Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K

ABSTRACT The PaEXG2 gene, encoding an exo-beta-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-beta-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.     

Experimental and clinical studies on plasma leptin in obese dogs

Leptin is a protein synthesized and secreted primarily by adipocytes, and the circulating leptin concentration is elevated in obese humans and rodents. Recently, we have established a sandwich enzyme-linked immunosorbent assay for canine leptin. In the present study, plasma leptin concentrations were measured in experimentally developed obese beagles and in clinically obese dogs. When 5 male beagles were given a high-energy diet for 3 months, all of them became obese and the plasma leptin concentration significantly increased from 2.4+/-1.2 to 4.9+/-0.9 ng/ml, positively correlating with body fat content estimated by the deuterium oxide dilution method (r=0.87). The leptin concentrations of plasma samples collected from 59 dogs in veterinary practices were compared with their body condition scores (BCS). The plasma leptin concentrations of obese dogs were 9.7+/-0.7 and 12.3+/-1.5 ng/ml at BCS=4 and BCS=5, respectively, which were significantly higher than those of optimal (BCS=3) dogs (2.7+/-0.3 ng/ml). There was no significant effect of sex and breed. A weak positive correlation (r=0.37) was found between the plasma leptin concentration and age, probably due to the lesser content of visceral fat in puppies younger than 1 year old. These results indicate that plasma leptin is a good index of adiposity in dogs regardless of breed, age and sex, and may be useful for quantitative assessment of obesity in small animal practice.     

Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.     

Protective Effects Against Erwinia amylovora Induced by hrp Mutants in the Whole Plant are Only Partially Mimicked in Cultivated Cells

A virulent strain of Erwinia amylovora, the causal agent of fire blight of Maloideae, and two of its non-virulent hrp mutants (a secretory and a regulatory mutant) were inoculated into apple cell suspensions either alone or in mixed inoculations. In single inoculations, death of 4- to 5-day-old apple cells occurred only when the concentration of the virulent strain of E. amylovora reached a threshold inoculum concentration of 10⁴CFUml^–1, while high concentrations of the hrp mutants were unable to kill apple cells. When mixed inoculated with the virulent parental strain, both hrp mutants protected apple cells from death caused by the virulent strain. The protective effect was associated with a decrease in the population level of the virulent strain and it was dependent on the non-virulent to virulent inoculum concentration suggesting a competition between the non-virulent mutant and the virulent strain. However, no differential protective ability between the two types of mutants could be noticed, contrary to previous results obtained with apple seedlings or apple flowers in which the regulatory mutant was significantly more effective than the secretory mutant. In conclusion, inoculation of apple cell cultures with E. amylovora does not seem to be a model suitable for investigating mechanisms leading to protection.     

Molecular and Pathogenicity Characteristics of Phytophthora nicotianae Responsible for Root Necrosis and Wilting of Pepper (Capsicum annuum L.) in Tunisia

Isolates of Phytophthora from pepper, produced in Tunisia, were characterised according to molecular and pathogenicity criteria. Polymerase chain reaction amplification of the ITS1 region in the ribosomal DNA resulted in different sized fragments. The pepper isolates and P. nicotianae yielded a fragment of 310bp that distinguished it from P. capsici with a fragment of 270bp. The ribosomal RNA gene amplicons of both internal transcribed spacers and the 5.8 S of the pepper Phytophthora and P. nicotianae were digested with 8 endonucleases. The patterns generated, with the 2 enzymes that cut, were identical for both taxa. This molecular analysis corroborated the morphological and biological characteristics and suggests strongly that the isolates of Phytophthora from pepper belong to the species P. nicotianae. Inoculation of pepper, tomato, eggplant and tobacco plants with the isolates of P. nicotianae from pepper showed they were highly pathogenic on pepper but not on tobacco, while their pathogenicity was weak on tomato and eggplant and was associated with atypical symptoms not observed in the field. These pathogenicity tests suggest that pepper isolates of P. nicotianae are particularly adapted to their host and may thus constitute a forma specialis of P. nicotianae.     

火鸡疫病:对兽医的新挑战

现代火鸡业,定位于产量高、品质好、价格低,且要与消费者对禽肉产品的需求相一致,预防火鸡疫病以保障人类健康是现代火鸡业的导向性目标.……     

Water spray as a potential thinning agent for date palm flowers (Phoenix dactylifera L.) c.v. ‘Lulu’

Flower or fruit thinning is a critical cultural practice in the date palm production chain that affects fruit development, quality and yield and regulates tree yearly bearing. Development of a save and economic thinning agent for date palm is critically required especially under harsh conditions. During 2004 and 2005 seasons, water spray for 3 min was applied at different times following mechanical pollination on ‘Lulu’ date palm cultivar growing under Al-Ain oasis conditions. The results showed that water spray following pollination generally decreased fruit set percentage to different extent depending of the time of application. In this respect, the most effective treatment was water spray after 4 h following pollination. This treatment significantly decreased fruit set percentage (48%) compared to the control (79%), as the mean of both seasons. However, the other treatments including water spray 1 h before pollination also decreased fruit set percentage but to a lesser extent. Fruit quality characteristics especially fruit and flesh weight, length and diameter were significantly increased by water spray treatments during the 2004 season, in contrast to the 2005 season. Thinning with water significantly decreased both bunch weight and total yield per tree at the Tamr stage especially when applied after 4 h following pollination. To the best of our knowledge, this is a pioneer study investigating the role of water as a potential thinning agent for date palm flowers. The use of water spray as a save and economic thinner needs, however, more research work to justify the time following pollination and duration of application which may vary upon cultivar, method of pollination and district.     

Fruit and seed diversity of domesticated carob (Ceratonia siliqua L.) in Morocco

The carob tree (Ceratonia siliqua L.) is an important economic resource for Morocco's rural populations. This species is used in reforestation actions and its cultivation in modern orchards is being undertaken to valorize marginal lands and substitute for drought sensitive species. However, little data is available on its intra-specific variability and its adaptability. Morphological characters of pods and seeds from 13 ecoregions of private-domesticated carob were used to assess phenotypic variation of this species. These stands extend from south-west to north-east and cover a wide range of Morocco's ecoregions. Pods length, width, thickness, seeds number, pulp weight, seeds yield and seeds length, width, thickness and weight were measured for 390 trees (30 trees per ecoregion). Statistically significant differences were found between ecoregions for all characters which were examined, what indicates a high phenotypic diversity. Principal component analysis (PCA) and hierarchical cluster of all ecoregions lead to identify two major and opposite groups (the northern ecoregions; and the central and south-western ecoregions). Ecoregions of the north of Morocco exhibited the largest and the thickest pods with the highest pulp weight while other ecoregions have relatively short pods but largest proportion of seed yield. Similarly, the northern ecoregions are characterized by the heaviest seeds. A correlation matrix between morphological characters, geographic parameters and precipitation exhibits a positive and a negative correlation of pods thickness and pulp weight with the latitude and the altitude, respectively. Seed yield and weight are negatively and positively correlated to pod width, pod thickness and pulp weight, respectively. In addition, seed weight is positively correlated with the latitude. The geographic pattern of the carob tree and its variability are discussed in this paper.     

Pathogenicity of peste des petits ruminants virus isolated from Egyptian goats in Egypt

2 Egyptian goats and Boscat rabbits were experimentally inoculated with peste des petits ruminants (PPR) local Egyptian strain (PPR, Egypt 87). The inoculated animals contracted the disease with minor clinical manifestations, accompanied by rise of neutralizing antibodies to PPR virus. Virus was isolated from ocular and nasal secretions, buffy coat, spleen, and liver. No contact infection was observed between inoculated and healthy goats.