SAGO PALM 27: 9−14 (2019)

colonization of roots in sago palm seedlings Associatedwith commercial Mycorrhizal Inocula
Koki Asano1, Toshiyuki Isoi2, Hirotatsu Murano2, Aidil Azhar1,3,
Yulius B. Pasolon4 and Hiroshi Ehara5,6*
1

Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
2

Faculty of Agriculture, Meijo University, 1-501 Shiogamaguchi, Tempaku-ku, Nagoya 468-8502, Japan
3

Vocational School, Bogor Agricultural University, Bogor 16680, Indonesia

4

5

Faculty of Agriculture, Halu Oleo University, Kendari 93132, Indonesia

International Center for Research and Education in Agriculture, Nagoya University, Nagoya 464-8601, Japan
6

Applied Social System Institute of Asia, Nagoya University, Nagoya 464-8601, Japan* Corresponding author: ehara@agr.nagoya-u.ac.jp

Keywords: Arbuscular mycorrhizas, arbuscule, colonization, hypha, sago palm, vesicle

Introduction

Sago palm (Metroxylon sagu Rottb.) extends acrossSoutheast Asia and northwestern Melanesia (Papua
New Guinea and the Solomon Islands). Among the

Arecaceae genera, the trunk of the sago palm
produces the largest amount of starch (over 200 kg

dry starch per plant), and it is used variously for fresh

food and processed food or floured for noodle making(Ehara et al., 2018). One of the oldest crops used byhuman beings, this species has been used as a foodsimilar to bananas and taro (Barrau, 1959) sinceancient times (Takamura, 1990).

Peat is an accumulation of partially decayed

vegetation or organic matter. Peatland has low pH,

and its parameters indicating natural soil fertilities are
low per unit of soil volume. Approximately 30 million

ha in Southeast Asia are estimated to be peatland(Chan, 2016). Sago palm is a useful plant resourcethat can adapt to tropical peat soil (Ehara et al., 2018).

Even in peatland, this species will produce acomparatively large quantity of starch, an estimated

164 –180 kg per plant on a dry weight basis,

according to Yamamoto et al. (2003). Although theyield of sago palm in peatland is lower than that inmineral soil (200 to 300 kg per plant [Ehara et al.,2018]), sago palm can be considered one of the mostvaluable peatland crops. Anugoolprasert et al. (2012a,b) reported from their laboratory level experiment andfield survey in southern Thailand that sago palm canmaintain the uptake of macronutrients, which may be
a major reason why sago palm can adapt to growing
in strongly acidic soil in a natural habitat.

Arbuscular mycorrhizas are mycorrhizas whose

hyphae penetrate plant cells, producing a structure ofarbuscules as a means of nutrient exchange.

Mycorrhizal associations exert great influence on
diverse agroecological and ecosystemic processes(Silvana et al. 2018). Inoculation with arbuscular

mycorrhizal fungi (AMF) is an interesting strategy for
improving crop yields because arbuscular mycorrhizal

symbiosis benefits crops in many ways (Gianinazzi etal., 2010), including enabling the efficient use offertilizers and soil nutrients (Javaid, 2009), protection
against drought stress (Porcel et al., 2007, Porcel andRuiz-Lozano, 2004) and diseases (Liu et al., 2007),
increased N-fixation in legumes (Barea and Azcon-

10

K. Asano et al.

Aguilar, 1983, Haselwandter and Bowen, 1996), andMaterials and Methods

symbioses, and, therefore, many crops could

green house (phytotron) under natural sunlight at

benefit varies greatly (Phosri et al., 2010), ranging

and 60 %, respectively.

improved soil physical properties (Hallett et al.,2009). Most plant species form mycorrhizal
potentially benefit from inoculation with the correct

AMF inoculum; however, the degree to which plantsfrom very little benefit to strong mycorrhizal
dependence (Janos, 2007). Arbuscular mycorrhizas

are key components of sustainable plant–soil
ecosystems because they play an essential role in the
nutrient acquisition, plant diversity, and nutrient
cycling of plants (Jeffries and Barea, 2000).

Phosri et al. (2010) investigated the application of

AMF technology for oil palm cultivation and reported

Plant materials

The experiment was conducted in an air-conditioned

Nagoya University in Japan. In the phytotron, the airtemperature and relative humidity were set at 28 ° C
Sago palm seedlings germinated from seeds

produced by a spiny mother palm in West Papua,Indonesia, were grown to the 8th leaf stage and used

for the experiment. During the nursery period,
seedlings were cultivated individually in 1/10000a

Wagner pots filled with vermiculite and Kimura B
culture solution containing (μ M) 36.5 (NH4)2SO4, 9.1

K2SO4, 54.7 MgSO4, 18.3 KNO3, 36.5 Ca(NO3)2, 18.2

that AMF has the potential to increase conventional

KH2PO4, and 3.9 FeO3 (Baba and Takahashi 1958). At

sustainable management and the practical use ofdiameter) filled with commercial black soil (Protoleaf,

agricultural productivity and is crucial for thefunctioning of agricultural ecosystems through more

AMF. Chan et al. (2002) detected that an endotrophic
mycorrhiza or AMF association existed in sago roots
with mycorrhiza identified as belonging to Glomalesof the Zygomycetes. According to Chan et al. (2010),

AMF inoculation should form an integral componentof the micropropagation process of sago palm

plantlets. Keys to the practical use of AMF are culture
technology and diagnosis technology for effects; inorder to offer it at low cost, a single culture technique

should be prepared. It is also important to clarify theenvironmental conditions in which AMF works
effectively. Further integrated studies are needed toutilize specific AMF for promoting sago palm growth.
In this study, we tried to use commercial mycorrhizal
inoculums, including Rhizophagus irregularis, which

is a well-known AMF, and their associatedcolonization in the roots of sago palm seedlings was
examined. Here, as a part of improving the cultivation
method and commercialization of sago palm withinterdisciplinary approaches, we investigated thepossibility of utilizing the existing AMF technology
for the cultivation of sago palm seedlings.

the 8th leaf stage, seedlings were transplanted
individually to 7 L plastic pots (20 cm depth, 12 cm

Japan) on August 27, 2018. When the seedlings weretransplanted, lateral roots of less than 1 mm diameter

were sampled and used for microscopic observation toconfirm whether mycorrhizal colonization existed.

After no mycorrhizal colonization was confirmed,

transplanted seedings were inoculated individuallywith two commercial mycorrhizal inocula, considering

the application rate recommended by the providers: (1)200 μ L (per plant) Mycogel including Rhizophagus
irregularis (HYPONex Japan, Japan) (corresponding
to 10000 spores) was mixed with 1 L tap water andapplied to a plant; (2) 14 g (per plant) of
Yukimotogoenotakumi including unpublishedmycorrhizal materials (Protoleaf, Japan) was mixed
with soil in a pot when the seedling was transplanted.

Besides, Yukimotogoenotakumi was a spore-form
inoculum. The amount of each inoculum applied wasdetermined in consideration of the instructions fromeach provider. The pots, which were placed

individually onto a plastic tray filled with tap water at

a depth of 3.5 cm, were placed in the phytotron. Water
was supplied to the tray daily to counter

evapotranspiration. Any additional nutrients were notColonization of Roots in Sago Palm Seedlings Associated with Commercial Mycorrhizal Inocula

11

applied for two months following transplanting. On

inoculation and at the end of experiment. The root

collecting lateral roots less than 1 mm diameter. The

at 80 ° C for 10 minutes, 3) rinsing with tap water, 4)October 24, 2018, one seedling each inoculated with
Mycogel or Yukimotogoenotakumi was sampled forlaterals roots were collected from approximately 5, 10,
or 15 cm deep from the soil surface and were observed

microscopically. When the seedlings were harvested at
the end of the experiment, 20 root samples
approximately 5 cm in length were taken at threedifferent depth layers from soil surface as follows:

depth of 5 cm: root approximately 5 cm in length
distributed in the soil layer 3 to 7 cm deep (5 ± 2 cm)from the soil surface; depth of 10 cm: root
approximately 5 cm in length distributed in the soil

layer 8 to 12 cm deep (10 ± 2 cm) from the soil
surface; depth of 15 cm: root approximately 5 cm inlength distributed in the soil layer 13 to 17 cm deep
(15 ± 2 cm) from the soil surface.

Analysis of soil and root samples

The soil sample that was used for the cultivation oftransplanted plants was air-dried at room temperatureandpreparedfor

analyzing the soil’ schemical properties bymeasured at a soil:wateror 1 N KCl ratio of 1:2.5 (w v-1) by a pH meter. The

total nitrogen and carbon concentrations were measured
by a C-N coder (JM1000CN, J-SCIENCE LAB, Japan).

The amount of available phosphorus was determined by

the Bray II method (Bray and Kurtz, 1945). The
exchangeable K , Ca , and Mg were measured by the2+ammonium acetate saturation method (1 N ammonium
acetate (NH4OAc) solution (pH 7.0)) (Schollenberger

and Dreibelbis 1930). All exchangeable cations were

determined by atomic absorption spectrophotometer(170-30 AA, Hitachi, Japan). The cation exchange
capacity was calculated from the amounts of K , Ca ,and Mg measured in the sample.
2+

soaking in 1 M HCl for 1 minute (until the root wascompletely bleached), 5) staining and incubating in asolution containing acid fuchsin:glycerol:lactic

acid:distilled water (1. 5 g:125 ml:1750 ml:125 ml) at
80 ° C for 10 minutes, 6) destaining in a solution

containing glycerol:lactic acid:distilled water (125
ml:1750 ml:125 ml) for 3 hours at room temperature.
Stained roots were observed under a compound
microscope to determine the presence or absence of

mycorrhiza. A camera attached to the microscope
recorded photomicrographs at high magnification.
Results and Discussion

Daytime air temperature in the phytotron ranged

from 29 to 31 ° C during the experiment, and the

maximum irradiance flux density inside the phytotron
was around 1000 μ mol m − 2 s − 1. The properties of thesoil used are shown in Table 1. The nutrient contentsⅡ

mesh. Soil pH was2+water, 2) soaking and incubation in 10 % KOH (w/v)

Table 1. Properties of soil usedsieving through a 2 mm+samples were treated as follows: 1) cleaning with tap+

2+

The lateral roots were sampled twice, before theof the soil were as follows: total nitrogen (4.4 g kg-1),

phosphorus (231.7 mg P2O5 kg-1), and exchangeable

potassium (207 mg K2O kg-1). The stained root samples

were examined under a compound light microscope,and the presence of AMF was assessed for eachsample. AMF were observed in the root samples ofsago palm seedlings by the presence of their vesicular

structures and the intracellular and intercellular hyphaein the roots (Fig. 1). The 20 sampled roots taken fromeach depth were cut into 1 cm lengths and stained. Fifty

samples were selected as well-stained ones among one
hundred samples and observed under the microscope.

In the seedlings inoculated with Mycogel, hyphae andarbuscules were observed in the samples taken from 10

K. Asano et al.

12

cm and 15 cm below the soil surface. On the other
hand,

in

the

seedling

inoculated

Many studies have focused on the use of AMF inwithsustainable agriculture as a mechanism forand vesicles were observed in the samples from 10 cm

been various studies on the use of AMF for tropical

Yukimotogoenotakumi, hyphae were observed in thesamples from 5 cm, 10 cm, and 15 cm, and arbusculesand 15 cm below the soil surface (Table 2, Fig. 1).

maintaining commercial crops efficiently (Andrews etal., 2012; Pellegrino et al., 2011). There have also
and subtropical crops (e.g., coffee, cacao, avocado,

1

1

2

1

33
2

1

Fig 1. Photomicrograph of root samples of sago palm seedlings inoculated with Mycogel (left)
1

and Yukimotogoenotakumi (right)
Left top: root from 5 cm below the soil surface, bar = 500 μ m; left middle: root from 10
cm below the soil surface, bar = 200 μ m; left bottom: 15 cm below the soil surface, bar =100 μ m; right top: root from 5 cm below the soil surface, bar = 200 μ m; right middle: root
from 10 cm below the soil surface, bar = 100 μ m; right bottom: 15 cm below the soil
surface, bar = 200 μ m; arrow 1: hypha, arrow 2: arbuscule, arrow 3: vesicle

2. Presence
orthe
absenceof hyphae, arbuscule, and vesicle in lateral roots taken from soils of
2 Tabledifferent
depth fromsoil surface
different depth from the soil surface

Inoculum
MycogelYukimotogoenotakumi

3+: presence, –: absence

Depth
5 cm
10 cm
15 cm
5 cm
10 cm
15 cm

Hyphae–+++++Arbuscule
–
+
+
–
+
+

Vesicle
–
–
–
–
+
+

Colonization of Roots in Sago Palm Seedlings Associated with Commercial Mycorrhizal Inocula

13banana, pineapple, and citrus) and forest trees, which

negated until around 200 ppm Olsen P in the soil

Aguirre-Medina et al., 2011, Colozzi-Filho andcomparatively high for associating with mycorrhizal

both Mycogel and Yukimotogoenotakumi were also

reported that mycorrhizal colonization was observed

showed the positive effects of mycorrhization on theirbiometric characteristics (Adriano-Anaya et al., 2011,
Siqueira, 1986; da Silveira et al., 2003; de Oliveira andde Olivera, 2005). Sago palm seedlings inoculated withconfirmed to be colonized by AMF as their vesicular
structures and their intracellular and intercellular

hyphae were observed in their roots in this experiment.

As mentioned earlier, a practical application at low costis absolutely essential for the utilization of AMF. To

put useful microorganisms into practice, the isolationand screening of target(s), its single culture, and theverification of its influence are needed. When the AMF
density in the soil is naturally high, the positive effectof AMF inoculation on the growth of crops will not be
expected. In such a case, it is necessary to actively
utilize indigenous AMF for crop production withcultivation management and/or some other practices;

the application of powdered coal is considered effectivein Japan (https://www.naro.affrc.go.jp/training/files/2005_14-04.pdf). The current result indicated thepossibility that an AMF symbiotic association could

exist in the roots of sago palm seedlings whencommercial inocula were used.

Understanding the environmental conditions thatwill promote the efficient influence of AMF is also a

subject for further study. The percentage of themycorrhiza colonization of roots in sago palm
seedlings was estimated to be a few percent inseedlings

inoculated

withMycogelorYukimotogoenotakumi in this experiment (data are notshown). Wood (1992) took a more cautious view andsuggested 5 ppm Olsen P (5 mg kg-1 available P2O5) asa general minimum for plants to benefit frommycorrhizal effects; he showed that the mycorrhizal

colonization of roots decreased rapidly as P levels rosefrom 5 to 10 ppm (5 to 10 available P2O5 mg kg-1).

However, dose–response curves presented by Stribley

et al. (1980) suggest that the mycorrhizal effect in thehighly mycorrhizal-dependent leek plant was not

solution. The available P2O5 in the soil used was about
231.7 mg kg -1 in this experiment, which might be

inocula, considering the former publications of Wood
(1992) and Stribley et al. (1980). Chan et al. (2010)

two months after transplanting without phosphateapplication in the plantlets inoculated with mycorrhiza
isolated from sago palm roots (in peat soil collected inSarawak, Malaysia; no property data available). In theformer publication of Chan (2016), there was no
significant difference in spore abundance among thefour different sites, where the available phosphorus
content in the soil was significantly different (1033 to1577 mg L -1); however, the mean value of spore

abundance looked to be high in soil that included lower

phosphorus among the four sites. Chan (2016) reported
the available phosphorus by concentration in dried soil;
therefore, their data cannot be compared with that of

the other publications referred to above. In any case,the mycorrhizal effect will be related to the phosphorus
content in the soil. Phosri et al. (2019) stated that there

are certainly doubts as to whether mycorrhizal inocula
have real potential to improve oil palm productivity inestablished plantations, especially if growers continue
high P input. A sustainable management had better beconsidered carefully for maintaining the undisturbed
soil microbiota in the case of sago palm growth as well.

As described above, it is possible that AMF’s

symbiotic association existed in the roots of sago palm

seedlings when commercial inocula were used.
Verification of the effect of commercial inocula on thegrowth of sago palm seedlings and environmental
conditions in which AMF works effectively associatedwith sago palm are future subjects that must be studied
to contribute to environmentally friendly cultivation in
intensive production.
Acknowledgments

This study was supported by JSPS KAKENHI

Grant Numbers JP20405020 and JP15H05245, for14which we express our gratitude.

K. Asano et al.

Tuinen, D. Redecker and D. Wipf 2010 Mycorrhiza

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