Citrus leprosis: a complex and multietiologic disease Pedro Luis Ramos-González Instituto Biológico de São Paulo [email protected] Citrus leprosis: historical review and distribution Florida, USA Rusty nail head 1907 Citrus leprosis: historical review and distribution Hawaii, USA Mexico Central America Paraguay, Argentina, Brazil Venezuela, Colombia Florida, USA 1907 1920-30 1960 1999 2000 2004 2011 2017 Viral genomes Locali et al. 2006 Viral particles Kitajima et al. 1972 Mites (genus Brevipalpus) Frezzi 1940 & Vergani 1945 Citrus leprosis: serious viral disease . 1990: control of vector mite costs Brazilian growers around US$ 80 million (Bastianel et al., 2010) . 2010: 10-20% cost of consumables 5-10% total expenditures (Fundecitrus, citrus grower, pers. comm.) São Paulo, 2017. Pictures by Daniel Junior de Andrade Citrus leprosis: symptoms . Mite preference . fruits>stems>leaves . Local necrotic or chlorotic lesions Citrus leprosis: two disease types C. sinensis. Enveloped-short bacilliform virions (50-60 nm X 110-120 nm). (Kitajima et al., 1974). C. sinensis. Rodlike virions (40-50 nm x 100-110 nm) in vesicles, in the nucleus and cytoplasm. Membrane-associated virions. (Kitajima et al., 1972). C. volkameriana. Short, bacilliform virions of ≈30 x 50 nm. Cytoplasm of symptomatic leaf tissues (Melzer et al., 2012). Nuclear type Cytoplasmic type Citrus leprosis: viral diversity Citrus leprosis-Nuclear Citrus leprosis-Cytoplasmic • Floating genera Order: Mononegavirales Family: Rhabdoviridae Genus: Cilevirus Genus: Higrevirus Genus: Dichorhavirus (Locali et al., 2012) (Melzer et al., 2013) (Dietzgen et al., 2014) (Afonso et al., 2016) • Single strand • Positive sense RNA • Bipartite genome • Tripartite genome • Single strand • Transmitted by Brevipalpus • Transmission not • Negative sense RNA mites • Bipartite genome confirmed • Transmitted by Brevipalpus • mites Citrus leprosis Nuclear type Country Virus Ref. Mexico OFV-citrus Roy et al., 2013 (orchid fleck virus) Cruz-Jaramillo et al., 2014 Colombia OFV-citrus Roy et al., 2014 Partial sequence Florida, 80% of nt identity Hartung et al., 2015 USA* > 92% aa identity OFV-citrus Panama ? Dominguez et al., 2001 Kitajima et al., 1972., Ramos- Brazil CiLV-N and CiCSV González et al., 2017; Chabi et (citrus leprosis virus N, citrus chlorotic spot virus al., submitted. *Knorr, 1948 Small RNA 61% of the genome fraction Hartung et al., 2015 Citrus leprosis Nuclear type in Brazil * RNA1 CiCSV 6.5 Kb 3´ N P MP M G 5´ RNA2 3´ L 5´ 5.9 Kb 51 < nt < 63 44 < aa < 64 RNA1 6.7 Kb 3´ N P MP M G 5´ RNA2 3´ L 5´ 6.0 Kb CiLV-N Four isolates, one species Ramos-González et al., 2017; Chabi et al., submitted. Citrus leprosis Nuclear type in Brazil • State of Piauí • Two isolates from an urban zone / Teresina • Altitude: 72 m • Average annual temperature: 27.1 °C • Large chlorotic spots • Sweet oranges • State of São Paulo • Four isolates from four counties (20-170 km) • Altitude: > 800 m • Average annual temperature: 18-20 °C, including frost • Small necrotic and chlorotic spots • Sweet oranges Citrus leprosis Nuclear type in Brazil Brevipalpus n. yothersi • Only association dorsal ventral microplates spermateca (Beard et al. 2015) Brevipalpus phoenicis ss • Confirmed viral transmission to healthy plants Ramos-González et al., 2017; Chabi et al., submitted Citrus leprosis Cytoplasmic type Country Virus Ref. Mexico to CiLV-C Bastianel et al., 2010 Argentina (citrus leprosis virus C) Colombia CiLV-C2 strain Co Roy et al., 2013 CiLV-C 99% Diversity and Brazil variability studies Argentina Panama in Brazil 1967 2006 2015 Locali et al., 2006 Pascon et al., 2006 Small RNA fraction Hartung et al., 2015 Citrus leprosis Cytoplasmic type CiLV-C (Locali, et al., 2006) 51 samples BZ, MX, CO, PA, PY, AR p29 mp cDNA-PCR CiLV-C2 ORF p29 (Roy et al., 2013) OFV-citrus ORF M (Roy et al., 2014) CiLV-C ORF mp (Locali et al., 2003) CiLV-C ORF p29 (Pereira et al. 2012) Clade CRD Clade SJP Citrus leprosis virus C: variability Nucleotide identity C. sinensis RNA1 Complete RNA2 siRNA sequencing genome > 99% strain SJP RNA1 B. yothersi RNA2 A1 isoline A. thaliana Isolate SJP01 Strains SJP-CRD ~ 85% nt identity Putative recombination Ramos-González et al., 2016 Citrus leprosis virus C: strain detection Strain-specific RT-PCR Mixed infections of CiLV-C strains detected. Ramos-González et al., 2016 Relevance: • Support for the putative RNA2 recombination predicted in silico • Reassorment between viral RNA components, synergism, trans- complementation... • New biological treats Citrus leprosis cytoplasmic type B. yothersi variability CiLV-C strain distribution Haplotype diversity: COI sequence Sánchez-Velázquez et al., 2015 CiLV-C / vector relationship adult larva protonymph deutonymph female male Brevipalpus yothersi (ex- B. phoenicis) AAP 4 h IAP 2 h Latent period 7 h Retention period 12 d Viruliferous 25-60% vulgarisPhaseolus mites / colony Ca. Cardinium • Non-transovarial transmission of CiLV-C Tassi et al., 2017 Citrus leprosis / vector relationship Citrus leprosis Cytoplasmic type B. yothersi CiLV-C • No viruses • Detection of inside Viral RNA mite’s cells negative strand ≠ in mites Kitajima & Alberti, 2014 Roy et al., 2015 Circulative non-propagative Citrus leprosis / vector relationship Citrus leprosis Cytoplasmic type Citrus leprosis Nuclear type B. yothersi B. phoenicis B. californicus CiLV-C CiLV-N OFV Roy et al., 2015 • No viruses • Detection of • Viruses inside inside Viral RNA mite’s cells mite’s cells negative strand • Long latent ≠ in mites periods Kitajima & Alberti, 2014 Roy et al., 2015 Circulative non-propagative Circulative propagative CiLV-C / plant interaction - Mite / plant interaction RT-qPCR After 7-10 days B. yothersi A1 isoline A. thaliana Col-0 Arena et al., 2015 Mock Mites Mites + CiLV-C Marker genes: 6 hai 12 hai 24 hai 8 dai SA: ICS1, EDS5, NPR1, TGA3, WRKY70, PR1, PR5 ROS JA/ET: JAR1, ETR1, EIN2, MYC2, VSP2, PDF1,2 production Gene silencing: RDR1, RDR6, DCL2, DCL4, DAB stain AGO1, AGO2, HEN1 ROS and cell death: NHL10, RBOHD Cell Reference genes: F-box and SAND death Trypan blue stain Arena et al., 2016 CiLV-C / plant interaction - Mite / plant interaction Working hypothesis: • Gene silencing • Host manipulation by mites • VOC emission • Metabolite quantity and quality changes • Virus as an effector • Role of each viral ORF Arena et al., 2016 CiLV-C / mite / plant interaction Primarily infested leaves Secondary infested leaves n= 26 plants (13 infested with non-viruliferous and 13 with viruliferous mites). 5 mites/leaf p < 0.05 (*) or < 0.01 (**) 3 leaves/plant Mite count: 13 dpi Arena et al., 2016 Citrus leprosis Cytoplasmic type Nuclear type OFV- CiLV-C CiLV-C2 HGSV2 CiLV-N CiCSV citrus B. yothersi B. papayensis B. californicus B. phoenicis B. n. yothersi Sweet oranges, grapefruit, lemon, lime, Sweet oranges, Sweet oranges, mandarin, sour orange, tangerines, Swinglea Hibiscus sp. C.volkameriana, Sweet oranges Sweet oranges sweet or navel lime, and glutinosa, Commelina Swinglea glutinosa, Hibiscus sp. Persian lime benghalensis Dieffenbachia sp. Camila Chabi Gabriela Arena Aline Tassi Elliot W. Kitajima Juliana Freitas- Astúa Collaborators Renato Bassanezi - Fundecitrus Daniel Junior de Andrade - UNESP / Jaboticabal Ricardo Harakava - IB Valdenice Morerira - CCSM Marines Bastianel - CCSM Marcos Machado -CCSM Mariani Rodrigues Mateo Postcam Citrus leprosis management • Reduction of the inoculum source: pruning • Use of mite- and virus-free seedlings • Avoiding the presence of alternative virus hosts in fields • Control of viruliferous mite populations by using acaricides Effect of gene silencing during CiLV-C infection in Arabidopsis Genotype 120 dcl2/4 rdr2/6 wt dcl2/4 rdr2/6 wt ) 100 leaves dpi 80 (10 60 symptomatic leaves of 40 20 Rosette Percentage 0 5 6 7 8 12 dpi ) dpi 2,5 2 * (15 titers n= 15 plants per mutant genotype 1,5 * Non symptoms Three independent experiments viral Inoculated leaves 1 observed neither 5 mites/leaf Systemic leaves leaves viral RNA detected 3 leaves/plant 0,5 by RT-qPCR Relative 0 wild type dcl2/4 rdr2/6 Cauline Citrus leprosis Nuclear type in Brazil N * * N * * I- CiLV-C (MP gene) RACE/ II- CiLV-C2 (p29 gene) NGS/ III-OFV-citrus (N gene) Total Secondary re- IV- OFV (N gene), bioinformatics VI- degenerate primer pair RNA sequencing for the detection of L gene In summary: viruses associated to Citrus leprosis Cytoplasmic type Nuclear type RdRP2 (motif) Protein L Cilevirus Blunervirus Higrevirus Negevirus Virgaviridae Tobamovirus Tobravirus Closteroviridae Dichorhavirus Nucleorhabdovirus Cytorhabdovirus Diagnostic tools: PCR primers VTB Sequência dos iniciadores MP-F: GCGTATTGGCGTTGGATTTCTGAC CiLV-C MP-R: TGTATACCAAGCCGCCTGTGAACT p29-F: CAGAAGGCCGAGGTTGTAAAG CiLV-C CRD p29-R: GTAGTGATCACTGAACTCGAATACC p29-F: GTAARCAAAAGGTCGAGGTTGTCC CiLV-C SJP p29-R: TCTGTTGTCTAGCAGCRAGTAATG p29-F: ATGAGTAACATTGTGTCGTTTTCGTTGT CiLV-C2 p29-R: TCACTCTTCCTGTTCATCAACCTGTT MP-F: CGATATTTGATCAATCCGTT MP-R: CACCTTAAAATTCGAGGGTT PfGSV RpdR-F : CTGTTGTGCCAAATCATCAA RpdR R : TTCATCGCAAGTTCATATACCT RpdR-F: TGTCGAACTTTGGTATGAGTCG SvRSV RpdR R:CCGGTTCGTCAAATAACTCC N-F: ATGGCTAACCCAAGTGAGATCGATTA OFV-citrus N-R: AGTTGCCTTGAGATCATCACATTGGT N-F: TGTCATAGCCGACATAAACACC OFV N-R: TGTAGAGCTTGCGAGATACAG L-F: ATATCACCGTTTAAGCAAGC ClCSV L-R: TCCTTGTTACAACTCCTTGC Cileviruses: diversity Floating genera Cilevirus Blunervirus Higrevirus Negevirus Virgaviridae Tobamovirus Tobravirus Closteroviridae RdRp2 (protein motif) .