Inheritance of southern root-knot nematode resistance in air-cured ...

Trop. plant pathol. (2017) 42:32–38 DOI 10.1007/s40858-016-0122-4

RESEARCH ARTICLE

Inheritance of southern root-knot nematode resistance in air-cured tobacco Zeinalabedin Shahadati-Moghaddam 1,2 & Nadali Bagheri 2 & Nadali Babaeian Jelodar 2 & Ghaffar Kiani 2 & Syed Abbas Hosseininejad 3

Received: 3 July 2016 / Accepted: 19 December 2016 / Published online: 5 January 2017 # Sociedade Brasileira de Fitopatologia 2017

Abstract The southern root-knot nematode (SRKN; Meloidogyne incognita) is one of the main pathogens for tobacco. The objectives of this study were to determine the genetic parameters and heritability of SRKN resistance in air-cured tobacco. For this purpose three resistant parents (Urmia3, KY9, K17) and two susceptible parents (Ergo, Burley TMV4) were crossed based on half diallel and analyzed by Hayman’s method for reaction to Meloidogyne incognita. Fifteen genotypes (parents and hybrids) were planted in the greenhouse in a completely randomized design with five replications and inoculated with 2500 J2 M. incognita race 2 one week after transplanting. Investigation of gall index (GI), number of egg masses (NEM) and average number of eggs per mass (AEM) showed significant differences among genotypes. Non-additive effects in AEM and additive effects in GI and NEM played an important role. Narrow-sense heritability was estimated as 0.75, 0.56 and 0.28 for GI, NEM and AEM, respectively, which revealed that AEM is not a suitable trait for nematode resistance identification. Regression analysis indicated that KY9 is an appropriate donor parent for SRKN resistance. Resistance to M. incognita race 2 is conditioned by a single partially dominant gene.

Section Editor: Rosana Rodrigues * Zeinalabedin Shahadati-Moghaddam [email protected]

1

Tirtash Research and Education Center, Behshahr, Iran

2

Plant Breeding and Biotechnology Department, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran

3

Iranian Research Institute of Plant Protection (IRIPP), Tehran, Iran

Keywords Nicotiana tabacum . Additive effects . Diallel . Gall index . Heritability

Introduction Southern root-knot nematode (SRKN; Meloidogyne incognita) is a serious pathogen in many crops, with yield losses of approximately 11% annually in the world (Agrios 2005). This pathogen causes a reduction in the growth and lowers the product quality with damage to roots in tobacco (Nicotiana tabacum L.). Development and use of resistant cultivars is one of the most effective and economical strategies for root-knot nematode management. Nematode resistance genes have been found in some crops such as tomato (Ernst et al. 2002), potato (Paal et al. 2004), pepper (Fazari et al. 2012) and sugar beet (Cai et al. 1997). There is diversity for resistance to root-knot nematode in tobacco genotypes (Ng’ambi et al. 1999b), which can be advantageous in producing resistant lines. Genetic response is an important factor to select the appropriate breeding method and create a cultivar with desired quality and quantity. This information can be obtained through techniques such as diallel cross (Jinks and Hayman 1953; Hayman 1954; Griffing 1956; Walters and Morton 1978; Mather and Jinks 1982). This method has been used to determine the genetic parameters of quality and quantity traits in crops and their resistance against pathogens (Pederson and Windham 1992; Zhang et al. 2007; Barros et al. 2011; Cardoso et al. 2014). Hayman’s method is one of the diallel analysis procedures for determining genetic parameters and heritability. This method is helpful to understand gene action and define the strategy of a breeding program to improve desired traits. Assumptions for diallel analysis include homozygous parents, diploid segregation, no multiple allelism and absence of epistasis.

Trop. plant pathol. (2017) 42:32–38

Moderate resistance to Meloidogyne incognita race 2 has been found in some genotypes of flue-cured tobacco (Ng’ambi et al. 1999a) and genetic control of resistance to M. arenaria race 1 by a single dominant gene has been reported (Ng’ambi et al. 1999b). The importance of general combining ability for cyst nematode (Globodera tabacum solanacearum) resistance in flue-cured tobacco has been expressed by Hayes et al. (1995) in a diallel perusal. Two partially dominant genes have been reported for SRKN resistance in cotton, with broad-sense and narrow-sense heritability for gall index having been determined as 0.82 and 0.65, respectively (Zhang et al. 2007). The importance of additive effects has been shown for SRKN resistance in white clover by Pederson and Windham (1992) in a six parental diallel method. Recently, QTL evaluation has led to the identification of chromosomal locations of nematode resistance in some crops (Pham et al. 2013; He et al. 2014; Huynh et al. 2016). He et al. (2014) identified two loci involved in root-knot nematode resistance on chromosomes 11 and 14 of upland cotton. QTL11 and QTL14 had negative effects on gall index and egg production, respectively. The authors reported additiveby-additive epistatic effects between the two resistant loci. The objectives of this study were to estimate the genetic parameters and heritability of SRKN resistance traits in air-cured tobacco and determine the best trait for resistance analysis.

Material and methods Plant materials Five pure genotypes of air-cured tobacco (Nicotiana tabacum L.), three SRKN resistant (KY9, K17, Urmia3) and two susceptible (Ergo, Burley TMV4) (Hosseini et al. 2011) were crossed in a half-diallel mating design at the Tirtash Research & Education Center, Behshahr, Iran. Parental and F1 seeds (fifteen genotypes) were planted in the greenhouse in 2015 based on a completely randomized design with five replications. Plastic pots (1.5 L) were used with sterilized sandy loam soil. Inoculation A population of M. incognita race 2 was isolated from fields of Mazandaran province, Iran and were proliferated on susceptible tomato plants (Solanum lycopersicum cv. Rutgers) in the greenhouse. Inoculum was collected from tomato roots according to Hussey and Barker (1973). One week after transplanting of tobacco plants, a hole near each plant in the pot was created. One mL of water suspension containing 2500 J2 nematodes/ mL was pipetted in each hole and the holes were covered with soil. The plants were irrigated daily by hand and the greenhouse temperature was controlled at 27 ± 1°C.

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SRKN screening Fifty days after inoculation plants were harvested by gently rinsing off the soil from the roots with running water. Root weight was measured and roots were rated for GI based on a 010 scale according to Bridge and Page (1980). Number of egg masses (NEM) on roots was counted and was calculated per 1 g of roots. Ten egg masses of each genotype were placed in 0.5% sodium hypochloride (1 mL) for 1 min and shaken according to Hussey and Barker (1973). Three samples (50 μL) were drawn from the suspension and were observed under a light microscope. Eggs were counted and the average number of eggs per mass (AEM) was calculated. Data analysis Data were subjected to analysis of variance in SAS 9.1 software. Bartlett’s test was applied to evaluate homogeneity of variance among parents and hybrids. Regression coefficients (Wr/Vr and T2 test) were used to assess diallel assumptions (parent homozygosity, diploid segregation and absence of epistasis). When the ANOVA results indicated a significant variation among genotypes, genetic analysis was done based on the diallel technique according to Hayman (1954) and Mather and Jinks (1982) using Microsoft Excel and Genes software (Cruz 2013). Heritability and degree of dominance were estimated according the Hayman’s method (Hayman 1954). Genetic variance components, E (environmental variance), D (additive effects of genes), F (covariance of additive and dominant effects), H1, H2 and h2 (dominant effects of genes) were calculated according to the following estimators: MSE r D ¼ Vp−E E¼

2ðp‐2ÞE p ð5p‐4ÞE H 1 ¼ Vp−4Wr þ 4Vr− p 4ðp2 ‐p þ 1ÞE H 2 ¼ 4Vr−4Vr− p2 4ðp2 ‐pÞE h2 ¼ 4ðMl1 −Ml0 Þ2 − p3 F ¼ 2V p −4Wr−

Where, MSE = mean square of error, r = number of replications, Vp = phenotypic variance; Wr = average of covariances between offsprings and non-recurrent parents, p = parents; Vr = average of offsprings variances in each parent, Vr = variance of the means of arrays; Ml1 = average of parents, Ml0 = average of offspring.

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Results ANOVA and diallel assumptions test Analysis of variance showed highly significant differences among genotypes (parents and hybrids) for all resistance traits (Table 1), which confirmed significant genotypic differences in nematode resistance among the genotypes. Galls were present at varying levels in the genotypes. The lowest GI related to KY9 (1.4) in parents and to KY9 × Urmia3 (2.6) in hybrids. Ergo (7.6) and Burley TMV4 (6.8) showed the higher GI. The lowest NEM was 4.2 for Urmia3 in parents and 16.6 for KY9 × Burley TMV4 in hybrids. Maximum NEM was 56.2 for Ergo and 53.6 for Burley TMV4. For AEM, Urmia3 (424) and KY9 × Burley TMV4 (432.8) had the lowest means in parents and hybrids, respectively. However, there was no significant differences among Ergo (458.9), Burley TMV4 (486.2), KY9 (459.3) and K17 (467.6) in AEM. Evaluation of diallel assumptions with the T2 test (T2df 4, 3 = 9.12) showed that values of T2 were not significant for GI (estimated T2 = 2.83), NEM (estimated T2 = 4.99) and AEM (estimated T2 = 0.4). Thus, there was no multiple allelism, no epistasis for all traits and diploid segregation was confirmed. Regression coefficients Wr/Vr were 1.03 ± 0.46 for GI, 1.12 ± 0.52 for NEM and 0.68 ± 0.39 for AEM, which had no significant differences from unity (Jinks and Hayman 1953). These results allowed the use of Hayman’s model for genetic analysis of these characters. Genetic parameters To estimate the genetic parameters for SRKN resistance in this set of parents and their hybrids, analysis was conducted following Hayman’s diallel model (1954). The additive and dominant variance components were both highly significant for all traits (Table 2). Additive gene action, which had higher value than dominant action, plays a major role in GI and NEM. But in AEM, for which H1 and H2 were higher than D, dominant effects was more important. Importance of additive effects in GI and NEM indicated that these traits can be helpful for SRKN resistance selection in tobacco breeding projects. These results were confirmed by narrow-sense heritability (GI = 0.75, NEM = 0.56). A greater D value than H1 Table 1 Analysis of variance for southern root-knot nematode resistance traits with 15 diallel cross genotypes of Nicotiana tabacum based on a completely randomized design and five replications

Sources of variation

DF

and H2 indicates partial dominance in GI and NEM. The average degree of dominance, (H1/D)0.5, confirmed the partial dominant nature of GI (0.60) and NEM (0.84). However, (H1/ D)0.5 for AEM was 1.89, indicating overdominance. Environmental variance (E) and h2 were not significant for GI and NEM (Table 2). Non-significant E variance revealed that these traits were not affected by environmental conditions. A highly significant E value showed that environmental variation was effective for AEM. h2 indicates heterozygous loci in parents. Because both parents were homozygous, h2 was not significant in all traits. F values were significant or highly significant for all traits. A positive F value indicates that dominant alleles were more predominant than recessive alleles in the parental lines. This was confirmed by KD/KR values greater than unity for all traits. The positive correlation between Wr/Vr and GI revealed that dominant alleles had negative effects (Table 2). Correlations between Wr/Vr and traits in NEM and AEM were not significant. The estimated minimum number of genes for M. incognita race 2 resistance suggests that there is a single partially dominant resistance gene controlling GI (1.00) and NEM (0.92). However, more than two genes are involved for AEM (2.87) (Table 2). High broad-sense heritability values were detected for GI (0.93) and NEM (0.94) (Table 2). However, broad-sense heritability was low for AEM (0.61). These values indicate that genetic control for GI and NEM was stronger than for AEM, most likely because environmental effects were highly significant for AEM (as previously described). Narrow-sense heritability was 0.75, 0.56 and 0.28 for GI, NEM and AEM, respectively. These values confirm the importance of additive effects for GI and NEM and non-additive effects for AEM, and indicate that GI and NEM are more suitable traits than AEM for nematode resistance studies. For all nematode resistance traits, the parental array points were scattered along the regression line in the Wr/ Vr graph. This indicates the existence of genetic diversity among the parents for the traits studied. Distribution of parental points in the Wr/Vr graph also indicates relative proportions of dominant and recessive alleles in the parents. Graphical representation revealed that the regression line intercepted the Wr axis above the point of origin in GI (Fig. 1) and NEM (Fig. 2), indicating the presence of

Mean squares Gall index

Number of egg masses

Average number of eggs per mass

1278** 44.8 17

2288** 867 6.4

Genotype

14

16.48**

Error CV%

60

0.6 18

**, significant at 1% probability level

Trop. plant pathol. (2017) 42:32–38 Table 2 Estimates of statistical indices and genetics parameters for southern root-knot nematode resistance traits in five tobacco varieties diallel crosses

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Parameter

Gall index

Number of egg masses

Average number of eggs per mass

D H1

9.6 ± 0.34**

727.6 ± 40**

336.2 ± 168**

3.5 ± 0.93** 2.1 ± 0.85**

509.8 ± 108** 329.6 ± 98**

1197.2 ± 454** 776.4 ± 411**

7.0 ± 0.85** 0.6 ± 0.67

672.7 ± 98** 1.6 ± 6.6

470.5 ± 412* −121.3 ± 278

(H1/D) KD/KR Hb Hn

0.1 ± 0.14 0.60 4.06

9.0 ± 16 0.84 3.47

173.4 ± 68** 1.89 2.18

0.93 0.75

0.94 0.56

0.61 0.28

Ng R (Wr+Vr)/trait

1.00 0.96**

0.92 0.76

2.87 −0.77

H2 F h2 E 0.5

D, genetic variation due to additive effects of genes H1, H2, genetic variation due to dominance effects of gene F, covariance of additive and dominant effects h2 , summation of dominance deviation over all loci E, environmental variation (H1/D)0.5 , average degree of dominance at each locus (0, no dominance; 1, complete dominance; >1, overdominance) KD/KR, ratio between the total number of dominant to recessive alleles in all parents Ng, minimum number of genes estimated as (maximum parent value – minimum parent value)2 /4D Hb, broad-sense heritability Hn, narrow-sense heritability R, correlation (Wr + Vr) with resistance traits *, ** significant at 5 and 1% probability levels, respectively

partially dominant genes. The regression line intercepted the Wr axis below the point of origin in AEM (Fig. 3), indicating overdominant gene action. These results are in agreement with results of degree of dominance [(H1/D)0.5]. Fig. 1 Linear regression of Wr/ Vr and limiting curve for gall index. Parents: P1, Urmia3; P2, Burley TMV4; P3, Ergo; P4, KY9; P5, K17

Discussion Additive effects in allelic interaction are important in plant breeding, as the phenotype is predictable by additive effects.

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Fig. 2 Linear regression of Wr/ Vr and limiting curve for number of egg masses. Parents: P1, Urmia3; P2, Burley TMV4; P3, Ergo; P4, KY9; P5, K17

GI and NEM had a main additive effect in our study. The existence of additive effects for nematode resistance has been reported in red clover (Call et al. 1997), sweet potato (Cervantes-Flores et al. 2008) and cotton (Zhang et al. 2007). There are reports that resistance to cyst nematode (Lamondia 2002; Crowder et al. 2003) and M. arenaria (Ng’ambi et al. 1999b) in tobacco is controlled by a single dominant gene. We also found out in this study that there is a single partially dominant gene for SRKN resistance. The number of nematode resistant genes in other plant has been reported to range from one to four (Fery and Dukes 1996; Cervigni et al. 2007; Zhang et al. 2007; Shrestha et al. 2012). Broad and narrow sense heritabilities are the proportions of phenotypic variance that are explained by genotypic and additive variance, respectively. Thus, narrow sense heritability is a more useful indicator in plant breeding. The moderate to Fig. 3 Linear regression of Wr/ Vr and limiting curve for average number of eggs per mass. Parents: P1, Urmia3; P2, Burley TMV4; P3, Ergo; P4, KY9; P5, K17

high narrow sense heritability for GI and NEM indicated that selections based on a single plant in early generations should be relatively effective in breeding for SRKN resistance under greenhouse conditions. High narrow sense heritability for nematode resistance was also reported in cotton (Zhang et al. 2007) and soybean (Cervigni et al. 2007). The first known SRKN gene in Nicotiana, designated as Rk, was derived from N. tomentosa. This gene confers resistance to races 1 and 3 of M. incognita and is inherited in a dominant fashion (Yi et al. 1998). The Rk gene does not provide resistance to M. incognita race 2. The “eggs per gram of root” parameter has been used in some studies of resistance to SRKN (Ng’ambi et al. 1999a; Starr et al. 2010). Separation of this trait into two parameters (eggs per gram of root = NEM × AEM) revealed NEM to be more useful than AEM. Vrain et al. (1994) did not observe


significant heritability for nematode numbers in the root of raspberry plants. It is possible that AEM is more affected by nematode features than by plant features, as the plant would not be able to control the AEM once female nematodes establish a successful feeding site at the root. Significant environmental variance reflects also the possible impact of additional factors besides plant genetics. Furthermore, there was a significant correlation between GI and NEM, while correlation among AEM and the other parameters was not significant, suggesting that the control mechanisms of AEM are distinct from those of GI and NEM. Comparing the diagrams for all traits revealed genotype KY9 to be located near the center of the coordinates, which indicates the presence of dominant alleles in this genotype. Hybrids with KY9 involved had the lowest value (= resistant) for all traits, indicating the power of KY9 in donating resistance factors. This genotype can be used in the production of male sterile hybrids with high SRKN tolerance. Moreover, KY9 can be crossed with high quality commercial varieties to introduce a line with suitable yield and nematode resistance. In conclusion, our results indicate that additive effects for southern root-knot nematode resistance are important and that resistance is controlled by a single, partially dominant gene. Also, gall index and number of egg masses are suitable traits for selection to nematode resistance in breeding projects. The average number of eggs per mass should not be used in such studies. Finally, KY9 is a suitable genotype as a donor parent for breeding projects. Acknowledgements We are thankful to A. Hosseini and Dr. J. Faghihi for technical assistance. This study was supported by the Tirtash Research and Education Center.

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