NMR Metabolite Profiling for the Characterization of Vessalico Garlic Ecotype and Bioactivity against Xanthomonas campestris pv. campestris.

The Italian garlic ecotype "Vessalico" possesses distinct characteristics compared to its French parent cultivars Messidor and Messidrôme, used for sowing, as well as other ecotypes in neighboring regions. However, due to the lack of a standardized seed supply method and cultivation protocol among farmers in the Vessalico area, a need to identify garlic products that align with the Vessalico ecotype arises. In this study, an NMR-based approach followed by multivariate analysis to analyze the chemical composition of Vessalico garlic sourced from 17 different farms, along with its two French parent cultivars, was employed. Self-organizing maps allowed to identify a homogeneous subset of representative samples of the Vessalico ecotype. Through the OPLS-DA model, the most discriminant metabolites based on values of VIP (Variable Influence on Projections) were selected. Among them, S-allylcysteine emerged as a potential marker for distinguishing the Vessalico garlic from the French parent cultivars by NMR screening. Additionally, to promote sustainable agricultural practices, the potential of Vessalico garlic extracts and its main components as agrochemicals against Xanthomonas campestris pv. campestris, responsible for black rot disease, was explored. The crude extract exhibited a MIC of 125 µg/mL, and allicin demonstrated the highest activity among the tested compounds (MIC value of 31.25 µg/mL).

Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

Susceptibility of gerbera and chrysanthemum varieties (Gerbera jamesoni and Chrysanthemum morifolium) to Fusarium oxysporum f.sp. chrysanthemi.

In 2002, gerbera plants (cv Kaliki) were observed exhibiting symptoms of a wilt in a soilless cultivation at Albenga area (Northern Italy). A similar wilt was also observed in the Sanremo area (Northern Italy) on cv Red Bull, Anedin and Gud finger grown in soil. The same observations were carried out in 2004 in SW Spain where gerbera plants showing wilt symptoms were observed in soilless crops. In all cases, the planting material originated from the Netherlands. Recently on the base of experimental trials F. oxysporum f. sp. chrysanthemi was recognized as the causal agent of wilts of gerbera both in Italy and in Spain. The aim of this experimental work was the evaluation of the resistance/susceptibility of available cultivars of chrysanthemum and gerbera to the Fusarium wilt. The pathogenicity of two isolates of Fusarium chrysanthemi obtained from infected gerbera (Gerbera jamesonii) and chrysanthemum (Chrysanthemum morifolium) plants was tested on several varieties both of gerbera and chrysanthemum in 2004-2006. In 2004 and 2005 respectively 54 and 30 cultivars of chrysanthemum and 57 and 55 of gerbera were tested, while in 2006 only 53 cultivars of gerbera were tested. The results showed that respectively in 2004 and 2005 67 and 33 % of chrysanthemum cultivars were highly resistant to F. chrysanthemi obtained from chrysanthemum while 57 and 53 % were highly resistant to strain isolated from gerbera. In 2004, 2005 and 2006 47, 65 and 75 % of gerbera cultivars were highly resistant to F. chrysanthemi obtained from chrysanthemum and 48, 56 and 72 % were highly resistant to the strain isolated from gerbera.

Soil fumigation with chloropicrin in Italy: experimental results on melon, eggplant and tomato.

Starting in 1999, seven experimental trials were carried out in Italy in order to evaluate the efficacy of chloropicrin (CP) (Tripicrin, a.i. 99% by wt) as alternative to methyl bromide for soil disinfestation. CP applied by shank injection at 30-40 g/m2 provided a satisfactory and consistent control of Fusarium wilt on melon and Verticillium wilt on eggplant, particularly in sandy soils. On melon against Fusarium wilt and on tomato against F. radicis lycopersici and Fusarium and Verticillium wilts CP at 40 or 60 g/m2 applied by drip irrigation at the concentration of 400 to 700 microliters/l or 850 microliters/l provided the best results. CP applied at 80 g/m2 with 60 mm of water (corresponding to 850 microliters/l) did not increase the efficacy of the soil fumigation treatment and, on tomato, was phytotoxic, causing sudden collapse of plants immediately after the transplant. The comparison of chloropicrin drip application under polyethylene, widely used in the past for methyl bromide soil fumigation, or under virtually gas impermeable film (VIF), to reduce atmosphere emissions, showed an increase of CP efficacy under VIF only reducing the dosages below 40 or 30 g/m2.

Identification of Fusarium oxysporum f. sp. basilici Isolated from Soil, Basil Seed, and Plants by RAPD Analysis.

Fifty-two isolates of Fusarium oxysporum, obtained from infected basil plants, seed, flower residues, and soil from different growing areas in Italy and Israel, were analyzed by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), coupled to a DNA extraction protocol from colonies grown on Fusarium-selective medium. In a pathogenicity assay, 35 isolates caused 32 to 92% disease on seedlings of the highly susceptible basil cultivar Fine verde, while 17 isolates were nonpathogenic on basil. Thirty of the F. oxysporum f. sp. basilici isolates obtained from soil or wilted plants gave identical amplification patterns using 31 different random primers. All tested primers allowed clear differentiation of F. oxysporum f. sp. basilici from representatives of other formae speciales and from nonpathogenic strains of F. oxysporum. RAPD profiles obtained from DNA of isolates extracted directly from cultures grown on Fusarium selective medium were identical to those obtained from DNA extracted from lyophilized mycelia.