Letty A. de Weger

Changes to Airborne Pollen Counts across Europe

A progressive global increase in the burden of allergic diseases has affected the industrialized world over the last half century and has been reported in the literature. The clinical evidence reveals a general increase in both incidence and prevalence of respiratory diseases, such as allergic rhinitis (common hay fever) and asthma. Such phenomena may be related not only to air pollution and changes in lifestyle, but also to an actual increase in airborne quantities of allergenic pollen. Experimental enhancements of carbon dioxide (CO) have demonstrated changes in pollen amount and allergenicity, but this has rarely been shown in the wider environment. The present analysis of a continental-scale pollen data set reveals an increasing trend in the yearly amount of airborne pollen for many taxa in Europe, which is more pronounced in urban than semi-rural/rural areas. Climate change may contribute to these changes, however increased temperatures do not appear to be a major influencing factor. Instead, we suggest the anthropogenic rise of atmospheric CO levels may be influential.

Microbial stimulation of plant growth and protection from disease

Certain microbes stimulate plant growth. The mechanisms used by such microbes to achieve this are: biofertilization (i.e. increasing the supply of mineral nutrients to the plant); biocontrol (i.e. elimination of the plants enemies including microbial pathogens, insects and weeds); and direct plant growth promotion (e.g. by delivering plant growth hormones to the plant). In all of these mechanisms, plant root colonization is crucial as the delivery system.

A two-component system plays an important role in the root-colonizing ability of Pseudomonas fluorescens strain WCS365

We describe the characterization of a novel Tn5lacZ colonization mutant of the efficiently colonizing Pseudomonas fluorescens strain WCS365, mutant strain PCL1210, which is at least 300- to 1,000-fold impaired in colonization of the potato root tip after co-inoculation of potato stem cuttings with a 1:1 mixture of mutant and parental cells. Similarly, the mutant is also impaired in colonization of tomato, wheat, and radish, indicating that the gene involved plays a role in the ability of P. fluorescens WCS365 to colonize a wide range of plant species. A 3.1-kb DNA fragment was found to be able to complement the observed mutation. The nucleotide sequence of the region around the Tn5lacZ insertion showed three open reading frames (ORFs). The transcriptional start site was determined. The operon is preceded by an integration host factor (IHF) binding site consensus sequence whereas no clear -10 and -35 sequences are present. The deduced amino acid sequences of the first two genes of the operon, designated as colR and colS, show strong similarity with known members of two-component regulatory systems. ColR has homology with the response regulators of the OmpR-PhoB subclass whereas ColS, the product of the gene in which the mutation resides, shows similarity to the sensor kinase members of these two-component systems. Hydrophobicity plots show that this hypothetical sensor kinase has two transmembrane domains, as is also known for other sensor kinases. The product of the third ORF, Orf222, shows no homology with known proteins. Only part of the orf222 gene is present in the colonization-complementing, 3.1-kb region, and it therefore does not play a role in complementation. No experimental evidence for a role of the ColR/ColS two-component system in the suspected colonization traits chemotaxis and transport of exudate compounds could be obtained. The function of this novel two-component system therefore remains to be elucidated. We conclude that colonization is an active process in which an environmental stimulus, through this two-component system, activates a so far unknown trait that is crucial for colonization.

Efficient and sensitive identification and quantification of airborne pollen using next-generation DNA sequencing

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen‐allergic patients. Current pollen monitoring methods are microscope‐based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost‐effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next‐generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.

Pseudomonas spp. with Mutational Changes in the O-Antigenic Side Chain of their Lipopolysaccharide are Affected in their Ability to Colonize Potato Roots

Microorganisms that efficiently colonize plant roots are ideal tools for the production of biocontrol agents, since many major and minor root pathogens are present at the root surface. In the past decade Pseudomonas spp. have received much attention as biocontrol agents (Kloepper et al 1980, Schippers et al 1986). The interest in this group of bacteria is mainly due to their antagonistic potentials in vitro towards pathogens combined with their ability to colonize the roots of many plants efficiently.

Impact of pollen.

The impact of pollen on human health is primarily evident in allergic diseases. Sensitized patients can respond to pollen by symptoms of nose, eyes and bronchi. Pollen threshold levels for sensitization are unknown; instead most studies focus on the prevalence of sensitization for different pollen species. The pollen thresholds for symptom development vary among the different studies. Factors that influence the threshold level of a pollen species for symptom development are discussed. (i) Differences in response are observed among individual patients, but also among (ii) ethnic populations, (iii) changes in response to pollen concentrations during the pollen season occur, (iv) the amount of allergens carried by the pollen grains can differ in per region, from day to day and from year to year, and finally (v) threshold levels are affected by environmental factors, like weather conditions (temperature, pressure and storms), and air pollutants.The diversity of factors that influence the health impact of pollen has hampered the definition of a straight forward relationship between pollen and the severity of symptoms. However, within the public, the policymakers and the pharmaceutical industry there is a need for a definition of threshold pollen levels. A first approach to meet this need could be to define preliminary threshold values for different regions, followed by a validation of these preliminary threshold levels with patient symptom scores that can be collected by using new information and communication technology (ICT).

Monitoring, Modelling and Forecasting of the Pollen Season

The section about monitoring covers the development of phenological networks, remote sensing of the season cycle of the vegetation, the emergence of the science of aerobiology and, more specifically, aeropalynology, pollen sampling instruments, pollen counting techniques, applications of aeropalynology in agriculture and the European Pollen Information System. Three data sources are directly related with aeropalynology: phenological observations, pollen counts and remote sensing of the vegetation activity. The main future challenge is the assimilation of these data streams into numerical pollen forecast systems. Over the last decades consistent monitoring efforts of various national networks have created a wealth of pollen concentration time series. These constitute a nearly untouched treasure, which is still to be exploited to investigate questions concerning pollen emission, transport and deposition. New monitoring methods allow measuring the allergen content in pollen. Results from research on the allergen content in pollen are expected to increase the quality of the operational pollen forecasts.

Use of a lux-based procedure to rapidly visualize root colonisation by Pseudomonas fluorescens in the wheat rhizosphere

The bioluminescently marked Pseudomonas fluorescens strain 5RL, has been used previously to follow colonisation of soy bean roots (De Weger et al. [1991] Appl. Environ. Microbiol. 57:36-41). In the present paper the method has been further developed and optimized for wheat roots and it is used to get a quick overview of the colonisation patterns of many different root systems at the same time. Colonisation was followed on wheat plants grown in our gnotobiotic sand system (Simons et al., 1996. Mol Plant Microbe Interact 9: 600–607) and the following results were obtained. (i) A spatio-temporal analysis of the colonisation of wheat roots showed that 4 days after planting the highest bacterial activity was observed at the upper part of the root. After 6 days the high bacterial activity at the upper part was further increased, whereas spot-like activities were observed on the lower root parts, possibly due to micro-colonies. (ii) Bacterial mutations causing lack of motility or auxotrophy for amino acids resulted in impaired colonisation of the lower root parts, indicating that motility and prototrophy for the involved amino acid(s) are important factors for wheat root colonisation by strain 5RL. (iii) Coinoculation of strain 5RL with other wild type Pseudomonas strains on the root influenced the colonisation pattern observed for strain 5RL. Colonisation was not visually affected when the competing strain was a poor root coloniser, but was severely reduced when the competing strain was a good root coloniser. The results show that the spatio-temporal colonisation of wheat root by P. fluorescens strain 5RL and derivatives is similar to that of strain WCS365 on tomato. The advantage of the use of lux-marked strains is that the results are obtained much quicker than when conventional methods are used and that the result is supplied as an image of the colonisation pattern of many different roots.

Detection of pollen grains in multifocal optical microscopy images of air samples.

Pollen is a major cause of allergy and monitoring pollen in the air is relevant for diagnostic purposes, development of pollen forecasts, and for biomedical and biological researches. Since counting airborne pollen is a time‐consuming task and requires specialized personnel, an automated pollen counting system is desirable. In this article, we present a method for detecting pollen in multifocal optical microscopy images of air samples collected by a Burkard pollen sampler, as a first step in an automated pollen counting procedure. Both color and shape information was used to discriminate pollen grains from other airborne material in the images, such as fungal spores and dirt. A training set of 44 images from successive focal planes (stacks) was used to train the system in recognizing pollen color and for optimization. The performance of the system has been evaluated using a separate set of 17 image stacks containing 65 pollen grains, of which 86% was detected. The obtained precision of 61% can still be increased in the next step of classifying the different pollen in such a counting system. These results show that the detection of pollen is feasible in images from a pollen sampler collecting ambient air. This first step in automated pollen detection may form a reliable basis for an automated pollen counting system. Microsc. Res. Tech., 2009.

The minimal clinically important difference of the control of allergic rhinitis and asthma test (CARAT): Cross-cultural validation and relation with pollen counts

Background:The Control of Allergic Rhinitis and Asthma Test (CARAT) monitors control of asthma and allergic rhinitis.Aims:To determine the CARAT’s minimal clinically important difference (MCID) and to evaluate the psychometric properties of the Dutch CARAT.Methods:CARAT was applied in three measurements at 1-month intervals. Patients diagnosed with asthma and/or rhinitis were approached. MCID was evaluated using Global Rating of Change (GRC) and standard error of measurement (s.e.m.). Cronbach’s alpha was used to evaluate internal consistency. Spearman’s correlation coefficients were calculated between CARAT, the Asthma Control Questionnaire (ACQ5) and the Visual Analog Scale (VAS) on airway symptoms to determine construct and longitudinal validity. Test–retest reliability was evaluated with intra-class correlation coefficient (ICC). Changes in pollen counts were compared with delta CARAT and ACQ5 scores.Results:A total of 92 patients were included. The MCID of the CARAT was 3.50 based on GRC scores; the s.e.m. was 2.83. Cronbach’s alpha was 0.82. Correlation coefficients between CARAT and ACQ5 and VAS questions ranged from 0.64 to 0.76 (P<0.01). Longitudinally, correlation coefficients between delta CARAT scores and delta ACQ5 and VAS scores ranged from 0.41 to 0.67 (P<0.01). Test–retest reliability showed an ICC of 0.81 (P<0.01) and 0.80 (P<0.01). Correlations with pollen counts were higher for CARAT than for ACQ5.Conclusions:This is the first investigation of the MCID of the CARAT. The CARAT uses a whole-point scale, which suggests that the MCID is 4 points. The CARAT is a valid and reliable tool that is also applicable in the Dutch population.