CC BY
29Translation of Russian References
Altukhova TV, Kostyuk AV, Spiridonov YuYa, Shestakov Dospekhov BA (1979) Metodika polevogo opyta [Methods of VG, Ginevskiy NK (2005). [How to protect the corn from field experiment]. Moscow: Kolos. 416 p. (In Russian) common ragweed]. Zashchita i karantin rasteniy 7:38-39 Kuznetsova SV, Bagrintseva VN, Guba EI (2011) [The (In Russian) effectiveness of the herbicides application on corn].
Bagrintseva VN, Kuznetsova SV (2010). [Dynamic of change
Kukuruza i sorgo 1:24-27 (In Russian)
of weed cenosis in corn self-pollinated line of maize]. T, . . . ,,,,„„,„ r„, , , . .
t. ■ s Kuznetsova SV, Bagrintseva VN (2015) [Weed plants in corn
Zernovoye khozyaystvo Rossii 6 (12):51-54 (In Russian)
Bagrintseva VN, Kuznetsova SV (2011) [Comprehensive assessment of herbicides for corn]. Grain growing of Russia
crops]. Zemledelie 6:44-45 (In Russian) Filev DS, Tsikov VS, Zolotov VI, Logachev NI (1980)
1(13) 31—34 (In Russian) Metodicheskiye rekomendatsii po provedeniyu polevykh
Veletsky IN (1989) Application technology of herbicides. [2^ opytov s kukuruzoy [Guidelines for conducting field
edition Revised and apdated. L.: Agropromizdat] Leningrad. experiments with corn]. VNII kukuruzy VASKhNIL.
176 p. (In Russian) Dnepropetrovsk. 54 p.
Plant Protection News, 2019, 2(100), p. 40-45
OECD+WoS: 4.01+AM http://doi.org/10.31993/2308-6459-2019-2(100)-40-45
COMPARATIVE EFFICIENCY STUDY OF HERBICIDES IN CORN CROPS
IN STAVROPOL TERRITORY S.V. Kuznetsova, V.N. Bagrintseva*, E.I. Guba All-Russian research scientific institute of corn, Pyatigorsk, Russia
*corresponding author, e-mail: maize-techno@mail.ru
In this work the herbicide mixture effect on the weed component of agrophytocenosis during the cultivation of corn hybrids was studied. Field tests were conducted in 2017-2018 in the conditions of the sufficient moisture zone of the Stavropol region on hybrids of different ripeness. For the experiment the tank mixture of herbicides was prepared with Dublon Super (0.4 kg/ha) + Egida (0.2 l/ha) and the herbicide Cordus Plus (0.44 kg/ha). The use of the preparation and tank mixture on the basis of several active ingredients allowed expanding the range of their action and to increase the effectiveness of the treatment against the herbicides. The biological efficiency of the studied herbicides was high, i.e. from 88.6 to 99.9 %. Up to 2.13 t/ha grain yield increments were obtained. Experimental data allowed us to recommend the studied preparations to protect corn from weeds and to obtain guaranteed yield increase.
Key words: corn, herbicides, weeds, biological efficiency, yield Received: 25.02.2019 Accepted: 30.05.2019
OECD+WoS: 1.06+IY http://doi.org/10.31993/2308-6459-2019-2(100)-45-51
Full-text Article
DETECTION OF MICROSPORIDIA INFECTING BEET WEBWORM LOXOSTEGE STICTICALIS (PYRALOIDEA: CRAMBIDAE) IN EUROPEAN PART OF RUSSIA IN 2006-2008
J.M. Malysh*, A.G. Kononchuk, A.N. Frolov
All-Russian Institute of Plant Protection, St. Petersburg, Russia
* corresponding author, e-mail: malyshjm@mail.ru
The beet webworm Loxostege sticticalis (L.) is a major insect pest that causes serious damage of agricultural crops in Russia, China and adjacent countries. Microsporidia are obligate intracellular parasites that negatively affect population density of many insect hosts including Lepidoptera. In particular, infection with microsporidia is an important mortality factor for L. sticticalis. Special methodology for the identification of microsporidia associated with terrestrial insects is required. In the present paper we report the results of screening beet webworm moths for microsporidia using two techniques, i.e. light microscopy (LM) and PCR. Adult moths were sampled in 2006-2008 in the European part of Russia: Rostov Region, Krasnodar Territory and Republic of Bashkortostan. Microsporidia infections were detected in insects collected from all sampling sites. Examination of smears by LM showed presence of microsporidian spores in 3.4 %% of samples (N=98). PCR analysis of the same dataset was positive in 6.7 %% of samples, including those containing and not containing spores. The higher infection rate determined by PCR is likely connected with the fact that only mature spores
can be unequivocally identified by LM, whereas PCR also allows detection of otherdevelopmental stages of microsporidia. Partial sequencing of an amplicon from Krasnodar Territory showed its close relatedness to Endoreticulatus poecilomonae from Poecilimon thoracicus Fieber (Orthoptera: Tettigoniidae).
Key words: microsporidia, beet webworm, diagnostics, fluorescent microscopy, DAPI, PCR, rDNA, Endoreticulatus Received: 04.02.2019 Accepted: 30.05.2019
Introduction
The beet webworm Loxostege (Pyrausta) sticticalis L. is an important outbreak pest, causing serious damage to the crops such as soybean, sugar beet, alfalfa, sunflower and other crops in Eurasia, including Northern China and steppe zones of European and Asian parts of Russia (Chen Xiao et al., 2008; Frolov et al., 2008). Recent studies have shown that the insect population density correlated with the prevalence of microsporidian infection in the previous generation, suggesting that microsporidia is an important factor of the regulation of beet webworm populations in nature (Frolov et al., 2008). Different species of microsporidia are able to infect beet webworm, including Nosema loxostegi (Issi et al, 1980), Tubulinosema sp., Nosema sp., Nosema ceranae (Malysh et al., 2018), Tubulinosema loxostegi (Malysh et al., 2013b) and Vairimorpha thomsoni (Malysh et al., 2013a).
The criterion of microsporidian infection rate is included into the specified forecast model of this dangerous agricultural pest (Malysh, 2006), substantiating the need for a reliable, fast and sensitive technique for detection of microsporidian infection. Light microscopic observation of microsporidian spores in insect tissue samples may reflect only a part of the infected individuals (Sokolova et al., 2004), but this technique is important to reveal the infection at the sporogonial stage of the parasite's development. Molecular detection is more sensitive and provides specified data on parasite prevalence, species composition and genetic polymorphism.
In the present paper, we report the results of screening of L. sticticalis adults collected in 2006-2008 at three sample sites located in European part of Russia for microsporidia using light microscopy (LM) and PCR and demonstrate infection with microsporidia belonging to the genus Endoreticulatus.
Materials
Adult moths of beet webworm were caught by net at three sampling sites in the European part of Russia: (a) pastures in Salsk District, Rostov Region, in July 2006; (b) meadows in Slavyansk District, Krasnodar Territory, in July 2007 and (c) a sugar beet field in Ufa District, Republic of Bashkortostan in August 2008 (Fig. 1). Moth were stored dried or fixed in ethanol (samples from Ufa) for 2-3 months at room temperature prior to analysis. For light microscopy (LM),
and Methods
moths were homogenized individually in 50 ^l of distilled water, smears were prepared and the homogenates were stored frozen at -22 °C for subsequent DNA extraction. Smears were examined using bright field light microscopy. The smears suspected for the presence of microsporidian spores were dried, fixed with methanol, stained with 5 ^M aqueous diamidine phenylenindole (DAPI) solution and examined
Figure 1. Loxostege sticticalis sampling sites: (1) Salsk District, Rostov Region, July 2006; (2) Slavyansk District, Krasnodar Territory, July 2007; (3) Ufa District, Republic of Bashkortostan August 2008
using Carl Zeiss Axioscope-2 equipped with epifluorescence and a digital camera (Tokarev et al., 2007).
For genomic DNA extraction, moth homogenates were re-homogenized with a plastic pestle in 1.5 mL microcentrifuge tubes in 100 ^l lysis buffer, containing 2 % CTAB, 1.4 M NaCl, 100 mM EDTA, 100 mM Tris-Cl (pH 8.0). After homogenization, 500 ^l lysis buffer with 0.2 % ß-mercaptoethanol and 10 ^l proteinase K (20 mg mL-1) were added to the samples and incubated for 3 hrs at 65 °C. DNA was further extracted with phenol-chloroform, precipitated with isopropanol and washed with 70 % ethanol (Sambrook et al., 1989). Dried DNA pellets were resuspended in 50 ^l of deionized molecular grade water. PCR was run using a BioRad iCycler in 10 ^l volume containing 5 ^l DNA template, PCR buffer, 0.25 mM dNTPs; 1 U Taq-polymerase (Sileks, Russia), and 0.5 pMol each of forward and reverse primers (Evrogen, Russia). We used 18f (forward) primer combined with one of the reverse primers (specific to small (ss) and large (ls) subunits of rRNA): ss530r, ss1492r or ls580r to obtain fragments ~500 bp, ~1200 bp or ~1800 bp long, respectively (Weiss, Vossbrinck, 1999). The PCR conditions consisted of an initial denaturation step (95 °C for 3 min), 30 amplification cycles (denaturation at 95 °C for 30 sec; annealing at 54 °C for 30 sec, elongation at 72 °C for 30-60 sec) and a final extension step (72 °C for 10 min). The PCR products were separated in 1 % agarose gels. One of the amplicons was gel purified, cloned in pAL-TA vector (Evrogen, Russia) and sequenced to confirm the specificity of the amplified band.
A new sequence was corrected manually in BioEdit and compared with Genbank entries using BLAST utility. Genbank (http://www.ncbi.nlm.nih.gov.nuccore/) was used to extract nucleotide sequence data for SSU rRNA gene of Endoreticulatus isolates (Table 1). The nucleotide sequences were aligned in BioEdit (Hall, 1999), nucleotide sequence similarity and pairwise genetic distances were calculated using built-in utilities of BioEdit. We used MEGA 7 software to align sequences of SSU rRNA gene of 7 isolates of genus Endoreticulatus from insects and Vittaforma corneae U11046 as the outgroup.
Phylogenetic analysis was undertaken using the Maximum Likelihood method in MEGA 7 with 500 iterations (Kumar et al., 2016). Hasegawa-Kishino-Yano (HKY) model (Hasegawa et al., 1985) was chosen using MEGA 7 built-in utility ("Find best DNA/protein models") to estimate the nucleotide substitution rate. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1799)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 42.09 % sites).
There were a total of 1150 positions in the final dataset. Table 1. Genbank-accessible isolates of genus Endoreticulatus from insects
# Microsporidia species/isolate Host Locality Genbank accession #
1 Endoreticulatus schubergi Euproctis chrysorrhoea (Lepidoptera: Noctuoidea) United States L39109
2 Endoreticulatus bombycis Bombyx mori L. (Lepidoptera: Bombycoidea) China AY009115
3 Endoreticulatus sp. Shengzhou Bombyx mori L. (Lepidoptera: Bombycoidea) Zhejiang, China JN688870
4 Endoreticulatus sp. Zhenjiang Bombyx mori L. (Lepidoptera: Bombycoidea) China FJ772431
5 Endoreticulatus sp. CHW-2004 Taiwan Ocinara lida Moore (Lepidoptera: Bombycoidea) Taiwan AY502944
6 Endoreticulatus sp. CHW-2004 Bulgaria Lymantria dispar L. (Lepidoptera: Noctuoidea) Bulgaria AY502945
7 Pleistophora sp.* Spodoptera litura Fabricius (Lepidoptera: Noctuoidea) Japan: Tokyo, Ogasawara LC052198
8 Endoreticulatus sp. Thaumetopoea processionea L. (Lepidoptera: Noctuoidea) Austria EU260046
9 Pleistophora sp. Sd-N-IW8201 Bombyx mori L. (Lepidoptera: Bombycoidea) Japan D85500
10 Pleistophora sp. OSL-2015-2 Spodoptera litura Fabricius (Lepidoptera: Noctuoidea) Japan:Tokyo, Ogasawara LC422312
11 Endoreticulatus poecilimonae Poecilimon thoracicus Fieber (Orthoptera: Tettigoniidae) Bulgaria KJ755827
12 Endoreticulatus sp. JMM-2007 Loxostege sticticalis (L.) (Lepidoptera: Pyraloidea) Russia, Krasnodar Territory MK929470
13 Endoreticulatus sp. Melnik Euproctis chrysorrhoea L. (Lepidoptera: Noctuoidea) Bulgaria KU900486
14 Endoreticulatus sp. Sofia Euproctis chrysorrhoea L. (Lepidoptera: Noctuoidea) Bulgaria KU900485
15 Endoreticulatus sp. WFH-2014b Listronotus bonariensis Kuschel (Coleoptera: Curculionoidea) New Zealand KJ755828
*Pleistophora was a collective genus name which was redefined with description of Endoreticulatus and other genera
Results
Examination of unfixed smears by LM revealed microsporidia spores in one moth in each of the three samples from 2006 (N=28), 2007 (N=25) and 2008 (N=36). Therefore, the prevalence of infection estimated by LM ranged from 2.8 % to 4.0 % (Table 2). DAPI staining confirmed the diagnosis, clearly demonstrating intensively stained double nuclei in these spores (Fig. 2). When PCR was applied for detection of microsporidia within the same samplings, positive signals (amplified band of expected size) were obtained for all LM-positive samples using each of the three primer combinations. In addition, one LM-negative sample per each of the three sampling appeared to be PCR-positive when 18f:530r (not shown) or 18f:1492r, but not 18f:ls580r primer sets, were used (Fig. 3). Thus, the prevalence rate of infection increased twofold, ranging from 5.6 % to 8 % (Table 1). Average prevalence rate for the whole dataset (N=89) was 3.5±0.35 % and 6.7±0.70 % when estimated using LM and PCR respectively (Table 2).
Figure 2. DAPI staining of microsporidia spores revealed in beet webworm adults from Rostov Region in 2006
Forward-end sequencing of one of the amplicons obtained using 18f:1492r primer set resulted in a ~462 bp sequence with 99.7 % sequence similarity with Endoreticulatus poecilimonae (Genbank accession # KJ755827) and ~94 % similarity with Endoreticulatus schubergi (# L39109) and Endoreticulatus bombycis (# AY009115). The majority of isolates from Lepidoptera was very similar to E. bombycis and E. schubergi
(Table 3). The variable region sequenced for E. poecilimonae h Endoreticulatus sp. JMM-2007 (294 bp from 858 to 1152 position) contained 17 SNPs which were different from all other isolates but identical in these two isolates. However, similarity of these two isolates is slightly below 100 % because of a single indel at position 1153 which was not reproduced in all other sequences assayed (Fig. 4) as well as in outgroup (not shown).
In the phylogram, E. bombycis and E. schubergi clustered with other isolates from Lepidoptera. Endoreticulatus sp. WFH 2014b from the Argentine stem weevil Listronotus bonariensis (# KJ755828) was in a basal position to this cluster while E. poecilimonae h Endoreticulatus sp. JMM-2007 formed a branch with 100 % bootstrap support forming a sister-group relationships withthe entire aforementioned group (Fig. 5).
Figure 3. Electrophoretic profiles of amplification products
using primers 18f:ss1492r (A) and 18f:ls580r (B) with samples of DNA, extracted from the beet webworm adults: "+" - LM-positive sample; "-" - LM-negative sample, "M" - molecular weight marker
Table 2. Microsporidia prevalence rates in beet webworm adults estimated using light microscopy and PCR
Sampling site, year Number of analyzed insects, N micro n Positive samples scopy % (n/N) estimated using PC n R % (n/N)
Rostov Region, 2006 Krasnodar Territory, 2007 Republic of Bashkortostan, 2008 Total/Average(±standard error) 28 25 36 89 1 1 1 3 3.6 4.0 2.8 3.5±0.35 2 2 2 6 7.1 8 5.6 6.7±0.70
Figure 4. Alignments of nucleotide sequences of SSU rRNA gene of Endoreticulatus isolates. Ovals show the positions of Endoreticulatus poecilimonae and Endoreticulatus sp. JMM-2007 which are identical in these two isolates
but different from the other ones
Table 3. Sequence similarity of SSU rRNA gene of Endoreticulatus isolates
*Iso-late #
1 2
3
4
5
6
7
8
9
10 11 12
13
14
15
16
Isolate names (Genbank accession #) Endoreticulatus schubergi (L39109)
Endoreticulatus bombycis (AY009115)
Endoreticulatus sp. Shengzhou (JN688870)
Endoreticulatus sp. Zhenjiang (FJ772431)
Endoreticulatus sp. CHW 2004 Taiwan (AY502944) Endoreticulatus sp. CHW 2004 Bulgaria (AY502945) Pleistophora sp. (LC052198) Endoreticulatus sp. (EU260046)
Pleistophora sp. OSL 2015-2 (LC422312)
Endoreticulatus sp. Melnik (KU900486) Endoreticulatus sp. Sofia (KU900485)
Pleistophora sp. Sd Nu IW8201
(D85500)
Endoreticulatus sp.
WFH 2014b (KJ755828)
Endoreticulatus sp. JMM 2007
(MK929470)
Endoreticulatus poecilimonae (KJ755827)
Vittaforma corneae (U11046)
1
ID 98.6 98.9 98.9 98.9 98.4 98.9 99.2
98.6 99.2 99.2 96.8
95.8
93.7
93.4
86.9
2
0.011 ID 99.7 99.7
99.7 98.6 98.9 98.9 98.6 99.4 99.4
97.1
95.8
94.2
93.9 86.4
Sequence similarity (lower diagonal)
8
3
0.008 0.003 ID 100 100 98.9 99.2
99.2 98.9 99.7 99.7
97.3
96.0
94.5
94.2 86.7
4
0.008 0.003 0.000 ID 100 98.9 99.2
99.2 98.9 99.7 99.7
97.3
96.0
94.5
94.2 86.7
5
0.008 0.003 0.000 0.000 ID 98.9 99.2
99.2 98.9 99.7 99.7
97.3
96.0
94.5
94.2 86.7
6
0.013 0.013 0.011 0.011 0.011 ID 99.2
98.6 98.9 99.2 99.2
96.8
96.0
93.9
93.7
86.1
7
0.008 0.011 0.008 0.008 0.008 0.008 ID 98.9 99.7 99.4
99.4 97.1 96.3 94.7
94.5 86.7
0.005 0.011 0.008 0.008 0.008 0.013 0.011 ID
98.6 99.4 99.4 97.1 96.0
93.7
93.4 86.4
and genetic distance (upper diagonal)
9 10 11 12 13 I 14 I 15 I 16
0.011 0.005 0.005 0.014 0.041 0.064 0.064 0.137
0.013 0.005 0.005 0.013 0.043 0.061 0.061 0.147
0.011 0.003 0.003 0.011 0.041 0.058 0.058 0.144
0.011 0.003 0.003 0.011 0.041 0.058 0.058 0.144
0.011 0.003 0.003 0.011 0.041 0.058 0.058 0.144
0.011 0.008 0.008 0.016 0.041 0.064 0.064 0.150
0.003 0.005 0.005 0.013 0.038 0.055 0.055 0.143
0.013 0.005 0.005 0.013 0.040 0.067 0.067 0.147
ID 0.008 0.008 0.016 0.041 0.058 0.058 0.146
99.2 ID 0.000 0.008 0.038 0.061 0.061 0.147
99.2 100 ID 0.008 0.038 0.061 0.061 0.147
96.8 97.6 97.6 ID 0.041 0.068 0.068 0.149
96.0 96.3 96.3 94.5 ID 0.076 0.076 0.167
94.5 94.2 94.2 92.1 92.9 ID 0.000 0.137
94.2 93.9 93.9 91.9 92.6 99.7 ID 0.137 86.4 86.4 86.4 84.9 84.8 87.2 87.0 ID
* as given in Table 1
Endoreticulatus schuberqi L3910Q
-f- DtfWBfrCldWuS Sp h UmXMIi
— Endoreticulatus bombycis A YO09115 Endoreticulatus sp CHW2004 Bulgaria AY502345 -f-nrirjmfira/Mïis sp WhH VU 14b <0755829
- Endoreticulatus JMM 2007
I Endoreticulatus poecilomonae KJI55S27 -m/nsae 1111(14ft
Figure 5. Molecular phylogenetic analysis by Maximum Likelihood method based on the Hasegawa-Kishino-Yano model (see Materials and Methods)
Discussion
It has been previously shown that the sensitivity of PCR is higher for detection of microsporidia (Sokolova et al., 2004) in comparison with LM. In our study, PCR showed presence of infection in samples both containing and not containing spores. Thus, the PCR is a reliable tool for microsporidia detection even when the spores have not been formed in mass yet to be visualized by LM. Notably, comparatively long DNA fragments (~1800 bp long, amplified using 18f:ls580r in the present study) are well preserved only within the spore
containing samples while shorter fragments can be amplified from samples not containing spores. Amplification of short fragments is useful for sensitive detection of the infection (Franzen, Müller, 2001) while longer fragments are required for precise species identification and elucidation of subtle genetic differences between closely related species (Tokarev, Issi, 2018). Meanwhile, microscopic examination of insect tissues remains an important part of the investigation process to estimate prevalence rate of patent infections, demonstrate spore morphology and collect spores for bioassays and other purposes.
Endoreticulatus is a genus of microsporidia infecting insects from different orders (Pilarska et al., 2015), but the majority of findings are associated with Lepidoptera. For example, E. schubergi was found in Lymantria dispar, Hyphantria cunea and Choristoneura fumiferana (Zwölfer, 1927; Cali, El Gary, 1991) and E. bombycis was described from Bombyx mori (Zhang et al., 1995). Several isolates of Endoreticulatus with unclear taxonomic position (lacking data sufficient for new species description) were reported from Euproctis chrysorrhoea (Pilarska et al., 2002), Ocinara lida (Wang et al., 2005), Thaumetopoeaprocessionea (Hoch et al., 2008) and Eilema complana (Pilarska, 2017). All these insect hosts belong to Bombycoidea or Noctuoidea. Therefore, the finding of an isolate of Endoreticulatus in L. sticticalis extends the host range of this parasite genus to Pyraloidea.
In the first round of bioinformatics analysis performed in 2008, there were no Genbank entries showing 100 % identity to the newly found rDNA haplotype. Among described species, only two (E. schubergi and E. bombycis) were genotyped and their SSU rRNA gene sequence similarity was about 94 % as compared to the microsporidium found in Krasnodar Territory. These results were briefly mentioned in a presentation given at 37th Annual Meeting of the Society of Invertebrate Pathology in 2010 and in a methodological paper (Tokarev et al., 2012) and a review (Malysh et al., 2013a), both papers published locally and with some mistakes. For example, in the review paper the spore morphotype from Krasnodar Territory is incorrectly indicated as the morphotype for Novosibirsk Region (Malysh et al., 2013a, Fig. 1A) and vice versa, the morphotype from the latter sampling site is designated as the one from the former sampling site (ibidem, Fig. 2A). More recent examination of all information accumulated after that time showed identical trends in microsporidia presence and
sensitivity of the two different detection methods using three independent datasets from three years of study. Moreover, high level of sequence similarity of the microsporidia from L. sticticalis to E. poecilimonae from the bellied bright bush-cricket Poecilimon thoracicus (# KJ755827) was also observed. Although the properly read DNA fragment was short, the sequenced region was variable among species and all other sequences showed similarity below 94 % when compared to the parasites of the bellied bright bush-cricket and the beet webworm (Table 3). Based on this, we suggest that the beet webworm microsporidium belongs to the same species as E. poecilimonae, or at least to a group of species which are more closely related to each other than to E. schubergi and E. bombycis (Fig. 5).
Further studies are needed to elucidate the genetic borders between the Endoreticulatus species from different hosts and localities.
The authors are thankful to anonymous reviewers for their valuable suggestions for manuscript improvement and Yuri Tokarev (All-Russian Institute of Plant Protection) for English translation.
The research is supported by Russian Foundation of Basic Research, grant # 17-04-00871.
References
Cali A, El Garhy M (1991) Ultrastructural study of the development of Pleistophora schubergi Zwölfer, 1927 (Protozoa, Microsporida) in larvae of the spruce budworm, Choristoneura fumiferana and its subsequent taxonomic change to the genus Endoreticulatus. J Eukaryot Microbiol 38:271-278
Chen X, Zhai B, Gong R, Yin M et al (2008) Source area of spring population of meadow moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae), in Northeast China. Acta Ecol Sin 28(4):1521-1535. http://doi.org/10.1016/ S1872-2032(08)60054-2 Franzen C, Müller A (2001) Microsporidiosis: human diseases
and diagnosis. Microbes Infect 3(5):389-400 Frolov AN, Malysh YuM, Tokarev YuS (2008) Biological features and population density forecasts of the beet webworm Pyrausta sticticalis L. (Lepidoptera, Pyraustidae) in the period of low population density of the pest in Krasnodar Territory. EntomolRev 88(6):666-675. http://doi. org/10.1134/S0013873808060055 Hasegawa M, Kishino H, Yano T (1985) Dating the human-ape split by a molecular clock of mitochondrial DNA. J Mol Evol 22:160-174. Hoch G, Verucci S, Schopf A (2008) Microsporidian pathogens of the oak processionary moth Thaumetopoea processionea (L.) (Lep., Thaumetopoeidae), in eastern Austria. Mitt Dtsch Ges Allg Angew Ent 16:225-228 Issi IV, Simchuk PM, Radishcheva DF (1980) [Microsporidiosis of beet webworm Loxostege sticticalis L. (Lepidoptera, Pyraloidae)]. Bulleten VIZR 48:3-6 (In Russian) Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870-1874. http://doi. org/10.1093/molbev/msw054 Malysh JM (2006) Features of the beet webworm biology during its low population density in Northern Caucasus. PhD thesis, St. Petersburg. 175 p. Malysh JM, Ignatieva AN, Artokhin KS, Frolov AN, Tokarev YS (2018) Natural infection of the beet webworm
Loxostege sticticalis L. (Lepidoptera: Crambidae) with three Microsporidia and host switching in Nosema ceranae. Parasitol Res 117(9):3039-3044. https://doi.org/10.1007/ s00436-018-5987-3 Malysh JM, Kononchuk AG, Nurzhanov AA, Frolov AN et al (2013 a) Morphological and genetic diversity of microsporidia infecting beet webworm Loxostege sticticalis (L.) (Pyraloidea, Crambidae) in Russia. Eurasian Entomol J 12(6):543-548
Malysh JM, Tokarev YS, Frolov AN, Issi IV et al (2013b) Tubulinosema loxostegi sp. n. (Microsporidia: Tubulinosematidae) from the beet webworm Loxostege sticticalis L. (Lepidoptera: Crambidae) in Western Siberia. Acta Protozool 52(4):299-308. http://doi.org/10.4467/1689 0027AP.13.027.1319 Pilarska D, Linde A, Solter L, McManus M, Takov D (2002) New data on the biology of the microsporidium Endoreticulatus schubergi infecting the browntail moth Euproctis chrysorhoea (Lepidopter: Lymantriidae). Acta Zool Bulg 54:55-62 Pilarska DK, Radek R, Huang WF, Takov DI et al (2015) Review of the genus Endoreticulatus (Microsporidia, Encephalitozoonidae) with description of a new species isolated from the grasshopper Poecilimon thoracicus (Orthoptera: Tettigoniidae) and transfer of Microsporidium itiiti Malone to the genus. J Invertebr Pathol 124:23-30. http://doi.org/10.1016/jjip.2014.09.007 Pilarska D, Takov D, Hylis M, Radek R, Fiala I, Solter L, Linde A (2017) Natural occurrence of microsporidia infecting Lepidoptera in Bulgaria. Acta Parasitol 62(4):858-869. http://doi.org/10.1515/ap-2017-0104 Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory.
Sokolova YuYa, Sokolov IM, Fuxa JR (2004) Identification of Microsporidia infections in nature: light microscopy or PCR? Protistology 3:273-281
Tokarev YS, Malysh JM, Frolov AN (2012) [Modern approaches to detection ofinsect pathogens when processing adults of Lepidoptera]. Metody fitosanitarnogo monitoringa i prognoza. St. Petersburg: Innovatsionnyy tsentr zashchity rasteniy. 15-21 (In Russian)
Tokarev YS, Issi IV (2018) Molecular phylogeny in modern systematics of microsporidia. Mikologiya i Fitopatologiya 52(1):5-21
Tokarev YS, Sokolova YY, Entzeroth R (2007) Microsporidia-insect host interactions: teratoid sporogony at the sites of host tissue melanization. J Invertebr Pathol 94:70-73. http://doi.org/10.1016/jjip.2006.08.006
Вестник защиты растений, 2019, 2(100), с. 45-51 OECD+WoS: 1.06+IY
Wang CY, Solter LF, T'sui WH, Wang CH (2005) An Endoreticulatus species from Ocinara lida (Lepidoptera: Bombycidae) in Taiwan. J Invertebr Pathol 89:123-135 Weiss LM, Vossbrinck CR (1999) Microsporidian molecular biology and phylogeny. In: Wittner M (Ed.) The microsporidia and microsporidiosis. Washington DC. 129-171 Zhang L, Du Y, Wan YJ (1995) A new species of Endoreticulatus (Protozoa, Microspora) from the larva of silkworm Bombyx mori. Xinan Nongye DaxueXuebao 17 (4):363-365 Zwölfer W (1927) Die Pebrie des Schwammspinner und Goldafters, eine neue wirtschaftlich bedeutungsvolle Infektionskrankheit. ZAngew Entomol 12:498-500
http://doi.org/10.31993/2308-6459-2019-2(100)-45-51
Полнотекстовая статья
ВЫЯВЛЕНИЕ МИКРОСПОРИДИИ, ЗАРАЖАЮЩИХ ЛУГОВОГО МОТЫЛЬКА LOXOSTEGE STICTICALIS (PYRALOIDEA: CRAMBIDAE) В ЕВРОПЕЙСКОЙ ЧАСТИ РОССИИ В 2006-2008 ГОДАХ
Ю.М. Малыш*, А.Г. Конончук, А.Н. Фролов
Всероссийский научно-исследовательский институт защиты растений, Санкт-Петербург
*ответственный за переписку, e-mail: malyshjm@mail.ru
Луговой мотылёк Loxostege sticticalis (L.) - опасный вредитель сельскохозяйственных культур в России, Китае и соседних странах. Микроспоридии - облигатные внутриклеточные паразиты, которые отрицательно влияют на плотность популяций многих видов насекомых, включая чешуекрылых. В частности, заражение микроспоридиями - важный фактор смертности L. sticticalis. Для идентификации микроспоридий наземных насекомых требуются специальные методические подходы. В настоящей работе мы описываем результаты мониторинга микроспоридий в имаго лугового мотылька с помощью двух методик - световой микроскопии и ПЦР. Имаго собирали в 2006-2008 в Европейской части России: Ростовской области, Краснодарском крае и в Республике Башкортостан. Заражение микроспоридиями выявлено в насекомых, собранных во всех точках сбора. Исследование мазков с помощью световой микроскопии показало присутствие спор микроспоридий в 3.4 % образцов (N=98). ПЦР-анализ этой же выборки был положителен для 6.7 % образцов, включая таковые, содержащие и не содержащие споры. Более высокие показатели зараженности, выявленные с помощью ПЦР, скорее всего, связаны с тем, что только зрелые споры могут быть однозначно идентифицированы с помощью световой микроскопии, тогда как ПЦР позволяет выявлять другие стадии развития микроспоридий. Частичное секвенирование ампликона из Краснодарского края показало его высокое родство с Endoreticulatus poecilomonae из Poecilimon thoracicus Fieber (Orthoptera: Tettigoniidae).
Ключевые слова: микроспоридии, луговой мотылёк, диагностика, флюоресцентная микроскопия, ДАФИ, ПЦР, рДНК, Endoreticulatus
Поступила в редакцию: 04.02.2019
Принята к печати: 30.05.2019
