Keywords

1 Introduction

The phyllosphere provides large surface area as a sustainable habitat for numerous microorganisms but remains a subject less explored as compared to the rhizosphere. However, in recent years, the role phylloplane colonizers play in plant growth and development, productivity, and raising the standards of plant defense mechanism has gained importance in research fraternity and attracted global attention towards their multifaceted potential in developing eco-friendly agronomic models for plant growth and protection.

A wide variety of microbes, predominantly bacteria, fungi, and yeast are known to colonize various plant surfaces. A number of factors drive the localization and colonization of microbial aggregates on the phylloplane. Trichomes, ridges, and stomatal openings on the leaf surface provide spaces for the microorganisms to aggregate and thrive (Lindow and Leveau 2002). The microbial life on the aerial parts of plants deals with complex system of microbe–microbe interactions, and interactions between the colonizers and host plant (Fig. 11.1). Many of them have been reported to produce phytohormones, promote plant growth and productivity, attenuate plant diseases, induce tolerance against biotic/abiotic stresses, and elicit defense response upon pathogenic attacks (Windham et al. 1986; Vorholt 2012). Phylloplane microorganisms could be very well considered as crucial strategic tools to devise bio-control strategies against a variety of phytopathogens because of the antimicrobial activity of non-pathogenic bacteria and fungi. They are known to facilitate water uptake by reducing leaf surface tension, act as stimulants of plant growth, and provide resistance against abiotic stress like soil salinity, drought, or floods (Lindow and Brandl 2003; Whipps et al. 2008). The enhancement of plant defense system by the action of non-pathogenic bacteria or fungi may act as model for development of sustainable and eco-friendly antimicrobial agents. A number of studies have well elucidated the inhibitory actions of microbes isolated from plant phylloplane. Studies suggest how Trichoderma species have been widely considered as leaf-associated microfungi and studied for their efficient antimicrobial properties (Kalita et al. 1996). Trichoderma viride was reported to alter the levels and expressions of phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase in rose plants. The enhanced activity of the defense enzymes provided protection against Diplocarpon rosae and reduced disease occurrence (Karthikeyan et al. 2007). Trichoderma harzianum OTPB3 could develop systemic resistance in Solanum lycopersicum against early and late blight disease, inhibiting the in vitro growth of Alternaria solani and Phytophthora infestans (Chowdappa et al. 2013). Microbial metabolites demonstrate antimicrobial properties and their application could significantly reduce the in vitro and in vivo growth of plant pathogens (Saleem and Paul 2015a, b, c). Phylloplane microbes are known to induce a defense response in plants upon pathogen invasion by activating the defense enzymes and phenolic compounds (Pradeep and Jambhale 2002).

The phylloplane colonizers may shift with respect to changes in the environment under which a plant is grown which may include sudden rise or drop in temperature conditions, change in humidity, loss of leaf surface wettability, or ageing and withering of leaves (Nix et al. 2008). Global warming is a growing concern of current times and has been implicated in detrimental aftereffects on the ecosystem. The impact of global warming/climate change may also lead to adverse effects on the plant–microflora relationship (Youssef et al. 2015). Increase in temperature, severity of solar radiations, atmospheric CO2, and fall in precipitation cause deficiency of nutrients and required moisture on the plant surface which eventually leads to a gradual loss of phyllosphere microbial populations which could be beneficial in the improvisation of plant defense system and protection against severe plant diseases (Tyagi et al. 2014). Insights into underlying problems concerned with microbial loss due to climatic change should help us work on the cause and build a proper management system for better plant growth, quality, and productivity. Principal requirements such as appropriate temperature, soil conditions, relative humidity, photoperiod, etc. should be taken into consideration to avoid loss of beneficial microbial communities from soil and plants. A deeper knowledge into the benefits conferred by phyllosphere microbiota may help us understand the different possibilities and dimensions in which application of leaf-associated microorganisms can be employed. There is a lacuna of information about the survival patterns of human pathogens and non-pathogenic microflora cohabiting plant surfaces under different climatic conditions. Intense studies are required to understand if different phylloplane colonizers respond to the climatic changes differently, and how the shifting pattern of microbial populations varies from one another.

2 Phylloplane

The surface of leaf acts as a suitable habitat for a wide array of microorganisms (Hirano and Upper 2000). The term Phyllosphere was proposed by Last (1955) and Ruinen (1956) to specify the leaf surface and its associated microenvironment. It provides the microflora with an ample space to survive and leaches out nutrients which contain sugars and amino acids as major sources of carbon and nitrogen, respectively, needed for their growth and development (Mercier and Lindow 2000). Various plants are the sources of sustenance for fluctuating microbial colonists owing to different physiological conditions (Lindow and Brandl 2003). The abaxial and adaxial leaf surfaces of different plants are known to carry a wide variety of epiphytic communities (Yadav et al. 2011).

Fig. 11.1
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Schematic representation of the inter-microbial and plant–microbial interactions

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3 Phylloplane Microbes or Epiphytes

Each part of the plant acts as a host to specific microbial communities. The most abundantly isolated microorganisms from the aerial plant parts belong to the leaf surface. They are usually sourced from air, soil, irrigation water, rain, insects, and are usually specific about the niches they thrive on the plant’s surface (Meyer and Leveau 2012).

Phylloplane harbors a significant variety of microflora termed as epiphytes surviving on the surface, whereas the microbes thriving in the internal tissues are called endophytes. Most prominently found microbes are bacteria along with fungi and yeast (Prabakaran et al. 2011). Microflora of the Phyllosphere is specialized at sustaining in the most inevitable conditions like exposure to solar radiations, temperature variations, biotic/abiotic stress, pollution, and lack of humidity (Kishore et al. 2005). The UV radiations act as a crucial basis for selection and survival of phylloplane microbiota and for selecting UV-resistant microbial species (Stockwell et al. 1999). The epiphytes have been found to modify the microenvironment of their host. Aging leads to the wearing of leaf cuticle, alteration in leaf surface wettability is also one of the various physiochemical changes the microbes have to face. Pseudomonas fluorescens have been reported to cause increase in the wettability of host leaves for their easy mobility and survival on phylloplane (Knoll and Schreiber 2000). The phyllosphere microbes are also specialized at producing phytohormones thereby promoting plant growth (Vastakaite and Buzaite 2011; Limtong et al. 2014), nitrogen and carbon dioxide fixation (Smith and Goodman 1999), cellulolytic activity (El-Said 2001), detoxification of pollutants, onset of systemic resistance in plants, production of defense enzymes/metabolites, and helping through the plants defense mechanisms against phytopathogens (Harman et al. 2004).

4 Inter-Microbial Interactions

The phylloplane microbiome is determined by different genetic and environmental factors, defining the survival of these microbes and shaping them into complex communities, forming microbial ecosystems where they interact and influence each other’s growth patterns (Gall 1970). The viability of epiphytic microorganisms decides the establishment of communication and interaction with one another in complex and specialized molecular and physiological mechanisms (Smid and Lacroix 2013). Control of pathogenic growth on the phylloplane is achieved by interactions among epiphytes (saprophytic in nature) and pathogenic microflora, subsequently reducing foliar diseases (Blakeman and Fokkema 1982). It is crucial to ponder upon the importance of these interactions to understand their implications on disease management and crop productivity. Microbial interactions are usually in the form of symbiosis, intra-cellular communications, or interactions upon direct surface contacts which may lead to inhibition of one microbe caused by the other (Gutjahr and Parniske 2013). Metabolic interactions, bio-control properties, secretion of toxins or metabolic compounds lead to the suppression or promotion of growth and help in the sustenance of microbial communities (Wolin et al. 1997). Various microbe–microbe interactions cause inhibition of the epiphytic colonizers due to competition for survival (Mercier and Lindow 2000). Most of the volatile fungal metabolites secreted by various leaf-inhabiting microfungi are known for their mycostatic activity against the growth of several foliar pathogens (Upadhyay 1981). Suppression of phytopathogens by phylloplane residents has managed to gain importance and may, thereby, help in affecting disease occurrence in plants (Patil and Kachapur 2000). Various bacterial–bacterial, bacterial–fungal and fungal–fungal antagonistic interactions have been studied for crop protection and plant health (May et al. 1997; Abdel-Sater 2001). A number of previous investigations have shown various bacterial species inhibiting the growth of certain microfungi and, on the other hand, promoting the growth of others (Schrey et al. 2012).

Pseudomonas species are gram negative, aerobic, polar flagella bearing rods (Srivastava and Shalini 2008). They have been widely acknowledged as significant antifungal agents with substantial investigations undertaken in the area of rhizosphere (Alemu and Alemu 2013). Chitinases are enzymes found in bacteria and other microorganisms that are useful in the biodegradation of polysaccharide “chitin” abundantly found as the main component of the exoskeleton of yeasts and fungi, and thus act as one of the major factors contributing to the bacterial antagonism against microfungi (Hamid et al. 2013). Bacillus cereus was found producing two antifungal chitinases against Botrytis elliptica causing leaf blight in Lily (Huang et al. 2005). Enterobacter agglomerans, a chitinolytic microbe, antagonizes the growth of Rhizoctonia solani that causes leaf spot and root rot diseases in tobacco plants (Gonzalez et al. 2011). Lactobacillus species are also known to antagonize the growth of Penicillium, Fusarium, and Aspergillus species (Magnusson et al. 2003). Erwinia herbicola and P. syringae isolated from soybean leaves had antimicrobial activity against Escherichia coli, P. syringae pv. glycinea, and Geotrichum candidum, respectively, thereby suppressing bacterial blight symptoms (Völksch et al. 1996). P. fluorescens, P. aeruginosa, and P. asplenii acted as potent inhibitor of R. solani causing sheath blight of Rice (Akter et al. 2014).

5 Antimicrobial Activity of Epiphytes

Microorganisms often tend to alter and restructure the colonization patterns on plant parts by antagonizing the growth and affecting the survival of other microbial communities (Beenish 2017). The bacterial–bacterial, bacterial–fungal, and fungal–fungal antagonistic interactions have been extensively investigated for the purpose of better agriculture, crop protection, and improving plant health (Mohamed and Sater 2001). These interactions lead to the inhibition of phytopathogens by phylloplane colonizers which are considered vital for their help in controlling plant diseases (Patil and Kachapur 2000). Earlier studies have reported bacterial species as potent inhibitors of certain microfungi and have also been observed promoting the growth of others (Schrey et al. 2012). A number of bacterial genera like Pseudomonas and human pathogens Serratia and Klebsiella have been found demonstrating antifungal activities against a variety of fungi (Kerr 1999). Pseudomonas species are also known to inhibit the activity of phytopathogens like Botrytis cinerea and Alternaria species (Swadling and Jeffries 1998). Thus, proving to be significant antagonist of prominently found pathogens, they could be used as a potent plant growth promoter and suppressor of plant diseases. Listeria denitrificans (E2), Pseudomonas fluorescens (C37 and C92), and Xanthomonas campestris (D119) retrieved from the phyllosphere of Lolium perenne (S24) showed inhibitory activity against Drechslera dictyoides (Drechsler) Shoemaker (Austin et al. 1977). Bacillus strains isolated from phylloplane were reported of possessing antimicrobial properties and were capable of reducing foliar diseases significantly (Halfeld-Vieira et al. 2008). The phylloplane bacterium Ochrobactrum anthropi BMO-111 was found effective in significantly reducing the occurrence of blister blight disease of tea, it antagonized the growth of causal pathogen Exobasidium vexans in vitro as well as on the plant (Sowndhararajan et al. 2013). Aureobasidium pullulans, Cladosporium cladosporioides, Epicoccum purpurascens, Fusarium oxysporum, and Myrothecium roridum caused the inhibition of Colletotrichum gloeosporioides whereas Aspergillus terreus, Cephalosporium roseo-griseum, and Penicillium oxalicum were capable of suppressing the growth of Puccinia psidii, a potent phytopathogen with extensive host range (Pandey et al. 1993). Trichoderma harzianum and Trichoderma pseudokoningii were observed suppressing the growth of Colletotrichum destructivum of cowpea (Akinbode and Ikotun 2011). Kawamata et al. (2004) isolated fungal microflora from phylloplane of rice plants and randomly selected them for investigation of antimicrobial activity against rice leaf blast. It was reported that the phylloplane isolates belonged to the species of Fusarium, Trichoderma, Cladosporium, Penicillium, Pestalotiopsis, and Epicoccum which caused disease suppression upon inoculation of plant with conidial/hyphal suspension of both phylloplane fungus and pathogen simultaneously. Fusarium sp. isolates significantly inhibited Magnaporthe grisea and formed inhibition zones 3–5 mm wide in vitro against the pathogen. A wide array of microfungal species are considered to be the major reason for foliar diseases. In order to cope up with fungal phytopathogens without affecting the plant’s vigor, it is crucial to make use of bio-control agents that may act against the pathogenic microorganisms, act as plant growth promoters, and do not deteriorate plant health. Various studies have provided ample information about the use of bacteria as an effective bio-control agent against different disease causing fungal phytopathogens, not leading to any deleterious effect on plant health (Thomashow and Weller 1996).

A variety of bacterial–fungal interactions have been studied previously. It is crucial to understand the biological mechanisms underlying this phenomenon which range from physical associations to molecular communications and antibiosis followed by toxic compounds and exudates released by the bacteria which may lead to the fungal growth inhibition (Warmink and Elsas 2009). Antibiosis is caused due to the varied metabolic products secreted by the microorganisms with antagonistic properties. Bacterial microflora has long been recognized for the synthesis of certain volatile compounds like HCN, benzaldehyde, acetaldehyde, and iron-binding compounds known as siderophores as well as lactic acid bacteria that produces cyclic dipeptides and phenyllactic acid which are implicated in imparting antimicrobial properties against different fungal microflora (Schnürer and Magnusson 2005; Weisskopf 2013). Saleem and Paul (2015a, b, c) concluded that Pseudomonas koreensis and Sphingobacterium daejeonense isolated from the phylloplane of Solanum lycopersicum significantly reduced the growth of cohabiting fungal pathogens. Various enzymes, bacteriocins, and lipopeptides produced by the Bacillus strains render it possible for the bacteria to antagonize the growth of various bacterial, fungal pathogens (Baruzzi et al. 2011). A key role is played by the antimicrobial peptides produced by microorganisms due to non-specific innate immunity response in inhibiting the growth of pathogens (Cruz et al. 2014). It can be concluded from previous studies that the bacterial–fungal contact inhibition also plays a vital role in limiting microbial growth and may lead to physiological changes in the microbes. In many cases, release of deleterious compounds by them may stimulate the diffusion of specific defensive compounds by the microfungal species as a line of protection against the inhibitory action of the antifungal compounds (Klett 2011). Human pathogens have been found colonizing the phylloplane of various plants, due to the high toxicity, Klebsiella pneumoniae and Serratia fonticola inhabiting the phylloplane of Solanum lycopersicum, were found antagonizing the growth of a variety of fungal pathogens like Cladosporium cladosporioides, Cladosporium herbarum, Fusarium oxysporum, etc. on the leaf surface (Beenish 2017).

6 Climate Change and Microbial Colonization

Global warming is a much researched topic, yet there are less explored aspects concerning its harmful effects on the agro-ecosystem, ecology of the phyllosphere, and correlation between phyllosphere colonization and plant's disease resistance (Aydogan et al. 2018). The envelope of gases surrounding our earth is getting adversely thick due to which it tends to contain more and more of heat within the atmosphere eventually causing a rise in temperature. Previous studies suggest loss of species among different microbial communities due to global warming (Kumar 2018). Composition of microflora colonizing the phylloplane may show variations among different plant species and is greatly affected by a number of factors including geographical location, season, and various environmental conditions such as temperature, rain, sun exposure, dryness, etc. (Whipps et al. 2008; Ding and Melcher 2016). Fig. 11.2 shows how abiotic changes, including fluctuations in weather conditions, are responsible for modification of the physicochemical conditions and microbial diversity on the phylloplane which may further add complexity to plant defense and agricultural systems. Studies suggest that the plants which are not tolerant to varying temperature conditions especially in soil polluted with heavy metals show difficulty in adapting to the changes and may demonstrate complex response towards the stress factors (Tyagi et al. 2014). Changes in the weather conditions including elevation in temperature, increased transpiration rates, and stomatal conductance whether long term or short term can affect crop quality and interrelationship dynamics between microbes colonizing the crops. It has been reported in a number of studies that any change in climatic conditions under which the plants are grown may disrupt the equilibrium required for feasible microbial sustenance which further leads to a shift of microbial population inhabiting the surface thereby causing alleviation in biodiversity and a rise in plant stress (Lindow and Brandl 2003). Earlier studies have led to the observation that any decrease or increase in the temperature and precipitation usually leads to the depletion of nutrients and altered moisture levels on the leaf surface which may modify the pathobiome by threatening microbial aggregation, abundance, and sustenance (Elad 2009). The irregular wettability on leaf surface usually results in intermittent growth of bacterial and fungal colonists, posing survival threats during dry season (Blakeman and Fokkema 1982). These changes consequently lead to the shifting of varieties of microorganisms from the plants. Some of the beneficial microbes produce antimicrobial compounds and induce systemic acquired resistance against pathogenic microflora by complex plant–microbe interactions upon stress recognition (Brader et al. 2017). Altered phyllosphere microbial populations may in turn strongly affect plant growth and its ability to combat diseases, making the plant more prone to pathogenic invasion (Elad 2009). Loss of plant beneficial microflora may significantly influence the plant’s metabolism, growth promotion, and disease suppression.

Fig. 11.2
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Schematic representation of impact of climate conditions on plants and microflora

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7 Plant–Microbe Interactions

The microbial colonization occurring on the host plants is followed by host–microbe interactions leading to either pathogenesis, resistance to diseases, or growth promotion that develops a cascade of secondary processes by the phylloplane colonizers (Knoll and Schreiber 2000). Microbes surviving on the plants benefit from metabolites and nutrients leached from the surface, utilizing sugars (glucose, sucrose, fructose) as carbon sources (Lindow and Brandl 2003). Frequent production of phytohormones by microbial flora has been vastly investigated and only since few years the role of phyllosphere microbes and their possible effects on the alteration of growth patterns of host has come into perspective (Vastakaite and Buzaite 2011; Limtong et al. 2014). Series of changes in the plant microhabitat and physiology could be an outcome of the colonizer's interactions with the host (Lindow and Brandl 2003). Synthesis of phytohormones has long been considered as a virulence factor. Pathogens on aerial parts of the plant may produce them at the site of infection or stress and quantities of phytohormones often play a crucial role in influencing the pathogenesis (Prasannath 2013). In studies undertaken previously, a rise in the levels of IAA has been noticed at the site and course of infection (Fu and Wang 2011).

Microfungal species associated with the phylloplane have been utilized as bio-control agents to prevent pathogens from infecting the host (Evueh and Ogbebor 2008). Many such fungi have shown to enhance plant growth promotion, crop yield and quality, resistance to stress factors, and the assimilation of nutrients (Windham et al. 1986). These fungi have also been reported to prevent a number of plant diseases such as foliar, root, or fruit diseases, along with invertebrates like nematodes (Shoresh et al. 2010). Microbes synthesize a range of biochemicals that cause localized or systemic resistance thereby accelerating the defense mechanism in host plants. Secretion of antimicrobial compounds on plant surfaces acts as a major line of defense by which plants deter potential pathogens (Lindow and Brandl 2003). These plant–microbe associations can cause significant modifications in the proteome and metabolism of the host (Harman et al. 2004). Foliar bacterial community is significantly affected and altered in terms of population, diversity, and richness upon powdery mildew infection. According to the study by Suda et al. (2009) shifting patterns of bacterial populations showed greater specificity towards plant species. Similarly, considerable changes in leaf-associated fungal and bacterial communities were observed upon powdery mildew infection in oak (Quercus robur) due to the causal pathogen Erysiphe alphitoides. The investigations further led to the revelation of pathobiotic network of different fungal and bacterial operational taxonomic units connected with each other and directly interacting with Erysiphe alphitoides. The study suggested that some of these might have been conferring protection to the oak phyllosphere by inhibiting E. alphitoides and thereby reducing the probabilities of powdery mildew occurrence in natural physiological and weather conditions (Jakuschkin et al. 2016). Severity of powdery mildew infection was negatively correlated with the richness and diversity of the fungal microflora present on phyllosphere of pumpkin (Cucurbita moschata). Phylloplane that was scarcely colonized by powdery mildew showed greater diversity and richness, whereas a significant decrease was seen in densely colonized leaves (Zhang et al. 2018). Investigations by Wang et al. (2019) suggested that Protomyces strain SC29 on the phylloplane of Arabidopsis activates defense responses by initiating yeast-specific MAMPs that can trigger plant immunity and can provide insights into the evolution of fungal virulence, and plant immunity against yeasts. SC29 treated Arabidopsis showed enhancement in immunity against Botrytis cinerea infection, associated with activation of MAPK3/6, camalexin, and SA-signalling pathways. Protomyces have been evident inhabitants of the phyllosphere as yeasts, but invade their hosts in the hyphal form. The inability of SC29 to cause disease on Arabidopsis, but the ability to persist on the leaf surface suggests a change to survival strategies where phylloplane as a microhabitat plays a key role.

8 Elicitation of Plant Defense Response by Fungal Metabolites and Ergosterol

Phylloplane fungi interact with plants in many ways and affect their growth, development, and protection. Fungi growing on the phylloplane can be classified into two groups: residents and casuals. Resident fungi multiply on the surface of healthy leaves without affecting the host. Casual fungi mostly land on the leaf surface due to environmental factors but are unable to grow and survive (Prabakaran et al. 2011). Endophytic fungi are crucial in implicating the functionality of the host plant’s endosymbiotic systems which could be nutritional, defense-related, or biotic/abiotic stress toleration (White et al. 2014). Various phylloplane microfungi have been well-known antagonists and are utilized as bio-control agents to prevent phytopathogens infecting the host. As suggested by earlier studies, secretion of antimicrobial biochemicals on aerial plant surfaces is thought to be one of the defensive strategies by which plants combat potential pathogens (Peláez et al. 1998). Phenols, peroxidase, and polyphenol oxidases are defense-related chemicals released by the plants against pathogens. Many of the volatile fungal metabolites secreted by various phylloplane microfungi are known for their antifungal activity against several foliar pathogens (Upadhyay 1981). Thakur and Harsh (2014) worked with ten phylloplane isolates of Chlorophytum tuberosum against its potent pathogen Colletotrichum dematium and found that volatile secretions of these leaf-inhabiting fungi could effectively reduce the mycelial growth of C. dematium. Trichoderma viride and Aspergillus flavus caused maximum growth inhibition of Alternaria brassicae causing leaf spot disease of mustard. The metabolites released by these phylloplane fungi arrested the hyphal growth of the pathogen (Yadav et al. 2011). Volatile and non-volatile compounds released by Trichoderma species may significantly inhibit the pathogen’s growth by antibiosis, competition, mycoparasitism, or direct contact inhibition (Kuberan et al. 2012; Parizi et al. 2012). Similarly, some Alternaria metabolites were also reported to exhibit antimicrobial properties (Lou et al. 2013). These metabolite acted as bio-control agent in controlling fungal disease in grapevine cultivation.

Ergosterol is of fungal origin, a sterol composition in cell membrane, and a common secretion of fungal metabolites (Weete et al. 2010). It acts as an elicitor of defense responses in plants (Felix et al. 1999). As per the study of Kauss and Jeblick (1996) ergosterol in plant cells effectively stimulates hydrogen peroxide (H2O2) production in the host. Application of ergosterol has been reported to activate the expression of defense genes in plants against pathogens thereby activating the defense signalling pathways (Lochman and Mikes 2006). It is reported to induce cross-talk between the salicylate and jasmonate signalling pathways, thus increasing the resistance against pathogenic attack. Ergosterol reportedly leads to alteration in the proton fluxes and membrane hyperpolarization mechanism in the motor cells of host plants (Amborabé et al. 2003). Cladosporium species have been reported to be amongst the dominant microfungi inhabiting phylloplane of Solanum lycopersicum (Saleem and Paul 2016). Granado et al. (1995) treated tomato plants with ergosterol extracted from Cladosporium fulvum and found that the plants could perceive ergosterol which elicited cellular alkalinization in them.

9 Intercellular Fluid Proteins

These are the proteins found in the apoplast or the intercellular space surrounding plant cells. This is known to be a dynamic environment where variety of metabolic, defensive, and translocation processes occur. Prior investigations have led to the understanding that apoplast is considered to be a primary location where initial resistance responses against pathogenic microbiota take place, e.g. generation of reactive oxygen species by extracellular peroxidases and oxidases, and strengthening of plant cell wall by crosslinking or callose deposition. As a result of the aforementioned processes, an array of proteins and amino acids are found in the leaf apoplastic fluid (O'Leary et al. 2014). Plant–pathogen interactions usually modify the intercellular fluid protein quantities. Rise in the quantities can be observed upon pathogen invasion. A number of new proteins such as P14 were observed in intercellular spaces or apoplast of Solanum lycopersicum phylloplane infected with Cladosporium fulvum, a potent fungal pathogen (De Wit et al. 1986).

10 Phenylalanine Ammonia Lyase (PAL) in Plant Defense

A number of researches have brought to our understanding that induction of resistance via biosynthesis and endogenous distribution of different phenolic compounds, expression of PAL, TAL, POX, PPO, and key enzymes offer a major contribution in the biological control of pathogens and insects, thus, promoting the standards of defense mechanism in plants (Mohamed et al. 2007; Mahmoud et al. 2012).

PAL acts as a key enzyme in the phenyl propanoid pathway. It catalyzes the synthesis of phenols and salicylic acid involved in defense against the pathogens, biotic stress response, and secondary plant metabolism and is associated with posttranslational phosphorylation (Nicholson and Hammerschmidt 1992). Induction of PAL activity in host tissues is followed by pathogen infection and several biotic and abiotic elicitors (Ebel 1986). Concentration of PAL activity may largely vary based on the developmental stage of plant, genotype, environmental factors, pathogen type, and plant–microbe interactions (Dixon and Paiva 1995). PAL activity indicates the degree of host resistance as its rapid accumulation reaches high levels during incompatible interactions when a defense response is induced within the host plant as compared to susceptible responses (compatible interactions) (Bhattacharyya and Warde 1988). Gupta and Kaushik (2002) in their study suggested the enhanced specific PAL activity in infected leaves as compared to healthy leaves of mustard.

11 Tyrosine Ammonia Lyase (TAL) in Plant Defense

TAL is a crucial enzyme in the phenyl propanoid pathway and member of the aromatic amino acid lyase family. TAL (transcription activator-like) effectors comprise a novel class of DNA-binding proteins. They are employed by gram negative Xanthomonas sp. which is pathogenic to plants. It generally transports various types of effector proteins through a type III secretion system into plant cells where they determine the virulence severity. TALs primarily localize to the nucleus where they associate with the target promoters thereby inducing gene expression in plants (Scholze and Boch 2011). It has high specificity for L-tyrosine, generates 4-coumaric acid as a protein cofactor and antibiotic precursor in microbes (Watts et al. 2006). Earlier studies report changes in the TAL activity in plants due to a number of factors including biotic/abiotic stress and pathogenic attack (Dixon and Paiva 1995). The increase in TAL activity in compost treated tomato plants due to induced systemic resistance was found to be related to biosynthesis of lignin from tyrosine (Fayzalla et al. 2009; Abdel-Fattah and Al-Amri 2012). A significant increase in TAL activity was recorded upon inoculation with plant pathogen Sclerospora graminicola which acted as a signal and led to induction of resistance in pearl millet seedlings (Hindumathy 2012).

12 Peroxidases (POX)

POXs are hem-containing glycoproteins and are found in microorganisms, plant and animal tissues. They are involved in a variety of plant physiological processes including cell wall modifications, developmental and defense-related mechanisms (Gaspar et al. 1991). Large multigene family encodes the class III plant peroxidases family (POX, EC 1.11.1.7), which further constitutes numerous peroxidase isoenzymes (Hiraga et al. 2001). Endoplasmic reticulum is responsible for targeting the plant peroxidases (class III) towards the outside of the plant cell or to the vacuole (Welinder et al. 2002). Plant peroxidases are synthesized extracellularly and are known to catalyze the generation of reactive oxygen species (ROS) coupled to oxidation of phytohormone indole-3-acetic acid and synthesis of biochemicals associated with plant defense mechanism: salicylic acid, aromatic monoamines, and chitooligosaccharides (Kawano 2003). Studies demonstrate that peroxidases are capable of generating reactive oxygen species during the massive oxidative burst in incompatible interactions which establishes an extremely toxic environment for pathogens to survive, thereby promoting plant protection (Gómez-Vásquez et al. 2004). POX enzymes are considered to play a vital role in defense against pathogenic attack and are also responsible for removal of toxic H2O2 from the host cells, thereby deterring oxidative damage and protecting the cells. Increase in POX activity is observed upon foliar infections or inoculations. Rise in POX activity was seen in three cucumber cultivars when the leaves were systemically infected with Cucumber mosaic virus (Wood and Barbara 1971). The rise in POX activity due to arbuscular mycorrhizal fungi is reported to be involved in lignification (Lagrimini and Rothstein 1987).

POX isoforms play a crucial part in plant defense mechanism. Previous researches have led to the observation that peroxidase expression levels and its isozyme patterns were significantly affected by biotic/abiotic stress, application of chemicals, and invasion by pathogens in a number of plants (Gasper et al. 1982). Two anionic isozymes of POX were induced in the diseased leaves of Tobacco plants infected with Tobacco mosaic virus, and were also later systemically induced in the leaves that remained distant and uninoculated (Lagrimini and Rothstein 1987). Another study demonstrated the presence of four types of POX isoforms in Pseudomonas syringae pv. tomato infected tomato plants while the control plants showed synthesis of only one type (Bashan et al. 1987). An early localized and systemic increase in peroxidase activity was observed upon Rhizoctonia infection in Norway spruce, and the prominent isoforms were found to be basic peroxidases (Nagy et al. 2004).

13 Polyphenol Oxidases (PPO)

PPOs have been playing a crucial role in the phenylpropanoid pathway (Kojima and Takeuchi 1989). They are present ubiquitously, which means they can be found in plants, animals, and microorganisms predominantly in bacteria and fungi. They are copper-containing enzymes, nuclear encoded and are known to catalyze the O2-dependent oxidation of mono and o-diphenols to o-diquinones. PPOs are highly reactive intermediates and their secondary reactions are responsible for the oxidative browning which is caused during plant senescence, pathogenic invasion, wounding, etc. (Thipyapong et al. 2004). It has been established clearly in different studies that PPOs play a key role in plant defense mechanism since they are largely present at the site of wounding, pathogenic ingression, or insect infestation. They can also be induced due to various abiotic and biotic injuries or signalling molecules (Constabel et al. 1995; Thipyapong and Steffens 1997). PPO activities were significantly increased in SA pre-treated onion plants inoculated with Stemphylium vesicarium (Abo-Elyousr et al. 2009). Ibrahim (2012) suggested that the resistance induced by SA against X. vesicatoria in tomato led to increased PPO activities. Previous studies provide us information as to how increase or decrease in PPO levels could be responsible in suppressing disease incidences, pathogen invasion, and multiplication in transgenic tomato plants. Significant reduction in bacterial growth was observed in tomato plants with enhanced PPO levels when they were inoculated with bacterial pathogen Pseudomonas syringae pv. tomato, whereas plants with suppressed PPO activity had higher disease incidence and showed a greater risk of infection (Constabel and Barbehenn 2008).

PPO isoforms are induced in tomatoes after infection with phytopathogens P. syringae pv. tomato and Alternaría solani. The expression and subsequent rise in isozymes levels upon pathogen attack suggest their part in inhibition of diseases (Thipyapong and Steffens 1997). A significant positive correlation was established between the degree of plant resistance and PPO level (Kavitha and Umesha 2008). Raised levels suggest a stronger defense response in host plants. Eight different types of PPO isozymes were induced in tomato plants that were treated with pathogens (Bashan et al. 1987). Similarly, involvement of PPO isozymes was observed in conferring resistance to wheat against A. triticana (Tyagi et al. 2000).

14 Age Related Resistance (ARR) in Plants

A plethora of factors could be responsible for a plant’s ability to protect itself from any pathogen ingress or infection; they may include environmental conditions—temperature, humidity, nature of the infected tissue, or the genotypic correlation between the host species and the pathogen. Apart from the aforementioned reasons the plant’s developmental stage also acts as a crucial factor that confers it the ability to fight against pathogenic attacks; however, it is far less frequently taken into consideration. Henceforth, investigating the implications of a plant’s development and its age on microbial colonization patterns and disease resistance could be a vital breakthrough in our knowledge of plant–microbe interactions. This development of plant resistance to pathogenic infections with respect to ageing has been recognized by various terms such as “ontogenic resistance,” “developmental resistance,” “mature seedling resistance,” “adult seedling resistance,” “age-related resistance,” etc. (Whalen 2005). According to Develey-Rivière and Galiana (2007), resistance developed in plants during the course of ageing could prove effective against a variety of pathogens, pathovar, or pathogenic strains, but when race-specific resistance is concerned, the resistance response is mainly dependent upon the functional regulation of genes related to plant resistance. Many of the plant–pathogen interactions are dependent on the developmental stage of the plant as the plant’s physiological age acts as a crucial factor in the induction of resistance response against the invading pathogen. Recent researches have made possible to understand how age can cause variations in the microbial colonization and thus may impact a plant’s defensive strategies thereof; the younger leaves of Solanum lycopersicum are heavily colonized and more defensive against diseases as compared to the ones approaching senescence (Saleem and Paul 2016). This may suggest of a rise in resistance with time as the plant ages, yet an increase in susceptibility, i.e. decrease in resistance could be seen towards senescence, plants already resistant to a pathogen raising their ability to control infection and colonization at a precise growth phase. Various observations have led to the understanding that there is no role of growth stage in development of resistance and a decrease in resistance response of a plant against any pathogen may occur as it matures because of increased susceptibility towards pathogens (Visker et al. 2003). Studies were carried out to determine the impact of antioxidants synthesized by leaves when a pathogenic attack takes place. Changes in quantities were observed as the leaves aged, which may help in mediating a defense response against pathogens (Dat et al. 2000).

Previous studies have also reported the involvement of phylloplane microbes in the biosynthesis of phytohormones which could further play a crucial role in aspects concerned with plant growth promotion, development, reduced susceptibility towards pathogens during plant–microbe interactions, and were found effective in developing ARR in the host plants (Mayda et al. 2000; Mauch-Mani and Mauch 2005; Saleem and Paul 2015a, b, c). The effects of salicylic, jasmonic, and abscisic acids along with ethylene, on the disease resistance capacity of the host plant have been well investigated (Develey-Rivière and Galiana 2007). Eventual development of ARR was also observed in Arabidopsis due to de novo synthesis of salicylic acid as the plants matured (Kus et al. 2002).

15 Systemic Acquired Resistance (SAR) in Plants

Previous investigations and extensive analysis of mechanisms related to the induction resistance responses provide essential insights into the wide range of strategies a plant could make use of in order to survive against pathogens and other trigger elements. Survival mechanism to attenuate disease occurrence may include activation of specific genes, synthesis of defense-related proteins, synthesis of antimicrobial compounds, etc. (Develey-Rivière and Galiana 2007). Systemic acquired resistance is a phenomenon which can be mediated by various factors where the plants can be induced by a number of conditions and agents including pathogen ingress, natural/synthetic/biochemicals, wounding, or application of plant extracts (Kessmann et al. 1994; Schweizer et al. 1998; Funnell et al. 2004; Hassan et al. 2009;). The exogenous application of biological or chemical agents on plants can elicit a defense response from the site of pathogenic invasion as well as the parts that were not affected by the pathogen. Non-pathogenic microbes with antimicrobial properties isolated from the phylloplane or any other niche can well be reckoned as effective bio-control agents and could play a significant part in the enhancement of plant defense strategies. A systematic acquired resistance was initiated in Solanum lycopersicum plants upon treatment with the bio-control bacteria Pseudomonas fluorescens WCS417. Earlier investigations mention that defense response in plants is initiated causing rise in the production and distribution of salicylic acid, both locally and systemically, the biosynthesis of salicylic acid acts as a signal responsible for the onset of SAR (Gaffney et al. 1993; Meuwly et al. 1995). Study of this phenomenon in tobacco (Nicotiana tabacum) could determine that effects of SAR persisted for a minimum of 20 days approximately (Ross 1961). Some of the investigations state that establishment of SAR is a salicylic acid dependent process while some studies stress on that, it is mediated by jasmonate-ethylene sensitive pathway. Pieterse et al. (1996) in their study revealed that systemic acquired resistance could be a defense response elicited in the plant when it encounters a pathogen. It was also reported that the resistance response is correlated with the biosynthesis and accumulation of salicylic acid along with distribution and is characterized by the activation of genes that encode pathogenesis-related proteins. Other inducers of systemic resistance include jasmonic acid, its derivative methyl jasmonate and ethylene (Pieterse and Van Loon 1999; Park et al. 2007). In the course of plant–microbe interactions, phylloplane microflora has also been implicated in playing a key role in inducing SAR in plants. Panstruga and Kuhn (2019) reported the efficacy of bacterial strain Bacillus subtilis UMAF6639 in initiating a defense response by the activation of jasmonate and salicylic acid dependent pathways. The microorganism originally isolated as a phyllospheric endophyte is responsible for induction of defense responses in melon plants thus offering them protection against cucurbit powdery mildew. The phenomenon generally leads to a notable rise in defense enzyme PAL and POX expressions and activities (Kurth et al. 2014). Investigations carried out by Podile and Prakash (1996) showed that Bacillus subtilis AF could significantly inhibit the occurrence of crown rot in Aspergillus niger infected soil, this suggests that the bacterium could be effectively considered as a bio-control agent. Similarly, as studies suggest that the exogenous application of biological agents on localized sites may confer resistance to the entire plant, a systemic resistance was initiated for protection against disease causing pathogens including Pseudomonas syringae pv. maculicola in Arabidopsis thaliana by local inoculation of a single leaf with avirulent P. syringae pv. tomato also found inhabiting the tomato phylloplane carrying the avrRpt2 avirulence gene (Cameron et al. 1994). Inoculums of Serratia marcescens strain B2 retrieved from the phylloplane of tomato plants were employed for the treatment of rice plants. The bacterium was effective in significantly inhibiting the in vitro growth of common phytopathogenic fungi including P. oryzae, Botrytis cinerea, Rhizoctonia solani AG-4, and Fusarium oxysporum f. sp. cyclaminis under greenhouse conditions (Someya et al. 2002).

16 Priming and Pathogenesis-Related (PR) Proteins

Priming has long been recognized as a state in which the plant prepares itself for a faster, better, and stronger defense response upon pathogenic invasion or any abiotic stress. This physiological state can be the preliminary step in the establishment of an appropriate defense mechanism and could be induced even by the application of microbial metabolites or synthetic compounds on plants (Conrath et al. 2006). Kauss et al. (1992) brought about the first systematic study of priming in plant cell suspension cultures. Priming is linked with a rise in accumulation or posttranslational modification of cellular signalling proteins in inactive state thereby playing a key role in signal amplification. Study by Van Peer et al. (1991) showed that the first evidence proving priming is an important factor in microorganism-mediated induced systemic resistance (ISR) was provided from experiments with carnation (Dianthus caryophyllus), where treatment with Pseudomonas fluorescens strain WCS417r mediated a rapid rise in phytoalexin levels upon inoculation with Fusarium oxysporum f.sp. dianthi. Frequent exposure to stress could initiate or modulate these otherwise dormant signalling proteins, followed by activating the signal amplification further leading to quicker and stronger activation of the defense mechanism and systemic resistance (Conrath et al. 2006). Earlier findings suggest an important role rhizobacteria play in activating the defense responses in Arabidopsis, it, however, does not directly affect the defense genes, but induce priming to enhance the expression of jasmonic acid and ethylene-inducible genes upon infection by Pseudomonas syringae (Van Wees et al. 1999; Verhagen et al. 2004). β-amino butyric acid (BABA) was reported to prime salicylic acid-inducible PR-1 expression in Arabidopsis (Zimmerli et al. 2001; Ton et al. 2009). Challenging Arabidopsis with BABA or avirulent bacteria generally initiates priming against P. syringae pv. tomato DC3000 correlating with raised levels of SA-dependent gene transcripts of PR1, PR2, and PR5 upon infection. The phenomenon indicates alterations in the regulatory mechanisms of defense gene expression (Slaughter et al. 2012). Elevation in the expression and quantities of phenylalanine ammonia lyase (PAL) and other PR proteins was also observed followed by the application of exogenous chemicals that may mimic the effect of pathogen invasion (Kohler et al. 2002). This clearly demonstrates that the plant prepares itself by accelerating the production of defense enzymes and biochemicals upon stimulation by chemicals, metabolites, or phytopathogens.

Proteins triggered in the plants upon pathogen invasion or similar conditions are called pathogenesis-related proteins (PRs). They are induced in the plants as a line of defense against the ingression (Van Loon et al. 1994). Pathogen invasion may refer to pathogen attack by various invaders including fungi, bacteria, viruses, insects, and herbivores. PR proteins may also be induced by the application of chemicals that resemble the effect of pathogenic attack e.g. ethylene, jasmonic acid, salicylic acid, along with that wounding of the tissue can also accumulate PR proteins at the site of injury or infection (Sels et al. 2008). Phytohormones have also been considered as major factors determining the onset of PR proteins. The PR proteins are considered to be exclusive products of SAR genes and their accumulation in the host plant is a sign of pathogen mediated systemic acquired resistance. Phytopathogens upon infecting the host tissue activate the defense-related genes which could be expressed in both infected and non-infected tissues as SAR is developed and helps in conferring protection to the entire plant (Van Loon et al. 2006). The PRs are regulated by the defense regulatory SAR and ISR-mediating hormones such as salicylic acid, jasmonic acid, and ethylene. It, henceforth, suggests that they could play a vital role in alleviating the effects of ingress by pathogens (Van Loon et al. 2006). ROS (reactive oxygen species) led biosynthesis of proteins and their induction by plant cell wall fragments has also gained importance, the proteins are highly toxic to invading pathogen due to their hydrolytic, proteinase-inhibitory, and membrane permeabilizing properties (Edreva 2005). Thus, it could be well perceived by previous studies that the term “pathogenesis-related proteins” was specified to indicate proteins triggered by a range of pathogens as well as stress conditions either stimulated by pathogens or induced by the application of biochemicals/agents that mimic the effect of pathogen infection initiating similar kind of stress (Van Loon et al. 1994).

17 Changes Induced in Total Phenols and Flavonoids by Microorganisms

The type, quality, and quantity of biochemical substances present on the phylloplane could be subjected to fluctuations and modifications as a result of the metabolic activity of microorganisms present on the leaf surface (Blakeman and Atkinson 1981; Morris and Rouse 1985). Earlier researches suggest that microorganism invasion on plants bears a direct association with the quantities of simple and polyphenols (Tyagi and Chauhan 1982). A number of studies revealed that the composition of phenolic content in a plant and degree of its resistance to diseases shared a positive correlation. Attenuation in the biosynthesis of phenolic compounds in plants was observed generally after attack by plant pathogens (Farkas and Kiraly 1962). Bhatia et al. (1972) studied the ability of tomato plants to resist infection caused by A. solani. It was found to be significantly higher in the plant variety with increased amount of phenolics in the leaf, stem, and roots as compared to the susceptible variety which comprised of lower quantities. Owing to this phenomenon, the investigations carried out by Saleem and Paul (2015a, b, c) on Solanum lycopersicum showed a distinct difference in the phenolic and flavonoid content of control plants and plants challenged with microfungi isolated from the phylloplane. The study demonstrated significant (p ≤ 0.05) changes in flavonoids and phenolic composition of leaves upon inoculation with Aspergillus candidus, A. niger, A. flavus, Fusarium oxysporum, Rhizoctonia solani, Trichoderma harzianum, Alternaria alternate, A. citrifolia, Cladosporium cladosporioides, C. herbarum, Curvularia lunata, and Penicillium expansum. Phenolics and the related oxidative enzymes have generally been considered as few of the most significant biochemical parameters determining the viability of plants, disease resistance, and differentiating between resistant and susceptible genotypes (Pradeep and Jambhale 2002). Phenolic compounds have attracted a number of studies previously, however, the role of flavonoids in plant defense has not been largely investigated, exceptions being catechins and proanthocyanidins (Feucht and Treutter 1999). Plants may synthesize increased amounts of flavonoids and phenolic compounds possessing effective antioxidant activities that may further lead towards devising various defensive and disease fighting strategies. Phenolic compounds may act as antimicrobial agents due to various factors which may include (a) binding of the quinone nucleus to –SH and NH2 groups in the bacterial cell or (b) disturbance caused in the electron transport chain (Owens Jr 1953).

Flavonoids involved in plant defense are generally “preformed” or “induced.” The preformed flavonoids are present in healthy plants having no instances of disease, while induced flavonoids are a consequence of plant–pathogen interaction (Treutter 2006). It is evident from previous studies that they could promote plant protection against pathogens, insects, and herbivores by increasing the toxicity and reducing their nutritive value followed by change in the palatability and decrease in digestibility (Harborne and Williams 2000). Flavonoids are generally considered as vital factors in the enhancement of plant resistance response against pathogenic microbiota, though the area of research has been explored scarcely. Studies suggest that antioxidative properties of flavonoids could be responsible for their antimicrobial properties which can be non-specific and helpful against ROS, which are generated by the host plant followed by pathogenic infection, and are a major governing factor for oxidative and pathological stress (Dai et al. 1996). Flavonoids rush to the site of ingress when a pathogen invades the plant’s tissues and a hypersensitive reaction is elicited by them ultimately causing programmed cell death. They could also be directly employed in suppressing the growth of pathogenic microflora by inhibiting the plant cell wall, digesting enzymes of the pathogens by chelation of metals essential for their activity (Treutter 2005). Blount et al. (1992) reported that flavonoids could act as potent antimycotic agents. The antimicrobial activity is largely based on inhibiting spore development, mycelial growth, or hyphae elongation. Studies revealed the presence of proanthocyanidins or small quantities of dihydroquercetin could render resistance to the barley mutants against Fusarium sp. (Skadhauge et al. 1997).

18 Conclusion

The role which associated microbial communities play in implicating the outcome of host–pathogen interactions is widely acknowledged, but little has been investigated in this regard. Not much information exists on how phyllosphere microbes affect the defensive strategies of the host plant. It is of great importance that leaf associated microbiota is capable of limiting infections, may act as barriers against pathogens, induces defense response, and could significantly contribute to the non-host resistance against non-adapted pathogens. Phylloplane microbes possess agricultural importance in acting as bio-control agents against a variety of pathogenic microorganisms. Intensive investigations may open channels leading to the application of beneficial phylloplane microbes in the development of bio-control for crop protection and plant health.

The chapter provides an insight into the functional correlation between phyllosphere microflora, changes in plant physiology, and implication on the plants defense response. It describes how immensely helpful phyllosphere microbes and their metabolites could act in designing bio-control strategies for combating diseases in agriculturally important plants and crops. The bacterial and fungal isolates could also be further studied individually or as consortium in providing resistance to plants against various biotic and abiotic stresses. It throws light on the molecular dynamics of how the human pathogens could provide resistance against phytopathogens by acting as bio elicitors of defense response in plants. The elevated concentration of defensive metabolites and enhancement of defense enzymes activity prove beneficial to the plant defense dynamics and help in understanding the role of phylloplane microbiota in improvising the disease resistance mechanism of plants and crops. Production of effective bio-control will prove to be environment friendly and cost effective as compared to the conventional approach and will be highly accepted for reducing the usage of chemical alternatives. A number of previous and current researches testify the extent of possibilities phyllosphere microbiome may open for designing models for crop protection. Leaf associated microorganisms may be instrumental in providing protection against diseases and may have broad-spectrum activity against various pathogens. Their antimicrobial and plant growth promoting properties are thus of conspicuous agronomic interest, however no comprehensive information is available and the concept remains less explored.

19 Future Prospects

Improvisation and maintenance of conditions under which plants or crops are grown in order to maintain the natural community structure of microbiome on plant surfaces. Intensive research in employment of epiphytes and human pathogens singly or in consortia could prove beneficial in the development of non-toxic and biodegradable antimicrobial agents. Betterment of quality, productivity, and stress tolerance in plants can be achieved. Foliar or root application of microbial metabolites may further help in reducing incidences of disease occurrence.