Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Tomo 57(1). ISSN (en línea) 1853-8665. Año 2025.

Original article

Antimicrobial and antioxidant properties of the woody endocarp of native and commercial walnuts from Argentina

Propiedades antimicrobianas y antioxidantes del endocarpio leñoso de nogales nativos y comerciales de Argentina

Ingrid Georgina Orce¹,

Sibila Ivonne Lencina^{1, 3},

Gabriela Inés Furque²,

Emilia Lorenzo³,

Gretel Rodriguez Garay⁴,

Fiamma Pereyra³,

María Rosa Alberto⁵,

Patricia Elizabeth Gomez Kamenopolsky^{1, 3},

Mario Eduardo Arena^5*

¹ Centro Regional de Energía y Ambiente para el Desarrollo Sostenible. CREAS (UNCA-CONICET) Prado 366. San Fernando del Valle de Catamarca. CP K4700AAP. Catamarca. Argentina.

² Universidad Nacional de Catamarca. Facultad de Ciencias Exactas y Naturales. Departamento de Química. Av. Belgrano al 300. San Fernando del Valle de Catamarca. CP K4700AAP. Catamarca. Argentina.

³ Universidad Nacional de Catamarca. Facultad de Ciencias Agrarias. Departamento de Química. Av. Maestro Quiroga 50. San Fernando del Valle de Catamarca. CP K4700AAP. Catamarca. Argentina.

⁴ Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Catamarca. Ruta 33 km 4.5 (4705). Valle Viejo. Catamarca. Argentina.

⁵ Instituto de Biotecnología Farmaceútica y alimentaria (INBIOFAL) Predio Universitario Ingeniero Herrera. Av. Kirchner 1900. CP 4000. San Miguel de Tucumán. Tucumán. Argentina.

* mario.arena@fbqf.unt.edu.ar

Abstract

Juglans australis is a tree from the Juglandaceae family found in the southernmost region of America. Its small edible nuts are not commercialized, and their bioactive characteristics are unknown. This study first reports the antioxidant, antiradical, and antibacterial activity of extracts from this native walnut against phytopathogenic bacteria and compared with its commercial counterpart, J. regia L. Different extracts from the woody endocarp (shells) were obtained using methanol and ethyl acetate. Methanolic extracts significantly inhibited phytopathogenic growth at all concentrations tested (0.1, 1, and 10 mg/mL). The best activity was reported against Xanthomonas. Highest total phenolics and the most significant antioxidant activity were determined in methanolic extracts (TPC: 121 mg gallic acid equivalent (GAE)/g of dried peel, FRAP: 58.6 mmol Trolox/100 g of peel dried and 9.7 mM Trolox/100 g of dried peel). Extracts from both species demonstrated congruent patterns. Gallic acid was the most abundant compound in the methanolic extract. However, extracts demonstrated superior efficiency, suggesting a potential synergistic effect among their components. Antioxidant and antimicrobial activity of methanolic extracts against Xanthomonas make them potential control agents.

Keywords: Juglans australis, phytopathogens, polyphenols, bioactive compounds, sustainable agriculture, Xanthomonas sp.

Resumen

Juglans australis es un árbol perteneciente a la familia Juglandaceae que se encuentra en la región más austral del continente americano. Aunque las nueces también son comestibles, son pequeñas y no se comercializan, sus características bioactivas son desconocidas. Este estudio constituye el primer informe sobre la actividad antioxidante, antirradical y antibacteriana de extractos de la nuez nativa frente a bacterias fitopatógenas, y su comparación con la especie comercial, J. regia L. Se obtuvieron diferentes extractos a partir del endocarpio leñoso (cáscaras) utilizando metanol y acetato de etilo. El extracto metanólico resultó ser la fracción más activa e inhibió significativamente el crecimiento de los fitopatógenos en todas las concentraciones analizadas (0,1, 1 y 10 mg/mL). La mejor actividad se registró para el género Xanthomonas. El mayor contenido de fenoles totales y la actividad antioxidante más significativa se determinó en el extracto metanólico (TPC: 121 mg de ácido gálico equivalente (GAE)/g de cáscara seca, FRAP: 58,6 mmol de Trolox/100 g de cáscara seca y 9,7 mM de Trolox/100 g de cáscara seca). Los extractos de ambas especies se comportaron de manera similar. Al analizar la composición química, el ácido gálico fue el compuesto más abundante en el extracto metanólico. Sin embargo, los extractos mostraron una eficiencia superior, lo que sugiere un posible efecto sinérgico entre sus componentes. La actividad antimicrobiana de los extractos metanólicos contra Xanthomonas, junto con su capacidad antioxidante, resalta su potencial aplicación como agentes de control de fitopatógenos.

Palabras claves: Juglans australis, fitopatógenos, polifenoles, compuestos bioactivos, agricultura sustentable, Xanthomonas sp.

Originales: Recepción: 23/06/2023 - Aceptación: 05/12/2024

Introduction

Phytopathogens cause significant economic loss in agriculture ⁽³⁵⁾. Even considering synthetic pesticides imply detrimental environmental and human health consequences, several products are still being used. In the last decade, eco-friendly compounds have emerged as alternative pesticides ^{(8, 19)}. These natural antimicrobial agents are safer and cheaper than chemical agents ⁽⁴⁸⁾ contributing to the economy and environment ^{(9, 19, 44)}.

Several plant bioactive compounds have shown antimicrobial activity ⁽¹⁴⁾. Phenolic-rich plant extracts have shown significant activity against phytopathogens ^{(22, 42)}, including Xanthomonas spp., a major threat to crops like rice and citrus, which have developed resistance to chemicals and antibiotics ^{(25, 29)}. Some studies report the effects of extracts as natural antimicrobials against Xanthomonas sp. ^{(3, 23, 24, 30)}.

Other pathogens, like Clavibacter michiganensis, are responsible for significant losses in tomato ⁽³²⁾, and the bacterium Erwinia amylovora mainly affects pome fruit trees like pear, apple, quince, and loquat ^{(5, 28)}. Carnaval et al. (2022) observed the inhibition effect of seriguela (Spondias purpurea L.) extract on Clavibacter michiganensis pv michiganensis and Xanthomonas phaseoli.

The genus Juglans includes over 20 species, being J. regia L. majorly significant due to its extensively studied nutritional and functional properties ^{(7, 15)}. Walnut by-products, including shells, are rich in bioactive phytochemicals with antimicrobial potential for medicine, food preservation, and agroindustry ^{(1, 4, 26, 27)}. Although their potential as biopesticides is recognized, the antimicrobial activity of these compounds is still unexplored. Meanwhile, shell biomass is often undervalued despite being a cost-effective, renewable resource.

On the other hand, Juglans australis is a native walnut tree from the Juglandacea family inhabiting the most austral region of South America, and the Northwestern subtropical rainforest in Argentina, locally known as “Yungas” ⁽¹¹⁾, in Jujuy, Salta, Tucumán, La Rioja, and Catamarca (figure 1). Its fruit is an indehiscent, subglobose drupe with a thick, adherent mesocarp and a rigid shell (endocarp) containing the embryo ⁽³⁹⁾ (figure 1).

Figure 1. Juglans australis tree in Ancasti (Catamarca) city, Argentina (left) and J. regia (right). Morphological comparison of native J. australis and commercial J. regia L. fruits.

Figura 1 Árbol y frutos de la especie y Juglans australis (izquierda) y Juglans regia (derecha), correspondientes a la localidad de Ancasti, provincia de Catamarca, Argentina.

Contrasting with the extensive knowledge about commercial walnuts, information on this native species remains scarce. To date, no research reports antimicrobial or antioxidant activity of Juglans australis; except for its activity against herpes virus ⁽³⁷⁾.

We aimed to analyze the antimicrobial and antioxidant properties of walnut shell extracts from the commercial J. regia and the native J. australis for future uses in agricultural management. These extracts would contribute to waste valorization while generating added value to the autochthonous species. Furthermore, extract chemical compositions allowed deeper comprehension of their bioactive activities.

Materials and methods

Sample collection

In 2021, samples of J. regia and J. australis walnut shells were collected in Ancasti, Catamarca, Argentina. Walnut shells were cleaned and dried under shaded conditions for a week. Selected samples were ground into small particles using a grinder.

Solvent extraction

Solvent extraction involved 50 g of the powdered walnut shells extracted with 250 mL of absolute methanol (MeOH) and ethyl acetate (AcOEt) for 45 min at room temperature and filtered through Whatman n° 4 ⁽⁴⁸⁾. The solvents were evaporated under a vacuum in a Büchi R-210 rotavapor. The extracts obtained were redissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.1, 1, and 10 mg/mL and stored in the dark at 4°C for further use. All extractions were done in duplicate.

Determination of total phenolic content and antioxidant activities

Total polyphenol content (TPC) was determined colorimetrically using Folin-Ciocalteu’s reagent at 765 nm. A standard curve was performed with gallic acid. The results were expressed as μg gallic acid/g dry weight (DW) ⁽⁴¹⁾. In vitro antioxidant activity was measured using the free radical elimination activity assay on 1,1, -diphenyl-2-picrylhydrazylradical (DPPH) ⁽¹⁰⁾, and ferric reduction capacity of plasma assay (FRAP) ⁽⁶⁾. Results are expressed in mmol Trolox equivalents/100 g dry weight (DW). All samples were analyzed in triplicate.

Identification of extract phenolic compounds by UHPLC-MS/MS

Phenolic compounds of extracts from walnut shells were identified by a UHPLC (Ultimate 3000 RSLC, Dionex - Thermo Scientific) equipped with a diode array detector and coupled to a TSQ Quantum ultra-triple-quadrupole mass spectrometer (TSQ Quantum Access Max, Thermo Scientific) and column Hypersil GOLD aQ (150 x 2.1 mm, 5 um) (Thermo Scientific). The mobile phase was a binary mixture of solvents: mobile phase A corresponded to ultrapure water/formic acid solution (Merck, Darmstadt, Germany) (0.1% v/v), and mobile phase B corresponded to an acetonitrile / formic acid solution (Merck, Darmstadt, Germany) (0.1% v/v). Gradient conditions were as follows: 0-18 min, 97% A; 18-21 min, 90% B; 21-26 min, 97% A. Electrospray source of the MS was performed in negative mode. Eluate was monitored at 250 nm, flow rate was 0.3 mL min-1, injection volume was 15 μL, and the column was maintained at 35°C. Polyphenols were tentatively identified according to their retention times, UV/Vis spectra, high-resolution MS, and MS/MS spectra by comparison with pure compounds. We searched for gallic acid, cumaric acid, caffeic acid, ferulic acid, rutin, and eriocitrin (Sigma-Aldrich, St. Louis, MO, USA). The linearity of each calibration curve was confirmed by plotting the ratio of peak areas of phenolic compounds to the internal standard against compound concentration. Data were analyzed via LC-MS Xcalibur workstation software (Version 2.6, Thermo Fisher Scientific).

Antibacterial activity

Antibacterial activity of the different walnut shell extracts was evaluated against Gram-negative bacteria Erwinia amilovora, Xanthomonas axonopodis pv. phaseolus, and X. campestris pv. campestris 8004 and a Gram positive bacteria, Clavibacter michiganensis. Bacterial suspensions were prepared in Tryptic Soy Broth (TSB). By microdilution, microtiter plate wells were filled with bacterial suspension (107 CFU/mL) and the extract solution (final concentrations 0.1; 1 and 10 mg/ mL). Pure gallic acid, the main extract component, was incorporated in the antimicrobial assays at three different concentrations: 34 ppm (higher concentration detected in the extracts), 100 ppm, and 500 ppm. The extracts and gallic acid suspensions were prepared by diluting stock solutions in DMSO. Vehicle controls were prepared with DMSO and each bacterial culture. The 1% vehicle did not affect bacterial growth and was used as negative control. Streptomycin sulfate was also used as positive control. The microplates were incubated at 28°C for 24 h, and growth was detected using a microplate reader (Multiskan Go, Thermo) at 560 nm ⁽⁴⁵⁾. Inhibition percentages were also calculated according to the Equation:

where

Ac = absorbance of the control sample (phytopathogens without extract and antibiotic)

As = absorbance of phytopathogens + extract samples.

All assays were conducted three times and analyzed in triplicate.

Statistical analysis

ANOVA and Tukey test evaluated differences between treatments using INFOSTAT (Student version, 2020e).

Results and discussion

Total Phenolic compounds and Antioxidants activities

Table 1, shows total phenolic content and antioxidant activity of organic extracts (ME: methanolic extract, EAE: ethyl-acetate extract) of J. australis and J. regia.

Tabla 1. Contenido fenólico total (TPC), actividades antirradicalias (DPPH) y reductoras (FRAP) de extractos metanólicos y de acetato de etilo obtenidos a partir del endocarpio de J. australis y J. regia.

Table 1. Total phenolic content (TPC), antiradical (DPPH) and reducing (FRAP) activities of methanolic and ethyl acetate of endocarp extracts from J. australis and J. regia.

Value is expressed as mean ± standard error. Different letters indicate statistically significant differences (p<0.05).

Los valores corresponden a la media ± error estándar. Letras diferentes indican diferencias estadísticas (p<0,05).

Solvent extraction capacities varied significantly (p < 0.05). The highest phenolic content and reducing activity were observed in the methanolic extract corresponding to J. australis (121 mg GAE/g d.w. and 56.8 mmol Trolox/100 g d.w., respectively). However, the strongest antiradical activity was measured in the methanolic extract of J. regia (p < 0.01).

Methanolic extracts had the highest total phenols content and the strongest antiradical and antioxidant activities. Extracts using polar solvents usually exhibit higher phenolic content and positively correlate with antioxidant potential ⁽⁴⁸⁾. Among different factors in the extraction process, total phenolic compounds in walnut shell varies from 1 mg/g shell to 32.76 mg GAE mg/g shell ^{(2, 26, 48)}. Here, we obtained 121 mg GAE mg/g shell in methanolic extracts of J. australis, significantly higher-more than two to ten times-than the reported ⁽¹⁹⁾.

When DPPH and FRAP activities were analyzed, methanolic extract of J. australis showed similar activity to J. regia and cited in the literature ⁽⁴²⁾. Yang et al. (2014) analyzed the antioxidant and antiradical properties of walnut-shell extracts with different polarity solvents, demonstrating that methanol also shows the strongest antioxidant activity and reducing power. These established methods are reliable indicators of antioxidant potential in the native walnut, comparable with commercial J. regia activities, making it a potential source of bioactive compounds.

Solvent selection is crucial for antioxidant isolation by extraction methods. The chosen solvent significantly affects extract yield and its antioxidant activity due to the varying polarities of the extracted compounds ⁽³¹⁾. Methanolic and ethyl acetate extracts are commonly used in phytochemical studies. Methanol is a polar solvent that effectively extracts water-soluble compounds, including phenolics, flavonoids, and alkaloids. Ethyl acetate, on the other hand, is a less polar solvent that targets more lipophilic compounds, such as terpenes, steroids, and some fatty acids.

This difference in composition might depend on the genotype and environmental conditions during development, and maturity at harvest ⁽²⁾.

Identification and quantification of phenolic compounds

Phenolic compounds of walnut shell extract in J. australis and J. regia were determined using the UHPLC-MS/MS method. Results observed for J. regia extracts coincide with previous reports ^{(2, 16, 18)}. The methanolic extract had the highest concentration of GLC (26.8 mg/L), followed by CMR (3.1 mg/L) and CFC (762 μg/L). Notably, rutin (RTN) was only detected in J. regia methanolic extract, albeit at a lower concentration (103 μg/L). In the ethyl acetate extract of J. regia, the most abundant were GLC (0.1 mg/L), followed by CMR (607 μg/L) and CFC (μg/L).

RTN in J. regia extracts suggests the potential unique properties of this species. Studies conducted on several parts of fruit and leaves consistently reveal that gallic acid is among the most abundant components in these extracts ^{(2, 16, 18)}. Fernandez Argulló et al. (2021) recently reported that gallic, ellagic, and ferulic acids were the major phenolic compounds in walnut wood waste extracts. However, our study did not detect ferulic acid.

On the other hand, Gallic acid (GLC), caffeic acid (CFC), and cumaric acid (CMR) were identified in both J. australis extracts (methanolic and ethyl acetate) (table 2). The methanolic extract presents the highest GLC content (34000 μg/L), followed by CMR (672 μg/L) and CFC (351 μg/L). In ethyl acetate extracts, GLC was most abundant (9000 μg/L), followed by CMR (269 μg/L) and CFC (103 μg/L). Table 2 shows GLC content was significantly higher than CFC and CMR compounds for both extracts. Ours is the first characterization and quantification of phenolic compounds in this native walnut.

Table 2. Phenolic compounds in walnut shell extracts, and quantitative analysis of phenolic components in methanolic and ethyl acetate extracts of J. regia and J. australis.

Tabla 2. Compuestos fenólicos en extractos de cáscaras de nuez, y análisis cuantitativo del contenido de componentes fenólicos en extractos de acetato de etilo y metanólico presentes en J. regia y J. australis.

ppb = parts per billion = μg/L; ppm = parts per million = mg/L.

Note: AcOEt: ethyl-acetate; MetOH: methanolic; ppb = parts per billion = μg/L; ppm = parts per million = mg/L, GLC: gallic acid; CFC: caffeic acid; CMR: cumaric acid; RTN: rutin. ND: not detectable.

ppb = partes por billón = μg/L; ppm = partes por millón = mg/L.

Nota: AcOEt: acetato de etilo; MetOH: metanol; LOD: Límite de detección; ppb = partes por mil millones = μg/L; ppm = partes por millón = mg/L; GLC: ácido gálico; CFC: ácido cafeico; CMR: ácido cumárico; RTN: rutin. ND: no detectable.

Since studies on phenolic compounds are mainly conducted in J. regia, more information on J. australis extracts is needed. Considering differences between species, essential in-depth studies would allow understanding phytochemical profiles while identifying lost or gained compounds during crop domestication.

Antibacterial activity and gallic acid effects on bacterial growth

The effects of J. regia and J. australis extract and gallic acid (main compound in all extracts) were evaluated against phytopathogen growth (figure 2). All extracts tested showed the highest inhibition against Xanthomonas (figure 2 A and B).

Each value is expressed as mean ± standard error. Different letters indicate statistically significant differences (p<0.01).

Cada valor se expresa como media ± error estándar. Las diferencias estadísticamente significativas se indican con letras diferentes (p < 0,01).

Figure 2. Antibacterial activity of shell extracts from walnuts.

Figura 2 Actividades antibacterianas contra bacterias fitopatógenos de los diferentes extractos de cáscara de nuez.

For Xcc 8004, methanolic extracts of J. regia (69.56%) and J. australis (72.31%) exhibited maximum inhibition at 10 mg/mL. Ethyl acetate extracts exhibited inhibitory activity against Xcc 8004, with J. regia demonstrating higher inhibition percentage (59.07%) than J. australis (41.61%).

All extracts from both Juglans sp. inhibited Xanthomonas axonopodis pv. phaseoli, exceeding 40% (figure 2B).

Considering Erwinia amylovora, methanolic extracts from both species diminished bacterial development in 37.64% (J. regia) and 35.92% (J. australis) at the highest tested concentration (figure 2C).

Once more, methanolic extracts at 10 mg/mL reached 54.65% and 52.60% inhibition against Clavibacter michiganensis, for J. regia and J. australis, respectively (figure 2D).

Gallic acid did not show substantial inhibition on the phytopathogens evaluated. At the highest concentration, it inhibited X. axonopodis (51.95%) (figure 2). Other studies mention antimicrobial activity of different parts of J. regia principally against pathogens of importance in human health ⁽³⁸⁾, but none on antimicrobial effect against phytopathogens. Here, we observe that the methanolic extract from walnut shells had the best antimicrobial activity against the phytopathogens assayed even at the minimum concentration (0.1 mg/mL).

Shell extracts have inhibitory effects against Xanthomonas sp. and the highest amount of gallic acid (table 2), probably involved in antibacterial activities. Gallic acid is extensively studied, and its mechanism of action as an effective antimicrobial is well known ^{(13, 21, 40)}.

Vu et al. (2017) report that gallic acid in walnuts can be found either in its free form or as part of hydrolyzable tannins. Nevertheless, gallic acid was less effective at inhibiting the phytopathogens assayed, suggesting a synergistic effect of the minor components.

Few studies have examined walnut shell extracts’ antimicrobial effects, typically requiring higher concentrations (1-100 mg/mL) ^{(31, 32, 44)}. Several reports used minimum bactericidal concentrations above 20 mg/mL for J. regia extracts ⁽⁴³⁾ with notable activity against gram-negative bacteria like E. coli and P. aeruginosa ⁽³⁶⁾. In our study, methanolic extracts effectively inhibited Gram-positive and Gram-negative phytopathogens at 10 mg/mL, particularly Xanthomonas spp, as previously reported with extracts from six walnut cultivars against Gram-positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (P. aeruginosa, E. coli, Klebsiella pneumoniae) ⁽³⁴⁾.

Recently, the search for new natural compounds has gained interest given antioxidant and antimicrobial properties. Native plants are valued for economic and ecological benefits, with preservation playing a vital role ⁽²²⁾.

The scarce information about using J. australis phytochemicals evaluates the effect of leaves and stem extracts on Herpes simplex virus ⁽³⁷⁾. Here, we could demonstrate the efficacy of the native extracts over various phytopathogens.

Conclusion

In this study, we first report antibacterial activity of extracts from J. australis against phytopathogenic bacteria, and first findings on their particular antioxidant and antiradical activities. This contributions could enhance regional value. This research first characterizes walnut shell extracts from J. australis. Our findings demonstrate that methanolic extracts exhibit significant antimicrobial activity against Xanthomonas sp., suggesting natural biocontrol alternatives to copper-based formulations.

Acknowledgments

This research received financial support from the Agencia Nacional de Promoción Científica y Técnica ANPCyT (PICT 2020-03408) and the Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET (PIBAA 0208CO).

The authors are grateful to the researcher, Dr. Osvaldo Delgado (PROIMI), for providing the bacterial strains used in the antimicrobial assays.

References

1. Acquaviva, R.; D’Angeli, F.; Malfa, G. A.; Ronsisvalle, S.; Garozzo, A.; Stivala, A.; Ragusa, S.; Nicolosi, D.; Salmeri, M. and Genoveseet, C. 2021. Antibacterial and anti-biofilm activities of walnut pellicle extract (Juglans regia L.) against coagulase-negative staphylococci. Nat Prod Res. 35(12): 2076-81.

2. Akbari, V.; Jamei, R.; Heidari, R.; Esfahlan, A. J. 2012. Antiradical activity of different parts of Walnut (Juglans regia L.) fruit as a function of genotype. Food Chemistry. 135: 2404-2410.

3. Bajpai, V. K.; Dung, N. T.; Suh, H. J.; Kang, S. C. 2010. Antibacterial activity of essential oil and extracts of Cleistocalyx operculatus Bbds against the bacteria of Xanthomonas spp. JAOCS, Journal of the American Oil Chemists Society. 87: 1341-1349.

4. Barekat, S.; Nasirpour, A.; Keramat, J.; Dinari, M.; Meziane-Kaci, M.; Paris, C.; Desobry, S. 2022. Phytochemical composition, antimicrobial, anticancer properties, and antioxidant potential of green husk from several walnut varieties (Juglans regia L.). Antioxidants. 12(1): 52.

5. Bennet, R. A.; Billing, E. 1978. Capsulation and virulence in Erwinia amylovora. Annals of Applied Biology. 89: 41-45.

6. Benzie, I. F. F.; Strain, J. J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay. Analytical Biochemistry. 239: 70-76.

7. Bernard, A.; Lheureux, F.; Dirlewanger, E. 2018. Walnut: past and future of genetic improvement. Vol. 14. Tree Genetics and Genomes. Springer Verlag.

8. Boiteux, J.; Espino, M.; Fernández, M. de los Á.; Pizzuolo, P.; Silva, M. F. 2019. Eco-friendly postharvest protection: Larrea cuneifolia-nades extract against botrytis cinerea. Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Mendoza. Argentina. 51(2): 427-437.

9. Boiteux, J.; Fernández, M. de los Á.; Espino, M.; Fernanda Silva, M. F.; Pizzuolo, P. H.; Lucero, G. S. 2023. In vitro and in vivo efficacy of Larrea divaricata extract for the management of Phytophthora palmivora in olive trees. Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Mendoza. Argentina. 55(2): 97-107. DOI: https://doi. org/10.48162/rev.39.112

10. Brand-Williams, W.; Cuvelier, M. E.; Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology. 28: 25-30.

11. Brown, A. D.; Grau, H. R.; Malizia, L. R.; Grau, A. 2001. In: Kappelle M, Brown AD (eds) Bosques nublados del neotrópico. Instituto Nacional de Biodiversidad. Costa Rica. p 623-659.

12. Carnaval, L. S. C.; Cerboneschi, M.; Tegli, S.; Yoshida, C. M. P.; Melo, E.; Santos, A. M. P. 2022. Potential agrifood application of seriguela (Spondias purpurea L.) residues extract and nanoZnO as antimicrobial, antipathogenic and antivirulence agents. Research, Society and Development 11: e37211125033. https://doi.org/10.33448/rsd-v11i1.25033

13. Cavalca, L. B.; Zamuner, C. F. C.; Saldanha, L. L.; Polaquini, C. R.; Regasini, L. O.; Behlau, F.; Ferreira, H. 2020. Hexyl gallate for the control of citrus canker caused by Xanthomonas citri subsp citri. Microbiology open. 9: 1-8.

14. Daglia, M. 2012. Polyphenols as antimicrobial agents. Current Opinion in Biotechnology. 23: 174-181.

15. Delaviz, H.; Mohammadi, J.; Ghalamfarsa, G.; Mohammadi, B.; Farhadi, N. 2017. A review study on phytochemistry and pharmacology applications of Juglans regia plant. Phcog Rev. 11: 145-52.

16. Fernández-Agulló, A.; Castro-Iglesias, A.; Freire, M. S.; González-Álvarez, J. 2020. Optimization of the extraction of bioactive compounds from walnut (Juglans major 209 x Juglans regia) leaves: Antioxidant capacity and phenolic profile. Antioxidants. 9: 4-6.

17. Fernández-Agulló, A.; Freire, M. S.; Ramírez-López, C.; Fernández-Moya, J.; González-Álvarez, J. 2021. Valorization of residual walnut biomass from forest management and wood processing for the production of bioactive compounds. Biomass Convers Biorefin. 11(2): 609-18.

18. Hu, Q; Liu, J.; Li, J.; Liu, H.; Dong, N.; Geng, Y.; Yang, L.; Wang, Y. 2020. Phenolic composition and nutritional attributes of diaphragma juglandis fructus and shell of walnut (Juglans regia L.). Food Sci Biotechnol. 29(2): 187-96.

19. Hussain, T.; Singh, S.; Danish, M.; Pervez, R.; Hussain, K.; Husain, R. 2020. Natural Metabolites: An eco-friendly approach to manage plant diseases and for better agriculture farming. In natural bioactive products in sustainable agriculture. https://doi.org/10.1007/978-981- 15-3024-1_3

20. INFOSTAT Analytical Software version 2020e. Universidad Nacional de Córdoba. Córdoba. Argentina.

21. Krol, E.; De Sousa Borges, A.; Da Silva, I.; Polaquini, C. R.; Regasini, L. O.; Ferreira, H.; Scheffers, D. J. 2015. Antibacterial activity of alkyl gallates is a combination of direct targeting of FtsZ and permeabilization of bacterial membranes. Frontiers in Microbiology. 6: 390.

22. Lorenzo, M. E.; Casero, C. N.; Gómez, P. E.; Segovia, A. F.; Figueroa, L. C.; Quiroga, A.; Werning, M. L.; Wunderlin, D. A.; Baroni, M. V. 2020. Antioxidant characteristics and antibacterial activity of native woody species from Catamarca, Argentina. Nat Prod Res. DOI: 10.1080/14786419.2020.1839461.

23. Luján, E. E.; Torres-Carro, R.; Fogliata, G.; Alberto, M. R.; Arena M. E. 2019. Fungal Extracts as Biocontrol of Growth, Biofilm Formation, and Motility of Xanthomonas citri subsp. citri. Global Journal of Agricultural Innovation, Research & Development. 6: 25-37.

24. Macioniene, I.; Cepukoit, D.; Salomskiene, J.; Cernauskas, D.; Burokiene, D.; Salaseviciene, A. 2022. Effects of Natural Antimicrobials on Xanthomonas Strains Growth. Horticulturae. 8: 7.

25. Mansfield, J.; Genin, S.; Magori, S.; Citovsky, V.; Sriariyanum, M.; Ronald, P.; Dow, M.; Verdier, V.; Beer S. V.; Machado, M. A. 2012. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol. 13: 614-629.

26. Masek, A.; Latos-Brozio, M.; Chrzescijanska, E.; Podsedek, A. 2019. Polyphenolic profile and antioxidant activity of Juglans regia L. leaves and husk extracts. Forests. 10: 988. doi:10.3390/f10110988

27. Mateș, L.; Rusu, M. E.; Popa, D. S. 2023. Phytochemicals and Biological Activities of Walnut Septum: A Systematic Review. J Antioxidants. 12(3): 604.

28. Merlin, E.; Lopez, J.; Sarmiento, H. 2014. Control del tizón del fuego en manzano. Folleto Técnico Núm. 73. INIFAP.

29. Miller, S. A.; Ferreira, J. P.; Lejeune, J. T. 2022. Antimicrobial use and resistance in plant agriculture: A one health perspective. Agriculture (Switzerland). 12(2): 1-27.

30. Mohana, D. C.; Raveesha, K. A. 2006. Antibacterial activity of Caesalpinia coriaria (Jacq.) Willd. against plant pathogenic Xanthomonas pathovars: an ecofriendly approach. Journal of Agricultural Technology. 2: 31.

31. Moure, A.; Cruz, J. M.; Franco, D.; Domínguez, J. M.; Sineiro, J.; Domínguez, H. 2001. Natural antioxidants from residual sources. Food Chem. 72: 145-171.

32. OEPP/EPPO. 2005. Clavibacter michiganensis subsp. michiganensis. Bull. OEPP-EPPO Bull. 35: 275-283.

33. Oliveira, I.; Sousa, A.; Ferreira, I. C. F. R.; Bento, A.; Estevinho, L.; Pereira, J. A. 2008. Total phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.) green husks. Food and Chemical Toxicology. 46: 2326-2331.

34. Pereira, A. P.; Ferreira, C. F. R.; Marcelino, F, Valentao, P.; Andrade, P.; Seabra, R.; Estevinho, L.; Bento A.; Pereira, J. A. 2007. Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobrançosa) leaves. Molecules. 12: 1153-1162.

35. Pontes, J. G. D. M.; Fernandes, L. S.; dos Santos, R.; Tasic, L.; Fill, T. P. 2020. Virulence factors in the Phytopathogen-Host Interactions: An overview. Journal of Agricultural and Food Chemistry. 68: 7555-7570.

36. Raaman, N.; Mathiyazhagan, K.; Jegadeesh, R.; Divakar, S.; Vennila, S.; Balasubramanian, K. 2011. Antimicrobial activities of different organic extracts of nut shells of Juglans regia (walnut). Herbal Tech Industry. 20: 22.

37. Ruffa, M. J.; Wagner, M. L.; Suriano, M.; Vicente, C.; Nadinic, J.; Pampuro, S.; Salomón, H.; Campos, R. H.; Cavallaro, L. 2004. Inhibitory effect of medicinal herbs against RNA and DNA viruses. Antiviral Chemistry and Chemotherapy. 15(3): 153-159. https://doi. org/10.1177/095632020401500305

38. Sandu-Bălan, A.; Ifrim, I. L.; Patriciu, O. I.; Ștefănescu, I. A.; Fînaru, A. L. 2024. Walnut by-products and elderberry extracts-sustainable alternatives for human and plant health. Molecules. 29(2). https://doi.org/10.3390/molecules29020498

39. Sharma, M.; Sharma, M.; Sharma. M. 2022. A comprehensive review on ethnobotanical, medicinal and nutritional potential of walnut (Juglans regia L.). Proceedings of the Indian National Science Academy. https://doi.org/10.1007/s43538-022-00119-9

40. Silva, I. C.; Polaquini, C. R.; Regasini, L. O.; Ferreira, H.; Pavan, F. R. 2017. Evaluation of cytotoxic, apoptotic, mutagenic, and chemo- preventive activities of semi-synthetic esters of gallic acid. Food and Chemical Toxicology. 105: 300-307.

41. Singleton, V. L.; Rossi, J. A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16: 144-158.

42. Soto-Maldonado, C.; Caballero-Valdés, E.; Santis-Bernal, J.; Jara-Quezada, J.; Fuentes-Viveros, L.; Zúñiga-Hansen ME. 2022. Potential of solid wastes from the walnut industry: Extraction conditions to evaluate the antioxidant and bioherbicidal activities. Electronic Journal of Biotechnology 58: 25-36.

43. Vieira, V.; Pereira, C.; Abreu, R. M.; Calhelha, R. C.; Alves, M. J.; Coutinho, J. A. P.; Ferreira, O.; Barros L.; Ferreira ICFR. 2020. Hydroethanolic extract of Juglans regia L. green husks: A source of bioactive phytochemicals. Food and Chemical Toxicology. 137: 111189. https:// doi.org/10.1016/j.fct.2020.111189

44. Villamil-Galindo, E.; Piagentini, A. 2024. Green solvents for the recovery of phenolic compounds from strawberry (Fragaria x ananassa Duch) and apple (Malus domestica) agro-industrial bio-wastes. Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Mendoza. Argentina. 56(1): 149-160. DOI: https://doi.org/10.48162/rev.39.131

45. Viola, C. M.; Torres-Carro, R.; Cartagena, E.; Isla, M. I.; Alberto, M. R.; Arena, M. E. 2018. Effect of wine wastes extracts on the viability and biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus Strains. Evidence-Based Complementary and Alternative Medicine. https://doi.org/10.1155/2018/9526878

46. Vu, T. T.; Kim, H.; Tran, V. K.; Vu, H. D.; Hoang, T. X.; Han, J. W.; Choi, Y. H.; Jang, K. S.; Choi, G. J.; Kim J. C. 2017. Antibacterial activity of tannins isolated from Sapium baccatum extract and use for control of tomato bacterial wilt. PLoS ONE. 12: 1-12.

47. Yabalak, E.; Erdogan Eliuz, E. A. 2021. Green synthesis of walnut shell hydrochar, its antimicrobial activity and mechanism on some pathogens as a natural sanitizer. Food Chemistry. 366. https://doi.org/10.1016/j.foodchem.2021.130608

48. Yang, J.; Chen, C.; Zhao, S.; Ge, F.; Liu, D. 2014. Effect of solvents on the antioxidant activity of walnut (Juglans regia L.) s.hell extracts. Journal of Food and Nutrition Research. 2: 621-626.