ABSTRACT
The diamond back moth, Plutella xylostella Linnaeus, 1758 (Lepidoptera: Plutellidae) is improperly controlled with synthetic insecticides in vegetable crops. This practice, which pollutes the environment, led to human and animal health problems, and P. xylostella resistance. Neem (Azadirachta indica A. Juss) is reported in the literature as a natural alternative to synthetic insecticides in the control of many insect pests including P. xylostella. The aim of our study is to evaluate the toxic and post- treatment effects of neem leaves extracts on P. xylostella larvae in comparison to synthetic insecticides namely Conquest plus 388 EC and Cydim Super. Results revealed that exposure of third instars larvae (L3) of P. xylostella on cabbage leaves treated with neem extracts for 24 h induced mortality rates between 1.67 and 6.67% (p > 0.05). Larvae consumed between 45.17 ± 4.48 and 210 ± 27.17 mm2 of cabbage leaves surfaces after 24 h exposure (p < 0.05). The emerging rates of P. xylostella adults were lower for extracts of neem and were between 19.44 ± 6.81 and 20.55 ± 5.38% compared to the controls and insecticide treatments which rates were between 34.07 ± 6.35 and 70.37 ± 10.25% (p < 0.05). The neem leaves extracts were more effective than synthetic insecticides in the control of P. xylostella. Therefore they can be considered as a new hope in developing a management program on P. xylostella.
Key words: Neem leaves extracts, Plutella xylostella, cabbage consumed, mortality.
Cabbage Brassica oleracea L. var. F1 KK cross is one of the most important market gardening, heat resistant vegetables grown in the world. It is eaten raw in salads or cooked. Its production generates substantial income for the producers and others stakeholders involved in the marketing system. However, its production limited by biotic pressure from insect pests particularly from the diamondback moth (DBM) Plutella xylostella Linnaeus, 1758 which causes a complete loss of the crop (Kim et al., 2001; Baek et al., 2005). P. xylostella also reduces marketability by causing damage to the cabbage through larvae consumption or by their frass. The major method to control that pest is the use of synthetic pesticides (Shelton et al., 1997; Améwuamé, 2006; Dovlo, 2007) leading often to serious environmental problems besides affecting the health of producers and consumers. They also reduce natural enemies population, induce insect resistance and increase the need for insecticide and production cost (Rodriguez et al., 2003; Regnault -Roger et al., 2004). To overcome resistance problem, producers in Togo increase frequency and pesticide doses application (Dovlo, 2007). An important alternative to synthetic pesticides is the use of botanical pesticides (Rodriguez et al., 2003; Sharma and Gupta, 2009; Defago et al., 2011) such as those extracted from the neem tree Azadirachta indica A. Juss, 1830 (Meliaceae) (Charleston et al., 2005). Different compounds of kernel of this tree have been recognised for their insecticidal properties, i.e. repellent, antifeedant and growth disruptive effects which have been found among several insect species treated with neem extracts (Isman, 2006; Mikami and Ventura, 2008; Mondédji et al, 2014; Shannag et al., 2014). Neem leaves extracts are also recognised for their insecticidal properties (Afshan, 2002; da Cruz et al., 2008). The neem seeds are not available over the year but the leaves are present at any time. Neem products (AgroneemTM, EcozinTM, Azatin, NeemixTM, Margosan O, Azatrol etc.) have been developed as commercial insecticides and are available in several countries (Liang et al., 2003; Shannag et al., 2014) but not in Togo (Améwuamé, 2006). Adults of Lepidoptera play important role in host plant selection (Schoonhoven, 1987; Bernays and Weiss, 1996) but larvae are responsible of damages (Liang et al., 2003; Charleston et al., 2005). Owing to such potential of neem leaves-based preparations to control insect populations, our hypothesis is that these preparations induce larvae mortality 24 h after ingestion or are antifeedant for them and affect the emergence rate of adults. The aim of this work was to assess treatment effects of neem leaves extracts (lethal and antifeedant effects) and post-treatment effect (emergence) on 3rd instars larvae of cabbage pest P. xylostella in laboratory.
Experimental conditions and cabbage plants production
All experiments were conducted at Laboratoire d’Entomologie Appliquée of the University of Lome in Togo. Cabbage plants (B. oleracea L. var. F1 KK cross) were potted in plastic buckets of 22 cm diameter and 20 cm height containing compost and grown outdoors for six to eight weeks. They were produced under natural rainfall and sunlight conditions but not treated. The plants were covered with pieces of untreated net to protect them from insects attack at 28±3°C, 75±5% RH and a photoperiod of 12:12 h LD. At the beginning of the experience, healthy plants with 7-8 fully expanded leaves were used.
Neem leaves extract preparation
Fresh leaves of A. indica were collected from trees in the field at the University of Lome (in southern Togo). These leaves were crushed and mixed with water or ethanolic solution (10%) to obtain aqueous extract of neem leaves called neem foliar water extract (NFW) or ethanolic extract of neem leaves called neem foliar ethanolic extract (NFEtOH) respectively. For the mixture, 1 kg of crushed leaves were added to 1.5 L of water or ethanolic solution (10%). The suspension was kept overnight (12 h) at the room temperature. The suspension was then strained through muslin cloth. The extracts effects were compared with controls (C), distilled water (CW), ethanolic solution (CEtOH) and two synthetic pesticides: Conquest plus 388 EC (Acetamiprid: 16 g/L, Cypermethrin : 72 g/L and Triazophos : 300 g/L) and Cydim Super (Cypermethrin : 36 g/L and Dimethoate 400 g/L). Conquest plus (CP), a cotton culture pesticide and Cydim Super (CS) are both used on crops by producers. Conquest plus 388 EC and Cydim Super were diluted in water to obtain doses of 517 and 1017 mg/L respectively.
Rearing of P. xylostella
P. xylostella larvae were collected from field cabbages in the Research Farm of the Agronomic School of the University of Lome in Togo. Moth was reared on cabbage plants of six to eight weeks age in bucket. Each bucket containing cabbage, was covered with a section of untreated net stretched with elastic. Ten males and ten females of P. xylostella were put on each pot for 24 h. Third instars larvae were obtained after 7 to 8 days.
Treatment effects
Lethal effect
Disks of cabbage leaves (diameter: 9 cm or area: 6358.5 mm2) were cut and soaked into solutions for 20 s except negative untreated control (C) disks. After air-drying for 1 h, each disk was inserted in a Petri dish (diameter: 9 cm) containing agar gel. Ten larvae of third instars were transferred onto treated or untreated leaf disks in Petri dishes. They were then introduced in incubator with a temperature of 25°C on night and 30°C on light, a photoperiod of 12:12 h LD and RH of 35 ± 5%. Dead and survived larvae were counted after 24 h. Each treatment was replicated 6 times and each replicate consisted of 10 larvae.
Antifeedant effect
To study antifeedant effect, the surface eaten by larvae during 24 h, was evaluated through a graph paper. Each cabbage leaf placed in the presence of the larvae was withdrawn after 24 h and deposited on the graph paper. The consumed surfaces were then reproduced and estimated.
Post-treatment effects
The survived larvae were individually transferred onto untreated cabbage leaf disks every 48 h until all larvae died or became nymphs. The nymphs were monitored until they all died or until the adult emergence. In experimental conditions, adults emerged from pupae after 4 days. This experiment aimed to know post-treatment effect on larvae development. The emergence rate (Re) was calculated by the formula:
NAe is the number of emerged adults and NLs is the number of survived larvae after 24 h of exposure to the treated and the untreated leaves.
Statistical analysis
Statistical analyses were performed using STATISTICA 5.5 software (StatSoft, Tulsa, OK, USA). The comparisons of the mean numbers were made using analysis of variance (ANOVA-1) followed by Student-Newman-Keuls (SNK) comparison tests when F of analysis of variance was significant at the 5% level.
Treatment effects
Lethal effect
Both neem extracts did not exhibit a significant lethal effect on larvae although the effect varied among the treatments. For instance, 1.67 ± 1.82 to 6.67 ± 2.31% of larvae died after 24 h on leaf disks. No significant differences in mortality were observed in 3rd instars larvae on treated cabbage foliar disks compared to the controls (F(6, 34) = 0.53; df = 6; p = 0.78) (Table 1).
Antifeedant effect
The antifeedant effects of the different products were indicated by foliar consumption. Both treated and untreated cabbage leaves were consumed by P. xylostella larvae. When larvae were introduced on treated leaf disk, they did not cause noticeable foliar damage like in treated and untreated controls or Conquest plus 388 EC treatments. A significant antifeedant effect on P. xylostella larvae was observed in Cydim Super treatment. The surface of cabbage leaf disk consumed when leaves were treated with neem extracts was not significantly different from that of water, ethanolic solution and untreated control. But there was less surface consumed in CS compared to the other treatments (F(6, 35) = 4.71; df = 6; p = 0.001) (Figure 1).
Post-treatment effects on emergence
Figure 2 shows that the emergence rate of adults of P. xylostella was significantly lower in NFEtOH and NFW treatments than in the controls and the other treatments (F(6, 35) = 11.25; df = 6; p = 0).
The neem tree (Azadirachta indica A. Juss) is known to be an important source of triterpenoids (Afshan, 2002; Siddiqui et al., 2004). Effects of various neem extracts or neem-based insecticides on P. xylostella larvae have been well documented (Perera et al., 2000; Liang et al., 2003). Generally, neem extracts or neem-based insecticides are effective against P. xylostella larvae with significant lethal and antifeedant effects followed by significant reduction in food consumption. But, the 3rd instars larvae were able to cause damage on treated cabbage leaves. Liang et al. (2003) reported that neem based insecticides such as AgroneemTM, EcozinTM and NeemixTM should be applied as early as possible when larvae are neonates, or at the second instars stage to prevent noticeable foliage damage in field. In this study, neem leaves extracts did exhibit lethal or antifeedant effect, 24 h after larvae exposure. But they have a post-treatment effect on larvae resulting in adult emergence regulation. Indeed, consumption of neem extracts-treated leaves affected the development of 3rd P. xylostella larvae in reducing adult emergence. Hence, neem leaves extracts showed growth regulating effects. Seeds oil extracts, water and ethanol neem leaves extracts are known to inhibit the growth of various insects species (Charleston et al., 2005; Aggarwal and Brar, 2006; Egwurube et al., 2010; Shannag et al., 2014). It was reported that neem leaves contain numerous chemicals with insecticide properties (Afshan, 2002; Siddiqui et al., 2004). Neem triterpenoids showed growth regulating effects towards many species of insects (Schmutterer, 1995; Afshan, 2002). This is probably the synergistic effect of the chemical compounds of neem which regulates the growth of insects. It is not only the action of azadirachtin, main active compound, synthesized and marketed. Gauvin et al. (2003) showed that there was no relationship between the amount of azadirachtin and insecticidal neem extracts.
Thus, the growth regulating effects appear to be due to the effects of all insecticides substances contained in extracts of neem and not just their concentration of azadirachtin. Amtul (2014) found that A. indica derived compounds were inhibitors of digestive alpha-amylase in Tribolium castaneum. This inhibition can cause digestive trouble or their death. Results of the current study demonstrated that neem leaves extracts regulated the growth of P. xylostella 3rd instars larvae after being fed on cabbage treated leaves during 24 h.
This study revealed that the two neem extracts did not exhibit significant lethal and antifeedant effects on P. xylostella 3rd instars larvae in laboratory. However, they could enhance the management of P. xylostella by regulating its development to adult stage. Thus, extracts of neem leaves can be included in the management strategy of P. xyllostella.
The authors have not declared any conflict of interest.
The authors thank the technical staff of the Unité de Recherche en Ecotoxicologie of Université de Lomé for helping in the design of this study. Special thanks to Dr Koffi Mensah AHADJI-DABLA from Faculty of Sciences (Université de Lomé) for revising the manuscript.
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