Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

‘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.     

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     

Secretion of Natural and Synthetic Toxic Compounds from Filamentous Fungi by Membrane Transporters of the ATP-binding Cassette and Major Facilitator Superfamily

This review provides an overview of members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) of transporters identified in filamentous fungi. The most common function of these membrane proteins is to provide protection against natural toxic compounds present in the environment of fungi, such as antibiotics produced by other microorganisms. In plant pathogenic fungi, these transporters can also be an important determinant of virulence on host plants by providing protection against plant defence compounds or mediating the secretion of host-specific toxins. Furthermore, they play a critical role in determining base-line sensitivity to fungicides and other antimycotic agents. Overexpression of some of these transporters can lead to the development of resistance to chemically-unrelated compounds, a phenomenon described as multidrug resistance (MDR). This has been observed in a variety of organisms and can impose a serious threat to the effective control of pathogenic fungi.     

Functional Analysis of ABC Transporter Genes From Botrytis cinerea Identifies BcatrB as a Transporter of Eugenol

The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expression of 11 ABC (BcatrA–BcatrK) and three MFS genes (Bcmfs1, Bcmfs2 and Bcmfs4) was studied. All genes showed a low basal level of expression, but were differentially induced by treatment with cycloheximide and the plant defence compounds camptothecin, eugenol, psoralen, resveratrol and rishitin. The latter compounds induced expression of BcatrB at a high level. Eugenol was more toxic to BcatrB gene-replacement mutants than to the control isolates. Eugenol also caused an instantaneous increase in mycelial accumulation of the fungicide fludioxonil, a known substrate of BcatrB. However, there was no difference in virulence between the wild-type and BcatrB gene-replacement mutants on Ocimum basilicum, a plant known to contain eugenol. The results indicate that BcatrB is a transporter of lipophilic compounds, such as eugenol, but its role in virulence remains uncertain.     

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.