Isolation and characterization of bacteria causing broccoli floret rot

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Isolation and characterization of bacteria
causing broccoli floret rot

• Dr. Gustavo Hernández-Guzmán
• CINVESTAV Irapuato

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Background The crop

• Broccoli crop and produce is very important for
  Guanajuato.
• Most important producer in México
• There are big investment, national and
  international
• Many farmers and their families depends on this
  crop

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Background Perils

• Agronomic Fertilization, plant density, cultural
  practices, etc.
• Environment Relative humidity
• Phytopatogens
• Fungi
• Alternaria
• Phoma
• Mildew
• Bacteria
• Pseudomonas
• Xanthomonas
• Erwinia

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Broccoli floret rot

• Etiology not well characterized
• But we know there are fungi and bacteria
  associated
• Agrichemicals are not so effective
• Weather conditions favour spread disease Rain
  season factor is very important.
• So, this means BIG problems

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Our objectiveTo study the microorganisms
associated to broccoli floret rot (BFR)

• Specific objectives
• Isolate and characterize the bacteria associated
  to BFR
• Investigate the phylogenetic relationships of
  isolates
• Study the disease stages of BFR

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Strategy

• Isolation and characterization
• Gram in vivo
• Growth in Non-selective media and selective to
  Pseudomonas, Xanthomonas and Erwinia
• Gram in vitro
• Colony phenotypes

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Strategy Sampling

• We sampled different places
• COTECO experimental fields
• Villa Verde Farm
• Huanímaro Gto.

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Strategy Isolation

• Affected plant tissue were selected in different
  stages of disease (small necrosis, chlorosis,
  necrosislt 1cm and necrosisgt2cm)
• Superficial disinfection with Sodium hipochloride
  0.5 (v/v)
• Individual tissue was grounded and solid
  particles separated by centrifugation
• Plating on non-selective media amended with
  cicloheximide

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Characterization

• Biochemical
• Gram in vitro
• API systems for Gram(-) or Gram () were
  applicable
• Pseudomonas LOPAT test
• Toxin bioassays
• Genetic
• PCR
• General Rep-PCR
• Alternative Sequencing of 16S intergenic
• Specific Search for specific genes. Vgr.
  Coronatine genes, pectate lyase, xanthomonadin
  genes, etc.

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Plant-pathogen interaction experiments

• Hypersensitive reaction
• Are those pathogenic to broccoli?
• Plantule infections
• Broccoli cultivars
• Sakata Marathon, Patriot, Patron
• Seminis Legacy, Grandeur
• Disease evaluation
• Disease kinetics
• Bacterial counts

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Advances

• We have three different phenotypes. Tested by
  microbiological techniques and API test
  (Analytical Profile Index. Biomèrieux)
• Fluorescent colonies. Pseudomonas spp. Appeared
  on approximately 90 of total counts
• Creamy, shiny and big colonies. Pantoea spp.
  (Erwinia spp.)
• Yellow colonies. Probably it is Xanthomonas spp.

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Pathogen screening on broccoli plants

• 60 isolates were tested
• Plants are susceptible at plantule and 6 week old
  plant stage
• We use different broccoli cultivars, but mainly
  Marathon was assayed

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Plant inoculation

• Modified from Silo-Suh. PNAS 2002
  99(24)15699704
• Phosphate buffer (PO4-) was used
• Bacterial suspensions were adjusted to A6000.1
  and then diluted 1/10 (A6000.01)
• Broccoli leaves were infiltrated with PO4- buffer
  and bacterial suspensions, using needless insulin
  syringes
• Symptoms were recorded daily until 6 dpi

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Disease symptoms

• Asymptomatic
• Localized small necrosis
• Large necrosis (hypersensitive reaction, HR)
• Chlorosis and necrosis

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Disease symptoms

• Asymptomatic
• A few cases, most of them are fluorescent bacilli
  (Pseudomonas)

NFB1
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Disease symptoms

• Localized small necrosis
• The most represented group of isolates

NFBX1
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Disease symptoms

• Large necrosis (hypersensitive reaction, HR)

NFBXS3
NFB10
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Chlorosis and necrosis

• Small necrotic area
• Evident chlorosis
• API test Pseudomonas spp
• Ribosomal 16S intergenic DNA sequencing is
  underway

NFB15
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Necessities

• Greenhouse It is very important for
  pathogenicity tests. We need a confined place
  with controlled humidity and temperature to test
  our isolates in broccoli florets
• Help to get reference strains US policies have
  stopped the trade of suspected plant pathogens,
  but for a full characterization of our bacterial
  isolates it is very important to get those
  references

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Underway and future work

• Pathogenicity tests
• Repetition of plantule experiments.
• Finish PCR characterization.
• Broccoli floret infection.
• Characterization of broccoli-Bacteria interaction.

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My special thanks

• Funds from COTECO and Fundación Guanajuato
  Produce.
• Agric. Engineer Martha Ramírez (Marbran company)
  my broccoli plant provider.
• Dr. Gabriela Olmedo, Dr. Rafael Rivera and Dr.
  Ariel Alvarez, for lab space at CINVESTAV
• And thanks to you for your precious time!
• Comments are greatly appreciated
• Sincerely
• Dr. Gustavo Hernández-Guzmán
• Visiting scientist
• CINVESTAV Irapuato
• P.O. Box 629
• Irapuato Gto. México
• ghernand_at_ira.cinvestav.mx
• ghernand67_at_yahoo.com.mx