Methods
Study Name: Morphological study of perennial Glycine. Experiment Type: Greenhouse Greenhouse Study Year: 1986 Latitude: 40 Degrees 08 Minutes N Longitude: 88 Degrees 12 Minutes W Elevation: 227 meters Year started: //1984 Year ended: //1986 Experiment length: 3 years
The squash technique was used to count chromosomes in root tips of germinating perennial Glycine following procedures described by R. Singh (2003. Plant Cytogenetics, Second Edition, CRC Press, Boca Raton, FL)
Chromosome counts were determined from root tips
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Seeds were placed on moist filter paper in Petri plates and germinated. If available, five seedlings of each accession were transplanted into 7- cm diameter clay pots containing 180 cm3 of sand and placed on a greenhouse bench. Depending on the accession, plants were grown for 3-12 months. Supplemental HID lighting was provided to maintain a 14-hour day length. One week before inoculation, seedlings of H. glycines-susceptible cv. Lee 74 soybean were transplanted into pots as described above. Plants were inoculated with 3,000 eggs and J2 obtained from females and cysts of Hg Type 0 raised on Lee 74 soybean (Assuncao et al., 2004) and maintained on a greenhouse bench for approximately 1 month. Supplemental heating and cooling were provided to maintain soil temperatures of 24-30?C. When mature females appeared on roots of Lee 74 soybean (1 month), the root system of each plant was dipped vigorously several times in a beaker containing 3 L of water to remove soil and females from the roots. Roots were placed on nested 850- and 250-um-pore sieves and sprayed with a strong stream of water to remove adhering females. The soil was suspended in the water and gravity sieving using the nested sieves was used to extract females from the soil suspension (Cobb, 1918). Females were collected from the 250-um-pore sieve and counted with the aid of a dissecting microscope. The thoroughly cleaned roots were placed in a drying oven at 70C for 48 hours. Roots were removed, allowed to equilibrate for 24 hours at room temperature and humidity, and weighed. A female index (FI) ((no. females per 0.1 g dry root of accession/no. females per 0.1 g dry root of Lee 74) x100) was calculated for each accession and a rating of resistance classified as: resistant (R): 0
Seeds of the perennial Glycine species were scarified by scoring seed coats on the opposite side of the hilum with a razor blade. Seeds were then germinated on moist filter paper inside petri dishes under light at 25C for 4 to 6 d. Germinated seedlings were planted in a 1:1 steam-sterilized soil:sand mix (1:1 v/v) at pH 7. The soil was a Drummer silt loam. All plants were grown in greenhouses under a photoperiod of 16 h of daylight at 24 +/- 3C/18 +/- 3C day/night temperature. For all evaluations, transplanted seedlings were grown for 2 to 3 wk before being inoculated. Seeds of the soybean checks were sown when the perennial accessions were transplanted. The seedlings were grown in a greenhouse and watered daily for 10 to 14 d before inoculating. Five plants of each accession were screened once in a series of 8 non-replicated runs. The cultivar Spencer was used as a susceptible check in each run. A selected set of 67 accessions, which were based on the result of the non-replicated runs with some accessions representing most species, was further screened in 2 replicated trials with 5 plants/per each of 4 replications. A final selected set of 15 accessions, which were based on those 67 accessions that performed well within some of the species, was screened in 2 trials with 3 replications each. A F. solani f. sp. Glycines isolate, Mont-1, was subcultured on 2% (w/v) water agar for 3 wk before infesting sorghum seed. Two hundred cm3 of sorghum seed were soaked in distilled water in a 1-L Erlenmeyer flask overnight, drained, and autoclaved on 2 consecutive days for 20 min at 121C. Ten mycelial plugs (4-mm diameter) were transferred from the water agar colonies. Cultures on sorghum grains were incubated under a 12 h-photoperiod with fluorescent light for 10 to 14 d at 25C. Nine entries of 5 seedlings or seeds were sown in 20- by 30-cm trays. Soil was infested by placing colonized sorghum seed below transplanted Glycine spp. and beneath soybean seeds at planting. A template was used to make 3 furrows that were 30 cm long, 2 cm deep, and 3 cm apart. 5 cubic centimeters of infested sorghum seed was evenly distributed in each furrow and about 800 cm3 of soil mix was added to cover the infested seed to a depth of 2 cm. Transplanted seedlings or seeds were added to each furrow directly over the infested sorghum seeds. Trays were placed in a greenhouse at 20 to 30C and watered daily. Disease severity was rated for each plant 21 d after planting.
Seeds of the perennial Glycine species were scarified by scoring seed coats on the opposite side of the hilum with a razor blade. Seeds were then germinated on moist filter paper inside petri dishes under light at 25C for 4 to 6 d. Germinated seedlings were planted in a 1:1 steam-sterilized soil:sand mix (1:1 v/v) at pH 7. The soil was a Drummer silt loam. All plants were grown in greenhouses under a photoperiod of 16 h of daylight at 24 +/- 3C/18 +/- 3C day/night temperature. For all evaluations, transplanted seedlings were grown for 2 to 3 wk before being inoculated. Seeds of the soybean checks were sown when the perennial accessions were transplanted. The seedlings were grown in a greenhouse and watered daily for 10 to 14 d before inoculating. Five seedlings of each accession were screened once in a series of eight nonreplicated runs that averaged 98 accessions per run. Cultivar Williams 82 was used as a susceptible check in each run. An isolate of S. sclerotiorum originating from infected plants from a field near Dekalb, IL, in 1994 was maintained the dark at 48C on potato dextrose agar. Mycelial plugs were transferred to acidified potato dextrose agar (APDA) and after 1 d, this culture was used as starter inoculum by transferring 3-mm-diam plugs from the margin of the colony to new APDA plates. After 1 to 2 d, 3-mm-diam plugs from the colony edges were used to inoculate plants. To inoculate the perennial plants, a single plug, mycelial-side down, was placed on the newest leaf petiole touching the main stem or in the plant whorl. For soybean plants, a single mycelial plug was placed mycelial-side down on a cotyledon approximately 2 mm from the stem of each seedling. Following inoculation with the plug method, all seedlings were lightly misted with water from a hand atomizer to increase humidity and then covered with a plastic dome that fit over individual flats. The dome-covered flats were placed about 1 m under black mesh shade cloth (80% light reduction) to prevent heat build up inside the domes. After 2 d, the domes and shade cloth were removed. The number of total seedlings that survived was counted daily usually beginning with the day the dome was removed until plants stopped dying, which was 4 to 5 d after the first rating. A selected set of 53 accessions, which were based on the results of the non-replicated runs with some accession representing most species, was further screened in two replicated trials with five plants in each of four replications. Soybean cultivars Asgrow A2242 (susceptible) and NK S19-90 (partially resistant) were used as checks. A final selected set of 12 accessions, which were based on those 53 accessions that preformed well within some of the species, was screened in two trials with three replications each.