Crop Science 40:841-843 (2000)
© 2000 Crop Science Society of America
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Methods for evaluating birdsfoot trefoil for susceptibility to foliar and shoot blight caused by Rhizoctonia Spp.
J.T. Englisha and
P.R. Beuselinckb
a Dep. of Plant Microbiology and Pathology, Univ. of Missouri, 108 Waters Hall, Columbia, MO 65211 USA
b USDA-ARS, Plant Genetics Research Unit, Columbia, MO 65211 USA
englishj{at}missouri.edu
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ABSTRACT
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Production of birdsfoot trefoil (Lotus corniculatus L.) is limited in humid, temperate regions by foliar and shoot blight caused by Rhizoctonia species. The objective of this study was to develop methods for quantifying blight symptoms on 14 plant introductions and three cultivars of birdsfoot trefoil. Susceptibility was evaluated in the greenhouse on the basis of shoot lesion formation and blight of leaves and apical meristems. On the basis of these symptoms, no entry of this limited collection of birdsfoot trefoil was completely resistant to infection. However, 4 d after inoculation, shoot lesion length differed significantly among entries. Additionally, lesion lengths varied between experiments in relation to environmental conditions. In contrast to shoot lesion length, foliar blight and the time to blight of apical meristems did not vary significantly among entries in either experiment. Shoot lesion development limits foliage production and survival under field conditions and should be considered an important factor in further screening of birdsfoot trefoil for disease resistance.
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INTRODUCTION
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BIRDSFOOT TREFOIL is a perennial legume that is important in forage systems in the USA. In northern regions, stands of birdsfoot trefoil will occasionally reseed, replacing plants lost to a crown rot complex or environmental stress (Beuselinck et al., 1984). In contrast, in warmer and more humid regions of the USA, natural reseeding and recruitment are insufficient to overcome losses to disease and environmental stress. In these areas, reduced stand persistence is associated not only with a crown rot complex, but with foliar and shoot blight caused by Rhizoctonia solani Kühn [teleomorph = Thanatephorus cucumeris A.B. Frank (Donk)] and binucleate Rhizoctonia spp. (Allison, 1951; English, 1992). Symptoms of these plant-canopy diseases include shoot lesions and blight of leaves and apical meristems.
Rhizoctonia spp., alone or in combination with other pathogens, severely reduce the life spans of leaves produced in dense canopies. English (1992) reported that the longevity of leaves produced during mid- to late-summer was 50% or less than the longevity of leaves produced in spring or early summer. Reduced leaf longevity was caused by blight and not senescence. Loss of leaves to blight in later portions of the growing season reduces forage availability and places stress on plants that are partitioning photoassimilate between seed production and root carbohydrate storage (Alison and Hoveland, 1989; Beuselinck and McGraw, 1988).
Field epidemics of Rhizoctonia foliar and shoot blight develop rapidly. The disease spreads within and among plants as infected shoots lodge against healthy shoots and foliage. Blight spreads rapidly from multiple initial infection points within a plant and causes a mosaic pattern of symptoms in field plant populations. Fungicides are not available or are impractical for control of Rhizoctonia blight. Breeding for resistance provides an alternative approach for limiting the impacts of the disease on plant growth, forage production, and stand persistence. However, before disease resistance can be evaluated, methods for plant inoculation and quantitative assessment of relevant disease symptoms must be established. The objective of this research was to develop methods to characterize foliar and shoot blight symptoms on birdsfoot trefoil infected by Rhizoctonia spp. Assessment methods were developed using a collection of birdsfoot trefoil accessions obtained from the USDA-GRIN system.
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Materials and Methods
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Seed of 17 randomly selected plant introductions (PIs) and cultivars of birdsfoot trefoil were obtained from the USDA Regional Plant Introduction Station, Pullman, WA (Table 1)
. Plants of each entry were grown from seed in a field at the University of Missouri, Agronomy Research Center, near Columbia, MO. Thirty ramets from each entry were produced by rooting stem cuttings using intermittent mist for 2 to 3 wk in plastic pots (9-cm diam x 7.5 cm deep) containing Promix (Premier Horticulture, Dorval, Quebec, Canada) and autoclaved sand (2:1 by volume). Over a period of 12 wk after rooting, lateral branches were cut back to 2 or 3 nodes to encourage additional axillary branching. Ramets were inoculated with Rhizoctonia spp. starting 12 wk after stem cuttings were rooted. The isolates of Rhizoctonia used as inoculum were selected from a collection of field isolates of R. solani and binucleate Rhizoctonia spp. All field isolates were recovered from diseased leaves and shoots of birdsfoot trefoil at the University of Missouri Horticulture and Agroforestry Research Center, near Columbia, MO, and the Agronomy Research Center. Three virulent isolates of R. solani from anastomosis group 2-2 (Sneh et al., 1991), and one virulent isolate of a binucleate Rhizoctonia sp. were chosen as representative of the collection. Preliminary inoculation experiments demonstrated that the chosen isolates were similar in virulence to the field collection (data not shown). The four isolates were stored on potato dextrose agar (PDA) in the dark at 20°C. For preparation of fresh inoculum, the isolates were transferred to fresh PDA in petri plates and allowed to grow for 2 d in an incubator in the dark at 24°C.
To prepare inoculum, asymptomatic shoots of field-grown birdsfoot trefoil were cut into 0.5-cm segments, saturated with water, and autoclaved in small glass beakers for 20 min. on each of two successive days. Approximately 100 sterilized shoot segments were transferred into a petri plate and inoculated with three 5-mm PDA plugs colonized by each of the four Rhizoctonia isolates. The isolates were combined in the experiments to represent the range of virulent pathogen types to which a plant would be exposed under field conditions. Inoculated segments were incubated at 25°C in the dark and mixed every 3 d to encourage complete colonization. After 7 d of incubation, several inoculated shoot segments were plated on PDA to confirm colonization by the isolates. It was not possible to determine the parity of shoot segment colonization by individual isolates.
Two colonized shoot segments were selected randomly from the petri plate and used to inoculate either a shoot at the base of the apical meristem or a leaf petiole. The colonized shoot segments were placed side-by-side and attached to an inoculation point with petroleum jelly. In preliminary experiments, neither the petroleum jelly alone nor attached non-colonized shoot segments caused any symptom development.
Two randomly selected shoots on each of five plants of a plant entry were inoculated at the base of the apical meristems. Two additional shoots of each of the five plants were selected randomly and inoculated on the petiole just above the attachment point of the lowest two leaflets. Inoculations at the apical mersitems and petioles were made simultaneously with time delays of at most five minutes. Although systemic acquired resistance has not been reported for L. corniculatus infected with Rhizoctonia spp., virtually simultaneous inoculations with highly virulent isolates were made to minimize concerns with such a possibility.
Inoculated plants were misted immediately and placed into a dew chamber for 48 h at 28°C. Each day thereafter, inoculated plants were moved to a mist chamber (a saturated atmosphere was maintained by a cool-air, sonicating humidifier) during daylight hours (minimum of 10 h light) and then were moved back into the dew chamber at night. The entire experiment was performed twice. Experiment 1 was conducted from 22 to 28 Feb. 1993 and Exp. 2 was conducted from 1 to 7 March 1993.
Disease development and symptom expression were evaluated by three variables: (i) shoot lesion length, (ii) number of blighted leaflets, and (iii) number of days for blight of apical meristem. Lesion length was determined as the length of necrosis that developed along a shoot from the point of inoculation below the apical meristem after 4 d. Number of blighted leaflets also was determined 4 d after inoculation of petioles. A leaflet was considered blighted if it was more than 50% necrotic. This value was used because in preliminary experiments, any leaf that showed blight symptoms always became completely blighted after 24 h further incubation. The maximum possible blight per inoculated leaf was five leaflets. The final disease expression variable was the number of days to blight of the apical meristem after shoot inoculation at the shoot apex. In numerous preliminary experiments, Rhizoctonia spp. were consistently the only pathogens reisolated from diseased shoots and leaves inoculated by these methods.
Data analyses were based on five replicate plants with 2 subsamples for each symptom or disease expression type per birdsfoot trefoil genotype. Differences in symptom development between experiments and among entries in each experiment were assessed by analysis of variance. Within experiments, differences among genotype means were separated by Duncan's multiple range test (P = 0.05).
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Results and Discussion
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Methodologies were developed that enabled, for the first time, quantitative assessment of Rhizoctonia blight development on birdsfoot trefoil. The inoculation methods emphasized simultaneous infection of multiple shoots and leaves of a plant, and thus effectively simulated disease processes as they occur in the field. Maintenance of inoculated plants in mist and dew chambers provided free water on plant tissues that is required for infection and colonization by Rhizoctonia spp. (Olaya and Abawi, 1994). Greenhouse temperatures varied during the 2-wk period of each experiment. For example, temperatures ranged between 28 and 30°C in Exp. 1, but in Exp. 2, they varied between 28 and 35°C. Both temperature ranges are encountered within plant canopies in the midst of the growing season when foliar and shoot blight are most severe.
Shoots of all birdsfoot trefoil entries were infected by Rhizoctonia spp., and shoot lesion development varied significantly among entries in both experiments. In Exp. 1, the maximum lesion length (0.53 cm) of PI 380896 (Table 2)
was more than 50 times greater than the minimum lesion length (0.01 cm) on PI 226798. However, only PIs 234692 and 226798 developed lesions significantly shorter than PI 380896. Significantly longer lesions were observed in Exp. 2 on all plant entries. In this experiment, the maximum lesion length (3.3 cm) occurred on PI 232098 and was three times the minimum lesion length (1.1 cm) on `Leo'. Only lesions of Leo and PIs 383689, 234692, and 255304 were significantly shorter than lesions of PI 232098. Four of the five entries (PIs 234692, 383689, 255304, and Leo) which developed the shortest shoot lesions in Exp. 1 also developed the shortest lesions in Exp. 2.
In both experiments, all entries of L. corniculatus were susceptible to foliar blight. Generally, within 2 d of infection, an entire leaflet was destroyed. Inoculation of leaf petioles resulted only in leaflet blight and did not lead to petiole collapse or leaflet wilt. Greater leaflet blight developed after 4 d in Exp. 2 than in Exp. 1. Average numbers of leaflets blighted were 2.5 and 4.0 in Exp. 1 and 2, respectively. However, number of blighted leaflets did not vary significantly among entries of either experiment.
The average number of days required for apical meristem blight was 4.8 and 2.2 in Exp. 1 and 2, respectively. However, no significant differences in time to blight were detected among plant entries of either experiment.
The methods developed for inoculating birdsfoot trefoil with Rhizoctonia spp. and for quantifying blight symptoms should prove useful in applied breeding efforts or for basic studies of plant physiological responses to disease. However, the significant differences observed in disease development between experiments emphasized the importance of controlling environmental conditions when evaluating blight development. In particular, the greater expression of disease symptoms observed in Exp. 2 was likely associated with the occurrence of higher greenhouse temperatures that enhance disease development.
Of the disease symptoms quantified, shoot lesion length may be the most useful in breeding efforts. Plant entries that exhibit rapid and extensive shoot lesion development lose not only the foliage attached to infected shoots, but they also are unable to generate additional leaves and lateral branches from necrotic apical or axillary meristems. Further testing of a broader collection of birdsfoot trefoil accessions will be needed to determine if significant resistance exists against shoot and foliar blight caused by Rhizoctonia spp.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge research support from the Clover and Special Purpose Legumes section of the Crop Germplasm Committee. The authors also thank Keith Emery and Susan Taylor for technical assistance and identification of anastomosis groups of Rhizoctonia solani used in these experiments.
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NOTES
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Journal Series Paper No. 12,694 of the Missouri Agric. Exp. Stn.
Received for publication April 16, 1999.
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REFERENCES
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- Alison M.W., Jr., Hoveland C.S. Birdsfoot trefoil management. I. Root growth and carbohydrate storage. Agron. J. 1989;81:739-745.[Abstract/Free Full Text]
- Allison J.L. Rhizoctonia blight of forage legumes and grasses. Plant Dis. Rep. 1951;35:372-373.
- Beuselinck P.R., McGraw R.L. Indeterminate flowering and reproductive success in birdsfoot trefoil. Crop Sci. 1988;28:842-845.[Abstract/Free Full Text]
- Beuselinck P.R., Peters E.J., McGraw R.L. Cultivar and management effects on stand persistence of birdsfoot trefoil. Agron. J. 1984;76:490-492.[Abstract/Free Full Text]
- English J.T. Modular demography of Lotus corniculatus infected by Rhizoctonia spp. Phytopathology 1992;82:1104.
- Olaya G., Abawi G.S. Influence of inoculum type and moisture on development of Rhizoctonia solani on foliage of table beets. Plant Dis. 1994;78:805-810.
- Sneh B., Burpee L., Ogoshi A. Identification of Rhizoctonia species. St. Paul, MN: APS Press, 1991.
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ABSTRACT
INTRODUCTION
