Correlation between hydrolytic enzyme activities measured in bean seedlings afterTrichoderma koningii treatment combined with pregermination and the protective effect againstPythium splendens

An experimental protocol consisting in the colonisation of pregerminated bean seeds dressed withTrichoderma sp. was used in order to study the mechanisms correlated with the protective effect againstPythium splendens. Seed dressed with TH-11 (T. koningii) for 24 h presented a higher protective effect and a higher level of seed colonisation as compared to those dressed with TH-13 (T. longibranchiatum). The levels of seed coat colonisation by TH-11 and TH-13 was shown to be correlated with the carboxymethylcellulase activity, as measured in the seed coats retreived from germinating dressed bean seeds. The seed coat colonisation was also associated with an increased activities of endo-1,3--glucanase and endochitinase measured in seed extracts, and an inhibitory effect of seed extracts onPythium sporangia germination. Pretreatment of TH-13-dressed seeds with a commercial cellulase improved all parameters mentioned above, thus suggesting a role of cellulase activity in the colonisation process and the linked protective effect. The possible role of hydrolytic enzymes in the protective effects is discussed.     

‘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.     

Effect of the Pietrain breed used as terminal boar on Post-weaning Multisystemic Wasting Syndrome (PMWS) in the offspring in four PMWS-affected farms

The aim of this study was to determine the potential risk factors for PMWS at the individual pig level and assess the effect of the Pietrain paternal genetic background of the animals in a cohort study. The survey was set up in four PMWS-affected farms with 2 repetitions (batches) per farm. A representative sample of 60 pigs per batch, stratified according to the paternal genetic background (Pietrain: yes vs. no), was randomly selected after farrowing. The representative cohort was divided into 8 batches and the pigs were individually monitored from birth to slaughter. Survival analysis was used to determine the factors related to the time to PMWS. The litter-cluster effect was taken into account using the marginal Cox model (robust estimation of the covariance matrix) and the gamma shared frailty model which were compared.No protective effect of the Pietrain breed on the time to PMWS and the proportion of affected pigs in the offspring was found in this study. Piglets showing low circovirus type 2 (PCV2) titres at 7 weeks-old with no subsequent seroconversion and piglets from PCV2 negative sows were most likely to be affected by PMWS (HR=7.0 and 2.8, respectively). Active infection of the pregnant dams with parvovirus was related to an increased risk of PMWS in the offspring (HR=2.3). Neck injuries due to poorly performed injections in the dams were associated with an increased risk of PMWS with the marginal model (HR=2.1). Oxytocin injection (dams) during farrowing was protective against PMWS in the offspring (HR=0.6).     

Characteristics for Practical Use of Attenuated Isolate UA-Fukushima of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis>

L₁₁A-Fukushima (L₁₁A-F) derived from attenuated isolate LuA of Tomato mosaic virus (ToMV) has the highest ability to cross protect against virulent ToMV among LuA and its derivatives and is stably inherited. Growth, yield, fruit quality and symptom attenuation of inoculated tomato plants did not differ significantly between L₁₁A-F and L₁₁A. The infectivity of progeny viruses in tomato infected with LuA-F was less than 4% of that with virulent ToMV. From these results, L₁₁A-F appears to possess the properties necessary for practical use. To manage L₁₁A-F strictly, a PCR-based assay to detect trace contamination of virulent ToMV in L₁₁A-F preparations was established. Received 10 June 2002/ Accepted in revised form 30 October 2002     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

Indole-3-acetic acid biosynthesis in <Emphasis Type="Italic">Aciculosporium take</Emphasis>, a causal agent of witches' broom of bamboo

 Aciculosporium take (Ascomycota; Clavicipitaceae) is a causal agent of witches' broom of bamboo plants. The symptoms of this disease are believed to be induced by plant hormones, particularly auxins. Indole-3-acetic acid (IAA) was identified in cultures of this fungus in an l-tryptophan-supplemented liquid medium. IAA production was confirmed on 30 isolates of A. take from various hosts and locations at levels up to 1 mg/l. The biosynthetic pathway of IAA in A. take culture was examined by analyzing intermediate products and by feeding experiments. The results showed that the indole-3-pyruvic acid pathway (l-tryptophan → indole-3-pyruvic acid → indole acetaldehyde → IAA) was the dominant pathway in A. take. Received: June 3, 2002 / Accepted: July 25, 2002     

Inconspicuous restraint of rice seedling growth by root-infecting fungi in soil of a rice paddy field

 Rice seedling growth, estimated by plant height and root development and discoloration, was better in pasteurized soil than in unpasteurized soil obtained from a flooded rice field. Rice seedlings also grew better in sterilized soil modified by adding roots harvested from the pasteurized soil than in soil modified by adding roots harvested from the unpasteurized soil. The results demonstrate that seedling growth in the rice field soil was inhibited by soil microorganisms, even though no typical symptoms such as seedling blight or damping-off appeared. Pythium aristosporum is suggested to be involved in the inhibition. Thus, it appears that inconspicuous restraint of rice seedling growth could occur in soils of rice paddy fields. Received: May 20, 2002 / Accepted: October 16, 2002 Acknowledgments The authors thank Dr. T. Ichitani, former professor at Osaka Prefectural University, for providing an isolate of Pythium aristosporum for comparison, and Mr. Mitsuaki Sato of Akita Prefectural College of Agriculture for technical assistance.     

Functional heterogeneity in resources within landscapes and herbivore population dynamics

Large mammalian herbivores are notorious for their propensity towards population irruptions and crashes, yet many herbivore populations remain relatively stable. I explore how resource heterogeneity within landscapes dampens population instability, using a metaphysiological modelling approach considering patch state distributions. Resource heterogeneity is functionally stabilizing through spreading consumption away from preferred resources before these become critically depleted. Lower-quality resources act as a buffer against starvation during critical periods of the seasonal cycle. Enriching resource quality is destabilizing, even if patch diversity is maintained, because food quantity then becomes the limitation. The potential consequences of landscape fragmentation are explored using the Serengeti ecosystem, characterised by broadscale resource gradients, as a hypothetical example. Further insights provided by the model are illustrated with specific examples concerning the effects of patch scales and waterpoint distribution. A metaphysiological modelling approach enables the basic consequences of landscape heterogeneity to be distinguished from further effects that may arise from specific patch scales and configurations, without the distracting detail of spatially explicit models.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.