Patrick M. Phipps

Enhancing Resistance to Sclerotinia minor in Peanut by Expressing a Barley Oxalate Oxidase Gene

Sclerotinia minor Jagger is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Based on evidence that oxalic acid is involved in the pathogenicity of many Sclerotinia species, our objectives were to recover transgenic peanut plants expressing an oxalic acid-degrading oxalate oxidase and to evaluate them for increased resistance to S. minor. Transformed plants were regenerated from embryogenic cultures of three Virginia peanut cultivars (Wilson, Perry, and NC-7). A colorimetric enzyme assay was used to screen for oxalate oxidase activity in leaf tissue. Candidate plants with a range of expression levels were chosen for further analysis. Integration of the transgene was confirmed by Southern-blot analysis, and gene expression was demonstrated in transformants by northern-blot analysis. A sensitive fluorescent enzyme assay was used to quantify expression levels for comparison to the colorimetric protocol. A detached leaflet assay tested whether transgene expression could limit lesion size resulting from direct application of oxalic acid. Lesion size was significantly reduced in transgenic plants compared to nontransformed controls (65%–89% reduction at high oxalic acid concentrations). A second bioassay examined lesion size after inoculation of leaflets with S. minor mycelia. Lesion size was reduced by 75% to 97% in transformed plants, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.

Sclerotinia blight resistance in Virginia-type peanut transformed with a barley oxalate oxidase gene.

Transgenic peanut lines expressing oxalate oxidase, a novel enzyme to peanut, were evaluated for resistance to Sclerotinia blight in naturally infested fields over a 5-year period. Area under the disease progress curve (AUDPC) for transgenic lines in single rows planted with seed from single-plant selections averaged 78, 83, and 90% lower than nontransgenic parents in 2004, 2005, and 2006, respectively. In addition, AUDPC in 14 transgenic lines planted with bulked seed in two-row plots averaged 81% lower compared with nontransgenic parents in 2005 and 86% lower in 16 transgenic lines in 2006. Six transgenic lines yielded 488 to 1,260 kg/ha greater than nontransgenic parents in 2005, and 10 lines yielded 537 to 2,490 kg/ha greater in 2006. Fluazinam (0.58 kg a.i./ha) fungicide sprays in 2008 and 2009 reduced AUDPC in transgenic and nontransgenic lines but AUDPC was lowest in transgenic lines. Without fluazinam, yields of transgenic lines averaged 1,133 to 1,578 kg/ha greater than nontransgenic lines in 2008 and 1,670 to 2,755 kg/ha greater in 2009. These results demonstrated that the insertion of barley oxalate oxidase in peanut conveyed a high level of resistance to Sclerotinia blight, and negated the need for costly fungicide sprays.

Assessment of Peanut Quality and Compositional Characteristics among Transgenic Sclerotinia Blight-Resistant and Non-Transgenic Susceptible Cultivars

This study presents the results of a comparison that includes an analysis of variance and a canonical discriminant analysis to determine compositional equivalence and similarity between transgenic, sclerotinia blight-resistant and non-transgenic, susceptible cultivars of peanut in 3 years of field trials. Three Virginia-type cultivars (NC 7, Wilson, and Perry) and their corresponding transgenic lines (N70, W73, and P39) with a barley oxalate oxidase gene were analyzed for differences in key mineral nutrients, fatty acid components, hay constituents, and grade characteristics. Results from both analyses demonstrated that transgenic lines were compositionally similar to their non-transgenic parent cultivar in all factors as well as market-grade characteristics and nutritional value. Transgenic lines expressing oxalate oxidase for resistance to sclerotinia blight were substantially equivalent to their non-transgenic parent cultivar in quality and compositional characteristics.

Comparative susceptibility of peanut genetically engineered for sclerotinia blight resistance to non-target peanut pathogens

Field trials were conducted from 2006 to 2008 at the Tidewater Agricultural Research and Extension Center (TAREC) in Suffolk, Virginia to determine whether Blight Blocker transgenic peanut lines showed possible increased or decreased susceptibility to common peanut pathogens. Disease susceptibility was evaluated for seven transgenic lines containing a barley oxalate oxidase gene and their corresponding parental cultivars (Perry, Wilson, NC 7). In addition to Sclerotinia blight, the peanut diseases evaluated included: i) early leaf spot caused by Cercospora arachidicola, ii) Cylindrocladium black rot caused by Cylindrocladium parasiticum, iii) southern stem rot caused by Sclerotium rolfsii, iv) tomato spotted wilt virus, and v) aflatoxin levels in seeds caused by Aspergillus flavus or A. parasiticus. Results demonstrated that the susceptibility of Blight Blocker transgenic lines to common peanut pathogens was similar to that of non-transgenic cultivars, while transgenic lines provided resistance to Sclerotinia blight caused by S. minor. Transgenic lines consistently provided high levels of resistance to S. minor in all three years, however, the barley oxalate oxidase had little or no effect on the disease susceptibility to other organisms on peanut. The results of this research should provide additional evidence needed to petition for deregulation of Blight Blocker peanut lines.

Comparison of Three Transgenic Peanut Lines with Their Parents for Agronomic and Physiological Characteristics

ABSTRACT Peanut (Arachis hypogea L.) is an important crop in the Virginia-Carolina (VC) region, but cool and wet falls may result in significant yield reductions due to Sclerotinia blight, caused by Sclerotinia minor (Jagger), a major disease in the region. Transgenic lines expressing a barley oxalate oxidase were previously shown to confer improved resistance to the disease. This research compared three blight resistant transgenic lines for oxalate oxidase, N70, P39, and W73 with their non-transgenic parents, NC 7, Perry, and Wilson, and high yielding check cultivars Bailey and CHAMPS. The objective was to ensure that the agronomic and physiological characteristics of the transformed lines were not negatively impacted by the transformation with oxalate oxidase before making recommendations for production. In 2009 and 2010, experimental plots were grown in two distinct fields for soil type and available water capacity for a total of four environments. The transgenic lines were statistically comparable wit...