The race for rugose resistance
Researchers are revisiting ancestral tomato varieties in search of genetic tools against ToBRFV.
May 18, 2021 By Greta Chiu
As the world continues their battle with COVID-19, vegetable researchers and seed providers find themselves racing to develop resistance against a different pathogen.
First identified in Israel in 2014, the tomato brown rugose fruit virus (ToBRFV, a.k.a. the rugose) is one of the most recent members of the tobamovirus genus to make headlines. Over the past several years, the virus has been reported in Mexico and the United States, as well as multiple countries in the EU including Spain, Italy and the Netherlands. According to the EPPO Global Database, Belgium reported its first case earlier this year in 2021, and China in 2019.
“Characteristics of tobamoviruses are that they tend to be very persistent in the environment, they are highly transmissible and easily spread mechanically, and there are very virulent strains that can be damaging to host crops,” said Cara McCreary, greenhouse vegetable IPM specialist at the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), during a webinar held in April 2020.
The tobamovirus genus contains 35 known species, including the tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). Resistance genes against TMV and ToMV have been bred into most commercial varieties of tomatoes. Known as Tm-1 and Tm-2, a different version (or allele) of the second gene known as Tm-22 has been cited as the more robust and stable of the three, but it doesn’t seem to work against ToBRFV.
“This Tm-22 gene was the gold standard against tobamoviruses in tomatoes for the last 40 years, and it worked very well until ToBRFV emerged,” says Dr. Jonathan Griffiths, research scientist at Agriculture and Agri-Food Canada (AAFC).
A paper published in scientific journal Plants in 2021 by Israeli scientists Zinger et al. documents how none of the tested tomato cultivars with
Tm-2, Tm-22, or either one of the two alleles stacked with Tm-1 were resistant to ToBRFV. This came as a surprise to the authors given the different targets of each gene.
Where Tm-1 inhibits proteins essential to viral replication, Tm-2 and its allele Tm-22 prevent the virus from spreading between plant cells.
Tm-22 can also trigger a hypersensitive response as part of the plant’s own defense mechanism, adds Griffiths. In the case of ToMV and TMV, this often leads to cell death wherever the virus is found, and effectively prevents it from replicating and spreading to healthy plant cells.
Back to square one
Similar to how Tm-1 and Tm-22 were once found, researchers are using a method called forward-genetics screening to identify candidate genes for rugose resistance. This approach takes a broad range of different tomato varieties and looks for ones unaffected by the virus, then identifies the specific gene or group of genes responsible, explains Griffiths.
To widen the search, they’re using ancestral varieties of tomatoes from areas such as South America and Chile. “Places where they’re undomesticated,” he explains, where they’ve been growing in the wild for thousands of years. This approach allows them to draw from a larger pool of genetic material and naturally occurring variations. “If you screen enough of them, you are likely to find some that are resistant. That let’s you identify what gene it is and then figure out the mechanism of resistance.”
The same Zinger et al. study identified a new gene that can provide tolerance to the rugose.
“Tolerance is good. You don’t get symptoms, your fruit looks okay, but the virus is still there. It’s still spreading and it can mutate and cause problems in the future,” explains Griffiths.
“But they also found a resistance allele,” he adds. Combined, the Tm-1 resistance gene and this new tolerance gene could provide resistance to the rugose together.
Not much is publicly known about the newly discovered gene. The authors of the research paper noted uncertainty around whether Tm-1 plays a direct role in rugose resistance or whether it involves other genes linked to Tm-1.
In the meantime, research efforts are not slowing down. As the authors of the study pointed out, they only screened 160 different tomato varieties – a relatively small population. These varieties were chosen for their genetic similarity to the cultivated tomato, which would help speed up breeding efforts for commercial production.
In Canada, Griffiths’ research group is casting a wider net by expanding the genetics screen to include thousands of different tomato varieties. This will give them much more genetic variation to work with and a greater chance at identifying additional genes for resistance. It means providing more tools to growers and breeders in developing varieties that are not only rugose-resistant, but also commercially viable.
As Griffiths explains, a single rugose-resistance gene might be linked to other traits that promote undesirable characteristics, such as lower fruit quality or undesirable taste. “But if you have multiple resistance genes, then you can find ways to eliminate these negative traits more easily.”
The research will also help the industry gain a better understanding of the virus and its potential weak points. But it also helps safeguard against potential mutations.
“In the event this virus mutates and overcomes the resistance, then you have other sources you can use to rapidly breed into commercial lines and restore resistance against the virus,” says Griffiths.
Currently, Griffiths is collaborating with Steven Loewen at the University of Guelph, as well as Travis Banks and Valerio Primomo at Vineland Research and Innovation Centre. Both research institutions have access to large numbers of varieties and lines which combine genetic diversity from ancestral and wild tomatoes in crosses with commercially adapted varieties. The process of growing, testing and evaluating for resistance and commercial viability is estimated to take about five years.
“This is a very sensitive project that we’re taking every precaution with because of how infectious the virus is and how easy it is to spread,” adds Griffiths. “We don’t want our research to end up contaminating a grower’s crop. In order to contain the virus, we have enacted multiple safeguards and protections including growth room restrictions to ensure we contain this virus.”
Part of those precautions involve testing at different research centres across Canada, located away from major horticultural production areas. The research teams are now in the process of shipping seed to begin screening the different varieties.
Also targeting the rugose, Griffiths’ research group is starting a separate project that will analyze the feasibility of detecting ToBRFV using greenhouse beehives. Early results have been promising, he says.
Bringing resistance to market
Since the emergence of the rugose, seed companies have been fast-tracking their breeding programs to incorporate rugose resistance into the pipeline.
“Breeding doesn’t happen overnight,” says Juan Labastida, marketing specialist at Enza Zaden in Mexico. Anything on the commercial market has been in progress for the past three to five years, at least.
The industry’s initial breeding efforts have largely targeted the unheated greenhouse market in the past few years. Areas include the Mediterranean, the Middle East and Mexico.
“It’s also where the biggest problems are and where the biggest volumes of tomatoes are grown,” says Freek Knol, Enza Zaden’s regional manager of greenhouse business in North America. “We’re going as fast as we can. Is it as fast as we’d like or as the industry likes? Probably not, but we have to do our homework.”
“The first step was identifying resistance loci in our programs and pipelines, and in our tomato breeding pools around the world, then selecting elite breeding lines establishing an accelerated conversion plan to bring resistant varieties to market as soon as possible,” says Carlos Buzio, who leads strategic marketing for the North American protected culture segment at Bayer’s vegetable seed divisions, De Ruiter and Seminis. “But with or without resistance, the product still has to perform.”
Both Enza Zaden and Bayer are conducting thorough testing and trialling in order to bring the best products to market – not only in resistance, but in yield, flavour and other desirable agronomic and consumer traits.
The International Seed Federation recommends the use of specific terms in describing how a variety interacts with a pathogen. It’s either susceptible or, in the case of resistant varieties, displays intermediate resistance (IR) or high resistance (HR) to a pathogen. The term ‘tolerance’ is reserved for describing plant responses to abiotic stress.
“A susceptible variety is when the virus can freely enter the plant and easily multiplies in [it],” says Knol. “With IR varieties, it’s either a matter of the plant slowing down the development of the virus or the plant being able to live with the virus in it.” IR varieties may be symptomless. Although the crop doesn’t develop signs of the rugose, it still harbours the virus.
“It’s essential to continue to follow best practices in phytosanitary hygiene even with IR varieties, as there may be a low level infection present, even though you can’t see it on the fruit,” adds Marjan Willett, protected market development lead for the Americas at Bayer Crop Science. “It’s important to understand what IR resistance does for your crop.”
But when it comes to HR varieties, the mechanism can be completely different, as is the case for Enza’s latest development. “In an HR variety, the plant will actively block the virus,” says Knol. “The virus will not be able to multiply after it is transferred to the plant, and if I go back to the plant two weeks later and start doing measurements, I will not find the virus in [it].”
That said, resistance genetics cannot be treated as a magic bullet. Crop health and integrated disease management are equally important.
Under controlled conditions, an HR variety may be completely immune to the rugose. However, invading pests, diseases and other stressors can affect the strength of the plant’s immune response. “You could potentially find some living virus on the (HR) plants, but in a very low dose,” says Knol.
Another caveat is that current resistance genes have only been trialled on known strains of the rugose. The emergence of new strains could put the industry back at square one. This emphasizes the importance of the work being carried out by Griffiths and his collaborators, as well as researchers and seed companies worldwide.
“It’s not just a question of Bayer or another company finding resistance and launching it into the market,” says Buzio. “That is not the solution long-term. We’re looking for multiple sources …. that offer more protection than one solitary source could potentially offer and also give us protection long-term to deal with changes in the virus that could happen as they’ve happened with other tobamoviruses. What we’re pursuing is a long-term strategy to maintain resistance against the rugose over the years to come.”
Bayer to release IR varieties this year
Bayer Crop Science plans to launch two varieties with intermediate resistance (IR) to the rugose this year. Both were bred specifically for the glasshouse environment in the northern hemisphere.
Currently being commercially trialled is a cherry plum (grape) tomato called ‘Navetta’ that launches in Canada this fall.
The second variety planned for release in 2021 will be a pink beef tomato known as ‘Yarmaru.’
The Bayer team expects to release tomatoes-on-the-vine and varieties in other tomato segments starting in 2022, with high resistance material in development.
Enza Zaden to ramp up HR variety testing
Last year, Enza Zaden’s tomato breeding team announced the identification of a gene which confers high resistance (HR) to the current strain of the rugose.
The gene is said to allow plant proteins to inhibit the virus before actively breaking it down. The identity of the gene remains undisclosed due to intellectual property rights.
HR varieties with this gene have been tested on non-heated, greenhouse tomatoes and they’ve held up in high rugose pressure areas in the Mediterranean. For heated greenhouse tomatoes including those meant for the Canadian greenhouse market, Enza Zaden will be ramping up their product development trials mid-to-late next year before they head into commercial testing.
What about greenhouse peppers?
There are L genes in commercial pepper varieties that are used for resistance against ToMV and pepper mild mottle virus (PMMoV), said Cara McCreary, OMAFRA greenhouse vegetable IPM specialist, in a webinar held last April. “So far, the rugose does not seem to be able to overcome those resistance genes, but more information is needed.”
Research has suggested that other genera from the Solanaceae family can act as potential hosts. Candidates include tobacco, weeds, petunias and certain ornamentals, though the range of plant hosts is still being established.
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