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) 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 × 10
6 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 × 10
7 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) 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 × 10
7 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:
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.
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].