Africa. METHODS: The whole transcriptomes of eight cassava varieties differing in resistance to CBSD were obtained at 1, 5 and 8 weeks after CBSV infection. RESULTS: Analysis of RNA-Seq data identified the overexpression of PAL1, PAL2, cinnamic acid and two chalcone synthase genes in CBSD-resistant cassava varieties, which was subsequently confirmed by RT-qPCR. The exogenous application of Acibenzolar-S-Methyl induced PAL1 gene expression to enhance resistance in the susceptible var. Kalawe. In contrast, the silencing of PAL1 by RNA interference led to increased susceptibility of the resistant var. Kaleso to CBSD. CONCLUSIONS: PAL1 gene of the phenylpropanoid pathway has a major role in inducing resistance to CBSD in cassava plants and its early induction is key for CBSD resistance.

A calmodulin-binding transcription factor links calcium signaling to antiviral RNAi defense in plants.

RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca2+ signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense.

Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

Genomic prediction models trained with historical records enable populating the German ex situ genebank bio-digital resource center of barley (Hordeum sp.) with information on resistances to soilborne barley mosaic viruses.

KEY MESSAGE: Genomic prediction with special weight of major genes is a valuable tool to populate bio-digital resource centers. Phenotypic information of crop genetic resources is a prerequisite for an informed selection that aims to broaden the genetic base of the elite breeding pools. We investigated the potential of genomic prediction based on historical screening data of plant responses against the Barley yellow mosaic viruses for populating the bio-digital resource center of barley. Our study includes dense marker data for 3838 accessions of winter barley, and historical screening data of 1751 accessions for Barley yellow mosaic virus (BaYMV) and of 1771 accessions for Barley mild mosaic virus (BaMMV). Linear mixed models were fitted by considering combinations for the effects of genotypes, years, and locations. The best linear unbiased estimations displayed a broad spectrum of plant responses against BaYMV and BaMMV. Prediction abilities, computed as correlations between predictions and observed phenotypes of accessions, were low for the marker-assisted selection approach amounting to 0.42. In contrast, prediction abilities of genomic best linear unbiased predictions were high, with values of 0.62 for BaYMV and 0.64 for BaMMV. Prediction abilities of genomic prediction were improved by up to ~ 5% using W-BLUP, in which more weight is given to markers with significant major effects found by association mapping. Our results outline the utility of historical screening data and W-BLUP model to predict the performance of the non-phenotyped individuals in genebank collections. The presented strategy can be considered as part of the different approaches used in genebank genomics to valorize genetic resources for their usage in disease resistance breeding and research.

Genetic analysis and QTL mapping for multiple biotic stress resistance in cassava.

In Sub-Saharan Africa cassava (Manihot esculenta Crantz) is one of the most important food crops where more than 40% of the population relies on it as their staple carbohydrate source. Biotic constraints such as viral diseases, mainly Cassava Mosaic Disease (CMD) and Cassava Brown Streak Disease (CBSD), and arthropod pests, particularly Cassava Green Mite (CGM), are major constraints to the realization of cassava's full production potential in Africa. To address these problems, we aimed to map the quantitative trait loci (QTL) associated with resistance to CBSD foliar and root necrosis symptoms, foliar CMD and CGM symptoms in a full-sib mapping population derived from the genotypes AR40-6 and Albert. A high-density linkage map was constructed with 2,125 SNP markers using a genotyping-by-sequencing approach. For phenotyping, clonal evaluation trials were conducted with 120 F1 individuals for two consecutive field seasons using an alpha-lattice design at Chambezi and Naliendele, Tanzania. Previously identified QTL for resistance to CBSD foliar symptoms were corroborated, and a new putative QTL for CBSD root necrosis identified (qCBSDRNc14AR) from AR40-6. Two QTL were identified within the region of the previously recognized CMD2 locus from this population in which both parents are thought to possess the CMD2 locus. Interestingly, a minor but consistent QTL, qCGM18AR, for CGM resistance at 3 months after planting stage was also detected and co-localized with a previously identified SSR marker, NS346, linked with CGM resistance. Markers underlying these QTL may be used to increase efficiencies in cassava breeding programs.

Dryland Cropping Systems, Weed Communities, and Disease Status Modulate the Effect of Climate Conditions on Wheat Soil Bacterial Communities.

Little knowledge exists on how soil bacteria in agricultural settings are impacted by management practices and environmental conditions in current and predicted climate scenarios. We assessed the impact of soil moisture, soil temperature, weed communities, and disease status on soil bacterial communities in three cropping systems: (i) conventional no-till (CNT) systems utilizing synthetic pesticides and herbicides, (ii) USDA-certified tilled organic (OT) systems, and (iii) USDA-certified organic systems with sheep grazing (OG). Sampling date within the growing season and associated soil temperature and moisture exerted the greatest effect on bacterial communities, followed by cropping system, Wheat streak mosaic virus (WSMV) infection status, and weed community. Soil temperature was negatively correlated with bacterial richness and evenness, while soil moisture was positively correlated with bacterial richness and evenness. Soil temperature and soil moisture independently altered soil bacterial community similarity between treatments. Inoculation of wheat with WSMV altered the associated soil bacteria, and there were interactions between disease status and cropping system, sampling date, and climate conditions, indicating the effect of multiple stressors on bacterial communities in soil. In May and July, cropping system altered the effect of climate change on the bacterial community composition in hotter conditions and in hotter and drier conditions compared to ambient conditions, in samples not treated with WSMV. Overall, this study indicates that predicted climate modifications as well as biological stressors play a fundamental role in the impact of cropping systems on soil bacterial communities.IMPORTANCE Climate change is affecting global moisture and temperature patterns, and its impacts are predicted to worsen over time, posing progressively larger threats to food production. In the Northern Great Plains of the United States, climate change is forecast to increase temperature and decrease precipitation during the summer, and it is expected to negatively affect cereal crop production and pest management. In this study, temperature, soil moisture, weed communities, and disease status had interactive effects with cropping system on bacterial communities. As local climates continue to shift, the dynamics of above- and belowground associated biodiversity will also shift, which will impact food production and increase the need for more sustainable practices.

Exploring dynamic protein-protein interactions in cassava through the integrative interactome network.

Protein-protein interactions (PPIs) play an essential role in cellular regulatory processes. Despite, in-depth studies to uncover the mystery of PPI-mediated regulations are still lacking. Here, an integrative interactome network (MePPI-Ux) was obtained by incorporating expression data into the improved genome-scale interactome network of cassava (MePPI-U). The MePPI-U, constructed by both interolog- and domain-based approaches, contained 3,638,916 interactions and 24,590 proteins (59% of proteins in the cassava AM560 genome version 6). After incorporating expression data as information of state, the MePPI-U rewired to represent condition-dependent PPIs (MePPI-Ux), enabling us to envisage dynamic PPIs (DPINs) that occur at specific conditions. The MePPI-Ux was exploited to demonstrate timely PPIs of cassava under various conditions, namely drought stress, brown streak virus (CBSV) infection, and starch biosynthesis in leaf/root tissues. MePPI-Uxdrought and MePPI-UxCBSV suggested involved PPIs in response to stress. MePPI-UxSB,leaf and MePPI-UxSB,root suggested the involvement of interactions among transcription factor proteins in modulating how leaf or root starch is synthesized. These findings deepened our knowledge of the regulatory roles of PPIs in cassava and would undeniably assist targeted breeding efforts to improve starch quality and quantity.

Estimating epidemiological parameters from experiments in vector access to host plants, the method of matching gradients.

Estimation of pathogenic life-history values, for instance the duration a pathogen is retained in an insect vector (i.e., retention period) is of particular importance for understanding plant disease epidemiology. How can we extract values for these epidemiological parameters from conventional small-scale laboratory experiments in which transmission success is measured in relation to durations of vector access to host plants? We provide a solution to this problem by deriving formulae for the empirical curves that these experiments produce, called access period response curves (i.e., transmission success vs access period). We do this by writing simple equations for the fundamental life-cycle components of insect vectors in the laboratory. We then infer values of epidemiological parameters by matching the theoretical and empirical gradients of access period response curves. Using the example of Cassava brown streak virus (CBSV), which has emerged in sub-Saharan Africa and now threatens regional food security, we illustrate the method of matching gradients. We show how applying the method to published data produces a new understanding of CBSV through the inference of retention period, acquisition period and inoculation period parameters. We found that CBSV is retained for a far shorter duration in its insect vector (Bemisia tabaci whitefly) than had previously been assumed. Our results shed light on a number of critical factors that may be responsible for the transition of CBSV from sub- to super-threshold R0 in sub-Saharan Africa. The method is applicable to plant pathogens in general, to supply epidemiological parameter estimates that are crucial for practical management of epidemics and prediction of pandemic risk.

Utilization of infectious clones to visualize Cassava brown streak virus replication in planta and gain insights into symptom development.

Cassava brown streak disease (CBSD) is a leading cause of cassava yield losses across eastern and central Africa and is having a severe impact on food security across the region. Despite its importance, relatively little is known about the mechanisms behind CBSD viral infections. We have recently reported the construction of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) infectious clones (IC), which can be used to gain insights into the functions of viral proteins and sequences associated with symptom development. In this study, we perform the first reporter gene tagging of a CBSV IC, with the insertion of green fluorescent protein (GFP) sequence at two different genome positions. Nicotiana benthamiana infections with the CBSV_GFP ICs revealed active CBSV replication in inoculated leaves at 2-5 days post inoculation (dpi) and systemic leaves at 10-14 dpi. We also constructed the chimera CBSV_UCP IC, consisting of the CBSV genome with a UCBSV coat protein (CP) sequence replacement. N. benthamiana infections with CBSV_UCP revealed that the CBSV CP may be associated with high levels of viral accumulation and necrosis development during early infection. These initial manipulations pave the way for U/CBSV ICs to be used to understand U/CBSV biology that will inform vital CBSD control strategies.

Bulked segregant RNA-sequencing (BSR-seq) identified a novel rare allele of eIF4E effective against multiple isolates of BaYMV/BaMMV.

KEY MESSAGE: A novel rare allele of the barley host factor gene eIF4E for BaMMV/BaYMV infection was identified in an Iranian landrace that showed broad resistance to barley yellow mosaic virus disease, and molecular markers facilitating efficient selection were developed. The soil-borne yellow mosaic virus disease caused by different strains of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) is a major threat to winter barley (Hordeum vulgare) production in Europe and East Asia. However, the exploration of resistant germplasm or casual genes for barley breeding is rather limited in relation to the rapid diversification of viral strains. Here, we identified an Iranian barley landrace 'HOR3298,' which represented complete resistance to BaYMV and BaMMV. In contrast to rym4 and rym5, which act as the predominant source in Europe and East Asia for breeding resistant cultivars over decades and which have been overcome by several virulent isolates, this landrace showed broad-spectrum resistance to multiple isolates of BaYMV/BaMMV in the fields of Germany and China. By employment of bulked segregant RNA sequencing, test for allelism, and haplotype analysis, a recessive resistance gene in 'HOR3298' was genetically mapped coincident with the host factor eukaryotic translation initiation factor 4E (eIF4E, causal gene of rym4 and rym5). The eIF4EHOR3298 allele encoded for a novel haplotype that contained an exclusive nucleotide mutation (G565A) in the coding sequence. The easily handled markers were developed based on the exclusively rare variation, providing precise selection of this allele. Thus, this work provided a novel reliable resistance source and the feasible marker-assisted selection assays that can be used in breeding for barley yellow mosaic virus disease resistance in cultivated barley.

Areca Palm Necrotic Ringspot Virus, Classified Within a Recently Proposed Genus Arepavirus of the Family Potyviridae, Is Associated With Necrotic Ringspot Disease in Areca Palm.

Areca palm (Areca catechu), one of the two most important commercial crops in Hainan, China, has been severely damaged by a variety of pathogens and insects. Here, we report a new disease, tentatively referred to as areca palm necrotic ringspot disease (ANRSD), which is highly epidemic in the main growing regions in Hainan. Transmission electron microscopy observation and small RNA deep sequencing revealed the existence of a viral agent of the family Potyviridae in a diseased areca palm plant (XC1). The virus was tentatively named areca palm necrotic ringspot virus (ANRSV). Subsequently, the positive-sense single-stranded genome of ANRSV isolate XC1 was completely determined. The genome annotation revealed the existence of two cysteine proteinases in tandem (HC-Pro1 and HC-Pro2) in the genomic 5' terminus of ANRSV. Sequence comparison and phylogenetic analysis suggested the taxonomic classification of ANRSV into the recently proposed genus Arepavirus in the family Potyviridae. Given the close relationship of ANRSV with another newly reported arepavirus (areca palm necrotic spindle-spot virus), the exact taxonomic status of ANRSV needs to be further investigated. In this study, a reverse transcription polymerase chain reaction assay for ANRSV-specific detection was developed and a close association between ANRSV and ANRSD was found.

Strategies for the Construction of Cassava Brown Streak Disease Viral Infectious Clones.

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate 'Kikombe', such an approach failed to deliver full-length clones for CBSV isolates 'Nampula' or 'Tanza', necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex.

Triticum aestivum heat shock protein 23.6 interacts with the coat protein of wheat yellow mosaic virus.

The role of heat shock proteins (HSPs) in viral replication has been described in numerous publications. Wheat yellow mosaic virus (WYMV) belongs to the genus Bymovirus (family Potyviridae), which causes yellow mosaic and dwarf symptoms in wheat (Triticum aestivum). In this study, the T. aestivum heat shock protein 23.6 (TaHSP23.6), which belongs to the small heat shock protein family, was shown to interact with the WYMV coat protein (CP) in a yeast two-hybrid screen. The co-localization and interaction between TaHSP23.6 and WYMV CP were additionally verified in Nicotiana benthamiana by co-localization assays and bimolecular fluorescence complementation (BiFC). Not only the transcription of TaHSP23.6 but also that of other HSP family members (TaHSP70, TaHSP90, and TaHSP101) was up-regulated in WYMV-infected leaves, as shown by semi-quantitative PCR assays. Interestingly, the expression levels of the T. aestivum heat stress transcription factor A2 (TaHSFA2) members were varied in response to WYMV infection. Thus, our results provide insights into the interaction between TaHSP23.6 and WYMV infection.

Maize Lethal Necrosis: An Emerging, Synergistic Viral Disease.

Maize lethal necrosis (MLN) is a disease of maize caused by coinfection of maize with maize chlorotic mottle virus (MCMV) and one of several viruses from the Potyviridae, such as sugarcane mosaic virus, maize dwarf mosaic virus, Johnsongrass mosaic virus or wheat streak mosaic virus. The coinfecting viruses act synergistically to result in frequent plant death or severely reduce or negligible yield. Over the past eight years, MLN has emerged in sub-Saharan East Africa, Southeast Asia, and South America, with large impacts on smallholder farmers. Factors associated with MLN emergence include multiple maize crops per year, the presence of maize thrips ( Frankliniella williamsi), and highly susceptible maize crops. Soil and seed transmission of MCMV may also play significant roles in development and perpetuation of MLN epidemics. Containment and control of MLN will likely require a multipronged approach, and more research is needed to identify and develop the best measures.

Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya.

BACKGROUND: Maize lethal necrosis is caused by a synergistic co-infection of Maize chlorotic mottle virus (MCMV) and a specific member of the Potyviridae, such as Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Johnson grass mosaic virus (JGMV). Typical maize lethal necrosis symptoms include severe yellowing and leaf drying from the edges. In Kenya, we detected plants showing typical and atypical symptoms. Both groups of plants often tested negative for SCMV by ELISA. METHODS: We used next-generation sequencing to identify viruses associated to maize lethal necrosis in Kenya through a metagenomics analysis. Symptomatic and asymptomatic leaf samples were collected from maize and sorghum representing sixteen counties. RESULTS: Complete and partial genomes were assembled for MCMV, SCMV, Maize streak virus (MSV) and Maize yellow dwarf virus-RMV (MYDV-RMV). These four viruses (MCMV, SCMV, MSV and MYDV-RMV) were found together in 30 of 68 samples. A geographic analysis showed that these viruses are widely distributed in Kenya. Phylogenetic analyses of nucleotide sequences showed that MCMV, MYDV-RMV and MSV are similar to isolates from East Africa and other parts of the world. Single nucleotide polymorphism, nucleotide and polyprotein sequence alignments identified three genetically distinct groups of SCMV in Kenya. Variation mapped to sequences at the border of NIb and the coat protein. Partial genome sequences were obtained for other four potyviruses and one polerovirus. CONCLUSION: Our results uncover the complexity of the maize lethal necrosis epidemic in Kenya. MCMV, SCMV, MSV and MYDV-RMV are widely distributed and infect both maize and sorghum. SCMV population in Kenya is diverse and consists of numerous strains that are genetically different to isolates from other parts of the world. Several potyviruses, and possibly poleroviruses, are also involved.

Wheat streak mosaic virus: a century old virus with rising importance worldwide.

Wheat streak mosaic virus (WSMV) causes wheat streak mosaic, a disease of cereals and grasses that threatens wheat production worldwide. It is a monopartite, positive-sense, single-stranded RNA virus and the type member of the genus Tritimovirus in the family Potyviridae. The only known vector is the wheat curl mite (WCM, Aceria tosichella), recently identified as a species complex of biotypes differing in virus transmission. Low rates of seed transmission have been reported. Infected plants are stunted and have a yellow mosaic of parallel discontinuous streaks on the leaves. In the autumn, WCMs move from WSMV-infected volunteer wheat and other grass hosts to newly emerged wheat and transmit the virus which survives the winter within the plant, and the mites survive as eggs, larvae, nymphs or adults in the crown and leaf sheaths. In the spring/summer, the mites move from the maturing wheat crop to volunteer wheat and other grass hosts and transmit WSMV, and onto newly emerged wheat in the fall to which they transmit the virus, completing the disease cycle. WSMV detection is by enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) or quantitative RT-PCR (RT-qPCR). Three types of WSMV are recognized: A (Mexico), B (Europe, Russia, Asia) and D (USA, Argentina, Brazil, Australia, Turkey, Canada). Resistance genes Wsm1, Wsm2 and Wsm3 have been identified. The most effective, Wsm2, has been introduced into several wheat cultivars. Mitigation of losses caused by WSMV will require enhanced knowledge of the biology of WCM biotypes and WSMV, new or improved virus detection techniques, the development of resistance through traditional and molecular breeding, and the adaptation of cultural management tactics to account for climate change.

Molecular insights into Cassava brown streak virus susceptibility and resistance by profiling of the early host response.

Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) are responsible for significant cassava yield losses in eastern sub-Saharan Africa. To study the possible mechanisms of plant resistance to CBSVs, we inoculated CBSV-susceptible and CBSV-resistant cassava varieties with a mixed infection of CBSVs using top-cleft grafting. Transcriptome profiling of the two cassava varieties was performed at the earliest time point of full infection (28 days after grafting) in the susceptible scions. The expression of genes encoding proteins in RNA silencing, salicylic acid pathways and callose deposition was altered in the susceptible cassava variety, but transcriptional changes were limited in the resistant variety. In total, the expression of 585 genes was altered in the resistant variety and 1292 in the susceptible variety. Transcriptional changes led to the activation of ß-1,3-glucanase enzymatic activity and a reduction in callose deposition in the susceptible cassava variety. Time course analysis also showed that CBSV replication in susceptible cassava induced a strong up-regulation of RDR1, a gene previously reported to be a susceptibility factor in other potyvirus-host pathosystems. The differences in the transcriptional responses to CBSV infection indicated that susceptibility involves the restriction of callose deposition at plasmodesmata. Aniline blue staining of callose deposits also indicated that the resistant variety displays a moderate, but significant, increase in callose deposition at the plasmodesmata. Transcriptome data suggested that resistance does not involve typical antiviral defence responses (i.e. RNA silencing and salicylic acid). A meta-analysis of the current RNA-sequencing (RNA-seq) dataset and selected potyvirus-host and virus-cassava RNA-seq datasets revealed that the conservation of the host response across pathosystems is restricted to genes involved in developmental processes.

Unusual occurrence of a DAG motif in the Ipomovirus Cassava brown streak virus and implications for its vector transmission.

Cassava is the main staple food for over 800 million people globally. Its production in eastern Africa is being constrained by two devastating Ipomoviruses that cause cassava brown streak disease (CBSD); Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), with up to 100% yield loss for smallholder farmers in the region. To date, vector studies have not resulted in reproducible and highly efficient transmission of CBSV and UCBSV. Most virus transmission studies have used Bemisia tabaci (whitefly), but a maximum of 41% U/CBSV transmission efficiency has been documented for this vector. With the advent of next generation sequencing, researchers are generating whole genome sequences for both CBSV and UCBSV from throughout eastern Africa. Our initial goal for this study was to characterize U/CBSV whole genomes from CBSD symptomatic cassava plants sampled in Kenya. We have generated 8 new whole genomes (3 CBSV and 5 UCBSV) from Kenya, and in the process of analyzing these genomes together with 26 previously published sequences, we uncovered the aphid transmission associated DAG motif within coat protein genes of all CBSV whole genomes at amino acid positions 52-54, but not in UCBSV. Upon further investigation, the DAG motif was also found at the same positions in two other Ipomoviruses: Squash vein yellowing virus (SqVYV), Coccinia mottle virus (CocMoV). Until this study, the highly-conserved DAG motif, which is associated with aphid transmission was only noticed once, in SqVYV but discounted as being of minimal importance. This study represents the first comprehensive look at Ipomovirus genomes to determine the extent of DAG motif presence and significance for vector relations. The presence of this motif suggests that aphids could potentially be a vector of CBSV, SqVYV and CocMov. Further transmission and ipomoviral protein evolutionary studies are needed to confirm this hypothesis.