Spot-inoculation
Spot-inoculation protocols have been developed for a variety of legumes but these techniques have not been easily transferred to pea. We developed a highly reliable rhizobial spot-inoculation technique by growing seedlings in growth pouches (see Materials and Methods) and placing a drop of the rhizobial inoculum on a lateral root located in the zone that is most susceptible to infection. We found that the substrate and the method of delivery of the inoculum were keys in the success of pea spot-inoculation. With the procedure presented in Material and Methods and in Figure 4, a nodule formed within ~0.5 cm from the intended location in 95-100% of the cases (Figure 5). Using this technique, it becomes possible to determine the exact age of the nodule from the time of inoculation to 42 days thereafter. We were able to apply what we learned with the rhizobial spot-inoculation and we targeted mycorrhizal infection.