Biocontrol Mechanisms of Endophytic Microorganisms

By Ajay Kumar

Biocontrol Mechanisms of Endophytic Microorganisms introduces endophytic microorganisms, colonization, diversity and distribution, describes the isolation and identification of endophytic microorganisms by traditional cultivation and by next generation sequencing technologies, and covers biocontrol mechanisms, bacterial priming, endophytic based methods, the significance on fungi, and metabolite based formulations. The book concludes with chapters on biofilms, microbiota and safety issues of microorganisms.

The intensive use of chemicals to control these plant pathogens has resulted in negative consequences such as the release of toxic chemicals in the environment, reduced soil fertility and human health problems. Therefore, environmentally-friendly and sustainable replacement of chemical fertilizers or pesticides is highly challenging.

• Contains exclusive information about research on immunogenetics going on all over the world
• Includes all the minute and recent details that will be the prerequisite requirement for any researcher who wants to work on immunogenetics and its applications
• Comes fully-equipped with pictures, illustrations and tables, delivering the information in a meticulous manner that makes it more attractive to readers

• Language: English
• Publisher: Academic Press
• Release date: Nov 23, 2021
• ISBN: 9780323884792

Book preview

Chapter 1

Colonization, diversity, and distribution of endophytic microbial communities in different parts of plants

Jasim Basheer,    Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic

Abstract

Endophytic microbes live inside plant tissues without harming or overriding any of the plant defense mechanisms. Their colonization has a mutualistic by getting themselves shelter and benefits the host in improving nutrient uptake and helps in plant growth under normal and stressed conditions. To achieve this, microbes must successfully colonize the plant endosphere through a series of controlled events both by plants and microbes. The diversity and selection of microbes are strictly controlled by the plants since the colonization process is highly energy demanding for the plants and will do only under specific conditions. Some endophytes can colonize a broad range of host with multiple plant growth potential and, thus, can be utilized as potential candidates for developing bioinoculants for sustainable agriculture. For this, it is necessary to study in detail about different aspects of colonization process and its dynamics. In the present chapter, different aspects of colonization, mechanisms involved in overriding defenses, and diversity are discussed.

Keywords

Endophytes; colonization; diversity; mechanisms; ETI; PTI; MAMPS

1.1 Introduction

Associations of plants and microbes are critical for them to overcome many challenges posed by the natural environment through their beneficial interactions (Afzal, Shinwari, Sikandar, & Shahzad, 2019; Santoyo, Moreno-Hagelsieb, del Carmen Orozco-Mosqueda, & Glick, 2016). The microbes can be divided into different groups based on their association with the plants as epiphytic (found externally on the leaf and stem surfaces); rhizospheric (found associated with roots in the rhizosphere region); or endophytic (which live inside the plant tissues) (Compant, Clément, & Sessitsch, 2010; John, Kumar, & Ge, 2020; Tichá, Illésová, & Hrbáčková, 2020). These associations have numerous mutualistic benefits to each other by providing help in mitigating various biotic and abiotic stresses (Miliute, Buzaite, Baniulis, & Stanys, 2015). Among these different classes of microbes, we are mainly focusing on the endophytic microbial communities being the most important contributor of plant growth.

First description of endophytes was done by a German botanist named Heinrich Friedrich Link in 1809 suggesting the presence of microbes inside plants. However, for a long time, the term endophytae was used only to describe pathogenic fungi due to their prevalence in diseased plants, thus, to the belief that healthy plants are sterile. But later in 19th century, reports of Galippe proved the presence of microbes inside the plants which pointed to study the beneficial role of soil microbes and their migration from soil to the plants (Hardoim, van Overbeek, & Berg, 2015). The term endophyte was coined by De Bary in 1866 using two Greek words endon which means within, and phyton which means plant for defining microbes that grow inside plants which was modified later by other researchers from their specific observations (Chanway, 1997). One of the significant observations was the isolation of pure culture of nitrogen fixing bacteria (later classified as Rhizobium leguminosarum) from the root nodules of Leguminosae plants by Martinus Willem Beijerinck in 1888 (Anyasi & Atagana, 2019). Another important one was from Albert Bernhard Frank who observed the mutualistic relationship between tree root and underground fungi which he later termed mycorrhiza (translates as fungus roots) where both parties benefited and, thus, the first observation of symbiosis (Domka, Rozpaądek, & Turnau, 2019). These observation enabled the researchers to study different aspects of these relationships which motivated Orlando Petrini in 1991 to modify the definition of endophytes to all organisms inhabiting plant organs that at some time in their life cycle can colonize internal plant tissues without causing apparent harm to their host and is evolving since then by updated observations (Petrini, 1991).

The importance of endophytes increases since nearly all species of the plants existing on the surface of the earth are colonized by one or more endophytic microbes (Ryan, Germaine, & Franks, 2008). Each one of these microbes thrives in the unique microenvironments based on the host’s metabolite capacity, making the endophytes a treasure trove of biosynthetic potency (Jasim, Benny, Sabu, Mathew, & Radhakrishnan, 2016). Due to the diverse biosynthetic capabilities, endophytes are reported to support the plant in growth enhancement and resistance to different stress factors (Chanway, Shishido, & Nairn, 2000; Cipollini, Rigsby, & Barto, 2012; Hardoim, van Overbeek, & van Elsas, 2008; Mei & Flinn, 2010; Rosenblueth & Martínez-Romero, 2006). Endophytes mostly originate from the rhizosphere and colonize the plant in some stages of the plant growth and share almost all the growth-promoting properties exhibited by rhizosphere microbes, thus, considered a subclass of rhizosphere bacteria by some researchers.

1.2 The rhizosphere as a microbial contributor

The rhizosphere is the area of soil that is in close contact with the plant root which has a dynamic role in maintaining the health and physiology of the plants and the microbiome associated with it (Singh, Singh, & Kumar, 2017; Singh, Singh, Singh, & Kumar, 2018; Kumar, Droby, Singh, Singh, & White, 2020). The process of colonization is highly complex which involves a cascade of processes from both host plant and microbial counterpart. Plants initiate the process of selection and attraction by communication with its associated microbes using chemical molecules present in the root exudates (de Weert et al., 2002; Rosenblueth & Martínez-Romero, 2006). The main components present in the root exudates are proteins, amino acids, and organic acids that play the key role in the process of selecting the microbial communities associated with the host plant. Oxalates are found to have such use in different studies which concludes that the levels of oxalates can even dictate the concentration of microbes attached to the plant. Microbes with defective oxalate utilization mechanism were found to have significant loss in colonization capability when studied in Burkholderia phytofirmans PsJN (Esmaeel, Miotto, & Rondeau, 2018).

Microbes use compounds from the exudates as quorum sensing molecules for the specific attachment to the host plant. Compounds with quorum sensing capability play an important role in the endophyte colonization and extending their support to the host. B. phytofirmans PsJN which has a knockdown quorum sensing gene was found to have lost the capability of colonizing and growth enhancement in Arabidopsis thaliana which confirms the importance of these compounds in the process (Esmaeel, Miotto, & Rondeau, 2018). N-Acyl-homoserine lactones (AHLs) are commonly found in Gram-negative bacteria and cyclic peptides are found in Gram-positive bacteria which act as quorum sensing molecules. The action of bacterial AHLs to trigger specific responses was the first to report in Phaseolus vulgaris and Medicago truncatula (Joseph & Phillips, 2003; Mathesius, Mulders, & Gao, 2003). Early responses in plants to these signals are currently believed to have significance either in recognizing pathogens to prepare themselves for the attack or to welcome mutualistic microbes for colonizing. There is a significant difference in the accumulation of over 150 different proteins as a response to AHLs Sinorhizobium meliloti (symbiotic bacteria) and Pseudomonas aeruginosa (pathogenic bacteria). AHL (with C4 and C6 side chains) produced by Serratia liquefaciens MG1 was found to have induction of systemic resistance proteins after inoculation on the roots of tomato plants sensing the presence of pathogens (Schikora, Schenk, & Hartmann, 2016). These reports confirm that plants are likely to be involved in quorum sensing for recognizing the difference between pathogens and beneficial microbes (Fig. 1.1). It is also demonstrated that some plant extracts have the capability of quorum quenching which helps them to protect against pathogens. There are reports which suggest that the capacity of endophytes to synthesize has LuxR homologs from the evidence that LuxR-LuxI type quorum sensing gene pairs isolated from several microbes of endophytic origin confirming their role in plant microbe crosstalk (Kandel, Joubert, & Doty, 2017).

Figure 1.1 Different roles of Quorum sensing compounds played during plant microbe interactions.

1.3 Attachment and colonization of endophytes

Attachment of microbe to the plant surface is the first and foremost step in the process of microbial colonization. The chemo-attracted microbial population in the rhizosphere migrates to the plant root surface and initiates colonizing potential entry sites like site of emergence of lateral root or sites of other injuries. In the case of bacteria, their cells synthesize exopolysaccharides (EPS) which helps in the attachment onto the root surface. In Gluconacetobacter diazotrophicus Pal5, EPS was found to be an important factor for its attachment and colonization to the root surface. After attachment, EPS prevents the cells from oxidative burst caused as an immune response to the invasion. EPS purified from the same strain (Pal5) was even capable of inducing the colonization and biofilm production in mutant strains (Meneses, Gonçalves, & Alquéres, 2017). Reports of Balsanelli, Tuleski, anf de Baura, (2013) demonstrated that, in maize, N-acetyl glucosamine residue of Lipopolysaccharides (LPS) binds to the root lectins which is crucial for the attachment and colonization of bacteria inside the roots. Endophytic fungi colonize the host either one of two methods: (1) vertical transmission in which the fungi in the maternal plants are transmitted to the offspring through the progeny seeds. The spores of the fungi present in the seeds will start to germinate and colonize once the seed starts to germinate (Gagic, Faville, & Zhang, 2018; Hodgson, de Cates, & Hodgson, 2014). (2) Horizontally transmitted ones mostly transmit via spores/hyphal fragmentation with the help of either biotic means by herbivores like insects or by abiotic means like wind and rain (Wiewióra, Żurek, & Pañka, 2015). The beginning of colonization after the attachment to the plant cell is the formation of the appressorium-like structures that help in the migration and colonization to the internal plant tissues (Esparza-Reynoso, Pelagio-Flores, & López-Bucio, 2020). During this process, unlike the pathogenic invasion, the cell integrity is not disturbed, which was proved by the microscopic observations of Trichoderma colonization in tomato roots (Yan & Khan, 2021).

1.4 Mechanisms involved in endophytic colonization

Endophytic microbes were highly successful in coevolving with their host plants which helped them to get equipped with all the necessary traits needed to internalize, colonize, and translocate into the intercellular spaces of the plant. Even though plant and endophytic interaction stays on the beneficial side, plants activate their immune system when sensing microbial presence (Zipfel & Oldroyd, 2017). They use innate immune responses for recognizing the signal molecules to trigger the defense mechanisms. The defense mechanism involves either microbe-associated molecular patterns (MAMPs) by recognizing the cell surface–localized pattern recognition receptors which activate the MAMP-triggered immunity; or by recognizing the molecules synthesized by microbes (effectors molecules) with the help of intracellular receptors which will activate the effector-triggered immunity (López-Gómez, Lara-Herrera, & Bravo-Lozano, 2012; Mendoza-Mendoza, Zaid, & Lawry, 2018; Zamioudis & Pieterse, 2012). Components of bacterial cell surfaces are distinct and unique from that of pathogens. MAMP was found to be triggered by the presence of bacterial flagellum (Butchart, Scharlemann, & Evans, 2012). However, studies conducted on flagellin-sensing system (flg22-Flagellin Sensing 2) derived from endophytic B. phytofirmans and pathogenic P. aeruginosa or Xanthomonas campestris in grapevine found to recognize the flagellin in a differential manner suggesting the recognition of endophytes (Trdá, Fernandez, & Boutrot, 2014). Studies of Chen, Marszałkowska, and Reinhold-Hurek (2020) explain the possibility of downregulated Mitogen-Activated Protein Kinase (MAPK) signaling pathway due to the slight upregulation of four different protein phosphatase 2C (PP2C) homologs during Azoarcus colonization (PGPB); however, in the case of Xoo infection, another PP2C homolog was downregulated leading to upregulated MAPK signaling leading to stronger PTI response when studied in detail in rice plants demonstrating its role in both beneficial and pathogenic interactions.

Another most important groups with host immune modulation are bacterial protein secretion systems (SSs) consist of large protein complexes that include Type I SS~Type VI SS, Sec and Tat in Gram-negative and Sec, Tat, secA2, Sortase, Injectosome, and Type VII SS in Gram-positive bacteria (Green & Mecsas, 2016; Tseng, Tyler, & Setubal, 2009). Among these, T3SS and T4SS are either present in low concentration or absent in endophytic bacteria, thus, have mild or no defense response, whereas present in pathogens leading to a stronger response (Green & Mecsas, 2016). There are lot of genomic and metagenomic studies concentrated on the abundance of T3SS and T4SS genes from endophytes, including Herbaspirillum frisingense GSF30(T), G. diazotrophicus PAI5, Azoarcus sp. BH72, Klebsiella pneumoniae 342, Azospirillum sp. B510, from diverse plants which lack the presence of T3SS genes; Herbaspirillum sp. lacks T4SS; and Azoarcus sp. strain BH72 lacks both T3SS and T4SS (Juhas, Van Der Meer, & Gaillard, 2009; Piromyou, Buranabanyat, & Tantasawat, 2011; Reinhold-Hurek & Hurek, 2011; Straub, Zabel, & Gilfillan, 2013). However, there are exceptions also found by having both the sets of genes present in Bradyrhizobium sp. SUTN9-2 isolated from Aeschynomene americana L., which was found crucial in colonization process (Piromyou, Buranabanyat, & Tantasawat, 2011).

Production of reactive oxygen species (ROS) is a nonspecific defense mechanism used by plants which will induce hypersensitive response thereby leading to programmed cell death against biotrophic pathogens (Apel & Hirt, 2004). Most of the endophytes protect themselves with the help of EPS, and the residual ROS is neutralized with the help of ROS-scavenging enzymes (Alquéres, Meneses, & Rouws, 2013; You & Chan, 2015). Quantification and diversity studies on the genes responsible to produce superoxide dismutase and glutathione reductase in metagenome of the endophytic bacterial communities in rice roots, Enterobacter sp. 638, G. diazotrophicus, etc. are significantly higher than free living and are found to be essential for colonization process (Liu, Carvalhais, & Crawford, 2017; Sessitsch, Hardoim, & Döring, 2012; Taghavi, van der Lelie, & Hoffman, 2010). It is also found that the transcript levels of these genes are upregulated when present inside the plants confirming their role in successful colonization (Liu, Carvalhais, & Crawford, 2017).

Another important mechanism influences the colonization of endophytes are the phytohormones because of their role in the plant defense signaling pathways. They have the capacity to regulate the structure of plant-associated microbiome involved in beneficial interactions, plant nutrition, and defense-related interactions (Liu, Carvalhais, & Crawford, 2017). Iniguez, Dong, & Carter, 2005) demonstrated the role of ethylene signaling pathway (ET) activation resulted in the suppression of colonization of both K. pneumoniae 342 (Kp342) (PGPB) and Salmonella enterica serovar Typhimurium (human enteric pathogen) in wild-type M. truncatula and when inoculated in ET insensitive M. truncatula leads to extensive colonization by Kp342. Similar studies are also conducted in the case of Jasmonic acid (JA), which suggests that its level is downregulated during colonization process to maintain plant favorable microbial densities inside the plant tissues. During early nodulation stages in Lotus japonicus suppression of JA-signaling pathway is observed by Nakagawa and Kawaguchi (2006). Similar results were also observed during the colonization of Azospirillum brasilense 245 on Arabidopsis roots where the JA-signaling was strongly downregulated (Spaepen, Versées, & Gocke, 2007). It is not the case in the case of rice varieties like Japonica and Indica rice cultivar where RT-PCR and proteomic analyses confirm the strong upregulation of markers like OsJAR1 and OsJAmyb during the endophytic colonization of Azoarcus olearius, Azospirillum B510, and G. diazotrophicus (Drogue, Sanguin, & Borland, 2013). Even though we have strong indication that the different defense mechanisms in the host plants are differentially modulated, the total mechanism involved is not yet clear (Plett & Martin, 2018). Endophytic microbes isolated from different plants are summarized in Table 1.1.

Table 1.1