Seasonal dynamics of ammonia-oxidizing microorganisms in freshwater aquaculture ponds

An annual investigation into the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in fresh water aquaculture ponds was performed by quantitative PCR of the amoA gene. The results showed that AOB were the main ammonia-oxidizing microorganisms in water, and significantly higher copy numbers of the AOB amoA gene were observed in the summer (Aug 2012), while no significant differences were detected among the other three seasons. AOA showed low abundances throughout the year. The predominance of AOB in aquaculture water was suggested to be related to photoinhibition. Both the AOB and AOA amoA genes in aquaculture pond sediments showed typical seasonal patterns. The maximum density of AOB was observed in the autumn (Nov 2012) and winter (Jan 2013), while the maximum density of AOA was observed in winter. The minimum densities of both AOA and AOB occurred in the summer. The concentration of the AOA amoA gene was higher than that of the AOB amoA gene in sediments by almost one order of magnitude, which indicates that AOA are the dominant ammonia-oxidizing microorganisms in the aquaculture pond sediments. Dissolved oxygen is suggested to be the key factor determining the predominance of AOA in pond sediments.     

Characterization of N<Subscript>2</Subscript>O emissions and associated microbial communities from the ant mounds in soils of a humid tropical rainforest

Tropical rainforest soils harbor a considerable diversity of soil fauna that contributes to emissions of N₂O. Despite their ecological dominance, there is limited information available about the contribution of epigeal ant mounds to N₂O emissions in these tropical soils. This study aimed to determine whether ant mounds contribute to local soil N emissions in the tropical humid rainforest. N₂O emission was determined in vitro from individual live ants, ant-processed mound soils, and surrounding reference soils for two trophically distinct and abundant ant species: the leaf-cutting Atta mexicana and omnivorous Solenopsis geminata. The abundance of total bacteria, nitrifiers (AOA and AOB), and denitrifiers (nirK, nirS, and nosZ) was estimated in these soils using quantitative PCR, and their respective mineral N contents determined. There was negligible N₂O emission detected from live ant individuals. However, the mound soils of both species emitted significantly greater (3-fold) amount of N₂O than their respective surrounding reference soils. This emission increased significantly up to 6-fold in the presence of acetylene, indicating that, in addition to N₂O, dinitrogen (N₂) is also produced from these mound soils at an equivalent rate (N₂O/N₂?=?0.57). Functional gene abundance (nitrifiers and denitrifiers) and mineral N pools (ammonium and nitrate) were significantly greater in mound soils than in their respective reference soils. Furthermore, in the light of the measured parameters and their correlation trends, nitrification and denitrification appeared to represent the major N₂O-producing microbial processes in ant mound soils. The ant mounds were estimated to contribute from 0.1 to 3.7% of the total N₂O emissions of tropical rainforest soils.     

Nitrogen Metabolism Genes from Temperate Marine Sediments

In this study, we analysed metagenomes along with biogeochemical profiles from Skagerrak (SK) and Bothnian Bay (BB) sediments, to trace the prevailing nitrogen pathways. NO₃ ^? was present in the top 5 cm below the sediment-water interface at both sites. NH₄ ⁺ increased with depth below 5 cm where it overlapped with the NO₃ ^? zone. Steady-state modelling of NO₃ ^? and NH₄ ⁺ porewater profiles indicates zones of net nitrogen species transformations. Bacterial protease and hydratase genes appeared to make up the bulk of total ammonification genes. Genes involved in ammonia oxidation (amo, hao), denitrification (nir, nor), dissimilatory NO₃ ^? reduction to NH₄ ⁺ (nfr and otr) and in both of the latter two pathways (nar, nap) were also present. Results show ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are similarly abundant in both sediments. Also, denitrification genes appeared more abundant than DNRA genes. 16S rRNA gene analysis showed that the relative abundance of the nitrifying group Nitrosopumilales and other groups involved in nitrification and denitrification (Nitrobacter, Nitrosomonas, Nitrospira, Nitrosococcus and Nitrosomonas) appeared less abundant in SK sediments compared to BB sediments. Beggiatoa and Thiothrix 16S rRNA genes were also present, suggesting chemolithoautotrophic NO₃ ^? reduction to NO₂ ^? or NH₄ ⁺ as a possible pathway. Our results show the metabolic potential for ammonification, nitrification, DNRA and denitrification activities in North Sea and Baltic Sea sediments.     

Ammonia-oxidizing archaea versus bacteria in two soil aquifer treatment systems

So far, the contribution of ammonia-oxidizing archaea (AOA) to ammonia oxidation in wastewater treatment processes has not been well understood. In this study, two soil aquifer treatment (SATs) systems were built up to treat synthetic domestic wastewater (column 1) and secondary effluent (column 4), accomplishing an average of 95 % ammonia removal during over 550 days of operation. Except at day 322, archaeal amoA genes always outnumbered bacterial amoA genes in both SATs as determined by using quantitative polymerase chain reaction (q-PCR). The ratios of archaeal amoA to 16S rRNA gene averaged at 0.70?±?0.56 and 0.82?±?0.62 in column 1 and column 4, respectively, indicating that all the archaea could be AOA carrying amoA gene in the SATs. The results of MiSeq-pyrosequencing targeting on archaeal and bacterial 16S rRNA genes with the primer pair of modified 515R/806R indicated that Nitrososphaera cluster affiliated with thaumarchaeal group I.1b was the dominant AOA species, while Nitrosospira cluster was the dominant ammonia-oxidizing bacteria (AOB). The statistical analysis showed significant relationship between AOA abundance (compared to AOB abundance) and inorganic and total nitrogen concentrations. Based on the mathematical model calculation for microbial growth, AOA had much greater capacity of ammonia oxidation as compared to the specific influent ammonia loading for AOA in the SATs, implying that a small fraction of the total AOA would actively work to oxidize ammonia chemoautotrophically whereas most of AOA would exhibit some level of functional redundancy. These results all pointed that AOA involved in microbial ammonia oxidation in the SATs.     

Abundance and diversity of ammonia-oxidizing archaea in a biological aerated filter process

Ammonia-oxidizing archaea (AOA) represent an important group of ammonia-oxidizing microorganisms that are able to convert ammonia to nitrite, a function which is crucial for the removal of nitrogen from wastewater. In this study, we investigated the abundance and diversity of AOA in a full-scale wastewater treatment plant (WWTP) which used a biological aerated filter (BAF) as the main processing mode. According to the quantitative PCR results, AOA clearly outnumbered ammonia-oxidizing bacteria (AOB) during the whole process. The abundance of AOA amoA genes in the filter layer of BAF was highest with the value varied from 6.32 × 10³ to 3.8 × 10⁴ copies/ng DNA. The highest abundance of AOB amoA genes was 1.32 × 10² copies/ng DNA, recorded in the effluent of the ACTIFLO® settling tank. The ratios of AOA/AOB in the WWTP were maintained at two or three orders of magnitude. Most AOA obtained from the WWTP fell within the Nitrosopumilus cluster. The abundance of AOA and AOB was significantly correlated with ammonium nitrogen concentrations and pH value. The community structure of AOA was significantly influenced by dissolved oxygen concentrations, pH value and chemical oxygen demand.     

Impact of carbon source amendment on ammonia-oxidizing microorganisms in reservoir riparian soil

Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) can be key players in ammonia biotransformation in the environment. Soil organic matter can affect the distribution of soil AOA and AOB. However, the link between organic matter and AOA and AOB communities remain largely unclear. The current study investigated the impact of organic carbon amendment on the abundance and composition of ammonia-oxidating microorganisms in reed-planted soil in a riparian zone of the Miyun Reservoir (Beijing). The results indicated that AOB outnumbered AOA in riparian wetland soil both before and after glucose application. Glucose application significantly increased the abundance of AOA , but had only a slight impact on the abundance of AOB. The addition of glucose had a strong impact on the community structures of both AOA and AOB. Moreover, phylogenetic analysis indicated that the obtained archaeal amoA gene sequences showed no close relationship with cultivated AOA species. Few Nitrosospira-like AOB sequences were detected in glucose-amended soil. This study may provide some new insight regarding soil ammonia-oxidizing microorganisms.     

Comparison of the abundance and community structure of ammonia oxidizing prokaryotes in rice rhizosphere under three different irrigation cultivation modes

The abundance, diversity and community structure of ammonia oxidizing archaea (AOA) and bacteria (AOB) in rice rhizosphere soils under three different irrigation cultivated modes, named continuous irrigation mode (C), intermittent irrigation mode (I) and semi-arid mode (M), respectively, were investigated using amoA gene as a molecular biomarker. Clone libraries and quantitative polymerase chain reaction results indicated the highest number of archaeal amoA gene copy was detected in M cultivation mode, then in I and C, whereas, their order of amoA gene copy numbers were I > M > C for AOB, and those were obvious higher than in the bulk soil. The ratios of AOA/AOB were greater than 1 for all samples, suggested the predominance of AOA throughout the period of rice growth in the three different irrigation cultivation modes. Diversity index (S_Chao1 and Shannon H) have an obvious variation in three different irrigation cultivation modes. For AOA, S_Chao1 was highest in M and lowest in I mode, whereas, Shannon H was highest in M cultivation mode and lowest in C mode. For AOB, mode M exhibited the highest diversity index (S_Chao1 and Shannon H), while C showed the lowest highest diversity, suggested long-term water input (continuous mode) may decrease diversity of ammonia oxidizers, whereas mode M may be more appropriate for them. In addition, AOA sequences fall within Nitrososphaera, Nitrosopumilus and Nitrosotalea cluster with proportion of 89.38, 8.85 and 1.77 %, respectively. AOB gene sequences belonged to the Nitrosomonas and Nitrosospira genera with proportion of 90.97 and 9.03 %, respectively. In addition, the abundances, diversity and community structure had an obvious temporal variation in three developmental stages of rice, further suggested rice growth obviously affected the ammonia oxidizing prokaryotes in their rhizosphere soil.     

Characteristics of ammonia oxidation potentials and ammonia oxidizers in mineral soil under <Emphasis Type="Italic">Salix polaris</Emphasis>–moss vegetation in Ny-Ålesund,Svalbard

Although nitrification is a unique and important process in the nitrogen cycle with respect to ammonium consumption and nitrate production, limited information on this process is available for high-Arctic soils. We elucidated the ammonia oxidation potentials (AOPs) and characteristics of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in mineral soils under climax vegetation, i.e., Salix polaris (polar willow)–moss vegetation, on a coastal hill in Ny-Ålesund, Svalbard. AOPs at 10 °C were determined by incubation with sufficient substrate (2 mM ammonium). The ammonia monooxygenase subunit A (amoA) genes of AOB and AOA were analyzed by using quantitative polymerase chain reaction and pyrosequencing. AOPs ranged from 1.1 to 14.1 ng N g^?1 dry soil h^?1—relatively low but of a similar order to the gross nitrification rates reported in another Svalbard study. AOP was positively correlated with thickness of the moss layer (P < 0.01), soil water content, and ammonium nitrogen content (P < 0.05). The population sizes of both AOB and AOA were not significantly related to AOP or edaphic factors. For AOB-amoA, six major operational taxonomic units (OTUs) were identified, all of which were classified into the Nitrosospira Mount Everest cluster. For AOA-amoA, six major OTUs were also identified, five of which were grouped with sequences from cold environments within clade A of the Nitrososphaera cluster, i.e., species known to have low, or no, AOP. It is, therefore, possible that the AOPs measured at the study site were driven mainly by psychrotolerant AOB.     

Soil N mineralization, nitrification and dynamic changes in abundance of ammonia-oxidizing bacteria and archaea along a 2000 year chronosequence of rice cultivation

Background and Aims

Soil mineralization, nitrification, and dynamic changes in abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were studied to validate our hypothesis that soil mineralization and nitrification decreased along the chronosequence of rice cultivation.

Methods

Paddy soils with a 300, 700 and 2000-year cultivation history (P300, P700 and P2000) were selected to study net mineralization and nitrification processes. Dynamic abundance of AOB and AOA was estimated by quantifying their respective amoA gene copies.

Results

The net mineralization rate was higher for P300 than P700 and P2000. Potential nitrification (N _p ) and average nitrification rates (V _a ) were similar for P300 and P700 soils, but the simulated potential nitrification rate (V _p ) and nitrification rate (k₁) was 72 % and 88 % higher for P300 than P700, respectively. V _a was about 70 % lower than for P2000 than P300 and P700. AOB amoA gene copies were higher for P300 than P700 and P2000, whereas AOA abundance did not show significant differences. AOB abundance showed a positive response to NH₄ supply but AOA did not.

Conclusions

Both N mineralization and nitrification were depressed with increased cultivation time. Archaea responded to mineralization positively rather than nitrification, which suggested that readily mineralized organic matter may play an important role in AOA.     

Temporal and spatial impact of <Emphasis Type="Italic">Spartina alterniflora</Emphasis> invasion on methanogens community in Chongming Island,China

Methane production by methanogens in wetland is recognized as a significant contributor to global warming. Spartina alterniflora (S. alterniflora), which is an invasion plant in China’s wetland, was reported to have enormous effects on methane production. But studies on shifts in the methanogen community in response to S. alterniflora invasion at temporal and spatial scales in the initial invasion years are rare. Sediments derived from the invasive species S. alterniflora and the native species Phragmites australis (P. australis) in pairwise sites and an invasion chronosequence patch (4 years) were analyzed to investigate the abundance and community structure of methanogens using quantitative real-time PCR (qPCR) and Denaturing gradient gel electrophoresis (DGGE) cloning of the methyl-coenzyme M reductase A (mcrA) gene. For the pairwise sites, the abundance of methanogens in S. alterniflora soils was lower than that of P. australis soils. For the chronosequence patch, the abundance and diversity of methanogens was highest in the soil subjected to two years invasion, in which we detected some rare groups including Methanocellales and Methanococcales. These results indicated a priming effect at the initial invasion stages of S. alterniflora for microorganisms in the soil, which was also supported by the diverse root exudates. The shifts of methanogen communities after S. alterniflora invasion were due to changes in pH, salinity and sulfate. The results indicate that root exudates from S. alterniflora have a priming effect on methanogens in the initial years after invasion, and the predominate methylotrophic groups (Methanosarcinales) may adapt to the availability of diverse substrates and reflects the potential for high methane production after invasion by S. alterniflora.     

In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

Characterization and comparison of potential denitrifiers in microbial mats from King George Island,Maritime Antarctica

Cyanobacterial microbial mats are highly structured communities commonly found in Antarctic inland waters including melt streams. These benthic microbial associations comprise a large number of microorganisms with different metabolic capacities, impacting nutrient dynamics where established. The denitrification process is a feasible nitrogen loss pathway and a biological source of nitrous oxide, a potent greenhouse gas that also promotes ozone depletion. Potential denitrifiers from five microbial mats were characterized using a PCR-DGGE (denaturing gradient gel electrophoresis) approach. Molecular markers encoding for key enzymes in the denitrification process (nirK, nirS and nosZ) were used. Fingerprints were obtained for the five sampled mats and compared for two successive years. Distance analysis showed that despite the sampled year, the denitrifying genetic potential was similar between most of the sites when represented in Euclidean space. The number of dominant denitrifiers detected for each sample ranged between 6 and 18 for nirK, 4–10 for nirS and 6–17 for nosZ. The seventy-two sequenced phylotypes showed 80–98 % identity to previously reported environmental sequences from water column, sediments and soil samples. These results suggest that Antarctic microbial mats have a large denitrification potential, previously uncharacterized and composed by both site-specific and common phylotypes belonging mainly to Alpha-, Beta- and Gammaproteobacteria.     

Co-expression of Beta-Glucosidase and Laccase in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> by Random Insertion with Enhanced Filter Paper Activity

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P _pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P _gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

Comparative study of antioxidant properties of extracts of various <Emphasis Type="Italic">Aloe</Emphasis> species

Nowadays, a search for sources of the most biologically active antioxidant compounds among various herbs is an urgent problem. In this connection, several succulent plants from the Aloe genus are of special interest, because their preparations are widely used in domestic medicine. In this study, we measured the antioxidant activity (AOA) of an alcoholic extract of leaves of 15 Aloe species by amperometric and chemiluminescent methods and performed a comparative analysis of the results. We observed a considerable difference between the AOA values for the several Aloe species and explained this fact. The most active antioxidant among the samples proved to be the A. pillansii representative of the Aloe genus. Both methods demonstrated that the extracts from leaves of this Aloe species exhibited the high AOA. The A. broomii and A. spinosissima also had rather high AOA along with the A. pillansii and A. arborescens. The newly discovered Aloe species could be as promising a source of biologically active compounds as the traditionally used A. arborescens and A. vera.     

Abundance,Activity and Community Structure of Denitrifiers in Drainage Ditches in Relation to Sediment Characteristics,Vegetation and Land-Use

Drainage ditches are ubiquitous yet understudied features of the agricultural landscape. Nitrogen pollution disrupts the nutrient balance of drainage ditch ecosystems, as well as the waterbodies in which they drain. Denitrification can help ameliorate the impact of N-fertilization by converting reactive nitrogen into dinitrogen gas. However, factors affecting denitrification in drainage ditches are still poorly understood. In this study, we tested how within-ditch and regional environmental conditions affect denitrifier activity, abundance, and community structure, to understand controls on denitrification at multiple scales. To this end, we quantified in situ denitrification rates and denitrifier abundance in 13 drainage ditches characterized by different types of sediment, vegetation and land-use. We determined how denitrification rates relate to denitrifier abundance and community structure, using the presence of nirS, nirK and nosZ genes as a proxy. Denitrification rates varied widely between the ditches, ranging from 0.006 to 24 mmol N m^?2 h^?1. Ditches covered by duckweed, which contained high nitrate concentrations and had fine, sandy sediments, were denitrification hotspots. We found highest rates in ditches next to arable land, followed by those in grasslands; lowest rates were observed in peatlands and nature reserves. Denitrification correlated to nitrate concentrations, but not to nirK, nirS and nosZ abundance, whereas denitrifier-gene abundance correlated to organic matter content of the sediment, but not to nitrate concentrations. Our results show a mismatch in denitrification regulators at its different organizational scales. Denitrifier abundance is mostly regulated at within-ditch scales, whereas N-loads, regulated by landscape factors, are most important determinants of instantaneous denitrification rates.     

The Ammonia Oxidizing and Denitrifying Prokaryotes Associated with Sponges from Different Sea Areas

Marine sponges have been suggested to play an important role in the marine nitrogen cycling. However, the role of sponge microbes in the nitrogen transformation remains limited, especially on the bacterial ammonia oxidization and denitrification. Hence, in the present study, using functional genes (amoA, nirS, nirK, and nxrA) involved in ammonia oxidization and denitrification and 16S rRNA genes for specific bacterial groups as markers, phylogenetically diverse prokaryotes including bacteria and archaea, which may be involved in the ammonia oxidization and denitrification processes in sponges, were revealed in seven sponge species. Ammonia oxidizers were found in all species, whereas three sponges (Placospongia sp., Acanthella sp., and Pericharax heteroraphis) harbor only ammonia-oxidizing bacteria (AOB), two sponges (Spirastrellidae diplastrella and Mycale fibrexilis) host only ammonia-oxidizing archaea (AOA), while the remaining two sponges (Haliclona sp. and Lamellomorpha sp.) harbor both AOB and AOA. S. diplastrella and Lamellomorpha sp. also harbor denitrifying bacteria. Nitrite reductase gene nirK was detected only in Lamellomorpha sp. with higher phylogenetic diversity than nirS gene observed only in S. diplastrella. The detected functional genes related to the ammonia oxidization and nitrite reduction in deep-sea and shallow-water sponges highlighted the potential ecological roles of prokaryotes in sponge-related nitrogen transformation.     

The effect of emerald ash borer-caused tree mortality on the invasive shrub Amur honeysuckle and their combined effects on tree and shrub seedlings

Invasive insects and plants are major threats to the health and viability of North American forests. Emerald ash borer (Agrilus planipennis) (EAB) may cause extensive changes to forest composition due to rapid ash (Fraxinus spp.) mortality. Invasive shrubs like Amur honeysuckle (Lonicera maackii) may benefit from EAB and have negative effects on woody seedlings. We predict that ash mortality has positive effects on seedling abundance, recruitment, and survival, but that these effects are influenced by L. maackii basal area and/or cover. We sampled 16 sites, representing a chronosequence of ash mortality throughout western Ohio. We tested whether L. maackii growth and fecundity varied in relation to ash decline. We also investigated effects of ash decline, stand basal area (BA), L. maackii BA and percent cover on woody seedling abundance, recruitment, and survival using linear mixed models evaluated with Akaike’s Information Criterion. These same responses were also investigated for four seedling groups: L. maackii, invasive plants (excluding L. maackii), shade tolerant natives, and shade intolerant natives. We found a significant positive relationship between ash decline and L. maackii BA growth. Lower seedling species richness corresponded with greater L. maackii BA and better ash condition. Greater L. maackii BA was also associated with lower seedling abundance and recruitment, as well as abundance and recruitment of shade-tolerant species, and recruitment of shade-intolerant species. Sites with poorer ash condition and greater L. maackii BA had more L. maackii seedlings. These findings indicate that the negative effects of L. maackii are more important to future forest composition than ash decline; however ash decline increases L. maackii growth, hence exacerbating the effects of this invasive shrub.