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DEVELOPMENT OF NOVEL BIOPESTICIDES AGAINST LEPIDOPTERA PESTS

Автор Доклада: 
Khurtsidze E., Keburia N., Gachechiladze N., Gaidamashvili M.
Награда: 
DEVELOPMENT OF NOVEL BIOPESTICIDES AGAINST LEPIDOPTERA PESTS

DEVELOPMENT OF NOVEL BIOPESTICIDES AGAINST LEPIDOPTERA PESTS

Khurtsidze Ekaterine, PHD, researcher
Keburia Nino, PHD, researcher
Gachechiladze Nino, PHD, Associate Prof.
Gaidamashvili Mariam, PHD, Asssociate Prof.
Faculty of Exact and Natural Sciences, Laboratory of Plant Physiology,
Iv. Javakhishvili Tbilisi State University


The insecticidal activity of Viscum album chitin-binding lectin (MChbL) against herbivore Lepidoptera pests Apamea sordens Hufn. and Agrotis segetum Schiff. larvae was investigated. MChbL exhibited proteinase inhibitory and chitinase activities and affected larval development and survival at different growth stages. MChbL produced 40% mortality of larvae when incorporated into a artificial diet at a level of 0.001% (w/w). MChbL affected larval gut proteolytic enzymes decreasing of total midgut protease activity. MChbL showed no inhibition activity to bovin trypsin and display possible degradation by mammalian proteolytic enzymes. Possible implication of mistletoe chitin-binding lectins as potential entomotoxic biopecticide for the control of Lepidoptera pests is discussed.
Key words: Biopesticides, Chitin-binding lectin, Insecticidal activity, Lepidoptera, Viscum album.

Insects are in competition with human agro-industry. Losses in agricultural production due to insect pests have been estimated at 15% of total production worldwide, especially in those countries which are the most dependent on agriculture for their subsistence. Apamea sordens Hufn. and Agrotis segetum Schiff. are serious herbivore Lepidoptera pests on various agricultural crops causing substantial crop losses throughout the world. They are responsible also for significant damage of stored seeds and post harvest loss of agricultural production. Intensive use of agrichemicals for the pest control results in increased pest resistance and subsequent growth of pesticides applied to the fields which are toxic to humans and domestic animals and harmful to the environment. Due to the environmental concerns of pesticide use and limited list of effective alternatives it is therefore urgent to develop novel biopesticides from natural sources that have low mammalian and environmental toxicity.

Lectins are among wide range of natural defense proteins found in plants [1]. They are heterogeneous group of proteins classified together on the basis of their ability to bind in a reversible way to well-defined simple sugars and/or complex carbohydrates. The main characteristic of these proteins is their ability to interact specifically with carbohydrates and to combine with glyco-components of the cell surface. While the physiological functions of plant lectins have not yet been fully elucidated, one possible function that of serving as a chemical defense against large array of insect pests is well documented [2,3]. Insecticidal activities were found to be associated mostly with the two main groups of plant lectins: monocot mannose-binding and chitin-binding lectin groups [4,5].

In this study the chitin-binding lectin (MChbL) from European mistletoe (Viscum album) was was examined for insecticidal activity against polyphagous Lepidoptera insects Apamea sordens (Noctuidae) and Pyrausta nubilalis (Pyralididae).

Materials and Methods
The fruits of European Mistletoe were harvested in mountainous region of East-Georgia, in winter (December-February) and stored at -15°C until use. Mistletoe chitin-binding lectin (MChbL) was prepared as described with some modifications [6]. The plant material was homogenized in medium consisting of 0.05 M Na-acetate buffer, pH 4.5, at ratio 1:3 (g/ml). The extracts were centrifuged at 5000g for 15 min; supernatant was filtered through Miracloth (Calbiochem., USA) and Watman GF/c filter. The soluble protein fractions were purified by affinity chromatography on the agarose (Serva) and chitin (Sigma) sorbents, dialyzed, lyophilized and stored until use.

Chitinase activity was measured according Hou by using 0.05% deacetyl glycol chitin (glycol chitosan, Sigma) as substrate in 50 mM Na-acetate buffer (pH5.0) [7]. Chitinase activity of MChbL was compared with enzyme activity of commercial chicken egg white Lysozyme preparate (Sigma). Trypsin Inhibitor Activity was determined by a continuous rate spectrophotometric assay and expressed as the inhibition of BAEE units. Soybean trypsin inhibitor from Glycine Max (Soybean) was used as standard.

The larvae of Apamea sordens Hufn. (Hadena basilinea Schiff. (F.) and Agrotis segetum Schiff. were obtained from Khashuri region (East Georgia). Larval cultures were reared continuously at 25±1°C and relative humidity of 65-75%, under a L16/D8 light regime. To examine the effects of MChbL on insect larvae, they were maintained in plastic boxes, with perforated plastic covers and reared on a control and experimental diet with or without lectin, respectively. The lectins were incorporated into natural diet daily at 0,001% (w/w). 10-15 larvae were used per treatment. Insect survival was estimated daily, the weights of larvae and pupae were measured and the duration of developmental stages was determined. The effect of MChbL on the development was assessed by determining the number and mass of surviving larvae.

Total gut protease activity was measured by FITC-casein assay. Fluorescein isothiocyanate was purchased from Sigma Chemical Co (USA). FITC labeled casein was prepared as follows: casein (10 mg) and FITC (4 mg) were dissolved in 2 mL of 0.1M sodium carbonate buffer (pH 9.0) containing 8M urea and left for 3 h at 20C. FITC labeled casein was separated by gel chromatography on a Sephadex G-25 column (10 mL) equilibrated with 10 mM phosphate buffered saline (pH 7.5) (PBS).

Midguts were isolated by dissecting the fifth instar larvae. The gut tissue was mixed with 3 volumes of 0,1M Gly-NaOH buffer (pH10.0) and allowed to stand for 15 min on ice to extract proteases. The gut luminal contents were recovered by centrifugation at 10000g for 10 min at 4°C. The resulting supernatant was analyzed for protease assays [8]. MChbL was preincubated with gut extract at 37°C for 15 min, prior to addition of the substrate. The enzyme solution (20 μl) was added to 40 μl of FITC-casein (1 μg/ml, in 0.1M Gly-NaOH buffer (pH10.0)) and incubated at 37°C for 1 h. The reaction was stopped by adding 5 μl of 60 % trichloroacetic acid (TCA). The fluorescence polarization of samples was measured with Ex: 490 nm and Em: 520 nm.

Total gut amylase activity was measured by using 1% soluble potato starch as substrate according to Bernfeld [9]. The whole midguts were homogenized in cold distilled water (1ml/gut), and centrifuged at 10 000g for 5 min at 4°C. The supernatant was collected and used as enzyme sources for enzymatic assays. The midgut extracts were pre-incubated with MChbL at 37°C for 15 min, prior to addition of the 10 μl of substrate solution. After 1 h incubation the reaction was stopped by the addition of 300 μl of DNS coloring reagent followed by developing color. The absorbance was read at 510 nm. Maltose was used as standard and one unit of activity was defined as the amount of enzyme that produces μM of maltose/min. Each assay was carried out in triplicate.

Results and Discussion
In long-term bioassays MChbL affected the larval development as well as their survival and showed increased pupation time for both insects. The results obtained are shown in Table 1.

Table 1. The effects of MChbL on the development of A. sordens and A.segetum larvae.

 

Groups

Days to reach pupation

Rate of pupation

(%)

Rate of adults emerging from pupa (%)

A. sordens

30±1

28,6%

33%

A. segetum

25±1

10%

5%

Control

23±1

41,4%

41,7%

The rates of pupae formation as well as the rate of adults successfully emerging from pupae fed on MChbL in all experimental groups were lower than those of control insects (41.4% and 41.7%, respectively).

The effects of MChbL on surviving of the larvae at different developmental stages is shown in Fig.1. Generally, in experimental groups the larval mortality observed were higher then that of control group. The mortality of third and fourth instar larvae fed on MChbL were 36% and 50%, respectively, compare to that of control insects (83%). The results suggest that the influence of lectins were much evident at the early stages of larval development. Apparently, this is related to the more sensitivity of glycosilated gut structures of young insects to carbohydrate-binding plant lectins. At the following stages of development lectin did not show significant influence on A. sordens larvae survival. Supposedly, larvae are less susceptible to deliterious effects of lectins at their late developmental stages. Pupae period and pupae weight were not significantly different among each treatment of both insects.

Effect of MChbL on the survival of experimental larvae when incorporated into an artificial diet at 0,001%
Figure 1. Effect of MChbL on the survival of experimental larvae when incorporated into an artificial diet at 0,001% (w/w).

In the following series of experiments larvae midgut enzyme exracts were prepared and inhibitory effects of MChbL on midgut proteases and amylases activity were determined. Proteolytic activity of the midgut extracts from fifth instar larvae was measured by fluorescence polarization spectroscopy using FITC-labeled casein as substrate. The results showed that MChbL influenced, in vitro, larval gut proteolytic enzymes activity (decrease of total protease activity of the midgut extracts was monitored). The highest inhibition was 60% at a concentration of 0.25μg/μl MChbL (Fig.2).
When incubated with the insect enzymes MChbL showed resistance to digestion and no inhibition of sugar-binding activity of lectin was observed. Resistance to degradation by pest metabolic systems is clearly beneficial for plant defensive proteins, production of which represents an effective strategy developed by some plants [10].

Effect of MChbL on proteolytic activity of midgut extract from A

Figure 2. Effect of MChbL on proteolytic activity of midgut extract from A. sordens and A. segetum measured by FITC-casein assay in the presence of 0.25μg/μl MChbL. control A, FITC-casein substrate solution; control B, substrate+midgut extract; 1-2, substrate+midgut extracts preincubated with MChbL.

Effects of MChbL on amylase activity were examined using soluble starch as a substrate. The results showed that MChbL had no inhibitory effect on midgut amylases of A. sordens and A. segetum larvae. In the chitinase enzyme assay weak chitinase activity of MChbL preparate was revealed (150μM/min/mg) (data not shown). MChbL showed no trypsin inhibitory activity towards bovin trypsin, indicating the possible digestibility of MChbL by mammalian gut enzymes.

The results obtained demonstrate that mistletoe chitin-binding lectin have obvious anti-nutritive effects on Lepidoptera pests. Apparently, lectin exerts its antinutritive effect on larvae at the early stages of development by interaction with midgut structures. The precise mechanism how the lectin exerts the insecticidal activity has not been fully elucidated, however, lectins with specificity for GlcNAc residues appear to reveal toxic effects to many insects [11]. Since glycoproteins are major constituents of insect gut structures, it is possible that specific interaction take place between the glycosilated gut structures and plant lerctins. [12]. It appears that surviving the hostile proteolytic environment of the insect midgut, specific binding to insect gut chitin components and alteration of glycosylated enzymes of digestive tract are basic prerequisites for MChbL lectin to exert its deleterious effects on insects. The insecticidal activity of MChbL may be attributed to the lectin-induced reduction in diet ingestion resulting starvation of larvae. Possible implication of mistletoe chitin-binding lectin as potential entomotoxic biopecticide for the control of polyphagous herbivore Lepidoptera pests is considered.


References:
1.Rudiger, H., Gabius, H.J., (2001), Plant lectins: Occurence, biochemistry, functions and applications. Glycoconjugate J., 18: 589-613.
2.Carlini, C.R., Grossi-de-Sá M.F. (2002), Plant toxic proteins with insecticidal properties. A review on their potentialities as biopesticides. Toxicon 20: 1515-1539.
3.Vasconcelos, I.M., Oliveira, J.T. (2004). Antinutritional properties of plant lectins. Toxicon, 44: 1737-1747.
4.Fitches, E., Woodhouse S.D., Edwards, J.P., Gatehouse, J.A. (2001), In vitro and in vivo binding of snowdrop (GNA) and jackbean (Con A) lectins within tomato moth (Lacanobia oleracea) larvae; mechanisms of insecticidal action. J. Insect Physiol., 47: 777-787.
5.Ohizumi, Y., Gaidamashvili, M., Ohwada, S., Matsuda, K., Kominami, J., Nakamura-Tsuruta, S., Hirabayashi, J., Naganuma, T., Ogawa, T., Muramoto K. (2009), Mannose-binding lectin from Yam (Dioscorea batatas) Tubers with insecticidal Properties against Helicoverpa armigera (Lepidoptera: Noctuidae). J. Agri. Food. Chem. 57 (7): 2896-2902.
6.Keburia N., Alexidze G. (2001), Galactose-specific Lectins from Mistletoe (Viscum album L.) Fruits: Isolation and Some Properties. Bull. Georg. Acad. of Sci., 164, 1:118-121.
7.Hou, W.C., Chen, I.C., Lin, Y.H. (1998), Chitinase activity of sweet potato (Ipomoca batata L.) Bot.Bull.Acad.Sin. 39, 393-97.
8.Harsulkar, A.M., Giri, A.P., Patankar, A.P., et al. (1999), Successive use of non-host plant proteinase inhibitors required for effective inhibition of Helicoverpa armigera gut proteinases and larval growth. Plant Physiol., 121, 497-506.
9.Bernfeld P., et al. (1955), Amylases [alpha] and [beta]. Methods in Enzymology. Acad. Press, NY, 149.
10.Brunelle F., Cloutier, C., Michaud, M. (2004), Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato. Arch. Insect biochem. Physiol. 55:103-113.
11.Macedo M., Freire M., Da Silva M., Coelho L, (2007), Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae). Comp. Biochem. and Physiol., Part A 146: 486-497.
12.Zhu-Salzman K., et al. (1998), Carbohydrate-binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II (GSH). Proc. Natl. Acad. Sci. USA 95: 15123-15128.  

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Limitation of phytopfagous insects pests abundance

Using of natural chemical matters for decresing of phytopfagous insects abundance is exeptionally important task. In this case the polution of environment of stable toxins as chlor-organic matters, which can accumulate in live animals, is not happened. This study is one of important insign to the application of ecologicaly safety agents against abundant phytopfagous insects pests. All the best in your work, Yu. V. Dubrosky
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