Commercial wheat fields at the grain-filling stage (from anthesis to physiological maturity) were visited in 136 locations across the High Valleys of Mexico. Each sample consisted of four leaves showing symptoms of blight and spotting. Of the above- mentioned localities, 25 locations were visited in Hidalgo, 45 in the State of Mexico, one in Morelos, five in Oaxaca, 15 in Puebla, and 45 in Tlaxcala. Samples were transported for processing to the Plant Pathology Laboratory at INIFAP-CEVAMEX, located in the municipality of Texcoco de Mora, State of Mexico.

A total of 136 leaf samples were examined under a stereomicroscope (LEICA EZ4D, manufactured in Heerbrugg, Switzerland). Leaf segments (2 cm) showing symptoms were selected and surface-sterilized through three washes-1% sodium hypochlorite, 70% alcohol, and sterile water-for one minute each. The leaf segments were then placed in a humid chamber for 48 hours and inspected every 24 hours. Conidial masses that developed on the leaf surfaces were transferred to sterile 10 mL test tubes containing 1 mL of sterile water and vortexed to homogenize the suspension. A 20 µL aliquot was then dispensed onto potato dextrose agar (PDA) plates using a micropipette. After 24 hours, germinated conidia were examined using a compound microscope (LEICA EZ DM500, manufactured in Heerbrugg, Switzerland). Each germinated conidium was transferred to a new PDA Petri dish (modified technique from Morales-Rodríguez et al., 2007).

The colonies obtained were identified using different culture media: PDA (for colony morphology and color), APZ (Potato Carrot Agar, to enhance colony growth), and SNA (Spezieller Nährstoffarmer Agar, used for the production of chlamydospores, microconidia, and macroconidia). Morphological identification was carried out using the keys of Leslie and Summerell (2006) and Zillinsky’s (1984) guide to identifying common diseases of small-grain cereals. Morphometric analysis was conducted on 100 structures (conidia, phialides, macroconidia, conidiophores, etc.) to group the fungi by isolate.

Isolation was carried out using two composite soil samples from the municipality of Bustamante, Nuevo León: M1B (67 morphotypes), consisting of six subsamples, and M2B (77 morphotypes), consisting of five subsamples. Both were collected from the rhizosphere of avocado trees and obtained and purified at the General Teheran Experimental Station, Montemorelos, N.L. - National Institute of Forestry, Agricultural and Livestock Research (INIFAP). The isolation technique involved serial dilutions (10⁻² and 10⁻³) in sterile water, followed by dispersal of 50 µL onto nutrient agar medium. After 24 and 72 hours of incubation, the cultures were examined under a compound microscope, and microcolonies were transferred to new Petri dishes containing nutrient agar. Based on the morphological characteristics of the isolated colonies, the organisms were identified as belonging to the actinobacteria group.

Two preliminary bioassays were conducted to identify actinobacteria with the highest inhibition potential (>20%). The first included 144 morphotypes distributed across 36 Petri dishes plus one control dish. The second assay involved 16 morphotypes distributed across four Petri dishes plus one control. A subsequent dual confrontation test was performed between seven actinobacterial morphotypes and three previously selected fungi, distributed across 63 Petri dishes plus three controls. The variable used in all tests was the percentage inhibition of mycelial growth (PIMG), calculated using the formula from Salazar et al. (2001): PIMG = [(Mycelial growth in the control − Mycelial growth with treatment) / Mycelial growth in the control] * 100. Mycelial growth was measured in millimeters as the radial growth of the fungal colony between the fungal inoculum disk and the corresponding actinobacteria disk.

Preliminary bioassays. In 65 × 15 mm Petri dishes, a fungal inoculum disk (selected based on the most frequently reported fungus in the samples) was placed at the center, and four different disks of actinobacterial morphotypes were positioned at the outer edges of the same dish (no replicates). Additionally, a control dish was prepared with only the fungal inoculum disk placed at the center. The distance between the fungus and each actinobacteria disk was 1.5 cm. Mycelial growth data were recorded until the control fully covered the entire area of the Petri dish.

Dual culture assay. This assay was conducted only with actinobacteria that showed the highest inhibition percentages in the preliminary bioassay (>40%). Each actinobacteria strain was paired with three fungal isolates-two obtained from the wheat samples and a third, Fusarium isolate 3 (Fusarium proliferatum), which was donated from the personal collection of Dr. Leticia Robles. This particular species was included because of its agricultural relevance, as it is associated with premature yellowing or early maturity in wheat (Rangel-Castillo et al., 2017). The actinobacteria and fungal disks were placed at opposite ends of the Petri dish, with a separation of 3 cm. Due to the rapid growth rate of the fungi during the assays, the transfer schedule was adjusted: the actinobacteria inoculum was placed first, followed by the fungal inoculum two days later, simulating a preventive treatment scenario.

A completely randomized experimental design was used for the dual culture assay, with three replicates. An analysis of variance (p ≤ 0.05) was then performed, followed by a mean comparison test (Tukey, p ≤ 0.05).

Seven fungal species were identified, three of which are associated with wheat leaf blight (Alternaria, Bipolaris, and Fusarium), characterized by chlorotic halos that extend along the leaf blade. Two types of rusts were also identified (both from the genus Puccinia), though they were not included in the experiment due to being obligate parasites. Additionally, two fungi associated with foliar spots, also known as septoria leaf blotch (Septoria and Zymoseptoria), were identified.

Alternaria genus. It was identified by the presence of conidia characteristic of the genus. Mata-Santoyo et al. (2018) report that Alternaria is associated with Bipolaris sorokiniana as a damage-enhancing agent or is identified as a saprophytic fungus in isolates obtained from wheat samples. However, Perelló et al. (2015) notes that Alternaria species have caused yield losses of up to 60% under favorable conditions, having been identified as pathogens responsible for foliar and grain diseases in wheat.

The conidia measured approximately 3-6 µm in thickness and 19-46.07 × 7.56-14.22 µm in length. Alternaria spp. colonies are dark green at maturity, while during active growth they display whitish to grayish tones. They are circular in shape, with a flat but cottony surface and filamentous edges (Figure 1).

Bipolaris Genus. The observed symptoms included dark brown necrotic lesions along the leaves (Figure 2A). Under humid chamber conditions, conidiophore formation was detected on the leaf surface, along with dark, dense mycelium. The conidia and conidiophores allowed identification of the genus Bipolaris (formerly Helminthosporium) (Mata-Santoyo et al., 2018). The observed conidia were oblong, olive-brown, septate, slightly curved, with smooth walls and a prominent basal scar (Figure 2B). Conidia measured approximately 34-63.12 × 10.06-18.5 µm, with three to nine septa per conidium. Conidiophores developed individually and measured on average

118.5-152 × 6.4-8.2 µm. Colonies were dark green, with a granular cottony texture, umbilicate elevation, and lobed margins (Figure 2C and D).

Figure 1

Figure 2

Fusarium Genus. The observed symptom was a chlorotic spot with a whitish center (Figure 3); however, reports indicate that at early developmental stages the spots are grayish green, and at more advanced stages they become oval and grayish brown (Zillinsky, 1984). Nevertheless, the conidial mass used to isolate Fusarium was obtained from significantly dried leaves.

Based on the morphology of the structures, the genus was identified by the presence of macroconidia, microconidia, chlamydospores, and the type of phialides. The morphological characteristics suggested two types of Fusarium spp. isolates. On PDA culture medium, the first colony exhibited a yellow to reddish coloration, with circular growth, a flat but cottony surface, and filamentous edges-features consistent with descriptions of F. graminearum (Leyva-Mir et al., 2017) (Figure 3); however, for future reference, it will be referred to as Fusarium isolate 1. Macroconidia recovered from SNA culture medium measured 21.01-63.14 × 3.08-4.85 µm, and chlamydospores measured

7.37-16.52 µm (Figure 3).

The second colony, grown on PDA culture medium, was whitish in color, with circular growth, slight ring formation, a cottony and flat surface (hereafter referred to as isolate 2) (Figure 3). Microconidia measured 5.55-11.2 × 1.73-3.79 µm, along with the presence of chlamydospores (Figure 3).

Figure 3

Puccinia genus. Species of the genus Puccinia are known as the causal agents of rusts and are considered “the most diverse and economically significant groups of phytopathogenic microorganisms worldwide in agricultural and forestry production” (Zuluaga et al., 2008). The characteristic symptom is the presence of pustules, which may contain two types of spores: urediniospores and teliospores. Puccinia 1 was observed with orange-red, spherical uredinia lacking ornamentation, measuring 22.53-28 µm in diameter. In contrast, Puccinia 2 showed uredinia distributed linearly along wheat leaves. These uredinia were yellow, slightly spherical, and measured 27.3-32.05 µm in diameter (Figure 4).

Septoria and Zymoseptoria genera. During sampling, various degrees of septoria severity were observed. The symptoms included chlorotic halos containing pycnidia distributed throughout the halo (Figure 5).

Figure 4

Figure 5

The presence of Septoria and Zymoseptoria can sometimes be distinguished under a stereomicroscope by a slight difference in pycnidium color: Zymoseptoria tends to range from black to grayish, while Septoria ranges from black to dark brown.

The Septoria colony is slightly circular with some irregularities, a slightly convex surface, and slightly filamentous edges. The conidia are short, straight didymospores, unornamented, and pale yellow in color. Conidia measured approximately 14.6-20.2 ×

2.86-3.48 µm.

In the case of Zymoseptoria, the colony was slightly circular with some irregularities, featuring a smooth surface and an umbilicate elevation (Figure 6). It is worth noting that Zymoseptoria colonies developed in two distinct forms: the first, at early stages, had a semi-liquid or viscous consistency with a pink coloration; the second, upon maturation, developed black mycelium with whitish segments. The conidia were filiform, unornamented, hyaline, and slightly curved, measuring 40-77.3 × 1.6-2.84 µm (Figure 5).

Figure 6

Once the main fungi present in the wheat samples were identified, disease incidence was determined based on the state in which each sample was collected (Table 1).

Table 1

State	Incidence by genus (%)
	Alternaria spp.	Bipolaris spp.	Fusarium spp.	Puccinia spp.
Hidalgo	20.0	4.0	52.0	92.0
Mexico	24.4	2.2	46.7	86.7
Morelos	0.0	0.0	0.0	100.0
Oaxaca	20.0	0.0	20.0	80.0
Puebla	13.3	0.0	60.0	80.0
Tlaxcala	8.9	0.0	35.6	88.9
Average	14.4	1.0	35.7	87.9

Considering the incidence of fungi in the evaluated samples, Puccinia and Fusarium were the most frequent, with 87.9% and 35.7%, respectively. Therefore, Fusarium was selected for the in vitro tests. The genera Septoria and Zymoseptoria could not be confirmed in all cases where symptoms were observed, and thus were not included in the incidence data.

A total of 144 actinobacteria were isolated from the soil samples M1B (67) and M2B

(77) collected in the municipality of Bustamante, Nuevo Leon. The actinobacteria with the highest percentage of mycelial inhibition (42.9%) corresponded to 17 out of 144 morphotypes, representing only 11.1% of the evaluated population. The rest showed no inhibitory effect. The selected morphotypes, according to their group, were: from M1B- M1, M4, M12, M14, M16, M33, M41, M55, M57, and M60; and from M2B-M3, M15,

M27, M45, M70, M73, and M74. In the second bioassay, the morphotypes showed variation in results compared to the first, with the second group showing a 25.1% decrease in PIMG (Table 2).

Table 2

Group		First bioassay			Second bioassay
	Morphotype	PIMG (%)	Average	Morphotype	PIMG (%)	Average
	1	45.5		1	47.4
	4	40.9		4	63.2
	12	40.9		12	52.6
	14	40.9		14	36.8
M1B	16	36.4	40.4	16	47.4	40.9
	33	40.9		33	57.9
	41	50.0		41	57.9
	55	36.4		55	21.1
	57	31.8		57	-15.8
	3	50.0		3	-5.3
	15	50.0		15	5.3
	27	31.8		27	-5.3
M2B	45	54.5	45.4	45	36.8	20.3
	70	31.8		70	21.1
	73	40.9		73	21.1
	74	59.1		74	68.4

The actinobacteria selected from the preliminary bioassays were MIB M4, MIB M33, MIB M41, M2B M60, and M2B M74, all of which showed a PIMG greater than 40%, confirming that some actinobacteria affect the mycelial growth of Fusarium morphotype 1 two days after inoculation (Figure 6).

While the inhibitory capacity of some isolates is notable, a higher degree of specificity is also observed, given that the PIMG varies depending on the Fusarium isolate exposed. Three morphotypes-M1B M4, M1B M33, and M2B M60-stood out statistically with the highest inhibition percentages; however, each was more effective against a different isolate. M1B M4 was most effective against Fusarium isolate 3, M1B M33 against isolate 2, and M2B M60 against isolate 1 (Table 3 and Figure 7).

Table 3

			PIMG by Fusarium isolation
Grupo	Morphotype	Fusariumisolation 1		Fusarium isolation 2		Fusarium isolation 3
	M1	8.0	e	6.2	e	8.8	d
	M4	43.8	b c	42.5	a b	66.9	a
M1B	M16	17.2	d	11.4	d	4.9	e
	M33	48.8	a b	47.1	a	47.9	b
	M41	38.3	c	31.3	c	43.4	b
M2B	M60	59.8	a	29.1	c	59.8	a
	M74	18.8	d	16.3	d	23.0	c

z Means with the same letter are not statistically different (Tukey, p ≤ 0.05).

Figure 7

In Table 4, it can be observed that Fusarium isolate 1 showed the highest growth rate starting on the third day after establishment, indicating that the difference in response among isolates of this genus depended on the growth rate of the fungus.

Table 4

Fungus				Growth rate (cm day-1)
	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9
Fusarium isolation 1	0.3	0.30	0.95	0.95	0.74	0.39	0.37	100	0.3
Fusarium isolation 2	0.51	0.52	0.53	0.44	0.39	0.33	0.28	0.29	0.28
Fusarium isolation 3	0.36	0.38	0.66	0.7	0.487	0.512	0.462	0.162	0.115