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Article

Initial Analysis of Plant Soil for Evidence of Pathogens Associated with a Disease of Seedling Ocotea monteverdensis

by
William D. Eaton
1,2,*,
Debra A. Hamilton
3,4,
Alexander Lemenze
5 and
Patricia Soteropoulos
6,7
1
Biology Department, Dyson College, Pace University, New York, NY 10038, USA
2
Department of Environment and Development, University for Peace, El Rodeo de Mora, San José 10701, Costa Rica
3
Vermont Cooperative Fish and Wildlife Research Unit, Rubenstein School of the Environment and Natural Resources, University of Vermont, Burlington, VT 05405, USA
4
Monteverde Institute, Monteverde, Puntarenas 60109, Costa Rica
5
Molecular and Genomics Informatics Core (MaGIC), Rutgers New Jersey Medical School, 205 South Orange Avenue, Newark, NJ 07103, USA
6
Genomics Center, Rutgers New Jersey Medical School, The State University of New Jersey, 185 South Orange Ave, MSB F653, Newark, NJ 07103, USA
7
Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, The State University of New Jersey, 185 South Orange Ave, MSB F653, Newark, NJ 07103, USA
*
Author to whom correspondence should be addressed.
Microorganisms 2025, 13(7), 1682; https://doi.org/10.3390/microorganisms13071682
Submission received: 30 June 2025 / Revised: 12 July 2025 / Accepted: 14 July 2025 / Published: 17 July 2025
(This article belongs to the Special Issue Plant–Microbe Interactions Revisited: Emerging Concepts and Biotechnological Innovations)
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Abstract

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.

1. Introduction

Restoration of tropical forests is essential, and the survival of seedlings is critical to the success of these efforts. Multiple factors may impact seedling survival, with fungal pathogens shown to be a significant, if not the greatest, threat to seedling survival [1]. Recent reports indicate that potential fungal pathogens are increasing in response to ongoing climate change, with expanding species ranges, greater diversity, and enhanced resilience [2]. It is believed that natural systems may be more vulnerable to increasing pathogens [3], and the warm, humid tropics may be especially susceptible. In light of these growing concerns, emerging models are beginning to reveal the complexity of these impacts [4]. Given the known challenges and costs associated with tropical forest restoration, identifying and protecting plants from these fungal pathogens is a priority. A crucial first step is to identify the specific types of fungal pathogens harming these restoration efforts.
A severely threatened tree species, Ocotea monteverdensis, is suspected of suffering high mortality rates due to a systemic pathogen that suddenly causes necrosis of seedling leaves and subsequent death with the onset of rains during the wet season. It is one of approximately 96 species of the Lauraceae family that comprises a large percentage of the diversity of the forests in the Monteverde region of Costa Rica. This tree is listed as globally “critically endangered” [5,6], as less than 1000 mature individuals remain within its principal occupancy range in Monteverde, where a large percentage of the mature forest of this region was cleared for timber, human development, or agricultural uses [6]. Only 5% of the remaining trees in this region are on permanently protected land [7].
Due to the highly threatened status of O. monteverdensis and its ecological importance as the preferred food source of large frugivorous birds such as the vulnerable three-wattled bellbird (Procnias tricarunculatus) [8,9], there have been multiple coordinated efforts to protect mature trees and plant seedlings and increase awareness by the Fundacion Conservacionista Costarricense (FCC) and the Monteverde Institute. The emphasis of these programs has been to augment existing forests and restore abandoned pastures with plantings of threatened tree species, such as O. monteverdensis, and to specifically advance the knowledge regarding the factors impacting the survival of O. monteverdensis.
There has been a limited number of studies concerning O. monteverdensis, even though its ecological importance in cloud and humid forests has been recognized [6,8,10]. The genetic variation within the limited population size is not known, and reproductive success to mature tree stage has not been determined. There are indications that O. monteverdensis is not regenerating its population sufficiently, which is thought to be due to decreased amounts of habitat appropriate for regeneration [11,12,13].
Collaborative research with the FCC restoration program found high mortality rates of O. monteverdensis seedlings planted in both forest fragments and abandoned pasture habitats. The seedling mortality continues past its fourth year post-planting, instead of reaching a plateau after two years, as found with other tree species [13,14]. Of special note is the observed sudden death of seedlings that starts with necrosis of most leaves shortly after the first rains of the May to June rainy season. Additional studies examining natural regeneration of this tree species have shown concerns regarding seedling success [13]. To date, even with this suspicion of pathogen involvement in seedling mortality, no work has been conducted on the root soil microbial community or activity, nor on the carbon and nitrogen components associated with healthy and diseased seedlings.
The current study was conducted to address the potential role of a pathogen in the mortality of young O. monteverdensis. While necrotrophic fungal pathogens are more common [15], the sudden, systemic death of O. monteverdensis seedlings suggests a soil-inhabiting pathogen. For this work, the soil carbon and nitrogen metrics were assessed, and the overall DNA sequence-based bacterial and fungal community compositions were determined within the root-associated and the plant bulk soil (soil within the dripline) of seedlings planted in reforested areas within the Monteverde premontane wet forest. In addition, the most common fungal and bacterial genera from each sample were determined, with any potential pathogens identified.

2. Materials and Methods

2.1. Sample Site and Collections

The Fundacion Conservacionista Costarricense (FCC, www.fccmonteverde.org) has made significant conservation contributions by distributing and planting over 300,000 seedlings from 140 native tree species on the property of nearly 500 landowners throughout the Bellbird Biological Corridor on the Pacific slope of Costa Rica. This has included the planting of over 5000 O. monteverdensis seedlings, with the monitoring of 950 individuals.
O. monteverdensis is a critically endangered tree that is endemic to Costa Rica, with its core population found in the tropical premontane wet life zone on the Pacific slope of the Cordillera de Tilarán (between 10°25′ N and 10°16′ N, and longitudes 84°42′ W and 84°55′ W) at elevations between 1200 and 1500 m asl [9,14,16,17]. O. monteverdensis is a large canopy tree that produces 3–4 cm-long avocado fruits every three years on average, with 64% synchrony within the species [8]. Once common in the Monteverde region, it was previously harvested for its fine red wood [9].
There is a suspicion that the high mortality of the seedlings within the first planted year may be caused by a pathogen. This is based on the observation that the seedling mortality post-planting is associated with a sequential pattern of chlorosis (Figure 1A), followed by development of brown spots (Figure 1B,C), then curling and wilting of the leaves (Figure 1D), followed by a sudden death and dropping of all its leaves, leading to plant death. This usually occurs after the start of the rainy season and involves seedlings that are in their first two years after planting.
The site used for this study is located at 1430 m asl in the premontane wet life zone and receives an average of over 2863.2 mm of precipitation per year [18]. In 2021, 6 seedlings of diseased and 6 of non-diseased O. monteverdensis were used for this study. The seedlings were raised from the same seed stock, in the same nursery with equivalent watering and seedling care, and with the same soil source used in the seedling bags. Healthy seedlings were planted in 2020 at the FCC’s Crandell Memorial Reserve, augmenting a reforestation effort of abandoned pasture done in 2008, and all were planted under 13-year-old O. monteverdensis, thereby resulting in light availability being reduced to a partial shade status. In addition, this method ensured all trees were planted in soil that had experienced the same land-use history, resulting in similar soil characteristics throughout the reforested area at the time of seedling planting. Of the 82 planted seedlings, 62 had survived at the time of sampling in 2021. They were planted with an average height of 20.3 cm with 13.8 leaves. After one year, the average height was 23.8 cm with 16.7 leaves. The 6 healthy and 6 diseased young O. monteverdensis used in this study were all 20–30 cm in height and separated from adjacent sampled seedlings by >2 m, had been propagated from seeds in the FCC nursery and maintained there for about 1 year, and had been planted in the study area 1 year prior to sampling.
For collection of soil from the 6 heathy and 6 diseased plants, 4 soil cores 10 cm deep were collected at the cardinal points around the plant at a distance from the plant equal to 75% of the dripline distance (between 10 cm and 16 cm from the plant base) using a 7.5 cm × 15 cm × 1.25 cm soil profiler. The 4 cores from individual plants were stored in sterile plastic bags. These samples are hereafter called the plant bulk soil samples. After collection of the soil from an individual plant, cuts were made in the soil around the same plant that were approximately a 3 cm-diameter distance from the plant base to allow easy removal of the plant and root system. This plant root soil sample was placed in a sterile bag. In the lab, the loose soil and the soil attached to the roots were collected and stored in sterile bags, and hereafter are called the plant root soil samples. This process resulted in the collection of 6 healthy and 6 diseased plant bulk soil and plant root soil samples to be used for analysis. All collection tools and gloves were disinfected with 70% ethanol between individual plants and between the collection of plant soil and plant root samples to inhibit cross-contamination. All field moist soil samples were passed through an ASTM No. 4 sieve (about 5 mm openings) previously disinfected with 70% ethanol.

2.2. Soil Environmental Metrics

Subsamples from each sample were analyzed at the Center for Tropical Agriculture Research and Education (CATIE) Laboratory in Turrialba, Costa Rica, for all C and N metrics. This included determination of the total organic C (TOC) mass by dry combustion analysis [19], the total N (TN) mass by the standard Kjeldahl method using a Thermofinigan EA12 autoanalyzer (San Jose, CA, USA), the respiration level as CO2 (Resp) by standard closed-vessel methods [20], and the levels of both nitrate (NO3−) and ammonium (NH4+) from 2 M KCl extracts using the spectrophotometric methods previously described [20].

2.3. DNA Extraction, Sequencing, and Bioinformatics

Total soil DNA was extracted from three 0.33 g replicate sub-samples from each soil sample using the MoBio PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA), with the concentration and purity determined as the A260/A280 ratio of the individual DNA samples using a NanoDrop 1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). The PCR amplification of DNA targeting the nuclear internal transcribed spacer (ITS) ribosomal RNA gene region of fungi [21] and the v3 and v4 of 16S ribosomal RNA gene region of bacteria [22] are now commonly used to identify microbial taxa. For the current project, previously described methods were used [23] in a 2-step PCR amplification process that included use of the specific PCR primer set 16Sv3F and 16Sv4R [24] for bacterial 16S rRNA amplification, and the primer set ITS4 and ITS1F [25] for fungal ITS region amplification. The PCR steps were the same for use of the 16S and the ITS primer sets. The first step of PCR amplification was performed on 2 µL of sample DNA in a reaction buffer containing 200 mM of Tris-HCl, 500 mM of KCl, a pH of 8.4, 50 mM of MgCl2, 10 mM of dNTPs mix (10 mM), 10 mM of forward primer, 10 mM of reverse primer, and 5 U/µL of Invitrogen Platinum Taq polymerase (5 U/µL). The PCR conditions were 95 °C for 5 min; 35 cycles of 94 °C for 40 s, 46 °C for 1 min, 72 °C for 30 s, and 72 °C for 5 min; and held at 4 °C. The second PCR step was performed using the purified 1st PCR product as a template with Illumina adapter tailed specific target primers, following the same protocol as mentioned before, except that 30 cycles were used. All raw DNA sequence reads were assessed for quality control (FASTQC v 0.12.0), then trimmed and filtered for quality (Cutadapt v 3.2). The 16S amplicon sequence variants that passed the quality filter were assigned taxonomic identification (DADA2 v 1.20.0) from phylum to genus based on the SILVA rRNA database (build 138) for bacteria. The fungal ITS amplicon sequence variants that passed the quality filter were taxonomically identified from phylum to genus using the Ribosomal Database Project Classifier (https://github.com/rdpstaff/classifier, accessed on 28 October 2021) with the UNITE database for fungi [26]. All DNA sequence data were submitted to the NCBI Sequence Read Archive (NCBI Bioproject numbers PRJNA1019742 and PRJNA646467). To normalize the effects of uneven numbers of sequences per sample, the number of times a specifically identified genus appeared in a sample was converted to mean proportion of the sequences (MPS) per sample, as previously recommended [27].
The total of all bacterial and fungal taxa identified by these DNA sequencing methods are hereafter referred to as the total bacterial and the total fungal communities in this study. A variety of resources was used to identify bacterial and fungal genera that were potential plant pathogens, and also to determine other potential functional groups of various bacterial and fungal genera (Table S1). The bacterial and fungal genera that were identified as potential plant pathogens are hereafter referred to as bacterial and fungal pathogen communities.

2.4. Data Analysis

All differences in the mean values of the soil sample TOC, TN, NH4+, NO3−, and respiration (CO2) were conducted using one-way ANOVA tests in SPSS (v.27, Armonk, NY, USA), with the sample year as the predictor variable. Prior to ANOVA, Levene’s test was performed in SPSS to determine homogeneity of the variances of the data. All data had p values > 0.05, suggesting that the use of the Tukey’s HSD post-hoc test was appropriate.
The MPS values for all microbial MPS values were 4th-root transformed, as recommended by Ref. [28]. The 4th-root-transformed MPS values were converted into Bray–Curtis similarity matrices and analyzed for differences in the composition of the total bacterial, bacterial pathogenic, total fungal, and fungal pathogenic communities using the analysis of similarity (ANOSIM) routine performed in PRIMER-E v.6 and PERMANOVA+ (Plymouth, UK) [28], which provided R and p values for the main and pairwise tests. The strength of any of these differences was determined using the Canonical Analysis of Principal Coordinates (CAP) method [29,30] in PRIMER-E with the PERMANOVA + add-on. The CAP axis R2 values ≥ 0.7 represent strong compositional differences, R2 = 0.5 to 0.69 represents moderate differences, R2 = 0.20 to <0.5 represents weak differences, and R2 < 0.20 suggests no differences. The differences in MPS values of the bacterial pathogens and fungal pathogens between the healthy and diseased plant bulk soils and plant root soils were determined using the Mann–Whitney U test in SPSS.
To further compare differences in microbial community composition between the different healthy and diseased plant soil samples, the Similarity Percentage (SIMPER) analysis in PRIMER-E routine was used on the 4th-root-transformed MPS data to demonstrate differences in the percent taxonomic dissimilarity. In addition, the richness (as number of genera) and diversity (as Shannon’s H index) were calculated, and differences in mean values of these metrics between the healthy and diseased bulk soil and root soil samples were determined using the Mann–Whitney U test in SPSS. Lastly, the differences in MPS of the more common (MPS > 1%) total fungal and total bacterial taxa were compared for the different paired healthy and diseased soil samples by Mann–Whitney analyses in SPSS. In addition, the potential functions of these taxa were determined using the resources mentioned above.
Distance-based linear model (DistLM) analyses in Primer E v.6 and PERMANOVA+ were used to analyze the relationships between the microbial community compositions and the soil environmental parameters in both healthy and diseased plant bulk and root soils [28,30]. Tests were conducted to determine the influence the environmental metrics had on the different microbial communities and the influence the communities had on the environmental metrics. In both cases, log (x + 1) environmental metrics and the 4th-root-transformed MPS values were used. For these analyses, a stepwise selection process was used, along with an AICc (Akaike Information Criterion Corrected) selection criterion, which is recommended for small datasets, such as the ones from this study, in which the number of samples (N) is small relative to the number (v) of predictor variables, such that N/v < 40 [30]. In all cases, 9999 permutations were used as recommended [28,30].

3. Results

3.1. Differences in Soil Carbon and Nitrogen Metrics

There were no differences (all p values > 0.09) in the levels of TOC or respiration between the diseased compared to the healthy plant root soils, but the levels of TN, NH4+, and NO3− were all greater (p range = 0.046 to 0.055) in the diseased plant root soils compared to those in the healthy plant root soils (Table 1). The levels of TN, NH4+, NO3−, and TOC were all greater (all p values < 0.001) in the diseased plant bulk soil compared to those in the healthy plant bulk soil, but there were no differences in respiration levels between the soils (Table 1).

3.2. Differences in Community Compositions

The ANOSIM results (Table 2) showed there were differences in the total bacterial community compositions between the healthy and diseased plant root (R = 0.566, p = 0.003) and plant bulk soils (R = 0.416, p = 0.0002). Similarly, there were differences in bacterial pathogen communities between the healthy and diseased plant root (R = 0.563, p = 0.004) and plant bulk soils (R = 0.485, p = 0.002). The CAP findings for these comparisons (Table 2) showed that all these differences were considered strong differences in community composition (CAP R2 range of 0.706 to 0.876, p values = 0.0001 and 0.0002). The ANOSIM results showed there were differences in the total fungal and fungal pathogen community compositions (Table 2) between the healthy and diseased plant root soils (R = 0.752, p = 0.004; and R = 0.554, p = 0.002). There were also differences (Table 2) in the fungal pathogen community composition between the healthy and diseased plant bulk soil (R = 0.231, p = 0.05), but not in the total fungal community composition between the healthy and diseased plant bulk soil (R = 0.046; p = 0.273). The CAP findings for these comparisons (Table 2) showed that the differences in the total fungal and fungal pathogen community compositions were strong between both the healthy and diseased plant root soils and the plant bulk soils (R2 = 0.801, p = 0.0022, and R2 = 0.855, p = 0.0008). The CAP results showed that between the healthy and diseased plant bulk soils the differences in the total fungal community compositions were very weak (R2 = 0.223; p = 0.0022) and the differences in community composition of the fungal pathogens were moderate (R2 = 0.599; p = 0.008).
There were no differences observed in the MPS (Figure 2) of the bacterial pathogen community between the healthy and diseased plant root soils (p = 0.873) or plant bulk soils (p = 0.109). There was also no difference in the MPS of the fungal pathogens between the healthy and diseased plant bulk soils (p = 0.749). However, the MPS of the fungal pathogens was greater (Figure 2) in the diseased plant root soils compared to the healthy plant root soils (45.35% vs. 15.25%; p = 0.016).
There were no differences in percent taxonomic dissimilarity (Figure 3) of the total bacterial or bacterial pathogen communities between the healthy and diseased plant bulk soil or the healthy and diseased plant root soil (41.97% vs. 42.19% and 36.42% vs. 36.52%, respectively). However, there were differences in the percent taxonomic dissimilarity (Figure 3) of the total fungal and fungal pathogen communities between the healthy and diseased plant root soils (55.65% vs. 63.66% and 50.16% vs. 61.34%, respectively). There were no differences in the richness or diversity of the total bacterial or bacterial pathogen communities (Table 3) between the healthy and diseased plant roots or plant bulk soils (all p values > 0.4). There were also no differences in the richness or diversity of the total fungal communities (Table 3) between the healthy and diseased plant bulk soils (p values > 0.26). However, the richness of the total fungal and the fungal pathogen communities was greater in the diseased compared to the healthy plant root soils (86.83 vs. 23.51, p = 0.0002; and 20.17 vs. 8.51, p = 0.004). Similarly, the diversity of the total fungal and the fungal pathogen communities was greater in the diseased compared to the healthy plant root soils (4.26 vs. 2.74, p = 0.0003; and 2.77 vs. 1.76, p = 0.005).
There were many differences in the MPS of specific bacterial genera (with MPS levels > 1%) between the healthy and diseased plant bulk soils (Table 4); however, Clostridum was the only potential bacterial plant pathogen [31] with greater MPS levels in the diseased compared to the healthy plant bulk soils (3.50% vs. 0.74, p = 0.001). There were few differences in the MPS of bacteria with an MPS > 1% in either the healthy or diseased plant root soils, although Clostridium was present at greater MPS levels in the diseased compared to the healthy plant root soils (3.18% vs. 0.36%, p = 0.019).
There were eight fungal genera with MPS levels > 1% in either the diseased or healthy plant bulk soils (Table 5), but the plant pathogen Ilyonectria was the only one of these genera with MPS levels that were different between the different plant bulk soils, with a greater MPS in the healthy compared to the diseased plant bulk soils (8.68% vs. 1.28%, p = 0.0039). There were 10 fungal genera with an MPS > 1% that were present in either the diseased or the healthy plant root soils (Table 5). The complex organic C compound-degrading (CCD) genus Apiotrichum was present at greater MPS levels in the healthy compared to the diseased plant roots (62.07% vs. 26.58%, p = 0.0163), while the CCD genus Saitozyma and the arbuscular mycorrhizal genus Glomus had MPS levels that were moderately (not statistically significantly) greater in the roots from healthy compared to diseased plants (9.74% vs. 4.17%, p = 0.0781; and 3.00% vs. 1.48%, p = 0.1093, respectively). Seven of the 10 genera had greater MPS values in the diseased compared to the healthy plant root soils. The plant pathogen Mycoshpaerella was present in the diseased plant root soils at a much greater MPS level than in the healthy plant root soils (11.65% vs. 3.20%, p = 0.0039), as were the plant pathogens Dipodascus, Didymella, and Acrocalymma, but at much lower MPS levels (1.97% to 2.76% vs. 0.01% to 0.37%, p = 0.0021 to 0.0225). There were two other genera worth noting. The MPS of the fungal Oomycete and algal parasite Rozella had a much greater MPS level in the diseased compared to the healthy plant root soils (13.3% vs. 0.65%, p = 0.0038). In addition, the MPS of the plant root-associated fungal genus Archaeorhizomyces also had much greater MPS levels in the diseased compared to the healthy plant root soils (11.26% vs. 0.23%, p = 0.0033).
The DistLM results (Figure 4a,b) showed there were no statistically valid relationships between the bacterial pathogen community and the environmental metrics in any habitats or in the fungal pathogen community in the healthy or diseased plant bulk soils. However, there were statistically valid relationships between the fungal pathogen community and some of the environmental metrics in the diseased and healthy plant root soils. Specifically, the levels of NO3− influenced 43.96% of the fungal pathogen community compositions in the diseased and healthy plant root soils (Figure 4a, AICc = 69.96, pseudo-F = 7.84, p = 0.0037), and the MPS levels of Mycosphaerella and Tremella combined greatly influenced 66.52% of the levels of TN, NO3−, and NH4+ in the diseased and healthy plant root soils (Figure 4b, AICc = 8.38, pseudo-F = 16.11, p = 0.002).

4. Discussion

The levels of the three nitrogen environmental metrics were all greater in the diseased plant root soils compared to the healthy plant root soils. In addition, the nitrogen metric values and the TOC were greater in the diseased plant bulk soils compared to the healthy plant bulk soils. Studies have shown that increases in the soil N and in either of the two forms of inorganic N can increase the risk of pathogens, causing diseases in plants through several mechanisms. Excess N can enhance the microbial pathogen’s metabolism by causing an increase in production of amino acids and gamma amino butyric acid, which are used by the microbe to enhance its growth rate and pathogenic processes, and also to increase the production and release of various microbial virulence factors [32]. Excess N also is known to decrease the thickness of plant cuticle and wax layers and reduce the amount of lignin produced by a plant, all of which enhance a pathogen’s ability to penetrate the plant surface layers [33]. Furthermore, excess N can inhibit production of plant metabolites that can be protective against pathogens, such as phytoalexins, antimicrobial proteins, amino acids, organic acids, and various defense-related enzymes [33]. It is possible that the greater levels of N in the diseased plant soils could be a result of the decomposition of the dead and dying plant roots. Regardless, given the potential pathogen-enhancing effects of excess N on plants and the greater levels of N found in the diseased plant roots, it is possible that high levels of TN and forms of inorganic N could be enhancing the pathogenesis of a yet unknown pathogen of O. monteverdensis.
There were clear differences in total bacterial and the bacterial pathogen community compositions between the heathy and diseased plant roots and plant soils. This suggests that no matter which pathogenic processes are occurring in association with the diseased O. monteverdensis, they appear to be influencing the overall bacterial community compositions. However, the greatest differences in composition found for both bacterial groups were between the healthy and diseased plant root soils. This suggests that the disease has a greater influence on the bacterial community near the roots, providing support that the disease is likely root-associated. That said, the only potential plant pathogen found in greater MPS levels in the diseased vs. healthy plant roots or plant bulk soil was Clostridium, which was found at MPS levels of about 3–3.5% in the soil and roots of diseased O. monteverdensis, but only at about 0.4–0.75% in the healthy plant soil and roots.
There have been six species of Clostridium recently identified from the roots and soil of kiwi plants (Actinidia chinensis var. deliciosa “Hayward”) with a withering and wilting disease, somewhat similar to what has been observed in O. monteverdensis [31]. It is interesting that the disease in kiwi plants was enhanced with heavy moisture, which is consistent with observations by the reforestation biologists in the FCC in Costa Rica, who have found that the disease in O. monteverdensis seems to occur in the rainy season. Given that Clostridium was the only potential bacterial pathogen found in greater levels in the diseased plants than in the healthy plants, it is the only possible bacterial candidate from this study that could be the causative agent for this O. monteverdensis disease.
There was also clear evidence of differences in the overall total fungal and fungal pathogen community compositions between the heathy and diseased plants. This indicates that the pathogenic processes associated with O. monteverdensis disease are influencing the soil fungal community compositions. Furthermore, as the greatest differences in community composition for both groups was between the healthy and diseased plant root soils, and there were minimal to no differences in these communities between the healthy and diseased plant bulk soils, this strongly supports the idea that this disease of O. monteverdensis is linked to a root-associated pathogen.
The known fungal pathogens Dipodascus, Didymella, Acrocalymma, and Mycosphaerella all had significantly greater MPS levels in the diseased plant root soils than in the healthy plant root soils, which were also greater than in the healthy or diseased plant bulk soils. Mycosphaerella is the fungus perhaps of greatest interest in this study, as its DNA was present in the diseased plant root soils at MPS levels far greater than those of the other pathogens or in the healthy plant root soils. Mycosphaerella is considered a plant pathogen; is very common, with over 10,000 species within the genus; and has members of the genus causing plant diseases involving chlorosis, which leads to leaf spot browning, leaf loss, and plant death [34,35] and also has been associated with the O. monteverdensis disease. It is also common for Mycosphaerella infection and disease to become enhanced during or following periods of heavy rain, which are thought to enhance spore growth and result in easier entry into plant stomata [36,37,38,39]. This pattern of enhanced disease after heavy rain has been observed with O. monteverdensis in the Monteverde area. Mycosphaerella coffeicola (the asexual form of Cercospora coffeicola) is a common pathogen of coffee, causing a leaf spot disease that begins as chlorotic spots on the leaf surface that develop into brown and necrotic lesions. These lesions become discolored with a lighter center and yellow halo, and sometimes fuse into large irregular areas, resulting in the leaves dropping [40,41]. These symptoms in plants have also been observed with the O. monteverdensis disease. In addition, similar to the disease occurrence in O. monteverdensis, the disease due to M. coffeicola in coffee plantations is enhanced after continual wetness for 36–72 h [41]. Lastly, it is also of interest that many of the diseased O. monteverdensis plants are seedlings that were planted in areas that had previously been forests before being cleared and used as coffee plantations, with some areas even still containing some abandoned coffee plants within the planted areas. It is certainly premature to suggest that Mycosphaerella is the cause of the O. monteverdensis disease, but it gives us a target to look for in future studies.
The DistLM showed that the levels of soil NO3− had the greatest influence on the fungal pathogen community compositions in the plant root soils, and that the MPS levels of Mycosphaerella and Tremella combined had the greatest influence on the levels of TN, NO3−, and NH4+ in the plant root soils. These results support the concepts of Mycosphaerella possibly being an etiologic agent of the disease and of inorganic N levels potentially also playing a role in the disease pathogenesis.
There were two other fungi of interest that warrant further mention. Archaeorhizomyces DNA was present at MPS levels much greater in the diseased plant root soils than in the healthy plant root soils. There is little known of this fungus, but it is known as a root-associated fungus that is involved in the decomposition of various plant materials, detritus, and debris found in the root zone, particularly those with low pH and high nutrient turnover, as is associated with diseased plants [42,43,44,45,46]. Archaerorhyzomyces is thought to be a decomposer of glucose, cellulose, and other carbohydrates released from dying plants into the root zone [47]. It has been suggested that Archaeorhizomyces may be replacing the ectomycorrhizal fungi in the plant roots during above-ground plant senescence and associated root decay, and may be involved in the decomposition of the dead and dying root material released during these periods of plant senescence [43,48,49]. This information and the more than 60× greater MPS levels of Archaeorhizomyces in the diseased plant roots suggests that while not the primary pathogen of the O. monteverdensis disease, it may be an indicator of the effects this disease has on the O. monteverdensis rhizosphere soil microbial community.
The other interesting fungal genus worth noting is Rozella, as the DNA of this fungus was present in the diseased plant root soils at much greater MPS levels in the healthy plant root soils. Rozella is an endoparasite of the Oomycota; the fungal phyla Blastocladiomycota, Monoblepharidomycota, Chytridiomycota, and Basidiomycota; and the green algal phylum Charophyta [50]. Very little is known of this fungus; however, it has been found to parasitize fungi within soils where there is an increase in the levels of decomposition of plant debris and detritus [51,52]. As such, it is thought to somehow be involved in the decomposition of dead plant roots and other rhizosphere plant debris, although the mechanisms are not known. Nonetheless, this activity could explain its increased abundance in the plant root soils of diseased O. monteverdensis compared to the levels in the plant bulk soil of these diseased plants. Thus, the increased Rozella abundance, like that of Archaeorhizomyces, could be a consequence or indicator of the disease in O. monteverdensis seedlings.

5. Conclusions

It is unclear what is causing the disease observed in O. monteverdensis in the Monteverde cloud forest region of Costa Rica. The evidence from this study strongly points to a root-associated pathogen, based on the greater difference in community composition of the different microbial groups being found between the diseased vs. healthy plant root soils. Although not definitive proof of the etiology of the disease, the data from this work on the fungus Mycosphaerella do provide the best evidence for a possible causative agent of the O. monteverdensis disease, as it was present in much greater levels in the diseased plant root samples than any other fungal pathogens and has members of the genus causing diseases in plants with similar symptoms to those of the O. monteverdensis disease, many of which are more common during or immediately after extremely wet periods, which is also the case with the O. monteverdensis disease. Furthermore, this is consistent with M. coffeicola, which causes a disease in coffee plants with symptoms similar to those observed with the O. monteverdensis disease, which develops after a 36–72 h period of extensive wetness. However, proof of the cause of this disease will require pure culture of a fungal taxa and replication of the disease in young O. monteverdensis. This work is planned for the future. While Mycosphaerella certainly may not be the causative agent of the disease, it is an interesting possibility that warrants further work. Also, the non-pathogenic fungi Archaeorhizomyces and Rozella may represent two genera that can serve as secondary indicators of the O. monteverdensis disease. It is also possible that the elevated levels of N may be enhancing the growth and/or pathogenic mechanisms of a potential pathogen or are reducing the effectiveness of some of the plant defense mechanisms, both of which could play a role in increasing the prevalence and/or pathogenic outcomes of a microbe associated with this plant disease in O. monteverdensis. Ongoing studies include the development of pure fungal cultures from the roots of diseased plants, the determination of the taxonomic identity of these cultures, and the use of these pure cultures in transmission studies with young O. monteverdensis seedlings to fulfill Koch’s postulates to determine the etiology of this disease.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13071682/s1, Table S1: Sources for identification of potential fungal and bacterial pathogens and functional groups.

Author Contributions

Conceptualization, W.D.E. and D.A.H.; methodology, W.D.E., D.A.H., A.L. and P.S.; investigation, W.D.E. and D.A.H.; formal analysis, W.D.E., A.L. and P.S.; resources, W.D.E., D.A.H., A.L. and P.S.; data curation, W.D.E.; writing—original draft, W.D.E.; writing—review and editing, W.D.E., D.A.H., A.L. and P.S.; visualization, W.D.E.; project administration, W.D.E.; funding acquisition, W.D.E., D.A.H. and P.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Pace University Dyson College Dean, the Dyson Faculty Research Grant Committee, and the Provost Office for research support; by the Fundacion Conservacionista Costarricense Fundacon (www.fccmonteverde.org); and by a grant (#2022-10) from the Fondation Franklinia (www.fondationfranklinia.org).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All DNA sequencing data were submitted to the NCBI-Sequence Read Archive (SRA) repository with SRA Project Code PRJNA1019742 and PRJNA646467. All other data are available upon request.

Acknowledgments

The authors thank Luisa Moreno, Monteverde Institute, for assistance in permit acquisition and laboratory access; the FCC for their restoration efforts and access to field sites; and Pace University student Wen Chen for assistance in sample collection. We especially wish to thank Patricia Leandro and the staff at the CATIE lab for their soil C and N analysis assistance. This work was conducted through the Costa Rican permit CONAGEBIO MINAE # R-047-2019-OT-CONAGEBIO.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Hazelwood, K.; Beck, H.; Paine, C.T. Negative density dependence in the mortality and growth of tropical tree seedlings is strong and primarily caused by fungal pathogens. J. Ecol. 2021, 109, 1909–1918. [Google Scholar] [CrossRef]
  2. Williams, S.L.; Toda, M.; Chiller, T.; Brunkard, J.M.; Litvintseva, A.P. Effects of climate change on fungal infections. PLoS Pathog. 2024, 20, e1012219. [Google Scholar] [CrossRef] [PubMed]
  3. Burdon, J.J.; Zhan, J. Climate change and disease in plant communities. PLoS Biol. 2020, 18, e3000949. [Google Scholar] [CrossRef] [PubMed]
  4. Yang, L.N.; Ren, M.; Zhan, J. Modeling plant diseases under climate change: Evolutionary perspectives. Trends Plant Sci. 2023, 28, 519–526. [Google Scholar] [CrossRef] [PubMed]
  5. IUCN. The IUCN Red List of Threatened Species. Available online: https://www.iucnredlist.org/ (accessed on 20 November 2024).
  6. Joslin, J.D.; Haber, W.A.; Hamilton, D. Ocotea monteverdensis. The IUCN Red List of Threatened Species, e.T48724260A117762662. Available online: https://www.iucnredlist.org/species/48724260/117762662 (accessed on 20 November 2024).
  7. Powell, G.V.; Bjork, R.D. Habitat linkages and the conservation of tropical diversity as indicated by seasonal migrations of the three-wattled bellbird. Conserv. Biol. 2004, 18, 500–509. [Google Scholar] [CrossRef]
  8. Hamilton, D.; Singleton, R.; Joslin, J.D. Resource tracking and its conservation implications for an obligate frugivore (Procnias tricarunculatus, the three-wattled bellbird). Biotropica 2018, 50, 146–156. [Google Scholar] [CrossRef]
  9. Haber, W.; Zuchowski, W.; Bello, E. An Introduction to Cloud Forest Trees: Monteverde, Costa Rica, 2nd ed.; Mountain Gem Publications: Monteverde, Costa Rica, 2000. [Google Scholar]
  10. Wheelwright, N.; Nadkarni, N. (Eds.) Monteverde: Ecology and Conservation of a Tropical Cloud Forest; Bowdoin’s Scholars’ Bookshelf: Brunswick, ME, USA, 2014. [Google Scholar]
  11. Guindon, C. The Importance of Forest Fragments to the Maintenance of Regional Biodiversity Surrounding a Tropical Montane Reserve, Costa Rica. Ph.D. Thesis, Faculty of the School of Forestry and Environmental Studies, Yale University, New Haven, CT, USA, 1997. [Google Scholar]
  12. Harvey, C.A.; Guindon, C.; Haber, W.; Hamilton, D.; Murray, K.G. Importancia de Los Fragmentos de Bosque, Los Arboles Dispersos and Las Cortinas Rompevientos Para la Biodiversidad Local y Regional in Evaluación y Conservación de Biodiversidad en Paisajes Fragmentados de Mesoamérica; Editorial INBio: Santo Domingo de Heredia, Costa Rica, 2008. [Google Scholar]
  13. Sciara, J.; Fite, C. Environmental Factors That Contribute to the Reproductive Ecology of Ocotea monteverdensis; Report to the CIEE Sustainability Program: Monteverde, Costa Rica, 2015. [Google Scholar]
  14. Hamilton, D.; Parshall, T.; Vargas Berocal, L.; Alvarado Mendez, R.; Goldsmith, G.; Donovan, T. Optimizing the restoration practitioner’s role: Evaluating seedling characteristics, maintenance, and cost-effective strategies for tropical forest restoration. Ecol. Rest. 2025. submit. [Google Scholar]
  15. Milici, V.R.; Comita, L.S.; Bagchi, R. Foliar disease incidence in a tropical seedling community is density dependent and varies along a regional precipitation gradient. J. Ecol. 2024, 112, 642–655. [Google Scholar] [CrossRef]
  16. Gonzalez, J.; Poveda, L. Lauraceae. In Manual de plantas de Costa Rica, Vol. VI: Dicotiledoneas (Haloragaceae-hytolaccaceae); Hammel, B.E., Grayum, M.H., Herrera, C., Zamora, N., Eds.; Missouri Botanical Garden Press: Louis, MO, USA, 2007. [Google Scholar]
  17. Tropicos.org. Tropicos v3.4.2 Missouri Botanical Garden, 4344 Shaw Boulevard—Saint Louis, Missouri 63110. 2017. Available online: https://www.tropicos.org/name/17804917 (accessed on 20 June 2025).
  18. LaVal, R. Datos Casa LaVal. Available online: https://monteverde-institute.org/research-and-innovation-2/#DC1 (accessed on 15 October 2023).
  19. Anderson, J.M.; Ingram, J.S.I. Tropical Soil Biology and Fertility: A Handbook of Methods, 2nd ed.; Cambridge University Press CAB International: Wallingford, UK, 1993. [Google Scholar]
  20. Alef, K.; Nannipieri, P. Methods in Applied Soil Microbiology and Biochemistry; Elsevier, Academic Press: London, UK, 1995. [Google Scholar]
  21. Gardes, M.; Bruns, T.D. ITS primers with enhanced specificity for basidiomycetes—Application to the identification of mycorrhizae and rusts. Mol. Ecol. 1993, 2, 113–118. [Google Scholar] [CrossRef] [PubMed]
  22. Caporaso, J.G.; Lauber, C.L.; Walters, W.A.; Berg-Lyons, D.; Lozupone, C.A.; Turnbaugh, P.J.; Fierer, N.; Knight, R. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. USA 2011, 4, 516–4522. [Google Scholar] [CrossRef] [PubMed]
  23. McGee, K.M.; Eaton, W.D.; Shokralla, S.; Hajibabaei, M. Determinants of soil bacterial and fungal community composition toward carbon-use efficiency across primary and secondary forests in a Costa Rican conservation area. Microb. Ecol. 2018, 10, 423. [Google Scholar] [CrossRef] [PubMed]
  24. Sundquist, A.; Bigdeli, S.; Jalili, R.; Druzin, M.L.; Waller, S.; Pullen, K.M.; El-Sayed, Y.Y.; Taslimi, M.M.; Batzoglou, S.; Ronaghi, M. Bacterial flora-typing with targeted, chip-based pyrosequencing. BMC Microbiol. 2007, 7, 108. [Google Scholar] [CrossRef] [PubMed]
  25. White, T.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR: Protocols and Applications—A Laboratory Manual; Innis, N., Gelfand, D., Sninsky, J., White, T., Eds.; Academic Press: New York, NY, USA, 1990. [Google Scholar]
  26. Liu, K.-L.; Porras-Alfaro, A.; Kuske, C.R.; Eichorst, S.A.; Xie, G. Accurate, rapid taxonomic classification of fungal large-subunit rRNA genes. Appl. Environ. Microbiol. 2012, 78, 1523–1533. [Google Scholar] [CrossRef] [PubMed]
  27. Weiss, S.; Xu, Z.Z.; Peddada, S.; Amir, A.; Bittinger, K.; Gonzalez, A.; Lozupone, C.; Zaneveld, J.R.; Vázquez-Baeza, Y.; Birmingham, A.; et al. Normalization and microbial differential abundance strategies depend upon data characteristics. Microbiome 2017, 5, 27. [Google Scholar] [CrossRef] [PubMed]
  28. Anderson, M.J.; Gorley, R.N.; Clarke, R.K. PERMANOVA+ for Primer: Guide to Software and Statistical Methods; PRIMER-E Ltd.: Plymouth, UK, 2008. [Google Scholar]
  29. Anderson, M.J.; Willis, T. Canonical analysis of principal coordinates: A useful method of constrained ordination for ecology. Ecology 2003, 84, 511–525. [Google Scholar] [CrossRef]
  30. Clarke, K.R.; Gorley, R.N. PRIMER v6: User Manual; PRIMER-E Ltd.: Plymouth, UK, 2006. [Google Scholar]
  31. Spigaglia, P.; Barbanti, F.; Marocchi, F.; Mastroleo, M.; Baretta, M.; Ferrante, P.; Caboni, E.; Lucioli, S.; Scortichini, M. Clostridium bifermentans and C. subterminale are associated with kiwifruit vine decline, known as moria, in Italy. Plant Pathol. 2020, 69, 765–774. [Google Scholar] [CrossRef]
  32. Sun, Y.; Wang, M.; Mur, L.A.J.; Shen, Q.; Guo, S. Unravelling the roles of nitrogen nutrition in plant disease defenses. Int. J. Mol. Sci. 2020, 21, 572. [Google Scholar] [CrossRef] [PubMed]
  33. Mur, L.A.J.; Simpson, C.; Kumari, A.; Gupta, A.K.; Gupta, K.J. Moving nitrogen to the centre of plant defence against pathogens. Ann. Bot. 2017, 119, 703–709. [Google Scholar] [CrossRef] [PubMed]
  34. Crous, P.W. Taxonomy and phylogeny of the genus Mycosphaerella and its anamorphs. Fungal Divers. 2009, 38, 1–24. [Google Scholar]
  35. Crous, P.W.; Kang, J.C.; Braun, U. A phylogenetic redefinition of anamorph genera in Mycosphaerella based on ITS rDNA sequence and morphology. Mycologia 2001, 93, 1081–1101. [Google Scholar] [CrossRef]
  36. Mondal, S.N.; Gottwald, T.R.; Timmer, L.W. Environmental factors affecting the release and dispersal of ascospores of Mycosphaerella citri. Phytopathology 2003, 93, 1031–1036. [Google Scholar] [CrossRef] [PubMed]
  37. Roger, C.; Tivoli, B.; Huber, L. Effects of temperature and moisture on disease and fruit body development of Mycosphaerella pinodes on pea (Pisum sativum). Plant Pathol. 1999, 48, 1–9. [Google Scholar] [CrossRef]
  38. Vicent, A.; Bassimba, D.D.M.; Intrigliolo, D.S. Effects of temperature, water regime and irrigation system on the release of ascospores of Mycosphaerella nawae, causal agent of circular leaf spot of persimmon. Plant Pathol. 2011, 60, 890–908. [Google Scholar] [CrossRef]
  39. Wakeham, A.J.; Kennedy, R. Risk assessment methods for the ringspot pathogen Mycosphaerella brassicola in vegetable Brassica crops. Plant Dis. 2010, 94, 851–859. [Google Scholar] [CrossRef] [PubMed]
  40. Nelson, S. Cercospora Leaf Spot and Berry Blotch of Coffee; Plant Disease; PD-41; University of Hawaii: Honolulu, HI, USA, 2008; Available online: https://www.ctahr.hawaii.edu/oc/freepubs/pdf/PD-41.pdf (accessed on 20 November 2024).
  41. Gaitán, A.L. Compendium of Coffee Disease and Pests; Amer. Phytopathol. Soc.: Saint Paul, MN, USA, 2015; pp. 27–28. [Google Scholar]
  42. Rosling, A.; Cox, F.; Cruz-Martinez, K.; Ihrmark, K.; Grelet, G.-A.; Lindahl, B.D.; Menkis, A.; James, T.Y. Archaeorhizomycetes: Unearthing an ancient class of ubiquitous soil fungi. Science 2011, 333, 876–879. [Google Scholar] [CrossRef] [PubMed]
  43. Rosling, A.; Timling, I.; Taylor, D.L. Archaeorhizomycetes: Patterns of distribution and abundance in soil. In Genomics of Soil- and PlantAssociated Fungi. Soil Biology; Horwitz, B., Mukherjee, P., Mukherjee, M., Kubicek, C., Eds.; Springer: Berlin/Heidelberg, Germany, 2013; pp. 333–349. [Google Scholar]
  44. Clemmensen, K.E.; Finlay, R.D.; Dahlberg, A.; Stenlid, J.; Wardle, D.A.; Lindahl, B.D. Carbon sequestration is related to mycorrhizal fungal community shifts during long-term succession in boreal forests. N. Phytol. 2015, 205, 1525–1536. [Google Scholar] [CrossRef] [PubMed]
  45. Carrino-Kyker, S.R.; Kluber, L.A.; Petersen, S.M.; Coyle, K.P.; Hewins, C.R.; DeForest, J.L.; Smemo, K.A.; Burke, D.J.; Anderson, I. Mycorrhizal fungal communities respond to experimental elevation of soil pH and P availability in temperate hardwood forests. FEMS Microbiol. Ecol. 2016, 92, fiw024. [Google Scholar] [CrossRef] [PubMed]
  46. Urbina, H.; Breed, M.F.; Zhao, W.; Gurrala, K.L.; Andersson, S.G.E.; Ågren, J.; Baldauf, S.; Rosling, A. Specificity in Arabidopsis thaliana recruitment of root fungal communities from soil and rhizosphere. Fungal Biol. 2018, 122, 231–240. [Google Scholar] [CrossRef] [PubMed]
  47. Pinto-Figueroa, E.A.; Seddon, E.; Yashiro, E.; Buri, A.; Niculita-Hirzel, H.; van der Meer, J.R.; Guisan, A. Archaeorhizomycetes spatial distribution in soils along wide elevational and environmental gradients reveal co-abundance patterns with other fungal saprobes and potential weathering capacities. Front. Microbiol. 2019, 10, 656. [Google Scholar] [CrossRef] [PubMed]
  48. Castro, H.F.; Classen, A.T.; Austin, E.E.; Norby, R.J.; Schadt, C.W. Soil microbial community responses to multiple experimental climate change drivers. Appl. Environ. Microbiol. 2010, 76, 999–1007. [Google Scholar] [CrossRef] [PubMed]
  49. Lindahl, B.D.; de Boer, W.; Finlay, R.D. Disruption of root carbon transport into forest humus stimulates fungal opportunists at the expense of mycorrhizal fungi. ISME J. 2010, 4, 872–881. [Google Scholar] [CrossRef] [PubMed]
  50. Letcher, P.M.; Powell, M.J. A taxonomic summary and revision of Rozella (Cryptomycota). IMA Fungus 2018, 9, 383–399. [Google Scholar] [CrossRef] [PubMed]
  51. Gleason, F.; Carney, L.; Lilje, O.; Glockling, S. Ecological potentials of species of Rozella (Cryptomycota). Fungal Ecol. 2012, 5, 651–656. [Google Scholar] [CrossRef]
  52. Marano, A.V.; Pires-Zottarelli, C.L.A.; Barrera, M.D.; Gleason, F.H. Diversity, role in decomposition and succession of zoosporic fungi and straminipiles on submerged decaying leaves in a woodland stream. Hydrobiologia 2011, 659, 92–109. [Google Scholar] [CrossRef]
Microorganisms 13 01682 g001aMicroorganisms 13 01682 g001b
Figure 1. Ocotea monteverdensis seedlings at different stages of disease. (A) Non-diseased, healthy seedling; (B) seedling demonstrating early stages of chlorosis (arrow); (C) seedling demonstrating later stages of chlorosis and brown spots (arrows); (D) seedling dying with curled leaves.
Figure 1. Ocotea monteverdensis seedlings at different stages of disease. (A) Non-diseased, healthy seedling; (B) seedling demonstrating early stages of chlorosis (arrow); (C) seedling demonstrating later stages of chlorosis and brown spots (arrows); (D) seedling dying with curled leaves.
Microorganisms 13 01682 g001aMicroorganisms 13 01682 g001b
Microorganisms 13 01682 g002
Figure 2. Differences in the mean proportion of DNA sequences (MPS) of the potential bacterial pathogens and fungal pathogens (identified as described in the Section 2.3) in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Figure 2. Differences in the mean proportion of DNA sequences (MPS) of the potential bacterial pathogens and fungal pathogens (identified as described in the Section 2.3) in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Microorganisms 13 01682 g002
Microorganisms 13 01682 g003
Figure 3. Results of the Similarity Percentage (SIMPER) analyses comparing the % taxonomic dissimilarity of the communities of total bacteria, bacterial pathogens, total fungi, and fungal pathogens (identified as described in the Section 2.3) in plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Figure 3. Results of the Similarity Percentage (SIMPER) analyses comparing the % taxonomic dissimilarity of the communities of total bacteria, bacterial pathogens, total fungi, and fungal pathogens (identified as described in the Section 2.3) in plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Microorganisms 13 01682 g003
Microorganisms 13 01682 g004
Figure 4. The results of the DistLM analyses determining the relationships between the microbial community compositions and the soil environmental parameters in the healthy and diseased plant root soils. (a) The influence of environmental metrics on the fungal pathogen communities in the two soils. (b) The influence of the fungal pathogen communities on the environmental metrics in the two soils.
Figure 4. The results of the DistLM analyses determining the relationships between the microbial community compositions and the soil environmental parameters in the healthy and diseased plant root soils. (a) The influence of environmental metrics on the fungal pathogen communities in the two soils. (b) The influence of the fungal pathogen communities on the environmental metrics in the two soils.
Microorganisms 13 01682 g004
Table 1. Mean levels of environmental variables in diseased and healthy plant root soils and diseased and healthy plant bulk soils from Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Table 1. Mean levels of environmental variables in diseased and healthy plant root soils and diseased and healthy plant bulk soils from Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Diseased PlantHealthy PlantMann–Whitney
MetricRoot SoilRoot Soilp Value
TN58.13 ± 11.2744.03 ± 11.180.052
TOC763.5 ± 184.78665 ± 98.560.093
NH4+6.12 ± 3.012.91 ± 2.040.055
NO3−60.19 ± 27.6229.25 ± 7.920.046
Resp196.50 ± 71.66207.53 ± 28.820.735
pH5.92 ± 0.245.72 ± 0.150.282
% Sand69.28 ± 4.8569.26 ± 2.480.998
% Silt21.20 ± 3.9220.15 ± 2.060.951
% Clay9.51 ± 1.3210.58 ± 1.020.43
BD2.5 ± 0.62.6 ± 0.90.761
Diseased PlantHealthy PlantMann–Whitney
MetricBulk SoilBulk Soilp Value
TN88.01 ± 9.5155.67 ± 7.840.0001
TOC1064.83 ± 80.90753.33 ± 118.210.0003
NH410.03 ± 4.643.20 ± 2.400.0009
NO389.70 ± 28.8022.15 ± 6.030.0001
Resp278.70 ± 51.35237.52 ± 75.580.2952
pH6.05 ± 0.225.89 ± 0.190.207
% Sand78.82 ± 4.2981.62 ± 3.320.592
% Silt11.48 ± 3.6714.88 ± 3.640.235
% Clay6.31 ± 1.246.91 ± 1.320.843
BD2.4 ± 0.12.5 ± 0.070.761
Table 2. The results of ANOSIM and CAP assessments to determine the differences in the taxonomic community compositions of the total bacteria, bacterial pathogens, total fungi, and fungal pathogens in soils from the plant root soil and plant bulk soil of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Table 2. The results of ANOSIM and CAP assessments to determine the differences in the taxonomic community compositions of the total bacteria, bacterial pathogens, total fungi, and fungal pathogens in soils from the plant root soil and plant bulk soil of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
ANOSIM ResultsTotal BacteriaBacterial PathogensTotal FungiFungal Pathogens
Global test resultsR = 0.373, p = 0.0002R = 0.366, p = 0.0003R = 0.347, p = 0.002R = 0.323, p = 0.0001
Pairwise comparisonsR Statp ValueR Statp ValueR Stat p ValueR Statp Value
Healthy vs diseased plant root soils0.5660.0030.5630.0040.7520.0040.5540.002
Healthy vs diseased plant bulk soils0.4190.00020.4850.0020.0460.2730.2310.05
CAP ResultsTotal BacteriaBacterial PathogensTotal FungiFungal Pathogens
ComparisonsCAP R2p ValueCAP R2p ValueCAP R2p ValueCAP R2p Value
Healthy vs diseased plant root soils0.7880.00010.8760.00020.8010.00220.8550.0008
Healthy vs diseased plant bulk soils0.7060.00010.7230.00020.2230.00220.5990.008
Table 3. Results of the richness and diversity analyses for the total bacteria, bacterial pathogens, total fungi, and fungal pathogens (identified as described in the Section 2.3) in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Table 3. Results of the richness and diversity analyses for the total bacteria, bacterial pathogens, total fungi, and fungal pathogens (identified as described in the Section 2.3) in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year.
Total Bacteria Bacterial PathogensTotal FungiFungal Pathogens
Diversity Index ComparisonsRichnessDiversityRichnessDiversityRichnessDiversityRichnessDiversity
Healthy plant root soils330 ± 1135.64 ± 0.2814.50 ± 3.792.51 ± 0.2323.51 ± 23.522.74 ± 0.718.51 ± 7.581.76 ± 0.80
Diseased plant root soils335 ± 1295.54 ± 0.4314.15 ± 4.032.54 ± 0.2486.83 ± 19.564.26 ± 0.2620.17 ± 3.872.77 ± 0.21
Mann–Whitney p value0.4680.6390.8710.7740.00020.00030.0040.005
Healthy plant bulk soil322 ± 575.67 ± 0.1614.33 ± 2.582.58 ± 0.1742.67 ± 24.293.44 ± 0.6615.50 ± 7.692.44 ± 0.61
Diseased plant bulk soil308 ± 1325.51 ± 0.4214.57 ± 4.402.55 ± 0.2959.67 ± 36.393.72 ± 0.7313.17 ± 5.602.40 ± 0.43
Mann–Whitney p value0.8130.4040.9110.8270.2620.5020.5710.898
Table 4. The mean proportion of DNA sequences (MPS) of the bacterial genera with MPS values > 1% in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year. Statistically different mean values (all p values < 0.03) are indicated by an *.
Table 4. The mean proportion of DNA sequences (MPS) of the bacterial genera with MPS values > 1% in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year. Statistically different mean values (all p values < 0.03) are indicated by an *.
% MPS Diseased% MPS HealthyM-W
Bacterial TaxaFunctionPlant Bulk SoilPlant Bulk Soilp Value
ClostridiumCCD, N-Fix, AMO, possible plant pathogen3.50 ± 2.820.74 ± 0.780.001 *
UdaeobacterProbable CCD3.45 ± 1.626.19 ± 1.980.005 *
GemmataProbable CCD3.42 ± 0.874.80 ±1.770.029 *
PseudolabrysCCD3.22 ± 1.540.94 ± 0.920.003 *
SolibacterCCD3.20 ± 1.746.52 ± 3.400.105
PedomicrobiumCCD3.09 ± 1.150.68 ± 0.200.001 *
VicinamibacterCCD2.65 ± 0.871.27 ± 1.100.015 *
BradyrhizobiumCCD, N-Fix, AMO2.37 ± 0.552.28 ± 0.830.355
BacillusCCD, N-Fix, AMO, possible plant pathogen1.84 ± 0.942.73 ± 1.210.015 *
MethyloligellaMethylotrophs1.81 ± 0.730.56 ± 0.600.004 *
RokubacterialesUnclear1.70 ± 0.670.44 ± 0.460.002 *
BlastocatelliaCCD1.55 ± 1.200.22 ± 0.080.001 *
Acidobacteria (subgroup 2)CCD1.51 ±2.356.38 ± 3.450.003 *
MycobacteriumCCD1.51 ± 0.560.42 ± 0.290.001 *
GaiellaCCD1.45 ± 0.670.48 ± 0.760.015 *
NitrospiraAMO1.45 ± 0.661.15 ± 0.360.105
PirellulaAMO1.28 ± 0.540.33 ± 0.51 0.002 *
ReyranellaUnclear1.22 ± 0.530.40 ± 0.380.003 *
ActinobacteriaCCD1.13 ± 0.540.11 ± 0.170.001 *
XiphinematobacterUnclear1.11 ± 0.923.05 ± 1.300.001 *
SolirubrobacterUnclear1.10 ± 0.490.17 ± 0.290.001 *
% MPS Diseased% MPS HealthyM-W
Bacterial TaxaFunctionPlant Root SoilPlant Root Soilp Value
XanthobacterCCD8.03 ± 4.106.77 ± 0.570.628
FlavobacteriumCCD6.80 ± 13.044.67 ± 1.780.138
UdaeobacterProbable CCD4.76 ± 3.194.01 ±1.811
SolibacterCCD4.65 ± 3.881.86 ± 0.360.731
Acidobacteria (Subgroup 2)CCD4.26 ± 3.721.79 ± 0.830.534
GemmataProbable CCD3.27 ± 2.323.66 ± 1.321
ClostridiumCCD, N-Fix, AMO, plant pathogen3.18 ± 2.820.36 ± 0.380.019 *
BacillusCCD, N-Fix, AMO, plant pathogen2.73 ± 0.612.31 ± 1.270.945
AcidobacteriaCCD2.64 ± 1.832.08± 0.400.628
RhizobiumCCD, N-Fix, AMO2.39 ± 2.720.0.33 ± 0.070.731
BradyrhizobiumCCD, N-Fix, AMO2.31 ± 0.652.86 ± 0.610.181
XiphinematobacterUnclear2.89 ± 2.001.59 ± 0.570.775
KtedonobacterUnclear1.94 ± 1.780.28 ± 0.180.153
ElsteralesUnclear1.81 ± 1.800.74 ± 0.750.836
BryobacterCCD1.79 ± 1.240.87 ± 0.230.253
AcidibacterUnclear1.33 ± 0.650.83 ± 0.31 0.138
AcidothermusCCD1.18 ± 0.721.00 ± 0.260.295
NitrospiraAMO1.18 ± 0.451.00 ± 0.270.181
BurkholderiaCCD, N-Fix, AMO, plant pathogen1.11 ± 0.923.05 ± 1.300.008 *
Table 5. The mean proportion of DNA sequences (MPS) of the fungal genera with MPS values > 1% in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year. Statistically different mean values (all p values < 0.05) are indicated by an *.
Table 5. The mean proportion of DNA sequences (MPS) of the fungal genera with MPS values > 1% in the plant root soils and plant bulk soils of diseased and healthy Ocotea monteverdensis planted in a Costa Rican primary forest for 1 year. Statistically different mean values (all p values < 0.05) are indicated by an *.
% MPS Diseased% MPS HealthyM-W
Fungal TaxaFunctionPlant Bulk SoilPlant Bulk Soilp Value
ApiotrichumCCD45.28 ± 29.1148.26 ± 29.960.3367
RozellaFungal, Oomycete, algae parasite4.17 ± 6.960.88 ± 0.910.259
MycosphaerellaPlant pathogen3.71 ± 2.824.93 ± 4.320.7478
SaitozymaCCD10.09 ± 26.883.15 ± 4.790.0782
GlomusARM1.54 ± 3.132.88 ± 3.710.1488
DactylonectriaPlant pathogen3.77 ± 8.390.01 ± 0.020.4418
IlyonectriaPlant pathogen1.28 ± 0.848.68 ± 5.160.0039 *
MonochaetiaPlant pathogen1.03 ± 2.520.02 ± 0.010.3173
% MPS Diseased% MPS HealthyM-W
Fungal TaxaFunctionPlant Root SoilPlant Root Soilp Value
ApiotrichumCCD26.58 ± 9.5262.07 ± 19.020.0163 *
RozellaFungal, Oomycete, algae parasite13.30 ± 7.220.65 ± 0.750.0038 *
MycosphaerellaPlant pathogen11.65 ± 4.643.20 ± 1.840.0039 *
ArchaeorhizomycesPlant root-associated11.26 ± 6.540.23 ± 0.500.0033 *
SaitozymaCCD5.17 ± 5.039.74 ± 5.000.0781
DipodascusPlant pathogen2.76 ± 4.660.01 ± 0.010.0021 *
DidymellaPlant pathogen2.37 ± 2.250.09 ± 0.210.0028 *
AcrocalymmaPlant pathogen1.97 ± 1.590.37 ± 0.660.0225 *
GlomusARM1.48 ± 1.163.00 ± 1.890.1093
TremellaCCD, fungal parasite1.01 ± 1.540.06 ± 0.150.0493 *
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MDPI and ACS Style

Eaton, W.D.; Hamilton, D.A.; Lemenze, A.; Soteropoulos, P. Initial Analysis of Plant Soil for Evidence of Pathogens Associated with a Disease of Seedling Ocotea monteverdensis. Microorganisms 2025, 13, 1682. https://doi.org/10.3390/microorganisms13071682

AMA Style

Eaton WD, Hamilton DA, Lemenze A, Soteropoulos P. Initial Analysis of Plant Soil for Evidence of Pathogens Associated with a Disease of Seedling Ocotea monteverdensis. Microorganisms. 2025; 13(7):1682. https://doi.org/10.3390/microorganisms13071682

Chicago/Turabian Style

Eaton, William D., Debra A. Hamilton, Alexander Lemenze, and Patricia Soteropoulos. 2025. "Initial Analysis of Plant Soil for Evidence of Pathogens Associated with a Disease of Seedling Ocotea monteverdensis" Microorganisms 13, no. 7: 1682. https://doi.org/10.3390/microorganisms13071682

APA Style

Eaton, W. D., Hamilton, D. A., Lemenze, A., & Soteropoulos, P. (2025). Initial Analysis of Plant Soil for Evidence of Pathogens Associated with a Disease of Seedling Ocotea monteverdensis. Microorganisms, 13(7), 1682. https://doi.org/10.3390/microorganisms13071682

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