Application of PCR and probe hybridization techniques in detection of airborne fungal spores in environmental samples

Specific PCR amplification and probe hybridization techniques were applied to examine the compositions of airborne fungi in samples from three different environments. The results from microscopic and CFU counting were compared to those of the molecular-based detections. The detection sensitivity for PCR amplifications was 9 to 73 spores and 1.3 to 19.3 CFUs per PCR reaction. The hybridization detection limit was 2 to 4 spores and 0.2 to 1.2 CFU. The hybridization method was more sensitive than PCR amplification and showed less variation among samples. Using specific PCR primers and probes we identified the presence of several fungal groups and species in the air samples. Specific detections through probe hybridization to PCR products amplified with universal or group-specific fungal primers have promising applications in the examination of air samples for environmental monitoring.     

Air sampling of fungal spores on filters. An investigation on passive sampling and viability

In this study, glycerol was tested as a collection substrate for passive bioaerosol sampling. Filters (mixed cellulose acetate and nitrate) were soaked in glycerol and exposed for an aerosol from three different fungal species: Penicillum commune, Aspergillus versicolor and Paecilomyces variotii. The passive sampling method was compared with a closed-face polycarbonate filter sampling method. Exposure was performed in an exposure chamber. The total number of spores was determined by microscopic techniques, and the cultivable number was determined by cultivation on Malt Extract Agar dishes. The glycerol soaked filter demonstrated a good correlation with the closed-face sampler with regard to the total count. Spores stored in a pumped filter cassette were not affected by storage for up to 7 days. On the other hand, the culturability of the spores was markedly decreased after 1 day when stored on glycerol soaked filters.     

Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, <Emphasis Type="Italic">Ips pini</Emphasis> (Coleoptera: Scolytidae)

In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.Electronic Supplementary Material  Supplementary material is available in the online version of this article at .     

Airborne chemical compounds on dairy farms

The chemical environment that dairy farmers are exposed to during milking was investigated. Volatile organic compounds (VOCs) were analysed and identified, and the levels of formaldehyde, ammonia and carbon dioxide were measured in eight farms in northern Sweden. Both stationary and personal samples were taken. A total of 70 VOCs were identified from the adsorbent samples, with p-cresol, 2-butanone, ethyl acetate, alpha-pinene and delta 3-carene occurring at the highest levels. All monitored levels were significantly lower for compounds having a stated highest occupational exposure level (OEL). Using multivariate techniques some differences in the composition of the workplace air between and within the farms were found. No difference was found between personal exposure and the surrounding environment in the cowshed.     

Toward Explaining Nitrogen and Phosphorus Trends in Chesapeake Bay Tributaries, 1992–2012

Understanding trends in stream chemistry is critical to watershed management, and often complicated by multiple contaminant sources and landscape conditions changing over varying time scales. We adapted spatially referenced regression (SPARROW) to infer causes of recent nutrient trends in Chesapeake Bay tributaries by relating observed fluxes during 1992, 2002, and 2012 to contemporary inputs and watershed conditions. The annual flow‐normalized nitrogen flux to the bay from its watershed declined by 14% to 127,000 Mg (metric tons) between 1992 and 2012, due primarily (more than 80% of the decline) to reduced point sources. The remainder of the decline was due to reduced atmospheric deposition (13%) and urban nonpoint sources. Agricultural inputs, which contribute most nitrogen to the bay, changed little, although trends in the average nitrogen yield (flux per unit area) from cropland and pasture to streams in some settings suggest possible effects of evolving nutrient applications or other land management practices. Point sources of phosphorus to local streams declined by half between 1992 and 2012, while nonpoint inputs were relatively unchanged. Annual phosphorus delivery to the bay increased by 9% to 9,570 Mg between 1992 and 2012, however, due mainly to reduced retention in the Susquehanna River at Conowingo Reservoir.     

Do mites phoretic on elm bark beetles contribute to the transmission of Dutch elm disease?

Dutch elm disease (DED) is a destructive vascular wilt disease of elm (Ulmus) trees caused by the introduced Ascomycete fungus Ophiostoma novo-ulmi. In Europe, this DED pathogen is transmitted by elm bark beetles in the genus Scolytus. These insects carry phoretic mites to new, suitable habitats. The aim of this study was to record and quantify conidia and ascospores of O. novo-ulmi on phoretic mites on the three elm bark beetle species Scolytus multistriatus, Scolytus pygmaeus, and Scolytus scolytus. Spores of O. novo-ulmi were found on four of the ten mite species phoretic on Scolytus spp. These included Elattoma fraxini, Proctolaelaps scolyti, Pseudotarsonemoides eccoptogasteri, and Tarsonemus crassus. All four species had spores attached externally to their body surfaces. However, T. crassus carried most spores within its sporothecae, two paired pocket-like structures adapted for fungal transmission. Individuals of Pr. scolyti also had O. novo-ulmi conidia and ascospores frequently in their digestive system, where they may remain viable. While E. fraxini and P. eccoptogasteri rarely had spores attached to their bodies, large portions of Pr. scolyti and T. crassus carried significant numbers of conidia and/or ascospores of O. novo-ulmi. P. scolyti and T. crassus, which likely are fungivores, may thus contribute to the transmission of O. novo-ulmi, by increasing the spore loads of individual Scolytus beetles during their maturation feeding on twigs of healthy elm trees, enhancing the chance for successful infection with the pathogen. Only S. scolytus, which is the most efficient vector of O. novo-ulmi in Europe, carried high numbers of Pr. scolyti and T. crassus, in contrast to S. multistriatus and S. pygmaeus, which are known as less efficient vectors. The high efficiency of S. scolytus in spreading Dutch elm disease may be partly due to its association with these two mites and the hyperphoretic spores of O. novo-ulmi they carry.