Cultural Growth Patterns and Incompatibility Reactions in Diaporthe and Phomopsis Populations

Comparative studies were carried out with cultures isolated from Phomopsis condiomata and/or Diaporthe ascomata found on naturally-infected living stems or on overwintered stem fragments of Arctium lappa L., Helianthus annuus L., Glycine max (L.) Merr., and Xanthium italicum Moretti, growing in or nearby the same fields in Vojvodina. Results underscored the heterogeneity of the Phomopsis anamorphs in the D. arctii (Lasch) Trav, complex, Intra-specific vegetative incompatibility groups were distinguished among Phomopsis and/or Diaporthe populations from A. lappa. More than one phytopathogenic Phomopsis and/or Diaporthe species were identified on X. italicum. P. longicolla Hobbs was isolated for the first time in Yugoslavia from soybean stem debris. The holomorph Diaporthe (Phomopsis) helianthi Muntanola- Cvetkovic, Mihaljcevic et Petrov showed distinguishing characteristics with respect to other congeneric fungi.     

A Coding Region in Diaporthe Helianthi Reveals Genetic Variability Among Isolates of Different Geographic Origin

A set of Diaporthe helianthi isolates collected in different geographic areas was studied in order to examine whether different genetic biotypes could be responsible for epidemiological differences shown by sunflower stem canker. D. helianthi causes serious losses in France and in former Yugoslavia, while the pathogen is only sporadically recorded in Italy in spite of conducive pedoclimatic conditions. Variability of a D. helianthi coding genomic region, evaluated by means of polymerase chain reaction (PCR), Southern blot hybridisation and restriction fragments length polymorphism (RFLP), showed a conserved homogeneous pattern shared by French and Yugoslavian isolates compared with the heterogeneous pattern of Italian isolates. These results are consistent with other investigations (IGS and ITS region variability) performed on the same set of isolates, allowing a correlation between D. helianthi biotypes, their geographic origin and sunflower stem canker epidemiology.     

Dermatophytes isolated from domestic animals in Barcelona,Spain

A comparative study was conducted with soybean material presenting symptoms of the Diaporthe/Phomopsis complex that was collected in two distant geographical regions of the world: Beltsville, MA, USA, and Vojvodina, Yugoslavia. Contrasting with earlier findings, great variability in the disease symptoms was observed, and one or more Phomopsis species could be isolated from lesions presenting similar characteristics. Among the thirty-three isolates obtained from the lesions the following species were identified: D. phaseolorum var. caulivora, P. phaseoli (teleomorph D. phaseolorum var. sojae, rare), P. longicolla (found for the first time on the soybean fields of Yugoslavia), Phomopsis sp., and one culture showing intermediate characters of D. phaseolorum var. caulivora and D. phaseolorum var. sojae. Much diversity was also found in the cultural characters of the the isolates from both localities, presumably indicating evolutionary and adaptation processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Novel family of cholesterol esterases produced by actinomycetes bacteria

Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly-Xaa-Ser-Xaa-Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.     

Screening and evaluation of antiparasitic and in vitro anticancer activities of Panamanian endophytic fungi

Many compounds produced by fungi have relevant pharmaceutical applications. The purpose of this study was to collect and isolate endophytic fungi from different regions of Panama and then to test their potential therapeutic activities against Leishmania donovani, Plasmodium falciparum, and Trypanosoma cruzi as well as their anticancer activities in MCF-7 cells. Of the 25 fungal isolates obtained, ten of them had good anti-parasitic potential, showing selective activity against L. donovani; four had significant anti-malarial activity; and three inhibited the growth of T. cruzi. Anticancer activity was demonstrated in four isolates. Of the active isolates, Edenia sp. strain F0755, Xylaria sp. strain F1220, Aspergillus sp. strain F1544, Mycoleptodiscus sp. strain F0194, Phomopsis sp. strain F1566, Pycnoporus sp. strain F0305, and Diaporthe sp. strain F1647 showed the most promise based on their selective bioactivity and lack of toxicity in the assays.     

Intraspecific Diversity within Diaporthe Helianthi: Evidence from Rdna Intergenic Spacer (IGS) Sequence Analysis

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe but recorded sporadically in Italy. The genetic diversity of D. helianthi isolates from different geographic origins (Argentina, France, Italy, Yugoslavia, Romania) was investigated using IGS sequences. A 400 bp fragment of the portion of the IGS region flanking the 5' end of the 18S gene was amplified from each isolate. The aligned nucleotide sequences showed intraspecific sequence homology from 99-100% among French/Yugoslavian isolates to 95-100% among Italian isolates. French/Yugoslavian isolates shared 90-92% sequence homology with Italian isolates. The phylogenetic tree obtained from the aligned data revealed three separate groups. Group 1 included all isolates from France and former Yugoslavia and one isolate from Argentina; Group 2 included all Italian isolates and one isolate from Argentina. The most distantly related isolate was that from Romania (Group 3). The average genetic distances among isolates within Group 1 and within Group 2 were 0.22 and 3.29 respectively. The analysis showed that all isolates originating from countries where severe outbreaks of the disease are reported annually (France and former Yugoslavia) form a well defined taxon characterized by relatively low variability. This group is distinct from the group formed by isolates originating from Italy, whose variability is relatively much higher. Results obtained revealed a marked differentiation among pathogen isolates, and members of Group 1 seem not yet to have spread into Italian sunflower-growing areas.     

Plasmid distribution in European <Emphasis Type="Italic">Diaporthe helianthi</Emphasis> isolates

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe, which has been sporadically recorded in Italy. A collection of 26 Diaporthe helianthi isolates deriving from different geographic origins was analysed in order to determine the presence of extra-chromosomal genetic determinants and their molecular diversity. Extra-chromosomal bands in total genomic DNAs were identified in every French and the Yugoslavian isolate and in only one Italian isolate, while no Romanian and Argentinean isolates resulted to host any plasmids. When tested for their chemicophysical nature, they were recognised as linear plasmids sized about 2.3 Kb. A more detailed analysis was performed on a plasmid purified from a French isolate (plasmid F). Its intracellular localisation resulted as mitochondrial. Plasmid F was also exploited as a probe in Southern hybridisation experiments, in which it recognised only plasmids present in the genomes of French and Yugoslavian isolates (countries were the disease has a heavy incidence) indicating a strong correlation to geographic origin. An RFLP hybridisation analysis performed on genomic DNAs revealed a homogeneous restriction pattern in all French and Yugoslavian isolates, suggesting molecular homology among plasmids present in those isolates.     

Screening of glucose isomerase-producing microorganisms

The ability to convert D-glucose into D-fructose was found in 14 out of 74 species of actinomycetes and bacteria tested. High intracellular glucose isomerase activity was displayed by Arthrobacter sp. and actinomycetes Streptomyces viridobrunneus, Streptomyces sp. 1 and Streptomyces sp. 32. The first showed maximal glucose-converting potential when cultured in both glucose and xylose media, while glucose isomerase activity of Streptomyces species could be found solely in medium supplemented with xylose. The ketose enzymatically formed from D-glucose was identified as D-fructose.     

Phylogenetic diversity of endophytic leaf fungus isolates from the medicinal tree Trichilia elegans (Meliaceae)

Various types of organisms, mainly fungi and bacteria, live within vegetal organs and tissues, without causing damage to the plant. These microorganisms, which are called endophytes, can be useful for biological control and plant growth promotion; bioactive compounds from these organisms may have medical and pharmaceutical applications. Trichilia elegans (Meliaceae) is a native tree that grows abundantly in several regions of Brazil. Preparations using the leaves, seeds, bark, and roots of many species of the Meliaceae family have been widely used in traditional medicine, and some members of the Trichilia genus are used in Brazilian popular medicine. We assessed the diversity of endophytic fungi from two wild specimens of T. elegans, collected from a forest remnant, by sequencing ITS1-5.8S-ITS2 of rDNA of the isolates. The fungi were isolated and purified; 97 endophytic fungi were found; they were separated into 17 morpho-groups. Of the 97 endophytic fungi, four genera (Phomopsis, Diaporthe, Dothideomycete, and Cordyceps) with 11 morpho-groups were identified. Phomopsis was the most frequent genus among the identified endophytes. Phylogenetic analysis showed two major clades: Sordariomycetes, which includes three genera, Phomopsis, Diaporthe, and Cordyceps, and the clade Dothideomycetes, which was represented by the order Pleosporales.     

Screening of glucose isomerase-producing microorganisms

The ability to convert D-glucose into D-fructose was found in 14 out of 74 species of actinomycetes and bacteria tested. High intracellular glucose isomerase activity was displayed by Arthrobacter sp. and actinomycetes Streptomyces viridobrunneus, Streptomyces sp. 1 and Streptomyces sp. 32. The first showed maximal glucose-converting potential when cultured in both glucose and xylose media, while glucose isomerase activity of Streptomyces species could be found solely in medium supplemented with xylose. The ketose enzymatically formed from D-glucose was identified as D-fructose.     

Selection and Characterization of Microorganisms Utilizing Thaxtomin A, a Phytotoxin Produced by Streptomyces scabies

Thaxtomin A is the main phytotoxin produced by Streptomyces scabies, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a Penicillium sp. isolate and a Trichoderma sp. isolate. One bacterial isolate was associated with the species Ralstonia pickettii, and the two other bacterial isolates were identified as Streptomyces sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism S. scabies and other Streptomyces species. The nucleotide sequences of the γ variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of Streptomyces mirabilis ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.     

蓝莓拟茎点枝枯病的病原

近期在山东省蓝莓种植区发现一种枝枯病害。为明确其致病菌,通过单孢分离方法和接种试验获得3个菌株。通过形态学特征观察和rDNA-ITS序列分析,确定该病原菌为乌饭树拟茎点霉Phomopsis vaccinii,即越橘间座壳Diaporthe vaccinii的无性阶段。这是乌饭树拟茎点霉所致蓝莓枝枯病在国内的首次发现。     

Xylanolytic enzyme activities produced by mesophilic and thermophilic actinomycetes grown on graminaceous xylan and lignocellulose

Mesophilic and thermophilic strains of actinomycetes were grown on media containing graminaceous xylan or lignocellulose. Aliquots of the culture fluids were sampled and assayed for enzyme activities involved in the degradation of hemicellulose. Xylanase, acetyl esterase and α-arabinofuranosidase activities could be detected after different times of incubation; their production was also dependent on the growth medium. The highest levels of xylanase activity were found in cultures of strains of Streptomyces, Actinomadura sp. and Saccharomonospora viridis. Streptomyces cyaneus produced the highest amount of arabinofuranosidase whereas acetyl esterase activities were highest in S. cyaneus, S. viridis and Pseudonocardia thermophila .     

海南近海30株抗B16细胞活性放线菌的16S rDNA多样性分析

从海南近海 ,包括文昌红树林、海口红树林以及洋浦港等地采集样品 ,经苯酚、SDS、加热等预处理 ,稀释涂布麦芽汁_酵母膏琼脂 (YE)、淀粉酪素琼脂 (SC)、葡萄糖天冬氨酸琼脂 (GA) ,或者直接将样品稀释涂布加有重铬酸钾的高氏一号琼脂 (Gause)和麦芽汁_酵母膏琼脂等进行平板分离。共获得 35 4株放线菌 ,其中有 76株具有不同程度的抗B16细胞毒活性。比较发现加热预处理法和重铬酸钾选择培养法对于广泛分离筛选抗肿瘤活性放线菌不失为一种快速、简便、行之有效的方法。YE、Gause培养基无论在分离到的放线菌总数 ,还是细胞毒活性菌株的比例上都显示了良好的效果。对 30株具有较强抗B16细胞活性的链霉菌进行了扩增性 16SrDNA限制性酶切片段多样性分析 (16SARDRA) ,表明这 30株链霉菌之间有较大的基因差异性。 0 5 0 6 4 2、0 6 0 386和 0 6 0 5 2 4等 3株菌序列分析进一步证明这 3株菌属于链霉菌属 ,其中菌株 0 5 0 6 4 2与其亲缘关系最近的Streptomycescattleya的相似性仅为 95 % ,因此可能是一个新种。     

Degradation of vanillic acid and production of guaiacol by microorganisms isolated from cork samples

The presence of guaiacol in cork stoppers is responsible for some cases of cork taint causing unpleasant alterations to wine. We have performed a characterization of the cork-associated microbiota by isolating 55 different microorganisms: eight yeast, 14 filamentous fungi or molds, 13 actinomycetes and 20 non-filamentous bacteria. A screening for degradation of vanillic acid and guaiacol production showed that none of the filamentous fungi could achieve any of these processes. By contrast, five of the eight yeast strains isolated were able to degrade vanillic acid, although it was not converted to guaiacol. Guaiacol production was only detected in four bacterial strains: one isolate of Bacillus subtilis and three actinomycetes, Streptomyces sp. A3, Streptomyces sp. A5 and Streptomyces sp. A13, were able to accumulate this compound in both liquid media and cultures over cork. These results suggest that guaiacol-mediated cork taint should be attributed to the degradative action of vanillic acid by bacterial strains growing on cork.     

Comparative genetic analysis of quantitative traits in sunflower ( Helianthus annuus L.) 1. QTL involved in resistance to Sclerotinia sclerotiorum and Diaporthe helianthi

Sclerotinia sclerotiorum and Diaporthe helianthi are important pathogens of sunflower ( Helianthus annuus L.). Two hundred and twenty F2-F3 families were developed from an intraspecific cross between two inbred sunflower lines XRQ and PSC8. Using this segregating population a genetic map of 19 linkage groups with 290 molecular markers covering 2,318 cM was constructed. Disease resistances were measured in field experiments during 3 years (1998, 1999 and 2000) for phomopsis and 2 years for S. sclerotiorum (1997 and 1999). QTL were detected using the interval mapping method at a LOD threshold of 3. A total of 15 QTL for each pathogen resistance were detected across several linkage groups, confirming the polygenic nature of the resistances. These QTL explained from 7 to 41% of the phenotypic variability. The QTL for phomopsis resistance, in the 3 years of tests, mapped in the same region, and this was also true for some forms of S. sclerotiorum resistance in the 2 years of tests. On linkage group 8, QTL affecting resistance to both S. sclerotiorum and D. helianthi mycelium extension on leaves colocalised, suggesting a common component in the mechanism of resistance for these two pathogens. The colocalisation of QTL and breeding for resistance to S. sclerotiorum and to D. helianthi by pyramiding QTL in sunflower are discussed.     

Identification and use of actinomycetes for enhanced nodulation of soybean co-inoculated with Bradyrhizobium japonicum

The utilization of actinomycetes as potential soybean (Glycine max (L.)) co-inoculants was evaluated. Soil samples from Carbondale and Belleville, Ill., were used to inoculate pre-germinated soybean plants to determine antibiotic sensitivity in the native Bradyrhizobium japonicum population. Sensitivity was in the order kanamycin > tetracycline > oxytetracycline > rifampicin > neomycin. Antagonism by five actinomycete cultures toward seven test strains of B. japonicum was also assessed. The ranking average inhibition (across all seven B. japonicum strains) by these actino mycetes was Streptomyces kanamyceticus = Streptomyces coeruleoprunus > Streptomyces rimosus > Streptomyces sp. > Amy colatopsis mediterranei. Ten antibiotic combinations were used to isolate antibiotic-resistant mutants of B. japonicum I-110 and 3I1B-110 via successive cycles of mutation. Eighty-one antibiotic-resistant strains were isolated and tested for symbiotic competency; nine of which were selected for further characterization in a greenhouse pot study. Few differences in nodule number were caused by these treatments. Nodule occupancy varied from 0% to 18.3% when antibiotic-resistant strains of B. japonicum were used as the sole inoculants. However, when three mutant strains of B. japonicum were co-inoculated with S. kanamyceticus, significant increases in nodule occupancy (up to 55%) occurred. Increases in shoot nitrogen composition (27.1%-40.9%) were also caused by co-inoculation with S. kanamyceticus.     

Identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione, a novel intermediate in the biosynthesis of Streptomyces meroterpenoids

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) produced by Streptomyces sp. strain KO-3988. All of the fur genes required for furaquinocin biosynthesis have been cloned, and the heterologous production of furaquinocin has been demonstrated in Streptomyces albus. Here, we report the identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione (8-amino-flaviolin) produced by the S. albus heterologous expression of the three contiguous genes encoding type III polyketide synthase (Fur1), monooxygenase (Fur2), and aminotransferase (Fur3) in the furaquinocin biosynthetic gene cluster. An S. albus transformant (S. albus/pWHM-Fur2_del3) harboring the fur gene cluster and lacking the fur3 gene did not produce furaquinocin, whereas furaquinocin production was restored when 8-amino-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del3. These results demonstrate that Fur3 aminotransferase is essential for furaquinocin biosynthesis and that 8-amino-flaviolin is an early-stage intermediate in furaquinocin biosynthesis. We propose that the biosynthetic pathway generating 8-amino-flaviolin is the common route for the biosynthesis of Streptomyces meroterpenoids.     

连作花生田根际土壤优势微生物的分离和鉴定

【目的】从不同连作年限的花生田根际土壤中分离优势微生物并进行鉴定,为研究花生连作后优势微生物的变化奠定基础。【方法】采用土壤稀释分离法从不同连作年限花生根际土壤中分离优势细菌、真菌和放线菌,结合菌株形态特征、培养性状、生理生化特征及16S rDNA序列分析对细菌、放线菌进行鉴定,通过形态特征、培养特征和分子鉴定方法对优势真菌进行鉴定。【结果】从连作花生田根际土壤中分离鉴定出7种优势细菌、7种优势真菌和7种优势放线菌。7种优势细菌分别为Leifsonia xyli、氯酚节杆菌(Arthrobacterchlorophenolicus)、黄色微杆菌(Microbacterium flavescens)、鞘氨醇单胞菌属(Sphingomonas sp.)、巴斯德菌属(Pasteurella sp.)、简单芽孢杆菌(Bacillus simplex)和巨大芽孢杆菌(Bacillus megaterium)。7种优势真菌分别为枝状枝孢菌(Cladosporium cladosporioides)、产紫青霉(Penicillium purpurogenum)、哈茨木霉有性型(Hypocrea lixii)、Exophiala pisciphila、微紫青霉(Penicillium janthinellum)、曲霉(Aspergillus sp.)和大丽轮枝菌(Verticillium dahliae)。7种优势放线菌分别为紫红链霉菌(Streptomyces violaceoruber)、华丽黄链霉菌(Streptomyces flaveus)、Streptomyces panaciterrae、不产色链霉菌(Streptomyces achromogenes)、假浅灰链霉菌(Streptomyces pseudogriseolus)、纤维素链霉菌(Streptomyces cellulosae)和金色链霉菌(Streptomyces aureus)。【结论】本研究是第一次系统的从连作花生根际土中分离鉴定优势微生物,种植花生后根际土壤中优势微生物的种类发生了明显变化,但变化没有规律。