Molecular identification of pathogen causing leaf rot of onion

Introduction

Initially small water-soaked spots appeared, then became small constricted area and then formed irregular translucent water-soaked oily lesion on leaves. Later became necrotic and hanging down of leaves at constricted area. Lower leaves bent down and necrotic area on the leaves formed and extended. The bent plants at the neck region have been observed. The leaves became sunken and soft at affected area, when the leaves were still green. At severe stage, leaves turned yellow and all the leaves were collapsed to the ground and the plants dies. Freshly cut infected portion of the plant showed milky white bacterial exudate in the ooze test.

Materials and Methods

Identification of bacteria by 16S rRNA methods through PCR technique

Identification of culture by molecular method PCR based approach was carried out to confirm the identification of the bacterium. The following procedure was followed for identification of unknown bacteria.

Materials required

• Forward and reverse primers (100 ng/μl.) • 10 mm deoxy nucleotide triphosphates (dNTPs viz., dATP, dGTP, dTTP)

Genomic DNA extraction

For extraction of genomic DNA from the test bacterial culture, the single colony from the fresh one day old culture was taken and inoculated in the 50 ml of nutrient broth and kept for the shaking incubation at 28±2 °C for 48h.

After 48h, 1.5 ml of culture was taken through pipette and transferred into 1.5 ml eppendorf tube. Then centrifuged for 2 min at 14,000 rpm at room temperature and added 550 μl of TE buffer + lysozyme (0.1 mg/ml) and resuspended by pipetting. Incubated the suspension for 30 min at 37 °C and then mixed the contents by flipping tube. Added 80 μl of CTAB/NaCl and mixed by flipping tube. Incubate for 10 min at 65 °C. Added 700 μl of Chloroform/Isoamylalcohol (24: 1) twice. Mixed the contents by flipping tubes for 15 sec. and centrifuged for 5 min at 14,000 rpm at RT. Transferred the aqueous layer (without disturbing or carrying over any of the white middle layer) to new 1.5 ml tube. This step was repeated several times when proteins are taken up with the aqueous layer. Added 2 volume of 2propanol (Isopropanol) and inverted several times to mix. Centrifuged for 15 min at 4 °C and stored at-20 °C for 3h. Gently drained out the supernatant and carefully added approx. 1 ml ice cold 70 percent ethanol. Collected the DNA by centrifugation for several minutes at 14,000 rpm at RT.

Carefully removed the supernatant and evaporated the remained ethanol in the laminar flow cabin. Dissolved the DNA in 50 μl sterilized distilled water or nuclease free water. Added 6 μl of RNase to DNA solution, to remove the contaminating RNA from precipitate. Dissolved DNA was incubated over night at 4 °C and then utilizad the bacterial DNA for molecular identification.

Primers

A representative isolate of the causal agent was subjected to PCR based identification by utilizing the primers of 4 different relevant bacterial pathogens viz., Acinetobacter haemolyticus, Acinetobacter calcoaceticus, Enterobacter cloacae strain ATCC 13047 and Pectobacterium carotovorum subsp. carotovorum. The primers of these bacteria were designed from nucleotide sequence available in the data base. The primers of relevant bacteria were presented below.

The primers of relevant bacteria were presented below.

Organism

PCR reaction

The PCR amplification was done with 4 primers from related bacterial pathogens, after confirmation the single primer was used for the isolates of different surveyed districts. PCR amplification of 16S rRNA primers were performed using 100 ng DNA template 1X PCR buffer 200 μM dNTPs mix 0.4 μM primers and 1 U Taq DNA polymerase (Merck, Banglore, India) and sterized sterile distilled water to make final volume of 20 μL. PCR amplification was conducted with a thermocycler (Eppendorf, Vapo project, Germany).

The amplification was carried out with an initial 94 °C for 4 min, followed by 30 cycles of 30 seconds, denaturation at 94 °C 45 seconds of primer annealing at 55 °C, 1 min extension at 72 °C. The final extension step was for 10 min at 72 °C.

Gel electrophoresis

One percent gel was prepared by adding 1gm of agarose to 100ml of 1x TAE buffer and kept in the micro wave oven for about 1-2 min for melting, then allowed to cool. After mild cooling of agarose, added 0.5 µl of ethydium bromide, then poured to the gel tray for solidification. A coumb was placed on the gel to make the wells, after solidification the coumb were taken out. Gel was taken out side from the gel casting unit and tank buffer (1x TAE) was poured into electrophoresis unit, then placed the gel in the electrophoresis unit. The DNA along with the dye and primers were added into the wells through micropipette and the temperature was set at 100 °C for 35 minutes. After 35 minutes the gel was taken and kept for the gel documentation and the gel image was taken in the Gensys software in Department of Biotechnology, COH-Bagalkote.

Results

16S rRNA identification of bacteria causing leaf rot disease of onion through PCR technique

The [3] were conducted an experiment on isolation and identification of Enterobacter cloacae from onion (Allium cepa L.,) leaf spot disease. They isolated the bacterial colonies on nutrient agar (NA) from infected leaves. They took pure single colonies which on gram staining, expressed as gram negative. Koch's postulate was proved on onion seedlings. They reconfirmed the culture as E. cloacae by molecular based identification through specific primers (Forward-16S RF-AGA GTT TGA TCG TGG, CTC AG, Reverse 16S RP-CGG TTA CCT TGT TAC GAC TT). To their knowledge, this is the first report of bacteria causing leaf spot of onion.

Hauben et al. (1998) [2] identified the phylogenetic position of phytopathogens within the Enterobacteriaceae. The almost complete 16S rDNA sequences of twenty-nine plantassociated strains, repressenting species of the genera Erwinia, Pantoea Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

Hafiz et al. (2013) [1] were conducted research on molecular and pathogenic diversity of Erwinia carotovora Spp. atroseptica associated with potato blackleg and soft rot. The isolates were identified as Eca by biochemical tests and their identity was further confirmed by polymerase chain reaction (PCR) using subspecies-specific primers Eca 1F and Eca 2R which amplified the Eca-specific band of 690 bp. Identification and characterization of Enterobacter complex of mulberry wilt disease in China, was done based on biolog metabolic profile, fatty acid methyl esterase analysis (FAME) and sequence analysis of partial 16S rDNA and rpoB genes. The 4 strains were identified as members of genus Enterobacter. The causal organism for mulberry wilt disease was Enterobacter cloacae. However, this study has found that other Enterobacter spp. (Enterobacter asburie, Enterobter morri and Enterobacter spp.) also cause mulberry wilt disease (Wang et al. 2010) [4] .

Conclusion

The pathogenicity test was proved that, the bacterium is pathogenic on onion seedlings, as isolated bacteria produced the peculiar disease symptoms of leaf rot disease. Through 16S rRNA molecular analysis, the test bacterium was identified as Enterobacter cloacae, which was amplified at 493bp.

Acknowledgement

AcknowledgmentsThe authors gratefully acknowledge the Dept. Plant Pathology for providing guidance during my research period and helping in publishing the research paper.

References

1. Hafiz FA, Muhammad J, Musharaf A, Ayesha B, Asad A, Shaukat HS, et al. Molecular and pathogenic diversity identified among isolates of Erwinia carotovora subsp. atroseptica associated with potato blackleg and soft rot. Pakistan Journal of Botany. 2013;45(3):1073-1078.
2. Hauben L, Moore ERB, Vauterin L, Steenackers M, Mergaert J, Verdonck L, et al. Phylogenetic position of phytopathogens within the Enterobacteriaceae. Systematic and Applied Microbiology. 1998;21:384397.
3. Kiran Kumar KC, Pradnyarani N, Jayasudha SM, Nataraja KD, Mesta RK. Isolation and identification of Enterobacter cloacae from onion leaf spot. National Symposium on Detection, Diagnosis and Management of Plant Diseases. 2017;54.
4. Wang GF, Praphat K, Xie GL, Zhu B, Li B, Liu B, et al. Bacterial wilt of mulberry (Morus alba) caused by Enterobacter cloacae in China. Plant Disease. 2008;92:483.