In hot weather, trees “sweat” to cool down by releasing water from their leaves or needles and then replacing it with water from the soil. But during long periods of high temperatures, trees may release more water than they can replace. To strengthen forests against heat stress and other environmental hazards, scientists have suggested adding fungi to the soil.
Soil fungi living on tree roots are called ectomycorrhizal fungi. They help trees absorb water by expanding their root networks, and trees help the fungi by providing them with sugars for energy. This beneficial partnership is known as symbiosis. Scientists at Fujian Agriculture and Forestry University in China tested whether symbiosis with ectomycorrhizal fungi could help the pine trees Pinus massoniana survive warmer temperatures.
For their experiment, the researchers grew pine seedlings with and without ectomycorrhizal fungi in a greenhouse under different temperature conditions. First, they grew fungi called Cenococcum geophilum for 45 days on a gel-like substance called agar. At the same time, they grew the pine seedlings for 30 days, then transferred them into pots with natural soil.
After 15 days in the pots, the researchers spread the gel-like agar containing the fungi over small 1 x 1 centimeter (cm) areas of the pine root tips to introduce the fungi to the seedlings, a process called inoculation. They assumed that the fungal network would expand from the starting area to colonize new root tips as the seedlings grew. The team’s inoculations included 6 different C. geophilum strains and 1 fungi-free control. To simulate heat stress, the researchers replicated each of the 7 inoculations at 25°C (77°F) and 35°C (95°F). They repeated each inoculation condition in 3 pots with 3 seedlings, for a total of 126 seedlings.
After 60 days, the researchers harvested the pine seedlings and used a microscope to count how many of their root tips had fungi. They divided the number of root tips with fungi by the total number of root tips to calculate a colonization rate. They considered seedlings with a colonization rate of 80% or higher to have successfully formed a symbiotic relationship with the fungi. Then, the researchers analyzed seedlings with an 80% colonization rate to investigate their symbiotic partnerships.
The team observed that pine seedlings grown under heat stress at 35°C were 24% to 59% smaller than those grown at 25°C, and their needles were yellow instead of a healthy green. However, the seedlings that formed a symbiotic relationship with the ectomycorrhizal fungi were greener and 80% heavier than the control seedlings.
Investigating the trees further, the team measured 3 chemical compounds related to plant stress, including malondialdehyde (MDA), proline, and nitric oxide. Plants release MDA when damaged, so high MDA levels mean a plant is struggling. In contrast, plants release proline and nitric oxide to repair cells or hold water, so high amounts of these compounds mean a plant is healing.
To measure MDA and proline levels in the pine needles, the researchers mixed them with different acids that change color depending on the sample’s MDA or proline content. To measure nitric oxide, the researchers mixed the needles with a protein that converts the compound into a chemical that changes color. They found that inoculated seedlings at 35°C had about 30% to 40% less MDA, 15% to 30% more proline, and 150% more nitric oxide in their roots than the control seedlings.
Finally, the researchers measured changes in gene activity in the inoculated pine seedlings. They identified active genes by analyzing RNA molecules from the pine seedling roots with a process called RNA sequencing or transcriptomics. They found that inoculating the seedlings with ectomycorrhizal fungi activated more genes related to root strength, water retention, cell defense, and general plant protection than were activated in the control seedlings.
By combining chemical analyses with growth measurements, the team concluded that a symbiotic partnership with the ectomycorrhizal fungi helped the pine seedlings survive heat stress. They demonstrated that through prolonged heat, inoculated seedlings maintained greener needles and lower levels of stress chemicals. They suggested that ectomycorrhizal fungi could be an ecological tool to help trees stay hydrated in a warming world. They recommended that future researchers conduct field trials with inoculated trees to translate these findings into forest management strategies.
