Despite all abovementioned positive attributes of EPNs and their mutualistic partner against the insect pests as a third partner, certain challenges are still hindering them from reaching broader biopesticides markets. Main issues that negatively affect wider applications of EPNs are high product price, limited product demand, insufficient knowledge of the end user, and low efficiency (Askary et al. 2017). Since these issues are interrelated, solving one of them can positively affect another issue (Abd-Elgawad 2019). For example, low efficiency may result in limited product demand. Also, effective-cost application can have an impact on EPN efficiency. Crucial scopes that can face such challenges and attain substantial progress in broader EPN marketing comprise EPN strain/species enhancement, improvement of production, formulation and application technology especially toward aboveground applications, conservation biocontrol, and other diverse factors. Most of these challenges have been addressed (Shapiro-Ilan et al. 2020). However, in the present review, we have tried to address them with an aim to solve such issues in various ways that grasp their effective approaches, identify research priorities, and highlight better techniques.
EPN strain/species enhancement
A native EPN species is more adaptive to local environmental conditions and has been found very successful in controlling local insect pests (Bhat et al. 2019). However, it is also well-established that EPN efficacy can be enhanced via discovery of new strain/species or nematode breeding/genetic engineering. The latter can be attained through direct selection, hybridization, or genetic manipulation (Baiocchi et al. 2017). On the other hand, if EPN species/strains that are already available do not meet efficacy requirements, a strict method for amelioration is to simply find a superior nematode strain or species. This is generally fulfilled by carrying out surveys, followed by Koch’s postulates, and subsequent virulence screening of EPNs that have been isolated in comparison to existing stocks (Shapiro-Ilan et al. 2020). Admittedly, the significant strain improvements made to EPN biocontrol have historically come through the discovery of new species/strains and then by classical genetics. Therefore, a few aspects to optimize EPN sampling and get more species/strains are addressed herein.
The extreme diversity of EPN sampling makes any generalization from a given case-study difficult. However, based on what has already been done in many related EPN surveys, it is clear that the percentage of samples positive for EPN is relatively low (Glazer 2015). This general decline in the positive samples demonstrates the desperate need to increase the number of nematode-positive samples whenever possible, and also by exploring untouched geographical areas and climatic conditions (Bhat et al. 2020). Hence, a hypothesis of rational sampling was recently tested (Abd-Elgawad 2020). It is based on combining four factors, i.e., advantageous sampling approach, site and time targeting, and utilizing repeated extraction cycles. In this respect, systematic and stratified random sampling from weed-infested soil under tree canopy during an outburst season of insect reproduction was adopted, and multiple Galleria-baiting technique was carried out. Consequently, as high as 61.7% of soil samples were EPN-positive. Collectively, these factors could successfully be used to significantly optimize sampling results (Abd-Elgawad 2020). Such a rational sampling could detect more EPN isolates from citrus orchards and allow applying different indices of dispersion to study their spatial distribution pattern. In this respect, contrary to the commonly used single Galleria cycle to check for nematode presence, multiple cycles of the Galleria-baiting with adequate last instar G. mellonella larvae per sample/cup were utilized in each cycle. The technique demonstrates that a single extraction cycle of the Galleria-bait method, used in many other surveys, is often insufficient for determining the presence of nematodes in particular soil samples or for extracting all the nematodes from soil samples. More importantly, it is expected that the data obtained in this manner can comprise EPN species/strains with differential pathogenicity among them. Clearly, this technique should be utilized to obtain EPN with high recovery value and possible differential pathogenicity.
The size of sampling unit
Nematologists have been using various sizes of sampling units. Obviously, if the area of a sampling unit is much larger or much smaller than the average size of EPN-infective juvenile aggregations and their clumps are regularly or randomly distributed; then, their population pattern is apparently random; factual non-randomness is not detected. Thus, when the size of the soil sampling unit steadily increase, the apparent EPN dispersion of a contagious population becomes random, contagious, and finally regular (Abd-Elgawad 2019). A standard size of sampling units is needed for accurate EPN-distribution which would help in grasping their best application patterns.
Improvement of production, formulation, and application technology
The production, formulation, and application technology represents one of the headlines that can significantly contribute in solving the main impediments for broader commercialization of EPNs (Shapiro-Ilan et al. 2020). Merits and demerits of each EPN production technology have been demonstrated (e.g., Shapiro-Ilan et al. 2014b). Issues of production concern comprise capital outlay, labor, technical expertise, nematode quality, and cost efficiency. In valuing the advantages of the methods among these issues, in vitro liquid culture and in vivo production are the extremes, and in vitro solid culture is intermediate between them. For instance, the level of capital outlay, production volume, and expertise requirement is the lowest for in vivo production (Shapiro-Ilan et al. 2014b). The major economic limitations for in vivo production are the cost of insects and labor. These issues have been addressed by mechanizing the process comprising insect production, inoculation, harvest, and packaging (Shapiro-Ilan et al. 2020). Moreover, adding host-cadaver macerate or EPN pheromones to EPN products could enhance desired behavior such as increased EPN dispersal for better pest control. On the other hand, a merit to in vivo set is the ease of adapting the process to new/different EPN species. Generally, the process can remain the same except for some slight modifications (e.g., temperature regimes). Since EPN products remain cost prohibitive in many markets, additional advances to increase production efficiency and to reduce costs are required. Hence, scaling up of in vivo method was suggested in developing countries like Egypt (Abd-Elgawad 2017). Basically, a small harvester and separator have been built for in vivo production. The production steps are infection, incubation, harvest, separation, and clean up (Askary and Ahmad 2017). Improvements are being made continuously in inoculation, harvest techniques, post-harvest nematode concentration, and cleaning of the system (Abd-Elgawad 2017). Each step has specific requirements, which must be optimized separately to ensure adaptation of a specific nematode to its best matching insect host. All parameters in the abovementioned 5 steps may be further optimized as a cottage industry. In this context, harvester dimensions and harvesting parameters require optimization for each specific host/nematode system for scaling up. Admittedly, such a local nematode production system can eliminate or reduce transport, packaging, formulation, and storage costs while providing fresh, locally adapted and effective strains of EPNs. Thus, this production system offers proper matching of nematode-host and ensures suitable affordability especially in developing countries for specific indigenous strain rather than “one size fits all” approach used elsewhere (Abd-Elgawad 2017). Factually, the system suits regions which lack the capital and expertise to develop a biopesticide production industry. The keys should be local small-batch custom production and quick turnover which can provide high-quality products (Shapiro-Ilan et al. 2020).
The highest production volume and least costs of EPNs are attained via liquid culture. Yet, lower EPN quality in terms of reduced host-finding, virulence, or longevity in liquid culture relative to in vivo production has been reported, though in other cases no differences were detected due to culture method (Shapiro-Ilan et al. 2020). Admittedly, media components and bioreactor parameters should be greatly considered for fine tuning because they can significantly affect EPN yields and quality in fermentation process of in vitro production.
Formulations of EPNs should be enhanced for easier handling, effective application, increased environmental persistence, and longer stability and shelf life. Formulations for aqueous EPN application vary widely and can be applied efficiently with practically all types of cultural application equipment. Nevertheless, the used equipment should properly comply with the cropping ecosystem. Therefore, a variety of parameters should be optimized in each case including nozzle/dripper type, volume, agitation, pressure and recycling time, environmental conditions, and EPN distribution pattern. In all cases, suitable agitation during application is critical. Other methods of formulation and application that could conceivably be expanded include baits, applying the nematodes in their infected hosts, microjet irrigation systems, trunk-sprayers, and subsurface injection (Shapiro-Ilan et al. 2020).
Foliage pests represent difficult targets for aqueous application of EPNs. Progress in this area was recently updated and should be exploited (Shapiro-Ilan et al. 2020). For instance, a surfactant and polymer formulation has been utilized to increase leaf coverage and improve aboveground application efficacy. Application of a sprayable fire gel could better control the lesser peach tree borer, Synanthedon pictipes. Other formulations developed for aboveground application include those based on chitosan, wood flour foam, or other adjuvants. Improved application equipment and advanced application techniques are being applied to enhance EPN efficacy to control the scarab beetle, Temnorhynchus baal, in strawberry fields. For this purpose, a device is used to pump the nematode suspension almost evenly at all drippers during trickle irrigation. Local heterorhabditid nematode strains that best match T. baal control (Shehata et al. 2019) could further optimize the techniques. On the other hand, splitting nematode applications in two at half rates approximately 5–10 days apart should be examined. Generally, more splitting may be done with an end in view that the applications synchronize with the most EPN-susceptible stage(s) of insect pest(s). These highly susceptible stages lend themselves the most for control by EPNs (Koppenhöfer et al. 2020b).
Biological pest control is often based on inundative release of mass-produced EPNs. However, weak points in this technique are pest control efficacy and cost of repeated application, often attributed to the poor establishment of EPNs. Additional approaches that decrease exposure to harmful biotic or abiotic stressors or increase EPN persistence, reproduction, or virulence will expand biocontrol efficacy (Abd-Elgawad 2017; Shapiro-Ilan et al. 2020). For example, EPN persistence can be improved through making the soil environment more conducive to EPN survival such as setting adequate soil pH or adding suitable soil amendments, e.g., mulch or crop residues (Campos-Herrera et al. 2019).
Moreover, EPNs utilize phased infectivity to bridge periods of time of environmental stress and lack of host availability. This supports the idea that these survival mechanisms are genetically encoded and are easily lost under conditions of continuous rearing practiced by some commercial producers. So, more emphasis should be placed on the long-term establishment of the EPN in the soil profile for expanded pest suppression through pest recycling and the selection of an EPN population which retains the genetic coding for extended persistence under low host density and unfavorable environmental conditions. In order for EPNs to retain their adaptation for field survival, several approaches can be utilized to help to keep such genes in laboratory culture. These include re-isolation from the field, establishing “wild populations” in easily accessible areas, and rational laboratory culturing to possess persistence (Shields 2015). Additionally, factors which affect EPN persistence and recycling levels such as host density, nematode species/strain, soil type, and ground cover should be considered (Shapiro-Ilan et al. 2020).
Other diverse factors to widen EPN utilization
Scientists should further advance biopesticides to occupy new positions. For example, IPM of Caribfly, Anastrepha suspensa, to boost guava production was established on adequate identification of conditions and practices that enhance effective application of EPNs using a cost-effective technique (Heve et al. 2018). Another avenue to wider utilization of EPNs is to control veterinary pests such as the gray flesh flies Parasarcophaga aegyptiaca as one of the external parasites, an important pest with wide distribution and significant role in causing serious diseases such as myiasis, which can invade various tissues of man and animals. Steinernema riobrave and H. floridensis might also be used as part of an integrated approach to control Yucatan strain of Rhipicephalus microplus (Canestrini) resistant to various classes of acaricides (Singh et al. 2019). These relatively neglected specialties need further applied research to maintain animal health using properly safe and environmentally friendly approaches. Moreover, applying the nematode-symbiotic bacteria or their byproducts to control arthropod pests (Abd-Elgawad 2017) or plant pathogens (Shapiro-Ilan et al. 2014a) have shown positive results that require following up to develop the methodology. Also, metabolites derived from Xenorhabdus and Photorhabdus bacteria have been recorded to suppress various serious plant pathogens in genera including Armillaria, Monilinia, Phytophthora, and Venturia (Shapiro-Ilan et al. 2020).
Other economical concepts may contribute to cut the costs short. The EPN producer can act at the same time as the distributor. Furthermore, the producer may nominate some of its employees with experience in EPN application (Abd-Elgawad 2017). Factually, those experts can recognize EPN issues such as the viability of IJs, contamination, and nematode fate/persistence. Admittedly, being responsible for producing, distributing, applying, and following up EPNs all by the same company will reduce costs and increase profit margins. Such a multiple service offered by the producer seems more attractive to the growers than an employee/company with a single job.
Clearly, in order to optimize their benefits, EPNs should operate in IPM programs in ways that make them complimentary or superior to chemical pest management and/or other agricultural inputs (Stevens and Lewis 2017). So, full and relevant lists of bio-insecticides with EPN species/strains that can act synergistically or additively with other agricultural inputs should be available. Strictly speaking, such lists should satisfy challenges to identify and broadly disseminate conditions under which the EPNs constitute a cost-effective, value-added approach to IPM. All the abovementioned lines of thinking represent current challenges that are substantial to seize more significant share of EPNs in the pesticide markets.