Evaluation of efficacy of four Cinnamomum species extracts and cinnamaldehyde to control anthracnose of mango fruit

Abstract

Anthracnose of mango is one of the major postharvest diseases of mango fruit caused by members of the Colletotrichum gloeosporioides species complex such as Colletotrichum siamense. Crude extracts from dry trunk bark of four Cinnamomum species (C. burmanni, C. iners, C. loureiroi, and C. verum), a commercial cinnamon powder, cinnamaldehyde, eugenol, and cinnamon oil were assayed for their antifungal activity against Colletotrichum siamense. The crude extract of C. verum at 500 mg L−1 showed the highest inhibition of mycelial growth. At a concentration above 10 g L−1 cinnamaldehyde, eugenol, and cinnamon oil showed 100% mycelial inhibition. Using the microdilution assay, C. burmanni and C. verum crude extracts were effective against Colletotrichum siamense spore germination and showed a minimum inhibitory concentration (MIC) value of 625 mg L−1 while the MIC value of cinnamaldehyde was 50 mg L−1. The direct bioautography of the C. verum extract and the fractions obtained by column chromatography over silica gel against Cladosporium herbarum revealed clear inhibition zones on TLC plates. The treatment of Colletotrichum siamense spores with this active fraction led to severe membrane damage which was observed by scanning electron microscopy. Comparative HPLC analyses of the Cinnamomum extracts and the active fraction of C. verum, cinnamon power, and the cinnamaldehyde and eugenol as standards indicated cinnamaldehyde as the major compound. The C. verum fraction reduced disease severity and disease incidence on inoculated mango fruit. Moreover, uninoculated mango dipped into C. burmanni and C. verum extracts reduced the naturally occurring disease while total soluble solid, titratable acidity, and weight loss of dipped mango were insignificantly different from the untreated fruit control.

Full text

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1 3
Eur J Plant Pathol
https://doi.org/10.1007/s10658-024-02897-y
Evaluation ofefficacy offour Cinnamomum species extracts
andcinnamaldehyde tocontrol anthracnose ofmango fruit
WiphaweeLeesutthiphonchai· OnumaPiasai· SrunyaVajrodaya·
SarinnaUmrung· JohannSchinnerl · SiegridSteinkellner ·
NetnapisKhewkhom
Accepted: 5 June 2024
© Koninklijke Nederlandse Planteziektenkundige Vereniging 2024
Abstract Anthracnose of mango is one of the major
postharvest diseases of mango fruit caused by mem-
bers of the Colletotrichum gloeosporioides species
complex such as Colletotrichum siamense. Crude
extracts from dry trunk bark of four Cinnamomum
species (C. burmanni, C. iners, C. loureiroi, and C.
verum), a commercial cinnamon powder, cinnamal-
dehyde, eugenol, and cinnamon oil were assayed for
their antifungal activity against Colletotrichum sia-
mense. The crude extract of C. verum at 500mg L−1
showed the highest inhibition of mycelial growth.
At a concentration above 10g L−1 cinnamaldehyde,
eugenol, and cinnamon oil showed 100% mycelial
inhibition. Using the microdilution assay, C. bur-
manni and C. verum crude extracts were effective
against Colletotrichum siamense spore germination
and showed a minimum inhibitory concentration
(MIC) value of 625mg L−1 while the MIC value of
cinnamaldehyde was 50 mg L−1. The direct bioau-
tography of the C. verum extract and the fractions
obtained by column chromatography over silica gel
against Cladosporium herbarum revealed clear inhi-
bition zones on TLC plates. The treatment of Colle-
totrichum siamense spores with this active fraction
led to severe membrane damage which was observed
by scanning electron microscopy. Comparative HPLC
analyses of the Cinnamomum extracts and the active
fraction of C. verum, cinnamon power, and the cin-
namaldehyde and eugenol as standards indicated cin-
namaldehyde as the major compound. The C. verum
fraction reduced disease severity and disease inci-
dence on inoculated mango fruit. Moreover, uninocu-
lated mango dipped into C. burmanni and C. verum
extracts reduced the naturally occurring disease while
total soluble solid, titratable acidity, and weight loss
of dipped mango were insignificantly different from
the untreated fruit control.
Keywords Anthracnose· Cinnamaldehyde·
Cinnamon· Colletotrichum siamense· Plant extract
Supplementary Information The online version
contains supplementary material available at https:// doi.
org/ 10. 1007/ s10658- 024- 02897-y.
W.Leesutthiphonchai· O.Piasai· S.Umrung·
N.Khewkhom(*)
Department ofPlant Pathology, Faculty ofAgriculture,
Kasetsart University, Bangkok, Thailand
e-mail: agrnpk@ku.ac.th
S.Vajrodaya
Department ofBotany, Faculty ofScience, Kasetsart
University, Bangkok, Thailand
J.Schinnerl
Department ofBotany andBiodiversity Research,
University ofVienna, Rennweg 14, 1030Vienna, Austria
S.Steinkellner
Department ofCrop Sciences, Institute ofPlant Protection,
University ofNatural Resources andLife Sciences,
Vienna, Austria

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Introduction
Natural compounds from plants, such as plant
extracts and essential oils, are potential alternatives
to synthetic chemical agents against plant pathogens.
The compounds are considered to be relatively safe
to use, environmentally friendly and biodegradable
(Tzortzakis & Economakis, 2007). Essential oil
obtained from the bark of Cinnamomum verum
J. Presl (synonym: C. zeylanicum Blume or Sri
Lankan cinnamon) (Lauraceae) contains three
main constituents including trans-cinnamaldehyde,
eugenol, and linalool. The major constituent of the
cinnamon bark oil distillate is trans-cinnamaldehyde
(Kowalska etal., 2021). Cinnamaldehyde (synonyms:
cinnamal, cinnamic aldehyde, phenylacrolein) is
on the list of additive foods classified as A (COE
No.102) and GRAS (21CFR 182.60) by the Food and
Drug Administration and as acceptable by FEMA
(Flavor and Extract Manufacturers’ Association)
and JECFA (Joint FAO-WHO Expert Committee on
Food Additives) (Hall & Oser, 1965; JECFA, 2000).
Apart from C. verum, other widespread Cinnamomum
species including C. burmanni (Nees & T.Nees)
Blume (Indonesian cinnamon) and C. loureiroi Nees
(Vietnamese cinnamon) are also natural sources
of cinnamaldehyde (Chen et al., 2014). Cinnamon
oil showed very powerful activities including
antimicrobial properties (Chericoni et al., 2005; He
et al., 2018; Shahina et al., 2022; Sukorini et al.,
2013; Wu etal., 2017).
Anthracnose, caused by Colletotrichum spp., is a
common postharvest disease that affects the shelf-life,
fruit quality, and marketability of many fruits, such
as mango (Ploetz, 2003). It is one of the most impor-
tant diseases of mango and causes major economic
losses. Colletotrichum can infect immature mango
and remain latent (Ploetz, 2003; Tovar-Pedraza etal.,
2020). Damages become visible as black and sunken
lesions postharvest. Eight species in the Colletotri-
chum gloeosporioides species complex have been
reported to cause anthracnose of mango including C.
asianum, C. fructicola, C. gloeosporioides, C. gros-
sum, C. queenslandicum, C. siamense, C. theobro-
micola, and C. tropicale (Li etal., 2019; Lima etal.,
2013; Manzano León et al., 2018; Mo et al., 2018;
Rattanakreetakul et al., 2023; Shivas et al., 2016;
Tovar-Pedraza etal., 2020; Weir etal., 2012).
Various fungicides are used worldwide to combat
anthracnose. Although pesticides are important to
ensure high agricultural productivity to feed the ever-
growing global human population, there is concern
about their hazardous side effects on human health,
their contribution to environmental pollution, and the
development of fungicide-resistant strains. Cinnamon
has been shown to affect many fungal stages
including vegetative structures, spores, the growth,
and the germination. Studies showed, among others,
a reduction in the fungal growth of Colletotrichum
gloeosporioides in avocado, banana, and rambutan
(Kyu Kyu Win et al., 2007; Regnier et al., 2010;
Sivakumar etal., 2002; Sivakumar & Bautista-Banos,
2014). In addition, the essential oils of C. zeylanicum
and Syzygium aromaticum (L.) Merr. & L.M.Perry
strongly inhibited the conidial germination and
mycelial growth of Colletotrichum gloeosporioides
isolates from papaya (Barrera-Necha etal., 2008). The
lipophilic crude extract of C. loureiroi inhibited the
spore germination of Colletotrichum gloeosporioides,
the causal agent of anthracnose on ‘Nam Dok Mai’
mango in Thailand (Khewkhom etal., 2009). Basil and
cinnamon oils were effective against Colletotrichum
acutatum causing anthracnose in Cat Hoa Loc mango,
a popular variety in Vietnam (Danh et al., 2021).
The underlying mechanisms are based on targeting
membrane integrity and permeability, leakage of
membrane and cell components, reactive oxygen
species, protease activity, gene expression, and sugar
content (Carmello et al., 2022; He et al., 2018; Lee
etal., 2020; Shahina etal., 2022; Zhang etal., 2021).
Although plants are rich with phytochemical com-
positions and bioactive compounds, different parts
and species of plants often contain different quanti-
ties of such compounds. Despite previous studies of
cinnamon, a comparison of antifungal activity among
four different cinnamon species against anthracnose
disease of mango is still lacking. Therefore, the aim
of this work was to identify and determine the anti-
fungal properties of the bark of C. burmanni, C. iners
(Reinw. ex Nees & T.Nees) Blume, C. loureiroi, C.
verum, cinnamaldehyde, and eugenol against Colle-
totrichum siamense.

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Materials andmethods
Fungal isolation and identification of Colletotrichum
siamense
The fungal pathogen Colletotrichum sp. was iso-
lated from ten anthracnose symptomatic fruits of
‘Nam Dok Mai’ ripe mango which was purchased
from three different wholesale fresh markets in
Bangkok, Thailand and stored at 25°C to investi-
gate the disease. Small area of peels (5 mm2) cut
from the edges of lesions were surface-sterilized
with 1% sodium hypochlorite for 3min, rinsed with
sterile distilled water, and dried with sterilized tis-
sue paper. Sterilized peels were plated on potato
dextrose agar (PDA) plates and incubated at 25°C.
The fastest growing isolate was purified by single
spore and maintained on potato dextrose agar (PDA;
Merck, Germany) at 28°C (Sivakumar etal., 2002).
Morphological characteristics of the Colletotri-
chum gloeosporioides species complex were used
to preliminary identify the isolates (Dugan, 2006;
Sutton, 1980). Multilocus analysis was performed
for Colletotrichum species identification. Five-day
culture was harvested and DNA extracted (Zelaya-
Molina et al., 2011). The PCR were performed
using PCRBIO Taq Mix (PCR Biosystems) and
primers listed in Table S1 to amplify the internal
transcribed spacer (ITS) (White etal., 1990), Glyc-
eraldehyde-3-phosphate dehydrogenase (GAPDH)
(Templeton etal., 1992), Actin (ACT) (Carbone &
Kohn, 1999), and Calmodulin (CAL) (Weir et al.,
2012) genes of isolates. DNA sequences of each
region were searched in the NCBI database using
the nucleotide BLAST. Phylogenetic multilocus
analysis was conducted using sequences from this
study and sequences in the NCBI database and the
accession numbers are given in TableS2. Multiple
sequences of ITS, GAPDH, ACT and CAL were
aligned using MUSCLE (Edgar, 2004) and concat-
enated in SeaView version 5.05 (Gouy etal., 2010).
The alignment was evaluated using the TCS web
server (Chang etal., 2015). Phylogenetic trees were
generated using PhyML (Guindon etal., 2010) and
Parsimony with 1000 bootstrap replicates. Bayes-
ian phylogenetic analysis was performed in BEAST
version 2.6.6 (Suchard etal., 2018).
Preparation of the fungal spore suspension
A spore suspension was prepared using an isolate of
Colletotrichum siamense grown on PDA under fluores-
cent light at room temperature on 9cm diameter Petri
dishes for ten to twelve days. A solution (20mL) of ster-
ile 0.9% NaCl (w/v) containing 5% dimethyl sulfoxide
(DMSO, v/v) was added to each plate, and the surface
was gently scraped with a sterile Drigalsky spatula to
release the spores. The resulting spore suspension was
filtered through a sterile two-layer filter paper (190mm
diameter, 11μm pore size) to remove any mycelial frag-
ments and centrifuged at room temperature for 5min to
allow the spores to settle. The supernatant was then care-
fully removed. The spores were re-suspended in 0.9%
NaCl solution (w/v) containing 5% DMSO and counted
using a hemocytometer (Boeco, Germany). The suspen-
sion was adjusted to a density of 1 × 105 spores mL−1.
Preparation of crude extracts from four Cinnamomum
species
Dry bark samples of C. burmanni, C. iners, C.
loureiroi, and C. verum were purchased from a tra-
ditional Thai pharmacy (Chao Krom Poh, Bangkok,
Thailand). The barks differed visually very clearly:
C. burmanni had a thick yellowish-brown bark, C.
iners had a grey-brown bark, C. loureiroi had a thick
grey-brown bark and C. verum showed a thin dark
red-brown bark (Fig.1). The dried and ground plant
material (500 g) of each sample was extracted with
methanol p.a. in the dark at room temperature for
72h. For the fruit dipping experiments, dried mate-
rial was extracted in ethanol. Cinnamon powder (Cey-
lon Zimt; Alnatura Produktionsund Handels GmbH,
Darmstadt, Germany) was extracted under the same
conditions. The extracts were placed in an ultrasonic
bath for 15min, then passed through a cellulose fil-
ter, and evaporated to dryness at 40°C under reduced
pressure. The dry extracts of the different Cinnamo-
mum species were suspended in water and this solu-
tion was repeatedly extracted with chloroform to sep-
arate the lipophilic compounds from the hydrophilic
plant constituents. The resulting chloroform phases
from each plant extract were pooled and the solvent
was evaporated to dryness. This step yielded 28.82g
of lipophilic crude extract from C. burmanni, 2.46g
from C. iners, 13.45g from C. loureiroi and 5.52g

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from C. verum. For the antifungal screening, stock
solutions of the lipophilic extracts were dissolved
in 20 mL of acetone (≥ 99.5%, Sigma-Aldrich) and
added to the cooled down PDA to achieve a final con-
centration of 500mg L−1.
Screening of antifungal activities against
Colletotrichum siamense
The antifungal activities of Cinnamomum spp.
crude extracts, cinnamon powder (Ceylon Zimt;
Alnatura Produktions und Handels GmbH), cinna-
maldehyde (≥ 95%, Sigma-Aldrich), eugenol (99%,
Sigma-Aldrich), and cinnamon oil (nature identi-
cal, Sigma-Aldrich) were evaluated using Colletotri-
chum siamense cultured on agar plates. Cinnamo-
mum burmanni, C. iners, C. loureiroi, and C. verum
crude extracts dissolved in methanol p.a. were each
mixed in with sterilized, slightly cooled down PDA
and adjusted to a final concentration of 500mg L−1.
Commercial cinnamon powder was prepared simi-
larly at concentrations of 500, 1000, 5000, 10,000,
20,000, and 30,000 mg L−1. Pure cinnamaldehyde,
eugenol, and cinnamon oil were prepared in concen-
trations of 50, 100, 500, 1000, 10,000, 20,000, and
30,000mg L−1. Pure media and PDA with methanol
(500mg L−1) served as negative controls and media
with the fungicide prochloraz (N-propyl-N-[2-(2,4,6-
trichlorophenoxy) ethyl] imidazole-1-carboxamide)
at 50 and 500mg L−1 were used as positive controls.
After thorough mixing, 15 mL of each medium was
poured into sterilized Petri dishes (9 cm in diam-
eter). After solidification of the prepared media,
mycelial plugs of 0.5mm in diameter were cut with
a cork borer from the growing margin of agar plates
containing Colletotrichum siamense and placed at the
center of the test plate. Eight replications were made
for each treatment and the cultures were incubated
at room temperature under 12h light and 12h dark
cycle. The diameter of the developed colonies was
measured after seven days incubation and the inhibi-
tion of mycelial growth was calculated according to
Kyu Kyu Win etal. (2007).
Microdilution assay
The evaluation of the effect of C. burmanni, C. iners,
C. loureiroi and C. verum crude extracts, cinnamal-
dehyde and cinnamon powder on Colletotrichum
siamense was performed according to Hadacek and
Greger (2000) using a standardized 96-well microtiter
plate test. The substances were dissolved and diluted
to a range of 1–2500 mg L−1 in acetone p.a., 1.7%
malt broth, and Tween80. The solution without cin-
namon preparations served as a control. Each dose
was repeated four times. The 96-well microtiter plates
(flat bottom clear polystyrene wells; Greiner, Krems-
muenster, Austria) were incubated at room tempera-
ture. Fungal growth was evaluated at 24, 48, and 72h
following method M27-A of the National Committee
for Clinical Laboratory Standards (1997), and the
minimum inhibitory concentration (MIC), the lowest
concentration of compound that completely inhib-
its spore germination, was monitored using a ste-
reoscopic microscope with 15× magnification. After
72 h incubation, the samples in wells at the MIC
value were further plated on PDA and incubated for
5days at 28°C to assure an inhibition.
For the spore germination inhibition, the experi-
ment was conducted as same as microdilution assay
Fig. 1 Bark of the four
Cinnamomum species

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but the spore suspension was incubated with C. bur-
manni and C. verum crude extracts, cinnamaldehyde,
and prochloraz for 24h. A control was distilled water.
The germination of spores was observed under a
compound microscope (Carl Zeiss, Jena, Germany)
with 400× magnification. A spore considered as ger-
minated when the the length of germ tube was more
than half of the width (Veloso etal., 2021).
Direct bioautography of Cinnamomum extracts
Bioautography was used to detect the active com-
pounds of crude extracts and the eluted fractions.
The C. verum crude extracts were separated using
open column chromatography and analyzed by thin
layer chromatography (TLC). The active compounds
were dissolved in MeOH and applied to TLC plates
(qualitative analysis: Merck no. 1.05715.001, F254,
0.25 mm and preparative separation: silica gel 60
Merck no. 1.05744, F254, 0.5 mm) using a dispos-
able glass micropipette for each sample. Plates were
developed in the solvent system n-hexane:diethyl
ether (Et2O) 3:2 and antifungal activity was recorded
directly on the developed TLC plate under a CAMAG
UV lamp at wavelengths of 254 and 366nm. The sil-
ica gel plates were sprayed with anisaldehyde reagent
(Sigma-Aldrich, Vienna, Austria) or a spore suspen-
sion with a final concentration 1 × 105 conidia mL−1 in
1.7% malt extract broth. Cladosporium herbarum was
used for this experiment because the spores are much
easier to visualize due to their dark color. Inoculated
plates were then placed in a 24 × 24 × 1.5 cm humid
chamber to monitor the effect of each compound on
fungal development after three days at 25 °C in the
dark. Distinct zones of fungal growth inhibition indi-
cated the presence of antifungal compounds.
Chromatographic separation and HPLC analyses
Open column chromatography was used for the
basic pre-fractionation of the crude extracts based
on the different polarities of the compounds. The
crude extract (20 mg) was subjected to a column
(length 70 cm, diameter 2 cm) filled with silica gel
60 (0.2–0.5mm) and eluted with organic solvent mix-
tures (100 mL each) consisting of n-hexane, ethyl
acetate (EtOAc), and methanol (MeOH) with increas-
ing polarities. The size of the collected fractions was
50mL. After evaporation of the solvent, the masses
of the fractions were weighed using an analytical bal-
ance and further analyzed by TLC and HPLC.
The following components were used in the TLC:
for preparative separations, silica gel 60 (Merck no.
1.05744), F254, plate (0.5mm thickness); for qualita-
tive analysis, silica gel 60 (Merck no. 1.05715.001),
F254, plates (0.25 mm thickness); all plates were
developed in n-hexane:diethylether (Et2O) 3:2. From
the bioautography, the fraction VI, which was one of
the fractions showing the clear zones, was subjected
to preparative TLC. The clear zone at the position
1 of fraction VI was scraped off the TLC plate and
dissolved in acetone. The solution was evaporated,
weighted, and stored in methanol at −20°C for fur-
ther analysis. This purification of fraction VI at the
position 1 was designated as VI1 and further used
for the mango infection assay and the treatment of
Colletotrichum spores observed by a scanning elec-
tron microscopy (SEM). SEM was performed at the
Kasetsart Agricultural and Agro-Industrial Product
Improvement Institute (KAPI), Kasetsart University,
Bangkok, Thailand. The samples were prepared as
described by Homrahud etal. (2016).
HPLC analyses were performed on an Agilent
1100 series with UV-diode array detection using a
Hypersil BDS-C18 (250 × 4.6 mm, 5 μm particle
size) column at a flow rate of 1.0mL min−1 and an
injection volume of 10 μL. A buffer consisting of
1.5mM tetrabutyl ammonium hydroxide and 15mM
o-phosphoric acid at a flow rate of 1mL min−1 (A)
and methanol (B) were used as eluents. The following
gradient was used: from 20 to 100% B in A within
20min, and 100% B was kept for 8min. The wave-
length of detection was set at 230nm. Cinnamomum
extracts and cinnamon powder were identified by
comparative HPLC using a commercial cinnamal-
dehyde (Sigma-Aldrich) and a commercial eugenol
(Sigma-Aldrich) as authentic standards.
Mango inoculation
Mangoes of ‘Nam Dok Mai’ cultivar were used to
evaluate disease incidence and disease severity of
Colletotrichum siamense after an application of C.
verum fraction VI1, which was previously obtained by
preparative TLC. Mangoes were purchased from the
wholesale fresh market, gently wiped in 70% ethanol
followed by sterile water, and stored at room tem-
perature. The fungal spore suspension was prepared

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as described above, adjusted to 1 × 105 spores mL−1,
50 μL were dropped onto sterile a Whatman fil-
ter paper disc (5 mm diameter), and then placed on
a healthy mango fruit for 5hours. Fraction VI1, 5%
methanol or distilled water was then applied on the
inoculation area in a volume of 100μL. Six mango
fruits were used per treatment and the experiment was
performed in triplicate using completely randomized
design (CRD). After 5–6 days incubation at room
temperature in the dark, the number of diseased man-
goes was counted. Disease incidence (DI) was calcu-
lated using the following formulas: DI (%) = Number
of disease fruit / Total number of fruits × 100. Man-
goes were divided into 6 groups for evaluation: free
of infestation, 1-5%, >5%, >10%, >20%, and > 40%.
Numbers of fruit in each category were recorded.
Disease severity (DS) was calculated using the fol-
lowing formula: DS (%) = Lesion area of mango fruit
in each treatment / Lesion area of mango fruit in con-
trol (fruits treated with distilled water) treatment ×
100.
Non-inoculated mango dipping in C. Burmanni, C.
verum extracts, and cinnamaldehyde
Seventy-two non-inoculated mangoes of ‘Nam
Dok Mai’ cultivar in green mature stage were used
to monitor the effect of C. burmanni extract, C.
verum extract, and cinnamaldehyde. The experi-
ment was conducted using CRD of 4 treatments and
3 replicates. After surface disinfection of the fruits
with ethanol and sterile water, they were dipped in
10,000 mg L−1of C. burmanni ethanolic extract, C.
verum ethanolic extract, or cinnamaldehyde. For the
dipping solution, the extracts were dissolved in dis-
tilled water. An untreated group was included as a
control. The fruits were incubated at room tempera-
ture until ripening approximately 5–6 days. Then,
the affected area and the total area of the mango
were measured. Disease severity was calculated as
described above.
Fruit quality analysis
Seventy-two mangoes were used to determine
whether dipping of fruits in the crude extracts
affect the fruit quality. Using CRD, the experi-
ment was performed with 4 treatments and 3 repli-
cates of 6 mangoes and included C. burmanni, C.
verum, or cinnamaldehyde, each at concentration of
10,000 mg L−1, and a non-treated control. Mangoes
were incubated at a room temperature for 3, 6, and
9days. Fruit qualities were assessed including weight
loss, total soluble solid (TSS) (%Brix) using a Brix
refractometer (Atago master-a alpha 0-32%), and
titratable acidity (TA) by titration with 0.1M NaOH
to an endpoint using 2 drops of 1%phenolphthalein
indicator (Sadler & Murphy, 2003). Percent TA was
calculated by volume of NaOH × concentration of
NaOH × equivalent weight acid × 100 / volume of
sample. Equivalent weight of predominant acid in the
sample was 0.064 for citric acid.
Statistical analysis
The experiments were conducted in completely ran-
domized design. Analysis of variance (ANOVA) and
Posthoc Test (Tukey HSD) were performed using a
web-based one-way ANOVA with Tukey HSD (Vasa-
vada, 2016) and one-way ANOVA Calculator and
Tukey HSD online platform (Statistics Kingdom,
2017).
Results
Identification of Colletotrichum siamense
Colonies of isolates showed generally dense myce-
lia with white to grayish color. The average myce-
lial growth at 25°C was approximately 10mm daily.
Conidia were hyaline, unicellular, cylindrical shape
with rounded ends and an inconspicuous hilum. The
length and the width of conidia ranged from 10.31 to
16.50μm and 3.19 to 5.52μm, respectively. Salmon
to orange colored spore masses were formed in mid-
dle of colony after incubated for 4–5days. Morpho-
logical features were similar to Colletotrichum gloe-
osporioides species complex (Dugan, 2006; Sutton,
1980; Weir etal., 2012).
The multilocus analysis of ITS, GAPDH, ACT,
and CAL revealed that the fungal isolate was closely
related to Colletotrichum siamense. The compari-
son of DNA sequences of each region to the NCBI
database showed 99.82% similarity with the ITS
from C. siamense, C. gloeosporioides, C. tropicale,
100% similarity with the GADPH gene from C. sia-
mense, 99.29% similarity with the ACT gene from C.

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siamense, and 99.86% similarity with the CAL gene
from C. siamense. Phylogenetic analysis of the Colle-
totrichum gloeosporioides complex species showed
that the Collectotrichum in this study clustered with
C. siamense (Fig.2).
Inhibition growth assay
The daily mycelial growth rate of Colletotrichum
siamense was 7.6mm in the control and 4.5 mm in
the C. verum crude extracts. The growth inhibition
relative to the control is shown in Fig.3. The crude
extracts of C. verum and C. burmanni at 500mg L−1
showed the highest growth inhibition at about 41%
compared to the control. Cinnamon powder at the
same concentration did not inhibit the growth of
Colletotrichum siamense. Cinnamaldehyde, euge-
nol, and cinnamon oil at 500 mg L−1 showed about
6–11% inhibition compared to the control. Levels of
1000mg L−1 cinnamaldehyde, 10,000mg L−1 euge-
nol, and 10,000mg L−1 cinnamon oil strongly inhib-
ited the growth of Colletotrichum siamense. Prochlo-
raz, which was used as a positive control, resulted in
the highest inhibition of Colletotrichum siamense.
Methanol, which was used as solvent for the four
crude extracts, inhibited fungal growth up to 3.3%.
Determination of the MIC values
The MIC values at 24, 48, and 72 h incubations
were 625 mg L−1 for C. burmanni, and C. verum,
1250 mg L−1 for C. iners and C. loureiroi, and
50mg L−1 for cinnamaldehyde (Table1). After 72 h
incubation, the samples with concentrations identi-
cal to the MIC value were plated on PDA to ensure
Colletotrichum siamense did not grow. The MIC val-
ues of cinnamon powder and control were higher than
2500mg L−1 so cinnamon powder did not inhibit the
growth of Colletotrichum siamense and were there-
fore not plated on PDA. The MIC of prochloraz as
positive control was 22.5mg L−1.
Inhibition of spore germination
The percent inhibition of Colletotrichum siamense
conidial spores treated with C. burmanni and C.
verum crude extracts, cinnamaldehyde, and prochlo-
raz at MIC concentrations and control is shown
Fig. 2 Phylogenetic tree
of Colletotrichum in this
study and Colletotrichum
gloeosporioides species
complex as well as Colle-
totrichum boninense as the
outgroup. Maximum-like-
lihood tree was calculated
based on concatenated
nucleotide sequences of
ITS, GADPH, ACT and
CAL genes. Numbers at
nodes show bootstrap val-
ues higher than 70% from
maximum-likelihood and
maximum parsimony and
Bayesian posterior probabil-
ity values higher than 0.95

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in Fig. 4a. Cinnamomum verum extract showed
86.50% inhibition of spore germination whilst
C. burmanni crude extract, cinnamaldehyde, and
prochloraz showed 100% inhibition. The nega-
tive control displayed 0% inhibition and all conidia
germinated and developed long germ tubes at 24h
incubation. By contrast, the C. verum extract only
contained few germinating conidia with germ tubes
reduced in length (Fig.4b). After these spores with
C. verum treatment were plated on PDA, no growth
could be observed.
Bioautography on TLC plates
Bioautography of the lipophilic extracts from C.
verum showed a clear inhibition zone of antifungal
compounds after spraying with a conidial suspen-
sion of Cladosporium herbarum (Fig. 5a). Spores
of C. herbarum had dark color so it was feasible to
observe the clear zone of antifungal compounds.
The compounds separated on TLC plates were also
visualized after spraying with anisaldehyde rea-
gent (Fig.5b). The fractions with a clear zone were
eluted with n-hexane: EtOAc (25:75) (fraction VI),
EtOAc:MeOH (95:5) (fraction VIII), EtOAc:MeOH
(90:10) (fraction IX), and EtOAc:MeOH (50:50)
(fraction XI). The bioautography of fraction VI of C.
verum that was eluted with 25:75 n-hexane: EtOAc
exhibited inhibition zones at retention factors of 0.79,
0.14, and 0.03 (Fig.5c).
Cinnamomum fraction treatment on inoculated
mango
The C. verum fraction VI1 from the preparative
TLC dropping on inoculated mango fruit signifi-
cantly reduced the disease incidence and the dis-
ease severity compared with water control. The
fraction VI1 showed about 19% disease incidence
Fig. 3 Percent growth inhibition of four Cinnamomum crude
extracts, cinnamon powder, cinnamaldehyde, eugenol, cinna-
mon oil, prochloraz served as positive control, methanol, and
pure PDA media as the negative control against Colletotrichum
siamense. The numbers under x–axis indicates the concentra-
tions in mg L−1 unit. The growth was measured at day 7 after
inoculation. Different lowercase letters represent significant
differences according to Tukey HSD (P < 0.05) and error bars
indicate ± standard deviation
Table 1 Minimum inhibitory concentration (MIC) of four
Cinnamomum crude extracts, cinnamaldehyde, and cinnamon
powder against Colletotrichum siamense spore germination at
24, 48, and 72h incubation time
Tested samples MIC (mg L−1)
C. burmanni 625
C. iners 1250
C. loureiroi 1250
C. verum 625
Cinnamaldehyde 50
Cinnamon powder >2500
Prochloraz 22.5
Control (acetone p.a. + malt broth +
Tween80)
>2500

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similar to the methanol treatment which was
approximately 25% disease incidence (Fig. 6). In
contrast, the disease incidence of the water con-
trol was almost 70%. The disease severity of the
fraction VI1 treatment showed 8 and 11% of fruits
with the severity scales of more than 20% and 40%,
respectively, and it was significantly different to
that of the water control which showed 11 and 58%
of fruits from the same severity scales; however, it
was similar to the severity of methanol treatment.
Effect of the Cinnamomum fraction on
Colletotrichum spores
The effect of the C. verum fraction VI1 on the
Colletotrichum siamense spores was observed
under a scanning electron microscope. The cell
wall of these spores treated with the fraction VI1
was not smooth and deformed while the cell wall
of Colletotrichum siamense spores was smooth and
intact (Fig.7).
Cinnamomum treatment on non-inoculated mango
fruit
Mango fruit dipped in C. burmanni and C. verum
extracts showed less naturally occurring disease than
the fruit dipped in cinnamaldehyde or non-treated
control (Fig.8). Thirty-seven percent and 30% of non-
inoculated fruit dipped in C. burmanni and C. verum
extracts, respectively, showed no symptoms, and
14% and 21% of fruit from both treatments showed
only mild symptoms (1–5% disease severity). Most
cinnamaldehyde treated fruit were sorted into 1–5%
and > 40% disease categories. Non-treated mango
showed the highest disease severity while prochloraz
displayed the lowest severity and 83% of dipped fruit
did not exhibit symptoms.
Quality of mango fruit after treatment with
Cinnamomum extracts and cinnamaldehyde
Fruit qualities including weight loss, TSS, and TA
of mango dipped in 10,000mg L−1 C. burmanni, C.
Fig. 4 (a) Percent inhibition of conidial germination treated
with Cinnamomum crude extracts (625 mg L−1), cinnamalde-
hyde (50mg L−1), prochloraz (22.5mg L−1) and distilled water
control. (b) non-germinated conidial spores in different treat-
ments and germinated conidia in control at 24 h incubation.
Scale bars represent 20μm

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verum, or cinnamaldehyde were similar to the non-
treated mango control (Table2). The weight loss of
all four treatments did not differ. Percent weight loss
at day 3 for each treatment was in the range from 5.31
to 7.10% and the loss was elevated on day 6, ranging
from 10.09 to 17.63%. Likewise, TSS and TA of four
treatments were similar. TSS of mango at day 0 was
13.67°Brix and TSS on day 3, 6, and 9 was in a range
of 16.67–20.13°Brix. TA at day 0 was 0.35% and TA
at day 3–6 was in a range of 0.12–0.23%. The acidity
Fig. 5 Thin layer chromatography plates of Cinnamomum
verum crude extracts separated in an open column chroma-
tography using a 3:2 n-hexane:diethylether solvent system and
sprayed with (a) a conidial suspension of Cladosporium her-
barum or (b) anisaldehyde reagent. (c) Bioautography test of
open column fraction VI eluted from n-hexane:ethyl acetate
(25:75) from methanolic extract of C. verum showing inhi-
bition zones at positions 1, 2, and 3 with retention factors of
0.79, 0.14, and 0.03, respectively
Fig. 6 Disease incidence (%) and disease severity (%) on
‘Nam Dok Mai’ mango fruit inoculated with Colletotrichum
siamense spores after treatment with methanol, destilled water
or C. verum fraction (VI1), respectively. Different letters to
the right of the bars denote significant differences according
to Tukey HSD at P < 0.05 and error bars indicate a standard
deviation

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was reduced to 0.03–0.04% on day 9 and similar in all
treatments.
Comparative HPLC analyses
The crude Cinnamomum extracts, along with the
commercially available cinnamon powder, and the
authentic standard cinnamaldehyde, and eugenol,
were analyzed using HPLC-PDA. Comparison of
retention and UV-spectra indicated the presence of
cinnamaldehyde in all plant extracts although the rel-
ative amounts in the extract of C. iners was clearly
lower (Fig.9). The other standard eugenol exhibited
a distinctly different retention time and could not be
identified in the extracts.
Discussion
Plant extracts with fungicidal and fungistatic activ-
ity could be suitable alternatives to synthetic fungi-
cides to control anthracnose in mango fruit. One of
advantages of extracts obtained from plants or plant
organs used as spices or vegetable is that they can be
used in the food industry without further approval
as they are categorized as “Generally Recognized
as Safe” by the U.S. Food and Drug Administra-
tion (Wen et al., 2016). The effectiveness of plant
extracts and other herbal products, including essential
oils, in treating other diseases has been successfully
demonstrated (Maqbool et al., 2010; Mohammadi
et al., 2016; Ranasinghe et al., 2002; Sivakumar &
Fig. 7 Scanning electron
microscope of Colletotri-
chum siamense spores
treated with (a) fraction VI1
of C. verum and (b) control
Fig. 8 Percent of ‘Nam Dok Mai’ mango fruit dipped in
10,000mg L−1 C. burmanni extract, 10,000 mg L−1 C. verum
extract, 10,000 mg L−1 cinnamaldehyde, non-treated control
or prochloraz at day 6 showing no disease and disease sever-
ity scales of 1–5%, >5%, >10%, >20%, and > 40%. Different
letters in the no disease bars show significant differences based
on Tukey HSD at P < 0.05

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Bautista-Banos, 2014). The results of our work show
that the four tested Cinnamomum crude extracts have
antifungal activity against Colletotrichum siamense,
indicating a potential as an alternative natural fungi-
cide to manage anthracnose. Extracts of C. burmanni
and C. verum at a concentration of 10,000 mg L−1,
when applied as a dip to mangoes, release a faint
smell for only 1day after treatment, as the volatile
compounds are easily released. After 6days of treat-
ment, the mango fruit ripens, and its smell and taste
return to normal. Addressing the growth inhibition
assay, Cinnamomum crude extracts inhibited the
growth of Colletotrichum siamense up to 41%. Cinna-
mon powder only showed a similar effect at a higher
concentration. It is possible that the cinnamon pow-
der is not only made from C. verum, but also mixed
with other Cinnamomum species such as Cinnamo-
mum cassia J. Presl and therefore has a lower effect.
Colletotrichum siamense was identified by both
morphological features and multilocus analysis. Mor-
phology or molecular identification using only ITS
would be deficient to identify Colletotrichum sia-
mense since it is morphologically indistinguishable to
Colletotrichum gloeosporioides species complex and
its ITS is almost identical within the species complex
(Weir etal., 2012). Colletotrichum siamense, which
causes anthracnose of mango in this study was con-
firmed by Koch postulates. The pathogen was re-iso-
lated from the inoculated mango fruit. Colletotrichum
siamense is one of the most common Colletotrichum
species which has been reported to be associated with
anthracnose of mango (Li et al., 2019; Rattanakree-
takul etal., 2023; Tovar-Pedraza etal., 2020).
Cinnamomum crude extracts inhibited the spore
germination of Colletotrichum siamense about
86.50–100%. Only few spores from C. verum treat-
ment germinated with relatively shorter germ tubes
after 24 h incubation while all spores in the con-
trol formed long germ tubes. Spores treated with C.
verum and subsequently plated on PDA did not grow.
It is known that Colletotrichum spores develop germ
tube within 3–6 hours (Mehta et al., 2021; Soares
etal., 2021) therefore, if after 24h, they were still not
developed, it can conclusively be reported that the
germination of the spores was inhibited by the crude
extract.
In our growth inhibition assay, the spore germina-
tion inhibition, and scanning electron microscopy, we
found that the extracts inhibit the growth and affect
Table 2 Weight loss, total soluble solids, and titratable acidity of mango fruit treated with C. burmanni, C. verum, cinnamaldehyde, or non-treated control
Numbers after ± are standard deviation. Different letters in columns of each parameter indicate significant differences according to Tukey HSD (P < 0.05)
Treatment on mango
fruit
Weight loss (%) Total soluble solid
(°Brix)
Titratable acidity
(%)
Day3 Day 6 Day 0 Day3 Day 6 Day9 Day 0 Day3 Day 6 Day9
Cinnamomum bur-
manni 7.10 ± 5.48 ab 17.63 ± 11.87 b 13.67 ± 0.76 a 19.00 ± 0.87 b 19.13 ± 0.76 b 19.07 ± 0.95 b 0.35 ± 0.02 a 0.22 ± 0.04 b 0.14 ± 0.03 b 0.03 ± 0.01 c
Cinnamomum verum 5.31 ± 13.03 a 10.09 ± 5.59 ab 16.67 ± 2.52 b 17.40 ± 0.87 b 18.27 ± 1.55 b 0.21 ± 0.04 b 0.12 ± 0.04 b 0.03 ± 0.01 c
Cinnamaldehyde 6.78 ± 7.27 ab 12.02 ± 6.70 ab 18.00 ± 1.11 b 20.13 ± 1.47 b 18.00 ± 0.80 b 0.21 ± 0.02 b 0.19 ± 0.05 b 0.04 ± 0.00 c
Non-treated control 5.79 ± 11.04 ab 12.75 ± 9.80 ab 19.20 ± 0.53 b 18.27 ± 1.10 b 19.00 ± 1.73 b 0.14 ± 0.02 b 0.23 ± 0.07 b 0.03 ± 0.00 c

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the spore germination of Colletotrichum siamense.
Our work showed deformed cell wall of the Colle-
totrichum siamense spores after treatment with the
cinnamaldehyde fraction obtained from the C. verum
extract. Similarly, damaged hyphae and spores were
reported in previous studies (He etal., 2018; Kumar
& Kudachikar, 2020; Wang et al., 2023). Moreover,
Wang et al. (2023) showed an insight mechanism
of cinnamon and clove essential oils towards the
damage of cell membrane integrity and organelles
of Colletotrichum gloeosporioides using electron
microscopes and RNA-seq. Interestingly, trans-cin-
namaldehyde was predicted to target the lanosterol
14α-demethylase (CYP51) in the ergosterol synthesis
of Colletotrichum gloeosporioides. Furthermore, cin-
namaldehyde damaging the membrane integrity and
permeability has been reported in other fungi (OuY-
ang etal., 2019; Shahina etal., 2018). OuYang etal.
(2019) exhibited that cinnamaldehyde altered the
cell wall permeability of Geotrichum citri-aurantii
by increasing the extracellular alkaline phosphatase
activity. Therefore, cinnamaldehyde is incorporated
during waxing process of citrus fruit to control the
postharvest diseases (Duan et al., 2021). Moreover,
Shahina et al. (2018) showed that essential oil from
Cinnamomum zeylanicum damages the cell walls
of Candida albicans by affecting the chitin con-
tent, which is a key component of fungal cell walls.
Fig. 9 HPLC chromato-
grams of Cinnamomum
burmanni, C. iners, C.
loureiroi, C. verum, cin-
namon powder, cinna-
maldehyde, and eugenol.
Wavelength of detection
was set at 230nm

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Cinnamon extract treatment also reduced the spore
production and leads to deformed hyphae of Fusar-
ium oxysporum f. sp. lycopersici and Alternaria alter-
nata (Carmello etal., 2022). Whether the mechanism
of trans-cinnamaldehyde towards Colletotrichum sia-
mense is similar to Colletotrichum gloeosporioides
remains open to be explored.
Frequently mentioned antifungal compounds in
Cinnamomun extracts are cinnamaldehyde and euge-
nol (Chericoni et al., 2005; Kowalska etal., 2021).
Cinnamaldehyde has been shown to be effective
against fungi, such as Botryodiplodia theobromae and
Gliocephalotrichum microchlamydosporum, which
cause a postharvest diseases, stem-end rot and brown
spot of rambutan (Sivakumar et al., 2002). Eugenol
is known to have strong antifungal activity against
Geotrichum citri-aurantii causing sour rot in citrus
(Wu etal., 2017). The performed HPLC analyses con-
firm both substances, although only small amounts of
eugenol were detectable. The analyzed extracts of the
Cinnamomum species exhibited some clear differ-
ences in the HPLC profiles, for example, the extract
of C. iners contained relatively low quantities of cin-
namaldehyde compared to the other analyzed Cin-
namomum species. However, our data is in line with
Chen et al. (2014) and Kowalska et al. (2021) who
also found that different Cinnamomum species and
different plant parts vary in the cinnamaldehyde con-
tent. It is challenging to compare the efficacy of cin-
namon against anthracnose to previous publications
as many studies have investigated cinnamon essen-
tial oils (Danh etal., 2021; He etal., 2018; Sarkhosh
etal., 2018; Wang etal., 2023) or cinnamon extracts
(Carmello et al., 2022; Kyu Kyu Win et al., 2007).
However, one certain discovery is that cinnamalde-
hyde is one of major components in both essential
oil and extracts. He et al. (2018) studied cinnamon
essential oil which had 86.16% trans-cinnamaldehyde
and Danh etal. (2021) used the essential oil contain-
ing 43.3% trans-cinnamaldehyde. Nonetheless, in this
study, high concentration (10,000mg L−1) of cinna-
maldehyde caused damaging effects on the surface of
mango fruits after dipping.
The similarity of mango fruit quality indicates
among the treatment with Cinnamomum extracts and
non-treatment on uninoculated mango assures the
postharvest application on mango. In our study, the
weight loss, TSS, and TA of treated and non-treated
mango were not different statistically. TSS was
similar from day 3 to day 9 while TA decreased over
the period. These factors which refer to sugar and
acid contents can be used to predict the quality and
sourness of mango (Malundo etal., 2001).
To conclude this work, the current results, together
with previous reports, indicate high antifungal capac-
ities of cinnamon extract and cinnamaldehyde. Com-
pared to commercial fungicides, specialized plant
metabolites are environmentally beneficial because of
their more rapid detoxification by naturally occurring
microorganisms.
Acknowledgements The authors thank the Agriculture
Research Development Agency (Public Organization), the Thai
Research Fund, an ASEA-UNINET scholarship, and the Office
of the Higher Education Commission of Thailand for funding
and the Kasetsart University Research and Development Insti-
tute (KURDI), Bangkok, Thailand for writing assistance.
Data availability The data generated or analysed in
this study are included in this published article and its
supplementaryinformation.
Declarations
Ethics approval The authors declare that ethical standards
have been followed and that no human participants or animals
were involved in this research.
Consent to participate Not applicable.
Consent for publication Consent for publication was
obtained from all co-authors.
Conflict of interest There are no conflicts of interest to declare.
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