Eduardo Fermino Carlos - Florida Citrus Mutual

Eduardo Fermino Carlos - Florida Citrus Mutual

Eduardo Fermino Carlos
scientific researcher,
http://www.centrodecitricultura.br/
Cordeirópolis, SP, Brazil
[email protected]
Cambuhy Farm, Jan 2005
Tree 1
Tree 2
Tree 3
sample 140) No symptoms (ns)
sample 141) initial vein clearing (ivc)
sample 146) leaf mottle (lm)
sample 142) initial mottling (im)
lm
ns
ivc
M
H
P
N 140 141 142 146
im
Primers: Colleta-Filho et al,
2005
Cambuhy Farm, Jan 2005
nfa
fa
Tree 2
Tree 1
Leaves: PCR +
Leaves: PCR -
sample 141) initial vein clearing (ivc)
sample 140) No symptoms (ns)
sample 142) initial mottling (im)
Fruits: fluorescent albedo (fa) under
UV light
VAN VUUREN (South Africa)
Fruits: non-flourescente albedo
(nfa)
Normative Law number 10, March 18th 2005 (2006; 2008?),
“Greening Task Force”:
Fundecitrus / Grower (2006)
1) Inspection
3) Help
2) Tagging
trees
CDA
Sampling
Centro APTA
Citros Sylvio
Moreira
analysis,
diagnosis,
official reports
Grower
CDA
notification
4) Block erradication (2008?)
4) Corrections
In 2005:
¾ Higher
¾ Only
risk in ~ 80 million trees !!!
for diagnostic ?
• ~ US$ 10.00 / PCR reaction
• Total cost of near US$ 800,000,000.00
(above millions !!! )
¾ Difficulties
to recognize the symptoms
Research actions
How to have a large scale reliable
diagnosis ?
¾ Phloem
bacterium
¾ Low concentration
¾ Not cultured yet !
Agrindus Farm, March 2005
A
B
B
A
C
C
D
D
P N
P P H2O
A B C D
Natal/Swingle
Jan to Mar of 2005,
Dissecting trees:
A) Yellow shoot
C) Blotchy mottle
tree 1
tree 2
P A B C D E
A B C DE
B) Blotchy mottle
tree 4
PA B C DE
tree 5
A B C DE
tree 6
A B C DE
D) nonsymptomatic
E) Roots
fotos: E.F.Carlos
Very didactical in 2005:
Blotchy mottled leaf
Asymetrical chlorosis
“pen method”
fotos: E.F.Carlos
Foto: E.F.Carlos
Training scouts for Fundecitrus and
CDA (2005)
Efficiency of HLB diagnosis
Samples (trees) processed at the Sylvio
Moreira Citrus Center
Fundecitrus transition
Samples for
HLB
New
law
% Negatives
% Positives
Jul
Aug
Sep
Oct
Nov
De
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
De
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
2005
2006
2007
the disease!
HLB (‘yellow shots’)
Not marketable
Fruit drop (‘greening’)
Diaphorina citri (Pedro
Yamamoto)
Fotos: E.F.Carlos
Diagnóstico de HLB (Huanglongbing), ou Greening
Amostras processadas no Centro APTA Citros Sylvio Moreira
Ano mes
Positivas
Negativas
Total %Positivas
Total 2005:
131,425
20,242
151,667
86.7
Total 2006
258,343
16,456
274,799
94.0
Total 2007
24,138
858
24,996
96.6
929
28
957
97.1
Fevereiro
2,975
16
2,991
99.5
Março
5,090
15
5,105
99.7
Abril
19,996
69
20,065
99.7
Maio
12,737
20
12,757
99.8
Junho
11,364
36
11,400
99.7
Julho
17,596
30
17,626
99.8
Parcial 2008
70,687
214
70,901
99.7
Total geral:
484,593
37,770
522,363
92.8
Janeiro
PCR quantitativo de
Tempo Real
f (PCR) = 2*c (até fase linear)
A
B
∆Ct = CtA – CtB
Log (quantificação)
DesPad
CV%
Produto menor, mais eficiente
Novais, C.M., Pires-Alves, M., Silva, F.F. PCR em tempo real. Revista Biotecnologia
Ciência & Desenvolvimento, n.33, julho/dezembro, p.10-13, 2004.
Estratégia para Liberibacters
em São Paulo (2006)
Taqman probes:
Ca. L. asiaticus
Ca L. asiaticus - AB008366
Ca L. asiaticus – São Paulo
Ca L. variante LSg2
101
150
AAAGTACCCAACATCTAGGTAAAAACCTAAACTTGATGGCAACTAGAGGC
AAAGTACCCAACATCTAGGTAAAAACCTAAACTTGATGGCAACTAGAGGC
AAAGTTCCCAAC--------------TTAA---TGATGGCAAATATAGGC
***** ******
***
********* ** ****
Ca. L. americanus
Variantes de Liberibacter:
Texeira et al (Plant Disease, v.89: 107, 2005)
Colleta-Filho et al (Plant Disease, v.89: 848, 2005)
Dragão
amarelo
A
Clorose
assimétrica
BeC
Sem sintoma
D
Experimento:
-5 plantas
-2 reações/amostra
-reações duplex
(18S+Ca.L.am.)
Carlos, et al, 2006)
Sonda: 18S
Sonda: Ca. L. americanus
Plantas com ~ 6 anos
A
B
C
C) Clorose assimétrica
B) Clorose assimétrica
D
D) sem sintomas
A) Dragão
amarelo
∆Ct = CtD – CtABC = 42 – 31 = 11 ciclos:
Detecção em folhas sem sintomas
ABC > D
2*11 = 2048 x
Different to CVC
Pruning does not work with HLB
foto: E.F.Carlos
HLB effect
Losses caused by HLB in São Paulo
¾
¾
¾
Officially: 484,593 (aug, 2008)
Volunteered: ~ 2.5 million trees removed
Total, around 3 million trees lost
Is that “impactant”?
¾
¾
¾
¾
Actual belt with ~ 200 million trees
So, around 1.5% lost in 4 years
Regular grove renew of 6 to 8% per year
New citrus area in the Southwestern SP
HLB em SP
Araraquara
2004
HLB em SP
Cafelândia
Santa Cruz do Rio Pardo
Itatinga
2004
2005
HLB em SP
Viradouro
Júlio Mesquita
Espírito Santo do Pinhal
Campos Novos Paulista
Cesário Lange
2004
2005
2006
Patrocínio Paulista
Pedranópolis
Capão Bonito
2004
2005
2006
2007
2008
Casos
aguardando
confirmação
HLB em SP
2004
2005
2006
2007
2008
Municipalities
Araraquara
São Carlos
Brotas
Sta. Rita do Passa Quatro
Matão
Descalvado
Boa Esperança do Sul
Others
Total
% Total
32.7
10.0
6.8
6.3
6.2
5.7
3.9
28.5
100.0
In HLB restraining
effort:
time is the most
precious factor
HLB = time
Muito obrigado,
Eduardo Fermino Carlos
[email protected]