Distribution of Leaf Tip Necrosis Genes with Expression of Lesion Mimic Genes and Resistance

Int. J. Life. Sci. Scienti. Res., 3(1): 808-816

JANUARY- 2017

Research Article (Open access)

Distribution of Leaf Tip Necrosis Genes and its Association with Expression of Lesion Mimic Genes and Resistance to Spot Blotch in Spring Wheat Shweta Singh1,2,*, V.K. Mishra2, R.N. Kharwar1, R. N. Ahirwar2, Smrati Sharma2,3, P.S. Yadav2, Ramesh Chand4 1 Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, India 2 Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University Varanasi, India 3 Department of Biotechnology, J C Bose Institute of Life Sciences, Bundelkhand University, Jhansi India 4 Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University Varanasi, India *

Address for Correspondence: Shweta Singh, Research Scholar, Department of Botany, Institute of Science, BHU, Varanasi, India Received: 06 November 2016/Revised: 23 November 2016/Accepted: 16 December 2016

ABSTRACT- The adult plant resistance (APR) gene Lr34 and Lr46 of wheat is associated with leaf tip necrosis (Ltn), and provide resistance against multiple diseases, viz. leaf rust, stripe rust, stem rust, powdery mildew and spot blotch.

APR gene Lr46 is also associated with Ltn. Lesion mimic (lm) mutants express hypersensitive responses in the absence of pathogens and also confer resistance to biotrophic pathogens, including leaf rust. However, association between spot blotch and Ltn is reported, but not with Lm and Ltn. Five hundred diverse lines of spring wheat including two hundred and ninety four wami population including were screened for Lr34 (CsLv34, 150 bp), Lr46 (STS1BL2, 600bp) and three lesion mimic genes, viz. lm (Xwmc85.1 and Xgwm264) on 1B, lm1 (Xbarc147 and Xwmc674) on 3BS, and lm2 (Xgwm513 and Xgwm149) on 4BL.Lr34alone was found in only one line, but in 31% of lines it was in combination with Lr46. In contrast, Lr46 was present singly in 63% of lines. The Lm genes appeared to increase spot blotch severity whereas Lr34 alone or with Lr46 provided moderate to high level of resistance. Lm expression was absent when both Lr34 and Lr46 were present indicating their masking effect on Lm genes. The presence of Lr46 alone showed variable Lm expression.

This suggests that the presence of Lr34 + Lr46 and/or absence of Lm genes enhance resistance to spot blotch.

Key-words- Leaf tip necrosis (Ltn), Lesion mimics (Lm), Spot blotch, Phenotypic marker, Adult plant resistance (APR) -------------------------------------------------IJLSSR-----------------------------------------------

INTRODUCTION The leaf tip necrosis (Ltn) is most popular trait among the wheat pathologists and breeders for its association with the durable rust resistance in adult plants. The adult plant resistance gene, Lr34 (formerly LrT2), was first described in a wheat lines PI58548 and was later shown to be present in a number of wheat cultivars [1-2]. Leaf tip necrosis is closely associated with Lr34 gene [3] and is being used as its phenotypic marker [4].

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DOI: 10.21276/ijlssr.2017.3.1.10

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Lr34 confers horizontal resistance or slow rusting resistance although under appropriate conditions it may confer resistance in seedlings to certain rust races. The efficianacy of Lr34 mediated APR also varies between different cultivar backgrounds and growth conditions. More importantly Lr34 has been recognized as major component of durable rust resistance as it can act synergistically with other leaf rust resistance genes. Lr34 encodes a putative adenosine triphosphate–binding cassette (ABC) transporter.

It belongs to the ABC subtype G (formerly PDR), subclass of transporters [5]. It was reported that developmentally regulated resistance is correlated with elevated Lr34 transcript levels in flag leaves of adult plants. There are several allelic forms of the Lr34 gene that have been identified in the wheat germplasm, with two alleles occurring at a high frequency [6]. Resistance allele (Lr34res) differs by only two amino acid changes from the other allelic forms associated with susceptibility (Lr34sus) and so

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gain-of its function of resistance. So it is assumed that the resistance conferred by Lr34res is the result of altered activity or substrate specificity. The presence of Lr34 and Lr46 in wheat is associated with leaf tip necrosis (Ltn), a type of necrosis that forms predominantly in the tips of wheat flag leaves, and is dependent on expression of Lr34res as well as on genetic. The gene Lr34 was postulated in few Indian bread wheat cultivars reported the association of leaf tip necrosis with the spot blotch of wheat [7] .

Spot blotch caused by Bipolaris sorokiniana has emerged an important problem in the last century in 9 million hectare what of South East Asia. This pathogen affects the crops of millions of small and marginal farmers of this region and cause severe damage. Annual average yield losses due to spot blotch are reported around 19.6 and 15.5 % in wheat [8]. There are many program have been initiated to improve the wheat against spot blotch [9-10]. But not much success can be achieved to reduce or minimize the damage due to spot blotch. Due to complex problems of pathogen and tropical environment of South Asia Breeding progress is slow. Substantial gain to spot blotch resistance as made by sleeting wheat lines with Ltn [7]. [11] reported the association of Lr34 and Lr46 with the spot blotch resistance. In present studied population genotyping with SSR markers shows the predominance of Lr46 alone or in combination of Lr34. This study was performed for variable level of spot blotch severity to understand the role of different gene combination in the spot blotch resistance.

Spot blotch spreading is very much influenced by environmental condition like temperature and radiation. It was reported that an increase in these two factors also shows additive effect in the susceptibility. Terminal heat stress increased severity and showed positive correlation in South Asia [10,12-13]. Temperature is an important non living factor that influenced the developmental growth stages of plant. The total temperature required for plant growth development can be calculated by accumulating degree days between the high and low temperature thresholds throughout the crop season [14]. Incidence and spread of spot blotch are influenced by the growth stages, and maturity duration of wheat [15]. Plants that spontaneously express necrotic lesions during development or in response to changes in the environment, without the presence of any pathogen, are denoted as lesion mimics exhibiting either constitutive or unregulated cell death like HR after pathogen infection. So it can be hypothesized that Lm in wheat facilitating the spot blotch development and its rapid spread in the host due to readily available necrotic tissue for colonization of the pathogen. If this is true then Lm free/least Lm wheat lines expected to have low susceptibility and Lm expression should also be suppressed

by Ltn like spot blotch so the objective of this study was to establish association between Ltn that is a trait of resistance, lm and spot blotch, phenotypic and genotypic expression and relation of different Ltn genes and distribution of different Ltn genes in present population. http://ijlssr.com

MATERIALS AND METHODS Screening of Germplasm: Five hundred diverse wheat germplasm including two hundred ninety four wami germplasm were evaluated for leaf rust caused by Puccinia recondite, spot blotch causal organism Bipolaris sorokiniana and Lesion mimic at the Research Farm of Banaras Hindu University, Varanasi during crop season 2013-14, 2014-2015 and 2015-16.

Sowing and maintenance of crop under field and polyhouse condition Banaras Hindu University falls in the rice wheat cropping system zone. The field was prepared by ploughed thrice after the harvest of rice crop. Fertilizers NPK were applied @ 60, 60 and 40 Kg/ha during land preparation. Five hundred wheat germplasm were sown in the alpha lattice design in two replication, 25 blocks and each block carrying 20 genotypes. Seeds were sown manually keeping row to row distance 25 cm and plant to plant 12 cm. The first irrigation was given at crown root initiation 20 days after sowing and 30 Kg Nitrogen was also given as top dressing after 3 days of irrigation while the last dose of nitrogen was given after flowering (GS45).

Scoring of leaf tip necrosis Leaf tip necrosis was scored on flag leaf in the field at the GS 47 to 73. Flag leaves showing necrosis from the tip and extended to 5cm were counted as Ltn. Leaf tip necrosis was not appeared in poly house therefore it was not scored.

Under poly house conditions genotypes were screened at GS 47-73 before ear emergence. Leaves were scored in two way, 1st it was scored as presence or absence of Ltn. 2nd when it was present it was scored as the level of their expression. The scale was used for it is 0 to 4 where 0= no Ltn, 1=25%, 2=50%, 3=75% and 4 more than 75%.

Scoring of lesion mimic The plants were observed from seedling to adult stage both in field and polyhouse for the expression of Lm symptoms.

Lesion mimic was recorded from seedling (GS25) to adult plant stage (GS60) in poly house. Lm phenotype was scored on flag leaf in the field and poly house at the GS 60.

The distribution of Lm on leaf i.e. apical, middle and basal part was also recorded. Leaves showing typical LM expression was scored with modification of 1- 6 rating scale of [16] in 1-9 scale. Where 0=no visible specks 1= 1-10%, 2=10.1 to 20%, 3= 20.1-30,4= 30.1-40, 5= 401-50, 6= 50.1-60, 7= 60.1-70, 8, 70.1-80, 9= above80%.

Isolation of pathogen All the genotypes produced leaf tip necrosis were collected for the presence of Bipolaris sorokiniana or other pathogen.

Three well development lesions were taken for the isolation of pathogen. A piece of 1mm2 leaf tissue was taken out from the one week old lesion. Isolation was performed by the method of [17].

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Inoculation of the pathogen A pure culture of the most aggressive isolate of Bipolaris sorokiniana (NABM MAT 1; NCBIJN128877, Banaras Hindu University, Varanasi, India) known to be highly aggressive was used to create artificial epiphytotic [7]. The isolate was multiplied on sorghum grain and a spore suspension adjusted to 10-4 spores/ml of water using haemocytometer was uniformly sprayed at GS 50 on Zadoks scale during evening hours [18].

Disease assessment Spot blotch score for each line was evaluated on five randomly tagged plants in the field at three different growth stages (GS) viz; GS 63 (beginning of anthesis to half complete), GS 69 (anthesis complete) and GS 77 (late milking) [18] following double digit scale (DD, 00-99) [19].

The first digit (D1) indicates vertical disease progress on the plant and the second (D2) indicates severity measured in diseased leaf area. For each score, the disease severity percentage was based on the following formula: % severity = (D1/9) (D2/9)100.

Area under the disease progress curve based on disease severity over time at GS63, GS69 and GS77 was calculated using the percent severity estimations corresponding to the disease ratings, as outlined by: AUDPC =

{(Y + Y i

) / 2} × (t

(i + 1)

(i + 1)

- t )] i

Where, Y = disease level at time ti i t(i + 1) - ti = Time (days) between two disease scores n = number of dates at which spot blotch was recorded

Use of molecular markers for the leaf rust resistance genes Molecular marker analysis A total of 2 SSR and STS markers associated with Ltn and 10 SSR markers associated with lesion mimic mutants, were used for screening of wheat germplasm viz; CsLv34(7DS) and STS1BL2 (1BL), Xwmc 674 (3BS); Xbarc1033 (3BS); Xbarc147 (3BS); Xgwm513 (4BL); Xgwm149 (4BL); Yao et al., 2009 Xbarc181 (1B); Xbarc61 (1B); Xgwm131 (1B); Xwmc85.1 (1B); Xgwm264.1 (1B) [20] .

DNA extraction, PCR amplification and gel electrophoresis Plant gnomic DNA extracted from the leaves of 15 days old seedling by following the modified CTAB method [21].

Isolated DNA was kept at -20°C for further use. Each Polymerase chain reaction (PCR) was performed in 15µl reaction volume containing 10ng genomic DNA, 1.5µl 10nM Tris-HCL (pH 8.8), 0.2µl dNTPs (MBI Fermentas), 0.2µl 2.5mM MgCl2 (MBI fermentas), 0.2µl Taq polymerase 5U/µl (MBI fermentas) and 0.6µl forward and reverse primers. The sequences of each primer are listed in Table 1. Amplification were performed in touchdown i.e., http://ijlssr.com

10 cycles were performed at 94°C for 1min, 65-55°C for 1min and 72°C for 1min. After completion of one cycle, the annealing temperature lowers down 1°C. Remaining 30 cycles were performed at 94°C for 30s, 55°C for 30s and 72°C for 30s with a final extension at 72°C for 7 min. The 15µl PCR products were mixed with 2µl loading dye (98% formamide, 0.3% of each bromophenol blue and xylene cyanol, and 10mM of EDTA). Electrophoresis was carried out in 2.5% agarose gel stained with EtBr and 1 x TAE gel electrophoresis buffer, prepared from 50 x TAE (242g Tris-base, 57.1ml Glacial acetic acid and 100ml 0.5M EDTA for 1L), was used for electrophoresis, at 75 W for 2 hours. Gels picture was visualized under UV light by using UVP gel doc.

RESULTS Experiment in Field and Poly house conditions Ltn were first observed on the leaves of plants. The expression of Ltn in all plant of same genotypes was uniform however it was un-uniform in few genotypes. The appearance of Ltn was recorded at late GS (47 to 73). Lm symptoms were first observed on the leaf at the time of booting stage and scored.The genotypes with good expression of Ltn show no or very less expression of lm (Fig. 1). Appearance and distribution of Ltn under poly-house was not like the field. Ltn in poly house was not expressed. There were no plant in poly house expressed Ltn.

Effect of Temperature on expression of Ltn During the experiment it was observed that no expression of Ltn was seen in poly house (DD 1107) however it was clearly expressed in field (DD 840). So it can be predict that Ltn and DD are negatively associated. At the time of experiment maximum temperature in field was 280C and in polyhouse it was above 400C. So it can also be predicted that at high temperature the Ltn did not expressed itself. So it is clear that gene of Ltn is thermolabile. Appearance and distribution of Lm under poly-house was different than the field. Under poly-house more than 70% genotypes expressed Lm at early (GS 25-30) (35 days after showing).

The Lm severity was higher in polyhouse than the field it may be due to absence of Ltn and presence of high temperature.

Leaf tip necrosis and its association with spot blotch In the field Ltn and spot blotch were recorded in some of different way as previously reported. All those lines which express Ltn very well were not resistant against spot blotch.

Some lines with Ltn were also susceptible for the disease, but some of the Ltn lines shows less severity or no severity of disease. The susceptible genotypes which have Ltn in their phenotype show the higher AUDPC (Table 2).

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Genotyping for Ltn lines

Lm, Ltn and Spot Blotch in field

Five hundred diverse lines including two hundred ninety four wami population of spring wheat were screened for Lr34 (CsLv34), Lr46 (STS1BL2). Genotyping of Ltn lines clearly showed the presence of two different genes i.e.Lr34, and Lr46 (Fig. 2). The frequency of these genes alone and in combinations is given in table 2.In the populationLr34 alone was found in only four line, but in 31% of lines it was in combination with Lr46 (Table 1). In contrast, Lr46 was present singly in 63% of lines. Lr34 alone or with Lr46 provided moderate to high levels of resistance.

The lines with Ltn which have less or no expression of lesion mimic were resistance or moderately resistance against spot blotch. Table 1, shows the distribution of Ltn in wheat genotypes and Table 2 shows slow or no Lm frequency on those genotypes where Ltn was present.

Leaf tip necrosis and its association with spot blotch The genotyping shows that Ltn with Lr46 does not shows the resistance against disease but if the gene Lr34 is present then they were shown good resistance against the disease.

Lr34 provides more resistance against spot blotch in comparison to Lr46 (Fig 1, Table 4).

Ltn, Lm and DD Lm and DD (degree days) were positively correlated whereas Ltn and DD are negatively correlated. Lesion mimic expression was recorded after 35 days of sowing in the poly house when it received the total DD 1101.1.

Whereas on the same genotypes LM appeared after 74 days under filed conditions with the DD 740 (Table 4.) but in case of Ltn in polyhouse at the 1101DD it does not appear and in field at 840 DD it was appeared very well (Fig 1, Table 3). Ltn is thermolabile where lm is thermostable.

Table 1. Distribution of LTN genes among 500 wheat genotypes Ltn gene (S)

Number of genotypes

% Germplasm

Ltn gene (S)

Lr34

4

0.8

Lr34

Lr46

315

63

Lr46

Lr34+Lr46

155

31

Lr34+Lr46

Table 2. List of wheat genotypes without Lesion mimic expression in the field and poly house, status of Lm gene, Ltn and yield related characters. (a- absent, p- present) Entry

Genotype

DH

AUDPC

1 2

VORONA/GEN PARA2//JUP/BJY/3/VEE/JUN/4/2*K AUZ KAUZ/RAYON RABE/2*MO88 OASIS/SKAUZ//4*BCN HUAYTU CIAT TARACHI F 2000 TAURUM CNDO/R143//ENTE/MEXI_2/3/A EGILOPSSQUARROSA (TAUS)/4/WEAVER/5/2*KAUZ KETUPA*2/PASTOR REH/HARE//2*BCN/3/CROC_1/ AE.SQUARROSA (213)//PGO/4/HUITES CROC_1/AE.SQUARROSA (205)//BORL95/3/PASTOR ATTILA/3*BCN*2//BAV92 TUKURU//BAV92/RAYON WBLL1*2/4/YACO/PBW65/3/KA UZ*2/TRAP//KAUZ PAVON KEA/BUC//FCT

87 86

3 4 5 6 7 8 9

10 11

12 13 14 15 16 17

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TKW

Lm field

417.22 564.45

PLOT YIEL D 119.5 55

lm

lm 1

lm 2

Ltn

A A

lm PolyHouse a a

31.8 24.8

p p

p a

P P

p P

82 86 86.5 83 86.5 86.5 84

447.66 438.09 365.81 406.73 453.03 366.48 351.92

125.5 114.5 110 116.5 107.5 63 148

25 24.8 22.6 30.2 30.4 21.6 30

a A A A A A A

a a a a a a a

p p p p p p p

p p a p a p p

P P P A P P P

a p p p p p p

86.5 82

573.21 523.77

104 147

20.3 32.4

A A

a a

p p

p p

P p

a a

84

490.81

107

29.8

A

a

p

p

A

a

86 82 84

396.05 562.22 461.24

130 128 134

25.4 28.3 28.4

A A A

a a a

p a p

a a p

P P P

a p p

87 80

436.91 505.13

106.5 127.5

22.8 33.2

A A

a a

p p

p p

P P

p p

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Int. J. Life. Sci. Scienti. Res., VOL 3, ISSUE 1 18 19

20

21

22 23 24 25 26 27 28

29

30

31 32 33 34

35

36

37

38

39

40 41 42 43 44

45 46

PRL/SARA//TSI/VEE#5 CNDO/R143//ENTE/MEXI_2/3/A EGILOPS SQUARROSA (TAUS)/4/WEAVER CROC_1/AE.SQUARROSA (205)//JUP/BJY/3/SKAUZ/4/KAU Z PASTOR//SITE/MO/3/CHEN/AEGILO PS SQUARROSA (TAUS)//BCN BARBET1 MILVUS2 ATTILA*2/PBW65 VOROBEY MILAN/KAUZ//PRINIA/3/BAV92 PASTOR/DHARWAR DRY MILAN/KAUZ/3/URES/JUN//KAUZ /4/CROC_1/AE.SQUARROSA (224)//OPATA CNO79//PF70354/MUS/3/PASTO R/4/CROC_1/AE.SQUARROSA (224)//OPATA CROC_1/AE.SQUARROSA (224)//OPATA/3/BJY/COC//PRL/B OW/4/BJY/COC//PRL/BOW CHEN/AE.SQ//2*OPATA/3/BAV9 2/4/JARU CHONTE WHEAR//2*PRL/2*PASTOR KAUZ//ALTAR 84/AOS/3/MILAN/KAUZ/4/HUIT ES CROC_1/AE.SQUARROSA (205)//BORL95/3/PRL/SARA//TSI /VEE#5/4/FRET2 CROC_1/AE.SQUARROSA (205)//BORL95/3/PRL/SARA//TSI /VEE#5/4/FRET2 ALTAR 84/AE.SQUARROSA (219)//OPATA/3/WBLL1/FRET2// PASTOR YAV_3/SCO//JO69/CRA/3/YAV79 /4/AE.SQUARROSA (498)/5/2*OPATA YAV_3/SCO//JO69/CRA/3/YAV79 /4/AE.SQUARROSA (498)/5/2*OPATA CHEWINK THB/KEA//PF85487/3/DUCULA/ 4/WBLL1*2/TUKURU WBLL1*2/3/SNI/TRAP#1//KAUZ *3/TRAP/4/KACHU PBW343*2/KUKUNA*2//WHEA R MILAN/KAUZ//PRINIA/3/BAV92/5/ TRAP#1/BOW//VEE#5/SARA/3/Z HE JIANG 4/4/DUCULA ASTREB/OAX93.10.1//SOKOLL BACANORA T 88

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84 83.5

557.8 520.75

132 162

30.2 36.6

A A

a a

p a

p p

P P

a p

86

409.88

151.5

30.6

A

a

p

p

P

p

82

425.06

151.5

36.8

A

a

a

a

A

p

84 82 82 86.5 84 74 81.5

502.9 540.06 439.33 366.05 443.83 211.85 239.14

124.5 130.5 127 121.5 115.5 157.5 159

27.2 29.2 27.8 25.4 30.6 34.2 36.9

A A A A 0 0 A

a a a a 0 a

p p p p p a p

p p p p p a p

P p P P P A P

p p p p p p p

84

522.66

118.5

28.4

A

a

p

p

P

82

454.32

110.5

29.4

A

a

a

a

P

a

82

434.26

132

32

A

a

a

a

P

a

86.5 80 82

528.53 548.34 536.55

141.5 110 144

31.2 31.6 32

A A A

a a a

p a p

a p p

A P P

p p p

84

498.15

103.5

24.4

A

a

p

a

a

p

84

538.83

101.5

26.2

A

a

p

p

P

a

81.5

345.74

135

34

A

a

p

p

A

p

82

177.66

159

31

A

a

p

p

P

p

83.5

487.04

104

27.8

A

a

p

p

P

p

84 82

214.75 585.43

158.5 147

28.4 33.4

A A

a a

a a

p a

P A

p p

85.5

395.99

133.5

33.6

A

a

a

a

A

a

81.5

375.74

140

32.8

A

a

p

a

P

p

82

373.77

119

31

a

a

p

p

P

p

82 80

211.14 534.14

131 105.5

31.6 24.2

A A

a a

p p

p a

P P

p p

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Int. J. Life. Sci. Scienti. Res., VOL 3, ISSUE 1 BECARD/KACHU C80.1/3*BATAVIA//2*WBLL1/5/ REH/HARE//2*BCN/3/CROC_1/ AE.SQUARROSA (213)//PGO/4/HUITES KFA/3/PFAU/WEAVER//BRAMB LING/4/PFAU/WEAVER*2//BRA MBLING MILAN/KAUZ//PRINIA/3/BAV92 QUAIU #3//MILAN/AMSEL TACUPETO F2001/SAUAL/4/BABAX/LR42// BABAX*2/3/KURUKU TRCH//INQALAB 91*2/KUKUNA CROC_1/AE.SQUARROSA (205)//KAUZ/3/SASIA/4/TROST ESDA//ALTAR 84/AE.SQUARROSA (211)/3/ESDA/4/CHOIX/5/WAXW ING

47 48

49

50 51 52

53 54 55

84 85.5

485.99 506.18

119.5 133.5

29 27.2

A A

a a

p p

p p

P P

p p

87

372.16

138

29.7

A

a

p

p

P

p

82 86.5 86.5

439.39 472.22 474.45

128 143.5 125

30.5 28.2 28.6

A A A

a a a

p p p

p p p

P A P

a p a

76

430.87

92.5

28.4

A

a

a

a

P

p

82

420.07

135

31.4

A

a

p

p

P

p

82

503.42

137

28.6

A

a

p

p

P

a

84

417.78

145

34.8

A

a

a

a

P

p

Table 3. Cumulative degree days in poly house and field Field Min-Max Da ys

Week No.

Month and Date

50 51 52 1 2 3 4 5

Dec 10-16 Dec 17-23 Dec 24-31 Jan1-7 Jan 8-14 Jan 15-21 Jan 22-28 Jan 29-Feb4

13.6-26 6.7-26 5-22.2 2.5-25 2.8-27 6.5-32.5 4-22.5 6.6-26

2 7 8 7 7 7 7 7

0 169.1 145.4 138.8 142.7 183.3 144.8 171

6 7 8 9

Feb 05-11 Feb 12-18 Feb 19-25 Feb 26Mar04 Mar 5-11 Mar 12-18

8-27.7 11.6-28 11.8-28 13.1-30

7 7 7 7

13.5-35 16.4-33.8

7 7

10 11

Max

Min

Digreedays

MinMax

0 73.5 58.8 50 39.9 82.4 41.3 63.7

18.3 51.3 22.1 24.4 21.3 62.85 23.05 47.35

0 0

177.6 179.1 176.2 199.6

79.5 93.8 92.3 98.8

58.55 66.45 64.25 79.2

219.1 232.5

101.7 128.6

90.4 110.55 740.05

Days

0 0 1

26-46 26-42 31-43 31-50 36-48

Poly House Max Min

Digree days

0 0 28 192 194 192 227 249

0 0 40 291 296 288 296 312

0 0 24 171.5 175 170 191.5 210.5

186 0 0 0

232 0 0 0

159 0 0 0

0 0

0 0

0 0 1101.5

Table 4. Correlation coefficient of Lesion mimics with Ltn and other yield related traits Character Ltn DH AUDPC PlotYield

2013-14 -0.87 -0.070* 0.088** -0.102**

2014-15 -0.99 -0.187** 0.141** -0.116**

2015-16 -0.85 -0.099** 0.193** -0.003

TKW

-0.085**

-0.075*

-0.050

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Fig 1. Association of lesion mimics with Ltn and spot blotch

Fig 2. Screening of Ltn genes in present studied population

Fig 3. Different pattern of expression of Ltn on wheat plant in the field http://ijlssr.com

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DISCUSSION Five hundred wheat accessions including two hundred ninety four wami populations were screened for Ltn and most of them possess Lr46genes alone or in combinations with Lr34. However, the expression of Ltn varied in the wheat genotypes. The Lr34 are known to reduce the leaf rust severity by reducing uredinial size. Thus, the wheat genotypes carrying both of Ltn i.e. Lr34 and Lr46 will serve an improvement of wheat against spot blotch in the warmer region of South Asia. There are several genotypes in this experiment identified without expression of Ltn although they had either the genes alone or its combination.

These genotypes need to be tested under diverse environment to validate the result. Expression of Ltn absent in poly house revealed the thermolabile nature of gene Lr34 and Lr46 both. It may be possible that at high temperature these genes could not able to translate their code into protein. So that there is no translation results in absence of Ltn protein expression. The Lr34 gene encodes an ABC transporter and has provided wheat, durable and broad spectrum resistance against multiple fungal pathogens for over many years. In wheat, transcriptional expression of Lr34 and Lr46 are highest in adult plants and confers with increased resistance and Ltn affecting the last emerging leaf. Ltn also is a type of PCD and the genes can only express in appropriate environmental condition.

Temperature is the important abiotic controlling element for Ltn gene expression. Plant growth stage is also a factor for these two gene expression in wheat.Lr34 and lr46 in this population shows additive effect against spot blotch resistance. So it is the need of future breeding program to develop the genotypes which carry both the genes for acquired the effective level of resistance against spot blotch and various diseases.

The increase in global heat or temperature also cause the threatened of escape of Ltn gene expression due to its thermolabile nature.

CONCLUSION Present investigation confirms that alone Lr34 or Lr46 does not provide desire level of resistance but both the genes in conjugation provide a good level of resistance against spot- blotch and mask the expression of lesion mimic. So in South Asia region the variety having both the genes is more useful for food safety and yield loss. Ltn inhibit the expression of lesion mimic phenotypically.

ACKNOWLEDGMENT The authors are thankful for the financial support to UGC and CGIAR and CYMMIT for research material.

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How to cite this article: Singh S, Mishra VK, Kharwar RN, Ahirwar RN, Sharma S, Yadav PS, Chand R: Distribution of Leaf Tip Necrosis Genes and its Association with Expression of Lesion Mimic Genes and Resistance to Spot Blotch in Spring Wheat. Int. J. Life. Sci. Scienti. Res., 2017; 3(1): 808-816. DOI:10.21276/ijlssr.2017.3.1.10 Source of Financial Support: UGC-CGIAR-CYMMIT, Conflict of interest: Nil

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