microorganisms
Article
Biofertilizer Activity of Azospirillum sp. B510 on the Rice
Productivity in Ghana
Elsie Sarkodee-Addo 1,† , Chihiro Tokiwa 1,†, Patrick Bonney 2, Daniel Asiamah Aboagye 3, Alex Yeboah 4 ,
Samuel Oppong Abebrese 4 , Ralph Bam 5, Eric Kwesi Nartey 3, Shin Okazaki 1 and Michiko Yasuda 1,*
1 Plant Microbiology Laboratory, Tokyo University of Agriculture and Technology, Saiwaicho 3-5-8,
Fuchu 183-8509, Tokyo, Japan; elsieaddo67@yahoo.com (E.S.-A.); s218271t@st.go.tuat.ac.jp (C.T.);
sokazaki@cc.tuat.ac.jp (S.O.)
2 West Africa Center for Crop Improvement, College of Basic and Applied Science, University of Ghana,
Accra PMB 30, Ghana; gillbonney@gmail.com
3 Department of Soil Science, School of Agriculture, University of Ghana, Accra P.O. Box LG 245, Ghana;
daaboagye003@st.ug.edu.gh (D.A.A.); enartey@ug.edu.gh (E.K.N.)
4 CSIR-Savanna Agricultural Research Institute, Tamale P.O. Box TL 52, Ghana;
lexisyeboah37@gmail.com (A.Y.); sam555oppa@yahoo.com (S.O.A.)
5 CSIR-Crops Research Institute, Kumasi P.O. Box 3785, Ghana; ralphbam@yahoo.com
* Correspondence: ysdmichi@cc.tuat.ac.jp; Tel.: +81-42-367-5847
† Elsie Sarkodee-Addo and Chihiro Tokiwa have contributed equally to this work.
Abstract: Rice production in Ghana has become unsustainable due to the extremely nutrient-poor
soils. It is caused by inadequate soil fertility management, including the inefficient application of





 fertilizers. A practical solution could be the biofertilizers, Azospirillum sp. B510. We performed field
trials in Ghana and Japan to compare the effects of B510 colonization on selected Ghanaian rice
Citation: Sarkodee-Addo, E.; Tokiwa,
C.; Bonney, P.; Aboagye, D.A.; Yeboah, varieties grown. The B510 inoculation significantly enhanced the rice cultivars’ growth and yield. The
A.; Abebrese, S.O.; Bam, R.; Nartey, phenotypic characteristics observed in rice varieties Exbaika, Ex-Boako, AgraRice, and Amankwatia
E.K.; Okazaki, S.; Yasuda, M. were mainly short length and high tillering capacity. These features are attributed to the host plant
Biofertilizer Activity of Azospirillum (cv. Nipponbare), from which the strain B510 was isolated. Furthermore, Azospirillum species has
sp. B510 on the Rice Productivity in been identified as the dominant colonizing bacterium of rice rhizosphere across a diverse range of
Ghana. Microorganisms 2021, 9, 2000. agroecologies in all major rice-growing regions in Ghana. Our results suggest that the utilization of
https://doi.org/10.3390/ B510 as a bio-fertilizer presents a promising way to improve rice growth, enhance soil fertility, and
microorganisms9092000 sustain rice productivity in Ghana.
Academic Editor: Francois Lefort Keywords: endophyte; plant growth promoting rhizobacteria; bio-fertilizer; microbial interaction;
rice cultivars
Received: 13 August 2021
Accepted: 15 September 2021
Published: 21 September 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
Rice (Oryza sativa L.) is a staple food source for over half the world’s population,
with regard to jurisdictional claims in
published maps and institutional affil- with a volume of over 700 million tons [1,2]. It is an annual crop considered a perennial
iations. cereal with great economic importance [3]. Globally, the average rice consumption in
2018 was about 488 million tons, and Asia accounts for about 90% of production and
consumption [4]. However, in Sub-Saharan Africa (SSA), rice consumption as food is
increasing rapidly. In recent decades, rice demand among consumers has proliferated
across Ghana due to the increased population, urbanization, and consumer-driven changes
Copyright: © 2021 by the authors.
away from traditional food staples such as maize and cassava [5–8]. The demand rate has
Licensee MDPI, Basel, Switzerland.
increased over the last two decades, and domestic rice production has not kept pace with
This article is an open access article
distributed under the terms and this surge, fulfilling less than 40% of the national consumption [9,10]. This observed low
conditions of the Creative Commons yield has been attributed to several factors including nutrient-poor soils resulting from
Attribution (CC BY) license (https:// inadequate soil fertility management, recurrent droughts during the growing season, and
creativecommons.org/licenses/by/ the standard of the machinery used in production [9,11]. Consequently, farmers have been
4.0/). forced to rely on the application of chemical fertilizers and herbicides in order to maximize
Microorganisms 2021, 9, 2000. https://doi.org/10.3390/microorganisms9092000 https://www.mdpi.com/journal/microorganisms
Microorganisms 2021, 9, 2000 2 of 14
output and fulfill the increasing demand for rice. Extensive use of agrochemicals during
crop production can lead to severe environmental problems, including residual soil, water,
and food contamination, along with loss of microbial diversity. These factors compromise
soil quality and thus pose a threat to human health and the environment [12].
The negative effects of the prolonged use of agrochemicals during crop production
has driven research to find an innovative alternative approach in which beneficial microbes,
such as plant growth-promoting rhizobacteria (PGPR), could be applied in the form of a
biofertilizer [13–16]. PGPR, as soil-borne bacteria, enhances soil productivity to support
plant growth [17]. The mechanisms of PGPR to promote plant productivity were reported
to include biological nitrogen fixation, phosphorus solubilization, and the production of
both siderophores and phytohormones [18,19]. These beneficial microorganisms belong to
the phyla, Proteobacteria, Actinobacteria, and Firmicutes, with the most common genera
being Azospirillum, Azotobacter, Bacillus, Pseudomonas, and Rhizobium [20–22]. It is well
documented that PGPR enhances plant growth and improves ecosystem diversity by
stimulating root growth [23] as well as increasing shoot biomass [24] and the uptake
of water and nutrients by plants [25]. Many studies have demonstrated a mutualistic
relationship between PGPR and crops, most notably in the Poaceae family, in which plant
growth and grain yield are significantly increased by the association [26,27]. Numerous
strains of the endophytic PGPR Azospirillum have been isolated from the roots and stems
of these crops [28].
Members of the genus Azospirillum have been demonstrated to enhance plant health
and crop productivity by several mechanisms, including metabolizing plant growth regu-
lators [29], boosting plants immunity against pathogens [30], and by acting as biological
nitrogen fixators [31]. The symbiotic association between Azospirillum and rice plants is
prevalent. The symbiotic association between Azospirillum and rice plants is widespread in
the paddy environment. Particularly during the vegetative phase, it is essential to growth
promotion and largely contributes to enhancing crop productivity [32]. Beneficial interac-
tion between microbes and crops profoundly impacts productivity and influences nutrient
cycling, thereby altering soil fertility, leading to improved plant health. The availability
of essential soil nutrients, such as nitrogen and phosphorus, is limited in tropical paddy
fields, restricting plant health and crop productivity. However, previous studies have
demonstrated a significant effect of PGPR on soil fertility under field conditions due to
their pronounced influence on nutrient mineralization and organic matter decomposition
and are not applied to replace fertilizers [20,33–35].
Azospirillum sp. B510, which was isolated from the stems of rice (Oryza sativa cv.
Nipponbare) in Japan [36], has been formulated into a commercial product and applied as
a biofertilizer in diverse crop production systems [37,38]. The application of Azospirillum
sp. strain B510 during rice production demonstrated a significant influence on immunity
against pathogens, plant growth, and yield output [39–41], which could be a potential
candidate for enhancing and sustaining rice production in Ghana. However, with limited
access to biofertilizer and the lack of farmers’ awareness of such farm inputs in Ghana, its
utilization could be challenging during crop production.
In the face of inefficient utilization of fertilizers, combined with their harmful effects
when used long-term, it is critical for crop production in Ghana that a method is found
to utilize naturally available resources such as PGPR. In this study, we investigated the
growth-promoting effect of B510 on Ghanaian rice cultivars in Japan and we performed an
inoculation test on rice productivity in Ghana. Considering the extremely nutrient-poor soil
conditions of paddy fields in Ghana, the field experiment carried out in Japan evaluated
the effects of both low nitrogen and standard nitrogen soil conditions. Multiple field
experiments were established to examine the impact of inoculation with B510. Moreover,
in this study, we examined the potential of B510 application to improve plant growth and
productivity in selected Ghanaian rice cultivars.
Microorganisms 2021, 9, 2000 3 of 14
2. Materials and Methods
2.1. Experimental Site Description and Field Management
The experimental field in Japan was conducted in the Experimental Farm of Field
Science Center, Tokyo University of Agriculture and Technology located Fuchu-honmachi,
Tokyo (35◦41′ N, 139◦29′ E, 46 AMSL). The soil of the paddy field is classified as alluvial
soil [42]. Phosphorus (P205) and potassium (K2O) fertilizers were applied at 30 kg ha−1 as
basal fertilizers. Ammonium sulfate (NH4SO4) was applied at 20 kg ha−1 as top-dressers at
five different times (at two weeks intervals). Rice seeds were sterilized using 0.1% Sumition
and 0.5% Sportac Stana SE (Sumitomo Chemical, Japan) for 24 h, then thoroughly washed
immediately in tap water at five separate times for 15 min. Seeds were later allowed to
imbibe in tap water for about 72 h (water is changed after every 24 h) till germination.
Germinated seeds were sown in nursery boxes on 7 May 2020. Seedling at the fourth leaf
stage was transplanted to the paddy field on 21 May. The planting area was 120 m2, with
a 30 cm × 30 cm planting distance (Figure S1). The field was submerged throughout the
experiments by irrigation.
The first field in Ghana was in the Ohayo farms located at Nsawam within the semide-
ciduous rainforest ecological zone (06◦8′48′′ N, 00◦53′58′′ W, 170 m). This area receives
bimodal rainfall (1300–1500 mm) annually, allowing for two cropping seasons. The soils
covering the area are classified as Acrisol and characterized as forest ochrosols with a fri-
able consistency in a fine granular structure (FAO/ISRIC/ISSS, 1998). Before transplanting,
seeds were soaked for 24 h in clean water and incubated in a jute sack (25 kg) filled to
half its capacity for 30–36 h for germination. Seeds were sown on prepared seedbed plots
(close to the field) of 1 m × 10 m and raised 5 cm above the original field level. Seedlings at
the third leaf stage were inoculated with the commercial formulation of B510 (30 mL/L)
and transplanted to the paddy field two days later at a spacing 25 cm × 20 cm, and a
planting density of 20 hills/m2. The experimental plots were laid out in a randomized
complete block design with three replicates arranged in a 2 × 2 × 4 factorial scheme. Field
management was by the local agronomic practices with a split fertilizer application rate of
30:30:30 kg ha−1 NPK and 30 kg ha−1 urea prior to the reproduction stage. The experiment
was under an irrigation system. The data were collected in November 2019.
The second field was at the Golinga irrigation area within the Guinea Savanna agro-
ecological zone (09◦21′06.0′′ N, 000◦57′01.0′′ W, 139 m), with unimodal rainfall (1100 mm),
flash floods, and drought; all of which significantly impact crop production (Ghana Meteo-
rological Agency, [43]). This site has one cropping season, and soils covering the area are
classified as Ferric Acrisols/Ferric Lixisols [44]. Seeds were sown in prepared seedbeds
on 8 August 2020. Seedlings at the fourth and fifth leaf stage were inoculated with the
commercial formulation of B510 and transplanted to the paddy field three days later. In a
split application, 60:60:60 kg ha−1 NPK was applied as a basal application to fertilized plots
a week after transplanting, and a top-dressing of 30 kg ha−1 nitrogen was applied at the
booting stage in the form of urea. Weeds were manually controlled (by hand-picking) when-
ever necessary, and this activity did not cause any effect on any experimental treatments.
The field was submerged throughout the experiments by irrigation.
2.2. Rice Cultivars
We obtained certified rice seeds from three different sources during these experi-
ments. For the field experiment in Japan, Ghanaian cultivars Ex-Amantin, Ex-Boako,
Bawku-Market, and Zebila of the sub-species group Indica were obtained from the Ge-
netic Resources Centre NARO (National Agriculture and Food Research Organization,
Tsukuba, Ibaraki, Japan). We used Japonica rice (Oryza sativa cv. Nipponbare) as a control
to confirm the positive PGPR effect of B510. At Nsawam, we used the commercial cultivar
Exbaika, which is tolerant against rice blast disease. Golinga seeds of four commercial
rice varieties AgraRice, Amankwatia, Exbaika, and Jasmine85, were obtained from the
Rice Development Sector, CSIR: Savanna Agriculture Research Institute, Tamale, Ghana.
Further characteristics of the rice cultivars used in this study are summarized in Table S1.
Microorganisms 2021, 9, 2000 4 of 14
2.3. Azospirillium Inoculation of Rice Seedlings
Rice seedlings were inoculated with Azospirillum sp. B510, using the commercial
bacterial solution Ine-Fighter® (Mayekawa Co., Ltd., Tokyo, Japan). Seedlings were watered
with 3.3 mL Ine-Fighter® solution diluted in 1 L of sterilized distilled water per nursery
box (approximately 2 × 107 CFU/plant). Nursery grown seedlings were inoculated with
B510 once, 2–10 days before transplanting, according to company’s instruction.
2.4. Colonization Assay Using Ds-Red-Tagged Azospirillum
Endophytic bacterial colonization and observation of lateral roots colonized by DsRed-
tagged B510 was performed according to the procedures followed by Naher et al. [41]. Rice
seeds were sterilized using 70% ethanol for 20 sec, followed by 5% sodium hypochlorite for
10 min and then thoroughly washed 5 times in sterilized distilled water. Sterilized seeds
were soaked in Rice Growth media containing 1 mM KNO3 and the bacterial suspension
was then dropped on the seeds (50 µL/seed, approximately 2 × 105 CFU/plant). Plants
were grown in a growth chamber (LPH-240SP; NK System, Osaka, Japan) under a regime
of 16 h light:8 h dark at 25 ◦C for 14 days. The lateral root surface was observed using an
Olympus IX71 fluorescence microscope (Olympus, Tokyo, Japan). The measurement of
colony-forming units (CFU/g) inside plant tissues was weighted and surface sterilized
using 70% ethanol for 15 s followed by 1% sodium hypochlorite for 30 s after which samples
were thoroughly washed 3 times in sterilized distilled water. A sterilized mortar and pestle
were used for crushing whole seedlings after adding 1 mL 0.85% sodium chloride; solutions
were spread on nutrient broth media containing 50 mg L−1 polymyxin B and 50 mg L−1
streptomycin at appropriate dilutions. After incubation at 28 ◦C for 3 days, the number of
antibiotic resistant colonies were counted.
2.5. Evaluation of Plant Growth and Yield
A range of agronomic characteristics was assessed at the vegetative and harvested
stages to evaluate the impact of strain B510 on the growth performance of rice plants: plant
height, tillers number, chlorophyll content (SPAD), root length, fresh and dry weight of
both shoots and roots, and the number of panicles and grain yield (harvested stage only).
Dry weight values were obtained by oven-drying at 60 ◦C for 72 h and weighted.
2.6. Statistical Analysis
Statistical analyses were performed using a one-way analysis of variance (ANOVA)
followed by the Student-Newman-Keuls (SNK) test in R software [45]. Different lower-case
letters represent significantly different values (p < 0.05).
3. Results
3.1. The Endophytic Colonization of Azospirillium sp. B510 in Japonica and Ghanaian Rice
In a previous study, some Indica rice cultivars showed a positive PGPR response to
inoculating with B510 under low nitrogen conditions [33]. However, it was unclear whether
this isolated strain from the Japonica rice cultivar could colonize Indica rice. Therefore, we
visualized the colonization of B510 in four selected Ghanaian rice cultivars (Ex-Amantin,
Ex-Boako, Bawku-Market, and Zebila) by performing an inoculation test using DsRED
tagged strain and estimated the colonization effect under a field condition in Japan [46].
The result revealed a red fluorescence in each cultivar’s lateral roots (Figure S1), indicating
that B510 had colonized the root surface. Besides, the display of colony-forming unit (CFU)
by the endophytic colonization of DsRED tagged B510 inside whole plants shows that the
greatest degree of B510-colonization was observed in the cultivars Zebila and Ex-Amantin,
respectively (Figure 1). However, no significant difference was found between the CFUs of
the Bawku-Market, Ex-Boako, and Nipponbare cultivars. These results indicated that B510
could colonize both the surfaces and inner tissues of Japonica and Ghanaian rice plants.
Microorganisms 2021, 9, x FOR PEER REVIEW 5 of 14 
 
between the CFUs of the Bawku-Market, Ex-Boako, and Nipponbare cultivars. These re-
sults indicated that B510 could colonize both the surfaces and inner tissues of Japonica 
Microorganisms 2021, 9, 2000 and Ghanaian rice plants. 5 of 14
200,000
a
ab
b
100,000 b
b
0
Am Ba Bo Ze NB
 
Figure 1. The endophytic colonization of Azospirillum sp. B510 on Japonica and Ghanaian rice.
FTighue reen d1o.p Thhytei cecnodloonpizhaytitoinc ocfoDlosnRiEzDa-ttiaogng eodf BA51z0osipnitrhilelutimss usepo. fBG5h1a0n aoinan Ja(Epxo-Anmicaan atinnd(A Gmh)a, naian rice. The 
eBnadwokpuh-Mytairck ectoal(oBna)i,zEaxt-iBoona kof( BDo)s, RanEdDZ-etbaiglag(Zede) )Ban5d10Ja pionn itchae(N tipispsouneb aoref (NGBh))arnicaei.aCno lo(Enixe-sAmantin (Am), 
Bwawerekcuo-uMntaerdkuestian (gBdai)l,u Etixon-Bmoeatkhod (sB.oV)a,l aunesdp Zreesbeniltae d(Zaree))t haenadv eJarapgoen±icSaD (Nfroipmp6ornebplaicraet e(Ns oBf )) rice. Colonies 
woenreep claonutneatechd. uDsififnegre ndtilleuttteiorsni nmdiectahteotdhse. sViganliufiecasn pt rdeifsfernetnecdes abreet wtheen atvreeartamgeen t±s (SSDtu dfernotm–  6 replicates of 
oNneew pmlaann–t Keeauclhs .( SDNiKff)etersetn, pt <le0t.t0e5r,sn i=n6d)i.cate the significant differences between treatments (Student–
N3e.2w. Emffaenct–oKf eAuzloss p(iSrNilliKu)m tesps.t,B p5 1<0 0I.n0o5c,u nla =ti o6n).o n the Growth-Promotion in Ghana Rice in a
Paddy Field in Japan
3.2. ETffheecte foffe cAt zoofsBp5ir1i0lliinuomcu slapt.i oBn5o1n0 Indoiccualraitcieoncu oltniv tahres Gharsobwetehn-Ppreovmi outisolyn dinem Gohna-na Rice in a 
Psatrdadteyd Ftioelbde ibne tJtaerpaunnd er low-nitrogen than standard-nitrogen conditions [34]. To address
whether plant growth was enhanced by B510 inoculation, we performed a field experiment
in whTihche weeffteracnt sopfla Bnt5e1d0t hiensoeceudllaintgiosnof orince Icnudltiicvaar sritcoep cadudltiievsaorfse ihthaesr nboene-nn itprorgeevniously demon-
s(t−raNte)do rtost abned baerdttenrit ruongdener(+ loNw) a-pnpitlricoagtieon tchonadni tsitoannsdaanrddc-onmitpraorgeednth ceoirnadgirtoionnoms [ic34]. To address 
wtrhaeittsh(eFrig purlean2t). gTrhoewJathpo wnicaas reicnehcaunltciveadr Nbyip pBo5n1b0a rienowcausluasteiodna,s wa ep opsietirvfeorcmonetrdo la field experi-
mtoeansts einss wtheheicffhe cwt oef tthreainnsopculalanttioend ptrhoece ssseebdy lsitnragisn Bo5f 1r0i.cTeh ceuplltainvtahresi gthot pinacdredmieens tof either non-
noitbrsoergveend a(-mNo)n ogra lslttahnedcualrtdiv anristwroags uninfluenced by B51+N conditions. The highest tiller numbeenr (w+aNs )r eacporpdleidcaintio
0ni ncoculatthe cvo.nNdipit
iioonnbsu at nindcr eased byponbare (Ficgourme p2Ba)r.ed their agro-
nWomhilisct ttirllaeirtns u(mFibgeur raem 2on).g Tthheec Jualtpivoanrsicwaa rsipcrei mcuarlitliyvianrfl uNenipcepdobnybnairtreo gwenasfe urtsileizda taiosn a;  positive con-
trwoel otbos earsvseedsas pthosei tievfefeincflt uoefn tcheeo fiBn5o1c0uinlaotciuolnat iponroinceEsxs- Bboya ksot;rpaaini cBle51n0u.m Tbheres ipnltahnet height incre-
m−eNntc oonbdsiteiorvnewde raemgroenatge stailnl Ethxe-B ocauklotivseaerdsl inwgassth uatnhinadflubeeennciendo cbulya teBd5w10it hinBo5c1u0 lation but in-
c(rFeiagsuered2 bCy). +N conditions. The highest tiller number was recorded in the cv. Nipponbare 
Conversely, B510 inoculated Ex-Amantin plants had significantly fewer panicles than
(Fthige unroen -2inBo)c. uWlatheidlscto tnitlrloelr pnluanmtsb(eFrig aumreo2nCg). tAhcec ocrudlitnivglayr, sg rwaians wperiigmhtarwilays ignreflauteernced by nitro-
giennt hferNtiilpizpaotniboanre; ,wEex -oBbosaekrov, aendd aZ peobisliaticvuelt iivnafrlsutehnacne− oNf Ba5n1d0n ionno-icnuolcautliaotend inp lEanxt-sBoako; panicle 
n(uFimgubreer2sD i)n.  Tthee g-rNai ncownedigihtitoonf iwnoecruel agterdeaptleasntt sinw Easxl-oBwoearkthoa sneneodnl-iinngoscu tlhaatetd hpaldan btseen inoculated 
wuintdhe Br t5h1e0− (NFicgounrdeit 2ioCn)(.E x-Amantin) and both −N and +N conditions (Bawku-Market).
The only evidence of a negative effect of B510 inoculation on shoot weight was observed in
the Bawku-Market grown cultivars in the −N area (Figure 2E).
 
CFU g-1 Fresh Weight
Microorganisms 2021, 9, 2x0 F0O0 R PEER REVIEW 6 6ooff 14 
 
A -Na
200 a a
B
a 30 -N, +B510
a a b
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ttiillllerr numbeerr ((B)),, paniiclle numbeerr ((C)),, graiin weiightt ((D)),, shoott weiightt (E) and root weight (F). The 
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<p 0<.005.0, 5n, =n 6=–96)–.9 ).
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uTrhee 2EDx)-.B Tohaek gorpaliann wtseiinghoct uolfa itneodcwulearteeds ipglnainfitcsa wntalsy ldoiwffeerr etnhtanco nmonp-airneodcutolactoedn tprolalnptlsa unnts-,
dwehr itche p-Nro vcoentdoitbioent h(Eexm-Aomstanetfifnec) taivned tbroetahtm -Nen atn, dre +vNea clionngdiatnionins c(rBeaawsekuin-Mroaorkt ewt)e. iTghet 
o(Fnilgyu ervei2dFe)n. ceT hoef rae nweagsatnivoes eigffneicfit coafn Bt 5d1i0ff einreonccuelabtieotnw oeenn shtroeoatt mweenigtshto wf tahse oZbesebrilvaedan idn 
tEhxe- ABamwaknuti-nMcaurlktievta grsr.oIwnno ccuullattievdarBsa iwn kthue-M -Nar akreetas (hFoiwguerde a2nE)a.p parent significant decrease
in roIont dNriypwpoenigbhatrec,o Bm5p1a0r eindowcuitlahtipolna nstisgtnriefaictaendtlwyi tihncfreeratisleizde ro(Foitg wureeig2hFt) .uTnhdeesre trhees u+lNts 
csoungdgietsiot nth, atnEdx f-eBrotialikzoerr etrsepaotnmdeendt preodsiutciveedl yrototB g5r1o0witnho c(Fuilgautiroen 2(Fw).h Cicohnwvearsselxyh, itbhiete rdooint 
wtheeighrat ionf yGiheladn)aciaonm rpicaer ecdulwtivitahrsth wearse sgproeantseer oinb spelravnetds tarmeaotendg wthiethc ufelrttivilaizresrE (xF-iAgumrea n2tFi)n. 
Tahned EBxa-wBkouak-Mo aprlkaentt.s inoculated were significantly different compared to control plants, 
which prove to be the most effective treatment, revealing an increase in root weight (Fig-
u3r.3e. 2CFh)a.r aTchteerriest iwc Rase snpoon sseigonf iCfiuclatnivta rdsiftfoeAreznoscpei rbilelutwmesepn. Btr5e1a0tmIneoncutsla toifo nthaem oZnegbFilear tailnizda tEioxn-
AmanPtriin ciuplatilvcaorms. pInoonceunlat taenda Blyaswisk(uP-CMAa)rkweat shpoewrfeodrm ane daptopadremnto snigstnriafticeahnot wdercirceeaisneo icn- 
ruoloat iodnrya wndeifgehrti lciozmatipoanreadp pwlictha tipolnanatfsf etcreteadtetdh ewriitche fceurtlitliivzaers ’(Fgirgouwreth 2-Fp)r.o Tmhoetsien gretsrualits 
saungdgyesiet ltdh.aMt Eoxr-eBoovaekr,oP rCesApownadseadp poliseidtivtoelcyo tmo pBa5r1e0 tihneoculatitvioanr’ s(wrehsipcho nwseas teoxhthibeidteidff einr- 
tehnet gtrreaaintm yeineltds,) pcaormtipcuarlaerdl ywtihthat tohfeB r5e1s0poinosec uolbastieornv.eSdt atmisotincga ltahnea clyusltiisvsahrso wExe-dAtmhaatntthine 
agnrodw Btahw-pkruo-mMoatriknegt.c haracteristics and yield parameters associated with the rice cultivars
 
Shoot weight (g) Plant height (cm)Panicle number/plant
Root weight (g) Grain weight /plant (g) Tiller number/plant
Microorganisms 2021, 9, x FOR PEER REVIEW 7 of 14 
 
3.3. Characteristic Response of Cultivars to Azospirillum sp. B510 Inoculation among 
Fertilization 
Principal component analysis (PCA) was performed to demonstrate how rice inocu-
lation and fertilization application affected the rice cultivars’ growth-promoting traits and 
Microorganisms 2021, 9, 2000 yield. Moreover, PCA was applied to compare the cultivar’s responses to the dif7feorfe1n4t 
treatments, particularly that of B510 inoculation. Statistical analysis showed that the 
growth-promoting characteristics and yield parameters associated with the rice cultivars 
responded differently among inoculated and fertilized samples. Two main components 
raecscpoounndteedd fdoirff 7e4re.7n%tl yofa tmheo nvgariinaboicluitlya toebdsearnvdedfe irnt itlhizee ddastaam, wpiltehs .PTCw1 oacmcoauinntcinogm fpoor n4e8n.8t%s  
aocfc othuen tteodtaflo vra7r4ia.7t%iono fatnhde PvCar2ia abcicliotyunotbinsegr vfoerd 2in5.9th%e (dFaitgau, rwe i3th). PInCo1cauclcaotiuonnt iwngithfo Br54180.8 a%f-
offecttheed troitcael gvraorwiatthio annadn ydiePldC 2reagcacrodulenstsin ogf fnoirtr2o5g.e9n% fe(Frtiigliuzraetio3)n.. IPnCoAcu ilnadtiiocnatewdi tphoBsi5t1iv0e 
affected r
ccoorrrreelalatitoio
icnes gbreotwwtehena npdlaynite hldeirgehgta, rrdoloets s of nitrogen fertilization. PCA indicated positivens between plant height, root lelennggththa anndds shhoooottd drryyw weeiigghhttb buuttn noottf foorr ttiilllleerr nnuumm--
bbeerr, ,p paanniciclelen nuummbbeer,r,p paanniciclelew weeigighht,t,g graraininw weeigighhtta annddr roooottd drryyw weeigighht.t.F Fuurrtthheerrmmoorree, ,P PCCAA 
ccoonnfifrirmmeeddt htheen negeagtaitvieveim impapcatcot foBf 5B1501i0n oincuoclautliaotnioonn othne tsheepsea rpaamreatmerest,ewrsi,t hwtihthe tehxec eepxtcioepn-
otfiorno ootf lreonogtt hle(nFgitghu (rFei3g)u. re 3). 
 
FFiigguurree3 3. . PPrriinncciippaall ccoommppoonneenntt aannaallyyssiiss ((PPCCAA)) ooff rriiccee ccuullttiivvaarrss wwiitthh oorr wwitithhoouutt BB551100 ininooccuullaattiioonn.. 
TThheep plalannttg grroowwththc chhaarraaccteterrisistticicss( s(shhoooottl elennggtthh,,p plalannttu uppppeerrw weeigighhtt,,r roooottl elennggthth, ,r orooottw weeigighht,t,t itlillelerr 
nnuumbbeerr,,p paanniciclelen nuummbbeerr, ,a annddp paanniciclelew weeigighht)t)w weererea asssesesssesedda at tt htheeh haravrvesetsetdeds tsatgage.e.R Riciceec ucultlitvivaarsrs 
aarree ddeennootetedd bbyyd dififefererennttc ocololorsrsa assg grerenen-N-Nipippoonnbbaarere( N(NBB),),y yelelloloww-E-Exx-B-Booaakkoo( B(Boo),),p puurprplele-Z-Zeebbiliala 
(Z(Zee),),r reedd-E-Exx-A-Ammaanntitnin( A(Amm),),a annddb bluluee--B Baawwkkuu-M-Maarkrkeet t( B(Baa).).T Thheel elennggththo offl alabbeeleleddl ilnineessa alolonngga anna axxisis 
ininddicicaatetesst htheem magangintuitduedeo focfo crorerlraetliaotniobne tbweteweneeonr igoirnigailnvaall uveasluoefsa otfr aai ttraanidt athnedp trhien cpiprianlcciopmalp coonmenpto.-
nent. 
We examined the impact of inoculation on plant morphology, yield, and phenotypic
characWteer iesxtiacms. iWneedo tbhsee rimvepdatcht aotfr iinceocullatitvioanrs owni pthlasnhto mrtocruplmhoslopgroyd, yuiceeld,h ainghde prhneunmotbyeprsic 
ocfhtailrlaecrtse,raisntdiccso. nWve rosbelsye,rtvheedc tuhlatitv raircse wcuitlhtivloanrsg wcuiltmh shporrotd cuuclemdsf epwroedruticleledr sh.iTgher enfourme,-
PbCe1rss oepf tairllaetresd, arincde cuolntivvearrsseliyn,t othtew cougltriovuaprss wiitth lcolneagr cduilsmtins cptirondubacesedd feownethr etilrlecrusl.m Thsiezre,-
rfeograer, dPlCes1s soefpfaeratitleizde ritcree acutmltievnatr.sT ihnetoc utwltiov agrsouwpitsh wcihtahr aclcetaer isdtiisctainllcytisohno rbtacsuedlm osnw thereeir 
Ncuiplmpo snibzaer, er,eEgxa-rBdoleaskso o, fa nfedrtZileizbeirla t,rweahtmerenast. ETxh-Ae mcualntitvinaras nwditBha wchkaur-aMctaeriksetitchalaldy lsohnogrt 
ccuullmss. wAmeroe nNgipthpeognrboaurep,s Ewx-eBoobaskeor,v aendd,  tZhebciulalt, iwvahresrEeaxs-B Eoxa-kAomaanndtiZne abnilda Boavwerklaup-Mpeadrkinet 
one group. Ex-Amantin and Zebila also coincided with the plot, suggesting similarity in
genotype (thus Indica). The assessment of morphological traits also confirmed the positive
effect of B510 inoculation on the growth of Nipponbare and Boako (as reflected by their
 higher grain weights) compared to the negative impact on Ex-Amantin and Bawku-Market
(as reflected in their lower grain weights).
Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 14 
 
had long culms. Among the groups we observed, the cultivars Ex-Boako and Zebila over-
lapped in one group. Ex-Amantin and Zebila also coincided with the plot, suggesting sim-
ilarity in genotype (thus Indica). The assessment of morphological traits also confirmed 
the positive effect of B510 inoculation on the growth of Nipponbare and Boako (as re-
Microorganisms 2021, 9, 2000 flected by their higher grain weights) compared to the negative impact on Ex-Aman8toifn1 4
and Bawku-Market (as reflected in their lower grain weights). 
3.43.. 4E. fEfefcfte cotf oAf zAozsopsirpiilrliullmu msps. pB.5B1501 I0nIoncoucluatliaotnio onno Gn rGorwotwht ahnadn Yd iYelidel dofo Cf oCmommmerecriacila Rl iRceic e
PrPordoudcutciotino nini nGGhahnaan a
WWe einivnevsetsigtiagtaetde dthteh eefefeffcetc ot fo Bf 5B1501 0ono nthteh eprpordoducutcivtiivtyit yofo fcocmommmerecricaila rlirciec ecuclutilvtiavrasr s
grgorwown nini nGGhhaannaa. .TTwwoo sseeppaarraattee fifieelldd ttrriiaallss wweerreec oconndduuctcetdedu suisnigngd idffiefrfeenretnrtic reicvea rviaetriieestiaens d
anudn udnerdderif dfeirfefenrteangt raog-eroco-elocoglyo. gMyo. Mrpohroplohgoilcoagliacanld aynide lydiereldsp roenspseosntsoesB t5o1 0B5in1o0 ciunloactuiolnatwioenr e
weevrea leuvaatleudaatetdth aet vtheeg evteagtievteataivned ahnadr vheasrtvedesstetadg set.age. 
InI nOhOahyaoy-foa-rfmar,m w,ew eevaelvuaaltueadt ethde tehfefeecftf oecf tBo5f10B i5n1o0cuinlaotciuolna toino nthoen mtohset mcuoltsitvcauteltdiv raictee d
cvr. iEcexbcav.ikEax ibna aik faieilnd atrifiael lmd atrniaagl emda unnadgeerd suimndilearr saigmroilnaormagicr opnroacmtiicceps roafc tthicee sloocaf lt hsme laollc-al
hoslmdearl lhfaorlmdeerrsf aorfm theer saorefat.h Tehaer efrae.shT hweefirgehsth owf Bei5g1h0t ionfocBu5l1a0teidn opcluanlattse idncprleaanstesdi nscigrenaifsie-d
casnigtlnyi (fiacpapnrtolyxi(mapapterloyx 1im.5 attimelyes1 h.5igtihmere)s inh icgohmerp)airniscoonm wpiatrhi snoonnw-initohcunloante-idn opclaunlatst e(dFipgularnet s
4A(F).i gTuhries 4sAig)n. iTfihciasnsti gefnfieficct arnetcoerffdeecdt  rienc othrde efdreisnh tphleafnrte swhepiglahnt towf Bei5g1h0t ionfoBcu51la0teindo pculalnattes d
wpasla antttsriwbuatseda ttori bthuete ndutmobtehre onfu pmanbiecrleosf ppearn piclalenst p(Feirgpulraen 4tB()F. iTguhere n4uBm).bTerh eofn puamnbicelreso f
pepra pnliacnlets frpoemr  pinlaonctuflraotemd irnicoec uplatnetds erixcheibpiltaendt sa esxighnibifiitceadnta isnicgrneiafiscea cnotminpcarreeadse wciothm npoanre-d
inwocituhlantoedn- pinlaoncutsl.a Nteodt polnalnyt sd. iNd otht eo nplayndicildesth inecpraenasicel,e bsuint cthreea wsee, ibguhtt tohfe sweeedigs,h atno fatstereibdus,tea n
ofa tytireibldu tiemopfryoiveledmiemnpt,r osvheomwendt ,as hsoigwneifdicaansitg ndififfcearnent cdeif fbeertewncee nb eitnwoeceunlaitneodc upllaatnedts palnandt s
noan-dinnoocunl-ainteodc upllatnetds (pFliagnutrse( 4FCig)u. Trehe4 Cgr).aiTnh we egirgahint rweceoirgdhetdr einco inrdoecdulianteidn opclaunlatste wdaps l1a.n2t s
timweas 1h.i2ghtiemr etshhanig hine rntohna-ninioncnuolant-eidn opclualnatse.d Cpollalnectsti.vCeolyll, eocutirv erleys,uolutsr frreosmul ttshfer ofmirsth feiefildrs t
trifiael lidn t2ri0a1l9i nsu2g0g19esstu tghgaet stthteh aritcteh yeireilcde oyfi etlhdiso cfothmismceormciaml ecruclitailvcaur lwtivaas rswigansifsiciganitfilyc aent-ly
haencheadn c(pe d< (0p.0<50) .b0y5) Bb5y1B0 5i1n0ocinuolactuiolant;i otnh;etrheeforerefo, rteh,et hsetrsatirna iBn5B105 1c0anca pnopteontetniatlilayll yimimprporvoev e
plpalnatn gtrgorwowtht hanadn dprpordoudcutcivtiivtyit ycuclutilvtiavrasr sini nGGhahnaan.a .
 
Figure 4. The effect of B510 inoculation on rice growth managed under field conditions in Nsawam,
FiGguhraen 4a.. TAhfie eelfdfetcets tofw Ba5s1p0e irnfoorcmuleadtioinn Oonh aryicoe- fgarromwatht N msaanwaagmedu usinndgecrv f.ieElxd- bcoainkdai.tiPolnans tisn wNesraewgaromw, n
Ghana. A field test was performed in Ohayo-farm at Nsawam using cv. Ex-baika. Plants were grown 
ini an naunrusersreyr fyiefildel danadn dseseedeldinlignsg ws wereer ienioncouclualtaetde dwwithit hB5B1501. 0A. fAtefrte 2r d2pdip, ti,hteh pelpanlatns tws wereer teratrnasnfesfrerrerde d
tot tohteh peapdaddyd fyiefildel.d G. rGarinai ynieylidel dwawsa ms meaesausruerde dafateftre 2r m2 monotnhtsh. sG. rGorwowtht hchcahraarcatectreisrtiisctsic assassessessesde dwwereer e
frefrsehs wh ewigehigth otfo tfhteh uepuppepr egrrgorwonw pnapratsr t(sA()A, n),unmumbebr eorf opfapnaicnliecsle (sB()B, a),nadn gdrgairna iwn ewigehigt hptepr eprlapnlat n(Ct ()C. ).
BaBra croclorlos:r sn:onno-inn-oincoucluatleadte cdocnotnrotrl o(lb(labclakc)k a)nadn idnioncoucluatleadte dwiwthit Bh5B1501 (0g(rgarya)y. V).aVlualeuse psrpersesnetnetde daraer tehteh e
average ± SD from 10 plants each 7 plots. Statistical analysis was performed, the asterisk indicates
the significant differences between treatments (Unpaired Student’s test, p < 0.05, n = 7).
 In the second field trial conducted at Golinga, we examined four commercial rice
varieties (AgraRice, Amankwatia, Exbaika, and Jasmine85) under a continuous irrigation
system, nitrogen fertilization and B510 inoculation. The results revealed that fertilization
significantly affected almost all the cultivars at the vegetative and reproductive stages
(Table S2). In addition, we observed some marginal increases in the plant growth at 56 and
Microorganisms 2021, 9, 2000 9 of 14
84 days after transplanting at the vegetative stage, which was equally significant under
the chemical fertilizer treatment (+N) compared to B510 inoculation without fertilizer
application (−N), among all four cultivars (Tables S2 and S3). For instance, in the +N
plants, the number of tillers and the leaf chlorophyll indices recorded among cultivars
were both significantly higher than the control plants (Table S3). The leaf chlorophyll
content recorded during this experiment was taken as a measure of the rice cultivars’
photosynthetic response to B510 inoculation and, therefore, can be used as a direct measure
of plant growth. However, the inoculation impact of B510 and the response of the rice
cultivars on leaf chlorophyll index varied. In inoculated Exbaika and Jasmine85 plants, a
significant increase was seen in the leafy chlorophyll index compared to non-inoculated
plants 56 days after transplanting. At 84 days after transplanting, the chlorophyll content
detected in inoculated Jasmine85 plants was significantly higher than inoculated Exbaika
plants (Table S3).
In assessing the impact of B510 colonization on the accumulation of dry matter compo-
nent in rice plants at the vegetative phase, the root length, root and shoot biomass of each
treatment was measured and estimated in comparison to the control plants. This result re-
vealed variable responses among the inoculated plants compared to +N and non-inoculated
plants within the 56 and 84 days after transplanting (Table S3). Generally, the average root
length recorded among all the treatments at 56 days after transplanting were approximately
4 cm longer than the length observed at 84 days after transplanting, whilst average root
biomass was approximately 15 g higher at 84 days after transplanting. Root length of
Amankwatia and Exbaika at 56 days after transplanting was enhanced significantly by
B510 inoculation compared to their respective control plants. The average root length of
AgraRice was significantly greater at 56 days in the fertilized and B510 inoculated plants
(approximately 27.5 cm) compared to the control plants (approximately 19 cm). AgraRice,
Amankwatia, and Exbaika root biomass were increased significantly by B510 inoculation
84 days after transplanting, compared to control plants. AgraRice and Jasmine85 shoot
biomass of B510 inoculated plants at 56 and 84 days after transplanting were significantly
higher than control plants. Amankwatia and Exbaika were significantly decreased by B510
inoculation after 56 days, compared to control plants. However, at 84 days, the shoot
biomass of inoculated Amankwatia was significantly higher than the control plants and
inoculated Exbaika plants also showed an increase in shoot biomass (although there was
no detectable significant difference in the latter). These results suggest that the overall
effect of B510 colonization on the growth of Ghanaian rice is positive, but the extent is
variable among cultivars.
Other yield parameters were considered at harvest after grain maturity, including plant
height, number of tillers, number of panicles, grain weight, harvest index, total biomass
and grain yield (Figure 5, Table S3). Variation was found between the cultivars’ response
to B510 inoculation, and the results were statistically significantly different. Inoculated
AgraRice plants exhibited a highly significant increase in grain yield (kg/m2) compared
with that of the control plants. Similarly, the grain yield of inoculated Amankwatia and
Exbaika plants was significantly greater than the control plants. Jasmine85 inoculated
rice plants differed in exhibiting a significant decrease in grain yield compared to the
control plants.
Microorganisms 2021, 9, x FOR PEER REVIEW 10 of 14 
 
Microorganisms 2021, 9, 2000 10 of 14
Figure 5. The effect of B510 inoculation on rice growth managed under field condit ions in Golinga,
FigGurhea n5.a T. Ahefi eeflfdectte sotf wBa5s10p einrfoocrumlaetdioinn oGno lriincge ag,rGohwatnha muasinnagg4edcu ultnidvaerrs fi(eAldm caonnkdwiatitoian,sA ing rGa,oElixn-gbaa,i ka,
Ghaannad. JAas mfieilnde 8te5s)t. wPlaasn tpsewrfeorremgerdo winn Ginolninugras,e rGyhfiaenlad ,uasnindg in4 occuulltaivteadrs B(5A1m0 aonnktwheatyiao, uAnggrsae, eEdxl-ing.
baiAkaf,t earn2dd Jpasi,mthineep8l5a)n. tPslwanetrse wtrearnes fgerrorewdnt oint hneufirseeldry.  Gfieraldin, ayniedld inwoacsulmateeads uBr5e1d0a oftne rth2em yoonutnhgs .sTeehde-bar
ling. After 2 dpi, the plants were transferred to the field. Grain yield was measured after 2 months. 
Thec oblaorr sc:onloorns-:t rneoante-dtrceoantetdro clo(wnthriotle )(,waphpitlei)e,d acphpelmieidc aclhfeemrtiilcizael rfe(grtrialiyz)earn (dgrianyo)c ualnadte dinwociuthlaBte5d10 w(bitlahc k).
B51V0a (lbuleascpkr).e sVeanltueeds aprreetsheenatevde raargee tShDe afrvoemragther SeeDr ferpolmica tthersepee rreaprleicaa. tSetsa tpisetri caarleaan. aSltyastiisstwicaasl apnearfloyrsmis ed,
wasd pifeferrfeonrmt leedtt,e drsif(fae,rebn, tc )leitntdericsa (tae, bsi,g cn) iifincdaincattdei sffiegrneinficceanbet tdwifefeenretnrceea tbmetewntese(nS ttruedaetmnte–nNtes w(Smtuadne–nKte–uls
New(SmNKan)–tKesetu, pls< (S0N.0K5,) nte=st9, )p. < 0.05, n = 9). 
4. D4.isDcuisscsuiossni on
In tIhnisth sitsusdtyu,d wy,ew fiersfitrlyst clylacrliafireidfi etdhatth oant oe noef tohfet hgeengeernae oraf PoGf PPGRP, BR5, 1B05 1e0nheannhcaendc epdlapnlta nt
grogwrothw tahnadn dinincrceraeasesedd tthhee yyiieelldd oof fs osmome see lseeclteecdteGdh aGnhaaiannairaicne cruicleti vcaurlst,ivinacrlsu, diinncgluEdxibnagik a,
ExbEaxi-kBao, aEkxo-B, Aoagkroa,R Aicger,aaRnidceA, amndan Akmwaatnika.wTahtieas. eTchuelstei vcaurlstisvhaarsr eshthaeref othlleo wfoilnlogwpinhgen pohtey-pic
notcharacteristpylpanict clehnagrathc
ictesroisftaicssh oofr tac ushlm and higand low tillerinogrt ccauplamc ia
hntdil lheriginhg tcilalperaicnity. Conversely, cultivars with a tallerties like Ex-Amagn tcinapaancdityB.a Cwoknuv-eMrsaerlkye, tcruelvtievaalresd a
witnhe ag attailvleerr peslapnotn lseentgothB 5a1n0di lnoowcu tliallteiorinng(F ciagpuareci3ti)e.sW liekeca Enxu-Asemtahnistifinn adnidng Btaowekausi-lMy aidrkenett ify
revweahliecdh oaf ntheegactuivlteiv raersspeovnaslue attoe dBw51o0u ilndorceusplaotniodnp (oFsiigtiuvreel y3t)o. WB5e1 0cacno lounseiz tahtiios nf.inOduinr gre stou lts
easailrye pidoetnentitfiya lwlyhbiechne ofifc tihalei ncuolrtidvearrtso erveadluucaetetdh ewroisukldin rveoslpvoedndin paosfiirtsivt-etlimy teoi nBo5c1u0l actoiloonntir-ial.
zatNiounm. Oeuroru rsesAuzltoss pairrei lpluomtensptieaclliyes bhenavefeicbiaeel nini osordlaetre dtoo rnedauwceo trhlde wriisdke inbvaosilsvefrdo imn ad fifirfestr-ent
timpel ainnotscuanladtisooni ltsr.iaTlh. eNyuhmavereobuese Anzuotsipliizreildluams sepsseecnietsia hl abvioe- fbeeretinli ziseorlsaitnedc roonp ap wroodruldctwiviidtye in
basvisa rfirooums cdoifufnerterinets p[4la7n].tsF aonrdin ssotialns.c Te,htehye hsapveec ibeesewn euutisliezdedin atsh eissssetnutdiayl wbieor-efeirstoilliazteerds firno m
crorpic perpoldauncttsivinityJa ipna vnaarinodusu ctioliuznetdriaess [b4i7o]-.f eFrotril iinzestrasnfcoer, rtihcee sppreocdieusc wtioen u.sTehde isne tbhaisc tseturidaya re
werreep iosrotleadteads fnroonm-p raicthe opgleannitcs rihni zJoapbaacnt earniad, ourtielinzdeodp ahsy bteios,-fcearptialbizleerosf fporro rmicoet ipnrgodthuecgtiroonw. th
Theasned byaicetledrioaf arriece repplaonrttsed[3 a7s,4 n8o].nT-phautsh,otgheenyica rrehiczoonbsaidcteerreiad, toor beendsaofpehaysteasn, icnaopcaubllaen otff or
profimelodtienvga tlhuea tgioronw. th and yield of rice plants [37,48]. Thus, they are considered to be safe 
as an inIoncuthlaenpt rfeosre fnietlsdt uedvya,luwaetioconn. firmed the growth-promoting attributes of Azospirillum
coIlno nthizea tpiroenseinntr sictuedpyro, wduec ctioonnfiirnmpeadd tdhye figreoldwsthu-npdreormdoitfifnerge natttrciublutitveas toiof nAmzoaspniargilelumme nt
colpornaicztaitcieosn.  Oinu rrircees uplrtosdinudctiicoante itnh aptaidndoycu fliaetlidosn uonf dBe5r1 0d,ieffiethreenr ta lcounlteivoartiinonc omabninagateimonenwti th
prancittircoegs.e On uferr rteilsiuzeltrs,  sinigdnicifiatcea nthtlayt iennohcaunlacetidonri ocef Bp5la1n0,t egirtohwert ahloanned oyri einld c-oremlabtiendatpioanra wmiethte rs
nitroovgeernt hfeerctoilnizteror,l stirgenatifmiceantt.lyM eannhyasntcuedi ersichea pvleanret pgorortwedths iamndil ayriereldsu-rletslaitnedri pcearparmodeutecrtsi on
ovearn tdheo tchoenrtcrrool ptrse. aAtmshernatf.f uMzaznamy astnuedtieasl. h[2a0v]e arnedpoIsrtaewda seimt ailla. r[3 r7e]surelptso irnte rdicteh patroindouccutiloatni on
ando foAthzeors pcirroilplusm. Aesihthraerffaulzoznaemoarni netv alr.i [o2u0s] caonmd bIsinawataio ents asli. g[3n7ifi] craenptolrytedn hthaantc eindotchuelagtriown th
of Aanzodspyiireillldumof eriitcheecro amlopnaer oedr itno vuanriinouocs ucloamtebdincaotniotrnosl ss.igIniaficreacnetnlyt esntuhdayn,cKedu mthaer gertoawl.th[4 9]
andre ypioerldte doft rhiacte icnoomcuplarteiodn to fuPnGinPoRcuallaotnede  ocronintrcoolsm. Ibnin aa rtieocnensti gsntuifidcya, nKtluyminacr reta sael.d [4p9l]a nt
rephoeritgehdt tuhpatt oin1o4c%ulactoimonp oarf ePdGtPoRth aelounei noorc iunl actoemd bwinhaetaitocno snitgrnoilf.icFaunrtlhye rinmcorerae,seitdh palsabnet en
heisghhotw unp itno m14a%iz ecoamndpasroerdg htou mtheth uant ibnotchulPaGtePdR wahloenate coornPtrGoPl. RFucrothmebrminoerde, wit ihthasn bitereong en
fertilizer increased plant dry weight compared to the control treatment [50].
 
Microorganisms 2021, 9, 2000 11 of 14
The B510 strain used in this study came from a source that has been considered to
enhance rice cultivars, exhibiting a short height and high tiller number phenotype. During
the ‘Green Revolution’ era (1940–1960), a semidwarf1 gene was inserted into the Japanese
rice cultivars from the cultivar IR-8 to reduce lodging and increase tillering capacity.
Significantly, the bacteria living inside the plant tissues of these cultivars evolved to survive
and become specific to their host plants, which explains the ability of B510 to enhance these
cultivars. As yet, an interaction existing between Japonica rice cultivars and Azospirillum
has not been reported. Although, malic acid secretion from rice roots has been reported as
a contributing factor to signaling and recruiting the bacteria during symbiosis [51]. Rice
roots secrete several organic acids, including malic acid, found in previous studies as a
bacterial substrate, enormously triggering chemotactic responses in B510 [40]. Therefore, to
investigate the interaction between Ghanaian rice cultivars and B510, we will quantify the
amount of organic acids secreted during colonization and analyze the varietal responses
to B510.
In our previous study, we explored the rice root microbiome from major rice-growing
regions in Ghana [52]. We analyzed the 16S rRNA amplicon sequences of bacteria within
the rice rhizosphere (cv. AgraRice and Jasmine 85) in diverse agroecological zones. We
obtained 64,180 microbiome sequences (denovoOTUs) from the extracted DNA of rice
roots in all sampling sites (DDBJ accession number; PRJDB9513, Table S4). Based on the
16S rRNA gene affiliation at the genus level, we identified 71 denovoOTUs belonging to
Azospirillum. The first four denovoOTUs possessed the greatest similarity to A. lipopherum
(denovo3368, similarity 100%), A. sp. TSO35-2 (denovo51236, similarity 100%), A. agricola
(denovo1560, similarity 99.21%), and A. brasilense (denovo28731, similarity 99.60%). This
result revealed Azospirillum species as the dominant strain colonized in the rice rhizosphere
of Ghana. Additionally, it is worth noting that A. sp. TSO35-2 shared the highest similarity
with the sequence from Ghana, an isolate from the rice paddy field in Tokyo, Japan [53].
The sequences of the inoculant used in this study (B510) also share high similarity to
TSO35-2. It was demonstrated that Azospirillum is ubiquitous in soils and can inhabit plant
tissues in diverse agro-ecologies across Ghana and Japan.
The application of bio-fertilizer by smallholder farmers in Ghana can increase farmers’
productivity and enhance soil fertility for sustainable production in the future. However,
the demand for bio-fertilizer in Ghana is limited in comparison to developed countries.
The low demand by farmers has been attributed to a lack of awareness, understanding of
what bio-fertilization are and how to apply them during crop production [54]. For instance,
Santos et al. [55] reported that farmers in SSA, including Ghana, use bio-fertilizers mainly
as an inoculant for leguminous plants, demonstrating insufficient awareness by farmers.
This contrasts with the occurrences in Argentina, South Africa, Brazil, and India, among
others, where they have embraced the use of biofertilizers with promising results [56].
In general, B510 application in rice cultivation provides an insight into bio-fertilization;
there are various plant responses that are important to the overall improvement of plant
growth and yield by cultivars with common phenotypic characteristics, even under dif-
ferent agroecological conditions and farm management. The findings of this study are
significant since the majority of research has focused on evaluating multiple PGPR in
the first trial. Our results indicate that the utilization of B510 as bio-fertilizer could be
an efficient approach in complementing chemical fertilizer requirements to sustain rice
productivity, particularly in Ghana. Importantly, their application could simultaneously
reduce production costs whilst restoring soil health and without harming the environment.
Further research might be conducted with other release rice varieties in order to clarify the
specificity mechanisms that exert beneficial effects on plant growth and development.
Microorganisms 2021, 9, 2000 12 of 14
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/microorganisms9092000/s1, Figure S1: The epiphytic colonization of Azospirillum sp. B510 on
Ghanaian rice, Table S1: Characteristics of rice (Oryza sativa L.) cultivars used in this study, Table
S2: Effect of Azospirillum sp. B510 inoculation on plant growth promotion in Ghanaian rice (Oryza
sativa L.) cultivars, Table S3: Effects of B510 on the reproductive stage of selected rice cultivars in field
conditions under fertilizer management, Table S4: The number of Azospirillum OTUs detected in the
rhizosphere of rice grown in Ghana.
Author Contributions: Conceptualization, S.O.; methodology, M.Y.; validation, E.S.-A., C.T., and
M.Y.; investigation, E.S.-A., C.T., M.Y., P.B., D.A.A., A.Y., and S.O.A; resources, S.O.A., R.B., E.K.N.,
and S.O.; data curation, E.S.-A and C.T.; writing—original draft preparation, E.S.-A.; supervision,
S.O.; project administration, S.O.; funding acquisition, M.Y. and S.O. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was supported by Grant-in-Aid for JSPS Fellows (20J40199 to M.Y.) and JSPS
Bilateral Program grant number (120199926 to S.O.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Tsuyoshi Isawa and Satoshi Shinozaki (Mayekawa Co., Tokyo, Japan)
for kindly providing Azospirillum sp. B510 and critical comments. We thank Yoshikazu Tamura
(Ohayo farm) for kind support for field experiment. We thank Sakiko Ueda (Woman Support Center,
Tokyo University of Agriculture and Technology) for technical support. We thank Kwame Sarppong
Appiah, Khondoker Dastogeer, and Dominic Vincent Agyekum for technical support. We thank
Safirah Tasa Nerves Ratu for kindly checking this manuscript. We thank all lab members for helping
with rice samplings.
Conflicts of Interest: The authors declare no conflict of interest.
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