Plant Soil (2013) 372:349–359 DOI 10.1007/s11104-013-1746-5
REGULAR ARTICLE
Effect of wheel traffic and green manure treatments on forage yield and crown rot in alfalfa (Medicago sativa) Deborah A. Samac & JoAnn F. S. Lamb & Linda L. Kinkel & Lindsey Hanson
Received: 8 November 2012 / Accepted: 26 April 2013 / Published online: 10 May 2013 # Springer Science+Business Media B.V. (outside the USA) 2013
Abstract Harvesting alfalfa damages crowns and increases the opportunity for entry of pathogens. Incorporation of green manures into soil increases population density of streptomycetes with broad pathogen antagonist activity. This study aimed to measure the impact of wheel traffic on forage yield and plant health and the effect of green manures to reduce disease. Buckwheat and sorghum-sundangrass were incorporated into soil 3 weeks before seeding alfalfa. Total bacteria, streptomycete, and pathogen antagonist densities were measured prior to planting green manures and alfalfa. Wheel traffic was applied 2 days after each forage harvest. Wheel traffic reduced forage yield 12 % to 17 % depending on year and location, significantly reduced plant counts, and increased
crown rot compared to the no traffic control. Cultivar had a significant effect on yield, plant counts, and crown rot. Streptomycete density and pathogen antagonists increased when fall-sown green manure crops were incorporated in spring. Forage yields were significantly higher in plots with greater antagonist density when traffic was applied. Green manure treatments did not affect plant counts or crown rot. Mechanical wheel traffic reduces forage yield and increases disease. Green manure crops may provide benefits in alfalfa production systems by increasing pathogen antagonists. Keywords Alfalfa . Buckwheat . Crown rot . Forage yield . Green manure . Lucerne . Medicago sativa . Streptomycete . Sorghum-sudangrass . Wheel traffic
Responsible Editor: Jesus Mercado-Blanco. D. A. Samac (*) : J. F. S. Lamb USDA-ARS-Plant Science Research Unit, 1991 Upper Buford Circle, St. Paul, MN 55108, USA e-mail:
[email protected] D. A. Samac : L. L. Kinkel : L. Hanson Department of Plant Pathology, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108, USA J. F. S. Lamb Department of Agronomy and Plant Genetics, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108, USA
Introduction Crown rot of alfalfa (Medicago sativa L.) is a serious problem in all alfalfa-producing areas. The disease reduces plant density as well as productivity and persistence. It is characterized by chronic loss of crown buds and decay of crown and root tissues (Leath 1990). The disease often results in asymmetric plant growth due to death of affected portions of the crown and development of secondary crown branches. Symptoms include longitudinal necrotic lesions of crown branches and dark brown to black wedgeshaped necrotic tissue of the crown and/or root cortex.
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In advanced stages of disease, the central core is rotted and hollow. Plants with severe symptoms are stunted, wilt, and eventually die. The organisms causing crown and root rot of alfalfa are a complex of soil-borne fungi that can differ substantially by geographic location. The most commonly isolated pathogenic species are Fusarium oxysporum, F. roseum, F. solani, Rhizoctonia solani, and Phoma medicaginis (Richard et al. 1980; Salter et al. 1994; Turner and Van Alfen 1983; Uddin and Knous 1991; Wilcoxson et al. 1977). Many other fungi have been associated with crown rot but not all were found to cause crown rot symptoms. In addition, Pythium species, several bacterial species, and the stem nematode (Ditylenchus dipsaci) can be involved. The pathogens gain entry into the alfalfa plants via colonization of roots early in the life of the plant and through wounds made by machinery, grazing animals, winter injury, and insect feeding (Richard et al. 1980; Turner and Van Alfen 1983; Wilcoxson et al. 1977). Damage of roots by larvae of the clover root curculio (Sitona hispidulus) is an important component of crown rot in many areas of the U.S. (Kalb et al. 1994). Pathogens may also enter through freshly cut stems with rot advancing through the crown into the taproot (Gossen 1994; Richard et al. 1980; Wilcoxson et al. 1977). Selection and breeding for crown rot resistance has been slow (Ariss et al. 2007; Miller-Garvin and Viands 1994; Richard et al. 1980; Salter et al. 1994; Wilcoxson et al. 1977) and no cultivars with resistance to crown rot are currently available. Due to the diversity of pathogenic organisms associated with crown rot, it may be difficult to develop cultivars with resistance that is effective across locations. Also, fungicides with the required persistent root and crown activity are not available. Alternative strategies for reducing damage from crown rot are needed. Incorporation of green plant material, known as green manure treatments, were previously suggested as a practical means by which damage from multiple pathogens can be reduced (Wiggins and Kinkel 2005a). Incorporation of sorghum-sudangrass as a green manure increased alfalfa stand counts and reduced severity of Phytophthora root rot in pot studies. There was evidence suggesting that spring incorporation of green plant material was more effective than incorporation of dead plant material
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from late summer seeding. Streptomycetes with broad pathogen antagonistic activity have also been shown to reduce damage to alfalfa from Phytophthora medicaginis (Xiao et al. 2002) and the root-lesion nematode (Pratylenchus penetrans) (Samac and Kinkel 2001), and protected potato from Verticillium wilt (Davis et al. 1996; Wiggins and Kinkel 2005b), potato scab (Ryan and Kinkel 1997), and Rhizoctonia stem canker (Scholte and Lootsma 1998). Streptomycete communities in soil have higher densities and greater antagonistic activity against Fusarium oxysporum and Rhizoctonia solani following green manure treatments (Chamberlain and Crawford 1999; Wiggins and Kinkel 2005a, b). Green manure treatments can also reduce weeds (Blackshaw et al. 2001) and improve soil structure and fertility (Abdallahi and N’Dayegamiye 2000) without removing land from crop production. Researchers evaluating crown rot in alfalfa speculated that crown morphology and depth of the crown below the soil surface could be important factors in crown rot resistance and that mechanical damage from heavy equipment wheel traffic would likely increase crown rot (Wilcoxson et al. 1977). Alfalfa fields are usually harvested every 28–35 days during the growing season, for typically three to four harvests per year in the Midwestern US. Fields are subjected to machinery wheel traffic at least three times at each harvest from the tractor, chopper or baler, and wagon, although not every plant may be subjected to wheel traffic. Standards for selecting and characterizing alfalfa cultivars for wheel traffic tolerance have not been established, but cultivars have been developed with standardized protocols for increased tolerance to grazing by cattle under continuous stocking, which causes mechanical damage to crowns. Grazing tolerance has been associated with an increased number of fall crown buds and maintenance of nonstructural carbohydrates under frequent defoliation (Brummer and Bouton 1991, 1992). Interestingly, resistance to Rhizoctonia root rot was greater in three out of four populations selected for grazing tolerance compared to the original populations (Smith and Bouton 1993), suggesting a relationship between plant morphology and/or physiology and disease resistance. The objectives of this research were to measure the effect of equipment wheel traffic on forage yield and plant health in four alfalfa cultivars, and determine the effect of two green manure crops, buckwheat
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(Fagopyrum esculentum L.) and sorghum sudangrass (Sorghum bicolor (L.) Moench × Sorghum sudanense (Piper) Stapf.), on streptomycete density, pathogen inhibitor density, plant health, and forage yield in two field sites over 2 years.
Materials and methods Plant materials Two species were chosen as green manure treatments to investigate the ability to alter soil microbial characteristics under field conditions, common buckwheat (Fagopyrum esculentum L.) and ‘Excel’ sorghumsudangrass (Sorghum bicolor (L.) Moench × Sorghum sudanense (Piper) Stapf.). A fallow treatment was included as a control. Four alfalfa entries, ‘Integrity’ (ABI), ‘Magnum V’ (Dairyland Seed Co.), and ‘SummerGold’ (Cal/West Seeds), and ‘Saranac’, a public variety released in 1964, were chosen for this study. The first three cultivars are resistant to bacterial wilt (Clavibacter michiganensis subsp. insidiosus), Fusarium wilt, (Fusarium oxysporum f. sp. medicaginis), Phytophthora root rot (Phytophthora medicaginis), Aphanomyces root rot (Aphanomyces euteiches), and anthracnose (race 1) (Colletotrichum trifolii), while Saranac is susceptible to all of these diseases except bacterial wilt. ‘Integrity’ was selected for cattle grazing tolerance under continuous stocking and is considered grazing tolerant. Experimental design The experimental design was a split plot arrangement of the treatments with four replications. Traffic or no traffic were the main plots, subplots were green manure treatments (buckwheat, sorghum-sudangrass, or fallow), and sub-subplots were the four alfalfa cultivars. The green manure plots were established at the University of Minnesota Sand Plain Experimental Farm, Becker, MN in August 2003 and at the Agricultural Experiment Station, St. Paul, MN in May 2004. The soil at the Becker site was a Hubbard loamy sand, sandy mixed Udorthentic Haploborall, with a pH 6.5 and 2.1 % organic matter. The St. Paul soil was aWaukegan fine-silty loam over sandy or sandy-skeletal, mixed, superactive, mesic Typic Hapludolls, pH 6.3, and 5.3 % organic matter. Soil
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pH was determined using a soil and water slurry (1:1; v/v) and organic matter was determined by loss on ignition by the Soil Testing Laboratory at the University of Minnesota. Buckwheat was seeded at a rate of 111 kg/ha and sorghum-sudangrass at a rate of 76 kg/ha in plots 7 m×4.6 m by broadcast seeding and raked into the soil. In both test locations the green manures were grown for 7 weeks before either killing frost (Becker) or incorporation of green material into soil (St. Paul) with a similar amount of biomass produced. The green manure treatments were incorporated (rotovated) into the soil approximately 3 weeks prior to establishing alfalfa in early April 2004 at Becker and late July 2004 at St. Paul. At Becker soil was amended with 28 kg/ha Sul-Po-Mag and 4.5 kg/ha boron to meet the nutrient levels recommended for alfalfa production in Minnesota (Rehm et al. 2000). No soil amendments were made at St. Paul. The four alfalfa cultivars were seeded using a Plotman plot planter (Wintersteiger, Inc., Salt lake City, UT) at a rate of 16.8 kg/ha in 0.9 m×5.2 m plots, with 5 rows drilled 12 cm apart on 4 May 2004 at Becker and on 16 August 2004 at St. Paul. The St. Paul site was rain fed, while the Becker site was irrigated to meet plant moisture needs using the checkbook method (Wright and Bergsrud 1991). Weeds were controlled by hand weeding. All plots were sprayed periodically with Pounce 25 WP (active ingredient permethrin: (3-Phenoxyphenyl)methyl (+/−) cis-trans-3-(2,2-dichloroethenyl)-2,2-dimethyl cyclopropanecarboxylate) to control potato leafhopper (Empoasca fabae) using a Model EX bicycle sprayer (R & D Sprayers, Opelousas, LA) maneuvered between rows to prevent mechanical damage of plants. Traffic treatments and forage yield assessment All plots were harvested at the bud stage when approximately 30 % to 40 % of the stems in each plot had flower buds. Plots were harvested on 15 June, 13 July, and 10 August 2005 and 1 June, 27 June, 25 July, and 21 August 2006 at Becker and on 3 June, 1 July, 28 July and 25 August 2005 and 31 May, 28 June, 26 July and 29 August 2006 at St. Paul. Hand grab samples (approximately 700 g) were taken, dried at 55 °C in forced air ovens and reweighed to calculate dry matter yields for each plot. Two days after each forage harvest, the traffic treatment was applied by
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driving once over all plants in the traffic treatment plots with a John Deere 5510 (2.6 Mg) at 6.4 km/h to simulate wheel traffic that occurs in alfalfa production fields after mowing the crop. At Becker, plots were not irrigated for 2 days after the traffic treatment. Microbial community analyses Soil cores were collected from each plot immediately prior to seeding green manure crops and prior to seeding alfalfa after incorporation of green manures. Three 2-cm×15-cm cores were removed from random locations within each green manure treatment or fallow treatment plot and combined. There were two replicate soil samples per plot. Soils were held at 4 °C until processed. The effect of green manure treatments on soil microbial density and antagonistic activity was assessed using a modified Herr’s assay (Herr 1959). Soil collected from Becker pre- and post-green manure amended plots was tested to determine total bacterial density, Streptomyces density, and Streptomyces colonies that had antagonistic activities against Fusarium oxysporum and Phoma medicaginis (inhibitory densities). These fungi, along with F. solani, were the most commonly isolated fungi from alfalfa crowns with symptoms of crown rot at the Becker and St. Paul locations. Streptomyces density and Streptomyces with antagonistic activities against other streptomycetes (inhibitor density) were measured in soil collected from the St. Paul field plots. Five grams of each soil sample was dried under a triple layer of sterile cheesecloth on the bench top overnight. Each sample was added to 50 ml sterile deionized water and shaken at 175 rpm for 1 h at 4 °C. Serial dilutions (0.1 ml) were plated onto water agar (WA) for determination of total bacterial and streptomycete densities. Plates were immediately overlaid with molten starch casein agar (SCA; Becker and Kinkel 1999) and were incubated at 28 °C. Total bacteria and streptomycete densities were recorded after 3 d. After densities for Becker soil samples were recorded, plates were then pathogenoverlaid to assess the capacity of streptomycetes to inhibit F. oxysporum and P. medicaginis. Colonized plates were overlaid with 5 ml molten potato dextrose water agar (PDWA; 2.4 g/l potato dextrose broth, 10 g/l bacto agar) containing a suspension of pathogen mycelium. The F. oxysporum f. sp. medicaginis strain 31F3 suspension was prepared by homogenizing a 7-
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d-old culture grown on a 100 mm×10 mm potato dextrose agar (PDA) plate in 100 ml sterile water using a blender, then adding 4 ml of the homogenate to 100 ml molten PDWA. The P. medicaginis strain 866 suspension was prepared in the same manner from a 21-d-old culture grown on PDA, then adding 8 ml of the homogenate to 100 ml PDWA. There were three replicate plates at each of two dilutions for each soil sample-pathogen combination. Plates were incubated at 28 °C for 3 d and the number of colonies exhibiting zones of pathogen growth inhibition were counted (inhibitor density). Antagonistic activity of streptomycete communities from the plots in St. Paul, MN was assessed similarly, except that after 3 d incubation, plates were overlaid with 10 ml of molten SCA and then spread with a suspension of spores from Streptomyces strain 2.12 or strain 4.20 (Davelos et al. 2004). Plates were incubated at 28 °C for 3 d and the number of colonies exhibiting zones of pathogen growth inhibition were counted (inhibitor density). The in vitro inhibition of these isolates is highly predictive of pathogen inhibition. All soil sampleStreptomyces overlay combinations were replicated twice at each of two dilutions. Crown rot assessment After the final forage harvest, plants were undercut and lifted from the plots. The plants in the center 3 m of each plot were removed for plant count and crown rot assessments. The amount of disease in 30 plants from each plot was estimated based on a 0 to 5 scale as illustrated by Turner and Van Alfen (1983). Briefly, a score of 0 = no sign of necrosis; 1 = necrosis encompassing less than 2 % of the crown; 2 = 2– 20 % of the crown with necrosis; 3 = 21–50 % of the crown and/or internal tap root necrotic; 4 = 51– 80 % of the crown and/or internal tap root necrotic, with partial decay of crown branches; 5 = greater than 80 % of the crown and/or internal tap root necrotic, decay of most or all crown branches, or a dead plant. Fungi were isolated from margins of symptomatic tissues by plating crown and root tissue pieces on acidified PDA. Identification of fungi in pure cultures was made from morphological characteristics and sequence of the rDNA internal transcribed sequence (ITS) as described previously (Castell-Miller et al. 2008).
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Statistical analysis Yield, plant counts, and crown root rot data were analyzed as a split–split–split plot arrangement of the treatments with four replicates using PROC GLM (SAS Institute, Cary, NC). Whole plots were the traffic treatments (with or without traffic), subplots were green manure treatments (buckwheat, sorghumsudangrass, or fallow), and sub-subplots were the four alfalfa cultivars. Locations were considered random and years, traffic treatments, green manure treatments, and alfalfa entries were considered fixed. Significance was declared at P
