Use of fiber interface direct mass spectrometry for the determination of volatile flavor release from model food systems.

Described in this paper is a fiber interface direct headspace mass spectrometric system for the real-time measurement of flavor release. The system was optimized for the detection of the garlic aroma volatile, diallyl disulfide, from water. Parameters investigated included interface temperature, flow rate through the fiber, flow rate through the sample vessel, and sample stir rate. The delay time for detection of sample after introduction into the sample vessel was determined as 43 s. The system proved to be reliable and robust with no loss in sensitivity or contamination of the mass spectrometer over a 6 month period. The technique was applied to a homologous series of aliphatic alcohols from C(2) to C(7). Results showed that as polarity decreased with increasing chain length the release of volatile into the headspace was faster and gave a higher maximum intensity. Release of the garlic aroma volatile from different commercial mayonnaise products clearly showed a decrease in the release of diallyl disulfide as fat content increased. These results demonstrate the potential of using this technique as a tool for understanding the complex interactions that occur between flavor compounds and the bulk food matrix.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.     

Long-term organic phosphorus mineralization in Spodosols under forests and its relation to carbon and nitrogen mineralization

In forest soils where a large fraction of total phosphorus (P) is in organic forms, soil micro-organisms play a major role in the P cycle and plant availability since they mediate organic P transformations. However, the correct assessment of organic P mineralization is usually a challenging task because mineralized P is rapidly sorbed and most mineralization fluxes are very weak. The objectives of the present work were to quantify in five forest Spodosols at soil depths of 0-15 cm net mineralization of total organic P and the resulting increase in plant available inorganic P and to verify whether net or gross P mineralization could be estimated using the C or N mineralization rates. Net mineralization of total organic P was derived from the net changes in microbial P and gross mineralization of P in dead soil organic matter. We studied very low P-sorbing soils enabling us to use lower extractants to assess the change in total inorganic P as a result of gross mineralization of P in dead soil organic matter. In addition, to enable detection of gross mineralization of P in dead soil organic matter, a long-term incubation (517 days) experiment was carried out. At the beginning of the experiment, total P contents of the soils were very low (19-51 μg g⁻¹) and were essentially present as organic P (17-44 μg g⁻¹, 85-91%) or microbial P (6-14 μg g⁻¹; 24-39%). Conversely, the initial contents of inorganic P were low (2-7 μg g⁻¹; 9-15%). The net changes in the pool size of microbial P during the 517 days of incubation (4-8 μg g⁻¹) and the amounts of P resulting from gross mineralization of dead soil organic matter (0.001-0.018 μg g⁻¹ day⁻¹; 0.4-9.5 μg g⁻¹ for the entire incubation period) were considerable compared to the initial amounts of organic P and also when compared to the initial diffusive iP fraction (<0.3 μg g⁻¹). Diffusive iP corresponds to the phosphate ions that can be transferred from the solid constituents to the soil solution under a gradient of concentration. Net mineralization of organic P induced an important increase in iP in soil solution (0.6-10 μg g⁻¹; 600-5000% increase) and lower increases in diffusive iP fractions (0.3-5 μg g⁻¹; 300-2000% increase), soil solid constituents having an extremely low reactivity relative to iP. Therefore, soil micro-organisms and organic P transformations play a major role in the bioavailability of P in these forest soils. In our study, the dead soil organic matter was defined as a recalcitrant organic fraction. Probably because gross mineralization of P from this recalcitrant organic fraction was mainly driven by the micro-organisms’ needs for energy, the rates of gross mineralization of C, N and P in the recalcitrant organic fraction were similar. Indirect estimation of gross mineralization of P in dead soil organic matter using the gross C mineralization rate seems thus an alternative method for the studied soils. However, additional studies are needed to verify this alternative method in other soils. No relationships were found between microbial P release and microbial C and N releases.     

Horizontal transmission of Mycobacterium avium subsp. paratuberculosis in cattle in an experimental setting: calves can transmit the infection to other calves

In September 2001, two subsequent transmission experiments both lasting 3 months were carried out to study cow-calf transmission of Mycobacterium avium subsp. paratuberculosis (Map) (Period 1), followed by calf-calf transmission of the infection (Period 2). Every 2 weeks, serum, heparinised blood and faecal samples were collected from all animals. After these experiments, the 20 calves were housed individually for more than 3 years to be able to detect the infection status and excretion pattern of each animal. In autumn 2004, the animals were inseminated, to observe a possible increase in faecal excretion of Map shortly before expected calving. One month before the expected calving date in 2005, animals were slaughtered and several tissues per cow and unborn calf were sampled for culture. The results indicate that horizontal cow-calf transmission is readily achieved (Period 1). At the highest infection pressure (six shedding cows of which three high shedders in Period 1) all five calves excreted Map in their faeces during Period 1 (shortly after infection), and four of these calves during Period 2 (when the shedding cows were absent). After that, excretion became less frequently. Horizontal calf-calf transmission did take place (Period 2), as the four donor-calves infected two receiver-calves. Transmission rates during the 3 months periods were quantified as a reproduction ratio R. The R [95% CI] of cow-calf and calf-calf transmission were estimated as 2.7 [1.1, 6.6] and 0.9 [0.1, 3.2] new infections per infectious animal during 3 months.     

The BIOEXPLOIT Project

The EU Framework 6 Integrated Project BIOEXPLOIT concerns the exploitation of natural plant biodiversity for the pesticide-free production of food. It focuses on the pathogens Phytophthora infestans, Septoria tritici, Blumeria graminis, Puccinia spp. and Fusarium spp. and on the crops wheat, barley, tomato and potato. The project commenced in October 2005, comprises 45 laboratories in 12 countries, and is carried out by partners from research institutes, universities, private companies and small-medium enterprises. The project has four strategic objectives covered in eight sub-projects. These objectives relate to (i) understanding the molecular components involved in durable disease resistance, (ii) exploring and exploiting the natural biodiversity in disease resistance, (iii) accelerating the introduction of marker-assisted breeding and genetic engineering in the EU plant breeding industry, and (iv) coordinating and integrating resistance breeding research, providing training in new technologies, disseminating the results, and transferring knowledge and technologies to the industry.     

Effect of temperature on bulb growth capacity and sensitivity to summer sprouting in Lilium longiflorum Thunb.

The production of Lilium longiflorum bulbs in The Netherlands, with its cool climate, has been a problem because of unsatisfactory bulb growth and the risk of premature sprouting of the daughter bulbs (summer sprouting). To investigate the possibilities of breeding a type of L. longiflorum that can be grown under cool climatic conditions, a collection of 27 L. longiflorum cultivars from Japan, The Netherlands and the United States was tested, together with two Asiatic hybrids and a L. speciosum cultivar, under low phytotron temperatures (10, 14, 17°C) and in field experiments.‘Mount Everest’, ‘Saeki’, ‘Indian Summer’ and ‘White American’ were among the best L. longiflorum cultivars, with less than 20% summer sprouting and good bulb production. This was in contrast to ‘Hinomoto’, ‘Ace’ and some American introductions, which showed more than 60% summer sprouting and low bulb production. The lower the phytotron temperature, the more summer sprouting occurred. The differences observed between the cultivars in the field experiments were in agreement with those observed in the phytotron. The genetic variation proved large enough to start a breeding program for L. longiflorum adapted to cool climate conditions.