Ekologija i suzbijanje Xanthomonas campestris pv. campestris

Abstract

U radu je proučavana ekologija i suzbijanje bakterije Xanthomonas campestris pv. campestris (Xcc), prouzrokovača crne truleži kupusnjača u Srbiji. Tokom 2014–2016. godine prikupljeni su uzorci obolelih listova sa kupusa, karfiola, brokolija, kelja i uljane repice sa različitih lokaliteta na teritoriji Republike Srbije, kao i uzorci zemljišta. Sakupljanje uzoraka sertifikovanog i naturalnog semena vršeno je u periodu od tri godine (2015–2017). Različitim postupcima izolacije je dobijeno 128 izolata: 82 poreklom iz lista, 26 iz zemljišta i 20 sa semena. Na osnovu testova patogenosti, morfoloških odlika i biohemijsko – fizioloških testova izolati su preliminarno identifikovani kao vrsta X. c. pv. campestris. Serološka ispitivanja su obuhvatila primenu direktne imunoenzimske metode na ploči (DAS-ELISA), koja je potvrdila antigensku uniformnost proučavanih sojeva Xcc na kupusnjačama u Srbiji. U molekularnoj analizi primenom m-PCR, izvedenog setom prajmera Zup 2309/Zup 2310, kao i DLH 120/DLH 125, kod svih proučavanih izolata, i kontrolnog referentnog soja NCPPB 1144, amplifikovani su fragmenti nukleinskih kiselina veličine 370 i 619 bp, respektivno, koji odgovaraju vrsti Xcc. Umnožavanjem sekvenci 16S rRNK gena, te njihovim poređenjem sa sekvencama iz NCBI baze, utvrđen je visok stepen sličnosti (100%) sa ostalim sojevima Xcc, deponovanim u ovu bazu. U cilju utvrđivanja epidemiologije i fluktuacije Xcc u zemljištu, rađeno je uzorkovanje zemljišta sa iste parcele, jednom mesečno u periodu od 24 meseca. Vremenski, bakterija Xcc je detektovana u periodu od septembra do novembra, pri opsegu srednjih dnevnih temperatura na 5 cm visine od -0,1–12,8 °C, kao i od aprila do juna sa opsegom temperatura 1,8–14 °C. Prisustvo Xcc u zemljištu nije dokazano u periodu od decembra do marta pri temperaturnom intervalu -5,1–1,5 °C, kao i u julu mesecu u kome su zabeležene srednje dnevne temperature na 5 cm od 14,6 °C. Ispitivanjem osetljivosti proučavanih sojeva, utvrđena je inhibitorna koncentracija od 300 ppm za sve ispitivane oblike bakra. Pri koncentraciji bakra od 200 ppm, zabeležen je razvoj 42 izolata kod bakar (II) – sulfata, 49 kod bakar – hidroksida i 60 izolata kod bakar – oksihlorida, od ukupno 114 izolata. Koncentracija 100 ppm ispitivanih oblika bakarnih jedinjenja, omogućila je razvoj svih ispitivanih izolata. Ispitivanjem specifičnosti izolata bakteriofaga prema vrsti Xcc, utvrđeno je da su izolati uskospecifični, ispoljavajući prema pomenutoj vrsti lipolitičku aktivnost. Ispitivanjem uticaja temperature na patogenost izolata u in vitro uslovima, ustanovljeno je da temperature u opsegu 24–32 °C obezbeđuju pojavu jasno vidljivih simptoma, dok je temperatura od 28 °C optimalna za razvoj oboljenja. Temperature od 16 °C i 36 °C uzrokuju sporiji razvoj simptoma. Utvrđivanjem efikasnosti baktericida primenom veštačke inokulacije Xcc u poljskim uslovima, najmanji intenzitet zaraze je zabeležen u tretmanu sa kombinacijom preparata bakar – hidroksid 0,5% + fosetil – Al 0,2% kao i u tretmanu sa bakar – hidroksidom 0,5% + mankozebom 0,25%. U uslovima spontane infekcije manji intenzitet zaraze u odnosu na druge ispitivane baktericide, zabeležen je u istim tretmanima sa navedenim kombinacijama preparata kao i u uslovima veštačke inokulacije. Većina ispitivanih tretmana u odnosu na kontrolni tretman je statistički značajna ili visoko statistički značajna. Analizom uticaja tretmana na prosečnu masu glavice, utvrđena je najveća masa od 1,94 kg tokom 2014. godine u tretmanu bakar – hidroksidom 0,5% + fosetil – Al 0,2%. U naredne dve godine ispitivanja u tretmanu sa bakar – hidroksidom 0,5% + mankozeb 0,25% zabeležena je najveća prosečna mase glavica 1,60 kg tokom 2015. godine i 1,54 kg u 2016 godini.

In this paper, the bacterium Xanthomonas campestris pv. campestris (Xcc) casual agent of black rot of Brassica vegetables in Serbia was studied. During 2014–2016, samples of infected leaves from cabbage, cauliflower, broccoli, kale and rapeseed from various localities in the territory of the Republic of Serbia and soil samples were collected. Collecting of samples of certified and natural seeds was performed over a period of three years (2015–2017). 128 isolates were obtained by different isolation procedures: 82 originating from leaves, 26 from soil and 20 from seeds. Based on pathogenicity tests, morphological characteristics and results of biochemical and physiological tests, the isolates were preliminarily identified as species X. c. pv. campestris. Serological research, included the application of the direct enzyme – linke immunosorbent assay (DAS-ELISA), and confirmed the antigenic uniformity of the studied Xcc strains in herbaceous vegetables in Serbia. In molecular test performed by the m-PCR with the primer set Zup 2309/Zup 2310 (370 bp), as well as DLH 120/DLH 125 (619 bp), in all studied isolates, and the control reference strain NCPPB 1144, nucleic acid fragments of sizes 370 and 619 bp were amplified, that corresponds to the identity of the strain Xcc. By determining and studying the sequences of the 16S rRNA gene, and comparing them with the sequences from the NCBI base, a high degree of similarity (100%) with other Xcc strains deposited in this base was also determined. In order to determine the epidemiology and fluctuation of the Xcc in the soil, soil sampling was performed from the same plot, once a month for a period of 24 months. Temporally, the bacterium Xcc was detected in the period from September to November, with a range of mean daily soil temperatures at 5 cm of -0.1–12.8 °C, as well as from April to June with a temperature range of 1.8–14 °C . The presence of Xcc in the soil was not proven in the period from December to March at the temperature interval -5.1–1.5 °C, as well as in the month of July, in which mean daily temperatures at 5 cm of 14.6 °C were recorded. By testing the susceptibility of the studied strains, an inhibitory concentration of 300 ppm was determined for all tested forms of copper. At a copper concentration of 200 ppm, the development of 42 isolates in copper (II) – sulfate, 49 in copper hydroxide and 60 isolates in copper oxychloride was recorded of 114 isolates in total. The concentration of 100 ppm of the tested forms of copper compounds enabled the development of all isolates. By examining the specificity of bacteriophage isolates according to the Xcc species, it was determined that the isolates are narrowly specific according to the mentioned species, exhibiting lipolytic activity. Examining the effect of the temperature on the pathogenicity of isolates in vitro, it was concluded that the temperatures in the range from 24 to 32 °C influenced the appearance of clearly visible symptoms, while a temperature of 28 °C is optimal for the development of the disease. Temperatures of 16 and 36 °C affected the poor development of the disease and the appearance of the symptoms. By determining the effectiveness of the bactericide using artificial inoculation Xcc in field conditions, the lowest infection intensity was recorded in the treatment with the combination of copper hydroxide 0.5% + fosetyl – Al 0.2% as well as in the treatment with copper hydroxide 0.5% + mancozeb 0,25%. In the conditions of spontaneous infection, a lower intensity of infection compared to the other tested bactericides was recorded in the same treatments with the mentioned combinations of the product as in the conditions of artificial inoculation. Most of the investigated treatments compared to the control treatment are statistically significant or highly statistically significant. By analyzing the effect of the treatment on the average weight of the cabbage head, the highest weight of 1.94 kg was determined in 2014 in the treatment with copper hydroxide 0.5% + fosetyl – Al 0.2%. In the next two years of testing with copper hydroxide 0.5% + mancozeb 0.25%, the highest average cabbage head weight was achieved: 1.60 kg in 2015 and 1.54 kg in 2016.