domingo, 7 de febrero de 2010

Organ-dependent induction of systemic resistance and systemic

Systemic induced resistance (SIR) is a
well-known host defense mechanism against pathogen attack
in herbaceous plants, but SIR has only recently been docu-
mented in conifers, We tested if inoculation of Austrian pine
(Pinus nigra Arnold) with Sphaeropsis sapinea (Fr.:Fr.) Dyko
and Sutton or Diplodia scrobiculata de wer Slippers and
Wingfield results in SIR or systemic induced susceptibility
(SIS) to subsequent colonization by S.sapinea. Induction atthe
stem base resulted in significant (P < 0.01) SIR in the upper
stem, and induction in the upper stem resulted in significant
(P < 0.05) SIR at the stem base, indicating that SIR is
bidirectional in Austrian pine. However, inoculation at the
stem base resulted in significant (P < 0.01) SIS in shoot tips,
demonstrating that, in the same host species, the expression of
resistance can be dent, resulting in either SIR or
SIS depending on the site of challenge infection. Systemic in-
duced resistance in the stem was associated with induced ligni-
fication, supporting a potential role for this defense mechanism
in disease resistance. Systemic induced susceptibility has been
documented before, but this is the first demonstration of or-
gan-dependent expression of both SIR and SIS in a tree or any
other plant
Keywords: Diplodia pinea, fungal pathogen, host defense,
HPLC, lignification, predisposition, secondary metabolism,
SIR, SIS, systemic induced resistance, systemic induced

Systemic induced resistance (SIR) is the induction of resis-
tance to pathogens in noninfected parts of a plant by prior in-
fections or activity by various organisms elsewhere in the plant
(sensu Bonello et al. 2001, Bonello and Blodgett 2003). There-
fore, the outcome of SIR is functionally analogous to immuni-
zation in mammals. Extensive research, mostly in herbaceous
species, particularly Arabidopsis (Durrant and Dong 2004),
has shown that SIR originates as the result of a hypersensitive
response that is mediated by the accumulation of one or more
of the phytohormones salicylic acid, jasmonic acid and ethyl-
ene. Expression of SIR is correlated with the accumulation of
pathogenesis-related (PR)-proteins and with the induction or
enhancement of secondary metabolic responses, such as accu-
mulation of soluble phenolics and cell wall lignification
(Sticher et al. 1997, Hei12001, Durrant and Dong 2004). Other
hormones, such as abscisic acid, may also playa role in SIR
(Dammann et al. 1997).
In addition to the large size and longevity of conifers, a dif-
ferentiated resin system and the presence of secondary tissues
set them apart from herbaceous plants. Thus, elucidation of
SIR in coniferous trees would significantly contribute to our
general understanding of how plants defend themselves
against injurious agents and provide insights into the ecology
of woody plants. Although the molecular basis of SIR is not
well characterized in trees, it is known that pathogenic infec-
tion results in localized accumulation of PR-proteins (Graham
and Bonello 2004), phenolics and terpenoids (the latter are of-
ten associated with of traumatic resin ducts) in sev-
eral conifers (Franceschi et al. 2005). Many free or condensed
phenolics have fungistatic or fungitoxic effects (Blanchette
and Biggs 1992) and are precursors in lignin biosynthesis. Al-
though induced cell-wall lignification is considered a major
disease resistance mechanism (Vance et al. 1980, Hammer-
schmidt and Kuc 1982, Hammerschmidt 1999), its potential
role in SIR has received little attention (Sticher et al. 1997).
Systemic induced resistance phenotypes occur in pine
(Enebak and Carey 2000, Bonello et al. 2001, Kosaka et al.
2001, Schmale and Gordon 2003, Enebak and Carey 2004).
There is evidence for the involvement of selected signaling
molecules in the localized and systemic induction of defense
responses in pine as well as in other conifers. For example, ex-
ogenously applied salicylic orjasmonic acid induces accumu-
lation of PR-proteins such as chitinase and a thaumatin-like
protein in pine seedlings (Davis et al. 2002, Piggott et al.
2004) and increased biosynthesis .
Materials and methods
may be associated with raumatic resin ducts in
several conifers (Franceschi et al. 2002, Martin et al. 2002,
Faldt et al, 2003, Hudgins et al. 2003, Martin et al. 2003,
Huber et al. 2004, Hudgins and Franceschi 2004, Miller et al.
2005). Secondary resin produced in traumatic resin ducts
(Wong and Berryman 1977) has been found to be more
fungistatic than constitutive resin in at least one case (Solheim
Bonello and Blodgett (2003) showed that, in the Austrian
pine (Pinus nigra Amold)-Sphaeropsis
sapinea (Fr.:Fr.)
Dyko and Sutton (syn. Diplodia pinea (Desm.) Kickx (Sutton
1980» pathosystem, fungal inoculations induced significant
local and systemic depletion or accumulation in the phloem of
several soluble and cell-waIl-bound phenolics, and a local in-
crease in lignin deposition. Furthermore, Luchi et al. (2005)
showed that wounding or inoculation of Austrian pine saplings
with S. sapinea and Diplodia scrobiculata de Wet, Slippers
and Wingfield (de Wet et al. 2003) induced an 8-fold increase
in systemic resin flow and systemic induction of traumatic
resin duct formation. However, these studies did not examine
if systemic induction of Austrian pine by S. sapinea inocula-
tion is correlated with SIR. Because no clear connection be-
tween alterations in secondary metabolism and the expression
of SIR has been established in conifers, the primary objective
of our study was to determine if and how these processes are
Sphaeropsis sapinea is an important agent of shoot blight,
crown wilt, and canker diseases of conifer species throughout
the world, in trees ranging in age from seedlings to fully ma-
ture (Gibson 1979, Palmer and Nicholls 1985, Farr et al. 1989,
Nicholls and Ostry 1990, Swart and Wingfield 1991, Stanosz
and Cummings Carlson 1996). Sphaeropsis sapinea and
D. scrobiculata are phylogenetic ally similar and were consid-
ered two different morphotypes of S. sapinea until recently (de
Wet et al. 2003). Sphaeropsis sapinea is an aggressive patho-
gen, whereas D. scrobiculata is less aggressive on most coni-
fers (Blodgett and Stanosz 1997, Blodgett and Stanosz 1999),
including Austrian pine (Blodgett and Bonello 2003). This
difference in aggressiveness between two phylogenetically
close fungal pathogens of Austrian pine provides an opportu-
nity to examine the nature of disease resistance in the host spe-
cies (Luchi et al. 2005). In the absence of genetically defined
host populations differing in susceptibility, it is postulated that
defense mechanisms expressed against the less aggressive
pathogen but not against the aggressive sister species are at the
basis of resistance.
We used the model pathosystem (Bonello and Blodgett
2003, Hammerschmidt 2003) comprising Austrian pine and
the two canker pathogens S. sapinea and D. scrobiculata to:
(1) test whether inoculation of Austrian pine with necrogenic
pathogens results in SIR or systemic induced susceptibility
(SIS), or both, following subsequent colonization by
S. sapinea; (2) determine whether SIR in Austrian pine is uni-
directional or bidirectional; and (3) correlate systemic resis-
tance and susceptibility to changes in the host's secondary me-
tabolism to identify potential disease resistance mechanisms.
Plant material
Dormant, 5-year-old Austrian pines were obtained from Ridge
Manor Nursery (Madison, OH) in spring 2001, and from
Willoway Nursery (Avon, OH) in spring 2005. Trees were se-
lected for uniformity in size and form to reduce experimental
variability. Trees were maintained in a greenhouse (40°1' N,
83°1' W) for the duration of the experiment, as described in
Bonello and Blodgett (2003). Because even moderate water
stress is known to affect canker development (Blodgett et al.
1997), the trees were watered twice daily to field capacity to
exclude water stress as a factor.
Study design
To assess the systemic effects of stem inoculation (stem induc-
tion) on resistance to S. sapinea, three experiments were con-
ducted (Figure 1). Experiments 1 and 2 tested the SIR phe-
nomenon in stems and whether induction is acropetal,
basipetal or both. In Experiment 3, we examined if stem induc-
tion results in resistance to shoot tip fungal challenge. Chemi-
cal analyses were performed on tissue samples to identify po-
tential mechanisms involved in systemic resistance or suscep-
Experiment 1 (lower stem induction, upper stem challenge)
Six treatment combinations were tested in two independent
trials, beginning on June 7 and June 21, 2001. One of three in-
duction treatments was applied to the stem of each tree. The in-
duction treatments were applied by removing a disk of bark
with a cork borer, followed by insertion of a sterile potato dex-
trose agar (PDA) plug (mock inoculation or wounding treat-
ment), or a PDA plug colonized by S. sapinea isolate 3AP or
D. scrobiculata isolate B1 (Blodgett and Bonello 2003). In
each trial, there were 10 trees per induction treatment. Un-
wounded control trees were omitted from these trials because
previous studies with Monterey and Austrian pines have
shown that unwounded control trees do not differ from trees
receiving a mock induction in terms of either SIR or secondary.
metabolism (Bonello et al. 2001, Bonello and Blodgett 2003).
Three weeks after induction, the trees were challenge-inoc-
ulated 25 em above the induction site by wounding with a cork
borer followed either by mock inoculation with sterile PDA
plugs, or inoculation with mycelial plugs from PDA cultures
of the S. sapinea isolate. One challenge inoculation treatment
was used per stem, and half the trees in each induction treat-
ment were either mock inoculated or inoculated with
S. sapinea. The lengths of lesions above the challenge points
were determined five weeks later and used as a measure of sys-
temic host resistance. Phloem samples were collected from the
margin of the challenge lesions (reaction zones), extracted and
processed for chemical analyses.
Experiment 2 (upper stem induction, lower stem challenge)
Experiment 2 was conducted in July 2005. There were two in-
duction treatments: mock induction and inoculation with
S. sapinea isolate 3AP, with five trees per treatment. In each in-
duction treatment, trees were inoculated at 30 em above the
soil in the same manner as described in Experiment 1. Three
weeks after induction, the trees were challenge-inoculated
25 cm below the induction site by wounding with a cork borer
followed by mock inoculation or inoculation with the
S. sapinea isolate. The lengths of challenge lesions below the
challenge points were measured three weeks later. In this case,
no chemical analyses were conducted.
Experiment 3 (lower stem induction, shoot tip challenge)
Experiment 3 was conducted in two independent trials (begin-
ning on June 7 and June 21, 2001). The induction treatments
were as in Experiment 1, except that an unwounded control
was also included. Challenge-inoculation treatments were per-
formed about 2 em below the shoot apex on expanding shoots
as described previously (Blodgett and Bonello 2003). One of
four randomly selected shoot tips on each tree was assigned to
the following challenge-inoculation treatments: unwounded
treatment, mock challenge, challenge with S. sapinea isolate
3AP and challenge with S. sapinea isolate 1SP (from Scots
pine). The latter challenge was conducted to determine if the
source of the isolate for the challenge affected the host re-
sponse in relation to the induction treatments. Results of this
challenge treatment did not differ from the challenge with
S. sapinea isolate 3AP, and are not shown. Three weeks after
the challenge-inoculation treatments, challenge lesions were
measured as previously described (Blodgett and Bonello
2003), and shoot tip samples were collected, extracted, and
processed for chemical analyses.
Chemical analices
Soluble and cell-wall-bound phenolics were analyzed by high-
performance liquid chromatography (HPLC), and lignin was
quantified spectrophotometrically as described by Bonello
and Blodgett (2003). All but two of the phenolic compounds
were positively identified with standards based on retention
time and UV spectrum.
Statistical analyses
When the assumptions of normality and variance homogeneity
could be satisfied by either the raw, log-transformed or square
root-transformed data, differences in lesion lengths and com-
pound concentrations among treatments were tested by
univariate ANOVA. Means were separated by Fisher's least
significant difference (LSD) multiple range test at P = 0.05.
When the assumptions of normality and variance homogeneity
could not be satisfied, non-parametric Kruskal-Wallis tests
were used. In Experiment 1,trial (two levels), induction treat-
ment (three levels) and challenge treatment (two levels) were
the factors. Comparisons were also made between fungal in-
duction treatment (S. sapinea and D. scrobiculata) and mock
induction giving two induction treatment levels. In Experi-
ment 3, trial (two levels), induction treatment (four levels) and
challenge treatment (four levels) were the factors. Compari-
sons were also made between fungal induction treatment
(S. sapinea and D. scrobiculata) and noninoculation (mock in-
duction and unwounded controls), giving two induction treat-
ment levels. All factors were treated as fixed factors in the
ANOVA. Degrees of freedom (DF) are indicated as subscripts
to the FDP statistics in the results.
Relationships among the various metabolites and among
metabolites and challenge lesion length were tested by
non-parametric Spearman correlations on individual tree data
and parametric Pearson's product moment correlations on
treatment means (Cipollini et al. 2004).
Induction treatment effects on systemic resistance and
susceptibility to challenge inoculations
Inoculation at the stem base with either S. sapinea or
D. scrobiculata was equally effective in inducing an SIR re-
sponse to a challenge-inoculation with S. sapinea at 25 em
above the induction site (Figure 1, Experiment 1) compared
with the mock induction (treatment: F2,28 = 7.241, P < 0.01;
trial: Fl,28 = 0.104, P = 0.750; treatment x trial: F2,28 = 2.566, P
= 0.099) (Figure 2A). On average, challenge lesions were 48%
(P < 0.01) and 37% (P < 0.05) smaller in stems induced with
S. sapinea and D. scrobiculata, respectively, compared with
the corresponding lesions in stems of mock-induced trees. In
Experiment 2, compared with mock-induced saplings, inocu-
lation of potted Austrian pine saplings in the upper stem at
30 em above soil with S. sapinea (Figure 1,Experiment. 2) in-
duced an SIR response to a challenge-inoculation with
S. sapinea in the stem 25 em below the induction site (F1,9 =
8.253, P < 0.05) (Figure 2B). On average, challenge lesions
were 38% smaller in stems of trees induced with S. sapinea
than the corresponding stem lesions of mock-induced trees. In
Experiment 3, inoculation at the stem base with either
S. sapinea or D. scrobiculata (Figure 1, Experiment 3) re-
sulted in SIS in elongating shoot tips challenge-inoculated
with S. sapinea about 2 em below the shoot apex compared
with shoot tips of mock-induced or untreated stems .
Hecho por: Willson A Mendoza C.
C.I: 16.959.604
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