Increased Hirsutella citriformis Conidia Shelf Life in Acacia and Hirsutella Gum Formulations

Abstract

Biological control by beneficial microorganisms is known to significantly reduce the effect of pests on crops yield. Among the biocontrol strategies is the use of entomopathogenic fungi such as Hirsutella citriformis, which has been applied to infect and kill hemipteran insect pests, including Diaphorina citri Kuwayama (Hemiptera: Liviidae) and Bactericera cockerelli Sulc. (Hemiptera: Triozidae). These biological agents are applied in the form of conidia that are often combined with other inert materials to facilitate application, protect conidia, and improve their shelf life. The aim of this study was to implement strategies for developing formulations to increase conidia shelf life. We evaluated gum produced from one strain and conidia from two different H. citriformis strains. Conidia were formulated by evaluating different concentrations of Acacia and Hirsutella gums to enhance conidia viability during storage at 4 °C or 25 °C. Results indicated that formulations maintained conidia viability for at least 90 d after storage at 25 °C (≥70% viability) and at least 120 d after storage at 4 °C, which was significantly (p ≤ 0.05) higher than that of the control, without observing changes in pH values. We also demonstrated 100% formulation purity from days 0 to 120, among all treatments. In conclusion, evaluated formulations maintained H. citriformis conidia viability for at least three months, when stored at 4 °C.

1. Introduction

Chemical pesticides currently account for 95% of the global pesticide market and prevent about 50% of crop losses, which have not decreased in the last decades. Reports of the undesirable effects of chemical pesticides on animal and human health have prompted the search for alternatives to chemical pesticides [1,2].

Biological control of insect pests reduces harmful chemicals and pests-associated crop yield loss by using beneficial organisms, including insects, plants, or microorganisms. One of the most relevant biological control strategies under development are biopesticides, involving formulations based on entomopathogenic bacteria, fungi, or viruses [3,4].

Entomopathogenic fungi are commonly used as biopesticides to control aphids, ticks, and other insect plague populations, affecting plants and animals [5]. Their application is in the form of conidia, which are commonly combined with other inert materials for protection from environmental changes and stabilization during storage [6]. Some polymers used as adherents in formulations have a dual purpose. For example, arabic gum (Acacia gum) is used as an emulsifier for safer and effective bioactive components delivery after an application [7] and to encapsulate entomopathogenic fungi [8]. It has also been reported that Hirsutella produces a protective exopolysaccharide against desiccation [9,10].

Among entomopathogenic fungi, Hirsutella is one of the most abundant and important fungal genera for pest insect control in the field. It includes about 90 species infecting and parasitizing a wide variety of invertebrates such as mites and insects, many of which are considered pests of economic importance [11]. In this regard, Hirsutella citriformis Speare is the only entomopathogenic fungus involved in Diaphorina citri Kuwayama natural epizootics, allowing for its dissemination [12,13,14]. H. citriformis has been isolated from Diaphorina citri carcasses in Mexican citrus localities [15,16,17], which has allowed for the identification of several strains of interest, as possible biocontrol agents [18]. It is a difficult-to-grow, complex fungus with a limited shelf life and well-known biological control potential against Diaphorina citri, which develops a low spore amount compared with other entomopathogenic fungi [19].

H. citriformis has been applied to control Diaphorina citri Kuwayama and Bactericera cockerelli Sulc. [13,15], which are Candidatus Liberibacter spp. bacterium vectors. This bacterium is associated with several diseases in tomato, chili, and potato and is the causing agent of the Huanglongbing disease (HLB) [18,20]. The main control method against D. citri and B. cockerelli is based on chemical insecticides. However, when the resistance of an insect pest to insecticides increases, the insecticide becomes ineffective and may also confer cross-resistance to other related chemical compounds [21,22]. For the biocontrol of such insects, high-efficacy formulations are based on entomopathogenic fungi, such as Metarhizium robertsii (formerly known as Metarhizium anisopliae) (Metchnikoff) Sorokin (1883), Isaria fumosoroseus Wize, and Beauveria bassiana (Bals. –Criv.) Vuill. [23,24]. The aim of the present study was to increase H. citriformis conidial viability, using formulations containing Acacia and H. citriformis gums as inert materials to improve the formulated conidia shelf life and support their stability for at least three months.

2. Material and Methods

2.1. Fungi

Hirsutella citriformis isolates (

Table 1. Hirsutella citriformis isolates.

H. citriformis Isolate	Isolation Location (MX)	Coordinates	Sampling Date	Host Plant
OP-Hir-3	Huimanguillo, Tabasco	17°51′14″ N, 93°25′13″ W	May 2018	Valence orange
OP-Hir-9	Othón P. Blanco, Quintana Roo	18°32′24″ N, 88°18′37″ W	August 2018	Persse lemon
OP-Hir-10	Xtepén, Uman, Yucatán	20°49′25″ N, 89°44′29″ W	November 2018	Murraya paniculata

) were kept in the Departamento de Microbiología e Inmunología in the Facultad de Ciencias Biológicas (FCB) at Universidad Autónoma de Nuevo León (UANL), México. They were grown in potato dextrose agar (Difco Laboratories, Sparks, MD, USA), containing 1% yeast extract (PDAY) (Difco Laboratories) at 25 ± 2 °C and a relative humidity of ~80% [18].

2.2. Hirsutella Citriformis Gum Production

We produced gum from H. citriformis strain OP-Hir-9, which was cultured in 1000 mL flasks with 250 mL of potato dextrose broth (Difco Laboratories), containing 1% yeast extract (PDBY) (Difco Laboratories). Culture media were inoculated with 3 cm² agar-grown H. citriformis, and flasks were incubated for 14 d at 25 ± 2 °C under agitation at 180 rpm, after which the flask content was centrifuged for 5 min at 10,000× g rpm for gum separation. Supernatant was then collected in a beaker, and isopropanol:gum at a 3:1 ratio (v/v) was kept without shaking for 6 h at 25 ± 2 °C. Next, gum was dried in an infrared balance (AD-4715; A&D Weighing Co., Milpitas, CA, USA) at 121 °C to determine dry weight, and dried gum was crushed in a mortar for storage, until use.

2.3. Conidia Production by Biphasic Hirsutella citriformis Culture

Conidia production by biphasic culturing was performed as previously reported [25], using oat grains for solid fermentation, with some modifications. The first phase consisted of a culture on PDAY in a Petri dish. Produced conidia were used as an inoculum for the second phase of the oat culture. Conidia were collected by adding 10 mL of sterile 0.85% saline solution to aerial mycelium, using a bacteriological loop. Collected conidia were then homogenized by stirring on a vortex for 5 min, and the inoculum was adjusted to 1 × 10⁶ conidia/mL. For solid fermentation on oat grains, we used high-density poly paper bags (two kilograms capacity). Oat grains (200 g) were washed three times to remove foreign particles and soaked for 24 h in 400 mL of purified water (Laboratorios Monterrey, S.A. de C.V. Monterrey, Nuevo León, México). Purified grade water was obtained by a multimedia filter, an activated carbon filter, reverse osmosis, ozonation, and ultraviolet light, and was selected instead of distilled water for its higher content of minerals. Oats were then mixed with 400 mg of oxytetracycline (Forrajera San Carlos, Ciudad Victoria, Tamaulipas, México) to avoid bacterial growth during the 24 h of soaking period.

Next, water was drained, and 4% wheat bran was added and sterilized twice in a 24 h period at 121 °C and 15 lb pressure for 30 min. After the second sterilization, and when oat/wheat bran temperature reached a temperature of 24 ± 2 °C, 16 mL of a suspension containing 1 × 10⁶ conidia/g was added as an inoculum, which was homogenized with a sterile spatula. Containers with H. citriformis-inoculated oat/wheat bran were incubated at 25 ± 2 °C for 21 d.

Conidia were then mechanically detached from the substrate, using a sterile spatula, in 250 mL of sterile distilled water and concentrated by centrifugation (Thermo Fisher Scientific, Waltham, MA, USA) at 10,000 rpm for 10 min, using 50 mL conical tubes. Supernatant was removed, keeping 5 mL to 10 mL of the precipitate from each conical tube, after which conidia were quantified in a Neubauer chamber and adjusted to 1 × 10⁷ conidia/mL with distilled water.

2.4. Effect of Formulation Ingredients on Hirsutella citriformis Conidia Viability

To evaluate each formulation ingredient’s effect on conidia viability, H. citriformis conidia (1 × 10⁷ conidia/mL) were cultured on PDAY medium plus 3% liquid vegetable oil (v/v) or 3% vegetable oil powder (w/v). We then determined conidia viability at least three times, as previously reported [25]. We also evaluated Hirsutella citriformis conidia viability, after adding 0.1% to 0.7% (w/v) of Acacia and 0.1% to 0.5% (w/v) of Hirsutella gums at 25 ± 3 °C or 4 ± 1 °C for 30 d. Treatments resulting in cytotoxicity at 30 d were discarded.

2.5. Formulations Preparation

Formulations were prepared as oil-in-gum emulsions, using conidia from OP-Hir-3 (isolated from Huimanguillo, Tabasco, México) and OP-Hir-10 (isolated from Xtepén, Uman, Yucatán, México) strains. Four different formulations were produced by combining different Acacia and Hirsutella gum concentrations (

Table 2. Hirsutella citriformis conidia formulation ingredients.

Strain	Acacia Gum	Hirsutella Gum	Formulation Code
OP-Hir-3	0.50%	-	FH3-1
	-	0.50%	FH3-2
	0.30%	0.20%	FH3-3
	0.20%	0.30%	FH3-4
Control	Water		FH3 Control
OP-Hir-10	0.50%	-	FH10-1
	-	0.50%	FH10-2
	0.30%	0.20%	FH10-3
	0.20%	0.30%	FH10-4
Control	Water		FH10 Control

) plus 3% vegetable oil powder. For this, emulsions were autoclaved for 15 min at 121 °C, under 15 lb of pressure. The emulsion ingredients were homogenized by stirring on a vortex at speed 5, and after cooling at 24 ± 2 °C, H. citriformis conidia from selected strains were added at 1 × 10⁷ conidia/mL (final concentration) [26].

2.6. Formulations’ Shelf Life

We determined Hirsutella citriformis formulations’ shelf life after storing at 25 ± 2 °C and 4 ± 1 °C. Evaluations were performed in triplicate at days 0, 30, 60, 90, 120, and 150 by counting germinated conidia on PDAY (72 h after sowing) [27].

2.7. Effect of pH on Formulated Conidia Stability

To determine the effect of pH changes on Hirsutella citriformis formulated conidia stability, samples from each formulation (Table 2) were mixed at a 1:10 ratio (w/v) with sterile distilled water, homogenized, and left to stand for one hour. The pH was determined from three different samples of each formulation, using a previously calibrated potentiometer. The pH evaluation of each sample was performed at least twice [28].

2.8. Purity Test

To evaluate the formulation purity after storage, PDAY were inoculated by extension with 100 µL of each formulation after storage and incubated at 25 ± 2 °C for 8 d. Evaluation was performed at least twice. In the event of the presence of other microorganisms, it was considered as a contaminated formulation. The contamination percentage [29] was determined using the following formula:

$$\% Sample purity=\frac{Number of colonies of interest}{Total colonies number }\times 100$$

2.9. Statistical Analysis

Results from different evaluations, except for storage temperatures comparison, were subjected to analysis of variance (ANOVA) (GraphPad Prism version 6.0; San Diego, CA, USA). Means were compared by the Tukey’s test (α = 0.05%), using the software IBM SPSS Statistics Version 21 (SPSS, Inc., Chicago, IL, USA).

Differences in conidia viability after formulations storage at different temperatures were analyzed by the Student t test, where we compared conidia germination percentages among the formulation treatments after 120 d.

3. Results

3.1. Effect of Formulation Ingredients on Conidia Viability

Hirsutella citriformis conidia showed a viability of 79.7 ± 7.04% and 77 ± 3.6% after exposure to vegetable oil powder or liquid vegetable oil, respectively, as compared with the control (70.7 ± 3.025% viability) (F_2,6 = 2.65; p ≥ 0.05). We discarded liquid vegetable oil to continue with the formulation elaboration, because it delayed conidia germination for up to 10 d.

3.2. Evaluation of Gum Concentration Regarding Conidia Viability

We evaluated Hirsutella citriformis OP-Hir-10 strain conidia viability at different concentrations of Acacia (from 0.1% to 0.7%) or Hirsutella (from 0.1% to 0.5%) gums and at 25 ± 3 °C and 4 ± 1 °C. Results showed that at 0 d of storage, conidia viability in all of the treatments and the control ranged from 90.44% to 93.49%. After 15 d, we observed 9.62% viability reduction (the lowest viability) with the 0.25% Acacia formulation stored at 4 °C (81.84 ± 2.29% viability), as compared with the control, whose viability was reduced by 19.85% (

Table 3. Effect of gum and temperature on Hirsutella citriformis OP-Hir-10 strain conidia germination after storage for up to 30 d ¹.

Treatments	Storage Period at 4 ± 1 °C			Storage Period at 25 ± 3 °C
	0 d	15 d	30 d	0 d	15 d	30 d
0.1% Acacia	92.96 ± 0.26 a	87.34 ± 0.64 a	87.77 ± 1.1 a	92.09 ± 0.73 a	87.99 ± 0.69 a	64.0 ± 1.62 a
0.2% Acacia	90.29 ± 1.36 a	86.26 ± 0.84 a	90.28 ± 0.88 a	91.43 ± 1.57 a	87.47 ± 1.98 a	61.36 ± 2.57 a
0.25% Acacia	90.56 ± 0.99 a	81.84 ± 2.29 a	85.96 ± 1.29 a	90.94 ± 1.27 a	86.49 ± 1.16 a	61.03 ± 3.93 a
0.5% Acacia	90.69 ± 1.38 a	90.15 ± 1.25 a	89.63 ± 2.65 a	92.86 ± 0.87 a	84.31 ± 0.64 a	67.43 ± 2.59 a
0.7% Acacia	90.44 ± 1.51 a	88.26 ± 1.91 a	90.68 ± 0.85 a	93.5 ± 0.78 a	88.62 ± 1.08 a	66.32 ± 2.05 a
0.1% Hirsutella	92.47 ± 1.16 a	87.99 ± 0.84 a	80.30 ± 0.85 a	94.58 ± 0.05 a	81.93 ± 0.9 a	60.72 ± 1.48 a
0.2% Hirsutella	94.29 ± 0.32 a	90.47 ± 2.73 a	86.40 ± 2.14 a	93.49 ± 1.29 a	83.12 ± 1.74 a	59.44 ± 2.91 a
0.25% Hirsutella	92.5 ± 0.88 a	90.38 ± 1.5 a	86.91 ± 2.07 a	89.09 ± 0.31 a	86.99 ± 1.24 a	68.76 ± 1.83 a
0.5% Hirsutella	93.17 ± 1.13 a	91.31 ± 0.99 a	93.20 ± 0.8 a	91.95 ± 1.36 a	87.86 ± 1.65 a	72.13 ± 0.9 a
Control	92.68 ± 0.32 a	74.28 ± 1.63 b	53.36 ± 3.58 b	91.59 ± 0.73 a	77.5 ± 1.49 b	31.34 ± 3.47 b

¹ Values followed by the same letter indicate no significant difference (Tukey α = 0.05). Data represent the means ± SEM of triplicate determinations from three independent experiments.

).

3.3. Formulated Conidia Shelf Life

Shelf life analysis results showed that formulated conidia from OP-Hir-3 and OP-Hir-10 Hirsutella citriformis strains maintained a high viability, as compared with the unformulated conidia, after storage at 25 ± 3 °C for up to 120 d (

Table 4. Effect of formulations and storage periods at 25 ± 3 °C on Hirsutella citriformis OP-Hir-3 strain conidia germination percentage ¹.

	Germination (Mean ± SEM)
Days	FH3-1	FH3-2	FH3-3	FH3-4	FH3-Control
0	95.5 ± 0.41 a	95.8 ± 0.69 a	96.2 ± 0.53 a	94.7 ± 0.61 a	91.8 ± 0.86 b
30	94.0 ± 0.37 a	93.7 ± 0.41 a	92.8 ± 0.86 ab	92 ± 0.53 b	90.2 ± 0.53 c
60	89.0 ± 0.98 a	87.7 ± 2.24 a	87.8 ± 1.63 a	87.0 ± 1.67 a	83.7 ± 1.67 a
90	77.7 ± 1.67 ab	79.2 ± 1.31 ab	80.3 ± 0.73 a	76.0 ±2.12 b	68.3 ± 1.22 c
120	61.3 ± 3.34 a	64.0 ± 2.53 a	67.5 ± 1.63 a	66.7 ±2.28 a	60.3 ± 0.76 a

¹ Values followed by the same letter indicate no significant difference (Tukey α = 0.05). Data represent means ± SEM of triplicate determinations from three independent experiments.

and

Table 5. Effect of formulations and storage periods at 25 ± 3 °C on Hirsutella citriformis OP-Hir-10 strain conidia germination percentage ¹.

	Germination (Mean ± SEM)
Days	FH10-1	FH10-2	FH10-3	FH10-4	FH10-Control
0	95.7 ± 0.21 a	95.0 ± 0.36 a	96.0 ± 0.36 a	95.7 ± 0.56 a	93.7 ± 0.21 b
30	93.2 ± 0.7 a	93.8 ± 0.48 a	94.0 ± 0.36 a	93.2 ± 0.6 a	92.0 ± 0.51 a
60	88.7 ± 1.88 a	86.0 ± 0.58 b	85.5 ± 0.43 b	85.5 ± 0.76 b	81.7 ± 1.14 c
90	81.3 ± 0.42 a	81.3 ± 0.42 a	80.8 ± 0.7 a	82.0 ± 0.63 a	69.2 ± 0.48 b
120	70.0 ±0.51 ab	69.8 ± 0.75 b	72.3 ±0.33 a	70.2 ± 0.6 ab	56.0 ± 1.53 c

¹ Values followed by the same letter indicate no significant difference (Tukey α = 0.05). Data represent means ± SEM of triplicate determinations from three independent experiments.

).

After 90 d of storage, conidia from the FH3-3 formulation of the OP-Hir-3 strain achieved the highest and most significant (F_4,25 = 10.19; p ≤ 0.05) percentage of viability, as compared with other formulations and the control, whereas conidia from the FH10-4 formulation of the OP-Hir-10 strain reached a high but not significant (F_4,25 = 101.43; p ≤ 0.05) germination percentage, as compared with other treatments, but it was significant as compared with the control.

Hirsutella citriformis conidia viability of OP-Hir-3 (

Table 6. Effect of formulations and storage periods at 4 ± 1 °C on Hirsutella citriformis OP-Hir-3 strain conidia germination percentage ¹.

Germination (Mean ± SEM)
Days	FH3-1	FH3-2	FH3-3	FH3-4	FH3-Control
0	95.50 ± 0.43 a	95.83 ± 0.7 a	96.17 ± 0.48 a	94.67 ± 0.71 a	91.83 ± 0.87 b
30	93.50 ± 0.76 a	94.50 ± 0.43 a	94.83 ± 0.48 a	93.50 ± 0.56 a	90.83 ± 0.48 b
60	92.83 ± 0.70 a	92.50 ± 1.06 a	92.33 ± 0.96 a	92.83 ± 0.70 a	85.33 ± 1.38 b
90	85.50 ± 1.33 a	84.83 ± 1.11 a	87.17 ± 0.83 a	85.50 ± 0.76 a	70.67 ± 1.28 b
120	79.50 ± 1.23 a	81.17 ± 0.60 a	82.00 ± 0.51 a	80.67 ± 0.56 a	68.67 ± 2.43 b

¹ Values followed by the same letter indicate no significant difference (Tukey α = 0.05). Data represent means ± SEM of triplicate determinations from three independent experiments.

) and OP-Hir-10 (

Table 7. Effect of formulations and storage periods at 4 ± 1 °C on Hirsutella citriformis OP-Hir-10 strain conidia germination percentage ¹.

Germination (Mean ± SEM)
Days	FH10-1	FH10-2	FH10-3	FH10-4	FH10-Control
0	95.67 ± 0.21 a	95.00 ± 0.36 a	96.00 ± 0.36 a	95.67 ± 0.56 a	93.67 ± 0.21 b
30	94.00 ± 0.26 a	94.33 ± 0.42 a	93.83 ± 0.31 a	93.83 ± 0.31 a	92.67 ± 0.49 b
60	92.00 ± 0.58 a	91.83 ± 0.48 a	91.67 ± 0.56 a	90.67 ± 0.49 a	85.67 ± 0.49 b
90	87.83 ± 0.48 a	87.33 ± 0.56 a	87.83 ± 0.75 a	86.67 ± 0.49 a	72.33 ± 0.33 b
120	80.67 ± 0.62 a	80.17 ± 0.91 a	82.17 ± 0.40 a	80.50 ± 0.67 a	61.50 ± 1.17 b

¹ Values followed by the same letter indicate no significant difference (Tukey α = 0.05). Data represent means ± SEM of triplicate determinations from three independent experiments.

) strains was similar among all formulation treatments, after storage at 4 ± 1 °C. Only the control showed a significant (F_4,25 = 18.18; p ≤ 0.05) and (F_4,25 = 118.2; p ≤ 0.05) difference, respectively. At 120 d of storage, conidia viability of OP-Hir-3 and OP-Hir-10 strains decreased by 16.5% and 17.3%, respectively. Viability reduction of conidia control ranged from 23.16% to 34.3%.

After evaluating if a higher temperature decreases conidia viability after 90 d of storage, results indicated a significantly lower viability of conidia in those formulations stored at higher temperatures. This was observed with OP-Hir-3 (Tabasco; Figure 1A) (F_4,11 = 3.356; p ≤ 0.05) and OP-Hir-10 (Yucatán; Figure 1B) (F_4,11 = 3.356; p ≤ 0.05) strains, showing that storage at 4 ± 1 °C decreased the loss of viability of the active ingredient.

3.4. Effect of pH on Formulated Conidia Stability

After analyzing the pH values of the OP-Hir-3 (Tabasco) strain-formulated conidia, we observed that treatments stored at 25 ± 3 °C (Figure 2A) remained stable after 90 d, showing no significant (F_1,11 = 4.844; p ≥ 0.05) difference as compared with the 0 d sample, except for the FH3-3 treatment. The FH3-3 treatment was formulated with both gums, which evidenced significant (F_1,11 = 4.844; p ≤ 0.05) differences between 0 d and 90 d. Furthermore, treatments showed significant (F_1,11 = 4.844; p ≤ 0.05) differences between 0 d and 120 d, except for the control, which evidenced no significant difference (F_1,11 = 4.844; p ≥ 0.05).

All treatments remained stable after 90 d of storage at 4 ± 1 °C (Figure 2B), as there was no significant (F_1,11 = 4.84; p ≥ 0.05) difference in pH values, compared with the initial sample. However, treatments presented significant differences between days 0 and 120, where pH values were lower, unlike the control, which did not show significant (F_1,11 = 4.844; p ≥ 0.05) differences after storage.

Furthermore, treatments of the OP-Hir-10 strain stored at 25 ± 3 °C remained stable after 90 d (F_1,11 = 4.844; p ≥ 0.05), as compared with the 0 d sample, except for the treatments FH3-2 and FH3-4, which were significantly (F_1,11 = 4.844; p ≤ 0.05) different after 0 d and 90 d of storage. All treatments, including the control, showed significantly (F_1,11 = 4.844; p ≥ 0.05) lower pH values after 0 d and 120 d of storage (Figure 2C).

We observed that the pH of treatments remained stable after 90 d of storage at 4 °C, as there was no significant (F_1,11 = 4.84; p ≥ 0.05) difference, as compared with the sample evaluated on the initial day. However, all treatments showed significantly lower pH values between 0 d and 120 d, including the control (Figure 2D).

3.5. Formulation Purity Test

Based on purity evaluations, formulated conidia were not contaminated (H. citriformis conidia was only detected; Figure 3). Results indicated a purity of 100% from 0 d to 120 d of storage for all treatments at 25 ± 3 °C and at 4 ± 1 °C.

4. Discussion

Biocontrol agents represent an alternative to chemical pesticides. They become natural enemies attacking pests, without affecting humans or animals. Entomopathogenic fungi are particularly important because of their pathogenicity routes, a variety of hosts, and their potential to control a wide range of pests. In this study, we observed that conidia viability and germination were not affected after analyzing conidia viability in response to formulation ingredients.

Some advantages of using oily formulations include the increase in the resistance to thermal changes, after evaluating different vegetable oils formulations, using the entomopathogenic fungus Metarhizium robertsii (Metchnikoff) Sorokin (1883), formerly known as M. anisopliae [30]. It has been reported that the use of oils in formulations favors spores’ survival by maintaining their humidity [31]. However, we observed a delayed effect on Hirsutella citriformis mycelial growth in the presence of the maize-based liquid vegetable oil. The typical composition of corn oil is 11% palmitic, 2% stearic, 24.1% oleic, 61.9% linoleic, 0.7% linolenic, and to a lesser extent, other fatty acids (˂ 1%) [32]. A high concentration (3.9 mmol/L) of palmitic acid was shown to inhibit mycelial growth of fungi such as Alternaria solani (Cooke) Wint., Colletotrichum lagenarium (Pass.) Ellis & Halst. and Fusarium oxysporum Schlecht. emend. Snyder & Hansen. Similarly, linoleic acid (2 mmol/L) was reported to reduce mycelium growth but oleic acid (3.2 mmol/L) did not inhibit mycelial growth of these fungi [33]. To date, studies on fatty acids antifungal mechanisms have focused on the inhibitory effect against human pathogenic fungi, whereas antifungal activities against entomopathogenic fungi are still unknown. Therefore, it can be inferred that fatty acids present in corn-based vegetable oils may interfere with Hirsutella citriformis mycelial growth.

Regarding conidia viability results, we showed that gums used in formulations at high concentrations reduced conidia viability at the two temperatures evaluated, in shelf life experiments. Number of viable conidia was initially calculated, inoculating a suspension of 1 cm² agar squares in Petri dishes. Conidia were incubated and the germination percentage was determined at least three times, thus assuring that conidia in the control were as viable as in all treatments. Nevertheless, each treatment was independently inoculated in Petri dishes. This may explain why the initial germination is different among treatments.

The formulation process may lead to an initial loss of viability, which is one of the disadvantages of using Hirsutella as a biological control agent, in addition to its slow growth and short lifetime [19]. This was shown in the germination at time 0 for both strains, where the germination percentage of the conidia was between 90.29% and 94.29% among treatments (Table 3). It was also observed that conidia germination in the untreated control was lower (viability between 91.8% and 93.7%), as compared with that of formulated treatments (Table 4, Table 5, Table 6 and Table 7). However, by comparing this value with the initial assay, we did not observe significant differences. It is possible that formulation ingredients maintained conidia viability by protecting the mucilaginous layer that covers conidia, preventing their early germination in a nutrient-deficient medium (distilled water) [25]. This protective effect was observed with the addition of the vegetable oil in powder.

After testing Trichoderma asperellum Samuels, Lieckf & Nirenberg formulations at higher storage temperatures than those used in our study, it was observed that supersaturation of adherents on conidia completely inhibited their viability [34]. Due to the low levels of viability obtained with high concentrations of the gums used as adherents, it was necessary to make modifications by decreasing gum concentrations and achieving a higher stability of active ingredients, which allowed us to have a stable formula for up to one month. If we compared results obtained with our formulations with other reports, ours improved H. citriformis conidia stability [35,36]. This is a very important issue since H. citriformis conidia are very sensitive to temperature and humidity changes [18]. After evaluating different gums as adherents (bovine gelatin, lemon pectin, modified corn starch) at low concentrations on Beauveria bassiana fungus, similar viability results to those reported by our research team after 15 d of storage were observed [37]. In field tests, Pérez-González et al. [25] reported that after applying conidia from Hirsutella citriformis strain INIFAP-Hir-2, which were formulated with Acacia and Hirsutella gums, the highest Diaphorina citri mortality was achieved with Hirsutella gum (80.0%), followed by the Hirsutella gum without conidia (control) (57.8%). In the same trial, conidia formulated with Acacia gum caused 37.8% mortality, whereas Acacia gum and negative controls induced 9% mortality. Therefore, this may indicate that conidia formulations including Hirsutella gums improve the efficacy of the active ingredient for D. citri biocontrol.

The most effective formulations were those that were kept stored at a cold temperature (4 °C), which maintained conidia viability above 70% after 120 d of storage, as compared with those stored at 25 °C, which remained viable for up to 90 d. This may be due to the use of additives to protect the active ingredient. Acacia and Hirsutella gums and vegetable oil powder may also provide protection to conidia. Interactions between storage temperature and bioformulate ingredients determine conidia longevity [37]. In a formulation of Beauveria bassiana conidia, in the presence of hydrogenated rapeseed oil granules, we obtained 84.7% viability when stored at 25 °C, and 92.3% when stored at 4 °C, after 45 d [38]. Furthermore, it was reported that after evaluating the efficacy of various sugars as additives in B. bassiana formulations, the use of gums or polysaccharides as additives for the active ingredient formulation, evidenced a nutritive effect and survival increase [38]. Formulations containing glucose as an additive were optimum for fungi growth and germination. Regarding shelf-life analysis, results revealed a significant variation in conidia germination, showing higher germination values (92%) after six months of storage at 30 °C. Similarly, a significant effect on the survival rate of polysaccharide-encapsulated Trichoderma harzianum Rifai conidia was previously reported [39].

Several studies have discussed the various storage conditions’ impact on conidial stability. In our study, storage of formulated conidia at 4 °C reduced conidia viability loss for up to 120 d, which agrees with previous reports, where B. bassiana was formulated using oily formulations and conidia, showing a higher germination percentage when stored at 4 °C than at room temperature [40]. This may be due to the cellular metabolism, since, at room temperature, conidia remain active, producing metabolites related to germination and consuming internal nutrients, which affects conidia viability and germination. At storage temperatures from 3 °C to 8 °C, fungal cells maintain a low metabolic rate and remain viable for prolonged periods of time [41,42]. Unformulated conidia had a lower shelf life compared with formulated treatments at 25 °C and 4 °C. These results indicate that the additives present in the treatments provide a protective effect on the active ingredient, improving its stability during the storage time.

Other studies have shown that pH influences the active ingredient during conidia germination and growth [43,44]. The pH tolerance ranges from 6.5 to 7.5 of other entomopathogenic fungi, such as Trichoderma spp. Persoon is known to favor its growth [45]. Other fungi have a wider pH tolerance range, such as Beauveria bassiana, whose reported pH tolerance values are between 5 and 13 [46]. Similarly, pH values reported for Hirsutella spp. Brandy growth and sporulation are between 6.0 and 9.2 [47] or between 5.5 and 7 [48]. In our study, even with differences in the stability of some treatments, the optimal range of pH was maintained for up to 90 d in all formulations in both storage conditions, indicating that pH in the aforementioned tolerance range did not affect conidia germination. In addition, formulations may contaminate if good production practices are not followed. However, it is known that formulations that have more than 90% purity show a greater stability and viability of the active ingredient [29]. This is because contaminants may reduce active ingredients’ efficacy due to the potential inhibitory effects of secondary metabolites [29,49,50].

5. Conclusions

The aim of the present study was to demonstrate that Hirsutella conidia shelf life increases after formulation. Vegetable oil powder used to formulate conidia did not reduce Hirsutella citriformis’ viability, since their germination was not affected. Similarly, Hirsutella gum may be used as an ingredient for increasing Hirsutella conidia shelf life, along with vegetable oil powder. In addition, we observed that formulations maintained conidia viability for at least 90 d after storage at 25 °C and at least 120 d after storage at 4 °C. It was evidenced that under storage at 4 °C, H. citriformis conidia loss of viability decreased. All formulations evaluated were within the optimum range of pH at 4 °C and 25 °C for 90 d. pH of the formulated conidia, which prevailed between 5.5 and 7.5, allowed for conidia germination, and all treatments maintained conidial purity (100%) up to 120 d at both storage temperatures.