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

Hydrolysable Tannins in Agriculture

Hydrolysable tannins, water-extracted from sweet chestnut (Castanea sativa Mill.) (CHT) and membrane concentrated, have several effects as antioxidant, antimicrobial, and metal complexing agents. Some patents described their use as nitrogen release modulators and iron complexing agent to fight plant chlorosis and to control legume seed-borne disease and nitrosamine and mycotoxin during plant and food processing. Biostimulating activity of raw CHT, placed near seed or transplant seedlings, was assessed on early plant growth (starter effect) and found related to earlier production of a larger plant fine root mass, with greater P early uptake. Increased resistance to nematodes, with CHT applications on tobacco, was investigated. Recent process stream fractioning permitted to identify some CHT fractions with antimicrobial and antioxidant effects which were tested for their potential in promoting selected aspects of plant yield, quality, and protection and maintaining and improving feed and food quality during processing. EU Life+2013 Evergreen found a method of application of a CHT fraction to protect tobacco and carrot plants in nematode-infested fields. A protective effect of CHT on some bacterial diseases of olive tree and kiwi was disclosed. Environmental and soil toxicities were also investigated finding very low impacts and the possibility to reduce Cu use in agriculture