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a INIA La Estanzuela, CC 39173, 70000 Colonia, Uruguay
b Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, St. Paul, MN 55108 USA
ehlke001{at}tc.umn.edu
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Abbreviations: DSI, disease severity index transforming the VD percentage rating into a five-class scale • DS x VD = (DSI + 1)/VD = a combined index • INIA, Instituto Nacional de Investigacion Agropecuaria • IR, percentage of internal rot in the root section visually rated as 0, 1, 5, 10, 20, 30, 40, 50% or greater • NLR, number of lateral roots • PDA, potato-dextrose agar • PRP, the percentage of resistant plants • SLR, size of lateral roots • VA, aerial vigor rating • VD, the total vertical discoloration from the inoculation site toward the crown (0.0–6.0 cm) • VTR, taproot vigor rating
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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The major constraint to the use of birdsfoot trefoil is its lack of persistence. In Uruguay, significant plant losses are observed in seed crops or pastures in 2-yr-old and older stands, especially following periods of environmental stress or under continuous grazing systems (Altier, 1997). The limited persistence of birdsfoot trefoil is generally attributed to the interaction of several abiotic and biotic factors, including climatic and edaphic stresses, diseases and pests, and management practices that produce a cumulative stress load (Leath, 1989). The use of birdsfoot trefoil would be increased if productive stands could be maintained under intensive management for several years.
Diseases are the major cause of premature stand decline and reduced productivity in most temperate forage legumes (Leath, 1989). Crown and root diseases are identified as the most important limitation to birdsfoot trefoil production and persistence (Drake, 1958; Henson, 1962; Hill and Zeiders, 1987; Hoveland et al., 1987; Miller et al., 1964; Pettit et al., 1966; Taylor et al., 1973). In Uruguay, Altier (1994) evaluated a space-planted nursery of birdsfoot trefoil that included two adapted cultivars (Estanzuela Ganador and San Gabriel) and a local population. By the end of the second year, 93% of the plants had died and 82% of the plant losses were due to crown and root diseases. A field survey also demonstrated that these diseases are widely distributed in farmer fields in diverse ecological regions of Uruguay (Altier, 1997).
Crown and root diseases are caused by a complex of soil organisms. Although several genera of fungi, including Rhizoctonia, Mycoleptodiscus, Macrophomina, and Phoma, have been isolated, Fusarium species make up the largest number of pathogens causing crown and root diseases of birdsfoot trefoil (Drake, 1961; Henson, 1962; Ostazeski, 1967). The species of Fusarium most frequently associated with crown and root rots of forage legumes is F. oxysporum, followed by F. avenaceum (Fr.) Sacc., F. solani (Mart.) Appel & Wollenw. emend. Snyd. & Hans., F. acuminatum Ell. & Ev., F. tricinctum (Corda) Sacc., and F. moniliforme Sheldon (Leath, 1989). Fusarium oxysporum is reported as the causal organism of fusarium wilt in birdsfoot trefoil (Gotlieb and Dorisky, 1983; Zeiders and Hill, 1988). More recently, Bergstrom and Kalb (1995) described a wilt organism of birdsfoot trefoil as a specific pathogen of this species, for which they proposed a new taxon, F. oxysporum f. sp. loti. In Uruguay, Altier (1994) studied the fungi associated with diseased birdsfoot trefoil plants in a spaced-plant nursery and showed the majority of fungi isolated from crown and root tissues were Fusarium spp. (72%). The two most frequently isolated species were F. oxysporum (54% of total) and F. solani (9% of total).
The crown and root rot or wilt of birdsfoot trefoil is complex, and resistant cultivars are not available. The variability shown by the pathogen species associated with this disease complex and the possible pathogen x environment interaction may reduce the effectiveness of breeding for increased resistance. The methods of breeding a forage legume cultivar with a high level of resistance that is effective across a broad geographic area are not straightforward (Leath, 1989). However, host plant resistance, if available, would be the most economical and environmentally sound means of managing crown and root disease of birdsfoot trefoil.
Genetic variation for Fusarium resistance in birdsfoot trefoil was reported by Ford (1959), and Henson (1962) suggested that progress could be made in increasing resistance to diseases caused by root rotting organisms if suitable inoculation techniques could be developed. More recently, Zeiders and Hill (1988) demonstrated that recurrent selection increased resistance to F. oxysporum wilt and associated root and crown rot in birdsfoot trefoil. They concluded that an initial step in breeding birdsfoot trefoil for greater persistence would be to screen potential source populations for resistance to F. oxysporum and concentrate breeding effort in the superior source populations.
Enhanced resistance to diseases caused by Fusarium species was successfully achieved in other tetraploid forage legumes and indicates the feasibility of this approach for birdsfoot trefoil. In alfalfa (Medicago sativa L.), Richard et al. (1980) and Wilcoxson et al. (1977) found an increased resistance to Fusarium crown and root rot after one cycle of phenotypic recurrent selection in the field and greenhouse. In diploid and tetraploid red clover (Trifolium pratense L.), Andersson and Kristiansson (1989) and Rufelt (1985) found a positive response in disease resistance after one to three cycles of recurrent selection in the greenhouse. These authors agreed that recurrent selection for fusarium root rot resistance would be of great value in developing more persistent legume cultivars. Recurrent phenotypic selection has been the most effective method employed for increasing the frequency of desirable genes when developing disease-resistant alfalfa populations (Elgin et al., 1988). Alfalfa and birdsfoot trefoil have the same ploidy level and autotetraploid genetic model, suggesting this method of selection should produce birdsfoot trefoil germplasm with increased levels of disease resistance.
The objectives of this study were to develop a greenhouse method to characterize birdsfoot trefoil germplasm for resistance to fusarium root disease, and to evaluate the effectiveness of one cycle of divergent phenotypic selection within a population adapted to Uruguay.
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Materials and methods |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Fungal isolates were initially stored on potato-dextrose agar (PDA) slants and later placed on silica gel crystals at 5°C for storage (Windels, 1992). Inoculum was produced by placing crystals on PDA in petri plates that were incubated at 22°C for 4 d under cool white light (12-h photoperiod; 20 µmol m-2 s-1). Mycelial plugs were taken from the colony margin and placed in potato-dextrose broth (Difco, Detroit, MI) in half-filled Erlenmeyer flasks (3 plugs per 125 mL).1 Flasks were placed on a rotary shaker (130 rpm) at room temperature (22–24°C) for 7 d. Conidia were strained through two layers of cheesecloth, quantified with a hemacytometer, and final concentrations were adjusted to 1 x 107 spores mL-1 with sterile 0.3% water agar.
Greenhouse Selection and Evaluation Method
Inoculation Procedure
Plants were grown from pregerminated seeds in a 1:1:1 autoclaved mixture of soil/sand/commercial substrate (Plantmax, Eucatex, Brazil) inoculated with commercial Rhizobium in 60 by 40 by 8 cm, 13 by 8 cell styrofoam seed starter trays (no. 40654, BROMYROS Co., Montevideo, Uruguay). Each of the 104 cells had a 4 by 4 cm square opening and a 0.5-cm-diam. hole in the bottom, with an approximate volume of 50 cm3. Two pregerminated seeds were placed in each cell, and later thinned to one plant per cell. Styrofoam trays were placed over 62 by 42 by 10 cm wooden frame boxes with a plastic bottom that contained the same 1:1:1 autoclaved mixture. Roots were allowed to grow through the bottom of the cell into the soil in the box below. Plants were grown under natural light in the greenhouse at 24°C (± 4°C). Plants were clipped to 5 cm when they reached 20 cm.
Twelve to 15 wk after seeding, shoots were clipped to 5 cm and plants were inoculated as follows. Roots were cut just below the styrofoam tray and above the soil surface of the box using a knife introduced between the tray and the box. After separating the tray from the box, roots that escaped cutting were cut with scissors or a scalpel, resulting in all roots being cut 
6 cm below the crown. The tray, containing plants with freshly wounded roots, was placed for 10 to 15 min in a large metal flat containing 2000 mL of a spore suspension adjusted to 1 x 107 spores mL-1. While the tray was soaking, 2.5 kg of sand and 600 mL of fungal inoculum were mixed together and spread uniformly in a 0.5-cm layer on the soil surface in the box. The styrofoam tray was set onto the inoculated box, allowing the freshly wounded roots to be in direct contact with fungal inoculum. Plants were kept in the greenhouse as described above until they were evaluated for disease.
Disease Assessment
Ten to 12 weeks after inoculation, plants were uprooted, washed, and individually evaluated for disease severity by using five different variables. Roots were split transversely at 1.5 or 3.0 cm above the inoculation site, and disease severity was visually rated as percentage of IR in the root section: 0, 1, 5, 10, 20, 30, 40, 50% or greater. Roots were split longitudinally from the transverse section down toward the inoculation site when there was no infected tissue (IR = 0%), or up toward the crown when the percentage of infected tissue was greater than zero (IR > 0%). The VD percentage rating was transformed to a disease severity index (DSI) five-class scale as follows: 0 = 0% (no disease), 1 = 1 to 5% (slight rot or discoloration), 2 = 10 to 20% (moderate rot or discoloration), 3 = 30 to 40% (severe rot or discoloration), 4 = 50% or more (almost or completely dead plant). Disease severity was measured as the total VD from the inoculation site toward the crown (0.0–6.0 cm). A combined index (DS x VD) was calculated by multiplying the class and vertical discoloration values: DS x VD = (DSI + 1)VD. The percentage of resistant plants (PRP) was calculated, considering plants scored 0 as resistant (DSI = 0, IR = 0%, VD < 1.5 cm).
Selection Protocol
One cycle of divergent phenotypic selection for reaction to F. oxysporum was conducted within the adapted cultivar San Gabriel using the greenhouse procedure previously described. Germinated seeds were transplanted on 7 July 1995. Plant tops were trimmed on 24 Oct. 1995, and roots were inoculated with F. oxysporum on 25 Oct. 1995 when the plants were 110 d old. Individual plants were rated for disease severity on 2 to 5 Jan. 1996 when they were 179 d old. A total of 1456 plants (14 styrofoam trays by 104 plants per tray) were screened.
From each tray, 24 plants were measured for vertical discoloration in the longitudinally split taproot to determine how the internal infection progressed from the inoculation site up to the crown and to measure the variability in disease reaction among the plants. The remaining plants were scored as described above. The roots were split transversely at 3.0 cm above the inoculation site. Since we wanted to save the selected plants, when IR percentage was >0% in the transverse section, the roots were not split longitudinally up toward the crown.
A total of 106 plants (7.3%) that scored 0 to 5% for IR were selected as polycross parents to produce seed for the resistant Cycle 1 population. A total of 26 plants (1.8%) that scored 30% or more for IR were selected as polycross parents to produce seed for the susceptible Cycle 1 population. Due to plant mortality from the disease evaluation procedure, the selection intensity was not equal in both directions. The selected plants were vegetatively propagated by stem cuttings and placed in the greenhouse with supplemental light for a 17-h day length to stimulate uniform flowering. Plants within each group were cross-pollinated in separate cages with native bees (Bombus spp., Megachile spp.) to produce the Cycle 1 seed of the resistant and susceptible populations, designated SG-R1 and SG-S1, respectively.
Evaluation of Selection Protocol
An evaluation experiment was conducted during 1996 to determine the effectiveness of one cycle of divergent phenotypic selection. The Fusarium isolates, the inoculation technique, and disease assessment method were the same as those used in the screening test.
The experiment included eight entries: the source population, San Gabriel; two cycle-1 selected populations, SG-R1 and SG-S1; two Uruguayan cultivars, Estanzuela Ganador and INIA Draco; two germplasm populations selected for Fusarium resistance in the USA, C534 and NY 9512; and one North American cultivar, Empire. Estanzuela Ganador is an adapted cultivar selected within materials of a Food and Agriculture Organization of the United Nations collection introduced from Argentina. It is more persistent and has higher forage yields than San Gabriel (Altier, 1997). INIA Draco (experimental line LE 65-56) is a new cultivar developed after two cycles of phenotypic recurrent selection for persistence under field conditions (Rebuffo and Altier, 1997). The germplasm NY 9512 was provided by Dr. D.R. Viands (Dep. of Plant Breeding and Biometry, Cornell University, Ithaca, NY) and had undergone two cycles of selection for resistance to fusarium wilt by Zeiders and Hill (1988). The parental sources of NY9512 trace to 15 North American varieties, six germplasm releases from the University of Guelph (Guelph, Ontario, Canada), and 22 plant introductions. The germplasm C534 was provided by Dr. R.R. Smith (U.S. Dairy Forage Research Center, Madison, WI) and had undergone two cycles of selection for resistance to F. oxysporum using New York isolates. The parentage of C532 include equal contributions from two cultivars Norcen (Miller et al., 1983) and Dawn (Beuselinck, 1994) and the USDA-Wis. Experimental population Trevig. Both of these selection protocols used Fusarium isolates that produce an acute wilting reaction allowing for visual assessment of the plants for disease reaction in the greenhouse screen.
The entries were seeded on 16 June 1996 in a randomized complete block design with five replicates. A replicate consisted of four 104-cell styrofoam trays and had 52 plants per entry. Plants were clipped on 30 Sept. 1996, and roots were inoculated with F. oxysporum on 1 Oct. 1996 when the plants were 107 d old. Individual plants were rated for disease severity from 16 to 19 Dec. 1996 when they were 183 d old.
Roots were split transversely at 1.5 cm above the inoculation site and scored as previously described. Plants were rated for the aerial vigor (VA), taproot vigor (VTR), number of lateral roots (NLR), and size of lateral roots (SLR) on a three-class scale with 1 = the least vigorous, least number, or smallest size. An analysis of variance for the five root rot variables (IR, VD, DSI, DS x VD, PRP) and the four vigor traits (VA, VTR, NLR, SLR) was conducted using the General Linear Model procedure (SAS Institute, 1982) and means were separated using Fisher's protected LSD test (P < 0.05). Entries were compared with respect to frequency distribution per disease severity class (DSI) and vertical discoloration range (VD = 0.0–1.4, 1.5–2.9, 3.0–4.5, and 4.6–6.0 cm) by the chi-square test. Linear correlation coefficients were calculated among IR, VD, VA, VTR, NLR, and SLR.
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Results |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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The mean VD and PRP for San Gabriel were similar in both the initial greenhouse disease assessment and the greenhouse evaluation experiments, demonstrating good repeatability of the experimental procedures. Since roots were transversely split at 1.5 cm rather than at 3.0 cm in the greenhouse evaluation, results for the other variables used to express root rot reaction are not directly comparable with the initial disease assessment test. The mean IR (15.4%) and DSI (1.52) were higher in the evaluation test as expected since the percentage of affected tissue was visually rated in a root section closer to the inoculation site.
Birdsfoot trefoil entries varied (P < 0.05) in reaction to root rot caused by F. oxysporum. The SG-R1 population showed a decrease in IR and DSI when compared with the source population San Gabriel (Table 1) . A consistent reduction in VD and the combined index (DS x VD) was observed, though it was not statistically significant and the PRP was twice that of the unselected population (Table 1). The most susceptible entry in the test was SG-S1.
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The three North American germplasms did not differ in root rot reaction. Previous selection in C534 and NY 9512 did not result in increased resistance to the composite of F. oxysporum isolates from Uruguay. This was expected since the populations were selected for resistance to F. oxysporum strains from New York recognized as F. oxysporum f.sp. loti, which is a specific wilt-inducing pathogen of birdsfoot trefoil (Bergstrom and Kalb, 1995). Furthermore, the three North American germplasms differed in root rot reaction from the currently grown cultivars San Gabriel and Estanzuela Ganador, but were similar in root rot reaction to INIA Draco and SG-R1 (Table 1).
In the SG-R1 population, the frequency of plants rated 0 or 1 increased and the frequency of plants rated 3 or 4 decreased for DSI; the opposite was observed in the SG-S1 population (Fig. 1A) . A similar tendency was observed when plotting VD frequencies (Fig. 1B). Chi-square tests showed that San Gabriel and SG-R1 differed in the frequency distribution of plants for DSI and VD (P < 0.01 and P = 0.02, respectively). INIA Draco, selected for persistence in the field, had a higher frequency of plants rated 0 or 1, and a lower frequency of plants rated 2, 3, or 4 for DSI when compared with Estanzuela Ganador, one of its parental sources (Fig. 1A). Vertical discoloration ratings also showed that INIA Draco had a higher frequency of plants with 0.0 to 1.4 or 1.5 to 2.9 cm of VD and lower frequency of plants with 4.6 to 6.0 cm of VD when compared with Estanzuela Ganador (Fig. 1B). When comparing selected and parental populations, the frequency distribution of plants for VD allowed for better discrimination among the germplasms than DSI (Fig. 1).
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Discussion |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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One cycle of divergent phenotypic selection was effective in increasing the level of resistance to F. oxysporum in San Gabriel, a population adapted to Uruguay. Although the differences between the source population and the selected Cycle 1 populations were not always statistically significant, the response was directionally consistent for all the traits evaluated. These results suggest that divergent phenotypic selection was successful in changing the frequency of genes that are involved in the response to fusarium root rot.
Our progress from selection for resistance to F. oxysporum agreed with the results of Zeiders and Hill (1988), who showed that recurrent selection increased resistance in birdsfoot trefoil. Recurrent phenotypic selection has been the most effective selection method employed for increasing the frequency of desirable genes when developing disease-resistant alfalfa populations (Elgin et al., 1988). Alfalfa and birdsfoot trefoil have the same autotetraploid genetic system. Additional cycles of selection using a higher selection intensity to improve the rate of progress should be conducted to assess the progress that can be made in increasing resistance to fusarium root rot.
Selection increased the frequency of plants in class 1 (IR = 1–5%), which indicates that the frequency of plants with long-term tolerance could be increased. As discussed by Zeiders and Hill (1988), the tolerance represented by a transitory Class 1 or by Class 2, which delayed disease development 60 to 90 d, is of little value in a perennial crop. Root rot measurements taken after the disease has had sufficient time for development (10–12 wk after inoculation) should discriminate among resistant plants (Class 0), tolerant plants (Class 1 and 2), and susceptible plants (Class 3 and 4). The selection of only Class 0 individuals as polycross parents in additional cycles of selection should result in a higher level of resistance.
The SG-R1 population should be field-tested to determine if the greenhouse selection was effective in increasing persistence. INIA Draco, selected for field persistence, showed increased resistance to F. oxysporum in the greenhouse when compared with San Gabriel and was similar to SG-R1. Hill and Zeiders (1987) demonstrated the importance of source population for improving the response of birdsfoot trefoil to F. oxysporum and stand persistence in the field. They found a positive association between the number of live plants 120 d after inoculation with F. oxysporum in the greenhouse and greater stands in the field, which suggests that F. oxysporum is an important factor in stand decline in birdsfoot trefoil in Pennsylvania.
Previous studies on fusarium root rot resistance in forage legumes tend to support a nonspecific type of resistance rather than gene-for-gene action (Richard et al., 1980). This type of resistance is important because breeding for resistance to a few Fusarium isolates could confer resistance to additional isolates. INIA Draco, selected for persistence under field conditions at INIA La Estanzuela, showed resistance to a composite of F. oxysporum isolates collected from different locations in Uruguay. This suggests the occurrence of a nonspecific type of resistance in INIA Draco.
The large variability in F. oxysporum isolates recovered from birdsfoot trefoil diseased roots and crowns justified the use of a composite of isolates to screen and characterize birdsfoot trefoil populations (Altier, 1997). The need for screening techniques useful in breeding for crown and root disease complex is obvious. The advantages of field selection over greenhouse selection are well established, because the plants are exposed to the array of pathogens naturally occurring in the field (Wilcoxson et al., 1977). However, the development of severe internal rot is generally slow, often taking a minimum of 2 yr to kill the taproot. The development of a reliable greenhouse screening technique adapted to a breeding program would have the advantage of giving the breeder the opportunity to run two cycles of selection per year.
The greenhouse evaluation method can be used as a standard test to screen and characterize birdsfoot trefoil populations for resistance to fusarium root rot. It gave reliable infection without escapes, and provided reproducible results between experiments. The method optimized efficiency by allowing the screening of large number of plants in a short time in limited space. Efficiency could be further increased by rating disease based on either percentage of internal rot or vertical discoloration. Vigor could be evaluated based on either the number or size of lateral roots. The evaluation of the improved populations under field conditions should be completed to validate the usefulness of the greenhouse procedure. Development of cultivars with increased levels of resistance to root rot caused by F. oxysporum could make a significant contribution towards increasing the persistence and production of birdsfoot trefoil.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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1 Mention of product and equipment names is for identification purposes only and does not imply a warranty or endorsement to the exclusion of other products that may be similar. 
Received for publication April 26, 1999.
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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