The drying or seedbed evil caused by Fusarium circinatum is a problem of importance in plant production in forest nurseries. The aim of this study was to assess Trichoderma harzianum as an alternative to prevent F. circinatum with the method of application and the use of two mixtures of substrates: (1) Peat moss, agrolite and vermiculite, and (2) Sawdust, pine bark and peat moss. Three forms of application of T. harzianum were made: to the substrate, to the seed and without the fungus, which gave a total of 12 treatments with completely at random factorial arrangement (2×3×2). The effect of the substrate was evaluated, the form of application of T. harzianum and its interaction on the pathogenicity and incidence of F. circinatum and the morphological properties of the seedlings. The results indicate that the morphological characteristics in the growth of the strain coincide with those described for F. circinatum and turned out to be pathogenic when causing disease. The pathogenicity of F. circinatum decreased in those treatments where T. harzianum was applied to the substrate, showing a positive response in the sawdust-based mixture and ensuring a better plant quality. The application of T. harzianum to the substrate and the use of sawdust-based mixture can serve as an option for the F. circinatum fungus, providing better plant quality indexes.

Key words: Biocontrol, plant quality, phytopathogen, Fusarium circinatum Nirenberg & O´Donnell 1998, antagonistic fungus, incidence.

La secadera o mal de almácigo causada por Fusarium circinatum es un problema importante en la producción de planta en viveros forestales. El objetivo de esta investigación fue evaluar a Trichoderma harzianum como alternativa para controlar de manera preventiva a F. circinatum con el método de aplicación y la utilización de dos mezclas de sustratos: (1) turba de musgo, agrolita y vermiculita, y (2) aserrín, corteza de pino y turba de musgo. Se utilizaron tres formas de aplicación de T. harzianum:al sustrato, a la semilla y sin el hongo; lo que dio un total de12 tratamientos con arreglo factorial completamente al azar (2×3×2). Se evaluó el efecto del sustrato, la forma de aplicación de T. harzianum y su interacción sobre la patogenicidad e incidencia de F. circinatum y las propiedades morfológicas de las plántulas. Los resultados indicaron que las características morfológicas en el crecimiento de la cepa coinciden con los descritos para F. circinatum y resultó ser patogénica, ya que causó enfermedad. La patogenicidad de F. circinatum disminuyó en aquellos tratamientos en los que se adicionó T. harzianum al sustrato, y presentó una respuesta positiva en la mezcla a base de aserrín con lo cual se aseguró una mejor calidad de planta. La aplicación de T. harzianum al sustrato y el uso de la mezcla a base de aserrín pueden servir como una alternativa para el control de F. circinatum, además de proveer mejores índices de calidad de planta.

Palabras clave: Biocontrol, calidad de planta, fitopatógeno, Fusarium circinatum Nirenberg & O´Donnell 1998, hongo antagonista, incidencia.

Mexico loses around 47 770 hectares of forest coverage per year due to anthropogenic activities; thus, the development of reforestation programs that allow their recovery (Gómez, 2021) has been sought. Therefore, quality plant from a forest nursery that guarantees sanity and morpho-phisiological characteristics necessary for its field establishment (Buamscha et al., 2012; Sáenz et al., 2014) is used. The quality of the plant is determined by physicochemical conditions that are suitable for its production.

One of the preferred species in reforestation programs is Pinus devoniana Lindl., from its adaptability and economic benefits. This species is generally produced in substrate based on Peat Moss, vermiculite and agrolite (55:35:10) with slow-release fertilizers (30-15-10); however, it has been observed that these mixtures favor the development of pests and diseases (Conafor, 2001; Sáenz-Romero, 2014).

Phytopathogenic fungi are the most harmful agents due to their fast reproduction, which makes it is necessary to perform constant samples to avoid diseases (Murace and Aprea, 2011). Fusarium circinatum Nirenberg & O'Donnell 1998 affects pine seedlings from the seed stage, damage to the embryo and hypocotyl, stem bending, root rot, discoloration of needles and death. It can favor plant losses of 40 to 100 % (González et al., 2008; García-Díaz et al., 2017).

There are alternative substrates made from by-products of the plant production process in forest nurseries, which can reduce the unit cost of the plant (Aguilera et al., 2016). For pathogen control there are options such as the use of the antagonist fungus of the Trichoderma Pers. 1794 genus (Okorski et al., 2014).

Studies focused on analyzing the effect of substrate mixtures on the pathogenicity of F. circinatum such as García-Díaz et al. (2017), who evaluated the pathogenicity of the fungus in three mixtures of substrates and during the germination stage of Pinus greggii Engelm. ex Parl. On the other hand, Robles et al. (2016) assessed the Fusarium oxysporum Schltdl. 1824 attack at the nursery stage and Fusarium solani (Mart.) Sacc. 1881 in Pinus spp. seedlings. Also, Moraga-Suazo et al. (2011) compared the effectiveness of the control exercised on F. circinatum with Trichoderma spp. and Clonostachys spp. in Pinus radiata D. Don.

Based on the above, the aim of this study was to assess the effect of Trichoderma harzianum Rifai 1969 as an alternative to prevent Fusarium circinatum, with the method of application and the use of two mixtures of substrate.

The experiment was conducted from May 9^th, 2021 to January 26^th, 2022 in the greenhouse of the Forest Sciences Division (Dicifo) of the Universidad Autónoma Chapingo (UACh) (Chapingo Autonomous University), in Texcoco, State of Mexico, located between 19˚29´34” N and 98˚53´38” W, at 2 240 masl. Inside the greenhouse, temperature remained at 24 °C during the study period.

A completely at random factorial experiment (2×3×2) was established with four replicates; the assessed factors were: (A) Mixture of substrate, with two levels (S1 and S2, described below), (B) T. harzianum application method, with three levels (application to the substrate, application to the seed and without applying), and (C) Presence of F. circinatum, with two levels (presence or absence of the fungus). The combined effect of the factors was evaluated, so there were 12 treatments (Table 1). The experimental unit consisted of 12 P. devoniana plants with 220 mL pipes. In total 48 trays were used. The response variables were incidence, severity and plant quality.

Table 1. Established treatments for the evaluation of substrate mixtures, application method of Trichoderma harzianum Rifai 1969, absence and presence of Fusarium circinatum Nirenberg & O´Donnell 1998 in Pinus devoniana Lindl. plants.

Treatments	Description
T1	S1+T. harzianum Rifai 1969to the substrate
T2	S1+T. harzianum Rifai 1969to the substrate+F. circinatum Nirenberg & O´Donnell 1998
T3	S2+T. harzianum Rifai 1969 to the substrate
T4	S2+T. harzianum Rifai 1969 to the substrate+F. circinatum Nirenberg & O´Donnell 1998
T5	S1+T. harzianum Rifai 1969 to the seed
T6	S1+T. harzianum Rifai 1969to the seed+F. circinatum Nirenberg & O´Donnell 1998
T7	S2+T. harzianum Rifai 1969 to the seed
T8	S2+T. harzianum Rifai 1969 to the seed+F. circinatum Nirenberg & O´Donnell 1998
T9	S1
T10	S1+F. circinatum Nirenberg & O´Donnell 1998
T11	S2
T12	S2+F. circinatum Nirenberg & O´Donnell 1998

S1 = Substrate 1 (peat moss+agrolite+vermiculite); S2 = Substrate 2 (pine sawdust+pine bark+peat moss).

An Anova was performed through the Glimmix of SAS procedure, version 13.1 (SAS Institute Inc., 2013) and multiple comparison of means through the Tukey test at a significance level of 0.05.

= Effect of the j^th level of the B factor (T. harzianum application method)

= Interaction of i^th level of the A factor with j^th level of the B factor with k^th level of the C factor

Two substrate mixtures were used, S1: peat moss, agrolite and vermiculite, and S2: pine sawdust from 15 days of sawing, composted pine bark and peat moss, 60:20:20 and Multicote^® 18-6-12 fertilizer was added, at a dose of 7 g L^-1 of substrate (Table 1). Two P. devoniana seeds were placed per tube, previously disinfected with 10 % sodium hypochlorite (NaClO) for 10 minutes, followed by 24 hours of soaking with sterile water and a second disinfection with 5 % NaClO for 3 min. Surface irrigation was carried out for a month and then every third day.

A 14 L of a T. harzianum (commercial strain PHC^® T22) solution was prepared at 1×10^-7 CFU/g dry weight. The application dose was 3.4 g L^-1 of water. The treatments with T. harzianum to the substrate were: T1, T2 (S1) and T3, T4 (S2). Those that received T. harzianum with seed soaking for 60 min. were: T5, T6 (S1) and T7, T8 (S2); while those without T. harzianum were the treatments: T9, T10 (S1) and T11, T12 (S2) (Table 1).

Strain SF11 of F. circinatum from the forest nursery of Amealco, state of Querétaro, provided by the Dicifo Forest Pathology Laboratory was used with seven days of growth at room temperature in Potato Dextrose Agar BD Bioxon^TM (PDA) culture medium; a solution of 1.6×10⁶ spores mL^-1 was prepared. In seedlings of two months and 20 days (July 2021), corresponding to treatments T2, T4, T6, T8, T10 and T12 (Table 1), 20 mL were applied directly to the substrate of each tube. 24 h after inoculation, the counting of plants with the presence of symptoms began.

The pathogenicity of F. circinatum was evaluated by its ability to cause disease with typical symptoms of secadera. The incidence was determined by the mortality of plants affected with symptoms for eight months. Severity was estimated using the Townsend and Heuberger (1943) diagrammatic scale (Figure 1) and the Formula 2:

Class 0 = Healthy or asymptomatic plant; Class 1 = Falling or distal yellowing needles; Class 2 = Partial plant wilting; Class 3 = Medium plant wilting; Class 4 = Advanced plant wilting; Class 5 = Dead plant.

Figure 1. Based on Townsend and Heuberger (1943), scale of symptoms caused by Fusarium circinatum Nirenberg & O´Donnell 1998 for severity estimation.

The degrees of infection according to the scale allowed us to estimate the severity and a Pearson correlation analysis was carried out between incidence and severity.

One plant with symptoms for each treatment inoculated with F. circinatum (a total of 24 plants) was selected for isolation and cultivation in PDA and stored at 28 °C for 24 h of light (model INCL-11 Prendo^® incubator) to obtain re-isolations of the pathogen and demonstrate Koch's postulates.

The morphological identification of F. circinatum was carried out by obtaining the isolation, sporulation and development of macroconidia, microconidia, phialides and coloration of the mycelium, in three culture media: PDA medium, to examine the morphology and pigmentation of the colony, Water Agar medium with carnation leaf for the formation of macroconidia and phialides on sporodochia, and for the development of circins the Synthetic Nutritive Agar medium (García-Díaz et al., 2017). 10 permanent mounts were made on slides with 100 % glycerin and methylene blue to observe and measure the structures of the fungus in a DM 2 500 Leica^® microscope. The keys of Barnett and Hunter (1998) and Leslie and Sumerell (2006) were used.

Eight months after sowing, five central plants were selected from each tray (20 plants per treatment, resulting in a total of 240); they were removed from the root ball and immersed in water to wash and not hurt the roots. Subsequently, measurements of Stem Diameter (D) (section in which it differs from the main root) (with TXD-a33 Lenfech^® digital caliper), and Height of the aerial part (A) (section in which it differs from the main root to the apex of the terminal bud) (with H-6 560 Cline^® steel rule) were made.

Each plant was sectioned at the neck level, and both sections, foliage and root, were placed in a paper bag labeled with the treatment data for drying in a Fe-143 Felisa^® oven at 70 °C for 72 h. The dry weight of the root (PSR) and the dry weight of the aerial part (PSA) were evaluated. The dry weight ratio of the aerial part to the dry weight of the root (PSA/PSR), Slenderness index (IE), plant height (cm) between diameter (mm) and Dickson quality index (ICD) were determined, with the equation PST/(A/D)+(PSA/PSR) (Dickson et al., 1960). The seedlings were weighed on a model Galaxy 2 000 OHAUS^® analytical balance.

F. circinatum symptoms in plants began in week 12 after inoculation; chlorotic needles, reddish brown color and drying of foliage, apical bending, root rot and death were present (Figure 2). These symptoms coincide with those reported by Flores-Pacheco (2017) and García-Díaz et al. (2017).

A = Rot at neck level; B = Chlorotic and twisted needles, tip bending; C = Descending death of the plant.

Figure 2. Symptoms observed in Pinus devoniana Lind. plants inoculated with Fusarium circinatum Nirenberg & O´Donnell 1998.

The analysis of variance revealed that there are no differences between the substrates (S1 and S2) and their interaction with the application of T. harzianum on the incidence. On the contrary, the application method of T. harzianum, the presence of F. circinatum and their interactions showed differences on the response variables at α=0.05 (Table 2 shows the Pr>F corresponding to the Anova of each variable).

Table 2. Determination of the effect of the mixture, application of Trichoderma harzianum Rifai 1969 and presence of Fusarium circinatum Nirenberg & O´Donnell 1998 on the incidence in Pinus devoniana Lindl. plants.

Table 3. Evaluation of incidence and severity of the disease caused by Fusarium circinatum Nirenberg & O´Donnell 1998 in Pinus devoniana Lindl. seedlings.

Number of treatment	Description	Incidence %	Severity %
T1	S1+T. harzianum Rifai 1969 to the substrate	5c	4.16d
T2	S1+T. harzianum Rifai 1969 to the substrate+F. circinatum Nirenberg & O´Donnell 1998	46b	26.16c
T3	S2+T. harzianum Rifai 1969to the substrate	3c	2d
T4	S2+T. harzianum Rifai 1969 to the substrate+F. circinatum Nirenberg & O´Donnell 1998	42b	25.16c
T5	S1+T. harzianum Rifai 1969to the seed	4c	2.83d
T6	S1+T. harzianum Rifai 1969 to the seed+F. circinatum Nirenberg & O´Donnell 1998	59a	33b
T7	S2+T. harzianum Rifai 1969to the seed	4c	2.73d
T8	S2+T. harzianum Rifai 1969to the seed+F. circinatum Nirenberg & O´Donnell 1998	58a	24.83c
T9	S1	4c	2.33d
T10	S1+F. circinatum Nirenberg & O´Donnell 1998	66a	33.99ab
T11	S2	3c	2.33d
T12	S2+F. circinatum Nirenberg & O´Donnell 1998	63a	37.5a

S1 = Substrate 1 (peat moss, agrolite and vermiculite); S2 = Substrate 2 (pine sawdust, pine bark and peat moss). Equal letters per column indicate that there are no significant differences (p≥0.05) and different letters indicate significant differences between treatments according to the Tukey test (p≤0.05). Values expressed as the mean of each response variable.

The analysis of disease incidence indicated that the low values (3 to 5 %) were in the treatments in the absence of F. circinatum, and the treatments with the highest incidence were those in which F. circinatum was inoculated, that is, 46 % (T2), 42 % (T4), 59 % (T6), 58 % (T8), 66 % (T10) and 63 % (T12). This demonstrates the importance of the pathogen on the loss of plant production. Plants with application of T. harzianum directly to the substrate presented 13 to 16 % less incidence, compared to application to the seed. Where T. harzianum was not applied, it was 20 % lower, a behavior similar to that of S1 and S2. The application method of T. harzianum was the one that had the greatest effect, due to a lower incidence of the disease when it was applied to the substrate (Table 4).

Table 4. Determination of the effect of the application method of Trichoderma harzianum Rifai 1969 on the incidence of the disease in Pinus devoniana Lindl. plants.

Equal letters per column indicate that there are no significant differences (p≥0.05) and different letters indicate significant differences between treatments according to the Tukey test (p≤0.05).

Table 5. Pearson correlation between incidence and severity of Fusarium circinatum Nirenberg & O´Donnell 1998 with P-value of <0.0001.

White colonies with fuchsia hues were obtained, turning strong violet, with violet pigmentation (Figure 3A and 3B), little formation of pale orange sporodochia (Figure 3C), macroconidia with 2-4 septa, typically three-septate, thin and curved (Figure 3E), 28-44×3.3-3.9 nm^-2, 39×3.8 nm^-2 on average and with a curved apical and poorly developed basal cell; finally, ovoid to allantoic shaped, unicellular microconidia, which develop on mono- and polyphialids (Figure 3D and 3F), with spiral-shaped sterile hyphae known as circins. These characteristics and measurements coincide with those recorded for F. circinatum by Flores-Pacheco (2017), García-Díaz et al. (2019) and O'Donnell et al. (2022).

A = Purple cottony mycelium; B = Development in three media; C = Sporodochia; D = Monophialides of the 40X microconidia; E = Allantoic macroconidia with poorly developed basal cell; F = Microconidia.

Figure 3. Characteristics of Fusarium circinatum Nirenberg & O´Donnell 1998.

The assessment of plant quality with the individual variables did not show significant differences between the treatments, but, on the other hand, in the ICD analysis, there are differences in the S2 treatments with values >1 compared to S1, which are smaller. Saenz et al. (2014) consider values ≥0.5 as high quality for cespitose species; the numbers found in this study were higher, so they are classified as high quality. In the ICD, no significant differences were observed in the application method of T. harzianum (Table 6).

Table 6. Plant quality indexes of Pinus devoniana Lindl. in two substrates with Trichoderma harzianum Rifai 1969 and Fusarium circinatumNirenberg & O´Donnell 1998.

No.	Treatment	*Alt* (cm)	*Diam* (mm)	*PSA* (gr)	*PSR* (gr)	IR	*PSA/PSR*	*ICD*
T1	S1+T. harzianum Rifai 1969 to the substrate	16.105ab	7.3abc	3.875abcd	1.09f	2.245ab	3.554a	0.859e
T2	S1+T. harzianum Rifai 1969 to the substrate+F. circinatum Nirenberg & O´Donnell 1998	13.47ab	6.775c	3.08d	0.995f	2.125ab	3.054abcd	0.843e
T3	S2+T. harzianum Rifai 1969to the substrate	15.555ab	7.375abc	4.43ab	2.12a	2.145ab	2.096fg	1.538ab
T4	S2+T. harzianum Rifai 1969 to the substrate+F. circinatum Nirenberg & O´Donnell 1998	14.215ab	7.79abc	4.425ab	1.765abcd	2.073ab	2.522cdefg	1.448abc
T5	S1+T. harzianum Rifai 1969to the seed	13.745ab	7.92ab	4.1abc	1.28ef	1.765ab	3.20ab	1.095cde
T6	S1+T. harzianum Rifai 1969to the seed+F. circinatum Nirenberg & O´Donnell 1998	15.645ab	7.24bc	3.415cd	1.11f	2.245ab	3.137abc	0.864e
T7	S2+T. harzianum Rifai 1969 to the seed	16.585ab	8.145ab	4.54a	1.9abc	2.14ab	2.44defg	1.439abc
T8	S2+T. harzianum Rifai 1969 to the seed+F. circinatum Nirenberg & O´Donnell 1998	12.56b	8.225ab	4.07abc	1.85abc	1.615b	2.231efg	1.628a
T9	S1	16.805ab	8.485a	4.255abc	1.54cde	2.043ab	2.849bdce	1.223bcde
T10	S1+F. circinatum Nirenberg & O´Donnell 1998	17.015a	7.2bc	3.525bcd	1.38ef	2.413a	2.598bcdef	1.005de
T11	S2	16.475ab	7.445abc	3.855abcd	2.04ab	2.315a	1.897g	1.478abc
T12	S2+F. circinatum Nirenberg & O´Donnell 1998	12.505b	7.205bc	3.345cd	1.635bcde	1.898ab	2.074fg	1.349abcd

S1 = Substrate 1 (peat moss+agrolite+vermiculite); S2 = Substrate 2 (pine sawdust+pine bark+peat moss); Alt = Height; Diam = Diameter; PSA = Air dry weight; PSR = Radical dry weight; IR = Robustness index; ICD = Dickson quality index. Equal letters per column indicate that there are no significant differences (p≥0.05) and different letters indicate significant differences between treatments according to the Tukey test (p≤0.05).

The analysis of variance showed that there are differences between S1 and S2. Substrate 2, based on sawdust, was significant for the ICD, with α=0.05. Table 7 shows the Pr>F corresponding to the Anova of each variable.

Table 7. Effect of the mixture of two substrates, with application of Trichoderma harzianum Rifai 1969 and presence of Fusarium circinatum Nirenberg & O´Donnell 1998 on the plant quality indexes of Pinus devoniana Lindl.

Based on Tukey's multiple comparison of means, sawdust-based substrate 2 (S2), showed significant differences for the ICD (Table 8).

Table 8. Multiple comparison test of means per substrate mixture for the Dickson quality index in Pinus devoniana Lindl. plants.

According to Tukey's test. Equal letters per row indicate that there are no significant differences (p≥0.05).

The results of this study indicate that Fusarium circinatum caused symptoms of apical bending, stem rot at the neck level and yellowish needles turning reddish, subsequently death, which coincides with the results of Flores-Pacheco (2017) and García-Díaz et al. (2017).

Gordon et al. (2015) point out that the use of peat moss is associated with greater susceptibility to F. circinatum, since they obtained a 97 % incidence when inoculating a pine isolate in corn plants, and in this research, such substrate (S1) inoculated in P. devoniana plants showed 66 %. Aguilera et al. (2016) mention that substrates with sawdust and pine bark are used with great success in nurseries in central Mexico, since sawdust is an abundant, economical by-product that allows the development of plants with good morphological characteristics. It can be added to this fact that the emergence of the disease is slightly lower; in this study, an incidence of 63 % was obtained with S2, compared to 66 % with S1, although statistically there were no differences. However, García-Díaz et al. (2017) report that the presence of the disease is lower in sawdust-based substrates.

The analysis of variance showed that the significant factor is the application of T. harzianum to the substrate, presenting low incidence and severity; these results coincide with those of García-Díaz et al. (2017), in which the damage caused by F. circinatum was lower in those treatments in which T. harzianum was applied to Pinus greggii plants. Likewise, what is stipulated agrees with what is stated by Moraga-Suazo et al. (2011), who mention that when applying Trichoderma to Pinus radiata plants the incidence is reduced by more than 20 %. Okorski et al. (2014) concludes that Trichoderma is used as a biocontrol agent for forest diseases.

The ICD analysis indicates that there are differences in the treatments of S2 by obtaining values greater than 1 with respect to S1, which are lower. This is considered within the “high quality” classification according to the parameters established for cespitose species, as mentioned by Rueda et al. (2012) and Sáenz (2014). The results agree with those of Vicente-Arbona et al. (2019), who when evaluating the ICD in P. greggii plants in different substrate mixtures obtained better results in that mixture with a formulation similar to S2 (pine sawdust, pine bark and peat moss) (Aguilera et al., 2016).

Fusarium circinatum was shown to be pathogenic in Pinus devoniana seedlings, it was observed that the application method with Trichoderma harzianum to the substrate can reduce the incidence and severity of the disease to be used preventively. It was observed that the plant quality based on the Dickson quality index for cespitose species had higher values in those individuals that developed in the sawdust-based substrate and the application of Trichoderma harzianum to the substrate.

The authors thank the Universidad Autónoma Chapingo (Chapingo Autonomous University), the Conahcyt sectoral fund and project A-S-67865 of the Conafor-Conahcyt Sectoral Fund, entitled “Monitoring, damage assessment, preventive management and control of drying and root rot caused by Fusarium spp., and the fungus flies Bradysia and Lycoriella” for their support to accomplish this project.

The authors of this work declare that they do not present any type of conflict of interest.

María Alejandra Ortega Cerón: field work, laboratory and writing of the manuscript; Elizabeth Hernández Acosta: research direction and review of the manuscript; Silvia Edith García Díaz: advice on laboratory and greenhouse research work and review of the manuscript; Antonio Villanueva Morales: statistical analysis, experimental design and drafting review of the manuscript.

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Effect	F-value	Pr>F
Application of T. harzianum Rifai 1969	11.32	0.0002
Fusarium circinatum Nirenberg & O´Donnell 1998	805.31	<0.0001
Mixture×Fusarium circinatum Nirenberg & O´Donnell 1998	1.21	0.2793
Application of T. harzianum Rifai 1969×Fusarium circinatum Nirenberg & O´Donnell 1998	12.23	<0.0001

Application method	Incidence %
Without Trichoderma harzianum Rifai 1969	8.5a
Trichoderma harzianum Rifai 1969 to the seed	7.8125a
Trichoderma harzianum Rifai 1969to the substrate	6b

Substrate Mixture	*ICD*
(S1) Peat moss, agrolite and vermiculite	0.9814b
(S2) Pine sawdust, pine bark and pear moss	1.4799a