By Elton Alisson | Agência FAPESP – Researchers at São Paulo State University (UNESP) in Rio Claro and São José do Rio Preto, Brazil, in collaboration with colleagues at the University of Groningen in the Netherlands, have identified a group of compounds that inhibit the action of Xanthomonas citri, the bacterium that causes citrus canker, a disease that attacks all commercially important citrus varieties and species.
The results of the study, supported by FAPESP under the aegis of a cooperation agreement with the Netherlands Organization for Scientific Research (NWO), were presented at the Biobased Economy Workshop held on April 5-6 at FAPESP’s headquarters in São Paulo.
“We identified dozens of compounds with the potential to combat Xanthomonas citri,” said Henrique Ferreira, a professor in the Biochemistry & Microbiology Department of UNESP’s Rio Claro Bioscience Institute (IBRC), during a presentation to the workshop.
Working in partnership with Luís Octávio Regasini, a professor in the Department of Chemistry & Environmental Sciences of UNESP’s São José do Rio Preto Bioscience, Letters & Exact Science Institute (IBILCE), Ferreira has focused in recent years on identifying compounds with a low environmental impact that can protect citrus plants from infection by X. citri or that can stop the disease from spreading to new areas. His research projects are also supported by FAPESP.
The bactericidal formulations that are currently used to prevent the propagation of citrus canker, which causes lesions on the leaves, fruit and stems and which may lead to defoliation and loss of fruit, are based on copper, a toxic element that builds up in the environment.
“These products contaminate the soil and water sources,” Ferreira said.
To search for an alternative, the group began screening compounds capable of killing X. citri by attacking vital processes, such as the cell division of the microorganism.
During cell division, each cell initially grows by replicating its genetic material and eventually divides into two daughter cells.
A crucial stage in the process is the assembly of the division septum, a structure consisting mainly of a protein called FtsZ, which forms a ring in the middle of the cell when it is about to divide.
“We chose this protein, which is essential for the cell division process in X. citri, as a target for anti-bacterial compounds,” Ferreira said.
Upon analyzing the compounds that inhibit FtsZ, the researchers noticed that they were all polyphenols, which are natural substances found in plants, such as flavonoids and tannins.
Based on this observation, they decided to test whether gallic acid was active against X. citri or interfered with cell division in this bacterium.
Gallic acid is an organic substance that is also known as 3,4,5-trihydroxybenzoic acid. It is widely present in nature and is found in many plants, such as tea leaves. It is considered a standard for the measurement of polyphenols, expressed as mg/g gallic acid equivalent.
“Gallic acid is involved in protection and regulation activities in plants and displays biological activity against various microorganisms,” Ferreira said.
To evaluate whether gallic acid could kill X. citri, the researchers first synthesized 15 esters of the compound, called alkyl gallates, and performed a series of tests with these substances to see whether they interfered with the bacterium’s ability to colonize citrus plants.
In one of these tests, the bacteria were exposed to the compounds and then inoculated into citrus leaves to measure the extent to which the alkyl gallates inhibited the microorganisms’ ability to infect the plants. The results were positive: the bacteria lost the ability to colonize citrus leaves.
“This is evidence for these esters’ potential to protect plants against infection by X. citri,” Ferreira said.
To determine the mechanism of action of these alkyl gallates, the researchers worked with colleagues at the University of Groningen, led by Professor Dirk-Jan Scheffers, to perform a number of experiments in which they used purified FtsZ protein from Bacillus subtilis.
Although this bacterium differs from X. citri in some respects, the two species share a conserved cell division process.
“That means observations of the effect of alkyl gallates on FtsZ from B. subtilis can be extrapolated to X. citri,” Ferreira said.
The results of the experiments showed that the compounds were also active against B. subtilis.
The alkyl gallates also disturbed the subcellular location of FtsZ from B. subtilis and affected the function of this protein, forming structures that could be easily observed by electron microscopy and sedimented in a high-speed centrifuge.
“These results suggest alkyl gallates act on the cell division machinery in X. citri, as well as B. subtilis,” Ferreira said.
Form of treatment
The researchers are now trying to find out how citrus plants can be treated with alkyl gallates.
To develop a method to immobilize the gallates on the plant surface, they have begun a research collaboration with Ulrich Schwanenberg, a professor at RWTH Aachen University in Germany.
This project is supported by FAPESP under a cooperation agreement with the German Federal Ministry of Education and Research (BMBF).
“The aim of the project is to produce hybrids of alkyl gallates and peptides capable of binding to the leaf surface,” Ferreira said.
Regasini has produced more than 100 promising compounds, he added. They include nine new compounds that differ from alkyl gallates and that display greater potential to combat citrus canker.
According to the data presented by Ferreira, the incidence of citrus canker has risen in recent years in citrus groves in São Paulo State. Approximately 75% of the orange juice exported by Brazil is produced in São Paulo, according to the National Association of Citrus Juice Exporters (CitrusBr).
Six months after a mid-2009 change to the state’s legislation on the control of citrus canker, establishing that only plants within 30 m of an infected plant must be eradicated, the incidence of the disease in São Paulo State had risen by more than 80%.
In 2013, another change to the law allowed growers to eliminate a contaminated plant and to spray plants within 30 m with copper.
“These changes to the legislation favored the presence of X. citri in São Paulo’s citrus groves,” Ferreira said.