New method detects pineapple viruses faster and more economically

Scientists from Embrapa Cassava and Fruits (BA) have developed a method capable of detecting the complex of viruses that cause pineapple wilt more quickly and cost-effectively. The technique, which uses short chains of DNA or RNA molecules (called primers, oligonucleotides) to track microorganisms, was patented as an invention in 2021 by the National Institute of Industrial Property (INPI). Of great interest for the manufacture of diagnostic kits by partner companies, it can benefit the entire pineapple production chain, since this is one of the worst threats to the crop worldwide, and still without definitive control or identified source of resistance.

Titled “Oligonucleotides, use, method and reaction composition and kit for detecting viruses associated with the cause of pineapple wilt”, the patent was published in the September 2021 issue of the Industrial Property Journal. Like all granted invention patents, there is a 20-year term from the filing date – as the start of the patenting process is called – which occurred on December 26, 2012.

Oligonucleotides, also called primers, are short chains of DNA or RNA molecules commonly used in molecular biology and medicine to screen for viral diseases and infections and as primers in DNA sequencing.

“The idea was to develop a test that would detect the three wilt viruses at the same time, without discriminating one by one. The intention is to know who has the virus and who does not. When indexing, it does not matter which virus is present; what matters is whether the plant is healthy or infected,” explains Eduardo Chumbinho de Andrade, a researcher at the Plant Virology Laboratory of Embrapa Cassava and Fruit, responsible for the invention and registration of the patent. 

"Pineapple wilt is a disease caused by a virus and transmitted by an insect. In addition to being essential for research work, detection kits are very important to ensure the health of planting material. Obtaining the patent reinforces the innovative nature of the research carried out by Embrapa Cassava and Fruits", says researcher Francisco Ferraz Laranjeira Barbosa, deputy head of Research, Development & Innovation at Embrapa Cassava and Fruits.

Method reduces the cost of testing and working time by 1/3

Indexing is the process that demonstrates the presence or absence of a virus in a plant and is carried out in the laboratory before the material is multiplied, guaranteeing the health of the seedlings. At Embrapa Mandioca e Fruticultura, the detection of the presence of viral DNA/RNA is done using the real-time polymerase chain reaction (PCR) technique (RT-qPCR), the same used in the so-called “gold test” to detect the new coronavirus and many other diseases caused by viruses in humans.

According to Andrade, wilt is caused by the viral complex called Pineapple Mealybug Wilt-Associated Virus (PMWaV), transmitted by the mealybug Dysmicoccus brevipes. The complex is made up of three different species of viruses, but which share regions of the genome that are very similar. “The position of the genes and even the sequence of some of them are also similar. So, we searched for all the sequences available up to that moment and made an alignment between them, placing one sequence under the other and looking for the most conserved regions between them, that is, stretches of the genome in which the sequences were identical, or with some specific variation”, details.

In the study of these sequences, two regions with high identity between them were identified. In them, the researcher designed oligonucleotides called degenerate. “Part of the degenerate sequence is the same for all viruses and part is variable. Then, it is possible to detect the three viruses in the sample, which reduces the test value and working time by 1/3. In other words, instead of carrying out three reactions [tests] and using three microtubes, among other components, the process can only be carried out once. If the result is positive, it is already known that that plant is infected and, in many cases, this information is sufficient”, says Andrade. “With this kit, the savings in material, financial resources and time in a large-scale indexing process are excellent”, he highlights. 

According to the researcher, the enzymes and reagents in the kit are already available on the market. “What exactly varies is this initiator. It is the differential of the reaction, which gives the specificity and which will indicate what will be detected. If you change the starter something else is detected. It will only bind to the sequence that is identical or complementary to it, guaranteeing the exclusive detection of pineapple viruses”, he points out.

Gene bank is the basis of molecular studies

In fact, the invention began to emerge for the researcher when it was necessary to index the BRS Ajubá pineapple – a variety that would be launched in 2009 – to send clean and healthy matrices to the biofactory that had won the bid at the time. “I had to index a large number of plants and do a test for each of the three wilt viruses. Since I had already worked with viruses from other crops, such as vein mosaic, begomovirus and cassava phytoplasma, and I was familiar with the approach of degenerate primers, I thought I could do the same in pineapple crops. Fortunately, we found conserved regions and managed to obtain two pairs of primers”, he recalls.

Although it was not used in the indexing of the BRS Ajubá, which was already in progress, the technique that generated the patent was later used to index a large part of Embrapa's Pineapple Active Germplasm Bank (BAG), which is where the research into new varieties and products, such as plant fibers and biomolecules for the pharmaceutical industry. The innovation was also applied in parallel to the new cryotherapy methodology developed by Embrapa Mandioca e Fruticultura as an important alternative for clonal virus cleaning for pineapple trees.

The granting of the patent coincided with the award, in October, by the Brazilian Association of Plant Tissue Culture (ABCTP), of a work on cryotherapy written by researchers from Embrapa Cassava and Fruit Farming Fernanda Vidigal Duarte Souza and Eduardo Chumbinho de Andrade, by professor from the Federal University of Recôncavo da Bahia (UFRB) Everton Hilo de Souza, and by UFRB scholarship holders Patrícia Araújo Guerra, Daniela de Andrade Silva Max and Rafaelle Souza de Oliveira.

Long-term objective is to benefit the entire pineapple production chain

Initially, the target audience for the invention is companies that sell pathogen detection kits, but it is expected that, over time, the entire pineapple production chain will benefit. 

Currently, most Brazilian farmers use field seedlings, which are cheaper and more accessible – often produced on the farm itself or from other producers – and without any guarantee of phytosanitary quality, while the ideal is to plant seedlings produced from healthy mother plants or seedlings obtained in biofactories using micropropagation techniques. “Unfortunately, there is still no great demand for quality material, for micropropagated seedlings, but when the Ministry of Agriculture, Livestock and Food Supply (MAPA) actually establishes standards for the production of mother plants, this is a demand that will increase significantly and, consequently, the interest in the patent will also increase”, believes Andrade.