Washington Tree Fruit Research Commission

Research Reports

Development of a molecular assay for screening post-harvest pathogens of apple and pear (2004)

FINAL PROJECT REPORT
WTFRC Project #PH-02-240
YEAR 0/0
Organization Project #
Title:Development of a molecular assay for screening post-harvest pathogens of apple and pear
PI:Peter Sholberg
Organization:AAFC-PARC Summerland
 PDF version of report

Co-PIs

Karen Bedford and Dan O’Gorman

AAFC-PARC, Summerland, BC

 

Objectives

  1. The objectives of this study were to develop: a) a PCR assay and b) a diagnostic DNA array for post harvest pathogens of apple and pear based on the β-tubulin gene that will allow rapid recognition of post-harvest pathogens, simultaneously recognize fungicide resistant strains, and quantitative assessment of pathogens in diverse samples.
                     

      2.     In  2002-2003 the objectives were to develop the specific primers and probes for PCR and DNA array assays through extraction, amplification, sequencing of the β-tubulin gene, alignment of sequences, primer and probe design, and testing of designed primers and probes for cross-reactivity and specificity. 

       3.  In  2003-2004 the objectives were to conduct direct testing of diverse samples using the designed assays and to refine the assays to provide quantification of post-harvest pathogens.

Significant findings

  • The β-tubulin gene was utilized to design PCR detection assays where the ribosomal gene was not useful.
    • Two multiplex PCR assays for Penicillium blue mold have been developed and tested that detect within one day (1) Penicillium expansum and other Penicillium spp.
    • A multiplex PCR assay for Botrytis gray mold has been developed and tested that detects within one day Botrytis spp..
    • A PCR assay for Mucor Piriformis has been developed and tested that will detect within one day the presence of M. piriformis.
  • DNA array probes targeting the β-tubulin gene have been designed and tested, to detect and identify:
    •  Penicillium expansum isolates and TBZ resistance.
    • Botrytis species and TBZ resistance.
    • Mucor Piriformis
  •  The DNA array was successfully used to distinguish living from dead cells.  This ensures that the DNA detected in a sample is truly representative of the number of viable pathogen cells capable of causing disease.

Methods

DNA extraction and amplification.    Fungal cultures grown in liquid culture until biomass was sufficient for DNA extraction were transferred into Bio 101 Fast DNA extraction kit in 2 ml tubes.   The extraction protocol supplied with the kit was followed and DNA was eluted in 100 µl volumes and stored at –20ºC until required.  DNA was amplified using the polymerase chain reaction (PCR) in 20 µl volumes containing: 0.5-1.0 μl DNA, 1x buffer, 2.0-3.0 µM MgCl2, 1.5-2.0 µM dNTP mix, 0.05-0.4 µM each forward and reverse primers, 1 unit of Taq DNA polymerase and for multiplex PCR, 2.5 units of Ultratherm polymerase.  Primer pairs used to amplify the β-tubulin gene were BT2MLev-up4/Bt-Lev-Lo1 and for the ribosomal spacer DNA, the primers were UN18S42/UN28S22 (For Species specific amplification using multiplex reactions please refer to table 1 for the specific primer names). Amplification was performed on a GeneAmp 2700 thermal cycler (Applied Biosystems) with variable cycle conditions depending on primers used.  PCR products were run out on a 1.5 % agarose gel (or 2.5% gel for multiplex PCR) stained with ethidium bromide.  The gel was visualized using a UV transilluminator and BioPhotonics Gel Print 2000i system linked to a Mitsubishi video copy processor P67UA.

DNA sequencing.  The sequence reaction mixture contained 15-20 ng of purified DNA, 1µl of 3.5 µM primer, 3 µl 10 x sequencing buffer and 1 µl of Big Dye terminator mix.  Sequencing reactions were carried out on a GeneAmp 2700 thermal cycler set to run for 25 cycles at 95ºC for 30 sec, 50ºC for 15 sec, and 60 ºC for 2.5 min. The same primer pairs used to amplify the DNA in the initial PCR reaction were also used in sequencing reactions using the ABI Big Dye Terminator Kit.  Electrophoresis of sequencing extension products was performed on an ABI 377 sequencer.   For each species multiple isolates have been sequenced to act as conformation and to check for genetic homogeneity between isolates.  Editing of ribosomal and β-tubulin DNA sequences were done using Sequence Navigator software (Applied Biosystems Inc).  Miscalled or missed bases were manually corrected using data from a reverse sequence reaction as conformation.  Once editing was complete, data were entered into our sequence database.  Because of difficulties encountered using sequencing Mucor piriformis isolates, ribosomal DNA was cloned following standard protocols.  Clones were screened using a PCR method and clones possessing the correct size of insert underwent direct sequencing.

DNA Primer and Array Probe development.  Final edited versions of the β-tubulin sequence data for each species were placed into groups.  β-tubulin sequences for identical and related species were also down loaded from Genbank and placed into corresponding groups.  Sequence data for each group was then aligned in ClustalX software.  Alignments were analyzed visually to identify regions exhibiting differences between species.  These regions of differences were then marked as possible sites for primer or probe selection.  To ensure consistent melting temperatures, and to prevent duplex and hairpin formation, final probe selection and design were carried out using Oligo 6 software (National Biosciences, Hamel, MN).    Probe sequences were entered into a Blast search of the Genbank database.  The Blast search checks for the occurrence of homologous sequences existing in other species with DNA sequences present in the database.
 
Testing of Primers and Probes.   Designed primers for Penicillium, and Botrytis were tested alone and in multiplex combinations using various temperature and cycling regimes for the ability to distinguish Penicillium and Botrytis species.  Designed primers for Mucor piriformis were tested alone in standard  PCR assays using various temperature and cycling regimes for the ability to distinguish the pathogen from other closely related spp. The cultures were detected and identified by characteristic band patterns on stained agarose gels.  The DNA array template was constructed by immobilizing the DNA probes on a nylon membrane by a 5'- amino modified spacer arm.  The probes were arranged on the membrane in an array pattern, grouping ITS probes (previously developed) and the β-tubulin speceies specific and TBZ resistance probes together.  The constructed arrays were used for testing in hybridization reactions with pure cultures. The cultures were detected and identified by positive hybridization signals, which appear as dark dots on exposed x-ray film.  Individual probes on the array were tested for strength of reaction and specificity of reaction.  The same set of cultures was used in testing the PCR and DNA array assays.  Additional PCR and DNA array hybridization tests were carried out with environmental and commercial samples.

Quantification of pathogen populations.   The quantification capabilities of the post harvest DNA array were investigated.  A 100 fold dilution of solutions containing spores were simultaneously plated for cell counts and processed for DNA extraction.    Plates were counted after 48 hrs to give an estimation of cell concentration for corresponding DNA extractions.  Amplification products from concentrations of 106, 105,104, 103, 102, and 101 and zero cells, were used as standards in hybridizations with unknown samples which were also plated out to determine population size per ml.  Hybridization results of the DNA concentration standards were used to estimate sample populations for a range of grayscale values (0-255). Grayscale values were then used to estimate cell numbers in  inoculated and environmental samples.

Detection of DNA from live verses killed cells.     Solutions containing spores of B. cierea, Mucor pirifomris and Penicillium expansum were used in reactions with the DNA array to determine detection of live cell vs. killed spores.  Spore from the three pathogens were washed from culture grown on agar plates with sterile water.  The spore counts were determined for each pathogen separately using a haemocytometer (Botrytis = 8,000 spores/ml, Mucor = 78,000 spores/ml and Penicillium = 90,000 spores/ml).  The spores from all three pathogens were combined and then added to beakers containing 100 ml of 100 ppm free Cl2, and 100 ml of sterile H20.   Three replications of each treatment were carried out.  One ml of each replicate was removed after one, 24, 48 and 96 hrs.  Samples were used for both plate counts and DNA extraction.  The DNA was amplified as described earlier and hybridized with the DNA post harvest array for detection and quantification of the pathogens.   

Results and discussion

PCR Assay.   Two multiplex PCR assays employing both ribosomal internal transcribed spacer (ITS) and β-tubulin primers (Table 1) used in different combinations have been developed to distinguish Penicillium expansum from other post-harvest pathogenic Penicillium species.  Testing of the assays   has so far been conducted on known isolates of Penicillium spp. obtained from Dr. Peter Sanderson (Washington State),  as well as Penicillium spp. isolates collected in British Columbia by our laboratory.  The Penicillium expansum isolates have been sequenced and characterized for resistance or sensitivity to TBZ.  Results of testing of the assay were shown in previous reports.  Each of the multiplex assays clearly distinguishes P.  expansum from other Penicillium species.  Depending on the combination of primers used the multiplex assays can simply distinguish P.  expansum from other Penicillium spp. or it can identify P. expansum, P. solitum, and P. commune; the multiplex assay used in  was unable to amplify P. aurantiogriseum. The PCR multiplex system was used to test a larger number of P. expansum isolates, and was successful in every case in distinguishing them from the other Penicillium spp. tested (reported earlier)


Botrytis cinerea and a second Botrytis spp. (B.tulipea-like) consistently present in British Columbian apples have also been successfully distinguished using a multiplex PCR assay employing designed primers (Table 1). The PCR primers were also used in amplifications to distinguish B. cinerea and the B.tulipea-like isolates from nine other Botrytis spp. B. fabae does however react with the B.cinerea primers while the B. tulipea-like primers appear to be 100% specific.  The PCR assay was also used in trials to help determine the pathogenecity of the two different Botrytis species.  In inoculations of apple using one or the other of the Botrytis spp., both were shown to cause rot, although lesions caused by B. cinerea were generally larger.  In inoculation of a mixture of both species, populations of both B.cinerea and B.tulipea-like were detected and identified by PCR as being present at the margin of the lesions. However, B.cinerea was consistently identified by PCR as being in greater numbers, indicating that it is more pathogenic.

The potential exists that a single multiplex PCR could be used for Penicillium and Botrytis spp. as the bands that appear on the gels for the two genera are distinct. Optimization of multiplex PCR assays becomes more difficult as more primers are used.  Further work is needed to explore the possibility of a Botrytis/Penicillium multiplex assay.

Previously developed primers for Bull’s Eye rot (De Jong et al.) have been used to assay pure cultures of Pezicula malicortis and to successfully assay fruit from a tree inoculated with a known isolate of Pezicula malicortis.  DNA extracted from developing cankers and from lesions appearing on the fruit both tested positive with the specific primers for Pezicula malicortis.  Isolation of the pathogen from these tissues, on agar plates, was used to confirm the results.

Attemps to design primers for Mucor piriformis from the β-tubulin gene were not successful.  Therefore primers were designed for this pathogen using ITS sequences from isolates in our collection, isolates received from Dr. Bob Spotts (Oregon State University, Hood River, OR), and one sequence obtained from GenBank which represented a South African isolate. Testing of these primers showed specificity for the pathogen using pure culture (Figure 1).

DNA array.    Probes specific for two Botrytis spp. (gray mold) and for TBZ resistance in B. cinerea and P. expansum were designed from  β-tubulin gene sequences (Table 1).  These and specific ITS probes for Mucor piriformis, Penicillium expansum and two Botrytis spp. were placed on a prototype array membrane for testing.  The prototype membrane was tested with the known strains of   P. expansum and B.  cinerea resistant or sensitive to TBZ, other Penicillium and Botyris spp., M. piriformis and other Mucor spp.  All of the Penicillium expansum isolates reacted with at least two of the three specific ITS probes.  TBZ resistant P. expansum and Botrytis isolates all reacted as expected.  The M. piriformis probes only reacted with M. piriformis isolates.  Other Mucor and Penicillium spp. tested did not react with any of the species specific probes providing confirmation of the specificity of the probes.  The various ITS region and β-tubulin gene probes clearly distinguished the two Botrytis spp. when hybridized at 55°C and at 58°C.  However, when hybridized at 55°C, the eight other Botrytis spp. also showed positive hybridization signals but were distinguished from the two apple pathogens by producing unique hybridization patterns with the various probes.  The hybridization temperature of 55°C appears to be optimum for the Penicillium and Mucor probes and therefore the Botrytis probes could still be modified to work more effectively at this temperature.


The post harvest DNA array was also used to detect and quantify pathogens from several environmental and commercial samples (Table 2).  An experiment was set up to investigate the detection and quantification of live vs. killed cells, because the detection of DNA by itself does not indicate the viability of the pathogen and its ability to cause disease.  Detection of DNA from dead cells would lead to an overestimate of the true pathogen population.  The results from this experiment showed detection values of live cells using the DNA array were in general agreement with both spore counts estimated with a haemocytometer, or from total plate counts of viable cells for all three pathogens (Figure 3; Table 3).  No Cl2 treated cells were detected with the DNA array after 48 hours (Table 3).
                                     


Table 1.  Primers and probes designed for PCR assays and DNA array  tests for post-harvest pathogens of apple and pear.

Post-harvest disease
Target species
Primer or Probe
Name
Blue mold
Penicillium expansum
ß-tubulin primer
Pex-bt-f

ß-tubulin primer
Pex-bt-r

TBZ sensitive probe   (Penicillium)
Pex-bt-H3-GAG

TBZ resistance probe (Penicillium)
Pex-bt-H1-GCG

TBZ resistance probe (Penicillium)
Pex-bt-H1-GTG

rDNA probe
PE-H2c

rDNA probe
PE-H3
Blue mold
Penicillium other
ß-tubulin primer
Pen-other-bt-f

ß-tubulin primer
Pen-other-bt-r
Blue mold
P. solitum
ß-tubulin primer
Psol-bt-f  1

ß-tubulin primer
Psol-bt-f  2

ß-tubulin primer
Psol-bt-r
Blue mold
P. commune
ß-tubulin primer
Pcom-bt-r
Blue mold
P. aurantiogriseum
ß-tubulin primer
Paur-bt-f

ß-tubulin primer
Paur-bt-r 1

ß-tubulin primer
Paur-bt-r 2

Grey mold
Botrytis cinerea
ß-tubulin primer
Bcin-bt-f

ß-tubulin primer
Bcin-bt-r 1

ß-tubulin primer
Bcin-bt-r 2

rDNA probe
BC-H2d

rDNA probe
BC-H3d

ß-tubulin primer
Bcin-133-H3

TBZ sensitive probe    (Botrytis)
Bot-95-H1 GAG

TBZ resistance probe  (Botrytis)
Bot-95-H2 GCG
Grey mold
Botrytis spp. from apple
ß-tubulin primer
Bstok-bt-f

ß-tubulin primer
Bstok-bt-r 1

ß-tubulin primer
Bstok-bt-r 2

rDNA probe
BT-H1d

rDNA probe
BT-H2d

ß-tubulin probe
Bstck-144-H4

Mucor rot
Mucor Piriformis
rDNA probe
Mpir-ITS-183H1

rDNA probe
Mpir-ITS-414H1

rDNA primer
Mpir-ITS-36f

rDNA primer
Mpir-ITS-108f

rDNA primer
Mpir-ITS-506r

rDNA primer
Mpir-ITS-585r


Table 2.   Summary of amplification and hybridization results using primers and probes designed for the detection of two Botrytis spp. (B. cinerea, B. tulipae-like) Penicillium expansum and Mucor piriformis.

Sample
PCR results
Hybridization results
Universal
 primers
Species-specific primers
Species specific probes
TBZ resistance probes
Pure culture samples

Penicillium expansum (R)
pos
pos
pos
pos
P. expansum (S)
pos
pos
pos
pos
P. commune
pos
pos
-
-
P. solitum
pos
pos
-
-
P. aurantiogrisium
pos
-
-
-
Botrytis cinerea (R)
pos
pos
pos
pos
B. cinerea (S)
pos
pos
pos
-
B.tulipea-like
pos
pos
pos
Not tested
B. fabea
pos
pos
pos
pos
B. streptothrix
pos
pos
pos
pos
B. porri
pos
-
pos
pos
B. squamosa
pos
-
pos
pos
B. aclada
pos
-
pos
pos
B. tulipea
pos
-
pos
pos
B. crystollina
pos
-
-
-
Botrytis sp.
pos
-
-
-
Mucor pyriformis
pos
pos
pos
NA
M. plumbeus
pos
-
-
-
M. hiemalius
pos
-
-
-
M. rouxii
pos
-
-
-
Environmental samples

Orchard litter
pos
pos
pos
-
Apple tissue
pos
pos
Not tested
Not tested
DPA (dump tank)
pos
pos
pos
pos
Dump tank water
pos
Not tested
pos
-
Flume water
pos
Not tested
pos
-

pos;  samples gave positive hybridization signals, but are distinguishable from either of the two Botrytis apple pathogens by generating unique hybridization patterns.


Figure 1.   Species specific PCR for Mucor piriformis targeting unique sites in the rDNA spacer region.  The image shows a species-specific band of approx 500 bp. generated from M. piriformis DNA.  Lane 1, is a 100bp ladder; lane 2, M. piriformis (57); lane 3, M. piriformis (563); lane 4, M. piriformis (1979); lane 5, M. plumbius (85); lane 6, M. hiemalis (87); lane 7, M. rouxii (1985); lane 8Mucor sp. (1979); lane 9, negative control.

                                                                   Post Harvest Array Template


Figure 2.     Template of the post harvest DNA array membrane. The template shows the arrangement of probes designed to simultaneously detect P. expansum and TBZ resistance; Botrytis cineria, B. tulipae-like sp. and Botrytis Genus TBZ resistance; and Mucor piriformis.  The probes target both ribosomal ITS DNA and the ß-tubulin gene sequences.  The ß -tubulin probes detecting TBZ resistance includes codon 198. These probes not only detect two point mutations, which lead to resistance but also identify P. expansum.  For Botrytis, the TBZ resistance probes are genus specific.  The ß -tubulin probe for Botrytis species identification are located in different region which does not include codon 198. The probes for Botrytis cinerea also reacts with B. fabae.  The ribosomal probes for Botrytis can distinguish between the two apple pathogens but are not completely species specific and will detect other Botrytis spp.  The ribosomal DNA probes are species specific for both P. expansum and M.  piriformis.


                       

Figure 3.     Quantification of live vs. killed cells.   Samples shown were taken at 1 hour after inoculating 100 ppm and 60 ppm C l2 solutions.  Standard curve was generated with gray values (0-255) obtained from positive hybridization reactions of known standards and was used to estimate spore concentrations in each sample.  Values for each pathogen are displayed separately (P = Penicillium; M = Mucor; and B= Botrytis) for H2O, 100 ppm and 60 ppm free Cl2 treatments.

Table 3.  Comparison of plate counts and DNA array detection of live vs. killed cells

Sampling times
H2O
100 ppm free Cl2
60 ppm free Cl2
DNA array
Plate count
DNA array
Plate count
DNA array
Plate count
1 hr
2.3x105
2.0x105
9x101
1.7x101
3.5x102
6.7x101
24 hrs
2.1x105
1.5x105
5x101
3.0x101
1.2x102
0
48 hrs
1.7x105
1.7x105
0
0
not tested
not tested
96 hrs
1.7x105
1.7x105
0
3.0x101
not tested
not tested
Values given for DNA array are spore equivalence derived from DNA extracted from a known concentration of spores.
* For plate count method values were determined by dilution plating and are given in cfu/ml.

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