Wheat streak mosaic virus： incidence in field crops, potential reservoir within grass species and uptake in winter wheat cultivars

Wheat streak mosaic virus（WSMV）has become a re-emerging pathogen in cereal crops in the Czech Republic.WSMV was first reported in the former Czechoslovakia in the early 1980s,and then no record of the virus was documented until 2009.The incidence of the virus was recorded in recent years in several winter wheat fields and many grass species.Here,we surveyed the incidence of WSMV in cereal crops.The results demonstrated the existence of the virus in winter wheat and volunteer wheat during each year of the monitoring period,which spanned from 2013–2016.Although the range of infected samples was low（6.4%of the total tested samples）,a high incidence of well-distributed virus was recorded.In at least six fields,the virus reached severe and potentially epidemic levels.In accordance with our previous report detailing WSMV infection of native grasses,we tested several grass species commonly grown in the Czech Republic.We found that some grass species acted as experimental hosts and possible reservoirs of the virus;these included Anthoxanthum odoratum（sweet vernal grass）,Arrhenatherum elatius（false oat-grass）,Lolium multiflorum（Italian rye-grass）,Bromus japonicus（Japanese chess）,Echinochloa crus-galli（barnyard grass）,Holcus lanatus（meadow soft grass）and Holcus mollis（creeping soft grass）.Some of these grass species are also important weeds of cereals,which may be the potential source of WSMV infection in cereal crops.Several widely used winter wheat cultivars were tested in the field after artificial inoculation with WSMV to evaluate virus titre by RT-qPCR.Overall,the tested cultivars had a low virus titre,which is associated with mild disease symptoms and may provide a good level of crop resistance to WSMV.

Molecular diversity of barley yellow dwarf virus-PAV from China and the Czech Republic

Wheat yellow dwarf disease(BYD),caused by different species of barley/cereal yellow dwarf viruses(B/CYDVs),is one of the most serious cereal diseases in China and the Czech Republic.Because genetic diversity of the virus directly influences disease epidemiology,the molecular diversity and population structure of 24 Chinese isolates and 16 the Czech Republic isolates of BYDV-PAV from different regions in two countries were analyzed by sequencing their coat protein(CP)and readthrough protein(RTP)domain(RTD)genes and comparing the sequences with six CP and 16 RTP sequences of BYDVPAV isolates from the NCBI database based on nucleotide identity position,phylogenetic analysis and nucleotide diversity.Nucleotide identities between the Chinese and the Czech Republic isolates for the CP were 76.6–99.4%,73.9–89.1%for RTD(ORF5),respectively.The Chinese and the other country isolates showed 74.7–99.2%nucleotide identity for RTP(ORF3+ORF5).Phylogenetic analysis of CP sequences showed that 20 Chinese isolates clustered in the same clade,but the other four Chinese isolates clustered in another clade with the isolates from the Czech Republic and other counties.The population of BYDV-PAV in China had greater nucleotide variability and was more divergent than that in the Czech Republic.Geographical and ecological factors but not hosts might contribute to the population differences in the two countries.

Virome identification in wheat in the Czech Republic using small RNA deep sequencing

High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA(sRNA)population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample.In the present sRNA deep-sequencing analysis of virus-infected wheat samples in the Czech Republic,samples were firstly tested for barley yellow dwarf viruses(BYDVs),wheat streak mosaic virus(WSMV)and wheat dwarf virus(WDV)using ELISA,RT-PCR and PCR.Subsequent sRNA sequencing of these samples yielded more than^60 million single-end 50-bp reads with high confidence for nine field samples of wheat.Overall,16.5%of reads were virus-specific and 83.5%were mapped to the host.More 21-nt reads(-7.7E+06 reads)were found than 24-nt(~6.20E+06 reads)or 22-nt(-4.30E+06 reads)reads.De novo assembly of the high-quality contigs revealed the presence of three earlier repoted viruses in the Czech Republic:BYDVs(31.48%),WSMV(24.23%)and WDV(2666%).We also showed the presence of cereal yellow dwarf virus(14.33%;two species CYDV-RPS and CYDV-RPV family Luteoviridae/Polerovirus)and wheat yellow dwarf virus(WYDV,3.30%;Luteoviridae).Phylogenetic analysis showed CYDV and WYDV grouped separately from BYDVs.Furthermore,several recombination breakpoints were found among the groups of yellow dwarf viruses(BYDVs,CYDV,and WYDV).Using RNA deep sequencing,we confirmed the presence of the three known viruses(BYDVs,WSMV,and WDV)and the first record of two species of CYDV and WYDV in wheat in the Czech Republic.

Quantitative analysis of the interaction of heterologous viruses with Plum pox virus in C5 HoneySweet transgenic plums

Stone fruits are an important crop in most parts of the world and are heavily challenged by several viruses including Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Apple chlorotic leaf spot virus (ACLSV). We validated the PPV resistance in C5 plum plants (commercially known as HoneySweet) grown in the Czech Republic for more than 16 years in a field trial experiment under natural environmental conditions. We quantified single (PPV-Rec) and mixed viruses (PPV-Rec+ACLSV, PPV-Rec+PDV and PPV-Rec+ACLSV+PDV) in C5 transgenic plums inoculated for the period 2016 to 2018. The accumulation of PPV-Rec was high (~5.43E+05 copies) compared with that of ACLSV (~8.70E+04 copies) in the inoculated graft of C5 transgenic plants. Leaves close to the inoculum sources showed a differential level of virus titre in single and mixed infections (~10 to ~5×10^2 copies). C5 plants with permanent virus pressure showed 103- to 105-fold fewer copies of viruses than those of the inoculated graft. We observed high accumulation of conserved miRNAs such as miR167, miR69 and miR396 in C5 plants co-infected with PPV, ACLSV and PDV that are associated with its resistance against viruses. Overall, i) C5 transgenic plums showed high resistance to PPV infection, and a low level (~32 copies) of PPV only accumulated in some grafted plants, ii) high accumulation of PPV was found in inoculated grafts in single PPV infection and mixed infections, iii) heterologous virus infection sustained by ACLSV or PDV did not suppress PPV resistance, and iv) high and low conserved microRNAs accumulated in C5 plants.

Two mutations in the truncated Rep gene RBR domain delayed the Wheat dwarf virus infection in transgenic barley plants

Wheat dwarf virus （WDV）, an important cereal pathogen, is closely related to Maize streak virus （MSV）, a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication- associated （RepA） gene （WDVRepA215） and the WDV RepA gene with a mutated retinoblastoma-related protein （RBR） interaction domain （WDVRepA215RBRre^t） were cloned into the plPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T1-generation plants infected by leafhoppers （Psammotettix alienus） fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBR^mut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBR^mut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.

Viral pathogens and plant parasitic nematode

This special focus is dedicated to three parts：i）One of the most ubiquitous viral pathogens of stone fruit tree,Plum pox virus（PPV）;ii）a re-emerging pathogen,Wheat streak mosaic virus（WSMV）of cereal crops in Central Europe;and iii）a less studied plant parasitic,cyst-forming nematode in cereal crops Heterodera avenae.