Quantification of Airborne Inoculum as an Aid in the Management of Leaf Blight of Onion Caused by Botrytis squamosa.

Botrytis leaf blight, caused by Botrytis squamosa, is a common and frequently damaging disease of onion crops, but the severity of epidemics varies widely from year to year. The disease is initiated and spread by airborne conidia. The relationship between airborne conidium concentration (ACC) and lesion development was studied in the field. A linear relationship was found between ACC and number of lesions per leaf: ACC values of 10 to 15 and 25 to 35 conidia m-3 were associated with 1 and 2.5 lesions per leaf, respectively. In 2000 and 2001, at three sites, four different criteria were used to start a fungicide spray program and their effect on epidemic development was compared with that of a grower's conventional schedule. The criteria were: at the fourth-true-leaf growth stage; according to an inoculum production index; when the ACC reached 10 to 15 conidia m-3; and when the ACC reached 25 to 35 conidia m-3. A nonsprayed control plot was included in the trial. Fungicide programs started when the ACC reached 10 to 15 conidia m-3 were as effective as the conventional program, but used fewer sprays. A fungicide spray program based on measurements of ACC and disease severity was evaluated in 2002 and 2003 in five and three commercial onion fields, respectively. At each site, half of the field was sprayed according to the grower's schedule and, in the other half, fungicide sprays were initiated when a threshold of 10 to 15 conidia m-3 or five lesions on the lower leaf (whichever came first) was reached. Overall, the number of fungicide applications was reduced by 75 and 56% in 2002 and 2003, respectively, without causing significant yield reduction. In both years, the reduction in number of fungicide applications was due mainly to the delay in initiation of the fungicide program.

A preliminary study of airborne microbial biodiversity over Peninsular Antarctica.

This study used PCR-based molecular biological identification techniques to examine the biodiversity of air sampled over Rothera Point (Antarctic Peninsula). 16S rDNA fragments of 132 clones were sequenced and identified to reveal a range of microorganisms, including cyanobacteria, actinomycetes, diatom plastids and other uncultivated bacterial groups. Matches for microorganisms that would be considered evidence of human contamination were not found. The closest matches for many of the sequences were from Antarctic clones already in the databases or from other cold environments. Whilst the majority of the sequences are likely to be of local origin, back trajectory calculations showed that the sampled air may have travelled over the Antarctic Peninsula immediately prior to reaching the sample site. As a result, a proportion of the detected biota may be of non-local origin. Conventional identification methods based on propagule morphology or culture are often inadequate due to poor preservation of characteristic features or loss of viability during airbome transfer. The application of molecular biological techniques in describing airbome microbial biodiversity represents a major step forward in the study of airborne biota over Antarctica and in the distribution of microorganisms and propagules in the natural environment.

The potential of optical canopy measurement for targeted control of field crop diseases.

There is increasing pressure to reduce the use of pesticides in modern crop production to decrease the environmental impact of current practice and to lower production costs. It is therefore imperative that sprays are only applied when and where needed. Since diseases in fields are frequently patchy, sprays may be applied unnecessarily to disease-free areas. Disease control could be more efficient if disease patches within fields could be identified and spray applied only to the infected areas. Recent developments in optical sensor technology have the potential to enable direct detection of foliar disease under field conditions. This review assesses recent developments in the use of optical methods for detecting foliar disease, evaluates the likely benefits of spatially selective disease control in field crops, and discusses practicalities and limitations of using optical disease detection systems for crop protection in precision pest management.

Molecular diagnostics for fungal plant pathogens.

Accurate identification of fungal phytopathogens is essential for virtually all aspects of plant pathology, from fundamental research on the biology of pathogens to the control of the diseases they cause. Although molecular methods, such as polymerase chain reaction (PCR), are routinely used in the diagnosis of human diseases, they are not yet widely used to detect and identify plant pathogens. Here we review some of the diagnostic tools currently used for fungal plant pathogens and describe some novel applications. Technological advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens and are now being applied to practical problems. Molecular methods have been used to detect several pathogens simultaneously in wheat, and to study the development of fungicide resistance in wheat pathogens. Information resulting from such work could be used to improve disease control by allowing more rational decisions to be made about the choice and use of fungicides and resistant cultivars. Molecular methods have also been applied to the study of variation in plant pathogen populations, for example detection of different mating types or virulence types. PCR-based methods can provide new tools to monitor the exposure of a crop to pathogen inoculum that are more reliable and faster than conventional methods. This information can be used to improve disease control decision making. The development and application of molecular diagnostic methods in the future is discussed and we expect that new developments will increase the adoption of these new technologies for the diagnosis and study of plant disease.

Methods for integrated air sampling and dna analysis for detection of airborne fungal spores.

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.

Antibody recognition of melphalan adducts characterized using immobilized DNA: enhanced alkylation of G-Rich regions in cells compared to in vitro.

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) indicated that in 0.01 and 0.1 M NaOH, loss of immunoreactivity to MP5/73 occurred with half-lives that were at least 2-fold longer than half-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 degrees C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan-treated cells, adducts recognized by Amp4/42 formed a smaller proportion of total adducts compared to DNA alkylated in vitro. The results presented here indicate that this was caused by a marked cellular influence on the overall sequence-dependent pattern of DNA alkylation or repair.

A monofunctional derivative of melphalan: preparation, DNA alkylation products, and determination of the specificity of monoclonal antibodies that recognize melphalan-DNA adducts.

Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was

Riboflavin and vitamin B-6 intakes and status and biochemical response to riboflavin supplementation in free-living elderly people.

Free-living elderly people aged > or = 65 y were recruited to assess riboflavin and vitamin B-6 intakes and status and the effect of riboflavin supplementation on biochemical indicators of these 2 vitamins. The status of riboflavin (erythrocyte glutathione reductase activation coefficient; EGRAC) and vitamin B-6 (plasma pyridoxal-5'-phosphate; PLP) were determined in a total sample of 92 subjects, from whom dietary intake data were obtained by using the diet history method (n = 83). Although dietary intakes of both vitamins were considered to be adequate according to current reference values, abnormal EGRAC and plasma PLP values were identified in 49% and 38% of subjects, respectively, with 21% having suboptimal status for both nutrients. A subgroup of subjects from the initial sample (n = 45) was assigned in a double-blind manner to receive either 1.6 or 25 mg riboflavin or placebo daily for 12 wk. In those subjects with a baseline EGRAC or plasma PLP value falling outside the currently accepted threshold value for adequacy, low-dose riboflavin supplementation improved status of the limiting nutrient significantly (P<0.0001 and P = 0.020 for EGRAC and plasma PLP responses, respectively). We conclude that a high proportion of healthy elderly people may have suboptimal status for these nutrients despite apparently adequate dietary intakes. Furthermore, we showed that riboflavin supplementation at physiologic doses corrects biochemical abnormalities of not only EGRAC, but also plasma PLP, confirming the biochemical interdependency of these vitamins and suggesting that riboflavin is the limiting nutrient.

Analysis of the relationships between environmental factors (aeroallergens, air pollution, and weather) and asthma emergency admissions to a hospital in Mexico City.

There have been several studies of the relationships between environmental factors, particularly air pollution, and attacks of asthma. Most of these studies have ignored the potential confounding effects of aeroallergens such as pollens and fungal spores. We report a statistical analysis of the relationships between emergency admissions for asthma to a hospital in Mexico City and daily average airborne concentrations of pollen, fungal spores, air pollutants (O3, NO2, SO2, and particulates) and weather factors. Asthma admissions had a seasonal pattern with more during the wet season (May-October) than the dry season (November-April). There were few statistical associations between asthma admissions and air pollutants for the three age groups studied (children under 15 years, adults, and seniors [adults over 59 years]) in either season. Grass pollen was associated with child and adult admissions for both the wet and dry seasons, and fungal spores were associated with child admissions during both the wet and dry seasons. The analysis was done with environmental data averaged over the day of admission and the 2 previous days. Our results suggest that aeroallergens may be statistically associated more strongly with asthma hospital admissions than air pollutants and may act as confounding factors in epidemiologic studies.