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.