Efficacy of Metarhizium anisopliae against Bactrocera cucurvitae under in-vitro conditions: A meta-analysis

doi:10.21203/rs.3.rs-5019233/v1

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Efficacy of Metarhizium anisopliae against Bactrocera cucurvitae under in-vitro conditions: A meta-analysis

https://doi.org/10.21203/rs.3.rs-5019233/v1

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This study evaluated the effectiveness of the entomopathogenic fungus Metarhizium anisopliae in controlling the cucurbit fruit fly Bactrocera cucurbitae under in-vitro conditions. This research aimed to develop sustainable and eco-friendly pest management strategies to replace traditional chemical pesticides. The experiment employed a randomized complete block design with five concentrations of M. anisopliae spore solution, replicated four times with 20 fruit flies each. The media for the fungus were prepared and purified, and fruit flies were collected using pheromone traps. The treatments were applied via the spray method, and data on mortality rates, mummification times, and toxicity levels were collected. Statistical analyses, including ANOVA and log-probit analysis, were also conducted to determine the lethal concentrations and durations. The results indicate a clear dose response relationship, with higher spore concentrations leading to significantly increased mortality rates and faster mummification. A concentration of 6.0 × 10⁵ CFU/mL was necessary for substantial population reduction, while the projected lethal concentrations for 50% and 99% mortality were approximately 2.65 × 10⁵ CFU/mL and 1.38 × 10⁵ CFU/mL, respectively. These findings suggest that M. anisopliae is a highly effective biocontrol agent against B. cucurbitae, and is a viable alternative to chemical pesticides. Further research is recommended to explore field applications and assess long-term ecological impacts.

Agronomy

Bactrocera cucurbitae

Metarhizium anisopliae

mortality-rates

fruit-fly

The cucurbit fruit fly, Bactrocera cucurbitae, is a major pest that causes significant damage to cucurbit crops, particularly squash. Infestations by this pest can lead to severe crop losses, posing a substantial threat to the agricultural sector and food security (Qazzaz et al., 2015). Therefore, safeguarding these crops against B. cucurbitae is critical for protecting farmers' livelihoods and ensuring stable food production.

Historically, chemical pesticides have been the primary tool used to combat B. cucurbitae. However, their widespread and prolonged use has raised serious concerns, including the accumulation of pesticide residues in harvested crops, harmful environmental impacts, and the development of pesticide-resistant pest populations. These issues underscore the need to explore alternative pest management strategies that are more sustainable and environmentally friendly (Abbas et al., 2017).

Entomopathogenic fungi (EPFs) have gained attention as promising alternatives to chemical pesticides due to their natural ability to target a wide range of insect pests and their widespread occurrence in various ecosystems (Vega et al., 2009). EPFs, such as Metarhizium anisopliae, are eco-friendly, and no known pests develop resistance to them (Hadi et al., 2013). Metarhizium anisopliae is particularly noteworthy for its effectiveness in controlling insect pests, its specificity for targeting insects, its minimal impact on nontarget organisms, and its biodegradability. These characteristics make it an appealing alternative to chemical pesticides, particularly for managing fruit fly populations such as B. cucurbitae in squash crops (Ekesi et al., 2002).

Evaluating the effectiveness of Metarhizium anisopliae against B. cucurbitae in controlled in vitro conditions allows for precise manipulation of variables and direct observation of the fungus's impact on the pest. These controlled studies provide crucial insights that can guide future field applications.

This study aimed to optimize pest management strategies by identifying the most effective concentrations and conditions for applying Metarhizium anisopliae under in-vitro conditions. This research seeks to contribute to the development of precise, eco-friendly, and sustainable methods for controlling B. cucurbitae during squash cultivation.

This experimental study was conducted from September to December 2023 in Mindanao State University-General Santos city.

2.1 Preparation of Materials

2.1.1 Materials used

The materials used were as follows: (1) pure culture of Metarhizium anisopliae (procured from the Regional Crop Protection Center, Tacurong City, last 2023); (2) rose bengal chloramphenicol agar; (3) potato; (4) dextrose; (5) agar; (6) sterile distilled water; (7) magnetic stirrer; (8) autoclaved; (9) petri plates; (10) sterile gauze pad; (11) wire loop; (12) laminar flow hood; (13) alcohol lamp; (14) autoclavable polypropylene plastic (02pp); (15) test tubes; (16) cotton plugs; (17) programmable incubator; (18) plastic gallon; (19) commercial methyl-eugenol pheromone solution; (20) fly net; (21) punctured plastic containers; (22) phycological saline solution (8.5% NaCl); (23) Neubauer hemocytometer; and (24) 50 mL spray bottle.

2.1.2 Methods

For the experiment, five (5) treatment levels were employed and replicated four (4) times. The experiment was arranged in accordance with a randomized complete block design (RCBD), with the blocking factor used as the container from which the corresponding fruit fly specimens were collected. For each experimental unit, 20 fruit fly specimens were assigned. The treatments were assigned as follows:

M1 = 0 CFU/mL

M2 = 1.5 × 10⁵ CFU mL-1

M3 = 3.0 × 10⁵ CFU mL-1

M4 = 4.5 × 10⁵ CFU mL-1

M5 = 6.0 x 10⁵ CFU mL-1

2.2 Media preparation

Metarhizium anisopliae was initially purified using semi selective media (rose-bengal chloramphenicol agar) and then subcultured using enrichment media (potato dextrose agar). The media was mixed in distilled water, cooked for 15 minutes at 85 degrees Celsius using a magnetic stirrer, and sterilized at 121 degrees Celsius for 15 minutes using vertical autoclave. The sterile media was cooled to 50 degrees Celsius and poured at a rate of 15 ml per sterile petri plate. After solidification, the Petri dishes were subsequently flipped to prevent contamination. Pure colonies were subsequently selected and grown on potato dextrose agar. To prepare the PDA, potato plants were initially peeled, and cooked in distilled water at 95 degrees Celsius for 20 minutes. The potato solution was homogenized, and impurities were removed by pouring the mixture over a sterilized gauze pad (folded eight times). Afterwards, 20 grams of dextrose and 14 grams of agar were added to every 1,000 ml of sterile distilled water, and the mixture was cooked for 15 mins at 95 degrees Celsius using a magnetic stirrer. Then, the media was sterilized at 121 degrees Celsius for 15 minutes. After cooling to 50 degrees Celsius, the media was poured at a rate of 15 ml per sterilized Petri dish. The plates were cooled and subsequently flipped.

2.3 Purification of the Fungal Culture

To purify M. anisopliae, one loopful of prepared sorghum culture was placed at three equidistant points on rose Bengal chloramphenicol agar (RBCA) plates and incubated for 7 days at 27 ± 0.5 degrees Celsius. Any subsequent colonies that were observably pure and characteristic of M. anisopliae were re-cultured on potato dextrose agar (PDA) plates repeatedly until a pure colony was obtained. The pure colonies were then transferred to PDA slants for storage at 4°C.

2.4 Collection of fruit fly specimens

To collect fruit fly specimens, a pheromone trap made of a 1-gallon plastic container filled with cotton containing commercial methyl-eugenol solution (pheromone) was hung in front of the Pest Clinic Laboratory of the College of Agriculture, Mindanao State University-General Santos. The trapped fruit flies were collected using a fly net and transferred to individual plastic containers (punctured to allow aeration).

2.5 Treatment preparation

To prepare the spore solutions and corresponding concentrations, slant cultures were first flooded with 10 mL of physiological saline each and scraped using an inoculating loop. The resulting spore suspension was then filtered through eight layers of sterile gauze. A serial dilution was performed up to 10^− 5, and the spores were counted using a Neubauer hemocytometer. The spore suspension was then adjusted to the appropriate concentrations by mixing with the appropriate amount of physiological NaCl solution. Fifty millilitres of each treatment was prepared.

2.6 Treatment application

To apply the treatments, sterile 50 ml plastic spray bottles were used. Sample specimens were sprayed such that no dripping occurred.

2.6.1 Processing

The research process began with the preparation of culture media, followed by the purification and large-scale production of Metarhizium anisopliae. After one week of inoculation, pure cultures of the fungus were obtained. Containers were then prepared to house the fruit fly specimens, and various concentrations of spore solutions were formulated for the treatments. Additionally, an alternative diet for the melon fruit fly was prepared. The experimental units were established, and the treatments were applied to the fruit flies. Posttreatment procedures included surface sterilization of deceased fruit flies for postmortem analysis, which was confirmed by the observation of mummification in the melon fruit fly caused by Metarhizium anisopliae.

2.6.2 Data collection

1. The final mortality rate (%) was calculated as the proportion of dead fruit fly specimens at the end of the experiment relative to the acclimatized population, as follows:

$$\:Final\:Mortality\:Rate\:\left(\%\right)=\:\frac{total\:number\:of\:dead\:fruit\:fly}{acclimitized\:population}x\:100$$

2. Average time to mummification – The time (in days) until the onset of mummification was recorded for dead fruit fly specimens given the characteristic shrinkage due to the loss of moisture and nutrients caused by mycosis:

3. Lethal concentration analysis-The lethal concentrations (LC50 and LC99) of the tubli root extract were projected by regressing the common logarithm (log base 10) of the test concentrations against the probit value of the response percentage (mortality rate). Lethal concentration analysis is expressible using the probit model:

$$\:P=\:\alpha\:+\:\beta\:\left[{log}_{10}\left(Concentration\right)\right]$$

where,

$\:P=5+\:{{\Phi\:}}^{-1}\left(p\right)$ , given p = corrected mortality rate, and $\:{{\Phi\:}}^{-1}\left(p\right)$ is the probit value of the corrected mortality rate. The corrected mortality rate of the non-uniform population was computed based on Sun-Shepard’s formula:

$$\:Corrected\:mortality\:\%=$$

$$\:\:\frac{Mortality\:\%\:in\:treated\:plot\:\pm\:Change\:\:\%\:in\:control\:plot}{100\:\pm\:change\:\%\:in\:control\:plot\:}$$

where,

$$\:Change\:\%\:in\:control\:plot=$$

$$\:\left(\frac{n\:after\:treatment-n\:before\:treatment}{n\:before\:treatment}\right)$$

Given that,$\:n=insect\:population$

$$\:{\alpha\:}=\text{e}\text{s}\text{t}\text{i}\text{m}\text{a}\text{t}\text{e}\text{d}\:\text{v}\text{a}\text{l}\text{u}\text{e}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{i}\text{n}\text{t}\text{e}\text{r}\text{c}\text{e}\text{p}\text{t},$$

$$\:{\beta\:}=\text{e}\text{s}\text{t}\text{i}\text{m}\text{a}\text{t}\text{e}\text{d}\:\text{v}\text{a}\text{l}\text{u}\text{e}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{s}\text{l}\text{o}\text{p}\text{e}$$

4. Lethal time analysis-lethal timeframes (LT50 and LT99) of tubli root extract were projected by the same log-probit analysis as lethal concentration analysis by regressing the common logarithm (log base 10) of different time periods (in days) against the probit value of the response percentage (mortality rate per day).

5. The relative toxicity – relative toxicity was taken as the comparison between the lethal time 50 and the treatment levels employed and computed as follows:

$$\:Relative\:toxicity=\:\frac{{LT}_{50\left(treatment\right)}}{{Lt}_{50\left(control\right)}}\:x\:100$$

The fiducial limits (upper and lower) were then computed as

$$\:{R}_{lower}=\:\frac{{Lower\:limit}_{treatment\:}}{{Upper\:limit}_{control}},\:{R}_{upper}=\:\frac{{Upper\:limit\:}_{treatment\:}}{{Lower\:limit}_{control}}.$$

2.6.3 Statistical analysis

One-way analysis of variance (ANOVA) following a randomized complete block design was performed using STAR software for average mortality rates and average time to mummification of the five treatments. A comparison of means was performed at the 5% significance level using Scheffe’s post hoc test to determine significant differences between treatment means.

On the other hand, to determine lethal concentration levels (LC50 and LC99) and lethal time levels (LT50 and LT90), treatment means for mortality rate were corrected using the Sun-Shepard formula (given mortality data with a non-uniform population):

$$\:Corrected\:\%=\:\frac{Mortality\:\%\:in\:treated\:plot\:\pm\:Change\:\:\%\:in\:control\:plot}{100\:\pm\:change\:\%\:in\:control\:plot\:}$$

where,

$$\:Change\:\%\:in\:control\:plot=\left(\frac{n\:after\:treatment-n\:before\:treatment}{n\:before\:treatment}\right)$$

Given that,$\:n=insect\:population$

The corrected data were then subjected to log-probit analysis using LdP (lethal dose probit) line software. Mortality rates were converted to probit values and regressed against the log10 of the test concentrations to obtain equations or the lines that predict lethal concentrations, lethal time, and relative toxicity.

This study explored the effectiveness of spore solutions containing different concentrations of Metarhizium anisopliae on the mortality and mummification rates of Bactrocera cucurbitae, revealing the significant impact of concentration on both metrics. Mortality rates increased with increasing spore concentration, with a significant population reduction observed at 6.0×10⁵ CFU/mL. This concentration was necessary to induce a substantial decrease in the population, aligning with the findings of previous studies by Onsongo et al. (2022), Toledo-Hernandez et al. (2018), and Prince et al. (2024) which reported varying mortality rates depending on the dose and conditions. Similarly, the number of dilutions decreased as the spore concentration increased, with the fastest mummification occurring at the highest concentration, consistent with the findings of Indriyanti et al. (2018). The study also established a strong concentration response relationship, indicating that it would take approximately 2.65×10⁵ CFU/mL to reach 50% mortality, with higher concentrations required for greater mortality. Additionally, time response analysis revealed that the lethal time to 50% mortality (LT50) decreased with increasing spore concentration, which was similar to the findings of El-Gendy et al. (2022) and Hussein et al. (2018), suggested that M. anisopliae is a highly effective biocontrol agent. These findings suggest that under the experimental conditions employed, M. anisopliae can achieve significant insecticidal effects at relatively lower concentrations than those observed in other studies, making it a viable, eco-friendly alternative to chemical pesticides for managing B. cucurbitae populations in squash cultivation.

3.1 Mortality rate

Table 1

Effects of different concentrations of *Metarhizium anisopliae* on the mortality rate of fruit flies (from *Bactrocera*).
Treatment level (CFU/mL)		Mortality rate
M1 = 0 CFU/mL		46.33 ^d
M2 = 1.5 x 10⁵ CFU mL^− 1		58.33 ^cd
M3 = 3.0 x 10⁵ CFU mL^− 1		73.67 ^bc
M4 = 4.5 x 10⁵ CFU mL^− 1		85.67 ^ab
M5 = 6.0 x 10⁵ CFU mL^− 1		96.00 ^a
F test		**
P value		.000
%CV		7.42
Treatment level (CFU/mL)	Days to Mummification
M1 = 0 CFU mL^− 1	22.67 a
M2 = 1.5 x 10⁵ CFU mL^− 1	15.00 b
M3 = 3.0 x 10⁵ CFU mL^− 1	11.67 c
M4 = 4.5 x 10⁵ CFU mL^− 1	9.00 d
M5 = 6.0 x 10⁵ CFU mL^− 1	6.00 e
F test	**
P value	0.000
%CV	3.01

Means with the same letter are not significantly different according to Scheffe’s test at the 5% level.

3.2 Time to Mummification

Table 2

Effects of different concentrations of *Metarhizium anisopliae* on the number of days until mummification of the fruit fly (*Bactrocera*).
Treatment level (CFU/mL)	Days to Mummification
M1 = 0 CFU mL^− 1	22.67 a
M2 = 1.5 x 10⁵ CFU mL^− 1	15.00 b
M3 = 3.0 x 10⁵ CFU mL^− 1	11.67 c
M4 = 4.5 x 10⁵ CFU mL^− 1	9.00 d
M5 = 6.0 x 10⁵ CFU mL^− 1	6.00 e
F test	**
P value	0.000
%CV	3.01

Means with the same letter are not significantly different according to Scheffe’s test at the 5% level.

3.3 Concentration Response Relationships between Metarhizium anisopliae and the Mortality Rate

Table 3

Projected lethal concentrations of *Metarhizium anisopliae* at the 5% significance level.
Lethal Concentration		Slope ± (standard error)	Chi-Square	P value*
Response Percentage	Concentration (CFU/mL)
50	2.65 x 10⁵	3.242 ± 0.577	1.677	0.4325
90	6.59 x 10⁵
99	1.38 x 10⁶
* Chi-square test for goodness of fit: measures goodness of fit to the weighted regression line with p > .05 indicating a good fit of the data to the line

Time Response Relationships between Metarhizium anisopliae and Mortality Rate

Table 4

Time response relationships between different levels of *Metarhizium anisopliae* and mortality rate, and the corresponding toxicity indices relative to those of the control.
Treatment	LT50	Toxicity Index
M1 = 0 CFU mL^− 1	4.676	100.00 b
M2 = 1.5 x 10⁵ CFU mL^− 1	3.400	137.53 ab
M3 = 3.0 x 10⁵ CFU mL^− 1	2.735	170.97 ab
M4 = 4.5 x 10⁵ CFU mL^− 1	2.275	205.54 a
M5 = 6.0 x 10⁵ CFU mL^− 1	2.106	222.03 a

Indices with the same letter indicate overlapping fiducial limits at the 5% significance level, suggesting nonsignificant differences in toxicity.

The corresponding toxicity indices also showed that a concentration of at least 4.5×10⁵ CFU mL^− 1 would induce more than twice the toxicity level in the control (margin of 105 between M1 and M4) and may reach an index of 222.03 as the concentration approaches the M5 level (see Fig. 2). However, a comparison of the fiducial limits at the 5% significance level indicated that the toxicity index did not increase significantly before it reached the M4 level at 4.5×10⁶ CFU mL^− 1. Overall, these findings suggested that toxicity increases with increasing spore solution concentration.

The study demonstrated that Metarhizium anisopliae is a potent biocontrol agent against Bactrocera cucurbitae under in-vitro conditions, with higher concentrations leading to greater mortality and faster mummification of fruit flies. The required lethal concentration was notably lower than that in previous research, highlighting its potential as a sustainable alternative to chemical pesticides in squash cultivation. To build on these findings, further research is recommended to test the effectiveness of M. anisopliae under field conditions, assess its impact on nontarget organisms and ecosystems, and optimize its application for practical use. Collaborating with agricultural stakeholders will be essential for successful implementation in real-world scenarios.

Acknowledgments

The researcher expresses deep gratitude to his mother, Guiamila Mangansakan Utto; his father, Datu Mingao Salibo Utto; and his brothers and sisters for their encouragement, support, and assistance, all of which were instrumental in the completion of this paper. To his thesis adviser, Dr. Aurelio Ampo, whose profound understanding of the study and dedication to its development inspired the researcher to pursue this work; to Prof. Dwight L. Mendez of the Agronomy Department and Prof. Musa M. Dicolano of the Agricultural Extension Department for their professional critiques, suggestions, and reading materials that greatly contributed to the improvement of this document; to his batchmates, Prof. Jeanneflor S. Atong and Prof. Shiela Mae A. Dionaldo; and above all, to the Almighty Allah, to whom all blessings and achievements are owed.

Funding

This research was self-funded by the author, without any external financial support.

Conflicts of interest/competing interests

The authors declare no conflicts of interest or competing interests related to this research.

Ethics Approval

This study did not require formal ethics approval according to the guidelines of the university or the supervising professor, as it did not involve any procedures or data collection that typically necessitate ethical review.

Consent to Participate

This study did not involve human participants; therefore, no consent to participate was needed.

Consent for Publication

This study did not involve human participants; therefore, no consent for publication was needed.

Availability of Data and Material

Due to the nature of this short-term study, the data and materials used are not publicly available but can be obtained from the corresponding author under specific conditions.

Code availability

No custom code or software was developed or used in this short-term study.

Authors' Contributions

Author A designed the study and wrote the manuscript; Author B conducted the experiments and analyzed the data; Author C contributed to the data interpretation and manuscript revision. All the authors read and approved the final manuscript.

Onsongo SK, Mohamed SA, Akutse KS, Gichimu BM, Dubois T (2022) The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana for management of the melon fly Zeugodacus cucurbitae: Pathogenicity, horizontal transmission, and compatibility with cuelure. Insects 13(10):859. https://doi.org/10.3390/insects13100859
Toledo-Hernández RA, Toledo J, Sánchez D (2018) Effect of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) on food consumption and mortality in the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae). Int J Trop Insect Sci 38(3):254–260. https://doi.org/10.1017/S1742758418
Prince M, McKinnon AC, Leemon D, Sawbridge T, Cunningham JP (2024) Metarhizium spp. isolates effective against Queensland fruit fly juvenile life stages in soil. PLoS ONE 19(1):e0297341. https://doi.org/10.1371/journal.pone.0297341
Indriyanti DR, Damayanti IB, Setiati N, Maretta YA (2018) Mortality and tissue damage of Oryctes rhinoceros larvae infected by Metarhizium anisopliae. ARPN 13(6):2279–2286
El-Gendy IR, Zawrah MF, El-Banobi MI (2022) The virulence effect of Metarhizium anisopliae (Met.) and Beauveria bassiana (Bals.) fungi against the peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae). Egypt J Biol Pest Control 32(1):43. https://doi.org/10.1186/s41938-022-00525-3
Hussein MA, Khaled AS, Ibrahim AA, Soliman NA, Attia SH (2018) Evaluation of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae on peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae). Egyptian Academic Journal of Biological Sciences, F. Toxicology & Pest Control, 10(1), 59–68. https://doi.org/10.21608/EAJBSF.2018.17213
Hamzah AM, Mohsin A, Naeem M et al (2021) Efficacy of Beauveria bassiana and Metarhizium anisopliae (Ascomycota: Hypocreales) against Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) under controlled and open-field conditions on bitter gourd. Egypt J Biol Pest Control 31:144. https://doi.org/10.1186/s41938-021-00490-7

The authors declare no competing interests.

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Efficacy of Metarhizium anisopliae against Bactrocera cucurvitae under in-vitro conditions: A meta-analysis

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Preparation of Materials

2.1.1 Materials used

2.1.2 Methods

2.2 Media preparation

2.3 Purification of the Fungal Culture

2.4 Collection of fruit fly specimens

2.5 Treatment preparation

2.6 Treatment application

2.6.1 Processing

2.6.2 Data collection

2.6.3 Statistical analysis

3. Results and Discussion

3.1 Mortality rate

3.2 Time to Mummification

3.3 Concentration Response Relationships between Metarhizium anisopliae and the Mortality Rate

4. Conclusions and Recommendations

Declarations

References

Additional Declarations

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