First Report of Powdery Mildew of Platanus × acerifolia and P. occidentalis Caused by Erysiphe platani in Greece.

London planetrees (Platanus × acerifolia, syn. P. × hispanica), American sycamores (P. occidentalis), and oriental planes (P. orientalis) are widely planted as urban shade trees throughout Greece and many other countries. In June 2012, typical symptoms of a powdery mildew were detected on all sycamores (10 trees) along a central avenue of Heraklion (Crete, Greece), with the disease affecting approximately 80% of the leaves of all infected trees. In August 2013, similar symptoms were observed on 20% of the leaves of all three London planes in a small grove in the Vrysses area of Lasithi (Crete, Greece). In both cases, the disease was severe, with white superficial colonies developing amphigenously on leaves, twigs, floral peduncles, inflorescences, and fruits. The colonies were initially distinct and circular but gradually enlarged and often coalesced to cover the entire leaf blade. Young leaves appeared curled and chlorotic, occasionally leading to defoliation. For the morphological description of the pathogen, samples from seven infected P. occidentalis and three P. × acerifolia trees were microscopically characterized. In all samples, the pathogen's mycelium was branched, septate, and hyaline, with lobed appressoria; conidiophores were erect, cylindrical, unbranched, and consisted of three to four (to five) cells; and conidia were single or in short chains (two to four), ellipsoid or doliiform, with a truncated base and rounded apex. Their dimensions were 24.3 to 48.6 × 15.8 to 27.9 µm (averaging 39.2 × 21.2 µm; n = 100), and their surfaces appeared reticulate. The teleomorph was never observed. Total fungal DNA was extracted from conidia harvested from affected leaves of one infected plant of each of P. occidentalis and P. × acerifolia planes, and the ITS1-5.8S-ITS2 region was PCR-amplified with universal primers 18S-ITS1 and 28S-ITS2 (2) and sequenced (GenBank Accession Nos. KM068123 and KM068124, respectively). A BLASTn search of GenBank revealed 100% identity of both samples to Erysiphe platani strains described on P. orientalis in Greece (JQ365943) and P. occidentalis in Brazil (KF499270). Based on the morphological and molecular analyses, the pathogen was identified as E. platani (Howe) U. Braun & S. Takam. (formerly known as Microsphaera platani Howe) (1). To prove pathogenicity and fulfill Koch's postulates, 10 1-year-old seedlings of each of P. occidentalis and P. × acerifolia hosts were artificially inoculated with conidia obtained from naturally infected plants of the corresponding species, with two methods: (i) five plants of each host were dusted with conidia from diseased leaves, and (ii) the remaining five seedlings of each plane were sprayed with a conidial suspension of the fungus (107 conidia ml-1), while five additional control plants of each species were treated only with sterile distilled water. All plants were maintained in the greenhouse at 25 ± 3°C, with 90% humidity. Powdery mildew symptoms, which appeared 9 and 15 days after inoculation on all dusted and sprayed plants, respectively, were similar to those observed on naturally infected trees, whereas no symptoms were observed on control plants. Although E. platani is known to infect plane species in several parts of the world (1), including oriental planes (P. orientalis and P. orientalis var. cretica) in Greece (3), this is the first report of E. platani causing disease of P. occidentalis and P. × acerifolia in Greece, underlining the need for appropriate control measures to prevent significant losses to the local ornamental industry. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, 2012. (2) I. A. Papaioannou et al. Eur. J. Plant Pathol. 136:577, 2013. (3) D. J. Vakalounakis and E. Klironomou. EPPO Bull. 25:463, 1995.

First Report of Pink Rot of Phoenix and Washingtonia Species Caused by Nalanthamala vermoesenii in Greece.

A disease resembling pink rot was first observed on Phoenix dactylifera in Heraklion (Crete, Greece) in the summer of 2007, and was later found to be relatively common in the same district on additional species (P. canariensis, P. theophrasti, Washingtonia filifera, W. robusta). Symptoms included chlorotic and necrotic leaves (dead tips), light-brown spots (1 to 2 mm in diameter) on the leaves and rachis, rot of the rachis, sheath, and trunk, and eventual death of infected plants. A pinkish-orange layer formed both on the surface and within the infected tissues. A hyphomycete was isolated from symptomatic petioles and the pinkish-orange layer of the sheath. Sixteen isolates were examined on potato dextrose agar (PDA). All formed salmon to grayish-red colonies with sparse aerial mycelium, hyaline conidiophores with penicillate branches and terminal phialides, and ovoid, single-celled conidia in long chains. Mean conidial dimensions were 3.5 (± 0.1) × 5.5 (± 0.1) µm (n = 60 each), for 1-week-old cultures of two single-spore isolates recovered from W. filifera. A BLASTn search of GenBank with sequences of rDNA ITS (JX456472 to JX456474) revealed 100% identity of three isolates to that of Nalanthamala vermoesenii (Biourge) Schroers, comb. nov. [syn. Penicillium vermoesenii Biourge; Gliocladium vermoesenii (Biourge) Thom] originating from several palm species in Spain, the Czech Republic, Australia, and the United States (GenBank AY554212 to AY554217). Therefore, our examination of morphological and molecular characteristics suggested that the fungus recovered from symptomatic trees was N. vermoesenii (3,4). Pathogenicity tests were performed on wounds (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface with a sterile scalpel) of petioles of mature leaves of eight 2-year-old seedlings each of P. canariensis, P. theophrasti, and W. filifera. A 6-mm agar plug from a 1-week-old PDA culture was placed on the artificial wound of each inoculated plant. For non-inoculated controls, sterile PDA plugs were placed on the artificial wounds of four seedlings per host. All plants were maintained in the greenhouse at 16 ± 5°C, with 95% humidity and a 12-h photoperiod. Petiole and stem rot, leaf necrosis, and production of pinkish-orange spore masses appeared at 5 weeks post-inoculation. Average lesion length was 2.75 ± 0.15, 3.28 ± 0.21, and 6.14 ± 0.53 cm for P. canariensis, P. theophrasti, and W. filifera, respectively, suggesting that the latter is more susceptible. The fungus was consistently reisolated from all three inoculated palm species, whereas no symptoms appeared on control plants. To our knowledge, this is the first report of N. vermoesenii infecting palms in Greece. The invasion of the plants by the fungus is probably favored by wounds, such as those caused by pruning or by feeding of the red palm wheevil Rhynchophorus ferrugineus Olivier, which is widespread in Greece (1). References: (1) D. C. Kontodimas et al. Entomol. Hellenica 16:11, 2006. (2) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (3) H.-J. Schroers et al. Mycologia 97:375, 2005. (4) J. Y. Uchida. Page 25 in: Compendium of Ornamental Palm Diseases and Disorders, APS Press, St. Paul, MN, USA, 2004.

First Report of Leaf Spot of Phoenix theophrasti Caused by Paraconiothyrium variabile in Greece.

In the spring of 2011, a severe leaf spot disease of Phoenix theophrasti was observed in the vicinity of Heraklion (Crete), Greece. Initial symptoms were small, round-ovoid spots of varying shades of brown on the leaves, later being transformed into oblong streaks (average dimensions 7.3 ± 1.0 × 3.3 ± 0.5 mm), surrounded by dark brown rings. As the disease progressed, the expanding streaks often coalesced to form enlarged necrotic lesions. Similar symptoms were also detected on petioles and leaf bases. Extended spotting and blighting occasionally resulted in leaf death. A filamentous fungus was consistently isolated onto potato dextrose agar plates from the periphery of the characteristic lesions, with cultures invariably producing brick to cinnamon colonies with sparse aerial mycelium, subglobose and dark brown superficial pycnidial conidiomata on pine needles, 1- to 3-celled hyaline conidiophores, and hyaline, subcylindrical to ellipsoidal, 1-celled, smooth- and thin-walled conidia, with average dimensions of 3.5 ± 0.6 × 1.7 ± 0.4 µm (n = 100). Total DNA of two isolates was extracted and used for PCR amplification and sequencing of the ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (4). Both sequences (GenBank Accession Nos. JX456476 and JX456477) were 100% identical to deposited Paraconiothyrium variabile ITS sequences (EU295640 to 48, JN983440 and 41, and JF934920), and were clustered together as a single group with these sequences with good support by phylogenetic analysis that included representatives of the relative P. brasiliense and P. africanum species. Based on the morphological, molecular, and phylogenetic analyses, the pathogen was identified as P. variabile Riccioni, Damm, Verkley & Crous (2). To prove pathogenicity, 10 P. theophrasti 2-year-old seedlings were sprayed with a conidial suspension of the fungus (107 conidia ml-1, 10 ml per plant), while five additional control plants were treated with sterile distilled water. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Characteristic leaf spots were evident 4 weeks post inoculation on the older leaves, and P. variabile was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. Paraconiothyrium variabile has been isolated from various woody host plants such as Prunus persica, P. salicina, and Malus sp. in South Africa (1,2), Actinidia chinensis and A. deliciosa in Italy (2), Laurus nobilis in Turkey (2), and Salix matsudana in China (3). To our knowledge, this is the first report of P. variabile naturally infecting and causing a leaf spot disease on a palm species. Palms are extensively used as ornamentals throughout Greece and the occurrence of P. variabile can potentially result in economic loss to the local ornamental industry. References: (1) M. Cloete et al. Phytopathol. Mediterr. 50:S176, 2011. (2) U. Damm et al. Persoonia 20:9, 2008. (3) H. Gao et al. Afr. J. Biotechnol. 10:4166, 2011. (4) M. P. Pantou et al. Mycol. Res. 109:889, 2005.

First Report of Petiole (Rachis) Blight of Washingtonia filifera Caused by Phoma glomerata in Greece.

In July 2007, a severe petiole (rachis) blight disease was observed on several California fan palms (Washingtonia filifera) in the vicinity of Heraklion (Crete), Greece. Typical symptoms included discolored (brown to reddish-brown), reversed V-shaped lesions on the petiole bases of the oldest (lowest) leaves, and elongated yellow to dark-brown stripes along the petiole. The lesions progressively expanded and penetrated the petioles, resulting in gradual discoloration (from tan to brown-black) of the internal petiole tissues, including the vascular tissue. The bases of infected petioles occasionally became fragile and burst open, while the corresponding leaf blades were characterized initially by yellowing and one-sided or uneven wilt and, later, desiccation and death with the entire leaves curving downwards. The disease gradually moved upward to younger leaves, severely debilitating but rarely killing the infected trees. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) plates from sections of diseased petioles, forming dense, dark green colonies with abundant light to dark brown, subglobose pycnidia (diameter ranging between 36.4 to 177.4 µm, and averaging 99.4 µm, n = 50) on the agar surface or immersed in the medium. Chlamydospores and numerous dictyochlamydospores were also observed, with the latter being initially light to dark brown and later becoming black. The numerous conidia were hyaline, ovoid to ellipsoid, and single-celled. Their dimensions were 5.3 to 7.3 × 2.4 to 4.9 µm, averaging 6.5 × 3.2 µm (n = 100). The ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (3), were amplified with PCR from total DNA extracted from two representative isolates, and sequenced (GenBank Accession Nos. KC802086 to KC802087). Using BLASTn, both sequences were 100% identical to Phoma glomerata ITS sequences (FJ427018, FJ427011, AF126816). Based on morphological and molecular analyses, the pathogen was identified as Phoma glomerata (Corda) Wollenw. & Hochapfel, also known as Peyronellaea glomerata (Corda) Goid. ex Togliani or Coniothyrium glomeratum Corda (1,2). To prove pathogenicity and fulfill Koch's postulates, petioles of the older leaves of eight W. filifera 2-year-old seedlings were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface), inoculated with agar discs from a 2-week-old PDA culture of the fungus, and sealed with Parafilm. For controls, sterile PDA plugs were placed on the artificial wounds of five more seedlings. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Petiole blight and leaf necrosis symptoms-identical to those observed in the infected plants-were evident 5 weeks post-inoculation, and P. glomerata was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. This is the first report of petiole blight of a palm species caused by P. glomerata in Greece. Due to the extensive use of palms as ornamentals in Greece, the occurrence of P. glomerata can potentially cause economic loss to the local ornamental industry. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) R. M. Hosford, Jr. Phytopathology 65:1236, 1975. (3) M. P. Pantou et al. Mycol. Res. 109:889, 2005.

First Report of Palm Rot of Phoenix spp. Caused by Neodeightonia phoenicum in Greece.

In July 2007, a severe rot was observed on Phoenix dactylifera and P. canariensis palms in the vicinity of Heraklion (Crete), Greece. Initial symptoms were pale, elongated spots that gradually turned to dark brown streaks extending along the leaf base and rachis. In early stages, the upper parts of the leaves usually remained unaffected. Eventually decay and premature death of leaves occurred, followed by terminal bud necrosis. Shoot blights and stalk rots were also observed. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) from leaf base necrotic lesions. Immersed pycnidial conidiomata on pine needles in culture were multiloculate and dark brown to black. Pycnidial paraphyses were absent. Conidiogenous cells were hyaline, cylindrical, and swollen at base. Conidia were thick-walled, ovoid to ellipsoid, with rounded apex and base; initially hyaline and aseptate, 15.2 ± 0.4 × 11.7 ± 0.3 µm, later becoming dark brown and 1-septate, 21.3 ± 0.4 × 11.8 ± 0.3 µm, with a striate appearance. Total DNA was extracted and used for PCR amplification and sequencing of the ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (1). The sequence (GenBank Accession No. JX456475) was found 99% identical to Neodeightonia phoenicum ITS sequences (GenBank Accession Nos. EU673338 to EU673340), and was clustered together as a single group with the above sequences with good support by phylogenetic analysis that included representatives of other Neodeightonia species and several other Botryosphaeriaceae members. Based on the morphological, molecular, and phylogenetic analyses, the pathogen was identified as N. phoenicum A. J. L. Phillips & Crous (2) (syn. Diplodia phoenicum (Saccardo) H. S. Fawcett & Klotz), formerly also known as Macrophoma phoenicum Saccardo and Strionemadiplodia phoenicum (Saccardo) Zambettakis. To prove pathogenicity, the petioles of the older leaves of seven 2-year-old seedlings of each of three palms, P. canariensis, P. theophrasti, and Washingtonia filifera were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface) and inoculated with agar discs from a 1-week-old PDA culture of the fungus. For controls, PDA discs without fungal mycelium were placed on the wounds of four seedlings of each host. Petiole rot, blight, and leaf necrosis were evident on all inoculated plants 6 weeks post inoculation and the pathogen was consistently reisolated from all three inoculated palm species, whereas no symptoms were observed on control plants. N. phoenicum has repeatedly and globally been reported on P. dactylifera (3). To the best of our knowledge, this is the first report of the occurrence of N. phoenicum infecting Phoenix species in Greece. Palms are extensively used as ornamental trees throughout Greece. A potential spread of palm rot caused by N. phoenicum might have a substantial economic impact and should be urgently addressed through appropriate disease management programs. References: (1) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (2) A. J. L. Phillips et al. Persoonia 21:29, 2008. (3) A. Zaid et al. Chapter XII in: Date palm cultivation, FAO Plant Production and Protection Paper 156 Rev. 1, 2002.

Molecular characterization of isolates of Beauveria bassiana obtained from overwintering and summer populations of Sunn Pest (Eurygaster integriceps).

AIM: To examine whether isolates of the entomopathogenic fungus Beauveria bassiana are more closely associated to their summer hosts compared with overwintering hosts, with recently developed molecular tools based on mitochondrial regions. METHODS AND RESULTS: Primers for the traditional ITS1-5.8S-ITS2 region and two mitochondrial intergenic regions, namely, nad3-atp9 and atp6-rns, were used. All amplified products were sequenced, aligned and Neighbour-Joining (NJ), parsimony and Bayesian phylogenetic inference analyses were performed. The isolates examined were grouped with very good support into three distinct groups, two of them showed geographical correlation, but no clear association to their host. CONCLUSIONS: The mitochondrial intergenic regions used were more informative than the nuclear ITS1-5.8S-ITS2 sequences. The sequence variability observed, that allowed the phylogenetic placement of the isolates into distinct groups, depended on the geographical origin of the isolates and can be exploited for designing group-specific and isolate-specific primers for their genetic fingerprinting. No clear associations with summer Sunn Pest populations were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on the genetic variability of biocontrol agents like B. bassiana are indispensable for the development of molecular tools for their future monitoring.

ISZm1068: an IS5-like insertion element from Zymomonas mobilis.

A new insertion sequence, designated ISZm1068, was isolated from Zymomonas mobilis strain CP4. This element consists of 1,068 bp and contains one major ORF which shows similarities both at the nucleotide and at the amino acid sequence level with the corresponding ORFs encoding the transposases of many IS5 family elements, in particular the IS1031 group. Moreover, the Z. mobilis ORF shares the conserved N2, N3 and C1 signature motifs of the IS4 and IS5 families. Six out of seven Z. mobilis wild-type strains were shown by hybridisation to contain a single copy of the ISZm1068 element. Nucleotide sequences of the insertion elements from these strains exhibited extremely high levels of identity, varying from 94.25 to 99.25%. ISZm1068 was shown to be active in Escherichia coli cells and led to plasmid replicon fusions within the host cell. Sequence analysis of rare cointegration and resolution derivatives suggests that ISZm1068 has putative imperfect inverted repeats at its extremities of 18 bp (IR-right) and 14 bp (IR-left), and that a 3-bp (5'-TCA-3') target sequence is duplicated upon insertion.

Molecular characterization of the host-adapted pathogen Verticillium longisporum on the basis of a group-I intron found in the nuclear SSU-rRNA gene.

Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb. nov. With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca. 2.5 kb was generated from all isolates of V. longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates. On the contrary, all the other phytopathogenic and nonphytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V. lecanii and two of Cordyceps sp., generated a product of ca. 1.65 kb. Sequence analysis of the SSU-rRNA gene of two typical isolates of V. longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae). The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements. Primers designed from the 839-bp intron sequence amplified only the V. longisporum. Phylogenetic analysis based on SSU-rDNA sequences showed that V. longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies.

Enzymes of the Entner-Doudoroff and pyruvate decarboxylation pathways in Zymomonas mobilis wild-type CP4 and mutant strains grown in continuous culture.

The osmotolerant Zymomonas mobilis strain suc40, (containing plasmid pDS3154-inaZ), which is capable of producing simultaneously ethanol and ice nuclei protein, was cultivated in a chemically defined complete sucrose medium, as well as in a sugar beet molasses medium in continuous culture. The strain exhibited the normal Monod's relationship between biomass and dilution rate, and between growth substrate concentration and dilution rate. Specific activities of a number of enzymes that appear to control important steps in the metabolic flux of the Entner-Doudoroff and pyruvate decarboxylation pathways were investigated over a range of growth rates in steady state cultures. With the exception of glucose-6-phosphate dehydrogenase and gluconate kinase, all of the enzymes exhibited a very similar pattern for the wild type Z. mobilis CP4 and for the osmotolerant mutants, independent of the media used; the enzyme patterns remained relatively constant over the studied growth range. The specific activity of glucose-6-phosphate dehydrogenase was increased 2-fold by decreasing dilution rate in sugar beet molasses. The specific activity of gluconate kinase was 2-fold lower at medium growth rates compared with that at either low or high growth rates. Pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase activities were significantly higher compared with those of the enzymes governing the early steps of the Entner-Doudoroff pathway. The present study, which was designed to determine the behaviour of important enzymes in sucrose metabolism of Z. mobilis suc40/pDS3154-inaZ grown in continuous culture showed that the microorganism required regulation of specific enzyme activities at the transcriptional level when sugar beet molasses were used as the growth medium.

Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture.

AIM: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. METHODS AND RESULTS: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. CONCLUSIONS: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.

Molecular identification of wine yeasts at species or strain level: a case study with strains from two vine-growing areas of Greece.

The composition of wine yeast populations, present during spontaneous fermentation of musts from two wine-producing areas of Greece (Amyndeon and Santorini) and followed for two consecutive years, were studied using a range of molecular techniques. Internal Transcribed Spacer (ITS) ribotyping was convincingly applied for yeast species identification, proving its usefulness as a reliable tool for the rapid characterization of species composition in yeast population studies. Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) was shown to be a convenient criterion for the detection of intraspecies genetic diversity of both Saccharomyces and non-Saccharomyces isolate populations. Similarly, polymorphism of amplified delta interspersed element sequences provided an additional criterion for S. cerevisiae strain differentiation. Comparative analysis of S. cerevisiae genetic diversity, using mtDNA restriction patterns and delta-amplification profiles, showed a similar discriminative power of the two techniques. However, by combining these approaches it was possible to distinguish/characterize strains of the same species and draw useful conclusions about yeast diversity during alcoholic fermentation. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were (i) almost complete absence of non-S.cerevisiae species from fermentations of must originating from the island Santorini, (ii) a well recorded strain polymorphism in populations of non-Saccharomyces species originating from Amyndeon and (iii) an unexpected polymorphism concerning S. cerevisiae populations, much greater than ever reported before in similar studies with wine yeasts of other geographical regions.

Characterization and replication properties of the Zymomonas mobilis ATCC 10988 plasmids pZMO1 and pZMO2.

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2.

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region.

The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region.

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: Intraspecific heterogeneity within the intergenic spacer region. Fungal Genetics and Biology 29, 19-27. The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.

Identification of group-I introns at three different positions within the 28S rDNA gene of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae.

Using a set of heterologous primers designed from the 3'-end of the 28S rRNA gene of Verticillium dahliae the corresponding gene region of 30 isolates of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae was amplified. The polymerase chain reaction products obtained could be classified into four groups varying in size from 1.0 to 2.2 kb. Sequence analyses of representative PCR products revealed the presence of five distinct introns, positioned in three different insertion sites. Fungal isolates 316 and 11 both harbored one intron each (374 and 337 bp in size, respectively), whereas isolate 33 harbored three introns (436, 334, and 412 bp) within the relevant 28S rRNA region. All five introns shared the conserved P, Q, R, S elements and all the other characteristic features of group-I introns in their deduced secondary structure; three (316-int, 33-int1, and 33-int3) belong to subgroup IC1 and two (33-int2 and 11-int) belong to subgroup IE. Further, reverse transcription polymerase chain reactions indicated that all these introns were absent from the mature RNA molecules. The appearance of the five introns at identical positions with those from other organisms belonging to various phyla is discussed.

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region.

The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.

Presence of a 20 bp insertion/deletion in the ITS1 region of Verticillium lecanii.

Polymerase chain reaction (PCR) amplification of the complete ribosomal RNA internally transcribed spacer (ITS) region of 36 isolates of Verticillium lecanii and related species gave a single 620 bp product in 31 isolates. Five isolates received as V. lecanii, however, gave a single product of 600 bp. Restriction fragment analysis of the PCR products from all isolates gave consistent patterns for the 31 isolates with a 620 bp product. The five isolates with the 600 bp product showed only minor discrepancies to these, generally related to the size of only one restriction fragment. The total ITS region was sequenced from 10 typical 620 bp isolates and one 600 bp isolate. Sequence variation between the isolates varied from 0 to 14.5%, and the 20 bp size discrepancy was found to relate to an insertion or deletion in the centre of the ITS1 region.

Biotransformation of the flavonoid tiliroside to 7-methylether tiliroside: bioactivity of this metabolite and of its acetylated derivative.

Incubation of kaempferol-3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) with Aspergillus nidulans gives the 7-methyl ether of tiliroside (2) which is a new compound. Its structure is determined by spectroscopic methods. Cytotoxic studies of 2 and of its acetylated derivative 2a were carried out in vitro against fourteen human leukemic cell lines. Results clearly show that compound 2 is ineffective against all leukemic cell lines tested. On the contrary, compound 2a exhibited cytotoxic activity against four of the cell lines (HL60, DAUDI, HUT78 and MOLT3) and additionally, a dose- and time-dependent effect on DNA synthesis.

Improvement of lysine production by analog-sensitive and auxotroph mutants of the acetylene-utilizing bacterium Gordona bronchialis (Rhodococcus bronchialis).

An acetylene utilizing Gordona (Rhodococcus) bronchialis strain, screened for the production of fine chemicals, was found to be capable of producing small amounts of lysine. Attempts to produce amino-acid analog-resistant and/or sensitive mutants and auxotrophs of this strain with increased lysine production were made following UV-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The bacterium exhibited surprisingly high resistance levels to the aforementioned mutagens which is attributed to highly effective inborn-repair systems. Natural resistance to high levels of S-(2-aminoethyl)-L-cysteine (AEC) (2%) was observed, in contrast with D, L-aspartic acid hydroxamate (AAH), L-lysine hydroxamate (LHX) and beta-fluoropyruvate (FP). A variety of amino-acid analog-resistant (AAHr, LHXr) or analog-sensitive (FPs) mutants were produced following UV-irradiation or MNNG treatment. Similarly, a large number of auxotrophs (68) of different types were also obtained. From these, one FPs mono-auxotroph and two poly-auxotrophs (with at least one requirement for the aspartic acid family) showed an increased lysine production (approximately 1.8 g/L) comparable (4 g/L) to that found in other bacteria capable of utilizing long-chain hydrocarbons (1).

Transposon mutagenesis and strain construction in Zymomonas mobilis.

Conjugative or mobilizable plasmids carrying the transposable elements Tn5, Tn501 or mini Mu were readily transferred from Escherichia coli donors into Zymomonas mobilis recipients with frequencies depending both on donor and recipient strain used. With the exception of pULB113 (RP4::mini Mu), all foreign plasmids exhibited high instability in Z. mobilis transconjugants under both selective and non-selective conditions. Transposition events and consequent mutagenesis occurred readily in Z. mobilis transconjugant strains, with Tn5 and Tn501 being far less successful than mini Mu. Transposon mutagenesis with the help of mini Mu resulted in the isolation of a large number of independent auxotrophs with polyauxotrophs, cysteine, methionine and isoleucine requiring-isolates being the most frequent. When chromosomal DNA from all these mutants was digested with various restriction enzymes and the resulting restriction patterns were hybridized with a mini Mu probe, the majority of these mutants appeared to have insertions at different sites of the chromosome. Thus, transposon mutagenesis by mini Mu is proven to be a simple and efficient tool for mutant production and the genetic analysis of Z. mobilis.