Root-Knot and Reniform Nematode Infection of Cotton Hairy Roots

1
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

2
Plant-parasitic nematodes of cotton

• 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

Root-Knot Nematode Meloidogyne incognita Renif
orm Nematode Rotylenchulus reniformis
3
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)

4
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
5
Primary objectives

• 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

6
Root-knot nematode inoculations
NaOCl extraction
Sugar floatation
Hatch chamber
Surface sterilization
Apply nematode / LMP suspension directly to the
root tips
Suspend in 1.5 low-melting-point agarose
7
RKN infection of cotton hairy roots
Galls
7 days after inoculation with infective RKN J2
10 days after inoculation with infective RKN J2
8
RKN infection of cotton hairy roots
17 days after inoculation roots stained with acid
fuchsin
9
RKN infection of cotton hairy roots
17 days after inoculation roots stained with acid
fuchsin
10
RKN fourth-stage juvenile
RKN third-stage juvenile
11
Gelatinous matrix exuded by RKN female (45 dai)
RKN egg production on cotton hairy root
culture (45 dai)
12
Reniform nematode inoculations

• Autoclaved sand
• Growth chamber
• Egg inoculum

Baerman funnel
Surface sterilization
Apply nematode / LMP suspension directly to the
root tips
Suspend in 1.5 low-melting-point agarose
13
Reniform nematode infection of cotton hairy roots
11 days after inoculation roots stained with
acid fuchsin
14
Reniform nematode on cotton hairy roots 24 days
after inoculation roots stained with acid fuchsin
15
32 days after inoculation
16
complete egg mass
mature female
32 days after inoculation
17
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

18
Acknowledgements

• USDA-ARS, MS State
• Dr. Russel Hayes
• Dr. Johnie Jenkins
• Mr. Kimber Gorley

• USDA-ARS, Lubbock
• Dr. Forest Robinson
• USDA-ARS, Stoneville
• Dr. Erik Sacks
• USDA-ARS, New Orleans
• Dr. Barbara Triplett