Construction and Genetic Analysis of Hybrid Strains between Apple and Tomato Pathotypes of Alternaria alternata by Protoplast Fusion

The genetic controls of host-specific toxin (HST) biosynthesis and the pathogenicity of A. alternata pathogens have been limited by the asexual nature of the life cycle of these fungi. We used a protoplast fusion system for A. alternata to analyze the genetics of HST production and its relation to the specific pathogenicity of these pathogens. Drug-resistant transformants were isolated by genetic transformation, using vectors conferring resistance to hygromycin B and geneticin, for the A. alternata apple pathotype (AM-toxin producer) and A. alternata tomato pathotype (AAL-toxin producer), respectively. Protoplasts of the respective transformants were fused by electrofusion. The majority of resultant stable fusants produced both AM- and AAL-toxins and were pathogenic to susceptible cultivars of both apple and tomato. Pulsed-field gel electrophoresis analysis demonstrated that these fusants (or hybrids) carried small 1.7-and 1.1-Mb chromosomes, characteristic of the parental strains of the apple and tomato pathotypes, respectively. Detection of the AMT gene, involved in AM-toxin biosynthesis, by polymerase chain reaction revealed that all fusants pathogenic to apple maintained this gene. Microfluorimetry analysis using propidium iodide staining suggested that the fusants might be diploid. Received 14 November 2000/ Accepted in revised form 11 December 2000     

Mating System and Pollen Contamination in a Pinus brutia Seed Orchard

Mating system parameters and pollen contamination were estimated in an 11-year-old Pinus brutia Ten. clonal seed orchard, located in Antalya, Turkey, with the aid of isozymes. Isozyme analysis was performed on both maternal (megagametophyte) and embryo tissues of seeds collected from the seed orchard and the surrounding natural stand. Fourteen loci encoding nine enzyme systems were assayed. Based on a multilocus estimator, the proportion of viable seeds originating from outcrossing (tˆ_m) in the seed orchard was found to be 0.947; the remainder (0.053) was due to selfing. We estimated that 85.7% of the orchard seed resulted from pollen contamination by trees in surrounding stands. This may be related to the young age and subsequent low pollen production of the orchard. Only 9% [i.e., 1.0−0.857 (contamination)−0.053 (selfing) = 0.090] of the orchard offspring was due to cross-pollination among clones within the orchard. Due to the high level of pollen contamination, it is estimated that genetic gain in this seed crop is less than 57% of that expected if all offspring resulted from random mating among orchard clones (i.e., no contamination).     

Comparison of extractive components in muscle and liver of three Loliginidae squids with those of one Ommastrephidae species

Extractive components were determined for the mantle muscle and liver of three species of Loliginidae squid, Sepioteuthis lessoniana, Loligo bleekeri, and L. edulis, and one species of Ommastrephidae, Todarodes pacificus, as a control. Total free amino acid levels and the major free amino acids in muscle, taurine, proline, glycine, alanine, and arginine, were significantly higher in Loliginidae squids than in T. pacificus. The main nucleotide was adenosine 5′-monophosphate, which did not differ significantly between species. Malate was the organic acid found in muscle in highest concentrations. The muscle of these species contained a large amount of trimethylamine oxide. A large amount of glycine betaine was also detected in the muscle, but showed no large species difference. From these results, the muscle of Loliginidae species is considered to have a much sweeter taste than that of T. pacificus. Compared with muscle, squid liver was characteristic with high contents of taurine, glutamate, bitter amino acids, succinate, propionate, trimethylamine, and glycine betaine, and with low contents of sweet amino acids, arginine, nucleotides, malate, and trimethylamine oxide. These results suggest that squid liver is characterized by a complicated taste containing umami, bitterness, sourness, fishy flavor, and less sweetness.     

Chromosome constitution of hybrid strains constructed by protoplast fusion between the tomato and strawberry pathotypes of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

To analyze the genetics of host-specific toxin production and its relation to the specific pathogenicity of a mitosporic fungus Alternaria alternata, we developed a protoplast fusion system. Protoplasts of drug-resistant transformants of the A. alternata tomato pathotype (AAL-toxin producer) and A. alternata strawberry pathotype (AF-toxin producer) were fused by electrofusion. Of five fusion strains examined, two strains were pathogenic on both tomato and strawberry host plants, whereas the rest of the fusion strains were pathogenic only on tomato. Pulsed-field gel electrophoresis analysis demonstrated that the hybrid strains pathogenic on both tomato and strawberry carry 1.0- and 1.05-Mb conditionally dispensable (CD) chromosomes derived, respectively, from the parental strains of the tomato and strawberry pathotypes. On the other hand, the fusion strains appeared to maintain only a single homologous chromosome derived from one of the parental strain in the case of essential chromosomes (A chromosomes). The results suggest that fusion strains between two different pathotypes of A. alternata might be haploid resulting from the deletion of extra sets of essential chromosomes in the fused nuclei, whereas the CD chromosomes derived from each parental strain could be maintained stably in a new genetic background with an expanded range of pathogenicity. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database under the accession numbers AB469331to AB469354.     

Transmission of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Electrophoretic analysis of the transmission pattern of mitochondrial plasmids in protoplast cell fusion between compatible monokaryons of Lentinula edodes indicates that three of the four plasmids carried in parental monokaryons are effectively transferred and replicated in the protoplast fusants. The two monokaryons, 1158a and 1569a, carried different plasmids that could be distinguished by a single restriction digest. Electrophoresis of intact plasmids and restriction analyses indicate that all but one of the fusants carry three of the four possible plasmids, indicating that transmission of plasmids in protoplast fusions is principally biparental in L. edodes. Thus, heterocytoplasmic cells of L. edodes can be effectively constructed by protoplast cell fusion. In addition, plasmids of the same homology group cannot coexist in the heteroplasmic cells after protoplast cell fusion. Contribution No. 382 of the Tottori Mycological Institute     

Variation of fruiting body productivity in protoplast fusants between compatible monokaryons ofLentinula edodes

Mycelial protoplasts of two compatible monokaryons ofLentinula edodes were prepared, and electro-fusion was undertaken. Sixteen dikaryons isolated after the electrofusion were tested for their fruiting abilities in comparison to the two dikaryons obtained by reciprocal mycelial mating. There were significant differences in fruiting body yield among the fusants, and some of them produced more fruiting bodies than did normally mated dikaryons. Although these dikaryons were divided into five genetically different groups by isozyme analyses and a cluster analysis, we could not find clear differences among the groups in terms of fruiting body yield, mycelial growth rate, or degree of wood decay. It appeared that fruiting body yield correlated positively with the mycelial growth rate but negatively with the degree of wood decay.     

Properties of extracts from defatted rice bran by its subcritical water treatment

Defatted rice bran was extracted with water and subcritical water at 50-250 degrees C for 5 min. The highest extract yield was achieved at 200 degrees C, at which the maximum amounts of protein and carbohydrate were also obtained. The total phenolic and furfural contents, radical scavenging activity, and antioxidative activity for the autoxidation of linoleic acid increased with increasing treatment temperature. The bran extracts exhibited emulsifying activity except for the extract prepared at 250 degrees C, which was concomitant with the disappearance of its high-molecular-mass substances. The extract prepared at 200 degrees C also had the highest emulsion-stabilizing activity.     

Genetic differentiation in <Emphasis Type="Italic">Pinus brutia</Emphasis> Ten. using molecular markers and quantitative traits: the role of altitude

Context   

Turkish red pine (Pinus brutia Ten.) is widespread in the eastern Mediterranean Basin. In the late 1970s, four common gardens were established along steep altitudinal transects extending from the coast to about 1,200 m in the Taurus Mountains (Antalya, Turkey).