N cessation
We observed an evident convergence in plant N and soil
inorganic
N concentrations between controls and previous N addition plots,
implying a rapid recovery in response to cessation of N inputs, a common
observation in cessation of N addition studies (O’Sullivan et al. 2011;
Shi et al. 2014; Storkey et al. 2015). Plant N uptake follows a positive
relationship with soil mineral N concentration (Hajari et al. 2014).
Declines of soil inorganic N pools resulted in the recovery in
aboveground biomass and tissue N following cessation of N inputs. These
findings suggest that plant and soil inorganic pools are not long-term
sinks for added N (Pilkington et al. 2005). Microbial biomass N was
significantly higher than untreated controls after 7 years of N
cessation. Although we did not measure microbial biomass N during the
fertilized period, research in other grasslands indicated that N
addition generally decreased microbial biomass and heterotrophic
respiration (Liu & Greaver, 2010; Sillen & Dieleman, 2012; Riggs et
al. 2015). Moreover, the N input adverse effects were more pronounced in
grasslands in long-term trials (>5yr) (Geisseler et al.
2016). Our findings imply that stopping N input for many years would
eliminate nitrogen-induced soil microbial biomass inhibition.
Although N addition effects on plant and soil inorganic N pools may be
relatively transient, AOB abundance and nitrification rate remain high
even though a reduction in inorganic N pools occurs following nutrient
inputs cessation. Few studies examined the legacy effect of N addition
on on nitrifiers in field soils. Most long-term investigations report
the lack of recovery in soil mineralization and nitrification, such as
20 years in a short grass steppe community (Vinton & Burke, 1995), 14
years in a Swedish pine forest (Chen & Hogberg, 2006), 12 years in a
Minnesota prairie-like acid grassland (Clark et al. 2009). This suggests
that the legacy effect of N addition on internal cycling may persist
many years after the cessation of N inputs. Changes in soil microbes are
possibly closely related to the functional process. Our SEM models (Fig.
5b) implied that soil microbial biomass N predominantly controlled
mineralization variability and soil nitrogen availability for ammonia
oxidizers. Other studies have confirmed the positive relationship
between soil nitrification rate and microbial biomass nitrogen (Baldos
et al. 2015; Wang et al. 2018; Li et al. 2019). The potential mechanism
is that higher soil microbial biomass stimulates soil AOB-induced
nitrification through increased nitrogen mineralization. A large portion
of the mineralizable nitrogen (55 – 89%) is derived from microbial
biomass nitrogen (Bonde et al. 1988). High soil microbial biomass
stimulates soil nitrogen mineralization (Li et al. 2019) and
mineralization ammonium is the substrate of soil autotrophic nitrifiers.
Therefore, soils with additional substrates enhanced soil ammonia
oxidizers, which could expedite nitrification.
Legacy effects of the microbial community due to N addition have been
recorded previously (Johnson, 1993; Bradley et al. 2006). Previous
studies in the same sites have shown that N addition acts as a
deterministic filtering factor favoring AOB but against AOA (Xiao et al.
2014). Even after 7 years of N cessation, high AOB suggests that
long-term impacts of nutrient addition on the AOB community may be
detrimental to nitrification, further triggering a series of
co-occurrence network changes in other groups. AOA was more likely to
co-occur with comammox Nitrospira than AOB in control plots,
indicating that AOA might share more similar niches or have more biotic
interactions with comammox Nitrospira . Ammonia oxidation kinetics
revealed that AOA and comammox Nitrospira possessed transporters with a
higher affinity for ammonia than AOB (Kits et al. 2017; Koch et al.
2019), which facilitated their prevalence in oligotrophic environments.
The AOB community dynamic responses enhance the robustness of microbial
co-occurrence networks, and the linked ecosystem functions could be less
vulnerable for the future ecosystem. Many of these species, such as
competitive AOB, may need elevated N to become established within a
typically low-nutrient community. This creates a situation where the
less competitive species cannot compete sufficiently to re-establish
themselves or become dominant again.