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Development of Powdery Mildew Resistant Tomato via CRISPR-Cas9

In tomato (Solanum lycopersicum), there are sixteen Mlo genes, with SlMlo1 being the major contributor to the susceptibility to the powdery mildew caused by Oidium neolycopersici. Natural loss-of-function slmlo1 mutants are available in tomato, however, introgression of such mutations is a lengthy process. The team of Vladimir Nekrasov from the Sainsbury Laboratory, Norwich Research Park in the UK aimed to generate a transgene-free genetically edited slmlo1 tomato using the CRISPR-Cas9 system.

The team targeted the SlMlo1 locus using the double sgRNA strategy. Transformants were analyzed and eight out of ten tested T0 transformants indicated the presence of mutations. Assays using the powdery mildew fungus revealed that all the generated T0 slmlo1 mutant plants were resistant to the pathogen, while wild-type plants were susceptible.

Furthermore, the slmlo1 mutant plants were morphologically similar to the wild type and also produced harvested fruit weight similar to the wild types. The team named the generated variety Tomelo. This study presents evidence for CRISPR-Cas9 being a highly precise tool for genome editing in tomato.

For more on this study, read the article in Nature.

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Brazil’s transgenic sugarcane stirs up controversy

  • Brazil’s transgenic sugarcane stirs up controversy

Copyright: Icaro Cooke Vieira for Center for International Forestry Research (CIFOR).

Speed read

  •  Sugarcane borer causes losses of around US$ 1.5 billion per year
  • Experts warn of lack of environmental impact studies in Brazil
  • Damage to biodiversity, including non-target insects, is among potential risks
 
[RIO DE JANEIRO] A genetically modified (GM) cane variety that can kill the sugarcane borer (Diatraea saccharalis) has been approved in Brazil,  to the delight of some scientists and the dismay of others, who say it may threaten Brazilian biodiversity.

Brazil is the second country, after Indonesia, to approve the commercial cultivation of GM sugarcane. The approval was announced by the Brazilian National Biosafety Technical Commission (CTNBio) on June 8.

Sugarcane borer is one of the main pests of the sugarcane fields of South-Central Brazil, causing losses of approximately US$1.5 billion per year.

“Breeding programmes could not produce plants resistant to this pest, and the existing chemical controls are both not effective and severely damaging to the environment,” says Adriana Hemerly, a professor at the Federal University of Rio de Janeiro, in an interview with SciDev.Net.

“Studies conducted outside Brazil prove that protein from genetically modified organisms harms non-target insects, soil fauna and microorganisms.”

Rogério Magalhães

“Therefore, the [GM variety] is a biotechnological tool that helps solve a problem that other technologies could not, and its commercial application will certainly have a positive impact on the productivity of sugarcane in the country.”

Jesus Aparecido Ferro, a member of CTNBio and professor at the Paulista Júlio de Mesquita Filho State University, believes the move followed a thorough debate that began in December 2015 — that was when the Canavieira Technology Center (Sugarcane Research Center) asked for approval to commercially cultivate the GM sugarcane variety.

“The data does not provide evidence that the cane variety has a potential to harm the environment or human or animal health,” Ferro told SciDev.Net.

To develop the variety, scientists inserted the gene for a toxin [Cry] from the bacterium Bacillus thuringiensis (Bt) into the sugarcane genome, so it could produce its own insecticide against some insects’ larvae.

This is a technology that “has been in use for 20 years and is very safe”, says Aníbal Eugênio Vercesi, another member of the CTNBio, and a professor at the State University of Campinas.

But Valério De Patta Pillar, also a member of the CTNBio and a professor at the Federal University of Rio Grande do Sul, points to deficiencies in environmental risk assessment studies for the GM variety — and the absence of assessments of how consuming it might affect humans and animals.

According to Pillar, there is a lack of data about the frequency with which it breeds with wild varieties. Data is also missing on issues such as the techniques used to create the GM variety and the effects of its widespread use.

Rogério Magalhães, an environmental analyst at Brazil’s Ministry of the Environment, also expressed concern about the approval of the commercial transgenic cane.

“I understand that studies related to the impacts that genetically modified sugarcane might have on Brazilian biodiversity were not done by the company that owns the technology,” said Magalhães in an interview with SciDev.Net. This is very important because Brazil’s climate, species, and soils differ from locations where studies might have taken place, he explained.

Among the risks that Magalhães identified is contamination of the GM variety’s wild relatives. “The wild relative, when contaminated with transgenic sugarcane, will have a competitive advantage over other uncontaminated individuals, as it will exhibit resistance to insect-plague that others will not have,” he explained.

Another risk that Magalhães warns about is damage to biodiversity. “Studies conducted outside Brazil prove that Cry protein from genetically modified organisms harms non-target insects, soil fauna and microorganisms.”

Magalhães added that some pests have already developed resistance to the Bt Cry protein, prompting farmers to apply agrochemicals that are harmful to the environment and human health.

This piece was originally published by SciDev.Net’s Latin America and Caribbean desk.

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Growing Produce

May 31, 2017

Is CRISPR/Cas9 a Viable Solution for the Control of Crop Insect Pests?

In my article “A Breakthrough In Biotechnology And The Future Of The Fruit Industry,” I described the precision genome editing technology, called CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats/CRISPR associated-nuclease) and its applications in crop plant improvement. Here, I would like to cover how the CRISPR/Cas9 technology has been repurposed as a gene drive system that may become a potentially viable solution for managing crop insect pests.The Gene Drive Process
What is CRISPR/Cas9-based gene drive and how can it be used for insect pest control?

Gene drive is a term that describes a process in which a particular gene or genetic element is spread quicker than what is expected according to genetic principles in a sexually reproducing population. CRISPR/Cas9-based gene drive is an engineered gene drive that can drive a rapid spreading of a CRISPR/Cas9-altered gene in a target population.

 

Figure 1. Spotted wing drosophila adult flies (A), genitalia (B) and dissected reproductive tissue (C) of wild type female, male (WT), and homozygous mutants (M) for the CRISPR/Cas9 interrupted sex lethal (Sxl) gene. Note that the mutants developed an intersexual phenotype with abnormal genitalia and abnormal ovaries. (Graphic: Adapted from Li and Scott 2016 Biochem Biophys Res Commun)

For insect pest control, genes involved in reproduction, development, feeding, and/or immune system are desirable targets. The vision is that fast spreading of the mutations created by CRISPR/Cas9 will skew sex ratios, reduce fitness, or restore susceptibility to insecticides in target populations.

In addition to agriculture insect pest control, controlling human and animal disease vectors such as mosquitos using CRISPR/Cas9 based gene drive has been envisioned as well. In fact, considerably more resources and efforts have been committed to this area to fight deadly diseases such as dengue, malaria, and Zika fever.

In practice, designing and constructing a specific CRISPR/Cas9 -based gene drive is a straightforward process. For delivering the gene drive into cells, embryo injection is commonly used. Through a series of selections in subsequent generations, insect carrying a desirable CRISPR/Cas9-based gene drive will be identified and raised. Like many other biological control agents, field trials in a contained environment will be conducted to evaluate both the intended and unintended effects. Before releasing these transgenic insects into the farm field or orchard for pest control, they also must be approved by the government, which will conduct an extensive regulatory review.

CRISPR/Cas9 Control of Insects
What studies have been done toward CRISPR/Cas9-based control of crop insect pests?

So far, several major insect pests have been investigated for the feasibility of pest control using CRISPR/Cas9-based gene drive, including drosophila, moth, beetle, grasshopper, and others. The progress has been encouraging. Below are two examples:

Spotted Wing Drosophila (SWD) (Drosophila suzukii). In a proof-of-concept study for the gene-drive-based control of SWD, a nasty pest for cherry and berry crops, the female specific sex lethal (Sxl) gene was targeted, which is a master regulator in female development. The study finds that if both of their Sxl alleles (copies of the gene) are interrupted by CRISPR/Cas9, the female flies will die in early developmental stages due to abnormal development of their reproductive system (Figure 1).

Figure 2. Male and female larvae of diamondback moth wild type and their abdominal-A gene mutants. The squares and arrows in red indicate the region of the testis in male and the disordered segments in mutants, respectively. (Graphic: Adapted from Huang et al. 2016 Insect Biochem Mol Biol)

 

Since most, if not all, females carrying the CRISPR/Cas9-based gene drive will become homozygous for the interrupted Sxl gene, the number of female flies will decline, which will eventually collapse the population.

Diamondback Moth (Plutella xylostella). The damage of diamondback moth to the cruciferous crops is a pressing problem worldwide. To explore a genetics-based control of diamondback moth, a moth gene, called abdominal-A that plays a crucial role in determining the identity and functionality of abdominal segments, was targeted by the CRISPR/Cas9 system in another proof-of-concept study. The study demonstrates that disrupting abdominal-A led to a range of inheritable defects, such as abnormal prolegs and malformed segments in both male and female larvae (Figure 2).

Implementation Timeline
How close are CRISPR/Cas9-based gene drives to a practical solution for crop pest control?

As mentioned earlier, rapid progress has been accomplished to make CRISPR/Cas9-based gene drives a solution for crop pest control. From a technical point of view, implementation could occur soon. However, considering the time needed for government approval and for evaluation of unintended effects on related species to minimize the risk of biosafety, it may take years to see commercial application of CRISPR/Cas9-based gene drives for insect pest control.

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SW FarmPress
California cotton
Researchers have found that crossing Bt cotton with non-Bt cotton can reverse issues of resistance by the pink bollworm to the transgenic efforts to control them.

Researchers able to reverse resistance of pink bollworm to Bt cotton

Collaborative effort by researchers in China and the University of Arizona find unique method to reverse resistance of pink bollworm to Bt cotton. Implications exist for transgenic corn and soybeans as well.

Farm Press Staff | May 09, 2017

Researchers with the University of Arizona and China discovered a surprising strategy to reverse pink bollworm resistance to genetically engineered cotton.

Cotton growers have been able to use genetically engineered cotton to fight the pink bollworm. This has happened as scientists have been able to produce pest-killing proteins from the widespread soil bacterium Bacillus thuringiensis, or Bt. Without adequate countermeasures, scientists have discovered that pests can quickly evolve resistance.

According Bruce Tabashnik, one of the authors of the study and a regents’ professor with the University of Arizona, these Bt proteins are considered environmentally friendly because they are not toxic to people and wildlife. They have been used in sprays by organic growers for more than 50 years, and in engineered Bt crops planted by millions of farmers worldwide on more than 1 billion acres since 1996.

Scientists from the U.S. and China discovered that by hybridizing genetically-engineered cotton with conventional cotton, it reduced the resistance of the pink bollworm. Details of the 11-year study that tested more than 66,000 pink bollworm caterpillars from China’s Yangtze River Valley are published in the Proceedings of the National Academy of Sciences.

Tabashnik says this is the first-ever case where scientists have seen substantial reversal to resistance developed by a pest.

The primary strategy for delaying resistance is providing refuges of the pests’ host plants that do not make Bt proteins, according to Tabashnik. This allows survival of insects that are susceptible to Bt proteins and reduces the chances that two resistant insects will mate and produce resistant offspring.

Before 2010, the U.S. Environmental Protection Agency required refuges in separate fields or large blocks within fields. Planting non-Bt cotton refuges is credited with preventing evolution of resistance to Bt cotton by pink bollworm in Arizona for more than a decade.

By contrast, despite a similar requirement for planting refuges in India, farmers there did not comply and the pink bollworm rapidly evolved resistance.

The ingenious strategy used in China entails interbreeding Bt cotton with non-Bt cotton, then crossing the resulting first-generation hybrid offspring and planting the second-generation hybrid seeds. This generates a random mixture within fields of 75 percent Bt cotton plants side-by-side with 25 percent non-Bt cotton plants, according to the University of Arizona.

“Because cotton can self-pollinate, the first-generation hybrids must be created by tedious and costly hand pollination of each flower,” said Tabashnik, “However, hybrids of the second generation and all subsequent generations can be obtained readily via self-pollination. So, the hybrid mix and its benefits can be maintained in perpetuity.”

Tabashnik calls this strategy revolutionary because it was not designed to fight resistance and arose without mandates by government agencies. Rather, it emerged from the farming community of the Yangtze River Valley. While most previous attention has focused on the drawbacks of interbreeding between genetically engineered and conventional plants, the authors point out that the new results demonstrate gains from such hybridization.

“For the growers in China, this practice provides short-term benefits,” Tabashnik added. “It’s not a short-term sacrifice imposed on them for potential long-term gains. The hybrid plants tend to have higher yield than the parent plants, and the second-generation hybrids cost less, so it’s a market-driven choice for immediate advantages, and it promotes sustainability. Our results show 96 percent pest suppression and 69 percent fewer insecticide sprays.”

There are implications in transgenic corn and soybeans as well.

“This study gives a new option for managing resistance that is very convenient for small-scale farmers and could be broadly helpful in developing countries like China and India,” explained coauthor Kongming Wu, who led the work conducted in China and is a professor in the Institute of Plant Protection in Beijing.

“A great thing about this hybrid seed mix strategy is that we don’t have to worry about growers’ compliance or regulatory issues,” Tabashnik said. “We know it works for millions of farmers in the Yangtze River Valley. Whether it works elsewhere remains to be determined.”

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Xin news

Chinese, U.S. scientists make first reversal of pest resistance to GM cotton – Xinhua | English.news.cn

Source: Xinhua| 2017-05-09 03:55:08|Editor: yan

WASHINGTON, May 8 (Xinhua) — The issue of pest resistance to genetically modified (GM) crops has received widespread attention, and researchers from China and the United States revealed on Monday the success of a surprising new strategy for countering this problem.

In a study published in the U.S. journal Proceedings of the National Academy of Sciences, they reported that hybridizing genetically engineered cotton with conventional cotton reduced resistance in the pink bollworm, a voracious global pest.

The findings were based on an 11-year study, in which researchers at the Chinese Academy Of Agricultural Sciences (CAAS) and the University of Arizona (UA) tested more than 66,000 pink bollworm caterpillars from China’s Yangtze River Valley, a vast region of southeastern China that is home to millions of smallholder farmers.

According to the study’s authors, this is the first reversal of substantial pest resistance to a crop genetically engineered to produce pest-killing proteins from the widespread soil bacterium Bacillus thuringiensis, or Bt.

“This study gives a new option for managing resistance that is very convenient for small-scale farmers and could be broadly helpful in developing countries like China and India,” study coauthor Kongming Wu, who led the work conducted in China and is a professor in the CAAS’s Institute of Plant Protection in Beijing, said in a statement.

Crops genetically engineered to produce insecticidal proteins from Bt kill some major pests and reduce use of insecticide sprays.

However, evolution of pest resistance to Bt proteins decreases these benefits.

The primary strategy for delaying resistance is providing refuges of the pests’ host plants that do not make Bt proteins. This allows survival of insects that are susceptible to Bt proteins and reduces the chances that two resistant insects will mate and produce resistant offspring.

Before 2010, the U.S. Environmental Protection Agency required refuges in separate fields or large blocks within fields.

Planting such non-Bt cotton refuges is credited with preventing evolution of resistance to Bt cotton by pink bollworm in Arizona for more than a decade.

By contrast, despite a similar requirement for planting refuges in India, farmers there did not comply and pink bollworm rapidly evolved resistance.

The new strategy used in China entails interbreeding Bt cotton with non-Bt cotton, then crossing the resulting first-generation hybrid offspring and planting the second-generation hybrid seeds.

This generates a random mixture within fields of 75 percent Bt cotton plants side-by-side with 25 percent non-Bt cotton plants.

“We have seen blips of resistance going up and down in a small area,” said senior author Bruce Tabashnik, a professor in the UA’s College of Agriculture and Life Sciences. “But this isn’t a blip. Resistance had increased significantly across an entire region, then it decreased below detection level after this novel strategy was implemented.”

Tabashnik called this strategy revolutionary because it was not designed to fight resistance and arose without mandates by government agencies. Rather, it emerged from the farming community of the Yangtze River Valley.

“For the growers in China, this practice provides short-term benefits,” Tabashnik added. “It’s not a short-term sacrifice imposed on them for potential long-term gains. The hybrid plants tend to have higher yield than the parent plants, and the second-generation hybrids cost less, so it’s a market-driven choice for immediate advantages, and it promotes sustainability.”

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From PestNet

Courthouse news update

https://www.courthousenews.com/cornell-researchers-set-use-gmo-moths-control-pests/

DAVID REESE

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 April 26, 2017

The diamondback moth costs farmers over $4 billion annually. (Photo: The Shelton lab at Cornell University)

(CN) – Researchers at Cornell University hope to tackle a small pest and a big problem that has plagued agriculture since the 1800s: controlling the destructive diamondback moth through genetic engineering.

The diamondback moth is a small creature, about the length of two grains of rice, but they are capable of inflicting billions of dollars of damage on cabbage and broccoli crops every year. In fact, the moth is Enemy No. 1 when it comes to the cabbage and broccoli family. Cornell University’s agriculture research division in Geneva, New York, is studying how to invade the invaders with genetically engineered male diamondback moths that mate with females, which then die before reaching adulthood.

The federal Animal and Plant Health Inspection Service said last week it is seeking public comment on Cornell’s plan to release sterile male diamondback moths from the United Kingdom on Cornell’s research grounds.

The diamondback moth (Plutella xylostella) is the world’s worst insect pest of brassica crops (cabbages, canola, broccoli, cauliflower and kale). The pest costs farmers $4 to $5 billion annually worldwide, according to the U.S. Department of Agriculture. The pest arrived on ships in the 1800s, according to Tony Shelton, head of the Shelton Lab at Cornell University’s agriculture research station.

Female diamondback moths can lay over 150 eggs during their short lifetime, and a generation can be produced in as little as two weeks. This invasive pest probably originated in Europe, but is now found throughout the world, including New York and other states that farm brassica vegetables.

Shelton is the leader of the diamondback moth project at Cornell University. The Shelton Lab is part of Cornell University’s Department of Entomology and is about 50 miles from the main campus at the New York State Agricultural Experiment Station in Geneva, New York, in the heart of the Finger Lakes.

Cauliflower

Damage to a cauliflower plant caused by diamondback moths. (Photo: The Shelton lab at Cornell University)

Shelton works with crucifer farmers globally, and this week he was off on a plane to Bangladesh. “Wherever crucifers are grown, diamondback moths are there,” he said in an interview.

Biological controls are part of the solution to controlling the tiny invaders, Shelton said. Best methods of controlling the pests are a combination of biological controls, plowing fields after harvest, and “judicious use of insecticides,” Shelton said. “Genetic control using a self-limiting method could make a valuable contribution of integrated pest management.”

Diamondback moths infest crucifer crops in all U.S. states, but most severe damage occurs in warmer areas where more generations of the moth are produced, Shelton said.

The proposed release of diamondback moths would be at the Cornell research station and would not exceed two years, according to the application. The release would be limited to an experimental field, up to 10 acres. The release site is surrounded by other agricultural fields within the research station’s 870 acres where diamondback moths occur naturally. Shelton’s team would release up to 10,000 male genetically engineered moths per release, up to 30,000 males per week.

The moths that Cornell University is looking to release onto its research grounds are genetically engineered to be sterile, and they also have a fluorescent red marker that shows up under special light. The field studies at Cornell are funded by the U.S. Department of Agriculture and Oxitec, the company that designs and breeds the diamondback moths to be released.

This biological approach to controlling pests has been used for about 50 years and is known as the sterile insect technique. In this technique, male insects are sterilized with radiation and then released to mate with female pest insects of the same species. Because the males have been sterilized, there are no offspring from the mating and this reduces the pest population over time with multiple releases, according to the USDA.

There is one problem with this technique in diamondback moths, though. The radiation used to sterilize the males can affect many other genes and the sterilization reduces the fitness of the male insects to the point they are actually less able to mate with the females.

Affiliated with Oxford University, Oxitec scientists developed a way to produce the same sterility effect using genetic control instead of radiation. By adding two self-limiting genes into the species, the moth offspring are unable to survive into adulthood. Scientists also added a color marker into the genetically engineered moths to be able to track and trace the insects in the environment and to distinguish Oxitec insects from the local pests.

Until now, farmers have sprayed crops to reduce infestation from diamondback larvae, but the moths have developed resistance to the poison, according to the USDA.


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Study identifies ways to encourage ‘refuge’ planting, slowing resistance to Bt crops

April 3, 2017 by Matt Shipman

Study identifies ways to encourage ‘refuge’ planting, slowing resistance to Bt crops
Credit: Alexander Steinhof

A new study from North Carolina State University finds a significant shortfall in the amount of “refuge” cropland being planted in North Carolina – likely increasing the rate at which crop pests will evolve the ability to safely devour genetically engineered Bt crops. However, the study also identified actions that may make farmers more likely to plant refuge crops in the future.

For about 20 years, have made use of Bt to limit crop damage from pests. Bt crops, including corn, are genetically engineered to produce proteins from the Bacillus thuringiensis (Bt) bacterium. These proteins are harmless to vertebrates, but toxic to a specific class of invertebrate crop pests.

To date, these Bt crops have been remarkably successful. However, insect pests have shown the ability to evolve resistance to Bt proteins. In order to slow down the development of Bt resistance, farmers who plant Bt crops are urged to plant a certain percentage of their fields with non-Bt crops – called refuge crops. In fact, in the case of Bt corn, farmers are required to plant a section of their fields with refuge crops.

That’s because refuge crops provide fodder for insect pests that are not resistant to Bt proteins. These pests are then able to breed with their Bt-resistant counterparts, diluting Bt resistance in the overall population.

But compliance with planting refuge crops is variable. Some growers plant too little of their fields with Bt crops, and some don’t plant refuge crops at all.

This raised some interesting questions for Dominic Reisig, an associate professor of entomology at NC State and an extension specialist at the Vernon James Research & Extension Center in eastern North Carolina. Reisig divides his time between conducting research and helping farmers deal with problems related to insect . Recently, Reisig began to wonder: How many growers aren’t planting sufficient refuge crops? Do growers understand the rationale behind refuge crops? What can influence whether growers plant refuge crops? And what factors affect a grower’s willingness to plant refuge crops?

To address these questions, Reisig talked with several hundred corn growers in more than a dozen counties in eastern North Carolina.

Reisig found that approximately 40 percent of corn growers who used Bt corn would not plant refuge crops in the next growing season, while another 25 percent weren’t sure. However, a majority of growers did understand the value of refuge crops – and felt they should be planting them.

Reisig also found that there was a high correlation between how much land was devoted to corn, cotton and soybeans in a county, and how likely farmers in that county were to plant refuge crops. The more land being devoted to crops, the more likely farmers were to plant refuge.

“Some of the resistance to planting refuge may be due to a lack of understanding about how important refuge crops are,” Reisig says. “But it’s also likely to be a function of the fact that many of the farms in counties with low refuge crop compliance are smaller operations. Growers may simply be trying to get more crop yield from their acreage – though there is little evidence of short-term benefit, and ample evidence of long-term risk from Bt-resistant pests.”

Reisig also found that better enforcement and peer pressure from other farmers weren’t seen as making farmers more likely to plant refuge crops. Instead, growers said that financial incentives – such as rebates on non-Bt seed – would make them more likely to plant crops, as would the availability of high-yield non-Bt seed.

“This study is really a starting point,” Reisig says. “We know this is a problem. I’m looking for partners in the social sciences to help me figure out how we can help growers make informed decisions and protect the long-term viability of their crops.”

Explore further: Armyworms develop resistance to Bt corn

More information: Dominic D. Reisig. Factors Associated With Willingness to Plant Non-Bt Maize Refuge and Suggestions for Increasing Refuge Compliance, Journal of Integrated Pest Management (2017). DOI: 10.1093/jipm/pmx002

Read more at: https://phys.org/news/2017-04-ways-refuge-resistance-bt-crops.html#jCp

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