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

Iden%ficazione  tramite  la  definizione  di  un  profilo  (iden%kit)  metabolico  

Sistema di identificazione Biolog

Metodi immunologici: saggio di agglutinazione su vetrino

Metodi immunologici: saggio di

immunofluorescenza

Metodi immunologici: saggio colorimetrico su “strip” di nylon o di nitrocellulosa

PCR-­‐RFLP  della  regione  ribosomiale  16s  di  Agrobacterium  spp.  

Metodi immunologici: saggio colorimetrico ELISA

Enzyme-­‐Linked  ImmunoSorbent  Assay    

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  

 Protocolli  di  PCR  disponibili  per  i  principali  baZeri  fitopatogeni  delle  specie  or:ve.  

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%  

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.