ISSN 1313 - 8820Volume 7, Number 1

March 2015

2015

Scope and policy of the journalAgricultural Science and Technology /AST/ – an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues.

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Editors and Sections

Genetics and Breeding

Atanas Atanasov (Bulgaria)Nikolay Tsenov (Bulgaria)Max Rothschild (USA)Ihsan Soysal (Turkey)Horia Grosu (Romania)Bojin Bojinov (Bulgaria)Stoicho Metodiev (Bulgaria)

Nutrition and Physiology

Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Bogdan Szostak (Poland)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)Martin Banov (Bulgaria)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2015

ISSN 1313 - 8820 Volume 7, Number 1March 2015

Mixed viral infections in tomato as a precondition for economic loss

N. Petrov*

Institute of Soil Science, Agro Technologies and Plant Protection Nikola Pushkarov, 7 Shosse Bankya, 1331 Sofia, Bulgaria

Abstract. Tomato is one of the economically important crops in Bulgaria. Low tomato yields in the country are mostly due to lack of knowledge on sustainable agronomic practices, lack of improved varieties, which are well suited for high yield and resistance to diseases, and also due to damage caused by pests and diseases. Many viruses infect the tomato crop. The object of this research was to identify viruses infecting tomato crops in Bulgaria. The presence or absence of a viral infection in tomato plants was carried out with DAS-ELISA serologic assays with specific polyclonal immunoglobulin G for the relevant plant virus. Most of the tomato plants were infected with one or two viruses and in rare cases with more than two viruses. We found that mixed viral infections caused more severe necrotic damages in the tomato fruits and increased the economic loss compared with mono infections.

Keywords: tomato, mixed plant viral infections

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 7, No 1, pp , 2015124 - 128

Introduction (Zitter, 1974). PVY, a member of the genus Potyvirus that infects tomato occurs in several pathotypes (Jones et al., 1991), which are transmitted by aphids in a non-persistent manner. Typical symptoms Tomato Spotted Wilt Virus (TSWV), Tomato Mosaic Virus of PVY in tomato include mosaic, vein chlorosis, mild mottling, dark (ToMV), Cucumber Mosaic Virus (CMV), Potato Virus Y (PVY) and brown necrosis on leaflets, severe necrosis, leaf crinkling, and Pepper Mild Mottle Virus (PMMV) infect different species of the drooping (Jones et al., 1991). family Solanaceae including tomatoes. They cause significant

The genus Tospovirus contains viruses, which are very losses and reduce product quality in tomatoes. These viruses can unstable, especially at pH values below 5.5. Viruses of this genus infect either singly or in combination and infection results in various have a characteristic membranous lipoprotein envelope and form symptoms, which range from a mild to severe mottling, leaf cytoplasmic inclusions in plant cells (Green and Kim 1991). TSWV is distortion, necroses of leaves and fruit to extreme plant stunting. the only known member of this genus that infects tomato. Mild CMV has been described as one of the five most important mutant strains of TSWV exist and have been inoculated in tomato for viruses infecting vegetable species worldwide (Palukaitis and cross-protection against more severe strains (Gonslaves and Garcia-Arenal, 2003). The virus causes severe mosaic symptoms, Providenti, 1989). The virus is known to cause chlorosis and yellow stunting, various types of necrosis, leaf deformation and leaf rings on tomato leaves and fruits. It is transmitted persistently by shoestring. Fruits are often malformed and necrotic lesions are thrips. Seed transmission also occurs (Jones et al., 1991; Ullman et common, thereby drastically reducing marketable yield (Palukaitis et al., 1996). al., 1992).

PMMV was first described in Italy in 1984. Since then, it has TMV is the type species of Tobamovirus genus. Another spread and become a significant pathogen of tomato and pepper species of this genus is ToMV, which is distinguished from TMV by its crops worldwide (Wetter, 1984). It is a member of the genus ability to produce local necrotic lesions in Nicotiana tabacum var. Tobamovirus (Fauquet, 2005). PMMV infects cultivated pepper White Burley and N. sylvestris (Green and Kim, 1991). ToMV strains plants through seed and soil transmission in the fields, causing include those which cause corky ring, crusty fruit, yellow streak and severe mosaic symptoms. PMMV is not transmitted by insects. It can aucuba symptoms (Jones et al., 1991). Consequently, it is not easy be seed borne, consequently, the seedlings can be infected by to correctly identify ToMV by basing on symptoms because it causes mechanical contamination from their seed coats during a variety of them. However, known common ToMV symptoms transplanting or other cultural procedures. This is a primary source include mosaic, systemic chlorosis, local necrotic lesions, leaf of infection. Foliar symptoms of PMMV consist of mottling and abscission, as well as systemic leaf and stem necrosis, which yellow/green mosaic, while fruit may be small, malformed and ultimately cause death (Brunt et al., 1990). The virus is transmitted mottled, with sunken or raised necrotic spots. Yield loss is by human activities, through seed, and from leaf and root debris considerable when young plants become infected. When the virus (Green and Kim, 1991). It is also readily sap-transmissible and contaminates once in a green pepper field by carrying over seed, it is cosmopolitan (Brunt et al., 1990). ToMV has been found as an extremely difficult to get rid of it. The virus in infected plants remains aerosol in fog in USA (Castello et al., 1995) and in nutrient solution as long as green pepper is cultivated continuously in that field, used for crop cultivation in Apulia, Italy (Pares et al., 1992). because the plants later become sources to infect newly In the VIDE database index of plant viruses (2006), for every 10 transplanted young seedlings. The virus is quite stable and highly virus species listed at least one is a Potyvirus. Potyviruses induce infectious and is easily spread from plant to plant during normal crop typical cylindrical, pinwheel-shaped inclusions in cells of infected maintenance. Also, the virus can persist in the previous crop in plants (Green and Kim, 1991). Some major viruses in this family that infected pepper debris such as leaves, stems or roots in soil for infect tomato include PVY, and Pepper veinal mottle virus (PVMV) several months (Agrios, 2005).

* e-mail: m_niki@abv.bg

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Therefore, is necessary to detect viruses on tomato crops in RNA (0,05-0,5 g) at 95°C for 5 min with 10 l PV Primer1 primer in order to take relevant measures to limit their spread and reduce a final volume of 10 l. cooling on ice to avoid renaturation losses from poor quality production caused by viral infections. The preparation 15 l of master mix: 5 l of 5 MMLV-buffer, 2 l of dNTPs object of this research was to identify viruses infecting tomato crops (2mM), 0.5 l of M-MuLV Reverse transcriptase (200 / l), 7.5 l in Bulgaria and to determine damages on plants caused by the virus H O. Incubation step at 42°С for 60 min. Master mix for the PCR is: 1 2

infections. l cDNA, 2.75 l 10 PCR buffer, 2.2 l MgCl (25 mM), 2.2 l dNTPs 2

(2 mM), 1 l PV Primer 1 (10 M), 1 l PV Primer 7 (10 M), 1 lPV Primer 8 (10 l), 1 l Taq DNA-Polymerase (5 / l), 12.85 l

H O. PCR was done in thermo cycler Auto-Q Server (LKB, K) with Material and met ods 2

following programme: initial denaturation step 3 min 95°C five cycles 30 sec 92°C, 30 sec 62°C, 90 sec 72°C five cycles 30 sec The tested tomato plants were from cv. Ideal.92°C, 30 sec 60°C, 90 sec 72°C five cycles 30 sec 92°C, 30 sec DAS-ELISA. The analysis was conducted by the method of 58°C , 90 sec 72°C, ten cycles 30 sec 92°C, 30 sec 55°C, 90 sec Clark and Adams (1977). We used a commercial kit of LOEWE 72°C final elongation 10 min 72°C.Biochemica GmbH, Sauerlach, Germany. ELISA plates were loaded

with antiserum (IgG) for the relevant virus, with dilutions (according to the instructions of the manufacturer) in 0.05 M carbonate buffer. The samples were incubated for 4 hours at 37°C, and the unbound esultscomponents were washed out with PBS-T buffer for 5 min. All samples were grounded in extraction buffer containing 1% PVP From all tested samples from tomato plants cv. Ideal we (polyvinyl pyrrolidone) at a ratio of 1:10. The plates were incubated received different symptoms. Most of them were mosaics, chlorosis at 4°C for 16 hours. Following the third wash step alkaline- and necrosis of the plant leaves, necrosis on the stems and different phosphatase conjugate for the relevant virus was added and the necrotic patterns on the fruits. For some of the tomato plants cv. Ideal plates were incubated for 4 hours at 37°C. The used substrate was there were mixed infections from PV , ToMV and CMV. These three p-nitrophenyl phosphate (p-nitrophenyl phosphate, Sigma) in viruses in mix infection caused uneven ripening of tomato fruits and diethanolamine buffer (pH 9.8) at a ratio of 1mg/ml. The reaction green islands on the surface of the fruits (Figure 1). When peeling proceeded in the light at room temperature and was stopped with 3N the skin of the fruit we noticed clear necrosis under the skin. Later on NaOH. The adsorption of the color reaction was measured at when the fruits ripen completely the necrotic spots grew in surface, multifunctional detector (DTX 880) at a wavelength of 405 nm. The became darker and deeper in the fruits, making the fruits unusable positive samples had optical density (OD) over the threshold (Cut- for fresh consumption or the canning industry (Figure 2). Thus off) which was two times the value of the negative control. damages of the tomato fruits from these plants strongly increased.

PV and CMV mono-infections of tomato plants caused only mosaic RNA extraction from potatoes infected with PVY. Extraction of and chlorotic symptoms of the leaves, but fruits remained

total RNA was performed with RN Easy Plant Mini Kit (Qiagen, symptomless. In contrast, ToMV mono-infection can cause necrotic Germany). Extraction was carried out according to the instructions of symptoms of the fruits. Not of less importance is early fall of the fruits the manufacturer. of the infected plants with these three viruses. Thus, both the

quantity and quality of the obtained production considerably Touch-Down RT-PCR. We used primers PV Primer 1, 7 and 8 decreased.

for P1 gene region of the virus (Petrov, 2012), with program For other tomato plants cv. Ideal there were mixed infections modification touch-down. Copy DNA synthesis: denaturation of total from TSWV and PMMV. The damages of the tomato plants were

Figure . S mptoms of tomato fruits infected with miinfection of PVY, CMV and ToMV

Figure . ecrosis of tomato fruits infected with mi infection of PVY, CMV and ToMV

Figure 4. DAS-ELISA results of tomato samples infected with mix infection of PVY, ToMV and CMVLegend: 1 – tomato leaf sample tested for PVY; 2 – negative control for PVY from the kit; 3 – positive control for PVY from the kit; 4 – negative control for PVY for Sample buffer used; 5 – tomato leaf sample tested for ToMV; 6 – negative control for ToMV from the kit; 7 – positive control for ToMV from the kit; 8 – negative control for ToMV for Sample buffer used; 9 – tomato leaf sample tested for CMV; 10 – negative control for CMV from the kit; 11 – positive control for CMV from the kit; 12 – negative control for CMV for Sample buffer used

2.5 PVY

OD

λ =

405

nm

ToMV

CMV2

1.553

K-

0.211 K-

0.112

K+

1.555

K+

1.997

K+

2.123

B-

0.092

B-

0.096B-

0.076

1.234

0.895

K-

0.189

1.5

1

0.5

01 2 3 4 5 6 7 8 9 10 11 12

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darkened, going into depth of these contaminated fruits.We found that in mix infections DAS-ELISA values of the tested

plant viruses were reduced compared with mono infections. Some tomato samples were infected with PVY, ToMV and CMV (Figure 2), where PVY had the highest virus titer 1.553 and repressed the other two viruses (Figure 4), but they were also present in the same plant in spite of their low virus titer compared with PVY. Thus DAS-ELISA values for ToMV was reduced to 1.234 and for CMV to 0.895. In spite of this reduction of DAS-ELISA values of the different viruses they remained over the cut off value (0.450) proving the presence of mixed viral infection. DAS-ELISA value of the used negative control for the sample buffer was 0.092 showing no virus contamination between samples. PVY in this case dominated as an aphid transmitted virus and suppressed the movement in the same plant of another aphid transmitted virus (CMV) and mechanically transmitted ToMV.

In the plants infected with PMMV and TSWV we received milder, but in the fruits these viruses caused severe necrotic patterns different results from the previous group of samples infected with the which significantly reduced the quality of the tomato fruits and three viruses. DAS-ELISA value of PMMV was higher in range – increased losses for the farmers (Figure 3). Necrosis grew and

Figure 3. Necrotic patterns on tomato fruits from cv. Ideal infected with mix infection of TSWV and PMMV

Figure 5. DAS-ELISA results for tomato samples infected with mix infection of TSWV and PMMVLegend: 1 – tomato leaf sample tested for TSWV; 2 – negative control for TSWV from the kit; 3 – positive control for TSWV from the kit; 4 – negative control for TSWV for Sample buffer used; 5 – tomato leaf sample tested for PMMV; 6 – negative control for PMMV from the kit; 7 – positive control for PMMV from the kit; 8 – negative control for PMMV for Sample buffer used

2.5

3.5

3 TSWV

OD

λ =

405

nm

PMMV

2

1.054

K-

0.188

K+

1.455

K+

1.825

B-

0.083

B-

0.091

3

K-

0.211

1.5

1

0.5

01 2 3 4 5 6 7 8

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3.000 (Figure 5). This was the maximum value for this method. (Gallitelli et al., 1988 Gallitelli, 2000). In spite of the high frequency PMMV DAS-ELISA value was three times higher than TSWV and great economic relevance of such mixed infections, there are no (1.054). So here a mechanically transmitted virus (PMMV) reports on the interactions between CMV and PV in this host. The suppressed the thrips transmissible TSWV. only available data in tomato focused on with

For confirmation of the results from DAS-ELISA for PV and for CMV and the Begomovirus sp. Abutilon mosaic virus (Wege and determination of the PV virus strain we performed Touch-down RT- Siegmund, 2007). PCR with specific primers for P1 gene region of the PV genome. The chosen fragment from P1 enclosed by PV Primer 1 and PV

N:O Primer 7 gave the product 640 bp for PV strain and the gene fragment enclosed by PV Primer 1 and PV Primer 8 445 bp

N/NTN Oproduct for PV strain or 280 bp for PV strain. We used three samples from PV infected tomato leaf, fruit and seed. From these

Virus diseases are difficult to control because of samples we extracted RNA with RNeasy plant mini kit. From this complex interrelationships among virus, host, vector, virus source, RNA we received cDNA with the PV Primer. This cDNA we used for and environment. Touch-down RT-PCR for determination of the different PV strains.

From all tested samples we received a single positive band of 443 bp N/NTNproduct, compared with DNA ladder, which corresponded to PV

necrotic group of virus strains (Figure 6). All the controls used in the PCR were clear (without any product)) showing that there were no contaminations between the different samples. The result confirmed

N/NTNthat tomato cultivar Ideal was infected with PV group strain. The N/NTN virus strain PV was identified in the leaves, fruits and seeds of

the infected tomato plant. This shows a smooth spread of the virus in all parts of the host plant, and especially in the seed which is a prerequisite for its mass distribution in the next year if it is used for planting the seeds of this infested plant.

onclusion

iscussion From all infected tomato plants from the cv. Ideal we received different results. There were some plants infected with one virus but most of them were infected with more than one virus. Mono infections did not affect so

major damage on fruit and loss of production.

plants mix-infected

The amount of damage that plant viruses cause to tomatoes varies, depending on the particular virus or combination of viruses present, the virulence of the virus strains, the susceptibility of the variety, the timing of infection, the abundance of insect vectors, and environmental conditions. Disease incidence can range from a few scattered plants in a field to total crop failure. Mixed infections may cause symptoms that are more severe than either virus might cause alone.

The earlier a plant becomes infected, the greater the loss. Control practices should begin before planting. Eradicate perennial and biennial weeds from the garden, field, greenhouse, and surrounding areas. Continue weed control during and after the growing season. se virus-resistant tomato varieties when feasible. Make sure that transplants are healthy and certified as disease free. Wash hands with soap and water before and after handling plants. Destroy infected plants found at transplanting and during the season. se a 2-year rotation between susceptible crops. se insecticides for the vectors.

In most cases in the literature scientists report mono infection of the plants. There were some reports of mixed infections in tomato. seriously quality and quantity of the fruit Mixed infections of PV and CMV, often carrying its satellite RNA production, but the infected plants were virus reservoirs. Mixed viral (CMV-satRNA), were detected in commercial fields of tomato crops infections in tomato led toduring the CMV outbreaks that occurred in Italy in the mid-1980s Testing the seeds and seedlings could prevent

Figure . Touch down T-PC for P gene region of PVY, p T strainLegend: ene uler p Plus D A Ladder p product from P gene region of PVY recei ed from infected tomato leaf sample p product from P gene region of PVY recei ed from infected tomato fruit sample p product from P gene region of PVY recei ed from infected tomato seed sample negati e control from PC

master mi negati e control from health tomato leaf negati e control from health tomato seed

443 bp

1 2 3 4 5 6 7

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distribution of these infections. Gonslaves D and Providenti R, 1989. A multidisciplinary Approach to Management of Tomato spotted wilt virus in Hawaii. Plant Disease, 73,375-382.Green SK and Kim JS, 1991. Characterisation and control of Acknowledgements viruses infecting peppers: a Literature Review. AVDRC, Technical Bulletin, 18, 60 pp.The study was financially supported by Bulgarian Science Fund Jones JB, Stall RE and Zitter TA, 1991. Compendium of Tomato with contract ДНТС/ИНДИЯ 01/1 for bilateral collaboration project Diseases. American Phytopathological Society. Library of Congress with India.Catalog Card No. 91-71246, 73 pp.Palukaitis P and Garcia-Arenal F, 2003. Cucumoviruses. Advances in Virus Research, 62, 241-323.References Palukaitis P, Roossinck MJ, Dietzgen RG and Francki RI, 1992. Cucumber mosaic virus. Advances in Virus Research, 41, 281-348Agrios GN, 2005. Plant Pathology. Fifth Edition. Elsevier Academic Pares RD, Gunn LV and Cresswell GC, 1992. Tomato mosaic virus Press, London.infection in a recirculating nutrient solution. Journal of Brunt A, Grabtree K and Gibbs A, 1990. Viruses of Tropical plants. Phytopathology, 135, 192-198.CAB International. UK., 707 pp.Petrov N, 2012. Potato virus Y (PVY) on cultivars of the family Castello JD, Lakshman DK, Tavantzis SM, Rogers SO, Bachand Solanaceae. Thesis for PhD, Institute of Soil Science, Agro

GD, Jagels R, Carlisle J and Liu Y, 1995. Detection of infectious Technologies and Plant Protection, Bulgaria.

Tomato mosaic virus in fog and clouds. Phytopathology, 85, 1409-Ullman DE, Cantone FA and Duer HL, 1996. Compartmentali-1412.zation, intracellular transport, and autophagy of Tomato spotted wilt

Clark MF and Adams AN, 1977. Characteristics of the microplate virus proteins in infected thrips cells. Phytopathology, 85, 644-654.

method of enzyme -linked immunosorbent assay for the detection of Wege C and Siegmund D, 2007. Synergism of a DNA and an RNA plant viruses. Journal of General Virology, 34, 475-483.virus: Enhanced tissue infiltration of the begomovirus Abutilon

Fauquet CM, Mayo MA, Maniloff J, Desselberger U and Ball LA, mosaic virus (AbMV) mediated by Cucumber mosaic virus (CMV).

2005. Virus Taxonomy. Eighth Report of the International Committee Virology, 357,10-28.

on Taxonomy of Viruses. Elsevier Academic Press, London.Wetter C, Conti M, Altschuh D, Tabillion R and van Regenmortel

Gallitelli D, 2000. The ecology of Cucumber mosaic virus and MHV, 1984. Pepper mild mottle virus a tobamovirus infecting pepper sustainable agriculture. Virus Research, 71, 9-21.cultivars in Sicily. Phytopathology, 74:405-410.

Gallitelli D, Di Franco A, Vovlas C and Kaper JM, 1988. Infezioni Zitter TA, 1974. Transmission of Pepper mottle virus from miste del virus del mosaico del cetriolo (CMV) e di potyvirus in susceptible and resistant pepper cultivars. Phytopathology, 65, 110-colture ortive di Puglia e Basilicata. Infectious Fitopatologia, 12, 57-114.64.

Review

Genetics and Breeding

Nutrition and Physiology

Production Systems

Molecular mechanisms and new strategies to fight stresses in egg-producing birds E. Shatskikh, E. Latypova, V. Fisinin, S. Denev, P. Surai

Gene action in the inheritance of date to ear emergence and time to physiological maturity in bread wheat crosses (Triticum aestivum L.)N. Tsenov, T. Gubatov, E. Tsenova

Productivity and stability of the yield from common winter wheat cultivars developed at IPGR Sadovo under the conditions of Dobrudzha region P. Chamurliyski, E. Penchev, N. Tsenov

Effectof black (stem) rust (Puccinia Graminis F.SP. Tritici) attack to the spike characteristics in Polishwheat (Triticum Polonicum L.)H. Stoyanov

Analysis of DNA polymorphism of CAST gene in Local Karnobat and Stara Zagora sheep breedsD. Hristova, S. Georgieva, S. Tanchev

Correlation between grain yield and yield components in winter barley varieties N. Markova Ruzdik, D.Valcheva, D.Vulchev, Lj. Mihajlov, I. Karov, V. Ilieva

Genetic diversity in different accessions of oat (Avena sativa L.)T. Savova, B. Dyulgerova, G. Panayotova

Interspecific hybridization in cotton and its use in breedingA. Stoilova, I. Saldzhiev

Influence of the direction of crossing on heterosis and transgression events in relation to the length of the vegetative period of Burley tobaccos variety group Y. Dyulgerski, T. Radoukova, L. Dospatliev

The performance of female dairy calves fed texturized starters with different protein sources E. Yavuz, N. Todorov, G. Ganchev, K. Nedelkov

Feeding value estimation of spring forage pea (Pisum sativum L.) in organic cultivationI. Nikolova, N. Georgieva, Y. Naydenova

Treatment of post harvest residues with cellulose decomposing preparationsI. Effect on grain yield from wheatG. Milev, I. Iliev, A. Ivanova

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Seasonal dynamics of important for Coriandrum sativum virus pathogensB. Dikova, H. Lambev

Crop relationship "yield-evapotranspiration" for green beanR. Kalaydzhieva, D. Davidov, A. Matev, V. Kuneva

Species composition and density of weeds in a wheat crop depending on the soil tillage system in crop rotationP. Yankov, M. Nankova, M. Drumeva, D. Plamenov, B. Klochkov

Assessment of Bulgarian Black Sea coastal water using the biological quality element phytoplanktonD. Petrova, D. Gerdzhikov

Evaluation on reaction of late maturing maize hybrids and lines to Fusarium ear rotM. Haddadi, M. Zamani

Contemporary state of macrophytobenthos along the Bulgarian coast of the Black SeaD. Petrova, V. Vachkova, D. Gerdzhikov

Bioconversion of nitrogen in eco-technical system for eggs productionA. Gencheva

Mixed viral infections in tomato as a precondition for economic lossN. Petrov

Storage and its effect on the antioxidant capacity of dried Bulgarian Chrysanthemum balsamita L.A. Popova, D. Mihaylova, I. Alexieva

Correcting the breadmaking quality of flour damaged by Sunn pest (Eurygaster integriceps) by using apple pectinI. Stoeva

Investigation the influence of dietary fiber on the rheological properties of alginate beadsZ. Manev, N. Petkova, P. Denev, D. Ludneva, S. Zhelyazkov

Occurrence of Pseudomonas syringae pv. tomato in BulgariaM. Stoyanova, K. Aleksandrova, D. Ganeva, N. Bogatzevska

Agriculture and Environment

Product Quality and Safety

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Preparation of papersPapers shall be submitted at the editorial office typed on standard typing pages (A4, 30 lines per page, 62 characters per line). The editors recommend up to 15 pages for full research paper ( including abstract references, tables, figures and other appendices)The manuscript should be structured as follows: Title, Names of authors and affiliation address, Abstract, List of keywords, Introduction, Material and methods,Results, Discussion, Conclusion, Acknowledgements (if any), References, Tables, Figures.The title needs to be as concise and informative about the nature of research. It should be written with small letter /bold, 14/ without any abbreviations. Names and affiliation of authorsThe names of the authors should be presented from the initials of first names followed by the family names. The complete address and name of the institution should be stated next. The affiliation of authors are designated by different signs. For the author who is going to be corresponding by the editorial board and readers, an E-mail address and telephone number should be presented as footnote on the first page. Corresponding author is indicated with *.Abstract should be not more than 350 words. It should be clearly stated what new findings have been made in the course of research. Abbreviations and references to authors are inadmissible in the summary. It should be understandable without having read the paper and should be in one paragraph. Keywords: Up to maximum of 5 keywords should be selected not repeating the title but giving the essence of study. The introduction must answer the following questions: What is known and what is new on the studied issue? What necessitated the research problem, described in the paper? What is your hypothesis and goal ?Material and methods: The objects of research, organization of experiments, chemical analyses, statistical and other methods and conditions applied for the experiments should be described in detail. A criterion of sufficient information is to be possible for others to repeat the experi-ment in order to verify results.Results are presented in understandable

tables and figures, accompanied by the statistical parameters needed for the evaluation. Data from tables and figures should not be repeated in the text.Tables should be as simple and as few as possible. Each table should have its own explanatory title and to be typed on a separate page. They should be outside the main body of the text and an indication should be given where it should be inserted.Figures should be sharp with good contrast and rendition. Graphic materials should be preferred. Photographs to be appropriate for printing. Illustrations are supplied in colour as an exception after special agreement with the editorial board and possible payment of extra costs. The figures are to be each in a single file and their location should be given within the text. Discussion: The objective of this section is to indicate the scientific significance of the study. By comparing the results and conclusions of other scientists the contribution of the study for expanding or modifying existing knowledge is pointed out clearly and convincingly to the reader.Conclusion: The most important conse- quences for the science and practice resulting from the conducted research should be summarized in a few sentences. The conclusions shouldn't be numbered and no new paragraphs be used. Contributions are the core of conclusions. References:In the text, references should be cited as follows: single author: Sandberg (2002); two authors: Andersson and Georges (2004); more than two authors: Andersson et al.(2003). When several references are cited simultaneously, they should be ranked by chronological order e.g.: (Sandberg, 2002; Andersson et al., 2003; Andersson and Georges, 2004).References are arranged alphabetically by the name of the first author. If an author is cited more than once, first his individual publications are given ranked by year, then come publications with one co-author, two co-authors, etc. The names of authors, article and journal titles in the Cyrillic or alphabet different from Latin, should be transliterated into Latin and article titles should be translated into English. The original language of articles and books translated into English is indicated in parenthesis after the bibliographic reference (Bulgarian = Bg, Russian = Ru, Serbian = Sr, if in the Cyrillic, Mongolian =

Мо, Greek = Gr, Georgian = Geor., Japanese = Jа, Chinese = Ch, Arabic = Аr, etc.)The following order in the reference list is recommended:Journal articles: Author(s) surname and initials, year. Title. Full title of the journal, volume, pages. Example:Simm G, Lewis RM, Grundy B and Dingwall WS, 2002. Responses to selection for lean growth in sheep. Animal Science, 74, 39-50Books: Author(s) surname and initials, year. Title. Edition, name of publisher, place of publication. Example: Oldenbroek JK, 1999. Genebanks and the conservation of farm animal genetic resources, Second edition. DLO Institute for Animal Science and Heal th, Netherlands.Book chapter or conference proceedings: Author(s) surname and initials, year. Title. In: Title of the book or of the proceedings followed by the editor(s), volume, pages. Name of publisher, place of publication. Example: Mauff G, Pulverer G, Operkuch W, Hummel K and Hidden C, 1995. C3-variants and diverse phenotypes of unconverted and converted C3. In: Provides of the Biological Fluids (ed. H. Peters), vol. 22, 143-165, Pergamon Press. Oxford, UK.Todorov N and Mitev J, 1995. Effect of level of feeding during dry period, and body condition score on reproductive perfor-

thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, September 11–14, Berlin, Germany.Thesis:Hristova D, 2013. Investigation on genetic diversity in local sheep breeds using DNA markers. Thesis for PhD, Trakia University, Stara Zagora, Bulgaria, (Bg).

The Editorial Board of the Journal is not responsible for incorrect quotes of reference sources and the relevant violations of copyrights.

Animal welfareStudies performed on experimental animals should be carried out according to internationally recognized guidelines for animal welfare. That should be clearly described in the respective section “Material and methods”.

Volume 7, Number 1March 2015

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