Lavender Harbors More Viruses than Previously Thought: First Report of Raspberry Ringspot Virus and Phlox Virus M in Lavandula × intermedia.

Plants of the genus Lavandula are thought to be rarely infected by viruses. To date, only alfalfa mosaic virus, cucumber mosaic virus, tobacco mosaic virus, and tomato spotted wilt virus have been reported in this host. In this study, we identified for the first time raspberry ringspot virus (RpRSV) and phlox virus M (PhlVM) in lavender using herbaceous indexing, enzyme-linked immunosorbent assay, and high-throughput sequencing. Nearly complete genome sequences for both viruses were determined. Phylogenetic and serological characterizations suggest that the obtained RpRSV isolate is a raspberry strain. A preliminary survey of 166 samples indicated RpRSV was spread only in the lavender cultivar 'Grosso', while PhlVM was detected in multiple lavender cultivars. Although RpRSV raspberry strain may have spread throughout Auckland and nearby areas in New Zealand, it is very likely restricted to the genus Lavandula or even to the cultivar 'Grosso' due to the absence or limited occurrence of the nematode vector. Interestingly, all infected lavender plants, regardless of their infection status (by RpRSV, PhlVM, or both) were asymptomatic. RpRSV is an important virus that infects horticultural crops including grapevine, cherry, berry fruits, and rose. It remains on the list of regulated pests in New Zealand. RpRSV testing is mandatory for imported Fragaria, Prunus, Ribes, Rosa, Rubus, and Vitis nursery stock and seeds for sowing, while this is not required for Lavandula importation. Our study revealed that lavender could play a role not only as a reservoir but also as an uncontrolled import pathway of viruses that pose a threat to New Zealand's primary industries.

Detection, Characterization, and Distribution of the First Case of Pepino Mosaic Virus in Aotearoa New Zealand.

Tomato (Solanum lycopersicum L., family Solanaceae) represents one of the most economically valuable horticultural crops worldwide. Tomato production is affected by numerous emerging plant viruses. We identified, for the first time in New Zealand (NZ), Pepino mosaic virus (PepMV) in greenhouse grown tomato crops using a combination of methods from electron microscopy and herbaceous indexing to RT-qPCR and high-throughput sequencing. Phylogenetic and genomic analysis of a near-complete PepMV genome determined that the detected strain belonged to the mild form of the CH2 lineage of the virus. Subsequently, a delimiting survey of PepMV was conducted, and PepMV was detected at four additional locations. PCR-derived sequences obtained from samples collected from different greenhouses and from herbaceous indicator plants were identical to the original sequence. Since PepMV has never been reported in NZ before, seed pathways are speculated to be the most likely source of entry into the country.

First report of Streptocarpus flower break virus in Streptocarpus hybrids in Aotearoa New Zealand.

Streptocarpus (Cape primrose, family Gesneriaceae) is a genus of plants native to Southern Africa commonly grown indoors for their foliage and trumpet-shaped flowers. In Aoteroa New Zealand (NZ) to date, no viruses have been reported to infect plants of the Gesneriaceae (Veerakone et al. 2015). In September 2022, a plant of Streptocarpus hybrid exhibiting necrotic rings was observed in a hobbyist's greenhouse in Auckland, NZ. High-Throughput Sequencing (HTS) using MinIONTM (Oxford Nanopore Technologies), was applied as a first screen (Liefting et al. 2021). Phylogenetic analysis was performed using Geneious Prime 2021 (Biomatters Ltd, NZ). A BLASTn search with 622,847 obtained reads resulted in 3,260 and 4,340 matches to the sequences of Streptocarpus flower break tobamovirus (SFBV) and Impatiens necrotic spot orthotospovirus (INSV), respectively. A near-complete (98.5%) genome sequence of SFBV was obtained (GenBank accession No. OQ970154), which shared 99.52% nucleotide identity to a SFBV type isolate from Germany (GenBank accession No. NC_008365). A phylogenetic tree was also generated (e-Xtra). To confirm the presence of both viruses, leaf tissue was rub inoculated onto herbaceous indicator plants as described by Tang et al. (2013). Chenopodium amaranticolor and C. quinoa plants developed local lesions while Nicotiana occidentalis plants showed local necrosis followed by systemic leaf puckering by 14 days post inoculation (dpi). Nicotiana benthamiana and N. clevelandii plants showed systemic chlorosis but N. tabacum plants did not exhibit any symptoms by 28 dpi. Samples from indicators and Streptocarpus were tested by RT-PCR (SFBV) or RT-qPCR (INSV), using in-house designed primers: SFBV-forward (5'-GTCATCAGCCGGAGAGGTTC-3'), SFBV-reverse (5'-AGGGCGAGTCTCTTCCTCTG-3'), INSV-forward (5'-CAATCAGAGGGTGACTTGGAA-3'), INSV-reverse (5'-GACTTTCCGAAGACTTGATGC-3') and INSV-probe (5'-CCATTGTCCTTTATCATTCCAACAAG-3'). RT-PCR products (across MP and CP regions) of the expected size (357 bp) were amplified from the Streptocarpus sample and symptomatic indicators. All amplicons were sequenced in both directions and found to be identical to the obtained HTS sequence. The presence of INSV was confirmed in all Streptocarpus and inoculated indicators except N. tabacum by INSV-specific RT-qPCR. A further 77 Streptocarpus plants were collected from a greenhouse in Auckland that holds a collection of multiple Streptocarpus cultivars from across NZ and overseas. Twenty-five plants, either displaying flower colour-break (only one plant) or asymptomatic (24 plants), tested positive for SFBV by RT-PCR. All amplicons were sequenced and found to be identical. SFBV was first described from naturally infected Streptocarpus plants in 1995 in the Netherlands (Verhoeven et al. 1995), and then in Germany (Heinze et al. 2006) and the United States (Pappu & Druffel 2007). While INSV has been found in NZ in several plant genera (Veerakone et al., 2015), to our knowledge, this is the first report of SFBV in NZ. SFBV was thought to be associated with colour breaking of Streptocarpus flowers (Verhoeven et al. 1995) but the virus was detected in asymptomatic Streptocarpus plants in this study and in California (Pappu & Druffel 2007). Given SFBV-infected plants were purchased from several sources, and leaf cuttings for propagation are shared among hobbyists, SFBV is likely to have spread throughout NZ. How this will affect production is unclear at this stage.

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand.

To protect New Zealand's unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

First report of Grapevine Algerian latent virus in carnation in New Zealand.

Carnation (Dianthus caryophyllus) is a popular ornamental plant widely used as a cut flower and in landscaping. In New Zealand, several viruses are known to infect plants of the genus Dianthus: arabis mosaic virus, carnation etched ring virus (CERV), carnation latent virus, carnation mottle virus, carnation necrotic fleck virus, carnation ringspot virus, carnation vein mottle virus and cucumber mosaic virus (Veerakone et al. 2015). In October 2020, a carnation sample with leaf chlorotic spots and distortion from a home garden in Auckland, New Zealand was submitted to the Plant Health and Environment Laboratory (PHEL) for virus testing. Leaf tissue of the sample was mechanically inoculated onto a range of herbaceous species using the method described in Tang et al. (2013). Chenopodium amaranticolor and C. quinoa plants developed local necrotic pinpoint spots while Nicotiana benthamiana, N. clevelandii, and N. occidentalis plants exhibited systemic leaf mosaic symptoms 7 days post-inoculation. The carnation plant and all five symptomatic indicator species tested positive for tombusviruses using an in-house designed generic RT-qPCR (available on request). Direct sequencing of the ~140 bp PCR product revealed the presence of grapevine Algerian latent virus (GALV). To further characterise the detected sequence, forward (5'-GTAGCGATGTATTGGGATAAGGA-3') and reverse (5'-TGCCGACACCCCGAAAGGT-3') primers were designed based on an alignment of the conserved region in the coat protein (CP) of 19 GALV isolates deposited in GenBank. Products of the expected size of 406 bp were amplified from all infected plants and their sequences found to be identical (GenBank accession No. OM891837). BLAST searches showed that the CP region of the sequence shared 97.0% (nucleotide) and 97.8% (amino acid) identity to the type isolate of GALV (GenBank accession no. NC_011535). GALV was first reported in Italy from a symptomless Algerian grapevine (Vitis vinifera) (Gallitelli et al., 1989), and is the only report of GALV in Vitis in the world. Since then, GALV has been reported in Germany, the Netherlands and Japan in several ornamental plant species including Alstroemeria sp. (Tomitaka et al., 2016), Gypsophila paniculata, Limonium sinuatum (Koenig et al., 2004, Fujinaga et al., 2009) and Solanum mammosum (Ohki et al., 2006). These infected ornamental host plants were reported to show various types of foliar symptoms, including chlorotic leaf spots. The GALV-infected carnation plant in this study was tested by PCR for all viruses that are known to infect D. caryophyllus in New Zealand, and CERV was identified. It is therefore unclear if the observed symptoms were induced by either GALV or CERV, or if they were the results of a synergistic interaction between GALV and CERV. Samples from a further 11 plants, comprised of nine symptomatic Dianthus spp. and two asymptomatic Alstroemeria spp. were collected from the same address and tested individually using the GALV-specific RT-PCR. GALV (along with CERV) was detected from all Dianthus plants while the Alstroemeria samples were negative. To our knowledge, this is the first report of GALV in New Zealand, and the first report in the host Dianthus in the world. Given the GALV-infected carnation plants were purchased from a local garden centre between 2007-2020, and plants from this garden centre have been widely distributed over this period of time to various customers, the virus is very likely to have spread throughout the country.

The Potential of Molecular Indicators of Plant Virus Infection: Are Plants Able to Tell Us They Are Infected?

To our knowledge, there are no reports that demonstrate the use of host molecular markers for the purpose of detecting generic plant virus infection. Two approaches involving molecular indicators of virus infection in the model plant Arabidopsis thaliana were examined: the accumulation of small RNAs (sRNAs) using a microfluidics-based method (Bioanalyzer); and the transcript accumulation of virus-response related host plant genes, suppressor of gene silencing 3 (AtSGS3) and calcium-dependent protein kinase 3 (AtCPK3) by reverse transcriptase-quantitative PCR (RT-qPCR). The microfluidics approach using sRNA chips has previously demonstrated good linearity and good reproducibility, both within and between chips. Good limits of detection have been demonstrated from two-fold 10-point serial dilution regression to 0.1 ng of RNA. The ratio of small RNA (sRNA) to ribosomal RNA (rRNA), as a proportion of averaged mock-inoculation, correlated with known virus infection to a high degree of certainty. AtSGS3 transcript decreased between 14- and 28-days post inoculation (dpi) for all viruses investigated, while AtCPK3 transcript increased between 14 and 28 dpi for all viruses. A combination of these two molecular approaches may be useful for assessment of virus-infection of samples without the need for diagnosis of specific virus infection.

Expression of genes associated with immunity in the endometrium of cattle with disparate postpartum uterine disease and fertility.

BACKGROUND: Contamination of the uterine lumen with bacteria is ubiquitous in cattle after parturition. Some animals develop endometritis and have reduced fertility but others have no uterine disease and readily conceive. The present study tested the hypothesis that postpartum cattle that develop persistent endometritis and infertility are unable to limit the inflammatory response to uterine bacterial infection. METHODS: Endometrial biopsies were collected several times during the postpartum period from animals that were subsequently infertile with persistent endometritis (n = 4) or had no clinical disease and conceived to first insemination (n = 4). Quantitative PCR was used to determine the expression of candidate genes in the endometrial biopsies, including the Toll-like receptor (TLR 1 to 10) family of innate immune receptors, inflammatory mediators and their cognate receptors. Selected proteins were examined by immunohistochemistry. RESULTS: The expression of genes encoding pro-inflammatory mediators such as interleukins (IL1A, IL1B and IL6), and nitric oxide synthase 2 (NOS2) were higher during the first week post partum than subsequently. During the first week post partum, there was higher gene expression in infertile than fertile animals of TLR4, the receptor for bacterial lipopolysaccharide, and the pro-inflammatory cytokines IL1A and IL1B, and their receptor IL1R2. The expression of genes encoding other Toll-like receptors, transforming growth factor beta receptor 1 (TGFBR1) or prostaglandin E2 receptors (PTGER2 and PTGER4) did not differ significantly between the animal groups. Gene expression did not differ significantly between infertile and fertile animals after the first week postpartum. However, there were higher ratios of IL1A or IL1B mRNA to the anti-inflammatory cytokine IL10, during the first week post partum in the infertile than fertile animals, and the protein products of these genes were mainly localised to the epithelium of the endometrium. CONCLUSION: Cattle may maintain fertility by limiting the inflammatory response to postpartum bacterial infection in the endometrium during the first week after parturition.

Bacterial lipopolysaccharide induces an endocrine switch from prostaglandin F2alpha to prostaglandin E2 in bovine endometrium.

Escherichia coli infection of the endometrium causes uterine disease after parturition and is associated with prolonged luteal phases of the ovarian cycle in cattle. Termination of the luteal phase is initiated by prostaglandin F(2alpha) (PGF) from oxytocin-stimulated endometrial epithelial cells. Compared with normal animals, the peripheral plasma of animals with E. coli infection of the endometrium had higher concentrations of lipopolysaccharide (LPS) and prostaglandin E(2) (PGE) but not PGF. Endometrial explants accumulated predominantly PGE in the culture medium in response to LPS, and this effect was not reversed by oxytocin. Endometrial cells expressed the Toll-like receptor 4/CD14/MD-2 receptor complex necessary to detect LPS. Epithelial and stromal cells treated with LPS had higher steady-state media concentrations of PGE rather than PGF. Arachadonic acid is liberated from cell membranes by phospholipase 2 (PLA2) enzymes and converted to prostaglandins by synthase enzymes. Treatment of epithelial and stromal cells with LPS did not change the levels of PGE or PGF synthase enzymes. However, LPS stimulated increased levels of PLA2 group VI but not PLA2 group IV C immunoreactive protein in epithelial cells. Endometrial cells expressed the E prostanoid 2 and E prostanoid 4 receptors necessary to respond to PGE, which regulates inflammation as well as being luteotropic. In conclusion, LPS detection by endometrial cells stimulated the accumulation of PGE rather than PGF, providing a mechanism to explain prolonged luteal phases in animals with uterine disease, and this PGE may also be important for regulating inflammatory responses in the endometrium.

Ovarian follicular cells have innate immune capabilities that modulate their endocrine function.

Oestrogens are pivotal in ovarian follicular growth, development and function, with fundamental roles in steroidogenesis, nurturing the oocyte and ovulation. Infections with bacteria such as Escherichia coli cause infertility in mammals at least in part by perturbing ovarian follicle function, characterised by suppression of oestradiol production. Ovarian follicle granulosa cells produce oestradiol by aromatisation of androstenedione from the theca cells, under the regulation of gonadotrophins such as FSH. Many of the effects of E. coli are mediated by its surface molecule lipopolysaccharide (LPS) binding to the Toll-like receptor-4 (TLR4), CD14, MD-2 receptor complex on immune cells, but immune cells are not present inside ovarian follicles. The present study tested the hypothesis that granulosa cells express the TLR4 complex and LPS directly perturbs their secretion of oestradiol. Granulosa cells from recruited or dominant follicles are exposed to LPS in vivo and when they were cultured in the absence of immune cell contamination in vitro they produced less oestradiol when challenged with LPS, although theca cell androstenedione production was unchanged. The suppression of oestradiol production by LPS was associated with down-regulation of transcripts for aromatase in granulosa cells, and did not affect cell survival. Furthermore, these cells expressed TLR4, CD14 and MD-2 transcripts throughout the key stages of follicle growth and development. It appears that granulosa cells have an immune capability to detect bacterial infection, which perturbs follicle steroidogenesis, and this is a likely mechanism by which ovarian follicle growth and function is perturbed during bacterial infection.

Expression and function of Toll-like receptor 4 in the endometrial cells of the uterus.

Prostaglandins have a central role in many endocrine functions in mammals, including regulation of the life span of the corpus luteum by prostaglandin F(2alpha) (PGF) and prostaglandin E2 (PGE), which are secreted by the uterine endometrium. However, the uterus is readily infected with bacteria such as Escherichia coli, which disrupt luteolysis. Immune cells detect E. coli by Toll-like receptor 4 (TLR4) binding its pathogenic ligand, lipopolysaccharide (LPS), although signaling requires accessory molecules such as CD14. The objective of this study was to determine the effect of E. coli or LPS on the function of bovine endometrial cells, and whether purified populations of epithelial and stromal cells express the molecules involved in LPS recognition. In addition, because the female sex hormones estradiol and progesterone modify the risk of uterine infection, their effect on the LPS response was investigated. Endometrial explants produced prostaglandins in response to LPS, with an increased ratio of PGE to PGF. Addition of LPS or E. coli to stromal and epithelial cells stimulated production of PGE and PGF and increased their cyclooxygenase 2 mRNA expression. The production of prostaglandins was abrogated by an LPS antagonist. In addition, estradiol and progesterone inhibited the production of PGE and PGF in response to LPS, indicating a role for steroid hormones in the response to bacterial infection. For the first time, Toll-like receptor 4 mRNA and CD14 mRNA and protein were detected in bovine endometrial stromal and epithelial cells by RT-PCR and flow cytometry. In conclusion, epithelial and stromal cells detect and respond to bacteria, which modulate their endocrine function.