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.

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.

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.

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.

Molecular diversity of ecologically distinct Mal de Río Cuarto virus isolates based on restriction fragment length polymorphism (RFLPs) and genome sequence analysis of segments 1, 7, 9 and 10.

Viruses of the species Mal de Río Cuarto virus (genus Fijivirus, family Reoviridae) cause significant economic losses in maize in Argentina. Genetic changes in the virus genome leading to better adaptation to diverse ecological conditions were postulated that would account for the increasing MRCV variability. The genomic differences between MRCV isolates from four ecologically different areas (Río Cuarto, RC; Pergamino, P; Jesús María, JM; and Tafí del Valle, TV) were studied. RT-PCR-amplified fragments comprising four genomic segments (Seg1, Seg7, Seg9 and Seg10) of MRCV isolates were compared by RFLPs and nucleotide sequences. The segments were chosen based on the proteins they encode: RNA-dependent-RNA polymerase, proteins putatively associated with tubular structures and viroplasm and the major outer capsid protein, respectively. Genetic comparison suggested that JM and TV isolates were genetically similar, but RC and P were different. Therefore, they were clustered in three genetic groups (JM = TV, RC and P). Together, nucleotide and amino acid sequence identities of the genomic segments were often above 96%. Seg1 was more variable (viral polymerase), whereas Seg7 (putative tubular structure) was the most conserved. Phylogeny analysis showed that MRCV isolates could be clustered in 'mountain area' and 'high production area' groups according to their geographical occurrence.

Wheat: A New Natural Host for the Mal De Río Cuarto Virus in the Endemic Disease Area, Río Cuarto, Córdoba Province, Argentina.

The fijivirus known as "Mal de Río Cuarto" that affects corn is endemic to the area of Río Cuarto, Cordoba Province, Argentina. One of the preferred species for the development of its vector, the insect Delphacodes kuscheli Fennah, is wheat. In this area, wheat plants with deformed leaves, spikes and spikelets, shortened internodes, leaves with serrated borders, and sterile spikelets were detected, suggesting the possibility that Mal de Río Cuarto Virus could also be infecting this crop. Samples originating in Río Cuarto, Sampacho, and La Carlota (Córdoba Province) that showed symptoms, were analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), polyacrylamide gel electrophoresis, and electron microscopy, confirming, for the first time, the occurrence of the disease in wheat. The frequency of the disease was assessed in random samples from 14 wheat plots located in the Department of Río Cuarto (Córdoba Province). The samples were analyzed using the DASELISA immunoenzymatic technique, and the disease was detected in the majority of the fields assessed, with levels of incidence that ranged between 2.5 and 24%. We must be aware of the presence of this virus in the wheat crop, where it appears to play a double role in the epidemiology of the disease, acting both as a virus reservoir and as a preferred host for the development of populations of the vector virus, D. kuscheli.