INNER WORKINGS

Portable DNA sequencer helps farmers stymiedevastating virusesLeah Shaffer, Science Writer

Armed with a battery-operated minicomputer, a hand-held DNA sequencer, and portable DNA-extraction ma-chine, researchers gathered in a cassava field in Tanzanialast August to chase down a major plant pest. Their planwas unprecedented: sequence the whole genome ofthe plant material to detect all potential viruses—anddo so in a single day on a farm. “We called it tree lab, wewere sitting under a tree,” says Laura Boykin, a compu-tational biologist at the University of Western Australia.

Because plant samples are typically sent overseasto test for viruses, there’s a big distance between thefarm and the laboratory—thousands of miles if you’rein eastern Africa. But within a few hours, a group ofresearchers, part of the Cassava Virus Action Project,determined what viruses infected the cassava crops onthe farm and, more importantly, alerted farmers theywould need to plant new crops that are resistant to theviruses they found.

The researchers call themselves an “action group”because they’re “taking the level of knowledge we getas scientists, down to the farmer,” says Peter Sseruwagi,a research scientist at Mikocheni Agricultural ResearchInstitute (MARI) in Tanzania. “We’re able to show thefarmer in a single day, what their crops are infectedwith.”

This is a big deal, especially in Africa, where cassava,a sweet potato–like starch, is intricately tied to lives andlivelihoods for many of the people on the continent.The two main cassava diseases, cassava brown streakvirus and cassava mosaic disease (CMD), are estimatedto cause a loss of $2 billion to $3 billion annually (1).CMD alone can cause loss of up to 40% of a crop (2).The work of the cassava action project, however,doesn’t only have an impact on African farmers. Itcan then pave the way to better crop science aroundthe world.

A group of farmers looks over a field of cassava in Mbinga, Tanzania, in 2016; unearthed cassava root ispictured (Inset). Researchers in the Cassava Virus Action Group have been able to increase yield by usingportable DNA sequencers and extractors that facilitate the early detection of diseased plants. Image credit: LauraBoykin (photographer).

Published under the PNAS license.

www.pnas.org/cgi/doi/10.1073/pnas.1901806116 PNAS | February 26, 2019 | vol. 116 | no. 9 | 3351–3353

INNER

WO

RKIN

GS

Diagnosing Sick PlantsCassava diseases are often spread by the whitefly,Bemisia tabaci, a notorious vector for more than 100plant viruses worldwide. In Florida for instance, re-searchers are grappling with a type of whitefly calledB. tabaci biotype Q that is resistant to insecticides andcan spread diseases to ornamental and vegetablecrops. Once plants have these diseases, they may ormay not be symptomatic, so they can easily spreadas farmers share what appear to be healthy plants.

To accurately detect a virus, plant virologists have acouple tools: If it’s a well-known virus, they can useELISA (enzyme-linked immunosorbent assay), whichdetects virus antigens using manufactured antibodies.But viruses often mutate in a way that the antibodies inELISA cannot detect. And disease is often not just fromone stable source: Cassava diseases have nine differentviruses (3) associated with them, explains Alana Jacobson,a plant pathologist at Auburn University.

Jacobson, who has studied African cassava viruseswith Sseruwagi, says they are still trying to understandthe distribution of cassava viruses, the different virusabundance in different locations, and what causes it tomutate. Detection tools such as ELISA are not equip-ped to identify a rapidly changing virus.

Researchers can also look for the DNA or RNAsignatures of the virus using PCR, which makes use ofmolecular primers that amplify certain sections of nu-cleotides. The primers serve as bookends and allowresearchers to cut out the section of DNA or RNA theywant to amplify or copy to identify a virus. But to selectthe correct primers, the researchers need some idea ofwhat sequence they’re looking for. If you have a rap-idly mutating virus or new viruses with a vastly differ-ent sequence, that primer isn’t going to bind with theDNA, which means you can’t detect it, explainsJacobson. With PCR, it’s a shorter segment and notthe “long read” of the full sequence, which meansthey could miss something. Also, researchers aroundeast Africa can’t always easily get to PCR equipment.And they need answers fast.

Taking ActionThe Cassava Virus Action Project is a partnershipamong primarily Ugandan, Tanzanian, and Kenyanresearchers, including Sseruwagi, and MARI directorJoseph Ndunguru. They ended up partnering withBoykin in 2013. But up until last year, they were relianton laboratories outside of the country to do much ofthe genetic sequencing work, says Sseruwagi.

Typically, they’d have to send plant samples to alab in the United Kingdom or Asia. Then it would take2 to 3 weeks, sometimes even a month or more, be-fore they would get results—and that doesn’t includethe time it takes to analyze those results. By the timethe findings get back to the farmer, 6 months or even ayear could pass, he says.

That changed in 2016, with news of a new se-quencing device used to track Ebola viruses (4). Thehandheld device, called MinION, made by OxfordNanopore Technologies, sequences DNA without theuse of primers. MinION identifies molecules using amicroscopic tube, called a nanopore, on a syntheticmembrane that is charged with an electric current.Drop a molecule of DNA through that tube, and thecurrent changes in a way that identifies the sequenceof nucleotides. It’s small, fast, and gives researchersthe code for the whole genome, not just one amplifiedsegment. MinION’s rival, Pacific Biosciences’ Sequeldevice also provides these “long-reads” of DNA se-quences with a reportedly higher degree of accuracy(5), but its $350,000 price tag keeps it out of reach formany researchers.

MinION, at $1,000, is much more affordable, andits portability is useful for researchers in Africa lookingto track human viruses. Why not, thought Boykin, trytracking plant viruses as well?

Fast ActingIn their first tests of the MinION device in 2017, theteam visited farms in Uganda, Tanzania, and Kenya tosee how the device, compared with PCR, detectedviruses. They confirmed plants that tested positivethroughMinION for cassava viruses were also found tobe positive through PCR. In a few cases, MinION de-tected the virus when PCR did not. The researchersconfirmed there was a virus because those plantsdeveloped symptoms 3 months later, says Boykin. TheMinION test took about 48 hours to get results, butthe researchers wanted it to be even faster. Themissing tool was the ability to extract DNA in the field,a solution Boykin first encountered when she gave atalk in New Zealand earlier last year. There, she met JoStanton, a researcher in the department of anatomy atthe University of Otago, who has partnered with a techfirm to produce what she calls the PDQeX DNA ex-traction system (“pretty damn quick extraction”).

It all came together last August and early Sep-tember as the team visited farms in Tanzania, Uganda,and Kenya to test their devices in the field. Theystarted in Tanzania, and by the time they finished inKenya, they had whittled the process down to 2 to3 hours. Conventional means would have takenmonths to get results, says Boykin.

Cassava diseases are often spread by the whitefly,B. tabaci, a notorious plant virus vector. Image credit:Monica Kehoe (photographer).

3352 | www.pnas.org/cgi/doi/10.1073/pnas.1901806116 Shaffer

And the farmers were “incredibly happy to seeresults,” she adds. “It’s a big trust-building thing withthem as well.” It also builds enthusiasm among Africanresearchers keen on tools that don’t rely on lots ofpower and fast Internet, both of which can be hard tofind in parts of Africa. The data analysis of the genomeis conducted by using another Oxford NanoporeTechnologies device, a book-sized computer calledMinIT, a module that plugs into MinION and can becontrolled via laptop or tablet. With battery-operatedgear, scientists can plug MinIT into their laptops in-stead of finding reliable Internet to download enor-mous data files from remote laboratories.

Whole sequencing can even help spot viruses be-fore they affect crop yields. Last summer, the re-searchers visited a region of Tanzania where a groupof farmers tended supposedly virus-resistant plants.First, the researchers checked the status of thosehealthy plants. “Wewere hoping that we wouldn’t findvirus, but we did,” says Boykin. Whether the plant hassymptoms or not, if the virus is present, even in lowlevels, farmers will need to replant with fully virus-freecuttings. Farmers can pick up virus-resistant plants atregional distribution centers, according to Boykin.

Disease-resistant varieties are going to lose thatresistance eventually, adds Boykin. That’s one of theadvantages of doing the whole-genome sequencing:They can characterize the viruses over time before theplant has symptoms. The more that researchers—anda given country’s agriculture agency—understandhow the virus changes and reacts to different man-agement strategies, the better equipped they’ll be tosave crops. “Being able to predict when the variety isgoing to break down is crucial,” says Boykin. Catch thevirus early, and they can then share virus-free plants toprevent the spread.

Sseruwagi says that many farmers in Africa areunaware of the diseases or pathogens they’re dealingwith. The first method of management is chemicalpesticides and insecticides, neither of which would fixa plant infected with a virus. “Cassava is a low-incomecrop and many farmers cannot afford to spray,” hesays, adding that “ensuring the health of plantingmaterials is of paramount importance.”

Other means of chasing down plant viruses are indevelopment as well. Jane Polston, a plant patholo-gist at the University of Florida, is working on de-veloping faster virus detection tests using recombinasepolymerase amplification (RPA), which functions at

room temperature and can work off crude DNA ex-tracts, unlike PCR. In laboratories already equippedwith PCR gear, techniques such as RPA or other formsof next-generation sequencing may do the trick. Com-panies such as Illumina (which recently purchased Pa-cific Biosciences) and 10X Genomics are providingtechnology to allow PCR equipment to produce longreads of genetic sequences that provide a more com-plete picture of a genome. According to an article inTrends in Genetics, researchers found the main down-side of MinION is its degree of error (approximately 15%in a 2017 test) compared with other equipment. How-ever, authors of that article note that the company’snewest technology lowers that error rate to 3% (5).

Scarce ResourcesFunding for such approaches is a challenge, saysSseruwagi. They would like to roll out more training forstudying not only cassava but also other crops. “Ournext steps [are] to look for funding that will allow us touse this on a routine basis,” he says.

“It’s frustrating to me because I think a lot ofpeople are just scared of what they don’t know,” saysBoykin, speaking of the funding challenges of gettingsupport for a new type of sequencing. But she has allthe motivation she needs when she sees farmers.

A little over a year ago, they visited Asha Mohammad’sfarm in Tanzania where there was almost no yieldbecause of the cassava diseases. Boykin and the otherCVAP researchers tested Mohammad’s farm and foundher crop infected with CMD, and the farmer and hercommunity replanted with new resistant varieties. Theteam visited again recently, and Mohammad’s yieldhad increased dramatically.

“It was the best day of my life. . . The woman wasbuilding a house for her family because she had somuch cassava that she was able to start selling it,”says Boykin. “We just need to do this, over and overagain.”

1 Boykin L, et al. (2018) Real time portable genome sequencing for global food security. F1000 Res 7:1101.2 Sseruwagi P, et al. (2018) The first transcriptomes from field-collected individual whiteflies (Bemisia tabaci, Hemiptera: Aleyrodidae): Acase study of the endosymbiont composition. Gates Open Res 1:16.

3 Jacobson AL, Duffy S, Sseruwagi P (2018) Whitefly-transmitted viruses threatening cassava production in Africa. Curr Opin Virol33:167–176.

4 Quick J, et al. (2016) Real-time, portable genome sequencing for Ebola surveillance. Nature 530:228–232.5 van Dijk EL, Jaszczyszyn Y, Naquin D, Thermes C (2018) The third revolution in sequencing technology. Trends Genet 34:666–681.

“It was the best day of my life. . . The woman wasbuilding a house for her family because she had somuch cassava that she was able to start selling it.”

—Laura Boykin

Shaffer PNAS | February 26, 2019 | vol. 116 | no. 9 | 3353