Not long after it introduced the Bollgard technology in 1996, Monsanto scientists began working on the development of a second Bacillus thuringiensis or Bt gene for the next generation of its transgenic cotton plants.

The purpose was twofold: 1) To broaden the spectrum of control provided when target pests fed on the Bt proteins in the genetically enhanced plants and 2) to extend the life of the technology by using a “two-gene” approach.

One of the lynchpins of the strategy was that those species not become resistant to the Cry 1A(c) gene insertion event in the original Bollgard since that would leave the second gene as the only effective protein in the Bollgard II technology and negate the two-gene strategy.

University of Arizona researchers have reported the first documented case of field-evolved resistance in Helicoverpa zea or bollworm to the Bt insecticidal protein in Bollgard cotton.

But other university entomologists and Monsanto representatives say they're not sure whether the study led by Bruce Tabashnik, professor of entomology at the University of Arizona, reflects what is actually occurring in the cotton field.

“Resistance is a decrease in pest susceptibility that can be measured over human experience,” Tabashnik said in a press release that was distributed in advance of the publication of his findings in the February issue of Nature Biotechnology magazine.

“When you use an insecticide to control a pest, some populations eventually evolve resistance,” said Tabashnik, who is considered to be a leading authority on Bacillus thuringiensis resistance. (The study used published data from monitoring studies of six major caterpillar tests of Bt crops in Australia, China, Spain and the United States.)

Resistance has always been a possibility for most control measures for insects. The development of resistance to pyrethroids in Heliothis virescens or tobacco budworm in the late 1980s and 1990s helped make Bollgard cotton one of the most rapidly adopted technologies in the history of agriculture.

Scientists at USDA, at land grant universities and the Environmental Protection Agency have been concerned that bollworms and tobacco budworms could develop resistance to the Bt toxin because of the “24/7” exposure of the pests to the Bt toxin.

For that reason, EPA required Monsanto and the cotton industry to develop resistance management plans to help maintain the effectiveness of the technology as long as possible. Growers were required to plant portions of their acres in non-Bt varieties to provide a reservoir of susceptible moths to mate with resistant insects.

Tabashnik first discussed his research at a meeting of the Entomological Society of America last fall. Most of the information was based on data developed by Randy Luttrell and Ibrahim Ali, entomologists with the University of Arkansas and from his computer modeling of their findings.

The University of Arizona researchers wrote in their report for the magazine article that Bt cotton and corn hybrids containing the Bacillus thuringiensis gene have been planted on more than 400 million acres since 1996, “generating one of the largest selections for insect resistance ever known.”

Nonetheless, the researchers said, most caterpillar pests of cotton and corn have remained susceptible to Bt crops. “The resistance occurred in one particular pest in one part of the United States,” Tabashnik said. “The other major pests attacking Bt crops have not evolved resistance. And most bollworm populations have not evolved resistance.”

But even if only a handful of bollworms fit into that category, that's a cause for concern, according to Walt Mullins, Monsanto's technical manager for Bollgard and Bollgard II.

“Obviously, anytime anyone claims resistance to Cry 1A(c), not only is it a scientific issue, a political issue and potentially a commercial issue, it also can become a regulatory issue for us,” he said.

Mullins says Tabashnik's findings include some of the early research generated by Luttrell when he was a professor of entomology at Mississippi State University and other entomologists and more recent work since Luttrell returned to the Department of Entomology at the University of Arkansas.

“Basically, his claim was that if you just add the LC50 values that were generated early on from baseline work that both Luttrell did and Stone and Sims did in the early 1990s and even prior to that, you will see what appears to be an apparent shift where the LC50 values are increasing,” Mullins notes.

“He also said his interpretation of the data was consistent with his own modeling efforts that would predict resistance in eight or nine years and that resistance had not occurred in North Carolina primarily because of the natural refuge that occurred in that state.”

Shortly after the release of the University of Arizona researchers' study, Luttrell was quoted as saying he did not believe the data developed at Mississippi State and the University of Arkansas supported such a conclusion. Luttrell said he “respectfully disagreed” with Tabashnik's interpretation.

“The response (the number of bollworms surviving on Bt cotton) was greater in 2002, which might make it appear we could be dealing with resistance,” he said. “But when you look closer, other factors have to be taken into account.”

The reaction of other Mid-South researchers to the Arizona study has been mixed.

“I don't think many in the Mid-South would agree with those conclusions,” said one entomologist. “Some of my colleagues say resistance has evolved, but I don't think that is the case,” said another.

The entomologists note Monsanto's Bollgard varieties have shown more damage from bollworms than tobacco budworms since before the commercial introduction in 1996. The reasons range from less expression of the protein in cotton flowers to “avoidance behavior” in the insects.

“Some research has shown that bollworms will take a bite in the upper part of the plant, don't like what they've found and spin down with their silking gland to an open flower,” Gus Lorenz, Extension entomologist in Arkansas was quoted as saying. “We know there's less expression there.

“So how much survivability is due to increased tolerance and how much is due to some type of behavioral modification? That question has yet to be answered. I do know we've had bollworms going through Bt cotton ever since it came on the market.”

Luttrell said that when anyone of Tabashnik's stature writes a paper like the one released by the University of Arizona researchers, “it has impact.” But he says he's not ready to agree resistance has happened.

Comparing the data from Luttrell's studies and those of other researchers such as Stone and Sims in the late 1980s and early 1990s with more recent research indicates some degradation of the protein over time, “but we expect that with bollworm and with Cry 1A(c),” Mullins said during a presentation at the Beltwide Cotton Conferences.

“During these same timeframes, USDA entomologists were running tests on both bollworms and budworms to get a discriminating dose (for mortality in the insects,) he said. “The discriminating dose tests did not indicate any resistance development at that period of time.”

Another factor that is important to understand, he said, is that the early research was conducted with a purified protein, “and we're talking about a decade and a half ago or even longer. We're not exactly sure how that would compare to what would be produced today. We could certainly question if there was some kind of protein degradation involved in these higher LC 50s.”

Different laboratories, different researchers at different times could also be involved,” he said. “One suggestion is that maybe the work Dr. Luttrell and Dr. Ali have done more recently could more fully explore the issues concerned in the shift,” says Mullins.

“One thing we do know is that we don't always get a consistent response in the genotype, particularly between these toxins and bollworm. We see a lot of genotypic variation from one strain to the next and who knows what other artifacts and reasons there might be.”

Monsanto continues to believe the Cry 1A(c) Bt gene is effective and — paired with the Cry 2Ab2 gene in Bollgard II — will help delay the development of resistance in target insects for a number of years.

Both Monsanto and Dow AgroSciences, which introduced its WideStrike Bt technology that features a two-gene approach two years ago, have received approval from EPA for a natural refuge option for Bollgard II and WideStrike cotton beginning with the 2008 seaon.

That means cotton producers in eligible regions will be able to take advantage of non-cotton crops and other wild host plants as a refuge for caterpillar pests and will not be required to plant a structured non-Bt refuge for their transgenic crops — and continue to delay resistance, company representatives say.

The natural refuge option applies to Bollgard II cotton planted in the states of Alabama, Arkansas, Florida, Georgia, Kansas, Kentucky, Louisiana, Maryland, Missouri, Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee, certain counties in Texas and Virginia.

The following counties in Texas are excluded from using the natural refuge option: Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, Loving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler. The natural refuge option also is not available in areas where pink bollworm is a significant pest.

(Note: EPA previously established prohibitions on the planting of Bt cotton in the Texas Panhandle counties of Carson, Dallam, Hansford, Hartley, Hutchinson, Lipscomb, Moore, Ochiltree, Roberts and Sherman as well as south of Highway 60 in Florida. These restrictions do not change with the approval of natural refuge for Bollgard II cotton.)

Cotton producers who plant the original Bollgard cotton varieties must continue to plant a structured refuge of 5 percent unsprayed or 20 percent sprayed non-Bt cotton as required by EPA's insect resistance management or IRM rules and specified in their technology use agreements.

EPA has announced cotton growers will be allowed to continue planting Bollgard varieties through 2010 with seed purchased through September 2009. Monsanto says only limited quantities of seed will be available at that time.

“Growers have told us they want to be able to continue to plant these highly popular varieties beyond those dates,” said Kevin Eblen, Monsanto's director of its Delta and Pine Land Business. “But we simply can't take the risk of allowing these single-gene varieties to remain in the field and allow insects to develop resistance.”