Progress 09/15/06 to 09/14/09
Outputs
OUTPUTS: Survey existing ranches: 2006-2007 Forage Legume Survey. During 2006-2007, 29 long-term kikuyugrass-legume pasture sites representing 23 soil types (10 Andisols, 4 Oxisols, 4 Inceptisols, 3 Ultisols, a Mollisol, and an Entisol) were sampled for soil and plant analyses. Legumes comprised 0 to 43% (mean = 10%) of the live herbage mass. Excessive stocking rates, particularly during droughty periods, appeared to be the primary factor behind declining and (or) low legume percentages in most of the low-legume pastures, rather than nutrient deficiencies or aluminum (Al) toxicity. Only three sites had a soil pH of less than 5.5 (range = 5.1 to 5.4). In no case did 1 M KCl exchangeable Al exceed 0.26 cmol/kg or greater than 6 percent saturation of the effective cation exchange capacity, thereby indicating little potential for Al toxicity to legumes. Evaluate forage peanut and barrel medic for tolerance to acidity: Pot studies using Al-toxic soil. Aluminum-toxic soil was collected from Kokee on the island of Kauai (medial ferrihydritic, isothermic Alic Hapludands). Four pH levels (4.5, 4.8, 5.5, and 6.6) were achieved by incubation of soil with varying amounts of dolomite. Manganese toxicity was not a problem in this soil. In one greenhouse experiment, one seedling of perennial peanut (cv. CIAT 17434) was transplanted into each pot and grown for 27 days. In a second study, one seedling of barrel medic (cv. Jemalong A-17) was transplanted per pot and grown for 42 days. Each trial followed a randomized complete block design with four pH treatments and five or six blocks. The longest root length and shoot dry weight increased significantly with increasing soil pH. Barrel medic appeared to be much more sensitive to an acid pH and/or Al-toxicity than perennial peanut. In a third study, forage legume seedling growth (10 week-old) and regrowth (5 weeks after clipping to 8-cm residual stubble) were measured for perennial peanut (cv. Amarillo) and two selections of barrel medic (accessions W6-6037 and W6-6092) in a greenhouse pot study. The results of this experiment suggest that perennial peanut is not very sensitive to a low pH (4.6) and high exchangeable Al or solution Al. In contrast, both medic selections were sensitive to soil acidity with accession W6-6037 more Al tolerant, provided the Al saturation and solution Al levels were not too high. Conduct genetic analysis of M. truncatula: Eighty-seven ecotypes of barrel medic were collected from Australia, Algeria, France, Greece, Italy, Spain, Portugal, Tunisia, Cyprus, Turkey, Malta, Libya, and Sweden. They were evaluated for their sensitivity to Al on an agarose medium containing 4 levels of Al (0, 50, 100, and 200 uM). Relative root growth was used as an indication of Al tolerance. We identified three Al sensitive accessions and three Al tolerant accessions. A total of 96 molecular markers were used for genetic mapping, and at least four makers showed polymorphism among these lines. PARTICIPANTS: This project involved collaborations across two universities in Hawaii and one in California. Lead investigator was Dr. Susan C. Miyasaka, a faculty member of the University of Hawaii - Manoa. Co-investigators included Dr. Bruce Mathews at the University of Hawaii - Hilo (an undergraduate institution). The ranch survey was conducted by Dr. Bruce Mathews and undergraduate students in AGRON 410 Soil-Plant-Herbivore Interrelations. Ms. Yawadee Srimake is the Graduate Research Assistant at the University of Hawaii - Manoa who is working towards her Ph.D. degree. She conducted research on genetic analysis of Medicago truncatula partly under the supervision of Dr. Douglas Cook at the University of California Davis. TARGET AUDIENCES: Target audience of this research are ranchers in Hawaii and other tropical environments. A poster presentation was made at a workshop for ranchers in Hawaii. Extension efforts to distribute aluminum-tolerant perennial peanut have helped ranchers plant seedlings of this acid-tolerant tropical legume. The other target audience of this research are scientists who are studying response to excess aluminum in legumes and other crop plants. Five presentations by Ms. Srimake and Dr. Miyasaka have been made at scientific conferences and meetings. PROJECT MODIFICATIONS: In this project, we completed project objectives, except for identifying genes involved in host response or nodulation response to excess Al. It has taken the Graduate Research Assistant Ms. Yawadee Srimake longer than expected to identify genotypes of Medicago truncatula that differ in Al-tolerance. She has developed the F1 generation by crossing Al-tolerant and Al-sensitive genotypes of M. truncatula. She is in the process of producing the selfed F2 generation from these crosses. Once she has developed a population that segregates for Al-tolerance, then she will be able to start identifying genes involved in Al-response in M. truncatula.
Impacts
Nitrogen is the most limiting plant nutrient for growth of many plants, including warm season grasses. Sustainability of warm season grass pastures is threatened by losses of nitrogen due to forage removal, erosion, leaching, or denitrification. Biological nitrogen fixation through legumes is a low-input method of providing nitrogen to warm season grasses in mixed pastures. In the tropics, acid soil pH and aluminum toxicity are two main factors that could limit biological nitrogen fixation. The identification of genetic variation underlying aluminum responses in barrel medic will provide information to legume breeders to help them improve aluminum tolerance in leguminous crop species. In our field surveys in Hawaii, it appears that drought stress and phosphorus deficiency are greater problems for persistence of pasture legumes than acid soils or aluminum toxicity. In our pot studies using an acid, aluminum-toxic soil, perennial peanut appears to be more acid soil tolerant than barrel medic. Previous studies have shown that perennial peanut is also tolerant to low phosphorus levels in soil. Seedlings of perennial peanut have been distributed to ranchers on the island of Hawaii for planting in their pastures.
Publications
- Miyasaka, S.C., C. Clayton, and D. Greenough. 2009. Acid Soil-Tolerance of Medicago truncatula. 2009 Model Legume Congress, Asilomar Conference Grounds, CA. Abstract P6, pp. 57. Srimake, Y., Penmetsa, R.V., D.R. Cook, S.C. Miyasaka. 2009. Responses to aluminum in ecotypes of Medicago truncatula. 2009 Model Legume Congress, Asilomar Conference Grounds, CA. Abstract P&, pp. 58. Srimake, Y., Penmetsa, R.V., D.R. Cook, S.C. Miyasaka. 2009. Response to aluminum in ecotypes of Medicago truncatula. 2009 Annual Meeting of the American Society of Plant Biologists (ASPB). Abstract P08051, pp. 145. Miyasaka, S. C., B. Mathews, P. Singleton, D. Greenough, and C. Clayton. 2007. Growth of Two Pasture Legumes in an Acid, Aluminum-toxic soil. Proc. Mealani Forage Field Day, Kamuela, HI. Srimake, Y. and S.C. Miyasaka. 2007. Comparison of Two Screening Methods for Aluminum Tolerance in Medicago Truncatula. In Annual meetings abstracts [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI. Miyasaka, S.C., D.R. Cook. 2005. Developing a method to screen mutants of Medicago truncatula for aluminum resistance. Model Legume Congress 2005, Pacific Grove, CA, Je. 5-9, 2005. p. 80.
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: Outputs: Activities: Pot studies: Forage legume seedling growth (10 wk) and regrowth (5 wk) following clipping to an 8-cm residual stubble were measured for perennial peanut (Arachis pintoi cv. Amarillo) and two selections of barrel medic (Medicago truncatula, W6-6037 and W6-6092) in a greenhouse pot study with Honolua silty clay (fine, parasesquic, isohyperthermic Ustic Palehumults, bulk density = 0.94 g cm3) and Kokee silty clay loam (medial, ferrihydritic, isothermic Alic Hapludands, bulk density = 0.80 g cm3) soils at two pH levels (4.6 and 5.6). Aluminum (Al) saturation of the effective cation exchange capacity at pH 4.6 for the Honolua and Kokee soils was 26 and 63%, respectively, while soil solution Al concentrations were 0.29 and 2.20 mg per L. At pH 5.6, Al saturation for both soils was 0.6% and solution Al was less than 0.1 mg per L. Soils for each soil type and pH combination were packed into 2.2 L pots with the appropriate amount of soil to simulate the field bulk density. The pH levels of 4.6 and 5.6 were attained for the Honolua soil (initial pH of 4.4) by mixing in the appropriate amount of dolomitic limestone. The Kokee soil had an initial pH of 4.6 therefore dolomitic limestone addition was only required to achieve a pH of 5.6. After the potted soils had gone through several wetting and drying cycles over a 3-mo period they were planted with four to five pre-germinated seeds for each soil type, pH, and legume combination with four replicates per combination. Perennial peanut seeds were inoculated with the cowpea type Bradyrhizobium strain CIAT 3101 at planting while the barrel medics were inoculated with Sinorhizobium meliloti. The seedlings were thinned to two per pot one week after planting. Genetic analysis: Activities: To conduct genetic analysis of M. truncatula for genes involved in response to excess aluminum (Al), seventy-five ecotypes of M. truncatula collected from Australia (2), Algeria (34), France (10), Greece (2), Spain (12), Portugal (6), Tunisia (5), Cyprus (2), Turkey (1), and Sweden (1) were used in this experiment. Six seedlings of each accession were grown in an agarose medium (0.5 mM CaCl2, pH 4.5) containing 3 levels of Al (0, 100, and 200 uM), under 18 hours light/ 6 hours dark. Root lengths of seedlings were measured at 24, 48 and 72 h after transfer. Experiment was repeated 3 times. Relative root growth (%) was used as an indication of Al tolerance. Twenty-five accessions were also tested for root cell viability using Evans blue and Al accumulation, using hematoxylin. Aluminum-tolerant ecotypes and Al-sensitive ecotypes are being grown for cross pollination. In addition, genotypes A17/TAP, A20, DZA 045, DZA 315 and F 83005 are being grown, because they have considerable genetic background information. Eighty-one primer sets been chosen for SSR markers analysis, to generate the genetic map of genes involved in aluminum tolerance. Primer sets were selected in order to cover approximately all eight M. truncatula chromosomes. PARTICIPANTS: Susan C. Miyasaka, University of Hawaii - Manoa; Bruce W. Mathews, University of Hawaii - Hilo; Paul W. Singleton, University of Hawaii - Manoa; Yawadee Srimake, University of Hawaii - Manoa; Cheryn Clayton, University of Hawaii - Manoa; Diana Greenough, University of Hawaii - Manoa; Dennis Matsuyama, University of Hawaii - Manoa; Douglas R. Cooke, University of California, Davis. TARGET AUDIENCES: Researchers; American Society of Agronomy; Crop Science Society of America; ranchers in Hawaii. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes: Increase of Knowledge: Pot studies: Seedling growth was very slow for the medic selections compared to perennial peanut regardless of soil type and pH. Medic W6-6037 consistently produced more dry matter (DM) than medic W6-6092. All seedlings of medic W6-6092 died in the Kokee soil at a pH of 4.6, likely due to a greater susceptibility of this medic to high solution Al concentration. However, shortly after clipping, medic W6-6037 failed to regrow in the Kokee soil at a pH of 4.6 and medic W6-6092 also died in the Honolua soil at a pH of 4.6. Regrowth of all the legumes was more vigorous than the seedling growth but perennial peanut was once again much more productive than the surviving medic selections. Root mass followed the same pattern as regrowth DM yield (data not shown). These results of this experiment suggest that perennial peanut is not sensitive to a low pH (4.6) and high exchangeable and solution Al, whereas both medic selections were sensitive to soil acidity with selection W6-6037 being more tolerant, provided that the Al level is not too high. This research also indicates that barrel medic is unlikely to be a viable forage in Hawaii's pasture systems due to its slow establishment and less than vigorous regrowth in the humid subtropical environment. Legumes need to be vigorous DM producers like perennial peanut in order to persist as an important player in Hawaii's tropical C4 grass-based pasture systems. Genetic analysis: Based on RRG result from100 uM Al-containing media, we identified six Al sensitive lines (RRG less than 20 %) and five Al tolerant lines (RRG greater than 70 %). The Al-tolerant ecotypes are PI577633, ESP031, W6 6037, DZA202, ESP 039, PRT 179 and the Al-sensitive ecotypes are ESP095, PI566889, PI537168, PI535648, PI535622, PI535614. We observed relatively high standard errors in some accessions, which might arise from maternal effects of the seeds in those populations. Both visual assays gave similar results. Correlations between RRG assay and visual assays were observed in certain ecotypes analyzed. Impact: Nitrogen is the most limiting plant nutrient for growth of many plants, including warm season grasses. Sustainability of warm season grass pastures is threatened by losses of nitrogen due to forage removal, erosion, leaching, or denitrification. Biological nitrogen fixation through legumes is a low-input method of providing nitrogen to warm season grasses in mixed pastures. In the tropics, acid soil pH and aluminum toxicity are two main factors that could limit biological nitrogen fixation. In our pot studies using an acid, aluminum-toxic soil, perennial peanut appears to be more acid soil tolerant than barrel medic. Medic W6-6037 appeared to be more Al-tolerant than medic W6-6092; however, both died when stressed with clipping in an acid soil. Seedlings of perennial peanut are being distributed to ranchers on the island of Hawaii for planting in their pastures.
Publications
- No publications reported this period
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: Field surveys in 2006-2007: Kikuyugrass (Pennisetum clandestinum) based pasture systems account for about 85% of the forage-livestock production in Hawaii. During 2006, 21 long-term kikuyugrass-legume pasture sites representing 17 soil types (7 Andisols, 4 Oxisols, 2 Ultisols, 2 Inceptisols, a Mollisol, and an Entisol) were sampled for soil and plant analyses. Legumes comprised 0 to 43% (mean = 14%) of the live herbage mass with tropical Greenleaf Desmodium (Desmodium intortum) and Kaimi (Desmodium canum) being most prevalent. Half of the pasture sites had less than 10% of the total herbage mass comprised of legumes. Excessive stocking rates, particularly during drought periods, appeared to be the primary factors behind declining and (or) low legume percentages in most of the low-legume pastures. Only three sites had a soil pH less than 5.5 (range = 5.1 to 5.4) and phosphorus deficiency was the major problem at these sites. Pot studies: Two greenhouse studies were conducted
using a Kokee soil series (Medial, Ferrihydritic, Isothermic Alic Hapludands) to determine the growth of two pasture legumes, perennial peanut (Arachis pintoi) and barrel medic Medicago truncatula). The aluminum-toxic soil was amended with varying levels of dolomite to achieve four pH levels (4.5, 4.8, 5.5, and 6.6). Length of the longest root of perennial peanut was reduced significantly at the lowest pH level, whereas that of barrel medic was reduced at the two lowest pH levels. Similarly, shoot dry weight of perennial peanut was reduced significantly at the lowest pH level whereas that of barrel medic was reduced at the two lowest pH levels. Apparently, perennial peanut is more tolerant of an acid, Al-toxic soil than barrel medic. Genetic analysis: To select aluminum tolerant plants, a quick, efficient screening method is needed. Our objective was to compare three screening methods for Al tolerance in Medicago truncatula. In the first method, one genotype and five ecotypes of M.
truncatula seedlings were germinated for 24 h, then grown in an agarose medium, containing 5 levels of Al. Root length was measured at 24, 48 and 72 h after transfer and relative root lengths (RRL, defined as root length at a particular Al level divided by that at no Al) were calculated. In the second method, seedlings were exposed to Al at the same levels as the first experiment for 12 h, then transferred to Al-free agarose medium, and root regrowth measured. In the third method, seedlings were grown in an Al-toxic soil at pH 4.8 and in limed soil at pH 5.5. Ranking of Al-tolerance based on RRL of M. truncatula cultivars differed depending on the Al-toxicity screening system. Dissemination of information: A public presentation was made to ranchers at the Mealani Forage Field Day in September 2007. A scientific presentation was given at the national meeting of the American Society of Agronomy in November 2007 in New Orleans.
PARTICIPANTS: Susan C. Miyasaka, University of Hawaii - Manoa; Bruce W. Mathews, University of Hawaii - Hilo; Paul W. Singleton, University of Hawaii - Manoa; Yawadee Srimake, University of Hawaii - Manoa; Cheryn Clayton, University of Hawaii - Hilo; Diana Greenough, University of Hawaii - Manoa; Douglas R. Cooke, University of California, Davis.
TARGET AUDIENCES: American Society of Agronomy; Crop Science Society of America; ranchers in Hawaii
Impacts
Nitrogen is the most limiting plant nutrient for growth of many plants, including warm season grasses. Sustainability of warm season grass pastures is threatened by losses of nitrogen due to forage removal, erosion, leaching, or denitrification. Biological nitrogen fixation through legumes is a low-input method of providing nitrogen to warm season grasses in mixed pastures. In the tropics, acid soil pH and aluminum toxicity are two main factors that could limit biological nitrogen fixation. In our field surveys in Hawaii, it appears that drought stress and phosphorus deficiency are greater problems for persistence of pasture legumes than acid soils or aluminum toxicity. In our pot studies using an acid, aluminum-toxic soil, perennial peanut appears to be more acid soil tolerant than barrel medic. Previous studies have shown that perennial peanut is also tolerant to low phosphorus levels in soil. Seedlings of perennial peanut are being distributed to ranchers on the
island of Hawaii for planting in their pastures.
Publications
- Y. Srimake, S.C. Miyasaka. 2007. Comparison of Two Screening Methods for Aluminum Tolerance in Medicago Truncatula. In Annual meetings abstracts [CD-ROM]. ASA, CSSA, and SSSA, Madison, WI.
- Miyasaka, S.C., B. Mathews, P. Singleton, D. Greenough, and C. Clayton. 2007. Growth of Two Pasture Legumes in an Acid, Aluminum-toxic soil. Proc. Mealani Forage Field Day, Kamuela, HI.
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