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UDC 632:633
MONITORING OF VIRUS SYMPTOMS IN WINTER WHEAT VARIETY SAMPLE FROM THE COLLECTION OF ALL RUSSIAN INSTITUTE OF PLANT INDUSTRY NAMED AFTER N.I. VAVILOV
Glinushkin A.P., Candidate of Biological Sciences Orenburg State Agrarian University
Beloshapkina O.O., Doctor of Agricultural Sciences Russian State Agrarian University - Moscow Timiryazev Agricultural Academy
named after K.A. Timiryazev
Vinogradov S.V., Candidate of Biological Sciences Federal State Budget Establishment Research Center «Bioengineering» of Russian Academy of Sciences
Nikolaev N.A., Researcher Orenburg State Agrarian University
Phone: +7 (912) 359-00-18, E-mail: [email protected]
ABSTRACT
Selection of variety samples and sowing of winter wheat collection of funds N. I. Vavilov AllUnion Research Institute of Plant Industry was conducted with the objectives of the definition: the productivity and quality of grain, phytosanitary characteristics (damage by pests (vectors), the prevalence of symptoms of viral, bacterial, fungal diseases), and promising accessions in the selection process. Identification of viruses, potentially affecting the wheat plants including be-longing to the genera Tritimovirus, Furovirus performed by visual agroecological monitoring and reverse transcription-polymerase chain reaction (RT-PCR). Of wheat plants with symptoms in the total RNA was isolated using the method described Rott and Jelkman (1990), which was used for RT-PCR as a template. Were selected for detection by using information on the field genebank primer coat proteins SBWMV, WSMV and synthesized by «Synthol» Moscow. As a result, a group of visually identified accessions with no signs of defeat viral diseases: Choc, Rafale (France); Leopold (Austria); Susquehanna, Norkan (USA), Cossack, Volga-Don (Volgograd); KNIISKH 756, Bystrica, villager, Krasnodar Scientific Research Institute of Agriculture 8 ( Krasnodar); Mironovskaya 34 Vasilina (Ukraine), Orenburg 14, Besouro 2 (Orenburg) Severodonskaya 14 (Rostov) and had a score of 0 on the proposed grade (tillering) - being the prime candidates for selection. In the steppe zone of the Southern Urals, the first time RT-PCR positive identification of pathogens - virus wheat streak mosaic (WSMV) and soil-wheat mosaic virus (SBWMV).
KEY WORDS
Collection of winter wheat; Viruses; Resistance; Identification.
Nearly half infectious diseases of plants which were identified over the last decade have a viral nature [1, 3, 4, 5]. In recent years, in the Orenburg Cisurals conditions, the spread of viral diseases, which affect cereals and particular grains with high degree of harmfulness, was registered. Combined infections of different origin and etiology cause even more tangible damage [5, 6, 7]. The reasons of the emergence of new species and strains of the viruses in agrocenoses and extension of the range of their host plants are agriculture globalization, grain cultivation intensification, the expansion of international seed material exchange which contributes to the introduction of viruses into new regions. Barley yellow dwarf virus, Bromegrass mosaic virus, Wheat streak mosaic virus, Russian wheat mosaic virus, Barley stripe mosaic virus, Triticum mosaic virus are considered to be widespread on
grain crops in Russia. U. G. Volkov with his co- authors [2] determined the spread of 10 viruses on cereals in the Primorsky region in the last years. More than 60 viruses, which infected cereals were diagnosed in Europe but in the world their number exceeds 90 [90]. These viruses can cause 70% and more winter wheat crop losses, and it is confirmed that more and more cereals viruses can spread by seeds. In 12 Ukraine regions Mishchenko L.T.[8] notes the presence of 6 viruses: Wheat streak mosaic virus(WSMV), Russian wheat mosaic (RWMV), Triticum mosaic virus (TrMV), Barley yellow dwarf virus (BYDV), Brome streak mosaic virus (BrMV), Barley stripe mosaic virus (BSMV). The sources of viral diseases infections are perennial grass and weeds, winter crops and their weed seeds. The transmission of pathogens is carried by insects, mites, nematodes, fungi, and by contact-mechanical way.
Together with chemical means of plant protection against insects-vectors and weeds as virus reservation it is necessary to pay attention to the biological and selective crossbreeding methods as a component of integrated protection from pathogenic complexes. The effectiveness of the last method largely depends on proper phytosanitary examination, based on agroecological examinations and accurate diagnosis.
The Purposes of Research: to conduct visual diagnosis of virus-like symptoms in the variety samples of the winter wheat collection, to determine the molecular-genetic methods of pathogens of viral diseases, to identify perspective variety samples for their use in breeding.
RESEARCH METHODS
As object of research more than 100 winter wheat varieties of different origin from the All Russian Scientific Research Institute of Plant Breeding after N.I .Vavilov collection which were evaluated in 2007 in the experimental field of Orenburg State Agrarian University on natural infectious background were used. The investigations were carried out in spring in the tillering period, in the period of viral disease development, in the collection nursery, in 4-field selection crop rotation; with the pilot area for each variety sample -2 m2. Agro techniques - is common for this area, seeding rate is 4.0 million of viable seeds per 1 hectare, the soil is the South black soil. The study of viruses was carried out by the methods of ecological monitoring and with the reverse polymerase chain reaction.
For mass valuation of prevalence (sustainability) of winter wheat variety samples in the collection the visual diagnostics was used, noting the typical virus (or virus-like) symptoms, the prevalence of viruses and their prevalence intensity were taken into account. We have developed and used the following gradation of the variety samples according to the manifestation of virus symptoms on the plants: 0 - group - conventionally immune - without affection signs; the 1-st group - resistant - plants with affection signs up to 5%; 2-nd group -middle resistant - plants with the affection signs up to 25%; 3-rd group - less resistant - from 25 to 50%; 4-th group - susceptible - more than 50%. This gradation, in our opinion, will allow doing more efficient selection of varieties for breeding work, immunological valuation and for other purposes.
Identification of viruses, potentially affecting the winter wheat plants, belonging to Tenuiviru, Furovirus genera, was performed by visual monitoring and reverse transcription-polymerize chain reaction (RT- pCr). Total RNA was isolated from plants of winter wheat with symptoms of infection according to the method, described by Rott and Jelkman (1990), which was used for RT -PCR as a matrix. Primers on protein’s membrane and WSMV and Soil-borne wheat mosaic virus (SBWMV) were selected for detection with the help of GenBank dates. RT-PCR product was visualized using electrophoresis in 1,5% agarose gel.
RESEARCH RESULTS
The creation of winter wheat collection from the funds of the All-Russian Research Institute of Plant Industry after N. I. Vavilov allowed doing different research work. Although the main purposes of the samples selection and seed collections were the determination of
their productivity and quality of grain, conducted in the period of 2006-2007, phyto-sanitary investigations covered registration of pests (vectors) number, the prevalence of symptoms of viral, bacterial, fungal diseases. Weeds component was not specially studied, because it was poorly presented, it was connected with the predecessor (black par). Estimated damaged by pests, sucking and gnawing, conducted observations on the evaluation of resistance to brown rust and powdery mildew, root rot, bacterioses, also took into account virus-like symptoms. Further all these observations were considered as background when separating the groups of in-fluence (manifestations) of harmful organisms; theoretically calculated shares of their harmfulness on the productivity and quality of grain. In the course of investigation a lot of potential transmission vectors of viruses on winter wheat: cicadas, aphids, trips were revealed. For example, the number of cicadas in sowing of the collection in spring in phase of bushing of winter wheat (1st half of May) was significant 31.5± 10,5 pieces, and on May 20, during moving, was installed a large number of 122±18 pieces for 25 strokes by entomological net. From sucking also presented the bugs, harmful bug, from munching - fleas, flies, grain beetles, grasshoppers, and other.
Our detailed surveys demonstrate that winter wheat variety samples had different degree of manifestation of the virus symptoms (table 1).
Table 1 - Prevalence (P%) of plants with the virus symptoms in the world collection of winter wheat (visual diagnostics, Orenburg State Agrarian University, 2007)
P% Group of resistance Variety samples (origin of group or variety sample)
75± 25 (from 50 to 100%) 4-th group of susceptible Garmil, Logor,Famulus (France); Kobra susceptible (Poland); Encore (England); McNair 1587, John (USA); Samara ( Czech Republic ) ; Verna, 193 P3, 145 P2, Kolos 47, Nasledni tca, 193 R1, Volnitca (Krasnodar), Zarnitza, Don 93 (Zernograd); Mayak 267, Bezor 1, Pionerskaya 32 (Orenburg); Mironovskaya 28, Mironovskaya 32, Mironovskaya 63, Khlebodarka (Ukraine); Rodnik Tarasovskiy, Donskaya 50 (Rostov)
37,5±12,5 (from 25 to 50%) 3-d group -less resistant Alcan (France); Avir (Netherlands); Cherokee, (from 25 up small resistant Kay (USA); Georg (Austria); Rialto, Sarasen to 50%) (England); Sarka, Livia (Czech Republic); Taras, Roti (German); Goryanka (Adygeya); Krasnodarskaya 90, 146 R6, Knyazhna, Lutescens 660, Khutoryanka, Deya (Krasnodar); Svetoch (Bezenchuk); Severodonetckaya ub; Tarasovskaya ost., Avgusta (Rostov).
15±10 (from 5 to 25%) 2-nd group -middle resistant Century, TAM107; Purcam, KS89WGRC6, Cimarron (USA); Chieftain, Alert (England) ;Saskia, Samanta (Czech Republic); Satsukei 131 (Japan); Bistritza, 177 R2, (Krasnodar); Ermak (Zernograd); Lutescens 321 (Voronezh); Lutescens 80 - 08 (Saratov); Mironovskaya 31(Ukraine); Prestizh, Rosiyanka Tarasovskaya (Rostov); Splav (Vladimir)
2,5±2,5 (not more 5%) 1-st group -high resistant Aviesan (Romania ); Bersy (Netherlands) KS90WGRC10, Andrews (USA); Livius, Hubertus (Austria); Louvre (France): Viscont (England); Don 85,Zernogradka 11; Rostovchanka 3 (Zernograd); Zimorodok (Krasnodar); Krista (Latvia); Lutescens 47-01 (Saratov); Mironovskaya 33, Mironovskaya 64 (Ukraine); Mironovskaya 5/5(Estonia); Moskovskaya 39 (Moscow); Orenburgskaya 105 (Orenburg)
The use of resistant and tolerant varieties, as well as measures for the maintenance of phytosanitary condition of sowings and agro technics on the proper level can reduce the severity of viral diseases and prevent the emergence of epitophetes.
The 0-group - with no affection signs, which gives hope to receive new varieties resistant to the disease, has practical breeding value. Among local varieties in this group there were :Orenburgskaya 14, Bezor 2; Krasnodar Scientific Research Institute of Agriculture 756; Bistritca; Selyanka, 146 R6, Krasnodar Scientific Research Institute of Agriculture 8, 145 R2 (Krasnodar; Kasachok, Volga- Don (Volgograd); Severodonetckaya 14 (Rostov);Mironovskaya 34, Vasilina (Ukraine);Leopold (Austria); Susquehanna, Norkan, KS89wGrC6 (USA); Choc, Rafale (France). It should be recognized, that inductively resistant variety samples with latent infection, tolerant and really resistant variety samples can be in this group.
To symptomatic characteristics of the infected by viruses plant belong: significant stagnation in growth, increased bushing, discolored leaves, which was light green, chlorotic, anthocyan, leaves with necrotic strokes and spotting, unusually located under the acute
angle. The examined plants were often affected by several diseases of different etiology at the same time, while different interactions of pathogens and negative abiotic factors led to the weakening, or more often to symptoms increasing.
In our opinion, it is necessary to take into account not only the obvious characteristics of viral destruction in the form of discoloration, deformation of leaves, stalks, typical necrosis and dwarfism, but also the number of stems (shoots) on one plant. The developed 5-point score scale was used for the unification of the investigation results: 0 points - standard number of common stems (up to7.5); 1 point - from 7,6 up to 12 stems; 2 points - 13-20 stems; 3 points - 21-50 stems; 4 points - more than 51 stems or absence of productive stems. Table 2 demonstrates a list of variety samples without visual symptoms of viral pathogens affection, but with increased bushiness, which is also characteristic of viruses and phytoplasm.
Table 2 - Gradation according to bushiness (stem forming) of winter wheat variety samples collection with plants without symptoms of virus affection (Orenburg State Agrarian University, 2007)
Number of stems per plant Scores and group of bushiness Variety samples, origin
34±5 4-scores more than 50 stems or absence of productive stems Willi (Austria)
15±3 2-scores 12 - 20 stems GornayaPolyana (Volgograd), Mosa (USA),Sofia (Czech Republic )
10,8±1 1-point from 7,5 up to 12 GornayaPolyana (Volgograd), Mosa (USA),Sofia (Czech Republic )
8,5±1 Brigadier, Amelio (France); Mandat (England); TCEP 9038, Nelson (USA);Bezor 3 (Orenburg) Don 95(Zedrnograd); Lutescens 31-98 (Saratov) NikaCubany, Ofeliya (Krasnodar);Povolzhskaya 86 (Samara); Resurs (Bezenchuk)
5,5 ± 2 0 score standard quantity Choc, Rafale (France); Leopold (Austria); Susquestandarthanna, Norkan (USA); Kasachok, Volga-Don(Volgo-grad); Krasnodar Scientific Research Institute of Agriculture 8 (Krasnodar); Mironovskaya 34, Vasilina (Ukraine);Orenburgskaya 14, Bezor 2 (Orenburg); Severodonskaya 14 (Rostov)
Absence or weak manifestation of the signs of affection of viruses even on the second graduation could be caused by climatic factors, genetic peculiarity of variety samples that are resistant to some pathogenic organisms and parallel inducing viruses resistance . The variety Norkan containing the gene of rust resistance Lr 24 [12], had no visible signs of viral infections that is why according to the results of valuation of the collection it is marked as promising viral resistant variety for breeding , though the specific resistance may be ineffective in the protection against other groups of, grade has the gene organisms. For example, Moscovskay 39 has gene resistant to rust pathogens Lr1, however, its virus-like symptoms demonstrated at the level of 5%.
Visual immunologic evaluation of variety samples often is incorrect because of the similarity of symptoms of the virus with other affections and because of the latent nature of the infections so, more precise diagnostics of viruses and their stamp is necessary.
The study and identification of viruses are carried out by different methods - agro-ecological monitoring, indicator analysis, light and electronic microscopy, immune fluorescence, immune-enzyme analysis (IEA), disk electrophoresis, reverse polymerase chain reaction (PCR), atomic absorption spectroscopy, fluorescence induction, electrical conductivity and complete laser muller-polarimetry [1,3,4,8]. Two ways that are more often used for identification and mass diagnosis of viral pathogen s should be noted: molecular (PCR) and serological (IEA) [3,4]. In the work for viruses identification we used the method (PCR), which really proved the presence of two viruses on winter wheat These specialized studies, aimed at the identification of viral diseases of cereals in the steppe zone of the South Urals, allowed to identify not detectable earlier viruses: WSMV and SBWMV.
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A B
Figurel. Electroforegramme of products RT-PCR using primers specific to WSMV and SBWMV (B): 1-molecular weight marker (Fermentas, GeneRuler TM100 bp DNA Ladder); 2- PCR without DNA marker; 3- RT PCR matrix; 4-6 RT- PCR on DNA preparations, isolated from the analyzed samples of winter wheat 1-3, respectively: 7 - RT PCR on the DNA preparations ex-tracted from the non-infected winter wheat plants; 8 -RT PCR on the DNA preparations extracted from WSMV (A) and SBWMV (B)
infected plants
The isolated wheat streak mosaic virus (WSMV) is stored in juice 4- 8days at a temperature of 20°C, so it can be transmitted by contact-mechanical way. The virus circulation in nature is done by a clamp (Acera tulipae and other), there are data about the distribution by seeds [10].
In the sample №2 (Fig. 1 B) terrestrial wheat mosaic virus (SBWMV) is de-tected, whose vector is the mushroom like microorganism of Plasmodiophoromycota- Polymyxa graminis. Zoospores of this fungus spread the virus, and it is stored in the soil for more than 20 years in disputes. That is why; the timing of grain crop cultivation in the crop rotation does not provide a protective effect.
Elimination of virus infection sources of wheat also has no practical value be-cause of the huge number of host-plants. In the complex of measures in struggle with viruses it is important the destruction of grain drops; mowing of the roadsides; space isolation. This is due to the fact drops, cereals in "inarables", as a rule, are strongly affected by the virus diseases; vectors distribute viruses on winter cereals. All this and a new identified group of soiltransmitted - of terrestrial viruses proves that for the protection of winter wheat from viral patocomplex, in addition to the phytosanitary activities on prevention their spread , selection and introduction of resistant varieties are effective protective measures.
The quality or specific resistance is of particular importance, because it may be associated with the isolation of the virus and its elimination in plants. But the high quantitative, field resistance and tolerance, which are more constant, are of increasing importance, although the breeding work with the polygene conditioned quantitatively resistant material is more complicated than with the immediately conditioned qualitative resistant material. It is desirable to unite different types of resistance in one variety. In abroad in the range of varieties of wheat, barley, triticale, oats there are different varieties, which differ by quantitative resistance and tolerance to the yellow dwarfism barley viruses, streak mosaic of barley, wheat streak mosaic, dwarfism wheat, and for the Soil-borne cereal mosaic virus (SBCMV), using wheat “Moulin” as a source of quan-titative resistant to it [9, 12]. Studies are conducted in the direction of of grain resistance to vectors of viruses .That is why begun in the southern Urals viro-logical investigation of the crop production and immunological assessment of a wide assortment of winter wheat, as the main grain crop of this region, can be of great help to breeders in the derivation of viruses resistant varieties and with complex resistance varieties.
CONCLUSIONS
From more than 100 examined accessions were identified as immune condi-tioned and high resistant: Choc, Rafale (France); Leopold (Austria); Susquehanna, Norkan (USA); Kazachok, Volga-Don (Volgograd); Krasnodar Scientific Research Institute of Agriculture
756, Bistritza, Selyanka KNIISKH 8,(Krasnodar); Mirovskaya 34, Vasilina (Ukraine);
Orenburgskaya 14, Bezor 2 (Orenburg); Severodonskaya 14 (Rostov), which are perspective
viruses resistant for breeding
Identification of viral pathogens on winter wheat, on results were identified the Wheat
streak mosaic virus and Soil-borne wheat mosaic virus was carried out by the method of the
reverse polymerase chain reaction.
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