CC BY
0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
MOLECULAR-GENETIC ANALYSIS OF AGROBACTERIUM TUMEFACIENS ISOLATES ISOLATED FROM WALNUT (JUGLANS REGIA L.) ORCHARDS
1G.G.Eshbekova, 2Z.F.Ismoilov
1PhD student, Samarkand State University, 2professor, Samarkand State University
https://doi.org/10.5281/zenodo.13828374
Abstract. In this study, molecular genetic analysis of isolates Agrobacterium tumefaciens isolated from crown gall infected seedlings in walnut orchards was carried out.
Keywords: Agrobacterium tumefaciens, crown gall, Juglans regia L., T-DNA, Ti plasmid, phylogenetic analysis.
Annotatsiya. Ushbu tadqiqotda, grek yongogi plantatsiyalarida bakterial rak bilan kasallangan ko chatlardan ajratib olingan Agrobacterium tumefaciens izolyatlarining molekulyar-genetik tahlili amalga oshirildi.
Kalit so^zlar: Agrobacterium tumefaciens, bakterial rak, Juglans regia L., T-DNK, Ti plazmida, filogenetik tahlil.
Аннотация. В статье проведен молекулярно-генетический анализ изолятов Agrobacterium tumefaciens, выделенных из зараженных корончатым галлом сеянцев ореха.
Ключевые слова: Agrobacterium tumefaciens, корончатый галл, Juglans regia L., Т-ДНК, Ti-плазмида, филогенетический анализ.
The main symptom of crown gall disease in walnut (Juglans regiaL), is galls in the crown of the tree. Crown gall is noted by the walnut industry as one of the main diseases affecting walnut seedlings and orchards in all walnut-growing regions [1,2]. When young seedlings are infected with this disease, the yield decreases in proportion to the degree of galls: when a walnut seedling occupies 25% of the main body circumference in the first four years of production, the yield can be reduced up to 12% [3, 4]. If these tumors are left untreated, they can kill even very large trees. In addition, when tumors become necrotic, the galls allow the entry of wood rot fungi and other pathogens that can cause additional damage to the tree. Also, the galls serve as a habitat for various insects that can cause secondary damage to the tree or serve as vectors for other pathogens. Necrotic tumors usually do not contain large numbers of virulent A. tumefaciens cells, but they may serve as a source of new infection if some are left in the soil during cleaning. After A. tumefaciens enters the soil, it can live for many years even in the absence of a host plant [5]. Interestingly, A. tumefaciens has been shown to spread systematically in walnut trees, which makes it difficult to maintain a stock of clean plants. Systemically infected trees may not show symptoms at the time of planting in the field or for several months after injury. As a result, it is necessary to reduce the risk of early infection with A. tumefaciens in order to prevent disease occurrence in young trees several months or years later [6,7].
Nowadays, walnut orchards have been established in several regions on a total area of more than 14.000 hectares. In these orchards, seedlings of world-famous varieties with their productivity, rapid growth and valuable fruits that meet world standards are planted and the introduction features are being studied. In particular, in 2017-2018, orchards were established based on the Chandler variety and Paradox Vlach rootstock in Jomboy, Bulungur, Payarik districts of Samarkand region [8].
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
In 2018-2020, the incidence rate of crown gall increased significantly in these walnut orchards and mechanical methods were used to combat the tumors. However, this method required labor and was also very expensive and in most of the seedlings, the smaller size galls were left untreated. As a result, by 2022, the percentage of seedlings infected with this disease in orchards reached 60-70% on average [9]. In this article, molecular genetic analysis of A. tumefaciens isolates from seedlings infected with crown gall was studied.
As a result of research, 34 isolates of A.tumefaciens were isolated from tumors formed on the crown of infected seedlings in the Jomboy orchard, 24 from the Bulungur orchard, 32 from the Payarik orchard and their molecular genetic and virulence characteristics were analyzed. Kalanchoe daigremontianaMill, Vlach rootstocks and carrot discs (Daucus carota) were used as indicator plants to determine the virulence of isolates (Fig. 1).
Fig.6 Tumor formation on plantlets infected with isolates: A-EG175; B-EG 219; C-
EG253
As a result, 63 of the isolates showed different levels of virulence, while 27 isolates showed no virulence. 12 virulent isolates (EG 106; EG 122; EG 144; EG 175; EG 207; EG 210; EG 219; EG 227; EG 238; EG 246; EG 253; EG 296) that showed virulence properties higher than 60% and 3 non-virulent isolates (EG 264; EG 283; EG 299) were examined by PCR to detect the VirD2 gene in T-DNA, which determines virulence. Results of PCR analysis shown that VirD2 gene was present in all virulent isolates, but do not have in non-virulent isolates.
16S rRNA gene sequencing analysis of 10 isolates (EG 106; EG 175; EG 207; EG 210; EG 219; EG 227; EG 238; EG 246; EG 253; EG 296) showed more than 60% virulence in Vlach rootstock was performed, a continuous contig was created in Bioedit program based on sequencing results. Then results were compared with the species in NCBI database based on BLAST program. The i solates were submitted to the database based on the relevant numbers (PQ098240, PQ098241, PP854426, PQ098242, PQ098243, PQ098244, PQ113704 PQ098245, PQ098246, PQ098247) (Table 1). Also, the phylogenetic characteristics of the EG219 strain, which showed high virulence were studied and analyzed.
Table 1. Comparative analysis of virulent strains sequences with closely related
species in the NCBI database
Strains submitted to the NCBI database
Closest species (based on 16S rRNA genes)
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Accession Accession Similarity
Strains number Strains number rate (%)
EG106 PQ098240 A.tumefacienscqsm_f1 MN826533 100%
EG175 PQ098241 A.tumefaciensstrain YN11 OM288134 100%
EG207 PP854426 A.tumefaciensstrain CY02 MK249671 99.89%
EG210 PQ098242 A.tumefaciens EBMB6 OQ932814 99.92%
EG219 PQ098243 A.tumefaciens FBG1034 OR838799 98.65%
EG227 PQ098244 A.tumefaciensstrain R-7Q HQ396794 99.87%
EG238 PQ113704 A.tumefaciensstrain AR2 MT279042 99.68%
EG246 PQ098245 A.tumefaciensstrain JZY4-60 MT102305 100%
EG253 PQ098246 A.tumefaciens IITA-TZ011 0M909260 99.89%
EG296 PQ098247 A.tumefaciens X-A284 OP678587 100%
Conclusion. 63 out of 90 isolates of A. tumefaciens from seedlings infected with crown
gall in walnut orchards located in Jomboy, Bulungur, Payariq districts were found to have different
levels of virulence. Among them EG219 strain, which showed high virulence, planned to use to
test the resistance of local walnut varieties and forms to crown gall disease.
ADABIYOTLAR:
1. l.Pacurar D. I., Thordal-Christensen H., Pacurar M. L., Pamfil D., Botez C., Bellini C. Agrobacterium tumefaciens: From crown gall tumors to genetic transformation // Physiological and Molecular Plant Pathology. 2011. Vol. 76. Is.2. P. 76-81.
2. Barton I.S., Fuqua C., Platt T.G. Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants // Environ. Microbiol. 2018. Vol. 20. Is. 1. P. 16-29.
3. Yakabe L. E., Parker S. R., Kluepfel D. A. Role of systemic Agrobacterium tumefaciens Populations in Crown Gall Incidence on the Walnut Hybrid Rootstock 'Paradox' // Plant Dis. 2012. Vol.96. Is.10. P.1415-1421.
4. Epstein, L., Kaur S., McKenna J.R., Grant J.A., Olson W.H., Reil W.O. Crown Gall can spread between walnut trees in nurseries and reduce future yields. California Agriculture // 2018. Vol. 62. Is.3. P.111-115.
5. 5.Yakabe L. E., Parker S. R., Kluepfel D. A. Effect of pre-plant soil fumigants on Agrobacterium tumefaciens, pythiaceous species, and subsequent soil recolonization by A. tumefaciens // Crop Protection. 2010. Vol. 29. Is.6. P. 583-590.
6. Strauss S.L., Stover J.K., Kluepfel D.A. Impact of biological amendments on Agrobacterium tumefaciens survival in soil // Applied Soil Ecology. 2015. Vol. 87. P. 39-48.
7. Ferdous M. L., Hossain M. N., Ali M. O., Islam M. S., Yasmin S. Morphological, biochemical and molecular identification of the wild strain of Agrobacterium tumefaciens from crown gall infected mango tree // Fundamental and Applied Agriculture. 2021. Vol. 6. Is.1. P. 43-49.
