Brevibacillus laterosporus, a bacterial biological control agent of Western Corn Rootworm

Background

A) An adult Western Corn Rootworm Beetle, Diabrotica virgifera virgifera. B) Root damage comparison between undamaged corn roots (right) and high-density larval infestation (left). Photo credit for this image belongs to L. J. Meinke, an entomologist at the University of Nebraska [1].

By Katya Naphtali

Underneath the umbrella of “pesticides”, there are biologically synthesized alternatives to chemicals called microbial pesticides. This alternative uses different microorganisms to target specific pests that threaten production. The actual material used as a pesticide is naturally formed, biodegradable, highly specialized to specific pests rather than having widespread impact with the added benefit that they can be used in small quantities with large effects .

This technique of pest control is commonly used to address damage incurred by the Western Corn Rootworm (CRW), which is specialized to parasitize corn monocropping productions . Damage incurred from CRW infestation of corn crops costs the United States a total of $2 billion annually (Wechsher et al. 2018)

A common management process to address CRW is the incorporation of microbial pesticides into corn GMO strains. A number of crops including cotton, soybeans, and corn are genetically engineered to contain genes sources from a soil bacteria, Bacillus thuringiensis, commonly referred to as Bt. This species is most commonly used for microbial pest control targetted at Coleopteran species (Fernandez-Cornejo et al.). The toxins produced by Bacillus bacteria attack cells in the larva’s midgut by causing the cells in their stomach lining to lyse (Paddock et al.) When genes for toxic protein production are incorporated into crops, the pests that parasitize these crops die from consuming these naturally synthesized toxic proteins (Fernandez-Cornejo et al.). These toxins can be incorporated into GMO crops, allowing for the corn plants themselves to produce these toxins and in effect protect themselves from CRW. Currently there are four strains of Bt corn that widely incorporated into corn productions (Paddock et al.)

Unfortunately, in some regions where these GMOs are overused, CRW has adapted resistance to this toxin (Levine et al. 2002). Currently there are populations of of CRW that have evolved to be resistant to every Bt toxin we are aware of (Paddock et al.) This drives researchers to search for similarly effective bacteria to replace their current technology.

Diabrotica virgifera virgifera, Western Corn Rootworm

This diagram depicts the yearly generational life cycle of Diabrotica virgifera virgifera, the Western Corn Rootworm. The photo credit for this diagram belongs to Davis-Vogel et al. 2018, who is a biochemist at Iowa State University[2].

Diabrotica virgifera virgifera, otherwise known as Western Corn Rootworm, is a species of Coleoptera (which are beetles) that specialize only in eating varieties of corn, Zea mays. These beetles are ¼” long and yellow with black stripes along the sides of their elytra. They only have a single generation a year, because their life cycle is dependent on corn developmental stages (Strnad et al.). Adult beetles lay their egg clusters below the soil surface for generation the following year, before dying themselves in the first winter frost . These eggs hatch during late May and early June into polypod larva that are long small and white but sport a scleritized brown head plate with strong mandibles. They spend most of their larval stage feeding on the root zone of soil. They tunnel through dirt until finding corn roots to feed on (Strnad et al.). This tunneling can kill an entire root or break the tips of them, this prevents the plant from recieving enough nutrients causing it to either fall over, or greatly damage growth. The plant can be weakened in terms of stunting growth, reducing the yield amount, or even reducing the size of each ear of corn .

These larva undergo three instars of molting over a 4-6 week period (Strnad et al.) & . Before they pupate within the soil to undergo a compleme metamorphosis of their bodies into their adult forms. 5-10 days after pupation, CRW emerge as adults coinciding with when the corn plants flower (Strnad et al.) & . CRW beetles still consume during their adult stages, mainly corn pollen and corn silk, sometimes their leaves as well. Corn silk and pollen are both are needed for corn to pollinate eachother, so adults reduce their ability to reproduce . Despite these negative impact, it is still less than the damage caused during their larval stage feeding on roots .

In laying their eggs for the next generation, adult CRW are relatively stagnant species and do not tend to move fields for laying their eggs. As a result a common pest management technique for CRW is crop rotation with soy beans (Paddock et al.). Each year switching corn out for soybean plants prevents CRW from parasitizing their roots, causing CRW larva populations to drop due to the lack of their niche corn food source. Unfortunately, in some cases CRW have adapted to crop rotation schedules and have begun laying their eggs in soybean fields in anticipation of crop rotation to corn the following Spring when their offspring hatch . This species adapts quickly due to its dependence on corn as its sole food source . As a result, other forms of pesticides are needed to be used alongside crop rotation .

Most commonly, crop rotation is paired with the use of Bt corn crops, that have been genetically modified to produce these pest specific toxins themselves. These genetic modifications allow farmers to use less chemical pesticides while still reducing the damage incurred by pests, and increasing their overall crop yield (Fernandes-Cornejo et al.). Unfortunately Bt is widely used which has resulted in the emergence of multiple resistant strains of CRW both in Northwest Iowa and Southeast Minnesota. While these resistant populations may not be able to move far without accidental human transportation, they fortell the immunity that may be seen in other regions that over-use specific resistant strains. As a result, new bacterial strains are being investigated to create new resistant corn GMO archetypes. Similar to crop rotation, a rotation of toxins may introduce more variety in pest control techniques that they will be less able to adapt to .

Brevibacillus laterosporus

A number of Bacillus species have been investigated for pesticidal properties due to their formation of specialized biotoxins. Since the 1960s the most commonly used biotoxin has been those sourced from various strains of Bt. These bacteria are produced industrially in fermentation tanks and have widespread use for various different orders and species of insects (Weinzier et al.) As pest species become more resistant to Bt toxins, researchers have taken interest in a different but similar species of Bacillus, Brevibacillus laterosporus. In recent years BL has become a bacterial species of more interest due to the speed in which it produces biotoxins, even if less powerful of a toxin than Bt produced .

Brevibacillus laterosporus (Bl) is a gram-positive, rod-shaped bacteria about 3-5um in length and 0.7-1.0um in width. This bacteria is facultatively anaerobic (Cai et al.) meaning it can survive in environments with or without oxygen. It also can either be found as individual bacteria or in short chains (Prassanna et al.) which can form flat white colonies (Ghazanchyan et al.). Due to its metabolic flexibility, Bl can be sourced from soil, plants or even directly from insects themselves . This bacteria reproduces by producing endospores. On one end of each spore, this species of bacteria can be identified by its production of canoe-shaped paraposal bodies (a crystallized shell) . Bl produces a number of products including enzymes and prebiotic materials that function as an insecticides (Orlova et al.) Some of these insecticidal products are sourced from the crystallized structures of these parasposal bodies that contain endotoxins . To extract these endotoxins, during cell lysis paraposal bodies detach from thel cell wall and can be removed as a precipitate. This material can then be utilized as a larvacide. Different strains of BL produce different shapes and structures of proposal bodies but their functional differences are unclear (Ruiu et al.) Additionally, some strains of Bl do not even form proposal bodies, but still are larvicidal (Oliveira et al.). While there are still many unknowns, Bl produces paraposal bodies and endotoxins at a higher rate than other species Bacillus making it optimal for industrial agricultural use (Ghazanchyan et al.).

Bl & Bt Larvicidal Properties

Describ. The photo credit for this diagram belongs to Davis-Vogel et al. 2018, who is a biochemist at Iowa State University[3].

Since Bt has been used nationally since the 1960s (Weinzierl et al.) there is more information on its role as an insecticide than for Bl. The endotoxins produced by Bt are fast acting, minutes after target insects consume Bt treated leaves, its endotoxins bind to receptors in the pest’s gut. These receptor sites in the gut wall are specific to toxin and species, allowing this form of pesticide to be highly specialized to either insect order or a handful of species. Once bound to this receptor site, over 1-2 hours this toxin begins lysing the wall of the gut. Once the gut wall is broken down, the toxin has already dissolved and spores from the Bt bacteria along with microbes from the insect’s own digestive system leak out into the rest of their body, and into their hemolymph. Once infected, the host insect stops eating and the insect dies within 1-2 days from septic infection in the blood (Weinzierl et al.).

Bl endotoxins work similarly through the gut of their target organisms, but less is known about how lethal it is compared to Bt. Initially sourced strains of Bl were collected from water sampling in 1916 (Laubach 1916) and these initial samples happened to have lower insecticidal abilities (Prassana et al.). As a result, studies comparing Bl to Bt strains assumed that Bl had weaker larvacidal abilities than Bt, and that Bl also had a more limited environmental range (Oliveira et al.).

In more recent years, more strains have been identified using 16S RNA sequencing (Prassana et al.) These strains have been found to functions as a broad spectrum antimicrobial agents with lethal impacts on Coleoptera as well as Lepidoptera, Diptera, and nematodes. With no pathogenic effect on non-target species (Ruiu et al.). Bl is toxic to larvae only through ingestion which allows the endotoxins to bind to their gut wall receptors (Ruiu et al. & Weinzier et al.) This toxin kills pests swiftly and dissolves within their gut in a matter of hours (Weinzier et al.) As a result, to effectively use Bl as a biocontrol, its genes need to be used in the creation of GMO crops or as a topical insecticide. For topical application, Bl mixtures must coat the entire crop plant, especially the undersides of leaves, due to the fact that it is deactivated by ultraviolet light from direct sunlight.

LETHAL/MORTALITY RATE What has been found is that Bl has an almost 100% effectiveness rate in the elimination of Coleopteran order pests depending on the strain. It was most effective in targeting beetles during their larval stage, especially during their second instar .

A different study found that Bl causes a 33-63% mortality rate in the larva of Colepotera, but up to a 90% mortality rate results on caterpillars (Anticarsia gemmatalis). Regardless of species, toxins had the strongest effects when collected during exponential growth phases which incurred a mortality rate of 90% compared to the lower range of toxins sourced during vegetative growth stages at 13-48%. (Oliveira et al.) Lastly, the lethal values of each bacteria are similar between Bt and Bl which indicates that Bl may be just as lethal as Bt it utilized correctly (Orlova et al.)

Additional Larvicidal Properties of Bl

INSECTICIDAL PROTEINS (ISPs) Bl produces proteins that are similar to those used from Bt. Specifically, Bl secretes insecticidal proteins (ISPs) into its surrounding environment, called the broth. From this broth, we can collect a supernatant of isolated ISPs that is highly toxic to CRW (Ruiu et al.) These ISPs include two types, ISP1A and ISP2A. These proteins are only toxic when consumed together with a complementary proten, they do not hold toxic properties individually. This protein combination has similar lethal effects on larva homologous to the ISPs produced by Bt during a vegetative growth stage (Ruiu et al.)

CHITINASE Prassana When this bacteria is in the presence of chitin, it produces extracellular chitinase to break it down outside of the cell in order to convert it into a carbon source it can metabolise within the cell (Prassana et al.) Chitin is a polysaccharide that can be found in the body structure of insects, mostly in their exoskeletons and the stronger portion of their skin (CHECK). Chitin cannot be found in other organisms such as humans, plants, and other insects making this a more targeted insecticide than chemicals that may have destructive impacts on the surrounding environment (Prassana et al.) These chitinases can be removed from the bacteria and turned into a mixture used as an insecticide. BL already possess insecticidal toxins, through the formation of proposal bodies, but in the presence of chitin, it has an additional toxin source (Prassana et al.) While the BL bacteria itself is adapted for moderate temperatures, surprisingly the chitinase sourced from it can remain active at up to 70oC and are more thermostable than predicted. This would indicate that chitinase mixtures sourced from BL are well suited for industrial production as they would still be effective in conditions and environments different from their adapted ecosystem (Prassana et al.) When this chitinase mixture is sprayed on leaves experimentally, this mixture had a faster mortality rate than bacteria that was not pre-exposed to chitin (Prassana et al.) When the larva of diamond back moths (plutella xylostella) were exposed to BL with a chitinase mixture, 50% of the larva died within 2.1 days compared to without the mixture in which it took 3.3 days to reach a 50% mortality rate. The use of the chitnase made the mortality rate 1.2 days shorter, This could be explained due to how chitin targets and weakens the inner gut lining of insects (Kramer 1997), both degrading the gut directly and allowing proposal body toxins to penetrate the gut as well (Flexner, Schnepf, Soberon etc). (Prassana et al.) Experiment: Induced chitnase production in some Bl bacteria and collected proteins. Then looked at impact of chitinase applied to un-induced bacteria to see if it had an additive effect on the insecticidal properties of BL. This chitin culture serum increased the lethal effect of BL application on leaves with a dosage effect. With a 500ul dose, 100% of the larva died within 5 days. With every other dosage, or conditions without citinase, 100% larva were still eliminated within 7 days. (Prassana et al.) Found that mortality was higher during the first instar while the 5th had the lowest mortality rate when exposed to chitinase. This may be due to the high feeding rates at earlier developmental stages as opposed to later instars where moths feed less and thus have less exposure to the insecticides spread on the leaves. (Prassana et al.)

Conclusion

Brevibacillus laterosporus holds the potential to be an effective specialized larvidcide. More research needs to be conducted on the specific impacts Bl may have on Western Corn Rootworm as well as creating variety in microbial pest control and variation similar to that of crop rotation. In combination with other IPM techniques, Bl could serve as an effective tool for managing Coleopteran pest species. While this strain is not as powerful as BT, it holds the potential to be as lethal to pests, and due to its low usage in agriculture thus far, few pests will be resistant to its toxins.

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2023, Kenyon College