The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.

Smut disease caused by the fungal pathogen Thecaphora frezii Carranza & Lindquist is threatening the peanut production in Argentina. Fungicides commonly used in the peanut crop have shown little or no effect controlling the disease, making it a priority to obtain peanut varieties resistant to smut. In this study, recombinant inbred lines (RILs) were developed from three crosses between three susceptible peanut elite cultivars (Arachis hypogaea L. subsp. hypogaea) and two resistant landraces (Arachis hypogaea L. subsp. fastigiata Waldron). Parents and RILs were evaluated under high inoculum pressure (12000 teliospores g-1 of soil) over three years. Disease resistance parameters showed a broad range of variation with incidence mean values ranging from 1.0 to 35.0% and disease severity index ranging from 0.01 to 0.30. Average heritability (h2) estimates of 0.61 to 0.73 indicated that resistance in the RILs was heritable, with several lines (4 to 7 from each cross) showing a high degree of resistance and stability over three years. Evidence of genetic transfer between genetically distinguishable germplasm (introgression in a broad sense) was further supported by simple-sequence repeats (SSRs) and Insertion/Deletion (InDel) marker genotyping. This is the first report of smut genetic resistance identified in peanut landraces and its introgression into elite peanut cultivars.

Aspergillus flavus is a soil fungus that commonly invades peanut seeds and often produces carcinogenic aflatoxins. Under favorable conditions, the fungus-challenged peanut plant produces and accumulates resveratrol and its prenylated derivatives in response to such an invasion. These prenylated stilbenoids are considered peanut antifungal phytoalexins. However, the mechanism of peanut−fungus interaction has not been sufficiently studied. We used pure peanut stilbenoids arachidin-1, arachidin-3, and chiricanine A to study their effects on the viability of and metabolite production by several important toxigenic Aspergillus species. Significant reduction or virtually complete suppression of aflatoxin production was revealed in feeding experiments in A. flavus, Aspergillus parasiticus, and Aspergillus nomius. Changes in morphology, spore germination, and growth rate were observed in A. flavus exposed to the selected peanut stilbenoids. Elucidation of the mechanism of aflatoxin suppression by peanut stilbenoids could provide strategies for preventing plant invasion by the fungi that produce aflatoxins.

No abstract

Aspergillus flavus and A. parasiticus fungi produce carcinogenic mycotoxins in peanut seeds, causing considerable impact on both human health and the economy. Here, we report nine genome sequences of Aspergillus spp., isolated from Georgia peanut seeds in 2014. The information obtained will lead to further biodiversity studies that are essential for developing control strategies.

New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double-stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell-to-cell and over long distances within the plant, and between host plants and parasites. In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicer-substrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem-loop primers and RT-PCR for DsiRNA detection. The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment. This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non-transformative option for mycotoxin contamination control.

The human Bcl-xL gene was transformed into peanut cultivar Georgia Green via microprojectile bombardment. Following selection on hygromycin-containing medium and regeneration, eighty hygromycin-resistant callus clusters were recovered. Southern blot analysis of ten fertile lines revealed multiple insertions of the Bcl-xL transgene in most lines. Western blot analysis of primary plants and T1 progenies demonstrated detectable levels of Bcl-xL expression in four transgenic lines. We could not detect Bcl-xL protein in other tested lines even though transcripts were identified by RT-PCR and northern blot. Three of the western-positive transgenic lines either were sterile or the progenies lost the expressive copy of Bcl-xL. Only T1 progenies from line BX25-4-2a-19 continued to express an intermediate level of Bcl-xL. This line demonstrated paraquat tolerance at the 5 microM level. Tolerance to salt of T1 and T2 seeds from seven other transgenic lines also was tested, but no tolerance was found in these lines. A high level of Bcl-xL transgene expression may be deleterious to plant growth and development even though the gene may confer tolerance to other abiotic and biotic stresses such as drought and pathogens.

A nonheme chloroperoxidase gene (cpo-p) from Pseudomonas pyrrocinia, a growth inhibitor of mycotoxin-producing fungi, was introduced into peanut via particle bombardment. The expression of the cpo-p gene is predicted to increase pathogen defense in peanut. Embryogenic peanut tissues were bombarded with gold particles coated with plasmid pRT66 carrying the cpo-p and hygromycin phosphotransferase (hph) genes, under the control of a double CaMV 35S and a single CaMV 35S promoter, respectively. Selection for hygromycinresistant somatic embryos was performed on a liquid medium containing 10-20 mg/L hygromycin 3-4 days after bombardment. The integration and expression of the cpo-p gene was confirmed by Southern, Northern and Western blot analyses. In vitro bioassay using crude protein extracts from transgenic T0, T1, and T4 plants showed inhibition of Aspergillus flavus hyphal growth, which could translate to a reduction in aflatoxin contamination of peanut seed.