The Distribution Characteristics of Trichoderma in Turf and Its Inhibitory Effect on Rhizoctonia solani

Abstract

Effective disease management is crucial for maintaining healthy turf. Trichoderma agents have emerged as a promising strategy for controlling turf diseases while reducing reliance on chemical fungicides. However, the distribution, diversity, and biocontrol potential of Trichoderma in turf ecosystems remain poorly understood. This study investigated Trichoderma strains isolated from rhizosphere soil of turf under different environmental and management conditions. Genetic distances were used to assess diversity, while co-culture assays evaluated inhibitory activity against Rhizoctonia solani. The Wilcoxon test was used for comparing diversity and antagonistic potential across environmental factors. The study identified Trichoderma brevicompactum and Trichoderma harzianum as the dominant species in turf ecosystems. Trichoderma diversity was highest in healthy turf under moderate management. However, strains from diseased turf showed stronger inhibitory effects on Rhizoctonia solani, suggesting that pathogen pressure and plant stress responses may enrich antagonistic Trichoderma. These findings provide valuable insights for the isolation and screening of Trichoderma species for effective biocontrol in turf management.

1. Introduction

Turf, a crucial component of urban ecosystems [1], serves as an important space for people to relax and engage in recreational activities in natural environments. However, as turf grows, it is frequently affected by various diseases. Critical diseases may destroy a large area of green cover and, furthermore, reduce the ecology and landscape function of turf [2,3].

Chemical fungicides are used to control turf diseases [4,5]. However, the long-term and heavy use of chemical fungicides may lead to serious environmental pollution, which further affects the health of environments [6,7,8] and contributes to the development of fungicide resistance of the pathogens [5,9].

Biological control strategies represent a promising alternative to chemical fungicides [10]. They utilize biological agents/metabolites to reduce the population of pathogens and disease severity [11]. Biological control offers several opportunities for improved disease control, especially where conventional approaches are limited or compromised [12]. In environments with frequent human interaction, such as turf, it is vital to adopt biological control methods to minimize human exposure to chemical pesticides. However, research on using biological control to manage turf diseases is limited, and there are few biological control products available for turf problems.

Trichoderma spp. is a famous bio-control fungus, which is cosmopolitan and ubiquitous [13]. It can control many plants’ fungal diseases, such as strawberry’s Armillaria root rot, eggplant’s wilt disease [14], tomato’s fusarium wilt disease [15], and mango’s fusarium wilt disease [16]. Limited studies have reported the biological control ability of Trichoderma against turf pathogens. It is reported that the organic composts enriched with T. atroviride on turf grass increased turf sustainability by promoting biological control activity against diseases [17]. Similarly, Lo reported that the application of T. harzianum strain 1295-22 showed high efficacy in controlling Pythium root rot, brown patch, and dollar spot of creeping bent grass [18]. The research on turf root zones also indicated the remarkable effects of Trichoderma against turf fungal pathogens [19].

In general, previous studies have confirmed the inhibitory property of Trichoderma isolated from turf against turf pathogens. However, the distribution characteristics of Trichoderma in turf remain unknown. This research gap highlights the lack of guidance on isolating and screening antagonistic Trichoderma species in turf. In this study, we concentrate on the distribution of Trichoderma in rhizosphere soil of turf under different environmental conditions, such as shaded/unshaded, diseased/healthy, different turf management levels, and different frequency of fungicide application. The diversity of Trichoderma and their antagonistic ability against Rhizoctonia solani, which causes turf brown patch disease, were evaluated. This study was conducted to fulfill the following two objectives: (i) evaluating the distribution characteristics of Trichoderma in turf; (ii) understanding the relationship between the Rhizoctonia solani inhibitory ability of Trichoderma and its habitat features.

2. Materials and Methods

2.1. Sample Collection

The samples were collected from eight turf or lawn sites in Beijing, China, comprising three golf courses, two park lawns, two urban green spaces, and one turf experimental field. To ensure representative coverage of different environmental factors, three sampling points were selected from each site. A total of 24 study points from 8 study sites were selected under the following environmental conditions: 1. shaded/unshaded; 2. diseased/healthy; 3. different turf management levels, namely, high, low, and middle management (Table S1). The management levels were characterized based on the frequency of fertilizer and broad-spectrum fungicide application, as well as the frequency of irrigation and mowing. The most commonly used broad-spectrum fungicides at the sampling sites were carbendazim, propiconazole, and thiophanate-methyl, while the most frequently applied fertilizer was NPK compound fertilizer. The turf management rating criteria were modified from the determination described by Kaufmann [20]. High-level management included frequent mowing, fertilization, pest control, and often used irrigation. Middle level management involved regular mowing with occasional weed control and fertilization. Low-level management could be considered, as it had no mowing, only occasional brushing and weed control.

As rhizosphere inhabitants, Trichoderma mediates interactions with other soil microorganisms, plants, and arthropods at multiple trophic levels. Rhizosphere soil is considered to be a favorable habitat [21]. In this study, the non-rhizosphere soil was shaken down from the plant root. The soil connected to the root surface was then forcefully shaken down to obtain the rhizosphere soil [22,23]. The samples were collected from June to September 2018, during the peak period of turfgrass disease occurrence. At each study point, 5 samples were collected from rhizosphere soil, mixed, packaged in sterile plastic bags, and stored at 4 °C. The Trichoderma in the samples were then isolated and identified during the subsequent 1 month.

2.2. Isolation of Trichoderma Strains

One gram of each sample was mixed with 10 mL of sterile water in a 15 mL centrifuge tube and fully vortexed (Vortex–Genie 2, Fisher scientific, Waltham, MA, USA). Tenfold serial dilutions were performed using nine milliliters of saline. A 100 μL aliquot from each dilution (10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶) was inoculated onto PDA (200 g potato, 20 g glucose, 20 g agar, and 1 L distilled water) containing two types of antibacterial compounds in Petri dishes. PDA1 was supplemented with 1 mL·L⁻¹ lactic acid to inhibit bacterial growth, while PDA2 was supplemented with a combination of three antibacterial agents (0.1 g·L⁻¹ streptomycin, 0.3 g·L⁻¹ chloramphenicol, and 0.02 g·L⁻¹ Rose Bengal). The inoculated plates were incubated at 25 °C for 7 d. All the experiments were conducted in triplicate. Growing mycelial cultures were purified by transferring to fresh PDA and Trichoderma strains were characterized based on macroscopic and microscopic morphology. All Trichoderma colonies were twice sub-cultured to purify on PDA and pure cultures were stored in 20% glycerol at −80 °C.

2.3. DNA Extraction, Amplification, Sequencing and Bioinformatics

The pure cultures of Trichoderma were grown on PDA for three days. Genomic DNA was extracted with the E.Z.N.A ^® Fungal DNA Kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer’s instructions. The ITS regions of the rRNA genes were amplified using the ITS1 (5′-TCCG TAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers. The nuclear translation elongation factor1 gene was amplified using the Ef728M (5′-CATCGAGAAGTTCGAGAAGG-3′) and Tef1αR (5′-GCCATCCTTGGGAGATACCAGC-3′) primers. The PCR reaction parameters were as follows: 5 min at 94 °C, followed by 35 cycles (94 °C for 1 min, 60 °C for 30 s, extension at 72 °C for 1 min) and a final 10 min extension period at 72 °C. The PCR products were sequenced by RuiBiotech (Beijing, China). Bidirectional sequences were assembled and then aligned using MEGA X [24]. The ITS and tef1 sequences were analyzed using BLAST search tool at NCBI (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov (accessed on 12 February 2025)). The Trichoderma species were identified based on the ITS sequence similarity value (when it has ≥76% sequence similarity with that in the dataset) and tef1 sequence similarity value (≥97%) [25]. The unclassified isolates, which did not match any reference strains in the database or were annotated to unknown species, were further investigated through phylogenetic analyses. Two-pair phylogenetic analysis of ITS and TEF1 sequence matrixes was computed using PhyliSuite with MrBayes mode and the maximum likelihood (ML) method [26].

ITS and TEF1 sequence data were combined for OTU analysis using FaBox [27]. When OTU has a higher taxonomic resolution than species, a single species may comprise multiple OTUs [28]. A distance matrix was generated from the alignment according to the Phylip software DNAdist [29] and then used for cluster analysis into OTUs by Mothur [30] at a 99% sequence similarity threshold. OTUs were the basic unit for diversity analysis in the present study, and the number of isolate sequences in each OTU indicated the abundance of OTUs at the study sites. ITS and TEF1 sequences were submitted to GenBank (MW454967–MW455047, PP908539–PP908619).

2.4. In Vitro Inhibition of Trichoderma Isolates Against R. solani

The R. solani strain was isolated from diseased turf. The strains were characterized based on macroscopic and microscopic morphology and its ITS sequence similarity was analyzed using the BLAST search tool as described above. In vitro inhibition tests were carried out by the dual culture technique [31]. Nine-millimeter diameter disks of Trichoderma and R. solani were obtained from the edge of actively growing three-day-old cultures in PDA. Each disk was placed at distances of 85 mm apart opposite each other in 90 mm diameter Petri plates containing PDA. Plates inoculated only with R. solani served as the control. The plates were incubated at 25 °C in darkness for 7 days. All the experiments were conducted in quadruplicate. Plates of dual cultures and controls were photographed on day 7, and the radius of each colony was measured using vernier caliper to determine the mycelial growth area for each isolate. The seven-day co-cultivation period is widely recognized as an optimal duration for assessing inhibition, as it allows sufficient interaction between microorganisms without nutrient exhaustion [32,33,34,35]. The percentage of inhibition of radial growth (PIRG) was calculated using the following formula [36]:

$$\mathrm{P}\mathrm{I}\mathrm{R}\mathrm{G}={\displaystyle \frac{\mathrm{R}\mathrm{c}-\mathrm{R}\mathrm{t}}{\mathrm{R}\mathrm{c}}}\times 100\%,$$

where Rc is the radial growth of the pathogen in the control (mm) and Rt is the radial growth of the pathogen in the presence of Trichoderma (mm).

2.5. Statistics Analysis

To identify the variation explained by environmental factors (shade, fungicide, disease, management), multivariate analysis of variance was performed using the normalized data and alpha diversity using the R Package Vegan 2.6-8. Furthermore, Shannon and Gini–Simpson indices were used to reflect alpha diversity. The relationship between the environmental factors and alpha diversity of Trichoderma among the sampled sites was analyzed using Wilcoxon test. The relationship between environmental factors and the inhibitory ability of Trichoderma against Rhizoctonia solani among the sampled sites was analyzed using t-test. To identify the relationship between the frequency of broad-spectrum fungicide use and alpha diversity or antifungal ability, the scale-appropriate correlation tests (Pearson, London, UK) were carried out. The differences in inhibitory effects of different strains against Rhizoctonia solani were analyzed using ANOVA for significance.

3. Results

3.1. Isolation and Identification of Trichoderma Species from Turf

Based on ITS sequence similarity against the published sequences and investigation through phylogenetic analyses (Figure S1), 81 isolates belonging to 11 species were identified as Trichoderma spp. The correspondence between the numbering of all Trichoderma isolates, their original ID, and collection sites are provided in Table S2. Sixteen Trichoderma strains were isolated from sampling point 24, which had the largest number of isolates. Sampling point 17 had the largest number of Trichoderma species with four species. In addition, some sampling points lacked Trichoderma strains, possibly due to environmental factors. Among the 81 isolates, T. brevicompactum was the most frequently isolated species, with a total of 37 isolates. T. harzianum was the second most common species, with 20 isolates. These two Trichoderma species constituted the majority of the isolates and were distributed across multiple sampling points. T. brevicompactum and T. harzianum were the most common species in turf (Figure 1).

3.2. Alpha Diversity Indices for Trichoderma Communities

The alpha diversity of Trichoderma in turf under different environmental factors was evaluated. With the change in environmental factors (disease, management, shade), the Gini–Simpson diversity index did not change significantly (p ≥ 0.05) (Figure 2D–F), while the Shannon diversity index was significantly affected by disease level (p < 0.01) (Figure 2A) and turf management level (p < 0.01) (Figure 2B).

The Shannon diversity index of Trichoderma communities from healthy turf was significantly higher than that from diseased turf. The Shannon diversity index in moderately managed turf was significantly higher than in both low-managed (p < 0.05) and high-managed turf (p < 0.01). Trichoderma is more diverse in healthy turf with middle-management level.

3.3. Relationship Between Turf Management Measures and Trichoderma Diversity

To further analyze the influence of turf management practices on the distribution of Trichoderma in turf, detailed information was obtained by consulting the turf managers. Unfortunately, some sampling points could not provide accurate data on irrigation or fertilization frequency, and these factors were only described in terms of intensity level. Turf with low management levels was rarely irrigated and never fertilized, which may have contributed to the low diversity of Trichoderma communities due to a lack of soil nutrients or water. Additionally, the diversity of Trichoderma communities was negatively correlated with the use of broad-spectrum fungicides. As the use of broad-spectrum fungicides increased, the Shannon index of Trichoderma communities decreased significantly (p < 0.01) (Figure 3A), suggesting that Trichoderma spp. was inhibited by broad-spectrum fungicides.

3.4. Inhibition Effect of Trichoderma Against R. solani

The Trichoderma isolates were co-cultured with R. solani for seven days. The results showed that R. solani covered the entire plate after seven days (Figure S2) but was inhibited when co-cultured with Trichoderma (Figure 4). When co-cultured with Trichoderma, the R. solani colony failed to cover the entire Petri dish, and its hyphae turned brown and twisted. Some Trichoderma strains, including strains 3, 4, 6, 7, 12, 14, 16, 26, 33, 35, 47, 48, 49, 50, 58, and 70, completely inhibited the growth of R. solani within seven days of incubation, and R. solani mycelium was not observed on the plates.

3.5. The Distribution of Inhibitory Trichoderma Against Rhizoctonia solani

Trichoderma’s inhibitory ability was evaluated using PIRG. Among the 81 Trichoderma isolates, the Trichoderma strains isolated from diseased turf had significantly (p < 0.01) higher PIRG than those isolated from healthy turf (Figure 5A). There was no significant (p ≥ 0.05) difference in the antagonistic ability of Trichoderma strains isolated from turf under different shading conditions (Figure 5B) or different turf management levels (Figure 5C), including different frequencies of broad-spectrum fungicide use (Supplementary Figure S3). Interestingly, not only did the Trichoderma strains isolated from diseased turf have high PIRG, but some strains from healthy turf also exhibited high inhibitory ability. To determine whether the highly inhibitory Trichoderma were selected due to competition with the pathogen, correlation tests between the Shannon index (Figure 6A) or Gini–Simpson index (Figure 6B) of Trichoderma communities and PIRG were conducted. However, there was no significant correlation between Trichoderma communities and PIRG, indicating that inhibitory Trichoderma strains were not only selected but may have accumulated due to other factors.

4. Discussion

Disease management is a challenging and crucial part of turf management. This study focuses on the distribution of Trichoderma in turf and its antagonistic ability. The results show that T. brevicompactum and T. harzianum were the most frequently isolated Trichoderma species from turf. Interestingly, T. harzianum was the most isolated Trichoderma species in natural grassland in Ma’s study [37] and T. longibrachiatum was the most isolated Trichoderma species in grassland in Dou’s study [38]. Joshi isolated 38 Trichoderma strains from the rhizosphere soil of sugarcane, with T. harzianum being the most prevalent (21 strains), followed by T. longibrachiatum (9 strains) [39]. Similarly, Anhar isolated Trichoderma from rice, with T. harzianum being the dominant species [40]. None of them isolated a large number of T. brevicompactum strains from natural grassland or agriculture ecosystems. Turf may be a special habitat for Trichoderma that differs from natural grassland and agricultural ecosystems. Trichoderma is more abundant in healthy turf with middle-management level. In this study, the diversity of Trichoderma community in healthy turf was significantly (p < 0.01) higher than that in diseased turf. The infection of the pathogen caused strong disturbance to the original microbial environment of plants [41]. The invasion increased the interaction of fungal communities in rhizosphere soil, further decreasing the diversity of fungal communities in rhizosphere soil [42]. As an important soil fungus, Trichoderma was also affected by pathogen infection, leading to a decrease in its community diversity in this study.

Frequent fertilization and irrigation not only bolster overall fungal diversity but also contribute to the greater Trichoderma community diversity observed in turf under moderate management. Fungal community is more sensitive to fertilization than bacterial community [43,44]. For rhizosphere soil microbial community, nitrogen addition [45] and the use of organic fertilizer [46] increase the diversity of fungal community. Soil moisture content emerges as a paramount determinant in shaping fungal diversity [47]. Proper irrigation and promoting a dry–wet cycle can improve the diversity of soil fungal communities [48]. Conversely, insufficient irrigation depletes soil moisture, which, in turn, diminishes fungal biomass [49]. In this study, turf under low management received minimal fertilization and irrigation, a condition that likely contributed to the reduced Trichoderma community diversity.

In addition, we found that the use of broad-spectrum fungicides has a significant negative effect on the diversity of the Trichoderma community. The frequent use of broad-spectrum fungicides led to the low diversity of Trichoderma community in turf with high management level. In this study, carbendazim, propiconazole, and thiophanate-methyl were the most commonly used fungicides to control various turf diseases. In a previous study, carbendazim, propiconazole, and thiophanatemethyl were tested for their ability to inhibit mycelial growth. At both the recommended and half-recommended rates, these fungicides exhibited 100% inhibition of mycelial growth in all Trichoderma species [50]. The use of broad-spectrum fungicides reduces the survival of beneficial microorganisms, such as Trichoderma. The extensive use of broad-spectrum fungicides may induce self-healing in turf [51]. Chemical control and biological control are two distinct effective strategies for managing turf diseases. The results of this study indicate that most Trichoderma strains are not effectively compatible with broad-spectrum chemical pesticides, suggesting that they may be more suitable for use as individual agents in turf disease management. Developing Trichoderma-based formulations with enhanced tolerance to various chemical pesticides, thereby enabling their combined application, represents a promising avenue for future research.

Previous studies have shown that microorganisms isolated from diseased plants tend to exhibit stronger antagonistic activity against those pathogens [52]. In this study, most Trichoderma were isolated from healthy turf, but the Trichoderma strains from diseased turf exhibited higher average inhibitory ability against R. solani. Since Trichoderma strains with strong inhibitory abilities were isolated both before and after pathogen infection, it can be inferred that, during pathogen invasion, environmental changes selectively enrich Trichoderma with strong antagonistic abilities from the initially diverse Trichoderma community. When pathogens invade, ecological niches of fungi with weaker competitive abilities are taken over by them, while more competitive Trichoderma strains are naturally selected. Interestingly, if inhibition was the only factor, the inhibitory ability of isolated Trichoderma strains should increase as pathogen invasion progresses. However, there is no significant negative correlation between Trichoderma diversity and antagonistic ability in this study. Antagonistic Trichoderma is not only selected in the process of competition but also may engage in plant–microorganism community interaction to induce the accumulation of antagonistic Trichoderma.

When the plant is infected by pathogens, it seeks help by reshaping microbial habitats and chemically inducing beneficial microorganisms to form a defense [53,54]. This initiative is called ‘cry-for-help’. The ‘cry-for-help’ hypothesis suggests that stressed plants release specific metabolites, particularly primary metabolites, which recruit beneficial microbes and constrain pathogen development [53]. The infection of Arabidopsis increases L-malic acid exudation, which leads to an increase in root colonization by resistance-inducing Bacillus subtilis [55]. Some special metabolites can attract the enrichment of beneficial microbes as signaling mechanisms [56]. The enrichment of highly inhibitory Trichoderma in the turf rhizosphere may be a response to a ‘cry for help’ from the turfgrass.

5. Conclusions

This study elucidates the distribution patterns of Trichoderma in turf and its inhibitory effects on Rhizoctonia solani. The findings indicate that Trichoderma brevicompactum was the most frequently isolated species in turf, suggesting that turf provides a distinct ecological niche compared to natural grasslands and farmlands. The highest diversity of Trichoderma was observed in moderately managed and healthy turf due to reduced pathogen competition, weaker inhibition by broad-spectrum fungicides, enough water, and nutrient availability. Notably, isolates from diseased turf exhibited stronger inhibitory effects against Rhizoctonia solani, suggesting that pathogenic stress selectively enriches antagonistic Trichoderma. These findings provide valuable insights into the ecological dynamics of Trichoderma in turf and offer practical guidance for the targeted isolation and application of Trichoderma strains in turf disease management.