Oranges could become genetically resistant to agricultural pests

Researchers plan to transfer genes linked to disease resistance from tangerines and other citrus fruits.

By Elton Alisson  |  Agência FAPESP – The orange, one of the most consumed fruits in Brazil and worldwide, may be about to become genetically more disease-resistant.

A group of researchers at the National Science & Technology Institute of Genomics for Citrus Breeding (INCT Citrus), one of the INCTs supported by FAPESP and the National Council for Scientific & Technological Development (CNPq) in São Paulo State, Brazil, plan to transfer genes related to disease resistance from tangerines and other citrus fruits to oranges.

A study resulting from the Thematic Project “Genomic platforms applied to citrus breeding”, funded by FAPESP via an agreement with CNPq to support INCTs in São Paulo State, was presented during the symposium “From basic to applied research: FAPESP’s support for citriculture research”, held on November 3, 2016, at FAPESP’s auditorium in São Paulo.

“We’ve already succeeded in characterizing several candidate disease-resistant genes in tangerines and other citrus fruits, and we’re now trying to transfer them to oranges in order to develop a modified plant using cisgenics, which involves the transfer of genes from related species that interbreed in nature, whereas transgenics uses genes from unrelated species,” said Marcos Antônio Machado, who heads the Sylvio Moreira Citriculture Center at the Campinas Agronomy Institute (IAC), also the home of INCT Citrus.

“We’re going to apply to CTNBio, Brazil’s biosafety watchdog, for permission to perform experimental assessments,” he told Agência FAPESP.

He said citrus genome sequencing had made it possible to identify the target genes for increasing the resistance of orange trees to agricultural pests.

Completed in 2014 by an international consortium of researchers from France, Italy, Spain and the United States, as well as Brazil’s IAC and EMBRAPA, the citrus genome sequencing project began in 2003, four years after sequencing of the genome of Xylella fastidiosa, the bacterium that causes citrus variegated chlorosis (CVC), a serious disease of sweet oranges and other citrus species.

The genome of the sweet orange (Citrus sinensis L. Osb.) is complex because it is highly heterozygous (its cells contain different alleles of one or more genes) and has many repeated sequences, making it difficult to assemble. Therefore, the researchers decided to sequence a less complex genome that could be used as a benchmark for all citrus genomes.

They chose the clementine (C. clementina), a hybrid of sweet orange and tangerine that is widely grown in Spain. C. clementina is normally diploid (having cells with two sets of chromosomes, one from each parent), but this genome sequence was generated from a haploid plant (containing cells with half the usual number of chromosomes).

Once they had completed the sequencing of the clementine genome, the researchers sequenced the genomes of nine candidate species and varieties related to C. sinensis in order to expand the basis of comparison within the group and to investigate how one species might have given rise to another during evolution.

Among the species and varieties analyzed were the Pineapple sweet orange (a highly productive variety similar to the Pera sweet orange and widely grown in Florida, USA, where it originated), the diploid clementine, the Ponkan tangerine (C. reticulata), the Mediterranean mandarin (C. deliciosa Tenore), the W. Murcott tangor, the Seville sour orange (C. aurantium), the Washington Navel sweet orange, the Chandler pummelo (C. maxima), and the Siamese sweet pummelo (C. grandis).

Their comparison of the genomes showed that C. sinensis L. Osb., the most widely grown fruit crop around the world, is not a pure species. 

Its main progenitors were C. maxima and C. reticulata, as well as some other unknown citrus species.

“The citrus reference genome has been of great help to us in understanding why the tangerine is resistant to CVC and why certain related groups of citrus are resistant to almost all the diseases that attack these plants,” Machado said.

“These varieties are interesting sources of genes that could increase resistance to pests and diseases.”

Disease resistance

According to Machado, thanks to seedling production in a protected environment initiated in 1990 with the support of the Sylvio Moreira Citriculture Center, together with the control of leafhoppers and the removal of old citrus groves, CVC is now considered to be under control by São Paulo’s orange industry (read more in Portuguese at: agencia.fapesp.br/23581).

The diseases that most threaten orange production in the state today, he said, are citrus greening or Huanglongbing (HLB), citrus sudden death (CSD), and black spot.

Researchers at the Center have used several approaches to help the industry combat these diseases, including the development of more disease-resistant varieties via traditional genetic improvement, molecular biology, novel epidemiological strategies, and appropriate management practices.

For example, under the auspices of another Thematic Project funded by FAPESP, they are developing new varieties and hybrid rootstocks and scions (orange varieties crossed with tangerines, mandarins or limes) that are more drought-tolerant and disease-resistant.

The materials are being registered with the Ministry of Agriculture and will undergo field testing in a few months’ time.

“We have a program called ‘Top Grade Citriculture’, whereby we supply materials to growers for them to do field testing,” Machado said. “This enables us to assess the results in terms of production and fruit quality. Growers benefit by gaining access to new material and preference in using it.”

Through the Xylella Fastidiosa Genome Project, also supported by FAPESP, the Citriculture Center’s researchers discovered some of the pathogenicity mechanisms of the bacterium that causes CVC.

That discovery resulted in the development of a novel technique to control CVC based on the use of N-acetylcysteine (NAC), a well-known drug used in human medicine as a decongestant and for detoxification and immune support.

NAC has now been licensed by CiaCamp, a startup founded by a scientist who did postdoctoral research at the Citriculture Center, as the basis for two products currently under development or being field-tested to evaluate their efficacy in combating citrus diseases through a project supported by FAPESP’s Innovative Research in Small Business (PIPE) program.

“We’re testing a biofertilizer and a spraying solution based on NAC for diseased plants with CVC, citrus cancer and greening, as well as healthy plants, and the results have been very promising,” said Simone Picchi, founder of CiaCamp, during the event.

“Fruit diameter increases in both CVC-infected and healthy plants when treated with the biofertilizer,” she added.

Other participants in the event included FAPESP’s CEO, Carlos Américo Pacheco, and its Scientific Director, Carlos Henrique de Brito Cruz. 

São Paulo State Secretary for Agriculture Arnaldo Jardim also attended, along with Orlando Mello de Castro, who heads the São Paulo State Agribusiness Technology Agency (APTA), Sérgio Augusto Morais Carbonell, Director General of IAC, members of the Brazilian Table Citrus Association (ABCM), and representatives of the companies Givaudan, Citograf Mudas, and Citrosuco.