Stephanie L. Parkes

An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea)

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.

Evolution of an Osmosensing Histidine Kinase in Field Strains of Botryotinia fuckeliana (Botrytis cinerea) in Response to Dicarboximide Fungicide Usage

ABSTRACT DNA sequence polymorphisms in the putative two-component histidine protein kinase encoded by the Daf1 gene have been identified within a sample of 5 sensitive and 27 dicarboximide-resistant field strains of Botryotinia fuckeliana (anamorph Botrytis cinerea). The gene of 3948 bp is predicted to encode a 1315-amino acid protein comprising an N-terminal region, an amino acid repeat region, which has been hypothesized to be the binding site for dicarboximide fungicide, and a C-terminal region encompassing kinase and response regulator domains. Two amino acid variants were distinguished among the sensitive strains characterized by alanine (group 1), or threonine (group 2), at position 1259 in the C-terminal region. All resistant strains could be classified into either group 1 or group 2 but, in addition, all showed changes in the second amino acid repeat region. On the basis of the differences in this repeat region, four classes of resistant strains were recognized; class 1 characterized by an isoleucine to serine mutation, class 2 by an isoleucine to asparagine mutation, class 3 by an isoleucine to arginine mutation (all at position 365), and class 4 by an isoleucine to serine mutation (position 365) as well as a glutamine to proline mutation (position 369). All classes showed similar low levels of resistance to iprodione and to vinclozolin, except for class 3 and class 4 strains, which show low resistance to iprodione but moderate (class 3) or high (class 4) resistance to vinclozolin. The classes as a group did not differ from sensitive strains in osmotic sensitivity measured as mycelial growth response, but some class 1 strains showed an abnormal morphology on osmotically amended medium. The evolution of the amino acid differences is discussed in relation to field observations. It is proposed that class 1 and class 2 strains arose by single mutations within the sensitive population, whereas classes 3 and 4 arose by single mutations within a resistant population.

Mating behaviour and genetics of fungicide resistance of Botrytis cinerea in New Zealand

Abstract The mating type of strains of Botrytis cinerea (teleomorph Botryotinia fuckeliana) from a diverse range of hosts and locations throughout New Zealand was determined by crossing with tester strains of known mating type. Most isolates were hetero‐thallic, 24 carrying the MAT 1–1 allele and 17 the MAT1–2 allele, but 9 were homothallic. The mating type alleles were widely distributed with respect to host, district, and fungicide resistance genes. Fungicide resistance phenotypes segregated as distinct characters consistent with the presence of the previously recognised genes Mbcl, conferring resistance to benzimidazole fungicides, and DafI, conferring resistance to dicarboximide fungicides. A new allele is recognised, DafIUR, encoding ultra‐low level dicarboximide resistance not associated with abnormal osmotic sensitivity. Pairing of single ascospore strains on malt extract agar amended with 0.68M NaCl demonstrated a mycelial incompatibility system in this fungus.

Use of nitrate non-utilising (Nit) mutants to determine vegetative compatibility in Botryotinia fuckeliana (Botrytis cinerea)

Nitrate non-utilising (Nit) mutants of six field strains and three single-ascospore strains of Botryotinia fuckeliana were selected by plating mycelial plugs onto a nitrate-containing minimal medium amended with chlorate. Mutants were characterised by growth responses on minimal medium amended with various sole nitrogen sources. For each parental strain two mutants were produced: nit1, defective in nitrate reductase apoenzyme; and NitM, defective in the molybdenum cofactor pathway. Complementation on nitrate minimal medium was observed between nit1 and NitM mutants from the same parent in all instances. However, complementation was not observed between any such combinations when the mutants were derived from different parents. It is concluded that a vegetative incompatibility system operates in B. fuckeliana resulting in multiple vegetative compatibility groups. One of the single-ascospore strains was derived from a cross between two of the field strains, indicating that new compatibility groups can be generated during sexual reproduction. Mycelial interaction zones were formed between all parental strains when they were paired on NaCl-amended medium, indicating congruence between mycelial incompatibility and vegetative incompatibility.

Self-incompatibility in Cordyline australis (Asteliaceae)

Abstract Controlled pollination among four individuals of Cordyline australis (cabbage tree, ti kouka) demonstrate that this species is self-incompatible, setting little or no seed when self-pollinated. Seed production per fruit in wild trees was less than half that observed following artificial pollination. Parthenocarpic fruit production is recorded in some wild populations.

Fungi associated with gorse and broom in New Zealand

Interest in the possibility of biological control of gorse and broom with fungal pathogens prompted a survey across New Zealand of fungi associated with diseased stem and leaf tissue of these weeds. New disease records made during the survey include: Ascochyta ulicis, Botryosphaeria dothidea, Gibberella avenacea, G. baccata, Glomerella cingulata and Septoria slaptonensis on gorse; Armillaria sp., Botryosphaeria dothidea, Colletotrichum acutatum, Gibberella avenacea and G. baccata on broom. Of the species found during the survey Pleiochaeta setosa on broom and Gibberella tumida on broom and gorse may have potential for development as mycoherbicides. Notes are provided on the biology of all species known from gorse and broom in New Zealand, including those found during the survey, others previously reported in the literature, and species deposited in Herbarium PDD.

Vegetative compatibility groups in Colletotrichum gloeosporioides (Glomerella cingulata) from apple and other fruits

Nitrogen non-utilising mutants were recovered from 32 strains of Colletotrichum gloeosporioides (Glomerella cingulata) isolated from h its of apple and other species. Mutants putatively defective in the nitrate reductase structural gene were recovered from all strains, and mutants putatively defective in molybdenum-containing cofactor synthesis from 17 strains. Complementation tests between these two classes of mutants on a medium containing nitrate as sole nitrogen source indicated the presence of at least 11 distinct vegetative compatibility groups, including at least six from apple.

Vegetative compatibility groups in the fungus Cryptosporiopsis actinidiae

Abstract Vegetative compatibility has been studied in the kiwifruit (Actinidia spp.) storage rot pathogen Cryptosporiopsis actinidiae by assessing the ability of nitrate non‐utilising mutants generated from the parental strains to complement on medium containing nitrate as sole nitrogen source. A total of 28 strains were allocated to 18 vegetative compatibility groups. One group contained four members, two contained three members, three contained two members, and the remainder contained single members. These numbers are consistent with the independent assortment of five vegetative compatibility genes during sexual recombination. Although a sexual stage has not yet been identified for this fungus, it is concluded that sexual reproduction plays an important role in its biology.