Transforming crop protection: The role of RNAi in mitigating barley yellow dwarf virus and aphid infestation in cereal crops.

SUMMARYWinter barley was sown by conventional-tillage (CT) and minimum-tillage (MT) cultivation over three seasons. Each cultivation treatment was split so that straw was incorporated into the soil during cultivation in one split, while the other did not receive straw. Aphid occurrences in autumn and incidence of barley yellow dwarf virus (BYDV) in spring were compared. Similar investigations on winter wheat were made over a further three seasons. The method of cultivation affected the number of aphids on barley and wheat plants in autumn and on wheat heads in summer; MT had fewest aphids. Soil incorporation of straw during cultivation had a similar effect. Method of cultivation affected the incidence of BYDV disease, with MT having least infection. Straw-treated cereal plots had fewer aphids and less BYDV than no-straw plots. Aphids and virus reached damaging levels only in the first barley crop. In this season, MT barley had significantly fewer aphids (48% fewer) and significantly less BYDV (71% less) than CT. Straw-treated plots within the CT system had rather similar aphid infestation and BYDV incidence as no-straw plots. Barley grown in the MT system with straw added had significantly fewer aphids (68%) than in the case when no straw was added. Overall, aphids in autumn were significantly fewer on MT relative to CT cereals in three of the six seasons and significantly fewer on straw relative to no-straw plots in two seasons. Aphids on wheat heads in summer were significantly fewer in MT relative to CT plots in one of the three seasons. Aphids on heads were also significantly fewer on straw-treated plots within each system of cultivation in two seasons, while MT wheat with straw had significantly fewer aphids/head than those without straw in one season. BYDV was lower each season in MT barley and wheat when compared with CT crops. These differences were significant for two of the three seasons in which each crop was grown. There was a lower incidence of virus in straw-treated plots than in no-straw plots. This effect was significant for one of the three seasons in which barley and wheat were grown. Plant and tiller density did not differ significantly between MT and CT barley either with or without straw incorporation. Plant density of wheat in autumn was significantly lower for straw-treated plots relative to no-straw plots in one season. Wheat head density was lower for MT relative to CT in one season, while MT with straw had significantly fewer heads than CT plots with straw in two seasons. BYDV significantly reduced grain yield only in 2001/02 when incidence of the disease was high. Grain yield in 2001/02 was 0·95 t/ha (16%) greater for MT barley, due to less BYDV, than for CT. Straw-treated CT barley outyielded no-straw plots by 0·45 t/ha, while the comparable value for the MT system was 0·3 t/ha. The combined effect of MT plus straw had a 1·24 t/ha (21%) yield advantage over CT without straw.It is concluded that MT cereals sown at the same time as CT crops in autumn have a lower risk of aphid infestation and BYDV infection than CT crops. The soil incorporation of straw further enhances the beneficial impact of MT in reducing aphids and virus.

Summary 1 In plant invasion ecology, viruses and other pathogens are often considered in terms of the enemy release hypothesis, which predicts that plants become invasive in new ranges if they escape pathogens from their home range. However, pathogens may sometimes facilitate host spread rather than hinder it. 2 Previously, we hypothesized that apparent competition mediated by barley and cereal yellow dwarf viruses (Luteoviridae: BYDVs, CYDVs) may have facilitated historical grassland invasion in California, USA, where Eurasian grasses displaced native grasses in the 18th and 19th centuries (the disease facilitation hypotheses). However, this could have happened only if the viruses were present during the invasion, which is unknown. 3 To investigate the historical ecology of BYDVs in California grasses, we analysed preserved virus infections in herbarium specimens and used the historical virus sequences to determine rough time estimates of relevant phylogenetic events. 4 The historical viral RNA sequences we identified in invasive and native grasses date from 1917 and are among the oldest recovered from plants thus far and the oldest from North America. 5 Herbarium evidence and phylogenetic analysis suggest that BYDVs were likely to have been present in wild grasses during the California grassland invasion and to have shared some functional characteristics with present-day isolates, supporting the disease facilitation hypothesis. 6 We found evidence of virus spread from California to Australia (or, less likely, from Australia to California) in the late 19th century, when much horticultural exchange occurred, as well as potential correspondence in the timing of virus diversification events and the beginning of extensive human exchange between the Old and New Worlds. 7 Synthesis. Increasing evidence indicates that viruses are important in the ecology of grasslands and may, in some cases, mediate apparent competition among species. Historical data provide essential insight into plant virus ecology and suggest the need to examine human influence on plant virus diversification and spread within natural ecosystems.

Barley yellow dwarf (BYD) is one of the most common diseases of cereal crops, caused by the phloem‐limited, cereal aphid‐borne Barley yellow dwarf virus (BYDV) (Luteoviridae). Delayed planting and controlling aphid vector numbers with insecticides have been the primary approaches to manage BYD. There is limited research on nitrogen (N) application effects on plant growth, N status, and water use in the BYDV pathosystem in the absence of aphid control. Such information will be essential in developing a post‐infection management plan for BYDV‐infected cereals. Through a greenhouse study, we assessed whether manipulation of N supply to BYDV‐infected winter wheat, Triticum aestivum L. (Poaceae), in the presence or absence of the aphid vector Rhopalosiphum padi L. (Hemiptera: Aphididae), could improve N and/or water uptake, and subsequently promote plant growth. Similar responses of shoot biomass and of water and N use efficiencies to various N application rates were observed in both BYDV‐infected and non‐infected plants, suggesting that winter wheat plants with only BYDV infection may be capable of outgrowing infection by the virus. Plants, which simultaneously hosted aphids and BYDV, suffered more severe symptoms and possessed higher virus loads than those infected with BYDV only. Moreover, in plants hosting both BYDV and aphids, aphid pressure was positively associated with N concentration within plant tissue, suggesting that N application and N concentration within foliar tissue may alter BYDV replication indirectly through their influence on aphid reproduction. Even though shoot biomass, tissue N concentration, and water use efficiency increased in response to increased N application, decision‐making on N fertilization to plants hosting both BYDV and aphids should take into consideration the potential of aphid outbreak and/or the possibility of reduced plant resilience to environmental stresses due to decreased root growth.

Host plant resistance in wheat to barley yellow dwarf viruses and their aphid vectors: a review.

A viral movement protein targets host catalases for 26S proteasome-mediated degradation to facilitate viral infection and aphid transmission in wheat

Summary Understanding environmentally dependent variation in interspecific interactions is needed for evaluating how agroecosystems respond to abiotic stressors, including climate change. Both biotic and abiotic conditions shape crop responses to stress events, but interactions between environmental conditions and insect borne plant pathogens remain poorly understood. We tested the hypothesis that drought stress, as applied by experimental water deprivation, drives conditional outcomes in host–pathogen and host–vector interactions using a cereal–aphid–virus association and greenhouse experiments. Under conditions of ample water supply, infection of wheat plants with Barley yellow dwarf virus (BYDV) resulted in reduced above‐ground growth, seed set, seed yields and seed germination compared with plants exposed only to non‐infected (non‐viruliferous) aphids or control plants not subjected to aphid infestation. However, when water was chronically limiting, infection with Barley yellow dwarf virus did not significantly affect plant performance. When wheat was subjected to acute drought stress, plants infected with Barley yellow dwarf virus surpassed both control plants and plants exposed to non‐infected aphids in all measured performance traits. Feeding experiments with aphid vectors (Rhopalosiphum padi) and subsequent life table analysis revealed that aphid fecundity improved by 47% when feeding on Barley yellow dwarf virus‐infected plants when water inputs were chronically low. However, when plants received ample water, aphid fecundity was enhanced by only 23% from feeding on BYDV‐infected plants. Synthesis and applications. Collectively, our experiments suggest that wheat– Barley yellow dwarf virus interactions shift along gradients of water stress severity and duration. When Barley yellow dwarf virus infection preceded water deprivation, plant performance was not reduced from virus infection, and infected plants recovered from severe stress events more readily than non‐infected plants. However, vector–pathogen mutualism resulting in enhanced reproduction of aphids on virus‐infected plants is likely to amplify direct plant injury from herbivory in the field. Our findings indicate that during periods of drought, management of Barley yellow dwarf virus infection may not be needed and infection could benefit wheat under conditions of acute water stress.

The barley yellow dwarf virus (BYDV) of cereals is thought to substantially increase the high-temperature tolerance of its aphid vector, Rhopalosiphum padi, which may enhance its transmission efficiency. This is based on experiments with North American strains of BYDV and R. padi. Here, we independently test these by measuring the temperature tolerance, via Critical Thermal Maximum (CTmax) and knockdown time, of Australian R. padi infected with a local BYDV isolate. We further consider the interaction between BYDV transmission, the primary endosymbiont of R. padi (Buchnera aphidicola), and a transinfected secondary endosymbiont (Rickettsiella viridis) which reduces the thermotolerance of other aphid species. We failed to find an increase in tolerance to high temperatures in BYDV-infected aphids or an impact of Rickettsiella on thermotolerance. However, BYDV interacted with R. padi endosymbionts in unexpected ways, suppressing the density of Buchnera and Rickettsiella. BYDV density was also fourfold higher in Rickettsiella-infected aphids. Our findings indicate that BYDV does not necessarily increase the temperature tolerance of the aphid transmission vector to increase its transmission potential, at least for the genotype combinations tested here. The interactions between BYDV and Rickettsiella suggest new ways in which aphid endosymbionts may influence how BYDV spreads, which needs further testing in a field context.

Summary Barley yellow dwarf virus (BYDV), the most economically important virus of small grains, features highly specialised relationships with its aphid vectors, a plethora of novel translation mechanisms mediated by long-distance RNA interactions, and an ambiguous taxonomic status. The structural and movement proteins of BYDV that confer aphid transmission and phloem-limitation properties resemble those of the Luteoviridae, the family in which BYDV is classified. In contrast, many genes and cis-acting signals involved in replication and gene expression most closely resemble those of the Tombusviridae. BYDV is in genus Luteovirus, family Luteoviridae. BYDV includes at least two serotypes or viruses: BYDV-PAV and BYDV-MAV. The former BYDV-RPV is now Cereal yellow dwarf virus-RPV (CYDV-RPV). CYDV is in genus Polerovirus, family Luteoviridae. Genus Luteovirus shares many features with family Tombusviridae. Physical properties: approximately 25 nm icosahedral (T = 3) virions. One major (22 kDa) and one minor (50-55 kDa) coat protein. 5.6-5.8 kb positive sense RNA genome with no 5'-cap and no poly(A) tail. Most grasses. Most important in oats, barley and wheat. Also infects maize and rice. Yellowing and dwarfing in barley, stunting in wheat; reddening, yellowing and blasting in oats. Some isolates cause leaf notching and curling. Key attractions: Model for the study of circulative transmission of aphid-transmitted viruses. Plethora of unusual translation mechanisms. Evidence of recombination in recent evolutionary history creates taxonomic ambiguity. Economically important virus of wheat, barley and oats, worldwide. Useful websites/meetings: International symposium: 'Barley Yellow Dwarf Disease: Recent Advances and Future Strategies', CIMMYT, El Batan, Mexico, 1-5 September 2002, http://www.cimmyt.cgiar.org/Research/wheat/Conf_BYD_02/invitation.htm http://www.cimmyt.org/Research/wheat/BYDVNEWS/htm/BYDVNEWS.htm Aphid transmission animation: http://www.ppws.vt.edu/~sforza/tmv/bydv_aph.html.

Barley yellow dwarf virus is a group of positive-sense single stranded RNA viruses with many species belonging to the family Luteoviridae and known to cause significant yield losses of cereal crops worldwide (Irwin and Thresh 1990). Oat (Avena sativa L.), a temperate cereal crop usually grown as forage in subtropical regions, has been recently reintroduced into Taiwan as a local winter forage source. In April 2017, yellowing, V-shape reddening, and purple symptoms were observed on 7.9% of the mature leaves of the oat plants grown in the experimental field of National Taiwan University (NTU) in Taipei city, Taiwan. We collected 37 leaf samples with suspected virus-infection symptoms in the experimental field for virus screening. The samples were expressing varying degrees of yellowing, browning, or typical barley yellow dwarf virus (BYDV)-associated V-shape reddening and purple symptoms. An additional five samples with no symptoms were also collected. These 37 samples belong to 22 oat lines, including two registered cultivars and 20 unreleased breeding lines. Some lines were replicated in the field and therefore were sampled more than once. A specific reverse transcription polymerase chain reaction assay (RT-PCR) for BYDV, covering the coat protein (CP) gene and partial RNA-dependent RNA polymerase regions, was performed with the primers Shu-F (5′-TACGGTAAGTGCCCAACTCC-3′) and Yan-R (5′-TGTTGAGGAGTCTACCTATTTG-3′) on the extracted total RNA of the samples (Malmstrom and Shu 2004). Among the examined samples, only the three expressing typical BYDV symptoms tested positive, whereas the other 34 samples with solely yellowing or browning symptoms and the five symptomless samples tested negative. The samples belonged to the cultivar Swan and two breeding lines, 15-39 and 16-47; the seeds of the three symptomatic oats were all imported from the United States. We further cross-examined the three positive samples with another RT-PCR assay, using specific primer pairs targeting the BYDV CP gene (for strains PAV, MAV, and GAV): Luteo1F (5′-TTCGGMSARTGGTTGTGGTCCA-3′) and YanR-new (5′-TGTTGAGGAGTCTACCTATTTNG-3′) (Mustafayev et al. 2013; Svanella-Dumas et al. 2013). The electrophoresis analysis of the RT-PCR amplicons revealed the expected 545-bp fragment from all. The amplicons were later cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) for sequencing analysis. All of the sequences obtained were identical (GenBank accession nos. MF618251, MK361042, and MK361043) and showed 99% similarity to the BYDV-PAV isolates from the United States (accession nos. EF521840 and DQ115532) in GenBank. Based on the phylogenetic analysis of CP gene sequences, the BYDV-PAV Taiwan isolates and these two U.S. isolates were in the same cluster. To further confirm the results, the BYDV-PAV specific enzyme-linked immunosorbent assay on the three symptomatic samples, using the commercialized kit (Agdia, Elkhart, IN), also aligned with the prior assay and showed positive reaction results. To our knowledge, this is the first report of BYDV-PAV in Taiwan. The phylogeny analysis of the virus suggests the BYDV-PAV might have been introduced through the seeds, which had gone through one cycle of multiplication at the border between Idaho and Oregon states prior to growing in Taiwan. Because the BYDV infection on oat presented a threat to cereal crop production in Taiwan, all oat plants in the NTU experimental field were eliminated immediately to prevent the spread of the disease. Agricultural agencies were informed of the incidence, and oat growers were educated to identify the BYDV symptoms on oat. Further field surveys of BYDV on the alleged vector insects will be carried to better understand the disease biology in Taiwan and to provide information on the disease system in the Southeast Asia region.

BackgroundPlant viruses maintain intricate interactions with their vector and non-vector insects and can impact the fitness of insects. However, the details of their molecular and cellular mechanisms have not been studied well. We compared the transcriptome-level responses in vector and non-vector aphids (Schizaphis graminum and Rhopalosiphum padi, respectively) after feeding on wheat plants with viral infections (Barley Yellow Dwarf Virus (BYDV) and Wheat dwarf virus (WDV), respectively). We conducted differentially expressed gene (DEG) annotation analyses and observed DEGs related to immune pathway, growth, development, and reproduction. And we conducted cloning and bioinformatic analyses of the key DEG involved in immune.ResultsFor all differentially expressed gene analyses, the numbers of DEGs related to immune, growth, development, reproduction and cuticle were higher in vector aphids than in non-vector aphids. STAT5B (signal transducer and activator of transcription 5B), which is involved in the JAK-STAT pathway, was upregulated in R. padi exposed to WDV. The cloning and bioinformatic results indicated that the RpSTAT5B sequence contains a 2082 bp ORF encoding 693 amino acids. The protein molecular weight is 79.1 kD and pI is 8.13. Analysis indicated that RpSTAT5B is a non-transmembrane protein and a non-secreted protein. Homology and evolutionary analysis indicated that RpSTAT5B was closely related to R. maidis.ConclusionsUnigene expression analysis showed that the total number of differentially expressed genes (DEGs) in the vector aphids was higher than that in the non-vector aphids. Functional enrichment analysis showed that the DEGs related to immunity, growth and reproduction in vector aphids were higher than those in non-vector aphids, and the differentially expressed genes related to immune were up-regulated. This study provides a basis for the evaluation of the response mechanisms of vector/non-vector insects to plant viruses.

SUMMARYThe control of barley yellow dwarf virus (BYDV) and its aphid vectors in minimum tillage (MT) and conventionally tilled (CT) winter barley by insecticide seed and foliar spray treatments was investigated in 2001, 2002 and 2003. Similar investigations were undertaken on winter wheat in 2004, 2005 and 2006. Aphids numbers in autumn and BYDV in spring on barley and wheat were significantly lower on MT relative to CT crops, in two of the six seasons. An insecticide spray at Zadoks growth stage (GS) 25 significantly reduced aphids and virus in both MT and CT crops in three of the six seasons of the study. An additional spray at GS 22 provided no benefit. Aphids were significantly fewer in three of the six seasons on crops grown from insecticide treated seeds, relative to untreated seeds. Both MT and CT barley sprayed at GS 25 had significantly fewer aphids than the seed treatment in one of the three seasons. Seed-treated MT and CT barley had significantly less BYDV than controls but significantly more than crops sprayed at GS 25. CT wheat grown from insecticide-treated seed had significantly less BYDV than controls. Overall, CT barley grown from insecticide-treated seed had 6-fold more BYDV than the sprayed crop, while untreated barley had 22-fold more than the spray treatment. In MT barley, the comparable values were 3- and 10-fold respectively. BYDV was almost exclusively the MAV strain. The grain yield for insecticide-sprayed CT barley was significantly greater in two of three seasons than that for untreated plots. In general, MT and CT barley receiving an insecticide spray had greater grain yield than barley grown from insecticide-treated seed, with differences being significant in one of three seasons. It is concluded that BYDV in MT and CT cereals is better controlled by applying a pyrethroid insecticide spray between GS 23 and 25, in autumn, than by treating the seed with a nitroguanidine-type insecticide. In MT crops, a single spray between GS 23 and 25 will give effective control of MAV-type BYDV.

Reliable detection and quantification of barley and cereal yellow dwarf viruses (YDVs) is a critical component in managing yellow dwarf diseases in small grain cereal crops. The method currently used is enzyme-linked immunosorbent assay (ELISA), using antisera against the coat proteins that are specific for each of the various YDVs. Recently, quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR) has been used to detect bacterial and viral pathogens and to study gene expression. We applied this technique to detect and quantify YDVs using primers specific for Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) coat protein genes because of the higher sensitivity of RT-PCR and the advantage of using a real-time PCR instrument. This Q-RT-PCR was used to detect BYDV and CYDV, and to examine disease development in a resistant wheatgrass, a resistant wheat line, a susceptible wheat line, and a susceptible oat line. BYDV-PAV and CYDV-RPV were detected as early as 2 and 6 h, respectively, in susceptible oat compared with detection by ELISA at 4 and 10 days postinoculation. BYDV-PAV RNA accumulated more rapidly and to a higher level than CYDV-RPV RNA in both oat and wheat, which may account for PAV being more prevalent and causing more severe viral disease than CYDV. Q-RT-PCR is reproducible, sensitive, and has the potential to be used for examining yellow dwarf disease and as a rapid diagnostic tool for YDVs.

The Median Latent Periods for Three Isolates of Barley Yellow Dwarf Virus in Aphid Vectors

A systematic search for viral infection was performed in the isolated Kerguelen Islands, using a range of polyvalent genus-specific PCR assays. Barley yellow dwarf virus (BYDV) was detected in both introduced and native grasses such as Poa cookii. The geographical distribution of BYDV and its prevalence in P. cookii were analyzed using samples collected from various sites of the archipelago. We estimate the average prevalence of BYDV to be 24.9% in P. cookii, with significant variability between sites. BYDV genetic diversity was assessed using sequence information from two genomic regions: the P3 open reading frame (ORF) (encoding the coat protein) and the hypervariable P6 ORF region. The phylogenetic analysis in the P3 region showed that BYDV sequences segregate into three major lineages, the most frequent of which (Ker-I cluster) showed close homology with BYDV-PAV-I isolates and had very low intra-lineage diversity (0.6%). A similarly low diversity was also recorded in the hypervariable P6 region, suggesting that Ker-I isolates derive from the recent introduction of BYDV-PAV-I. Divergence time estimation suggests that BYDV-PAV-I was likely introduced in the Kerguelen environment at the same time frame as its aphid vector, Rhopalosiphum padi, whose distribution shows good overlap with that of BYDV-Ker-I. The two other lineages show more than 22% amino acid divergence in the P3 region with other known species in the BYDV species complex, indicating that they represent distinct BYDV species. Using species-specific amplification primers, the distribution of these novel species was analyzed. The high prevalence of BYDV on native Poaceae and the presence of the vector R. padi, raises the question of its impact on the vulnerable plant communities of this remote ecosystem.

RNAinterference (RNAi) technology is considered an alternative tool to develop more environmentally friendly broad-spectrumpesticides in agriculture. In this approach, sequence-specific knockdown of gene targets in pests and pathogensusing double-strandedRNA(dsRNA) is utilized. Two different dsRNAapplicationmethods, host induced gene silencing (HIGS) and spray induced gene silencing (SIGS) are being followed. HIGS involves developing transgenic plants that produce the intended dsRNA which will be delivered into the pests when they feed or grow on the transgenic plants, while in SIGS the dsRNAs applied topically on the plants will be taken up by the target organisms. Once the dsRNA is in the target organism, the host RNAi cellular machinery will be used to silence the target genes. SIGS has been applied now against many pests and diseases in different crops and has given promising results. With the development of tools that facilitate economic production of large scale dsRNA and improve the stability and longevity of the sprayed dsRNAs on the plant surface, SIGS is a promising technology that could be adopted across crops and against different pests and pathogens. In this research update, we provide a summary of the recent developments in the area of SIGS with an emphasis on the examples of fungal pathogen control.