Method for diagnosing papaya blight

Quite susceptible to viruses, the papaya tree (Carica papaya L.) has been suffering major losses in production, which can lead to the destruction of all affected plants. In general, plants attacked by viruses show low vegetative development, lower production yield, poor product quality and shorter productive longevity.

The intensity of the damage caused by viruses logically depends on the host's resistance, the virulence of the pathogen and the environmental factors favorable to the development of the disease.

Papaya ringspot virus (PRSV), papaya yellowing lethal virus (PYLV) and papaya sticky disease virus (PSDV) are the main viruses that infect papaya in Brazil.

Currently considered one of the main diseases of papaya trees, papaya blight has been causing losses of 30 to 40% in production, and can affect up to 100% of the plants in orchards in the states of Bahia, Espírito Santo, Pernambuco, Ceará, Goiás and Rio Grande do Norte. Recently, blight has also been observed in commercial plantations in the state of Paraíba, causing mixed infection in papaya trees together with PRSV.

The disease is characterized by intense exudation and spontaneous oozing of latex with a very fluid consistency from the fruits, petioles and stems of plants infected by the virus. Due to oxidation, the oozing latex darkens, giving it a 'honeyed' or 'blurred' appearance, making the fruits unsuitable for sale. Light spots may also occur on the skin and pulp of affected fruits.

In young plants, before fruiting, symptoms of necrosis may occur on the edges of the youngest leaves, due to the exudation and oxidation of latex. Recently, new symptoms associated with the virus were observed in plantations infected with the honeydew disease, located in the states of Bahia and Pernambuco: exudation and oxidation of latex on the edges of the petals of papaya flowers in the flowering and/or fruiting phase, followed by necrosis.

Papaya plants infected with PSDV also exhibited symptoms of latex exudation and oxidation in the leaf veins of the apex and middle third.

Disease management

Currently, the diagnosis of papaya blight is carried out by monitoring the orchard by observing the characteristic symptoms, which become more evident during the fruiting phase. Training technicians linked to extension agencies, technical assistance, phytosanitary defense and leading producers to recognize these symptoms is of fundamental importance for controlling papaya blight.

Other recommended control measures for the disease are: the use of healthy seedlings and seeds, obtained from orchards where the disease did not occur; the installation of the orchard in areas where the virus is not present; keeping the orchard free of weeds to avoid the multiplication of insects, possible vectors of the disease, and disinfecting the cutting instruments used in cultural treatments and harvesting, with detergent or bleach, before working with the next plant.

The long period between planting seedlings and fruiting has contributed to the spread of the disease, due to the lack of an early, rapid and safe method of diagnosis. This gap ends up considerably increasing the sources of inoculum and delaying control actions.

The eradication of plants infected by honey should be the most effective measure to be adopted. This practice is common among the most enlightened farmers (Nascimento et. al., 2001).

Diagnosis of honeydew: method in use

In the laboratory, disease detection is performed by the presence of dsRNAs in samples of leaves, roots and flowers. To do this, dsRNA is extracted based on the method described by Dodds et al. (1984), with modifications in the volumes used in the extraction, washing and final elution, followed by its electrophoretic analysis in agarose or polyacrylamide gels.

In this method, it takes an average of 48 hours until the analysis results are available. The aim of this work was to simplify the method of detecting honeydew in the laboratory, with a substantial reduction in the time and costs of diagnosis.

Diagnosis of honeydew: proposed method

The methodology described is based on the collection and subsequent electrophoretic analysis, in agarose gels, of latex from papaya trees at different stages of development, which enables early diagnosis in adult plants before fruiting and even at the sowing stage.

Latex collection:

• In fruiting plants: Latex collection from plants in the fruiting phase is done by pricking the fruits with a needle or toothpick. The latex, in a volume of approximately 90 microliters, should be collected in microtubes containing or not sodium phosphate buffer (0,01 M, pH 7,0), since dsRNA is detected equally in pure latex or in buffer collected. In the field, the collected latex should be kept at 4ºC and stored at -20ºC.

• In adult plants without fruiting: In plants where there are no fruits, the latex is collected from the petiole, stem, axil or leaf veins, directly into microtubes containing 30 microliters of sodium phosphate buffer (0,01 M, pH 7,0). In the field, the collected latex should be kept at 4ºC and stored at -20ºC.

• In seedlings: In seedlings, latex is extracted by sectioning the petiole, stem or by grinding 0,5 g of new leaves. The material should be collected directly in microtubes containing 30 mL of sodium phosphate buffer (0,01 M, pH 7,0) and subjected to microcentrifugation at 11.750 g for 10 min. In the field, the collected latex should be kept at 4ºC and stored at -20ºC.

Analysis of collected material

Sample analysis is performed by electrophoresis, with the direct application of 30 mL of latex diluted in 0,01 M phosphate buffer, pH 7,0 (1: 1.5) or without dilution, in 1,2% agarose gel, stained with ethidium bromide, with the run carried out between 80 and 100 V, for three to four hours in TBE buffer (Tris Borate EDTA).

Advantages of the proposed method

• It is characterized by its speed and accuracy in diagnosing plants infected with papaya blight, even in seedlings from two months of age, making it an important tool in the preventive control of the disease.

• It reduces the time to detect the disease, as well as the costs of laboratory analyses, by eliminating high-value reagents used in traditional dsRNA extraction methods. The average time spent diagnosing the disease using this method was reduced to approximately five hours.

• It allows for rapid assessment of the susceptibility of papaya genotypes to the virus.

• Allows you to check the presence of honeydew in new areas.

• In the laboratory, it promotes a considerable reduction in environmental contamination caused by the use of phenolic compounds and other toxic reagents used in other dsRNA extraction methods.

* By Tuffi Cerqueira Habibe e Antonio Souza do Nascimento (Embrapa Cassava and Fruit Growing)

Article published in issue 30 of the magazine Cultivar Hortaliças e Frutas (2005)