Unprecedented methodology in plant health allows early detection of HLB

A methodology never used in the vegetable area in Brazil has been the main strategy of the State Agricultural Defense Agency of Bahia (adab) to maintain the state free from huanglongbing (HLB), also known as greening, or “fruit yellowing”, the most destructive citrus disease in the world and still without definitive control. These are sentinel routes, whose objective is the early detection of the bacteria that cause HLB (Candidatus Liberibacter (Ca.L.) in nymphs and adults of the psyllid insect Diaphorina citri, main transmitter vector of these microorganisms.

Bahia is the fourth largest citrus producer in Brazil and the entry of the disease could have a major impact on the state's economy. The methodology is part of the HLB BioMath 3 project, led by researcher Francisco Laranjeira, deputy head of Research & Development at Embrapa Cassava and Fruit Growing, and integrates the Plant Health portfolio from Embrapa, with monitoring from the Ministry of Agriculture, Livestock and Supply (Map).

Laranjeira explains that the sentinel route technique is widely used in ecological studies and consists of determining a straight line that crosses a certain region for sampling. In the case of Bania, the routes used were defined by Adab.

To the general coordinator of Plant Protection of the Department of Plant Health and Agricultural Inputs (DSV) from Mapa, Graciane Castro, “this is an extremely innovative and promising strategy to add to other actions to prevent the spread of the pest in the country. Mapa is in negotiations with Embrapa Mandioca e Fruticultura to establish a partnership in the sense of validating and expanding this type of strategy at a national level”, he states.

The DSV is responsible for regulating official prevention and control measures and establishes annual goals relating to phytosanitary surveys to detect and delimit the pest and the control actions that must be applied in the areas where it is found. Currently, considering the importance of citrus farming for the states, HLB control actions are carried out in 13 of them by state Agricultural Defense agencies, in conjunction with the Federal Superintendencies of Agriculture, Livestock and Supply (SFA) of the Map in each state.

 Sentinel routes involve more than 50 municipalities in Bahia

Detected in Brazil in 2004, HLB is officially confirmed in the states of São Paulo, Minas Gerais and Paraná — the three largest citrus producers in the country — and an isolated case has already been detected in Mato Grosso do Sul, causing numerous problems for citrus growers. For now, the techniques most used to combat the disease are the planting of certified healthy seedlings, the elimination of infected plants — which has a high cost for the producer — and vector control. In Bahia, citrus farming is an economic activity of great social importance and the entry of HLB can cause great losses.

As part of the HLB BioMath project, since 2011, four positive and three negative sentinel routes have been created by Adab, which are covered quarterly by a team of eight people, including agricultural engineers, agricultural inspectors, technicians and inspection assistants. The positive routes have a greater probability (high risk) of finding infectious insects as they are in the largest citrus producing regions and possible transit of clandestine seedlings, i.e., without a Phytosanitary Certificate of Origin (CFO) or that were produced in states with HLB occurrence. They are located in the West, Chapada Diamantina, Recôncavo Baiano and Litoral Norte and cover 44 municipalities. The negative ones (low risk) are on Linha Verde, on the South Coast and on Estrada do Feijão, totaling eight municipalities. These regions do not have commercial citrus orchards, but they have several real estate and hotel developments, in addition to many vegetable gardens, which sell the ornamental myrtle plant (Murraya paniculata), widely cultivated in gardens and public squares, and which is an alternative host for HLB.

“When putting together a contingency plan to control diseases, you need to think about three things: early detection of infectious vectors and sick plants or plants with symptoms and, then, a plan to manage the problem in a given region. For now, we are still in the research phase, but it is research that, at the same time, is surveillance. The results will tell us how we will consider it in the end”, highlights Laranjeira.

State agricultural inspector Suely Xavier de Brito Silva, coordinator of the Adab Citrus Phytosanitary Project and the work on the sentinel routes, states that the partnership with Embrapa Mandioca e Fruticultura has been fundamental to the success of the undertaking: “It is a defense work, but organized and planned by research. We are executors and, at the same time, clients of the results of ongoing research. The sooner the presence of bacteria is detected, the faster actions can be taken and losses can be minimized. As a defense agency concerned with the surveillance of orchards and the transit of propagative materials, we have established the necessary preventive and, in the future, containment measures for when the pest reaches our territory, always in partnership with Embrapa”.

In addition to the quarterly capture of the insect vector on sentinel routes — three and a half times more abundant on positive routes than on negative routes — Adab intends to implement and monitor two or three Regional Management Areas (Armas) in Recôncavo, Chapada Diamantina and Litoral Norte to record the population fluctuation of the vector and feed a Phytosanitary Alert System, similar to that of the Citrus Defense Fund (fundecitrus), based in Araraquara (SP), is already used in the São Paulo citrus belt. The team is using a drone for aerial surveys and characterization of the citrus landscape. “We intend to establish a biweekly monitoring routine and popularize the need to adopt management practices on a regional scale. However, first, it was necessary to validate some of the 41 georeferenced points per area, which were selected using Google Earth maps”, explains Suely.

 Sample evaluation

Under the supervision of the researcher Eduardo Chumbinho de Andrade, the insects captured by Adab on the sentinel routes are evaluated at the Virology Laboratory of Embrapa Mandioca and Fruticulture, via real-time PCR (qPCR), to verify the presence of the bacteria in the psyllids, in addition to the quality, integrity and concentration of the DNA of the material . The protocol is the same used by Fundecitrus and standardized by Mapa.

The detection technique used is extremely sensitive. “Even if the amount of bacteria found in the insect is low, the qPCR test will detect it”, says Andrade. Since the beginning of the work, almost 10 thousand samples collected have already been processed. In 2013, three samples of the insect collected in Seabra, in Chapada Diamantina, were positive for the bacteria, two from myrtle and one from an orange tree. “After that, we contacted Adab, which carried out new sampling within a radius of four kilometers around the three plants and eradicated those containing the insects. We carried out new analyses, but nothing was found. Despite the detection of the bacteria in the psyllid, from the perspective of Federal Legislation in its Normative Instruction 53/2008, it cannot yet be considered that the pest is present in the state of Bahia. It’s tireless work, but very important for Bahia’s citrus industry”, he highlights.

 Legislation update

In Bahia, the state ordinance 243, of August 13, 2011, establishes the obligation to produce citrus seedlings in a protected, screened nursery, but the use of this protection is not yet a reality in the state. “The screens are made with a very small mesh that prevents the entry of leafhoppers, aphids and the psyllid itself that transmits the HLB bacteria. The screen is an excellent strategy to protect the production of seedlings from various diseases and we, at Adab, invest in awareness raising activities so that citrus growers start using seedlings grown in screen environments”, says Suely.

In Brazil, federal ordinance 32, of 29/01/2020, aimed to submit for public consultation a proposal for a Normative Instruction relating to the review of the then official program to combat the plague, created in 2003 and already outdated. The suggestions sent to the public consultation were consolidated by Mapa in the Ordinance No. 317, of May 21, 2021, which establishes the National Program for the Prevention and Control of the disease called Huanglongbing (HLB) – PNCHLB and aims to strengthen the agricultural production system for hosts of the pest, according to the official list of Quarantine Pests Present, establishing the criteria and procedures for the prevention and containment of Candidatus Liberibacter spp.

 Scientific production

 Part of the routine of the sentinel routes was recorded by Adab agronomist Antonio Lopes, in his thesis for the Professional Master's Course in Agricultural Defense, a partnership between Embrapa and the Federal University of Recôncavo da Bahia (UFRB), defended in 2015.

 The subject was also highlighted in the international journal Neotropical Entomology, in the form of the article “First Report of HLB Causal Agent in Psyllid in State of Bahia, Brazil”, which became a eBook published by UFRB in 2021.