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CROP BREEDING, GENETICS & CYTOLOGY

Associations among Forage Quality Traits, Vigor, and Disease Resistance in Alfalfa

^a EMBRAPA-Cerrados, Rodovia BR 020 Km 18, Planaltina-DF, Brazil 73301-970
^b Dep. of Plant Breeding, 523 Bradfield Hall, Cornell Univ., Ithaca, NY 14853-1902 USA
^c Dep. of Animal Science, 325 Morrison Hall, Cornell Univ., Ithaca, NY 14853-4801 USA

drv3{at}cornell.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Major objectives for alfalfa (Medicago sativa L.) breeding include improving forage quality and levels of disease resistance. Little is known about the associations between forage quality characteristics, including pectin (the main component of neutral detergent-soluble fiber [NDSF]), and disease resistance and vigor in alfalfa. Our objectives were to determine, in two alfalfa populations, the (i) correlations among NDSF and other forage quality traits; (ii) correlations of forage quality traits with plant persistence, vigor, and levels of resistance to diseases; and (iii) heritabilities and expected gains from selection for the aforementioned traits. Simple, phenotypic, and additive genetic correlation coefficients were estimated from half-sib (HS) progeny tests of two alfalfa populations (NY9505 and NY9515). In both populations, NDSF was negatively correlated with acid detergent fiber (ADF), lignin, and neutral detergent fiber (NDF), and positively correlated with true in vitro dry matter digestibility (IVDMD). Vigor was positively correlated with ADF, lignin, and NDF, and negatively correlated with IVDMD and crude protein (CP) only in NY9515. Of 144 correlation coefficients computed between forage quality traits and resistances to five major alfalfa diseases, only six were significant, and they were low in magnitude (-0.22 ± 0.11 to 0.30 ± 0.14). In an alfalfa improvement program, selection for forage quality is not expected to indirectly affect levels of disease resistance, but it may reduce vigor.

Abbreviations: ADF, acid detergent fiber • AN, anthracnose • ASI, average severity index • BW, bacterial wilt • CP, crude protein • FW, fusarium wilt • HS, half-sib • IVDMD, true in vitro dry matter digestibility • NDF, neutral detergent fiber • NDSF, neutral detergent-soluble fiber • NIRS, near infrared reflectance spectroscopy • PRR, phytophthora root rot • VW, verticillium wilt

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
MAJOR OBJECTIVES of alfalfa breeding in the USA include improving forage quality and multiple disease resistance. Improvement in forage quality has been accomplished mostly by selecting for higher crude protein (Phillips et al., 1982; Teuber and Phillips, 1988) or lower fiber concentrations (Coors et al., 1986; Sumberg et al., 1983). Breeding for NDSF has been considered an alternative strategy for developing alfalfa with superior quality characteristics (Hatfield, 1996; Van Soest, 1995; Viands, 1995a, 1995b). Pectin, the predominant component of NDSF in alfalfa, is rapidly and completely fermented by rumen microorganisms, its fermentation does not decrease the ruminal pH, and it is not subject to metabolic turnover in the plant (Hatfield and Weimer, 1995; Van Soest et al., 1991). Recently, significant genetic variation in NDSF was reported for alfalfa, suggesting that this trait could be exploited in a breeding program (Fonseca et al., 1999).

When breeding alfalfa with higher forage quality, other plant characteristics inadvertently can be altered. Correlations between forage quality, and morphological and agronomic traits have been reported. Positive correlations were reported for IVDMD with leaf/stem ratio and number of vascular bundles (Shenk and Elliot, 1970, 1971). Negative correlations were reported for lignin with leaf/stem ratio and stem height (Kephart et al., 1989, 1990). Johnson et al. (1994) found positive correlations for CP with leaf/stem ratio and lodging, and negative correlations for CP and regrowth height. Moderate or no associations have been reported for yield with NDF, ADF, lignin, IVDMD, and CP (Coors et al., 1986; Gil et al. 1967; Hill and Barnes, 1977; Hill, 1981; Kephart et al., 1989; Shenk and Elliot, 1970; Sumberg et al., 1983). Studies on NDSF and associations with other quality constituents and plant characteristics have not been conducted.

Very few studies have addressed the association between levels of disease resistance and forage quality traits in alfalfa. Some researchers have suggested, without supporting data, that plants selected for higher lignin and NDF are more vigorous and persistent in the field than plants selected for lower lignin and NDF (Buxton and Casler, 1993; Viands, 1995a, 1995b). Thus, selection for higher CP and lower fiber concentrations may adversely affect the level of disease resistance, plant vigor, and persistence.

Our research objectives were to determine, in two alfalfa populations, the (i) correlation coefficients among NDSF and other forage quality traits; (ii) correlation coefficients of forage quality traits with persistence, vigor, and levels of resistance to diseases; and (iii) heritabilities and expected gains from selection for forage quality, disease resistance, vigor, and persistence.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Plant Material and Experimental Design
Two genetically broad-based alfalfa populations, NY9505 and NY9515, from the forage breeding program at Cornell University were evaluated for forage quality, disease resistance, vigor, and persistence. Both populations were mixtures of germplasm with fall dormancy rating 4, typical of cultivars used in the northeastern USA. These two populations were bred primarily for multiple disease resistance. Population NY9505 also was selected for its higher hemicellulose concentration.

A random sample consisting of 75 HS families from each population was used to evaluate forage quality and resistance to 5 alfalfa diseases: bacterial wilt (BW), caused by Clavibacter michiganense subsp. insidiosum Mc.Cull.; fusarium wilt (FW), caused by Fusarium oxysporum Sch. ex Fr. f. sp. medicaginis J.L. Weimer; verticillium wilt (VW), caused by Verticillium albo-atrum Reinke & Berthier; phytophthora root rot (PRR), caused by Phytophthora megasperma Drechs. f. sp. medicaginis Kuan & Erwin (Pmm); and anthracnose (AN), caused by Colletotrichum trifolii Bain. & Essary (Fox et al., 1991; Viands and Pennypacker, 1996). For each disease evaluation, check cultivars (Fox et al., 1991) resistant and susceptible to the disease being evaluated were used to monitor whether disease reactions of the HS progenies were typical. Similarly, `WL 322HQ' and `Vernal' were used as checks in the forage quality evaluations. All experiments were designed as three-replicate randomized complete blocks with 20 to 35 plants per half-sib row in each replicate. Only the HS progenies were included in the computation of correlation coefficients and heritability estimates.

General Procedures
For each evaluation, seeds of both populations and of check cultivars were planted into sterilized cedar flats (35 by 51 by 8 cm for AN and 35 by 51 by 13 cm for all others) filled with sterilized medium-grade vermiculite no. 3. At planting, Rhizobium melliloti Dang., soluble 10-10-20 N-P-K fertilizer, and soluble trace elements were applied on all flats. Flats were fertilized weekly with liquid 10-10-20 N-P-K fertilizer. For BW, FW, and forage quality evaluations, 7- to 8-wk-old seedlings were transplanted from the greenhouse to the field with a mechanical transplanter with spacings of 20 cm within the row and 90 cm between rows. The FW and BW evaluations were planted on a Dalton Channery silt loam (coarse-silty, mixed, mesic Aeric Fragiaquept) soil with 0 to 3% slope, on 1 and 8 June 1995, respectively. The quality evaluations were planted on a Williamson silt loam (coarse-silty, mixed, mesic Typic Fragiochrept) soil with 2 to 6% slope on 21 June 1995. Fields were limed and fertilized as recommended for New York, and insects and weeds were controlled when necessary. Four weeks after transplanting, the number of live plants was counted as a measure of the initial stand. In fall 1995, the field was overseeded with common timothy (Phleum pratense L.) to simulate a forage mixture typical of the northeastern USA. The grass was separated from the alfalfa when the latter was sampled for forage quality analyses.

Forage Quality Experiments
Forage from each HS family row was sampled at 6-wk intervals, three times in 1996 beginning in the first week of June (Harvests 1, 2, and 3), and twice in 1997 beginning in the second week of July (Harvests 2 and 3). Plants were at early bud stage at Harvest 1 in 1996, late bud stage at Harvests 2 and 3 in 1997, and early flower stage at Harvests 2 and 3 in 1996. A 400-g aboveground fresh weight sample, composed of randomly selected stems from each HS row, was harvested with manual clippers at a stubble height of 5 cm. All samples were dried for 72 h in a forced air oven at 55°C, sequentially ground through a 2-mm screen in a Wiley mill (A.H. Thomas Co., Philadelphia, PA), through a 1-mm screen in a Udy cyclone mill (Udy Corp., Boulder, CO), and stored in 0.2-L plastic bags for laboratory analysis (Whirl-Pak by NASCO, Fort Atkinson, WI).¹

The NDF, ADF, lignin, IVDMD, CP, and NDSF concentrations were determined by near infrared reflectance spectroscopy (NIRS). Spectra for 2430 samples, 486 from each harvest, were collected on a Pacific Scientific 6350 scanning monochromator (Pacific Scientific, Silver Springs, MD). In each harvest, 56 to 60 samples (12–13% of the total) were randomly selected for the NIRS calibration set. Sequential NDF, ADF, and lignin analyses were performed according to Van Soest et al. (1991) as modified by Komarek et al. (1994). Sodium sulfite and heat-resistant -amylase (ANKOM Tech. Corp., Fairport, NY) were used during the NDF refluxing. True IVDMD was determined by incubating the samples for 48 h at 39.5°C in an ANKOM Daisy II #IV100 in vitro system according to the basic procedure described by Goering and Van Soest (1970). Nitrogen concentration was determined by Kjeldahl in a Tecator Kjeltec Auto 1030 digestion–distillation system (Tecator AB, Höganäs, Sweden). Crude protein was calculated as N times 6.25. Neutral detergent-soluble fiber analyses were performed according to Hall et al. (1997) with minor modifications described by Fonseca et al. (1999). Using the Infrasoft International software program, equations were developed for each harvest or forage quality trait (CAL, version 1.5, ISI, Port Matilda, PA)

In the second production year, just prior to Harvest 2 in 1997, HS family plots were visually evaluated for vigor and field persistence (percentage of survivors). The rating scale for vigor ranged from 1 to 5, where 1 = least and 5 = most amount of vegetative growth.

Disease Evaluations
For all disease evaluations, the inoculum preparation, and the characterization of reaction through ASI and percentage of resistant plants were performed according to Fox et al. (1991). Three isolates of F. oxysporum f. sp. medicaginis for root-soak inoculation were equally mixed and adjusted to 1.6 x 10⁶ spores mL^-1 in 10 L of tap water. After digging, the seedling roots were washed and soaked for 30 min in the inoculum suspension. After 3 mo of field growth, individual plants were dug, and their taproots were transversely sectioned to evaluate disease reaction. The rating scale ranged from 0 to 5, where 0 = no internal taproot discoloration (resistant reaction) and 5 = dead plant. The difference between initial and final stands represented plants that died due to disease infection. Resistant and susceptible check cultivars were Agate and MNGN-1, respectively.

Bacterial wilt inoculum consisted of 500 g of ground and frozen diseased alfalfa roots, which were thawed and suspended in 10 L of tap water. Subsequent experimental techniques were similar to those described for FW. Resistant and susceptible check cultivars were Vernal and Narragansett, respectively.

Verticillium wilt inoculum consisted of proportional mixtures of conidial suspensions from six New York isolates of V. albo-atrum that were adjusted to 8 x 10⁶ spores mL^-1. Six-week-old seedlings were inoculated through the stubble (Grau et al., 1991). Plants were grown in a growth chamber at 20 ± 2°C, in a light/dark regime of 16:8 h, and at a light intensity of 345 µmol m^-2 s^-1 provided by both inflorescent and incandescent bulbs. Six weeks after inoculation, the plants were individually evaluated for severity of foliar symptoms. The rating scale ranged from 1 to 5, where 1 = no chlorosis of leaves (resistant reaction) and 5 = dead plant. Resistant and susceptible check cultivars were Oneida VR and Saranac, respectively.

Inoculum of seven New York isolates of P. megasperma was prepared by culturing the fungus for 2 wk in V-8 (Campbell's Soup Co., Camden, NJ) broth under continuous light. Fungal mats were separated from broth by filtering through cheesecloth. The same number of fungal mats per isolate was blended for 20 s in a proportion of three and one-half mats per 210 mL of water. Inoculation was done by pouring 210 mL of the inoculum per flat at the stem base of 2-wk-old seedlings. Flats were immersed to 5 cm below the substrate surface in water baths in a greenhouse for 9 d. Heating mats were used to keep the water temperature at 27°C. Before inoculation, the numbers of live plants were counted to determine the initial stand of each experimental unit. Three weeks after inoculation, the plants were dug and evaluated for severity of root symptoms. The rating scale ranged from 1 to 6, where 1 = no root lesions, many small rootlets on taproot (resistant reaction) and 6 = dead plant. Resistant and susceptible check cultivars were Agate and Saranac, respectively.

Anthracnose inoculum consisted of proportional mixtures of spore suspensions from five New York isolates of C. trifolii (Race 1) that were adjusted to 10⁶ spores mL^-1. Before inoculation, the number of live plants was counted to determine the initial stand. Fifteen-day-old seedlings were inoculated by spraying to runoff 50 mL of spore suspension per flat. Inoculated flats were placed in a dark mist chamber for 3 d at 23°C before returning to the greenhouse. Two weeks after inoculation, the plants were evaluated for severity of symptoms. The rating scale ranged from 1 to 5, where 1 = no stem lesions (resistant reaction) and 5 = dead plants. Resistant and susceptible check cultivars were Saranac AR and Saranac, respectively.

Heritability, Correlations, and Expected Gains
Analysis of variance for each disease in each population was performed using the mean disease scores and the percentage of resistant plants. Analysis of variance for each quality trait was performed with data averaged across all five harvests. Half-sib families were considered random, and harvests represented fixed effects. Components of variance were estimated by equating mean squares with their expected values (Schultz, 1955) and solving for the appropriate component. Narrow-sense heritability on a HS progeny-means basis, and associated standard errors, were computed for all traits and for each population according to Hallauer and Miranda (1981) and Nguyen and Sleper (1983). For all possible combinations of traits, simple (r_S), phenotypic (r_P), and additive genetic (r_A) correlation coefficients were computed according to Hallauer and Miranda (1981), Johnson et al. (1955), and Snedecor and Cochran (1980). Generally, values from individual plots were used for computing simple correlation coefficients, family means for phenotypic correlation coefficients, and estimated half-sib variance and covariance components for additive genetic correlation coefficients. Their standard errors were computed according to Mode and Robinson (1959) and Scheinberg (1966). Correlation coefficients were considered significant when they were more than twice their standard errors. Expected gains from direct and indirect selection were computed according to Hallauer and Miranda (1981) and Nguyen and Sleper (1983). All statistical analyses were performed using JMP—Statistics Made Visual software, version 3.2.1 (SAS Institute Inc., Cary, NC).

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
For all disease and forage quality experiments, the check cultivars were significantly different and ranked (data not presented) as typical of previous evaluations. Significant variation among HS families within each population was observed for all diseases, quality traits, and vigor, except for the PRR evaluation in population NY9515 (Tables 1 and 2) . Variation for persistence in the field in the second production year was not significant for either population. Correlations using ASI to characterize disease resistance were opposite in sign but similar in magnitude to those using percentage of resistant plants. Thus, only correlations using ASI are discussed here.

In both populations, the additive genetic correlation coefficients for NDSF and CP were not significant. Plant proteins, except extensins (present in small concentrations), are not covalently linked to the cell wall matrix, i.e., not lignified (Van Soest, 1994). Although closely associated with the cell wall, most pectins also are not covalently linked to the cell-wall matrix (Hatfield, 1996; Van Soest, 1986). Thus, NDSF and CP may not be correlated because they are independent and separate from the lignified cell-wall matrix. Alternate or simultaneous cycles of selection for NDSF and CP may be an effective breeding strategy when developing alfalfa cultivars for higher forage quality.

Vigor was positively correlated with ADF, lignin, and NDF and negatively correlated with CP and IVDMD in NY9515. These results confirm the preliminary observations reported by Viands (1995a, 1995b), whereby alfalfa populations bred for higher NDF concentration were much more vigorous after two production years than those bred for lower NDF concentration. However, in NY9505, the correlations between vigor and forage quality traits were not significant, suggesting that alfalfa can be bred for improvements in both forage quality and yield. Variability among HS families was not detected for persistence; thus correlation coefficients were not computed (Tables 1 and 2). The possible association of persistence with both lignin and NDF concentrations suggested by Buxton and Casler (1993) and Viands (1995a, 1995b) remains to be confirmed.

Association among Disease Resistance, Forage Quality, and Vigor
Most correlation coefficients involving disease resistances, and forage quality or vigor, were not significant. The exceptions were the negative simple and phenotypic correlation coefficients for CP vs. FW in population NY9505 (-0.16 ± 0.07 and –0.22 ± 0.11, respectively), positive simple and phenotypic correlation coefficients NDSF vs. FW in NY9505 (0.20 ± 0.07 and 0.25 ± 0.11, respectively), and positive phenotypic and additive genetic correlation coefficients for NDF vs. AN in NY9515 (0.23 ± 0.11 and 0.30 ± 0.14, respectively). However, since these estimates were low in magnitude, selection for higher CP and NDSF, and lower NDF concentrations is not likely to affect the resistance levels of FW and AN. A small negative simple correlation between FW and vigor (-0.17 ± 0.07) was observed in NY9505, indicating that selection for FW resistance might increase vigor even in the absence of the disease. In contrast, positive phenotypic and genetic correlations were found between AN and vigor in NY9515 (0.22 ± 0.11 and 0.31 ± 0.15, respectively), indicating that selection for AN resistance might decrease vigor in some populations. These estimates were not sufficiently high to expect a significant effect on vigor when selecting for FW or AN resistance.

Although lignin and associated phenolics are involved in providing plant resistance to diseases (Buxton and Casler, 1993), we did not find an association between lignin and levels of disease resistance. This association may be more apparent at later stages of development, when more lignified tissue is present in the plants and the forage is of lower quality.

Heritability and Expected Gains
Narrow-sense heritability estimates ranged from moderate to high (0.35 ± 0.18 to 0.95 ± 0.16) for the traits measured in the two populations (Tables 1 and 2). Of the 18 heritability estimates, 17 were significant for NY9505 and 15 were significant for NY9515. In general, heritability estimates for all traits were consistently higher in NY9515. The difference between these populations was attributed to the larger additive genetic component estimates for NY9515. Considering the genetic correlation coefficients and the heritability estimates, indirect selection (data not shown) was not more effective than direct selection. Expected gains from direct selection ranged from moderate to high for most of the disease and quality traits (Tables 1 and 2).

The heritability estimates for NDSF concentration were similar to those of the other forage quality traits. Thus we anticipate selection for NDSF to be as effective as that experienced by alfalfa breeders for the other quality traits (Coors et al., 1986; Hill, 1981; Shenk and Elliot, 1970; Teuber and Phillips, 1988). Because of the moderately high and negative correlation coefficients for NDSF with ADF and NDF concentrations, we suggest selecting plants first on the basis of either NDF or ADF, followed by selection for NDSF concentration, which has a more complex assay.Falconer 1981

	ACKNOWLEDGMENTS

 
Special thanks to Drs. Jill Miller-Garvin and James B. Robertson for discussions and assistance. We thank Drs. Peter Schofield and Mike Van Amburgh for the use of their laboratory equipment and assistance. We also thank Edward Thomas for his assistance on greenhouse and laboratory experimental procedures.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Part of a dissertation submitted by the senior author in partial fulfillment of the requirements for a Ph.D. degree at Cornell University. Joint contribution of EMBRAPA-Cerrados and Dep. of Plant Breeding, Cornell Univ.

¹ Use of specific products does not constitute an endorsement or recommendation by Cornell University and does not imply approval to the exclusion of other suitable ones.

Received for publication July 1, 1998.

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