Keywords

11.1 Introduction

Plants have evolved finely regulated complex of metabolic processes to sustain homeostatic balance as well as constitutive and inducible defense mechanisms to help in both wound healing and defense against attack by microbes and herbivores. The constitutive defenses include static structures, such as lignified cell walls, mineral or organic crystals that create physical barriers as well as wide variety of organic compounds. The inducible defenses are also multitudinous, involving gene activation-linked de novo enzyme syntheses and various metabolites called phytoalexins. The role of phytoalexins in defense mechanisms was intensively studied (Van Etten et al. 2001), while to constitutive compounds has been paid less attention. In this chapter, we focus on constitutive mechanisms that might be used for controlling phytopathogenic fungi with special regard to organic substances, which might serve either as botanical fungicides or as lead compounds for molecular design.

Contrary to phytoalexins that are synthesized de novo after the plant is exposed to microbial attack, i.e., produced in response of elicitors or stressors, the phytoanticipins are not performed in the tissue or released from preexisting plant constituents, but are plant antibiotics presented in tissue prior to infection, serving as the basis of pest tolerance. Several thousands of such molecules of different structure have been identified; however, few of them met practical application. These compounds represent heterogeneous chemical structures, and significant part of them is synthesized via polyketide, isoprenoid, shikimate, and phenylpropanoid pathways (Pedras and Yaya 2015). The progress in separation and analytical techniques has allowed the rapid identification of plant secondary metabolites. The screening of their biological activities combined with molecular genetic techniques elucidated various roles in defense mechanisms (Mazim et al. 2011; Carere et al. 2016).

Present control technologies of plant pathogenic fungi decouple the pathogen’s life cycle mainly in two points of ontogeny. The applied chemicals either destroy spores, preventing the infection or inhibit the biotrophic thallus, anticipating the formation of new infective propagules. Although the tolerance of cultivated plants can be enhanced by diverse methods, the possibilities of biocontrol, as well as the enhancement of plant resistance with chemical treatment, are limited; none of these approaches resulted in the economically acceptable level of control for long term of application in recent plant cultivation technologies, contrary to modern synthetic pesticides. Nowadays, the only tool for creditable control of cultivated plants is the use of synthetic chemicals. However, the calculability of yield sureness has been worldwide threatened by the emergence of acquired tolerance to this group of chemicals as well as by anxious feelings for undesirable side effects. All these are major causes of concerns as even 10% disease incidence can cause economic loss threatening the net return. This situation urges the development of efficient alternative control agents. One approach to discover newer antimicrobial compounds is to search for their presence in natural sources exploiting the defense strategies of plants against their pathogens. Microbial species or strains that do not invade the plant are usually more sensitive to the components of performed barriers than a viable pathogen of this plant. Consequently, the introduction of alien phytoanticipins and precursors of phytoalexins into the proper host/parasite system can represent a prospective tool for disease management (Piasecka et al. 2015).

The possible use of botanicals in pest control technologies intrigued big expectations hitched up by social movements. Indeed, in some special cases, these preparations performed well.

However, in comparative studies, the new generation of synthetics surpassed the botanicals at some orders of magnitude (Table 11.1). The use of natural compounds as lead molecules is seemingly more prospective, and the new techniques of molecular design help to map the parts of the active molecule that respond for the desired biological effect. In past decades, the losses caused by peronosporaceous pathogens are increasing, and only a few synthetics are available to control them at an economically acceptable level. Unfortunately, the populations of pathogens rapidly adopt to these highly active monosite inhibitors. Some natural compounds in model experiments exhibited notable antiperonospora effect, especially in their abiotrophic stages of ontogeny (Deepak et al. 2007).

Table 11.1 Antisporulant activity of commercial fungicides and reference substances
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Some natural compounds in model experiments exhibited notable antiperonospora effect, especially in their abiotrophic stages of ontogeny, among them the known Na+ ion channel activator ceveratrum alkaloids effectively inhibited the systemic invasion of the parasitizing thallus as well (Oros 2010). These amphiphilic steroid alkaloids are thought to act by direct incorporation into the microbial membrane disrupting its structural and functional integrity. Examination of the effect of veratridine on the alkali metal salt tolerance of Plasmopara halstedii showed that this steroid alkaloid dramatically impaired the tolerance of microbes to Li+, Na+, Cs+, and, especially, to K+. Modifying its structure synthetically, the sporicidal activity was successfully increased about thousand times (Oros and Ujváry 1999). The non-steroidal analogues of ceveratrum alkaloids designed by molecular modeling have an anti-oomycetes activity that depends significantly on the chemical structure and is confined to certain biotrophic and abiotrophic developmental forms of P. halstedii (Table 11.2).

Table 11.2 Effect of steroid alkaloids and non-steroid analogues on sunflower downy mildew and on the asexual spores of Plasmopara halstedii
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Interestingly, the main structural features of these non-steroidal compounds presented here bear a certain resemblance to known commercial fungicides such as fenpropimorph and fenpropidin as well as to the experimental diaryltetrahydropyridines (Takayama et al. 1995). Thus, the new compounds, on the one hand, refine the structure–fungicidal activity relationship for substituted piperidines and, on the other hand, define an extended structural scaffold for new fungicide development (Ujváry and Oros 2002). The ecological role of the botanical steroid alkaloids is not fully known. Nevertheless, it can be assumed that these substances have multiple functions in the wild plants among them to protective against herbivores and diseases (Wink 1993). In this context, it is interesting that digitonin, α-solanine, and their aglycones showed activity against the asexual spores of P. halstedii and S. sclerospora even though it is generally believed that cleavage of the glycoside bond of plant glycoalkaloids represents a deactivation process utilized by glycoalkaloid-resistant fungi. It should be emphasized, however, that P. halstedii and S. sclerospora are specific and obligate pathogens, and their host plants have not been shown to contain glycoalkaloids; thus, these pathogens are unlikely to have evolved such deactivation mechanism.

From now on, we summarize results of the past two decades searching for promising candidate botanicals useful in pest management practices. The selected for screening plants are attractive for humans, because of their characteristic organoleptic properties (smell and taste). Most of them are cultivated plants being part of the human diet. Their features are well known, and the marketed samples refer to traditionally accepted standards, that is important, as these plants exceptionally rich in secondary compounds of divergent structure (Table 11.3). The composition can largely vary within samples. Chemotypes—chemically distinct entities within plant species on genetic variation—are exceptionally frequent for secondary compounds and can influence the quality of plant materials, which property has been largely used in chemotaxonomy.

Table 11.3 Inhibitory effect of plant preparations on the growth of phytopathogenic fungi
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From the agroindustrial point of view, the herbs have special advantages as their effects on mammalians are well known. Thus, the risk of elaboration of botanical preparation for pest control is significantly lower and less risky than the introduction of the plant with unknown biological effects in details. The use of pure compounds is more favorable; however, their production in industrial scale frequently meets difficulties and unprofitable. Thus, the herbal preparations may have a place in pest control technologies. Moreover, the protective effect can be resulted by synergic joint action of several secondary metabolites that phenomenon needs further studies.

11.2 Standard Operating Protocols

The growth response of 25 filamentous fungi pathogenic to 100 herbal preparations and seven culinary mushrooms was compared in model experiment applying poisoned agar technique following, in general, the route of Walker et al. (1937).

The herbal preparations of plant species listed in Table 11.3 were either home prepared of the plants collected in Protected Landscape Area of Buda Mountains (N 47°33′00″, E 18°52′60″) following traditional manners or purchased in drug store (Herbaria Co., Budapest). The desiccated plants were stored protected of light at ambient conditions over silica gel. The dry material was micronized before use.

The test fungi listed in Fig. 11.1 were maintained on potato dextrose agar slants at 22–25 °C (CM0139B, OXOID, Basingstoke) amended with two gL−1 casein digest (Difco, Detroit, USA), vitamins, and mineral salts (Oros and Naár 2018). All strains were isolated from various sources in Hungary and deposited in the Mycology Collection (WDCM824) of PPI.

Fig. 11.1
figure 1figure 1figure 1

Major secondary metabolites with approved antifungal effect of the most potent culinary and medical herbs tested. R1—gluocose, R2—galactose-glucose[xylose]-galactose-glucose. The presence of as minimum as one of listed compounds has been demonstrated in proper marketed spice or herb at more than 10% of active ingredients

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Toxicity test: The conidia of fungi for inoculation of agar plates were washed up with sterile distilled water containing 0.05% Tween 20 of 8-day-old colonies grown up on PDA slants.

The herbal preparation was mixed with the agarized medium (2500 mg in 100 mL) and poured into Petri dishes (20 mL into a 90-mm-diameter dish). Then these plates were overlayered with 5 mL sterile agar (1.5 gL−1 in distilled water) and after solidification were inoculated with conidial suspensions (105 cell per mL) using a multipoint inoculator, and subsequently incubated at 20–22 °C. The intensity of colony growth was evaluated after 24 and 48 h by the following four-grade scale: 0 = no growth, 1 = growth on the limit of visual apperception, 2 = apparent but retarded growth as related to the untreated control, and 3 = the colony is not visually distinguishable from the untreated control and −1 = stimulation.

  • Data Analysis Fisher’s test was applied to evaluate the significance of differences between variants at p = 0.05 level. The basic data matrix (107 preparations × 25 target strains × 2 evaluations) comprising response values by the scale of evaluation was subsequently analyzed with multivariate statistical methods following previously described scheme to elucidate the number of factors affecting the selective response of target fungi to toxic principles (Magyar and Oros 2012).

Potency mapping (PM) and spectral component analysis (SCA) were employed to disclose differences between both antifungal activity of preparations and sensitivity responses of test strains following Lewi (1976). The SCA separates the basic data matrix into two part; the first is a vector proportional to overall strength of responses (PM), while the second is a matrix of spectral components (Spectral Map, SPM) characterizing the spectrum of activity or sensitivity.

PCA was carried out on the correlation matrix calculated of basic data matrix, and only the components having an eigenvalue greater than one were included into the evaluation of data to demonstrate potential number of factors influencing on sensitivity responses of target fungi. Moreover, principal component regression analysis (PCRA) was employed to reveal changes in weight of influencing factors during the incubation, i.e., time dependence of the growth inhibitory effect.

Box plot analysis was applied to demonstrate time-dependent alterations in sensitivity responses. Cluster analysis (CA) combined with SCA was used to reveal relationships among the spectrum of sensitivity responses of phytopathogenic fungi to preparations.

Statistical functions of Microsoft Office Excel 2003 (Microsoft, Redmond, USA) and Statistica5 program (StatSoft 5.0., Tulsa, USA) were used for analysis of data. The graphical presentation of result of data analysis was edited uniformly in MS Office PowerPoint 2003.

11.3 Results

The conidia of all strains germinated and start to form well-distinguishable colonies within 24 h after inoculation, and the intensity of radial growth corresponded to character of species on untreated control plots. The differences between parallels did not surpass 1 mm, so their growth was near synchronous.

The germination of conidia of all strains was inhibited by various degree by herbs after 24 h of inoculation with the exception of Alternaria that start to form colony growing on Clematis vitalba: Therefore at given dose, all herbs exhibited outstanding antifungal effect being the Hyssopus officinalis the least active (Table 11.3). However, this situation changed dramatically after 24 h when the only ten herbs inhibited the growth of all strains (Table 11.4). The loss of activity varied within large limits, and no pattern could be recognized about the taxonomic position of plants (details of the analysis of SMP are not shown). The increase of inhibition as compared to untreated control was observed in 99 cases of 2675 pairs; the more than half of such cases were observed in Ranunculales, Caryophyllales, Myrtales, and Rosales (7, 8, 7, and 23, respectively), and no cases occurred in culinary fungi and moss. The relationship between the initial activity of herbal preparations and activation process needs further studies, although, seemingly the moderately active herb suffered the major deterioration of their antifungal effect.

Table 11.4 Similarity of hidden variables influencing the performance of growth response of target strains
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The sensitivity response of strains varied in large limits; however, none of them was inhibited completely by all preparations. With exception of Colletotrichum musae and Gliocladium catenulatum, all strains activated as minimum as one of herbs, taking into the consideration the 99 of 2675 pairs, so this process seems to be highly specific and depends on target fungus. Clustering the fungi based on daily changes in their response to herbal preparation (A24-A48), two big clusters have been separated (Fig. 11.2). The strains of soil origin and the insect pathogen were separated of those isolated of foliage. The abilities to either deteriorate or activate the antifungal effect seemingly were not related to taxonomic position of target fungi, as, for example, Geotrichum candidum and Trichotecium roseum formed a close cluster, or two Glomerella cingulata anamorphs (Sour Cherry 1 and 2) have been linked into two different subclusters. The clusters A and B forming a supercluster comprise more sensitive strains than C, D, and E; moreover, the latter are more heterogeneous in respect of the origin of strains. Thus, one can suppose that former environmental adaptation takes more influence on their sensitivity responses to herbs than traits formed during phylogeny.

Fig. 11.2
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Relationship between spectrum of deterioration of inhibitory substances and growth response of test fungi. Prior to SCA, the growth responses were converted to probit values. Potency mapping technique was used to calculate potential overall sensitivity of strains (growth response to strength of herbal action). The similarity of the sensitivity spectra of strains was analyzed applying unweighted pair group averages method based on correlation matrix of spectral variables. Subclasses were sorted at p < 0.05 level

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The principal component analysis revealed high number of factors determining the action of herbal preparations. The response of fungi was influenced during conidial germination and germ tube elongation, i.e., start of colony formation (first day of evaluation) by sixteen principal components (PCs) having an eigenvalue greater than one, which comprised 95% of total variation, and among them four hidden factors were seemingly responsible at 70% of the inhibitory effect of herbs. After subsequent incubation (second-day evaluation), the growth response of the same set altered as it was delineated above (see Table 11.3); the PCA elucidated 13 relevant PCs comprising 93% of total variation, where three of them related to 73% of inhibition of colony formation.

This time-dependent reduction of the number of PCs (hidden variables) that influences significantly the performance of strains growing on poisoned agar plates indicates that some factors were eliminated of the medium. Indeed, comparing sets of data recorded at first and second evaluations by means of PCRA sorted out eight PCs in both sets (Table 11.4), which were correlated significantly and explaining majority of acting hidden factors (72 and 81%, respectively). In both sets were separated five PCs which did not show similarity (explaining 19 and 12% of total variation, respectively). The increase of the weight of similar hidden variables as well as decrease of their number (three PCs of 3.7% weight) as compared to the first evaluation might indicate the changes in the level of active compounds in the medium resulted by metabolic activity of target fungi. As the activity of various herbs was affected by strain-dependent manner, only some general aspects of the character of major hidden variables could be postulated. Plotting strains as PC variables by intercorrelating the major PCs of two sets (Fig. 11.3) elucidated remarkable selectivity of interaction between herbs and strains. The first pair (Fig. 11.3a) negatively influenced the performance of herbs, so it can be most probably related to metabolic degradation of active principles, while the second pair (Fig. 11.3b) affected positively, which may indicate the increase of importance of permanent target sites in expression of antifungal effect (characterized by intensity of growth inhibition).

Fig. 11.3
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Similarities between hidden variables influencing the performance of sensitivity responses of target strains at various phases of their ontogeny. The Roman numerals indicate strains listed in Fig. 11.2. The size of pies is proportional to potential capacity of strain to deactivate the growth inhibitory effect of herb incorporated into the medium. The path coefficients of the fitness of regression lines in graphs A and B are 0.7099 and 0.7845, respectively

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11.4 Exploitation of Findings

The anthracnose caused by Glomerella anamorph has caused increasing losses in Hungarian sour cherry orchards since 2006. The pathogen rapidly acquired tolerance to most effective triazole fungicides. Because of the short tolerance period (maximum 6 days), the protection of sour cherry fruit is a special problem, and use of rapidly deteriorating fungicide is requested. The botanical preparations can stand this prerequisite. The possible use of ten herbs proved to be most active among tested ones (shiitake, galangal, cinnamon, yellow mustard, clove, oregano, summer flavory, wasabi root, wood ear, pomegranate) had been examined to control anthracnose of almond, bilberry, cherries, green pepper, grape, and tomatoes.

However, the promising results in model experiments could not be reproduced in large scale in the sour cherry orchard, where the situation was similar to those observed in the case of pathogenic Glomerella anamorphs (Oros et al. 2010). The only pomegranate preparation acted at acceptable way at 1 kg ha−1 rate in model experiments that means the preparation manufactured of aborted flowers can control the pathogen, while the others either should be applied at irrational for control doses or proved to be phytotoxic in effective dose, i.e., their therapeutic value lagged behind highly active synthetic monosite inhibitors (Table 11.5).

Table 11.5 Most important relationships to be evaluated for development and application of pest control agents
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Unfortunately, 5 of 35 lines of anthracnose pathogen proved to be highly tolerant to prospected new biofungicide (Fig. 11.4). Most probably, in the case of other host/pathogen pairs, similar results can be expected that shows the limit of development of botanical fungicides based on crude preparations.

Fig. 11.4
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Growth response of Glomerella anamorphs to various doses of pomegranate. The standard preparation made of micronized aborted pomegranate flowers was incorporated into PDA prior to pouring into Petri dishes (5-mm-thick layer) at rate mimicking the concentration of the spray to be used in field conditions. The strains in black bordered box (A) are various Glomerella anamorph (Colletotrichum species), while those of group B were isolated of sour cherry fruits collected in orchards of Fruitculture Research Institute (47°40′22.2″ N, 21°41′24.7″ E). The positions of the proper strains are identical on plates

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11.5 Discussion

The use of chemical fungicides is costly and potentially harmful to the environment. The trend toward the environmentally friendly pesticides has led to the search for new antifungal agents from various sources, including medicinal herbs, however, to plants of culinary use have paid less attention. Alternative control with herbal preparations showing the greatest antifungal potential could provide economical, safe, and non-hazardous tools for management of cultivated plants and increase food quality from sustainable production (Khaskheli et al. 2016). Most probably all plants have phytoanticipins of diverse molecular structure and size of simple myrcene or phenylethanol to steroid alkaloids, oligocarbohydrates, proteins, etc. There are increasing number of studies dealing with the isolation and chemical characterization of such molecules as well as their role in host–pathogen interactions. Several and successful efforts have been made to introduce compounds of plant origin (strychnine, rotenone, cevadine, pyrethrins) to use against pests; however, the botanicals of similar activity or formers active against phytopathogenic fungi have not been marketed yet. Here we investigated only the heat-tolerant compounds of low molecular weight.

There are increasing number of studies dealing with the isolation and chemical characterization of phytoanticipins as well as their role in host–pathogen interactions. Nevertheless, it seems to be clear that the defense molecules either predisposed or induced cannot be regarded as the agents of a single defense mechanisms. Very little detailed information is at our disposal about the multiple mechanisms for plant resistance against pests and pathogens, and these are still a matter of debate.

The Biopesticides and Pollution Prevention Division in the Office of Pesticide Programs of Environmental Protection Agency of USA encourages the development of biopesticides as well as the use of safer pesticides, including biopesticides. Since generally accepted that biopesticides tend to pose fever risks than conventional ones, EPA generally requires much less data to register them than latter. In fact, new biopesticide is often registered less than a year, compared with an average of more than three years for those based on synthetic chemicals. However, using any chemical in pest control management the same requirements have to be taken into the consideration, when these preparations aimed to be applied at large scale! Moreover, the selectivity of action also has to be evaluated by the same manner, independently of the character of active ingredient, and this requirement is more strict than those used in the case of pharmaceuticals (Table 11.5). For example, the bees meet regularly the essential oil flavonoids that are mighty attractants for pollinating animals. However, the dose in concerted activities is very low. It is well known; the essential oils might be detrimental for humans in elevated doses when inhaling for long-term exposure. Numerous reports support that content of preformed antifungal compounds correlates with disease resistance, for example, the fall of preformed antifungal compounds in strawberry fruits was correlated with a decline in natural disease resistance against Botrytis cinerea (Terry et al. 2004). Analogies of medicine frequently used as some botanical preparations are traditionally applied against dermatomycoses. However, the decision on therapeutic value is different: In medicine, some iatrogenic effect might be accepted, for example, the drug applied more harmful to cancer cells than regular cells, or the use of arsenic derivatives to eliminate parasitic protozoans. In these cases usually, the ration of ED50 or LD50 values is used. Contrarily, the adverse effects in the case of host/parasite pairs are rarely accepted, and the ration of maximum tolerated dose by host plant and minimum inhibitory dose for pathogen should be taken (Table 11.5); moreover, the decisionmaker should take account of suspected knowledge of users when recommends dose for practical applications, i.e., the three- to fivefold overdose cannot harm the exposed cultivated plant.

The separation and identification of active principles of herbs important as well as the use of well-identified molecules have advantages. However, the crude extracts and herbal material per se often differ in the activity being the latter more effective (Al-Sohaibani et al. 2011). The preparation may destroy the active principle, or separate synergically interacting substances (Kapoor et al. 2008). The content of single molecules and their ratios often differ batch to batch, which shows similar affectivity due to synergic interaction of component. This fact indicates that the use of homemade crude preparations may have advantages in special regards in microscale applications.

Dramatic advancement in biology can be seen within the last fifty years. The contemporary plant biology, which led through meristem culture to the clonal propagation as well as these procedures led to tissue culture techniques, which were utilized to grow cells in suspension cultures with subsequent ability to regenerate whole plants that created a whole new era in plant biology. Some efforts have been made, and there is an increasing interest to introduce alien genes coding performed defense molecules into cultivated plants. These new properties also should be approved by selectivity criteria that are identical to those requested in the case of synthetic pesticides (Table 11.2). The experiences are contradictory: Unexpected adverse effect has manifested both in biocoenoses and in pests themselves, mainly due to acquired tolerance in populations of target organisms, like Lepidopteras to thuringiensis toxins or innumerable weed species to herbicides. No doubt about that microbes of agricultural interests will also rapidly adapt to new properties. The introduction of toxic substances alien to edible plants can also induce serious damages as it was demonstrated in the case of galanthus toxin—all this underlines that the soft practice of EPA cannot be kept when the registration of biopreparations for large-scale use takes place. Nevertheless, the intentions to improve the plant resistance by rationally designed genetic manipulations using biotech methods are promising together with to develop botanical preparations to combat losses in agriculture (Table 11.6).

Table 11.6 Expected application of gene engineering to approve efficiency of pest control by biorational way
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Some questions need answers, first of all problems of unwanted exposures. In spite of intensive studies on defense molecules our knowledge regarding their mode of action and the flow of signal transmitters from the pathogen to the plant cells is still poor. The protective functions are highly diversified, and the variegation of defense mechanism shows multifunctional character. The exposed population, being not uniform genetically, is a mixture of strains as well as the ratio of different isomers can vary in botanical preparation depending on source and mode of manufacturing, the strain-specific action and stereometric-dependent response may limit the usefulness of herbal preparations. Nevertheless, studies on the biological activities of herbs are increasingly important in the search for natural and safe alternative pesticides in recent years. There is a lot of to be done before their use in large scale. More in-depth knowledge of potentially useful plants can provide results of economic importance for food and even pharmacological industry.

The abundant use of antimicrobial agents resulted in the emergence of drug-resistant bacteria, fungi, and viruses both in medicine and agriculture. To overcome this threat, there is necessary to find new, effective antimicrobial agents with novel modes of actions. The plant defense molecules are promising candidates for lead compounds. Some compromise among yield sureness, quality, and number of products is requested for making the biorationally designed and carefully selected new varieties.