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Microbiology

For people and plants

Expectorant medicine may prove effective in controlling orange disease

Xylem (green) colonized by Xylella fastidiosa

ALESSANDRA DE SOUZA / IACXylem (green) colonized by Xylella fastidiosaALESSANDRA DE SOUZA / IAC

It came to biologist Alessandra de Souza as she gave expectorant cough syrup to her son who had the flu: could the same medicine be used to treat the orange tree disease that is a constant focus of her work? The inspiration is less unlikely than it seems when one considers flu symptoms in children and the anatomy of an orange tree.  Xylella fastidiosa, the bacterium that causes citrus variegated chlorosis (CVC), also known as amarelhinho for the yellow spots it leaves on the plant’s leaves and fruit, takes over the plant and forms a biofilm that unites the invading microorganisms.  Interrupting biofilm production while it is just starting to form may be the best way to treat the disease that causes serious losses to Brazil’s orange harvest, says biologist Marie-Anne Van Sluys of the University of São Paulo (USP), in a report published in the Special Issue FAPESP 50 Years. And that is precisely the objective of Souza, a researcher at the Sylvio Moreira Citricuture Center of the  Campinas Institute of Agronomy (IAC) in the city of Cordeirópolis in interior São Paulo State.

And she seems to be right on track, according to the findings in master’s research done by her student Lígia Muranaka, published in the journal PLoS One in 2013. “The pathogenesis of Xylella is similar in gene expression and mechanisms to that of the bacteria that cause infections in humans,” says Souza.  Because of this, she has already tested several types of antibiotics such as tetracycline and neomycin.   “The Xylella is susceptible to these medicines,” she says, “but they are too expensive to be used in agriculture.”  The researcher explains that the formation of the biofilm inside the plant allows the bacteria to communicate among themselves and behave as a single organism. This peculiarity ends up clogging the plant xylem, where the microorganisms are found, and impedes the passage of nutrients and water from the roots to the plant’s crown If this is the mechanism that acts in the disease, then perhaps it is also the basis for an economically viable solution that causes no environmental impact.

The compound N-acetylcysteine (NAC)–active ingredient in the cough syrup Souza gave her son, which is an old favorite among those well-versed in treating respiratory problems–is a mucolytic agent – in other words, it breaks up mucus.  “It breaks down the biofilm and destroys the protein structure of several bacteria that infect humans, such as Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa, ” she says. The medicine had never before been used on plants, but knowing through studies of genome function that many of the proteins that promote adhesion among the X. fastidiosa bacteria inside orange trees form connections among themselves as a result of the cysteine, her group worked on the basis of the assumption that the medication might be effective in fighting the chlorosis. 

Live bacteria, with fluorescent mutation, using confocal microscopy

Richard Janissen / Unicamp, INFABIC microscopeLive bacteria, with fluorescent mutation, using confocal microscopyRichard Janissen / Unicamp, INFABIC microscope

In the orange grove
It worked in in vitro experiments, but theory or action in bacteria cultivated on glass plates in the laboratory is one thing.  Applying the knowledge to bacteria active in actual orange trees is quite a different matter.  In the first experiment with whole plants, Souza’s group applied NAC to orange trees grown using a hydroponics system, where the roots are directly exposed to the medication.  The results were promising:  the number of yellow-spotted leaves and the quantity of bacteria diminished in the medicated plants.  But in order to maintain control, they had to supply medicine to the plant on a nearly continual basis.  If it was removed, the symptoms returned within three months.

A more realistic experiment (“after all, orange trees don’t grow in hydroponics,” Souza cautions), one in which plants were irrigated with a solution that included NAC and in some cases had the drug injected into its roots, showed similar results.   But the positive action of the mucolytic agent was promising enough for the team that also included researchers from the University of Campinas  (Unicamp) and the Federal University of São Carlos (UFSCar) to look for a more effective application mode. 

“We established a non-profit partnership with an organic fertilizer company,” says Souza.  The manufacturer, Agrolatino, developed a way to add NAC to granular fertilizer so that the medicine could be released gradually.  This time, the symptoms diminished even more, and for a longer period of time – around eight months after the application.  This solution may be viable for controlling citrus variegated chlorosis in actual plantations, but Souza sees even more potential for improvement. “We’re working on how to make the release time even slower by using nanoecapsulated NAC.”  The field effectiveness of this type of treatment still needs to be evaluated and is therefore being tested in partnership with the citrus industry. 

Inside the xylem
The path traversed by Souza began with the ambitious initiative to sequence the genome of X. fastidiosa, just as she completed her master’s degree and began work at the IAC with Marcos Machado, coordinator of one of the work groups of the FAPESP $12 million project that came to be a sign of the coming-of-age of Brazil’s scientific community. Souza is one of the 116 authors of the article published in the July 2000 issue of the journal Nature that contained the findings from Brazil’s first genomic project, in which she found the material for her doctoral thesis that studied the genes involved in the pathogenesis and formation of the biofilm of this bacteria. From there came the title of the talk she gave at the Brazilian Phytopathological Conference in Ouro Preto in October 2013:  “Genome of Xylella fastidiosa: 13 years after the “moment of glory” where are we?” The very short answer is that the investment in a controversial undertaking, focused on an organism (even one considered to be bad) at that time more widely known by orange producers than researchers, continues to bear fruit.  And its repercussions continue to be felt in various fields of science.

While it tests in practice how to control the scourge of citrus producers that as recently as 2009 continued to affect 35% of Brazil’s orange groves, the same figure as a decade before, Souza found partners on the physics side so that she could better understand how X. fastidiosa forms the biofilm that allows it to infect plants.  The study led by Mônica Cotta of Unicamp is independent of the  work conducted at the IAC, yet complementary. “We now have a complete adhesion model that allows us to understand what the Xylella does on all the surfaces,” she says.  The scale is very different from that of the orange groves that hold Souza’s attention.  The main tools used in the physics work are sophisticated microscopes such as the atomic power and the confocal spinning disk microscopes, the latter housed at the National Institute of Photonics Applied to Cell Biology (INFABIC) headed by Hernandes Carvalho of Unicamp.

The fruit from diseased plants is smaller

HELVECIO DELLA COLETTA FILHO / IACThe fruit from diseased plants is smallerHELVECIO DELLA COLETTA FILHO / IAC

By using this equipment, Cotta’s group is able to see how the bacteria behave on a variety of surfaces, especially with regard to formation of the biofilm.  She is close to answering the chance question posed by Souza when they met back in 2007:  why do they still “stand” at the end of the laboratory growth cycle, after 30 days?  “They” refer to the small cylindrical rods that are actually supported on one of the ends in certain situations.  What is important, however, is that the physics approach has shown that cultivating these bacteria on glass plates to examine under the electron microscope is not enough to fully understand them, since the conditions in which they live make all the difference.

Cotta demonstrated her expertise in microscopy and used more natural substrates – two types of cellulose –, in addition to the glass. “First, the bacterium adheres and then it secretes the exopolysaccharides to form a capsule and then the biofilm,” she explains.  A September 2013 article published in the journal PLoS One, the main part of the doctoral thesis by team member Gabriela Lorite, shows that the substrate causes significant variations in both the shape as well as the edges of the biofilm. “It loves silicon, but doesn’t like cellulose,” says Cotta, who has built her career on studying materials and seems to have become particularly attached to the organism that introduced her to the world of biology.  Among the two types of cellulose produced in the laboratory, cellulose acetate is rougher and less comfortable for the Xylella, which is unable to cover the entire surface. Ethyl cellulose, however, has fewer irregularities and the bacteria adhere better.  “They have a different kind of  roughness, which we could liken to the difference between the Alps and the Mantiqueira Mountain Range,” Cotta explains. 

Physical adhesion
But roughness is not the most important variable in adhesion, and the techniques associated with microscopy allow minute manipulations to detail the process.  By using an atomic force microscope, Cotta has been able, for example, to capture a protein the Xylella produces at the very beginning of the infection cycle. By prodding the bacteria with this substance, researchers are able to induce them to adhere and then measure the strength of the interaction between the organism and the substrate.  “We’ve determined that the bacteria like some regions better than others.”  Experiments with silicon and ethyl cellulose indicate that at least one of the proteins almost always attaches itself to the substrate. The same does not occur with cellulose acetate, where adhesion was only observed in 20% of the cases. The study’s more general conclusion is that the Xylella have a greater tendency to attach themselves to electrically more uniform surfaces that are positively charged, as well as to hydrophilic surfaces (that attract water).

The studies conducted by Cotta’s group up to now constitute the beginning of the understanding of how the biofilm establishes itself and spreads through the orange tree xylem.   By taking advantage of the characteristics that give the bacteria its name and allow its dynamic to be observed in real time – its  fastidiousness lends itself to slowness –, Cotta has many plans. They include conducting additional research into how genetic expression influences biofilm formation, using the brightness given off by the green fluorescent protein (GFP) to better view its dynamic and, in line with Souza’s work, enhancing her understanding of how the NAC works on the biofilm’s properties as it forms and develops.  This will allow Cotta to elaborate upon Van Sluys’ suggestion that the initial moments of infection are crucial.  

Typical lesions from CVC on the leaves

ALESSANDRA DE SOUZA / IACTypical lesions from CVC on the leavesALESSANDRA DE SOUZA / IAC

The higher resolution with which the physicists work is also helping to redefine the initial moments from the experimental standpoint.  When Cotta told Souza that the biofilm was already visible six hours after introducing the bacteria into the culture medium, albeit with only a few bacteria, the biologist was incredulous.   After all, she’d had to wait five days from incubation in order to see a cluster of bacteria.  

Cotta emphasizes that the advances achieved are only possible due to the constant, although sporadic, interaction between biologists and physicists.  As one of her students remarked after visiting the IAC: “They think differently.” This different way of thinking is what generates new questions and new perspectives and helps find innovative answers. For Cotta, the opportunity to use multiuser equipment thanks to a substantial investment by FAPESP is crucial.  “We’re learning to interact with other areas in addition to using the equipment,” she says.  

Another aspect that the Unicamp physicist sees as important is the fact that both laboratories are led by women.  “And chatty ones at that,” she adds.  The connections that have proven to be so fruitful came out of lunchtime conversations in which mutual interests arose and work relationships were developed.  Besides that is the fact that they are all jugglers, constantly balancing personal and professional life, motherhood, friendships and collaborative efforts.  “Ideas are born of experience,” says Cotta, recalling the initial inspiration that came when her colleague gave her child cough syrup. 

Projects
1. Biological characteristics of Xylella fastidiosa in biofilm: the importance of adhesion genes in pathogenesis and adaptation (2004/14576-2); Grant Mechanism Young Investigator Award; Coord. Alessandra Alves de Souza/IAC; Investment R$205,432.59 (FAPESP)
2. Chemical and structural analysis of Xylella fastidiosa biofilms (2010/51748-7); Grant Mechanism Regular Line of Research Project Award; Coord. Mônica Alonso Cotta/Unicamp; Investment R$187,405.53 (FAPESP).

Scientific articles
MURANAKA, L. S. et al. N-Acetylcysteine in agriculture, a novel use for an old molecule: focus on controlling the plant-pathogen Xylella fastidiosa. PLoS One. V. 8, No. 8, e72937. Aug. 2013.
LORITE, G. S. et al. Surface physicochemical properties at the micro and nano length scales: role in bacterial adhesion and Xylella fastidiosa biofilm development. PLoS One. V. 8, No. 9, e75247. Sept. 2013.

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