Evaluation of Argentinean medicinal plants and isolation of their bioactive compounds as an alternative for the control of postharvest fruits phytopathogenic fungi

Abstract

One of the main problems that fruit health goes through in recent years is the difficult eradication of their fungal pathogens after harvesting. This concern to the whole world because it represents huge losses of production, fruit export restrictions and consumers distrust. One of the alternatives to solve this problem could be the exploration of plants and their active compounds, which have proven to be antifungal against human pathogens, but now applied to the treatment of fruits health. In this work, eighteen plant species that grow in Argentina were evaluated against four phytopathogenic fungi that greatly affect the postharvest stage of fruits commercially important to our country. All the species studied were at least active against one fungus of the panel, while three of them displayed high antifungal properties inhibiting the growth of selected pathogens. In addition, bio-guided fractionation of these most active extracts, led to the isolation of some compounds which proved to be responsible for their antifungal activity. Although they are known compounds and were previously isolated from other natural sources, this is the first time that they are evaluated for their phytopathogenic activities against this panel of fungi.

Keywords
Bioactive compounds; Fruits; Phytopathogenic fungi; Postharvest

Introduction

Plant diseases caused by phytopathogenic fungi are responsible for economic losses arising mainly from crop yield reduction, but also resulting from diminished product quality and safety; sometimes they also represent a risk for human and animal health due to food contami nation and the accumulation of toxic residues in the environment. Penicillium digitatum (Pers.) Sacc, Botrytis cinerea (Pers.: Fr.), Monilinia fructicola (G. Wint.) honey and Rhizopus stolonifer (Ehrenb.: Fr.) Vuill, are four of the main phytopathogenic fungi that affect Argentine fruit exports and their producers (^{Pergomet et al., 2018}).

Since regulations on the use of new and existing fungicides are becoming more and more stringent, it urges to identify and develop new chemical entities with antifungal activity. Different non-toxic naturally occurring compounds (^{Fu et al., 2017}), semisynthetic derivatives (^{Zhang et al., 2015}), chitosan-based formulations (^{Novaes Azevedo et al., 2014}) and plant extracts (^{Gatto et al., 2016}) including essential oils (^{Mohammadi and Aminifard, 2012}) have emerged as promising alternatives to synthetic, and many times toxic, fungicides.

This communication provides results, based on in vitro studies, about the possible use of Argentine medicinal plants (or their bioactive compounds) as fruit post harvested antifungals. The species have been chosen based on their antifungal activities against human pathogens cited in the literature. All of them were at least active against one fungus of the panel, meanwhile three of them (Solidago chilensis Meyen., Asteraceae, Drimys winteri J.R.Forst. & G.Forst., Winteraceae and Polygonum stelligerum Cham., Polygonaceae) displayed high antifungal properties inhibiting the growth of selected pathogens. The bio-guided fractionation of S. chilensis hexane extract led to the isolation of a diterpene-lactone known as solidagenone (1) which has been previously isolated from Solidago canadiensis L. (^{Anthonsen, 1966}) and recently evaluated by its antiproliferative potential (^{Gomes et al., 2018}). The sesquiterpene-dialdehyde polygodial (2) was found to be responsible for D. winterii hexane extract activity, in accordance with previous reports (^{Muñoz-Concha et al., 2007}; ^{Derita and Zacchino, 2011}). Two flavonoids [a flavanone, pinostrobin (3) and a chalcone, flavokawin B (4)] were isolated as the bioactive compounds of P. stelligerum ethyl acetate extract, which were previously isolated from Boesenbergia pandurata, Myrica pensilvanica, Polygonum spp. and Piper spp. (^{Burke and Nair, 1986}; ^{Hodgetts, 2001}; ^{López et al., 2006}; ^{Derita and Zacchino, 2011}).

Materials and methods

Plants were collected, mostly, from farms and side of roads in areas surrounding litoral region of Santa Fe province (Argentina), between March 2015 and February 2016. Each vegetal material was identified by Marcos Gabriel Derita and a voucher specimen was deposited at the Herbarium of the FCA-UNL "Arturo Ragonese" (SF Herbarium), Kreder 2805-(3080HOF)-Esperanza, Argentina (^{Supporting information and materials}, Table 1). After collected, plants were dried and separated in leaves, flowers, fruits, bark or the whole plant according to the extracts that had to be prepared. Air-dried samples (100 g) were powdered and successively macerated (3 × 24 h each) with hexane, ethyl acetate and methanol or ethanol, using mechanical stirring to obtain the corresponding extracts, after filtration and evaporation.

Table 1
Antifungal activities of compounds isolated from Solidago chilensis, Drimys winteri and Polygonum stelligerum against four post-harvest phytopathogenics fungi: Minimum Inhibition Concentrations (MIC) and Minimum Fungicidal Concentrations (MFC) are expressed in µg/ml.

Compound	MICs/MFCs (µg/ml)
	Pd	Bc	Mf	Rs
Solidagenone (1)	1000/I	1000/I	31.2/125	62.5/250
Polygodial (2)	250/1000	250/1000	31.2/125	31.2/125
Pinostrobin (3)	1000/1000	1000/1000	62.5/125	62.5/125
Flavokawin B (4)	1000/1000	1000/1000	62.5/125	62.5/125
IMZ	3.9/3.9	0.5/0.5	0.12/0.12	7.8/7.8
CBZ	15.6/15.6	0.12/0.25	0.5/0.97	31.2/62.5

• Pd: P. digitatum CCC-102; Bc: B. cinerea CCC-100; Mf: M. fructicola INTA-SP345; Rs: R. stolonifer LMFIQ-317.Commercial antifungals imazalil (IMZ) and carbendazim (CBZ) were used as positive controls. I: inactive (MIC or MFC > 1000 µg/ml).

For the evaluation of the antifungal activity, standardized strains from the Micology Reference Center (CEREMIC, Rosario, Argentina), the National Institute of Agricultural Technology (INTA, San Pedro, Argentina) and the Department of Microbiology of the Chemical Engineering Faculty, National University of Litoral (UNL, Santa Fe, Argentina), were used. The inocula of spore suspensions were obtained according to the Clinical & Laboratory Standards Institute (CLSI, 2008) reported procedures and adjusted to 1 × 10⁴ Colony Forming Units (CFU)/ml. Minimum Inhibition Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values were determined by using broth microdilution techniques according to the CLSI guidelines for filamentous fungi (document M38-A2, ^{CLSI 2008}). Commercial antifungal agents' imazalil (IMZ) and carbendazim (CBZ) were used as positive controls.

The three most active extracts were submitted to column chromatography and preparative thin layer chromatography in order to obtain the compound/s responsible/s for their antifungal activities.

Results and discussion

Results showed that at least one extract of each selected plant was active against at least one pathogen of the panel. Some of them were selective and displayed activities only against one fungus but some others demonstrated to be active against the four pathogens under study. Most of them proved to be fungistatic (MIC ≤ 1000 µg/ml) and some of them were also fungicides (MFC ≤ 1000 µg/ml).

Hexane and ethyl acetate extracts of S. chilensis were active against M. fructícola and R. stolonifer (MIC between 62.5 and 250 µg/ml; MFC between 500 and 1000 µg/ml), slightly active against B. cinerea (MIC = 1000 µg/ml) and inactive against P. digitatum; while its ethanolic extract and lyophilization product were fungistatic and fungicidal against P. digitatum (MIC = 250 µg/ml and MFC = 1000 µg/ml) and less active against the rest of the fungi. Hexane extract of D. winterii showed significant activity against M. fructícola and R stolonifer (MIC and MFC between 62.5 and 250 µg/ml) and displayed moderate growth inhibition of P. digitatum and B. cinerea (MIC = 500 µg/ml). Regarding C. officinalis, all its extracts showed high antifungal potency against M. fructícola (MIC between 125 and 500 µg/ml and MFCs between 500 and 1000 µg/ml); the ethyl acetate extract inhibited the growth of P. digitatum (MIC = 500 µg/ml) and the hexane extract showed MIC = 125 µg/ml for R. stolonifer. Hexane extract of E. japonica fruits was active against all the fungi evaluated except B. cinerea, displaying MIC and MFC between 250 and 1000 µg/ml, while the rest of the extracts slightly inhibited the growth of M. fructícola with MIC = 1000 µg/ml. Among the extracts of E. japonica seeds, the most active ones were hexane and ethyl acetate with MIC and MFC between 250 and 1000 µg/ml, while the methanolic extract scarcely inhibited the growth of R. stolonifer with MIC = 1000 µg/ml. M. azadirachta flowers and fruits extracts were moderately active against M. fructícola and R. stolonifer (MIC and MFC between 500 and 1000 µg/ml), except for the ethyl acetate extract of flowers that presented a greater spectrum of action, inhibiting the four fungi of the panel with MICs between 500 and 1000 µg/ml. All the extracts of S. grisebachii flowers showed important fungistatic activities against the four pathogens of the panel, with MICs between 250 and 1000 µg/ml; although its fungicidal activity was poor, this plant constitutes one of those with the widest spectrum of fungistatic action among all evaluated in this work. Regarding P. caerulea fruits, both extracts were selectively active against B. cinerea with MIC and MFC between 500 and 1000 µg/ml. Hexane and methanolic extracts of E. grandiflorum flowers showed moderate antifungal activity against B. cinerea and M. fructícola with MIC and MFC between 125 and 1000 µg/ml. Hexane extract of C. intybus was active against B. cinerea and M. fructícola displaying MIC and MFC between 125 and 1000 µg/ml; while the methanolic extract was only fungistatic against the same fungi with MIC between 500 and 1000 µg/ml. P. madagascariensis ethyl ether extract, was found to be active against the whole panel inhibiting the growth of the four fungi with MIC between 125 and 1000 µg/ml. Regarding P. stelligerum, all the extracts were at least fungistatic against all de fungi tested (MIC between 62.5 and 1000 µg/ml) being the ethyl acetate extract the most active particularly against M. fructícola and R. stolonifer (MIC = 62.5 and MFC = 125 µg/ml). The hexane and ethyl acetate extracts of T. aeranthos showed moderate fungistatic activity with MIC between 500 and 1000 µg/ml against P. digitatum, B. cinerea and M. fructícola, but were inactive against R. stolonifer. Finally, C. bonariensis hexane extract was inactive against B. cinerea and P. digitatum but slightly fungicidal against M. fructícola and R. stolonifer (MFC = 1000 µg/ml).

Antifungal activities of pure compounds isolated through bio-guided fractionation of the most active plant extracts are shown in Table 1. Hexane extracts of S. chilensis and D. winterii, led to the isolation of solidagenone (1) and polygodial (2), respectively. From P. stelligerum ethyl acetate extract, pinostrobin (3) and flavokawin B (4) were obtained. Compound 1 was not only fungistatic (MIC = 31.2 µg/ml and 62.5 µg/ml for M. fructícola and R. stolonifer respectively) but also fungicide (MFC = 125 µg/ml and 250 µg/ml for the same fungi); meanwhile it barely inhibited the growth of P. digitatum and B. cinerea with MIC = 1000 µg/ml. Compound 2 showed the highest antifungal activity among all, with MIC = 250 µg/ml and MFC = 1000 for both fungi P. digitatum and B. cinerea, two of the main pathogens that greatly affect post-harvested citrus and berries. Polygodial (2) also showed interesting activities against M. fructícola and R. stolonifer with MIC = 31.2 µg/ml and MFC = 125 µg/ml, being the most promising compound to go on with ex vivo and in vivo studies. In addition, both flavonoids 3 and 4 displayed the same activities against the four fungi evaluated, resulting specific on the inhibition of M. fructícola and R. stolonifer with MIC = 62.5 µg/ml and MFC = 125 µg/ml.

After this evaluation, we can state that plants which have been shown to be antifungal against human pathogens or those that are used in traditional medicine for treatments associated with fungal infections could also have a very promising use as fruit health controllers. In particular, the species that have been most active in this work (S. chilensis, D. winterii and P. stelligerum) are being studied more deeply in terms of their application in ex vivo models and formulation technologies.

• Ethical disclosures
  Protection of human and animal subjects
  The authors declare that no experiments were performed on humans or animals for this study.
  Confidentiality of data
  The authors declare that no patient data appear in this article.
  Right to privacy and informed consent
  The authors declare that no patient data appear in this article.

Acknowledgment

The authors gratefully acknowledge to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) for financial support (PIP N° 2015-0524, PICT N° 2015-2259 and Nº 2016-1833). MGDL and MIS are also thankful to CONICET for their fellowships.

Appendix A Supplementary data

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.bjp.2019.05.007.

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Publication Dates

• Publication in this collection
  09 Dec 2019
• Date of issue
  Sep-Oct 2019

History

• Received
  15 Mar 2019
• Accepted
  2 May 2019