Co-infection with a wheat rhabdovirus causes a reduction in Mal de Río Cuarto virus titer in its planthopper vector.

Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) causes one of the most important diseases in maize (Zea mays L.) in Argentina and has been detected in mixed infections with a rhabdovirus closely related to Maize yellow striate virus. In nature both viruses are able to infect maize and several grasses including wheat, and are transmitted in a persistent propagative manner by Delphacodes kuscheli Fennah (Hemiptera: Delphacidae). This work describes the interactions between MRCV and rhabdovirus within their natural vector and the consequences of such co-infection regarding virus transmission and symptom expression. First- and third-instar D. kuscheli nymphs were fed on MRCV-infected wheat plants or MRCV-rhabdovirus-infected oat plants, and two latency periods were considered. Transmission efficiency and viral load of MRCV-transmitting and non-transmitting planthoppers were determined by real-time quantitative polymerase chain reaction analysis (RTqPCR). Vector transmission efficiency was related to treatments (life stages at acquisition and latency periods). Nevertheless, no correlation between transmission efficiency and type of inoculum used to infect insects with MRCV was found. Treatment by third-instar nymphs 17 days after Acquisition Access Period was the most efficient for MRCV transmission, regardless of the type of inoculum. Plants co-infected with MRCV and rhabdovirus showed the typical MRCV symptoms earlier than plants singly infected with MRCV. The transmitting planthoppers showed significantly higher MRCV titers than non-transmitting insects fed on single or mixed inocula, confirming that successful MRCV transmission is positively associated with viral accumulation in the insect. Furthermore, MRCV viral titers were higher in transmitting planthoppers that acquired this virus from a single inoculum than in those that acquired the virus from a mixed inoculum, indicating that the presence of the rhabdovirus somehow impaired MRCV replication and/or acquisition. This is the first study about interactions between MRCV and a rhabdovirus closely related to Maize yellow striate virus in this insect vector (D. kuscheli), and contributes to a better understanding of planthopper-virus interactions and their epidemiological implications.

A molecular framework for the identification of planthopper vectors (Hemiptera: Delphacidae) of central Argentina.

Planthoppers are important worldwide crop pests as well as vectors of numerous diseases. Different species transmit Mal de Río Cuarto virus, which causes the most economically important corn disease in central Argentina. Epidemiological studies rely on the accurate identification of the species present in the field. Presently, morphological identification of planthoppers requires taxonomic expertise and there are no taxonomic keys for females and nymphs. Nevertheless, no molecular protocols are available for accurate species identification of most frequent delphacid species from central Argentina. In this context, the aim of this study was to evaluate the utility of the cytochrome oxidase I gene (COI) as a DNA barcode and its digestion with restriction enzymes (Restriction Fragment Length Polymorphism, RFLP) for the identification of the most common species of planthoppers in central Argentina. We amplified and sequenced a 843 bp fragment of the COI gene of taxonomically identified specimens and evaluated its use as a DNA barcode. Restriction enzymes were also selected for digesting the COI fragment via RFLP. The high interspecific variability (20.79%; ± 2.32%) and low intraspecific divergence (0.12%; ± 0.17%) observed in the studied species, demonstrate the effectiveness of the COI gene for species identification of major vector delphacids affecting corn crops in Argentina. Moreover, the digestion of this COI gene fragment with Bfa I and Apo I enzymes allows a fast and cost-effective species identification method when numerous specimens need to be processed. Both molecular techniques developed here, allow the accurate identification of planthopper species at regional scale. These new tools would assist traditional identification of these insects, especially for aiding non-experts in morphological taxonomy.

Wolbachia Occurrence in Planthopper (Hemiptera: Delphacidae) Vectors of Cereal Viruses in Argentina.

Maize (Zea mays L.) and wheat (Triticum aestivum L.) are the most important cereal crops for the Argentinean economy and are affected by several diseases. Different planthopper species transmit causal agents of some of those diseases, including Mal de Río Cuarto virus, barley yellow striate mosaic virus, and the recently proposed maize yellow striate virus. Many planthopper species are sap feeders and therefore are expected to host bacteria providing essential nutrients lacking in the diet. Previous studies have evidenced that some of these bacterial symbionts are involved in the virus transmission. Wolbachia is a group of obligate intracellular bacteria infecting numerous arthropod species and causing reproductive alterations in their hosts. These bacteria have been detected in planthopper species, considered rice pests in various regions of the world. To date, Wolbachia infection status of planthopper species of Argentina is unknown. Amplification by PCR and sequencing of 16S rDNA, wsp- and ftsZ-specific genes demonstrated Wolbachia infection in Caenodelphax teapae (Fowler), Delphacodes kuscheli Fennah, Pyrophagus tigrinus Remes Lenicov & Varela, Tagosodes orizicolus (Muir), and Toya propinqua (Fieber). This is the first report of Wolbachia in delphacid vectors of viruses affecting maize and wheat. An understanding of the bacterial diversity harbored by these insect vectors could lead to new options for future management of diseases of economically important crops in a developing country.

Three Members of the Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species Complex Occur Sympatrically in Argentine Horticultural Crops.

The whitefly, Bemisia tabaci (Gennadius), is a cryptic species complex that attacks >600 different species of plants and transmits several plant viruses causing severe economic losses. Until 2010, the B. tabaci complex comprised 24 distinct putative species. Recently, at least 15 new species have been reported. The objective of this study was to identify B. tabaci species present in bean, melon, and tomato crops in Argentina by applying phylogenetic analyses and pairwise comparison of genetic distances of mitochondrial cytochrome c oxidase subunit I (mtCOI) sequences. The 39 proposed whitefly species were identified with both analyses, and the presence in Argentina of one indigenous species, New World 2 (NW2), and two introduced species, Middle East-Asia Minor one (MEAM1) and Mediterranean, was confirmed. Common bean crop presented the three whitefly species detected, with NW2, MEAM1, and Mediterranean being present all together under field conditions. Also, Mediterranean was the only species identified in tomato, whereas MEAM1 was found in melon. To the best of our knowledge, Mediterranean is a recent invasive species in open-field agriculture in the American continent and in greenhouse tomato in Argentina. Additionally, we provide the first report of MEAM1 in common bean and melon. These findings raise several questions on the future scenario of B. tabaci and the viruses it transmits in Argentina.

Species within the Bemisia tabaci (Hemiptera: Aleyrodidae) complex in soybean and bean crops in Argentina.

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex that contains some of the most damaging pests in tropical and subtropical regions. Recent studies suggested that this complex is composed of at least 24 distinct species. We use the approach from these studies to consider the identity of B. tabaci in Argentina. Previous studies have suggested the presence of a B. tabaci presumably indigenous to the Americas and referred to as the BR biotype in Argentina. We placed the entity referred to as the BR biotype within the B. tabaci cryptic species complex using whiteflies collected in soybean and bean crops in northern and central Argentina. The whiteflies were assigned using the mitochondrial cytochrome oxidase (mtCOI) gene. Four unknown haplotypes plus two Argentina sequences from GenBank formed a cluster that was basal to the rest of the New World sequences. These sequences diverged from the consensus sequence across the range of 3.6 to 4.3%. Applying the species assignment rules of recent studies suggests that the individuals from Argentina form a separate species. A fifth unknown haplotype fell within the New World putative species and formed a distinct cluster with haplotypes from Panama. These results suggest that Argentina has two indigenous species belonging to the B. tabaci cryptic species complex. Rather than using mtCOI sequencing for all B. tabaci collected, a simple random amplified polymorphic DNA-polymerase chain reaction diagnostic was used and tested along with previously published primers designed to work specifically with the BR biotype from Brazil. These primers were either unable to distinguish between the two indigenous members of the complex in Argentina or indicated a difference when none was evident on the basis of mtCOI sequence comparison.