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Article

Antifungal Efficacy and Convenience of Krameria lappacea for the Development of Botanical Fungicides and New Alternatives of Antifungal Treatment

by
Martin Zabka
Crop Research Institute, Drnovska 507, 161 06 Prague, Czech Republic
Agronomy 2022, 12(11), 2599; https://doi.org/10.3390/agronomy12112599
Submission received: 31 August 2022 / Revised: 14 October 2022 / Accepted: 20 October 2022 / Published: 22 October 2022
(This article belongs to the Special Issue Chemical Diversity, Yield and Quality of Aromatic Plant)
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Abstract

:
The support of trends in agriculture with limited or restricted use of pesticides is linked to the difficulty of protection against pathogenic and toxigenic fungi. Therefore, it is a great challenge to find alternatives to these dangerous fungi. These alternatives include using safe antifungal plant substances of medicinal or aromatic plants as components of botanical pesticides. Within 69 plant species, only 13 were selected as potentially of interest. However, the species Krameria lappacea, whose extraction yield (economic factor) achieved 17.6% and minimum inhibitory concentrations (MIC50) 0.11–1.24 mg mL−1, was found to be enormously advantageous. Extraordinary efficacy on a set of dangerous filamentous fungi, comparable to expensive essential oils or active phenolic compounds, was demonstrated. In the most effective extract fraction, two main substances from the group of neolignans, analogues of kramerixin, were detected by using GC-MS and LC-MS analysis, and their molecular structure was determined. The advantage of K. lappacea was discussed on the basis of the mode of action and chemical properties of the detected neolignans. K. lappacea could be a suitable source for environmentally friendly preparations, thanks to its high yield in simple extraction, excellent antifungal activity, broad antifungal spectrum, harmlessness, and assumed lower volatility of active compounds.

1. Introduction

Modern agriculture, strict inspections of final foods, and environmental protection are linked to the current innovative trends, whereas consumption of synthetic fungicides is restricted and inspected. Very marked changes are currently occurring regarding the support of organic farming with zero tolerance of synthetic pesticides. Even though these trends are becoming more popular on the market and among ordinary consumers worldwide, farming in such a situation is not simple given the higher infection pressure and development of fungal pathogens. Toxigenic and pathogenic fungi currently pose a very serious health risk, mainly due to their ability to produce highly toxic secondary metabolites. Apart from their potential toxigenic and health risks to the consumer, fungi are one of the major factors capable of significantly decreasing the yield and quality of food and agricultural products. In terms of food safety, species of the Fusarium, Penicillium, and Aspergillus genera represent the most significant groups of toxigenic and pathogenic fungi, mainly on account of their worldwide distribution and ability to produce a great majority of the known mycotoxins [1,2,3]. In addition, the Aspergillus and Fusarium genera in particular are known to include species which are even able to cause very dangerous systemic human and animal mycoses [4,5,6]. Pathogenic and toxigenic fungi are mostly controlled by applying synthetic fungicides, but this can be very complicated in many cases due to high toxicity to mammals or other side effects, along with residual persistence. This fact is obvious in the case of antifungal treatment of stored products such as food [7,8,9,10]. Treatment of the mentioned human mycoses can be considered the most problematic and questionable issue. In these cases, synthetic fungicides are often the only means of suppressing the pathogenic fungal species. Antifungal treatment is commonly connected with direct toxicity and various side effects of the synthetic fungicides used [11,12]. In addition, the number of resistant fungal pathogenic and toxigenic fungal species is increasing [13,14,15,16]. Therefore, the need for new antifungal substances and alternative treatments is becoming more and more obvious in many areas. One of the most promising and ecologically safe possibilities could be based on taking advantage of a plant’s natural antifungal properties. Thanks to long evolution, plants possess an effective defence system, making them the richest natural source of bioactive compounds that could provide natural alternatives to synthetic chemical fungicides. The promising biological activities of many plant extracts or essential oils has recently become a focal point of research dealing with seeking new, environmentally safe botanical fungicides based on plant active substances. Many previous studies have demonstrated promising fungicidal effects [17,18,19,20]. Their mild toxicity confirms the correctness of the hypotheses stating the necessity to study the effects of plant extracts on significant toxigenic fungal pathogens. The primary goal of this study was to find the most suitable candidate, among various significant medicinal or aromatic and commercially used plant species, having potential during production of botanical pesticides intended for environmentally friendly inhibition of dangerous and problematic filamentous fungi. This study focuses on significant toxigenic plant and human fungal pathogens, with a primary focus on Fusarium oxysporum, F. verticillioides, Penicillium expansum, P. brevicompactum, Aspergillus flavus, and A. fumigatus. As the supporting selective factor during commercial production of botanical pesticides, the final yield was compared for all 69 tested species of plant candidates. By using targeted experiments, a group of 13 usable species with high antifungal efficacy was selected. From the aspect of yield and, especially, antifungal efficacy, we established the dominance of the significant and commercially valuable species Krameria lappacea (Dombey) from Burdet and B.B. Simpson. K. lappacea is a slow-growing hemiparasitic shrub reaching a height of up to one meter. Its procumbent branches, growing outward along the ground are covered with little hairs. Branches bear yellowish-white oblong-ovate leaves approximately one centimeter in length [21]. In order to gain more understanding of the origin of the antifungal activity of the extract of K. lappacea, chemical analyses of the active fractions and identification of the key antifungal-active compounds were performed by using chromatographic and mass spectrometry methods.

2. Materials and Methods

2.1. Plant Material and Extraction

Fresh plant material from each of the selected species (Table 1) was collected in flowering season. The plant material was shade-dried at 40 °C. Samples were subsequently homogenized by means of cutting mill (CM-1000; Laarmann, Roermond, The Netherlands) into particles with a size of 2–5 mm. The dry powder was extracted with 100% pure methanol (500 mL of MeOH for 100 g of plant powder) for 24 h. The crude extracts were separately filtered and evaporated under reduced pressure in a rotary evaporator (R-200; Büchi, Flawil, Switzerland). The yield was determined by percentage ratio to the dry weight of the original plant material. The crude extracts were stored at 7 °C until further assay.

2.2. Fungal Strains

All target pathogenic and toxigenic fungal strains were obtained from the collection of pathogenic fungi maintained in the Crop Research Institute (Prague, Czech Republic).
F. oxysporum (MZL/21215) and F. verticillioides (MZL/100415) strains were isolated originally from an infected corn-cob, whereas P. brevicompactum (MZL/270215), P. expansum (MZL/280912), A. flavus (LS/25702), and A. fumigatus (LS/2206) were isolated from contaminated stored corn. Strains were preserved on slant agar (potato carrot agar) at 4 °C. Subcultivations on Petri dishes and other manipulations with these strains were carried out in the Bio Security Level 2 (BSL 2) laboratory, given the BSL of the Fusarium and Aspergillus species used in our experiment.

2.3. Experimental Design Used for Determination of Inhibitory Effect

The inhibitory effect of methanol extracts on the growth of fungi was tested by the agar dilution method. Dried plant extracts were dissolved in an equal volume of methanol. The dissolved extracts were properly diluted in potato dextrose agar (PDA) at concentration 2 mg mL−1. The final concentration of the solvent (methanol) in PDA was 0.75% (v/v). The prepared Petri dishes (9.0 cm diameter) were aseptically inoculated with assay disc (0.4 cm) cuts from the periphery of a seven-day-old culture of the target fungi. The control sets were subsequently prepared by using an equal volume of methanol without extracts. Incubation was carried out in the dark at 21 °C for seven days. The percent inhibition of the radial growth of the target fungi was calculated according to the following formula: Percent inhibition = (DC − DT) / DC × 100, where DC is the colony diameter of the control sets and DT is the colony diameter of the treated sets. Extracts whose inhibitory effect on mycelial growth was higher than 50% at the basic concentration 2 mg mL−1 were chosen for further testing for evaluation of median inhibitory concentration (MIC50). The value of MIC50 was determined by the method of graded concentration of the plant extracts (0.10, 0.25, 0.50, 1.00, 1.50, 2.00 mg mL−1) in the PDA. Cultivation was carried out in the same way as before (in the dark at 21 °C, for 7 days). The MIC50 was regarded as the concentration of plant extract that results in a 50% inhibition of visible growth when compared with control sets [17,22]. The fungicide propiconazole (high purity grade-Pestanal® from Sigma Aldrich, St. Louis, MO, USA) was used as a reference compound.

2.4. Statistical Analysis

Probit analysis was applied to assess the MIC50 values for each effective compound associated with 95% confidence limits (CI95) [23]. The EPA Probit Analysis Program (Version 1.5) was used for statistical evaluation. The MIC values were statistically calculated and associated with Chi square values significant at the p < 0.05 level. MIC50 were assessed for each extract showing a basic fungal growth inhibitory effect higher than 50% at the basic concentration of 2 mg/mL. The Welch’s T-test was used for the most effective extracts.

2.5. Purification and Preparation of K. lappacea Extract Fractions

For the purposes of the purification and separation of a large amount of extract, silica gel column chromatography was utilised. A silica gel (Merck Silica gel 60, 70–230 mesh ASTM) column (50 cm × 4 cm diameter) was prepared. A sample of crude K. lappacea extract was then loaded with a Pasteur pipette. The column with sample was then washed with dichloromethane: methanol mobile phase. Different fractions for bioassays were obtained with a step gradient (from 100:0 v/v up to 80:20 v/v) [24,25]. The obtained fractions were then evaporated to dryness and preserved at 4 °C until the bioassays. The inhibitory effect of the acquired fractions was tested by using the same method used to test the original extracts mentioned above. Fractions demonstrating the highest antifungal activity were subsequently examined by using GC/MS and LC/MS analytical methods.

2.6. Derivatization

The aliquots of obtained extracts were evaporated to dryness in 100 µL ethylacetate-acetone (95:5, v/v). Derivatization of extracts was performed by using 0.5 mL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA): trimethylsilane (TMS) (99:1, v/v) at 70 °C. Samples were then cooled to room temperature, the derivatization agent was removed by using a nitrogen stream, and samples were analyzed by using GC/MS.

2.7. GC/MS Analysis

GC/MS analyses were performed by using a Scion SQ gas chromatogram with MS detection equipped with a CP-8400 autosampler and MS workstation 8.0 software (Bruker, Bremen, Germany). Separations of non-derivatized and derivatized extracts were separately performed by using an Rxi-5ms column (30 m × 0.25 µm, 0.25 mm ID; Restek, Bellefonte, PA, USA). Helium 5.0 (99.999% purity; Linde, Prague, Czech Republic) was used as the carrier gas with the constant flow 1 mL/min. The injector was operated in split/splitless mode, with the splitless time 1 min. The injector temperature was 240 °C and the EI source, transferline and manifold temperatures were 250, 280, and 50 °C, respectively. The GC oven temperature program started from 60 °C (for 1 min), then heated up to 120 °C at 25 °C/min, and finally to 240 °C at 2.5 °C/min, and was held isothermally for 28 min. The injection volume was 1 µL.
The mass spectra were recorded at 3 scans/min under electron impact 70 eV. For qualitative analysis, the full scan mode (50–750 amu) was used. Mass spectrometry workstation software (8.0, Bruker, Germany) equipped with the NIST 08 library was used for verification of structure identity.

2.8. UHPLC-ToFMS Analysis

UHPLC-ToF analyses were performed by using a Waters Acquity UPLC System (Waters; Prague, Czech Republic) consisting of Acquity UPLC Sample Manager, Acquity UPLC Solvent Manager, Acquity UPLC Column Heater and Waters LCT Premier XE orthogonal accelerated ToFMS (Water MS; Manchester, UK). MassLynx V4.0 software was used for data processing.
For the ionization of analytes, an ESI interface was employed (operating in the positive ion mode) by using the following parameters: cone voltage, 40 V; capillary voltage, +2800 V; ion source block temperature, 120 °C; nitrogen desolvation gas temperature, 350 °C; desolvation gas flow, 800 L/h; cone gas flow, 50 L/h. Full scan spectra were acquired in the range of 100–1000 m/z, with a scan time of 0.15 s and an interscan delay of 0.01 s. Mass accuracy was maintained by lock spray by using leucine-enkephalin (5 ng/µL; 5 µL/min).
The aliquots of obtained extracts were evaporated to dryness and reconstituted in acetonitrile-acetone (95:5, v/v). Analytes were separated on an Acquity UPLC C18 column (50 mm × 2.1 mm × 1.7 µm) with the mobile phase consisting of (A) formic acid-water (0.1:99.9, v/v), and (B) formic acid-acetonitrile (0.1:99.9, v/v). A linear gradient elution program was employed as follows (min/%B): 0/5; 15/80; 18/99 followed by 1.5 min step with 100% B and 2.0 min equilibration step. The mobile phase flow rate was 0.4 mL/min, the column temperature was 40 °C and the injection volume was 5 µL.
For verification of the compounds’ identity, the parameters set for the Elementary Composition editor were: mass measurement, 5 mDa; i-FIT (norm) error, 5; CHNO algorithm.

3. Results

The observed percentage yield of extracts from individual plant species of aromatic and other medicinal plants is listed in Table 1. The lowest percentage yield of the extract was in the case of the species P. hirta with a value of 2.1%. The highest yield of 21.6% was measured in the species P. fruticosa. K. lappacea showed extract yield of 17.6%.
The inhibitory effects of all 69 different plant methanolic extracts on the mycelial growth of target pathogenic and toxigenic fungi are listed in Table 2.
The results showed that all 69 plant extracts influenced fungal growth. In the end, 13 of them were evaluated as sufficiently effective. Methanolic extracts obtained from Achillea ageratum, Alpinia purpurata, Angelica archangelica, Angostura trifoliata, Asarum europaeum, Foeniculum vulgare, Guaiacum officinale, Krameria lappacea, Leuzea carthamoides, Mentha arvensis, Mentha longifolia, Mentha suaveolens, and Tabebuia impetiginosa exerted a growth inhibition ratio higher than 50% against the mycelial growth of target fungal species, at least in the case of one target fungal species, at the basic experimental concentration of 2 mg mL−1. These 13 extracts were chosen for subsequent experiments to assignment of their MIC50 values. The MIC50 values are presented in Table 3.
K. lappacea and F.vulgare were the only species with the efficacy against all target fungal species. K. lappachea was then evaluated as a significantly higher effective by using the Welch’s T-test comparison. The difference in fungal inhibition between K. lappachea (mean = 0.37; SD = 0.437) and F. vulgare (mean = 0.95; SD = 0.418) was significant (t(10) = 2.3166; p = 0.4307). On the basis of a statistical comparison of MIC50 values, the species K. lappacea was rated the most effective, not only in regard to the broadness of the spectrum of inhibited species of fungi, but chiefly because it had the highest inhibitory efficacy, which is represented by the lowest MIC50 values. In the case of the very dangerous species A. fumigatus, a value of 0.11 mg mL−1 was statistically proven.
Separation of the extract of K. lappacea into individual fractions by using the silica gel column chromatography method enabled selection and separation of the antifungally inactive or less active fractions from fractions with extreme antifungal activity (Table 4).
Of a total of seven fractions differing in the polarity of the contained substances, two were identified whose inhibitory effect far exceeded the 50% level in a concentration of just 0.5 mg mL−1. Specific 3% and 5% fractions achieved a significantly higher inhibitory effect, ranging between 55.7% and 95.5% at this concentration. The abovementioned inhibitory effect naturally differed depending on the varying sensitivity of the target pathogens. Other fractions were not effective across the entire spectrum of target fungi, or did not demonstrate any antifungal activity on any pathogen. In the 3% fraction, the most abundant compound was identified as the methylated form of deoxykramexin (MW 264; trimethylsililated derivate MW 336) (Figure 1). GC/MS analysis of the significantly effective 5% fraction revealed a high abundance of the methylated form of dihydrogen deoxykramerixin (MW 266; trimethylsililated derivate MW 338) (Figure 2). The molecular properties were also obtained by using LC/MS analysis.

4. Discussion

Target fungal species were inhibited in the case of K. lappacea in very similar extremely low concentrations. Nevertheless, such significant efficacy is achieved, for instance, only by the most effective and mostly expensive essential oils or several antifungal phenolic compounds [18,22]. The percentage yield during the extraction process was also the highest in the case of K. lappacea compared to the aforementioned effective species (Table 1 and Table 3). In relation to plants from the Krameria family, the most frequently mentioned is the antifungally effective kramerixin, which has significant antifungal effects and whose efficacy is comparable to that of the problematic synthetic fungicide amphotericin, which is frequently used for medical purposes [26,27,28,29]. The anti-microbially very potent kramerixin and its molecular analogues are members of the group of neolignans [30,31]. On the basis of our findings, it is clear that the antifungal efficacy of the examined extract from the root of Krameria lappacea is most likely not caused by kramerixin, but rather by its methylated forms. In the case of the most antifungally effective fractions, these substances were detected as the substances with the greatest abundance. These majority substances have not often been described from the aspect of their antifungal efficacy. The efficacy of kramerixin and its analogues was chiefly investigated mostly against yeast fungi and not against more resistant filamentous fungi. Similar modifications of kramerixin, including the use of synthetic methods, are being intensively investigated during the search for new active substances for medical purposes as well [26,32]. However, antifungal efficacy against the complex of filamentous pathogenic and toxigenic fungi, as in our study, has seldom been mentioned in previous literature. Standard antifungals primarily act against the ergosterol component of cellular membranes, ergosterol synthesis or the mechanism of inhibition of RNA [33]. The mode of action of neolignans, such as the present analogues of kramerixin, is different, which broadens their potential in the field of antifungal treatment. This concerns blockage of 1,3-β glucan-synthase and chitin synthase and is thereby targeted at important components specific for fungi [26,34]. It therefore fulfils one factor for utilisation and does not target components occurring in higher-animal cells. In our study, the high efficacy against hygienically, agriculturally, and medically important species of the resistant filamentous species Aspergillus, Fusarium, and Penicillium was demonstrated. Obviously, individual inhibition levels based on MIC values of plant extracts were influenced also by sensitivity of the target fungi. The same effect was observed for example in other studies of different antifungals against a similar spectrum of fungal species. It was found that most of the efficient substances exhibited the highest efficacy against A. fumigatus. On the contrary, most substances in the mentioned studies exhibited the least efficacy against A. flavus. In certain cases, efficacies against A. flavus even several-fold lower than in the other target fungi [17,18,35] were found. The described difference in the sensitivity of the individual species of filamentous fungi used in the study was also similarly observed in the case of synthetic fungicide propiconazole used in this study as a reference compound. These species were selected on the basis of how extremely dangerous and problematic they are in practice, specifically in agriculture, in the protection of agricultural products and during the production of safe foods and other products. It must be mentioned again that some target species in this study are also dangerous human pathogens [36,37]. The extract from the root of K. lappacea offers the advantage of high antifungal efficacy and harmlessness to human health, because it is also significantly used in traditional medicine, chiefly in South America [38,39]. Various parts of K. lappacea are described to be used during stomach ailments, diarrhoea and inflammation of the oropharynx. Examples of its use for strengthening and protecting the teeth and against oral ulcers, bleeding and inflammation of the oral cavity, are also known [40,41,42,43]. This plant is currently included in the European pharmacopoeia. The photo-protective and antioxidant effects of this plant have also been described [44]. The stability and non-volatility of the antifungally active substances in the extract of K. lappacea represent other practical advantages compared to the frequently discussed essential oils with their active phenolic substances, such as thymol, carvacrol or eugenol [18,22]. Due to their high volatility, their efficacy quickly falls after application, which is a limiting factor [35,45]. The increasing support for eco-friendly trends in agriculture and the production of harmless agricultural products and foods is linked to limitation or strict restriction of synthetic pesticides. It is therefore necessary to find new alternative methods of protection against harmful pathogenic and toxigenic filamentous fungi. Substances and extracts from plants are one of the promising and markedly popular sources. In our study, we assessed and compared the yield and antifungal efficacy of many potential plant species. On the basis of the data we acquired about yield and the level of antifungal efficacy, it is possible to consider the species K. lappacea an especially suitable candidate for the purposes of natural antifungal protection. This is supported by the very high extraction yield compared to biomass, the broad antifungal spectrum, and high level of efficacy against dangerous filamentous fungi. Furthermore, the nature of the key detected antifungal substances of the extract reinforces the idea of safe and advantageous use in inhibiting the occurrence of dangerous filamentous fungi.

Funding

This study was supported by the Ministry of Agriculture of the Czech Republic, institutional support No. MZE-RO0418.

Data Availability Statement

Not applicable.

Conflicts of Interest

The author declares no conflict of interest.

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Agronomy 12 02599 g001 550
Figure 1. MS analysis of majority peak in 3% fraction 56.61 min, m/z 264 and 57.65 min, m/z 336 (trimethylsililated) corresponding to methylated form of deoxy-kramerixin.
Figure 1. MS analysis of majority peak in 3% fraction 56.61 min, m/z 264 and 57.65 min, m/z 336 (trimethylsililated) corresponding to methylated form of deoxy-kramerixin.
Agronomy 12 02599 g001
Agronomy 12 02599 g002 550
Figure 2. MS analysis of majority peak in 5% fraction 50.172 min, m/z 266 and 50.98 min, m/z 338 (trimethylsililated) corresponding to methylated form of dihydrogen-deoxy-kramerixin.
Figure 2. MS analysis of majority peak in 5% fraction 50.172 min, m/z 266 and 50.98 min, m/z 338 (trimethylsililated) corresponding to methylated form of dihydrogen-deoxy-kramerixin.
Agronomy 12 02599 g002
Table
Table 1. Plants used in this study, their part used, origin, and yield of extracts.
Table 1. Plants used in this study, their part used, origin, and yield of extracts.
SpeciesFamilyPlant Part Assayed Yield (%)Origin
Acanthopanax senticosus (Rupr. & Maxim.) HarmsAraliaceaeRoots5.4Cicenice, Czech Republic
Acer campestre L.AceraceaeLeaves9.8Prague, Czech Republic
Acer capillipes Maxim.AceraceaeLeaves12.5Prague, Czech Republic
Acer platanoides L.AceraceaeLeaves9.3Prague, Czech Republic
Achillea ageratum L.AsteraceaeStem11.8Prague, Czech Republic
Achillea collina HeimerlAsteraceaeStem7.0Prague, Czech Republic
Achillea nobilis L.AsteraceaeStem10.9Prague, Czech Republic
Aegopodium podagraria L.ApiaceaeStem5.6Dobre, Czech Republic
Ajuga chamaepitys (L.) SchreberLamiaceaeStem14.8Prague, Czech Republic
Alpinia purpurata K. Schum. ZingiberaceaeRoots7.0Cicenice, Czech Republic
Anethum graveolens L.ApiaceaeStem12.4Prague, Czech Republic
Angelica archangelica L.ApiaceaeFruits6.1Cicenice, Czech Republic
Angostura trifoliata (Willd.) T.S.EliasRutaceaeBark11.7Cicenice, Czech Republic
Asarum europaeum L.ArisrolochiaceaeStem7.5Vranov, Czech Republic
Astragalus glycyphyllos L.FabaceaeStem9.1Prague, Czech Republic
Bistorta officinalis DelarbrePolygonaceaeRoots7.2Cicenice, Czech Republic
Borago officinalis L.BoraginaceaeStem4.5Prague, Czech Republic
Buddleja davidii Franch.BuddlejaceaeStem21.0Prague, Czech Republic
Cinchona officinalis L. RubiaceaeBark10.4Cicenice, Czech Republic
Citrus × sinensis (L.) OsbeckRutaceaePericarp8.3Cicenice, Czech Republic
Citrus aurantium L. RutaceaePericarp5.6Cicenice, Czech Republic
Citrus bergamia (Risso) Wright & ArnRutaceaePericarp9.1Cicenice, Czech Republic
Daucus carota L.ApiaceaeStem8.1Prague, Czech Republic
Dracocephalum moldavica L.LamiaceaeStem12.7Prague, Czech Republic
Foeniculum vulgare Mill.ApiaceaeSeeds5.9Prague, Czech Republic
Galega officinalis L.FabaceaeStem14.8Prague, Czech Republic
Galium sylvaticum L.RubiaceaeStem5.2Vranov, Czech Republic
Geum urbanum L.RosaceaeRoots13.6Cicenice, Czech Republic
Gonolobus condurango Decne.ApocynaceaeBark12.6Cicenice, Czech Republic
Guaiacum officinale L.ZygophyllaceaeXylem5.3Cicenice, Czech Republic
Harpagophytum procumbens (Burch.) DC ex Meissn.PedaliaceaeRoots5.2Cicenice, Czech Republic
Hyssopus seravschanicus (Dub.) PazijLamiaceaeStem11.0Prague, Czech Republic
Inula magnifica LipskyAsteraceaeStem11.8Prague, Czech Republic
Krameria lappacea (Dombey) Burdet & B.B.SimpsonKrameriaceaeRoots17.6Cicenice, Czech Republic
Lamium argentatum(Smejkal) Henker ex G. H. LoosLamiaceaeStem15.1Vranov, Czech Republic
Lathyrus tuberosus L.FabaceaeStem13.5Znojmo, Czech Republic
Lavandula angustifolia Mill.LamiaceaeStem9.4Prague, Czech Republic
Lavandula canariensis Mill.LamiaceaeStem6.2Prague, Czech Republic
Leuzea carthamoides DC.AsteraceaeRoots2.3Cicenice, Czech Republic
Lotus corniculatus L.FabaceaeStem7.3Dobre, Czech Republic
Lythrum virgatum L.LythraceaeStem9.6Prague, Czech Republic
Melilotus albus Medik.FabaceaeStem10.7Dobre, Czech Republic
Mentha arvensis L.LamiaceaeStem6.2Prague, Czech Republic
Mentha longifolia (L.) L.LamiaceaeStem9.2Prague, Czech Republic
Mentha suaveolens Ehrh.LamiaceaeStem16.9Prague, Czech Republic
Nepeta pannonica L.LamiaceaeStem8.5Prague, Czech Republic
Ononis arvensis L.FabaceaeStem12.6Prague, Czech Republic
Orlaya grandiflora (L.) Hoffm.ApiaceaeStem11.4Prague, Czech Republic
Picramnia excelsa (Swartz) Planch.SimaroubaceaeXylem10.7Cicenice, Czech Republic
Plantago lanceolata L.PlantaginaceaeStem18.6Prague, Czech Republic
Potentilla anserina L.RosaceaeStem7.3Prague, Czech Republic
Potentilla fruticosa L.RosaceaeStem21.6Prague, Czech Republic
Potentilla hirta L.RosaceaeStem2.1Prague, Czech Republic
Potentilla reptans L.RosaceaeStem12.2Prague, Czech Republic
Quercus robur L. FagaceaeBark4.6Cicenice, Czech Republic
Rhamnus frangula L.RhamnaceaeBark9.7Cicenice, Czech Republic
Rheum officinale L.PolygonaceaeRoots8.0Cicenice, Czech Republic
Salix alba L. SalicaceaeBark3.5Cicenice, Czech Republic
Salvia officinalis L.LamiaceaeStem14.0Prague, Czech Republic
Stachys palustris L. LamiaceaeStem8.0Prague, Czech Republic
Stachys recta L.LamiaceaeStem8.3Prague, Czech Republic
Symphytum officinale L. BoraginaceaeRoots7.5Cicenice, Czech Republic
Tabebuia impetiginosa (Mart. ex DC.) Standl.Bignoniaceaebark11.2Cicenice, Czech Republic
Tanacetumparthenium (L.) Schlutz-Bip.AsteraceaeStem12.3Prague, Czech Republic
Teucrium botrys L.LamiaceaeStem4.6Prague, Czech Republic
Teucrium capitatum L.LamiaceaeStem9.7Prague, Czech Republic
Uncaria tomentosa (Willd. ex Schult.) DCRubiaceaeBark8.3Cicenice, Czech Republic
Urtica dioica L. UrticaceaeRoots3.6Cicenice, Czech Republic
Valeriana officinalis L.ValerianaceaeRoots11.7Prague, Czech Republic
Table
Table 2. Inhibition effect of plant extracts on pathogenic and toxinogenic fungi at concentration 2 mg mL−1.
Table 2. Inhibition effect of plant extracts on pathogenic and toxinogenic fungi at concentration 2 mg mL−1.
Plant Extract% Inhibition of Target Fungi (Mean ± SE)
Fusarium
oxysporum		Fusarium
verticillioides		Penicillium
brevicompactum		Penicillium
expansum		Aspergillus
flavus		Aspergillus
fumigatus
Acanthopanax senticosus31.22 ± 0.0533.33 ± 0.0812.20 ± 0.0822.64 ± 0.0913.54 ± 0.0544.44 ± 0.05
Acer campestre13.82 ± 0.0917.43 ± 0.00−8.16 ± 0.055.66 ± 0.054.71 ± 0.145.26 ± 0.00
Acer capillipes24.39 ± 0.0018.35 ± 0.05−4.08 ± 0.009.43 ± 0.008.24 ± 0.0028.07 ± 0.05
Acer platanoides12.20 ± 0.0014.68 ± 0.00−4.08 ± 0.009.43 ± 0.000.00 ± 0.055.26 ± 0.00
Achillea ageratum35.34 ± 0.0027.59 ± 0.0020.00 ± 0.0032.08 ± 0.0032.22 ± 0.0551.72 ± 0.05
Achillea collina19.83 ± 0.0020.69 ± 0.0011.11 ± 0.0535.85 ± 0.0523.33 ± 0.0027.59 ± 0.00
Achillea nobilis22.41 ± 0.0017.24 ± 0.006.67 ± 0.0026.42 ± 0.0026.67 ± 0.0041.38 ± 0.05
Aegopodium podagraria0.00 ± 0.05−5.75 ± 0.05−2.22 ± 0.0513.21 ± 0.0515.56 ± 0.0531.03 ± 0.05
Ajuga chamaepitys−1.72 ± 0.05−12.64 ± 0.0915.56 ± 0.0518.87 ± 0.0517.78 ± 0.0518.97 ± 0.05
Alpinia purpurata76.19 ± 0.0065.93 ± 0.1721.95 ± 0.0952.83 ± 0.0548.96 ± 0.0572.73 ± 0.00
Anethum graveolens22.41 ± 0.0027.59 ± 0.0020.00 ± 0.0026.42 ± 0.0016.67 ± 0.0815.52 ± 0.05
Angelica archangelica82.01 ± 0.0573.33 ± 0.0048.78 ± 0.0064.71 ± 0.0950.00 ± 0.0088.89 ± 0.05
Angostura trifoliata58.20 ± 0.0546.67 ± 0.0048.78 ± 0.0037.74 ± 0.0016.67 ± 0.1963.54 ± 0.05
Asarum europaeum63.43 ± 0.0560.75 ± 0.0083.33 ± 0.0555.10 ± 0.0542.86 ± 0.0057.81 ± 0.00
Astragalus glycyphyllos18.70 ± 0.2422.94 ± 0.000.00 ± 0.057.55 ± 0.0518.82 ± 0.0010.53 ± 0.00
Bistorta officinalis23.81 ± 0.0011.85 ± 0.05−9.76 ± 0.0011.32 ± 0.05−33.33 ± 0.052.02 ± 0.24
Borago officinalis3.25 ± 0.0914.68 ± 0.0018.37 ± 0.125.66 ± 0.05−3.53 ± 0.058.77 ± 0.05
Buddleja davidii12.20 ± 0.0021.10 ± 0.05−6.12 ± 0.059.43 ± 0.008.24 ± 0.0012.28 ± 0.09
Cinchona officinalis23.28 ± 0.0511.11 ± 0.1424.39 ± 0.0515.09 ± 0.002.08 ± 0.33−26.26 ± 0.05
Citrus × sinensis17.99 ± 0.0912.59 ± 0.12−17.07 ± 0.003.77 ± 0.008.33 ± 0.0521.21 ± 0.00
Citrus aurantium12.17 ± 0.0514.07 ± 0.05−14.63 ± 0.0515.09 ± 0.002.08 ± 0.056.06 ± 0.00
Citrus bergamia14.81 ± 0.0916.30 ± 0.09−14.63 ± 0.0520.75 ± 0.00−1.04 ± 0.0526.26 ± 0.05
Daucus carota21.55 ± 0.0513.79 ± 0.148.89 ± 0.0524.53 ± 0.0533.33 ± 0.0034.48 ± 0.09
Dracocephalum moldavica21.95 ± 0.0016.51 ± 0.052.04 ± 0.009.43 ± 0.0010.59 ± 0.053.51 ± 0.05
Foeniculum vulgare65.85 ± 0.0060.55 ± 0.0597.96 ± 0.0569.81 ± 0.0578.95 ± 0.0094.74 ± 0.00
Galega officinalis24.39 ± 0.0022.94 ± 0.00−4.08 ± 0.005.66 ± 0.058.24 ± 0.00−7.02 ± 0.05
Galium sylvaticum10.45 ± 0.0022.43 ± 0.0514.58 ± 0.0920.41 ± 0.0023.81 ± 0.0923.44 ± 0.05
Geum urbanum20.63 ± 0.149.63 ± 0.0917.07 ± 0.0518.87 ± 0.09−10.42 ± 0.058.08 ± 0.05
Gonolobus condurango26.98 ± 0.0022.22 ± 0.142.44 ± 0.0528.30 ± 0.05−10.42 ± 0.0524.24 ± 0.00
Guaiacum officinale64.02 ± 0.0547.41 ± 0.0519.51 ± 0.0020.75 ± 0.003.13 ± 0.0051.52 ± 0.00
Harpagophytum procumbens21.69 ± 0.0513.33 ± 0.080.00 ± 0.0924.53 ± 0.051.04 ± 0.0523.23 ± 0.09
Hyssopus seravschanicus21.55 ± 0.0518.39 ± 0.0913.33 ± 0.0026.42 ± 0.0026.67 ± 0.0024.14 ± 0.05
Inula magnifica35.77 ± 0.0532.11 ± 0.0516.33 ± 0.0516.98 ± 0.0524.71 ± 0.0549.93 ± 0.00
Krameria lappacea87.30 ± 0.0090.37 ± 0.0563.41 ± 0.0052.83 ± 0.0569.38 ± 0.0091.92 ± 0.00
Lamium argentatum12.07 ± 0.082.30 ± 0.096.67 ± 0.0020.75 ± 0.0013.33 ± 0.0017.24 ± 0.00
Lathyrus tuberosus−2.99 ± 0.006.54 ± 0.0912.50 ± 0.0820.41 ± 0.0810.71 ± 0.0020.31 ± 0.00
Lavandula angustifolia19.51 ± 0.1431.19 ± 0.008.16 ± 0.00−9.43 ± 0.0918.82 ± 0.007.02 ± 0.05
Lavandula canariensis14.66 ± 0.00−6.90 ± 0.00−13.33 ± 0.0032.08 ± 0.0023.33 ± 0.0017.24 ± 0.00
Leuzea carthamoides55.03 ± 0.0937.78 ± 0.1478.05 ± 0.0069.81 ± 0.0518.75 ± 0.0037.37 ± 0.05
Lotus corniculatus8.62 ± 0.05−2.30 ± 0.12−13.33 ± 0.009.43 ± 0.0012.22 ± 0.0525.86 ± 0.05
Lythrum virgatum6.50 ± 0.0512.84 ± 0.05−4.08 ± 0.000.00 ± 0.058.24 ± 0.00−5.26 ± 0.14
Melilotus albus6.90 ± 0.00−2.30 ± 0.09−4.44 ± 0.0518.87 ± 0.0523.33 ± 0.0037.93 ± 0.00
Mentha arvensis51.72 ± 0.0951.72 ± 0.0057.78 ± 0.0566.04 ± 0.0032.22 ± 0.0555.17 ± 0.09
Mentha longifolia32.76 ± 0.0013.79 ± 0.0046.67 ± 0.0058.49 ± 0.0530.00 ± 0.0072.41 ± 0.05
Mentha suaveolens25.86 ± 0.0918.39 ± 0.0946.67 ± 0.0054.72 ± 0.0032.22 ± 0.0556.90 ± 0.05
Nepeta pannonica31.90 ± 0.0910.34 ± 0.0026.67 ± 0.0045.28 ± 0.0533.33 ± 0.0032.76 ± 0.00
Ononis arvensis15.52 ± 0.0512.64 ± 0.056.67 ± 0.0041.51 ± 0.0527.78 ± 0.0551.72 ± 0.12
Orlaya grandiflora29.27 ± 0.0828.44 ± 0.008.16 ± 0.0018.87 ± 0.0517.65 ± 0.0531.58 ± 0.00
Picramnia excelsa20.11 ± 0.255.93 ± 0.09−2.44 ± 0.005.66 ± 0.09−12.50 ± 0.1418.18 ± 0.08
Plantago lanceolata−6.90 ± 0.05−14.94 ± 0.050.00 ± 0.0020.75 ± 0.0021.11 ± 0.4717.24 ± 0.00
Potentilla anserina−8.94 ± 0.175.50 ± 0.24−2.04 ± 0.0516.98 ± 0.0918.82 ± 0.007.02 ± 0.05
Potentilla fruticosa−3.25 ± 0.0511.01 ± 0.090.00 ± 0.05−1.89 ± 0.0021.18 ± 0.0512.28 ± 0.09
Potentilla hirta−3.25 ± 0.0512.84 ± 0.05−4.08 ± 0.009.43 ± 0.0018.82 ± 0.008.77 ± 0.19
Potentilla reptans13.82 ± 0.057.34 ± 0.096.12 ± 0.055.66 ± 0.0916.47 ± 0.1912.28 ± 0.09
Quercus robur17.99 ± 0.092.22 ± 0.0812.20 ± 0.005.66 ± 0.050.00 ± 0.0015.15 ± 0.00
Rhamnus frangula36.51 ± 0.004.44 ± 0.0019.51 ± 0.0030.19 ± 0.05−13.54 ± 0.059.09 ± 0.00
Rheum officinale46.56 ± 0.0530.37 ± 0.052.44 ± 0.1237.74 ± 0.0012.50 ± 0.0027.27 ± 0.14
Salix alba6.35 ± 0.08−0.74 ± 0.09−12.20 ± 0.057.55 ± 0.05−18.75 ± 0.142.02 ± 0.09
Salvia officinalis60.34 ± 0.0547.13 ± 0.0546.67 ± 0.0033.96 ± 0.0535.56 ± 0.0582.76 ± 0.05
Stachys palustris13.82 ± 0.0512.84 ± 0.09−2.04 ± 0.059.43 ± 0.001.18 ± 0.147.02 ± 0.09
Stachys recta10.34 ± 0.091.15 ± 0.05−6.67 ± 0.0018.87 ± 0.0916.67 ± 0.008.62 ± 0.09
Symphytum officinale0.00 ± 0.00−4.44 ± 0.002.44 ± 0.0524.53 ± 0.05−14.58 ± 0.09−24.24 ± 0.36
Tabebuia impetiginosa50.79 ± 0.0034.81 ± 0.0926.83 ± 0.0037.74 ± 0.00−3.12 ± 0.0036.36 ± 0.00
Tanacethum parthenium30.17 ± 0.0033.33 ± 0.0520.00 ± 0.0035.85 ± 0.0530.00 ± 0.0036.21 ± 0.05
Teucrium botrys28.46 ± 0.0528.44 ± 0.00−8.16 ± 0.5213.21 ± 0.057.06 ± 0.0519.30 ± 0.09
Teucrium capitatum25.20 ± 0.0530.28 ± 0.054.08 ± 0.0511.32 ± 0.0511.76 ± 0.0015.79 ± 0.00
Uncaria tomentosa11.64 ± 0.055.19 ± 0.05−4.88 ± 0.0513.21 ± 0.05−26.04 ± 0.096.06 ± 0.00
Urtica dioica22.75 ± 0.09−2.22 ± 0.4929.27 ± 0.0935.85 ± 0.05−28.13 ± 0.0013.13 ± 0.05
Valeriana officinalis48.78 ± 0.0028.44 ± 0.0034.69 ± 0.0537.74 ± 0.0034.12 ± 0.0943.86 ± 0.09
Table
Table 3. Medium inhibitory concentration of the most effective plant extracts against target fungal species (mg mL−1).
Table 3. Medium inhibitory concentration of the most effective plant extracts against target fungal species (mg mL−1).
Plant SpeciesTarget Fungal Species
Fusarium oxysporumFusarium verticillioidesPenicillium brevicompactumPenicillium expansumAspergillus flavusAspergillus fumigatus
MIC50 (CI95) a
Chi b		MIC50 (CI95) a
Chi b		MIC50 (CI95) a
Chi b		MIC50 (CI95) a
Chi b		MIC50 (CI95) a
Chi b		MIC50 (CI95) a
Chi b
Achillea ageratum>2>2>2>2>20.96 (0.75–1.50)
2.226
Alpinia purpurata0.30 (0.24–0.38)0.84 (0.61–1.07)>20.74 (0.44–2.27)>20.17 (0.13–0.20)
3.1775.019 1.615 0.962
Angelica archangelica0.20 (0.15–0.29)0.17 (0.12–0.21)>20.28 (0.19–0.44)>20.39 (0.26–0.50)
1.5790.401 2.219 1.487
Angostura trifoliata1.76 (1.36–2.98)>2>2>2>20.29 (0.21–0.45)
2.251 1.173
Asarum europaeum1.68 (1.01–5.05)1.87 (1.53–2.31)1.58 (1.03–1.73)1.78 (1.57–2.22)>21.85 (1.73–2.05)
2.0781.7900.6121.337 0.868
Foeniculum vulgare1.44 (1.27–1.67)1.22 (1.01–1.50)0.71 (0.63–0.79)1.14 (0.98–1.34)0.89 (0.49–1.10)0.27 (0.23–0.32)
0.1651.5170.5944.8461.6252.716
Guaiacum officinale0.84 (0.62–1.57)>2>2>2>20.44 (0.34–0.72)
2.743 0.842
Krameria lappacea0.14 (0.07–0.19)0.12 (0.07–0.16)0.38 (0.30–0.49)1.24 (0.74–1.61)0.25 (0.20–0.33)0.11 (0.08–0.14)
2.1900.5061.3173.0192.1750.190
Leuzea carthamoides1.98 (1.63–2.31)>21.46 (1.22–1.86)1.32 (1.13–1.54)>2>2
0.6562.9160.4911.291
Mentha arvensis1.99 (1.66–2.62)1.89 (1.60–2.82)1.85 (1.69–2.14)1.84 (1.66–2.14)>21.86 (1.53–2.50)
3.5321.7252.7850.113 1.053
Mentha longifolia>2>2>20.82 (0.60–1.23)>20.92 (0.76–1.22)
0.468 3.472
Mentha suaveolens>2>2>20.66 (0.44–1.74)>21.27 (1.03–1.69)
2.941 0.030
Tabebuia impetiginosa1.60 (1.13–1.85)>2>2>2>2>2
3.150
Propiconazole *0.69 (0.47–0.93) *0.52 (0.35–0.69) *0.75 (0.52–1.01) *0.53 (0.42–0.63) *3.16 (2.19–5.23) *0.49 (0.40–0.59) *
0.5340.3613.3391.0011.5311.827
* Fungicide reference standard (µg/mL). a Median inhibitory concentration (MIC50) with 95% confidence intervals. b Chi-square value, significant at p < 0.05 level.
Table
Table 4. Inhibition effect of K. lapachea extract fractions on pathogenic and toxinogenic fungi at concentration 0.5 mg mL−1.
Table 4. Inhibition effect of K. lapachea extract fractions on pathogenic and toxinogenic fungi at concentration 0.5 mg mL−1.
Column Fraction MeOH (%)% Inhibition of Target Fungi (Mean ± SE)
Fusarium
oxysporum		Fusarium
verticillioides		Penicillium
brevicompactum		Penicillium
expansum		Aspergillus
flavus		Aspergillus
fumigatus
0.1%NINININININI
0.5%NINININININI
1%13.2 ± 0.0518.7 ± 0.0512.6 ± 0.01NINI23.9 ± 0.05
3%86.9 ± 0.1084.2 ± 0.0074.5 ± 0.0069.3 ± 0.0570.0 ± 0.0595.5 ± 0.00
5%85.0 ± 0.0595.2 ± 0.0079.5 ± 0.0063.1 ± 0.0055.7 ± 0.1093.0 ± 0.00
10%9.0 ± 0.007.9 ± 0.00NININI16.8 ± 0.05
20%NINININININI
NI—No Inhibition.
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Zabka, M. Antifungal Efficacy and Convenience of Krameria lappacea for the Development of Botanical Fungicides and New Alternatives of Antifungal Treatment. Agronomy 2022, 12, 2599. https://doi.org/10.3390/agronomy12112599

AMA Style

Zabka M. Antifungal Efficacy and Convenience of Krameria lappacea for the Development of Botanical Fungicides and New Alternatives of Antifungal Treatment. Agronomy. 2022; 12(11):2599. https://doi.org/10.3390/agronomy12112599

Chicago/Turabian Style

Zabka, Martin. 2022. "Antifungal Efficacy and Convenience of Krameria lappacea for the Development of Botanical Fungicides and New Alternatives of Antifungal Treatment" Agronomy 12, no. 11: 2599. https://doi.org/10.3390/agronomy12112599

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