Antifungal Activity of Propyl Disulfide from Neem - MDPI

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Article

Antifungal Activity of Propyl Disulfide from Neem(Azadirachta indica) in Vapor and Agar Diffusion Assaysagainst Anthracnose Pathogens (Colletotrichum gloeosporioidesand Colletotrichum acutatum) in Mango Fruit

Muhammad Rafiullah Khan 123 Vanee Chonhenchob 23 Chongxing Huang 1 and Panitee Suwanamornlert 4

Citation Khan MR

Chonhenchob V Huang C

Suwanamornlert P Antifungal

Activity of Propyl Disulfide from

Neem (Azadirachta indica) in Vapor

and Agar Diffusion Assays against

Anthracnose Pathogens

(Colletotrichum gloeosporioides and

Colletotrichum acutatum) in Mango

Fruit Microorganisms 2021 9 839

httpsdoiorg103390

microorganisms9040839

Academic Editor Gary A Strobel

Received 14 March 2021

Accepted 13 April 2021

Published 14 April 2021

Publisherrsquos Note MDPI stays neutral

with regard to jurisdictional claims in

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iations

Copyright copy 2021 by the authors

Licensee MDPI Basel Switzerland

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https

creativecommonsorglicensesby

40)

1 School of Light Industry and Food Engineering Guangxi University Nanning 530004 Chinakhan87gxueducn

2 Department of Packaging and Materials Technology Kasetsart University Bangkok 10900 Thailand3 Center for Advanced Studies for Agriculture and Food KU Institute for Advanced Studies

Kasetsart University (CASAF NRU-KU Thailand) Bangkok 10900 Thailand4 College of Integrative Medicine Dhurakij Pundit University Bangkok 10210 Thailand paniteetipdpuacth Correspondence vaneeckuacth (VC) huangcx21163com (CH)

Abstract Microorganisms causing anthracnose diseases have a medium to a high level of resistanceto the existing fungicides This study aimed to investigate neem plant extract (propyl disulfide PD) asan alternative to the current fungicides against mangorsquos anthracnose Microorganisms were isolatedfrom decayed mango and identified as Colletotrichum gloeosporioides and Colletotrichum acutatum Nexta pathogenicity test was conducted and after fulfilling Kochrsquos postulates fungi were reisolated fromthese symptomatic fruits and we thus obtained pure cultures Then different concentrations of PDwere used against these fungi in vapor and agar diffusion assays Ethanol and distilled water wereserved as control treatments PD significantly (p le 005) inhibited more of the mycelial growth ofthese fungi than both controls The antifungal activity of PD increased with increasing concentrationsThe vapor diffusion assay was more effective in inhibiting the mycelial growth of these fungi thanthe agar diffusion assay A good fit (R2 0950) of the experimental data in the Gompertz growthmodel and a significant difference in the model parameters ie lag phase (λ) stationary phase (A)and mycelial growth rate further showed the antifungal efficacy of PD Therefore PD could be thebest antimicrobial compound against a wide range of microorganisms

Keywords natural plant extract antimicrobial microorganisms neem propyl disulfide mangopostharvest decay anthracnose

1 Introduction

Mango (Mangifera indica L) is an important tropical fruit due to its favorable flavorrich nutrition and high marketing value However this fruit is highly susceptible tovarious pathogens leading to quality deterioration and significant economic losses Avariety of Colletotrichum spp are responsible for causing anthracnose in various fruitswith a causal agent of anthracnose in mango being Colletotrichum gloeosporioides Pathogensinfect immature mango fruit as a latent infection and the lesions progressively appear afterstorage and ripening [12]

Anthracnose can be controlled to a great extent by the applications of syntheticfungicides such as benomyl carbendazim thiabendazole prochloraz dithiocarbamate andazoles Nonetheless numerous adverse effects are associated with synthetic fungicidessuch as toxicity environmental pollution and potential risks to human health [2ndash5] Thesenegative effects have increased consumer awareness of food safety Furthermore thedevelopment of middle to high levels of resistance of pathogens to the existing fungicideshas compelled the researchers to find other alternatives

Microorganisms 2021 9 839 httpsdoiorg103390microorganisms9040839 httpswwwmdpicomjournalmicroorganisms

Microorganisms 2021 9 839 2 of 13

Recently natural plant extracts have become of great interest to researchers due to theirvolatile nature aroma antioxidant and antimicrobial properties [67] Further plant diseasemanagement with plant essential oils has been recognized as one of the best approaches [8]A considerable number of essential oils exhibited antimicrobial activities against differentpathogens [910] Plant extracts such as aroma compounds (trans-cinnamaldehyde citraland phenylacetaldehyde [11] Mentha piperita L essential oil (MPEO) [12] volatile organiccompounds [2] essential oil from Cymbopogon citratus (DC ex Nees) Stapf [13] and es-sential oil extracted from Cympopogon citratus and Lippia rehmannii [14] have suppressedanthracnose in mango fruit Feng et al [3] evaluated the antifungal activity of camp-tothecin (a naturally occurring quinoline alkaloid having a high pesticidal activity) againstC gloeosporioides causing anthracnose disease in mango Dessalegn et al [15] evaluatedthe plant defense-inducing chemicals (PDIC) combined with postharvest treatments withinorganic salts and hot water to prevent mangorsquos anthracnose

Neem plant (Azadirachta indica) is a rich repository of more than 300 primary andsecondary metabolites and is a powerful natural pesticide [16] Many bioactive compoundshave been isolated from neem plants and have antifungal antibacterial antioxidant andother biological properties [17ndash19] Thus far neem extracts are primarily used in pharma-ceutical or traditional medicines few studies are reported against postharvest pathogensFor example the extract of neem leaf was used to inhibit the growth of Botrytis cinereaAspergillus flavus and Aspergillus niger fungi causing anthracnose in wild mango [20] andAspergillus viridae Penicillium digitatum and Rhizopus sp causing anthracnose in tomato [21]

Propyl disulfide is one of the active compounds in neem seed and is a potent pesticidein grain storage Recently we worked on the antifungal activity of propyl disulfide onmango fruits against Lasiodiplodia theobromae and Neofusicoccum parvum causing the stem-end rot in mango fruit Propyl disulfide was much more effective in controlling the growthof these pathogens [22] Interestingly when the Gompertz growth model was appliedthe data were a good fit in the model and propyl disulfide had a positive effect on thestationary phase and lag phase of the microbial growth [22]

As anthracnose is one of the major diseases in postharvested fruits we continuedour study to work on propyl disulfide from neem against the major pathogens causinganthracnose in mango with the aim to find a potential leading compound for fungicidedevelopment A pathogenicity test was conducted to confirm the major pathogens ofmango causing anthracnose Furthermore a growth model was also applied to see theaccuracy and good fit of the experimental and theoretical results

2 Materials and Methods21 Plant Materials and Chemicals

Propyl disulfide (purity ge 97 FG oil phase) (Figure 1) a volatile antifungal com-pound of neem extract (Azadirachta indica) was obtained from Sigma-Aldrich (St Louis MOUSA) Ripe mangoes (Mangifera indica) brought from the local market were stored in thelaboratory at ambient temperature for decay symptoms as shown in Figure 2

Figure 1 Structure of propyl disulfide

22 Isolation and Identification of Fungi

Fungi were isolated and identified according to the method of Khan et al [22] Brieflyfresh and healthy mangoes were washed with clean tap water to remove any soil debris thesurface sterilized with NaOCl (1) and stored at ambient temperature for fungal growthSmall pieces of active lesions (triplicate) from decayed mango (Figure 2) were separatedwith the help of a sterile blade and washed with NaOCl (1) for 3 min rinsed with steriledistilled water dried and transferred to Petri dishes containing potato dextrose agar (PDA)

(one piece per plate) The Petri dishes were sealed with Parafilm and incubated for 7 days atambient temperature (26 plusmn 3 C) Upon the mycelial growth a small colony was reisolatedand transferred to a new Petri dishes incubated and checked visually and morphologicallyunder the stereo and compound microscopes This process was repeated several timesto obtain pure cultures and based on the colony and conidial features these fungi wereidentified as C gloeosporioides and C acutatum the causal microbes of anthracnose

Figure 2 C acutatum and C gloeosporioides isolated from the decayed mangoes and their respectivecolonies and conidia Bar = 10 microm

23 Pathogenicity Test

A pathogenicity test was conducted to establish Kochrsquos postulates following theprotocols of Khan et al [22] and Wanjiku et al [23] Fresh and healthy mangoes werewashed with clean tap water to remove any soil debris the surface sterilized with NaOCl(1) and kept in trays A sterile cork borer (1 cm diameter) was used to wound the fruitand mycelial disc obtained from the edge of actively growing pure cultures (7 days oldobtained in Section 22) was placed on the wound Trays were covered with plastic filmmoisturized and stored at room temperature of 26 plusmn 3 C Finally after the pathogenicitytest reisolation from the symptomatic fruit was carried out the fungal colonies and conidialfeatures were compared to the original isolates (Figure 2) Actively growing pure cultureswere transferred to PDA slants screw test tubes (plugged with cotton) or in Petri dishes(wrapped with parafilm to reduce drying) containing agar medium After the establishmentof culture these were stored in room temperature 26 plusmn 3 C or refrigerated temperature of4 C by following the protocol of Karabıccedilak et al (2016) [24] as stock cultures and werechecked periodically for contamination and desiccation Before each experiment fungiwere grown in fresh PDA and incubated at room temperature

24 Antifungal Activity of Propyl Disulfide

A two-fold serial dilution method was used to prepare different concentrations bydissolving 25 50 100 200 400 and 800 g Lminus1 of PD in absolute ethanol (AR grade MerckDarmstadt Germany) Antifungal activity of the PD was conducted in both vapor andagar diffusion assays [2225]

For the vapor phase assay a sterile filter paper (47 mm diameter) was fixed withthe Petri dishrsquos inside lid Then a 5 mm mycelium plug taken from actively grown pureculture was positioned upside down in the center of a 90 mm Petri dish containing PDAFrom each concentration of PD 50 microL was placed on the attached sterile filter paper Filterpaper with ethanol and sterile distilled water were used as control treatments Petri disheswere immediately sealed with Parafilm to prevent vapor loss and all plates were stored atambient temperature 26 plusmn 3 C until mycelial growth reached the margin of the Petri dishin control treatments Each concentration was conducted in five replicates

Similarly 50 microL from each concentration was used in agar diffusion assay here thePD was mixed with PDA in Petri dish After solidification a 5 mm of mycelium plug takenfrom actively grown pure culture was kept upside down in each Petri dish and sealedimmediately with Parafilm Ethanol and sterile distilled water were incorporated in PDAas control treatments All the plates were incubated at ambient temperature 26 plusmn 3 C untilthe mycelia reached to the edge of Petri dish in control treatments Each treatment wasconducted in five replicates The radial mycelial growth was measured (in mm) with aVernier caliper in two perpendiculars and the mean diameter was obtained every 24 h

A minimum inhibitory concentration (MIC) was defined as the lowest concentrationof the antifungal compound that prevents visible mycelial growth of the fungi In thisstudy MIC of PD was taken into account with respect to its different concentrations againstthe fungal radial growth (mm) and compared to distilled water and ethanol following theprotocol of Khan et al [22] and Suwanamornlert et al [25] Percent growth inhibition (PI) ofeach concentration of the propyl disulfide was calculated with the following formula [322]

PI =[(C minus I)minus (T minus I)

(C minus I)times 100 (1)

where C = mycelial growth diameter of the control I = initial mycelial plug diameter(5 mm) and T = mycelial growth diameter of the pathogen exposed to propyl disulfide ateach concentration

25 Gompertz Model for Fungal Growth

Gompertz growth model was applied to find the mango fungal growth model and itsparameters [2226]

ln(DtD0) = A expminusexp

[(Vm e

A

)(λ minus t) + 1

](2)

where Dt (mm) the average colony diameter at time t D0 (mm) average colony diameterat the initial time A maximum colony diameter at stationary phase Vm maximum growthrate (1time) λ the lag phase (time) and e = exp (1) The model parameters were measuredby regression analysis using Microsoft Excel The value of mean square error (MSE) and thecoefficient of determination (R2) were used to determine the goodness-of-fit of the model

26 Statistical Analysis

Data were subjected to analysis of variance with Duncanrsquos new multiple range test at asignificance level of p le 005 using SPSS version 16 software (SPSS Inc Chicago IL USA)

3 Results31 Anthracnose Rot and Characteristics of Isolated Fungi

The symptoms of anthracnose caused by Colletotrichum spp were dark brown andblack lesions (Figure 2) The cultural and morphological investigations revealed thatC acutatum and C gloeosporioides were causing anthracnose disease in mango fruit Thesefungi were differentiated by their colony colors growth rates the presence or absence of ateleomorph and conidial morphologies Mycelium of C acutatum were white to light greyand orange growth was relatively slow and reached the edge of Petri dish in about 14 daysConidia of C acutatum were fusiform and cylindrical with a narrow end On the otherhand the mycelium of C gloeosporioides was whitish to orange becoming deep orange ordark gray with time having a regular or an irregular margin with both submerged andaerial topography The mycelial growth rate of C gloeosporioides was relatively fast andreached the edge of the Petri dish in about 10 days C gloeosporioides mostly producedhyaline one-celled ovoid to oblong slightly curved or dumbbell-shaped conidia withthickheaded and round end with well developed hyaline conidiophores (Figure 2)

In both vapor and agar diffusion assays the antifungal activity of PD increased with itsconcentration In vapor phase assay both controls and PD (25 g Lminus1) were nonsignificantlydifferent while at 50 g Lminus1 and above PD significantly inhibited the mycelium growthof C acutatum Among the control treatments ethanol was significantly more effectivethan distilled water (Figure 3A) Regarding the C gloeosporioides both controls and PD(25 g Lminus1 and 50 g Lminus1) were nonsignificantly different while at 100 g Lminus1 and above PDsignificantly (p le 005) inhibited the mycelium growth of C gloeosporioides (Figure 3B)

Figure 3 Effects of different concentrations of propyl disulfide in the vapor phase assay on themycelial growth of C acutatum at day 14 (A) and C gloeosporioides at day 10 (B) Different small lettersin each concentration represent a significant difference among the treatments The mycelial growthpresented is the mean of five replicates plusmn SD at p le 005

Similarly in the agar diffusion assay PD was significantly effective than both controlsat all concentrations in C acutatum and 100 g Lminus1 and above in the case of C gloeosporioidesAgain in both tested fungi the antifungal efficacy of PD increased with increasing itsconcentrations (Figure 4AB) However in agar diffusion assay no significant differencewas found in distilled water and ethanol in C acutatum and C gloeosporioides (Figure 4AB)

Figure 4 Effects of different concentrations of propyl disulfide in the agar diffusion assay on themycelial growth of C acutatum at day 14 (A) and C gloeosporioides at day 10 (B) Different small lettersin each concentration represent a significant difference among the treatments The mycelial growthpresented is the mean of five replicates plusmn SD at p le 005

Interestingly in the vapor phase assay the percent growth inhibition of PD was higher(p le 005) for C gloeosporioides than C acutatum (Table 1) while in the agar diffusion assaythe percent growth inhibition of PD was significantly higher (p le 005) for C acutatum thanC gloeosporioides (Table 2) The minimum inhibitory concentration of PD was higher than800 g Lminus1 in both fungi However both fungi responded differently when exposed to thedifferent concentrations of PD in both the vapor and agar diffusion assays (Tables 1 and 2)

Table 1 Percentage of mycelial growth inhibition () of major pathogens isolated from mangoexposed to various concentrations of propyl disulfide in vapor phase compared to control stored atambient temperature (26 plusmn 3 C)

Concentrations(g Lminus1)

C acutatum(Day 14)

C gloeosporioides(Day 10)

800 2923 aB (088) 4387 aA (170)400 2341 bB (289) 3353 bA (175)200 1934 cB (206) 2893 cA (193)100 1796 cA (078) 1773 dA (055)50 1174 dB (254) 1551 dA (170)25 526 eA (200) 663 eA (239)

MIC gt80 gt80Different small letters within the column and capital letters within the row represent a significant differencebetween different concentrations and within fungi at each concentration respectively Means are separated by theDMRT test (p le 005) Each point is the average of five replicates standard deviation (SD) is given in parenthesesMIC = minimum inhibitory concentration

Table 2 Mycelial growth inhibition () of major pathogens isolated from mango exposed to variousconcentrations of propyl disulfide incorporated in the potato dextrose agar (PDA) medium (agardiffusion assay) at ambient temperature (26 plusmn 3 C)

C acutatum(Day 14)

800 3519 aA (284) 3393 aA (110)400 2515 bA (107) 1843 bB (213)200 1993 cA (150) 1136 cB (218)100 933 dA (109) 695 dB (173)50 864 dA (171) 344 eB (115)25 715 dA (266) 295 eB (082)

MIC gt80 gt80Different small letters within the column and capital letters within the row represent a significant differencebetween different concentrations and within fungi at each concentration respectively Means are separated by theDMRT test (p lt 005) Each point is the average of five replicates standard deviation (SD) is given in parenthesesMIC = minimum inhibitory concentration

Figures 5 and 6 show the growth curves of C acutatum and C gloeosporioides against thedistilled water ethanol and PD while their model parameters (A Vm λ) are summarizedin Tables 3 and 4 for the vapor and agar diffusion assays respectively The model R2095 showed a good fit of the experimental data to the model equation A decrease inthe mycelial growth during the stationary phase (A) indicated a higher antifungal PDactivity than distilled water and ethanol In the vapor phase assay the maximum growthrate (Vm) was significantly (p le 005) different in all treatments for both C acutatum andC gloeosporioides (Table 3) whereas in agar diffusion assay the maximum growth rate(Vm) was significantly (p le 005) different in PD than distilled water and ethanol for bothC acutatum and C gloeosporioides (Table 4) Propyl disulfide also significantly (p le 005)enhanced the lag phase (λ) of both fungi from 132 to 153 d as compared to 089 to 099 din ethanol and 088 to 096 d in distilled water in the vapor and agar diffusion assaysrespectively (Tables 3 and 4)

Figure 5 Effect of PD in the vapor diffusion assay on the growth of C acutatum (A) and C gloeospo-rioides (B) The mycelial growth presented is the mean of five replicates plusmn SD at p le 005 Dt isthe average colony diameter at time t and D0 is the average colony diameter at initial time fitted(distilled water ethanol and propyl disulfide) with the modified Gompertz model

Figure 6 Effect of PD in the agar diffusion assay on the growth of C acutatum (A) and C gloeosporioides(B) The mycelial growth presented is the mean of five replicates plusmn SD at p le 005 Dt is the averagecolony diameter at time t and D0 is the average colony diameter at initial time fitted (distilled waterethanol and propyl disulfide) with the modified Gompertz model

Table 3 Modified Gompertz model parameters for vapor diffusion assay

AntifungalAgents C acutatum C gloeosporioides

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

Distilled water 279 a

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

Propyl disulfide 247 c

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

Different superscript letters within each column are significantly different (p le 005) The standard deviation (SD) is given in parenthesesbelow the average

Table 4 Modified Gompertz model parameters for agar diffusion assay

A(mm)

vm(1d)

λ(d) MSE R2 A (mm) vm

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

Propyl disulfide 240 b

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

Anthracnose caused by Collectrotrichium spp is a major postharvest disease of man-goes that results in substantial economic losses In addition in light of the developmentof medium to high levels of resistance of these pathogens to the existing fungicides thepresent work is the first report on the anthracnose rot while using an active volatile com-pound (PD) from neem to develop a potential new fungicide Colletotrichum spp infectsthe mango fruit by producing appressoria from germinating spores that penetrate into thesurface of the fruits The fungus usually attacks the fruit in the early stages and typicallyremains dormant until the fruit ripens Upon ripening dark spots develop enlargingto form lesions that may amalgamate and cover almost the fruitrsquos entire surface [2] Inthis study pathogens were identified based on their cultural morphological and conidialstructures such as shape size and colony color Similar characteristics were previouslyreported by [2728] in mango fruit Pathogenicity test results revealed that the inoculatedmango fruits showed the anthracnose symptoms and the causal agents of the fruit rotwere reisolated cultured and identified as C gloeosporioides and C acutatum thus fulfillingKochrsquos postulates

Propyl disulfide was effective in inhibiting the mycelial growth of these fungi Fur-thermore the slower mycelial growth in ethanol than the distilled water suggested itsantimicrobial efficacy against mangorsquos anthracnose Antimicrobial activity of ethanolagainst anthracnose was found in guava [29] The mycelial growth inhibition was higherin the vapor phase assay than in the agar phase assay which was probably due to theaccumulation of the antifungal compoundrsquos vapors and its slow release in the Petri dishrsquosheadspace This shows that fumigation which slowly releases the active compound makesthe fungi more susceptible to treatment than the direct contact method This effect was alsofound in L theobromae and N parvum of stem-end rot pathogens when exposed to propyldisulfide [22] In another study conducted by our research group [25] it was found that

the vapor diffusion assay of thymol carvacrol and trans-cinnamaldehyde had strongerantifungal activities than agar diffusion assay on the longan pathogens ie Lasiodiplodiaspp Phomopsis spp Pestalotiopsis spp and Geotrichum candidum

Compared to our previous work [22] in the present study we found that PD wasmore prominent in preventing the stem-end rot pathogens than anthracnose In stem-endrot the 800 g Lminus1 PD growth inhibition was 7170 and 7066 in vapor phase assay and6347 and 6815 in the direct contact assay (agar diffusion assay) for L theobromae andN parvum respectively [22] While in the current study the percent growth inhibitionof 800 g Lminus1 PD was 2923 and 4387 in vapor phase assay and 3519 and 3393 inagar diffusion assay for C acutatum and C gloeosporioides respectively (Tables 1 and 2)In present study an interesting finding was that in vapor phase assay the percent growthinhibition of the 800 g Lminus1 PD was higher (p le 005) for C gloeosporioides (4387) thanC acutatum (2923) (Table 1) while in agar diffusion assay the percent growth inhibition of800 g Lminus1 PD was higher (p le 005) for C acutatum (3519) than C gloeosporioides (3393)(Table 2) This also confirmed that each fungus responded differently when exposed to thedifferent concentrations of PD However in all cases the percentage growth inhibition ofthe PD against the tested fungi increased with increasing the concentration (Tables 1 and 2)This suggests that antifungal activity of PD is a dose-dependent activity as previouslyfound by [20] that 60 aqueous solution of neem leaf extract was more effective than40 and 20 in inhibiting the mycelial growth of Botrytis cinerea Aspergillus flavus andAspergillus niger in mango fruit It also indicates that the growth reduction of the majorpathogens isolated from mango is attributed to PD rather than ethanol Mycelial growth ofC gloeosporioides was also inhibited by using aroma compounds ie trans-cinnamaldehydecitral and phenylacetaldehyde [11] Feng et al [3] reported an effective inhibition ofC gloeosporioides in mango while using camptothecin (CPT-1) a naturally occurring quino-line alkaloid with significant cantineoplastic and pesticidal activities Mycelial growth ofC gloeosporioides and other pathogens such as Alternaria citri Diaporthe citri Geotrichumcitri-aurantii Penicillium digitatum Penicillium italicum causing postharvest decay in citrusfruit were inhibited by the use of pinocembrin-7-glucoside (P7G) extracted from Ficus hirtaVahl fruit [30]

The current study revealed that MIC value of the PD compound was higher than800 g Lminus1 in both vapor and direct contact assays However the antifungal activity variedwith the fungi as can be seen in both vapor and agar diffusion assays that stem endrot pathogens (L theobromae and N parvum) were more vulnerable [22] than anthracnosepathogens (C acutatum and C gloeosporiodies) (Tables 1 and 2) This shows that the antifungaleffect of the active compound depends on the target microorganisms

The antifungal activity of PD could be attributed to the sulfur compound as sulfurcompounds are well-known for microbial growth prevention According to Koul [31] thephysiological toxicity and behavioural interaction makes PD a potential grain protectantagainst the insect system The author fumigated grain pest ie Sitophilus oryzae andTribolium castaneum with 1 10 or 20 mg Lminus1 of propyl disulfide and diallyl disulfide andstated that the high deterrence index induced by propyl disulfide shows its higher potentialeffectiveness than diallyl disulfide Lyer and Williamson [32] accredited the antifungalproperties of neem extracts to inhibit protease activity of dermatophytes induced by theneem extract Ramos et al [33] stated that antiadhesive mechanism of the neem extract onthe cell surfacersquos hydrophobicity and biofilm formation could affect the colonization of theC albicans Zhong-hui et al [34] and Okemo et al [35] suggested that the neem extractrsquoskilling ability against different pathogenic microorganisms depends on extraction time con-centration and cell wall constituents Kumar and Kudachikar [11] stated that the possibleantifungal mechanisms of natural plant extracts against pathogens could be attributed tothe disruption of membrane integrity and cellular componentsrsquo leakage Zakawa et al [20]used neem leaf extracts and prevented the mycelial growth of Botrytis cinerea Aspergillusflavus and Aspergillus niger fungi causing postharvest rot in mango Suleiman [21] also

used neem leaf extract and prevented the mycelial growth of Aspergillus viridae Penicilliumdigitatum and Rhizopus spp causing anthracnose postharvest rot in tomato

A good fit (R2 0950 of the experimental data in the model equation and a significantdifference in the model parameters ie lag phase (λ) stationary phase (A) and mycelialgrowth rate further showed the greater antifungal efficacy of PD than distilled water andethanol Therefore it can be stated that this compound could also be a useful fumigantmaterial against a wide range of pathogens

5 Conclusions

Anthracnose is one of the major diseases in mango fruit causing considerable eco-nomic losses To some extent the disease could be successfully prevented by using theexisting fungicides however the medium to high levels of resistance of the pathogensto the current fungicides have compelled researchers to find other suitable alternativesTherefore the present research was carried out to test PDrsquos fungicidal effect from neemplantsrsquo seeds against anthracnose in the mango Anthracnose causing fungi were firstisolated and subcultured many times to obtain a pure culture A pathogenicity test wasconducted to confirm the causal agents to fulfill the Kochrsquos postulates and major pathogenswere identified as C acutatum and C gloeosporioides causing anthracnose in mango Theresults revealed that PD more effectively inhibited the mycelial growth of C acutatum andC gloeosporioides than distilled water and ethanol Furthermore a significant difference inthe Gompertz growth model parameters and a good fit of the model suggested that PDcould be the best alternative to the existing fungicides in controlling anthracnose in mangoand other fruits and could decrease the huge economic losses in the fresh produce industry

Author Contributions Conceptualization and design MRK VC CH methodology MRKwritingmdashoriginal draft MRK supervision CH and VC statistical analysismodelling MRKand PS review and editing VC and CH funding acquisition CH and VC All authors have readand agreed to the published version of the manuscript

Funding This research was funded by the Guangxi Science and Technology Plan Project (ProjectNo 2018AB45007) and Postdoctoral Project of the Guangxi University and Center for AdvancedStudies for Agriculture and Food KU Institute for Advanced Studies Kasetsart University (CASAFNRU-KU) Bangkok Thailand

Institutional Review Board Statement Not applicable

Informed Consent Statement Not applicable

Data Availability Statement Not applicable

Conflicts of Interest The authors declare no conflict of interest

References1 Xu X Lei H Ma X Lai T Song H Shi X Li J Antifungal activity of 1-methylcyclopropene (1-MCP) against anthracnose

(Colletotrichum gloeosporioides) in post-harvest mango fruit and its possible mechanisms of action Int J Food Microbiol 2017 2411ndash6 [CrossRef]

2 Konsue W Dethoup T Limtong S Biological control of fruit rot and anthracnose of postharvest mango by antagonistic yeastsfrom economic crops leaves Microorganisms 2020 8 317 [CrossRef] [PubMed]

3 Feng G Zhang XS Zhang ZK Ye HC Liu YQ Yang GZ Chen C Chen M Yan C Wang LY et al Fungicidalactivities of camptothecin semisynthetic derivatives against Colletotrichum gloeosporioides in vitro and in mango fruit PostharvestBiol Technol 2019 147 139ndash147 [CrossRef]

4 Zhang Z Yang D Yang B Gao Z Li M Jiang Y Hu M β-Aminobutyric acid induces resistance of mango fruit topost-harvest anthracnose caused by Colletotrichum gloeosporioides and enhances activity of fruit defense mechanisms Sci Hortic2013 160 78ndash84 [CrossRef]

5 Hu M Yang D Huber DJ Jiang Y Li M Gao Z Zhang Z Reduction of post-harvest anthracnose and enhancement ofdisease resistance in ripening mango fruit by nitric oxide treatment Postharvest Biol Technol 2014 97 115ndash122 [CrossRef]

6 Dong J Zhang L Liu Y Xu N Zhou S Yang Q Yang Y Ai X Thymol protects channel catfish from Aeromonas hydrophilaInfection by inhibiting aerolysin expression and biofilm formation Microorganisms 2020 8 636 [CrossRef]

7 Topa SH Palombo EA Kingshott P Blackall LL Activity of cinnamaldehyde on quorum sensing and biofilm susceptibilityto antibiotics in pseudomonas aeruginosa Microorganisms 2020 8 455 [CrossRef]

8 Tzortzakis NG Methyl jasmonate-induced suppression of anthracnose rot in tomato fruit Crop Prot 2007 26 1507ndash1513[CrossRef]

9 Abdelhamid AG Yousef AE Natural antimicrobials suitable for combating desiccation-resistant Salmonella enterica in milkpowder Microorganisms 2021 9 421 [CrossRef]

10 Munekata PES Pateiro M Rodriacuteguez-Laacutezaro D Domiacutenguez R Zhong J Lorenzo JM The role of essential oils againstpathogenic Escherichia coli in food products Microorganisms 2020 8 924 [CrossRef] [PubMed]

11 Kumar A Kudachikar V Efficacy of aroma compounds for post-harvest management of mango Anthracnose J Plant Dis Prot2019 [CrossRef]

12 De Rima Oliveira KA Berger LRR De Amorim Araujo S Camara MPS De Leite Souza E Synergistic mixtures of chitosanand Mentha piperita L essential oil to inhibit Colletotrichum species and anthracnose development in mango cultivar TommyAtkins Food Microbiol 2017 66 96ndash103 [CrossRef]

13 Oliveira PDL De Rimaacute Oliveira KAacute Vieira WAS Cacircmara MPS De Leite Souza E Control of anthracnose caused byColletotrichum species in guava mango and papaya using synergistic combinations of chitosan and Cymbopogon citratus (DC exNees) Stapf essential oil Int J Food Microbiol 2018 266 87ndash94 [CrossRef] [PubMed]

14 Linde JH Combrinck S Regnier TJC Virijevic S Chemical composition and antifungal activity of the essential oils of Lippiarehmannii from South Africa S Afr J Bot 2010 76 37ndash42 [CrossRef]

15 Dessalegn Y Ayalew A Woldetsadik K Integrating plant defense inducing chemical inorganic salt and hot water treatmentsfor the management of post-harvest mango anthracnose Postharvest Biol Technol 2013 85 83ndash88 [CrossRef]

16 Nicoletti M Murugan K Neem the tree of 21st century Pharmacologyonline 2013 3 115ndash12117 Kumar D Rahal A Malik KJ Neem extract In Nutraceuticals Efficacy Safety and Toxicity Ramesh C Ed Gupta Chennai

India 2016 pp 585ndash597 [CrossRef]18 Hossain MA Al-Toubi WAS Weli AM Al-Riyami QA Al-Sabahi JN Identification and characterization of chemical

compounds indifferent crude extracts from leaves of Omani neem J Taibah Uni Sci 2013 7 181ndash188 [CrossRef]19 Serrone PD Toniolo C Nicoletti M Neem (Azadirachta indica A Juss) oil A natural preservative to control meat spoilage

Foods 2015 4 3ndash14 [CrossRef]20 Zakawa NN Channya KF Magga B Akesa TM Antifungal effect of neem (Azadirachta indica) leaf extracts on mango fruit

post-harvest rot agents in Yola Adamawa state J Pharmacogn Phytochem 2018 7 23ndash2621 Suleiman MN Antifungal properties of leaf extract of neem and tobacco on three fungal pathogens of tomato (Lycopersicon

Esculentum Mill) Adv Appl Sci Res 2011 2 217ndash22022 Khan MR Suwanamornlert P Sangchote S Chonhenchob V Antifungal activity of propyl disulphide from neem against

Lasiodiplodia theobromae and Neofusicoccum parvum causing stem end rot in mango J Appl Microbiol 2020 129 1364ndash1373[CrossRef] [PubMed]

23 Wanjiku EK Waceke JW Wanjala BW Mbaka JN Identification and pathogenicity of fungal pathogens associated withstem end rots of avocado fruits in kenya Int J Microbiol 2020 [CrossRef]

24 Karabıccedilak N Karatuna O Akyar I Evaluation of the viabilities and stabilities of pathogenic mold and yeast species using threedifferent preservation methods over a 12-year period along with a review of published reports Mycopathologia 2016 [CrossRef][PubMed]

25 Suwanamornlert P Sangchote S Chinsirikul W Sane A Chonhenchob V Antifungal activity of plant-derived compoundsand their synergism against major post-harvest pathogens of longan fruit in vitro Int J Food Microbiol 2018 271 8ndash14 [CrossRef][PubMed]

26 Rosas-Gallo A Ramiacuterez-Corona N Palou E Loacutepez-Malo A Modeling Penicillium expansum growth response to thymeessential oil at selected water activities and pH values using surface response methodology Procedia Food Sci 2016 7 93ndash96[CrossRef]

27 Phoulivong S Cai L Chen H McKenzie EHC Abdelsalam K Chukeatirote E Hyde KD Colletotrichum gloeosporioidesis not a common pathogen on tropical fruits Fungal Divers 2010 44 33ndash43 [CrossRef]

28 Wei Y Pu J Zhang H Liu Y Zhou F Zhang K Liu X The laccase gene (LAC1) is essential for Colletotrichum gloeosporioidesdevelopment and virulence on mango leaves and fruits Physiol Mol Plant Pathol 2017 1ndash10 [CrossRef]

29 Ponzo FS Benato EA De-Silva BMP Cia P Ethanol on the post-harvest control of anthracnose in lsquoKumagairsquo guava Bragantia2018 77 160ndash167 [CrossRef]

30 Chen C Chen J Wan C Pinocembrin-7-Glucoside (P7G) reduced postharvest blue mold of navel orange by suppressingPenicillium italicum growth Microorganisms 2020 8 536 [CrossRef]

31 Koul O Biological activity of volatile di-n-propyl disulfide from seeds of neem Azadirachta indica (Meliaceae) to two species ofstored grain pests Sitophilus oryzae (L) and Tribolium castaneum (Herbst) J Econ Entomol 2004 97 1142ndash1147 [CrossRef]

32 Lyer SR Williamson D Efficacy of some plant extracts to inhibit the protease activity of Trichophyton spesies Geobios 1991 83ndash6

33 Ramos De Resende A Ludke Falcao L Salviano Barbosa G Helena Marcellino L Silvano Gander E Neem (Azadirachtaindica A Juss) components Candidates for control of Crinipellis perniciosa and Phytophthora spp Microbiol Res 2007 162 238ndash243[CrossRef]

34 Zhong-hui P Yu-qun Z Zhong-qiong Y Jiao X Ren-yong J Yang L Fan Y Antibacterial activity of 9-octadecanoicacid-hexadecanoic acid-tetrahydrofuran-3 4-diyl ester from neem oil Agric Sci China 2010 9 1236ndash1240 [CrossRef]

35 Okemo PO Mwatha WE Chhabra SC Fabry W The kill kinetics of Azadirachta indica A juss (Meliacae) extracts onStaphylococcus aureus Escherichia coli Pseudomonas aeruginosa and Candida albicans Afr J Sci Technol 2001 2 113ndash118

Introduction
Materials and Methods
- Plant Materials and Chemicals
- Isolation and Identification of Fungi
- Pathogenicity Test
- Antifungal Activity of Propyl Disulfide
- Gompertz Model for Fungal Growth
- Statistical Analysis
- - Results
  - - Anthracnose Rot and Characteristics of Isolated Fungi
    - - Discussion
      - Conclusions
      - References

Page 2: Antifungal Activity of Propyl Disulfide from Neem - MDPI

Recently natural plant extracts have become of great interest to researchers due to theirvolatile nature aroma antioxidant and antimicrobial properties [67] Further plant diseasemanagement with plant essential oils has been recognized as one of the best approaches [8]A considerable number of essential oils exhibited antimicrobial activities against differentpathogens [910] Plant extracts such as aroma compounds (trans-cinnamaldehyde citraland phenylacetaldehyde [11] Mentha piperita L essential oil (MPEO) [12] volatile organiccompounds [2] essential oil from Cymbopogon citratus (DC ex Nees) Stapf [13] and es-sential oil extracted from Cympopogon citratus and Lippia rehmannii [14] have suppressedanthracnose in mango fruit Feng et al [3] evaluated the antifungal activity of camp-tothecin (a naturally occurring quinoline alkaloid having a high pesticidal activity) againstC gloeosporioides causing anthracnose disease in mango Dessalegn et al [15] evaluatedthe plant defense-inducing chemicals (PDIC) combined with postharvest treatments withinorganic salts and hot water to prevent mangorsquos anthracnose

Neem plant (Azadirachta indica) is a rich repository of more than 300 primary andsecondary metabolites and is a powerful natural pesticide [16] Many bioactive compoundshave been isolated from neem plants and have antifungal antibacterial antioxidant andother biological properties [17ndash19] Thus far neem extracts are primarily used in pharma-ceutical or traditional medicines few studies are reported against postharvest pathogensFor example the extract of neem leaf was used to inhibit the growth of Botrytis cinereaAspergillus flavus and Aspergillus niger fungi causing anthracnose in wild mango [20] andAspergillus viridae Penicillium digitatum and Rhizopus sp causing anthracnose in tomato [21]

Propyl disulfide is one of the active compounds in neem seed and is a potent pesticidein grain storage Recently we worked on the antifungal activity of propyl disulfide onmango fruits against Lasiodiplodia theobromae and Neofusicoccum parvum causing the stem-end rot in mango fruit Propyl disulfide was much more effective in controlling the growthof these pathogens [22] Interestingly when the Gompertz growth model was appliedthe data were a good fit in the model and propyl disulfide had a positive effect on thestationary phase and lag phase of the microbial growth [22]

As anthracnose is one of the major diseases in postharvested fruits we continuedour study to work on propyl disulfide from neem against the major pathogens causinganthracnose in mango with the aim to find a potential leading compound for fungicidedevelopment A pathogenicity test was conducted to confirm the major pathogens ofmango causing anthracnose Furthermore a growth model was also applied to see theaccuracy and good fit of the experimental and theoretical results

2 Materials and Methods21 Plant Materials and Chemicals

Propyl disulfide (purity ge 97 FG oil phase) (Figure 1) a volatile antifungal com-pound of neem extract (Azadirachta indica) was obtained from Sigma-Aldrich (St Louis MOUSA) Ripe mangoes (Mangifera indica) brought from the local market were stored in thelaboratory at ambient temperature for decay symptoms as shown in Figure 2

Figure 1 Structure of propyl disulfide

22 Isolation and Identification of Fungi

Fungi were isolated and identified according to the method of Khan et al [22] Brieflyfresh and healthy mangoes were washed with clean tap water to remove any soil debris thesurface sterilized with NaOCl (1) and stored at ambient temperature for fungal growthSmall pieces of active lesions (triplicate) from decayed mango (Figure 2) were separatedwith the help of a sterile blade and washed with NaOCl (1) for 3 min rinsed with steriledistilled water dried and transferred to Petri dishes containing potato dextrose agar (PDA)

[(Vm e

A

)(λ minus t) + 1

](2)

C acutatum(Day 14)

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 3: Antifungal Activity of Propyl Disulfide from Neem - MDPI

[(Vm e

A

)(λ minus t) + 1

](2)

C acutatum(Day 14)

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 4: Antifungal Activity of Propyl Disulfide from Neem - MDPI

[(Vm e

A

)(λ minus t) + 1

](2)

C acutatum(Day 14)

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 5: Antifungal Activity of Propyl Disulfide from Neem - MDPI

C acutatum(Day 14)

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 6: Antifungal Activity of Propyl Disulfide from Neem - MDPI

C acutatum(Day 14)

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 7: Antifungal Activity of Propyl Disulfide from Neem - MDPI

C acutatum(Day 14)

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 8: Antifungal Activity of Propyl Disulfide from Neem - MDPI

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 9: Antifungal Activity of Propyl Disulfide from Neem - MDPI

A(mm)

vm(1d)

λ(d) MSE R2 A

(mm)vm

(1d)λ

(d) MSE R2

(001)081 a

(001)096 b

(001)0091 0905 286 a

(001)094 a

(000)091 b

(000)0041 0949

Ethanol 264 b

(003)066 c

(001)099 b

(000)0077 0911 282 a

(002)087 b

(002)094 b

(001)0044 0938

(002)076 b

(002)132 a

(012) 0244 0750 236 b

(002)074 c

(002)138 a

(009) 0189 0763

A(mm)

vm(1d)

(1d)λ

(d) MSE R2

(001)084 a

(001)093 b

(001)0056 0939 285 a

(002)097 a

(001)088 b

(002)0052 0934

Ethanol 263 a

(001)082 a

(001)094 b

(000)0084 0903 283 a

(002)096 a

(001)089 b

(000)0039 0952

(001)069 b

(001)153 a

(017) 0064 0924 249 b

(001)072 b

(001)133 a

(000) 0225 0722

4 Discussion

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 10: Antifungal Activity of Propyl Disulfide from Neem - MDPI

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 11: Antifungal Activity of Propyl Disulfide from Neem - MDPI

5 Conclusions

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 12: Antifungal Activity of Propyl Disulfide from Neem - MDPI

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

Page 13: Antifungal Activity of Propyl Disulfide from Neem - MDPI

Introduction
- - Results
    - - Discussion
      - Conclusions
      - References

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