Detection of the Pinewood Nematode, Bursaphelenchus xylophilus, Using a Real-Time Polymerase Chain Reaction Assay

ABSTRACT The pinewood nematode, Bursaphelenchus xylophilus, has caused significant damage to pine plantations both in East Asia and North America and is an important quarantine organism. A real-time polymerase chain reaction (PCR) assay was developed to detect B. xylophilus. A set of primers and probe specific for B. xylophilus was designed to target the ribosomal DNA internal transcribed spacer region. Optimal primer concentration, Mg(2+) concentration, and extension temperature were 400 nM, 3.0 mM, and 60 degrees C, respectively. The assay was highly specific and sensitive, detecting as little as 0.01 ng of B. xylophilus DNA. The real-time PCR assay also successfully detected B. xylophilus in field samples, and it should be very useful for quarantine purposes.     

Some diagnostic, biologic and morphologic characteristics of Francisella tularensis strains isolated from the ticks Ixodes ricinus (L.) in the Prague agglomeration

Four infectious agents were isolated from the ticks Ixodes ricinus (L.) collected in the recreational area and park-forest of Prague. On the basis of cultivation, staining, biochemical, serologic properties, pathogenicity for animals and histological tests they were identified as Francisella tularensis with the following features: they are short, gram-negative rods of approximate dimensions of 0.3 X 0.8 micron, growing in enriched media after 3-4 day incubation at 37 degrees C. They form small circular, at first transparent, later greyish turbid colonies with regular rims. They are little active biochemically. They are susceptible to streptomycin and some broad spectrum antibiotics. They react positively with tularemic serum, but in lower titres than those in which this serum reacts with standard antigen. The microbes are highly pathogenic for mice, guinea-pigs, young rats, in which a massive bacteriemia occurs before death, but they do not kill rabbits. They multiply well in chick embryo, but do not grow in cell or tissue cultures. The most important histologic changes were observed in liver and spleen of mice. No pathologic changes were found in brain, lungs, heart, kidneys. Necroses were found in liver and in their marginal zones the microbes were present. Conspicuous were changes in numerous hepatocytes which became enlarged due to microbial multiplication and finally transformed into "sacs" packed with microbes. Histological and electronoptical examination showed that these are intracellular parasites fringed with a light lytic zone. Discussed is the problem to what extent the properties of the isolated strains are typical of F. tularensis as well as the importance of their detection from the aspect of epidemiology and differential diagnostics.     

Molecular characterization of a phytoplasma associated with Euonymus bungeanus witches’ broom in China

Euonymus bungeanus plants exhibiting symptoms of abnormal branches, small leaves and phyllody, which is indicative of E. bungeanus witches’ broom (EbWB) disease, have recently been found in Beijing, China. A phytoplasma from symptomatic E. bungeanus plants was identified by 16S rRNA polymerase chain reaction (PCR) using the phytoplasma‐specific universal primer pair R16mF2/R16mR1. Inoculation of healthy E. bungeanus plants by grafting with diseased scions was also performed. The rp and secY genes of the EbWB phytoplasma were cloned and sequenced as was the 16S rRNA gene. Sequence and phylogenetic analyses of 16S rRNA, rp and secY genes indicated that the phytoplasma associated with E. bungeanus belongs to the 16SrV‐B, rpV‐C and secY‐C subgroup, the same subgroup as the jujube witches’ broom (JWB) phytoplasma that is widely distributed among jujube trees in China. Comparative analyses based on virtual restriction fragment length polymorphism (RFLP) showed that the EbWB phytoplasma is more closely related to another 16SrV‐B subgroup strain: RPWB (Robinia pseudoacacia witches’ broom). To the best of our knowledge, this is the first report of a witches’ broom phytoplasma in E. bungeanus in China, and the findings add a new cultivated plant species to the already broad natural host range of 16SrV‐B subgroup phytoplasmas.     

Ethylene formation from ethyl moiety of ethionine

In plants, ethylene is formed in the presence of light and flavin mononucleotide from ethionine and S-ethylcysteine. The ethylene is formed from the ethyl moiety of ethionine.     

Effects of formic acid and potassium diformate on the fermentation quality,chemical composition and aerobic stability of alfalfa silage

The objective of this study was to evaluate the efficacy of potassium diformate (KDF) as a potential additive for alfalfa silage. Fresh alfalfa was untreated or treated with formic acid (4 g/kg fresh weight, FW) or three concentrations of KDF (4, 5.5 or 7 g/kg FW). After 60 days of ensiling, the addition of formic acid and greater levels of KDF (5.5 and 7 g/kg) effectively reduced silage pH and inhibited the undesirable bacteria, indicated by lower butyric acid, ethanol, ammonia N concentrations and microbial populations (including enterobacteria, yeasts, moulds and clostridia). Additives decreased the dry‐matter loss, and more water‐soluble carbohydrates were preserved in the silages with formic acid or potassium diformate than in the control. Alfalfa silages treated with formic acid at 4 g/kg FW or potassium diformate at 5.5 or 7 g/kg FW were classified as the highest quality silage based on the higher Flieg's point (above 70) and remained stable for more than 9 days during aerobic exposure. Potassium diformate is recommended as an effective additive for alfalfa silages at a level of 5.5 or 7 g/kg FW under the humid and hot conditions of southern China.     

Effects of Cu2+-exchanged montmorillonite on intestinal microflora, digestibility and digestive enzyme activities of Nile tilapia

A total of 360 Nile tilapia at an average initial body weight of 3.9 g were randomly allocated to four treatments, each of which had three replicates of 30 fish per tank and used to investigate the effects of Cu²⁺‐exchanged montmorillonite (Cu‐MMT) on intestinal microflora, digestibility and digestive enzyme activities. The dietary treatments were: (i) basal diet, (ii) basal diet + 1.5 g kg^?1 MMT; (iii) basal diet + 30 mg kg^?1 copper as CuSO₄ (equivalent to the copper in the Cu‐MMT treatment group), or (iv) basal diet + 1.5 g kg^?1 Cu‐MMT. The results showed that supplementation with Cu‐MMT significantly improved growth performance as compared with the control and fish fed with Cu‐MMT had higher growth performance than those fed with MMT or CuSO₄. Supplementation with Cu‐MMT reduced (P < 0.05) the total intestinal aerobic bacterial counts and affected the composition of intestinal microflora with Aeromonas, Vibrio, Pseudomonas, Flavobacterium, Acinetobacter, Alcaligence, Enterobacteriaceae decreasing as compared with the control. Addition of MMT improved (P < 0.05) the apparent digestibility of dry matter and crude protein, and the activities of intestinal amylase, lipase, and alkaline phosphatase as compared with the control. Addition of Cu‐MMT improved (P < 0.05) the apparent digestibilities of dry matter, crude protein, ether extract, and crude ash, and the activities of total protease, amylase, lipase, and alkaline phosphatase as compared with the control. While supplementation with CuSO₄ had no (P > 0.05) effect on intestinal microflora, digestibility, and digestive enzyme activities the results showed that Cu‐MMT exhibited antibacterial activity in vivo resulting in a positive effect on digestive enzyme activities, and then promoted the digestion and absorption of dietary nutrition.