Biochemical basis of radiation-sensitivity in mutants of Neurospora crassa

The available UV-sensitive mutants of Neurospora crassa were examined for their ability to excise and photoreactive cytosine-containing dimers invivo. All strains exhibited in vivo photoreactivation, including upr-1, which was originally thought to be deficient in photoreactivation. Two strains, uvs-2 and upr-1 were shown to be deficient in excision repair; uvs-3 was shown to contain a residual amount of excision capabilit. The remaining strains, uvs-1, uvs-5, and uvs-6, were normal in their ability to excise dimers. Based on these results, tentative analogies were drawn between the Neurospora mutants and the known classes of UV-sensitive mutants in E. coli. Accordingly, the N. crassa mutants were classified as uvs-1, -lon; uvs-2, -uvr; uvs-3, -uvr (rec?); uvs-5, -lon; uvs-6, -rec; and upr-1, -uvr. A comparison was made between the biochemical responses and the available published data on mutation induction in the Neurospora mutants. Althoughsome relationships were seen between repair defects and mutation induction, too little data were available for any definitive conclusions.     

Root Exudates of Various Host Plants of Rhizobium leguminosarum Contain Different Sets of Inducers of Rhizobium Nodulation Genes

Rhizobium promoters involved in the formation of root nodules on leguminous plants are activated by flavonoids in plant root exudate. A series of Rhizobium strains which all contain the inducible Rhizobium leguminosarum nodA promoter fused to the Escherichia coli lacZ gene, and which differ only in the source of the regulatory nodD gene, were recently used to show that the regulatory nodD gene determines which flavonoids are able to activate the nodA promoter (HP Spaink, CA Wijffelman, E Pees, RJH Okker, BJJ Lugtenberg 1987 Nature 328: 337-340). Since these strains therefore are able to discriminate between various flavonoids, they were used to determine whether or not plants that are nodulated by R. leguminosarum produce different inducers. After chromatographic separation of root exudate constituents from Vicia sativa L. subsp. nigra (L.), V. hirsuta (L.) S.F. Gray, Pisum sativum L. cv Rondo, and Trifolium subterraneum L., the fractions were tested with a set of strains containing a nodD gene of R. leguminosarum, R. trifolii, or Rhizobium meliloti, respectively. It appeared that the source of nodD determined whether, and to what extent, the R. leguminosarum nodA promoter was induced. Lack of induction could not be attributed to the presence of inhibitors. Most of the inducers were able to activate the nodA promoter in the presence of one particular nodD gene only. The inducers that were active in the presence of the R. leguminosarum nodD gene were different in each root exudate.     

Combination Patterns of Major R Genes Determine the Level of Resistance to the M. oryzae in Rice (Oryza sativa L.)

Rice blast caused by Magnaporthe oryzae is the most devastating disease of rice and poses a serious threat to world food security. In this study, the distribution and effectiveness of 18 R genes in 277 accessions were investigated based on pathogenicity assays and molecular markers. The results showed that most of the accessions exhibited some degree of resistance (resistance frequency, RF >50%). Accordingly, most of the accessions were observed to harbor two or more R genes, and the number of R genes harbored in accessions was significantly positively correlated with RF. Some R genes were demonstrated to be specifically distributed in the genomes of rice sub-species, such as Pigm, Pi9, Pi5 and Pi1, which were only detected in indica-type accessions, and Pik and Piz, which were just harbored in japonica-type accessions. By analyzing the relationship between R genes and RF using a multiple stepwise regression model, the R genes Pid3, Pi5, Pi9, Pi54, Pigm and Pit were found to show the main effects against M. oryzae in indica-type accessions, while Pita, Pb1, Pik, Pizt and Pia were indicated to exhibit the main effects against M. oryzae in japonica-type accessions. Principal component analysis (PCA) and cluster analysis revealed that combination patterns of major R genes were the main factors determining the resistance of rice varieties to M. oryzae, such as ‘Pi9+Pi54’, ‘Pid3+Pigm’, ‘Pi5+Pid3+Pigm’, ‘Pi5+Pi54+Pid3+Pigm’, ‘Pi5+Pid3’ and ‘Pi5+Pit+Pid3’ in indica-type accessions and ‘Pik+Pib’, ‘Pik+Pita’, ‘Pik+Pb1’, ‘Pizt+Pia’ and ‘Pizt+Pita’ in japonica-type accessions, which were able to confer effective resistance against M. oryzae. The above results provide good theoretical support for the rational utilization of combinations of major R genes in developing rice cultivars with broad-spectrum resistance.     

Genetic analyses of reddish-brown polyoxin-resistant mutants of Bipolaris maydis

In the southern corn blight fungus, Bipolaris maydis, five polyoxin-resistance genes (Pol1 to Pol5) have been reported. Pol2 and Pol5 are pleiotropic for not only polyoxin resistance but also reddish brown colonies. Here, we used a comparative genomics approach to identify Pol2 and Pol5 at a molecular genetics level. Our analysis revealed that nucleotide sequence variations in the genes for hydroxymethylbilane synthase (HMBS) and ferrochelatase (FECH) were linked to the phenotypes of Pol2 and Pol5, respectively. Further variations in the nucleotide sequences of these genes were also found in other strains of Pol2 and Pol5. Complementation tests with the wild type genes confirmed that these mutations at Hmbs and Fech were responsible for the polyoxin resistance in the Pol2 and Pol5 mutants. The deletion mutants of these genes (ΔHMBS and ΔFECH) were conditionally lethal without exogenous heme. The heme contents of Pol2 and Pol5 mutants were lower than that in the wild type, suggesting that the mutations in hmbs and fech reduced the functions of HMBS and FECH, although neither was completely inactivated. These results suggested Pol2 and Pol5 encode HMBS and FECH, members of enzymes in the heme-biosynthetic pathway of this fungus.     

Role of the exoprotease InA in the pathogenicity of Bacillus thuringiensis in pupae of Hyalophora cecropia

Two geographical biotypes of Nomuraea rileyi (from Ecuador and the United States) were topically bioassayed against seven lepidopteran species, i.e., Anticarsia gemmatalis, Heliothis zea, Heliothis virescens, Heliothis subflexa, Pseudoplusia includens, Spodoptera exigua, and Trichoplusia ni. There was an average difference of 1.7-fold in mortality in how cultures of the same insect species from different sources responded to topical applications of either biotype of N. rileyi. Regression equations and LC₅₀ values were obtained for each insect species and fungal biotype combination. Larvae of S. exigua were equally susceptible to both biotypes of N. rileyi. Although larvae of A. gemmatalis were moderately susceptible to the Ecuadoran biotype, they were relatively nonsusceptible to the Mississippian biotype. Species of Heliothis (H. zea, H. virescens, and H. subflexa) were about equally susceptible to the Mississippian biotype. Larvae of H. subflexa and H. virescens, however, were significantly less susceptible than H. zea to the Ecuadoran biotype. When the integumental barrier was breached via intrahemocoelic injections, larvae of H. virescens were as susceptible as H. zea larvae to blastospores of either biotype of N. rileyi.     

Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily

To infer the phylogenetic relationships of Xylaria species associated with termite nests within the genus Xylaria and among genera of the subfamily Xylarioideae, β-tubulin, RPB2, and α-actin sequences of 131 cultures of 114 species from Xylaria and 11 other genera of the subfamily were analyzed. These 11 genera included Astrocystis, Amphirosellinia, Discoxylaria, Entoleuca, Euepixylon, Kretzschmaria, Nemania, Podosordaria, Poronia, Rosellinia, and Stilbohypoxylon. We showed that Xylaria species were distributed among three major clades, TE, HY, and PO, with clade TE—an equivalent of the subgenus Pseudoxylaria—encompassing exclusively those species associated with termite nests and the other two clades containing those associated with substrates other than termite nests. Xylaria appears to be a paraphyletic genus, with most of the 11 genera submerged within it. Podosordaria and Poronia, which formed a distinct clade, apparently diverged from Xylaria and the other genera early. Species of Entoleuca, Euepixylon, Nemania, and Rosellinia constituted clade NR, a major clade sister to clade PO, while those of Kretzschmaria were inserted within clade HY and those of Astrocystis, Amphirosellinia, Discoxylaria, and Stilbohypoxylon were within clade PO.     

Identification and origin of host-associated volatiles attractive to Prorops nasuta, a parasitoid of the coffee berry borer

Previous studies have shown that prepupae/pupae and dust/frass from Hypothenemus hampei (Ferrari) reared on coffee berries were attractive to its parasitoid Prorops nasuta Waterston, while the same biological materials obtained from an artificial diet were not. In this study, we identified the volatiles from prepupae/pupae and dust/frass from H. hampei reared on Coffea canephora Pierre ex Froehner berries and artificial diet by gas chromatography coupled with mass spectrometry (GC?CMS), evaluated their behavioural activity, and investigated the possible origin of the kairomones in the H. hampei dust/frass attractive to P. nasuta. The GC?CMS analysis indicated that 24 peaks were consistently present in the headspace volatiles of dust/frass from H. hampei reared on coffee berries, 18 of these peaks were identified. Nineteen compounds were found in the dust/frass from H. hampei reared on an artificial diet. The compounds 1-octen-3-ol, limonene, n-tridecane, n-tetradecane, longifolene, n-pentadecane and n-heptadecane were common in dust/frass from H. hampei reared on coffee berries and artificial diet. Parasitoids were more attracted to 3-octanone, limonene, longifolene and n-dodecane compared to clean air. In contrast, P. nasuta preferred clean air to n-tetradecane. The rest of the compounds did not influence the behaviour of parasitoids. Two compounds were released by prepupae/pupae from H. hampei obtained from coffee berries, whereas six compounds were emitted by prepupae/pupae from the artificial diet. The compounds n-hexadecane and n-heptadecane were found in both types of prepupae/pupae. Parasitoids were more attracted to n-hexadecane than to clean air. In contrast, females did not show any preference for n-heptadecane or clean air. Among the compounds identified from dust/frass from H. hampei attractive to P. nasuta, only longifolene was found in the healthy C. canephora berry volatiles. Four species of fungi were isolated from the dust/frass of H. hampei, including Fusarium solani (Mart.) Sacc., Penicillium crustosum Thom, Aspergillus aculeatus lizuka and Mucor sp. Among the compounds identified in the dust/frass from H. hampei attractive to P. nasuta females, only 3-octanone was detected in the volatiles from F. solani and P. crustosum.     

Mitochondrial Four-Point Crosses in ASPERGILLUS NIDULANS : Mapping of a Suppressor of a Mitochondrially Inherited Cold-Sensitive Mutation

Four-point mitochondrial crosses were conducted in heterokaryons of Aspergillus nidulans. The mutations used were (oliA1), conferring resistance to oligomycin, (camA112), conferring resistance to chloramphenicol; (cs-67), conferring cold-sensitivity, and ( sumD16), a suppressor of (cs-67). Initially, the crosses were conducted by observing the segregation of extranuclear markers in heterokaryotic sectors emerging from the original point of heterokaryosis. This showed that (camA112), (cs-67) and (sumD16) were linked but were probably all unlinked to (oliA1). Second, four-point crosses were conducted using a double marker selection technique, in which (camA112 ) and (oliA1) were always set in repulsion and the frequency of the phenotypes produced by the segregation of the mutant and wild-type alleles of (cs-67) and (sumD) were observed in (camA112 oliA1) recombinants. From these results we concluded that (camA112 ), (cs-67) and (sumD16) were linked and probably mapped in the order given. It was observed that the two nuclear types of conidia from a heterokaryon often had a dissimilar frequency distribution of the segregants of a mitochondrial cross.     

Variation in cuticular lipid profiles of black butterflies of the genus Papilio in Japan

Insect (epi)cuticular lipids characterize sex and species and often play an important role in mating behavior. We previously revealed that two black-colored swallowtail butterflies, Papilio polytes and Papilio protenor, show sexual dimorphism and species specificity in cuticular lipid composition and that P. polytes males use specific monoene components for mate discrimination. These findings suggest that their closely related species may have different profiles of cuticular lipids. We examined the cuticular lipid compositions of five Papilio species (P. bianor, P. maackii, P. helenus, P. macilentus, and P. memnon), closely related and sympatric to P. polytes and P. protenor, and discussed whether it is possible to discriminate between sexes, and between species based on their chemical profiles. The cuticular lipids consist mainly of C23–C31 aliphatic hydrocarbons, in which n-tricosane, n-heptacosane, and n-nonacosane are predominant. Several aliphatic ketones, aliphatic acids, and oxygenated terpenoids were also identified as major components shared by several species. There were no components exclusive to a particular species. Conspecific males and females shared most of the components but were mostly distinguishable based on their composition. Moreover, P. helenus males, P. polytes females, and P. protenor females had two different phenotypes of lipid composition. Although unrelated to the genetic lineage, the seven species were classified into four clusters based on their lipid profiles. The first cluster was composed of only P. memnon. The other six species were broadly classified into three clusters consisting of subclusters for each species: 1) P. polytes, P. helenus, P. macilentus, and several P. protenor females; 2) P. bianor and P. protenor; and 3) P. maackii and several P. helenus males. These results indicate that cuticular lipid profiles characterize the species and sex of the Papilio species and may be responsible for mate discrimination among them.     

Evaluation of Asteraceae Plants for Control of Meloidogyne incognita

Of the 56 species and 43 genera of Asteraceae tested, 9 were highly resistant or immune to Meloidogyne incognita and did not form root galls. Twenty-six species and six cultivars had 25% or fewer roots galled and were considered moderately resistant to M. incognita. Pre-planting Cosmos bipinnatus (F190), Gaillardia pulchella, Tagetes erecta, Tithonia diversifolia, or Zinnia elegans (F645) reduced root galling and M. incognita J2 in and around Ipomoea reptans. Amendment of soils with roots, stems, or leaves of G. pulchella was effective in controlling M. incognita on I. reptans. Tissue extracts of G. pulchella were lethal to various plant-parasitic nematodes but were innocuous to free-living nematodes. Root exudates of G. pulchella were lethal to J2 of M. incognita and were inhibitory to the hatch of eggs at the concentration of 250 ppm or higher. Gaillardia pulchella could be used to manage M. incognita as a rotation crop, a co-planted crop, or a soil amendment for control of root-knot nematode.     

Detection of resistance genes and susceptibility patterns in Bacteroides and Parabacteroides strains

Susceptibility to five antimicrobials was determined for Bacteroides spp. (n = 52) and Parabacteroides distasonis (n = 8). All isolates were susceptible to metronidazole. The resistance rates to ampicillin, cefoxitin, tetracycline and clindamycin were 98%, 9.6%, 65.3% and 19.2% of the Bacteroides strains, respectively. The genes cepA, cfiA, cfxA, tetQ, ermF and nim were found in 69.2%, 17.3% 9.6%, 50%, 7.7% and 3.8% for these strains respectively. All P. distasonis strains were resistant to ampicilin. Cefoxitin, tetracycline and clindamycin resistance rates were 75%, 87.5% and 50%, respectively. The ermF and nim genes were absent and 37.5%, 12.5%, 12.5% and 87.5% of this strains possessed cepA, cfiA, cfxA and tetQ genes, respectively. Ten cfiA gene positive strains of Bacteroides and Parabacteroides were submitted to E-test with imipenem and amoxicillin–clavulanate. The resistance rate to imipenem was 4.1% and 8.3% to amoxicillin–clavulanate. This feature is for the first time described in Brazil.     

RAPD and FAME analyses of Astragalus species growing in eastern Anatolia region of Turkey

Fatty acid (FAs) and RAPD profiles were used to examine phenotypic and genetic relationships between eight Astragalus species including Astragalus maximus Willd. var. maximus, Astragalus coadunatus Hub. Mor. & Chamb., Astragalus kurdicus Boiss. var. kurdicus, Astragalus lagurus Willd, Astragalus christianus L., Astragalus cicer L., Astragalus atrocarpus Champ & Matthews and Astragalus onobrychioides Bieb., which were wildly growing in eastern Anatolia region of Turkey. All of the eight Astragalus species tested in this study were separated based on the presence and composition of 45 different FAs. Four of the Astragalus species including A. coadunatus, A. lagurus, A. christianus, and A. atrocarpus were rich in terms of FA contents containing at least 22–31 different FAs. The relative proportions of two fatty acids, 16:0, and 18:1:ω8c were higher in these four Astragalus species. The remaining species have limited number of FAs with unique FAMEs profiles. Six of the 10 decamer primers examined were selected to find out genetic polymorphism in Astragalus species. A total of 98 polymorphic bands were observed, ranging in size from 250 bp to 3000 bp. The RAPD results suggested that A. atrocarpus, A. onobrychioides and A. kurdicus are closely related and completely different from the other species. Six genetically distinct groups were found among the species of Astragalus. High genetic variations among Astragalus species growing wildly in eastern Anatolia region of Turkey may imply the differences in their origins. The results in the present study suggested that both RAPD and FA analyses are useful for differentiation of Astragalus species.     

Significance of Four Methionine Sulfoxide Reductases in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen and emergence of antibiotic resistance in clinical staphylococcal isolates raises concerns about our ability to control these infections. Cell wall-active antibiotics cause elevated synthesis of methionine sulfoxide reductases (Msrs: MsrA1 and MsrB) in S. aureus. MsrA and MsrB enzymes reduce S-epimers and R-epimers of methionine sulfoxide, respectively, that are generated under oxidative stress. In the S. aureus chromosome, there are three msrA genes (msrA1, msrA2 and msrA3) and one msrB gene. To understand the precise physiological roles of Msr proteins in S. aureus, mutations in msrA1, msrA2 and msrA3 and msrB genes were created by site-directed mutagenesis. These mutants were combined to create a triple msrA (msrA1, msrA2 and msrA3) and a quadruple msrAB (msrA1, msrA2, msrA3, msrB) mutant. These mutants were used to determine the roles of Msr proteins in staphylococcal growth, antibiotic resistance, adherence to human lung epithelial cells, pigment production, and survival in mice relative to the wild-type strains. MsrA1-deficient strains were sensitive to oxidative stress conditions, less pigmented and less adherent to human lung epithelial cells, and showed reduced survival in mouse tissues. In contrast, MsrB-deficient strains were resistant to oxidants and were highly pigmented. Lack of MsrA2 and MsrA3 caused no apparent growth defect in S. aureus. In complementation experiments with the triple and quadruple mutants, it was MsrA1 and not MsrB that was determined to be critical for adherence and phagocytic resistance of S. aureus. Overall, the data suggests that MsrA1 may be an important virulence factor and MsrB probably plays a balancing act to counter the effect of MsrA1 in S. aureus.     

Experimental investigations on the host range of Sarcocystis podarcicolubris

Lizards experimentally infected with S. podarcicolubris sarcocysts were fed to different species of the family Colubridae. Only species of the genus Coluber proved to be suitable definitive hosts and passed sporocysts in their feces. No sporo- cysts were shed by the species of the genus Elaphe, Coronella and Malpolon that were tested. Fecal sporocysts from different species of the genus Coluber were orally administered to numerous lizards of the genera Lacerta, Podarcis and Algyroides. In all experimental lizards, except the controls, sarcocysts of S. podarcicolubris developed.     

Comparative Analysis of FLC Homologues in Brassicaceae Provides Insight into Their Role in the Evolution of Oilseed Rape

We identified nine FLOWERING LOCUS C homologues (BnFLC) in Brassica napus and found that the coding sequences of all BnFLCs were relatively conserved but the intronic and promoter regions were more divergent. The BnFLC homologues were mapped to six of 19 chromosomes. All of the BnFLC homologues were located in the collinear region of FLC in the Arabidopsis genome except BnFLC.A3b and BnFLC.C3b, which were mapped to noncollinear regions of chromosome A3 and C3, respectively. Four of the homologues were associated significantly with quantitative trait loci for flowering time in two mapping populations. The BnFLC homologues showed distinct expression patterns in vegetative and reproductive organs, and at different developmental stages. BnFLC.A3b was differentially expressed between the winter-type and semi-winter-type cultivars. Microsynteny analysis indicated that BnFLC.A3b might have been translocated to the present segment in a cluster with other flowering-time regulators, such as a homologue of FRIGIDA in Arabidopsis. This cluster of flowering-time genes might have conferred a selective advantage to Brassica species in terms of increased adaptability to diverse environments during their evolution and domestication process.     

Genetics of Vegetative Incompatibility in Cryphonectria parasitica

Vegetative incompatibility in the chestnut blight fungus, Cryphonectria parasitica, in Europe is controlled by six unlinked vic loci, each with two alleles. Four previously identified vic loci (vic1, vic2, vic3, and vic4) were polymorphic in European vegetative compatibility (vc) types. Two new loci, vic6 and vic7, also were identified among European vc types. In one cross, vic genes segregated independently at five loci, and 194 progeny were assigned to 32 vc types; none of these loci were linked. A total of 64 vc types were identified from all crosses. All 64 genotypes possible from six vic loci, each with two alleles (2⁶ = 64), were identified and assigned to vc types. Based on our model, vc types v-c 5 and v-c 10, which had been used in previous genetic studies, differ by only five vic genes. Future studies of vc types in C. parasitica can use knowledge of vic genotypes for analysis of population genetic structure based on vic allele frequencies and to determine the effect of each vic gene on virus transmission between vc types.     

Identification and characterization of four new tra cistrons on the E. coli K12 sex factor F.

Thirty-eight amber-suppressible transfer-deficient mutants of the Flac element F13-1 have been analyzed in conjugational complementation tests. Many of the mutations were in tra genes that have been identified previously (including traI), but several were in four new genes, traN, traU, traV, and traW. Results for representative mutations in the new genes were confirmed by complementation tests using λtra transducing phages and these also allowed the genes to be mapped quickly and simply. Approximate physical locations were found by complementation with a series of chimeric plasmids carrying various EcoRI fragments of the F transfer region. All four genes are located in the transfer operon, the order being traA … traBtraVtraWtraCtraUtraNtraF … traD. While traU, traV, and traW mutants were resistant to male-specific phages, traN mutants were sensitive.     

Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18

A method of transductional complementation was developed in Pseudomonas aeruginosa to identify the cistrons involved in the conjugal transfer of the wide host range R plasmid R18. This used the P. aeruginosa bacteriophage E79tv-2 and has led to the identification of eight tra cistrons encoded by this plasmid. Plasmids mutant in six cistrons, traA, traB, traC, traD, traE, and traG were resistant to donor-specific phage (Dps^?) while traF and traH mutant plasmids retained phage sensitivity. Some traB mutants were unable to inhibit the replication of phage G101 (Phi(G101)^?) while some were also deficient in entry exclusion (Eex^?). Two traB mutants which were also Eex^? were suppressible by an amber suppressor. Three tra mutants selected directly as being Phi(G101)^? were found to be also Dps^?Eex^? and mutant in traB. These data suggest a relationship between traB, Eex, and Phi(G101). In order to facilitate future genetic comparison of the tra genes of R18 and other wide host range plasmids and the role of the host in conjugation, R18 DNA was compared with that of RP4, by restriction enzyme fragment patterns and found to be identical.     

Genetic complementation of rhizobial nod mutants with Frankia DNA: artifact or reality?

Two divergent reports have been published on the genetic complementation of rhizobial nod mutants using Frankia DNA. In 1991 putative Frankia cosmid library clones were reported to restore normal nodulation properties to Rhizobium leguminosarum biovar viciaenodD::Tn5, but no supporting sequence data were published. In 1992 a second group reported a failure to find any evidence of functional complementation of various rhizobial nod mutants by Frankia DNA (nodA, nodB and nodC). Complementation tests of nine Nod^? R. leguminosarum bv. viciae or Sinorhizobium meliloti Tn5 mutants (nodA ^? , nodB ^? , nodC ^? , nodD ^? , nodF? ^? , nodL ^? , nodH ^? ) were thus performed using a Frankia gene library in pLAFR3 to clarify this situation. Rhizobial transconjugants obtained by tri-parental matings were screened for restoration of the nodulation phenotype on their host plants, Vicia sativa subsp. nigra or Medicago sativa. Nodulation was observed on plants inoculated with transconjugants of the R. leguminosarum bv. viciaenodC::Tn5 mutant. The Nod⁺ rhizobial transconjugants were isolated and analysed. The Nod⁺ phenotype of these transconjugants was found to be due to Tn5 excision/transposition. No functional complementation was found with any of the mutants used, suggesting that rhizobial complementation of nod mutants with Frankia DNA is unlikely to occur.