In-Depth Characterization of Crown Gall Disease of Tobacco in Serbia
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection and Bacterial Isolation
2.2. Molecular Characterization
2.2.1. DNA Isolation
2.2.2. Multiplex PCR Detection of Agrobacterium Species/Biovars
2.2.3. Detection of Virulence Genes
2.2.4. Multi-Locus Sequence Analysis (MLSA)
2.3. Phenotypic Characterization
2.3.1. Pathogenicity
2.3.2. Biochemical Tests
3. Results
3.1. Symptoms and Bacterial Isolation
3.2. Molecular Characterization
3.2.1. Multiplex PCR Detection of Agrobacterium Species/Biovars
3.2.2. Detection of Virulence Genes
3.2.3. Multi-locus Sequence Analysis (MLSA)
3.3. Phenotypic Characterization of Tobacco Isolates
3.3.1. Pathogenicity
3.3.2. Biochemical Tests
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Primer | Primer Sequence | Region | Fragment Length (bp) |
---|---|---|---|
UF/ B1R | 5′-GTAAGAAGCGAACGCAGGGAACT-3′ 5′-GACAATGACTGTTCTACGCGTAA-3′ | Chromosomal (23S rRNA) gene, A. tumefaciens/biovar 1 | 184 |
UF/ B2R | 5′-TCCGATACCTCCAGGGCCCCTCACA-3′ | Chromosomal (23S rRNA) gene, A. rhizogenes/biovar 2 | 1066 |
UF/ AvR | 5′-AACTAACTCAATCGCGCTATTAAC-3′ | Chromosomal (23S rRNA) gene, A. vitis | 478 |
UF/ ArR | 5′-AAAACAGCCACTACGACTGTCTT-3′ | Chromosomal (23S rRNA) gene, A. rubi | 1006 |
A/ C | 5′-ATGCCCGATCGAGCTCAAGT-3′ 5′-TCGTCTGGCTGACTTTCGTCATAA-3′ | Ti and Ri plasmid virD2 gene | 224 |
CYT/ CYT | 5′-GATCG(G/C)GTCCAATG(C/T)TGT-3′ 5′-GATATCCATCGATC(T/C)CTT-3′ | Ti plasmid ipt gene | 427 |
VCF3/ VCR3 | 5′-GGCGGGCGYGCYGAAAGRAARACYT-3′ 5′-AAGAACGYGGNATGTTGCATCTYAC-3′ | Ti and Ri plasmid virC gene | 414 |
PGF/ PGR | 5′-GGGGCAGGATGCGTTTTTGAG-3′ 5′-GACGGCACTGGGGCTAAGGAT-3′ | Chromosomal pehA gene, A. vitis | 466 |
atpD (800F/ 1350R) | 5′-GGCCAGGACGTTCTGTTCTT-3′ 5′-CTTGAAGCCCTTGATCGTGT-3′ | F0-F1 ATP synthase subunit beta | 465 |
dnaK (720F/ 1400R) | 5′-GAAGACTTCGACATGCGTCT-3′ 5′-GCCGAGCAGCTTGTTGTC-3′ | Heat shock protein, 70 kDa | 480 |
glnA (144F/ 900R) | 5′-GTCATGTTCGACGGCTCCT-3′ 5′-CCTTGGCATGCTTGATGAT-3′ | Glutamine synthetase | 474 |
rpoB (2040F/ 2718R) | 5′-GAAAACGACGACGCCAAC-3′ 5′-GCGCAGAAGCTTTTCTTCC-3′ | Beta subunit RNA polymerase | 534 |
27F 1495R | 5′-GAGAGTTTGATCCTGGCTCAG-3′ 5′-CTACGGCTACCTTGTTACGA-3′ | 16S rRNA | 1550 |
Species | Strain | Isolation Source | Country | Acc. No. |
---|---|---|---|---|
A. tumefaciens | HAMBI 105 | soil | USA | CP139997 |
Gle002 | walnut | USA: California | CP048564 | |
Yol001 | walnut | USA: California | CP048477 | |
Yol002 | walnut | USA: California | CP048473 | |
12D1 | - | - | CP033031 | |
183 | almond | Tunisia | CP029044 | |
O54/95 | cherry | - | CP124967 | |
BIM B-1315G | root endosphere of soybean | Belarus: Minsk | CP061003 | |
A. larrymoorei | AF3.44 | Ficus benjamina | USA: Florida | CP072167 |
CFBP5477 | - | Italy | CP124733 | |
A. leguminum | CFBP4996 | - | - | CP120211 |
A. fabrum | C58 | USA | AE007869 | |
1D132 | - | CP033022 | ||
A. vaccinii | B7.6 | blueberry | Poland | CP054150 |
A. pusense | 76 | hyphae Fusarium oxysporum f. sp. cucumerinum | China: Beijing | CP053856 |
M. huakuii a | NZP2235 | Lotus japonicus | New Zealand | CP139858 |
Isolate | ||||||||
---|---|---|---|---|---|---|---|---|
Test | DA5 | DA17 | DA21 | DA34 | DA40 | DA52 | DA65 | C58 |
Gram reaction | − | − | − | − | − | − | − | − |
Catalase | + | + | + | + | + | + | + | + |
Oxidase | − | − | − | − | − | − | − | + |
Production of fluorescent pigment | − | − | − | − | − | − | − | − |
Fermentation of: | ||||||||
glycerol | + | + | + | + | + | + | + | − |
d-arabinose | + | + | + | + | + | + | + | + |
l-arabinose | + | + | + | + | + | + | + | + |
d-ribose | + | + | + | + | + | + | + | + |
d-xylose | + | + | + | + | + | + | + | + |
d-adonitol | + | + | + | + | + | + | + | + |
methyl-β-d-xylopyranoside | + | + | + | + | + | + | + | − |
d-galactose | + | + | + | + | + | + | + | − |
d-glucose | + | + | + | + | + | + | + | + |
d-fructose | + | + | + | + | + | + | + | + |
d-mannose | + | + | + | + | + | + | + | + |
l-rhamnose | + | + | + | + | + | + | + | + |
dulcitol | + | + | + | + | + | + | + | + |
inositol | + | + | + | + | + | + | + | + |
d-mannitol | + | + | + | + | + | + | + | + |
d-sorbitol | + | + | + | + | + | + | + | + |
aesculin ferric citrate | + | + | + | + | + | + | + | + |
salicin | + | + | + | + | + | + | + | + |
d-cellobiose | + | + | + | + | + | + | + | + |
d-maltose | + | + | + | + | + | + | + | + |
d-lactose | + | + | + | + | + | + | + | + |
d-melibiose | + | + | + | + | + | + | + | + |
d-sucrose | + | + | + | + | + | + | + | + |
d-trehalose | + | + | + | + | + | + | + | + |
d-raffinose | + | + | + | + | + | + | + | + |
d-turanose | + | + | + | + | + | + | + | + |
d-lyxose | + | + | + | + | + | + | + | + |
d-tagatose | + | + | + | + | + | + | + | + |
d-fucose | + | + | + | + | + | + | + | − |
l-fucose | + | + | + | + | + | + | + | − |
d-arabitol | + | + | + | + | + | + | + | − |
l-arabitol | + | + | + | + | + | + | + | + |
erythritol | − | − | − | − | − | − | − | − |
l-xylose | − | − | − | − | − | − | − | − |
l-sorbose | − | − | − | − | − | − | − | + |
methyl α-d-mannopyranoside | − | − | − | − | − | − | − | − |
methyl α-d-glucopyranoside | − | − | − | − | − | − | − | − |
N-acetylglucosamine | − | − | − | − | − | − | − | − |
amygdalin | − | − | − | − | − | − | − | − |
arbutin | − | − | − | − | − | − | − | − |
inulin | − | − | − | − | − | − | − | − |
d-melezitose | − | − | − | − | − | − | − | − |
amidon (starch) | − | − | − | − | − | − | − | − |
glycogen | − | − | − | − | − | − | − | − |
xylitol | − | − | − | − | − | − | − | − |
gentiobiose | − | − | − | − | − | − | − | − |
potassium gluconate | − | − | − | − | − | − | − | − |
potassium 2-ketogluconate | − | − | − | − | − | − | − | − |
potassium 5-ketogluconate | − | − | − | − | − | − | − | − |
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Iličić, R.; Jelušić, A.; Barać, G.; Nikolić, D.; Stošić, N.; Scortichini, M.; Milovanović, T.P. In-Depth Characterization of Crown Gall Disease of Tobacco in Serbia. Agronomy 2024, 14, 851. https://doi.org/10.3390/agronomy14040851
Iličić R, Jelušić A, Barać G, Nikolić D, Stošić N, Scortichini M, Milovanović TP. In-Depth Characterization of Crown Gall Disease of Tobacco in Serbia. Agronomy. 2024; 14(4):851. https://doi.org/10.3390/agronomy14040851
Chicago/Turabian StyleIličić, Renata, Aleksandra Jelušić, Goran Barać, Dušan Nikolić, Nemanja Stošić, Marco Scortichini, and Tatjana Popović Milovanović. 2024. "In-Depth Characterization of Crown Gall Disease of Tobacco in Serbia" Agronomy 14, no. 4: 851. https://doi.org/10.3390/agronomy14040851
APA StyleIličić, R., Jelušić, A., Barać, G., Nikolić, D., Stošić, N., Scortichini, M., & Milovanović, T. P. (2024). In-Depth Characterization of Crown Gall Disease of Tobacco in Serbia. Agronomy, 14(4), 851. https://doi.org/10.3390/agronomy14040851