HUANG-LONG-BING (HLB), AWARENESS.

Transcription

1 HUANG-LONG-BING (HLB), a destructive, newly-emerging, century-old disease of citrus: HISTORY, TRANSMISSION, ETIOLOGY, DISTRIBUTION, DIAGNOSTIC, DAMAGE, CONTROL, and AWARENESS. Josy Bové, emeritus professor of microbiology, University of Bordeaux 2 and INRA, France.

2 I. HISTORY and EARLY ACHIEVEMENTS 1. CHINA. 1. Disease mentionned as «huanglongbing» as early as Lin Kung Hsiang (1956): first transmission of «huanglongbing» by graft-inoculation. 3. Huang long bing (Yellow shoot disease) becomes the official name of the disease in SOUTH AFRICA. A.P. McClean and P.C. Oberholzer (1965): the African psyllid, Trioza erytreae, is a vector. 3. INDIA. S. P. Capoor (1967): Diaphorina citri, the Asian psyllid, is a vector in India. 4. PHILIPPINES. A.L.Martinez and J.M.Wallace (1967): Diaphorina citri is also a vector in the Philippines. 5. France. D. Laflèche and J. Bové. (1970): the agent of HLB is not a virus, but a bacterium.

3 Yellow shoot disease = huang long bing [Guilin-Yangshuo region (Guangxi), 2005]

4 GREENING! 1 normal orange 3 greening - affected sweet oranges South Africa

5 I.5. CAUSAL AGENT OF HLB: not a virus, but a bacterium!

6 II. TRANSMISSION of HLB 1. TRANSMISSION by GRAFTING GRAFT INOCULATION GRAFT PROPAGATION 2. TRANSMISSION by PSYLLID INSECTS Trioza erytreae Diaphorina citri 3. TRANSMISSION by DODDER (Cuscuta)

7 II.2. TRANSMISSION by PSYLLID INSECTS IN NATURE: Trioza erytreae, the African psyllid, transmits Ca. L. africanus: McCLEAN and OBERHOLZER, 1965 Diaphorina citri, the Asian psyllid, transmits Ca. L. asiaticus: CAPOOR et al.,1967; MARTINEZ and WALLACE, 1967; Ca. L. americanus: YAMAMOTO et al., 2006 EXPERIMENTALLY: Trioza erytreae transmits Ca. L. asiaticus: MASSONIE et al., 1976 Diaphorina citri transmits Ca. L. africanus: LALLEMAND et al., 1986

8 Diaphorina citri, the Asian psyllid vector of HLB (Martinez & Wallace, 1967; Capoor et al., 1967) Reported in D. citri HLB Brasil U.S.A

9 Diaphorina citri nymphs and adults on Mexican lime (Citrus aurantifolia) shoots in Saudi Arabia. Nymphs, when feeding on HLB-trees, may become infected. They yield infected adults, which are able to immediately transmit the HLB bacterium.

10 Trioza erytreae adult on young lemon leave, Nelspruit, South Africa

11 Yellow eggs on Lemon Lemon Lemon leaf Lower side Nymph nests Nymphs of Trioza erytreae make a nest on lower leaf side and produce bumps on upper leaf side. Todalia sp. Upper side Nymph bumps Nymph bumps Lemon

12 ECTOPARASITES, ENDOPARASITES and HYPERPARASITES of CITRUS PSYLLIDS. Trioza erytreae HYPERPARASITE Aphidencirtus cassatus Diaphorina citri ECTO- PARASITE T. dryi Tamarixia T. radiata P. pulvinatus Psyllaephagus harrisoni ENDOPARASITE

13 Nymphs of psyllids parasitized by the ectoparasite Tamarixia: hole in the nymph. Parasitized nymphs Tamarixia Non- parasitized nymph

14 Biological control of the two psyllids in Reunion island was excellent because: 1. Initially, the two psyllids were present in the absence of psyllid- parasites. 2. Psyllid-parasites (Tamarixia spp.), free of psyllidhyperparasites, were imported, reared and released. 3. Tamarixia dryi, the parasite of Trioza erytreae, was able to parasite another psyllid, Trioza eastopi, breeding on a non-citrus weed, Litsea chinensis. Thus, the population of T. dryi was stronly increased, resulting in the disappearance of Trioza erytreae (Aubert & Quilici, 1984).

15 III. ETIOLOGY Until the late 1960s, the HLB agent was considered a virus. 1970: bacteria were observed by electron microscopy in the sieve tubes of HLB-afffected sweet orange seedlings by Dominique Laflèche & J. M. Bové in France, and further studied by Monique Garnier et al. in the ensuing years. 1971: the HLB bacterium possessed a cell wall, and was not mycoplasma-like. 1984: Monique Garnier et al. demonstrated by electron microscopy that the HLB organism was a Gram-negative bacterium.

16 CHARACTERIZATION of the HLB bacterium The HLB bacterium has not yet been obtained in culture. On the basis of molecular characterizations, there are 3 HLB bacteria and they belong to the α-proteobacteria (Gram negative bacteria); they have been named: Candidatus Liberibacter africanus for the African HLB bacterium, Candidatus Liberibacter asiaticus for the Asian HLB bacterium, also present in São Paulo State, Brasil, and Florida, U.S.A. Candidatus Liberibacter americanus for the American HLB bacterium, the major HLB agent in São Paulo State. The word Candidatus indicates that the bacteria are not available in culture, and have been characterized only by molecular techniques. In the Cape region of South Africa, Calodendron capensis (Rutaceae) is infected with Ca. L. africanus, subsp. capensis

17 IV. DISTRIBUTION of African ( ), Asian ( ), and American ( ) Huanglongbing.

18 MEDITERRANEAN REGION: Free of HLB, but T D : Trioza erytreae, : Diaphorina citri Madeira 1994 T T Canarias 2002 T T D T ~1995 DDDD S-E DDDD Iran D D D

19 Municipalities with Huanglongbing in São S o Paulo State August São Paulo HLB Municipality August Brotas - Aguaí - São Simão - Casa Branca - Boa Esperança do Sul - Santa Rita do Passa Quatro -Avaré 15 municipalities with HLB São Paulo - Araraquara -Matão - São Carlos - Itirapina -Rincão - Luiz Antônio - Tambaú - Taquaritinga

20 May 2007 Colômbia Barretos municipalities with HLB *** Municipalities with comercial Citrus groves C. Liberibacter americanus C. Liberibacter americanus and asiaticus C. Liberibacter asiaticus Results from Centro de Citricultura Barretos and Colombia municipalities São Paulo

21 Characteristics of African, Asian, and American Huanglongbing (HLB) AFRICAN HLB ASIAN HLB AMERICAN HLB Pathogen Ca. L. africanus Ca. L. asiaticus Ca. L.asiaticus (10%) Ca. L. asiaticus Ca. L.americanus (90%) Geographic distribution East Africa, South Africa, Cameroun, Madagascar, Reunion, Mauritius, Saudi Arabia/Yemen Indian subcontinent, South China, South-East Asia Reunion, Mauritius, Saudi Arabia/Yemen São Paulo State, Brazil. Florida,USA. Vector Trioza erytreae Diaphorina citri Diaphorina citri Diaphorina citri Approximate temperature ( C) for symptom expression and insect vector development Heat sensitive HLB Heat tolerant HLB : : Heat tolerant HLB (Ca.L.asiat.) Ca.L.american.? Heat tolerant HLB

22 V. DIAGNOSTIC 1. Symptomatology 2. Electron microscopy 3. DNA hybridization 4. Polymerase Chain Reaction (PCR) 1. Conventional PCR Amplification of 16S rdna Amplification of rpl (ribosomal protein) gene Multiplex 2. Nested PCR 3. Real Time (RTi) PCR

23 Yellow shoot disease = huang long bing [Guilin-Yangshuo region (Guangxi), 2005]

24 South Africa

25 South Africa

26 South Africa

27 OK HLB BRASIL J. Bové, INRA, Bordeaux, Fran

28 BRASIL J Bové INRA Bordeaux France

29 LEAF SYMPTOMS of HLB: Blotchy mottle 1. MOTTLE, but not BLOTCHY! 2. BLOTCHY MOTTLE ON - sweet orange leaves, - mandarin leaves, - other citrus leaves.

30 MOTTLE, but NOT BLOTCHY

31 MOTTLE, but NOT BLOTCHY PETRI S VARIEGATION in TUNISIA on sour orange

32 BLOTCHY MOTTLE! South Africa

33 BLOTCHY MOTTLE

34 São Paulo

35 BLOTCHY MOTTLE South Africa

36 BLOTCHY MOTTLE Behai, CHINA

37 Behai, CHINA

38 Blotchy mottle on LEMON, South Africa

39 Upper side Lower side Leaf # 1 clean Lower side is clean: mottle on upper side is meaningful Leaf # 2 Lower side is dirty: mottle on upper side is meaningless dirty

40 INDIA, Coorg

41 Deficiency symptoms MANGANESE ZINC

42 BLOTCHY MOTTLE No SYMETRY Zn DEFICIENCY ~ SYMETRY BLOTCHY MOTTLE No SYMETRY

43 Fruit drop South Africa

44 Silvery finger mark South Africa

45 On normal fruit, coloration starts at stylar end

46 On HLB-affected fruit, coloration starts at pedoncular end: color inversion! PCR + KARMITAR, NEPAL

47 COLOR INVERSION starts at site of peduncle-insertion on fruit Orange-stained vascular bundles

48 LOPSIDED FRUIT with orange-stained vascular bundles and brownish aborted seeds PCR + KARMITAR, NEPAL

49 China MANDARINS Bali Thailand J Bové INRA Bordeaux France

50 V. DIAGNOSTIC (Continued) 1. Symptomatology 2. Electron microscopy 3. DNA hybridization 4. Polymerase Chain Reaction (PCR) 1. Conventional PCR Amplification of 16S rdna Amplification of rpl (ribosomal protein) gene Multiplex 2. Nested PCR 3. Real Time (RTi) PCR

51 MULTIPLEX PCR DETECTION of Ca. L. americanus and/or Ca. L. asiaticus M1 Am As Af Am+As E M2 Controls Samples americanus asiaticus africanus Courtesy of D. Teixeira, E. Martins Am: Positive Control for C. L. americanus (1027 bp); As: Positive Control for C. L. asiaticus (703 bp); Af: Positive Control for C. L. africanus (669 bp); Am + As: Positive Control for C. L. americanus + C. L. asiaticus; Samples E1, 881 and 882: positive for C. L americanus; Samples 797, 811 and 835: positive for C. L. asiaticus; Sample 123: positive for C. L. americanus and C. L. asiaticus. M1 and M2: 1 Kb and 100 bp markers.

52 October 1999 No control! January 2005

53 Florida grove (Valencia + Hamlin), 67,274 trees planted late 2001/ early First survey, Jan./ Febr., Removed trees: 1099 (1.63%) Second survey, October, Removed trees: 9580 (14.2%)

54 VII. CONTROL HLB was reported in São Paulo State in March Control started in july 2004, and is based on: 1. Elimination of liberibacter inoculum, by removal of symptomatic trees. 2. Reduction of psyllid (D. citri) populations by insecticide treatments with ground sprayers and airplanes. 3. Production of healthy trees for resets in closed, insect-proof nurseries. Only preventive control is available. There are no alternative control methods.

55 Identification of symptomatic trees: Young trees: by ground inspection (2 inspectors per raw)

56 Identification of symptomatic trees : Young trees: - by ground inspectors Adult trees: - by platforms with 2 inspectors (2005). - by platforms with 4 inspectors (2006 )

57 Platform with 4 inspectors (2006)

58 After identification, symptomatic trees have to be removed as quickly as possible.

59

60 SYSTEMATIC CONTROL OF THE PSYLLID

61 3. By law, since 2003, nurseries must be insect-proof.

62 São Paulo State

63 RESULTS «S. A.»FARM ( trees) ERADICATION OF INFECTED TREES (HLB) ERADICATED TREES / / /07

64 Fazenda «A» Fazenda Santana - Evolução HLB ,00 Plantas Erradicadas Infection comes from a neighbour farm Only 2 inspections 20,00 15,00 10,00 5,00 Acumulado (%) (1) 2005 (4) 2006 (2) 2007 (2) Período (ano) 0,00 Santana Pt HLB Santana % Errad

65 CONTROL: CONCLUSIONS HLB is worse than Citrus Canker. HLB is like fire! If not controlled, HLB will eventually destroy the citrus industry. Three years of experience in São Paulo State give evidence that HLB can be controlled preventively. Preventive control of HLB requires two actions: removal of symptomatic trees AND insecticide treatments of the psyllid vector population. HLB will not be controlled by applying only one of these actions. Preventive control of HLB is difficult and requires high quality work. As soon as control measures become «relaxed», the disease starts increasing again, and more agressive control measures must be applied. Control of HLB is a joint effort of all growers. Prevent HLB from coming

66 AWARENESS HLB is destructive. HLB spreads fast. Once an insect vector is present, HLB follows soon (Florida) or later (Brasil). In Brasil and Florida, HLB was present, unrecognized, for several years (~ 5 to 10 years) before it was reported. Therefore, when eventually reported, the disease was so widespread that its eradication had become in-feasible. Once established, HLB could never be eradicated. Once HLB is established, one has to live with the disease by preventive control measures. There are no alternative control measures. Therefore, HLB is much more serious than citrus canker for which alternative control measures exist: copper sprays.

67 AWARENESS (continued) HLB is a serious threat for the Mediterranean citrus industry because of the presence of Trioza erytreae in several nearby islands, and Diaphorina citri is in Iran. Biological control of the African psyllid has been highly successful in Reunion island, and could be envisaged for the Atlantic Ocean islands. Is HLB present in the Atlantic Ocean islands? Search for HLB in the islands, and eventually on the mainland, depends on: Frequent surveys by well-trained inspectors able to recognize the disease symptoms, the psyllid vectors, and the Trioza bumps on citrus leaves. Use of PCR techniques to confirm or infirm suspicious symptoms.

68 MOLTES GRACIES MUCHAS GRACIAS MUITO OBRIGADO MERCI BEAUCOUP THANK YOU VERY MUCH