A Symbiotic Approach to Generating Stress Tolerant Crops.

Studies were undertaken to determine if fungal endophytes from plants in stressful habitats could be commercialized to generate climate resilient crop plants. Fungal endophytes were isolated from weedy rice plants and grasses from South Korea and the USA, respectively. Endophytes (Curvularia brachyspora and Fusarium asiaticum) from weedy rice plants from high salt or drought stressed habitats in South Korea conferred salt and drought stress tolerance to weedy rice and commercial varieties reflective of the habitats from which they were isolated. Fungal endophytes isolated from grasses in arid habitats of the USA were identified as Trichoderma harzianum and conferred drought and heat stress tolerance to monocots and eudicots. Two T. harzianum isolates were exposed to UV mutagenesis to derive strains resistant to fungicides in seed treatment plant protection packages. Three strains that collectively had resistance to commonly used fungicides were used for field testing. The three-strain mixture (ThSM3a) increased crop yields proportionally to the level of stress plants experienced with average yields up to 52% under high and 3-5% in low stress conditions. This study demonstrates fungal endophytes can be developed as viable commercial tools for rapidly generating climate resilient crops to enhance agricultural sustainability.

Increased fitness of rice plants to abiotic stress via habitat adapted symbiosis: a strategy for mitigating impacts of climate change.

Climate change and catastrophic events have contributed to rice shortages in several regions due to decreased water availability and soil salinization. Although not adapted to salt or drought stress, two commercial rice varieties achieved tolerance to these stresses by colonizing them with Class 2 fungal endophytes isolated from plants growing across moisture and salinity gradients.Plant growth and development, water usage, ROS sensitivity and osmolytes were measured with and without stress under controlled conditions.The endophytes conferred salt, drought and cold tolerance to growth chamber and greenhouse grown plants. Endophytes reduced water consumption by 20-30% and increased growth rate, reproductive yield, and biomass of greenhouse grown plants. In the absence of stress, there was no apparent cost of the endophytes to plants, however, endophyte colonization decreased from 100% at planting to 65% compared to greenhouse plants grown under continual stress (maintained 100% colonization).These findings indicate that rice plants can exhibit enhanced stress tolerance via symbiosis with Class 2 endophytes, and suggest that symbiotic technology may be useful in mitigating impacts of climate change on other crops and expanding agricultural production onto marginal lands.

Symbiotic regulation of plant growth, development and reproduction.

The growth and development of rice (Oryzae sativa) seedlings was shown to be regulated epigenetically by a fungal endophyte. In contrast to un-inoculated (nonsymbiotic) plants, endophyte colonized (symbiotic) plants preferentially allocated resources into root growth until root hairs were well established. During that time symbiotic roots expanded at five times the rate observed in nonsymbiotic plants. Endophytes also influenced sexual reproduction of mature big sagebrush (Artemisia tridentata) plants. Two spatially distinct big sagebrush subspecies and their hybrids were symbiotic with unique fungal endophytes, despite being separated by only 380 m distance and 60 m elevation. A double reciprocal transplant experiment of parental and hybrid plants, and soils across the hybrid zone showed that fungal endophytes interact with the soils and different plant genotypes to confer enhanced plant reproduction in soil native to the endophyte and reduced reproduction in soil alien to the endophyte. Moreover, the most prevalent endophyte of the hybrid zone reduced the fitness of both parental subspecies. Because these endophytes are passed to the next generation of plants on seed coats, this interaction provides a selective advantage, habitat specificity, and the means of restricting gene flow, thereby making the hybrid zone stable, narrow and potentially leading to speciation.

Phytolacca americana from contaminated and noncontaminated soils of South Korea: effects of elevated temperature, CO(2) and simulated acid rain on plant growth response.

Chemical analyses performed on the invasive weed Phytolacca americana (pokeweed) growing in industrially contaminated (Ulsan) and noncontaminated (Suwon) sites in South Korea indicated that the levels of phenolic compounds and various elements that include some heavy metals (Al, As, B, Cd, Co, Cu, Fe, Mn, Ni, Pb, and Zn) were statistically higher in Ulsan soils compared to Suwon soils with Al being the highest (>1,116 mg/l compared to 432 mg/l). Analysis of metals and nutrients (K, Na, Ca, Mg, Cl, NH(4), N, P, S) in plant tissues indicated that accumulation occurred dominantly in plant leaves with Al levels being 33.8 times higher in Ulsan plants (PaU) compared to Suwon plants (PaS). The ability of PaU and PaS to tolerate stress was evaluated under controlled conditions by varying atmospheric CO(2) and temperature and soil pH. When grown in pH 6.4 soils, the highest growth rate of PaU and PaS plants occurred at elevated (30 degrees C) and non-elevated (25 degrees C) temperatures, respectively. Both PaU and PaS plants showed the highest and lowest growth rates when exposed to atmospheric CO(2) levels of 360 and 650 ppm, respectively. The impact of soil pH (2-6.4) on seed germination rates, plant growth, chlorophyll content, and the accumulation of phenolics were measured to assess the effects of industrial pollution and global-warming-related stresses on plants. The highest seed germination rate and chlorophyll content occurred at pH 2.0 for both PaU and PaS plants. Increased pH from 2-5 correlated to increased phenolic compounds and decreased chlorophyll content. However, at pH 6.4, a marked decrease in phenolic compounds, was observed and chlorophyll content increased. These results suggest that although plants from Ulsan and Suwon sites are the same species, they differ in the ability to deal with various stresses.

Stress tolerance in plants via habitat-adapted symbiosis.

We demonstrate that native grass species from coastal and geothermal habitats require symbiotic fungal endophytes for salt and heat tolerance, respectively. Symbiotically conferred stress tolerance is a habitat-specific phenomenon with geothermal endophytes conferring heat but not salt tolerance, and coastal endophytes conferring salt but not heat tolerance. The same fungal species isolated from plants in habitats devoid of salt or heat stress did not confer these stress tolerances. Moreover, fungal endophytes from agricultural crops conferred disease resistance and not salt or heat tolerance. We define habitat-specific, symbiotically-conferred stress tolerance as habitat-adapted symbiosis and hypothesize that it is responsible for the establishment of plants in high-stress habitats. The agricultural, coastal and geothermal plant endophytes also colonized tomato (a model eudicot) and conferred disease, salt and heat tolerance, respectively. In addition, the coastal plant endophyte colonized rice (a model monocot) and conferred salt tolerance. These endophytes have a broad host range encompassing both monocots and eudicots. Interestingly, the endophytes also conferred drought tolerance to plants regardless of the habitat of origin. Abiotic stress tolerance correlated either with a decrease in water consumption or reactive oxygen sensitivity/generation but not to increased osmolyte production. The ability of fungal endophytes to confer stress tolerance to plants may provide a novel strategy for mitigating the impacts of global climate change on agricultural and native plant communities.

Phytotoxicity of Phytolacca americana leaf extracts on the growth, and physiological response of Cassia mimosoides.

We examined the allelochemical effects of control soil, native soil (treated soil), and leaf extracts of Phytolacca americana (pokeweed) on the germination rate and seedling growth of Cassia mimosoides var. nomame. We also studied the resulting changes in root-tip ultrastructure and peroxidase isozyme biochemistry. P. americana leaf extract inhibited seed germination, seedling growth, and biomass when compared to control and treated soil. Root and shoot growth in treated soil was stimulated relative to control soil, but root growth was inhibited by 50% in the leaf extract treatment. Biomass of C. mimosoides seedlings grown on leaf extract was reduced sevenfold when compared to the control seedlings. The amounts of total phenolic compounds in the leaf extract, treated soil, and control soil were 0.77, 0.14, and 0.03 mg l(-1), respectively. The root tips of C. mimsoides treated with leaf extracts of P. americana showed amyloplasts and large central vacuoles with electron-dense deposits inside them when compared to control root tips. The activity of guaiacol peroxidase (GuPOX) in whole plant, roots, and shoots of C. mimosoides increased as leaf extract increased; maximum activity was observed in extract concentrations of 75% and higher. Root GuPOX activity was three times higher than in shoots. Therefore, we conclude that inhibition of C. mimosoides growth is related to the phenolic compounds in the P. americana leaf extract and the ultrastructure changes in root-tip cells and increased GuPOX activity is a response to these allelochemicals.

Phytotoxic effects and chemical analysis of leaf extracts from three Phytolaccaceae species in South Korea.

We analyzed phenolic compounds and other elements in leaf extracts and compared morphology of three species of the Phytolaccaceae family found in South Korea. To test allelochemical effects of the three Phytolacca species, we also examined seed germination and dry weight of seedlings of Lactuca indica and Sonchus oleraceus treated with leaf extracts. The concentrations of total phenolic compounds were exotic Phytolacca esculenta (3.9 mg/l), native Phytolacca insularis (4.4 mg/l), and exotic Phytolacca americana (10.2 mg/l). There was no significant difference in concentrations between P. esculenta and P. insularis, but the concentration of total phenolics in P. americana was two times higher than either P. esculenta or P. insularis. Analysis of aqueous extracts by HPLC showed seven phenolic compounds (gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, m-hydroxybenzoic acid, p-coumaric acid, and cinnamic acid). Total phenolics in P. americana were eight to 16 times higher than either P. esculenta or P. insularis, respectively. P. americana inhibited seed germination and dry weight of the two assay species. The phytotoxic effects of the two Phytolacca species were different, despite the fact that P. esculenta and P. insularis had similar levels of total phenolic compounds. We also found that P. americana had invaded Ullung Island, which suggested that P. americana had excellent adaptability to the environment. The three species of Phytolaccaceae in South Korea can be distinguished by their different allelopathic potentials and morphologies.