Fighting citrus diseases

Researchers produce first transgenic orange plant resistant to Xylella fastidiosa

Imagem: ALESSANDRA ALVES DE SOUZA/IAC Genetically modified plant (left) is less severely affected by Xylella infection than normal orange trees (right)Imagem: ALESSANDRA ALVES DE SOUZA/IAC

Xylella fastidiosa is an opportunistic bacterium that causes citrus variegated chlorosis (CVC). When it infects orange trees through the bite of a leafhopper insect, it immediately begins to multiply and obstruct the channels that transport water and nutrients from the root to the top of the plant. This results in leaves with yellow spots and small, hard fruit that ripen faster and are not suitable for sale. Together with researchers from the University of California at Berkeley, biologists Raquel Caserta and Alessandra Alves de Souza, both of the Sylvio Moreira Center for Citrus Culture at the Campinas Institute of Agriculture (IAC) in Cordeirópolis, São Paulo, were able to obtain a variety of genetically modified orange plants that is resistant to the pathogen. The strategy consisted of introducing a gene from the bacterium itself into the plant’s genome: rpfF is responsible for producing a protein of the same name that reduces the movement of Xylella. In the initial tests, the bacterium’s reduced mobility stopped it from spreading throughout the orange tree, restricting its colonization to only a few parts of the plant.

In Xylella, the RpfF protein controls production of the diffusible signal factor (DSF), a molecule involved in regulating the population density and behavior of the microorganism. After invading the plant and adhering to the wall of the xylem (the set of tubes that bring water and nutrients from the soil up to the leaves), the bacterium multiplies and remains attached to its descendants, forming a network or biofilm that allows them to communicate between each other and behave like a single organism. When the biofilm clogs most of the plant’s vessels, DSF concentration rises and signals the bacteria to stop moving and spreading throughout the orange tree.

In the experiment, the researchers introduced the Xylella rpfF gene into two varieties of sweet orange: pineapple and Hamlin. The goal was to make the plant produce DSF on its own, reducing the bacteria’s ability to establish itself in the xylem. Next, they infected the plants with Xylella and monitored them over 18 months. The orange trees which produced DSF exhibited a milder version of the disease, since Xylella colonized only part of the tubes (approximately 30%). “Some plants do not even show symptoms of the disease,” explains de Souza.

Inserting the Xylella rpfF gene into orange trees could also be effective against Xanthomonas citri, which causes citrus canker. The bacterium enters the plant through openings in the leaves or lesions on the stem or fruit. It multiplies at the infection site and spreads through the healthy tissue, damaging the leaves and fruit. “Inserting the rpfF gene into the orange tree made it produce DSF and interrupted the expression of Xanthomonas genes which are involved in the virulence and pathogenicity of the bacterium,” adds Caserta.

Xylella was one of the worst diseases to affect orange groves in São Paulo during the 1990s; seedlings had been produced in the open air and the plants were exposed to the bacterium’s insect vector. At that time, the disease affected 34% of the state’s orange groves and caused approximately US$100 million (R$327 million) a year in damage to São Paulo citrus production. With support from FAPESP, sequencing of Xylella’s genome and investigation of the biology of the bacteria led to the development of a model to manage CVC which was based on planting seedlings grown in protected nurseries, pruning or removing contaminated plants, and controlling the vectors. Implementation of this model caused the number of orange groves in São Paulo State affected by the bacterium to plummet from 43% in 2008 to 3% in 2017.

Even though damage to the groves was limited, other strategies still need to be developed to combat Xylella, which besides oranges also affects grapevines, olive trees, and almond trees in other countries. Xylella has been causing damage to olive groves in the Apulia region of southern Italy, and recently arrived in Spanish grape and almond plantations. In November, de Souza presented her results in orange plantations at a congress in Palma de Mallorca, Spain. Alongside agricultural engineers Helvécio Della Coletta Filho of IAC and João Spotti Lopes of the Luiz de Queiroz College of Agriculture of the University of São Paulo (ESALQ-USP), she and Italian researchers are participating in a project that funds research and stimulates the transfer of innovative bacteria control solutions to industry, as part of Horizon 2020, the European Union’s main research promotion program. According to de Souza, the transgenic technique used in the orange grove could be adapted for other plants.

Imagem: 2 LÉO RAMOS CHAVES Healthy fruit: with proper management, the number of orange groves in São Paulo State infected with Xylella dropped from 43% in 2008 to 3% in 2017Imagem: 2 LÉO RAMOS CHAVES

Joint strategies
At ESALQ-USP, agricultural engineer Francisco Alves Mourão Filho’s team has been working for roughly 20 years on producing genetically modified orange trees that are resistant to disease. The team also included agricultural engineer Beatriz Mendes, who today is retired. Their most promising results were orange trees with greater resistance to Xanthomonas. The plants received a gene from the moth Trichoplusia ni, which produces an antimicrobial compound. The plants are still being evaluated, but already are showing milder symptoms of the disease. For Mourão Filho, the production of transgenic varieties needs to be accompanied by other management measures that help control the disease in the field.

Despite the promising results, there is still a long road ahead before the modified oranges at the IAC can leave the greenhouses and move into the fields. The next step, according to de Souza, is to file a request for Planned Release into the Environment with the Brazilian National Technical Commission for Biosecurity (CNTBIO), the agency responsible for assessing the safety of genetically modified organisms in Brazil. If everything goes well, the new variety may be on the market as early as 2022.

Interaction between Xylella fastidiosa/insect vector/host plant and approaches to controlling citrus variegated chlorosis and citrus canker (No. 13/10957-0); Grant Mechanism Thematic Project; Principal Investigator Alessandra Alves de Souza (IAC); Investment R$1,246,847.60.

Scientific article
CASERTA, R. et al. Ectopic expression of Xylella fastidiosa rpfF conferring production of diffusible signal factor in transgenic tobacco and citrus alters pathogen behavior and reduces disease severity. Molecular Plant-Microbe Interactions. V. 30 (11), p. 866–75. Nov. 2017.