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M.B.Udalov*, A.V.Poskryakov*, G.V.Benkovskaya*, I.A.Tulaeva**,
M.K.Barinov**, and A.G.Nikolenko*
*Institute of Biochemistry and Genetics, USC RAS, 450054,
Russia, Bashkortostan, Ufa, prospekt Oktyabrya, bild 69, e-mail: udalov-m@yandex.ru,
YFor Correspondence
**All-Russia Institute of Plant Protection, 196608, Russia,
St. Petersburg-Pushkin, 3 Podbelsky shosse, e-mail: barimaksim@yandex.ru
INTRODUCTION The spider mite Tetranychus urticae - one of the major phytophagous pests, alterating cultivated plants. Among the host plants of this pest there are various vegetables and decorative glass-house cultures, fruit-trees, grape and cotton-plants. High fecundity, exceptional ecological plasticity of mite and its capability to give more than one generation during the season makes it an extraordinary harmfull object. On this reason mite capable at short periods to form the high level acaricides resistance that vastly reduces efficiency of the chemical method of plant protection (Smirnova, 1968, Smirnova et all. 1972, Kornilov et all. 1975). In connection with aforesaid extremely actual is searching for simple and reliable methods, allowing reveal the trend to resistance forming in natural populations of the pest.
The biochemical mechanisms of resistance more varied and hang from chemical nature of toxicant. However, in any event, change of sensitivity is followed by the change of pest populations genetic structure.
In this work is shown the intercoupling between resistance forming in common spider mite to two acaricides of different chemical nature, demitan and talstar, and changes in the resistant individuals genome, as well as possibility of the detection these typical changes by means of amplification of the free sequences DNA in the polymerase chain reaction (RAPD-PCR). This method, for instance, is successfully used with morphometric methods for comparison between morphological different strains of spider mite (Hance et all. 1998) and is the most perspective for complex genome estimation inwardly and between populations, including for resistance study.
MATERIALS AND METHODS
Rearing conditions
The breeding and selection spider mite were conducted in laboratory of
ecotoxicology of VIZR. Two strains of common spider mite Tetranichus
urticae Koch were used for study, resistant to demitan and talstar
(RD and RT, accordingly), as well as sensitive checking strain (SS). The
material for under investigation strains was received from production
glasshouses of the Leningrad area.
The families of the mite were kept is insulated one from another. For this were used first leaves of the young beans plants decomposable on humid cotton wool in glass Petry dishes under constant temperature +25° C and photoperiodic 18:6 h L:D
Used acaricides
Demitan - 20% suspension concentrate. The active substance is phenasahine
(4-tret-butilfenethylhinazolin-4-il). Demitan - contact acaricide, acting
on movable stages of the mite and possessing by expressed ovicide effect.
Talstar - 10 % emulsive concentrate. The active material - bifentryne (2-methyl-/1,1-bifenil/-3-il)-methyl-3(2-hlor-3,3,3-tri-fluoro-1-propenil)-2,2-dimethyl cyclopropanecarboxylate). Talstar - contact insectoacaricide with broad spectrum of the action.
Methods of the treatment and selection of resistant
individuals
Treatment with acaricides was realized by method of the submersion
of leaf bits from beans with mite in solution of the preparation with
discriminative concentrations (0.0036% on acting material for demitan
and 0.002% for talstar). The account of mortality was conducted through
day after treatment by demitan and on the third day after treatment by
talstar.
Constant family selection in RD and RT strains was winnowed In the course of breeding. After treatment with discriminative concentration females from the families in which was noted minimum of mortality, were transferred for the further breeding separately. The offspring of each female was tested and analyzed individually. Analogically was winnowed selection of susceptible strains (SS), with that only difference that for the further breeding were selected females from families, in which was noted maximum of mortality.
DNA extraction and PCR reaction
DNA extraction and PCR-amplification were performed in the laboratory
of adaptive biochemistry of insects, Institute of Biochemistry and Genetics
of Russian academy of sciences, Ufa. Total DNA extraction carried out
in 1.5 ml tubes by described by Chomezynski et all. (1987) with
some modifications. Pulled mites (30 females from one clone) were homogenized
in 400 µl of extraction buffer, containing 4 M guanidinum thiocyanate,
25 mM sodium citrate, 100 mM 2-mercaptoethanol and 0.5% sodium sarkosyl,
TRIS-HCl-buffer pH 8.0 up to 0.1 M. Samples were incubated at 0° Cfor
20 min, vortexed energically for 15 min with 400 µl phenol, pH 8.0/400
µl chloroform: isoamil alcohol (24:1 v/v) and cenrifugated for 15 min
at 10000 g. The supernatant (400 µl) was transferred to a new tube, vortexed
again for 5 min with 200 µl chloroform and cenrifugated for 5 min. After
centrifugation, 400 µl of the supernatant was transferred to a new tube
and DNA was precipitated with 400 µl cold (-20° C) 96% EtOH for 24 h.
Following the cenrifugation for 20 min the precipitate was washed with
200 µl 70 % EtOH, dried and dissolved in 50 µl of bidistilled water. DNA
samples stored at -20° C.
Polymerase chain reaction (PCR) were carried out in a total volume of 30 µl, containing 2 µl of mite DNA solution. The reaction buffer consists of 10 pM of primer (Sintol, Russia), 250 mkM each of dNTP, (Fermentas, Lithua), 1*Taq buffer (10 mM TRIS-HCl, pH 8.8, 50 mM KCl, 2.5 mM MgCl) and 2.5 U Taq polymerase (Silex, Russia). Reaction mix was topped with a drop of sterile mineral oil. PCR amplification of RAPD markers was performed in thermal cycler "Cycloterm" (Russia) under the following cycle conditions: five cycles of 94° C - 1 min for denaturing the DNA, 34° C- 2 min for annealing and 72° C- 2 min for elongation; 25 cycles of 94° C- 1 min, 42° C- 2 min, 72° C- 2 min. An additional 7 min at 72° C was allowed for last strand elongation.
After amplification, 10 µl product was separated by electrophoreses in 1.5% agarose gels (18*10 cm) in 1xTAE buffer for 1 h at 70 mA. Gels were stained with ethidium bromide and the DNA was visualized by fluorescence UV-light (312 nm) transilluminator TM-36 and photographed.
Data analyses
Amplification products were scored as discrete and binary stares (present
/absent or 1/0) for each individual samples. A data matrix of "individuals´bands"
containing the band scoring information was calculated using the simple
matching coefficient described by Sokal et all. (1958) and were given
by:
were a is the presence of the marker in both bands, d - is absence in both bands and b and c are markers present in one and absent in the other bands (Hance et all. 1998). This dataset was used to calculate the genetic diversity among individual trees using the Statistica (v.5.0) software package. The clustering was performed on Ward's method, as a distance measure were used Euclidean distances.
Coefficient biodiversity between strain was calculated by Shannon-Weaver index (Chalmers et all. 1992):
were Pi -frequency of i-allele in strain.
RESULTS AND DISCUSSION
Toxicological features of investigated strains
In spite of strictly individual nature of the breeding during 20 generations,
in strains detected the genetic heterogeneity (Fig.
1). In the course of family testing there periodically proceeds the
isolation of susceptible families from resistant strains and resistant
families - from susceptible ones. From tables is seen that practically
in each generation of the selection in resistant strains are present the
families with level of mortality 30-60% or below. One of the spider mites
biological particularities is the male emergence from nonfertilized eggs
and, accordingly, their haploidy, as well as dominant nature of the resistance
sign inheritance (Helle, 1962, Schulten et all. 1968). In consequence
if in strains are present the females, heterozygous on sign of resistance,
then half a males in their offspring will carry the dominant allele, but
another half - the recessive one. Under sibmating condition (crossbreeding
of individuals from offspring of one female) this promotes the conservation
an heterozygous females in strains and, consequently, periodic appearance
of susceptible individuals in resistant females offspring.
RAPD-PCR results
We analyzed RAPDs in search of molecular-genetic marker, allowing differentiate
these strains of the mites. Performed screening of several primers has
allowed to choose one oligonucleotide sequence (5'-GTGCTCGGC-3'), suitable
for revealing the difference between mite individuals of different strains
(Fig.2).
On the first stage we had chose 3 RAPD-fragments, allowed separate the DNA samples from mites of the different strains. The fragments A are present only in resistant individuals genomes. This fragments allow to separate the susceptible strain from resistant ones and differ the resistant strains one from another. The fragment B allows to reveal SS-strains.
On the second stage we performed cluster analysis for revealing possible heterogeneity of these strains on molecular-genetic level. On figure 3 is presented dendrogram of genetic relations for 6 analyzed DNA samples. On given dendrogram is seen clear isolation of cluster, including only susceptible strain (SS1-SS5) samples. Two other clusters contain as samples from resistant to demitan strain (RD1-RD4), as from resistant to talstar strain (RT1-RT7). This, on our glance, speaks about the genetic heterogeneity of investigated strain as evidenced by isolation of susceptible families from resistant strains and resistant families - from susceptible ones. The genetic distances on the dendrogram between families in each cluster also speak of heterogeneity of considered strains.
Such a heterogeneity in selected strains speaks about insufficiency of the use for checking in the course of selection only toxicological method.
The reduction of biodiversity level in resistant strains (comparatively susceptible one) is, in our opinion, the result of acaricides selection (Fig.4). Herewith in the course of selection the elimination of individuals with susceptible genotypes occurs that brings about reduction of the genetic diversity in populations.
Under artificial populations control it is necessary to know that any one of them has its biological optimum of biodiversity, established throughout the evolution. The mentioned optimum is limited with frames of minimum and maximum biodiversity level which are revealed in the course of perennial observations. Without knowledge of the optimum biodiversity borders in the populations there is impossible to realize some work with any population, whether reproduction or reduction of its individuals number. In particular, the biodiversity level reduction in spider mite populations (where for control of its number acaricides are using ) is a signal of the resistance development.
CONCLUSIONS In spite of uneven nature of the resistance forming in the laboratory reared common spider mite, we had received two strains with resistance factor 38,4 (RD) and 54,5 (RT). The DNA analysis of mites from these strains has shown that simultaneously with resistance forming qualitative and/or quantitative changes of the object's genome occured, well distinguished on RAPD-spectrum.
In our study analysis was conducted on one mite strain of each variant only i.e. on one SS, RD or RT genotype. The confirmation of the data obtained on the other strains, wich are resistant to insecticides mentioned, will allow to perform the molecular-biological analysis for other strains or populations of the mite, and at finding of similar marker bands to draw a conclusion about the resistance of strain or about resistant genotypes presence in analysed population.
This method of the DNA investigation can be used and at analysis of any other mite strain. When use the other primers and on the other strains line and species the other marker bands can be received, but under corresponding confirmation of obtained results all of these can be recommended for practical use.
REFERENCES
Smirnova, A.A., 1968, The condition of the question about resistance of spider mite on cotton plant to phosphororganic acaricides. // Tez. dokl. sovesch. on mite resistance to acaricides., 3-6.
Smirnova, A.A., Kornilov, V.G. and Sukhoruchenko, G.I., 1972, The resistance development to phosphororganic acaricides in common spider mite on cotton plant and chemical actions on fight with resistant populations. // Works VIZR., V. 35, 189-208.
Kornilov, V.G. and Sikina, N.B., 1975, Change of susceptibility in Tetranychus urticae Koch. to pesticides in glasshouse facilities of Leningrad area // Tez. dokl. 4 sovesch. on pest resistance to chem. sred. zasch. rast., 39-40.
Hance, T., Neuberg, P. and Noel-Lastelle, C., 1998, The use of fecundity, lobe biometry and the RAPD-PCR technique in order to compare strains of Tetranychus sp., Experimental & Applied Acarology, V. 22. 649-666.
Chomezynski, P. and Sacchi, N., 1987, Single-step method of RNA isolation by acid guanidinum thiocyanate-phenol-chlorophorm extration // Anal. Biochem., V.162. 156-159.
Chalmers, K.J., Waugh, J.I., Sprent, A.J., Simons. A.J. and Powell, W. 1992. Detection of genetic variation between and within populations of Gliricidia sepium and G. maculata using RAPD markers, Heredity, 69, 465-472.
Helle, W., 1962, Genetics of resistance to organophosphorus compounds and its relation to diapause in Tetranychus urticae Koch. (Acari)., T. Pl.-ziekten, V. 63. 155-195.
Schulten, G.G.M., 1968, Genetics of organophosphate resistance in the two-spotted spider mite (Tetranychus urticae Koch.)., Koninklijk Instituut Voor de Tropen, Amsterdam, 1-57.
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Mark
E. Whalon
Robert M. Hollingworth
Area Editors:
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Herbicide
Jonathan Gressel
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