Eumicota – Funghi Veri – Ascomiceti lezione diagnosi.pdfEumicota – Funghi Veri – Ascomiceti...
Transcript of Eumicota – Funghi Veri – Ascomiceti lezione diagnosi.pdfEumicota – Funghi Veri – Ascomiceti...
Eumicota – Funghi Veri – Ascomiceti La riproduzione sessuata, la formazione dell’asco e le ascospore, e tipi di ascocarpi o ascomi
Tipi di ascocarpi prodotti dagli Ascomiceti (ripr. sessuata)
Assenza di ascocarpo: Aschi nudi
cleistotecio
Peritecio Apotecio
Pseudotecio
Isolamento da matrici vegetali su substrati selettivi o semiselettivi e successiva purificazione delle colonie
Detec%on of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop using post-‐enrichment DAS-‐ELISA
² Two sensi:ve, specific and userfriendly serological methods were used to detect the pathogen in latently infected tubers and stems: double-‐an:body sandwich (DAS)-‐ELISA and indirect ELISA on nitrocellulose membrane (NCM) aNer enrichment of the plant extracts in a semi-‐specific broth.
² BW detec:on probabili:es were higher for tubers than for stems; a 99% detec:on probability was obtained by analysing 400 stems sec:ons or 250 tubers using DAS-‐ELISA. Detec:on of BW infec:on in symptomless plants 20 days before harvest using post-‐enrichment DAS-‐ELISA is a reliable and user-‐friendly technique that can easily be used by na:onal plant protec:on services and seed programmes in developing countries
ANALISI QUANTITATIVA IDENTIKIT Q TM BOTRYTIS CINEREA
Il kit è un prodoZo nuovo ed unico nel suo genere che offre l'opportunità di s:mare in maniera accurata il livello di infezione da B. cinerea. Il kit impiega un an:corpo monoclinale, che rileva efficacemente B. cinerea in estrab di uva e di altri frub. Le reazioni specifiche con altri componen: degli estrab dei frub sono così basse da poter rilevare e diffferenziare infezioni a livelli assai ridob (0 -‐ 10%).
U%lizzatori del kit: • produZori di uve • industrie enologiche • produZori e trasformatori di fruZa • industrie di conservazione della fruZa • organizzazioni di ricerca • industrie di fitofarmaci • ibridatori • osservatori di malabe delle piante • laboratori di analisi fitopatologica
Kit Alert per la ricerca rapida in campo di patogeni fungini. Tempo di analisi: 16 -‐ 20 minu% Phytophtora spp. 8 Pythium spp. 8 Rhizoctonia spp. 8 Phytophtora, Pythium, Rhizoctonia, 12 (3x4)
Detection and quantification of Fusarium oxysporum f. sp. cucumerinum in environmental samples using a specific quantitative PCR assay
The rapid and reliable identification and quantification of pathogens is essential for the management of economically important plant diseases.
Engineer transgenic plants for the purpose of early detec:on of plant pathogen infec:on, which was accomplished by employing synthe:c pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infec:on
A number of synthe:c promoters consis:ng of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.
(a) Scheme of synthe%c promoter construct as tetramers of certain regulatory elements (4 X RE) were placed upstream of CaMV35S minimal promoter (min 35S containing the TATA box).
(b) Scheme of enhanced synthe%c promoter construct of 4 X RE were placed between B (-‐415 -‐90) and A1 (-‐90 -‐46) domains of CaMV35S promoter.
Results: synthe:c promoters containing pathogen and/or defence signalling inducible cis-‐ac:ng regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system
Mul%plex PCR to detect four different tomato-‐infec%ng pathogens
PCR assay to detect infec:ous agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants
Single PCR allows the amplifica%on of only one DNA template whereas Mul%plex PCR reduces %me and work since one can amplify more than one template in a single tube
v To amplify them all at the same :me, a gradient PCR, with temperatures increasing from 50 to 60 °C was applied to each template of the pathogens under study.
v To facilitate Taq polymerase access to DNA templates, gDNA was digested with a restric:on enzyme that did not digest the PCR product fragments.
v For the posi:ve control, the plasmid cocktail was used as template
v The most cri:cal factor in op:miza:on of the mul:plex PCR assay was the rela:ve concentra:on of the primer sets
Op:mized mul:plex assay electrophore:c paZern of genomic DNA from an infected plant with C. michiganensis subsp. michiganensis
From leN to right, first half, PCR amplifica:on with generic primers ITS4/ITS5 is shown for a healthy and a C. michiganensis subsp. michiganensis-‐infected plant using 4 or 100 ng of HindIII digested-‐genomic DNA. The genomic DNA samples were also amplified using the primer mix as shown in the second half. M stands for molecular weight markers (1-‐kb DNA ladder in basepairs) and C+ for posi:ve control (plasmids cocktail and primer mix)
Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants
Current pathogen detec:on methods and technologies are largely constrained to those occurring post-‐symptoma:cally. Recent efforts were made to generate plant sen%nels (phytosensors) that can be used for sensing and repor:ng pathogen contamina:on in crops.
Time-‐course analyses of fluorescent protein FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments.
Tobacco Arabidopsis
leaves were infiltrated with salicylic acid for PR1 and SARE, ethephon (an ethylene releasing chemical) for ERE and methyl jasmonate for JAR regulatory element containing constructs or with Pseudomonas syringae pv. tomato. Expression of the pporRFP reporter was visualized 72 h following the treatment using an epifluorescent microscope
These stable transformed plants allow in situ monitoring of biological properties during their immediate contact with pathogens. They could be used for stable phytosensing for early detection and rapid report of bacterial pathogen infection.These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.