1. Analysis of Grape Cultivars for Pierce’s Disease Resistance with a Xylella-
specific QRT PCR Probe
Reyna Valdez, Amanda Markham and Csilla Buday
Dr. Lisa Morano, Research Mentor, Department of Natural Sciences, UHD
INTRODUCTION
Pierce’s Disease (PD) is caused by Xylella fastidiosa, a gram
negative bacterium that infects grapevines as well other
agricultural plants especially in the Gulf Coast region
(Hopkins and Purcell, 2003) (Figure 1). X. fastidiosa causes
disease by invading the xylem vessel of a plant and blocking
the transport of water and mineral nutrients to the rest of the
plant. Plants affected by X. fastidiosa display symptoms such
as smaller fruit and leaves as well as premature leaf
abscission and eventually death (Figure 2). PD is transmitted
from one grapevine to another by its insect vector, the
glassy-winged sharpshooter (GWSS) (Texas PD Task Force,
2004). PD is found throughout many states although Texas
Gulf Coast has a high recurrence rate due to the warm
temperatures which are thought to be optimal for both the
bacterium and the insect (Hopkins and Purcell, 2002). This
means PD is a particularly serious issue for the current Texas
wine grape growers and the future of the Texas wine
industry. Several potential disease resistant varieties have
been planted in a vineyard in Industry, Texas. It is a goal of
the Texas AgriLife Extenstion office to evaluate whether
these varieties are PD resistant and will therefore be an
option for growers in extreme PD areas. The varieties
planted range from wild species selected hundreds of years
ago to novel varieties bred at UC Davis (Table 1). The goal
of this project was to use both ELISA and QRT PCR
analysis using a X. fastidiosa specific probe to determine
which varieties contain significant levels of X. fastidiosa
and are therefore less resistant.
METHODS
Vineyard Sampling
Fifty-eight samples of grape petioles were collected from
Paradox House Vineyard in Industry, Texas. Seven to 10
replicates of 8 different varieties were evaluated for X.
fastidiosa levels from an experimental vineyard in Industry,
Texas. These varieties are known to have some level of PD
resistance (Table 1).
ELISA Testing
To evaluate for X. fastidiosa levels several petioles were
sampled in fall of 2014 when bacterial levels are known to be
highest. Petioles were washed and allowed to air dry. To
prepare samples for ELISA, 0.1g of petioles were cut into
smaller pieces and crushed with mortar and pestle in ELISA
buffer. Positive and negative controls and other steps were
performed as outlined by the ELISA kit (Agdia, Inc.).
Absorbance measurements of a 96 well-plate at 650nm were
performed using a spectrophotometer (Beckman Coulter
DU800, Fullerton, CA).
QRT PCR
Quantitative Real-Time PCR investigations using X.
fastidiosa-specific TaqMan (Schaad et al. 2002). 2 µl of X.
fastidiosa DNA 23 µl of master mix containing1 µM forward
primer, 1 µM reverse primer and the TaqMan probe. QRT
PCR reactions were run as follows: 95°C for 30 s, 40 cycles
at 95°C for 1 s, and 62°C for 20 s.
ABSTRACT: Pierce’s Disease (PD) is a plant disease of grapevines caused by the gram negative bacterium Xylella fastidiosa. This plant pathogen causes a blockage in the xylem vessel preventing water flow
and killing the vine. X. fastidiosa is transferred by an insect vector and both the vector and the bacteria are found at particularly high levels along the Texas Gulf Coast. One strategy to deal with this deadly disease is
to plant PD resistant plants. Texas AgriLife Extension scientists have planted an experimental vineyard in Industry, Texas with grape varieties bred by scientists in Texas, Florida and California that may have PD
resistance. PD resistant vines should maintain low levels of X. fastidiosa. The objective of this study is to evaluate grape isolates for the levels of X. fastidiosa using both ELISA and QRT PCR. The ELISA method
requires a high bacterial concentration for a positive result. QRT PCR can be a more sensitive technique but is difficult to optimize. Here, we show results of an X. fastidiosa specific probe that shows promise for
analyzing X. fastidiosa samples from the vines in the experimental vineyard.
RESULTS
• ELISA test results show that Victoria, U0502-10, U0502-
20, U0502-26, U0502-38 scored slightly positive for X.
fastidiosa (Table 2).
• QRT-PCR using a X. fastidiosa specific probe (Figure 3)
shows a positive result on DNA from X. fastidiosa bacterial
cultures. The blue line represents isolate TRA FLA 420,
green line represents isolate GIL BEC 632 (Figure 4).
DISCUSSION
ELISA results showed that some resistant grape varieties
may actually harbor X. fastidiosa. It is possible these
varieties will not have good resistance the bacterium. This
ELISA test is not a particularly sensitive test and requires
about 104 cells to show a clear positive it is important to
confirm ELISA results with the more sensitive QRT PCR
method. QRT PCR using standard primers has not been
successful for us. This is our first attempt to use a X.
fastidiosa specific probe in a QRT PCR reaction. The
method is now working on DNA from X. fastidiosa
cultures. Our next goal will be to optimize the method on
grape samples from the experimental vineyard so that we
may verify the ELISA data.
REFERENCES
• Hopkins, D. L. & Purcell, A. H. (2002). Xylella fastidiosa: Cause of
pierce's disease of grapevine and other emergent diseases. The
American Phytopathological Society, 86(10), 1056.
• The Texas Pierce's Disease Task Force. (2004, December). Grape
growing. Wine Business Monthly, XI(12), 34. DOI:
www.winebusiness.com
• Golino, D. 2009. FPS Grape Program Newsletter. Foundation Plant
Services, UC Davis. October 2009. 40 pgs.
• Kamas, J., Stein, L., and M. Nesbitt. 2010. Pierce’s Disease Tolerant
Grapes. AgriLife Extension, Texas A&M. October 2010. 4 pgs.
• Kliman, T. 2010. The Wild Vine: A Forgotten Grape and the Untold
Story of American Wine. Random House, New York.
• Mortensen, J. 1987. Blanc Du Bois: A Florida bunch Grape for White
Wine. Agricultural Experiment Station Institute of Food and
Agricultural Sciences, University of Florida. Circular S-340. 5 pgs.
• Schaad, N. W., Opgenorth, D. and P. Gaush. 2002. Real-time
Polymerase Chain Reaction for One-Hour On-Site Diagnosis of
Pierce’s Disease of Grape in Early Season Asymptomatic Vines.
Phytopathology. 92(7):721-728.
ACKNOWLEDGEMENTS
• Funding for supplies in this project were provided by a
Texas Pierce’s Disease Research and Education Grant,
USDA-APHIS.
• Cultures of X. fastidiosa for positive controls and QRT
PCR optimizations are from Dr. Mark Black, Texas
AgriLife Extension.
• We would like to thank Jim Kamas and Andrew Labay
from Texas AgriLife Extension for their assistance in this
project.
Grape Variety Evaluated Genetics/Heritage
Breeding History and
Resistance
Norton A selection of the wild species
Vitis aestivalis.
This selection of wild American
grapes was grown as early as the
1700s in the US because of
excellent disease resistance
(Kliman, 2010)
Blanc Du Bois (BDB) A complex grapevine cross with
parentage that has a grandparent
of the Vitis vinifera variety
(Golden Muscat) and other
grandparents that are the wild
Vitis aestivalis, a variety Pixiola,
and open pollinated.
Bred by the University of Florida
in the late 1960s and released in
the 1980s for excellent resistance
to Pierce’s Disease (bacterial
disease) and Powdery Mildew
(fungal disease). (Mortensen,
1987).
Victoria (Victoria Red) Cross of a Vitis vinifera variety
(Exotic) by another variety
(Ark1123) which is a French
American hybrid (V. vinifera x
wild American species).
Bred by the University of
Arkansas in 1971 for Pierce’s
Disease (PD) resistance. This
variety has survived some PD area
for decades (Kamas, 2010)
U0502-10 Cross of AB1-138 (which is a
Vitis arizonica and V. vinifera)
crossed again to V. vinifera
variety Chardonnay. This variety
is Chardonnay like (87.5% V.
vinifera) with resistance from V.
arizonica.
Bred by UC Davis for Pierce’s
Disease resistance in 2009. The
resistance is from one gene from
Vitis arizonica that is back crossed
to V. vinifera for good flavor
(Golino, 2009).
U0502-20 Another cross of AB1-138 to V.
vinifera variety Chardonnay
(87.5% V. vinifera).
“
U0502-26 Another cross of AB1-138 to V.
vinifera variety Chardonnay
(87.5% V. vinifera).
“
U0502-35 Another cross of AB1-138 to V.
vinifera variety Cabernet
Sauvignon (87.5% V. vinifera).
“
U0502-38 Another cross of AB1-138 to V.
vinifera variety Chardonnay
(87.5% V. vinifera).
“
Table 1. Summary of grape varieties, genetic cross or
heritage and the history behind each variety. Replications of
these varieties were planted at the same time in an
experimental vineyard in Industry, Texas to determine their
ability to resist Pierce’s Disease (PD).
Figure 2. Grapevine leaf form
experimental vineyard showing leaf
scorch, a symptom of PD.
Table 2. Summary of ELISA test
results on experimental grape varieties.
Figure 1. Gram stain of X.
fastidiosa. Gram negative bacillus,
forms clumps within the xylem
(photo from Morano Lab).
Figure 4. QRT PCR results showing Taq probe is working
on DNA extracted from X. fastidiosa cultures.
Summary ELISA Test Results
Positive Varieties Negative varieties
Victoria BDB
U0502-10 Norton
U0502-20 U0502-35
U0502-26
U0502-38
Figure 3. A) TaqMan Probe. B) Probe
binding to target DNA. C) Dye gets
released form the DNA strand created
by Taq (from www.bio.davidson.edu).
A
B
C