The interest in management
fire blight prevention and control increased
considerably in last years due to pathogen
spread almost throughout the country. The
objective of this paper focuses on testing of
some plant extracts such as Salvia
officinalis, Thymus serpyllum, Pelargonium
odoratissimum, Hedera helix, Ocimum
basilicum, Levisticum officinale, Tagetes
patula, Galium verum and different
pesticides against an Erwinia amylovora
strain isolated from quince dameged
samples. In this study, the biological
material was represented by quince shoots,
harvested from pomological collection of
"Vasile Adamachi" farm, which belongs to
University of Agricultural Science and
Veterinary Medicine Iaşi. The research were
performed in the Phytopathology laboratory.
Following the experiment performed note
that among the eight plant extracts a good
antibacterial activity has Pelargonium
odoratissimum, Ocimum basilicum and
Salvia officinalis and of the three pesticides,
Alcupral 50 PU. Contrary to expectations
worst results were obtained after testing
Aliette 80 WG and Hedera helix products.
The results were estimated statisticaly, by
performing ANOVA test

Chiriac, Irina-Paraschiva

Ulea, E.

Cercetari Agronomice in Moldova

Ulea, Eugen

ALSE Repository

SUSCEPTIBILITY OF ERWINIA AMYLOVORA STRAIN TO PLANT EXTRACTS AND PESTICIDES   69  Cercetări Agronomice în Moldova Vol. XLV , No. 2 (150) / 2012   STUDY REGARDING EFFECTIVENESS OF SOME PLANT EXTRACTS AND DIFFERENT PESTICIDES AGAINST AN ERWINIA AMYLOVORA (BURRILL.) WINSLOW ET AL. STRAIN ISOLATED FROM QUINCE  Irina-Paraschiva CHIRIAC1*, E. ULEA1  *E-mail: chiriacirina84@yahoo.com   Received  April 7, 2012                                                       1 University of Agricultural Sciences and Veterinary Medicine “Ion Ionescu de la Brad” of Iaşi, Romania ABSTRACT. The interest in management fire blight prevention and control increased considerably in last years due to pathogen spread almost throughout the country. The objective of this paper focuses on testing of some plant extracts such as Salvia officinalis, Thymus serpyllum, Pelargonium odoratissimum, Hedera helix, Ocimum basilicum, Levisticum officinale, Tagetes patula, Galium verum and different pesticides against an Erwinia amylovora strain isolated from quince dameged samples. In this study, the biological material was represented by quince shoots, harvested from pomological collection of "Vasile Adamachi" farm, which belongs to University of Agricultural Science and Veterinary Medicine Iaşi. The research were performed in the Phytopathology laboratory. Following the experiment performed note that among the eight plant extracts a good antibacterial activity has Pelargonium odoratissimum, Ocimum basilicum and Salvia officinalis and of the three pesticides, Alcupral 50 PU. Contrary to expectations worst results were obtained after testing Aliette 80 WG and Hedera helix products. The results were estimated statisticaly, by performing ANOVA test.  Key words: Fire blight; Plant extracts; Hedera helix, Alcupral 50 PU.   REZUMAT. Studiu privind eficacitatea unor extracte din plante şi pesticide asupra unei suşe de Erwinia amylovora (Burrill.) Winslow et al., izolata de pe gutui. Interesul pentru managementul prevenirii şi combaterii focului bacterian al rozaceelor a crescut considerabil în ultimii ani, ca urmare a răspândirii patogenului pe aproape întreg teritoriul ţării noastre. Obiectivul acestei lucrări are în vedere testarea unor extracte din plante precum Salvia officinalis, Thymus serpyllum, Pelargonium odoratissimum, Hedera helix, Ocimum basilicum, Levisticum officinale, Tagetes patula, Galium verum şi a unor molecule active, asupra unei suşe de Erwinia amylovora, izolată din lăstarii de gutui, ce prezentau simptome tipice atacului de foc bacterian. Materialul luat în studiu a fost recoltat din ferma “Vasile Adamachi” din cadrul Universităţii de Ştiinţe Agricole şi Medicină Veterinară Iaşi, iar cercetările  Irina-Paraschiva CHIRIAC, E. ULEA   70 s-au efectuat în laboratorul de cercetare al disciplinei de Fitopatologie. În urma experimetului efectuat se observă că, dintre cele opt extracte de plante, cea mai bună activitate antibacteriană o prezintă Pelargonium odoratissimum, Ocimum basilicum şi Salvia officinalis, iar dintre cele trei pesticide, Alcupral 50 PU. Contrar aşteptărilor, cele mai slabe rezultate s-au obţinut în urma testării produselor Aliette 80 WG şi Hedera helix. Rezultatele au fost interpretate statistic, folosind testul ANOVA.  Cuvinte cheie: focul bacterian; extracte din plante; Hedera helix; Alcupral 50 PU.  INTRODUCTION  This disease was first reported in Romania in 1992, in two counties from the Southeast region: Piteşti (Mărăcineni) and Brăila (Severin, 1996). Fire blight disease spread rapidly, in 1993 and since has been reported in nine counties (Constantinescu et al., 1994) and in 2000 all counties across Romania have reported the presence of the disease (Vlad, 2003). Most economically relevant apple and pear cultivars are highly susceptible to fire blight, a risk even more intensified in modern high-density orchards (Longstroth, 2001). Erwinia amylovora is a pathogen that affects plants from the Rosaceae family and has a wide host range. Fire blight has been described in around 200 species included in 40 rosaceous genera (van der Zwet and Keil, 1979). Among the four subfamilies of the Rosaceae, Maloideae (syn. Pomoideae), Rosoideae, Amygdaloideae (syn. Prunoideae) and Spireoideae only in the last fire blight has not been yet described. Chemical control relies upon the use of antibiotics (such as streptomycin) and copper compounds which prevent bacterial multiplication and further infection. Unfortunately, the antibiotics have lead to the selection of resistant bacterial populations and therefore their use is strictly limited or even forbidden in a number of countries (Cesbron et al., 2000). To control fire blight, many researchers have tested various natural components such as plant extracts and essential oils to determine their antibacterial activity against the pathogen Erwinia amylovora since 1989 in Europe, and later also to check ability to induce resistance. Aim of this study was to test in vitro antibacterial activity of some plant exctracts compared to three different pesticides against E.A. - A.G. strain, isolated from pomological collection of "Vasile Adamachi" farm.  MATERIAL AND METHOD  From quince samples with tipical fire blight symptoms, harvested from pomological colection of “Vasile Adamachi” farm, we isolated after performing the patogenicity test on immature pear fruits (Billing et al., 1960), a strain of Erwinia amylovora called E.A.- A.G.  In order to obtain plant extracts, flowers, leaves and stem portions of Salvia officinalis, Thymus serpyllum, Pelargonium odoratissimum, Hedera helix, Ocimum basilicum, Levisticum officinale, Tagetes patula and Galium verum were used. These were placed in SUSCEPTIBILITY OF ERWINIA AMYLOVORA STRAIN TO PLANT EXTRACTS AND PESTICIDES   71 glass jars with grain alcohol 40%. Dishes were kept at 28º C and dark for two weeks. After 14 days, the plant extracts obtained were stored at 4º C and darkness. Chemical control was performed using three different active substances: fosetyl aluminum (Aliette 80 WG), copper hydroxide (Champ 77 WG) and copper oxychloride (Alcupral 50 PU). Culture media and growth conditions. Bacteria was grown at 28°C on Nutrient Sucrose Agar (Billing et al., 1961) and King’s B (King et al., 1954) medium and examinated for fluorescence under ultraviolet light. Nutrient Sucrose Agar: NaCl 5 g, beef extract 1 g, peptone 5 g, agar 30 g, yeast extract 2 g, sucrose 50 g, NaOH 6 mL, distilled water 1000 mL, pH ajusted to 7.2 and sterilised by autoclaving at 121° for 20’ (Billing et al., 1961). King B medium: peptone 20g, glycerol 10 mL, K2HPO4 1,5g, MgSO4 · 7 H2O 1,5 g, agar 15 g, distilled water 1000 mL, pH ajusted to 7.0 – 7.2 and sterilised by autoclaving at 121° for 20’ (King et al., 1954). Microbiological media plates were prepared using Masterclave 09 plate maker and an aliquot portion of 16mL of media was poured using APS 320 automated Petri plate filler (AES). In each Petri dish was added 1mL of each plant extract and pesticide used and mixed together. All Petri dishes were sown with E.A. – A.G., Erwinia amylovora strain and than incubated at 28°C. Antimicrobial activity was assessed by measuring the growth inhibition zones diameters. The results were estimated statisticaly, by performing ANOVA test.  RESULTS AND DISCUSSION  Bacterial colonies outlines were recorded after their appearance in Petri dishes, each 12 hours using different color markers. Figure 1 presents data concerning bacterial colony diameter after 42 hours, because then no differences were recorded. Analyzing the figure below, we can see differences between the bacterial colonies diameter on NSA and King B medium mixed with the same products.  Although fire blight has been known for more than 200 years, most, if not all developed bactericides tested against this disease have shown no satisfactory and reliable to be used in the spray methodology, recommended for field application. There are many reasons behind, the main one being the nature of the disease and lack of effective, commercially available, environmentally safe and non-phytotoxic systemic bactericides (Psallidas and Tsiantos, 2000). Of the three active molecules tested in vitro against E.A. – A.G. strain, copper oxychloride (Alcupral 50 PU) was able to inhibit the bacterial activity. This is confirmed not only by reduced diameter of bacterial colonies but also of their color, which is dark brown. The effectiveness of copper against various pathogens is attributed to the availability of copper ions that inactivate many different enzymes and other proteins essential to vital cell membrane function. Under the same conditions, copper hydroxide (Champ 77 WG) showed a weak antibacterial activity comparing to copper oxychloride (Alcupral 50 PU). Irina-Paraschiva CHIRIAC, E. ULEA   72  Figure 1 - The diameter of bacterial colony formed by E.A. - A.G. strain on NSA and King B culture medium mixed with pesticides  Fosetyl-aluminum is an acidic systemic fungicide marketed under the name Aliette and used primarily against different species of the genus Phytophthora. However, because of its systemic property, fosetyl aluminum was thought to be a good bactericide against fire blight. The results of experiments conducted in both the USA and Europe showed that its effectiveness was inconsistent. Norelli and Aldwinckle (1993) reported that the use of Aliette gave poor control of fire blight under both high or low inoculum pressure and it failed to control blossom blight in spring when applied the previous autumn. Following the experiment performed in vitro, we found low efficiency of this product to destroy the bacterium Erwinia amylovora. Figure 2 presents the results on the effectiveness of antibacterial plant extracts tested. On both culture media mixed with the extract obtained from Pelargonium odoratissimum, E.A. - A.G. strain has formed small colonies. Mosch et al. (1989) also tested, the extract of Pelargonium odoratissimum in vitro and the results were satisfactory. Antibactericid effect of this extract was confirmed by the results in control of Erwinia amylovora strain isolated from pear shoots (Chiriac and Ulea, 2012). On NSA culture medium, Pelargonium odoratissimum, Ocimum basilicum and Salvia officinalis, showed a very good antibacterial activity. Analyzing the data in the figure above we see that the extracts obtained from Pelargonium odoratissimum, Ocimum basilicum and Salvia officinalis showed antibacterial activity better than, that obtained from Hedera helix. Six of the 11 products tested in vitro showed a lower bacterial colony diameter on NSA medium, compared with those of the same environmental conditions on King B.  SUSCEPTIBILITY OF ERWINIA AMYLOVORA STRAIN TO PLANT EXTRACTS AND PESTICIDES   73  Figure 2 - The diameter of bacterial colony formed by E.A. - A.G. strain on NSA and King B culture medium mixed with plant extracts  Table 1 - The results of ANOVA test  Anova: Single Factor       SUMMARY       Groups Count Sum Average Variance   Alcupral 50 PU 2 1.46 0.73 0.1058   Pelargonium odoratissimum 2 1.56 0.78 0.005   Ocimum basilicum 2 2.06 1.03 0.0578   Salvia officinalis 2 2.3 1.15 0.125   Galium verum 2 2.5 1.25 0.125   Levisticum officinale 2 2.43 1.215 0.06845   Tagetes patula 2 2.1 1.05 0.045   Thymus serpyllum 2 2.29 1.145 0.02645   Champ 77 WG 2 2.49 1.245 0.01445   Aliette 80 WG 2 3.19 1.595 0.11045   Hedera helix 2 5.56 2.78 0.3528   ANOVA       Source of Variation SS df MS F P-value F crit Between Groups 6.115 10 0.6115 6.491507 0.00237 2.853625 Within Groups 1.0362 11 0.0942   Total 7.1512 21      The results were estimated statisticaly, by performing ANOVA test in Microsoft Office Excel 2007 (Table 1). The p-value tells us that the lowest significance level (0.01) attainable is only 0.00237 and F > F crit., so in this situation we are able to reject the null hypothesis. Therefore, there is very strong evidence to suggest that the means are not all equal. According to statistics the products tested in vitro show significant differences to inhibit E.A. – A.G. strain. Irina-Paraschiva CHIRIAC, E. ULEA   74  CONCLUSIONS  The highest antibacterial efficiency was found after testing copper oxychloride (Alcupral 50 PU) on both Nutrient Sucrose Agar and King B culture media. Following the experiment performed in vitro, Pelargonium odoratissimum, Ocimum basilicum and Salvia officinalis, showed good antibacterial activity. Six of the 11 products tested in vitro showed a lower bacterial colony diameter on NSA medium, compared with those of the same environmental conditions on King B. The weak antibacterial activity was recorded for the extract obtained from Hedera helix and Aliette 80 WG pesticide.  REFERENCES  Billing E., Crosse J.E., Garrett C.U.E., 1960 - Laboratory diagnosis of fire blight and bacterial blossom blight of pear. Plant Pathol. 9: 19–25. Billing E., Baker L.A.E., Cross J.E., Garrett C.M.E., 1961 - Characterization of English isolates of Erwinia amylovora (Burrill.). Winslow et al. Journal of Applied Bacteriology 24: 195-211. Cesbron S., Thomson S.V., Paulin J. P., 2000 - Acibenzolar-S-methyl induces the accumulation of defense-related enzymes in apple and protects from fire blight European Journal of Plant Pathology  Vol. 106 No. 6 pp. 529-536. Chiriac Irina, Ulea E., 2012 - In vitro susceptibility of Erwinia amylovora (Burrill) Winslow et al. strain isolated from pear to several plant extracts and different pesticides, Cercetări Agronomice în Moldova, Vol. XLV , No. 1 (149) / 2012, pp. 69. Constantinescu F., Severin V., Bibanu L., Chira E., 1994 - Protectia Plantelor (Plant protection), vol IV, nr. 14, Editura “Genesis” Cluj-Napoca, pp. 24-28. King E.O., Ward M., Raney D.E., 1954 - Two simple media for the demonstration of pyocyanin and fluorescein, J. Lab. Clin. Med. 44: 301-307. Longstroth M., 2001 - The 2000 fire blight epidemic in southwest Michigan apple orchards. Compact Fruit Tree 34: 16–19. Mosch J., Klingauf F., Zeller W., 1989 - On the effect of plant extracts against fireblight (Erwinia amylovora). Acta Horticulturae 273, 355–361. Norelli J., Aldwinckle H., 1993 - Orchard evaluation of chemical and biological spray materials to control fireblight. Acta Horticulturae 338, 363–368. Psallidas P.G., Tsiantos J., 2000 - Chemical control of fire blight, In Fire Blight: the Disease and its Causative Agent, Erwinia amylovora. Ed. J L Vanneste. Wallingford, United Kingdom: CAB International, pp. 199-234. Vlad F., 2003 - Epidemiologia şi prevenirea focului bacterian al rozaceelor (Erwinia amylovora), (Epidemiology and prevention of fire blight), Teza de doctorat, Bucureşti, pp. 9. Severin V., 1996 - Focul bacterian al rozaceelor (Fire bligh) (Erwinia amylovora), Edit. Ceres, Bucureşti. pp.67. van der Zwet T., Keil H.L., 1979 - Fire blight - A bacterial disease of Rosaceous plants. Agriculture Handbook 510. US Department of Agriculture. Washington. 200 pp. van der Zwet T., 1996 - Present worldwide distribution of fire blight. Acta Horticulturae 411: 7–8.  

Study regarding effectiveness of some plant extracts and different pesticides against an Erwinia amylovora (Burrill.) Winslow et al., strain isolated from quince

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Study regarding effectiveness of some plant extracts and different pesticides against an Erwinia amylovora (Burrill.) Winslow et al., strain isolated from quince

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