Field response of three Tephritid fruit ies to three food-based attractants and suppression of Bactrocera zonata (Saunders) using Mazoferm– Spinosad in guava ecosystem in Sudan

Fruit ies of the genus Bactrocera are the most damaging pests of horticultural crops, leading to severe economic losses hindered exportation. Bactrocera dorsalis (Hendel) and Bactrocera zonata (Saunders) were reported in Sudan in 2005 and 2011 respectively affecting most of the fruits and vegetables in Sudan threatening income of poor farmers. Only Male Annihilation Technique (MAT) is applied in Sudan to manage the two Bactrocera species. A led experiment was conducted to evaluate the response of B. dorsalis, B. zonata and Zeugodacus cucurbitae to three food-based attractants using McPhail traps in two sites in Gezira state, Sudan. Also, other trial was undertaken to determine the effect of spray of Mazoferm and Spinosad combination to control B. zonata. The results showed that food-based attractants lured both sexes of the above mentioned fruit ies and females represented (55-86%). At the rst site, B. zonata responded in high numbers to Mazoferm followed by Torula yeast and GF-120 respectively while it responded equally to the Mazoferm and Torula yeast in the second site. B. dorsalis responded positively to Mazoferm followed by Torula yeast and GF-120 while Z. cucurbitae was attracted to Mazoferm, GF-120 and Torula for each attractant respectively. Spray of Mazoferm combined with Spinosad signicantly reduced population of B. zonata (FTD) population and suppressed infestation level of guava fruits (fruit ies/Kg of fruits) when compared to unsprayed orchard. Bait Application Technique is an environmentally friendly approach that reduces infestation levels, lessen contamination and safeguard produce.


Introduction
Guava (Psidium guajava L.) belonging to the family Myrtaceae is one of the most important tropical fruit trees in the world enriching the diet of hundreds of millions of people with high nutritive and health values (Morton, 1987;El-Bulk et al, 1997). In Sudan, guava is a popular fruit representing source of income to farmers all around the year (Ali et al,. 2014). Guava fruits are produced all around Sudan and most are consumed locally. For its long fruiting period and softness, guava fruit are a highly preferred susceptible host to many insects and diseases and among them are fruit ies. Fruit ies are a group of insect belonging to the family Tephrtidae of the order Diptera with various genera of economic importance among them are Ceratitis, Zeugodacus, Dacus and Bactrocera.
Fruit ies are subjected to strenuous efforts worldwide regarding their control for their vast invasions, highly reproduction and huge damage they caused. Different types of protein hydrolysates baited in different types of traps or sprayed in combination with insecticides are widely used since a century ago for monitoring and controlling fruit ies ( (Ekesi and Tanga, 2016). Food-based attractants, caught both sexes of fruit ies but they are highly preferred by females because they are highly required by females for the development of the ovaries and to increase eggs maturity (Bateman and Morton 1981;Mazor et al., 1987, Epsky andHeath, 1998;Robacker and Flath, 1995;Robacker and Heath, 1996;Hull and Cribb, 2001). Other sources of protein such as ammonia which are released by human urine, chicken and duck feces was found as a potential attractant for Anastrepha spp (Hedström, 1988 andPin ˜ero et al., 2009) and three species of Ceratitis, four species of Bactrocera as well as two species of Dacus (Mahmoud et al. 2012a). Mahmoud et al. 2012a, b reported that, Nulure, Torula yeast, Mezoferm, human urine, water extract of mango, guava, apple and grape attracted B. dorsalis, C. cosyra, C. capitata, C. quinaria Dacus ciliates Loew, D. vertebratus Bezzi and Z. cucurbitae. Mazoferm E802 and Torula yeast were found very effective to attract B. dorsalis and captured more ies than the standard Nulure (Ekesi et al. 2014).
Field control of fruit ies using GF-120 combined with Spinosad is widely used since many years ago (Vayssières et al 2009). Recently, mixture of Mazoferm and Spinosad was found very effective in suppression population of B. invadens/ dorsalis in mango elds in Kenya (Ekesi et al. 2014). The use of Spinosad; an insecticide derived from the metabolites of the soil bacterium Saccharopolyspora spinosa, has proofed high mortality for different insect pests without affecting human health and natural enemies (Thompson et al. 2000).
The Peach Fruit Fly (PFF), Bactrocera zonata (Saunders) is an alien invasive pest of horticultural crops known to be native to Asia with a wide range of host plants. The pest was detected in central Sudan in 2011 (Salah et al. 2012) and since that time it became the key fruit y which displaced native species and competed strongly with the highly invasive B. dorsalis which infested Sudan in 2005 and spread to different states (Mahmoud, et al. 2020a).
Spatial distribution studies in Sudan, revealed the movement of B. zonata southward and eastward endangering the Republic of South Sudan, Ethiopia and Eretria respectively while temporally B. zonata existed all around the year due to the availability of its hosts (Mahmoud, et al., 2017a). Peach Fruit Fly was reported to attack more than 50 host plants and among them are peach, mango, guava, papaya, orange, grape fruit (edible hosts), Jujube and Ivy gourd (wild hosts) (Mahmoud et al. 2017). In Sudan, B. zonata infestation level of mango reached 15% while 100% loss was recorded in guava (Mahmoud et al. 2016).
B. zonata is a newly introduced pest to Sudan; no any studies were conducted to evaluate the effectiveness of different food-based attractants for its monitoring and suppression. Thus this study was conducted to evaluate the eld response of Bactrocera species specially B. zonata to three different foodbased attractants viz., Torula, Mazoferm and GF-120, and to determine the effectiveness of Mazoferm in combination with Spinosad in attract and kill tool to control this alien invasive fruit ies in guava ecosystem.

Suppression trials
Effect of Mazoferm combined with Spinosad on controlling fruit ies was evaluated for two consecutive fruiting seasons of guava (November 2015 -April 2016) and (October 2016 to January 2017) respectively at Fadasi area, Gezira State, Sudan.
Half hectare farm consisted of hundred guava trees was used to be treated with the combination of Mazoferm and Spinosad and same area with same number of trees was selected on a distance of 1 Km as untreated one. The spacing between trees was 6 X 6m. The two selected farms did not receive any control measures before and the trial was conducted with full cooperation of the growers.
Before starting the suppression trial, pre-spray assessment for the population of fruit ies was conducted through distributing 4 McPail traps equipped with 15 g of Torula yeast (Standard attractant) diluted in 300 ml of water. Traps were distributed 20 m apart from each other in the middle of each farm. Traps were settled as described above and the same procedure was applied after each spray to assess the performance of Mazoferm and Spinosad combination on reducing the population of fruit ies. Mazoferm E802Ð + Spinosad bait spray was applied for eight consecutive weeks to the orchard that was selected as (treated) for two successive seasons while the second orchard remained untreated (control). The application rate of Mazoferm E802 was 400 ml active ingredient per hectare with Spinosad (Tracer 4 EC; Dow Agro Sciences, Indianapolis, IN) at 100 g active ingredient per hectare at an output volume of 5 litres per hectare (50 ml per tree). The Mazoferm E802Ð Spinosad bait spray was applied as spot application using Knapsack sprayer to 1 m 2 of the canopy of every tree using a quadrate.
Caught fruit ies in traps were collected, preserved in 70% alcohol, sorted out, sexed and recoded as fruit y/trap/day, on other hand the level of infestation was assessed where ambient amount of fruits were collected from the trees and from the ground from the two farms. Collected fruit were weighed, kept on ventilated container on surface of 20 cm sterilized sand up to the emergence of puparia. Emerged puparia were transferred to other container on humidi ed sand up to the emergence of adult ies which were sorted out, sexed and recorded as fruit ies/ kg of fruit.

Statistical analysis
To evaluate the e cacy of food-bait attractants on trapping fruit ies, data of trapping ies/trap/day of each week was transformed using the formula (( +1) and the results of all weeks of each treatment for each site was combined and subjected to ANOVA statistical analysis and Tukey test was applied to separate the means between different treatments, weeks and the interaction between treatments and weeks.
For fruit ies suppression trial, T-test analysis (PROC TTEST) was conducted to determine the difference between populations of fruit ies, level of infestation and harvest loss % due to infestation by B. zonata for treated and control orchards. At Fadasi site, signi cant differences between the mean numbers of total daily captured ies of B. zonata per trap was recorded among weeks (Pr>F Week <0.0001) treatments (Pr>F Treatment < 0.0001), and interaction between treatments and weeks (Pr>F Week*Treatment <0.0001). The same trend of signi cance was also reported for females and males. All test products attracted a higher percentage of females and represented 67.2, 61.3 and 68.4% of total caught ies for Mazoferm, Torula and GF-120 respectively and it was obviously that traps equipped with water only didn't attract any fruit y ( Table 1).
G. El l At G. El l, the test baits attracted B. zonata, B. dorsalis and Z. cucurbitae in different numbers. Table 2 showed the results of response of B. zonata to different test attractants at this site, signi cant difference was observed in the mean number daily captures of B. zonata per trap among weeks, treatments and also among the interaction between treatments and weeks Pr>F Treatment <.0001, Pr>F Week <.0001 and Pr>F Week*Treatment <.0075. Statistically same number of B. zonata was attracted to both Torula and Mazoferm 1.2 and 1.1 while GF-120 attracted the least number 0.3 y/trap/day (Table  2.).

B. zonata
Sex wise, the catch of attractants was found signi cantly different in the mean numbers of males and/ or females and also between catches for different weeks and also the interaction between treatments and weeks (Pr>F Treatment <.0001, Pr>F Week <.0001 and Pr>F Week*Treatment<.0001 for both sexes.
Mazoferm and Torula attracted same number of males as well as same number females greater than that attracted to GF-120. The percentage of captured females for each attractant were 81.8, 75 and 66.7% to Mazoferm, Torula and GF-120 respectively.

B. dorsalis
The results indicated that, B. dorsalis responded positively to the test attractants and there was signi cant differences among the number of fruit ies for weeks (Pr>F week<.0001), treatments (Pr>F Treatment <.0001 and their interaction Pr>F week*Treatment<.0075. Mazofrm attracted the highest number of B. dorsalis followed by Torula yeast and G-F120 with 10.8, 4.4, 1.6 ies/trap/day respectively (Table 3). The same trend of attraction was observed for both females and males and the percentage of attracted females for the three test attractants were 68.5, 68.2, 56.3 % for Mazoferm, Torula and GF-120 respectively (Table 3).

Z. cucurbitae:
This fruit y was not found infesting guava but was attracted form cucurbits growing wildly around the farm of the study.
Z. cucurbitae was attracted positively to the three test products but its attraction to Mazoferm was greater than that to Torula and GF-120 which were statistically same.

Suppression trials:
Effect of bait spray on the population and level of infestation of B. zonata In the both seasons, according to the T-test results, the difference between the means of the daily caught B. zonata per trap for the two treatments is not equal to zero at 0.05 probability because the value is greater than Pr> T = 0.0502 in the rst season and Pr> T= 0.0029 in the second season respectively. The result in this study indicated that using Mazoferm + Spinosad is very effective to reduce population of B. zonata.
According to (Fig 1 and 3) the population of B. zonata was very high in both sites during the period before application of Mazoferm combined with Spinosad there were 30 FTD for control and > 20 FTD for the treatment. After application of the second spray of Mazoferm plus Spinosad, the population of B. zonata decreased drastically and due to the prolonging of spraying the population went down till it reached zero FTD and then started to increase slightly before the application of the last three sprays where the population decreased again to very low levels.
The T-Test for the level of infestations revealed signi cant difference between treated and untreated orchards (Pupa of B. zonata/kg of fruit) for the two consecutive seasons with (Pr > |t| 0.0005) and (Pr > |t| 0.0049) respectively. As indicated in (Fig 2 and 4), the number of pupa/Kg fruit of the untreated farm is greater than that of the treated farm for the two consecutive seasons. It is very clear that, the decrease of infestation levels in the rst farm is attributed to the application of the Mazoferm and Spinosad combination.

Discussion
Fruit ies are polyphagous pests and their control is very di cult unless there is integrated procedures. Different management techniques were used worldwide to reduce the devastating effect caused by fruit ies. Field sanitation, the use of natural enemies, applying of the male annihilation technique have been used widely for the suppression of tephritid fruit ies; Cornelius et al., (2000) ; Vargas et al., (2003); Mahmoud et al., (2012a) and Ekesi et al., (2014) reported that, the most important factor that affect the attractiveness of fruit ies is the type of protein. As demonstrated in the results of this study, the tested food based attractants were found potent to attract both sexes of B. zonata and other species of fruit ies with different levels due to type of attractant. Mazoferm and Torula were more effective than GF-120 on trapping B. zonata especially females in the two study sites. Nulure was excluded in this experiments because under Sudan condition a thick layer of fungus was found growning on the product which prohibit the emission of the attractant (Mahmoud et al., 2012a).
The high number of females attracted to the three protein hydrolysates in this study is in accordance with ndings of (Vargas et al., 2002) where he and others reported the positive response of females of C. capitata to USB ® yeast hydrolysate enzymatic, Mazoferm ® E802, Nu-Lure ® Insect Bait, or Provesta ® 621 autolyzed yeast extract) he added that, the protein-starved females responded more than protein-fed females.
Studies conducted by many authors regarding the attractiveness of different protein hydolystes to different species of fruit ies proofed that most of the baits are highly attractive to fruit ies but due to the high price of most of them, use Mazoferm because it was considered the cheapest (Moreno and Mangan, 1995).
With regard to its attractiveness, Ekesi et al., (2014) put a high emphasis on using Mazoferm to control B. dorsalis in Africa and from the results obtained the new invasive species (B. zonata) can be added to the list of fruit ies that are attracted to it.
The attractiveness of GF-120 in this study is very low when compared with Torula and Mazoferm. The same result was obtained by   Torula yeast was found to be very effective to attract B. dorsalis (Mahmoud etal 2012 andEkesi et al. 2014) but due to its high price it comes second to be recommended to be used for trapping fruit ies.
In Sudan, controlling fruit ies depend on the use of different pesticides and the use of the male annihilation technique with methyl eugenol for controlling B. dorsalis. Not any use of bait or spray of food attractant is practiced and according to cited literature regarding effectiveness of Mazoferm, its cheapness, availability as animal feed and due to its results obtained in this study, using Mazoferm as bait spray is considered as right choice for farmers for the mentioned reasons in addition to its environmental friendly characteristics. The obtained results regarding reduction of population of B. zonata are in agreement with that achieved by (Vayssie `res et al. 2009) who recorded 8% mango fruit infestation by B. dorsalis and C. cosyra using GF-120 (which is also a combination of Spinosad and a bait derived from Mazoferm E802) compared to 48% infestation in the control. Ekesi et al. 2009 also reported mango fruit infestation ranging from 28 to 30% by B. dorsalis in orchards receiving six treatments of GF-120 baits spray compared with 60% fruit infestation in an untreated mango orchard and Ekesi et al. 2014 reported that using Mazoferm in combination with Spinosad is cost effective and increase farmers pro tability.

Conclusion
In summary, results of this study revealed that, the three test baits were attractive to B. zonata, B. dorsalis and Z. cucurbitae when compared to water only. Mazoferm gained the highest attraction level to B. zonata and the other fruit ies with speci c attraction to females. Mazofem has the ability to reduce the population of fruit ies to zero level when sprayed in a combination with Spinosad and it increase the production signi cantly. Based on the above results of the existing study, Mazoferm can be used as costless attractant and in combination with Spinosad for controlling B. zonata in an Area-wide management strategy that can suppress y populations.

Declarations Competing interests
Authors would like to proof that there is no any competing of interest Yee W, Chapman P. Effects of GF-120 Fruit Fly Bait concentrations on attraction, feeding, mortality, and control of R Tables