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ALFALFA

Improved Seedling Health, Yield, and Stand Persistence with Alfalfa Resistant to Aphanomyces Root Rot

Univ. of Kentucky, Lexington, KY 40546-0091 USA

pvincell{at}ca.uky.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Methods Results Discussion REFERENCES

 
Alfalfa (Medicago sativa L.) breeders have made substantial progress in recent years to develop cultivars with resistance to Aphanomyces root rot (ARR, caused by Aphanomyces euteiches), yet data showing the agronomic benefits of this resistance under natural field conditions are limited. Two trials were seeded into naturally infested soils with alfalfa cultivars ranging from susceptible to highly resistant to ARR. The trials provided a test of the hypothesis that ARR-resistant cultivars would provide improved performance. In one test, the combination of high rainfall during the 4 wk following seeding on a soil with a slow percolation rate led to a severe outbreak of ARR, with symptoms typical of a syndrome commonly observed in commercial alfalfa fields in Kentucky. Under these conditions, the ARR-resistant cultivars provided dramatically improved seedling health, yield, and persistence. Cultivars having a resistance (R) or high resistance (HR) rating provided the most consistent performance. In the other test, a near-normal rainfall amount on a deep soil with good internal drainage led to moderate disease pressure. In that case, the ARR-resistant cultivars exhibited a slight improvement in seedling health, but yield trends were not as clear as in the former trial. Based on these findings and previous research, we conclude that the use of cultivars with R or HR ratings to ARR may solve a common stand-establishment problem in spring-seeded alfalfa in Kentucky.

Abbreviations: ANOVA, analysis of variance • ARR, Aphanomyces root rot • HR, high resistance • LR, low resistance • MR, moderate resistance • NARB, National Alfalfa Review Board • PRR, Phytophthora root rot • R, resistance • S, susceptible

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Methods Results Discussion REFERENCES

 
FOR DECADES, pathologists have known that A. euteiches Drechs. can be found in the necrotic roots of seedlings and mature plants of alfalfa (Linford, 1927; Schmitthenner, 1964). Studies in the greenhouse and growth chamber have clearly demonstrated the pathogenicity of this oomycete to seedlings of alfalfa under controlled conditions (Delwiche et al., 1987; McKeen and Traquair, 1980; Schmitthenner, 1964). Such studies have been conducted using growing media other than natural field soil, which is useful for studying pathogenicity but may not provide insight into potential crop loss in natural field soil. As a result of these and other studies (Holub and Grau, 1990a; Holub and Grau, 1990b), commercial alfalfa breeders have focused attention on developing cultivars that are resistant to ARR. These cultivars first started to become commercially available in the early 1990s and have since become common; currently, 76% of the available certified alfalfa cultivars with a fall dormancy rating of 3 to 4 have at least a moderate resistance (MR) rating to A. euteiches Race 1 (Alfalfa Council, 1999, p. 9; Hudelson and Grau, 1998).

Given this ongoing breeding effort, it is important to test the hypothesis that alfalfa cultivars with ARR resistance provide superior agronomic performance to that of susceptible cultivars under natural field conditions. Few studies have addressed this hypothesis. Holub and Grau (1990c) were the first to show significant improvement in alfalfa yield from resistance to ARR under field conditions, using rooted stem cuttings that were planted into several naturally infested soils. This experimental approach is valid for measuring the reactions of mature shoots, but it did not provide data on the possible agronomic benefit of ARR resistance in seedling plants. Wiersma et al. (1995) reported a small but significant trend of increasing improvement in seedling vigor and yield with increasing ARR resistance when all cultivars tested were pooled into five resistance classes that are recognized by the National Alfalfa Review Board (NARB) of the Association of Official Seed Certifying Agencies. They concluded their paper by recommending the use of cultivars with R or HR ratings to ARR and Phytophthora root rot (PRR, caused by Phytophthora medicaginis) for maximum agronomic performance. Studies on the possible agronomic benefits of resistance to ARR have not been reported outside of Wisconsin.

For several decades, Kentucky producers have experienced a common stand-establishment disease syndrome when spring-seeded alfalfa was followed by extended periods of wet weather (P. Vincelli and W.C. Nesmith, unpublished data, 1980–2000). When this syndrome is severe, affected seedlings exhibit severe stunting of the internodes, petioles, and leaf blades; the cotyledons are commonly yellow and may exhibit reddening, especially on the abaxial surface; leaf blades exhibit a bluish-green cast; and affected seedlings do not wilt or collapse but remain erect. Commonly, the syndrome affects most or all of the planting. It is similar to ARR (Grau, 1990), and since research began on this syndrome more than a decade ago, growing evidence has suggested that A. euteiches might have been responsible for many of the observed outbreaks (Vincelli, 1992; Vincelli et al., 1994; P. Vincelli and W.C. Nesmith, unpublished data, 1985–1995). However, we lacked rigorous proof that the syndrome could be avoided by sowing ARR-resistant alfalfa cultivars. In a previous study (Vincelli et al., 1995), there was little agronomic benefit to the use of ARR-resistant cultivars over a period of 5 yr although no outbreaks of this seedling syndrome occurred in those trials. Thus, in the mid-1990s, there was no research base for specifically recommending the use of ARR-resistant cultivars in Kentucky or the region.

In this paper, we report the results of investigations, which tested the hypothesis that agronomic performance of alfalfa would be enhanced from the use of ARR-resistant cultivars under natural field conditions.

	Methods

TOP NOTES ABSTRACT INTRODUCTION Methods Results Discussion REFERENCES

 
Two trials were conducted that included alfalfa cultivars representing a wide range of levels of susceptibility and resistance to ARR. The resistance ratings for each cultivar were based on those approved by the NARB; for one experimental entry, they were based on information provided by the breeder. Using a disc drill seeder, both trials were sown into a prepared seedbed with soils that were naturally infested with both A. euteiches and P. medicaginis Hansen et Maxwell, another root-rot fungus known to attack alfalfa. The seed of all entries were pretreated with metalaxyl fungicide [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester] and Rhizobium meliloti Dang. inoculant, either by the seed company or by the investigators. The cultivars were sown at 22.4 kg seed ha^-1, and the plots were arranged in a randomized complete block design with four replicates. Both tests were managed according to the University of Kentucky Cooperative Extension Service recommendations.

Eden Shale Farm near Owenton
The site, on a Heitt silt loam soil, had been in a mixture of alfalfa and orchardgrass (Dactylis glomerata L.) almost continuously since 1950; it was planted to no-till corn in 1995. Plots measuring 1.2 by 5.8 m were seeded on 18 Apr. 1996. Whole-plot visual assessments of the overall seedling health were made visually 6 wk after seeding using a scale of 0 to 5, where 0 = plants are extremely stunted (1.3 cm in height) and 5 = very good seedling vigor throughout the plot (7.6–10.2 cm tall). Symptomatic seedlings were carefully dug and washed, and the roots with necrosis symptoms representative of the collected seedlings were tested for oomyceteous pathogens of alfalfa using previously described methods (Vincelli et al., 1991). Soil samples (20 samples that were 1.9 cm wide and 10 cm deep) were also collected on several occasions to test for oomyceteous pathogens (Vincelli et al., 1994). Whole-plot assessments of alfalfa ground cover were made on several dates by visually estimating the percent of ground cover in 5% increments. A swath measuring 0.9 by 4.6 m was harvested from the center of each plot with a sickle-bar mower on 11 July 1996, 22–24 Aug. 1996, and 21 May 1997. Because of heavy weed infestation in some plots, the yields from the first harvest date (11 July 1996) were not reported, and the samples collected from each plot during the latter two harvests were hand-separated into alfalfa and all other plant matter; subsamples were dried at 32–38°C and weighed to allow for calculation of the amount of alfalfa dry matter in each plot.

UKREC Farm in Princeton
The site, on a Crider silt loam soil, had a history of alfalfa in rotation with cool-season grasses but had been out of alfalfa 3 yr before seeding. Plots measuring 1.5 by 4.6 m were seeded on 10 Apr. 1997. Whole-plot assessments of seedling health were made visually 2 mo after seeding. Due to less disease, seedling health was rated on a scale of 0 to 5, which was different from the one used at Eden Shale; in this case, 0 = plants are dead or nearly dead throughout plot and 5 = plants are generally 51 to 61 cm tall with robust leaflets, without chlorosis, and with a soil surface that is completely obscured by plant growth. The health of the border area adjacent to each end of each plot, which was planted to a uniform alfalfa variety throughout the trial area, was assessed for use as a covariate using the same rating scale because field observations at that time suggested an unusually high degree of spatial variability in disease severity. Symptomatic seedlings and soil samples were collected and tested for oomyceteous pathogens. A swath measuring 1.5 by 3.1 m was harvested with a sickle-bar forage plot harvester from the center of each plot. The first cutting in 1997 was delayed until full bloom to allow the alfalfa to reach maturity; otherwise, harvests were taken when the alfalfa was in the bud to early flower stage. Fresh weight samples were collected at each harvest to calculate the percent of dry matter production. The percent of ground cover with alfalfa was estimated visually in all plots on two occasions.

Data Analysis
Single degree-of-freedom contrasts were used to compare the mean performance of two populations of alfalfa cultivars: Those with a substantial amount of ARR resistance (an MR rating or higher—hereafter referred to as ARR-resistant cultivars) vs. those without reported resistance. Additionally, we were interested in the performance of individual cultivars as representatives of different classes of resistance ratings. Thus, cultivars were compared using the Waller–Duncan k-ratio t-test.

	Results

TOP NOTES ABSTRACT INTRODUCTION Methods Results Discussion REFERENCES

 
Eden Shale Test
A severe outbreak of seedling disease developed during the 4-wk period following seeding, with symptoms closely matching those of previously recorded outbreaks of stand establishment problems in commercial spring-seeded alfalfa fields in Kentucky. During that period, a total of 34 cm of precipitation fell at the test site at approximately regular intervals, and the average air temperature was 16.1°C at a weather station approximately 8 km away. Plants in the most severely affected plots were typically 2.5 cm or less in height 6 wk after seeding and remained that height for most of the summer. A. euteiches was readily detected in the necrotic roots of symptomatic plants and in soil samples. P. medicaginis was also detected in roots of some plants.

Alfalfa cultivars without reported resistance to ARR were severely diseased (Table 1) . The damage in these cultivars was generally uniform across all replicates of the trial, with the variances in seedling health scores ranging from 0.03 to 1.36 for these seven cultivars. As a group, the mean seedling health of ARR-resistant cultivars was higher (P < 0.0001) than that of cultivars without reported resistance. Among the classes of ARR resistance represented in the test, only the R or HR classes contained cultivars with seedling health scores that were all equal to that of the top performer. The yields from the first harvest (11 July 1996) are not included in Table 1 because the level of weed infestation may have differed among cultivars exhibiting different levels of seedling health, which could have confounded the detection of effects that were due to ARR resistance. Following the first cutting, the vigor of alfalfa (measured by the percent of alfalfa ground cover 12 d after cutting) was 32% higher (P < 0.0001) in ARR-resistant cultivars than in those without reported resistance. All but one (`Rushmore') of the 10 cultivars in the R and HR classes of ARR resistance provided ground cover that was equal to that of the top performer. Collectively, the ARR-resistant cultivars outyielded those without reported resistance by 0.12 Mg ha<sup>-1</sup> (P < 0.0001) on 22–24 Aug. 1996. With two exceptions (`DK127' and Rushmore), the cultivars with an R or HR rating to ARR provided yields equal to the top performer. Several cultivars with substantial resistance to PRR, but virtually no reported resistance to ARR (`Apollo', `Fortress', `Gem', and `WL 252HQ'), exhibited poorer seedling health, less ground cover, and lower yields than the top performers.

Princeton Test
A moderate outbreak of ARR developed during the 4-wk period following seeding. During that period, a total of 11.7 cm of precipitation fell at irregular intervals, and the average air temperature was 13.3°C. The cultivars without reported resistance to ARR were only moderately affected by the disease; plants in these cultivars were typically 8 to 15 cm tall 2 mo after seeding, compared with 51 to 61 cm in the healthiest plots of resistant cultivars. A. euteiches was readily detected in the rotted roots of symptomatic plants, but no other pathogens were detected in root samples. Both A. euteiches and P. medicaginis were detected in soil samples.

In contrast to the Eden Shale test, the variability in the seedling health scores among cultivars without reported resistance to ARR was exceptionally high. The variances in scores for these four cultivars ranged from 0.75 to 1.90, and visual observations suggested that the level of spatial variability was substantially higher than that observed in commercial alfalfa fields where we have diagnosed ARR. Due to this spatial variability, we evaluated the results of covariance analyses for each dependent variable, where the covariate was the mean seedling health of the border entry of alfalfa planted at both ends of each plot. Covariance analysis improved the precision of estimating the cultivar effects on seedling health in 1997 and the percent of ground cover in April 1998, as measured by substantial increases in R² values when compared with standard analysis of variance (ANOVA). For yield data, however, covariance analysis did not improve precision; therefore, we present yield data that are analyzed without the covariate.

As a group, the ARR-resistant cultivars exhibited significantly (P < 0.0001) better seedling health and greater ground cover than the cultivars without reported resistance (Table 3) . With the exception of Gem, the cultivars without reported resistance to ARR exhibited significantly lower seedling health and poorer ground cover than the top performer in the test. As a group, the ARR-resistant cultivars provided significantly higher yield in the seeding year (0.87 Mg ha^-1 more; P < 0.01) and higher total yield over 3 yr (1.72 Mg ha^-1 more, P < 0.05) than the mean yield of the cultivars without reported resistance. However, the differences in the yields of individual cultivars were not of sufficient magnitude to be statistically significant (P > 0.20) when the cultivars were treated as an ANOVA main effect.

	Discussion

TOP NOTES ABSTRACT INTRODUCTION Methods Results Discussion REFERENCES

Not surprisingly, the magnitude of the benefit of ARR resistance was higher under the conditions of substantially greater disease pressure observed at Eden Shale. A heavy rainfall following seeding and a soil with a slow percolation rate [permeability rates of 0.51–1.52 cm h^-1 at a depth of 15–43 cm for the Heitt silt loam (USDA-SCS, 1976)] probably were key factors in inducing the high disease pressure observed at Eden Shale. It is worth noting that the severity of the damage at Eden Shale was typical of what we commonly have observed in commercial problem fields while on diagnostic visits. Under severe disease pressure, substantial increases in seedling health and crop performance were observed in cultivars with R and HR ratings to ARR, and the detrimental effects of the disease on stand health and agronomic performance were evident in susceptible cultivars 1 yr after seeding. This is the first report of such dramatic effects past the seeding year following an outbreak of ARR from a natural epidemic.

The Princeton trial was conducted on a Crider silt loam soil, a deep, well-drained soil series [permeability rates of 2.0–5.1 cm h^-1 to a depth 152 cm (USDA-SCS, 1966)]. The excellent internal drainage of the soil and near-normal rainfall during the period following seeding resulted in only moderate disease pressure. The slightly cooler conditions that occurred at Princeton may have also contributed to the reduced disease pressure compared with Eden Shale (Holub and Grau, 1990b). In the Princeton trial, the ARR-resistant cultivars provided significant improvements in seedling health 2 mo after seeding and in ground cover 1 yr after seeding. Also, the ARR-resistant cultivars collectively outyielded those without reported resistance both in the seeding year and for the total yield over 3 yr. However, the yields of the individual cultivars tested were not found to differ statistically, using a multiple comparison procedure. Several factors may help explain this. First, ARR was less of a yield determinant in Princeton than in Eden Shale, given the lower disease pressure at the former site. Another contributing factor may be the unusually great spatial variability in disease severity observed in the rather small area of the Princeton trial, which in our experience is uncommon in commercial fields with outbreaks of ARR. Although the precise cause for the spatial variability in disease severity observed in the Princeton trial is unknown, it may have reduced the statistical precision, helping to mask possible yield differences among cultivars having different levels of ARR resistance. It is also worth noting that following the disease outbreak at the Princeton site, weather conditions for the remainder of the first growing season were generally very conducive to the growth and recovery of alfalfa. This is in contrast to the Eden Shale test, where rainfall was substantially below normal during much of the period following the initial disease outbreak—conditions that can be expected to exacerbate the plant damage caused by root-rotting pathogens.

We found that cultivars within a given class of resistance to ARR did not always provide equivalent performance under the high disease pressure observed at Eden Shale. For example, the two entries at Eden Shale with an MR rating to ARR differed substantially by several measures of plant health. An extreme example of this intraclass variation was seen in the performance of `Saranac AR'. The resistance level of Saranac AR to ARR has not been characterized although it is reasonable to assume that it either has an S or LR rating, as is typical for cultivars that are not deliberately screened for ARR resistance. Nevertheless, Saranac AR was only moderately affected by the disease in 1996 and was the top-yielding cultivar in 1997. Such performance was exceptional among cultivars known or suspected to be susceptible, and it suggests that Saranac AR possesses some low resistance, tolerance, or ability to recover from severe outbreaks of ARR that may be expressed under certain conditions. Our results at Eden Shale serve as a reminder that while disease resistance ratings approved through the NARB can be useful guides when selecting an alfalfa cultivar, they are not a substitute for a regional and local evaluation of the agronomic performance of individual alfalfa cultivars under conditions of natural disease pressure.

Based on the prevalence of A. euteiches in Kentucky agricultural soils (Vincelli et al., 1994), the results summarized here, and the relative lack of other significant management practices for ARR at this time, we now recommend ARR-resistant cultivars when sowing alfalfa in the spring. Because of their more consistent performance under high disease pressure, we recommend using cultivars with an R or HR rating for ARR resistance, especially on soil series with low percolation rates in the subsoil.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Methods Results Discussion REFERENCES

 
Paper no. 00-12-92 of the Kentucky Agric. Exp. Stn. journal series.

Received for publication January 6, 2000.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Methods Results Discussion REFERENCES

 

• Alfalfa Council. 1999. Fall dormancy and pest resistance ratings for alfalfa varieties. Alfalfa Council, Davis, CA.
• Delwiche P.A., Grau C.R., Holub E.B., Perry J.B. Characterization of Aphanomyces euteiches isolates recovered from alfalfa in Wisconsin. Plant Dis. 1987;71:155-161.
• Grau C.R. Aphanomyces root rot. In: Studeville D.L., Erwin D.C., eds. Compendium of alfalfa diseases, 2nd ed St. Paul, MN: APS Press, 1990:10-11.
• Holub E.B., Grau C.R. Specificity of resistance to Aphanomyces euteiches in seedling alfalfa. Plant Dis. 1990;74:164-168 a.
• Holub E.B., Grau C.R. Ability of Aphanomyces euteiches to cause disease of seedling alfalfa compared with Phytophthora megasperma f. sp. medicaginis. Phytopathology 1990;80:331-335 b.
• Holub E.B., Grau C.R. Productivity and survival of alfalfa ramets in soil naturally infested with Aphanomyces euteiches and Phytophthora megasperma. Can. J. Plant Pathol. 1990;12:83-91 c.
• Hudelson, B.D., and C.R. Grau. 1998. Development of a standardized test for evaluating alfalfa for resistance to Aphanomyces euteiches Race 2. p. 29. In Proc. N. Am. Alfalfa Improvement Conf., 36th, Bozeman, MT. 2–6 Aug. 1998.
• Linford M.B. Additional hosts of Aphanomyces euteiches, the pea root rot fungus. Phytopathology 1927;17:133-134.
• McKeen W.E., Traquair J.A. Aphanomyces sp., an alfalfa pathogen in Ontario. Can. J. Plant Pathol. 1980;2:42-44.
• Schmitthenner A.F. Prevalence and virulence of Phytophthora, Aphanomyces, Pythium, Rhizoctonia, and Fusarium isolated from diseased alfalfa seedlings. Phytopathology 1964;54:1012-1018.
• USDA-Soil Conservation Service (SCS). 1966. Soil Survey of Caldwell County, Kentucky. USDA-SCS.
• USDA-Soil Conservation Service (SCS). 1976. Soil Survey of Carroll, Gallatin, and Owen Counties, Kentucky. USDA-SCS.
• Vincelli P.C. Potential for seedling disease of alfalfa caused by Aphanomyces euteiches in a Kentucky soil. Plant Dis. 1992;76:622-626.
• Vincelli P.C., Eshenaur B.C., Bachi P.R., Nesmith W.C. Diagnosing root rots of alfalfa in Kentucky caused by Phytophthora megasperma f. sp. medicaginis and Aphanomyces euteiches. Plant Diagnostics Q. 1991;12:112-115.
• Vincelli P., Lauriault L.M., Henning J.C. Yields of alfalfa varieties selected for Aphanomyces resistance in Kentucky. Agron. J. 1995;87:748-752.[Abstract/Free Full Text]
• Vincelli P., Nesmith W.C., Eshenaur B.C. Incidence of Aphanomyces euteiches and Phytophthora medicaginis in Kentucky alfalfa fields. Plant Dis. 1994;78:645-647.
• Wiersma D.W., Grau C.R., Undersander D.J. Alfalfa cultivar performance with differing levels of resistance to Phytophthora and Aphanomyces root rots. J. Prod. Agric. 1995;8:159-264.

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