Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction

ABSTRACT Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were beta-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.     

Comparison of ITS profiling,REP- and ERIC-PCR of Salmonella Enteritidis isolates from Poland

Thirty-one Salmonella Enteritidis strains isolated from chickens, broilers and hens were analysed by genotypic typing including REP-PCR. ERIC-PCR and ITS profiling (PCR-ribotyping). Analysis of DNA banding patterns generated by REP-PCR revealed the presence of 22 different genotypes, which were grouped by dendrogram analysis into three distinct lineages (maximum similarity approx. 50%). Each isolate of S. Enteritidis analysed by ERIC-PCR generated an individual DNA pattern. Again, these isolates could be divided into three distinct genomic groups (maximum similarity approx. 60%) by their ERIC-PCR fingerprints. REP- and ERIC-PCR were found to be more discriminatory for typing of S. Enteritidis than ITS profiling. Amplification of the 16S-23S rDNA spacer region gave nine different profiles of DNA, subdivided into two closely related groups by dendrogram analysis. In summary, data obtained by genotyping methods for S. Enteritidis isolates from regions located in the south-west and the central parts of Poland revealed an enormous heterogeneity among analysed samples, and proved that REP- and ERIC-PCR are highly discriminatory techniques, which can be used, in addition to conventional methods, in epidemiological studies of S. Enteritidis infections.     

Identification of mechanisms of natural resistance to African trypanosomiasis in cattle

Natural resistance to African trypanosomiasis in certain Bos taurus cattle in West Africa, called trypanotolerance, may hold solutions for control of this economically crippling disease. Comparison of immune responses between trypanotolerant and trypanosusceptible cattle have shown some differences in antibody response, complement level and cytokine expression, but it is not known whether these differences are the cause of resistance.Two experiments were carried out to assess the contribution of the immune and haemopoietic systems to trypanotolerance. The production of haemopoietic chimaeras from trypanotolerant and susceptible twin calves and comparison of their responses after infection with singleton calves, allowed an assessment of the role of the haemopoietic system in trypanotolerance. An in vivo depletion of CD4 cells in the two breeds allowed an appraisal of the role of T and B lymphocytes in trypanotolerance. The results of the two experiments suggest that natural resistance comprises at least two mechanisms, an innate mechanism that controls parasite growth, and another, involving the haemopoietic system, that is able to limit anaemia. This supports the hypothesis that innate mechanisms in trypanotolerant cattle are more efficient in controlling disease, making them less reliant on antibody responses.     

Risks to the aquatic ecosystem from the application of Metarhizium anisopliae for locust control in Australia

Laboratory tests of Metarhizium anisopliae var acridum Driver & Milner, at a dose of 1.3 x 10(6) conidia ml-1, had no adverse effects on nymphs of mayfly, Ulmerophlebia sp or 8-week-old fry of the rainbow fish, Melanotaenia duboulayi Castelnau. This dose was toxic to the cladoceran, Ceriodaphnia dubia Richard, causing 100% mortality in 48 h. When this test was repeated at doses of up to 6.7 x 10(3) conidia ml-1, there was only 5% mortality after 192 h. Spraying of artificial water sources with a very high dose of the fungus as an aqueous spray resulted in 80-130 conidia ml-1 at 15 cm depth in the first 24 h after spraying. The conidia rapidly settled out and were absent from the top 15 cm layer of water after about 50 h. A similar experiment using the oil formulation as used in field control resulted in a 2- to 20-fold lower level of conidia in the water. Finally, sampling actual water sources in spray areas revealed a very low level of contamination of the water, with a maximum mean level of 29 conidia ml-1 in the first 24 h after treatment. Thus the level of conidia likely to enter water during control campaigns is a small fraction of that required to kill cladocerans, the only sensitive non-target organism tested. It is concluded that the biopesticide is very unlikely to pose any hazard to aquatic organisms.     

Changes in abundance and infectivity of powdery mildew conidia from cucumber plants treated systemically with lithium chloride

Systemic treatment of cucumber plants with lithium chloride reduced the numbers of conidia produced by colonies of powdery mildew, Sphaerotheca fuliginea , growing on leaves, and lowered the infectivity of conidia produced from those leaves when they were applied to leaves of untreated plants. Production of conidiophores was lower in both lithium-treated and calcium-deprived plants, and lithium slightly decreased the calcium content of leaves. When the lithium-containing growth medium was supplemented with phosphate, conidiophore production was still markedly reduced, although leaves had normal levels of calcium. Fungal development was not correlated with either the calcium or phosphorus content of leaves. It is concluded that, although severe calcium deficiency can inhibit fungal development, the inhibitory effects of lithium are not mediated through alterations in calcium or phosphorus uptake by host tissues.     

Plant-parasitic nematodes of Croatia1

Investigations on the distribution of nematodes were carried out mostly in the northern and eastern parts of Croatia up to 1991. Sampling was performed in the following crops: maize, wheat, barley, sugarbeet, soybean, sunflower, rape, tobacco, potato, alfalfa, blackberry, onion, carrot, cucumber, lettuce, tomato, capsicum, oak, chrysanthemum, tulip, rose, grapevine, peach, apple, plum, pear, cherry, poplar, willow, clover, nettle, grass, meadow, woodland, many species of weeds, various glasshouse crops and vegetation in a nature reserve. 63 genera of nematodes with 81 species were detected. The most numerous representatives of plant parasitic nematodes were: Anguina, Aphelenchoides, Aphelenchus, Criconemoides, Ditylenchus, Gracilacus, Helicotylenchus, Hemicycliophora, Heterodera, Longidorus, Macroposthonia, Meloidogyne, Paratylenchus, Pratylenchus, Rotylenchulus, Rotylenchus, Trichodorus, Tylenchorhynchus, Tylenchus and Xiphinema.     

Nematological problems,production systems and Mediterranean environments1

The characteristic phytonematological problems in areas with Mediterranean climate are analysed. The problems are, in order of economical importance, root-knot nematodes, cyst nematodes, virus vectors, endoparasitic nematodes, stem nematodes, citrus nematode and ectoparasitic nematodes. The interest in getting alternative control techniques supported by production systems is stated. Nematodes were already differentiated in the Pliocene, at the end of the Tertiary, the period in which the Mediterranean climate appeared. Two structural elements are distinguished in the nematofauna and are defined as a characteristic temperate environment group and a tropical group. These two groups are found in Mediterranean environments according to environmental characteristics conditioned by geomorphology and, above all, since the Neolithic, by agrarian systems through cultural techniques. The importance of agrarian production systems, as an alternative control for plant parasitic nematodes in the Mediterranean region, is pointed out. Control alternatives are considered that are based on the capability of such systems to keep the environmental and spatial heterogeneity, that allows the nematode populations regulation and spatio-temporal isolation, avoiding, in this way, the epidemic development and dispersion of pathogenic species.