Changes in pests, plants and their interactions as drivers of emerging plant health risks: tomato yellow leaf curl disease epidemics in

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1 Changes in pests, plants and their interactions as drivers of emerging plant health risks: tomato yellow leaf curl disease epidemics in Spanish protected cultivation, a study case. Enrique Moriones Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-CSIC) La Mayora Experimental Station Algarrobo-Costa, Málaga, Spain

2 >end 1980 s Bemisia tabaci emergence warm and temperate regions Whitefly-transmitted transmitted viruses CYSDV EMERGING VIRUSES (since late 1980s) Begomoviruses (Fam. Geminiviridae) Criniviruses (Fam. Criniviridae) Ipomoviruses (Fam. Potyviridae) Torradoviruses (Picorna-like) ToCV TToV CVYV

3 PARADIGM: Tomato yellow leaf curl disease (TYLCD) Presence of TYLCD CAUSAL AGENTS Genus: Begomovirus, Family: Geminiviridae C4 IR V2 Rep ~2,8Kb CP TrAP REn ssdna genome

4 Infection at early growth stages: 100% yield loss

5 TYLCD: complex of viruses associated ToLCV 833 TYLCKaV TYLCGuV TYLCCNV TYLCTHV TYLCKaV (bipartite) TYLCGuV TYLCCNV TYLCTHV (bipartite) TYLCMalV TYLCMalV TYLCAxV TYLCAxV 0.1 -ES -Sar -Sic -Gez 911 -Mld 932 -IL -IR TYLCMLV (Tomato Yellow Leaf Curl Virus) TYLCMLV

6 biological differences exist (Navas-Castillo et al Plant Disease 83: 29-32) ToLCV TYLCKaV-[TH:Kan1] TYLCKaV 833 TYLCGuV-[CN:G3] TYLCCNV-[CN:Gua] TYLCTHV-[TH:1] TYLCGuV TYLCCNV TYLCTHV TYLCMalV-[ES:421:99] TYLCMalV TYLCAxV-[ES:Sn8:00] TYLCaXV -ES[ES:1:92] -[IT] -Sic[IT] -Gez[SD] 911 -Mld[IL] 932 -[IL] -IR TYLCGezV x Bean leaf crumple disease 0.1 TYLCMLV TYLCMLV Resistance break down -Mld -IL (Squash-blot hybridization) Tomás et al MPMI (in press)

7 TYLCD emergence, SPAIN as study case: MIGRATION High genetic stability of the population (8 years period) nucleotide genetic diversity (intergenic region) (Nei, 1987) PROVINCE ALMERÍA 0,0000 0,0028 0,0052-0,0080 0, MÁLAGA ,0018 0,0058 0, MURCIA 0,0066 0,0043 0,0085-0,0101 0,0085 0,0096 0,0314 (Sánchez-Campos et al Phytopathology 92: )

8 ES -Mld DISPLACEMENT of by : Tomato - Southern Spain ( ) YEAR NO FIELDS VISITED NO SAMPLES COLLECTED INCIDENCE NO (%) POSITIVE SAMPLES FOR: TYLCD (%) (68,6) 398 (39,8) 71 (7,1) 219 (21,9) (81,3) 103 (17,0) 231 (38,2) 158 (26,1) (96,7) 10 (1,8) 403 (72,7) 123 (22,2) Sánchez-Campos, et al Phytopathology 89:

9 Host range PLANT HOST Common bean Tomato Rotation tomato common bean tomato: purifying selection for Vector transmission BIOTYPE B. TABACI N O PERCENTAGE INFECTED PLANTS EXPERIMENTS B 4 11,8 c 33,7 b Q 4 40,1 ab 50,0 a Sánchez-Campos, et al Phytopathology 89: Navas-Castillo et al Plant Disease 83:

10 ES -Mld -IL Incidence of -IL in TOMATO among TYLCD-infected samples % -IL L-infecte ed sampl les Murcia Almería Málaga

11 Population study: TYLCD in Western Mediterranean Basin ( ) Systematic survey: 1,900 samples from Italy and Spain (tomato / common bean) (Collaboration : GP Accotto. IVA, Torino, Italy) Random selection: 111 isolates analyzed Rep 780 nt (30% genome) IR V2 C4 CP (García-Andrés, et al Virology 359: )

12 ES:T50:03-F ES:B117:03-F2 100 ES:B109:03-F2 ES:B104:03-F1 ES:B130:03-F3 ES:T31:03-F5 ES:B428:03-F4 ES:B145:03-F2 ES:B152:03-F2 ES:B115:03-F2 ES:B101:03-F ES:B99:03-F1 ES:B110:03-F2 ES:B143:03-F1 ES:B121:03-F3 61 ES:B123:03-F3 ES:B106:03-F1 ES:B416:03-F2 ES:B98:03-F1 ES:B422:03-F3 ES:B134:03-F1 ES:T64:03-F7 62 ES:B423:03-F3 ES:B432:03-F4 ES:B147:03-F2 ES:B138:03-F1 ES:B142:03-F1 ES:B119:03-F3 TYLCMalV-[ES:421:99] ES:B111:03-F [IT:Sic:05] IT:T529:02-F2 IT:T535:02-F2 IT:T570:02-F4 IT:T573:02-F4 IT:T568:02-F4 -[IL] ES:T223:03-F ES:T15:03-F3 ES:T249:03-F5 ES:T239:03-F5 ES:T323:03-F1 ES:T398:03-F9 ES:T387:03-F8 ES:T245:03-F5 ES:T162:03-F1 ES:T333:03-F2 ES:T190:03-F3 ES:B437:03-F5 ES:T206:03-F7 ES:T409:03-F9 ES:T3:03-F2 ES:T84:03-F8 ES:T158:03-F1 ES:T365:03-F6 ES:T383:03-F7 ES:T318:03-F1 ES:T6:03-F2 ES:T80:03-F8 ES:T341:03-F3 ES:T177:03-F2 ES:T305:03-F9 ES:T268:03-F6 ES:T70:03-F7 ES:T400:03-F9 -[ES:Alm] ES:B434:03-F4 ES:T327:03-F2 ES:T337:03-F3 ES:T167:03-F1 ES:T196:03-F3 ES:T281:03-F8 ES:T48:03-F1 ES:T16:03-F3 ES:T28:03-F4 ES:T188:03-F3 100 ES:T362:03-F5 ES:T377:03-F7 ES:T357:03-F5 98 ES:B122:03-F3 81 ES:B139:03-F1 56 ES:T21:03-F4 -Mld[ES:72:97] 62 IT:T538:02-F2 76 IT:T310:99-F4 IT:T511:02-F1 IT:T561:02-F3 97 IT:T548:02-F3 90 IT:T284:99-F1 IT:T289:99-F IT:T306:99-F3 74 IT:T292:99-F2T IT:T303:99-F3 -[IT:Sar] 70 IT:T287:99-F1 IT:T315:99-F4 IT:T313:99-F4 IT:T293:99-F2 88 IT:T304:99-F3 -Sic[IT] IT:T500:02-F1 64 IT:T558:02-F3 100 IT:T286:99-F1 IT:T506:02-F1 100 ES:T36:03-F5 ES:T57:03-F6 60 -ES[ES:92:1] -IL - Mld -Sar -Sic -ES TYLCMalV Great genetic diversity: 6 virus types present: : IL/ Mld : Sar / Sic / ES TYLCMalV Geographic and host STRUCTURATION O Italy y( (tomato) O Spain (common bean) O Spain (tomato) (García-Andrés, et al Virology 359: ) 0.05

13 Mixed infections occur in natural populations: risk of RECOMBINATION -IL ToLCV-[AU] -Mld[ES:72:97] -[IT:Sic:05] T535/02 P2 T570/02 P4 T573/02 P4 T529/02 P2 T568/02 P4 pgem-t pgem-t Within single plants RFLP differentiation BglII digestion Bgl II digestion 1Kb 1Kb 1Kb -Sar - -Sic Sic Uncut cut -[ES:Alm] 577 SICILY -Sar TYLCMalV-[ES:421:99] -[IT:Sar] T284/99 P1 991 T561/02 P3 T548/02 P3 T538/02 P2 T310/99 P4 T511/02 P1 999 T303/99 P3 -Sic T306/99 P3 T289/99 P2 T292/99 P2 T304/99 P3 T286/99 P1 T315/99 P4 T287/99 P1 T313/99 P4 0.1 (García-Andrés, et al Virology 359: ) -ES[ES:1:92] T506/02 P1 -Sic[IT:Sic] T500/02 P1 T558/02 P3 T293/99 P2 [Ty304:99] 1kb kb -Sar -Sic [Ty570:02] Sar -Sic -IL

14 TYLCD emergence, SPAIN as study case: RECOMBINATION Mld -IL -ES TYLCMalV TYLCAxV (Monci et al Virology 303: ) (García-Andrés et al Virology 350: )

15 Emergence of recombinants: increased host range / virulence Changes in host range (detection by molecular hybridization) Parental viruses Recombinant viruses -ES TYLCMalV TYLCAxV Tomato Common bean Solanum nigrum Changes in virulence Symptom severity Symptom severity tomato 5 S. nigrum common commom bean bean tomato Solanum nigrum (Monci et al Virology 303: ) Days post-inoculation (García-Andrés, et al Virology 350: ) ES TYLCAxV -IL

16 Emergence of recombinants: Resistance break down -Mld vs. -IL Resistance break down 1.0 C3 C2 C1 C4 IR V2 V1 Plant number -Mld -IL A I II I (Squash-blot hybridization) Region I TGMV ToLCKav ToLCBV -IR Region II TGMV -IL 772 ToLCKaV -IR 950 ToLCBV 0.1 -MILD -IL MILD (Navas-Castillo et al J Gen Virol 81: )

17 ES:T50:03-F ES:B117:03-F2 100 ES:B109:03-F2 ES:B104:03-F1 ES:B130:03-F3 ES:T31:03-F5 ES:B428:03-F4 ES:B145:03-F2 ES:B152:03-F2 ES:B115:03-F2 ES:B101:03-F ES:B99:03-F1 ES:B110:03-F2 ES:B143:03-F1 ES:B121:03-F3 61 ES:B123:03-F3 ES:B106:03-F1 ES:B416:03-F2 ES:B98:03-F1 ES:B422:03-F3 ES:B134:03-F1 ES:T64:03-F7 62 ES:B423:03-F3 ES:B432:03-F4 ES:B147:03-F2 ES:B138:03-F1 ES:B142:03-F1 ES:B119:03-F3 TYLCMalV-[ES:421:99] ES:B111:03-F [IT:Sic:05] IT:T529:02-F2 IT:T535:02-F2 IT:T570:02-F4 IT:T573:02-F4 IT:T568:02-F4 -[IL] ES:T223:03-F ES:T15:03-F3 ES:T249:03-F5 ES:T239:03-F5 ES:T323:03-F1 ES:T398:03-F9 ES:T387:03-F8 ES:T245:03-F5 ES:T162:03-F1 ES:T333:03-F2 ES:T190:03-F3 ES:B437:03-F5 ES:T206:03-F7 ES:T409:03-F9 ES:T3:03-F2 ES:T84:03-F8 ES:T158:03-F1 ES:T365:03-F6 ES:T383:03-F7 ES:T318:03-F1 ES:T6:03-F2 ES:T80:03-F8 ES:T341:03-F3 ES:T177:03-F2 ES:T305:03-F9 ES:T268:03-F6 ES:T70:03-F7 ES:T400:03-F9 -[ES:Alm] ES:B434:03-F4 ES:T327:03-F2 ES:T337:03-F3 ES:T167:03-F1 ES:T196:03-F3 ES:T281:03-F8 ES:T48:03-F1 ES:T16:03-F3 ES:T28:03-F4 ES:T188:03-F3 100 ES:T362:03-F5 ES:T377:03-F7 ES:T357:03-F5 98 ES:B122:03-F3 81 ES:B139:03-F1 56 ES:T21:03-F4 -Mld[ES:72:97] 62 IT:T538:02-F2 76 IT:T310:99-F4 IT:T511:02-F1 IT:T561:02-F3 97 IT:T548:02-F3 90 IT:T284:99-F1 IT:T289:99-F IT:T306:99-F3 74 IT:T292:99-F2T IT:T303:99-F3 -[IT:Sar] 70 IT:T287:99-F1 IT:T315:99-F4 IT:T313:99-F4 IT:T293:99-F2 88 IT:T304:99-F3 -Sic[IT] IT:T500:02-F1 64 IT:T558:02-F3 100 IT:T286:99-F1 IT:T506:02-F1 100 ES:T36:03-F5 ES:T57:03-F6 60 -ES[ES:92:1] -IL - Mld -Sar -Sic -ES TYLCMalV Emergence of recombinants: Population structure change (Spain common bean) Displacement of by TYLCMalV in common bean O Italy (tomato) O Spain (common bean) O Spain (tomato) (García-Andrés, et al Virology 359: ) 0.05

18 Recombination as source of genetic diversity During co-infections of a single plant -ES + -Mld Virus population analysis [days post-infection (dpi)] Tomato plants Moneymaker TY-1S MmA MmB 20 dpi dpi dpi dpi 400 dpi 400 dpi

19 RFLP analysis 25 clones/date Moneymaker TY-1S MmA MmB 20 dpi dpi dpi dpi 400 dpi 400 dpi V2 REn C4 TrAP CP Rep IR Dra III Sph I Eco NI Pml I Sac II Kpn I Bgl II Bgl I Kpn I -Mld -ES Hind III -Mld -ES R1 R2 R3 R4 Sequence analysis (García-Andrés, et al Virology 365: ) Restriction Mm A Mm B Ty-1S Haplotype pattern 20 dpi 60 dpi 130 dpi 400 dpi 400 dpi 400dpi I II R1 III R2 IV V VI VII VIII IX R3 X XI R4 XII Incidence of recombinants 0/25 0/25 12/25 12/25 7/25 13/25

20 Factors driving emergence of TYLCD-associated viruses in SPAIN migration -ES -Mld -IL recombination TYLCMalV TYLCAxV -ES -ES + -Mld -ES + -Mld + -IL -ES -ES + + -Mld -IL + + -IL -Mld + + TYLCMal TYLCMal + TYLCAxV

21 WILD RESERVOIRS: Solanum nigrum populations as a study case (Málaga, Spain, ) IR C4 V2 Rep nt CP TrAP REn (García-Andrés, et al Virology 350: ) AYVV ES:Sn29:00 ES:Sn4-1:03 ES:Sn15-1:03 ES:Sn30-1:00 ES:Sn8-1:00 -ES[ES:1:92] ES:Sn16:00 ES:Sn34:00 ES:Sn10-1:00 ES:Sn2:02 TYLCMalV-[ES:421:99] 970 ES:Sn27: ES:Sn28-1:00 ES:Sn13: ES:Sn6-1: Mld[ES:72:97] ES:Sn10-2:00 ES:Sn11:00 ES:Sn5-1:00 -[ES:Alm] ES:Sn8-2:00 ES:Sn3-1:01 ES:Sn35:03 ES:Sn25: ES:Sn5-2:00 ES:Sn15-2:03 ES:Sn30-2:00 ES:Sn15-3:00 ES:Sn32:00 ES:Sn3-2: ES:Sn12:01 ES:Sn8-3:00 ES:Sn5-3:00 ES:Sn4-2:03 ES:Sn30-3:00 ES:Sn1:03 ES:Sn28-2:00 ES:Sn19:03 ES:Sn6-2:00 TYLCAxV-[ES:Alg:00] 939

22 TYLCD emergence: use of GENETIC RESISTANCE Resistance genes can be selective: TYLCD complex ToLCV TYLCKaV-[TH:Kan1] TYLCGuV-[CN:G3] TYLCCNV-[CN:Gua] TYLCTHV-[TH:1] TYLCMalV-[ES:421:99] TYLCAxV-[ES:Sn8:00] -ES[ES:1:92] -[IT] -Sic[IT] -Gez[SD] 911 -Mld[IL] 932 -[IL] -IR TYLCMLV TYLCKaV TYLCGuV TYLCCNV TYLCTHV TYLCMalV TYLCaXV TYLCGezV TYLCMLV Ty-1 RESISTANCE: near isogenic lines (UPV, Valencia): Anastasia (resistant, R) R heterozygous (Ty1ty1) Susceptible (ty1ty1) R homozygous (Ty1Ty1) Moneymaker V V V V V V V V V V Plant number C squash blot (30 days post inoculation) García-André et al Virus Res 146:

23 vs. in COMMERCIAL HYBRIDS resistant to TYLCD (Ty-1 gene?): Yamile Yanira Dulcinea Anastasia Moneymaker 15 dpi 30 dpi 45 dpi change in virus population structure resistance breakdown???

24 EVOLUTION OF POPULATION STRUCTURE: Effect of use of resistance Susceptible Resistant (Ty-1 gene) >1998 Samplings Year Variety Nº of samples Nº (%) samples positive for Susceptible (100) Susceptible (100) Susceptible (66,1) 12 (18,5) 10 (15,4) Resistant 62 4 (6,5) 41 (66,1) 17 (27,4) 2001 Susceptible 63 0 (0) 48 (98) 1 (2) Resistant 67 0 (0) 55 (100) 0 (0) (García-Andrés, et al Virus Res 146:66-72) 2002 Susceptible (8,9) 77 (85,6) 5 (5,5) Resistant (0) 43 (100) 0 (0)

25 Main conclusions - Several factors are driving emergence of virus diseases (migration, transmission, virus recombination, human manipulation,..) - Emergence can result in great epidemiological alterations - Understanding emergence can help durable virus control

26 Virus emergence FUTURE WORK Understanding virus-hosts interactions associated with of host range Understanding mechanisms associated with recombination Understanding virus-vector interactions and emergence

27 who performed the work.. SPAIN (CSIC, Málaga). Enrique Moriones. Jesús Navas-Castillo phd students. Sonia Sánchez-Campos (population studies). Susana García-Andrés (population studies, resitance studie). Francisco Monci (recombination studies). Diego M. Tomás (recombination studies, resistance studies) ITALY (IVV, Torino) Gian Paolo Accotto (population p studies)

28 Plant Virus Laboratory Plant Virus Laboratory Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora Algarrobo-Costa, Málaga, SPAIN