Belowground heterogeneity and fungal variances

Heterogeneity within the soil (physical and chemical variances) is another important factor that may influence ECM fungal diversity and richness, and may be considered while studying below-ground microbial diversity; fungi inclusively. For instance, soil parameters including organic matter, pH, phosphate, ammonium and C:N); which have been linked with ECM fungal distribution (Natel and Neumann, 1992; Coince et al., 2013), showed strong spatial patterns to a scale less than 1 m in the soil (Jackson and Caldwell, 1993). In relation to soil physicochemical heterogeneity, a study on belowground bacterial distribution was conducted, and revealed spatial dependency of different bacterial taxa on a scale range of 2 to 4 mm (Grundmann and Debouzie, 2000). This may not be relatable to ECM fungi given that they integrate a larger soil volume due to their mycelial nature, and may show no or minimum disparities in their distribution in the given range. However, sampling effort with a reasonable distance between samples is desirable to address issues like soil heterogeneity to have a fair picture of ECM communities as representatives of a given area. Therefore, in this study, appropriate sampling efforts have been established while keeping in view the interconnected issues; for instance, sampling size, inter-sample distances, and soil parameters that may influence the estimation of ECM species richness.

Soil samples were taken via using a “soil borer” that could extract soil samples at a depth of 30 cm. In order to address soil vertical heterogeneity, the top layer of the soil (approximately 0 – 10 cm) was separated from the mineral soil/lower layer (10 – 30 cm) (Coince et al., 2013), to identify mycorrhizal taxa that have more affinity for either soil layer. Besides, mycorrhizal fungal diversity may be proportional to the distribution of roots below ground, which is possibly not predictable. Therefore, four duplicate soil samples were taken from each tree while maintaining a 1 m distance from the trunk. All duplicate samples (soil cores) were taken at equal distances from each other, but in a way that could represent the root distribution belowground (Fig. 2). Samples taken from each tree ( 4 soil cores) were combined to make a composite sample for each tree. In total, 200 composite samples (100 trees x 2 soil layers) were prepared out of 800 soil samples (4 cores x 100 trees x 2 soil layers) taken from 100 lodgepole trees.

After sampling handling and fungal identification

After sampling, the samples were kept on ice in a cooler on-site, later at –20 ^oC until processed. Soil analysis of subsamples were carried out in the laboratory to determine soil parameters including soil organic matter content, soil pH, soil texture, total nitrogen, and phosphorus contents. Mycorrhizal fungal communities in soil samples were identified using molecular techniques. Ideally, it is advised to use both molecular and morphological techniques to conduct species-level identification. However, speaking of morphology-based identification, some obvious and well-determined differences between fungal species, for example, shape and color could easily be identified by the untrained eye, yet this may not be enough to recognize variances among fungal species. Because, advanced and accurate identification of fungal species involve a keen understanding of types of hyphae and their arrangement, which requires significant experience and specialization in the field (Horton and Bruns, 2001). Therefore, most of the reported studies opt for the molecular technique to recognize species-level variances within fungal communities. Besides molecular identification, fungal biomass will be quantified by measuring ergosterol as an account of variation in soil properties that may have caused by droughts. After fungal identifications, ECM fungal species were pure-cultured and sub-cultured for the following experiments (Fig. 3).

Objective 2

To study the plant growth responses towards ECM fungal inoculation under drought stress and optimal growth condition.

This study was conducted to determine whether compositional differences between ECM fungal communities associated with drought and non-drought lodgepole pine stands differentially affect plant growth and drought resistance, and how lodgepole pine seedlings respond morphologically and metabolically to ECM fungal communities isolated from two different regions with significantly different environmental conditions.

Plant material and growth substrate

Pinus contorta seedlings were grown from seeds in nursery trays under controlled environmental growth conditions. The seeds were surface sterilized using 0.05% KMnO4 for 30 min, followed by 3 washes with sterile water, and immersed in sterilized water for 1 h. Sterilized seeds were transferred to a sterilize gauze and grown under dark conditions at 25  °C. The germinated seeds were transplanted to seedling trays filled with sterilized vermiculite, and allowed to grow for 2 months. During this period, the seedlings were watered biweekly, and fertilized with 10 mL half-strength Hoagland solution every week (Hoagland, 1950). After 2 months of germination, we selected 405 uniform and healthy seedlings and transplanted them into plastic pots (10 cm in depth and diameter) filled with a mixture of sterilized soil medium containing soil, sand and vermiculite (1:1:1 v/v/v). Three seedlings were transplanted to each pot, for a total of 135 pots (9 treatments x 15 replications), which were thinned out to 1 seedling per pot after 4 week of growth during the assimilation period under optimal growth conditions.

ECM fungal inoculation

The fungal strains were originally isolated from the soil cores taken from each tree and cultivated on potato dextrose agar medium for making pure cultures of ECM fungi. After 12 days of growth, 4 blocks of media from each pure culture was inoculated in a 300 mL PDA liquid medium, and allowed to culture shake for 2 weeks in an incubator (25 °C with 120 rpm). The fungal mycelia was homogenized using a blender and inoculated to pots as mycelium suspension at 30 mL of homogenized material for each fungal specie. Each pot with inoculation treatment received all the culturable ECM fungal strains as a “community”, since forest tree seedlings with multiple ECM fungal symbionts withstand a wide range of planting sites than those having one species of ECM fungi (Dunabeitia et al., 2004). However, many of the ECM fungal species may not be cultivable in the laboratory, therefore, we may get a reduced number of fungal species cultured than the one identified using the molecular technique. There are different ways to root inoculate ECM fungi; adding “sampled soils” containing ECM communities into the potting medium as inoculum is the most common and convenient method among all. However, this method comes with uncertainties and difficulties in many ways and may result in uninterpretable outcomes. For instance, soils beside desired microbials (ECM fungi in our case) contain other microbial entities including bacteria, nematodes, and other genres of fungi (saprophytic and pathogenic). In such cases, it will be difficult to track plant responses that are solely associated with ECM fungi, which might be influenced by other microbes, particularly, when they are interlinked, and their presence may cause many known and unknown effects imposed on both seedlings and ECM fungi. In order to address this issue, “pure culture inoculation” was carried out using fungal mycelium from pure fungal cultures.

Beside several ways of inoculum application, there are other factors that could be of some consideration, for instance, the time of inoculation particularly in studies conducted under controlled environmental conditions. The inoculum can be applied before sowing or seedling plantation, and after seedling emergence or seedling plantation (Repáč, 2011); which solely depends on experiment objectives, duration of the experiment and the type of inoculant (soil, spores, or mycelium). In our case, the inoculation was carried out as mycelium suspension (10 mL of each mycelium suspension/fungal strain), and applied as early as 1 week after the seedlings are transplanted into the sterile rooting medium. Early inoculation of ECM communities may minimize the acclimation period or transplanting shock the seedlings may face, and positively influence the establishment of seedling roots (Rudawska and Leski, 2021). Other studies reported four weeks of acclimation period given to seedlings to adjust to the new environment (Wang et al., 2021).

Drought stress simulation

The most challenging part of this study is to establish adequate levels of drought stress for lodgepole pine seedlings. There are several methods reported to simulate drought stress in pot experiments. The most basic method used is to passively dry pots by withholding irrigation, however, this method facilitates fast drying taking into account that pots dry really fast, and may not mimic the natural and occasional drought conditions (Poorter et al., 2012). Another method that includes the use of PEG (polyethylene glycol) solution; polyether compounds, to induce drought stress by reducing water movement due to increased osmotic pressure. Although this method has suitably been used on agricultural crops and mostly; in seed germination assays, and their use on forest trees has been limited. However, using PEG may interfere with ion uptake (Yeo and Flowers, 1984) and oxygen diffusion to roots (Mexal et al., 1975) by influencing the osmotic pressure. Marchin et al. (2020) used a method, perhaps a modified and simplified version of the one initially proposed by (Haan and Barfield, 1971). In this method, soil water content of pots was controlled by capillary irrigation via placing the drought pots (base drilled and fitted with nylon mesh to give passage to air and water but to prevent root penetration) above a low water permeable slid column. The solid column was placed in a plastic container containing water/nutrient solution with different water tables as water deficit treatments. The water availability was inversely proportionate with the depth of the water table; the more the depth of water table the less was the water available for the pots. However, as of my knowledge, this method has rarely been reported in experiments regarding soil microbial setups, including mycorrhizal fungi. Also, it may not be a good fit for large experimental formats. Saying that, we lack the knowledge and information required to use this technique as a drought simulator. However, in our study, we will be using a common method which is more of a natural way of inducing drought stress, by reducing water supply in drought treatments in relation to the field capacity of the soil medium being used as the rooting medium. Wang et al. (2021) recently used this method in their study on ECM fungal effects on Pinus tabulaeformis seedling growth under drought stress. However, this method needs a lot of care as it's very important to maintain the water content according to the drought treatment. To do so, pots need to be weighed every day and watered accordingly in order to make sure that the soil water content is up to the set levels. Besides, the soil moisture content will be measured every 3 to 4 days by using a “soil moisture meter” and adjusted by air-drying or irrigating the drought pots in order to maintain the soil water content.