Carrot slice test: A reliable method for evaluating the tumorigenicity of Pseudomonas savastanoi pv. nerii

Summary Pathogenicity studies on oleander plants take a long time and require signi ﬁ cant costs to con ﬁ rm the identi ﬁ cation of isolates and to verify their pathogenicity. In this study, the carrot slice test was used as a rapid method for Pseudomonas savastanoi pv . nerii . The test is suitable for the investigation of tumorigenicity of 25 Pseudomonas savastanoi pv. nerii isolates from different parts of Hungary. On carrot slices, the ﬁ rst characteristic knots were observed 7 days after inoculation and were fully developed 16 days after inoculation. The one-year oleander plants used to con ﬁ rm the pathogenicity of the bacteria were inoculated with a bacterial suspension to con ﬁ rm the reliability of the carrot slice test. Characteristic symptoms of knots were observed on all oleander plants 46 days after inoculation. The results showed that the carrot slice method is a straightforward, rapid, and reliable method for testing and con ﬁ rming tumor formation of Pseudomonas savastanoi pv. nerii without plants.


Introduction
The oleander (Nerium oleander L.) is a popular ornamental plant because of its abundant and long-lasting fl owering.
Native to the Mediterranean region, it is popular in Central and Western Europe both as a greenhouse plant and as a terrace plant.Oleander knot disease caused by Pseudomonas savastanoi pv.nerii [1,2] is the most common and dangerous disease of oleander (Nerium oleander L.).The typical symptoms are knots or galls on stems, twigs, leaves, and seedcases [2,3] which are incited by phytohormones [4].The severity of the disease is mainly due to the systematic spread of the pathogen in the plant, which makes prevention and control very diffi cult [5].

Bacterial strains
Infected plant samples were collected between 2018 and 2023 from hobby gardeners and public places in Hungary.[35].In addition, the bacterial culture was stored at -20 ºC and -70 ºC in 20% glycerol preservative liquid for longer storage (3g Beef extract, 5 g pepton, 20 g glycerol, 1 l H 2 O).

Tumorigenicity test of carrot slices
The carrot taproots, which were bought in the supermarket, were washed under running water, next sterilised in 70% ethanol for 5 min, then washed with sterile distilled water, and fi nally dried on sterile paper.After sterilization, the carrot taproots were peeled, the top and bottom segments were cut off, and were sliced with a sterile scalpel to a thickness of 0.5 cm -1 cm.Carrot slices were inoculated separately per isolate with pure bacterial cultures using a toothpick and placed in sterile Petri dishes on moistened sterile fi lter paper.Each carrot slice was punctured once in the middle.For each isolate (Table 1), 3 slices inoculated with bacterial culture and 2 slices inoculated with sterile distilled water were used as negative controls.Petri dishes were placed in a plant growth chamber (PHCBI MLR-352H-PE) at 80% -85% relative humidity and 25 °C.Knots were observed until 20.days after inoculation and results were visually assessed [16,23,31].

Pathogenicity test on oleander plants
One-year-old home-grown oleander plants were used to verify the safety of the carrot slice assays.Internodes between the fi rst and second, and third and fourth leaves of oleander plants were injected with a sterile syringe needle containing bacterial suspension at a concentration of 5 × 10 7 cells mL -1 , which was determined using a spectrophotometer set at 560 nm.Sterile distilled water was used as a negative control.
Inoculated plants were kept in a greenhouse at a relative humidity above 90% for 1 week and observed 60 days after inoculation.

Identifi cation of re-isolated pathogens
The carrot slice and parts of oleander plants were surfacesterilized with 75% ethanol and small pieces of tissues were cut

Results and discussion
All tested strains caused small, green tumors on the endodermis of the carrot slides.It was not possible to distinguish our isolates on the basis of typical knot symptoms.
The fi rst young knots on carrot slices were observed 7 days after inoculation and the typical knots developed 16 days after inoculation (Figure 1).Positive control strains showed the same symptoms as our isolates.Negative control carrot slices inoculated with sterile distilled water showed no symptoms.
The pathogens were re-isolated from the knots and confi rmed by PCR.The reaction of the other two pathotypes of P. savastanoi (Ps pv.retacarpa and Ps pv.fraxini) showed a slight delay as the symptoms appeared 9 days after inoculation, but there was no difference in symptoms.In previous studies using carrot slices to test tumorigenicity, the same results were obtained [16,23].
In the pathogenicity test on oleander plants, the fi rst knots were formed on stems 28 days after inoculation.After 46 days the typical knots were observed on all infected plants (Figure 2).Negative control plants inoculated with sterile distilled water showed no symptoms.The pathogens were re-isolated from the knots and PCR confi rmed that Pseudomonas savastanoi pv.nerii was responsible for the symptoms.

Conclusion
The results showed that the inoculation of carrot slices technique can be successfully applied to the tumorigenicity test of all Pseudomonas savastanoi pathovar.The tumorigenicity technique with the use of carrot slices may also be applicable to other tumor-forming bacteria.
, an Agrobacterium radiobacter (B.01178) strain, an A. rubi (B.01336) strain [29,30], a P. savastanoi (B.01823) strain from National Collection of Agricultural and Industrial Microorganisms (NCAIM, Hungarian University of Agriculture and Life Sciences, Budapest, Hungary), a P. savastanoi pv.retacarpa (CFBP5513) strain and a P. savastanoi pv.fraxini (CFBP5062) strain from the French Collection for Plant-associated Bacteria (CFBP), which is a collection of the International Center for Microbial with a sterile scalpel and macerated in Sterile Distilled Water (SDW).A spoonful of the resulting suspension was streaked onto King B agar[35].The plates were incubated at room temperature (RT) for 48 hours -72 hours and pure cultures of bacterial isolates were analyzed by PCR (Polymerase chain reaction).The amplifi cation and sequencing of 16S rDNA was achieved using 63F (5'-CAGGCCTAACACATGCAAGTC-3') and 1389R (5'-ACGGGCGGTGTGTACAAG-3') universal primer pair[32].The Polymerase Chain Reaction (PCR) conditions were the following: initial denaturation at 94 °C for 5 min, followed by 35 cycles at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 90s.The fi nal extension was at 72 °C for 10 min.Amplifi cation was verifi ed on a 1% (w/v) agarose gel in 1 × TBE buffer.The PCR products were cleaned with the High Pure PCR Product Purifi cation Kit (Roche Diagnostics GmbH).The nucleotide sequence of the PCR-amplifi ed DNA fragment was determined and compared with sequences from the National Center for Biotechnology Information (NCBI Genebank) database, using the Basic Local Alignment Search Tool (BLAST) program.
The tumorigenicity tests on carrot discs and pathogenicity tests on oleander plants resulted in similar tumors on the plant parts.The carrot slice technique is a quite simple and responsible method for testing the tumorigenicity of P. savastanoi pv.nerii without plants.It requires less space and resources, the carrot taproot is easily accessible and the time required for a pathogenicity test can be signifi cantly reduced.This is the fi rst report where carrot slices were used for testing the tumorigenicity of P. savastanoi pv.nerri, P. savastanoi pv.fraxini and P. savastanoi pv.retacarpa.

Figure 1 :
Figure 1: Typical knots on carrot slice 14-16 days after inoculation: Legend: The fi rst row shows symptoms of other Pseudomonas savastanoi pathotypes, the second row shows symptoms of Agrobacterium spp.and absence of knot formation on sterile distilled water inoculated carrot slice, and from the third our Pseudomonas savastanoi pv.nerii isolates on carrot slices 14 days after inoculation.