GrainGenes

ITEMS FROM UKRAINE

INSTITUTE OF PLANT PRODUCTION N.A. V.YA. YURJEV

National Centre for Plant Genetic Resources of Ukraine, Moskovs'kiy pr., 142, Kharkiv, 61060, Ukraine.

Morphological traits of polyploid wheat forms of the subgenus Boeoticum E.Migusch. Et Dorof. [p. 92-95]

Elena V. Tverdokhleb and Svitlana V. Rabinovych.

The use of synthetic wheats with the G genome seems to be a fruitful way for genetic improvement of cultivated wheat because of genes controlling a number of valuable traits including disease and pests resistance, high protein content in the grain, high groat quality, specific starch quality (waxy), and tolerance to soil acidity.

According Dorofeev et al. (1979), species and forms with the G genome constitute a separate branch of evolution of the genus Triticum and are included in subgenus Boeoticum E.Migusch.et Dorof., which consists of the sections Monococcon Dum., Timopheevii A. Filat. et Dorof., Kiharae Dorof. et E.Migusch. Species with the G genome are found in the last two sections.

Section Timopheevii (genomes A^bG) includes wild emmer T. araraticum Jakubz., cultivated emmer T. timopheevii Zhuk., and also a naked analogue of T. timopheevii named T. militinae Zhuk. et E.Migusch. The free threshing ability of the last species is caused by the Q gene.

Section Kiharae (A^bGD ) includes artificially obtained synthetic forms. Triticum kiharae Dorof. et E.Migusch. (A^bGD) is an amphidiploid (T. timopheevii/Ae. tauschii) synthesized in Japan and named in honor of the Japanese geneticist H. Kihara. This species is homologue of T. spelta and has all the genes for resistance that are in T. timopheevii.

Triticum miguschovae (A^bGD) is the first homologue of bread wheat and was synthesized by E.G. Zhirov (1980) in the Krasnodar Institute of Agriculture n.a. P.P.Luk'yanenko, Russian Federation, from cross of T. militinae and Ae. tauschii subsp. strangulata. This species is named in honor of E.F. Migushova.

In 1940, Bulgarian geneticist D. Kostov obtained a hexaploid amphidiploid by crossing T. monococcum and T. timopheevii, naming it T. timococcum Kost (genome A^bA^bG). At the Kihara Institute, Japan, by crossing T. timopheevii and Ae. umbellulata, Amphidiploid 217 was created (genome A^bGU).

Natural octoploid (2n = 56) wheat species do not exist. One of the first a such forms, obtained by P.M. Zhukovskyi in 1944 and named T. fungicidum Zhuk. (A^bA^uBG), was from the cross 'T. persicum/T. timopheevii' followed by a colchicine treatment. In 1959, the French botanist H. Heslot obtained and described with R. Ferrari the octoploid form T. timonovum Heslot et Ferrari as an autopolyploid of T. timopheevii (genome A^bA^bGG). In 1981, N. Navruzbekov of the Daghestan Experimental Station of the N.I. Vavilov Institute of Plant Industry, Russia, obtained an octoploid wheat by crossing two naked tetraploid (2n = 28) species, T. militinae and T. persicum, and named the resulting amphidiploid T. flaksbergeri Navr. (genome A^bA^uBG) in honor of K.A. Flaksberger.

We studied the morphological traits connected with plant productivity in 10 polyploid species and forms of subgenus Boeoticum. The material was obtained from the N.I.Vavilov Institute of Plant Industry. The base species are T. timopheevii and T. militinae, both 2n = 28 and A^bG genomes. However, as Navruzbekov found in 1979, T. militinae seems to be created by hybridization of T. timopheevii with T. persicum and may have a modified genome.

The addition of the D genome from Ae. tauschii to genomes of both species resulted in T. kiharae and T. miguschovae and lead to lengthening of spike internodes and a decrease in the number of spikelets and, consequently, grains/spike.

The addition of the U genome from Ae. umbellulata to T. timopheevii resulted in AD 217 (2n = 42, genomes A^bGU), decreased plant height, spike length, grain weight/spike, and the length and width of the flag and penultimate leaf blades. The addition of subgenome A^b1 from T. monococcum, resulting in T. timococcum (2n = 42, genomes A^bA^b1G), decreased tiller number but increased spike length, spikelets/spike, amd also the length and width of the flag and penultimate leaf blades.

Chromosome doubling of the T. timopheevii genome resulted in T. timonovum (2n = 56, genomes A^bA^bGG) increased spike length and grains/spike, and length and width of the flag and penultimate leaf blades but decreased plant height, which may explain the reaction on of this species to drought during the development of the upper internode.

The specific influence of the different genomes added those of T. timopheevii and T. militinae should be understood when they and their derivatives are used in breeding and experiments.

References.

• Zhirov EG. 1980. Synthesis of new hexaploid wheat. Bull Appl Bot, Genet Breed 68(1):14-16 (In Russian).
• Dorofeev VF, Filatenko AA, Migushova EF, Udachin RA, and Yakubtsiner MM. 1979. Wheat. Cultivated flora of USSR. Leningrad, Kolos, 348 pp. (In Russian).

Phytosanitary state of winter wheat with different sowing dates at spring tillering stage. [p. 93-95]

N.V. Kuzmenko, Yu.G. Krasilovets, M.I. Nepochatov, and V.A. Tsyganko.

Winter wheat is one of the staple cereal crops in Ukraine and a general level of production is ensured. To date, harmful organisms are one of the limiting factors. At the first growth stages, insect pests of the stalk and root roots are most injurious, controlled most by sowing date. At present, global climate changes are causing an urgent need to adapt sowing dates for winter wheat, thus the search for optimal sowing dates for wheat are quite topical.

The investigations were conducted in a nine-course rotation stationary field at the Laboratory for Plant Production and Cultivar Investigations of the Yurjev Plant Production Institute of UAAS (Eastern-Steppe of Ukraine). During 2001-05, we studied the degree of damage in winter wheat plants with flies and the intensity of root rot development depending on sowing dates. Winter wheat was sown on 10, 20, and 30 September in 2001-04 and 1, 10, and 20 September in 2004-05.

Our results showed that during 2001-05 in winter wheat field at during autumn tillering, the dominant flies were Oscinella spp. (55-70%) and Phorbia securis Tiens. (11-32%). During spring tillering, crop damage also was caused by Opomyza florum F. (37.5-45.5%). Mayetiola destructor and Leptochylemyia coarctata Fll. were observed but were fewer in number. Among the root roots, Helminthosporium rot (B. sorokiniana) dominated in dry years and Fusarium rot in humid years.

The average number of plants/m² at spring tillering of winter wheat for sowing dates from 10-30 September was nearly similar between 2001-04, 455-495 (Table 1). However, wheat sown on the first date had more tillers compared to the second and third dates, 10 and 29% less, respectively. On the 10 September sowing date, there was a higher density of fly larvae than the later sowing dates 20 (1.5% less) and 30 (2.4% less) September. For winter wheat sown on the first date, we observed a higher degree of pest damage of plants and tillers compared with plants sown on the last date. When sowing on 20 September, plants and tiller damage were insignificantly less than for those sown on 10 September. The damage index at the first sowing date was 14.2%, 13.0% at the second, and 8.9% at the third. Tiller damage was 5.0, 4.2, and 2.5% for the first, second, and third sowing dates, respectively. Thus, plant damage was 1.6 times greater for plant sown on the first sowing date compared to those sown on 30 September. Tiller damage was twice as high for plants sown on 10 September. The biological effectiveness of the second sowing date was 16.0% and 50.0% for the third sowing date. Correlation analysis revealed a strong positive correlation (r = 0.9) between the total number of tillers/m² and the number of tillers that were undamaged by fly larvae; 1,953 healthy tillers at the 1st sowing date, 1,773 at the 2nd sowing date, and 1,415 at the third date. The occurrence and development of root rots in the spring tillering stage were the least for plants sown on 30 September compared to the first and second dates. The biological effectiveness of the second sowing date was 16.0% and 50.0% for the third compared to plants sown on the first date.

The incidence of root rots at the 30 September sowing date was 27.5%, whereas at the earlier date former dates it was 31.9 (10 September) and 32.0% (20 September). Disease intensity, as indicated by the number of plants undamaged by root rots/m², was 12.8% greater at the second sowing date and 15% greater at the first sowing date when compared to the last sowing date.

We concluded that the influence of these different factors depends on tiller number/m² as well as on the occurrence and intensity of root rot development (positive correlation r = 0.3­0.4). Shifting the sowing date for winter wheat can increase grain yield from 6.26 t/ha (first sowing date), to 6.51 (second sowing date), and 6.93 t/ha (third sowing date); a difference in grain yield between the first and third dates of 0.67 t/ha.

In 2004-05, a considerably larger number of harmful flies was noted at spring tillering compared to previous years. Shifting the first sowing date to 1 September, the fly density was 27.6 larvae/m². At 10 September, the number was 31% lower and 63% lower by 20 September. The maximum damage on plants and tillers was observed at the first sowing date, 44.8% (plants) and 22.0% (tillers) and the minimum at the third date, 21.4% (plants) and 7.3% (tillers). The biological effectiveness of the second sowing date was 40.5% and for the third was 66.8% compared to the first date. A larger number of healthy plants/m² (24.5% more) was noted for the third sowing date compared with the first.

The highest values for the spread and intensity of root rots were for the 1 September sowing date (38.0% spread, 18.4% intensity); the lowest values were for 10 September (29.6 spread, 18.4% intensity). Biological effectiveness for the reduction in disease intensity for plants sown on 10 September was 54.9% greater than those sown on 1 September and 33.2% greater for those sown on 20 September. Shifting the first sowing date from 1 September to 10 September increased the number of undamaged plants/m² to 300.

Winter wheat sown between 10 and 20 September out yielded that sown on 1 September by 1.28 and 1.45 t/ha, respectively. Taking into account the phytosanitary state of winter wheat being cultivated for the Forest-Steppe zone of the Eastern part of Ukraine, we found that the optimal sowing date for winter wheat was 10-20 September.

V.N. KARAZIN KHARKOV NATIONAL UNIVERSITY

Biology Faculty, Department of Plant Physiology and Biochemistry, Svoboda sq. 4, Kharkov, 61077, Ukraine.

Associative nitrogen fixation in the rhizosphere of near-isogenic VRN lines of soft winter wheat. [p. 95-97]

V.V. Zhmurko, O.A. Avksentyeva, and A.M. Samoilov.

Introduction. Associative nitrogen fixation is the process of fixing atmospheric N₂ by microorganisms of the rhizosphere. Today, N₂-fixing bacteria are thought to provide plants with some nitrogen compounds (Bashan et al. 2004), synthesize growth stimulating substances (Cassanet al. 2001; Zakharova et al. 1999), increase nitrate assimilation by bacterial nitratereductase (Patyka et al. 2002), influence on root cell membrane penetration (Katupitiya et al. 1995), and prevent plants from pathogens (Patyka et al. 2002). Plants provide the bacteria with essential nutritive substances in the form of the root exudates that contains different carbohydrates, amino and organic acids, vitamins, and other bioactive substances used by bacteria for their metabolism. Genetic and physiological investigations of associative nitrogen fixation are not numerous and need supplementation with experimental data.

Our research studied associative nitrogen fixation in the rhizosphere of soft winter wheat NILs with Vrn1-Vrn3 genes for determining the type and rates of development. This paper presents the results of the total number of diazotrophic bacteria and Azospirillum brasilense measurement, a study of nitrogenase activity, and the species structure of the rhizosphere.

Materials and methods. The NILs of the soft winter wheat cultivar Mironovskaya 808 (one dominant Vrn gene), a spring type with the genotype Vrn112233 , and a winter wheat with the genotype vrn112233 were used. Plants were grown in the field after a spring sowing in 2005-06. Nitrogen-fixing bacteria were cultured from the plant roots during flowering and/or spike maturation. Monolayer and multilayer agar plates; liquid culture; and Federov-Kalininskaya, Rotter, Dobereiner, modified media with Congo-red (CR), and a simple synthetic media not containing a source of nitrogen but with different sources of carbohydrates were used. Colonies of each type were streaked onto nutrient agar to check for purity and were identified according to the standard biochemical, cultural, and cytological methods (Bergy 1994). The total number of the rhizosphere diazotrophic bacteria was counted using the Federov-Kalininskaya medium in liquid culture by means of MacCredy's tables. The number of the specific diazotrophic bacteria of wheat­Azospirillum brasilense was counted and identified using Dobereiner's, CR, and BMS media. Nitrogenase activity was measured by acetylene reduction. The data tables present averages and standard errors.

Results and discussion. Studying the time of transition of the Mironovskaya 808 NILs demonstrated that spring wheat lines with Vrn112233 come into the tillering phase 27-29 days earlier than the line vrn112233. Two of the lines flowered 31­33 days earlier than the other. The winter-type line with vrn112233 after a spring sowing only tillered (Table 1). These results confirmed our earlier results (Zhmurko et al. 2004) and those of Zakharova et al. (1999) about the effect of Vrn genes on the rate of plant development.

Associative nitrogen fixation activity may correlate with the general physiological status of a plant and determine productivity (Zakharova et al. 1999). We studied two characteristics of productivity, the number and weight of grains/ear. Two of the NILs greater than the third (Table 2). We believe that the Vrn genes determine both the rate of development ad productivity.

The main characteristics of associative nitrogen fixation show that the total number of diazotrophic bacteria, the number of Azospirillum brasilense and nitrogenase activity (NA) in the rhizosphere of two of the Vrn112233 lines were higher than those of the other Vrn112233 line and the winter wheat vrn112233 (Table 3). We do not believe that associative N₂-fixation activity is determined by Vrn1­Vrn3 genes.

Within the species f diazotrophic bacteria of the rhizosphere, we did not find any correlation between the species structure and the genotype. The species structure (microcenosis) may be typical for T. aestivum and not determined by Vrn genes. The results show the high diversity of associative N₂-fixing bacteria (Table 4). We isolated 15 species belonging to seven families, six orders, four classes, and two phyla. The majority of the nitrogen-fixing bacteria isolated from wheat rhizosphere belong to the Pseudomonadaceae, Bacillaceae (four species), Bradyrhizobiaceae (two species) and Rhodospirillaceae. Population diversity of the rhizosphere is higher than that of the rhizoplane (a small zone around the roots). We identified and classified the following species of diazotrophic bacteria in the rhizosphere of the NILs: Azospirillum brasilense, Azotobacter vinelandii, Agrobacterium sp., Agromonas oligotrophica, Azomonas agilis, Arthrobacter sp., Bacillus subtilis, Bacillus mesentericus, Bacillus macerans, Bacillus polymyxa, Enterobacter aerogenes, Herbaspirillum seropedicae, Pseudomonas sp., Pseudomonas fluorescens, and Xanthobacter autotrophicus. We identified only three species in the rhizoplane, Bacillus subtilis, Azospirillum brasilense, and Agrobacterium sp.

Thus, the NILs with Vrn genes are characterized with different nitrogenase activity and number of associative N₂-fixing bacteria. According to their level, the NILs with Vrn genes may be arranged: Vrn112233 > Vrn112233 > vrn112233 Vrn112233. We have reason to believe that Vrn genes determine the type of development and the process of associative nitrogen fixation.

One of the possible mechanisms of the determination may be the following: in accordance with our previous results (Zhmurko et al. 2004) these lines are differed in an intensity of a carbohydrates metabolism. Two of the Vrn112233 lines have a more intensive reflux of carbohydrates and other organic substances from leaves to the acceptor zones than vrn112233 and the other Vrn112233 lines have. Consequently, carbohydrates are transported faster to the roots influencing growth and development of associative N₂-fixing microflora (bacteria) and, therefore, the total number of diazotrophic bacteria and nitrogenase activity increase.

Acknowledgment. This work is supported by the grant 6-07 of the fundamental researches fund of V.N. Karasin Kharkov National University.

References.

• Bashan Y, Hoguin G, and de-Bashan LE. 2004. Azospirillum-plant relationships: physiological, molecular, agricultural and environmental advances. Can J Microbiol Rev 50(8):521-577.
• Bergy's Manual of Determinative Bacteriology (9th ed; transl. Eng) Moscow: Myr, 1994, Vol. 1, 2.
• Cassan F, Bottini R, Schneider G, and Piccoli P. 2001. Azospirillum brasilense and Azospirillum lipoferum hydrolyze conjugates of GA₂₀ and metabolize the resultant aglycones to GA₁ in seedlings of rice dwarf mutants. Plant Physiol 125:2053-2058.
• Katupitiya S, Millet J, et al. 1995. A mutant of Azospirillum brasilense Sp7 in flocculation with a modified colonization pattern and superior nitrogen fixation in association with wheat. Appl Environ Microbiol 61(5):1987-1995.
• Patyka VP, Volkohon VV, Nadkernichnaya OV, et al. 2002. Biological nitrogen fixation: yesterday, today and tomorrow. Plant Physiology on the Verge of Millennium. Pp. 212­226.
• Stelmakh AF. 2001. The genetics of wheat development rates, the contribution of Institute of Selection and Genetics through 30 years, The works in fundamental and applied genetics to the 100 aniversary of Genetics. Kharkov: Shtrikh. 280 pp.
• Zakharova EA, Shcherbacov AA, et al. 1999. Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Eur J Biochem 259(3):572-576.
• Zhmurko VV, Avksentyeva OA, Gerashenko OU, and Stelmakh AF. 2004. Manifestation of Vrn gene effects in isogenetic winter wheat lines. Ann Wheat Newslet 50:176-178.