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1 Introduction

Nematodes are filiform roundworms belonging to phylum Nematoda commonly found in plants, animals, and soil. They have the ability to utilize the various organic sources for the production of energy (Akhtar and Panwar 2011). Some plant parasitic nematodes usually feed on plant cells by choosing and establishing a single feeding site known as sedentary feeders, while others are migratory feeders which means they move from site to site on the root and rarely feed on plant single cell. In general, the plant parasitic nematodes are documented as the utmost vicious pests for several economically important crops worldwide. Bowers et al. (1996) reported that the nematode had the ability to alter the root exudates in qualitative and quantitative fashion, which may influence the activity of beneficial and pathogenic microbes in the rhizosphere. The estimated average annual yield loss of various crops by plant parasitic nematodes is about 12.3 % (Sasser and Freckman 1987), but it varies from 8.8 to 14.6 % from developed to developing countries (Nicol et al. 2011; Palomares-Rius and Kikuchi 2013). Among the sedentary feeders, Meloidogyne species are predominant and are considered as the most damaging genera throughout the world. About 95 % of the total nematode populations are represented only by four major species such as M. incognita, M. javanica, M. arenaria, and M. hapla.

Suppression of plant diseases in the presence of a pathogen, suitable host plant, and favorable climatic conditions is known as soil suppressiveness (Mazzola et al. 2004; Weller et al. 2007). It is directly associated with the nature and fertility level of the soil and the types of soil microorganisms. However, the level of disease suppressiveness is directly proportional to the level of soil microbial activity, meaning the larger the active microbial biomass, the greater the soil capacity to use carbon, nutrients, and energy, thus lowering their availability to pathogens (Kumar et al. 2012). Any treatment to increase the microbial activity in the soil enhanced the suppression of pathogens by increasing competition for nutrients, but overall it is a very tough task to control all types of soilborne pathogens by suppressive soils. To control the diseases caused by plant parasitic nematodes, frequent use of chemical nematicides has been increased in the past few decades globally (Gupta and Dikshit 2010; Leng et al. 2011). But these chemical nematicides possess several toxic effects on the human health, soil microbiota, and environment. Thus, several cultural practices have been adopted for the management of nematodes, but gradually the annual losses observed in the quality and quantity of crop yields revealed that there is a decisive need to develop a new eco-friendly way to control the plant parasitic nematodes. In this regard, biological control strategies provide an alternative tool for management of plant parasitic nematodes over the conventional chemical control strategies (Mazzola 2007). The biological control of nematodes could be achieved either by managing the natural habitats to marmalade by increasing the activity of native fungi or by introducing new beneficial rhizospheric fungi or by the combination of both (Timper 2011). Nevertheless, the augmentation of the beneficial microorganisms in the agricultural fields and their potential benefits on the various crops is feasible through the adoption of various management practices such as reduced tillage, crop rotation, and lowering the micronutrient uses.

The rhizosphere is the immediate microenvironment surrounding the plant roots which provides novel environments for microbes due to change in increased levels of nutrients and intense microbial population (Giri et al. 2005; Gupta et al. 2012; Yadav et al. 2015). The rhizoplane and the surrounding rhizosphere soil are colonized and occupied by a wide range of microorganisms. Of the various microorganisms present, opportunistic fungi and arbuscular mycorrhizal (AM) fungi play a key role in the biocontrol of diseases caused by plant parasitic nematodes. Consequently, the plant parasitic nematode and beneficial rhizospheric fungi share common ecological niche and also influenced the plant growth and yield attributes in various means (Akhtar and Siddiqui 2008; Akhtar and Panwar 2011). Because of multifaceted nature, it is very hard to generalize the overall underground interaction processes taking place between the plant parasitic nematodes, opportunistic fungi, and AM fungi. The aim of this chapter is to provide an overview of the biocontrol potential of opportunistic as well as AM fungi on the growth and improvement of various crop plants and population of plant parasitic nematodes in different pathosystems. The chapter also focuses on the cost-effective technologies used for the mass propagation of opportunistic fungi and AM fungi and their ample application in the expansion of practical control system desired for the sustainable agricultural practices.

2 Opportunistic Fungi

Fungi have the immense miscellany in their metabolic pathways and offer numerous important classes of commercial compounds having nematicidal activity (Li et al. 2007; Anke 2010) and limit the nematode densities by the production of nematotoxic compounds due to their parasites and antagonistic or predatory actions between fungi and plant parasitic nematodes (Lopez-Llorca and Jansson 2007; Akhtar and Panwar 2011). Lopez-Llorca and Jansson (2007) found that the opportunistic fungi either directly parasitize the nematodes or secrete some nematicidal metabolites which may affect the viability of one or more stages of the nematode life cycle or having deleterious effects on reproductive structures of a nematode. The secondary reproductive stage of the nematode is highly susceptible against the opportunistic fungi. The obese females are highly prone to fungal attack similarly like the parasitism of egg masses. The opportunistic fungi when come in contact with nematode eggs grow more rapidly and parasitize the eggs during initial embryonic developmental stages. This may reduce the parasitic actions of nematode juveniles. Among the various known opportunistic fungi, P. lilacinus and P. chlamydosporia have been extensively studied by several previous researchers for their nematophagous knack and biocontrol potentiality (Khan et al. 2004; Kiewnick and Sikora 2006; Siddiqui and Akhtar 2009a, b; Akhtar and Panwar 2011; Azam et al. 2013).

2.1 Paecilomyces lilacinus

Paecilomyces lilacinus (Thom) Samson is a mutual Hyphomycetes and is ubiquitously distributed especially in warmer climates (Samson 1974). It is encompassed in the group of frequently tested biocontrol agents against the plant parasitic nematodes (Brand et al. 2010; Pau et al. 2012; Azam et al. 2013). It is basically a saprophyte but could also compete for extensive range of substrates (Holland et al. 2003; Pau et al. 2012).

Jatala (1986) reported that P. lilacinus infects eggs and females of plant parasitic nematodes and destroyed the embryo within 5 days under laboratory conditions. He found that the infection of nematode eggs starts in a gelatinous matrix with the development of fungal hyphae which latter surrounds the entire nematode eggs. The colonization of nematode eggs occurred through the diffusion of egg cuticle by the fungal hyphal network by enzymatic or mechanical actions. His experiments clearly indicated that P. lilacinus grow well between 15 and 30 °C. It also had the adaptability to grow in a wide range of soil pH which made it a pretty modest organism in most of the cultivated fields. The suppression of plant parasitic nematode by P. lilacinus is ascribed by disintegration of the embryo, inhibition of hatching, and parasitism of adult females (Fig. 11.1). However, after establishment of P. lilacinus in soil, it grows faster and spread rapidly within a short span in the introduced area as dominant species. Moreover, the production of secondary metabolites such as chitinases, leucinotoxins, and proteases has also been associated with P. lilacinus infection (Park et al. 2004).

Fig. 11.1
figure 1

Cross section of tomato root infected with root-knot nematode; (a) showing presence of nematode, egg masses, abnormal phloem, and abnormal xylem in the cortical region; (b) showing conidia of P. lilacinus surrounding the nematode eggs and egg masses; (c) disruption of eggs and egg masses by P. lilacinus hyphae; (d) complete disintegration of nematode eggs by P. lilacinus hyphae

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2.2 Pochonia chlamydosporia

Pochonia chlamydosporia (Goddard) Zare and Gams is a well-known nematophagous fungus and ubiquitously distributed in all parts of the world. It is naturally occurring as a facultative parasite of females, eggs, cyst, and plant parasitic nematodes (Lopez-Llorca et al. 2008; Manzanilla-Lopez et al. 2013). In the rhizosphere, this fungus could settle the host root as endophytes preferably with the plants belonging to families Gramineae and Solanaceae and provide numerous benefits to host plant defense against the soilborne pathogens (Macia-Vicente et al. 2009a, b). P. chlamydosporia have been extensively studied for its biocontrol potential against plant parasitic nematodes (Kerry and Hirsch 2011; Manzanilla-Lopez et al. 2013). The efficacy of this potential biological fungus against the plant parasitic nematode is affected by three major factors: (1) the amount of fungus in the rhizosphere, (2) the rate of development of eggs in the egg masses, and (3) the size of galls in which female nematode develops.

The population of P. chlamydosporia could be identified on the basis of position and shape of conidia, the plethora of dictyo-chlamydospores, and the development of conidia either in heads or chains (Zare and Gams 2004). P. chlamydosporia infects the nematode eggs through the expansion of aspersoria at the tip or lateral position of hyphae, which encompasses tightly to the surface of eggshells (Fig. 11.2), and finally penetrated into eggshells by the formation of an infection peg (Holland et al. 1999). A postinfection bulb leads to the expansion of mycelia within the eggs that caused almost the complete devastation of their contents (Tikhonov et al. 2002; Esteves et al. 2009a). Khan et al. (2004) reported that the eggshells and juvenile cuticles both have been physically disrupted, and the fungal hyphae willingly multiplied inside the eggs and juveniles due to enzymatic activity and biosynthesis of diffusible toxic metabolites. P. chlamydosporia are reported to secrete serine, protease, and chitinase responsible for the major structural changes inside the nematode eggs which may result in the disintegration of lipid and vitelline layers. Application of P. chlamydosporia as soil inoculants could reduce the natural nematode population up to 90 % under field condition (Bordallo et al. 2002), but the fungus differs in virulence toward nematode competence to colonize the root and production of chlamydospore (Bordallo et al. 2002; Yang et al. 2007; Macia-Vicente et al. 2009a, b). All these specific features make P. chlamydosporia a successful biocontrol agent under different pathosystems (van Damme et al. 2005; Rumbos et al. 2006; Esteves et al. 2009b).

Fig. 11.2
figure 2

Classical and electron microscopic images of root-knot nematode infected by P. chlamydosporia; (a) egg of a nematode infected by P. chlamydosporia hyphae; (b) complete disintegration of nematode egg by P. chlamydosporia hyphae; (c) electron microscopic view of P. lilacinus hyphae covering the nematode egg; (d) disruption of nematode egg by P. chlamydosporia hyphae

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3 Arbuscular Mycorrhizal Fungi

Arbuscular mycorrhizal (AM) fungi are the key components of soil microbial populations with ubiquitous distribution in almost all the agroclimatic conditions of the world and form symbiosis with most of the land plants, in any kind of terrestrial ecosystem (Akhtar and Siddiqui 2008). Currently, AM fungi have been cited in the phylum Glomeromycota (Redecker and Raab 2006), and over 200 morphospecies of Glomeromycota have been described (Schüßler 2008). AM fungi have been categorized on the basis of extra-radical mycelium and branched haustoria-like structure within the cortical cells, termed as arbuscules. These arbuscules are the core sites for the nutrient exchange (Fig. 11.3), where the fungi supply water and nutrients like N and P to plants and in turn receive carbon from plants (Bonfante and Genre 2010).

Fig. 11.3
figure 3

Microscopic view of colonization pattern of AM fungi inside the tomato root; (a) showing hyphae of AM fungi; (b) showing formation of arbuscles; (c) visualization of AM spores inside the cortical tissue; (d) AM spores with hyphae stained with Melzer’s reagents

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Due to their unique ability and adaptability in different agroclimatic conditions, the AM fungi improved plant health by the acquisition of essential mineral nutrient and water from soil and enhanced production of growth regulations, tolerance toward various abiotic conditions, and mutualistic relationship with additional rhizospheric microorganisms existing in the same ecological niche (Akhtar and Siddiqui 2008; Akhtar 2011).

4 Efficacy and Biocontrol Strategies of Beneficial Rhizospheric Fungi

Persistence of plant parasitic nematodes is the most serious problem worldwide because they nourish and multiply their population on live host plants and also actively migrate inside the plants and aerial parts or in the rhizosphere. Among all the available options, chemical control has been extensively used against the plant parasitic nematode, due to its nonselective nature. However, use of chemicals to control plant parasitic nematodes has been restricted in many countries due to their environmental toxicity and ability to leach into the soil. They may cause the hazardous effect on the soil microbial flora and fauna as well as on the environment (Akhtar 1997). In the beginning, most of the fumigants were effectively used to control the plant parasitic nematodes due to their nematicidal properties, but later the detection of their remains in soil, water, and edible crops has caused awareness among the global scientific community concerned about the safety of human health and the environment (Alphey et al. 1988). Methyl bromide was the first fumigant which was widely used against the pathogens causing soilborne diseases, but it has been now banned and completely withdrawn from the market by imposing an international agreement in most of countries worrying about the environment safety (Oka et al. 2000).

Nowadays, several control measures such as the use of green manure, organic or inorganic soil amendments, crop rotation, resistant variety cultivation, unplanted treatment, and biological control have been used to limit the population of plant parasitic nematodes in the soil. But, unfortunately, all these control methods have led to limited success (Barker and Koenning 1998). Integrated pest management provides a working methodology for pest management in sustainable agricultural systems. With the increasing cost of inorganic fertilizers and the environmental and human health hazards associated with the use of pesticides, opportunistic and AM fungi may provide a more suitable and environmentally acceptable alternative for sustainable agriculture. Several comprehensive reviews have been published time to time exploring the possibilities of using AM fungi (Barea et al. 2005; Akhtar and Siddiqui 2008; Smith and Read 2008; Akhtar and Panwar 2011) and opportunistic fungi in the biocontrol of plant diseases (Atkins et al. 2005; Hildalgo-Diaz and Kerry 2008). We have summarized some recently published results of interaction studies between opportunistic fungi, AM fungi, and plant parasitic nematodes in tabular forms (Tables 11.1, 11.2, and 11.3).

Table 11.1 Effect of Paecilomyces lilacinus on the plant growth and reproduction of plant parasitic nematodes
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Table 11.2 Effect of Pochonia chlamydosporia on the plant growth and reproduction of plant parasitic nematodes
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Table 11.3 Effect of AM fungi on the plant growth and reproduction of plant parasitic nematodes
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5 Mass Propagation Strategies of Opportunistic Fungi and AM Fungi

5.1 Mass Production of Opportunistic Fungi

Several media have been extensively used for the mass production of opportunistic fungi. For the mass production of P. lilacinus potato dextrose broth (Rangaswami 1972), Richard’s medium, 10 % molasses (Rangaswami 1972), and semi-selective medium (Mitchell et al. 1987) can be used. The highest mycelium weight and spore production were achieved by using the semi-selective medium followed by 10 % molasses medium (Prabhu et al. 2008). Corn meal agar and potato dextrose agar media have also been used for the mass production of P. lilacinus (Robl et al. 2009). Similarly, the mass production of Pochonia spp. was achieved by using shrimp agar medium (Moosavi et al. 2010). Besides this wheat, bran and barley grain were also used for the mass production of Pochonia spp. (de Leij and Kerry 1991; Crump and Irving 1992). For the large-scale commercial production, liquid fermentation method is generally used because of difficulties to improve spore production on solid medium (Khan and Anwer 2011).

5.2 Mass Production of AM Fungi

AM fungi have the unique ability to improve the uptake of water and mineral nutrients from the soil and also to guard the plants against the pathogen attack (Smith and Read 2008). AM fungi also scavenge the available P through their extra-radical hyphae and upsurge the secretion of various amino acids (such as serine and isoleucine) and defense-related proteins (Akhtar and Siddiqui 2008; Akhtar et al. 2011), which augments their importance toward the modern and profitable agronomic practices. Due to their obligate nature, the AM fungi could not be cultured in vitro, which may limit the mass production of AM fungal propagules. In the conventional method of propagation, the AM fungi are propagated through the pot or pan culture usually with single spore culture, swiftly spread on the substrate, and finally colonize the root of host plants (Akhtar and Abdullah 2014). This method is quite useful for the production of clean fungal inoculum with high potentiality in a short span of time. Similarly, aeroponic culture systems allow the production of cleaner spores and enable even nourishment of AM fungi-colonized plants (Jarstfer and Sylvia 1999). Propagation of any AM fungal strains on root-organ cultur e permitted the propagation of monoxenic strains that could be used either directly as inoculum or as a starter inoculum for the mass production of AM fungi. A very simple and low-cost technique of single spore pot culture has been developed by Panwar et al. (2007). It permits undistributed growth of the mutualistic partners and visualization of germinating AM fungal spores and their mass multiplication. Moreover, the mass production of AM fungal inoculum requires control and optimization of both host growth and fungal development. The microscopic sizes of AM fungi, together with the complex identification processes, also contribute to the drawbacks of inoculum propagation.

Nevertheless in vitro bulk production of AM fungal inoculum is a promising approach, offering clean, viable, contamination-free fungal propagules. The cost of in vitro inoculum may appear expensive compared to the greenhouse-propagated fungal inoculum, but its use as starting inoculums is a warranty of purity (Akhtar and Abdullah 2014). The main purpose of this cultural method is to provide pure, clean, and reliable material as starter inoculum for the fundamental and applied research. There were several reports which indicate that mycorrhizologists were able to produce 25 spores/ml in 4 months’ incubation time (Chabot et al. 1992), while the other workers claimed for the production of 3250 spores/ml in 7 months (Douds 2002). Recently another work justifies the production of more than 2400 spore/100 g of soil after 120 days from single spore culture (Panwar et al. 2007).

6 Conclusions

The present chapter provides an overview on the interactions between opportunistic fungi, AM fungi, and plant parasitic nematodes. Use of opportunistic and AM fungi will not only reduce the load of nematicides in agricultural practices but also increase the plant vigor through the uptake of essential mineral nutrients and also reduce the nematode buildup in the plant and soil. Moreover, use of these biocontrol agents has an eco-friendly approach toward the environment as well as human health. The protection of nematode diseases by the application of these biocontrol agents is a complex process which may depend upon the molecular interactions between hosts, biocontrol agents, and pathogenic microorganisms. Application of single or mixed inoculum of opportunistic fungi, AM fungi were found to be effective in controlling the nematode diseases under greenhouse, pot, and field conditions in various agroclimatic conditions. An overview of the recent cost-effective technologies used for the mass propagation of these beneficial rhizospheric microorganisms is discussed. The success of mass propagation of indigenous biocontrol agents depends upon its selective nature toward edaphic, environment, and other rhizospheric biota, but it is still a challenge to develop these biocontrol agents in the sustainable agricultural practices to understand real underground mechanisms involved between the host, biocontrol agents, and pathogenic microorganisms.