Genetic variability and evolutionary dynamics of atypical Papaya ringspot virus infecting Papaya

Papaya ringspot virus biotype-P is a detrimental pathogen of economically important papaya and cucurbits worldwide. The mutation prone feature of this virus perhaps accounts for its geographical dissemination. In this study, investigations of the atypical PRSV-P strain was conducted based on phylogenetic, recombination and genetic differentiation analyses considering of it’s likely spread across India and Bangladesh. Full length genomic sequences of 38 PRSV isolates and 35 CP gene sequences were subjected to recombination analysis. A total of 61 recombination events were detected in aligned complete PRSV genome sequences. 3 events were detected in complete genome of PRSV strain PK whereas one was in its CP gene sequence. The PRSV-PK appeared to be recombinant of a major parent from Bangladesh. However, the genetic differentiation based on full length genomic sequences revealed less frequent gene flow between virus PRSV-PK and the population from America, India, Colombia, other Asian Countries and Australia. Whereas, frequent gene flow exists between Pakistan and Bangladesh virus populations. These results provided evidence correlating geographical position and genetic distances. We speculate that the genetic variations and evolutionary dynamics of this virus may challenge the resistance developed in papaya against PRSV and give rise to virus lineage because of its atypical emergence where geographic spread is already occurring.

High Mutation Frequency and Significant Population Differentiation in Papaya Ringspot Virus-W Isolates

A total of 101 papaya ringspot virus-W (PRSV-W) isolates were collected from five different cucurbit hosts in six counties of Oklahoma during the 2016–2018 growing seasons. The coat protein (CP) coding region of these isolates was amplified by reverse transcription-polymerase chain reaction, and 370 clones (3–5 clones/isolate) were sequenced. Phylogenetic analysis revealed three phylogroups while host, location, and collection time of isolates had minimal impact on grouping pattern. When CP gene sequences of these isolates were compared with sequences of published PRSV isolates (both P and W strains), they clustered into four phylogroups based on geographical location. Oklahoman PRSV-W isolates formed one of the four distinct major phylogroups. The permutation-based tests, including Ks, Ks *, Z *, Snn, and neutrality tests, indicated significant genetic differentiation and polymorphisms among PRSV-W populations in Oklahoma. The selection analysis confirmed that the CP gene is undergoing purifying selection. The mutation frequencies among all PRSV-W isolates were within the range of 1 × 10−3. The substitution mutations in 370 clones of PRSV-W isolates showed a high proportion of transition mutations, which gave rise to higher GC content. The N-terminal region of the CP gene mostly contained the variable sites with numerous mutational hotspots, while the core region was highly conserved.

Transmission and pathogenicity of papaya virus E: insights from an experimental papaya orchard

A study was conducted to investigate epidemiological aspects of papaya virus E (PpVE), a cytorhabdovirus commonly found in papaya (Carica papaya L.) plantings of Ecuador. Besides papaya, PpVE was found in three Fabaceae weeds, including Rhynchosia minima, Centrosema plumieri and Macroptilium lathyroides; the latter being the species with the highest virus prevalence. Greenhouse experiments showed that in M. lathyroides, single infections of PpVE induce only mild leaf mosaic, whereas in mixed infections with cowpea severe mosaic virus, PpVE contributes to severe mosaic. In papaya, PpVE did not induce noticeable symptoms in single or mixed infections with papaya ringspot virus. Transmission experiments confirmed that whiteflies (Bemisia tabaci) transmit PpVE in a semi-persistent, non-propagative manner.

Generation of mild recombinants of papaya ringspot virus to minimize the problem of strain-specific cross protection

Papaya ringspot virus (PRSV) causes severe damage to papaya (Carica papaya L.) and is the primary limiting factor for papaya production worldwide. A nitrous acid-induced mild strain PRSV HA 5-1, derived from Hawaii strain HA, has been applied to control PRSV by cross protection for decades. However, the problem of strain-specific protection hampers its application in Taiwan and other geographic regions outside Hawaii. Here, sequence comparison of the genomic sequence of HA 5-1 with that of HA revealed 69 nucleotide changes, resulting in 31 aa changes in which 16 aa are structurally different. The multiple mutations of HA 5-1 are considered resulting from nitrous-acid induction since 86% of nucleotide changes are transition mutations. The stable HA 5-1 was used as a backbone to generate recombinants carrying individual 3’ fragments of Vietnam severe strain TG5, including NIa, NIb, and CP3’ regions, individually or in combination. Our results indicated that the best heterologous fragment for the recombinant is the region of CP3’, with which symptom attenuation of the recombinant is like that of HA 5-1. This mild recombinant HA51/TG5-CP3’ retained high levels of protection against the homologous HA in papaya plants and significantly increased the protection against the heterologous TG-5. Similarly, HA 5-1 recombinants carrying individual CP3’ fragments from Thailand SMK, Taiwan YK, and Vietnam ST2 severe strains also significantly increase the protection against the corresponding heterologous strains in papaya plants. Thus, our recombinant approach for mild strain generation is a fast and effective way to minimize the problem of strain-specific protection.

Crescimento inicial e índices fisiológicos de acessos de mamoeiro inoculados com Papaya ringspot vírus / Initial growth and physiological indexes of papaya accessions inoculated with Papaya ringspot virus

Objetivou-se quantificar o crescimento vegetal e avaliar os índices fisiológicos de diferentes acessos de mamoeiro inoculados com o vírus PRSV-P e assim identificar acessos superiores frente a mancha anelar. O experimento foi realizado na Casa de Vegetação da Embrapa Mandioca e Fruticultura, localizada em Cruz das Almas - BA, em blocos inteiramente casualizados. Foram testados 24 acessos e uma cultivar, com três repetições e dois controles de cada acesso. Três plantas foram inoculadas com PRSV-P e duas deixadas como controles negativos. As avaliações de crescimento foram feitas aos 15, 30 e 60 dias após inoculação. Foram mensurados a altura de planta, diâmetro do caule, número de folhas e área foliar e os índices fisiológicos Taxa de crescimento absoluto (TCAP), Taxa de crescimento relativo da planta (TCRP), Taxa de crescimento absoluto foliar (TCAF) e Taxa de crescimento relativo foliar (TCRF). Os dados foram submetidos ao teste F da análise de variância. As médias das cultivares foram agrupadas pelo teste de Scott-Knott a 5% de probabilidade e as médias dos tratamentos (controle x inoculados) foram comparadas pelo teste de Tukey a 5% de probabilidade. A análise quantitativa de crescimento e os índices fisiológicos mostraram ser ferramentas eficientes para identificar diferenças no crescimento de diferentes materiais. O acesso CMF026 demonstrou maior estabilidade para as características número de folhas e área foliar ao longo das avaliações, sendo menos afetada para essas características pelo vírus da mancha anelar.

The Existence of Papaya ringspot virus-Papaya Strain on Cucumber in Gianyar, Bali

Yellow mosaic symptoms were identified from cucumber production systems in Gianyar and were similar to symptoms of PRSV infection. Further research was conducted to determine diseases incidence and molecular characteristic of PRSV. Ninety leaf samples were collected from Gianyar by purposive sampling and disease incidence calculations were based on symptoms in the field. Detection and identification were done using a RT-PCR with specific primers of CP PRSV-P, CP PRSV-W and DNA sequencing. Disease incidences in the fields ranged between 5.81–66.87%. Specific DNA band 470 bp was successfully amplified from several cucumber leaf samples collected from Ubud, Payangan, Tegallalang, Sukawati, Gianyar, and Blahbatuh; but no DNA were amplified from all samples when using CP PRSV-W specific primer. Nucleotide and amino acid analysis showed nucleotides homology to isolates from Ubud, Payangan, Tegallalang, Sukawati, Gianyar, and Blahbatuh, i.e. 98.9–99.5% and 99.1–100%, respectively. Results indicated that genetic variation of PRSV-P from Gianyar was low. Furthermore, the nucleotides homology of PRSV-P isolates from Ubud, Payangan, Tegallalang, Sukawati, Gianyar, and Blahbatuh were with PRSV-P isolates which infected cucumbers from Nganjuk (LC311783) and Brebes (LC311784), while from native papaya collected in Bali Bali (LC223115) were 97.2–98.4% and 98.6–100%, respectively. Phylogenetic analysis confirmed that PRSV-P isolates from Indonesia were in the same cluster with Thailand isolates. The results showed that sources of PRSV-P inoculums spreading into new areas.

Genetic characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential under greenhouse and field conditions

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf patches radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in mixed-infected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90–120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.