Symbiotic Fungi: Principles and Practice (Soil Biology)

24 Auxin Production by Symbiotic Fungi 391
l Drying gas (nitrogen) pressure: 18 psi (pounds per square inch, equivalent to 124
kPa) at 250 C, nebulizing gas (air): 50 psi (345 kPa)
l Pressure of collision gas (argon): 1.4 mTorr
l Needle voltage: 4400 V
l Shield voltage: 600 V
l Capillary voltage: 40 V
l The mass spectrometer was operated with negative Electrospray ionization in
multiple reaction monitoring mode.
l Mass transition for IAA: m/z 173.9/ m/z 130.0 (collision energy (CE) 9.0 eV)
l Mass transition for D 5-IAA: m/z 178.8/ m/z 134.0 (CE 11.5 eV)
A calibration curve of the ratio of peak areas of unlabeled standards to peak area
of deuterium labeled IAA was used for quantification.
Remark: Because IAA occurs in common media components, extracts of media
in which no fungus had been cultured should always be analyzed as negative
controls.
If any IAA is found in the pure medium, its concentration has to be subtracted
from the IAA concentrations in culture filtrates of the fungus. Obviously, the use of
media which do not contain detectable amounts of IAA is preferable.
24.5 Examples of IAA Production by Fungi
24.5.1 Piriformospora indica
Piriformospora indica is an endophytic fungus which increases plant growth in
many plants of different families (Varma et al. 1999). In contrast to arbuscular
mycorrhizal fungi, P. indica grows well on artificial media, and it is also able to
colonize cruciferous plants including A. thaliana. In many interactions increased
shoot growth was accompanied by increased root biomass. The mechanism by
which this beneficial effect is achieved has long been debated. In our bioassay,
A. thaliana inoculated with P. indica showed increased root branching and reduced
main root length (‘‘bushy roots’’), whereas the roots of uninoculated plants reached
the bottom of the Petri dish after 2 weeks (Figs. 24.2a, b). Similarly enhanced root
branching was observed after inoculation of the mycorrhizal plant Lotus japonicus
with P. indica (Figs. 24.2c, d). The phenotype resembled very much typical auxin
effects. Therefore we compared the effect of pure externally applied auxin in
different concentrations (Figs. 24.2e, f, for details see Sirrenberg et al. 2007) with
the effect of P. indica culture filtrates and its ethyl acetate extracts. The pure culture
filtrate as well as the extracts inhibited main root length while increasing root
branching. The IAA content in the culture filtrate was determined by HPLC-MS
and GC-MS to be 1.36 +/ 0.36 mM (after 4 weeks of growth in M+ medium at
23 C on a rotary shaker (Sirrenberg et al. 2007).