Engineering of a Single Chain Variable Fragment Antibody Specific for the Citrus tristeza virus and its Expression in Escherichia coli and Nicotiana tabacum

Citrus tristeza virus (CTV) is one of the most destructive citrus virus diseases in the world. The construction of an engineered antibody, EMBL accession number AJ278109, able to specifically recognize its antigen, i.e. the coat protein of CTV, directly on infected plant material without any purification or manipulation of the entire woody plant. The potential uses of this engineered antibody are discussed.     

Suppression of Verticillium Wilt in Eggplant by Some Fungal Root Endophytes

One hundred and twenty-three fungal isolates were obtained from 225 root segments of eggplants, melon, tomato, strawberry and Chinese cabbage, grown as bait plants in a mixed soil made up of samples from different fields in Shizuoka, Japan. Isolates belonging to Mycelium radicis atrovirens (MRA), including Phialocephala fortinii, were the most prevalent in all the five bait plants. Eleven of the 123 isolates, after being inoculated onto axenically reared eggplant seedlings, almost completely suppressed the pathogenic effects of a post-inoculated, virulent strain of Verticillium dahliae. Seven of these 11 isolates had come from the roots of eggplant and included Heteroconium chaetospira, P. fortinii, and unidentified species of Fusarium, Penicillium, Trichoderma and MRA. P. fortinii, H. chaetospira, a non-sporulating isolate with white mycelium (SWM) and MRA were easily reisolated from root segments. Hyphae of H. chaetospira, P. fortinii and SWM colonized the root tissues of eggplant without causing apparent pathogenic symptoms. The mechanisms by which these endophytes confer resistance to infection by V. dahliae are unknown but the effectiveness of these fungi in a laboratory setting indicates that they have potential as biocontrol agents and merit further investigation.     

Effects of relative humidity, light intensity and photoperiod on the colony development of Erysiphe sp. on Rhododendron

Growth and reproduction by powdery mildew pathogens is generally inhibited by decreasing relative humidity. With Erysiphe sp. on Rhododendron cv. Elizabeth, the initial stages of colony development were adversely affected by reducing the relative humidity from 100% to 70 and 85%. No significant effects on secondary or tertiary hyphal development were detected. Light intensity and photoperiod both had considerable effect on the induced resistance response of the host. Over the initial 5 days of colonization there were no significant differences between any of the treatments. After 13 days, however, expansion of fungal colonies at 180 photosynthetic active radiation (PAR) was limited solely to the area initially infested by primary hyphae. By comparison, in colonies grown at 80 PAR regardless of day length, secondary and tertiary hyphae had extended beyond the area first colonized. These effects resulted in differing morphologies, small colonies of densely packed hyphae formed at 180 PAR compared with open spreading colonies at 80 PAR.     

Biological and sequence comparisons of Potato virus Y isolates associated with potato tuber necrotic ringspot disease

The biological and molecular relationships between a large number of Potato virus Y (PVY) isolates were examined, concentrating mainly on isolates associated with potato tuber necrotic ringspot disease (PTNRD). Following detailed analysis of the coat-protein gene, four main groups were identified which broadly corresponded to the phenotype of the different isolates. The groups comprised the ordinary strain (PVY^O), the necrotic strain (PVY^N), the C strain (PVY^C) and a group of recombinant (between ordinary and necrotic) isolates. In the latter group, all members were associated with PTNRD. However, four nonrecombinant isolates were also identified which were associated with PTNRD or tuber necrosis. Three were from tubers showing PTNRD symptoms in the field, while the fourth originated from symptomless tubers, but could cause necrotic rings on tubers under glasshouse conditions. The results show that although coat-protein recombination is always found associated with the PTNRD phenotype, some nonrecombinant isolates have very similar biological properties.     

Comparison of viral RNA populations of pathogenically distinct isolates of Citrus tristeza virus : application to monitoring cross-protection

The population of sequence variants of Citrus tristeza virus (CTV) isolates of different geographic origins and pathogenicity properties was characterized by single-strand conformation polymorphism (SSCP) analysis of cDNA of the genes p18, p13, p20 and p23. The mild isolates analysed here usually yielded a SSCP profile with two DNA bands, suggestive of a predominant sequence variant, whereas the SSCP profile of the most virulent isolates contained more than two DNA bands, indicating that their viral populations are likely to be more complex. The set of SSCP profiles of the four genes allowed identification of individual isolates, but no profile characteristic of a geographic area or a biogroup was found. Sweet orange plants singly inoculated with a mild or with a severe isolate yielded the SSCP profile characteristic of each isolate, whereas the SSCP profile of plants successively inoculated with both isolates was a composite of the two individual profiles. The SSCP profile of plants singly inoculated remained constant, but the profile of doubly inoculated plants varied with time. Plants in which the SSCP profile of the severe isolate became predominant showed stem pitting, and those in which the predominant profile corresponded to the mild isolate remained symptomless. The results indicate that SSCP analysis can be used to study changes in RNA populations of doubly inoculated plants and to monitor cross-protection between mild and severe isolates.     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Genetic variability within Phaeoisariopsis griseola from Central America and its implications for resistance breeding of common bean

The genetic and virulence variability of 112 isolates of Phaeoisariopsis griseola , collected from various locations in Central America, were studied using seven random amplified polymorphic DNA (RAPD) primers and 12 common-bean differential genotypes. Broad molecular diversity ( H  = 0·92) among isolates was found using RAPD markers. Fifty pathotypes were identified on 12 differential bean genotypes, 29 of which were represented by only one isolate. Only 18 pathotypes were found in two or more countries. Pathotype 63-63 was the most virulent and caused leaf spots on all 12 common-bean differential genotypes. Comparison of virulence phenotypes and RAPD profiles to known Andean P. griseola isolates confirmed that all isolates belonged to the Mesoamerican group. Pairwise comparison between individual RAPD loci showed that the majority were in gametic phase linkage disequilibrium, revealing that P. griseola maintains a genetic structure that is consistent with asexual reproduction. The molecular and virulence diversities of P. griseola isolates from Central America imply that using single resistance genes to manage angular leaf spot is inadequate and stacking resistance genes may be necessary to manage the disease effectively.     

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.