Biological control of green mould on <Emphasis Type="Italic">Agaricus bisporus</Emphasis> by a native <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain from mushroom compost

Fifty bacterial isolates obtained from compost were tested in vitro against the causal agents of green mould in Agaricus bisporus. Isolate B-38 which induced 48.08% in vitro growth inhibition of T. harzianum T54 and 52.25% of T. aggressivum f. europaeum T77 was identified as Bacillus subtilis, based on 16S rDNA sequence and used in mushroom growing room experiments. B. subtilis B-38 did not decrease mycelial growth rate of Agaricus bisporus A15 in mushroom compost in glass tubes. After applying prochloraz-manganese, B. subtilis B-38 and B. subtilis QST 713, no significant differences in BE values among treatments were found concerning both total yield and the weight of healthy mushrooms. Statistical analyses showed that only inoculation significantly influenced the healthy mushroom yield. In plots inoculated with T. harzianum T54 disease incidence was significantly lower after treatments with prochloraz-manganese (11.81%), B. subtilis QST 713 (12.26%) and B. subtilis B-38 (14.19%) compared to the control (28.16%), as well as in plots inoculated with T. aggressivum f. europaeum T77 11.88%, 12.2% and 15.03%, respectively, in comparison with the control (23.47%). Statistically significant differences were not found among the efficacy values of tested bio-fungicides based on B. subtilis and the commercial fungicide prochloraz-manganese suggesting the use of B. subtilis B-38 and B. subtilis QST 713 as good alternatives to chemical fungicides.     

Viruses affecting tomato crops in Serbia

In a two-year survey (2011–2012), 3220 samples were collected and analyzed in order to determine the presence and distribution of viruses in tomato crops at 56 localities of 18 districts in Serbia. Out of 12 viruses tested, Cucumber mosaic virus (CMV), Potato virus Y (PVY), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) were detected in 42.1, 40, 11, 8.6, 2.3 and 1.3% of the total tested samples, respectively. The results revealed that CMV was prevalent in 2011 and PVY in 2012. CMV and PVY, apart from being predominant, were also the most widespread viruses. In general, single infections were the most frequent type of infection. Additionally, the most common mixed infections were double infections and the most prevalent combination was CMV and PVY. In 2011, the incidence of diseases and the percentage of all infection types were significantly higher than in 2012. Furthermore, in 2011, regardless of total single infections being prevalent compared to mixed infections, two prevailing viruses were commonly detected in mixed infections. The additional molecular testing of ELISA-negative samples using virus specific primers did not reveal the presence of Pepino mosaic virus (PepMV), Tomato yellow leaf curl virus (TYLC), Tomato infections chlorosis virus (TICV) and Tomato chlorosis virus (ToCV).     

Biochemical and physiological responses to long‐term Citrus tristeza virus infection in Mexican lime plants

Reactions that occur when a plant is subjected to Citrus tristeza virus (CTV) infection often result in triggering of numerous defence mechanisms to fight the infection. The reactions vary according to virus strain, host genotype, time of exposure to the infection and environmental conditions. To date, no study has examined in detail the consequences of 10‐year exposure to CTV infection on the biochemical and physiological status of susceptible Mexican lime plants (Citrus aurantifolia). To understand the reaction of such plants, changes in nutrient status, total proteins, enzyme activity involved in scavenging of reactive oxygen species, photosynthetic and transpiration rates, chlorophyll content, membrane permeability and water content were analysed in plants infected with different CTV isolates and in healthy plants. The activity of superoxide dismutase and polyphenol oxidase significantly decreased in the infected leaves, and membrane permeability was lower in the infected plants. Macro‐ and micronutrient elements were significantly changed: concentrations of leaf nitrogen, zinc, magnesium and iron were elevated but potassium concentration depressed in comparison to noninfected control leaves. Levels of other analysed nutrient elements, enzymes, photosynthesis and stomatal conductance, chlorophyll content and relative water content were unchanged. Clear physiological changes were found among infected and noninfected control plants but none between plants infected with different CTV isolates. The data suggest that some of the defence mechanisms investigated here were suppressed due to the continuous and long‐term pressure of biotic stress.     

First description of postweaning multisystemic wasting syndrome (PMWS) in wild boar (Sus scrofa) in Croatia and phylogenetic analysis of partial PCV2 sequences

This report describes the first case of postweaning multisystemic wasting syndrome (PMWS) in wild boar in Croatia. During the winter season of 2004, eight wild young piglets (of approximately 2 to 5 months of age) were found dead in a fenced hunting area. Polymerase chain reaction (PCR) was carried out on mesenteric lymph nodes and all animals yielded positive results. In one of these animals diagnosis of PMWS was established based on the three key diagnostic criteria including the clinical manifestation, moderate lymphoid lesions consisting of lymphocyte depletion and granulomatous inflammation, and detection of the presence of PCV2 genome within the lymphoid lesions by in situ hybridisation (ISH). Three additional wild piglets had also mild PMWS-like lesions and a low amount of PCV2 was also found. No PMWS-like lesions or PCV2 genome were detected in the rest of the wild piglets studied. Three PCR-positive isolates were partially sequenced, which confirmed the diagnosis of PCV2 and demonstrated that the three sequences were genetically identical. The phylogenetic analysis of a representative PCV2 isolate indicated that its sequence (DQ875444) is grouped in a separate branch with Hungarian isolate (AY256460) and differs from any of the annotated sequences.     

Estimation of Eucalyptus productivity using efficient artificial neural network

Prediction of plantation productivity from environmental data is challenged by the complex relationships defining the growth and yield processes. Artificial neural network (ANN) can be used to model complex nonlinear interrelations but are challenged by the choice of the training algorithm and the structure and size of the network. The objective of the present study is to find an efficient ANN to estimate the eucalyptus productivity from biotic, abiotic and silvicultural data. We defined the efficiency of an ANN as the processing time to supply an accurate solution. We increase the efficiency of the network in two steps: one outside the ANN, through principal component analysis (PCA), and one inside the ANN, through dedicated pruning methods. To test different network configurations, we used data from 507 Eucalyptus plantations, 3 to 7 years old, located in Minas Gerais, Brazil. An ANN configuration is a combination of the number of neurons in the hidden layers, training algorithm and pruning method. Each ANN was trained five times to assess the impact of the initial weights on the results, which led to a factorial experiment with 9000 combinations. The most accurate result was supplied in 38.81 s by an ANN using the data trimmed with PCA, trained with the Scaled Conjugate Gradient algorithm with four neurons and Magnitude-Based Pruning method. However, an accuracy loss of less than 1% (i.e., the second most accurate result) was obtained in 1.7 s using the same ANN configuration, but no pruning. Our results indicate that an efficient prediction of Eucalyptus productivity does not use all the data or the most complex training algorithms. The proposed efficient ANN supplies the most precise results compared with the existing models. Furthermore, the efficient ANN produces more accurate results from environmental variables than some models that contain dimensional variables. Our study suggests that modeling the yield of Eucalyptus plantations should be executed using a large set of environmental variables, as no clear edaphic or climatic variables supplied accurate results.

     

Phage displayed peptides and anti-idiotype antibodies recognised by a monoclonal antibody directed against a diagnostic antigen of Mycoplasma capricolum subsp. capripneumoniae

A monoclonal antibody (Mab 4.52) raised against Mycoplasma capricolum subsp. capripneumoniae (Mccp) cell lysate was used as a template to obtain substitute antigens recognised by its paratope. Two approaches were investigated: a 17-mer random peptide library displayed on the surface of a filamentous phage was screened by panning on the immobilised Mab 4.52 and anti-idiotype antibodies were generated by immunising a chicken with the F(ab')(2) fragments of the antibody. Analysis of the peptide sequences displayed by the isolated phages identified two peptides. Both contained two cysteine residues and had identical or similar amino acids in positions 5 (P), 8 (I/L) and 13 (L). The fusion phages were also recognised by Mab 4.52 in enzyme-linked immunosorbent assay (ELISA) and binding was shown by surface plasmon resonance. One of the peptides was a markedly better inhibitor (67%) of the binding of Mab 4.52 to its original antigen than the other (20%) at 1mg/ml. After absorption, to remove isotypic and allotypic reactivities, the anti-idiotype IgY was specifically recognised by Mab 4.52 in ELISA and was able to inhibit its binding to the original antigen, whereas anti-idiotype antibodies raised against a bluetongue virus-specific antibody had no effect. In spite of unequivocal binding of the anti-idiotype antibodies and the fusion phages to the paratope of Mab 4.52, goat antisera appeared not to react with either of the surrogate antigens. In contrast, the test sera bound to the original antigen suggesting that Mab 4.52 does not recognise exactly the same antigenic site as antibodies in the goat antisera.     

Trichinella pseudospiralis in pig from Croatia

Trichinella spiralis and Trichinella britovi are species that are frequently found in domestic pigs and various sylvatic animals in Croatia. During routine trichinoscopy, non-encapsulated larvae were detected in the muscle tissue of a domestic pig. Artificial digestion revealed a larvae burden of 602 muscle larvae per gram of tissue. Tissue section analysis confirmed the presence of non-encapsulated larvae. Multiplex PCR identified the larvae as T. pseudospiralis. This observation is consistent with the reports of a local veterinary inspector who described the presence of non-encapsulated Trichinella in four individual cases over the last 2 years. This is the first report of T. pseudospiralis in Croatia and one of very few cases of T. pseudospiralis infection described in domestic pigs. The detection of non-encapsulated larvae stresses the need for implementation of artificial digestion instead of trichinoscopy for the detection and identification of Trichinella infections.     

Nutritive quality and energy yield of high oil,opaque-2 and waxy maize hybrids compared to normal maize hybrids

Grain yield, chemical composition, protein quality and digestible energy were measured for 18 maize varieties (opaque-2, high oil, waxy hybrids, normal hybrids) cultivated in Yugoslavia. Protein quality assessment was based on amino acid composition, and true protein digestibility, biological value, net protein utilization and utilizable protein as determined in nitrogen balance experiments with rats. The protein concentration in dry matter varied from 8.63–18.81% while the fat concentration varied from 4.45–16.13%. There were only minor differences in ash and crude fibre, while starch and sugar varied inversely to fat and protein concentration. Lysine levels were approximately 30% higher for the opaque-2 varieties compared with normal maize, while leucine levels were about 30% lower. Yields were extremely high for all varieties with the highest value being 10.3 tonnes/ha. Due to the high yields, protein production/ha was high in range of 822–977 kg. This resulted in high yields of amino acids/ha. Protein utilization was very high in the opaque-2 varieties, which had the highest lysine contents. The biological values were close to 80 in these varieties, whereas they were only 60–65 in the normal maize varieties.     

Biological properties of a naturally attenuated infectious bursal disease virus isolated from a backyard chicken flock

The biological properties of an infectious bursal disease (IBD) virus isolated from bursas collected during an outbreak in a village chicken flock in Macedonia are described. The mortality rate was 50%. Two viruses coexisted in the bursas of infected chickens (IBDVwt and IBDVtc). The virus termed IBDVtc grows on chicken embryo fibroblast (CEF) cells from the first passage. Specific pathogen free chickens inoculated with IBDVtc at passage level 4 did not develop any clinical signs of disease. Some discrete bleeding on the leg muscles was seen and the bursa of Fabricius revealed pathological lesions similar to those caused by classical strains. However, the bursa recovered quickly (bursa lesion score 2) by 14 days post infection (PI). We also found evidence of bursal repopulation by means of perinuclear antigen staining. Strong CD3 influx was evident at 4 days PI, and at 33 days PI the CD3+ cell finding was comparable to the control. The mean antibody titre was 9.2 log 2 at 14 days PI. The amino acid composition of VP2 in IBDVwt (222 Ala, 242 Ile, 253 Gln, 256 Ile, 279 Asp, 284 Ala, 294 Ile and 299 Ser) is described. The same sequence was found in IBDVtc, except for two point mutations, at Gln253→His and Ala284→Thr. Such amino acid substitution is responsible for partial attenuation and the ability of the strain to replicate in cell culture. None of the commercial vaccine viruses has a similar arrangement of amino acids in the variable domain of IBDV. This strongly suggests that IBDVtc originates from a very virulent strain. To the best of our knowledge, this is the first report of a concomitant infection of chickens with highly pathogenic IBDV and its mutant counterpart.