Toward Effective Biocontrol of Oomycete Plant Pathogens: Traits and Modes of Action of Biocontrol Agents, and Their Screening Approaches

Abstract

Oomycete plant pathogens often cause severe diseases in forest trees and agricultural crops, posing threats to natural ecosystems and reducing crop yields. The use of biocontrol agents (BCAs) in agrosystems has proven to be an excellent and eco-friendly alternative to chemical pesticides for protecting plants from oomycete pathogens. However, the range of commercially available biopesticides that growers need is still limited, and in many cases, their practical application cannot yet compete with conventional chemicals. To accelerate the development of effective biocontrol products, it is crucial to efficiently identify BCAs that exhibit robust and consistent biocontrol efficacy against oomycete diseases. In this paper, we conduct a thoughtful review of prior biocontrol studies, report the characteristics of BCAs commonly linked with their suppressive effect against oomycete diseases, discuss their mode of action concerning pathogen ecology, and offer recommendations for the development of high-throughput screening procedures.

1. Introduction

Oomycetes, often referred to as “water molds,” are a group of eukaryotic filamentous microorganisms that are phylogenetically related to diatoms and brown algae in the Stramenopila group [1]. To date, over 1,200 oomycete species have been identified, with their distribution spanning a wide range from freshwater environments like rivers, lakes, and streams to terrestrial habitats such as soil and plant surfaces [2]. They fulfill diverse ecological roles, functioning as saprophytes, parasites of plants, animals, and other fungi, and symbionts with other microorganisms [3, 4]. The majority of oomycetes identified so far are classified as detrimental plant pathogens causing significant ecological and economic impacts on a broad spectrum of plant species, including crops, ornamentals, and native vegetation [5].

Plant pathogenic oomycetes have independently evolved into two primary lineages: the Saprolegniales and the Peronosporales [6]. Within the Saprolegniales, necrotrophic pathogens from the genus Aphanomyces are the most representative members [7]. Conversely, the order Peronosporales comprises the most economically significant hemibiotrophs and necrotrophs from the genera Phytophthora and Pythium, as well as obligate biotrophs such as the pathogens of white blister rusts (i.e., genus Albugo) and downy mildews (i.e., Bremia, Peronospora, Hyaloperonospora, Plasmopara, and Pseudoperonospora) [8]. Phytophthora infestans, the causal agent of the potato late blight that led to the Irish famine of the 1800s, is one of the most destructive pathogens in the Peronosporales lineage. Yield losses in potato production due to this pathogen can reach up to 80% in a single season, resulting in substantial economic losses to farmers, estimated at € 5.2 billion annually [9]. Other economically significant pathogens in the order Peronosporales include Plasmopara viticola, the causal agent of grapevine downy mildew, Pythium ultimum, the causal agent of root rot and damping-off in many plants, and Phytophthora sojae, the causal agent of root and stem rot in soybean [10].

The use of chemical pesticides such as metalaxyl, azoxystrobin, and copper sulfate, along with the introduction of resistant cultivars have been the main strategies to manage oomycete diseases [11, 12]. However, the continued use of chemical pesticides has led to unintended adverse effects on non-target organisms and the emergence of new pathogen genotypes resistant to these fungicides [13]. As an alternative to overcoming these limitations, significant effort has been channeled toward biological control. The use of naturally occurring plant-protective microorganisms, also known as biocontrol agents (BCAs), to control oomycetes holds the potential to reduce the risk of resistance development in pathogens, owing to their multiple modes of action [14]. BCAs have been the subject of extensive research and are currently regarded as one of the most crucial tools in integrated pest management strategies [15]. This review provides a comprehensive overview of studies focused on the biocontrol of oomycete pathogens. It offers insights for enhancing screening strategies for microbial BCAs targeting oomycetes and discusses the modes of action of BCAs in relation to the oomycete life cycle.

2. Mode of infection of plant pathogenic oomycetes

Oomycetes have developed unique strategies for reproduction and host infection, enabling them to bypass host immunity and adapt to a changing environment. Infections in plants by oomycete pathogens are typically initiated by asexual sporangia, which emerge at the apices of somatic hyphae or on the tips of branched sporangiophores [16]. Depending on the species and environmental conditions, sporangia can germinate directly by forming a germ tube or indirectly through the production of asexual motile zoospores, as depicted in Figure 1 [17].

Figure 1: Lifecycle of plant pathogenic oomycetes and microbial biocontrol mechanisms during infection stages. ISR: induced systemic resistance; AB: antibiosis; CO: competition; SD: signaling disruption; HP: hyperparasitism

For most oomycete species, particularly soil-borne pathogens, indirect germination is deemed the primary mode of infection, occurring under conditions of low temperatures and nutrient levels and high humidity [18, 19]. Zoospores, released from sporangia, navigate through flooded soil, water on leaf surfaces, and hydroponic media toward their host plants, guided by chemotactic and electrical stimuli [20]. Numerous studies have identified factors present in root exudates, such as organic acids and isoflavones, that play a role in host recognition and induce various infection behaviors like zoospore encystment and cyst germination in various soil-borne oomycetes [21, 22]. The chemotactic responses of zoospores vary depending on the oomycete species and the composition of the root exudate. Most Pythium species exhibit positive chemotaxis to a variety of amino acids, but their response to specific sugars such as sucrose or maltose varies by species [23]. The zoospores of P. sojae, the causal agent of soybean stem and root rot, are strongly attracted to the legume-specific isoflavones daidzein and genistein at concentrations as low as 0.01 μM, nearly their concentration in the legume rhizosphere, whereas other Phytophthora species do not exhibit this attraction [24]. These preferences for specific chemicals in the chemotactic response of zoospores are believed to play a crucial role in determining host specificity [25, 26].

Zoospore movement has also been reported to be coordinated by a behavior dependent on population density known as autoaggregation or autotaxis, where zoospores swim collectively in response to surrounding zoospores [27]. This population density-dependent behavior has also been observed in other microorganisms, such as bacteria and fungi [28, 29]. Although the signaling molecules involved in the coordinated zoospore movement have not yet been identified, certain chemical compounds released by zoospores of several Phytophthora species have been reported to induce aggregation and synchronized movement of zoospores onto host plants [30].

After migrating to the host plant, zoospores transform into non-motile cysts by shedding their flagella and developing a primary cell wall. They then release a mucilaginous matrix that enables the cysts to adhere to the host surface and eventually form a germ tube that penetrates either directly or by forming an appressorium-like structure [17].

Sporangia of most airborne oomycetes can also germinate directly to form a germ tube and penetrate host cell surfaces, likely when environmental conditions are unsuitable for zoospore production and survival, such as warm temperatures and low humidity [31]. Direct sporangial germination has independently evolved in various genera, including Peronospora, Hyaloperonospora, and Vientotia [32]. In certain cases, the sporangia of specific downy mildew pathogens such as Hyaloperonospora arabidopsidis and Peronospora destructor only germinate directly, reducing their dependence on the water content required for zoospores to infect their host [8, 33]. Most aerial oomycetes infect the leaves and enter the host through the stoma or directly through the epidermis [10]. Additionally, some species, including floricolous downy mildews (belonging to the genus Peronospora), many downy mildews, and white blister rusts, infect the reproductive organs of their hosts, enabling transmission by seeds or pollinating insects [6]. Other species that infect the reproductive organs, such as graminicolous downy mildews (e.g., Peronosclerospora or Sclerophthora), have the ability to transform flowers into shoots, resulting in the formation of witches’ brooms [6]. Such a strategy provides the pathogen with a distinct advantage by exposing it to wind and rain, thereby ensuring more efficient spore dispersal [34].

3. Biological control of phytopathogenic oomycetes

Numerous studies to date have investigated the potential application of plant-protective microorganisms as BCAs against oomycete pathogens, with some already commercialized as biofungicides, such as PRESTOP^® (Lallemand Plant Care), Trianum-P (Koppert), and Sonata^® ASO (Bayer CropScience), for managing oomycete diseases (Table 1).

Table 1: Approved microbial biopesticides for oomycetes plant disease control

Product name / Manufacter	Biocontrol agent	Target pathogen
Serenade®/ Agraquest Inc.	Bacillus subtilis QST 713	Downy mildews, Pythium spp., Phytophthora spp.
Companion®/ Growth Products Ltd	Bacillus subtilis GB03	Pythium spp., Phytophthora spp.
Integral® / BASF	Bacillus subtilis MBI 600	Pythium spp.
SonataTM /AgraQuest, Inc.	Bacillus pumilus strain QST 2808	Bremia lactucae, Peronospora spp., Phytophthora spp., Plasmopara viticola, Pseudoperonospara cubensis
Lalstop® / Lallemand Inc.	Streptomyces griseoviridis K61	Pythium spp., Phytophthora spp.
Actinovate® / Natural Industries Inc.	Streptomyces lydicus WYEC108	Pythium spp., Phytophthora spp., Aphanomyces spp.
Prestop® / Verdera Oy	Gliocladium catenulatum JII446	Pythium spp., Phytophthora spp.
SoilGard® 12/ Certis Biologicals	Gliocladium virens GL–21	Pythium spp.
Bioten® / Isagro	Trichoderma asperellum ICC012, Trichoderma gamsii ICC080	Pythium spp., Phytophthora spp.
RootShield® / Bioworks	Trichoderma harzianum T–22, Trichoderma virens strain G–41	Pythium spp., Phytophthora spp.
Plant helper® /AmPac Biotech	Trichoderma atroviride	Phytophthora spp.
Polyversum® / Bioprepáty	Pythium oligandrum M1	Phytophtora spp., Pythium spp.

Despite the development of these products, the current repertoire of commercially available biofungicides that can deliver satisfactory protective effects against a variety of oomycete diseases is still limited, so there remains a need to expand the options available to growers seeking alternative or complementary control measures to agrochemicals and conventional cultural practices [35, 36]. Therefore, additional biocontrol research is crucial for the development of practical and effective biofungicide products. These products offer a sustainable and environmentally friendly approach to managing oomycete diseases in agriculture.

The subsequent sections discuss previous studies on microorganisms that exhibit antagonistic properties against oomycetes, their beneficial properties, and successful screening methods used to isolate effective BCAs.

3.1 Successful BCAs for the management of oomycete diseases

3.1.1 Bacillus

The genus Bacillus represents a large group of gram-positive bacteria extensively utilized as BCAs against a variety of plant pathogens, including bacteria, nematodes, fungi, and oomycetes [37]. Their success as BCAs is attributed to traits associated with biocontrol mechanisms, such as the secretion of potent antimicrobial metabolites and the stimulation of plant immunity [38]. Additionally, their exceptional stress tolerance, conferred by endospore production, is a significant advantage that enables them to stably colonize soil and plants under stress conditions such as drought and ultraviolet radiation [39].

Owing to their exceptional biocontrol properties, Bacillus species have been extensively researched for their potential as BCAs against oomycete diseases, and several Bacillus strains have already been commercialized as biopesticides (Table 1). Many of these biocontrol Bacillus strains have been discovered in soil and plant rhizospheres. Seed treatment with two B. subtilis strains, R33 and R13, isolated from the pepper rhizosphere, reduced disease severity against Phytophthora blight in peppers caused by Phytophthora capsica, by approximately 87% and 71%, respectively [40]. Another B. subtilis strain, BSIPR35, discovered in Iranian soil, was reported to exhibit strong antifungal activity against Phytophthora pistaciae and an excellent ability to withstand conditions of high salinity and temperature [41]. Drench application of this strain could reduce the mortality rate of pistachio seedlings caused by P. pistachiae by 80% in greenhouse experiments.

Bacillus species isolated from the interior of plant tissues have also been extensively studied for their potential as BCAs against oomycete diseases [37]. Their endophytic nature enhances their ability to colonize host plants by evading environmental stress and competition with native microbes residing on the plant surface, potentially leading to a sustained biocontrol effect [42]. The endophytic B. subtilis strain Tpb55, isolated from tobacco leaves, was identified as a BCA against tobacco black shank caused by Phytophthora nicotianae [43]. This strain was found to colonize tobacco roots for over a month post-inoculation and to produce volatile organic compounds (VOCs) that induced abnormal hyphal growth in P. nicotianae. In field trials, root-dip treatment with this strain suppressed tobacco black shank by up to approximately 60%. A recently identified endophytic strain of B. velezensis, KOF112, derived from grapevine stem xylem, reduced the disease severity of grapevine downy mildew, likely by inhibiting zoospore release from zoosporangia and inducing the production of lytic enzymes in grape leaves [44].

3.1.2 Pseudomonas

The genus Pseudomonas, a group of gram-negative bacteria ubiquitous in environments including soil, rhizosphere, and phyllosphere, is renowned as a BCA against various plant pathogens [45]. While only a few Pseudomonas-based biofungicides effective against oomycetes, such as PSEUDO-PEP (Peptech Biosciences Ltd.) and Striker (Katyayani), are commercially available in India, numerous studies have demonstrated the potential of this genus as a BCA against soil-borne oomycete diseases. Species within the Pseudomonas fluorescens complex, including P. fluorescens, P. protegens, P. koreensis, and P. chlororaphis, are particularly effective, as highlighted in the literature, due to their ability to produce potent antimicrobial compounds (e.g., 2,4-diacetylphloroglucinol (DAPG), phenazines, pyrrolnitrin, and hydrogen cyanide) and their high competitive fitness [46, 47]. The P. fluorescens strain DR54, isolated from sugar beet, has been shown to protect cucumber seedlings from P. ultimum by producing an antifungal cyclic lipopeptide, identified as viscosinamide [48]. Soil application of another P. fluorescences strain, K-Hf-L9, sourced from mixed cropping field soil, reduced pea root rot caused by Aphanomyces euteiches under greenhouse conditions. In vitro assays demonstrated that K-Hf-L9 could inhibit the mycelial growth and zoospore germination of A. euteiches [49]. As Pseudomonas species are susceptible to drought, their foliar application may be less effective against airborne oomycete diseases. However, seed and root treatments with Pseudomonas strains capable of stimulating plant immunity have been reported to suppress downy mildew [50, 51].

3.1.3 Streptomyces

The genus Streptomyces, a gram-positive, filamentous member of the phylum Actinobacteria, is widely distributed in soil and rhizosphere environments, representing a significant proportion of the soil and rhizosphere microbiota [52]. Known for producing a variety of secondary metabolites and lytic enzymes with antimicrobial activity, this genus has become a popular choice for the biocontrol of soil-borne pathogens [53]. Additionally, their ability to produce highly stress-resistant spores makes them suitable candidates for formulation into biofungicides [54]. Several antagonistic Streptomyces strains have been commercialized as biofungicides to control soil-borne pathogens, including oomycetes such as Pythium spp., Phytophthora spp., and Aphanomyces spp. (Table 1). Numerous studies to date have reported the excellent biocontrol performance of Streptomyces against soil-borne oomycete pathogens [53, 55]. Soybean seed coating with an antagonistic Streptomyces sp. strain S11, isolated from the rhizosphere of healthy soybean, was reported to effectively reduce the severity of soybean root rot caused by P. sojae by 57.1% and increase root and shoot dry weights by 140% and 108%, respectively, compared to the diseased control [56]. In another example, the antibiotic-producing Streptomyces sp. strain GS93-23, discovered in soil in the United States, exhibited in vitro antagonistic activity against multiple oomycete pathogens and significantly reduced the severity of root rot in alfalfa grown in soil naturally infested with Phytophthora medicaginis [57].

3.1.4 Trichoderma

The genus Trichoderma, comprising filamentous fungi, has received significant attention as a BCA. These fungi exhibit rapid growth and produce an abundance of asexual spores. Moreover, they possess an exceptional ability to efficiently colonize diverse environments, including soil, rhizosphere, and phyllosphere [58]. These characteristics make Trichoderma advantageous for commercialization and use as a biofungicide against soil-borne and airborne diseases. Within this genus, T. asperellum, T. atroviride, and T. harzianum have been extensively studied for their efficacy in controlling oomycete pathogens [35, 59]. These species are known to inhibit the growth and reproduction of oomycetes by producing a variety of secondary metabolites or by parasitizing the hyphae and reproductive organs of oomycetes [60, 61]. Xing et al. [62] reported that a volatile pyrone, 6-pentyl-2H-pyran-2-one, from T. erinaceum LS019-2, exhibits potent inhibitory activity against the hyphal growth and sporangia germination of Peronophythora litchii. Benhamou and Chet [63] demonstrated that T. harzianum isolate T-203 could parasitize and enzymatically degrade the hyphae of P. ultimum. Through these direct actions, Trichoderma BCAs protect plants from infection by soil- and airborne oomycete pathogens. Zegeye et al. [64] found that the foliar application of an antagonistic strain of T. viride isolate ES-1 could significantly reduce potato late blight. T. asperellum isolates 659-7, PR10, PR11, and PR12, selected from over 200 fungal isolates from Cameroonian soils, were able to parasitize Phytophthora megakarya, one of the causal agents of black pod disease in cacao [65]. Foliar application of these isolates to cacao trees showed a significant control effect on the incidence of black pod disease in a long-term field trial.

3.1.5 Pythium

Certain Pythium species have been reported to suppress diseases caused by other oomycete species (Table 2). Pythium oligandrum is the best known biocontrol species within this genus, with numerous studies confirming its biocontrol efficacy against many oomycete diseases [66, 67], and one of the effective strains, namely M1, has been commercialized. P. oligandrum has the ability to colonize plant roots like pathogenic species, but it does not cause damage to plant tissues [68]. In addition, this species is well known as a mycoparasite that can parasitize hyphae of many fungal pathogens, including phytopathogenic oomycete species [69]. Pythium nunn, discovered by Kobayashi et al. [70] in Japan, is another mycoparasitic species and has been reported to suppress damping off of cucumber seedlings caused by P. ultimun var. ultimum.

Table 2: Microbial biocontrol agents reported to antagonize oomycete plant pathogens in biocontrol studies

Biocontrol agent	Host	Target pathogen	Main mechanism of action proposed	Reference
Actinobacteria	–	Phytophtora infestans	Antibiosis	Santos et al. [71]
Actinomycetes	Cucumber	Pythium aphanidermatum	Antibiosis	El‐Tarabily et al. [72]
Bacillus altitudinis JSCX–1	Soybean	Phytophthora sojae	Antibiosis, ISR	Lu et al. [73]
Bacillus amyloliquefaciens JDF3, Bacillus subtilis RSS–1	Soybean	Phytophthora sojae	Antibiosis, ISR	Liu et al. [74]
Bacillus amyloliquefaciens PP19, Exiguobacterium acetylicum SI17	Litchi	Peronophythora litchii	Antibiosis	Situ et al. [75]
Bacillus pumilus B048	Soybean	Phytophthora sojae	Antibiosis	Fu et al. [76]
Bacillus pumilus, Bacillus subtilis, Bacillus cereus, Bacillus mycoide, Paenibacillus polymyxa	Pea	Aphanomyces euteiches	Antibiosis	Wakelin et al. [77]
Bacillus subtilis	Red pepper	Phytophthora capsici	Antibiosis	Lee et al. [40]
Bacillus subtilis KS1	Grapevine	Plasmopara viticola	Antibioisis	Furuya et al. [78]
Bacillus subtilis Tpb55	Tobacco	Phytophthora nicotianae	Antibiosis	Han et al. [43]
Bacillus subtilis, Bacillus pumilus, Burkholderia cepacia, Enterobacter kobei	–	Phytophthora nicotianae	Antibiosis	Malvi et al. [79]
Bacillus velezensis Ba168	Tobacco	Phytophthora nicotianae	Antibiosis	Guo et al. [80]
Bacillus velezensis SDTB038	Potato	Phytophtora infestans	Antibiosis	Yan et al. [81]
Bacillus velezensis KOF112	Grapevine	Plasmopara viticola, Phytophthora infestans	ISR, Antibiosis	Hamaoka et al. [44]
Bacillus asahii CE8	Cucumber	Pseudoperonospora cubensis	Unknown	Sun et al. [82]
Burkholderia cenocepacia	Burrowing clover	Phytophthora cinnamomi	Unknown	Colavolpe et al. [83]
Chaetomium globosum, Chaetomium lucknowense, Chaetomium cupreum	Pomelo	Phytophthora nicotianae	Antibiosis, Mycoparasitism	Hung et al. [84]
Enterobacter sp. DP14, Bacillus licheniformisHS10, Bacillus pumilus DS22	Cucumber	Pseudoperonospora cubensis	ISR	Zheng et al. [85]
Glomus mosseae, Trichoderma harzianum	Papaya	Phytophthora nicotianae	ISR	Sukhada et al. [86]
Hanseniaspora uvarum MP1861	Tomato	Phytophthora nicotianae	Antibiosis	Liu et al. [87]
Lysobacter enzymogenes 3.1T8	Cucumber	Pythium aphanidermatum	Unknown	Folman et al. [88]
Paenibacillus polymyxa B2, B5, B6	Arabidopsis	Phytophthora palmivora, Pythium aphanidermatum	Unknown	Timmusk et al. [89]
Paenibacillus polymyxoides P2–5	Rhododendron	Phytophthora cinnamomi	Antibioisis	Liu et al. [90]
Paenibacillus sp. B2	Grapevine	Plasmopara viticola	ISR, Antibiosis	Hao et al. [91]
Penicillium daleae, Metarhizium anisopliae, Penicillium herquei	Rhododendron, Camellia	Phytophthora ramorum	Unknown	Widmer and Dodge [92]
Pseudomonas aeruginosa	Cucumber	Phytophthora capsici	Antibiosis	Zohara et al. [93]
Pseudomonas azotoformans UQ4510An	Tomato	Phytophthora capsici	ISR	Arkhipov et al. [94]
Pseudomonas fluorescens PRA25, Pseudomonas cepacia AMMD, Corynebacterium sp. 5A	Pea	Pythium aphanidermatum	Unknown	Parke et al. [95]
Pseudomonas corrugata CCR04, CCR80 Flavobacterium sp., GSE09, Chryseobacterium indologenes ISE14	Red pepper	Phytophthora capsici	Unknown	Sang et al. [36]
Pseudomonas corrugata, Pseudomonas fluorescens	Cucumber	Pythium aphanidermatum	Antibiosis, Signaling disruption	Paulitz et al. [96]
Pseudomonas fluorescens	Avocado	Phytophthora cinnamomi	Antibiosis	Sumida et al. [97]
Pseudomonas fluorescens K–Hf–L9, Pantoea agglomerans PSV1–7, Lysobacter capsici K–Hf–H2	Pea	Pythium aphanidermatum	Unknown	Godebo et al. [40]
Pseudomonas fluorescens SS101	Tomato	Phytophtora infestans	Antibiosis	Tran et al. [98]
Pseudomonas fluorescens SS101	Hyacinth	Pythium ultimum var. sporangiiferum, Pythium intermedium, Phytophthora infestans	Antibiosis	De Souza et al. [99]
Pseudomonas putida MGP1	Papaya	Phytophthora nicotianae	ISR	Shi et al. [100]
Pseudomonas spp.	Potato	Phytophthora infestans	Antibiosis	De Vrieze et al. [101]
Pythium nunn	Cucumber	Pythium ultimum	Mycoparasitism	Kobayashi et al. [70]
Pythium nunn	Sweet orange	Phytophthora parasitica	Unknown	Fang and Tsao [102]
Pythium oligandrum	–	Phytophthora parasitica	Mycoparasitism	Horner et al. [69]
Pythium oligandrum	Tomato	Phytophthora parasitica	ISR	Picard et al. [103]
Streptomyces OB21, Streptomyces BA15	Pea	Aphanomyces euteiches	Antibiosis	Oubaha et al. [55]
Streptomyces rochei	Pepper	Phytophthora capsici	Antibiosis	Ezziyyani et al. [104]
Streptomyces sp. S11	Soybean	Phytophthora sojae	Antibiosis	Arfaoui et al. [56]
Streptomyces spp.	Soybean, Alfalfa	Phytophthora sojae	Antibiosis	Xiao et al. [57]
Trichoderma asperellum AFP, Trichoderma asperellum MC1, Trichoderma brevicompactum MF1, Trichoderma harzianum CH1	–	Phytophthora capsici	Antibiosis	Das et al. [105]
Trichoderma viride ES1	Potato	Phytophtora infestans	Mycoparasitism	Zegeye et al. [64]
Trichoderma asperellum	–	Phytophthora ramorum	Mycoparasitism	Widmer [106]
Trichoderma asperellum Tv–1	Rhododendron	Phytophthora cinnamomi	Mycoparasitism	Liu et al. [90]
Trichoderma harzianum	Gypsophila	Pythium aphanidermatum	Antibioisis	Sivan et al. [107]

ISR: Induced systemic resistance.

3.2 Biocontrol mechanisms

3.2.1 Antibiosis

Antibiosis refers to the process where bioactive compounds are produced that disrupt the normal growth or metabolism of other microorganisms [108]. The inhibition of growth and reproduction of oomycete pathogens through antibiosis is a prevalent mode of action of BCAs, as documented in the literature (Table 2). Several studies have identified key antimicrobial metabolites that play pivotal roles in the biocontrol of oomycetes. For example, 2,4-DAPG and massetolide A, synthesized by Pseudomonas spp. [98, 99], actinomycin D and borrelidin, synthesized by Streptomyces spp. [109, 110], fengycin and lichenysin, synthesized by Bacillus spp. [111, 112], and gliovirin and gliotoxin, synthesized by Trichoderma spp. [113, 114], have all been demonstrated to contribute to biocontrol mechanisms against oomycete diseases.

Additionally, antimicrobial VOCs emitted by BCAs have been proposed as potential biocontrol mechanisms against oomycete diseases. Gfeller et al. [115] demonstrated that potato-associated Pseudomonas produced 1-undecene and sulfur compounds on the surface of potato leaves, inhibiting the development of P. infestans.

3.2.2 Competition for nutrients

Nutrient deficiency can cause severe damage to cellular functions, leading to the death or stunted growth of microorganisms [116]. Therefore, competition for limited nutrients has been proposed as one of the primary modes of action of BCAs against plant pathogens [14]. Although the significance of nutrient competition in the biocontrol of plant pathogens is not well understood experimentally, and there are only a few examples available in the literature, some BCAs have been found to control oomycete diseases through nutrient competition. Iron, an essential element for almost all microorganisms, plays a critical role in oomycete pathogenicity [117]. Under iron-deficient conditions, many microorganisms secrete low-molecular-weight iron chelators, known as siderophores, to solubilize and acquire iron [118]. Competition for iron by siderophores has been implicated in the biocontrol of oomycete diseases by several BCAs. P. fluorescens strain 3551 exhibited biocontrol activity against cotton damping-off caused by P. ultimum. Indeed, Tn5 insertion mutants, which lacked the capacity to generate a siderophore, demonstrated a significant reduction in effectiveness compared to the original strain 3551 [119].

3.2.3 Hyperparasitism

Hyperparasitism, also known as mycoparasitism, is recognized as a significant disease control mechanism found in certain BCAs such as Trichoderma spp., P. oligandrum, non-pathogenic Fusarium spp., and Streptomyces spp. These BCAs parasitize the hyphae, or survival structures, of plant pathogens, effectively preying on them [69, 106, 120, 121]. Oomycete cell walls are primarily composed of β-1,3-glucan, β-1,6-glucan, and cellulose [122]. Additionally, some oomycetes contain minor amounts of chitin in their cell walls [123]. During mycoparasitism, BCAs secrete various potent hydrolytic enzymes, such as cellulase, chitinase, and proteases, leading to the degradation of oomycete cell walls and cellular components and resulting in the death of hyphae, chlamydospores, oospores, and sporangia [124, 125]. Despite numerous studies reporting in vitro mycoparasitic activity of BCAs against oomycete pathogens, only a few studies demonstrate mycoparasitism as a determinant mechanism of in planta antagonism against oomycete pathogens [126]. As mycoparasitic BCAs may antagonize plant pathogens in planta in conjunction with other modes of action such as antibiosis, it is challenging to ascertain the exact importance of mycoparasitism in the biocontrol effect [127]. One of the few studies demonstrating that mycoparasitism is the primary mechanism of biocontrol of oomycete diseases is that of Harman et al. [126], where they found that a seed treatment with a mycoparasitic isolate of T. hamatum, which lacked antifungal activity, could control damping-off in pea and radish caused by Pythium spp.

3.2.4 Enhancement of plant immunity

The enhancement of plant immunity by BCAs has been widely recognized as a biocontrol mechanism [128]. It is well known that plants possess innate immunity to shield themselves from harmful organisms attacks [129]. Certain BCAs can induce or prime plant defense responses, such as the accumulation of antimicrobial substances and structural modifications in cell walls, thereby indirectly preventing plant infection by oomycete pathogens [130]. Four rhizobacterial strains, Burkholderia gladioli TRH423-3, Miamiensis avidus TRH427-2, Acinetobacter genomospecies KRJ502-1, and Bacillus cereus KRY505-3, identified by An et al. [131], were found to significantly reduce the severity of late blight symptoms on tomato leaves caused by P. infestans. The drench application of these strains induced callose deposition on leaves, preventing leaf infection by P. infestans. Pseudomonas azotoformans UQ4510An, a rhizobacterium, has been used to protect tomato plants from Phytophthora blight caused by P. capsici [94]. This strain has demonstrated the ability to prevent pathogen infection through a blend of direct antagonism and swift immune response activation by activating multiple defense-related signaling pathways. BCAs that stimulate plant immunity could be highly beneficial in situations where technical constraints or costs prohibit the delivery of BCA to specific plant parts. This is because the application of BCA to one part of the plant can systemically activate immune responses, thereby suppressing pathogen attacks at remote locations [132].

3.2.5 Other unique mechanisms

In addition to the aforementioned mechanisms, the unique activities of BCAs have been identified as potential biocontrol mechanisms against oomycete pathogens. Certain chemicals released by host plants are known to attract zoospores or induce the infection behavior of oomycete pathogens. The elimination or degradation of such signaling chemicals has been reported as an effective mechanism to disrupt plant infection by oomycete pathogens, thereby suppressing disease incidence [133, 134]. For instance, Zhou and Paulitz [135] suggested that biocontrol strains of Pseudomonas reduced the attraction, encystment, and germination of zoospores of P. aphanidermatum by metabolizing certain compounds released from cucumber roots.

Hypovirulent pathogens, which exhibit reduced or no virulence against host plants, have been extensively studied for their use in the biocontrol of various plant diseases. Hypovirulence is often caused by infection with certain mycoviruses (hypoviruses) that result in reduced sporulation and defective growth of host fungi [136]. Hypoviruses can be transmitted horizontally from a hypovirulent strain to a virulent pathogen through hyphal fusion [137]. Therefore, artificial inoculation of a mycovirus-mediated hypovirulent strain can reduce the population of pathogens in host plant tissues or fields. The biocontrol of chestnut blight in Europe through the introduction of a hypovirulent Cryphonectria parasitica strain is a prime example of successful biocontrol using hypovirulent strains of fungal pathogens [138]. Oomycete pathogens have also been reported to harbor mycoviruses [137], some of which exhibit a hypovirulent phenotype [139]. The use of such hypovirulent oomycete strains could represent a novel strategy to control oomycete diseases, although, to our knowledge, there are no reports of successful examples of this approach.

4. Screening approaches for the selection of effective BCAs

The efficient selection of BCAs that provide adequate disease control is crucial for the development of successful biofungicides. This section aims to offer a thorough review of screening methods that have resulted in the identification of effective BCAs against oomycetes.

In vitro screenings are frequently used in biocontrol studies for the initial screening of antagonistic BCAs against a variety of plant diseases. This approach allows for the rapid and concurrent screening of numerous microbial strains within a defined timeframe and space [140]. The dual culture assay, which evaluates the ability of potential BCAs to produce inhibitory compounds effective against the mycelial growth of pathogens on solid agar media, is a common choice for preliminary in vitro screening of BCAs against oomycetes. Many previous biocontrol studies targeting oomycete pathogens have often used nutrient-rich media such as potato dextrose agar, V8 juice agar, and cornmeal agar for the dual culture assay [141, 142], as either the oomycete pathogens or the BCAs, or both, can actively grow on these media. However, the type or composition of the medium used for the dual culture assay must be carefully considered. The production of antimicrobial metabolites is generally influenced by the availability of carbon, nitrogen, and phosphate, particularly being upregulated under conditions of limited phosphorus and nitrate availability [143]. It is also well known that the types of antimicrobial metabolites produced by the same microbial strain can vary under different nutrient conditions [144]. Therefore, even if selected BCA strains produce a specific potent antimicrobial compound in a particular nutrient-rich medium, they may not produce that compound or may produce it at very low concentrations in soil or in planta conditions, which can lead to discrepancies between the in vitro antifungal activity and in planta disease control activity of BCAs.

In research focused on the identification of BCAs for combating obligate parasitic oomycetes pathogens, such as downy mildew pathogens that are non-culturable on media, two alternative methodologies can be used for large-scale screening of BCA strains. The initial method involves a dual culture assay using culturable oomycetes as test pathogens in place of obligate pathogen species. Hamaoka et al. [44] identified the biocontrol strain KOF112 of B. velezensis, which demonstrated efficacy against the downy mildew pathogen of grapevine, Plasmopara viticola (Table 2). This was based on its antifungal activity against culturable P. infestans and other fungal pathogens on agar plates. The secondary method involves in vivo bioassays using host plant tissues infected with the target pathogen. BCA candidate strains are applied to detached host plant tissues, such as leaf discs, infected with the pathogen. Following incubation, their inhibitory effects on hyphal growth, production of infection structures (e.g., sporangia and zoospore), and symptom development are observed. Sun et al. [82] utilized two in vivo bioassays (i.e., the leaf disc method and the separate leaf method), in conjunction with a sporangia release inhibition assay, to screen antagonistic bacteria with control activity against the cucumber downy mildew pathogen. Through these screening procedures, they identified a biocontrol strain of Bacillus sp. CE8 that demonstrated superior control efficacy against the pathogen compared to the chemical fungicide metalaxyl-mancozeb in field trials.

Beyond the aforementioned strategies, such as screening based on the in vitro antagonistic capability of BCAs against pathogens, marker-based in vitro screening is also frequently employed in preliminary mass screenings. This approach analyzes marker characteristics that are closely associated with antagonism. The most commonly sought markers in such screenings include the production of lytic enzymes (e.g., cell wall-degrading glucanases, cellulases, and proteases) [145, 72] or the production of siderophores [40, 85]. In their study, Situ et al. [75] selected bacterial strains based on criteria such as enzymatic activity (e.g., protease, chitinase, cellulase, and glucanase), siderophore production capability, and antifungal activity. They successfully identified two biocontrol strains, B. amyloliquefaciens PP19 and Exiguobacterium acetylicum SI17, from these selected strains. These strains demonstrated excellent biocontrol efficacy against litchi downy mildew caused by Peronophythora litchii.

For the successful biocontrol of oomycete diseases, it is considered crucial to identify BCAs that can inhibit multiple stages in the life cycle of the oomycete pathogen, rather than merely inhibiting mycelial growth. As such, numerous prior studies on oomycete biocontrol have evaluated the efficacy of microbial strains in inhibiting sporangia formation, zoospore germination, and zoospore release and motility as a criterion for selecting effective BCAs. For example, Zohara et al. [93] reported that three antagonistic Pseudomonas strains, chosen from 30 bacterial strains based on their in vitro ability to inhibit mycelial growth, sporangia formation, and the release and motility of P. capsici zoospores, demonstrated excellent protection against damping-off in cucumber plants. In a separate study, three strains were selected from 604 bacterial strains based on their ability to inhibit mycelial growth, zoospore germination, and the chemotaxis of zoospores to aspartic acid. These strains exhibited significant biocontrol activity against P. aphanidermatum in cucumber plants [96].

5. Conclusion

As illustrated in this review, certain microbial BCAs have shown to provide a level of crop protection that is comparable to or superior to that of commonly used chemical pesticides against oomycete diseases under field conditions. However, the creation of new and practical biocontrol products against oomycetes remains a complex task. This complexity arises from the absence of efficient, cost-effective, and high-throughput screening procedures that facilitate the identification of BCAs that consistently deliver satisfactory performance in planta. To establish such screening procedures, it is essential to reveal the modes of action and biocontrol traits through a comprehensive comparative analysis of various unsuccessful and successful BCAs. A review of existing literature indicates that the inhibitory activity against sporangia germination, encysted zoospore germination, and/or chemotaxis of zoospores toward host plants may be a more suitable trait than the inhibitory activity against hyphal growth for the screening of effective BCAs against oomycete pathogens. Furthermore, the optimization of formulation and application methods, along with a deeper understanding of the functional interactions between the various players in the soil microbiome, will also contribute to the development of successful biocontrol products. By adopting a multidisciplinary approach and collaboratively addressing these challenges, we can pave the way for a future where biocontrol assumes a central role in promoting agricultural sustainability.

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