{"id":144861,"date":"2014-02-18T19:31:49","date_gmt":"2014-02-18T22:31:49","guid":{"rendered":"http:\/\/revistapesquisa.fapesp.br\/?p=144861"},"modified":"2017-03-10T13:28:13","modified_gmt":"2017-03-10T16:28:13","slug":"people-plants","status":"publish","type":"post","link":"https:\/\/revistapesquisa.fapesp.br\/en\/people-plants\/","title":{"rendered":"For people and plants"},"content":{"rendered":"
\"Xylem

ALESSANDRA DE SOUZA \/ IAC<\/span>Xylem (green<\/em>) colonized by Xylella fastidiosa<\/em>ALESSANDRA DE SOUZA \/ IAC<\/span><\/p><\/div>\n

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.\u00a0 Xylella fastidiosa<\/i>, the bacterium that causes citrus variegated chlorosis (CVC), also known as amarelhinho<\/i> for the yellow spots it leaves on the plant\u2019s leaves and fruit, takes over the plant and forms a biofilm that unites the invading microorganisms.\u00a0 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\u2019s orange harvest, says biologist Marie-Anne Van Sluys of the University of S\u00e3o Paulo (USP), in a report published in the Special Issue FAPESP 50 Years<\/i>. And that is precisely the objective of Souza, a researcher at the Sylvio Moreira Citricuture Center of the\u00a0 Campinas Institute of Agronomy (IAC) in the city of Cordeir\u00f3polis in interior S\u00e3o Paulo State.<\/p>\n

And she seems to be right on track, according to the findings in master\u2019s research done by her student L\u00edgia Muranaka, published in the journal PLoS One <\/i>in 2013. \u201cThe pathogenesis of Xylella<\/i> is similar in gene expression and mechanisms to that of the bacteria that cause infections in humans,\u201d says Souza.\u00a0 Because of this, she has already tested several types of antibiotics such as tetracycline and neomycin.\u00a0\u00a0 \u201cThe Xylella<\/i> is susceptible to these medicines,\u201d she says, \u201cbut they are too expensive to be used in agriculture.\u201d\u00a0 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\u2019s 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.<\/p>\n

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 \u2013 in other words, it breaks up mucus.\u00a0 \u201cIt breaks down the biofilm and destroys the protein structure of several bacteria that infect humans, such as <\/span>Staphylococcus aureus, Enterococcus faecalis <\/i>and<\/span> Pseudomonas aeruginosa, <\/i>\u201d 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 <\/span>X. fastidiosa <\/i>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.\u00a0<\/span><\/p>\n

\"Live

Richard Janissen \/ Unicamp, INFABIC microscope<\/span>Live bacteria, with fluorescent mutation, using confocal microscopyRichard Janissen \/ Unicamp, INFABIC microscope<\/span><\/p><\/div>\n

In the orange grove
\n<\/b>It worked in in vitro<\/i> experiments, but theory or action in bacteria cultivated on glass plates in the laboratory is one thing.\u00a0 Applying the knowledge to bacteria active in actual orange trees is quite a different matter.\u00a0 In the first experiment with whole plants, Souza\u2019s group applied NAC to orange trees grown using a hydroponics system, where the roots are directly exposed to the medication.\u00a0 The results were promising:\u00a0 the number of yellow-spotted leaves and the quantity of bacteria diminished in the medicated plants.\u00a0 But in order to maintain control, they had to supply medicine to the plant on a nearly continual basis.\u00a0 If it was removed, the symptoms returned within three months.<\/p>\n

A more realistic experiment (\u201cafter all, orange trees don\u2019t grow in hydroponics,\u201d 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.\u00a0\u00a0 But the positive action of the mucolytic agent was promising enough for the team that also included researchers from the University of Campinas\u00a0 (Unicamp) and the Federal University of S\u00e3o Carlos (UFSCar) to look for a more effective application mode.\u00a0<\/span><\/p>\n

\u201cWe established a non-profit partnership with an organic fertilizer company,\u201d says Souza.\u00a0 The manufacturer, Agrolatino, developed a way to add NAC to granular fertilizer so that the medicine could be released gradually.\u00a0 This time, the symptoms diminished even more, and for a longer period of time \u2013 around eight months after the application.\u00a0 This solution may be viable for controlling citrus variegated chlorosis in actual plantations, but Souza sees even more potential for improvement. \u201cWe\u2019re working on how to make the release time even slower by using nanoecapsulated NAC.\u201d\u00a0 The field effectiveness of this type of treatment still needs to be evaluated and is therefore being tested in partnership with the citrus industry.\u00a0<\/span><\/p>\n

Inside the xylem
\n<\/b>The path traversed by Souza began with the ambitious initiative to sequence the genome of X. fastidiosa<\/i>, just as she completed her master\u2019s 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\u2019s scientific community. Souza is one of the 116 authors of the article published in the July 2000 issue of the journal Nature<\/i> that contained the findings from Brazil\u2019s 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:\u00a0 \u201cGenome of Xylella fastidiosa<\/i>: 13 years after the \u201cmoment of glory\u201d where are we?\u201d 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.\u00a0 And its repercussions continue to be felt in various fields of science.<\/p>\n

While it tests in practice how to control the scourge of citrus producers that as recently as 2009 continued to affect 35% of Brazil\u2019s orange groves, the same figure as a decade before, Souza found partners on the physics side so that she could better understand how <\/span>X. fastidiosa <\/i>forms the biofilm that allows it to infect plants.\u00a0 The study led by M\u00f4nica Cotta of Unicamp is independent of the\u00a0 work conducted at the IAC, yet complementary. \u201cWe now have a complete adhesion model that allows us to understand what the <\/span>Xylella<\/i> does on all the surfaces,\u201d she says.\u00a0 The scale is very different from that of the orange groves that hold Souza\u2019s attention.\u00a0 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.<\/span><\/p>\n

\"The

HELVECIO DELLA COLETTA FILHO \/ IAC<\/span>The fruit from diseased plants is smallerHELVECIO DELLA COLETTA FILHO \/ IAC<\/span><\/p><\/div>\n

By using this equipment, Cotta\u2019s group is able to see how the bacteria behave on a variety of surfaces, especially with regard to formation of the biofilm.\u00a0 She is close to answering the chance question posed by Souza when they met back in 2007:\u00a0 why do they still \u201cstand\u201d at the end of the laboratory growth cycle, after 30 days?\u00a0 \u201cThey\u201d refer to the small cylindrical rods that are actually supported on one of the ends in certain situations.\u00a0 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.<\/span><\/p>\n

Cotta demonstrated her expertise in microscopy and used more natural substrates \u2013 two types of cellulose \u2013, in addition to the glass. \u201cFirst, the bacterium adheres and then it secretes the exopolysaccharides to form a capsule and then the biofilm,\u201d she explains.\u00a0 A September 2013 article published in the journal <\/span>PLoS One<\/i>, 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. \u201cIt loves silicon, but doesn\u2019t like cellulose,\u201d 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.\u00a0 Among the two types of cellulose produced in the laboratory, cellulose acetate is rougher and less comfortable for the <\/span>Xylella<\/i>, which is unable to cover the entire surface. Ethyl cellulose, however, has fewer irregularities and the bacteria adhere better.\u00a0 \u201cThey have a different kind of\u00a0 roughness, which we could liken to the difference between the Alps and the Mantiqueira Mountain Range,\u201d Cotta explains.\u00a0<\/span><\/p>\n

Physical adhesion
\n<\/b>But roughness is not the most important variable in adhesion, and the techniques associated with microscopy allow minute manipulations to detail the process.\u00a0 By using an atomic force microscope, Cotta has been able, for example, to capture a protein the Xylella<\/i> 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.\u00a0 \u201cWe\u2019ve determined that the bacteria like some regions better than others.\u201d\u00a0 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\u2019s more general conclusion is that the Xylella<\/i> 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).<\/p>\n

The studies conducted by Cotta\u2019s group up to now constitute the beginning of the understanding of how the biofilm establishes itself and spreads through the orange tree xylem.\u00a0\u00a0 By taking advantage of the characteristics that give the bacteria its name and allow its dynamic to be observed in real time \u2013 its\u00a0 fastidiousness lends itself to slowness \u2013, 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\u2019s work, enhancing her understanding of how the NAC works on the biofilm\u2019s properties as it forms and develops.\u00a0 This will allow Cotta to elaborate upon Van Sluys\u2019 suggestion that the initial moments of infection are crucial. \u00a0<\/span><\/p>\n

\"Typical

ALESSANDRA DE SOUZA \/ IAC<\/span>Typical lesions from CVC on the leavesALESSANDRA DE SOUZA \/ IAC<\/span><\/p><\/div>\n

The higher resolution with which the physicists work is also helping to redefine the initial moments from the experimental standpoint.\u00a0 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.\u00a0\u00a0 After all, she\u2019d had to wait five days from incubation in order to see a cluster of bacteria. \u00a0<\/span><\/p>\n

Cotta emphasizes that the advances achieved are only possible due to the constant, although sporadic, interaction between biologists and physicists.\u00a0 As one of her students remarked after visiting the IAC: \u201cThey think differently.\u201d 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.\u00a0 \u201cWe\u2019re learning to interact with other areas in addition to using the equipment,\u201d she says. \u00a0<\/span><\/p>\n

Another aspect that the Unicamp physicist sees as important is the fact that both laboratories are led by women.\u00a0 \u201cAnd chatty ones at that,\u201d she adds.\u00a0 The connections that have proven to be so fruitful came out of lunchtime conversations in which mutual interests arose and work relationships were developed.\u00a0 Besides that is the fact that they are all jugglers, constantly balancing personal and professional life, motherhood, friendships and collaborative efforts.\u00a0 \u201cIdeas are born of experience,\u201d says Cotta, recalling the initial inspiration that came when her colleague gave her child cough syrup.\u00a0<\/span><\/p>\n

Projects<\/strong>
\n<\/span>1.<\/b> Biological characteristics of Xylella fastidiosa in biofilm: the importance of adhesion genes in pathogenesis and adaptation (2004\/14576-2<\/a>); Grant Mechanism<\/b> Young Investigator Award; Coord.<\/b> Alessandra Alves de Souza\/IAC; Investment<\/b> R$205,432.59 (FAPESP)
\n2.<\/b> Chemical and structural analysis of Xylella fastidiosa <\/b>biofilms (2010\/51748-7<\/a>); Grant Mechanism<\/b> Regular Line of Research Project Award; Coord.<\/b> M\u00f4nica Alonso Cotta\/Unicamp; Investment<\/b> R$187,405.53 (FAPESP).<\/p>\n

Scientific articles<\/em>
\n<\/span>MURANAKA, L. S. et al<\/i>. N-Acetylcysteine in agriculture, a novel use for an old molecule: focus on controlling the plant-pathogen Xylella fastidiosa<\/i><\/a>. PLoS One<\/b>. V. 8, No. 8, e72937. Aug. 2013.
\nLORITE, G. S. et al<\/i>. Surface physicochemical properties at the micro and nano length scales: role in bacterial adhesion and Xylella fastidiosa<\/i> biofilm development<\/a>. PLoS One<\/b>. V. 8, No. 9, e75247. Sept. 2013.<\/p>\n","protected":false},"excerpt":{"rendered":"Expectorant medicine may prove effective in controlling orange disease","protected":false},"author":3,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_exactmetrics_skip_tracking":false,"_exactmetrics_sitenote_active":false,"_exactmetrics_sitenote_note":"","_exactmetrics_sitenote_category":0,"footnotes":""},"categories":[159],"tags":[209],"coauthors":[1601],"class_list":["post-144861","post","type-post","status-publish","format-standard","hentry","category-science","tag-biology"],"acf":[],"_links":{"self":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/144861","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/comments?post=144861"}],"version-history":[{"count":0,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/144861\/revisions"}],"wp:attachment":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/media?parent=144861"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/categories?post=144861"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/tags?post=144861"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/coauthors?post=144861"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}