Enrico Biondi

Detection of potato brown rot and ring rot by electronic nose: From laboratory to real scale

A commercial electronic nose (e-nose) equipped with a metal oxide sensor array was trained to recognize volatile compounds emitted by potatoes experimentally infected with Ralstonia solanacearum or Clavibacter michiganensis subsp. sepedonicus, which are bacterial agents of potato brown and ring rot, respectively. Two sampling procedures for volatile compounds were tested on pooled tubers sealed in 0.5-1 L jars at room temperature (laboratory conditions): an enrichment unit containing different adsorbent materials (namely, Tenax(®) TA, Carbotrap, Tenax(®) GR, and Carboxen 569) directly coupled with the e-nose (active sampling) and a Radiello(™) cartridge (passive sampling) containing a generic Carbograph fiber. Tenax(®) TA resulted the most suitable adsorbent material for active sampling. Linear discriminant analysis (LDA) correctly classified 57.4 and 81.3% total samples as healthy or diseased, when using active and passive sampling, respectively. These results suggested the use of passive sampling to discriminate healthy from diseased tubers under intermediate and real scale conditions. 80 and 90% total samples were correctly classified by LDA under intermediate (100 tubers stored at 4°C in net bag passively sampled) and real scale conditions (tubers stored at 4°C in 1.25 t bags passively sampled). Principal component analysis (PCA) of sensorial analysis data under laboratory conditions highlighted a strict relationship between the disease severity and the responses of the e-nose sensors, whose sensitivity threshold was linked to the presence of at least one tuber per sample showing medium disease symptoms. At intermediate and real scale conditions, data distribution agreed with disease incidence (percentage of diseased tubers), owing to the low storage temperature and volatile compounds unconfinement conditions adopted.

Identification of volatile markers in potato brown rot and ring rot by combined gc-ms and ptr-ms techniques: Study on in vitro and in vivo samples

Ralstonia solanacearum (Rs) and Clavibacter michiganensis subsp. sepedonicus (Cms) are the bacterial causal agents of potato brown and ring rot, respectively, and are included in the A2 list of quarantine pathogens in Europe. Identification by GC-MS analysis of volatile organic compounds from Rs or Cms cultured on different nutrient media was performed. GC-MS and PTR-MS analysis were carried out also on unwounded potato tubers infected with the same pathogens. Infected tubers were produced by experimental inoculations of the plants. In in vitro experiments, Rs or Cms emitted volatile compounds, part of which were specific disease markers of potato (2-propanol and 3-methylbutanoic acid), mainly originating from bacterial metabolism (i.e., amino acid degradation, carbohydrate and fatty acid oxidation). In potato tubers, pathogen metabolism modified the volatile compound pattern emitted from healthy samples. Both bacteria seem to accelerate metabolic processes ongoing in potatoes and, in the case of Rs, disease markers (1-hepten-3-ol, 3,6-dimethyl-3-octanone, 3-ethyl-3-methylpentane, 1-chloroctane, and benzothiazole) were identified.

Potential Applications and Limitations of Electronic Nose Devices for Plant Disease Diagnosis

Electronic nose technology has recently been applied to the detection of several plant diseases and pests, with promising results. However, in spite of its numerous advantages, including operational simplicity, non-destructivity, and bulk sampling, drawbacks include a low sensitivity and specificity in comparison with microbiological and molecular methods. A critical review of the use of an electronic nose for plant disease diagnosis and pest detection is presented, describing the instrumental and procedural advances of sensorial analysis, for the improvement of discrimination between healthy and infected or infested plants. In conclusion, the use of electronic nose technology is suggested to assist, direct, and optimise traditionally adopted diagnostic techniques.

Characterization of volatile organic compounds emitted by kiwifruit plants infected with Pseudomonas syringae pv. actinidiae and their effects on host defences

Key messageSpecific VOC emissions from infected plants allow their recognition and elicit defence responses in neighboring plants, which are, however, insufficient to induce resistance.AbstractA wide range of volatile organic compounds (VOCs) is released during plant–pathogen interactions both by the pathogens and the hosts. Some of these VOCs are specific for the different diseases and are known to play a role in the pathogenicity or in plant defence responses. Besides, disease-specific VOCs may serve as markers for diagnostic protocols, which allow a non-destructive and rapid screening of bulk samples of plant material. This work aimed to verify the feasibility of a VOC-based diagnosis and to investigate the possible biological role of VOCs released during the plant–pathogen interactions. The volatile emissions from Pseudomonas syringae pv. actinidiae in axenic cultures and from inoculated in vitro kiwifruit plants were characterized by gas chromatography–mass spectrometry (GC–MS) and proton transfer reaction–time-of-flight-mass spectrometry (PTR–ToF-MS). By GC–MS, several putative biochemical markers, such as 1-undecene, were identified. PTR–ToF-MS resulted highly effective in screening the plant material for latent infections. To develop a more user-friendly, portable and less expensive diagnosis system, two different electronic nose models were tested for the early diagnosis of P. syringae pv. actinidiae in asymptomatic plant material. Our experiments demonstrated the feasibility of the electronic nose-based screening of infected plant material. Concerning the biological role of the VOCs released during the plant–pathogen interactions, the exposure of healthy plants to VOCs from infected ones influences the plant growth and induces the stimulation of protective responses. However, after the infection, P. syringae pv. actinidiae is able to selectively inactivate the induced plant defences.

Draft whole genome sequence analyses on Pseudomonas syringae pv. actinidiae HR negative strains detected from kiwifruit bleeding sap samples.

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

EVALUATION OF DIFFERENT PCR PRIMERS FOR IDENTIFICATION OF TUMORIGENIC BACTERIA ASSOCIATED WITH GRAPEVINE CROWN GALL

Grapevine crown gall caused by Allorhizobium vitis, or less frequently by Agrobacterium tumefaciens complex and Rhizobium rhizogenes, may seriously impact production in nurseries and vineyards worldwide. Although rapid and efficient detection and identification of tumorigenic bacteria is facilitated by PCR-based methods, high genetic diversity of these pathogens may hinder use of these methods in the disease diagnosis. Therefore, reliability of 11 primer pairs targeting fragments located on Ti plasmid or chromosomal DNA was tested on extensive collection of All. vitis, A. tumefaciens complex and R. rhizogenes strains isolated from grapevine throughout the world. Only primers VCF3/VCR3 targeting virC gene located on Ti plasmid clearly and accurately identified all tested tumorigenic strains associated with grapevine crown gall. Moreover, this primer pair coupled with primers specific for chromosomal pehA gene (PGF/PGR) in duplex PCR, may be recommended as the method of choice for routine preliminary identification of tumorigenic strains and differentiation of All. vitis.

Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe

To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8‐year period and to assess the relative contribution of mutation and recombination in shaping their diversity.