<Emphasis Type="Italic">Penicillium viticola</Emphasis>, a new species isolated from a grape in Japan

A new monoverticillate Penicillium species (subgenus Aspergilloides Dierckx), Penicillium viticola, was isolated from a grape cultivated in Yamanashi Prefecture, Japan. Morphologically, P. viticola is characterized by the production of slightly roughened conidia, slightly roughened stipes, and rapid growth on 25% glycerol nitrate agar (G25N). This species is phylogenetically close to Penicillium angulare S.W. Peterson, E.M. Bayer & Wicklow, but differs with respect to colonial characteristics and conidia and penicilli morphology.     

Secondary metabolites in taxonomy of the <Emphasis Type="Italic">Penicillium</Emphasis> fungi

A correlation was established between species specificity and synthesis of specific secondary metabolites by the Penicillium fungi. Strains of the subgenus Aspergilloides usually synthesize metabolites of polyketide nature. Most strains of the subgenus Furcatum produce clavine ergot alkaloids and metabolites of diketopiperazine nature. The only clavine ergot alkaloids and diketopiperazine alkaloids produced by strains of the subgenus Biverticillium are rugulovasines and rugulosuvines, respectively. Species designations of the strains of the subgenus Penicillium isolated from permafrost soil, the Mir orbital complex, and sites undergoing anthropogenic load were refined based on the marker secondary metabolites. Changes in the taxonomic position of some strains in the genus Penicillium are suggested.     

Genetic and phenotypic analyses of Pythium isolates from reed suggest the occurrence of a new species,P. phragmiticola,and its involvement in the generation of a natural hybrid

A set of isolates closely related to the reed pathogen Pythium phragmitis was found among Pythium strains obtained from reed (Phragmites australis) soil and plant samples in Germany and Switzerland. These isolates consistently differed from P. phragmitis at 6, 6 and 3 nucleotide positions in the rDNA internal transcribed spacer region, the β-tubulin, and the cytochrome oxidase II gene, respectively. They are formally described here as Pythium phragmiticola sp. nov. An analysis of phenotypic traits like oospore size, growth and aggressiveness to reed showed that P. phragmiticola is almost indiscernible from P. phragmitis, as most features assessed overlapped considerably between species. Phylogenetic analyses provided evidence that P. phragmiticola is the second parent of a previously described Pythium hybrid involving P. phragmitis. The alloploid status of the hybrid, and the parents' reproductive separation was confirmed by molecular evidence and the high ratios of aborted oospores. In natural environments there obviously are two species closely associated with reed, P. phragmitis and P. phragmiticola which are homothallic, but can outcross, resulting in sterile, vegetatively propagated interspecific hybrids. Driving forces leading to the radiation of these two species are unknown, but might be connected with divergent parasitic adaptation to different host tissues or species.     

Penicillium fusisporum and P. zhuangii,Two New Monoverticillate Species with Apical-Swelling Stipes of Section Aspergilloides Isolated from Plant Leaves in China

Two new Penicillium species isolated from plant leaves are reported here, namely, P. fusisporum (type strain AS3.15338^T = NRRL 62805^T = CBS 137463^T) and P. zhuangii (type strain AS3.15341^T = NRRL 62806^T = CBS 137464^T). P. fusisporum is characterized by fast growth rate, apical-swelling monoverticillate penicilli, verrucose stipes, fusiform to oblong conidia about 3.5–4×2–2.5 µm and cinnamon-colored sclerotia. While P. zhuangii presents a moderate growth rate, it also bears apical-swelling monoverticillate penicilli but its stipes are smooth-walled, and produces ovoid to globose smooth-walled conidia about 3–3.5 µm. Both species belong to section Aspergilloides, and P. fusisporum is related to “P. thomii var. flavescens”, while P. zhuangii is morphologically similar to P. lividum. Phylogenetic analyses of sequences of calmodulin and beta-tubulin genes both show that the two new taxa form distinct monophyletic clades.     

Physiological, Ecological, and Phylogenetic Characterization of Stappia, a Marine CO-Oxidizing Bacterial Genus

Bacteria play a major role in marine CO cycling, yet very little is known about the microbes involved. Thirteen CO-oxidizing Stappia isolates obtained from existing cultures, macroalgae, or surf samples representing geographically and ecologically diverse habitats were characterized using biochemical, physiological, and phylogenetic approaches. All isolates were aerobic chemoorganotrophs that oxidized CO at elevated (1,000 ppm) and ambient-to-subambient concentrations (<0.3 ppm). All contained the form I (OMP) coxL gene for aerobic CO dehydrogenase and also the form II (BMS) putative coxL gene. In addition, some strains possessed cbbL, the large subunit gene for ribulose-1,5-bisphosphate carboxylase/oxygenase, suggesting the possibility of lithotrophic or mixotrophic metabolism. All isolates used a wide range of sugars, organic acids, amino acids, and aromatics for growth and grew at salinities from 5 to 45 ppt. All but one isolate denitrified or respired nitrate. Phylogenetic analyses based on 16S rRNA gene sequences indicated that several isolates could not be distinguished from Stappia aggregata and contributed to a widely distributed species complex. Four isolates (of strains GA15, HI, MIO, and M4) were phylogenetically distinct from validly described Stappia species and closely related genera (e.g., Ahrensia, Pannonibacter, Pseudovibrio, and Roseibium). Substrate utilization profiles, enzymatic activity, and membrane lipid composition further distinguished these isolates and supported their designations as new Stappia species. The observed metabolic versatility of Stappia likely accounts for its cosmopolitan distribution and its ability to contribute to CO cycling as well as other important biogeochemical cycles.     

Diversity and enzyme activity of <Emphasis Type="Italic">Penicillium</Emphasis> species associated with macroalgae in Jeju Island

A total of 28 strains of 19 Penicillium species were isolated in a survey of extracellular enzyme-producing fungi from macroalgae along the coast of Jeju Island of Korea. Penicillium species were identified based on morphological and β-tubulin sequence analyses. In addition, the halo-tolerance and enzyme activity of all strains were evaluated. The diversity of Penicillium strains isolated from brown algae was higher than the diversity of strains isolated from green and red algae. The commonly isolated species were Penicillium antarcticum, P. bialowiezense, P. brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and P. terrigenum. While many strains showed endoglucanase, β-glucosidase, and protease activity, no alginase activity was detected. There was a positive correlation between halo-tolerance and endoglucanase activity within Penicillium species. Among 19 Penicillium species, three species–P. kongii, P. olsonii, and P. viticola–have not been previously recorded in Korea.     

NephrotoxigenicPenicillium species occurring on farm-stored cereal grains in western Canada

The incidence of nephrotoxigenicPenicillium species on farm-stored cereals in western Canada was determined by morphological and metabolite profile examination. Of the 142 isolates examined 102 were toxin producers with 61P. aurantiogriseum and 27P. freii. Other nephrotoxigenic species includedP. tricolor (6 isolates),P. verrucosum Chemotype II (4 isolates) andP. viridicatum Westling (4 isolates). The nephrotoxigenicPenicillium species profile for western Canada appears to differ from that of Denmark whereP. verrucosum,P. cyclopium,P. freii and, to a lesser extent,P. aurantiogriseum,P. polonicum, andP. viridicatum predominate.     

Genomics of Sponge-Associated Streptomyces spp. Closely Related to Streptomyces albus J1074: Insights into Marine Adaptation and Secondary Metabolite Biosynthesis Potential

A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts.     

A New record of four <Emphasis Type="Italic">Penicillium</Emphasis> species isolated from <Emphasis Type="Italic">Agarum clathratum</Emphasis> in Korea

Agarum clathratum, brown algae, play important ecological roles in marine ecosystem, but can cause secondary environment pollution when they pile up on the beach. In order to resolve the environment problem by A. clathratum, we focus to isolate and identify Penicillium because many species are well known to produce extracellular enzymes. A total of 32 Penicillium strains were isolated from A. clathratum samples that collected from 13 sites along the mid-east coast of Korea in summer. They were identified based on morphological characters and phylogenetic analysis using β-tubulin DNA sequences as well as a combined dataset of β-tubulin and calmodulin. A total of 32 strains were isolated and they were identified to 13 Penicillium species. The commonly isolated species were Penicillium citrinum, P. roseomaculatum, and Penicillium sp. Among 13 Penicillium species, four species – P. bilaiae, P. cremeogriseum, P. madriti, and P. roseomaculatum – have not been previously recorded in Korea. For these four new species records to Korea, we provide morphological characteristics of each strain.     

A mycological survey of the South Adriatic Sea

A total of 498 fungal isolates belonging to 33 genera and 90 species were identified in the course of a 2-yr survey of the southern part of the territoreal waters of the Yugoslav Adriatic Sea. The best represented genus was Penicillium (32.12% of the total number of isolates), the P. chrysogenumnotatum series being the most abundant, followed by P. verrucosum var. cyclopium, and P. brevicompactum The genus Aspergillus represented 22.28% of the total. A. versicolor being the most ubiquitous. The Dematiaceae as a group, with 148 isolates included in 14 genera and 26 species, accounted for 29.71% of the total; the dominant genera were Cladosporium, Alternaria, and Ulcladium, with 14 25, 5.42 and 4.21%, respectively of the total number of isolates. Hyphomycetes with hyaloconidia, with 46 isolates included in 9 genera and 20 species, constituted about 8.83%, the best represented genus in this group being Fusarium, with 10 identified species. The Sphaeropsidales accounted for 5.42% of the total number of isolates, the genus Phoma being the most abundant.The number of isolates obtained from surface waters accounted for 85% of the total. The decreasing number of isolates in the water column was particularly evident in the case of the most ubiquitous species, a fact which may be explained by the influence of the contact of water masses with the atmosphere and the lower salinity of the surface waters. The distance from the shore as well as the human factor is reflected in the number of isolates and species. The ecological factors influenced not only the fungal population densities, but also their quality.     

Metallopolia, a new subgenus of Polia (Noctuidae,Noctuinae, Hadenini), with the description of two new species and a new subspecies

A new subgenus of Polia, Metallopolia subgen. n., two new species (Polia (Metallopolia) dysgnorima sp. n. and P. (M.) metagnorima sp. n.) from China (Sichuan, Yunnan, Gansu), and a new subspecies, P. (M.) subviolacea kanchenjunga ssp. n. from Nepal and SE Tibet (Sikkim) are described. Habitus, antennae, male and female genitalia of all Metallopolia species are characterised, depicted and compared with the related species.     

Characterization of the anti-fungal activity of a Bacillus spp. associated with sclerotia from Sclerotinia sclerotiorum

Sclerotinia sclerotiorum fruiting bodies (sclerotia) were found to harbour bacteria that possess anti-fungal activity. Among 1,140 bacterial isolates collected, 32 were found to inhibit the growth of four common fungal pathogens of canola, S. sclerotiorum, Rhizoctonia solani, Alternaria brassicae and Leptosphaeria maculans. One of these broad-spectrum isolates, LEV-006, was found to be closely related to Bacillus subtilis based on 16S rRNA analysis. The anti-fungal activities were purified and found to be associated with a low molecular weight peptide complex consisting mostly of the cyclic lipopeptide fengycin A and B, as revealed by matrix-assisted laser desorption/ionization time-of-flight and post-source decay analysis, as well as two proteins of 20 and 55 kDa. Peptide mass fingerprinting revealed that the 55-kDa protein was similar to vegetative catalase 1; however, when the enzyme was expressed in Escherichia coli, it exhibited catalase but not anti-fungal activity. The sequences of several peptides from the 20-kDa protein were obtained and indicated that it was a unique anti-fungal protein.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

Development and characterization of microsatellite markers for Fusarium virguliforme and their utility within clade 2 of the Fusarium solani species complex

Clade 2 of the Fusarium solani species complex contains plant pathogens including Fusarium virguliforme and closely related species Fusarium brasiliense, Fusarium crassistipitatum, Fusarium tucumaniae, which are the primary causal agents of soybean sudden death syndrome (SDS), a significant threat to soybean production. In this study, we developed microsatellite markers from a F. virguliforme genome sequence and applied them to a F. virguliforme population collection of 38 isolates from Michigan and four reference strains from other locations. Of the 225 detected microsatellite loci, 108 loci were suitable for primer design, and 12 of the microsatellite markers were determined to be highly polymorphic, amplifying on average 5.7 alleles per locus. Using these markers, F. virguliforme isolates were partitioned into three distinct clusters, but isolates were not grouped based on relatedness of sampling sites. In addition, 11 out of 12 markers were demonstrated to be highly transferrable to other closely related species.     

Integrative taxonomy and preliminary assessment of species limits in the Liolaemus walkeri complex (Squamata,Liolaemidae) with descriptions of?three?new species from Peru

Species delimitation studies based on integrative taxonomic approaches have received considerable attention in the last few years, and have provided the strongest hypotheses of species boundaries. We used three lines of evidence (molecular, morphological, and niche envelopes) to test for species boundaries in Peruvian populations of the Liolaemus walkeri complex. Our results show that different lines of evidence and analyses are congruent in different combinations, for unambiguous delimitation of three lineages that were “hidden” within known species, and now deserve species status. Our phylogenetic analysis shows that L. walkeri, L. tacnae and the three new species are strongly separated from other species assigned to the alticolor-bibronii group. Few conventional morphological characters distinguish the new species from closely related taxa and this highlights the need to integrate other sources of data to erect strong hypothesis of species limits. A taxonomic key for known Peruvian species of the subgenus Lioalemus is provided.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

Diversity and biocontrol potential of endophytic fungi in Brassica napus

This study was conducted to isolate endophytic fungi from oilseed rape (Brassica napus), to identify the fungal endophytes based on morphology and ITS (ITS1-5.8S rDNA-ITS2) sequences, and to evaluate their efficacy in suppression of the plant pathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea. Selected endophytic fungal isolates were further tested for promoting growth of oilseed rape in potting experiments. A total of 97 endophytic fungal isolates were obtained from roots (35), stems (49) and leaves (13) of B. napus. Forty fungal species were identified and most species (80%) belong to Ascomycota. The species composition is highly diversified with Simpson’s diversity index reaching 0.959. Alternaria alternata is the dominant species accounting for 12.4% of the isolates. Twenty-four isolates exhibited antifungal activity against S. sclerotiorum in dual cultures on potato dextrose agar forming inhibition zones of 3–17 mm in width. The culture filtrates of Aspergillus flavipes CanS-34A, Chaetomium globosum CanS-73, Clonostachys rosea CanS-43 and Leptosphaeria biglobosa CanS-51 in potato dextrose broth exhibited consistent and effective suppression of oilseed rape leaf blight caused by S. sclerotiorum. Fusarium oxysporum CanR-46 was detected capable of production of volatile organic compounds highly inhibitory to S. sclerotiorum and B. cinerea. Moreover, A. alternata CanL-18, Fusarium tricinctum CanR-70 and CanR-71r, and L. biglobosa CanS-51 exhibited growth-promoting effects on oilseed rape. These results suggest that B. napus harbors diversified endophytic fungi, from which potential biocontrol agents against S. sclerotiorum and B. cinerea, and for promoting growth of B. napus can be screened.     

Differential Response to Root-Knot Nematodes in Prunus Species and Correlative Genetic Implications

Responses of 17 Prunus rootstocks or accessions (11 from the subgenus Amygdalus and 6 from the subgenus Prunophora) were evaluated against 11 isolates of Meloidogyne spp. including one M. arenaria, four M. incognita, four M. javanica, one M. hispanica, and an unclassified population from Florida. Characterization of plant response to root-knot nematodes was based on a gall index rating. Numbers of females and juveniles plus eggs in the roots were determined for 10 of the rootstocks evaluated against one M. arenaria, one M. incognita, one M. javanica, and the Florida isolate. These 10 rootstocks plus Nemaguard and Nemared were retested by growing three different rootstock genotypes together in containers of soil infested individually with each of the above four isolates. Garfi and Garrigues almonds, GF.305 and Rutgers Red Leaf peaches, and the peach-almond GF.677 were susceptible to all isolates. Differences in resistance were detected among the other rootstocks of the subgenus Amygdalus. The peach-almond GF.557 and Summergrand peach were resistant to M. arenaria and M. incognita but susceptible to M. javanica and the Florida isolate. Nemaguard, Nemared, and its two hybrids G x N no. 15 and G x N no. 22 were resistant to all but the Florida isolate. In the subgenus Prunophora, Myrobalan plums P.1079, P.2175, P.2980, and P.2984; Marianna plum 29C; and P. insititia plum AD.101 were resistant to all isolates. Thus, two different genetic systems of RKN resistance were found in the subgenus Amygdalus: one system acting against M. arenaria and M. incognita, and another system also acting against M. javanica. Prunophora rootstocks bear a complete genetic system for resistance also acting against the Florida isolate. The hypotheses on the relationships between these systems and the corresponding putative genes of resistance are presented.     

Three new <Emphasis Type="Italic">Penicillium</Emphasis> species from marine subaqueous soils

During investigation of the mycobiota in the Sea of Okhotsk, three new Penicillium species were isolated from subaqueous soils collected in the Sakhalin Gulf and in the northeastern part of the Sakhalin shelf, near the Piltun Bay. According to Raper and Thom’s classification, the newly described species P. piltunense, P. ochotense, and P. attenuatum belong to subsection Divaricata. Two analyses were performed to reveal the phylogenetic relationships of the putative new species with other Penicillium species. Phylogenetically, the new species are related to the members of the P. canescens group and share some morphological and physiological features with them. However, they differ in having a larger colony diameter that makes them similar to P. atrovenetum, P. coralligerum, and P. antarcticum. A detailed analysis based on ITS, combined β-tubulin and calmodulin datasets, and morphological features revealed that the new species formed a distinct group inside the P. atrovenetum subclade. Moreover, P. attenuatum differs from the other two species macro- and micromorphologically and may represent a distinct phylogenetic lineage.