Effects of Inoculation of Plant Growth-Promoting Bacteria on Plant Growth, Bacterial Endophytic Community Structure and Colonization

概要

（Format No. 13）
SUMMARY OF DOCTORAL THESIS
Name: Ahsanul Salehin
Title: Effects of Inoculation of Plant Growth-Promoting Bacteria on Plant Growth,
Bacterial Endophytic Community Structure and Colonization
(植物成長促進細菌の接種が植物成長、内生細菌の群集構造および植物内定着に及ぼす影響)
Sweet potato (Ipomoea batatas L.) grows well even in infertile and nitrogen-limited
fields, and endophytic bacterial communities have been proposed to be responsible for
this ability. Plant-growth-promoting bacteria are considered eco-friendly and are used
in agriculture, but their application can interact with endophytic communities in many
ways. In this study, a commercial biofertilizer, OYK, consisting of a Bacillus sp., was
applied as PGPR (about 8E+9 CFU/mL) to two cultivars of sweet potato, and the effects
on indigenous endophytic bacterial communities in field conditions were examined.
One milliliter of OYK solution was diluted to 4L with sterilized distilled water, and
twelve seedlings of each cultivar were dipped in the solution for 60 h. The same
numbers of the seedlings were soaked in distilled water as a control. At harvest, the
fresh weights of the shoots and tubers of each sweet potato plant were measured. The
surface of each tuber sample was washed with running tap water for 10 min and cut
longitudinally with a sterilized knife at its middle part after wiping off the water with
a sterilized paper towel. Then, the cut surface was stamped on modified MR agar
medium, with and without the supplementation of ammonium nitrate as a nitrogen
source in a petri dish. Originally, 269 bacterial colonies appeared on the agar plates in
total, of which 232 strains were successfully isolated. Based on their morphological
relative abundance, 1–3 representative isolates were selected from each group,
comprising 30–81% of the original isolates; as a result, 109 isolates were selected, in
total, for further analysis. Among the 109 selected endophytic bacterial isolates, 101
strains were successfully sequenced for the partial 16S rRNA gene. The isolates
belonged to 25 bacterial genera in 9 classes, which showed 97–100% homology.
Although the inoculated OYK was not detected and significant
plant-growth-promoting effects were not observed, the inoculation changed the
endophytic bacterial composition, and the changes differed between the cultivars, as
follows: Novosphingobium in α-Proteobacteria was dominant; it remained dominant in
Beniharuka after the inoculation of OYK, while it disappeared in Beniazuma, with an
increase in Sphingomonas and Sphingobium in α-Proteobacteria as well as
Chryseobacterium and Acinetobacter in Flavobacteria. The behavior of Bacilli and
Actinobacteria also differed between the cultivars. The Shannon diversity index (H)
increased after inoculation in all conditions, and the values were similar between the
cultivars. Competition of the inoculant with indigenous rhizobacteria and endophytes
may determine the fates of the inoculant and the endophytic community. When the
commercial biofertilizer, OYK, consisting of a Bacillus sp., was applied to two cultivars
of sweet potato, the inoculation changed the culturable indigenous endophytic bacterial
communities, differently between the cultivars, and increased the diversity of the
bacterial communities, although the inoculated OYK was not detected and significant
plant-growth-promoting effects were not observed. Competition of the inoculant with
indigenous rhizobacteria and endophytes may determine the fates of the inoculant and
the endophytic community. Origin of the inoculant, which was isolated from soil, was
expected as the possible reasons for the lack of the endophytic potential.
As many endophytic Bacillus strains have already been reported in several plants,
it is assumed that endophytic bacteria have some colonization strategies in interaction

with plants. For efficient and practical use of PGPR, it is essential to understand its
colonizing behavior and abilities to compete with co-existing bacteria. Though several
studies have been reported on the effects of co-inoculation with multiple bacteria on
plant growth, their effects on colonization have not been extensively studied yet. Then
we designed a new experiment intending to evaluate the colonization properties of
Bacillus sp. OYK, which was isolated from a soil, in relation to its origin by comparing
it with those of the other Bacillus sp. strains isolated from plant endosphere and
rhizosphere, and then to elucidate the effects of co-inoculation of the endophytic
Bacillus sp. strain with the other endophytes on their colonization and plant
growth-promoting activities.
In addition to Bacillus sp. OYK, three strains of Bacillus sp.: two strains (Bacillus
sp. RF-12 and RF-37) isolated from the rhizosphere of sweet potato and another one
(Bacillus sp. F-33) as an endophyte of the same plant cultivated in Japan, and three
strains of endophytes: Herbaspirillum sp. Sal 6, Klebsiella sp. Sal 1, and Enterobacter
sp. Sal 3, isolated from Nepalese sweet potato were used in this study.
Surface-sterilized seeds of tomato (Solanum lycopersicum L. cv. Chika F1 hybrid, Takii
& Co., Ltd., Kyoto, Japan) were sown in the sterilized vermiculite in a Leonard jar
supplied with the sterilized Hoagland solution, and 1ml of the four each Bacillus sp.
strains were inoculated (at ca. 108 CFU/ml) onto the seed zone. After cultivation in a
phytotron at 28/25°C (16h/8h, day/night), plant growth parameters and population of
the inoculants in the root, shoot, and rhizosphere were determined. Statistical analysis
of the data on the plant growth and population of the inoculant obtained in each
twice-repeated experiment was performed using the MSTAT-C 6.1.4 software package.
Data were subjected to Tukey’s test after one-way ANOVA. In addition, effects of
co-inoculation and time interval inoculation of Bacillus sp. F-33 with the other
endophytes were examined. All Bacillus sp. strains promoted plant growth except for
Bacillus sp. RF-37, and populations of the rhizospheric and endophytic Bacillus sp.
strains were 1.4–2.8 orders higher in the tomato plant than that of Bacillus sp. OYK.
The plant growth promotion by Bacillus sp. F-33 was reduced by co-inoculation with
the other endophytic strains: Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and
Herbaspirillum sp. Sal 6., though the population of Bacillus sp. F-33 maintained or
slightly decreased. When Klebsiella sp. Sal 1 was inoculated after Bacillus sp. F-33, the
plant growth-promoting effects by Bacillus sp. F-33 were reduced without a reduction
of its population, while when Bacillus sp. F-33 was inoculated after Klebsiella sp. Sal 1,
the effects were increased in spite of the reduction of its population. Klebsiella sp. Sal 1
colonized dominantly under both conditions.
In expansion to plant growth-promoting properties, the colonization potential
should be considered as important criteria when assessing their suitability for
commercial development. The lower population of Bacillus sp. OYK, which was isolated
from soil, than the other Bacillus sp. strains, which were isolated from either the
rhizosphere or endosphere of plant samples, suggests the importance of the origin of
the strains for their colonization. The plant growth promotion and colonization
potentials were independently affected by the co-existing microorganisms. Further
studies are necessary to evaluate the colonization potential of PGPR under field
conditions where diverse microorganisms exist.

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