Identification of deep bark canker agent of walnut and study of its phenotypic,pathogenic, holotypic and genetic diversity in Iran

The deep bark canker of the walnut is a serious threat to walnut plants that had an increasing verge since recent years. This disease is caused by Brenneria rubrifaciens bacteria. Therefore, this research was carried out with an aim to identify the deep bark canker agent. The suspected samples having symptoms were collected from the bark and branches of walnut trees. After purification, the phenotypic and biochemical specification of separations was assessed. Even the genetic diversity of pathogen population was conducted with BOX-polymerase chains reaction (PCR) technique and BOXAIR primer. Based on the phenotypic and biochemical specifications, 30 isolates were identified as B. rubrifaciens. In PCR reaction, the specific amplicons of size 537 and 671?bp in the case study strains were amplified via specific primer pairs. The present study is a prime report on outbreak of this disease in Iran and bacterial pathogenicity evidence of B. rubrifaciens on fruit in the world.     

Use of hen lysozyme for protection against bacterial contamination ofin vitro embryo cultures

Summary Curative treatments with antibiotics and hen egg white lysozyme (HEWL) were used to salvage embryo cultures contaminated withBaccillus subtilis. The use of HEWL gave good control ofBaccillus subtilis, but no control ofErwinia. HEWL was better than antibiotics, being much less phytotoxic. The antibiotics piperacillin, ampicillin and imipenem were also found to be ineffective againstErwinia. HEWL, at a final concentration of 1 mg per mL, was used as a preventive and curative agent for routine use in embryo culture ofTriticum aestivum and other Triticeae, as it cured from 30% to 50% of bacterial contamination problems over a one year period. Standardin vitro culture precautions remained essential, as certain bacteria were not controlled by HEWL.     

A Saccharifying Pectate trans-Eliminase of Erwinia aroideae

A saccharifying pectate trans-eliminase was found in the cells of Erwinia aroideae. This enzyme differs from known pectate trans-eliminase in the next two points. It degrades pectic acid liberating 4,5-unsaturated digalacturonic acid from the chain end of the molecule. It does not require calcium ion.

Some properties of 4,5-unsaturated digalacturonic acid, the main product of the saccharifying pectate trans-elimination, were also described in this paper.     

Reconfirmation of antimicrobial activity in the coelomic fluid of the earthwormEisenia fetida andrei by colorimetric assay

A novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was used in the assessment of antimicrobial activity in earthworm in the presence of phenazine methosulphate (PMS) as an electron coupling reagent. This activity was purified from the coelomic fluid of the earthworm (ECF),Eisenia fetida andrei (Oligochaeta, Lumbricidae, annelids) using a series of column chromatography techniques and was tested against three Gram-negative strains ofEscherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and three Gram-positive strains ofStaphylococcus aureus, Bacillus megaterium, Arthrobacter sp., respectively. Only the pigment-free eluate of coelomic fluid of the earthworm (ECFPE) showed activity againstB. megaterium amongst three isolated active fractions. The anion (DEAE-52) exchange effluent of the ECFPE was reported to have the strongest activity againstP. aeruginosa amongst the three active fractions. The 20% acetonitrile eluate (AE) by Sep-Pak C₁₈ cartridge was also tested and showed fair resistance againstE. coli, P. aeruginosa andArthrobacter sp., respectively.     

Pectolytic Enzymes of Exo-Types

A bacterium identified as Pseudomonas sp. was found to be a better source of oligogalacturonide transeliminase (OGTE) than Erwinia aroideae.

The OGTE of Pseudomonas sp. differed from that of Erwinia aroideae in the following respects: (1) The activity was maximal with tetramer and followed by trimer, dimer and polymers. (2) The OGTE of Pseudomonas sp. degraded the saturated uronides as rapidly as, or a little more rapidly than, the corresponding unsaturated uronides. (3) Calcium ion stimulated considerably the OGTE activity.

Both oxidized and reduced acid-soluble pectic acids were resistant to the action of the OGTE.

With the purified enzyme preparation, 4-deoxy-5-keto-d-glucuronic acid was the end product of the OGTE action on oligo- and polygalacturonides. 4,5-Unsaturated galacturonic acid is probably the intermediate in the formation of 4-deoxy-5-keto-d-glucuronic acid.     

Physical characterization of a glucose-dehydrogenase-bearing plasmid from ketoacid-producingErwinia herbicola

Erwinia herbicola (ATCC 21998), a facultative anaerobe, has two plasmids: pVQ1 and pVQ2. Curing with mitomycin C indicated that pVQ2 was cryptic but pVQ1, a 7.4-kb plasmid, bears a 4.3SacI fragment which strongly hybridized to the C-terminal region of the glucose dehydrogenase gene ofAcinetobacter calcoaceticus. A restriction map of plasmid pVQ1 is presented.The authors are with the Department of Biotechnology, Regional Research Laboratory, Canal Road, Jammu Tawi-180 001, India;     

N2-Fixierung in der phytopathogenen Gattung Erwinia

Von 18 Erwinia herbicola- und 16 Enterobacter agglomerans-Stammen wiesen vier Stämme von Erwinia herbicola und zwei von Enterobacter agglomerans hohe Stickstoff-Fixierungsaktivitäten in Flüssigkeitskultur auf und wuchsen auf N₂ als einziger Stickstoffquelle unter anaeroben Bedingungen. Stickstoff-Fixierungsaktivität in Erwinia herbicola konnte erst nach einigen Tagen der Inkubation unter N₂ nachgewiesen werden. Die für Erwinia herbicola gemessenen spezifischen Nitrogenaseaktivitäten übertreffen in der Höhe die für andere Enterobacteriaceae bisher gefundenen Werte. Die Berechnung des Verhältnisses von Stickstoff-Fixierungsaktivität (C₂H₂-Reduktion) zu effektiv assimiliertem Stickstoff (Proteingehalt) ergab, daß von Erwinia herbicola nur 35 bis 50 % der Nitrogenaseaktivität für das Wachstum genutzt wurden. Die nif+-Enterobacter agglomerans- und Erwinia herbicola-Stämme lassen sich mit den klassischen Diagnoseverfahren kaum gegeneinander abgrenzen. Sie unterscheiden sich jedoch darin, daß die untersuchten Erwinia herbicola-Stämme nur unter streng anaeroben Bedingungen Nitrogenaseaktivität zeigen, während die Enterobacter agglomerans-St'imme auch auf Agaroberflächen unter Luft beträchtliche Stickstoff-Fixierungsaktivität aufweisen. Bisher konnte bei phytophatogenen Erwinia amylovora- und Erwinia carotovora-Stämmen keine Stickstoff-Fixierungsaktivität nachgewiesen werden. Wir danken der Deutschen Forschungsgemeinschaft für die gewährte Unterstützung im Schwerpunktprogramm ?Stoffwechsel der anorganischen Schwefel- und Stickstoff-Verbindungen”.     

Purification and characterization of a lactonase from<Emphasis Type="Italic"> Erwinia cypripedii</Emphasis> 314B that hydrolyzes (<Emphasis Type="Italic">S</Emphasis>)-5-oxo-2-tetrahydrofurancarboxylic acid

A bacterium, strain 314B, able to assimilate (S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii. A lactonase hydrolyzing (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l--hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE. The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively. The maximum activity was observed at pH 6.5–7.5 and 35–45 °C. The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on (R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested.     

8-Aza Analogues of Deaza Purine Nucleosides. Synthesis and Biological Evaluation of 8-Aza-1-deazaadenosine and 2′-Deoxy-8-aza-1-deazaadenosine

Abstract

The syntheses of 7-amino-3-(β-D-ribofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (8-aza-1-deazaadenosine) (2) and 7-amino-3-(2-deoxy-β-D-erythro-pentofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (2′-deoxy-8-aza-1-deazaadenosine) (3) by glycosylation of the anion of 7-chloro-3H-1,2,3-triazolo[4,5-b]pyridine are described. The anomeric configuration as well as the position of glycosylation were determined by ¹H, ¹³ NMR, UV and N.O.E. difference spectroscopy. The cytotoxicity of these nucleosides against several murine and human tumor cell lines is discussed. Compounds 2 and 3 proved to be good inhibitors of adenosine deaminase.     

Characteristics of five bacteriophages of yellow-pigmented enterobacteria

Four bacteriophages (C2, C2F, E3, and E16P) belonging to morphological group C3 and one belonging to morphological group A3 (E16B) were purified by deuterium oxide gradient centrifugation and cesium chloride gradient centrifugation. Morphological group C3 phages had a densityd=1.534–1.541 and group A3 phage (E16B) had a densityd=1.492 in CsCl. Phages of morphological group C3 isolated onEnterobacter sakazakii (C2, C2F) and onErwinia herbicola (E3, E16P) were compared withSalmonella newport phage 7-11 with respect to host-range, genome size, antigenic relatedness, and ultraviolet and heat susceptibility. Phages C2 and C2F could multiply inEnterobacter cloacae, E. sakazakii, Erwinia herbicola, E. rhapontici, andLevinea malonatica; whereas phages E3, E16P, and 7-11 could multiply on these same species and onEscherichia coli and severalSalmonella serotypes. Molecular weights of phage DNAs were determined to be 58×10⁶ (C2), 60×10⁶ (7-11), 67×10⁶ (E3), and 39×10⁶ (E16B).All studied phages of morphological group C3 (includingSalmonella newport phage 7-11) were neutralized by anti-phage C2 serum. Despite differences in neutralization kinetics and in ultraviolet and heat sensitivities, these phages of morphological group C3 constitute one phage species. Phage E16B (morphological group A3) had a host-range limited toEnterobacter cloacae, Erwinia herbicola, andE. rhapontici; it was antigenically unrelated to the preceding phage group C3, and showed ultraviolet and heat susceptibility close to that of coliphage T4.     

Hydroxyl Radical-Mediated Oxidation of Serotonin: Potential Insights into the Neurotoxicity of Methamphetamine

Abstract: When incubated with a hydroxyl radical (HO^?)-generating system (ascorbic acid/Fe²⁺-EDTA/O₂/H₂O₂), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3′-bis(2-aminoethyl)-5-hydroxy-[3,7′-bi-1H-indole]-2,4′,5′-3H-trione (7). The latter slowly cyclizes to 3′-(2-aminoethyl)-1′,6′,7′,8′-tetrahydro-5-hydroxyspiro[3H-indole-3,9′-[9H]pyrrolo[2,3-f]quinoline]-2,4′,5′(1H)- trione (9). A minor amount of T-4,5-D dimerizes to give 7,7′-bi-(5-hydroxytryptamine-4-one) (7,7′-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO^?-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO^? formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO^?-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO^? formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO^? might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.     

X-ray crystal, and molecular, structure of 1,2,3-tri-O-acetyl-4,5-dideoxy-4-C-[(R)-phenylphosphinyl]-α- -lyxofuranose: The first x-ray analysis of a pentofuranose having phosphorus in the hemiacetal ring

X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure of 1,2,3-tri-O-acetyl-4,5-dideoxy-4-C-[(R)-phenylphosphinyl]-α- -lyxofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the E₃ conformation, with a tendency towards the ³T₂ form, the substituents at P-5 and C-5 are linked bisectionally, the acetoxyl group at C-2 and the methyl group at C-4 are linked quasiequatorially, and the acetoxyl group at C-3 is linked axially.     

Temperate Bacteriophage ZF40 of Erwinia carotovora: Phage Particle Structure and DNA Restriction Analysis

Structural organization of the temperate bacteriophage ZF40 of Erwinia carotovora was studied. Phage ZF40 proved to be a typical member of the Myoviridae family (morphotype A1). Phage particles consist of an isometric head 58.3 nm in diameter and a contractile 86.3-nm-long tail with a complex basal plate and short tail fibers (31.5 nm). Phage tail sheath, a truncated cone in shape, is characterized by specific packaging of structural subunits. The ZF40 phage genome is 45.8 kb in size, as determined by restriction analysis, and contains DNA cohesive ends. The ZF40 phage ofErwinia carotovora is assumed to be a new species of bacteriophages specific for enterobacteria.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Stereoselective Synthesis of the Optically Active Samin Type of Lignan from L-Glutamic Acid

The optically active samin type of lignan, (1R,2S,5R, 6S)-6-(2-methoxy-4,5-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octan-2-ol, was stereoselectively synthesized from L-glutamic acid via (2R,3R)-2-[(1S and R)- 1-[(tert-butyldimethylsilyl)oxy]-1-(2-methoxy-4,5-methylenedioxyphenyl)methyl]-3-[(tert-butyldiphenylsilyl)oxy]methyl-1,4-butanediol.     

Effect of Mannich bases on some plant pathogenic fungi

3-(2-Pyridyl)-3-iminoisatin, 1-piperidinomethyl-3-(2-pyridyl)-3-iminoisatin, and 1-acetyl-3-(2-pyridyl)-3-iminoisatin affect spore germination ofAlternaria alternata, A. carthemi, Curvularia lunata, Fusarium oxysporum f.sp.cieri andF. udum and influence the development of powdery mildew (Erysiphe pisi) on pea under glasshouse condition as well as conidial germination ofE. pisi on excised pea leaves. Spore germination was inhibited in the sequence 1-acetyl-3-(2-pyridyl)-3-iminoisatin > 1-piperidinomethyl-3-(2-pyridyl)-3-iminoisatin > 3-(2-pyridyl)-3-iminoisatin followed the order accordingly. The powdery mildew development and conidial germination ofE. pisi 1-piperidinomethyl-3-(2-pyridyl)-3-iminoisatin > 1-acetyl-3-(2-pyridyl)-3-iminoisatin > 3-(2-pyridyl)-3-iminoisatin. The chemicals were compared with commonly used antifungal fungicides.     

Behavior of bacteriophage P1 inErwinia carotovora subsp.carotovora

Bacteriophage P1KMclr100 was tranferred toErwinia carotovora subsp.carotovora. P1 was stably maintained as detected by hybridization and transfer of kanamycin resistance. Lysogens ofE. carotovora failed to produce any viable P1 phage. Although total DNA from P1 lysogens ofE. carotovora hybridized to³²P-labeled P1 probe, we were not able to detect P1 DNA as an extrachromosomal element. Attempts to use bacteriophage P1 as a vector for transposon Tn5 insertion mutagenesis inE. carotovora were not successful. Our results indicate that lytic replication of P1 DNA does not occur in P1 lysogens ofE. carotovora and that P1 DNA is probably integrated into the bacterial chromosome.Journal paper 10085 from the Purdue Agricultural Experiment Research Station.     

STRUCTURE AND BIOLOGICAL ACTIVITY OF A SERIES OF CONFORMATIONALLY RESTRICTED ANALOGUES OF GABA

—Microelectrophoretic methods were used to study the effects on spinal neurones of a series of conformationally restricted analogues of GABA, most of which are structurally related to musci-mol (3-hydroxy-5-aminomethylisoxazole). 3-Hydroxy-5-(l-aminoethyl)isoxazole and 3-hydroxy-5-(2-aminoethyl)isoxazole were GABA-like depressants comparable in effectiveness with GABA. The inhibitors of GABA uptake 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol and nipecotic acid (piperidine-3-carboxylic acid) reversibly enhanced the depressant action of GABA. 3-Hydroxy-5-dimethylaminomethly-isoxazole, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepm-3-ol, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol, and nipecotic acid reversibly antagonized the postsynaptic action of glycine. A structure-activity correlation was made in an indirect attempt to elucidate some comformational requirements for interaction of GABA with its postsynaptic receptor and the binding site of its uptake system. The results seem to indicate that different conformations of GABA are required for these interactions.     

Detection of pectolytic activity andpel homologous sequences inFrankia

Using a cup-plate pectin agar assay, pectolytic activity was detected in nodule filtrates obtained fromAlnus rugosa (DuRoi) Spreng,A. glutinosa (L.) Gaertn andA. crispa (Ait.) Pursh seedlings after infection with twoFrankia strains (ACN1 ^AG , CpI1). Pectolytic activity was also detected in cultures filtrates of the same twoFrankia isolates afterin vitro-cultivation on Qmod pectin liquid medium. When Southern blots of Frankia total DNAs from 3 isolates ofF. alni subsp.Pommerii (ACN1 ^AG , ArI3, and CPX32b) and 3 isolates ofF. elaeagni (EUN1 pec, SCN 10a and TX31e ^HR ) were hybridized withPelBDA probes fromErwinia chrysanthemi, positive signals were found in all 7 Frankiae tested.     

Marine Bacterial Sulfated Fucoglucuronomannan (SFGM) Lyase Digests Brown Algal SFGM into Trisaccharides

Abstract Three kinds of trisaccharides were prepared by digesting fucoidan from the brown alga Kjellmaniella crassifolia, with the extracellular enzymes of the marine bacterium Fucobacter marina. Their structures were determined as Δ^4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp, Δ^4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp(6-O-sulfate), and Δ^4,5GlcpUA1-2(L-Fucp(2,4-O-disulfate)α1-3)D-Manp(6-O-sulfate), which indicated the existence of a novel polysaccharide in the fucoidan and a novel glycosidase in the extracellular enzymes. In order to determine the complete structure of the polysaccharide and the reaction mechanism of the glycosidase, the fucoidan was partially hydrolyzed to obtain glucuronomannan, which is the putative backbone of the polysaccharide, and its sugar sequence was determined as (-4-D-GlcpUAβ1-2D-Manpα1-)_n, which disclosed that the main structure of the polysaccharide is (-4-D-GlcpUAβ1-2(L-Fucp(3-O-sulfate)α1-3)D-Manpα1-)_n. Consequently, the glycosidase was deduced to be an endo-α-D-mannosidase that eliminatively cleaves the α-D-mannosyl linkage between D-Manp and D-GlcpUA residues in the polysaccharide and produces the above trisaccharides. The novel polysaccharide and glycosidase were tentatively named as sulfated fucoglucuronomannan (SFGM) and SFGM lyase, respectively.