Sexual Crosses Research Articles

The TNL gene Rdr1 confers broad-spectrum resistance to Diplocarpon rosae.

Black spot disease, which is caused by the ascomycete Diplocarpon rosae, is the most severe disease in field-grown roses in temperate regions and has been distributed worldwide, probably together with commercial cultivars. Here, we present data indicating that muRdr1A is the active Rdr1 gene, a single-dominant TIR-NBS-LRR (Toll/interleukin-1 receptor-nucleotide binding site-leucine rich repeat) (TNL)-type resistance gene against black spot disease, which acts against a broad range of pathogenic isolates independent of the genetic background of the host genotype. Molecular analyses revealed that, compared with the original donor genotype, the multiple integrations that are found in the primary transgenic clone segregate into different integration patterns in its sexual progeny and do not show any sign of overexpression. Rdr1 provides resistance to 13 different single-spore isolates belonging to six different races and broad field mixtures of conidia; thus far, Rdr1 is only overcome by two races. The expression of muRdr1A, the active Rdr1 gene, leads to interaction patterns that are identical in the transgenic clones and the non-transgenic original donor genotype. This finding indicates that the interacting avirulence (Avr) factor on the pathogen side must be widespread among the pathogen populations and may have a central function in the rose-black spot interaction. Therefore, the Rdr1 gene, pyramided with only a few other R genes by sexual crosses, might be useful for breeding roses that are resistant to black spot because the spread of new pathogenic races of the fungus appears to be slow.

Characterization of Fusarium spp. associated with pineapple fruit rot and leaf spot in Peninsular Malaysia

AbstractPineapple (Ananas comosus) is one the important fruit crops planted in Malaysia, and this study was conducted to determine Fusarium spp. associated with diseases of the fruit crop as Fusarium is prevalent in tropical countries. Our objective was to identify and characterize Fusarium spp. associated with pineapple fruit rot and leaf spot mainly found on the fruits and leaves in Peninsular Malaysia. Fusarium isolates (n = 108) associated with pineapple fruit rot and leaf spot were characterized by morphological, molecular and phylogenetic analyses, a mating study and pathogenicity testing. TEF‐1α sequence analysis identified Fusarium proliferatum, Fusarium verticillioides, Fusarium sacchari and Fusarium sp. Mating was successful only between tester strains of F. proliferatum and F. verticillioides. Sexual crosses with standard tester strains showed that 82 isolates of F. proliferatum produced fertile crosses with mating population D (Gibberella intermedia) and three isolates of F. verticillioides were fertile with the tester strain of mating population A (Gibberella moniliformis). All isolates were pathogenic, causing pineapple fruit rot and leaf spot, thus fulfilling Koch's postulates.

High-density genetic mapping identifies the genetic basis of a natural colony morphology mutant in the root rot pathogen Armillaria ostoyae

Filamentous fungi exhibit a broad spectrum of heritable growth patterns and morphological variations reflecting the adaptation of the different species to distinct ecological niches. But also within species, isolates show considerable variation in growth rates and other morphological characteristics. The genetic basis of this intraspecific variation in mycelial growth and morphology is currently poorly understood. By chance, a growth mutant in the root rot pathogen Armillaria ostoyae was discovered. The mutant phenotype was characterized by extremely compact and slow growth, as well as shorter aerial hyphae and hyphal compartments in comparison to the wildtype phenotype. Genetic analysis revealed that the abnormal phenotype is caused by a recessive mutation, which segregates asa single locus in sexual crosses. In order to identify the genetic basis of the mutant phenotype, we performed a quantitative trait locus (QTL) analysis. A mapping population of 198 haploid progeny was genotyped at 11,700 genome-wide single nucleotide polymorphisms (SNPs) making use of double digest restriction site associated DNA sequencing (ddRADseq). In accordance with the genetic analysis, a single significant QTL was identified for the abnormal growth phenotype. The QTL confidence interval spans a narrow, gene dense region of 87kb in the A. ostoyae genome which contains 37 genes. Overall, our study reports the first high-density genetic map for an Armillaria species and shows its successful application in forward genetics by resolving the genetic basis of a mutant phenotype with a severe defect in hyphal growth.

A Novel Environmental Azole Resistance Mutation in Aspergillus fumigatus and a Possible Role of Sexual Reproduction in Its Emergence

ABSTRACTThis study investigated the dynamics of Aspergillus fumigatus azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR46/Y121F/T289A genotype, 2% (1/45) harbored the TR46/Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR463/Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR463 mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR46 repeats. Importantly, in the laboratory, we recovered the TR463 mutation from a sexual cross between two TR46 isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases.

Development of pollination and in vitro germination techniques to improve the hybridization in Hydrangea spp.

Hydrangea is a genus of ornamental plants which is gaining new markets mainly as a fresh or dried cut flower, but it is also important as a pot plant and for landscaping. To expand its market, new hybrids should be developed. To increase the hybridization efficiency, some techniques were developed and tested: i) evaluation of two pollination systems; ii) comparison among fruit-cut systems before in vitro cultivation to develop embryos and to allow the growth of new genotypes; iii) evaluation of seed disinfection systems for in vitro germination; iv) sowing systems using seeds and fruits from stocks cultivated in two environments. To increase inter- and intra-specific hybridization, pollination by dispersion of previously collected pollen on the top of a corymb by a brush was more effective than pollination using the corymb itself as a brush. A longitudinal cut system can be considered the best treatment to be applied on fruits before in vitro cultivation to allow growth of seedlings. Sterilization of seeds can be done by immersion in a solution of commercial bleach for 5 minutes on MS culture medium with PPM®. When stocks are cultivated in greenhouses, in vitro contamination is lower and seeds have a better rate of germination. The results of these experiments were applied in a breeding program on Hydrangea using sexual crosses.

Diminishing-returns epistasis among random beneficial mutations in a multicellular fungus.

Adaptive evolution ultimately is fuelled by mutations generating novel genetic variation. Non-additivity of fitness effects of mutations (called epistasis) may affect the dynamics and repeatability of adaptation. However, understanding the importance and implications of epistasis is hampered by the observation of substantial variation in patterns of epistasis across empirical studies. Interestingly, some recent studies report increasingly smaller benefits of beneficial mutations once genotypes become better adapted (called diminishing-returns epistasis) in unicellular microbes and single genes. Here, we use Fisher's geometric model (FGM) to generate analytical predictions about the relationship between the effect size of mutations and the extent of epistasis. We then test these predictions using the multicellular fungus Aspergillus nidulans by generating a collection of 108 strains in either a poor or a rich nutrient environment that each carry a beneficial mutation and constructing pairwise combinations using sexual crosses. Our results support the predictions from FGM and indicate negative epistasis among beneficial mutations in both environments, which scale with mutational effect size. Hence, our findings show the importance of diminishing-returns epistasis among beneficial mutations also for a multicellular organism, and suggest that this pattern reflects a generic constraint operating at diverse levels of biological organization.

Effect of linoleic acid on reproduction and yeast–mycelium dimorphism in the Dutch elm disease pathogens

Elm populations from North America and Europe were devastated by Dutch elm disease (DED), which is a vascular disease caused by fungi from the genus Ophiostoma (Ascomycota). These pathogens feature a yeast–mycelium dimorphism that may be related to virulence by facilitating colonization of the host xylem. Cyclooxygenases (COX) have been proposed to modulate yeast–mycelium dimorphism of DED fungi, and homologs of cox genes have been found in the nuclear genome of O. novo-ulmi subsp. novo-ulmi. Linoleic acid, a substrate for COX, was reported to stimulate the formation of asexual and sexual reproduction structures in DED strains grown on complex media. We hypothesized that linoleic acid also induced mycelium production in liquid shake culture conditions. Linoleic acid was found to enhance the production of reproductive structures in sexual crosses conducted on a complex medium (elm sapwood agar), but was not sufficient for these structures to form on a minimal medium. In liquid shake cultures grown in a minimal medium, the addition of linoleic acid stimulated mycelial formation. Our results suggest that linoleic acid plays a role in reproduction and dimorphism in the DED pathogens.

Open and Closed Breeding of Crops

Mutual suiting relationship between breeding way and widening germplasm was first analyzed. Source of utilization germplasm was the more the widener from only using variation in a variety to among family even bigger scope, especially transformation gene breeding could use superfine gene of living things in the world and create germplasm resources that could not obtain in conventional breeding. Secondly contradiction and its movement between ecological and yield ability character, among yield ability characters or ecological characters exist all; this verified that explaining all of phenomenon was “net system of character composition of yield performance”, “theoretical genetics and breeding science of crops” theory system setting up by author. Thirdly new concepts, closed breeding and open breeding were put forward on basis of relationship of unity of opposition among above characters (genes) and supplying way of necessary energy in character express. Theory of closed and open breeding was a generalization of most essential distinction for different breeding way, also theory basis of correct evaluation these. It made people abandon tradition concept that only had variation and also emergence of new variety, and also made people refute mythology on basis of theory that depending on closed breeding could obtain higher yield variety. Openness of sexual cross breeding etc. decided that it will forever be foothold in an invincible position, develop always and any breeding way will not all replace it.

A RALDH-like enzyme involved in Fusarium verticillioides development

Retinaldehyde dehydrogenases (RALDHs) convert retinal to retinoic acid, an important chordate morphogen. Retinal also occurs in some fungi, such as Fusarium and Ustilago spp., evidenced by the presence of rhodopsins and β-carotene cleaving, retinal-forming dioxygenases. Based on the assumption that retinoic acid may also be formed in fungi, we searched the Fusarium protein databases for RALDHs homologs, focusing on Fusarium verticillioides. Using crude lysates of Escherichia coli cells expressing the corresponding cDNAs, we checked the capability of best matches to convert retinal into retinoic acid in vitro. Thereby, we identified an aldehyde dehydrogenase, termed CarY, as a retinoic acid-forming enzyme, an activity that was also exerted by purified CarY. Targeted mutation of the carY gene in F. verticillioides resulted in alterations of mycelia development and conidia morphology in agar cultures, and reduced capacity to produce perithecia as a female in sexual crosses. Complementation of the mutant with a wild-type carY allele demonstrated that these alterations are caused by the lackof CarY. However, retinoic acid could not be detected by LC–MS analysis either in the wild type or the complemented carY strain in vivo, making elusive the connection between CarY enzymatic activity and retinoic acid formation in the fungus.

Fusarium paranaense sp. nov., a member of the Fusarium solani species complex causes root rot on soybean in Brazil

Isolates of Fusarium obtained from soybean plants showing symptoms of root rot collected in subtropical southern and tropical central Brazil were characterized based on phylogenetic analyses, sexual crossing, morphology, and pathogenicity tests. A novel species within the Fusarium solani species complex (FSSC) causing soybean root rot is formally described herein as Fusarium paranaense. This species can be distinguished from the other soybean root rot pathogens in the FSSC, which are commonly associated with soybean sudden death syndrome (SDS) based on analyses of the combined DNA sequences of translation elongation factor 1-α and the second largest subunit of RNA polymerase II and on interspecies mating compatibility. Bayesian and maximum parsimony phylogenetic analyses showed that isolates of F. paranaense formed a distinct group in clade 3 of the FSSC in contrast to the pathogens currently known to cause SDS, which are in clade 2. Female fertile tester strains were developed that can be used for the identification of this new species in the FSSC based on sexual crosses. All isolates were heterothallic and belonged to a distinct mating population. Fusarium tucumaniae, a known SDS pathogen, was found in the subtropical southern region of the country.

Virulence, Host-Selective Toxin Production, and Development of Three Cochliobolus Phytopathogens Lacking the Sfp-Type 4'-Phosphopantetheinyl Transferase Ppt1.

The Sfp-type 4'-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.

Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues.

Production of novel value-added compounds in transgenic crops has become an increasingly viable approach in recent years. However, in many cases, product yield still falls short of the levels necessary for optimal profitability. Determination of the limiting factors is thus of supreme importance for the long-term viability of this approach. A significant challenge to most metabolic engineering projects is the need for strong coordinated co-expression of multiple transgenes. Strong constitutive promoters have been well-characterized during the >30 years since plant transformation techniques were developed. However, organ- or tissue-specific promoters are poorly characterized in many cases. Oilseeds are one such example. Reports spanning at least 20 years have described the use of certain seed-specific promoters to drive expression of individual transgenes. Multi-gene engineering strategies are often hampered by sub-optimal expression levels or improper tissue-specificity of particular promoters, or rely on the use of multiple copies of the same promoter, which can result in DNA instability or transgene silencing. We describe here a flexible system of plasmids that allows for expression of 1-7 genes per binary plasmid, and up to 18 genes altogether after multiple rounds of transformation or sexual crosses. This vector system includes six seed-specific promoters and two constitutive promoters. Effective constitutive and seed-specific RNA interference gene-suppression cloning vectors were also constructed for silencing of endogenous genes. Taken together, this molecular toolkit allows combinatorial cloning for multiple transgene expression in seeds, vegetative organs, or both simultaneously, while also providing the means to coordinately overexpress some genes while silencing others.

Molecular Characterization of Natural Hybrids Formed between Five Related Indigenous Clade 6 Phytophthora Species.

Most Phytophthora hybrids characterized to date have emerged from nurseries and managed landscapes, most likely generated as a consequence of biological invasions associated with the movement of living plants and germplasm for ornamental, horticultural and agricultural purposes. Presented here is evidence for natural hybridization among a group of five closely related indigenous clade 6 Phytophthora species isolated from waterways and riparian ecosystems in Western Australia. Molecular characterization of hybrids consisted of cloning and sequencing two nuclear genes (ITS and ASF), sequencing of two further nuclear loci (BT and HSP) and of two mitochondrial loci (COI and NADH). Additionally, phenotypic traits including morphology of sporangia and optima and maxima temperatures for growth were also determined. In most cases the nuclear genes were biparentally and in all cases the mtDNA were uniparentally inherited, indicating hybrid formation through sexual crosses. Some isolates bear the molecular signature of three parents suggesting additional hybrid events, although it cannot be determined from the data if these were sequential or simultaneous. These species and their hybrids co-exist in riparian ecosystems and waterways where their ability for rapid asexual proliferation would enable them to rapidly colonize green plant litter. The apparent ease of hybridization could eventually lead to the merging of species through introgression. However, at this point in time, species integrity has been maintained and a more likely scenario is that the hybrids are not stable evolutionary lineages, but rather transient hybrid clones.

Rare interspecific breeding in Pseudo-nitzschia (Bacillariophyceae)

The biological species concept defines species based on sexual incompatibility between strains and the F1 sub-viability or inviability. However, to date, there is only a limited number of studies that formally deal with sexual incompatibility in unicellular protists and hence the rigorous application of biological species concept is fuzzy in these organisms. Here, we investigated interbreeding between two species of the planktonic pennate diatom Pseudo-nitzschia, P. arenysensis and P. pseudodelicatissima. We observed hybridization between these two species in controlled laboratory condition. The F1 generation showed: i) low viability; ii) morphological, ultrastructural, and morphometric features that resembled those of one of the parental strains (P. pseudodelicatissima); iii) intermediate maximum cell size to the one observed in intraspecific sexual crosses in the parental species. Our results may suggest that interbreeding between these two species is possible although likely rare. We invite a larger body of experimental evidence in unicellular protists to assess the applicability of the biological species concept to these organisms.

Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains.

The recent discovery and functional characterization of opposite mating-type loci in the industrial penicillin producer Penicillium chrysogenum demonstrated their regulatory role in sexual as well as asexual development. Subsequent experiments further showed that a sexual life cycle can be induced in P. chrysogenum that was for long believed to reproduce exclusively by asexual propagation. Finally, crossing of wild type and production strains resulted in the generation of recombinant ascospore isolates. We predict from these recent findings that recombinant progeny for industrial applications can be obtained by sexual crossings and discuss experimental difficulties that occur when parental strains with karyotype heterogeneity are used for mating.

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus’s Role in Virulence

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Evaluation of spontaneous generation of allelic variation in soybean in response to sexual hybridization and stress

Espinosa, K., Boelter, J., Lolle, S., Hopkins, M., Goggi, S., Palmer, R. G. and Sandhu, D. 2015. Evaluation of spontaneous generation of allelic variation in soybean in response to sexual hybridization and stress. Can. J. Plant Sci. 95: 405–415. Intra-cultivar variation reported in pure lines of soybean has been hypothesized to result from genetic mechanisms contributing to de novo genetic variation. In this study we have detected allele switching by following segregation patterns of Aconitase-4 isozyme in sexual crosses and pure lines. In sexual crosses, one F2plant showed a switch at the Aconitase-4 (Aco4) locus from the expected heterozygous genotype Aco4-ac to Aco4-ab. In the pure lines grown in a honeycomb planting design and treated with an accelerated aging test, multiple cases of allele switching were detected at the Aco4 locus. Both single and double switches were detected that were stable and heritable. These findings indicate that the generation of endogenous variation continues in pure lines as a result of intrinsic genetic mechanisms. With a long-term goal of understanding the genetic nature of the changes, we genetically mapped the Aco4 gene to a 3.3 cM region on Chromosome 11. The corresponding physical region is ∼293 kb with 39 predicted genes. Of these, Glyma.11g080600 is of particular interest, as it shows 93% and 88% identity to Medicago truncatula and Arabidopsis aconitase genes, respectively. Further characterization of the soybean Aco4 gene may shed light on genetic mechanisms responsible for allele switching.

Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco.

The outcome of simultaneously increasing SPS and GS activities in transgenic tobacco, suggests that sucrose is the major determinant of growth and development, and is not affected by changes in N assimilation. Carbon (C) and nitrogen (N) are the major components required for plant growth and the metabolic pathways for C and N assimilation are very closely interlinked. Maintaining an appropriate balance or ratio of sugar to nitrogen metabolites in the cell, is important for the regulation of plant growth and development. To understand how C and N metabolism interact, we manipulated the expression of key genes in C and N metabolism individually and concurrently and checked for the repercussions. Transgenic tobacco plants with a cytosolic soybean glutamine synthetase (GS1) gene and a sucrose phosphate synthase (SPS) gene from maize, both driven by the CaMV 35S promoter were produced. Co-transformants, with both the transgenes were produced by sexual crosses. While GS is the key enzyme in N assimilation, involved in the synthesis of glutamine, SPS plays a key role in C metabolism by catalyzing the synthesis of sucrose. Moreover, to check if nitrate has any role in this interaction, the plants were grown under both low and high nitrogen. The SPS enzyme activity in the SPS and SPS/GS1 co-transformants were the same under both nitrogen regimens. However, the GS activity was lower in the co-transformants compared to the GS1 transformants, specifically under low nitrogen conditions. The GS1/SPS transformants showed a phenotype similar to the SPS transformants, suggesting that sucrose is the major determinant of growth and development in tobacco, and its effect is only marginally affected by increased N assimilation. Sucrose may be functioning in a metabolic capacity or as a signaling molecule.

Bacterial community affects toxin production by Gymnodinium catenatum.

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell−1) was similar to the parent cultures (169–206 fmol STX cell−1), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell−1) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell−1). Specific toxin production rate (fmol STX day−1) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell−1 day−1) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.

Inheritance of mefenoxam resistance in Phytophthora nicotianae populations from a plant nursery

Three sexual crosses involving isolates of P. nicotianae with differing sensitivity to mefenoxam were established to study the inheritance of mefenoxam resistance. Mefenoxam sensitivity was determined by measuring the mycelial growth on both mefenoxam-amended clarified V8 agar and non-amended agar and then calculating the relative growth. When both parents had the same phenotype (both were resistant or both were sensitive), all F 1 progeny had the parental phenotype and no segregation for a major effect gene was observed in sensitivity to mefenoxam. However, variations in the mycelial growth of progeny indicated the segregation of minor-effect genes. When the cross involving the mefenoxam-resistant isolate 3A4 and a sensitive parent, the F 1 progeny segregated for mefenoxam resistance in a ratio of 1:1 (resistant: sensitive), indicating that the mefenoxam resistance is controled by a single dominant gene. Mating type was not linked to the mefenoxam-resistance gene locus. One RAPD marker linked in trans to the MEX locus was obtained by bulked segregant analysis using RAPD markers and was converted to a sequence characterized amplified region marker (SCAR). The SCAR maker identified in this study is a limited but useful tool for differentiating homozygous resistant isolates from sensitive isolates of P. nicotianae.