Tunisian Journal of Plant Protection Vol. 10, No. 2, 2015...Messgo-Moumene, D. Merzouk, Z. Houmani, and K. Moumene. (Algeria) 131. Comparison of Four Trapping Systems for the Control

Tunisian Journal of Plant Protection Vol. 10, No. 2, 2015


Tunisian Journal of Plant Protection (SIS‐Indexed;SIS‐Impacted)

http://www.iresa.tn/tjpp

Volume 10, Number 2, December 2015

Contents 95. Improvement of the Production of Entomopathogenic Proteases of Bacillus

thuringiensis. K. Ennouri, R. Ben Ayed, H. Ben Hassen, H. Azzouz, and M.A. Triki. (Tunisia)

105. Comparison between Insecticide Effects of Wild and Cultivated Rosemary Essential Oils on Stored Product Insects. S. Khalil, K. Zarrad, A. Ben Hammouda, Y. Ayed Lakhal, S. Rguez, W. Tayeb, A. Laarif, and I. Chaieb. (Tunisia)

117. Valorization of Three Plant Species of Arid Areas in Biological Control of

the Desert Locust Schistocerca gregaria. S. Messgo-Moumene, D. Merzouk, Z. Houmani, and K. Moumene. (Algeria)

131. Comparison of Four Trapping Systems for the Control of the Medfly

Ceratitis capitata. M. Tlemsani and S. Boulahia-Kheder. (Tunisia) 141. A Contribution to the Knowledge of Platypus cylindrus in Tunisian Cork

Oak Forests. A. Bellahirech, L. Bonifácio, M. Lurdes Inácio, E. Sousa, and M.L. Ben Jamâa. (Tunisia/Portugal)

151. Efficacy of Diatomaceous Earth Based Formulation on Date Moth

Ectomyelois ceratoniae. S. Yousfi and J. Mediouni-Ben Jemâa. (Tunisia)

Photo of the cover page: Platypus cylindrus (Courtesy Amani Bellahirech)


In Loving Memory

of

Prof. Mohamed Chérif

)2015-1963( األستاذ محمد الشريفالمرحوم

Our colleague the late Prof. Mohamed Chérif was a simple man coming from a

modest family. After getting the Engineer Diploma in Horticulture from the Higher Agronomic Institute of Chott-Mariem (ISA ChM) with the mention “very good” and obtaining the Presidential Prize in 1987, he left the homeland for Canada to continue his studies. In 1993, he went back home with the PhD degree in Plant Pathology and was recruited by the National Agronomic Institute of Tunisia (INAT, University of Carthage). Very devoted in his work, he climbed the professional scale to reach in 2008 the rank of University Professor. In the same year, he was nominated as General Director of the Technical Center of Citrus (CTA) at the Cap-Bon region in the North-East of Tunisia. Along with his new position responsibility, he maintained all his activities inside the Plant Protection Department of INAT by teaching, researching and advising master theses and doctorate dissertations, until his early desperation.

Prof. Mohamed Chérif had a gold heart who knows listening without judging, confident in the human spirit, serious and frank, living for his work and his family. Sometimes obstinate, but he was always self-confident, combative and very active. He was specially combative against his disease. Supported and accompanied in his last battle by his family and the medical entourage, he was an example of serenity. Aware about the degree of his disease, its evolution and his near end, he left the life without regret, without remorse.

TJPP


In Loving Memory

of

Prof. Mohamed El Mahjoub األستاذالمرحوم

)2015- 1944( محجوبمحمد ال

Horticultural and Plant Protection Community has recently lost one of its eminent and famous scientists. Sudden Mohamed El Mahjoub's death on 11 December 2015 came as great shock to his wife Leila, his children Monia, Hajer, and Walid, to his brother Mokhtar, to his colleagues and students. Prof. M. El Mahjoub was a simple man coming from a modest family in Benihassen composed of his father Ali, his mother Nejia and his brother.

Prof. M. El Mahjoub made a substantial and significant contribution to the plant pathology of horticultural crops during his varied and productive 39 year research career. He was first recruited on 1971 as principal engineer at the National Institute of Agronomic Research of Tunisia (INRAT), then he left Tunisia for France (1981-1985) where he obtained his degree of Doctor of Science in Natural Sciences (specialty Plant Pathology) on 1985 from the Faculty of Sciences and Techniques at the University of Western Brittany, Brest, France. Back to Tunisia, he moved from INRAT to the Higher Agronomic Institute of Chott-Mariem (ISA-ChM) (Sousse) where he worked from April 1986 to September 2009. He taught General Mycology, Fungal Taxonomy, Phytopathology, Epidemiology, and Biological control of fungal diseases courses to benefit of engineering and pre-doctoral master's degrees. He also supervised many PhD, Masters of Science and Projects of End of Engineering Studies at ISA-ChM.

El Mahjoub's research spanned plant diseases caused mainly by soilborne and seedborne fungi and bacteria. He left a legacy in the scientific literature of more than 150 publications and many technical papers which almost invariably connected detailed studies on detection, characterization and biology of fungal pathogens to their chemical, genetic and biological control, useful for academic training, research studies and profession.

Administratively, Prof. M. El Mahjoub served as Director of Plant Protection Department, Director of Studies at ISA-ChM, then as Coordinator of the Regional Pole of Agricultural Research-Development in the Centre-East before being nominated as General Director of ISA-ChM (University of Sousse). During 2003-2008, he coordinated a research unit entitled "Biotechnology and Protection of Horticultural Species". He coordinated the Committee of Theses and Masters in Agronomic Sciences, specialty Plant Protection and Environment at ISA-ChM.

Prof. M. El Mahjoub will be much missed by his family, his research team, his friends and his scientific associates throughout Tunisia. For me, he was the teacher, the supervisor, the leader, the scientific summit, the friend, and the spiritual Father.

Prof. M. El Mahjoub, you are gone but your memory will live in our hearts and minds forever.

Prof. Mejda Daami-Remadi


Acknowledgement of Reviewers

Tunisian Journal of Plant Protection gratefully appreciates the volunteer help of reviewers which evaluate, with care and competence, papers proposed for publication in the 10th Volume, 2015. They are listed below in recognition of their contribution.

Abdellaoui, Khemais, ISAChM, Tunisia Al-Heneidy, Ahmed, UC, Egypt Ammar, Mohamed, INAT, Tunisia Auger, Philippe, INRA, France Banni Mohamed, ISAChM, Tunisia Barbouche, Naïma, INAT, Tunisia Belkadhi, Mohamed Sadok, IRAM/CTCPG, Tunisia Benazoun, Abdessalam, IAVHassenII-CHA, Morocco Ben Halima-Kamel, Monia, ISAChM, Tunisia Ben Hamouda, Mohamed Lahbib, INAT, Tunisia Ben Jamâa, Mohamed Lahbib, INRGERF, Tunisia Boughalleb-M'Hamdi, Naïma, ISA-ChM, Tunisia Boukhris-Bouhachem, Sonia, INRAT, Tunisia Boulahya-Khedher Synda, INAT, Tunisia Braham, Mohamed, CRRHABChM, Tunisia Chaabane-Boujnah, Hanène, INAT, Tunisia Chaieb, Ikbal, CRRHABChM, Tunisia Chérif Ameur, FST, Tunisia Chermiti, Brahim, ISAChM, Tunisia Dellagi, Alia, AgroParisTech, France Gharbi, Naceur, IO, Tunisia Glida-Gnidez, Habiba, ESAK, Tunisia Grissa-Lebdi, Kaouthar, INAT, Tunisia Hajlaoui, Mohamed Rabeh, INRAT, Tunisia Ippolito, Antonio, UB, Italy, Kharrat, Mohamed, INRAT, Tunisia Laarif, Asma, CRRHABChM, Tunisia Mazih, Ahmed, IAV HassenII, Morocco Mediouni-Ben Jamâa, Jouda, INRAT, Tunisia Mekki, Mounir, ISAChM, Tunisia Namsi, Ahmed, CRRAO, Tunisia Nasraoui, Bouzid, INAT, Tunisia Pasqualini, Edison, UB, Italy Rhouma, Ali, IO/IRESA, Tunisia Schiffers, Bruno, ULg, Belgium Triki, Mohamed Ali, IO, Tunisia Zappalà, Lucia, UC, Italy Ziedan, El-Sayed H.E., NRC, Egypt

Special thanks go to Prof. Malik Laamari (University of Betna, Batna, Algeria) and

Prof. Brahim Chermiti (ISA Chott-Mariem, University of Sousse, Tunisia) for writing for Tunisian Journal of Plant Protection the Guest Editorial in Issues No. 1 and No. 2 of Vol. 10 (2015), respectively.


Guest Editorial

SYNPIP 2015: Think IPM for a Sustainable Agriculture

Integrated pest management (IPM) should be supported by social and economic authorities allowing long term viable and sustainable agriculture.

The willingness of convinced agronomists is however not sufficient for the development of IPM which implies in addition that producers, distributors, retailers, economists and consumers should be motivated and willing to act together.

For instance, the distributor must strive to decrease the risks associated with chemical control by promoting new and specific environment friendly molecules with low persistence and minor risks to human health and beneficial organisms.

In a framework of IPM, the farmer should focus more precisely on the compatibility of the different control methods used and their side effects on the environment. The mastership of non target side effects consists in stabilizing populations' levels of different organisms acting in the agro-system in a way where cultivated plant, auxiliary fauna, pests and diseases externalize their optima (not maxima) to guarantee maximum productivity (not production) of the crop. The farmer should be able to accurately assess non intentional impacts of chemicals used in the crop on the plant itself (phytotoxicity, residues,…) as well as on the agricultural pests and their natural enemies. Taking into account the tropic and geographic relationships

among organisms, the agronomist should be able to establish the control practices that will allow defining the best possible management procedure of the agro-system including modifications on the plant, auxiliary fauna and the environment levels.

From an economic perspective, the economist should be capable to evaluating time, equipments and running costs necessary for the monitoring of the requirements of IPM.

Finally, if the consumer is convinced that IPM products have superior quality, he should be ready to pay the price. This means that governmental structures jointly with professional groups should set up production units of biological control agents with emphasis on local strains which are well adapted to local climatic conditions. Moreover, efficient and recognized control structures should be created allowing a rapid analysis of products commercialized under the IPM label.

The consumer is also invited to modify his habits to finally understand that, for example, a slightly blemished fruit is not necessarily unsuitable for consumption.

In this complex context, agronomic researchers have a major role in conceiving sustainable solution of phytosanitary constrains while keeping in mind the economic and ecologic background of our country.


SYNPIP 2015 in its first edition was a great opportunity to present innovative and sustainable solutions for our farmers, so share ideas and to discuss future perspectives in relationship with

the Tunisian plant protection policies. It was also an important occasion for many young agronomists to meet their peers, to exchange expertise and to build future collaborations and partnerships.

Prof. Brahim Chermiti, ISA Chott-Mariem,

University of Sousse, Tunisia

Tunisian Journal of Plant Protection 95 Vol. 10, No. 2, 2015

Improvement of the Production of Entomopathogenic Proteases of Bacillus thuringiensis

Karim Ennouri and Rayda Ben Ayed, Centre de Biotechnologie de Sfax, PB 1177, 3018 Sfax, Tunisia, Hanen Ben Hassen, Laboratoire de Physiques, Mathématiques et Applications, Faculté des Sciences de Sfax, Université de Sfax, Tunisia, Hichem Azzouz, Centre de Biotechnologie de Sfax, PB 1177, 3018 Sfax, Tunisia, and Mohamed Ali Triki, Laboratoire Ressources et Amélioration Génétiques de l’Olivier, du Pistachier et de l’Amandier, Institut de l’Olivier, Université de Sfax, Tunisia _________________________________________________________________________ ABSTRACT Ennouri, K., Ben Ayed, R., Ben Hassen, H., Azzouz, H, and Triki, M.A. 2015. Improvement of the production of entomopathogenic proteases of Bacillus thuringiensis. Tunisian Journal of Plant Protection 10: 95-103. Bacillus thuringiensis (Bt) is a spore forming bacterium that produces an insecticidal crystalline protein (ICP) making it a successful biopesticide. The ICPs are also referred to as Cry proteins and contain delta-endotoxins which cause mortality of insects belonging to different orders such as Diptera, Coleoptera and Lepidoptera. Bt subspecies produce also proteases which affect their entomotoxicity toward targeted insects as proteolytic activities are strongly associated with Bt crystal protein. Statistical techniques were applied to optimize the fermentation medium composition for the production of bacterial proteases in shake-flask cultures. An experimental statistical design was performed to evaluate the effects of different components on the concentration of proteolytic enzymes. Preliminary results showed that starch and K2HPO4 are able to increase Bacillus sp. protease production. In order to obtain more accurate results, interactions between ingredients were also studied. In concordance with coefficient of determination (R²) value, considered as the most important criterion for predictive model success, the best model demonstrated the effect of interactions and allowed precise prediction of protease production. In fact, K2HPO4, KH2PO4, MgSO4, FeSO4 as well as Soybean meal × starch and MnSO4 × starch interactions were shown to have active action on protease production. This method revealed that limited number of experiments allowed useful results. Keywords: Bacillus thuringiensis, coefficient of determination, production, protease, statistical design __________________________________________________________________________

The development of insecticide resistance and the growing awareness of ecological and environmental problems caused by classical chemical insecticides

Corresponding author: Karim Ennouri Email: [email protected]

Accepted for publication 09 December 2015

have spurred research into biologically-based approaches and environmentally benign alternatives. Among the compounds identified throughout this research are proteins isolated from microbes, predators, and plants and exhibiting specific toxicity to insect pests.

The most prominent insecticidal proteins are the Bacillus thuringiensis crystal (Cry) delta-endotoxin proteins. These proteins are produced by Bt by


forming crystalline inclusions during sporulation (20). Cry proteins combine to receptors and incorporate into the midgut epithelial cell membranes of insects by forming pores and subsequently causing cellular lysis and midgut epithelium lethal damage (4).

Proteases are enzymes that beak peptide bonds. They are classified based on their structure or properties of their active sites. Bacillus proteases are predominantly extracellular and can be concentrated in the fermentation medium. The main objective of many studies focused on screening proteases is the increase of their activity levels (11). Proteases are logical candidates for use as insecticidal agents. Proteolytic enzymatic activity can target and destroy essential proteins and tissues in insect pests, or other animals, leading to their mortality.

Besides, proteases have evolved in plants to protect themselves against herbivorous insects. For insect microbial pathogens, proteases habitually play considerable pathogenicity role towards host insects. Thus, selection of the accurate organisms may play a key role in high yielding of desirable enzymes.

On the other hand, it is a well-known fact that extracellular protease production by microorganisms is greatly influenced by media components, especially carbon or nitrogen sources (14), and metal ions (1). Isolation and characterization of new promising strains, using carbon and nitrogen sources, are a continuous process (2). In order to obtain the optimal reaction conditions, it is generally required to study numerous parameters.

The use of mathematical methods and experimental designs enable the determination of the influence of several parameters with minimum trials. Likewise, these methods could be applied in numerous fields. The basic approach

has been derived by statisticians (19). This methodology allows researcher to collect information with minimum experiences, mainly in process optimization.

Therefore, we present in this work, an original pattern of medium ingredients’ optimization for particular bacterial enzymes production by using experiment matrices. This study was performed using a strain exhibiting high proteolytic activities for which some fermentation conditions such as carbon and nitrogen sources and effects of various metal ions on protease production were investigated. A modified medium was defined for high protease production. MATERIALS AND METHODS Bacillus thuringiensis strain.

The strain used in the present study belonged to B. thuringiensis subsp. kurstaki and was kindly provided by Dr. Hichem Azzouz (Centre de Biotechnologie de Sfax, Tunisia). This strain is known for its toxicity against Lepidoptera insects (8) and was routinely grown on Luria Bertani (LB) agar medium and stored at 4°C until use. Microorganism and cultivation media.

For fermentation medium, an economic complex medium (9) was used containing the following components (g/l): starch, 25; soybean meal, 20; MgSO4, 0.3; MnSO4, 0.02; FeSO4, 0.02; K2HPO4, 1; KH2PO4, 1. The CaCO3 (20 g/l) was added for keeping pH stability. All media used in this study were adjusted to pH 7.0 ± 0.01 before medium autoclaving. Culture conditions.

For pre-inoculum preparation, a loopful of Bt grown on LB medium was used to inoculate a 3 ml of sterilized LB medium and incubated in a rotary shaker


(New Brunswick incubator shaker model INNOVA 44, USA), at 30°C and 200 rpm overnight (14-18 h). For inoculum preparation, 250 ml Erlenmeyer flasks containing 50 ml of LB medium were inoculated with 1% (v/v) of the pre-inoculum and incubated in a rotary shaker at 30°C and 200 rpm for 6 h. The volume of culture inoculum was calculated on the basis of a final absorbance of 0.15 measured at 600 nm. The 250 ml shake flasks including 25 ml of complex low-cost medium were incubated with estimated inoculum volume. In such media, the initial optical density (OD) was not measured after inoculation but calculated on the basis of the OD measured in the inoculum. Samples taken regularly from the incubated cultures were subjected to microscope observation. The fermentation process was considered as ended when 90% (or more) of the Bt cells were destroyed, liberating the crystals and spores. Protease assay.

The proteolytic activity of the enzyme was determined using casein as a substrate. Casein was dissolved in 0.1 M Tris-HCl buffer (pH 7.0) at a concentration of 1%. The assay mixture consisted of enzyme solution suitably diluted with 0.1 M Tris-HCl buffer (pH 9.0). The reaction mixture was incubated at 60°C for 20 min and the reaction was terminated by the addition of 5 ml of 5% trichloroacetic acid (TCA) and then centrifuged at 5000 g for 10 min to discard the resulting precipitate. Protease activity was estimated as liberated tyrosine from the supernatants according to an adapted Lowry method. One unit of enzyme activity was defined as the amount of enzyme resulting in the liberation of 1 µg of tyrosine per min at 60°C under reaction conditions. The obtained values were the mean of

triplicate runs of two different experiments. Statistical experimental design.

The significance of the various media constituents towards protease production was further tested using Plackett-Burman statistical experimental designs (16). This method is based on the existence of Hadamard matrices, which are square matrices of order N with entries at two levels, +1 and −1. These matrices are orthogonal such that for each column the number of +1 is equal to the number of −1. This experimental design is suitable for screening the effect of a large number of factors in an experiment and ideal for the determination of main effects. The design assumes that there are no interactions between different medium constituents, Xi, in the range of variables under consideration and a linear approach is considered sufficient for screening:

where Y is the estimated target function or response and ˇi are the regression coefficients. With this experimental design, N factors can be screened with only N+1 experiments and screening up to 100 variables (5) is possible with the help of this design. In the present work, the effect of 7 medium’s components on protease production was assessed.

All the experiments were performed in triplicate and at two different times and the responses considered for analysis represent mean of these responses. The effect of each variable was determined by the standard equation:

where R(H) represents all responses when component was at high levels, R(L) represents all responses when component was in low levels, N is the total number of runs.


The standard error (SE) of the concentration effect is the square root of the variance of an effect and the significance level (P-value) of each concentration effect is determined using student’s t-test:

where E(xi) is the effect of the variable xi.

The coefficient of determination (R²) describes the degree of co-linearity between simulated and measured data. Similarly, R² describes the proportion of the variance in measured data explained by the model. R² ranges from 0 to 1, with higher values indicating less error variance, and typically values greater than 0.5 are considered acceptable (18). Although R² have been widely used for

model evaluation, these statistics are oversensitive to high extreme values (outliers) and insensitive to additive and proportional differences between model predictions and measured data (13). RESULTS

Table 1 represents the independent variables and their respective high and low concentrations used in the optimization study, whereas Table 2 represents the experimental design for 12 trials with two concentration levels for each variable, which was followed by the optimization of medium’s components for protease production. The variables X1–X7 represent the medium constituents.

Table 1. Medium components (variables) and their respective high and low concentration levels used in experimental design

Variable Medium component

Lower level (g/l)

Higher level (g/l)

X1 KH2PO4 1.5 0.5 X2 K2HPO4 1.5 0.5 X3 MgSO4 0.1 0.5 X4 MnSO4 0 0.002 X5 FeSO4 0 0.002 X6 Starch 25 35 X7 Soybean meal 20 30

Table 3 represents the results of statistical experiment with respect to protease production, the effect, standard error, t and P-value (confidence level) of each component. The components were screened at the confidence level of 95% on the basis of their effects. If the component showed significance at or above 95% confidence level and its effect was negative, it indicated that the component was effective in protease

production but the amount required was lower than the indicated as low (-) concentration in studied statistical experiment. If the effect was positive, a higher concentration than the indicated high value (+) concentration was required during further optimization studies. According to Table 2, protease concentrations ranged between 565 IU/ml and 2386 IU/ml.


Table 2. Protease concentrations according to experimental design

The confidence levels of all

components were below 95% in protease production and hence, were considered insignificant (Table 3). Starch, considered as principal source of carbon, and K2HPO4 enhanced protease production whereas soybean meal (source of nitrogen), KH2PO4, MnSO4, FeSO4 and MgSO4 inhibited proteolytic enzyme production.

In addition, the regression is not liner (according to Table 3) because the amount of F-statistic for determination of effective factors on protease production is 0.32 with the significance level of P = 0.908. The results presented in Table 3 show the regression model with R2 = 0.362 and adjusted R2 = 0 which means that constructed model explains two plausible cases: the first one is that there is no linear competitive advantage between the seven components and the second is that the production is totally influenced by other factors.

The relationships between nutrients having positive effect on protease production have been investigated using a linear multivariate approach. Thus, in order to increase secretion of proteolytic enzymes by Bt strain, our major focus is to investigate the relationships between parameters having positive effect on protease production by this strain. In this study, only starch and K2HPO4 allowed an improvement of proteolytic enzyme production. Relationships between starch and most significant ingredients will be more studied. Then the regression equation, with adequate interactions, becomes: Proteases (IU/ml) = 17254 + 434 KH2PO4 + 181 K2HPO4 + 1574 MgSO4 - 172945 MnSO4 + 18920 FeSO4 - 535 Starch - 621 Soybean meal - 63.9 (MgSO4 × Starch) + 6040 (MnSO4 × Starch) + 20,1 (starch × soybean meal).

The variable with confidence level above 95% is considered as a significant factor. According to Table 4,

KH2PO4 (g/l)

K2HPO4 (g/l)

MgSO4 (g/l)

MnSO4 (g/l)

FeSO4 (g/l)

Starch (g/l)

Soybean meal (g/l)

Proteases (IU/ml)

1.5 1.5 0.1 0.02 0 25 20 1986 ± 58

0.5 1.5 0.5 0.02 0 35 30 1922 ± 64

0.5 0.5 0.5 0.02 0.02 25 30 565 ± 31

1.5 1.5 0.1 0.02 0.02 25 30 1204 ± 55

1.5 0.5 0.5 0.02 0 35 20 1394 ± 47

0.5 0.5 0.1 0 0 25 20 1822 ± 26

1.5 1.5 0.5 0 0.02 35 20 1145 ± 28

1.5 0.5 0.5 0 0 25 30 1047 ± 34

1.5 0.5 0.1 0 0.02 35 30 2063 ± 57

0.5 1.5 0.5 0 0.02 25 20 2386 ± 44

0.5 0.5 0.1 0.02 0.02 35 20 1563 ± 20

0.5 1.5 0.1 0 0 35 30 1445 ± 36


soybean meal, starch and MnSO4 had significantly affected the protease production independently for a significance level α = 0.05. In addition, only two 2-factor interaction terms,

namely MnSO4 × starch and Soybean × starch, among these variables also significantly affect protease production for α = 0.05.

Table 3. The prediction model from regression analysis

Table 4. The prediction model (with interactions) from regression analysis

Predictor Coefficient SE t P Rank Constant 17253.6 612.8 28.16 0.023 KH2PO4 434.47 35.08 12.39 0.051 6 K2HPO4 181.44 35.08 5.17 0.122 8 MgSO4 1574.3 602.1 2.61 0.233 10 MnSO4 -172945 12042 -14.36 0.044 5 FeSO4 18920 1754 10.79 0.059 7 Starch -534.52 21.31 -25.09 0.025 1 Soybean meal -620.66 24.08 -25.77 0.025 1 MgSO4 × starch -63.92 19.89 -3.21 0.192 9 MnSO4 × starch 6039.8 397.7 15.19 0.042 4 Soybean × starch 20.0795 0.7955 25.24 0.025 1 S = 45.9256 R² = 99.9% R²(adj) = 99.2% Analysis of variance

Source DF SS MS F P Regression 10 2903780 290378 137.67 0.066 Residual Error 1 2109 2109 Total 11 2905889

Predictor Coefficient SE t P Rank Constant 2377 1694 1.4 0.233 KH2PO4 -144.1 393.1 -0.37 0.732 5 K2HPO4 272.2 393.1 0.69 0.527 2 MgSO4 -676.6 982.7 -0.69 0.529 3 MnSO4 -10616 19655 -0.54 0.618 4 FeSO4 -5748 19655 -0.29 0.784 6 Starch 8.69 39.31 0.22 0.836 7 Soybean meal -34.15 39.31 -0.87 0.434 1 S = 680.853 R² = 36.2% R²(adj) = 0.0%

Analysis of variance Source DF SS MS F P Regression 7 1051643 150235 0.32 0.908 Residual Error 4 1854245 463561 Total 11 2905889


Data given in Table 4 show the regression model with R2 = 0.999 and adjusted R2 = 0.992. Furthermore, the signs of the parameters in the model are examined. Positive signs mean that the production value will go in the same direction as the parameter, and negative ones imply opposite effect. Therefore, enzyme secretion by Bt will be improved with the increase of concentrations of FeSO4, MgSO4, KH2PO4, K2HPO4, and interactions starch × soybean meal and starch × MnSO4. DISCUSSION

The goodness of fit of a linear regression model is universally measured by using the simple coefficient of determination R². This evaluation has a tendency to rise and to reach its upper limit as the number of explanatory variables in the model increases. Such an uncomfortable feature is controlled to a large extent through application of a correction for degrees of freedom (DF). This leads to another measure of the goodness of fitted model known as adjusted R². Both the high adjusted R² value and the close to zero P-value in the analysis of variance (ANOVA) (3, 6, 15) show that this model has a satisfactory goodness of fit.

The use of R² also called the multiple correlation coefficient, is well established in classical regression analysis (17). It represents the proportion of variance explained by the regression model which makes it useful as a success measure of predicting the dependent variable from the independent ones. Generally, R² value is accepted as a

standard criterion for the predictive success of the models (10).

Multivariate models are able to assess a large number of variables and interrelations and are therefore in defining and predicting more successfully biological processes such as protease production.

Results shown in Table 4 demonstrated that proteolytic enzyme secretion by Bt will be increased due to the enhancement of FeSO4, MgSO4, KH2PO4, and K2HPO4 concentrations and also due to interactions starch × soybean meal and starch × MnSO4. In fact, several medium components such as nitrogen and carbon sources influence the metabolic and the biochemical behavior of the microbial strain and subsequently the metabolite production pattern, such as protease secretion (7, 12). However, starch and MgSO4 interaction seems to decrease the production of proteases. Based on the obtained results, positive individual effect of starch was converted in negative effect when interactions were applied.

In planning a factorial experiment, prior knowledge may suggest that some interactions are potentially important. However, results from the current study clearly demonstrated that interaction effects allowed significant improvement of the model accuracy. The focal question is how to select a factorial design that allows investigation of key effects and the set of interactions specified by the researcher. In this study, R² has been considered as the most important criterion for best selection of interactions between medium components.

__________________________________________________________________________ RESUME Ennouri K., Ben Ayed R., Ben Hassen H., Azzouz H. et Triki, M.A. 2015. Amélioration de la production des protéases entomopathogènes par Bacillus thuringiensis. Tunisian Journal of Plant Protection 10: 95-103.


Bacillus thuringiensis (Bt) est une bactérie formant des spores et qui produit un cristal protéique insecticide (CPI) qui en fait un biopesticide mondialement réputé. Les CPI sont également appelés protéines Cry et contiennent les delta-endotoxines, qui causent la mortalité des insectes appartenant à différents ordres tels que Diptère, Coléoptère et Lépidoptère. Les sous-espèces de Bt produisent des protéases qui affectent leur entomotoxicité envers les insectes cibles car les activités protéolytiques sont étroitement associées aux protéines insecticides de Bt. Des techniques statistiques ont été appliquées pour optimiser la composition du milieu de fermentation pour la production de protéases bactériennes dans les cultures en flacons agités. Un plan statistique expérimental a été utilisé pour évaluer les effets de différents composants du milieu de culture sur la concentration d’enzymes protéolytiques. Les résultats préliminaires ont montré que l’amidon et K2HPO4 sont impliqués dans l’augmentation la production de protéases chez Bacillus sp. Afin d’obtenir des résultats plus détaillés, les interactions entre les ingrédients ont été étudiées. En concordance avec les valeurs du coefficient de détermination (R²), considéré comme le critère le plus important pour la validation des modèles prédictifs, le meilleur modèle a démontré l'effet des interactions et peut ainsi prédire avec précision la production de protéases. En effet, K2HPO4, KH2PO4, MgSO4, FeSO4, les interactions (tourteau de soja × amidon) et (MnSO4 × amidon) ont une action active sur la production des protéases. Cette méthode a révélé qu’un nombre limité d'expériences a permis d’obtenir des résultats utiles. Mots clés: Bacillus thuringiensis, coefficient de détermination, modèle, production, protéase _________________________________________________________________________

ملخصإنتاج البروتياز تحسين . 2015. محمد علي التريكيوھشام عزوز وحنان بن حسان و رائدة بن عيادوكريم النوري،

.الممرضة للحشرات Bacillus thuringiensisبكتيريا للTunisian Journal of Plant Protection 10: 95-103.

البروتين ( امبيدا بيولوجي التي تنتج لألبواغالمكونة ت نوع من البكتيريا يھ) Bt) Bacillus thuringiensis البكتيريا

- تحتوي على الدلتا التيCry أيضا البروتينات CPIتسمى . عالميةالشھرة الذو ))CPI( حشراتمبيد للالكريستال ال ثنائيات األجنحة مختلفة مثلرتب والتي تتسبب في القضاء على الحشرات من ) delta-endotoxines(توكسين أوندو

)Diptera(، األجنحة ات غمديو)Coleoptera ( األجنحة وقشريات)Lepidoptera .( ينتج النوع الفرعيBt لبروتياز ا المبيدةBt مرتبطة ارتباطا وثيقا ببروتيناتعالية في الحد من تكاثر الحشرات ألن أنزيمات البروتياز النجاعة الذو لتخمير إلنتاج األنزيم البروتيني وسط ا تطبيق أساليب إحصائية من شأنھا الترفيع في جودةتم اإلطارفي ھذا . لحشراتل

لتخمير على تركيز وسط اتم استخدام مثال إحصائي تجريبي لتقييم تأثيرات مختلف عناصر . قنيناتالبكتيري في . Btلدى ن على زيادة اإلنتاج البروتيني ايساعد K2HPO4وقد أظھرت النتائج األولية أن النشا و. واإلنزيماتوتينات البر

الذي يعتبر أھم R²دراسة التفاعالت بين جميع المكونات حيث اتضح أن المؤشر توللحصول على نتائج أكثر تفصيال، تمإنتاج بأنه أفضل نموذج يبين تأثير التفاعالت وبالتالي يمكن التنبؤ بدقة ت ثبُ فالمعايير للتحقق من صحة النماذج التنبؤية،

FeSO4 و MgSO4 و KH2PO4 و K2HPO4: اذكرھ األتيوتبعا للنتائج المتحصل عليھا فان المكونات . البروتيازھذه الطريقة . ات البروتينيةلھا تأثير فعال على إنتاج اإلنزيم) النشا× MnSO4(و ) النشا× الصوجا/الصويا(والتفاعالت

.كشفت أن عددا محدود من التجارب يمكن أن يثمر نتائج ناجعة

Bacillus thuringiensis ،R²إنتاج، بروتياز، نموذج، :يةاحتكلمات مف__________________________________________________________________________ LITERATURE CITED 1. Adinarayana, K., Ellaiah, P., and Prasad, D.S.

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Comparison between Insecticide Effects of Wild and Cultivated Rosemary Essential Oils on Stored Product Insects Sabrine Khalil, Khaoula Zarrad, Amel Ben Hammouda, Yasmine Ayed-Lakhal, UR13AGR09-Production Horticole Intégrée au Centre-Est Tunisien, Centre Régional des Recherches en Horticulture et Agriculture Biologique, Chott Mariem, Université de Sousse, Tunisia, Safa Rguez, Laboratoire des Substances Actives, Centre de Biotechnologie de Borj-Cedria, Technopôle Borj Cédria, Tunisia, Wafa Tayeb, Laboratoire de Biochimie, Faculté de Médecine de Monastir, Université de Monastir, Tunisia, Asma Laarif, UR13AGR09-Production Horticole Intégrée au Centre-Est Tunisien, Centre Régional des Recherches en Horticulture et Agriculture Biologique, Chott Mariem, Université de Sousse, Tunisia, and Ikbal Chaieb, Laboratoire de Protection des Végétaux, Institut National de la Recherche Agronomique de Tunisie, Université de Carthage, Tunisia __________________________________________________________________________ ABSTRACT Khalil, S., Zarrad, K., Ben Hammouda, A., Ayed Lakhal, Y., Rguez, S., Tayeb, W., Laarif, A., and Chaieb I. 2015. Comparison between insecticide effects of wild and cultivated rosemary essential oils on stored product insects. Tunisian Journal of Plant Protection 10: 105-115. This study was conducted to evaluate the insecticidal potential of Rosmarinus officinalis essential oils (wild and cultivated plants) against two stored product insects i.e. Tribolium castaneum and Trogoderma granarium. GC-MS analyses showed that both essential oils contain 4 major constituents which are L-camphor, 1,8-cinerole, L-borneol, and bornyl acetate. Fumigant toxicity tests showed that the two rosemary essential oils were more toxic to T. granarium than to T. castaneum adults. The corresponding LC50 values of wild and cultivated rosemary essential oils against T. castaneum were 65.5 μl/l air and 180 μl/l air, respectively, in contrast to 19.75 μl/l air and 18.75 μl/l air recorded towards T. granarium adults. Pest repellent activity was found to be dependent upon essential oil concentration and exposure time. The essential oil from wild rosemary plants was more repulsive against adults of T. castaneum than that from cultivated ones. Applied at the dose 0.25 µl/cm2, repellency achieved was of about 65 and 45% after 120 min of exposure to essential oils from wild and cultivated rosemary, respectively. These results suggested that R. officinalis essential oils may be potentially active in controlling the two stored product insects. Keywords: Essential oil, fumigant, repellency, Rosmarinus officinalis, Tribolium castaneum, Trogoderma granarium __________________________________________________________________________ Corresponding author: Sabrine Khalil Email: [email protected] Accepted for publication 01 October 2015

The khapra beetle, Trogoderma granarium (Coleoptera, Dermestidae), is among the most feared stored grain pests worldwide (4). It is a serious pest of


stored grains and cereal products in many warm and arid climates (4, 6). Likewise, the red flour beetle, Tribolium castaneum (Coleoptera, Tenebrionidae), is also one of the most important cosmopolitan pest insects leading to great damage on stored grains and products throughout the world (11).

The control of these pests in storage systems mainly depends on fumigants (9). However, chemical control has been associated with dangers like environmental pollution, human toxicity, development of insecticide resistance and adverse effects on non-target organisms (23, 25, 27). Essential oils and their derivatives are recognized as an alternate means of controlling many harmful insects, and being rapidly degradable in the environment and harmless to non-target organisms (22).

Insecticidal activities of essential oils against stored product pests were cited in several studies. In fact, essential oil of Artemisia herba alba were found to be toxic to Oryzaephilus surinamensis and T. castaneum (3). The essential oils from Eucalyptus camaldulensis, E. intertexta and E. sargentii were found to be able to control three major stored-product beetles namely Callosobruchus maculatus, Sitophilus oryzae and T. castaneum (18). Furthermore, the insecticidal activity of the essential oil of the rhizomes of Acorus calamus used at different rates (30, 50 and 70 μl per 1150 ml glass jars) was shown to be effective against T. granarium infesting wheat grains (17). Rosemary (Rosmarinus officinalis) oil has been traditionally used as a medicine for colic, nervous disorders and painful menstruation (7, 13). Rosemary oil is relatively effective against insect and mite pests. It has been

demonstrated that the aromatic vapor of rosemary oil exhibited ovicidal and larvicidal effects toward several stored product pests (20, 24).

The aim of this study was to determine the chemical composition of R. officinalis (wild and cultivated) essential oils and to assess their insecticidal potential against two stored-product insects i.e. T. castaneum and T. granarium. MATERIALS AND METHODS Plant material.

Cultivated R. officinalis plants were harvested during February from an organic rosemary crop of the Regional Center of Research on Horticulture and Organic Agriculture of Chott-Mariem (CRRHAB), Tunisia. Wild R. officinalis plants were collected from the ‘Hmadda’ in the region of Akouda-Sousse, Tunisia. Extraction of essential oil.

The essential oils were extracted from fresh leaves of wild and cultivated rosemary plants by hydrodistillation using a Clevenger apparatus. The obtained essential oils were stored in a refrigerator at 4°C before being used in the bioassays. GC-MS analysis of the essential oils.

The GC-MS analyses were performed using an HP-5972 mass spectrometer with electron impact ionization (70 eV) coupled with an HP-5890 series II gas chromatograph. An HP-5MS capillary column (30 m × 0.25 mm coated with 5% phenyl methyl silicone, and 95% dimethyl polysiloxane, 0.25 µm film thicknesses) was used. The oven temperature was programmed to rise from 50 to 240°C at a rate of 5°C/min. The transfer line temperature was 250°C.


Helium was used as carrier gas with a flow rate of 1.2 ml/min and a split ratio of 60:1. Scan time and mass range were 1 s and 40e300 m/z, respectively. Essential oil volatile compounds were identified by calculating their retention index (RI) relative to (C9-C18) n-alkanes (Analytical reagents, Labscan, Ltd, Dublin, Ireland) and data for authentic compounds available in the literature and in our data bank. Insect rearing.

T. castaneum and T. granarium adults were obtained from the rearing colony initiated in the laboratory of entomology at the Regional Center of Research on Horticulture and Organic Agriculture of Chott-Mariem (CRRHAB), Tunisia. Insects were reared on wheat flour diet. The rearing conditions were: 28 ± 1°C, 60% RH and photoperiod of L: D 16 h: 8 h.

Fumigant toxicity bioassays.

The insecticidal activity of the tested essential oils against T. castaneum and T. granarium adults was assessed by vapor-phase toxicity bioassay using closed container method. Groups of 10 insects were released into the bottom of a plastic container (40 ml). Paper discs were treated with different concentrations of the essential oils i.e. 25, 50, 100 and 200 μl/l air and 5, 12.5, 25 and 50 μl/l air were tested against T. castaneum and T. granarium, respectively. Control insects were kept under the same conditions without any treatment.

Mortality was determined 24 h after treatment. When no leg or antennal movements were observed, insects were considered as dead. The mortality was calculated using the Abbott correction

formula (1). All bioassays were performed in 5 replications. These bioassays were designed to determine the median effective dose causing 50% of mortality (LC50) and the effective dose causing 90% of mortality (LC90) using Probit analyses (10). Repellency test.

Repellent activity of rosemary leaf essential oils were evaluated against T. castaneum (this species is more suitable in repellency test than T. granarium). We used an area preference method according to Jilani and Saxena (15). Two concentrations of the tested materials (0.12 and 0.25 μl) were dissolved into 500 μl of acetone, respectively. Whatman filter papers (2 cm in diameter) were cut in half and were treated with prepared solutions or with 500 μl of acetone uniformly as possible. After drying for 10 min, treated and control half discs were taped together. Twenty T. castaneum individuals were released in the center of the filter paper in Petri dishes which were then covered. The number of insects present on the control (C) and treated (T) areas was recorded after 15, 30, 60 and 120 min. Percentage repellency (PR) was calculated as follows: PR = ((Nc-Nt)/(Nc+Nt)) × 100, where Nc: Number of insects present in the untreated area after the exposure interval, and Nt: Number of insects present on the treated area after the exposure interval. Five replications were used for each concentration. The mean number of insects on the treated portion of the filter paper was compared to the number recorded on the untreated portion. Results were presented as the mean of percentage repellency. Statistical analysis.


Data are presented as means. One-way analysis of variance was performed for data using Statistical Package for Social Sciences (SPSS, Chicago, III; version 11.0). The Duncan’s Multiple Range test was applied to detect significant differences of mortality among concentrations at P ≤ 0.05 level. Probit analysis (10) was used to estimate LC50 and LC90 values. RESULTS Essential oil yield.

Yield of essential oil extracted from R. officinalis leaves varied depending on nature of plants used for extraction. In fact, the essential oil from wild rosemary was yielded at 0.305% in contrast to 0.053% obtained from cultivated one. Essential oil composition.

Table 1 summarizes the composition of wild and cultivated

rosemary leaf essential oils. According to the GC-MS results, 55 compounds have been identified in the essential oil from wild rosemary leaves where the major compounds detected were L-camphor (16.29%), 1,8-cinerol (16.21%), bicyclo[3.1.1]hept-3-en-2-one (14.86%), bornyl acetate (14.54%), L-borneol (6.02%), bicyclo[3.1.1]heptan-3-one (4.63%), 1-methyl-2-(1-methyl (4.01%) and β-caryophyllene (3.59%) are major. However, 65 compounds were detected in essential oils from cultivated rosemary leaves and the major compounds were 1,8-cinerol (16.27%), L-camphor (15.72%)%), L-borneol (14.05%), α-terpinolene (8.20%), L-linalool (6.69%), benzen, 1-methyl-2-(1-methylethyl (3.57%), camphene (3.52%), delta-3-carene (3.28%), 1,2-benzenedicarboxylic (2.68%), nopol (2.46%), terpinene-4-ol (1.95%), caryophylene oxide (1.29%) and bornyl acetate (1.21%).

Table 1. Chemical composition of essential oil from wild and cultivated rosemary leaves

N° Compound RT (min) Wild rosemary Cultivated rosemary

1 β-Pinene 5.20 0.52 1.70 2 α-Pinene 5.36 - 2.09 3 Verbenene 5.44 0.39 - 4 Myrcene 6.04 - 1.32 5 Delta-3-Carene 6.06 3.28 - 6 1,8-Cineol 6.95 16.27 16.21 7 Trans- β-Ocimene 7.30 - 0.08 8 γ-Terpinene 7.53 7.53 0.71 9 γ-Terpinene 7.70 0.41 - 10 1-Methyl-2-(1-methyl 8.01 - 4.01 11 Benzen, 1-methyl-2-(1-methylethyl 8.13 3.57 - 12 Terpinolene 8.22 0.20 0.74 13 1(7), 3, 8-o-Menthariene 9.71 0.06 0.06 14 Camphene 9.88 - 0.07 15 Bicyclo[2.2.1]heptan-2 10.28 - 0.06 16 Cis-3-Hexenol 10.52 10.52 0.14 17 1-Methyl-4-(1-methylethen 11.06 - 0.37 18 Filifolone 11.11 1.42 -


19 1-Octen- 3- ol 11.49 0.37 20 Trans-sabinene Hydrate 11.56 - 0.38 21 α-Campholene aldehyde 12.06 - 0.06 22 Cyclopentene 12.16 0.36 - 23 L-Camphor 12.68 15.72 16.29 24 Bicyclo[3.1.1]heptan-3-one 13.05 - 4.63 25 L-Linalool 13.15 6.69 - 26 1-β-Pinene 13.26 - 0.11 27 Bornyl Acetate 13.74 1.21 14.54 28 Terpinene-4-ol 14.05 1.95 - 29 β-Caryophyllene 14.09 - 3.59 30 1-Methoxyl-2-methyllidenecyclopropan 14.19 - 0.66 31 Trans-carpophyllene 14.26 0.76 - 32 Verbenol 14.42 0.68 - 33 Bicyclo[3.1.1]hept-2-ene-2-carboxa 14.44 - 0.31 34 3,6-Octadienoic acid, 3,7-dimethyl 14.63 - 0.35 35 Benzeneacetaldehyde 14.66 0.48 - 36 Sabinyl acetate 14.78 - 0.23 37 Estragole 15.11 - 1.11 38 Sabinol 15.17 1.20 - 39 α-Humulene 15.29 - 0.34 40 2-Cyclohexen-1-one 15.41 0.62 - 41 Myrthenylacetate 15.50 - 1.12 42 Bicyclo[3.1.1]hept-3-en-2-one 15.87 - 14.86 43 α-Terpinolene 15.90 8.20 - 44 L-Borneol 16.08 14.05 6.02 45 γ-Compholenol 16.65 - 0.39 46 Delta-Cadienne 16.70 1.06 - 47 1,4-Cyclooctadienne 16.90 - 0.08 48 Spiro[5.5]undec-1-ene 17.00 0.18 - 49 α-Campholene aldehyde 17.20 1.09 0.36 50 Myrthenol 15.52 17.52 1.20 51 Nopol 17.65 2.46 1.53 52 Trans –(+)-Carveol 17.66 - 0.18 53 2-Cyclohexen-1-one 17.83 0.57 - 54 Para-Cymen-8-ol 17.95 0.41 - 55 5,9-Undecadien-2-one 18.03 0.41 - 56 Geraniol 18.29 - 0.37 57 Camphene 18.47 3.52 0.54 58 Piperiteone 19.06 0.95 0.25 59 Cis Jasmone 19.25 - 0.10 60 Bensenemethanol 19.50 - 0.12 61 7-Oxabicyclo[4.1.0]heptane, 1-meth 19.77 - 0.07 62 Carophylene oxide 19.91 1.29 0.60 63 Chrysanthenone 20.14 - 0.42 64 Methyl Eugenol 20.25 1.13 - 65 Spiro[2.4]heptane, 4-methylene-(c 20.38 - 0.06 66 Ocimene 20.43 0.15 - 67 12-Oxabicyclo[9.1.0]dodeca-3,7-die 20.76 0.42 0.10 68 12-Oxabicyclo[9.1.0]dodeca-3,7-die 20.69 - 0.10 69 4-Methyl-2-(3-methyl-2-butenyl)-fu 20.90 - 0.08 70 4-Lethyl-2-(3-methyl-2-butenyl)-fu 20.94 0.40 - 71 Naphtalene 21.05 0.11 - 72 2-Trideuteromethoxy-3-Methylpyrazi 21.31 - 0.13 73 1-β-Pinene 21.53 - 0.06


74 Cis-3-Hexenyl Benzoate 21.83 0.08 - 75 9-Aristolen-1.alpha.-ol 22.16 0.11 - 76 Phenol, 2-methyl-5-(1-methylethyl) 22.34 - 0.08 77 Eugenol 22.35 0.36 - 78 Bicyclo[4.4.0]Dec-1-En

22.49 0.11 -

79 Phenol 22.56 0.08 - 80 1H-Imidazole, 2-ethyl-4,5-dihydro 22.65 - 0.11 81 Alpha-cadinol 22.71 0.10 - 82 (-)-Anymol 23.03 0.23 - 83 2-Acetylcyclopentanone 23.09 - 0.05 84 Bicyclo[4.4.0]Dec-1-En 23.27 0.39 - 85 1(2H)-Naphthalenone 24.16 0.08 - 86 Benzenemethanol, 3,5-dimethyl- (CA 24.39 - 0.06 87 10,10-Dimenthyl-2,6-dimethylenebicy 24.41 0.81 - 88 Phosphonous dichloride 24.85 0.09 0.13 89 3-(1-Ethyl-4-methoxycyclohexa-2,4-

(1RS, 2SR, 3SR, 4SR, 6SR)-1,2;3,4-diep 25.02 1.18 -

90 Trans- α-Bergamotene 25.02 - 0.11 91 7-Oxabicyclo[4.1.0]heptane,1-meth 25.54 - 0.08 92 Caryophyllenol 25.56 0.40 - 93 1-Isopropenyl-4-methylcyclohexanec 25.83 - 0.16 94 Pyridine, 3,4-dimethyl- (CAS) 26.07 - 0.09 95 Nerolidol Z and E 26.39 - 0.06 96 Methyl ethyl cyclopentene 26.63 - 0.05 97 Benzaminine 27.72 - 0.09 98 (8S, 5R)-8-methyl-5-2-propenyl 28.30 - 0.05 99 2-Hexadecen-1-ol 28.31 0.07 - 100 1,2-Benzendicarboxylic acid 28.68 2.68 0.11 101 Acetamide, N-methyl-N-[4-[4-methox]] 28.85 - 0.06 102 2-Methyl-1-(methylamino)-1-cyanopr 29.85 - 0.11

Fumigant activity.

Results showed that fumigant toxicity of the tested essential oils varied depending on targeted insect species and oil concentration used. In fact, as shown in Table 3, essential oil from wild rosemary leaves was more toxic toward T. castaneum and T. granarium than that extracted from the cultivated ones. Probit analysis showed that for T. castaneum,

the corresponding LC50 values were 65.5 μl/l air and 180 μl/l air and CL90 values were 145 μl/l air and 330 μl/l air for wild and cultivated rosemary essential oils, respectively. Concerning T. granarium toxicity, the corresponding LC50 values were 19.75 μl/l air and 18.75 μl/l air and CL90 values were 39 μl/l air and 41 μl/l air for wild and cultivated rosemary, respectively (Table 2).


Table 2. Variation in Tribolium castaneum and Trogoderma granarium adults’ mortality depending on nature of rosemary plants and essential oil concentration used and LC50 and LC95 values determined

Targeted insect

Essential oil plant source

Concentration (μl/l air)

Mortality (%)

LC50 (μl/l air)

LC90 (μl/l air)

Tribolium castaneum

Wild rosemary

0 0 a

65.5 145 25 12 b 50 74 c 100 78 c 200 92 d

Cultivated rosemary

0 0 a

180 330 25 12 ab 50 16 b 100 24 b 200 56 c

Trogoderma granarium

Wild rosemary

0 0 a

19.75 39 25 22.5 b 50 27.5 b 100 80 c 200 82.5 c

Cultivated rosemary

0 0 a

18.75 41 25 22.5 b 50 42.5 c 100 85 d 200 87.5 d

For each targeted insect species and for each rosemary plant nature, values followed by similar letters are not significantly different according to Duncan’ Multiple Range test (at P < 0.05).

Repellent activity.

Wild and cultivated rosemary leaf essential oils were found to be repellent to T. castaneum adults (Table 3). The highest concentration (0.25 µl/cm2) of the two oils led to percentage

repellency of 30 and 27.5% after 15 min of exposure and had increased to up to 65 and 45% after 120 min of exposure, respectively, using essential oils from wild and cultivated rosemary leaves.


Table 3. Percentage repellency of essential oils from rosemary leaves against Tribolium castaneum adults recorded after various exposure durations

Essential oil plant source

Exposure duration

(min)

Dose (μl/cm2)

Repellency (%)

Repellency class

Wild

rosemary

15 0.12 -40 Class 0 0.25 30 Class 2

30 0.12 -27.5 Class 0 0.25 47.5 Class 3

60 0.12 -5 Class 0 0.25 57.5 Class 3

120 0.12 -7.5 Class 0 0.25 65 Class 4

Cultivated rosemary

15 0.12 -35 Class 0 0.25 27.5 Class 2

30 0.12 -37.5 Class 0 0.25 22.5 Class 2

60 0.12 -40 Class 0 0.25 45 Class 3

120 0.12 -25 Class 0 0.25 45 Class 3 DISCUSSION

The aim of this study was to investigate the chemical composition of R. officinalis essential oils and to evaluate their insecticide potentialities with a comparison between wild and cultivated rosemary in Tunisia. GC-MS analysis showed that both essential oils contained L-camphor, 1,8-cineole, L-borneol and bornyl acetate as four major components with a remarkable difference in the percentage of bornyl acetate and L-borneol. In fact, essential oil from wild rosemary leaves contained 14.54% of bornyl acetate and 6.02% of L-borneol, while that extracted from cultivated rosemary leaves yielded 1.21% and 14.05% of these components, respectively. Several studies reported on the chemical composition of the essential oils from R. officinalis belonging to different regions in the world. The Moroccan R. officinalis essential oil was characterized by high amounts of α-pinene (37.0-40.0%), cineole (58.7-

63.7%) and camphor (41.7-53.8%) (8). Other investigation from Lebanon, indicated that eucalyptol (50%), α-pinene (14%), camphor (14%), borneol (5%) and camphene (4%) are the principal components in R. officinalis essential oil (12). Bekkara et al. (5) found that in Algeria, their essential oil from wild rosemary was characterized by high amounts of α-pinene (23.1%), camphor (15.3%) and β-pinene (12.2%), but the main components of cultivated rosemary essential oils were camphor (13.8%), α-pinene (12.6%), cineole (11.8%) and borneol (10.8%). These variations can be attributed to climatic, seasonal and geographic conditions, harvest period, chemotypes and extraction procedures (19).

Concerning the fumigant activity against T. castaneum and T. granarium, essential oil from wild rosemary leaves was found to be more toxic than that from the cultivated one. This result can be explained by the relationship that may


exist between the mode of action of essential oils and their chemical composition. The fumigant activity of R. officinalis has been evaluated against other insect species. Indeed, Iskiber et al. (14) reported that rosemary essential oil was highly toxic and they showed that T. confusum adults’ mortality reached 90% at the dose 6.5 g/l. Additionally, fumigant toxicity of R. officinalis essential oil was investigated against Sitophilus granarius where 93.93% mortality was achieved after exposure for 96 h at 20 μl/l air (26). Moreover, rosemary essential oil exhibited high insecticidal toxicity toward Acanthoscelides obtectus adults (21).

In term of repellent activity against T. castaneum, our results clearly showed that wild rosemary essential oil was more repellent than that from cultivated one. In this context, Al-Jabr (2) also reported the repellent effect of R. officinalis essential oil against Oryzaephilus surinamensis

and T. castaneum. Besides, important repellent activity against Rhyzopertha dominica reaching 55 and 71% was achieved using rosemary essential oils (16).

Results from this study revealed an interesting potential of R. officinalis essential oils in controlling T. castaneum and T. granarium, and especially those extracted from wild plants. Thus, these essential oils can be considered as natural insecticide and could be used as means to manage many pests of stored products. However, further studies are needed to better explain the relationship between the chemical composition of the essential oils and their modes of action against the targeted insect pests. ACKNOWLEDGMENTS This work was supported by grant from the Ministry of Higher Education and Scientific Research of Tunisia.

__________________________________________________________________________ RESUME Khalil S., Zarrad K., Ben Hammouda A., Ayed Lakhal Y., Rguez S., Tayeb W., Laarif A. et Chaieb I. 2015. Comparaison de l’effet insecticide des huiles essentielles du romarin sauvage et cultivé contre des insectes de denrées stockées. Tunisian Journal of Plant Protection 10: 105-115. Ce travail a été entrepris dans le but d’évaluer le potentiel insecticide des huiles essentielles de Rosmarinus officinalis (sauvage et cultivé) contre deux insectes des denrées stockées, Tribolium castaneum et Trogoderma granarium. L’analyse chimique effectuée par chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS) a montré que les deux huiles essentielles contiennent 4 composés majoritaires qui sont le L-camphre, le 1,8-cinéole, le L-bornéol et l'acétate de bornyle. Les essais effectués ont montré que les deux huiles essentielles du romarin ont une activité fumigène plus importante sur les adultes de T. granarium que sur ceux de T. castaneum. Les concentrations létales correspondantes CL50, respectivement pour les huiles essentielles du romarin sauvage et cultivé, étaient de 65,5 μl/l air et 180 μl/l air pour T. castaneum alors qu’elle étaient de 19,75 μl/l air et 18,75 μl/l pour T. granarium. L’huile essentielle du romarin sauvage était plus répulsive contre les adultes de T. castaneum que celle du romarin cultivé. L'activité répulsive a varié selon la concentration de l’huile et la durée d’exposition. Appliqués à la dose 0,25 µl/cm2, l’effet répulsif des huiles essentielles du romarin sauvage et cultivé a atteint 65 et 45% environ respectivement après 120 min d’exposition. Ces résultats suggèrent que l’huile essentielle du R. officinalis pourrait être potentiellement active dans la lutte contre ces deux insectes des denrées stockées.


Mots clés : Fumigation, huile essentielle, répulsion, Rosmarinus officinalis, Tribolium castaneum, Trogoderma granarium __________________________________________________________________________

ملخص قبال إسماء العريف وأاالكحل وصفاء رقاز ووفاء التايب و-سمين عيادامال بن حمودة ويأصبرين وخولة زراد وخليل، .محاصيل مخزونةحشرات ضد المزروعو البّريلزيوت األساسية لإلكليل لفاعلية المبيدة مقارنة بين ال. الشايب

Tunisian Journal of Plant Protection 10: 105-115.

ضّد ، البّري والمزروع ،)Rosmarinus officinalis( لإلكليلأجريت ھذه الدراسة لتقييم قدرة الزيوت األساسية وخنفساء Tribolium castaneum خنفساء الطحين الحمراءھما ن للمحاصيل المخزونةيحشرتقصد تشخيص تركيبتھا GC-MS تقنيةوقع تحليل الزيوت األساسية بواسطة . Trogoderma granariumالحبوب و Cinerole-1,8و L-Camphor ساسا من مادة أالمزروع تتكون و البّريتبين أن الزيوت األساسية لإلكليل . الكيميائية

L-Borneol وBornyl Acetate .ر أنٌھا أكثر فاعليّة ضديأظھرت الزيوت األساسية لنبتة اإلكليل عن طريق التبخ Trogoderma granarium بالحشرة مقارنة .Tribolium castaneum وبلغت الجرعات القاتلةCL50 65.5

كما بلغت ،Tribolium castaneumل بالنسبة لإلكليل المزروع ضد /مكل 180ل بالنسبة لإلكليل البّري و /مكلوع ضد ربالنسبة لإلكليل المزل /مكل 18.75بالنسبة لإلكليل البّري و ل/مكل CL50 19.75 الجرعات القاتلة

Trogoderma granarium. 0.25 عند جرعةف .ة تركيز الزيت ومّدة التعّرضقوّ كانت الفاعليّة الطاردة مرتبطة ب على والمزروع البّري إلكليلى الإبالنسبة بعد ساعتان من التعّرض% 45و 65بلغت النسبة المئويّة للطرد ، 2سم/مكل

لمحاصيل ا مكافحة خنافسفي لنبتة اإلكليل األساسي الزيت ه يمكن استعمالالمتحّصل عليھا أنّ تشير النتائج .التوالي .المخزونة

، Rosmarinus officinalis ،Tribolium castaneumطرد، زيت أساسي، تبخير، : كلمات مفتاحية

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Valorization of Three Plant Species of Arid Areas in Biological Control of the Desert Locust Schistocerca gregaria

Saïda Messgo-Moumene, Djamel -Eddine Merzouk, Zahia Houmani, Laboratoire de Recherche des Plantes Médicinales et Aromatiques, Département de Biotechnologies, Faculté des Sciences de la Nature et de la Vie, Université de Blida 1, BP 270, Route de Soumaa, Ouled Yaïch, 09100, Blida, Algeria, and Khaled Moumene, Laboratoire de Recherche en Acridologie, Institut National de la Protection des Végétaux, Route Hacen Badi, El Harrach, Algeria __________________________________________________________________________ ABSTRACT Messgo -Moumene, S., Merzouk, D.E., Houmani, Z., and Moumene, K. 2015. Valorization of three plant species of arid areas in biological control of the desert locust Schistocerca gregaria. Tunisian Journal of Plant Protection 10: 117-130. The present work aims to evaluate the in vitro biocide effect of aqueous and ethanol extracts of plants collected from arid Algerian Sahara i.e. Calotropis procera, Schouwia purpurea, and Zizyphus lotus on the fifth larval instar (L5) of Schistocerca gregaria. These extracts obtained by decoction and solvent were tested respectively crude for the first one and at concentrations of 12.5% and 50% for the second one by contact and by ingestion. Significant mortality was recorded the fifth day after treatments, using ethanol at 50% by ingestion (52.5%) and crude aqueous C. procera extracts (45%) when applied by contact and ingestion and with S. purpurea ethanol extracts at 50% when used by contact (47%). Morphological changes and molting inhibition were detected following treatment by contact and ingestion while antifeeding effects and structural changes of L5 larvae mesenteron were observed for ingestion mode. In contrast, negligible mortality rates, similar to those of the untreated control and no changes affecting the behavior or L5 larval mesenteron structure were recorded using ethanol and aqueous extracts from Z. lotus according to the two application modes. Thus, the ethanol extract of C. Procera exhibited in vitro acridicide potential toward S. gregaria L5 larvae. It would be interesting to test this extract under semi-natural and natural environments and to identify the active ingredients responsible for the biocontrol of desert locusts. Keywords: Acridicide potential, aqueous extracts, Calotropis procera, ethanol extracts, Schistocerca gregaria, Schouwia purpurea, Zizyphus lotus __________________________________________________________________________

Since time immemorial, humanity has been confronted with a dangerous and devastating enemy, the desert locust Schistocerca gregaria. This insect is

Corresponding author: Saïda Messgo Moumene Email: [email protected]

Accepted for publication 03 November 2015

capable, under the influence of a density increase, of moving from a solitary form, quite harmless for cultures and pastures, to a gregarious and very harmful form.

The desert locust constitutes in Africa an extremely serious threat for agriculture. Its invasion area covers Africa, the North of Ecuador, the Middle East, the Arabian and India-Pakistan Peninsulas and sometimes, Mediterranean


Europe. This represents in total 57 countries and 20% of land masses. Outside the invasion periods, the desert locust withdraws in recession period in the most arid regions of its scatter area where it is often unnoticed (23).

Algeria, like other concerned countries, is periodically confronted with these invasions (24). Studies conducted by several researchers on the desert locust in Algeria as those of Volkonsky, during the colonial period over three consecutive years (1939, 1940 and 1942), have shown that Algeria has really potential areas of survival and multiplication of the desert locust, extending over the whole Sahara and according to eco-meteorological conditions, movements of locust populations take place each year between Algeria and Sahelian countries (38).

In order to be efficient, it is necessary to act rapidly at the level of primary outbreaks of reproduction to avoid formation of uncontrollable swarms. The preventive fight is recognized by the international community as the only lasting strategy to control invasions of desert locusts. This method consists in watching closely and permanently gregarigenous areas and destroying, through quick interventions on limited surface areas, larval bands and the first locust groupings which have started the gregarization process (16).

Pesticides represent the only means used to stop multiplication and spreading of locusts. However, even by using chemicals in a controlled way, the risk on environment and human health is still present. Awareness of these problems relating to the environment and ecology has led organizations and research institutions to focus more on biological control in its different forms to fight locusts. One of these forms relies on the use of acridicide, acridifuge or

antiappetant substances produced by plants to protect crops from desert locus outbreaks (6, 10, 20, 29).

The aim of the present study is to evaluate the potential of aqueous and ethanol extracts from three plants (Calotropis procera, Schouwia purpurea, and Zizyphus lotus) from gregarization biotope to control the desert locust (S. gregaria). MATERIALS AND METHODS

This study has required the use of fifth larval instar (L5) of the desert locust and the aerial parts of three wild plants namely C. procera, Z. lotus, and S. purpurea, largely distributed in the locust biotope of the Algerian Northerly Sahara.

The larvae come from a mass breeding of desert locusts conducted and maintained at the breeding room of the locust control department of the National Institute of Plant Protection of El-Harrach (INPV), Algeria. The individuals have been collected from the region of Taghit (Oued Zouzfana), governorate of Bechar, in April 2013, following spring infestation.

The samples of the three desert plant species have been collected from the region of Sillet, Oued Amded, governorate of Tamanrasset during spring 2013. Mass breeding of the desert locust.

Our experimental trials have required breeding of S. gregaria larvae and adults which was conducted in plane-parallel cages of wood structure (1.50 × 0.70 ×0.55 m) for adults rearing but much smaller cages (0.55 × 0.55 ×0.55 m) were used for larvae. The base of the cage is of plywood and the rest consists in thin mesh metal screen. A small trapdoor at the front face allows access inside the


cage, the bottom of which has circular openings where are placed nest boxes filled with regularly humidified sand in the case of adults. Breeding is maintained at a temperature of 30-34°C with relative humidity of 60-65%. Lamps of 75W allow continuous lighting. Feeding consists essentially in turf leaves (Stenotaphrum americanum) and wheat bran (Triticum turgidum). Renewal of food, cleaning and humidification of nest boxes as well as their verification for seeking egg sacks are daily performed. Preparation of plant extracts.

After being dried at room temperature of the laboratory, the aerial parts of the three collected plants were ground into fine powder and kept in hermetic paper bags to be used for preparation of extracts whereby two extraction modes have been required for extraction of active ingredients of the three studied plants.

Ethanol plant extracts. Ethanol extracts were prepared using Wolff’s method (39) which consists in filling a filter paper cartridge with 40 g of plant material, then introducing it in the glass cylinder of the Soxhlet-type extractor (200 ml) equipped with an adapter and a bubble cooler.

This set is placed on a 500 ml ball containing 350 ml of ethanol and maintained at a boiling temperature. Through heating, the solvent steam rises into the extractor pipe, condenses at the level of the water cooler and drops into the glass cylinder containing the porous cartridge. The solvent bathes the solid which is inside the cartridge and loads into a most soluble compound. When the level of the liquid solvent reaches the summit of the siphon, the cartridge gets empty and the solvent gets down into the

ball. The most soluble compound in the solvent is therefore progressively concentrated in the ball. After an average of ten siphoning, the plant material is depleted and the experience is interrupted (24).

The substance extracted, filtered and freed from solvent has been evaporated under vacuum at 35°C with a Büchi-type rotating evaporator. After complete elimination of the solvent, the dry residue has been recuperated with 10 ml distilled water to constitute crude extract (1).

Aqueous plant extracts. Extraction by decoction consists in mixing 50 g of plant dry powder with 500 ml of distilled water in a 1-liter flask and boiling it out in a Soxhlet-type extractor for 90 min (22). After cooling and filtration, the extraxts were kept at 4°C until use. Study of the toxicity of plant extracts.

Toxicity tests. These tests focused on the counting of the mortality of treated insects after a period of time from the beginning of the experiment. Two modes of application of plant extracts have been retained for this study, one by contact and the other by ingestion. The effect of extracts of the three plants was evaluated based on mortality, morphological alteration, behavior change, and structure of the digestive tract. These parameters were noted on a total of 480 emerging individuals where 10 larvae were used per type of plant extract and per concentration tested. Each individual treatment was replicated thrice. For a long-term follow-up and in order to avoid mass effects, especially interferences or perturbations, L5 larvae have been individually placed into plastic boxes (0.12 × 0.16 × 0.24 m), with an insect-


proof cover of the same size as the boxes. They have been maintained under the same conditions of temperature and humidity as those of mass breeding of adults and larvae. A volume of 10 ml has been retained for the tested plant extracts, either by contact or ingestion. Also, two concentrations, 50 and 12.5%, have been chosen for ethanol extracts, after a preliminary test and according to the number of locusts used.

Contact toxicity. It consists in making a contact between the insect body and the tested extracts. The spraying mode is used (28) and it consists in performing treatment approaching as possible as the natural pest control conditions.

The three plant extracts have been directly sprayed on S. gregaria L5 larvae in order to assess their activity by contact. The experiment has been followed until death of treated individuals.

Ingestion toxicity. It consists in poisoning food with the tested extracts. The ingestion mode consists in feeding L5 larvae putted in starvation during 24 h in order to let empty their digestive tract, with determined surface fragments from the food plant. For this study, insects were fed with treated turf separately from the three plant extracts (8), with two controls, water for decocted extracts and diluted methanol (50%) for ethanol extracts. Evaluation of mortality, behavior change and cytotoxicity.

The rates of mortality have been daily assessed and the malformations have been described for each plant extract according to the concentrations and the modes of their application. Antifeedancy

tests permit the determination of food consumption reduction caused by the treatment (13).

The cytotoxicity consists in identifying the effect of plant extracts on morphology of insects. Histological studies were performed on individuals exhibiting necrosis symptoms (11).

The structure of the digestive tract of the larvae treated with the different plant extracts was compared with that of the control. It is first based on macroscopic observations of the individuals’ digestive tracts. The digestive tracts were sampled by cutting the elytra and the femurs. A large incision was performed at the level of the ventral part of the individual up to the cephalic capsule.

The digestive tract parts showing morphological changes have been stained, according to pathologic sections, based on the technique of Martoja and Martoja (25). Therefore, the microscopic observations performed were focused on changes or alterations of the structure of the digestive epithelium of the treated individuals as compared to the controls. Statistical analysis.

To compare the biocide activity of the three plant extracts tested at different concentrations using two modes of application toward S. gregaria L5 larvae, statistical analyses have been performed with the software SYSTAT vers. 12, 2009 SPSS, by determining the variance by means of GLM (Generalized Linear Model). The differences have been considered as significant at P < 0.05. Correlations between the different plant extract

Documents

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