Microbial antagonists against plant pathogens in Iran: A review

The purpose of this article was to give a comprehensive review of the published research works on biological control of different fungal, bacterial, and nematode plant diseases in Iran from 1992 to 2018. Plant pathogens cause economical loss in many agricultural products in Iran. In an attempt to prevent these serious losses, chemical control measures have usually been applied to reduce diseases in farms, gardens, and greenhouses. In recent decades, using the biological control against plant diseases has been considered as a beneficial and alternative method to chemical control due to its potential in integrated plant disease management as well as the increasing yield in an eco-friendly manner. Based on the reported studies, various species of Trichoderma, Pseudomonas, and Bacillus were the most common biocontrol agents with the ability to control the wide range of plant pathogens in Iran from lab to the greenhouse and field conditions.


Introduction
Increasing human population in the world demands more food (70 to 100%) by 2050 to supply human needs (Godfray et al. 2010). Furthermore, different pests and diseases cause annual economic losses (20 to 40%) in agricultural products by decreasing the crop yield, destroying the quality, and pollution of products with toxic chemicals (Guo et al. 2013). Therefore, growers have generally concentrated on the intensive use of chemicals for the management of pests and diseases which induce several problems, including resistance to pesticides, hazardous effects on human health, loss of beneficial soil microorganisms, entrance of residual toxic material in the food chain, and reduction in macro-microorganism biodiversity (Sindhu et al. 2016). These problems make enhanced attempts for developing ecofriendly microbe-based pesticides or biopesticides which use biological control agents (BCAs) as active ingredients and basically act different from common chemical pesticides (Sindhu et al. 2009).
Biological control, which attracted broad considerations in the past few decades, is defined as a bioeffector strategy that uses other living organisms for controlling insects, mites, weeds, and phytopathogens (Flint et al. 1998). Biocontrol agents either with antagonistic activities, or modifying effects on plant physiology and anatomy, mostly reduce the negative effects of pathogens. The advantages of beneficial microbes for associated plants are establishment of antagonistic microorganisms, prevention of phytopathogens, overall improvement of plant health, plant growth promotion, enhanced nutrient availability and uptake, and increased resistance to both biotic and abiotic stresses in the hosts (Vinale et al. 2014).
The first published studies on biological control of plant pathogens in Iran were presented in 1992. Trichoderma spp. and Gliocladium spp. were the first biocontrol agents applied against Athelia rolfsii (Sclerotium rolfsii), Rhizoctonia solani, and Fusarium solani, the causal agents of diseases on groundnut, bean, and apple, respectively (Asghari and Myee 1992; Bazgir et al. 1992; Karampour and Okhovat 1992). In the twenty-first century, with the improvement of biological control of plant pathogens throughout Iran, different biocontrol agents have been applied against the various pathogens in vitro, in greenhouse and field conditions. A large number of fungal and bacterial biocontrol agents have been found as the most important agents for plant disease management with identification of their role in plant pathogen management (Ramadan et al. 2016). Trichoderma, Pseudomonas, and Bacillus species have mostly been used for biological control of phytopathogens in Iran (Peyghami Furthermore, because of increasing the stability of biological agents, the bioformulation progress has recently been evaluated in Iran (Karimi and Sadeghi 2015). The current study is a comprehensive review of applying fungal and bacterial antagonists for biological control of various plant diseases caused by fungal, bacterial, and nematodes in Iran during a period of 26 years.

Mechanisms of biocontrol agents for the management of phytopathogens
A key factor for attaining an effective prevention of phytopathogens in their hosts is the knowledge about their mechanism of action. Understanding the mechanisms in the biological control process can allow the establishment of favorable conditions in the interaction between phytopathogen and biocontrol agent that is important in performing a successful biological control strategy in a specific pathosystem (Handelsman and Stabb 1996). The microorganisms operating for biocontrol of phytopathogens have different modes of action (Nega 2014). In the present study, the most common mechanisms of interspecies antagonisms include direct antagonism, mixed-path antagonism, and indirect antagonism (Pal and McSpadden 2006;Parveen et al. 2016), which lead to biological control of plant pathogens, have been addressed. Microbial biocontrol agents take care of plants against pathogens via different modes. These agents could induce resistance or initial enhanced resistance against pathogens without direct confrontation with the phytopathogen. Also, competitions for nutrients and spaces are additional indirect interactions with phytopathogens (Köhl et al. 2019). These agents might directly interact with the pathogens using hyperparasitism (Ghorbanpour et al. 2018) or antibiosis (Raaijmakers and Mazzola 2012). Without these agents in soil and tissues of plants, the pathogens easily attack plants and could weaken or kill considered hosts ( Figure 1). These modes will be discussed in the following sentences.

Parasitism
Mycoparasitism, direct parasitism or hyperparasitism, is the ability of fungal antagonistic agents to parasite other fungi Over time, affected plants will show the weakness in the development and symptoms of diseases. Right: in the presence of antagonists with different biocontrol mechanisms, such as competition, parasitism, and antibiosis, the pathogens will not be able to progress in the host, and thus, the plant can grow and develop well rather than the absence of antagonists in soil and tissues of hosts.
for utilizing them as food. Mycoparasitism causes either complete death of fungal propagules or destruction and lysis of their structure (Maloy 1993). Mycoparasitism depends upon the sequential occurrence of the following events: coming into close contact with fungal pathogen, mutual recognition between antagonist and pathogen, lytic enzyme secretion by antagonist, penetration into the host, active growth of antagonist into the host, and exit (Spadaro and Gullino 2004;Talibi et al. 2014). Various chemical compounds can be implicated in these processes, such as lectins, during the initial contact and recognition and cell wall-degrading enzymes (CWDEs), such as β-1,3-glucanases, chitinases, proteinases, and lipases, during the penetration process (Vos et al. 2015). Wisniewski et al. (1991) who studied biological control of Botrytis cinerea by yeast antagonist Meyerozyma guilliermondii (Pichia guilliermondii) demonstrated that lectin-like interaction resulted in firm attachment of antagonist's cell to B. cinerea. Lysis of fungal cell wall also occurred due to the action of extracellular β-1,3-glucanase enzyme secreted by the antagonistic yeast. Trichoderma species are specific mycoparasitic fungi with the species of T. atroviride, T. virens, and T. reesei confirming that mycoparasitism is their ancestral lifestyle (Kubicek et al. 2011).
One of the main components in mycoparasitism event is CWDEs including endochitinases, β-1,3-glucanases, and proteases that are extracellular enzymes secreted by Trichoderma (Vos et al. 2015). After initial pathogen recognition by Trichoderma, hyphae wind around the pathogen's hyphae by forming hook, the appressorium permeates into the pathogen cell, and chitin is broken down by enzymes such as chitinase and glucanase (Ghorbanpour et al. 2018). Subsequently, mycoparasitic's hyphae release antibiotic compounds which penetrate the affected pathogen's hyphae and resynthesize the host cell wall inhibited by these compounds (Toghueoa et al. 2016).

Antibiotic
Antibiotic is a secreting secondary metabolite with low molecular weight that is deleterious to the other microorganisms at low concentrations (Fravel 1988

Cell wall degradation enzymes
Microorganisms which produce enzymes are able to hydrolyze chitin, proteins, cellulose, and hemicellulose and also may play a role in the suppression of plant pathogens. Chitin and β-1,3-glucans are major constituents of many fungal cell walls (Lam and Gaffney 1993). Trichoderma strains with antagonistic potential have been mainly characterized by their ability to secrete enzymes such as chitinases, glucanases, and proteases that hydrolyze the cell walls of pathogens (López-Mondéjar et al. 2011). Geraldine et al. (2013) reported that N-β-acetylglucosaminidase and β-1,3glucanase are the key components of Trichoderma species action in biocontrol of Sclerotinia sclerotiorum in the field. Serratia marcescens which produces chitinases was found to suppress the growth of Botrytis spp., R. solani, and Fusarium oxysporum (Ningaraju 2006).

Competition for available resources
Microorganisms' challenge for available resources is named competition. For instance, when pine stumps were inoculated by spores Phlebiopsis gigantea (Phlebia gigantea), the spores prevent from Heterobasidion annosum infections. Considering that the pathogen is non-established on the pine, the severity of root rot disease could be decreased by the biocontrol agent (Cook and Baker 1983). Despite the possibility of existing antagonistic relationship (e.g., antibiosis) between the two fungi, the achievement of available resource sites may be the first mechanism in competition (Maloy 1993). Carbon sources such as glucose and fructose are one of the important action modes in yeasts Papiliotrema laurentii (Cryptococcus laurentii) and Sporobolomyces roseus, which can control B. cinerea in decreasing its colonization and sporulation (Ghorbanpour et al. 2018). In the biological control of P. digitatum by Debaryomyces hansenii, competition plays an important role in obtaining nutrients in occupied sites (Droby et al. 1998). Furthermore, arbuscular mycorrhiza due to the creation of physiological and anatomical modifications can limit the progression of pathogen. These changes involve root lignification, creation of a thick cell wall using pectin, chitinase activation, and transfer of pathogenesis-related protein-1a to the infected area of root (Malik et al. 2016).

Siderophore
Low-molecular weight chelators with a very high and specific affinity for Fe(III) are called siderophores (Barbeau et al. 2002). Aerobic and facultative anaerobic microorganisms with the ability of siderophore production may have an important role in microorganism interactions (Haggag and Mohamed 2007). Siderophores have been known to play a significant role in phytopathogen prevention by several bacteria as BCAs which prevent the growth, development, and metabolic activity of phytopathogens by iron chelation (Haggag Wafaa et al. 2000). Different species of Trichoderma as biocontrol antagonists release more effective siderophores that chelate iron (Fe 3+ ) and prevent growth and development of other fungal pathogens (Naher et al. 2014). Iron competition can be a limiting factor in alkaline soils for microbial growth and development (Leong and Expert 1989). Siderophores produced by some bacteria, such as fluorescent pseudomonads, have very high dependency for iron, as a result, sequestering these limited resources from other microflora can inhibit their growth and development (Loper and Buyer 1991). In several studies, it has been reported that Pseudomonas fluorescens with siderophore biosynthesis plays an important role in the prevention of pathogen (Costa and Loper 1994). Rahnella aquatilis with siderophore production can inhibit B. cinerea and P. expansum postharvest pathogens (Calvo et al. 2007). The siderophore pulcherrimin produced by Metschnikowia pulcherrima and Monilinia fructicola yeasts was applied for biological control of postharvest apple pathogens B. cinerea, Alternaria alternata, and P. expansum (Saravanakumar et al. 2008). In particular, several species of Streptomyces detach iron by siderophore production in a way that some pathogens, owing to a lack of siderophore production, cannot take these ions for growth (Kloepper et al. 1980).

Induction of host resistance
Plant growth promoting rhizobacteria can protect plants against pathogens using induction of systemic resistance (ISR) (Sikora 1992). P. fluorescens with stimulating ISR can prevent the early penetration of Heterodera schachtii to roots (Oostendorp and Sikora 1989 The direct promotion of plant growth by plant growth promoting bacteria through the production of phytohormones has been called phytostimulation (Bloemberg and Lugtenberg 2001). The enzyme 1-aminocyclopropane-1carboxylate (ACC) deaminase is a phytostimulation that is the most studied one. Some bacterial endophytes producing ACC deaminase have been shown to enhance plant growth, such as Arthrobacter spp., Bacillus spp., P. putida, Rhodococcus spp.

Reduction in the population of biocontrol agents
Phytopathogens may significantly alleviate the growth of biocontrol agents by using the nutrition resources within their occupied spaces more rapidly as well as by modifying their efficacy. This was found in several fungal root pathogens which can colonize the wheat rhizosphere despite the presence of P. fluorescens biocontrol agent (Mazzola and Cook 1991). Decline in the population of P. fluorescens occurs in the existence of some Pythium species. In this instance, infection by Pythium species leads to the limitation of the root surface which is available for P. fluorescens colonization and to the reduction of population of potential antagonists. Fedi et al. (1997) reported that a plant pathogenic P. ultimum with modification of gene expression of P. fluorescens tends to decrease biocontrol agent population. The competition in the rhizosphere for nutrients released from root wounds caused by P. ultimum was limited by the reduction of population size. Because of the importance of microbial community in number and diversity, competition and microorganism-microorganism interactions may also happen in phyllosphere (Vorholt 2012). On the other hand, existence of these microbial communities may also impress the efficacy of BCAs. Understanding the rhizosphere, phyllosphere, and endosphere microbial community structure and their interactions in these niches can contribute to the betterment of biocontrol (Bardin et al. 2015).

Improving the biocontrol agent effects
The use of combinations of BCAs may be a better method for developing biocontrol positive effects (Duffy and Weller 1995). Combined biocontrol agents with high level of biocontrol protection have been investigated for better efficacy and prevention of several phytopathogens (Mihajlović et al. 2017). It has been confirmed that natural prevention of Fusarium wilt in France (Châteaurenard soil) was related to the different mechanisms in which multiple microorganisms singly or together restricted the pathogen activation (Alabouvette et al. 1998). However, given that the application of biological control against soilborne pathogens will not be a good replacement of methyl bromide fumigation, these two methods could act together in integrated pest management (Akrami et al. 2011).

Biological control in Iran
A complete list of all pathogens and the antagonists used against them is provided in            In vitro and greenhouse Mousavi Mirak (2014) Microbial antagonists against plant pathogens in Iran  419