Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.     

Acidic derivatives of the fungicide fenpiclonil: effect of adding a methyl group to the N-substituted chain on systemicity and fungicidal activity

A new acidic derivative of the fungicide fenpiclonil was synthesized containing a methyl group on the alpha-position of the carboxyl function of N-carboxymethyl-3-cyano-4-(2,3-dichlorophenyl)pyrrole. The phloem mobility of the resulting N-(1-carboxyethyl)-3-cyano-4-(2,3-dichlorophenyl)pyrrole was comparable with that of the former compound, but was higher at external pH 5.0. Unlike the derivatives previously synthesized, it was comparable with fenpiclonil in its fungicidal activity against the pathogenic fungus Eutypa lata.     

Synthesis and phloem mobility of acidic derivatives of the fungicide fenpiclonil

A series of derivatives of the phenylpyrrole fungicide fenpiclonil was synthesized in which a carboxyl group was present at various sites of this non-phloem-mobile molecule. Using the Kleier model, all these acidic analogues were predicted to be moderately phloem-mobile, especially the N-substituted derivatives. One of these latter molecules, N-carboxymethyl-3-cyano-4-(2,3-dichlorophenyl)pyrrole, exhibited some fungicidal activity on the pathogenic fungus Eutypa lata, and was then tested as a phloem-mobile pesticide in the Ricinus system. The compound was indeed mobile in the sieve tubes and was not degraded to fenpiclonil in the phloem sap under our experimental conditions. Its concentration in the sap was closely correlated to the percentage of the undissociated form of the molecule in the external medium, and was similar under acidic conditions (external pH 4.6-5.0) to that of the herbicide glyphosate.     

Acetylcholinesterase-Inhibiting Activity of Pyrrole Derivatives from a Novel Marine Gliding Bacterium, Rapidithrix thailandica

Acetylcholinesterase-inhibiting activity of marinoquinoline A (1), a new pyrroloquinoline from a novel species of a marine gliding bacterium Rapidithrix thailandica, was assessed (IC₅₀ 4.9 μM). Two related pyrrole derivatives, 3-(2′-aminophenyl)-pyrrole (3) and 2,2-dimethyl-pyrrolo-1,2-dihydroquinoline (4), were also isolated from two other strains of R. thailandica. The isolation of 3 from a natural source is reported here for the first time. Compound 4 was proposed to be an isolation artifact derived from 3. The two isolated compounds were virtually inactive in the acetylcholinesterase-inhibitory assay (enzyme inhibition < 30% at 0.1 g L⁻¹).     

Effect of Phenylpyrroles on Glycerol Accumulation and Protein Kinase Activity of Neurospora crassa

The effect of phenylpyrroles on glycerol synthesis in Neurospora crassa has been investigated and compared with the mode of action of vinclozolin (a dicarboximide). The results indicated that fenpiclonil, fludioxonil and vinclozolin at concentrations which inhibit growth by 50% induce accumulation of glycerol in the mycelium of N. crassa. Furthermore a protein kinase (PK-III) possibly involved in the regulation of the glycerol synthesis is inhibited by phenylpyrroles, whereas vinclozolin is without effect. This implies that the target sites of phenylpyrroles and dicarboximides in the osmosensing signal transmission pathway are different. Comparative experiments with enzymes from human and animal sources revealed that PK-III could be a protein kinase Cδ. It is suggested that inhibition of PK-III activity may result in an increased concentration of a non-phosphorylated regulatory protein which may activate a MAP-kinase cascade of reactions resulting in increased glycerol synthesis. © 1997 SCI.     

Natural products in plant protection

Natural products can be used to control pests and diseases in crops. These products include anorganic compounds, but also a variety of plant extracts. In the beginning of this century active microbial extracts were discovered as well. Synthetic crop protection chemicals were developed from about 1940 onwards and sustained progress in modern agriculture. The first generation chemicals have aspecific modes of toxic action and are in many instances deleterious to the environment. The second generation chemicals have specific modes of action and meet modern environmental requirements. A disadvantage of these chemicals is the potency of target organisms to acquire resistance. This condition urged agrochemical industry to develop chemicals with new modes of action. Such chemicals can be developed by using natural bioactive products as leads in synthesis programmes. This paper decribes the progress that was made in the development of natural bioactive compounds in pest and disease control.