An immunocytochemical procedure for protein localization in various nematode life stages combined with plant tissues using methylacrylate-embedded specimens

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access.     

Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.     

Effect of temperature, wetness duration, and planting density on olive anthracnose caused by Colletotrichum spp

The influence of temperature, wetness duration, and planting density on infection of olive fruit by Colletotrichum acutatum and C. simmondsii was examined in laboratory and field experiments. Detached olive fruit of 'Arbequina', 'Hojiblanca', and 'Picual' were inoculated with conidia of several isolates of the pathogen and kept at constant temperatures of 5 to 35°C in humid chambers. Similarly, potted plants and stem cuttings with fruit were inoculated and subjected to wetness periods of 0 to 48 h. Infection occurred at 10 to 25°C, and disease severity was greater and the mean latent period was shorter at 17 to 20°C. Overall, C. acutatum was more virulent than C. simmondsii at temperatures <25°C. When temperature was not a limiting factor, disease severity increased with the wetness period from 0 to 48 h. Disease severity was modeled as a function of temperature and wetness duration; two critical fruit incidence thresholds were defined as 5 and 20%, with wetness durations of 1.0 and 12.2 h at the optimum temperature. In the field, anthracnose epidemics progressed faster in a super-high-density planting (1,904 olive trees/ha) than in the high-density plantings (204 to 816 olive trees/ha) and caused severe epidemics in the super-high-density planting even with the moderately resistant Arbequina. Data in this study will be useful for the development of a forecasting system for olive anthracnose epidemics.     

Resistance to Pseudomonas syringae in a collection of pea germplasm under field and controlled conditions

A total of 242 Pisum accessions were screened for resistance to Pseudomonas syringae pv. pisi under controlled conditions. Resistance was found to all races, including race 6 and the recently described race 8. Fifty‐eight accessions were further tested for resistance to P. syringae pv. syringae under controlled conditions, with some highly resistant accessions identified. Finally, a set of 41 accessions were evaluated for resistance to P. syringae pv. pisi and pv. syringae under spring‐ and winter‐sowing field conditions. R2, R3 and R4 race‐specific resistance genes to P. syringae pv. pisi protected pea plants in the field. Resistance sources to race 6 identified under controlled conditions were ineffective in the field. Frost effects were also evaluated in relation to disease response. Results strongly suggest that frost tolerance is effective in lowering the disease effects caused by P. syringae pv. pisi and pv. syringae under frost‐stress conditions, even in the absence of disease resistance genes, although the highest degree of this protection is reached when frost tolerance and disease‐resistance genes are combined in the same genetic background.     

Chlorophyll fluorescence as a marker for herbicide mechanisms of action

Photosynthesis is the single most important source of O₂ and organic chemical energy necessary to support all non-autotrophic life forms. Plants compartmentalize this elaborate biochemical process within chloroplasts in order to safely harness the power of solar energy and convert it into usable chemical units. Stresses (biotic or abiotic) that challenge the integrity of the plant cell are likely to affect photosynthesis and alter chlorophyll fluorescence. A simple three-step assay was developed to test selected herbicides representative of the known herbicide mechanisms of action and a number of natural phytotoxins to determine their effect on photosynthesis as measured by chlorophyll fluorescence. The most active compounds were those interacting directly with photosynthesis (inhibitors of photosystem I and II), those inhibiting carotenoid synthesis, and those with mechanisms of action generating reactive oxygen species and lipid peroxidation (uncouplers and inhibitors of protoporphyrinogen oxidase). Other active compounds targeted lipids (very-long-chain fatty acid synthase and removal of cuticular waxes). Therefore, induced chlorophyll fluorescence is a good biomarker to help identify certain herbicide modes of action and their dependence on light for bioactivity.     

Low temperatures enhance winter wilt of pepper plants caused by Pythium sp.

Pepper is the main vegetable crop grown in the Arava region of southern Israel. It is grown in the winter in nethouses and greenhouses. Low temperature wilt of mature pepper plants has been known for years in this region. The incidence of plant wilting was usually low when the soil was pretreated with methyl bromide. In recent years methyl bromide usage has been banned and disease incidence has increased. The causal agent of this phenomenon was unknown until the current study. Pythium sp. was the most common microorganism genus isolated from wilted plant roots. Young pepper plants were artificially inoculated with Pythium isolated from wilted plants and maintained at temperatures of 20°, 14°, 10.5° and 8.6°C. Significant wilting was observed in plants grown at 8.6°C, with symptoms starting 2?weeks after inoculation. At 10.5°C wilting developed more slowly and inoculated plants maintained at 14° and 20°C did not exhibit any wilting symptoms. The unique variation in sporangium morphology and the sequence of the ribosomal internal transcribed spacer (ITS) suggest that a new species of Pythium is involved. The fungicide metalaxyl-M was found effective in controlling the disease in pot experiments. The relationship between low temperatures and high disease incidence can explain the high disease incidence in the Arava Valley of Israel during the cold winters of 1999?C2000, 2004?C2005 and 2006?C2007.     

Relationships between agronomic factors and epidemics of Phytophthora branch canker of citrus in southwestern Spain

Phytophthora branch canker, caused by Phytophthora citrophthora, has been an increasing problem in clementine (Citrus reticulata) production in Spain during last years. The disease was particularly severe in the new citrus-growing areas of the southwestern coastal areas in Huelva Province. Recent studies revealed that disease emergence was not related to either genetic drift or host specificity changes in P. citrophthora population. Therefore, the possible association of agronomic factors with the disease was investigated. A total of 110 orchards were selected arbitrarily from the main citrus-growing areas in Huelva Province. The presence of branch cankers together with agronomic factors including soils, cultivars, rootstocks, irrigation, pruning, techniques to improve fruit production, fungicide treatments, presence of brown rot of fruit and frost damage were recorded. Logistic regression analysis was used to detect correlations between the agronomic factors studied and disease prevalence. Phytophthora branch canker was significantly associated with mature clementine orchards. Sweet orange and hybrid cultivars as well as young clementine orchards were less affected by the disease. Although disease was less frequent in Salorthid soils, alternative high resolution procedures are required to draw conclusions about the effect of soil properties on disease prevalence. As in other Phytophthora-induced diseases, soil flooding during the rainy season was correlated positively with the prevalence of branch cankers. Improving fruit production by branch scoring showed a strong positive correlation with Phytophthora branch canker. This is the first time that girdling has been associated with Phytophthora disease epidemics on a fruit tree crop, but further research is needed to determine the cause of this relationship. Cultural practices including pruning, regulated deficit irrigation, additional phosphonate sprays, and abiotic and disease factors such as frost damage and presence of brown rot of fruit were not significantly correlated with disease prevalence.     

Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa

Moniliophthora perniciosa is a fungus that causes witches?? broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this prompted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were used to study the variability of 70?M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geographical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.     

Experimental comparison of aerial larvicides and habitat modification for controlling disease-carrying Aedes vigilax mosquitoes

BACKGROUND: Microbial and insect‐growth‐regulator larvicides dominate current vector control programmes because they reduce larval abundance and are relatively environmentally benign. However, their short persistence makes them expensive, and environmental manipulation of larval habitat might be an alternative control measure. Aedes vigilax is a major vector species in northern Australia. A field experiment was implemented in Darwin, Australia, to test the hypotheses that (1) aerial microbial larvicide application effectively decreases Ae. vigilax larval presence, and therefore adult emergence, and (2) environmental manipulation is an effective alternative control measure. Generalised linear and mixed‐effects modelling and information‐theoretic comparisons were used to test these hypotheses. RESULTS: It is shown that the current aerial larvicide application campaign is effective at suppressing the emergence of Ae. vigilax, whereas vegetation removal is not as effective in this context. In addition, the results indicate that current larval sampling procedures are inadequate for quantifying larval abundance or adult emergence. CONCLUSIONS: This field‐based comparison has shown that the existing larviciding campaign is more effective than a simple environmental management strategy for mosquito control. It has also identified an important knowledge gap in the use of larval sampling to evaluate the effectiveness of vector control strategies. Copyright © 2011 Society of Chemical Industry