Several inoculation techniques have been used:
- Mixing either crushed sporocarps or dry spores directly into soil or container medium
- Mixing spores with a moistened carrier, such as vermiculite, kaolin or sand, broadcasting onto soil and then mixing into the nursery soil or the growing medium of containers
- Dusting dry spores onto soil around young seedlings and leaching them into the root zone
- Suspending in water and drenching, irrigating, or injecting into growth substrate
- Dusting or spraying on roots of nonmycorrhizal seedlings
- Mixing with the pelletizing matrix and encapsulating or coating seeds before sowing
Marx et al. (1978) broadcast spores of Pisolithus tinctorius added to moist vermiculite onto soil and mixed inoculum with the soil using handtools. The spore quantities were 108, 324, or 648 mg of spores.m~2 of soil surface. Rincon et al. (2001) mixed dry spores of P. tinctorius and Scleroderma verrucosum included in vermiculite with potting substrate and filled containers with 103-108 spores per 175 ml of substrate.
Perhaps the most used method is application of spore water suspension to growth substrate. Marx and Bryan (1975) poured basidiospores of P. tinctorius (10 g suspended in 500 ml of distilled water with 1 drop of Tween 20) into microplots which were heavily watered to wash the basidiospores into the soil. Theodorou (1984) inoculated seeds of Pinus radiata with basidiospores (5.15 x 104 spores per seed) of Rhizopogon luteolus 1-2 days before sowing. Suspension of spores of R. luteolus (4.46 x 107 spores.mP1) was sprayed onto soil, and the spores were mixed with the soil by raking and rolling and by watering 1 day after sowing of inoculated seeds (i.e., 2 days after soil inoculation).
In experiments of Roldan et al. (1996) and Querejeta et al. (1998), water suspension of spores of Pisolithus arhizus was applied three times, 1 month apart, 12 weeks after sowing of Pinus halepensis to 300-ml plastic bags filled with 3:1 soil/peat mixture. Each plant received 5 x 105 spores per application. Molina et al. (1997) applied spore slurries of six Rhizopogon spp. to container-grown Pseudot-suga menziesii and Pinus ponderosa seedlings over two inoculations. For each inoculation, 10 ml of diluted spore suspension was pipetted onto the peat-vermiculite substrate. Chen et al. (2006) inoculated Eucalyptus urophylla seedlings 2 weeks after transplanting to plastic pots with 10 ml of spore suspension at a rate 106 spores per seedling. The spore slurry was added to a 2-3 cm deep hole near the plant using a 5-ml pipette. Parlade et al. (1996b), Rincon et al. (2001), and Hortal et al. (2008) inoculated 1-month-old container-grown seedlings with spore suspension of several ECM fungi at the rate of 102-108 spores per seedling. Nunez et al. (2006) applied spore suspension of Tuber melanosporum by injecting manually into each seedling's pot substrate (400 ml of light and dark peat and vermiculite, 2:1:1) approximately 7.5 x 105 spores at 3-8 cm depth. In the work of Becerra et al. (2009), 1 ml of spore suspension of two Alpova species containing 106 spores was inoculated at the base of Alnus acuminata seedlings.
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