Plant-growth promoting fungi from coastal herbs

Background:

Tetragonia tetragonioides in English is called New Zealand spinach, because it can be cultivated and serve as leaf vegetable. In nature, it occurs on sand beaches and adapted to the major stress factor at marine sand coasts, namely sand burial by wind. This plant belongs to the exceptional plants that lack mycorrhiza. We hypothesize that the protective function of mycorrhiza fungi is taken over by endophytic fungi, i.e., fungi that grow within living plants without causing disease. This symbiosis is similar to the bacteria in the human gut, but the species and detailed roles of the microbes in both niches are poorly known and have high potential for application in plant or human health.

Collaboration:

The internship is envisaged for one undergraduate student for 2 months between June and November 2026. The grant of NT$30,000 per person per month is fully envisaged for the student’ s living expenses and study fees.

Experiment:

We will go to collect fresh samples of the plant at the sea coasts at different places in Taiwan, and isolate and identify endophytic fungi from the roots by the protocols we have already adopted for this plant from our previous research on endophytic fungi (Yeh & Kirschner 2023, 2025). The methods for axenic germination of T. tetragonioides have been developed by our master student who will do growth experiments with this plant with and without endophytic fungi. In the internship we want to transfer this experiment to seedlings of rice and tomato as done by Pham et al. (2019).

In addition to comparing the growth performance of the seedlings with and without fungi, we also want to compare the growth under different salt stress regimes, with 0M, 0.2M, 0.5M, 1M, 1.5M, and 2.5M salt concentrations in the growth substrate. Sea water has an average NaCl concentration of 3.5% roughly corresponding to 0.5M; 2.5M is the approx. concentration at which the halotolerant T. tetragonioides can still survive.

Plant-fungus co-cultivation:

Seeds are sterilized by using 5 % (v/v) sodium hypochlorite solution with a few drops of Tween 20 for 10 min, and washed four times with sterilized water. The seeds (or rhizome buds) are placed onto Murashige-Skoog (MS) medium supplemented with 1.5% sucrose and 0.8% agar. After 2 days, two healthy seedlings (or germinating buds) are placed on agar containing 1.5 % (v/v) phosphate-citric buffer (pH 5.5) at 2 cm apart from the margin of the plate. The test fungus is inoculated at 1 cm apart from the opposite margin of the plates for two weeks in a growth chamber for an additional 5 days with a 16 h:8 h light-dark cycle at 25 oC. The amount of root formation, length of shoots, length of primary roots, and chlorophyll contents of negative control and fungus-cocultivated seedlings are measured after 5 days of incubation. The hair roots of seedlings are viewed under a microscope and photographed. From their images, area and pixel value statistics are calculated with ImageJ software. Chlorophyll content is extracted. Chlorophyll a and b contents are measured at wavelengths of 662 and 647 nm, respectively. Absorbance is recorded using an UV-visible spectrophotometer. Total chlorophyll is to be expressed as the content (mg) per plant and plant weight. All the tests are repeated three times.

Test for salt tolerance in fungi and fungus-plant co-cultivation:

MMN (Melin-Norkrans) medium is used for pre- growing seedlings and test fungi and as control. For testing salt tolerance, in this medium the above-mentioned concentrations of NaC1; are applied. Fungi are inoculated onto the center of the agar plates for monitoring the colony growth at defined intervals. A similar method is applied for measuring the dry weight of mycelium biomass after incubation in liquid MMN medium. The co-cultivation with seedlings and fungi is done as mentioned above. The growth of seedlings on these media without fungus is also tested and quantitatively assessed as mentioned above.

Pham, M.T., Huang, C.-M. & Kirschner, R. 2019. The plant‐growth promoting potential of the mesophilic wood‐rot mushroom Pleurotus pulmonarius. Journal of Applied Microbiology 127(4): 1157-1171.

Yeh, Y.-H., & Kirschner, R. 2025. Study of endophytic fungi of Ipomoea pes-caprae reveals the superiority of in situ plant conservation over ex situ conservation from a mycological view. Scientific Reports 15:2040, 10 pp. https://doi.org/10.1038/s41598-025-86508-8