Divergent molecular and growth responses of young "Cabernet Sauvignon" (Vitis vinifera) plants to simple and mixed infections with Grapevine rupestris stem pitting-associated virus.

Grapevine rupestris stem pitting associated virus (GRSPaV) is one of the most widely distributed viruses; even so, little is known about its effect on Vitis vinifera. To provide new insights, the effects of single and mixed GRSPaV infections on the V. vinifera cultivar "Cabernet Sauvignon" were studied by evaluating growth parameters, such as measurements of the total plant length, the number and distance of internodes and the number of leaves per shoot. In addition, parameters relating to gas exchange, i.e., the stomatal conductance, net photosynthetic rate, internal CO2 concentration and leaf transpiration, were also assessed. All the measurements were performed in one- and two-year-old plants with a single GRSPaV infection or mixed infections of GRSPaV and Grapevine fanleaf virus (GFLV). The results show that the plant phytosanitary status did not significantly alter the growth and gas exchange parameters in one-year-old plants. However, in two-year-old plants, single GRSPaV infections increased shoot elongation, which was accompanied by the overexpression of genes associated with the gibberellic acid response pathway. The gas exchange parameters of these plants were negatively affected, despite exhibiting higher LHCII gene expression. Plants with mixed infections did not have modified growth parameters, although they presented a greater reduction in the primary photosynthetic parameters evaluated with no change in LHCII expression. The results presented here confirm the co-evolution hypothesis for V. vinifera and GRSPaV during the early stages of plant development, and they provide new evidence about the effects of GRSPaV and GFLV co-infections on the "Cabernet Sauvignon" cultivar.

Exoenzymes and metabolites related to the nematicidal effect of rhizobacteria on Xiphinema index Thorne & Allen.

AIMS: To identify enzymes and metabolites in the rhizobacteria filtrates that have a nematicidal effect on Xiphinema index and perform molecular characterization of the strains evaluated. METHODS AND RESULTS: A series of four bacteria selected for their nematicidal potential were considered for in vitro, biochemical and molecular studies. The direct effect of the bacterial filtrates was evaluated in vitro on X. index juveniles and adults. Hydrogen sulphide and hydrogen cyanide liberation and protease, chitinase, collagenase and lipase activity were verified in the strains. Up to five housekeeping genes and one ITS 16S-23S rRNA were analysed. All bacterial filtrates presented 54-100% mortality when evaluated during up to 72 h of nematode exposure. Strains presented protease activity; two of them (strains FB833T and FR203A) showed reliable collagenase and chitinase activities, respectively, and three of them showed strong lipolytic activity (FB833T, FR203A and FS213P). Strain Bacillus megaterium FB133M had no lipase activity and presented the lowest nematicidal effect. Bacillus amyloliquefaciens FR203A had the largest lethal effect. CONCLUSION: The rhizobacteria strains evaluated in this study possess nematicidal compounds, which may offer an interesting alternative for X. index control. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of exoenzymes and metabolites associated with nematicidal effect of rhizobacteria on X. index, which can be a possible alternative for control of this plant-parasitic nematode.

First Report of Alfalfa mosaic virus Infection in Viburnum tinus in Chile.

Viburnum tinus L., commonly known as laurustinus, is an ornamental shrub that is widely used as a garden plant and flower crop. V. tinus is popular because of its desirable characteristics such as evergreen foliage, tolerance to pruning, winter blooms, and its adaptation to cold temperate zones. It is also relatively easy to grow and is commonly used as a windbreak. Infection of this ornamental species by Alfalfa mosaic virus (AMV) has been associated with yellow mottling or variegated leaf coloring, including light green and white, and has been referred to as the "Viburnum Calico" (1,4). In April 2011, at the onset of winter in the Southern Hemisphere, intense yellow spotting and mottling was observed on V. tinus leaves in the San Joaquin Campus at Pontificia Universidad Catolica de Chile. Presence of Aphis spiraecola Patch was observed on the shrubs, however, in the area it is also common to find other aphid species such as Toxoptera aurantii (Boyer de Fonscolombe) and A. fabae Scopoli. Leaf tissue samples from 10 asymptomatic and 10 symptomatic plants were examined for the presence of AMV by tissue-blot immunoassay with a commercially available polyclonal antibody (Agdia Inc., Elkhart, IN) along with the Goat affinity purified anti-rabbit IgG conjugated (Whole Molecule) (Molecular Probes, Invitrogen Corp., Carlsbad, CA). First-strand cDNA synthesis and PCR were performed with specific primers CP-AMV1 and CP-AMV2 (3). AMV was detected in all symptomatic leaves and also in two of the asymptomatic tissue analyzed by tissue blot assay. Reverse transcription (RT)-PCR produced 753-bp amplicons in all samples that were positive to AMV by tissue printing. No amplification product was observed when water control or seronegative samples were used as templates in the RT-PCR assays. Two amplicons were directly sequenced in both directions to confirm the identification of AMV in the leaf samples. The sequences obtained were homologous and BLASTN analysis of the submitted sequence (GenBank Accession No. JN040542) showed 99% nucleotide sequence identity to an AMV isolate described from Nicotiana tabacum L. (GenBank Accession No. FJ527749). These results demonstrate the presence of AMV in V. tinus in Chile. This pathogen has also been described to be affecting V. tinus in France (1) and V. lucidum Mill. in Spain (2). In Chile, V. tinum is increasingly grown as an ornamental plant. Therefore, care should be taken to ensure that the propagative materials of V. tinum are devoid of AMV infection to prevent further spread of this virus. References: (1) L. Cardin et al. Plant Dis. 90:1115, 2006. (2) M. Cebrián et al. Plant Dis. 92:1132, 2008. (3) M. Finetti-Sialer et al. J. Plant Pathol. 79:115, 1997. (4) H. E. Williams et al. Phytopathology 61:1305, 1971.

Serological and Molecular Assays for Rapid and Sensitive Detection of Iris yellow spot virus Infection of Bulb and Seed Onion Crops.

Iris yellow spot virus (IYSV) has spread rapidly in the United States and has become an important economic constraint to the production of both bulb and seed onion crops. Symptoms caused by IYSV may be confused with those caused by other fungal and bacterial pathogens and virus-specific, reliable, sensitive, and rapid detection methods would improve the diagnosis. Antiserum was produced to Escherichia coli-expressed nucleocapsid protein of IYSV and an indirect format of the enzyme-linked immunosorbent assay (ELISA) was developed. IYSV could be detected in onion tissue at dilutions of up to 1:1,000. An IYSV-specific primer pair was designed and used in a real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for the rapid detection of IYSV. Compared with standard RT-PCR, real-time RT-PCR was more rapid and sensitive. A commercially available RNA extraction kit and a total nucleic acid extraction method were compared for the quality of the templates obtained for use in real-time RT-PCR and there was no difference in limits of detection. Availability of ELISA- and PCR-based rapid and sensitive detection methods would facilitate accurate virus diagnosis and aid in better understanding of the epidemiology of the disease and in development of management strategies.

First Report of a Lettuce-Infecting Sequivirus in Chile.

Sequiviruses are isometric aphidborne plant viruses. Dandelion yellow mosaic virus (DaYMV), genus Sequivirus, was isolated from dandelion and lettuce in Europe. Lettuce mottle virus (LeMoV), a putative sequivirus, is often found in mixed infections with Lettuce mosaic virus (LMV) in Brazil (3). DaYMV, LeMoV and LMV cause similar mosaics in field-grown lettuce. Differences in biology and sequence suggest that DaYMV and LeMoV are distinct species (2). Forty-two and 101 lettuce samples with mosaic symptoms collected from two locations near Santiago during a survey of lettuce viruses in Chile in 2002 and 2003, respectively, were analyzed for the presence of LeMoV using reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted (1) and used for RT-PCR with the specific LeMoV primers pairs Lmo3 (5' ACATGAGCACTAGTGAGG 3') and Lmo4 (5' AGATAGAGCCGTCT GGCG 3') (2). One of the 42 and three of the 101 samples produced the expected 300-bp fragment. Isometric particles of 30 nm diameter, typical of a sequivirus, were visualized by transmission electron microscopy. These samples were tested using RT-PCR for the presence of LMV and Cucumber mosaic virus (CMV), but no mixed infections were observed. One isolate, Ch36, was reamplified with the degenerate primer pairs DALE 1 (5' GARTTCAACATGCACGCCAG 3') and DALE 2 (5' TTTTTCTCCCCATYCGTCAT 3') which amplify part of the putative replicase gene (2) and produced a 563-bp fragment that was cloned on pGEM-T Easy (Promega, Madison, WI) and sequenced. The Ch36 product (EMBL Accession No. AM039965) showed 97% amino acid identity with LeMoV from Brazil, 79% with DaYMV, 72% with the sequivirus Parsnip yellow fleck virus, and 34% with the waikavirus Maize chlorotic dwarf virus. To our knowledge, this is the first report of a sequivirus in field lettuce in Chile, and although the virus was found at low incidence, this report extends the range of LeMoV to the western side of the Cordillera de Los Andes. The impact of LeMoV needs to be further analyzed in Chile, Brazil, and possibly other South American countries. References: (1) Y. D. Bertheau et al. DNA amplification by polymerase chain reaction (PCR) 1998. In: Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica on potatoes. M. C. N. Perombelon and J. M. van der Wolff, eds. Scott. Crop Res. Inst. Occasional Publ., Dundee, 1998. (2) A. S. Jadão. Caracterização parcial e desenvolvimento de oligonucleotídeos específicos para detecção de sequivirus infectando alface. Ph.D. thesis. FCA-UNESP-Botucatu, Brazil, 2004. (3) O. Stangarlin et al. Plant Dis. 84:490, 2000.

First Report of Lettuce big-vein virus and Mirafiori lettuce virus in Chile.

Lettuce big-vein disease (BVD) is a serious virus disease of lettuce. Recent evidence has brought into question the role of Lettuce big-vein virus (LBVV) in the etiology of BVD, and suggested that Mirafiori lettuce virus (MiLV) and not LBVV is the causal agent of BVD (1,2). Lettuce plants (Lactuca sativa L.) with symptoms similar to those of BVD were observed during the winter of 2003 in open-field and hydroponic-grown lettuce plants located in the Chacabuco Province of central Chile. Symptomatic plants exhibited leaves with chlorotic vein banding that became ruffled and distorted. Symptoms were usually accompanied by reduced plant size and absence of head formation. Roots from symptomatic plants were analyzed by light microscopy-acid fuchsin staining. Zoosporangia and resting spores of Olpidium brassicae were identified on the basis of their morphology and structure. Additionally, soil transmission experiments were performed with 50 healthy lettuce seedlings replanted into contaminated soil collected from lettuce fields having symptomatic crops. After 3 weeks, one-half of the seedlings showed differing degrees of big-vein symptoms, and the presence of spores of O. brassicae was confirmed in the roots by light microscopy. Seedlings raised in sterilized soil showed no symptoms after the same period of time. On the basis of nucleotide sequences of LBVV and MiLV from the GenBank database, primers specific to the coat protein genes of each virus were designed as follows: MiLVV-CP1: 5'-CAAATCTGTCCACAATTCC-3'; MiLVV-CP2: 5'-TCTCACTTGAAAACCTTCC-3'; MiLVV-CP3: 5'-TTGCAACGTGATGAAACC-3'; MiLVV-CP4: 5'-AAAGAAGAGAAGCCTGTTCC-3'; LBVV-CP1: 5'-AAGCTTTCCGTACTGTCC-3'; LBVV-CP2: 5'-CCTTGATACAGTTTTTGACC-3'; LBVV-CP3: 5'-GTATGCTGATTTCTGTAGACC-3'; LBVV-CP4: 5'-TAGATGTTCTTCGCCACC-3'. The primers were used in reverse transcription-polymerase chain reaction (RT-PCR) assays with dsRNA as a template. Amplicons of the expected size were obtained in each of the five symptomatic plants analyzed, using each of the designated primer sets: MiLVV-CP1/MiLVV-CP2: 562 bp; MiLVV-CP3/MiLVV-CP4: 743 bp; LBVV-CP1/LBVV-CP2: 485 bp; and LBVV-CP3/ LBVV-CP4: 570 bp. No amplicons were obtained from healthy lettuce plants. The identity of both viruses was verified by cloning and sequencing of the amplicons. Nucleotide sequences were compared with those in the GenBank database. Sequences derived from the Chilean isolates resulted in identities of 87 to 97% for MiLV and 97 to 99% for LBVV. All samples analyzed were from the Chacabuco Province where 43% of the lettuce crops in Chile are grown. Thus, the impact that BVD may have on lettuce availability for local consumption may be significant. To our knowledge, this is the first report of Lettuce big-vein disease and Mirafiori lettuce virus infecting lettuce and the first report of BVD in Chile. References: (1) H. Lot et al. Phytopathology 92:288, 2002. (2) P. Roggero et al. Eur. J. Plant Pathol. 109:261, 2003.

Distribution and Characterization of Citrus tristeza virus in South Florida Following Establishment of Toxoptera citricida.

The incidence of Citrus tristeza virus (CTV) was found to increase significantly in southern Florida within 2 years after the establishment of its most efficient vector, Toxoptera citricida (Kirkaldy). Increased incidence of both mild and severe strains was documented, with the incidence of severe strains increasing more than mild strains. Molecular probes capable of differentiating mild, quick decline and various types of stem-pitting strains demonstrated that trees often were infected with more than one strain of CTV, with trees containing up to five different strains. Some CTV strains detected in the southeast urban corridor of Florida and in commercial groves in southwest Florida were found to react with probes specific for stem-pitting strains known from elsewhere in the world. The implications of the presence of these CTV strains in Florida and their possible presence in citrus budwood scion trees are discussed.