root-knot and reniform nematode infection of cotton hairy roots
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Root-Knot and Reniform Nematode Infection of Cotton Hairy Roots. Martin J. E. Wubben and Franklin E. Callahan USDA-ARS Crop Science Research Laboratory Genetics and Precision Agriculture Research Unit Mississippi State, MS. Plant-parasitic nematodes of cotton. Sedentary root parasites - PowerPoint PPT PresentationTRANSCRIPT
Root-Knot and Reniform Nematode Infection of Cotton Hairy Roots
Martin J. E. Wubben and Franklin E. Callahan
USDA-ARSCrop Science Research Laboratory
Genetics and Precision Agriculture Research UnitMississippi State, MS
Plant-parasitic nematodes of cotton
Root-Knot NematodeMeloidogyne incognita
Reniform NematodeRotylenchulus reniformis
• Sedentary root parasites
• Require living cells to siphon nutrients from the host root
• Inflict significant annual yield losses
• Incorporation of genetic resistance into elite cultivars is ongoing
• Molecular aspects of their interaction with cotton remain largely unstudied
Advantages of an in vitro system• Provide a controlled environment
- limit abiotic stresses- eliminate secondary infections- requires a growth chamber and not a greenhouse (saves space)
• Feeding reniform nematodes can more easily be collected for functional genomic studies
• Candidate resistance gene screening
• Increase throughput for testing alternative biotech-driven control methods (example: RNAi)
Cotton hairy roots• Established for soybean, tomato, tobacco, coffee and others in studying nematode parasitism
• Arabidopsis lacks resistance to root-knot and reniform nematode
• Cheaper and easier to generate transgenic hairy roots versus whole plant transformation
Hairy root initiation on cotton cotyledons
• Optimize hairy root growth conditions for propagation and nematode inoculation
• Optimize protocol for inoculum collection and surface-sterilization of infective nematode life-stages
• Verify nematode life-cycle completion on the susceptible hairy root culture DP90
Primary objectives
Root-knot nematode inoculations
NaOClextraction
Sugarfloatation
Hatchchamber
Surfacesterilization
Suspend in 1.5 %low-melting-point
agarose
Apply nematode / LMP suspension
directly to the root tips
7 days after inoculation with infective RKN J2
10 days after inoculation with infective RKN J2
RKN infection of cotton hairy roots
Galls
RKN infection of cotton hairy roots
J2
J3
17 days after inoculationroots stained with acid fuchsin
RKN infection of cotton hairy roots
17 days after inoculationroots stained with acid fuchsin
RKN fourth-stage juvenile RKN third-stage juvenile
Gelatinous matrix exuded by RKN female(45 dai)
RKN egg production on cotton hairy root culture
(45 dai)
Reniform nematode inoculations
Surfacesterilization
Suspend in 1.5 %low-melting-point
agarose
Apply nematode / LMP suspension
directly to the root tips
Baermanfunnel
- Autoclaved sand- Growth chamber- Egg inoculum
11 days after inoculation roots stained with acid fuchsin
Reniform nematode infection of cotton hairy roots
Reniform nematode on cotton hairy roots 24 days after inoculation roots stained with acid fuchsin
live feedingnematodes
32 days after inoculation
maturefemale
completeegg mass
32 days after inoculation
Research in progress• Hairy root lines from root-knot and reniform
nematode resistant plants are being propagated
• Determine best method for measuring nematode reproduction (galls, eggs, females, ….)
• Histopathology of susceptible and resistant interactions
• Candidate gene over-expression in susceptible root culture
Acknowledgements
USDA-ARS, MS StateDr. Russel Hayes
Dr. Johnie JenkinsMr. Kimber Gorley
USDA-ARS, LubbockDr. Forest Robinson
USDA-ARS, StonevilleDr. Erik Sacks
USDA-ARS, New OrleansDr. Barbara Triplett