Growing plants on oily, nutrient-poor soil using a native symbiotic fungus

Abstract

The roots of land plants associate with microbes, including fungal symbionts that can confer abiotic stress tolerance. Bitumen extraction following oil-sand surface mining in the Athabasca region of Alberta, Canada removes plant nutrients but leaves a petrochemical residue, making the coarse tailings (CT) hostile to both plants and microbes. We isolated an endophyte strain of the Ascomycete Trichoderma harzianum we call TSTh20-1 (hereafter, TSTh) from a dandelion that was naturally growing on CT. TSTh colonization allowed tomato, wheat, and remediation seed mixtures to germinate and their seedlings to flourish on CT without the use of fertilizer. Compared to control plants, TSTh increased germination speed, percent germination, and biomass accumulation. TSTh also improved plant water use efficiency and drought recovery. TSTh-colonized plants secreted twice the level of peroxidase into CT as did plants alone. Over two months, plants colonized with TSTh doubled the petrochemical mobilization from CT over plants alone, suggesting a peroxide-mediated mechanism for petrochemical degradation. TSTh grew on autoclaved CT, bitumen, and other petrochemicals as sole carbon sources. Further, TSTh is a micro-aerobe that could metabolize 13C-phenanthrene to 13CO2 in 0.5% oxygen. TSTh has excellent potential for contributing to revegetating and remediating petrochemical contamination.

Fig 1. Trichoderma harzianum TSTH20-1 (TSTh) culture and conidiation morphologies.

TSTh20 growing on A) 100% potato dextrose agar, B) synthetic nutrient-poor agar (SNA). C) TSTh conidiophore on 10% PDA. Bar represents 5 μm. Inset: TSTh conidiophores on 100% PDA. Bar represents 20 μm.

Fig 2. Plant growth is improved with Trichoderma harzianum TSTh20-1.

A) Two week old control (left) or TSTh-inoculated (right) tomato seedlings were transplanted to coarse tailings (CT), then given ultrapure water for an additional 2 weeks. B) Equal numbers of white clover and slender wheat grass seeds were scattered on CT (left, middle) or potting mix (right). Left- and right-hand pots were treated with NPK. TSTh-treated seeds on CT (middle) were given ultrapure water. Pots were photographed at 3 weeks.

Fig 3. TSTh improved drought recovery for tomato seedlings.

Six-week-old tomato seedlings were deprived of water for 2 d to induce wilt, then watered heavily. The graph shows leaf water content prior to and during recovery. Plants colonized with TSTh (ovals) wilted less and recovered faster than those without (triangles). The image shows recovery at 30 min. ‘With TSTh’ regression: y = 0.165 ln(x) + 88.9, r² = 0.9988. ‘Without TSTh’ regression: y = 0.016 x + 87.79, r² = 0.981.

Fig 4. Secretion of reactive oxygen species.

A) When grown on coarse tailings, both axenic plants and those colonized with TSTh secreted cell-free peroxidases into the soil. TSTh colonization was associated with a more than 2-fold increase in peroxidase secretion. B) TSTh was assayed with diaminobenzidine. As well as the H₂O₂ positive control, TSTh grown on 10W30 motor oil and on diesel oil were positive for ROS, whereas TSTh grown on glucose was not. Cultures were grown until sporulation (arrows).

Fig 5. Trichoderma harzianum TSTh20-1 (TSTh) growing for 14 d (A, B) or 7 d (C, D) on diesel and 10W30 oil, respectively.

TSTh in A, B had been growing for 5 weeks on petrochemicals, whereas C, D had been growing for 16 weeks. Box in A indicates tiny spore aggregates (ca 10⁵ spores) on diesel. Box in B indicates a larger spore aggregate (ca 5x10⁶) on 10W30. Growth and sporulation was much improved in Fig 5C, D.

Fig 6. Plants colonized with Trichoderma harzianum TSTh20-1 (TSTh) mobilized petrochemicals across a broad size range.

Plants colonized with TSTh (area under the green [top] trace) had twice the petrochemical mobilization as plants alone (area under blue [middle] trace), and tailing sand (TS; area under red [bottom] trace). Petrochemicals larger than C14 (arrow) are not volatile.

Fig 7. Trichoderma harzianum TSTh20-1 (TSTh) metabolized ¹³C-phenanthrene to ¹³CO₂ under micro-aerobic conditions.

After two weeks growth and for more than nine weeks, TSTh generated several-fold more ¹³CO₂ than the media-only control treatment (solid line) or that plus two standard errors (dashed line).