Utilization of biopesticides as sustainable solutions for management of pests in legume crops: achievements and prospects

The legume family is represented by nearly 400 genera and 10,000 species which harbors various pulse crops including chickpea (Cicer arietinum L.), pigeon pea (Cajanus cajan L.), field pea (Pisum sativum L.), lentil (Lens esculenta L.), green gram (Vigna radiata L.), black gram (Vigna mungoL.), cowpea (Vigna ungiculata L.), faba bean (Vicia faba), lathyrus (Lathyrus sativus), and rajmash (Phaseolus vulgaris L.), which are grown for both seeds and grains in different parts of the world (http://www.botany.hawaii.edu/faculty/carr/fab.htm). Capable of releasing nitrogen into the soil, these crops are cultivated to obtain dry seeds that are rich in proteins and important minerals and micronutrients (Bohra et al., 2015, Nene, 2006). Frequent attacks by pathogens, insect pests, and nematodes cause impairments to the plants at various stages of crop growth. The average annual loss in legumes due to pests and pathogens was estimated to be up to 20% (Dhaliwal et al., 2010). In other words, the efforts dedicated to protect the crop may witness substantial increment in food legume production. Up to 100% losses have been reported in various legume crops in Asia and Africa in case of the conditions that favor diseases and pests (Vijay et al., 2015). In India, a considerable extent of yield in pigeon pea and chickpea is lost due to pod borer [Helicoverpa armigera (Hubner)]. In pigeon pea, spotted pod borer [Maruca vitrata (Fabricius)] remains the second most important pest causing up to 84% reduction in crop yield, amounting to a loss of nearly US$30 million in monitory terms (Margam et al., 2011). Similarly, more than 40% damage to the pods in pigeon pea is caused by pod fly [Melanagromyza obtusa (Malloch)] (Singh et al., 2013). The other pathogens that plague legume production include wilt, dry root rot, Phytophthora blight, collar rot, stem/white rot, Macrophomina blight, and yellow vein mosaic virus (YVMV). Losses caused by wilt in legumes may vary from 0 to 100% depending on the crop stage (Pande et al., 2013).

Besides being an expensive affair, the application of hazardous pesticides has detrimental effects on the environment, negatively impacting upon soil fertility and soil microfauna. Also, extended use of chemical pesticides not only often causes to the development of resistance (to pesticides) in insect pests, pathogens, and nematodes but also leads to carcinogenic, teratogenic, and mutagenic effects in human and animals as well. A range of strategies aiming to contain various pests and pathogens of grain legumes are currently available, which includes development of resistant varieties, genetically engineered plants, and use of pesticides and cultural and physical methods.

This review offers an update on the current status of biological control applications especially microbial organisms and their formulations in crops with an emphasis on legume crops. Future prospects of sustainable use of biological control agents (BCAs) to improve performance of legume crops are also discussed.

An increase in selection pressure consequent to indiscriminate application of chemical pesticides leads to emergence of pesticide resistance. In such conditions, alternate options of pest/disease management are much sought. During the past two decades, an urgent need was realized for management strategies that are safe vis-a-vis the environment and the land. Farmers are shifting towards eco-friendly technology for the management of pests, i.e., BCAs or BCA-based formulations, referred to as biopesticides. Examples include Trichoderma spp., Pseudomonas spp., Bacillus spp., Agrobacterium radiobacter, nonpathogenic Fusarium spp., Coniothyrium spp. and Aspergillus niger, Bacillus thuringiensis (Bt), Metarhizium spp., Beauveria bassiana, and nuclear polyhedrosis virus (NPVs), which are popularly used in plant protection (Keswani et al. 2015; Mishra et al., 2015). According to a recent report (NAAS, 2013), nearly 1400 BCA products were sold and 175 biopesticide active ingredients and 700 products were registered worldwide for their commercialization. In India, only 15 biopesticides have been registered so far under the Insecticides Act 1968 (Table 1). A growing body of research articles report on the identification and efficacy of different BCAs against a number of pests and pathogens; however, their slow embrace is evident from the fact that only 2% biopesticides are currently used for crop protection worldwide. On the positive side, the usage of BCAs has witnessed an increasing trend (Ranga Rao et al., 2007; Singh et al., 2012). The rise is notably high at international level. Though literature on BCAs is growing worldwide (Fig. 1), the filing of patents on BCA technology is not in sync with the number of publications.

S. no.	Name of the biopesticide	Use for
1.	Bacillus thuringiensis var. israelensis	Diamondback moths
2.	Bacillus thuringiensis var. kurstaki	Diamondback moths
3.	Bacillus thuringiensis var. galleriae	Helicoverpa armigera
4.	Bacillus sphaericus	Diamondback moths
5.	B. firmus	Diamondback moths
6.	Trichoderma viride	Root rots and wilts
7.	Trichoderma harzianum	Root rots and wilts
8.	Pseudomonas fluorescens	Bacterial and fungal pathogen
9.	Beauveria bassiana	Mango hoppers and mealy bugs and coffee pod borer
10.	NPV of Helicoverpa armigera	Helicoverpa on chickpea
11.	NPV of Spodoptera litura	Spodoptera litura
12.	Neem based biopesticides	Insect white fly
13.	Cymbopogon	Insect
14.	H. bacteriophora	Borers
15.	Trichogramma parasitoid	Sugarcane borers

Development of a stable and economically viable bioformulation remains central to biological control. Of the various carrier-based formulations available worldwide, alginate pellet- and talc-based formulations of BCAs have emerged as the most important carrier for the application in the management of crop diseases (Lewis et al., 1985). During initial phase of biological control development, it was viewed as an interaction of a single BCA with a pathogen in the rhizosphere (Wilson and Backman, 1999). Still, the majority of the reports on BCA document one target disease. This, however, has yielded inconsistent performance given that a single agent might not remain active in all soil conditions. Integrating microbes in a biological control formulation may serve as more effective management strategy in longer term (Duffy and Weller, 1995). Few biological control formulations based on combination of two or more BCAs are available in the market. Notwithstanding their significant role in sustainable agriculture, consortia-based formulations have not received adequate attention. Production of BCAs for sustainable agriculture relies on cost-effectiveness of the procedure and viability of a potential strain. Further, enabling mass production with high level of microbial count and viability also assumes greater significance.

BCAs interact with a variety of factors to control diseases and insect pests of crop plants. Therefore, an improved understanding of the BCA mechanisms can facilitate optimization of control in addition to permitting search for more efficient strains. Also, knowledge about the mechanisms underlying BCA-driven pest management may allow us to select and construct the BCAs with greater effectiveness. Studied by different researchers (Adams, 1990; Lo et al., 1998), these mechanisms encompass (i) antibiosis, (ii) competition, (iii) mycoparasitism/hyperparasitism, and (iv) induced resistance.

Soils and plant surfaces constitute nutrient limited environment, thereby putting pressure on a microbe to compete for the available nutrients (Pal and Gardener, 2006). Both BCAs and the pests compete with each another for nutrients and space to establish in the environment. This process involves an indirect interaction between BCAs and pathogens, eventually resulting in pathogen exclusion by means of diminishing food base and physical occupation of site (Lorito et al., 1994). Also, BCAs compete for the essential micronutrients such as iron (Fe) and manganese (Mn) especially in highly oxidized and aerated soils. However, the competition for micronutrients exists due to the BCAs having more efficient uptake system for the substances than the pests. While competing with pathogen for physical occupation of site, BCAs reduce or delay the root colonization by the pathogen. Owing to a rapid colonization of root surface of plants by the BCAs, the failure of pests and pathogens to establish on host helps lessening the severity of diseases that affect roots of plants.

Fungi that are parasitic on other fungi are usually referred to as mycoparasites (Baker and Cook, 1974). Parasitism requires the host fungus to be recognized by the mycoparasite followed by production of hydrolytic enzymes and antibiotics. Direct parasitism or lysis of the mycelium of a fungal pathogen by mycoparasitic fungus is known as hyperparasitism. Weindling (1932) observed Trichoderma lignorum parasitizing the hyphae of Rhizoctonia solani. The mycoparasitism of Trichoderma species enabled by appressoria-like structures to penetrate the target fungus cell wall (Chet, 1987) to contributes towards the development of biological control strategies (Harman et al., 2004). A range of pests with different kinds of parasitisms were observed viz., simple-, super-, multiple-, hyper-, auto-, and cleptoparasitisms. Larval parasitoid, Campoletis chlorideae, shows simple parasitism on H. armigera (Pillai et al., 2016). Similarly, Maruca obtusa is parasitized by Euderus lividus at larval-pupal stages (Moudgal et al., 2005).

Induced resistance varying from local to systemic in nature is the most indirect form of antagonism. With regard to systemic acquired resistance, salicylic acid (SA) and non-expresser of pathogenesis-related genes 1 (NPR1) are key players. Trichoderma harzianum when inoculated into the root system or on leaves of crops to manage the Botrytis cinerea on leaves spatially separated from the site of application of the BCA (Desmukh et al., 2006). While inducing resistance in plants, different kinds of compounds are released by Trichoderma spp. into the zone where the interaction occurs. Proteins with enzymatic or other activity constitute the foremost class in this regard. For instance, fungal proteins such as xylanase, cellulases, and swollenins are secreted by Trichoderma spp. (Martinez et al., 2001). Similarly, Trichoderma endochitinase can also enhance defense, probably through induction of plant defense-related proteins.

Physical thickening of cell walls in plants caused by BCAs contributes to induced resistance via diverse mechanisms that involve lignification, callose deposition and accumulation of antimicrobial low-molecular-weight substances (e.g., phytoalexins), and synthesis of chitinases, glucanases, peroxidases, and other pathogenesis-related (PR) proteins. Several endophytes such as Bacillus and Paenibacillus have been reported to inhibit soil-borne phytopathogens of pulse crops by production of siderophore, hydrolytic enzymes, antibiotics, and hydrogen cyanide (Senthilkumar et al., 2009). Florescent pseudomonad bacterium produces phenazine (Toohay et al., 1965), phloroglucinol (Howell and Stipanovic, 1980), siderophores (Sakthivel et al., 1986), and pyrrolnitrin (Burkhead and Geoghegan, 1994).

Considerable efforts have been made to manage important pests of legumes by incorporating BCA-colonized natural substrates into the rhizosphere. Bacterial endophytes like Bacillus, Paenibacillus, and Pseudomonas show antifungal activity against major pathogens like Rhizoctonia solani (Kuhn.), Rhizoctonia bataticola (Taub.) Butler, Fusarium udum Butler., F. oxysporum f. sp. cireri (Padwick) Snyd. & Hans., and Sclerotium rolfsii Sacc. infecting pulse crops (Senthilkumar et al., 2009). Among the most pronounced antagonistic fungi, Trichoderma species have been extensively investigated as potential BCAs in pulse-based ecosystem. These are demonstrated to be effective against wilt and root rot (root, collar, and stem) diseases conditioned by different Fusarium spp., R. solani, R. bataticola, Sclerotium rolfsii, Sclerotinia sclerotiorum (Lib.) de Bary, Phytophthora drechsleri f. sp. cajani Tucker, and Pythium spp. in different pulses and other field crops (Chaudhary et al., 2004). The fungi belonging tohyphomycetes such as Metarhizium anisopliae, Beauvaria bassiana, Nomuraea, Verticillium, and Paecilomyces have been employed as biopesticides in legume crops. In soybean, the application of Paecilomyces lilacinus, Pochonia chlamydosporia, Aspergillus nidulans var. dentatus, and T. harzianum at 2 g/kg soil can effectively reduce nematode (Rotylenchus reniform) population along with promoting plant growth (Gurjar et al., 2012; Singh and Prasad, 2014). Apart from controlling diseases in pulses, BCAs are also reported to enhance nodulation.

Researchers at Indian Institute of Pulses Research (IIPR), India, has identified several native potential strains of Trichoderma spp. (T. harzianum, T. asperellum, T. longibrachiatum, and T. reesei) and plant growth-promoting rhizobacteria (PGPRs) isolated from rhizospheres in major pulse-growing areas in India and evaluated these for their antagonistic potential against a variety of pathogens (Fig. 2a, b). Accordingly, mass production technology has been developed and popularized among the pulse-growing farmers in different agro-ecosystems (Chaudhary et al., 2004; Mishra et al., 2015, 2016).

BCAs are formulated by several means including dry formulations such as dusts, granules, and microgranules; seed dressing formulations such as powder for seed dressing; dry formulations for dilution in water including dispersible granules and wettable powders; and liquid formulations for dilution in water such as emulsions and suspension concentrates (Knowles, 2005, 2006). Globally, biopesticides are currently available in the market as wettable powder, liquid, and granular formulations (Singh et al., 2012, 2014). In pulses, researchers at IIPR employed 2% wettable powder formulations for seed treatment and soil application (Chaudhary et al., 2004).

Biological control formulations and their consortia are delivered through several means relying primarily on survival nature and mode of infection of the pathogen. These diverse modes of application include seed treatment, soil/foliar application, or through workable combination of different methods. A brief description about these methods is provided here.

A series of experimentation and concerted efforts has led to commercialization of biological control products. The implementation of biological control products in managing plant diseases and insect pests is gaining ground. However, these still represent only 1% of the total plant protection products in comparison to fungicides accounting for 15% of total chemicals used in agriculture (Fravel, 2005). On the plus side, recent entry of several small- and large-scale entrepreneurs into commercial production of BCAs can be viewed as expanding scope of these in the world market.

Pulses are grown mostly by the resource poor and small-scale farmers. Cultivation of these crops mostly in harsh and unpredictable environments renders these prone to attack by a wide range of pests and pathogens at various stages of the crops. The use of chemical pesticides as a control measure is not deemed environmentally safe, and at the same time, affordability of these chemicals by the marginal farmers also remains limited. Therefore, employing BCAs in pulses is safer in view of the fact that these crops are consumed directly as seeds.

In current agricultural scenario, the use of BCAs and their formulations is of utmost important in grain legumes for biotic stress management. However, their full potential remains to be seen because of the limited attention these have received so far in terms of commercial scale production of BCAs and formulations. In addition, commercially available formulations have not been accessible by the pulse growers. The major stumbling block impeding the progress in this regard is inadequate awareness about its application and advantages. This underlines the scope of making these BCA-based formulations and techniques popular at farmers’ field.

The quality of BCAs and their different formulations and efficiency of the antagonist strain are utmost important. It is measured in terms of inoculum potential (propagules/unit weight and the aggressiveness), shelf life, and ease of application and purity of the formulation. Generally, the biological control formulations available in the market are of very poor quality with meager shelf life (Singh et al., 2012), which demand strict regulation through quality control agencies. Also, biological control agents despite of performing well under controlled environments may fail to yield the same results in the field conditions. Further, storage and marketing of microbial biopesticides is another issue that greatly limits the extensive use of BCAs in today’s agriculture. It is a high time to educate the dealers and users (cultivators) on importance of appropriate storage conditions, shelf life, and mode of action of BCAs.

The pathogenic variability reported in soil-borne phytopathogens (especially in Fusarium spp. causing wilt) in many pulse crops poses a serious challenge aiming to develop potential BCAs. Further, before selecting BCA for developing its formulations, it should be thoroughly screened against a large number of phytopathogens or a large number of isolates/strains/races of phytopathogens.

Increasing development and deployment of such cost-efficient and environment-friendly techniques are the methods-of-choice to check the rising pest and pathogen problem and to sustain production of pulse crops in the face of growing agricultural adversities.

Grain legumes remain important crops not only from nutritional security perspective, but also for their contribution to the health and fertility of the soil. Cultivation of these crops faces a number of biotic and abiotic stresses, reflected in the form of deteriorated yield and quality. The use of chemicals, as a control measure, is not commercially viable. More importantly, it has considerable damaging consequences on the environment, soil and microorganisms. In this respect, BCAs have shown immense potential, thus increasingly playing a significant role in agricultural production systems. Management of pests and pathogens of legumes with BCAs offers several benefits over the traditional protection measures. We advocate application of BCAs in grain legumes crops in view of their potential to deliver sustained gains in crop productivity.