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Getting to the Root of ‘Flordaguard’
Rootstock Resistance:
Rootstock Alternatives and Current Efforts
Mary Ann D. MaquilanGraduate student
Horticultural Sciences Department
UF Stonefruit Field DayApril 22, 2014PSREU, Citra
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Peach trees are composite genetic systems
–Genetically distinct scion• Unique fruit quality
• Low-chill adaptation
–Genetically distinct rootstock
• Locally-adapted
• Nematode-resistant
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• Four most common RKN species:
1. M. incognita (MI) – the Southern RKN2. M. javanica (MJ) – the Javanese RKN3. M. arenaria (MA) – the peanut RKN4. M. hapla (MH) – the Northern RKN
Root-knot nematodes (RKN), Meloidogyne spp., are parasitic to many agricultural crops
http://nematode.net/NN3_frontpage.cgi?navbar_selection=speciestable&subnav_selection=Meloidogyne_javanica
Light micrographs of the anterior end of second-
stage juveniles
Three major species present in most areas with tropical and Mediterranean climates
MI MJ MA MH
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Meloidogyne floridensis (MF)common name: peach root-knot nematode
Occurs only in Florida - first detected by Dr. Ralph Sharpe in 1960s in Gainesville, FL
Initially identified as M. incognita Race 3
Characterised as a new species based on morphology and unique esterase isozymepattern (Handoo et al., 2004; Carneiro et al., 2000)
Wide host range and overcomes nematode resistance of ‘Nemaguard’, ‘Okinawa’, ‘Guardian’ and ‘Nemared’ rootstocks
Problem: the peach root-knot nematode infects known resistant peach rootstocks
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Comparison of esterase dehydrogenase profiles ofMeloidogyne floridensis n. sp. (Mf) with that of M. incognita (Mi) and M. javanica (Mj).
Source: Carneiro, R. M. D. G., M. R. A. Almeida, and P. Queneherve. 2000. Enzyme phenotypes of Meloidogyne spp. populations. Nematology 2:645–654.
http://pubs.ext.vt.edu/444/444-107/444-107.html
Adult female (J4) and egg mass
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Root-knot nematode infestation life-cycle
Endophytic phase in the root
Exophytic phase in the soil
I. InvasionJ2 juveniles penetrate the root and migrate intercellulary to reach the vascular cylinder
II. Induction Induces feeding cells, becomes sedentary
III. Nutrient AcquisitionVascularization of feeding site, nematode matures to adult
J1 in eggs
Eggs
J2
J3J4
Female
Male
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Tree stunting and reduced vigor
Damage Caused by RKN in Peach
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Damage Caused by RKN in Peach
• Early defoliation and reduced foliage resulting in the production of unpalatable “stress fruits”
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Damage Caused by RKN in Peach
Galled roots
http://www.freshfromflorida.com/pi/enpp/triology/5001/triology_5001_nematology.html
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No Quick Solutions…
Can’t eliminate nematodes
No post-plant nematicidesavailable for many crop-nematode combinations
No single practice will control nematodes, so two or more control methods must be used
RKN-resistant cultivars –effective against nematode genotypes
Fallow
Sanitation Resistance
Sanitation
Rotation
SuccessfulNematode
Control
Fallow
Solarization
Rotation
ChemicalsResistance
Nematode Management
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• Three commonly used rootstock Nemaguard, Guardian, Nemared, are susceptible to Meloidogyne floridensis
• Nemaguard and Nemared require more winter chill for proper fruiting
• Flordaguard has improved root-knot nematode resistance and low-chill adaptation– Used as standard rootstock
for low-chill peach production in root-knot nematode infested non-alkaline soils
http://hos.ufl.edu/extension/stonefruit/stone-fruit-varieties
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How do we manage the disease while maintaining production and staying
competitive?
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Current Efforts
I. Molecular characterization of resistance in Flordaguard Understand the genetic nature of resistance to
MF in ‘Flordaguard’ peach Identify SSR markers associated with resistance
against MF in various segregating populations
II. Evaluation of horticultural performance of peach rootstocks Identify potential rootstocks with resistance
to MF
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Resistance spectrum of main Prunus RKN-resistant sources (Claverie et al., 2004; Van Ghelder et al., 2010; Cao et al., 2011)
Rootstock MA MI MJ MF RKN resistance genotype
Almond (P. dulcis)
‘Alnem1’ R S R S RMja / RMja
Peach (P. persica)
‘Shalil’ R R S S RMia / rMia
‘Nemaguard’, ‘Nemared’ R R R/S* S RMiaNem / RMiaNem
(G x N)15 = ‘Felinem” R R R/S* S RMiaNem / rMiaNem
Myrobalan plum (P. cerasifera)
Accession P.2175 R R R R Ma / maJapanese plum (P. salicina)
Accession J.222 R R R R Rjap / rjap
Wild peach (P. kansuensis)
‘Honggengansutao’ -- I** -- -- PkMi tested only on MI; possibly an allele of RMia
*R/S – variable behaviour in function of M. javanica isolates**I = Immune
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Wild peach (P. kansuensis)
Flordaguard(P. persica)
‘Okinawa’ (P. persica)
‘UF Sharp’ (P. persica)
Parental Genotypes for the Crosses
rr
RR
rr
RR
and hybrids with P. kansuensisRr
and hybrids with P. kansuensisRr
325 cu
300 cu
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Manual pollinations (late Jan – early March)
Pollen collection (late Jan – early Feb)
Fruit harvesting & seed extraction (April – May)
Seed stratification and
germination (Jun – Aug)
Growing plants in greenhouse(Sept – Nov )
Generating the mapping populations for phenotyping
Screening for MF resistance(Dec - Mar)
1
2
34
5
6
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Parent 1 (P1) Parent 2 (P2)
DNA isolation
Screening parental genotypes with molecular markers
Marker A
Marker B
Marker C
Marker D
P1 P2
Screening of parental genotypeswith SSR markers
Markers A, C, and D can differentiate the two parental genotypes
https://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechSTS.shtml
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Current Efforts…
II. Evaluation of horticultural performance of peach rootstocks Identify potential rootstocks with resistance
to MF
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Flordaguard Barton Okinawa MP-29 P-22
Peach Rootstocks
Scion: UFSun
1 2 3 4 5 6 7 8 9 10
Rutgers Tomato
Peach rootstock field evaluation for Resistance to M. floridensis
**
*
*
*
*
**
*
**
*
*
*
**
*
*
****
***
**
*
**
*Replaced
trees
FG = 21P-22 = 6Ok = 4MP-29 = 3B = 3
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• Location: Citra, Florida
• Plot size: 200 ft x 130 ft
• Design: RCBD with 5 subsamples per treatment
• No. of rows (replicates): 10
• Spacing: 4ft between trees, 20ft between rows
• Treatments: Flordaguard (R), Barton (R), Okinawa (S), and 2 new USDA rootstocks (R)
Experiment Design
Photo courtesy of Dr. Andrew Nyczepir, USDA-ARS Byron, Georgia
Plants established in microplots
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Setting up the microplots
Auger was used to
excavate holes
Trenches at plot borders for installation of sprinkler irrigation system
Microplots were laid out in rows and
fumigated soil was backfilled into pots
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Budding on ‘Barton’ and ‘MP-29’
Barton
MP-29
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Nematode inoculation for resistance evaluation
Total of 45,000 juvenile nematodes inoculated per plant
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2013 Scion and rootstock trunk circumferences (2 inches above and below graft union) of grafted trees in field microplots at Citra, FL.
0
2
4
6
8
10
12
Okinawa P-22 Flordaguard Barton MP-29
ScionRootstock
a
a
a
a
bb
bc
bc
cc
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Scion and rootstock trunk circumferences (2 inches above and below graft union) of grafted trees in field microplots
02468
1012141618
Okinawa P-22 Flordaguard Barton MP-29
ScionRootstock
aa
aa
b
b
bc c cc
field-budded
nursery-budded
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0
0.2
0.4
0.6
0.8
1
1.2
MP-29 Barton Flordaguard P-22 Okinawa
ScionRootstock
Relative growth rates of scion based on trunk circumference (2 inches above graft union) at the end of 2013 growing
season in field microplots
a
aa
ab
b b b b b
nursery-budded
field-budded
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Pearson correlations between the scion trunk circumference and selected growth (n = 50)
*, P=0.02; **, P=0.001.
Parameter Correlation coefficient, r
Scion trunk Pruning weight 0.83**circumference Tree height -0.28**
Tree spread 0.88**Scion trunk relative growth rate -0.33*
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More studies underway…
II. Testing of ‘Flordaguard’ for durability of resistance against four main root-knot nematodes – M. arenaria, M. incognita, M. javanica, and M. floridensis
III. Histological characterization of resistance in ‘Flordaguard’
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Terminal root fragments, 10 mm long
1, 2, 3, 5, 8, 12, 17, 23, 30, 40 dpost-inoculation
Examine for nematode penetration and root galling
Chemical fixation with 2% glutaraldehyde
overnight
Processing – dehydration through graded ethanol series and clearing
Infiltration and embedding with epoxy resin
2 d
Sectioning, 5 um
Staining
Microscope examinationsStained slides
Unstained slides for phenolic compound examination
Histological characterization of resistance
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• UF Stonefruit breeding program• Continued efforts to develop improved rootstocks for the
peach industry
• Nurserymen• Maintain nematode-free nursery stock• Ascertain rootstocks’ trueness-of-type
• Growers• Monitor nematode numbers• Report any signs of nematode infestation• Send soil/root samples to nematode diagnostics lab to
identify nematode species
Nematode management requires a concerted effort…
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Seedlings UF
Beau
tyO
kina
wa
Nem
agua
rdP.
kan
suen
sis
Tard
y N
onpa
reil
Citra orchardClones Co
mm
’lnu
rser
y
Com
m’l
orch
ard
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Com
m’l
orch
ard
2
BPPCT008
UDP98-025
CPSCT034
* * * * * * * *
* * **
237/237
112/112
133/133
* ** * * *
MARKERS
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Assessing trueness-of-type
OUTCROSS!
TRUE-TO-TYPE (red color fades when exposed to warm temperatures)
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Resistance-breaking nematodes?
True-to-type ‘Flordaguard’ peach rootstock, confirmed from genotyping analyses
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Collecting root and soil samples for nematode diagnostics
Send samples to: UF Nematode Assay Lab nematology.ifas.ufl.edu
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Acknowledgment
Florida Department of Agriculture and Consumer Services (FDACS) Specialty Crop Block Grant
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Lineage of ‘Flordaguard’ Rootstock