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Cucumber mosaic virus infecting ‘Cavendish’ banana in Argentina

  • Dariel Cabrera Mederos
  • Margarita Jaramillo Zapata
  • José Villarreal Filipovich
  • Claudia Nome
  • Carolina Torres
  • Orelvis Portal
  • Fabián Giolitti
Article

Abstract

Banana plants (Musa sp.) showing severe mosaic symptoms in leaves were observed in Laguna Naineck, Formosa, Argentina. Electron microscopy observations of leaf dip preparations revealed isometric particles ca. 30 nm in diameter, which were identified as cucumber mosaic virus (CMV) by enzyme-linked immunosorbent assay (ELISA) test and reverse transcription-polymerase chain reaction (RT-PCR). CMV was mechanically transmitted to healthy Nicotiana glutinosa plants. Sequence analysis of coat protein gene showed highest nucleotides identity with other CMV isolates from United States of America, Serbia, Russia, Democratic Republic of Congo, Iran and Brazil, ranging between 96.9–98.3%. Phylogenetic analysis indicated that the virus isolate named Laguna Naineck (MH716245) belongs to the CMV group IA. To our knowledge, this is the first report of CMV in banana in Argentina. Moreover, constitutes the first Argentine CMV isolate available in the international nucleotide sequence databases.

Keywords

Cucumber mosaic virus Banana crops Sequence analysis 

In Argentina, the banana production area, located in the northern provinces, covers approximately 5400 ha. The banana is the most consumed fruit in Argentina and approximately 20% of the volume consumed corresponds to national production (Tapia and Fagiani 2015; Molina 2016). Viruses are assuming greater importance in Musa cultivation due to the current worldwide movement of its germplasm. Among viruses reported to infect banana, banana streak virus (BSV), banana mild mosaic virus (BanMMV), cucumber mosaic virus (CMV) and banana bract mosaic virus (BBrMV) have been detected in this crop in south America (Bhat et al. 2016; Reichel et al. 2003; Eiras et al. 2004; Quito-Avila et al. 2013). CMV is one of the most important plant viruses, infecting more than 1000 plant species worldwide (Jacquemond 2012). Both subgroups I and II has been detected in horticultural crops from Argentina (Atencio et al. 1997), however, there are no sequences of any Argentine CMV isolates available in international nucleotide sequence databases. Moreover, no information is known about the status of viruses infecting banana in the North of Argentina, which is the main growing region in this country.

In July 2017, Cavendish banana (Musa sp. AAA group) cv. ‘Congo’ showing mosaic symptoms in leaves was observed (Fig. 1a) and collected in Laguna Naineck, department Pilcomayo, Northeast of Argentina. These symptoms were similar to those induced by viruses in banana crop (Selvarajan 2015).
Fig. 1

Mosaic and streak symptoms associated with cucumber mosaic virus infection of banana in Laguna Naineck, Argentina (a). Electron micrographs of negatively stained virus particles (~30 nm in diameter) with 2% uranyl acetate (b)

Symptomatic banana leaf tissues were analyzed by transmission electron microscopy; leaf-dip preparations were made with borate buffer 0.05 M pH 7.0 and stained with 2% uranyl acetate (Sigma-Aldrich, USA) (Kitajima and Nome 1999). The observations revealed the presence of isometric particles ca. 30 nm in diameter (Fig. 1b). In these observations, particles belonging to other virus families were not detected. Leaf extracts obtained from symptomatic leaves were serologically analyzed for CMV using polyclonal and monoclonal antibodies. Firstly by ImmunoStrip® and after, by Triple Antibody Sandwich (TAS-ELISA) test against CMV subgroups I and II (Agdia, USA). Reactions were positive to CMV and CMV subgroup I, respectively.

Leaf extracts obtained from infected banana plant were used to inoculate healthy Nicotiana glutinosa plants, which was performed according to Singh et al. (1995). Plants of N. glutinosa showed symptoms, similar to those induced by this virus (data not shown). In the naturally infected and experimentally inoculated plants, virus infections were confirmed by RT-PCR.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Germany), according to the manufacturer’s instructions. The reactions to amplify the CP gene were performed in two steps. First-strand cDNAs were synthesized using Moloney Murine Leukemia virus reverse transcriptase enzyme (M-MLV) (Promega, USA) according to the manufacturer’s instructions and 3’CP as the initial primer (Rizos et al. 1992). The reaction was incubated for 4 min at 99 °C, followed by 60 min of incubation at 42 °C. PCR was carried out using Kapa Hiffi DNA polymerase enzyme (Kapa Biosystems, USA) and primers: 5’CP (5’-CTCGAATTCGGATCCGCTTCTCCGCGAG-3′) and 3’CP (5’-GGCGAATTCGAGCTCGCC GTAAGCTGGATGGAC-3′), which amplifies the 3′ portion of RNA 3 in CMV isolates (Rizos et al. 1992). PCR reactions was performed using 2 μl of cDNA, 1 U of Kapa Hiffi DNA polymerase enzyme (Kapa Biosystems, USA), 300 μM dNTPs each and 0.3 μM of each primers in a final reaction volume of 50 μl. The PCR conditions included an initial incubation for 2 min at 95 °C, followed by 34 cycles of denaturation at 94 °C for 45 s, annealing at 40 °C for 45 s, extension for 1 min at 72 °C and final extension at 72 °C for 10 min.

The products obtained by RT-PCR of ~800 bp (data not shown) were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, USA) and then directly sequenced in both directions at Macrogen Inc., Seoul, Korea. The sequence determined in this study was deposited in international nucleotide sequence databases with Accession number MH716245. Preliminary sequence data analysis of the CP gene was performed with the NCBI blast tool to search for related sequences. CMV complete CP sequences from different origins, with identities greater than 75%, were selected from GenBank database for further analysis. Sequence alignment of CMV isolates was carried out using MUSCLE (Edgar 2004), by mean of SeaView software v. 4.4.2 (Gouy et al. 2009). The model of base substitution was estimated using the jModelTest v. 2.1.9 software (Darriba et al. 2012), according to the Akaike Information Criterion. Phylogenetic trees were obtained by using Bayesian inference in Mr. Bayes 3.2.6 (Ronquist et al. 2012).

The values of the sequence identities between the Argentine CMV isolate and others from different geographic regions and subgroups ranged from 76.4 to 98.3% and from 81.2 to 98.6% at the nucleotides and amino acids levels, respectively (Table 1). According to literature data, the members of the same subgroup share more than 90% homology (Palukaitis and García-Arenal 2003). In this study, the nucleotide homology between isolates from I and II subgroups was approximately 75.3–78%. Argentine isolate was phylogenetically most related to the members of subgroup IA (≥98% nt homology), which coincides with the sequence homology values reported in the same subgroup (Gallitelli 2000). However, they were distinct from the subgroups IB and II isolates. Members of subgroup II showed the lowest nucleotide homology (76–77%) with the Argentine CMV isolate (Table 1).
Table 1

Nucleotide (nt) and amino acid (aa) sequence identities of an Argentine cucumber mosaic virus isolate (MH716245) with isolates from other geographic regions

Host

Subgroup

Origin

GenBank

Identities (%)

nt

aa

Mertensia virginica

IA

USA

GU362669

98.3

98.1

Anemone sp.

IA

USA

FJ375723

98.3

98.1

Cucumis melo

IA

Serbia

KT270566

98.3

98.6

Canna sp.

IA

Russia

KY595419

98.3

98.1

Solanum lycopersicum

IA

Serbia

KC847071

98.3

98.1

Cucumis melo

IA

USA

D10538

98.1

98.1

Nicotiana tabacum

IA

USA

U20668_

98.1

98.1

Iris sp.

IA

Iran

KM262647

98.1

98.1

Musa balbisiana

IA

DR Congo

KX216873

98.1

98.1

Musa balbisiana

IA

DR Congo

KX216870

98.1

98.1

Musa balbisiana

IA

DR Congo

KX216868

98.1

98.1

Musa acuminata

IA

DR Congo

KX216872

98.1

98.1

Musa acuminata

IA

DR Congo

KX216869

98.1

98.1

Musa acuminata

IA

DR Congo

KX216871

98.1

98.1

Chrysanthemum

IA

Brazil

AY380533

98.0

97.7

Solanum lycopersicum

IB

Italy

Y16926

95.1

97.2

Nicotiana tabacum

IB

China

AB008777

93.8

96.7

Capsicum sp.

IB

Italy

HE962480

93.7

96.7

Musa sp.

IB

Nigeria

KU976478

93.4

95.4

Musa sapientum

IB

Indonesia

AB069971

92.8

96.7

Capsicum sp.

II

Australia

M21464

77.0

82.1

Musa sp.

II

China

AF268598

76.7

81.7

Lactuca saligna

II

USA

AF127976

76.7

81.2

Lupinus angustifolius

II

Australia

AF198103

76.4

81.7

Origin and GenBank accession numbers of the nucleotides sequences of CMV isolates used in phylogenetic analysis are presented

Bayesian phylogenetic tree of nucleotide sequences showed three distinct clusters corresponding to subgroups IA, IB and II. Reference CP gene sequences of CMV isolates Tfn for subgroup IB (Y16926), Fny for subgroup IA (D10538) and Q for subgroup II (M21464) were used for subgrouping (Fig. 2). Within subgroup I, the Argentine CMV isolate showed close phylogenetic relationships with isolates from United States of America and Serbia (GU362669, FJ375723 and KT270566). The phylogeny of CMV shows no clear correlation with the provenance of the isolates (Fig. 2), which suggest that CMV world population seems to be thoroughly mixed (Ohshima et al. 2016).
Fig. 2

Majority rule consensus tree obtained from the Bayesian analysis of coat protein nucleotide sequences of an Argentine cucumber mosaic virus isolate (MH716245) and other 25 CMV isolates available at GenBank database. Branch lengths are proportional to genetic distances and branch significance is indicated at nodes for posterior probability values higher than 0.5. For each isolate, the GenBank accession number and geographic origin are indicated. The CP gene sequence of Peanut stunt virus (PSV) (U15730) was used as an outgroup for comparison

From an epidemiological point of view, the results presented are not only important in Argentina, but also for neighboring countries. The banana producing region of Argentina where the virus was detected is separated only by the Pilcomayo river from the Paraguay. This geographical scenario, linked to the absence of sexual reproduction in vegetative propagated plants and the subtropical conditions that favour the development of vector aphids, enables viruses to establish long-term infections and to be transmitted with high efficiency. Moreover, the occurrence of CMV in banana from Northeast Argentina is possibly associated with the wide host range of this virus in perennial weed and cultivated species and constitutes an alert due to the propagation methods used by regional producers. To our knowledge, this is the first report of the occurrence of CMV infecting banana in Argentina and the unique viral disease detected in banana in this country. In 2018, it was developed the Phytolab technology in Misión Tacaaglé (Formosa province), which estimates the field establishment of in vitro-produced banana plants to restore 200 ha per year. The presence of this virus suggests the monitoring of virus-free propagative materials, which is required for the establishment of new plantations through asexual or in vitro propagation.

Notes

Acknowledgements

The authors thank Ricardo Lopez, Ricardo Vailatti and Jorge Balonga (CEDEVA, Formosa) for support during the visits from the banana plantations of Misión Tacaaglé and Laguna Naineck areas.

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Copyright information

© Australasian Plant Pathology Society Inc. 2018

Authors and Affiliations

  1. 1.Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP)Instituto Nacional de Tecnología Agropecuaria (INTA)CórdobaArgentina
  2. 2.Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Buenos AiresArgentina
  3. 3.Universidad de San Pablo-TTucumánArgentina
  4. 4.Centro de Validación de Tecnologías Agropecuarias (CEDEVA Misión Tacaaglé)FormosaArgentina
  5. 5.Departamento de Biología, Facultad de Ciencias AgropecuariasUniversidad Central ‘Marta Abreu’ de Las VillasSanta ClaraCuba

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