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Revista mexicana de fitopatología
versão On-line ISSN 2007-8080versão impressa ISSN 0185-3309
Rev. mex. fitopatol vol.34 no.3 Texcoco Set. 2016
https://doi.org/10.18781/r.mex.fit.1604-1
Phytopatological Notes
Detection of Iris yellow spot virus in onion plants from Tepalcingo, Morelos state, Mexico
1Campo Experimental Zacatepec, INIFAP. Km. 0.5 Carretera Zacatepec-Galeana. C. P. 62780, Colonia Centro Zacatepec, Morelos, México.
2Universidad Politécnica del Estado de Morelos. Bulevard Cuahunahuac #566, C.P. 62550, Colonia Lomas del Texcal, Jiutepec, Morelos, México.
3Posgrado en Fitosanidad-Fitopatología. Colegio de Postgraduados, km 36.5 carretera México-Texcoco, Montecillo, Texcoco, C.P. 56230 México.
In Morelos state, recently have been observed viral diseases in onion (Allium cepa); one of them is caused by the Iris yellow spot virus (IYSV) which belong to the Bunyaviridae family and the Tospovirus gender and is transmitted by Thrips tabaci Lindeman (Thysanoptera: Thripidae) to onion plants. In 2012, there was a 100 % incidence of IYSV and severity of over 90 % on 2,500 ha of commercial crop. The objective of this research was to identify the presence of IYSV through real time RT-PCR and sequencing of the virus. To accomplish this, leaves were sampled from commercial fields at Tepalcingo, Morelos, from transplanting to harvest. Total RNA extraction was done with TRIzol® Reagent. Virus detection was done using specific primers for the nucleoprotein gen of IYSV giving as a result the amplification of a specific product through real-time RT-PCR, and an expected strip of 896 bp, which after sequencing confirmed 99 % identity with the nucleoprotein gen of the virus. The IYSV virus was detected in onion plants from Tepalcingo, Morelos and the sequence obtained was registered in the database of the GenBank (JX946658).
Keywords: Real time RT-PCR; Tospovirus; Thrips tabaci; Onion yellow spot
En el estado de Morelos recientemente se han observado enfermedades virales en cebolla (Allium cepa); una de ellas es la mancha amarilla causada por el Iris yellow spot virus perteneciente a la familia Bunyaviridae del género Tospovirus, el cual se trasmite a la cebolla por Thrips tabaci Lindeman (Thysanoptera: Thripidae). En el 2012 la incidencia de esta enfermedad fue de 100 % en las 2,500 ha cultivadas en la entidad con una severidad superior a 90 %. El objetivo de este trabajo fue detectar mediante RT-PCR en tiempo real y secuenciar la presencia de IYSV. Para su identificación se tomaron muestras de hojas de cebolla con manchas amarillentas alargadas, desde el trasplante hasta la cosecha en Tepalcingo, Morelos. La extracción de ARN total se realizó utilizando TRIzol® Reagent. La detección del virus se realizó con primers específicos al gen de la nucleoproteína de IYSV dando como resultado la amplificación de un producto específico mediante RT-PCR en tiempo real y una banda esperada de 896 pb la cual mediante secuenciación confirmó 99 % de identidad con el gen de la nucleoproteína de este virus. El IYSV fue detectado en plantas de cebolla en Tepalcingo, Morelos y su secuencia se registró en la base de datos del GenBank (JX946658).
Palabras clave: RT-PCR en tiempo real; Tospovirus; Thrips tabaci; Mancha amarilla de la cebolla
The onion plant is an economically important plant, with 53 million tons of bulbs harvested from the nearly 3 million hectares planted worldwide (Gent et al., 2006). In Latin America, Mexico is the largest onion producer with 77,328 t harvested in 7, 400 ha; it is also the tenth exporter worldwide (FAO, 2011). It is the fifth most important vegetable crop; in Morelos, 10 % of the country’s production is harvested, which makes it the fourth largest producer on a national scale (SIAP, 2012).
Some of the diseases that affect the crop include viral diseases, particularly the Iris yellow spot virus (IYSV) (Gent et al., 2006).
The IYSV infects various species of the genus Allium, including onions (Allium cepa), causing the onion yellow spot. This virus belongs to the genus Tospovirus of the family Bunyaviridae (Gent et al., 2006). Its presence has been detected since 1981 in Brazil and the United States (Cortês et al., 1998.). IYSV has exteded to important onion-producing regions worldwide (Bulajić et al., 2009; Córdoba et al., 2005; Du Toit et al., 2004; Gent et al., 2006; Gera et al., 2004; Nischwitz et al., 2007; Pozzer et al., 1999; Schwartz et al., 2002; Smith et al., 2006).
In Mexico this virus has been identified in onion plantations and greenhouses in Zacatecas (Velásquez and Reveles, 2011; Velásquez et al., 2016).
IYSV is transmitted by Thrips tabaci, main pest of this crop (Riley et al., 2011). Kritzma et al. (2001) found a relation between the populations of T. tabaci and the incidence of infection of IYSV, and showed that the highest concentrations of the virus are near the neck of the plant, which is the site for the feeding and protection of thrips.
The incidence of IYSV increases after the bulb begins to form, by 40 %, compared with con 3 % before this stage (Fichtner et al., 2004; Hammon 2004), and it often reaches levels of 50 to 60 % (Kritzman et al., 2001). In countries such as Brazil it has reached levels of 100 % causing complete losses of both seeds and bulbs (Pozzer et al, 1999).
The damage caused by the virus leads to the drying of the leaves and stops bulb growth. Therefore, the main economic impact of this is related to the reduction in bulb size and consequently the reduction of yield (Gent et al., 2004). Likewise, the infection makes the onion plant more vulnerable to adverse conditions such as drought, excess irrigation, high temperatures, damage of leaf miners and thrips, and others (Velásquez et al., 2016).
In Mexico, this plant is produced in two agricultural cycles, the most important of which is autumn-winter, in which 60 % of the total production is obtained, although the symptoms of IYSV have been observed in the two cycles mentioned (Osuna and Ramírez, 2013). The aim of this study was to detect the presence of IYSV, using RT-PCR in real time, in onion plants from Tepalcingo, Morelos
RNA extraction was performed on a total of five onion plants in the phase of bulb formation with symptoms attributed to IYSV, as observed in Figure 1, using TRIzol® Reagent (Invitrogen). The cDNA synthesis was carried out using 1 µg of total RNA with the package QuantiTect Reverse Transcription kit® (QIAGEN) using primers IYSV-465c and IYSV-239f, specific for the detection of IYSV (Pappu et al., 2008). The reaction of PCR in real time was carried out using the package QuantiFast® SYBR® Green PCR (QIAGEN) with 10 ng of cDNA. Each sample was analyzed three times. The real time PCR program consisted of an initial step at 95 °C for 10 min, followed by 40 cycles with three steps: 95 °C 10 s, 61 °C 15 s, 72 °C 10 s. The increase in optimization was registered between 72 and 95 °C for the dissociation curve. The partial sequencing of the gene N of the nucleoprotein was carried out using the primers IYSV917L and IYSV56U (Robène-Soustrade et al., 2006). The program CLC Sequence Viewer 6.7 was used to carry out the phylogenetic analysis between the partial sequence of the gene of the nucleoprotein of IYSV identified in Tepalcingo, Morelos, with other samples reported in the databases of the GenBank NCBI (National Center for Biotechnology Information). The analysis was carried out using the algorithm UPGMA with 100 replications.
The symptoms found in the onion crops related to IYSV were yellow or white chlorotic spots, dry and long. Mature leaves with these symptoms presented long spots of different sized, which covered up to 70 % of their surface (Figure 1).
Figure 1. Symptoms related to the infection by Iris yellow spot virus in onion plants: a) and b) chlorotic spots in early stages of the infection; c) long hay-colored stains; d) coalescence of damages, mainly on the base of the leaves.
To confirm the presence of the IYSV, a PCR was carried out in real time on plants with symptoms, obtaining a specific amplification (Figure 2A). The dissociation analysis showed only one peak which corresponded to the amplification of a fragment of a gene of the nucleocapsid of the virus (Figure 2B), which shows that the amplification was specific to this test.
Figure 2. Detection of the Iris yellow spot virus (IYSV) by PCR in real time. A. Amplification of a fragment of the gene of the nucleocapsid of the virus, from damages in onion leaves. B. Specific dissociation curve of the amplification of the gene of the nucleocapsid of the same virus.
The sequence of the gene of the nucleocapsid of the IYSV detected in onion plants was registered in GenBank with the access number JX946658. The homology analysis confirmed a 99 % identity with previously reported sequences of the gene of the nucleoprotein of the IYSV. The phylogenetic analysis showed that the virus in Tepalcingo, Morelos (JX946658) has a higher similarity with those reported in Europe and Asia. The analysis shows that there is no association between the sequence of the virus and its geographic location, which limits its traceability (Figure 3). The viral genomes of RNA naturally present a high mutation rate during the replication process (Elena et al., 2008). However, the global IYSV analysis has so far shown a restricted gene flow due to the geographic confinement, giving two genotypes as a result: IYSV-NL and IYSV-BR, the former being, up to now, the only one present in the American continent, whereas the latter is found mainly in Asian isolations (Iftikhar et al., 2014).
Figure 3. Phylogenetic relations of the Iris yellow spot virus sequences registered in the GeneBank of different parts of the world, in regard to the sequence obtained in onion plants from Morelos, Mexico (JX946658_IYSV_MEX).
According to Pappu et al. (2008), the RT-PCR technique in real time has been proven to be quick and highly efficient to detect the presence of IYSV in onions.
Acknowledgements
To the onion farmers in Morelos, Mexico and the project supported by FOMIX MOR-2010-01 code 148902.
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Received: April 01, 2016; Accepted: June 15, 2016
Este es un artículo publicado en acceso abierto bajo una licencia Creative Commons
