The effect of arbuscular mycorrhizal fungal isolates on the development and oleoresin production of micropropagated Zingiber officinale

BRAZILIAN JOURNAL OF PLANT PHYSIOLOGY
The official journal of the
RESEARCH ARTICLE
Brazilian Society of Plant Physiology
The effect of arbuscular mycorrhizal fungal isolates on
the development and oleoresin production of
micropropagated Zingiber officinale
Maicon F. da Silva1, Rosete Pescador1*, Ricardo A. Rebelo2 and Sidney L. Stürmer3
1Laboratório de Biotecnologia e Micropropagação Vegetal, Departamento de Ciências Naturais, Universidade
Regional de Blumenau, CP 1507, 89010-971 Blumenau, SC, Brasil. 2Laboratório de Química Orgânica, Departamento
de Química, Universidade Regional de Blumenau, 89010-971 Blumenau, SC, Brasil. 3Laboratório de Botânica,
Departamento de Ciências Naturais, Universidade Regional de Blumenau, 89010-971 Blumenau, SC, Brasil.
*Corresponding author: rosetep@furb.br
Received: 05 April 2008; Returned for revision: 23 May 2008; Accepted: 24 June 2008
We have investigated the effects of phosphate fertilization and inoculation with isolates of arbuscular mycorrhizal fungi
Scutellospora heterogama SCT120E, Gigaspora decipiens SCT304A, Acaulospora koskei SCT400A, Entrophospora
colombiana SCT115, and an assemblage (Mix) of all four isolates on growth, development and oleoresin production of
micropropagated Zingiber officinale. After 120 and 210 d of growth, the Mix and phosphorus addition significantly
increased shoot height relative to control plants. Phosphorus addition was the only treatment resulting in significantly
large shoot dry biomass relative to control after 120 d. No statistical differences were observed between treatments for
shoot dry biomass after 210 d and for fine and coarse root biomass at both harvests. Inoculation with S. herogama and
G. decipiens resulted in larger yields of oleoresin, corresponding to 3.48% and 1.58% of rhizome fresh biomass
respectively. Based on retention index and mass spectrometry, we have characterized the following constituents present
in ginger rhizomes: ar-curcumene, zingiberene, ?-cadinene, bisabolene, ?- or ?-cadinene and farnesol. Two other
constituents were characterized as possible members of the gingerol class. Results suggest that the screening and
inoculation of arbuscular mycorrhizal fungi in ginger plants is a feasible procedure to increase the oleoresin production
of Z. officinale and consequently increase the aggregate value of ginger rhizome production.
Key words: fungal assemblage, ginger, oleoresin, secondary metabolites
Efeito de isolados de fungos micorrízicos arbusculares no desenvolvimento e produção de óleo-resina em plantas
micropropagadas de Zingiber officinale: Avaliou-se o efeito da adubação fosfatada e da inoculação de isolados de
fungos micorrízicos arbusculares [Scutellospora heterogama SCT120E, Gigaspora decipiens SCT304A, Acaulospora
koskei SCT400A, Entrophospora colombiana SCT115 e uma mistura (Mix) de todos os quatro isolados] sobre o
crescimento e desenvolvimento vegetativo e produção de óleo-resina em plantas micropropagadas de gengibre
(Zingiber officinale). Verificou-se que, aos 120 e 210 d após o cultivo, as plantas dos tratamentos Mix e sob adição de
fósforo tiveram maior altura em relação às plantas-controle. A adição de fósforo foi o único tratamento que resultou em
aumento significativo na biomassa seca da parte aérea relativamente ao tratamento-controle, após 120 d de cultivo. A
biomassa seca da parte aérea, aos 120 d, bem como a biomassa de raízes finas e grossas, aos 120 e 210 d, não
responderam aos tratamentos aplicados. A inoculação com S. herogama e G. decipiens foram os únicos tratamentos que
proporcionaram incrementos significativos de produção de óleo-resina, respectivamente 3,48 e 1,58% em relação à
biomassa fresca de rizomas de gengibre. Baseado no índice de retenção e espectrometria de massa, caracterizaram-se os
seguintes constituintes presentes nos rizomas de gengibre: ar-curcumeno, zingibereno, ?-cadineno, bisaboleno, ?- ou ?-
cadineno e farnesol. Outros dois constituintes foram caracterizados como possíveis membros da classe dos gingeróis.
Braz. J. Plant Physiol., 20(2):119-130, 2008

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120
M.F. SILVA et al.
Os resultados sugerem que a seleção e a inoculação de fungos micorrízicos arbusculares em plantas de gengibre é uma
alternativa viável para aumentar a produção de óleo-resinas e, conseqüentemente, aumentar o valor agregado da
produção de rizomas nessa espécie.
Palavras-chave: assembléia de fungos, gengibre, metabolismo secundário, óleo-resina
INTRODUCTION
vulgare Mill. (fennel). For Mentha arvensis L. (mint)
mycorrhizal colonization significantly increases oil
Several aspects of the mycorrhizal symbiosis
content and yield relative to non-mycorrhizal plants
resulting from the biotrophic interaction between
(Gupta et al., 2002). Freitas et al. (2004) also observed that
arbuscular mycorrhizal fungi (AMF) and roots of
inoculation with AMF resulted in increments of 89% in
micropropagated plants have been reported in the
the essential oil and menthol contents of mint. To our
literature. In this association, AMF improve uptake of soil
nutrients such as P, N, Zn2+, Cu2+, K+, thereby influencing
knowledge, the influence of AMF inoculation on ginger
plant growth and survival, plant nutrition, tolerance to
oleoresin production and yield has not been studied.
water stress and to adverse environmental conditions
Ginger (Zingiber officinale Roscoe) is an
(Smith and Read, 1997). For micropropagation systems,
economically important plant largely cropped for its
the acclimatization stage represents a developmental
variety of uses, especially for its medicinal and flavoring
phase where plants are subject to environmental stress
potentials (Onyenekwe and Hashimoto, 1999). Ginger has
due to poor root, shoot and cuticular development.
been used as a spice since ancient times (Goyal and
Inoculation with AMF represents a biological solution
Korla, 1993) and among others its carminative, diuretic,
that can result in growth enhancement at this stage
and expectorant properties are well known in medical
(Hooker et al., 1994). Several papers have reported the
research (Cost, 1989). Ginger rhizomes contain both
positive effect of AMF inoculation on growth parameters,
aromatic and pungent components responsible for its
root morphology and survival rates of micropropagated
potent aroma and use in food and beverages, and these
bananas (Declerck et al., 2002), potatoes (Vosátka and
are mainly monoterpenoids such as geraniol, linalool and
Gryndler, 2000), strawberries (Borkowska, 2002),
geranial (Sekiwa-Iijima et al., 2001). Concentration of
grapevine (Schellenbaum et al., 1991), apple (Locatelli
oleoresins in the dry rhizome ranges from 1.5 to 3%
and Lovato, 2002), Prunus (Monticelli et al., 2000), and
(Onyenekwe and Hashimoto, 1999; Zancan et al., 2002)
artichokes (Fortunato et al., 2005).
and the study of these oils is of paramount importance as
However, little is known about the effect of AMF upon
they determine ginger flavor, which ultimately determines
either plant secondary metabolic pathways or the
quality and international market price of ginger
production and yield of secondary compounds of their
(Onyenekwe and Hashimoto, 1999; Sekiwa-Iijima et al.,
hosts (Copetta et al., 2006). For instance, studies have
2001).
demonstrated that AMF can influence phytohormone
In this investigation, the biotechnological processes
levels of jasmonate (Hause et al., 2002), terpenoids and
of plant micropropagation and mycorrhizal inoculation
carotenoids (Akiyama and Hayashi, 2002; Fester et al.,
were used to study the effect of AMF on ginger growth
2002) and phenols (Zhu and Yao, 2004). In addition, the
and oleoresin production. Ginger is an alternative crop
association with AMF has altered essential oil yield and
for small and medium growers in the Itajai Valley in Santa
quality of several plants. Kapoor et al. (2002) observed
Catarina state, in the South of Brazil, and oil extraction
that inoculation with AMF Glomus macrocarpum and G.
represents a means of increasing the aggregate value of
fasciculatum increased significantly the concentration of
this crop for small families. Our approach rests on the
limonene and ?-phellandrene, respectively, relative to
needs to produce homogenous, disease-free and high
non-mycorrhizal control plants of Anethum graveolens L.
quality medicinal plants for essential oil exploitation on a
Kapoor et al. (2002b, 2004) also observed enhanced
commercial scale. This is particularly important for ginger
concentration and quality of essential oils on mycorrhizal
as conventional plant propagation using rhizomes might
Coriandrum sativum L (coriander) and Foeniculum
transmit pathogens from one growth cycle to another
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MYCORRHIZAL FUNGAL AFFECTING THE DEVELOPMENT AND OLEORESIN PRODUCTION OF Zingiber officinale 121
(EPAGRI, 1998; Debiasi et al., 2004). The hypothesis that
needed. At this moment, the shoots were discarded and
different AMF isolates will influence yield and quality of
the soil together with the root ball was thoroughly
oleoresin of micropropagated ginger plants under
homogenized to produce the mycorrhizal soil inoculum
greenhouse conditions was tested.
for each isolate.
Plant growth experiment: In vitro micropropagated ginger
MATERIAL AND METHODS
plants were transferred to plastic pots (400 mL) containing
a sterilized mix of river sand:soil (1:1, pH 5.5). The substrate
Plant material: In vitro ginger plants obtained from the
was previously sterilized twice at 121ºC for 1 h in an
Laboratory of Biotechnology and Plant Micropropagation
autoclave. At transplanting, one plant was established per
of the Universidade Regional de Blumenau (Brazil) were
pot and each pot was inoculated with 10 mL of AMF soil
used in the experiment. Buds were excised from
inoculum. Fungal treatments included Scutellospora
commercially grown ginger rhizomes obtained from a field
heterogama (Sh), Gigaspora decipiens (Gd), Acaulospora
crop in the municipality of Ilhota, Santa Catarina state
koskei (Ak), Entrophospora colombiana (Ec) and an
(Brazil) and superficially disinfected according to the
assemblage of all four isolates (Mix). Control (Ctl) and
methodology of Debiasi et al. (2004). After this process,
Phosphorus (P) treatments were inoculated with 10 mL of
buds were placed on Murashige-Skoog medium
non-mycorrhizal soil inoculum from Sorghum bicolor pot
(Murashige and Skoog, 1962) supplemented with sucrose
culture. Phosphorus was added. The experiment was
(30 g L-1), agar (7 g L-1) and 10 µM 6-benzylaminopurine,
carried out in a greenhouse under growth conditions as
with pH adjusted to 5.8 before autoclaving. Plant cultures
described above for up to seven months. Plants were
were maintained in growth chambers in 20 mL tubes for 50
distributed in a completely randomized design, with seven
d, at a temperature of 25 ± 2ºC and a light intensity of 50
treatments and 15 replicates.
µmol m-2 s-1.
Plant harvest and measurements: After 120 d of growth,
Mycorrhizal inoculation: Single cultures of four AMF
five plants per treatment were used for measurement of
species were obtained from the germplasm bank of the
shoot height (SH) and biomass production. Weight of
Laboratory of Botany of the Universidade Regional de
shoot biomass (SB) was obtained by drying leaves at
Blumenau to bulk up inoculum for use in the plant growth
50oC for 4 d. Roots were first divided into fine roots and
experiment. Fungal isolates were Scutellospora
coarse roots (> 3 mm diameter). Half of the fresh weight of
heterogama (Nicol. & Gerdemann) Walker & Sanders
fine roots and coarse roots was dried to obtain root
(isolate SCT120E), Gigaspora decipiens Hall & Abbott
biomass (RB) and coarse root biomass (CB). The
(isolate SCT304A), Acaulospora koskei Blaszkowski
remaining half of fine roots was stained according to
(isolate SCT400A) and Entrophospora colombiana
Koske and Gemma (1989) and percentage of mycorrhizal
Spain & Schenck (isolate SCT115). Soil inoculum of each
root colonization estimated by the grid line methods of
isolate (containing hyphae, spores and colonized pieces
Giovannetti and Mosse (1980). Spores (AMF) were
of root) was individually mixed with a substrate
extracted from a 30 mL soil subsample after wet sieving
composed of a sterilized soil:sand mix (v/v, 1:1, pH 5.5),
(Gerdemann and Nicolson, 1963) followed by
placed in plastic pots of 1.5 kg and seeded with Sorghum
centrifugation on a sucrose gradient (20%/60%) and
bicolor L. Pots containing only sterilized soil:sand mix
counted under a compound microscope.
were also set up to obtain appropriate non-mycorrhizal
After 210 d, the measurements of SH, SB, RB, CB, and
inoculum for use in the plant growth experiment. The
AMF root colonization and sporulation were repeated,
soil:sand mix was sterilized twice (121oC for 1 h each) with
except for rhizomes which were weighed fresh and used
a 24-h interval between autoclaving sessions. Plants were
for the chemical evaluation of oleoresin.
grown under greenhouse conditions for three to four
months [(16 h daylength, temperature between 20-25oC,
Extraction and Chemical analysis of oleoresins: After 210
supplemented with fluorescent light (irradiance
d of growth, rhizomes and shoots within each treatment
equivalent to 16.2 µmol m-2 s-1)] and watered daily or as
were pooled to obtain oleoresin: nine replicates were used
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122
M.F. SILVA et al.
for the Ctl, Mix, Ec and Sh treatments and 10 replicates for
RESULTS
the P, Gd and Ak treatments. Rhizomes were first washed
with distilled water, blot dried and weighed to obtain total
Vegetative development: After 120 d, shoot height (SH) of
fresh weight. After this procedure, they were sliced and
the ginger plants was 8.18 cm for P and 8.98 cm for Mix
dehydrated in an oven with air circulation (33 + 3oC, 48 h) to
treatments and these were the only treatments differing
reduce the moisture content to 7-9 %, as determined by the
statistically from Ctl plants (Table 1). At 210 d, plants
azeotropic distillation method (Cecchi, 1999). Oleoresins
inoculated with Mix and Ak and added P produced
were extracted from 2 g of rhizomes using 90 mL of acetone in
significantly longer shoots than Ctl plants. Plants
a Soxhlet extractor for 3 h. The extracts were concentrated in
associated with Sh produced the shortest shoots at both
a rotary evaporator at 55oC under reduced pressure. The
harvests (Table 1). Shoot biomass (SB) was significantly
yield for each extraction was determined by the quotient of
higher in plants of P treatment than for Ctl plants after 120 d,
oleoresin mass and rhizome fresh weight. Shoots were
but no differences between treatments and Ctl were
detected for SB after 210 d (Table 1). Fine and coarse root
weighed fresh and dried out to obtain SB before using the
biomass did not differ significantly among treatments at
same procedures for oleoresin extraction.
both 120 and 210 d of growth (Table 1).
The analysis was carried out by gas chromatography
(GC) on a Shimadzu-14B instrument and by gas
Mycorrhizal colonization and AMF sporulation: No
chromatography coupled to a mass spectrometer (GC-
mycorrhizal colonization was detected in Ctl and P roots
MS) using a HP5890A/5970 instrument. Gas
while root colonization ranged from 5.4 to 59% in plants
chromatography was performed using the following
inoculated with Mix or individual AMF isolates (Table 2).
conditions: sample injection (1 µL obtained by diluting
The largest values observed for ginger mycorrhizal
ca. 10 µg of extract in 1 mL hexane); fused silica capillary
colonization was for Mix at 120 d and Gd at 210 d.
column (DB-5, 30 m ´ 0.25 mm, 0.25 µm film thickness);
Mycorrhizal colonization significantly decreased from
helium as carrier gas, flow rate 1 mL min-1; split mode 1:50;
120 to 210 d for Mix and Sh and significantly increased
injector temperature 250oC and FID 280oC; oven
between harvests for Gd. Ginger root colonization by Ak
temperature gradient programmed from 60oC (5 min) to
and Ec remained the same between harvests (Table 2).
190oC (2 min) at 5oC min-1, then raised to 280oC (15 min) at
Number of spores per 30 mL of soil for Ak, Ec and
10oC min-1. Coupled GC-MS at 70 eV was performed using
AMF present in the Mix tended to increase from 120 to
a computerized system associated with a mass selective
210 d, although the differences were not statistically
detector under the same analytical conditions as
significant (Table 2). Spore numbers of Gd slightly
previously described. The identification of the
increased from 120 to 210 d while those of Sh decreased.
components was made by a computer library search
based on matching of MS spectra, followed by
Chemical analysis of ginger rhizome oleoresins: Data
fragmentation pathway analysis and, when required, on
related to oleoresin extraction from shoots are not shown as
the basis of retention indexes with reference to a
our procedure resulted in extraction of chlorophyll. After 210
homologous series of linear saturated hydrocarbons (C -
d, plants grown with P and Ak produced the heaviest
10
C ) (Adams, 2007).
rhizomes, weighting 0.35 and 0.33 g, respectively (Table 3).
30
The yield of oleoresin based on the rhizome fresh weight
Statistical analysis: All dependent variable data (spore
was < 1% for Ctl and Ec plants and the largest value (3.48%)
counts, shoot height and biomass) were checked for
was observed for plants associated with Sh (Table 3). For all
homogeneity of variance according to Levene´s test. After
other treatments, yield ranged from 1.02% to 1.58%.
that, ANOVA was carried out and if the F test was
The GC profile from the Mix treatment (Figure 1G) was
significant, treatment means were compared by the ad hoc
selected to determine the retention index (RI) of the
Tukey’s test with significance at ? 0.05. Percentage values
oleoresin main components from ginger rhizome (Table 4).
of mycorrhizal colonization were transformed to arc sin
We chose the Mix treatment as the standard due to the
square root prior to analyses. All statistical analyses were
equilibrated presence of compounds in all regions of the
performed using the software JMP® (SAS Inst. Inc., 1995).
chromatogram and therefore as representative of all
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MYCORRHIZAL FUNGAL AFFECTING THE DEVELOPMENT AND OLEORESIN PRODUCTION OF Zingiber officinale 123
;
samples. The different treatments could be grouped into
three categories according to their chromatographic
ogama
(g)
0.012a
±
profiles: (i) Group 1 including treatments Gd, P, Sh and Ctl,
210 d
(Figures 1A,B,D,E) characterized by the presence of two
e letter within

heter
0.225
0.025 ± 0.374a
0.137 ± 0.055 a
0.043 ±0.053 a
0.251 ±0.398 a
0.173 ± 0.111 a
0.035 ± 0.011 a
major resinous compounds with a calculated RI of 1977 and
biomass*
2818 (Table 4); (ii) Group 2 including treatments Ak and Mix
root
(Figures 1C,G) characterized by the presence of a single
0.029a
0.055a
±
±
dominant resinous compound (calculated RI of 2598)
Scutellospora
120 d
=
Coarse
associated with volatile constituents (calculated RI below
Sh
0.010 ± 0.017 a
0.048
0.039 ± 0.016 a
0.009 ± 0.002a
0.021 ± 0.019 a
0.038
0.029 ± 0.017 a
1703), the former corresponding to ca. 29% of the sample
a
(Table 4); (iii) Group 3 including treatment Ec (Figure 1F)
characterized by a complex mixture with dominance of
0.010a
0.006a
volatile constituents (calculated RI < 1703). Monoterpenes
(g)
±
210 d
were not detected in any of the samples analyzed.
= Phosphorus;
0.015 ±
0.020 ± 0.002 a
0.010
0.007 ± 0.007
0.012 ± 0.006 a
0.013 ± 0.007 a
0.018 ± 0.010 a
We determined the constituents of the rhizome extract
biomass
using both the retention index and mass spectrometry of
a
the treatment Mix (Table 4). The following ginger rhizome
roots
sesquiterpenes were identified: ar-curcumene,
alues are means ± SD.
Fine
± 0.006 a
120 d
zingiberene,
V
?-cadinene, bisabolene, ?- or ?-cadinene and
.
farnesol. Two compounds were characterized as possible
0.011 ± 0.005a
0.015 ± 0.004 a
0.012
0.006 ± 0.004 a
0.007 ± 0.001
0,023 ± 0.03a
0.011 ± 0.011 a
representatives of gingerols. We were not able to
a
establish the molecular structure of some oleoresin
compounds, but they are resinous compounds based on

colombiana
0.046
±
the calculated RI (Table 4).
210 d
0.061
0.085 ± 0.021 a
0.093 ± 0.038 a
0.048 ± 0.031 a
0.061 ± 0.002 a
0.092 ± 0.036 a
0.071 ± 0.003 a
DISCUSSION
ophospora
ferent treatments after 120 and 210 d of growth. Means followed by the sam
Entr
In this study we have demonstrated that inoculation
ab
c
=
with AMF isolates and P addition influenced mainly the
Ec
0.17
0.029 abc
0.000 abc
;
shoot height and dry biomass production of ginger
< 0.05). Ctl = Control; Mix = mix of all four isolates; P
Shoot dry biomass (g)
±0.012
P
120 d
microplants. Treatments P, Mix, and Ak improved height
0.056 ± 0.02 bc
0.100 ± 0.019 a
0.092 ±
0.054
0.080 ±
0.082 ± 0.020 abc
0.070 ±
relative to control plants while the addition of P was the

koskei
s test,
only treatment that influenced dry shoot biomass after
c
a
120 d. These results indicate that the use of mycorrhizal
ukey’
2.35
inoculation is a feasible approach to replace or decrease
±
Acaulospora
the use of phosphorus fertilizer during ginger plantlet
210 d
ferent (T
=
7.90 ± 2.32 ab
production. Besides lowering cost of fertilizer
3.54 ± 1.36 c
9.22 ± 1.51 a
4.16 ± 1.17
5.46 ± 1.58 bc
10.14
5.56 ± 0.74 bc
Ak
;
consumption, inoculation with effective mycorrhizal
fungal isolates represents a more ecologically sound
approach to sustainable ginger production. Considering
Shoot height (cm)
the production of shoot dry biomass in dill, Kapoor et al.

decipiens
120 d
(2002) observed that the addition of phosphate resulted
3.38 ± 1.50 b
8.18 ± 1.28 a
8.98 ± 1.36 a
4.44 ±1.60 b
6.10 ± 1.85 ab
6.40 ± 2.48 ab
6.46 ± 0.68 ab
egetative development of ginger submitted to the dif
in higher yield compared to inoculation with several
V
species of Glomus. Similar results were observed by
Gigaspora
t
l
i
x
k
C
P
M
Sh
Gd
Ec
Taylor and Harrier (2001) evaluating the effect of nine
=
able 1.
Treatments
A
*Coarse roots have diameter > 3mm.
species of AMF in microplants of strawberry where they
T
columns are not statistically dif
Gd
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124
M.F. SILVA et al.
Table 2. Spore numbers and percentage of mycorrhizal root colonization in micropropagated ginger plants inoculated
with different AMF isolates singly or in an assemblage (Mix) after 120 and 210 d. See further details in legend to Table 1.
Treatments
Mycorrhizal colonization (%)
Spore numbers (in 30 mL soil)
120 days
210 days
120 days
210 days
Mix
43.45 ± 21.30a
23.40 ± 8.46 b
154 ± 37.42a
260 ± 129.34 a
Sh
14.76 ± 9.04 a
5.75 ± 1.30 b
105 ± 121.24 a
60 ± 49.58 a
Gd
17.80 ± 10.00 b
58.95 ± 6.13 a
49 ± 32.22a
53 ± 41.42 a
Ak
29.82 ± 12.47 a
28.42 ± 16.04a
123 ± 90.76a
298 ± 166.45 a
Ec
26.50 ± 13.75 a
5.39 ± 3.85a
25 ± 37.14a
97 ± 136.02 a
Table 3. Rhizome fresh biomass and levels of oleoresin after 210 d of micropropagated ginger plants. See further details
in legend to Table 1.
Treatments
Fresh biomass (g)
Content of total extracted oils (g)
Yield of oleoresin (%)
Ctl
0.1454 ± 0.2333
0.0130
0.99
P
0.3471 ± 0.1836
0.0469
1.35
Mix
0.2730 ± 0.1994
0.0251
1.02
Sh
0.1000 ± 0.0240
0.0348
3.48
Gd
0.2166 ± 0.2113
0.0340
1.58
Ak
0.3331 ± 0.2445
0.0344
1.02
Ec
0.1466 ± 0.1488
0.0096
0.72
Table 4. Ginger oleoresin chemical composition of the rhizome from the Mix treatment after 210 d. Mix = mix of all four
isolates. RI = retention index.
Retention time (min)
Constituent
RI Calculated
RI Literature
Yield (%)
23.401
Ar-curcumene
1487
1479
4.8
23.744
Zingiberene
1500
1493
9.4
23.888
?-Cadinene
1505
1513
2.1
24.095
Bisabolene
1514
1505?/1529?
3.9
24.499
Cadinene
1529
1522?/1537?
5.3
28.795
Farnesol
1703
1714
1.6
35.016
-
1977
-
2.0
35.276
-
1991
-
1.6
36.087
-
2042
-
1.5
37.816
-
2164
-
2.1
40.415
Gingerol (1)
2397
-
7.2
40.551
Gingerol (1)
2411
-
0.9
42.306
-
2598
-
29.1
45.268
-
2818
-
19.1
(1) Compound structurally related to gingerol.
found that some fungal species suppressed plant growth
and host in the mycorrhizal symbiosis, the isolate
compared to non-mycorrhizal control plants. However,
efficiency is under genetic control, and is also affected by
the levels of root mycorrhizal colonization indicate that
the plant species, fungal species and environmental
all AMF isolates were able to establish a compatible
conditions (Declerck et al., 1995). The soil pH could
symbiosis with the ginger root system that was not
represent one of the environmental factors selecting
necessarily translated into larger height or shoot biomass
AMF species; hence the substrate pH used in the
yield. Despite the absence of specificity between fungus
experiment was 5.5, which is considered the optimum for
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MYCORRHIZAL FUNGAL AFFECTING THE DEVELOPMENT AND OLEORESIN PRODUCTION OF Zingiber officinale 125
A
B
C
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126
M.F. SILVA et al.
D
E
F
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MYCORRHIZAL FUNGAL AFFECTING THE DEVELOPMENT AND OLEORESIN PRODUCTION OF Zingiber officinale 127
G
Figure 1. Chromatographic profiles obtained from oleoresin extracts of Zingiber officinale after gas chromatography.
(A) = Gigaspora decipiens, (B) = Phosphorus, (C) Acaulospora koskei, (D) = Scutellospora heterogama, (E) = Control,
(F) = Entrophospora colombiana, (G) = Mix. Note figures have different scales.
ginger crop (Epagri, 1998) and all isolates originated from
allocates carbon to the production of coarse roots, which
soils with pH < 5.0. After 210 d, the production root
further help the formation of the rhizome, rather than to
biomass of mycorrhizal plants was not statistically
fine roots. It is well established that mycorrhizal
different from the control plants. One explanation for this
colonization occurs in fine roots and arrest of fine root
result is the natural dormancy period experienced by
production leads to lower levels of mycorrhizal
ginger during winter (Mello et al., 2000), where ginger
colonization. Although no comparison was performed
shoots become senescent due to the low temperatures.
between 120 and 210 d for root production, the overall
The harvest of the experiment was coincident with
biomass of coarse roots increased and the biomass of fine
wintertime, while the optimum growth of ginger occurs
roots remained constant between harvests (Table 1).
between 25 and 30oC during summer time.
Our hypothesis that different AMF isolates influence
Mycorrhizal root colonization of ginger after 120 d
oleoresin yield was supported by the data obtained after
was significantly higher than after 210 d except for plants
growing ginger plants for 210 d. For most treatments the
inoculated with Gd. Variations in root colonization
levels of total oils extracted were 2-4 fold higher than
between harvest dates could also be related to ginger
control and inoculation with Sh increased the oleoresin
plant dormancy affecting fungal nutrition and root
yield threefold compared to all other treatments. In
morphology. At this phase, plants loose their shoots
comparison with P treatment, inoculation with Gd and Sh
thereby decreasing the production of carbon compounds
promoted a higher yield of oleoresin. Kapoor et al. (2002)
by photosynthesis, the only source of carbon for fungal
also observed that inoculation with Glomus
growth and development (Siqueira et al., 1985). Reduction
macrocarpum and Glomus fasciculatum significantly
of carbon allocation for fungal growth is perceived as a
increased the concentration of essential oil in dill and
stressful condition that reduces mycorrhizal colonization
carum relative to P fertilization and control plants.
and triggers sporulation (Pearson and Schwiger, 1993).
Copetta et al. (2006) tested three AMF isolates and
Indeed, sporulation increased from 120 to 210 d for all
observed that only one isolate (Gigaspora rosea)
isolates except Sh, although values were not statistically
significantly increased the amount of essential oil in
significant. Moreover, at this phenological stage, ginger
basil. Phosphorus is one of the main nutrients involved in
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128
M.F. SILVA et al.
the synthesis of secondary metabolites as their
with RI 1416 which is found in treatments Sh, Gd and Ec.
production demands ATP (Sangwan et al., 2001).
The concentration of compounds related to
Although P shoot concentration was not measured, the
oleoresins of Mix plants indicated that two unidentified
increase in availability of P through mycorrhizal
compounds (calculated RI of 2598 and 2818) were the
association would probably underlie the increase of
major constituents followed by zingiberene (calculated RI
secondary metabolites such as oleoresins. Maffei et al.
of 1500) (Table 4). The essential oil of ginger is a mixture
(1989) state that the synthesis of secondary metabolites
of monoterpenes and sesquiterpenes that have volatile
is also dependent on plant age and developmental stage.
compounds responsible for the aroma where zingiberene
Inoculation with different AMF isolates and addition
is the major component (Zancan et al., 2002). We did not
of P resulted in different compounds being detected in
observe the production of monoterpenes in this study
t h e e x t r a c t s . T h i s f i n d i n g l e n d s s u p p o r t t o o u r
despite the fact that these compounds are commonly
hypothesis that different AMF isolates would result in
found in the oleoresin of ginger rhizomes (Wu et al., 1990;
qualitative differences in ginger oleoresin production.
Onyenekwe and Hashimoto, 1999; Jiang et al., 2006).
We were able to distinguish three groups among
According to Castro et al. (2004), some of the
treatments according to chromatographic profiles:
monoterpene compounds are chemical components
group 1 characterized by the presence of two resinous
developed by plants as defense mechanisms against
compounds, group 2 characterized by the presence of
herbivores and pathogens. However, in the present
one resinous compound plus a volatile compound and
study, ginger plants were grown under greenhouse
group 3 described as a complex mixture of volatile
conditions in sterilized substrate and no signs of
compounds. Similarly, Copetta et al. (2006) in their work
pathogenesis were detected during the experiment.
on basil inoculated with Glomus mosseae, Gigaspora
Under these circumstances, it is possible that production
margarita and Gigaspora rosea found that essential oil
of monoterpenes was not induced as the inoculum
concentration was modulated according to each fungal
potential of soil pathogens was not high due to soil
isolate. Camphor and á-terpineol concentrations were
sterilization. The oleoresin contains volatile substances
significantly higher in basil inoculated with Gigaspora
responsible for the pungency of ginger rhizome and some
rosea compared to the other two isolates. Differential
of the constituents are 4-, 6-, 8-, 10- and 12-gingerol. In
production in quantity and quality of essential oils was
our study, two compounds structurally related to
also reported by Freitas et al. (2004), Kapoor et al. (2004)
gingerol were detected but we were not able to determine
and Khaosaad et al. (2006), in other host plants. At the
the exact nature of the constituent.
m o m e n t w e a r e u n a b l e t o p r o p o s e a m e c h a n i s m
explaining how different AMF isolates influence ginger
CONCLUSIONS
essential oil production. However, it is interesting to
n o t e t h a t G d a n d S h p e r t a i n i n g t o t h e f a m i l y
For a rhizome-producing plant such as ginger, the
G i g a s p o r a c e a e , a n d t h e r e f o r e c l o s e l y r e l a t e d
effect of AMF inoculation and phosphate addition on
phylogenetically, resulted in similar HPLC profiles. The
height and shoot biomass production has to consider the
analysis of chromatograms obtained from rhizome
phenological state of the plant. Dormancy in ginger
extracts of mycorrhizal ginger plants also suggests that
seems to influence carbon allocation to roots that in turn
the isolate Ak had a more consistent influence on the
might affect AMF root colonization.
composition of oleoresin obtained in the treatment Mix
Different isolates of AMF were equivalent to P nutrition
compared to the other fungal isolates. This is observed
in terms of oleoresin yield, indicating that inoculation with
by the similarity existing between the chromatograms of
AMF (i) can replace P fertilization when the aim is to produce
Ak and Mix, except for the peak with a calculated RI of
oleoresin and (ii) is a feasible technique for the production
2818, absent in the former (Figure 1); indeed Ak was the
of ginger plants with increased quantities of oleoresin and
main sporulator in the Mix treatment (data not shown).
oleoresins with different composition, depending on the
Neither of these treatments show the first constituent
associated AMF isolate.
Braz. J. Plant Physiol., 20(2):119-130, 2008

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