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DIAGNOSTICS OF POTATO BACTERIAL PATHOGEN
DICKEYA DIANTHICOLA
Karlov Alexander - PhD Student, Laboratory of Plant Protection RTSAU.
Tel. (499) 976-12-79; e-mail: [email protected].
Ignatov Alexander - DSc, Institute of Phytopathology, Academy of Agricultural Sciences; Center "Bioengineering" RAS. E-mail: [email protected] Karlov Gennadii Ilyich - DSc; tel. (499) 977-70-01; e-mail: [email protected].
Pekhtereva Erna Sharifovna - PhD, Institute of Phytopathology.
Academy of Agricultural Sciences. E-mail: [email protected] Matveeva Evgenia - PhD Mr., Institute of Phytopathology, Academy of Agricultural Sciences. E-mail: [email protected]
Schaad Norman - Ph.D; Foreign diseases and weeds science research unit, USDA-ARS, Fort Detrick, Maryland, USA. E-mail: [email protected] Varitsev Yuri - PhD, Potato Research Institute by A.G. Lorch, Academy of Agricultural Sciences. E-mail: [email protected] Dzhalilov Fevzi Seid Umerovich - DSc; tel. (499) 976-12-79, e-mail: [email protected].
Abstract: the host range and diagnostic methods where tested for bacterium Dickeya dianthicola, the causative agent of blackleg ofpotato, that has been recently been found in Russia. Artificial inoculation has identified additional host plants of D. dianthicola: tomato, tobacco, and iris. This should be considered at planning of protective measures. We obtained specific to D. dianthicola polyclonal antibodies, which provided in specific ELISA sensitivity about 105 cells / ml of the tuber extract. Specific probe ADE3 designed for the primers ADE1/ADE2 could be used to identify bacteria of the genus Dickeya by real-time PCR.
Key words: black leg of potato, Dickeya dianthicola, enzyme-linked immunoenzyme assay, real-time PCR.
Black leg is the most harmful bacterial disease of potatoes, and appears as a necroti-zation of plant stem in field and soft rot of tubers stored after harvest [2, 4].
This disease is caused by three separate but closely related species of pectolytic bacteria of family Enterobacteriaceae:
- Pectobacterium carotovorum subsp. carotovorum (Pcc, syn. Erwinia carotovora subsp. carotovora) [15];
- P atrosepticum (Pa, syn. E. carotovora subsp. atroseptica) [16];
- Dickeya spp. (D, syn. E. chrysanthemi or P chrysanthemi) [22].
The first two species are common pathogens of potato at the territory of the former USSR [2, 5, 7, 9, 10, 11]. Bacteria of the genus Dickeya differ significantly from other pathogens causing soft rots. For the first time these bacteria were described in the early 1950-s and called Erwinia chrysanthemi so as their first host was Chrysanthemum plant. In the 1980-s the bacteria were found to cause diseases of other crops, including potatoes [18].
Some later, E. chrysanthemi was transferred to a new genus Dickeya and divided into six species [22]. Bacteria of the genus Dickeya can damage a wide range of plants in diverse climatic conditions [23].
Since 2004, strains Dickeya spp. were causing significant economic losses in potato production in Western Europe. D. dadanthii and D. zeae (biovar 3) the most harmful pathogens of potatoes in hot-climate areas. D. dianthicola (biovars 1 and 7) is more adapted to the moderate climate and widely distributed in Europe [22]. Recently, this pathogen was found in Spain [20] and Finland [17]. European Organization of Plant Quarantine and Protection (EPPO) included the phytopathogenic bacteria of genus Dickeya into the list A2 of Plant Quarantine organisms [24].
In the last few years researchers have found an emergence of new genetic group of highle aggressive strains that are proposed to call D. solani [23].
In 2009, analyzing infected potato tubers from the Lipetsk region, we found for the first time in Russia bacteria of the genus Dickeya. A number of microbiological and molecular tests has proved similarity of the isolates to species D. dianthicola [6]. Potato seeds were the main source of infection. In many countries, the infection of potato by Dickeya was associated with planting material imported from the Netherlands [23]. In Russia, the risk may increase after entry to Word Trade Organization (WTO), and increasing import of planting material produced in countries with different phytosanitary situation. We need a reliable methods of detection with high sensitivity and specificity for this pathogen to reduce the possible damage from introduction of this emerging pathogen.
The aim of the work was to clarify the range of potential host plants of Russian strains of Dickeya sp. and develop methods of molecular and serological diagnostics of the pathogen.
Materials and methods
Bacteria were isolated from infected potato samples, obtained from several regions of Russian Federation in 2009-2010 on Potato agar (PDA) or nutrient broth with yeast extract (NBY) in Petri dishes. The plates were incubated for 2-3 days at 27°C. Flat, slightly translucent colonies were sub-cultured on Logan’s medium, and checked for pectate layer liquefaction. The selected pectolytic isolates were stored in 15% glycerol at -70°C and in sterile tap water at room temperature. All the isolates were analyzed with biochemical and physiological tests according to Laboratory guide for identification of plant pathogenic bacteria [13]. Reduced carbohydrates formed from sucrose and indole synthesis were tested as well. Pathogenicity of strains was evaluated by the ability to rot potato slices at 28 ° C in 24 and 48 h after infection.
To determine the virulence of the bacteria, we carried out inoculation of plants from different families, including carnation (Dianthus caryophyllus L., cv. “Margarita”), dahlia (Dahlia variabilis Desf., cv. “Figaro F1”), barley (Hordeum vulgaris L., cv. “Mikhailovsky”), wheat (Triticum aestivum L., cv. “Moskovskaya 39”), clover (Trifolium repens L., cv. “Red”), flax (Linum usitatissimum L., cv. “Smolensk”), white lupine (Lu-pinus albus L., cv. “Gamma”), oats (Avena sativa L., cv. “Horse”), vetch (Vicia sativa L., cv. “L'govskaya 22”), rape (Brassica napus L., cv. “Griffin” grade), triticale (Triticale Wittm. & A. Camus, cv. “Valentin”), iris (Iris germanica L., cv. “Rimefire”), potato (So-lanum tuberosum L., cv. “Luck”), tomato (Lycopersicon esculentum L., cv. “Belyi naliv”), tobacco (Nicotiana tabacum L., cv. “Samsun”).
For inoculation, the bacterial suspension with concentration 108 cells / ml was infiltrated by sterile syringe into leaf axils. Four plants of each species were inoculated by each strain, including D. dianthicola D9, D33, and P atrosepticum Eca 393, sterile tap water was used as a negative control. If symptoms appeared on the inoculated plant, re-isolation and pathogen identification was done to fulfill the Koch’s triad.
Specific polyclonal antiserum was obtained by immunization of rabbits with bacterial suspension prepared for D. dianthicola strains D9 and D17 grown on YDC medium for
48 h at 27°C according to Allan and Kelman [12]. Chinchilla rabbits (age 4-5 months) were immunized according to the method developed at Potato Research Institute (Moscow) for bacterial antigens. Immunoglobulins were isolated on chromatographic column filled with Protein A of Staphylococus aureus immobilized on sepharose as affine sorbent [3]. Antibodies were adjusted to a concentration of 1 mg/ml and stored as 1 ml aliquots at -20°C.
The antibodies titer was measured by indirect immuno-anzyme assay (ELISA) using anti-rabbit donkey antibodies labeled by peroxidase (Research Institute by Gamaleya, Moscow).
The obtained antibodies were conjugated with horseradish peroxidase by method of Nakane [19]. For P atrosepticum we used a commercial ELISA kit produced by the Potato Research Institute.
The sensitivity of antibodies was determined by ELISA against the strains of Dickeya sp., Pa, Pcc, and strain of Clavibacter michiganensis subsp. sepedonicus Cms204 as a negative control. All bacteria were applied at concentration of 108 cells / ml.
Sensitivity of the ELISA was evaluated against homologous strain of D. dianthi-cola D9.
The suspension of D9 was prepared from one-day age culture in the buffer and in potato tuber extract at range of concentrations up to 109 cells / ml and applied in reaction against the antibody adsorbed on standard 96-wells plate in series of ten-fold dilutions down to concentration of 10 cells / ml.
ELISA was performed by “double sandwich" method [1] and repeated 3 times. Optical density of ELISA enzymatic reaction product was measured at 450 nm using a microplate photometer (Bio-Rad, model 680).
DNA from bacteria was isolated by commercial kit “Proba-GS ("DNA-Technology", Moscow) according to manufacturer's recommendations. Collection of DNA for strains of all species of genus Dickeya was kindly provided by Dr. van der Wolf (Plant Research International, Wageningen, the Netherlands) [6].
Real-time PCR analysis was made with published primers ADE1/ADE2, specific to the pectatlyase (pelB) gene of Dickeya spp. [14], and original probe ADE3 (FAM-gcg ccg tcg tgc tgc aca tat ttt tcg ccg-BHQ1). Strains of D. dianthicola (D9, D17) were used as positive control, and strains Pcc and Pca (Ecc 3, Ecc 36, and Eca 393) - as negative one. DNA concentration was adjusted to 10 ug / ml.
MasterMix ("Dialat Ltd", Moscow) and «iCycler iQ5» (Bio-Rad Laboratories, USA) were used for PCR with following temperature-time profile: initial denaturation - 95°C, 9 min, 40 cycles of: denaturation at 94°C - 1 min, annealing - 57°C - 1 min and elongation - 72°C - 2 min.
The data were analyzed by MANOVA and compared by Duncan’s tests using SPSS 15.
Results and Discussion
Potato plants with typical symptoms of black leg collected at different regions of Russia in 2009-2010 were used for isolation of bacterial isolates. Colonies obtained on potato agar gentian violet had pale white center with a transparent mucous border. The same colonies on NBY medium were flat and almost transparent. Typical of Dickeya spp. colonies were sub-cultured on Logan’s medium with sodium polypectate. Strains of Pectobacterium and Dickeya softened the pectate layer. Among majority of Pectobacterium colonies we have found a number of bacteria, identified as Dickeya spp. by biochemical profile.
All the isolates were tested for hypersensitivity reaction (HR) in tobacco leaves, and HR-positive ones were used for pathogenicity test on potato slices. Classic PCR with specific primers for pelB gene ADE1/2 was applied to confirm identification of the Dickeya
spp. isolates (table 1), isolates of Pcc gave no PCR product with ADE1/2, although, they did not differ in reaction of indole reduced compounds synthesis, and in pectolytic activity.
T a b l e 1
Strains of genera Pectobacterium and Dickeya used in this work
Genus, Species Strain Origin, time of isolation Host plant
Collection of Laboratory of plant protection of RGAU-MSKHA by K.A. Timiryazev
Dickeya sp. D9Tr Moscow, 2010 Tomato
Dickeya sp. D231 Moscow region, 2010 Potato
Dickeya sp. D232 Moscow region, 2010 Potato
Dickeya sp. D233 Moscow region, 2010 Potato
Dickeya sp. D234 Moscow region, 2010 Potato
Dickeya solani D.Fil Voronezh region, 2010 Potato
Collection of Russian Research Institute of Phytopathology
Dickeya dianthicola D33 Nizhni Novgorod region, 2009 Potato
Dickeya dianthicola D9 Nizhni Novgorod region, 2009 Potato
Dickeya dianthicola D8 Nizhni Novgorod region, 2009 Potato
Dickeya dianthicola D17 Nizhni Novgorod region, 2009 Potato
Dickeya sp. D3B1 Voronezh region, 2010 Potato
Dickeya sp. D3B2 Voronezh region, 2010 r Potato
Dickeya sp. D3B3 Voronezh region, 2010 Potato
Dickeya sp. D1 Voronezh region, 2010 Potato
Dickeya sp. D9B Voronezh region, 2010 Potato
Artificial inoculation of carnations, dahlias, barley, wheat, clover, flax, lupines, oats, vetch, canola, and triticale did not produce any visible symptoms (table 2).
T a b l e 2
Evaluation of pathogenicity and range of host plants infected by D. dianthicola and P atrosepticum at artificial inoculation
Вид растения D. dianthicola, D9, D33 D. solani D Fil P. atrosepticum, Eca 393, Eca21, Eca31a Контроль (H2O)
Potato + + + -
Carnation - - - -
Tobacco + + - -
Tomato + + - -
Dahlia - - - -
Iris + + + -
Barley - - - -
Wheat - - - -
Clover - - - -
Flax - - - -
White lupine - - - -
Oats - - - -
Вид растения D. dianthicola, D9, D33 D. solani D Fil P. atrosepticum, Eca 393, Eca21, Eca31a Контроль (H2O)
Vetch - - - -
Rape - - - -
Triticale - - - -
Mustard - - - -
Note:. “+” Typical symptoms of wilting and necrosis, “-“ no reaction.
Iris plants and potatoes were affected by strain D9, D33, and Eca 393. In 4-5 days after inoculation, potato plants had a dark blurring spreading stem spot. Bacteria spread through the vascular system, caused wilting of the leaves, which eventually dried up completely.
Iris plants in 2-3 days turned black around the point of inoculation with bacterial exudates, leaves softened, and further spreading of the pathogen was accompanied by darkening of the entire leaf surface (fig. 1).
Plants of tomato and tobacco were not damaged by the strain Eca 393, but affected by D9 and D33. Plant stems of tomato were broken in the site of inoculation in 5-6 days, and leaves were wilted. Plant stem of tobacco darkened and lost turgor, the bacteria spread up from the site of inoculation in 3-4 days (see fig. 1).
Experiments conducted recently in Spain showed that Dickeya spp. can cause disease of potatoes, corn, onions, chicory, and African violet [21]. The obtained results allow us to include in this list tobacco, tomato, and iris. This improves our understanding of the pathogen
a) b)
c) d)
Fig. 1. Symptoms caused by D. dianthicola after artificial plant inoculation: a - iris, b - potato, c - tobacco, d - tomato
circulation in nature shows the importance of spatial isolation of potato from other host plant species.
ELISA results showed that D. dianthicola, P. atrosepticum and P carotovorum subsp. carotovorum were serologically distinct. The strains of D. dianthicola and Pa reacted only with homologous antiserum and did not cross-reacted. Pcc strains gave negative results agains both two antibodies (table 3). Thus, it was established that those three pathogen causing blackleg of potato are serologically different and can be easily distinguishable by ELISA.
Sensitivity of ELISA in detection of D. dianthicola showed no difference for buffer- and tuber-extract (table 4). In both variants, detection thresholds were around
T a b l e 3
ELISA reaction of the black leg pathogens against different antibodies
(optical density at 450 nm)
Species Strain Antibodies against:
D. dianthicola P. atrosepticum
D9 D17 Eca 393 Eca 31a Eca 21
D. dianthicola D9 D8 D17 D33 0,832 c 0,871 d 0,759 b 0,827 c 0,657 b 0,638 b 0,662 b 0,650 b 0,026 a 0,021 a 0,020 a 0,025 a 0,016 a 0,021 a 0,015 a 0,028 a 0,018 abc 0,015 ab 0,010 ab 0,026 abc
P. atrosepticum Eca 21 Eca 393 Eca 31a 0,020 a 0,030 a 0,047 a 0,023 a 0,020 a 0,017 a 0,300 b 0,841 c 0,320 b 0,118 c 0,300 d 0,128 c 0,229 d 0,546 e 0,256 d
P. carotovorum subsp. Carotovorum P39 P3-2 P3-3 0,013 a 0,038 a 0,030 a 0,013 a 0,023 a 0,020 a 0,008 a 0,030 a 0,026 a 0,009 ab 0,053 a 0,014 ab 0,009 a 0,010 ab 0,015 ab
Negative control-C.michiganensis ssp. sepedonicus Cms204 0,037 a 0,019 a 0,024 a 0,034 ab 0,021 abc
Note. Values with the same letter are not significantly different by Duncan’s test (95% probability).
T a b l e 4
ELISA sensitivity against D. dianthicola strain D9 at buffer and tuber extract
Concentration of D. dianthicola, CFU/ml (Factor A) Optical density at 450 nm, A450 (Factor B) Average for Factor A LSD05 = 0,080
buffer tuber extract
106 2.934c 2.117c 2.525c
105 0.745b 0.710b 0.727b
104 0.266a 0.288a 0.277a
103 0.257a 0.227a 0.242a
102 0.234a 0.223a 0.228a
Concentration of D. dianthicola, CFU/ml (Factor A) Optical density at 450 nm, A450 (Factor B) Average for Factor A LSD05 = 0,080
buffer tuber extract
101 0.220a 0.228a 0.224a
100 0.217a 0.229a 0.223a
10-1 0.230a 0.239a 0.234a
Negative control - phosphate Buffer 0.235a 0.237a 0.236a
Average for Factor B Ff < F05 0.593 0.500
LSD05 for pairs wise difference - 0,253.
105 cells / ml. This is indicated a high specificity of the obtained antibodies and the absence of reaction inhibitors in potato tuber extracts. Thus, Dickeya sp. could be detected by ELISA directly in tuber extract without labor-intensive isolation of bacterial pure culture.
The real-time PCR assay was developed based on the sequence of DNA gene pelB of Dickeya sp. and other enterobacteria available in the genebank (www.ncbi.nlm .nih.gov). Fluorescent
bye-labeled probe ADE3 was selected and the assay sensitivity and specificity has been evaluated in analysis of DNA of different species within genera Dickeya and Pectobacterium.
The threshold crossing values for 20 strains of Dickeya including D9 were in average at 25th cycle (with range 2137 cycles) (table 5), ie we observed the genus-specific reaction.
Such result shows that the probe ADE3 in combination with the primers ADE1/ADE2 can be used for reliable detection of bacteria of genus Dickeya, and can be included in diagnostic kit. Reliable identification of species within the genus Dickeya still available only by sequencing fragments of dnaX gene [6].
The threshold cycle value (Ct) in real time PCR at different bacterial cell
T a b l e 5
Threshold crossing values Ct for real-time PCR of Dickeya strains with primers ADE1/ADE2 and probe ADE3
Strains Dickeya spp. Threshold crossing cycles, Ct
2132 23,67 ± 1,47
2124 22,26 ± 0,03
2126 21,26 ± 0,16
2117 23,16 ± 0,25
2115 24,83 ± 0,15
2118 23,64 ± 0,02
2127 33,07 ± 0,19
2121 28,04 ± 0,50
2122 24,46 ± 0,16
2120 22,01 ± 0,54
2133 23,68 ± 0,01
2094 22,51 ± 0,01
2128 37,57 ± 0,41
2222 22,38 ± 0,11
2131 23,14 ± 0,11
2119 25,82 ± 0,03
2125 23,01 ± 0,04
2116 24,51 ± 0,19
2129 22,88 ± 0,11
D9 26,84 ± 0,32
Negative control - Pcc n.r.*
Negative control - Pa n.r.*
* No reaction.
concentration revealed a reliable decrease of Ct for increasing bacteria concentrations. The obtained curve (fig. 2) was used for calculation of PCR efficiency [8] with obtained
Fig. 2. Correlation between Ct and concentration of D. dianthicola cells
value 1.98 (incensement of copy number per cycle). Thus, the developed primer/probe combination has efficiency close to maximum for PCR and can be used for practical application.
Conclusions
1. The host plants for D. dianthicola include tomato, tobacco and iris. The isolation of potato fields from these species should be considered as a protective measure.
2. ELISA kit was developed for detection of D. dianthicola, and provided a high specificity and sensitivity around 105 cells / ml in the tuber extract.
3. Matched fluorescent probe ADE3, which in combination with primers ADE1/ADE2 can be used for rapid detection of bacteria of the genus Dickeya PCR in real time.
References
1. Varitsev Yu.A., Belov G., Uskov A., Varitseva G.P., Zavriev S.K., Arshava N.V, Zaitsev, V.V Guidelines for the diagnosis of causative agents of blackleg (Erwinia carotovora (Jones) Bergey et al.) and potato ring rot (Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth.) Skaptas-son et Burk.) by enzyme immunoassay, immunofluorescent microscopy and polymerase chain reaction. M.: Institute of potatoes, 2003.
2. Generalova I.V Biological characteristics of pathogens in the blackleg
Belarus in connection with selection for disease resistance and development of control measures: abstract. Candidate. thesis. Minsk, 1977.
3. Egorov A.M., Osipov A.P., Dzantiev B.B., Gavrilova E.M. Theory and practice of immuno-nofermentnogo analysis. Moscow: Higher School, 1991.
4. Israel VP. Bacterial diseases of plants. M.: Kolos, 1979.
5. Kapustin M.N., Knyazeva V.P. Ways to spread pathogens wet (soft) rot // Plant Protection, 1986. N 10. S. 32-33.
6. Karlov A., Zotov VS., Pekhtereva E.S., Matveeva E.V., Dzhalilov F.S., Fesenko I.A., Karlov G., Ignatov A.N. Dickeya dianthicola - new for Russia bacterial pathogen of potato // Proceedings of the TAA, 2010. No. 3. S. 134-141.
7. Lazarev A.M. Biological features of the causative agent of black leg of potato in the North-West zone of the RSFSR and the methods of diagnosis: abstract. Candidate. thesis. Leningrad, 1985.
8. Rebrikov D.V, Samatov G.A., Trofimov P.A., Semenov P.A., Savilova A.M., Kofiadi I.A., Abramov D. “Real time” PCR. M. BINOM. Laboratory of Knowledge, 2009.
9. Sanaa El Khatib Ramadan. Pathogenesis of black leg, depending on the virulence properties of the pathogen: abstract. candidate. thesis. M.: TAA, 1977.
10. Ho Li Yuan. Some features of the biology of the causative agent of black leg of potato: abstract. candidate. thesis. M., 1961.
11. Schneider Y. Bacterial diseases of potato caused by bacteria of the genera Pectobacterium, Pseudomonas and Bacillus: avtoref. doctor. M., 1972.
12. Alan E., Kelman A. Immunofluorescent stain procedures for detection and identification of Erwinia carotovora var. atroseptica // Phytopathol., 1977. 67. 1305-1312.
13. Bradbury J.F. Guide to plant pathogenic bacteria. Wallingford, UK: CABI, 1986.
14. Darrasse A., Priou S., Kotoujansky A., Bertheau Y. PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases // Appl. Environ. Microbioology, 1994. V. 60. P. 1437-1443.
15. De Haan E.G., Dekker-Nooren T.C. E.M., Van den Bovenkamp G.W. Speksnij-der A.G.C.L., Van der Zouwen P.S., Van der Wolf J.M. Pectobacterium carotovorum subsp. caroto-vorum can cause potato blackleg in temperate climates // European Journal of Plant Pathology, 2008. 122, 561-569.
16. Gardan L., Gouy C., Christen R., Samson R. Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavas-culorum sp.nov. and Pectobacterium wasabiae sp. nov // International Journal of Systematic and Evolutionary Microbiology, 2003, 53, 381-391.
17. Joutsjoki T., Laurila J., Pirhonen M., Lehtinen A., Hannukkala A. Diagnostics and incidence of black leg caused by Erwinia bacteria in Finland. Proceedings of the 16th Triennial Conference of the EAPR. Bilbao, Spain, 2005. 717-718.
18. Lumb V.M., PerombelonM.C.M., ZutraD. Studies of a wilt disease of the potato plant in Israel caused by Erwinia chrysanthemi // Plant Pathology, 1986. 35. 196-202.
19. Nakane P.K. New developments in tissue enzyme-labeled immunoassays // Immunoassays: clinical laboratory techniques for the 1980s, Alan R., Liss Inc., New York, 1980. 157-169.
20. Palacio-BielsaA., CambraM.A., LopezM.M. Characterization of potato isolates of Dickeya chrysanthemi in Spain by a microtitre system for biovar determination // The Annals of Applied Biology, 2006. 148. 157-164.
21. Palacio-Bielsa A., Mosquera MER, Alvarez MAC, Rodriguez IMB, Lopez-Solanilla E., Rodriguez-Palenzuela P Phenotypic diversity, host range and molecular phylogeny of Dickeya isolates from Spain // Eur. J. Plant Pathol., 2010. 127:311-324.
22. Samson R., Legendre JB, Christen R., Fischer Le SauxM., Achouak W., Gardan L. Transfer of Pectobacterium chrysanthemi (Burkholder et al. In 1953) Brenner et al. 1973 and Brenneria paradisiacal to the genus Dickeya gen. nov. as Dickeya chrisanthemi comb. nov. and Dickeya paradisiacal comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., and Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov., and Dickeya zeae sp. nov // International Journal of Systematic Evolution and Microbiology, 2005. 55. 1415-1427.
23. Tsror L., Erlich O., Lebiush S, Hazanovsky M., Zig U., Slawiak M. et al. Assessment of recent outbreaks of Dickeya sp. (Syn. Erwinia chrysanthemi) slow wilt in potato crops in Israel // European Journal of Plant Pathology, 2008. 123: 311-320.
24. The European Plant Protection Organization, 2011 http://www.eppo.org/QUARANTINE/ listA2.htm
This work was supported by the Federal Program “Scientific and scientific-pedagogical staff Innovative Russia”, a government contract and grant P2380 ISTC № 3431 and 3978.
ДИАГНОСТИКА БАКТЕРИАЛЬНОГО ПАТОГЕНА КАРТОФЕЛЯ
DICKEYA DIANTICOLA
Аннотация: исследовали круграстений-хозяев и возможность диагностики недавно обнаруженного в России возбудителя черной ножки картофеля — бактерии Dickeya dianthicola. При искусственном заражении было показано, что помимо картофелярастениями-хозяевами D. dianthicola являются томат, табак и ирис. Это необходимо учитывать при планировании защитных мероприятий. Были получены специфичные к D. dianthicola антитела, которые обеспечивали при диагностике методом ИФА высокую специфичность и порог чувствительности 105 клеток/мл в клубневом экстракте. Подобрана проба ADE3, которая в сочетании с праймерами ADE1/ADE2 может быть использована для выявления бактерий рода Dickeya методом ПЦР в реальном времени.
Ключевые слова: черная ножка картофеля, Dickeya dianthicola, иммуноферментный анализ, ПЦР в реальном времени.
Автор для корреспонденции: Джалилов Февзи Сеид-Умерович - д. б. н., зав. лабораторией защиты растений РГАУ-МСХА имени К. А. Тимирязева; e-mail: [email protected].
