OBTAINING HIGH-QUALITY PLANTING MATERIAL OF FOREST BERRY PLANTS BY CLONAL MICROPROPAGATION FOR RESTORATION OF CUTOVER PEATLANDS

The results of studies on the improvement of technology for producing highquality planting material of half-high blueberry and Arctic bramble by the method of clonal micropropagation are presented in the current paper. Creation of forest berry plantations in peat extraction areas allows reducing environmental damage and significantly increasing the efficiency of the timber industry. In recent decades, there has been an increasing interest in the creation of forest berry plantations on drained and cutover peatlands in Russia and other countries. It is necessary to use varietal planting material for the successful cultivation of forest berry plants on an industrial scale. Clonal micropropagation is the most effective of the vegetative methods for obtaining planting material, which allows receiving a huge amount of healthy planting material all year round in the conditions of a small laboratory area. Chloride-free ecosterilizer and bleaching agent based on sodium hypochlorite “Belizna” with an exposure of 15 and 20 min showed high efficiency in sterilization of explants of half-high blueberry and Arctic bramble. The highest viability of explants of the studied forest berry crops was observed when sterilized with a 0.1 % mercuric chloride solution and 15 min exposure, and its sharp decrease at 20 min exposure. At the stage of micropropagation, with an increase in the concentration of cytokinin 6-BAP from 0.5 to 1.0 mg/L on the nutrient Woody Plant Medium the number of shoots in regenerated plants of half-high blueberry (Northcountry and Northblue cultivars) and Arctic bramble (Anna and Sofia cultivars) increased. The effect of the concentration of IBA-derived auxin on the number and length of roots of regenerated plants was observed at the in vitro rooting stage.

Notes on the fifty operations

At the beginning of his report, the author describes in detail the environment in which he operates. The instruments immediately before the operation are boiled in water for 5 minutes, toothed instruments (tweezers, etc.) are calcined on an alcohol lamp; during the operation, they lie in boiled water. For seams, silk is used, boiled in a 5% carbolic solution and stored in a mixture of equal parts of a 1% (? Ref.) Solution of mercuric chloride and absolute alcohol; is used to apply a catgut disinfected previously lying in the course of 12 hours 0.1% mercuric chloride solution and then kept for several days in a mixture of 1 part ol juniperi and 2 parts alcohol. Sponges are rarely used - they are replaced by tampons from aseptic gauze, which are put into 1/2 solution of mercuric chloride at the time of the operation. The operator and his four assistants put on decontaminated rubber aprons; the sleeves are rolled up above the elbows; hands are washed with green soap and a brush, then with absolute alcohol and mercuric chloride solution. The patient is given a general bath on the eve of the operation and a laxative is given; then, when it is already chloroformed, the hair on mons Veneris is shaved off, the abdominal wall is thoroughly washed with green soap with a brush, sulfuric ether and 1 solution of mercuric chloride. After that, during the operation itself, the author does not use any disinfectant liquids; the abdominal cavity, if necessary, is washed with boiled water. After the operation, the edges of the abdominal wound are washed with mercuric chloride and pulverized with idoform; then sutures are applied, 3-4 deep, covering the entire thickness of the abdominal walls, and many superficial.

First Report of Root Rot on Atractylodes macrocephala (Largehead Atractylodes Rhizome) Caused by Ceratobasidium sp. in China

Atractylodes macrocephala is a perennial herbaceous plant (family Asteraceae) native to China. The biennial root, Largehead Atractylodes Rhizome (LAR), is the most commonly used Chinese herbal medicine to prevent early pregnancy loss due to miscarriage. From summer 2010 to spring 2012, symptoms of root rot were observed on LAR in Xianfeng county, Enshi city, Hubei Province, China. White mold on the root of LAR could be observed at an early growth stage in the field and the white mold spread over the entire plant after 10 days, which differs from root rot of LAR caused by Fusarium oxysporum and Rhizoctonia solani, neither of which are characterized as having mycelium spreading over the whole plant (4). Where root rot symptoms were present, rhizome yield was reduced by 15% on average, with up to 40% yield loss in some fields. Under humid conditions in mid-June, the disease in the field spread quickly and the rhizomes of LAR were completely rotted. After rainfall and increasing temperature from 16 to 35°C, white mycelium appeared and plants withered within a few weeks. In April 2011 and 2012, a fungus was consistently recovered from symptomatic rhizome samples after they were surface sterilized with 0.1% mercuric chloride solution and plated onto potato dextrose agar (PDA). Pale gray colonies with short aerial mycelia and brown sclerotia formed on PDA after 7 days incubation at 28°C. Binucleate cells were observed using light microscopy and the characteristics were matched with morphological characteristics of a Ceratobasidium sp (3). Genomic DNA of the culture was extracted, and the rDNA-internal transcribed spacer sequence (GenBank Accession No. JQ926741) showed 99% identity to Ceratobasidium sp (GenBank No. H269825.1). Mycelial plugs of the culture taken from PDA were inoculated onto 40 rhizomes of 1-year-old seedlings and plants were incubated with a 16-h photoperiod at 28°C and 90% relative humidity in an artificial climate chamber where they developed typical disease symptoms after 2 days. Ten rhizomes of 1-year-old seedlings and were treated with PDA plugs only. All seedlings inoculated with the pathogen were withered and the rhizomes were completely covered with gray mycelium 2 days after inoculation, which was similar to the symptoms observed in the field. After 7 days, the symptoms were more severe than those observed in the field, with seedlings rotted completely. The main stalk of all inoculated plants was covered with gray mycelia in 4 days, and the stalk became withered, which was similar to the symptoms observed in the field. No symptoms were observed on control seedlings and plants. Koch's postulates were fulfilled by successful reisolation of Ceratobasidium sp. from diseased seedlings. The pathogenicity tests were carried out twice. Ceratobasidium sp. has been reported to cause root rot of canola in Washington (2). It has also been observed on Rehmannia in China (1). To our knowledge, this is the first report of Ceratobasidium sp. causing root rot on LAR. References: (1) B. B. Chen et al. Chin. J. Chin. Material Medica (In Chinese) 9:1137, 2011. (2) K. L. Schroeder et al. Plant Dis. 96:591, 2012. (3) B. Sneh et al. Page 39 in: Identification of Rhizoctonia Species. The American Phytopathological Society, 1991. (4) S. X. Zang et al. J. Agric. Univ. Hebei (In Chinese) 28:73, 2005.

First Report of Stem Blight of Eleocharis dulcis Caused by Phoma bellidis in China

Eleocharis dulcis is a perennial herbaceous plant in the family Cyperaceae, which is native to China and India where it grows well in moist-to-wet soils. It is commonly used as a fruit or a vegetable. From August 2009 to December 2010, symptoms were observed on E. dulcis stems in Tuanfeng County, Hubei, China, with the crop area affected estimated to be more than 1,300 ha per year. Corm yield was reduced by 20% on average with as much as 60% yield losses in some fields. Lesions were initially small, red-brown, and oval or circular that enlarged to produce apical necrosis and extending until the stems withered, usually within 2 months. To obtain isolates, diseased tissue was disinfested for 1 min in 0.1% mercuric chloride solution, rinsed with sterilized water, and plated on potato dextrose agar. Isolates with similar morphological characteristics were consistently recovered. Three isolates, CTF-3, CTF-10, and CTF-11, were used to further evaluate characteristics of the pathogen. After 7 days, white colonies were 76 to 80 mm across on oatmeal agar (OA) with sparse aerial hyphae and a slight salmon color in the conidial masses. Pycnidia produced on OA were globose to subglobose, usually with one slightly ostiolar papilla, olivaceous to olivaceous black, and 93 to 245 μm in diameter. Conidia were hyaline, unicellular, ellipsoidal, mostly with two polar guttules, and 3.6 to 6.2 × 2.0 to 3.3 μm. Chlamydospores were absent. Growth of the isolates on malt extract agar (MEA) was slower than on OA, and the colony diameters at 7 days were 60 to 65 mm. The reactions with 1M NaOH were both positive on OA and MEA where the cultures initially changed to yellow green and gradually turned to red. The pathogen was identified as Phoma bellidis Neerg. based on descriptions in Boerema et al. (2). Pathogenicity tests were performed with the three isolates in the laboratory by spraying conidial suspensions (1 × 106 conidia/ml) containing 0.1% Tween 20 until runoff (30 ml per plant) onto stem surfaces of 50-day-old, 60 cm tall plants. For each isolate, there were 50 stems from five replicate plants that had multiple stems per plant. Control plants were treated with sterilized water containing 0.1% Tween 20 only. Plants were incubated with a 16-h photoperiod at 28°C and 90% relative humidity in an artificial climate chamber. Five days after inoculation, typical red-brown spots were observed on all inoculated stems but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of P. bellidis from diseased stems. The pathogenicity tests were repeated twice more with the same results. P. bellidis has only been reported previously on Bellis spp. from England, Denmark, Italy, the Netherlands, and Switzerland (1,2). Furthermore, there are only a few fungal diseases known to be associated with E. dulcis, and none so far that involve species of Phoma (3,4). To our knowledge, this is the first report of P. bellidis infecting E. dulcis worldwide. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:27, 2010. (2) G. H. Boerema et al. Phoma Identification Manual: Differentiation of Specific and Infra-Specific Taxa in Culture. CABI Publishing, Wallingford, UK, 2004. (3) P. L. Lentz. Am. Midl. Nat. 67:184, 1962. (4) L. Pan et al. J. Changjiang Vegetables (in Chinese) 14:10, 2010.

Developmental Time, Mortality and Weight of Immature FIeshfIy Bercaea cruentata (Diptera: Sarcophagidae) Larvae Exposed to Mercury

The total developmental time of the fleshfly, Bercaea cruentata (Meigen) reared on ground beef media treated with mercuric chloride solution (Hg concentrations of 0, 100, 200, 300, 400 and 500 ug/g dry ground beef), from first-instar larvae to adult was 20.5, 20.5, 20.9, 20.9, 20.9 and 21.1 days and 20.3, 20.3, 20.7, 20.4, 20.5 and 20.8 days for males and females, respectively. There was no significant correlation (p > 0.05) between mercury concentrations and developmental time (larvae, pupae and total), mortality (larvae, pupae and total) and weights (pupae and adults). The immature stages of the fleshfly B. cruentata proved to be highly tolerant to mercury.

Effects of preservatives and acidification on the stable isotope ratios (15N:14N, 13C:12C) of two species of marine animals

When animal tissues are prepared for stable isotope ratio analysis, they may or may not be treated with acid prior to analysis to remove carbonates and are loaded into tin or silver weigh boats for quantitative combustion. The effects of these methodological variations are poorly known. The effects of various preservation methods on isotopic compositions are also poorly known. We tested the effects of four preservation methods, (i) formalin, (ii) formalin followed by a transfer to ethanol (formalin/EtOH), (iii) saturated mercuric chloride solution, and (iv) freezing/freeze-drying, on the carbon and nitrogen isotopic composition of the muscle tissue of juvenile winter flounder (Pleuronectes americanus) and the tails (including exoskeleton) of mud shrimp (Crangon septemspinosa). Freezing and freeze-drying were the only preservation methods that did not affect stable isotope ratios of carbon and nitrogen. Formalin, formalin/EtOH, and saturated mercuric chloride solution produced significant increases in δ15N values (0.5-1.4‰) and decreases in δ13C values (0.6-2.3‰) compared with frozen samples. There was also an increase in the variability of δ15N and (or) δ13C values. We also tested the effects of acidification by comparing samples that were acidified either by fuming with concentrated HCl or by the direct application of 1 N HCl containing 1.0% platinum chloride (a combustion catalyst) to unacidified samples. Neither concentrated HCl fumes nor HCl/platinum chloride had a significant effect on the δ15N or δ13C values of either species compared with unacidified samples. Therefore, acidification may be unnecessary in the preparation of some marine animals. Finally, we compared the effects of two types of sample boats: tin and silver. We found no significant effect of boat material on the δ15N or δ13C values of either species.

Analysis of Penicillin G in Milk by Liquid Chromatography

A liquid chromatographic (LC) method that was previously developed for penicillin G residues in animal tissues has been adapted to milk and milk products. After protein precipitation with sodium tungstate, samples are applied to a C18 solid-phase extraction cartridge, from which penicillin is eluted, derivatized with 1,2,4-triazole-mercuric chloride solution, and analyzed by isocratic liquid chromatography (LC) on a C18 column with UV detection at 325 nm. Quantitation is done with reference to penicillin V as an internal standard. Penicillin G recoveries were determined to be >70% on standards fortified at 3-60 ppb. Accuracy approached 100% using the penicillin V internal standard. The detection limit for penicillin G residues was 3 ppb in fluid milk. Samples may be confirmed by thermospray/LC at concentrations approaching the detection limit of the UV method.

Evaluation of Electron Capture Gas Chromatographic Method for Determination of Methyl Mercury in Freezer-Case Seafoods

A method was recently adopted by AOAC for determination of methylbound mercury in canned and fresh-frozen seafood by electron capture gas chromatography. That method was applied to the analysis of commercially prepared freezer-case seafoods. None of the commercially added ingredients produced electron capture responses that interfered in the analysis for methyl mercury. Recoveries of 95.7-114% were obtained in fortification studies of methyl mercury at 0.2 and 1.0 ppm levels. The applicability of aqueous methyl mercuric chloride solution for fortification studies was demonstrated.

Postoperative Mercury Poisoning

Three mercury poisonings (two fatal), following the use of mercuric chloride solution for peritoneal lavage during operations for the removal of cancerous growths, are described. The concentrations of mercury measured in the tissues from these cases are consistent with those found in poisoning by injection, inhalation and ingestion.