Exploring the Trends in Actinobacteria as Biological Control Agents of Phytopathogenic Fungi: A (Mini)-Review

Abstract

Biological control has been considered a sustainable alternative to combat phytopathogens. The increase of studies in the past few years involving Actinobacteria as biological control agents of phytopathogenic fungi has motivated us to search for which Actinobacteria genus that have been studied in the last five years and explore their mechanisms of antifungal activity. The accesses were carried out on three multidisciplinary digital platforms: PubMED/MedLine, Web of Science and Scopus. Actinobacteria from genus Amycolatopsis, Curtobacterium, Kocuria, Nocardioides, Nocardiopsis, Saccharopolyspora, Streptoverticillium and especially Streptomyces showed a broad antifungal spectrum through several antibiosis mechanisms such as the production of natural antifungal compounds, siderophores, extracellular hydrolytic enzymes and activation of plant defense system. We observed the formation of a methodology based on antagonistic compounds bioactivity to select efficient Actinobacteria to be used as biological control agents against phytopathogenic fungi. The use of multifunctional Actinobacteria has been proven to be efficient, not only by its natural protective activity against phytopathogenic fungi but also because of their ability to act as plant growth-promoting bacteria.

Introduction

To avoid agricultural losses it is necessary to manage plant diseases. The management can be done by resistant cultivars farming and crop rotation, but mostly, it is made through the application of synthetic chemical pesticides in crop plants and seeds. The continuous and intensive use of these synthetic chemical pesticides in the agriculture industry can increase environmental pollution by its accumulation in soil, water, and air and induce the emergence of drug resistant pathogens that threaten human and other animals' health. Their toxicity can also suppress beneficial microbes affecting the native plant growth [1–7].

Sustainable agriculture is an emerging area of research [8]. Biological control has been considered a sustainable alternative to combat phytopathogens and promoting plant growth [5]. It is a kind of strategy, for managing plant diseases, that aims to reduce the excessive use of synthetic chemical pesticides and induce de use of organisms or chemicals derived from microorganisms, plants and animals that have a natural antagonistic activity [3, 9]. In this scene, Actinobacteria play an important role as biological control agents (BCAs) to manage plant diseases caused by microorganisms. The well-known ability of Actinobacteria species to produce bioactive secondary metabolites that suppress or inhibit pathogens growth and the increase of studies in the past few years involving Actinobacteria as BCAs against phytopathogenic microorganisms has motivated us to search for which Actinobacteria genus have been studied against phytopathogenic fungi and explore their mechanisms of antifungal activity.

Methods

This work is a narrative literature review. The accesses were carried out in July 2022 on three multidisciplinary digital platforms: PubMED/MedLine, Web of Science and Scopus. The search strategy applied on the three platforms included the following keywords and boolean operators: (Actinobacteria OR Actinobacterias OR Actinomycete OR Actinomycetes) AND ("Biological Control Agents" OR "Biological Control Agent" OR Biopesticides OR "Biological Pesticides" OR Biopesticide OR "Biological Control") AND (phytopathogen*). For the analysis, only english original articles, published between 2017 to 2022 and made available in PDF extension, were considered. After eliminating duplicate articles, a total of thirty-five articles were analyzed. Review articles and extra references were used to support discussion.

The Potential of Actinobacteria as Antifungal Agent

Actinobacteria is a phylum of Gram-positive bacteria that constitute one of the largest bacterial phyla and have a distinguishing feature, which is high guanine + cytosine DNA content. Actinobacteria phylum is constituted by Bifidobacterium, Corynebacterium, Frankia, Gardnerella, Gordonia, Leifsonia, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Rhodococcus, Salinispora, Streptomyces, Thermobifida and Tropheryma genus. Most Actinobacteria have a mycelial lifestyle (except Bifidobacterium), are aerobic bacteria (except Propionibacterium and Gardnerella which are anaerobic and facultative anaerobic, respectively) chemoheterotrophic and free-living. Filamentous Actinobacteria generally reproduce by sporulation, growing by tip extension and branching of the hyphae, but they can also perform vegetative reproduction through mycelial fragmentation [10]. Figure 1 is an example of the diversity of filamentous morphology from colonies and microstructures of Actinobacteria that belong to the genus Streptomyces.

Fig. 1.

Diversity of morphological characteristics of filamentous Actinobacteria from genus Streptomyces grown under laboratory conditions. (a, b and c) Streptomyces colonies on International Streptomyces Project-2 medium (morphological characteristics: whitish, grayish, stiff and powdery). (d, e and f) Streptomyces spores on Water Agar medium (400X magnification) (morphological characteristics: rod shaped spores that are distributed in long chains or forming whorls of small chains). (g, h and i) Streptomyces colonies on Starch Casein Agar medium (morphological characteristics: whitish, yellowish, stiff and powdery) (Author´s image)

Actinobacteria are ubiquitous bacteria that are abundantly present in soil [11–13]. They represent a high proportion of the microbial flora of the rhizosphere and some of them are viewed as soil saprophytes with crucial role in nutrient cycling [10]. Actinobacteria belong to the second most abundant group of endophytes (organisms that live inside plants) with a mutualistic relation with plants. Beneficial endophytic Actinobacteria can control phytopathogenic fungi and promote plant growth in a similar way to free-living Actinobacteria [2]. Both have potential to produce several secondary metabolites that act as antifungal compounds, act as elicitors of plant defense system, increase the availability of nutrients in soil to be absorbed to plants, produce phytohormones that promote direct plant grown and enable the plant to cope up with environmental stress [2, 4, 6, 14–21]. Thus, Actinobacteria play a beneficial role for sustainable agriculture either through prevention of losses and/or increasing agricultural yields.

Genera of Actinobacteria That Have Been Used Against Phytopathogenic Fungi

Actinobacteria from eight different genera (Fig. 2) showed a broad antifungal spectrum (Table 1).

Fig. 2.

Genus of Actinobacteria that have been used against phytopathogenic fungi (Amycolatopsis 8%; Curtobacterium 3%; Kocuria 3%; Nocardioides 3%; Nocardiopsis 5%; Saccharopolyspora 3%; Streptoverticillium 3% and Streptomyces 75%). The image was created by the authors with MicroSoft Excel 2016

Table 1.

Antagonistic strains of Actinobacteria and susceptible phytopathogenic fungus

Genus	Antagonistic strain	Susceptible phytopathogenic fungus	References
Amycolatopsis	Amycolatopsis 1119	Pythium ultimum, Rhizoctonia solani and Fusarium oxysporum	[22]
	Amycolatopsis sp. BX17	Fusarium graminearum	[13]
	Amycolatopsis lexingtonensis CIAD-CA13 and Amycolatopsis lurida CIAD-CA30	Alternaria alternata and Sclerotium rolfsii	[23]
Saccharopolyspora	Saccharopolyspora shandongensis CIAD-CA15	Sclerotium rolfsii	[23]
Curtobacterium	Curtobacterium sp. XA15-35	Botrytis cinerea and Sclerotinia sclerotiorum	[5]
Nocardioides	Nocardioides albertanoniae CIAD-CA20	Fusarium oxysporum, Alternaria alternata and Sclerotinia sclerotiorum	[23]
Nocardiopsis	Nocardiopsis dassonvillei MB22	Bipolaris sorokiniana	[24]
	Nocardiopsis aegyptica H14	Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani	[25]
Kocuria	Kocuria rhizophila PT10	Botrytis cinerea and Fusarium graminearum	[26]
Streptoverticillium	Streptoverticillium morookaense	Ustilaginoidea virens, Rhizoctonia solani and Bipolaris maydis	[3]
Streptomyces	Streptomyces flavotricini 25, Streptomyces globisporus subsp. globisporus C28, Streptomyces globisporus 7, Streptomyces senoensis 420 and Streptomyces pactum Act12	Sclerotium rolfsii	[27]
	Streptomyces sp. SN0280	Phytophthora capsici	[14]
	Streptomyces sp. IISRBPAct1, IISRBPAct25 and IISRBPAct42	Sclerotium rolfsii	[28]
	Streptomyces spp. SS1, SS5 and SS8	Magnaporthe oryzae and Rhizoctonia solani	[2]
	Streptomyces spp.	Fusarium solani and Rhizoctonia solani	[1]
	Streptomyces AV04, AV05, F4, F5 and H2	Fusarium verticillioides	[29]
	Streptomyces corchorusii stain AUH-1	Fusarium oxysporum f. sp. niveum	[30]
	Streptomyces chumphonensis AM-4	Penicillium digitatum and Penicillium italicum	[11]
	Streptomyces sp. FJAT-31547	Fusarium oxysporum	[16]
	Streptomyces albus PFK4 and PFBOT7 and Streptomyces gandoceansis PFEL2	Fusarium oxysporum and Colletotrichum gloeosporioides	[4]
	Streptomyces sp. DAAG3-11, DGS1-1, DDPA2-14 and DGS3-15	Sclerotinia sclerotiorum	[31]
	Streptomyces sp. SCA3-4	Fusarium oxysporum f. sp. cubense Tropical Race 4	[15]
	Streptomyces sp. MR14	Pyricularia oryzae, Exserohilum sp., Colletotrichum gloeosporioides, Colletotrichum acutatum, Alternaria brassicicola, Alternaria alternata, Alternaria solani, Alternaria mali and Cladosporium herbarum	[32]
	Streptomyces MG788011 and MG788012	Botrytis cinerea	[8]
	Streptomyces palmae CMU-AB204T	Aspergillus niger, Candida albicans, Ganoderma boninense and Mucor racemosus	[6]
	Streptomyces sp. M4	Alternaria brassicicola, Alternaria solani, Fusarium oxysporum, Colletotrichum acutatum and Colletotrichum gloeosporioides	[18]
	Streptomyces violaceusniger JBS5-6	Fusarium oxysporum Race 4, Colletotrichum acutatum, Curvularia fallax, Fusarium oxysporum sp. cucumebrium, Pyricularia oryzae, Colletotrichum gloeosporioides, Fusarium graminearum, Botryosphaeria dothidea, Curvularia lunata, Colletotrichum fragariae and Botrytis cinerea	[17]
	Streptomyces nigrogriseolus GanoSA1	Ganoderma boninense	[33]
	Streptomyces albidoflavus OsiLf-2	Magnaporthe oryzae	[34]
	Streptomyces sp. YYS-7	Fusarium oxysporum f. sp. cubense race 4	[19]
	Streptomyces sp. CACIS-1.5CA	Colletotrichum sp., Alternaria sp., Aspergillus sp., Botrytis sp., Rhizoctonia sp. and Rhizopus sp.	[9]
	Streptomyces spp. M2A2	Rhizoctonia solani	[35]
	Streptomyces albidoflavus H12	Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani	[25]
	Streptomyces sp. (PNM-149)	Colletotrichum gloeosporioides	[20]
	Streptomyces sp. H4	Colletotrichum fragariae	[21]
	Streptomyces cangkringensis CIAD-CA07, Streptomyces misionensis CIAD-CA27 and Streptomyces kanamyceticus CIAD-CA45	Fusarium equiseti, Fusarium oxysporum, Alternaria alternata and Sclerotium rolfsii	[23]
	Streptomyces exfoliatus	Aspergillus flavus	[36]

Actinobacteria from genus Amycolatopsis, Nocardioides and Nocardiopsis, share a chemotaxonomic characteristic, the lack of mycolic acids in their peptidoglycan but differ on the rest of composition. Amycolatopsis cell walls contain meso-2,6-diaminopimelic acid, arabinose and galactose [37], i.e., cell wall chemotype IV [10], Nocardioides cell walls contain LL-2,6-diaminopimelic acid and glycine, i.e., cell wall chemotype I [38] and Nocardiopsis cell walls contain meso-2,6-diaminopimelic acid but no diagnostically important carbohydrates [39]. Like Nocardioides, both Streptoverticillium and Streptomyces have cell wall chemotype I and unlike aforementioned genera, cell walls of Actinobacteria from genus Curtobacterium are deprived of 2,6-diaminopimelic acid but contain ornithine in peptidoglycan composition [10].

Actinobacteria can be catalase-positive, e.g., Nocardiopsis [39] and Saccharopolyspora [40]; coagulase-negative, e.g., Kocuria [41]; non acid-fast, e.g., Nocardioides [38] and Nocardiopsis [39]; form rod and/or coccoids shape spores, e.g., Nocardioides [10], Saccharopolyspora [40] and Kocuria [41], that can be distributed in long chains or form whorls of small chains, e.g., Streptoverticillium and Streptomyces [10].

Actinobacteria are widespread. Most genera can be found in both marine and terrestrial environments [39, 42–44]. Curtobacterium have been isolated as endophytes from many plants [5]. Nocardiopsis are able to prevail under different environmental conditions and have been known for their halotolerant nature, which allows them to be found in hypersaline habitats [39]. Both strains of Nocardiopsis (Table 1) were isolated from Algeria environments, marshes and Sahara soil. Saccharopolyspora, isolated from soil [23] and Kocuria, isolated from roots [26] (Table 1) can also be found in clinical samples [40] and as part of the normal skin and oral microbiota of humans [41], respectively. According to Barka et al. [10] soil populations are dominated by the genus Streptomyces. They represent over 95% of the Actinobacteria strains isolated from soil. Most strains represented in Fig. 2 and listed in Table 1 are from the genus Streptomyces and most of them were isolated from soil and rhizosphere (Fig. 3). Interestingly, the only three strains isolated from marine environments [11, 20, 21] also belong to this genus.

Fig. 3.

Environments from which Streptomyces strains have been isolated (Sponges 4%; Corals 4%; Molluscs 4%; Marshes 4%; Roots 7%; Leaves 4%; Soil and rhizosphere 73%). The image was created by the authors with MicroSoft Excel 2016

Nocardiopsis [24, 25, 39], Amycolatopsis [43] and Streptomyces [10, 37] have been known as producers of various bioactive secondary metabolites, including anticancer, antiviral, antibacterial, antifungal, immunosuppressive compounds, among others. Amycolatopsis species possess 25 biosynthetic gene clusters [37] that encode a variety of secondary metabolites [43], while Streptomyces species contain 637 biosynthetic gene clusters [37]. In fact, Streptomyces species produce more than two-thirds of the medically and agriculturally important secondary metabolites [45]. In terms of biological control of phytopathogenic fungi, secondary metabolites have high applicability, as they act by inhibiting essential primary metabolic processes of microorganisms [46]. According to Quinn et al. [42], the bioactive secondary metabolites are produced during the growth phase of Actinobacteria and although they are not strictly necessary for growth or reproduction, they can give Actinobacteria a competitive advantage. The production of secondary metabolites such as natural antifungal compounds, siderophores and extracellular hydrolytic enzymes, is considered a mechanism of Actinobacteria antifungal activity at the same time that is considered a plant growth promoting trait [47].

Mechanisms of Antifungal Activity

Antimicrobial activity of Actinobacteria indirectly enhances plant growth. Naturally, the inhibition of phytopathogens infection allows the plant to grow healthily. Some of the best known mechanisms of Actinobacteria antifungal activity are: the production of natural antifungal compounds [6, 16–18, 21]; competition with phytopathogens for iron (Fe) through the production of an Fe(III)-specific ligand named siderophore [4, 28]; production of extracellular hydrolytic enzymes, such as chitinases and glucanases, which can degrade fungal cell wall [34] and the expression of genes that may be related to the activation of plant defense system [2]. Several mechanisms of antifungal activity presented by Actinobacteria play a key role in phytopathogenic fungi biocontrol strategy [5]. Actinobacteria antifungal activity can involve more than one of these mechanisms acting at the same time, which make them potent BCAs.

Kocuria rhizophila PT10 is a powerful Actinobacteria producer of extracellular hydrolytic enzymes amylase, lipase and protease [26]. According to the authors, this set of enzymes are potentially associated with its ability to inhibit pathogens growth. In silico analysis associated PT10 capacity of hydrolytic enzyme production to the presence of putative genes in its genome, especially those related to the glycoside hydrolase family, e.g. amylase, lipase, cellulase and chitinase. The authors indicate that glycoside hydrolase proteins play an important role in the hydrolysis of the fungal cell wall. Guesmi et al. [26] suggest that bacteria and fungi that produce glycoside hydrolase proteins have great potential to be applied in the biological control of phytopathogenic fungi, which could make Kocuria rhizophila PT10 an efficient BCA.

Curtobacterium sp. XA 15–35 also showed the ability to lyse the fungal cell wall through the production of hydrolytic enzymes chitinase and protease. Furthermore, the XA 15–35 genome carries surfactin biosynthesis (Sfp), surfactin synthase (SrfC) and iturin A biosynthesis (ItuD) genes [5]. According to the authors the detection of those three functional genes on XA 15–35 genome could be related to the nonribosomal biosynthesis of two cyclic lipopeptides, surfactin and iturin, known by their antibacterial and antifungal properties and their use as BCA’s on different crops [48, 49]. Therefore, the production of surfactin and iturin can be another antifungal mechanism played by Curtobacterium sp. XA 15–35.

Nonribosomal peptide synthetases (NRPSs) are enzymes responsible for the production of nonribosomal peptides, which are classified as secondary metabolites. These include siderophores and natural antibiotics, e.g. surfactin, iturin and fengycin. NRPS enzymes can be found in bacteria, eukarya and archaea domains, with certain abundance in Actinobacteria phylum [50]. NRPS genes were detected in the genome of Streptomyces sp. CACIS-1.5CA, as well as polyketide synthase (PKS) type I and II genes [9]. Polyketides are natural metabolites that comprise the basic chemical structure of several biomolecules of biotechnological interest, such as natural antifungal compounds [46]. PKS type I and II genes were also detected in the genome of other Streptomyces, Streptomyces sp. SCA3-4 [15].

Many Streptomyces strains showed as producers of extracellular hydrolytic enzymes. Streptomyces spp. IISRBPAct1, IISRBPAct25 and IISRBPAct42 produced protease, cellulase and amylase [28], whereas Streptomyces spp. PFK4, PFBOT7 and PFEL2 produced protease, chitinase, lipase and β-1, 3-glucanase [4] and all those Streptomyces strains produced siderophores as a further mechanism of antifungal activity. In addition to the production of extracellular hydrolytic enzymes, Streptomyces albidoflavus OsiLf-2 [34] showed a very interesting mechanism of antifungal activity. Rice leaves sprayed with OsiLf-2 enhanced the production of intracellular hydrogen peroxide (H₂O₂), a critical secondary messenger of defense signaling in plants as well as enhanced the deposition of callose around the stomata. Callose is a plant polysaccharide produced in the early stages of phytopathogen invasion and acts as an effective barrier in plant cell walls. According to Gao et al. [34] the production of callose by OsiLf-2 suggests that it can contribute to the defense system in rice plants. Streptomyces albidoflavus OsiLf-2 also stimulated the rice plant defense system through the plant´s hormones salicylic acid and/or jasmonic acid pathway. According to Pateal et al. [2] it is generally accepted that the induction of plant systemic responses to phytopathogens is mediated by jasmonic acid and ethylene, whereas the salicylic acid pathway is the predominant mechanism by which phytopathogens induce systemic resistance (ISR).

The triggering of plant defense system activation by the action of secondary metabolites produced by Actinobacteria or by interaction with endophytic Actinobacteria can be verified through the expression of genes related to plant defense [2, 22]. According to Maier et al. [51] systemic acquired resistance (SAR) of plants is a disease resistance state that is induced after infection with necrotizing phytopathogens. It is regulated by increasing levels of salicylic acid, which result in the induction of a heterogeneous group of SARs markers, termed pathogenesis-related (PR) proteins, that supposedly confer resistance against biotrophic phytopathogenic microorganisms.

Endophytic Streptomyces spp. SS1, SS5 and SS8 modulated defense responses in rice plants that were under pathogen attack [2]. SS1, SS5 and SS8 strains increased the expression of non-expressor of pathogenesis-related genes 1 (NPR1), pathogenesis-related 10a gene (PR10a) and lipoxygenase-2 gene (LOX2). LOX gene encodes lipoxygenase, which is the first enzyme in the biosynthesis of the jasmonic acid pathway [22]. In contrast, rice plants bacterized with SS1 and SS5 strains that were not challenged with phytopathogenic fungi showed down-regulation of those pathogen-related genes. According to Patel et al. [2], this type of regulatory behavior caused by the colonization of plants with bacteria that induce systemic responses is generally observed and indicates that Streptomyces spp.SS1 and SS5 subside the defense-related genes expression in healthy plants. Likewise, bacterial treatment with rhizospheric Amycolatopsis 1119 enhanced the expression of PR and LOX genes, as well as ascorbate peroxidase (APX) and GLU defense-related genes in cucumber plants [22]. In Amycolatopsis 1119 case, the authors indicate that the relative increase in APX transcription and the subsequent increase in ascorbate peroxidase activity in bacterial treated cucumber plants not challenge with phytopathogens also showed that Amycolatopsis 1119 stimulated ISR at the transcription level without pathogen exposure need. In both cases, Actinobacteria showed potential to decrease the plant's time response to phytopathogenic microorganisms attack. That ability characterizes another mechanism of antagonistic activity developed by Actinobacteria as BCAs that can be applied against phytopathogenic fungi.

Many authors study the side effects caused by the application of secondary metabolites produced by Actinobacteria isolates in the microstructures of phytopathogenic fungi [3, 17, 30]. Extracts of Streptomyces violaceusniger JBS5-6 significantly inhibited the hyphal growth and spore germination of Fusarium oxysporum Race 4 by destroying membrane integrity and the ultrastructure of cells [17]. The crude extract of Streptomyces corchorusii AUH-1 significantly inhibits ergosterol biosynthesis and accelerates lipid peroxidation in Fusarium oxysporum f. sp. niveum membranes, which can affect the structure and function of the fungi cells [30]. Antifungal metabolite compound produced by Streptoverticillium morookaense provoke severe morphological abnormalities in hyphae structure of Ustilaginoidea virens and Bipolaris maydis, also affecting spore germination of these two phytopathogenic fungi [3]. In general, the authors observed that there is an inversely proportional relationship between the concentration of the antifungal compound and its power antagonistic activity. The number of germinated spores and mycelium growth decreased with increasing concentrations of the antifungal metabolites, which characterizes a dose-dependent behavior [3, 13, 19, 20, 27, 34, 36]. In Table 2 we gather some of these antifungal compounds.

Table 2.

Antagonistic strains of Actinobacteria and their antifungal compounds

Actinobacteria strain	Antifungal compounds	References
Streptomyces spp. SS1, SS5 and SS8	2,4-ditert-butylphenol	[2]
	1‐ethylthio‐3‐methyl‐1,3‐butadiene
	2-(chloromethyl)-2-cyclopropyl oxirane
Streptomyces palmae CMU-AB204T	(Z)-5-(2-methylphenyl)-4-pentenoic acid	[6]
	(Z)-7-(2-methylphenyl)-6- heptenoic acid
	Anguinomycin A
	Leptomycin A
	Actinopyrone A
Streptomyces sp. SN0280	(4E,8E)-4,10,12- trimethyl-4,8-diene-13-oxo tetradecanoic acid	[14]
Streptomyces sp. M4	Salvianolic acid B	[18]
Streptomyces violaceusniger JBS5-6	5-hydroxymethyl-2- furancarboxaldehyde	[17]
Streptomyces sp. FJAT-31547	1-Nonadecene; among others	[16]
Streptomyces sp. (PNM-149)	Methyl anthranilate	[20]
Streptomyces sp. H4	Dibutyl phthalate; among others	[21]
Streptomyces sp. YYS-7	2,2’-methylenebis [6-(1,1-dimethylethyl)-4-methyl-	[19]
	2,4-Di-tert-butylphenol; among others
Streptomyces sp. SCA3-4	N-hexadecanoic acid	[15]
	2,4-bis (1,1-dimethylethyl)- phenol
	(Z)-13-docosenamide
	Oleic acid
	Tetradecanoic acid; among others
Streptomyces sporoclivatus DAAG3-11	Azalomycins F4a and Azalomycins F5a	[31]
Streptomyces cavourensis DGS1-1	Bafilomycin B1
Streptomyces pratensis DGS3-15	Actinolactomycin
	Dimeric dinactin
	Tetranactin
	Dinactin
Streptomyces capitiformicae DDPA2-14	Maremycin G and Maremycin analogue

The production of Actinobacteria antifungal compounds is related to growth conditions and that dynamic depends on a number of factors such as pH, temperature, salt concentrations and carbon sources [15, 19]. Palafox-Félix et al. [13] tested the antifungal metabolites secretion of the Actinobacteria Amycolatopsis BX17 in a culture medium supplemented with glucose. The authors observed a decrease of 35% on the inhibition of the mycelial growth of Fusarium graminearum in a medium supplemented with glucose compared to glucose-free medium. According to Palafox-Félix et. al [13] the inhibition decrease suggests that in the absence of glucose Amycolatopsis BX17 metabolized amino acids to synthesize antifungal compounds, whereas in the presence of glucose carbon flux was directed to the synthesis of energy and cell growth.

Plant Growth-Promoting

In addition to the antimicrobial activity of Actinobacteria, which indirectly enhances plant growth, the ability of some strains of Actinobacteria to synthesize phytohormones and solubilize essential micronutrients is considered to be a direct plant growth promotion trait. [4, 5, 22, 28]. According to Djemouai et al. [7] Actinobacteria’s multifunctional enzymatic machinery makes them strongly involved in nutrient cycling and soil quality.

Phosphorus is one of the key plant nutrients; therefore, its availability directly affects plant growth and, consequently, crop production. In soil, phosphorus is found mostly unavailable to plants. Microorganisms that have the ability to solubilize phosphate, through mechanisms that include production of organic acids, can release free phosphate to be absorbed by plants [47]. Beyond their antifungal activity Curtobacterium sp. XA 15–35 [5] and Streptomyces spp. PFK4, PFBOT7 and PFEL2 [4] were able to solubilize inorganic phosphate. Likewise Streptomyces spp. IISRBPAct1, IISRBPAct25 and IISRBPAct42 were able to solubilize zinc, which is also an essential micronutrient for plant growth [28]. The nutrient solubilization trait of Actinobacteria also includes the production of siderophores, since their production improves the bioavailability of Fe to the plants by influencing its mobility and solubility in the soil [47].

Streptomyces spp. PFK4, PFBOT7 and PFEL2 were also able to produce other important plant growth promotion traits. They produced 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase or ACCd) as well as ammonia and indole-3-acetic acid (IAA), an auxin-class phytohormone [4]. ACCd is an enzyme that decreases the production of plant ethylene by metabolizing ACC into α-ketobutyric acid and ammonia, therefore, under stressful conditions, Actinobacteria that produce ACCd, like Streptomyces spp. PFK4, PFBOT7 and PFK2, decrease the level of ethylene, which allows plant elongation and growth. IAA is known for controlling several fundamental cellular mechanisms of plants (e.g., cell division, differentiation, and elongation) and enhances root hair formation, which improves plant nutrient absorption capacity from soil [47]. In addition to the production of ACCd and IAA Streptomyces albidoflavus OsiLf-2 also produced gibberellic acid [34].

The search for growth-promoting traits is generally used for screening and selecting effective plant growth-promoting bacteria (PGPB) for agricultural applications [22]. The PGPB strategy aims to reduce the use of high amounts of chemically synthesized fertilizers and increase the application of bio fertilizers. As it has been found, Actinobacteria have the power to act on two fronts, as potent BCAs as well as PGPBs, which in addition to being an eco-friendly strategy can have potentially economic advantages.

Application of Actinobacteria in the Biological Control of Phytopathogenic Fungi

Agricultural soils are a reservoir of diverse and persistent phytopathogenic fungi [1] that are a major threat to crop production due to their capability to induce plant diseases [30]. Their development in the field, especially in tropical regions, can cause great losses as well as quality reduction of several kinds of crops. It is estimated that phytopathogenic fungi cause 70–80% of the diseases present in crops worldwide [7]. Postharvest losses of fresh fruits and vegetables, caused by fungal infections, along with the food supply chain is also an important point of concern [9, 11, 21]. The management of these disease agents is crucial not only economically but for the maintenance of food security as well, especially now, when the world’s population has reached eight billion people. In this context, biological control strategies have emerged due to the high efficiency and considerably lower environmental contamination and health risks [5, 35].

Extracts of Streptomyces violaceusniger JBS5-6 were able to inhibit the infection caused by Fusarium oxysporum Race 4 to banana seedlings, reducing the disease index of banana wilt disease [17]. Streptomyces sp. FJAT-31547 reduced the disease incidence of Fusarium wilt caused by Fusarium oxysporum in tomato plants by 80.59% [16]. The antifungal metabolite produced by Streptoverticillium morookaense reduced the negative effects of Ustilaginoidea virens, causative agent of rice false smut, in susceptible rice seeds. Compared with carbendazim, a systemic fungicide usually used to protect rice plants, the antifungal metabolite produced by Streptoverticillium morookaense was more effective in emerging health, and increased fresh and dry weights and vigor of seedlings [3]. Streptomyces griseocarneus R132 suppressed the germination of Fusarium oxysporum and controlled the development of anthracnose caused by Colletotrichum gloeosporioide in Capsicum annuum plants and fruits. R132 strain showed a severity index of plant wilt similar to non-infected plants and also effectively promoted plant growth, especially increasing shoot dry mass [52]. Fermentation extracts from Streptomyces chumphonensis AM-4, a marine isolate, were able to control green mold caused by Penicillium digitatum and blue mold caused by Penicillium italicum in fresh citrus fruits [11]. Other examples of Actinobacteria BCAs are listed in Table 3.

Table 3.

Actinobacteria as biological control agents

Actinobacteria	Pathogenic fungi	Host and disease	References
Streptomyces spp. IISRBPAct1, IISRBPAct25 and IISRBPAct42	Sclerotium rolfsii	Black pepper basal wilt	[28]
Streptomyces spp. M2A2	Rhizoctonia solani	Rice sheath blight	[35]
Streptomyces sp. H4	Colletotrichum fragariae	Strawberry fruits anthracnose	[21]
Streptomyces sp. MR14	Fusarium moniliforme	Tomato wilt	[32]
Streptomyces sp. YYS-7	Fusarium oxysporum f. sp. cubense tropical race 4	Banana wilt	[19]
Nocardiopsis dassonvillei MB22	Bipolaris sorokiniana	Wheat root rot	[24]
Streptomyces sp. SCA3-4	Fusarium oxysporum f. sp. cubense tropical race 4	Banana wilt	[15]
Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 consortium	Fusarium oxysporum f. sp. radicis-lycopersici	Tomato infection	[25]
	Rhizoctonia solani	Carrot infection

Under field conditions Streptomyces MG788011 and MG788012 were able to prevent the chocolate spot disease symptoms caused by Botrytis cinerea in Faba at the same time that increased the yields of the plants [8]. The authors tested three different types of treatments: coating the seeds with Streptomyces spp. spores, spraying mycelia and spraying the crude metabolites produced by MG788011 and MG788012 over the plants, being the latter the most efficient of them. Spraying the crude metabolites produced by MG788011 and MG788012 over the plants enhanced the dry weight by approximately 2 g and 8 g, respectively. Also under field conditions, spore suspension of Streptomyces albidoflavus OsiLf-2 significantly decreased the disease index of rice blast caused by Magnaporthe oryzae in 19,6% when compared to the untreated control [34]. OsiLf-2 strain achieved an efficient percentage, close to that presented by the treatment with tricyclazole. The treatment with foliar spraying showed a higher disease control efficiency. The authors suggest that inoculation onto leaves will not be affected by soil microbes, then the spraying treatment could be more efficient.

On the other hand, Shariffah-Muzaimab et al. [33] pointed out the importance of providing the prior establishment of the BCAs population in treatments based on soils application. The authors observed the need for more than one application of Streptomyces nigrogriseolus GanoSA1 to control basal stem rot disease symptoms caused by Ganoderma boninense in oil palm nurseries and plantations. Zambrano et al. [35] also indicated that the preventive treatment of the soil with Streptomyces spp. M2A2 as well as during the development of rice plants reduced the progress of rice sheath blight symptoms caused by Rhizoctonia solani.

Many authors have observed that the number of laboratorial data compared to the number of field studies that provide information on the protective activity of Actinobacteria against phytopathogens is scarce. Among the thirty-five analyzed articles only two tested the antagonistic activity of Actinobacteria isolates in the field. Djemouai et al. [7] pointed out that there is a lack of retrospective literature discussing the effectiveness of Actinobacteria in soil-borne pathogens biocontrol compared to non-Actinobacteria, such as Pseudomonas and Bacillus, which hinders the development of control strategies using Actinobacteria as biological control agents. This gap between laboratory and field may also be linked to the action of environmental weathering on the stability of secondary bioactive metabolites produced by Actinobacteria as well as the inability of antagonistic strain of Actinobacteria, selected in vitro conditions, to adjust under natural conditions [24, 32]. Therefore, the in vitro antifungal activity does not guarantee the application of the Actinobacteria as BCA under field conditions. The mechanism of antifungal action of Actinobacteria and the effectiveness of their actions as BCAs depends on the dynamics between Actinobacteria, plant, phytopathogenic fungi and abiotic conditions (i. e., temperature, pH, soil moisture, etc.) [53] (Fig. 4). Djemouai et al. [7] exalt the need for in-depth understanding on that relationship to ensure safe and sustainable crop production worldwide. We observed the formation of a general methodology based on the bioactivity of antagonistic compounds to select efficient Actinobacteria to be applied as BCAs against phytopathogenic fungi (Fig. 5) and despite the peculiarities, Actinobacteria continue to prove their value as important BCAs against phytopathogenic fungi and as value PGPBs to many plants.

Fig. 4.

The dynamics between Actinobacteria, plants, phytopathogenic fungi, and abiotic conditions define the BCA and PGP activity. The image was created by the authors with personal collection photos and images available in Pixabay

Fig. 5.

Scheme of methodology based on the bioactivity of antagonistic compounds to select efficient Actinobacteria to be applied as BCAs against phytopathogenic fungi (First step: Actinobacteria isolation through serial dilution. Second step: screening of Actinobacteria through a confrontation assay. Third step: in vitro assays i. e. effect of the antifungal compounds on mycelia growth, conidia germination and hyphal structures. Fourth step: identification of the Actinobacteria through morphological, molecular and physiological parameters. Fifth step: identification of antifungal compounds i. e. extraction of bioactive antagonistic compounds, temperature range, pH levels, light exposure, gas chromatography coupled with mass spectrometry (GC–MS). Sixth step: in vivo assays, from greenhouse to the field). The image was created by the authors with icons available in Flaticon

Conclusions and Future Perspectives

The improvement of studies in the use of microorganisms as biological control agents is notable. Many authors attribute interesting features to microbial pesticides like low toxicity and low resistance generation. In past few years the number of publications related to Actinobacteria as protective agents against phytopathogenic fungi has taken a leap and some actinobacterial BCAs have been developed into commercial formulations, such as Actinovate® (Streptomyces lydicus WYEC 108) and Mycostop® (Streptomyces sp. K61). The findings in the analyzed articles corroborated to attest the high efficiency of these multifunctional bacteria as biological control agents and growth promoting microorganisms, especially Streptomyces sp. In times where much is discussed about food security due to the increase in the world population in a context of economic crisis, drastic climate changes and changes in habits in a post-pandemic society, it is extremely important to continue developing technologies that work together with nature. The development of studies that aim to use Actinobacteria to reduce the use of pesticides and chemical fertilizers meets the need to strengthen sustainable agriculture. The use of Actinobacteria in the integrated management of phytopathogenic fungi, as well as on other crop pests, is promising. The development of selection methodologies based on Actinobacteria antagonistic activity and plant growth promote traits, as well as the elucidation of those mechanisms, is shown to be the most accurate way to reduce this gap between laboratory and field.

Author Contributions

All authors contributed to the study conception and design. The first draft of the manuscript was written by Heloísa Giacomelli Ribeiro and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Case Number: 88887.463460/2019-00.

Availability of Data and Materials

The data and material that support the findings of this study are openly available in PubMED/MedLine, Web of Science and Scopus at https://pubmed.ncbi.nlm.nih.gov/, https://clarivate.com/webofsciencegroup/ and https://www.scopus.com/, respectively.

Declarations

Conflict of interest

The authors declare no conflicts of interest regarding the publication of this manuscript.