Effects of encapsulated Satureja hortensis/calcium propionate against fire blight in pear cv. Spadona

Abstract

Amylovoran (amsC gene) is one of the major virulence factors of a devastating plant pathogen of Erwinia amylovora. Herein, the antibacterial potential of the combined encapsulated plant bioactive compound (Satureja hortensis) and organic acid salt (calcium propionate) was evaluated against E. amylovora, for the first time. To this goal, savory essential oil and calcium propionate were encapsulated using the spray drying technique and then the effects of this formulation on gene expression (amsC) and control of fire blight (inhibiting the amylovoran production) were evaluated. The results of GC/MS analysis showed that O-cymene (25%), terpinolene (18.5%), and carvacrol (2.9%) were the most important compounds in savory essential oil. The highest encapsulation efficiency (96.25%) was provided by optimizing the ratio of maltodextrin to modified starch at 75:25 as the shell of the as-prepared microcapsules. The biocontrol efficiency of this formulation was investigated at three different concentrations T1 (1 g/l formulation), T2 (2 g/l formulation), and T3 (3 g/l formulation), and all treatments significantly impaired the amylovoran production in shoots of pear cv. Spadona. Moreover, among all treatments, T3 caused the greatest decrease in amylovoran gene expression. The reduction of gene expression by T3 was twice more than T2. T3 caused a 2-fold decrease in the expression of the amylovoran gene compared to the internal control gene (16 S rRNA) compared to T2. Also, T2 caused a 2-fold decrease in the expression of the amylovoran gene compared to the internal control gene (16 S rRNA) rather than T1.

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Journal of Plant Pathology (2023) 105:869–885
https://doi.org/10.1007/s42161-023-01385-2
ORIGINAL ARTICLE
Effects ofencapsulated Satureja hortensis/calcium propionate
againstfire blight inpear cv. Spadona
RouhollahFaramarziDozein1· ElahehMotamedi2· SaeedTarighi3· EhsanOskoueian4· AramBostan5
Received: 5 November 2022 / Accepted: 13 April 2023 / Published online: 30 May 2023
© The Author(s) under exclusive licence to Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2023
Abstract
Amylovoran (amsC gene) is one of the major virulence factors of a devastating plant pathogen of Erwinia amylovora.
Herein, the antibacterial potential of the combined encapsulated plant bioactive compound (Satureja hortensis) and organic
acid salt (calcium propionate) was evaluated against E. amylovora, for the first time. To this goal, savory essential oil and
calcium propionate were encapsulated using the spray drying technique and then the effects of this formulation on gene
expression (amsC) and control of fire blight (inhibiting the amylovoran production) were evaluated. The results of GC/MS
analysis showed that O-cymene (25%), terpinolene (18.5%), and carvacrol (2.9%) were the most important compounds in
savory essential oil. The highest encapsulation efficiency (96.25%) was provided by optimizing the ratio of maltodextrin
to modified starch at 75:25 as the shell of the as-prepared microcapsules. The biocontrol efficiency of this formulation was
investigated at three different concentrations T1 (1g/l formulation), T2 (2g/l formulation), and T3 (3g/l formulation), and
all treatments significantly impaired the amylovoran production in shoots of pear cv. Spadona. Moreover, among all treat-
ments, T3 caused the greatest decrease in amylovoran gene expression. The reduction of gene expression by T3 was twice
more than T2. T3 caused a 2-fold decrease in the expression of the amylovoran gene compared to the internal control gene
(16S rRNA) compared to T2. Also, T2 caused a 2-fold decrease in the expression of the amylovoran gene compared to the
internal control gene (16S rRNA) rather than T1.
Keywords Essential oil· Erwinia amylovora· Calcium propionate· Gene expression· Microencapsulation· Satureja
hortensis
Introduction
Fire blight disease caused by Erwinia amylovora bacteria
is the most important disease of pome fruits (apple, quince,
and pear) all over the world (Burrill 1881; Sarojini 2012).
The bacteria can enter plants through natural openings or
wounds on plants and then can spread through xylem ves-
sels to infect and kill the entire plant (Yuan etal. 2022a, b).
So far, pruning the diseased twigs in combination with
copper-based formulation and antibiotics are considered
the common strategies for managing the fire blight disease
(Singh and Khan 2019). The use of copper compounds over
a long period may pose risks to mammals, birds, aquatic, and
soil organisms (Hafez Yaseret al. 2021). Antibiotics may lead
to the appearance of pathogen-resistant strains (Kim etal.
2022). A large number of studies conducted in numerous tri-
als around the world in the last two decades have identified
several natural antimicrobial compounds that have the ability
to control fire blight disease(Abdallah etal. 2023). Although,
* Elaheh Motamedi
motamedi.elaheh@gmail.com
* Saeed Tarighi
starighi@um.ac.ir
1 Mashhad Branch, Agricultural Biotechnology Research
Institute ofIran (ABRII), Agricultural Research, Education
andExtension Organization (AREEO), Mashhad, Iran
2 Department ofNanotechnology, Agricultural Biotechnology
Research Institute ofIran (ABRII), Agricultural Research,
Education andExtension Organization (AREEO), Karaj, Iran
3 Department ofPlant Protection, Faculty ofAgriculture,
Ferdowsi University ofMashhad, Mashhad, Iran
4 Mashhad Branch, Agricultural Biotechnology Research
Institute ofIran (ABRII), Agricultural Research, Education
andExtension Organization (AREEO), Mashhad, Iran
5 Food Nanotechnology Department, Research Institute
ofFood Science andTechnology (RIFST), Mashhad, Iran

870 Journal of Plant Pathology (2023) 105:869–885
1 3
some bio-control methods (Gwinn 2018), controlling fire
blight is still a challenging task.
The most important control stage of fire blight disease
is the removal of the bacterial agent during the blooming
stage of pome fruits. Essential oils are naturally occurring
odorous, volatile, and oily liquids which are biologically
active and produced by secondary metabolism in aromatic
plants (Amzad etal. 2022). During the last two decades,
there has been a significant increase in pesticide formula-
tions that used natural materials (i.e., essential oils or plant
extracts). Simultaneous use of chemical compounds and bio-
active plant compounds caused either triggering the systemic
acquired resistance (SAR) such as acibenzolar-S-methyl
(ASM) or suppression of shoot blight such as prohexodi-
one-calcium (Kokošková and Pavela 2007). Salicylic acid
(SA) has stimulated various enzymes involved in the plant’s
defense system (Ebrahimi-Zarandi etal. 2022). An inter-
esting alternative to bactericide application for controlling
plant disease involves the use of some organic and inorganic
salts (Askame etal. 2011). The use of different salts for
controlling various postharvest diseases of potato and other
crops were assessed in some studies (Askame etal. 2011;
Hervieux etal. 2002; El Asbahani etal. 2015). Although the
potential of antimicrobial activity of essential oils and their
use as free agents is high in agriculture and other areas, their
application can be hindered by their inherent characteristics
including high volatility, susceptibility to degradation in
aqueous conditions, and hydrophobicity (Singh and Pulik-
kal 2022). To overcome the drawbacks, the effectiveness of
a novel approach whereby the essential oils were placed in
micro-capsulation was examined, which indicated supply-
ing the protection, avoiding the evaporation of the oils, and
improving their stability, long-term durability, and immis-
cibility in aqueous phases (Cadena etal. 2018; Partheniadis
etal. 2019; El Asbahani etal. 2015). Recently, for detec-
tion of the several plant pathogens, real-time PCR assays
were employed because this method is rapid, automated,
sensitive, possibly quantitative, and reduces post-PCR han-
dling (Laforest etal. 2019). Some PCR (polymerase change
reaction) and real-time PCR techniques are accessible for
identifying and quantifying E. amylovora targeting the
pEA29 plasmid and chromosomal DNA (Pirc etal. 2009;
Yuan etal. 2022a, b). The methods based on quantifying the
chromosomal DNA could be considered the first choice for
the screening tests (Kaluzna etal. 2013). The objectives of
this study include screening the plant bioactive compounds
and organic acid salts against E. amylovora (ATCC 49946),
preparing the optimal formulation, evaluating the efficacy of
the microencapsulation against E. amylovora on pear shoots,
and investigating the population of microorganisms using
real-time PCR.
From 1989 to 2022, several studies using plant essential
oils, including Satureja hortensis essential oil, have been
conducted on fire blight in the laboratory, greenhouse, and
garden (Scortichini and Rossi 1989, 1991; Sahin etal. 2003;
Adiguzel etal. 2007; Razzaghi-Abyaneh etal. 2008; Sharma
etal. 2009; Mihajilov-Krstev etal. 2010; Karami-Osboo etal.
2010; Konecki etal. 2013; Akhlaghi etal. 2020; Bastas 2020;
Sun etal. 2021; Proto etal. 2022). However, up to now, no
research has been done on the antimicrobial, antioxidant, and
molecular effects of organic acid salts on fire blight disease.
In this study, for the first time, the encapsulated mixture of
savory essential oil (Satureja hortensis) and calcium propion-
ate organic acid salt was developed, and its efficiency was
approved at the microbial and molecular level in the labora-
tory and greenhouse.
Materials andmethods
Materials
Organic acid salts included potassium acetate, calcium
acetate, magnesium acetate, potassium lactate, calcium lac-
tate, magnesium lactate, potassium citrate, calcium citrate,
magnesium citrate, and calcium propionate were purchased
from Mobtakeran Chemistry Company (Iran). Further,
the essential oils evaluated in this study included Satureja
hortensis, Origanum majorana, Mentha spicata, and Tra-
chyspermum ammi were bought from the local market. The
bacterial stock (Erwinia amylovora ATCC49946) was pre-
pared by the Iranian Biological Resource Center (IBRC).
Two-year-old pear (Pyrus communis) trees, susceptible cul-
tivar Spadona, were obtained from seedling companies in
Mashhad for extracting nucleic acid (RNA or mRNA). Spe-
cific primers were designed by the company Takapouzist. The
experiments were conducted in the laboratory of the Agri-
cultural Biotechnology Research Institute of Iran (ABRII)
and laboratory of the Research Institute of Food Science and
Technology (RIFST) in Khorasan Razavi Province, Mash-
had, during 2021–2022.
Experimental design
Screening 4 of the essential oils (EOs) and 10 of the
organic acid salts (OASs) was performed on E. amylovora
(ATCC49946) in the laboratory by colony forming unit
(CFU), minimum inhibitory concentration (MIC) and mini-
mum bactericidal concentration (MBC). Then, the formula-
tions were prepared, and the best formulation was chosen
for the spray drying technique. In the greenhouse, the pear
seedlings (2-year-old pear) cv. Spadona were planted, and
the microencapsulated powder (formulation of EOs + OASs)
was sprayed on the pear seedlings every 20 days for a
period of 2 months. Then, bacterial water suspension (~108
CFU/ml) of E. amylovora (ATCC49946) was sprayed on

871Journal of Plant Pathology (2023) 105:869–885
1 3
2-year-old pear (P. communis) seedlings, susceptible cul-
tivar Spadona (at a humidity of 60%, the temperature of
25 to 28°C, and light level of 7000 to 100,000lx). In order
to investigate the expression of the amylovoran gene, the
samples were taken from the border between the healthy and
infected parts and immediately immersed in RNAlater® Sta-
bilization solution, placed in a container containing ice, and
then transported to the laboratory in the shortest possible
time. Finally, the expression level of amylovoran gene after
applying the treatments (S: streptomycin, T1: 1g/l formula-
tion, T2: 2g/l formulation, and T3: 3g/l formulation) was
checked by real-time reverse transcription PCR (qRT-PCR).
Extracting theessential oil
100g of the dried aerial parts of the plants (Satureja horten-
sis, Origanum majorana, Mentha spicata, and Trachysper-
mum ammi) were cut and distillated for 3h by Clevenger
apparatus. The as-prepared essential oils were dried using
anhydrous sodium sulfate and maintained at 4°C (Elyemni
etal. 2019). The percentage of essential oil was calculated
by using the following formula.
GC‑MS analyses
The analysis of the essential oil (S. hortensis) was carried
out by gas chromatography coupled with mass spectrometry
(GC-MS). The device (GC-MS- Hewlett Packard HP 6890
model) has a polar column (30m×0.25mm, film thickness,
0.25μm). Helium was used as the carrier gas with a flow
rate of 1 ml min− 1. The initial temperature was programmed
from 50 to 200°C, with a gradient of 5°C min− 1. Ionization
was by electron impact at 70eV and the ion source tempera-
ture was 175°C. The identification of the compounds was
based on a comparison of the retention time with those of
authentic standards, comparing their linear retention indices
relative to the series of n-hydrocarbons. The percentage of
each compound according to the area under its curve in the
spectrum of the chromatogram obtained from the device was
performed by the method of normalizing the surface of the
curve (Mariem etal. 2018; Ainane etal. 2018).
MIC andMBC
The antibacterial activities of the essential oils (EOs) and the
organic acid salts (OAS) were screened by determining the
minimum inhibitory concentrations (MICs) and minimum
bactericidal concentrations (MBCs) against E. amylovora
Essential oil content (%)
=Volume of the distilled oil (ml)
Mass of the material which was distilled (g)
×
100
(ATCC49946). First, organic acid salts (100mg/ml) and
essential oils (20mg/ml) were each serially diluted in ster-
ile tryptic soy broth (TSB) in 96 well plates. Each well was
inoculated with 50µl of the standardized cell suspension
(5.3 × 108 CFU/ml) and incubated at 28°C overnight. After
24h, 30µl of Resazurin (0.015g/100 ml distilled water) was
added in 96 well plates and incubated at 28°C for 10–15min,
and the highest dilution where no growth occurred was
recorded as the MIC. To determine MBC, 100µl of broth
from wells containing no growth were plated on NA (nutrient
agar) or TSA (tryptone soy agar), and incubated overnight,
at 28°C. After 24h, the highest dilution, where no survivors
were observed, was recorded as the MBC. Streptomycin (100
ppm) and distilled water were used as the positive and nega-
tive controls, respectively, in both of the above methods. Each
experiment was performed three times and the results were
recorded (Gkanatsiou etal. 2019).
Disc diffusion
The paper disc diffusion method was used for screening
the antibacterial activity of the essential oil, which was
performed by nutrient agar (Akhlaghi etal. 2020). Bac-
teria grown overnight were diluted to a concentration of
5 × 108 CFU/ml. Sterile cellulose discs of 6mm diameter
were impregnated with DMSO (dimethyl sulfoxide), EOs, or
OAS (100 and 1000µg ml− 1 after dissolving in DMSO) and
placed on TSA (tryptone soy agar) or NA (nutrient agar),
solid medium previously amended with 5 × 108 CFU ml− 1
of bacteria. DMSO and 50–100 ppm of streptomycin were
used as the negative and positive controls, respectively. The
inoculated plates were incubated at 28°C, for 24h. The anti-
microbial activity was evaluated by measuring the clearance
zone around the discs, the values of which were expressed as
log CFU mm− 2. All the experiments were done in triplicate.
Assessing thecolony forming units (CFU)
The colony-forming units (CFU) method was applied to
assess the number of viable cells after treatment with formu-
lation (EOs + OASs). First, overnight culture was prepared
from E. amylovora bacteria. Stock solutions E. amylovora
ATCC49946 with a concentration of 5 × 108 CFU/ml were
diluted serially in TSB (tryptic soy broth) medium. After
that, 10µl of each bacterial dilution was applied directly on
TSA or NA medium in triplicate, followed by incubation at
28°C, for 24h, and the number of colonies obtained was
counted to assess CFU. Also, it was done in the following
way to calculate the number of bacteria in different organs
after infection and treatment with the formulation. First, the
samples (collecting microorganisms from the organ) were
carefully taken from the border between the healthy and

872 Journal of Plant Pathology (2023) 105:869–885
1 3
infected parts of the target organ. Next, 0.5 to 1g of the
infected sample (branch, leaf, or fruit infected with bacteria)
was accurately weighed with a scale. Two-fold dilution was
carried out and plated on NA medium and then incubated
at 28°C, for 24h. Finally, the growth kinetics of both nor-
malizers and treated cells were recorded visually (Gayder
etal. 2020).
Designing thewall andcore material
ofmicroencapsulation
Maltodextrin (MD) is a partially hydrolyzed product of
starch that can be used to encapsulate essential oils. MD can
enhance the color, aroma, and taste of products, improve the
solubility and stability of core materials, and slowly release
the core materials for a long time to achieve certain spe-
cific uses (Truong etal. 2022). Therefore, the development
of MD-based microcapsules is a key research field in the
encapsulation of essential oils. On the other hand, Hi-Cap
100 derived from waxy maize is widely used for microen-
capsulation of bioactive compounds by spray drying, offer-
ing advantages such as neutral aroma and good protection
against oxidation (Truong etal. 2022). Calcium propionate
(C6H10CaO4) is a food additive presented in many foods,
especially baked goods. It acts as a preservative to help
extend shelf life by interfering with the growth and repro-
duction of microorganisms (Wigati etal. 2022). In this study,
the blends of the wall (maltodextrin and Hi-Cap-100) and
core (calcium propionate and savory essential oil) materials
were described in the experimental design (Table1).
Preparing theemulsion
To prepare the emulsions (Table1) a mechanical stirrer was
utilized. The emulsifier (Tween 20, 4.5g) was dissolved in
110 ml of distilled water and stirred at 8000rpm until com-
plete dissolution (approximately 1h). Then, 28g of malto-
dextrin and 0g of Hi-Cap 100 (S1), 21g of maltodextrin
and 7g of Hi-Cap 100 (S2), 14g of maltodextrin and 14g
of Hi-Cap 100 (S3), 7g of maltodextrin and 21g of Hi-Cap
100 (S4) and 0g of maltodextrin and 21g of Hi-Cap 100
(S5) were added to the solution. After that, 4.2g essential
oil of S. hortensis and 2.8g calcium propionate were added,
which was topped up with distilled water to 150 ml (the ratio
of core wall material was chosen 1:4). Finally, homogenizing
was conducted at ambient temperature by a digital Ultra-Tur-
rax IKA T25, at 10000rpm for 30min and the samples were
immediately transferred to the next stage (spray drying pro-
cess). The droplet-size distributions of the emulsions were
obtained by using laser diffraction measurements, a Malver
Mastersizer 2000 equipped with a Hydro 2000 SM disper-
sion unit (Malvern Instruments Ltd., Worcestershire, Eng-
land) (Clausse etal. 2018; Kausadikar etal. 2015; Kfoury
etal. 2019; Tomazelli Júnior etal. 2018b).
Spray drying process
To obtain the microcapsules, the emulsions were spray-
dried by using a laboratory scale dryer (BUCHI mini Spray
Dryer B -191). The diameter of the drying chamber was
800mm×620mm/cone 60º. The emulsion was fed into the
drying chamber using a peristaltic pump, at an ambient tem-
perature (25°C), drying airflow rate of 40kg/h, the feed flow
rate of 2.5 ml/min, and atomizer speed (14,000–16,000rpm) on
the powder properties. The inlet temperature was set at 150°C
and the outlet temperature was maintained at 80°C. The humid-
ity ratio was less than 50%. The resultant powder was collected
and kept at 4°C, in an opaque container (Partheniadis etal.
2019; de Oliveira etal.2022).
Particle morphology andsize distribution
The microcapsules solution (EOs + OASs) was observed
using an optical microscope (LX400, LABOMED, USA).
A droplet of the emulsion was placed between the slide and
the cover glass and photographs were taken in transmission
mode under 100 magnification (Aicha etal. 2021). The par-
ticle size of microcapsules was determined by using the laser
diffraction system (ANALYSETTE 22 Nano Tec) fitted with
a small sample dispersion unit (MS1) connected to a disper-
sion unit controller. The results were indicated as the volume
mean diameter, D (4,3), and size distribution provided in the
software of MAS. The analysis was conducted in triplicate
for obtaining an average. The scanning electron microscope
(SEM) was used for analyzing the surface morphology of
the dried powders. First, the sample was placed on a sample
Table 1 Different ratios of the
wall and core compounds
Tween 20® at 0.5-3% based on the dry basis were used as the emulsifiers
Wall and core materials/treatments S1 S2 S3 S4 S5
Wall materials Maltodextrin 100 75 50 25 0
Hi-Cap 100 0 25 50 75 100
Core materials Savory essential oil 20 16 18 14 12
Distilled water 80 64 72 56 48
Calcium propionate 0 20 10 30 40

873Journal of Plant Pathology (2023) 105:869–885
1 3
holder previously covered with double-sided carbon tapes
and then coated with a thin layer of gold by a sputter coater.
Observation and photography were performed in a scanning
electron microscope (XL30; Philips, Eindhoven, The Neth-
erlands) operated at an acceleration voltage of 20kV, The
Quanta has a tungsten-based electron optical column with a
resolution of 3.5nm and an ion resolution of 10nm. Meas-
urements were taken in the vacuum at different magnifica-
tions (Bajac etal. 2022).
Microencapsulation efficiency
The dried powder (5g) was dissolved in distilled water
(150 mL), and distilled for 3h, in a Clevenger apparatus to
determine the total oil content of the microcapsules. Two ml
of n-hexane was added for extracting the essential oil from
the water phase. The solution was slowly brought to boil
water and allowed to distill. The resulting oil was collected
in a pre-weighed Erlenmeyer, and ethyl ether was allowed
to evaporate at room temperature for 24h (Tomazelli Júnior
etal. 2018a). A modified method described by Tomazelli
Júnior (Tomazelli Júnior etal. 2018a) was selected for
determining the surface oil. The dried powder was added to
n-hexane (1:2.5 w/v), and stirred at 3000rpm, for 10min.
The resultant mixture was filtered and washed three times
with n-hexane. To evaporate all the solvent, the filter was
maintained in a desiccator connected to a vacuum pump.
The oil encapsulation efficiency (OEE) was calculated by
the following equation (Bajac etal. 2022).
Efficacy ofthemicroencapsulation againstE.
amylovora onpear shoots
The procedure explained by Hafez Yaser etal. (2021) was
used with a few changes. The design was conducted in a
randomized complete block design (RCBD) with 2-year-
old pear seedlings (P. communis) trees, susceptible cul-
tivar`Spadona`. Seedlings were individually planted in
20-liter pots with a mixture of soil and sand, and grown in
a greenhouse. Treatments consisted of a polymeric struc-
ture containing effective bioactive compounds (savory
essential oil) and organic acid salts (calcium propionate),
at three different levels T1: one gram per liter of encap-
sulated powder, T2: two grams per liter of encapsulated
powder, and T3: three grams per liter of encapsulated pow-
der), and positive control (T4: 100mg/liter streptomycin
treatment), control seedlings (T5: no contamination and no
spray treatment), and only infected seedlings (E amylovora
Microencapsulation Efficiency
(%)=
Total oil −Surface oil
Total oil
×
100
108 CFU/ml) (T6) giving a total of six treatments in a
randomized complete block design (RCBD). After 24h,
the shoots were inoculated by using the needle approxi-
mately 3cm of their length from the tip (the needle was
poked into the seedlings) and covered with several 100µl
droplets of bacterial water suspension (~108 CFU/ml). The
temperature and air humidity were maintained at about
25–28°C and 70–90%, respectively, in the greenhouse.
The presence of blight symptoms (lesions) on the shoots
was recorded (infected shoots were measured with a ruler)
in the fifth and fifteenth days after inoculating. Three
independent biological replicates were performed for each
sample (72 in total). Detached shootlets tip of pear were
used for laboratory tests (Hafez Yaser etal. 2021).
Disease severity was estimated by evaluating 25 seed-
lings randomly in the middle 2 rows in each treatment
with a score of 9. It was calculated when the disease index
in the control treatment was more than 75% (Choi etal.
2022). The average disease severity for each treatment was
calculated using the following formula:
where DS: disease severity, ni: number of infected plants
with the same score, Vi: disease score from 1 to 9 for each
treatment, N: total number of evaluated plants, and V: the
highest disease score (9) were considered.
Evaluating thepopulation ofmicroorganisms
RNA extraction
RNA extraction was performed using the kit (Pars tous-
Iran). 20–30mg of the plant tissue (the border between the
healthy and infected part of the target organ) was transferred
to a 1.5 ml micro-centrifuge tube, and 200 µL of PL solu-
tion (Lysis Buffer) was added. Cutting the plant tissue into
small pieces increases the yield of genomic RNA (mRNA)
and reduces lysis incubation time. 20µl of proteinase K was
added, mixed well by vortexing, and incubated at 56°C,
for 10min. After the lysis of tissue, 200µl of PTB solution
(Tissue Binding Buffer) was added, vortexed, and incubated
for 10min (56°C). Next, ethanol (98%, 200µl) was added,
and the lysate was carefully transferred to the spin column.
Then, 500µl of PW1 (Wash Buffer 1) was added and spun
for 1min at 13000rpm. 700µl of PW2 (Wash Buffer 2) was
added and spun for 1min at 13000rpm. Finally, 50–100µl
of preheated PE (Elution Buffer) was added and waited for
3min at room temperature. Then, the RNA solution was
stored at -70°C (Pirc etal. 2009; Yuan etal. 2022a, b).
DS = Σ(ni ×𝜈i∕N×𝜈)×100

874 Journal of Plant Pathology (2023) 105:869–885
1 3
cDNA synthesis
Based on the instructions of the manufacturer, total RNA
was retro-transcribed by gene-specific primer and random
primers of Reverse Transcriptase (Thermo Fisher Scientific).
The mixture of template RNA (5 ng), buffer (10µl), enzyme
(2µl), and distilled water (3µl) was quickly vortexed. Then,
it was incubated for 10 and 60min at 25 and 47 ºC, respec-
tively. Finally, the reaction was stopped by heating at 85
ºC, for 5min, and chilling on the ice or at 4 ºC. The cDNA
was used as a template for qRT-PCR reactions. The PCR
reactions were performed in technical triplicates by using
the Light Cycler 480 SYBR Green I Master reagent and
the Light Cycler® 480 Instrument (Roche, Italy) in 96-well
reaction plates (Kharadi and Sundin 2022).
Real‑time reverse transcription PCR (qRT‑PCR)
The E. amylovora quantification reliability was increased
by a rapid and sensitive real-time PCR quantification sys-
tem to target chromosomal DNA (Bereswill etal. 1995; Pirc
etal. 2009; Powney etal. 2011). The amsC region gene was
chosen as the target for primer design. All three real-time
PCR assays were examined in silico regarding specific-
ity, and by amplifying the plant DNA was extracted from
12 samples of different trees with shoot blight symptoms.
Real-time PCR was used to test diluted bacterial suspen-
sions. These primer sequences of the target gene amsC were
designed by Primer3 Plus software (F:5´-GTC GAC ACG
GCG ATA AAA CT-3´.R:5´- CTG GCA TGG ATG ATT CAC
AG-3´. 193bp. Wang 2011)). Further, 16 SrRNA was used
as an external control (F:5´-TCC CTC AAA ACC GCT CTG
AC-3´.R:5´-AGG CTC AAC GTC TGG AAC TG-3´. 134bp.
FN666575.1). The real-Time PCR reaction was performed
in a final volume of 20µl using SYBR® Green Real Time
PCR Master Mix (Parstous, IR) based on the instruction of
the manufacturer. The thermocycling conditions included
an initial denaturation at 95 ºC for 5min, followed by 35
cycles of 95 ºC for 10s, 58 ºC for 30s, and 72 ºC for 2min,
and final extension at 72 ºC for 4min. All experiments were
conducted with three replication.
The 16S rRNA gene was used as an endogenous control.
CT data were analyzed quantitatively via the comparative
CT method to generate relative fold change values among
control (non-infected and non-treatment), infected (contami-
nation of seedlings with E amylovora 108 CFU/ml without
applying treatments), and treatments (T1, T2, T3, and strep-
tomycin). Each quantitative real-time PCR analysis was per-
formed in triplicate.
The Ct (Cycle threshold) of amsC was normalized to the
chosen reference gene Ct of 16S rRNA. Relative quantifica-
tion was carried out according to the delta-delta Ct model
shown in Eqs. (1) and (2) (Livak and Schmittgen 2001).
In addition, efficiency correction was implemented.
Finally, fold change was implemented by Eq. (3). The
derived ratio values describe the relative expression change
of the target gene relative to the 16S rRNA reference gene
expression (Pfaffl 2001).
The statistical analysis of the relative gene expression was
done by REST (Relative Tool) with PCR efficiency values
of ≥ 0.8 (Lehman etal. 2008; Bahadou etal. 2018).
Statistical analyses
Bioinformatics analyses of this study were done by using the
Primer Plus software and biotechnology sites. Reset 2009
software was used for real-time PCR statistical data analysis.
Screening data were analyzed by using SAS software.
Results
Yield ofessential oils
The percentage of essential oil is given in Table2. The dis-
tillation of 100g of S. hortensis produced 1.8 ml of essen-
tial oil, which is equivalent to 1.8% of the gross weight of
the aerial parts. However, 100g of O. majorana produced
an average of 0.4 ml of essential oil or 0.40% of the gross
weight of the aerial parts. The essential oil yields varied
from 0.4 (O. majorana) to 1.8% (S. hortensis). The average
yield of essential oil of each species was calculated based on
the dry plant material obtained from the aerial parts (stems,
leaves, and seeds) of the plants. Several factors including the
age of the plants, the nature of the soil and climate, part of
the plant subjected to extraction, the period of the harvest,
etc. could lead to the variations (Elyemni etal. 2019).
(1)
R=2−(ΔCtta rget−ΔCt ref erence)
(2)
R=2−ΔΔCt
(3)
Fold change
=
(Etarget )
ΔCt targe(control−sample)
(E
reference
)
ΔCt reference(control−sample
)
Table 2 Essential oil content of aerial parts of the plants
Botanical name of the plant Part of plant Percentage of
essential oil
(% V/W)
Satureja hortensis Aerial part 1.8 ± 0.02
Trachyspermum ammi Seeds 1.4 ± 0.02
Mentha spicata Aerial part 1.0 ± 0.2
Origanum majorana Aerial part 0.4 ± 0.06

875Journal of Plant Pathology (2023) 105:869–885
1 3
Chemical composition analysis byGC‑MS
Essential oils are complex mixtures of volatile compounds
extracted from a large number of plants (El Asbahani etal.
2015). Gas chromatography-mass spectrometry (GC-MS) was
used for identifying and quantifying the S. hortensis oil in this
study. GC-MS analysis showed the presence of 37 components
(Table3), the most abundant of which are o-Cymene (25.6%)
and Terpinolene (18.5%). Moreover, chromatographic analyses
of the essential oils can identify 37 compounds which repre-
sented approximately 99.53% for S. hortensis.
GC-MS is considered one of the most advanced and
widely used instruments in the field of analysis of active
plant compounds (Lebanov etal. 2021). The information
about the molecular weight, elemental structure, geom-
etry, and spatial isomer of a molecule can be obtained
using GC-MS (Kiralan etal. 2021). The basis of the mass
spectrometer method, the molecule ionization process, and
its scope are affected by the ionization process (Sánchez-
Hernández etal. 2021). The total ion chromatogram of
the GC‒MS analysis of S. hortensis essential oil was rep-
resented in Fig.1a. The GC-MS analysis gave a spectrum
characteristic of O-cymene (Fig.1b). The x- and y axes indi-
cated the m/z ratio and abundance, respectively (the com-
pounds were isolated in the diagram of the ions based on the
ratio of mass to charge (m/z).
Screening theantimicrobial activity
The inhibition zones were given in Table4. All essential oils
showed significant activity against E. amylovora. The tested
EOs indicated the various antibacterial activity against E.
amylovora which included: і) strong means inhibition
zone ≥ higher than 20mm, іі) moderate means inhibition
zone ≤ in the range of 12– to 20mm, and ііі) low means
inhibition zone ≤ lower than 12mm antimicrobial activi-
ties. Overall, the oil of S. hortensis (39.3mm) displayed the
maximum, and M. spicata (17.3mm) revealed the minimum
growth inhibition (conc. of 1000µg/disc), respectively.
Table 3 Identified compounds in the essential oil of S. hortensis
presented as the relative amounts based on the peak area by GC-MS
chromatograms
***Effective compounds below 1% were not reported in this study
Number Retention/Time Name of the compounds Area (% sum)
1 4.30 Octane 2.1
2 7.96 2-Thujene; β-Thujene 6.5
3 8.24 α-Pinene 7.0
4 9.86 Bicyclo[3.1.1]heptane, 3.6
5 10.30 β-Pinene 9.7
6 10.94 α-Phellandrene 2.9
7 11.40 Terpinolene- 18.5
8 11.80 o-Cymene 25.6
9 11.88 D-Limonene 1.9
10 11.95 β-Phellandrene 1.1
11 17.67 Terpinen-4-ol 2.9
12 24.28 Carvacrol acetate, 2.9
13 25.97 Caryophyllene 4.5
14 26.55 Aromandendrene 1.1
15 30.88 Spatulenol; 1.0
16 31.04 Caryophyllene oxide 2.7
Fig. 1 a Total ion chromatogram of the GC‒MS analysis of S. hortensis essential oil, bMass spectrum of the compound 8, O-Cymene (RT:
11.80) in S. hortensis essential oil

876 Journal of Plant Pathology (2023) 105:869–885
1 3
MIC andMBC
The minimum bactericidal concentration (mg/ml medium)
and minimum inhibitory concentration (mg/ml medium) of
the essential oils were shown in Table5. All of the essen-
tial oils showed significant activity against E. amylovora.
Among them, S. hortensis essential oil revealed the highest
bacteriostatic (1.8mg/ml) and bactericidal (5.0mg/ml)
activities against E. amylovora. However, M. spicata essen-
tial oil displayed the minimum bacteriostatic (12.5mg/ml)
and bactericidal (15.2mg/ ml) activities. The results of the
study of Janaćković etal. (2022), conducted on the effective-
ness of Ambrosia essential oil against the bacterium Erwinia
showed that the rates of MIC and MBC were equal to 0.047
and 0.063, respectively (Janaćković etal. 2022), which was
better compared to the results of MIC (1.8) and MBC (5.0)
in the present study.
Disk diffusion
Organic acid compounds such as acetic acid and propionic
acid were effective growth inhibitors of E. amylovora, which
may be related to the uptake of free acid and its intracel-
lular accumulation (Konecki etal. 2013). Ten organic acid
salts (OASs) were evaluated at a concentration of 50–200
ppm for the inhibitory activity against E. amylovora in vitro
(Table5). Several salts significantly inhibited the growth
of E. amylovora. Calcium propionate, magnesium acetate,
potassium acetate, potassium citrate, and magnesium citrate
strongly inhibited the growth of E. amylovora. The other
Table 4 Inhibition zone diameter (mm) of four medicinal plants used
at different concentrations against Erwinia amylovora (ATCC49946)
Streptomycin at the concentration of 1µg/disk was used as the posi-
tive control (PC). Analyses were performed in triplicate
SEM Standard error of the mean
Inhibition zone diameter (mm)
Essential oils 500 (µg/disc) 1000 (µg/disc) Rank
Satureja hortensis 29.4 ± 0.350 39.3 ± 0.342 1
Trachyspermum ammi 19.8 ± 0.436 28.6 ± 0.309 2
Origanum majorana 16.5 ± 0.326 21.3 ± 0.505 3
Mentha spicata 11.5 ± 0.301 17.3 ± 0.345 4
Streptomycin 43.0 ± 0.490 45.4 ± 0.231 PC
SEM 0.22 0.20
Table 5 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the plant essential oils and the
organic acid salts against Erwinia amylovora (ATCC49946)
Analyses were performed in triplicate. S.E.M: Standard error of the mean. Streptomycin at the concentration of 1µg/disk was used as the posi-
tive control (PC)
Essential Oils Concentration (mg/ml))
MIC MBC Rank
Satureja hortensis 1.8 ± 0.625 5.0 ± 0.288 1
Trachyspermum ammi 3.7 ± 1.250 10.0 ± 0.110 2
Origanum majorana 5.9 ± 1.389 12.4 ± 0.152 3
Mentha spicata 12.5 ± 0.315 15.2 ± 0.250 4
Streptomycin 0.1 ± 0.015 0.14 ± 0.035 PC
SEM 0.51 0.08
Organic acid salts Concentration (mg/ml))
MIC MBC Rank
Calcium propionate 2.6 ± 0.012 33.3 ± 0.058 1
Magnesium acetate 5.2 ± 0.09 50 ± 0.036 2
Magnesium citrate 7.0 ± 0.047 50 ± 0.066 3
Potassium acetate 7.0 ± 0.023 50 ± 0.055 3
Potassium citrate 7.0 ± 0.078 50 ± 0.023 3
Calcium acetate 19.3 ± 0.07 33.3 ± 0.065 4
Calcium lactate 31.2 ± 0.08 33.3 ± 0.052 5
Potassium lactate 34.3 ± 0.025 50 ± 0.074 6
Magnesium lactate 34.3 ± 0.025 50 ± 0.087 6
Calcium citrate 43.7 ± 0.092 50 ± 0.052 7
Streptomycin 0.06 ± 0.077 0 + 0.3 ± 0.044 PC
SEM 9.21 4.35

877Journal of Plant Pathology (2023) 105:869–885
1 3
salts displayed only weak inhibitory effects. Streptomycin
at the concentration of 50 ppm was used as the positive con-
trol (Table5). The effectiveness of organic acid salts was
compared to streptomycin in Table5; The best effectiveness
compared to streptomycin belonged to the organic acid salt
of calcium propionate which showed the highest bacterio-
static (2.6mg/ml), and bactericidal (33mg/ml) activities
against E. amylovora. Conversely, calcium citrate showed
the minimum bacteriostatic (43.7mg/ml) and bactericidal
(50mg/ ml) activities.
As shown in Fig.2, S. hortensis (c) and M. spicata (d)
essential oils and calcium propionate (e) and calcium acetate
(f) salts possessed antibacterial activity against E. amylo-
vora. Distilled water (a) had no inhibition on the selected
bacteria (E. amylovora). The zone of inhibition of S. horten-
sis (c) and M. spicata (d) essential oils against E. amylovora
were larger than the zone of inhibition for calcium propion-
ate and calcium acetate. Streptomycin (b) had the highest
antibacterial activity against E. amylovora.
Microencapsulation efficiency andparticle size
ofthepowders
Table6 indicated the surface oil and total oil contents and
microencapsulation efficiency. Total and surface oil con-
tents are the main parameters influencing the encapsulation
efficiency (EE) of the encapsulated powders. Therefore,
the surface oil content of the samples was highly correlated
with encapsulation efficiency. Further, the experimental data
showed that the lower surface oil content caused the higher
encapsulation efficiency (Ding etal.2020). The maximum EE
(97.02%) (Exp. No: S2) was obtained for wall material compo-
sition containing 75% MD, 25% Hi-cap 100, and 3% Tween 20.
However, the minimum EE (85.49%) (Exp. No: S5) belonged
to the mixture of 0% MD, 100% Hi-cap 100, and 1% Tween 20.
Particle size distribution can be done using the values
of D10, D50, and D90 state that there are 10%, 50%, and 90%
of the total particle size distribution, respectively (Subroto
etal.2020). In other words, Table6 represented the cal-
culated mean, mode, and equivalent spherical diameter at
a cumulative percentage of 10% (D10), 50% (D50%), and
90% (D90). For instance, in S2 sample (Maltodextrin: Hi-
Cap. 75:25), 10% of the particles had sizes below 0.9μm,
50% of the particles were below 2.6μm, and 50% of the
particle sizes were above 2.6μm; Then, 90% of the parti-
cles had sizes above 97.9μm. The results of particle size
distribution analysis showed that the average diameter of
microencapsulated particles S2 (D90 = 96.23μm) was the
smallest size in the prepared samples (Table6).
The results ofobservation ofmicrocapsules
byoptical microscope
The morphology of EOs + OASs microcapsules was investi-
gated based on their optical micrograph (Fig.3). As can be
seen in Fig.3, the optical micrograph showed that most of
Fig. 2 Some images of the antibacterial screening of essential oils
and organic acid salts by means of the disc diffusion method (DDM).
a: control negative (distilled water). b: positive control (streptomycin.
500µg/disc). c: essential oil of S. hortensis (500µg/disc). d: essential
oil of M. spicata (500µg/disc). e: calcium propionate (4 mg/disc). f:
calcium acetate (4mg/disc)

878 Journal of Plant Pathology (2023) 105:869–885
1 3
the microcapsules were spherical with no visible aggregation.
According to the light microscope images, it seemed that the
higher the amount of modified starch (Hi-cap), the greater the
adhesion of the microcapsule particles to each other, which
was probably due to the nature of the substance Hi-cap (Lavelli
and Sereikaitė 2022). Also, the results showed that sample
S2 (the ratio of maltodextrin to Hi-cap = 75: 25) had the low-
est amount of particle adhesion and compaction, while the
particles were highly spreadable and the appearance of the
particles was smooth (Fig.3). While the highest amount of
wrinkling, adhesion, particle compression, and particle break-
age were observed in sample S5 (The ratio of maltodextrin to
Table 6 Influence of particle
size of the encapsulated
powders on encapsulation
efficiency during spray drying
and values of D10, D50, D90 of
typical treatments
Formulation MD:Hi-cap 100 Surface oil Total oil EE (%) Particle
size
(µm)
S1 0:100 0.0059 0.0458 87.11019/0 ± d4.5
S2 75:25 0.0022 0.0739 97.02023/0 ± a2.7
S3 50:50 0.0033 0.0473 93.02022/0 ± b5.26
S4 25:75 0.0055 0.0473 88.37019/0 ± c6.75
S5 0:100 0.0065 0.0448 85.49019/0 ± d7.55
D10 D50 D90
S1 3.8 80.7 313.5
S2 0.9 2.6 97.9
S3 0.8 1.9 617.3
S4 0.7 2.1 97.4
S5 0.6 1.8 122.3
Fig. 3 Optical microscope
(x100 magnification) of formu-
lation (S. hortensis and calcium
propionate) before spray drying.
The ratio of maltodextrin to
modified starch (Hi-Cap 100):
0:100 (S1), 75:25 (S2), 50:50
(S3), 25:75 (S4), and 0:100 (S5)

879Journal of Plant Pathology (2023) 105:869–885
1 3
Hi-cap = 0: 100). The images obtained from the optical micro-
scope of the present samples (S1-S5) were consistent with the
results of Lavelli and Sere.
Scanning electron microscopy (SEM)
SEM images are the best technique at which invisible worlds
of micro and nanoparticles can be seen (Proto etal. 2022).
Microstructures of the spray-dried powders produced by
combining different proportions of carrier agents showed
both rough and smooth surfaces as the usual character of
spray-dried powders, along with the sizes of the microparti-
cles (Bajac etal. 2022). SEM images play an important role
not only in confirming the particle size data obtained but
also in evaluating the characteristic morphological features
explaining the functional characteristics of the particles.
Fig. 4 Scanning electron micrographs of formulation (S. hortensis and calcium propionate) before spray- drying. The ratio of maltodextrin to
modified starch (Hi-Cap 100): 0:100 (S1), 75:25 (S2), 50:50 (S3), 25:75 (S4), and 0:100 (S5)

880 Journal of Plant Pathology (2023) 105:869–885
1 3
Having a polydisperse size is a characteristic feature of the
spray-dried particles, which is usually related to the material
used, formulation, as well as atomization process (Fig.4).
In the investigations carried out by scanning electron micro-
scope, all the produced microcapsules (in all five tested
treatments) showed spherical shapes and a smooth surface,
although some of them also had broken up or displayed
wrinkles. The average size of encapsulated powder parti-
cles in 5 treatments were between 2.77 and 7.5μm (Fig.4).
The observed morphology of the investigated treatments was
consistent with that reported by Ding etal. 2020.
Incidence andseverity
The main effect of S. hortensis EOs and calcium propionate
OAS (formulation) was significant on shoot blight severity
at the greenhouse conditions (Fig.3). Tagged shoots seed-
lings in the greenhouse were observed for disease response
7–17 days after inoculating. All of the treatments (T1, T2,
T3, and streptomycin) reduced the incidence. However, the
treatments increased the severity. Infectivity, which was
measured by the mean percentage of the total shoot length
affected by visible fire blight lesions (percent lesion length)
as well as by the percentage of infected shoots or seedlings,
was recorded when lesion extension ceased. The severity of
fire blight on pear shoots was determined by measuring the
length of shoot lesion/total length of shoot × 100 (Fig.5).
After two weeks from inoculation with E. amylovora,
the clear symptoms of the pathogen could be clearly seen
in the seedlings. None of the seedlings treated with for-
mulation (S. hortensis and calcium propionate) at lethal
concentrations became infected (in this experiment,
streptomycin with a concentration of 100 ppm was evalu-
ated as a positive control). Therefore, cross-infection was
neglected in subsequent analyses. The use of formulation
(calcium propionate and savory microencapsulation pow-
der) could offer an effective strategy for controlling the
fire blight related to their reducing effect on virulence
factors of E. amylovora. Disease progress was measured
after 14 days (Fig.6). Bar graph showed the average lesion
length resulting from inoculation trials with Erwinia
amylovora. One study in 2022 demonstrated that deletion
proQ (RNA-Binding Protein ProQ) caused E. amylovora
avirulence in an apple shoot (Yuan etal. 2022a, b).
Fig. 5 Effect of treatments
(T1, T2, T3, and streptomycin)
on incidence and severity of
the fire blight infections on
Spadona pear shoots inoculated
with E. amylovora; Infected:
just infected with suspension of
E. amylovora (5.3 × 108 CFU/
ml); T1: 1g/l. T2: 2g/l, and
T3: 3g/l of microcapsules (S.
hortensis and calcium propion-
ate); streptomycin concentration
is 100 ppm
63.75
36.25
27.5
16.25
10
37
64
73
82
92
0
20
40
60
80
100
120
infect T1 T2 T3 Streptomycin
percentage of incidence and severity
Treatments
Spadona pear shoots 2021-2022
Incidenceseverity
12.6
7.2
5.4
3.6
1.6
0
2
4
6
8
10
12
14
16
infecT1T2T3strep
average lesion length (cm)
treatments
Fig. 6 The shoots of Spadona pear were inoculated with bacterial
suspensions containing approximately 10^8 CFU ml–1. Vertical bars
represent the standard errors of the means

881Journal of Plant Pathology (2023) 105:869–885
1 3
Gene expression inpear shoots
We used the amsC gene as proxies for assessing the effects
of the formulation (Eos + OASs) in cells (E. amylovora) on
amylovoran biosynthesis gene expression. In E. amylovora,
amsC encoded amylovoran production (Salm and Geider
2004). In the last decade, quantifying E. amylovora by real-
time PCR is usually used for research purposes (Koczan
etal.2009; Dreo etal. 2012; Jin et al. 2022; Salm and
Geider 2004). Based on the results of the gene expression,
three treatments (1g/l, 2g/l, and 3g/l powder of micro-
capsules) were effective on the expression of amylorane
gene (amsC) compared to only inoculated (just infected
with E. amylovora, without apply treatments), streptomycin
and control (seedling no infected with E. amylovora and
without apply any treatments). All three treatments 1 (red
color), 2 (blue color), and 3 (yellow color) reduced the
expression of amylovoran gene (amsC) in shoots of pear
cv. Spadona after treating (green color) with streptomycin
(positive control) in Fig.7. Further, among the treatments,
the third treatment (T3) caused the greatest decrease in
amylovoran gene expression than only inoculated and con-
trol (Fig.7). Analysis of REST 2009 software (fold change)
showed that the reduction of gene expression by treatment
three (3g/l) was twice more than treatment 2 (2g/l). T3
(3g/l) caused a 2-fold decrease in the expression of the
amylovoran gene compared to the treatment of 2g/l (T2).
Also, T2 (2g/l) caused a 2-fold decrease in the expres-
sion of the amylovoran gene compared to T1 (1g/L). Yuan
etal. in 2022 showed that deletion Pro Q (a cytoplasmic
Fig. 7 (up) Relative fold change in amsC expression. Different
effects of the treatments 1–3 and streptomycin (positive control) on
the relative expression of amylovoran gene (amsC) on the shoots of
the susceptible cultivar, Spadona pear, after E. amylovora infec-
tion; T1 = 1 g/l, T2 = 2 g/l, and T3 = 3 g/l of microcapsule powder,
S = Streptomycin (100 ppm); only inoculated; E. amylovora (5.3 × 108
CFU/ml); control: no treated with T1, T2, T3, S and no infected with
E. amylovora solution (5.3 × 108 CFU/ml). The relative expression
software tool was used (REST 2009), (bottom) Photographs showing
the fire blight severity in terms of shoot blight

882 Journal of Plant Pathology (2023) 105:869–885
1 3
protein with RNA chaperone) decreased the amylovoran
(a heteropolymer with glucose, galactose, and pyruvate)
production in E. amylovora. The decrease in amylovoran
gene expression by the formulation (calcium propionate
and savory essential oil) was in great statistical significance
with Yuan etal. 2022a, bresearch.
Discussion
E. amylovora is considered a major risk to the pear trees in
the world (Yuan etal. 2022a, b). However, a large number
of studies indicated that chemical methods such as copper
compounds, antibiotics, etc. not only cause environmental
problems but also cannot control fire blight. It seems that
in addition to positive effects on the environment, increas-
ing the inductive resistance of plants, and food health, the
use of effective plant compounds (phenol, flavonoid, tan-
nin, alkaloid, etc.) causes a relative inhibition of fire blight
(Bereswill etal. 1995; Elyemni etal. 2019; Bahadou etal.
2018; Dreo etal. 2012; Gwinn 2018; Mikiciński etal. 2012;
Janaćkoviće etal. 2022). This study aims to evaluate the
effect of microcapsules containing the plant essential oil
(Savory) and organic acid salt (Calcium propionate) on
shoots in two-year-old pear (Spadona cultivar) infected with
pear fire blight.
Hervieux etal. (2002) evaluated 11 inorganic and 12
organic salts at a concentration of 0.2M for identifying
the inhibitory activity against H. solani invitro in one test
(Hervieux etal. 2002). Several salts significantly inhibited
the mycelial growth and spore germination of H. solani.
Aluminum salts, phosphate salts, potassium sorbate, sodium
benzoate, sodium bicarbonate, sodium carbonate, sodium
citrate, and sodium metabisulfite completely or almost
completely inhibited the spore germination. However, the
other salts displayed only weak inhibitory effects (Hervieux
etal. 2002). The present study developed the study of
Hervieux etal. (2002) and used organic salts as part of
the final formulation for combating the fire blight disease.
On the other hand, based on the study of Valadabadi and
Yousefi (2022), screening the dead bacteria was examined
using essential oil (Valadabadi etal. 2022). Therefore,
savory essential oil formed the second part of the nucleus
of microcapsules in the present study. The yield of essential
oil (S. hortensis) was determined 1.8% (V/W), which was
consistent with the results of the study of Valadabadi and
Yousefi (1.93%). Based on the results of GC/MS analysis,
37 compounds existed in the savory essential oil, the major
components of which was recorded o-Cymene (25.6%)
and Terpinolene (18.5%). The results were consistent with
those of obtained by Chambre etal. (2020) indicating that
γ-terpinene (42.30%), carvacrol (32.83%), and p-cymene
(8.05%) were the major components in savory essential
oil. Moreover, Mohammadhosseiniet al. (2016) reported
that γ-terpinene, carvacrol, and p-cymene were the major
compounds of Iranian S. hortensis essential oils (Chambre
etal. 2020; Mohammadhosseini etal. 2016).
The inlet temperature of the spray-dryer and the type and
ratios of carrier materials could control the morphology and
microstructures of the powders (Veiga etal. 2019). Increas-
ing the MD concentration from 25 to 75% resulted in the
production of more spherical powders (Fig.2). Such results
were reported in one study, where maltodextrin made spheri-
cal and smooth powder particles due to its sugar units with a
low-molecular weight and high flexibility (Tomazelli Júnior
etal. 2018b).
Aćimović etal. (2015) reported that shoot blight inci-
dence was best controlled by injecting SS (streptomycin)
and then acibenzolar-S-methyl (ASM) and PH (potassium
phosphites), both of which induced the resistance in leaves.
At the first date of disease rating, SS and PH in 2012 and
all the injected compounds in 2013 showed better fire blight
suppression on shoots than on flowers, which was consistent
with the results of this study (Aćimović etal. 2015).
Comparing the relative quantification of E. amylovora
concentration in a sample with the standard curve provided
additional information on the probability of its isolation in
pure culture and the possibility of confirming the results
with other less sensitive methods. Quantification can only be
accurate if the target copy number is constant and does not
vary among the isolates when the concentration is normal-
ized to cell or CFU number (De Bellis etal. 2007). A fFur-
ther consideration of the accuracy of quantification causes
the similarity of PCR amplification efficiencies between the
samples and the standard curve.
Conclusion
Amylovoran production was significantly impaired when E.
amylovora came in contact with different treatments (EO/
OAS) leading to a halt in amylovoran production. Based
on the results, the effect of EO/OAS on E. amylovora indi-
cated that the phenolic compounds and organic acid salts in
the phytotoxic firstly changed the membrane permeability
allowing different molecules to permeate inside the cells.
Infection of young shoots is the main source of dissemina-
tion of fire blight disease in spring. In fact, controlling fire
blight was mainly obtained using effective plant compounds
for blooms for suppressing the pathogen infection. Spray-
ing the pear shoots and leaves indicated that the phytotoxic
(microcapsules of EO/OAS) could reduce the disease devel-
opment. The results suggested that amylovoran production
inhibitors can be useful in controlling fire blight disease. It
seemed that the antimicrobial activity of the microcapsules
(EO/OAS) against E. amylovora was promising.

883Journal of Plant Pathology (2023) 105:869–885
1 3
Acknowledgements We thank Agricultural Biotechnology Research
Institute of Iran (Karaj), Ferdowsi University of Mashhad and Research
Institute of Food Science and Technology (Mashhad) for the material
and spiritual support in conducting this research.
Data availability Datasets related to this article will be available on
request.
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