Abstract
During the 2016 growing season, melon fruits were severely affected by rind necrosis and rot and nearly 80% of the fruits were unmarketable in a field in Koçarlı district of Aydın. In order to determine occurrences of the fruit infections in the following year, surveys were conducted in 2017. Isolations were made from the symptomatic fruits and bacterial growths were observed consistently. In this study, we identified the causal agents of melon fruit necrosis and rot. Pathogenicity of the bacterial strains was determined on melon fruit by needle injection method and thirteen strains were further characterized with phenotypic characterization, analysis of 16S rRNA gene sequences and specific PCR assays. As a result of the pathogenicity tests, brown spots and necrosis formation was observed initially and in the later stage, inoculated fruit burst out by the fermentative strains. The oxidative strains produced limited necrosis on the rind. Fermentative bacterial agents isolated from the melon necrosis and rot were identified as Enterobacter cloacae, Klebsiella pneumoniae and Kosakonia cowanii. Enterobacter cloacae strains were further tested with tDNA-PCR using T5A/T3B primers. The oxidative bacteria were phenotypically grouped within the fluorescent pseudomonads.
Homology search of the oxidative strains using the sequence information of partial 16S rRNA gene identified the strains as Pseudomonas spp. The oxidative strain 413-Si2-L2 and the reisolate 06R2K were the most similar to strains of Pseudomonas putida and P. reidholzensis including the type strains. The strain, 413-MKV-3 and the reisolate 03R2K were the most related to Pseudomonas fluorescens, P. baetica, P. syringae, and Pseudomonas sp. strains.
The ability of Enterobacter cloacae, Klebsiella pneumoniae and Kosakonia cowanii to cause disease on melon fruit was shown for the first time in this study by fulfilling Koch postulate. Furthermore, although limited rind necrosis was produced, pathogenicity of the fluorescent pseudomonads on melon fruit was also confirmed.





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Alves, M. S., da Silva Dias, R. C., Dias de Castro, A. G., Riley, L. W., & Moreira, B. M. (2006). Identification of clinical isolates of indole-positive and indole-negative Klebsiella spp. Journal of Clinical Microbiology, 44, 3640–3646.
Arai, T., Enomoto, S., & Goto, S. (1970). Determination of Pseudomonas aeruginosa by biochemicaltest methods. Japanese Journal of Microbiology, 14, 285–290.
Aysan, Y., Mirik, M., Ala, A., Sahin, F., & Cinar, O. (2003). First report of Pseudomonas viridiflava on melon in Turkey. Plant Pathology, 52, 800.
Bishop, A. L., & Davis, R. M. (1990). Internal decay of onions caused by Enterobacter cloacae. Plant Disease, 74, 692–694.
Brady, C. L., Venter, S. N., Cleenwerck, I., Engelbeen, K., de Vos, P., Wingfield, M. J., et al. (2009). Isolation of Enterobacter cowanii from Eucalyptus showing symptoms of bacterial blight and dieback in Uruguay. Letters in Applied Microbiology, 49, 461–465.
Brady, C., Cleenwerck, I., Venter, S., Coutinho, T., & De Vos, P. (2013). Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): Proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter. Systematic and Applied Microbiology, 36, 309–319.
Brisse, S., van Himbergen, T., Kusters, K., & Verhoef, J. (2004). Development of a rapid identification method for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates. Clinical Microbiology and Infection, 10, 942–945.
Braun-Kiewnick, A. and Sands, D.C. (2001). Pseudomonas in: Schaad, N.W., Jones, J.B. And Chun, W. (Eds.) Laboratory Guide for Identification of Plant Pathogenic Bacteria (pp. 84–120). APS press: St. Paul, Minnesota.
Clementino, M. M., De Filippis, I., Nascimento, C.R., Branquinho, R., Rocha, C.L, Martins, O.B. (2001). PCR analysis of tRNA intergenic spacer, 16S-23S internal transcribed spacer, and randomly amplified polymorphic DNA reveal inter- and intraspecific relationships of Enterobacter cloacae strains. Journal of Clinical Microbiology, 39, 3865–3870.
Drancourt, M., Bollet, C., Carta, A., & Rousselier, P. (2001). Phylogenetic analysis of Klebsiella species delineate Klebsiella and Raoultella gen. Nov., with description of Raoutella ornithinolytica comb. nov., Raoutella terrigena comb. nov. and Raoultella planticola comb. nov. International Journal of Systematic and Evolutionary Microbiolology, 51, 925–932.
Edwards, P. R., & Fife, M. A. (1956). Cyanide media in the differentiation of enteric bacteria. Applied Microbiology, 4, 46–48.
Farmer, J. J. I. I. I., Davis, B. R., Hickman-Brenner, F. W., McWhorter, A., Huntley-Carter, G. P., Asbury, M. A., et al. (1985). Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. Journal of Clinical Microbiology, 21, 46–76.
Frank, J. A., Reich, C. I., Sharma, S., Weisbaum, J. S., Wilson, B. A., & Olsen, G. J. (2008). Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology, 74, 2461–2470.
Frasson, D., Opoku, M., Picozzi, T., Torossi, T., Balada, Smits, T.H.M et al. (2017). Pseudomonas wadenswilerensis sp. nov. and Pseudomonas reidholzensis sp. nov., two novel species within the Pseudomonas putida group isolated from forest soil. International Journal of Systematic and Evolutionary Microbiology, 67, 2857–2861.
Furtado, G. Q., Guimarães, L. M. S., Lisboa, D. O., Cavalcante, G. P., Ariel, D. A. A., Alfenas, A. C., et al. (2012). First report of Enterobacter cowanii causing bacterial leaf spot on Mabea fistulifera, a native forest species in Brasil. Plant Disease, 96, 1576.
Grimont, P.A.D., and Grimont, F. (2005). Genus XII. Enterobacter. Vol 2. Part B. Brenner, D.J., N.R.Krieg, J.R. Staley (Eds.), in: Bergey’s Manual of Systematic Bacteriology, (pp. 661-669), 2nd Edn, springer: New York.
Hinton, D. M., & Bacon, C. W. (1995). Enterobacter cloacae is an endophytic symbiont of corn. Mycopathopathologia, 129, 117–125.
Hopkins, D. L., & Elmstrom, G. W. (1977). Etiology of watermelon rind necrosis. Phytopathology, 67, 961–964.
Horuz, S., Çetinkaya-Yıldız, R., Mirik, M., & Aysan, Y. (2014). Occurrence, isolation, and identification of Acidovorax citrulli from melon in Turkey. Plant Protection Science, 50, 179–183.
Inoue, K., Sugiyama, K., Kosako, Y., Sakazaki, R., & Yamai, S. (2000). Enterobacter cowanii sp. nov., a new species of the family Enterobacteriaceae. Current Microbiology, 41, 417–420.
Kido, K., Adachi, R., Hasegawa, M., Yano, K., Hikichi, Y., Takeuchi, S., Atsuchi, T., & Takikawa, Y. (2008). Internal fruit rot of netted melon caused by Pantoea ananatis(=Erwinia ananas) in Japan. Journal of General Plant Pathology, 74, 302–312.
Krawczyk, K., & Borodynko-Filas, N. (2020). Kosakonia cowanii as the new bacterial pathogen affecting soybean (Glycine max Willd.). European Journal of Plant Pathology, 157, 173–183.
Lehman D. (2005) Triple sugar iron agar protocols. American Society For Microbiology. https://www.asmscience.org/content/education/protocol/protocol.2842
McDevitt S. (2009). Methyl red and Voges-Proskauer test protocols. American Society For Microbiology. https://www.asmscience.org/content/education/protocol/protocol.3204
Nishijima, K. A., Couey, H. M., & Alvarez, A. M. (1987). Internal yellowing, a bacterial, disease of papaya fruits caused by Enterobacter cloacae. Plant Disease, 71, 1029–1034.
Nishijma, K. A., Alvarez, A. M., Hepperly, P. R., Shintaku, M. H., Keith, L. M., Sato, D. M., et al. (2004). Association of Enterobacter cloacae with rhizome rot of edible ginger in Hawaii. Plant Disease, 88, 1318–1327.
Pruesse, E., Peplies, J., & Glöckner, F. O. (2012). SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics, 28, 1823–1829.
Rosenblueth, M., Martínez, L., Silva, J., & Martínez-Romero, E. (2004). Klebsiella variicola, a novel species with clinical and plant-associated isolates. Systematic and Applied Microbiology, 27, 27–35.
Santana, M. A., Rodiguez, M., Matehus, J., Faks, J., Bocsanczy, A., Gerste, A., et al. (2012). A new bacterial disease of cassava in Venezuela caused by Enterobacter cloacae. International Journal of Agriculture and Biology, 14, 183–189.
Schaad, N. W., Jones, J. B., & Chun, W. (2001). Laboratory guide for identification of plant pathogenic Bacteria (3rd ed.). St. Paul, Minnesota, USA: APS Press.
Schroder, B. K., Waters, T. D., & du Toit, L. J. (2010). Evaluation of onion cultivars for resistance to Enterobacter cloacae in storage. Plant Disease, 94, 236–243.
Shields, P. and Cathcart, L. (2011). Motility test medium protocol. American Society For Microbiology. https://www.asmscience.org/content/education/protocol/protocol.3658
Stock, I., & Wiedemann, B. (2001). Natural antibiotic susceptibility of Klebsiella pneumoniae, K. oxytoca, K. planticola, K. ornitinolytica and K. terrigena strains. Journal of Medical Microbiology, 50, 396–406.
Takahashi, Y., Takahashi, K., & Kawano, T. (1997). Bacterial leaf rot of Odontioda orchids caused by Enterobacter cloacae. Annual Phytopathological Society of Japan, 63, 164–169.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.
Thomas, C. E. (1976). Bacterial rind necrosis of cantaloupe. Plant Disease Reporter, 60, 38–40.
Welsh, J., & McClelland, M. (1991). Genomic fingerprints produced by PCR with consensus tRNA gene primers. Nucleic Acids Research, 19, 861–866.
Online Sources: FAOSTAT: http://www.fao.org/faostat/en/#data/QC/visualize Last accessed January 16th 2021. https://www.tarimorman.gov.tr/GKGM/Belgeler/Uretici_Bilgi_Kosesi/Dokumanlar/kavun-karpuz.pdf
Acknowledgements
This work supported by Aydın Adnan Menderes University Scientific Research Fund (ADU-BAP) Project No. ZRF-17026. Author thanks graduate student Emine Şenay for collection of the samples, help with biochemical tests and undergraduate student Erman Öğütcü for collection of the samples and photography.
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Özdemir, Z. Identification of Enterobacteriaceae members and fluorescent pseudomonads associated with bacterial rind necrosis and rot of melon in Turkey. Eur J Plant Pathol 160, 797–812 (2021). https://doi.org/10.1007/s10658-021-02282-z
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DOI: https://doi.org/10.1007/s10658-021-02282-z


