William D. Eaton

Seedlings of the ecologically important, critically endangered tree Ocotea monteverdensisis experience high mortality in the Monteverde, Costa Rica, cloud forests at the onset of the wet season, yet there are no studies suggesting the disease etiology. Here, healthy and diseased plant root and bulk soils were analyzed for various carbon and nitrogen (N) metrics and respiration levels, and DNA sequence-based bacterial and fungal community compositions. All nitrogen metric levels were greater in diseased vs. healthy plant root soils, which could enhance pathogen growth and pathogenic mechanisms. Greater DNA percentages from several potential pathogens were found in diseased vs. healthy plant root soils, suggesting this disease may be associated with a root pathogen. The DNA of the fungus Mycosphaerella was at greater levels in diseased vs. healthy plant root soils than other potential pathogens. Mycosphaerella causes similar diseases in other plants, including coffee, after onset of the wet season. The O. monteverdensis disease also occurs in seedlings planted within or near former coffee plantations at wet season onset. Distance-based linear model analyses indicated that NO₃⁻ levels best predicted the pattern of fungal pathogens in the soils, and Mycosphaerella and Tremella best predicted the patterns of the different N metrics in the soils, supporting their possible roles in this disease.

Seedlings of the ecologically important and “critically endangered” tree, Ocotea monteverdensisis, are experiencing high mortality in the cloud forests of Monteverde, Costa Rica at the onset of the wet season, yet no previous work has been conducted to determine the disease etiology. Here, healthy and diseased Plant Root and adjacent Bulk soils were analyzed for total organic carbon and nitrogen (N), respiration, nitrate, and ammonium, and DNA sequence-based bacterial and fungal community composition. All N metric levels were greater in Diseased vs. Healthy Plant Root soils, which could enhance pathogen growth and pathogenic mechanisms. Greater DNA percentages from several potential pathogens were found in Diseased vs. Healthy Plant Root soils, suggesting a root pathogen etiology. The DNA percentage of the fungus Mycosphaerella was present at greater levels in the Diseased Plant Root soils than other potential pathogens (about 11.7% vs. < 3.2%). Mycosphaerella causes similar diseases in other plants, including coffee, often after onset of the wet season. Similarly, the O. monteverdensis disease occurs in seedlings planted within or near former coffee plantations often at wet season onset. Although fungal pure culture isolation and transmission studies are needed, this is the first evidence of a potential etiologic agent of this disease.

Leguminous Inga trees are thought to enhance soil carbon (C) accumulation following reforestation, through mostly unknown mechanisms. This study amplified soil DNA using the ITS1F and ITS4 primers for PCR and Illumina MiSeq methods to identify fungal taxa, and traditional C analysis methods to evaluate how planted 4-, 8-, and 11-year-old Inga punctata trees affected soil fungal community compositions and C utilization patterns compared to old-growth I. punctata trees and an adjacent unplanted pasture within the same reforestation zone in Monteverde, Costa Rica. Along the tree age gradient, the planted I. punctata trees enhanced the tree soil C capture capacity, as indicated by increased levels of soil biomass C, Respiration, and efficiency of organic C use (with lower qCO₂ values), and development of increasingly more abundant, stable, and successionally developed fungal communities, including those associated with the decomposition of complex organic C compounds. The level and strength of differences coincided with differences in the time of separation between the pasture and tree age or between the different tree ages. Fungal taxa were also identified as potential indicators of the early and late stages of soil recovery. Thus, planting I. punctata should be part of future reforestation strategies used in this region of the Monteverde Cloud Forest in Costa Rica.

Leguminous Inga trees are thought to enhance soil carbon (C) accumulation following reforestation through mostly unknown mechanisms. This study amplified soil DNA using the ITS1F and ITS4 primers for PCR and Illumina MiSeq methods to identify fungal taxa, and traditional C analysis methods to evaluate how planted 4, 8, 11-year-old, Inga punctata trees affected soil fungal community compositions and C utilization patterns compared to old growth I. punctata trees and adjacent unplanted pasture within the same reforestation zone in Monteverde, Costa Rica. Along the tree age gradient the planted I. punctata trees enhanced the tree soil C-capture capacity as indicated by increased levels of soil Biomass-C, Respiration, and efficiency of organic C use (as lower qCO2 values), and development of increasingly more abundant, stable and successionally developed fungal communities, including those associated with decomposition of complex organic C compounds. The level and strength of differences coincided with differences in time of separation between the pasture and tree age or between the different tree ages. Fungal taxa were also identified as potential indicators of early and late stages of soil recovery. Thus, planting I. punctata should be part of future reforestation strategies used in this region of Costa Rica.

This study assessed whether a natural regeneration or active tree‐planting reforestation strategy better restored the C and N‐cycle processes and associated microbiota within soils after 18 years in a Premontane Wet Life zone site in Monteverde, Costa Rica, compared to adjacent old secondary forest and pasture soils (both >60 years). Our findings apply to small‐scale restoration sites (4⁺, NO₃⁻, Biomass‐C, and efficiency of organic C use. Both strategies appeared to positively influence the recovery of the levels and community compositional stability of the Actinobacterial, Acidobacterial, N‐fixing (N‐Fixer) bacterial, ammonium‐oxidizing bacterial, and complex organic C‐degrading fungal communities. The main differences between the two strategies were that the tree‐planted and pasture soils had similar compositions of the Actinobacterial, N‐Fixer, and Fungal complex organic C degrader, while the natural regeneration and pasture soils had similar compositions of these groups and the Acidobacteria. However, the community compositions of all five microbial groups were different between restored forest and the old secondary forest soils. These results suggest that while the soil ecosystems from both reforestation strategies are recovering, after 18 years, there is still more recovery to occur. Lastly, possible indicators of post‐restoration soil ecosystem enhancement included increasing constancy of critical microbial group composition, efficiency of organic C conversion to biomass, Biomass‐C, NH₄⁺, NO₃⁻, and levels of Acidothermus, Acidobacteria subgroups 2, 3, and 5, Archaeorhizomyces, Anaeromyxobacter, Bradyrhizobium, Nitrosomonas, Flavobacterium, and Nitrospira.

Leguminous tree root nodule nitrogen-fixing bacteria are critical for recuperation of soil C and N cycle processes after disturbance in tropical forests, while other nodule-associated bacteria (NAB) may enhance nodule development and activity, and plant growth. However, little is known of these root nodule microbiomes. Through DNA analysis, we evaluated the bacterial taxa associated with the root nodules of the 1-year-old, 2-year-old, 13-year-old, and old growth Inga punctata trees in a cloud forest. Bradyrhizobium diazoefficiens was the dominant taxon found in all nodules at 63.16% to 85.71% mean percent sequences (MPS) of the total nodule bacterial DNA and was found in the youngest nodules examined (1 year old), suggesting that it is the primary nodular bacteria. There were 26 other NAB genera with collective MPS levels between 7.4% to 12.2%, while 15 of these genera were found in the Bulk Forest soils at collective MPS levels of 4.6%. These bacterial community compositions were different between the NAB and Bulk Forest soils, suggesting the NAB became concentrated within the root nodules, resulting in communities with different compositions from the Bulk Forest soils. Twenty-three of the 26 NAB genera were previously identified with the potential to perform 9 plant growth promoting (PGP) activities, suggesting their importance in root nodule development and plant growth. These NAB communities appeared to successionally develop over time into more complex taxonomic communities, which is consistent with the outcome of advanced microbial communities following succession. The presence of both B. diazoefficiens and the NAB communities in the nodules across all ages of tree roots, and the potential for PGP activities linked with most of the NAB genera, suggest the importance of B. diazoefficiens and the NAB community for nodule development and enhanced development and growth of I. punctata throughout its lifespan, and most critically in the younger plants.

Background and Aims Leguminous tree root nodules have N-fixing, nodulating bacteria important for soil C and N cycle recovery following forest disturbance, and nodule-associated bacteria (NAB), which often provide plant growth promoting (PGP) activities. However, composition and function of these microbiomes within tropical leguminous forest trees is understudied. Methods Root nodules were collected from 1, 2, 13-year-old and old growth Inga punctata trees within a tropical cloud forest. Nodule DNA was extracted, from which bacterial taxa were identified through Illumina DNA sequencing methods. Results Bradyrhizobium diazoefficiensDNA sequences represented 63% to 86% and NAB sequences represented 7% to 12% of the total bacterial taxa in the nodules. Collectively, 26 of the NAB taxa were identified with 9 PGP activities. The 1 year-old nodules had the greatest percentages of PGP NAB taxa, which decreased with tree age, though the percentage of total NAB DNA increased with tree age, as did the NAB community complexity. Conclusions Bradyrhizobium diazoefficiensappears to be the root nodule-forming bacteria, while the NAB genera with known PGP activities appear to provide important functions that benefit both the nodule microbiome and I. punctata. The greater percentage of PGP NAB in the youngest nodules suggests their importance in early growth and development of I. punctata and its root nodules, the great presence of NAB DNA in all nodules suggests the importance of NAB throughout the lifespan of I. punctata, and the increasing NAB community complexity with tree age suggests the microbiome undergoes parallel succession with the tree.

Aims This study was designed to determine if planting the leguminous tree Inga punctata in formerly cleared former tropical premontane wet forests changed the Nitrogen (N)-fixer and Lignin Degrader community compositions resulting in increased accumulation of soil carbon (C) and N components. Methods Soils were collected from old growth (> 50 years old), 4, 8 and 11-year-old I. punctata trees, and assessed for differences in various soil C and N metrics, and the mean proportion of sequences (MPS, as “relative abundance” via 16S rRNA V3, V4 sequencing), composition, stability and evenness of the community of N-fixer and Lignin Degrader genera. Results The N-fixer MPS decreased, and Lignin Degraders increased, while evenness and stability of both increased along the tree soil age gradient. The Lignin Degrader MPS were positively correlated with TN, NO₃⁻, TOC, and Biomass C, and negatively correlated with N-fixer MPS and soil NH₄⁺. The N-fixer MPS were negatively correlated with TN, NO₃⁻, TOC, Biomass C and Lignin Degrader MPS, and positively correlated with NH₄⁺. Evenness, stability, and diversity increased for both groups along the tree soil age gradient. Conclusions This is the first evidence that I. punctata facilitates changes the tree soil N-fixer and Lignin Degrader communities over time by increasing the diversity, evenness and stability of both groups, and increases the accumulation of tree soil C and N. This suggests I. punctata facilitates soil recovery such that it can serve as C and N sinks, and support its future use in reforestation management strategy.