Introduction
Sporobolus (formerly
Spartina) species have been used in Asia, Europe, and North America for salt marsh restoration, increasing bird and fisheries habitat, providing pasture for livestock, reclaiming lost salt marshes, and stabilising coastlines (
Broome et al. 1988;
Cooke and Lefor 1990;
Warren et al. 2002).
Sporobolus have extensive root networks that provide infrastructure and stability to salt marsh sediments (
Broome et al. 1988).
Sporobolus pumilus (Roth) is commonly used in salt marsh restoration, due to root branching traits and far-reaching rhizome growth (
Shepard et al. 2011).
Sporobolus pumilus has low seed germination rates; seeds are easily washed away by tidal forces, making seedling transplants the method of choice for restoration (
Cooke and Lefor 1990). Little is known about seed predators of this species, which may further decrease the chance of
S. pumilus survival using restorative seed stock. In the Bay of Fundy region of the Canadian Maritimes, mega-tidal dynamics and winter ice scouring compound these challenges. Successful restoration is difficult and needed to provide long-term functioning and conservation of salt marsh ecosystems (
Callaway 2005).
Re-examination of salt marsh sites 10 years postrestoration reveals that restored areas can revert back to tidal flats, even when seedling transplants were used. Tissue analysis from native and restored
S. pumilus indicated the restored plants were not forming symbioses with arbuscular mycorrhizal fungi (AMF), which restricted establishment and growth (
Cooke and Lefor 1990). AMF affect the competitive ability of salt marsh plants by increasing access to sediment phosphorous and nitrogen (
Daleo et al. 2008). The inability to re-establish the mutualism between
S. pumilus and AMF may point to the absence of AMF in infilled sediments, which our project works to identify and remedy. In Nova Scotia (NS), Canada, salt marsh restoration practices have been successful after dyke breaches and culvert expansion, with 100% vegetation cover (primarily
Sporobolus alterniflorus (Loisel.)) 3 years postbreach, but these projects have not examined local AMF (
Bowron et al. 2012;
van Proosdij et al. 2010); long-term plant survival is unknown.
We previously isolated and identified the cosmopolitan halotolerant AMF species
Funneliformis geosporum (T.H. Nicolson & Gerd.) (
Hildebrandt et al. 2001;
Landwehr et al. 2002) from a salt marsh in Wolfville, NS; this obligately symbiotic fungus may be of critical importance to
S. pumilus establishment and growth (
d’Entremont et al. 2018).
Funneliformis geosporum may produce 80% of all AMF spores found in salt marshes (
Hildebrandt et al. 2001). Currently,
F. geosporum remains understudied, but likely plays a crucial role in the
Sporobolus root endophytic community in the salt marshes of Wolfville Harbour (
d’Entremont et al. 2018). Use of this obligate mutualistic fungus in simulated salt marshes may reveal the importance of this species to salt marsh ecosystem structure. Controlling environmental parameters, such as light intensity and tidal inundation, while still producing seminatural conditions may provide evidence to support the role of
F. geosporum (
Callaway et al. 1997). Mesocosms are useful tools for testing factors and restoration techniques prior to large-scale implementation, offering great potential for evaluation (
Callaway et al. 1997).
The objectives of this study were to: (i) identify whether inoculation of S. pumilus with F. geosporum increased survival and growth, compared with a sterile control, under simulated salt marsh conditions (tidal mesocosm); (ii) determine if F. geosporum inoculant outperformed the use of natural sediment in improving survival and growth of S. pumilus under simulated salt marsh conditions; and (iii) determine whether AMF-inoculated rhizome- or seed-derived S. pumilus survived better under simulated salt marsh conditions. We predicted that using rhizome-propagated plants inoculated with F. geosporum would outperform all other treatments under our simulated salt marsh conditions due to plant life stage and increased AMF colonisation.
Discussion
Simulated salt marsh trial 1: rhizome-derived plants, sterile sand (proof of concept)
Survival of AMF trap-pot inoculated
S. pumilus was much higher than the control, indicating that
F. geosporum may help offset the shock of the highly saline salt marsh conditions for these plants. Previous research has shown improved salt stress tolerance by plants when colonized by AMF (
Giri et al. 2007). Early survival is fundamental to healthy plant community establishment and sustainability (
Cooke and Lefor 1990); higher early survival reduces the planting density needed for
S. pumilus in salt marsh restoration projects, saving both time and money. Our trial also demonstrated that inoculation increased the growth and final length of
S. pumilus under simulated salt marsh conditions, which are also factors that contribute to the successful establishment of salt marsh ecosystems; healthy vegetation slows tidal waters, leading to increased sedimentation and salt marsh growth (
Fagherazzi et al. 2006).
We also found a significant growth difference between the AMF-inoculated
S. pumilus plants and the uninoculated control group after the 21-d treatment period. Evidence of AMF influence was already present, with significantly greater shoot lengths prior to exposure to simulated salt marsh conditions in our tidal mesocosm. Natural salt marsh sediment contains nutrients that may not be present in autoclaved sand, which may have aided in growth along with the AMF, although the chemical composition was not tested for either treatment. Autoclaving sand increases the nutrient availability of the substrate (
Badía and Martí 2003), but we expect the nutrients provided by the natural salt marsh sediment inoculant to be greater even though the nutrient profile of the sand may have been positively altered during autoclaving. For this reason, our control group was biased but served as a proof of concept trial, showing us that the inoculant, whether it be nutrients or AMF, does improve growth over no treatment at all. Conversely, the salt marsh sediment also introduces possible stressors such as salts (and potentially pathogens), which are naturally present in the sediment used to create the inoculant. The AMF present in the inoculant may remedy the increase in salt, while maintaining the benefits of the added mineral nutrients; if this is occurring, the mechanism remains unknown (
Estrada et al. 2013). Our results show AMF inoculant significantly increased the size of the treated
S. pumilus, which increased their survival and growth under simulated salt marsh conditions.
Simulated salt marsh trial 2: rhizome-derived plants, natural salt marsh sediment
This trial compared our AMF inoculant against natural salt marsh sediment containing naturally occuring nutrients and microorganisms surrounding
S. pumilus roots. By using this as a treatment, we can assess whether our inoculant outperforms natural sediment, where
S. pumilus would naturally grow. Mixed natural sediment and autoclaved sand (in a 1:1 ratio) were used to give the control treatment the same texture as the AMF inoculant treatment. Sediment characteristics, including porosity (
Martin et al. 2012), may play a role in the colonisation of
S. pumilus roots by AMF; therefore, similar sediment types were sought in our comparison of natural sediment against and propagated AMF inoculant.
As in mesocosm trial 1, the survival of S. pumilus inoculated with our AMF propagated treatment was higher than the natural sediment control (90% vs. 57%, n = 84).These treatments differed only regarding fungal load, indicating that AMF may be the driver of this survival. Shoot lengths of AMF-inoculated S. pumilus were significantly higher than the control group despite similar sediment treatments and lower average AMF colonisation than the previous trial (17% for the inoculated and 9% for the control, n = 84). The significantly larger size of inoculated S. pumilus persisted throughout the 48-d mesocosm trial, although the overall length increase of the two groups did not differ significantly. This experiment indicates that using a propagated inoculant may be beneficial, rather than rearing S. pumilus in natural sediment, for early plant establishment in salt marsh restoration.
Simulated salt marsh trial 3: seedlings, natural salt marsh sediment
In this trial we tested
S. pumilus seedlings as opposed to rhizome-derived plants (trials 1 and 2). Conditions of the second experiment were repeated to determine if the same trends would be found for seedlings. Although Nova Scotian salt marsh restoration projects have not focused on active planting, many salt marsh restoration projects elsewhere have focused on transplanting
S. pumilus propagated from collected seed stock due to its ease and low cost; many of these projects failed (
Cooke and Lefor 1990). Thus, a comparison between seedlings and rhizome-derived plants was completed to determine if one method of salt marsh plant propagation may be superior, for restoration purposes.
The AMF-inoculated group had higher survival than the control group, even though the average AMF colonisation, prior to the mesocosm growth trial, was low for both groups (7% and 3% respectively, n = 60, as assessed 21-days after treatment). Trial 3 duration was only 27 d, due to the rapid death of both S. pumilus test groups. There was no difference in S. pumilus size after the 21-d inoculation period nor the 27-d mesocosm growth trial. Interestingly, although the groups didn’t differ in length, the inoculated plants grew significantly more than the control, when looking at their final size in comparison to their initial size. In many ways, the AMF-inoculated plants outperformed the control groups in all three of our trials.
Comparison of the survival of seedlings (trial 3) to the rhizome-derived
S. pumilus (used in trials 1 and 2) showed that rhizome-derived plants had better survival rates, possibly due to differences in the AMF colonisation abilities of adult tissue to juvenile tissue, although this was not tested. Previous research on the seagrass (submerged angiosperm)
Cymodocea nodosa showed that vegetative progeny from adult clones had a better chance of substrate colonisation than seedling
C. nodosa (
Balestri and Lardicci 2012). These results are mirrored by our experiment which showed higher survival for rhizome-derived
S. pumilus, indicating that future restoration projects using
S. pumilus should use AMF-inoculated rhizome-derived plants.