Biological control can be defined as the use of one type of organism to reduce the population density of another. Biological control has been used for approximately two millennia, and has been widely used in pest management since the end of the nineteenth century . The following types of biological control can be distinguished: natural, conservative, inoculative (or classical), and augmentative. Natural biological control involves the reduction of pest organisms by their natural enemies and has been occurring since the evolution of the first terrestrial ecosystems, 500 million of years ago . It takes place in all of the world’s ecosystems without any human intervention, and, in economic terms, is the greatest contribution of biological control to agriculture . Conservation biological control consists of human actions that protect and stimulate the performance of naturally occurring enemies . In inoculative biological control, natural enemies are collected in an exploration area (usually the area of origin of the pest) and then released in new areas where the pest was accidentally introduced. In augmentative biological natural control, natural enemies are mass-reared in biofactories for release in large numbers to obtain immediate pest control .
2.1.1. Entomophagous agents: parasitoids and predators
Parasitoids can be defined as insects that are only parasitic in their immature stages, kill their host in the process of development, and have free-living adults that do not move their hosts to nests or hideouts .
All stages of the diamondback moth are attacked by numerous parasitoids and predators, with parasitoids being the more widely studied. Over 90 parasitoid species attack the diamondback moth . Egg parasitoids belonging to the polyphagous genera Trichogramma and Trichogrammatoidea contribute little to natural control and require frequent mass releases. Larval parasitoids are the most predominant and effective. Many of the effective larval parasitoids belong to two major genera, Diadegma and Cotesia; a few Diadromus spp., most of which are pupal parasitoids, also exercise significant control . The majority of these species come from Europe where the diamondback moth is believed to have originated . In countries near Brazil, such as Argentina, P. xylostella larval parasitoids collected in the field include the species Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae), Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae), and C. plutellae (Kurdjumov) (Hymenoptera: Braconidae) .
Seven species of parasitoids were observed in a P. xylostella population on cabbage crops in the Brasilia region of Brazil, with the two most common species being Diadegma liontiniae (Brethes) (Hymenoptera: Ichneumonidae) and Apanteles piceotrichosus (Blanchard) (Hymenoptera: Braconidae). Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae) and Actia sp., previously more abundant, had become very minor parasitoids. Six species of hyperparasitoids emerged from D. liontiniae and A. piceotrichosus, showing a high diversity of natural enemies in this region of recent colonization by P. xylostella .
In organically farmed kale in Pernambuco, Brazil, seven natural enemies of P. xylostella were observed: three parasitoids, C. plutellae Kurdjumov (Hymenoptera: Braconidae), Conura pseudofulvovariegata (Becker) (Hymenoptera: Chalcididae) and Tetrastichus howardi (Olliff) (Hymenoptera: Eulophidae), and four predators, Cheiracanthium inclusum (Hentz) (Araneae: Miturgidae), Pheidole sp. Westwood (Hymenoptera: Formicidae), and nymphs and adults of Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) .
Several studies have been conducted in Brazil to examine whether these entomophagous agents of the diamondback moth could be used as a biological control for this pest in crucifer crops.
Parasitoids of the genus Trichogramma are among the entomophagous agents that have already been studied for P. xylostella. The species T. pretiosum Riley (Hymenoptera: Trichogrammatidae), Tp8 strain, can parasitize approximately 15 P. xylostella eggs in the first or second generation when reared in this host under laboratory conditions, with 100% emergence, and 10 to 11 days for adult emergence . Eggs of two P. xylostella populations, one reared on kale leaves and the other on broccoli leaves, were exposed to the T. pretiosum Tp8 strain, and the number of parasitized eggs was 5.8–9.4 on kale and 3.2–8.4 on broccoli . Furthermore, the optimal way to mass rear this parasitoid in the laboratory is to use eggs glued to blue, green, or white colored cards .
The impact on non-target species, particularly Trichogramma, of insecticides for P. xylostella control should be analyzed because some are toxic to these parasitoids in crucifers. Endosulfan and etofenprox, classified as class-4 toxic products, are extremely toxic to the parasitoids. Triflumuron, classified as a non-toxic product, is selective for these parasitoids in the eggs of P. xylostella . The combination of chemicals or natural insecticidal products from vegetables with certain cultivars of crucifers enables more effective management of the diamondback moth, particularly in the case of the interaction between pyroligneous extract and cabbage. However, the interaction among cultivars and products can be detrimental to the effectiveness of T. pretiosum and T. exiguum, and thus requires a careful evaluation to minimize the impact on these natural enemies . Bioinsecticides based on B. thuringiensis for controlling P. xylostella can influence the parasitoid T. pretiosum in the moth’s eggs. The application of isolates of B. thuringiensis on P. xylostella larvae influenced the parasitism of T. pretiosum in eggs of subsequent pest generations .
Another parasitoid of P. xylostella larvae, which has been studied in Brazil, is O. sokolowskii. The duration of the immature stage of these parasitoids can range from 12.9 to 31.6 days at 28 and 18°C, respectively, and the number of adults emerged per pupa of P. xylostella varies between 7.3 and 12, with a sex ratio of between 0.86 and 0.91 . During a year, the number of generations of O. sokolowskii is always higher than that of P. xylostella, suggesting that O. sokolowskii could develop up to 24 generations per year while the diamondback moth could reach 20 annual generations . Furthermore, the O. sokolowskii parasitoid is able to disperse and parasitize P. xylostella throughout a kale field up to 24 meters from the release point .
Another larval parasitoid studied in Brazil for P. xylostella is A. piceotrichosus, which was collected in the Rio Grande do Sul State. Its immature stage was observed to last 14.6 to 15.5 days and its adult longevity was found to be 12.7 to 13.4 days .
Among the stink bug predators, P. nigrispinus has great potential for use in P. xylostella control. P. nigrispinus has been reported preying on P. xylostella in crucifer crops , and, furthermore, this predator consumed on average 10.9 larvae or 5.5 pupae in 24 h . Adults of Orius insidiosus (Say) (Hemiptera: Anthocoridae) has been reported consuming 5.9 diamondback moth eggs in 24 h .
2.1.2. Entomopathogens: Bacteria
The occurrence of P. xylostella populations of resistant to certain active ingredients, like synthetic and biological insecticides, has caused a considerable increase in research directed at developing tactics for Integrated Pest Control based on economic, social, and ecological parameters [21,45-47).
Recent studies on control strategies and population reduction of P. xylostella using microorganisms has been increasingly cited in the scientific community, with emphasis on the entomopathogenic bacterium B. thuringiensis Berliner (1911) [48-51,39].
This entomopathogen can be easily found in different environments [52,53], and it is characterized by a variety of strains, each forming one or more protein crystals (Cry) and cytolytic toxins  that have insecticidal activity and determined its efficiency as a control on certain agricultural pests. Another type of insecticidal protein that can be synthesized by some strains of B. thuringiensis is “Vegetative Insecticidal Proteins” (Vip), whose insecticide action spectrum operates in different insect species .
A long history of intensive research has established that their toxic effect is due primarily to their ability to form pores in the plasma membrane of the midgut epithelial cells of susceptible insects [56,57]. The presently available information still supports the notion that B. thuringiensis Cry toxins act by forming pores, but most events leading to their formation, following binding of the activated toxins to their receptors, remain relatively poorly understood .
Strains of B. thuringiensis can produce from one to five toxins that represent a large variability in toxicity and interfere in the expression levels and the spectrum control of insects, and differ in their specificity to certain species . For example, the Cry proteins are show high toxicity to insects of the orders Lepidoptera, Coleoptera, Hymenoptera, Diptera, Orthoptera, and Mallophaga, and to other organisms such as nematodes and mites [60,54,61].
Among the different protein crystals identified in insect control, 59 toxins were tested against 71 Lepidoptera species . The broadest range of toxins was tested against P. xylostella (43 toxin types), which was one of only 12 species that were tested against 15 toxins or more .
In Brazil, P. xylostella is controlled using entomopathogenic bacteria in phytosanitary applications of formulation products properly registered for a particular crop, most commonly biological products containing B. thuringiensis var. kurstaki, which expresses Cry1Aa, Cry1Ab, and Cry1Ac toxins  (Table 1).
Commercial products based on Bacillus thuringiensis recommended for controlling the population of Plutella xylostella in different brassica crops.
Source: . WP = Wettable Powder; WG = Water-Dispersible Granules; SC = Suspension concentrate.
However, the low variability in the number of toxins related to formulated biological products, combined with a high number of applications in the field, puts selection pressure on the population of P. xylostella and, consequently, expression of resistance of this pest to protein crystals has been observed since the 1990s [20,24].
The development of resistance in P. xylostella populations is related to the binding of these toxins with the intestinal epithelium, which occurs through the same membrane receptors [19,22].
Some alternative methods of resistance management of this pest towards B. thuringiensis toxins can reduce resistance and even make it possible to break the resistance to biological products [22,64].
According to , mixed formulations of different bacteria or isolates of B. thuringiensis that have a wide variety of Cry toxins, organized in isolation or together, have the ability to reduce selection pressure and, consequently, the development of new cases of resistance in populations of P. xylostella.
To improve the biological control of P. xylostella using this entomopathogenic bacterium, several studies have initially focused to on the characterization of new strains of B. thuringiensis, with the objective of discovering more efficient insecticides and implementing them in new formulations [65,66,51]. In a study conducted by  using stored grains and different strains of B. thuringiensis from soils of several regions of Brazil, there was high mortality (98–100%) of second-instar larvae of P. xylostella. These results have demonstrated that a high variability of Cry genes in the same strain can constitute a substantial tool for resistance management of this pest, with subsequent use in the synthesis of new biological products.
In pathogenicity tests, the strains behave in different ways, and few of them are able to cause total mortality in the insects analyzed. In research conducted by , approximately 19% of the strains tested caused total mortality to second-instar larvae of P. xylostella between 24 and 48 hours.
In this case, in addition to pathogenicity and virulence tests, researchers should analyze the sublethal effects of these strains on the remaining individuals, an important parameter in the toxicological evaluation of B. thuringiensis strains [67,68].
Many biological characteristics of P. xylostella may be influenced by the sublethal effects of these toxins, causing discernible changes in insect behavior, such as appetite loss, decreased movement with subsequent paralysis, change in the tegument color from bright green to dark yellow or dark brown, and loss of reaction to touch [69,51].
According to  and , the most pronounced biological changes observed between phytosanitary applications with strains and commercial products based on B. thuringiensis were in the viability of larvae and pupae and the weight of pupae. The biological characteristics less influenced by these strains were related to the caterpillar and pupal period and sex ratio .
The behavior of strains or commercial products based on B. thuringiensis that result in individuals surviving phytosanitary application, but that provide sublethal effects in subsequent generations, may be a significant tool for Integrated Pest Management , the objective of which is to improve management of the pest through interactions with other control methods, such as biological control with predators and parasitoids, which will reduce the population density due to sublethal effects caused by strains of B. thuringiensis. The remaining pests may be a food source and host for other insects considered beneficial to agriculture, and can help maintain and assist the populations of these arthropods in different crops.
The Integrated Management of P. xylostella based on biological control with the entomopathogenic bacterium B. thuringiensis is an important method for reducing the population density of this pest in brassica crops. However, the use of this control must be well planned, because there are populations of this pest resistant to biological products, necessitating the use of certain methods of resistance management to eliminate these harmful individuals and, perhaps, prevent future problems with the development of resistant populations that can undermine the whole program of rational control of this pest.