Development of an FTP-LAMP assay based on TaqMan real-time PCR and LAMP for the specific detection of Xylella fastidiosa De Donno and mulberry strains in both plants and insect vectors

We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal)

Comparison of conventional and novel molecular diagnostic methods for detection of Xylella fastidiosa from insect vectors

The efficiency of three diagnostic methods, i.e. PCR, real-time PCR and LAMP, for detection of Xylella fastidiosa (Xf) genomic DNA from Philaenus spumarius (Ps) and Neophilaenus campestris (Nc) insect vectors was evaluated using three total nucleic acids (TNA) extraction methods (EM). In addition, a new real-time LAMP technology, Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOSLAMP), originally developed for human virus diagnoses, was optimized and assessed for detection of Xf in insect vectors. EM1 consisted of entire insects heated in an extraction buffer (EB) containing Tris-EDTA and TRITON-X100. In EM2, TNAs were extracted only from excised heads of insects, and heated again in the EB of EM1. EM3 consisted of grinding entire insects, heads and bodies recuperated from EM2, with a CTAB buffer. The molecular analyses conducted on 100 specimens of Ps and 50 of Nc, collected from a Xf-infected olive orchard (Lecce province, Italy), showed that 29% of specimens (40 Ps and four Nc) were positive to the presence of Xf. The comparison between the three methods revealed that EM3 is the most efficient for extracting Xfgenomic DNA from insect vectors, of which 44 specimens were positive for Xf in each of the diagnostic methods used, including the newly optimized FLOS-LAMP assay. In general, the real-time PCR and LAMP assays were more competent than the conventional PCR for detection of Xf in insect vectors, independently from the EM used. The newly optimized FLOS-LAMP technique had a detection limit of 1 fg μL-1 of Xfgenomic DNA, compared to the 10 fg μL-1 for conventional LAMP. The high sensitivity of the FLOS-LAMP was evident through the greater number of overall Xf-infected insect vectors detected (60%), compared to those for LAMP (45%,), real-time PCR (28%) and PCR (10%). FLOS-LAMP, being a more sensitive and specific assay, together with EM3, were the most appropriate approaches for an accurate detection of Xf in insect vectors

DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population

Background: Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations. Results: In this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85 % ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ε-LCY, and Lycopene beta-cyclase, β-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes. Conclusion: We demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions. The availability of this tool together with TILLING technique will expand the polymorphisms in candidate genes of agronomically important traits in wheat

Development of singleplex and multiplex real-time (Taqman®) RT-PCR assays for the detection of viruses associated with fig mosaic disease

Singleplex and multiplex real-time (TaqMan®) RT-PCR assays were developed to detect seven fig-infecting viruses, i.e., fig leaf mottle-associated virus 1 (FLMaV-1), fig leaf mottle-associated virus 2 (FLMaV-2), fig mild mottle-associated virus (FMMaV), fig mosaic virus (FMV), fig latent virus 1 (FLV-1), fig cryptic virus 1 (FCV-1) and fig fleck-associated virus (FFkaV). The sensitivity of the newly developed TaqMan® assays was compared with the corresponding conventional RT-PCR (RT-PCR) using 10◦ to 10− 6 serial dilutions of both cDNA and crude fig extracts. The results showed that the Taqman® RT-PCR assays were generally 102 to 103 -fold more sensitive than the RT-PCR assays, except in the case of FLV-1 detection, where the two techniques had the same sensitivity. In the multiplex Taqman® RT-PCR, only a maximum of five viruses could be detected simultaneously in naturally infected fig trees, regardless of which combination of the virus-specific probes and primers were used. Both the RT-PCR and Taqman® RT-PCR assays were used in a large-scale survey of 100 field-grown fig trees in Egypt. The results showed the presence of all seven viruses under study, mostly occurring as mixed infections (63 %). The prevalence of infections observed in the tested samples were as follows: FMV (62 %), FFkaV (59 %), FLMaV-2 (32 %), FLV-1 (16 %), FLMaV-1 (14 %), FCV-1 (7%) and FMMaV (4%). FMV was invariably associated with diseased trees that presented mosaic-like symptoms. In the few cases where the mosaic-affected trees were found to be free of FMV, they were found to be infected with a mixture of two or more other viruses

Additional file 1: of DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population

Supplemental file S1. β-LCY gene sequences. (DOCX 12 kb

First report of pseudopithomyces mori causing leaf spot on mandarin in Italy

In July 2023, a leaf spot-like disease was observed on approximately 15% plants in a mandarin (Citrus reticulata) orchard in Calabria, Southern Italy. The symptomatic leaves displayed irregular, dark brown lesions surrounded by a yellow halo. Fifteen symptomatic leaves from five trees were collected and surface sterilized in 2% NaOCl. Lesion pieces (3 × 3 mm) were plated on potato dextrose agar (PDA) with antibiotics and incubated at 24 °C for 5 days. Three isolates morphologically identical were obtained from different plants, and purified as required. The colonies on PDA were cottony, first hyaline, then salmon pink (front) to orange (reverse). Conidia were ellipsoid, light brown, with 3–4 transverse septa, usually divided by a longitudinal septum, and 23.5 ± 5.5 × 13.69 ± 4.79 μm. The morphological features were consistent with those of Pseudopithomyces sp. (Ariyawansa et al. 2015). Identification was confirmed for the representative strain AP77 by sequencing the internal transcribed spacer (ITS) region and portion of translation elongation factor 1 alpha (TEF1) gene with primers ITS1/ITS4 and EF-983 F/EF-2218R, respectively (Jayasiri et al. 2019). Sequences were deposited in GenBank with accession numbers OR911598 (ITS) and OR921209 (TEF1). BLASTn analyses showed their 99% homology with Pseudopithomyces mori strain MFLUCC 18-1630 (GenBank No. MW183777 and MW063153, respectively). Phylogenetic analyses of the concatenated sequences confirmed the clustering with P. mori strain MFLUCC 18-1630 (Tennakoon et al. 2022). The pathogenicity was tested on 10 healthy citrus leaves, which were slightly injured with a sterile scalpel, inoculated by a drop of suspension (106 conidia/ml) or sterile water (control), and maintained at 24 °C in a humid chamber. Seven days after inoculation, lesions like those observed in the field occurred only on inoculated leaves. The same fungus was reisolated from infected leaves, fulfilling Koch’s postulates. To our knowledge, this is the first report of leaf spot on mandarin leaves caused by P. mori in Italy

Fruit-set and SSR markers of fig cultivars from Puglia region, Southeastern Italy

Fig cultivars may produce one or two crops per year of which the first crop is defined as breba whereas the second is the main fig crop. More than 100 different fig genotypes are present in the fig repository at the Experimental Station “P. Martucci” belonging to the University of Bari ‘Aldo Moro’ and they produce both brebas and figs. In order to investigate the biological behavior of local varieties, a set of 18 fig genotypes was studied for fruit-set. Breba-set resulted very variable, ranging from no brebas at all to around 90%. Fig-set was generally low-medium in many genotypes and particularly low in cultivars defined as “San Pedro-type” producing mainly brebas. We coupled this analysis with the screening of SSR markers. Results showed that 94.9% of tested primer pairs were found polymorphic, of which 50% showed multiple discrete bands and 50% presented a single band. This allowed us to discriminate the genetic variability of fig genotypes

Characterization of edible fig germplasm from Puglia, southeastern Italy: Is the distinction of three fig types (Smyrna, San Pedro and Common) still valid?

Fig. (Ficus carica L.) is a gynodioecious species with two major sex types: the caprifig (hermaphroditic), which has male flowers and short-styled female flowers, and the fig (female) with only long-styled female flowers. Many fig varieties require pollen to allow flower fertilization and fruit development in a process known as caprification. Fig varieties produce one or two crops per year; the first is the breba and the second, the main crop. Puglia is characterized by a wide germplasm of both edible (female) figs and (male) caprifigs. Over 100 different fig genotypes, mainly collected in Puglia, are located in the fig repository at the P. Martucci experimental station, University of Bari 'Aldo Moro'. The tendency towards caprification of the three fig types (Common, Smyrna and San Pedro) is still unclear. To investigate the biological behavior of 24 fig genotypes, both caprification trials and microsatellite analysis were used. Fruit-set of brebas was very variable, whereas fruit-set of main crop was medium-high in the Common type varieties and low in the San Pedro types. Almost all genotypes were physiologically biferous. We compared these results with those obtained from molecular and phylogenetic analyses. Out of 49 SSR markers tested, 39 amplified one or two PCR products, and 31 were polymorphic. Phylogenetic analysis of the 24 fig genotypes revealed a clear distinction between Common and San Pedro type figs. Greater understanding of fig biological caprification is important to distinguish fig types into those requiring caprification and those that do not require caprification