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Soft rot disease caused by Klebsiella aerogenes on Austrocylindropuntia subulata in Iran

Authors:
Esmaeil Basavand at Vali Asr University of Rafsanjan
Esmaeil Basavand
Pejman Khodaygan at Vali Asr University of Rafsanjan
Pejman Khodaygan
Valiollah Babaeizad at Sari Agricultural Sciences and Natural Resources University
Valiollah Babaeizad
Heshmat Rahimian at Sari Agricultural Sciences and Natural Resources University
Heshmat Rahimian
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Abstract

During the surveys in the major cactus-growing regions of Mazandaran Province of Iran, symptoms of soft rot were observed on the Eve's pin (Austrocylindropuntia subulate) plants. Cactus samples with typical symptoms including soft rot, black colored and water-soaked lesion at the entire of plants stem were collected and transferred to the laboratory for further assays. Isolation of causal agent from the infected stems and phenotypical and biochemical tests were performed using conventional microbiological methods. Based on isolates reactions in phenotypic tests, the isolates were found to belong to Enterobacteriaceae. The characteristics of the representative isolate were determined by amplification and sequencing of rpoB gene. In conclusion, the causal pathogen from the affected cactus was identified as Klebsiella aerogenes based on its pathogenicity, physiological and biochemical properties and molecular test. This is the first attempt to confirm the occurrence of soft rot disease of A. subulata caused by K. aerogenes in Iran.
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Indian Phytopathology
https://doi.org/10.1007/s42360-020-00201-6
NEW REPORTS
Soft rot disease caused byKlebsiella aerogenes onAustrocylindropuntia
subulata inIran
EsmaeilBasavand1· PejmanKhodaygan1 · ValiollahBabaeizad2· HeshmatollahRahimian2·
HosseinAliMirhosseini3
Received: 30 September 2019 / Revised: 9 December 2019 / Accepted: 27 January 2020
© Indian Phytopathological Society 2020
Abstract
During the surveys in the major cactus-growing regions of Mazandaran Province of Iran, symptoms of soft rot were observed
on the Eve’s pin (Austrocylindropuntia subulate) plants. Cactus samples with typical symptoms including soft rot, black
colored and water-soaked lesion at the entire of plants stem were collected and transferred to the laboratory for further assays.
Isolation of causal agent from the infected stems and phenotypical and biochemical tests were performed using conventional
microbiological methods. Based on isolates reactions in phenotypic tests, the isolates were found to belong to Enterobacte-
riaceae. The characteristics of the representative isolate were determined by amplification and sequencing of rpoB gene. In
conclusion, the causal pathogen from the affected cactus was identified as Klebsiella aerogenes based on its pathogenicity,
physiological and biochemical properties and molecular test. This is the first attempt to confirm the occurrence of soft rot
disease of A. subulata caused by K. aerogenes in Iran.
Keywords Klebsiella aerogenes· Austrocylindropuntia subulate· Soft rot disease· rpoB
During 2018, soft rot symptoms on cactus (Austrocylindro-
puntia subulata) were found in the major cactus-growing
regions of Mazandaran Province, Iran. Symptoms included
soft rot black color and water-soaked lesions at the entire
of plant stem, that initiated symptoms were as small water-
soaked and then enlarged quickly to the whole stem (Fig.1).
In order to identify the causal agent, the plants were trans-
ferred to the laboratory. Infected tissues of diseased plants
after cutting into 4mm in length pieces were surface-ster-
ilized in 0.5% sodium hypochlorite for 30s and rinsed 3
times in sterilized water. Tissues were kept into sterilized
distilled water (SDW) for 25min, and the resulting suspen-
sion was streaked onto plates containing sucrose nutrient
agar (SNA) and modified eosin methylene blue (EMB) con-
taining 1% glycerol and the plates incubated at 26–28°C
(Borkar 2017). After 4–5days, single white mucoid colonies
on SNA and green metallic colonies formed on EMB media
were selected for investigation. The bacterial isolates were
eventually purified by repetitive streaking onto SNA medium
(Schaad etal. 2001) and were kept at –80°C in a glycerol
mixture (40% v/v) for further use. One of the isolates were
deposited in the Culture Collection of Microorganisms at
the Vali-E-Asr University of Rafsanjan (VRU 1925), Iran.
Selected isolates were non fluorescent on King’s B, and were
negative in tests for oxidase, hydrolysis of tween 80, urease,
3’-Ketolactose, phenylalanine deaminase, lecithinase, NaCl
4 and 6% tolerance, indole, arginine dihydrolase, tyrosinase
activity, but positive in tests for H2S from peptone, thiosul-
fate and cysteine, RSS, gas from glucose, nitrate reduction
and hydrolysis of casein, starch, esculin, gelatin, and arbu-
tin. Litmus milk was turned alkaline and digested by the
isolates. All of the tested isolates isolates used d-sorbitol,
d -xylulose, d -raffinose, succinic acid, d-lactose, proline,
d-galactose, fructose, maltose, l-serine, sucrose, l-valine,
l-arabinose, fumarate, melibiose, glucose, mannitol,
malonate, but not l-tartrate, d-cellobiose, d-tartrate, sor-
bose, l-ornithine, l-arginine as carbon source for growth.
The isolates induced a hypersensitive reaction (HR) on
geranium (Pelargonium × hortorum) 24h post inoculation.
* Pejman Khodaygan
pkhodaygan@vru.ac.ir
1 Department ofPlant Pathology, Vali-E-Asr University
ofRafsanjan, Rafsanjan, Iran
2 Department ofPlant Pathology, Sari Agricultural Sciences
andNatural Resources University, Sari, Iran
3 Department ofPlant Protection, Gorgan University
ofAgricultural Sciences andNatural Resources, Gorgan, Iran
Indian Phytopathology
1 3
For pathogenicity test, a bacterial suspension was adjusted
to approximately 1 × 106CFU/ml from cultures grown on
nutrient agar medium for 24h. Under glasshouse condition,
one-year-old stem of A. subulata was inoculated with the
bacterial suspension using a sterile insulin syringe and cov-
ered by the plastic bags. Controls were injected with SDW.
The inoculated isolates were caused soft rot symptoms on A.
subulata seedling after one week in the site of injection in
pathogenicity test. The inoculated bacteria were re-isolated
from the symptoms and no symptoms were observed on the
controls (Fig.1).
Molecular identification was conducted by amplifying the
RNA polymerase beta subunit region (rpoB) in representa-
tive isolate using the rpoBCM7-F/CM31b-R primer pair
(Brady etal. 2008). Extraction of genomic DNA from isolate
BK1 was carried out using a CTAB based method described
by Ausuble etal. (1992) with slight modification. In PCR
assay, Universal PCR Kit Ampliqon Taq DNA Polymerase
Master Mix Red (Odense, Denmark) was used according to
the manufacturer’s recommendation. The PCR amplifica-
tion was carried out in 25µl reaction mixture containing
50ng total DNA and 1µl of each primer (10pmolµl−1).
PCR was performed in T-100 (Applied Biosystem) ther-
mal cycler. The amplification product of the expected size
(1000bp) was detected by 1% agarose gel stained with Red
Safe Nucleic Acid Staining Solution (iNtRON Biotechnol-
ogy, South Korea). The PCR products were submitted to
Bioneer Corporation (South Korea) to be sequenced. Raw
sequences were directly edited using BioEdit v. 7.0.5.2 and
compared with those deposited in GenBank and then com-
pared to those available in the NCBI GenBank using the
blast in algorithm. However, the blast search showed that
the nucleotide sequence of cactus isolate (accession num-
ber: MN520290) was 100% similar to K. aerogenes strain
(AR-0018) available in GenBank. In conclusion, the causal
pathogen isolated from the affected cactus was identified
as K. aerogenes based on its physiological and biochemi-
cal properties and molecular test. More recently, species
of Klebsiella bacterium has increasing importance as plant
pathogen and reported in different crops worldwide (Vasebi
etal. 2018). Based on our knowledge, this is the first occur-
rence of soft rot disease caused by K. aerogenes on A. subu-
lata in Iran.
Acknowledgements This research was supported by, Vice-Chancellor
of Research and Technology grant (AGR95PP5938) at the Vali-E-Asr
University of Rafsanjan, Iran.
References
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Schaad NW, Jones JB, Chun W (2001) Laboratory guide for identifica-
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Vasebi Y, Faghihi MM, Seyahooei MA, Bagheri A (2018) First report
of eggplant’s meristem Rotting and necrosis caused by Klebsiella
pneumonia in Iran. In: Proceedings of the 23rd Iranian Plant Pro-
tection Congress, Gorgan University of Agricultural Sciences and
Natural Resources, p. 542.
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Fig. 1 a Symptoms of bacterial
soft rot caused by K. aero-
genes on Austrocylindropuntia
subulata. b Typical soft rot
symptoms (arrow) 1 week after
inoculation of K. aerogenes bac-
terium on the K. aerogenes on
Austrocylindropuntia subulata
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... This study identified two pathogenic isolates, S9 and S11, as K. michiganensis. Klebsiella is a genus of the Enterobacteriaceae that has been reported in past studies as the cause of soft rot disease in various plants (Basavand et al., 2020;Chandrashekar et al., 2023;Fan et al., 2016). Furthermore, K. michiganensis was reported as the cause of soft rot disease on lettuce in Iran (Parvin et al., 2023). ...
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First report of eggplant’s meristem Rotting and necrosis caused by Klebsiella pneumonia in Iran
  • Y Vasebi
  • M M Faghihi
  • M A Seyahooei
  • A Bagheri
Schaad NW, Jones JB, Chun W (2001) Laboratory guide for identification of plant pathogenic bacteria. APS Press, Minnesota Vasebi Y, Faghihi MM, Seyahooei MA, Bagheri A (2018) First report of eggplant's meristem Rotting and necrosis caused by Klebsiella pneumonia in Iran. In: Proceedings of the 23 rd Iranian Plant Protection Congress, Gorgan University of Agricultural Sciences and Natural Resources, p. 542.
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Ausuble F, Brent FM, Kingestone RE, Moor DD, Smith JA, Seideman JG, Struhl K (1992) Curr Protoc Mol Biol. Wiley Interscience, New York Borkar SG (2017) Laboratory techniques in plant bacteriology. CRC Press, New York Brady C, Cleenwerck I, Venter S, Vancanneyt M, Swings J, Coutinho T (2008) Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis (MLSA). Syst Appl Microbiol 31:447-460