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Article

Viability of Entomopathogenic Fungi in Oil Suspensions and Their Effectiveness against the Agave Pest Scyphophorus acupunctatus under Laboratory Conditions

by
Teodulfo Aquino-Bolaños
,
Yolanda Donají Ortiz-Hernández
*,
Angélica Bautista-Cruz
and
Marco Aurelio Acevedo-Ortiz
Instituto Politécnico Nacional, CIIDIR-Oaxaca, Hornos 1003, Santa Cruz Xoxocotlán 71230, Mexico
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(6), 1468; https://doi.org/10.3390/agronomy13061468
Submission received: 12 April 2023 / Revised: 23 May 2023 / Accepted: 23 May 2023 / Published: 25 May 2023
(This article belongs to the Special Issue Advances in Plant–Fungal Pathogen Interactions)
Versions Notes

Abstract

:
Oaxaca, Mexico, is home to over 30 species of the genus Agave, and its cultivation is of great economic and social importance for the mezcal industry, which depends on its production. The incidence of the pest Scyphophorus acupunctatus causes severe losses and damage. Agrochemicals are used for its control, but a viable alternative is microbial control. The objectives of this study were to determine the natural occurrence of the entomopathogenic fungi (EPF) Beauveria bassiana and Metarhizium anisopliae, isolated from S. acupunctatus in agave crops, and to evaluate the effect of vegetable oil in water emulsions containing conidia from the native fungi against adults of S. acupunctatus under laboratory conditions. Viability of the fungal isolates was determined at a concentration of ×108 conidia/mL in avocado (Persea americana), mamey (Mammea americana), chia (Salvia hispanica), or olive (Olea europaea) oil in water emulsions at two concentrations, 20% and 40%. The most effective oil emulsion on S. acupunctatus adults was determined under laboratory conditions. Naturally occurring fungi in 900 field collected insects was 2.21%. Ninety-six hours after preparing a 20% emulsion in P. americana oil, B. bassiana and M. anisopliae had a viability of 75% and 66.5%, respectively, while the control conidia suspended in distilled water remained viable for only 48 h. Twenty-four hours after applying M. anisopliae conidia in a 40% P. americana oil emulsion, effectiveness was 100% on S. acupunctatus, followed by M. anisopliae in 20% P. americana oil emulsion with 75% effectiveness. At 72 h post-application, all fungus in oil emulsions achieved an accumulated insect mortality of 100%, while the control showed no effect on adult S. acupunctatus. The most promising combination was 40% P. americana oil emulsion, which achieved 50% viable B. bassiana or M. anisopliae conidia up to 96 h after preparation, and its accumulated effectiveness on S. acupunctatus adults was 87.5% after 24 h.

1. Introduction

Mexico is home to over 75% of the world’s agave species, distributed in arid and semi-arid regions, and extensively cultivated in Oaxaca, Sonora, Morelos, Puebla, Guerrero, San Luis Potosí, Zacatecas, Durango, Michoacán, Tamaulipas, and Guanajuato. These perennial plants are part of agroforestry systems of great cultural, agronomic, ecological, and social importance [1,2]. They are used to obtain syrup, fiber, construction materials, and bioplastics, and to control soil erosion, among other uses [3,4]. The agave agroindustry generates jobs for 85,000 workers and 17,000 producers who cultivate more than 111,420 hectares to produce “tequila” and “mezcal”, alcoholic beverages with national and international demand; sales were over a 1 billion USD in 2017 [5,6,7].
In the Central Valleys and Sierra Sur of the state of Oaxaca, Mexico, more than 10,000 hectares of Agave spp. are cultivated [8], and 200,000 tons of agave are destined for the mezcal industry [9,10]. Agave angustifolia, known as “maguey espadín”, is the most widely produced species in the area [5]. However, as with other agaves, yield of the plants for mezcal production is affected by environmental problems and by incidence of aggressive pathogens, such as the bacterium Pectobacterium carotovora, for which Scyphophorus acupunctatus is a vector [11,12].
S. acupunctatus (Coleoptera: Curculionidae), the agave weevil, is a year-round pest, that causes severe damage to agave plants and affects their growth [13]. The larvae bore into the plant’s stem, causing weakening, and larvae and adults feed on the leaves and stems, causing plant death [14]. Furthermore, the weevil transmits the bacteria P. carotovorum, which causes putrescence. The loss of plant tissue due to damage caused by S. acupunctatus and P. carotovorum is 10% in A. angustifolia and up to 90% in A. tequilana [15].
S. acupunctatus is controlled with contact pesticides such as malathion and endosulfan, which pollute the environment, and their residues remain in both the soil and agave plants. Misuse of these products increases resistance of insect pest populations and causes intoxication to farmworkers [16,17,18]. Biological control with entomopathogenic agents is an alternative for management of this coleopteran because S. acupunctatus larvae and adults are susceptible to infection caused by entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPNs) [19,20,21].
Application of EPF in the field using spray technology requires support adjuvants, such as sunscreens and mineral or vegetable oils in suspensions of conidia to protect them against adverse abiotic factors such as high temperature [22], rapid desiccation [23], and UV radiation [24]. Vegetable oil provides some protection from the physical damage to the propagules caused during preparation in suspensions with distilled water, which diminishes their effectiveness in field applications [25]. It also increases adhesion of conidia on the insect cuticle and plant surface, thus increasing contact time to facilitate germination and penetration [26]. In addition, because of their viscosity, the evaporation of oils is slower than water and they contribute to maintaining the viability of EPF conidia [17].
Prepared emulsions of soybean oil [27] and water with Beauveria bassiana (strain ESALQ-PL63) or Metarhizium anisopliae (strain ESALQ-1037), which were originally isolated from ants, were exposed to a temperature of 36 °C. After 6 h, viability decreased to less than 7% for both species, while in water suspension they showed decreasing viability from 60 to 7% [27]. When M. anisopliae (IP 46) conidia formulated in pure canola vegetable oil was heated (45 ± 0.2 °C), almost 50% remained viable after 48 h [22]. Indeed, the biocompatibility of some vegetable oils with EPF has been reported [28], and in some cases, the mixtures have had a repellent effect against Acari, such as ticks [29] or a toxic effect on insects.
Isolated native EPF are effective agents for controlling local insect pests, and oil adjuvants are not aggressive for conidial viability in aqueous suspension. Oil emulsions might extend the viability of conidia and increase the mortality of S. acupunctatus adults isolated from Agave spp. The objectives of this study were (1) to determine the natural occurrence of EPF in S. acupunctatus populations collected from agave crops and (2) to evaluate the effect of conidia from the native fungi B. bassiana or M. anisopliae in vegetable oil emulsions on conidia viability and the effectiveness of the fungi against S. acupunctatus adults under laboratory conditions.

2. Materials and Methods

2.1. Presence of Entomopathogenic Fungi (EPF) in Scyphophorus Acupunctatus Adults

S. acupunctatus adults were collected from mature agave plants on plantations located in Ocotlán, Oaxaca, Mexico [16°47′29″ north latitude, 96°40′29″ west longitude, 1500 m altitude, 600–800 mm annual precipitation, average annual temperature of 26.9 °C, subhumid warm climate]. The agave plants exhibited a degree of damage of 3 (more than six lesions on the leaf) or 4 (necrotic lesions on the plant’s base and healthy core of the plant “piña”), according to the classification scale reported [11]. From June 2021 to May 2022, 12 samplings were conducted. Every 30 days, 75 live adult insects were collected, and inmediately divided into two populations, each of which was placed in plastic containers.
A total of 900 adult specimens were collected and kept in a 500 mL cylindrical plastic container without a lid; the aperture was covered with a fine mesh fixed with an elastic band. The insects were fed with small pieces of ripe agave leaves and maintained under controlled conditions in the entomology laboratory of the Interdisciplinary Research Center for Integral Regional Development (IPN-CIIDIR Oaxaca), at a temperature of 21 ± 2 °C to 23 ± 2.16 °C, with a relative humidity of 45 ± 8.68 to 56 ± 8.36% and 10 h light.
Positive samples (insects with signs of infection by EPF) were separately placed in humid chambers [30] and incubated at 24 ± 2 °C and 50–85% relative humidity for 10 days to promote mycelium growth on the insects. The insect cadavers were then observed with a stereoscopic microscope at 200× magnification to determine mycelium growth (without distinguishing the EPF genera) and the percentage of natural EPF occurrence on the insect sample was calculated with Equation (1).
Natural occurrence of EPF = (ISI + IMG/TIS) × 100
where, ISI = Individuals with Signs of Infection, IMG = Individuals with Mycelium Growth, and TIS = the Total of Individuals Sampled.
To confirm Koch’s postulates [31] and the actual death of S. acupunctatus adults, insects identified as infected by EPF were individually deposited in humid chambers and observed for mycelial growth. Once isolated, the EPF were seeded on potato dextrose agar Sabouraud (PDA) culture medium. The conidia were tested in bioassays against adult insects.

2.2. Culture of the Entomopathogenic Fungi

In the Biological Control Laboratory of CIIDIR, two isolated EPF were identified with the support of taxonomic keys and of the morphological characteristics of the species described as M. anisopliae and B. bassiana [32]. EPF were cultured on a solid Sabouraud potato dextrose agar medium in Petri dishes (80 × 10 mm) and incubated at 26 ± 1 °C for 15 days. The conidia were harvested manually with a microbiological inoculation loop, concentrated in sterile distilled water (DW) at a ratio of 1 × 108 conidia/mL, and placed in a beaker before preparation in vegetable oil emulsions.

2.3. Preparation of Conidia Suspension in Oil Emulsions

The native fungi M. anisopliae or B. bassiana (isolated from S. acupunctatus) were suspended in oil emulsions made with one of the following vegetable oils: avocado (Persea americana Miller) Laboratorios Hersol®, olive (Olea europaea Linnaeus) Español®, chia (Salvia hispanica Linnaeus), and Ines® or mamey (Mammea americana Linnaeus). A dispersant 0.1% Tween 80 was used to separate and disperse the mycelia in water, and then serial dilutions were made in DW plus 0.1% Tween until a concentration of 1 × 108 conidia/1 mL was achieved.
Using a 10–100 µL micropipette (Pipet-Lite XLS), 20/40 vol-% concentrations of vegetable oil emulsions were each placed in a glass Petri dish (55 mm diameter). A volume of 0.5 mL of the aqueous suspension containing 1 × 108 conidia was then added to the glass Petri dishes and both liquids were mixed with a magnetic stirrer for 3 min. The conidia obtained were counted in a Neubauer chamber, and we worked with a concentration of 1 × 108 conidia/1 mL.

2.4. Bioassay of the Viability of M. anisopliae and B. bassiana Suspended in Vegetable Oil Emulsions

One mL of oil emulsion with the suspended conidia was incubated at 26 ± 1 °C for germination, and their viability was determined every 24 h post-preparation for four days. The suspension was placed on slides and a coverslip was used to facilitate observation of the spores; 100 conidia were counted per slide. The conidia in oil emulsion were observed under a light microscope at 200× magnification and were considered viable when they had germ tubes that were longer than their diameters. Viability was also determined by germination of conidia on insects and their growth in a potato dextrose agar (PDA) culture medium.
The treatments with M. anisopliae or B. bassiana were applied. Two controls were included: M. anisopliae (T1) or B. bassiana (T2) in 1 mL sterile DW without vegetable oil (Table 1). Sixteen treatments used vegetable oil (P. americana, O. europaea, S. hispanica and M. americana) at two oil concentrations (20% or 40%) with B. bassiana or M. anisopliae. For each treatment, there were twelve replications, and one insect was used per repetition. The conidia in oil emulsions at two oil concentrations were prepared as follows: (1) 20% (0.2 mL vegetable oil + 0.8 mL DW) with suspension of 1 × 108 conidia, and (2) 40%, (0.4 mL vegetable oil + 0.6 mL DW), with suspension of 1 × 108 conidia.

2.5. Bioassay of the Effectiveness of M. anisopliae or B. bassiana Conidia Suspended in Oil Emulsions on S. acupunctatus Adults under Laboratory Conditions

Effectiveness was defined as the proportion of S. acupunctatus adults that died after exposure to the native fungi B. bassiana or M. anisopliae, which were applied at a concentration of 1 × 108 conidia/insect in 2 mL of a P. americana oil emulsion. Effectiveness was measured every 24 h. The viability of conidia proved to be the highest in P. americana oil emulsion and, thus, this emulsion was later used to test its effectiveness against pest insects. Two oil concentrations were prepared: (1) 20% P. americana oil emulsion consisting of 0.4 mL oil + 1.6 mL of a suspension of conidia in DW and (2) 40% P. americana oil emulsion consisting of 0.8 mL of oil + 1.2 mL of a suspension of conidia in DW. Eight treatments were tested with twelve replications each (Table 2). The treatments comprising four P. americana oil emulsions at 20% or 40% with B. bassiana (T1 and T2) or M. anisopliae (T3 and T4), two P. americana oil emulsions at 20% (T5) or 40% (T6) without conidia, an absolute control (T7) of DW with no conidia or vegetable oil, and a treatment of only vegetable oil (T8).
Adult S. acupunctatus were placed individually in 300 mL cylindrical plastic jars with a piece of agave as food, and 1 mL of oil emulsion was then applied on the insects and food. Twelve replications per treatment were conducted, and one insect was used per repetition. Insect mortality was checked every 24 h post-application for 7 days. The insects showing signs of infection by EPF were placed in humid chambers and the reason of the death was confirmed by the appearance of mycelial growth on the cadaver, seen through a light microscope at a 200× magnification.

2.6. Statistical Analysis

Cumulative viability of the EPF conidia in the oil emulsions for each treatment was expressed as a percentage, based on the number of replications that had mycelium germination divided by the total number of replicas. Mortality was quantified every 24 h for 96 h. Data of the accumulated percentage of insect mortality every 24 h during 72 h were normalized by arcsine transformation √(x/100) and processed through an analysis of variance (ANOVA). Statistical differences between the mean mortality of each treatment were established by a Tukey test (α = 0.05). All analyses were performed with the statistical software SAS® [33].

3. Results and Discussion

3.1. Natural Occurrence of Entomopathogenic Fungi on S. acupunctatus

The natural occurrence of EPF in the sample of 900 insects was 2.21%. The native EPF isolated from positive adult S. acupunctatus insects collected from Agave spp. plants in Oaxaca, Mexico, were identified as M. anisopliae and B. bassiana. This percentage is low, compared to the 18.1% of two entomopathogenic fungi (1% for M. anisopliae and 17.1% for B. bassiana) found in insects in agricultural soils in chaparral habitat associated with Agave lechuguilla in Saltillo, Coahuila, Mexico [34]. The isolation of B. bassiana had been previously reported in mummified cadavers of S. acupunctatus adults collected from Agave cocui plants in Estado Falcón, Venezuela [35]. Our study is of great importance since there are no previous reports of any species of Metarhizium spp. isolated from insects associated with Agave spp.

3.2. Viability of B. bassiana and M. anisopliae Suspended in Oil Emulsions

The maximum mean viability of conidia suspended in vegetable oil emulsion 96 h after preparation can be seen in (Table 1). Twenty-four hours after preparing emulsions of vegetable oil with suspended conidia, three treatments exceeded 80% viability, the highest of which was T8 (B. bassiana + DW + 40% O. europaea) with 100%, and T3 and T17 with 85%, while T2 (Control) maintained 62.5%. After 48 h, conidia suspended in DW were completely dehydrated, and the treatments that showed viability higher than 60% were T3 (B. bassiana + DW +20% P. americana) with 87.5% and T5 and T9 both with 62.5%. At 72 and 96 h, mycelial growth was observed in treatment T3 (B. bassiana + DW + 20% P. americana) with 75% viability and T5 (M. anisopliae + DW + 20% P. americana) with 67%. These results suggest that P. americana oil contributed more than the other vegetable oils to preventing dehydration of EPF conidia and maintained viability at 23 ± 4 °C for up to 96 h. Hence, the experimental results support the proposal of using EPF conidia in oil–water emulsion (mainly with P. americana oil) at a concentration of 20%, which maintains viability for up to 96 h. Comparing avocado oil and olive oil, the omega 3/omega 6 ratio is higher in avocado oil than in olive oil [36], which may make it more biocompatible with EPF. Some of the benefits could be protection against physical damage to cells and repair of conidia damage from adverse environmental factors, such as UV radiation, heating, or aging, among others [37].

3.3. Effectiveness of B. bassiana and M. anisopliae Suspended in P. americana Oil Emulsions on S. acupunctatus Adults under Laboratory Conditions

Accumulated effectiveness of the EPF in oil–water emulsions on S. acupunctatus adults at 24–72 h after application is presented in (Table 2). Twenty-four hours post-application, effectiveness of treatments T4 (M. anisopliae + 60% DW + 40% P. americana) and T8 (40% P. americana) were 100% and 87.5% for treatments T6 (60% DW + 40% P. americana) and T3 (M. anisopliae + DW + 20% P. americana). Effectiveness of these four treatments was statistically similar, but different from the other treatments. After 48 h, the accumulated effectiveness of treatments T5 and T6 increased to 100%. It was not until after 72 h that treatments T1, T2, and T3 accumulated 100% effectiveness on S. acupunctatus. The absolute control T7 (DW without EPF) did not cause mortality in adult insects and was statistically different from all the other treatments. However, at 48 h, two treatments were found to be 100% toxic/lethal to adults of S. acupunctatus T8 (oil P. americana) and T4 (M. anisopliae + P. americana 40%).
The effectiveness of B. bassiana suspended in 20% P. americana oil-in-water emulsion on S. acupunctatus adults (100%) is similar to another report [38], in which B. bassiana applied in sunflower oil to control the coffee berry borer Hyphotenemus hampei resulted in 100% mortality of adult insects after 120 h. Mortality of Leptinotarsa decemlineata larvae after application of a dispersion of mineral oil formulation with B. bassiana (GHA) at a concentration of 254 conidia/mm2 was 58.6% [39].
The effectiveness of M. anisopliae applied in 40% P. americana oil-in-water emulsion (T4) on S. acupunctatus was even higher in less time (100% in 24 h) compared with other reported results [40], who found a synergistic effect between the combination of M. anisopliae (ATCC-Nr. 90448) and some vegetable oils. They found that effectiveness on white flies (Trialeurodes vaporariorum and Bemisia tobaci) increased up to three times when combined. With sunflower oil, effectiveness was 100%, but 8 days post-application. B. bassiana against the coffee berry borer caused 100% mortality within 6 days [38]. Hence, based on these reports, mortality of insects caused by EPF conidia can be a good alternative as a control method against insect pests. The high mortality (100%) of S. acupunctatus by application of only P. americana oil (T8) is in accord with the toxic effect of avocado oil on insects, which has been reported in previous studies, but this effect is not usually evident so quickly on the first day post-application. For example, application of avocado idioblast cell oil against Spodoptera exigua (Hübner) caused 100% mortality after seven days [41]. Against Drosophila suzukii (Matsumura), application of P. americana essential oil had a pesticidal effect [42].
Against the coleoptera Conotrachelus psidii, the effectiveness of the fungi M. anisopliae and B. bassiana at 1 × 108 conidia/mL applied with sunflower oil + Tween 80 was 57.3 to 84.3% and 77.3 to 95.3%, respectively. However, this mortality is a value accumulated over 20 days post-application [43]. A rate of 5 × 106 conidia/mL of M. anisopliae was formulated into a water-in-oil emulsion, whose composition was coconut oil (19%), soybean oil (28.5%), Tween 20 (2.5%), sterile DW (45.25%), glycerin (4%), and water-soluble wax (0.75%). The formulation was stored at 20 ± 1 °C, maintaining 50% conidia viability for 4.6 months, and the effectiveness against Bemisia tabaci and Tetranychus cinnabarinus was 58 to 100% within a period of 3 to 4 days after application [17]. The viability and effectiveness results of our study were below reported values. However, it is important to mention that the difference in storage temperature was a distinctive factor in the execution of our experiments with respect to others [44].

4. Conclusions

Conidia from naturally occurring fungi B. bassiana and M. anisopliae isolated from S. acupunctatus adults collected from agave plantations and suspended in P. americana oil emulsion proved to be pathogenic to adult insects and highly effective for their control under laboratory conditions, probably due to the contribution of the toxicity of avocado oil. This is the first report of the pesticidal effect of avocado oil (P. americana) on S. acupunctatus adults collected from Agave spp. Fungal formulation will serve as a more environmentally friendly alternative to control the agave weevil, S. acupunctatus.

Author Contributions

Conceptualization, T.A.-B.; Data curation, T.A.-B. and Y.D.O.-H.; Formal analysis, T.A.-B. and Y.D.O.-H.; Validation, T.A.-B.; Writing—original draft, T.A.-B. and Y.D.O.-H.; Writing—review and editing, T.A.-B., Y.D.O.-H., A.B.-C. and M.A.A.-O. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Instituto Politécnico Nacional through project SIP-IPN.

Data Availability Statement

Not applicable.

Acknowledgments

We thank the Instituto Politécnico Nacional (IPN) of Mexico and Entomology Laboratory of IPN-CIIDIR Oaxaca (Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Oaxaca IPN). Thanks to the anonymous reviewers for improving the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Table
Table 1. Viability percentage for B. bassiana and M. anisopliae (concentration of 1 × 108 conidia/mL) suspended in emulsions of four vegetable oils at concentrations of 20% or 40%, at four different times.
Table 1. Viability percentage for B. bassiana and M. anisopliae (concentration of 1 × 108 conidia/mL) suspended in emulsions of four vegetable oils at concentrations of 20% or 40%, at four different times.
TreatmentEPF
(1 × 108 Conidia)		Oil Concentration (%)Time after Formulation
(h)
24487296
None
T1B. bassiana050 ± 0.6737.5 ± 0.6700
T2M. anisopliae062.5 ± 0.6712.5 ± 0.6700
P. americana oil emulsion
T3B. bassiana2087.5 ± 0.5287.5 ± 0.5275 ± 0.5275 ± 0.62
T44062.5 ± 0.6737.5 ± 0.6737.5 ± 0.670
T5M. anisopliae2050 ± 0.6762.5 ± 0.6762.5 ± 0.6762.5 ± 0.67
T64075 ± 0.6237.5 ± 0.6237.5 ± 0.620
O. europaea oil emulsion
T7B. bassiana2037.5 ± 0.6737.5 ± 0.6737.5 ± 0.670
T84010050 ± 0.5250 ± 0.520
T9M. anisopliae2012.5 ± 0.6762.5 ± 0.6762.5 ± 0.670
T104050 ± 0.6737.5 ± 0.6737.5 ± 0.670
S. hispanica oil emulsion
T11B. bassiana2012.5 ± 0.6712.5 ± 0.6712.5 ± 0.670
T124025 ± 0.67000
T13M. anisopliae2037.5 ± 0.6725 ± 0.6725 ± 0.670
T144050 ± 0.67000
M. americana oil emulsion
T15B. bassiana2062.5 ± 0.67000
T164050 ± 0.6725 ± 0.6725 ± 0.670
T17M. anisopliae2087.5 ± 0.5250 ± 0.5237.5 ± 0.520
T18400000
Table
Table 2. Cumulative effectiveness of B. bassiana and M. anisopliae (1 × 108 conidia/insect) applied in emulsions of P. americana oil at concentrations of 20% or 40% on S. acupunctatus adults.
Table 2. Cumulative effectiveness of B. bassiana and M. anisopliae (1 × 108 conidia/insect) applied in emulsions of P. americana oil at concentrations of 20% or 40% on S. acupunctatus adults.
TreatmentComposition (%)Time after Application (h)
244872
T1B. bassiana + P. americana 2014.2 b57.1 b100 a
T2B. bassiana + P. americana 4071.4 ab87.5 a100 a
T3M. anisopliae + P. americana 2087.5 a87.5 a100 a
T4M. anisopliae + P. americana 40100 a100 a100 a
T580 DW + P. americana 2042.8 ab100 a100 a
T660 DW + P. americana 4087.5 a100 a100 a
T7100 DW (absolute control)0 c0 c0 c
T8100 P. americana (positive control)100 a100 a100 a
For statistical analysis, the original dates were transformed into arcsine √(x/100). Means with different letters in each column are statistically different (Tukey, p ≤ 0.05). DW: distilled water.
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Aquino-Bolaños, T.; Ortiz-Hernández, Y.D.; Bautista-Cruz, A.; Acevedo-Ortiz, M.A. Viability of Entomopathogenic Fungi in Oil Suspensions and Their Effectiveness against the Agave Pest Scyphophorus acupunctatus under Laboratory Conditions. Agronomy 2023, 13, 1468. https://doi.org/10.3390/agronomy13061468

AMA Style

Aquino-Bolaños T, Ortiz-Hernández YD, Bautista-Cruz A, Acevedo-Ortiz MA. Viability of Entomopathogenic Fungi in Oil Suspensions and Their Effectiveness against the Agave Pest Scyphophorus acupunctatus under Laboratory Conditions. Agronomy. 2023; 13(6):1468. https://doi.org/10.3390/agronomy13061468

Chicago/Turabian Style

Aquino-Bolaños, Teodulfo, Yolanda Donají Ortiz-Hernández, Angélica Bautista-Cruz, and Marco Aurelio Acevedo-Ortiz. 2023. "Viability of Entomopathogenic Fungi in Oil Suspensions and Their Effectiveness against the Agave Pest Scyphophorus acupunctatus under Laboratory Conditions" Agronomy 13, no. 6: 1468. https://doi.org/10.3390/agronomy13061468

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