Chronopathological aspects of disease incidence in rice (Oryza sativa L.)

Rice seedlings maintained under uncontrolled glasshouse conditions were inoculated with conidial suspensions of a fungal pathogen, Helminthosporium oryzae, at various times during the 24 h. Significant increase in the percent germination and germ tube length of conidia were observed in the rice samples inoculated at 02:00 and 06:00h. The 24 h temporal variation in leaf temperature was positively correlated with variation in stomatal movements. The results indicate a 24 h rhythm in the behavior of the fungal pathogen on the host in relation to the conditions of the growing environment. In all the inoculated seedlings, the appearance of a large number of brown leaf spots was confined to the light span. Among the plants inoculated, earlier initiation of brown leaf spot appearance, maximum number of leaf spots, and highest disease severity were observed when plants were inoculated at 02:00h. There was a positive correlation between disease severity of the host and in vivo values of percent germination of conidia and germ tube length of the pathogen in plants inoculated between 02:00 and 06:00h. The findings of this study implicate that light intensity and temperature could play a predominant role in controlling disease susceptibility rhythms in plants.     

Differential responses in vitro of rice cultivars to Italian lineages of the blast pathogen Pyricularia grisea (Cooke) Sacc. 1. Oxidative burst

Suspension cultured cells of six rice cultivars differing in their sensitivity to blast were treated with mycelial wall hydrolysates prepared from seven isolates belonging to different Pyricularia grisea lineages. Soon after elicitor addition, rice cells produced significant amounts of superoxide anion, which was rapidly converted into diffusible peroxide. Maximal effects were achieved at 50 mg L-1 elicitor. In all cases, a 7 to 13-fold increase in the basal rate of reactive oxygen species production was found. Neither differential effects among strains nor clear relationships between lineage and the resulting oxidative burst were evident. Interestingly, a good correlation was found between basal (and elicited) levels of peroxide generation and the overall tolerance of rice cultivars to the pathogen. About two days after elicitation, cell death occurred proportional to the amount of hydrogen peroxide released. Peroxide was required to trigger loss of cell viability, but the latter was not due to a direct toxic effect, suggesting the induction of programmed cell death. Results represent the first data aimed to develop in vitro tests for pathogenicity prediction of Italian blast lineages toward rice cultivars.     

Evidence on inhibition of Listeria monocytogenes by divercin V41 action

AIMS: The aim of this study was to investigate the role of divercin V41 in inhibition and prevention of Listeria monocytogenes. METHODS AND RESULTS: Carnobacterium divergens V41 deficient in bacteriocin production was isolated and characterized by enzyme-liked immunosorbent assay, multiplex polymerase chain reaction and bacteriocin diffusion test. Carnobacterium divergens V41 (divercin+) and Carnobacterium divergens V41C9 (divercin-) were grown in the presence of L. monocytogenes in smoked salmon model medium. Carnobacterium divergens V41, but not C. divergens V41C9, was able to inhibit growth of L. monocytogenes. The results indicate that inhibition of L. monocytogenes in the presence of C. divergens V41 is because of the production of divercin V41 and not to a nutritional advantage. CONCLUSIONS: Carnobacterium divergens V41 may be a promising agent in food safety. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates a potential use of a bacteriocin producing lactic acid bacteria in the area food protection.     

Comparison of sample collection methods for the PCR detection of oral anaerobic pathogens

AIMS: To provide evidence that DNA-PCR diagnostics of oral pathogens based on standard sample collection by paper point insertion from the depth of the periodontal pocket can be replaced by a novel non-invasive collection method based on swab technique from the gingiva. METHODS AND RESULTS: In this study we compared the results from two collection methods performed in 35 patients with chronic adult periodontitis. Statistical analysis showed a highly significant association of diagnostic results between both collection techniques. CONCLUSIONS: The Pocket-out method represents a reliable alternative to the standard collection technique for PCR diagnosis of oral pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to its simplicity and non-invasiveness, the Pocket-out collection could be performed in any physician office, or even by the patient himself. With respect to the putative association between periodontal disease and various systemic illnesses, this method could be integrated with various screening programs of oral pathogens.     

Endotoxic activity of lipopolysaccharides isolated from emergent potential cystic fibrosis pathogens

Improved antimicrobial therapies against the classical spectrum of pathogenic bacteria which colonise the lungs of cystic fibrosis (CF) patients has resulted in improved life expectancy and quality of life. Bacterial species that are resistant to a broad range of antibiotics including Stenotrophomonas maltophilia and Alcaligenes xylosoxidans have now emerged as potential new pathogens to fill the niche. At present, it is unclear from clinical data whether these microbes are commensal or pathogenic. In this study we have quantified the inflammatory potential of lipopolysaccharide (LPS) from eight species of Gram-negative organisms which have been cultured with increasing frequency from CF patients. Inflammatory responses induced by LPS from whole human blood and a human-derived monocyte cell line (THP-1) were assessed. Enzyme-linked immunosorbent assays were used to detect interleukin-6, interleukin-8, and tumour necrosis factor alpha (TNF). A bioassay was also used to assess TNF activity. With the exception of S. maltophilia, LPS extracted from all of the bacteria tested upregulated, by varying degrees, expression of each of the proinflammatory cytokines assayed. This study represents the first comprehensive report of the endotoxic potential of a new wave of microbes which are associated with CF.     

Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review

Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known toxin producers, including the foodborne pathogen, B. cereus. This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products. In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host. In dairy products, the occurrence of Bacillus spp. is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization. Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures. Evaluation of toxin gene presence and toxin expression in Bacillus spp. other than B. cereus has not been thoroughly investigated. However, the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.     

The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis

The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.     

Subcellular localization of β-1,3-glucanase in Puccinia recondita f.sp. tritici-infected wheat leaves

An antiserum raised against the purified 33-kDa β-1,3-glucanase of wheat (Triticum aestivum L.) was employed to investigate the ultrastructural localization of the enzyme in wheat leaves infected with Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. and Henn. using a post-embedding immunogold labelling technique. In both compatible and incompatible interactions, β-1,3-glucanase was detected in the host plasmalemma and in the domain of the host cell wall near the plasmalemma of the mesophyll cells, but higher concentrations of the enzyme were detected in infected resistant wheat leaves than in infected susceptible ones. β-1,3-Glucanase was also found in the secondary thickening of xylem vessels and in the walls of guard cells, epidermal cells and phloem elements, while no labelling was observed in host organelles, viz. vacuoles, mitochondria, endoplasmic reticulum, Golgi bodies, nuclei and chloroplasts. A low concentration of the enzyme was detected on the intercellular hyphal wall and in the hyphal cytoplasm. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β-1,3-glucanase was found in host cell wall appositions, in the extracellular matrix in the intercellular space, and in electron-dense structures of host origin which occurred in the incompatible interaction only. Received: 22 July 1997 / Accepted: 16 August 1997     

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene ( Rns ) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.