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

Efficacy of Metarhizium anisopliae against Bactrocera cucurvitae under in-vitro conditions: A meta-analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of Metarhizium anisopliae against Bactrocera cucurvitae under in-vitro conditions: A meta-analysis Andal U. Salibo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5019233/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 × 10 5 CFU/mL was necessary for substantial population reduction, while the projected lethal concentrations for 50% and 99% mortality were approximately 2.65 × 10 5 CFU/mL and 1.38 × 10 5 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 Figures Figure 1 Figure 2 Figure 3 1. Introduction 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. 2. Materials and methods 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 × 10 5 CFU mL-1 M3 = 3.0 × 10 5 CFU mL-1 M4 = 4.5 × 10 5 CFU mL-1 M5 = 6.0 x 10 5 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. 3. Results and Discussion 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 6.0×10 5 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 2.65×10 5 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 10 5 CFU mL − 1 58.33 cd M3 = 3.0 x 10 5 CFU mL − 1 73.67 bc M4 = 4.5 x 10 5 CFU mL − 1 85.67 ab M5 = 6.0 x 10 5 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 10 5 CFU mL − 1 15.00 b M3 = 3.0 x 10 5 CFU mL − 1 11.67 c M4 = 4.5 x 10 5 CFU mL − 1 9.00 d M5 = 6.0 x 10 5 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 10 5 CFU mL − 1 15.00 b M3 = 3.0 x 10 5 CFU mL − 1 11.67 c M4 = 4.5 x 10 5 CFU mL − 1 9.00 d M5 = 6.0 x 10 5 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 10 5 3.242 ± 0.577 1.677 0.4325 90 6.59 x 10 5 99 1.38 x 10 6 * 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 10 5 CFU mL − 1 3.400 137.53 ab M3 = 3.0 x 10 5 CFU mL − 1 2.735 170.97 ab M4 = 4.5 x 10 5 CFU mL − 1 2.275 205.54 a M5 = 6.0 x 10 5 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 4.5×10 5 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 4.5×10 6 CFU mL − 1 . Overall, these findings suggested that toxicity increases with increasing spore solution concentration. 4. Conclusions and Recommendations 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. Declarations 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. References 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 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5019233","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348477530,"identity":"03096e5f-5707-4f3a-be6b-ddcdb014e82a","order_by":0,"name":"Andal U. Salibo","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1090-8381","institution":"Mindanao State University- Maguindanano","correspondingAuthor":true,"prefix":"","firstName":"Andal","middleName":"U.","lastName":"Salibo","suffix":""}],"badges":[],"createdAt":"2024-09-02 14:41:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-5019233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5019233/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63989416,"identity":"10a0d189-a49a-4491-9b95-2f217afaaa5b","added_by":"auto","created_at":"2024-09-04 15:05:13","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":708748,"visible":true,"origin":"","legend":"\u003cp\u003eThe use of culture media and mass production of \u003cem\u003eMetarhizium anisopliae \u0026nbsp;\u003c/em\u003eled to the assignment and application of treatments.\u003c/p\u003e\n\u003cp\u003ePreparation of media (a); initial purification of \u003cem\u003eM. anisopliae\u003c/em\u003e(b); preparation of slant cultures and mass production of \u003cem\u003eM. anisopliae \u003c/em\u003e(c); pure cultures of \u003cem\u003eM. anisopliae\u003c/em\u003e 1 week after inoculation (d); setup of containers for fruit fly specimens (e); treatment preparation for different levels of spore solution (f); setup of experimental units (g); surface sterilization of dead fruit flies for postmortem inspection (h); mummification of melon fruit flies due to \u003cem\u003eM. anisopliae\u003c/em\u003e (i).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5019233/v1/15974a070130939663df9b5d.jpeg"},{"id":63989415,"identity":"11df5277-f39f-44a5-bd23-e00fba3443b4","added_by":"auto","created_at":"2024-09-04 15:05:13","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39570,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration response Relationships between \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e and Fruit Fly Mortality.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5019233/v1/28af27447ffa104475abf6d6.jpeg"},{"id":63989417,"identity":"538d511b-fb4e-4b5b-8c7d-860eba686231","added_by":"auto","created_at":"2024-09-04 15:05:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66037,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the time response relationships of different concentrations of Metarhizium anisopliae spore solution and fruit fly mortality. These results appear to conform with those from the study of Onsongo et al., (2022), who noted that the LT50 of melon fly \u003cem\u003e(Zzeugodacus cucurbitae\u003c/em\u003e) was within 2.61-4.53 days. Notably, however, in their study, the time to mortality did not vary as much between male and female recipients but was heavily influenced by transmission, as donors died sooner than recipients. Furthermore, the authors applied \u003cem\u003eM. anisopliae \u003c/em\u003eon a dry basis, allowing insects to pass over a dry contaminating device, suggesting that there is possibility of exposure to higher doses of\u003cem\u003e M. anisopliae\u003c/em\u003e. This finding falls into perspective, because relatively the same periods of mortality are possible for lower concentrations of \u003cem\u003eM. anisopliae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5019233/v1/e7729ccaa1e1f0b5d783c032.jpeg"},{"id":63990135,"identity":"1446feb4-f976-47ac-a1d3-ec75e03b07d2","added_by":"auto","created_at":"2024-09-04 15:13:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1356477,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5019233/v1/3bcc1a8a-e112-4120-b79e-522bcce9e9aa.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEfficacy of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMetarhizium anisopliae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBactrocera cucurvitae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under in-vitro conditions: A meta-analysis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe cucurbit fruit fly, \u003cem\u003eBactrocera cucurbitae\u003c/em\u003e, 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 \u003cem\u003eB. cucurbitae\u003c/em\u003e is critical for protecting farmers' livelihoods and ensuring stable food production.\u003c/p\u003e \u003cp\u003eHistorically, chemical pesticides have been the primary tool used to combat \u003cem\u003eB. cucurbitae\u003c/em\u003e. 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).\u003c/p\u003e \u003cp\u003eEntomopathogenic 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 \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e, are eco-friendly, and no known pests develop resistance to them (Hadi et al., 2013). \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e 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 \u003cem\u003eB. cucurbitae\u003c/em\u003e in squash crops (Ekesi et al., 2002).\u003c/p\u003e \u003cp\u003eEvaluating the effectiveness of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e against \u003cem\u003eB. cucurbitae\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eThis study aimed to optimize pest management strategies by identifying the most effective concentrations and conditions for applying \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e under in-vitro conditions. This research seeks to contribute to the development of precise, eco-friendly, and sustainable methods for controlling \u003cem\u003eB. cucurbitae\u003c/em\u003e during squash cultivation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eThis experimental study was conducted from September to December 2023 in Mindanao State University-General Santos city.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of Materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Materials used\u003c/h2\u003e \u003cp\u003eThe materials used were as follows: (1) pure culture of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Methods\u003c/h2\u003e \u003cp\u003eFor 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:\u003c/p\u003e \u003cp\u003eM1\u0026thinsp;=\u0026thinsp;0 CFU/mL\u003c/p\u003e \u003cp\u003eM2\u0026thinsp;=\u0026thinsp;1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU mL-1\u003c/p\u003e \u003cp\u003eM3\u0026thinsp;=\u0026thinsp;3.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU mL-1\u003c/p\u003e \u003cp\u003eM4\u0026thinsp;=\u0026thinsp;4.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU mL-1\u003c/p\u003e \u003cp\u003eM5\u0026thinsp;=\u0026thinsp;6.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Media preparation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.3 Purification of the Fungal Culture\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo purify \u003cem\u003eM. anisopliae\u003c/em\u003e, 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\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 degrees Celsius. Any subsequent colonies that were observably pure and characteristic of \u003cem\u003eM. anisopliae\u003c/em\u003e 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\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Collection of fruit fly specimens\u003c/h2\u003e \u003cp\u003eTo 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Treatment preparation\u003c/h2\u003e \u003cp\u003eTo 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\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.6 Treatment application\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo apply the treatments, sterile 50 ml plastic spray bottles were used. Sample specimens were sprayed such that no dripping occurred.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Processing\u003c/h2\u003e \u003cp\u003eThe research process began with the preparation of culture media, followed by the purification and large-scale production of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e. 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 \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.6.2 Data collection\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e 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:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Final\\:Mortality\\:Rate\\:\\left(\\%\\right)=\\:\\frac{total\\:number\\:of\\:dead\\:fruit\\:fly}{acclimitized\\:population}x\\:100$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e 2. Average time to mummification \u0026ndash; 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:\u003c/p\u003e \u003cp\u003e3. 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:\u003c/p\u003e \u003cdiv id=\"Equb\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:P=\\:\\alpha\\:+\\:\\beta\\:\\left[{log}_{10}\\left(Concentration\\right)\\right]$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003ewhere,\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:P=5+\\:{{\\Phi\\:}}^{-1}\\left(p\\right)\\)\u003c/span\u003e \u003c/span\u003e, given p\u0026thinsp;=\u0026thinsp;corrected mortality rate, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\Phi\\:}}^{-1}\\left(p\\right)\\)\u003c/span\u003e\u003c/span\u003e is the probit value of the corrected mortality rate. The corrected mortality rate of the non-uniform population was computed based on Sun-Shepard\u0026rsquo;s formula:\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:Corrected\\:mortality\\:\\%=$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:\\:\\frac{Mortality\\:\\%\\:in\\:treated\\:plot\\:\\pm\\:Change\\:\\:\\%\\:in\\:control\\:plot}{100\\:\\pm\\:change\\:\\%\\:in\\:control\\:plot\\:}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere,\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:Change\\:\\%\\:in\\:control\\:plot=$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equf\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equf\" name=\"EquationSource\"\u003e\n$$\\:\\left(\\frac{n\\:after\\:treatment-n\\:before\\:treatment}{n\\:before\\:treatment}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eGiven that,\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:n=insect\\:population\\)\u003c/span\u003e\u003c/span\u003e\u003cdiv id=\"Equg\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equg\" name=\"EquationSource\"\u003e\n$$\\:{\\alpha\\:}=\\text{e}\\text{s}\\text{t}\\text{i}\\text{m}\\text{a}\\text{t}\\text{e}\\text{d}\\:\\text{v}\\text{a}\\text{l}\\text{u}\\text{e}\\:\\text{o}\\text{f}\\:\\text{t}\\text{h}\\text{e}\\:\\text{i}\\text{n}\\text{t}\\text{e}\\text{r}\\text{c}\\text{e}\\text{p}\\text{t},$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equh\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equh\" name=\"EquationSource\"\u003e\n$$\\:{\\beta\\:}=\\text{e}\\text{s}\\text{t}\\text{i}\\text{m}\\text{a}\\text{t}\\text{e}\\text{d}\\:\\text{v}\\text{a}\\text{l}\\text{u}\\text{e}\\:\\text{o}\\text{f}\\:\\text{t}\\text{h}\\text{e}\\:\\text{s}\\text{l}\\text{o}\\text{p}\\text{e}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e 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).\u003c/p\u003e \u003cp\u003e5. The relative toxicity \u0026ndash; relative toxicity was taken as the comparison between the lethal time 50 and the treatment levels employed and computed as follows:\u003c/p\u003e \u003cdiv id=\"Equi\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equi\" name=\"EquationSource\"\u003e\n$$\\:Relative\\:toxicity=\\:\\frac{{LT}_{50\\left(treatment\\right)}}{{Lt}_{50\\left(control\\right)}}\\:x\\:100$$\u003c/div\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe fiducial limits (upper and lower) were then computed as\u003cdiv id=\"Equj\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equj\" name=\"EquationSource\"\u003e\n$$\\:{R}_{lower}=\\:\\frac{{Lower\\:limit}_{treatment\\:}}{{Upper\\:limit}_{control}},\\:{R}_{upper}=\\:\\frac{{Upper\\:limit\\:}_{treatment\\:}}{{Lower\\:limit}_{control}}.$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003e2.6.3 Statistical analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eOne-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\u0026rsquo;s post hoc test to determine significant differences between treatment means.\u003c/p\u003e \u003cp\u003eOn 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):\u003cdiv id=\"Equk\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equk\" name=\"EquationSource\"\u003e\n$$\\:Corrected\\:\\%=\\:\\frac{Mortality\\:\\%\\:in\\:treated\\:plot\\:\\pm\\:Change\\:\\:\\%\\:in\\:control\\:plot}{100\\:\\pm\\:change\\:\\%\\:in\\:control\\:plot\\:}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere,\u003cdiv id=\"Equl\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equl\" name=\"EquationSource\"\u003e\n$$\\:Change\\:\\%\\:in\\:control\\:plot=\\left(\\frac{n\\:after\\:treatment-n\\:before\\:treatment}{n\\:before\\:treatment}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eGiven that,\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:n=insect\\:population\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThis study explored the effectiveness of spore solutions containing different concentrations of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e on the mortality and mummification rates of \u003cem\u003eBactrocera cucurbitae\u003c/em\u003e, 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\u0026times;10\u003csup\u003e5\u003c/sup\u003e 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. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), Toledo-Hernandez et al. (2018), and Prince et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) 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. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The study also established a strong concentration response relationship, indicating that it would take approximately 2.65\u0026times;10\u003csup\u003e5\u003c/sup\u003e 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. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Hussein et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), suggested that \u003cem\u003eM. anisopliae\u003c/em\u003e is a highly effective biocontrol agent. These findings suggest that under the experimental conditions employed, \u003cem\u003eM. anisopliae\u003c/em\u003e 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 \u003cem\u003eB. cucurbitae\u003c/em\u003e populations in squash cultivation.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Mortality rate\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of different concentrations of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e on the mortality rate of fruit flies (from \u003cem\u003eBactrocera\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTreatment level (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMortality rate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eM1\u0026thinsp;=\u0026thinsp;0 CFU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.33 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eM2\u0026thinsp;=\u0026thinsp;1.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.33 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eM3\u0026thinsp;=\u0026thinsp;3.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.67 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eM4\u0026thinsp;=\u0026thinsp;4.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.67 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eM5\u0026thinsp;=\u0026thinsp;6.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eF test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e%CV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment level (CFU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDays to Mummification\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u0026thinsp;=\u0026thinsp;0 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e22.67 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2\u0026thinsp;=\u0026thinsp;1.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e15.00 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM3\u0026thinsp;=\u0026thinsp;3.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e11.67 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4\u0026thinsp;=\u0026thinsp;4.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e9.00 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM5\u0026thinsp;=\u0026thinsp;6.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e6.00 e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%CV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMeans with the same letter are not significantly different according to Scheffe\u0026rsquo;s test at the 5% level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Time to Mummification\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of different concentrations of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e on the number of days until mummification of the fruit fly (\u003cem\u003eBactrocera\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment level (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDays to Mummification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u0026thinsp;=\u0026thinsp;0 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.67 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2\u0026thinsp;=\u0026thinsp;1.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.00 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM3\u0026thinsp;=\u0026thinsp;3.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.67 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4\u0026thinsp;=\u0026thinsp;4.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.00 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM5\u0026thinsp;=\u0026thinsp;6.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.00 e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%CV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMeans with the same letter are not significantly different according to Scheffe\u0026rsquo;s test at the 5% level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Concentration Response Relationships between Metarhizium anisopliae and the Mortality Rate\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProjected lethal concentrations of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e at the 5% significance level.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eLethal Concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSlope \u0026plusmn; (standard error)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChi-Square\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP value*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse Percentage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (CFU/mL)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.65 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3.242\u0026thinsp;\u0026plusmn;\u0026thinsp;0.577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1.677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.4325\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.59 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.38 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e* Chi-square test for goodness of fit: measures goodness of fit to the weighted regression line with p\u0026thinsp;\u0026gt;\u0026thinsp;.05 indicating a good fit of the data to the line\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTime Response Relationships between\u003c/b\u003e \u003cb\u003eMetarhizium anisopliae\u003c/b\u003e \u003cb\u003eand Mortality Rate\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTime response relationships between different levels of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e and mortality rate, and the corresponding toxicity indices relative to those of the control.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLT50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eToxicity Index\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u0026thinsp;=\u0026thinsp;0 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.00 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2\u0026thinsp;=\u0026thinsp;1.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e137.53 ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM3\u0026thinsp;=\u0026thinsp;3.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e170.97 ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4\u0026thinsp;=\u0026thinsp;4.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e205.54 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM5\u0026thinsp;=\u0026thinsp;6.0 x 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e222.03 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIndices with the same letter indicate overlapping fiducial limits at the 5% significance level, suggesting nonsignificant differences in toxicity.\u003c/p\u003e \u003cp\u003eThe corresponding toxicity indices also showed that a concentration of at least 4.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). 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\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Overall, these findings suggested that toxicity increases with increasing spore solution concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions and Recommendations","content":"\u003cp\u003eThe study demonstrated that \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e is a potent biocontrol agent against \u003cem\u003eBactrocera cucurbitae\u003c/em\u003e 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 \u003cem\u003eM. anisopliae\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003eAcknowledgments\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eFunding\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis research was self-funded by the author, without any external financial support.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eConflicts of interest/competing interests\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe authors declare no conflicts of interest or competing interests related to this research.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eEthics Approval\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis 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.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eConsent to Participate\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis study did not involve human participants; therefore, no consent to participate was needed.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eConsent for Publication\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis study did not involve human participants; therefore, no consent for publication was needed.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eAvailability of Data and Material\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eDue 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.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eCode availability\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNo custom code or software was developed or used in this short-term study.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eAuthors\u0026apos; Contributions\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAuthor 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOnsongo SK, Mohamed SA, Akutse KS, Gichimu BM, Dubois T (2022) The entomopathogenic fungi \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e and \u003cem\u003eBeauveria bassiana\u003c/em\u003e for management of the melon fly \u003cem\u003eZeugodacus cucurbitae\u003c/em\u003e: Pathogenicity, horizontal transmission, and compatibility with cuelure. Insects 13(10):859. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects13100859\u003c/span\u003e\u003cspan address=\"10.3390/insects13100859\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToledo-Hern\u0026aacute;ndez RA, Toledo J, S\u0026aacute;nchez D (2018) Effect of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (Hypocreales: Clavicipitaceae) on food consumption and mortality in the Mexican fruit fly, \u003cem\u003eAnastrepha ludens\u003c/em\u003e (Diptera: Tephritidae). Int J Trop Insect Sci 38(3):254\u0026ndash;260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S1742758418\u003c/span\u003e\u003cspan address=\"10.1017/S1742758418\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrince M, McKinnon AC, Leemon D, Sawbridge T, Cunningham JP (2024) \u003cem\u003eMetarhizium spp\u003c/em\u003e. isolates effective against Queensland fruit fly juvenile life stages in soil. PLoS ONE 19(1):e0297341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0297341\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0297341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIndriyanti DR, Damayanti IB, Setiati N, Maretta YA (2018) Mortality and tissue damage of \u003cem\u003eOryctes rhinoceros\u003c/em\u003e larvae infected by \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e. ARPN 13(6):2279\u0026ndash;2286\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Gendy IR, Zawrah MF, El-Banobi MI (2022) The virulence effect of \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (Met.) and \u003cem\u003eBeauveria bassiana\u003c/em\u003e (Bals.) fungi against the peach fruit fly, \u003cem\u003eBactrocera zonata\u003c/em\u003e (Saunders) (Diptera: Tephritidae). Egypt J Biol Pest Control 32(1):43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s41938-022-00525-3\u003c/span\u003e\u003cspan address=\"10.1186/s41938-022-00525-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussein MA, Khaled AS, Ibrahim AA, Soliman NA, Attia SH (2018) Evaluation of entomopathogenic fungi, \u003cem\u003eBeauveria bassiana\u003c/em\u003e and \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e on peach fruit fly, \u003cem\u003eBactrocera zonata\u003c/em\u003e (Saunders) (Diptera: Tephritidae). \u003cem\u003eEgyptian Academic Journal of Biological Sciences, F. Toxicology \u0026amp; Pest Control, 10\u003c/em\u003e(1), 59\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21608/EAJBSF.2018.17213\u003c/span\u003e\u003cspan address=\"10.21608/EAJBSF.2018.17213\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamzah AM, Mohsin A, Naeem M et al (2021) Efficacy of \u003cem\u003eBeauveria bassiana\u003c/em\u003e and \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e (Ascomycota: Hypocreales) against \u003cem\u003eBactrocera cucurbitae\u003c/em\u003e (Coquillett) (Diptera: Tephritidae) under controlled and open-field conditions on bitter gourd. Egypt J Biol Pest Control 31:144. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s41938-021-00490-7\u003c/span\u003e\u003cspan address=\"10.1186/s41938-021-00490-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"there was no primay institution that sponsored only the university professor as required me to do the research as part of my study.","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bactrocera cucurbitae, Metarhizium anisopliae, mortality-rates, fruit-fly","lastPublishedDoi":"10.21203/rs.3.rs-5019233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5019233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the effectiveness of the entomopathogenic fungus \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e in controlling the cucurbit fruit fly \u003cem\u003eBactrocera cucurbitae\u003c/em\u003e 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 \u003cem\u003eM. anisopliae\u003c/em\u003e 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 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL was necessary for substantial population reduction, while the projected lethal concentrations for 50% and 99% mortality were approximately 2.65 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL and 1.38 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU/mL, respectively. These findings suggest that \u003cem\u003eM. anisopliae\u003c/em\u003e is a highly effective biocontrol agent against \u003cem\u003eB. cucurbitae\u003c/em\u003e, and is a viable alternative to chemical pesticides. Further research is recommended to explore field applications and assess long-term ecological impacts.\u003c/p\u003e","manuscriptTitle":"Efficacy of Metarhizium anisopliae against Bactrocera cucurvitae under in-vitro conditions: A meta-analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 15:05:08","doi":"10.21203/rs.3.rs-5019233/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d64f9eff-27d6-415b-b173-cedd7d93d742","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36973235,"name":"Agronomy"}],"tags":[],"updatedAt":"2024-09-04T15:05:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-04 15:05:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5019233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5019233","identity":"rs-5019233","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}