Clonostachys: Ecology, Multitrophic Biocontrol Mechanisms, and Applications in Sustainable Crop Protection

Abstract

Clonostachys species are functionally diverse filamentous fungi with strong potential for sustainable crop protection. They suppress plant pathogens, plant-parasitic nematodes, and insect herbivores, while also promoting plant growth. These fungi are widely distributed in soils and plant-associated habitats, reflecting their ecological adaptability and multitrophic interactions. Despite their promising agricultural potential, the practical use of Clonostachys species as microbial agents remains limited. This review summarizes current knowledge on the taxonomy, ecology, and functional diversity of Clonostachys species, with emphasis on the mechanisms underlying their biocontrol activity. We also discuss their multitrophic interactions, potential risks of opportunistic pathogenicity, and key challenges for practical application. Finally, future perspectives are presented to support the development of reliable Clonostachys-based strategies for sustainable agriculture.

1. Introduction

Ensuring stable agricultural production amid rising global food demand remains a major challenge for modern agriculture [1]. Crop plants are continuously threatened by fungi, bacteria, viruses, nematodes, and insect pests, which significantly reduce crop productivity and quality [2]. These organisms cause substantial yield losses worldwide and remain major barriers to sustainable food production [3]. Therefore, developing effective and sustainable pest management strategies has become a critical priority in modern agriculture [4].

For decades, agricultural pest management has relied heavily on chemical pesticides because of their rapid effectiveness and ease of application. However, their extensive use has raised concerns regarding environmental contamination, harmful effects on beneficial organisms, and the emergence of resistant pest populations [5, 6]. Increasing public awareness and stricter regulations have therefore encouraged the development of environmentally sustainable alternatives for plant protection [7].

Biological control has emerged as a promising strategy for sustainable crop protection [8]. Biological control agents (BCAs) are beneficial organisms that suppress plant pathogens and other harmful pests, thereby protecting plants [9]. Compared with chemical pesticides, BCAs are generally considered more environmentally friendly and compatible with integrated pest management programs [10].

Among fungi, species of the genus Clonostachys, particularly Clonostachys rosea (formerly Gliocladium roseum/G. catenulatum), have gained attention as multifunctional BCAs because of their mycoparasitic, entomopathogenic, nematophagous, and plant growth-promoting activities [11, 12]. These fungi are widely distributed in natural and agricultural environments, inhabiting diverse niches such as soil, rhizosphere, phyllosphere, and endosphere habitats [13, 14]. Recent studies further indicate that Clonostachys species promote plant growth [15] and improve plant resilience under various environmental conditions [13, 16]. They also interact with a wide range of organisms across multiple biological kingdoms, including insect pests and other plant-associated microorganisms [11, 17]. Despite their ecological and agricultural importance, knowledge of the diversity, ecology, and functional capabilities of this genus remains fragmented across research fields.

This review summarizes current knowledge on the taxonomy, ecological distribution, life cycle, and functional diversity of Clonostachys species. We discuss their roles as BCAs, plant growth-promoting fungi, and, in some contexts, plant pathogens. We further examine the mechanisms underlying their interactions with plant pathogens, plant-parasitic nematodes, insect pests, and plant hosts. By integrating recent ecological, physiological, and molecular findings, this review highlights the potential of Clonostachys species as multifunctional agents for sustainable crop protection.

2. Taxonomy and ecology of the Clonostachys species

2.1 Taxonomy and species diversity

The genus Clonostachys belongs to the family Bionectriaceae within the order Hypocreales [14]. Historically, several species now classified as Clonostachys had been assigned to the genus Gliocladium based on morphological traits such as penicillate conidiophores and slimy conidial masses. However, molecular phylogenetic analyses have substantially refined the taxonomy and clarified their evolutionary relationships [14, 18].

Recent revisions indicate that approximately 60–70 Clonostachys species are currently accepted [14, 19], although more than 100 species names have historically been proposed [20]. Species boundaries continue to be refined through multilocus and phylogenomic approaches, revealing previously unrecognized taxa and highlighting the genetic and functional diversity of the genus [14, 20].

C. rosea is the most extensively studied species and serves as a model for understanding the genetic diversity, functional traits, and biocontrol potential of the genus [21, 22, 23]. Additional species identified through multilocus and phylogenomic analyses, including C. chloroleuca [24], C. byssicola [25], C. rhizophaga [26], C. reniana [27], and C. solani [28], further illustrate the taxonomic complexity and species richness of Clonostachys.

2.2 Ecological distribution and habitat

Clonostachys species are widely distributed in terrestrial and freshwater habitats in both natural and agricultural ecosystems [14]. Soil is their primary habitat, including the rhizosphere, where they mainly function as saprophytes that decompose organic matter [11]. Some species also act as necrotrophic parasites of fungi [13], nematodes [29], and insects [30].

These fungi are frequently isolated from the interior and surfaces of belowground and aboveground tissues of woody and herbaceous plants [25, 26, 31]. They are often dominant members of fungal communities associated with these plant microhabitats [13, 32, 33]. Clonostachys species can colonize plant surfaces and internal tissues and may produce conidia on plant surfaces under favorable conditions [34, 35]. Epiphytic and endophytic strains can influence host physiology and microbial communities occupying the same niche [11, 28, 36], although their precise ecological roles remain unclear.

Artificial inoculation with rhizospheric, epiphytic, or endophytic Clonostachys strains has been reported to promote plant growth and reduce pest damage, suggesting beneficial effects on host health [15, 29]. Across these habitats, Clonostachys species engage in diverse ecological interactions, including antagonism against fungi [23, 37], associations with plant hosts [15, 38], and interactions with nematodes and insects [21, 30, 39], collectively influencing microbial dynamics and plant health.

2.3 Life cycle and reproductive characteristics

The life cycle of Clonostachys species follows the typical ascomycete pattern and includes sexual and asexual stages [14, 19]. The sexual stage (teleomorph) involves the formation of perithecial ascomata on plant-derived substrates such as bark, twigs, wood, or leaves [14, 19, 20]. These perithecia are usually globose to ovoid and may occur singly or in groups. Inside, clavate to subcylindrical asci produces hyaline, one-septate ascospores that germinate to form hyphae and new mycelial networks [20]. However, the sexual stage is relatively rare in natural environments [11, 14, 19].

In contrast, the asexual stage (anamorph) predominates in nature and involves conidiophore production from aerial hyphae [14, 19] (Fig. 1). Conidiophores show diverse branching patterns, including penicillate, verticillate, gliocladium-like, and acremonium-like forms. They produce unicellular, hyaline conidia in chains or mucilaginous masses [19, 20]. These conidia function as the main dispersal units and rapidly germinate on suitable substrates to establish vegetative hyphae and new colonies [40].

Figure 1: Microscopic morphology of Clonostachys chloroleuca. Representative asexual reproduction structures, including (A) branched conidiophores, (B) phialides, and (C) conidia.

3. Clonostachys as a biocontrol agent of plant pests

Certain Clonostachys species are well-documented parasites of fungi, nematodes, and insects and therefore function as natural enemies of these organisms. Consequently, they have been extensively studied as BCAs against plant-pathogenic fungi, plant-parasitic nematodes, and insect herbivores.

3.1 Biocontrol of fungal pathogens

C. rosea (formerly classified as G. roseum) was first reported to parasitize Ceratocystis fagacearum, the causal agent of oak wilt, in the late 1950s [41]. Since then, other species, including C. chloroleuca, C. pseudochroleuca, C. rhizophaga, and C. byssicola, have also been shown to exhibit mycoparasitism [13, 26, 42]. Among them, C. rosea has been most extensively investigated as a BCA against fungal pathogens across diverse crop systems [13] (Table 1). Its efficacy has been demonstrated against pathogens affecting aerial tissues [26, 40], roots and crowns [43], seeds [44], and postharvest products [45], indicating broad-spectrum biocontrol activity [11].

Table 1: Biocontrol efficacy of Clonostachys species against fungal pathogen

Speciesa	Target pathogens	Host plant	System	Biocontrol effect	Mechanism(s)	References
	Foliar pathogens
C. rosea (67-1, IK726, PG-88-710)	Botrytis cinerea	Tomato, strawberry, rose	In vitro/ household/ field	Reduced disease severity, pathogen growth, and sporulation; improved yield; enhanced host resistance	Mycoparasitism, antibiosis, competition, ISR	[37, 49, 50, 53, 54]
C. rosea (IK726)	Bipolaris sorokiniana, Pyrenophora teres, Rhynchosporium commune	Barley	Greenhouse	Reduced disease severity and sporulation	Competition	[55]
C. rosea (ACM941)	Fusarium graminearum	Wheat	Greenhouse/field	Reduced disease severity	Antibiosis, competition	[54]
C. rosea (IK726)	Zymoseptoria tritici	Wheat	Greenhouse	Reduced disease severity (genotype-dependent)	Competition, ISR	[57]
Clonostachys species	Hemileia vastatrix	Coffee	In vitro/ greenhouse	Reduced disease severity and spore germination	Mycoparasitism	[26]
Clonostachys species	Alternaria grandis	Potato	Greenhouse	Reduced disease severity and sporulation	Mycoparasitism	[61]
	Soil-borne and vascular pathogens
C. rosea (J1446, 67-1)	Fusarium oxysporum	Cucumber, tomato	In vitro/ greenhouse	Reduced disease severity	Mycoparasitism, competition	[43, 68, 80, 81]
C. rosea (f. catenulata)	Sclerotinia sclerotiorum	Various crops	In vitro/ greenhouse	Reduced disease severity and sclerotial viability	Mycoparasitism, antibiosis	[69, 71, 73]
C. rosea	Rhizoctonia solani	Potato, rice, ornamentals	In vitro/ greenhouse	Reduced disease severity and improved yield	Mycoparasitism, competition	[76, 77, 78]
C. rosea (19B/1)	Eutypa lata, Phaeomoniella chlamydospora	Grapevine	In vitro/ greenhouse	Reduced pathogen colonization and disease incidence at infection sites	Mycoparasitism	[23, 79]
C. rosea f. catenulata J1446	Plasmodiophora brassicae	Canola	Greenhouse/field	Reduced root infection and disease severity	Mycoparasitism, antibiosis, and ISR	[66]
	Post-harvest and seed-borne pathogens
C. rosea (IK726)	Fusarium avenaceum, Fusarium caeruleum (Syn. Fusarium coeruleum)	Potato	Storage	Reduced dry rot incidence	Mycoparasitism, antibiosis	[45]
C. rosea (IK726)	Bipolaris sorokiniana	Barley	Storage	Reduced seed-borne infection and viability	Mycoparasitism, competition	[83]
C. rosea (IK726)	Alternaria dauci, Alternaria radicina	Carrot	In vitro/ storage	Reduced seed-borne infection and improved seedling establishment	Mycoparasitism, competition, and antibiosis	[84]
C. rosea (IK726)	Fusarium culmorum	Wheat	In vitro	Reduced seed-borne infection and disease severity; improved seedling establishment	Mycoparasitism, competition, and antibiosis	[44]
C. rosea (IK726)	Ascochyta spp.	Pea	In vitro	Reduced seed-borne infection and improved seedling establishment	Competition, antibiosis	[85]
C. rosea (ACM941)	Didymella pinodes (Syn. Mycosphaerella pinodes)	Pea	Greenhouse/field	Reduced root rot severity and improved plant establishment	Competition, antibiosis	[86]

ᵃ Parenthetical codes following fungal species names indicate strain or isolate designations

3.1.1 Efficacy against foliar pathogens

The most extensively studied examples of foliar disease suppression by Clonostachys involve gray mold caused by Botrytis cinerea [46, 47, 48]. C. rosea consistently suppresses gray mold on leaves [37], flowers [49], stems [50], and other aerial tissues across multiple crops [51]. Its ability to colonize and persist on leaf surfaces and crop residues is considered essential for disease suppression.

Disease reduction has been reported in roses [49], tomatoes [46, 50, 52], and strawberries under controlled and field-relevant conditions [48, 53, 54]. Recent studies also indicate that combining C. rosea with succinate dehydrogenase inhibitor (SDHI) fungicides improves control efficacy while reducing chemical inputs [47].

C. rosea has also shown efficacy against several cereal foliar diseases, including spot blotch caused by Bipolaris sorokiniana, net blotch caused by Pyrenophora teres, barley scald caused by Rhynchosporium commune [55], Fusarium head blight caused by Fusarium graminearum [56], and Septoria tritici blotch caused by Zymoseptoria tritici [57].

Additional studies further support the potential of Clonostachys species as BCAs against diverse foliar pathogens [58, 59, 60]. For example, Kapeua-Ndacnou et al. [26] isolated mycoparasitic strains of C. rosea, C. rhizophaga, and C. byssicola from healthy coffee tissues. Foliar application enabled endophytic colonization for more than 1 month and significantly reduced coffee leaf rust caused by Hemileia vastatrix. Similarly, da Silva et al. [61] reported that spray application of C. chloroleuca, C. pseudochroleuca, and C. rhizophaga strains reduced early blight severity in potato caused by Alternaria grandis by more than 80%.

Commercial products further support the practical applicability of Clonostachys species. Formulations based on C. rosea strain J1446, including Prestop® and Lalstop® G46 WG (Lallemand Plant Care, Canada), are widely used to manage gray mold and other foliar pathogens in vegetable and horticultural crops [12, 62, 63]. Emerging delivery systems, such as insect-mediated dispersal using VectoriteTM with CR-7 (Bee Vectoring Technologies International Inc., Canada), may further improve field-level disease control [64].

3.1.2 Efficacy against soil-borne and vascular pathogens

Substantial evidence supports the efficacy of Clonostachys species against soil-borne pathogens [11, 17], although fewer studies have investigated these interactions compared with foliar diseases. Available reports indicate activity against important pathogens such as Fusarium, Sclerotinia, and Rhizoctonia species affecting roots and crown tissues.

Several studies have demonstrated suppression of cucumber Fusarium wilt caused by Fusarium oxysporum. C. rosea strain J1446 effectively colonized cucumber roots and crowns and suppressed disease development when applied before pathogen infection [43, 65]. This strain was commercialized as Prestop® for the management of multiple soil-borne diseases [66, 67]. Similarly, strain 67-1 reduced Fusarium wilt severity by approximately 70% alone and achieved near-complete control when combined with dazomet fumigation [68].

Clonostachys species also show strong activity against Sclerotinia sclerotiorum [40, 69, 70, 71, 72]. These fungi can intensively colonize pathogen sclerotia [69, 73], reducing inoculum levels in soil [40, 74, 75]. Activity against Rhizoctonia solani has also been reported in potato [76], rice [77], and ornamental crops [78]. Additional reports indicate suppression of vascular pathogens such as Eutypa lata and Phaeomoniella chlamydospora, suggesting the ability of Clonostachys species to colonize woody and vascular tissues [23, 79].

Commercial products further demonstrate their practical value. In addition to Prestop®, Clonotri (MS Biotech Ltd., Italy), a consortium containing Clonostachys, Trichoderma, and Glomus, significantly suppressed tomato Fusarium wilt caused by F. oxysporum f. sp. lycopersici under greenhouse conditions [80, 81].

3.1.3 Efficacy against postharvest and seed-borne pathogens

Compared with foliar and soil-borne diseases, fewer studies have examined the use of Clonostachys species against postharvest and seed-borne pathogens. Nevertheless, available evidence demonstrates promising biocontrol potential. C. rosea strain IK726 reduced dry rot incidence in stored potato tubers caused by Fusarium avenaceum and Fusarium caeruleum (syn. F. coeruleum) by approximately 45% under simulated storage conditions [45].

Clonostachys species can also persist on and within seeds [36, 43, 82]. Seed treatment with Clonostachys strains effectively reduced diseases caused by seed-borne pathogens. For example, barley seed treatment with strain IK726 reduced seedling blight caused by Bipolaris sorokiniana by more than 80% [83]. The same strain also reduced seedling mortality caused by Alternaria dauci and A. radicina in carrot [84]. Similar efficacy has been reported against Fusarium culmorum in wheat [44], Ascochyta spp. in pea [85], and Didymella pinodes (syn. Mycosphaerella pinodes) in pea [86].

Commercial formulations also support practical application. LALFRESH S (Lallemand Plant Care, Canada), based on C. rosea strain J1446, has been developed for postharvest control of fruit rot pathogens such as Monilinia laxa and M. fructicola in stone fruits [87]. Other products derived from the same strain, including Prestop® and LALSTOP G46 WG® (Lallemand Plant Care, Canada), are used for pre- and postharvest disease management, particularly against Botrytis and related pathogens [12, 62].

3.2 Efficacy against plant parasitic nematodes

C. rosea is a well-known nematophagous fungus that parasitizes and kills various nematode species [21, 39]. Consequently, it has been extensively studied for the control of plant-parasitic nematodes (PPN), including Meloidogyne, Helicotylenchus, Heterodera, Pratylenchus, Ditylenchus, and Trichodorus species [29, 88, 89] (Table 2). The most extensively studied interactions involve root-knot nematodes, particularly M. incognita, one of the most economically important soil-borne pests [90]. Under greenhouse and field conditions, application of C. rosea consistently reduced gall formation, egg production, and nematode populations. Reported reductions in nematode density and reproductive parameters generally range from approximately 50% to more than 80%, depending on the host system and experimental conditions. These suppressive effects are often accompanied by improved plant growth and vigor [21, 91, 92, 93]. Despite the well-documented efficacy of nematophagous Clonostachys species, only a limited number of strains have been commercialized as bionematicides. Examples include VectoriteTM and EndoFine® (contains C. rosea strain 88-710, developed by Adjuvants Plus Inc., Kingsville, Ontario N9Y2Y8), which are used in Canada for managing PPN and soil-borne pathogens, and Kamoi® (contains strain CPQBA 040-11/DRM 07, developed by Agrivalle Brasil Indústria e Comércio de Produtos Agrícolas S.A.) in Brazil [39].

3.3 Biocontrol of insects

Clonostachys species, including C. rosea [94], C. rogersoniana [95, 96], C. krabiensis [97], C. aranearum [18], and C. chuyangsinensis [18], have been reported to infect arthropods, including insects and spiders. Among them, C. rosea has attracted particular attention because of its broad-spectrum entomopathogenicity and potential as a BCA against herbivorous insects [11, 12] (Table 2). Mortality rates of up to 97% have been reported in model insects such as Galleria mellonella and Tenebrio molitor [17].

Entomopathogenic Clonostachys species have shown efficacy against several economically important insect pests. Significant reductions in survival and population development have been reported in sap-sucking insects, including whitefly (Bemisia tabaci) [40, 98] and aphids (Myzus persicae and Aphis fabae) [99], under laboratory conditions. However, most evidence has been derived from controlled experiments, and field-level efficacy remains insufficiently validated.

Although field evidence remains limited, support for the ecological relevance of entomopathogenic Clonostachys species is gradually increasing. C. rosea has been reported to naturally infect mango hopper (Amritodus atkinsoni), representing one of the earliest records of field occurrence [100]. Additional studies demonstrated activity against Dubas bug (Ommatissus lybicus) [30] and tomato leaf miner (Tuta absoluta) [101], reducing insect survival and feeding damage under laboratory and greenhouse conditions. Similar suppressive effects have also been observed against stored-product pests, including Trogoderma granarium, Tribolium castaneum, and Callosobruchus maculatus, where fungal treatments reduced adult survival and reproductive capacity [94].

Despite these promising findings, fully commercialized Clonostachys-based products specifically targeting insects remain limited. Nevertheless, some formulations developed for disease control also exhibit insect-suppressive effects. For example, EndoFine®, when coapplied with Beauveria bassiana (BotaniGard®) through bumble bee-mediated delivery, reduced populations of whitefly (Trialeurodes vaporariorum) and tarnished plant bug (Lygus lineolaris) while simultaneously suppressing gray mold [102].

Table 2: Biocontrol efficacy of Clonostachys species against nematodes and insects

Speciesa	Target pests	Host plant	System	Biocontrol effect	Mechanism(s)	References
	Nematodes
C. rosea (PHP1701, TNAU CrN01)	Meloidogyne incognita	Tomato	In vitro/ greenhouse/ field	Reduced gall formation, egg production, and population density; improved crop growth	Antibiosis and enzymatic degradation	[21, 91]
C. rosea (IK726)	Pratylenchus, Heterodera, Helicotylenchus, and Trichodorus	Wheat, carrot	In vitro	Reduced nematode populations	Antibiosis, parasitism	[29, 88]
C. rosea	Ditylenchus destructor	Potato	In vitro	Reduced nematode populations	Antibiosis	[89]
	Insects
C. rosea (GY24)	Galleria mellonella, Tenebrio molitor	-	In vitro	High mortality	Entomopathogenic activity and antibiosis	[17]
C. rosea (CR 02)	Bemisia tabaci	Potato, bean	In vitro/ greenhouse	Increased nymph and adult mortality	Entomopathogenic activity	[40, 98]
C. rosea (A80, A82)	Myzus persicae and Aphis fabae	-	In vitro	Reduced survival and fecundity	Plant-mediated effect	[99]
C. rosea (ICIPE 707)	Thrips tabaci	Onion	Greenhouse	Reduced survival and fecundity	Plant-mediated effect	[82]
C. rosea	Amritodus atkinsoni	Mango	In vitro	High mortality	Plant-mediated effect	[100]
C. rosea	Ommatissus lybicus	Date palm	Semi-field	Population suppression	Entomopathogenic activity	[30]
C. rosea (G133)	Tuta absoluta	Tomato	In vitro/ greenhouse	Reduced larval survival and feeding damage	Entomopathogenic activity	[101]
C. rosea (A80, A82)	Trogoderma granarium, Tribolium castaneum, and Callosobruchus maculatus	-	In vitro	Reduced adult survival and reproduction	Antibiosis	[94]

ᵃ Parenthetical codes following fungal species names indicate strain or isolate designations

4. Mechanisms underlying the biocontrol activity of Clonostachys species

Although parasitism has been considered the primary mechanism underlying the biocontrol activity of Clonostachys species, evidence shows that their efficacy results from multiple interacting mechanisms. These mechanisms act directly on target organisms or indirectly through plant-mediated processes and may function simultaneously or synergistically depending on environmental and biological conditions (Fig. 2).

Figure 2: Protective effects of Clonostachys species against plant pests through multiple actions

4.1 Mycoparasitism

Mycoparasitism is a central mechanism of Clonostachys species, particularly in C. rosea and C. chloroleuca, which are regarded as necrotrophic mycoparasites against a broad range of fungal pathogens [103]. This process involves sequential stages of host recognition [103, 104], directed hyphal growth, attachment, and penetration [105, 106], followed by intracellular degradation [107, 108] and host cell death [109, 110].

Upon contact, Clonostachys species form hyphal coils and appressorium-like structures that facilitate host penetration [37, 111]. This interaction is supported by secretion of cell wall–degrading enzymes (CWDEs), including chitinases, β-1,3-glucanases, and proteases [112, 113], which degrade fungal cell walls and are induced by pathogen-derived substrates [112]. Functional studies further indicate that increased expression of CWDE-related genes enhances mycoparasitic efficiency and disease suppression [114].

Following penetration, Clonostachys species rapidly colonize and overgrow pathogen structures, often causing extensive degradation of mycelia and survival structures such as sclerotia [37, 73]. These processes are regulated by signaling pathways, including MAP kinase (MAPK) [42, 105, 106] and cAMP signaling networks [103], which coordinate host sensing, enzyme secretion, and metabolic adaptation [107, 108].

4.2 Antibiosis

Antibiosis is an important mechanism underlying the biocontrol activity of Clonostachys species, particularly C. rosea, through the production of inhibitory secondary metabolites [22, 29, 115]. Unlike mycoparasitism, antibiosis does not require direct physical contact and can suppress target organisms through diffusible compounds and, in some cases, volatile organic compounds (VOCs) [21]. These metabolites include nitrogen-containing compounds, polyketides, and terpenoids, reflecting the strong biosynthetic capacity of the genus [116].

Genomic and functional studies have shown that biosynthetic gene clusters, including polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), are strongly induced during interactions with pathogens such as F. graminearum and B. cinerea, often before physical contact occurs. Disruption of key genes, including PKS22 and PKS29 in C. rosea, significantly reduced metabolite production and antagonistic activity, directly linking these compounds to biocontrol efficacy [107, 117].

These metabolites inhibit development of fungal pathogens by suppressing spore germination and infection processes [55, 115, 118]. Their effects also extend to plant-parasitic nematodes [21, 29] and insect pests [17, 22], where metabolites and culture filtrates induce mortality and reduce pest fitness. In some pathosystems, antibiosis appears to play a greater role than direct parasitism, particularly when Clonostachys species function as endophytes and produce bioactive compounds within plant tissues [11, 26, 28].

4.3 Competition for nutrients and space

Competition for nutrients and space is another important noncontact mechanism by which Clonostachys species suppress pathogen establishment and development [28]. These fungi exhibit rapid growth and efficient nutrient acquisition, enabling early colonization of plant surfaces and residues and limiting the availability of essential resources, such as carbon and nitrogen, to competing pathogens [119].

In plant-associated systems, early occupation of infection sites, including flowers, roots, and wounds, restricts pathogen germination and spread [29, 49, 118]. In crop residues and organic substrates, interactions are often dominated by competition for resources, where Clonostachys species reduce pathogen biomass through competitive displacement [120]. Experimental studies further indicate that environmental conditions influence niche overlap with pathogens, such as Fusarium species, thereby affecting competition intensity for carbon sources and space [119].

4.4 Plant-mediated effects: induced resistance and growth promotion

Root colonization by Clonostachys species enables stable, nonpathogenic associations with host plants and forms the basis of plant-mediated biocontrol [62]. Following epiphytic or endophytic colonization, some strains act as plant-activating agents that induce or prime plant immunity, resulting in stronger or faster defense responses against pathogen attack [17, 121]. Induced systemic resistance (ISR) is mainly associated with jasmonic acid (JA) and ethylene (ET) signaling pathways and is often accompanied by salicylic acid (SA)-mediated responses. These responses activate defense-related genes, increase defense enzyme activity, and reinforce structural barriers [121, 122, 123].

Evidence from multiple pathosystems supports this role. In Pinus radiata, C. rosea reduced lesion development caused by Fusarium circinatum by up to 50% and strongly induced defense-related genes, although responses varied among strains and host genotypes [121, 125]. Similar activation of defense enzymes and PR proteins has also been reported in oat [126], wheat [127], and tomato [46, 52], suggesting a conserved ISR response across diverse hosts.

In addition to induced resistance, Clonostachys species promote plant growth through phytohormone-associated effects, such as auxin production [128], modulation of metabolic and photosynthesis-related gene expression [16], and emission of VOCs that enhance root development and biomass accumulation [38]. These effects are often strain- and host-dependent and may become more pronounced under stress or disease conditions.

Recent studies also suggest the involvement of cross-kingdom RNA exchange, in which small RNAs derived from the host plant or Clonostachys species regulate gene expression in the interacting partner. This emerging mechanism may complement classical signaling pathways and contribute to plant defense and growth regulation [129, 130].

5. Opportunistic pathogenicity and ecological trade-offs of Clonostachys species

Although Clonostachys species are generally regarded as beneficial fungi, some strains can exhibit opportunistic pathogenicity under specific host or environmental conditions. For example, C. rosea has been associated with root rot in soybean [131] and faba bean [132], whereas C. rhizophaga has been reported to cause root disease in chickpea [133], lentil [134], and water chestnut [135]. In most cases, symptoms are mild and influenced by host condition and environmental factors, supporting their classification as opportunistic rather than aggressive pathogens. This context dependency reflects substantial strain-level variability and host specificity, in which the same species may act as a beneficial antagonist in some systems but display pathogenic traits in others [136]. Such ecological plasticity is consistent with their role as generalists capable of shifting between beneficial and detrimental interactions [28].

These dual roles likely reflect trade-offs in their metabolic and genomic repertoire. Enzymes involved in mycoparasitism and colonization [28], as well as secondary metabolites associated with antagonistic activity, may also contribute to host tissue colonization or damage under certain conditions [110, 116]. Consequently, Clonostachys species may suppress or, in some cases, facilitate pathogen activity depending on the interacting species and environmental context [136].

These findings highlight important biosafety considerations [11]. Reliable agricultural application of Clonostachys species requires careful strain selection and evaluation, including assessment of host specificity, non-target effects, and environmental stability [11, 137].

6. Challenges, future perspectives, and conclusions

Despite their broad functional capacity, the field performance of Clonostachys species can vary with environmental conditions, host genotype, and interactions with resident microbiota. In addition, opportunistic behavior reported in some strains raises important biosafety concerns, indicating that beneficial traits do not always guarantee safety across all hosts and environments. These challenges highlight the need for integrative approaches combining genomic, metabolomic, and ecological analyses to identify strains with stable colonization ability, consistent biocontrol performance, and minimal nontarget effects. Careful strain selection, multihost pathogenicity testing, and postrelease monitoring are essential for reliable application. Advances in formulation strategies, including multistrain systems and microbiome-informed approaches, are expected to improve efficacy, consistency, and scalability under field conditions. Integrating Clonostachys-based solutions into sustainable and climate-resilient agricultural systems may reduce reliance on chemical inputs while maintaining crop productivity under environmental stress. Overall, Clonostachys species represent a versatile and promising group of fungi for sustainable crop protection. However, successful application will depend on integrating mechanistic understanding with strain selection, formulation development, and field-level validation.

CRediT authorship contribution statement

　　 Alchemi Putri Juliantika Kusdiana: Writing–original draft, Writing–review & editing, Visualization, Conceptualization. Masafumi Shimizu: Writing–review & editing, conceptualization, and supervision.

Use of Generative AI and AI-Assisted Technologies

The authors used Grammarly to improve English grammar and readability. The authors reviewed and edited the output and take full responsibility for the content of the manuscript.

Funding

This research was supported by Science and Technology Research Partnership for Sustainable Development, Japan Science and Technology Agency/Japan International Cooperation Agency, and JPMJSA2006.

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