Epidemiology and detection of stolbur phytoplasma affecting grapevine in Spain. A. BATLLE, J. Sabaté, and A. Laviña. Dpt. Protecció Vegetal. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra Cabrils s/n, 08348 Cabrils (Barcelona), Spain. Publication no. P-2005-0023-CRA. In Spain two Grapevine yellow diseases have been reported, Flavescence dorée in the Northeast of Spain, which is considered eradicated, and Bois Noir caused by Stolbur phytoplasma. Several leafhopper and planthopper species are suspected to be involved in the transmission of Stolbur phytoplasma, but only Hyalesthes obsoletus has been found to be a transmitter of stolbur in grapevine. However, in the surveys conducted in Spain, this species was only found in a few plots and low populations were detected, suggesting that the vector in these areas could be another species. The results of samplings conducted the last five years showed that individuals of Cicadelidae, Fulgoridae and Cercopidae were captured from the beginning of February until November in weeds at the edges and inside the grapevine plots. The species identified as positive for stolbur phytoplasma after PCR analysis were among them: Agallia laevis, Adarrus taurus, Euscelis obsoletus, Hardya tenuis, Hyalesthes obsoletus, Laodelphax striatellus, Peragallia sinuata and Psammotettix striatus. Within the grapevine plots, the species found in higher number of individuals were: Scaphoideus titanus, Empoasca vitis, A. laevis and E. obsoletus. Transmission trials have indicated that the latter, E. obsoletus, could also be responsible for the transmisión. Due to the low concentration of this phytoplasma in grapevine plants, nested-PCR is the most suitable technique for its detection. Traditional nucleic acid extraction and nested PCR is a time-consuming procedure that entails risks of contamination. Here we compare the results obtained of stolbur phytoplasma detection using immunocapture-PCR, PCR-dot blot and nested-PCR, with universal and specific primers. Results demonstrate that a combined method employing immunocapture-PCR-dot blot is as, or more, sensitive than nested PCR and offers a possible way of simplifying the diagnosis of phytoplasma pathogens through PCR.
Molecular recognition of an Urapan disease in Colombia: Possible phytoplasmosis disease. J. J. Filgueira (1), L. Franco-Lara (1), E. Salcedo (1), S. L. Gaitan (1), A. M. Rodríguez, and E. Boa (2). (1) Universidad Militar “Nueva Granada”, Facultad de Ciencias (Biología) Cra. 11 No 101-80 Bogotá, Colombia, firstname.lastname@example.org; (2) CABI Bioscience, Egham, Surrey TW20 9TY, UK. Publication no. P-2005-0024-CRA.Since the introduction of Urapan in Colombia 50 years ago (commonly referred to as Fraxinus chinensis), it has been mainly used as street tree although it is also found in rural areas. In the 1990s Urapan trees in Bogotá were observed with leaf loss and dieback. We have detected phytoplasmas in samples examined using the DAPI test, light and electron microscopy, DotBlot hybridization and PCR using universal primers for 16S rDNA P1/Tint and R16/R2 (Filgueira et al., 2004). A PCR phytoplasma product of 1200 bp obtained with R16/R2, was sequenced and showed a 94% homology with Ash Yellows (AshY3) gene 16sRNA (GenBank accession number: AF105315). The presence of this phytoplasma and symptoms like yellowing of leaves, little leaves, unusual patterns of regrowth in crowns and tufted appearance is consistent with Ash yellows disease, early reported by Griffiths et al., (2001). DAPI test showed bacilliform fluorescence bodies located only into phloem tubes of petiole leaf sections but these were not observed in healthy control plants. Observation under light microscopy showed in symptomatic plants, individual spherical structures in the phloem tubes. This observation that was corroborated by transmission electron microscopy, where particles of 0.1 to 3.1 µm in diameter were revealed corresponding to pleomorphic wall-less organisms with typical phytoplasma features. DotBlot hybridization a probe was produced from Ash Y phytoplasma. In the assays no hybridization was observed with nucleic acid preparations from healthy plants but it was observed with symptomatic plant DNA samples, indicating sequence homology with this phytoplasma.
Alternative hosts and epidemiology of European stone fruits yellows phytoplasma (ESFYP) in Spain. A. LAVIÑA, J. Sabaté, M. García-Chapa, and A. Batlle. Dpt. Protecció Vegetal. Institut de Recerca i Tecnologia Agroalimentàries (IRTA). Ctra Cabrils s/n. 08348 Cabrils (Barcelona), Spain. Publication no. P-2005-0025-CRA.The European Stone Fruit Yellows Phytoplasma (ESFYP) is the causal agent of Apricot Chlorotic Leaf Roll (ACLR) and other decline diseases of the Genus Prunus. Symptoms associated to this Phytoplasma were observed in Spain in plum, peach, apricot, nectarine and cherry trees. The symptoms were early blooms and shoots during winter, which were responsible for a lack of fructification and substantial losses and chlorosis irregularly distributed in the tree during autumn. The aim of this study was to survey different Prunus (Prunus salicina, P. cerasifera, P. persica and P. avium), wild rosaceous plants (Prunus mahaleb, Crataegus monogyna and Rubus sp.) and insects surrounding the areas affected by ESFYP in order to study the alternative hosts plants, and potential vectors of ESFYP. The PCR-RFLP technique using universal or specific primers was used for phytoplasma detection in plant tissues and in insects. The ESFYP is the only one detected in Prunus orchards of all areas examined. P. mahaleb trees were found affected by ESFYP nearby Prunus orchards, they could play an important role in the spread of the disease. Insects were captured on sticky yellow or blue (in winter) traps placed within or near the ESFY-infected plots, and also with an aspirator. The traps were replaced weekly from February to July. Sundry species of leafhoppers and planthoppers were detected in the affected fruit fields. Among them Zyginia sp., Idiocerus and Empoasca sp. and two Cacopsylla species were captured in all affected fruit fields, C. pruni and C. pulchella. C. pruni vector of the European stone fruit yellows in other countries were found infected by ESFYP and could also transmit the phytoplasma in Spain. C. pulchella and some individuals of Zygina sp. and Empoasca sp. were found infected by a phytoplasma belonging to Apple Proliferation Group (AP).
Characterization of the disease to the coconut lethal yellowing in Cuba. R. E. Llauger (1), J. Cueto (1), E. L. Peralta (2), M. Alonso (1), O. Coto (1), M. Dollet (3), W. Rohde (4), D. Becker (4), M. Rodríguez (1), J. Juncal (1), R. Rodríguez (5), and V. González (6). (1) Instituto de Investigaciones en Fruticultura Tropical (IIFT) email@example.com; (2) Instituto Nacional de Investigaciones de la Caña de Azúcar (INICA); (3) Centre de coopération internationale en recherche agronomique pour le développement (CIRAD/EMVT); (4) Max-Planck-Institut für Züchtungsforschung (MPIZ); (5) Instituto de Investigaciones de Ecología y Sistemática (IES); (6) Empresa del Coco Baracoa. Publication no. P-2005-0026-CRA.The lethal yellowing (LY) of the coconut palm (Cocos nucifera L.) associated with phytoplasma presence is one of the most important coconut diseases in the Caribbean region. The objectives for this study were to establish the bases for the management of the LY based on the diversity and distribution of phytoplasmas associated with LY of coconut palm, and the identity and distribution of possible vectors in Cuba. Isolates were collected from different regions of the country and diversity was compared using RFLP analysis and universal primers P1 and P6 (Deng & Hiruki, 1991), digested with the restriction enzymes EcoRI, RsaI y BamHI. Also PCR amplification was undertaken using the primers LYF1/LYR1 (Harrison et al., 1994), followed by digestion with the enzymes EcoRI, RsaI y AluI. Isolates also were analyzed to determine the extent of diversity using Inverse Sequence-Tagged Repeat analysis, and diversity was characterized based on restriction patterns produced by digestion with the enzymes EcoRI, RsaI y BamHI. Differences in restriction profiles were obtained using LYR1/LYF1 primers and the enzyme Rsa for isolates collected in the Baracoa and Pylon regions. The number of polymorphic markers detected using each primer was relatively low. The presence of phytoplasma in coconut was demonstrated in all counties evaluated, although the percentage infection was low. The Green Indian, the Hybrid Green, and the Creole coconut were the cultivars most often infected, at 31.25, 15.62 and 18.75%, respectively. Nymphocixia caribbea (Fennah) (Homoptera: Cixiidae) was associated with all coconut palm plantations, and may be a vector of the LY phytoplasma in Cuba.
Infectivity titration of different strains of Xanthomonas axonopodis in Key Lime plants. M. NARANJO (1), M. Luis (1), and B. I. Canteros (2). (1) Instituto de Investigaciones en Fruticultura Tropical. IIFT, Ciudad de la Habana, Cuba; (2) Estación Experimental Agropecuaria Bella Vista, Instituto Nacional de Tecnología Agropecuaria, INTA, Bella Vista, Corrientes 3432, Argentina. Publication no. P-2005-0027-CRA.Citrus canker is caused by pathovars of Xanthomonas axonopodis (Hasse) Vaut. This disease is currently spreading in the most important citrus growing state of Brazil and in Florida. The probability of the disease to spread to other countries of America is high. There are different studies of the relationship between inoculum level and disease intensity in bacterial diseases. This work had the objective to determine the curve of infectivity titration of known concentrations of A, B and C strains of Xanthomonas that infect citrus, in plants of Key Lime kept in greenhouses with temperature and light control. The half of three leafs were inoculated by infiltration-injection. Leaves were 14-21 days old. The concentrations inoculated were 500 000; 50 000; 5 000; 500 and 50 cells per milliliter. The assays were done in three different times. Infectivity titration curves were obtained. The values of infectivity (lesions per sq cm) obtained and the number of cells per milliliter inoculated were log transformed and linearized. The regression slopes of A and C were similar whereas the B type gave the lowest numbers for all doses. The regression equations allowed to obtain a prediction that can be used in different experiments to quantify the number of X. axonopodis cells in suspensions of different origins.
Actual situation of viruses or related diseases in Cuba. J. M. PÉREZ (1), I. Peña (1), L. Batista (1), K. Velázquez (1), R. Pérez (1), N. del Valle (2), R. Cueto (1), and M. Aranguren (1). (1) Instituto de Investigaciones en Fruticultura Tropical, Ciudad de la Habana, Cuba; (2) Empresa de cítricos Cítricos Ciego, C. de Avila, Cuba. Publication no. P-2005-0028-CRA.In modern citriculture, the replacement of sour orange rootstock by others, potentially sensitive to various pathogens, makes an integrated management program for graft-transmissible diseases necessary. This work presents current results on the presence of the diseases as well as the biological, serological and molecular methods used to diagnose them. Regarding Tristeza, the predominance of asymptomatic virus-carrying plants is maintained. However there are isolated plants that present typical symptoms. The study of viroids in citrus areas allowed the identification of different species such as Citrus exocortis viroid, Citrus bent leaf viroid, Hop stunt viroid, Citrus dwarfing viroid and Citrus viroid IV, which are spread in different plantations. Psorosis and Concave gum diseases are propagated in old sweet orange and mandarin trees affected by scaling or concavities on the trunk. The presence of Citrus psorosis virus was identified by TAS-ELISA. Epidemiological studies of Blight, carried out for twenty years, showed a major disease dissemination during the first five years. The spatial distribution was random, with a higher tendency to be aggregated along the rows. The results obtained have allowed to establish preventive and management programs and also to ensure the health of the new plantations.
Efficacy of fungicides for control of powdery mildew on delphinium. S. N. Wegulo and M. VILCHEZ. Dept. Plant Pathology, University of California, Riverside, 92521. Publication no. P-2005-0029-CRA.Powdery mildew (pm), caused by the fungi Golovinomyces cichoracearum, Erysiphe polygoni, and Podosphaera xanthii, is a serious disease of delphinium. In 2002, eleven products including conventional, reduced risk, organic, and biological fungicides were tested for efficacy against delphinium powdery mildew (dpm) in a field experiment at the South Coast Research and Extension Center in Irvine, CA. Test seedlings of a susceptible cultivar were inoculated in the greenhouse with pm spores from field-collected plants and transplanted 10 d later into beds on 16 Sept. Bed spacing was 1 m with 0.25 m between plants in a row. Fungicides were applied at 7 or 10 d intervals according to label rates from 29 Oct to 20 Dec using a hand-held carbon dioxide-pressurized sprayer. A randomized complete block design with 4 replications was used. Disease severity (ds) (0-5 scale) and incidence (percent) were measured on 22 Nov, 9 Dec, and 24 Dec. Propiconazole and chlorothalonil applied every 10 d reduced ds by up to 95 and 77 percent, respectively. Jojoba oil and potassium bicarbonate applied every 7 d reduced ds by up to 54 and 45 percent, respectively. The results from this study suggest that environmentally benign products such as Jojoba oil and potassium bicarbonate can control dpm, but not as effectively as the best conventional fungicides.
Effects of fungicides on downy mildew of snapdragons. S. N. Wegulo (1), M. VILCHEZ (1), and S. Tjosvold (2). (1) University of California, Riverside, CA 92521; (2) UCCE, Watsonville, CA 95076. Publication no. P-2005-0030-CRA.Downy mildew caused by Peronospora antirrhini can result in major economic losses in snapdragon production. In 2003, 7 fungicides with different rates, tank-mixes or rotation schemes were evaluated under field conditions for efficacy in disease control in 2 trials (Oxnard and Irvine, CA). Fungicides were applied at 10-day intervals. The most effective treatments were fenamidone (fen) + mancozeb (man) tank mix, man, pyraclostrobin (pyr) + man tank mix, fosetyl-Al (fos-A) + man tank mix, and fen, with disease severity values (dsv) less than 1 on a 0-5 scale. Treatments with intermediate efficacy were man alternated with azoxystrobin (azo) and dimethomorph (dim), fos-A, pyr, dim, and azo alternated with dim and fose-A (dsv range = 1 – 2.1). Treatments with low efficacy or those similar to the control were azo alternated with fos-A and pyr, azo, trifloxystrobin (dsv range = 2.5 – 3.8). These results can help growers to choose the best fungicides and fungicide tank mixes and rotation schemes to use in their IPM programs.
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