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Volume 10, Number 1March 2018

2018

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2018

ISSN 1313 - 8820 (print)ISSN 1314 - 412X (online)

Volume 10, Number 1March 2018

Evaluation of lentil cultivars and lines for resistance to Fusarium oxysporum f.sp. lentis

1 2 2 1 1M. Koleva *, Y. Stanoeva , I. Kiryakov , A. Ivanova , P. Chamurlyiski

1Department of Crop Science and Plant protection, College Dobrich, Shumen University “Bishop Konstantin Preslavski”, 9300 Dobrich, Bulgaria

2Dobroudzha Agricultural Institute, 9500 General Toshevo, Bulgaria

(Manuscript received 10 July 2017; accepted for publication 18 January 2018)

Abstract. Lentil (Lens culinaris Medik.) is one of the most important food legume crops in the farming and food systems of many countries, including Bulgaria. In recent years lentil fields increased in our country. Fusarium wilt caused by Fusarium oxysporum f.sp. lentis (F.o. f.sp. lentis) is a key yield-limiting factor worldwide. The major approach to control the disease is through resistance breeding. The aim of this investigation was to screen lentil cultivars and lines for resistance to F.o. f.sp. lentis. The investigations were carried out during 2013-2015. Thirty two lentil accessions were sown in 1m rows, in three replications. Barley grains (100g/m) inoculated with mix of four isolates of F.o. f.sp. lentis were used as inoculum. Disease reaction was estimated during flowering and pod filling stage according to 9 degree scale. The disease incidence (DI) of every accession was calculated according to McKinney index. Cluster analysis was used to group the accessions according to DI. 'Genotype' x 'Year' interaction was estimated by Two-way ANOVA. The results showed that middle disease incidence (MDI) was the highest in 2014 and the lowest in 2015. Our investigation confirms the results of many authors that disease incidence of F.o. f.sp. lentis on lentil was determined by the interaction of the factors 'Genotype'x'Year'. The cluster analysis grouped the accessions into two major classes. No resistant accessions were found. Six accessions showed MDI from 48.59 to 58.11 and they can be used in a genetic-improvement breeding program for fusarium wilt resistance.

Keywords: lentil, fusarium wilt, resistance, lens culinaris, F.o. f.sp. lentis

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. , No , pp 5 - , 20110 1 2 28 8DOI: 10.15547/ast.2018.01.006

Introduction

Lentil (Lens culinaris Medik.) is one of the most important food legume crops in the farming and food systems of many countries, including Bulgaria. In recent years lentil fields increased in our country. The productivity of lentil is affected by a number of biotic and abiotic stresses of which Fusarium wilt (Fusarium oxysporum f.sp. lentis-Fol) is the most important yield-limiting factor (Bayaa et al., 1997; Mohammadi et al., 2012; Pouralibaba et al., 2016; Al-Husien et al., 2017). Fusarium wilt occurs during seedling and reproductive stages of the crop and can cause up to 100% yield losses under favorable conditions for disease development (Khare, 1981). Disease symptoms include stunting, wilting, marked reduction of root system, and internal vascular discoloration of the lower stem (Tosi and Cappelli, 2001). Management practices for disease control include long-term crop rotation with nonsusceptible hosts and removal and destruction of infected crop debris (Tosi and Cappelli, 2001). Seed treatment with carbendazim, captan (Maheshwari et al., 2008), benzimidazoles and tiram (Tosi and Cappelli, 2001) can reduce disease incidence and plant mortality. Breeding resistant cultivars is the major approach to control the disease and many resistant lines and cultivars are released in different countries (Pandya et al., 1980; Mihov et al., 1987; Bayaa et al. 1997; El-Ashkar et al. 2004; Mohammadi et al., 2012). Sources of resistance to wilt have been found in the cultivated lentil (Bayaa and Erskine, 1990; Mohammadi et al., 2012; Pouralibaba et al., 2016) and lentil wild relatives (Bayaa et al., 1995). Resistance of lentil to Fol is controlled by major genes (Eujayl et al., 1998). For many years, it was believed that no races/pathotypes are existing in the Fol population (Bayaa et al., 1997), but pathogenic and genetic variability of the pathogen has been reported from Algeria, India and Iran (Belabid and Fortas,

2002; Belabid et al., 2004). In 2016 Pouralibaba et al. (2016) developed a differential set with four lentil accessions and identified seven Fol pathotypes.

Fusarium wilt in Bulgaria is caused by Fol and Fusarium gibbosum but Fol has predominant distribution (Stanoeva, 2007). Cultural and pathogenic characteristics of the pathogens, the effect of the year, genotype and inoculum dose on the disease incidence (DI), and the reaction of lentil accessions to the pathogen under field condition have been investigated but no resistant varieties and breeding lines were found (Stanoeva, 2007). Since then systematic screening of the germplasm has not been undertaken.

The aim of this investigation is to evaluate lentil cultivars and lines to Fol with a view to their use for breeding purposes.

Material and methods

The investigations were carried out during 2013-2015 at Dobrudzha Agricultural Institute, General Toshevo. Thirty two lentil accessions (14 cultivars and 18 lines) were sown in 1m rows in three replications. In every two rows, line ILL 4650 was used as a susceptible check (Erskine and Bayaa, 1996). Four isolates of Fol were used (F97.4, F99.3, F1.27 and F5.23). They were storage on filter pepper in 4-5˚C (Correl et al., 1986), then cultivated on Potato Dextrose Agar (PDA) and multiplied in PD Broth. Sterilized barley

6seeds were placed in plastic bags with spore suspension (1x10 , x25 ml/kg seeds), separately for every isolate (Stanoeva et al., 2006). The bags were kept in darkness for two weeks in 25˚C. Barley seeds (100g/m) with the different isolates were mixed and integrated in the rows before sowing (Stanoeva et al., 2006).

The reaction of the plant to the disease were estimated in

25

* e-mail: koleva_magdalena@abv.bg

26

flowering and pod filling according to 9 degree scale (Bayaa et al., 1995), where:

l 1-no symptoms (Immune); l 3-yellowing of the lower leaves (Resistant); l 5-yellowing in 50% of leaves (Middle Resistant); l 7-yellowing of whole plant or partial wilting (Susceptible); l 9-the whole plant is wilted and/or dried. Disease incidence (DI) was calculated according to the Mc

Kinney index (McKinney, 1923) for every accession:DI=∑(ab)100/NK, Where: a - infection class frequencies; b - number of plants of each class; N - total of observed plants; K - highest value of the evaluation scale. Cluster analysis was used to group the accessions according to

the DI. 'Genotype'x'Year' interaction was estimated by Two-way ANOVA. The program used is STATISTICA 7.

Results and discussion

Line ILL 4605 had DI=100 during the three years of investigation (Table 1). In 2013 the line X04L24-112 had the lowest DI (48.37), followed by cultivar Stela (DI=55.34) and line X99L26-11 (DI=56.06). Line X98L27-112 showed the lowest DI (45.00) in 2014 followed by cultivar Naslada (DI=49.30). In 2015 the lowest DI had line X98L31-13 (29.25) followed by lines X00L45-111 (DI=35.42) and X04L24-112 (DI=36.67). Generally the DI of the investigated accessions were the highest in 2014 (73.06) and the lowest in 2015 (49.63). MDI is in the range of 48.59 (line X98L27-112) and 97.08 (ILL 101 x ILL 162) during the period of investigation. No immune accessions were found.

Cluster analysis grouped the accessions in two classes according to DI (Figure 1). The first class included two accessions

(ILL 4605 and ILL 101 x ILL 162) which showed MDI=100 and MDI=97.08 (Table 1), respectively and could be determined as Very susceptible. The second class is divided into two subclasses. The first subclass included 25 accessions with MDI from 64.52 (X98L014) to 90.07 (Palmira) and could be classified as Susceptible. The other six accessions (X98L27-112, X98L31-13, Naslada, X99L26-11, Stela, X04L24-112) formed the second subclass with MDI from 48.59 to 58.11 (Table1, Figure 1) and could be classified as Middle resistant. Screening 196 Spanish landrace for resistance to Fol Pouralibaba et al. (2015) used similar method for disease scoring. The authors identified 12 accessions which showed more advance symptoms than the highly resistant ones, and less affected by the disease than the susceptible lines and called this resistance 'incomplete'. The authors determined this kind of resistance as polygenic where each gene added a small amount of resistance. Therefore the resistance in the six accessions in this investigation can be classified as incomplete too.

The analysis of variance of 33 accessions after inoculation with Fol showed significant 'genotype' x 'year' interaction, and significance of the factors 'genotype' and 'year' (Table 2). These results were expected and determined by many authors in lentil and Fol (Bayaa et al., 1997; Stanoeva et al., 2006, Mohammadi et al., 2012). Cultivars Naslada and Tadzhikskaya 95 were of the first reported cultivars resistant to Fol (Mihov et al., 1987). Stanoeva et al. (2006) reported MDI=48.46 for Naslada and MDI=63.40 for Tadzhikskaya 95 using the same Fol isolates during 2001-2004. In this investigation cultivar Naslada showed moderately resistant phenotype (MDI=52.57) and cultivar Tadzhiskaya 95 showed susceptible phenotype (MDI=74.07). The differences in the results by Mihov et al. (1987), Stanoeva et al. (2006) and the results of our investigation are probably due to: the effect of the factor 'year', the virulence of the used Fol population and the methods for disease scoring, because the past the resistance to Fol used to be scored by percentage of dead plants.

Table 1. Disease incidence (DI) and Middle disease incidence (MDI) of 33 lentil (Lens culinaris) accessions after inoculation with Fusarium oxysorum f.sp. lentis

No MDI MDI2013 2014 2015

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

X98L27-112

X98L31-13

Naslada

X99 L 26-11

Stela

X04L24-112

X98L014

X00 L 45-111

ХМ95L33-22

Stanka 1

X04 ML053

X04L17-22

X98 L33-23123

X99L20-11351

Zornitza

Ilina

Mutant 17 ММ

Nadezhda

X04 ML057

M89 LT4-1162441

Tadzhikskaya 95

X94L39-11246

Ilina-1232

X00L38-313

Stanka 2

Yanitza

Elitza

Bella

X03L4-1344

X94L62-421203

Palmira

ILL 101 x ILL 162

ILL 4650

Average

64.00

59.00

63.25

56.06

55.34

48.37

68.34

66.00

73.79

74.00

87.08

66.37

70.11

56.41

80.91

71.71

79.54

74.37

76.88

78.37

64.14

82.64

71.71

75.41

71.71

84.60

82.10

89.06

88.46

96.04

95.89

91.25

100.00

67.08

85.20

85.00

73.43

69.68

86.47

77.25

99.00

87.50

81.20

86.37

89.06

86.53

96.70

96.13

100.00

100.00

73.06

55.41

55.19

66.17

88.40

53.67

74.37

50.29

67.64

67.14

68.18

75.45

80.00

63.67

78.19

100.00

100.00

49.63

71.66

72.36

72.66

74.07

74.26

74.44

74.90

75,62

77.65

78.88

84.52

85.00

85.47

90.07

97.08

100.00

45.00

59.58

49.30

69.64

73.81

89.28

69.44

93.57

80.31

62.50

62.75

93.26

77.00

80.00

79.25

77.25

80.10

36.78

29.25

45.17

37.83

41.33

36.67

55.78

35.42

45.00

66.18

55.34

45.90

61.19

77.19

54.13

65.37

55.18

48.59

49.28

52.57

54.51

56.83

58.11

64.52

65.00

66.37

67.56

68.39

68.51

69.43

71.20

71.43

71.44

71.61

YearNo AccessionsAccessions

2013 2014 2015

Year

27

On the basis of their vegetative compatibility, Stanoeva (2007) grouped 88 Fol isolates, derived from different regions of Bulgaria, into 76 vegetative compability groups (VCGs). The Fol isolates used in this investigation belong to different VCGs (Stanoeva, 2007). The vegetative compatibility was a suitable polymorphic sign for determine genetic diversity in Fol population (Correll et al., 1987) but it was difficult to use it for breeding purposes. Other investigations need to be made to identify race diversity in Bulgarian Fol population using the differential set developed by Pouralibaba et al. (2016). The results will probably be able to explain better all preliminary investigations on Fol and lentil breeding strategies for resistance to

the pathogen should be planned according to the prevalent pathotypes present in the country.

Conclusion

Six lentil accessions (two cultivars and four lines) have had moderately resistant phenotype after inoculation with Fusarium oxysporum f.sp. lentis. These accessions can be used for breeding purposes. Significant 'genotype' x 'year' interaction and significance of the two factors separately have been determined after inoculation

0

Tadzhikskaya 95Stanka 1Stanka 2

IlinaX98 L33-23123

M89 LT4-1162441Ilina-1232X98L014

YanitzaElitzaBella

X03L4-1344Palmira

NadezhdaX04 Ml057

Mutant 17 MMZornitza

X94L39-11246XM95L33-22

X94L62-421203X00 L 45-111

X04L17-22X00L38-313

X04 Ml053Naslada

X98L27-112Stela

X99 L 26-11X98L31-13

X04L24-112ILL 101 x ILL 162

ILL 4650

5 10 15 20 25

Linkage Distance

Figure 1. Dendrogram derived from cluster analysis showing relationship among the 33 lentil cultivars and lines according to disease incidents after inoculation with Fusarium oxysporum f. sp. lentis isolates during 2013-2015

Table 2. Analysis of variance of disease incidents of 33 lentil cultivars and lines after inoculation with Fusarium oxysporumf.sp. lentis

**Significant at P

28

of 33 lentil accessions with Fusarium oxysporum f.sp. lentis.

References

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density associated with vescular wilt on lentil. Phytopathologia Mediterranea, 35, 24-32.Eujayl I, Erskine W, Bayaa B, Baum M and Pehu E, 1998. Fusarium vascular wilt in lentil: Inheritance and identification of DNA markers for resistance. Plant Breeding, 117, 497-499.Khare MN, 1981. Diseases of lentil. In: Lentils (Eds. Webb and G. Hawtin), Commonwealth Agricultural Bureaux, Slough, UK pp. 163-172.Maheshwari SK, Bhat Nazir A, Masoodi SD and Beig MA, 2008. Chemical control of lentil wilt caused by Fusarium oxysporum f. sp. lentis. Annals of Plant Protection Sciences, 16, 419-421.McKinney HH, 1923. Influence of soil, temperature and moisture on infection of wheat seedlings by Helminthosporium sativum. Journal of Agriculture Research, 26, 195-217.Mihov M, Stoeva I and Ivanov P, 1987. Resistance of lentil to Fusarium wilt. Plant Science, 24, 45-51.Mohammadi N, Puralibaba H, Mohammadi E, Ahari AB and Pakdaman B, 2012. Advance lentil lines screened for resistance to Fusarium oxysporum f. sp. lentis under greenhouse and field conditions. Phytoparasitica, 40, 69-76.Pandya BP, Pandey MP and Singh JP, 1980. Development of Pant L 406 lentil resistant to rust and wilt. LENS, 7, 34-37. Pouralibaba HR, Rubiales D and Fondevilla S., 2016. Identification of pathotypes in Fusarium oxysporum f. sp. lentis. European Journal of Plant Pathology, 144, 539-549.Stanoeva Y, Penchev E and Kiryakov I, 2006. Study on the effect of inoculum dose in field testing of lentil accessions resistance to the cause agent fo fusarium wilt (Fusarium oxysporum f.sp. lentis). Field Crop Studies, III-2, 305-310.Stanoeva Y, 2007. Investigations on Fusarium wilt on lentil (Lens culinaris Medik.) in Bulgariq. Thesis for PhD, General Toshevo, Bulgaria (Bg).Tosi L and Cappelli C, 2001. First report of Fusarium oxysporum f. sp. lentis of lentil in Italy. Pland Disease, 85, 562.

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Achievements and problems in the weed control in grain maize (Zea mays L.)G. Delchev, M. Georgiev

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Yield and coefficient of ecological valence of spring barley in the regions of Sadovo and Karnobat, BulgariaN. Neykov, T. Mokreva

Agronomic performance of mutant lines of winter two-rowed barleyB. Dyulgerova, D. Valcheva, N. Dyulgerov

Phenotypic diversity in six-rowed winter barley (Hordeum sativum L.) varietiesN. Dyulgerov, B. Dyulgerova

Evaluation of rye specimens in maturity stage on the base of mathematical – statistical analysisV. Kuneva, E. Valchinova, A. Stoyanova

Evaluation of lentil cultivars and lines for resistance to Fusarium oxysporum f.sp. lentisM. Koleva, Y. Stanoeva, I. Kiryakov, A. Ivanova, P. Chamurlyiski

Registration of a new sunflower hybrid - SevarP. Peevska, M. Drumeva, G. Georgiev

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The effect of novel xylanase on feeding value of diet containing cereal by-products for broilersJ.M. Abdulla, S.P. Rose, V. Pirgozliev

Effect of dietary garlic powder and probiotics supplementation on growth performance of male Ross 308 broilersH. Lukanov, I. Pavlova, A. Genchev

Slaughter traits of Pharaoh Japanese quails А. Genchev, H. Lukanov, I. Penchev

Blood count in dogs with mammary gland carcinomaTs. Hristov, R. Binev

Production Systems

Economic efficiency of fattening on different genotypes slow-growing and fast-growing broiler chickens M. Oblakova, Y. Popova, P. Hristakieva, N. Mincheva, M. Lalev

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Effect of nutmeg extract supplementation on some productive traits and economic efficiency of common carp (Cyprinus carpio L.) cultivated in recirculation system G. Zhelyazkov, S. Stoyanova, I. Sirakov, K. Velichkova, Y. Staykov

Agriculture and Environment

Influence of biomanipulation on the living communities and the water quality in the Strezhevo hydroecosystem, R. MacedoniaR. Nastova, V. Kostov, N. Gjorgovska, V. Levkov

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Residue analysis of difenoconazole in apple fruits grown in Republic of MacedoniaV. Jankuloska, I. Karov, G. Pavlovska

Organoleptic properties of white yam (Dioscorea rotundata poir) as affected by autoclaving timeM. Ahmed, Y.B. Kiri, M.S. Abubakar

Influence of Goji berries on oxidative changes, microbiological status and chemical properties of sausagesA. Mitev, A. Kuzelov, E. Joshevska

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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 f o r A n i m a l S c i e n c e a n d H e a l t h , 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”.

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