3.1 Introduction

Mycorrhiza is a combination of two classical Greek words, “mushroom” and “root.” Mycorrhiza represents a symbiotic association of the underground mycelia of fungi with plant roots without harming the plant. Mycorrhizal fungi are responsible in improving growth of host plant species due to increased nutrient uptake, production of growth promoting substances, and tolerance to biotic and abiotic stresses (Sreenivasa and Bagyaraj 1989). The Arbuscular Mycorrhizal Fungi (AMF) are widely distributed in natural and agricultural environments and have been found associated with more than 80% of land plants, liverworts, ferns, woody gymnosperms and angiosperms, and grasses (Smith and Read 2008). Recently, Basidiomycete fungi belonging to the order Sebacinales, including Piriformospora indica as well as Sebacina spp., have been shown to colonize the roots of a variety of agricultural crops and to provide similar benefits to plants in terms of growth promotion, nutrient acquisition, and protection from abiotic and biotic stress (Varma et al. 2012; Gill et al. 2016).

Plant parasitic nematodes (PPNs) represent one of the largest sources of uncontrollable biotic stress experienced by plants, causing as much as US$173 billion in annual losses of crops worldwide (Elling 2013). They influence nearly all crops to some degree. The majority of crop damage is caused by the tylenchid nematodes, root-knot nematodes (RKN), and cyst nematodes (Bird 2004). The most damaging nematodes have sedentary endoparasitic lifestyles (Hussey and Roncadorl 1982; Vercauteren et al. 2002). The two main sedentary nematodes are the cyst nematodes (Heterodera and Globodera) and the root-knot nematodes (Meloidogyne) (Baum et al. 2007). In sedentary nematodes, the J2 larval worm stage invades the plant near the tip of a root and infects through the epidermal and cortex tissue and migrates to the developing vascular cells. The J2 nematodes inject their secretions into and around the plant cells to form the large feeder cell(s) (Caillaud et al. 2008). The feeding cells of cyst nematodes merge through the breakdown of neighboring cell walls to form the feeding structure known as the syncytium, through which the nematodes feed throughout their development (Ali et al. 2015). Feeding cells of root-knot nematodes (giant cells) form by repeated nuclear division in the absence of cell division (Abad et al. 2003). On the formation of feeding cells the juvenile nematode rapidly becomes sedentary because of their somatic muscles atrophy. The juveniles feed, enlarge, and molt three times to the adult stage. The large feeding cells formed by these nematodes plug the vascular tissue of the plant increasing susceptibility to water stress (Grundler and Hofmann 2011). Female sedentary endoparasites enlarge considerably into a saclike shape and are capable of laying large numbers of eggs. They are typically laid outside the nematode in a gelatinous egg mass, but in cyst nematodes most eggs are retained inside the female body which becomes melanized to encase and protect the eggs. Both types of nematodes have the same basic feeding strategy, but many cyst nematodes have an obligate sexual cycle (Cotton et al. 2014), whereas common species of RKN can reproduce largely by parthenogenesis (Ritz and Trudgill 1999).

3.2 Control of Plant Parasitic Nematodes

One of the main methods for control of PPN has been the use of resistant crop varieties. However, known resistance alleles are limited, breeding resistant varieties require large time and resource investments, and many PPN have already evolved to overcome plant resistance. Other agricultural practices such as crop rotation and the use of organic amendments have also been employed with some success (Timper 2014). Nematicides were once widely used to control PPN, but these chemicals are often associated with harmful environmental and health effects. For example, methyl bromide, one of the most important chemical fumigants used to control nematodes and other pests, affects a wide range of organisms, including beneficial microorganisms and humans, and is a chemical that contributes to the depletion of the Earth’s ozone layer (Carpenter et al. 2001). In recent decades, concerns about the environmental and health hazards of using chemical nematicides and limited availability and durability of resistant crop varieties have led to increased interest in development of biological control agents, including fungi, as a component of crop protection (Grosch et al. 2005). Root symbionts such as AMF can compete with plant pathogens for nutrients and space by producing antibiotics, by directly parasitizing pathogens, or by inducing resistance in the host plants (Schouteden et al. 2015). Thus, these microbes have great potential for the biocontrol of nematodes and other soil-borne pathogens (Berg et al. 2007).

3.3 Role of AMF in Biocontrol of Nematodes

The biocontrol effect of AMF on soil-borne pathogens has been observed in a wide range of plant species and against many pathogens, many of them soil-borne fungi causing root rot or wilting (Azcon Aguilar and Barea 1996; Harrier and Watson 2004). However, they have also shown potential against both necrotrophic and biotrophic aboveground pathogens (Fritz et al. 2006) as well as nematode pests (Veresoglou and Rillig 2012; Schouteden et al. 2015). AMF have been shown to control PPN in a variety of temperate agricultural crop plants (Pinochet et al. 1996) such as tomato and carrot (Sousa et al. 2010), soybean (Oyekanmi et al. 2007), as well as tropical crops such as banana (Hol and Cook 2005). Although there are many research reports on the biocontrol effect of AMF, their actual use as biological control agents in the field is still not a routine agricultural practice (Salvioli and Bonfante 2013). This is partially due to variability in performance, depending on the AMF isolate, pathogen, plant species, and environmental conditions (Dong and Zhang 2006; Veresoglou and Rillig 2012; Salvioli and Bonfante 2013; Bajaj et al. 2017).

3.4 Mechanism of Biocontrol of Nematodes by AMF

The potential modes of action of AMF against nematodes include direct effects of AMF on the pathogen such as competition for space or nutrients or inhibition or indirect plant-mediated responses. The latter includes enhanced or altered plant growth, morphology and/or nutrition, biochemical changes associated with plant defense mechanisms, and changes in plant root exudates that promote antagonistic microbiota that leads to increased tolerance to nematodes (Whipps 2004; Schouteden et al. 2015). However, it has been observed that a threshold level of AMF colonization is a pre-requisite for many these plant responses (Cordier et al. 1998; Slezack et al. 2000). AMF also have the ability to induce systemic resistance against plant parasitic nematodes in roots (Elsen et al. 2008). The different mechanisms cannot be considered as completely independent from each other, and biocontrol probably results from a combination of these mechanisms (Vierheilig et al. 2008; Cameron et al. 2013). In addition, the relative importance of a specific mechanism can vary depending on the specific AMF–pathogen–plant interaction.

3.5 Increased Nutrient Uptake

The mutualistic relationship of AMF with plants increases the uptake of water and mineral nutrients, such as P, N, Ca, Cu, Mn, S, Zn, and Fe (Parniske 2008; Bajaj et al. 2014; Balliu et al. 2015), and in exchange the fungus receives photosynthetic carbon for their survival from their host (Gianinazzi et al. 2010). AMF protect the plant from both biotic and abiotic stresses (Chadha et al. 2015; Bajaj et al. 2015). Nematode damaged plants frequently show impaired water uptake through roots and deficiencies of N, B, Fe, Mg, and Zn, particularly. Cotton fields with better nutrient status were able to tolerate higher populations of when infested with Rotylenchulus reniformis, the sedentary semi-endoparasitic nematode, in their roots (Pettigrew et al. 2005). Cotton plants which were colonized with AMF, also showed increased Zn uptake, which contributed to tolerance against Meloidogyne incognita by reducing the detrimental nutrient deficiency imposed by RKN (Kantharaju et al. 2005). Regression analysis of nematode population densities against the mineral content in rice also revealed a positive correlation between the migratory ectoparasitic Helicotylenchus spp. and Mg. However, a negative correlation was observed between the migratory endoparasitic nematode Pratylenchus zeae and Zn or Fe, and between Meloidogyne incognita and Mg and Ca (Coyne et al. 2004). These observations indicate that the nutrient status of the host plant can affect PPN population densities in both positive and negative ways.

3.6 Altered Root Morphology

In addition to increased nutrient uptake, mycorrhiza-colonized plants have enhanced root growth and branching (Gamalero et al. 2010; Gutjahr and Paszkowski 2013). Increased root growth may help the plant to counterbalance suppression of root growth caused by PPN. For example, this ability of AMF was observed in the banana tree where decreased root branching caused by the migratory endoparasitic nematodes was overcome by colonization with the Glomeromycete Funneliformis mosseae (Elsen et al. 2003).

3.7 Competition for Nutrients and Space

The PPN and fungi share similar physiological requirements and ecological niches. Thus, there can be competition for nutrients and space between these two groups of organisms, especially when critical nutrient sources such as carbon are limited (Vos et al. 2014). Several studies have demonstrated nutrient competition between AMF and fungal pathogens with respect to carbon (Hammer et al. 2011; Vos et al. 2014), but there is not much evidence for direct competition with nematodes (Jung et al. 2012). Similarly, since AMF and PPN both reside in and derive their nutrition from roots, they may also compete for space (Jung et al. 2012). The suppression of growth by PPN could be because the arbuscules of mycorrhiza are formed in the cortex, the same region where migratory PPN feed. This is not the case for cyst nematodes which feed on syncytia, with the feeding cells confined within the endodermis and thus less affected by AMF (Schouteden et al. 2015).

3.8 AMF-Induced Systemic Resistance

Systemic biological control of several pathogens has been reported to result from indirect effects resulting from changes in the host plant (Shoresh et al. 2010; Vos et al. 2012a; Song et al. 2011). Recently, it has been reported that the induction of systemic plant defense responses by AMF occurs because MAMP (microbe-associated molecular patterns) are conserved between beneficial and pathogenic fungi (Zamioudis and Pieterse 2012). Thus, AMF may be considered as putative pathogens by plants (Paszkowski 2006). When the plant’s pattern recognition receptors recognize MAMP, a MAMP-triggered immunity response (MTI) is activated which forms the first line of defense of the plant, inhibiting invasion of other pathogens (Jones and Dangl 2006). The systemic nature of the mycorrhiza-induced resistance was observed in banana colonized by G. intraradices against the migratory burrowing nematode R. similis (Elsen et al. 2008). On other hand, in Ammophila arenaria, no systemic resistance against P. penetrans, a lesion nematode, was observed after colonization by native AMF (De la Peña et al. 2006).

3.9 Altered Roots Exudates

The symbiosis of plants with AMF often changes the biochemical composition and level of production of roots exudates. This, in turn, impacts the hatching, mobility, chemotaxis, and host localization by nematode juveniles (Vos et al. 2012a, b). Changes in root exudates could involve compounds such as sugars and organic acids (Hage-Ahmed et al. 2013), amino acids (Harrier and Watson 2004), flavonoids and strigolactones (Steinkellner et al. 2007), plant hormones, and phenolics (McArthur and Knowles 1992). There is ample evidence that root exudates can alter the rhizosphere microbiome (Lakshmanan et al. 2014). While few studies address this topic, it is possible that root exudates induced by AMF and other root symbionts promote rhizosphere communities antagonistic to nematodes (Vos et al. 2012a, b). The level of colonization and the particular symbiont involved also impacts root exudates in the rhizosphere (Kobra et al. 2009; Lioussanne et al. 2008), and it is believed that a threshold level of colonization is also required for this mechanism of biocontrol (Paulitz 2000; Chatterton and Punja 2011).

3.10 Role of Piriformospora indica in Biocontrol of Nematodes

Piriformospora indica, a Basidiomycetes of the order Sebacinales, is an endophytic symbiotic fungus which was isolated from rhizosphere of the xerophytic woody shrubs from the Thar deserts of Rajasthan, India (Verma et al. 1998; Varma et al. 2013, 2014). It has plant growth promotional activity while providing benefits of biotic and abiotic stress tolerance to the host plant (Gill et al. 2016). It also protects the plants from pathogens and herbivores (Verma et al. 1998; Deshmukh et al. 2006; Daneshkhah et al. 2013; Bajaj et al. 2015). Like AMF, it has an extensive range of hosts, colonizing members of the bryophytes, pteridophytes, gymnosperms, angiosperms (both monocots and dicots), and orchids. In the majority of plant species investigated, there are two distinct phases in the colonization of plants by P. indica. In initial stages of infection, P. indica acts as a biotroph, but later on acts as a necrotrophic, killing some cells of the plant root through apoptosis (Zuccaro et al. 2011) and essentially forming a saprophytic association (Deshmukh et al. 2006) However, in orchids, the fungus forms a symbiotic association with the plant that promotes root growth (Ye et al. 2014). Piriformospora indica increases nutrient uptake, particularly of phosphorus (Singh et al. 2000; Malla et al. 2004), and improves plant growth and stress tolerance by inducing phytohormones (Gill et al. 2016; Siddhanta et al. 2017). Studies have revealed that it also can enhance the production of plant secondary metabolites (Bagde et al. 2010, 2014; Das et al. 2012, 2013; Kumar et al. 2012; Prasad et al. 2008, 2013; Bajaj et al. 2014).

3.11 Biotic Stress Tolerance

P. indica-infested plants are more resistant to biotic stresses. In barley infected with macroconidia of the necrotrophic fungal pathogen Fusarium culmorum, P. indica-infested plants were more tolerant to the devastating effect of F. culmorum root disease (Harrach et al. 2013). Root and shoot fresh weights were reduced only twofold in P. indica-colonized plants, compared with the 12-fold in controls with F. culmorum alone. Similar results were observed for the root pathogen Crocus sativus, which shows a hemibiotrophic nourishment strategy (Waller et al. 2005). These results show that P. indica exerts beneficial activity against major crop pathogens that cause enormous worldwide economic losses. Deshmukh et al. (2006) reported comparable biological activities of the treatments in terms of biomass increase and protection against biotrophic stress of Blumeria graminis, powdery mildew fungus in barley. Colonization of barley roots with P. indica induced systemic resistance against the biotrophic leaf pathogen. Analysis of a number of Arabidopsis mutants showed that jasmonate signaling is important for P. indica-induced resistance (Stein et al. 2008). A subset of defense-related genes are expressed earlier and more strongly induced by leaf pathogens in root endophyte-colonized barley plants than in control plants (Molitor et al. 2011). Hence, the mechanisms of P. indica-induced resistance seem to be similar to the well-characterized induced systemic resistance described for plant growth-promoting rhizobacteria-colonized plants (van Wees et al. 2008).

3.12 Biocontrol of Nematodes by P. indica

Daneshkhah et al. (2013) reported that colonization of P. indica on Arabidopsis roots in vitro antagonized the infection and development of cyst nematodes. In other fungi, this antagonistic activity can be elucidated by production of secondary fungal metabolites and enzymes such as chitinases that feature toxicity against parasitic nematodes (Shinya et al. 2008). Endophytic fungi are able to produce large amounts of toxic chemicals in vitro (Vu 2005), some of which may have direct nematicidal activity. Further studies are needed to determine if P. indica produces compounds with direct toxicity to nematodes, although its genome sequence showed few genes with known functions in fungal secondary metabolism (Zuccaro et al. 2011). Daneshkhah et al. (2013), however, noted that cell-wall extracts of P. indica alone significantly decreased nematode infection and development. P. indica may also impact production of plant secondary metabolites that deter nematodes. P. indica root colonization affected J2 infection, especially during the biotrophic phase. In this phase, the expression of MYB51, a plant gene involved in the biosynthesis of antimicrobial indole glucosinolates (Clay et al. 2009), is induced in roots of P. indica-treated plants (Jacobs et al. 2011). In roots inoculated with P. indica, it was observed that expression of CBP60g and SID2, markers of the salicylic acid-mediated signaling pathway, were upregulated (Jacobs et al. 2011). Therefore, salicylic acid-mediated signaling may also be involved in significant inhibitory effect on H. schachtii, since salicylic acid was revealed to inhibit growth of H. schachtii (Wubben et al. 2008).

3.13 Case Study: Piriformospora indica Antagonizes the Soybean Cyst Nematode in Planta

Field soil was collected from an agricultural field with no soybean cyst nematode infestation, mixed with 30% sand and autoclaved twice. Mycelium of P. indica at concentrations of 0% (w/w), 2.5% (w/w), and 5% (w/w) was thoroughly mixed with soil and placed into clay pots in a controlled greenhouse trial in order to analyze its possible effects on growth, development, and pest resistance towards the SCN (Bajaj et al. 2015, 2017).

Root colonization was observed by staining the roots with lactophenol cotton blue, and intracellular chlamydospores of P. indica were observed confined to the root cortex. Levels of root colonization ranged from 45% to 50% in 2.5% and 5% P. indica treatments at 8 weeks after planting. No colonization was observed in 0% P. indica treatment. Soybean showed a positive interaction with P. indica, as demonstrated by increased shoot biomass and shoot length of inoculated plants as compared to control plants. However, the overall biomass of colonized roots was lower than that of the uncolonized control roots. P. indica not only induces development of the vegetative aerial part of the plant, but also is responsible for early maturation with respect to flowering in soybean (Table 3.1).

Table 3.1 Effects of P. indica on soybean
Full size table

The number of SCN eggs per cc soil, a common screening measure of SCN severity in agricultural fields, was significantly lower in the P. indica amended pots. There was a decrease of 29.7% in the 2.5% P. indica treatment and 36.7% in the 5% P. indica treatment. Egg density per cc soil was also significantly reduced between the 2.5% to the 5% P. indica treatments. Egg density calculated as number of eggs/cc soil/gram root wet or dry weight also showed a trend, although not significant, of decreasing egg density with increasing P. indica in soil.

Although the mechanism of nematode inhibition in this study is unknown, P. indica may either directly inhibit nematode development, as discussed above, or may control plant responses that impact nematode colonization and development. One possible plant response is altered carbon-partitioning in the plant. The fungus has been shown to control expression of a Nicotiana attenuate homolog of Hsl-Pro-l, a locus initially identified as having a role in resistance to the beet cyst nematode (H. schachtii Schmidt) (Cai et al. 1997), but now thought to be involved in more generalized responses to both abiotic and biotic stresses and in repartitioning of carbon resources within the plant (Schuck et al. 2012). Enzymes such as sucrose synthases and invertases may also impact development of cyst nematodes by altering plant sink strengths and changing systemic sugar partitioning to decrease syncytial sugar levels. Higher sugar levels in roots were shown to contribute to enhanced nematode development (Cabello et al. 2013) and have major nutritional value for this obligate parasite. Thus, allocation of sugars to shoot growth over root growth could impact the availability of sugars to root cyst nematodes. In contrast to AMF, P. indica decreased root growth and branching. However, decreased root growth may also reduce infection by the SCN (Bajaj et al. 2015) by lowering the number of potential sites for nematode infection (Schouteden et al. 2015).

3.14 Conclusions

AMF and other beneficial fungi such as P. indica confer many of the same benefits to plant hosts including improved nutrient uptake, increased plant growth, enhanced abiotic and biotic stress tolerance, induced systemic resistance against pathogens, and production or induction of plant protective secondary compounds or root exudates. However, there may also be significant differences in the mechanisms of nematode antagonism by these two groups of fungi. Instead of increasing root growth and proliferation like AMF, P. indica causes cell death in roots and directs resources to shoot growth, leading to smaller root:shoot ratios. Reduced root growth and volume may promote biocontrol of nematodes both by diverting sugars on which juvenile nematodes feed from root to shoot and also by providing less root surface area for infection. While further studies are needed to investigate nematode inhibitory compounds produced by AMF, to our knowledge, the majority of known mechanisms of AMF protection against nematodes are plant mediated. In contrast, compounds and cell-wall components of P. indica have been shown to directly inhibit infection and development of nematodes in roots. Both groups of fungi offer promising avenues for successful biocontrol of PPN.