Powdery Mildew of Prairie Gentian: Characteristics,Molecular Phylogeny and Pathogenicity

In March 1999, we found prairie gentian (Eustoma grandiflorum) infected with powdery mildew in a greenhouse in Oita Prefecture, Japan. Morphological observation revealed that the causal fungus belongs to the mitosporic genus Oidium subgenus Pseudoidium [teleomorph: Erysiphe sensu Braun and Takamatsu (2000)]. Precise taxonomic position of the fungus, however, is uncertain due to lack of the perfect stage. We determined the nucleotide sequence of the rDNA ITS region of the fungus. Comparison of the sequence with those obtained from DNA databases of this fungal group revealed that the sequence is identical to those of powdery mildews from garden four-o'clock (Mirabilis jalapa) and broad bean (Vicia faba). Inoculation of an isolate from garden four-o'clock caused mildew on prairie gentian and broad bean, suggesting that the prairie gentian mildew originates from garden four-o'clock or broad bean. Molecular phylogenetic analysis indicated a close relationship of this fungus to Erysiphe baeumleri on Vicia spp. and E. trifolii on Trifolium pratense. From these results, we propose that prairie gentian mildew diverged from a Fabaceae-parasitic ancestor. Received 14 March 2002/ Accepted in revised form 28 May 2002     

Simultaneous detection of multiple mycotoxins in broiler feeds using a liquid chromatography tandem-mass spectrometry

Mycotoxins are secondary fungal metabolites that are typically present in grain and feed ingredients used for animal feeds. An analytical method using LC-ESI-MS/MS was developed to quantify nine mycotoxins, consisting of aflatoxin B₁ (AFB₁), AFB₂, AFG₁, AFG₂, T-2 toxin, deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA) and ochratoxin A (OTA) in broiler feeds. In total, 100 samples of broiler feeds were collected from poultry farms in Central Thailand. The survey found that AFB₁ and ZEA were the most prevalent mycotoxins in the feed samples at percentages of 93% and 63%, respectively. The limit of detections (LODs) of investigated mycotoxins was 0.20–0.78 ng/g. AFB₂, DON, AFG₁, NIV and T-2 toxin were also detectable at low contamination levels with percentages of 20%, 9%, 7%, 5% and 1%, respectively, whereas OTA and AFG₂ were not detected in any of the feed samples. These results suggest that there is a very low level of risk of the exposure to mycotoxins in feeds obtained from broiler farms in Central Thailand.     

Pharmacokinetics of marbofloxacin in Green sea turtles (Chelonia mydas) following intravenous and intramuscular administration at two dosage rates

Limited pharmacokinetic information to establish suitable therapeutic plans is available for green sea turtles. Therefore, the present study was conducted to evaluate the pharmacokinetic characteristics of marbofloxacin (MBF) in the green sea turtle, Chelonia mydas, following single intravenous (i.v.) or intramuscular (i.m.) administration at two dosages of 2 and 4 mg/kg body weight (b.w.). Blood samples were collected at assigned times up to 168 hr. MBF in plasma was extracted using liquid–liquid extraction and analyzed by a validated high-performance liquid chromatography (HPLC). MBF was quantifiable from 15 min to 96 hr after i.v. and i.m. administrations at two dose rates. A noncompartmental model was used to fit the plasma concentration of MBF versus time curve for each green sea turtle. The t_1/2λz value, similar for both the dosages (22–28 hr), indicated that the overall rate of elimination of MBF in green sea turtles is relatively slow. The average i.m. F% ranged 88%–103%. MBF is a concentration-dependent drug and the AUC/MIC ratio is the best PK/PD predictor for its efficacy. The MBF dosage of 4 mg/kg appeared to produce an appropriate value of the PK-PD surrogate that predicts antibacterial success for disease caused by susceptible bacteria. In contrast, i.m. administration of MBF at a dosage of 2 mg/kg b.w. was not found to produce a suitable PK-PD surrogate index. However, further studies of multiple doses and plasma binding proteins are warranted to confirm an appropriate dosage regimen.     

Pharmacokinetic profiles of amoxicillin trihydrate in freshwater crocodiles (Crocodylus siamensis) after intramuscular administration at two doses

The aim of the present study was to elucidate the pharmacokinetic profiles of amoxicillin trihydrate (AMX) in Siamese freshwater crocodiles (Crocodylus siamensis). Crocodiles were administered a single intramuscular injection of AMX, at a dose of either 5 or 10 mg/kg body weight (b.w.). Blood samples were collected at preassigned times up to 120 hr. The plasma concentrations of AMX were measured using a validated liquid chromatography tandem-mass spectrometry method. AMX plasma concentrations were quantifiable for up to 72 hr (5 mg/kg b.w.) and 96 hr (10 mg/kg b.w.). The elimination half-life (t_1/2λz) of AMX following dosing at 5 mg/kg b.w. (8.72 ± 0.61 hr) was almost identical to that following administration at 10 mg/kg b.w (8.98 ± 1.13 hr). The maximum concentration and area under the curve from zero to the last values of AMX increased in a dose-dependent fashion. The average binding percentage of AMX to plasma protein was 21.24%. Based on the pharmacokinetic data, susceptibility break point, and the surrogate PK-PD index (T > MIC, 0.25 μg/ml), intramuscular administration of AMX at dose of 5 mg/kg b.w. every 4 days might be appropriate for the treatment of susceptible bacterial infections in freshwater crocodiles.     

The fate and tissue disposition of deoxynivalenol in broiler chickens

To evaluate the fate of deoxynivalenol (DON) in broilers, DON was administered either intravenously or orally to broilers at a dose of 1 mg/kg BW. Concentrations of DON in plasma were measurable up to 4 hr and 2 hr after intravenous and oral administration, respectively. Following intravenous administration, the values for the elimination half-life, the volume of distribution and the clearance were 1.25 ± 0.25 hr, 7.55 ± 2.03 l/kg and 4.16 ± 0.42 l/hr/kg, respectively. The oral bioavailability was 15.46 ± 4.02%. DON was detectable in all tissues examined after oral administration. These results suggest that DON is able to penetrate into the various tissues in broilers, though poorly absorbed from their gastrointestinal tract.     

Rapid and Sensitive Detection of Vibrio alginolyticus by Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the rpoX Gene

Vibrio alginolyticus is a major bacterial pathogen causing disease in marine animals. The present study aimed to develop a loop-mediated isothermal amplification (LAMP) coupled with a lateral flow dipstick (LFD) for rapid and simple visual detection of V. alginolyticus–specific amplicons. The biotin-labeled LAMP amplicons from the targeted portion of a gene encoding rpoS-like sigma factor (rpoX) were generated at 60°C for 1 h and then hybridized with a fluorescein isothiocyanate–labeled probe for 5 min for visual detection with LFD. In pure cultures, the detection limit of the LAMP–LFD technique for V. alginolyticus was 1.8 × 10² CFU/mL while that of PCR was 1.8 × 10³ CFU/mL. In spiked whiteleg shrimp samples Penaeus vannamei, the sensitivity for V. alginolyticus detection was 2 × 10³ CFU/g (equivalent to 4 CFU per reaction) while PCR was 10 times less sensitive. The LAMP–LFD method for V. alginolyticus correctly identified 21 isolates of V. alginolyticus but did not recognize 23 non-V. alginolyticus Vibrio isolates and 15 non-Vibrio isolates. In summary, this LAMP–LFD method targeted to the rpoX gene is a convenient assay for specific identification of V. alginolyticus with high sensitivity.

Received November 11, 2014; accepted March 29, 2015     

COMPARATIVE STUDIES ON THE IRON AND ZINC CONTENTS ESTIMATION USING ATOMIC ABSORPTION SPECTROPHOTOMETER AND GRAIN STAINING TECHNIQUES (PRUSSIAN BLUE AND DTZ) IN MAIZE GERMPLASMS

Malnutrition arising from dietary deficiency of critically important mineral micronutrients such as iron (Fe) and zinc (Zn) is a serious problem affecting nearly half of the world's population. Screening for micronutrients rich breeding lines includes the estimation of grain micronutrient contents in the seed samples using atomic absorption spectrophotometer (AAS). Though the protocol produces precise results, it is usually costly and time consuming. The nutritionist started searching for newer methods like staining the particular plant tissue with dyes so the initial screening of among vast germplasm lines becomes easier. A set of four hundred and thirty genotypes were used in this experiment wherein the reliability and the cost effectiveness of the use of the staining method in place of the estimation using AAS was investigated. The intensity of the color developed was directly proportional to the amount of iron and zinc in the grains and were scored as 1, 2, 3, 4, and 5. The genotype ‘UBC 7’ has got the maximum level of iron (65.43 mg kg^?1) with about 64 lines is found to contain higher levels of iron. The genotype ‘UMI 421’ had the maximum level of zinc (60.15 mg kg^?1) along with ten lines showing richer zinc levels. The genotypes namely ‘UMI 1069’, ‘UMI 630’, ‘UMI 473’, ‘UMI 327’, and ‘UMI 382’ are found to be having considerably higher levels of iron and zinc in the kernels. These lines may be used for the introgression of the traits concerning micronutrient enhancement into the popular elite cultivars. When compared to the estimation with AAS, the staining methods with Prussian Blue and Dithizone (DTZ: 1,5-diphenyl thiocarbazone) are found be very cost effective and precise in identifying and categorizing the lines as micronutrient rich/deficit. The varying color intensities serve as a reliable selection criterion for grain micronutrient contents in maize and other cereal crops. These protocols, could, therefore, be effectively used as the initial screening methodology and so the genotypes might be identified for higher grain micronutrient contents among huge population in a short period of time.     

Production and optimization of indole acetic acid by indigenous micro flora using agro waste as substrate

Indole Acetic Acid (IAA) producing bacterium was isolated from the Rhizosphere soil and identified as Rhizobium sp. and Bacillus sp., Optimization of Indole acetic acid production was carried out at different cultural conditions, such as pH, temperature and substrate with Rhizobium sp., Bacillus sp. and Rhizobium sp., produced higher amount of Indole acetic acid (6.1 mg mL(-1)) than the Bacillus sp., (4.4 mg mL(-1)) at pH 7 and 37 degrees C in the Bengal gram substrate. Partial purification of Indole acetic acid was done by Thin Layer Chromatography (TLC). In conclusion Rhizobium sp., appear to be a suitable soil microorganism for high level of IAA production.     

Hairy Vetch Incorporated as Green Manure Inhibits Sulfathiazole Uptake by Lettuce in Soil

Veterinary antibiotics like sulfonamides are frequently detected in arable lands and they can potentially contaminate food crops. It is thus of great importance to identify strategies to reduce food crops’ uptake of antibiotics. For the first time, using a pot culture experiment, sulfathiazole (STZ) uptake by lettuce (Lactuca sativa L.) grown in antibiotic-contaminated soils (10 and 100 mg STZ kg^?1 soil) and treated with (in)organic amendments, namely chemical fertilizer (NPK), compost, and hairy vetch, was investigated. Subsequent enhanced plant growth was witnessed when using hairy vetch treatment. The amount of antibiotic uptake was significantly reduced to 5 and 33% with hairy vetch application compared to compost or NPK application at 10 and 100 mg kg^?1 STZ, respectively. The total amounts of accumulated STZ in plant parts increased as the levels of STZ contaminated in soils were increased. STZ was much more abundant in the roots than the leaves. Within 30 days, the extractable STZ in the treated soils—especially with hairy vetch—diminished considerably to concentrations that are frequently detected in arable soils. We conclude that utilization of green manure (cover crop—hairy vetch) is a viable strategy for safer crop production in antibiotic-contaminated soils.     

Long-Term Inorganic Fertilization Effect on the Micronutrient Density in Soil and Rice Grain Cultivated in a South Korean Paddy Field

Soil fertility exerts a direct influence on the essential micronutrient contents of food crops. The focus in this study was on the role that long-term inorganic fertilization (nitrogen (N), phosphorous (P) and potassium (K)) plays in increasing the micronutrient output of the paddy cropping system. After more than 45 years of inorganic fertilizer application, the combined application of fertilizers (PK > NP > NK) substantially increased As (arsenic), B (boron), Cd (cadmium), Co (cobalt), Cr (chromium), Cu (copper), Fe (iron), Mn (manganese), Mo (molybdenum), Ni (nickel), Se (selenium), V (vanadium) and Zn (zinc) density in the soil and rice grain. Optimized and continuous application of PK fertilizers increased the overall micronutrient densities in rice milling fractions (grain and bran). Micronutrient concentrations were usually the highest in bran. Both grain and bran were rich in Fe, Mn and Zn. Correlation analysis indicated that soil pH and organic matter exert a significant and direct effect on the micronutrient concentration of rice. Although long-term fertilization enhanced the proportion of micronutrients in rice grain, the levels were still much lower than the recommended dietary intake levels for micronutrients. We therefore suggest high consumption levels of brown rice (with micronutrient-dense bran layer) because they may increase the daily intake level of micronutrients and meet the nutritional requirements that people need for sound health.