**9. Conclusions**

The study of rehabilitation of masonry involves a team of specialists from historians, architects, structural engineers, geotechnical and chemical technicians, etc. That is, it cannot be considered only as a structural problem.

The seismicity of the site and the abandonment of the old buildings have caused the collapse of most of the old buildings, leading to the loss of cultural values that have been part of the local history. Therefore, the rehabilitation of old buildings should be considered a state policy, in order to preserve the few buildings that remain for the future.

It is emphasised that in the region with near-source earthquake, historic buildings that have been standing are made up of ceramic solid bricks; only very few of adobe and stone have managed to survive due to the high demand for ductility of earthquakes near-fault.

Modelling by MEF applying nonlinear constitutive models provides an effective tool for the simulation and verification of historic masonry heritage buildings, so it is necessary to research the formulation of efficient constituent models for thick masonry.

The monitoring through environmental vibration measurement has been a useful tool to evaluate the level of recovery of construction, allowing in the future to evaluate the state of conservation of the same. Model calibration is possible from frequency identification.


**2.3. Cation exchange capacity**

One of the important properties of the clay minerals is that contains cations that can be exchanged for any guest species of anion or cation by treating clay mineral with such clay mineral. The exchangeable cations are held on the outside of silica-alumina clay mineral structural units and the exchange does not affect the layout of the silica-alumina units [11]. The ion exchange capacity phenomenon is measured in terms of milli-equivalents per gram or per 100 g. The commonly used cations used to evaluate the cation exchange capacity of the clay mineral includes; Mg2+, Ca2+, K+ , Na+ , H+ and NH4 + [16].

**Figure 3.** Typical structure of 2:1 clay [19].

Different clay minerals portray different cation capacity depending on the substitution within the lattice structure [11]. Type 1:1 clay minerals such as kaolinite have limited substitution between their lattice structure compared to type 2:1 clay minerals such as smectite, vermiculite and sepiolite and consequently they have lower cation exchange capacity [10]. Several studies have indicated that cation exchange capacity of the clay mineral decreases after modification by inorganic species [2, 20, 21]. This is because modification involves the ion exchange reaction between the exchangeable cations in the clay interlayer and the guest species. Low CEC of modified clays suggests the irreversibility of cationic exchange and thus intercalated metallic polycations are hardly exchanged [21]. **Table 1** summarizes the cation exchange capacity of different raw and modified clay minerals.


The specific behavior of aromatic amines in the coordination and extraction processes of isolation and separation of platinum and other metals is discussed using the example of 2(4)-aminopyridines (2(4)-AP). As intrasphere ligands, 2(4)-AP have a high electron-donor capacity due to the pumping of an easily polarizable π-electron density. The chemistry of the extraction of platinum metals, iridium in particular, is considered: depending on the conditions, ion associates, coordination-solvated compounds or compounds containing an amine in the inner and outer coordination sphere of the metal are extracted. In the extraction of simple singly charged anions, there is a violation of the exchangeextraction series established for a large set of aliphatic amines. Soft anions (according to Pearson), for example, SCN- and I-, are best extracted, while for aliphatic amines such an anion is hard СlO4 . In the coordination compounds of platinum metals, 2(4)-AP acts as an electron donor, is coordinated by heterocyclic nitrogen with a redistribution of electron density not only to the accepting metalcomplexing agent, but also further along the N-Me-X chain (X is an acido ligand in the composition of the complex), which leads to even greater covalence of the molecule as a whole.

**Keywords:** 2(4)-aminopyridines, platinum metals, extraction, complex formation, coordination compounds

#### **1. Introduction**

In recent years, interest has increased in the study of the extraction properties of high-molecular-weight aromatic amines, primarily because 2(4)-octylaminopyridines turned out to be good extractants for the isolation and separation of platinum metals [1–3]. Particularly interesting is the question of the specificity with respect to platinum metals of aromatic amines, as ligands, which differ from aliphatic amines in that the lone pair of electrons of the nitrogen atom largely acquires an π-donor character. Compared to aliphatic amines, aromatic amines demonstrate a number of new properties in coordination and extraction chemistry [2, 4]. All this determined the interest in this class of extractants, typical representatives of which are 2(4)-octylaminopyridines (2(4)-OAP).

Research carried out by the authors [5–9], allow us to get an idea of the specifics of the behavior of 2(4)-aminopyridines in the coordination and extraction processes of isolation and separation of metals.

#### **2. Specificity 2(4)-aminopyridinesas ligands**

The specificity of 2(4)-aminopyridines as ligands is due to the nature of the nitrogen atom in aromatic amines, which can be judged from the results of studies of halides [8] and coordination compounds of 2-OAP with nickel, palladium, and platinum [9]. In metal complexes, 2-OAP, as an intrasphere ligand, has a high electrondonating capacity due to the pumping of an easily polarizable π-electron density. The mobility of the electron density in the 2-OAP molecule depending on the requirements of the acceptor is evidenced by the delocalization of the positive charge of the proton in the cation (outer sphere ligand), which is the higher, the greater the polarizability of the anion [8].

An idea of the mobility of the electron density in a 2(4)-OAP molecule can be obtained within the framework of the theory of limiting structures (the theory of resonance) [10], giving an account of a certain formalism of this theory. The conclusions obtained in the framework of the theory of resonance and the theory of perturbations of molecular orbitales (PMO), which have a deep quantum-chemical substantiation [11], are quite adequate.

Conventionally, the 2-OAP molecule (**Figure 1**) [2], as well as the 4-OAP molecule (**Figures 2** and **3**) [5], can be represented as an average between the amine and pyridonimine limiting structures.

The contribution of the pyridoniminine structure increases the electron density on the heterocyclic nitrogen and decreases the electron density on the amine nitrogen. This contribution can be estimated if the energy of the N1s level of heterocyclic and amine nitrogen atoms is known. **Figure 4** shows, as an example, the experimental X-ray electron spectra of the N1s level of 4-OAP with band separation obtained on a

*2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

**Figure 2.**

*Limiting (resonance) structures of 4-OAP molecules and the effective charges on nitrogen atoms calculated for them.*

#### **Figure 3.**

*Limiting (resonant) structures in protonated 4-OAP and effective charges on nitrogen atoms calculated for them.*

Riber SIA-200 X-ray photoelectron spectrometer. It can be seen that the nitrogen atoms are not equivalent, the lower level refers to heterocyclic nitrogen.

The energy of the N1s level correlates with the effective charge on the nitrogen atom. Satisfactory correlation of these values for a large group of nitrogen-containing compounds of various structures was obtained in [12]. Effective charges on nitrogen atoms for limiting structures can be calculated using the concept of ionic nature (**Figures 2** and **4**) [10]. From the charge balance equations for nitrogen atoms, the contribution pyridoniminine structure in 2-OAP and 4-OAP molecules: 9 [2] and 48.6%, respectively.

Thus, in the first approximation, it can be assumed that 2(4)-OAP molecules represent a resonant structure with a contribution from the pyridoniminine component. This leads to an increased basicity of heterocyclic nitrogen compared to pyridine

#### **Figure 4.** *X-ray spectrum of 4-OAP. Peak area: 1–50.63, 2–49.37%.*

due to the pumping of electron density from the amino group in the ortho and para positions of the pyridine ring and partial delocalization of the charge in the cation. Since the "depth" of the resonance is higher in the case of 4-OAP, its basicity exceeds that of 2-OAP by two orders of magnitude.

All this points to the "soft" nature of 2(4)-AP as ligands. If we use Pearson's classification [13], then free 2(4)-AP should be attributed to "soft" bases (inner sphere ligand), protonated to "soft" acids (outer sphere ligand). Soft and intermediate bases include other aromatic amines, while aliphatic amines are "hard" bases.

Factors such as the energetic and spatial arrangement of the top donor orbital of the nitrogen atom are thought to be responsible for the "soft" or "hard" behavior of the amine. From the standpoint of the quantum theory of perturbed molecular orbitals (PMO), one can consider the energy of the metal–ligand interaction and the resulting extraction chemistry depending on the nature of the amine, metal, and extraction conditions [14].

The formation of a coordination-solvated compound or associate, where the metal is present in the composition of the acid complex, depends on the result of the competitive process of complexation of the amine and proton, on the one hand, and the metal, on the other. In the first approximation, the quantitative side of this process is expressed by the main equation of the PMO theory [14]. An ionic associate or a coordination-solvated compound is formed depending on the relative contribution of the Coulomb or covalent component to the metal-nitrogen interaction energy.

If the contribution of the covalent component is much greater than that of the Coulomb component, then the coordination of the amine by the metal in the presence

#### *2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

of a proton is possible. The higher the energy of the donor orbital and the lower the energy of the acceptor orbital of the amine and metal, the greater the contribution of the covalent component. These energy parameters of the interacting orbitals within the PMO are characterized by the orbital electronegativity of the donor and acceptor according to Klopman [13]. In addition, the covalent component increases with the length of the amine donor orbital.

If we talk about the nature of the amine, then in the presence of a proton, only amines with a low orbital electronegativity of the lone electron pair (OELEP), which depends on the valence state of the nitrogen atom in the amine molecule, can be coordinated by the metal. The OELEP of nitrogen decreases with an increase in the ρand π-character of an unshared pair of electrons, that is, with a decrease in the energy of the donor orbital and with an increase in its population [13]. Consequently, the OELEP of nitrogen decreases as one goes from aliphatic amines to anilines and further to heteroaromatic amines. In the same series, the softness of amines and their ability to extract platinum metals in the form of coordination-solvated compounds increase.

Of no less interest is the behavior of protonated 2(4)-AP, which acts as an outersphere ligand with respect to acid complexes of platinum and other rare metals.

#### **3. Chemistry of metal extraction**

2(4)-OAP extract metals from acidic and slightly acidic solutions. The extraction of iridium and other platinum metals has been studied most fully [2, 6].

Iridium (III) is extracted with a 0.1 M solution of 2-OAP in chloroform from dilute hydrochloric acid solutions with distribution coefficients D = 100–200, however, a nonequilibrium minimum appears on the curve D = f (рН) at pH 2 (the contact time of the phases is 30 min). During extraction from 1 to 6 M HCl equilibrium is established slowly: the value of D increases by almost an order of magnitude with an increase in the duration of phase contact up to 50 hours. Iridium (IV) is reduced to iridium (III) during extraction. 4-OAP extracts iridium (IV) with high distribution coefficients from more acidic solutions [3].

When 2-OAP is introduced directly into the aqueous phase (0.1 M solution in acetone) and the solution is heated to boiling in the presence of a tin (II) chloride catalyst for 30–40 min followed by extraction of the resulting compounds with chloroform ("heterogeneous" extraction), iridium is extracted with an unusually high partition coefficient for this element. The maximum extraction is observed from 1 to 2 M HC1 and reaches 99.9% for a single extraction.

Other platinum metals, as well as Au, under conditions optimal for the extraction of iridium, are extracted much worse (**Table 1**), and gold is quantitatively, and silver and palladium are partially concentrated at the phase boundary. The distribution coefficient of non-ferrous metals and iron, from which iridium usually needs to be separated, under these conditions by 3–5 orders of magnitude lower than iridium. Of these elements, only copper in the form of Cu (II) passes into the organic phase in a noticeable amount. At a high concentration of SnCl2 in the aqueous phase, the organic phase contains tin.

Since alkylated 2(4)-AP strong organic bases, they are able to extract halide and other metal acid complexes in the form of ion associates.

On the other hand, 2(4)-OAP can be considered as a potentially coordinatingactive reagent due to the presence of heterocyclic aromatic nitrogen. In addition, during extraction, the formation of chelates due to the NH2 group in α-position to the

**Table 1.**

*Distribution coefficient of some metals in the extraction of 2-OAP under iridium extraction conditions: Ir, Pt, Au, Ag 1104 -1105 M; Rh, Pd 5104 ; 0.05 M 2-OAP, 0.1 M SnCl2, heating for 40 min at 100°C, phase contact for 15 min, organic phase – Chloroform.*

heterocyclic nitrogen. In the course of extraction, one or another mechanism is realized depending on the conditions [13].

*Extraction of ion associates***.** In the form of ionic associates, platinum metals are extracted from HC1 solutions at a certain ratio of the concentration of components and the duration of phase contact [2]. Palladium (II) is extracted by 2-OAP predominantly in the form of an associate (OAPH+ )2[PdCl4] only from concentrated solutions of HC1 and when organic diluents are used solvents with a strong protondonating ability. Platinum is extracted in the form of such a complex already from acidic solutions of HC1. Ir (III, IV) are predominantly extracted in the form of ionic associates of the composition (OAPH+ )2[IrCl6] and (OAPH+ )3[IrCl6] from 1 to 6 M HC1, especially with a short duration of phase contact.

According to this mechanism, Pd (II) is extracted from salicylate solutions with 4 heptylaminopyridine [15], and from oxalate solutions with 4-dodecylaminopyridine [16]. Ro (III) is extracted from citrate solutions with 2-dodecylaminopyridine [17], and from Ru (III) succinate solutions with 2-OAP [18]. From acetate solutions, 2-OAP extracts Ir (III) [19] from malonate – Au (III) [20]. 2-OAP and other metals are extracted in the form of ionic associates from chloride, malonate, succinate, salicylate, citrate media: To (IV) [21], Zr (IV) [22], V (V) [23], Mo (VI) [24], Cr (VI) [25], Bi (III) [26], Ga (III) [27], Tl (III) [28], Sm (III) [29], Hg (II) [30].

Anions of inorganic acids are also extracted as ionic associates [8].

*Extraction of coordination-solvated compounds.* Сu (II) is extracted with a solution of 2-OAP in chloroform from a neutral medium in the presence of at least 1 g-ion/ l chloride ion, apparently in the form of a neutral coordination-solvated complex. In the case of Pd (II), a neutral diamine complex of the composition Pd(OAP)2C12 is formed during extraction from solutions with a concentration of HC1 ≤ 3 M, Pt (II) in the form of Pt(OAP)2Cl2 from weakly acid solutions of HC1 (pH >1.5); the phase contact duration is 30 min [2]. Most often, the organic phase contains compounds with 2(4)-OAP in the inner and outer coordination spheres of the metal ("mixed" extraction mechanism).

*Mixed extraction mechanism.* Iridium, under conditions optimal for its extraction, is extracted in the form of compounds containing 2-OAP in the inner and outer coordination spheres of the metal [7]. In addition to 2-OAP, the extractable compounds include SnCl2, which is added to overcome the kinetic inertness of the initial

*2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

complex iridium chlorides [6]. Complexation in the aqueous phase and subsequent extraction of the resulting compounds are described by the following Equations [7]:

$$\left[\mathrm{IrCl\_6}\right]^{3-}\mathrm{\tiny{m}} + \mathrm{m}[\mathrm{SnCl\_3}]^{-}\mathrm{\tiny{\tiny{\tiny{\tiny{\tiny{\tiny{\tiny{\tiny{\tiny{\tiny{\tiny{\tiny{\Gamma\_{\mathrm{Br}}}}}}}}}}\mathrm{I}}\mathrm{Cl\_{6-m}}}\mathrm{I\_{6-m}}\right]^{3-}\mathrm{\tiny{\tiny{\tiny{\tiny{\rm{\tiny{\rm{\tiny{\rm{\tiny{\rm{\tiny{\rm{\rm{\tiny{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\rm{\cdots}}}}}}}}}}}}}}}}}}}}}}}}}}}}}} $$

$$\begin{aligned} \left[\text{Ir}(\text{SnCl}\_3)\_\text{m}\text{Cl}\_{(6-\text{m})}\right]^{\mathfrak{J}-}\text{w} + \text{xO}\text{AIIH}^+\text{w} &\to \left[\text{Ir}(\text{SnCl}\_3)\_\text{m-x}\text{ (OAlI)}\_\text{x}\text{Cl}\_{(6-\text{m})}\right]^{\mathfrak{x}-\mathfrak{J}}\text{w} \\ + \text{xH}\_\text{w} + \text{xCl}\text{B} &\to \left[\text{SnCl}\_3\right]^-\text{w} \end{aligned} (2)$$

$$\begin{aligned} & \left[ \text{Ir} (\text{SnCl}\_3)\_{\text{m}-\text{x}} \left( \text{OAII} \right)\_{\text{x}} \text{Cl}\_{(\text{6-m})} \right]^{\text{x}-\text{3}} + (\text{x} - \text{3}) \text{OAII} \cdot \text{HCl}\_{\text{o}} \\ & \rightarrow (\text{OAIII}^{+})\_{\text{x}-\text{3}} \left[ \text{Ir} (\text{SnCl}\_3)\_{\text{m}-\text{x}} \left( \text{OAII} \right)\_{\text{x}} \text{Cl}\_{(\text{6-m})} \right]^{\text{x}-\text{3}} + (\text{3} - \text{x}) \text{Cl}^{-} \text{w} \end{aligned} \tag{3}$$

Here x = (0–2), m = 1–6; component concentration interval: 1�10�5 -1�10�3 g-at/l Ir; 0.05–0.2 M; **SnСl2** ≤ 0.1 M 2-OAP in acetone; 1–6 M HCl.

The ratio of ligands in the inner coordination sphere of iridium is determined by the concentration of the components in the specified range, as well as the temperature and duration of heating the solution before extraction. At a low concentration of 2-OAP, along with coordination-solvated compounds, anionic iridium chlorotin complexes are extracted that do not contain 2-OAP in the inner coordination sphere of the metal. The ratio between these two types of extractable compounds under these conditions can be estimated from the results of a physicochemical study of the extraction of iridium in the presence and absence of OAP in the aqueous phase upon heating [7].

The given chemistry of iridium extraction is confirmed by the study of extracts by high-voltage electrophoresis on paper and iridium compounds isolated from the extract using physicochemical and spectral methods of analysis [7].  The total content of the organic component (C, H, N), according to elemental analysis, is 41.95%, which indicates a high molecular weight of the anionic part of the associate; indirectly indicates the presence of tin. Direct evidence for the presence of tin in the complex is the Mossbauer spectrum of the compound on 119Sn nuclei, which is characteristic of the [SnCl3] � ligand in the iridium coordination sphere (chemical shift 1.65 mm/s, quadrupole splitting 2.33 mm/s). Significant quadrupole splitting in the Mossbauer spectrum of the compound indicates the presence of 2-OAP in the inner coordination sphere of the metal.

This conclusion most convincingly follows from the data of PMR spectroscopy of substances before and after electrophoresis: in the spectrum of the substance after the separation of the cationic part, signals from the protons of the hetero ring and the octyl radical are clearly recorded; 2-OAP is indeed part of the anionic part of the associate and is coordinated by iridium. If we take into account the results of elemental analysis (32.38% C, 4.70% H, 4.87% N), then the probable composition of the compound is (OAPH+ )[Ir(OAP)2(SnCl3)3C1]�, possibly impurity of the complex (OAPH+ )2[Ir(OAP)(SnCl3)2Cl3] 2�.

Compounds containing OAP in the inner and outer coordination spheres of the metal can be extracted without preliminary heating of the metal solution with OAP in the aqueous phase, if its kinetic inertness is relatively low. In particular, the results of the study of platinum extracts using electron spectroscopy and thin layer chromatography [2] can be explained if the presence of the associate (OAPH+ )[Pt(OAP)C13] – is assumed in the organic phase.

In principle, more than two molecules of 2(4)-AP can enter into the coordination sphere of a metal. In this case, the formation of complexes containing the metal in the

**Figure 5.**

*Electrical conductivity of 1 104 M aqueous solution of K2[PdCl4] depending on the metal/amine molar ratio with the addition of: 1–2-AP; 2–4-AP.*

#### **Figure 6.**

*Electrical conductivity of 1 10–4 M aqueous solution of K2[PtCl4] depending on the metal/amine molar ratio with the addition of: 1–2-AP; 2–4-AP.*

cationic form is not excluded. Under the conditions of extraction of iridium, such compounds should precipitate at the phase boundary, which is observed in the extraction of palladium, as well as gold and silver, and only if 2-OAP is present during heating in the aqueous phase [2].

Another proof of the possibility of the formation of cationic complexes are the results of conductometric and spectrophotometric studies of complex formation 2(4)- AP with Pd (II), Pt (II) in aqueous solutions at concentrations of reagents 1105 - 1104 M, simulating the extraction conditions (**Figures 5** and **6**). The conductometric curves χ = f (CAm/CMe) show breaks at CAm/CMe = 2 and 4.

Thus, the chemistry of 2(4)-AP metal extraction can be quite complex. Depending on the nature of the metal and extraction conditions, associates containing 2(4)-AP only in the cationic part, and the metal in the anionic part, associates with OAP in the inner and outer coordination spheres of the metal, neutral coordination-solvated compounds can pass into the organic phase; the formation of cationic complexes is also not excluded.

#### **4. Interionic interactions in 2(4)-AP associates**

Extraction of hydrochloric acid with a solution of 2(4)-OAP in chloroform according to the neutralization mechanism is described by the equation:

*2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

**Table 2.**

*Distribution constants of 2-AP and 4-OAP salts between chloroform and water (25 2° С, μ 1) and thermodynamic characteristics of anion hydration in infinitely dilute solutions at 298°K (\* calculated by correlation dependence ΔSh = f (R, Аo ), where R radius of ions in water.*

Кex = KАmH+Cl-(KaКD) 1 , where Кex is the HCl extraction constant, KАmH+Cl- is the chloride distribution constant, Ka is the ionization constant of the protonated amine, КD is the amine distribution constant.

2-OAP chloride with an intramolecular hydrogen bond passes into the organic phase and contains practically no water molecules, which apparently explains the low over stoichiometric extraction only from 12 M HCl [1]. On the contrary, for 4-OAP, a high over stoichiometric extraction is observed already from 6 M HCl, since in this case the chelate cycle based on the intramolecular hydrogen bond is not formed [5].

In the extraction of simple singly charged anions, there is a violation of the exchange-extraction series established for a large set of aliphatic amines. This conclusion follows from the data on the distribution constants of 2-aminopyridine [8] and 4- OAP salts between chloroform and water (**Table 2**), according to which, according to the extractability of 2(4)-OAP, singly charged anions are arranged in a row:

$$\mathrm{F^{-}} < \mathrm{Cl^{-}} < \mathrm{NO\_{3}}^{-} < \mathrm{Br^{-}} < \mathrm{ClO\_{4}}^{-} < \mathrm{SCN^{-}} < \mathrm{I^{-}}$$

Soft anions (according to Pearson) are best extracted: SCN and I, while for aliphatic amines such an anion is hard СlO4. In addition, it is well known that for aliphatic amines there is a linear correlation between the exchange constants of singly charged anions and the extraction constants of monobasic acids with the heat of hydration of the anion or the free energy of hydration. In the case of 2(4)-OAP, such a correlation is observed separately in the series Br < SCN < I and F < Сl < NO3 < СlO4 , but not for the entire series as a whole (**Figures 7** and **8**).

The study of 2-OAP halides by PMR, IR and X-ray electron spectroscopy showed [8] that they all have a structure similar to chloride (**Figure 9**):

The specificity of the interionic interaction in 2(4)-OAP associates manifests itself in a decrease in the polarization of the n-electron cloud of the aromatic cation, depending on the nature of the anion, on the one hand, and the formation of a chelate cycle based on hydrogen bonds in the case of 2-OAP with another. The data of IR spectroscopy indicate that the strength of the chelate ring in the case of 2-OAP decreases on passing to an anion with better extractability [8]. Consequently, the selectivity of the extraction of soft anions is due to the redistribution of the electron density in the aromatic cation, depending on the nature of the anion. Degree of

**Figure 7.** *Dependence of distribution constants of 2-aminopyridine salts on free energy anion hydration.*

**Figure 8.** *Dependence of distribution constants of 4-OAP salts on free energy anion hydration.*

indignation π-electron cloud of an aromatic cation can be quantified by the degree of charge delocalization (α) in the cation according to the data of X-ray electron or NMR spectroscopy [8]. For 2-OAP it is 90, 56, 54 and 48% in the series I, Br, Cl, [GaCl4] , and for 4-OAP it is 90, 79, 65% in the series I, Br, Cl, respectively.

The distribution constants of 2-aminopyridine halides increase with increasing α.

Other processes involving aromatic cations show similar phenomena. In particular, on the surface of micelles RPy+ X (RPy+ long chain alkyl pyridinium ion; X anions of different nature) in an aqueous solution, an interaction with charge transfer was found for soft anions, which increases in the series Br < SO3 2 < N3 < I < S2O3 2 according to an increase in the softness of the anion. Similarly, the interaction in the series Cl < Br < I is observed for ion pairs in chloroform and is absent in the case of hard СlO4 . Charge transfer in ion pairs and on the surface of micelles is absent in the case of hard tetraalkyl- and tetraphenylammonium cations with soft Br and I [31]. The charge transfer is due to the mixing of the wave functions of nearby excited ones with the wave function of the ground state. Since in an ion pair the ground state is charged, and the excited neutral, then it should be recognized that in the associates of a soft cation, for example, an OAPH+ or RPy+ cation with a soft anion, there is a covalent contribution (delocalization energy in terms of MO). This contribution is absent in associates with a hard cation, for example, the cation of an

*2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

**Figure 9.** *3D structure of 2-OAP chloride.*

aliphatic amine. This is also confirmed by the results of the study of 2(4)-OAP associates.

#### **5. Structure of coordination compounds**

The extraction of platinum metals by 2(4)-OAP in the form of coordinationsolvated compounds is highly selective with respect to non-ferrous metals, in particular with respect to nickel. Therefore, it is of interest to study the structure of coordination compounds of the isovalent and isoelectronic series of metals with the composition MeCl2(OAP)2, where Me = Ni, Pd, Pt, i.e. complexes that pass into the organic phase during the coordination extraction of Pd and Pt with a solution of 2- OAP in chloroform.

The complexes were synthesized according to specially developed procedures [9]. Their composition was confirmed by the results of elemental analysis and the properties of the complexes. The formal oxidation state of the central atom of the complexes

#### **Table 3.**

*Binding energy (eV 0.1) of internal electrons of metal and ligands in Ni, Pd, Pt complexes with 2-OAP.*

is +2, which follows from X-ray electron spectroscopy data from the ionization energies of the Ni 2p3/2, Pd 3d5/2 and Pt 4f7/2 levels (**Table 3**).

The chlorine ion is a part of the coordination sphere of the central atom, which is due to the relatively high energy of the 2p3/2 level C1 in comparison with the corresponding energy values for ionically bound chlorine in the 2-OAPHC1.

Formally, 2-OAP and 2-AP are ambidentate ligands with two donor nitrogen atoms, the heterocyclic nitrogen of the pyridine ring and the nitrogen of the amine group located in the α-position. The results of electron, IR, and NMR spectroscopy testify to the mode of coordination of 2-OAP by the metal [9]. 2-OAP and 2-AP are coordinated by Pd and Pt at the nitrogen of the heterocycle; The α-amino group does not interact directly with the metal. However, the spatial arrangement of the amino group, as well as the fact that coordinated chlorine has an excess negative charge, contribute to the formation of a chelate cycle due to the intramolecular H-bond, as in the case of associates (**Figures 10** and **11A**). The chelate cycle is absent in Pd and Pt complexes with 4-AP (**Figure 11B**).

**Figure 10.** *3D structure of the Pd complex with 2-OAP.*

*2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

**Figure 11.** *3D structures of Pt(II) complexes: A – 2-AP, B – 4-AP.*

The extraction of coordination-solvated complexes can be considered from the point of view of the formation of electron-donor-acceptor complexes by neutral halide complexes with the electron-donor OAP molecule. The results of X-ray electron spectroscopy indeed show that the binding energy of the N1s electrons of the nitrogen atom decreases upon passing from free 2-OAP to the complex for all the studied metals; 2-OAP is primarily an electron donor, and the energies of the N1s electrons of the aromatic and aliphatic nitrogen atoms are equalized during complexation. Based on the change in the energy of the N 1 s level of OAP during complex formation, the acceptor ability of Ni is significantly higher than the acceptor ability of Pd and Pt in the corresponding halides.

It is interesting to compare the electron ionization energy from the 2p3/2 level of chlorine in the compounds MeCl2(OAP)2 and MeCl2(NH3)2, where Me = Pd, Pt [32], in compounds in which the central atom in one case forms a bond with a heterocyclic nitrogen 2-OAP, and in another with ammonia nitrogen (the most rigid aliphatic amine). In complexes with 2-OAP, these values are much smaller; the electron density initially localized on the donor nitrogen atom is not only and not so much directly redistributed to the accepting complexing metal, but also further along the N—Me— C1 chain, which leads to an even greater covalence of the molecule as a whole. It is noteworthy that, in this respect, the complexes of palladium with OAP are similar to the complexes with triphenylphosphine and diphenylthiourea [33] other soft ligands.

#### **6. Conclusion**

The specific behavior of aromatic amines is considered.in coordination and extraction processes for the isolation and separation of platinum and other metals on the example of 2(4)-aminopyridines (2(4)-AP). Toas intrasphere ligands2(4)-APhave a high electron-donating capacity due to the pumping of an easily polarizable πelectron density. In a protonated amine, electron density mobility is accompanied by delocalization of the positive proton charge over the ligand molecule, depending on the requirements of the acceptor. The degree of delocalization is the higher, the greater the polarizability of the anion. Chemistry of extraction of platinum metals2

(4)-AP, iridium in particular, can be quite complex. Depending on the nature of the metal and the extraction conditions, associates containing 2(4)-AP only in the cationic part, and the metal in the anionic part, associates with 2(4)-octylaminopyridine in the inner and outer coordination spheres of the metal, coordination neutral - solvated compounds; the formation of cationic complexes is also not excluded.

In the extraction of simple singly charged anions, the exchange-extraction series established for a large set of aliphatic amines is violated. Mild anions (according to Pearson), SCN- and I-, for example, are extracted best. For aliphatic amines, this anion is hard СlO4-. In coordination compounds of platinum metals, 2(4)-APact as an electron donor, coordinate on heterocyclic nitrogenwith the redistribution of the electron density not only to the accepting metal-complexing agent, but also further along the chain N—Me—X (X-acid ligand in the complex), which leads to an even greater covalence of the complex.

### **Author details**

Liliya Sergeevna Ageeva\*, Nikolai Alekseevich Borsch and Nikolay Vladimirovich Kuvardin Southwest State University, Kursk, Russian Federation

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

#### **References**

[1] Borshch NA, Petrukhin OM. 2-Octyla minopyridine - a new extraction reagent. J. Analyt. Chem. 1978;**33**(9):1805-1812

[2] Borsch NA. Development of methods for the extraction isolation of iridium a nd other platinum metals. Moscow: Institute of Geochemistry and Analytical Chemistry, Academy of Sciences of the USSR; 1978

[3] Seregina NF, Petrukhin OM, Formanovsky AA, Zolotov YA. 4-Octyla minopyridine - extraction reagent for iridium. Reports of the Academy of Scie nces of the USSR. 1984;**275**(2):385-387

[4] Petrukhin OM, Borshch NA, Zolotov YA. International Solvent Extraction Conference. Vol. 3. Liege: ISEC; 1980. p. 200

[5] Borshch NA, Ageeva LS, Frolova AY. Effect of 4(2)-octylaminopyridine d istribution in a water (HCl)/chloroform system on the extraction of metal ions f rom chloride solutions. Russian Journal of Physical Chemistry A. 2019;**93**(5): 828-834. DOI: 10.1134/S00360244190 50066

[6] Borshch NA, Petrukhin OM. Extrac tive preconcentration of iridium and rh odium with 2-octylaminopyridine. Journal of Analytical Chemistry. 1978; **33**(11):2181-2190

[7] Borshch NA, Petrukhin OM, Sokolov AB, Marov II. Study of the chemistry of iridium extraction with 2 octylaminopyridine. J. Inorganic Chem. 1981;**26**(3):734-743

[8] Borsch NA, Maltseva NG. Specificity of interionic interaction in associates of 2-octylaminopyridine and some aspects of selective extraction of inorganic anions with aromatic amines. Journal of

Inorganic Chemistry. 1982;**27**(9): 2355-2363

[9] Borshch NA, Petrukhin OM, Zolotov Yu A, Zhumadilov EG, Nefedov VI, Sokolov AB, et al. Coordination compounds of platinum (II), palladium (II) and nickel (II) with 2 octylaminopyridine. Journal of Coordination Chemistry. 1981;**7**(8): 1242-1249

[10] Pauling L. The Nature of the Chemical Bond. 3rd ed. New York; 1960. p. 97

[11] Dewar M, Dougherty R. Perturbation theory of molecular orbitals in organic chemistry. The Journal of Chemical Physics. 1977;**106**:695-711

[12] Nordberg R, Albridge RG, Bergmark T, Ericson U, Hedman J, Nordling G, et al. Ark. Kemi. Vol. 28. Almquist & Wiksells Boktryckeri; 1967. p. 257

[13] Klopman GJ. Chemical reactivity and the concept of charge- and frontiercontrolled reactions. Journal of the American Chemical Society. 1968;**90**:223

[14] Klopman GN. Reactivity and Ways of Reactions. Moscow: Mir; 1977. p. 384

[15] Khogare BT, Anuse MA, Piste PB, Kokare BN. Development of a solvent extraction system with 4 heptylaminopyridine for the selective separation of palladium (II) from synthetic mixtures, catalysts and water samples. Desalination and Water Treatment. 2015;**57**:1-11. DOI: 10.1080/ 19443994.2015.1124054

[16] Umrao B. Shep, Rucha K. Pawar, Balasaheb R. Arbad, Solvent extraction of palladium (II) from oxalate medium by 2-dodecylaminopyridine. Chemistry & Biology Interface. 2016;**6**(6):379-387

[17] Shep UB, Bagal MR, Arbad BR. 2- Dodecylaminopyridine assisted solvent extraction system for selective separation of rhodium (III) ion-pair complex from synthetic mixtures. J. Materials and Environmental Sciences. 2017;**8**(8):2894-2902

[18] Suryavanshi VJ, Patil MM, Zanje SB, Kokare AN, Gaikwad AP, Anuse MA, et al. Development of liquid-liquid extraction and separation method for ruthenium (III) with 2- Octylaminopyridine from succinate media: Analysis of catalysts. Russian Journal of Inorganic Chemistry. 2017; **62**(2):257-268. DOI: 10.1134/ S003602361702019X

[19] Suryavanshi VJ, Patil MM, Zanje SB, Kokare AN, Kore GD, Mansing A, et al. Extraction of iridium(III) by ion-pair formation with 2-octylaminopyridine in weak organic acid media. Separation Science and Technology. 2016;**51**(10): 1690-1699. DOI: 10.1080/ 01496395.2016.1177076

[20] Suryavanshi V, Kokare A, Zanje S, Mulik A, Pawar R, Patil M, et al. Ion-pair based liquid–liquid extraction of gold (III) from malonate media using 2 octylaminopyridine as an extractant: Analysis of alloys, minerals, and drug samples. Turkish Journal of Chemistry. 2018;**42**:1032-1044. DOI: 10.3906/kim-1712-34

[21] Kore GD, Patil SA, Anuse MA, Kolekar SS. An extractive studies on behavior of thorium (IV) from malonate media by 2-octylaminopyridine: A green approach. J. Radioanalyt. and Nucl. Chem. 2016;**310**(1):329. DOI: 10.1007/ s10967-016-4857-7

[22] Noronha LE, Kamble GS, Kolekar SS, Anuse MA. Solvent extraction of zirconium (IV) with 2-octilaminopyridine from succinate media analysis of real samples. Ind. J. Chem. Technology. 2013; **20**(7):252-258

[23] Noronha LE, Kamble GS, Kolekar SS, Anuse MA. Extractive separation of vanadium.(V) from succinate medium by solvent extraction using 2-noctylaminopyridine. Int. J. of Analyt. And Bioanalyt. Chemistry. 2013;**3**(7): 27-35

[24] Noronha LE, Kamble GS, Kolekar SS, Anuse MA. Recovery of molybdenum (VI) from hydrochloric acid medium by solvent extraction with 2-n-octylaminopyridine. Inter. J. Chem. Science Technology. 2013;**3**(1):15-24

[25] Mane CP, Mahamuni SV, Kolekar SS, Han SH, Anuse MA. Hexavalent chromium recovery by liquid–liquid extraction with 2-octylaminopyridine from acidic chloride media and its sequential separation from other heavy toxic metal ions. Arabian Journal of Chemistry. 2016;**9**(2):1420-1427. DOI: org/10.1016/j.arabjc.2012.03.021

[26] Mane CP, Anuse MA. Studies on liquid-liquid extraction and recovery of bismuth (III) from succinate media using 2-Octylaminopyridine in chloroform. J. Chinese Chem. Soc. 2008; **55**:807-817. DOI: 10.1002/ jccs.200800121

[27] Sandip VM, Prakash PW, Anuse MA. Liquid–liquid extraction and recovery of gallium (III) from acid media with 2 octylaminopyridine in chloroform: Analysis of bauxite ore. Journal of the Serbian Chemical Society. 2010;**75**(8): 1099-1113. DOI: 10.2298/ JSC090630072M

*2(4)-Aminopyridines as Ligands in the Coordination and Extraction Chemistry of Platinum… DOI: http://dx.doi.org/10.5772/intechopen.106376*

[28] Sandip VM, Prakash PW, Anuse MA. Rapid liquid-liquid extraction of thallium (III) from succinatte media with 2-octylaminopyridine in chloroform as the extractant. Journal of the Serbian Chemical Society. 2008; **73**(4):435-451. DOI: 102298/ JSC0804435M

[29] Mandhare AM, Han SH, Anuse MA, Kolekar SS. Arab liquid–liquid anion exchange extraction studies of samarium (III) from salicylate media using high molecular weight amine. Journal of Chemistry. 2015;**8**(4):456-464. DOI: 10.1016/j.arabjc.2011.01.026

[30] Mane CP, Mahamuni SV, Gaikwad AP, Shejwal RV, Kolekar SS, Anuse AM. Journal of Saudi Chemical Society. 2015;**19**(1):46-51. DOI: 10.1016/ j.jscs.2011.12.016

[31] Rau A, Mukerjec RJ. Physical Chemistry. 1966;**70**:2144

[32] Nefedov VI. Structure of Molecules and Chemical Bond. Vol. 1. Moscow: VINITI; 1973. p. 148

[33] Petrukhin OM, Nefedov VI, Salyn OV, Shevchenko VNJ. Inorgan. Chem. 1974;**19**:1418-1420

### *Edited by Satyanarayan Pal*

This book discusses the chemistry and applications of pyridine derivatives. The library of pyridine derivatives is growing steadily with numerous synthetic analogues already described and the identification of new, naturally occurring pyridine-based compounds. The book includes ten chapters organized into two parts. The first part focuses on the numerous types of reactions that arise from pyridine derivatives. The second part examines the pharmaceutical applications of pyridine derivatives as well as their usefulness as sensors for metal cations and extracting agents for platinum group metals.

Published in London, UK © 2023 IntechOpen © lesichkadesign / iStock

Exploring Chemistry with Pyridine Derivatives

Exploring Chemistry with

Pyridine Derivatives

*Edited by Satyanarayan Pal*


*Article* **Screening Wild Pepper Germplasm for Resistance to** *Xanthomonas hortorum* **pv.** *gardneri*

**Zoltán Gábor Tóth 1 , Máté Tóth 1 , Sándor Fekete 2 , Zoltán Szabó 1 and Zoltán Tóth 1,\***

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Bacterial spot disease on peppers is caused by four species of the genus *Xanthomonas*. This disease causes black spot lesions not only on the leaves but also on the fruit, leading to yield and quality loss. *Xanthomonas* species cause major disease outbreaks in tropical, subtropical and humid continental regions worldwide. Bacterial blight caused by xanthomonads occurs on both greenhouse- and field-grown peppers and is particularly important in areas characterized by hot and humid environmental conditions. As pesticides are currently not sufficiently effective in the control of bacterial spot, the development of pepper varieties resistant to *Xanthomonas* species, including X. *hortorum* pv. *gardneri*, is of primary importance for sustainable production. In our research, 119 lines of *Capsicum baccatum* from the USDA ARS gene bank (Griffin, GA) and MATE (Hungarian University of Agriculture and Life Sciences) were tested against strains of *X. hortorum* pv. *gardneri* under greenhouse conditions. Four accessions of the wild pepper species *C. baccatum* appeared to be resistant to seven strains of *X. hortorum* pv. *gardneri* in greenhouse trials. The resistant genotypes of *X*. *hortorum* pv. *gardneri* identified in this study can be used for the resistance gene pyramidation against different bacterial spotted *Xanthomonas* species in pepper.

**Keywords:** sustainability; pepper breeding; bacterial spot resistance; *Xanthomonas hortorum* pv. *gardneri*

**1. Introduction**

Pepper (*Capsicum* spp.) is one of the most important vegetables and spices worldwide because of its versatility, its high nutritional value, as well as its role in reducing human micronutrient deficiencies [1]. The chili pepper genus is currently known to consist of 43 species [2] that are native to temperate and tropical Central and South America, Mexico and the West Indies, five of which are domesticated: *C*. *annuum*, *C*. *chinense*, *C*. *frutescens*, *C*. *baccatum*, and *C*. *pubescens* [3]. Despite the crucial agronomic importance of this crop, diseases are still one of the most important damaging factors in pepper production, and only few can be controlled by chemical treatments. However, environmental and consumer concerns, as well as the risk of crop pathogen insensitivity, underline the importance of developing resistant pepper varieties as a more effective way of reducing the impact of disease [4,5]. *Capsicum* germplasm collections represent an immense genetic wealth resource this purpose, but screening for disease resistance is often a lengthy, labor-intensive, expensive and mostly complex process.

Bacterial spot disease of peppers occurs in both processing and fresh market pepper fields, particularly where growing conditions are characterized by high humidity. Known for its aggressiveness, the pathogen *X*. *hortorum* pv. *gardneri* causes large, star-shaped necrotic lesions on the foliage, stem and fruit. Diseased leaves drop prematurely, resulting in extensive defoliation, thus reducing plant productivity and exposing fruits to potential sunscalding. Stem lesions occur as narrow, light brown, longitudinally raised cankers. Fruit with such large lesions are non-marketable in both the fresh and processed markets.

**Citation:** Tóth, Z.G.; Tóth, M.; Fekete, S.; Szabó, Z.; Tóth, Z. Screening Wild Pepper Germplasm for Resistance to *Xanthomonas hortorum* pv. *gardneri*. *Sustainability* **2023**, *15*, 908. https://doi.org/10.3390/su15020908

Academic Editor: Balázs Varga

Received: 29 November 2022 Revised: 26 December 2022 Accepted: 30 December 2022 Published: 4 January 2023

**Copyright:** © 2023 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:// creativecommons.org/licenses/by/ 4.0/).

Bacterial spot is caused by four bacterial species of the genus *Xanthomonas*: *X*. *euvesicatoria*, *X*. *vesicatoria*, *X*. *perforans* and *X*. *hortorum* pv. *gardneri* [6]. *X*. *hortorum* pv. *gardneri* was first described in 1957 [7]. The pathogen *X*. *hortorum* pv. *gardneri* was recognized as a causative agent of bacterial spot epidemics in the year 2000. Currently, there are few chemicals available that are effective against these pathogens, and even these are ineffective under high disease pressure and have a negative impact on the environment. Since the overuse of copper has led to the development of resistance in the bacterial population, there is a need to find new sustainable and environmentally friendly alternatives [8,9]. The growing number of bacterial spot diseases caused by emerging varieties of xanthomonads makes it increasingly urgent for growers to find practical and sustainable solutions. The use of varieties resistant to bacterial leaf spot offers a potential tool for disease control in field conditions. The spread of new *Xanthomonas* species over the last few years has made the discovery of new sources of resistance a major priority. Thus, host resistance is considered to be a critical element of disease management strategies.

Resistance breeding programs successfully developed commercial pepper lines with hypersensitive and quantitative resistance to various *Xanthomonas* species [10]. Genes regulating resistance have been found in *X*. *euvesicatoria* [11], *X. perforans* species T3 [12], *X*. *perforans* species T4 [13], and *X*. *vesicatoria* [14], and six dominant resistance genes have been identified in pepper to date, namely *Bs1*, *Bs2*, *Bs3*, *Bs4C*, *Bs7* and *BsT* [15]. However, the persistence of resistance can change rapidly due to the changing geographical distribution of the pathogen and the rapid emergence of new pathogenic variants [16]. In the case of *X*. *hortorum* pv. *gardneri*, only one resistance gene source has been described so far, although in this case the infection was caused by a single *Xantomonas* strain (USVLXG1) and resistance was not tested with other *Xanthomonas* isolates [17]. The introduction of resistance genes with different mechanisms of action could provide a durable and sustainable solution to this ongoing host–pathogen arms race.

Commercial pepper varieties carrying recessive resistance genes, namely *bs5* and *bs6*, have not proven effective against *X*. *hortorum* pv. *gardneri*. Based on current publications, the recessive resistance gene *bs8* identified in *C*. *annuum* accession PI 163192 is the only one that was shown to confer resistance to *X*. *hortorum* pv. *gardneri* [18]. With the increasing frequency of *Xanthomonas* outbreaks around the world, there is a growing rationale to identify new sources of resistance in pepper to emerging bacterial species, including *X*. *hortorum* pv. *gardneri* and to incorporate these new resistance genes into breeding programs.

Preliminary results are presented below, which are aimed at screening wild pepper cultivars for heritable resistance to *X*. *hortorum* pv. *gardneri*. The aim of our current work was to identify new sources of resistance to *X*. *hortorum* pv. *gardneri* in pepper and to identify potential candidate genes involved in resistance to bacterial spot disease. A diverse USDA panel of pepper accessions were screened for resistance to *X*. *hortorum* pv. *gardneri*, and highly resistant accessions were identified in our work.

#### **2. Materials and Methods**

#### *2.1. Plant Materials*

The work was carried out on a collection of *C. baccatum* accessions originating from the USDA ARS genebank. We tested 119 accessions including 97 *C. baccatum* var. *pendulum*, 2 *C. baccatum* var. *pratermissum*, 7 *C. baccatum* var. *baccatum,* and 2 *C. baccatum* var. *umbilicatum*, and in 11 cases the variant was not specified (Table S1). In order to improve germination, the seeds were pre-soaked for four hours before they were sterilized with 10% calcium hypochlorite (Ca(ClO)2) solution for twenty minutes with a few drops of Tween20. The seeds were rinsed five times with sterile Milli-Q Water. After that, all of the seeds were placed on MS medium [19] with 20 g sucrose (MS20) in sterilized glass storage jars until the cotyledon stage. Afterwards, the plants were placed into pots filled with Klasmann Traysubstrate soil mixed with 50 g NPK and microelement fertilizer (14% N + 7% P + 21% K + 1% Mg + 1% microelements (B, Cu, Mn, Fe, Zn) to 70 L of soil.

The plants were then grown under controlled greenhouse conditions at 24 ± 2 ◦C, with 14/10 h photoperiods, and 50–70% relative humidity until they reached the six-mature-leaf stage. A cultivated variety *C. annuum* Fö "Fehérözön" was used as a susceptible control.

#### *2.2. Bacterial Isolates and Plant Infiltration*

To find a resistant phenotype, two *Xg* strains were used for plant infiltration—LMG962 from the Belgian Coordinated Collections of Microorganisms (BCCM) and SRB, which is a field isolate from Serbia [7]. Bacteria were cultivated in YDC [20] medium for two days in an incubator at a temperature of 30 ◦C. After two days of incubation, bacteria were collected and diluted with sterile Milli-Q Water to a final concentration of 106 cfu/mL. The infiltration was performed in planta with a needle-free syringe into the intercellular spaces on the abaxial side of the first true leaves. After infiltration, the plants were placed in plant growth chambers at 25 ◦C with a 16/8 h photoperiod, at 50–70% relative humidity.

The response to infection was monitored continuously, but the final evaluation was made after 7 days. The plants that showed complete resistance to both isolates were also tested with five further *Xg* strains: Xg51, Xg152, Xg153, Xg156, Xg177 (Table 1), which were donated by Brian Staskawicz (Innovative Genomics Institute University of California, Berkeley).

**Table 1.** *Xg* strains used in this study and their origin.

1 means that the exact origin, host, year, isolation ID or collector of the isolate has not been established.

#### *2.3. Bacterial Growth Test*

In order to determine how the presence of resistance affected bacterial growth in resistant plants, leaf disc samples were collected from the infiltrated area at different times. For the test of the LMG962 isolate, four resistant and three susceptible plants were used in two replicates at 25 ◦C and 30 ◦C. The photoperiod and humidity were the same as for the accession testing. Sampling was performed at 0, 2, 4, 6, 8, 10, 12 days post inoculation (dpi) from the inoculated area. In the case of resistant plants, re-isolation was also performed 30 days after infection to monitor possible changes in bacterial counts. The isolated leaf discs were macerated in 100 µL sterilized water and prepared in dilution lines and were scattered on YDC medium. After two or three days, the colonies were counted. The cfu/cm2 of leaves was calculated by counting the number of adult colonies.

#### **3. Results**

#### *3.1. Evaluation of C. baccatum Accessions for the Susceptibility and Resistance to X. hortorum pv. gardneri*

One hundred and nineteen *C*. *baccatum* accessions were inoculated with two highly virulent strains (LMG962 and SRB) of *X*. *hortorum* pv. *gardneri* and evaluated for symptoms. Disease evaluation started one week after inoculation and the earliest typical disease symptoms were observed on the leaves 3–6 days after inoculation on the infected pepper plants. The average incubation period varied between 4 and 7 days until the initial abnormal discoloration appeared, while the length of time required to develop complete leaf necrosis in sensitive individuals varied between 6 and 10 days.

Susceptible symptoms typically started with chlorotic lesions, which developed into angular, water-soaked lesions and eventually into brown or black necrotic lesions. Phenotyping of the control line "Fehérözön" (highly susceptible) conformed to the expected disease reactions. In our studies, we found that *C*. *baccatum* accessions differed in terms of

the earliness and intensity of symptoms. The vast majority of the accessions were susceptible to both strains, showing a similar phenotype to "Fehérözön" used as a control. A small part of the accessions showed partial resistance to one of the *X*. *hortorum* pv. *gardneri* strain. Based on the evaluation of their reactions, four of the 119 *C*. *baccatum* genotypes showed strong resistance to both *Xg* isolates used in the first selection round (Figure 1). disease reactions. In our studies, we found that *C*. *baccatum* accessions differed in terms of the earliness and intensity of symptoms. The vast majority of the accessions were susceptible to both strains, showing a similar phenotype to "Fehérözön" used as a control. A small part of the accessions showed partial resistance to one of the *X*. *hortorum* pv. *gardneri* strain. Based on the evaluation of their reactions, four of the 119 *C*. *baccatum* genotypes showed strong resistance to both *Xg* isolates used in the first selection round (Figure 1).

*3.1. Evaluation of C. baccatum Accessions for The Susceptibility and Resistance to X. hortorum*

One hundred and nineteen *C*. *baccatum* accessions were inoculated with two highly virulent strains (LMG962 and SRB) of *X*. *hortorum* pv. *gardneri* and evaluated for symptoms. Disease evaluation started one week after inoculation and the earliest typical disease symptoms were observed on the leaves 3–6 days after inoculation on the infected pepper plants. The average incubation period varied between 4 and 7 days until the initial abnormal discoloration appeared, while the length of time required to develop complete leaf

Susceptible symptoms typically started with chlorotic lesions, which developed into angular, water-soaked lesions and eventually into brown or black necrotic lesions. Phenotyping of the control line "Fehérözön" (highly susceptible) conformed to the expected

*Sustainability* **2023**, *15*, x FOR PEER REVIEW 4 of 9

necrosis in sensitive individuals varied between 6 and 10 days.

**3. Results**

*pv. gardneri*

**Figure 1.** Symptoms of bacterial spot 7 days after inoculation with strain LMG962 in susceptible Fö (**a**,**b**) and the resistant phenotype on Cbp2 plants infected with strain LMG962 (**c**,**d**) and SRB (**e**,**f**). Adaxial and abaxial view. **Figure 1.** Symptoms of bacterial spot 7 days after inoculation with strain LMG962 in susceptible Fö (**a**,**b**) and the resistant phenotype on Cbp2 plants infected with strain LMG962 (**c**,**d**) and SRB (**e**,**f**). Adaxial and abaxial view.

In the second phase, all four previously resistant phenotypes of *C*. *baccatum* accessions were challenged with five additional *Xg* isolates. Based on our results, all four accessions also showed strong resistance (Table 2) to all five additional *Xg* isolates (Xg51, Xg152, Xg153, Xg156, Xg 177). In the second phase, all four previously resistant phenotypes of *C*. *baccatum* accessions were challenged with five additional *Xg* isolates. Based on our results, all four accessions also showed strong resistance (Table 2) to all five additional *Xg* isolates (Xg51, Xg152, Xg153, Xg156, Xg 177).

**Table 2.** *C. baccatum* var. *pendulum* accessions showing partial or complete resistance to the *X*. *hortorum* pv. *gardneri* strains used.

+: resistant response to infection, −: sensitive response to infection.

In the resistant plants, the infected area turned light green, but at 30 dpi, it was no longer possible to distinguish between infiltrated and untreated areas. Small necrotic spots *C. Baccatum pendulum*

appeared on the leaves at 10–12 dpi typically along the leaf veins, but these lesions increased very little if at all after 30 days (Figure 2). No difference was observed between the emerging phenotype in resistant plants according to the bacterial strain used for inoculation. increased very little if at all after 30 days (Figure 2). No difference was observed between the emerging phenotype in resistant plants according to the bacterial strain used for inoculation.

In the resistant plants, the infected area turned light green, but at 30 dpi, it was no longer possible to distinguish between infiltrated and untreated areas. Small necrotic spots appeared on the leaves at 10–12 dpi typically along the leaf veins, but these lesions

*Sustainability* **2023**, *15*, x FOR PEER REVIEW 5 of 9

**Species Variety Accession SRB LMG962 XG51 XG152 XG153 XG156 XG177**

+: resistant response to infection, −: sensitive response to infection.

*torum* pv. *gardneri* strains used.

PI 441541 + − PI 441542 + − PI 370010 − + PI 441543 + - PI 441578 + − PI 441552 − + PI 441533 − + PI 441520 + −

**Table 2.** *C. baccatum* var. *pendulum* accessions showing partial or complete resistance to the *X*. *hor-*

Cbp1 + + + + + + + Cbp2 + + + + + + + Cbp3 + + + + + + + Cbp4 + + + + + + +

**Figure 2.** Symptoms of bacterial spot 30 days after inoculation with strain LMG962, SRB and Xg51 in a resistant accession plant. **Figure 2.** Symptoms of bacterial spot 30 days after inoculation with strain LMG962, SRB and Xg51 in a resistant accession plant.

#### *3.2. Evaluation of the Bacterial Growth Test 3.2. Evaluation of the Bacterial Growth Test*

We determined the effect of the identified resistance gene on bacterial growth in the plant and the correlation of visual symptoms with bacterial population levels. The growth in the populations of different bacterial races was monitored for 12 days after inoculation and incubation at 25 °C and 30 °C. "Fehérözön" was used as a negative control. Compared to the almost equal initial values, the bacterial growth curve in planta at 25 °C indicated that resistant plants reached a maximum of 4.75 log at 4 dpi and then showed a minimal decrease with no significant change in the following 6–12 days. In contrast, in the case of the sensitive plants, a value of 6.7 log at 4 dpi was obtained and a steady increase was detected until leaf drop at 8 dpi (Figure 3). In the majority of cases, the infected leaves of the susceptible plants had fallen off by the eighth day and sampling could not be continued. At 30 °C, the resistant plants reached a maximum value of 5.55 log at 6 dpi and then a slight decrease was observed. Susceptible plants showed a value of 7.1 log at 6 dpi until We determined the effect of the identified resistance gene on bacterial growth in the plant and the correlation of visual symptoms with bacterial population levels. The growth in the populations of different bacterial races was monitored for 12 days after inoculation and incubation at 25 ◦C and 30 ◦C. "Fehérözön" was used as a negative control. Compared to the almost equal initial values, the bacterial growth curve in planta at 25 ◦C indicated that resistant plants reached a maximum of 4.75 log at 4 dpi and then showed a minimal decrease with no significant change in the following 6–12 days. In contrast, in the case of the sensitive plants, a value of 6.7 log at 4 dpi was obtained and a steady increase was detected until leaf drop at 8 dpi (Figure 3). In the majority of cases, the infected leaves of the susceptible plants had fallen off by the eighth day and sampling could not be continued. At 30 ◦C, the resistant plants reached a maximum value of 5.55 log at 6 dpi and then a slight decrease was observed. Susceptible plants showed a value of 7.1 log at 6 dpi until leaf drop (Figure 4). In susceptible plants, the temperature had no significant effect on the growth curve of *Xg* bacteria, but significantly affected the tolerance of plants to infection, because inoculated leaves fell off earlier at higher temperatures. Plants kept at 25 ◦C were also sampled at 30 dpi and the results show that no significant change in the cfu value in the leaf occurred. In the case of plants kept at 30 ◦C, it was not possible to collect a sample after one month, due to early aging of the plant and leaf fall. *Sustainability* **2023**, *15*, x FOR PEER REVIEW 6 of 9 leaf drop (Figure 4). In susceptible plants, the temperature had no significant effect on the growth curve of *Xg* bacteria, but significantly affected the tolerance of plants to infection, because inoculated leaves fell off earlier at higher temperatures. Plants kept at 25 °C were also sampled at 30 dpi and the results show that no significant change in the cfu value in the leaf occurred. In the case of plants kept at 30 °C, it was not possible to collect a sample after one month, due to early aging of the plant and leaf fall.

**Figure 3.** Time course of bacterial population growth at 25 °C after infiltration of leaves of resistant and susceptible plants by LMG962 strains. **Figure 3.** Time course of bacterial population growth at 25 ◦C after infiltration of leaves of resistant and susceptible plants by LMG962 strains.

**Figure 4.** Time course of bacterial population growth at 30 °C after infiltration of leaves of resistant

*C. baccatum* is the most popular and widely cultivated chili pepper in South America. There is a wild form called *C*. *baccatum* var. *baccatum* and a domesticated form (*C*. *baccatum* var. *pendulum*) [21]. Brazil is considered to be the center of the genetic diversity of *Capsicum spp*., and at least 116 accessions already described are found in Brazil [22,23]. It is expected that new species [2] and new resistance genes for pepper breeding will be found in this important and untapped genetic resource. Pathogens are increasingly threatening the cultivation and yield of most *Capsicum* species worldwide. In this context, the search for new sources of resistance remains a major challenge today and *Capsicum* germplasm

Previous searches for resistance to *X*. *hortorum* pv. *gardneri* concentrated on the *Xg* isolates which are most prevalent in the US. In contrast to previous research, in our present work we also used the isolates most commonly found in Europe and the US. The results of this screening study in which pepper accessions were inoculated with seven *Xg*

collections represent a promising target for this purpose.

and susceptible plants by LMG962 strains.

**4. Discussion**

and susceptible plants by LMG962 strains.

**Figure 3.** Time course of bacterial population growth at 25 °C after infiltration of leaves of resistant

leaf drop (Figure 4). In susceptible plants, the temperature had no significant effect on the growth curve of *Xg* bacteria, but significantly affected the tolerance of plants to infection, because inoculated leaves fell off earlier at higher temperatures. Plants kept at 25 °C were also sampled at 30 dpi and the results show that no significant change in the cfu value in the leaf occurred. In the case of plants kept at 30 °C, it was not possible to collect a sample

after one month, due to early aging of the plant and leaf fall.

#### **4. Discussion 4. Discussion**

*C. baccatum* is the most popular and widely cultivated chili pepper in South America. There is a wild form called *C*. *baccatum* var. *baccatum* and a domesticated form (*C*. *baccatum* var. *pendulum*) [21]. Brazil is considered to be the center of the genetic diversity of *Capsicum spp*., and at least 116 accessions already described are found in Brazil [22,23]. It is expected that new species [2] and new resistance genes for pepper breeding will be found in this important and untapped genetic resource. Pathogens are increasingly threatening the cultivation and yield of most *Capsicum* species worldwide. In this context, the search for new sources of resistance remains a major challenge today and *Capsicum* germplasm collections represent a promising target for this purpose. *C. baccatum* is the most popular and widely cultivated chili pepper in South America. There is a wild form called *C*. *baccatum* var. *baccatum* and a domesticated form (*C*. *baccatum* var. *pendulum*) [21]. Brazil is considered to be the center of the genetic diversity of *Capsicum* spp., and at least 116 accessions already described are found in Brazil [22,23]. It is expected that new species [2] and new resistance genes for pepper breeding will be found in this important and untapped genetic resource. Pathogens are increasingly threatening the cultivation and yield of most *Capsicum* species worldwide. In this context, the search for new sources of resistance remains a major challenge today and *Capsicum* germplasm collections represent a promising target for this purpose.

Previous searches for resistance to *X*. *hortorum* pv. *gardneri* concentrated on the *Xg* isolates which are most prevalent in the US. In contrast to previous research, in our present work we also used the isolates most commonly found in Europe and the US. The results of this screening study in which pepper accessions were inoculated with seven *Xg* Previous searches for resistance to *X*. *hortorum* pv. *gardneri* concentrated on the *Xg* isolates which are most prevalent in the US. In contrast to previous research, in our present work we also used the isolates most commonly found in Europe and the US. The results of this screening study in which pepper accessions were inoculated with seven *Xg* strains indicated that there were significant differences in resistance between PI lines from the USDA ARS pepper germplasm collection. Screening was carried out in two stages. As a first step, 119 accessions from the *C. baccatum* collection were challenged with two different strains of *X*. *hortorum* pv. *gardneri* to determine the extent and types of resistance in *C. baccatum*. Sensitive symptoms of bacterial spot were observed in 107 accessions and *Xg* was recovered from all plants, indicating that none of the accessions carried a source of resistance to this pathogen. Eight accessions showed partial resistance to one of the *Xg* isolates and strong resistance was identified in four PI lines (Cbp1, Cbp2, Cbp3 and Cbp4). In the second stage, screening was carried out on Cbp1, Cbp2, Cbp3 and Cbp4 accessions using the five potentially important isolates in Europe and the US. Complete screening was carried out under controlled conditions in growth chambers using a method (leaf screening of young plants with a concentrated bacterial suspension) that clearly indicated the presence of the pathogen and the rapid onset of symptoms.

The screening results showed two main types of resistance (Table 2) in the tested plant population. Species-specific resistance was the most common and was present in 6% of all accessions tested. Resistance to all species used (potential species-nonspecific resistance) was very rare and was present in 3% of accessions tested.

#### **5. Conclusions**

Species shifts in bacterial spot pathogen populations are very common [10,24] and pose a serious problem and challenge for pepper breeding programs due to the decline in resistance persistence [25]. Therefore, there is an increasing need to identify new sources of resistance to these pathogens. The resistance germplasm of *X*. *hortorum* pv. *gardneri*

identified in this study can be of significant importance for both breeders and researchers and is highly relevant for introgression into pepper lines and in terms of gene pyramidation efforts against various *Xanthomonas* species. The resistance genes that we identified in *C. baccatum* might also have significant potential for resistance inhibition in other pepper species such as *C*. *annuum*. Interspecific crosses provide the opportunity to pass genes from one species to another, but in most cases, this can present several difficulties. A successful wide hybridization among species is highly dependent on the genetic distance between species. According to our current knowledge, two options are available for introgressing *C*. *baccatum* genes into *C*. *annuum*. The first option is using *C*. *chinense* and *C*. *frutescens* as bridge species [26], while the second option is to carry out direct crosses between *C*. *annuum* and *C*. *baccatum* combined with in vitro embryo rescue [27]. Our future goal is to use both methods to introduce our newly identified valuable resistance gene into *C*. *annuum*. Using the four resistant parental lines, crosses are underway with the aim of creating F1 and F2 progeny to determine the inheritance of disease resistance.

The new sources of resistance identified in this study provide a basis for further work on breeding disease-resistant varieties that are resilient to the changing population structure of pathogens. The current study showed that the different genotypes of *C*. *baccatum* differed in their response to wilt caused by the pathogen *Xanthomonas*, which also demonstrated that the pepper PI collection offered a valuable public source of resistance for pepper breeders to develop varieties resistant to bacterial spot. In conclusion, the information in this study can be summarized in that the use of a resistant genotype, together with cultural practices and sanitary control measures, is considered the most feasible and sustainable way to control *X*. *hortorum* pv. *gardneri* wilt.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/su15020908/s1, Table S1: Screening of *C. baccatum* germplasm to *X. hortorum* pv. *gardneri*.

**Author Contributions:** Conceptualization, Z.G.T. and Z.T.; methodology, Z.G.T. and Z.T.; software, Z.T.; validation, Z.G.T., Z.S. and Z.T.; formal analysis, Z.G.T., M.T., Z.S. and Z.T.; investigation, Z.G.T. and Z.T.; resources, Z.G.T. and Z.T.; data curation, Z.G.T. and Z.T.; writing—original draft preparation, Z.G.T. and Z.T.; writing—review and editing, Z.G.T., S.F., Z.S. and Z.T.; visualization, Z.T.; supervision, S.F. and Z.T.; project administration, Z.G.T. and Z.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**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.

#### **References**

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