Both-strand gene coding in a plastome-like mitogenome of an enoplid nematode.

The phylum Nematoda remains very poorly sampled for mtDNA, with a strong bias toward parasitic, economically important or model species of the Chromadoria lineage. Most chromadorian mitogenomes share a specific order of genes encoded on one mtDNA strand. However, the few sequenced representatives of the Dorylaimia lineage exhibit a variable order of mtDNA genes encoded on both strands. While the ancestral arrangement of nematode mitogenome remains undefined, no evidence has been reported for Enoplia, the phylum's third early divergent major lineage. We describe the first mitogenome of an enoplian nematode, Campydora demonstrans, and contend that the complete 37-gene repertoire and both-strand gene encoding are ancestral states preserved in Enoplia and Dorylaimia versus the derived mitogenome arrangement in some Chromadoria. The C. demonstrans mitogenome is 17,018 bp in size and contains a noncoding perfect inverted repeat with 2013 bp-long arms, subdividing the mitogenome into two coding regions. This mtDNA arrangement is very rare among animals and instead resembles that of chloroplast genomes in land plants. Our report broadens mtDNA taxonomic sampling of the phylum Nematoda and adds support to the applicability of cox1 gene as a phylogenetic marker for establishing nematode relationships within higher taxa.

Expression of Hairpin-Enriched Mitochondrial DNA in Two Hairworm Species (Nematomorpha).

Nematomorpha (hairworms) is a phylum of parasitic ecdysozoans, best known for infecting arthropods and guiding their hosts toward water, where the parasite can complete its life cycle. Over 350 species of nematomorphs have been described, yet molecular data for the group remain scarce. The few available mitochondrial genomes of nematomorphs are enriched with long inverted repeats, which are embedded in the coding sequences of their genes-a remarkably unusual feature exclusive to this phylum. Here, we obtain and annotate the repeats in the mitochondrial genome of another nematomorph species-Parachordodes pustulosus. Using genomic and transcriptomic libraries, we investigate the impact of inverted repeats on the read coverage of the mitochondrial genome. Pronounced drops in the read coverage coincide with regions containing long inverted repeats, denoting the 'blind spots' of short-fragment sequencing libraries. Phylogenetic inference with the novel data reveals multiple disagreements between the traditional system of Nematomorpha and molecular data, rendering several genera paraphyletic, including Parachordodes.

Prevalence of Recombinant Strains of Potato Virus Y in Seed Potato Planted in Idaho and Washington States Between 2011 and 2021.

Potato virus Y (PVY) has emerged as the main reason for potato seed lot rejections, seriously affecting seed potato production in the United States throughout the past 20 years. The dynamics of PVY strain abundance and composition in various potato growing areas of the United States has not been well documented or understood up to now. The objective of this study was to find out the prevalence of PVY strains in potato fields in the Pacific Northwest (PNW), including seed potato production systems in the State of Idaho and commercial potato fields in the Columbia Basin of Washington State between 2011 and 2021. Based on the testing of >10,000 foliar samples during Idaho seed certification winter grow-out evaluations of seed potato lots and seed lot trials in Washington State, a dramatic shift in the PVY strain composition was revealed in the PNW between 2011 and 2016. During this time period, the prevalence of the ordinary, PVYO strain in seed potato dropped 8- to 10-fold, concomitantly with the rise of recombinant strains PVYN-Wi and PVYNTNa, which together accounted for 98% of all PVY positives by 2021. In Idaho seed potato, PVYNTNa strain associated with the potato tuber necrotic ringspot disease (PTNRD) was found to increase threefold between 2011 and 2019, accounting for 24% of all PVY positives in 2019. Mild foliar symptoms induced by recombinant PVY strains may be partially responsible for the proliferation of PVYN-Wi and PVYNTNa in potato crops. A spike of another PTNRD-associated recombinant, PVY-NE11, was recorded in the PNW between 2012 and 2016, but after reaching a 7 to 10% level in 2012 to 2013 this recombinant disappeared from the PNW potato by 2019. Whole genome sequence analysis of the PVY-NE11 suggested this recombinant was introduced in the United States at least three times. The data on PVY strain abundance in the PNW potato crops suggest that virus management strategies must consider the current dominance of the two recombinant PVY strains, PVYN-Wi and PVYNTNa.

Strain-Specific Resistance to Potato virus Y (PVY) in Potato and Its Effect on the Relative Abundance of PVY Strains in Commercial Potato Fields.

Potato virus Y (PVY) is a serious threat to potato production due to effects on tuber yield and quality, in particular, due to induction of potato tuber necrotic ringspot disease (PTNRD), typically associated with recombinant strains of PVY. These recombinant strains have been spreading in the United States for the past several years, although the reasons for this continuing spread remained unclear. To document and assess this spread between 2011 and 2015, strain composition of PVY isolates circulating in the Columbia Basin potato production area was determined from hundreds of seed lots of various cultivars. The proportion of nonrecombinant PVYO isolates circulating in Columbia Basin potato dropped ninefold during this period, from 63% of all PVY-positive plants in 2011 to less than 7% in 2015. This drop in PVYO was concomitant with the rise of the recombinant PVYN-Wi strain incidence, from less than 27% of all PVY-positive plants in 2011 to 53% in 2015. The proportion of the PVYNTN recombinant strain, associated with PTNRD symptoms in susceptible cultivars, increased from 7% in 2011 to approximately 24% in 2015. To further address the shift in strain abundance, screenhouse experiments were conducted and revealed that three of the four most popular potato cultivars grown in the Columbia Basin exhibited strain-specific resistance against PVYO. Reduced levels of systemic movement of PVYO in such cultivars would favor spread of recombinant strains in the field. The negative selection against the nonrecombinant PVYO strain is likely caused by the presence of the Nytbr gene identified in potato cultivars in laboratory experiments. Presence of strain-specific resistance genes in potato cultivars may represent the driving force changing PVY strain composition to predominantly recombinant strains in potato production areas.

Biological and molecular characterization of a tomato isolate of potato virus Y (PVY) of the PVYC lineage.

An isolate of potato virus Y (PVY), PVY-H14, was collected on the island of Oahu, Hawaii, from tomato plants exhibiting stunting and necrotic lesions on leaves. PVY-H14 triggered the hypersensitive resistance response in potato cultivars King Edward and Maris Bard, typical of a PVYC strain, and was unable to infect systemically the four tested cultivars, Desiree, Maris Bard, King Edward, and Russet Norkotah. Phylogenetic analysis of H14 and the whole genomes of 31 PVY isolates of non-recombinant strains of PVY placed PVY-H14 in the same clade with PVYC and several unclassified PVY isolates from tomato and tobacco.

Recombinants of bean common mosaic virus (BCMV) and genetic determinants of BCMV involved in overcoming resistance in common bean.

Bean common mosaic virus (BCMV) exists as a complex of strains classified by reactions to resistance genes found in common bean (Phaseolus vulgaris); seven BCMV pathotypes have been distinguished thus far, numbered I to VII. Virus genetic determinants involved in pathogenicity interactions with resistance genes have not yet been identified. Here, we describe the characterization of two novel field isolates of BCMV that helped to narrow down these genetic determinants interacting with specific P. vulgaris resistance factors. Based on a biological characterization on common bean differentials, both isolates were classified as belonging to pathotype VII, similar to control isolate US10, and both isolates exhibited the B serotype. The whole genome was sequenced for both isolates and found to be 98 to 99% identical to the BCMV isolate RU1 (pathotype VI), and a single name was retained: BCMV RU1-OR. To identify a genetic determinant of BCMV linked to the BCMV pathotype VII, the whole genome was also sequenced for two control isolates, US10 and RU1-P. Inspection of the nucleotide sequences for BCMV RU1-OR and US10 (both pathotype VII) and three closely related sequences of BCMV (RU1-P, RU1-D, and RU1-W, all pathotype VI) revealed that RU1-OR originated through a series of recombination events between US10 and an as-yet-unidentified BCMV parental genome, resulting in changes in virus pathology. The data obtained suggest that a fragment of the RU1-OR genome between positions 723 and 1,961 nucleotides that is common to US10 and RU1-OR in the P1-HC-Pro region of the BCMV genome may be responsible for the ability to overcome resistance in bean conferred by the bc-2(2) gene. This is the first report of a virus genetic determinant responsible for overcoming a specific BCMV resistance gene in common bean.

Molecular and Serological Typing of Potato virus Y Isolates from Brazil Reveals a Diverse Set of Recombinant Strains.

In Brazil, Potato virus Y (PVY) currently presents a significant problem for potato production, reducing tuber yield and quality. Recombinant tuber necrotic isolates of PVY had been reported to occur in the country but no systematic study of the PVY isolate diversity was conducted thus far. Here, a panel of 36 PVY isolates, randomly collected in Brazil from potato between 1985 and 2009, was subjected to a systematic molecular and serological typing using reverse-transcription polymerase chain reaction and a series of PVYO- and PVYN-specific monoclonal antibodies. The data collected were combined with biological characterization of the same isolates in tobacco. Of the 36 isolates tested, 3 were typed as PVYO, 10 as PVYN:O/N-Wi, 21 as PVYNTN, and 2 as "unusual" or inconclusive. Of the 10 isolates from the recombinant PVYN:O/N-Wi strain group, 1 isolate, MAF-VOY, was found to have an unusual serological profile identical to the nonrecombinant PVYO-O5 strain group. The 21 tested PVYNTN isolates included 1 isolate that did not induce vein necrosis in tobacco and 2 isolates with an unusual serological profile (i.e., displaying negative reactivity to one commercial PVYN-specific monoclonal antibody). Whole genome sequences were determined for four PVY isolates from Brazil, representing PVYO, PVYNTN, and PVYN-Wi strains. The genome of the MAF-VOY isolate was found to be recombinant, having characteristic N-Wi structure with two recombinant junctions and carrying a single mutation in the capsid protein at position 98, which led to an unusual O5 serological reactivity. Taken together, the data obtained suggest that the two recombinant strains, PVYNTN and PVYN:O/N-Wi, now are apparently dominant in the Brazilian potato crop. The data also suggest that recombinant isolates in Brazil often have unusual serological reactivity which may hamper their correct identification by conventional typing based on enzyme-linked immunosorbent assay.

Identification of the molecular make-up of the Potato virus Y strain PVY(Z): genetic typing of PVY(Z)-NTN.

Potato virus Y (PVY) strains were originally defined by interactions with different resistance genes in standard potato cultivars. Five distinct strain groups are defined that cause local or systemic hypersensitive responses (HRs) in genetic background with a corresponding N gene: PVY(O), PVY(N), PVY(C), PVY(Z), and PVY(E). The nucleotide sequences of multiple isolates of PVY(O) and PVY(N) differ from each other by ≈8% along their genomes. Additionally, complete genome sequences of multiple recombinant isolates are composed of segments of parental PVY(O) and PVY(N) sequences. Here, we report that recombinant isolate PVY-L26 induces an HR in potato 'Maris Bard' carrying the putative Nz gene, and is not recognized by two other resistance genes, Nc and Ny(tbr). These genetic responses in potato, combined with the inability of PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate from the PVY(Z) strain group and provide the first information on genome structure and sequence of PVY(Z). The genome of PVY-L26 displays typical features of European NTN-type isolates with three recombinant junctions (PVY(EU-NTN)), and the PVY-L26 is named PVY(Z)-NTN. Three typical PVY(NTN) isolates and two PVY(N) isolates, all inducing vein necrosis in tobacco, were compared with PVY-L26. One PVY(NTN) isolate elicited HR reactions in Maris Bard, similar to PVY-L26, while two induced a severe systemic HR-like reaction quite different from the quasi-symptomless reaction induced by two PVY(N) isolates. 'Yukon Gold' potato from North America produced HR against several PVY(NTN) isolates, including PVY-L26, but only late and limited systemic necrosis against one PVY(N) isolate. Consequently, according to symptoms in potato indicators, both PVY(Z) and PVY(NTN) isolates appeared biologically very close and clearly distinct from PVY(O) and PVY(N) strain groups.

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains.

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.

Immunodetection of Two Curtoviruses Infecting Sugar Beet.

Beet leafhopper-transmitted curly top virus is a serious problem in many different crops in the semiarid western United States, including sugar beet, tomatoes, and beans. Curly top is caused by a genetically diverse complex of phloem-limited curtoviruses. Due to the phloem restriction of curtoviruses and the lack of a convenient laboratory host-vector system for curly top virus propagation and purification, no commercial immunodetection tests are available for curtoviruses. Routine diagnostics for curly top rely either on visual symptoms or on polymerase chain reaction (PCR) tests. Lack of an enzyme-linked immunosorbent assay (ELISA) system is one of the factors hampering development and screening of the curly top resistant germplasm in, for instance, sugar beet and bean breeding programs. To fill in this gap, we developed an ELISA-based detection system for curtoviruses which utilizes virus-specific antibodies generated against bacterially expressed capsid protein (CP) of Beet mild curly top virus. Bacterially expressed CP was affinity purified and used as an antigen for antibody production in two animal species. Specificity of the resulting antisera was tested in Western blots and various triple-antibody sandwich (TAS)-ELISA formats with sugar beet, bean, and Nicotiana benthamiana leaf tissue. We demonstrate reliable detection of two curtoviruses in different crops in TAS-ELISA format, suitable for large-scale screening of germplasm in breeding programs.

Inhibition of host ER glucosidase activity prevents Golgi processing of virion-associated bovine viral diarrhea virus E2 glycoproteins and reduces infectivity of secreted virions.

Recently, it was shown that replication of bovine viral diarrhea virus (BVDV) is sensitive to inhibitors of host ER glucosidases. Consistent with these findings, we report that incubation of BVDV-infected MDBK cells with the glucosidase inhibitor n-butyl-deoxynojirimycin (nB-DNJ) reduced BVDV yields by 70- to 100-fold (n = 27), while having no effect on MDBK cell viability. However, the 70- to 100-fold reduction in infectious virus was associated with only a 2-fold reduction in genomic RNA synthesis and secretion of enveloped virus particles. Analysis of secreted virions showed that in the absence of glucosidase inhibitor, approximately 50% of the virion-associated BVDV E2 glycoprotein was resistant to endoglycosidase H (endo H) digestion, whereas intracellular E2 was completely sensitive to endo H digestion. In the presence of glucosidase inhibitor, virion-associated E2 and intracellular E2 were completely sensitive to endo H digestion. Taken together, these results suggest that BVDV is secreted through a Golgi-mediated pathway and that host ER glucosidase activity is required for production of infectious virions and Golgi processing of envelope E2 protein during virus egress.

Serological Differentiation of the Citrus Tristeza Virus Isolates Causing Stem Pitting in Sweet Orange.

Citrus tristeza virus (CTV) complex comprises a number of isolates or strains producing several economically important disease syndromes in commercial Citrus spp. The stem pitting syndrome is the most important, and causes substantial losses in many citrus-producing regions of the world. In an attempt to develop a serological tool to rapidly differentiate stem pitting isolates of CTV, we evaluated many combinations of trapping and detecting antibodies in an indirect double-antibody sandwich (I-DAS) enzyme-linked immunosorbent assay (ELISA). Two combinations of trapping and detecting antibodies were found suitable for differentiating stem pitting isolates in extracts of infected sweet orange plants. One used a polyclonal serum raised against bacterially expressed CTV coat protein (CP) for trapping and a conformational monoclonal antibody 3E10 for detection, and the other used two polyclonal antisera generated against bacterially expressed CTV CP. Seventy-six CTV isolates from 20 countries, including 35 that cause stem pitting in sweet orange plants, were analyzed in I-DAS-ELISA using different combinations of polyclonal and monoclonal antibodies for trapping and as intermediate detecting antibodies. The ELISA format developed produces a strong positive signal for CTV isolates that cause stem pitting in sweet orange plants and a negative ELISA signal for CTV isolates that do not cause stem pitting. When combined with data on a universal ELISA format, i.e., reacting with a broad range of CTV isolates, these selective ELISA formats allowed reliable serological differentiation of CTV isolates that caused stem pitting in infected sweet orange plants.