When the rainfall increases in the coming weeks, two biologists from São Paulo State are planning to take the compounds they have been developing over recent years to the orange groves. They’ll be looking to see if these materials, used in outdoor conditions, can guard against the bacteria Xanthomonas citri subsp. citri, responsible for citrus canker, a long-standing disease in Brazilian cultivation, and present in some 20% of plantations.
André Alexandrino, a partner in the startup BionFarm in São Carlos, SP, plans to evaluate the inhibitors that block an enzyme of this bacteria, without which the disease cannot propagate. Henrique Ferreira, of São Paulo State University (UNESP), Rio Claro campus, will deploy into the field with formulae based on eugenol, a dark-brown oil extracted from cloves (Syzygium aromaticum). The two compounds demonstrated good results in the greenhouse and resulted in an integrated project between universities, companies, private investors, and citrus growers.
A global issue, citrus canker takes the form of brown markings on all types of citrus fruits and leaves (oranges, tangerines, and lemons), with some varieties more susceptible than others. Other species of Xanthomonas cause diseases in some 400 plant species, including those that produce grapes, passionfruit, tomatoes, cabbage, broccoli, and cauliflower.
This canker was tackled in Brazil through elimination of blighted plants from 1957, the year in which it was identified in the country, to 2017, when eradication was replaced by other types of disease control, such as the planting of healthy seedlings and more resistant varieties, the use of windbreaks, and destruction of contaminated fruits. The bacteria, which propagates among the plants via rainwater, is also countered with copper-based pesticides; this, though, does not fully solve the issue.
“The price of agricultural pesticides made with copper is still unbeatable, but we are preparing ourselves for when European consumers no longer wish to buy citrus fruits treated with copper,” says Ferreira, of the Center for Research in the Biology of Bacteria and Bacteriophages, one of the Research, Innovation, and Dissemination Centers (RIDCs) supported by FAPESP. Although efficient, this type of agrochemical can accumulate in the soil and plants.
Ferreira saw this firsthand at the end of 2021, when an organic lemon exporter asked for an alternative to sodium hypochlorite (bleach) because a buyer in Europe no longer wanted it used to wash the fruits. Over two weeks at UNESP, he tested and validated the bactericide effect of a solution with 5% eugenol, previously accepted by importers; the solution was immediately applied to the washing of lemons prior to packaging, as detailed in a March 2021 article in Food Processing and Preservation. The results led to eugenol’s inclusion in products Ferreira was already testing against citrus canker.
The tenacity of the disease drives research across several countries, among the most recent by a group from the Dr. Panjabrao Deshmukh Krishi Vidyapeeth University in India, which in September 2023 wrote in the Journal of Plant Disease Sciences that the extract of two plants used in Indian traditional medicine, the amla or Indian gooseberry (Emblica officinalis), and the babchi (Psoralea corylifolia), successfully eliminated X. citri in a laboratory setting. One month later, a team from South China Agricultural University (SCAU) reported on eight compounds that inhibited the disease in citrus leaves in the Journal of Agricultural and Food Chemistry.
Leaves from orange trees with citrus canker, a long-standing blight for global citrus fruit cultivationLéo Ramos Chaves / Pesquisa FAPESP
Integrated project
Ferreira says that eugenol and other essential oils, such as that of oregano (Origanum vulgare) and lemongrass (Cymbopogon spp.), which also presented good results in the greenhouse, constitute the second generation of compounds that he began to test against X. citri some 15 years ago. Gallate, derived from an acid deployed by the plant against insect attacks, represents the first generation and has proven effective in keeping bacterial multiplication in check, as detailed in Frontiers in Microbiology in April 2015.
In addition, Ferreira developed other compounds with colleagues from two universities in the Netherlands, and is working on the formula with specialists from Santa Clara Agrociência, a company producing agrochemicals in Ribeirão Preto, in the interior of São Paulo State. “We have more than 15 formulae tested and validated in the greenhouse, and undergoing economic feasibility studies,” he says.
Alexandrino is optimistic and says that the enzyme inhibitors could compete with copper in terms of price. The efficacy of the two types of compound has been demonstrated to be equal in greenhouse testing, as reported in the journal Microbiology Spectrum in May this year. In partnership with his brother Daniel, he successfully gained the support of two investors, and a large-scale citrus producer is set to provide them with an area for testing.
Development of these inhibitors began around 2009. Looking for relevant X. citri proteins in the evolution of the disease, chemist and pharmacist-biochemist Maria Teresa Marques Novo Mansur, of the Federal University of São Carlos (UFSCar), identified the enzyme phosphomannose iseromase/GDP-mannose pyrophosphorylase (GDP stands for guanosine diphosphate), also known as XanB.
Described in detail in the Journal of Proteomics in 2017, this enzyme helps to produce xantham gum, which facilitates the bacteria’s interaction with the plant. During his doctoral work at UFSCar, concluded in 2020, Alexandrino examined the gene that produces XanB. “After genetic analyses and in vivo testing, we saw that without this enzyme the bacteria didn’t cause the disease in citrus fruits,” says Mansur.
One of her team, chemist Mariana Barcelos, liaised with chemist Carlos Henrique Tomich Paula da Silva, of the University of São Paulo (USP) School of Pharmaceutical Science at Ribeirão Preto, to design molecules capable of inhibiting the enzyme. Over the course of two years, using molecule bases and computer modeling, Tomich and Barcelos, during her master’s work with him, modeled the three-dimensional structure of the enzyme and found the points of connection with other molecules, known as attachment sites.
They then selected the most promising inhibitors, all derived from carbohydrates—three are amino-sugars, and the fourth a phenolic glycoside—which block the interaction of the bacteria with the plant. As these were already in commercial production for other purposes, it was possible to import them, and Alexandrino evaluated them: “We now have some 15 molecules, but not all are feasible because of the production cost.”
The field testing should go on for two years. “The efficacy of the new compounds tends to be lesser than when tested in the greenhouse, because wind and rain facilitate the entry of bacteria into plants, and the sun can speed up the degradation of applied defenses,” warns agricultural engineer Franklin Behlau, of the Brazilian Fund for Citrus Protection (FUNDECITRUS), who is participating in the two lines of research.
Though satisfied with the results to date, Behlau has a concern over the efficacy of the compounds: “It’s not enough to act against the bacteria only when the compounds are applied just before it arrives. In the citrus grove, the effect has to linger for a number of weeks between one application and another. Copper is good at this, even in the wind and rain.”
If testing is successful when concluded, the formal evaluations required by official regulators, for registration and authorization for the sale of new products, should begin.
The story above was published with the title “The quest for alternatives to copper” in issue 344 of October/2024.
Projects
1. Sustainable citrus farming through controlled release of antibacterial compounds from microgel-based formulations (n° 21/10839-4); Grant Mechanism Thematic Project, agreement with the Dutch Research Council (NWO); Principal Investigator Henrique Ferreira (UNESP); Investment R$2,962,257.89.
2. Center for Biology Research on Bacteria and Bacteriophages (n° 21/10577-0); Grant Mechanism Research, Innovation, and Dissemination Centers (RIDC); Principal Investigator Shaker Chuck Farah (USP); Investment R$28,895,915.64.
3. Differential proteomic analysis in Xanthomonas axonopodia: Proteins and genes of biotechnological interest (n° 07/50910-2); Grant Mechanism Young Investigator Award; Principal Investigator Maria Teresa Marques Novo Mansur (UFSCar); Investment R$990,036.24.
4. Functional analysis of the xanB and xylA genes potentially involved in the pathogenicity of Xanthomonas citri subsp. citri (n° 20/05529-3); Grant Mechanism Regular Research Grant; Principal Investigator Maria Teresa Marques Novo Mansur (UFSCar); Investment R$314,088.22.
5. Planning and biological activity of phosphomannose isomerase (PMI) inhibitors of Xanthomonas sp. for treatment and prevention of citrus canker (n° 23/01921-4); Grant Mechanism Regular Research Grant; Principal Investigator Carlos Henrique Tomich de Paula da Silva (USP); Investment R$172,698.26.
Scientific articles
ALEXANDRINO, A. V. et al. GDP-mannose pyrophosphorylase is an efficient target in Xanthomonas citri for citrus canker control. Microbiology Spectrum. Vol. 12, no. 6. May 9, 2024.
BATTAGIN, T. S. et al. Syzygium aromaticum (clove) essential oil: An alternative for the sanitization of citrus fruit in packinghouses. Food Processing and Preservation. e15496. Mar. 27, 2021.
CARNIELLI, C. M. et al. Xanthomonas citri subsp. citri surface proteome by 2D-Dige: Ferric enterobactin receptor and other outer membrane proteins potentially involved in citric host interaction. Journal of Proteomics. Vol. 151, pp. 251–63. Jan. 16, 2017.
KRÓL, E. et al. Antibacterial activity of alkyl gallates is a combination of direct targeting of FtsZ and permeabilization of bacterial membranes. Frontiers in Microbiology. Vol. 6. Apr. 28, 2015.
MORE, K. K. et al. Evaluation of plant extracts against Xanthomonas citri causing citrus canker. Journal of Plant Disease Science. Vol. 18, no. 1. Aug. 8, 2023.
WANG, X. et al. Innovative strategy for the control of citrus canker: Inhibitors targeting the type III secretion system of Xanthomonas citri Subsp. citri. Journal of Agricultural and Food Chemistry. Vol. 71, no. 43. Oct. 13, 2023.
