68 research outputs found
Method for determining Macrophomina phaseolina
No full textThe present invention relates to a method for determining the presence of the phytopathogenic fungus Macrophomina phaseolina in a biological sample of interest. The present invention further relates to a kit for implementing the method of the invention
Development of a rapid PCR-Nucleic Acid Lateral Flow Immunoassay (PCRNALFIA) based on rDNA IGS sequence analysis for the detection of Macrophomina phaseolina in soil
No full textThe ‘Nucleic Acid Lateral Flow Immunoassay’ (NALFIA) using a generic ‘Lateral Flow Device’ (LFD), combined with
PCR employing labelled primers (PCR-NALFIA), enables to circumvent the use of electrophoresis, making the
diagnostic procedure more rapid and easier. If the specific amplicon is present in the sample, a coloured band,
with an intensity proportional to the amplicon concentration, will develop on the LFD strip in addition to the
control band.
Species-specific primers for M. phaseolina based on the rDNA intergenic spacer (IGS) were developed and their
specificity was checked and confirmed using 20 isolates of M. phaseolina and other 16 non-target fungi.
A DNA extraction protocol based on a bead-beating technique using silica beads, skimmed milk and PVP was
also developed.
The M. phaseolina specific primers MP102F/MP102R, 5′ labelled with biotin and FITC respectively, were used
in the PCR-NALFIA assay to identify the pathogen starting from mycelium or microsclerotia. Microsclerotia of M.
phaseolina (1, 10, 100 and 200) were manipulated under a stereomicroscope and their DNA was extracted using
microsclerotia alone or mixed with different types of soil. The resulting DNA, used for the PCR-NALFIA assay,
provided positive results for all the samples tested. A semi-quantitative grey-scale reference card based on the
PCR-NALFIA assay using intervals corresponding to microsclerotia soil number was developed. For this purpose,
the normalized pixel grey volumes obtained after a densitometric analysis of the test line intensity generated by
the LFD dipsticks were used
METODO PER DETERMINARE LA PRESENZA DI MACROPHOMINA PHASEOLINA
No full textLa presente invenzione riguarda un metodo per determinare la presenza
del fungo fitopatogeno Macrophomina phaseolina in un campione
biologico di interesse. Inoltre, la presente invenzione si riferisce ad un kit
per la realizzazione del metodo oggetto della presente invenzione
Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster
No full textLupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01–0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitiveseeddetectionassaytoscreenseedlotsbeforeplanting. PCR-baseddetectionsystemsexhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific C. lupini primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of C. lupini, 23 different Colletotrichum species, and 21 different organisms isolated from seeds of Lupinusalbus cv. Multitalia, L.luteus cv. Mister, and L.angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/10,000 infected seed
First report of Colletotrichum godetiae causing grape (Vitis vinifera) berry rot in Italy
No full textIn October 2016, rotting grape berries were detected on grapevine (Vitis vinifera) in Livorno (Nugola, Tuscany, Italy). Symptoms on grape berries skins varied from circular brown spots to rotting fruits. Both berries and petioles were covered with creamy salmon-colored masses of conidia. Rotten grape berries loss turgor and turn into ‘mummies’ (e-Xtra 1) over time. Symptoms suggested that a member of the genus Colletotrichum could be involved. Single spore cultures were obtained from conidial masses and grown in the laboratory at 25°C with a 12 hour light period on potato dextrose agar (PDA). Monoconidial isolates had light grey cottony aerial mycelium with colony color ranging from whitish to dark grey, while the reverse ranged from whitish to salmon-pink. Conidia were hyaline and unicellular, cylindrical or clavate and often with a light median constriction. However, Colletotrichum spp. are often difficult to distinguish morphologically. Total genomic DNA was extracted from monoconidial isolate SS354. The ITS region of rDNA and partial GAPDH, CHS-1, HIS3, ACT and TUB2 genes were amplified and sequenced according to Damm et al. (2012). Sequences were deposited in GenBank (Accession No. KY293406 for ITS, KY293407 for TUB, KY293403 for CHS, KY293405 for HIS3, KY293402 for ACT and KY293404 for GAPDH). The multilocus phylogenetic analysis carried out with the obtained and reference sequences (Damm et al. 2012) revealed that the SS354 isolate clustered within C. godetiae (e-Xtra 2). Pathogenicity tests were performed in laboratory by inoculating detached grape berries with or without petioles at the petiole insertion point with 20 μL of a conidial suspension (105 conidia/mL) of the isolate SS354. Grape berries without petiole developed symptoms similar to those observed in the field. Fungal colonies re-isolated from the lesions on berries were morphologically identical to isolate SS354. Control grape berries inoculated with sterile water remained healthy as well as grape berries with petioles inoculated with the pathogen. This suggests that C. godetiae is able to infect wounded grape berries. However, information regarding other infection routes were not searched, as this was not the aim of this work. This is the first report of C. godetiae causing grape berry rot in Italy. The phylogenetic analysis reveals that C. godetiae SS354 is closely related to C. godetiae RB118, the causal agent of anthracnose on grapevine in UK (Baroncelli et al. 2014). Since C. godetiae is polyphagous, cross-infections between grape and other crops are possible. Remarkably, Cacciola et al. (2012) reported C. clavatum (syn. C. godetiae) as the prevalent Colletotrichum species associated with epidemic outbreaks of olive anthracnose in Italy. However, at present no information regarding cross-infection of C. godetiae between grapevine and olive are available. Due to the high economic and social value of wine production in Italy (in 2013 only in Tuscany the production of grapes accounted for 8 million tons), a monitoring plan based on simple molecular identification tools should be advisable.Total genomic DNA was extracted from one monoconidial isolate (SS354) and the ITS region of rDNA was amplified, using the universal primers ITS4 and ITS5, then sequenced. The resulting sequence was 100% identical to those of C. acutatum species complex obtained by a BLAST search in GenBank. Based on Damm et al. (2012) five other loci were used to further characterise the isolate: partial GAPDH, CHS-1, HIS3, ACT and TUB2 gene sequences were amplified and sequenced. Sequences were deposited in GenBank (Accession No. KY293406 for ITS, KY293407 for TUB, KY293403 for CHS, KY293405 for HIS3, KY293402 for ACT and KY293404 for GAPDH). The multilocus phylogenetic analysis carried out with the obtained sequences and reference sequences (Damm et al. 2012) revealed that the SS354 clustered within C. godetiae (e-Xtra 2). Pathogenicity tests were performed in laboratory. 20 μL of a conidial suspension (105 conidia/mL) of C. godetiae SS354 was inoculated at the petiole insertion point on detached grape berries with or without petioles. Grape berries without petiole developed primary symptoms similar to those observed in the field. Fungal colonies re-isolated from lesions were morphologically identical to C. godetiae SS354. Control grape berries inoculated with sterile water remained healthy as well as grape berries with petioles inoculated with the pathogen. This latter evidence suggests that C. godetiae is able to infect grape berries from wounded tissues. However, information regarding other infection routes were not searched, as this was not the aim of this work. This is the first report of C. godetiae causing anthracnose on grapevine in Italy. The phylogenetic analysis reveals that C. godetiae SS354 is very close to C. godetiae RB118, the causal agent of anthracnose on grapevine in UK (Baroncelli et al., 2014). Since C. godetiae is a polyphagous pathogen, cross-infections between grape and other crops are possible. Remarkably, Cacciola and colleagues (2012) reported C. clavatum (synonymous to C. godetiae) as the prevalent Colletotrichum species associated with epidemic outbreaks of olive anthracnose in Italy. However, at present no information regarding cross-infection of C. godetiae between grapevine and olive are available. Due to the high economic and social value of wine production in Italy, in 2013 grapes production accounted for 8 million tonnes with 2.6 million hectoliters of wine production only in Tuscany, a monitoring plan based on simple molecular identification tools should be advisable
MP102-LABINABAG: development of a prototype molecular diagnostic kit for the on-site detection of Macrophomina phaseolina using a portable PCR platform
No full textThe early diagnosis of plant pathogens from plant and soil samples needs a simple, rapid and cost-effective test, without the use of sophisticated and expensive equipment and reagents. In this study, the soil-borne pathogen Macrophomina phaseolina is used to demonstrate the potential for on-site molecular detection of the prototype diagnostic kit developed. This was achieved using a rapid and simple protocol comprising of a magnetic bead-based nucleic acid extraction, a portable PCR platform and a final NALFIA assay with a generic lateral flow device (LFD). The portable PCR platform powered by an external battery pack and all materials and
reagents for an accurate on-site detection of M. phaseolina within 2 h were included in a professional bag. The DNA extraction method from
infected plant and soil material can be completed within 45 min, and does not require centrifugation steps, organic solvents, or the use of liquid nitrogen for sample homogenization. A procedure for lyophilizing PCR reagents was developed to allow their storage and transportation at room temperature and their use in the field. Formulation development, trial freeze drying cycle and post-process analyses were performed in collaboration with the Biopharma Technology Company. DNA extracts were tested by PCR, which is completed in just 1 h, using patented primers for M. phaseolina labelled with biotin and FITC at the 5′ end. The results obtained were comparable to those of PCR testing in the laboratory. Research to develop this platform for the detection of other important plant pathogens is ongoing in our laboratory.
This work was supported by the University of Pisa, Bando Dimostratori Tecnologici 2018, D.R.1937/18 del 6.11.2018, Progetto MP102-
LABINABA
The Rapid Identification of Anoplophora chinensis (Coleoptera: Cerambycidae) From Adult, Larval, and Frass Samples Using TaqMan Probe Assay
No full textA molecular diagnostic method using TaqMan probe qPCR is presented for the identification of Anoplophora chinensis (Förster) (Coleoptera: Cerambycidae) from whole body insects (adults and larvae) and frass samples stored under different conditions. The results showed a perfect amplification of DNA from all samples; the repeatability and reproducibility of the protocol were very good, with standard deviations of inter-run and intrarun variability less than or equal to 0.5. The assay allowed to discern all A. chinensis samples from those of the other non-target wood-borer species, with 100% correspondence to the homologous sequences. No amplification or cross reactions were observed with A. glabripennis (Motschulsky) (Coleoptera: Cerambycidae), which is the most related species among those tested. The protocol was validated by an internal blind panel test which showed a good correspondence between the results obtained by different operators in the same lab. The analytical sensitivity for the lab frass with the Probe qPCR, namely the lowest amount of A. chinensis DNA that can be detected (LoD), was 0.64 pg/μl with a Cq of 34.87. The use of indirect evidence for the identification of a pest is an important feature of the method, which could be crucial to detect the presence of wood-boring insects.
This diagnostic tool can help prevent the introduction of A. chinensis into new environments or delimit existing outbreak areas thanks to indirect frass diagnosis
Morphological and Molecular Identification of Dactylonectria macrodidyma as Causal Agent of a Severe Prunus lusitanica Dieback in Italy
No full textDuring the summer of 2016, severe dieback was observed on young potted Prunus lusitanica (Portugal laurel) plants in a nursery in the Pistoia province (Tuscany, Italy). Cylindrocarpon-like isolates were consistently recovered from diseased plant tissues. The combination of morphological and molecular traits, including sequence data of histone 3 and β-tubulin genes (HIS3, TUB2) and internal transcribed spacers (ITS), allowed the identification of Dactylonectria macrodidyma (Halleen, Schroers & Crous) L. Lombard & Crous (asexual form Cylindrocarpon macrodidymum) as the causal agent of the disease. Pathogenicity tests reproduced disease symptoms observed in the nursery after six months fulfilling Koch’s postulates. D. macrodidyma is a soilborne plant pathogen and is to be considered of great economic importance on P. lusitanica, especially under favorable conditions such as stress and/or reduction of plant vitality. The increasingly frequent reports of the disease caused by the pathogen in various nurseries suggest that pot cultivation, together with prolonged drought periods, may play a role in favoring infections. To the best of our knowledge, this is the first report worldwide of the occurrence of dieback on Prunus lusitanica caused by D. macrodidyma
Genome Sequence of Fusarium graminearum ITEM 124 (ATCC 56091), a Mycotoxigenic Plant Pathogen
No full textFusarium graminearum is among the main causal agents of Fusarium head blight (FHB), or scab, of wheat and other cereals, caused by a complex of Fusarium species, worldwide. Besides causing economic losses in terms of crop yield and quality, F. graminearum poses a severe threat to animal and human health. Here, we present the first draft whole-genome sequence of the mycotoxigenic Fusarium graminearum strain ITEM 124, also providing useful information for comparative genomics studies
spp. plants
No full textThis paper provides a first report of Aleurocanthus camelliae, the Camellia spiny whitefly, from Italy. The pest was found on plants of Camellia spp. grown in the nursery. Brief morphological and biological information is provided on this whitefly, as well as some considerations on the phytosanitary measures to be adopted to reduce the potential risk of its spread on ornamental plants in Europe and the EPPO region
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