Journal of biological and health sciences http://biotecnia.unison.mx

Universidad de Sonora

ISSN: 1665-1456

Streptomycetes isolated from the rhizosphere of agricultural soils in the Valle del Mezquital, Mexico: in vitro production capacity of growth- promoting metabolites and antagonists against phytopathogenic fungi

Yamily Elianeth Castañeda-Cisneros1

, Germán Zafra2

, Yuridia Mercado-Flores1

, Miguel Angel Anducho-

Reyes1 , María del Rocío Ramírez-Vargas1 and Alejandro Téllez-Jurado1*

1 Laboratorio de Aprovechamiento Integral de Recursos Bióticos, Universidad Politécnica de Pachuca, Carretera Pachuca-Cd. Sahagún, km 20, Ex-Hacienda de Santa Bárbara. Zempoala, Hidalgo, C.P.43830, México.

2 Grupo de Investigación en Bioquímica y Microbiología. Escuela de Microbiología. Universidad Industrial de Santander, Bucaramanga, 680003, Colombia.

Streptomicetos aislados de la rizosfera de suelos agrícolas del Valle del Mezquital, México: capacidad de producción in vitro de metabolitos promotores de crecimiento y de antagonismo contra hongos fitopatógenos

ABSTRACT

The Valle del Mezquital, Mexico, is one of the main crop- producing areas in the country. This valley is irrigated by wastewater, primarily from Mexico City. This characteristic creates unique environmental conditions that can impact microorganisms in agricultural soils. In this study, acti- nomycetes were isolated from the rhizosphere of agricultural soils to characterize them and determine their potential as plant growth promoters and inhibitors of phytopathogenic fungi. Thirteen strains of the Streptomyces genus were iso- lated, and in vitro studies revealed that all could produce indoleacetic acid, siderophores, and organic acids. Strep- tomyces thinghirensis and Streptomyces lateritius solubilized phosphates, while Streptomyces lusitanus produced HCN. Previously unreported strains with antagonistic activity aga- inst plant pathogenic fungi were identified, the main ones being Streptomyces pseudogriseolus, Streptomyces atrovirens, Streptomyces lateritius, Streptomyces nigra, and Streptomyces griseoplanus. The results obtained provide new knowledge into Streptomycetes that have not been previously studied and may offer tools for the biological control of diseases cau- sed by phytopathogenic fungi, as well as strategies for en- hancing productivity under conservation tillage conditions. Keywords: Actinomycetes; Conservation agriculture; Was- tewater irrigation.

RESUMEN

El Valle del Mezquital, México, es una de las principales zonas productoras de diferentes cultivos en el país. Este Valle es irri- gado por las aguas residuales provenientes principalmente de la Ciudad de México, esta característica genera condi- ciones ambientales únicas que pueden incidir sobre los mi- croorganismos de los suelos agrícolas. En el presente trabajo, se aislaron actinomicetos de la rizósfera de suelos agrícolas con el fin de caracterizarlos y determinar su potencial como promotores del crecimiento vegetal y de inhibición de hon- gos fitopatógenos. Se aislaron 13 cepas del género de Strep- tomyces, a través del estudio in vitro se observó que todas

*Author for correspondence: AlejandroTéllez Jurado e-mail: alito@upp.edu.mx

Received: May 22, 2025

Accepted: July 2, 2025

Published: August 13, 2025

fueron capaces de producir ácido indol acético, sideróforos y ácidos orgánicos. Streptomyces thinghirensis y Streptomyces lateritus fueron capaces de solubilizar fosfatos y Streptomyces lusitanus de producir HCN. Se identificaron cepas no repor- tadas con anterioridad con capacidad antagónica contra hongos fitopatógenos siendo los principales Streptomyces pseudogriseolus, Streptomyces atrovirens, Streptomyces la- teritius, Streptomyces nigra y Streptomyces griseoplanus. Los resultados obtenidos aportan nuevos conocimientos sobre Streptomycetes que no han sido estudiados previamente y pueden ofrecer herramientas para el control biológico de enfermedades causadas por hongos fitopatógenos, así como estrategias para mejorar la productividad en condiciones de labranza de conservación.

Palabras clave: Actinomicetos, Agricultura de conservación, Riego con aguas residuales.

INTRODUCTION

Actinomycetes constitute a heterogeneous group of mi- croorganisms capable of utilizing simple or complex carbon sources and organic molecular compounds such as acids, polysaccharides, lipids, proteins, and aliphatic hydrocarbons. They also utilize ammonium, nitrates, amino acids, peptones, and a large number of proteins as nitrogen sources (Leveau and Bouix, 2000).

Direct mechanisms occur when bacteria synthesize me- tabolites that facilitate plant growth or increase the availabi- lity of nutrients required for plant metabolism, and improve plant nutrition. They include nitrogen fixation, synthesis of phytohormones, vitamins and enzymes, phosphorus solubilization, nitrite production, nitrate accumulation, and siderophore secretion (Gómez et al., 2012). In addition to these characteristics, they can also exhibit antagonistic acti- vity, which is an interaction between microorganisms where one interferes with the other, causing the loss or reduction of activity of one of them. This is the basis for true biological control of phytopathogenic microorganisms in plants.

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Volume XXVII

DOI: 10.18633/biotecnia.v27.2666

The Valle del Mezquital (VM) is a unique ecosystem located southeast of Hidalgo, covering 642,653 ha, 60 km northwest of the Mexico City Metropolitan Area. The VM is the region in the world with the second-highest use of was- tewater in the agricultural sector. Paradoxically, the economic development of the area is closely linked to the use of this resource in agriculture (Durán-Álvarez et al., 2021). Conven- tional agricultural practices applied in the VM are based on intensive tillage techniques, residue burning, and plowing (Zhang et al., 2018), which has generated problems of soil alkalization and desertification. In the VM, there are experi- mental platforms whose objective is to apply agricultural soil recovery practices. These platforms have been in existence for around 20 years, and strictly adhere to the conservation of agriculture practices. Furthermore, analyzing the biota of agricultural soils provides a better understanding of the biogeochemical cycles that occur. Additionally, it is possible to infer the potential relationships between microorganisms and their impact on crop productivity (Castañeda-Cisneros et al., 2024).

Some authors have described how irrigation water has a significant impact on soil microbiota, with prevailing species including Proteobacteria, Firmicutes, and Actinobacteria (Jiang et al., 2024), which primarily specialize in degrading toxic contaminants. Therefore, the actinomycetes present in the rhizosphere of crops in the area, as well as the potential impact of wastewater irrigation on these microorganisms, are of interest. This study aimed to study actinomycetes isolated from agricultural soils irrigated with wastewater and subject to conservation agriculture in the VM. The production of mo- lecules involved in plant growth by each microorganism was studied, as well as their possible antagonistic effect against phytopathogenic microorganisms present in the VM.

MATERIAL AND METHODS

Microorganisms, strain propagation, and conservation The method used for isolating actinomycetes was by serial plate dilutions (Hayakawa, 2008). One g of dry soil from each sample was vigorously shaken in 9 mL of sterile distilled water and heated at 50 °C for 30 min (Oskay et al., 2004). This solution (10-1) was diluted five times (10-5) using sterile dis- tilled water. A 50 µL aliquot was taken and distributed onto Petri dishes containing ISP2 (4 g/L yeast extract, 10 g/L malt extract, 4 g/L dextrose, and 20 g/L bacteriological agar at pH

7.3) or ISP3 (20 g/L oats, 1 mL trace salt solution [0.1 g FeSO47 H2O, 0.1 g MnCl24 H2O, 0.1 g ZnSO47 H2O] in 100 mL of disti- lled water, and 18 g/L bacteriological agar at pH 7.2) media. All strains were propagated both media. The media were supplemented with nystatin (50 µg/mL) and nalidixic acid (25 µg/mL) to inhibit fungal and bacterial contamination. The plates were incubated at 28 °C for 28 d and replated. All plates were reseeded monthly and kept refrigerated until use. The strains Streptomyces atrovirens, Streptomyces lateritius, Strep- tomyces sp. A-C7, Streptomyces pseudogriseolus, Streptomyces heliomycini, Streptomyces anulatus, Streptomyces virginiae, Streptomyces nigra CB-8, Streptomyces griseoplanus B4-M10,

Streptomyces flavogriseus, Streptomyces thinghirensis, Strep- tomyces lusitanus and Streptomyces griseoaurantiacus, were used (Castañeda-Cisneros et al., 2020).

In vitro plant growth promotion activities Phosphate solubilization

The qualitative activity of phosphate solubilization was analyzed on Pikovskaya (PVK) agar (Pikovskaya, 1948). Each strain was inoculated in the center of the plate by puncturing it with a sterile toothpick. A PVK agar plate served as a con- trol. A clear zone around the colony was considered positive after 14 d of incubation at 28 °C. The phosphate solubilization index (SI) was calculated from the ratio of the total diameter (colony plus halo area) to the colony diameter, according to a formula reported by Chouyia et al. (2020) expressed in mm.

Organic acid production

Phosphate-solubilizing actinomycetes were transferred to Sandar-Rao and Shina (SRSM-1) agar (Sandar and Shina, 1963). Each strain was inoculated onto the center of the plate using a sterile toothpick and incubated at 28 °C for 14

d. An uninoculated SRSM-1 agar plate served as a control. A color change in the solubilization halo from purple to yellow indicated a positive result. Results were reported using the phosphate solubilization index, expressed in mm. Strains were classified based on their SI as low (SI < 2), intermediate (2 < SI < 4), and high (SI > 4) solubilization (Elias et al., 2016).

Siderophore production

Siderophore production was determined on Chromium Azurol S (CAS) agar, according to the method described by Schwyn and Neilands (1987) and modified by Hu and Xu (2011). Strains were inoculated by pinprick in the center of the plate and incubated at 28 °C for 7 d. An uninoculated plate was used as a control. A colony with the formation of an orange-yellow halo was considered positive for siderophore production. Activity diameters were reported using the fo- llowing scale: 0 = negative; 1 = 1–10 mm halo; 2 = 11–20 mm

halo; 3 = 21–30 mm halo; 4 = 31–40 mm halo; and 5 = 41–50 mm halo (Sreevidya et al., 2016).

Hydrogen cyanide production

The qualitative determination of hydrogen cyanide (HCN) was evaluated using the method described by Lorck (1948). Strains were streaked onto modified Bennett agar plates (Jo- nes, 1949) (glycine 4.4 g/L, meat extract 1 g/L, enzymatically hydrolyzed casein 2 g/L, yeast extract 1 g/L, and bacteriolo- gical agar 15 g at pH 7.3). A Whatman No. 1 filter paper was placed under the lid of the Petri dish, previously soaked in 0.5

% picric acid in 2 % sodium carbonate for 1 minute. All plates were sealed with parafilm and incubated at 28 °C for 7 d. A color change expressed a positive indicator of HCN produc- tion in the filter paper; the scale was negative (-) = no color change; weak producer (+) = yellow to light reddish brown; Moderate producer (++) = yellow to medium reddish brown, and heavy producer (+++) = yellow to dark reddish brown (Sreevidya et al., 2016).

Indoleacetic acid production

Indoleacetic acid (IAA) production was estimated using the Gordon and Weber (1951) method. Actinomycete strains were grown on ISP2 agar and incubated at 28 °C for 10 d. One mm diameter discs were cut from each plate using a sterile scalpel and transferred to Erlenmeyer flasks containing 100 mL of ISP2 broth supplemented with 0.2 % L-tryptophan. The flasks were maintained at 28 °C with continuous shaking at 125 rpm. After 15 d of incubation, the resulting cultures were centrifuged at 11,000 rpm for 15 min. The reaction consisted of placing 1 mL of supernatant with 2 mL of Salkowski rea- gent in test tubes, which were incubated for 25 min in the dark at 28 °C. The appearance of a pink-red color indicated the production of IAA (Naik and Gupta, 2020). Absorbance was measured at a wavelength of 530 nm, and the concen- tration was reported using a calibration curve with IAA as the standard, expressed in micrograms per milliliter (μg/mL). Distilled water was used as the reaction blank.

Ammonia production

To evaluate the capacity of actinomycetes as ammonia producers, the methodology proposed by Cappucino and Sherman (1992) was followed. The strains were inoculated into Erlenmeyer flasks with 10 mL of peptone water (1 g/L peptone and 8.5 g/L NaCl at pH 7) and maintained at 28 ºC with continuous shaking at 120 rpm for 15 d. Then, 0.5 mL of Nessler reagent was added to each culture, and the develo- pment of a yellow to brown color was considered a positive result.

Antagonistic activity against plant pathogenic fungi

The in vitro antagonistic activity of actinomycetes against 10 plant pathogenic fungi that impact crops was evaluated. These fungi were identified and donated by Dr. Issac Juan Luna Romero of the Plant Pathology Laboratory of the Na- tional Polytechnic Institute: Colletotrichum lindemuthianum, Pestalotia spp., Helminthosporium maydis, Curvularia spp., Co- lletotrichum spp., Fusarium spp., Alternaria dauci, Phytophtho- ra spp., Sclerotinia sclerotiorum, and Fusarium oxysporum. The strains were grown on Potato Dextrose Agar (PDA) at 28 °C for 10 d or until complete sporulation. Each fungus was ino- culated by puncturing it with a sterile bacteriological loop in the center of a PDA plate, and seven different actinomycetes were plated around it. Control plates were prepared without inoculating the actinomycetes to evaluate fungal growth. The plates were kept at 28 °C for 10 d. In the antagonism experiments, inhibition was identified by the absence of contact between the actinomycetes and the fungus. This was

Data analysis

The mean and standard deviation were calculated based on the replicates of each experiment. Data were analyzed using one-way analysis of variance (ANOVA). Significant differences between means were determined using Tukey’s method with a 95 % confidence interval, as calculated with Minitab 18.1 software (Minitab Inc., State College, PA, USA). All analyses were performed in triplicate.

RESULTS

Production of metabolites involved in plant growth

The first stage of the study focused on analyzing the in vitro properties of Streptomyces strains isolated from agricultural soils in the Valle del Mezquital. Of the 13 isolated strains, only 2 (15.38 %) (Fig. 1A) were able to utilize tricalcium phosphate as an insoluble source (Fig. 1B). S. thinghirensis had the hig- hest phosphate SI at 3.4, followed by S. lateritius with an SI of

2. The strains were transferred to SRSM-1 medium (PVK plus bromocresol purple) to determine the secretion of organic acids. Eleven Streptomyces strains (84.62%) (Fig. 1A) were ob- served to acidify the medium, as indicated by the formation of yellow halos around the colonies (Fig. 1C). The maximum acidification zone was obtained with S. thinghirensis (SI = 5). At the same time, S. lateritius (SI = 4), S. griseoplanus B4-M10 (SI = 3.2), S. griseoaurantiacus (SI = 2.83), S. anulatus (SI = 2.69), and S. lusitanus (SI = 2.67) showed intermediate indices (Table 1). The 13 strains (Fig. 1A) studied produced siderophores on CAS agar. Different colors were observed in the halos due to the availability of Fe since most of this ion is chelated to CAS (blue); that is, the strain takes up Fe by synthesizing sidero- phores (orange or purple), which are molecules with a higher affinity for Fe than CAS itself (Fig. 1D). S. griseoplanus B4-M10 showed the highest activity with a siderophore diameter of 35 mm (siderophore rating of 4), followed by S. flavogriseus and S. nigra CB-8 with diameters of 24 and 22 mm (sidero- phore rating of 3), respectively (Table 1). The strain with the lowest activity diameter was S. atrovirens, with a diameter of 4 mm (siderophore rating of 1).

On the other hand, only one of the strains studied tested positive for HCN (Fig. 1A). S. lusitanus was found to be a mo- derate producer (++) of HCN (Fig. 1E). Regarding IAA produc- tion (Fig. 1F), all 13 strains exhibited this activity (Fig. 1A). The strains with the highest IAA production were S. thinghirensis and S. griseoplanus B4-M10, with 231.73 and 229.54 µg/mL of IAA, respectively. At the same time, S. griseoaurantiacus produced the lowest concentration (28.79 µg/mL). Finally, ammonia production was observed in 100 % (Fig. 1A) of the

strains tested using Nessler’s reagent, where the color turns

expressed as a percentage of inhibition based on the diffe- rence between the diameter of the fungal mycelium on the control plates and the diameter of the fungal mycelium on the plates containing the different actinomycetes. The results

yellowish-brown, and its intensity depends on the NH3 centration (Fig. 1G).

Evaluation of Antagonistic Activity

con-

were expressed as described by Kumar et al. (2014) as: - = no activity; + = weak activity (< 25 % inhibition); ++ = moderate activity (25-50 % inhibition); and +++ = strong activity (> 50

% inhibition).

A preliminary qualitative assay evaluated the inhibitory acti- vity of 13 actinomycete strains on the mycelial growth of 10 phytopathogenic fungi impacting agriculture. The strain that exhibited the greatest antagonistic activity was Streptomyces

A

B

C

D

E

F

G

Figura 1. Número total de cepas productoras de moléculas promotoras del crecimiento vegetal y algunos resultados de las pruebas cualitativas en placa de las actividades ensayadas. A = Número de cepas productoras de moleculas promotoras del crecimiento vegetal; B = Solubilización de fosfatos; C = Producción de ácidos orgánicos; D = Producción de sideróforos; E = Producción de cianuro de hidrógeno; F = Producción de AIA y, G

= Producción de amoniaco.

Figure 1. Total number of strains producing plant growth-promoting molecules and some results of qualitative plate tests for the activities tested. A = Number of strains producing plant growth-promoting molecules; B = Phosphate solubilization; C = Organic acid production; D = Siderophore production; E = Hydrogen cyanide production; F = IAA production; and G = Ammonia production.

Tabla 1. Producción de metabolitos detectados de las 13 cepas de Streptomicetos aislados de los suelos agrícolas del Valle del Mezquital.

Table 1. Production of metabolites detected from the 13 strains of Streptomycetes isolated from the agricultural soils of the Valle del Mezquital.

IS1 índice de solubilización en agar PVK; IS2 índice de solubilización en agar SRSM-1 (PVK+ púrpura de bromocresol). Escala para la clasificación de acuerdo al IS (Marra et al., 2015); bajo (IS<2); intermedio (2< IS <4); alto (IS>4). - = Sin actividad; + = con actividad.

SI1 Solubilization Index on PVK agar; SI2 Solubilization Index on SRSM-1 agar (PVK + bromocresol purple). Scale for classification according to IS (Marra et al., 2015): low (IS<2); intermediate (2< IS<4); high (IS>4). - = no activity, + = with activity.

sp. A-C7 exhibited antagonistic activity against all 10 phyto- pathogenic fungal strains. S. griseoaurantiacus exhibited antagonistic activity against nine phytopathogenic fungi, except for Fusarium spp. did not show this activity, while S. pesudogriseolus showed antagonistic activity against seven phytopathogenic fungi, except Fusarium spp., Phytophtora spp., and F. oxysporum. The S. lusitanus strain only showed antagonistic activity against C. lindemuthianum. The results obtained from this step are presented in Table 2.

From the in vitro qualitative experiment, actinomycetes with the highest antagonistic potential (+++) were selected and subjected to quantitative tests for 10 d. The results of the dual culture assays between actinomycetes and fungi are shown in Table 3. It was detected that C. lindemuthianum was inhibited by 11 actinomycetes (84.62 %), of which Strep- tomyces sp. A-C7 showed the maximum inhibition activity with 95.18 %, followed by S. griseoaurantiacus with 91.33 %,

S. griseoplanus B4-M10 with 88.69 %, and S. heliomycini with

81.23 %. S. atrovirens and S. anulatus strains were unable to

affect the radial growth of the other. It was also observed that A. dauci, the cause of leaf blight in carrots, was inhibited by six actinomycetes (46.15 %), the most effective of which was Streptomyces sp. A-C7 (69.62 %) and S. pseudogriseolus (60.76 %). The species S. atrovirens, S. lateritius, S. griseoplanus B4-M10, and S. flavogriseus exhibited low inhibition percen- tages, ranging from 15 % to 30 %.

Streptomyces sp. A-C7 and S. pseudogriseolus were the candidates with the highest antifungal spectrum potential. Streptomyces sp. A-C7 actively suppressed the growth of all 10 fungi studied, reaching inhibition percentages greater than 65 % for 8 phytopathogenic strains: C. lindemuthianum, Pestalotia spp., H. maydis, Curvularia spp., Colletotrichum spp.,

A. dauci, Phytophthora spp., and S. sclerotiorum. Meanwhile, lower inhibition values were observed for Fusarium spp. and

F. oxysporum, with 34.16 % and 32.53 %, respectively (Figure 2A). On the other hand, S. pseudogriseolus had inhibitory effects on 6 of the fungi analyzed: C. lindemuthianum, Pesta- lotia spp., H. maydis, Curvularia spp., A. dauci, and S. sclerotio- rum (Figure 2B).

Tabla 2. Actividad antagónica in vitro de diferentes actinomicetos aislados de suelos agrícolas del Valle del Mezquital, Hidalgo contra hongos fitopatógenos.

Table 2. In vitro antagonistic activity of different actinomycetes isolated from the Valle del Mezquital agricultural soils, Hidalgo, against phytopathogenic fungi.

Hongos: 1 = C. lindemuthianum; 2 = Pestalotia spp.; 3 = H. maydis; 4 = Curvularia spp.; 5 = Colletotrichum spp.; 6 = Fusarium spp.; 7 = A. dauci; 8 = Phytophtora spp.; 9 = S. sclerotiorum; 10 = F. oxysporum. - = Sin actividad; + = actividad débil; ++ = actividad moderada, and +++ = actividad fuerte.

Fungus: 1 = C. lindemuthianum; 2 = Pestalotia spp.; 3 = H. maydis; 4 = Curvularia spp.; 5 = Colletotrichum spp.; 6 = Fusarium spp.; 7

= A. dauci; 8 = Phytophtora spp.; 9 = S. sclerotiorum; 10 = F. oxysporum. - = no activity; + = weak activity; ++ = moderate activity, and +++ = strong activity.

Tabla 3. Inhibición de los hongos fitopatógeno por los actinomicetos aislados de suelos agrícolas del Valle del Mezquital en estudio.

Table 3. Phytopathogenic fungi inhibition by actinomycetes isolated from agricultural soils of the Valle del Mezquital under study.

Hongos: 1 = C. lindemuthianum; 2 = Pestalotia spp.; 3 = H. maydis; 4 = Curvularia spp.; 5 = Colletotrichum spp.; 6 = Fusarium spp.; 7 = A. dauci; 8 =

Phytophtora spp.; 9 = S. sclerotiorum; 10 = F. oxysporum; - = sin inhibición.

Fungus: 1 = C. lindemuthianum; 2 = Pestalotia spp.; 3 = H. maydis; 4 = Curvularia spp.; 5 = Colletotrichum spp.; 6 = Fusarium spp.; 7 = A. dauci; 8 =

Phytophtora spp.; 9 = S. sclerotiorum; 10 = F. oxysporum; - = no inhibition.

C. lindemuthianum, which causes bean anthracnose, was inhibited by 11 of the 13 actinomycetes; only S. atrovirens and S. anulatus could inhibit its growth. A. dauci, the cause of carrot leaf blight, was inhibited by six streptomycetes. Streptomyces sp. A.C7 exhibited the greatest inhibition with

69.62 %, followed by S. pseudogriseolus with 60.76 %.

DISCUSSION

Evaluation of plant growth promotion capacity Streptomyces is considered the most abundant actinomycete and possibly the most important due to its ability to produ- ce a wide range of bioactive compounds, antibiotics, and extracellular enzymes. Chouyia et al. (2020) reported the phosphate solubilizing activity of Streptomyces roseocinereus and Streptomyces natalensis with maximum SI of 1.75 and 1.17, respectively. Boubekri et al. (2021) found an SI of 3.17 for

S. anulatus P16 in a medium supplemented with phosphate rock as the sole source of insoluble phosphate. Compared with the present work, the S. anulatus A5-M14 strain did not show phosphorus solubilizing capacity; only S. lateritius and

S. thinghirensis showed the ability to solubilize phosphates, with solubilization indices of 2 and 12, respectively. Regar- ding S. thinghirensis, SI is higher than that reported by both authors. The potential of S. thinghirensis to efficiently solubi-

lize phosphates has been previously described by Djebaili et al. (2020). Rehan et al. (2021) analyzed an S. thinghirensis HM3 strain isolated from agricultural soils in Saudi Arabia and found it capable of growing on PVK agar. However, the for- mation of a visible solubilization halo was not evident, they found visible phosphate traces. Among the mechanisms associated with phosphate solubilization by bacteria, acidifi- cation, chelation, phosphatase enzymes, and the production of organic acids may be involved (Mohammed, 2020).

Low molecular weight organic acids originate from metabolizing high molecular weight compounds, such as carbohydrates, peptides, and lipids. Among the organic acids produced by Streptomyces are succinic, formic, acetic acid, and the most predominant ones, such as oxalic, citric, and gluconic acid (Vargas-Hoyos et al., 2021), which are possibly responsible for the decrease in pH and, therefore, the solubi- lization of minerals.

Siderophores sequester Fe from the rhizosphere, inhi- biting pathogen growth or metabolic activity. Jarmusch et al. (2021) reported that the Streptomyces sp. S29 strain was effective in Fe chelation due to the secretion of siderophores, which inhibited the growth of filamentous fungi. On the other hand, Manigundan et al. (2020) reported that strains with higher secretion of siderophores can promote plant growth

A

B

Figura 2. Confrontación dual de la cepa A-C7 de Streptomyces sp. contra los hongos fitopatógenos (2A): a) C. lindemuthianum; b) Pestalotia spp.; c) H. maydis; d) Curvularia spp.; e) Colletotrichum spp.; f ) Fusarium spp.; g) A. dauci; h) Phytophtora spp.; i) S. sclerotiorum y j) F. oxysporum. La cepa S. pseudogriseolus A7-M9 (2B) se confronta contra

a) Pestalotia spp.; b) H. maydis; c) Curvularia spp.; d) A. dauci y e) S. sclerotiorum, mientras que la cepa de S. griseoauranticus se confronta contra f ) H. maydis; la cepa S. heliomycini se confronta contra g) S. sclerotiorum y finalmente S. flavogriseus se confronta contra h)

A. dauci.

Figure 2. Dual confrontation of the Streptomyces sp. strain A-C7 against phytopathogenic fungi (2A): a) C. lindemuthianum; b) Pestalotia spp.; c) H. maydis; d) Curvularia spp.; e) Colletotrichum spp.; f ) Fusarium spp.; g) A. dauci; h) Phytophtora spp.; i) S. sclerotiorum;

j) F. oxysporum. The S. pseudogriseolus A7-M9 strain (2B) is confronted against a) Pestalotia spp.; b) H. maydis; c) Curvularia spp.; d) A. dauci and e) S. sclerotiorum, while the S. griseoauranticus strain is confronted against f ) H. maydis; the S. heliomycini strain is confronted against g) S. sclerotiorum and finally S. flavogriseus is confronted against

h) A. dauci.

and yield better crops. In the present study, the generation of siderophores by S. lateritius, S. flavogriseus, and S. lusitanus was detected; this ability has not been previously reported in these microorganisms, making them an additional option for developing new biotechnological products.

Hydrogen cyanide (HCN) is a volatile secondary metabo- lite, a byproduct of glycine metabolism, which is highly de- pendent on glycine availability and environmental Fe levels (Short et al., 2018). HCN production by Streptomyces sp. has been reported. Anwar et al. (2016) evaluated HCN secretion in six actinomycetes obtained from the rhizosphere of wheat and tomato plants grown in Punjab province, Pakistan. Pas- sari et al. (2015) found that 68 % of endophytic isolates from medicinal plants produced the metabolite, and most belon- ged to the genus Streptomyces. In the present work, only S. lusitanus could produce HCN; no evidence was found in the literature of S. lusitanus as an HCN producer. The fact that S. lusitanus produces HCN could indicate its potential role in controlling diseases of fungal or bacterial origin.

Among auxins, indole-3-acetic acid (IAA) is the phyto- hormone most produced by microorganisms; it is synthesized through the metabolism of L-tryptophan (Widawati, 2020).

This metabolite regulates essential biological processes, primarily in plant growth and development, including cell expansion, division, differentiation, fruit development, leaf formation, and trophic responses. Of the microorganisms studied, those that presented the highest IAA production were S. thinghirensis, S. griseoplanus B4-M10, and S. lateritius. The IAA concentrations in the present study are higher than those reported by other authors; Harikrishnan et al. (2014) selected IAA-producing microorganisms from the rice rhizosphere, they found that Streptomyces sp. VSMGT1014 produced 15.96 µg/mL of IAA, while Detraksa (2018) isolated 95 strains from the sugarcane rhizosphere, and only eleven isolates showed the capacity to produce IAA in a range of

4.76 to 29.02 µg/mL. In the present study, S. thinghirensis was the strain with the highest production of IAA with 231.73 µg/ mL. This result contrasts with that described by Djebaili et al. (2020) who evaluated two strains of S. thinghirensis, the first, J4, showed a biosynthesis capacity of 12.8 µg/mL of IAA, and the second, K23, did not produce this auxin. On the other hand, Rehan et al. (2021) analyzed S. thinghirensis HM3 and detected 86.66 µg/mL of IAA.

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3

2

Nitrogen (N2) is essential for all living organisms. It is the most abundant element in the atmosphere, but biochemica- lly, it is not available to plants and most microorganisms, as it is a non-reactive form. The accessible forms are ammonia (NH3), ammonium (NH +), nitrates (NO −), and nitrites (NO ) (Shomi et al., 2021). The release of NH3 gas by bacteria in vitro is associated with the use of substrates rich in prote- ins or amino acids, involved in amino acid catabolism and deamination reactions (Vlassi et al., 2020). NH3 production by Streptomyces is considered an efficient way to overcome rhizosphere competitors due to its biosynthetic simplicity and low metabolic cost. Avalos et al. (2020) demonstrated that Streptomyces can produce high levels of NH3 that affect the growth of gram-positive and negative bacteria over long distances. Borah and Thakur (2020) reported that different Streptomyces isolates can produce NH3, an essential metabo- lite in suppressing the growth of plant-pathogenic fungi. The 13 strains studied showed the ability to produce ammonia, which suggests that they are involved in N bioavailability processes in plants.

Antagonistic and Inhibitory Activity of Isolated Strains The antagonism exhibited by actinomycetes is mainly due to the production of lytic enzymes, antibiotics, and parasitism. Different Streptomyces species produce antibiotics, and some strains are used in the biological control of plant diseases mainly caused by fungi (Law et al., 2017). The results obtained were heterogeneous regarding the antagonistic activity of the 13 Streptomyces strains studied. C. lindemuthianum was inhibited by 11 of the Streptomyces strains; only S. atrovirens and S. anulatus could not inhibit this fungus. There are no recent reports on the growth inhibition of C. lindemuthianum with the 11 strains studied. Regarding the growth inhibition of Pestolia spp., only Streptomyces spp. A-57 and S. pseudogri- seolus were able to inhibit it. Regarding the growth inhibition of H. maydis, Curvularia spp., and A. dauci, the antifungal and antibacterial activity of S. pseudogriseolus has already been reported; Alekhya and Gopalakrishnan (2014) reported that Streptomyces species are capable of inhibiting the growth of F. oxysporum, Macrophomina phaseolina, and Rhizoctonia bataticola, which are pathogens of chickpea and sorghum; Fatmawati et al. (2018) found high antagonistic activity aga- inst E. coli, Staphylococcus aureus, and F. oxysporum. However, in this study, S. pseudogriseolus A7-M9 showed no effect on F. oxysporum. Furthermore, Vatsa-Portugal et al. (2017) reported that S. anulatus S37 induces an early plant response against various diseases and pests. Other S. anulatus strains have also shown antagonistic and inhibitory activity against Phytophtora sp. (Kunova et al., 2016), F. oxysporium (Djebaili et al., 2021), and S. sclerotiorum (Kunova et al., 2016). On the other hand, Vijayabharanthi et al. (2014) found that S. griseoplanus displayed a broad spectrum of activity against entomopathogens. Different strains of this microorganism have also shown activity against bacterial diseases and pests (Kumar et al., 2024). For their part, Vurukonda et al. (2021) observed that S. atrovirens showed antagonistic activity

against Colletotrichum spp. Regarding S. lateritius, it showed inhibitory activity against Fusarium spp. (Gromovykh et al., 2005), while S. heliomycini against Fusarium graminearum, and S. griseoaurantiacus showed antagonistic activity against Sclerotium rolfsii in pepper (Qiu et al., 2024).

The results of the present study indicated that strains isolated from the soils of the Mezquital Valley exhibit signi- ficant antagonistic and inhibitory activity, likely due to the growth conditions to which they are subjected. It was ob- served that several of the Streptomyces strains isolated had not been reported for their antagonistic or inhibitory activity, as is the case primarily with S. pseudogriseolus, S. virginiae,

S. thinghirensis, and S. griseoaurantiacus. Environmental growing conditions likely influence this activity.

CONCLUSIONS

The strains isolated from the agricultural soil under study showed the ability to secrete metabolites related to plant growth, such as siderophores, IAA, and organic acids. Only two strains (S. thinghirensis and S. lateritius) showed the ability to solubilize phosphates, and one strain (S. lusitanus) showed the ability to produce HCN. Previously undescribed strains with antagonistic and inhibitory capacity were iden- tified, such as S. pseudogriseolus, S. atrovirens, S. lateritius, S. nigra, S. griseoplanus, S. flavogriseus, and S. griseoaurantiacus, representing new options for the generation of formulations for the biological control of diseases caused by phytopatho- genic fungi.

ACKNOWLEDGMENTS

We thank the National Council of Sciences, Humanities, and Technology for supporting the doctoral scholarship awarded No. 467187.

CONFLICTS OF INTEREST

The authors declare that they have no conflict of interest.

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