Dehalogenation of 4-chlorobenzoate

Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg²⁺ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 °C. The apparent K _m value for 4-chlorobenzoyl-coenzyme A was 2.4–2.7 µM. V _max was 1.1 × 10^–7 M sec^–1 (2.2 µmol min^–1 mg^–1 of protein). The NH₂-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.Abbreviations CBA chlorobenzoate - CoA coenzyme A - HBA hydroxybenzoate - DTT dithiothreitol - HPLC high performance liquid chromatography - PAGE polyacrylamide gel electrophoresis     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Entomopathogenic fungi as a potential control agent against the lesser mealworm, <Emphasis Type="Italic">Alphitobius diaperinus</Emphasis> in broiler houses

We tested the pathogenicity of 18 Metarhizium anisopliae (Metschn.) Sorokin isolates and 22 Beauveria bassiana (Balsamo) Vuillemin isolates against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae and adults. The efficacy of the most virulent isolate—M. anisopliae K—was evaluated in containers with a concrete bottom covered with wood shavings, under simulated poultry house conditions. Application of conidia of this isolate to the shavings or directly to the concrete bottom reduced the yield of larvae in 8–15 time compared with the control. In another test, the mortality of mature larvae placed on previously inoculated shavings or bottom reached 80–90% within 14 days, compared with 14% in the control. The residual activity of conidia kept at 28°C retained its initial level during 14 days post-inoculation, but declined after three weeks. Based on our data M. anisopliae has considerable potential for the control of A. diaperinus.

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.     

Genetic variability in relocated Père David’s deer (<Emphasis Type="Italic">Elaphurus davidianus</Emphasis>) populations—Implications to reintroduction program

Since 1985, China has established three breeding herds of Père David’s deer: the Beijing Père David’s Deer Park (39°07′N, 116°03′E), the Dafeng Père David’s Deer Nature Reserve (33°05′N, 120°49′E) and Shishou (Tianezhou) Père David’s Deer Nature Reserve (29°49′N, 112°33′E), through reintroductions of about 30–40 founders. Since establishment, all three populations have grown steadily. However, genetic backgrounds in those populations are still unknown. We studied the genetic diversity in Père David’s deer and genetic consequences of population relocations in China. We revealed that genetic diversity was extremely low in Père David’s deer populations in China. Only a single mtDNA D-loop haplotype was found in the deer, furthermore, only five polymorphic microsatellite loci were screened out from 84 pairs of species-transferred primers. Genetic makeup in the three Père David’s deer populations were significantly different (P < 0.01). H _E and allelic richness in the Tianezhou population were the highest (0.54, 2.60, n = 31), Beijing population (0.52, 2.4, n = 125) showed the second highest measures, while the Dafeng population (0.46, 2.39, n = 39) measured lowest. Our results suggest that effective management of a species of low genetic diversity like the Père David’s deer should consider the genetic background of each founder to make sure genetic variations are preserved in both source population and relocated population. Now, the Tianezhou population is the most appropriate source population in China when establishing new Père David deer populations in the wild.

Effects of temperature on the immature development and emergence of five species of <Emphasis Type="Italic">Trichogramma</Emphasis>

Five constant temperatures between 14 and 30°C were used to evaluate their effect on the development time and adult emergence of five Trichogramma species found parasitizing eggs of the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) on soybeans in subtropical Southern Brazil. Host eggs were parasitized at 20°C and then transferred to the study temperatures to follow development and emergence of parasitoids. All five species were able to develop and emerge within the range of temperatures evaluated, and the effect of temperature on development rates could be described by linear regression. Trichogramma acacioi Brun, Moraes & Soares and T. rojasi Nagaraja & Nagarkatti were the most cold-tolerant species, with lower threshold temperatures of 8.1 ± 0.16°C and 9.2 ± 0.16°C, respectively. Trichogramma atopovirilia Oatman & Platner was the least cold-adapted species, with a lower threshold of 10.2 ± 0.13°C. Degree-day accumulation ranged from 153.8 DD for T. atopovirilia to 190.7 DD for T. acacioi. Adult emergence was higher than 90% for T. atopovirilia and T. pretiosum at all temperatures, whereas T. lasallei Pinto emergence dropped to 71.3% at 14°C and to 58.3% at 26°C, both significantly lower than the emergence of T. pretiosum and T. atopovirilia. Significantly less T. acacioi adults emerged at 30°C than either T. pretiosum or T. atopovirilia. The sex-ratio was not affected within the range of temperatures studied, and varied from 0.65 to 0.88 (female/(male + female)). Differences among Trichogramma spp. densities in the field can be attributed to slower development rates and/or reduced emergence of adults, both at low and high temperatures.

<Emphasis Type="Italic">Hox</Emphasis> cluster duplication in the basal teleost <Emphasis Type="Italic">Hiodon alosoides</Emphasis> (Osteoglossomorpha)

Large-scale—even genome-wide—duplications have repeatedly been invoked as an explanation for major radiations. Teleosts, the most species-rich vertebrate clade, underwent a “fish-specific genome duplication” (FSGD) that is shared by most ray-finned fish lineages. We investigate here the Hox complement of the goldeye (Hiodon alosoides), a representative of Osteoglossomorpha, the most basal teleostean clade. An extensive PCR survey reveals that goldeye has at least eight Hox clusters, indicating a duplicated genome compared to basal actinopterygians. The possession of duplicated Hox clusters is uncoupled to species richness. The Hox system of the goldeye is substantially different from that of other teleost lineages, having retained several duplicates of Hox genes for which crown teleosts have lost at least one copy. A detailed analysis of the PCR fragments as well as full length sequences of two HoxA13 paralogs, and HoxA10 and HoxC4 genes places the duplication event close in time to the divergence of Osteoglossomorpha and crown teleosts. The data are consistent with—but do not conclusively prove—that Osteoglossomorpha shares the FSGD. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Cloning of a novel levoglucosan kinase gene from Lipomyces starkeyi and its expression in Escherichia coli

Levoglucosan, cellulosic pyrolysate, is converted to glucose-6-phosphate by a specific levoglucosan kinase in fungi. A novel cDNA of levoglucosan kinase gene (lgk) from yeast Lipomyces starkeyi YZ-215 was isolated by RACE method. The 1,445 bp cDNA fragment (lgk) harbouring the kinase gene exhibited one open reading frame (ORF) composed of 1,317 bp flanked by a 14 bp 5′-UTR and a 114 bp 3′-UTR, including a 25 bp poly(A) tail. The ORF encoded a 439 amino acid polypeptide with a 48.4 kDa predicted molecular mass. Analysis of amino sequence revealed that the kinase belonged to the bacterial anhydro-N-acetylmuramic acid kinase (AnmK) family, and kinase-like proteins existed in some fungi, especially in filamentous fungi such as Aspergillus. The kinase gene was transformed into Escherichia coli BL21 (DE3), recombinant E. coli could grow in M9 minimal medium with levoglucosan as a sole carbon source when induced by IPTG. In addition, the recombinant kinase was overexpressed, purified and characterized. The kinase was stable at pH 7–10 and showed maximum activity at 30°C and pH 9.0 as natural kinase, but presented higher thermostability. Kinetic constants (apparent K _m values) for LG and ATP were 105.3 ± 12.5 and 0.20 ± 0.02 mM, respectively. Furthermore, the kinase showed substrate specificity for LG. This novel levoglucosan kinase gene would be useful in constructing recombinant microbial strains for the efficient bioconversion of cellulosic pyrolysate to ethanol.

Type 1 ribosome-inactivating proteins from <Emphasis Type="Italic">Phytolacca dioica</Emphasis> L. leaves: differential seasonal and age expression,and cellular localization

The expression of type 1 ribosome-inactivating proteins (RIPs) in Phytolacca dioica L. leaves was investigated. Fully expanded leaves of young P. dioica plants (up to 3 years old) expressed two novel RIPs, dioicin 1 and dioicin 2. The former was also found in developing leaves from adult P. dioica within about two and a half weeks after leaf development, and the latter continuously synthesized, with no seasonal or ontogenetic constraint. Fully expanded leaves from adult P. dioica expressed four RIPs (PD-Ls1–4) exhibiting seasonal variation. RIPs were localized in the extracellular space, in the vacuole and in the Golgi apparatus of mesophyll cells. Dioicin 1 and dioicin 2 showed rRNA N-β-glycosidase activity and displayed the following properties, respectively: (1) Mr values of 30,047.00 and 29,910.00, (2) pIs of 8.74 and 9.37, and (3) IC₅₀ values of 19.74 (0.658 nM) and 6.85 ng/mL (0.229 nM). Furthermore, they showed adenine polynucleotide glycosylase activity and nicked pBR322 dsDNA. The amino acid sequence of dioicin 2 had 266 amino acid residues, and the highest percentage identity (81.6%) and similarity (84.6%) with PAP-II from Phytolacca americana, while its identity with other RIPs from Phytolaccaceae was around 40%. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Substrate specificity of a recombinant chicken <Emphasis Type="Italic">β</Emphasis>-carotene 15,15′-monooxygenase that converts <Emphasis Type="Italic">β</Emphasis>-carotene into retinal

The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k _cat of 1.65 min⁻¹ and a K _m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol⁻¹). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₀ seems to be essential for enzyme activity.     

Immobilization of the recombinant invertase INVB from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on Nylon-6

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for of immobilized and free re-INVB, respectively. values for immobilized and free enzymes were 6.1 × 10² and 1.2 × 10⁴ s⁻¹, respectively, and immobilized re-INVB showed of 158.73 μmol h min⁻¹ mg⁻¹. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). k _cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K _m became twice of that of the wild type.     

Personal Experience of Schizophrenia and the Role of <Emphasis Type="Italic">Danwei</Emphasis>: A Case Study in 1990s Beijing

Danwei as a cornerstone of Chinese urban society has received great research attention. The relationship between the Danwei and psychiatric patients, however, remains unclear. This article aims to shed light on the subject with an integrated micro–macro approach. It introduces a historical understanding of mental health in urban China under the “economic state in transition” framework. A detailed case study in clinical sociology is provided to reveal the many social factors affecting the experience of a schizophrenic patient and his significant others. A changing role of the Danwei is hypothesized and validated with qualitative data. The Danwei was shown to have changed significantly before the mid-1990s, yet it still played a major role in urban workers’ lives, including those of psychiatric patients, and even more so in people’s expectations. This lends support to the need for a more responsive public policy to address various social issues brought about by economic reform, with the learning of worldwide experiences including “community care,” “social support” and “social rehabilitation.” Implications for social research, policymaking and professional practice are discussed.

Aggregations of juvenile whale sharks (<Emphasis Type="Italic">Rhincodon typus</Emphasis>) in the Gulf of Tadjoura,Djibouti

A total of 23 whale sharks were identified over a 5 d period in the Arta Bay region of the Gulf of Tadjora, Djibouti. Most of the sharks aggregating in this area were small (<4 m TL) males. Individuals were identified using photographs of distinctive scars and spot and stripe patterns on the sides of the animals. Of these, 65% had scarring that was attributable to boat or propeller strikes. Most of the whale sharks we encountered were feeding on dense accumulations of plankton in shallow water just off (10–200 m) the shoreline. This food source may account for the aggregation of sharks in this area. One 3 m male shark was tagged with an ARGOS (Splash) satellite tag for 9 d. During this time the shark traversed to the shoreline on the opposite side of the Gulf (a distance of 14 km) and then returned to the Arta Bay area before retracing his path to the other shore. The shark spent most of the daylight hours at the surface, while at night dives were more frequent, deeper and for longer durations.

Purification and properties of fluoroacetate dehalogenase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 8341

The degradation of fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases capable of hydrolyzing the stable C–F bond have been studied. Pseudomonas fluorescens DSM 8341 was originally isolated from soil and readily degrades fluoroacetate, thus it was thought that its dehalogenase might have some desirable properties. The enzyme was purified from cell-free extracts and characterised: it is a monomer of 32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg²⁺, Mn²⁺ and Fe³⁺), but inhibited by others (Hg²⁺, Ag²⁺). The enzyme is specific for fluoroacetate, and the K _m for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date.     

A novel alkalo- and thermostable phospholipase D from <Emphasis Type="Italic">Streptomyces olivochromogenes</Emphasis>

A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75°C and the enzyme was stable from pH 7 to 13 and from 55 to 75°C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca²⁺-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 μmol min⁻¹ mg⁻¹, respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50°C. The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry.     

Genetic discrimination of middle Mississippi River <Emphasis Type="Italic">Scaphirhynchus</Emphasis> sturgeon into pallid,shovelnose, and putative hybrids with multiple microsatellite loci

The pallid sturgeon (Scaphirhynchus albus), which is protected under the US endangered species act, and shovelnose sturgeon (S. platorhynchus), which is legally harvested in some locations, are sympatric throughout the range of pallid sturgeon. There is considerable morphological overlap between the species making discrimination problematic. The inability to reliably differentiate between species across all life stages has hampered pallid sturgeon recovery efforts. Furthermore, the two species are believed to hybridize. This study used allele frequency data at multiple microsatellite loci to perform Bayesian and likelihood-based assignment testing and morphological measures and meristics to discriminate pallid, shovelnose, and putative hybrid sturgeons from the middle Mississippi River. Bayesian model-based clustering of the genetic data indicated that two natural genetic units occur in the region. These units correspond to morphologically identified pallid and shovelnose sturgeon. Some individuals were morphologically intermediate and many of these failed to strongly assign genetically as either pallid or shovelnose sturgeon, suggesting they may be hybrids. These data indicate that pallid sturgeon and shovelnose sturgeon are genetically distinct in the middle Mississippi River (F _ST = 0.036, P < 0.0001) and suggest that hybridization between pallid sturgeon and shovelnose sturgeon has occurred in this region with genetic distance estimates indicating the greatest distance is between pallid and shovelnose sturgeon, while hybrid sturgeon are intermediate but closer to shovelnose. This study demonstrates that assignment testing with multiple microsatellite markers can be successful at discriminating pallid sturgeon and shovelnose sturgeon, providing a valuable resource for pallid sturgeon recovery and conservation.

Suppression of Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic microorganisms

Fusarium wilt of watermelon commonly occurs in locations where the crop has been grown for many seasons. Its occurrence results in a severely decreased watermelon crop. The goal of this study was to assess the capability of a new product (bio-organic fertilizer) to control the wilt in Fusarium-infested soil. Pot experiments were conducted under growth chamber and greenhouse conditions. The results showed that the fertilizer controlled the wilt disease. Compared with control pots, the incidence rates of Fusarium wilt at 27 and 63 days following treatment of the plants with the bio-organic fertilizer at a rate of 0.5% (organic fertilizer + antagonistic microorganisms, including 3 × 10⁹ CFU g⁻¹ Paenibacillus polymyxa and 5 × 10⁷ CFU g⁻¹ Trichoderma harzianum) were reduced by 84.9 and 75.0%, respectively, in both the growth chamber and greenhouse settings. The activities of antioxidases (catalase, superoxide dismutase and peroxidase) in watermelon leaves increased by 38.9, 150 and 250%, respectively. In the roots, stems and leaves, the activity of β-1,3-glucanase (pathogenesis-related proteins) increased by 80, 1140 and 100% and that of chitinase increased by 240, 80, and 20%, respectively, while the contents of malondialdehyde fell by 56.8, 42.1 and 45.9%, respectively. These results indicate that this new fertilizer formula is capable of protecting watermelon from Fusarium oxysporum f.sp. niveum. The elevated levels of defense-related enzymes are consistent with the induction and enhancement of systemic acquired resistance of plant.