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Studies on Nitrogen Utilization Efficiency and Involvement of Nitrogen-fixing Endophytic Bacteria in the Growth of lesser yam(Dioscorea esculenta L.)

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Rezaei Abdul Qayom

学位(専攻分野の名称)

博 士(国際農業開発学)

学 位 記 番 号

甲 第 743 号

学 位 授 与 の 日 付

平成 29 年 3 月 20 日

学 位 論 文 題 目

Studies on Nitrogen Utilization Efficiency and

In-volvement of Nitrogen-fixing Endophytic Bacteria

in the Growth of lesser yam (Dioscorea esculenta L.)

論 文 審 査 委 員

主査 教

授・博士(農学)

志和地 弘 信

授・博士(農学)

入 江 憲 治

授・博士(農学)

山 田 隆 一

授・博士(農芸化学)

田 中 尚 人

博士(農学)

遠 城 道 雄

論 文 内 容 の 要 旨

Yams (Dioscorea spp.) are an important crop around

the world, especially in West Africa, where yams are

highly regarded food products that are closely

inte-grated into social, cultural, economic, and religious

aspects of life. Yams are widely grown in a range of

soils under either improved or subsistence cultivation

techniques. Yam yields are thought to be decreasing

because of low soil fertility caused by shorter fallow

periods and the use of more marginal land for yam

production with increasing demands on agriculture to

feed increasing human populations.

Application of organic fertilizers such as farmyard

manure, compost, or bio-fertilizer can be necessary to

maintain soil fertility in yam cropping systems. Thus,

additional inputs and costs such as manure, compost,

or fertilizers are difficult to introduce to yam-based

cropping systems in developing countries. So reduced

fertilizer costs can benefit yam production. The

man-agement of fertilizer application is a crucial issue for

yam production in tropical and sub-tropical regions

and should be studied. However, comparisons of

studies on the effects of soil fertilization in yam

pro-duction have been complicated.

1. Efficiency of Chemical Fertilization for the

Growth and Yield in Yams

Yams are an important crop and staple food in

tropical regions and have very high soil fertility

de-mands. However, comparisons of studies on the

effects of soil fertilization in yam production have been

complicated by the use of many species/varieties, soil

types, precipitation, and different agricultural and

agro-ecological management practices in various

reports. Generally, the yield of yam under traditional

cultivation, such as fallow production systems in

tropical regions, is low. Adequate application of both

organic and inorganic fertilizer increases the yield and

improves yam crop productivity. Therefore,

applica-tion of NPK (nitrogen : phosphorus : potassium)

com-pound fertilizer during yam production is expected to

improve yield under low fertility condition. But

ad-ditional production costs result in higher prices in

underdeveloped countries that often have to import

fertilizer. Nitrogen fertilizers such as urea and

am-monium sulfate are relatively cheaper than NPK

fertilizers and are often used in yam crop systems.

However, the most appropriate amounts of urea or

ammonium sulfate to apply and the best timing for

application have not clear in previous reports. Also,

there have been no reports on the effectiveness of

single applications of nitrogen fertilizer under fallow

production systems with poor soil organic matter

con-ditions. Further experiments are needed to study the

role of nitrogen for the growth and yield of yam plants

in low-fertility soil.

─ 49 ─

(2)

2. Effect of Nitrogen Fertilizer on Growth of the

Lesser Yam (Dioscorea esculenta L.)

The effect of fertilization on lesser yam growth is not

clearly understood. Increased lesser yam production

in Okinawa prefecture, Japan, is desired due to

in-creasing demand for use in commercial health

sup-plements. However, fertilization guidelines for

pro-duction are not yet available in Japan. This study

aimed to reveal the effects of nitrogen application on

growth of lesser yam in Okinawa from April 2014 to

January 2015. Growth with urea application (N

treat-ment) and without urea (control) were compared.

N treatment had a positive effect on growth and

tuber yield in soil with poor organic matter. Yellowish

leaves were observed in control plants from 120 days

after planting (DAP), but plants grew until 240 DAP.

It was possible to grow lesser yams under poor fertile

soil conditions. The d

15

N values of the N treated

plants suggested that nitrogen was absorbed from the

urea ; however, the source of nitrogen in the control

plants was not identified. The quantity of nitrogen

required for a stable yield of lesser yam is thought to

be less than that required for water yam (D. alata).

3. Nitrogen-fixing Endophytic Bacteria is

In-volved with the Lesser Yam (Dioscorea esculenta

L.) Growth under Low Fertile Soil Condition

The relationship between nitrogen absorption and

growth of lesser yam (Dioscorea esculenta L.) is not

clearly understood. The results of our previous

ex-periment that lesser yam was able to grow under poor

fertile soil conditions without nitrogen fertilization ;

however, the source of nutrition was not identified.

This study, which was conducted from April 2015 to

January 2016 aimed to determine whether

nitrogen-fixing endophytic bacteria are associated with lesser

yam. Growth with urea (N treatment), with cow

manure (CM treatment) and without nitrogen

(con-trol) was compared. N and CM treatment had a

positive effect on growth and tuber yield in soil with

poor organic matter. Control plants presented with

yellowish leaves, but grew until harvest and had a

tuber yield comparable with other treatments.

Changing natural isotope values (d

15

N) under N and

CM treatment suggested that nitrogen was absorbed

from the urea and cow manure, respectively ;

how-ever, the source of nitrogen in the control plants was

not identified. Isolation of nitrogen-fixing endophytic

bacteria using an acetylene reduction activity test and

phylogenetic analysis of 16S rRNA gene sequences

revealed five species of in the stem and roots of the

lesser yam plants. These species were shown to be

related to species in the genera Azospirillum, Devosia,

Klebsiella, Rhizobium and Xanthomonas. The

Rhizo-bium sp. was found in stem and the others were

observed in the roots of lesser yam. All these species

are known as nitrogen fixing bacteria and associate

with plants growth. This is the first report to identify

nitrogen-fixing endophytic bacteria associated with

lesser yam.

4. Discussion

Lesser yam can be grown without inorganic input

(Noralyn and Malab, 2013), with yields under

inor-ganic fertilizer showing no significant differences with

yield under organic fertilization and no fertilizer,

respectively. Under low fertile soil conditions,

N-treated water yam plants were found to absorb N from

the urea ; however, but nitrogen source in control

plants (no N application) were not identified Takada et

al. (in press). The nitrogen absorption phenomenon in

plants can be used to explain changing stable nitrogen

isotope (d

15

N) values, which in water yam suggest

fixing of atmospheric nitrogen. In our previous

ex-periment, comparisons of lesser yam and squash

revealed that lesser yam plants were able to grow in

extremely poor soil conditions (Rezaei et al., 2016),

while squash plants, which are unable to fix

atmos-pheric nitrogen, died under similar conditions. These

findings suggest that lesser yam plants absorb

ni-trogen. Sweet potato is able to grow under limited

nitrogen conditions as a result of nitrogen-fixing

endophytes associated with plant growth

(Terakado-Tonooka et al., 2013). It is therefore possible that

there are similar endophytes associated with lesser

yam.

A total of five nitrogen-fixing endophytic bacteria

were isolated in the roots of the control plants through

ARA tests and 16S rRNA gene sequences analysis.

Moreover, the gene of a Rhizobium sp., a typical root

─ 50 ─

(3)

nodule bacteria (Devvrat et al., 2008), was identified in

a stem sample. Four of the endophytic bacteria were

isolated from the roots, and found to be related to

species of the genera Azospirillum, Devosia, Klebsiella

and Xanthomonas.

As reported by Raul et al., (2002), Devosia is a

nitrogen-fixing root nodule bacterium found in the

aquatic legume Neptunia natans (L.f.) Druce.

More-over, Rodrigues et al., (2015) revealed that

Azospi-rillum is associated with cereals and grasses, having

now been described in 17 species. Klebsiella and

Xanthomonas have been found in sweet potato (Asis

and Adachi, 2003 ; Khan and Doty, 2009).

The findings suggest that control plant were able to

survive through symbiosis with nitrogen-fixing

bacte-ria. Farther studies are now needed to confirm the

rate of nitrogen-fixing and contribution of these

bacteria to lesser yam growth. Isolation of more

bacteria using different bacterial isolation media is

also warranted.

和文要旨

ヤムイモは,単子葉植物ヤマノイモ科(Dioscoreaceae),

ヤマノイモ属(Dioscorea spp.)の食用種の総称であ

る。ヤムイモ栽培は,人類の農耕の歴史とともに行われ

てきたが,熱帯・亜熱帯地域では,現在でも焼き畑など

の原始的な在来農法で行われている。元来,ヤムイモは

痩せた土地でも栽培できるのが経験的に知られた作物で

あるが,ヤムイモの需要が増加しているアフリカでは,

ヤムイモ生産において近代的な栽培技術を導入するため

に,有機質肥料や化学肥料の施用が試みられている。し

かし,イモの収量に及ぼす施肥の効果は未だ明確ではな

い。

本研究では東南アジア原産のトゲイモ(D. esculenta)

について,まず,窒素施用の影響を調べたところ,トゲ

イモの生育は窒素によって促進されるが,無施肥の窒素

がほとんど含まれない土壌でも維持された。同じ条件で

栽培したカボチャは枯死したことから,トゲイモが低肥

沃土壌でも生育する能力を持つことが明らかになった。

次に,トゲイモの窒素吸収源の推定をするために,植

物体内における窒素の安定同位体自然存在比の経時的推

移を調べた。その結果,植物体の d

15

N 値が生育の進行

につれて減少したことから,無施肥の植物体は土壌以外

から窒素を得ており,窒素固定細菌などと共生し,窒素

を得て生育している可能性が示唆された。そこで,内生

窒素固定細菌の探索を目的に,アセチレン還元活性法を

用いて窒素固定能をもつ細菌の選抜と分離を行い,16S

rRNA 遺伝子解析により分離細菌の近縁種の決定を行っ

た。その結果,Rhizobium, Azospirillum, Devosia,

Kleb-siella および Xanthomonas 属の窒素固定細菌が発見さ

れた。Rhizobium と Devosia 属は根粒菌として知られ

ており,Klebsiella と Xanthomonas 属はサツマイモに

内生する窒素固定細菌である。これらのことから,低窒

素土壌下で生育するトゲイモは内生する窒素固定細菌か

ら窒素の供給を受けていると推定された。ヤムイモにお

ける窒素固定菌の共生の発見は世界で初めての報告であ

る。

審 査 報 告 概 要

ヤムイモは多くの種において痩せた土地でも栽培でき

ることが経験的に知られている。本研究ではトゲイモ

(D. esculenta)が窒素のほとんど含まれない土壌でも

生育することを確認し,その要因を解析した。その結

果,植物体内における窒素の安定同位体自然存在比の経

時的推移は植物体が土壌以外から窒素を得ていることを

示し,窒素固定細菌などの関与が考えられた。次に,ア

セチレン還元活性法を用いて窒素固定能をもつ細菌の選

抜と分離を行い,16S rRNA 遺伝子解析により分離細菌

の近縁種の決定を行った。その結果,Rhizobium,

Azo-spirillum, Devosia, Klebsiella および Xanthomonas 属

の窒素固定細菌が発見された。これらのことから,低窒

素土壌下で生育するトゲイモは内生する窒素固定細菌か

ら窒素の供給を受けていると推定された。ヤムイモにお

ける窒素固定菌の共生の発見は世界で初めての報告であ

る。これらの知見と発見は学術的に価値の高いものと認

め,審査員一同は学位請求者に博士(国際農業開発学)

の学位を授与する価値があると判断した。

─ 51 ─

参照

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