Harmful viruses on stone fruit crops and sanitation methods

Relevance. Viral diseases can significantly reduce the yield of stone fruit crops. More than 30 viruses have been characterized on stone fruits crops, among which the most harmful are Plum pox virus (PPV), Prunus dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Cherry leaf roll spot virus (CLRV), Apple chlorotic leaf spot virus (ACLSV). Viral diseases monitoring is essential for controlling the viruses prevalence and choosing a control strategy. In the absence of healthy plants of a certain variety, health improvement is carried out using laboratory methods, including thermotherapy. Sanitation methods need to be improved in relation to the culture characteristics and the virus type. Of considerable interest is the development of techniques that reduce the viruses concentration when growing stone fruit trees in the field.Methods. During 2016–2020 using the ELISA (“Loewe” diagnostic kits) diagnostics of viruses on varieties and clonal rootstocks of cherry, sweet cherry and plum (660 plants) was carried out in the conditions of the Moscow region. For plant health in 2019–2021 used thermotherapy for 3 months. To study the effect of Pharmayod (“Farmbiomed”) on viruses in open ground, 24 plum plants of 5 varieties were treated with this drug at a concentration of 0.3 ml/l.Results. The total prevalence of viruses on cherries varieties was 44%, sweet cherries — 40%, plums — 59%, on clonal rootstocks — 46, 55 and 56% respectively. The highest incidence of PNRSV and PDV viruses has been established. Cherry plants of 11 varieties, free from the main harmful viruses, 4 varieties of sweet cherries, 12 varieties of plums and 9 forms of clonal rootstocks were revealed. The use of the Pharmayod on plum trees in the open field contributed to a decrease in the infection index of the studied viruses. Plum plants in a heat chamber were characterized by a higher survival rate and growth parameters in comparison with cherry and sweet cherry. After the completion of thermotherapy, a significant decrease in the index of infection in plants was noted.

Vitrification Cryo-Foil for Apple Cryopreservation and the Seesaw Effect Between Shoot Recovery and Virus Eradication

Improvements of existing cryopreservation protocols are necessary to facilitate long-term preservation of plant germplasm and the cryotherapy-effect of pathogen eradication. This study reported a vitrification (V) cryo-foil/plate methods for cryopreservation of shoot tips and cryotherapy effect in ‘Pink Lady’ apple. In V cryo-foil/plate protocols, shoot tips were first attached onto aluminum foils/plates using calcium alginate before other procedures. Shoot tips cryopreserved by V cryo-foil required 6.1 weeks to fully recover and 53% of shoot regrowth was obtained, comparable to the Dv cryopreservation. Similar regrowth levels were produced between applying V cryo-foil and Dv cryopreservation to another 4 Malus genotypes. Histological observations in shoot tips cryopreserved by Dv and V cryo-foil found only those with surviving apical dome and leaf primordia (LPs) could recover after cryopreservation. In apical meristem of shoot tips cryopreserved by Dv and V cryo-foil, higher surviving probability was detected from the V cryo-foil protocol, and the young LPs showed the highest level of surviving. Virus detection in cryo-derived plants showed apple stem grooving virus and apple chlorotic leaf spot virus were all preserved after cryopreservation, and higher eradication efficiency of apple stem pitting virus (70%) was produced by Dv than the 55% of V cryo-foil. These results supported applying V cryo-foil as an improvement to the widely applied Dv method in shoot tip cryopreservation, and also revealed a seesaw mode between shoot recovery and cryotherapy effect. Once the seesaw moves to increase the recovery after cryopreservation, the cryotherapy-effect on the other side would be decreased.

In silico Structural and Functional Annotation of Tomato Chocolate spot Virus

Aims: The study aims to predict in-silico the structural and functional annotation of Tomato Chocolate Spot Virus (TCSV) retrieved from Uniprotkb with the accession number C7EXM3. Study design: To use the In-silico approach for the structural and functional annotation of the Tomato Chocolate Spot Virus. Place and Duration of Study: The research was conducted at the Bioinformatics Unit, Chevron Biotechnology Centre, Modibbo Adama University Yola, Nigeria. Between August 2021 to September 2021. Methodology: The sequence of the Tomato Chocolate Spot Virus was retrieved from Uniprotkb with accession number C7EXM3, Physicochemical characteristics were computed using the ProtParam tool. The sever SOPMA was used for secondary structure analysis (Helix, Sheets and Coils). The tool CELLO v2.5 was used to predict the subcellular localization of the protein. Four different Homology Modelling tools (trRosetta, Lomet, RaptorX and IntFOLD5) were used to predict the 3D structure of the protein, the quality of the predicted proteins was assessed used PROCHECK. Three tools (InterProScan, NCBI conserved domains and Phobius) were used to get the possible function(s) of the protein. Results: ProtParam tool computed various Physical and Chemical properties such as Molecular weight (MW) 20396.96 Daltons, isoelectric point (pI) of 6.92. Instability Index 41.94, and Grand Average Hydropathy (GRAVY) -0.503. SOPMA was used for calculating the secondary structure parameters of the protein as Helices (Hh) 43.48%, Extended strands (Ee) 18.48%, Random coils (Cc) 38.04%. CELLO v2.5 was used for subcellular localization of the protein, it predicted that the protein can be both Nuclear and Cytoplasmic with the reliability of 1.653 and 1.504 respectively. Different Homology modelling tools were used to obtain the best 3D structure of the protein. Furthermore, PROCHECK was used to assess the quality of the models obtained. Model from trRosetta was found to be the best because of the quality of the Ramachandran Plot obtained from PROCHECK which has more than 90% of amino acid in the most favourable regions. NCBI-CDD and interproScan predicted that protein is a DNA double-strand break repair Rad50 ATPase, which is involved in the early steps of DNA double-strand break (DSB) repair. Furthermore, the Phobius server predicted the protein to be non-cytoplasmic in its domain, which means they help target proteins to their final destinations. Conclusion: The study has helped in obtaining the 3D structure of the protein Tomato Chocolate Spot Virus from different Modelling tools, as well as the possible function of the protein.

First Detection and Molecular Characterization of Apple Stem Grooving Virus, Apple Chlorotic Leaf Spot Virus, and Apple Hammerhead Viroid in Loquat in Spain

Loquat (Eriobotrya japonica) is an important crop in Spain. To date, only one viral species, apple stem pitting virus (ASPV), has been detected in Spanish loquat orchards. In this study, the presence of additional viruses infecting this crop in Spain was investigated. RT-PCR and high-throughput sequencing (HTS) of symptomatic loquat plants led to first-time detection and characterization of apple stem grooving virus (ASGV), also known as citrus tatter leaf virus (CTLV), and apple chlorotic leaf spot virus (ACLSV) from Spain with description of nearly complete genomic sequences. The frequency of ACLSV infection was the highest, with over 30% of the samples testing positive and were also detected as coinfections with ASGV and ASPV, although most of the samples infected were symptomless. Studies on all the full-length sequences available in the databases were performed in order to establish the phylogenetic relationships of the Spanish isolates of these two viral species. Moreover, apple hammerhead viroid (AHVd) was also detected to infect loquat, the first host different from apple reported for this viroid to date.

First Report of Capsicum chlorosis virus Infecting Chromolaena odorata L. in Yunnan, China

Capsicum chlorosis virus (CaCV) is a negative sense ssRNA virus belonging to the genus Orthotospovirus in the family Tospoviridae. It was first discovered in Australia, and then reported in other places including Thailand, China, India, Greece, and United States (Zheng et al.2011; Melzer et al.2014; Chrysoula et al. 2018; Abudurexiti et al. 2019). CaCV infects plants of the families Amaranthaceae, Apocynaceae, Chenopodiaceae, Cucurbitaceae, Amaryllidaceae, Fabaceae and Solanaceae (Basavaraj et al. 2017; Basavaraj et al. 2020). Chromolaena odorata L. (commonly known as Feiji cao in China) is an invasive weedy herb that belongs to the genus Eupatorium (family Asteraceae), and is native to Central America. In May 2020, serrated chlorotic ring and chlorotic ringspots resembling symptoms of orthotospovirus infection (Supplementary Figure 1) was observed on the leaves of C. odorata plants in Honghe County, Yunnan. Three symptomatic leaf samples were collected and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was performed using antisera targeting Tomato spotted wilt virus (TSWV), Calla lily chlorotic spot virus (CCSV), Capsicum chlorosis virus (CaCV), and Tomato zonate spot virus (TZSV) (Proteintech Group, Inc., China). Buffer solution and healthy leaves were used as a blank and negative controls, respectively. All three symptomatic samples showed positive reactions with only CaCV antiserum (OD450 of 0.315-0.345 relative to 0.078 and 0.076 for healthy plants and the blank control, respectively. The total RNA extracted from the positive samples were further analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using generic primers gL3637 (CCTTTAACAGTDGAAACAT) and gL4435c (CATDGCRCAAGARTGRTARACAGA) which were designed to amplify partial L segment encoding the RNA-dependent RNA polymerase (RdRP) of orthotospoviruses (Chu, et al. 2001). The expected ~800 bp DNA fragment was amplified from all three positive samples by RT-PCR. The amplified DNA was cloned and sequenced. BLAST search of the partial L RNA sequence (GenBank acc. nos. MW964378 to MW964380) revealed that they shared 86.2-97.4% nucleotide (nt) and 97.2-100% amino acid (aa) sequence identities with different isolates of CaCV available in GenBank with CaCV chili isolates (KU941834 to KU941836) from India sharing the highest aa identity of 100%. This confirmed the presence of CaCV in the symptomatic C. odorata plants. The 825 bp complete nucleocapsid protein (NP) of CaCV was also amplified from the samples using primers CaCV-F: ATGTCTAMCGTYAGGCAAC and CaCV-R: TYACACYTCWATAGAWGTACTAG) (Basavaraj et al. 2020), cloned, and sequenced to obtain complete S fragment-nucleocapsid protein (NP) with a size of 825 bp (MW964381 to MW964383). The pairwise comparisons of three fragments showed 85.1-98.3% nt and 87.6-99.6% aa sequence identities with different isolates of CaCV. Maximum-Likelihood phylogenetic trees inferred from the partial RdRP and complete NP aa sequences showed that the C. odorata isolates (CaCV-YN) clustered closely with CaCV tomato isolate from Taiwan and tomato (Yuxi-2013) isolate from China, respectively (Supplementary Figure 1). To our knowledge, this is the first time CaCV has been detected in C. odorata. This study will serve as an important reference for the study of host range of CaCV. Further studies will be required to determine whether thrips could transmit CaCV between C. odorata and other hosts of the virus.

First report of papaya ring spot virus (PRSV) infecting jute (Corchorus olitorius) in India

Jute is the most important bast fibre crop of the world, which is mainly cultivated in India, Nepal, Bangladesh, China, Indonesia and South American countries. The fibre is utilized for making apparels, ropes, bags, carpets etc (Biswas et al. 2014). This bio-fibre is gaining importance due to growing environmental consciousness worldwide. In June 2019, we noticed jute plants (less than 2%) showing virus like symptoms viz., downward curling, puckering, angular brownish to yellowish spots etc in a farmer’s field at Amdanga Block, North 24 Paraganas, West Bengal, India. To identify the virus, five symptomatic leaves from five different plants were used for high throughput sequencing (HTS). We extracted total RNA from each leaf which was subjected to construction of cDNA libraries. Sequencing was done on Illumina Hiseq 4000 (CytoScan, Thermo Fisher). Approximately 46 million 105 nt paired end reads were generated. Raw reads were trimmed and filtered to perform de novo assembly as described previously by (Grabherr et al. 2013). The obtained contig was 10,326 bp nucleotides (nt) long and in BLASTn against GenBank showed highest identity with papaya ring spot virus (PRSV) with the contig covering 99.6% of the viral genome. The obtained contig shared 99.33% sequence similarity with PRSV strain P (Accession No. MT470188). The selected leaf samples were also tested by double-antibody sandwich (DAS)- enzyme linked immunosorbent assay (ELISA) for papaya ring spot virus (PRSV) along with some common viruses, viz., Potato leaf roll virus (PLRV), Watermelon mosaic virus, Cowpea mosaic virus and Cucumber mosaic virus with the help of commercial diagnostic kits (Agdia). However, only the test with PRSV gave positive reaction for the symptomatic samples. The major symptoms of PRSV on papaya are severe mosaic, chlorosis, reduced lamina with curling and puckering (Gonsalves et al. 2010). To confirm PRSV infection, five symptomatic leaf samples (used for HTS) were collected and whole RNA was extracted from the samples using RNeasy plant minikit (Qiagen, USA). Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted by using isolated RNA. One pair of PRSV specific primer (PSRV1F: 5' TTAAATCTGATTCGTC 3' PRSV 1R: 5'GAAATTCACGCAAAGTCGA3') was developed by using primer BLAST software and was used in RT-PCR assays. Amplified fragments were cloned and sequenced and all the fragments shared 98% sequence identity with PRSV. One of the amplicons was deposited in NCBI (Accession No. MN615832). Crude sap was prepared by homogenizing PRSV-infected jute leaf tissues in 0.1 M sodium phosphate buffer and 2% carborundum dust was added as abrasive (Holkar et al. 2018). The sap was then gently rubbed on to the healthy papaya leaves for inoculation. Typical PRSV like symptoms appeared in inoculated leaves 10 days post inoculation which confirmed the presence of PRSV-P. PRSV was detected by RT-PCR as well as (DAS)-ELISA from all inoculated infected papaya leaf tissues, but could not be detected from uninoculated healthy papaya tissues. To the best of our knowledge, this is the first report of PRSV-P infecting jute in India. References: Biswas, C. et al. 2014. Plant Dis. 98(4): 565. https:// doi.org/10.1094/PDIS-08-13-0826-PDN. Gonsalves, D. et al. 2010. The Plant Health Instructor. https:// doi.org/10.1094/PHI-I-2010-1004-01 Holkar, S. K. et al. 2018. Crop Protection. 108:110-119. https://doi.org/10.1016/j.cropro.2017.12.013 Grabherr, M. et al. 2011. Nat Biotechnol. 29(7): 644-652.

Viral complexes in the Allium cepa L.- Frankliniella occidentalis P. interaction by DAS-ELISA in Baja California, Mexico

The onion (Allium cepa L.) is one of the main horticultural crops produced in Baja California, Mexico. In this crop, it has been observed presumptive viral symptoms, which are able to cause losses in yield and quality. Therefore, the objective of this study was to detect the associated virus in onion in high producing culture zones in Baja California. Plant material samples (symptomatic and asymptomatic) and trips insects were collected from commercial farming or areas. Commercial anti-serum were used for the detection of Iris yellow spot virus (IYSV), Tomato spot wilt virus (TSWV), Leek yellow spot virus (LYSV), Onion yellow dwarf virus (OYDV) and Garlic common latent virus (GarCLV). Seven indicator plants species were mechanically inoculated with onion sap (symptomatic and asymptomatic). The results showed the presence of IYSV, TSWV, OYDV and GarCLV in symptomatic onion plants. The plant samples showed a 77 % of incidence in the form of viral complexes and in a 23 % only the presence of LYSV was detected. The trips identified as Frankiniella occidentalis P. harbored IYSV, TSWV, LYSV, OYDV and GarCLV in the form of complexes in La Trinidad and San Quintin. The indicator plants did not show sympotoms of virosis. It is evident that F. occidentalis P. is responsible for the transmision of the analyzed viruses in this study in cultured onions. There are not previous reports for the detection of Orthotospovirus, Potyvirus and Carlavirus in Baja California onions.