Quantitative real-time PCR and high-resolution melting (HRM) analysis for strain-specific monitoring of fluorescent pseudomonads used as biocontrol agents against soil-borne pathogens of food crops

Fluorescent pseudomonads colonizing roots of crop plants and producing antifungal metabolites are regarded as a reliable alternative to chemical fungicides against soil-borne phytopathogens. Key factors in successful pathogen control are presence and activity at the appropriate concentration, time, and place of biocontrol agents. Thus, quantification methods to monitor population dynamics are pivotal to the development of reliable application protocols. Real-time PCR is nowadays the most widespread cultureindependent technique for the detection and enumeration of different target sequences. Here, its implementation with high resolution melting analysis as a powerful tool to accurately discriminate microbial inoculants is discussed

Transmissible tolerance to European stone fruit yellows (ESFY) in apricot: cross-protection or a plant mediated process?

European Stone Fruit Yellows (ESFY) is an emerging disease caused by 'Candidatus Phytoplasma prunorum' ('Ca. P. prunorum') affecting stone fruits, as apricots. Resistant apricot cultivars are unknown, but it has been demonstrated that individual plants can recover from the disease, behaving as completely tolerant to ESFY. The status of tolerance is transmissible by grafting to successive apricot individuals, but it is not clear whether recovery corresponds to a transmissible tolerance that depends on a plant-mediated reaction or if it is due to a cross-protection promoted by a transmissible protective agent i.e. hypovirulent strain/s of 'Ca. P. prunorum'. Results achieved after prolonged field experiments support the first hypothesis. Two groups of apricot plants derived from a common recovered mother (one 'Ca. P. prunorum'-free after heat-treatment and the second not heat-treated, i.e. harbouring potential protective strain/s of the phytoplasma), behaved similarly: no plants from either of the two groups developed stable ESFY symptoms after natural infections. Corresponding groups of plants, derived from symptomatic mothers, developed a high percentage of diseased plants after natural infection. No potential protective 'Ca. P. prunorum' hypovirulent strains were detected in the asymptomatic apricot plants. The summarized evidence supports a host-defence induction, likely of epigenetic feature. The present long-term study in apricot represents an uncommon empiric proof supporting the theory of inducible resistance to pathogens in plants

Genomic-assisted characterisation of <i>Pseudomonas</i> sp. strain Pf4, a potential biocontrol agent in hydroponics

<p>In an attempt to select potential biocontrol agents against <i>Pythium</i> spp. and <i>Rhizoctonia</i> spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus <i>Enterobacter</i>, whereas nine strains belonged to the genus <i>Pseudomonas</i>. In <i>in vitro</i> assays, one strain of <i>Pseudomonas</i> sp., Pf4, closely related to <i>Pseudomonas protegens</i> (formerly <i>Pseudomonas fluorescens</i>), showed noteworthy antagonistic activity against two strains of <i>Pythium aphanidermatum</i> and two strains of <i>Rhizoctonia solani</i> AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for <i>in vivo</i> treatments on lamb’s lettuce against <i>R. solani</i> root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵ CFU g⁻¹ of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that <i>Pseudomonas</i> sp. Pf4 has the potential to produce an arsenal of secondary metabolites (<i>plt</i>, <i>phl</i>, <i>ofa</i> and <i>fit-rzx</i> gene clusters) very similar to that of the well-known biocontrol <i>P. protegens</i> strain Pf-5.</p