Fundamental Studies on Arbuscular Mycorrhizal Fungi and Endophytic Nitrogen-Fixing Bacteria Colonized in the Sago Palm (Metroxylon sagu Rottb.) Roots

概要

報告番号

※

第

主

号

論

文

の

要

旨

Fundamental Studies on Arbuscular Mycorrhizal Fungi and

論文題目
氏

名

Endophytic Nitrogen-Fixing Bacteria Colonized in the Sago
Palm ( Metroxylon sagu Rottb.) Roots

ASANO Koki

論 文 内 容 の 要 旨
Sago palm ( Metroxylon sagu Rottb., Arecaceae) is an underexploited starchproducing crop in Southeast Asia and Melanesia. They have high adaptability to
grow in various soils, including peat soil with a high-water table, under nonfertilized conditions. Nursery technology is currently under development toward
active cultivation and industrialization. To fight against the future concern of the
price increase of chemical fertilizer, the maximum utilization of plant growth promoting microbes during nursery is an attractive solution. However, there is
little information about the nutrient response and symbiotic relationships with
plant growth-promoting microbes. In this study, the nutrient response of sago palm
seedlings (Chapter 2), fundamental in formation of arbuscular mycorrhizal fungi
(AMF) (Chapter 3.1) and nitrogen-fixing bacteria (NFB) (Chapter 3.2), and growthpromoting effect of AMF (Chapter 4) were investigated.
Based on the three chapters of the study, the results can be summarized below.
(1) The growth of sago palm seedlings was influenced by the amount of nutrient
supply for about one year, and among three major elements, nitrogen was found to
be the most influential element. Phosphorus absorption and translocation were
also affected (lower P concentration in roots) , while K absorption and translocation
were not restricted by reducing nutrient application. The restriction of nutrient
supply proceeded in leaf morphogenesis , such as leaflet area expansion and
photochemical reaction process prior to the depression of new leaf formation and
leaf gas exchange.
(2) Indigenous AMF in sago palm roots were surveyed in two fields in East
Malaysia (mineral soil of clay loam and shallow peat soil). Furthermore, the

relationships with soil physicochemical properties were investigated in detail in
five sago palm fields in Trang province (clay loam, sandy clay loam) and Nakhon
Si Thammarat Province (sandy clay, sandy clay loam, and clay soils) in South
Thailand. AMF colonization rate in the roots and AMF spore density in bulk soils
(only in the Thai sample) ranged from 7.3 to 73.2% and 9.7-111 (cm - 3 ), respectively.
Amplicon sequencing analysis revealed that Glomus , Claroideoglomus , and

Paraglomus were detected in both soil and roots of all samp ling sites. Glomus was
the most dominant genus in the roots, while the relative abundance of

Claroideoglomus and Paraglomus was comparatively high in the soil. Interestingly,
genus Acaulospora in the roots showed higher occurrence (25.2%) in shallow peat
soil (pH 4.1) and suggested that they may have tolerant against strong acidity and
high-water level in the soil. Among the sampling sites in South Thai, the AMF
colonization rate and spore density were positively correlated with sand content
and gaseous phase ratio and negatively correlated with volumetric water content .
In addition, AMF spore density showed a negative correlation with total P content.
AMF spore density was positively correlated with alpha di versity indices (number
of species, Shannon diversity index, and Simpson diversity index) but not with
alpha diversity in root AMF. Furthermore, AMF spore density was positively
related to the alpha diversity of soil AMF (observed ASV numbers and Shannon
diversity). Soil bulk density, soil texture, organic matter content, total carbon
content, volumetric water content, pH, EC, exchangeable cation content, and total
nitrogen content appeared to affect AMF community structure in both roots and
soil. It seems that AMF abundance and diversity in both roots and soil are
significantly suppressed by oxygen availability limited by the pore size and water
content of the soil, together with total P content. In other words, higher AMF
colonization and diversity are expected in the low P level of sandy soil without
waterlogging. There will have a seasonal change in AMF colonization degree and
the shift of taxonomic composition by the water table fluctuation.
(3) Amplicon sequencing of 16S rRNA and nifH gene revealed the root endophytic
bacterial and NFB diversity and taxonomic composition. Shannon diversity and
Simpson’s evenness of bacteria and NFB among sampling sites were not different
(although the root sample in shallow peat soil showed low value). Soil bulk density,
clay content, volumetric water content, pH, EC, exchangeable cation contents, and
total nitrogen seem to affect the community structure of both endophytic bacteria
and NFB. As a result of phylogenetic analysis of NifH amino acid sequences
(translated from nifH gene amplicon), NifH neighboring to Bradyrhizobium ,

Burkholderia ,
Desulfovibrio ,

Cupriavidus ,
and

Clostridium ,

Spirochaeta

were

highly

Geobacter ,
detected.

Anaeromyxobacter ,
Surprisingly,

NifH

neighboring Burkholderia xenovorans was dominant (> 30% relative abundance) in
the strong acidity (pH 4.1) of shallow peat soil in East Malaysia. The relative
abundance of possible endophytic NFB in sago palm roots ranged from 3.0 to 64.4%
(average 21.5%). The relative abundance of aerotolerant NFB ( Bradyrhizobium ,

Burkholderia , Cupriavidus , and Frankia genera) was negatively correlated with
the

relative

abundance

of

anaerobic

NFB

( Clostridium ,

Geobacter ,

Anaeromyxobacter , Desulfovibrio , and Spirochaeta ) . The change of root endophytic
nitrogen-fixing players by the soil water fluctuation was suggested.
(4) The colonization of AMF by the application of commercial AMF inocula,
Mycogel (HYPONex Japan, Japan) containing Rhizophagus irregularis, and
Yukimotogoenotakumi (Protoleaf, Japan) was confirmed in the roots of sago palm
seedlings. The growth-promoting effect of AMF application to sago palm seedlings
was tested by the inoculation of commercial AMF inoculum containing Glomus sp.
Leaf emergence speed and Soil Plant Analysis Development (SPAD) value of sago
palm seedlings between control and AMF treatments were not different for 61 to
121 days after treatment. However, the fourth measured leaf area and the number
of leaflets in the fifth leaf were significantly larger in AMF -treated plants than in
control.
Nowadays, the development of the nursery-raising technique of sago palm
seedlings is expected for future expansion and stable production of starch
resources. Active utilization of AMF and NFB is a useful method for achieving lowinput fertilizer management. To maximize the plant growth -promoting effect of
AMF, soil texture, water levels, and phosphate content should be well examined,
considering the result in Chapter 2. Co-inoculation of AMF and NFB may be a
valuable strategy for getting a synergy effect. Further growth evaluation against
plant growth-promoting microbes under several soil physical and chemical
conditions will help us establish an eco-friendly nursery-raising method.