root-knot and reniform nematode infection of cotton hairy roots

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