Primer reporte de ocurrencia en el Perú del virus de la Hoja Blanca, comprobada molecularmente

1. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/313369685
Occurrence and Molecular Detection of Rice hoja blanca virus (Genus Tenuivirus
) in Peru
Article in Plant Disease · March 2017
DOI: 10.1094/PDIS-12-16-1797-PDN
CITATION
1
READS
96
6 authors, including:
Some of the authors of this publication are also working on these related projects:
Biotechnology in tuberous crops for pest and disease resistance View project
CGIAR Research Program on Root, Tubers and Bananas View project
Ana M. Leiva
Consultative Group on International Agricultural Research
12 PUBLICATIONS 51 CITATIONS
SEE PROFILE
Maribel Cruz G.
Latin American Fund for Irrigated Rice
14 PUBLICATIONS 41 CITATIONS
SEE PROFILE
Wilmer J. Cuellar
Consultative Group on International Agricultural Research
131 PUBLICATIONS 1,487 CITATIONS
SEE PROFILE
All content following this page was uploaded by Wilmer J. Cuellar on 27 February 2020.
The user has requested enhancement of the downloaded file.

2. Occurrence and molecular detection of Rice hoja blanca virus
1
(Genus Tenuivirus) in Peru
2
C. Bolaños1
, A. M. Leiva1
, J. Saavedra2
, C. Bruzzone2
, M. Cruz3
, W.J. Cuellar1
*
3
1
Agrobiodiversity Research Area (AgBio), International Center for Tropical Agriculture (CIAT),
4
AA6713, Km 17, Recta Cali-Palmira, Colombia. *email: w.cuellar@cgiar.org
5
2
Hacienda El Potrero, Av. Mesones Muro 1835, Jaén, Cajamarca, Peru.
6
3
Fondo Latinoamericano para el Arroz de Riego (FLAR), Cali, Colombia.
7
8
Rice hoja blanca virus (RHBV; Genus Tenuivirus), transmitted by the insect Tagosodes orizicolus
9
(family Delphacidae), causes Hoja Blanca disease (HBD) in rice (Oryza sativa L.) which has been
10
reported in all countries of major rice production in tropical and sub-tropical America.
11
Symptoms usually include chlorotic streaks on the leaves, plant dwarfing and panicle sterility
12
and associated yield losses can reach up to 25-75% in susceptible cultivars (Morales and
13
Jennings 2010). In Peru, the main rice producing areas are concentrated in the Coastal region
14
where rice yields can reach up to 10.6 tons per hectare (t/ha), followed by areas located in the
15
amazon basin region with average yields of 7.6 t/ha. (MINAGRI, 2015). HBD and the insect
16
vector are known to occur in Peru (Casanova et al. 1970; Morales and Jennings 2010), however,
17
exact locations, incidences and the identity of the virus in Peru are currently unknown. During a
18
field visit in the provinces of Utcubamba (Latitude -5.658533, Longitude -78.616637) and Jaén
19
(Latitude -5.614058, Longitude -78.635361) located in the border between the departments of
20
Amazonas and Cajamarca, characteristic HBD symptoms were observed in 4 out of 200 plants of
21
the variety FEDEARROZ 60. (Supplementary Fig. 1). Symptomatic plant samples and 89
22
collected insects were stored dried with silica gel for later analysis. A Double antibody sandwich
23
(DAS)—enzyme-linked immunosorbent assay (ELISA) was carried out using a polyclonal
24
antiserum as previously reported (Zeigler and Morales 1990) and RHBV was detected in
25
symptomatic plants and in 3.3% of insect samples. To confirm by sequencing the presence of
26
RHBV, total RNA was extracted from 50 mg of dried leaf tissue using Trizol (Chomczynski et al.
27
1993) and cDNA was synthesized from 2 µg of RNA using random hexamer primers and the M-
28
Page 1 of 3

3. MLV reverse transcriptase enzyme, following the manufacturer instructions (Invitrogen).
1
Sequences were obtained using primers RHBV-R3-F: 5’-CCTTTCCAGTTTCATCTACCATC-3’ and
2
RHBV-R3-R: 5’-AACAATGACGATGCCAGTTGCTGA-3’ to amplify a region of RNA3 encoding the
3
NS3 protein, and primers RHBV-R4-F: 5’-CTCAGGCCTCAATCATTACC-3’ and RHBV-R4-R: 5’-
4
ACTTTCAGGATAATAGACAG-3’ to amplify a region of RNA4 encoding the NS4vc protein. PCR
5
products were sent directly for sequencing (Macrogen). Nucleotide sequences obtained, 980 nt
6
for RNA3 and 466 nt for RNA4 (GenBank accession numbers KY369921 and KY369922,
7
respectively), share 96-99% identity to RHBV isolates from Costa Rica and Colombia and less
8
than 86% identity to other tenuiviruses reported in the region (Supplementary Fig. 1). RNA
9
samples obtained from pools of dried T. orizicolus insects were also positive for RHBV by RT-
10
PCR. This is the first sequence report of RHBV in Peru; the PCR primers described here have
11
been successfully tested for detection of RHBV in all rice production areas in Colombia (where
12
the disease is endemic), and they are recommended as an alternative to DAS-ELISA. Virus
13
surveillance protocols are encouraged for early detection and monitor changes in the incidence
14
of HBD in other rice production areas.
15
16
REFERENCES
17
Casanova, P., et al. 1970. Rev. Per. Entomol. Agrico. 13: 96 (in Spanish)
18
Chomczynski, P., et al. 1993. Biotechniques. 15: 532
19
MINAGRI, 2015. Sistema Integrado de Estadistica Agraria (SIEA), Ministerio de Agricultura y
20
Riego, Peru (in Spanish)
21
http://minagri.gob.pe/portal/download/pdf/herramientas/boletines/boletineselectronicos/est
22
adisticaagrariamensual/2015/bemsa_setiembre15.pdf
23
Morales, F., Jennings, P. 2010. CAB Reviews. 5: 1
24
Zeigler, R., and Morales, F. 1990. Phytopathology. 80: 559
25
Page 2 of 3

4. Supplementary Figure 1. A, Samples collected in Peru. 1=Not infected, 2 and 3=Infected samples showing
characteristic symptoms of HBD. B, Detection of RHBV by DAS-ELISA; absorbance values were recorded
after 1 hour incubation. NC=Not infected control of rice cultivar BB50; 1=asymptomatic plant, 2, 3=
symptomatic samples collected during this work; PC=Infected control of rice cultivar BBC50 inoculated with
RHBV (Colombian isolate). C) Phylogenetic analysis of sequences obtained place the tenuivirus isolate from
Peru together with other isolates of RHBV from Colombia and Costa Rica. They form a separate group from
the tenuiviruses Urochloa hoja blanca virus (UHBV) and Echinocloa hoja blanca virus (EHBV). Maize stripe
virus (MSpV) was used as an outgroup. Evolutionary analyses were conducted in MEGA6 using the
neighbor-joining method (bootstrapping of 1000 replicates).
3
1 2
A
C L07940-Costa_Rica
AF004658-Colombia
KY369921_Peru_3
L75930_EHBV-Costa_Rica
U82447_UHBV-Costa_Rica
M57426_MSpV
52
100
64
0.05
L14952-Costa_Rica
AF004657 -Colombia
KY369922_Peru_4c
L48441_EHBV-Costa_Rica
U82446_UHBV-Costa_Rica
L13438_MSpV
80
99
68
0.05
RHBV RHBV
B
RNA3 RNA4
ABSORBANCE
405nm
0
0.2
0.4
0.6
DAS-ELISA RHBV
NC 1 2 3 PC
Page 3 of 3
View publication stats
View publication stats