Compost Teas: 24 hour, EM, IM, BD & fermented plant extracts

[Steve Diver <steved AT ncat.org>]

Two items on AM fungal colonization of brasscias / crucifers;
factors affecting fungal mycelial root penetration... parasitic
vs. true symbiosis; insights from non-mycorrhizal plants.

Regards,

Steve Diver

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1.

Meeting a non-host: the behaviour of AM fungi
Manuela Giovannetti and Cristiana Sbrana
Mycorrhiza (1998) 8 :123–130
http://mycorrhiza.ag.utk.edu/giovannetti_1998_mycorrhiza.pdf


Abstract

Arbuscular mycorrhizal (AM) fungi are obligately
biotrophic organisms that live symbiotically with
the roots of most plants. The establishment of a
functional symbiosis between AM fungi and host
plants involves a sequence of recognition events
leading to the morphological and physiological
integration of the two symbionts. The developmental
switches in the fungi are triggered by host signals
which induce changes in gene expression and
a process leading to unequivocal recognition
between the two partners of the symbiosis. It
has been calculated that about 80% of plant
families from all phyla of land plants are hosts
of AM fungi. The remaining plant species are
either non-mycorrhizal or hosts of mycorrhizas
other than the arbuscular type. Non-host plants
have been used to obtain information on the
factors regulating the development of a functional
symbiosis.


Selected excerpts:

The following stages of the life cycle of AM fungi
are analysed in detail: spore germination,
presymbiotic mycelial growth, differential branching
pattern and chemotropism, appressorium formation,
root colonization.

>>>

AM fungi are obligate biotrophs living symbiotically in
the roots of host plants. For the establishment of the
symbiosis, the key events in the life cycle of AM fungi
are spore germination and the pre-symbiotic mycelial
growth phase, differential hyphal branching, appressorium
formation, root colonization and arbuscule development
(Giovannetti et al. 1994). This sequence does not occur
when AM fungi are challenged with non-host roots and
thus the life cycle of these obligate symbionts is
interrupted.

>>>

Root colonization

After the differentiation of appressoria, AM fungi
usually colonize host roots by forming intercellular
and intracellular hyphae and intracellular arbuscules.
Two main anatomical types have been identified in
arbuscular mycorrhizas (Arum and Paris), their
occurrence depending on the host plant genome
(Smith and Read
1997).

Many authors have described the colonization of
non-host roots with intercellular development of
fungal hyphae, often associated with the formation
of vesicles (Malloch and Malloch 1981; Taber and Strong
1982; Giovannetti and Lioi 1990; Càzares and Smith 1992;
Càzares and Trappe 1993; Guerrero 1996; Treu et al.
1996). Vesicles in arbuscular mycorrhizal host roots
usually occur after arbuscule development. In contrast,
vesicles in non-host roots have been detected in the
absence of arbuscules.

Ultrastructural observations of non-host Brassica roots
colonized by Glomus mosseae showed that intracellular
colonization occurred only when cells were dead, i.e
. when no plasma membrane was present (Glenn et al.
1985). Moreover, AM fungi have been described as
being able to colonize different organs or tissues of
host and non-host plants and to form swellings and
intraradical vesicles (Park and Linderman 1980;
Stasz and Sakai 1984; Warner 1984; Giovannetti
and Lioi 1990).

The absence of both appressoria and arbuscules in these
interactions between AM fungi and plant tissues suggests
the lack of any recognition event leading to the establishment
of a functional symbiosis (Figs. 6, 7) and instead a rather
parasitic type of colonization.


Selected Reference Citations:

Glenn MG, Chew FS, Williams PH (1985) Hyphal penetration
of Brassica (Cruciferae) roots by a vesicular-arbuscular
mycorrhizal fungus. New Phytol 99 : 463–472

Hirrel MC, Mehravaran H, Gerdemann JW (1978)
Vesicular-arbuscular mycorrhizae in the Chenopodiaceae
and Cruciferae: do they occurr? Can J Bot 56 :2813–2817

Vierheilig H, Ocampo JA (1990a) Role of root extract and
volatile substances of non-host plants on vesicular-arbuscular
mycorrhizal spore germination. Symbiosis 9 :199–202

Vierheilig H, Ocampo JA (1990b) Effect of isothiocyanates
on germination of spores of G. mosseae. Soil Biol Biochem
22:1161–1162


2.

Regulation of the Vesicular-Arbuscular Mycorrhizal Symbiosis

R T Koide, and ­R P Schreiner

Annual Review of Plant Physiology and Plant Molecular Biology
Vol. 43: 557-581 (June 1992)
http://dx.doi.org/10.1146/annurev.pp.43.060192.003013


Exerpts:

One of the best pieces of evidence supporting the
idea that mycorrhizal fungi can increase phosphorus
uptake is that nonmycotrophic species appear to
have evolved alternative means of increasing their
ability to acquire phosphorus. These include a high
degree of root hairiness (81, 86), production of
proteoid roots and accompanying chelators and acids
(70), rhizosphere acidification (71, 99), and high
rhizosphere phosphatase activity (86).

Mustards (Brassicaceae) may also reduce competition
for phosphate with other plant species by producing
allelopathic substances (31, 85).

>>>

Mycorrhizal fungal hyphae often grow on or around the
roots of nonmycotrophic species without significant
penetration (summarized in 149). Could this lack of
penetration result from a lack of positive stimuli or the
lack of sufficient nutrients? The presence of roots of
mycotrophic species may, in some cases, increase
the penetration and limited internal growth of VAM fungi
in the roots of nonmycotrophic species. This increase
occurs in members of the Chenopodiaceae (2, 105, 163),
the Brassicaceae (summarized 149), and in
Lupinus (108, 155). This observation is consistent with
the lack of some positive cues in these nonmycotrophic
species, which can be supplied by nearby roots of
mycotrophic species. An alternative explanation, however,
is simply that when the fungus infects a host root, it can
acquire a carbon source and thereby have more energy
with which to attempt to infect the nonmycotroph.

It seems probable that different evolutionary scenarios have
led to a range of mechanisms responsible for the
nonmycotrophic status. Some of these mechanisms may,
indeed, involve the failure to provide correct chemical cues
leading to the symbiotic state. Another possible reason
for nonmycotrophy in some species, however, may be
the production of compounds that directly inhibit mycorrhizal
fungi. The plant taxa utilizing anti-fungal compounds would
obviously be poor research tools for the investigation of
chemical signals necessary for full expression of the symbiosis.
Use of such taxa may not allow one to detect positive signals
because of the presence of active fungal inhibitors. For
example, Glenn et al (63) found that when VAM fungal
hyphae came into close proximity to Brassica roots,
the number of germ tube branches and hyphal tufts was
reduced compared to when they approached roots of
tobacco or tomato. These data are consistent with the lack
of a stimulatory compound in mustards as they proposed,
but they are also consistent with the presence of an
inhibitor. A more tractable approach toward elucidating
the signals and recognition events necessary for establishment
of the VAM symbiosis would be to employ either mutants
of mycotrophic plant species or mutant mycorrhizal fungi
that are, for some reason, incapable of engaging in the
symbiosis.

>>>

Production of Anti-fungal Compounds by Nonmycotrophic Species

Research on the mechanisms responsible for the nonmycotrophic
status of plants has often focused on members of the Brassicaceae
because they produce mustard oils (isothiocyanates) that have
potent insecticidal (94), antibiotic (I03), allelopathic (31,164),
and fungicidal (44, 69, 80, 106, activities. Isothiocyanates are
enzyme-mediated (myrosinase, a thioglucosidase) hydrolysis
products of sulfur-containing secondary metabolites called
glucosinolates; they are often volatile but may also be
nonvolatile (89, 106, 125). In addition to isothiocyanates,
glucosinolates may yield a variety of other compounds when
hydrolyzed, including nitriles, thiocyanates, and others depending
on the structure of the glucosinolate and the conditions of
hydrolysis (33).

Glucosinolates and myrosinase appear to coexist in a type
of idioblast called a myrosin cell. In the root, these cells are
apparently confined to the cortex (72, 79, 151). The water
soluble glucosinolates appear to be compartmented in the
vacuole while myrosinase appears to be associated with
various membranes in the cytoplasm (72, 96, 151). This
arrangement referred to as the isothiocyanate "bomb" (96).
Compartmentation is obviously necessary if production of
toxic compounds upon hydrolysis is to be controlled.

A question remains about whether sufficient concentrations
of isothiocyanates exist in the rhizosphere and on the
rhizoplane to account for observed inhibition of mycorrhizal
fungi by intact roots. It is usually assumed that only mechanical
disruption of myrosin cells due to herbivory, for example, will
allow the enzyme and substrate to mix and release isothiocyanates
and other potentially toxic compounds. The "bomb," however,
may be a bit leaky. That is, a small level of hydrolysis may
occur in the absence of tissue disruption. Constitutive production
of volatile isothiocyanates actually serves as an attractant for
some specialized isothiocyanate-resistant insects (73). Tang
Takenaka (148) and R. P. Schreiner & R. T. Koide (unpublished)
have shown that isothiocyanates are released into the rhizospheres
of Carica papaya and Brassica kaber, respectively, even in
the absence of root damage. Because isothiocyanates are rather
nonpolar compounds, they may accumulate on the rhizoplane,
particularly those that are nonvolatile. Volatile isothiocyanates
would be expected to have farther-reaching effects but, being
nonpolar, would still tend to accumulate in the rhizosphere.
Thus, despite having a low rate of constitutive release,
isothiocyanates may accumulate sufficiently to have significant
biological activity (148). Mustard plants themselves appear to
be less susceptible to the ill effects of toxic hydrolysis
products than species outside the Brassicaceae (85). In several
instances mustards have been shown to have no significant
effect on infection of host plants growing in the same soil
(19, 111, 114, 120; R. P. Schreiner & R. T. Koide, unpublished
data). The production of nondiffusing or chemically unstable
isothiocyanates is consistent with this observation. There is
evidence that anti-fungal substances produced by mustards
may be degraded by rhizosphere microorganisms or rendered
ineffective by adsorption onto soil particles (113). In a few
cases, however, the presence of mustards has been shown
inhibit infection in host species growing nearby (46, 75, 119),
a finding consistent with the production and accumulation
of significant concentrations of anti-fungal compounds.

Glenn et al (62) concluded that glucosinolates were not involved
in the lack of infection of Brassica species by mycorrhizal fungi.
They examined a number of Brassica cultivars differing in
glucosinolate concentrations and also attempted to vary
glucosinolate concentration by placing seedlings on agar
with or without sulfur. Nearly all combinations of sulfur
treatment and cultivar resulted in penetration of roots by fungi,
but arbuscular infections never developed. Glucosinolate
concentrations, however, were only examined in separate
hydroponically grown ~6 week old plants, not in the actual
test seedlings. Moreover, even if grown on sulfur-deficient
media, seedlings would probably not be glucosinolate
deficient because adequate sulfur may be supplied by the
cotyledons. Indeed, the ultrastructural work of Glenn
et al (62) indicated that tnycorrhizal fungal penetrations
occurred only in dead cortical cells, those that would be
incapable of producing isothiocyanates. In addition, hyphae
growing near healthy cells had retracted cytoplasm,
consistent with the presence of inhibitory compounds.
Moreover, it is not clear how the concentration of the
nontoxic glucosinolates (80, 106, 159, 161) they
measured is related to the concentration of the active
isothiocyanates.

Tester et al (149) concluded that isothiocyanates were
probably ineffective in preventing mycorrhizal infections
because species producing them had been reported to be
infected by VAM fungi and because of the belief that
intact tissues do not release isothiocyanates. As indicated
above, however, isothiocyanate release from intact tissues
does occur. Furthermore, the mycorrhizal infections of
isothiocyanate-producing species they reported, for example
Carica papaya, were very slight, lacked arbuscules, and
did not result in a host growth response even in poor soils
(82, 121; but see 116, which indicates the presence of
arbuscules in Carica).

The importance of isothiocyanates to the nonmycotrophic
status of many species may be variable because different
plant species produce different combinations of isothiocyanates
and because variation in biological activity among the many
isothiocyanates is great (44, 106, 161). It is also possible
that some mycorrhizal fungal isolates have developed a
limited capacity to detoxify some isothiocyanates, as have
certain insects (156, 160). Certainly there does appear to
be variation among mycorrhizal fungal species in their
susceptibility to antifungal compounds (108). Isothiocyanate
production tissue specific (48), and it is not clear whether
all isothiocyanate-producing species make substantial quantities
in the roots. Moreover, the poorly developed infections or
attempted infections in several Brassica species are consistent
with an isothiocyanate-mediated resistance, since myrosin cells
only make up a small proportion of the cells in a given tissue
(72, 79, 151). VAM fungi could penetrate quite a few cortical
cells before encountering a myrosin cell.

There does appear to be evidence for the production of
inhibitory compounds by mustards that are effective in deterring
mycorrhizal fungi. Extracts of radish and cabbage plants applied
to alfalfa roots significantly reduced mycorrhizal infection and
reduced germination of Glomus mosseae spores (113). The
volatile fraction of cabbage root extracts alone was also shown
have inhibitory activity on Glomus mosseae spore germination
(46, 158). Vierheilig & Ocampo (159) also demonstrated that
reaction of the glucosinolate sinigrin with myrosinase produced
a strong volatile inhibitor of VAM fungal spore germination.
From these studies and others presenting consistent results
(154), it is clear that some isothiocyanates are capable of
inhibiting mycorrhizal fungi.

We indicate here that there is currently little reason to dismiss
isothiocyanates as effective anti-mycorrhiza compounds. The
use of mutant mustard plants incapable of producing
isothiocyanates would help to determine with greater surety
whether such compounds are involved in the nonmycotrophic
status of many mustards.


Selected Reference Citations:

149. Tester, M., Smith, S. E., Smith, F. A. 1987. The
phenomenon of "nonmycorrhizal" plants. Can. J. Bot.
65:419-31

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