Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

Different Cell Types Affect the Transition from Juvenile to Mature Phase in Citrus Plants Regenerated through Somatic Embryogenesis.

Robust protocols for the regeneration of somatic embryos in vitro are essential for the efficient use of the most modern biotechnologies. Unfortunately, in perennial trees such as Citrus, plants regenerated from juvenile tissues usually exhibit strong, undesirable juvenile characters such as thorny habit and delayed flowering and fruit production. In this work, we tested whether the cell types (nucellar and stigma/style) used to regenerate Citrus plants through somatic embryogenesis affected the transition from the juvenile to mature phase. The results show that regenerants from nucellar cells presented persistent juvenile characters, whereas plants originating from stigma/style explants transited to the mature phase more rapidly. Our observations support the hypothesis that the totipotent cells originated from different cell types are not equivalent, possibly by maintaining memory of their previously differentiated state.

A non-targeted metabolomics study on Xylella fastidiosa infected olive plants grown under controlled conditions.

In the last decade, the bacterial pathogen Xylella fastidiosa has devastated olive trees throughout Apulia region (Southern Italy) in the form of the disease called "Olive Quick Decline Syndrome" (OQDS). This study describes changes in the metabolic profile due to the infection by X. fastidiosa subsp. pauca ST53 in artificially inoculated young olive plants of the susceptible variety Cellina di Nardò. The test plants, grown in a thermo-conditioned greenhouse, were also co-inoculated with some xylem-inhabiting fungi known to largely occur in OQDS-affected trees, in order to partially reproduce field conditions in terms of biotic stress. The investigations were performed by combining NMR spectroscopy and MS spectrometry with a non-targeted approach for the analysis of leaf extracts. Statistical analysis revealed that Xylella-infected plants were characterized by higher amounts of malic acid, formic acid, mannitol, and sucrose than in Xylella-non-infected ones, whereas it revealed slightly lower amounts of oleuropein. Attention was paid to mannitol which may play a central role in sustaining the survival of the olive tree against bacterial infection. This study contributes to describe a set of metabolites playing a possible role as markers in the infections by X. fastidiosa in olive.

A new inclusive MLVA assay to investigate genetic variability of Xylella fastidiosa with a specific focus on the Apulian outbreak in Italy.

The Olive Quick Decline Syndrome by Xylella fastidiosa subspecies pauca is among the most severe phytopathological emergencies nowadays. In few years, the outbreak devastated olive groves in Apulia (Italy), potentially endangering the entire Mediterranean basin. This research aimed to develop a multiple locus VNTR analysis assay, a molecular tool to differentiate between populations of the pathogen. It has already been successfully applied to different X. fastidiosa subspecies from various plant hosts. The previously published TR loci, together with a set of new design, have been tested in silico on the genome of the Apulian De Donno strain. The resulting selection of 37 TR loci was amplified on the genomic DNAs of the Apulian strains AND from representatives of X. fastidiosa subspecies, and directly on DNA extracted from infected plants. The assay clearly discerned among subspecies or even sequence types (ST), but also pointed out variants within the same ST so as to provide more detailed information on the dynamics and pathogen diffusion pathways. Its effective application even on total DNAs extracted from infected tissues of different host plants makes it particularly useful for large-scale screening of infection and for the strengthening of containment measures.

Seasonal Fluctuations of Sap-Feeding Insect Species Infected by Xylella fastidiosa in Apulian Olive Groves of Southern Italy.

A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves.