Update presentation by Dr Hoat from PPRI Vietnam

1. UPDATE REPORT
CASSAVA MOSAIC VIRUS DISEASE (SLCMV)
IN VIETNAM
Plant Protection Research Institute, Vietnam
Laos, 9/2019

2. • Cassava production areas in
seven agro-ecological zones in
Viet Nam. Each dot represents
1,000 ha cassava.
• About 600,000 ha.

3. Pseudococcus
jackbeardsleyi
Red mite
Phenacoccus
manihoti
B. tabaci
Paracoccus
marginatus
Ferrisia virgata

4. Anthracnose disease
(Colletotrichum
gloeosporioides)
Cassava Bacterial Blight
(Xanthomonas campestris
pv. Manihotis)
Root rot
Cassava Witches Broom
disease
Cassava Mosaic Disease
(Sri Lankan Cassava
Mosaic Virus)

5. Pink Mealybug
(Phenacoccus manihoti)
in Tay Ninh Vietnam
In 2012
Outbreak
in 2008
Anagyrus lopezi

6. In 2010
Cassava Witches Broom
in Quang Ngai Province,
Vietnam

7. A B
C D
E F
E F
Typical symptoms of Cassava Witches Broom
disease

8. Cassava witches’-broom (AY787139) - - - - - - - - - - - - - - - - - - - -
 Cassava witchesCassava witches’’--broom isolatebroom isolate KontumKontum ((JN381550)JN381550) - - - - - - -
 Cassava witchesCassava witches’’--broom isolate Dongbroom isolate Dong NaiNai ((JN381549)JN381549) - - - - - -
 Cassava witchesCassava witches’’--broom isolatebroom isolate BaBa RiaRia--VungTauVungTau ((JN381547)JN381547)--
 Cassava witchesCassava witches’’--broom isolatebroom isolate QuangQuang NgaiNgai ((JN381548)JN381548) - - - -
'Brassica napus' phyllody phytoplasma (JN193482) - - - - - - - - - - - - - -
Sesamum indicum phytoplasma (JN006072) - - - - - - - - - - - - - - - - - - -
Australian grapevine yellows ( L76865) - - - - - - - - - - - - - - - - - - - - - - - -
Stolbur phytoplasma (AF248959) - - - - - - - - - - - - - - - - - - - --
Spartinum witches’-broom (X92869) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Apple proliferation (AF248958) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Faba bean phyllody (EF193354) - - - - - - - - - - - - - - - - - - - - - - - - - -
Papaya yellow crinkle (Y10097) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Hibiscus witches’-broom (AF147708) - - - - - - - - - - - - - - - - - 16SrXV
Weeping tea tree witches’-broom (AF521672) - - - - - - - - - - - - - - - - - - - - - - - - - - - 16SrXXV
Cassava frogskin disease (AY737646) - - - - - - - - - - - -- - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - 16SrIII
Luffa witches’-broom (AF086621) - - - - - - - - - - - -- - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - 16SrVIII
Rice yellow dwarf (AB052873) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Sugarcane grassy shoot (AM261831) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
98
100
96
82
84
100
100
95
100
100
0.01
Acholeplasma laidlawii (M23932)
16SrXI
16SrII
16SrX
16SrXII
16SrI
Nhóm 16S
Cassava witches’-broom (AY787139) - - - - - - - - - - - - - - - - - - - -
 Cassava witchesCassava witches’’--broom isolatebroom isolate KontumKontum ((JN381550)JN381550) - - - - - - -
 Cassava witchesCassava witches’’--broom isolate Dongbroom isolate Dong NaiNai ((JN381549)JN381549) - - - - - -
 Cassava witchesCassava witches’’--broom isolatebroom isolate BaBa RiaRia--VungTauVungTau ((JN381547)JN381547)--
 Cassava witchesCassava witches’’--broom isolatebroom isolate QuangQuang NgaiNgai ((JN381548)JN381548) - - - -
'Brassica napus' phyllody phytoplasma (JN193482) - - - - - - - - - - - - - -
Sesamum indicum phytoplasma (JN006072) - - - - - - - - - - - - - - - - - - -
Australian grapevine yellows ( L76865) - - - - - - - - - - - - - - - - - - - - - - - -
Stolbur phytoplasma (AF248959) - - - - - - - - - - - - - - - - - - - --
Spartinum witches’-broom (X92869) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Apple proliferation (AF248958) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Faba bean phyllody (EF193354) - - - - - - - - - - - - - - - - - - - - - - - - - -
Papaya yellow crinkle (Y10097) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Hibiscus witches’-broom (AF147708) - - - - - - - - - - - - - - - - - 16SrXV
Weeping tea tree witches’-broom (AF521672) - - - - - - - - - - - - - - - - - - - - - - - - - - - 16SrXXV
Cassava frogskin disease (AY737646) - - - - - - - - - - - -- - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - 16SrIII
Luffa witches’-broom (AF086621) - - - - - - - - - - - -- - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - 16SrVIII
Rice yellow dwarf (AB052873) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Sugarcane grassy shoot (AM261831) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
98
100
96
82
84
100
100
95
100
100
0.01
Acholeplasma laidlawii (M23932)
16SrXI
16SrII
16SrX
16SrXII
16SrI
Nhóm 16S
Phylogenetic tree analysis for identification of phytoplasma causing Cassava Witches
Broom disease in Vienam

9. Transmission of Cassava Witches Broom
disease:
1. Planting material 2. Vectors of CaWB:
unidentified

10. June 2017
SLCMV was
detected firstly in
Tay Ninh province
PPSD of Tay Ninh
Province

11. Collection of infected leaves
Collection of whitefly in the infected plants

12. PC1
PC2
NC1
NC2
M
Primer F1/R2
1 2 3 4 5 6 7 8 9 10
SLCMV (AJ890229)
ICMV (AY998122)
SLCMV (KT861468)
ICMV (AY769966)
SlCMV (KU550961)
SLCMV (KP455486)
H1
H11
ICMV (KU308385)
SLCMV (Cambodia)
ICMV (KU550960)
SLCMV (AJ890226)
JMIV (AM296493)
JMV (JN692494)
ICMV (EU113300)
JMIV (JN704612)
JMIV (JN704614)
AYMV (JN807769)
CLCuV (LC080677)
ChiLCMV (HM587709)
ToLCBV (KM383761)
RaLCV (KF218188)74
91
98
99
99
99
67
98
0.02
1 2
3 4
5 6
7 8
9 10

14. Identification the transmission of SLCMV via planting material
PCR -++

15. Plant was inoculated by
SLCMV-infected whiteflies
Plant was inoculated by
SLCMV-free whiteflies
PCR -+

16. Host plant
SLCMV-infected
plant
Environment
Human
“Vectors”Inoculum
SLCMV-infected
whitefly
Whitefly
MANAGEMENT OF CASSAVA MOSAIC VIRUS DISEASE
IN VIETNAM

18. TT Province
Total infected area (ha) Total destroyed
area (ha)Total Light Medium High
1 Tay Ninh 25,064.4 16,550.0 4,184.2 4,33.2 143.2
2 Binh Phuoc 320.0 240.0 80.0 0.0 -
3 Binh Duong 1,828.5 481.0 786.5 561.0 -
4 Ba Ria-Vung Tau 31.0 31.0 0.0 0.0 -
5 Ho Chi Minh 40.2 40.2 0.0 0.0 -
6 Long An 234.0 170.0 17.0 47.0 5.0
7 An Giang 15.3 15.3 0.0 0.0 -
8 Đong Nai 616.7 580.4 32.0 4.3 42.4
9 Phu Yen 615.7 136.5 244.4 234.8 25.2
10 Ninh Thuan 223.1 134.1 83.0 6.0 31.6
11 Binh Thuan 370.0 15.0 213.0 142.0 -
12 Gia Lai 1,817.8 1.376.4 423.0 18.4 73.8
13 Kon Tum 365.0 290.0 56.0 20.0 -
14 Đak Lak 394.5 110.5 7.0 277.0 -
15 Ha Tinh 170.3 170.3 0.0 0.0 -
16 Quang Ngai - - - - -
Total 31,865.5 20,100.7 6,144.1 5,620.7 321.2
Total of CMV-infected growing area (ha) (10/9/2019)

19. Effect of SLCMV on cassava yield in Tay Ninh province, 2019
Variety
On yield On starch content On stalk and leaves yield
Yield
(tons/ha)
Reduction
percentage (%)
Starch content
Reduction
percentage (%)
Stalk and leaves
yield (tons/ha)
Reduction
percentage (%)
Healthy HL-S11
15.30-24.53 1.02-7.30 5.88-39.14
SLCMV- HL-S11
Healthy KM94
12.65-19.23 2.48-18.97 14.48-37.68
SLCMV- KM94
Healthy KM419
15.08-34.55 4.49-8.94 3.62-38.97
SLCMV- KM419
Healthy KM140
22.74-45.74 5.69-10.16 8.66-45.65
SLCMV- KM140

20. IDENTIFICATION OF BIOTYPES OF WHITEFLY (B. TABACI)
BY NUCLEOTIDE SEQUENCE OF ITS MITOCHONDRIAL COX1
GENE

21. Begomoviruses are transmitted from plant to plant only by the vector
whitefly Bemisia tabaci
1 mm
Bemisia tabaci Med
The mode of transmission is
● circulative,
● non-replicative
transmission

22. COXF1/COX1R 2195Bt/CO12/Bt-sh2
2195Bt (5’-TGRTTTTTTGGTCATCCRGAAGT-3’)
CO12/Bt-sh2 (5’-TTTACTGCACTTTCTGCC-3’)
(Mugerwa ET AL., 2018)
COXF1(5’-TGRTTYTTTGGTCATCCVGAAGT-3’)
COX1R(5’-TTACTGCACTTTCTGCCACATTA-3)
(Yayaka et al., 2019)
M 1 2 3 4 5 M 1 2 3 4 5
M: 100 bp DNA marker
1-4: Whitefly samples
5: No DNA negative control

23. Bemisia tabaci Asia II 1
The whitefly Bemisia tabaci has long been considered
one species composed of several biotypes, but now it is reclassified as a
complex of at least 24 species
Bemisia tabaci New World
Bemisia tabaci Mediterranean
current classification
Bemisia tabaci Middle-East Asia Minor1
species
Bemisia tabaci
species
Bemisia tabaci Asia 1
biotypes
biotype H
biotype M
biotype K
biotype P
biotype A
biotype C
biotype Q
biotype J
biotype B
Former classification
Viet Nam

24. CONFIRMATION THE PRESENCE OF ACMV & EACMV FROM CASSAVA
SAMPLES COLLECTED FROM TAY NINH PROVINCE

25. M
CMBRep-F/ACMVRep-R
CMBRep-F/EACMVRep-R
500 bp
300 bp
700 bp
Samples:
TN1-TN10: Cassava leaf
samples collected from
Tay Ninh in 2019
11: Positive control
(collected from Tay
Ninh in 2017 and was
positive with SLCMV)
12: Negative control
(Healthy leaf)
Primers:
CMBRep-F: (5’-CATCAAATGC GTTGT ACCA-3’
ACMVRep-R: (5’- CAGCG GCAGT AAGTC CGA-3’
EACMVRep-R: (5’-GGTTT GCAGA GAACT ACATC-3’)
PCR program:
94oC-4, 30 cycles (94oC-30’’, 55oC-30’’, 72oC-30’’), 72oC-4’
TN1
TN2
TN3
TN4
TN5
TN6
TN7
TN8
TN9
TN10
NC
PC
TN1
TN2
TN3
TN4
TN5
TN6
TN7
TN8
TN9
TN10
NC
PC

26. >1st_BASE_3554870_AC1_CMBRep_F
AGGACTCAGTCCAGATGTCCACACAGATAATTGTGTGGACCTAATGACCTAGCCCACATTGTTTTGCCTGTACGACTATCGCCCTC
GATGACTATACTATTAGGTCTCAAAGGCCTCGCAGAGGCACCCATGACGTTCTCGGACACCCACTCCTCAAGTTCATCTGGAACTT
GGTCAAATGAAGAGGCTGAGAAGGGAGACACATATACCTCGGGAGGAGGTGTAAAAATCCTATCTAAATTAGCATTTAGATTATG
AAATTGTAAAACATAATCCTTGGGTGCTAACTCCTTAATGACTCTAAGAGCCTCGGACT
>1st_BASE_3554871_AC7_CMBRep_F
AGGACTCAGTCCAGATGTCCACACAGATAATTGTGTGGACCTAATGACCTAGCCCACATTGTTTTGCCTGTACGACTATCGCCCTC
GATGACTATACTATTAGGTCTCAAAGGCCTCGCAGAGGCACCCATGACGTTCTCGGACACCCACTCCTCAAGTTCATCTGGAACTT
GGTCAAATGAAGAGGCTGAGAAGGGAGACACATATACCTCGGGAGGAGGTGTAAAAATCCTATCTAAATTAGCATTTAGATTATG
AAATTGTAAAACATAATCCTTGGGTGCTAACTCCTTAATGACTCTAAGAGCCTCGGACT
>1st_BASE_3554872_AC10_CMBRep_F
AGGACTCAGTCCAGATGTCCACACAGATAATTGTGTGGACCTAATGACCTAGCCCACATTGTTTTGCCTGTACGACTATCGCCCTC
GATGACTATACTATTAGGTCTCAAAGGCCTCGCAGAGGCACCCATGACGTTCTCGGACACCCACTCCTCAAGTTCATCTGGAACTT
GGTCAAATGAAGAGGCTGAGAAGGGAGACACATATACCTCGGGAGGAGGTGTAAAAATCCTATCTAAATTAGCATTTAGATTATG
AAATTGTAAAACATAATCCTTGGGTGCTAACTCCTTAATGACTCTAAGAGCCTCGGACT

27. >1st_BASE_3554873_EAC3_CMBRep_F
AGTCCAGATGTCCACACAGATAATTGTGTGGACCTAATGACCTAGCCCACATTGTTTTGCCTGTACGACTATCGCCCTCGATGACTATACTATTAGGTCTCAAAGGCCTCGCAG
AGGCACCCATGACGTTCTCGGACACCCACTCCTCAAGTTCATCTGGAACTTGGTCAAATGAAGAGGCTGAGAAGGGAGACACATATACCTCGGGAGGAGGTGTAAAAATCCT
ATCTAAATTAGCATTTAGATTATGAAATTGTAAAACATAATCCTTGGGTGCTAACTCCTTAATGACTCTAAGAGCCTCTGGCTTACTGCCTGTGTTAAGTGCTGCGGCGTAAGCG
TCGTTTGCTGACTGCTGTCCCCCTCTTGTAGATCGTCCATCGATCTGAAACTCACCCCAGTCGATGGTGTCTCCATCCTTGTCGATGTAGGACTTGACGTCTGAGCTGGACTTA
GCGCTCTGTATGTTGGGGTGGAAACTGGTGCTACTGCTTGGGTGTACACAATCGAATTGCCGATTGTTTGTGATTGTGAGCTTCCCTTCGAACTGAAGCAGAGCATGGAGGTG
AGGTTCCCCATTCTGATGTAGTTC
>1st_BASE_3554874_EAC4_CMBRep_F
AGTCCAGATGTCCACACAGATAATTGTGTGGACCTAATGACCTAGCCCACATTGTTTTGCCTGTACGACTATCGCCCTCGATGACTATACTATTAGGTCTCAAAGGCCTCGCAG
AGGCACCCATGACGTTCTCGGACACCCACTCCTCAAGTTCATCTGGAACTTGGTCAAATGAAGAGGCTGAGAAGGGAGACACATATACCTCGGGAGGAGGTGTAAAAATCCT
ATCTAAATTAGCATTTAGATTATGAAATTGTAAAACATAATCCTTGGGTGCTAACTCCTTAATGACTCTAAGAGCCTCTGGCTTACTGCCTGTGTTAAGTGCTGCGGCGTAAGCG
TCGTTTGCTGACTGCTGTCCCCCTCTTGTAGATCGTCCATCGATCTGAAACTCACCCCAGTCGATGGTGTCTCCATCCTTGTCGATGTAGGACTTGACGTCTGAGCTGGACTTA
GCGCTCTGTATGTTGGGGTGGAAACTGGTGCTACTGCTTGGGTGTACACAATCGAATTGCCGATTGTTTGTGATTGTGAGCTTCCCTTCGAACTGAAGCAAAGCATGGAGGTG
AGGTTCCCCATTCTGATGTAGTTC
>1st_BASE_3554875_EAC7_CMBRep_F
AGTCCAGATGTCCACACAGATAATTGTGTGGACCTAATGACCTAGCCCACATTGTTTTGCCTGTACGACTATCGCCCTCGATGACTATACTATTAGGTCTCAAAGGCCTCGCAG
AGGCACCCATGACGTTCTCGGACACCCACTCCTCAAGTTCATCTGGAACTTGGTCAAATGAAGAGGCTGAGAAGGGAGACACATATACCTCGGGAGGAGGTGTAAAAATCCT
ATCTAAATTAGCATTTAGATTATGAAATTGTAAAACATAATCCTTGGGTGCTAACTCCTTAATGACTCTAAGAGCCTCTGGCTTACTGCCTGTGTTAAGTGCTGCGGCGTAAGCG
TCGTTTGCTGACTGCTGTCCCCCTCTTGTAGATCGTCCATCGATCTGAAACTCACCCCAGTCGATGGTGTCTCCATCCTTGTCGATGTAGGACTTGACGTCTGAGCTGGACTTA
GCGCTCTGTATGTTGGGGTGGAAACTGGTGCTACTGCTTGGGTGTACACAATCGAATTGCCGATTGTTTGTGATTGTGAGCTTCCCTTCGAACTGAAGCAGAGCATGGAGGTG
AGGTTCCCCATTCTGATGTAGTTC
>1st_BASE_3554876_EAC8_CMBRep_F
AGTCCAGATGTCCACACAGATAATTGTGTGGACCTAATGACCTAGCCCACATTGTTTTGCCTGTACGACTATCGCCCTCGATGACTATACTATTAGGTCTCAAAGGCCTCGCAG
AGGCACCCATGACGTTCTCGGACACCCACTCCTCAAGTTCATCTGGAACTTGGTCAAATGAAGAGGCTGAGAAGGGAGACACATATACCTCGGGAGGAGGTGTAAAAATCCT
ATCTAAATTAGCATTTAGATTATGAAATTGTAAAACATAATCCTTGGGTGCTAACTCCTTAATGACTCTAAGAGCCTCTGGCTTACTGCCTGTGTTAAGTGCTGCGGCGTAAGCG
TCGTTTGCTGACTGCTGTCCCCCTCTTGTAGATCGTCCATCGATCTGAAACTCACCCCAGTCGATGGTGTCTCCATCCTTGTCGATGTAGGACTTGACGTCTGAGCTGGACTTA
GCGCTCTGTATGTTGGGGTGGAAACTGGTGCTACTGCTTGGGTGTACACAATCGAATTGCCGATTGTTTGTGATTGTGAGCTTCCCTTCGAACTGAAGCAGAGCATGGAGGTG
AGGTTCCCCATTCTGATGTAGTTC

29. 1 8 12 15 16 17
CMBRep/F-ACMVRep-EACMVRep/R
M 19 29 NC
Samples:
+ From 1-29: Cassava leaves collected
from Tay Ninh in 2018
+ NC: Negative control (Healthy leaf)
CONFIRMATION THE PRESENCE OF ACMV AND EACMV FROM CASSAVA SAMPLES
COLLECTED FROM TAY NINH PROVINCE WITH MORE SPECIFIC PRIMER PAIRS
500 bp
300 bp
700 bp

31. EikenChemical Co., Ltd., JPN

32. LAMP procedure
• Break the leaf surface with a wooden toothpick
• Wash the toothpick in a reaction mixture
• Keep the tube in hot water for 30 min
• Observe by bare eyes or under UV

33. Labor: Easy Laborious
Time: Fast Slow
Sensitivity: High Thấp/Low
Cost: Inexpensive Expensive
On-site: Yes No
LAMP to detect genome DNA

34. Detection of DNA by loop-mediated isothermal
amplification (LAMP)
Negative: No florescence Positive:
Greenfluorescence

35. Merit of LAMP
cutting from a diseased
plant
cutting from a healthy
plant
(ii) On-site diagnosis also for cuttings

36. PCR vs LAMP
PCR LAMP (dried kit)
sensitivity very high extremely high
primers two four or six
polymerase Taq (kept in –20℃) Bst (dried)
temperature
92oC
55oC (Cycling)
72oC
60-68℃ (one temp)
detection electrophoresis eyes (light or UV)

37. PCR LAMP (dried kit)
sensitivity very high extremely high
primers two four or six
polymerase Taq (kept in –20℃) Bst (dried)
temperature
92oC
55oC (Cycling)
72oC
60-68℃ (one temp)
detection electrophoresis eyes (light or UV)
PCR vs LAMP
DNA extraction by a
centrifuge is NOT
needed
-20℃ freezer is NOT
needed
Thermal cycler is NOT
needed
Electrophoresis
apparatus is NOT
needed

39. Root rot disease in Binh Duong province

40. Fall Armyworm (Spodoptera frugiperda (Smith)) [Lepidoptera: Noctuidae]
Male adult Female adult
Egg mass
Pupa
Mature larvaCorn leaf damage

41. Molecular Identification of Fall Armyworm

42. Thanks for your attention