Development of a versatile tool for the simultaneous differential detection of Pseudomonas savastanoi pathovars by End Point and Real-Time PCR

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas savastanoi </it>pv. <it>savastanoi </it>is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars <it>nerii </it>and <it>fraxini</it>, respectively. When artificially inoculated, pv. <it>savastanoi </it>causes disease also on ash, and pv. <it>nerii </it>attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including <it>P. savastanoi</it>, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these <it>P. savastanoi </it>pathovars, in multiplex and <it>in planta</it>.</p> <p>Results</p> <p>Specific PCR primers and probes for the pathovars <it>savastanoi</it>, <it>nerii </it>and <it>fraxini </it>of <it>P. savastanoi </it>were designed to be used in End Point and Real-Time PCR, both with SYBR^®Green or TaqMan^®chemistries. The specificity of all these assays was 100%, as assessed by testing forty-four <it>P. savastanoi </it>strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars <it>phaseolicola </it>and <it>glycinea </it>of <it>P. savastanoi </it>and bacterial epiphytes from <it>P. savastanoi </it>host plants were also assayed, and all of them tested always negative. The analytical detection limits were about 5 - 0.5 pg of pure genomic DNA and about 10²genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants.</p> <p>Conclusions</p> <p>Here for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of <it>P. savastanoi </it>pv. <it>savastanoi</it>, pv. <it>nerii </it>and pv. <it>fraxini</it>. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and <it>in vivo</it>. Compared with the other methods already available for <it>P. savastanoi</it>, the identification procedures here reported provide a versatile tool both for epidemiological and ecological studies on these pathovars, and for diagnostic procedures monitoring the asymptomatic presence of <it>P. savastanoi </it>on olive and oleander propagation materials.</p

Characterization of microalga Chlorella as a fuel and its thermogravimetric behavior

.Microalgae are photosynthetic microorganisms living in marine or freshwater environment. In this study, samples of Chlorella spp. and Nannochloropsis from two different origins were analysed to settle a preliminary characterization of these microorganisms as intermediate energy carriers and their properties compared to a conventional lignocellulosic feedstock (pine chips). Both microalgae samples were characterized in terms of elemental composition (CHONS and P) and thermogravimetric behavior. This was investigated through non-isothermal thermogravimetric analysis in nitrogen atmosphere at heating rate of 15 °C min−1 and temperature up to 800 °C. Solid residues produced at 300 °C and 800 °C from TGA were also analysed to determine the ultimate composition of chars. Activation energy, reaction order and pre-exponential factor were calculated for the single step conversion mechanism of 1 g of Chlorella spp. and compared to literature data on Chlorella protothecoides and Spirulina platensis. Calculated kinetic parameters, given as intervals of several determinations, resulted to be: pre-exponential factor (A) 1.47–1.62E6 min−1, activation energy (E) 7.13–7.92E4 J mol−1, reaction order (n) 1.69–2.41. 1.2 kg of Chlorella spp. was then processed in a newly designed batch pyrolysis pilot reactor, capable of converting up to 1.5 kg h−1 of material, and pyrolysis liquid collected, analysed and compared with a sample of fast pyrolysis from pine chips. This preliminary investigation aimed at carrying out a first characterization of algae oil and optimise the operational aspects of the reactor, tested with the first time with this unconventional feedstock. The algae pyrolysis oil exhibited superior properties as intermediate energy carrier compared to pyrolysis oil from fast pyrolysis of pine chips, in particular higher HHV and carbon content and lower oxygen and water content. These data can potentially be used in the design and modelling of thermochemical conversion processes of microalgae