biology
Article
Enhancement of the Aroma Compound 2-Acetyl-1-pyrroline in
Thai Jasmine Rice (Oryza sativa) by Rhizobacteria under
Salt Stress
Kawiporn Chinachanta 1,2 , Arawan Shutsrirung 2 , Laetitia Herrmann 3,4 , Didier Lesueur 3,4,5,6
and Wasu Pathom-aree 7,*
1 Doctor of Philosophy Program in Environmental Soil Science, Faculty of Agriculture, Chiang Mai University,
Chiang Mai 50200, Thailand; kawiporn.ch@cmu.ac.th
2 Department of Plant and Soil Sciences, Faculty of Agriculture, Chiang Mai University,
Chiang Mai 50200, Thailand; arawan.s@cmu.ac.th
3 Alliance of Bioversity International and Centre International of Tropical Agriculture (CIAT), Asia Hub,
Common Microbial Biotechnology Platform (CMBP), Hanoi 10000, Vietnam; L.Herrmann@cgiar.org (L.H.);
didier.lesueur@cirad.fr (D.L.)
4 School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment,
Deakin University, Melbourne, VIC 3125, Australia
5 Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD),
UMR Eco&Sols, Hanoi 10000, Vietnam
6 Eco&Sols, Université de Montpellier (UMR), CIRAD, Institut National de la Recherche Agricole, Alimentaire
et Environnementale (INRAE), Institut de Recherche pour le Développent (IRD), Montpellier SupAgro,





 34060 Montpellier, France
7 Research Center of Microbial Diversity and Sustainable Utilization, Department of Biology, Faculty of Science,
Citation: Chinachanta, K.; Chiang Mai University, Chiang Mai 50200, Thailand
Shutsrirung, A.; Herrmann, L.; * Correspondence: wasu.p@cmu.ac.th; Tel.: +66-53943346-48
Lesueur, D.; Pathom-aree, W.
Enhancement of the Aroma Simple Summary: The major aroma compound (2-acetyl-1-pyrroline) of the world-famous Thai
Compound 2-Acetyl-1-pyrroline in jasmine rice, variety KDML105, has declined due to high soil salinity and agrochemical input. In
Thai Jasmine Rice (Oryza sativa) by this work, the rhizobacteria from rice were investigated for the aroma compound’s production, as
Rhizobacteria under Salt Stress.
well as their potential for increasing the compound content in Thai jasmine rice seedlings under
Biology 2021, 10, 1065. https://
saline conditions. Our results provide evidence that the addition of aroma compound-producing
doi.org/10.3390/biology10101065
rhizobacteria increases the aroma content in the rice seedlings under salt stress. Sinomonas sp. strain
Academic Editors: Ana Alexandre ORF15-23 which colonize the rice roots, is a promising rhizobacteria in promoting the aroma level
and Kathrin Wippel of the Thai jasmine rice grown under salt stress and could be developed as a bioinoculant for Thai
jasmine rice cultivation in a salt-affected area.
Received: 27 August 2021
Accepted: 13 October 2021 Abstract: Thai jasmine rice (Oryza sativa L. KDML105), particularly from inland salt-affected ar-
Published: 19 October 2021 eas in Thailand, is both domestically and globally valued for its unique aroma and high grain
quality. The key aroma compound, 2-acetyl-1-pyrroline (2AP), has undergone a gradual degrada-
Publisher’s Note: MDPI stays neutral tion due to anthropogenic soil salinization driven by excessive chemical input and climate change.
with regard to jurisdictional claims in Here, we propose a cheaper and an ecofriendly solution to improve the 2AP levels, based on the
published maps and institutional affil- application of plant growth-promoting rhizobacteria (PGPR). In the present study, nine PGPR iso-
iations. lates from rice rhizosphere were investigated for the 2AP production in liquid culture and the
promotion potential for 2AP content in KDML105 rice seedlings under four NaCl concentrations
(0, 50, 100, and 150 mM NaCl). The inoculation of 2AP-producing rhizobacteria resulted in an in-
crease in 2AP content in rice seedling leaves with the maximum enhancement from Sinomonas sp.
Copyright: © 2021 by the authors. ORF15-23 at 50 mM NaCl (19.6 µg·kg−1), corresponding to a 90.2% increase as compared to the
Licensee MDPI, Basel, Switzerland. control. Scanning electron microscopy confirmed the colonization of Sinomonas sp. ORF15-23 in
This article is an open access article
the roots of salinity-stressed KDML105 seedlings. Our results provide evidence that Sinomonas sp.
distributed under the terms and
ORF15-23 could be a promising PGPR isolate in promoting aroma level of Thai jasmine rice KDML105
conditions of the Creative Commons
Attribution (CC BY) license (https:// under salt stress.
creativecommons.org/licenses/by/
4.0/).
Biology 2021, 10, 1065. https://doi.org/10.3390/biology10101065 https://www.mdpi.com/journal/biology
Biology 2021, 10, 1065 2 of 16
Keywords: 2-acetyl-1-pyrroline; 2AP; aromatic rice; Thai jasmine rice; Oryza sativa; plant
growth-promoting rhizobacteria; salinity stress
1. Introduction
The famous Thai jasmine rice (Oryza sativa) (Kao Dok Mali 105; KDML105) has enjoyed
an international reputation for its unique aroma and premium grain quality [1]. The rice
cultivar KDML105 is mainly cultivated in the northeastern region of Thailand, particularly
in the Thung Kula Rong-Hai (TKR) area, which covers 2.1 million rai (1 hectare = 6.25 rai)
and provides the highest grain quality with a unique aroma [2–4]. Aromatic rice vari-
eties including KDML105 usually give low yield, but its premium price generates higher
profit for the farmers compared to nonaromatic rice [3–5]. Thai jasmine rice KDML105
is considered a drought- and salt-sensitive cultivar [6–8]. Some environmental factors
together with cultivation practices have been shown to affect rice aroma. In general, rice
yield and quality are negatively affected by salinity stress [9–14]. Soil salinity with an
electrical conductivity (EC) value higher than 8.0 dS·m−1 was reported [15,16] to reduce
grain yield and boiled rice quality but increase the level of aroma compounds in the aro-
matic rice grains [9,17,18]. Unfortunately, recent excessive soil salinity and water shortage
negatively affect both yield and grain quality of KDML105 rice, particularly levels of the
aroma compound 2-acetyl-1-pyrroline (2AP) [1].
The salinization problem in agriculture, including the TKR region, is magnified by high
agro-chemical inputs together with increased incidents of drought as the consequence of
climate change [19–21]. Recently, a progressive reduction in 2AP aroma was observed due
to the shift toward farming practices which rely heavily on the use of chemicals (fertilizers,
herbicides, and pesticides). The overuse of chemical fertilizers is leading to a depletion in
rice grain quality, particularly the aroma quality [19,22,23]. For example, the application
of nitrogenous fertilizers, the use of pesticides, and usage regimes in agriculture from
traditional to modern were reported as causes of aroma loss in Indian aromatic rice [22]. In
addition, the best-quality Tarori basmati rice, particularly the aroma score, was achieved
using 100% farmyard manure [23].
The aroma compound, 2-acetyl-1-pyrroline (2AP), has been identified as the major
aromatic compound in aromatic rice, including the KDML105 cultivar, and it is recog-
nized as a criterion for premium- or good-quality aromatic rice, thereby determining its
price [24,25]. The 2AP compound in aromatic rice has been proven to synthesize from
proline, a key nitrogen precursor, and it can be detected in all aboveground plant tissues
of the aromatic rice plant [26]. Biosynthesis of 2AP has also been recorded in a wide
range of organisms including plants and microorganisms [5,9,27–29]. Rice rhizobacterial
strains of Pseudomonas, Enterobacter, and Acinetobacter were reported as producers of 2AP.
These rhizobacteria, particularly high 2AP-producing isolates, increased the 2AP level
in the grains of aromatic rice variety Basmati-370 [29]. Recently, Enterobacter hormaechei
(AM122) and Lysinibacillus xylanilyticus (DB25) having the property of synthesizing 2AP
were isolated from the rhizosphere of aromatic rice cultivars in India. The inoculation of
these bacteria in consortium resulted in a significant improvement in vegetative growth,
yield, and 2AP content over mono inoculation and control [5].
The application of beneficial microbes offers a sustainable alternative way to restore
the rice aroma, as well as increase yield [5,29]. Our previous investigation in the TKR area
indicated that much higher soil microbial populations was recorded in areas of organic
rice farming (ORF) than conventional rice farming (CRF). These high microbial population
in the ORF system provided higher soil organic matter content and higher total nitrogen
content in the soil, as well as 3.5 times higher 2AP content in rice grains than those in the
CRF system [30]. Therefore, the use of 2AP-producing PGPR, especially in the ORF system,
to promote rice growth and 2AP level, as well as minimize the rice production cost, could
be of interest.
Biology 2021, 10, 1065 3 of 16
The benefits of PGPR have been widely recognized in both normal and salt-affected
soil [21,31–33]. In general, PGPR exhibit various mechanisms to enhance plant growth,
e.g., phytohormone production, biological nitrogen fixation, phosphate and potassium
solubilization, and siderophore production [34]. Under salt-stress conditions, salt-tolerant
PGPR (ST-PGPR) are able to help plants to enhance osmotic adjustment and detoxification
of reactive oxygen species by antioxidants [21,31,33,35–37]. ST-PGPR are now being used as
bioinoculants for enhancing crop yields, protection from phytopathogens, and improving
soil health [38,39]. Growth promotion and alleviation of salt stress by ST-PGPR inoculation
has been recorded in various bacterial genera, both Gram-positive and Gram-negative,
e.g., Bacillus, Pseudomonas, and Streptomyces [31,32,40,41]. ST-PGPR Bacillus nanhaiensis
strain TY0307 exhibited promising ability in promoting salt tolerance, proline accumulation,
growth, and yield of rice under salt-stress conditions [41]. Suksaard et al. [42] demonstrated
that Nocardiopsis sp. 1SM5-02, Pseudonocardia sp. 3WH5-01, Streptomyces sp. 2SH3-07, and
Streptomyces sp. 3SH5-05, isolated from sea water and mangrove sediments, enhanced
the growth of KDML105 rice seedlings under saline (100–200 mM NaCl) and non-saline
conditions. Similarly, inoculation of salt-sensitive rice cultivars with Brevibacterium linens
RS16 isolated from coastal soil significantly improved the salt tolerance and growth of
salt-stressed rice (O. sativa) [12]. In addition, plant growth-promoting Streptomyces isolate
S2-SC16 from mangrove was reported to not only promote the growth of the KDML105 rice
seedlings but also have the ability to restrict the infection of plant fungal pathogen Fusarium
fujikuroi [43]. Recently, salt-tolerant bacteria belonging to the genera Bacillus, Exiguobac-
terium, Enterobacter, Lysinibacillus, Stenotrophomonas, Microbacterium, and Achromobacter
were able to alleviate salt stress and promote the growth of rice (O. sativa) seedlings in a
pot experiment under greenhouse condition [44].
From the above investigations, we opine that the use of 2AP-producing ST-PGPR
could benefit rice growth, as well as production of the 2AP aroma compound, under salinity
stress. To the best of our knowledge, the use of 2AP-producing ST-PGPR for enhancing the
aromatic quality of KDML105 rice has not been studied. In the present investigation, the
previously screened PGPR isolates capable of solubilizing phosphate and potassium, as
well as producing siderophore [45], were selected to investigate their 2AP production in the
culture broth. All isolates were then used to inoculate Oryza sativa KDML105 rice seedlings
to evaluate their potential to promote the production of a major aromatic compound,
2AP, in the seedlings under salt stress. We hypothesized that the inoculation of the 2AP-
producing PGPR isolates obtained from the KDML105 rice rhizosphere would enhance the
aroma level of this rice cultivar.
2. Materials and Methods
2.1. Rice Rhizobacterial Isolates
Nine PGPR were isolated from the rhizospheric soil of Thai jasmine rice (Oryza sativa)
KDML105 using a conventional serial dilution and plating technique on nutrient agar.
These isolates exhibited different levels of phosphate and potassium solubilization, as well
as siderophore production, determined in a previous study [45] (Table 1). Quantitative
determination of solubilized P and K was performed using a spectrophotometer and atomic
absorption spectrophotometer, respectively. Siderophore type was determined by ferric per-
chlorate assay for hydroxamate type and Arnow assay for catecholate type [45]. Rhizobacte-
rial isolates were obtained from either organic rice farming (ORF) or conventional rice farm-
ing (CRF). The five ORF strains were Sinomonas sp. ORF4-13, Enterobacter sp. ORF10-12,
Micrococcus sp. ORF15-19, Micrococcus sp. ORF15-20, and Sinomonas sp. ORF15-23. The
four CRF strains were CRF5-8 (unidentified), Sinomonas sp. CRF14-15, Burkholderia sp.
CRF16-3, and Bacillus sp. CRF17-18.
Biology 2021, 10, 1065 4 of 16
Table 1. Phosphate and potassium solubilization, as well as siderophore production, by the nine selected KDML105 rice
rhizobacterial isolates.
Phosphate Potassium
(Ca-P) (K-Feldspar) Siderophore
Farming Practice Rhizobacterial Genus Solubilization Solubilization
Production
Isolate
Soluble P Soluble K Hydroxamate-Type
(mg·L−1) (mg·L−1) (µmol·L−1)
Organic farming ORF4-13 Sinomonas sp. 35.6 37.3 110.8
ORF10-12 Enterobacter sp. 31.6 39.4 162.5
ORF15-19 Micrococcus sp. 11.6 34.6 64.2
ORF15-20 Micrococcus sp. 25.0 30.7 83.3
ORF15-23 Sinomonas sp. 20.9 49.5 22.5
Conventional farming CRF5-8 unidentified 31.6 44.8 2.5
CRF14-15 Sinomonas sp. 36.6 38.6 1.7
CRF16-3 Burkholderia sp. 17.5 43.0 618.3
CRF17-18 Bacillus sp. 35.0 28.7 0.5
Adapted from Chinachanta and Shutsrirung [45].
2.2. Determination of 2AP Production by Rhizobacterial Isolates in Liquid Culture
One loopful of each 3 day old culture of each rhizobacterial isolate was transferred
to 150 mL of nutrient broth (NB) (Hi Media, Mumbai, India) in a 500 mL Erlenmeyer
flask and incubated with shaking (120 rpm) at room temperature (28–35 ◦C) for 7 days.
The supernatant of each culture was obtained by centrifugation at 10,000 rpm for 10 min
(4 ◦C) and primarily screened for the production of 2AP by sniffing [46]. To confirm 2AP
production in the liquid culture, each positive strain (by sniffing) was further cultivated
as described above in 2 L of NB medium for 7 days. After that, the 2 L culture broth
was concentrated to about 200 mL using a rotary vacuum evaporator. The pH of the
culture liquid was adjusted to pH 8.0 and then extracted with diethyl ether (2 × 70 mL).
The diethyl ether layer was collected and concentrated to approximately 3–4 mL, using a
Vigreux distillation column, and injected into a GC–MS for 2AP analysis [46].
2.3. Effect of Rhizobacterial Inoculation on 2AP Level in Rice Seedlings Grown under Salt Stress
In the present study, the ability of nine selected rhizobacterial isolates in promoting
the 2AP level in KDML105 rice leaves under various concentration of NaCl was evaluated
at harvesting time of 30 days after transplanting.
2.3.1. Experimental Design
The experiment was conducted using a completely randomized design (CRD) in
a factorial scheme (10 × 4) with three replications. In total, there were nine selected
rhizobacterial isolates plus one uninoculated control (ten treatments), and four NaCl
concentrations (0, 50, 100 and 150 mM NaCl). Each treatment had its corresponding control
with the same concentration of NaCl and no rhizobacterial inoculation.
2.3.2. Preparation of Rhizobacterial Isolates
All rhizobacterial isolates were grown in 25 mL of nutrient broth (NB) for 3 days at
37 ◦C with shaking at 120 rpm. The biomass of rhizobacteria was collected by centrifugation
at 10,000 rpm for 15 min to separate the culture broth and the cells. Cells were suspended
in 100 mL of sterile distilled water to obtain the concentration of 106 colony-forming units
(CFU) per mL (OD600~0.2). This cell suspension was used for rice seed biopriming and
rice seedling inoculation. Sterile distilled water was used as a negative control (without
rhizobacterial inoculation).
Biology 2021, 10, 1065 5 of 16
2.3.3. Rice Seedlings
Oryza sativa L. KDML105 rice seeds, from the Community Enterprise of Thamo
Organic Agricultural Group, Surin Province, were surface-sterilized in a mixture of
0.2% Tween-80 and 2% sodium hypochlorite for 3 min. Seeds were then washed three
times with 70% ethanol followed by rinsing five times with sterile water. Surface sterile
seeds were soaked (seed biopriming) in the cell suspension of each rhizobacterial isolate ac-
cording to the treatment, and were then incubated in the dark at 25 ◦C for 24 h. Bio-primed
seeds were then placed at an equal distance on sterilized wet tissue paper in a petri dish
(20 seeds per plate) using sterile forceps (five replicates per treatment) and kept in the plant
growth chamber in the dark at 25 ◦C. Four days after germination, uniform seedlings were
selected and transplanted into the growth pouch containing modified Hoagland nutrient
solution prepared according to Premsuriya et al. (2017) and Nawara et al. (2020) [47,48].
Then, the cell suspensions (~106 CFU·mL−1) were inoculated to the seedlings according to
the treatments. Rice seedlings were initially irrigated with 1⁄4 strength Hoagland solution
for 5 days, and the solution was replaced twice during this period. Then, the seedlings
were irrigated with 1⁄2 strength Hoagland solution for 2 days. After that, the irrigation
medium was changed to a full-strength Hoagland solution [49] supplemented with four
salinity levels (0, 50, 100, and 150 mM NaCl). The average EC values of an irrigation
medium were 2.06, 7.69, 13.78, and 19.51 dS·m−1, respectively. The full-strength Hoagland
solution was filled twice a week to maintain the volume of an irrigation medium. The
uninoculated (controls) and inoculated seedlings were grown in a climate-controlled room
(12:12 light–dark photoperiod at 25 ± 3 ◦C under a light level of approximately 5.8 klux).
The rice seedlings from each pouch were harvested at 30 days after transplanting. Leaves
of KDML105 seedlings from each treatment (three replications) were used to determine the
2AP level.
2.4. Quantification of 2-Acetyl-1-pyrroline in KDML105 Rice Leaves
Rice leaves were dried in an oven at 60 ◦C for 3 days until the moisture content was
reduced to 14% before being sent to Salana Organic Village (Social Enterprise) Co., Ltd.,
Nakhon Pathom, Thailand, for the determination of the 2AP content. The analysis of 2AP
was made by static headspace gas chromatography coupled with a nitrogen/phosphorus
detector (SHS-GC–NPD) by the method of [50].
2.5. Survival of Rhizobacterial Isolates
This experiment was conducted to evaluate the effect of different concentrations of
NaCl on the population of the rhizobacterial isolates 30 days after transplanting. The
presence of the PGPR isolates in the fresh rice roots was evaluated by microscopic analysis.
The initial population of all isolates inoculating to the rice seedlings at transplanting time,
was approximately 106 CFU·mL−1 (OD600~0.2). One milliliter of sample from each treat-
ment with four NaCl levels (0, 50, 100, and 150 mM NaCl) was taken after the end of the
experiment. Serial dilutions were prepared, and 0.1 mL aliquots (10−3–10−5) were spread
on nutrient agar (NA). The experiment was carried out with three replicates for each treat-
ment. The survival of rhizobacterial isolates under salt-stress conditions was determined
by observing the number of viable cells after exposure to NaCl. After incubation at 37 ◦C
for 72 h, colonies that appeared on NA plates were counted. The number of colonies of
NaCl-stressed rhizobacteria was compared with that of untreated rhizobacteria (without
NaCl) and presented as the percentage survival using the following equation:
% Survival = CFU (with NaCl)CFU (without NaCl × 100,) (1)
CFU = colony forming unit.
2.6. Microscopic Analysis of Rhizobacterial Isolates
To confirm the colonization of the inoculated Sinomonas sp. ORF15-23 and Bacillus sp.
strain CRF17-18 in the KDML105 rice roots under different NaCl concentrations, the
Biology 2021, 10, x FOR PEER REVIEW 6 of 16 
 
2.6. Microscopic Analysis of Rhizobacterial Isolates 
Biology 2021, 10, 1065 To confirm the colonization of the inoculated Sinomonas sp. ORF15-23 and Bacillu6so sfp16. 
strain CRF17-18 in the KDML105 rice roots under different NaCl concentrations, the mi-
crostructure of the rice roots from all treatments was examined under a scanning electron 
mmiiccrroossctroupcet u(SreEMof)t huesirnicge lraoteortsalf rroomot aslalmtrepaletms oenf tasllw thaes einxoamcuilnaetdedu insdoelartaess.c Tanhnei rnogoetsle wcteroren 
cmuti cirnotsoc o1p cem(S sEeMgm) uensitns ganladte wraelrreo optresfaimxepdl eisn o4f%a lgl tluhetairnaoldcuehlaytdede issoolluattieosn.  Tohveerrnoiogthstw aenrde 
wcuatshinetdo w1 citmh s0e.1g mMe nstosdainudmw cearceopdryelfiatxee dbuinffe4r% tghlruetea rtaimldeesh yfodre 3s0o lumtiionn eoavcher. nOigshmtiaunmd 
twetarashoexdidew bituhff0e.r1 (1M%)s owdaius musecdac foodry plaotset bfiuxaffteiornt.h Arefetetri ma esserfioers o3f0 dmehinyderaacthio. nO stsempisu imn 
atceetrtoaonxei, dtheeb suafmfeprl(e1s% w)ewrea dsruiesded inf oar cpriotisctafil pxaotiinotn d.rAyefrte arnad smeroiuesntoefdd oenh yaldurmatiinounmst setpusbisn, 
sapcuettotenre-c, othaetesda mwpitlhe sgwoledr,e adnrdie dviienwaecdr iutincdalepr oloinwt-dvrayceuruamnd scmanounnintegd eolenctarlounm miniucrmossctoupbes ,  
(sJpEuOtLte, rm-coodaetel dJSwMi5th91g0oLlVd,, aTnodkyvoie, wJaepdanu)n adt eFralcouwlt-yv aocf uSucimensccea, nCnhiinagngel Mecatrio Unnmiviecrrsoistcyo. pe
(JEOL, model JSM5910LV, Tokyo, Japan) at Faculty of Science, Chiang Mai University.
2.7. Statistical Analysis 
2.7. STtwatois-twicayl A AnNalOysVisA together with Turkey’s HSD at the 1% probability level [51] was 
applieTdw foo-rw aanyalAyNzinOgV cAoltloegcteetdh edrawtai tuhsTinugr kSetayt’isstHixS D9 (aAtnthaley1ti%calp Srobftawbailriety, Ilnecv.e, lT[a5l1la]hwaas-s
saepep, FliLed, UfoSrAa)n. a lyzing collected data using Statistix 9 (Analytical Software, Inc., Tallahassee,
FL, USA).
3. Results 
33..1.R 2e-sAuclettsyl-1-pyrroline Production Potential of Rhizobacterial Isolates 
3.1. 2-Acetyl-1-pyrroline Production Potential of Rhizobacterial Isolates
The presence of 2AP was detected in all the culture liquid samples from the rhizo-
bacterTihale isporleasteens cbeyo sfn2ifAfiPngw. Hasodweetveectre, dwhinena ltlhteh eexcturaltcutsr efrloiqmu iedacsha mcupltluesref rloiqmuidth we erhrei- 
ezxoabmacinteerdia floirs o2lAatPe susbiyngsn ai fGfiCng–.MHSo, wseevveenr ,owuth oefn ntihnee erxhtirzaocbtsacfrteormiale iascohlactuelst ushreowliqeudi da pweearke 
wexitahm tihnee dsafmore 2rAetPenutsiionng taimGeC a–sM thSe,  s2eAvPen stoauntdoafrndi.n Ne orh pizeoabka wctaesr idaletiesoctlaedte sfosrh tohwe endegaapteivaek 
cwonitthrothl e(Fsiagmureer e1tae)n, taios nwteimll easa sfothr es2trAaiPnss taCnRdFa5r-d8. aNnod pBeaackillwusa sspd.e tCecRtFe1d7f-o1r8.t hSeignneigfiactainvet 
dciofnfetrreonlc(eFsi g(up r≤e 01.a0)0,1a)s inw 2eAllPa spfroordsutcrtaiionns eCxRisFte5d-8 aamnodnBga tchilelu tsesstpe.dC isRoFla1t7e-s1,8 a.nSdi gtnhiefi 2cAanPt 
pdrioffdeurecnticoens (ips ≤sum0.0m0a1r)iizned2A inP Tpraobdleu c2t. ioTnhee xmisatexdimaummo n2gAtPh eptreosdteudctiisoonl awteass, arnecdotrhdeed2A inP 
Spirnoodmuocntaios nspi.s OsuRmF1m5-a2r3i z(e3d72i.n8 µTagb· le−21 . The maximum 2A·kg−,1 Figure 1b), followed
P bpyr Eodntuecrtoiboanctwer assp.r eOcRorFd1e0d-1i2n, 
BSuinrkomhoolndearsias ps.pO. RCFR1F51-62-33(, 3a7n2d.8 Mµgicrkogcocc,uFsi gsupr.e O1Rb)F,1f5o-l1lo9w wedithb yvaElnuteesro obfa c3t3er0.s2p, .2O54R.F91, 0a-n1d2 ,
2B5u4r.0kh µogld·kergia− sp. CRF254.0 g·kg−
1, 1r,ersepsepcetci
1v6tie
-l3vey
,
l.y 
and Micrococcus sp. ORF15-19 with values of 330.2, 254.9, and
µ .
  
(a) (b) 
FFiigguurree 11.. CChhrroomaattooggrraamss ooff GGCC––MSS aannaallyyssiiss ooff 22AAPP pprroodduuccttiioonn ffoorr ccoonnttrrooll nnuuttrriieenntt bbrrootthh wwiitthhoouutt PPGGPPRR iissoollaatteess ((aa)) aanndd 
SSiinnoommoonnaass sspp.. OORRFF1155--2233 ((bb)).. 
Table 2.. 2--Acettyll--1--pyrrolliine (2AP) production by selected rhizobacterial isolates. 
FFaarrmiing Prraaccttiiccee RRhihziozobbaacctteerriiall Isollaattee 2A2APP PProrodduuccttiion ((µgg··kkgg−−1)) 
 UUninninooc 1cuullaatteedd ccoonnttrrooll NNDD 1 
OORRFF44--13 3030.9.911 ±± 00.4.433 ee 
OORRFF1100--1122 333030.2.244 ±± 11.7.700 bb 
OOrrggaanniicc ffaarrmminingg OORRFF1155--1199 252454.0.044 ±± 33.8.899 cc 
ORF15-20 96.94 ± 1.49 d
OORRFF1155--2203 9367.92.47 6± ±1.47.99 9da 
ORF15-23 37A2v.e7r6a g±e 72.1969.9 a8 
 CRF 5-8 AveragNe D216.98 
Conventional farming CRF14-15 12.00 ± 1.53 f
CRF16-3 254.95 ± 2.48 c
 CRF17-18 ND
Average 133.48
Biology 2021, 10, 1065 7 of 16
Table 2. Cont.
Farming Practice Rhizobacterial Isolate 2AP Production (µg·kg−1)
F-test **
LSD(0.05) 6.37
CV% 1.88
1 ND = not detectable; ** significant at the 0.001 probability level.
3.2. Effect of Rhizobacterial Isolate Inoculation on the 2AP Level of KDML105 Rice Seedlings
The inoculation effects of nine rhizobacterial isolates were evaluated in O. sativa
KDML105 rice seedlings under normal (0 mM NaCl) and saline conditions (50, 100, and
150 mM NaCl). Uninoculated seedlings at 0, 50, 100, and 150 mM NaCl were considered as
control-0, control-50, control-100, and control-150, respectively. The term ‘control’ was used
for the uninoculated seedling treatments of all salinity levels. These abbreviations are used
throughout this publication. Statistical analysis showed a significant interaction (p ≤ 0.01)
between PGPR isolates and NaCl concentrations for all the study variables (Table 3).
Table 3. Significance level and Turkey’s HSD values for studied variables.
Source of Variance The 2AP Content
PGPR isolates (A) **
NaCl concentration (B) *
A × B **
Turkey’s HSD (0.01) for (A × B) 1.0995
CV% 3.6756
** Significant at the 0.01 probability level; * significant at the 0.05 probability level.
The 2AP level in rice is widely used as one important indicator which reflects the high
quality of aromatic rice. In the present study, the inoculation of PGPR isolates clearly en-
hanced the 2AP level in the leaves of KDML105 seedlings under both normal (0 mM NaCl)
and saline conditions (50, 100, and 150 mM NaCl) (Figure 2a). Two-way ANOVA indicated
that PGPR isolates and salinity effects on the 2AP content of rice were significant (p < 0.001).
Under normal conditions, the application of PGPR isolates significantly increased the 2AP
content of the fresh rice leaves in the range of 15.6–69.8% (10.65–15.64 µg·kg−1) as com-
pared to control-0 (9.21 µg·kg−1) (Figure 2a).
Interestingly, under salinity stress, inoculation of the PGPR isolates also significantly
increased the 2AP content in rice leaves in the range of 3.5–193.7% (4.41–19.61 µg·kg−1)
as compared to each respective control. Among all the tested strains, the maximum
2AP content was observed upon inoculation of Sinomonas sp. ORF15-23 at 50 mM NaCl
(19.6 µg·kg−1) with an increase of 90.20% as compared to control-50 (10.3 µg·kg−1) and
Burkholderia sp. CRF16-3 at 150 mM NaCl (9.4 µg·kg−1) with a 193.7% increase as compared
to control-150 (3.2 µg·kg−1). The 2AP content of all treatments increased with the increase in
NaCl concentration up to 50 mM; however, beyond 50 mM NaCl, (100 and 150 mM NaCl),
the 2AP content decreased with the increase in NaCl concentration. At 100 and 150 mM NaCl,
strains ORF10-12 and CRF16-3 gave the highest 2AP levels with values of 12.87 and
9.43 µg·kg−1, equivalent to 107.2% and 193.7% increases, as compared to control-100 and
control-150, respectively (Figure 2a,b).
BBioiloolgoygy2 022012,11, 01,01, 0x6 F5OR PEER REVIEW 88 ooff 1166  
25
0 50 100 150
20
15
10
5
0
Controls ORF 4-13 ORF10-12 ORF15-19 ORF15-20 ORF15-23 CRF 5-8 CRF 14-15 CRF 16-3 CRF 17-18
Rhizobacterial isolates  
(a) 
200.00
180.00
160.00
140.00
120.00
100.00
80.00
60.00
40.00
20.00
0.00
0 50 100 150
NaCl concentration (mM)
ORF 4-13 ORF10-12 ORF15-19 ORF15-20 ORF15-23 CRF 5-8 CRF 14-15 CRF 16-3 CRF 17-18  
(b) 
FFigiguurere2 .2.2 -2A-Acecteytly-l1--1p-ypryrrorloinlien(e2 A(2PA)Pc)o cnotenntetnint finre sfrheKshD KMDLM10L51r0ic5e rliecaev leesav(ae)s, a(an)d, apnedrc epnetracgeentinagcree ainsceriena2seA iPn( b2A) aPt (3b0)d aaty 3s0 
adftaeyrst raafntesrp tlaranntisnpglaunntidnegr usanldineer csaolnindeit icoonnsd(i0ti,o5n0s, 1(00,0 5,0a,n 1d001,5 a0nmd M15N0 maCMl) .NaCl). 
33.3.3.. EEfffeecctto off DiifffeerreennttN NaaCCllC Coonncceennttrraattioionnsso onnt thheeS Suurrvvivivaallo offR Rhhizizoobbaaccteterriaial lI sIsoolalatetessa att3 300D Daayyss 
aaftfteerrI Innooccuulalattioionn 
NNinineei sisoolalatteessw weerreet teessteteddf foorrt thheeirira abbiliiltiytyt toos suurrvviviveeu unnddeerrd dififffeerreennttc coonncceenntrtraatitoionnsso off 
NNaaCCll( (00,,5 500,,1 10000,,a anndd1 15500m mMMN NaaCCl)l)i ninH Hooaagglalanndds soolulutitoionn, ,3 300d daayyssa aftfeterri ninooccuulalatitoionni nintoto 
tthhee rricicees seeeeddlilninggss.. IInn tthhiiss ssttuuddyy,,w weei ninvveesstitgigaatteeddt htheeg grroowwththo offr hrhizizoobbaaccteterriaiai sisoolalatetessi nin 
NNAAm meeddiiaa ffoorr 7722h h,,a afftteerri ninccuubbaattiioonn aatt 3377 ◦°CC,, ttoo rreeaacchh tthhee lloogg pphhaassee ooff bbaacctteerriiaall ggrroowwtthh.. 
TThheen nuummbbeerro offC CFFUUo offa allllt htheer hrhizizoobbaaccteterriaialli sisoolalatetesss shhoowweedda ad deeccrreeaassininggt rtreennddw witihtha ann 
ininccrreeaasseei nins saaltltc coonncceenntrtraatitoionn( T(Taabblele4 4).). 
  Figure 3 shows the percentage of surviving bacteria, demonstrating a significant
difference in salt tolerance ability between isolates cultured in Hoagland’s solutions
containing 0–150 mM NaCl. In general, each rhizobacterial isolate was able to tolerate
all tested NaCl levels with the remaining number of approximately 106 CFU·mL−1 at
150 mM NaCl (Table 4). The growth of rhizobacterial isolates was slightly decreased from
0 to 50 mM NaCl but sharply decreased thereafter. In order to evaluate the relationship
between salt-stress levels and the survival of the rhizobacterial strains, the number of
treated rhizobacteria (under NaCl stress) was compared with that of untreated rhizobacte-
ria (without NaCl) and presented as the survival percentage. The survival rate showed
a decreasing trend with an increase in salt concentration. At 50 mM NaCl, the survival
percentage was 36.1–67.2%, while, at 100 and 150 mM NaCl, these values were 0.3–7.6%
and 0.1–0.7%, respectively (Figure 3). Sinomonas sp. isolate ORF15-23 showed the highest
cell number at most NaCl levels (Table 4).
 
2AP content (µg·g−1)
Percentage increase of 2AP
Biology 2021, 10, x FOR PEER REVIEW 9 of 16 
 
Table 4. Number of viable rhizobacteria cells under different NaCl concentrations in Hoagland so-
lution at 30 days after inoculation. 
Number of Viable Cells 
(CFU·mL−1) 
Rhizobacteria Isolates NaCl Concentrations (mM) 
0 50 100 150 
Organic rice farming 
ORF4-13 5.8 × 108  3.7 × 108 6.0 × 106 2.0 × 106 
ORF10-12 3.5 × 108 2.2 × 108 2.7 × 107 2.3 × 106 
ORF15-19 2.0 × 109 7.2 × 108 4.3 × 107 2.3 × 106 
Biology 2021, 10, 1065 ORF15-20 1.4 × 109 8.8 × 108 4.3 × 106 1.0 × 106 9 of 16
ORF15-23 2.2 × 109 1.5 × 109 6.1 × 106 4.8 × 106 
Conventional rice farming 
Table 4. NCRumF5b-e8r of viable rhi8z.o0b ×a c1te0r8i a cells8u.5n d× e1r0d7 ifferent N6a.1C ×l  c1o0n6c entration4s.8in ×H 1o0a6 gland
solutionCatR3F01d4a-1y5s after inocula1t.i2o n×. 109 5.5 × 108 4.5 × 106 3.1 × 106 
CRF16-3 1.2 × 109 7.0 ×N 1
8
u0m ber of Vi3ab.3l e×C 10
6 6
ell s 3.0 × 10  
CRF17-18 4.7 × 108 2.0 × 108 (CFU·mL7−.01 )× 106 2.5 × 106 
Rhizobacteria Isolates NaCl Concentrations (mM)
Figure 3 shows the percentage of surviving bacteria, demonstrating a significant dif-
ference in salt tolerance ability bet0ween isolates cu50ltured in Hoag1la0n0d’s solutions 1c5o0ntain-
ing 0–150 mM NaCl. In general, eachO rrghaiznoicbraiccetefrairaml iinsgolate was able to tolerate all tested 
NaCl leveOlsR wF4it-h13 the remainin5g.8 n×um10b8er of app3.r7o×xim10a8tely 106 6C.0FU×·m10L6 −1 at 1502 m.0 M×  1N06aCl 
(Table 4).O TRhFe1 0g-r1o2wth of rhizo3b.5a×cte1r0i8al isolate2s. 2w×as1 0s8lightly d2e.c7r×ea1s0e7d from 02 .t3o ×501 0m6 M 
NaCl but OshRaFr1p5-l1y9 decreased th2e.0re×af1t0e9r. In orde7r.2 to× e1v0a8luate the4 .r3e×lat1i0o7nship bet2w.3e×en1 0s6alt-
stress leveOlRs Fa1n5d-2 t0he survival o1f. 4th×e 1r0h9izobacter8i.a8l× st1r0a8ins, the n4u.3m×be1r0 6of treated1 .r0h×iz1o0b6ac-
9 9
teria (undOeRrF N15a-2C3l stress) was2 .2co×m1p0ared with1. 5th×at1 0of untrea6te.1d×
6 6
 rh10izobacteri4a. 8(w×i1th0 out 
NaCl) and presented as the survivCaoln pvenrctieonntaalgreic.e Tfharem siungrvival rate showed a decreasing 
trend withC aRnF i5n-8crease in salt c8o.0n×ce1n0t8ration. A8t .550× m1M07  NaCl, th6.e1 s×ur1v0i6val perce4n.8ta×g1e0 w6 as 
36.1–67.2%CR, Fw1h4-i1le5, at 100 and1 .125×0 1m0M9  NaCl, 5t.h5e×se1 0v8alues we4r.5e ×0.130–67.6% and3 .10.×1–100.67%, 
respectiveClyR F(F1i6g-3ure 3). Sinomo1n.2as× sp10. 9isolate O7R.0F×151-2038  showed3 .t3h×e h1i0g6hest cell3 n.0u×mb10e6r at 
most NaCClR leFv17e-l1s8 (Table 4). 4.7 × 108 2.0 × 108 7.0 × 106 2.5 × 106
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 50 100 150
Rhizobacterial isolates  NaCl concentrations (mM )
ORF 4-13 ORF10-12 ORF15-19 ORF15-20 ORF15-23
CRF 5-8 CRF 14-15 CRF 16-3 CRF 17-18  
FiFgiugruer e3.3 S. uSruvrivviavla loof frhrhizizoobbaaccteterriaial liissoollaatteess uunnddeerr ddiiffffeerreenntt ccoonncceennttrraattiioonnsso offN NaaCCl li ninH Hoaogalgalnadn’ds’sso -
solluuttiioonnssa aftfeterr3 300d dayaysso foifn ioncouclualtaiotino.nT. hTeheer erorrrobra brsarrse prerepsreenstenthte thstea nstdaanrddadrdev diaetvioiantioofnm oef amsueraesmureen-ts
mfeonrttsh froere trherpelei craetpiolincsa.tions. 
3.4. Microscopic Observations of Root Colonization
The potential of rice root colonization by the different rhizobacterial isolates was
 determined by scanning electron microscopy (SEM). The SEM images showed, as ex-
pected, no root colonization in the uninoculated controls seedlings at 0, 50, 100, and
150 mM NaCl (Figure 4). Dense root colonization by all nine tested rhizobacterial isolates
was clearly observed in inoculated seedlings (data not shown). However, the colonization
density declined with the increasing salinity level, and no colonization was observed at
150 mM NaCl except for the seedling roots inoculated with Sinomonas sp. isolate ORF15-23
and Bacillus sp. strain CRF17-18. In addition, the inoculated roots of both non-stressed and
salinity-stressed seedlings were colonized by Sinomonas sp. strain ORF15-23 and Bacillus sp.
Strain CRF17-18 (Figure 4).
Survival percentage 
Biology 2021, 10, x FOR PEER REVIEW 10 of 16 
 
3.4. Microscopic Observations of Root Colonization 
The potential of rice root colonization by the different rhizobacterial isolates was de-
termined by scanning electron microscopy (SEM). The SEM images showed, as expected, 
no root colonization in the uninoculated controls seedlings at 0, 50, 100, and 150 mM NaCl 
(Figure 4). Dense root colonization by all nine tested rhizobacterial isolates was clearly 
observed in inoculated seedlings (data not shown). However, the colonization density de-
clined with the increasing salinity level, and no colonization was observed at 150 mM 
NaCl except for the seedling roots inoculated with Sinomonas sp. isolate ORF15-23 and 
Bacillus sp. strain CRF17-18. In addition, the inoculated roots of both non-stressed and 
Biology 2021, 10, 1065 10 of 16
salinity-stressed seedlings were colonized by Sinomonas sp. strain ORF15-23 and Bacillus 
sp. Strain CRF17-18 (Figure 4). 
Control Control (50 mM NaCl) Control (100 mM NaCl) Control (150 mM NaCl)
ORF15-23 ORF15-23 (50 mM NaCl) ORF15-23 (100 mM NaCl) ORF15-23 (150 mM NaCl)
Salt crystals
CRF17-18 CRF17-18 (50 mM NaCl) CRF17-18 (100 mM NaCl) CRF17-18 (150 mM NaCl)
 
FFiigguurree 44.. RRoooott ccoololonnizizaatitoionn oof fSSininomomononasa sspsp. s.trsatrinai nOROFR1F51-253-2 a3nadn BdaBciallcuilsl ussp.s pst.rasitnra CinRFC1R7F-1187 -i1n8 KinDMKDLM10L5 1s0e5edsleiendglsi negxs-
aemxaimneinde wdiwthi tah faiefildel-dem-emissisiosino nscsacnanninnign geleelcetcrtornon mmicircoroscsocoppee (s(sccaalele bbaarr,, 55000000×× mmagagnnifiificcaatitoionn).) .TThhee fifigguurreess aarree rreepprreesseennttaattiivvee 
ooff tthhrreeee rroooott ssaampplleess ppeerr ttrreeaattmeenntt.. 
44.. DDiissccuussssiioonn 
22--AAcceettyyll--11--ppyyrrrroolliinnee ((22AAPP)),, aa nniittrrooggeenn--ccoonnttaaiinniinngg ccoommppoouunndd,, iiss tthhee mmaajjoorr ccoommppoouunndd 
rreessppoonnssiibbllee ffoorr tthhee uunniiqquuee aarroommaattiicc cchhaarraacctteerriissttiicc iinn rriiccee iinncclluuddiinngg tthhee TThhaaii jjaassmmiinnee 
rriiccee//TThhaai iaraormomataicti cricreic Ke DKMDLM1L0150 c5ulctuivltairv [a5r2[–5525–]5. 5T]h. isT ahriosmaarotimc caotimc pcoomunpdo huansd ahlsaos baelesno 
rbeepeonrtreedp otort ebde ptorobdeupcreodd buyce ad wbiydae rwaindgeer oafn mgeicorfomorigcraonoisrgmasn, iisnmclsu, dininclgu dpilnangtp-glaronwt-gthro-pwrtoh--
mprootminogt rinhgizorhbiazcotebraicat (ePriGaP(RP)G [P5,R27) ,[259,]2.7 A,2 p9]r.evAioupsr esvtuioduys bsyt uDdeyshbmyuDkehs ehtm alu. k[2h9]e rteaplo. r[t2e9d] 
trheapto rritceed rhthizaotbraicceterrhiaiz coobualcdt eprriaodcuouceld thper o2AduPc ceotmhepo2uAnPdc womithp ovuanludesw riathngvianlgu fersorman 6g.5ing−1 3–
1fr1o9m.3 6µ.g53·k–g1−119. .H3 iµggh·ekrg 2A.PH pigrohderuc2tAioPnp wroadsu ocbtisoenrvwedas ionb rsheirzvoebdaicnterrhiaiz oobbtaacitneerida forbotmai nthede 
rfhroizmostphheerrhei zoofs aprhoemreaotifc arriocme caotimc rpiacerecdo mtop naorendartoomnaotniac rroimcea. tAicllr iicseo.laAtlelsi sforolamte soufrro mstuoduyr 
wsthuidcyh wwheirceh owbtearienoebdt afrinoemd tfhroem artohme aatriocm riacteic rrhiiczeorshpihzoersep hsheroewsehdo wa ehdigahheirg 2hAerP2 AcoPntceonnt- 
ttheannt  tthhains tehairslieearr lrieeprorertp. oArt .sAimsiilmari loabrsoebrsveartvioanti ownaws arsepreoproterdte dbyb yDDhohnonddgge eeet taal.l .[[55]],, wwhhoo 
ffoouunndd tthhee mmaaxxiimmuumm 22AAPP pprroodduuccttiioonn iinn EE.. hhoorrmmaaeecchheeii aanndd LL.. xxyyllaanniillyyttiiccuuss iissoollaatteedd ffrroomm tthhee 
aarroommaattiicc rriiccee rrhhiizzoosspphheerree.. IInn tthhiiss ssttuuddyy,, ffoorr tthhee ffiirrsstt ttiimmee,, wwee pprroovviiddee eevviiddeennccee tthhaatt mmeemm--
bbeerrss ooff tthhee hhiigghh--GGCC GGrraamm--ppoossiittiivvee bbaacctteerriiaa MMiiccrrooccooccccuuss aanndd SSiinnoommoonnaass are able to producethe 2AP compound. Members of the genera Bacillus (low-GC Gram-po asritei vaeblbea tcot eprriao)dauncde 
Burkholderia (Gram-negative bacteria) are also added to the list of 2AP-producing bacteria.
This observation supports the view that the ability to produce the 2AP compound is widely
 distributed among bacteria. Our finding and previous reports strongly suggest that the
rhizosphere of aromatic rice is a good source for 2AP producing rhizobacteria. Since a
gradual decrease in the 2AP level in KDML105 rice grains is eminent due to excessive soil
salinity driven by chemical inputs and climate change, this 2AP-producing PGPR might
provide a sustainable alternative solution to cope with the aroma deterioration problems.
In the present study, all PGPR isolates from the ORF system produced 2AP in the
range of 30.91–372.76 µg·kg−1. However, 2AP production was found in only half of the
tested PGPR isolates from the CRF system (0–254.95 µg·kg−1). It was interesting to note
that, on average, PGPR isolates obtained from the ORF system gave higher 2AP production
(216.98 µg·kg−1) in the culture medium than PGPR isolates obtained from the CRF system
Biology 2021, 10, 1065 11 of 16
(133.48 µg·kg−1). In addition, only one CRF isolate (Burkholderia sp. CRF16-3) showed
high 2AP production (254.95 µg·kg−1), while the highest 2AP value (372.76 µg·kg−1) was
obtained from an ORF isolate (Sinomonas sp. ORF15-23). These observations support the
view that agricultural practices play a role in aromatic level of aromatic rice. Our previous
study suggested that the high soil bacterial population and total soil nitrogen from an ORF
system of the TKR area were responsible for the 3.5-fold higher 2AP content of KDML105
rice grains in this system than the CRF [30]. Appropriate soil environments and sufficient
N supply in the ORF system might promote a higher 2AP production by PGPR isolated
from this system. Although PGPR have been extensively investigated, few studies have
correlated agricultural practices with the plant-growth-promoting capacity of rhizobacteria
isolated from different systems, e.g., organic vs. conventional farming systems. This aspect
requires further research effort before a definitive conclusion can be reached.
The high 2AP level in the KDML105 rice leaves led to a high 2AP level in the rice
grains grown in clay and sandy soil [56]. Therefore, in the present study, it was expected
that the enhancement of the 2AP level in the leaves of KDML105 rice seedlings by ST-PGPR
inoculation might lead to high 2AP in the rice grains. A previous study revealed that
secondary metabolites including 2AP are mostly produced in rice plant during the booting
stage [57]. In addition, previous studies suggested that the 2AP was translocated from
the leaves to the grain during later stages of rice growth [58–60]. The 2AP content in rice
continuously increased from the seedling stage to the maximum level around the booting
stage and the enhancement of 2AP content at or after booting stage was reported to increase
2AP accumulation in mature grains [59]. Therefore, the enhancement of 2AP by ST-PGPR
inoculation during the seedling stage might also increase the 2AP level in mature grains
similar to inoculation at the booting stage. The determination of 2AP level in leaves might
be useful for prediction of the 2AP level in the rice grains.
The inoculated aromatic rice variety Basmati-370 had around a 1.14–1.41-fold higher
2AP level than the uninoculated control [29]. The highest 2AP-producing strain provided
the highest 2AP in the aromatic rice, while the 2AP-nonproducing strains had no significant
effect on the 2AP contents of Basmati-370. Our results indicated that the inoculation of
2AP producing PGPR markedly enhanced the 2AP level in the rice seedling leaves under
both normal (15.64–69.82% increase) and saline conditions (3.54–193.77% increase). The
inoculation of 2AP-producing Enterobacter hormaechei (AM122) and Lysinibacillus xylanilyti-
cus (DB25) in consortium resulted in a significant improvement in 2AP content of Basmati
rice over mono inoculation and control [5]. Interestingly, the highest 2AP-producing
PGPR, Sinomonas sp. ORF15-23, provided the maximum 2AP content in the seedling
leaves at 50 mM NaCl (19.6 µg·kg−1), with a percentage increase of 90.20% as compared
to control-50. It was observed that, on average, the 2AP level of the inoculated seedling
leaves showed much higher values than those of the uninoculated controls at all tested
NaCl concentrations. However, as compared to each respective control, the 2AP value of
the inoculated seedling leaves showed a sharp increase within a certain range of NaCl
concentrations (0–50 mM NaCl with an EC value of 2.06 to 7.69 dS·m−1, respectively).
These 2AP values slightly increased beyond this NaCl range (100 and 150 mM NaCl with
EC values of 13.78 and 19.51 dS·m−1, respectively). According to the salinity tolerance,
rice is considered a salt-sensitive crop, and the rice seedling and reproductive stages are
regarded as the most sensitive periods to salt stress, in which the growth and yield of rice
are greatly affected by salinity with a threshold EC value of 3 dS·m−1 for most cultivated
varieties [14,61]. Various studies revealed that the rice biomass, grain yield, and quality
were drastically affected by salinity beyond 8 dS·m−1 [17,18,62]. Although, the 2AP content
in the seedling leaves at 150 mM NaCl was much lower than in those at 50 mM NaCl, the
percentage increase was higher. This phenomenon suggests that the maximum percentage
increase in 2AP production at 150 mM was mainly due to an excessive salinity stress. As
compared to the uninoculated control, our results clearly indicate that the application of
PGPR isolates could effectively enhanced the 2AP synthesis in rice seedling leaves when
the EC value was <8 dS·m−1; however, beyond this EC value, the PGPR isolates showed
Biology 2021, 10, 1065 12 of 16
only slight upregulation of the 2AP synthesis. This phenomenon implies that the highly
positive interaction between the PGPR isolates and the seedlings for rice 2AP synthesis
could be expected when the EC value of the medium was less than the threshold value
(~8 dS·m−1).
The key aspects dominating the success of PGPR inoculation are their survival number
in the rhizosphere environment and the establishment of the introduced bacteria in the
plant roots. In the present study, the number of PGPR isolates slightly decreased from
0 to 50 mM NaCl but sharply decreased thereafter (>50 mM NaCl). Although the popu-
lation decreased beyond 50 mM NaCl, each isolate remained around 106 CFU·mL−1 at
150 mM NaCl. Therefore, these PGPR isolates could be considered as salt-tolerant PGPR
(ST-PGPR). Deshmukh et al. concluded that the optimal number of rhizobacterial iso-
lates which showed a highly positive influence on rice 2AP level is 105–106 CFU·mL−1,
whereas 102–104 CFU·mL−1 showed less influence on the 2AP level [29]. This obser-
vation suggested that the ST-PGPR tested in this study could probably survive under
salt-stressed soil, such as the TKR region. As these strains were reported to solubilize
P and K, as well as produce siderophore, they could be applied for the enhancement of
rice growth as well as 2AP level in the KDML105 rice grains [30]. A study showed that
Streptomyces isolate S3, which produces IAA and siderophore and solubilizes phosphate,
enhanced the growth of KDML105 rice seedlings under drought stress, as phosphorus
and iron are essential for plant growth [7]. Therefore, the nine strains obtained in this
study have high potential application in the rice field. Members of the genus Sinomonas are
Gram-positive bacteria in the phylum Actinobacteria [63]. Currently, there are 10 validly
described species on the List of Prokaryotic names with Standing in Nomenclature (LSPN)
[https://www.bacterio.net/genus/sinomonas (accessed date 13 September 2021)] [64].
A search through the Online Risk Group Database provided by ABSA International (the
Association for Biosafety and Biosecurity) found no record regarding the risk of members
of the genus Sinomonas as animal, human, or plant pathogens or select agents by both
the Center for Disease Control (CDC) and the United States Department of Agriculture
(USDA). In addition, to our best knowledge, there are no publications reporting the risk of
Sinomonas species toward the environment or human health.
The adhesion of bacteria to the seeds is the first step in rhizobacteria–plant interactions
for successful colonization [65,66]. Colonization of beneficial bacteria is of central impor-
tance for growth-promoting activity [65]. All PGPR in this study exhibited root colonization
ability in KDML105 seedlings as examined by SEM analysis. Sinomonas sp. strain ORF15-23
was able to colonize both non-stressed and salinity-stressed seedlings of KDML105 rice.
The SEM results of Dhondge et al. [5] revealed that cells of L. xylanilyticus and E. hormaechei
were consistently distributed and adhered to the root surface of inoculated seedlings in
clusters as microcolonies. The formation of a microcolony by rhizobacteria on the surface
of the root indicates successful colonization [67]. Exposure to excessive salinity was found
to decrease maize and wheat root attachment by Azospirillum brasilense [68]. This finding is
in accordance with the results of the present study, which showed that the colonization
density declined with an increase in salinity level, clearly indicating a negative impact of
salt stress on rhizobacterial population (Figure 4). However, the most salt-tolerant isolate,
Sinomonas sp. ORF15-23, maintained its high density upon root attachment at all NaCl
levels, and this might be the reason for its greatest ability in promoting the 2AP level of
rice seedling leaves.
5. Conclusions
In conclusion, the present investigation revealed that most of the tested PGPR isolates
are able to produce 2AP (a key volatile aroma compound in Thai jasmine rice KDML105) in
liquid medium in vitro. The inoculation of these PGPR isolates, particularly Sinomonas sp.
strain ORF15-23, markedly enhanced the 2AP level in the leaves of rice seedlings under
salinity stress up to 150 mM, which could lead to a high 2AP level in the rice grains,
thus promoting the economic value of KDML105 aromatic rice. In addition, all PGPR
Biology 2021, 10, 1065 13 of 16
isolates are considered salt-tolerant PGPR, as they survived in 150 mM NaCl at high cell
density. Sinomonas sp. strain ORF15-23 was also able to colonize the roots of both non-
stressed and salinity-stressed seedlings. Our findings clearly indicated that 2AP-producing
rhizosphere PGPR isolates are potential candidate for promoting the growth and aromatic
compound level of Thai jasmine rice KDML105 under salinity stress. Further investigation
under field conditions is needed for the development of these promising PGPR isolatess,
especially Sinomonas sp. strain ORF15-23, as a bioinoculant for KDML105 rice cultivation
in salt-affected areas.
Author Contributions: K.C. carried out the study as part of her PhD thesis, discussed the results,
and wrote the manuscript; writing—original draft preparation, K.C. and A.S.; conceptualization
and discussion, K.C., A.S., L.H. and D.L.; funding acquisition, K.C. and A.S.; supervision and
writing—review and editing, W.P.-a. All authors have read and agreed to the published version of
the manuscript.
Funding: This research study was funded by Chiang Mai University. K.C. is grateful to the Graduate
School, Chiang Mai University, for the TA/RA scholarship for 2018–2020. W.P.-a. acknowledges the
partial support from Chiang Mai University through the Research Center of Microbial Diversity and
Sustainable Utilization.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing does not apply to this article as no datasets were gener-
ated or analyzed during the current study.
Acknowledgments: The authors are indebted to the farmers of Community Enterprise of Thamo
Organic Agricultural Group, Surin Province, and Na So Rice Farmers Group (Nature Conservation
Club), Kut Chum, Yasothon Province, for their help during field sampling, as well as the Alliance
of Biodiversity International and Center International of Tropical Agriculture (CIAT), Asia Hub,
Common Microbial Biotechnology Platform (CMBP), Hanoi, Vietnam.
Conflicts of Interest: The authors declare no conflict of interest.
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