Biocontrol of Calonectria causing root rot in Eucalyptus

Biocontrol of Calonectria causing root rot in Eucalyptus | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Biocontrol of Calonectria causing root rot in Eucalyptus H. Andrés Villar , Sara García , Elena Tavares , Eduardo Abreo doi: https://doi.org/10.1101/2025.09.02.673731 H. Andrés Villar 1 National Institute of Agriculture Research (INIA)-Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: villarandres{at}gmail.com Sara García 1 National Institute of Agriculture Research (INIA)-Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elena Tavares 2 UPM Biofore , Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eduardo Abreo 1 National Institute of Agriculture Research (INIA)-Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Calonectria spp. are among the most important pathogens affecting Eucalyptus production, causing severe root rot and significant losses in nurseries. In this study, we established a collection of locally isolated strains of Trichoderma spp. and Bacillus spp., complemented with commercial formulations, to evaluate their potential as biocontrol agents (BCAs) against Calonectria . Leaf bating in vitro assays were conducted using naturally colonized, non-sterilized substrates to closely simulate nursery conditions. Several locally isolated Trichoderma strains (T3, T5, T6, and T7), as well as two commercial products (PC1 and PC2), consistently reduced the incidence of Calonectria infection, indicating their capacity to decrease pathogen inoculum levels in the substrate. The reproducibility of these effects across independent trials underscores the robustness of Trichoderma as reliable BCAs. Altogether, these findings highlight the potential of locally isolated Trichoderma strains as promising alternatives for managing Calonectria root rot in Eucalyptus nurseries, while reinforcing the need for further trials under commercial greenhouse conditions to confirm their efficacy in real production systems. Introduction The genus Eucalyptus represents a major component of the forestry sector in Uruguay, with E. grandis and E. dunnii accounting for the largest planted areas in the country ( MGAP-DGF, 2025 ). The cultivation of these species has contributed substantially to the national economy. However, several biotic factors have constrained its productivity, particularly root and stem diseases caused by soilborne pathogens ( Tellechea, 2010 ). Among these, Calonectria species are recognized as serious threats to young Eucalyptus plants, causing root rot, stem lesions, and damping-off, often resulting in significant plant losses and reduced plantation establishment rates ( Alfenas et al., 2015 ; Bose et al., 2023 ). This pathogen is one of the most frequently found in Uruguayan forestry nurseries (Martinez et al., 2021). Management of Calonectria in nurseries typically relies on cultural practices and chemical fungicides. However, the use of fungicides is often limited by environmental regulations, concerns over human and environmental health, the development of pathogen resistance and the reduced number of active ingredients, while cultural management is insufficient due to lack of resistant plant genotypes and the pathogen’s survival ability. The utilization of contaminated substrate or soil represents the main source of inoculum (Lui and Chen, 2017), which makes it a critical point in the disease cycle to be targeted. This stage is particularly suitable for control through biological control agents (BCAs) that can suppress pathogen populations. Trichoderma and Bacillus species are among the most widely studied BCAs for the management of soilborne plant pathogens, due to their well-documented antagonistic activity and the greater feasibility of formulation for biological control applications ( Meher et al., 2020 ; Liu et al., 2022 ; Zhang et al., 2023 ; Yang et al., 2024 ). Both genera possess multiple mechanisms of action, including mycoparasitism, antibiosis, competition for nutrients and space, and induction of systemic resistance in host plants (Guzman-Guzman et al, 2023; Zhang et al., 2023 ). Their efficacy has been demonstrated against various pathogens in forestry, horticultural, and agricultural systems, and commercial formulations of both genera are available in many countries. Specifically, various authors have shown that Calonectria can be inhibited by the used of Trichoderma and Bacillus strains (Vitale et al., 2011; Kong and Hong, 2017 ; Carvalho et al., 2018 ; Paz et al., 2018 ; Samavat, 2022 ; Zang et al., 2023 ). The performance of BCAs can be influenced by environmental conditions, substrate and plant characteristics and pathogen populations, making the selection of locally adapted strains essential for effective disease suppression ( Hoitink and Boehm 1999 ; Villar et al., 2022 ). In this context, the isolation and evaluation of native Trichoderma and Bacillus strains from Eucalyptus nursery environments can increase the likelihood of identifying antagonists with enhanced adaptability and efficacy under local conditions. Including commercially available BCAs in comparative assays provides a performance benchmark and may identify candidates for immediate application in production systems. The aim of this study was to isolate, identify and evaluate the potential of local Trichoderma and Bacillus strains to reduce Calonectria inoculum under controlled conditions. Materials and Methods Calonectria The Calonectria isolate 3V from the Biofore Uruguay and INIA Las Brujas-Uruguay collection was used. This isolate was originally obtained from nursery substrate and characterized by its capacity of causing root rot ( Villar et al., 2025 ). Trichoderma and Bacillus A collection of Trichoderma and Bacillus strains was generated from plant and substrate material obtained from commercial Eucalyptus nurseries representing the environments where they are intended to act. The process was carried out in two steps: i) Field sampling. Three-month-old seedlings of E. dunnii and E. grandis with superior growth and sanitary status were sampled from three commercial Eucalyptus nurseries located in the Paysandú region, Uruguay. A total of 125 seedlings were collected (25 for each clone–nursery combination). Additionally, commercial substrate (BCS: 70% composted by pine bark and 30% peat moss; produced by Biofore, Uruguay) widely used in commercial nurseries was also sampled. ii) Isolation. Trichoderma and Bacillus strains were isolated from the collected seedlings (endophytic - stem and root- and rhizospheric) and substrate. For endophyte isolation, stems and roots were washed with tap water, cut into ∼1 cm segments, and surface-sterilized for 120 s in 3% sodium hypochlorite followed by 90 s in 70% ethanol. Samples were rinsed three times in sterile distilled water, and three 100 µL aliquots from the final rinse were plated on potato dextrose agar (PDA; Oxoid, UK) to verify surface sterilization. Stem and root segments were plated on PDA acidified (2 drops of lactic acid per 100 mL) for Trichoderma isolation or Luria–Bertani (LB) agar for Bacillus isolation, with five stem and five root segments per plate. A total of 25 plates were prepared and incubated at 25 °C. For isolation from rhizosphere and substrate, the root of each seedling was gently shaken to remove loosely adhering soil, while retaining the soil firmly attached to the roots. Root samples (2.5 g, including adhering soil) were transferred to Erlenmeyer flasks containing 100 mL of sterile saline solution. For substrate-derived isolates, 10.0 g of the BCS were subsampled and suspended in 90 mL of sterile saline solution. All suspensions were agitated at 180 rpm for 20 min. Serial dilutions were prepared up to 10 −3 , and 100 µL aliquots of each dilution were plated in triplicate onto acidified PDA for Trichoderma isolation, and LB agar (with thermal shock of 80°C for 30 min) for Bacillus . Plates were incubated at 25 °C and examined daily starting 48 h after plating. Colonies with morphological characteristics consistent with Trichoderma or Bacillus were subcultured to obtain pure isolates, which were confirmed to genus level by microscopic observation of conidiophores/conidia ( Trichoderma ) or cell/spore morphology ( Bacillus ). Additionally to the newly isolated strains, three commercially available biological fungicides containing Trichoderma or Bacillus were included in the evaluation. Inoculum production Trichoderma Inoculum was produced by culturing the strains on PDA plates. Strains were grown in 90 mm Petri dishes at 25 °C. After 7 days, fungal conidia were harvested by scraping the agar surface, suspended in sterile distilled water, and quantified by conidial counting in a Neubauer chamber. Bacillus Inoculum was produced via submerged fermentation. Colonies from 2-day-old LB agar plates were transferred into Erlenmeyer flasks containing 2XSG broth (composition) and incubated for 72 h at 28 °C with shaking. Serial dilutions (10 ?6 , 10 ?7 , 10 ?8 ) were plated on LB agar in triplicate, incubated at 28 °C for 48 h, and colony-forming units (CFU) were enumerated. Calonectria Calonectria 3V was grown in 90 mm Petri dishes with Malt Extract culture medium (Oxoid, UK) at 25 °C for 7 days, after which the agar was cut into small pieces, suspended in sterile distilled water, and blended for 2 min in a previously sterilized blender (Blanco and Romero, 2017). Serial dilutions were performed to confirm a target concentration of ∼1 × 104 CFU/mL. Biocontrol assays Biocontrol efficacy was assessed using the bait plant technique described by Gonçalves et al. (2001) , an effective method for quantifying Cylindrocladium in soils with varying inoculum densities. Treatments consisted of the co-inoculation of Trichoderma or Bacillus strains (individually) with the Calonectria isolate in plastic trays (13 × 17 cm) containing 200 g of BCS commercial nursery substrate. Trichoderma suspensions were standardized to final concentrations of 5 × 10 7 conidia/mL ( Trichoderma ) or 5 × 10 7 CFU/mL ( Bacillus ). Commercial products were adjusted to the same concentrations prior to use. Inoculations with the Calonectria strain were performed using suspensions adjusted to ∼1 × 10 4 CFU/mL. Each biocontrol tray received 5.0 mL of antagonist suspension and 5.0 mL of pathogen suspension at the concentrations described above, plus 80 mL of sterile water to achieve usual substrate humidity levels. Trays were incubated at 25 °C for 5 days (antagonist–pathogen interaction period). After 5 days, 30 leaf segments of E. globulus (∼1.5 × 0.5 cm, previously disinfected by 5 min in 3% sodium hypochlorite) were partially buried in the substrate and trays were re-incubated at 25 °C to allow pathogen infection. From the fourth day after leaf placement, the number of infected leaves per plastic trays was recorded. Control treatments consisted of a disease control with pathogen inoculation only (Only P -5 mL, 1 × 10 4 CFU/mL-) and a healthy control without pathogen inoculation. Disease assessment was conducted by a single evaluator on the same day for all treatments, which limited the number of treatments per trial to a maximum of 11. Consequently, four independent biocontrol assays were conducted using the same experimental technique but evaluating different sets of strains in each trial. In trials 1 and 2, Trichoderma strains were assessed, while in trials 3 and 4, Bacillus strains were tested. In total, 10 Trichoderma and 12 Bacillus strains were evaluated. From the second trial onward, two additional reference controls were included to estimate pathogen inoculum reduction: Reference 1 (Only P –30%), with 30% less inoculum than the control Only P, and Reference 2 (Only P –70%), with 70% less inoculum than the control Only P was applied. Strain T3 was systematically included in all experiments as a bridging strain to enable cross-trial comparisons. All treatments, both those tested in a single trial and those systematically included across trials (e.g., T3 and reference controls), were replicated five times. For each trail, a different batch of substrate was used and not sterilized to better represent nursery production conditions. Substrate was microbiologically characterized using a representative sample, by quantifying total culturable fungi (in acidified PDA) and bacteria and actinomycetes (in R2A; Oxoid, UK) through the same dilution–plating technique used for substrate-derived isolates described above. From this analysis, bacteria, actinomycetes, and fungi were recovered. Bacteria represented the most abundant group (10 6 CFU g −1 ), followed by actinomycetes (10 5 CFU g −1 ) and fungi (10 4 CFU g −1 ) ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Microbiological characterization of the commercial substrate used in biocontrol assays. Trichoderma identification Three strains that showed the greatest antagonistic capacity were subjected to molecular identification by amplification and sequencing of translation elongation factor 1-a (TEF1). Genomic DNA was extracted using the CTAB method described by Murray and Thompson (1980) . Trichoderma strains were cultured on PDA medium at 25 °C for 4 days. Mycelium was then harvested with a sterile scalpel, transferred into 1.5 ml Eppendorf tubes, and homogenized using sterile plastic pestles prior to the chemical DNA extraction steps. Amplification of the translation elongation factor 1-a (TEF1) was performed following the protocol described by Kubicek et al. (2003) . The PCR was prepared in 50 µl of total volume, 0.25 µl of 5U Dream Taq polymerase (Thermo Fisher Scientific), 5 µl of 2.0 mM of each dNTP solution (Thermo Fisher Scientific), 5 µl of amplification buffer (Thermo Fisher Scientific), and 1.0 µl of DNA (approximately 50 ng/µL), 2.5 µl of each primer EF1-T (5′-ATGGGTAAGGATGACAAGAC-3′) and EF2 (5′-GGA(G/A)GTACCAGT(G/C)ATCATGTT-3′). PCR reaction was run with an annealing temperature of 55 °C. The PCR products were analyzed on agarose gel 1% yielding an amplicon of about ≈900bp. The amplification products were sequenced by Macrogen Inc. (Seoul, Korea). Sequence similarity searches were performed with BLAST network service in NCBI GenBank ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ). Ex-type cultures sequences ( Bissett et al. 2015 ) and representative sequences of the closest matching species from Genbank were selected to be included in the phylogenetic analysis ( Table 2 ). The sequence alignment was done using Clustal W algorithm within the free software MEGA 12 ( Tamura et al. 2021 ). Manual adjustments were made to improve the alignments. Maximum parsimony (MP) and maximum likelihood (ML) phylogenetic analyses were implemented in MEGA 12. For MP analysis, we used the addition of 1000 random replicates and tree bisection and reconnection (TBR) were selected as branch swap ping algorithm. Gaps were treated as missing character, and all characters were unordered and of equal weight. Branches of zero length were collapsed. To estimate branch support, MP bootstrap values were determined using 1000 bootstrap replicates ( Felsenstein 1985 ). For the ML analysis, the evolutionary history was inferred by using the method based on the Kimura 2-parameter model, with 1000 replicates of bootstrap test. MP and ML analyses generated nearly identical topologies for each data set; thus, only the topology from the ML analysis is presented in the main text. View this table: View inline View popup Download powerpoint Table 2: Name and Genbank accession number for the transcription elongation factor (TEF) of reference strains of Trichoderma species used in phylogenetic analysis Statistical analysis The incidence of infection (proportion of infected leaves and non-infected leaves) was analysed using pairwise proportion comparisons ( pairwise.prop.test ) based on the Chi-squared test, with Holm correction for multiple comparisons. For each evaluation day, p -value matrices were generated and grouping letters were assigned to treatments according to their statistical diferences ( p ≤ 0.05). All analyses were performed in R software (version 4.4.2, R Core Team, 2024). Results Trichoderma and Bacillus collection A collection of 20 Trichoderma and 46 Bacillus strains was obtained from surveys conducted in commercial Eucalyptus nurseries. From this collection, strains isolated from different sources (endophytes from stem or root, rhizosphere, and commercial substrate), as well as from different Eucalyptus species, clones, and nurseries, were selected for biocontrol assays to capture the widest possible diversity of origins for both genera ( Table 1 ). In addition, commercially available products registered in Uruguay as biological fungicides based on Trichoderma (PC1, PC2) and Bacillus (PC3) strains were included ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Strains of Trichoderma and Bacillus selected for the four biocontrol assays Biocontrol Among the selected isolates, several Trichoderma strains reduced the percentage of pathogen-infected leaves ( Figure 1 ). In Trial 1, strains T3 and T5 showed significantly lower infection levels compared to the control. In Trial 2, all Trichoderma strains tested decreased infection incidence caused by Calonectria , with T6, T7, PC2, and PC1 achieving the strongest reductions. The infection levels recorded for these strains were comparable to those obtained with T3 and with the treatment where the pathogen was inoculated at a 70% lower concentration than the control (Only P -70%). Download figure Open in new tab Figure 1. Percentage of infected leaves the in four independent trials according to Trichoderma or Bacillus strains. In trials 1 (A) and 2 (B) were evaluated Trichoderma strains, and trials 3 (C) and 4 (D) Bacillus strains. Bars represent the mean percentage of infected leaves for each treatment. Different letters above the bars indicate statistically significant differences between treatments (p < 0.05). In contrast, none of the Bacillus strains were able to reduce the incidence of infected leaves. Notably, in all cases where strain T3 (used as a bridging strain) was inoculated, the percentage of infected leaves was significantly lower than the diseased control (Only P), and equal to or lower than that observed in the Only P – 70% treatment. Trichoderma identification The TEF sequences analysis involved three of local strains of Trichoderma that showed the greatest antagonistic capacity (T3, T5 and T7), 23 Trichoderma sequences from GeneBank, and Nectria eustromatica as out group. The analysis involved 26 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 490 positions in the final dataset. The phylogenetic tree showed that the 3 isolates belonged to two species of Trichoderma ( Figure 2 ). Strains T3 and T5 formed a clade with Trichoderma asperellum , and T7 grouped with Trichoderma asperelloides. Download figure Open in new tab Figure 2. Maximum likelihood tree based on partial sequences of the translation elongation factor 1 alfa (tef1alfa) of local sequences and sequences retrieved from genBank including ex-type strain sequences (T). Bootstrap values of 1000 replicates of the ML/MP analysis are shown below branches. only values greater than 70 are presented. Nectria eustromatica was used as outgroup. Discussion The search for local microbial control agents is justified since sustainable alternatives to chemical active ingredients demands the selection of adapted microbial strains, with higher chances of effective control in productive environments. In this work, we set up a collection of locally isolated Trichoderma spp. and Bacillus spp. strains obtained from different sources, to which commercially available formulations were added for comparison. Among these, both locally isolated Trichoderma strains (T3, T5, T6, and T7) and commercial products (PC1 and PC2) consistently achieved significant reductions in Calonectria infection, indicating their ability to decrease pathogen inoculum levels in the substrate. These results are consistent with previous reports highlighting Trichoderma spp. as promising antagonists of soilborne pathogens ( Harman, 2005 ; Muhammad et al., 2018 ). Several studies have reported Trichoderma strains as antagonists of Calonectria (Vitale et al., 2011; Kong and Hong, 2017 ; Carvalho et al., 2018 ). Interestingly, strain T3 consistently reduced the incidence of infected leaves across all trials in which it was included, demonstrating the reproducibility of its biocontrol effect. Although some variation was observed in its efficacy, infection levels were always equal to or lower than those obtained with the disease control treatment in which the pathogen inoculum was reduced by 70% (Only P –70% treatment), which could be taken as an indication of inoculum reduction by at least 70% when T3 was applied. Variability observed around 70% reduction may be attributed to differences among substrate batches used in each trial. Even though commercial substrates are standardized, factors such as storage time, temperature, and humidity can lead to diferences in their microbiological, chemical, and physical properties, potentially influencing pathogen development and biocontrol performance. Further studies are required to identify the specific conditions that enable this strain to fully express its antagonistic potential. Moreover, other strains performed at levels comparable to the Only P (–70%) control suggests that they may achieve medium-to-high reductions (>70%) in pathogen inoculum. Importantly, these performances were achieved in a non-sterilized substrate, meaning that the biocontrol strains not only antagonized the pathogen but also successfully competed with the naturally occurring microbial community. According to the substrate sample evaluated, this community was abundant, with total culturable bacteria reaching 10 6 , actinomycetes 10 5 , fungi 10 4 CFU g −1 . These findings strengthen the case for their application as promising and effective management alternatives under nursery production conditions. The inclusion of commercial products based on Trichoderma with biocontrol capacity brings the adoption of biological control in Eucalyptus nurseries closer to reality, since it would not require going through the entire process of product development and registration. However, this does not preclude the use of locally isolated strains identified in this study, but rather broadens the spectrum of available microbial resources, with strains from different Trichoderma species. The coexistence of commercial formulations and newly isolated strains could provide complementary tools, strengthening the foundation for the gradual incorporation of biological control into nursery disease management programs in Uruguay. In contrast, none of the Bacillus strains tested here significantly reduced the number of leaves infected, even though Bacillus spp. are widely recognized as effective biocontrol agents, also against Calonectria ( Paz et al., 2018 ; Samavat, 2022 ; Zang et al., 2023 ). The absence of activity under our experimental conditions should not be interpreted as definitive lack of potential. Several key factors may have limited their observed efficacy. For instance, the temperature of 25 °C was used because it corresponds to the average conditions registered in the greenhouse. However, the optimal temperature range for Bacillus species growth is generally reported as 28–35 °C ( Errington and van der Aart, 2020 ; Ajuna et al., 2020 . Additionally, suboptimal formulation or inoculum density, the lack of biofilm-enhancing conditions, low competition ability in the studied substrate, among others, are known to influence colonization and antagonistic activity, may have compromised outcome ( Fessia et al., 2022 ). Therefore, their biocontrol potential cannot be ruled out, and should be re-evaluated under optimized conditions. Regarding the identification of T. asperellum and T. asperelloides , these are closely related sister species within the T. asperellum sensu lato group ( Samuels and Ismaiel, 2010 ). Their observed biocontrol performance is consistent with the well-established role of T. asperellum sensu lato as one of the most widely employed biocontrol species, due to its versatile antagonistic mechanisms that include mycoparasitism, competition for resources, production of antifungal enzymes (e.g., chitinases and glucanases), and induction of host defense responses (Rivera Méndez et al., 2020; Mukherjee, 2022; Abbas et al., 2022 ). Although no specific reports were found on T. asperellum or T. asperelloides directly antagonizing Calonectria , both species have been repeatedly associated with antagonism against other soilborne pathogens in diverse plant–pathogen systems ( Phoka et al., 2020 ; Rivera Méndez et al., 2020; Boukaew et al., 2024 ; Hasna et al., 2025), highlighting their strong antifungal potential against this type of pathogens. To our knowledge, this is the first report of strains belonging to T. asperellum and T. asperelloides exhibiting antagonistic activity against Calonectria pathogenic species of Eucalyptus. In summary, this study identified several Trichoderma strains with promising antagonistic capacity. One of them, T. asperellum strain T3 isolated from Eucalyptus stems performed consistently across independent trials. These results were obtained under in vitro conditions using a method that goes beyond traditional dual culture assays, since it quantified pathogen inoculum and was carried out in a non-sterilized commercial substrate with a natural microbiota, under temperatures representative of greenhouse conditions. This approach brings the evaluation closer to real production scenarios, thereby strengthening the evidence of their potential efficacy in nurseries. Altogether, these findings suggest that the selected strains are likely to maintain consistent performance under nursery conditions. Nonetheless, greenhouse trials remain necessary to confirm their effectiveness under real production conditions. Funding sources UPM Biofore Uruguay and the National Institute of Agriculture Research (INIA) Acknowledgments We gratefully acknowledge UPM Biofore and the National Institute of Agriculture Research (INIA) for funding this project, as well as the staff members of both institutions involved in its execution. Funder Information Declared National Institute of Agriculture Research (INIA)-Uruguay , INIA SA47 UPM Biofore, Uruguay , AV PhD Scholarship References ↵ Abbas , A. , Mubeen , M. , Zheng , H. , Sohail , M. A. , Shakeel , Q. , Solanki , M. K. , Iftikhar , Y. , Sharma , S. , Kashyap , B. K. , Hussain , S. , Zuñiga Romano , M.C. , Moya-Elizondo , E. A. , & Zhou , L. ( 2022 ). Trichoderma spp. genes involved in the biocontrol activity against Rhizoctonia solani . Frontiers in Microbiology , 13 , 884469 . doi: 10.3389/fmicb.2022.884469 OpenUrl CrossRef PubMed ↵ Ajuna , H. 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