Detection of phytoplasma infections in declining Populus nigra ‘Italica’ trees and molecular differentiation of the aster yellows phytoplasmas identified in various Populus species

A disease of Populus nigra‘Italica’ associated with foliar yellowing, sparse foliage, stunting, dieback, and decline was observed in south-western Germany; a witches’ broom disease of Populus alba that is known in other countries was also detected in Hungary and Germany. The aetiology of the diseases was studied by fluorescence microscopy and polymerase chain reaction (PCR) amplification. Using fluorescence microscopy, phytoplasmas could be detected only in P. alba. However, most diseased trees of P. nigra‘Italica’ tested phytoplasma-positive by PCR. In some of the trees the phytoplasma numbers were so low that nested PCR was required to detect the infection. Very low phytoplasma numbers were also observed in diseased Populus tremula. The identity of phytoplasmas from P. nigra‘Italica’ sampled in Germany and France, P. alba and also P. tremula was examined by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA. In all poplars, phytoplasmas of the aster yellows group were detected. However, three different RFLP groups were identified that consisted of (1) French strains from P. nigra‘Italica’, (2) German strains from P. nigra‘Italica’ and (3) strains from P. alba and P. tremula. The profile observed in the last group was probably the result of sequence heterogeneity in the two 16S RNA genes.     

Mycosphaerella species associated with leaf disease of Eucalyptus globulus in Ethiopia

Eucalyptus spp. are among the most widely planted exotic trees in Ethiopia. Several damaging leaf pathogens are known from Eucalyptus spp. worldwide. Of these, Mycosphaerella spp. are among the most important, causing the disease known as Mycosphaerella leaf disease (MLD). Characteristic symptoms of MLD include leaf spot, premature defoliation, shoot and twig dieback. Recent disease surveys conducted in Ethiopian Eucalyptus plantations have revealed disease symptoms similar to those caused by Mycosphaerella spp. These symptoms were restricted to E. globulus trees growing in several localities in south, south western and western Ethiopia. The aim of this study was to identify the fungi associated with this disease. This was achieved by examining ascospore germination patterns, anamorph associations and sequence data from the Internal Transcribed Spacer (ITS) region of the rRNA operon, for representative isolates. Several different ascospore germination patterns were observed, suggesting that more than one species of Mycosphaerella is responsible for MLD on E. globulus in Ethiopia. Analysis of sequence data showed that three Mycosphaerella spp., M. marksii, M. nubilosa and M. parva were present. This is the first report of these three species from Ethiopia and represents a valuable basis on which to build further studies in the region.     

Sweating sickness: relative curative effect of hyperimmune serum and a precipitated immunoglobulin suspension and immunoblot identification of proposed immunodominant tick salivary gland proteins

Although of low morbidity, sweating sickness is readily induced in calves by infestation with positive Hyalomma truncatum adult ticks. This epitheliotrophic disease has no specific cure except by the administration of hyperimmune serum obtained from animals which have recovered and are subsequently immune to the disease. Treatment with hyperimmune serum, however, has associated problems of donor availability, possible serum contamination and i.v. administration of a relatively large volume. This paper compares the treatment and cure of sweating sickness using unrefined hyperimmune serum and that of an experimental suspension. The latter proved relatively inefficient probably due to a low concentration of effective immunoglobulins. Immunoblot analyses of the sera of affected animals, using tick salivary glands as antigen during the course of the trial revealed 4 tick salivary gland proteins with molecular masses of between 27-33 kDa. These are proposed as being associated with sweating sickness immunodominance.     

Electrophoretic and immunological analyses of almond (Prunusdulcis l.) genotypes and hybrids

Aqueous extracts from sixty almond samples representing various genotypes and interspecies hybrids of almond, including almond-peach, were analyzed for protein and peptide content using electrophoresis, Western immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Nondenaturing nondissociating polyacrylamide gel electrophoresis (NDND-PAGE) of the aqueous extracts indicated that a single major storage protein (almond major protein -- AMP or amandin) dominated the total soluble protein composition. Denaturing SDS--PAGE analyses of the aqueous extracts revealed that the AMP was mainly composed of two sets of polypeptides with estimated molecular masses in the ranges of 38--41 kDa and 20--22 kDa, regardless of the source; however, distinct variations in the intensity and electrophoretic mobility of some bands were noted between samples. In addition to AMP, several minor polypeptides were also present in all the genotypes, and variations were seen in these as well. Regardless of the genotype, AMP was recognized in Western blots by rabbit polyclonal anti-AMP antibodies, mouse monoclonal anti-AMP antibodies (mAbs), and serum IgE from patients displaying strong serum anti-almond IgE reactivity. As with protein staining results, antibody reactivity also revealed common patterns but displayed some variation between samples. An anti-AMP inhibition ELISA was used to quantify and compare aqueous extracts for various samples. All samples (n = 60) reacted in this assay with a mean +/- standard deviation (sigma n) = 0.82 +/- 0.18 when compared to reference aqueous extract from Nonpareil designated as 1.0.     

Mechanisms of evolution in Rickettsia conorii and R. prowazekii

Rickettsia conorii is an obligate intracellular bacterium that causes Mediterranean spotted fever in humans. We determined the 1,268,755-nucleotide complete genome sequence of R. conorii, containing 1374 open reading frames. This genome exhibits 804 of the 834 genes of the previously determined R. prowazekii genome plus 552 supplementary open reading frames and a 10-fold increase in the number of repetitive elements. Despite these differences, the two genomes exhibit a nearly perfect colinearity that allowed the clear identification of different stages of gene alterations with gene remnants and 37 genes split in 105 fragments, of which 59 are transcribed. A 38-kilobase sequence inversion was dated shortly after the divergence of the genus.     

In vitro digestibility of α-casozepine, a benzodiazepine-like peptide from bovine casein, and biological activity of its main proteolytic fragment

α-Casozepine is a peptide, corresponding to the sequence 91-100 of the bovine α(s1)-casein, displaying anxiolytic activity in the rat. The α(s1)-casein tryptic hydrolysate containing this peptide decreases stress effects after oral administration in various species including man. Therefore, the stability of this peptide toward gastric and pancreatic proteases has been assessed by using pepsin, chymotrypsin/trypsin, Corolase PP, pepsin followed by chymotrypsin/trypsin or pepsin followed by Corolase PP. α-Casozepine was slowly degraded by chymotrypsin, much more sensitive to pepsin and Corolase PP but not completely destroyed after 4 h kinetics. The bonds in the region 91 to 95 of the α-casozepine were totally resistant to hydrolysis by all studied proteases. Surprisingly, a fragment, corresponding to the sequence 91-97 and found in all the hydrolysis media in significant amount, possessed an anxiolytic activity in three behavioral tests measuring this parameter. This peptide could participate in the in vivo activity of α-casozepine.     

The pathogenic potential of endophytic Botryosphaeriaceous fungi on Terminalia species in Cameroon

In Cameroon, native Terminalia spp. represent an important component of the forestry industry, but limited information is available regarding the fungal pathogens that affect them. The Botryosphaeriaceae are endophytic fungi and latent pathogens that can result in wood stain, cankers, die‐back and death of trees, particularly when trees are under stress. The aim of this study was, therefore, to identify and characterize the Botryosphaeriaceae occurring as endophytes of Terminalia spp. in Cameroon, as part of a larger project to identify potential pathogens of these trees in the country. Samples were collected from three Terminalia spp. in the Central, Southern and Eastern Regions and the resultant Botryosphaeriaceae were identified using morphology and DNA sequence comparisons for the ITS and tef 1‐α gene regions. Furthermore, inoculation trials were conducted to consider the relative pathogenicity of the isolates collected. The majority of isolates (88%) represented species of Lasiodiplodia, including L. pseudotheobromae, L. theobromae and L. parva. The remaining isolates were identified as Endomelanconiopsis endophytica. Pathogenicity trials on young T. mantaly and T. catappa trees revealed that L. pseudotheobromae was the most pathogenic species followed by L. theobromae.     

Characterization of Botryosphaeriaceae from plantation‐grown Eucalyptus species in South China

The Botryosphaeriaceae is a species‐rich family that includes pathogens of a wide variety of trees, including Eucalyptus species. Symptoms typical of infection by the Botryosphaeriaceae have recently been observed in Eucalyptus plantations in South China. The aim of this study was to identify the Botryosphaeriaceae associated with these symptoms. Isolates were collected from branch cankers and senescent twigs of different Eucalyptus spp. All isolates resembling Botryosphaeriaceae were separated into groups based on conidial morphology. Initial identifications were made using PCR‐RFLP fingerprinting, by digesting the ITS region of the rDNA operon with the restriction enzymes CfoI and KspI. Furthermore, to distinguish isolates in the Neofusicoccum parvum/N. ribis complex, a locus (BotF15) previously shown to define these species, was amplified and restricted with CfoI. Selected isolates were then identified using comparisons of DNA sequence data for the ITS rDNA and translation elongation factor 1‐alpha (TEF‐1α) gene regions. Based on anamorph morphology and DNA sequence comparisons, five species were identified: Lasiodiplodia pseudotheobromae, L. theobromae, Neofusicoccum parvum, N. ribis sensu lato and one undescribed taxon, for which the name Fusicoccum fabicercianum sp. nov. is provided. Isolates of all species gave rise to lesions on the stems of an E. grandis clone in a glasshouse inoculation trial and on the stems of five Eucalyptus genotypes inoculated in the field, where L. pseudotheobromae and L. theobromae were most pathogenic. The five Eucalyptus genotypes differed in their susceptibility to the Botryosphaeriaceae species suggesting that breeding and selection offers opportunity for disease avoidance in the future.     

Detection and stability of the major almond allergen in foods

Almond major protein (AMP or amandin), the primary storage protein in almonds, is the major allergen recognized by almond-allergic patients. A rabbit antibody-based inhibition ELISA assay for detecting and quantifying AMP in commercial foods has been developed, and this assay, in conjunction with Western blotting analyses, has been applied to the investigation of the antigenic stability of AMP to harsh food-processing conditions. The ELISA assay detects purified AMP at levels as low as 87 +/-16 ng/mL and can detect almond at between 5 and 37 ppm in the tested foods. The assay was used to quantify AMP in aqueous extracts of various foods that were defatted and spiked with known amounts of purified AMP or almond flour. In addition, AMP was quantified in commercially prepared and processed almond-containing foods. Neither blanching, roasting, nor autoclaving of almonds markedly decreased the detectability of AMP in subsequent aqueous extracts of almonds. Western blots using both rabbit antisera and sera from human almond-allergic patients confirm a general stability of the various peptides that comprise this complex molecule and show that the rabbit antibody-based assay recognizes substantially the same set of peptides as does the IgE in sera from almond-allergic patients.     

Biochemical characterization of amandin,the major storage protein in almond (Prunus dulcis L.)

The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.