The objective of this experiment is to determine how plant growth and rhizosphere carbon availability change when roots are infected by parasitic nematodes.  Furthermore, we want to know how changes in the quantity and quality of rhizosphere carbon influence the structure and function of the soil microbial community. 

Clover will be grown in sand with or without the presence of a parasitic nematode.  Rhizosphere carbon solutions will be collected from these systems periodically for chemical analysis.  These solutions will drip from the plant into columns containing soil.  After application of the solutions, the microbial communities in the soils will be comparatively analyzed.  Changes in rhizosphere chemistry will be linked to any microbial responses. 

Future experiments involving grass and nematode complexes will provide complementary data and an emerging understanding of how plant-parasitic nematodes modify the rhizosphere environment and influence microbial diversity and function in grasslands. 

(1) Nematode infection will affect plant biomass negatively in this experiment, where nutrients are not limiting and there is no positive microbial feedback from the activity of rhizosphere organisms.  We recognize that in the soil environment, in the presence of a natural rhizosphere microbial community, lower levels of nematode plant parasitism may have a positive effect on plants by enhancing microbial activity and nutrient availability in the rhizosphere.  Furthermore, in a nutrient limiting environment, root damage by nematodes may illicit a 'compensatory growth' response below ground, such that nutrient acquisition by the plant is enhanced.  However, we predict that the overall effect of the nematodes in our system will be negative. 

(2) Rhizosphere carbon sources (including root exudation) will change quantitatively and qualitatively when plants are infected with nematodes.  This hypothesis will be evaluated on the basis of (1) each individual plant and (2) per unit of plant biomass.  We predict that although plant biomass is reduced by nematode infection, rhizodeposition per unit of plant biomass will be enhanced due to root damage and leaking. 

(3) Microbial communities receiving rhizosphere solutions from nematode infected plants will have higher activity and biomass, reflecting increased carbon supply.  These microbial communities will also show changes in substrate utilization patterns, reflecting qualitative changes in rhizosphere chemistry.  We predict that microbial responses will be specific to particular microbial groups, as reflected in analysis of community PLFAs and plate counts of important heterotrophic groups (psuedomonads, fungi).

Heterodera trifolii, the clover cyst nematode, is a sedentary endoparasite of clover roots.  Juveniles enter the roots around the root hairs and can invade up through the root, causing cell damage and death.  At its final site in the root, the juvenile's feeding induces an initial feeding cell to enlarge, and adjacent cells are incorporated by wall dissolution to form a multi-nucleate cell complex called a syncytium.  The development of the nematode into an enlarged female often causes the root to split open, exposing the female to the outside.  When the female dies, its body forms a protective sheath around the eggs that is known as a cyst.  The eggs within the cyst are contained within a gelatinous matrix.  The generation time (egg to egg) of this nematode can range from 20 to 70 days, depending on temperature and other environmental conditions. 

Root penetration, feeding, cell enlargement, and rupture of the cortex are all aspects of nematode infection that could trigger pulses of rhizodeposition. 

Clover plants will be subject to infection by H. trifolii while growing in "Microcosms".  Rhizosphere solutions will be collected from the plants and chemically analyzed.  Solutions will also be allowed to drip onto soil to stimulate the soil microbial communities. 

(1) Top Chamber:  70 ml syringe barrel, containing muffled (carbon-free) sand, with a small layer of muffled gravel on the bottom, and a small layer of cotton wool in between.  The cotton wool is to prevent sand/nematode/root escape and the gravel is to promote drainage through the system.  Units will be autoclaved.  Germinated seeds will be transferred into this top 'plant' chamber.  Each plant chamber will be equipped with a single glass tube, 3 cm in length, embedded into the sand, to facilitate nematode introduction to root systems.  These chambers will be contained on a rack with a polycarbonate cover that has ports for pumping in sterile air and nutrient solutions. 

(2) Lower chamber:  Also syringe barrels.  Will contain fresh Sourhope soil (4mm sieved, plant material removed).  Fresh soil will be mixed 1:1 by weight with dry, muffled, autoclaved sand.  Addition of sand is to improve drainage, reduce the volume of soil that solutions are being applied to.  Solution dripping out of these lower chambers will be collected in a 'waste' bottle and disposed.  Application of solutions to soils will commence when plants are introduced to system.  These chambers will be contained on a rack directly below the top chambers. 

(3) Rhizosphere solution flow:  Between the two chambers will be a 3-way leur lock stopcock that will normally be set in a position that allows any solution that drips out of the plant chamber to drip into the soil chamber.  In order to collect rhizosphere solutions for chemical analysis, the stopcock will be set so that solution comes out of the side port, through a section of tubing, draining into a poly bottle.  To collect solutions, pumps will be set to a high flow rate and bottles will be left overnight to obtain approximately 60 ml of solution.  Solution volume will be estimated by weighing the bottles after collection of solutions and subtracting the mass of the empty bottle. 

Photos of system:
 

In order to quantify potential changes in rhizosphere chemistry it is necessary to attempt to manipulate plants in an aseptic environment, where there are no micro-organisms present to modify the compounds being released by the roots.  The challenge in this experiment is to design treatments that allow for this to some extent, while at the same time acknowledging the importance of the Rhizobium/Trifolium association and the difficulties presented in introducing aseptic nematodes to the system.  Our treatment selection attempts to address this challenge. 

Design: Randomized Complete Block (8 blocks)
Treatments (each replicated 8 times):
(1) Control Trifolium plants (no nematodes)
(2) Trifolium + Heterodera
(3) Trifolium + Rhizobium
(4) Trifolium + Heterodera and Rhizobium
(5) Heterodera only
(6) Non-plant Blanks
(7) (8) Extra plants for mid-experiment harvests to assess nematodes (degree of infection, life stage, population growth).  Will receive Rhizobium.

Sterile clover plants will be established from para-acetic acid sterilized seeds in petri plates on sand.  The appropriate treatments will be inoculated with Rhizobium at the time of seed germination.  Several days after germination, seedlings will be transferred to the microcosm system, one plant per plant chamber.  Plants will be grown for approximately 12 weeks, or until the root system is developed.  All plants will be supplied with 0.5 mM N nutrient solution at a flow rate of 12 ml/day.  This flow rate will represents an application of solution to the soils that is higher than the Sourhope average daily rainfall of 2.3 mm/day (which converts to 1.85 ml/day applied to chambers).  Plants and soils will be maintained at a growth room temperature of 20C, with a 16h photoperiod.  During the light part of the cycle, air temperature will be set at 5C.  The polycarbonate cover for the top chamber will elevate the temperature within so that plant growth is occurring at a slightly higher temperature.  The chambers, including the root-containing top chamber, will be kept in the dark by covering with plastic sheeting.  Temperature and PAR will be measured throughout.  At the time of nematode addition, the dark temperature for the chamber will be lowered to 12C to ensure cool conditions for nematode establishment.  After one week, temperature will be returned to the original settings.

Once plants are established (8-12 weeks), nematodes will be added to the appropriate treatments.  Nematodes will be surface sterilized briefly with antimicrobial solutions.  Nematode density in solution will be estimated and approximately 100 jjuvenile nematodes will be added to each unit, 2 times over the first 4 days of the experiment.  Rhizosphere solutions will be collected every 7 days over the next 30-45 days.  The number of nematodes in the solutions will be assessed, to evaluate the impact of solution collection. 

At harvest the following measurements will be made:
 

Schedule:
 

References: 

Bardgettt RD, Cook R, Yeates GW, and Denton CS.  1999.  The influence of nematodes on below-ground processes in grassland ecosystems.  Plant and Soil.  212:23-33.

Cook R and GW Yeates.  1993.  Nematode pests of grassland and forage crops.  In:  K Evans, DL Trudgill, & JM Webster (Eds.)  Plant Parasitic Nematodes in Temperate Agriculture.  Oxon, UK: CAB International, pp. 305-350.

Denton CS, Bardgett RD, Cook R, and Hobbs PJ.  1999.  Low amounts of root herbivory positively influence the rhizosphere microbial community in a temperate grassland soil.  Soil Biology & Biochemistry. 31:155-165

Pederson GA and Quensberry KH.  1998.  Clovers and other forage legumes.  In:  KR Barker, GA pederson, & GL Windham (Eds.)  Plant and Nematode Interactions.  Madison, WI: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, pp399-425.

Yeates GW, Bardgett RD, Mercer CF, Saggar S, and Feltham CW.  1999. The impact of feeding by five nematodes on 14C distribution in soil microbial biomass and nematodes: initial observations.  Proceedings of the New Zealand Society for Parasitology. 

Yeates GW, Saggar S, Denton CS, & Mercer CF.  1998.  Impact of clover cyst nematode (Heterodera trifolii) infection on soil microbial activity in the rhizosphere of white clover (Trifolium repens) - a pulse-labelling experiment.  Nematologica 44:81-90.