Antibacterial and antifungal activity of phenylboronic acid on tomato plant pathogens

Pronalaženje prikladne alternative malom broju postojećih antimikrobnih sredstava za suzbijanje biljnih patogena, vitalan je zadatak u suvremenoj poljoprivredi. U tu svrhu se provode intenzivna istraživanja kako bi se otkrili ekološki prihvatljivi i učinkoviti spojevi koji mogu suzbiti patogene otporne na primjenjivane antimikrobne aktivne tvari. Učinkovitost fenilboronske kiseline (PBA) protiv nekoliko fitopatogenih bazidiomicetnih vrsta u smislu zaštite drvne građe, poznata je od ranije, ali do sada nije testirana u suzbijanju uzročnika bolesti poljoprivrednih kultura. Cilj disertacije je testirati antibakterijski i antimikotički učinak PBA na patogene u uzgoju rajčice i kontaminantnog patogena svježih plodova rajčice u uvjetima in vitro i in vivo. Dobiveni rezultati potvrđuju letalan učinak PBA na fitopatogenu gljivu Alternaria alternata i bakteriju Pseudomonas syringae pv. tomato pri niskoj koncentraciji (0,05 %) u uvjetima in vitro. U usporedbi s tim, borna kiselina (BA) je na patogenu gljivu imala fungistatički učinak, dok je rast bakterijskih patogena inhibiran pri pri 6 puta većoj koncentraciji od primijenjene koncentracije PBA. Bakterija Escherichia coli je ispirana s plodova rajčice otopinama PBA i BA, a otopine nakon ispiranja su nanošene na hranjive podloge odmah ili 120 min kasnije. Broj razvijenih kolonija na mediju s 0,05 % (1/2 MIC) PBA bio je oko 40 % manji u odnosu na kontrolu (ispranje sterilnom destiliranom vodom), a taj broj se 2,5 puta smanjio nakon inkubacije u trajanju od 120 min. Podjednako smanjenje broja živih stanica bakterije postignuto je s 3 puta većom koncentracijom BA. Preventivnim tretiranjem biljaka rajčice cv. Rutgers s 0,05 % (1 MIC) PBA, provedenom 7 dana prije inokulacije gljivom Alternaria alternata ili bakterijom Pseudomonas syringae pv. tomato, zabilježeno je značajno smanjenje površine lezija koncentrične pjegavosti (za 42 – 61 %) i bakteriozne pjegavosti (za 55 - 71 %) u odnosu na kontrolnu skupinu biljaka koje nisu preventivno tretirane. Niska koncentracija PBA značajno je reducirala simptome koncentrične i bakteriozne pjegavosti u odnosu na učinak BA, pri čemu značajne razlike u učinku na fiziologiju zaraženih biljaka tretiranih s PBA, odnosno s BA, nisu zabilježene. Dokazana je profilaktička aktivnost PBA i BA na uzročnike bolesti rajčice, pri čemu je PBA pokazala značajniji učinak na supresiju simptoma bolesti rajčice od BA. Također, dokazano je da PBA, kao i BA, nemaju štetan utjecaj na zdravlje biljaka u baktericidnim i fungicidnim koncentracijama. Ova disertacija je prvo istraživanje antibakterijskog i antimikotičkog učinka PBA na uzročnike bolesti zeljaste domaćinske biljke, a testiranje je provedeno usporedno s istraživanjem učinka medicinski značajne BA. Ostvarenjem ciljeva i prihvaćanjem hipoteza disertacije, postignut je preduvjet za nastavak istraživanja PBA kao potencijalnog sredstva za suzbijanje ekonomski značajne fitopatogene bakterije i gljive u uzgoju rajčice, ali i za razmatranje PBA kao potencijalnog dekontaminanta svježih plodova rajčice od patogene bakterije Escherichia coli. Budući da nije štetna za zrele biljke rajčice, kao ni za sisavce, a otpornost na testiranu fitopatogenu gljivu i bakteriju nije zamijećena, navedene spoznaje kvalificiraju ekološki prihvatljivu PBA kao prikladan antimikrobni spoj.Finding a suitable alternative to the limited number of existing antifungal agents aimed to control plant pathogens is a vital task in modern agriculture. Therefore, intensive research is being conducted to identify environmentally friendly and effective compounds that can control plant pathogens resistant to the antimicrobials used. Various studies cite phenylboronic acid (PBA) as a commercially available, environmentally and pharmaceutically acceptable compound known as the simplest boric acid (BA) derivative that in certain concentrations has bactericidal and fungicidal effects on several species of human fungi and bacteria. Previous studies demonstrate antifungal effect of PBA on basidiomycete fungi that cause wood decay, but it has not been tested in controlling plant pathogens in agriculture. This fact puts PBA in the context of a potential plant protection compound and explains the need for research on agriculturally interesting herbaceous host pathogens. Because tomato is globally one of the most famous crops whose cultivation is hampered by damage caused by attack of resistant pathogens, it was selected as the herbaceous host on which the bactericidal and fungicidal effect of PBA was investigated in this research. For testing this compound, the economically important pathogens, after isolation from symptomatic tomato leaves, were molecularly identified and morphologicaly characterized. The aim of the dissertation is to test, in vitro and in vivo, the antibacterial and antifungal effect of PBA and BA on two tomato pathogens (fungus Alternaria alternata and bacterium Pseudomonas syringae pv. tomato). The obtained results confirm the lethal effect of PBA on the fungus A. alternata and the bacterium P. syringae pv. tomato at low concentration (0.05 %) in vitro. In comparison, BA had a fungistatic effect on the pathogenic fungus, while the growth of bacterial pathogen was inhibited at 6 times higher concentration of BA than that of the applied PBA. The severity of symptoms caused by infection of economically important tomato plant pathogens was determined experimentally alongside with quantification of physiological parameters of treated plants. Also, it has been shown that PBA, like BA, does not have a detrimental effect on plant health in bactericidal or fungicidal concentrations when applied in the late phenophase of development. By preventive in vivo application on tomato cv. Rutgers 7 days before inoculation with fungus A. alternata or P. syringae pv. tomato, low PBA concentration (0,05 %, 1 MIC) significantly reduced the symptoms of early blight (42 - 61 %) and bacterial speck (55 – 71 %) compared to the control group of plants that were not treated. Prophylactic activity of PBA and BA on tomato pathogens has been demonstrated, with PBA showing a stronger effect on the suppression of tomato disease symptoms than BA. In addition to economically important phytopathogens in tomato growing (A. alternata and P. syringae pv. tomato), the negative aspect in tomato production is caused by the human pathogen - bacterium Escherichia coli, which contaminates fruits through manuring and irrigation with contaminated water. The bactericidal effect of PBA on E. coli washed off from tomato fruits was investigated by the in vitro experiment. E. coli was washed off from tomato fruits with PBA and BA solutions and these bacterial suspensions were transferred to nutrient media immediately or 120 min later. Inactivation of E. coli from tomato fruits (97 %) was achieved by 120 min exposition of the bacterium to 0,05 % PBA (1/2 MIC) solution is equivalent to the effect of 3-time higher concentrations of BA. The number of bacteria that developed colonies in 0,05 % (1/2 MIC) PBA was about 40 % lower compared to the control, washed with water, and this number decreased 2,5 times after incubation for 120 min. This dissertation is the first study of the bactericidal and fungicidal effect of PBA on pathogens of herbaceous host plant. By achieving the goals and accepting the hypotheses of dissertation, the precondition for continuing research on PBA as a potential plant protection compound for the control of economically important plant pathogenic bacterium and fungus in tomato was achieved, as well as for considering PBA as a potential decontaminant of fresh tomato fruits from human pathogen – bacterium E. coli. Since PBA is not harmful to mature tomato plants and mammals, and no resistance of plant pathogenic fungi and bacteria has been observed, the mentioned findings effectively qualify PBA as a suitable antimicrobial compound. Additional advance of PBA is its environmental friendliness