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.     

Evaluation of resistance to Phytophthora cinnamomi in seed-grown trees and clonal lines of Eucalyptus marginata inoculated in lateral branches and roots

Seed-grown trees and six clonal lines of 3·5–4·5-year-old Eucalyptus marginata (jarrah) growing in a rehabilitated bauxite mine site in the jarrah forest were underbark-inoculated on lateral branches (1995) or simultaneously on lateral branches and lateral roots (1996) with isolates of Phytophthora cinnamomi in late autumn. Individual seedlings from which the clonal lines were derived had previously been assessed as either resistant (RR) or susceptible (SS) to P. cinnamomi . At harvest, the acropetal lesion and colonization lengths were measured. Overall, the length of colonization in roots and branches was more consistent as a measure of resistance than lesion length, because colonization length recorded the recovery of P. cinnamomi from macroscopically symptomless tissue ahead of the lesion which, on some occasions, was up to 6 cm. In both trials, one RR clonal line was able to contain the P. cinnamomi isolates consistently, as determined by small lesion and colonization lengths in branches and roots. In contrast, the remaining two RR clonal lines used in both trials were no different from the SS line in their ability to contain lesions or colonization. These latter two RR lines may therefore not be suitable for use in rehabilitation of P. cinnamomi -infested areas. Differences in lesion and colonization lengths among P. cinnamomi isolates occurred only in the 1995 trial. Colonization and lesion lengths in branches were up to eight times greater in 1996 than in 1995, but the relative rankings of clonal lines were consistent between trials. Although colonization was always greater in branches than roots, the relative rankings of the lines were similar between branch and root inoculations. Branch inoculations are a valid option for testing the resistance and susceptibility of young jarrah trees to P. cinnamomi .     

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.     

Identification and characterization of Pepino mosaic potexvirus in tomato

At the beginning of 1999, a new virus disease occurred in protected tomato crops in The Netherlands. Initial diagnostic tests revealed the presence of a potexvirus but serological tests ruled out the presence of Potato X potexvirus (PVX). Tests for other potexviruses reported from solanaceous crops provisionally identified the virus as Pepino mosaic potexvirus (PepMV). The virus was purified, and an antiserum was produced, which showed strong reactions with both the type isolate of PepMV from pepino and two other isolates from tomato. Host range and symptomatology of the pepino and tomato isolates of PepMV revealed clear differences from PVX. However, differences were also observed between the pepino and tomato isolates of PepMV. Sequence alignment of DNA fragments of 584 bp derived from the RNA polymerase cistron showed almost 95% identity with the pepino isolate, whereas the identity with PVX appeared to be < 60%. Together, these results identified PepMV as the causal agent of the new virus disease in tomato. Based on the differences from the type isolate from pepino ( Solanum muricatum ), the isolates from tomato should be considered as a distinct strain of PepMV for which the name tomato strain is proposed.     

Effects of aqueous extracts and decomposition of Mikania micrantha H.B.K. debris on selected agronomic crops

Laboratory and greenhouse experiments were conducted to determine the effects of aqueous extract and the debris of Mile-a-Minute ( Mikania micrantha H.B.K.) on the germination and growth of four test species, viz. tomato ( Lycopersicon esculentum Mill.), Chinese cabbage ( Brassica chinensis L.), corn ( Zea mays L.) and long bean ( Vigna sesquipedalis L.). Germination percentage, radicle length and fresh weight of two crops, Chinese cabbage and tomato, decreased progressively when plants were exposed to increasing concentrations (12.5, 25, 37.5 and 50 g L⁻¹) of aqueous extract of Mikania , but did not affect those of corn and long bean seedlings. The emergence of tomato and Chinese cabbage was greatly reduced when Mikania debris was incorporated into the soil, irrespective of the amount of debris and the decomposition period. In contrast, the emergence of corn and long bean seedlings was not affected. The application of fertilizer (NPK 15 : 15 : 15) enhanced the fresh weight of all seedlings tested as compared with those not supplied with the fertilizer. Four compounds were identified in the leaf extract of Mikania , viz. caffeic acid, p -hydroxybenzaldehyde, resorcinol and vanillic acid.     

Effect of phytoplasmal infection on photosystem II efficiency and thylakoid membrane protein changes in field grown apple (Malus pumila) leaves

We have studied the effect of the apple proliferation phytoplasmal infection on some features of the thylakoids from field grown apple (Malus pumila) leaves. Changes in photosynthetic pigments, soluble proteins, ribulose-1,5-bisphosphate carboxylase, nitrate reductase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll and carotenoids were reduced in phytoplasma-infected leaves. Similar results were also observed for soluble proteins and ribulose- 1,5-bisphosphate carboxylase activity. The in vivo nitrate reductase activity was significantly reduced in infected leaves. When various photosynthetic activities were followed in isolated thylakoids, phytoplasmal infection caused marked inhibition of whole chain and photosystem II activity while the inhibition of photosystem I activity was only marginal. The artificial exogenous electron donors, diphenyl carbazide and hydroxylamine significantly restored the loss of photosystem II activity in infected leaves. The same results were obtained when F_v/F_m was evaluated by chlorophyll fluorescence measurements. The marked loss of photosystem II activity in infected leaves could be due to the loss of 47, 33, 28–25, 23 and 17 kDa polypeptides. It is concluded that phytoplasmal infection inactivates the donor side of photosystem II. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water-splitting complex was diminished significantly in infected leaves.     

Combined effect of generally regarded as safe (GRAS) compounds andTrichoderma harzianum on the control of postharvest diseases of rambutan

Botryodiplodia theobromae, Colletotrichum gloeosporioides andGliocephalotrichum microchlamydosporum are the causal fungi of the rambutan postharvest diseases stem-end rot, anthracnose and brown spot, respectively. Two different treatments of rambutan fruits(Nephelium lappaceum) against the three pathogens were compared: potassium metabisulphite (250 ppm) or cinnamaldehyde (30 ppm), each combined withTrichoderma harzianum (TrH 40). The application of TrH 40 and potassium metabisulphite effectively controlled the incidence and severity of the three postharvest diseases and maintained the overall quality and color of the fruit under low temperature storage at 13.5°C and 95% r.h. for 18 days. The greatest effect of this treatment was shown onG. microchlamydosporum. Cinnamaldehyde affected the growth and germination of TrH 40, whereas potassium metabisulphite did not. http://www.phytoparasitica.org posting Nov. 4, 2001.