In inoculation programs, since hyphae cannot grow from the inoculum to roots, inoculum must be placed in the rooting zone of seedlings where roots can grow into the inoculum (Marx and Kenney 1982). In principle, natural and vegetative inoculum can be applied by three methods:
- Mixed with the rooting medium
- Banded or layered below seeds or seedlings
- Suspended in water and poured onto seedlings or dipping seedlings into the slurry before planting (except of beads)
Bareroot and container-grown seedlings and cuttings of numerous tree species were inoculated with vegetative inocula of many ECM fungi in greenhouse and nursery experiments. Pisolithus tinctorius (Marx and Bryan 1975; Marx et al. 1982; Marx and Cordell 1989; Vijaya and Srivasuki 1999; Rincon et al. 2001) and genus Laccaria (Mortier et al. 1988; Kropacek and Cudlin 1989; Gagnon et al. 1995; Garbaye and Churin 1997; Parlade et al. 1999; Baum et al. 2002; Gange et al. 2005; Machon et al. 2006) have been the most often tested fungi in inoculation experiments. ECM seedlings or cuttings are produced through inoculation of different growing substrates. Soil and peat mixed with vermiculite or other substrate components - the standard substrates for production of planting stock of forest tree species, are convenient and most frequently used for ECM inoculation.
Unequivocally, the most preferred technique of application of organic matter and vegetative inoculum is mixing it with a growth substrate. Amount of inoculum mixed with substrate in pot experiments is most commonly expressed as a ratio of inoculum and substrate by volume. Organic matter (humus, litter, rotten wood) is added to substrate in a larger ratio (1:4 to 1:1) (Kropp 1982; Parke et al. 1983; Repac 1996a) than vegetative inoculum. Vermiculite-peat inoculum was mixed with potting substrate at the proportion 1:4 (Rincon et al. 2001), 1:10 (Hortal et al. 2009), as well as 1:64 (Rincon et al. 2001). Baum et al. (2009) expressed a ratio of soil:vermiculite-peat inoculum mixture (10:1) by weight. Parlade et al. (1999) and Baum et al. (2000, 2002) carried out inoculation by mixing alginate beads with potting substrate in the proportion 1:20 (v:v). Before filling containers, Gagnon et al. (1991) mixed alginate beads with the substrate so that each seedling received a volume of 17 ml of beads. Gagnon et al. (1995) mixed liquid inoculum with substrate in a cement mixer to get a final volume of 30 ml of mycelial slurry per seedling.
In nursery bed experiments, vegetative inoculum was broadcast evenly over nursery bed (0.5-2.8 l.m~2) and incorporated into the upper 10-12 cm of substrate with handtools (Marx et al. 1978; Mortier et al. 1988; Duponnois and Garbaye 1991; Repac 1996b; Garbaye and Churin 1997). Marx and Bryan (1975) reported application of P. tinctorius inoculum to fumigated soil at a ratio 1:8 (v:v) in upper 10 cm layer of plot.
Cline and Reid (1982) injected 20 ml of mycelial slurry into a root zone of container-grown seedlings at a depth of 6-8 cm using a glass syringe. Garbaye et al.
(1988), Machon et al. (2006), and Gange et al. (2005) added 50 ml of vermiculite-peat inoculum and 20 ml of mycelial slurry, respectively, into containers in contact with the roots of transplanted seedlings. Aucina et al. (2007) placed a layer of pine or oak litter on the surface of the nursery bed soil with seedlings to influence the growth and ECM communities of seedlings. Kropacek and Cudlin (1989) and Repac (2007) evenly spread alginate-bead inoculum below surface of substrate immediately before seed sowing.
Perhaps the most capable way of expression of amount of vegetative inoculum is in a dry weight of mycelium, enabling the comparison of inoculum rate between experiments of different patterns. Unfortunately, to ascertain quantity of mycelium in vermiculite-peat carrier is almost impossible. Nevertheless, Mortier et al. (1988) reported 1-2 g of mycelium (dry weight) per m2 in this type of inoculum. Mortier et al. (1988), Duponnois and Garbaye (1991), Vijaya and Srivasuki (1999), and Repac (2007) referred 2 g, 2-10 g, 4 g, and 5 g of mycelium in dry weight per m2, respectively, in alginate-bead inoculum. In the pot experiments, each seedling received either 6.82 mg (Gagnon et al. 1991) or 12 mg (Gagnon et al. 1995) of dried mycelium or 20-80 mg of mycelium in fresh weight (Parlade et al. 1999).
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