Effect of Ozone on Inactivation of Purified Pepper Mild Mottle Virus and Contaminated Pepper Seed

Pepper mild mottle virus (PMMoV) is a major viral pathogen of pepper (Capsicum spp.). PMMoV is readily mechanically transmitted and is seed transmissible. Trisodium phosphate (TSP) treatment is commonly used to reduce the level of viable PMMoV in contaminated seed. Ozone is efficacious in disinfecting fungal-contaminated seed and disrupting bacterial and viral pathogens on various substrates. The purpose of this study was to evaluate efficacy of ozone and chemical treatment on PMMoV viability. Treated pepper seed infectivity was evaluated via bioassay with Nicotiana benthamiana. Symptoms of PMMoV infection were not observed in bioassays of TSP-treated seed. Sufficient viable PMMoV remained on ozone-treated seed to cause infection, which was confirmed by ELISA. Neither treatment affected seed germination. Ozone treatment of purified PMMoV was assessed to determine the extent, if any, of PMMoV inactivation by ozone. At the low PMMoV concentration (0.01 mg/ml), 14-h ozone exposure eliminated infectivity as determined by N. benthamiana bioassays with ELISA confirmations. At the higher PMMoV concentration (0.1 mg/ml), ozone treatment was insufficient to prevent infection. Ozone inactivation of purified PMMoV was quantified via bioassay using the local lesion host N. glutinosa and quantitative real-time PCR. Ozone exposure reduced lesion counts and PMMoV concentration, and PMMoV degradation increased with exposure time. Although PMMoV infection was eliminated at the low PMMoV concentration, bioassays using naturally infected seed and purified PMMoV preparations at relatively higher concentrations demonstrated that ozone is not efficacious as a standard treatment to sufficiently reduce levels of infective PMMoV in contaminated pepper seed. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Impatiens necrotic spot virus and Tomato spotted wilt virus Diagnosed in Phalaenopsis Orchids from Two Florida Nurseries

In October 2006 (Arcadia, FL) and January 2007 (Sorrento, FL), several white Phalaenopsis orchids with large chlorotic/necrotic ringspot symptoms were sent to the Division of Plant Industry, Gainesville, FL. Symptomatic leaf tissues were tested with the Agdia immunostick-comb (Agdia, Elkhart, IN) for Impatiens necrotic spot virus (INSV), Tomato spotted wilt virus (TSWV), Cucumber mosaic, and Tobacco mosaic virus. Plants from the nursery in Sorrento, FL tested positive for TSWV, while those from the nursery in Arcadia, FL tested positive for INSV. Symptomless leaves from the infected plants tested negative for the viruses with the immunostick-comb. The plants also were tested for TSWV and INSV by double-antibody (DAS)-ELISA (Agdia Inc.) with the same results. Total RNA was extracted from one symptomatic orchid leaf from each nursery. Reverse transcription (RT)-PCR was performed with the universal tospovirus primer set BR60and BR65 (1). PCR bands of the expected size were amplified from each leaf. PCR products were sequenced directly. The orchid leaf that tested positive for TSWV by ELISA produced a 495-bp sequence with 97% identity to several isolates of the TSWV nucleocapsid protein gene listed in GenBank (Accession Nos. AY744479, AY8770391, DQ376185, and AF02659). The orchid leaf that tested positive for INSV by ELISA produced a 396-bp sequence with 98 to 99% identity to several isolates of the INSV nucleocapsid protein gene (Accession Nos. D00914, DQ425096, X66972, and AD109100). Although these viruses have been reported a few times in orchids previously (2,3), to our knowledge, this is the first time they have been reported in this host in Florida. In addition, white Phalaenopsis spp. appears to be a local lesion host and not a systemic host for these viruses. References: (1) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (2) J. S. Hu et al. Plant Dis. 77:464, 1993. (3) S. T. Koike and D. E. Mayhew. Orchids 70:746, 2001.

Host-induced avirulence of hibiscus chlorotic ringspot virus mutants correlates with reduced gene-silencing suppression activity

Post-transcriptional gene silencing (PTGS) and virus-encoded gene-silencing suppressors are defence and counterdefence strategies developed by host and pathogens during evolution. Using a green fluorescence protein-based transient suppression system, the coat protein (CP) of Hibiscus chlorotic ringspot virus (HCRSV) was identified as a strong gene-silencing suppressor. CP suppressed sense RNA-induced but not dsRNA-induced local and systemic PTGS. This is different from another virus in the genus Carmovirus, Turnip crinkle virus (TCV), the CP of which strongly suppresses dsRNA-induced PTGS. HCRSV CP domain deletion mutants lost their suppression function, indicating that the complete CP is essential for suppression of PTGS. When CP was expressed from a Potato virus X (PVX) vector, it was able to enhance the symptom severity and to increase the accumulation of PVX RNA. Here, it is proposed that HCRSV CP suppresses PTGS at the initiation step, which is different from TCV CP. In addition, a previous study demonstrated that CP mutants resulting from serial passage of HCRSV in its local lesion host also showed a significantly reduced suppression function, indicating that host-induced mutations that lead to avirulence of HCRSV in kenaf correlate with its reduced ability to suppress PTGS.

Mutation of Phe50 to Ser50 in the 126/183-kDa proteins of Odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion

Sequence comparison of a non-biologically active full-length cDNA clone of Odontoglossum ringspot virus (ORSV) pOT1 with a biologically active ORSV cDNA clone pOT2 revealed a single nucleotide change of T→C at position 211. This resulted in the change of Phe50 in OT2 to Ser50 in OT1. It was not the nucleotide but the amino acid change of Phe50 that was responsible for the inability of OT1 to replicate. Time-course experiments showed that no minus-strand RNA synthesis was detected in mutants with a Phe50 substitution. Corresponding mutants in Tobacco mosaic virus (TMV) showed identical results, suggesting that Phe50 may play an important role in replication in all tobamoviruses. Complementation of a full-length mutant OT1 was demonstrated in a co-infected local-lesion host, a systemic host and protoplasts by replication-competent mutants tORSV.GFP or tORSV.GFPm, and further confirmed by co-inoculation using tOT1.GFP+tORSV (TTC), suggesting that ORSV contains no RNA sequence inhibitory to replication in trans. Surprisingly, a small number of exact revertants were detected in plants inoculated with tOT1+tORSV.GFPm or tOT1.GFP+tORSV (TTC). No recombination was detected after screening of silent markers in virus progeny extracted from total RNA or viral RNA from inoculated and upper non-inoculated leaves as well as from transfected protoplasts. Exact reversion from TCT (OT1) to TTT (OT2), rather than recombination, restored its replication function in co-inoculated leaves of Nicotiana benthamiana.

Covariation in the Capsid Protein of Hibiscus Chlorotic Ringspot Virus Induced by Serial Passaging in a Host That Restricts Movement Leads to Avirulence in Its Systemic Host

ABSTRACT Hibiscus chlorotic ringspot virus (HCRSV) from naturally infected Hibiscus rosa-sinensis L. loses virulence in its experimental systemic host Hibiscus cannabinus L. (kenaf) after serial passages in a local lesion host Chenopodium quinoa. Here we report the genetic changes responsible for the loss of virulence at the molecular level. A remarkable covariation of eight site-specific amino acids was found in the HCRSV capsid protein (CP) after serial passages in C. quinoa: Val49→Ile, Ile95→Val, Lys270→Arg, Gly272→Asp, Tyr274→His, Ala311→Asp, Asp334→Ala, and Ala335→Thr. Covariation of at least three of the eight amino acids, Val49, Ile95, and Lys270, caused the virus to become avirulent in kenaf. Interestingly, the nature of the covariation was consistent and reproducible at each serial passage. These data indicate that the nonsynonymous substitutions of amino acids in the HCRSV CP after serial passages in C. quinoa are not likely to be random events but may be due to host-associated positive selection or accelerated genetic drift. The observed interdependence among the three amino acids leading to avirulence in kenaf may have implications for structural or functional relationships in this virus-host interaction.

Frequent Homologous Recombination Events between Molecules of One RNA Component in a Multipartite RNA Virus

ABSTRACT Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.

Tobacco Mosaic Virus Subliminal Infection of African Violet

`Wild White' African violet (Saintpaulia ionantha H. Wendl.) was previously reported to be probably immune to tobacco mosaic virus (TMV) infection. In this study, 15 other S. ionantha cultivars were mechanically inoculated with 200 μg TMV/ml sodium phosphate buffer. Two weeks postinoculation, tissue was harvested and assayed for TMV infection by a) TMV-specific enzyme-linked immunosorbent assay and b) bioassay on the local lesion host, `Samsun NN' tobacco (Nicotiana tabacum L.). There was evidence of TMV infection in directly inoculated tissue of each of the 15 S. ionantha cultivars but not in noninoculated tissue or in mock-inoculated control plants. The small amount of virus recovered from inoculated tissue was shown to be the result of de facto viral infection and not the detection of residual inoculum. Postinoculation treatment with ultraviolet light significantly enhanced virus recovery in directly inoculated tissue. These results suggest that S. ionantha is not immune to TMV infection and that this host undergoes an asymptomatic subliminal infection by TMV.

SCREENING OF FIELD PEA BREEDING LINES FOR PEA SEED-BORNE MOSAIC VIRUS

Pea seed-borne mosaic virus (PSbMV) was first identified in Canadian field pea (Pisum sativum L.) breeding lines in 1974. Since then, an extensive program has been underway to eradicate this virus from the breeding lines. At the Morden Research Station, nearly 2000 breeding lines were evaluated. The virus was assayed by infectivity tests using the local lesion host Chenopodium amaranticolor, and by a gel immunodiffusion test. PSbMV was detected in 1361 lines. The level of infection within lines varied from 1 to 3%. Due to the restricted extent of the virus in the breeding lines, it was possible to continue the breeding program without a serious loss in germplasm.