Establishing single species (monospecific) cultures of AMF is a task that requires patience and attention at every step during the process. The method used in CICG to establish monospecific cultures is through the inoculation of spores into pre-germinated brachiaria (Brachiaria brizantha) seedlings. Therefore, the origin of the spores (from the field or from trap cultures) is extremely important for the success of monospecific cultures. In some cases, the spores can come directly from the field soil, although the percentage of success in this case can be low or null. An exception occurs when spores are from soils with low organic matter (e.g., sand dune soils) or spores are of exceptionally good quality (no evidence of parasitism, clear spore content). When the spores used to establish monospecific cultures come from trap cultures, the success rate is high. This is because the spores obtained from trap cultures were produced in the last 3-4 months and are therefore healthy and all of the same age. In addition, they have all the important phenotypic characters for accurate species identification.
The spores are extracted via wet sieving and sucrose gradient (20%-60%) from the field or trap cultures just 2-3 days before inoculation. Afterwards, the spores are separated by morphotypes, removing them from the Petri dish using a Pasteur pipette (with an extruded tip), and placed in a watch glass. Until the time of inoculation, they are kept clean in this watch glass, which is placed in a Petri dish and kept in the refrigerator (4o C). This time in the refrigerator is important to reduce the infectivity of any piece of hypha that may eventually be present with the spores. The spores are examined daily and spores that show any change in morphology or have evidence of parasitism are removed.
Before inoculation, a little water is added to the watch glass and the spores stirred up with the use of a needle, and any foreign particles, bits of hyphae or old spores are removed.
IMPORTANT: The watch glass must be carefully inspected to eliminate any pieces of hypha that may be present with the spores or floating in the water. This is because hyphae of some AMF species are highly infective and, if inoculated together with spores, can be one source of contamination, which is often only detected after several cultivation cycles.
Monospecific cultures are established in black plastic cones with three grooves and a capacity of 270 ml of substrate, into which 3 plantlets of the host species are transplanted (see below). These cones are first sterilized by keeping them in 10% bleach for 3 days, washed under running water and stored in clean plastic bags. The cones are filled with the standard substrate used in CICG: a mixture (1:1) of soil and quartz sand (particle size = 0.9 mm) sterilized with 5% of vermicompost. The cones are then labeled with the species name, isolate code and date. A hole is made in the center of each cone using a glass rod previously sterilized with 70% alcohol and passed through a flame.
Every week, a pot (500 ml) containing the standard substrate is prepared by sowing 30-40 brachiaria seeds. This amount is enough to start 10-13 pure cultures per week. Our host plant is brachiaria, as it resists transplanting, develops well under the conditions of our greenhouse and is not attacked by ants. You can also use other host plants such as sorghum, millet, or even a legume; the important thing is for the plant to have a well-developed root system and to grow well under greenhouse conditions. The contents of this pot are gently placed on a tray or in a clean container with tap water approximately one hour before establishing monospecific cultures. Afterwards, groups of 3 seedlings are separated, which will be inoculated with the clean AMF spores that are in the watch glass.
The three seedlings are placed together and the roots rinsed under tap water to place them all together. Afterwards, the spores are collected using a Pasteur Pipette (a clean pipette for each fungal isolate) and pipetted along the root system of the three seedlings. The spores stick to the roots and excess water runs off the end of the roots.
The number of spores to be pipetted into each set of three seedlings depends on the AMF genus. For gigasporoid (e.g.Gigaspora, Scutellospora, Racocetra) and glomoid (e.g., Paraglomus, Glomus, Septoglomus, Funneliformis) spores that germinate quickly, about 50-70 spores are inoculated, while for Acaulospora and Entrophospora we use at least 100 spores.
The amount of spores will certainly depend on the amount of spores that we can extract from a trap culture. Some species sporulate in low amounts in trap cultures and if there is interest in obtaining isolates of these species, a smaller number of spores is eventually used.
The seedlings are then immediately transplanted to the center of the cone and the glass rod used to accommodate the seedlings in the hole and compact the substrate around the roots so that the seedlings stand upright. Afterwards, CICG’s standard substrate is placed gently on the top of the cone and around the seedlings, leaving a space for watering. All cones are watered and transported to the greenhouse which contains only monospecific cultures. Although the standard substrate is very poor in nutrients, fertilization does not take place within the first 30 days to stimulate spore germination and mycorrhizal colonization. Usually only after 45 days is added 50 ml per cone of Hoagland’s nutrient solution with 10% P only.
Adding the standard substrate to the top of the cone is an efficient way to avoid cross contamination and allows several monospecific cultures to be grown at the same time in the greenhouse, with minimal care when watering.