Detection and molecular characterization of Polymyxa betae , transmitting agent of sugar beet rhizomania disease , in Iran

The plasmodiophorid Polymyxa betae is considered as the only natural transmitting agent of Beet necrotic yellow vein virus (BNYVV), the most devastating agent of sugar beet fields throughout the world. To evaluate for the first time the genetic diversity of P. betae isolated from different autumn and spring sugar beet fields, and also to detect the presence of virus in these isolates, susceptible sugar beet plants (cv. Regina) were grown in soil samples collected from 10 different regions of Iran. P. betae detection was carried out using root microscopic observations, DAS-ELISA, and PCR-based methods. Results showed the presence of plasmodiophorids in all soil samples. Complementary assays also revealed the presence of viruses in soil samples collected from Khorasan Razavi, Fars, Hamadan and Kermanshah regions. The genetic diversity was evaluated through comparing glutathione-S-transferase nucleotide sequences amplified by PCR with the Internal Transcribed Spacers region. Results showed no significant differences in nucleotide sequences between virus-bearing and virus-free isolates of P. betae. Additional key words: Beta vulgaris; cystosori; ITS; GST; BNYVV; virus-bearing; virus-free. * Corresponding author: hasanzadeh.fatemeh1662@gmail.com Received: 18-04-13. Accepted: 11-07-14. Abbreviations used: BNYVV (Beet necrotic yellow vein virus); ELISA (enzyme-linked immunosorbent assay); GSH (thiol group of the glutathione); GST (glutathione-S-transferase); ITS (internal transcribed spacers); PCR (polymerase chain reaction). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(3): 787-794 http://dx.doi.org/10.5424/sjar/2014123-4385 ISSN: 1695-971X eISSN: 2171-9292 RESEARCH ARTICLE OPEN ACCESS 788 F. Hasandazeh Davarani et al. / Span J Agric Res (2014) 12(3): 787-794 conventional or transgenic breeding, offers a new approach in its continuing struggle against rhizomania to the sugar beet industry (McGrann et al., 2009). Germplasm screening for resistance to P. betae infection demands a quick diagnostic test to select those plants that show low symptoms of carrying the parasite when grown in infested soil (Mutasa et al., 1993). Polymyxa species are obligate parasites of plant roots and their detection involves skilled and tedious microscopic examination (Mesbah et al., 1997). In recent years, molecular techniques have been developed to study and detect Polymyxa species (Mutasa et al., 1993, 1995, 1996; Ward et al., 1994, 2004). Such procedures also have the potential to distinguish isolates of the parasite from different host plant or virus transmission specif icities. Through comparing internal transcribed spacers (ITS) sequences of Olpidium brassicae, S. subterranea; P. brassicae, Ligniera spp. and Polymyxa graminis, Legrève et al. (2002) clearly distinguished P. graminis from the rest. The specif ic rDNA sequence type of P. graminis varies depending on the host range, temperature requirements, and geographical origin. Therefore, Legrève et al. (2002) suggested the classif ication of these sequences into different subgroups (formae speciales). Glutathione-S-transferase (GST) is an enzyme which has been found in all living species, including plants, animals, fungi, and bacteria. It belongs to a super family of multi-functional enzymes that detoxify endobiotic and xenobiotic compounds by conjugating glutathione (GSH) to a hydrophobic substrate (Öztetik, 2008). Previous study showed that GST was expressed at high levels in P. betae, and could overcome host plant defense mechanisms (Mutasa-Gottgens et al., 2000). Kingsnorth et al. (2003) found that P. betae GST is an immunogenic protein. Safarpour et al. (2012) cloned the coding sequences of the GST gene from an Iranian P. betae isolate and developed an efficient procedure for producing a polyclonal antibody against this Iranian isolate. The objectives of this study were: (i) to detect P. betae in spring and autumn sowing sugar beet fields based on quantitative ELISA, specific PCR assay, and microscopic observation; (ii) to investigate the presence of BNYVV in Polymyxa-infected isolates using the ELISA method; iii) to determine variability of Iranian P. betae isolates (virus-bearing and virus-free) based on rDNA and GST sequences. Material and methods Detection of Polymyxa betae in infested soil samples In order to detect P. betae, susceptible sugar beet plants, cv. Regina (as a bait plant) were grown in soil samples collected from 10 different regions of Iran including Markazi, West Azarbaijan, Khoozestan, Golestan, Hamadan, Alborz, Kerman, Kermanshah, khorasan Razavi, and Fars (Fig. 1). Soil samples were taken from five different parts of the field in each region and were mixed thoroughly. To provide favorable growth conditions for P. betae, the soil samples (250 cm in a pot) were dried and sieved to 3 mm, then were heated to 40°C for 15 h to encourage P. betae germination (Beemster & De Heij, 1987). Five weeks after cultivation, microscopic examination was conducted to confirm the infection. Rootlet samples were taken out from each plant and were washed to remove soil debris, then stained with lactophenol containing 0.1% cotton blue or acid fuchsine. Infection of P. betae was checked by observing the amount of resting spore clusters in pieces of the rootlets (about 1 cm long) of each plant under a light microscope. Detection of P. betae and BNYVV by ELISA To detect P. betae and BNYVV, DAS-ELISA test was carried out as described by Kingsnorth et al. (2003) and Asher et al. (2002) respectively, with slight modifications. Lateral root samples collected from Regina plants were used for P. betae detection and BNYVV quantification. The plant samples were prepared by crushing 0.2 g fresh roots in liquid nitrogen followed by 10-fold dilution (w/v) in extraction buffer (PBS buffer containing 2% PVP-25 and 0.2% skimmed dried milk). Specific detection of P. betae was achieved with polyclonal antibody against recombinant expressed protozoan GST (Safarpour et al., 2012). Also the BNYVV was specifically detected using anti-BNYVV polyclonal antibodies (kindly provided by Mr. Saeed Darabi, Research Center for Agriculture and Natural Resources, Fars Province, Iran). DAS-ELISA tests were conducted using negative (recombinant immunodominant membrane protein; 100 μg mL, kindly provided by Dr. Fatemeh Shahryari, Agricultural Biotechnology Research Institute of Iran) and positive (GST recombinant protein; 100 μg mL) controls for P. betae. Soil samples collected from Shiraz, Iran, contained the A type of BNYVV (Amiri et al., 2003). Sugar beet Regina’s cultivar was used as both negative and positive controls in all tests. To produce negative control plants, seedlings of the cultivar Regina were transplanted into the sand and for positive controls, the seedlings were transplanted into a uniform mixture of infested soil and sand (3:7 v/v). All samples were tested in triplicate. To quantify the expressed GST from P. betae, a standard curve with 3500 μg mL of purified recombinant GST (10 to 0.5 μg mL Tris-HCl buffer) was plotted in triplicate and was analyzed in parallel (Table 1). Total protein concentration of the GST standard was determined using the Bradford (1976) assay.


Introduction
Rhizomania, which causes both abnormal rootlet proliferation and sugar yield loss, is the most destructive disease in sugar beet (Beta vulgaris L.) growing areas in Europe, Asia, and America (McGrann et al., 2009).The causal agent of the disease is Beet necrotic yellow vein virus (BNYVV) and the natural vector of virus is Polymyxa betae Keskin (Fujisawa & Sugimoto, 1977).Polymyxa belongs to the order Plasmodiophora containing many genera, among which Plasmodiophora brassicae (the causal agent of brassica club root), Spongospora subterranea (the causal agent of powdery scab of potato), and P. betae are of agroeconomic importance as pathogens or virus vectors (Kanyuka et al., 2003;Rush, 2003).The vector can survive in the soil for years and transmit other different viruses including Beet soil-borne mosaic virus (Wisler, 2001), Beet soil-borne virus, and Beet virus Q (Prillwitz & Schlösser, 1992).
With no acceptable pesticides available to restrict the vector, the control of rhizomania is now achieved almost exclusively through using resistant cultivars.A single dominant resistance gene, Rz1, has been used to manage the disease worldwide in recent years, although this gene confers only partial resistance (McGrann et al., 2009).New variants of BNYVV have evolved which are able to cause signif icant yield penalties on resistant cultivars (McGrann et al., 2009).A breeding strategy for durable resistance to BNYVV is to combine virus resistance genes (e.g., Rz1, Rz2) with resistance genes to P. betae vector (Barr et al., 1995;Asher et al., 2009;Pavli et al., 2011).Combined virus and vector resistance, achieved either by

Detection and molecular characterization of Polymyxa betae, transmitting agent of sugar beet rhizomania disease, in Iran
Fatemeh Hasanzadeh Davarani 1 *, Saeed Rezaee 2 , Seyed B. Mahmoudi 3 , Peyman Norouzi 3 , Mohammad R. Safarnejad 4 and Hossein Safarpour 5 conventional or transgenic breeding, offers a new approach in its continuing struggle against rhizomania to the sugar beet industry (McGrann et al., 2009).Germplasm screening for resistance to P. betae infection demands a quick diagnostic test to select those plants that show low symptoms of carrying the parasite when grown in infested soil (Mutasa et al., 1993).
Polymyxa species are obligate parasites of plant roots and their detection involves skilled and tedious microscopic examination (Mesbah et al., 1997).In recent years, molecular techniques have been developed to study and detect Polymyxa species (Mutasa et al., 1993(Mutasa et al., , 1995(Mutasa et al., , 1996;;Ward et al., 1994Ward et al., , 2004)).Such procedures also have the potential to distinguish isolates of the parasite from different host plant or virus transmission specificities.Through comparing internal transcribed spacers (ITS) sequences of Olpidium brassicae, S. subterranea; P. brassicae, Ligniera spp.and Polymyxa graminis, Legrève et al. (2002) clearly distinguished P. graminis from the rest.The specif ic rDNA sequence type of P. graminis varies depending on the host range, temperature requirements, and geographical origin.Therefore, Legrève et al. (2002) suggested the classification of these sequences into different subgroups (formae speciales).
Glutathione-S-transferase (GST) is an enzyme which has been found in all living species, including plants, animals, fungi, and bacteria.It belongs to a super family of multi-functional enzymes that detoxify endobiotic and xenobiotic compounds by conjugating glutathione (GSH) to a hydrophobic substrate (Öztetik, 2008).Previous study showed that GST was expressed at high levels in P. betae, and could overcome host plant defense mechanisms (Mutasa-Gottgens et al., 2000).Kingsnorth et al. (2003) found that P. betae GST is an immunogenic protein.Safarpour et al. (2012) cloned the coding sequences of the GST gene from an Iranian P. betae isolate and developed an efficient procedure for producing a polyclonal antibody against this Iranian isolate.
The objectives of this study were: (i) to detect P. betae in spring and autumn sowing sugar beet fields based on quantitative ELISA, specific PCR assay, and microscopic observation; (ii) to investigate the presence of BNYVV in Polymyxa-infected isolates using the ELISA method; iii) to determine variability of Iranian P. betae isolates (virus-bearing and virus-free) based on rDNA and GST sequences.

Detection of Polymyxa betae in infested soil samples
In order to detect P. betae, susceptible sugar beet plants, cv.Regina (as a bait plant) were grown in soil samples collected from 10 different regions of Iran including Markazi, West Azarbaijan, Khoozestan, Golestan, Hamadan, Alborz, Kerman, Kermanshah, khorasan Razavi, and Fars (Fig. 1).Soil samples were taken from five different parts of the field in each region and were mixed thoroughly.To provide favorable growth conditions for P. betae, the soil samples (250 cm 3 in a pot) were dried and sieved to 3 mm, then were heated to 40°C for 15 h to encourage P. betae germination (Beemster & De Heij, 1987).Five weeks after cultivation, microscopic examination was conducted to confirm the infection.Rootlet samples were taken out from each plant and were washed to remove soil debris, then stained with lactophenol containing 0.1% cotton blue or acid fuchsine.Infection of P. betae was checked by observing the amount of resting spore clusters in pieces of the rootlets (about 1 cm long) of each plant under a light microscope.

Detection of P. betae and BNYVV by ELISA
To detect P. betae and BNYVV, DAS-ELISA test was carried out as described by Kingsnorth et al. (2003) and Asher et al. (2002) respectively, with slight modifications.Lateral root samples collected from Regina plants were used for P. betae detection and BNYVV quantification.The plant samples were prepared by crushing 0.2 g fresh roots in liquid nitrogen followed by 10-fold dilution (w/v) in extraction buffer (PBS buffer containing 2% PVP-25 and 0.2% skimmed dried milk).Specific detection of P. betae was achieved with polyclonal antibody against recombinant expressed protozoan GST (Safarpour et al., 2012).Also the BNYVV was specifically detected using anti-BNYVV polyclonal antibodies (kindly provided by Mr. Saeed Darabi, Research Center for Agriculture and Natural Resources, Fars Province, Iran).DAS-ELISA tests were conducted using negative (recombinant immunodominant membrane protein; 100 µg mL -1 , kindly provided by Dr. Fatemeh Shahryari, Agricultural Biotechnology Research Institute of Iran) and positive (GST recombinant protein; 100 µg mL -1 ) controls for P. betae.Soil samples collected from Shiraz, Iran, contained the A type of BNYVV (Amiri et al., 2003).Sugar beet Regina's cultivar was used as both negative and positive controls in all tests.To produce negative control plants, seedlings of the cultivar Regina were transplanted into the sand and for positive controls, the seedlings were transplanted into a uniform mixture of infested soil and sand (3:7 v/v).All samples were tested in triplicate.To quantify the expressed GST from P. betae, a standard curve with 3500 µg mL -1 of purified recombinant GST (10 to 0.5 µg mL -1 Tris-HCl buffer) was plotted in triplicate and was analyzed in parallel (Table 1).Total protein concentration of the GST standard was determined using the Bradford (1976) assay.

DNA extraction
DNA was extracted from the dried lateral root plants, grown in each soil of the different origins, according to Liu et al. (2000) with slight modifications.Lateral roots (1 g) were frozen in liquid nitrogen and were dis- rupted to a fine powder in lysis buffer [400 mM Tris-HCl (pH 8.5), 60 mM EDTA (pH 8.5), 150 mM NaCl, 1% SDS] using mortar and pestle.All centrifugation steps were conducted at 11000 × g for 5 min at 4°C.

Cloning and sequencing of the P. betae GST gene
PCR (polymerase chain reaction) amplification of GST gene was performed using forward (5'-CAACGT CGACAAGGGACCAAGGTCAATGC-3') and reverse (5'-CAACGCGGCCGCTTATTTTGGACCGGCTGC-3') primers developed by Safarpour et al. (2012).PCR was performed in a volume of 50 μL containing 0.2 μM primers, 1U Taq DNA polymerase (Promega, Madison, USA), 0.2 mM dNTPs (Promega, Madison, USA), 10X PCR buffer (Promega, Madison, USA) and 23 mM MgCl 2 .The cycle parameters in the PCR program were as follows: 95°C for 5 min; 35 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 90 s; a final extension at 72°C for 5 min.PCR products were analyzed on 1% agarose gel and DNA was then eluted from the gel using the Agarose Gel DNA Extraction Kit (Roche, Ind, USA).PCR products were subjected to ligation with a TA cloning vector pTZ57R/T, 2886 bp (Fermentas, Lithuania).The ligated product was transformed into E. coli DH5α and plated onto LB agar Amp-X gal-IPTG plates.Plates were left at 37°C for 12-14 h.On the next day, blue and white colonies were observed and positive white colonies were further grown for plasmid isolation.Intact clones containing the right sequence were selected after digestion, PCR amplification, and subsequent sequencing analysis using M13 sequencing primers.

PCR amplification, cloning and sequencing of P. betae rDNA
A fragment of rDNA between ITS1, 5.8s gene and ITS2 regions from P. betae was amplif ied using specific primers for polymyxa detection [Psp1 (5´-TAGACGCAGGTCATCAACCT-3´) and Psp2rev (5´-AGGGCTCTCGAAAGCGCAA-3´)] as described by Legrève et al. (2003).PCR was performed in a volume of 20 μL containing 1 μL DNA template (30 ng), 1 μL of each forward and reverse primers (30 ng), 11.2 μL ddH 2 O, 2 μL 10X PCR buffer, 1.6 μL 10 mM dNTP, 2 μL 25 mM MgCl 2 and 0.2 μL Taq DNA polymerase (Promega).Cycling conditions were as follows: 95°C for 2 min; 36 cycles of 96°C for 45 s, 50°C for 45 s and 72°C for 1 min; and a final synthesis at 72°C for 10 min.The PCR products were run on 1% agarose gel followed by recovery using DNA Extraction Kit (Vivantis, Malaysia).PCR products were then subjected to ligation with a TA cloning vector pTG19/T (Vivantis, Malaysia).The ligated products were transformed into E. coli DH5α and intact clones containing the right sequence were selected by colony PCR using M13 universal primers.Finally, subsequent sequencing analysis was conducted as mentioned above.

P. betae phylogenetic tree based on rDNA region
Sequence polymorphism of P. betae isolates ribosomal DNA, originated from each soil sample, was analyzed by CLUSTALW and was adjusted manually.The most appropriate model was determined using Bayesian Information Criterion (BIC) implement in MEGA version 5.0 program.The alignments selected by the lowest BIC were used to calculate genetic distance by k2+G method (Tamura et al., 2011).Phylogenetic analyses were done using the neighbor-joining method within the MEGA 5.0.Robustness of inferred evolutionary relationships were assessed by 1,000 bootstrap replicates.P. betae sequence [GenBank Acc.No. Y12827 (Ward & Adams, 1998)] was compared with P. betae isolates collected from the different geographical regions of Iran.Several P. graminis subspecies and other plasmodiophorids were also used for sequence comparison including: P. graminis f. sp.temperata (Y12824), P. graminis f. sp.tropicalis (Y12825), P. graminis f. sp.colombiana (AJ010424), P. graminis f. sp.tepida (Y12826), Ligniera sp.(AJ010425), and Plasmodiophora brassicae (Y12831)].

Observation of P. betae sporosori in rootlets of sugar beet
Based on cystosori morphology (irregular clusters) and cystosori individual size (4-7 μm diameter), the parasite was identified as P. betae (Fig. 2).P. betae cystosori were greatly detected in the roots of 'Regina' plants grown in sand and soil mixture, but were absent in roots of the plants grown in sand alone.

ELISA-based assay of P. betae and BNYVV
To determine P. betae concentration and BNYVV content using ELISA method, lateral roots of the test plants were used.The ELISA displayed significant differences in P. betae concentration and BNYVV based on optical density (OD) in different soil samples.The ELISA results showed the presence of P. betae in all soil samples (Fig. 3).Soil samples collected from Khorasan Razavi, Fars, Golestan and Kerman showed the highest P. betae contamination.Using specif ic BNYVV antibodies, ELISA results confirmed the presence of virus in samples collected from Khorasan Razavi, Fars, Hamadan and Kermanshah, but other samples were detected as virus-free (Fig. 3).

P. betae variability in rDNA sequence and phylogenetic analysis
Using P. betae-infected sugar beet DNA and Psp1 and Psp2rev primers, PCR fragments of about 454 bp were produced.ITS1 + 5.8S + ITS2 rDNA sequences from all P. betae isolates (A, AZ, D, G, H, K, KE, KH, M and S) were determined and aligned with rDNA sequence of P. betae isolate from the UK (Acc.No. Y12827, Ward & Adams, 1998).Results showed a very high homology among them (Fig. 4).A few single nucleotide changes were detected in ITS1+5.8S+ITS2region leading to a very close phylogenetic distance among different P. betae isolates of Iran.However, P. betae could be distinctly separated from P. graminis (Group 2).Within Group 2, two subgroups were detected: one comprised of f.sp.temperata and f.sp.tropicalis and the other comprised of f.sp.colombiana and f.sp.tepida.Two other plasmodiophorids including Ligniera sp. and Plasmodiophora brassicae were considered as out-group.

Comparison of P. betae isolates using GST sequence
Using all DNA samples, GST-specific primer set produced a PCR product of about 903 bp length.The sequence of two isolates were determined and deposited in Genbank (AGC94564.1 [Fars] and AGC94563.1 [Golestan]).After comparing with other GST genes in the Genbank, two exon regions between 1-343 and 655-903 nucleotides with an intron of 312 bp between them were identified.Because of no homology in blast searches, no difference was observed between the two Iranian sequences in GST exon and intron regions, but their exon regions differed in three nucleotides and five amino acids compared to other P. betae GST isolates in the GenBank (AJ132355).There was little sequence homology among P. betae-GST gene and other organisms such as Pseudomonas sp.DJ77T, Sphingomonas paucimobilis, Proteus mirabilis, Haemophilus influenza, Rhizobium leguminosarum, and blue green algae Cocomyxa sp.

Discussion
Rhizomania is one of the most widespread sugar beet diseases which is caused by BNYVV and results in severe yield loss in the absence of effective control methods (Pavli et al., 2011).Natural virus transmission occurs through the root parasite P. betae, a protist that dwells in the soil and survives in the form of sporosori (Fujisawa & Sugimoto, 1977).The virus is carried internally by 10%-15% of P. betae resting spores in the soil (McGrann et al., 2009).Currently, management of rhizomania disease is based on genetic resistance to BNYVV.However, for durable disease management, the decrease in inoculum potential of the vector as well as genetic resistance to the virus should be considered.The major objective of the present study was the detection and characterization of P. betae in 10 sugar beet sowing regions in Iran.Results confirmed the presence of this plasmodiophorid in all collected samples.Microscopic observation of cyctosors in infected roots was used for preliminary studies.Although it is a simple method for P. betae detection in plants, it is also time consuming and inaccurate.In an earlier study (Mesbah et al., 1997), this method was used for detection and quantification of P. betae.
To overcome these limitations, complementary approaches such as DAS-ELISA and PCR amplification analysis were performed on these samples.Results obtained from DAS-ELISA method confirmed the initial results obtained using microscopic observations.ELISA is ideal for usage in P. betae screening in sugar beet germplasm.Unlike PCR-based methods, antibodies detect the actual amount of antigen without amplification, and in addition are more likely able to differentiate between live and dead pathogen biomass (Kingsnorth et al., 2003).ELISA sensitivity, as determined by an ability to detect low levels of P. betae infection, enhances their utility for screening f ield samples.Complementary analysis for detection of BNYVV in selected samples confirmed the presence of virus in the fields of Fars, Khorasan Razavi, Kermanshah, and Hamadan regions while remaining regions were virusfree.Therefore, to prevent the spread of virus into clean areas, both soil and plant residues translocation from infected lands should be prohibited.
Genetic variation of the collected P. betae isolates, including virus-bearing and virus-free, was compared with another isolates of P. betae and P. graminis by comparing the sequence of rDNA (ITS1 and ITS2) regions and GST gene.Result showed a high level of homology among virus-bearing and virus-free P. betae isolates from different regions in Iran.However, only few nucleotide changes could be detected in sequenced P. betae ITS1 + 5.8 + ITS2 from different locations.Apparently, such deletions, insertions, and other mutations, which could appear due to higher genomic diversification, were not detected and as a result were not correlated to their geographic spread.Therefore, the lack of rDNA genetic diversity does not exclude differences in the ability to multiply, take up and transmit BNYVV which indicates more work is required to unravel variation in these virus vector properties.These results also showed major phylogenetic distance between the two plasmodiophorid isolates that may be re-  lated to their different hosts (as the main hosts for P. graminis are Poaceae which separates them from P. betae) which corresponds with Legrève et al. (2003) study.P. graminis isolates were very diverse in their ITS rDNA sequences (Legrève et al., 2002;Kanyuka et al., 2003).The lack of diversity in this region of P. betae has already been observed in other studies (Pferdmenges & Varrelmann, 2009;Smith et al., 2011Smith et al., , 2013)).Our work with Iranian isolates supports the finding that this part of the genome is highly conserved in P. betae.
Result showed that specific GST primer can be used for quick detection of P. betae using PCR method.Analyses of PCR product on agarose gel showed that the size of GST gene was 903-bp.Indeed, the results showed that P. betae-GST sequence was quite different from fungal GSTs sequence (Cavalier-& Chao, 2003).
In conclusion, based on the results obtained from DAS-ELISA analysis, P. betae was detected in all autumn and spring sowing sugar beet regions as well as newly cultivated regions (Golestan).On the other hand, phylogenetic analysis revealed that there is low genetic diversity among virus-bearing and virus-free P. betae isolates collected from different regions of Iran.

Figure 2 .Figure 3 .
Figure 2. Photomicrographs of sporosori of P. betae detected in roots of sugar beet 'Regina' planted in the soil sample from Fars.

Table 1 .
Quantification of P. betae using ELISA

technique Negative GST recombinant protein (µg mL -1 ) control
* IMP: immunodominant membrane protein.**Average of three replications.Detection of Polymyxa betae in Iran 789 Figure 1.Geographical distribution of sugar beet fields where the Polymyxa betae samples used in this study were collected.* Only a research field.