Evaluation of some agricultural waste extracts against mosquito larvae , and some types of microorganisms as insecticidal and antibiotic agents

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INTRODUCTION
Mosquitoes (Diptera: Culicidae) are among the most serious insect pests of medical importance.They are vectors of various disease agents some of which cause millions of cases of illnesses and deaths in human and animal each year.Among these diseases, malaria, yellow fever, dengue and dengue hemorrhagic fever, filariasis and Rift Valley fever at endemic and epidemic areas in many countries (WHO, 1991, Lerdthusnee, et al., 1995and Madani, et al., 2003).The use of chemical insecticides for controlling pests and vector of diseases is undesirable, save and yet effective methods of control are being sought.The use of natural products of plants origin is a new trend that preserves the environment and can be applied effectively by using techniques more sui Table for developing countries (Abbassy, 1998;Mohamed et al., 2003 andKamel et al., 2005).
Infectious diseases are still one of the leading causes of death in the world.Although conventional drugs provide effective treatment for some infections, antibiotic resistance continues to grow among key microbial pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus spp and Enterobacteriaceae (Bax et al., 2000;Bhavnani and Ballow, 2000).
The plant fungal diseases have been controlled by chemical fungicides.The development of resistant strains of pathogens against various chemical fungicides (Lin, 1981 andWitte, 1998) and their harmful effects on soil biosphere and causing health hazards for humans and animals which found to pose carcinogenic risk due to their residual toxicity (Anonymous, 1998 andSarmamy, 2001) make the use of these chemicals limited.
Plants are the gifts of nature used to cure number of human diseases (Deepa et al., 2012).Plant extracts show antibacterial effects (Sarmamy and Al-Juboory, 2005) and antifungal activity against wide range of fungi (Aba Alkhail, 2005;Basm and Khalil, 2007;Sarmamy and Saleem, 2009).Some authors using agricultural waste materials and plastic waste to decrease the cost of production of natural insecticides, antimicrobial agent and convert the waste material to benefit one (Bakr et al., 2008, El-Maghraby et al., 2012and Fahim, et al., 2013).
The present study was undertaken to: a) test the potency of several waste plant extracts against the larval stage of mosquito (Culex pipiens) and some types of microorganisms (bacteria & fungus b) analyze the joint action toxicity resulting from mixing botanical extracts with conventional insecticides, c) evaluate the latent effect of waste extract on the developmental stages of Culex pipiens.

Test microorganisms and culture preparation:
Gram negative bacteria Escherichia coli, Pseudomonas aeruginosa, Morganella sp.Gram positive bacteria Staphylococcus aureus, and Micrococcus sp. were obtained from "Culture Collection of Antibiotic Resistant Microbes (CCARM)" Military Hospital Tabuk.The yeast Candida albicans was obtained from (CCARM)" Military Hospital Tabuk, Aspergillus niger was isolated from infected onion bulbs, Alternaria alternata and Fusarium sp.Isolated from infected tomato fruits while Penicillium sp.isolated from infected citrus fruits and pure cultures of the isolated fungi were identified on the basis of morphological and microscopically characteristics according to the key of (Bessy, 1968 andBarnett andHunter, 1972).The bacterial strains were cultured on nutrient agar (NA) medium at 37 • C, and fungal strains on potato dextrose agar (PDA) medium at 28 • C (Imtiaj and Lee. 2007).The yeast strain Candida albicans has been maintained at 4 • C on Sabouraud Dextrose Agar (SDA) plates and subcultured at 25 • C in Sabouraud Dextrose Broth (SDB) before each experiment to ensure viability and purity (Sha Fighi et al., 2012).

Antimicrobial assay Determination of antibacterial and antifungal (yeast) Candida albicans activities:
Antimicrobial activity of the crude extracts was determined by Agar well assay methods as described by (Collins et al., 1995, NCCLS 1999, Moshi et al., 2006and Rojas et al., 2006).The inoculum size of each group of bacteria and yeast were prepared by using nutrient broth to give a concentration of 1×10 8 bacteria and 1×10 6 yeast per milliter.The suspension (100µl) was spread onto the surface of Mueller Hinton Agar (MHA) medium.Wells (5 mm in diameter) were cut from the agar with a sterile borer and 50µl extract solutions were delivered into them.Negative controls were prepared using sterile distilled water, gentamycin, was used as positive reference standards to determine the sensitivity of each microbial species tested.The treated plates were stored in a refrigerator at 4 • C for at least six hours to allow diffusion of the extracts into the agar while arresting the growth of the test microbes (Ndyetabura et al., 2010).The plates were then incubated for 24-48 hours at 37 • C. Antimicrobial activity was determined by measuring the diameters of inhibition zones (DIZ) in mm.All tests were performed in triplicates and the developing inhibition zones were compared with those of the reference discs.The means and standard deviations (±SE) of (DIZ) was done.

Determination of antifungal activity:
Concentrations of 0, 2, 5, 10, 15 and 20% of the raw extracts were prepared and added to the sterilized (PDA) medium, mixed well then 20 ml of the mixture (PDA medium + plant extract) were poured in each 9 cm sterilized Petri dishes.The medium without extract was served as control 0%.Mycelial discs of fungi were prepared using a cork borer (5mm in diameter) from the margin of 5 days old culture of the tested fungi and placed at the center of Petri dishes after solidification of PDA medium.Each treatment was replicated three times.Plates were incubated in an incubator at 28 • C. Fungal growth after 48, 96 and 168 hrs was measured by taking the mean of the two diameters taken at right angles for each colony (Sarmamy et al., 2011).

Tested compounds:
The tested plant wastes were washed to avoid dusts and dirt then lift to dry under shade in the laboratory.Dried part of plants (Pomegranate peel and Apricot kernel) were cut into small pieces and ground in an electric grinder.Hundred grams of the resulting powdered materials of each plant were exhaustively extracted with ethanol absolute, following the method described by Kamel et al., (2005b).
Provided by collecting from Tabuk area and transferred to the research laboratory of Biology Department-Science Collage-Tabuk University where self-perpetuating colonies were established and maintained during the present study, according to the method described by Kamel et al., (2005a).Late third larval instars were used for toxicological studies.

Toxicological studies: Efficiency of plant extracts:
Preliminary, toxicological bioassay tests were carried out to the selected plant extracts on tested insects as a modification for the method described by (Wright, 1971 andKamel et al., 2005b) their LC 50 and LC 95 values were determined as well as their slope function, according to Finney, 1971 andWHO, 1981).Bioassay tests were carried out to Deltamethrin and Altosid on the tested insect.

Joint action of selected waste extracts:
The selected waste plant extracts were mixed with each other, and with either IGR or Pyrethroid at a level of their corresponding LC 25 values.The tests were carried out as mentioned before.The combined action of the different mixtures was expressed as the co-toxicity factor which was estimated according to the equation given by (Kamel et al., 2005a).

Effect of sublethal treatments of selected waste plant extract and Deltamethrin on some biological activities of Culex pipiens:
The 3 rd instar larvae of Culex pipiens (500 larvae) were exposed to sub lethal dose of the most potent plant extract and Deltamethrin.Survived larvae after twentyfour hours post treatment were gently washed and transferred to labeled pans.Treated larvae and controls were kept under laboratory conditions were maintained under laboratory conditions till adult emergence.The percentage of pupation and adult emergence were calculated.

RESULTS AND DISCUSSION Antimicrobial studies:
The antimicrobial activity of the ethanolic extract of Pomegranate (Punica granatum) and Apricot (Prunus armeniaca) were determined against the linear growth of four fungi Fusarium spp, Alternaria alternata, Aspergillus niger and Penicillium spp.(Plate 1) and determination of (DIZ values) for five bacteria used Escherichia coli, Pseudomonas aeruginosa, Morganella sp, Micrococcus sp, Staphylococcus aureus and yeast (Candida albicans) at different concentrations, (Plate 2).

Effect of Pomegranate (Punica granatum) extract on studied fungi
The different Pomegranate extract concentrations affected significantly the mycelial growth of the studied fungi.In the case of Fusarium sp and Alternaria alternata (Table 1) all pomegranate extract concentrations (0.5-5%) had antifungal activity against two fungi and reduced their mycelia growth (6.33-19mm) and (7.33-13 mm) compared with control 34.66 mm and 30.66 mm after 3 days of inoculation.The highest antifungal activity was recorded at concentration of 2 % and no growth occurred at concentration 5% at 9 day for the two fungi respectively.Results presented in (Table 2) show that Penicillium sp was more sensitive more than Aspergillus niger to pomegranate extract.The mycelial growth was 22.33 mm and 59 mm, respectively in comparison with 83 mm and 85.66 mm for the control at 9 day of the two fungi, respectively.Time of incubation affected the growth of mycelia significantly.(Figs. 1 and 2) show that all concentrations of pomegranate extract at the three times affected mycelia growth of Fusarium sp and Alternaria alternata and the inhibitory effect significantly increased with the increase in the concentration.It seems that the interactions between time of incubation and extract concentrations (Figs. 3 and 4).At day 3, it was observed that pomegranate extract significantly reduced the liner growth of Penicillium sp and Aspergillus niger with the increase in the concentrations.The linear growth continued to increase after 6 days of inoculation with different concentrations and at day 9, the highest antifungal activity was recorded at concentration of 10 % and 20 % for the two fungi, respectively.It seems that Penicillium sp and Aspergillus niger are more sensitive to high concentrations of pomegranate extract while no growth occurred at 20% and 30% of the two fungi, respectively.

Effects of Apricot (Prunus armeniaca) extract on studied fungi
Results presented in (Table 3) show that the extract of apricot at all concentrations affected significantly the growth of mycelia of Fusarium sp, Alternaria alternata and Aspergillus niger except Penicillium sp was resistant to apricot extract at all tested concentrations.The mycelial growth of previous sensitive fungi reduced to (8.33-69.33mm), (18.33-50 mm) and (47.66-74.33mm) at 6 day in comparison with 75.33 mm, 52.66 mm and 75 mm for control of three fungi, respectively. Figs.
(5-8) show the interaction between time of incubations and apricot extract concentrations, at day 9, the highest effect was in concentration 40 % with 21.66 mm, 22.33 and 52.33 mm compared with 83 mm, 84.33 mm and 85 mm for control of three fungi, respectively.Apricot extract at 50 % concentration, continued to be most effective inhibitor of the growth of the studded fungi while no growth occurred.Figs.5-8: Effect of interactions between conc. of Apricot extract and time of incubation on mycelia growth of Fusarium sp (Fig. 5), Alternaria alternata (Fig. 6), Penicillium sp (Fig. 7) and Aspergillus niger (Fig. 8).
Extract of Pomegranate were more effective on studied fungi than apricot extract and caused significant reduction in mycelial growth for the fungi.This may be due to the differences between the chemical components such as volatile oils, tannin and amygdalin (Mertens et al., 2006 andPaaverurve &Raal, 2010).The antimicrobial activities of phenolic compounds may involve multiple modes of action.For example, degrade the cell wall, interact with the composition and disrupt cytoplasmic membrane, damage membrane protein, interfere with membrane integrated enzymes (Nychas, 1995;Sha Fighi et al., 2012 andHassan et al., 2013).The results are in agreement with the study of (Sarmamy et al., 2011) who concluded that ethanol extract of pomegranate was more effective on A. niger and Penicillium sp.

Effects of Apricot (Prunus armeniaca) and pomegranate (Punica granatum) extract on bacteria and Candida albicans:
The antibacterial activity of seven different concentrations of Apricot and pomegranate extract (1,2,5,10,20,30,40 and 50 %) and Gentamycin (10 µl), the later used as positive control against Escherichia.coli, Pseudomonas aeruginosa, Morganella sp, Micrococcus sp, Staphylococcus aureus and antifungal activity of Candida albicans were recorded after 24 hr. of incubation.In this study it is observed that apricot extract did not show any antibacterial effects against used bacteria or antifungal effect against Candida albicans however, pomegranate extract inhibited the growth of all used bacteria and Candida albicans and the inhibition zones ranged between 12-33 mm.Table (4) shows that high antibacterial effect was recorded on Morganella sp and Micrococcus sp, followed by Staphylococcus aureus and less effect was recorded for Escherichia coli, Pseudomonas aeruginosa and Candida albicans.As shown in (Fig. 9 and Plate 2).The inhibition zone increased proportionally with the increase in pomegranate concentration.The high inhibitory effect was observed with extract concentration between (5-40 %) of Morganella sp, Micrococcus sp and Staphylococcus aureus with DIZ values of (21.33-30.66mm), (22-33.67mm) and (23.33-32 mm), respectively.The DIZ values for Escherichia coli and Pseudomonas aeruginosa were between (12.33-23.33mm) and (5.5-22.67 mm) at concentration of (10 -40 %), respectively.For Candida albicans the DIZ values were between (4.33 -17.33 mm) at concentration (10 -40 %).
These findings are in accordance with the observations of (McCarrell et al., 2008), who found that aqueous extract of pomegranate rind inhibits growth of S. aureus and P. aeruginosa.Similarly, (Al-Zoreky, 2009) has reported that metanolic extract of pomegranate fruit peels is a potent inhibitor for S. aureus, Listeria monocytogenes, E. coli and Yersinia enterocolitica.Ahmet Duman et al., 2009 also reported the in vitro antibacterial activity of pomegranate extract against bacteria P. aeruginosa, S. aureus, E. coli showing inhibition zones ranging from 13-26 mm, however Similar results were recorded in our study.(Hassan et al., 2013) reported that Punica granatum significantly contributed to antibacterial activity, these included E. coli, Staphylococcas aureus and Streptococcus faecalis.(Nuamsetti et al., 2012) found that extract of Pomegranate fruit peels exhibited inhibitory activity against bacteria Bacillus subtilis, Salmonella typhimurium and E. coli.Moreover, Punicalagin showed strong activity against Candida spp.(Endo et al., 2010).In contrast, the study of (Abtahi et al., 2008) reported the antibacterial activity of bitter apricot extract against several bacterial strains and this might be brought by amygdalin, alkaloids, flavanoids, tannins and phenolic compounds.
In the present study, extract of pomegranate has been tested against bacteria (S. aureus and P. aeruginosa) as well as against pathogenic yeast, C. albicans.The S. aureus is responsible for a wide variety of diseases, including pneumonia, skin and soft tissue infections, and diabetic foot infections (Shorr, 2007).Similarly, P. aeruginosa is a common pathogen associated with burn wound infections, keratitis, and respiratory tract infections (Marquart et al., 2005).Under the conditions employed here, the bacteria were found to be sensitive to extract.

Insecticidal studies: Evaluation of the larvicidal activity of waste plant extracts:
The waste plant extracts studied are available in large quantities to reduce the cost of production.The other principal criterion in the selection of plant species was their continuous usage due to their medicinal properties.Improvement the insecticidal properties was achieved on the following experiments.

Larvicidal activity of waste plant extracts against Culex pipiens larvae:
The results are represented in Table ( 5) and the regression lines are presented in (Fig. 10).The confidential limits of each of the tested plant extract were statistically calculated for LC 50 and LC 95 at P= 0.05.The LC 50 values for ethanolic extract of pomegranate peel and apricot kernel are 1253.9and 138.7ppm, respectively.The present data showed that despite the differences in potency of pomegranate peel and apricot kernel.They were found to possess parallel regression lines.This may suggest that these extracts have the same mode of action against the tested insect larvae (Busvine, 1971).The results showed in (Table 6) and (Fig. 11) represent the susceptibility of Culex pipiens to traditional insecticides (Deltamethrin & Altosid), although the potency of extracts are less than chemical insecticides but they more safe (Mann and Koufman, 2012) and conversion of waste material to natural beneficial insecticide (El-Maghraby, et al., 2012).

Joint action of waste plant extracts on Culex pipiens larvae:
The waste plant extracts were individually mixed with each other or with Altosid and Deltamethrin in a ratio of 1:1 at the level of LC 25 of each.Thus, a total of 5 binary mixtures are prepared.The data in the (Table 7) and (Fig. 12) indicated that, potentiation effect was induced in all mixtures except Pomegranate extract with Altosid insecticide give additive action.Similarly variations on the joint action effect on the plant extracts and insecticides were demonstrated by (Barakat et al., 1984 -85;Mesbah et al., 1990 andEl-Bokl andMoawad, 1997).Moreover, several authors have used the plant extracts to synergize the insecticidal activities of the traditional insecticides (Mohan et al., 2010 andMansour et al., 2011).Variation in the joint action effect of the different insecticides binary mixtures could be attributed to the site and mode of action of the mixture components.Therefore the potentiation and additive effects may be attributed to that the mixture components which have the same site or/and mode of actions (El-Bokl and Moawad, 1997).

Effect of larval treatment with Apricot kernel extract of the development of Culex pipiens:
The purpose of this experiment is evaluating the potency of extract by latent effect on developmental stages of Culex pipiens.The results represent in (Table 8) showed decrease in pupation and adult emergence in Apricot kernel extract and Deltamethrin treatments comparing with control.Both treatments showed highly significant reduction in the percentage of pupation.Estimation of adult emergence percentage is based on the ratio of the total number of emerged adults to the total number of pupae.The results showed highly significant reduction in Apricot extract while showed significant reduction in Deltamethrin treatments comparing with control.
Effect of Apricot kernel ethanolic extract on developmental stages of Culex pipiens is resembled to Deltamethrin effect on pupation but the apricot extract more effective on adult emergence than Deltamethrin.These results may be due to the similarity between deltamethrin structure and some components of extract which phytochemical analyzed by (Talabani et al., 2012).
The authors concluded that the pomegranate extract is the promising agent against bacteria and fungi while the apricot extract is effective for mosquitoes and some pathogenic fungi.The authors recommended that the promising extracts need more investigations to know the mode of action and their physiological effect on target mosquito and microorganism.Also, semi field studies operate to know their effect on mosquitoes breading sites.4).

Fig. 9 :
Fig. 9: Effect of different concentrations of Pomegranate extract on bacterial cultures and C. albicans.

Fig. 12 :
Fig. 12: Joint action analysis for the waste plant extracts together either with Deltamethrin or Altosid (IGR) at LC25 levels.

Plate 2 :
Inhibition zones of Pomegranate extract on bacteria and C. albicans.G and C indicate positive and negative control.(1-7) indicate different concentrations of Pomegranate extract on tested bacteria as shown in Table (

Table 1 :
Effect of Pomegranate extract on the growth of Fusarium sp and Alternaria alternata.Each value is the mean of 3 replicates ± S.E.
Table2: Effect of Pomegranate extract on the growth of Penicillium sp and Aspergillus niger.Each value is the mean of 3 replicates ± S.E.

Table 3 :
Effect of Apricot extract on the growth of Fusarium sp, A. alternata, A. niger and Penicillium sp.Each value is the mean of 3 replicates ± S.E.

Table 4 :
Bacteria and Candida albicans sensitivity to different concentrations of Pomegranate extract.Each value is the mean of 3 replicates ± S.E.

Table 5 :
Larvicidal activity of tested plants against Culex pipiens larvae: Susceptibility of Culex pipiens larvae to waste extracts

Table 7 :
Joint action analysis for the waste plant extracts with Deltamethrin or Altosid (IGR) or with each other mixed at LC 25 levels.

Table 8 :
Efficiency of Apricot kernel extract and Deltamethrin on pupation and adult emergence of Culex pipiens: