Here we describe two Brome mosaic virus (BMV) isolates from the Wielkopolska region of Poland. The BMV-Sr and BMV-Sz isolates were collected from maize [Zea mays L. ssp. Indentata] and triticale [× Triticosecale Wittm. ex A. Camus] plants respectively. Newly emerged BMV isolates, similarly to the BMV-M2 strain derived from an Arkansas isolate, have a wide host range that includes species in the Poaceae and Fabaceae families. Furthermore, immunological reactions of icosahedral virions from non-inoculated cowpea [Vigna unguiculata L.] leaves with a specific immunoglobulin confirmed that each of these isolates can systemically infect cowpea. We characterized the BMV-Sr and BMV-Sz genomic sequences encoding the replication protein (1a), polymerase (2a), complete movement protein (3a) and coat protein (CP) genes. The 1a, 2a, 3a and CP gene sequences showed 98.9, 97.6, 98.4 and 97.7 % nucleotide sequence identity between the Polish isolates respectively. Phylogenetic analysis based on these four genes confirmed the species identity of the isolates. A phylogenetic tree based on RNA3 (3a and CP genes) showed independent clustering of the Polish isolates. Amino acid sequence analysis of the 3a protein revealed that both Polish isolates are characterized by a single amino acid mutation at the 81st position, when compared with the Russian isolate. It had been previously reported that four amino acid mutations in this region determined BMV systemic infection in cowpea. Our results indicate that a single non-synonymous substitution in the cell-to-cell movement protein may be crucial for the nature of cowpea infection.

Wheat dwarf virus (WDV) is considered as one of the most common viruses on cereal crops. Recently, severe outbreaks of WDV have been observed especially on winter wheat in southwestern part of Poland. Moreover, the presence of genetically different WDV-barley-specific and WDV-wheat-specific forms (WDV-B and WDV-W, respectively) was confirmed. In this study, a loop-mediated isothermal amplification assay (LAMP) was developed for the first time for efficient and rapid detection of WDV-B and WDV-W in infected plants. The reaction was performed using a set of three primer pairs: WDVF3/WDVB3, WDVFIB/WDVBIP and WDVLoopF/WDVLoopB specific for coat protein coding sequence. The amplified products were analyzed by direct staining of DNA, gel electrophoresis and real-time monitoring of the amplification curves. The sensitivity of optimized reaction was tenfold higher in comparison with conventional PCR. LAMP assay developed here is a useful and practical method for the rapid detection of different WDV isolates and can be implemented by phytosanitary services.

From 2005 to 2007 in Southern Wielkopolska, Lower Silesia, and Malopolska regions, maize (Zea mays) plants showing leaf mosaic and stunting symptoms were found. ELISA tests using commercial polyclonal antisera against Maize dwarf mosaic virus (MDMV) obtained from Bioreba (Basel, Switzerland) and Loewe (Munich, Germany) gave positive results in 71 samples. However, the ELISA response for symptomatic plants, in most cases, was low, with OD values ranging from 0.05 to 0.18. Therefore, only eight plants with relatively high virus concentration were chosen for further identification assays. Examination of leaf extracts with an electron microscope revealed the presence of potyvirus-like particles. Symptomatic leaves were positive for MDMV by using immunosorbent electron microscopy (ISEM) with antiserum raised against the Spanish isolate of MDMV (supplied as positive MDMV control from A. Achon, Centre Vdl-Irta, Lleida, Spain). A set of test plants, including sweet corn, dent corn, sorghum (Sorghum vulgare), and true millet (Panicum miliaceum), were mechanically inoculated with extracts from symptomatic plants in 0.05 M phosphate buffer plus 1% β-mercaptoethanol. Inoculated plants developed symptoms typical of MDMV in 2 to 5 weeks (1,2). For further investigations, three virus isolates were chosen. To confirm the identification of MDMV, reverse transcription (RT)-PCR was performed with total RNA isolated from infected plants with primers 3MDF (5′ GAT GAG TTR AAY GTY TAT GCA CGA C 3′), a forward primer in the 3′ region of NIb gene and either 1MDR (5′ RTG CAT RAT TTG TCT GAA AGT TGG 3′) or 3MDR (5′ ACC AVA CCA TYA TWC CAC TC 3′), reverse primers in the 3′ region of the coat protein gene (A. Zare, Shiraz University, personal communication). 3MDF corresponds to nucleotides 8306 to 8330, 3MDR is complementary to nucleotides 8791 to 8813, and 1MDR is complementary to nucleotides 8917 to 8939 of the MDMV genome (GenBank Accession No. AJ001691). The RT-PCR products obtained were analyzed by agarose gel electrophoresis. Amplicons of the expected sizes (635 and 560 bp) were obtained with RNA from symptomatic plants, but not from asymptomatic plants. The sequence of the 576-bp PCR product was deposited in GenBank (Accession No. EU240460). In alignments done with BlastN ( www.ncbi.nlm.nih.gov/blast ), the highest nucleotide sequence identities were 99% with Spanish MDMV isolates (“SP” AM110758, “SP” AJ416645, and “S1” AJ416635), 91% with the Hungarian isolate “Sc/H, sweet corn” AJ542536, 90% with “MDMV-A” U07216, and 87% with an Israeli MDMV (AF395135). On the basis of these findings, the virus isolated from diseased maize plants was identified as MDMV. The significance of MDMV detection is noteworthy because maize has become an important crop in Poland in recent years and acreage is increasing systematically. References: (1) M. A. Achon et al. Eur. J. Plant Pathol. 102:697, 1996. (2) A. J. Gibbs. Maize dwarf mosaic virus. Page 752 in: Viruses of Plants. Descriptions and Lists from the VIDE database. A. A. Brunt et al., eds. CAB International, Wallingford, UK, 1996.

Barley yellow dwarf is a threat to cereal crops worldwide. Barley yellow dwarf virus—PAS (BYDV-PAS) was detected for the first time in Poland in 2015, then in 2019. In the spring of 2021, in several locations in Poland, winter wheat and barley plants with dwarfism and leaf yellowing were collected. Reverse transcription—polymerase chain reaction results revealed BYDV presence in 47 samples and excluded wheat streak mosaic virus infections. Next, immuno-captured polymerase chain reactions confirmed only one case of co-infection caused by BYDV and wheat dwarf virus. Moreover, restriction fragment length polymorphism analysis showed that BYDV-PAS was predominant. The preliminary results were confirmed using sequencing. Infected cereal plants originated mainly from northwestern Poland. The complete coding sequence of coat protein (CP) and a fragment of RNA-dependent RNA polymerase (RdRp) genes of 14 Polish isolates were determined and deposited in the GenBank database. The nucleotide and deduced amino acid sequences of local isolates were compared with others reported to date, indicating their high similarity, from 75.4% to 99.5% and from 81.1% to 100% nucleotide sequence identity, in RdRp and CP, respectively. Phylogenetic analysis, based on the CP gene, revealed the presence of 3 main groups. The Polish isolates clustered together within the Ia group.

In the spring of 2019, many plants, mainly winter wheat, were observed to have dwarfism and leaf yellowing symptoms. These plants from several regions of Poland were collected and sent to the Plant Disease Clinic of the Institute of Plant Protection-National Research Institute in Poznań to test for the presence of viral diseases. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) results showed numerous cases of Wheat dwarf virus (WDV) and a few cases of plant infections caused by Barley yellow dwarf viruses (BYDVs). WDV was detected in 163 out of 236 tested winter wheat plants (69.1%), in 10 out of 27 tested winter barley plants (37%) and in 6 out of 7 triticale plants (85.7%) while BYDVs were found, respectively, in 9.7% (23 out of 236) and in 18.5% (5 out of 27) of tested winter forms of wheat and barley plants. Infected plants came mainly from the regions of Lower Silesia and Greater Poland. Furthermore, individual cases of infections were also confirmed in the following districts: Lubusz, Opole, Silesia, Kuyavia-Pomerania and Warmia-Masuria. Results of Duplex-immunocapture-polymerase chain reaction (Duplex-IC-PCR) indicated the dominance of WDV-W form in wheat and WDV-B form in barley plants. Moreover, results of reverse transcription-polymerase chain reaction (RT-PCR) connected with restriction fragment length polymorphism (RFLP) analysis, performed for 17 BYDVs samples, revealed 8 BYDV-PAS, 4 BYDV-MAV and 2 BYDV-PAV as well as the presence of two mixed infections of BYDV-MAV/-PAS and one case of BYDV-MAV/-PAV. Next, RT-PCR reactions confirmed single BYDV-GAV infection and the common presence of BYDV-SGV. To the best of our knowledge, in 2020 the viruses were not a big threat to cereal crops in Poland.