Zareen Khan

Enhanced phytoremediation of volatile environmental pollutants with transgenic trees.

Small, volatile hydrocarbons, including trichloroethylene, vinyl chloride, carbon tetrachloride, benzene, and chloroform, are common environmental pollutants that pose serious health effects. We have developed transgenic poplar (Populus tremula × Populus alba) plants with greatly increased rates of metabolism and removal of these pollutants through the overexpression of cytochrome P450 2E1, a key enzyme in the metabolism of a variety of halogenated compounds. The transgenic poplar plants exhibited increased removal rates of these pollutants from hydroponic solution. When the plants were exposed to gaseous trichloroethylene, chloroform, and benzene, they also demonstrated superior removal of the pollutants from the air. In view of their large size and extensive root systems, these transgenic poplars may provide the means to effectively remediate sites contaminated with a variety of pollutants at much faster rates and at lower costs than can be achieved with current conventional techniques.

Diazotrophic endophytes of native black cottonwood and willow

Poplar and willow are economically-important, fast-growing tree species with the ability to colonize nutrient-poor environments. To initiate a study on the possible contribution of endophytes to this ability, we isolated bacteria from within surface-sterilized stems of native poplar (Populus trichocarpa) and willow (Salix sitchensis) in a riparian system in western Washington state. Several of the isolates grew well in nitrogen-limited medium. The presence ofnifH, a gene encoding one of the subunits of nitrogenase, was confirmed in several of the isolates including species ofBurkholderia, Rahnella, Sphingomonas, andAcinetobacter. Nitrogenase activity (as measured by the acetylene reduction assay) was also confirmed in some of the isolates. The presence of these diazotrophic microorganisms may help explain the ability of these pioneering tree species to grow under nitrogen limitation.

Characterization of bacterial endophytes of sweet potato plants.

Endophytic bacteria associated with sweet potato plants (Ipomoea batatas (L.) Lam.) were isolated, identified and tested for their ability to fix nitrogen, produce indole acetic acid (IAA), and exhibit stress tolerance. Eleven different strains belonging to the genera, Enterobacter, Rahnella, Rhodanobacter, Pseudomonas, Stenotrophomonas, Xanthomonas and Phyllobacterium, were identified. Four strains were shown to produce IAA (a plant growth hormone) and one strain showed the ability to grow in nitrogen free medium and had the nitrogenase subunit gene, nifH. To determine if IAA production by the endophytes had any role in protecting the cells against adverse conditions, different stress tests were conducted. The IAA producer grew well in the presence of some antibiotics, UV and cold treatments but the response to pH, osmotic shock, thermal and oxidative treatments was the same for both the IAA producer and the no IAA producer. To determine if IAA produced by the strains was biologically relevant to plants, cuttings of poplar were inoculated with the highest IAA producing strain. The inoculated cuttings produced roots sooner and grew more rapidly than uninoculated cuttings. These studies indicate that endophytes of sweet potato plants are beneficial to plant growth.

Degradation, Phytoprotection and Phytoremediation of Phenanthrene by Endophyte Pseudomonas putida, PD1

Endophytes have been isolated from a large diversity of plants and have been shown to enhance the remediation efficiency of plants, but little information is available on the influence of endophytic bacteria on phytoremediation of widespread environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs). In this study we selected a naturally occurring endophyte for its combined ability to colonize plant roots and degrade phenanthrene in vitro. Inoculation of two different willow clones and a grass with Pseudomonas putida PD1 was found to promote root and shoot growth and protect the plants against the phytotoxic effects of phenanthrene. There was an additional 25-40% removal of phenanthrene from soil by the willow and grasses, respectively inoculated with PD1 when compared to the uninoculated controls. Fluorescent microscopy using fluorescent protein tagging of PD1 confirmed the presence of bacteria inside the root tissue. Inoculation of willows with PD1 consistently improved the growth and health when grown in hydroponic systems with high concentrations of phenanthrene. To our knowledge this is the first time that the inoculation of willow plants has been shown to improve the degradation of PAHs and improve the health of the host plants, demonstrating the potential wide benefit to the field of natural endophyte-assisted phytoremediation.

Biodegradation of Trichloroethylene by an Endophyte of Hybrid Poplar

ABSTRACT We isolated and characterized a novel endophyte from hybrid poplar. This unique endophyte, identified as Enterobacter sp. strain PDN3, showed high tolerance to trichloroethylene (TCE). Without the addition of inducers, such as toluene or phenol, PDN3 rapidly reduced TCE levels in medium from 72.4 μM to 30.1 μM in 24 h with a concurrent release of 127 μM chloride ion, and nearly 80% of TCE (55.3 μM) was dechlorinated by PDN3 in 5 days with 166 μM chloride ion production, suggesting TCE degradation.

Bacterial and Yeast Endophytes from Poplar and Willow Promote Growth in Crop Plants and Grasses

Endophytic associations with plants have a beneficial effect for many different plant species with some of them being host specific. Several endophytes isolated from poplar and willow were screened for their effects on commercially important crops including corn, tomato, pepper, squash, sunflower, and grasses. Most of these endophytes produce growth hormones such as indoleacetic acid (IAA) and have the nitrogenase gene required for nitrogen fixation. The effects of these isolates on plant growth and yield were evaluated under greenhouse conditions. We found that inoculated plants not only had better viability and earlier flowering and fruiting, they also had increased plant growth and fruit yields when grown in nitrogen-limited soil. In a particular variety of perennial rye grass, the endophytes increased the total nitrogen content of the plants, indicative of nitrogen fixation, in addition to promoting plant growth. The use of specific endophytes may be preferable to the use of chemical fertilizers because of the monetary and environmental costs, contributing to more sustainable agricultural systems.

Variable Nitrogen Fixation in Wild Populus

The microbiome of plants is diverse, and like that of animals, is important for overall health and nutrient acquisition. In legumes and actinorhizal plants, a portion of essential nitrogen (N) is obtained through symbiosis with nodule-inhabiting, N2-fixing microorganisms. However, a variety of non-nodulating plant species can also thrive in natural, low-N settings. Some of these species may rely on endophytes, microorganisms that live within plants, to fix N2 gas into usable forms. Here we report the first direct evidence of N2 fixation in the early successional wild tree, Populus trichocarpa, a non-leguminous tree, from its native riparian habitat. In order to measure N2 fixation, surface-sterilized cuttings of wild poplar were assayed using both 15N2 incorporation and the commonly used acetylene reduction assay. The 15N label was incorporated at high levels in a subset of cuttings, suggesting a high level of N-fixation. Similarly, acetylene was reduced to ethylene in some samples. The microbiota of the cuttings was highly variable, both in numbers of cultured bacteria and in genetic diversity. Our results indicated that associative N2-fixation occurred within wild poplar and that a non-uniformity in the distribution of endophytic bacteria may explain the variability in N-fixation activity. These results point to the need for molecular studies to decipher the required microbial consortia and conditions for effective endophytic N2-fixation in trees.

Genome sequence of the plant growth promoting endophytic yeast Rhodotorula graminis WP1

Author(s): Firrincieli, Andrea; Otillar, Robert; Salamov, Asaf; Schmutz, Jeremy; Khan, Zareen; Redman, Regina S; Fleck, Neil D; Lindquist, Erika; Grigoriev, Igor V; Doty, Sharon L

Comparison of Trichloroethylene Toxicity, Removal, and Degradation by Varieties of Populus and Salix for Improved Phytoremediation

Trichloroethylene (TCE), a chlorinated organic solvent, is one of the three most common and widespread groundwater contaminants worldwide. Exposure to this contaminant has been linked to liver and kidney toxicity and cancer in humans and animals [1]. The Salicaceae family, which includes poplar and willow trees, has been very successful in phytoremediation efforts involving chlorinated solvents including TCE. Previous phytoremediation studies investigated removal of this contaminant using one or two genotypes, but a screening of a large number of genotypes in this family of plants had not yet been conducted. In this study, nine Populus and twelve Salix varieties were chosen for their previous success in phytoremediation efforts or local native significance, and experiments were conducted to compare toxicity, uptake, and degradation of TCE. Results from the toxicity screening demonstrated that hybrid poplar clones Crandon (Populus alba x grandidentata), Nisqually-1 (P. trichocarpa), D.Pa (P. alba) and willow clone S365 (Salix discolor) had the highest mass gain and best health at the highest TCE concentration. Percent removal of TCE in one week ranged from 19.2 to 44.9 with Crandon, P. deltoides hybrid 91x0403, poplar clone H11-11 (P. trichocarpa x deltoides), Salix sitchensis clone B, and Nisqually-1 all removing more than 40 percent of the TCE from hydroponic solution. In terms of TCE degradation, S. sitchensis clone B, poplar hybrid 91x0403, and hybrid poplar H11-11 had the most TCE metabolite, trichloroethanol, per gram of fresh plant weight, with the best performer, S. sitchensis clone B, having eight times the amount than the lowest genotype tested. These results indicated that there are significant differences in the ability of poplar and willow genotypes to remove and degrade TCE, and that many different genotypes could be successfully utilized for phytoremediation of TCE. This data set could be applied toward choosing the appropriate genotypes for the phytoremediation of TCE in a given location and climate.

Enhanced Degradation of TCE on a Superfund Site Using Endophyte-Assisted Poplar Tree Phytoremediation

Trichloroethylene (TCE) is a widespread environmental pollutant common in groundwater plumes associated with industrial manufacturing areas. We had previously isolated and characterized a natural bacterial endophyte, Enterobacter sp. strain PDN3, of poplar trees, that rapidly metabolizes TCE, releasing chloride ion. We now report findings from a successful three-year field trial of endophyte-assisted phytoremediation on the Middlefield-Ellis-Whisman Superfund Study Area TCE plume in the Silicon Valley of California. The inoculated poplar trees exhibited increased growth and reduced TCE phytotoxic effects with a 32% increase in trunk diameter compared to mock-inoculated control poplar trees. The inoculated trees excreted 50% more chloride ion into the rhizosphere, indicative of increased TCE metabolism in planta. Data from tree core analysis of the tree tissues provided further supporting evidence of the enhanced rate of degradation of the chlorinated solvents in the inoculated trees. Test well groundwater analyses demonstrated a marked decrease in concentration of TCE and its derivatives from the tree-associated groundwater plume. The concentration of TCE decreased from 300 μg/L upstream of the planted area to less than 5 μg/L downstream of the planted area. TCE derivatives were similarly removed with cis-1,2-dichloroethene decreasing from 160 μg/L to less than 5 μg/L and trans-1,2-dichloroethene decreasing from 3.1 μg/L to less than 0.5 μg/L downstream of the planted trees. 1,1-dichloroethene and vinyl chloride both decreased from 6.8 and 0.77 μg/L, respectively, to below the reporting limit of 0.5 μg/L providing strong evidence of the ability of the endophytic inoculated trees to effectively remove TCE from affected groundwater. The combination of native pollutant-degrading endophytic bacteria and fast-growing poplar tree systems offers a readily deployable, cost-effective approach for the degradation of TCE, and may help mitigate potential transfer up the food chain, volatilization to the atmosphere, as well as direct phytotoxic impacts to plants used in this type of phytoremediation.