Detection and typing of tomato yellow leaf curl viruses

The denomination Tomato yellow leaf curl virus (TYLCV) comprises several viruses that cause severe damage to tomato crops in warm and temperate regions worldwide. TYLCV viruses are widespread in the Mediterranean Basin, in which two species have been reported: Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV, previously TYLCV-Is). The availability of methods convenient for the diagnosis of these viruses is essential. We have investigated several alternatives for reliable detection and differentiation of TYLCSV and TYLCV. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) proved to be very useful for large-scale diagnosis in field situations, but lacked discriminating capacity and sensitivity in the stages of infection in which low virus titre is present. The DNA-based methods are suited to laboratory operations and plant disease clinics, where accuracy of detection and discrimination of viruses is required. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was the most reliable method to discriminate between TYLCSV and TYLCV, but is not suited to high sample turnover. For large-scale testing, tissue print hybridization assay provides a reliable and sensitive alternative to PCR.     

Construction of a cDNA library of Bemisia tabaci for use in the ''yeast two-hybrid screen'' method

Bemisia tabaci was reported for the first time in the Mediterranean part of Croatia in 2000. It was found in glasshouses in the agricultural area between the towns of Trogir and Omis, on the following crops: Euphorbia pulcherrima , Thunbergia grandiflora , Cucumis sativus (cucumber), Solanum melongena (aubergine), Phaseolus spp. (beans), Ficus carica (fig), Rubus spp. and several weeds of the families Asteraceae and Solanaceae . In 2001, monitoring for the pest was organized all over the country, in each of the 21 counties. In each county, there were several monitoring points so that all the major vegetable and flower producers were included. A special effort was made to record the spread of B. tabaci in the region where it was first found, bearing in mind that optimal conditions for outdoor spread exist along the Adriatic coast. Yellow sticky traps and visual inspection are used to monitor for B. tabaci . Eradication measures are being applied, and regulatory measures have been taken to prevent further spread of B. tabaci to continental parts of Croatia.     

Systemic spread of Plum pox virus (PPV) in Mariana plum GF 8-1 in relation to shoot growth

Infection of Prunus spp. by Plum pox virus (PPV) is characterized by an uneven distribution of the virus within the tree and branches. In order to gain a better understanding of this distribution, a method for modelling tree growth was used. PPV spread was followed within susceptible Mariana plum clone GF 8-1 shoots for 4 months after inoculation. Shoot growth was unaffected by the presence of the virus. Symptoms appeared on leaves produced in the most actively growing parts of the shoots, i.e. at the beginning of the season. PPV was detected in leaves other than those showing symptoms. The proportion of leaves with detectable virus decreased from the zone showing symptoms, with 100% ELISA-positive responses, to the shoot tip with no detectable virus in leaves produced between 111 and 127 days after inoculation. Furthermore, a higher proportion of positive ELISA results was obtained below the zone showing symptoms (77%) compared with 50% above. PPV was detected in 95% of the most vigorous shoots 71 days after inoculation compared with 37% of slower-growing, later-produced shoots.     

Effect of air temperature, rain and drought on hot water weed control

The influence of rain and drought before, and air temperature during, weed control with hot water was studied in laboratory experiments on the test weed Sinapis alba (white mustard). The plants were grown in a greenhouse and treated outdoors. There was no difference in weed control effect when S. alba plants at the four‐leaf stage were treated at the air temperatures 7°C and 18^°C. The effective energy dose for a 90% fresh weight reduction was 465 kJ m^?2 for both air temperatures. Weed control of S. alba at the four‐ to six‐leaf stage in rainfall above the rainwater run‐off level increased the required effective energy dose by 20% (i.e. 120 kJ m^?2) compared with dry plants. A short period of drought just before treatment on S. alba at the two‐ to four‐leaf stage increased the plant fresh weight reduction, which was 22% at low energy dose (190 kJ m^?2) and 44% at high energy dose (360 kJ m^?2). Hot water weed control should thus be carried out when the plants are drought stressed and avoided when the plants are wet. The air temperature seems to be of little importance in the range 7–18^°C.     

Characterization of phytoplasmas detected in Chinaberry trees with symptoms of leaf yellowing and decline in Bolivia

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in foliage samples from Chinaberry ( Melia azedarach ) trees displaying symptoms of yellowing, little leaf and dieback in Bolivia. A ribosomal coding nuclear DNA (rDNA) product (1·8 kb) was amplified from one or more samples from seven of 17 affected trees by PCR employing phytoplasma-universal rRNA primer pair P1/P7. When P1/P7 products were reamplified using nested rRNA primer pair R16F2n/R16R2, phytoplasmas were detected in at least one sample from 13 of 17 trees with symptoms. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 products indicated that trees CbY1 and CbY17 harboured Mexican periwinkle virescence (16SrXIII)-group and X-disease (16SrIII)-group phytoplasmas, respectively. Identification of two different phytoplasma types was supported by reamplification of P1/P7 products by nested PCR employing X-disease-group-specific rRNA primer pair R16mF2/WXint or stolbur-group-related primer pair fSTOL/rSTOL. These assays selectively amplified rDNA products of 1656 and 579 bp from nine and five trees with symptoms, respectively, of which two trees were coinfected with both phytoplasma types. Phylogenetic analysis of 16S rDNA sequences revealed Chinaberry yellows phytoplasma strain CbY17 to be most similar to the chayote witches'-broom (ChWBIII-Ch10) agent, a previously classified 16SrIII-J subgroup phytoplasma. Strain CbY1 resembled the Mexican periwinkle virescence phytoplasma, a 16SrXIII-group member. The latter strain varied from all known phytoplasmas composing group 16SrXIII. On this basis, strain CbY1 was assigned to a new subgroup, 16SrXIII-C.     

Seasonal distribution of phytoplasmas in Australian grapevines

The distribution and persistence of phytoplasmas were determined in Australian grapevines. Phytoplasmas could be detected using the polymerase chain reaction (PCR) from shoots, cordons, trunks and roots throughout the year, and phytoplasmas appear to persistently infect Australian grapevines from year to year. Phytoplasmas were not always detected in samples from the same sampling area from one sampling period to the next. Phytoplasma detection by PCR was improved by sampling from shoots, cordons and trunks, especially during October (early spring). The diseases expressed by the 20 grapevines used in the distribution and persistence studies were monitored. Australian grapevine yellows disease (AGY) was expressed by 17/20 grapevines at some time during the study, whilst only 4/20 and 15/20 grapevines expressed restricted growth disease (RG) and late season leaf curl disease (LSLC), respectively. All grapevines with RG and LSLC also had AGY. The three diseases were persistently expressed in some grapevines and remission of disease was observed in others. The results of PCR detection in the same grapevines indicated that phytoplasmas were more frequently detected in AGY-affected grapevines that also expressed RG and LSLC compared with grapevines expressing AGY alone. Phytoplasmas were detected in symptomless plant material but less frequently compared with AGY-affected material.     

Purification and some properties of Papaya meleira virus, a novel virus infecting papayas in Brazil

'Meleira', or 'sticky disease', is currently the most damaging papaya disease in the mid-eastern Brazilian growing regions. Consistent disease transmission via latex injection, presence of similar isometric particles in the laticiferous vessels of diseased plants, and detection of double-stranded DNA in naturally and experimentally infected papaya trees suggest that a virus is the causal agent. Conclusive evidence for viral aetiology was previously lacking, mostly because every attempt to purify the putative virus from infected papayas had failed. Following the successful purification and partial characterization of the meleira virus, healthy papaya seedlings injected with purified virus particles later developed typical symptoms of the disease. Negatively stained, isometric, full and 'empty' purified virus particles measured 42 and 38 nm, respectively. The viral genome was a single dsRNA molecule of about 12 kbp. Several capsid proteins, ranging in size from 14·4 to 45 kDa, were consistently revealed by PAGE. Papaya meleira virus (PMeV) appears to represent a novel group of viruses, with no known similar counterpart among known plant-, vertebrate-, invertebrate- or prokaryote-infecting viruses.