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Published online 1 March 2007
Published in Crop Sci 47:529-534 (2007)
© 2007 Crop Science Society of America
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CROP BREEDING & GENETICS

Genetic Enhancement of Resistance to Alternaria Leaf Blight in Sunflower through Cyclic Gametophytic and Sporophytic Selections

T. Shobha Rani and R. L. Ravikumar*

Dep. of Genetics and Plant Breeding, Univ. of Agricultural Sciences, Dharwad, Dharwad 580 005, Karantaka, India

* Corresponding author (ravikumarrl{at}yahoo.co.in).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Only partial resistance has been reported so far in Helianthus species for leaf blight caused by Alternaria helianthi (Hansf.). Therefore, in the present study one to two cycles of recurrent selection based on sporophytic and gametophytic selection was effected to increase resistance to leaf blight in the base population. The base population was synthesized by random mating five diverse early generation breeding lines. The gamete selection was practiced by applying pathogen culture filtrate to stigma and style 1 h before pollination. The selection response was measured by recording observations on percent disease index (PDI) at flowering, 15 d after flowering and at physiological maturity. In both gametophytic and sporophytic selection cycles significant reduction in PDI was observed. The populations improved through gametophytic selection were more promising as the pollen selection could allow high selection intensity and absence of dominance effects. In sporophytic selection the rare favorable allelic combination is hardly detected. The progress in resistance in two cycles of selection was not very high for their use in crop improvement. There is a need to increase the number of cycle of selection to maximize the desirable alleles to achieve high level of resistance. The gametophytic selection combined with the conventional sporophytic selection can be considered as an effective tool in improving populations for partial resistance.

Abbreviations: PDI, percent disease index.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
SUNFLOWER (Helianthus annuus L.) has become a major oil seed crop in India within a short period of its introduction. But the productivity of the country is low; several biotic and abiotic factors affect the crop. Among biotic stresses Alternaria leaf blight is the most important disease of sunflower causing significant yield losses in India and other tropical countries (Acimovic, 1969; Balasubrahmanyam and Kolte, 1980; Hiremath et al., 1990). In India, the disease is particularly severe during the rainy season (Hiremath et al., 1990). The disease is known to infect all the parts of the plant viz., leaf, petiole, stem, all the flower parts, and seeds. The pathogen causes brown spots on the leaves, stem, petals, and sepals, which lead to premature defoliation, stem breakage, and death of the plant under severe infection (Anilkumar et al., 1974; Morris et al., 1983). The conidia of A. helianthi, germinate on the leaf surface by producing several germ tubes (Tubaki and Nishihara, 1969). The germinated conidia form appresoria and enter the cells by direct penetration through the cuticle and epidermis. The entry through stomata and wounds without formation of appresoria was also observed. The penetration is accompanied by a chemical degradation of surrounding tissue (Allen et al., 1983). The development of resistant cultivars offer the most technically feasible and economic means of disease control, as fungicides are not economical, particularly among farmers of developing countries including India. The studies over the years identified only partial resistance to Alternaria leaf blight in cultivated or closely related species (Carson, 1985; Ravikumar et al., 1995; Shobharani and Ravikumar, 2003). The resistance in sunflower is due to difference in the infection frequency of the pathogen (Herr and Lipps, 1981; Sackston, 1981). There are limited studies on the heritability of Alternaria leaf blight resistance, which indicate resistance is quantitative (Ravikumar et al., 1995).The heritability estimates based on F2/F3 parent offspring regression indicate that, Alternaria leaf blight resistance appears to be additive and only moderate gains can be expected by selection (Kong et al., 1996). They have further reported that the presence of significant genotype x environment interactions complicates the process. There is a need to enhance the level of resistance for genetic analysis.

Partial resistance in general is a quantitative trait complexly inherited. Several genes with small effects act together to provide resistance to plants (Diaz-Lago et al., 2002; Garcia et al., 2003). Therefore, any enhanced resistance in sunflower must be an increase in this partial resistance. Recurrent selection scheme by constantly removing susceptible genotypes followed by recombination of the remaining phenotypes (Parlevliet, 1983) is the best way to increase partial resistance. Recurrent selection has been used successfully to increase resistance in oat (Avena sativa L.) to crown rust (caused by Puccinia coronata var. avenae Fraser and Ledingham) (Diaz-Lago et al., 2002); in bean (Phaseolus vulgaris L.) to soil-borne diseases (Garcia et al., 2003); in soybean [Glycine max (L.) Merr.] to phytophthora rot (caused by Phytophthora megasperma Drecks f. sp. glycinia Kuan and Erwin) (Walker and Schmitthenner, 1984); in barley (Hordeum vulgare L.) to leaf rust (caused by Puccinia hordei Otth.) (Parlevliet and Ommeren, 1988; Reinhold et al., 1993).

The selection intensity employed and the heritability estimate of the trait influence the extent of response to selection. The population size mainly determines the selection intensity and the dominance effect on trait expression, the heritability (Ottaviano and Mulcahy, 1986). In this direction, selection of a trait at male gametophytic generation/pollen offers several advantages such as larger population size, ease of handling of a larger population, and haploid state, which avoid masking effects of the dominant over recessive alleles (Ottaviano and Mulcahy, 1986). The idea of gametophytic selection was supported by the evidence of considerable genetic overlap. The studies over the past 25 yr using both protein electrophoresis and mRNA-cDNA hybridization technique have shown that a large portion of the genome of the pollen (20000–23000 genes) is transcribed and translated in both sporophyte and gametophytic stages of the life cycle (Tanksley et al., 1981; Hamilton and Mascarenhas, 1997). The screening of pollen grains and anther gene expression showed that proteins known to accumulate during seed desiccation and to be inducible by water stress are also expressed during pollen or anther dehydration, and expression of these genes was enhanced by imposed water stress (Ruiter et al., 1999). The evaluation of the correlated response to the gametophytic selections has allowed the detection of significant improvement for many sporophytic traits (Simon and Sanford, 1986; Sari-Gorla et al., 1992; Hormaza and Herrero, 1996; Clarke et al., 2004). The direct proof to alter the frequency of genes (Touraev et al., 1995), allele (Ravikumar and Patil, 2004), and CP4EPSPS transgene (Walker et al., 2006) in the progeny through gamete selection has been provided.

In sunflower we demonstrated the positive association between resistance to Alternaria leaf blight at sporophytic stage and pollen tolerance to pathotoxin (Ravikumar and Chikkodi, 1998). We have also demonstrated a successful transmission of pollen-selected leaf blight resistance to sporophytic progeny as well as to succeeding generations (Chikkodi and Ravikumar, 2000; 2003). The pollen can be easily combined with conventional crop improvement programs. The application of a pollen selection in sunflower population improvement is also reported (Shobha Rani and Ravikumar, 2006). In the present study, we further demonstrate the superiority of practicing gametophytic and sporophytic selection together in comparison to sporophytic selection alone in cyclic selection to enhance Alternaria leaf blight resistance in sunflower populations.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The schematic diagram showing synthesis and evaluation of different populations is given in Fig. 1 .


Figure 1
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  		Figure 1. Schematic diagram showing synthesis and evaluation of base and improved populations.

 
Synthesis of Base Population
We used five genetically distinct near-homozygous breeding lines in the S2 generation (180-47, 180-48, 873-05, 1229-4, and 1229-17) to synthesize the base population of sunflower. The lines 180-47 and 180-48 were derived by selfing the selected plants of germplasm collection, Acc. No. 180. Similarly the line 873–05 is a derivative from germplasm Acc. No. 873, and 1229-4 and 1229-17 were derived from germplasm Acc. No. 1229. The germplasm collection Acc. No. 180, 873, and 1229 showed moderate resistance to Alternaria leaf blight in our earlier experiments (Ravikumar et al., 1995). The selected lines were grown together, in which each genotype was planted in five rows of 6 m during winter 1999. Ten plants were randomly selected in each genotype and were treated with 100 µg g–1 of gibberellic acid (GA3) to induce male sterility (Seetharam and Kusuma-Kumari, 1974). At flowering an equal quantity of pollen from remaining male fertile plants of all the genotypes was mixed to pollinate male sterile plants. The pollination heads were covered with bags to prevent pollen contamination. Hand pollination was done using pollen mixture for 8 to 10 d until all the flowers in all the male sterile plants were pollinated. The seeds from male sterile plants were harvested and an equal quantity of seeds from all the plants was mixed to serve as the base population. About two-thirds quantity of the seeds of base population was preserved. The remaining one-third quantity of seeds (5000–6000 seeds) was used to serve as base population for subsequent recurrent selection program.

Nearly 5000 plants of base population were grown in field during the rainy season of 2000 following standard agronomic practices. Fungicides were not applied. These agronomic practices were repeated for all the field experiments during the course of this study. The moderate temperature and high relative humidity that prevailed during the rainy season were favorable for disease development. From such a large population 207 plants were selected for recording observations on Alternaria leaf blight at three stages, that is, at flowering (Stage I), 15 d after flowering (Stage II), and physiological maturity (Stage III) using standard procedure (Mayee and Datar, 1986). From these observed plants, the plants with lowest PDI were selected for recombination to develop Cycle I (C1) population.

Synthesis of C1 and C1G1 Populations
The progenies of the selected plants were grown with a row length of 9 m per progeny during winter season of 2000. Six plants in each progeny were made male sterile as described earlier. Three male sterile plants in each progeny were used for gametophytic selection cycle and the remaining for sporophytic selection as described below. Gametophytic selection (pollen selection) cycle was effected by applying pathogen culture filtrate (pathotoxin) to stigma and style 1 h before pollination as described earlier (Chikkodi and Ravikumar, 2000). It is expected that only resistant pollen grains would be able to germinate and fertilize. The remaining three male sterile plants in each progeny were used for sporophytic selection C1. Equal quantity of pollen mixed from all the male fertile plants of progenies were used to pollinate all the male sterile plants. The seeds from toxin-treated male sterile plants of all the progenies were mixed in equal quantity to produce C1G1 population (C1 population with gamete selection). The seeds mixed in equal quantity from untreated male sterile plants formed C1 of sporophytic selection.

Evaluation of C1 and C1G1 Population
Half the quantity of seeds in the C1 and C1G1 populations was used for disease resistance evaluation along with base population. The remaining half was preserved for another round of evaluation along with next cycle of populations. The base (C0), C1, and C1G1 populations were grown in a plot size of 50 by 5 m side by side during the rainy season of 2001. Along all the borders and after every 10 rows, the susceptible check Morden was planted. Three hundred plants from each population were selected for recording observations on PDI at three developmental stages (Mayee and Datar, 1986). The population means of PDI of different populations were compared using Fisher's test (Fisher and Yates, 1963). Fifteen plants with relatively least PDI were selected from each population for recombination to develop second cycle (C2) populations.

Synthesis of C2 and C2G2 Populations
The plant to row progenies of selected plants of C1 and C1G1 were grown during winter season of 2001. From each progeny five plants were made male sterile. The male sterile plants in progenies of C1 were manually pollinated without gamete selection using a pollen mixture from all the fertile plants of the progenies of C1. The seeds formed on male sterile plants of all the progenies were mixed in equal proportion to form C2 population. Similarly, the progenies of C1G1 were intermated using gamete selection as described earlier, producing seeds of C2G2 population.

Evaluation of Base and Improved Populations
A field study comprising of five populations viz., C0, C1, C2, C1G1, and C2G2 was conducted during rainy season of 2002 in trials with two replications. The susceptible cultivar Morden was grown both along the borders and after every 10 rows within each plot. Each population was grown in a plot size of 25 by 6 m per replication. To give equal opportunity for all the plants during selection, the plot in each replication was divided into six equal-sized subplots and from each subplot an equal number of plants was randomly selected for recording observations. Two hundred fifty plants per replication for each population and together 500 plants for each population were selected for recording observations on PDI at three stages and seed yield per plant (Mayee and Datar, 1986).

Statistical Analysis
In 2002, the analysis of variance (ANOVA) was performed following a randomized block design using the data on five populations and two replications. In 2001, the mean of the C0 was compared with the mean of C1 population and the mean of C1 population was compared with the mean of C1G1 population using Fisher's t test to test the effect of different selection procedures.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The variability in the base population and initial frequency of resistant alleles determine the success for initiation of selection program. The selection differential values (Table 1) showed that the selected plants for recombination had low PDI values indicating higher resistance to disease. The base population recorded a wide range of variability for PDI at all three stages. Similarly, the C1 and C1G1 also recorded high variability during 2001 and the selected plants for intermating had lower mean PDI values compared to respective population means. Even after one cycle of sporophytic (C1) or gametophytic (C1G1) recurrent selection, the populations recorded high variability for disease resistance alleles. The mean of selected plants was less than their respective population means suggesting that resistance is heritable.


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  		Table 1. Mean percent disease index (PDI) and selection differential in three cyclic populations of sunflower.

 
The comparison of mean values of C1 and C1G1 populations grown during the year 2001 revealed that the mean PDI value of C1G1 population (one cycle of improvement with pollen selection) was significantly lower than C1 population (one cycle of improvement without pollen selection) at all three stages (Table 2). The results indicate that gametophytic selection significantly increases resistance. The resistant population obtained at high selection intensity through pollen selection (C1G1 population) is higher than that obtained at low intensity (i.e., sporophytic selection alone). Therefore the gametophytic selection for resistance can thus serve to regulate the frequency of resistance alleles in the populations by removing a large amount of susceptible alleles produced in the population. Such skewing of the population or progeny toward desirable traits consequent to gametophytic selection has been observed by several workers (Simon and Sanford, 1986; Chikkodi and Ravikumar, 2000; Clarke et al., 2004). The frequency distribution of C1 and C1G1 populations also support the observations made based on mean values of populations. The C1G1 population was skewed toward low PDI values and resistance compared to C1 population (Fig. 2 ). The C1G1 population had more number of plants with low PDI values, consequently more number of resistant plants reducing the mean values.


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  		Table 2. Mean performance of base and improved populations for percent disease index (PDI) after one cycle of improvement in sunflower, 2001.

 

Figure 2
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  		Figure 2. Frequency distribution of plants for percent disease index in sunflower populations (2001) (a) at flowering, (b) at 15 d after flowering, (c) at physiological maturity. C0, base populations; C1, Cycle I population without pollen selection; C1G1 = Cycle I population with pollen selection.

 
The ANOVA suggested significant variation among selection cycle populations in 2002 (Table 3). The comparison of means of five populations grown during the rainy season of 2002 showed significant reduction in mean PDI values from C0 to C1 of sporophytic selection and from C1 to C2. The improvement in resistance was significant after one cycle of recurrent selection itself (Table 4). The results are consistent with the results observed during the year 2001. The effectiveness through one cycle of recurrent selection was observed for disease resistance in other crops also; scab (caused by Gibberella zeae Petch) resistance in wheat (Triticum aestivum L.) (Jiang et al., 1991), powdery mildew [caused by Sclerospora graminicola (Sacc.) Schrot] resistance in pearl millet [Pennisetum glaucum (L.) R. Br.] (Rattunde et al., 1993) and barley (Parlevliet and Ommeren, 1988). The best way to accumulate minor genes would be through a recurrent selection scheme over many cycles (Parlevliet, 1983). A significant improvement was made for Alternaria leaf blight resistance from cycle to cycle. The second cycle of selection also significantly reduced the mean PDI values. Overall, a significant reduction in mean PDI values was observed from C0 to C2 through C1 at all the three stages. These results corroborate with the findings of Menkir and King (1999) for rust (caused by Puccinia sorghi Schw.), Promson et al. (1990) for downy mildew [caused by Peronosclerospora maydis (Racib.) C.G. Shaw], and Graham et al. (1993) for leaf blight [caused by Exserohilum turcicum (Pass) Leonard and Suggs] in maize (Zea mays L.) who observed improved resistance with two cycles of recurrent selection. The marked response to selection for Alternaria leaf blight resistance indicates a large amount of additive genetic variability in the source population for resistance.


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  		Table 3. Mean sum of square of analysis of different selection cycle populations in 2002 field trials.

 

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  		Table 4. Mean performance of different selection cycle populations for percent disease index (PDI).

 
When gametophytic selection was combined with sporophytic selection, the magnitude of response to selection for disease resistance continued to be higher for gametophytic selection over respective sporophytic selection cycle. In general, the C1G1 and C2G2 populations had lower PDI values compared to C1 and C2 populations, respectively. Consistently the incidence of disease (PDI values) was lower in gametophytic selection compared to sporophytic selection alone after progressive cycles of selection. The improved populations C2 and C2G2 were skewed toward resistance with disproportionately more resistant plants compared to C1 and C1G1 populations, respectively. The accelerated improvement in disease resistance in gametophytic selection may be due to strong selection pressure as the pollen selection could allow the choice of wide range of rare favorable allelic combination that would hardly be detected for the selection on the sporophyte alone. Because of this, gametophytic selection is much more effective in purging undesirable alleles. The results clearly indicate that by combining sporophytic and gametophytic selection it is possible to enhance the effect of recurrent selection (Kovacs and Barnabas, 1992; Landi et al., 1989; Chikkodi and Ravikumar, 2000). The limitations of population size and masking effect of dominance of sporophytic selection could be overcome by adopting gametophytic selection with sporophytic selection together in recurrent selection programs. The frequency distribution of base and improved populations also support the observations made based on the mean. The improved populations C2 and C2G2 were skewed toward resistance with more resistant plants compared to C1 and C1G1 populations, respectively (Fig. 3 ). Similarly C1 population was skewed toward resistance compared to C0 population. We have observed the same response in our earlier studies in sunflower involving a different population (Shobha Rani and Ravikumar, 2006).


Figure 3
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  		Figure 3. Frequency distribution of plants for percent disease index (a) at flowering, (b) at 15 d after flowering, (c) at physiological maturity. C0 = base population; C1 = Cycle I population without pollen selection; C2 = Cycle II population without pollen selection; C1G1 = Cycle I population with pollen selection; C2G2 = Cycle II population with pollen selection.

 
The results clearly demonstrate that the strong selection pressure applied from cycle to cycle has improved the resistance and the frequency of desirable alleles. However, the improvement in resistance was not very high. The main disadvantage commonly mentioned with the use of polygenic resistance is the relatively long time required for its accumulation (Jenkins et al., 1954). At this rate of progress for recurrent selection for resistance per cycle, additional recurrent selection cycles could be required to attain high level of resistance. The repeated recurrent selection cycles ranging from 4 to 8 for improvement of partial resistance has been reported in other crops (Walker and Schmitthenner, 1984; Reinhold et al., 1993). Therefore, this approach may require extended cycles of selection to maximize the frequency of desirable alleles of resistance in sunflower.

The selection and intermating of resistant progenies has increased the resistance to Alternaria blight. However, overall variance was decreased with improved resistance in the populations (Avey et al., 1982). The genetic variance of the population would however be with the continued improvement of the population mean for resistance and the shift of frequency distributions in the desired directions. But the improved populations still carries potential variability for further improvement through recurrent selection. By combining sporophytic and gametophytic selection in population improvement one could enhance the effectiveness of desired trait improvement. But in the present and our previous study (Shobha Rani and Ravikumar, 2006), the magnitude of improvement per cycle was slow for Alternaria leaf blight resistance in sunflower. The main reason for slow progress could be due to low level of resistance available in the base population. To overcome this disadvantage, we propose to synthesize new base population involving interspecific lines with higher level of resistance (Sujatha, personal communication, 2006), combined with more number of cycles of selection.

The results of this study and our earlier results (Shobha Rani and Ravikumar, 2006) clearly demonstrated that resistance to Alternaria leaf blight can be improved through recurrent selection. By combining both sporophytic and gametophytic selection in recurrent selection the progress would be faster. The improved population can be used as gene source for Alternaria leaf blight resistance. The superior plants in the improved populations are selfed and they are in the S2 generation.


   ACKNOWLEDGMENTS
 
R.L. Ravikumar thanks the Indian Council of Agricultural Research for the grant and Dr. N.B. Singh, ADG (O&P), ICAR for the keen interest and help throughout the study.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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Received for publication December 30, 2005.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





This Article
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Right arrow Full Text (PDF) Free
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Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rani, T. S.
Right arrow Articles by Ravikumar, R. L.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Rani, T. S.
Right arrow Articles by Ravikumar, R. L.
Agricola
Right arrow Articles by Rani, T. S.
Right arrow Articles by Ravikumar, R. L.
Related Collections
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Right arrow Plant Disease
Right arrow Crop Genetics

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