Keywords

8.1 Introduction

During the last 40 years, the world population has increased by 90%, while food production has increased only by 25% per head. It is estimated that 39% more production is needed worldwide to feed an additional 1.5 billion mouths by 2020 and the production needed to be doubled by 2050. However, attack by pest and diseases causes a loss to the tune of 40% of the gross crop production. Further, with the rapid change in climatic factors, plant pathogens are becoming more aggressive, breaking the plant resistance, and inhibit the crops to reach its optimum yield. Current practices for integrated disease management are largely based on genetic host resistance and synthetic chemicals. Continuous use of those chemicals in controlling plant diseases has negative effects on the environment, causes pollution in the biosphere and harms the human beings. Further, those chemicals themselves are acting as selective agents, making the pathogens more resistant, and help these pathogens to persist as they are slowly becoming resistant to these agents. Thus, there was a necessity to execute new methods which would supplement conventional strategies for plant disease control and are competent to minimize adverse effects of chemical pathocides on human health and the environs. Control of plant diseases using biological agents like live microbial cells, or byproducts produced by them, is a powerful alternative way, called biological control. Biological control is eco-friendly, and the diversified microbial world provides endless resources for biologically active molecules which can stably inhabit the environment as nondominant species but maintain their effectiveness in suppression of plant pathogens. For instance, in the 1880s, the cottony cushion scale in citrus was the major threat to citrus industry in California. Vedalia beetle (Rodolia cardinalis Mulsant), a predatory insect, was introduced in California to cease the effect of the pest (Icerya purchasi Maskell). That was the first success story of the biological control. Since this success, scientists have developed diverse techniques to manage a variety of pests and pathogens using diverse biological agents. In recent years, they diverted their attention towards the potential of beneficial microbes. Therefore, dynamic research efforts for developing and exploring innovative tools for the control of diseases have become imperative.

8.2 Why Eco-friendly Management Is Important to Control Plant Pathogen?

Control of the diseases is very important for securing human food sources and agriculture-based industries. There are two main ways to manage diseases and pests, using chemicals (chemical control) and by predators or parasites (natural control/biological control). Controlling of diseases in economically important crops with chemicals has long been practiced in agricultural settings, and use of this method is more acceptable by the farming community, as it is typically less expensive and immediate than natural control methods. But extensive use of those chemicals for an extended period has long lasting negative effects on the environment, including human life and other living organisms existing in the ecological niches. Being detrimental to both beneficial and harmful organisms, they can damage the ecological balance and also contaminate the food chain through bioaccumulation of toxic residues. In this way the chemicals become worse for the organisms belonging to the higher tropic levels (Fig. 8.1).

Fig. 8.1
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Harmful chemicals enter the food chain and deposition increased tissues of the organisms belonging to the higher tropic level

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The term ‘biocontrol agent’ (biopesticide), as a generic definition, has been applied with a narrow focus on preparations containing living microorganisms, through to a wider definition that includes botanical compounds and semiochemicals (e.g. pheromones) (Kiewnick 2007). The biocontrol agents and the process of biological control have several other benefits.

  1. 1.

    Biocontrol agents are safer both for the environment and the persons who are applying them and avoid environmental pollution (soil, air and water) by leaving no toxic residues.

  2. 2.

    It is comparatively easier to manufacture biocontrol agents, sometimes less expensive than chemical agents.

  3. 3.

    The biggest advantage of using biocontrol agents is that it can eliminate the specific pathogens effectively from the site of infection and can be used in combination with biofertilizers.

  4. 4.

    Biocontrol agents are very effective for a large number of soil-borne pathogen where using of chemical fungicide is not possible.

  5. 5.

    Biocontrol agents do not cause any toxicity to the plants; rather these increase crop yields by enhancing the root and plant growth through the encouragement of beneficial microflora in rhizosphere. It also helps in the mobilization of plant nutrients and makes it available to the plant.

  6. 6.

    Biocontrol agents avoid problems of resistance and also induce systemic resistance among the crop species.

  7. 7.

    Biological control is self-regulating, does not require any intricate management and helps to preserve the ecosystem.

However, despite the fascinating advantages of biocontrol of plant diseases, there might be few adverse effects on humans and the environment. Increasing the population of a certain biological agents artificially could be the reason of paying unexpected concerns. An organism that has been introduced from another area to destroy a pathogen in a new habitat may itself become a pathogen or predator for some beneficial organisms present in natural habitat or crops. Other than that it has the following limitations.

  1. 1.

    Biocontrol agents work slowly and less effectively in comparison to the chemical pesticides, as their efficacy almost completely depends on environmental conditions.

  2. 2.

    Biocontrol agents are mainly used against specific diseases as a preventive measure, not as a curative measure.

  3. 3.

    The antagonists and shelf life of biocontrol agents are short. For example, the shelf life of Pseudomonas fluorescens is 3 months and of Trichoderma viride is 4 months only. To maintain the effective level of biocontrol agents in cropping area, periodical checking is needed and this requires skilled persons.

  4. 4.

    Skilled persons are also required for multiplying and supplying the biocontrol agents without contamination.

  5. 5.

    At present, biocontrol agents are available only in a few places and in less quantities.

8.3 Groups of Biological Control Agents

After the development of the first commercial biological agent, a range of microorganisms, including virus, bacteria, actinomycetes, fungi, oomycetes, protozoa, etc., were identified for the purpose of plant disease management. Many organisms are found to be very effective against a variety of plant diseases. A few of those organisms are now being used for successful disease management in plants at fields and greenhouse conditions (Table 8.1).

Table 8.1 Groups of successful biocontrol agents that effectively control diseases in economically important crop species
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8.4 Plant Extract

Plants are capable of synthesizing an overwhelming variety of small organic molecules, the secondary metabolites, which help the plants overcome from pathogen infection. Identification of novel effective secondary metabolites as fungicide or insecticide is essential to inhibit increasing resistance rates of the pathogens. The botanical extracts are more effective as insecticidal compounds (Table 8.2). But nowadays plant extracts are being used as effective biocontrol agents for inhibiting fungal diseases of plants. The plant extracts from Cymbopogon proximus, Allium sativum, Carum carvi, Eugenia caryophyllus and Azadirachta indica were found to have inhibitory effects on some phytopathogens including Botrytis cinerea, Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani (Alkhail 2005). The methanolic plant extracts from Salvadora persica, Lantana camara, Thymus vulgaris, Ziziphus spina-christi and Zingiber officinale have antifungal properties against Fusarium oxysporum, Rhizoctonia solani and Pythium aphanidermatum (Hussin et al. 2009). Ethyl acetate extracts of Lantana camara showed inhibitory effects against Colletotrichum gloeosporioides which causes anthracnose in papaya (Carica papaya L.).The mother tincture extract of Myroxylon balsamum showed antifungal activity against the filamentous fungi Fusarium guttiforme and Chalara paradoxa, causing pineapple fusariosis.

Table 8.2 Botanical pesticides used to control different pests and pathogens
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8.5 Different Mechanisms of Biological Control

8.5.1 Direct Antagonism

8.5.1.1 Parasitism

Parasitism is an interactive mechanism in which two phylogenetically unrelated organisms live together over a prolonged period of time. In this type of relationship, one organism, usually benefitted, called the ‘parasite’ and the other called the ‘host’, is harmed. For instance, Trichoderma is a parasite of a range of fungi and oomycetes in the soil, which produce toxic metabolites and cell wall-degrading enzymes and inhibit the growth of others.

8.5.1.2 Hyperparasitism

Hyperparasites are the agents that are parasites of harmful plant pathogens. A classic example is the Hypovirus, a hyperparasitic virus on Cryphonectria parasitica, a fungus causing chestnut blight. The hypovirulence of Hypovirus reduces the disease-producing capacity of C. parasitica (Tjamos et al. 2010). Some strains of fungi have hyperparasitic activity against other fungi. The fungus Ampelomyces quisqualis grows on mildew pathogen; similarly Nectria inventa and Gonatobotrys simplex are parasites of Alternaria (Kiss et al. 2004). The fungus Phlebiopsis gigantea is used to control Heterobasidion annosum, a fungal pathogen that causes rots in freshly cut stumps of pine trees that can spread subsequently to intact trees by root-to-root contact (Pratt et al. 1999). The fungal species, Acremonium alternatum, Acrodontium crateriforme, Cladosporium oxysporum and Gliocladium virens, have the capacity to parasitize powdery mildew pathogens and be used as biocontrol agent (Heydari and Pessarakli 2010).

8.5.1.3 Commensalism

Commensalism is a unidirectional association between two unrelated species by living together, in which one population (commensals) benefits from these relationships, while the other (the host) is not harmed. Microbes present in the rhizosphere control soil-borne pathogens through competition for nutrients and production of antibiotics and help the plants survive pathogen infection (Kumar et al. 2016a, b). On the other hand, the microbes have an important role on the growth of the plant by increasing solubilization of minerals or by synthesizing amino acids, vitamins and growth regulators that stimulate the plant growth.

8.5.2 Mixed-Path Antagonism by Synthesis of Allochemicals

8.5.2.1 Siderophores

Siderophores are ligands with low molecular weight having high affinity to sequester iron from the micro-environment. It has the ability to sequester ferric ion and competitively acquire iron from iron-limiting microenvirons, thereby preventing growth of other microorganisms. Two major classes of siderophores, classified on the basis of their functional group, are catechols and hydroxamate. A mix of carboxylate-hydroxamate group of siderophores is also reported (Hider and Kong 2010) (Table 8.3). Numerous strains of Streptomyces spp. have been reported as siderophore producers, namely, S. pilosus (Muller et al. 1984; Muller and Raymond 1984), S. lydicus (Tokala et al. 2002) and S. violaceusniger (Buyer et al. 1989). Biological control of Erwinia carotovora by several siderophore-producing and plant growth-promoting Pseudomonas fluorescens strains A1, BK1, TL3B1 and B10 was reported for the first time as an important mechanism of biological control (Kloepper et al. 1980). On the other hand, increased efficiency of iron uptake by the commensal microorganisms is thought to dislocate pathogenic microorganisms from the possible infection sites by aggressive colonization in plant rhizosphere. Sneh et al. (1984) and Elad and Baker (1985) showed a direct correlation between in vitro inhibition capacity of chlamydospore germination of F. oxysporum and siderophore synthesis in fluorescent pseudomonads.

Table 8.3 Examples of siderophores produced by various bacteria and fungi
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8.5.2.2 Antibiosis

The term ‘antibiosis’ came from the term antibiotics, which refers to organic substances produced by microorganisms that affect the metabolic activity of other microbes and inhibit the growth (Roshan et al. 2013). The result of antibiosis is often death of microbial cells by endolysis and breakdown of the cell cytoplasm. Agrobacterium radiobacter K-84, produced commercially as Agricon 84, was first recognized as a valuable control agent of crown gall since 1973. It is very effective against A. tumefaciens attacking stone fruit (e.g. plums and peaches), but not effective against A. tumefaciens strains that attack grapes, pome fruit (e.g. apples) and some ornamentals. A variety of antibiotics have been identified, including compounds such as 2,4-diacetylphloroglucinol (DAPG), amphisin, oomycin A, hydrogen cyanide, pyoluteorin, phenazine, tensin, pyrrolnitrin, cyclic lipopeptides and tropolone produced by pseudomonads and kanosamine, oligomycin A, xanthobaccin and zwittermicin A produced by Streptomyces, Bacillus and Stenotrophomonas spp. (Kumar et al. 2014) (Table 8.4). For instance, antibiotic 2,4-diacetyl phloroglucinol is reported to be involved in the suppression of Pythium spp., iturin suppresses the pathogens Botrytis cinerea and Rhizoctonia solani, and phenazine carboxylic acid antagonist the pathogen Rhizoctonia solani in rice (Padaria et al. 2016) and phenazines control Gaeumannomyces graminis var. tritici in wheat.

Table 8.4 Selected examples of antibiotics produced by biocontrol bacteria
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8.5.2.3 Volatile Substances

Apart from the production of antibiotics, some biocontrol agents are also known to produce volatile compounds as tools for pathogen inhibition. Common volatile compounds are hydrocyanic acid (HCN), certain acids, alcohols, ketones, aldehydes and sulphides (Bouizgarne 2013). HCN production is reported to play a role in disease suppression (Wei et al. 1991), for instance, Haas et al. (1991) reported HCN production by strains of P. fluorescens that helped in the suppression of black root rot of tobacco. Reports on the production of HCN by beneficial microbes in order to minimize the deleterious effect of pathogenic fungi and bacteria are available (Ahmad et al. 2008; Gopalakrishnan et al. 2011a, b, 2014).

8.5.2.4 Lytic Enzyme Production

Many microorganisms secrete and excrete lytic enzymes that can hydrolyse a wide range of polymeric compounds, including hemicellulose, cellulose, chitin, DNA and proteins (Table 8.5). These extracellular hydrolytic enzymes play an important role in the suppression of plant pathogens. Chitinase secreted by Streptomyces sp., Paenibacillus sp. and Serratia marcescens was found to be inhibitory against Sclerotium rolfsii, Botrytis cinerea and Fusarium oxysporum f. sp. cucumerinum. Similarly, modifying plant growth substratum with chitosan inhibits the root rot in tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici. β-1,3-Glucanase produced by Actinoplanes philippinensis and Micromonospora chalcea was found to hydrolyse Pythium aphanidermatum in cucumber (El-Tarabily 2006).

Table 8.5 Examples of lytic enzymes produced by biocontrol bacteria
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8.5.2.5 Unregulated Waste Products

Few soil microbes release a range of unregulated waste products or harmful gases, e.g. ethylene, methane, nitrite, ammonia, hydrogen sulphide, other volatile sulphur compounds, carbon dioxide, etc., and suppress the growth of other pant pathogenic bacteria. This interaction between two species is called ammensalism. Bacillus megaterium produces ammonia and has an inhibitory effect on the growth of Fusarium oxysporum (Shobha and Kumudini 2012).

8.5.2.6 Detoxification and Degradation of Virulence Factor

Biological control by detoxification involves production of a protein that binds with the pathogen toxin and detoxifies pathogen virulence factors, either reversibly or irreversibly, ultimately decreasing the virulence potential of pathogen toxin. For example, the biocontrol agents Alcaligenes denitrificans and Pantoea dispersa are able to detoxify albicidin toxin produced by Xanthomonas albilineans. Similarly, strains like B. cepacia and Ralstonia solanacearum can hydrolyse fusaric acid, a phytotoxin produced by various Fusarium spp. The protein has the ability to bind reversibly with the toxins of both Klebsiella oxytoca and Alcaligenes denitrificans, as well as irreversibly with the toxin albicidin in Pantoea dispersa.

8.5.3 Indirect Antagonism

8.5.3.1 Competitive Root Colonization

From the microbial perspective, living plant surfaces and soils are often nutrient-restricted environments. Nutrient limitation is an important mode of action of some biological control agents. Carbon plays an important role for competition of root colonization for nutrients such as Trichoderma spp. (Sivan and Chet 1989). Carbon competition between pathogenic and non-pathogenic strains of F. oxysporum is one of the main mechanisms in the suppression of Fusarium wilt (Alabouvette et al. 2009). The disease suppression of bacterium Erwinia amylovora causes fireblight by the closely related saprophytic species E. herbicola due to competition of the nutrient on the leaf surface. Competition between rhizosphere bacteria and Pythium ultimum, a common cause of seedling damping-off for the same carbon source, has resulted in an effective biological control of the latter organism in several crops. Germination of the conidia of Botrytis cinerea is inhibited by Pseudomonas species due to competition for amino acids. This mechanism may not be useful in suppressing biotrophs such as powdery mildews and rusts, because they do not require exogenous nutrients for host infection.

8.5.3.2 Plant Growth Promotion Through SAR and ISR

Chemical stimuli are produced by some biocontrol agents, i.e. non-pathogenic plant growth-promoting rhizobacteria (PGPR) and fungi (PGPF), or by soil- and plant-associated microbes. Such stimuli can either induce a sustained change in the plants which increase the capacity of tolerance to infection by pathogens or induce the local and/or systemic host defences of the whole plant against broad-spectrum pathogens. This phenomenon is known as induced resistance. Two types of induced resistance are distinguished in plants, systemic acquired resistance (SAR) and induced systemic resistance (ISR). The first of the two pathways is mediated by salicylic acid (SA) which is frequently produced after pathogen infection and induces the expression of pathogenesis-related (PR) proteins that include a variety of enzymes. The second method is mainly jasmonic acid (JA) and/or ethylene mediated following the applications of some nonpathogenic rhizobacteria (Fig. 8.2). The SAR-induced resistant was observed when Trichoderma harzianum was inoculated in roots and leaves of grapes, and it provides control of diseases caused by Botrytis cinerea from the site of application of T. harzianum (Deshmukh et al. 2006). It was found that the biocontrol agent P. fluorescens strain CHAO induces accumulation of salicylic acid and by inducing SAR-associated proteins confers systemic resistance to a vira1 pathogen in tobacco. Colonization of Glomus intraradices on the roots of Oryza sativa conferred resistance through induction of defence-related genes (Campos-Soriano et al. 2012). Penicillium simplicissimum enhanced the resistance of barley to Colletotrichum orbiculare by inducing salicylic acid accumulation, formation of active oxygen species, lignin deposition and activation of defence genes. In addition, Fusarium equiseti and Phoma sp. elicited Arabidopsis thaliana systemic resistance against Pseudomonas syringae pv. tomato and Pythium oligandrum against Ralstonia solanacearum. However, different ISR elicitors like secondary metabolites and proteins involved in mycoparasitism and antibiosis have also been identified. Secondary metabolites like trichokinin, alamethicin, harzianopyridone, harzianolide and 6-pentyl-α-pyrone have antagonist effects at high doses but in low doses act as ISR inducers. Expression of endochitinase Ech42 of Trichoderma atroviride was found to act as an ISR inducer in barley, resulting in an increased resistance to Fusarium sp. infection. Similarly, chitinase Chit42 of T. harzianum expression increased resistance in potato and tobacco against the foliar pathogens, B. cinerea, Alternaria solani and A. alternata, and soil-borne pathogen, Rhizoctonia solani.

Fig. 8.2
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Interaction within the plants, pathogenic microorganisms and biocontrol

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8.6 Genomic Approaches of Biocontrol Agent

Recent advances in molecular technologies have brought a revolution in microbial worlds and unzipped the immense diversity in microbial population helping scientific community to find out novel biocontrol agents (Kumar et al. 2014; Sharma et al. 2016). Utilizing bioinformatics tools and inexpensive sequencing techniques has led to the assembly of genomic data for microbial biocontrol agents and exploring the untapped and novel microbial isolates for important secondary metabolites and enzymes.

Seventy-eight percent of the genes functionally associated with antagonism were found to be distributed in Trichoderma species, described as the best fungal biocontrol agent till date. This is followed by Coniothyrium, Pythium and Clonostachys with 6, 5 and 4%, respectively. The way of antagonism is different in different microbes and sometimes depends on the pathogens. The genes associated with antagonism are diverse and involved in antibiosis, signalling, parasitism or transport. Of the identified genes, 44% are related to mycoparasitism, and 26% were for the antibiosis, whereas ISR-, signalling- and competition-related genes represent only 12, 11 and 5%, respectively. The role of different glucanases and chitinases during mycoparasitism is demonstrated with the functional characterization by gene-by-gene study in Trichoderma spp. (Daguerre et al. 2014). However, molecular mechanisms involved in the antagonism are not well known for all the cases. Now metatranscriptomic analyses appear as a more powerful tool as they provide generous information on different aspects of the antagonism allowing for comparison from the early stages to the later ones. The use of metatranscriptomic analyses prior to functional characterization seems to be the most sensible strategy. However, functional characterization is needed for verifying and ensuring the molecular mechanisms of antagonism.

The use of advanced molecular technique and genomic approaches in the identification of novel biocontrol agent is in its initial stages, but in the near future, latent biochemical products may arise as the key of antagonism of major phytopathogens as well as PGP in crops. For example, a total number of six genera of actinobacteria, viz. Corynebacterium, Mycobacterium, Arthrobacter, Frankia, Rhodococcus and Streptomyces, have been sequenced and analysed for potential secondary metabolite and gene diversity (James and William 2013).

8.7 Commercially Available Eco-friendly Biological Agents

Formulation of biopesticide based on a variety of microorganisms, e.g. nematodes, protozoa, fungi, bacteria, viruses, etc., is known as microbial pesticides or biocontrol agents. Predominantly five microbes, P. fluorescens, B. subtilis, Gliocladium spp., Verticillium lecanii and Trichoderma spp., are used for the purpose of commercial microbial pesticides. Several biopesticides are commercially available (Table 8.6) globally. However, in India only 35 microbes have been included in the Insecticides Act (1968) till now for commercial production of biocontrol agent, since the first biopesticide was notified in the Gazette of India dated 26 March 1999. In India, Singh (2006) identified novel Trichoderma strain with enhanced nematicidal, fungicidal and growth promotion property and used for developing biocontrol agent. The technology was transferred to Department of Agriculture, Government of UP, for its commercial production. Later on, this technology has also been transferred to Gujarat State Fertilizer and Chemicals Limited (GSFC), Gujarat Green Revolution Company Limited (GGRC) and Balaji Crop Care Pvt. Ltd., Hyderabad, for commercial production. The products ‘Sardar Eco Green Biofungicide’ and ‘TRICHA’ based on a potential strain of Trichoderma harzianum NBRI-1055 are in market for controlling phytopathogenic fungi. A talc-based formulation of Trichoderma viride strain 2953 has recently been transferred to Balaji Crop Care Pvt. Ltd., Hyderabad, for large-scale production.

Table 8.6 List of biocontrol products commercially available for the control of plant pathogens
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8.8 Socio-economic Impact, Ethical Issues Winding with the Biocontrol

Global assessment of biocontrol agents’ commercial availability in markets shows that the percentage of users and land have steadily increased since the late 1990s and the projected growth is continuing at a rate of 15.6% per year (Glare et al. 2012). Lehr (2010) reported that the global sales of biocontrol agents were estimated at US$ 396.48 million in 2003 and have continued to increase with projections to reach up to US$ 1.068 billion by 2010. With the successful implementation of biological agents in field for integrated plant disease management, demand for commercial biocontrol agent is increasing within the growers. There are approximately 225 microbial biocontrol agents which were manufactured in 30 member countries and registered by the Organization for Economic Development and Cooperation (Kabaluk and Gazdik 2007) for commercialization. The rest of the global market share is distributed among the countries within the Oceania at 20%, Latin and South American countries at 10% and less than 5% each accredited to Asia and India (Thakore 2006). The chances of future market expansion within the latter countries are likely to be variable. Organic and conventional producers are anticipating the use of alternative biocontrol products that pose a lower-risk exposure to human health than synthetic chemicals. Worldwide evolutionary exploration with the microbial products and the illustrating actions of the government personnel within the country, the growers and the industry have led to changes in strategy, management and research initiatives. On the other hand, legislation concurrently is supporting to make new policy that encouraged the registration of lower-risk pest control products.

Quality of the inoculants available in the market, however, needs to be carefully monitored as the formulation available in the market should contain sufficient population of the biocontrol microbes to produce an economic gain. Many countries such as the Netherlands, Thailand, Russia, France, Australia, Canada and the UK have regulations for inoculant quality which lead to improvements in the quality of commercial inoculants (Bashan et al. 2014). Canada and France have set norms that formulated products should have 106 viable cells per seed with no detectable contaminants (Catroux et al. 2001). However, that is not the case in developing countries as most of the inoculants produced are of poor or sub-optimal quality. Brockwell and Bottomley (1995) observed that most of the inoculants produced in the world is of relatively poor quality and 90% of all inoculants has no practical effect on the productivity of crops for which it is used. Further, the presence and nature of contaminants encountered in inoculants may represent a risk for humans, plants and for the environment, which remains to be assessed. Hence, quality of inoculants available in the market needs to be closely monitored, and make sure that farmers use the quality inoculants so that they will have trust on biocontrol.

8.9 Future Prospects for Biocontrol

In the past five decades, an increasing number of chemical fertilizer and biocidal molecules were the main cause for a substantial increase in crop production and quality. Because of environmental issues and health concerns, continuous and extensive use of those molecules has raised serious debate, and often various biological control methods based on natural pest and pathogen-suppressing organisms are being recommended as a substitute. Globally the registrations of microbial biocontrol agents are increasing significantly. The changes in legislation in the country level, development of new policies and management structures to address the reduction of chemical uses are the expanding scope of biocontrol agents. On the other hand, the researchers worldwide have been supported to discover new biocontrol agents to reinforce for entering in the industry. Being practical, at present biocontrol agents are not comparable to chemical pesticides in meeting efficacy which is needed for market expectations, but they still have a promising future if knowledge and methods of various fields of biotechnology are utilized. The availability of recent molecular technologies has significantly facilitated for surveying and identification of candidate agents, and helped to interpret the modes of action after field applications. These new technologies like proteomics and functional genomics will give new possibilities for insights in ecological constraints and will help to see hitherto unseen possibilities to determine the physiological status and expression of crucial genes present within the biocontrol agents during mass production, formulation, storage and application.